Kuehl, Michael W.

Abstract Chromosomal translocations involving the immunoglobulin heavy-chain (IgH) locus at 14q32 are genetic hallmarks of human multiple myeloma (MM) and are likely primary oncogenic events in myeloma transformation. Common recurrent translocation partner genes of IgH account for 40–50% of cases and these rearrangements result in the transcriptional dysregulation of various proto-oncogenes such as CCND1, CCND3, FGFR3/MMSET, c-MAF, MAFB, MUM 1(IRF-4), c-MYC and it is becoming clear that these dysregulated genes play important roles in the pathogenesis and the treatment outcome of MM. Although the frequency of other partner genes of IgH translocations may be rare (<1%), it is possible that many genes not currently identified as translocation partners are functionally converged into several signal pathways and play important roles as well. Through global gene expression profiling (GEP) studies of a large population of newly diagnosed MM, we have recently reported that dysregulation of any one of the three D-type cyclins is seen in almost all newly diagnosed MM and most patients with high levels of CCND2 expression was highly correlated with expression of c-MAF, MAFB or FGFR3/MMSET. In order to identify novel translocation partner genes in MM we combined GEP and fluorescence in situ hybridization (FISH) analysis in a large population of newly diagnsoed myeloma. We investigated whether IGH-CCND2 and IGH-MAFA translocations are seen in patients exhibiting high levels of CCND2 expression but not expressing c-MAF, MAFB, or FGFR3/MMSET. We also looked for a breakpoint and targeted gene at a non-random breakpoint at the 11q23 locus in 20 MM patients with 11q23 abnormalities detected by conventional G-banding. Here we report evidence of an IGH-CCND2 fusion in a newly diagnosed case of MM and an IGH-MAFA fusion in a newly diagnosed MM and an MGUS and also mapped the 11q23 breakpoint to within 100 kb in a newly diagnosed MM and a human myeloma cell line, NCU-MM-1 and identified an IGL Pou2AF1 fusion in the NCU-MM-1 line. Importantly these translcoatios do not involve the MLL or ATM genes. These three genes were all highly expressed as detected by GEP or quantitive RT-PCR in the samples in which these genes were translocated to IG loci. Although the frequency of these 3 translocations are very rare, they further the concept of a critical role of CCND2 expression in myelomegenesis and identifed the transcriptional coactivator Pou2AF1, which specifically associates with either OCT1 or OCT2, in the B-cell response to antigens and required for the formation of germinal centers, as a potential new translocation target in myeloma. Finally, our strategy of combining GEP signatures and cytogenetic analyses appears to be a practical means of identifying new IG- mediated translocations in myeloma.

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 Multiple myeloma (MM) is a tumor of isotype-switched plasma cells that accumulate in the bone marrow, replacing normal plasma cells and leading to bone destruction and marrow failure. It is often preceded by a stable monoclonal gammopathy (MG) with limited (<10%) bone marrow plasmacytosis that progresses stochastically to MM at a rate of between 1–2% per year. Primary genetic events thought to be associated with disease initiation are present at similar frequencies in both MG and MM. In approximately 40% of cases, there are recurrent chromosome translocations involving the immunoglobulin heavy chain gene that have the hallmarks of being mediated by errors in switch recombination (90%) or somatic hypermutation (10%). These involve recurrent loci that fall into three categories: Cyclin D (15% CCND1 at 11q13, <1% CCND2 at 12p13, 2% CCND3 at 6p21), MAF (5% c-maf at 16q23, 2% MAFB at 20q12, <1% MAFA at 8q24.3), and MMSET (usually with FGFR3 at 4p16, 15%). Approximately one-half of cases are hyperdiploid (48–75 chromosomes) with trisomies involving chromosomes 3, 5, 7, 9, 11, 15, 19 and 21. A unifying feature of MM is dysregulated expression of a cyclin D gene, either directly by Ig gene translocation, by transactivation by MAF (CCND2), or by unknown mechanisms (MMSET – CCND2, Hyperdiploid – CCND1). Interestingly, about 2% of patients express no cyclin D, and one-half of these patients have bi-allelic deletion of RB, highlighting the critical role of the CCND/RB axis in the pathogenesis of MM. Deletion of chromosome 13 is seen in one-half of MM but is much more common in the tumors that express CCND2 (>80%) than in those that express CCND1 (~30%). Although with largely redundant functions, CCND1 has additional ability to antagonize RB not shared by CCND2, suggesting that CCND2 tumors may be more critically dependent on the absolute level of RB protein. A variety of secondary genetic events associated with disease progression occur with a similar frequency in hyperdiploid and non-hyperdiploid tumors (17p loss or p53 mutations, RAS mutations, secondary Ig translocations, MYC translocations, and inactivation of p18INK4c or RB1). In contrast, a promiscuous array of mutations that activate the NFKB pathway are less common in hyperdiploid MM, which, nonetheless, has high levels of presumably ligand-dependent NFKB activity and appears particularly dependent on the bone marrow microenvironment. Both some of the primary (MAF, MMSET) and secondary (17p deletion) genetic events are associated with a poor prognosis with melphalan-based regimens and appear to benefit from the early introduction of bortezomib.

Abstract It has been suggested that errors in the repair of DNA double strand breaks (DSB) can result in gross chromosomal rearrangements (GCR), including chromosomal amplifications, deletions, inversions, and translocations. To study repair of DNA DSB in vivo, we established a subclone of the U937 cell line designated F5, in which a single copy of the rare-cutting meganuclease I-SceI recognition site has been integrated into chromosome 7. We transfected an I-SceI expression vector into these cells and analyzed misrepair events. We recovered no GCR, but instead found that most DSBs were repaired by small (<50bp) indels (91.8%). We also noticed some DSBs were repaired by large (<50bp) deletions (6.4%) and rare DSBs were repaired by large (100-500 bp) insertions (1.8%). Surprisingly, the nucleotide sequence of these insertions matched distant regions of the genome. To determine if this templated sequence insertion (TSI) was peculiar to I-SceI induced DNA DSB, we repeated the experiment using TALENs to produce DNA DSB. Again, we found insertions that were derived from distant genomic regions. To determine whether this form of DNA repair was restricted to experimentally induced DNA DSB, we analyzed structural variations identified by whole genome sequence (WGS) of two myeloma cell lines (KP6, MC1286PE1), and identified 15 independent TSIs. These TSIs were verified by Sanger sequencing, and lengths of inserted fragment were 38 to 666 bp (median 189 bp). Six insertions were noted to be identical between the 2 cell lines, suggesting that these TSIs were germline polymorphic events. These TSIs were compared to a catalog of known structural variations identified by WGS of 52 healthy individuals; 13 of 15 TSI junctions were identified, suggesting that these represented polymorphic insertions in the human genome. Two TSIs were potential somatic mutations in the KP6 cell line, including one that had 85bp insertion at the junction of an acquired t(6;12) chromosomal translocation. Six TSIs showed the hallmarks of L1 retrotransposon-mediated insertions, with a 5’-TTTT/A-3’ integration site, a target site duplication (TSD), a polyA tract at the insertion site, and a polyadenylation signal. However, the inserted sequence was neither a LINE nor a SINE, but instead was a transcribed, genic region mapping to a distant genomic region. The presence of these L1 ORF1 hallmarks strongly suggests that these insertions were caused by the L1 integrase and reverse-transcriptase acting on nuclear pre-mRNA. A second class of TSI showed no preference for a 5’-TTTT/A-3’ integration site, no TSD, no poly A tract, but instead showed several bp of microhomology at the insertion junctions. This second class is similar to TSI detected at I-SceI induced DNA DSB, and we predict that these TSI were generated by reverse transcription of pre-mRNA creating a “patch” for a spontaneous DNA DSB that occurred in a germ cell. To determine if TSI at I-SceI induced DNA DSB could be repaired by RNA-templated sequences, we cotransfected murine RNA and an I-SceI expression vector into F5 cells. Approximately 10% of the TSI recovered from these experiments contained mouse sequences, indicating that they were derived from mouse RNA. To further support the contention that TSIs were derived from RNA, we again used I-SceI to induce a DNA DSB, and treated the cells with reverse transcriptase inhibitors. The TSI frequency was reduced by 70%. Finally, a subset of insertions were due to telomere repeat sequences, lending further support for the hypothesis that RNA could serve as a template for the repair of DNA DSB. TSI is novel form of potentially mutagenic DNA DSB repair, which should be considered as an alternative to a balanced chromosomal translocation in the interpretation of reciprocal interchromosomal structural variations identified by short-read deep sequencing. Disclosures: No relevant conflicts of interest to declare.

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