Faculty Bibliography

Osteolytic bone disease is a major cause of morbidity in patients with multiple myeloma. Our understanding of the pathophysiology of multiple myeloma has increased substantially during the past decade. However the underlying mechanisms of bone destruction and the treatments available have, until recently, received relatively little specific attention. In this review, we provide an overview of the RANK/RANKL/osteoprotegerin system; we describe its interaction with other cellular mechanisms, through which malignant plasma cells drive osteolysis, and explain how bisphosphonates can be used to block this action. We also review the supporting evidence for bisphosphonates as the treatment of choice for patients with bone complications related to multiple myeloma, and discuss possible developments for targeted therapy in the future.

BACKGROUND:High-dose therapy with supporting autologous stem-cell transplantation remains a controversial treatment for cancer. In multiple myeloma, first-line regimens incorporating high-dose therapy yield higher remission rates than do conventional-dose treatments, but evidence that this translates into improved survival is limited. METHODS:In this multicenter study, the Medical Research Council Myeloma VII Trial, we randomly assigned 407 patients with previously untreated multiple myeloma who were younger than 65 years of age to receive either standard conventional-dose combination chemotherapy or high-dose therapy and an autologous stem-cell transplant. RESULTS:Among the 401 patients who could be evaluated, the rates of complete response were higher in the intensive-therapy group than in the standard-therapy group (44 percent vs. 8 percent, P<0.001). The rates of partial response were similar (42 percent and 40 percent, respectively; P=0.72), and the rates of minimal response were lower in the intensive-therapy group than in the standard-therapy group (3 percent vs. 18 percent, P<0.001). Intention-to-treat analysis showed a higher rate of overall survival (P=0.04 by the log-rank test) and progression-free survival (P<0.001) in the intensive-therapy group than in the standard-therapy group. As compared with standard therapy, intensive treatment increased median survival by almost 1 year (54.1 months [95 percent confidence interval, 44.9 to 65.2] vs. 42.3 months [95 percent confidence interval, 33.1 to 51.6]). There was a trend toward a greater survival benefit in the group of patients with a poor prognosis, as defined by a high beta2-microglobulin level (more than 8 mg per liter). CONCLUSIONS:High-dose therapy with autologous stem-cell rescue is an effective first-line treatment for patients with multiple myeloma who are younger than 65 years of age.

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.

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).

The t(14;18) is present in a significant proportion of diffuse large B-cell lymphomas (DLBCLs), however, the prognostic effect of the translocation and the relationship with transformed follicular lymphoma remains controversial. To clarify these uncertainties, interphase fluorescence in situ hybridization (FISH) was used to determine the incidence of the t(14;18) in nodal DLBCL, and this was correlated with BCL2 expression, germinal center (GC) immunophenotype, and patient outcome. FISH was performed on paraffin-extracted nuclei from 137 de novo nodal DLBCLs. Eighteen of 137 de novo DLBCLs were t(14;18) positive. The t(14;18) was most commonly associated with the subset of DLBCLs that expressed a GC phenotype, defined as CD10+, BCL6+ (GC-type DLBCL): positive in 14 of 47 (30%) cases, compared with 4 of 89 (5%) in the non-GC group (Pearson's chi(2) = 28.4; P < 0.0001). All cases with a translocation expressed BCL2 protein, however, 40 expressed BCL2 protein without a t(14;18). GC-type DLBCL patients with a t(14;18) had a significantly worse survival compared with GC-type DLBCL patients without the translocation (2-year survivals were 29 and 63%, respectively; P = 0.006). Of the cases without the translocation, BCL2 protein expression did not affect survival. In contrast, in the non-GC group of DLBCLs, BCL2 protein expression reduced the 2-year overall survival from 64% in the BCL2-negative group to 38%, with a median survival of 15.0 months (P = 0.02). In conclusion, the t(14;18) is common in DLBCLs, particularly in GC-type DLBCLs, where the presence of the translocation has a poor prognostic effect. BCL2 protein expression defines a group of non-GC DLBCL patients with a poor prognosis.

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.

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.

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.