Faculty Bibliography

The t(4;14) translocation is found in approximately 10% of myeloma patients and results in the deregulation of at least two genes, MMSET and fibroblast growth factor receptor 3 (FGFR3), with the formation of a fusion product between MMSET and the immunoglobulin heavy chain (IgH) locus and overexpression of FGFR3. We have analysed a series of 80 patient samples, comprising 67 multiple myeloma (MM) cases and 13 monoclonalgammopathy of undetermined significance (MGUS) cases, using RT-PCR to detect IgH-MMSET fusions. The t(4;14) translocation was detected in 7/67 (10%) myeloma cases and all seven expressed FGFR3 which was not seen in t(4;14)-negative myeloma cases. In the MGUS cases, a similar proportion of t(4;14)-positive cases was found (2/13; 15%), but none of these expressed FGFR3. All patients with detectable FGFR3 expressed both the FGFR3 IIIb and FGFR3 IIIc isoforms, the result of alternative splicing in the ligand binding domain, and exon-deleted variants of FGFR3. We also identified a cryptic splice site in MMSET which results in a 277 amino acid deletion downstream of the breakpoint on der(4). FGFR3 mutation analysis revealed no mutations in the presenting myeloma or MGUS samples. However, we also had access to paired presentation and relapse samples which had been taken from a patient 13 months apart. Both samples had the t(4;14) translocation and overexpressed FGFR3, but only the relapse sample possessed the K650E mutation in the kinase domain of FGFR3. This suggests that targeted mutation in the translocated FGFR3 gene when under the control of the immunoglobulin promoters can occur and may provide one mechanism for disease progression.

Molecular and cellular markers associated with malignant disease are frequently identified in healthy individuals. The relationship between these markers and clinical disease is not clear, except where a neoplastic cell population can be identified as in myeloma/monoclonal gammopathies of undetermined significance (MGUS). We have used the distinctive phenotype of chronic lymphocytic leukemia (CLL) cells to determine whether low levels of these cells can be identified in individuals with normal complete blood counts. CLL cells were identified by 4-color flow cytometric analysis of CD19/CD5/CD79b/CD20 expression in 910 outpatients over 40 years old. These outpatients were age- and sex-matched to the general population with normal hematologic parameters and no evident history of malignant disease. CLL phenotype cells were detectable in 3.5% of individuals at low level (median, 0.013; range, 0.002- 1.458 x 10(9) cells/L), and represented a minority of B lymphocytes (median, 11%; range, 3%-95%). Monoclonality was demonstrated by immunoglobulin light-chain restriction in all cases with CLL phenotype cells present and confirmed in a subset of cases by consensus-primer IgH-polymerase chain reaction. As in clinical disease, CLL phenotype cells were detected with a higher frequency in men (male-to-female ratio, 1.9:1) and elderly individuals (2.1% of 40- to 59-year-olds versus 5.0% of 60- to 89-year-olds, P =.01). The neoplastic cells were identical to good-prognosis CLL, being CD5+23+20(wk)79b(wk)11a(-)22(wk)sIg(wk)CD38-, and where assessed had a high degree (4.8%-6.6%) of IgH somatic hypermutation. The monoclonal CLL phenotype cells present in otherwise healthy individuals may represent a very early stage of indolent CLL and should be useful in elucidating the mechanisms of leukemogenesis.

Recently there have been substantial improvements in our understanding of the biology of myeloma. These findings have important implications for aetiological studies aimed at defining the causative factors for myeloma. Myeloma is closely related to monoclonal gammopathy of unknown significance (MGUS), which is now recognized to be very common in the older population. The epidemiology of these conditions is presented and discussed in the context of the genetic factors governing both the risk of developing MGUS or of transformation to myeloma. Biological studies support a role for aberrant class switch recombination early in the natural history of myeloma suggesting that factors in the environment may interact with this mechanism to increase myeloma risk. Case-control and cohort studies have identified several known and suspected environmental exposures. These exposures include high doses of ionizing radiation, and occupational exposure in the farming and petrochemical industries. The data supporting these associations are presented and discussed in the context of the molecular mechanisms underlying these exposures. In particular DNA damage occurring as a consequence could readily interact with the class switch recombination process to increase the risk of chromosomal translocations, oncogene deregulation and malignant transformation. A further hypothesis, which has been extensively investigated, is the role of chronic immune/antigenic stimulation and the risk of myeloma. This concept is difficult to explain in the context of our current immunological concepts. The data supporting the association and how molecular epidemiological studies using genetic variants in cytokine genes are allowing us to revisit this concept are discussed in detail.

An autoimmune mechanism in the pathogenesis of myelodysplastic syndrome (MDS) is suggested by response to immunosuppression, with CD8+ T-lymphocytes implicated in the haematopoietic suppression. We therefore sought evidence for human leucocyte antigen (HLA) restriction and variant frequency differences in selected polymorphisms at the loci for the immunomodulatory cytokines, tumour necrosis factor alpha (TNF-alpha), lymphotoxin-alpha (LT-alpha) and interleukin 10 (IL-10) in patients with MDS and acute myeloid leukaemia (AML) compared with normal controls. DNA from 150 MDS/AML patients [24 AML, 53 refractory anaemia (RA), 25 RA with excess blasts (RAEB), four RAEB in transformation (RAEBt), 21 sideroblastic leukaemia, 22 chronic myelomonocytic leukaemia] was screened. Control data was from Scottish blood donors (HLA class I/II), healthy General Practitioner-based subjects (TNF-alpha/LT-alpha) and published values (IL-10). HLA class I/II haplotypes were determined using sequence-specific primers. Polymorphisms were assayed at TNF-alpha -308, LT-alpha +252 and IL10 -824, -597 and -1082 loci. Variant frequencies of common haplotypes at HLA class I and II, high-/low-producer TNF-alpha/LT-alpha and IL-10 loci were not different between patients and controls or within the French-American-British, International Prognostic Scoring System or cytogenetic subgroups and were not associated with altered survival for MDS/AML patients. TNF2 allele frequency was greater in the MDS/AML cohort (chi2 = 6.593, P < 0.05) but the biological significance was uncertain in the absence of an increased high-producer TNF-alpha/LT-alpha haplotype frequency. We can find no genetic influence for these polymorphisms in HLA class I/II, TNF-alpha/LT-alpha and IL-10 loci on either predisposition or disease progression in MDS/AML.

Translocations at the immunoglobulin heavy chain locus (14q32) are now considered the commonest karyotypic change in multiple myeloma. These translocations are thought to be intimately involved in the pathogenesis of this disease. The heavy chain locus is strongly transcriptionally active in B and plasma cells and transfer of a potential oncogene to 14q32 would result in its dysregulation. Molecular characterization suggests that the majority of these breakpoints cluster in switch regions within the heavy chain locus. Switch regions are normally involved in the regulated process of isotype switching so that in myeloma the rearrangements are believed to be a result of so-called illegitimate (aberrant) switch recombination and are likely to be an early event in myeloma development. A legitimate switch recombination event occurs between two switch regions producing a hybrid switch; this is necessary for class switching to proceed on a productive allele. In this review we describe the process of isotype switching and how illegitimate class switching may be related to the pathogenesis of multiple myeloma.

We previously reported that 2 polymorphisms in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene at positions C677T and A1298C were associated with lower risk of adult acute lymphocytic leukemia (ALL). In the present study, we have examined whether polymorphisms in other folate-metabolizing genes play a role in ALL susceptibility. Polymorphisms in methionine synthase (MS A2756G), cytosolic serine hydroxymethyltransferase (SHMT1 C1420T), and a double (2R2R) or triple (3R3R) 28-bp tandem repeat in the promoter region of thymidylate synthase (TS) were studied and found to modulate ALL risk. In a univariate analysis, SHMT1 1420CT individuals exhibited a 2.1-fold decrease in ALL risk (odds ratio [OR] = 0.48; 95% confidence interval [CI], 0.25-0.91), whereas the 1420TT genotype conferred a 3.3-fold reduction in risk (OR = 0.31; 95% CI, 0.10-0.90). Similarly, TS 2R3R individuals exhibited a 2.8-fold reduction in ALL risk (OR = 0.36; 95% CI: 0.16-0.83), while the TS 3R3R genotype conferred an even greater level of protection (OR = 0.25; 95% CI, 0.08-0.78). However, no significant associations were evident for the MS 2756AG polymorphism (OR = 0.79; 95% CI, 0.38-1.7). In addition, potential interactions between the SHMT1 and TS or MS genes were observed. TS 3R3R individuals who were SHMT1 1420CT/TT had a 13.9-fold decreased ALL risk (OR = 0.072; 95% CI, 0.0067-0.77). Further, MS 2756AG individuals who were SHMT1 1420CT/TT had a 5.6-fold reduction in ALL risk (OR = 0.18; 95% CI, 0.05-0.63). This study suggests an important role for uracil misincorporation and resultant chromosomal damage in the pathogenesis of ALL, and that genetic interactions involving low penetrance polymorphisms in folate-metabolizing genes may increase ALL risk.

The AML1 gene encodes a transcription factor that, together with its heterodimeric partner CBFB, regulates a number of target genes that are essential for normal hemopoiesis. In acute myeloid leukemia (AML), AML1 is disrupted not only by chromosomal translocations but also by mutations in the runt domain, which binds both DNA and CBFB. Acquired mutations have been described predominantly in the AML FAB type M0. To date, most patients appear to have biallelic disease, suggesting a complete lack of normal AML1 function. Inherited loss of function mutations thought to lead to haploinsufficiency also have been described in patients who have a familial disorder with predisposition to AML (FPD/AML), indicating the role of AML1 in megakaryopoiesis. Using single-strand conformation polymorphism analysis, we studied the AML1 runt domain in 41 patients with M0 AML and identified potentially pathologic mutations in five (12%). Biallelic disease could be confirmed in only one patient, using loss of heterozygosity studies. At least three of the mutations would lead to truncated proteins similar to those reported in FPD/AML, suggesting that haploinsufficiency plays a role in the pathogenesis of this minimally differentiated type of leukemia. The incidence of acquired mutations in AML patients with acute megakaryoblastic leukemia (FAB type M7) was the same as that reported in other non-M0 patients, with only one mutation detected in 20 (5%) patients studied.

Diffuse large B-cell lymphomas (DLBCL) are a heterogeneous group of tumours, varying in clinical features, immunophenotype and cytogenetics. The aim of this study was to investigate the prognostic significance of BCL6 gene rearrangement at the 3q27 locus in patients with primary nodal disease, and to examine interrelationships with immunophenotype and International Prognostic Index (IPI). We have developed a fluorescent in situ hybridization (FISH)-based technique for the retrospective analysis of the effect of BCL6 gene rearrangements on survival, using nuclei extracted from paraffin-embedded tissue. FISH results were obtained in 111 presentation cases of nodal DLBCL. The IPI was calculated and each case was stained immunocytochemically for BCL6, BCL2 and CD10. 3q27 rearrangements were detected in 25% of cases. BCL2 protein and a germinal centre (GC) phenotype (defined as CD10+, BCL6+) were expressed in 56% and 41% of cases respectively. In multivariate analysis, rearrangement of 3q27 and BCL2 expression and the absence of a GC phenotype were associated with a poor prognosis. These factors can be used in conjunction with the IPI to improve risk stratification in nodal DLBCL.

It is generally considered that most cancers arise following the accumulation of several genetic events and that as a consequence its incidence increases with age. We report a cytogenetic subgroup of acute myeloid leukaemia whose incidence is independent of age. This observation indicates that acute myeloid leukaemia can develop via multiple pathways, and underlines the importance of cytogenetics in understanding this disease.