Faculty Bibliography

The c-FMS gene encodes the macrophage colony-stimulating factor receptor (M-CSFR or CSF1-R), which is a tyrosine kinase growth factor receptor essential for macrophage development. We have previously characterized the chromatin features of the mouse gene; however, very little is known about chromatin structure and function of the human c-FMS locus. Here we present a side-by-side comparison of the chromatin structure, histone modification, transcription factor occupancy and cofactor recruitment of the human and the mouse c-FMS loci. We show that, similar to the mouse gene, the human c-FMS gene possesses a promoter and an intronic enhancer element (c-fms intronic regulatory element or FIRE). Both elements are evolutionarily conserved and specifically active in macrophages. However, we demonstrate by in vivo footprinting that both murine and human c-FMS cis-regulatory elements are recognised by an overlapping, but non-identical, set of transcription factors. Despite these differences, chromatin immunoprecipitation experiments show highly similar patterns of histone H3 modification and a similar distribution of chromatin modifying and remodelling activities at individual cis-regulatory elements and across the c-FMS locus. Our experiments support the hypothesis that the same regulatory principles operate at both genes via conserved cores of transcription factor binding sites.

Glutathione S-transferase P1 (GSTP1) is a phase 2 drug metabolism enzyme involved in the metabolism and detoxification of a range of chemotherapeutic agents. A single nucleotide polymorphism (Ile105Val) results in a variant enzyme with lower thermal stability and altered catalytic activity. We hypothesized that patients with the less stable variant have a decreased ability to detoxify chemotherapeutic substrates, including melphalan, and have an altered outcome following treatment for multiple myeloma. We have assessed the impact of GSTP1 codon 105 polymorphisms in 222 patients entered into the Medical Research Council (MRC) myeloma VII trial (comparing standard-dose chemotherapy with high-dose therapy). In the standard-dose arm, patients with the variant allele (105Val) had an improved progression-free survival (PFS) (adjusted hazard ratios for PFS were 0.55 for heterozygotes and 0.52 for 105Val homozygotes, compared with 105Ile homozygotes; P for trend =.04); this was supported by a trend to improved overall survival, greater likelihood of entering plateau and shorter time to reach plateau in patients with the 105Val allele. No difference in outcome by genotype was found for patients treated with high-dose therapy. However, the progression-free survival advantage of the high-dose arm was seen only in patients homozygous for 105Ile (P =.008).

Gastric marginal zone lymphoma (GMZL) is strongly associated with Helicobacter pylori infection, which induces a chronic inflammatory response. Inflammation can result in DNA damage related to its severity, the cellular antioxidant capacity, and the integrity of DNA repair mechanisms. Interleukin-1 (IL-1) polymorphisms have been shown to be important mediators of inflammation, while glutathione S-transferase GST T1 and GST M1 polymorphisms are believed to affect cellular antioxidant capacity. We aimed to determine whether polymorphisms at the IL-1 and GST T1 and GST M1 loci modulate the risk of developing GMZL. Blood and biopsy samples were obtained for a historical series of 66 GMZL cases, whereas blood samples were available from 163 healthy controls. Genotypes were obtained for GST T1, GST M1, IL-1 RN, and IL-1B-31 using PCR-based techniques. H pylori infection was found in 86.0% of cases, whereas in the control population only 37.4% tested positive. The IL-1 RN 2/2 genotype was significantly associated with risk of GMZL (odds ratio [OR], 5.51; 95% confidence interval [CI] 2.16-14.07), but not the IL-1B-31 genotype. Likewise, the GST T1 null genotype was strongly associated with risk of GMZL (OR, 9.51; 95% CI 4.57-19.81), but not the GST M1 genotype. Evidence was found of effect modification between the IL-1 RN and GST T1 genotypes (P =.02). The combination of the IL-1 RN 2/2 and GST T1 null genotype was most strongly associated with risk of GMZL (OR, 32.29; 95% CI 6.92-150-63). These results support the hypothesis that the risk of developing GMZL is influenced by inter-individual variation in the cellular inflammatory immune responses to H pylori infection, and to antioxidative capacity.

The FAS (TNFRSF6/CD95/APO-1) gene is silenced in many tumor types, resulting in an inability to respond to proapoptotic signals. The FAS promoter is polymorphic, including a G to A substitution at -1377 bp and an A to G substitution at -670 bp, which occur within SP1 and signal transducers and activators of transcription 1 transcription factor binding sites, respectively. In a case-control study of adult acute myeloid leukemia (AML), we show a significantly increased risk of AML associated with heterozygotes (GA) and homozygote variants (AA) at position -1377 bp (32.3% in cases versus 22.0% in controls; odds ratio, 1.69; 95% confidence interval, 1.32-2.16). Extended haplotype analysis revealed that the -1377A/-670A haplotype was significantly associated with disease (3% versus 0.5%; odds ratio, 6.72; 95% confidence interval, 3.13-14.51). These data suggest that variation in the FAS gene promoter may affect FAS gene expression and modulate apoptotic signaling, contributing to an increased risk of AML.

PURPOSE/OBJECTIVE:We sought to determine whether the -6 exon 13 T>C polymorphism in the DNA mismatch repair gene hMSH2 modulates susceptibility to acute myeloid leukemia after therapy and particularly after O(6)-guanine alkylating chemotherapy. We also determined the extent of microsatellite instability (MSI) in therapy-related acute myeloid leukemia (t-AML) as a marker of dysfunctional DNA mismatch repair. EXPERIMENTAL DESIGN/METHODS:Using a novel restriction fragment length polymorphism, verified by direct sequencing, we have genotyped 91 t-AML cases, 420 de novo acute myeloid leukemia cases, and 837 controls for the hMSH2 -6 exon 13 polymorphism. MSI was evaluated in presentation bone marrow from 34 cases using the mononucleotide microsatellite markers BAT16, BAT25, and BAT26. RESULTS:Distribution of the hMSH2 -6 exon 13 polymorphism was not significantly different between de novo acute myeloid leukemia cases and controls, with heterozygotes and homozygotes for the variant (C) allele representing 12.2 and 1.6%, respectively, of the control population. However, the variant (C) hMSH2 allele was significantly overrepresented in t-AML cases that had previously been treated with O(6)-guanine alkylating agents, including cyclophosphamide and procarbazine, compared with controls (odds ratio, 4.02; 95% confidence interval, 1.40-11.37). Thirteen of 34 (38%) t-AML cases were MSI positive, and 2 of these 13 cases were homozygous for the variant (C) allele, a frequency substantially higher than in the control population. CONCLUSIONS:Association of the hMSH2 -6 exon 13 variant (C) allele with leukemia after O(6)-guanine alkylating agents implicates this allele in conferring a nondisabling DNA mismatch repair defect with concomitant moderate alkylation tolerance, which predisposes to the development of t-AML via the induction of DNA mismatch repair-disabling mutations and high-grade MSI. Homozygosity for the hMSH2 variant in 2 of 13 MSI-positive t-AML cases provides some support for this model.

The introduction of microarrays offers the opportunity to examine the expression of many thousands of genes in a single experiment. Investigations in leukaemia and lymphoma have led to the identification of a number of subgroups, with a defined gene expression pattern, not previously identified by morphology, cytogenetics or molecular techniques. In many cases these expression patterns can be linked to the tumour cells normal developmental counterpart, and represent distinct disease subgroups with different clinical presentations and outcomes. The technology has also identified genes that may be important in tumour cell biology including key genes in cell proliferation, adhesion, apoptosis, and the development of drug resistance. These early studies demonstrate that genetic microarrays will be useful in classifying haematological malignancies, predicting response to treatment, predicting prognosis, and identifying novel targets for therapy.

Fluorescence in situ hybridization (FISH) has been used to demonstrate the t(14;18) in up to 100% of follicular lymphoma (FL) cases, however, there is little reproducible data using fixed tissue. The aim was therefore to develop a robust FISH method for the demonstration of translocations in archival tissue. The technique was evaluated by comparison with multiplex polymerase chain reaction (PCR), capable of detecting the majority of known breakpoints. Twenty-eight paired frozen and fixed cases of FL and 20 reactive controls were analyzed. The t(14;18) was detected in 23 of 28 cases using PCR on frozen material and 8 of 20 in paraffin. Using FISH, 24 of 26 frozen and 26 of 28 paraffin cases had a demonstrable translocation. All 20 reactive nodes were negative for the t(14;18) by PCR. Using FISH, one of the reactive cases had occasional cells with a translocation FISH pattern, demonstrable in frozen and paraffin samples. This is consistent with the presence of the t(14;18), which is well described in normal individuals. Both PCR and FISH are highly effective for t(14;18) analysis in unfixed tissue. When only paraffin blocks are available, FISH is the method of choice, and a result was achieved in 100% of cases. The method is applicable to the retrospective analysis of a range of translocations.

Our objective was to test the hypothesis that the risk of childhood leukemia is associated with allergies or a family history of allergy. We used a German population-based case-control study with self-reported information on allergies of the children and their first-degree relatives. Our study included a total of 1,130 cases of acute lymphoblastic leukemia (ALL), 164 cases of acute myeloid leukemia (AML) and 2,957 controls. A major finding of our study is that hay fever, neurodermatitis and contact eczema are underrepresented within the group of children with ALL, with respective odds ratios (OR) of 0.45 (95% confidence interval [CI] 0.31-0.66) for hay fever, of 0.49 (CI 0.34-0.71) for neurodermatitis and of 0.62 (CI 0.39-0.99) for eczema, respectively. Atopic diseases, comprising hay fever, neurodermatitis and asthma, are much stronger related with a reduced risk of ALL than other allergies (OR 0.52, CI 0.40-0.67 vs. OR 0.89, CI 0.66-1.21). The strongest association is seen with an atopy in the index child; however, ALL risk is also reduced if one of the parents or a sibling had an atopic disease. No such consistent pattern is seen for AML. Our data suggest that atopy or a family history of atopy are associated with a reduced risk of childhood ALL. Recall bias remains a concern, but sensitivity analysis provided some evidence that the protective effect is unlikely to be attributable to this bias in its entirety.

Although many leukaemia-associated nuclear oncogenes are well characterized, little is known about the molecular details of how they alter gene expression. Here we examined transcription factor complexes and chromatin structure of the human c-FMS gene in normal and leukaemic cells. We demonstrate by in vivo footprinting and chromatin immunoprecipitation assays that this gene is bound by the transcription factor AML1 (RUNX1). In t(8;21) leukaemic cells expressing the aberrant fusion protein AML1-ETO, we demonstrate that this protein is part of a transcription factor complex binding to extended sequences of the c-FMS intronic regulatory region rather than the promoter. The AML1-ETO complex does not disrupt binding of other transcription factors, indicating that c-FMS is not irreversibly epigenetically silenced. However, AML1-ETO binding correlates with changes in the histone modification pattern and increased association of histone deacetylases. Our experiments provide for the first time a direct insight into the chromatin structure of an AML1-ETO-bound target gene.