Braggio, Esteban

Abstract Introduction: Only few genetic studies have been performed in PCNSL, partly due to the rarity of the tumors and the limited amount of available tissue. Unlike nodal DLBCL, PCNSL is poorly understood in terms of its precise histogenetic origin and molecular pathogenesis. It is still a matter of debate whether PCNSL differs from nodal DLBCL with respect to their molecular features and pathogenesis and also if there is a genomic signature PCNSL-specific. Materials and Methods: To determine the pattern of genetic alterations, 17 immunocompetent (EBV-, HIV-) PCNSL were analyzed by aCGH. Seven cases were analyzed using fresh tumor samples and 10 using formalin-fixed paraffin embedded (FFPE) tissues. B-cell differentiation status was characterized by immunostains for CD10, MUM-1, and BCL-6. The aCGH profile from PCNSL cases was compared with aCGH data obtained from 59 nodal DLBCL. Results: We found a complex karyotype with a median of 21 copy-number abnormalities (CNA) per case (range 10-49). Overall, 18 regions (10 losses and 8 gains) were affected in >20% of cases. Loss of 9p21 (CDKN2A) was the commonest CNA (82% of cases). Other common CNAs were loss of 6q23.3 (TNFAIP3; 59%), 6p21 (HLA genes; 53%), 6q21 (PRDM1; 47%) and gain of 12q21-q24 (53%), 7q21-q31 (35%) and 19q13 (35%). Five novel recurrent focal CNAs were found in PCNSL; loss of 3p21.31, 3q26.32, 10p15.3-p14, 12q24.31 and 16q12.1-q21 (29% of cases each). All of them, excluding –16q12.1-q21, were found either in less than 5% or absent in nodal DLBCL, thus suggesting to be unique to PCNSL. Recurrent homozygous deletions were found in CDKN2A, TMEM30A, TOX and CD58, the latter two involved in T-cell development and activation. CD58 was also recurrently affected by small focal deletions in the Ig-like C2-type domain. Furthermore, DNA sequencing analysis showed missense mutations in the Toll/IL-1 receptor domain of MYD88 in 35% of PCNSLs. Another 34 genes, including B2M, ETV6, MHC class II genes, PRDM1, and TNFRSF10A were homozygously deleted. Bioinformatics analysis including the most commonly affected genes showed an enrichment of networks associated with immune response, NF-kB pathway, proliferation, regulation of the apoptosis and lymphocyte differentiation and activation. Conclusions: We showed evidence of a highly complex genome and identified a subset of genes with potential relevance in PCNSL pathogenesis. The genomic profile described here reinforces the existence of a specific molecular signature in PCNSL, thus helping to genetically differentiate this entity from the nodal DLBCL and related lymphomas. Additionally, the results obtained from FFPE samples are encouraging and larger archival tissue collections can now be analyzed in order to complement the still fragmented knowledge we have of the genetic basis of the disease. 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 5081. doi:1538-7445.AM2012-5081

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 Peripheral T-cell lymphomas (PTCLs) are aggressive malignancies of mature T lymphocytes with 5-year overall survival rates of only ∼ 35%. Improvement in outcomes has been stymied by poor understanding of the genetics and molecular pathogenesis of PTCL, with a resulting paucity of molecular targets for therapy. We developed bioinformatic tools to identify chromosomal rearrangements using genome-wide, next-generation sequencing analysis of mate-pair DNA libraries and applied these tools to 16 PTCL patient tissue samples and 6 PTCL cell lines. Thirteen recurrent abnormalities were identified, of which 5 involved p53-related genes (TP53, TP63, CDKN2A, WWOX, and ANKRD11). Among these abnormalities were novel TP63 rearrangements encoding fusion proteins homologous to ΔNp63, a dominant-negative p63 isoform that inhibits the p53 pathway. TP63 rearrangements were seen in 11 (5.8%) of 190 PTCLs and were associated with inferior overall survival; they also were detected in 2 (1.2%) of 164 diffuse large B-cell lymphomas. As TP53 mutations are rare in PTCL compared with other malignancies, our findings suggest that a constellation of alternate genetic abnormalities may contribute to disruption of p53-associated tumor suppressor function in PTCL.

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 longitudinal evolution of a myeloma genome from diagnosis to plasma cell leukemia has not previously been reported. We used whole-genome sequencing (WGS) on 4 purified tumor samples and patient germline DNA drawn over a 5-year period in a t(4;14) multiple myeloma patient. Tumor samples were acquired at diagnosis, first relapse, second relapse, and end-stage secondary plasma cell leukemia (sPCL). In addition to the t(4;14), all tumor time points also shared 10 common single-nucleotide variants (SNVs) on WGS comprising shared initiating events. Interestingly, we observed genomic sequence variants that waxed and waned with time in progressive tumors, suggesting the presence of multiple independent, yet related, clones at diagnosis that rose and fell in dominance. Five newly acquired SNVs, including truncating mutations of RB1 and ZKSCAN3, were observed only in the final sPCL sample suggesting leukemic transformation events. This longitudinal WGS characterization of the natural history of a high-risk myeloma patient demonstrated tumor heterogeneity at diagnosis with shifting dominance of tumor clones over time and has also identified potential mutations contributing to myelomagenesis as well as transformation from myeloma to overt extramedullary disease such as sPCL.

Abstract Emerging evidence indicates that tumors can follow several evolutionary paths over a patient's disease course. With the use of serial genomic analysis of samples collected at different points during the disease course of 28 patients with multiple myeloma, we found that the genomes of standard-risk patients show few changes over time, whereas those of cytogenetically high-risk patients show significantly more changes over time. The results indicate the existence of 3 temporal tumor types, which can either be genetically stable, linearly evolving, or heterogeneous clonal mixtures with shifting predominant clones. A detailed analysis of one high-risk patient sampled at 7 time points over the entire disease course identified 2 competing subclones that alternate in a back and forth manner for dominance with therapy until one clone underwent a dramatic linear evolution. With the use of the Vk*MYC genetically engineered mouse model of myeloma we modeled this competition between subclones for predominance occurring spontaneously and with therapeutic selection.

AbstractTo identify rational therapeutic combinations with cytarabine (Ara-C), we developed a high-throughput, small-interference RNA (siRNA) platform for myeloid leukemia cells. Of 572 kinases individually silenced in combination with Ara-C, silencing of 10 (1.7%) and 8 (1.4%) kinases strongly increased Ara-C activity in TF-1 and THP-1 cells, respectively. The strongest molecular concepts emerged around kinases involved in cell-cycle checkpoints and DNA-damage repair. In confirmatory siRNA assays, inhibition of WEE1 resulted in more potent and universal sensitization across myeloid cell lines than siRNA inhibition of PKMYT1, CHEK1, or ATR. Treatment of 8 acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML) cell lines with commercial and the first-in-class clinical WEE1 kinase inhibitor MK1775 confirmed sensitization to Ara-C up to 97-fold. Ex vivo, adding MK1775 substantially reduced viability in 13 of 14 AML, CML, and myelodysplastic syndrome patient samples compared with Ara-C alone. Maximum sensitization occurred at lower to moderate concentrations of both drugs. Induction of apoptosis was increased using a combination of Ara-C and MK1775 compared with using either drug alone. WEE1 is expressed in primary AML, ALL, and CML specimens. Data from this first siRNA-kinome sensitizer screen suggests that inhibiting WEE1 in combination with Ara-C is a rational combination for the treatment of myeloid and lymphoid leukemias.

While no specific genetic markers are required in the diagnosis of multiple myeloma (MM), multiple genetic abnormalities and gene signatures are used in disease prognostication and risk stratification. This is particularly important for the adequate identification of the high-risk MM group, which does not benefit from any of the current therapies, and novel approaches need to be proposed. Fluorescence in situ hybridization (FISH) has been employed for establishing risk-based stratification and still remains the most used genetic technique in the clinical routine. The incorporation of gene expression profiling (GEP) in the study of MM has shown to be a very powerful test in the patient stratification, but its incorporation in clinical routine depends on some technical and logistic resolutions. Thus, FISH still remains the gold standard test for detecting genomic abnormalities and outcome discrimination in MM.

Abstract The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their antitumor effects remains unclear. We investigated the role of cereblon (CRBN), a primary teratogenic target of thalidomide, in the antimyeloma activity of IMiDs. CRBN depletion is initially cytotoxic to human myeloma cells, but surviving cells with stable CRBN depletion become highly resistant to both lenalidomide and pomalidomide, but not to the unrelated drugs bortezomib, dexamethasone, and melphalan. Acquired deletion of CRBN was found to be the primary genetic event differentiating isogenic MM1.S cell lines cultured to be sensitive or resistant to lenalidomide and pomalidomide. Gene expression changes induced by lenalidomide were dramatically suppressed in the presence of CRBN depletion, further demonstrating that CRBN is required for lenalidomide activity. Downstream targets of CRBN include interferon regulatory factor 4 (IRF4) previously reported to also be a target of lenalidomide. Patients exposed to, and putatively resistant to, lenalidomide had lower CRBN levels in paired samples before and after therapy. In summary, CRBN is an essential requirement for IMiD activity and a possible biomarker for the clinical assessment of antimyeloma efficacy.