ALBERT

Description: Introduction Among recently discovered B cell activators responsible for signaling events leading to B-cell activation and maturation, of particular interest are the Toll-like receptors (TLRs). TLR expression is heterogeneous and variable among B-cells. In addition, abnormal TLR levels/signaling may play an important role in the pathogenesis of lymphoma, particularly in splenic marginal zone lymphoma (SMZL), since TLR pathways are recurrently targeted by genetic changes in this lymphoma. Methods Frozen spleen tissue specimens from patients with SMZL (n=13) and splenic diffuse red pulp lymphoma with villous lymphocytes (SDRPL, n=5) were analyzed for the expression of TLR1 to TLR10 in comparison to control cases (traumatic spleen, n=7) using multi-parametric flow cytometry and quantitative mRNA Taqman assay. To identify the B-cell subset, the samples were also stained with anti-CD19 and anti-CD3. All cases were studied by morphological, immunological, cytogenetic and molecular analysis. Results The TLR profile obtained at protein level by flow cytometry was closely related to that obtained at mRNA level. The SMZL/SDRPL B-cells expressed all TLRs, but with variable levels of protein expression (low for TLR1, TLR2, TLR3, TLR8, TLR9, TLR10, and high for TLR4, TLR5, TLR6 and TLR7). On the other hand, distinct TLRs profiles were observed according lymphoma subtypes. The SDRPL cases showed indeed a significant TLR2 under-expression and TLR4/TLR7 over-expression in comparison to SMZL cases and control B-cells. SMZL cases exhibited a significant TLR4/TLR8 over-expression in comparison to control B-cells. These TLR profiles were not associated with specific cytogenetic features (presence of del7q or trisomy 3) and/or immunological profile (expression of the CD11c/CD27/isotype of immunoglobulin). But, in both entities, TLR4 expression was higher in cases with mutated IGHV than in cases with unmutated IGHV. The overexpression of TLR7 MyD88-dependent signaling molecules has been reported as pathogenic mechanism for autoimmune diseases; however no more autoimmune disease or circulating auto-antibodies were associated with SDRPL cases as compared to SMZL cases. As previously reported, all cases but one SMZL case presented unmutated MyD88 (L265P mutation), and MyD88 protein evaluated here by flow cytometry was similarly expressed in both entities. While TLR7 stimulation is known to induce CD38 expression, all SDRPL cases were CD38 negative; while in SMZL cases, higher TLR7 expression was observed in CD38 positive as compared to CD38-negative cases. This may suggest the existence of an abnormal TLR7 pathway in SDRPL as opposed to SMZL. Conclusion TLR profiling should allow a better understanding of the mechanisms involved in SMZL/SDRPL pathogenesis. Present data suggest an abnormal TLR pathway involving NF-kB and/or MyD88 in the SDRPL entities. Disclosures: No relevant conflicts of interest to declare.

Description: Chromosomal translocations involving the MYC oncogene (8q24) are known to occur in Burkitt lymphomas/leukemias (BL) but also in 5–30% of diffuse large B-cell lymphomas which are usually highly aggressive (8q24 DLBCL). Two recent conventional cytogenetics (CC) studies showed that secondary chromosomal abnormalities have a negative prognostic impact, especially13q abnormalities (frequently described as a deletion and usually associated with a complex karyotype, i.e. 〉 3 chromosomal alterations), mainly in childhood mature B-cell lymphomas, and in a less extent 7q, 3q, 22q and chromosome 17. M-FISH was applied to 120 (74% adults and 26% children) high grade B-cell non-Hodgkin lymphomas (86% BL, 14% 8q24 DLBCL) carrying MYC rearrangement and complex karyotype and/or 13q abnormality in order to find recurrent and/or cryptic chromosomal alterations. Abnormal metaphases were available in 96 (80%) cases. We described ‘new’ (not seen in CC) chromosomal rearrangements in 50 (52%) cases, refined those seen by CC in 28 (29%) cases and confirmed CC abnormalities in 18 (19%) cases. M-FISH allowed to characterize 55 structural 13q abnormalities in 42 patients (21 ‘new’ alterations): 24 der(13q) leading to partial del(13q) and partial gain of different partner chromosomes [mainly 1q or 7q], 15 del(13q) [of 2 minimal regions : q14 & q31q34], 13 gains (only 2 seen by CC) and 3 balanced translocations. Combined results of CC and M-FISH showed that the most frequent abnormalities among patients with complex karyotype involved 1q, 7q, Xq, 3q, 18q, 6q, 17p and chromosome 22 (excluding 8q24 translocations). 79 1q abnormalities were detected in 54 patients (26 % ‘new’): mostly gains (minimal amplified region: q22q31) due to unbalanced translocations with chromosomes 13, 22, 7 (59%) or duplications (25%). 55 partial 7q gains were observed in 42 patients (22% ‘new’), mostly +7 or unbalanced translocations with 13q, 6q or 1q. The other most common abnormalities were: t(14;18) in 8q24 DLBCL, del(6q), der(3q) with various partners leading to partial loss of 3q, monosomy 17 or del(17p) and numerical changes of chromosomes 22 (monosomy) and X. In conclusion, this study confirms the contribution of M-FISH in refining CC results in highly aggressive 8q24 B-cell lymphomas: ‘new’ rearrangements were identified especially in 1q (leading to partial 1q gains), 18q, 6q (leading to partial 6q deletions), 13q ; partial 13q gains were underestimated by CC and both 13q deletions and gains were more heterogeneous than expected. We are characterizing these prognostic additional chromosomal abnormalities with SNP-CHIPS 50K array (Affymetrix) to look for candidate genes and/or cellular pathways involved in Burkitt lymphomagenesis in cooperation with oncogenic effect of MYC. °on behalf of the GFCH (Groupe Francophone de Cytogénétique Hématologique) and the BCG-Ho (Belgian Cytogenetic Group of Hematology and Oncology).

Description: B-PLL is defined by the presence of prolymphocytes in peripheral blood exceeding 55% of lymphoid cells. The diagnosis, mainly based on clinical and morphological data, can be difficult because of overlap with other B-cell malignancies. Because of the rarity of the disease, only case reports and small series describe its cytogenetic features. Few prognostic markers have been identified in this aggressive leukemia usually resistant to standard chemo-immuno therapy. We report here the cytogenetic and molecular findings in a large series of B-PLL. We also studied the in vitro response to novel targeted drugs on primary B-PLL cells. The study included 34 cases with a diagnosis of B-PLL validated by morphological review performed by three independent expert cytologists. The diagnosis of mantle cell lymphoma was excluded by karyotype (K) and FISH using CCND1, CCND2 and CCND3 probes. Median age at diagnosis was 72 years [46-88]. K was complex (≥3 abnormalities) in 73%, and highly complex (HCK≥5) in 45%. Combining K and FISH data, the most frequent chromosomal aberrations were: translocation targeting the MYC gene [t(MYC)] (21/34, 62%), 17p deletion including TP53 gene (13/34, 38%), trisomy 18/18q (10/33, 30%), 13q14 deletion (10/34, 29%), trisomy 3 (8/33, 24%), trisomy 12 (8/34, 24%) and 8p deletion (7/31, 23%). Whole-Exome Sequencing analysis of paired tumor-control DNA was performed in 16 patients. The most frequently mutated genes were TP53(6/16, 38%), associated with del17p in all, MYD88 (n=4), BCOR (n=4), MYC (n=3), SF3B1 (n=3), FAT1 (n=3), SETD2 (n=2), CHD2 (n=2), CXCR4 (n=2), BCLAF1 (n=2) and NFASC (n=2). Distribution of the chromosomal aberrations is shown in Fig 1. The main group of patients (21/34, 62%) had a t(MYC) that was associated with a higher % of prolymphocytes (86 vs 76, p=0.03), CD38 expression (90% vs 15%,p

Description: Primary plasma cell leukemia (PCL) is a rare plasma cell malignancy. Consequently, few large reports have been published. Presented is a cytogenetic analysis of 40 patients with primary PCL compared with 247 newly diagnosed patients with stage III multiple myeloma (MM). Cytogenetic abnormalities were observed in 23 of 34 patients, with usually complex hypodiploid or pseudodiploid karyotypes. Analysis of rearrangements of the 14q32 region revealed significant differences with high cell mass MM—a higher incidence of t(11;14) (33% vs 16%; P 

Description: The t(14;18)(q32;q21) involving the MALT1 and IGH genes is a recurrent abnormality in MALT lymphomas. So far, molecular genetic characterization of the t(14;18)/IGHMALT1 has only been performed in 2 cases and revealed a fusion of the entire coding region of MALT1 to the IGH locus. We herein report the molecular genetic analyses of 2 new cases of MALT lymphoma harboring the t(14;18)/IGH-MALT1 using fluorescence in situ hybridization (FISH) and we determined the molecular characteristics at the IGH-MALT1 junctions using long-distance PCR (LD-PCR). The first case, a 71-year-old female, presented with an extranodal MALT lymphoma of the conjunctiva, stage IEA. The second case, a 53-year-old-male patient, was diagnosed as having a MALT lymphoma originating from the lung. FISH with PAC clones 117B5 and 59N7 revealed a translocation involving MALT1. Further FISH assays with probes hybridizing to MALT1 and IGH showed the t(14;18)(q32;q21)/IGH-MALT1. By FISH with specific probes for the P53, P16, RB1, and ATM genes no deletions of these genes were found. The IGH-MALT1 fusion was confirmed by LD-PCR on patients’ DNA with nested primers for the MALT1 and IGH genes. Cloning and sequencing of the purified PCR products revealed a fusion of sequences upstream of the coding region of MALT1 to the JH segment of the IGH locus in both cases. The breaks on chromosome 18 were located in the 5′ non-coding region of MALT1, only 13 nucleotides apart from each other. The breaks at IGH were located in the JH4 joining segment in both cases and showed features of a V(D)J-mediated recombination. Deletion and “de novo” nucleotides additions at the point of joining were observed in case 1. Furthermore, a detailed analysis of the “de novo” nucleotides additions in this case revealed the presence of DH segments of the DH gene D3-10 in the JH/MALT1 junction. Our findings indicate that the pathomechanism underlying the t(14;18)/IGH-MALT1 in MALT lymphomas is probably based on an illegitimate V(D)J recombination at IGH, similar to other IGH-associated translocations, such as the t(14;18)/IGH-BCL2 in follicular lymphomas and the t(11;14)/CCND1-IGH in mantle cell lymphomas and that the events leading to the t(14;18)/IGH-MALT1 might take place during an initial DH-to-JH or a later VH-to-DJH joining.

Description: Abstract 582 Chromosomal translocations (t) are usually analyzed as one group, and are associated with poor prognosis in chronic lymphocytic leukemia (CLL). Translocations involving immunoglobulin (IG) genes are recurrent, but uncommon (〈 5%) in CLL. The two most frequent IG-partners are BCL2 (18q21) and BCL3 (19q13). On the behalf of the Groupe Francophone de Cytogenetique Hematologique (GFCH), we report an extensive analysis of 75 t(14;18)-CLLs, and a comparison to our previously published series of 29 t(14;19)-CLLs (Chapiro et al, Leukemia 2008). The 75 t(14;18)-CLLs or variant BCL2-t have been collected between 1985 and 2009. The morphological and immunological reviews were performed by KM, CS, and HM-B. All karyotypes were reviewed by the GFCH. Fluorescence in situ hybridization analyses were performed to detect IG and BCL2 rearrangements, trisomy 12, and deletions of 11q22 (ATM), 17p13 (TP53), 6q21, 13q14 (D13S319). IGHV mutation analyses were performed by referring laboratories. Statistical analyses were carried out using the Fisher's exact test, and continuous data using the Mann-Whitney test. Overall survival (OS) and treatment free survival (TFS) calculated from diagnosis were estimated using the Kaplan-Meier method, and the statistic significance was determined using log-rank test. Among BCL2-CLL, the sex ratio was 57M/18F, the median age at diagnosis was 66 years; of 68 patients with available data, 63 (93%) presented with Binet stage A; median lymphocytosis was 14.6×109/l. There were 47/75 (63%) “classical” CLL and 28/75 (37%) “atypical” CLL, with more than 10% of lymphoplasmacytoid cells and/or large cells. All tested cases (58/58) were CD10-, 69/73 (94%) were CD5+, and 44/63 (70%) were CD38-; 57/68 (84%) had a Matutes score 〉 4, 7/68 (10%) a score = 3, 4/68 (6%) a score 〈 3. We observed 62 t(14;18) and 13 variant translocations [11 t(18;22), 2 t(2;18)]. The karyotype was complex (〉 3 abnormalities) in 15/74 (20%) cases, and the BCL2-t was isolated in 25/74 (34%) cases. There were 33/75 (44%) tri12, 32/68 (47%) del13q14, 1/72 (1%) delTP53, 0/72 (0%) delATM, 0/59 (0%) del6q21. Of 42 analyzed cases, 33 (78%) were mutated. Finally, the median time from diagnosis to first therapy was 24 months (m). Comparisons with the BCL3-CLL showed no difference in sex ratio, age, and Binet stages. The lymphocytosis was lower in BCL2-CLL (14.6 vs 24.4 x109/l, p

Description: Abstract 1594 Background: Follicular Lymphoma (FL) is the most frequent low-grade NHL. Clinical course is heterogeneous, some patients (pts) presenting an indolent clinical course with overall survival (OS) over 15 y and others developing a more aggressive disease with shorter survival. The Follicular Lymphoma International Prognostic Index (FLIPI) or FLIPI-2 are commonly used to predict pts outcome, but fail to identify pts with a really poor prognosis. At diagnosis, few FL pts present with detectable leukemic phase (FL-LP) and this characteristic has been seldom described. Aim: The aim of the study was to describe the clinical features and outcome of FL-LP pts. Results: Among 499 pts diagnosed with FL according to the WHO criteria in the Centre Hospitalier Lyon Sud (transformation and grade 3b excluded) and treated between 01/1992 and 01/2012, 37 (7.4%) had characteristic FL-LP detected by cytological blood smears analysis with confirmation by flow cytometry (kappa/lambda clonality). Median age was 58 y and FLIPI score repartition was 4 pts in low, 16 in intermediate, and 17 in high risk groups. Splenomegaly was present in 23 pts, high tumour burden (GELF criteria) in 11, B symptoms in 8, and ECOG PS〉1 in 3. Seven pts had anaemia, 17 platelets UNL, and 11 LDH 〉UNL. The circulating lymphoma cells expressed the CD10 in 29/37 cases and surface Ig expression was detected in 31/35 cases, mainly IgM or IgG isotype. The median count of circulating lymphoma cells was 1.95.109/L, ranging from 0.6 to 129.109/L, 15 pts having count 〉4.109/L and 6 pts 〉10.109/L. Cytogenetic data were available for 21 pts, 20 carried the t(14;18), or its variant t(18;22), 16 of them having complex karyotype. Two pts were on watchful waiting for 24 and 82 m and 35 received a chemotherapy regimen at diagnosis including rituximab in 27 cases. Overall response rate was 83% (29/35) with 23 CR/CRu. Median progression-free survival (PFS) was 29 m. PFS and OS estimates were 37% and 86% at 5 y and 31% and 68% at 10 y, respectively. Splenomegaly (P=.035), high tumour burden (P=.017), lymphoma cells count greater than 4.109/L (P=.028), β2-m〉UNL (P=.036), and thrombocytopenia (4.109/L (HR 5.92; P=.000916) as independent prognostic factors. After progression, 12 pts received high-dose therapy (HDT) with HSCT as salvage with a long second PFS (68% at 10 y) compared to 8.3 m median PFS in first line. Pts not receiving HDT at salvage had a median second PFS of 27 m. To further evaluate the impact of FL-LP on pts outcome, a 1:3 matched analysis was performed. The FL-LP 37 pts were successfully matched with 111 newly diagnosed FL without FL-LP according to FLIPI score, age, treatment type (abstention vs chemotherapy, with or without rituximab) and treatment period (before or after 2000). In these 111 matched pts, 5- and 10-y OS was 97% and 91%, respectively. Considering all 148 pts, high FLIPI score, presence of FL-LP, and β2-m〉UNL were all significantly associated with a worse PFS. In a Cox regression model for PFS (120/148 pts with complete data), high FLIPI score (P=.0034; HR=2) and presence of FL-LP (P=.0085; HR=2.2) remained independently associated with shorter PFS. High FLIPI score and presence of FL-LP were also associated with a shorter OS. Interestingly pts with less than 4.109/L of circulating lymphoma cells had a similar PFS than those without. When circulating lymphoma cells 〉4.109/L as variable (see abstract figure) instead of FL-LP were tested in the Cox regression model for PFS including β2-m〉UNL and FLIPI score as variables, the most significant predictor for a shorter PFS was circulating lymphoma cells 〉4.109/L (P=.0004; HR=3.56) as compared to FLIPI score (P=.051; HR=1.6) and β2-m (P=.09; HR=1.52). Conclusion: The presence of circulating lymphoma cells in FL is a rare event and is associated with shorter PFS independently of FLIPI score and β2-m level. A validation of these findings on pts from the PRIMA study is on progress. However, this population is not homogenous and pts with circulating lymphoma cells 〉4.109/L have a poorer outcome. Although ∼1/3 of the pts experience long term PFS, these pts should be monitored carefully during and after first line treatment to consider HSCT as a therapeutic option to achieve a sustained response. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Ibrutinib, a first-in-class, oral, covalent inhibitor of Bruton's tyrosine kinase, is approved in many countries for treatment-naïve (TN) and previously treated CLL, supported by improved response rates, progression-free survival (PFS), and overall survival (OS) vs chlorambucil in RESONATE-2™ (NCT01722487; Burger JA, et al. N Engl J Med. 2015;373:2425-2437) and ofatumumab in RESONATE™ (NCT01578707; Byrd JC, et al. N Engl J Med. 2014;371:213-223). We analyzed survival outcomes for ibrutinib vs RW treatments for TN and relapsed/refractory (R/R) CLL by del11q status, an adverse prognostic factor linked with poor patient outcomes despite conventional chemoimmunotherapy. An adjusted comparison restricted to patients with confirmed del11q status was conducted using patient-level data from RESONATE-2™ and RESONATE™ and RW databases from 2 countries. Methods The Lyon-Sud RW database holds medical records for CLL patients diagnosed between 1980 and 2017 from the Centre Hospitalier Lyon-Sud, France; the Chronic Lymphocytic Leukemia Registry (CLLEAR) RW database holds medical records for CLL patients diagnosed between 1988 and 2017 from 7 academic centers in the Czech Republic. TN CLL database patients were selected using RESONATE-2™ criteria (which excluded those aged 〈 65 years or del17p positive). For the R/R group, patients ≥ 18 years of age or those with del17p were allowed per RESONATE™ inclusion criteria. PFS and OS outcomes by del11q status were compared between the ibrutinib arms of RESONATE-2™/RESONATE™ and physicians' choice (PC) treatment in the pooled RW databases (excluding ibrutinib). A multivariate Cox proportional hazards model was fitted on pooled RCT/RW data to estimate adjusted hazard ratios (HRs) for effect of ibrutinib vs RW PC treatment using age, sex, and treatment line as covariates. The unit of observation for the RW databases was the treatment line (rather than patient) number; RW patients receiving multiple lines of therapy contributed to multiple observations, and baseline was defined as the line-specific treatment start date. Results For the RW TN cohort, 466 treatment lines were analyzed; 134 (28.8%) patients were del11q positive. In RESONATE-2™, 29/136 (21.3%) TN patients were del11q positive. The table below shows adjusted HRs for PFS and OS for patients with/without del11q. For the del11q-positive subgroup, adjusted HRs for ibrutinib vs RW PC first-line (1L) therapy were 0.02 (95% confidence interval [CI], 0.00-0.17; p = 0.0002) for PFS and not estimable for OS (no deaths on ibrutinib). For the del11q-negative subgroup, adjusted HRs were 0.34 (95% CI, 0.2-0.57; p 〈 0.0001) for PFS and 0.74 (95% CI, 0.37-1.49; p = 0.3957) for OS. For second-line (2L) PC treatment in the RW R/R cohort, 385 treatment lines were analyzed; 135 (35.1%) were from del11q positive patients. In RESONATE™, 13 (37.1%) 2L patients were del11q positive. For the del11q-positive subgroup, adjusted HRs for ibrutinib vs RW PC of 2L therapy were 0.03 (0.00-0.21; p = 0.0003) for PFS and 0.08 (0.01-0.46; p = 0.0050) for OS. For the del11q-negative subgroup, adjusted HRs were 0.27 (0.14-0.51; p 〈 0.0001) for PFS and 0.50 (0.24-1.05; p = 0.0670) for OS. For 2L and beyond (2L+) treatment in the RW R/R cohort, 727 treatment lines were analyzed; 291 (40.0%) were from del11q positive patients. In RESONATE™, 63/195 (32.3%) 2L+ patients were del11q positive. For the del11q-positive subgroup, adjusted HRs (95% CIs) for ibrutinib vs RW PC of 2L+ therapy were 0.13 (0.08-0.20; p 〈 0.0001) for PFS and 0.15 (0.08-0.26; p 〈 0.0001) for OS. For the del11q-negative subgroup, adjusted HRs were 0.20 (0.14-0.29; p 〈 0.0001) for PFS and 0.33 (0.21-0.52; p 〈 0.0001) for OS. Concl usions Adjusted comparisons of the registration trial (RESONATE-2™/RESONATE™) and RW patient-level data suggest that OS and PFS improvements with ibrutinib vs PC therapies are pronounced for patients with CLL harboring del11q compared with those who are del11q negative. The findings inform physicians of the comparative effectiveness of ibrutinib in the RW for patients with this high-risk cytogenetic abnormality. Funding Source: This project was sponsored by Janssen Pharmaceutica NV, and Pharmacyclics LLC, an AbbVie Company. The real-world databases are independently owned. Writing assistance was provided by Emma Fulkes of PAREXEL and funded by Janssen Pharmaceutica NV. Disclosures Salles: Takeda: Honoraria; Acerta: Honoraria; Epizyme: Honoraria; Pfizer: Honoraria; Amgen: Honoraria; AbbVie: Honoraria; Gilead: Honoraria; Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Servier: Honoraria; Janssen: Honoraria; Merck: Honoraria; Morphosys: Honoraria. Besson:Janssen Pharmaceutica NV: Employment. Doyle:Janssen Pharmaceutica NV: Employment. Garside:Janssen Pharmaceutica NV: Employment. Spacek:Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria. Doubek:Novartis: Consultancy; AbbVie: Consultancy, Research Funding; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Affimed: Research Funding; Gilead: Consultancy, Honoraria, Research Funding.