Ahmann, Greg

AbstractIdentifying biomarkers associated with disease progression and drug resistance are important for personalized care. We investigated the expression of 121 curated genes, related to immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs) responsiveness. We analyzed 28 human multiple myeloma (MM) cell lines with known drug sensitivities and 130 primary MM patient samples collected at different disease stages, including newly diagnosed (ND), on therapy (OT), and relapsed and refractory (RR, collected within 12 months before the patients’ death) timepoints. Our findings led to the identification of a subset of genes linked to clinical drug resistance, poor survival, and disease progression following combination treatment containing IMIDs and/or PIs. Finally, we built a seven‐gene model (MM‐IMiD and PI sensitivity‐7 genes [IP‐7]) using digital gene expression profiling data that significantly separates ND patients from IMiD‐ and PI‐refractory RR patients. Using this model, we retrospectively analyzed RNA sequcencing (RNAseq) data from the Mulltiple Myeloma Research Foundation (MMRF) CoMMpass (n = 578) and Mayo Clinic MM patient registry (n = 487) to divide patients into probabilities of responder and nonresponder, which subsequently correlated with overall survival, disease stage, and number of prior treatments. Our findings suggest that this model may be useful in predicting acquired resistance to treatments containing IMiDs and/or PIs.

Abstract Background: Waldenström macroglobulinemia (WM) is a plasma cell dyscrasia characterized by a monoclonal IgM paraproteinemia. The most common genomic abnormality in the clonal cells of WM is loss of portions of the long arm of chromosome 6 (6q-), however a minimal area of deletion has yet to be identified. We decided to further clarify the area of deletion on the 6q-arm in WM along with its prevalence. Methods: Patients (n=41) were required to have a serum IgM paraproteinemia of 1.5 g/dl or greater and a monoclonal lymphoplasmacytic infiltrate. We used interphase fluorescence in situ hybridization (FISH) combined with cytoplasmic immunoglobulin (cIg) staining with an AMCA conjugated anti-IgM antibody along with cytomorphology for the detection of the clonal cells on cytospin slides. Ten probes were used including two commercial (CEP 6 and Telomere 6q) and eight Bacteria Artificial Chromosome (BAC). BAC clones were selected based on a specific gene encompassed in the insert and/or localization on the q arm. Genes of interest included BLIMP1, c-myb, BTF, SHPRH, LATS1 and FOP. BAC’s were grown in selective media; DNA was extracted and directly labeled with a SpectrumGreen or SpectrumOrange fluorophore by standard nick translation. Primers were designed to confirm the insert contained the gene of interest and probes were tested on normal metaphases to reaffirm localization to the correct area of the chromosome and rule out any cross hybridization. Results: Deletions of 6q were noted in 21 (51%) patients. Of the 19 abnormal patients with all probes tested 1 (5%) case had ten probes deleted, 7 (37%) cases had nine probes, 1 (5%) had eight, 4 (21%) had seven, 3 (16%) had six and 3 (16%) had four probes deleted. Only 3 cases retained the end of the q arm indicating an interstitial deletion while the remaining 16 cases indicate terminal deletions. Deletions of the BAC containing the gene SHPRH at 6q24 were seen in each of the 19 abnormal cases. The majority of patients exhibited little heterogeneity for any given deletion having greater than 80% involvement of the clone. Conclusion: This study demonstrates that large deletions of the long arm of chromosome 6 in WM are frequent and are usually terminal deletions rather than interstitial and have a common area of deletion of the q24 portion of the q arm. Using the given number of probes localizing along the q arm makes it highly unlikely of any areas of noncontiguous deletion. The presence of deletions in the majority of the clonal cells may suggest that clones with 6q- emerge early in the course of the disease and is selected because of growth advantage or apoptosis resistance. Whether 6q deletions are important for disease pathogenesis has yet to be determined. Clinical prognosis studies and microarray data analysis are pending.

Abstract Table 1. Samples to p53 deletions p 53 deletion FISH aCGH FISH and aCGH MGUS 142 - - SMM 108 - - Newly diagnosed MM - 182 - Relapsed/refractory MM 62 156 19 HMCLs - 48 - Total 312 386 19 Background: Inactivation of p53 by mutation or allelic loss is a rare event in multiple myeloma (MM) at the time of disease diagnosis and believed to be more common in the late stages of the disease. Here we defined the prevalence of p53 deletions in monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), newly diagnosed MM, relapsed MM and human myeloma cell lines (HMCLs). Indirect mechanisms of p53 inactivation such as amplification of MDM2, or deletion of CDKN2A (p14ARF) were also investigated. Patients and Methods: A total of 631 MM samples and 48 HMCLs were analyzed for p53 abnormalities (Table 1) and compared with a cohort of plasma cell leukemia (PCL) previously published by us. Interphase fluorescence in situ hybridization (FISH) and highresolution array-based comparative genomic hybridization (aCGH) were used to detect p53 deletions. MDM2 amplification and p14ARF loss were evaluated by aCGH. Overall survival (OS) was estimated through Kaplan Meier method. Results: The prevalence of p53 deletions was 1.4%, 3.7%, 8.9%, 20% and 87.5% in MGUS, SMM, newly diagnosed MM, relapsed MM and HMCLs respectively (Figure 1). Of interest patients in first relapse showed a p53 deletion prevalence of 18% in comparison with 33% for patients in second relapse or more. We obtained p53 deletion status in 8 patients noted to have deletion at the time of relapse and in whom we had previous samples stored. In 7 of 8 cases deletions had been acquired (absent in the previous samples). The median OS for relapsed MM patients with or without p53 deletion were 4.2 months and 37.8 months respectively (p <0.001). MDM 2 amplification and p14ARF loss was detected in 0.4% and 2% of new diagnosed MM, 0.6% and 5.1% of relapsed MM and 2% and 29% of HMCLs respectively. Discussion: Emergence of p53 deletion/mutations denotes progression genetic events in MM. Importantly we show for the first time the overriding importance of p53 deletions as prognostic markers in relapsed MM. A molecular staging system that is based on the presence of p53 inactivation may be more powerful than classifying patients based on the loss of p53. Other abnormalities involving p53 pathway regulators like MDM2 gain and p14ARF loss are infrequent events in MM, even in advanced disease. Figure 1. Prevalence of p53 deletions Figure 1. Prevalence of p53 deletions

Abstract INTRODUCTION The bone marrow plasma cell labeling index (PCLI) and the beta 2 microglobulin (β2M) when combined provide a powerful staging system highly predictive of outcomes in myeloma patients (Greipp et. al., Blood, 81:3382–87, 1993). This staging system lacks information on cytogenetics and it has been demonstrated that deletion 13 (Δ13) by conventional cytogenetics is associated with poor outcomes. In conventionally treated patients Δ13 by FISH is an independent prognostic variable. We analyzed the importance of Δ13 by FISH in a cohort of patients receiving high dose therapy and assessed its value relative to known prognostic parameters, PCLI and β2M. PATIENTS AND METHODS 238 patients undergoing high dose therapy between January 1990 and September 2001 were studied. The minimum follow-up of patients was 34 months. The median age of the cohort 56 years. Interphase fluorescence in situ hybridization with Probes LSI13/D13S319 was performed with simultaneous immunofluorescent detection of bone marrow plasma cells by the presence of cytoplasmic immunoglobulin. PCLI was done by a slide-based immunofluorescence method with antibodies to bromodeoxyuridine. RESULTS Of the 238 patients studied 215 had a successful FISH analysis and 212 of these had a bone marrow labeling index. Deletion 13 was absent in 104 patients (48%) and present in 111 patients. The overall survival and time to progression for patients lacking and having deletion 13 was 47.8 vs 26.4 (OS) and 19.0 vs 11.7 (TTP)months respectively. A Cox model was constructed with the PCLI, the presence or absence of deletion 13 and serum β2M. Although all three variables were independently predictive of survival and time to progression, the β2M was the weakest of these three variables. The hazard function for survival for PCLI, deletion 13 and β2M was 1.4, 1.6 and 1.1 respectively. A staging system was constructed assigning 1 point for the presence of deletion 13 or a PCLI ≥ 1%. Patients fell into 3 categories of 0 points (N=69), 1 point (N=104) and 2 points (N=39). The overall survival for the three groups respectively were 56.5, 36.6 and 13.7 months (figure). The time to progression for the three groups were 22.3, 15.4 and 8 months (P<0.001). When analysis was limited to those patients transplanted within 12 months of diagnosis the results remained significant (P<0.003) with median survivals of stage 0, 1 and 2 of 72.5, 59.1 and 15.4 months respectively. TTP for stage 0, 1 & 2 in this same cohort of patients transplanted < 12 mos after diagnosis is 30.6, 21.5 & 8.2 months respectively. CONCLUSION Deletion 13 by FISH is an independent predictor of survival and time to progression providing information independent of the β2M and the PCLI. Combining the PCLI with FISH del 13 identifies patients who following transplant have a median time to progression of 8 months and a median OS of 13.7 months. This group may not benefit from autologous transplant and are candidates for novel therapeutic approaches. Figure Figure

Abstract Events mediating transformation from the pre-malignant MGUS to MM is largely unknown. Previous studies have highlighted RAS mutation as a progression event. However, this abnormality is present in only a small subset of patients. Global gene expression differences between MGUS and MM have been reported but provided little coherent biological insights. To define progression events, we examined the differences in gene expression profiles (GEP, Affymetrix U133A genechip) between 101 MM and 22 MGUS using a powerful functional genomic tool, Gene Set Enrichment Analysis (Subramanium et al PNAS 2005), that allow extraction of biological information based on enrichment of genes associated with a phenotype within gene-sets derived experimentally or extracted from database. We found that a predominant theme relating to MM progression is the over-expression of proliferation related genes in MM highlighted by the enrichment of cell cycle related gene-sets. Another common theme is the enrichment of gene-sets relating to MYC activation. Seven of eight gene-sets related to MYC activation in the database are significantly enriched (p-value <0.05 and false detection rate <0.05). Leading edge (enriched genes) analysis of enriched MYC-related and proliferation-related gene-sets revealed a core, overlapping signature of 8 genes specific to MYC and not proliferation. These genes are further verified as MYC transcriptional targets using Metacore, a network analysis tool. In addition, 7 of the 8 genes had been validated as MYC target genes by chromatin immunoprecipitation previously (www.myccancergene.org). We validated this signature in independent publicly available cell lines and mouse gene expression experiments where this signature is present in mammary epithelial cell lines engineered to over-express MYC but not E2F3, SRC and BCAT, and mouse embryonic fibroblast over-expressing MYC but not HER2 or with RB knocked-out. In addition, analysis of proliferation index in these datasets showed that there is dissociation of proliferation from this signature. This showed conclusively that our signature is MYC specific and not related to proliferation. Of interest, the signature is also present in cell lines with activating RAS mutation, which also have high expression of MYC, suggesting that MYC activation is a downstream effect of activated RAS signaling. Indeed, in our patient cohort, most patients with RAS mutation express this signature. When applied to 2 large GEP MM patient cohorts, this signature was not present in normal plasma cells, rarely present in MGUS, and present in a significant proportion of MM and almost all the human myeloma cell lines. When analyzed in the context of other B-cell malignancies, the signature is very strong in Burkitt’s lymphoma, present in most myeloma but absent in chronic lymphocytic leukemia, Waldenstrom Macroglobulinemia, and normal B-cells. Our analysis therefore suggests that MYC activation may be a unifying pathological event in MGUS to MM progression. As MYC translocations occur in only about 13% of newly diagnosed MM patients, activating RAS mutations and other trans-mechanisms may mediate MYC activation. Our recent demonstration that sporadic MYC activation in a mouse strain prone to monoclonal gammopathy led to MM development provides important functional proof.

Key Points IMiDs inhibit TrxR-mediated intracellular decomposition of H2O2 and caused oxidative stress in MM cells. MM cells with lower antioxidative capacity were more vulnerable to lenalidomide-induced H2O2 accumulation and its associated cytotoxicity.

Abstract Detection of specific chromosomal abnormalities by FISH and metaphase cytogenetics allows risk stratification in multiple myeloma; however, gene expression profiling (GEP) based signatures may enable more specific risk categorization. We examined the utility of 2 GEP-based risk stratification systems among patients undergoing initial therapy with lenalidomide in the context of a phase 3 trial. Among 45 patients studied at baseline, 7 (16%) and 10 (22%), respectively, were high-risk using the GEP70 and GEP15 signatures. The median overall survival for the GEP70 high-risk group was 19 months versus not reached for the rest (hazard ratio = 14.1). Although the medians were not reached, the GEP15 also predicted a poor outcome among the high-risk patients. The C-statistic for the GEP70, GEP15, and FISH based risk stratification systems was 0.74, 0.7, and 0.7, respectively. Here we demonstrate the prognostic value for GEP risk stratification in a group of patients primarily treated with novel agents. This trial was registered at www.clinicaltrials.gov as #NCT00098475.

Abstract Hyperdiploid multiple myeloma (H-MM) is the most common form of myeloma. In this gene expression profiling study, we show that H-MM is defined by a protein biosynthesis signature that is primarily driven by a gene dosage mechanism as a result of trisomic chromosomes. Within H-MM, four independently validated patient clusters overexpressing nonoverlapping sets of genes that form cognate pathways/networks that have potential biological importance in multiple myeloma were identified. One prominent cluster, cluster 1, is characterized by high expression of cancer testis antigen and proliferation-associated genes. Tumors from these patients were more proliferative than tumors in other clusters (median plasma cell labeling index, 3.8; P < 0.05). Another cluster, cluster 3, is characterized by genes involved in tumor necrosis factor/nuclear factor-κB signaling and antiapoptosis. These patients have better response to bortezomib as compared with patients within other clusters (70% versus 29%; P = 0.02). Furthermore, for a group of patients generally thought to have better prognosis, a cluster of patients with short survival (cluster 1; median survival, 27 months) could be identified. This analysis illustrates the heterogeneity within H-MM and the importance of defining specific cytogenetic prognostic factors. Furthermore, the signatures that defined these clusters may provide a basis for tailoring treatment to individual patients. [Cancer Res 2007;67(7):2982–9]

The mechanisms underlying aneuploidy in multiple myeloma (MM) are unclear. Centrosome amplification has been implicated as the cause of chromosomal instability in a variety of tumors and is a potential mechanism causing aneuploidy in MM. Using immunofluorescent (IF) staining, centrosome amplification was detected in 67% of monoclonal gammopathies, including monoclonal gammopathy of undetermined significance (MGUS). We also investigated the gene expression of centrosome proteins. Overall, gene expression data correlated well with IF-detected centrosome amplification, allowing us to derive a gene expression-based centrosome index (CI) as a surrogate for centrosome amplification. Clinically, MM patients with high CI (> 4) are associated with poor prognostic genetic and clinical subtypes (chromosome 13 deletion, t(4; 14), t(14;16), and PCLI > 1%, P < .05) and are shown here to have short survival (11.1 months versus 39.1 months, P < .001). On multivariate regression, a high CI is an independent prognostic factor. Given that centrosome amplification is already observed in MGUS and probably integral to early chromosomal instability and myeloma genesis, and patients with more extensive centrosome amplification have shorter survival, the mechanisms leading to centrosome amplification should be investigated because these may offer new avenues for therapeutic intervention.