Martyn T. Smith

Date: February 12, 2003

Using new technologies to develop biomarkers of leukemia and lymphoma

New technologies, such as real-time PCR, microarrays and protein chips, offer great potential for advances in biomarker-based toxicology and epidemiology. I will describe the application of a number of new biotechnologies to the development of biomarkers for leukemia and lymphoma in children and adults. The goal of this research is to help elucidate the etiology of these diseases and to then prevent them.

Reciprocal chromosome translocations that form oncogenic fusion genes often play a critical role in the development of leukemias and lymphomas. Various specific translocations and inversions including t(8;21), t(15;17), inv(16), and t(11q23) are associated with particular types of acute myeloid leukemia (AML). In acute lymphoblastic leukemia (ALL), particularly in childhood ALL translocations t(12;21) and t(1;19) are common, whereas the translocation t(14;18) is found in follicular lymphoma. These changes could occur early in the disease process, in some instances before birth, and their detection at the molecular level could be useful in predicting future risk of these cancers.

We used multiplex long-distance PCR to sequence patient-specific genomic translocation breakpoints from more than 30 patients with childhood ALL or AML. We then analyzed neonatal blood spots called Guthrie cards for the presence of these genomic translocations by nested PCR. The presence of t(12;21), t(8;21), t(15;17) and inv(16) could be detected in approximately half of the Guthrie cards, in some cases over 10 years prior to diagnosis. These findings show that chromosome rearrangements are probably the initiating events in leukemia and have the ability to persist for at least 10 years without triggering acute disease. It is possible that even longer latency periods extending into adulthood could occur. Future intervention may be based on monitoring individuals harboring pre-leukemic clones who are at increased risk of progression to full-blown disease e.g. those with predisposing genetic polymorphisms, such as NQO1 C609T.

In order to screen people for leukemia and lymphoma-related chromosomal changes we have developed and refined real-time reverse transcriptase-PCR methods that permit the quantification of t(12;21), t(15;17), inv(16), t(11q23), t(14;18), t(8;21) and t(1;19) fusion gene expression at very low levels (1 transcript per 100,000 cells). This has enabled us to examine the levels of these translocations in adult and cord blood of control individuals and in people exposed to the known leukemogen benzene in the workplace. This data has been compared to that obtained by molecular cytogenetics using fluorescence in situ hybridization (FISH).

Real-time PCR is also a robust technology for genotyping. We have used this technology to examine genetic susceptibility to leukemia and lymphoma in the case-control setting using individual genotyping and DNA pools. We have shown that the NQO1 C609T inactivating polymorphism confers susceptibility to benzene toxicity, adult AML and infant leukemia. We have also shown that polymorphisms in genes involved in folate metabolism confer susceptibility to ALL. Recent studies have also shown a possible relationship between functional polymorphisms in the leptin and leptin receptor genes and risk of non-Hodgkins lymphoma.

Apart from real-time PCR and FISH we are also using cDNA microarrays and proteomics to develop new biomarkers of early effect for leukemia and lymphoma. Preliminary findings using these technologies are extremely promising and will be presented.

ACKNOWLEDGEMENTS: I am grateful to our many collaborators including P. Buffler, X. Ma, J. Wiemels and J. Wiencke on childhood leukemia; N. Rothman, R. Vermuelen, Q. Lan and G-L. Li on benzene; E. Holly on lymphoma; and, G. Morgan on adult leukemia. I am especially grateful to my lab members C. McHale, L. Zhang, C. Skibola, W.Guo, R. Turakulov, J. Curry, M. Forrest & L.Gunn who significantly contributed to this work. Supported in part by NIH grants P42 ES04705, P30 ES01896 and RO1 ES06721.

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