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: 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: Mantle cell lymphoma (MCL) represents a subtype of B-cell lymphoma associated with a very unfavourable clinical outcome. Currently no therapy can be considered as standard, and new therapeutic approaches are needed. Forodesine is a potent inhibitor of purine nucleoside phosphorylase (PNP), whose major role is to catalyze the cleavage of inosine, deoxyinosine guanosine, and deoxyguanosine (dGuo) to their corresponding base and sugar 1-phosphate by phosphorolysis. In the presence of deoxycytidine kinase, PNP inhibition leads to an increase in the concentration of dGuo triphosphate (dGTP), followed by inhibition of DNA synthesis and cell death by apoptosis. When combined with dGuo, forodesine has been shown to have in vitro cytotoxic activity on T-cell (T-ALL, T-PLL) and on B-cell malignancies (CLL, B-ALL), and Phase I/II trials are on going in CLL and CTCL patients. Here, we report the first data on in vitro activity of forodesine in MCL. Primary MCL cells, derived from six patients, were exposed to forodesine (0, 2, 20 μM) in combination with dGuo (0, 10, 20 μM), for 48 hrs. Cells were cultured in X-VIVO 10 medium (Cambrex) with 10% FBS. Cell viability was assessed by flow cytometry with the Annexin V - propidium iodide assay. Four patient samples (67%) showed an increase in the number of Annexin V positive cells ranging from 1.9 to 5.3 times compared to untreated cells. The effect was larger for 20 μM forodesine compared with 2 μM. There was no effect of dGuo alone and only a minimal effect of increasing dGuo concentration from 10 μM to 20 μM. Cell lines did not appear to be ideal models to evaluate the efficacy of forodesine in vitro. Three established MCL cell lines (Granta-519, Rec, JeKo1) were treated with escalating doses of forodesine, but the results were not reproducible, while the same cells showed expected IC50 values between 25–30 μM when exposed to bendamustine for 72 hrs. In conclusion, the in vitro data reported here with 4/6 MCL patients primary samples sensitive to forodesine and the results from various groups on other T- and B-cell malignancies suggest that clinical trials of forodesine in MCL may be warranted.

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: 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: Abstract 2648 Follicular lymphoma (FL) is an indolent lymphoma and the second most common type of non-Hodgkin lymphoma in the Western world. It is characterized by the t(14;18) chromosomal translocation, which is present in up to 90% of cases. About 40% of FL cases eventually transform into a more aggressive lymphoma (tFL), most commonly diffuse large B-cell lymphoma (DLBCL). To identify the secondary chromosomal abnormalities that contribute to the development of FL, and to its transformation, we undertook a large study using the Affymetrix 250k NspI SNP array to identify copy number abnormalities (CNAs) in 198 FL and 79 tFL samples, 75% of which have concurrent gene expression profiling studies using Affymetrix U133+2 or A+B arrays for correlative analysis. There were 22 recurrent chromosomal abnormalities that were present in over 10% of FL cases, including gains of 7, 12, 18, 21, X, 1q, 2q, 5p, 6p, 8q, 17q and loss of 6q. We also identified 20 smaller CNAs that occurred in over 5% of FL cases, the most frequent being loss of chromosome 1p36.33-p36.31 including TNFRSF14, loss of chromosome 10q23.1-q25.1 encompassing several possible cancer-related genes such as PTEN, gain of chromosome 2p16.1-p15 including REL, and gain of chromosome 8q24.13-q24.3 including MYC. Univariate Cox regression models were used to analyze the CNA regions that occurred in at least 10 FL cases as predictors of overall survival. Four recurrent CNAs were predictive of survival in univariate analysis below the p=0.05 significance level, and two were found to be borderline significant. A gain of X or the p arm of X was predictive of poor survival. Additionally, two losses on 6q (6q13–15 and 6q23.3–24.1) were associated with poor survival. The 6q23.3–24.1 loss contains TNFAIP3, which encodes a negative regulator of the NF-kB pathway, and is a frequent site of homozygous loss. Additionally, a gain of chromosome 8 that includes the MYC gene, and a loss of chromosome 9 that includes CDKN2A, were borderline predictors of poor survival. Patients with FLs that have 7 or more abnormalities had worse survival than those with fewer abnormalities. We also compared the CNAs found in tFL samples to FL samples and identified 26 abnormalities that were at least 5 times more frequent in tFL and present in at least 5% of tFLs. A gain of 3q27.3-q28 containing 5 genes including BCL6 and LPP, for example, was found in 11% of tFL case, but only 2% of FL cases. We also found differences in the deletion of Beta-2 Microglobulin (B2M) between FL and tFL. The B2M locus is deleted in 8% of FLs, but in 21% of tFLs. B2M, a subunit of the MHC class I molecule, is known to be repressed by mechanisms such as mutation and deletion in de novo DLBCL, as a way for the tumor to evade immune surveillance. HLA-A- B, and/or -C were deleted in 5% of FLs and almost 9% of tFLs. CD58, which plays a role in T- and NK-cell immune responses, was deleted in 3% of FLs and 11% of tFLs. Overall, 19% of FLs and 37% of tFLs had an abnormality in CD58, B2M, and/or HLA class I, indicating that evasion of immune surveillance is important in transformation to a more aggressive disease. We also compared CNAs from tFL cases to those found in de novo GCB-DLBCL cases and identified several that differed markedly between the 2 diseases, such as a gain of chromosome 21 which was present in 21% of tFL cases but only 3% of DLBCL cases. In conclusion, FL, tFL, and de novo GCB-DLBCL share common CNAs, but the prevalence of the individual lesions differ among the 3 entities. Functional validation of potential candidate genes will determine important pathways in the development and progression of FL, and identify possible targets for therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

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

Description: Background IMGN529 is an antibody drug conjugate (ADC) consisting of an anti-CD37 antibody with direct anti-tumor activity conjugated via a thioether linker to the cytotoxic maytansinoid antimicrotubule agent DM1. IMGN529 has shown pre-clinical (Deckert et al, Blood 2013) and clinical activity in lymphoma (Stathis et al, ASH 2014; NCT01534715). Here, we assessed the anti-tumor activity of IMGN529 on a large panel of B cell and T cell human lymphomas to identify potential biomarkers of response. Methods Fifty-four lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), n.=27; mantle cell lymphoma (MCL), n.=10; anaplastic large T-cell lymphoma, n.=5; marginal zone lymphomas, n=6, others, n=6] were exposed to increasing doses of IMGN529 or to the unconjugated DM1 for 72h. Cell proliferation was measured using the MTT. Apoptosis induction was defined by at least 1.5-fold increase in caspase 3/7 signal activation with respect to controls using the Promega ApoTox-Glo Triplex Assay. CD37 surface expression was assessed by cytofluorimetry. Gene expression profiling (GEP) was done with the Illumina HumanHT-12 Expression BeadChips on untreated cell lines followed by GSEA (NES 〉 |2|, P

Description: Background. The PI3K/AKT/mTOR pathway is an important therapeutic target in lymphomas. PQR309 is a dual PI3K/mTOR inhibitor that has shown in vitroanti-lymphoma activity (Tarantelli et al, ASH2015) and is in phase 2 trial (NCT02249429, , NCT02723877, NCT02669511). PQR620 is a novel mTORC1/2 inhibitor that has shown preclinical activity in solid tumor models (Beaufils et al, AACR 2016). Here, we present the in vitro and in vivo anti-lymphoma activity of PQR620 as single agent and also the in vivo results of PQR620 or PQR309 containing combinations with the BCL2 inhibitor venetoclax. Materials and Methods. The drug concentration causing 50% inhibition of cell proliferation (IC50) was obtained in lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), no.=26; mantle cell lymphoma (MCL), no.=8; anaplastic large T-cell lymphoma, no.=5; others, no=5] exposed to increasing doses of PQR620 for 72h using a Tecan D300e Digital Dispenser on 384well plates. For in vivo experiments, NOD-Scid (NOD.CB17-Prkdcscid/J) mice were subcutaneously inoculated with 10 x106 (RIVA) or with 5 x106(SU-DHL-6) cells. Results. PQR620 had a median IC50 of 250 nM (95%CI, 200-269 nM) when tested on 44 lymphoma cell lines. Activity was higher in B cell (no.=36) than in T cell tumors (no.=8) (median IC50s: 250 nM vs 450 nM; P=0.002). At 72h, anti-tumor activityof PQR620 was mostly cytostatic and apoptosis induction was seen only in 6/44 cell lines (13%), Sensitivity to PQR620 or apoptosis induction did not differ between DLBCL and MCL, and they were not affected by the DLBCL cell of origin, by TP53 status or by the presence of MYC or BCL2 translocations. The activity of PQR620 as single agent underwent in vivo evaluation in two DLBCL models, the germinal center B cell type DLBCL (GCB-DLBCL) SU-DHL-6 and the acivated B cell-like DLBCL (ABC-DLBCL) RIVA. Treatments with PQR620 (100mg/kg dose per day, Qdx7/w) started with 100-150 mm3 tumors and were carried for 14 (SU-DHL-6) or 21 days (RIVA). In both models, PQR620 determined a 2-fold decrease of the tumor volumes in comparison with control, with significant differences in both SU-DHL-6 (D7, D9, D11, D14; P 〈 0.005) and RIVA (D14, D16, D19, D21; P 〈 0.005). Based on the previously reported synergy between the dual PI3K/mTOR inhibitor PQR309 and venetoclax (Tarantelli et al, ASH 2015), we evaluated the combination of the PQR620 or PQR309 with the BCL2 inhibitor venetoclax (100 mg/kg, Qdx7/w) in the SU-DHL-6 model. Both the venetoclax combination with the dual PI3K/mTOR inhibitor and the venetoclax combination with mTORC1/2 inhibitor were superior to the compounds given as single agents, leading to the eradication of the xenografts. The combination of PQR620 with venetoclax showed highly significant differences either versus control or single agents during all days of the experiment (D4, D7, D9, D11, D14; P 〈 0.001). Similarly, the combination of PQR309 with venetoclax showed highly significant differences versus venetoclax (D7, D9, D11, D14; P 〈 0.001) and PQR309 (D7, D9, D11; P 〈 0.005) alone. Conclusions. The novel mTORC1/2 inhibitor PQR620 had in vitro and in vivo anti-lymphoma activity as single agent. In vivo experiments showed that both PQR620 and the dual PI3K/mTOR inhibitor PQR309 can strongly benefit from the combination with the BCL2 inhibitor venetoclax. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Fabbro:PIQUR Therapeutics AG: Employment. Cmiljanovic:PIQUR Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees.