Kevin Boyd

A compendium of myeloma-associated chromosomal copy number abnormalities and their prognostic value

To obtain a comprehensive genomic profile of presenting multiple myeloma cases we performed high-resolution single nucleotide polymorphism mapping array analysis in 114 samples alongside 258 samples analyzed by U133 Plus 2.0 expression array (Affymetrix). We examined DNA copy number alterations and loss of heterozygosity (LOH) to define the spectrum of minimally deleted regions in which relevant genes of interest can be found. The most frequent deletions are located at 1p (30%), 6q (33%), 8p (25%), 12p (15%), 13q (59%), 14q (39%), 16q (35%), 17p (7%), 20 (12%), and 22 (18%). In addition, copy number-neutral LOH, or uniparental disomy, was also prevalent on 1q (8%), 16q (9%), and X (20%), and was associated with regions of gain and loss. Based on fluorescence in situ hybridization and expression quartile analysis, genes of prognostic importance were found to be located at 1p (FAF1, CDKN2C), 1q (ANP32E), and 17p (TP53). In addition, we identified common homozygously deleted genes that have functions relevant to myeloma biology. Taken together, these analyses indicate that the crucial pathways in myeloma pathogenesis include the nuclear factor-κB pathway, apoptosis, cell-cycle regulation, Wnt signaling, and histone modifications. This study was registered at http://isrctn.org as ISRCTN68454111.

A novel prognostic model in myeloma based on co-segregating adverse FISH lesions and the ISS: analysis of patients treated in the MRC Myeloma IX trial.

The association of genetic lesions detected by fluorescence in situ hybridization (FISH) with survival was analyzed in 1069 patients with newly presenting myeloma treated in the Medical Research Council Myeloma IX trial, with the aim of identifying patients associated with the worst prognosis. A comprehensive FISH panel was performed, and the lesions associated with short progression-free survival and overall survival (OS) in multivariate analysis were +1q21, del(17p13) and an adverse immunoglobulin heavy chain gene (IGH) translocation group incorporating t(4;14), t(14;16) and t(14;20). These lesions frequently co-segregated, and there was an association between the accumulation of these adverse FISH lesions and a progressive impairment of survival. This observation was used to define a series of risk groups based on number of adverse lesions. Taking this approach, we defined a favorable risk group by the absence of adverse genetic lesions, an intermediate group with one adverse lesion and a high-risk group defined by the co-segregation of >1 adverse lesion. This genetic grouping was independent of the International Staging System (ISS) and so was integrated with the ISS to identify an ultra-high-risk group defined by ISS II or III and >1 adverse lesion. This group constituted 13.8% of patients and was associated with a median OS of 19.4 months.

Aberrant global methylation patterns affect the molecular pathogenesis and prognosis of multiple myeloma

We used genome-wide methylation microarrays to analyze differences in CpG methylation patterns in cells relevant to the pathogenesis of myeloma plasma cells (B cells, normal plasma cells, monoclonal gammopathy of undetermined significance [MGUS], presentation myeloma, and plasma cell leukemia). We show that methylation patterns in these cell types are capable of distinguishing nonmalignant from malignant cells and the main reason for this difference is hypomethylation of the genome at the transition from MGUS to presentation myeloma. In addition, gene-specific hypermethylation was evident at the myeloma stage. Differential methylation was also evident at the transition from myeloma to plasma cell leukemia with remethylation of the genome, particularly of genes involved in cell-cell signaling and cell adhesion, which may contribute to independence from the bone marrow microenvironment. There was a high degree of methylation variability within presentation myeloma samples, which was associated with cytogenetic differences between samples. More specifically, we found methylation subgroups were defined by translocations and hyperdiploidy, with t(4;14) myeloma having the greatest impact on DNA methylation. Two groups of hyperdiploid samples were identified, on the basis of unsupervised clustering, which had an impact on overall survival. Overall, DNA methylation changes significantly during disease progression and between cytogenetic subgroups.

Homozygous Deletion Mapping in Myeloma Samples Identifies Genes and an Expression Signature Relevant to Pathogenesis and Outcome

Purpose: Myeloma is a clonal malignancy of plasma cells. Poor-prognosis risk is currently identified by clinical and cytogenetic features. However, these indicators do not capture all prognostic information. Gene expression analysis can be used to identify poor-prognosis patients and this can be improved by combination with information about DNA-level changes. Experimental Design: Using single nucleotide polymorphism–based gene mapping in combination with global gene expression analysis, we have identified homozygous deletions in genes and networks that are relevant to myeloma pathogenesis and outcome. Results: We identified 170 genes with homozygous deletions and corresponding loss of expression. Deletion within the “cell death” network was overrepresented and cases with these deletions had impaired overall survival. From further analysis of these events, we have generated an expression-based signature associated with shorter survival in 258 patients and confirmed this signature in data from two independent groups totaling 800 patients. We defined a gene expression signature of 97 cell death genes that reflects prognosis and confirmed this in two independent data sets. Conclusions: We developed a simple 6-gene expression signature from the 97-gene signature that can be used to identify poor-prognosis myeloma in the clinical environment. This signature could form the basis of future trials aimed at improving the outcome of poor-prognosis myeloma. Clin Cancer Res; 16(6); 1856–64

Mapping of Chromosome 1p Deletions in Myeloma Identifies FAM46C at 1p12 and CDKN2C at 1p32.3 as Being Genes in Regions Associated with Adverse Survival

Purpose: Regions on 1p with recurrent deletions in presenting myeloma patients were examined with the purpose of defining the deletions and assessing their survival impact. Experimental Design: Gene mapping, gene expression, FISH, and mutation analyses were conducted on patient samples from the MRC Myeloma IX trial and correlated with clinical outcome data. Results: 1p32.3 was deleted in 11% of cases, and deletion was strongly associated with impaired overall survival (OS) in patients treated with autologous stem cell transplant (ASCT). In patients treated less intensively, del(1)(p32.3) was not associated with adverse progression-free survival (PFS) or OS. The target of homozygous deletions was CDKN2C, however its role in the adverse outcome of cases with hemizygous deletion was less certain. 1p22.1-21.2 was the most frequently deleted region and contained the candidate genes MTF2 and TMED5. No mutations were identified in these genes. 1p12 was deleted in 19% of cases, and deletion was associated with impaired OS in univariate analysis. The target of homozygous deletion was FAM46C, which was mutated in 3.4% of cases. When cases with FAM46C deletion or mutation were considered together, they were strongly associated with impaired OS in the intensive treatment setting. Conclusion: Deletion of 1p32.3 and 1p12 was associated with impaired OS in myeloma patients receiving ASCT. FAM46C was identified as a gene with potential pathogenic and prognostic significance based on the occurrence of recurrent homozygous deletions and mutations. Clin Cancer Res; 17(24); 7776–84. ©2011 AACR.

XBP1s levels are implicated in the biology and outcome of myeloma mediating different clinical outcomes to thalidomide-based treatments

Immunoglobulin production by myeloma plasma cells depends on the unfolded protein response for protein production and folding. Recent studies have highlighted the importance of IRE1alpha and X box binding protein 1 (XBP1), key members of this pathway, in normal B-plasma cell development. We have determined the gene expression levels of IRE1alpha, XBP1, XBP1UNSPLICED (XBP1u), and XBP1SPLICED (XBP1s) in a series of patients with myeloma and correlated findings with clinical outcome. We show that IRE1alpha and XBP1 are highly expressed and that patients with low XBP1s/u ratios have a significantly better overall survival. XBP1s is an independent prognostic marker and can be used with beta2 microglobulin and t(4;14) to identify a group of patients with a poor outcome. Furthermore, we show the beneficial therapeutic effects of thalidomide in patients with low XBP1s/u ratios. This study highlights the importance of XBP1 in myeloma and its significance as an independent prognostic marker and as a predictor of thalidomide response.

The impact of extramedullary disease at presentation on the outcome of myeloma

This study was conducted to compare the presenting features and outcome of newly-diagnosed myeloma with and without extramedullary (EM) manifestations and to determine the optimum treatment. Seventy-five (16.3%) patients with EM involvement at diagnosis were compared with 384 cases without EM disease. EM patients had a more favourable International Staging System and a different distribution of myeloma isotypes. When adjusted according to the independent risk factors, patients in the EM group treated with chemotherapy alone had significantly shorter overall survival (OS) compared to those without EM disease receiving similar treatment. High-dose treatment (HDT) was associated with significantly improved OS in both groups; however, it had more impact on OS among EM group, overcoming the negative prognostic impact of presenting EM disease. Patients in the EM group treated with HDT have a similar outcome to those without EM manifestations treated with HDT. HDT should form an integral component of first-line treatment for patients with EM disease whenever possible.

The addition of cyclophosphamide to lenalidomide and dexamethasone in multiply relapsed/refractory myeloma patients; a phase I/II study

We report the results of a Phase I/II dose escalation study to determine the maximum tolerated dose (MTD) of cyclophosphamide when combined with lenalidomide and dexamethasone in relapsed/refractory myeloma. Thirty‐one patients were enrolled in cohorts of 3, at five dose levels of cyclophosphamide to a maximum of 700 mg on days 1 and 8 of a 28‐d cycle. Patients received lenalidomide 25 mg days 1–21 and dexamethasone 20 mg orally days 1–4 and 8–11. The MTD was 600 mg cyclophosphamide, days 1 and 8. Grade 3/4 haematological complications occurred in 26% of patients, grade 3/4 infection in 3% (both at 700 mg cyclophosphamide), with thromboembolic complications in 6% of patients. Overall complete response (CR) rate was 29%, very good partial response rate 7% and partial response rate 45% giving an overall response rate of 81%. After 21 months median follow‐up, projected 2‐year progression‐free survival was 56%, with 80% overall survival at 30 months. Ten further patients were treated at MTD with a 40% CR rate. No dose reductions for any study drugs or deaths occurred during cycles 1–9. Lenalidomide, cyclophosphamide and dexamethasone is a safe, effective combination in relapsed myeloma inducing a high response rate, warranting further investigation in phase III trials.

The potential role of epigenetic therapy in multiple myeloma.

This review describes the role that epigenetic changes play in the pathogenesis of cancer, concentrating on the plasma cell malignancy multiple myeloma, and highlights recent findings regarding the efficacy of epigenetic therapeutic agents in laboratory studies and clinical trials. DNA methylation is altered in a wide range of cancers with hypermethylation of CpG islands associated with silencing of tumour suppressor genes. Genes found to be silenced by methylation in myeloma samples include VHL, TP53, CDKN2A, and TGFBR2. Myeloma is linked to the overexpression of a histone methylatransferase (MMSET) and inactivating mutations of a histone demethylase (UTX), suggesting that the regulation of histone methylation is a potential therapeutic target. Abnormal expression of histone deacetylases (HDACs) has been widely described in solid tumours and haematological malignancies. In myeloma, histone deacetylase inhibitors show promising results both in laboratory‐based cell culture studies and in clinical trials, where they demonstrate particularly good therapeutic outcome when administered in combination with other standard chemotherapeutic agents. The study of epigenetics shows great promise for understanding the alterations in gene expression that underlie malignancies and provides exciting novel drugable targets.

The Clinical Impact and Molecular Biology of del(17p) in Multiple Myeloma Treated with Conventional or Thalidomide-Based Therapy

Hemizygous deletion of 17p (del(17p)) has been identified as a variable associated with poor prognosis in myeloma, although its impact in the context of thalidomide therapy is not well described. The clinical outcome of 85 myeloma patients with del(17p) treated in a clinical trial incorporating both conventional and thalidomide‐based induction therapies was examined. The clinical impact of deletion, low expression, and mutation of TP53 was also determined. Patients with del(17p) did not have inferior response rates compared to patients without del(17p), but, despite this, del(17p) was associated with impaired overall survival (OS) (median OS 26.6 vs. 48.5 months, P < 0.001). Within the del(17p) group, thalidomide induction therapy was associated with improved response rates compared to conventional therapy, but there was no impact on OS. Thalidomide maintenance was associated with impaired OS, although our analysis suggests that this effect may have been due to confounding variables. A minimally deleted region on 17p13.1 involving 17 genes was identified, of which only TP53 and SAT2 were underexpressed. TP53 was mutated in <1% in patients without del(17p) and in 27% of patients with del(17p). The higher TP53 mutation rate in samples with del(17p) suggests a role for TP53 in these clinical outcomes. In conclusion, del(17p) defined a patient group associated with short survival in myeloma, and although thalidomide induction therapy was associated with improved response rates, it did not impact OS, suggesting that alternative therapeutic strategies are required for this group.