Faculty Bibliography

We have evaluated the use of allele-specific PCR (AS PCR) on DNA pools as a tool for screening inherited genetic variants that may be associated with risk of adult acute myeloid leukemia (AML). Two DNA pools were constructed, one of 444 AML cases, and another of 823 matched controls. The pools were validated using individual genotyping data for GSTP1 and LTalpha variants. Allele frequencies for variants in GSTP1 and LTalpha were estimated using quantitative AS PCR, and when compared to individual genotyping data, a high degree of concordance was seen. AS primer pairs were designed for nine candidate genetic variants in DNA repair and cell cycle/apoptotic regulatory genes, including Cyclin D1 [codon 870 splice site variant (A>G)]; BRCA1, P871L; ERCC2, K751Q; FAS -1377 (G>A); hMLH1 -93 (G>A) and V219I; p21, S31R; and the XRCC1 R194W and R399Q variants. For six of these assays, there was at least 95% concordance between AS PCR genotyping and an alternative approach carried out on individual samples. Furthermore, these six AS PCR assays all accurately estimated allele frequencies in the pools that had been calculated using individual genotyping data. A significant disease association was seen with AML for the -1377 variant in FAS (odds ratio 1.76, 95% confidence interval 1.26-2.44). These data suggest that quantitative AS PCR can be used as an efficient screening technique for disease associations of genetic variants in DNA pools made from case-control studies.

PURPOSE/OBJECTIVE:We have developed and evaluated a CNS-targeted chemotherapy regimen based on the pharmacokinetic properties of the individual drugs in the combination. PATIENTS AND METHODS/METHODS:In a twin-track study, 16 patients with secondary CNS lymphoma (SCNSL) and 8 with primary CNS lymphoma (PCNSL) were treated with IDARAM which comprised idarubicin 10 mg/m(2) i.v., days 1 and 2; dexamethasone 100 mg, 12-h infusion, days 1, 2 and 3; cytosine arabinoside (ARA-C) 1.0 g/m(2), 1-h infusion, days 1 and 2; methotrexate 2.0 g/m(2), 6-h infusion, day 3 (with folinic acid rescue); and cytosine arabinoside 70 mg plus methotrexate 12 mg, intrathecally, days 1 and 8. Two cycles were delivered at 3-weekly intervals. After response assessment, patients received adjuvant cranial radiotherapy (40 Gy over 20 fractions). RESULTS:The series comprised 24 patients, 11 male and 13 female. Their median age was 53 years (range 21 to 73 years). Grade 4 neutropenia and thrombocytopenia occurred in the majority of patients treated. Of the eight PCNSL patients, seven achieved complete remission (CR). Four remained in CR at the time of this report with a median duration of follow-up of 25 months (range 11 to 42 months). Of the 16 SCNSL patients, 12 achieved CR. Seven patients remained in CR at the time of this report with a median duration of follow-up of 24 months (range 18 to 57 months). CONCLUSION/CONCLUSIONS:This study suggests that IDARAM is an effective regimen in both PCNSL and SCNSL and is suitable for further development and evaluation.

Understanding haematological malignancies at the protein level is important as the development of targeted treatments must be based on knowledge regarding the molecular pathogenesis of the tumour, inherited genetic variation and the mode of action of drugs. 'Proteomics' describes the analysis of the entire proteome of a cell or tissue and incorporates multiple technologies including Western blotting, two-dimensional gel electrophoresis, mass spectrometry, and ProteinChip-based technology. Although there are a limited number of studies to date in haematology those performed highlight the potential future impact of these technologies in the discovery of novel markers, proteins associated with drug resistance and the identification of tumour biomarkers which may facilitate the development of a rapid diagnostic test easily applicable in the clinical setting. Rapid large-scale analysis of the proteome in normal pathways and disease offers the opportunity of identification of potential diagnostic/prognostic markers and proteins associated with the malignant phenotype. This review discusses the current situation regarding the use of these technologies and the potential opportunities their future use may offer in the field of haematology.

We describe the characterization of the genomic DNA breakpoints of two multiple myeloma (MM) patients with t(11;14). IGH translocation events are present in many MM tumors, and it is proposed that they occur early in the pathogenesis, based on the assumption that the translocations are simple reciprocal events mediated by errors in class-switch recombination (CSR). We provide evidence from two patients that the translocation event can be more complex, with DNA from chromosome band 11q13 joined to apparently already recombined hybrid (Smu/Sgamma) switch region sequences. In one case, there was also evidence that a further rearrangement had occurred at the t(11;14) recombination site, resulting in an inversion of 40 bp of the 5'Smu flanking sequence. This suggests that primary IGH arrangements in MM may be more complex than previous myeloma models have suggested, but that they essentially occur through illegitimate CSR events.

In this chapter, we apply the molecular epidemiological paradigm of biomarkers of exposure, early effect and susceptibility to causal models of leukaemia and lymphoma. The aim is to enhance the development of biomarkers for use in studying the causes of these haematopoeitic cancers in the general population. Two causal models of acute myeloid leukaemia are discussed in detail: chemotherapy-induced and benzene-induced acute myeloid leukaemia. Specific chromosomal changes found in acute myeloid leukaemia may serve as useful biomarkers of early effect in these models, and genetic variants in glutathione S-transferases, NQO1 and DNA-repair enzymes may serve as useful biomarkers of susceptibility. Several causal models of lymphoma exist in which biomarkers could be developed and validated. These include human immunodeficiency virus (HIV) immunosuppression, families with inherited disorders and workers exposed to petroleum products, pesticides or organochlorines. Biomarkers of early effect could include markers of DNA double-strand breaks and aberrant V(D)J recombination, and susceptibility may be related to polymorphisms in genes controlling DNA repair and immunological status. We predict that biomarkers of susceptibility will continue to be studied in the case-control format, perhaps in large pooled studies, but that for biomarkers of early effect, there will be a move away from the study of diseased populations to the study of individuals 'at risk' in the causal models described above.

To define specific pathways important in the multistep transformation process of normal plasma cells (PCs) to monoclonal gammopathy of uncertain significance (MGUS) and multiple myeloma (MM), we have applied microarray analysis to PCs from 5 healthy donors (N), 7 patients with MGUS, and 24 patients with newly diagnosed MM. Unsupervised hierarchical clustering using 125 genes with a large variation across all samples defined 2 groups: N and MGUS/MM. Supervised analysis identified 263 genes differentially expressed between N and MGUS and 380 genes differentially expressed between N and MM, 197 of which were also differentially regulated between N and MGUS. Only 74 genes were differentially expressed between MGUS and MM samples, indicating that the differences between MGUS and MM are smaller than those between N and MM or N and MGUS. Differentially expressed genes included oncogenes/tumor-suppressor genes (LAF4, RB1, and disabled homolog 2), cell-signaling genes (RAS family members, B-cell signaling and NF-kappaB genes), DNA-binding and transcription-factor genes (XBP1, zinc finger proteins, forkhead box, and ring finger proteins), and developmental genes (WNT and SHH pathways). Understanding the molecular pathogenesis of MM by gene expression profiling has demonstrated sequential genetic changes from N to malignant PCs and highlighted important pathways involved in the transformation of MGUS to MM.

Autoimmune associated bone disease and intestinal inflammation are closely linked with deregulation and hyperactivation of autoreactive CD4 T cells. How these T cells are activated and mediate disease is not clear. Here we show that in the Interleukin 2-deficient mouse model of autoimmunity spontaneous osteopenia and colitis are caused by increased production of the ligand for receptor activator of NFkappaB (RANKL). RANKL acting via its receptor, receptor activator of NFkappaB (RANK), increases bone turnover and promotes intestinal dendritic cell (DC) survival in vivo. Modulation of RANKL-RANK interactions with exogenous recombinant osteoprotegerin (Fc-OPG) reverses skeletal abnormalities and reduces colitis by decreasing colonic DC numbers. This study identifies a common causal link between bone disease and intestinal inflammation and establishes the importance of DC in mediating colonic inflammation in vivo.

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.