1. Therapeutic Application of Intra-Vascular Nitrite for Sickle Cell Disease (CC/NHLBI)

Abstract

Sickle cell disease is an autosomal-recessive disorder and the most common genetic disease affecting African Americans. Approximately 0.15% of African Americans are homozygous for sickle cell disease and 8% have sickle cell trait. Hemoglobin S polymerization leads to red cell rigidity, microvascular obstruction, inflammation, and end-organ ischemia-reperfusion injury and infarction. Previously published data indicate that up to 50% of sickle cell patients have endothelial dysfunction due to impaired bioavailability of endogenous nitric oxide (NO) due in large part to scavenging of NO by cell-free plasma hemoglobin. These data suggest that therapies directed at restoring NO bioavailability might prove beneficial. We have recently discovered that the nitrite anion, available currently for human use as a component of the cyanide antidote kit, is a vasodilator in vivo by generating NO in tissues with lower oxygen tension and pH. The mechanism involves a novel physiological function of human hemoglobin as an oxygen- and pH-dependent nitrite reductase. To date we have observed that nitrite infusions in animal models significantly reduce liver and cardiac ischemia-reperfusion injury and infarction in mouse models, prevent cerebral vasospasm after subarachnoid hemorrhage in primates, and decrease pulmonary hypertension in newborn hypoxic sheep. This protocol is designed as a phase I/II trial to address the hypothesis that nitrite infusions will vasodilate the circulation in patients with sickle cell disease at rest and during vaso-occlusive pain crisis, inactivate circulating cell-free plasma hemoglobin, reduce pulmonary artery pressures, and reduce ischemia-reperfusion injury.

2. Molecular Profiling of Response to Proteasome Inhibition by Bortezomib (PS341) in a Clinical Trial of Mantle Cell Lymphoma (NHLBI/NCI)

Abstract

Mantle cell lymphoma (MCL) is a currently incurable, rare form of lymphoma with a short median survival of 3 years. Bortezomib is the first clinically available member of a drug class with a novel principle of action, inhibition of the proteasome. Recently, bortezomib monotherapy has shown promising clinical activity in patients with MCL. The effect of proteasome inhibition on tumor cells is incompletely understood. We hypothesize that proteasome inhibition may be particularly effective in MCL by targeting the pathogenic mechanism that deregulates cell cycle control in this disease. The molecular hallmark of MCL is a t(11;14) translocation that leads to constitutive overexpression of cyclin D1 and thereby drives proliferation of the malignant clone. Our previous gene expression profiling study identified a gene expression signature of proliferation as the dominant feature predicting survival. To address the mechanisms underlying bortezomib responsiveness we chose a novel clinical trial design in which patients will receive bortezomib monotherapy for two cycles followed by six cycles of EPOCH-R chemotherapy and bortezomib maintenance therapy. This trial design will allow us to integrate molecular profiling of biopsy samples before and during bortezomib monotherapy. We will use whole genome gene expression profiling to assess the effects of proteasome inhibition and determine what features of the tumor influence clinical response to this regimen. Additional studies will examine levels and activity of key proteins such as the cell cycle inhibitor p27kip-l, components of the NF-κB pathway, and proteins involved in apoptosis such as bc1-2 and caspases. These studies should allow us to develop tailored treatment regimens in MCL based on a molecular predictor of response to bortezomib.

3. A Phase I/II Pilot Study to Evaluate the Treatment of Intraocular Lymphoma with the BL22 Immunotoxin (NEI/NCI)

Abstract

Primary central nervous system lymphoma (PCNSL) is a rare, B-cell extranodal non-Hodgkin lymphoma that can originate in the brain, spinal cord, leptomeninges, and eyes. Primary intraocular lymphoma (PIOL) defines a subset of PCNSL in which lymphoma cells are initially present only in the eyes without evidence of CNS involvement. Present treatment strategies for PCNSL include the use of combined chemotherapy and radiation regimens and chemotherapy-only regimens using methotrexate as a primary agent. The best method of treatment for patients with PIOL or recurrent PCNSL involving only the eyes is unknown and recurrences after treatment are the rule. The treatment of ocular disease with radiation and systemic chemotherapy is associated with toxicity that may limit the future use of these therapies for the treatment of CNS disease. Local therapies such as ocular radiation and intravitreal chemotherapy are effective and avoid the toxicity associated with systemic therapy; however, these therapies are known to cause delayed ocular complications that can cause loss of vision although the intraocular tumor has been eradicated. A major limitation to the study of new treatments for PIOL is the lack of an animal model and the difficulties associated with designing clinical trials for rare diseases. Antibody-mediated immunotherapy has been a significant advance in the treatment of lymphoma. BL22, a monoclonal antibody coupled with a highly lethal cellular toxin, is an immunotoxin that is cytotoxic to CD22+ B cells that are found in patients with PCNSL and PIOL. Using the collaborative resources of the NEI and NCI we propose to develop an animal model of PIOL to investigate the intraocular use of BL22 as a therapeutic agent for PIOL and translate the data into a phase I/II pilot clinical trial for the treatment of patients with recurrent PIOL or PCNSL limited to the eyes.

4. A Phase I Treatment Trial of the Circadian Sleep Disturbance in Smith-Magenis Syndrome (SMS) (NHGRI/CC-Pharmacy)

Abstract

Smith-Magenis syndrome (SMS) is a rare contiguous gene syndrome caused by interstitial deletion of chromosome 17p11.2. This rare microdeletion syndrome is estimated to occur in 1/25,000 births and has physical, cognitive, and neurobehavioral consequences. The striking neurobehavioral phenotype includes sleep disturbance, stereotypies, and self-injurious and maladaptive behaviors. Sleep disturbance, characterized by daytime sleepiness, early sleep onset, and early morning awakening, is the strongest predictor of maladaptive behavior in children with SMS. Diminished nocturnal sleep is virtually universal in SMS, representing a major challenge to patient and family.

One of the likely contributing factors to the sleep disturbance is an inverse circadian pattern of the sleep-promoting hormone melatonin in which plasma melatonin is high during the day and low at night, opposite the normal pattern. The underlying reason for this daytime melatonin secretory pattern is unknown. It is distinctive to persons with SMS and is not found anywhere else within the animal and plant kingdoms. SMS therefore offers a rare and unique human syndrome for study of melatonin function. There is currently no effective treatment for the sleep disturbance in SMS. Furthermore, in the United States there are currently no controlled treatment trials under way with the specific goal of correcting the disturbed sleep pattern in this disease.

The overall goal of this project is to improve the quality of nocturnal sleep and decrease the need for daytime sleep by restoring a normal circadian pattern of melatonin levels in SMS patients. We predict that the inverse pattern of release can be corrected by the combination of nonpharmacological suppression of daytime melatonin release and pharmacological replacement of nocturnal melatonin. We propose two methods to achieve the overall goal. First, controlled artificial bright light, used at intensities previously shown to suppress melatonin, will be used to reduce daytime levels of melatonin in SMS patients and to correct the nighttime sleep disturbance. Second, Clinical Center Pharmaceutical Development Services will develop a delayed, time-release capsule that will elevate nocturnal melatonin levels specifically during the second half of the night, thus improving sleep quality. A more normal sleep pattern is predicted to follow the effects of the proposed therapeutic interventions: (a) decreased daytime sleepiness by reducing daytime levels of melatonin, and (b) increased nocturnal sleep by increasing nocturnal levels of melatonin. In addition, it is anticipated that negative behaviors that are associated with accumulated sleep debt will diminish as sleep quality improves.

5. Preclinical Nonhuman Primate Studies of an In Vivo Selectable Vector Intended for Use in a Planned Clinical Trial of Gene Therapy for Chronic Granulomatous Disease (NIAID/NHLBI)

Abstract

Chronic granulomatous disease (CGD) is an inherited immune deficiency of phagocyte oxidase associated with significant infection, morbidity, and mortality. Allogeneic bone marrow transplantation is curative but is available only to patients with an unaffected HLA-matched sibling donor and is associated with risk of graft failure or Graft-versus-Host Disease. Gene therapy for CGD is an appealing alternative. Knockout mice with CGD have been cured by gene therapy using an approach with ex vivo-transduced hematopoietic stem cells in a setting of ablative elimination of resident bone marrow stem cells. In past clinical trials of ex vivo gene therapy for CGD conducted by Malech and colleagues at the NIH Clinical Center, full functional correction of small numbers (less than 0.3%) of circulating neutrophils was achieved in a setting where no marrow conditioning was used, but the effect lasted only a few months and was below a level required for expected clinical benefit. Gene therapy as a field has seen its first clinically beneficial application in the cure of patients with another immunodeficiency, X-linked severe combined immunodeficiency (XSCID). The clinical benefit observed in XSCID gene therapy is thought to be due to the fact that the therapeutic transgene, IL2 receptor common gamma chain, confers a profound selective survival and growth advantage to gene-corrected T lymphocytes. Correction of the oxidase gene in CGD provides no selective growth or survival advantage to stem cells or neutrophils, but the xscm trial suggests that an alternative strategy for CGD gene therapy incorporating an in vivo selection strategy might enhance and prolong the functional correction achieved previously in the clinic to a level that provides clinical benefit. We have focused on a selection approach that incorporates the gp91phox therapeutic gene corrective for X-linked CGD together in the same vector with a benzyl guanine-resistant mutant form of human methyl guanine methyltransferase (MGMT), a DNA alkylation repair enzyme. We have shown that this vector will enhance gene marking using the appropriate selection regimen both in vitro and in vivo in a NOD/SCID mouse model transplanted with transduced human X-CGD patient stem cells. However, before such a vector can be brought to the clinic its applicability, safety, and performance in a nonhuman primate model in the autologous ex vivo gene transfer setting is required. Although the mutant MGMT selection model is of considerable interest to many in the field, no one to date has tested this selection model in nonhuman primates. Our bench-to-bedside collaboration combines the expertise of the Dunbar laboratory in the rhesus macaque autologous transplantation and gene transfer model with the Malech laboratory experience in development and study of vectors for gene therapy of CGD and the conduct of clinical trials for gene therapy of CGD. Funding requested would support a trial of in vivo selective enhancement of gene marking in a total of four nonhuman primate subjects using a bicistronic retrovirus vector incorporating gp91phox and mutant MGMT.

6. Isolation and Characterization of Circulating Endothelial Cells in Primary Pulmonary Hypertension: Implications for Early Diagnosis and Novel Therapeutic Targets (CC-CCMD/CIT)

Abstract

Primary pulmonary hypertension (PPH), a subgroup of plexogenic pulmonary arteriopathy (PPA), is a rare disorder associated with severe morbidity and high mortality rates. There are no routine screening tests or validated markers of disease activity in PPH or the broader group of PPA. Therefore, patients usually present at advanced stages of disease. The pathogenesis of PPH and other forms of PPA remains unclear. Current thinking focuses on a "two-hit" hypothesis: (1) genetic susceptibility and (2) a triggering stimulus that initiates pulmonary vascular injury, resulting in endothelial cell dysfunction. Loss of function mutations in the bone morphogenetic protein receptor 2 (BMPR2) gene have been implicated in the pathogenesis of PPH. Endothelial cell dysfunction in PPH has been associated with decreases in both endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production.

Endothelial cells are normally shed into the circulation and are a valuable source of clinical material for studying diseases characterized by endothelial cell dysfunction. Unfortunately, no clear methodology exists for isolating clinically relevant numbers of circulating endothelial cells (CECs). In the bench phase of the project we plan to use flow cytometry to develop a methodology for isolating clinically relevant numbers of viable CECs. We hypothesize that CECs can be used to define a subset of differentially regulated biomarkers in PPH and other forms of PPA that may lead to earlier diagnosis and methods for measuring responses to therapy. We also hope to identify novel targets for future therapeutic interventions.

In the clinical phase of the project, we will recruit the following subject groups: (1) patients with newly diagnosed PPH and other forms of PPA, (2) patients with pulmonary hypertension (PH) ascribed to a nonvascular injury process, and (3) normal individuals (controls). All subjects will undergo right heart catheterization. CECs drawn peripherally and from the pulmonary artery catheter will be characterized for disease phenotype by cell surface markers and oligonucleotide microarrays. Total RNA for microarrays will be prepared from CECs by cell sorting and subjected to amplification. We plan to follow response to therapy by restudying the same parameters in patients with PPH or PPA after therapeutic intervention.

7. Evaluation of the Humanized MiK-â-1 Monoclonal Antibody Directed Toward the IL-2/IL-15Rβ Subunit (CD122) that Blocks IL-15 Action: Effect on IL-15 Induced Immunopathogenic Virus-Specific T Cells in Patients with HTLV-1-Associated Neurologic Disease (NINDS/NCI)

Abstract

The human T-cell lymphotropic virus type I (HTLV-1) is an exogenous human retrovirus that infects approximately 10–20 million people worldwide. The majority of infected individuals remain healthy lifelong asymptomatic carriers while 0.25–3% develop a rare inflammatory disease of the central nervous system termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). In patients with HAM/TSP, increased HTLV-1 provirus load and augmented immune responses to HTLV-1 have been reported in peripheral blood and cerebrospinal fluid (CSF). One of the most striking features of the cellular immune response in HAM/TSP patients is the highly increased numbers of HTLV-1-specific CD8+ cytotoxic T cells (CTL), which are lower or absent in asymptomatic carriers. The immunology of HAM/TSP, in particular the persistent expansion of these antigen-specific memory CD8+ T cells, offers unique opportunities to study antigen-specific immune response in humans. Recently, dysregulation of the newly described cytokine interleukin-15 (IL-15) has been implicated in the pathogenesis of a number of immune-mediated diseases, including HAM/TSP. Collaborative research by the Viral Immunology Section, NINDS, and the Metabolism Branch, NCI, suggests that upregulation of IL-15 may sustain the persistent expansion of virus-specific CD8+ T cells in HAM/TSP. Based on fundamental immunological and virological information that has been defined in the pathogenesis of HAM/TSP, a major goal of this proposal is to translate this information into the development of immunotherapeutic treatments. The prospect of downmodulating the activated cellular immune process that has been defined in HAM/TSP provides a rational approach for the treatment of this disorder. The recent development of Hu MiK-1, a monoclonal antibody that blocks the action of IL-15, offers the unique opportunity to study the maintenance and dysregulation of long-term immunologic memory in a human immune-mediated disease. We propose the application of Hu MiK-â-1 as a proof-of-principle clinical trial in patients with HAM/TSP. We will assess the effects of intravenously administered Hu MiK-â-l on the cellular immune response with particular focus on virus-specific memory CD8+ T cells. Secondary outcomes to be measured will be clinical responses, including toxicity. As IL-15 overexpression has been demonstrated in a wide variety of autoimmune disorders including rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, and psoriasis, the successful application of Hu MiK-â-1 in HAM/TSP has significant and broad therapeutic implications. The proposed study is unique in providing an opportunity to test, in humans, the hypothesis that IL-15 is required for the maintenance of memory CD8+ cells because HAM/TSP may be the sole human disease wherein sufficient numbers of antigen-specific memory CD8+ T cells can be readily detected. This proposal represents longstanding collaborative efforts between the NINDS and NCI in the understanding and treatment of rare diseases that have direct application to more common human autoimmune, hematologic, and neurologic disorders.

8. Molecular Profiling and Drug Discovery for Patients with PTEN Hamartomatous Tumor Syndromes (PHTS) (NCI/NHGRI)

Abstract

PTEN is a tumor suppressor gene that negatively regulates the phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathway, and its function is commonly lost in human cancers through somatic mutations or gene silencing. Germline mutations of PTEN are rare (~1 in 200,000) and are the cause of hamartomatous syndromes such as Cowden syndrome (CS) or Bannayan-Riley-Ruvalcalba syndrome (BRRS), collectively referred to as PTEN hamartomatous tumor syndromes (PHTS). PHTS are inherited in an autosomal-dominant fashion. Although the manifestations of PHTS can vary widely, they are commonly characterized by the development of multiple benign and malignant tumors that decrease quality of life and increase morbidity through repetitive diagnostic and surgical procedures. No medical options exist for these patients. Despite the importance of PTEN as a tumor suppressor, the molecular consequences of PTEN loss in tissues from PHTS patients have not been described. We propose to profile the molecular changes in cells and tissues from these patients and to develop new therapies to counteract the loss of PTEN. These approaches will be tested in cell lines established from PHTS patients and in PTEN^+/- mice that recapitulate tumor formation in PHTS patients prior to translation into therapeutic clinical trials for patients with PHTS. Clinical trials for PHTS patients that develop from studies in PTEN-null model systems will incorporate traditional response criteria, metabolic imaging, and analysis of readily accessible tumor tissue such as skin tumors. Analysis of accessible tumors from PHTS patients undergoing treatment will allow validation that the treatment compensated for PTEN loss, thus completing the bench-to-bedside cycle. This approach will provide the first rational therapies for a rare patient population without medical options and could be highly valuable for credentialing drugs that target the PTEN/Akt pathway prior to evaluation in traditional oncology clinical trials.

9. Immunotherapy for Myelodysplastic Syndrome (NHLBI/VRC/NCI)

Abstract

Myelodysplastic syndrome (MDS) has an incidence of approximately 30,000/year in the United States. It is a clonal (preleukemic) disorder of the hematopoietic stem cell that terminates in death from marrow failure or acute leukemia. For patients with MDS progressing to acute leukemia, current chemotherapeutic treatment approaches can at best induce brief remissions in a proportion of patients. (1) Allogeneic stem cell transplantation is potentially curative through a T-cell-mediated graft-versus-leukemia (GVL) effect, but its application is restricted by high procedural mortality and the availability of a suitably matched donor. Nevertheless, the success of the GVL effect in eliminating MDS suggests that T-cell-based cellular immunotherapy could be effective in MDS. (2) The antigenic targets on MDS cells that elicit the GVL effect are not known, but recently attention has focused on peptides derived from two proteins: proteinase 3 and Wilms tumor-1 (WT1). Two groups recently reported clinical responses in a small group of patients with MDS and other myeloid malignancies following WT1 vaccination associated with significant increases in circulating WT1-specific T cells.

These findings and preliminary clinical observations suggest that peptide-based vaccination is worth exploring further in patients with MDS. Patients with MDS progressing to acute leukemia who do not have a suitable donor do not have a chance of cure with a stem cell transplant, and chemotherapy only produces brief responses. In this patient group, vaccine treatment following chemotherapy to reduce tumor burden offers the possibility of an autoimmune-based disease control.

Little is known about the optimum ways to deliver peptide vaccines in MDS. Peptide vaccination, while clinically feasible and relatively straightforward from a regulatory standpoint, is probably an inefficient method of boosting T-cell responses to tumor antigens. Recent studies by Dudley et al. (NCI Surgery Branch) suggest that immune responses may be amplified if antigen-specific T cells are first primed in vitro and transfused into a lympho-depleted recipient. Their innovative strategy of treating melanoma patients with a combination of fludarabine and cytoxan to produce profound lymphocyte depletion prior to infusion of melanoma-specific T cells resulted in sustained high percentages of circulating melanoma-specific T cell clones exhibiting tumor infiltration and causing prolonged tumor regression.

A similar strategy could also be applied to patients with MDS. Studies by Rezvani (Hematology Branch) show that the low but detectable frequencies of PR1- and WT1-specific T cells in patients with myeloid malignancies have a memory (antigen experienced) phenotype. Thus PR1 and WT1 vaccination would involve boosting an established T-cell response rather than the more complicated initiation of a naive T-cell response with dendritic cells. Indeed, the massive expansions of WT1- and PR1-specific T cells described by ourselves and others after transplantation or after vaccination is testament to the readiness with which these antigen-specific T cells can be expanded. It is therefore proposed to boost antigen-specific cytotoxic T cell (CTL) responses in MDS patients by priming apheresis lymphocyte collections with PR1 and WT1 peptides, giving lymphoablative and tumor reductive chemotherapy, reinfusing the primed cells, and giving repeated peptide vaccinations.

The joint project will be an inter-institute collaboration between NHLBI Hematology Branch (Dr. Barrett, Dr. Rezvani, and Dr. Sloand), the NCI Surgery Branch (Dr. Rosenberg), and the NIAID Vaccine Research Center (Dr. Douek and Dr. Price). The components of the study are (1) basic research into antigen-specific T-cell interactions with overexpressed proteins in MDS; (2) translational research to develop techniques to prime apheresis products with peptides, monitor antigen-specific T-cell responses, and measure antigen expression by molecular and antibody-based approaches; and (3) a clinical trial incorporating lymphoablation, reinfusion of antigen-primed T cells, and peptide vaccination. Aside from any clinical responses from such a "proof-of-principle" trial, much would be learned about the kinetics of antigen-specific T-cell expansion in the lymphoablated host, which would prove useful in the design of future trials. Importantly, the trial would determine whether the generation of large numbers of antigen-specific T cells results in clinical responses.

10. Intermediate Phenotype and Genetic Mechanisms for Psychosis and Cognitive Disturbance in 22q11.2-Hemideletion Syndrome

Abstract

22q11.2 (DiGeorge MIM# 188400, velocardiofacial MIM# 192430) syndrome is a hemizygous microdeletion on 22q11.2 of typically 3Mb, encompassing approximately 30 genes and mediated by aberrant homologous recombination and unequal crossing-over events between intrachromosomal flanking low-copy repeats (LCRs). The incidence is 1:4,000 live births. While somatic symptoms include congenital cardiovascular and craniofacial abnormalities, recurrent infections, and hypocalcemia, the most prevalent group of symptoms are neuropsychiatric and include cognitive dysfunction with mild mental retardation, behavioral difficulties, and psychosis. The syndrome is associated with a lifetime prevalence of schizophrenia-like illness (phenotypically mostly similar to sporadic schizophrenia) of approximately 25 times that of the general population, making the presence of this hemideletion the strongest known risk factor for the development of schizophrenia excepting the presence of a monozygotic twin with the illness. The 22q11 region is implicated in the risk architecture of schizophrenia by several linkage studies and harbors a number of proposed susceptibility genes, including genes for catechol-o-methyltransferase (COMT) and proline dehydrogenase (PRODH).

The neural basis of these pronounced neurocognitive and psychiatric abnormalities is unknown. This work proposes to (a) study a group of exceptionally high functioning, normal intelligence, psychosis-free individuals with 21q11.2 syndrome using a hierarchical multimodal imaging approach to define the intermediate systems-level phenotype of the disease combined with deletion mapping techniques and (b) study the functional effects of single nucleotide polymorphisms in genes in the hemideleted regions that have been implicated in schizophrenia, taking advantage of the unique fact that the hemizygous deletion allows immediate construction of molecular haplotypes and of potential epistatic allelic effects. This work is expected to (a) elucidate the pathophysiology of the CNS manifestations of the 22q11.2 syndrome and yield a brain intermediate phenotype that will allow studies in small and atypical deletion individuals in an effort to define individual genes responsible for neurocognitive deficit and increased risk for psychosis, (b) facilitate the identification of functional mechanisms underlying increased risk for schizophrenia for individual susceptibility genes in the deletion and for interacting risk alleles within the deleted locus, and (c) result in a clinical protocol in which the results from (a) and (b) can be applied to a prospective study evaluating early diagnostic and interventional approaches based on genetic risk and intermediate phenotype ascertainment in this group of patients at high risk for the development of psychosis.

11. Childhood Cancer and Plexiform Neurofibroma Tissue Microarray for Molecular Target Screening and Clinical Drug Development

Abstract

Cancer drug discovery is now focused on identifying molecularly targeted drugs that are more selective and specific for proteins and signaling pathways directly involved in tumorigenesis. This new paradigm alters the approach to clinical drug development for childhood cancers because the emphasis is on therapeutic targets present in common epithelial-derived adult cancers, which have a different pathogenesis. The pharmaceutical industry is unlikely to undertake target discovery programs for low-incidence cancers such as childhood cancers. Therefore, a more pragmatic approach to the development of molecularly targeted drugs in children is required. We propose to manufacture childhood cancer tissue microarrays (TMA) to aid in screening and selecting the most appropriate molecularly targeted agents for testing in childhood cancers. Twenty representative specimens for each of 17 histologically distinct pediatric solid tumors or plexiform neurofibromas will be collected. Using a robotic arrayer, 0.6 mm cores from each tissue block will be donated into one of three recipient paraffin TMA blocks. Standard immunohistochemical techniques will be applied to 5-µm sections from the TMA block using validated antibodies directed against specific protein targets of interest. The results of the TMA target assessment will guide in the selection of molecularly targeted drugs for pediatric phase I clinical trials in the POB and the Children's Oncology Group Pediatric Phase I/Pilot Consortium and the selection of candidate tumors for activity testing in phase II clinical trials. If clinical responses are observed in phase I or II trials in tumor types that express the target on the TMA, we will return to the original paraffin blocks, perform laser capture microdissection, and produce reverse-phase lysate protein microarrays to assess the activation state of targeted signaling pathways and investigate the role of the target protein in tumorigenesis of the responsive childhood cancers.