ALBERT

Description: Introduction: About 2 to 10% of patients (pts) diagnosed with Chronic Lymphocytic Leukemia (CLL) develop diffuse large B-cell lymphoma (DLBCL, so-called Richter transformation (RT)) over long-term follow-up. The outcomes of pts with RT are variable and poorly understood and there is no consensus on the best therapeutic approach. The aim of this study was to analyze the clinical characteristics, outcomes and factors predictive of survival in a large series of RT from the French Innovative Leukemia Organization (FILO). Methods: Biopsy-confirmed RT (limited to DLBCL and excluding Hodgkin lymphoma) diagnosed from 2001 to 2018 were identified from eight FILO centers. Clinical and biological characteristics of CLL and RT at diagnosis, including cytogenetics, clonal relation with the pre-existing CLL, Epstein-Barr virus (EBV) status, cell of origin (COO) analyzed by immunohistochemistry and RT score (Tsimberidou AM et al, J Clin Oncol, 2006) were analyzed as well as treatment and outcomes. Overall survivals (OS) were defined as time from CLL and RT diagnosis to death from any cause and analyzed using the Kaplan-Meier method. Statistical analyses were performed with SAS version 9.4. Results: A total of 70 CLL pts who developed RT were identified. The median age at CLL diagnosis was 62 years old (range 35-82), and 50 (71.4 %) were male. The median time to transformation was 5.5 years (range 0 to 22 years), with 12 simultaneous diagnosis of CLL and RT. Prior to RT, 20 (29%) pts had not been treated for CLL, 50 received one (n=21) or more (n= 29) line of treatment ; 6 pts had received a novel agent (ibrutinib, idelalisib or venetoclax). The median age at RT diagnosis was 68 years old (range 42-88). All biopsies were centrally reviewed; 38/58 pts (66%) had elevated LDH (〉1.5N) ; 35/65 pts (54 %) had bulky disease (≥ 5 cm); 10/54 (18.5%) pts had del(17p) or TP53 mutation ; 9/42 pts (21%) had a complex karyotype (at least 3 abnormalities). The CLL and RT were clonally related in 27/27 (100%) tested pts. COO by Hans algorithm was non germinal center B cell-like (GCB) in 26/28 pts (93%). EBV was positive or detected in 5/40 (12.5%) pts. The median of Ki67 positivity was 70% (range 30% to 100%). The RT score (based at RT diagnosis on ECOG performance status 2-4, LDH 〉1.5 x normal, platelets5 cm and 〉1 prior therapy for CLL) was : low risk in 17 pts (31%), low-intermediate risk in 10 pts (19%), high-intermediate risk in 14 pts (25%) and high risk in 14 pts (25%). The most common first-line treatment of RT was immunochemotherapy (n=57, 87%) including R-CHOP-like regimen (n=48, 73%). Autologous or allogeneic transplantation was performed for 7 pts (11%). Response to first-line treatment was complete or partial response in 26 pts (40%), and stable disease or progression in 39 pts (60%). After a median follow-up of 8 years, 51/64 pts (80%) have died. The main causes of death were progressive DLBCL (n=36, 71%), infection (n=8, 16%) or progressive CLL (n=2, 4%). The median OS of the cohort from CLL and RT diagnosis (Figure 1) were 7.8 years and 9.5 months, respectively. In univariate analysis, patients with TP53 disruption at CLL stage, low platelets count, elevated LDH, elevated beta2-microglobulin, high ECOG score, high RT score, EBV positivity and absence of response to first-line RT treatment had worse OS. The ECOG score, platelets count and TP53 disruption remain significant in multivariate Cox-regression. Last, we compared the clinical and biological parameters of two Richter groups defined as: (i) short-term survivors (48 months, n = 18). Long survival was significantly associated with elevated platelets count, low LDH, low ECOG, low RT score and response to RT first-line treatment. Discussion: The clinical outcomes of RT patients is poor and novel treatment options are needed. However, a group of long-term survivors was identified, characterized by elevated platelets count, low LDH, low ECOG, low RT score and response to immunochemotherapy. Disclosures Leblond: Astra Zeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Thieblemont:Roche: Honoraria, Research Funding; Gilead: Honoraria; Novartis: Honoraria; Kyte: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Cellectis: Membership on an entity's Board of Directors or advisory committees. Cymbalista:Janssen: Honoraria; Gilead: Honoraria; AstraZeneca: Honoraria; Sunesis: Research Funding; Roche: Research Funding; Abbvie: Honoraria. Guièze:Abbvie: Honoraria; Janssen: Honoraria; Gilead: Honoraria; Roche: Honoraria. Broseus:Janssen: Honoraria; Gilead: Honoraria; Novartis: Research Funding. Feugier:gilead: Honoraria, Research Funding, Speakers Bureau; janssen: Honoraria, Research Funding, Speakers Bureau; abbvie: Honoraria, Research Funding, Speakers Bureau; roche: Honoraria, Research Funding, Speakers Bureau.

Description: Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease in which BCR signaling capacity plays a central role in disease progression. Previously, we have shown that BCR engagement allows the identification of two groups of patients with a strong correlation between the in vitro cell survival response and the biological criteria (IgVH somatic hypermutations, ZAP 70 expression ) or the clinical stage. One group, corresponding to non evolutive patients, showed absence of a substantial BCR signaling (Non responder). In contrast, BCR signaling promoted in vitro cell survival in the group of Responder cases corresponding to evolutive patients. In the latter only, response to BCR ligation was associated with induction of NFkB and NFAT regulated genes, such as Cyclin D2 and CD23. Since NFAT and NFkB transcription factors, regardless of the patients clinical status, have been described as constitutively nuclear in B-CLL cells, the aim of our study was to investigate whether the functional implication of these factors was different between our two groups. First, we confirmed by confocal microscopy and band shift analysis that localization of NFAT and NFkB was uniformely nuclear independently of the CLL status and the BCR response capacity. By RQ-PCR , we also investigated the expression of the various isoforms of NFAT (NFAT1, 2, 3 and 4) and showed that their expression profile was similar to normal B cells. Despite the nuclear localization of NFkB and NFAT, DNA binding capacity was only enhanced in cases responsive to BCR stimulation. Finally, using specific Tat-peptide inhibitors of NFAT and NFkB nuclear transport upregulation of cyclin D2 and CD23 was abrogated and survival advantage was lost in responder cases thereby confirming the crucial role of these factors upon BCR stimulation. However, in non responder cases in which no CD23 or cyclin D2 induction was present, this inhibition had no impact on cell survival upon BCR stimulation . Nonetheless, these cells retained the ability to respond to ionomycin or PMA reflecting functional PKC/calcium-calcineurin signaling pathways. Altogether, our results showed that NFAT and NFkB were activated in responder cells only despite their homogenous expression in all cases. In conclusion, the inability of the cells from stable B-CLL cases to activate the NFAT and NFkB pathways upon BCR signaling is not related to a functional defect or abnormal expression of these factors but likely reflects an early signaling defect. Moreover, our results show that the use of peptide inhibitors may restore a stable profile in B-CLL cells from evolutive patients.

Description: Background Cytogenetic abnormalities are of key importance for predicting clinical course and response to therapy in patients with chronic lymphocytic leukemia (CLL). Trisomy 12 (tri12), the third most frequent chromosomal aberration in CLL patients (10-20%), is associated with an intermediate prognostic risk but represents a clinical heterogeneous entity. Recently, next generation sequencing have revealed recurrent mutations in genes that were unknown to be involved in CLL pathogenesis, including NOTCH1, MYD88, SF3B1, XPO1 and BIRC3. In patients harboring tri12, NOTCH1 mutations have been shown to be present in up to 25% of cases and to confer unfavorable outcome explaining in part the clinical heterogeneity of tri12 patients. To better understand the genetic basis and prognosis of tri12 patients, we performed a multicenter retrospective study combining extensive mutational and cytogenetic analysis. Methods Patients carrying tri12 were identified using fluorescence in situ hybridization (FISH) and/or chromosome banding (CB). Main clinical and biological characteristics were collected and included in univariate analysis of prognostic factors, comprising age, Binet stage (A vs. B-C), splenomegaly, lymphocyte doubling time (LDT), LDH, beta2microglobulin (B2M), CD38 expression, IGHV mutational status, percentage of interphase nuclei positive (INP) for tri12, additional FISH (del13q, del11q, del17p) or chromosomal aberrations and presence of complex karyotype (〉 2 CB abnormalities). Search for mutations was performed by Sanger direct sequencing for TP53 (exons 5-10), NOTCH1 (exon 34), MYD88 (exons 14-16), SF3B1 (exons 14-16) and XPO1 (exons 14-15). Primary and secondary endpoints were time to first treatment (TFT), response to therapy, time to next treatment (TNT) and overall survival (OS). Results The study population comprised a total of 177 untreated patients including 112 and 75 patients with stage A and B-C CLL, respectively. The median age at diagnosis was 62 years old (range, 31-87) and 33% of patients were female. B2M was superior to 4 mg/L in 30/92 (32%) patients and LDH elevated in 65%. CD38 expression was positive (〉30%) in 58% and IGHV status was unmutated in 60%. Among the whole study population, all patients were positive for tri12 by FISH and 158/165 by CB. Tri12 was associated by CB with tri19 in 21 patients (13.2%), tri18 in 12 patients (7.5%), tri3 in 1 patient (

Description: Background : Expression of ZAP-70 protein has been shown to be correlated with mutational status of immunoglobulin heavy-chain variable region (IgVH) genes, a major prognostic factor in CLL. We investigated whether the detection of ZAP-70 protein by flow cytometric analysis using unconjugated and conjugated monoclonal antibodies (mAbs) could be applied securely in the workup of patients with CLL. Methods: Flow cytometric analysis of ZAP-70 protein was performed using the method described by Crespo et al (N Engl J Med2003;348:1764) with minor modifications. Both fresh and cryopreserved mononuclear cells from CLL patients and healthy donors were fixed and permeabilized using Fix and Perm kit (Caltag Laboratories), incubated with anti-ZAP-70 mAb (clone 2F3.2, Upstate Biotechnology) and then revealed with goat antimouse FITC mAb (Immunotech). Finally cells were incubated with CD3-APC, CD56-APC and CD19-PC5. We also tested 3 mAbs conjugated to various fluorochromes: 2F3.2-FITC (Upstate), 1E7.2-PE (eBioscience), 1E7.2-PE or -Alexa 488 (Caltag). ZAP-70 protein detection in B-cells was expressed either as a percentage of its expression in the T and NK-cells or as a ratio (R) of T-cell mean cell fluorescence (MCF) to B-cell MCF. Western blotting of protein lysates from purified B-cells was carried out to control results obtained by cytometry in 55 samples. Mutational status was defined using a cutoff of 98%. Results: In 13 healthy donors, the mean percentage of ZAP-70 protein expression obtained by flow cytometry with unconjugated mAb (clone 2F3.2) was 4.69% ± 1.93 [range 2–9%] and the R ratio was 6.64 ± 1.54 and 〉 4.8. In 83 B-CLL samples, ZAP-70 expression was determined using the same method and compared to IgVH mutational status. Results in table below show a 75% concordance between gene mutations and ZAP-70 expression when considering a percentage of positive B-cells 〉 20%. A better concordance (81%) is obtained with a threshold T-cell MCF/ B-cell MCF at 4 determined by Youden’s index. To note the high concordance (90%) between unmutated status and ZAP-70 + expression (19/21). Comparison with at least 1 of the 3 conjugated mAbs has been performed for 63 samples, with discordant results in our laboratories. 62 mutated IgVH samples 21 unmutated IgVH samples ZAP-70 B-Cells + ≤ 20 % : 43 〉 20% : 19 T-cell MCF/B-cell MCF ≥ 4 : 48 〈 4 : 19 Conclusions: Our data document the concordance between IgVH gene mutational status and ZAP-70 protein expression measured by flow cytometry, particularly in ZAP-70 negative samples. We found that the indirect method of labelling with unconjugated anti-ZAP-70 mAb remains until now, in our hands, the gold standard method compared to the available dyes conjugate mAbs.

Description: New treatment approaches in CLL aim at lowering as much as possible the level of minimal residual disease (MRD) to prolong the duration of response and eventually overall survival. Such a goal should be achieved by combining chemotherapy and monoclonal antibodies. The present phase II study was designed to evaluate the efficacy and the safety of first line therapy with 3 courses of oral fludarabine and cyclophosphamide (FC : F 40 mg/m² and C 250 mg/m² D1 to D3 every 4 weeks) followed by consolidation treatment with alemtuzumab in previously untreated patients (pts) aged 65 to 70 years with Binet stages B and C CLL. Pts achieving complete response (CR) and partial response (PR) after FC were eligible for the second part of the study i.e. consolidation with alemtuzumab administered subcutaneously (SC) at the dose of 10 mg thrice a week for 8 weeks. CMV antigenemia was monitored every week and alemtuzumab was discontinued in pts with more than 3 positive nuclei. MRD was evaluated before and during alemtuzumab treatment using a highly sensitive 6 color flow cytometry technique. Since severe infectious toxicity has been reported by others with similar strategies, FC regimen was limited to 3 courses and the duration of alemtuzumab treatment to 8 weeks with stringent stopping rules for CMV in this trial targeting a population of elderly pts in first line treatment. From June 2004 to June 2006, 42 pts have been enrolled in the study of whom 37 are assessable so far for induction treatment with FC. CR was achieved in 11 pts (30%) and PR in 17 pts (46%) for an overall response rate of 76% (28 pts). FC regimen was discontinued in 5 pts (13.5%) and stable disease was observed in 3 pts (8%). All the responding pts but one (one woman with onset of breast cancer) proceeded to alemtuzumab consolidation after a 2 month rest period. Alemtuzumab therapy has been completed in 16 pts, discontinued in 5 pts because of CMV reactivation as defined above (none of them developed CMV disease) and is still on going in 6 pts. Blood MRD assessment by flow cytometry was centralized in one center (RL) and could be performed in 19 pts with a set of 14 pts having been tested sequentially before alemtuzumab, after 4 weeks and after 8 weeks of treatment. In most cases, there was an impressive decrease of MRD level following alemtuzumab therapy and residual tumor cells were no longer detected. Detailed results of MRD study will be presented at the meeting. In conclusion, 3 courses of FC yielded a high response rate in previously untreated elderly CLL patients and SC alemtuzumab consolidation could thereafter be administered safely in most of them, resulting in a striking reduction of blood MRD.

Description: Introduction: Median age of Chronic Lymphocytic Leukemia (CLL) patients is 72 years with 40% older than 75 and 22.8% over 80 years. Important therapeutic progresses have been made, including chemo-immunotherapy as well as the recent use of targeted therapies, leading to progression-free-survival (PFS) and overall survival (OS) improvements. Although the elderly represents the largest subgroup of CLL patients, they are underrepresented in clinical studies and little is known about their clinical characteristics, treatment and outcome. Consequently, results from trials cannot be directly translated into clinical practice for these patients. Bairey et al (Ann Hematol, 2011) reported a series of 214 patients (80 years or older) diagnosed in Israel between 1979 and 2009 with a mean age of 84. However, in this cohort, 56% of the patients had a Rai stage 0 and only 53 received treatment. Median survival was 56 months. Methods: We performed a retrospective study of CLL patients aged 80 or more at initiation of first line therapy. Patients were treated between 2003 and 2013, in 17 hospitals affiliated to the French Innovative Leukemia Organisation (FILO). We report here a cohort of 201 such CLL patients, and describe their clinical and biological characteristics, treatment options and outcome. Results: Patients' median age was 83.4 years (80-92), 29% were older than 85 years, and the sex ratio was 60% male/40% female. Performance status (97%≤ 2) and nutritional status (median Corporal Mass Index of 25.3 kg/m²) were preserved. The median Cumulative Index Rating Scale (CIRS) comorbidity score was 5. More precisely in term of fitness, 57.8% patients were characterized as "go-go" with a CIRS ≤ 6 and organ comorbidities 3, and CD38 was positive in 43,4% of the 129 cases tested (64%). Cytogenetic data were available for 42% of the patients. Isolated abnormalities were deletion 13q (38.1%), trisomy 12 (21.4%), deletion 17q (10.7%) or deletion 11q (7.1%). Besides, associated chromosomic abnormalities were detected, mainly by fluorescence in situ hybridization (FISH) and complex karyotypes (1.2%). At treatment initiation, Binet stage was either A (27.2%), B (28.7%) or C (41.5%). Therapies consisted mainly in Chlorambucil (65.5%), Bendamustine (10.5%) and Rituximab (44.3%). Indeed, therapy regimens were composed of Chlorambucil alone (45.3%) or chemo-immunotherapy (48.3%) including Rituximab+Chlorambucil (22.7%), Rituximab+Bendamustine (10.4%), Rituximab+Cyclophosphamide+Dexamethasone (5.5%) or Rituximab+Fludarabine+Cyclophosphamide (5.5%). In term of tolerance, 20.2% of the patients required hospitalization and 10% of these cases were febrile neutropenia. Finally, 31.8% required a dose reduction of chemotherapy. The Overall Response Rate was 65.9% with 31.4% of clinical complete remission. The median OS and PFS (from treatment initiation) were 48.6 and 18 months, respectively (cf. Survival curves). Afterwards, an important number of patients (41.3%) remained fit enough to receive a second line treatment. In univariate analysis, only comorbidities evaluated by the CIRS had a significant impact on survival (p=0.03). Indeed patients identified as fit by a CIRS score ≤ 6 and no organ comorbidity 〉 3 had a better outcome. Conclusion: We report a large series of elderly CLL patients, who received first line treatment at a median age of 83. Median OS was about 4 years, which is less than normal population of the same age. Our results suggest that treatment (including immunochemotherapy) is feasible, even in this very old population. Different bias are possible in this retrospective study including the selection of only fit patients, the low percentage of geriatric evaluation, and the possible undertreatment of this population since chlorambucil was the most frequent treatment. In the future, prospective trials should target this population. Oncogeriatric evaluation and new targeted therapies should be part of such future trials. Figure 1. Survival curve 1: Overall Survival Figure 1. Survival curve 1: Overall Survival Figure 2. Survival curve 2: Progression Free Survival Figure 2. Survival curve 2: Progression Free Survival Disclosures Dupuis: ROCHE: Speakers Bureau; ABBVIE: Membership on an entity's Board of Directors or advisory committees. Aurran-Schleinitz:CSLBehring: Honoraria; Janssen: Honoraria. Cymbalista:Karyopharm: Honoraria; Gilead: Honoraria; Roche: Honoraria; Janssen: Honoraria, Research Funding. Dilhuydy:Roche: Honoraria, Other: Travel reimbursement; Janssen: Honoraria, Other: Travel reimbursement; Mundipharma: Honoraria. Cazin:GILEAD,: Honoraria; ROCHE: Consultancy; MUNDIPHARMA: Honoraria, Research Funding; NOVARTIS: Honoraria. Leblond:Roche: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; GSK: Consultancy, Honoraria, Speakers Bureau; Mundipharma: Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau. Cartron:Sanofi: Honoraria; Gilead: Honoraria; Celgene: Honoraria; GSK: Honoraria; Roche: Consultancy, Honoraria.

Description: Abstract 1946 Poster Board I-969 STATEMENT OF PURPOSE: Rituximab (RTX) is a chimeric antibody targeting CD20, a membranous B-cell specific protein, currently used in the treatment of B-cell malignancies and various autoimmune diseases. Despite its clear efficacy in non-Hodgkin's lymphoma, clinical and biological responses are highly variable between patients. Therefore, predictive factors are necessary to adapt therapy to each individual as early as possible. MATERIAL AND METHODS: We studied a set of biomarkers in a cohort of 39 patients with indolent non-Hodgkin's B-cell lymphoma (low grade follicular lymphoma or marginal zone lymphoma) and a low tumour burden, treated with single agent RTX as follows: 4 weekly 375 mg/m2 infusions, associated with short-course oral steroids. All patients benefited from FDG-PET analysis before treatment and 10 weeks later, allowing their classification into 3 early response groups: complete isotopic remission, partial response and no response. Dosage of rituximabemia, BAFF and Human Anti-Chimeric Antibodies (HACA) was performed with in-house ELISAs before each infusion and 2 months later. Polymorphisms of FcγRIIIa[158], FcγRIIa[131] and C1qA[276] genes were determined using allele-specific PCR or direct sequencing. Results were compared using Student's t-Test or two-tailed Fischer's exact test when appropriated. RESULTS: Referring to PET analysis, 18 patients were classified as complete isotopic remission, and 21 as partial or no response. Mean age was significantly lower in the first group (54 y+/-19 vs 66+/-13, p=0.022), as well as initial SUVmax (6.3+/-3.4 vs 10.8+/-6.8, p=0.024). Serum levels of RTX were highly variable between patients at each timepoint, but were not statistically different between the different response groups. Basal levels of BAFF were not statistically different between lymphoma patients and a control population, and did not vary after RTX-induced B cell depletion. No patient developed a HACA response during the study. Homozygous FcγRIIIa158V/V genotype was significantly associated with a complete response (p=0.037). Conversely, FcγRIIa[131] and C1qA[276] polymorphisms were not associated with the response. Genetic polymorphisms seemed to modify RTX pharmacokinetics, since homozygous FcγRIIa[131]H/H, FcγRIIIa[158]V/V and C1qA[276]G/G subjects displayed lower levels of RTX than others, without reaching statistical significance. CONCLUSIONS: Younger age, lower initial SUVmax and FcγRIIIa[158]V/V genotype are associated with a better early response to RTX in indolent lymphoma patients, as assessed by PET analysis. FcγR polymorphisms affect RTX kinetics probably through a modification of the efficacy of antibody dependant cell cytotoxicity. Determination of these factors could help in adapting individually therapeutic protocols. Disclosures: No relevant conflicts of interest to declare.

Description: Antigen-driven signals are involved in the pathogenesis and progression of CLL with a particular occurrence within the lymph node microenvironment. We have previously shown that ex-vivo BCR engagement promoted simultaneous down-regulation of CXCR4 and CD62L membrane expressions in CLL cells from patients at risk of disease progression only. Functionally, cells down-regulating CXCR4 and CD62L in response to BCR triggering displayed both a reduction in migration toward CXCL12 and in adhesion to lymphatic endothelial cells. We studied a cohort of 73 previously untreated patients, 36 of whom were IGHV mutated and 37 were IGHV unmutated. The distribution of the extent of CXCR4 down-regulation was identical among CXCR4 responders, whether IGHV mutated (median 49% [Q1:28-Q3:62]) or unmutated (median 43% [Q1:31-Q3:66]). We also studied the expression of another chemokine receptor, CXCR5, which is also expressed at the membrane of CLL cells and plays an important role in the homing and trafficking of lymphocytes to the lymphoid follicles. Sustained antigenic stimulation of CLL cell samples (n=25) promoted down-regulation of CXCR4 and CXCR5 in a strictly correlated manner (r= 0,9) i.e. in the same subsets of cells. The extent of BCR-dependent decrease of the two proteins was strikingly representative of the heterogeneous capacity of the B-CLL cells to respond to BCR engagement in a given patient. Moreover, treatment with the specific PKD inhibitor CID755673 blocked significantly both CXCR4 and CXCR5 BCR-mediated decrease (n=11), demonstrating that PKDs specifically target these 2 molecules, whereas CD62L down-regulation was not significantly affected by the PKD inhibitor. At the functional level, treatment with the PKD inhibitor restored CLL cell migration capacity in response to CXCL12. PKD phosphorylation/activation in response to BCR stimulation, which involvesPI3K-d, was required for CXCR4-phosphorylation and its down-regulation. We then studied the clinical relevance of CXCR4 down-regulation after BCR engagement. The capacity to down-regulate CXCR4 was significantly related to shorter PFS (p=0.043). This cohort allowed exploring the link with IGHV mutational status. 36/37 unmutated IGHV cases had a significant CXCR4 down-regulation after in vitro BCR stimulation, in concordance with the ultimately constant progression of the disease. Conversely, IGHV mutated cases fell into two groups: a) one group of 14/36 patients, with very low (

Description: Zeta-chain Associated Protein of 70 kDa (ZAP-70) is the second member of the Spleen tyrosine Kinase (Syk) family, and plays a key role in T-cell Receptor signalling. An alternative variant of ZAP-70 was described in 2004 by Hirokawa et al. producing a shorter protein named Truncated ZAP-70 Kinase (TZK). ZAP-70 has also been found expressed in some B-cell subsets and in various B-cell malignancies. In CLL, the expression of ZAP-70 retains a prognostic significance; however, its function is not completely understood. Recently, whole genome sequencing identified mutations affecting the spliceosome such as SF3B1 mutations that are associated with poor outcome in CLL. In this work, we aimed at identifying novel alternative transcripts of ZAP-70 in normal lymphocytes and in CLL cells. We also explored the potential link between ZAP70 alternative transcripts and SF3B1 mutations. Peripheral blood mononuclear cells (PBMC) were obtained from normal subjects and CLL patients. T cells, NK cells and monocytes were purified by negative selection from 11 normal subjects. We explored the transcriptional profile of ZAP-70 by standard PCR using 4 combinations of primers spanning over the 14 exons of the transcript. PCR products were revealed by gel electrophoresis, purified and sequenced by Sanger method. The expression of ZAP-70 was studied by real-time quantitative PCR using SYBR Green® technique after designing specific primers for the four different transcripts individualized. The expression of the full length and truncated forms of FOXP1 was studied by real-time quantitative PCR as previously described (EHA2015 abst#4919). SF3B1 mutations were detected by Sanger sequencing of the exons 14, 15, and 16. In Jurkat cell line, we identified two new variants of ZAP-70 corresponding to the alternative splicing of exon 3 and exons 3 and 4. We named them Δ3 and Δ3-4 respectively after confirmation by Sanger sequencing. To detect both Δ3 and Δ3-4 in the same reaction, we designed a PCR encompassing exons 1 to 5, and we were able to detect ZAP-70, Δ3 and Δ3-4 in peripheral blood unselected normal cells. SYBR Green ® technique with new sets of primers allowed us to explore ZAP-70, TZK and the two new variants in selected NK cells and T lymphocytes. As previously described, ZAP-70 was more expressed in NK cells than in T cells, and at lower level in CLL cells. TZK was also detected in T-lymphocytes and NK cells, Δ3 and Δ3-4 were found at very low levels in both cell types and were absent from ZAP-70 negative CLL cells. We studied ZAP-70 transcripts in 13 ZAP-70 positive CLL. All alternative transcripts were present with a pattern of expression different to that of T-cells. We have shown recently that in all cases of CLL with SF3B1 mutation, a high expression of the FOXP1 truncated variant was observed as compared to a very low level in unmutated samples. This variant was highly expressed in the samples carrying five different mutations of SF3B1 (Y623C, R625H, K700E, G740V and G742D). Among our 13 ZAP70 positive patients, 2 cases harboured the SF3B1 Y623C mutation. In these two cases, the balance between the four ZAP-70 transcripts detected showed no obvious difference with wild-type SF3B1 ZAP-70 positive cases. In conclusion, we studied the transcription profile of ZAP-70 in various cells types and identified two novel alternative transcripts of ZAP-70 (Δ3 and Δ3-4). These transcripts were detected at various levels in all ZAP-70-positive cell types. Interestingly, we found that the presence of SF3B1 mutation impacted the splicing of FOXP1 transcription factor but not the balance between ZAP-70 transcripts We are currently expanding the cohort of ZAP-70 cases with other types of SF3B1 mutations. Mutations affecting the spliceosome such as SF3B1 mutations do not seem to impact transcription of ZAP-70. Disclosures Troussard: Roche: Honoraria; Janssen: Honoraria. Cymbalista:Janssen: Honoraria, Research Funding; Gilead: Honoraria; Roche: Honoraria; Karyopharm: Honoraria. Letestu:Alexion: Honoraria, Research Funding; Roche: Honoraria.

Description: Introduction: Autoimmune hemolytic anemia (AIHA) and immune mediated thrombocytopenia (ITP) are frequent complications of chronic lymphocytic leukemia (CLL)/small lymphocytic leukemia (SLL) that may evolve independently or occur at any stage of disease progression. Ibrutinib (Imbruvica®), a first-in-class BTK inhibitor, is a once-daily single-agent approved by the US FDA for CLL patients (pts) who had received ≥1 prior therapy, and for CLL pts with deletion 17p. Comprehensive efficacy and safety results from the interim analysis of the phase III RESONATE (PCYC-1112) study have previously been reported, demonstrating that improved progression-free survival (PFS) and overall survival were seen with ibrutinib (ibr) as compared to ofatumumab (ofa) in pts with previously treated CLL/SLL. Interim analysis data from the Phase III RESONATETM study are presented for pts with autoimmune complications, including that of a case report in which recurrent AIHA/ITP episodes resolved following initiation of ibr. Methods: History of AIHA and ITP along with status on study entry and resolution date when applicable were collected from 386 enrolled pts in both arms (ibr n=195; ofa n=191) who received study treatment. Ibr was administered at 420 mg once daily until PD or unacceptable toxicity. Ofa was administered at 300 mg followed by 2000 mg dose for up to 12 doses. Treatment emergent adverse events of ITP and AIHA are summarized for treated pts based on randomized arm as of the interim analysis. Pts with uncontrolled AIHA or ITP, defined as declining counts in the 4 weeks prior to randomization or requirement for steroids 〉20 mg/daily were excluded per study eligibility criteria. In addition, detailed medical history was reviewed for RESONATE patient MXC, as this patient was diagnosed with rapidly progressing CLL at its inception complicated by recurrent AIHA/ITP episodes over a 10-year course of CLL treatment. Results: In the RESONATE trial, median age was 67 years with 40% ≥70 years, and median number of prior therapies was 3 (ibr) vs 2 (ofa). In all treated pts (ibr n=195; ofa n=191), 29 (15%) pts in ibr arm had a history of AIHA with 20 (10%) ongoing at study entry, compared to 30 (16%) pts in the ofa arm with only 9 (5%) ongoing at study entry. 18 (9%) pts in ibr arm had a history of ITP with 12 (6%) ongoing at study entry, compared to 20 (10%) pts in ofa arm with 10 (5%) ongoing at study entry. Nine ibr and 8 ofa pts reported both AIHA and ITP at baseline, including patient MXC. No pts on the ibr arm developed treatment-emergent AIHA or idiopathic thrombocytopenic purpura. Two pts on the ofa arm developed AIHA, 1 of which was Grade 3/4. Two pts on the ofa arm developed idiopathic thrombocytopenic purpura, both of which were Grade 3/4. Case history showed that patient MXC underwent first-line fludarabine followed by alemtuzumab as consolidation treatment for rapidly progressing CLL diagnosed in 2004. Patient had unmutated IGHV status and deletion 17p. In 2005, the patient underwent autologous peripheral blood stem cell transplant. First AIHA episode was noted in 2007, followed by ITP in 2008 despite prior steroid and IVIG treatment. Recurrent episodes of AIHA (n=6) and ITP (n=3) transpired over the course of CLL, not always related to simultaneous disease progression. After several treatment failures during the 3rd AIHA episode and 2nd ITP episode, splenectomy was performed to obtain temporary clinical control of autoimmune events, and low dose steroids were successfully administered. In March 2013, patient was randomized to ibr as part of the RESONATE trial, steroid use was discontinued, and no further episodes of AIHA/ITP have been observed since ibr initiation. The patient showed Coombs test negativity after only a few weeks following ibr treatment, and no CLL progression has been observed to date. Conclusions: Efficacy and safety of ibrutinib has been evaluated in CLL/SLL pts including pts with ongoing AIHA/ ITP, both frequently noted complications of this disease. Data from the Phase III RESONATE study suggest that these CLL disease-related autoimmune complications did not limit ibrutinib treatment. This is supported by the lack of AIHA and ITP adverse events on the ibrutinib arm despite 19% having a history of these complications and further exemplified by a case report from the RESONATE study, where sequential episodes of severe AIHA/ITP ceased following ibrutinib initiation in the setting of disease control. Disclosures Montillo: Janssen: Honoraria. O'Brien:Amgen, Celgene, GSK: Consultancy; CLL Global Research Foundation: Membership on an entity's Board of Directors or advisory committees; Emergent, Genentech, Gilead, Infinity, Pharmacyclics, Spectrum: Consultancy, Research Funding; MorphoSys, Acerta, TG Therapeutics: Research Funding. Hillmen:Pharmacyclics, Janssen, Gilead, Roche: Honoraria, Research Funding. Dearden:Roche, GSK, Gilead, Janssen, Napp: Honoraria. Brown:Sanofi, Onyx, Vertex, Novartis, Boehringer, GSK, Roche/Genentech, Emergent, Morphosys, Celgene, Janssen, Pharmacyclics, Gilead: Consultancy. Barrientos:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Mulligan:Roche, Abbvie : Consultancy, Honoraria. Furman:Pharmacyclics: Consultancy, Speakers Bureau. Cymbalista:Janssen, Roche, GSK, Gilead, Mundipharma: Honoraria. Plascencia:Pharmacyclics: Employment. Chang:Pharmacyclics: Employment. Hsu:Pharmacyclics: Employment. James:Pharmacyclics: Employment. Byrd:Pharmacyclics: Research Funding.