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                        UNITED STATES OF AMERICA 

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                 DEPARTMENT OF HEALTH AND HUMAN SERVICES

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                          PUBLIC HEALTH SERVICE

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                      FOOD AND DRUG ADMINISTRATION

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                     CENTER FOR BIOLOGICS EVALUATION
                                RESEARCH

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                      BIOLOGICAL RESPONSE MODIFIERS
                           ADVISORY COMMITTEE

                                 MEETING

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                                 TUESDAY
                             MARCH 24, 1998

                The meeting took place in Plaza II and III,
          Doubletree Hotel, Rockville, MD at 8:30 a.m., Julie M.
          Vose, M.D., Chair, presiding.

          PRESENT:

          JULIE M. VOSE, M.D., Chair
          GAIL DAPOLITO, Executive Secretary
          W. FRENCH ANDERSON, M.D., Member
          HUGH AUCHINCLOSS, JR., M.D., Member
          ELLIN R. BERMAN, M.D., Member
          VIRGINIA C. BROUDY, M.D., Member
          PAMELA M. HARTIGAN, Ph.D., Member

          RICHARD HONG, M.D., Member
          MICHAEL S. KATZ, Patient Representative
          EUGENIE S. KLEINERMAN, M.D., Member
          KATHERINE E. KNOWLES, Consumer Representative
          ABBEY S. MEYERS, Consumer Representative (by phone)

                                                                          2

          PRESENT (cont'd)

          WILLIAM M. O'FALLON, Ph.D., Member
          JONATHAN SILVER, M.D., Member
          ALTON FLOYD, Ph.D., Temporary non‑voting Member
          PATRICIA KEEGAN, M.D., FDA Representative
          CAROLE B. MILLER, M.D., FDA Representative
          JAY P. SIEGEL, M.D., FDA Representative
          KAREN WEISS, M.D., FDA Representative

          EDA BLOOM, Ph.D., FDA Speaker
          JUDITH KASSIS, Ph.D., FDA Speaker
          STEPHEN LITWIN, M.D., FDA Speaker
          EDWARD MAX, M.D., FDA Speaker
          PHILIP NOGUCHI, M.D., FDA Speaker
          JAMES BERENSON, M.D., Sponsor Represenative
          CINDY JACOBS, Ph.D., M.D., Sponsor Representative

          MONIKA KRIEGER, Ph.D., Sponsor Representative

          ALSO PRESENT:

          KEN ANDERSON, M.D.
          KEITH STEWART, M.D.
          JOE TARNOWSKI, Ph.D.

          BOB VESCIO, M.D.
          MIKE WHITE, Ph.D.

                                                                          3

                               A‑G‑E‑N‑D‑A

          OPENING AND ADMINISTRATIVE REMARKS
            Executive Secretary Gail Dapolito . . . . . . .  4
            Chairperson Julie Vose  . . . . . . . . . . . .  7

          OPEN COMMITTEE DISCUSSION:  Premarketing Approval
          Application BP94‑001/03, for Ceprate SC System,
          CellPro Incorporated

            Presentation by CellPro, Inc.
              I.  Introduction
                    Monika Krieger, Ph.D. . . . . . . . . .  7
                      Director, Regulatory Affairs, CellPro

             II.  Description of PCR Assay for Detection of   

                  Tumor Cells Results of Phase I/II Study
                    James Berenson, M.D.  . . . . . . . . .  9
                      Chief of Medical Oncology, West Los
                      Angeles VA Medical Center

            III.  Results of Phase III Study
                  Additional Tumor Purging Results

                    Cindy Jacobs, Ph.D., M.D. . . . . . . . 17
                      Vice‑President, Clinical Research CellPro

          FDA Perspective
              I.  Introduction
                    Stephen Litwin, M.D., OTRR, CBER  . . . 67

             II.  Description of PCR‑Based Myeloma Cell Assay
                    Edward Max, M.D., OTRR, CBER  . . . . . 75

            III.  Safety and Efficacy
                    Stephen Litwin, M.D.  . . . . . . . . . 87

          Overview of Research Programs Laboratory of Cellular

          Immunology Laboratory of Developmental Biology

              Philip Noguchi, M.D.  . . . . . . . . . . . .167
               Division Director, Cellular and Gene Therapies

              Eda Bloom, Ph.D.  . . . . . . . . . . . . . .170
               Lab Chief, Laboratory of Cellular Immunology

              Judith Kassis, Ph.D.  . . . . . . . . . . . . . 
               Laboratory of Developmental Biology

                                                                          4

     1                    P‑R‑O‑C‑E‑E‑D‑I‑N‑G‑S

     2                                             (8:38 a.m.)

     3                MS. DAPOLITO:  Good morning.  I would like

     4    to welcome Dr. Vose to the 22nd meeting of the

     5    Biological Response Modifiers Advisory Committee.  I

     6    am Gail Dapolito, and I am the designated federal

     7    official for today's proceedings.  I would like to

     8    begin by introducing the committee members and

     9    consultants seated on our panel today.  I will begin

    10    on my left.  Dr. Mike O'Fallon, Mayo Clinic; Dr.

    11    Jonathan Silvers is here from the National Institutes

    12    of Health; Dr. Virginia Broudy, University of

    13    Washington, School of Medicine;  our industry

    14    representative, Dr. Alton Floyd of Trigon

    15    Technologies; Dr. Eugenie Kleinerman, MD Anderson

    16    Cancer Center; Dr. Richard Hong, University of

    17    Vermont; our Chair, Dr. Julie Vose, University of

    18    Nebraska; Dr. Ellin Berman, Memorial Sloan‑Kettering

    19    Cancer Center; Dr. Pamela Hartigan, VA Medical Center,

    20    Westhaven; Dr. Hugh Auchincloss, Harvard Medical

    21    School; Ms. Katherine Knowles, Health Information

    22    Network.  Ms. Knowles has graciously accommodated our

    23    very last minute request to join us as a consumer

    24    representative today.  We thank her very much.  Next

    25    is our patient represenative, Mr. Michael Katz,

                                                                          5

     1    International Myeloma Foundation, and Dr. French

     2    Anderson, University of California.  

     3                The Center for Biologics Evaluation and

     4    Research is represented today by Dr. Patricia Keegan,

     5    Dr. Karen Weiss, and Dr. Jay Siegel, who will join us

     6    later.  

     7                Dr. Vose, with your permission, I will now

     8    read the conflict of interest statement.  The

     9    following announcement is made part of the pubic

    10    record to preclude even the appearance of a conflict

    11    of interest at this meeting.  Pursuant to the

    12    authority granted under the Committee charter, the

    13    Commissioner of FDA has appointed Ms. Katherine

    14    Knowles as a temporary voting consumer representative,

    15    and Dr. Alton Floyd as a temporary non‑voting industry

    16    representative.  In addition, the Director of the

    17    Center for Biologics Evaluation and Research has

    18    appointed the following individuals as temporary

    19    voting members:  Mr. Michael Katz and Dr. Jonathan

    20    Silver.  Based on the agenda made available, it has

    21    been determined that all financial interests in firms

    22    regulated by the Center for Biologics Evaluation and

    23    Research, which have been reported by the

    24    participating members and consultants as of this date

    25    present no potential for an appearance of a conflict

                                                                          6

     1    of interest at this meeting with the following

     2    notations to preclude even the appearance of a

     3    conflict of interest.

     4                Mr. Michael Katz, a patient

     5    representative, reported that he is a volunteer member

     6    of the Executive Board of Directors of the

     7    International Myeloma Foundation, a non‑profit

     8    organization.  The International Myeloma Foundation is

     9    being reimbursed by CellPro for the production costs

    10    of an informational booklet on bone marrow

    11    transplantation.  Mr. Katz participated in a previous

    12    meeting between the IMF and a representative from

    13    CellPro regarding bone marrow transplantation.  Mr.

    14    Katz received no personal remuneration for attending

    15    this meeting.

    16                Dr. Carol Miller has recused herself from

    17    today's Committee discussions.  The following members

    18    and consultant have no interests to disclose:  Dr.

    19    French Anderson, Dr. Hugh Auchincloss, Dr. Ellin

    20    Berman, Dr. Virginia Broudy, Dr. Alton Floyd, Dr.

    21    Pamela Hartigan, Dr. Richard Hong, Dr. Eugenie

    22    Kleinerman, Ms. Katherine Knowles, Ms. Abbey Meyers,

    23    Dr. Michael O'Fallon, Dr. Jonathan Silver, and Dr.

    24    Julie Vose.  In the event that the discussions involve

    25    other products or firms not already on the agenda for

                                                                          7

     1    which the FDA participants have a financial interest,

     2    the participants are aware of the need to exclude

     3    themselves from such involvement and their exclusion

     4    will be noted for the public record.  I turn it over

     5    to you, Dr. Vose.

     6                CHAIRPERSON VOSE:  Thank you.  I'd like to

     7    welcome everyone to the hearings today.  And I think

     8    that we will go ahead and ‑‑ is there anyone today

     9    that would like to come forward for the open public

    10    comment?  Not seeing anyone at this time, we will go

    11    ahead with the hearings today.  First would be to

    12    initiate the discussion on the pre‑market approval for

    13    Ceprate, CellPro Incorporated.  And we will go ahead

    14    with the presentation by CellPro.

    15                DR. KRIEGER:  Good morning.  On behalf of

    16    everyone at CellPro, I would like to thank the FDA for

    17    inviting us to speak this morning and present the

    18    results of the Phase III Study.  This study was

    19    designed to expand the label indication for the

    20    CellPro Ceprate SC stem cell concentration system to

    21    include the use of peripheral blood.

    22                The first slide here shows the instrument,

    23    the CellPro instrument system with the disposables

    24    attached to it.  This system is currently in use at

    25    300 medical centers around the world and more than

                                                                          8

     1    6,000 patients have been treated with cells that have

     2    been selected using the CellPro system.

     3                Our presentation today will be divided

     4    into three parts.  Dr. James Berenson, Professor of

     5    Medicine at UCLA will present the first part.  He will

     6    discuss the Phase I/II study, which was the basis for

     7    our Phase III trial.  He will present the development

     8    of the tumor detection assay which was used in our

     9    study as well as the clinical trial results from the

    10    Phase I/II study.  

    11                Then Dr. Cindy Jacobs from CellPro, our

    12    Vice President of Clinical Research, will present the

    13    results of the Phase III study, and she will also

    14    summarize the literature on additional tumor purging

    15    results using the Ceprate SC in patients with multiple

    16    myeloma, breast cancer, and lymphoma.  

    17                After the presentation, if there are

    18    questions, we have a number of people with us prepared

    19    to answer questions.  We have four clinical

    20    investigators with us who were part of the Phase III

    21    trail; Dr. Berenson, Dr. Vescio, Dr. Stewart, and Dr.

    22    Anderson.  We also have two statisticians with us who

    23    can answer questions; Dr. Mike White of CellPro and

    24    Dr. Brent Blumenstein of the Fred Hutchinson.  In

    25    terms of questions on the PCR assay, we have some

                                                                          9

     1    additional people; Dr. Jerry Radich from Fred

     2    Hutchinson, Dr. Amy Ross from MRD Diagnostics.  And

     3    then if there are questions on the clinical trials,

     4    there are, of course, those of us from CellPro,

     5    including Dr. Jacobs, Dr. Sing, and myself.

     6                Okay, I would like to now turn the meeting

     7    over to Dr. Berenson.

     8                DR. BERENSON:  Good morning.  Multiple

     9    myeloma is a bone marrow‑based malignancy

    10    characterized by monoclonal plasma cells, these fried

    11    egg‑appearing cells in this patient's bone marrow. 

    12    This is the second most common hematological

    13    malignancy with 15,000 new cases per year.  Although

    14    a variety of different chemotherapeutic and biologics

    15    have been tried with varying response rates. none of

    16    these have translated to cure with a median survival

    17    of about 30 months.  The use of high‑dose therapy

    18    followed by either allogeneic or autologous bone

    19    marrow support has a higher response rate than the

    20    conventional regimens.  Whether it leads to an

    21    improvement in survival was not clear until the

    22    publication of a recent randomized study from the

    23    French Intergroup, in which 200 patients were

    24    randomized to either receive conventional therapy or

    25    conventional therapy followed by high‑dose with

                                                                         10

     1    Melphalan and TBI followed by in this case autologous

     2    bone marrow transplant.  In this study published in

     3    the New England Journal of Medicine, there was an

     4    improvement in overall survival as shown here in the

     5    high‑dose arm compared to the conventional arm, and a

     6    recent update of this study presented to the ASH

     7    Meeting in San Diego in December continues to show the

     8    improvement in overall survival in the arm that

     9    underwent the high‑dose followed by bone marrow

    10    support.  

    11                Although one thinks of myeloma as a bone

    12    marrow‑based disease, there is evidence for peripheral

    13    blood involvement in most if not all patients, the

    14    most specific of which is based on molecular

    15    techniques developed in our laboratory and others.  So

    16    that, therefore, it becomes not only of biologic, but

    17    possibly clinical importance to determine where in B

    18    cell differentiation this tumor may begin.  In fact,

    19    we know that CD34 is expressed on the pluripotent stem

    20    cell, and cells selected for this in the peripheral

    21    blood are capable of engrafting patients following

    22    myeloablative chemotherapy.  However, we know that

    23    this antigen is also expressed on early B cells in

    24    this cascade.  So, therefore, it became important to

    25    determine if any CD34 expressing cells were also part

                                                                         11

     1    of the malignant clone.

     2                Now we are fortunate in multiple myelomas

     3    to have an excellent marker for the tumor, and that is

     4    based on the antibody that is produced by those

     5    monoclonal plasma cells and the specificity resides in

     6    the variable region, the amino terminal portion of the

     7    molecule which gives rise to its antigen recognition

     8    specificity.  Three parts of that portion of the

     9    molecule called hyper‑variable regions are the areas

    10    of the molecule with directly contact antigen.  These

    11    have also been called complimentarity determining

    12    regions.

    13                Thus, a molecular marker is possible in

    14    myeloma to determine malignancy based on the sequence

    15    at the gene level which gives rise to that portion of

    16    the molecule.  Now the variable region is made up of

    17    50 different genes which have been divided into 7

    18    different families.  Thus, one can develop a set of

    19    primers here in the 5 prime region in the leader part,

    20    and then in the 3 prime region, one can develop a set

    21    of primers based on C‑gamma for IgG‑producing myeloma

    22    or C‑alpha for IgA producing myeloma.  And using PCR

    23    technique on RNA turned into DNA by reverse

    24    transcriptase, one can obtain the sequence of this

    25    portion of the molecule.

                                                                         12

     1                Now importantly, those CDR's are again

     2    very specific in terms of determining malignancy, and

     3    that is because they are the portions of the molecule

     4    which bind to antigen.  The CDR3 region is ont only

     5    made of a short stretch of nucleotides called

     6    diversity segment, but also is accompanied by the

     7    addition of non‑germ line nucleotides called end‑

     8    region addition.  So it has additional specificity. 

     9    Thus, by obtaining that sequence that is expressing

    10    that part of the molecule, one has an excellent

    11    molecular marker. 

    12                Here is an example of one of the sequences

    13    that we obtained.  In fact, this was one of the first

    14    ones many years ago, and this is what we called WAD#1,

    15    the patient sequence, and this is the most homologous

    16    germ line sequence called V1DP5.  You can see in most

    17    parts of the molecule, there is identity between the

    18    patient sequence and the germ line sequence indicated

    19    by the dots.  But in those regions that bind to

    20    antigen by that process of somatic mutation, so the

    21    final antibody produced can more avidly bind to

    22    antigen, there are differences, again called somatic

    23    mutations.  So this becomes an excellent specific

    24    tumor marker that we can develop primers for to detect

    25    malignant cells in the CDR1 and an accompanied 3 prime

                                                                         13

     1    primer encompassing that CDR3, and if you recall, that

     2    consists of that D segment plus the addition of non‑

     3    germ line nucleotides.  Thus, the combination of the

     4    CDR1 and the CDR3, pairs of those primers, becomes

     5    very specific at detecting only malignant cells in

     6    mixed population.

     7                So in order to first answer the question

     8    of whether CD34 was expressed onto any malignant

     9    cells, CD34 cells were first enriched using the

    10    Ceprate device followed by flow sorting using a

    11    Ceprate antibody against CD34.  And using patient‑

    12    specific primers as I showed were generated in a

    13    similar manner to the previous slide, we could not

    14    find any amplified PCR product in the CD34 purified

    15    population, and yet the bone marrow from that same

    16    patient containing the tumor cells diluted out even

    17    10,000‑fold, we were able to find amplified product,

    18    suggesting indeed that CD34 was not expressed on the

    19    malignant clone.

    20                So this then provided the rationale for

    21    our Phase II study in which patients with advanced

    22    myeloma underwent high‑dose chemotherapy followed by

    23    the use of autologous CD34 selected peripheral blood

    24    stem cells.  The rationale, of course, the bone marrow

    25    has a lot of tumor and the blood has a little tumor,

                                                                         14

     1    and theoretically selecting for CD34 will reduced the

     2    tumor burden that will be given back to the patient

     3    following the myeloablative chemotherapy.

     4                The regimen used is outlined here.  We

     5    mobilized stem cells using intravenous

     6    cyclophosphamide, four days of oral prednisone, and

     7    daily injections of G‑CSF.  The stem cells were

     8    collected and then selected using the Ceprate device

     9    and stored away.  Patients were then given high doses

    10    of Busulfan at 15/kg and cyclophosphamide at 120/kg. 

    11    They then received back the stem cells, which again

    12    were CD34 selected.  GM‑CSF was used to enhance

    13    recovery.  And in this particular Phase II trial, we

    14    used interferon and dexamethasone for one year post‑

    15    transplant as maintenance treatment.

    16                Now the tumor detection assay developed by

    17    Bob Vescio in our group is outlined in the next few

    18    slides.  Again, primers were made that were

    19    complimentary to the unique regions in the CDRs.  We

    20    employed a 60‑cycle PCR using our sample DNA, in this

    21    case either CD34 selected or unselected autograft

    22    material, which was serially diluted in normal, in

    23    this case, placental DNA to a final concentration

    24    containing the worth of 100,000 cells of DNA, which is

    25    .6 micrograms.  And then the products were

                                                                         15

     1    electrophoresed and stained appropriately.

     2                Now the quantitative nature of the assay

     3    was because we actually did serial replicates at each

     4    of these dilutions.  I will outline this briefly for

     5    you.  This is in cartoon form, of course.  In this

     6    case, LK for leukophoresis DNA in the undiluted

     7    sample, there is only leukophoresis DNA.  With serial

     8    dilution in placental DNA, we have, of course, less

     9    and less of our sample DNA and more and more of our

    10    normal DNA.  And as you see out here, very little

    11    sample DNA and much normal DNA.  So theoretically, a

    12    tumor containing sample, all of these reactions would

    13    be possible.  All of these may be positive. And slowly

    14    this number would become zero out of 5.  And then one

    15    can convert this to a percentage contamination in the

    16    undiluted sample using Poisson distribution analysis.

    17    And this is based on the fact that we can detect one

    18    copy of the target DNA, in this case that tumor‑

    19    specific immunoglobulin gene primer pairs, in our PCR

    20    tube.  Again, as I showed you, five replicates done at

    21    each serial dilution.  And then we can use the old

    22    computer using Poisson distribution analysis to

    23    determine the percentage of tumor in the undiluted

    24    sample.

    25                And here is an example of one of the

                                                                         16

     1    patients actually from the Phase III trial, and here

     2    we are looking at post mobilization chemotherapy

     3    peripheral blood mononuclear cells.  In part A here is

     4    the undiluted blood, and we serially dilute this out. 

     5    So you can see the reactions using patient‑specific

     6    primers.  All five of these are positive, five of

     7    these, et cetera.  And as we move out here slowly,

     8    these become negative.  And again one then can convert

     9    using Poisson distribution analysis to determine the

    10    percentage of tumor in the undiluted sample and

    11    multiply that by the amount of leukophoresis cells

    12    obtained and obtain a number of tumor cells per

    13    kilogram in the leukophoresis product and then in the

    14    CD34 selected absorbed product.  

    15                These are the results from the clinical

    16    samples from the Phase II trial in which 18 patients

    17    were analyzed.  11 out of the 18 contain tumor in the

    18    unselected fraction as you see here, ranging from

    19    about 4,000/kg to a high of 2 million.  Following CD34

    20    selection in the absorbed fraction, the tumor cell

    21    numbers were reduced below the level of sensitivity of

    22    this assay, which is 1:700,000 normal cells in the 8

    23    cases you see.  In the three in which we could obtain

    24    numbers, you can see they range from 83 to a high of

    25    2,000, with a log reduction ‑‑ in most cases, we

                                                                         17

     1    couldn't give a quantitative number.  It was a greater

     2    than because we couldn't obtain any tumor cells in the

     3    CD34 selected fraction.  In the three cases that we

     4    could, there was approximately a 3 log reduction in

     5    tumor burden following CD34 selection.

     6                Now despite that excellent reduction in

     7    tumor burden, there was very quick engraftment in that

     8    neutrophil recovery by a median of 12‑days occurs, and

     9    platelets to 20,000 and 50,000 occurred at 12 and 13

    10    days respectively.  Importantly in this Phase II

    11    study, we also establish a threshold dose of CD34

    12    required, and that was 2 million cells per kilogram. 

    13    In patients who received less than that threshold

    14    dose, there was a delay in neutrophil recovery and the

    15    patients had a delay in platelet recovery as well and

    16    required, as you see here, more red cell transfusions

    17    and platelet transfusions.  This established our goal

    18    for a threshold dose of CD34 for the Phase III trial,

    19    which will now be presented by Dr. Cindy Jacobs. 

    20    Thank you.

    21                DR. JACOBS:  For the Phase III study, I

    22    will first present the study design and then the

    23    patient characteristics and processing results, next

    24    the primary and secondary endpoints, and the long‑term

    25    follow‑up results at one year post‑transplant.

                                                                         18

     1                This is a controlled Phase III study

     2    randomizing patients to either have their stem cells

     3    CD34 selected using the Ceprate system or unselected. 

     4    The 15 participating sites are listed here in order of

     5    accrual, with UCLA, Toronto Hospital, Dana Farber

     6    Cancer Institute, Washington University, and the

     7    University of South Florida occurring the majority of

     8    the patients.

     9                This shows the scheme of treatment. 

    10    Patients were registered if they had a diagnosis of

    11    multiple myeloma and stable or responsive disease

    12    after 3 cycles of chemotherapy.  They were excluded if

    13    they had progressive disease or greater than 3 months

    14    of ablative therapy or a total of greater than six

    15    months of chemotherapy.  All patients were mobilized

    16    using cytoxin, prednisone, and G‑CSF.  The patients

    17    were randomized just prior to starting leukophoresis

    18    and PBPC collection to have their stem cells either

    19    Ceprate selected or unselected using standard

    20    procedures.  All patients received high‑dose

    21    chemotherapy consisting of Busulfan and cytoxin, and

    22    then either received their CD34 selected stem cells or

    23    their unselected stem cells.  All patients received

    24    GM‑CSF post‑transplant.  The primary study period was

    25    six months, and we have annual follow‑up that is

                                                                         19

     1    ongoing.  

     2                There were two primary study objectives,

     3    one for safety and one for efficacy.  The safety

     4    objective was to demonstrate equivalent neutrophil

     5    engraftment for both arms.  The efficacy objective was

     6    to demonstrate a reduction in tumor cells in the

     7    Ceprate selected arm.  

     8                There were four main secondary safety

     9    endpoints; time to neutrophil engraftment, time to

    10    platelet engraftment, percent of patients with

    11    infections, and overall survival at 6 months.  There

    12    were a number of other secondary endpoints.  The first

    13    six listed here were additional secondary safety

    14    endpoints.  The next three listed are additional

    15    secondary efficacy endpoints.  And the last two were

    16    long‑term follow‑up endpoints.

    17                I would like to now show the patient

    18    characteristics and processing results.  There were

    19    134 patients that were registered on the study.  Three

    20    of those patients were not randomized to the study. 

    21    So of the 131 patients registered and randomized onto

    22    the study, 67 were in the CD34 selected arm and 64

    23    were in the unselected arm.

    24                Of the 131 patients randomized, all were

    25    infused except for one patient.  This patient had

                                                                         20

     1    inadequate mobilization of stem cells.  This patient

     2    ended up withdrawing consent for transplant on this

     3    study.  So there were 130 patients that were infused.

     4                This is a summary of the demographic

     5    characteristics.  The arms were comparable for the

     6    demographic characteristics except for one, gender. 

     7    There were more females in the CD34 selected arm than

     8    in the unselected arm.  For the other parameters ‑‑

     9    race, age, weight, and years since first diagnosis ‑‑

    10    the arms were comparable.  Although not shown here,

    11    the arms were also comparable for other disease

    12    characteristics as well as prior cancer‑related

    13    therapy.

    14                Of the 40 various laboratory parameters

    15    that we measured at baseline and at randomization, all

    16    were comparable between the arms except for four, and

    17    they are listed here.  For the white blood cell count

    18    and platelet count, the CD34 selected arm had

    19    significantly lower counts in the unselected arm.  For

    20    the median white blood cell count, the CD34 selected

    21    arm had approximately 16,000 compared to 26,000 with

    22    a P value of .04.  The median platelet count for the

    23    CD34 selected arm was 110,000 versus 152,000, with a

    24    P value of less than .01.  

    25                The CD4/CD8 ratio was also significantly

                                                                         21

     1    lower in the CD34 selected arm compared to the

     2    unselected arm with a P value of .03.  And the number

     3    of patients that had reactivity to CMV were higher in

     4    the CD34 selected arm than in the  unselected arm with

     5    a P value of .01.

     6                This is intended to keep you awake.  For

     7    the first two parameters, the clinical relevance of

     8    this will be discussed later.  For the latter two

     9    parameters, the clinical relevance is unknown at this

    10    point.  However, the overall trend for these four

    11    parameters was in favor of the unselected arm.

    12                Before going over the processing results,

    13    I would like to briefly go over each step in each of

    14    the arms.  For the CD34 selected arm, the product to

    15    start is incubated with the CD34 antibody.  Then the

    16    cells are processed using the Ceprate system yielding

    17    a CD34‑enriched product and a CD34‑depleted product.  

    18                For the CD34‑enriched product, it is cryo‑

    19    preserved until transplant was thawed just prior to

    20    infusion.  For the unselected arm, the product to

    21    start was cryopreserved until transplant and then

    22    thawed just prior to infusion.  

    23                Let's first look at the number of

    24    leukophoresis.  The majority of the patients required

    25    two leukophoreses.  However, if you look at the

                                                                         22

     1    overall general distribution, you can see there were

     2    a few less patients in the CD34 selected arm requiring

     3    two and a few more requiring three and four when

     4    compared to the unselected arm.  This overall

     5    distribution was statistically significant.  

     6                So we did additional analyses to look at

     7    factors influencing the number of leukophoresis and

     8    there were two important factors.  First of all, this

     9    study required that a minimum of two leukophoreses

    10    were obtained, and leukophoresing continued until all

    11    patients had 5 x 108 nucleated cells per kilogram. 

    12    For the CD34 selected arm, there was an additional

    13    criterion for post‑processing to have at least 4 x 106

    14    nucleated cells per kilogram.  This is really based on

    15    achieving a minimum of 50 percent CD34 cells, so the

    16    minimum target of 2 x 106 CD34 cells per kilogram would

    17    be achieved.  18 of the 67 patients or 27 percent of

    18    the patients required an additional leukophoresis

    19    because of this criterion.  However, this is where,

    20    again, the baseline differences between the arms also

    21    appeared.  As with the WBC or the hematologic values

    22    at randomization, which occurred just prior to

    23    leukophoresing, the trend toward ‑‑ there was a trend

    24    toward the CD34 selected arm requiring an additional

    25    leukophoresis than the unselected arm to at least

                                                                         23

     1    achieve the first criterion of 5 x 108 nucleated cells

     2    per kilogram in the peripheral blood product.  So both

     3    of these played a factor.

     4                This slide summarizes the processing

     5    results so you can see the median of 2 leukophoreses

     6    in both arms with overlapping ranges.  The number of

     7    CD34 cells at the various processing steps are here. 

     8    Both arms were comparable for the number of CD34 cells

     9    in the initial product with the median of 10 x 106 CD34

    10    cells per kilogram in the CD34 selected arm and 8.7 in

    11    the unselected arm.  The overall yield in this trial

    12    was 60 percent.  So in the enriched product, the

    13    median CD34 cells infused was 5.4 x 106 per kilogram

    14    compared to the 8.7 x 106 for the unselected arm.

    15                Let's now review the primary safety and

    16    efficacy endpoints.  The primary safety endpoint was

    17    successful neutrophil engraftment on or by day 14. 

    18    For the CD34 selected arm, 94 percent of the patients

    19    achieved this and 100 percent in the unselected arm. 

    20    Of the four patients who did not achieve neutrophil

    21    engraftment by day 14, they are listed here.  Three of

    22    those patients engrafted on day 15.  One of the

    23    patients was not infused, and this is the patient you

    24    saw previously that had inadequate mobilization and

    25    withdrew consent for transplant on the study.  Since

                                                                         24

     1    this was an intent to treat analysis, this patient was

     2    considered a failure to engraft.  The intent to treat

     3    analysis for the study did show that the arms were

     4    equivalent in successful neutrophil engraftment.

     5                For the primary efficacy endpoint, an

     6    attempt was made for all patients to obtain a clonal

     7    immunoglobulin sequence and then to assess the number

     8    of tumor cells in the initial PBPC product.  To that

     9    end, 42 percent of the patients in the CD34 selected

    10    arm and 30 percent in the unselected arm did have

    11    their clonal immunoglobulin sequence obtained.  Thus,

    12    for those patients, we could look at the number of

    13    tumor cells in the initial PBPC product.  The median

    14    number of tumor cells in the CD34 selected arm was 2.6

    15    million.  For the unselected arm, it was 2.3 million. 

    16    Both of these parameters were comparable between the

    17    arms prior to processing.

    18                The reasons that a clonal immunoglobulin

    19    sequence were not obtained are listed here.  Although

    20    the reasons varied, the arms were comparable for the

    21    various reasons that a clonal immunoglobulin sequence

    22    was not obtained.  

    23                In looking at the Ceprate arm then prior

    24    to and after processing, in the initial PBPC product,

    25    the median, as you saw before, of tumor cells was 2.6

                                                                         25

     1    million with a range of zero to 363 million tumor

     2    cells.  In the CD34 enriched product, the median was

     3    zero.  This is mainly because the majority of the

     4    patients had no detectable tumor cells.  The range

     5    still showed zero to 1.2 million tumor cells.  In the

     6    CD34 depleted product, the median number of tumor

     7    cells was approximately 9 million with the range of

     8    zero to 375 million similar to the PBPC product.  

     9                If you now take these numbers for each

    10    individual patient and calculate the log depletion of

    11    tumor cells for before to after processing, the median

    12    log of tumor cells was 3.1.  The range was 1.6 to 6

    13    logs of tumor cell depletion.  For those patients that

    14    had no detectable tumor cells in their enriched

    15    product, we calculated for each patient the lower

    16    limit of detection and considered there was residual

    17    tumor cell and used that value to calculate the log

    18    depletion.  So the log depletion is underestimated for

    19    those patients.

    20                We also, of the 28 patients ‑‑ there were

    21    four patients who had no detectable tumor cells in the

    22    initial PBPC product and no detectable tumor cells

    23    after processing, and those four patients are not in

    24    this analysis.  

    25                We did a couple of analyses looking at

                                                                         26

     1    mass balance of tumor cells.  This figure represents

     2    one of them.  We looked at linear regression, taking

     3    the number of tumor cells in the enriched product plus

     4    the number of tumor cells in the depleted product and

     5    compared it to the number of tumor cells in the

     6    initial PBPC product.  This shows the linear

     7    regression line.  One would expect the intercept of 0

     8    and a slope of 1.  The slope of this line was 1.02 and

     9    an r coefficient of .98.  

    10                I would like to now review the main

    11    secondary safety results.  Based on neutrophil

    12    engraftment, the median day was 12 in both arms with

    13    overlapping ranges.  Based on platelet engraftment,

    14    the median was day 11 in the CD34 selected arm versus

    15    day 9 in the unselected arm.  This two‑day difference

    16    was statistically significant.  I think you can better

    17    see it in the Kaplan Meier curve, where you are

    18    looking at the CD34 selected arm in the solid line and

    19    the unselected arm in the dash line.  You can see

    20    there is a slight delay here.  

    21                In this protocol, we had prospectively

    22    planned to do an analysis looking for factors

    23    influencing engraftment just as we had done in the

    24    previous Phase III trial.  And as we had done in the

    25    previous Phase III trial, we looked at various

                                                                         27

     1    clinical variables and processing variables and did a

     2    multi‑variate analysis looking for which factors were

     3    the most significant in influencing platelet

     4    engraftment and platelet recovery.  Platelet

     5    engraftment is platelet transfusion independence. 

     6    Platelet recovery is platelet transfusion independence

     7    and a platelet count of greater than 20,000.  The

     8    analysis for both of these is the same, so they will

     9    be presented together.  

    10                The multivariate analysis showed two

    11    significant factors, CD34 cells per kilogram in the

    12    infused product and the platelet count at

    13    randomization.  Since we had already put in the trial

    14    to look at all engraftment, we did the same analysis

    15    for neutrophil engraftment as well.  Only one factor

    16    came out in neutrophil engraftment and that was CD34

    17    cells per kilogram in the infused product.  

    18                Now we were already sensitive to the

    19    number of CD34 cells because in the previous Phase

    20    I/II trial, we already knew that the target should be

    21    2 x 106 CD34 cells per kilogram.  So we did look at the

    22    population of patients, regardless of arm in this

    23    study, evaluating that.  For the 130 patients, five

    24    patients we did not have CD34 values.  So 108 patients

    25    here received greater than 2 x 106 CD34 cells.  The

                                                                         28

     1    remaining 17 patients had less than 2 x 106 CD34 cells. 

     2    And you can see here the significant delay in platelet

     3    engraftment.  Of the 17 patients that received less

     4    than 2 x 106, there were 10 in the Ceprate arm and 7

     5    in the unselected arm.  

     6          We then looked at comparing the arms for the 108

     7    patients with greater than 2 x 106, and there was no

     8    significant difference in time to platelet

     9    engraftment.

    10                We also looked at adjusting for CD34 cells

    11    per kilogram and platelet count at randomization.  If

    12    you also adjust for those factors, there is no

    13    significant difference between the arms.

    14                Incidence of infections was a main

    15    secondary safety endpoint.  As you can see here, the

    16    number of patients with at least one infection from

    17    day zero to day 100 was comparable between the arms. 

    18    Also out to day 6 months was comparable.  Severe or

    19    life‑threatening infections from day zero to day 100

    20    were comparable and out to 6 months were also

    21    comparable.

    22                The main secondary endpoint of survival is

    23    actually shown here as the number of deaths.  There

    24    were only 7 deaths that occurred six months post‑

    25    transplant.  Two in the CD34 selected arm, 5 in the

                                                                         29

     1    unselected arm.  The two in the CD34 selected arm died

     2    of underlying malignancy.  Of the five in the

     3    unselected, one died of infection, one of VOD, and the

     4    other three of underlying malignancy.  So the arms

     5    were comparable.

     6                Let's now look at the other secondary

     7    safety results.  Number of transfusions were evaluated

     8    to day 100.  Platelet transfusion events, the median

     9    was 3 in the CD34 selected arm versus 2.  This was

    10    statistically significant.  Since platelet transfusion

    11    independence was a criteria for platelet engraftment

    12    and recovery, we looked at the number of platelet

    13    transfusion events for those patients greater than 2

    14    x 106, and there was no significant difference between

    15    the arms.  We also adjusted for platelet count at

    16    randomization.  And if you adjust for platelet count

    17    at randomization, again there is no significant

    18    difference between the arms for platelet transfusion

    19    events.  There was no difference between the arms as

    20    far as red blood cell units given.

    21                The incidence of bleeding events were

    22    comparable between the arms.  There were no new

    23    bleeding events reported after day 100.  

    24                The immunity reconstitution was evaluated

    25    in this study for all patients looking at

                                                                         30

     1    immunophenotyping and serum quantitative

     2    immunoglobulin levels.  We also looked at lymphocyte

     3    proliferation and viral serologies in a subgroup of

     4    patients.  For cell‑mediated immunity, there were no

     5    differences between the arms at baseline, day 100 or

     6    6 months visits for total lymphocytes, CD3 cells, CD8,

     7    and CD56 cells.  There was also no difference between

     8    the arms at baseline day 100 and 6 months for PHA ConA

     9    and tetanus for the lymphocyte proliferation assays. 

    10    There was a significant difference in CD4 counts at

    11    day 100 and 6 months.  For example, at day 100, the

    12    median CD34 count in the selected arm was 211 compared

    13    to 298 in the unselected arm.  The relevance of this

    14    significance is unknown.  

    15                For humoral immunities, there were no

    16    differences between the arms for the quantitative

    17    immunoglobulin levels assessed or reestablishment of

    18    antibody reactivity to various viruses.  If you look

    19    at late infections, there were no differences between

    20    the arms in incidence of infections after day 100 to

    21    the 6‑month visit for the arms, and there were no

    22    differences between the arms in the type of late

    23    infections either.

    24                Let's look at the secondary efficacy

    25    results.  We looked at the number of tumor cells

                                                                         31

     1    infused not only for the Ceprate selected arm but the

     2    unselected arm for those patients that we obtained a

     3    clonal immunoglobulin sequence.  You have seen these

     4    numbers before with the median of zero.  The

     5    unselected arm, the median was 2.3 million, and this

     6    was significant.

     7                Here we see the number of patients that

     8    had no detectable tumor cells in the initial

     9    leukophoresis prior to processing or in the initial

    10    product.  There were four patients in the selected arm

    11    that had no detectable tumor cells or 14 percent, and

    12    also 4 in the unselected arm.  There was no

    13    significant difference between the arms prior to

    14    processing.  After processing, 54 percent of the

    15    patients had no detectable tumor cells and this was

    16    significantly different.

    17                Incidence of infusional toxicities were

    18    also evaluated.  The incidence of hypertension was

    19    significantly lower in the CD34 selected arm compared

    20    to the unselected arm.  The incidence of bradycardia

    21    was also lower in the CD34 selected arm compared to

    22    the unselected and approached significance.  There was

    23    no difference in heart block or atrial arrhythmias. 

    24    So the reduction in hypertension and bradycardia is

    25    similar to our previous approval for bone marrow.  

                                                                         32

     1                Let's look a the long‑term follow‑up

     2    results.  We looked at hematologic values at 6 months

     3    for ANC, platelet counts of less than 50,000,

     4    hemoglobin of less than 10 grams, and there was no

     5    difference.  The arms were comparable.  We had one

     6    patient in the CD34 selected arm that received the

     7    back‑up depleted product at day 119 for platelet count

     8    of less than 20,000.  This patient was platelet

     9    transfusion independent.  And even after infusion of

    10    the back‑up, platelet counts remained below 20,000.

    11                Although the curve is immature, this is a

    12    Kaplan Meier for overall survival, and there is no

    13    difference between the arms.  And again, although the

    14    curve is immature, this is the progression pre‑

    15    survival for the selected arm and the unselected arm

    16    and there is no difference between the arms at this

    17    point.  

    18                So in summary, this study successfully met

    19    the primary safety and efficacy endpoints, the safety

    20    being successful neutrophil engraftment by day 14 and

    21    the efficacy for a significant reduction of tumor

    22    cells in the Ceprate arm.  There were no statistically

    23    significant differences between the arms for time to

    24    neutrophil engraftment, incidence of infections, and

    25    overall survival.  However, there was a significant

                                                                         33

     1    difference in median time to platelet engraftment,

     2    which was 11 in the Ceprate arm and 9 days in the

     3    unselected arm.  When evaluating this further, the

     4    time to platelet engraftment and the number of

     5    platelet transfusion events were influenced by the

     6    number of  CD34 cells infused and the platelet count

     7    at randomization.  There were no significant treatment

     8    arm effects after adjusting for these factors and

     9    there were also no significant differences if you

    10    evaluate patients who received at least 2 x 106 CD34

    11    cells per kilogram.

    12                There were no statistical significant

    13    differences between the arms in the number of other

    14    secondary safety endpoints.  Some of them I have not

    15    presented like days of hospitalization and incidence

    16    of adverse events.  

    17                The secondary efficacy endpoints, there

    18    was a reduction in the number of tumor cells infused. 

    19    I didn't present this, but there was a median 74‑fold

    20    enrichment of CD34 cells and a reduced proportion of

    21    patients with hypertension post‑infusion.

    22                I would now like to just briefly summarize

    23    the additional tumor purging results from other

    24    investigative studies that are published using the

    25    Ceprate system.  In your briefing document, we have

                                                                         34

     1    presented results from the Phase I/II multiple myeloma

     2    study that Dr. Berenson showed you.  We also went back

     3    and looked at the breast cancer purging in the

     4    previous Phase I/II study from the prior approval. 

     5    Since these data are presented in the publications, I

     6    am not going to present it separately.  

     7                Let's just first go to the publications

     8    looking at tumor purging for multiple myeloma

     9    patients.  Of the studies, three of them looked at log

    10    tumor depletion.  The Schiller publication does

    11    contain some of the Phase I/II data.  This showed a

    12    range of 2.5 to greater than 4.5 log tumor depletion. 

    13    Looking at the number of contaminated products purged

    14    below detection after the Ceprate system, it was 63

    15    percent, 140, or 100 percent depending on the samples.

    16                Of the publications looking at tumor

    17    purging for patients with breast cancer, both

    18    peripheral blood and bone marrow have been evaluated. 

    19    The log depletion, the median was 2 with a range of 1

    20    to greater than 4 for both bone marrow and peripheral

    21    blood.  For the number of contaminated products purged

    22    below detection, it was 18 for bone marrow and 67

    23    percent here for peripheral blood and 100 percent here

    24    in the Vogel article.  

    25                This slide shows tumor purging for

                                                                         35

     1    patients with non‑Hodgkin's lymphoma, again evaluating

     2    both peripheral blood and bone marrow.  There were two

     3    publications that actually quantitated the log tumor

     4    depletion showing a range from 1 to 3 logs of tumor

     5    depletion.  For the number of contaminated products

     6    purged below detection, there was 89 percent, 60, 80,

     7    and 75 percent.

     8                Thus, based on the Phase I/II study, the

     9    Phase III study, and the additional investigative

    10    studies in the literature, CellPro would like to

    11    expand our indication to include peripheral blood

    12    progenitor cells in addition to the autologous bone

    13    marrow.  We would like to include a selection of

    14    peripheral blood results in greater than 100‑fold or

    15    two log reduction in the number of tumor cells present

    16    in the autograft.  And we will also recommend for

    17    peripheral blood that at least 2 x 106 CD34 cells per

    18    kilogram be collected after selection.  That is it.

    19                CHAIRPERSON VOSE:  Thank you, Dr. Jacobs. 

    20    I would just like to announce that Abbey Meyers, the

    21    consumer representative, is joining us by phone before

    22    we start into questions.  And I would like to open it

    23    up for questions of the sponsor from the committee.

    24    I guess we can't turn up the lights apparently, so we

    25    are in the dark.  They are permanently dim.  Dr.

                                                                         36

     1    Auchincloss?

     2                DR. AUCHINCLOSS:  Can I come back to Dr.

     3    Jacobs, and in particular to your comparison between

     4    the number of tumor cells before and after processing. 

     5    The slope of the curve that you showed us was 1 or

     6    essentially 1, but there are individual points,

     7    obviously, that are well outside of the line.  And in

     8    particular, you have at least two that appear to have

     9    105 cells prior to processing and then 108 cells

    10    after.  What does that say about the accuracy of the

    11    assay?

    12                DR. JACOBS:  You mean at ‑‑ there were no

    13    patients that actually had more tumor cells after

    14    processing.  There were three outliers there, but none

    15    of the patients had a total of more tumor cells after

    16    processing.

    17                DR. AUCHINCLOSS:  No.  I am not saying in

    18    the product that you infused, but when you calculate

    19    the number in the enriched plus depleted, there were

    20    108 cells calculated, whereas there were 105 cells in

    21    the original product.  How did you gain three logs

    22    worth of cells?

    23                DR. JACOBS:  Mike, do you know?  I am not

    24    quite sure I am understanding.  Heather, can you put

    25    the slide with the linear regression line?  If you

                                                                         37

     1    would just put it in the middle of the carousel, I can

     2    get it to that slide.  I am not quite sure ‑‑ keep

     3    going forward just a little bit.  There you go. 

     4                DR. AUCHINCLOSS:  So if you just focus on

     5    those two points in the upper left‑hand area, you

     6    originally start with a calculation that says that you

     7    have 105 tumor cells.  And that is ‑‑ at the end, it

     8    measures out to 108 tumor cells in your combined

     9    enriched and depleted product. And my concern is what

    10    does this say about the accuracy of the individual

    11    numbers that you have if you can go from 105 to 108

    12    cells in one preparation?

    13                DR. VESCIO:  Well, my name is Dr. Vescio,

    14    and I am one ‑‑ I guess the person that oversaw the

    15    tumor quantitation part of the trial.  And I think as

    16    with any assay that one develops, there is going to be

    17    some variability.  And we did ‑‑ as was submitted to

    18    the Board, we did an initial set of experiments to

    19    verify the accuracy of the assay and we found that in

    20    general the assay was accurate within a half a log or

    21    actually in the majority of cases, less than half a

    22    log, 0.2 logs by the standard deviation.  Obviously

    23    these points here, there was more variability than one

    24    would have ‑‑ than that would have suggested.  But

    25    again, it is just a matter of the variability between

                                                                         38

     1    the assays.  These are all done by hand and there is

     2    going to be, as expected, some variability.  But if

     3    you look at overall, the results were very comparable

     4    and what one would expect comparing tumor burden in

     5    the combined enriched and depleted product versus the

     6    leukophoresis product itself.  So I think obviously

     7    one did not create tumor cells out of air.  But I

     8    think it just shows that at least just for these two

     9    cases, there was a little bit of variability in the

    10    assay.

    11                DR. AUCHINCLOSS:  Well, as I understand

    12    it, the central issue here is the degree of accuracy

    13    in the assay.

    14                DR. VESCIO:  Sure.

    15                DR. AUCHINCLOSS:  And for you to suggest

    16    that it is accurate to within half a log I guess

    17    doesn't fit with a picture that says ‑‑ there are two

    18    patients up there at least, and actually there are

    19    several others ‑‑ but there are two at least there

    20    where it is not accurate within 103.

    21                DR. JACOBS:  Actually, with this analysis,

    22    if you are adding enriched and depleted, that half log

    23    variability for both would probably be a full log of

    24    variation.  

    25                DR. VESCIO:  And I think that if ‑‑ you

                                                                         39

     1    know, again, there is always possibility for some

     2    error.  We could only run the samples once.  And I

     3    think that if one looked at, again, when the assay was

     4    verified, all the results were done there and those

     5    were done in quadruplicate.  And in those particular

     6    cases, the assay variability was less than a half a

     7    log.  We also did some controls looking at beta‑actin

     8    quantitatively and also in all of those cases, the

     9    variability was less than a half a log. So I think

    10    although I agree in these particular two examples out

    11    of the numerous cases that were done in assay, those

    12    fell outside of the variability that one would have

    13    seen.  But I think the majority of the patients

    14    followed what one would have expected. And for this

    15    very complicated assay, I think that in general it ‑‑

    16    one would expect the variability to be balanced on

    17    both ends, and I am not sure that it really would have

    18    influenced the results.  

    19                CHAIRPERSON VOSE:  Dr. Silver?

    20                DR. SILVER:  I'm not sure I understand

    21    fully the details of the collection process, but the

    22    essence of my question is the enriched product

    23    contains maybe 50 percent or 20 percent or some

    24    percentage of CD34 negative cells which are not

    25    selected against in this process ‑‑ contaminating the

                                                                         40

     1    enriched population.  Is there any reason that you

     2    would not expect the number of tumor cells in that

     3    half of the cells which are not CD34 positive to be

     4    depleted if half of the enriched product is basically

     5    a random sampling of the non‑CD34 cells, and that

     6    includes the tumor cells, then wouldn't you expect

     7    about half of the product to contain the original

     8    number of tumor cells?

     9                DR. VESCIO:  That is a very good point and

    10    in fact that is basically what is seen.  Really there

    11    was some additional elimination of tumor cells.  But

    12    if you actually look at the percent contamination

    13    within the product, it is not that dramatic.  It is

    14    maybe a half log or maybe a log less in tumor cells

    15    within the 34‑enriched product.  But the effectiveness

    16    of the separation procedure is primarily by reducing

    17    the total quantity of cells that one gives back to the

    18    patient, since one gives back to the patient two logs

    19    less of autograft cells with the CD34 selection.  So

    20    I think when we measure actually tumor within the

    21    product, the percent contamination of the enriched

    22    product is only about as one would have expected,

    23    about a half to one log less by that procedure.

    24                DR. JACOBS:  If you look at the Ceprate

    25    procedure, what we look at is probably about a two log

                                                                         41

     1    depletion due to the loss of the contaminating cells

     2    while you are purifying or you are selecting the CD34

     3    cells.  And a log of 3 for the tumor purging ‑‑ I

     4    mean, there may be some additional tumor purging

     5    depending on the type of tumor cell going through

     6    actually easier in the wash in the depleted.  I don't

     7    know, Jim, if you have anything more to add from the

     8    Phase I/II study and what you have seen in tumor

     9    purging.

    10                DR. SILVER: Could you clarify, do you wash

    11    the column before you elute or do you just collect the

    12    cells that are CD34 positive and then elute them?

    13                DR. JACOBS:  You wash or you elute the

    14    column as far as the depleted cells.  And then to get

    15    the CD34 selected cells off, there is a little

    16    mechanical stir bar that gently washes the cells off

    17    when you put them, RPMI in this case, to the column.

    18                DR. BERENSON:  Well, I think the only

    19    comment I would make is that the sensitivity of the

    20    assay is about where the tumor contamination is.  You

    21    saw in several cases that the unselected product,

    22    approximately 20 percent in both the Phase II and

    23    Phase III, did not contain tumor and that is why we

    24    had to put greater than signs in those patients that

    25    went completely to negative.  But obviously we can't

                                                                         42

     1    tell you the exact number.  But again, your point is

     2    well taken that we are going from approximately 1

     3    percent positive to 80 percent positive or 99 percent

     4    negative to approximately 20 percent negative, which

     5    is less than a log per se.  But again, we have more

     6    than two logs simply by cell number.  The additional

     7    close to approaching the log approximates the three

     8    logs that Dr. Vescio found, very close to what we

     9    would expect.  But I would not argue against that the

    10    column itself could add additional stickiness in terms

    11    of possibly the cell sticking on.  We don't know.  I

    12    mean, that really has not been looked into.  But three

    13    logs is pretty much what we would expect and that is

    14    what we got.

    15                DR. SILVER: I thought two logs is what you

    16    would expect from your selecting 1 percent of the

    17    cells?

    18                DR. BERENSON:  No, that is incorrect.  As

    19    you know ‑‑ as Dr. Jacobs presented, we do actually

    20    give back less than 1 percent of the cells.  And in

    21    addition, we go from 99 percent, if you will, of CD34

    22    negative to approximately 30 percent CD34 negative. 

    23    So adding that additional approximated log, we would

    24    expect about 2.2 logs, 2.3 plus .7, about 3 logs,

    25    which is what we got.  

                                                                         43

     1                DR. O'FALLON:  Well, just let me comment

     2    on this figure.  I find it hard to believe that the

     3    correlation coefficient is as high as you are quoting. 

     4    It seems unlikely to be .98 with those two points that

     5    we have already attended to being that far away from

     6    the line.  

     7                DR. WHITE:  I am Mike White from CellPro. 

     8    I don't know what to say, Dr. O'Fallon.  We ran the

     9    analysis in SAS.  This analysis is not meant to be

    10    predictive.  This is meant to be a descriptive

    11    analysis of what is going on.  We aren't trying to

    12    suggest that there is any sort of prediction attached

    13    to the regression.  It was a zero intercept model

    14    where we are only estimating the slope of the

    15    regression.  And as Dr. Jacobs presented, the slope

    16    was essentially one.  We have also done other analyses

    17    look at the ratio of the number of tumor cells in the

    18    PBPC product to the sum of the number in the depleted

    19    and enriched product.  Again, in this case when you

    20    look at the log ratio, you would expect it to be zero. 

    21    We tested it to see if it was significantly different

    22    from zero.  It was not significantly different from

    23    zero.  That is not to say that it is equal to zero,

    24    but simply that we did not see any trend in the data

    25    for loss of tumor cells in the depleted plus the

                                                                         44

     1    enriched product.  And that is really all we were

     2    trying to show there.  Not so much is the slope 1, but

     3    is there any trend toward loss of cells.  And that is

     4    what we were trying to get at in these analyses.  

     5                CHAIRPERSON VOSE:  Dr. Berman?

     6                DR. BERMAN:  Is the claim that there is

     7    reduced toxicity from the separated product, that is

     8    just based on the fact that there is less DMSO?  It is

     9    a 7.5 percent versus 10 percent, right?

    10                DR. JACOBS:  Also there was a reduction in

    11    the volume of infusate also that made a difference as

    12    well.

    13                CHAIRPERSON VOSE:  Mr. Katz?

    14                MR. KATZ:  The four patients that had a

    15    higher percentage of tumor cells in the purified

    16    product, does it follow that it is still better for

    17    them to have that product infused versus the original?

    18                DR. JACOBS:  If you take the percent and

    19    then you multiply it by the number of nucleated cells

    20    in the infused product, two of those four patients had

    21    the 1.6 log depletion, the other one was close to 2

    22    logs, and the other two were greater than 2 logs. So

    23    even what you give back is still going to be roughly

    24    1.5 to 2.5 logs even for those patients.

    25                MR. KATZ:  Thank you. 

                                                                         45

     1                CHAIRPERSON VOSE:  Just to get back to the

     2    MRD assay for a minute.  The CDR3 assay is such a

     3    difficult assay to perform it so individualized for

     4    patients, and there is a wide variety, as you have

     5    shown, for patients that couldn't be amplified or

     6    other reasons that in the end you end up with a fairly

     7    small number of patients that could actually be used

     8    for that.  So do you want to comment on that relative

     9    to the small number of patients that could actually be

    10    used for your analysis?

    11                DR. JACOBS:  Actually, one of the problems

    12    that we had was the criteria ‑‑ the inclusion

    13    criteria.  The patients had to have responsive or

    14    stable disease after a minimum of three cycles of

    15    chemotherapy.  The bone marrow sample for which the

    16    sequence was attempted to be obtained was right at

    17    baseline just prior to going on to the study.  So in

    18    many of the patients, obviously, with responsive

    19    disease, it was more difficult to obtain the clonal

    20    sequence in those patients.  I think we would have

    21    gotten a greater percentage of patients if we had had

    22    like de novo patients, where on their initial

    23    diagnosis we were able to obtain a bone marrow sample. 

    24    So we were at a little bit of a disadvantage in this

    25    study.

                                                                         46

     1                DR. BERENSON:  Julie, in the Phase II

     2    trial, we actually obtained bone marrow earlier on. 

     3    So in that trial in the patients we attempted, we

     4    could actually obtain sequences with primers that were

     5    usable in about 70 percent of the cases.  And you saw

     6    the data was quite consistent with Phase III. 

     7    Obviously that wasn't a blinded kind of trial because

     8    it didn't come under FDA jurisdiction.

     9                DR. JACOBS:  Actually, that was a heated

    10    discussion with the investigators as well because they

    11    wanted to use ‑‑ if they had a bone marrow sample from

    12    diagnosis, to use it.  But we were very conservative

    13    and we said no because then we could have injected

    14    bias into the number of patients from arm versus the

    15    other that could have had their clonal sequence.  So

    16    we were very strict on it had to be the bone marrow

    17    sample at the time that the patient was registered and

    18    had informed consent.

    19                CHAIRPERSON VOSE:  Well, I think that is

    20    the correct thing to do.  It is just a little

    21    concerning that in the end we only ended up with a

    22    fairly small number of patients that could be

    23    analyzed, even though that was the crux of it.

    24                DR. JACOBS:  Right.

    25                CHAIRPERSON VOSE:  Additional questions

                                                                         47

     1    for the sponsor?  Dr. Auchincloss?

     2                DR. AUCHINCLOSS:  The FDA document that we

     3    are going to be looking at a little bit later on talks

     4    about three patients in whom ‑‑ these are 008, 009,

     5    and 0013 ‑‑ in whom the reduction in total nucleated

     6    cells was approximately equal to the reduction in

     7    tumor cells.  I just want your comment on those three

     8    patients that we will be hearing about.  Does that

     9    imply that tumor cells were, in fact, sticking to the

    10    column there?

    11                DR. JACOBS:  Well, we don't have any

    12    evidence for that.  I mean, we haven't looked at that. 

    13    What was the site number?  The first two numbers on

    14    the patient ‑‑ 

    15                DR. AUCHINCLOSS:  They are all from 2600

    16    or 260.  

    17                DR. JACOBS:  Okay.  Actually, those

    18    patients were from a Dana Farber, and there was

    19    nothing that was ‑‑ I mean, Ken, if you would like to

    20    comment as far as processing results for those

    21    patients abnormal.  I think those patients were just 

    22    ‑‑ two of them were the ones that were on the lower

    23    end of tumor purging, 1.5 to 2.5 logs.

    24                DR. AUCHINCLOSS:  And again, I am just

    25    asking because I assume we will hear about these

                                                                         48

     1    later.  I wanted to hear any comments.  But again,

     2    does it suggest to you that there is some variability

     3    in the assay?

     4                DR. JACOBS:  Well, I mean, again I think

     5    we realize the variability of the assay is at least

     6    half a log.  

     7                DR. AUCHINCLOSS:  And one other question

     8    for you.  That is in your progression free survival

     9    curve, have you made any effort to determine whether

    10    those who have not shown progression free ‑‑ sorry ‑‑

    11    those who have shown progression free survival came

    12    from the group that got zero tumor in the ‑‑ 

    13                DR. JACOBS:  No.  Right now there is no

    14    correlation although the numbers are small.  We will

    15    be looking at that, but there is none at this point.

    16                DR. AUCHINCLOSS:  Is it fair to say,

    17    therefore, that you have no evidence that this

    18    procedure of tumor cell depletion does a patient any

    19    good?

    20                DR. JACOBS:  At this point, no, this study

    21    wasn't designed to show that obviously with the

    22    numbers.

    23                DR. AUCHINCLOSS:  I understand that. 

    24                DR. JACOBS:  But obviously the principle

    25    is giving back tumor cells is not good.  And we do

                                                                         49

     1    have plans to follow this study for long‑term,

     2    although we may or may not see a significant

     3    difference with the number of patients that we have in

     4    this study.

     5                DR. AUCHINCLOSS:  Well, there is ‑‑ yes,

     6    I agree with you.  There is sort of a general sense

     7    that it is probably not good to give back tumor cells. 

     8    That seems like a reasonable supposition.  On the

     9    other hand, but putting tumor depletion in your

    10    labeling, there is an implication, I think, that you

    11    are doing somebody some good.  Is that your feeling?

    12                DR. JACOBS:  Well, I mean when this was

    13    originally discussed as a primary efficacy endpoint,

    14    it came from the BRMAC meetings that happened in 1994

    15    that at least if there were no safety issues that

    16    depleting tumor cells would be an endpoint for a

    17    pivotal study.

    18                DR. AUCHINCLOSS:  I am familiar with the

    19    meeting that you are referring to.  Also, that meeting

    20    suggested that it would be tumor specific.  Do you

    21    agree with that point also made by the committee at

    22    that point?

    23                DR. JACOBS:  Right now our Phase I/II and

    24    Phase III, that is why we chose multiple myelomas

    25    because of the specificity of the assay, so we could

                                                                         50

     1    show proof of principle.  The only other publications

     2    that we have were with breast cancer and non‑Hodgkin's

     3    lymphoma.

     4                DR. AUCHINCLOSS:  My only point being that

     5    if one simply took the word of the committee in 1994,

     6    you wouldn't label, based on the current trial, for

     7    more than multiple myeloma.  

     8                CHAIRPERSON VOSE:  Dr. Silver.

     9                DR. SILVER: One other question about the

    10    assay, the PCR assay.  Probably the major concern is

    11    that you might be missing some cells ‑‑ that the assay

    12    might not be sensitive to a single positive cell.  Was

    13    the assay tested for its ability to detect a single

    14    copy of DNA on more than just one of the primer pairs

    15    because all of the primer pairs are different for each

    16    patient?

    17                DR. VESCIO:  I think because of the ‑‑

    18    there is no gold standard for getting a sample of say

    19    myeloma bone marrow in these patients, particularly

    20    patients that have been treated for three or four

    21    months.  There is no way of having a standard that one

    22    could insure and be absolutely positive that one could

    23    detect just one copy of tumor cell DNA within a tube. 

    24    However, in the majority of cases ‑‑ actually, in the

    25    case when we compared tumor contamination within the

                                                                         51

     1    bone marrow, we initially looked at tumor

     2    contamination in that original bone marrow specimen,

     3    and it was consistent with the amount of contamination

     4    that one found on the cytospins of those same bone

     5    marrow specimens.  So one could then assume that,

     6    indeed, we were able to detect one copy of the target

     7    gene.  We have also subsequently quantified the

     8    presence of the beta‑actin gene within cells from

     9    various patients and did indeed show in those three

    10    patients from the leukophoresis and the 34‑enriched

    11    specimens, that we were able to in all of those cases

    12    detect one copy of the beta‑actin gene by the assay

    13    itself.  So I think there is no way of absolutely

    14    proving in an individual patient that one could detect

    15    one copy per gene.  In this particular analysis, it is

    16    a relative comparison anyway.  And so if by some

    17    chance there are some patients where the detection is

    18    less than one or we need more than one copy of the

    19    gene per cell, it should not influence the results,

    20    because again it is a relative comparison.  The

    21    absolute numbers may be a little bit off in those

    22    cases if that occurs.  But the percent of tumor log

    23    depletion would not have been changed by the

    24    sensitivity of the assay.

    25                DR. SILVER: Since you cloned the gene for

                                                                         52

     1    each patient and sequenced it, you could have done a

     2    reconstruction experiment where you dope in a certain

     3    number of molecules of the clone target into a pool of

     4    .6 micrograms of genomic DNA or placental DNA and see

     5    what the sensitivity of the assay is.  Was that done

     6    on ‑‑ 

     7                DR. VESCIO:  We did not do that.  You can

     8    ‑‑ that can be done.  The problem with even that

     9    particular technique is that it is different to dope

    10    genomic DNA with plasma DNA, which may be more easily

    11    amplifiable than the actual genomic DNA.  I think

    12    genomic DNA is more difficult to amplify in the first

    13    place.  So some groups have now subsequently looked at

    14    that, but again I think the results looking at the

    15    original tumor contamination within that primary bone

    16    marrow specimen was pretty convincing that we were,

    17    indeed, able to detect one copy of the target cell

    18    within a PCR tube.

    19                DR. BERENSON:  One thing to reemphasize is

    20    that we, as part of the validation, looked at, as you

    21    know, multiple bone marrows as well as cell lines. 

    22    And certainly we were accurate using three different

    23    techs who were blinded within this .5.  In most cases,

    24    it was within .2 or .3.  So to somehow argue that this

    25    assay is more off than that would be, I think,

                                                                         53

     1    misleading, given the fact that we have taken fresh

     2    bone marrows from myeloma patients where we knew the

     3    percent plasma cells as well as tumor cell lines. 

     4    Somehow you would have to argue not only is the assay

     5    inaccurate, but that it is biased somehow toward the

     6    CD34 arm, which I think that is a lot to fathom.  

     7                DR. SILVER: One last relating question. 

     8    When you see a significant or a major drop in the

     9    percent of tumor cells in the enriched product, one

    10    concern is that you are getting a false ‑‑ and you

    11    don't see any tumor cells ‑‑ the concern is that you

    12    are getting a false negative to say something

    13    inhibiting the PCR or the sensitivity of the PCR down

    14    to one molecule in those assays.  Did you do any

    15    mixing experiments where you mixed a sample from the

    16    enriched cells in which you couldn't detect tumor with

    17    DNA from cells in which you knew there was tumor

    18    present or tumor DNA present to see if you inhibited

    19    the ability to detect a small number of copies.

    20                DR. BERENSON:  Well, I think the beta‑

    21    actin really is against what you say.  That Dr. Vescio

    22    has run beta‑actin primers on several different

    23    patients and indeed has shown an accuracy of the assay

    24    for detecting DNA to within .5, in fact in most cases

    25    it was within .2 to .3 logs.  I don't know, Bob, if

                                                                         54

     1    you have another comment.

     2                DR. VESCIO:  Yes.  I think one of the

     3    things to insure that indeed these results were

     4    reproducible, one of the additional mechanisms and

     5    procedures that we did to insure that the results were

     6    reproducible is that we always processed the 34‑

     7    enriched specimen and the leukophoresis specimen and

     8    some bone marrow specimens that were diluted to a

     9    point where the bone marrow became at the limits of

    10    detectability, and those were all processed at the

    11    same time in the same PCR machine.  So, again, we

    12    showed in those cases the results ‑‑ the bone marrow

    13    controls were consistent to what one would have

    14    expected.  And again, the 34‑enriched and the

    15    leukophoresis products were processed at the same time

    16    in the same machine, and we had this beta‑actin

    17    control that was always positive in the enriched

    18    specimen and also in the leukophoresis specimen to

    19    show that the DNA was, in fact, qualitatively

    20    sufficient for the procedure itself.  So I think with

    21    all those controls, we feel very confident of the

    22    results that were obtained.  

    23                CHAIRPERSON VOSE:  Dr. O'Fallon?

    24                DR. O'FALLON:  Three questions.  Was

    25    randomization of the patients stratified in any way? 

                                                                         55

     1    I couldn't detect that from reading your description.

     2                DR. JACOBS:  The patients were stratified

     3    for age greater than 55 and less than 55.  

     4                DR. O'FALLON:  Okay.

     5                DR. JACOBS:  That was the only

     6    stratification that we used.

     7                DR. O'FALLON:  Were they stratified within

     8    the individual centers, then, separately?

     9                DR. JACOBS:  Yes.

    10                DR. O'FALLON:  Okay.  Several of the

    11    centers had very small numbers of participants.  Did

    12    any of the analyses change if you analyzed only those

    13    centers which had more than ‑‑ 

    14                DR. JACOBS:  No. There was no variation or

    15    no site effect that we could see when we did those

    16    analyses.

    17                DR. O'FALLON:  Okay, the last question. 

    18    One of your ‑‑ well, your primary safety endpoint is

    19    based on a type of analysis which is somewhat unusual

    20    in terms of you are specifying a hypothesis of a 12

    21    percent reduction in responsiveness in the one group. 

    22    You have four subjects that did not engraft according

    23    to schedule, and you quote a P value of .019.  Since

    24    the numbers were small enough, I thought I could try

    25    to replicate that P value, but I was unsuccessful. 

                                                                         56

     1    Perhaps you could describe to me a little bit what you

     2    did there.

     3                DR. WHITE:  Mike White from CellPro.  The

     4    reference for that analysis is the Blackwelder paper

     5    on demonstrating equivalents.  Rather than specifying

     6    your standard null hypothesis, you specify a null

     7    hypothesis that a difference exists.  The difference

     8    we specified was that the proportion of patients with

     9    successful neutrophil engraftment in the selected arm

    10    was at least 12 percent less than in the unselected

    11    arm.  When we did the analysis, we used that as our

    12    null hypothesis. I used the normal approximation to

    13    the binomial.  The analysis we reported was the

    14    analysis without adjustment for continuity.  I also

    15    did an analysis adjusting for continuity and also used

    16    ‑‑ I looked at other alternatives.  I wanted to say

    17    that I used an exact binomial test, but I did not use

    18    an exact binomial test on that one.  That would be

    19    pretty tough to do.  But the P values were pretty

    20    consistent.  It is a tough analysis to do because of

    21    the non‑null hypothesis.  You can't go to things like

    22    Fisher's exact test, obviously.  And I agree the

    23    numbers were small.  We are in the tail of the

    24    binomial.  I tried to replicate it with other analyses

    25    and came up with a consistent result.

                                                                         57

     1                CHAIRPERSON VOSE:  Dr. Siegel?

     2                DR. SIEGEL:  Yes.  Just as a point of

     3    information or perhaps a couple on a couple of issues

     4    recently, we certainly accept and encourage the idea

     5    of a null hypotheses with pre‑specified differences. 

     6    The primary endpoint, though, as you referred to it is

     7    not one that the Agency considers appropriate as a

     8    measure of neutrophil engraftment, and that is

     9    consistent with advice and discussion of this

    10    committee.  We prefer not to look primarily and do not

    11    look primarily at engraftment by day 14, but rather at

    12    time to engraftment analyses, which are more sensitive

    13    to across the board shifts of a couple of days, which

    14    might be of interest.  And also of important secondary

    15    interest at percent who have engrafted or failed to

    16    engraft at later time points, 30 days or so, as a sign

    17    of possible tailing in the distribution, where some

    18    therapies may actually increase the number of

    19    engraftment failures.  Those issues were discussed

    20    with the company.  All of those things were measured

    21    as secondary endpoints, so we didn't wind up getting

    22    into loggerheads specifically on that issue.  You have

    23    heard all those endpoints presented and we will hear

    24    more about them.

    25                I also wanted to say regarding Dr.

                                                                         58

     1    Silver's question just to add in that ‑‑ since I don't

     2    think it was mentioned ‑‑ that there is implicit in

     3    the limiting dilution analysis an individual test by

     4    test assessment of whether in fact the probe has the

     5    sensitivity for a single copy of DNA.  When you do a

     6    limiting dilution analysis and you look at how the

     7    percents of positivity drop off from dilution to

     8    dilution, if you have sensitivity down to a single

     9    cell, you will see a distribution that is a function

    10    of the Poisson distribution and of the actual

    11    frequency.  And if you have a less sensitive test, you

    12    may see ‑‑ you will see a different pattern of fall‑

    13    off as you dilute the cell number out.  It is not a

    14    highly sensitive assessment given the numbers of

    15    replications in this, but it is not a highly sensitive

    16    assessment of whether there is a single copy

    17    sensitivity.  But it does provide some evidence ‑‑ in

    18    this case, some positive evidence that there was that

    19    level of sensitivity.

    20                CHAIRPERSON VOSE:  Additional questions? 

    21    Mr. Katz?

    22                MR. KATZ:  Yes.  I have two technical

    23    clarifications I wanted to ask about.  One, has there

    24    been any look at the different disease types of the

    25    various patients ‑‑ you know, IgA, IgG, light

                                                                         59

     1    chain/heavy chain?  Is there any reason to expect that

     2    that would make a difference in how effective it would

     3    be?

     4                DR. JACOBS:  Actually, no, that was not

     5    done.  I don't know if you would expect any

     6    differences.  I would rather have ‑‑ 

     7                DR. BERENSON:  Well, I think the problem,

     8    Mike, of course is there is not enough patients to

     9    answer that particular question.  I don't know the

    10    exact number that were IgA and IgG on that CD34

    11    selected.  Do you offhand, Bob?  It is probably, if I

    12    could guess, approximately two‑thirds were IgG and

    13    one‑third are IgA.  But we just don't have those

    14    numbers.  It is too small.

    15                MR. KATZ:  Well, I guess the question

    16    would be that some other therapies seem to be

    17    different, whether it is Kappa or lambda or light

    18    chain or God knows what.

    19                DR. BERENSON:  Depending on whose trial

    20    you read.

    21                MR. KATZ:  What?  

    22                DR. BERENSON:  I don't know, Ken, if you

    23    have a comment on this.  But there is a lot of

    24    variability of results. For example, with Interferon,

    25    some trials suggesting differences as you have

                                                                         60

     1    suggested with different isotypes.  Other trials do

     2    not suggest that.  Ken, do you want to make a comment

     3    on that?

     4                DR. ANDERSON:  Yes. I would just echo what

     5    Jim has said.  I think that this trial was not

     6    designed to test the efficacy of high dose therapy

     7    versus conventional therapy or subgroup analysis

     8    within that.  The efficacy endpoint, as you heard, was

     9    tumor cell depletion.

    10                MR. KATZ:  Well, I guess this is a

    11    technical question, though.  Is there any reason to

    12    believe that the different ‑‑ is isotypes the word ‑‑

    13    that different types would make any difference in the

    14    effectiveness of the selection technology?

    15                DR. ANDERSON:  No, there is none.  I think

    16    to get right to your point, if you look at B cell

    17    lineage differentiation or myeloma, the malignant B

    18    cell, and you look at CD34 antigen expression, it does

    19    not vary, Mike, with the subtype of myeloma.

    20                MR. KATZ:  Okay.  One other technical

    21    clarification.  This has been tested with a trial that

    22    uses busulfan and cytoxin as the transplant regimen. 

    23    That is, in fact, the minority of transplants that are

    24    done in the country, right, or in the world?  Most

    25    transplants are done with melphalan or possibly with

                                                                         61

     1    TBI.  Is there any reason to question what this ‑‑ how

     2    this might behave in patients undergoing those

     3    regimens?

     4                DR. BERENSON:  Well, that has actually

     5    been looked at by the IBMTR, and there is no

     6    difference in outcome.  Obviously that is not a

     7    randomized group, but at least in their registry, the

     8    IBMTR has looked at different regimens and shown no

     9    difference, whether it is BUCY, TBI‑containing, or

    10    melphalan.  So it does not seem to influence outcome. 

    11    Obviously that is registry data and fraught with all

    12    of those problems, but that is, I guess, about the

    13    best we have.  We certainly have no randomized data

    14    comparing BUCY to melphalan or TBI.  

    15                MR. KATZ:  Well, I am not asking the

    16    question in terms of the effectiveness of the

    17    transplant.  What I am asking is that presumably this

    18    is going to be approved or the proposal is to approve

    19    it for any of these regimens to be used as a purging

    20    technique with any of the regimens.  So the question

    21    is would you have any reason to believe that

    22    engraftment might be different or that there might be

    23    other issues with people that have different

    24    myeloablation?

    25                DR. BERENSON:  Myeloablative chemotherapy? 

                                                                         62

     1    Is that what you are asking, Mike?

     2                MR. KATZ:  Yes.

     3                DR. BERENSON:  I have no reason to expect

     4    that given all the other trials that have been done by

     5    CellPro.  I don't know if Ken or Keith has any other

     6    comments.

     7                DR. ANDERSON:  I would just echo those

     8    comments.  As Jim showed you all, the only randomized

     9    trial in myeloma, and we are fortunate to have one

    10    that showed a benefit for high‑dose therapy versus

    11    conventional therapy, is from France.  And by chance

    12    it had TBI and melphalan as the ablative regimen. 

    13    There are four other randomized trials; one in the

    14    United States, one in Scandinavia, one in England, and

    15    one in Spain which will be using different ablative

    16    regimens and in some cases some chemotherapy only,

    17    which are asking the high‑dose versus conventional

    18    therapy question and will be available within the next

    19    several years.  But for today, the issue is that in

    20    this trial, great care was taken to make sure there

    21    were no differences in the conventional chemotherapy

    22    and the intensity that was received or the ablative

    23    regimen that was given.  So that prior to the two‑arm

    24    experiment, if you will, the prior therapy, including

    25    ablation, was not significantly different in the two

                                                                         63

     1    groups in order to allow a fair comparison.

     2                CHAIRPERSON VOSE:  I think ‑‑ I was just

     3    going to say that I agree with your comment that I

     4    think there is a concern about TBI especially

     5    containing regimens and highly selected cell products. 

     6    There is some data of concern about that area and I

     7    think we do have to keep that in consideration in the

     8    labeling.  So I think that is a concern.

     9                DR. JACOBS:  In the final clinical report

    10    that we submitted to the FDA, we did a literature

    11    search of publications, looking at then the time to

    12    neutrophil and time to platelet engraftment, which is

    13    very comparable to what we see in the Phase III study. 

    14    Obviously a variety of regimens were used, not only

    15    for multiple myeloma but for breast cancer and

    16    lymphoma.  So there is a consistent pattern as far as

    17    time to neutrophil and platelet recovery.  

    18                The other point is that the type of

    19    regimen is usually specified by the investigators, and

    20    we do have a Phase III randomized trial in Europe and

    21    their preference was melphalan and TBI.  So it really

    22    is ‑‑ the trial here was not to look really at a

    23    specific regimen, but just at the unselected and

    24    selected infusion.

    25                CHAIRPERSON VOSE:  Dr. Berman?

                                                                         64

     1                DR. BERMAN:  Did you break down your

     2    groups in terms of stage of disease at diagnosis?  I

     3    am particularly questioning why four patients weren't

     4    able to be mobilized correctly?  Were those people

     5    with advanced stage disease at the start?

     6                DR. JACOBS:  There was actually only one

     7    patient that ‑‑ there was only one patient that didn't

     8    mobilize adequately.  The other three patients were

     9    randomized.  One had progressive disease that was

    10    found during baseline assessment.  One patient elected

    11    not to go to transplant on this study but had an

    12    allogeneic transplant.  And one patient actually died

    13    of sepsis during the mobilization prior to being

    14    randomized.  

    15                DR. BERMAN:  But just to get back to the

    16    question of staging of disease in the two arms, did

    17    you look at that?  Were they comparable?

    18                DR. JACOBS:  Yes, they were comparable.

    19                CHAIRPERSON VOSE:  Dr. Hartigan?

    20                DR. HARTIGAN:  In your looking at the

    21    baseline factors that were responsible for the time to

    22    platelet engraftment, you found that CD4 counts and

    23    platelet count at baseline were significantly related. 

    24    Did you look at the interaction between the two?

    25                DR. WHITE:  Which interaction are you

                                                                         65

     1    talking about?  The interaction between platelet count

     2    at randomization and CD34 count infused?

     3                DR. HARTIGAN:  Yes.

     4                DR. WHITE:  Yes, we did.  There was no

     5    interaction between them.  We have also done some

     6    further investigation of what is happening with these

     7    two factors, and we have looked at patients who took

     8    a longer time to engraft and ranked their CD34 count

     9    as well as their platelet count.  What happens is that

    10    they really are somewhat independent in their effect

    11    on time to engraftment.

    12                CHAIRPERSON VOSE:  Additional questions? 

    13    I had one.  Jim, you mentioned that the patients had

    14    Interferon maintenance post‑transplant, is that

    15    correct?

    16                DR. JACOBS:  Not in the Phase III.

    17                DR. BERENSON:  That was true in Phase II.

    18                CHAIRPERSON VOSE:  In Phase II, but not

    19    this study?

    20                DR. JACOBS:  No.

    21                DR. BERENSON:  Yes, Julie.  In the Phase

    22    II, we did employ Interferon, which was also done in

    23    the French Intergroup.  Most of us these days are a

    24    little disconcerted using it based on some recent

    25    randomized trials that you are aware of.  So we did

                                                                         66

     1    not use any maintenance in this Phase III.

     2                CHAIRPERSON VOSE:  In the Phase II study,

     3    was there any difficulty as far as toxicity for the

     4    patients outside what would normally be expected?

     5                DR. BERENSON:  In the Phase II study?

     6                CHAIRPERSON VOSE:  Phase II.

     7                DR. BERENSON:  There was difficulty for

     8    them to tolerate the Interferon definitely, both

     9    count‑wise as well as constitutional symptom‑wise. 

    10    But that has been the experience of others using

    11    unselected transplants as well.  And again, Keith or

    12    Ken, if you have other comments.

    13                DR. ANDERSON:  I would just echo that

    14    there is no standard maintenance therapy now in

    15    myeloma.  The large meta‑analysis that has been done

    16    looking at Interferon really has not shown an

    17    overwhelming effect for its utility.  In the

    18    randomized trial of conventional therapy versus

    19    standard therapy that is just about finished accrual

    20    in the United States, we are randomizing responding

    21    patients who get either conventional or high‑dose

    22    therapy to Interferon or not, hoping once and for all

    23    to answer this question.  But since there wasn't a

    24    standard at the time the Phase III was designed, there

    25    was no maintenance built into this trial.

                                                                         67

     1                CHAIRPERSON VOSE:  Okay.  Thank you. 

     2    Additional questions or comments?  Okay.  Why don't we

     3    take a 10‑minute break, and then we will resume with

     4    the FDA's perspective.  Thank you.

     5                (Whereupon, at 10:07 a.m. off the record

     6    until 10:23 a.m.)

     7                CHAIRPERSON VOSE:  If everyone can take

     8    their seats.  We would like to go ahead with the FDA

     9    perspective, and Dr. Litwin will present that.

    10                DR. LITWIN:  Thank you for the music. I am

    11    Dr. Stephen Litwin, and I will open the CBER review.

    12    This is a supplement to an existing license device,

    13    the Ceprate SC stem cell selectional concentration

    14    system.  The sponsor, as you very well know, is

    15    CellPro.  The product essentially are a collection of

    16    CD34 selected cells selected from autologous

    17    peripheral blood which will be hematologically re‑

    18    engrafted after myeloablative chemotherapy in patients

    19    with multiple myeloma.  

    20                This was a highly interactive review, and

    21    I would just like to briefly mention the contributions

    22    of Wendy Shores, who looked at the transferability of

    23    the device for peripheral blood as opposed to bone

    24    marrow, for which it is licensed, and some other

    25    aspects of yields and such.  And Dr. Edward Max, who

                                                                         68

     1    will very shortly present a critical review of the PCR

     2    technique.  Also, Dr. Gupta, who did all the

     3    statistical analyses for us.

     4                The current package insert indication for

     5    the device is for CD34 selection of autologous bone

     6    marrow.  The clinical indication is a lowered

     7    incidence of DMSO infusion‑associated complications,

     8    mostly cardiorespiratory.  And there was a caveat in

     9    the current indication that the infusion of less than

    10    1.2 million CD34 cells per kilogram is associated with

    11    delayed platelet engraftment.

    12                The proposed indication is that the

    13    labeling be extended from autologous bone marrow to

    14    autologous bone marrow and peripheral stem cells.  The

    15    clinical benefit would have as additionally a 100‑fold

    16    reduction, that is a 2 log or greater reduction in the

    17    number of tumor cells present in the hematologic

    18    autograft.  And to the caveat about a minimum number

    19    of CD34 for bone marrow is added a further labeling

    20    that the infusion of less than 2 million CD34 positive

    21    cells per kilogram of peripheral blood cells or cells

    22    collected from peripheral blood is associated with

    23    delayed or slower platelet engraftment.

    24                A few words about the CBER review.  Rather

    25    than repeat a lot of the descriptive elements which

                                                                         69

     1    Dr. Jacobs has so carefully presented, the CBER review

     2    will focus after a very short descriptive two slides

     3    on three elements; the collection and the processing

     4    of the cells and the impact that that has had on

     5    various outcomes, the efficacy analysis, which Ed Max

     6    and myself will present, and finally the safety

     7    analysis, which will mainly focus on engraftment.

     8                A few words about the experimental design. 

     9    131 multiple myeloma patients were enrolled.  All of

    10    them had to have a diagnosis of intermediate or high

    11    cell mass.  All of them had to lack progressive

    12    disease at any time during their course.  And all of

    13    them had to have had no more than three months prior

    14    alkylator chemotherapy.  After mobilization, they were

    15    randomized in a balanced fashion.  The Ceprate

    16    selected arm patients numbering 67 underwent

    17    leukophoresis and followed on the same day by the

    18    processing through the Ceprate device.  The control

    19    arm of 64 subjects simply underwent leukophoresis, and

    20    the two underlined cell products here are the ones

    21    that were infused into the patients.  After high‑dose

    22    chemotherapy and the autologous transplant, safety was

    23    followed for the first 100 days, particularly post‑

    24    transplant.  The two periods of time of major interest

    25    to CBER were the collection and the processing of the

                                                                         70

     1    autologous cells during which time the efficacy was

     2    determined by examining a subset, that is the 28

     3    patients out of the 67 or 66 in the Ceprate selected

     4    arm, for whom an immunoglobulin sequence could be

     5    developed.  And the second safety period, that is the

     6    period 100 days post‑transplant.

     7                The efficacy endpoint was reduction by 2

     8    logs or more of tumor cells in the infusate after the

     9    Ceprate selection.  And the safety endpoint was

    10    comparable neutrophil and platelet engraftment.

    11                The next six slides deal with the cell

    12    collection and processing.  You should keep in mind,

    13    first of all, that the guidelines for the collection

    14    and the end of collection of cells for the two arms

    15    differed.  And secondly, that the amount of CD34

    16    progenitor cells mobilized from the blood in

    17    individual patients in the selected arm varied

    18    greatly, and both of these had an impact on the

    19    outcomes.

    20                There were two leukophoreses minimum

    21    requirement.  The first criterion or guideline applied

    22    both to the selected and the unselected arms, and that

    23    was that there was a requirement that a minimum of 5

    24    x 108 total nucleated cells per kilogram be in the

    25    leukophoresis referred to as the PBPC product.  The

                                                                         71

     1    second criterion applied only to the selected arm. It

     2    required a minimum of 4 x 106 total nucleated cells

     3    per kilogram in the Ceprate selected, that is the

     4    enriched product.  The assumption was that half of

     5    these cells would be CD34 positive, and therefore

     6    every patient in the selected arm would receive at

     7    least 2 million CD34 positive cells.  This did not

     8    work out in every case.

     9                Looking at the leukophoreses to begin

    10    with, there were more leukophoreses in the selected

    11    arm, 3.0 mean, with a standard error of the mean of

    12    0.2, as opposed to the unselected arm, in which they

    13    were 2.3 with a standard error of the mean of 0.1. 

    14    The median was the same and the range was slightly

    15    longer for the selected arm.  

    16                It would be anticipated that the total

    17    nucleated cells per kilogram would also be increased

    18    in the selected arm because there were an increased

    19    number of leukophoreses.  This is not as marked as one

    20    would have anticipated, but the trend is present.

    21                The next slide looks ‑‑ oh, I am sorry. 

    22    This is the slide you have seen already but in

    23    different colors. This is a distribution of the

    24    leukophoreses.  You can see that 50 percent of the

    25    control patients required no more than 2

                                                                         72

     1    leukophoreses, whereas 36 percent of the selected arm

     2    patients were able to be satisfied with two

     3    leukophoresis.  And this difference, that is, a

     4    requirement for more leukophoreses in the selected

     5    arm, is present at all points of the distribution. 

     6                This looks at the progenitor cells, the

     7    CD34 cells that were collected.  If we look at the

     8    upper portion of this, you can see that three analyses

     9    can be done.  If we just look at the comparison of the

    10    leukophoresis product, there were more CD34 positive

    11    cells collected in the selected arm, presumably

    12    because of the increased number of leukophoreses, 14.3

    13    million, as opposed to the control or unselected arm,

    14    11.7.  If on the other hand we look at the two steps

    15    that each patient in the selected arm underwent, that

    16    is, the leukophoresis and then the Ceprate selection,

    17    there was a reduction of about 50 percent of the CD34

    18    cells lost during the selection procedure.  And the

    19    number of infused enriched product, that is, Ceprate

    20    selected cells, was 7.1 million.  And finally we can

    21    compare in the darkened boxes the number of actually

    22    infused CD34 positive cells.  There were less in the

    23    selected arm, 7.1 million per kilogram of body weight,

    24    as opposed to the unselected arm, 11.7.  It should be

    25    noted that these numbers, the numbers of infusate CD34

                                                                         73

     1    cells, are relatively high for autologous transplants.

     2                The same analysis can be made using the

     3    data for CFU per kilogram.  I won't go into this

     4    except to say that the trend is in the same direction,

     5    but the data are much less dramatic.  The reasons for

     6    this may be that the CD34 measurements were done at a

     7    central site, whereas the CFU measurements were done

     8    at individual sites and the assay is known to be

     9    highly variable.  

    10                The third impact of the cell collection

    11    strategy was that there were 14 percent of the

    12    patients or 17 patients who received less than 2

    13    million CD34 cells per kilogram.  They are listed

    14    here.  There were 10 in the selected arm and 7 in the

    15    unselected arm. The numbers of CD34 infused are shown. 

    16    If we look at the first column under selected for ANC

    17    engraftment, keeping in mind that the median day to

    18    ANC engraftment is 12 days, it is possible that there

    19    is some prolongation.  But this is ‑‑ it is not

    20    possible to statistically analyze this.  If we look at

    21    platelet recovery, there are four patients who had

    22    delayed platelet recovery, that is, longer than 20

    23    days, which is beyond the 95th percentile of the

    24    patients in the unselected group. Three of the four

    25    patients are in the selected arm and one in the

                                                                         74

     1    unselected arm.  And the same three patients who had

     2    a delayed platelet recovery, when examined by platelet

     3    count ‑‑ and these are platelet counts at the 6‑month

     4    visit after transplantation ‑‑ the same three patients

     5    continued to have platelet numbers that were

     6    substantially less than would be predicted for the

     7    remainder of the group.  The last patient actually was

     8    the only patient in the study group who could be

     9    considered as an engraftment failure for platelets. 

    10    This was an individual who continued to have platelets

    11    under 20,000 after transplantation, and on day 119 was

    12    given the depleted fraction, that is, the pass‑through

    13    fraction, which was used as a back‑up in the case of

    14    engraftment failure.  And this patient at 180 days

    15    still continued to have low platelet counts.  In the

    16    unselected arm, the single patient seems to have a

    17    normal platelet level.

    18                In summary, then, the impact of the

    19    collection processing strategy on the outcomes were

    20    three‑fold.  There were more leukophoreses and more

    21    total nucleated cells collected from selected arm

    22    patients.  On the other hand, there were less CD34

    23    cells infused into these selected arm patients,

    24    presumably due to loss during the selection process. 

    25    And there were 17 patients who were infused with less

                                                                         75

     1    than 2 million CD34 positive cells, a limited number

     2    of whom, 4 exactly, had delayed platelet recovery.

     3                I am going to introduce now Dr. Ed Max,

     4    who is going to give us a critical review of the

     5    assay.

     6                DR. MAX:  I'm using the slides rather than

     7    the video.  I am just going to go through our analysis

     8    of the PCR assay, which forms an essential element to

     9    the efficacy determination in this trial.  As Dr.

    10    Berenson mentioned, the PCR provides a potential for

    11    a unique assay for the patient's own myeloma because

    12    the multiple myeloma cell will have an immunoglobulin

    13    rearrangement involving a V, a D, and a J.  So if a

    14    sense primer is positioned within the V region and an

    15    anti‑sense primer positioned within the D/J region,

    16    then one can develop an assay that would be specific

    17    for the patient's immunoglobulin gene.  The sponsor

    18    reports one multiple myeloma cell per 100,000

    19    nucleated cell sensitivity of the assay, and the

    20    strategy for quantification was to make successive

    21    dilutions of a half log starting with 100,000 cells

    22    and use five duplicates at each dilution, and then to

    23    calculate the number of myeloma cells in the starting

    24    population by Poisson statistics based on the number

    25    of the five duplicate PCR amplifications that were

                                                                         76

     1    positive at each dilution.

     2                I am going to very briefly go through the

     3    kind of data that was submitted to us so you can see

     4    the kind of nitty gritty of the assay.  This just

     5    represents an individual patient's immunoglobulin

     6    gene.  Here is the sense primer.  Here is the anti‑

     7    sense primer.  For each patient, the germ line gene

     8    corresponding to the myeloma gene was looked at in

     9    comparison to the myeloma sequence and the design of

    10    the primers included an effort to maximize the somatic

    11    mutation in the primer so that the primers would have

    12    less likelihood of amplifying a product of the same

    13    immunoglobulin V region germ line.

    14                The DNA samples for the various cell

    15    samples accumulated during the trial were encoded with

    16    letters here chosen by a random code.  And we are

    17    going to look at the results for one patient, the

    18    first patient in the trial, where the leukophoresis

    19    was coded with a V and the CD34 enriched sample was

    20    encoded with a P.

    21                This just shows that for each patient

    22    there was an attempt to optimize the conditions of

    23    amplification and a particular amplification protocol

    24    was chosen.  I am showing you now the protocol for an

    25    individual gel, where you can see the P sample was

                                                                         77

     1    dilution A, which is undiluted sample representing DNA

     2    from 100,000 cells.  Five replicates here ‑‑ here is

     3    dilution B and here is dilution C.  As mentioned, each

     4    sample had a beta‑actin control, an internal control

     5    for the DNA integrity.  We will come back to that

     6    because we had some concerns about that control.  Here

     7    is the V sample and bone marrow samples were run as a

     8    positive control.

     9                I am going to show you the actual

    10    appearance of the gel.  Here is a sample of five

    11    replicates.  Four for this P sample were detected at

    12    the starting dilution, and then here is the next

    13    dilution where it went down to 2 and then to zero.

    14                These gel lanes were observed by three

    15    independent observers who identified the number of

    16    lanes in each dilution which were positive, and those

    17    observers generally agreed with each other and most of

    18    the time agreed with us.  We looked at every lane of

    19    every gel submitted for the CD34 enriched and the

    20    starting PBPC product and samples of the CD34 depleted

    21    product.  

    22                The number of positive wells at each

    23    dilution was then entered into a Poisson statistical

    24    analysis program called DIL‑SOLVE.  Here are the

    25    numbers.  I am sure it is too small for the people at

                                                                         78

     1    the back to see.  For this particular sample that we

     2    looked at before where the initial undiluted sample

     3    gave 4 wells out of 5 positive.  The next one was 2. 

     4    On the basis of this, the program calculated a number

     5    of target cells per well at the starting concentration

     6    representing 100,000 cells.  And also calculated an

     7    expected number of lanes positive for each of the

     8    dilutions.  And in general, these were quite

     9    remarkably close to what was observed.  And in

    10    general, the progression of the numbers of positive

    11    wells at the successive dilutions were pretty much as

    12    one would expect for Poisson distribution, although

    13    there were several samples where the positive wells

    14    trailed down from 5 slowly over successive dilutions,

    15    indicating probably some problem with the assay. 

    16    Although, of course, some statistical fluctuation

    17    would be expected.

    18                This slide illustrates the basis of the

    19    calculation.  I would just like to go through it in

    20    detail just to show you for this one patient.  We are

    21    looking now at the three samples, V, P, and C

    22    representing the PBPC product, the starting product,

    23    the CD34 enriched product, and the CD34 depleted

    24    product.  There were three sorts of numbers that

    25    entered into the final determination of the number of

                                                                         79

     1    myeloma cells in each sample.  First, the total number

     2    of nucleated cells were counted on the basis of a

     3    hemocytometer.  As you can see for this patient, there

     4    was a very substantial number of depletion in going

     5    from the starting nucleated cells in the PBPC product

     6    to the CD34 enriched sample.  And this ranged from

     7    approximately 200 to 400 depletion.  So what this

     8    means is that as was pointed out earlier, if the

     9    column did not selectively bind to the myeloma cells,

    10    one would expect approximately a 200‑fold depletion of

    11    myeloma cells just on the basis of the fact that the

    12    total nucleated cells in the CD34 enriched sample were

    13    very extensively depleted.  But then, of course, the

    14    PCR assay was done to detect whether there was any

    15    further depletion, and in fact there was for most of

    16    the samples, although not all of the samples.  There

    17    were three or four samples where the ratio of myeloma

    18    cells per total nucleated cell was either about the

    19    same in the CD34 enriched or actually slightly higher. 

    20    Two of these patients represented the individuals that

    21    did not achieve the 2 log depletion of myeloma cells

    22    in the CD34 enriched sample.

    23                So then the DNA was prepared from each of

    24    these samples and was quantified.  So the first

    25    measurement that influenced the final assay result was

                                                                         80

     1    the total nucleated cell count based on the

     2    hemocytometer.  The second was the DNA assay.  And

     3    that was performed using a fluorometric assay.  Based

     4    on that DNA determination, an amount of DNA

     5    corresponding to 100,000 cells and successive

     6    dilutions of those cells were, as previously

     7    mentioned, alloquoted and dispensed in replicates into

     8    the various tubes.  This shows the dilution and here

     9    are the number of positive wells out of the 5 ‑‑ 4 out

    10    of 5 and 2 out of 5 as shown earlier.  Based on these

    11    numbers using the Poisson distribution analysis, a

    12    calculation was made of the percentage of myeloma

    13    cells in the total nucleated cell count.  This number

    14    was then multiplied by the number that had been

    15    determined on the basis of the hemocytometer reading

    16    to give you a final estimate of the number of tumor

    17    cells in that sample per million cells.

    18                So as you can see here, the percentage of

    19    tumor cells was, in this case, substantially depleted

    20    from .02 percent to .0013 percent.  And when these

    21    numbers were multiplied together, you get a very small

    22    number, which for this particular patient represented

    23    a 3.745 log depletion of tumor cells.

    24                This is a summary of all the patients. 

    25    You have seen these numbers before.  28 patients, a

                                                                         81

     1    subset of the total number of patients in the

     2    randomized to the CD34 selected arm, had a product

     3    which was capable of being amplified.  24 of these

     4    patients had a product that showed evidence of the

     5    multiple myeloma cell in the PBPC product.  Of these,

     6    in 11 of the patients, the myeloma cells were

     7    undetectable after Ceprate selection, or 46 percent.

     8                Now this represents an amended version of

     9    a figure that was submitted by CellPro showing for

    10    each patient the starting log number of tumor cells in

    11    the PBPC product, and this shows the CD34 enriched

    12    sample after processing through the Ceprate.  The

    13    original figure actually showed those 11 patients as

    14    going to zero.  We have replotted these using the

    15    calculation based on the detection sensitivity because

    16    the assay is, of course, limited by the detection

    17    sensitivity of the PCR assay.

    18                This slide actually shows the final

    19    results just plotted as a log tumor depletion.  What

    20    I have plotted here in black represents the number of

    21    multiple myeloma cells in the CD34 enriched samples. 

    22    And the open circles just represent those patients for

    23    whom no myeloma cell could be detected in the product. 

    24    What I have used here as a calculation to estimate the

    25    log depletion is that number of cells which represents

                                                                         82

     1    the minimal detection level of the PCR assay.  So, in

     2    fact, these are potential underestimates.  You can see

     3    that the average log depletion is in the range that

     4    CellPro has stated.  There are definitely two patients

     5    who did not meet the goal of 2 log removal of myeloma

     6    cells in the assay.

     7                Now I would like to mention one concern

     8    that FDA had apart from the question of the assay

     9    accuracy and the log error, which CellPro has

    10    estimated as about a half a log.  An additional issue

    11    we felt that raised some concern is that the control

    12    that CellPro used, the beta‑actin control, as an

    13    internal control for the integrity or the

    14    amplifiability of each DNA sample was inadequate.  And

    15    the reason for that is that amplifying the undiluted

    16    DNA at 60 PCR cycles for beta‑actin, this is basically

    17    a non‑quantitative assay.  It is insensitive to

    18    potential template problems that might have decreased

    19    the amplifiability of that sample, either due to poor

    20    DNA quality or the presence of PCR inhibitors.  60

    21    cycles basically overwhelms the system.  

    22                Therefore, CellPro has not ruled out the

    23    worst case scenario, and that is that systematic

    24    template problems specific to the CD34 selected sample

    25    might have led to an undercount of multiple myeloma

                                                                         83

     1    cells in that sample, and such an undercount would

     2    simulate myeloma cell depletion by the Ceprate device.

     3                We raised this issue to the sponsor, and

     4    we have had kind of a back and forth over the past

     5    couple of months about how worrisome this concern

     6    might be.  Whether it is just a theoretical issue or

     7    whether it is ruled out by the data that they present. 

     8    Their first response was that no multiple myeloma

     9    signal has been lost due to any hypothesized

    10    systematic undercount since the myeloma cells assay in

    11    the two fractions, that is the CD34 enriched plus the

    12    CD34 depleted sample, add up to the myeloma cells in

    13    the starting PBPC.  We looked at this and actually it

    14    is difficult to make a case that that is correct when

    15    you look at individual patients.  I will just show you

    16    this patient we have looked at before.  Here the

    17    starting PBPC product showed 13 million myeloma cells. 

    18    And in the CD34 depleted sample, there were 94 million

    19    assayed.  Obviously one can't sum these two fractions

    20    and get 94.  Here in this patient, we have the

    21    opposite problem as was pointed out earlier.  This

    22    represents, I guess, one of those two patients that

    23    kind of fell outside the cluster in the log plot shown

    24    earlier.  Here there were 363 ‑‑ I am sorry, this

    25    represents one of those patients.  Here is the

                                                                         84

     1    opposite problem.  We had 363 million in the PBPC

     2    product, and the sum of the two fractionated samples

     3    is quite low.  So clearly analysis of individual

     4    patients will not show the kind of conservation of

     5    myeloma cells that might have ruled out the possible

     6    systematic undercount of myeloma cells in the CD34

     7    depleted sample.

     8                Well, CellPro came back with another

     9    suggestion which was to average over all the patients. 

    10    They looked at the ratio of PBPC product, the

    11    starting, plus this sum.  Theoretically, this sum

    12    should equal the PBPC product, so this ratio would be

    13    1.  The log would therefore be zero.  And so if this

    14    was examined over all patients, they found that this

    15    value was not significantly different from zero, that

    16    is, the 95 confidence limits included zero as expected

    17    if there was no myeloma signal loss.  We felt that

    18    this kind of analysis really failed to test whether

    19    the data are inconsistent with an undercount of

    20    myeloma cells.  And, in fact, if you examine or if you

    21    assess that possibility and multiply the number of

    22    determined myeloma cells in the CD34 enriched fraction

    23    by 250 and add that to the myeloma cells in the CD34

    24    depleted sample, one finds that this log is still not

    25    significantly different from zero. So the data do not

                                                                         85

     1    exclude a 250‑fold undercount.  

     2                The reason, of course, that this addition

     3    is insensitive to multiplying one of these addends by

     4    250 is the fact that the number of myeloma cells

     5    measured in the CD34 enriched sample was just very,

     6    very small compared to the error in these numbers,

     7    which CellPro has estimated at .5 log, but is perhaps

     8    larger than that.  

     9                So in view of our concern, CellPro

    10    submitted to us data on a single patient in which they

    11    tried to address the concern about possible systematic

    12    undercount of myeloma cells in this CD34 enriched

    13    sample.  They did a quantitative PCR amplification of

    14    beta‑actin using the same kind of strategy of dilution

    15    analysis and Poisson statistics, showing for that

    16    single patient, amplification efficiency was nearly

    17    equivalent for the starting PBPC and the CD34 enriched

    18    sample.  However, in the data they submitted to us,

    19    the efficiency was only about 5 percent of what was

    20    predicted, and perhaps even less than that.  We had

    21    some question about their assumptions for these

    22    numbers.  CellPro suggested that this low efficiency

    23    might have been due to a PCR protocol that was

    24    optimized for shorter amplification products, products

    25    in the range of 100 to 200 base pairs versus the

                                                                         86

     1    product that they amplified for beta‑actin, which was

     2    650 base pairs, and might have required longer PCR

     3    extension time.

     4                So I would like to summarize our final

     5    assessment and hope that the Advisory Committee will

     6    consider our concerns.  The PCR assay has demonstrated

     7    that the Ceprate selection leads to a substantial

     8    depletion of myeloma cells.  Usually it is greater

     9    than 100‑fold, that is, in 22 of the 24 evaluable

    10    patients based on the numbers they submitted.  We

    11    still remain somewhat concern that the possibility of

    12    a systematic undercount of myeloma cells has not

    13    completely been ruled out by CellPro, and the reasons

    14    are the beta‑actin control that they presented to us

    15    was only on one patient and it did give an

    16    unexpectedly low beta‑actin amplification on that one

    17    patient.  Finally, we have sort of a theoretical

    18    concern that even that beta‑actin amplification

    19    doesn't really test the integrity of the myeloma cell

    20    DNA because it is done on the bulk CD34 enriched

    21    sample, which is obviously made up mostly of non‑

    22    myeloma cells.  It is conceivable that DNA deriving

    23    from the myeloma cells might have some impairment in

    24    its amplifiability.  But FDA feels that testing for

    25    this would not really be feasible.

                                                                         87

     1                So in conclusion, there is no reason to

     2    believe that such an undercount would have occurred. 

     3    It is a theoretical possibility that CellPro failed to

     4    provide adequate controls against.  It is only a

     5    theoretical concern and the data that they submitted

     6    certainly supports depletion for most patients that

     7    reaches the target of 100‑fold depletion.  

     8                DR. LITWIN:  I'm going to add some

     9    additional data on the efficacy analysis.  I will try

    10    not to repeat what Dr. Max has gone into in great

    11    detail.  These are simply the numbers of tumor cells. 

    12    This is the number in the leukophoresis product on the

    13    28 patients in whom a probe was available, and this is

    14    the number in the Ceprate selected enriched product. 

    15    The distribution was asymmetric and the values are

    16    given as quartiles, 25th, median 50, and 75th.  If we

    17    look at the numbers, you could see that the median

    18    patient would have between 2 million and 3 million

    19    tumor cells in the first leukophoresis product.  All

    20    of these data are done on the first leukophoresis

    21    product.  The numbers for the post‑device depletion

    22    product are much lower.  This is equivalent to about

    23    700 cells if accurate.

    24                The determination for the depletion was

    25    done as a log geometrically.  These are the figures

                                                                         88

     1    with the same distribution expressed for the

     2    leukophoresis product and the Ceprate selected

     3    product.  The median depletion was 3.10 logs.  The

     4    mean was 3.29.  This met the prospectively discussed

     5    endpoint of greater than 2 logs depletion.  

     6                The next slide shows the distribution of

     7    the log depletion.  This is very much similar to the

     8    vertical slide that Ed showed.  Most patients had

     9    between ‑‑ the log depletion is shown under the X

    10    axis.  Most patients had between 3 and 4 logs

    11    depletion or 2 and 3, but there were some patients who

    12    had less than that and there were a number of patients

    13    who had more.  These are the 24 patients in whom the

    14    log depletion was measured.

    15                Finally, I think most of this has been

    16    discussed.  I will only emphasize the fact that 11 of

    17    the 24 patients could be purged to non‑detectability,

    18    and 13 continued to have detectable tumor.  The

    19    average ‑‑ we also looked at the log depletion of

    20    total nucleated cells.  The average depletion was

    21    about two‑fold as opposed to the 3‑plus log depletion

    22    we have just discussed for tumor cells.

    23                I will turn now to the safety analysis

    24    starting with neutrophil engraftment.  CBER chose to

    25    determine the engraftment parameters for neutrophils

                                                                         89

     1    and platelets using the mean and median days to

     2    duration of engraftment and the Kaplan Meier analysis

     3    rather than for the neutrophils the landmark analysis

     4    that Dr. Jacobs has presented.  And I think Dr. Siegel

     5    has already commented on the previous communications

     6    between us and the sponsor and the reasons that we

     7    consider this more useful.  

     8                The selected arm had 7/10ths of a day

     9    longer mean duration to neutrophil engraftment, 12.4

    10    days as opposed to 11.7 days.  The median day of

    11    engraftment was 12 days for both arms at the point

    12    that 50 percent of the engraftment events had

    13    occurred.  Both arms were 12 days.  The Kaplan Meier

    14    analysis looks a little unusual because of the highly

    15    compacted period of time over which the engraftment

    16    occurred.  Testing using non‑parametric tests for the

    17    differences between the two arms shows a low P value,

    18    the null hypothesis being that there is no difference

    19    to suggest that there is a possibility or a difference

    20    between the two arms favoring the unselected arm.  

    21                Turning to platelet safety endpoints, I

    22    think the data are more dramatic.  The median day ‑‑

    23    we use platelet recovery rather than platelet

    24    engraftment, which is, I think, some of the data that

    25    you saw shown by Dr. Jacobs.  Because CBER considers

                                                                         90

     1    it a more suitable endpoint with respect to the fact

     2    that it is not as dependent on transfusions.  The data

     3    is very similar between the two.  The median day of

     4    platelet recovery differed by one day.  It was one day

     5    longer in the selected arm.  The mean day differed by

     6    several days.  The P values once again are low.  The

     7    null hypothesis is that there is no difference, and

     8    this strongly suggests that the difference we see,

     9    that is, the prolongation of engraftment of platelets

    10    by a few days in the selected arm is real.  The next

    11    slide shows the Kaplan Meier analysis.  You can see

    12    that when 50 percent of the events had occurred, the

    13    two arms are pretty much the same.  If we follow it

    14    further out, it is obvious that ‑‑ the red, by the

    15    way, is the selected arm.  I know it is hard to see. 

    16    It is obvious that a number of patients in the

    17    selected arm had probably several days prolonged

    18    engraftment.  And at a little over 40 days, all of the

    19    patients in the control arm had platelet recovery,

    20    whereas there are a number of patients who continued

    21    out.  We have not ‑‑ we stopped this graph for the

    22    sake of description at 80 days, but there was a

    23    patient who I described already who at 119 days still

    24    lacked platelet recovery.  And this was the single

    25    patient who was given the back‑up or depleted product. 

                                                                         91

     1                The analyses of this indicates that the P

     2    values are once again, strongly supporting a

     3    difference between the arms, that is, a prolonged

     4    engraftment for platelet recovery ‑‑ prolonged

     5    platelet recovery in the selected arm.

     6                There were a number of platelet safety

     7    endpoints.  The platelet transfusion events and days

     8    of platelet transfusion events are listed.  The mean

     9    showed a difference.  the selected arm was 4.4 days,

    10    the unselected arm 3.2.  The median, which you have

    11    seen already, I believe, is 3 and 2 days.  The ranges

    12    are shown below.

    13                A number of other secondary endpoints that

    14    were related to engraftment were also done.  They

    15    included incidents of infection day 0 to 100,

    16    incidence of severe infection, days of

    17    hospitalization, days of rehospitalization, number of

    18    days of growth factor given post‑transplant.  All of

    19    these secondary endpoints, as summarized by Dr.

    20    Jacobs, were the same in both arms.

    21                Late engraftment events were also looked

    22    at, and these are platelet late engraftment events. 

    23    CBER did an analysis by taking from the list files all

    24    patients who at 100 days after transplantation ‑‑ I am

    25    sorry, 6 months after transplantation had 100,000 or

                                                                         92

     1    less platelets.  When there was more than one

     2    determination, as there often was, we took the first

     3    determination.  These 17 patients that we had were

     4    further subset.  There were 17 altogether, 13 in the

     5    selected arm.  By having equal to or less than 75,000,

     6    et cetera, for 50 and 20.  The preponderance of

     7    selected arm patients, about three‑quarters, is

     8    noticed here.  

     9                The next slide continues this analysis. 

    10    We were concerned with the possibility that the data

    11    that we were looking at in terms of late engraftment

    12    or late recovery or incomplete recovery ‑‑ it is a

    13    little hard to judge at six months ‑‑ was related to

    14    risk factors.  And the two that were considered were

    15    infusion of less than 2 million CD34 cells per

    16    kilogram.  This is simply the control down here.  And

    17    having equal to or less than 75,000 platelets at the

    18    time of mobilization.  The latter data are not shown. 

    19    They don't ‑‑ they are not very clearcut.  But we did

    20    both analyses.  If we just look at the upper section,

    21    you can see once again the preponderance of selected

    22    arm subjects.  If we look at those patients who have

    23    less than or equal to 50,000 ‑‑ now this is 6 months

    24    post‑transplant ‑‑ all four subjects were infused with

    25    less than 2 million cells, and in fact all four were

                                                                         93

     1    selected arm subjects.  If we look at those who have

     2    less than 75,000, of which there were 8 subjects, 6 of

     3    the 8 were infused with less than 2 million cells.  2

     4    of the 8 had over that.  And of these 6, 5 out of the

     5    6 were in the selected arm.

     6                These data suggest that the infusion of

     7    less than 2 million per kilogram of CD34 positive

     8    cells could be a risk factor, particularly in

     9    conjunction with the Ceprate selection.

    10                Continuing the safety analysis, there was

    11    a central review of progression of disease.  Patients

    12    were evaluated at baseline at 100 days, at 6 months,

    13    and at 12 months.  At baseline, almost all the

    14    patients were either in remission or were stable. 

    15    There are no substantial differences in the numbers of

    16    patients here.  At 12 months, there were 19 patients

    17    in each arm who showed disease progression, 29 percent

    18    and 30 percent respectively.

    19                A similar analysis was done for deaths. 

    20    At 100 days, 6 months, and 12 months, once again there

    21    are no significant differences in the number of deaths

    22    between the groups, 12 percent for the selected arm

    23    and 9 percent for the unselected arm respectively.

    24                During the course of the CBER analysis, a

    25    number of labeling issues arose, and three are listed

                                                                         94

     1    here.  The one of major concern, which I suspect is

     2    already obvious, is the question of whether infusion

     3    of less than 2 million CD34 positive cells per

     4    kilogram is a risk factor.  Four pieces of information

     5    bear on this.  The 17 patients of the 131 who were

     6    studied who were infused with less than 2 million

     7    cells had a limited number, 4 specifically, of slow

     8    platelet recovery.  I have just presented the late

     9    engraftment data, which is 6 months after

    10    transplantation.  There is published literature that

    11    strongly supports the fact that patients infused with

    12    less than 2 to 5 million CD34 positive cells had a

    13    slower rate of engraftment than did those infused with

    14    above that.  The sponsor has presented the data

    15    already on logistic regression analysis which shows

    16    that among those variables which they concluded were

    17    connected with engraftment events, the two that were

    18    most prominent were the number of CD34 positive cells

    19    infused and the number of platelets at the time of

    20    mobilization.

    21                There were two other issues.  We asked the

    22    sponsor to provide a literature search on the

    23    expression of CD34 on malignant cells.  They supplied

    24    that of 66 publications.  The publications show

    25    essentially what I suspect most of the Committee and

                                                                         95

     1    the audience is already aware of, that is, that CD34

     2    is seen for the most part on undifferentiated immature

     3    cells and cells of a leukocytic series.  That aside

     4    from leukemic cells, it is rarely seen among cells of

     5    solid tumors.  However, there are several reports,

     6    including one last year in 1997 in Blood, which does

     7    report very strong data on the appearance of CD34 on

     8    myeloma cells. So that it can happen and it does

     9    happen apparently.  

    10                Finally, the question of reduction of

    11    tumor burden in other than multiple myeloma cancers. 

    12    The literature review of the 15 papers has already

    13    been alluded to by Dr. Jacobs.  The sponsor also in

    14    response to our request has provided Phase I and II

    15    data on two studies.  One was from myeloma and the

    16    other from breast cancer.  They both are consistent

    17    with a lot of the data recorded. The myeloma study

    18    indicated that about half the patients could be purged

    19    to non‑detectability and that there was a reduction of

    20    tumor burden by 3 to 4 percent in that report.  And

    21    the second paper on 15 patients out of a larger group

    22    of breast cancer patients reported a reduction of

    23    tumor burden by one and a half logs, but that may be

    24    very dependent on the methodology.

    25                The last two slides I would like to

                                                                         96

     1    summarize my comments and reviewers comments.  Primary

     2    endpoints were satisfied as prospectively designated

     3    using data from single protocol CP0060‑02.  There was

     4    a marginal delay in neutrophil engraftment and a

     5    definitive one to two day delay in platelet recovery. 

     6    The term marginal as used here is based on our data,

     7    which indicates that there is a 7/10th's of a day

     8    delay in mean and that there is a low P value for

     9    supporting differences in the Kaplan Meier analysis

    10    for neutrophil engraftment.  Late engraftment

    11    suggested a limited number of selected subjects had

    12    poor or delayed platelet recovery.  The infusion of 2

    13    million CD34 positive cells per kilogram may be a risk

    14    factors.  There were concerns, which Dr. Ed Max has

    15    discussed, about the quantitative aspects of the PCR‑

    16    based assay of the tumor cells.  

    17                The disadvantages of the proposed use of

    18    Ceprate selection includes a small increase in the

    19    number of leukophoresis, a decreased number of

    20    progenitor cells which could be infused, and possibly

    21    slower and incomplete engraftment, that is, referred

    22    to as late engraftment problems.  The major advantage

    23    is reduction of tumor cells in the autografts.  There

    24    were limited data provided on tumor purging in

    25    clinical settings other than multiple myeloma and

                                                                         97

     1    follow‑up studies of disease progression and patient

     2    survival should continue after FDA action.  Thank you.

     3                CHAIRPERSON VOSE:  Thank you, Dr. Litwin. 

     4    Let's go ahead and open it up for any questions from

     5    the Committee.  Dr. Auchincloss?

     6                DR. AUCHINCLOSS:  May I ask Dr. Max about

     7    his concerns, which statistically look perfectly

     8    reasonable.  But is there any biologic example where

     9    such systematic undercounting of cells has occurred by

    10    PCR analysis as versus positive selection?

    11                DR. MAX:  I don't know of any example. And

    12    as I said, we have no particular reason.  We would

    13    assume that the cells would behave more or less

    14    identically ‑‑ the cells that have stuck to the column

    15    and the cells that have passed through.  FDA prefers

    16    not to base approvals on unproven assumptions,

    17    however.

    18                DR. AUCHINCLOSS:  But then you went on to

    19    say that there was no way they could prove their

    20    assumption as I understood you.  That the test of the

    21    multiple myeloma cell DNA was impossible to perform.

    22                DR. MAX:  I think that test is maybe not

    23    quite impossible, but unfeasible to ask to look at

    24    every patient.  What I think is feasible and would

    25    rule out let's say PCR inhibitors, for instance, is to

                                                                         98

     1    look at the beta‑actin in a quantitative fashion as

     2    they have done for one patient to assess effects that

     3    might be having an action on the myeloma cells as well

     4    as the bulk DNA.  And we understand that they have

     5    looked at with a different pair of primers and two

     6    additional patients and have gotten more satisfactory

     7    results, which maybe CellPro would care to describe. 

     8    But FDA has not seen those data.  

     9                DR. AUCHINCLOSS:  Does the fact that they

    10    did in fact in those three patients end up with the

    11    same proportion of tumor cells as in the initial

    12    sample ‑‑ does that lead you to conclude that, in

    13    fact, they can measure tumor cells when they are

    14    there?

    15                DR. MAX:  Well, maybe I should clarify

    16    what they have done.  It is not a question of assaying

    17    tumor cells in the original sample.  It is a question

    18    of assaying the beta‑actin, which is an internal

    19    control for ‑‑ 

    20                DR. AUCHINCLOSS:  No, but I am talking now

    21    about multiple myeloma cell detection.  And they were,

    22    in fact, detecting a similar proportion to their

    23    starting population in three patients.  That was part

    24    of the complaint about how accurate the assay really

    25    was.  But there they are apparently measuring what

                                                                         99

     1    would seem like a reasonable number of cells.  So they

     2    must be able to detect multiple myeloma cells if they

     3    are there.  

     4                DR. MAX:  I am not sure I follow.  

     5                DR. AUCHINCLOSS:  No.  That may be a lousy

     6    argument.  Let me conclude by just asking you this. 

     7    Is it fair to characterize your concern as raising a

     8    remote possibility, this degree of systematic

     9    undercounting?  

    10                DR. MAX:  I would say since we don't know

    11    how remote it is, I would say it is a theoretical

    12    possibility.

    13                DR. BROUDY:  I have a question for Dr. Max

    14    as well.  Part of the concern was that the number of

    15    tumor cells in the CD34 positive and then the CD34

    16    negative fractions did not always add up to the number

    17    of tumor cells in the starting fraction, but you

    18    presented very clearly how they calculated that by

    19    multiplying the number of cells times a percentage of

    20    tumor cells.  Did the total numbers of cells always

    21    add up?  Could part of the disparity in the numbers be

    22    due to systematic or random errors in counting? 

    23    Perhaps they undercounted or overcounted and then

    24    multiplied and that is why things didn't add up?

    25                DR. MAX:  Yes.  I think ‑‑ I can't give

                                                                        100

     1    you an absolute ‑‑ I can't tell you quantitatively,

     2    but there were some cases where there was a failure to

     3    add up just in terms of the total number of nucleated

     4    cells.  And that is why I emphasized that there are

     5    really three independent determinations in two

     6    samples.  So we are talking about six numbers that

     7    combine together for that final log removal.  And that

     8    is the DNA assay, which has its own potential

     9    problems, the counting on a hemocytometer, and the PCR

    10    assay.  I think it is probable that the ‑‑ based on my

    11    own experience that the error in the PCR assay is the

    12    one that is most worrisome.  But really we have no way

    13    of knowing.

    14                CHAIRPERSON VOSE:  Dr. Anderson?

    15                DR. ANDERSON:  Yes.  We have seen the data

    16    where it doesn't add up.  Let me just ask, of all the

    17    various patients, how many times did it add up?

    18                DR. MAX:  Well, I would say that there are

    19    only two or three patients where the numbers added up

    20    in a way that you would hope to see if the assay was

    21    quantitative.  There were ‑‑ I don't have the numbers

    22    completely memorized, but there were about maybe half

    23    a dozen patients in which the counts in the depleted

    24    sample were, let's say, in the range of 7 to 10 times

    25    that in the starting sample.  There was one patient

                                                                        101

     1    where the amount measured in the depleted sample was

     2    200‑fold and another close to 1000‑fold of what had

     3    been present in the starting sample.  And then there

     4    were similarly on the side of errors in the other

     5    direction where the myeloma cells were much lower in

     6    the depleted sample, and therefore the total of

     7    depleted and enriched was much lower.  Those numbers

     8    were also ‑‑ I don't, again, have the numbers in front

     9    of me, but I would say there were three or four

    10    patients where the numbers looked the way you would be

    11    very happy to see if they were your own experimental

    12    results.  

    13                DR. ANDERSON:  But what this sounds like

    14    is FDA doing what FDA ought to do, which is to really

    15    look at very strict criteria to determine if there is

    16    a systematic error that basically discounts the

    17    results. And I gather your conclusion is that having

    18    analyzed it very thoroughly, that the sponsors have

    19    not proven their point, but the FDA has not disproven

    20    it either?

    21                DR. MAX:  Well, what I would like to

    22    emphasize is that what we feel is the error in the

    23    assay is larger than one would hope.  On the other

    24    hand, the margin of error that would discount the

    25    sponsor's results is such that this assay may be

                                                                        102

     1    sufficient to prove their case.  So that if instead of

     2    a half log error, let's say the error was more like a

     3    log, then there might be more than the two patients ‑‑

     4    two patients fell below ‑‑ on their best estimate fell

     5    below the target of 2 logs removal. Now if you asked

     6    how many patients fell such that their 95 percent

     7    confidence interval was below their target, obviously

     8    that would be greater than 2.  And depending on their

     9    log error ‑‑ if their log error was sufficiently high,

    10    then that number would rise significantly.

    11                DR. ANDERSON:  What would be the

    12    possibility that the same set of errors could have

    13    resulted randomly in two patients being under, but

    14    that in fact none of the patients were under?

    15                DR. MAX:  I am not sure I ‑‑

    16                DR. ANDERSON:  In other words, what I am

    17    saying is because of the randomness in the numbers, it

    18    is theoretically possible that 64 out of 64 all had a

    19    depletion rather than 62 out of 64.

    20                DR. MAX:  Yes.  I think ‑‑ yes, that is

    21    certainly true.  I mean if one, instead of asking what

    22    is the 95 percent confidence limit that they were all

    23    above, you could say ‑‑ 

    24                DR. ANDERSON:  What are the chances that

    25    they are all below.

                                                                        103

     1                DR. MAX:  Below.  And that would be quite

     2    low.  

     3                DR. ANDERSON:  Okay.

     4                DR. MAX:  But I don't think that their

     5    error is so great ‑‑ that we have evidence that their

     6    error is so great.  The data that was shown by Dr.

     7    Berenson, I believe, of the log log plot would

     8    indicate that there is some correlation between the

     9    myeloma in the starting PBPC sample and the CD34

    10    depleted sample, which implies that these are not

    11    random numbers.  

    12                DR. ANDERSON:  I guess where I am coming

    13    from, and then I will stop here, is that based on that

    14    plot, what you just mentioned, where there are the two

    15    outliers to the upper left that you pointed out and

    16    there appeared to be one outlier on the right, the

    17    rest of them all seem to be reasonably close to the

    18    line, which implies that ‑‑ and there are enough

    19    points on that line ‑‑ that it would imply that the

    20    assay is good enough.

    21                DR. MAX:  That is what I think is true. 

    22    That is, of course, excluding this theoretical

    23    possibility of the undercount in the CD34 enriched

    24    cells.  Because that undercount would not have

    25    affected that curve.

                                                                        104

     1                DR. ANDERSON:  Right.

     2                CHAIRPERSON VOSE:  Additional questions? 

     3    Mr. Katz?

     4                MR. KATZ:  Well, I guess if I were a

     5    patient that was imminently considering a transplant,

     6    I would be looking at what is the potential benefit of

     7    this to me and what is the potential risk of it to me. 

     8    It seems from the discussion here that we will not

     9    agree conclusively on benefit.  And there is an area

    10    of benefit that hasn't even been discussed that

    11    mentally appeals to me just listening to it in saying

    12    that we are measuring tumor cells, cells that have

    13    gone all the way down the chain of differentiation and

    14    whatever else happens to make them a myeloma cell. 

    15    But if there is evidence to say that progenitor cells

    16    that aren't yet identifiable by this assay or however

    17    as myeloma cells might actually be selected out as

    18    CD34 negative, that would be appealing.  But it seems

    19    like no one is going to be able to prove that one way

    20    or the other.  So I would be more concerned about the

    21    risk side of this.  I think your recommendation in the

    22    document says it correctly in that there should be a

    23    commitment to study the outcomes because clearly no

    24    one has any data on the real outcomes in terms of

    25    survival because the patients really are looking at

                                                                        105

     1    that, not at specifically what is happening in

     2    engraftment.  As long as they are not going to die

     3    during the transplant.  But also that the risk factors

     4    are addressed.  Because I am not hearing ‑‑ it sounds

     5    like there may be some need to very closely monitor

     6    these late engraftment events.  I am not sure what is

     7    implied in an approval in terms of a commitment to

     8    monitor that very closely. I am not sure what

     9    constitutes a reportable event to the FDA if this gets

    10    approved.  And also from listening to the discussion,

    11    it seems like maybe there are some strictures around

    12    if you look at the harvest and you conclude that you

    13    are not going to inject or infuse 2 million cells,

    14    that maybe we shouldn't be using strictly the enriched

    15    product if that is a risk.  I don't know.

    16                So I guess my concern to the group would

    17    be ‑‑ and I think we are focused on whether we can

    18    absolutely prove the benefit, and I don't think we

    19    can.  If we can control the risk, it seems like

    20    something that should be available to the patient

    21    community and something that should be available for

    22    study if we can manage the risk.

    23                CHAIRPERSON VOSE:  I think a lot of those

    24    issues relate to things such as the study was not

    25    designed to look at overall survival, so we cannot

                                                                        106

     1    really say that.  A lot of the risks things and

     2    follow‑up information that you mentioned are things

     3    that we can discuss when we discuss the labeling

     4    issues and also the post‑marketing studies that we

     5    would suggest.

     6                DR. FLOYD:  I have a number of questions. 

     7    First I would like to make a couple of comments about

     8    devices in general.  We are talking here about a

     9    device that is affecting a biological specimen.  The

    10    first question that popped to my mind when I read

    11    through this is that everything I learned as a young

    12    biologist being trained in growing up seems not to

    13    have been considered in the design of this experiment. 

    14    Now I say that because one of the things we have done

    15    here is we have subjected half of a population ‑‑

    16    essentially half of a population to a treatment, but

    17    we haven't subjected the other half.  Basically, the

    18    unselected arm of this experiment simply had

    19    leukophoresis period.  Their cells were not subjected

    20    to essentially a dummy device, if you will.  The only

    21    way this experiment could have made sense from my

    22    perspective on evaluating a device is if those cells

    23    had gone through the same Ceprate machine that had a

    24    capsule that did not have a CD34 binder on whatever

    25    the binding medium is in this case.  That kind of

                                                                        107

     1    experiment would have gotten rid of many of the

     2    questions that keep coming up in this particular

     3    submission as well as the others that I have sat on

     4    for this particular group.  I would like to see what

     5    happens with a dummy device, if you will, because

     6    otherwise we don't have any way to evaluate the effect

     7    here.  It is a real issue.

     8                The other issue that has not been

     9    discussed that I have seen, and frankly I haven't gone

    10    back and done a literature search on all literature

    11    about binding devices, and I know there is a

    12    fantastically large literature in that area, one of

    13    the other pieces of information that I have not seen

    14    here whatsoever is what the actual repeatability of

    15    devices provided by the company are with respect to

    16    capture.  In other words, if I open 10 different

    17    packages of a capture cartridge, what is the range of

    18    variability in capture of each of those cartridges and

    19    what is the range of release using whatever mechanism

    20    they are using.  As I understood it here, we are using

    21    a stirring bar of some sort to dislodge the bound

    22    cells.  What range of human variability do we have in

    23    real patient populations of release from that kind of

    24    mechanism?  Those are the kinds of concerns I have. 

    25    And at this point, I haven't heard any answers to any

                                                                        108

     1    of those things.  

     2                CHAIRPERSON VOSE:  If someone from the

     3    company would like to comment on the second issue

     4    regarding variability between actual devices?  

     5                DR. TARNOWSKI:  I am Joe Tarnowski from

     6    CellPro.  I am Vice President and Chief Technical

     7    Officer.  Regarding your question about

     8    reproducibility, there are two answers to your

     9    question.  First you said a controlled study using a

    10    non‑binder.  We have done studies with mobilized

    11    normal donors where we passed material through our

    12    column without the antibody, and what we find is no

    13    difference in the distribution of the cell population

    14    as it passes through the column.  You do have some

    15    hang‑up in the tubing pathways, et cetera, where you

    16    have some loss of cells in general, and this is just

    17    a physical manipulation.  So I think the answer to

    18    your control arm is that we have done those in

    19    laboratory experiments and we wouldn't predict any

    20    difference there.

    21                As far as reproducibility of the system,

    22    we find that capture is very high in terms of we

    23    capture about 75 percent to 80 percent of the cells

    24    using the monoclonal antibody.  We see some cells that

    25    pass through in the unbound fraction, and these

                                                                        109

     1    probably are lower density cells that have lower CD34

     2    antigen distribution on its surface.  Those cells

     3    don't seem to be readily captured by our antibody

     4    because of affinity differences and they pass through

     5    and are in the depleted bag.  The release mechanism is

     6    very efficient.  We use a non‑chemical, non‑

     7    interactive release.  We use a physical stirring

     8    mechanism.  And the bond that breaks between the cell

     9    and the antibody is quite efficient.  In the

    10    laboratory, we see a very tight range of about 50 to

    11    60 percent using mobilized normal donors.  In the

    12    patient population, of course, the distribution is a

    13    little bit broader because it is dependent on the

    14    incidence of CD34 positive cells in the starting

    15    material.  We find that when patients have a

    16    mobilization of .5 percent or greater, our efficiency

    17    and recovery is much higher than the low mobilizers. 

    18    So some of the data here in terms of the wide ranges

    19    you might see is based on the incident at which you

    20    can mobilize. And I think that makes good sense for

    21    the label claim that we are saying 2 million per kilo

    22    is a good target that you want to choose.  Any other

    23    questions that I might be able to answer?

    24                DR. FLOYD:  Well, I am not surprised at

    25    all at your comments about the biological way the

                                                                        110

     1    device works.  What I am suggesting here is that in

     2    this kind of study, there are always these unanswered

     3    questions about why you get delayed engraftment. 

     4    Whether it is 7/10th's of a day, one day, 10 days,

     5    whatever.  What else is happening to this sample? 

     6    What else binds?  All of us who have been involved in

     7    research and the research laboratory know that when

     8    you deal with cells and/or proteins and/or

     9    glycoproteins and all sorts of other compounds,

    10    anytime they are subjected to a place where they can

    11    stick by and clump or whatever, most likely they will. 

    12    In this particular case, we have a group of patients,

    13    all of whom have obviously signed consent agreements

    14    to undergo a study.  One group, however, has had the

    15    standard traditional clinical treatment, that is,

    16    leukophoresis and then re‑engraftment, if you will. 

    17    The other group has gone through a device.  And what

    18    I am suggesting here is that the only way to tighten

    19    up this data is to run both groups through a device,

    20    one of which is CD34 positive and the other one which

    21    is not.  And I don't see that kind of study here, and

    22    it is the kind of study that I would really like to

    23    see happen.

    24                DR. SIEGEL:  But if we were to do that

    25    study, presumably in the ones that went through ‑‑

                                                                        111

     1    clearly in the ones that go through the CellPro, you

     2    would infuse those that adhere to the column and then

     3    eluted.  If you were to do that in the control arm,

     4    there would be virtually no cells there and it would

     5    be reasonable to assume that those patients would fail

     6    to engraft.  Would you suggest that there would be

     7    value in the dummy device in infusing the population

     8    that did not adhere and comparing that to the results

     9    of the population that did adhere in the CellPro

    10    device?

    11                DR. FLOYD:  Precisely.

    12                CHAIRPERSON VOSE:  But I think the idea is

    13    to compare it to the standard of care, which is what

    14    they have done.  They have compared it to the standard

    15    of care that is currently used.  I understand the

    16    rationale and scientific behind that, but you need to

    17    compare it to what is done in the patient population.

    18                DR. BROUDY:  And could I make just a brief

    19    comment.  That is that I have fewer concerns about the

    20    column perhaps than you do.  I think it is very

    21    clearly shown in the literature that the speed of

    22    engraftment is related to the CD34 content infused in

    23    peripheral blood progenitor cell transplant.  And I

    24    think the reason they engrafted perhaps slightly

    25    slower, particularly for platelets, is that fewer CD34

                                                                        112

     1    cells were infused, and that has been clearly shown

     2    even in populations of cells that have not gone

     3    through a column or some other device.  So I am not

     4    concerned that the column is damaging the cells and I

     5    would be less concerned about the need for that type

     6    of control.

     7                CHAIRPERSON VOSE:  Dr. Auchincloss?

     8                DR. AUCHINCLOSS:  A question for Dr.

     9    Litwin.  Again on the delayed platelet engraftment. 

    10    It looked to me as if the people who got into trouble

    11    were the people who were already in trouble if you

    12    will.  I mean before this study ever started, they

    13    were the ones who had low platelet counts to begin

    14    with . I guess my question for you is a) is that a

    15    reasonable interpretation of the data; and b) would

    16    you conclude from that that maybe more than 70,000

    17    platelets or whatever the cut‑off point was should be

    18    mentioned as a safety feature in taking part in this

    19    kind of cell transplant?

    20                DR. LITWIN:  We looked at immediate

    21    platelet engraftment and we found limited numbers of

    22    patients with slow platelet recovery.  The majority of

    23    the data that I think I showed you was this late event

    24    that is six months afterward.  We looked at two risk

    25    factors.  What is clear is a risk factor is infusion

                                                                        113

     1    of less than 2 million CD34.  We also looked at having

     2    75,000 platelets at time of mobilization, and the data

     3    were just too few to reach any conclusion whatsoever. 

     4    I think I included that in the briefing, but I did not

     5    mention ‑‑ 

     6                DR. AUCHINCLOSS:  But my point is that the

     7    ones that were infused with less than 10 x 106 ‑‑

     8                DR. LITWIN:  2 million per kilogram.

     9                DR. AUCHINCLOSS:  Looked like they were

    10    the ones who you could have predicted they were going

    11    to end up with a smaller number of cells because they

    12    already had evidence of diminished bone marrow

    13    reserve.

    14                DR. LITWIN:  That may very well be.  We

    15    have no way of distinguishing that.

    16                DR. AUCHINCLOSS:  The question then is

    17    should you label this to protect yourself from going

    18    to those patients, i.e., have a platelet count of

    19    100,000 instead of 70,000?

    20                CHAIRPERSON VOSE:  That is actually pretty

    21    well documented in the literature even for unselected

    22    transplant patients.  That if they have a platelet

    23    count below 100,000 when you start mobilization.

    24                DR. AUCHINCLOSS:  I think this study went

    25    for 70,000.

                                                                        114

     1                CHAIRPERSON VOSE:  Yes.  100,000 is kind

     2    of more standard.  

     3                DR. WEISS:  And this is an issue we tried

     4    to address in one of the questions, but it is still

     5    confusing.  And I was wondering if maybe the sponsor

     6    could address this.  Because there are patients that

     7    entered the study, but they weren't randomized until

     8    after they were ‑‑ until just at the start of

     9    leukophoresis, and the minimum platelet count at the

    10    time that they collected platelets was supposed to be

    11    30,000 platelets.  That is the minimum number.  And

    12    maybe Dr. Jacobs can address and try to clarify.

    13                DR. JACOBS:  There were two criteria for

    14    starting or initiating leukophoresis, and that was a

    15    white blood cell count greater than 1,000 and the

    16    platelet counts had to be greater than 30,000.  There

    17    were only two patients that were below 30,000 that I

    18    gave exception to to go ahead and randomize because

    19    their white blood cell counts were clearly climbing. 

    20    And there was one at I think 20,000 platelet counts

    21    and another one maybe at 26,000 or 27,000 platelet

    22    counts.  And probably one of our clinicians could kind

    23    of address the standard as far as starting

    24    leukophoresis and taking patients to transplant with

    25    platelet counts.  

                                                                        115

     1                DR. ANDERSON:  Ken Anderson from Boston. 

     2    I would comment on that.  I think the main finding of

     3    this study and supported broadly in the literature in

     4    a variety of cancers is that this threshold of 2

     5    million CD34 positive cells per kilogram is necessary

     6    to get satisfactory engraftment long‑term of all

     7    lineages.  In terms of trying to identify factors

     8    beforehand that might predict for inability to collect

     9    those cells, I think the best evidence is that prior

    10    treatment is where it is at.  And in particular, we

    11    have learned over the years that certain classes of

    12    drugs, alkylating agents and nitrosoureas in

    13    particular, which are known to be stem cell toxins,

    14    can clearly ‑‑ or other factors such as extensive

    15    radiation ‑‑ can abrogate our ability to collect

    16    sufficient numbers of cells later.  So, in fact, when

    17    this trial was designed, that was considered and that

    18    is why there was a limitation on the amount of

    19    alkylating agent exposure patients could have had

    20    prior to entering this trial, these three cycles.

    21                DR. AUCHINCLOSS:  So your recommendation

    22    to the FDA would be to include that in the labeling

    23    and the warning of the product?

    24                DR. SIEGEL:  There are just a couple of

    25    things I would like to comment on here.  One is that

                                                                        116

     1    the platelet counts that you have been looking at in

     2    these analyses and that you referred to as baseline

     3    platelet counts are the platelet counts after

     4    mobilization with cyclophosphamide and G‑CSF, and in

     5    general are lower than the platelet counts on study. 

     6    And it is those platelet counts after mobilization at

     7    the time of randomization and leukophoresis that seem

     8    to correlate with and be somewhat predictive of who is

     9    going to have delayed platelet engraftment.  Just a

    10    point of information.  They still could be used to

    11    determine whether or not to use this device.  

    12                DR. AUCHINCLOSS:  That is all I am looking

    13    for.  What is the predictor of who is not going to

    14    come up with 2 million cells.  

    15                DR. SIEGEL:  Right.  But it should be

    16    clear that that is what we are talking about, not the

    17    platelet count when you present for the conditioning

    18    regimen.

    19                The labeling that we have for use in this

    20    device with bone marrow also indicates that when low

    21    members of CD34 cells are infused, there is a delay in

    22    platelet engraftment. And this study is consistent

    23    with that.  And in that study also, there was more

    24    delay in platelet engraftment in the treated arm than

    25    in the non‑treated arm, suggesting strongly that it is

                                                                        117

     1    related to use of the device.  It is not listed as a

     2    contraindication.  I think people need to way ‑‑ to

     3    look at the data and whether or not a delay in

     4    platelets is likely to be a critical issue in that

     5    indication.  

     6                Finally I would note, and I think the

     7    record is clear on this ‑‑ it was stated in the

     8    company's ‑‑ I think it probably has been clear since

     9    then ‑‑ something that seemed a little misleading

    10    although true in the company statement that there is

    11    no significant treatment arm effect on platelet

    12    engraftment after you adjust for CD34 cells received

    13    and for a baseline platelet.  And while that may be

    14    true, it needs to be noted that the treatment arm is

    15    an important determinant here of the number of CD34

    16    cells received.  That in fact it halved the number. 

    17    So that statement might imply that there aren't

    18    effects beyond the fact that the device reduces the

    19    number of CD34 cells, but it doesn't suggest that the

    20    device, per se, is not responsible ‑‑ or the use of

    21    the device for those delays in platelets.

    22                And that reminds me actually of another

    23    point regarding Dr. Floyd's and Dr. Broudy's comments,

    24    which is that there aren't hard data as to whether per

    25    CD34 cell the device does better or less well in

                                                                        118

     1    engraftment.  There is a very soft suggestion I

     2    noticed in the data that was up that if you looked

     3    amongst those who had less than 2 million cells at a

     4    platelet cut point of 50,000 ‑‑ and as you saw, there

     5    are many cut points and there are a lot of

     6    multiplicity issues ‑‑ but there are about 5 of the 10

     7    in the treatment arm that had delays or problems and

     8    1 in 7 in the other arm, which I am sure is not

     9    significant, but I don't think we can say one way or

    10    the other for sure whether all the effects are

    11    attributable to reduction in CD34.  Even if we could,

    12    it is important to note that we couldn't say that if

    13    you just did more leukophoreses and got more CD34

    14    cells, the problem would go away.  That is probably

    15    something one shouldn't presume.  Reductions in CD34

    16    cells may also correlate with issues regarding bone

    17    marrow reserve, prior chemotherapy, and the actual

    18    functional efficacy of the CD34 cells, and it exists

    19    as a theoretical and not terribly unlikely possibility

    20    that patients who have fewer CD34 cells and don't

    21    engraft well that if you kept whipping them with some

    22    drug or more leukophoresis until you got more CD34

    23    cells, they might still not engraft as well as those

    24    people who had a lot of cells to begin with.  

    25                DR. BROUDY:  I'd just like to make a

                                                                        119

     1    couple of brief comments.  About your concern about

     2    whether patients with extensive prior alkylator

     3    therapy, there should be a comment in here.  I guess

     4    my feeling is no.  And the reason is that it is the

     5    clinician's judgment and also because there are many

     6    predictors one could use, and one is the number of

     7    CD34 cells per ml less than 2,000 CD34 cells per ml

     8    prior to mobilization, for example.  This predicts a

     9    group of patients who will mobilize poorly.  So there

    10    are many, many predictors one could use, and I guess

    11    I would prefer not to see them all spelled out, but I

    12    think one should spell out the 2 x 106 CD34 cells per

    13    kilogram because I think it is very clear that

    14    patients infused with fewer than that do engraft more

    15    slowly.

    16                I would like to make one other comment in

    17    response to what Jay has said, and that is that CD34,

    18    even though we are going to propose to use it I think

    19    here as a committee, is an imperfect measure because

    20    it is a very small subset of the CD34 cells that

    21    actually engraft.  What you are measuring with the

    22    CD34 cells is the progenitor cells mainly, whereas it

    23    has clearly been shown in the mouse model by Weissman

    24    and the Systemics folks and some others quite recently

    25    that it is actually the stem cells and not the

                                                                        120

     1    progenitor cells that are required for early

     2    engraftment in the first 14 days.  And those cells we

     3    have no way to measure.  And CD34 does not really

     4    measure those cells.  So it is an imperfect measure

     5    anyway.

     6                CHAIRPERSON VOSE:  It is a surrogate. 

     7    Unfortunately we don't have a better one to use at the

     8    present time. I agree that it is going to be

     9    impossible to list all of the factors as far as

    10    putting what we should and shouldn't do for selecting

    11    patients, and we have to use our best surrogate, which

    12    right now is the CD34.

    13                DR. AUCHINCLOSS:  But if I can just ask

    14    you a little bit further on that.  I believe that 2 x

    15    106 is the right number, but you only know that after

    16    you put them through the column whether you have it. 

    17    So to tell people, hey, if you didn't get 2 million,

    18    you are in trouble and they have already used the

    19    device ‑‑

    20                CHAIRPERSON VOSE:  You need to continue to

    21    do additional leukophoresis.  

    22                DR. AUCHINCLOSS:  Well, that is the

    23    question that Jay just brought up.  I am not so sure

    24    that just continuing to do leukophoresis is really

    25    going to provide you with what you want.  So don't you

                                                                        121

     1    think it is wise to tell people this device may get

     2    you into trouble if you set out with a set of

     3    predictors that tell you you are not going to get very

     4    many CD34 positive cells out of it.

     5                DR. SIEGEL:  Well, you can continue to

     6    leukophorese and you also have the option as to

     7    whether in those future leukophoreses, you separate

     8    them or not.

     9                DR. AUCHINCLOSS:  You wouldn't separate

    10    them perhaps.  And I guess there are ways around it. 

    11                DR. SIEGEL:  But you also have the back‑up

    12    there.  

    13                CHAIRPERSON VOSE:  You have the back‑up.

    14                DR. AUCHINCLOSS:  But I agree with you

    15    fundamentally that it comes down to clinical judgment.

    16                CHAIRPERSON VOSE:  Each clinical situation

    17    is so different that it is hard to generalize I think. 

    18    Dr. Silver?

    19                DR. SILVER: I have a question about the

    20    back‑up cells.  Is there any advantage to being

    21    infused with the back‑up cells?  This is really a

    22    clinical question.  If they have been depleted of

    23    CD34, are they useless or what function do they ‑‑ and

    24    was there any data in this study that in those few

    25    patients that did get back‑up cells, was there any

                                                                        122

     1    immunologic benefit?

     2                DR. STEWART:  Keith Stewart from Toronto. 

     3    Only one patient was infused with the back‑up, which

     4    was in Toronto, and it was ineffective.

     5                DR. BROUDY:  I guess I would say if they

     6    recover 60 percent of the CD34 cells, some percent of

     7    the CD34 cells will still be left in the flow‑through.

     8                CHAIRPERSON VOSE:  Additional questions?

     9                DR. AUCHINCLOSS:  Yes, one big question

    10    for Jay really and the FDA.  I think it is a rerun of

    11    the conversation we had in this Committee a hear and

    12    a half ago, the last time we looked at one of these

    13    issues.  As highlighted when Dr. Litwin started his

    14    presentation, he said this device is already approved. 

    15    And my question for you basically is as a device, I

    16    believe this device does what the company says it

    17    does.  It removes tumor cells and gives CD34 cells

    18    that are safe.  You are really asking them to give us

    19    efficacy or give us a sense of efficacy in clinical

    20    treatment.  Do you really think it is wise to judge

    21    this on a clinical treatment efficacy as opposed to it

    22    is a device that does what the device says it is

    23    doing?

    24                DR. SIEGEL:  Well, that is a tough

    25    question to answer.  We haven't, obviously, asked nor

                                                                        123

     1    have we seen evidence of efficacy in clinical

     2    treatment in the sense that that would be ‑‑ would

     3    constitute survival and outcome data I would think. 

     4    In fact, the data you are looking at is our data as to

     5    whether it does a specific function, remove CD34

     6    cells.  This device and most devices, although

     7    obviously it is variable from device to device, but

     8    most devices carry with them a certain ‑‑ well, I

     9    don't know if it is most devices, but certainly some

    10    devices carry with them a certain downside to their

    11    use.  And I think we have seen that aside from the

    12    issues of platelet engraftment impairment and a very

    13    minor but statistically significant neutrophil

    14    engraftment impairment, there is the issue of more

    15    leukophoreses.  It just took more to meet the

    16    standards that were imposed before.  So I think the

    17    correct question in the context of this device is is

    18    the overall picture appropriate?  Can what we presume

    19    about the function of removing CD34 cells is that

    20    adequate against what we know about the impact on

    21    engraftment and number of leukophoreses to consider

    22    this device safe and effective for use?  Devices do

    23    have an effectiveness standard.  It isn't always

    24    applied in quite the same manner as drugs because the

    25    indications often are not the same as for drugs.  And

                                                                        124

     1    it is hard to general about what we should be asking

     2    of a device except to say that I think it should be in

     3    context of a risk benefit analysis as it is for all

     4    our products.

     5                DR. AUCHINCLOSS:  Jay, you need to be

     6    clear that there has been efficacy asked of the

     7    company in the sense that this product was originally

     8    approved based on diminished first dose infusement

     9    effects.  Correct?  And basically they are coming back

    10    to you now and saying in 1994, your committee said

    11    that a 2 log or maybe more than that reduction in

    12    tumor cells could also be used as a surrogate marker

    13    for efficacy.  Now be careful about what the committee

    14    really said in 1994.  We can read back to that at some

    15    point if we need to.  My point is I think that that

    16    marker for efficacy is probably flawed.  I don't think

    17    this device is actually providing benefit to patients,

    18    at least as far as I can tell so far.  And I think you

    19    get yourself into trouble by asking them to show

    20    efficacy in a clinical sense, whereas what you really

    21    can do is say, all right, make a device that does what

    22    your device says it does and let clinicians decide

    23    whether it is worth using it.

    24                DR. SIEGEL:  You are suggesting that we

    25    did ask them to show efficacy?

                                                                        125

     1                DR. AUCHINCLOSS:  Yes.  I think you have

     2    asked them to show efficacy for something that I am

     3    not sure is ‑‑ 

     4                DR. SIEGEL:  What sort of efficacy did we

     5    ask them show?  Efficacy in ‑‑ 

     6                DR. AUCHINCLOSS:  I thought the entire

     7    response that the company was coming up to was we will

     8    show you 2 log tumor reduction and that will be taken

     9    as a surrogate marker for efficacy.  I mean, isn't

    10    that ‑‑ 

    11                DR. SIEGEL:  Right.  But in fact if you

    12    are saying we should ask them to show that the device

    13    does what it says it does, then if the device says

    14    that it reduces tumors ‑‑ 

    15                DR. AUCHINCLOSS:  I am not going to ask

    16    them to show me anything except that they reduce tumor

    17    number.

    18                DR. SIEGEL:  Well, that is all we asked.

    19                DR. AUCHINCLOSS:  And I don't want to use

    20    the device because I don't think that does anybody any

    21    good.

    22                CHAIRPERSON VOSE:  I think Abbey Meyers

    23    would like to ask a question on the phone here if we

    24    can hear her.  

    25                MS. DAPOLITO:  Abbey, can you hear us?

                                                                        126

     1                MS. MEYERS (telephonically):  Yes, I can

     2    hear you.  Can you hear me?  

     3                MS. DAPOLITO:  Go ahead and we will see

     4    what we can do.  

     5                MS. MEYERS (telephonically):  Okay.  It is

     6    along the lines of what Jay was just saying.  

     7                CHAIRPERSON VOSE:  She was asking about

     8    there are many different types of malignancies, and is

     9    the device going to need to go through an approval

    10    process for each type of malignancy?

    11                DR. SIEGEL:   I think that is part of one

    12    of our questions to the committee.  I don't think we

    13    have a determination on that.

    14                CHAIRPERSON VOSE:  It is a question for

    15    later in the discussion, yes.

    16                DR. SIEGEL:  I would like to interject

    17    here that Dr. Max has a flight to catch shortly on

    18    Government business.  It was a very difficult

    19    scheduling problem and we are certainly most

    20    appreciative of his efforts in that regard.  But if

    21    there are further questions specifically of Dr. Max,

    22    he won't be available later in the day.

    23                CHAIRPERSON VOSE:  Maybe we could just ‑‑

    24    I was going to say we could go through many of the

    25    questions.  I think they address a lot of the things

                                                                        127

     1    that we are talking about.  

     2                DR. SIEGEL:  I think Bob Vescio should

     3    address that with some additional data regarding the

     4    beta‑actin just to reassure you.  

     5                DR. VESCIO:  Yes.  First of all, I want to

     6    apologize for not having the data sooner.  There were

     7    a lot of time constraints and unfortunately my

     8    clinical responsibilities were such that I couldn't

     9    get this data in time.  But I was hopeful that maybe

    10    if you have ‑‑ I can show you actually the beta‑actin

    11    gels that were run in a quantitative fashion to again

    12    address whether there was any quantitative degradation

    13    in the DNA from one patient ‑‑ from the 34 enriched

    14    product versus the leukophoresis product. 

    15                Basically what was done is we took 3

    16    patients ‑‑ it is carousel 2, slide 51 ‑‑ 3 patients

    17    took their leukophoresis DNA and their CD34 enriched

    18    DNA and basically quantified the amount of beta‑actin

    19    that was amplifiable using this Poisson PCR.  This is

    20    not the right slide.  Carousel 2, slide 51.  

    21                Basically what we found ‑‑ this is an

    22    example of one of the patients.  Because the assay was

    23    set up and the PCR conditions were set up to amplify

    24    a small PCR product, when we took the initial beta‑

    25    actin primers that were used as a positive control and

                                                                        128

     1    run with all the assays, those PCR primers were not

     2    really optimized to detect just one copy of the gene

     3    per cell and that was why the percent contamination

     4    rate was quite low.  I redesigned primers that were

     5    more comparable to this PCR product size looking for

     6    tumor burden, and in fact kind of fortuitously these

     7    particular primers actually amplified two germ line

     8    genes within the patient, one of about 225 base pairs

     9    in size and one of about 112 base pairs in size.  I

    10    don't have a pointer, but you can see there on the

    11    slide.  And basically in this particular example ‑‑

    12    this is an H dilution, so this represents DNA from 30

    13    cells, DNA on an I dilution and there are five

    14    replicates, DNA from 10 cells, 3 cells, 1 cell, .3,

    15    and .1.  And as you can see here, there is ‑‑ as one

    16    might expect when one starts getting to a statistical

    17    chance of having one cell within the PCR tube,

    18    occasionally one will find an amplifiable product and

    19    occasionally one will find no beta‑actin gene present. 

    20    Again, one can score the positive reactions from this

    21    upper band which represents one germ line gene, and

    22    also at the same time as a comparison control the

    23    number of replicates positive bands looking at this

    24    lower gene product.  In this particular case, 124

    25    percent of the cells had an amplifiable beta‑actin

                                                                        129

     1    gene looking at the upper product and 88 percent of

     2    the cells had an amplifiable beta‑actin gene with 100

     3    percent obviously being what one would expect.

     4                This is the analysis for the same patient

     5    on the 34 enriched product, and again you can see very

     6    similar findings.  And as one might expect as one

     7    starts getting to 1 cell of DNA within the PCR tube,

     8    one starts to have positive and negative results, and

     9    that again falls off.  And again in this particular

    10    example, the calculated contamination rate was 78

    11    percent for the upper band and 217 percent for the

    12    lower band.  

    13                I have two more slides that we can just

    14    whip through just again for comparison.  Again, here

    15    is the results for the leukophoresis product.  Here is

    16    158 percent.  Here was 145 percent.  And for the 34

    17    enriched product, here was somewhat lower 32 percent. 

    18    Here the bands are a little bit weak, but it was 127

    19    percent.  And the third patient was also quite

    20    comparable and I have the gels if the panel would like

    21    to look at those. 

    22                So I think if one looked at all ‑‑

    23    basically in this particular case, there are 3

    24    patients.  If you looked at the 34 enriched fraction,

    25    looked at the leukophoresis fraction, the fact that

                                                                        130

     1    there is actually two genes that are amplified and

     2    assessable in each of these time points.  Again, all

     3    the findings were within the half log error rate of

     4    the assay.  Again, I hope that this kind of allays

     5    some of the fears that the panel may have that the DNA

     6    was qualitatively or systematically degradaded in the

     7    34 enriched product versus the leukophoresis product.

     8                CHAIRPERSON VOSE:  Why don't we go ahead

     9    and go through the questions.  And then if it is not

    10    answered at the end of the questions, we can have that

    11    additionally, Abbey.  Because I think many of the

    12    questions address what we are talking about here. 

    13    Let's turn to the questions, then.  The first one

    14    really has to do with what we have been talking about

    15    as far as the validation and performance of the assay

    16    system to detect the tumor in the phoresis product. 

    17    Do we have any additional concerns or questions

    18    regarding the discrepancies that we have noted or the

    19    lack of internal control?  Dr. Silver, are you

    20    satisfied now with the information that has been

    21    presented? Dr. Auchincloss?

    22                DR. AUCHINCLOSS:  I didn't hear exactly

    23    how you phrased the question.  

    24                CHAIRPERSON VOSE:  Just are you satisfied

    25    with the information as far as the ‑‑ do you still

                                                                        131

     1    have concerns regarding the assay system, the

     2    discrepancies we have discussed or lack of internal

     3    control?

     4                DR. AUCHINCLOSS:  Well, as I think has

     5    been brought out here already, there are two different

     6    concerns.  One is is the assay really accurate to

     7    within half a log.  And I suspect the answer to that

     8    is no.  But that doesn't terribly bother me because I

     9    do not believe that there is much evidence ‑‑ there is

    10    any evidence, I guess, that there is a systematic

    11    error that would lead to the incorrect conclusion that

    12    they are depleting tumor cells by at least 2 logs.  So

    13    I think that part is probably true.  I am not sure if

    14    your assay isn't as sensitive as you think it is that

    15    you necessarily are infusing as many products that are

    16    tumor free as you think you are, but that is neither

    17    here nor there from the point of view of what the

    18    company says it is trying to do.  So I believe that

    19    with the data I have seen that their product does what

    20    they say it does to in general remove at least 2 logs

    21    of tumor and probably more.

    22                CHAIRPERSON VOSE:  Is everyone satisfied

    23    that the data suggests that there is at least 2 log

    24    removal?

    25                DR. BROUDY:  Yes, I certainly am.  I would

                                                                        132

     1    have to say that I think Dr. Max's very careful

     2    analysis of all the data, how it was generated and

     3    looking at all the gels served to actually convince me

     4    that the company has done a very good job in studying

     5    as large a number of patients as was possible that

     6    they could amplify the product from that it does

     7    remove at least 2 logs of tumor cells.  And I think

     8    they are to be commended for having done this very

     9    carefully and having analyzed the run‑through fraction

    10    as well.

    11                CHAIRPERSON VOSE:  It is actually a very

    12    carefully performed study.  It is unfortunate that

    13    there couldn't have been a higher number of patients

    14    that could be amplified to add to that number.  But

    15    that is unfortunately a difficulty of the analysis. 

    16    Any additional comments?  Okay.  Let's move to the

    17    next question.  There typically are not reports of

    18    CD34 antigen expressed from myeloma cells, but there

    19    recently has been at least one report of that.  To

    20    what extent does this cause any safety or efficacy

    21    concerns of this selection device in patients with

    22    multiple myeloma?  Anyone want to comment on that? 

    23                DR. BROUDY:  Well, I think it also depends

    24    on the cell surface density.  What you can detect by

    25    flow cytometry is probably about 1,000 molecules per

                                                                        133

     1    cell.  I am not sure how many molecules need to be

     2    detected for a cell to be retained by this particular

     3    device.  But reassuring is the fact that in no

     4    patients in whom we saw the data was there any

     5    evidence that the myeloma cells were preferentially

     6    selected, at least in this group of 28 patients.  So

     7    we couldn't exclude that possibility, but it would be 

     8    a lesser concern of mine.

     9                CHAIRPERSON VOSE:  I think certainly the

    10    number of patients that would have this problem would

    11    be very small and probably a minority of cells as you

    12    discussed.  So in this particular patient population,

    13    I wouldn't say it is a huge problem, although

    14    certainly it is a concern for a generalizability to

    15    other malignancies.

    16                DR. SIEGEL:  If they were preferentially

    17    selected ‑‑ if they were CD34 positive and

    18    preferentially selected, it would still be almost

    19    inconceivable ‑‑ I better put that almost ‑‑ that you

    20    would actually wind up infusing more tumor cells than

    21    you would infuse if you didn't separate them.  It is

    22    unlikely that you are going to ‑‑ certainly highly

    23    unlikely that you are going to infuse more than you

    24    took out in leukophoresis.  It is not impossible.  So

    25    I guess one issue that we talk around and that is

                                                                        134

     1    somewhat implicit in this question is what if you did

     2    somebody who was CD34 positive and you purified those

     3    tumor cells.  So basically what you would be infusing

     4    would be a product that was enriched that had half of

     5    the original number of CD34 cells and half or maybe

     6    more of the original number of tumor cells, but then

     7    didn't have a lot of other things like T cells, for

     8    example.  Is there any reason that if that were done 

     9    ‑‑ if you did, in a sense, enrich for tumor cells,

    10    that that might be or would be a safety concern?

    11                CHAIRPERSON VOSE:  Of course, if we don't

    12    know if taking them out makes any difference, how do

    13    we know if addition them makes any difference?  But I

    14    think there is a theoretical concern about this is

    15    going to be somewhat of a depleted product with immune

    16    effector cells, and if you are going to put in a

    17    product that is very enriched with tumor cells without

    18    immune effector cells, that could theoretically be a

    19    problem.  There is no way to know that.  We don't have

    20    any data.  

    21                DR. ANDERSON:  Yes, I agree.  Basically

    22    you could imagine all kinds of theoretical

    23    possibilities that you would select out on your column

    24    a specific CD34 population that is more malignant than

    25    the standard and therefore make it worse.  But all

                                                                        135

     1    these things come down to basically getting data and

     2    the point of the FDA is to look at the risk and to

     3    look at the benefit and to put appropriate labeling so

     4    that physicians are aware of the risk, and then one

     5    looks to see what happens in patients and a continued

     6    analysis.  And in the use of the product, these

     7    theoretical possibilities will be worked out. We can't

     8    answer everything.

     9                CHAIRPERSON VOSE:  Dr. Silver?

    10                DR. SILVER: It seems to me it would be

    11    appropriate ‑‑ possibly appropriate in the literature

    12    to say ‑‑ to note that some tumors or some myelomas

    13    have been reported or one at least has been reported

    14    to be CD34 positive.  And in that case, the claim of

    15    100‑fold reduction would probably not apply.

    16                CHAIRPERSON VOSE:  To put that

    17    specifically in the labeling?

    18                DR. SILVER:  Yes.

    19                CHAIRPERSON VOSE:  We will leave that up

    20    to our FDA colleagues.  Any additional comments on

    21    that?  Okay.  The next question relates to safety

    22    outcomes for engraftment.  Discuss the effects of the

    23    Ceprate device on neutrophil and platelet engraftment. 

    24    Does anyone have comments on that as far as clinical

    25    relevance to what we have seen today?  Well, from my

                                                                        136

     1    standpoint, I would say that neutrophil engraftment

     2    was certainly adequate in both arms and platelet

     3    engraftment, although it appeared to be slightly

     4    slower in the Ceprate arm was really not clinically

     5    significantly different and did not represent a

     6    problem as far as patients are concerned.  There were,

     7    however, several outliers, and that needs to be looked

     8    at with respect to the number of cells infused and

     9    things like that that we have already discussed. 

    10    Additional comments?  Okay.  Question number 4 ‑‑ this

    11    was a question that our FDA colleagues wanted us

    12    actually to vote on.  Are the findings of additional

    13    leukophoresis procedures, platelet transfusions, and

    14    in certain patients an impaired platelet engraftment

    15    acceptable given the potential benefits of a 2 to 3

    16    log tumor depletion?  Can we have some discussion on

    17    that first?  This is kind of the $64,000.00 question. 

    18                MR. KATZ:  I think that what we are

    19    talking about in terms of additional leukophoresis

    20    once you've got the set‑up to do that ‑‑ you've got

    21    the catheter and all the other good stuff ‑‑ and

    22    transfusions is kind of kidstuff compared to going

    23    through a transplant.  So I think that if there was

    24    even a prayer that it was going to improve the outcome

    25    and survival, I don't think patients would look at

                                                                        137

     1    that very unfavorably.  

     2                CHAIRPERSON VOSE:  And typically I think

     3    it was only one extra leukophoresis for most patients. 

     4                MR. KATZ:  Yes.  Once you've got the

     5    plumbing installed, it is pretty easy.

     6                CHAIRPERSON VOSE:  Dr. Broudy? 

     7                DR. BROUDY:  I guess I would just like to

     8    point out that at least some of the extra

     9    leukophoreses were dictated even pre‑column just to

    10    achieve the target minimal number of mononuclear

    11    cells.  So I believe that probably some fraction of a

    12    leukophoresis extra was required to generate the

    13    number of cells required to go through the column. 

    14    But most of it was just that that patient population

    15    happened to have perhaps slightly poorer bone marrow

    16    reserve for some reason that we don't know at the

    17    present time.  I guess I would vote yes on this.  I am

    18    quite convinced that they deplete at least 2 to 3 logs

    19    of tumor, and while the major part of the clinical

    20    problem is residual tumor in the patient, at least

    21    this potentially offers a step forward.  So I would

    22    vote yes on this.

    23                CHAIRPERSON VOSE:  Additional discussion?

    24                DR. SIEGEL:  I would interject that

    25    perhaps we could rephrase your comment to say that

                                                                        138

     1    this is not the $64,000.00 question, but the log 4.8

     2    dollar question.  

     3                CHAIRPERSON VOSE:  Thank you.  Okay.  So

     4    let's take this ‑‑ 

     5                DR. AUCHINCLOSS:  Let me ‑‑ I guess I need

     6    education.  It sounds good, a 2 log reduction in

     7    tumor.  It feels good.  Is there any evidence

     8    anywhere, even a suggestion, that it is good?

     9                DR. BROUDY:  Well, I think this is by

    10    implication from the gene marking studies done by Dr.

    11    Brenner, a former member of our committee, in which he

    12    clearly showed that infused gene marked peripheral

    13    blood tumor cells can contribute to relapse. So I

    14    think though much of the problem in myeloma and

    15    perhaps some of the clinicians here who deal more with

    16    myeloma than I do since I do mostly lymphomas ‑‑ you

    17    know, a major part of the problem is relapse in the

    18    patient.  But at least this has been demonstrated in

    19    Childhood ALL that infused tumor cells can contribute

    20    to relapse.  So I think it is desirable that we infuse

    21    fewer.  Does anyone want to comment?

    22                DR. AUCHINCLOSS:  Do you think you need to

    23    go to zero or do you think you need to be less?

    24                DR. BERENSON:  Let me comment on that

    25    based on the Phase II.  Obviously that trial was not

                                                                        139

     1    designed with all the caveats of the Phase III.  But

     2    several of the patients with extremely high tumor

     3    burden, for example the one that had 200 million tumor

     4    cells before the column, she is now in remission four

     5    years out from that transplant. And I certainly

     6    believe the 3 logs we removed may have contributed to

     7    that.  I can't prove that to you.  And we have two

     8    other patients who had high tumor burden as well that

     9    are out now at this point.  One of them nearly five

    10    years out.  I can't prove that to you, but those are

    11    just anecdotal cases.  But you wanted at least an

    12    inkling that there may be some help here.  

    13                MR. KATZ:  Well, I think this debate about

    14    whether removing the tumor cells at this stage is a

    15    bit of a red herring because the whole issue of

    16    whether you should transplant rather than pop

    17    melphalan prednisone pills, you can't prove that

    18    either.  We wouldn't have trials ongoing for

    19    transplant versus standard and early versus late

    20    transplants.  So I think we are basically saying

    21    should we allow investigation of the logical extension

    22    of the transplant philosophy, which is knock out as

    23    many of the cells in the body as you can. Am I

    24    thinking incorrectly about that?

    25                DR. AUCHINCLOSS:  Yes, in my view in the

                                                                        140

     1    sense that there is nothing that I am wondering about

     2    ‑‑ and all I am doing is wondering out loud ‑‑ that

     3    prevents further investigation.  Believe me, I am very

     4    much in favor of further investigation.  The question

     5    is does the FDA want to put its stamp of ‑‑ they can

     6    label that this reduces tumor burden in the infused

     7    cells, which I think carries with it the implication,

     8    at least the way the FDA is handling this review, that

     9    that is good for you.  I am not sure that I believe

    10    that it is good for you. I believe that they

    11    accomplish it.  I just don't know that I believe that

    12    the FDA wants to say ‑‑ 

    13                CHAIRPERSON VOSE:  We have no clinical

    14    evidence that it is good for you, and the labeling has

    15    to say that it can do X, Y, Z that it says it can do. 

    16    Unfortunately, we are not going to be able to police

    17    every physician who is going to use it.  And I agree

    18    with you that that is a little bit of a concern.  But

    19    we have to specifically, I think, say that we don't

    20    have any evidence that it is good for you.  

    21                DR. ANDERSON:  Let me take a brief crack,

    22    Hugh, at trying to answer you.  This is basically

    23    looking at the philosophy behind doing the initial

    24    gene marking trials.  The basic principle ‑‑ see if

    25    you would agree with this ‑‑ is that one does not have

                                                                        141

     1    to get the tumor burden to zero. One has to get it

     2    below the level that the body's immune system can

     3    counteract it.  The difficulty is that we don't know

     4    what that level is.  Now the justification for going

     5    forward with the marker trials initially was to get

     6    the sensitivity of the assay down into a level where

     7    one can start getting an answer or hopefully getting

     8    an answer as to what level of tumor burden below which

     9    the body can handle itself.  So if you buy that

    10    argument ‑‑ you are nodding yes, so I gather you buy

    11    it up to this point ‑‑ then the logic is that the

    12    closer you get to that level, the better off you are,

    13    although if you are above it at all, then the

    14    possibility is that you are going to have relapses,

    15    just not quite as rapidly.  So the issue is if you

    16    can't get below the level, then is simply putting off

    17    a relapse by a week or two weeks or three weeks, does

    18    that help, and the answer is no.  But at what point do

    19    you drop below the level, and the only way to find out

    20    is to keep dropping the tumor burden down until you

    21    get statistically relevant data.  Do you agree with

    22    all that?

    23                DR. AUCHINCLOSS:  I do.  But I am not sure

    24    it speaks to the question of whether or not at this

    25    point the FDA would want to imply that it knows this

                                                                        142

     1    level of reduction is important.

     2                DR. ANDERSON:  No, it can't.  And that is

     3    ‑‑ I think our journal is quite correct.  All the

     4    labeling can do is say what happens and not imply. 

     5    Now the informed consent ought to make clear that a

     6    reduction in added tumor cells does not imply an

     7    improvement in either the survivability, time to

     8    relapse, et cetera.  

     9                DR. AUCHINCLOSS:  You are talking about

    10    the physician's informed consent just in the ordinary

    11    course of doing the procedure, not something related

    12    to study.  Because at this point, you are talking this

    13    out of study.  

    14                DR. ANDERSON:  Well, that is true.  A

    15    valid point.  Which means it has to be in the

    16    labeling.  And which is then a valid point whether the

    17    labeling should err on the side of being conservative

    18    and specifically state that there is no evidence that

    19    this will reduce.

    20                DR. AUCHINCLOSS:  Jay, what do you think? 

    21    What is your reaction to this?

    22                DR. SIEGEL:  Well, there is some sort of

    23    implication that if you indicate something as a marker

    24    of efficacy.  Clearly, I think the labeling we would

    25    write would make it very clear, as we would expect

                                                                        143

     1    physicians to make clear to patients that there are no

     2    data indicating an impact in outcome.  This is the

     3    dilemma that we brought to his committee, and we got

     4    very cogent comments and advice, much of which has

     5    been discussed, some hasn't.  There was discussion, of

     6    course, of the difference between a tumor cell that is

     7    in the circulation and that is leukophoresed versus a

     8    tumor cell that might be, if you will, engraftable and

     9    give rise to tumor, the latter being something that

    10    can't be measured.  There was a lot of discussion of

    11    the issue of the extent to which we know that tumor in

    12    the product contributes to disease.  One view

    13    expressed was that to the extent there is also disease

    14    coming back from the patient, there was kind of a dual

    15    arm approach to get better therapies for what is in

    16    the patient and better therapies for what is in the

    17    marrow, and that you shouldn't hold the latter hostage

    18    to the former.  If you don't allow marrow quality to

    19    improve until you've got treatment quality to improve

    20    to the extent to where marrow quality matters, you

    21    won't get marrow quality to improve.

    22                None of these I am forwarding as consensus

    23    statements or even as FDA opinions.  These were all

    24    things that were said.  I think what we took home from

    25    that, with all of that said, and what we talked about

                                                                        144

     1    with this company as this trial was designed was that

     2    reductions of tumor, particularly in hematologic

     3    malignancies, but reductions of tumor in general of

     4    specifically substantial amounts, and I don't know

     5    that there was consensus, but clearly we weren't

     6    talking about getting rid of half of the tumor cells

     7    as likely to matter, but substantial reductions of

     8    tumor were something that was to be considered a

     9    desirable outcome.  Not something that was to be

    10    considered necessarily a proven measure of patient

    11    benefit, but a desirable outcome.  And we specifically

    12    asked the committee how much problem with engraftment

    13    are you ‑‑ should we be willing to accept in a product

    14    that gives that desirable outcome. Because nobody had

    15    any problem with something that would do that and have

    16    no downside.  The answer was, well it depends.  It

    17    depends on the type of tumor, the likelihood that the

    18    marrow contributes, the log reduction, the sensitivity

    19    of the assay, the proportion of patients who were

    20    reduced to below that sensitivity, the amount of

    21    delay, and the clinical significance of both the

    22    duration and the number of patients who did have delay

    23    of engraftment and the quality of the data and whether

    24    or not there were any data suggesting any impact on

    25    survival.  So that is what leads us to where we are

                                                                        145

     1    and to this question.  Now we have it depends and here

     2    we are and we need to make some decisions.  I don't

     3    know if that answers your question, but that is all

     4    I've got to go on.

     5                DR. AUCHINCLOSS:  One thing to say is that

     6    you remember that meeting pretty well because I have

     7    spent the morning looking through those minutes and

     8    you got it absolutely perfect.  That was not a

     9    committee meeting that I was part of.  That was before

    10    my time.  But I guess in a general sense the way I

    11    look at this is that the downside for this particular

    12    device and this particular disease looks like it is

    13    really on the verge of trivial and I think they are

    14    reducing tumor burden by at least 2 logs.  I guess

    15    that means to go for it.

    16                CHAIRPERSON VOSE:  Okay.  Why don't we

    17    vote on this question.  Are the findings of additional

    18    leukophoresis products, platelet transfusions, and

    19    impaired platelet engraftment acceptable given the

    20    potential benefits of a 2 to 3 log tumor reduction? 

    21    Everyone that thinks this is acceptable, please raise

    22    your hand.  It looks unanimous to me.  And Dr. Floyd

    23    doesn't vote.  Okay.  Any other comments?  Okay, good. 

    24                The next question, if approved, how should

    25    labeling address the risk factors associated with

                                                                        146

     1    engraftment delays?  Should labeling advise that the

     2    number of CD34 cells post‑selection be determined and

     3    recommend infusion of selected product only for

     4    patients who have at least 2 x 106 CD34 cells per kilo? 

     5    And should there be any further studies done or other

     6    analysis of risk factors?  From my standpoint, I would

     7    say that the labeling should say that, at least 2 x

     8    106 per kilo just as we did for the bone marrow say

     9    1.2.

    10                DR. HONG:  What would be the option?

    11                CHAIRPERSON VOSE:  The option would be ‑‑

    12                DR. HONG:  Just to pool everything?

    13                CHAIRPERSON VOSE:  Pool everything

    14    together.  But specifically, I don't think you want to

    15    say ‑‑ you don't want to use the number that we use

    16    for bone marrow because in this circumstance, it would

    17    be inadequate.  So you need to specify whether you are

    18    using bone marrow or stem cells for your product.

    19                DR. SIEGEL:  We are also asking in this

    20    question about the platelet.  We have already heard

    21    comments in response to this question.  We are not

    22    looking for a vote.  But if there are any other

    23    comments on either of those that we might take in

    24    terms of thinking through as we work on this, I would

    25    appreciate them.

                                                                        147

     1                DR. WEISS:  Or maybe a clarification.  I

     2    am not so sure that we presented ‑‑ we actually did

     3    not present any analysis of platelet count.  I think

     4    somebody asked earlier of Dr. White whether or not

     5    there is a correlation between platelet count at the

     6    start of the mobilization or at the start of the

     7    phoresis and the CD34 that you end up afterwards, and

     8    Dr. White said, no, there was none.  But Dr.

     9    Auchincloss, I think, asked a very appropriate

    10    question.  Are there ways before you actually start

    11    this whole procedure to try to predict and whether or

    12    not there are ways to look at it and other types of

    13    analyses.  Are there other types of things that can be

    14    done to try to get a handle on that particular

    15    question?

    16                CHAIRPERSON VOSE:  Eugenie?

    17                DR. KLEINERMAN:  I assumed that was a

    18    question, and I just would like to reiterate what Dr.

    19    Broudy said.  I would like to see that put in the

    20    label, but I wouldn't use it as a cut‑off.  I think

    21    you need to give the clinician some latitude.  There

    22    are all sorts of parameters that one takes into

    23    account and limiting it to patients who have 75,000

    24    platelets I think may be a mistake.

    25                MR. KATZ:  I guess a question ‑‑ you

                                                                        148

     1    raised the issue before ‑‑ I think it was down there 

     2    ‑‑ about whether additional leukophoresis would do any

     3    good if you were getting that kind of a yield that was

     4    below the 2 million.  I guess the question that comes

     5    to mind is would you get a warning ‑‑ is there a

     6    predictable pattern of how you accumulate the cells

     7    and would you know that earlier in the collection and

     8    would there be any merit to sort of taking someone off

     9    the column early in the collection if that happened?

    10                CHAIRPERSON VOSE:  The problem from my

    11    standpoint is that patient variability is so high that

    12    it is really difficult to tell that except on a

    13    person‑by‑person basis. I am not sure that we can

    14    dictate something like that in a label.  I don't know

    15    what anybody else thinks.

    16                DR. BROUDY:  Maybe they should say

    17    something like a sufficient number of peripheral blood

    18    mononuclear cells should be collected such that more

    19    than 2 x 106 will be infused after the Ceprate column,

    20    and then making some assumptions that there are going

    21    to be 50 percent recovery, for example, from the

    22    Ceprate column.  Because clearly you need to collect

    23    more than the 2 x 106 CD34 cells, at least twice as

    24    many as that, before the column, and then not making

    25    any comments about the platelet count or the number of

                                                                        149

     1    CD34's at the start or the extent of alkylator

     2    pretreatment and leaving all these things up to the

     3    clinician.  But I do think that number of 2 x 106

     4    CD34's per kilo post‑column should be in there, and

     5    maybe the company has some thought about how that

     6    could be phrased to make sure that safety issue is

     7    met.  

     8                DR. KRIEGER:  We already have that in the

     9    labeling that ‑‑ 

    10                DR. BROUDY:  Could you read that to us?

    11                DR. KRIEGER:  The labeling that we have

    12    proposed and submitted to the FDA already has in it

    13    the caveat that they should collect at least 2 x 106

    14    CD34 positive cells per kilogram.  That was the first

    15    slide.  So this is the labeling that we have proposed

    16    and it was also the labeling that Dr. Litwin showed. 

    17    The next slide actually shows the number.  So we have

    18    shown here ‑‑ you see we have recommended that a

    19    sufficient amount of peripheral blood be harvested to

    20    yield 2 x 106 CD34 cells per kilogram.

    21                DR. BROUDY:  But that doesn't give any

    22    guidance to the clinician about how many should be

    23    collected prior to the column.

    24                DR. KRIEGER:  We can put something in it

    25    similar to what we did in the clinical trial where we

                                                                        150

     1    say they collect 4 x 108 total nucleated cells.  

     2                DR. JACOBS:  And we could do that either

     3    in the labeling or a cautionary warning as well in the

     4    labeling and it may be more appropriate for a

     5    cautionary warning for patients that may not have 5 x

     6    108 total nucleated cells per kilogram at the time of

     7    processing.  

     8                CHAIRPERSON VOSE:  I guess I am a little

     9    bit concerned about using the total nucleated cells

    10    because that doesn't always necessarily correlate. So

    11    I have a little concern about using that.

    12                DR. SIEGEL:  Indeed the design of the

    13    trial was such that they targeted 4 x 108 per kilogram

    14    and 10 of the patients didn't have 2 million.  

    15                CHAIRPERSON VOSE:  Yes, Dr. Silver?

    16                DR. SILVER: I wasn't sure what the role of

    17    the FDA is in this.  Is it a foregone conclusion that

    18    there would be a sentence following the first

    19    paragraph saying some sort of attempt to inform the

    20    clinician that there is no evidence at the moment

    21    whether reduction of tumor cells has a clinical

    22    benefit?

    23                DR. SIEGEL:  Is your question what the

    24    role of the FDA is in terms of what goes into the

    25    label?  It is proposed by the sponsor, but we have to

                                                                        151

     1    approve it.  We play a significant role in determining

     2    what is said where.  What in particular were you

     3    asking about?  Was there to be a warning to the

     4    clinician about the ‑‑

     5                DR. SILVER: It seems to me appropriate

     6    that there be a note that says it is not clear whether

     7    a 2 log reduction in tumor in the transplant has a

     8    beneficial clinical effect.  In the absence of such a

     9    statement, the claim proposed by the sponsor could be

    10    misleading, it seems to me. It sort of implies that it

    11    is obvious that there is such a benefit, and it is not

    12    really so obvious.  So it seems to me that there

    13    should be some disclaimer to that effect.

    14                DR. SIEGEL:  You are advising that there

    15    be a prominent statement about the lack of information

    16    about that?

    17                DR. SILVER: Yes.

    18                CHAIRPERSON VOSE:  Typically, if the Phase

    19    III trial is described and it is described as not

    20    being adequately sized to show benefit or lack of

    21    benefit thereof is important information to have in

    22    there.  Let's move on to the next question, which has

    23    to do with the generalizability of this information. 

    24    Should this study just simply be ‑‑ should the label

    25    be restricted just to patients with multiple myeloma

                                                                        152

     1    or is it generalizable to other tumors, or should

     2    there be post‑marketing studies of other tumor types? 

     3    Some discussion on that?  Abbey, would you like to

     4    comment on that since that was your question to begin

     5    with?

     6                MS. DAPOLITO:  You are breaking up, Ms.

     7    Meyers.  We can't hear you.

     8                MS. MEYERS (telephonically):  Can you hear

     9    me now?

    10                MS. DAPOLITO:  Try again.

    11                MS. MEYERS (telephonically):  Hello?

    12                MS. DAPOLITO:  Yes, can you hear me?  

    13                MS. MEYERS (telephonically):  Yes. 

    14                MS. DAPOLITO:  Try again.

    15                CHAIRPERSON VOSE:  I think the gist of

    16    what Abbey said was that she didn't think that they

    17    should have to do studies in all different types of

    18    malignancies.  The labeling should just say that it

    19    separates the cells and not otherwise be specific. 

    20    Virginia?

    21                DR. BROUDY:  I guess I would differ from

    22    Ms. Meyers opinion on that.  I would recommend

    23    personally that the label say depleted myeloma cells,

    24    because that is what has been I think elegantly and

    25    convincingly shown in this carefully done study by the

                                                                        153

     1    company.  A potpourri of other trials were shown at

     2    the end, and they were characterized by much smaller

     3    patient numbers in the studies of, for example,

     4    lymphoma peripheral blood depletion studies.  And I

     5    would like to see those studies done, particularly

     6    with BCL2 gene.  That could be analyzed or looking for

     7    breast cancer cells.  I would like to have it

     8    convincingly shown that this device also depletes

     9    breast cancer cells and lymphoma cells before

    10    generalizing this to other tumors.

    11                CHAIRPERSON VOSE:  Dr. Silver?

    12                DR. SILVER: I think I disagree on two

    13    grounds.  First of all, I don't think it was very

    14    convincingly shown in this study because of all the

    15    statistical problems and some patients in which the

    16    data wasn't consistent.  But it is overwhelmingly

    17    likely from a biological point of view that a tumor

    18    that doesn't have CD34 on it won't be selected in this

    19    column and the column allows you to reduce the number

    20    of cells you infuse by 100‑fold.  So almost certainly

    21    the number of tumor cells that are going to go back

    22    for a CD34 negative tumor is going to be down by about

    23    a factor of 100.  And given that, I don't think it is

    24    appropriate to force the company to do additional

    25    studies.  I think a statement saying something like

                                                                        154

     1    for tumors which are CD34 negative, this result might

     2    be generalizable to tumors that are CD34 negative. 

     3    Because it is biologically very likely and a lot of

     4    work and a lot of money would have to be spent to

     5    prove it in each individual case.

     6                CHAIRPERSON VOSE:  Dr. Anderson?

     7                DR. ANDERSON:  Since it is difficult to

     8    hear Abbey ‑‑ Abbey, I am going to ask the next

     9    question that you would ask.  So I am now the voice of

    10    Abbey Meyers.  Will the label be such that third party

    11    payors will pick up the cost if this device is used in

    12    diseases other than myeloma?  That was Abbey's next

    13    question.

    14                CHAIRPERSON VOSE:  Yes, I am sure it would

    15    have been knowing her so well.  I guess that question

    16    has to go to Jay.  

    17                DR. SIEGEL:  Well, obviously we are here

    18    seeking guidance from the committee.  This is a

    19    complex area.  I think it is fair to say, among other

    20    things, that although one can arguably say and I think

    21    quite correctly say that more could be done in a

    22    variety of other tumors and perhaps more should be

    23    done in a variety of other tumors, that even at best

    24    it would be very hard to do in most tumors anything

    25    close to what was done here.  PCR has a level of

                                                                        155

     1    sensitivity that is not apt to be ‑‑ for tumor

     2    detection that is not apt to be found with monoclonal

     3    antibodies, histochemical staining and other

     4    approaches that might be used.  Certainly numbers

     5    could be done better and I think even relatively small

     6    numbers provide some assurances regarding the

     7    possibilities that Dr. Silver mentioned.  The concerns

     8    ‑‑ if you look at a column that depletes cells by 2.2

     9    logs ‑‑ that depletes a total number of cells by 2.2

    10    logs, and then if you then assume you are CD34

    11    negative and note that the number of CD34 negative

    12    cells goes down from almost 100 percent to about 30

    13    percent, that is another half a log.  You are starting

    14    with about a 2.7 log.  And there is a reasonable a

    15    priori assumption if the tumor is CD34 negative that

    16    it will be reduced by about that much.  However, we

    17    all know that there are cells that have non‑specific

    18    sticking to all sorts of devices and columns and I am

    19    not sure I am so comfortable buying that.  So I guess

    20    what we are looking for is solid data or reasonably

    21    solid data in one tumor and is there enough elsewhere

    22    to make us reasonably comfortable with the type of

    23    assumption Dr. Silver mentioned.  Certainly we can

    24    write a label in such a way ‑‑ we always ‑‑ you know,

    25    related issues come up at every meeting and my answer

                                                                        156

     1    always is that we try to be informative about the data

     2    rather than definitive in labeling.  Certainly one of

     3    our options is to write an option that would both ‑‑

     4    that would not restrict use to myeloma but would

     5    indicate the amount of data and the relative paucity

     6    of data in other tumor types.  Another option would be

     7    one that would limit the indication.  And I am not

     8    really here to tell you but to get input from you as

     9    to what might be more appropriate.  

    10                DR. ANDERSON:  All right.  I am going to

    11    follow on this because basically if Abbey were here,

    12    she would do this.  What is the experience with what

    13    third party payors will do based on the labeling?  If

    14    the labeling specifically says myeloma and then is

    15    sort of wishy washy about other things, will third

    16    party payors pay it for breast cancer, or does the

    17    labeling have to be specific that it is payable for

    18    other indications.

    19                CHAIRPERSON VOSE:  They won't even hardly

    20    pay for breast cancer transplants anyway.

    21                DR. ANDERSON:  I am sorry?

    22                CHAIRPERSON VOSE:  They won't hardly even

    23    pay for breast cancer transplants anyway, so that is

    24    probably a moot issue.  Personally, I think that there

    25    is enough concern about CD34 positivity in cells.  In

                                                                        157

     1    breast cancer it has been reported and also in some

     2    very early populational lymphoma cells.  I think there

     3    is some concern there that we have to be careful about

     4    not just having a totally generalizable statement.  We

     5    have to have some concern in the labeling in some way

     6    to reflect that.

     7                DR. KLEINERMAN:  Julie, in terms of

     8    paying, are we not looking at approving this for stem

     9    cell transplant, not just ‑‑ I mean, as a device to

    10    use for stem cell transplant and not just as a device

    11    to use to remove 2 logs of tumor?  So, theoretically

    12    if it is approved as a device to select CD34 cells for

    13    stem cell transplant, you could use it with breast

    14    cancer and the log tumor reduction should be non‑

    15    considered when it comes to third party payors.  I

    16    mean, that is the way I would read it.  That you are

    17    approving it as a selection device for stem cells, as

    18    we have done with another device.  And in addition,

    19    you may get this benefit.

    20                DR. SIEGEL:  Well, it would depend on how

    21    we wrote the labeling, I guess.  That would be

    22    certainly a way that one could view it.

    23                DR. KLEINERMAN:  Then you could leave it

    24    up to the clinician as to whether they want to

    25    consider that.

                                                                        158

     1                CHAIRPERSON VOSE:  Ms. Knowles?

     2                MS. KNOWLES:  Yes.  I am aware of some

     3    examples in the Seattle area actually where there have

     4    been women who have sued their third party carriers to

     5    get this kind of treatment.  Actually to have it done

     6    for them.  They have been successful, but it has not

     7    been without a lot of effort on their part.

     8                CHAIRPERSON VOSE:  Abbey, did you have

     9    another comment?  

    10                DR. ANDERSON:  Tell Abbey she has to

    11    shout.  

    12                CHAIRPERSON VOSE:  Shout louder.  Jay, did

    13    you hear Abbey's question?

    14                DR. SIEGEL:  If we approve it, will it ‑‑

    15    I didn't catch the end.  

    16                CHAIRPERSON VOSE:  The patent problem.  If

    17    they approve it, will it be able to get on the market?

    18                DR. ANDERSON:  Is that an issue we can

    19    deal with?

    20                CHAIRPERSON VOSE:  I think that is

    21    something we cannot address.

    22                DR. SIEGEL:  I think the device is

    23    currently on the market.  Is it not on the market?  I

    24    think if you want any more in‑depth answer to that,

    25    you should ask the company to respond.

                                                                        159

     1                CHAIRPERSON VOSE:  I think that is not

     2    appropriate for our meeting today.

     3                DR. AUCHINCLOSS:  Can I suggest a change

     4    in the wording for the proposed labeling in that

     5    portion that talks about tumors?  I would say

     6    selection of PBPC can result in greater than 100‑fold

     7    reduction in number of tumor cells present in the

     8    autograft if the tumor is CD34 negative.

     9                DR. BROUDY:  But how about saying that it

    10    has been demonstrated to result in a 2 log depletion

    11    of myeloma cells?  That is what has been demonstrated.

    12    And I guess my concern is that breast carcinoma cells

    13    and epithelial cells are sticky cells, and I would

    14    just like to ask that the company do a study that

    15    quantitates the fold log reduction in breast cancer

    16    cells and lymphoma cells given the immense

    17    applicability of this potential device.  I don't think

    18    that is asking too much.  They have convinced me

    19    already that it is safe.  That CD34 selected

    20    peripheral blood ‑‑ mobilized peripheral blood

    21    progenitor cells selected by this device are safe. 

    22    That they result in rapid neutrophil engraftment and

    23    only slightly delayed platelet engraftment.  So I

    24    wouldn't necessarily require that they replicate all

    25    of those data, but I would like to see a carefully

                                                                        160

     1    done analysis of depletion of tumor cells for breast

     2    cancer cells and lymphoma cells.  Because those other

     3    studies were just not as carefully done from the brief

     4    overview we had and from my own reading as this

     5    particular study.  

     6                CHAIRPERSON VOSE:  Dr. Berman?

     7                DR. BERMAN:  I would disagree.  I think

     8    that what the company has shown is that it is an

     9    extractable material, that is, the CD34 cells are safe

    10    going in.  I think it is up to the clinical trials,

    11    all of which will be done in breast and lymphoma, to

    12    prove whether it is effective or not.  But it is not

    13    up to the company.  It is up to the remainder of the

    14    investigative community.  And it will be used or not

    15    depending on those studies.  But I think to require it

    16    for the company is wrong.  I think it is up to us to

    17    prove it or disprove it.  All we know is that the CD34

    18    population is safe, and by the way, it can also

    19    effectively reduce the log contamination.

    20                DR. BROUDY:  Oh, I wouldn't necessarily

    21    require it of the company.  Perhaps I misspoke there. 

    22    But I am concerned that the labeling not say that it

    23    reduces tumor cells in general.  Because what we have

    24    seen in my view convincingly is that it depletes

    25    myeloma cells by 2 logs.

                                                                        161

     1                DR. BERMAN:  But that will go into the

     2    labeling.  The data are in myeloma cells. I am

     3    assuming that that will go in. 

     4                DR. AUCHINCLOSS:  That will be in later. 

     5    But for this particular up‑front portion, there will

     6    be this sentence that makes the general statement. 

     7    How about a compromise.  Has been demonstrated to

     8    accomplish a 2 log reduction in tumor for a CD34

     9    negative tumor and then require of the company a Phase

    10    IV post‑marketing trial for breast cancer or your

    11    other tumor of choice.

    12                CHAIRPERSON VOSE:  I think I have to agree

    13    with Dr. Broudy. I mean the information we have, the

    14    good data that we have, such as it is, is in myeloma,

    15    and I don't think it is generalizable to the other

    16    tumors.  There is too many differences in their

    17    physical properties. There are too many issues

    18    regarding CD34 positive stem cells in lymphoma, for

    19    example.  I think that is a concern.

    20                DR. AUCHINCLOSS:  Well, there is no

    21    question it is not absolutely generalizable, right? 

    22    I mean, you wouldn't do this for a CD34 positive

    23    tumor, and probably there are other tumors.  What we

    24    are trying to get away from is the Abbey concern.  We

    25    really would like not to make this disease specific. 

                                                                        162

     1    So if you made a general statement here, but not one

     2    that implies absolute generalizability, and then ask

     3    the company to come back and do additional studies,

     4    isn't that the best solution all around?  

     5                CHAIRPERSON VOSE:  Other comments?

     6                DR. KLEINERMAN:  Yes, I would agree with

     7    that.  I think we need to try to keep it as

     8    generalizable.  Because I think we need to keep in

     9    mind that we want all these studies done, but part of

    10    the ability to do these studies is for patients to be

    11    able to pay for them.  And I think that is a real

    12    concern.  We can design studies, but if we can't get

    13    patients to enter them, we will never know the answer. 

    14    So I think it is important that the label be designed

    15    in some kind of specific yet general enough way,

    16    either just by using a peripheral stem cell transplant

    17    that it is safe and effective in selecting CD34 cells. 

    18    So that it can be picked up by third party payors, so

    19    these other studies can be done.

    20                CHAIRPERSON VOSE:  But yet we do have to

    21    have some sort of caution, I think, somewhere

    22    regarding that the information we have is in myeloma.

    23                MR. KATZ:  I would think that the third

    24    party payors would seize on the disclaimer that we

    25    were talking about before about no proof of clinical

                                                                        163

     1    effect on the overall outcome, which I think is an

     2    important element of it.  I think they would go

     3    straight to that.  They wouldn't worry about the

     4    earlier statement.  Wouldn't they?

     5                CHAIRPERSON VOSE:  You never know with

     6    those guys.  Okay, any other discussion on that?

     7    Why don't we move to the last question then.  Data

     8    collection for overall and disease‑free survival in

     9    the study will continue if approved in addition to

    10    follow‑up from the ongoing trial.  Should post‑

    11    marketing studies for evaluation of the effect of the

    12    Ceprate device on measures of time to relapse or

    13    reduction in recurrence rates on patients with myeloma

    14    be sought or other malignancies?  Would the committee

    15    recommend any additional post‑marketing studies?  Dr.

    16    Auchincloss, you must have something.

    17                DR. AUCHINCLOSS:  Well, I just mentioned

    18    one.  This is where I put in ‑‑ I mean, for sure we

    19    want to know what the survival and recurrence of

    20    disease is.  That is assumed.  But another tumor, I

    21    think, is critical.  And breast cancer is the obvious

    22    one, right?

    23                CHAIRPERSON VOSE:  Well, if you are going

    24    to do a post‑marketing study, I would say that we at

    25    least need to do one that is large enough to be able

                                                                        164

     1    to see a difference in disease‑free survival or

     2    survival or something.

     3                DR. AUCHINCLOSS:  Now you are talking

     4    something big from the company's point of view.  If

     5    you start requiring a post‑marketing study of the

     6    company that is powered to be able to show

     7    progression‑free benefit, wouldn't that calculate to

     8    be something like 800 patients?

     9                CHAIRPERSON VOSE:  At least.  I didn't ‑‑

    10                DR. AUCHINCLOSS:  I don't know that I

    11    think that is fair to ask of the company in return for

    12    the amount of labeling we have given them so far.  

    13                DR. SIEGEL:  But think of the labeling

    14    they could get if they showed something.

    15                CHAIRPERSON VOSE:  But then why bother

    16    doing a post‑marketing study at all?  What are we

    17    asking them for?

    18                DR. AUCHINCLOSS:  To prove what they say

    19    in their label is true.  That is what we are asking

    20    them to do.  In this case it would be to generalize it

    21    to more than one cancer.  But I am not sure it is up

    22    to the company at this point to prove that 2 log or

    23    more tumor reduction is a good thing.  

    24                DR. BERMAN:  That is the crux of the

    25    issue.  I think it is to the rest of us as the

                                                                        165

     1    clinical investigators to prove it is a good thing or

     2    a bad thing.  So I would not require it of the

     3    company.  I think it is up to us. All the company has

     4    shown is that it is a safe product that can engraft

     5    promptly, and there is a log reduction in myeloma. 

     6    Whether that is of any efficaceousness is up to us,

     7    and that will be answered 5 years from now when the

     8    randomized large studies have been done.  But it is

     9    not up to the company to show that.

    10                DR. SIEGEL:  Do you anticipate the

    11    randomized large studies will be done?  

    12                DR. BERMAN:  Yes, I do.  

    13                CHAIRPERSON VOSE:  This could be a big

    14    undertaking.  I don't know how such a large study

    15    could be done unless it was supported by the

    16    companies.

    17                DR. BERMAN:  Well, support is another

    18    question.  Requiring them to perform it is something

    19    else.  

    20                CHAIRPERSON VOSE:  Additional discussion

    21    on post‑marketing studies?  

    22                DR. JACOBS:  Could I just add one thing? 

    23    We did have an additional 59 patients randomized onto

    24    the study.  So we have close to 200, but still not

    25    enough.  We also have a Phase III study that is

                                                                        166

     1    randomized in multiple myeloma looking at mephalan and

     2    TBI in Europe. So we possibly could look at 300

     3    multiple myeloma patients.  That is probably the best

     4    that a small company can afford to do.  It may or may

     5    not show what is needed.  But there will be follow‑up

     6    on 300 patients.

     7                CHAIRPERSON VOSE:  I think one thing also

     8    to consider in the follow‑up besides the disease‑free

     9    survival is the long‑term immunologic reconstitution

    10    of these patients as well.   I think that is an

    11    important issue that we need to look at.  Because

    12    there have been concerns about that as well.  But I

    13    don't think that should really hold up anything that

    14    we are doing today.

    15                DR. SIEGEL:  You are talking about in the

    16    patients in the study?

    17                CHAIRPERSON VOSE:  Right.  Any additional

    18    questions or comments?  Okay.  I think we are done. 

    19    We are going to take a 45‑minute lunch break and then

    20    we are going to resume.  Dr. Noguchi is going to start

    21    off this afternoon.  Let's resume about 1:30.

    22                (Whereupon, at 12:46 p.m., the meeting was

    23    adjourned for lunch to reconvene at 1:41 p.m.)

    24

    25

                                                                        167

     1              A‑F‑T‑E‑R‑N‑O‑O‑N   S‑E‑S‑S‑I‑O‑N

     2                                             (1:41 p.m.)

     3                CHAIR VOSE:  Dr. Siegel, thank you.

     4                We are still in the open session right

     5    now.  Yes, we are going to proceed with Dr. Phil

     6    Noguchi, who is going to do the overview of the

     7    Laboratory or Cellular Immunology and Laboratory of

     8    Developmental Biology.

     9                DR. NOGUCHI:  I'll just go ahead and get

    10    started here, and I want to personally thank all the

    11    members for allowing us the opportunity to tell you a

    12    little bit about our research.

    13                The division that I represent is called

    14    the Division of Cell and Gene Therapies, and just as

    15    an example of some of the product classes we have,

    16    basically, this is unlike taking just a single

    17    product, such as we've seen this morning with the

    18    CellPro device, where you are actually taking the

    19    cells and you may expand them, take them from the

    20    body, expand them with interleukin 2, people are

    21    looking at other types of mesenchymal stem cells, some

    22    are even using sertoli cells for immunosuppression.

    23                We also cover all the gene therapies using

    24    a variety of vectors to genetically alter cells, and

    25    we are getting into xenotransplantation using both

                                                                        168

     1    fetal tissues and trasnsgenic animals as well.

     2                Now, in order to also just give you the

     3    other breadth of it, we cover a number of different

     4    diseases, among them genetic diseases, some of the

     5    more neurological, debilitating ones.

     6                Cardiovascular disease, it's interesting,

     7    we think that gene therapies for that are going to be

     8    a very big area over the next year or so, and, of

     9    course, cancer and AIDS.

    10                Now, the numbers of INDs is actually quite

    11    interesting.  Over the last three years, we received

    12    in the area of cell and gene therapy about 100 to 120

    13    INDs, which translates to about one out of every four

    14    INDs that comes to the center is a cell or a gene

    15    therapy IND, so we are seeing a lot of investigational

    16    activity and we hope that soon we'll be able to bring

    17    to this committee some actual products.

    18                Now, the divisional structure, just

    19    briefly going over that, Dr. Gerry Marti is in the

    20    Office of the Director, there will be three

    21    individuals who will be reviewed from the Laboratory

    22    of Cellular Immunology.  This group has recently been

    23    reorganized to focus on issues of xenotransplantation,

    24    including both immunology and viral aspects.

    25                We've reorganized Molecular and

                                                                        169

     1    Developmental Biology into one group, and Dr. Judy

     2    Kassis will also be reviewed today, and she'll be

     3    talking more about her specific interest in gene

     4    therapies.

     5                And then the last laboratory, which is not

     6    being reviewed but has been reviewed previously, is

     7    the Laboratory of Molecular Tumor Biology.

     8                Now, we always appreciate the opportunity

     9    for you to tell us how we are doing in our research,

    10    and given that we'd like to just focus on both what we

    11    are currently covering and some of the areas that we

    12    think will be coming down the pike.  There will be

    13    other kinds of gene therapy vectors that we'll be

    14    looking at.  We know lentiviral vectors are coming. 

    15    Dr. Anderson, is he still here, is already talking

    16    about in utero types of protocols, where we'll do gene

    17    therapy in utero for some genetic diseases.  There

    18    will be cell tissue and organ regeneration.  We

    19    already have some animal cloning, and we've already

    20    stated that human cloning, should it ever happen,

    21    would be an FDA regulated product.

    22                So, basically, for this division what we

    23    can say is, what can be imagined will be done, and Dr.

    24    Eda Bloom will present her program now.

    25                DR. BLOOM:  Thank you.

                                                                        170

     1                It is my great pleasure to follow Phil and

     2    give you a more descriptive introduction,

     3    particularly, to the Laboratory of Cellular

     4    Immunology, and to give you an overview of both our

     5    regulatory and our research work.

     6                To reiterate, the members of the

     7    laboratory that have been site visited include myself,

     8    Dr. Carolyn Wilson and Dr. Parris Burd, and our little

     9    Laboratory of Cellular Immunology is named as one that

    10    has been standing for a while, it is less descriptive

    11    of what the laboratory does now than what it once was. 

    12    And, in fact, as you will see, the cohesive element

    13    that holds the three of us very tightly together in a

    14    collaborative network is that of xenotransplantation

    15    and the porcine endogenous ‑‑ and, clearly, the

    16    porcine endogenous retrovirus, but the idea of

    17    xenotransplantation and whatever endogenous retrovirus

    18    we need to look at.

    19                The regulatory work in the laboratory is

    20    representative of that which is done at CBER.  It

    21    includes review work, policy formation.  We also,

    22    however, do research related and have done research

    23    related to adverse events and safety, as well as doing

    24    research applying our expertise, both in the

    25    anticipation of new products, and also in the

                                                                        171

     1    exploration of products that are currently under

     2    development and under IND.  And, of course, as any

     3    group in an academic or similar atmosphere, we provide

     4    other services to CBER through various committee

     5    services.

     6                The regulatory review activities in a

     7    little more depth include, of course, the product

     8    review of INDs, and I'm going to use the word "we" a

     9    lot, and when I use the word "we" I mean to say that

    10    one or more of us within the  laboratory, that is, one

    11    or more of Dr. Burd, Dr. Wilson or myself. 

    12                We also mentor and advise IND reviewers. 

    13    We have chaired and been members on various license

    14    application committees, as well as reviewing numerous

    15    post‑market license supplements, which, as you may

    16    know, also have their own set of clocks and their own

    17    set of criteria that need to be met.

    18                In addition, we have been inspectors of

    19    manufacturing facilities to assure that such

    20    facilities conduct their manufacturing under good

    21    manufacturing procedures.

    22                And, as you can see today, and have seen

    23    in the past, we make various scientific regulatory

    24    presentations to the FDA Advisory Committee panels.

    25                This is a little bit of a rehash of a

                                                                        172

     1    slide that Phil just showed.  The only difference is

     2    that, and this is totally cribbed from him, is that it

     3    also shows the growth of the cell and gene therapy

     4    INDs relative to the rest of the center.

     5                The next slide focuses a little bit more

     6    on our laboratory per se, and as you can see, the

     7    reviewers in our laboratory provide a substantial

     8    share of the IND review within the division,

     9    especially, relative to the number of reviewers that

    10    we do provide.

    11                As far as our policy activities are

    12    concerned, we have been drafting points to consider,

    13    now currently called guidance documents, for industry

    14    to be helpful in product manufacture.  We have been

    15    involved in the drafting of the Public Health Service

    16    Guidance Document in xenotransplantation.  We have

    17    also participated in both CBER and department‑wide

    18    committees on xenotransplantation, prevented various

    19    invited talks on the spectrum of regulatory subjects

    20    that the whole division deals with.

    21                We have organized the FDA/NIH gene therapy

    22    conferences, which some of you I'm sure are familiar

    23    with, that have been held very successfully the past

    24    two years, '97 and '96.  We have organized a Cystic

    25    Fibrosis Foundation conference, organized and led

                                                                        173

     1    CBER‑wide viral vector working groups, which has

     2    served as a template for other working groups dealing

     3    with the types of products that our division deals

     4    with, and we have been panel members on public fora,

     5    such as the Commissioners Roundtable for Autologous

     6    Cells Manipulated Ex Vivo.

     7                The laboratory responses to regulatory

     8    issues that our laboratory has been involved in

     9    include addressing the unexpected toxicities that

    10    arose in clinical trials of interleukin 12.  We have

    11    designed paradigms and performed experiments for

    12    testing the presence of replication competent

    13    retroviruses, and particular emphasis on gene therapy

    14    products, and this slide says "developing," but, in

    15    fact, standards have been developed, a standard viral

    16    stock has been developed for use in safety testing,

    17    and we have developed technologies, isolated and

    18    characterized the endogenous infectious agent or

    19    agents, probably more precisely agents, that are

    20    present in pig cells.

    21                Now, I'm going to spend just a couple more

    22    minutes and go through each investigator's program.

    23                Dr. Carolyn Wilson, program is Viral and

    24    Cellular Factors Influencing Retroviral Infection. She

    25    has two major studies within her program.  In the

                                                                        174

     1    first study, she uses murine leukemia virus in order

     2    to study the viral receptor interactions, and one of

     3    the important emphases of her studies is, in fact, on

     4    how the retrovirus can enter the cells, an important

     5    first step in viral infection.  In addition, she has

     6    been studying virus variation selection, using the now

     7    famous or infamous porcine endogenous retrovirus.

     8                This next slide summarizes the relevance

     9    of her particular project to the regulatory mission of

    10    CBER.  Notably, her xenotransplantation studies, using

    11    the porcine endogenous virus, has enabled a certain

    12    amount of risk assessment and, thereby, permitting FDA

    13    to make recommendations to sponsors of ongoing

    14    protocols using pig tissue for transplantation into

    15    humans.

    16                Her studies in retroviral in the murine

    17    system have enabled studies expected to help develop

    18    new generations of retroviral vectors that, perhaps,

    19    may provide a safer or more efficacious way of

    20    delivering genes in gene therapy.

    21                And finally, her development of detection

    22    assays for recombinant complications ‑‑ complication,

    23    we don't mean that ‑‑ for replication competent

    24    retrovirus have enabled the means by which we are able

    25    to detect replication competent virus in retroviral

                                                                        175

     1    vectors and other products which may include such

     2    contaminants, such as monoclonal antibodies that are

     3    produced in mice, as well as in xenografts.

     4                Just to briefly summarize progress by Dr.

     5    Wilson since the site visit, she has had an additional

     6    manuscript accepted for publication, she is currently

     7    expressing gp70, the envelope glycoprotein on

     8    amphitrobic murine leukemia virus in a vaccinia system

     9    and will be purifying this for use in binding studies,

    10    and in her virus variation selection program she has

    11    also had a very important paper accepted for

    12    publication, detailing the production of porcine

    13    endogenous virus by primary pig cells.  She has

    14    isolated in a sequencing that envelopes CDNA for this

    15    virus.

    16                Dr. Parris Burd's program has focused on

    17    the molecular biology of immunologically active cells. 

    18    He has worked on cytokine networks in health and

    19    disease, and in this study he has emphasized

    20    interleukin 12, interleukin 13 and chemokines.  He has

    21    also developed PCT methods for analysis of archived

    22    histopathologic materials, which has clear and obvious

    23    relevance to studying potential reasons for adverse

    24    events using cytokines.

    25                In the porcine endogenous retrovirus

                                                                        176

     1    studies, Dr. Burd is our resident molecular biologist,

     2    where Dr. Wilson is the resident retrovirologist, and

     3    his expertise has been necessary in the studies across

     4    species viral infectivity and activation.

     5                As far as the relevance of Dr. Burd's

     6    research to the CBER mission, cytokine networks, as

     7    you on this committee certainly are aware, are part of

     8    the causal chain in disease processes, and provide key

     9    sites for the intervention and therapeutic approaches.

    10                Also, we have, certainly, our share of

    11    adverse events that are referable to cytokine

    12    treatments.

    13                In the porcine endogenous retrovirus

    14    study, it is clearly a prominent safety feature at the

    15    moment for use of porcine endogenous ‑‑ excuse me, for

    16    porcine xenografts, and Dr. Burd's establishment of a

    17    PCR method to assess the virus transfer to humans is

    18    something that will have a lot of relevance to our

    19    ability to regulate these products.

    20                Since the site visit, Dr. Burd has had two

    21    manuscripts accepted for publication, and he has

    22    submitted three additional manuscripts.

    23                On the regulatory front, he has

    24    spearheaded an initiative to foster development of

    25    gene therapies for rare genetic disorders, and, in

                                                                        177

     1    particular, this program will effect outreach and

     2    education to the public and education of new

     3    investigators in the handling of therapies that may

     4    involve very few patients.

     5                He has also been the co‑organizer of an

     6    international conference on vectors for gene therapy

     7    that was held ‑‑ where is this, I think it was this

     8    month in Brussels.

     9                My own program is centered around the

    10    cellular and molecular regulation of cytolytic

    11    lymphocytes, and I have those years up there because

    12    that's the time that elapsed since I was site visited

    13    at CBER, not the time period during which these

    14    projects particularly took place.

    15                I have had three subtopics in my program. 

    16    One is the activation and regulation of human natural

    17    killer cells, which is actually an extension of the

    18    project that I embarked upon in the early 1970s.

    19                More recently, we've been studying the

    20    regulation of natural killer cells by oxidation

    21    reduction, and not just the regulation of their

    22    functional activity, but the regulation of their

    23    elimination through apoptosis as well.

    24                In order to do this, we've been looking

    25    for effects on signaling pathways and have found

                                                                        178

     1    effects on signaling pathways as well as cell cycle

     2    progression as being key regulatory points by

     3    oxidation reduction.

     4                A project that's winding down in my

     5    laboratory is the alteration of cytolytic T

     6    lymphocytes and the effect of age on CTL activity and

     7    generation.  I list it here because it provides an

     8    important predecessor to the study that we are

     9    currently emphasizing, which would be the cellular

    10    immunity and safety issue in xenotransplantation, and

    11    where Dr. Wilson is the retrovirologist and Dr. Burd

    12    provides the molecular biology, I provide the cellular

    13    immunology and cell biology to that project.

    14                The relevance of my particular program to

    15    the regulatory issues at CBER has been substantial, I

    16    like to think.  Immune cells, as you are, again, I'm

    17    sure aware, comprise a large proportion of the somatic

    18    cellular therapies that are reviewed by CBER, in

    19    particular, lymphokine‑activated killer cells provide

    20    the prototype, and these are cells that you probably

    21    know are derived from natural killer cells.  Cytolytic

    22    T cells are another subclass of somatic cell

    23    therapies, and gene therapy is also used frequently,

    24    lymphocytes as delivery systems.

    25                My research program has led, or is leading

                                                                        179

     1    to, appropriate tests for lot release for certain of

     2    our products and has been important in the development

     3    of policy and particular guidelines that we have

     4    published for development of cellular therapies, as

     5    well as currently in xenotherapies.

     6                Finally, within our program we have

     7    provided scientific data that have aided in the

     8    understanding of the adverse reactions to IL‑12.

     9                Finally, the progress in my own projects

    10    since the site visit is that I have had three

    11    manuscripts accepted for publication and an additional

    12    one is being submitted.  We have tantalizing results

    13    that are, unfortunately, too preliminary to talk

    14    about, but, nevertheless, I have to mention them,

    15    regarding the effect of cytokines and various immune

    16    responses on the expression of porcine endogenous

    17    virus by lymphocytes.

    18                The regulatory work since November has

    19    blessed me with two license supplements for review,

    20    and I have acquired 16 new INDs to review. 

    21                And, with that, I'd like to introduce Dr.

    22    Judy Kassis, who will talk about her program and

    23    progress since her site visit.

    24                DR. KASSIS:  Okay.  I'm going to keep this

    25    really short.  My program is designed to study the

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     1    control of gene expression in transgenic animals, and

     2    we are the Division of Gene Therapy, and I think it's

     3    important for us to understand how transgenes are

     4    regulated in an organism.  And, in order to have safe

     5    and effective gene therapies, like I just said, it is

     6    important to be able to predict how the therapeutic

     7    transgene will be expressed.

     8                I study transgenic drosophila because it's

     9    a very fast system, it's a very easy system to study,

    10    and the system is, basically, one of an integrated

    11    transgene.  You can think of this as a retrovirus,

    12    it's transposon with two repeated sequences, and you

    13    put your gene of interest in between them, and this

    14    transposon can get incorporated anywhere in the

    15    genome.  And, what I'm interested in knowing is, how

    16    the genomic location influences the expression of this

    17    transgene.

    18                So, there are two factors which can

    19    contribute to the control of the integrated transgene. 

    20    One is the regulatory DNA in the transgene, and one is

    21    regulatory DNA flanking the transgene.  For instance,

    22    this transgene carries information to be expressed in

    23    the lung, but since it's inserted near enhancers which

    24    cause it to express in the heart, it will be expressed

    25    in all three tissues, and we'd like to understand how

                                                                        181

     1    to better control transgene expression.

     2                Also, during development and

     3    differentiation, genes are packaged into active and

     4    inactive chromatin, and, basically, we want to prevent

     5    a situation where a transgene, which we want to be

     6    active, is packaged into inactive chromatin, so we

     7    need to understand how this inactivation occurs.

     8                So, in drosophila there's a group of genes

     9    called the polycomb group, which are involved in

    10    keeping genes off.  In this schematic, early in

    11    development this gene is turned on by segmentation

    12    genes in this region of the embryo.  Then, the

    13    polycomb group genes somehow recognize that this gene

    14    is off in these two regions of the embryos, and they

    15    stay with this gene packaging it into inactive

    16    chromatin so that this gene is repressed throughout

    17    development, so that in the in tact fly this gene is

    18    only expressed in this part of the fly.

    19                If the polycomb group genes are mutated,

    20    this gene is expressed in other parts of the fly, and

    21    then you end up with flies with legs in place of

    22    antennae, and legs in place of wings.

    23                So, I study a piece of regulatory DNA from

    24    a gene called engrailed, which has two unusual

    25    activities.  First, when you make a transgenic

                                                                        182

     1    drosophila we use a marker, we use the mini‑white

     2    gene.  We inject white eyed flies, and when you inject

     3    this gene and you get a transgenic fly, the flies then

     4    have colored eyes.

     5                The name of the gene was named for the

     6    mutation, when this gene is mutant the flies eyes are

     7    white, but the gene product makes the flies have

     8    colored eyes.

     9                This vector inserts in the genome randomly

    10    and homozygous flies have two copies of this

    11    transgene.  Therefore, they have darker eye color than

    12    heterozygous flies.

    13                When you include this piece of engrailed

    14    DNA in this construct, the transgene now inserts in

    15    the genome in a more selective manner, and this piece

    16    of engrailed DNA causes the white transgene to be

    17    turned off.

    18                So, the way this works is, for a normal

    19    transformant, without the engrailed piece, you recover

    20    a transformant that has yellow eyes.  You make it

    21    homozygote, it has red eyes.  For the engrailed piece,

    22    if you recover an engrailed transformant with yellow

    23    eyes, if you make it homozygote, now it's two copies

    24    near each other in the genome and it has ‑‑ the

    25    transgene become repressed and you get white eyes.

                                                                        183

     1                If you have two copies far away from each

     2    other, you get, again, red eyes, but these two copies

     3    can either be very close to each other in cis or in

     4    trans, to get the transgene repression.

     5                I'll just show a couple of slides.  This

     6    is a normal transformant, this is a homozygote.  This

     7    has two copies, it's got a darker eye color.  For the

     8    transformant with the engrailed fragment, this has the

     9    heterozygote, this is a homozygote, the eye color is

    10    completely repressed, and now this I'd like to show

    11    because it gives a very dramatic example of the effect

    12    of the position of insertion in the genome on

    13    transgene expression.

    14                In this case, the heterozygote has a

    15    represssion in part of the eye, just based on where it

    16    is inserted in the genome, and when you make the fly

    17    homozygous you get a patterned eye expression, only

    18    part of the eye color is repressed.

    19                We'd like to understand how this type of

    20    thing happens.

    21                And, the model for this is that the model

    22    for selective insertion is the same as the model for

    23    pairing sensitive silencing.  They are DNA binding

    24    proteins which interact with the fragment, the

    25    engrailed DNA, proteins bind to that, they recognize

                                                                        184

     1    other proteins found in the genome, causing the

     2    transposon to be pulled to a particular region of the

     3    genome, and then you get selective insertion.  For the

     4    silencing, this complex forms and silences the

     5    transgene expression.

     6                Now, we've been dissecting this pairing

     7    sensitive silencer, and at the site visit I told about

     8    the isolation of a protein which binds to this

     9    conserved site, this sequence one, and the progress in

    10    my laboratory now is that we have candidates for DNA

    11    binding proteins which bind to this conserved site

    12    two.  Both sites one and two are necessary for pairing

    13    sensitive silencing.

    14                So, in summary, what I've found is a

    15    drosophila homolog of the mammalian transcription

    16    factor YY1 is necessary for pairing sensitive

    17    silencing.  YY1 is encoded by pleiohomeotic, which is

    18    a member of the polycomb group of genes. 

    19    Pleiohomeotic is the first polycomb group protein

    20    shown to find a DNA, but we think that four additional

    21    proteins may also be involved in pairing sensitive

    22    silencing.

    23                It's important to realize that this

    24    silencing fragment, we don't know what all the

    25    proteins are that cause this silencing, and we'd like

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     1    to be able to predict this to keep such a fragment out

     2    of transgenes.

     3                I just want to make the point that all

     4    cloned polycomb group genes have human homologs, and

     5    I want to show one more slide which shows a model of

     6    silencing.  Here I show these two chromosomes coming

     7    together, which turns off the transgene.  It turns out

     8    that in particular locations in the transgene, in the

     9    genome, you can get interactions between these

    10    elements on different ‑‑ on widely separated insertion

    11    sites in the same chromosome, and it turns out that

    12    it's recently been shown by Jim Burchler's group that

    13    if you put six copies of a transgene in, that doesn't

    14    even have an obvious one of these silencing fragments,

    15    the transgenes will then come together and silence

    16    even the endogenous gene in a mechanism called co‑

    17    repression, and this is mediated by the polycomb group

    18    genes.

    19                So, I think the polycomb group, the action

    20    of these silencer proteins is very important for us to

    21    understand, to be able to predict how transgenes will

    22    be regulated in the organisms.

    23                That's all.

    24                CHAIR VOSE:  Thank you.

    25                Gail?

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     1                EXECUTIVE SECRETARY DAPOLITO:  We just

     2    need a minute to clear the room.  I think if the

     3                can vouch for everybody in the audience,

     4    and FDA, yes, and Dr. Siegel, you can vouch for

     5    everybody else as FDA on this side.

     6                (Whereupon, the open session was

     7    concluded.)

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