Fonseca, Rafaël

Abstract The plasma cell malignancy Multiple Myeloma (MM) is a rare but deadly form of cancer with 5 year survival rates of ∼30%. The disease exacts an especially heavy burden on African American (AA) patients. In fact, African Americans are twice a likely to be diagnosed with and die from MM as compared to Caucasian Americans. Although socio-economic factors likely influence this disparity, the role of biology has not been ruled out. We have profiled ∼250 MM tumors for somatic copy number alterations using array-based Comparative Genomic Hybridization (aCGH) and Gene Expression Profiling as part of the Multiple Myeloma Genomics Initiative. To determine if somatic alterations may play a role in the disparity in incidence and/or severity of MM in AA, we compared the frequency of specific chromosomal aberrations in genes and genomic regions previously found to be involved in progression and poor outcome in MM among a cohort of one hundred ninety-six (196) patients including 16 African Americans and 180 Caucasians. The frequency of occurrence (expressed as a percentage) was determined for each of the following aberrations: 1p deletion, homozygous deletion of p18, chromosome 13 deletion, and chromosome 13 monosomy. No significant difference was detected in the frequency of chromosome 13 deletion and chromosome 13 monosomy between the two groups. However, significant differences were observed for chromosome 1p alterations. Homozygous deletion at the p18 locus at 1p occurred in 12.5% (2/16) of tumors from AA patients and 2.8% (5/180) of CA patient tumors (p = 0.045). We further stratified tumors by hyperdiploid or non-hyperdiploid status using a-CGH profiles to determine ploidy status. Most significantly, p18 homozygous deletion was found in 25% (2/8) of AA patients with non-hyperdiploid tumors and in only 1.2% (1/83) of CA patients with non-hyperdiploid tumors (p=3.2 × 10-4). A nominally significant difference among non-hyperdiploid patients was also present for overall 1p deletion, which was observed in 2/8 (25%) of AA non-hyperdiploid tumors and 33/83 (40%) of CA with non-hyperdiploid tumors (p=0.05). No differences were observed for 1p deletion or p18 homozygous deletion among hyperdiploid tumors between AA and CA patients. These results suggest that deletions at 1p are more common among African American MM patients. Furthermore, homozygous deletion encompassing the p18 locus is more significantly common among AA MM patients, especially AA patients with non-hyperdiploid tumors. As 1p deletion and p18 homozygous deletion have been linked to poor outcome, our results suggest that the increased frequency of these alterations in tumors from AA patients may in part explain the disparity in unfavorable outcomes seen among AA MM patients Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4822.

Abstract Microarray technologies are powerful assays, which can identify copy-number abnormalities (CNA) underlying the pathogenesis of cancer. Several commercial arrays are available but it is unclear which is the best platform. We did a side-by-side comparison between two leading arrays; the Affymetrix SNP6 and Agilent 1M, to compare data compression, noise, gene and exon coverage, and CNA detection. Five samples were run on both platforms and analyzed as recommended by the manufacturer. We first performed an in silico comparison to define the number of probes per serial 5k interval, number of probes per gene and exon, and the coefficient of variation of different smoothing windows. Comparing the CV of the two arrays showed a similar level of variation between a 3 and 5 probe averaging on the 1M and SNP6, respectively. Using these smoothing levels the 1M interrogates 81.2% of the genes in the genome compared to 69.3% on the SNP6. At the exon level the 1M has at least one probe in 64.8% compared to 41.2% on the SNP6. We observed a similar level of data compression (∼10%) on the two arrays but the SNP6 had nearly twice the noise (SD 0.38 versus 0.21). Finally, there was a marked increment in the number of CNA identified by 1M (mean of 78.5) than SNP6.0 (59.5). In this comparison the Agilent 1M out performed the Affymetrix SNP6. Even with half the number of probes, the 1M was superior in CNA detection, gene/exon coverage and showed less signal variation. A comparison with previous platforms showed that these two arrays did not identify new gene amplifications but did detect new deletions, however, neither platform detected several known deletions detected by other methods. Therefore, we designed a custom, exon-centered, 2×400K array (Agilent) with the goal of identifying additional deletions. The design outline was: a) all the probes from the Agilent CGH 44K array; b) 3 probes per exon across the human exome; c) high resolution tiling of 1450 cancer implicated genes; and d) tiling the 4kb flanking all MIRs. To improve performance, we only included probes with performance scores higher than 0.8 and 0.85 in queries b) and c), respectively. Lastly, 15000 probes from the 1M array that map to exons missed by our design parameters were also included. Our custom array has half the probes (415K) of the 1M (960K) and 4.5x less than the SNP6 (1.85M) but shows the best gene and exon coverage. When the minimum number of probes needed for calculating a CNA is considered, 86% of genes are interrogated compared to 81.2% (1M) and 69.3% (SNP6). The difference is even more marked at the exon level with 84% covered by at least one probe and at least 63.2% interrogated by two probes compared to 5.3% (1M) and 18.0% (SNP6). The promising in silico comparison of this exon-centered array, its more stringent design parameters in combination with significantly reducing the cost per run should make this a very promising array design for CNA analysis of tumor samples in the future Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4957.

AbstractA panel of members of the 2009 International Myeloma Workshop developed guidelines for risk stratification in multiple myeloma. The purpose of risk stratification is not to decide time of therapy but to prognosticate. There is general consensus that risk stratification is applicable to newly diagnosed patients; however, some genetic abnormalities characteristic of poor outcome at diagnosis may suggest poor outcome if only detected at the time of relapse. Thus, in good-risk patients, it is necessary to evaluate for high-risk features at relapse. Although detection of any cytogenetic abnormality is considered to suggest higher-risk disease, the specific abnormalities considered as poor risk are cytogenetically detected chromosomal 13 or 13q deletion, t(4;14) and del17p, and detection by fluorescence in situ hybridization of t(4;14), t(14;16), and del17p. Detection of 13q deletion by fluorescence in situ hybridization only, in absence of other abnormalities, is not considered a high-risk feature. High serum β2-microglobulin level and International Staging System stages II and III, incorporating high β2-microglobulin and low albumin, are considered to predict higher risk disease. There was a consensus that the high-risk features will change in the future, with introduction of other new agents or possibly new combinations.

Abstract Multiple myeloma (MM) is a cancer of the plasma cells in the bone marrow. Although rare, MM is a deadly form of cancer with a 5 year survival rate of only 37%. African Americans (AA) are twice as likely to be diagnosed with and die from MM as compared to European Americans (EA). Socioeconomic factors may contribute to this disparity, however, biological factors may also be an influence. We have identified somatic copy number alterations in MM as a part of the Multiple Myeloma Genomics Initiative by profiling 250 MM tumors using array-based comparative genomic hybridization (aCGH). Using this cohort, we identified 16 African American and 180 European American patients using a MMRC database that indicated the racial demographics of each patient. To determine if somatic alterations may play a role in the disparity in incidence and/or severity of MM in AA, we compared the frequency of specific chromosomal aberrations in genes and genomic regions found to be involved in progression and poor outcome. The number of events of occurring was calculated and a univariate analysis was conducted using the Fisher's exact test. P-values were used to determine statistical significance. Results revealed significant differences for chromosome 1 p alterations. Specifically, homozygous deletion at the p18 locus at 1 p occurred in 12.5% (2/16) of tumors from AA patients and 2.8% (5/180) of EA patient tumors (p = 0.045). Further stratification of tumors by hyperdiploid or non-hyperdiploid status revealed significant differences in p18 homozygous deletion. Homozygous deletion of p18 was found in 25% (2/8) of AA patients and only 1.2% (1/83) of EA patients with non-hyperdiploid tumors (p=3.2 × 10-4). A nominally significant difference among non-hyperdiploid patients was also present for overall 1 p deletion, which was observed in 2/8 (25%) of AA non-hyperdiploid tumors and 33/83 (40%) of EA with non-hyperdiploid tumors (p=0.05). No differences were observed for 1 p deletion or p18 homozygous deletion among hyperdiploid tumors between AA and EA patients. We then combined our AA dataset with an independent dataset of 8 AA MM patients generated at the Mayo Clinic, Scottsdale AZ and repeated the analysis. Interestingly, very little or no aberrations were found in the dataset from the Mayo Clinic, however, differences of p18 homozygous deletion and 16q deletion among AA and EA remained statistically significant. To determine the correlation between hemizygous and biallelic loss of p18 and poor prognosis we used the 70 gene signature (Shaughnessy, et al., 2007) as a surrogate to identify patients with poor prognosis within the Multiple Myeloma Genomics Initiative dataset. Genomic identification of significant targets in cancer (GISTIC) was used (Beroukhim et al., 2007) to identify regions of aberrant copy number that were over-represented among samples. A relationship between poor prognosis and biallelic loss was found to be statistically significant (p = 0002) while samples with hemizygous loss showed no relationship (p = 0.57). Although the size of the current AA cohort is small, these results suggest that homozygous deletions at the p18 locus may be more common among African American MM patients with non-hyperdiploid tumors which may help to explain the disparity in AA patients with MM. Array CGH is currently being conducted on an additional 40 tumors from AA MM patients for a more robust analysis. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):B64.

Abstract Multiple myeloma (MM) is a plasma cell malignancy of the bone marrow, which evolves from a premalignant stage called monoclonal gammopathy of undetermined significance (MGUS). In some patients, an intermediate stage referred to as smoldering multiple myeloma (SMM) is clinically recognized, with the full-bore malignancy termed MM. We conducted a study to assess differential CpG methylation at 1,500 genic loci during MM progression and profiled CD138+ plasma cells from MGUS, SMM, and MM specimens; human myeloma cell lines; and normal plasma cell (NPC) samples. We showed that the number of differentially methylated loci (DML) increased with tumor grade, and the vast majority were due to hypomethylation. Hierarchical clustering analysis revealed samples that coclustered tightly with NPC. These cases, referred to as “normal-like,” contained significantly fewer DML when compared with their non–normal-like counterparts and displayed overall methylation levels resembling NPC. This study represents one of the first methylome interrogation studies in MM and points toward global hypomethylation at genic CpG loci as an important and early mechanism driving myelomagenesis. Determining the set of critical genes and pathways based on the myeloma methylome is expected to lead to an improved understanding of biological mechanisms involved in myelomagenesis. Cancer Res; 70(17); 6934–44. ©2010 AACR.

AbstractThe objective of this case-control study was to compare the efficacy and toxicity of lenalidomide plus dexamethasone (len/dex) versus thalidomide plus dexamethasone (thal/dex) as initial therapy for newly diagnosed myeloma. We retrospectively studied 411 newly diagnosed patients treated with len/dex (228) or thal/dex (183) at the Mayo Clinic. The differences were similar in a matched-pair analysis that adjusted for age, sex, transplantation status, and dexamethasone dose. The proportions of patients achieving at least a partial response to len/dex and thal/dex were 80.3% versus 61.2%, respectively (P < .001); very good partial response rates were 34.2% and 12.0%, respectively (P < .001). Patients receiving len/dex had longer time to progression (median, 27.4 vs 17.2 months; P = .019), progression-free survival (median, 26.7 vs 17.1 months; P = .036), and overall survival (median not reached vs 57.2 months; P = .018). A similar proportion of patients in the 2 groups experienced at least one grade 3 or 4 adverse event (57.5% vs 54.6%, P = .568). Main grade 3 or 4 toxicities of len/dex were hematologic, mainly neutropenia (14.6% vs 0.6%, P < .001); the most common toxicities in thal/dex were venous thromboembolism (15.3% vs 9.2%, P = .058) and peripheral neuropathy (10.4% vs 0.9%, P < .001). Len/dex appears well-tolerated and more effective than thal/dex. Randomized trials are needed to confirm these results.

AbstractA paucity of validated kinase targets in human multiple myeloma has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in myeloma tumor lines bearing common t(4;14), t(14;16), and t(11;14) translocations to identify critically vulnerable kinases in myeloma tumor cells without regard to preconceived mechanistic notions. Fifteen kinases were repeatedly vulnerable in myeloma cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. Whereas several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly G protein–coupled receptor kinase, GRK6, appeared selectively vulnerable in myeloma. GRK6 inhibition was lethal to 6 of 7 myeloma tumor lines but was tolerated in 7 of 7 human cell lines. GRK6 exhibits lymphoid-restricted expression, and from coimmunoprecipitation studies we demonstrate that expression in myeloma cells is regulated via direct association with the heat shock protein 90 (HSP90) chaperone. GRK6 silencing causes suppression of signal transducer and activator of transcription 3 (STAT3) phosphorylation associated with reduction in MCL1 levels and phosphorylation, illustrating a potent mechanism for the cytotoxicity of GRK6 inhibition in multiple myeloma (MM) tumor cells. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma.

Purpose Thalidomide and lenalidomide are immunomodulatory drugs (IMiDs) that produce high remission rates in the treatment of multiple myeloma. Pomalidomide is a new IMiD with high in vitro potency. We report, to our knowledge, the first phase II trial of pomalidomide administered in combination with low-dose dexamethasone for the treatment of relapsed or refractory multiple myeloma. Patients and Methods Pomalidomide was administered orally at a dose of 2 mg daily on days 1 through 28 of a 28-day cycle. Dexamethasone 40 mg daily was administered orally on days 1, 8, 15, and 22 of each cycle. Responses were recorded using the criteria of the International Myeloma Working Group. Results Sixty patients were enrolled. Thirty-eight patients (63%) achieved confirmed response including complete response in three patients (5%), very good partial response in 17 patients (28%), and partial response in 18 patients (30%). Responses were seen in 40% of lenalidomide-refractory patients, 37% of thalidomide-refractory patients, and 60% of bortezomib-refractory patients. Responses were seen in 74% of patients with high-risk cytogenetic or molecular markers. Toxicity consisted primarily of myelosuppression. Grade 3 or 4 hematologic toxicity consisted of anemia (5%), thrombocytopenia (3%), and neutropenia (32%). One patient (1.6%) had a thromboembolic event. The median progression-free survival time was 11.6 months and was not significantly different in patients with high-risk disease compared with patients with standard-risk disease. Conclusion The combination of pomalidomide and low-dose dexamethasone is extremely active in the treatment of relapsed multiple myeloma, including high response rates in patients refractory to other novel agents.