ALBERT

Description: Key Points The commonest lesions in anaplastic large cell lymphomas are losses at 17p13 and at 6q21, concomitant in up to one-quarter of the cases. PRDM1 (BLIMP1) gene (6q21) is inactivated by multiple mechanisms and acts as a tumor suppressor gene in anaplastic large B-cell lymphoma.

Description: Anaplastic large-cell lymphomas (ALCLs) are a group of clinically and biologically heterogeneous diseases including the ALK+ and ALK− systemic forms. Whereas ALK+ ALCLs are molecularly characterized and can be readily diagnosed, specific immunophenotypic or genetic features to define ALK− ALCL are missing, and their distinction from other T-cell non-Hodgkin lymphomas (T-NHLs) remains controversial. In the present study, we undertook a transcriptional profiling meta-analysis of 309 cases, including ALCL and other primary T-NHL samples. Pathway discovery and prediction analyses defined a minimum set of genes capable of recognizing ALK− ALCL. Application of quantitative RT-PCR in independent datasets from cryopreserved and formalin-fixed paraffin-embedded samples validated a 3-gene model (TNFRSF8, BATF3, and TMOD1) able to successfully separate ALK− ALCL from peripheral T-cell lymphoma not otherwise specified, with overall accuracy near 97%. In conclusion, our data justify the possibility of translating quantitative RT-PCR protocols to routine clinical settings as a new approach to objectively dissect T-NHL and to select more appropriate therapeutic protocols.

Description: Key Points A recurrent gain of a region of chromosome 11 (11q24.3) occurs in up to one-quarter of cases of diffuse large B-cell lymphoma. ETS1 and FLI1 genes are overexpressed and determine proliferation, survival, and differentiation arrest of the lymphoma cells.

Description: Marginal zone lymphoma (MZL) is a B-cell non-Hodgkin lymphoma (B-NHL) derived from marginal-zone B-cells. The current WHO classification recognizes three types of MZLs, which comprise extranodal MZL (EMZL), nodal MZL (NMZL), and splenic MZL (SMZL). Four recurrent and mutually exclusive chromosomal translocations: t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21), and t(3;14)(p14.1;q32) have been described in EMZLs, but not in NMZLs or SMZLs, with frequencies ranging from 0–40% depending on differing anatomic sites and geographic regions. At least three of these translocations, t(11;18) API2/MALT1, t(1;14) IgH/BCL10, and t(14;18) IgH/MALT, result in constitutive activation of nuclear factor-kB (NF-kB), a transcription factor complex regulating multiple cellular processes including cell growth and survival. Approximately 25% of MZL however, lack any recognizable recurrent genetic alteration. Recently, biallelic deletions of the chromosomal region 6q23.3-q24.1 containing the tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20), which is a negative regulator of NF-kB, were detected in ocular adnexal MZLs, suggesting a role for A20 as a tumor suppressor in this disease entity. Here, we investigated a panel of 32 cases representing all three types of MZLs by PCR amplification and sequencing of the A20 coding exons. FISH was also performed using locus specific probes for A20 and Blimp1. Five 1–2 bp deletions and a single bp insertion, all leading to premature stop codons, were identified in 5/20 (25%) non-splenic MZLs and 1/12 (8.3%) SMZLs. The somatic nature of the mutations was confirmed by analyzing matched normal DNA in 4 cases. All A20 mutations were predictive of severely truncated polypeptides lacking functionally relevant domains. Genetic loss of A20 was detected by FISH in 4/20 (20%) non-splenic MZLs, including 1 with biallelic loss, but not in SMZLs. Two of the four A20 deletions seen by FISH were also observed by array-CGH. Deletions encompassing Blimp1 were not detected in cases with A20 deletions. No evidence for epigenetic silencing of A20 via promoter methylation of CpG islands was found in 10 MZLs analyzed. Thus, biallelic inactivation of A20, either via deletion of both alleles or frameshift mutations of one allele and loss of the second allele was identified in 3/20 (15%) non-splenic MZLs, while an additional 4/20 (20%) non-splenic MZLs displayed monoallelic A20 inactivation by mutations (N=3) or deletion (N=1). When we determined the relationship between A20 inactivation and trisomy 18 or the translocations associated with constitutive activation of NF-kB, A20 inactivation appeared mutually exclusive with these cytogenetic aberrations, with the exception of one case that had trisomy 18. In summary, our results indicate that A20 is mono- or biallelically inactivated in approximately one third of MZLs, arising in both nodal (3/9, 33.3%) and extranodal sites (4/11, 36.4%), and in a minority of SMZLs (1/12, 8.3%). Evidence of a classic two-hit mechanism was observed in 3/20 (15%) non-splenic MZL. In addition, detection of MZLs with monoallelic inactivation suggests a haploinsufficient role of A20 as a tumor suppressor. Thus, A20 inactivation may represent a common mechanism for constitutive activation of the NF-kB pathway, which, in turn, may contribute to lymphomagenesis by stimulating cell proliferation and survival. The identification of genetic lesions in MZLs that deregulate NF-kB activity offers the possibility of targeted therapeutic intervention.

Description: Multiple myeloma (MM) is characterized by a high genomic instability that involves both ploidy and structural rearrangements. Nearly half of MM tumors are non-hyperdiploid and are frequently associated with 13q deletion and chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus on chromosome 14q32. The remaining tumors are hyperdiploid, showing low prevalence of both IGH translocations and chromosome 13 deletions. Our study was aimed at providing insights into the genomic heterogeneity associated with plasma cell neoplasms by defining the genome-wide pattern of genetic lesions in a representative and stratified panel of MM patients. To this end, genome-wide profiling data of 45 plasma cell dyscrasia patients (41 MM and 4 plasma cell leukemia) were generated on GeneChip® Human Mapping 50K Xba SNP arrays, and the local DNA copy number variations were calculated using the DNAcopy Bioconductor package. The patients were clustered using the non-negative matrix factorization (NMF) algorithm to identify, within the natural grouping of profiles, the strongest clusters on the basis of their genomic characteristics. We identified three consistent clusters, characterized byrecurrent gains of odd-chromosomes, suggestive of the hyperdiploid status (Group A),high frequency of chromosome 13 deletion and 1q gains (Group B), orhigh frequency of chromosomes 13, 14, 16 and 22 deletions and losses of 1p and 4p regions, together with some cases showing 1q gains (Group C). To determine whether peculiar transcription fingerprints characterized these groups, gene expression profiles of 40 out of 45 corresponding samples generated on GeneChip® HG-U133A arrays were analyzed using the Prediction Analysis of Microarray (PAM) software. The multi-class analysis identified 229 transcripts (corresponding to 195 genes), which specifically marked the three groups. In particular, Group A was characterized by the overexpression of genes involved in the translational machinery or thought to be involved in MM pathogenesis such as the HGF, the tumor necrosis factor ligand TRAIL, DKK1, and c-KIT. Upregulation of the CKS1B gene was present in Group B and C, most likely reflecting the high frequencies of 1q gains in tumors within group B and C and its consequent deregulation. Group C was marked by the specific downregulation of genes mainly mapped to 1p arm: AMPD1, CSDE1, AKR1A1 and the PRKACB kinase, suggesting a relationship with the recurrent 1p loss within the group. Our data further supported the notion that structural abnormalities in multiple myeloma are associated with gene expression imbalances, and provide novel analytical approaches for the identification of genetic lesions and molecular patterns of the disease.

Description: Abstract 3578 Introduction: Chronic lymphocytic leukemia (CLL) patients bearing 13q14 deletion are known to experience a more favorable clinical course. Recent studies, focusing on patients with loss of 13q as the sole cytogenetic aberration at diagnosis (del13q-only cases), showed that the number of malignant cells carrying this genetic lesion correlates with a more aggressive clinical behavior. However, whether the size of the 13q deletion may also influence the clinical outcome remains to be elucidated. Patients and Methods: Probes for chromosome 13q (LSI-RB1, LSI-D13S319), 11q (LSI-ATM), 17p (LSI-p53) and chromosome 12 (CEP12) were utilized on nuclei collected at diagnosis from: i) a multi-institutional CLL cohort (342 del13q-only cases) and ii) a consecutive unselected single-institution cohort of 265 cases. RB1 deleted cases (delRB1) were defined as having at least 5% of deleted nuclei. Time to treatment (TTT) intervals, as well as Rai staging, IGHV mutational status, CD38 and ZAP70 expression, B2-microglobulin levels, all evaluated at diagnosis, were also available for all cases that entered the study. Genome wide DNA profile was performed in a pilot series of 90 CLL samples using Affymetrix GeneChip Human SNP6 arrays. Results: According to genome wide DNA analysis, delRB1 occurred in a proportion of del13q-only cases (36/90; 40%), always comprising the deleted region detected with the LSI-D13S319 probe (that covers the miR-15a/16-1 cluster and the DLEU2 gene) and characterized by a larger chromosome loss (median size 2.07 Mb vs. a median size of 0.86 Mb for the canonical del13S319). Maximally selected log-rank statistics identified the 70% of nuclei bearing del13S319 as the most appropriate cut-off value capable of separating del13q-only cases into two subgroups with different TTT distributions. Consistently, del13q-only cases with at least 70% of nuclei bearing del13S319 showed a significantly shorter TTT than del13q-only cases with less than 70% deleted nuclei (p=0.0001). Del13q-only cases were then divided in four subsets according to the percentage of nuclei bearing del13S319 with or without a concomitant delRB1: del13S319 70% + delRB1 (group 4), 39 cases. The median TTT of group 1 (not reached) was significantly longer than the median TTT of group 2 (92 months, p=0.012), group 3 (68 months, p

Description: Abstract 51 Chronic lymphocytic leukemia (CLL) is a malignancy of CD5+ mature B cells that includes two distinct subtypes marked by the differential presence of immunoglobulin gene mutations and a distinct clinical course. The pathogenesis of CLL is largely unknown: in contrast to other types of B cell malignancies, CLL is not associated with recurrent, balanced chromosomal translocations, nor genes have been found that are specifically targeted by somatic mutations, with the exception of ATM and p53 in 6–12% of cases. Instead, more than 80% of CLL patients carry genomic deletions of chromosomal regions 13q14, 11q22–23, and 17p13, or trisomy 12. Of these, the 13q14.3 deletion, encompassing the DLEU2/miR-15a/miR-16-1 cluster (Calin and Croce, Nat Rev Cancer 2006), has been recently shown to promote the development of CLL in mice (Klein et al., Cancer Cell 2010), suggesting its pathogenetic role in the human disease. To determine the extent of somatic genetic lesions (mutations and gene copy number changes) that are present in the CLL genome, we have integrated next generation whole-exome sequencing analysis (Nimblegen/Roche FLX454) and genome-wide high-density single nucleotide polymorphism array analysis (Affymetrix SNP 6.0) in 5 cases representative of the two CLL immunogenetic subgroups and their paired normal DNAs. Candidate tumor-specific nonsynonymous mutations were verified by Sanger sequencing in the same tumor/normal pairs, and all genes validated as mutated were then screened in an independent panel of 48 CLL DNAs by PCR amplification/direct sequencing of their entire coding region. The whole-exome sequencing approach revealed a total of 36 mutations, corresponding to 36 distinct genes at an average of 7 mutations/case (range, 5–9 mutations/case). The majority of these events were represented by single base pair substitutions (N=34, of which 30 missense mutations and 4 nonsense mutations), while frameshift insertion/deletions were rare (N=2 deletions of 4 and 32 base pairs, respectively; 5.5%). Analysis of the mutation features showed a prevalence of transitions versus transversions (64% vs 36%) and an elevated G+C over A+T ratio (66% vs 44%), analogous to the mutation spectrum observed in the genome of epithelial tumors such as colorectal, pancreatic and brain cancer. SNP array analysis in 4 of the 5 “discovery” cases confirmed the presence of the 13q14 deletion in 2 samples and identified 25 additional regions of copy number changes, corresponding to ∼7 lesions/case (range: 3 to 10) and mostly represented by deletions (N=16/27, ∼60%). When screened in the extended CLL panel, several of the 36 genes identified through the whole exome sequencing approach appeared to be mutated in at least one additional patient. Overall, these data indicate that the coding genome of CLL contains on average ∼14 somatic gene alterations per case. When classified based on functional annotation, most of these lesions appeared to converge on discrete signaling pathways, which likely represent important pathogenetic and possibly therapeutic targets in CLL. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction. PQR309 is a novel oral dual PI3K/mTOR inhibitor (Cmiljanovic et al, AACR 2015), being now evaluated as single agent in a phase I study in patients with relapsed or refractory lymphomas (NCT02249429). We previously reported activity of PQR309 as single agent in a large panel of lymphoma cell lines, and early combination data in two diffuse large B-cell lymphoma (DLBCL) cell lines (Tarantelli et al, ASH 2014). Here, we present extended combination data that might enhance the anti-proliferative effect of the single drug. Methods. A panel of DLBCL cell lines derived from activated B-cell like (ABC) or germinal center B (GCB) subtype, with or without BCL2 or MYC deregulation, were used: RIVA (ABC, BCL2 amplification), SU-DHL-2 (ABC), TMD8 (ABC) and U2932 (ABC, BCL2 amplification); DOHH2 (GCB, BCL2 and MYC translocations), SU-DHL-6 (GCB, BCL2 translocation), KARPAS422 (GCB, BCL2 translocation), OCI-LY18 (GCB, BCL2 and MYC translocations), SU-DHL-10 (GCB), OCI-LY1 (GCB, BCL2 translocation). Cell lines were exposed to increasing doses of PQR309 alone or in combination with increasing doses of other agents for 72h and synergism was assessed with the Chou-Talalay combination index (CI): 1.1, no benefit. Results. The combination of PQR309 with the BCL2 inhibitor venetoclax (ABT199) gave positive results in 6/8 cell lines, with strong synergism in three (U2932, median CI=0.1, SU-DHL-6, 0.14, Karpas422, 0.14) and synergism in the remaining three (TMD8, 0.5, RIVA, 0.56, DOHH2, 0.65). No benefit was observed in SU-DHL-2 and OCI-LY18. The combination of PQR309 with the BTK inhibitor ibrutinib was synergistic in 3/4 ABC-DLBCL (RIVA, 0.42; U2932, 0.6; TMD8, 0.57), while it was of no benefit in SU-DHL-2. The combination of PQR309 with the immunomodulator lenalidomide was of benefit in 4/4 ABC-DLBCL: synergistic in three (RIVA, 0.38; U2932, 0.4; TMD8, 0.5) and additive effect SU-DHL-2 (1.01). The combination of PQR309 with the HDAC-inhibitor Panobinostat was synergistic in 5/6 (KARPAS422, 0.21; U2932, 0.65; SU-DHL-6, 0.67; DOHH2, 0.8; SU-DHL-2, 0.83), but not in the TMD8 (1.14). In our models, synergism was observed in only 2/5 cell lines (TMD8, 0.6; DOHH2, 0.89) exposed to both PQR309 and anti-CD20 monoclonal antibody, while not in KARPAS422, SU-DHL-6, or U2932. Finally, since we had previously observed an up-regulation of the PIM1 kinase after exposure to PQR309 (Tarantelli et al, ICML 2015), potentially acting as a mechanism of adaptive resistance, we evaluated the addition of the PIM inhibitor AZD1208 to PQR309 in four DLBCL cell lines. The combination showed synergism in two (OCI-LY1, 0.29; SU-DHL-10, 0.57), an additive effect in TMD8 (0.95) and no benefit in the SU-DHL-2 (1.15). Conclusions. The novel dual PI3K/mTOR inhibitor PQR309 showed synergism when combined with additional targeted agents in different DLBCL models, including some derived from double hit lymphomas. In particular, the combination with the BCL2 inhibitor venetoclax showed very good results, especially in cell lines bearing BCL2 gene deregulation due to chromosomal translocation or genomic amplification, and the combination with a PIM inhibitor might overcome early adaptive resistance to PQR309. These data provide the basis for future pre-clinical and clinical studies. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Stathis:PIQUR Therapeutics AG: Research Funding. Fabbro:PIQUR Therapeutics AG: Employment. Wicki:PIQUR Therapeutics AG: Employment, Research Funding. Cmiljanovic:PIQUR Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees. Bertoni:PIQUR Therapeutics AG: Research Funding; Oncology Therapeutic Development: Research Funding.

Description: BRAF V600E somatic mutation, found in all the classic HCL, does not fully explain all the distinctive morphologic, phenotypic, and immunogenetic features of HCL. Differently from other indolent B-cell neoplasms, HCL has a highly stable genomic profile. HCL is highly sensitive to purine analogs, including cladribine that has demethylating activity (Spungeon et al, 2009). We hypothesized that epigenetic changes might contribute to disease pathogenesis, and we investigated the genome-wide promoter methylation of a well characterized series of classical HCL. Methods DNA was extracted from circulating cells including at least 70% of neoplastic cells, of 11 BRAF V600E mutated HCL, 7 splenic marginal zone (SMZL, all negative for BRAF V600E mutation), and 29 chronic lymphocytic leukemia (CLL) and from 6 normal CD19+ B-cell (NBCs) samples. IGHV genes were mutated in all HCL and SMZL, and in 16/29 CLL cases. Samples were analyzed with the Illumina Infinium HumanMethylation27 arrays. Signal intensities and beta values were exported from Illumina Beadstudio 2.0 software. Probes interrogating CpG residues inside CpG islands (CGIs) or outside (non-CGI) were analyzed separately. Unsupervised analysis was performed using hierarchical clustering with Euclidean distance and Ward linkage. Supervised analyses were performed using a t-test on the continuous beta values, followed by the Benjamini-Hochberg multiple test correction. Probes showing q-value