Ahmann, Gregory

SummaryWhile proteasome inhibition is a validated therapeutic approach for multiple myeloma (MM), inhibition of individual constitutive proteasome (c20S) and immunoproteasome (i20S) subunits has not been fully explored owing to a lack of effective tools. We utilized the novel proteasome constitutive/immunoproteasome subunit enzyme‐linked immunosorbent (ProCISE) assay to quantify proteasome subunit occupancy in samples from five phase I/II and II trials before and after treatment with the proteasome inhibitor carfilzomib. Following the first carfilzomib dose (15–56 mg/m2), dose‐dependent inhibition of c20S and i20S chymotrypsin‐like active sites was observed [whole blood: ≥67%; peripheral blood mononuclear cells (PBMCs): ≥75%]. A similar inhibition profile was observed in bone marrow–derived CD138+ tumour cells. Carfilzomib‐induced proteasome inhibition was durable, with minimal recovery in PBMCs after 24 h but near‐complete recovery between cycles. Importantly, the ProCISE assay can be used to quantify occupancy of individual c20S and i20S subunits. We observed a relationship between MM patient response (n = 29), carfilzomib dose and occupancy of multiple i20S subunits, where greater occupancy was associated with an increased likelihood of achieving a clinical response at higher doses. ProCISE represents a new tool for measuring proteasome inhibitor activity in clinical trials and relating drug action to patient outcomes.

SummaryWe constructed a multiple myeloma (MM)‐specific gene panel for targeted sequencing and investigated 72 untreated high‐risk (del17p) MM patients. Mutations were identified in 78% of the patients. While the majority of studied genes were mutated at similar frequency to published literature, the prevalence of TP53 mutation was increased (28%) and no mutations were found in FAM46C. This study provides a comprehensive insight into the mutational landscape of del17p high‐risk MM. Additionally, our work demonstrates the practical use of a customized sequencing panel, as an easy, cheap and fast approach to characterize the mutational profile of MM.

Abstract The plasma cell malignancy Multiple Myeloma (MM) is a rare but deadly form of cancer with 5 year survival rates of ∼30%. Epidemiological studies have suggested that MM exacts an especially heavy burden on African American (AA) patients. We examined a multi-ethnic cohort of MM tumors to assess potential differences in the frequency of molecular events in tumors derived from either African American (AA) or European American (EA) patients. The frequency of 14q32 translocation breakpoints at the IgH locus was compared among a series of data from 115 AA MM patients from three studies and EA MM from the Eastern Cooperative Oncology Group (ECOG). In addition, we analyzed matching genome-wide copy number and transcriptional data among 45 AA MM tumors and 196 EA MM tumors to determine differences in the frequency of molecular events in tumors from these populations. No differences were found for specific translocation subtypes; however, we detected a statistically significant difference in the overall frequency of IgH translocations between the two groups, which occurred more frequently in EA patients (52% vs. 40%; p = 0.032). When assessing genomic copy number events previously shown to be associated with poor outcome in MM, frequencies of alterations were not significant after correcting for multiple testing. Furthermore, there was not a significant difference in the association of high-risk disease using expression profiling. Our study represents the first comprehensive assessment of the frequency and distribution of molecular alterations in MM tumors from both AA and EA patients and could help shed light on possible biological features associated with population differences in incidence and outcome in MM. Citation Format: Angela S. Baker, Esteban Braggio, Susana Jacobus, Sungwon Jung, Dirk Larson, Terry Therneau, Angela Dispenzieri, Scott A. Van Wier, Gregory Ahmann, Joan Levy, Louise Perkins, Seungchan Kim, Kim Henderson, David Vesole, S. Vincent Rajkumar, Dianne F. Jelinek, John Carpten, Rafael Fonseca. Characterization of myeloma tumors from a multi-ethnic cohort using cytogenetics and genomic analysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2021. doi:10.1158/1538-7445.AM2013-2021

Key PointsIn this study, African American MM patients have a significantly lower frequency of IgH translocations than European American patients.

Abstract Previous epidemiological studies have suggested that African Americans (AA) are twice as likely to be diagnosed with and to die from Multiple Myeloma (MM) as compared to European Americans (EA). In recent years, however, mortality rates have become more comparable between these groups. Although socioeconomic factors could play a major role to this potential disparity, biological factors may also be responsible for influencing these differences in either incidence and/or mortality. Here we examined a multi-ethnic cohort of MM tumors to assess potential differences in the frequency of molecular events in tumors derived from either African American (AA) or European American (EA) patients. First, we compared the frequency of 14q32 translocation breakpoints at the IgH locus among a series of data from 115 AA MM patients from three studies and EA MM from the Eastern Cooperative Oncology Group (ECOG). Furthermore, we analyzed matching genome-wide copy number and transcriptional data among 47 AA MM tumors and 196 EA MM tumors to determine differences in the frequency of molecular events in tumors from these populations. Univariate analyses were conducted using the Fisher's Exact Test and p-values were calculated to measure statistical significance. We observed statistically significant differences in the frequency of IgH translocations and somatic copy number alterations. Although we did not detect differences for specific translocation subtypes, we saw a statistically significant difference in the overall frequency of IgH translocations between the two groups, which occurred more frequently in EA patients (p = 0.032). When assessing genomic copy number events previously shown to be associated with poor outcome in MM, chromosome 13 monosomy was more frequent in hyperdiploid MM from AA patients and significant after correcting for multiple testing using the Bonferoni correction method (p = 0.001). Moreover, when using the Benjamini-Hochberg multiple testing correction method, chromosome 13 monosomy remained significantly different among hyperdiploid AA patients (p = 0.0121). Chromosome 1q amplification was also significant among hyperdiploid tumors from EA patients when using this correction method (p = 0.036). No differences in the frequency of hyperdiploid status or gene expression-based subtypes were detected when comparing tumors from AA and EA patients. Our study represents the first comprehensive assessment of the frequency and distribution of molecular alterations in MM tumors from both AA and EA patients and could help shed light on possible biological features associated with population differences in incidence and outcome in MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5064. doi:1538-7445.AM2012-5064

Abstract Multiple Myeloma (MM) is an incurable plasma-cell disorder progressing from indolent monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) to relapse MM and finally plasma cell leukemia (PCL). Alterations affecting TP53 are among the principal genetic progression events associated with disease progression. We analyzed results from gene expression profiling (GEP), array-based comparative genomic hybridization (aCGH), fluorescent in situ hybridization (FISH) and mutational data on the largest cohort published to date of 806 patients from all MM stages and 48 human myeloma cell lines (HMCLs). The prevalence of monoallelic TP53 deletions by aCGH is 1.4%, 3.7%, 8.9% and 25%, in MGUS, SMM, newly diagnosed and relapsed MM respectively. It reaches 53% in HMCLs and 75% in secondary PCL. No biallelic TP53 deletion were observed in patients and only 8% in HMCLs. In relapsed MM, the prevalence of TP53 deletions increases from 12/75 patients (16%) at first, to 9/31 patients (29%) in second and 5/7 patients (71%) in third relapse or more. We studied by FISH the status of TP53 in 113 patients from early stages progressing to relapse. Overall 15/113 patients had TP53 deletion, we confirmed that 10/15 patients acquired TP53 deletion at late stages. Next, we investigated the mutational status of TP53 in 59 relapsed MM patients, 5/59 samples (8.5%) had TP53 mutations, 4/5 mutations were located in the DNA binding domain. Presence of TP53 deletion conferred poor overall survival in relapsed MM patients (4.2 vs. 37.8 months, p = 0.015). A cohort of 239 patients had GEP-aCGH paired analysis we used a validated 70-gene model to stratify patients based on calculations obtained by GEP. The cohort was divided into quartiles and we observed the prevalence of TP53 in the low and high-risk groups. TP53 deletion was observed in 33/239 patients (14%) by aCGH, 2/33 (6%) had GEP values in the low-risk and 13/33 (39%) in the high-risk group. Next, we used a previously proposed cut-off value predicting TP53 deletion when GEP values are less than 733. We tried to validate this value as a surrogate for direct detection of TP53 deletion, and used the positive predictive value (PPV) of such level as a potential clinically useful marker. The fact that aCGH is the gold standard approach to define copy-number alterations, the proposed cut-off value failed to predict TP53 deletion as the PPV was low at 18% and the sensitivity of 69%. Finally, we studied by aCGH the status of MDM2 and CDKN2A, two key regulators of TP53. MDM2 gain and CDKN2A loss were infrequent events in patients even in advanced disease. In 48 HMCLs, only one had MDM2 copy gain (2%) while 14 cell lines had CDKN2A loss (29%). In MM, emergence of TP53 deletion/mutations in MM and its increase in aggressive stages of disease remain the overriding genetic factor for poor prognosis determination and a marker of progressive genetic events. Based on GEP, the 733 cut-off value did not accurately predict TP53 deletion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 219. doi:10.1158/1538-7445.AM2011-219

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 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.