ALBERT

Description: Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B cell receptor immunoglobulins (BcR IG). Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR IG stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR IG stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. In order to address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29,856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed 'satellites', were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

Description: Chronic lymphocytic leukemia (CLL) is preceded by monoclonal B cell lymphocytosis (MBL), characterized by the presence of monoclonal CLL-like B cells in the peripheral blood, yet at lower numbers than those required for the diagnosis of CLL. MBL is distinguished into low-count (LC-MBL) and high-count (HC-MBL), based on the number of circulating CLL-like cells. While the former does not virtually progress into a clinically relevant disease, the latter may evolve into CLL at a rate of 1% per year. In CLL, genomic studies have led to the discovery of recurrent gene mutations that drive disease progression. These driver mutations may be detected in HC-MBL and even in multipotent hematopoietic progenitor cells from CLL patients, suggesting that they may be essential for CLL onset. Using whole-genome sequencing (WGS) we profiled LC-MBL and HC-MBL cases but also CLL patients with stable lymphocytosis (range: 39.8-81.8*109 CLL cells/l) for 〉10 years (hereafter termed indolent CLL). This would refine our understanding of the type of genetic aberrations that may be involved in the initial transformation rather than linked to clinical progression as is the case for most, if not all, CLL driver mutations. To this end, we whole-genome sequenced CD19+CD5+CD20dim cells from 6 LC-MBL, 5 HC-MBL and 5 indolent CLL cases; buccal control DNA and polymorphonuclear (PMN) cells were analysed in all cases. We also performed targeted deep-sequencing on 11 known driver genes (ATM, BIRC3, MYD88, NOTCH1, SF3B1, TP53, EGR2, POT1, NFKBIE, XPO1, FBXW7) in 8 LC-MBL, 13 HC-MBL and 7 indolent CLL cases and paired PMN samples. Overall similar mutation signatures/frequencies were observed for LC/HC-MBL and CLL concerning i) the entire genome; with an average of 2040 somatic mutations observed for LC-MBL, 2558 for HC-MBL and 2400 for CLL (186 for PMN samples), as well as ii) in the exome; with an average of non-synonymous mutations of 8.9 for LC-MBL, 14.6 for HC-MBL, 11.6 for indolent CLL (0.9 for PMN samples). Regarding putative CLL driver genes, WGS analysis revealed only 2 somatic mutations within NOTCH1, and FBXW7 in one HC-MBL case each. After stringent filtering, 106 non-coding variants (NCVs) of potential relevance to CLL were identified in all MBL/CLL samples and 4 NCVs in 2/24 PMN samples. Seventy-two of 110 NCVs (65.5%) caused a potential breaking event in transcription factor binding motifs (TFBM). Of these, 29 concerned cancer-associated genes, including BTG2, BCL6 and BIRC3 (4, 2 and 2 samples, respectively), while 16 concerned genes implicated in pathways critical for CLL e.g. the NF-κB and spliceosome pathways. Shared mutations between MBL/CLL and their paired PMN samples were identified in all cases: 2 mutations were located within exons, whereas an average of 15.8 mutations/case for LC-MBL, 8.2 for HC-MBL and 9 for CLL, respectively, concerned the non-coding part. Finally, 16 sCNAs were identified in 9 MBL/CLL samples; of the Döhner model aberrations, only del(13q) was detected in 7/9 cases bearing sCNAs (2 LC-MBL, 3 HC-MBL, 2 indolent CLL). Targeted deep-sequencing analysis (coverage 3000x) confirmed the 2 variants detected by WGS, i.e. in NOTCH1 (n=1) and FBXW7 (n=1), while 4 subclonal likely damaging variants were detected with a VAF 10 years display similar low genomic complexity and absence of exonic driver mutations, assessed both with WGS and deep-sequencing, underscoring their common low propensity to progress. On the other hand, HC-MBL comprising cases that may ultimately evolve into clinically relevant CLL can acquire exonic driver mutations associated with more dismal prognosis, as exemplified by subclonal driver mutations detected by deep-sequenicng. The existence of NCVs in TFBMs targeting pathways critical for CLL prompts further investigation into their actual relevance to the clinical behavior. Shared mutations between CLL and PMN cells indicate that some somatic mutations may occur before CLL onset, likely at the hematopoietic stem-cell level. Their potential oncogenic role likely depends on the cellular context and/or microenvironmental stimuli to which the affected cells are exposed. Disclosures Stamatopoulos: Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses. Ghia:Adaptive: Consultancy; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Honoraria, Research Funding.

Description: B-Cell Receptor (BCR) triggering and responsiveness play a crucial role in the survival and expansion of Chronic Lymphocytic Leukemia (CLL) clones. In the recent past, several groups including ours have investigated the activation status of the signaling pathways originating from the leukemic BCR. Specifically we found that around 50% of CLL patients display a biochemical signature characterized by constitutive phosphorylation of ERK1/2 (pERK(+)) and constitutive nuclear translocation of NF-ATc1. These cases are unable to respond in vitro to BcR stimulation and are resistant to spontaneous apoptosis, thus resembling B lymphocytes previously anergized in vivo. Similar biochemical and functional features have been recently demonstrated in B leukemic cells persisting in the blood in patients treated with the BTK inhibitor, Ibrutinib, thereby making anergy an attractive target on the way to obtain eradication of the disease. CLL-associated B cell anergy can be specifically targeted by using different MAPK-inhibitors that have been shown to induce apoptosis selectively in the group of pERK(+) CLL. These data suggested that MAPK signalling can be efficiently inhibited in CLL for therapeutic purpose and that the phosphorylation status of ERK1/2 may represent a reliable biomarker to predict and monitor treatment response. However, even if the tested compounds were shown to be extremely efficient in inhibiting ERK1/2 phosphorylation in vitro, a lack of clinical activity was reported for many of them when tested in patients, mostly with solid tumors. In the present work, we used Trametinib, a specific MEK1/2 inhibitor, recently approved as a single-agent for the treatment of V600E mutated metastatic melanoma, and we investigated, at preclinical level, its activity in both primary CLL samples and a xenograft leukemic mouse model. Trametinib treatment completely inhibited constitutive ERK1/2 phosphorylation in 10 pERK1/2(+) samples at 3uM after 30 minutes treatment. Additionally, in 23 patients Trametinib treatment for 48 hours reduced cell viability in the cells from all 12 pERK1/2(+) patients (28,2% ± 3,5 mean survival) tested as compared to those from the pERK(-) group (11 cases, 58,1% ± 3,8 mean survival, p〈 0,0001). To strengthen our in vitro data, we evaluated the effect of Trametinib administration in the xenograft Rag2-/-gc-/- mouse model subcutaneously transplanted with the CLL cell line MEC1, characterized by specific features of anergy. Mice were subcutaneously injected with 10x106 cells and then challenged with Trametinib (oral gavage with 1mg/kg or with vehicle alone) starting from day 21 after tumour injection for 14 days. The effect of the inhibitor was monitored by tumour volume growth. Trametinb administration delayed tumour growth (p

Description: The IGHV4-34 gene is very frequent (~10%) in the B cell receptor immunoglobulin (BcR IG) gene repertoire of chronic lymphocytic leukemia (CLL). Over 30% of IGHV4-34 CLL cases can be assigned to different subsets with stereotyped BcR IG. The largest is subset #4 which represents ~1% of all CLL and ~10% of IGHV4-34 CLL and is considered a prototype for indolent disease. The BcR IG of a great majority (~85%) of IGHV4-34 CLL cases carry a significant load of somatic hypermutation (SHM), often with distinctive SHM patterns. This holds especially true for stereotyped subsets and is suggestive of particular modes of interactions with the selecting antigen(s). In detail, subsets #4 and #16, both involving IgG-switched cases (IgG-CLL), exhibit the greatest sequence similarity in SHM profiles, whereas they differ in this respect from IgM/D subsets #29 and #201. Prompted by these observations, here we explored the extent that these subset-biased SHM profiles in different IGHV4-34 stereotyped subsets were reflected in distinct demographics, clinical presentation, genomic aberrations and outcomes. Within a multi-institutional series of 20,331 CLL patients, 1790 (8.8%) expressed IGHV4-34 BcR IG. Following established bioinformatics approaches for the identification of BcR IG stereotypy, 573/1790 IGHV4-34 CLL cases (32%) were assigned to stereotyped subsets; of these, 340 cases (19% of all IGHV4-34 CLL and 60% of stereotyped IGHV4-34 cases) belonged to subsets #4, #16, #29 and #201, all concerning IGHV-mutated CLL (M-CLL). Clinicobiological information was available for 275/340 patients: #4, n=150; #16, n=44; #29, n=39; and #201, n=42. Comparisons between subsets revealed no differences in gender and age distribution. Interestingly, however, 36-43% of each subset cases were young for CLL (defined as patients aged ≤55 years), which is higher compared to general CLL cohorts, where young patients generally account for ~25% of cases. In contrast, significant differences were identified between subsets regarding: (i) disease stage at diagnosis, with 〉90% of IgG subsets #4 and #16 diagnosed at Binet stage A versus 83% in subset #201 and 74% in subset #29 (p=0.029); (ii) CD38 expression, ranging from 1% in subset #4 to 10% in subset #201 (p=0.013); (iii) the distribution of del(13q), peaking at a remarkable 92% in subset #29 versus only 37% in subset #16 (p

Description: Key Points Low-count and high-count monoclonal B-cell lymphocytosis (MBL) have distinct immunogenetic signatures, with only the latter resembling CLL. Rather than a true premalignant condition, low-count MBL may merely reflect immune senescence or result from persistent antigen stimulation.

Description: Key Points HS1 protein activation is differentially regulated by LYN kinase in CLL subsets. Dasatinib targets cytoskeletal activity, BCR signaling and survival of a sizable portion of patients with activated LYN/HS1.

Description: Background: Chronic lymphocytic leukemia (CLL) is characterized by phenotypic and functional defects of immune cells, which often emerge into increased susceptibility to infections and autoimmunity, and also contribute to immune evasion of cancer cells. The BTK inhibitor ibrutinib exerts its anti-tumor activity via the targeting of key pathways in CLL cells. In addition, ibrutinib has also shown immune modulatory properties suggesting the ability to partially restore immune functions in CLL. Currently, available data are mainly limited to the activity exerted by ibrutinib on conventional T cells, whereas little is known on the effects induced on other non-neoplastic immune cell populations. Aim: The aim of this study was to perform a comprehensive and longitudinal analysis of the immune changes occurring in multiple lymphoid populations in a broad cohort of CLL patients treated with ibrutinib. Methods: We included 22 CLL patients with progressive disease (P-CLL) and eligible to ibrutinib therapy. Peripheral blood samples were collected from patients at baseline and after 1, 6 and 12 months of treatment with ibrutinib. For comparison, we also analyzed 7 healthy donors (HD) and 10 treatment-naïve CLL patients with stable disease not requiring treatment (S-CLL). The percentages and the absolute numbers of CLL cells, T cells, γδ (Vδ1 and Vγ9Vδ2) T cells, T regulatory cells (Tregs), natural killer (NK) and NK-T cells, as well as the expression of activation markers and immune checkpoint molecules were assessed by flow cytometry. The cytotoxic function of Vγ9Vδ2 T cells was evaluated using the CD107 assay. Statistical analyses were carried out by paired t-test. Results: Median age of enrolled patients was 70 years (range 42-80). The median lymphocyte count at study entry was 35.7 x 109/L (range 1.8-178) and the median number of previous treatment regimens was 2 (range 0-5). After 12 months of ibrutinib, 20 out of 22 (91%) patients achieved at least a partial response. The mean absolute number of CLL cells started to decrease by month 6 and became significantly lower than the baseline value by month 12. We also observed a parallel reduction of the total count of CD4+ T cells, CD8+ T cells and Tregs which reached statistical significance for the CD4+ T-cell compartment at the 12-month timepoint. Overall, ibrutinib treatment had no impact on the absolute numbers of Vδ1 and Vγ9Vδ2 T cells, NK and NK-T cells over time. In our cohort, we observed no change in the differentiation subset distribution of conventional CD4+ and CD8+ T cells, Tregs, Vδ1 and Vγ9Vδ2 T cells after 6 and 12 months of ibrutinib treatment. At baseline, we observed in the P-CLL cohort a significantly higher surface expression of the early activation marker CD69, both in the leukemic cell compartment and on T cells, NK and NK-T cells compared to S-CLL and HD. CD69 expression significantly decreased on CLL cells, T cells and NK-T cells already after 1 month of ibrutinib treatment, and on NK cells after 6 months (Figure 1A-D). The expression of the costimulatory molecule NKG2D was not modulated by ibrutinib treatment in any immune cell compartment. Among checkpoint molecules, the expression of CD96 was significantly reduced after 12 months of ibrutinib treatment on T lymphocytes and on NK, NK-T, and Vγ9Vδ2 T cells, whereas TIGIT, PD-1, TIM-3 and BTLA were not modulated (Figure 1E-H). In addition, when we restricted the analysis to patients showing a response in terms of lymphocyte count (i.e. 〉50% reduction in 6 months) (11 out of 22 patients) we observed a recovery of CD16 surface expression on NK cells (Figure 1I, blue graph), a reduced expression of the co-inhibitory molecule TIGIT on Tregs (Figure 1J, blue graph) and a normalization in the mean values of CD4+ and CD8+ T cells, all becoming significant after 12 months of treatment. From a functional standpoint, we observed, after 12 months of treatment, an improvement in the cytotoxic function of Vγ9Vδ2 T cells in response to IL-2 and zoledronic acid, which was not associated to a modulation of their proliferative ability. Conclusions: Our data suggest that in CLL patients the anti-tumor activity of ibrutinib is paralleled by a dampening of immune exhaustion features, which is more evident in patients showing a more profound decrease in leukemic cell counts, and by a recovery of Vγ9Vδ2 T cell cytotoxic functions. These ibrutinib-induced effects might be exploited in the context of cellular immunotherapeutic strategies. Disclosures Mauro: Abbvie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Shire: Consultancy, Research Funding; Jannsen: Consultancy, Research Funding; Roche: Consultancy, Research Funding. Scarfo:AstraZeneca: Honoraria; AbbVie: Honoraria; Janssen: Honoraria. Gaidano:Astra-Zeneca: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sunesys: Consultancy, Honoraria; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Foà:Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Boccadoro:Amgen: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Mundipharma: Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; AbbVie: Honoraria. Coscia:Abbvie: Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Easily reproducible animal models that allow for study of the biology of chronic lymphocytic leukemia (CLL) and to test new therapeutic agents have been very difficult to establish. We have developed a novel transplantable xenograft murine model of CLL by engrafting the CLL cell line MEC1 into Rag2−/−γc−/− mice. These mice lack B, T, and natural killer (NK) cells, and, in contrast to nude mice that retain NK cells, appear to be optimal recipient for MEC1 cells, which were successfully transplanted through either subcutaneous or intravenous routes. The result is a novel in vivo model that has systemic involvement, develops very rapidly, allows the measurement of tumor burden, and has 100% engraftment efficiency. This model closely resembles aggressive human CLL and could be very useful for evaluating both the biologic basis of CLL growth and dissemination as well as the efficacy of new therapeutic agents.

Description: Introduction: Chronic lymphocytic leukemia is the most frequent adult leukemia that remains incurable, despite the use of innovative compounds targeting crucial signaling pathways of the disease. Additional non-toxic, targeted therapies are urgently needed in order to improve the curability of CLL as well as to increase the therapeutic index of existing drugs through rational combinations. Biomimetic high-density lipoprotein-like nanoparticles (HDL NP) have comparable size, shape, and surface chemistry as natural, spherical HDLs. HDL NPs modulate cellular cholesterol homeostasis, and potently induce apoptotic cell death in B cell lymphoma cell lines after tightly binding to the high-affinity HDL receptor, scavenger receptor type B-1 (SR-B1). Based on these data, we investigated SR-B1 expression and tested the nanoparticles' effect on primary human CLL with the hypothesis that HDL NP binding to SR-B1 and modulation of cellular cholesterol homeostasis in the leukemic cells would produce a therapeutic response. In addition, we explored the possibility of enhancing its therapeutic effect through the combination of the HDL NP with BCL2-inhibitor ABT-199 and the BTK-inhibitor, ibrutinib, in a series of fresh leukemia cells from patients with CLL. Methods: Fresh human CLL cells from peripheral blood were obtained from patients with CLL (N = 20) after signing an informed consent as part of a investigational study approved by the IRB. Samples were also obtained from healthy volunteers. Normal and leukemic B cells were negatively purified using a B-lymphocyte enrichment kit (RosetteSep; StemCell Technologies) and cell lysate was utilized to evaluate SR-B1 expression by Western blotting utilizing a monoclonal antibody (AbCam). HDL NPs were added to the samples at a concentration of 30nM and 100nM and allowed to incubate for 72 hours in vitro. In parallel experiments, ABT-199 (0.003µM) and Ibrutinib (1µM) were added either alone or in the presence of the HDL NP to the cell culture. Flow cytometry was performed using Annexin/PI staining to measure cell death. Results: Among the 11 CLL patients' samples tested, 8 (72%) expressed the SR-B1 receptor as detected by WB. In this series, SR-B1 expression was independent of any known biological features, including IGHV gene mutational status. When purified leukemic cells (n=9) were incubated for 72 hours in the presence of HDL NPs, a dose response pro-apoptotic effect was evident with a higher percentage of Annexin V positive cells compared to untreated controls at a concentration of 30nM (52.6% ± 6.7% range vs 28.6% ± 2.6%), peaking at 100nM (74.2% ± 4.6%) (p

Description: Abstract 1924 Poster Board I-947 According to the 2008 WHO Classification, PSMZLs mainly comprise splenic marginal-zone lymphoma (SMZL) and splenic diffuse B cell lymphoma/leukemia unclassifiable (SDLU). Until recently, histological examination of splenectomy specimens was considered as a prerequisite for the diagnosis of SMZL though, due to the recent advancement in chemo-immunotherapy therapies, splenectomy is no longer performed in most cases. We evaluated the diagnostic efficiency of BMB examination in the documentation of the diagnosis of PSMZLs and their differential diagnosis from mimickers, including other small B-cell lymphomas, and tested whether BMB may serve as a substitute for spleen microscopic examination in the differential diagnosis of SMZL from SDLU. The study group included 108 BMB samples from patients with a diagnosis of PSMZL based either on examination of splenectomy specimens (n= 46), or clinical presentation, cell morphology and flow cytometry analysis (n= 62). Histological and immunohistochemical results are summarized in Table 1. Molecular analysis of immunoglobulin genes showed that 10/21 analyzed CD27+ cases carried IGHV genes with more than 98% germline identity (unmutated), whereas 5/8 analyzed CD27- cases carried mutated IGHV genes. Furthermore, 7/14 analyzed SIgD+ cases carried mutated IGHV genes. Splenectomy specimens were available in 46/108 cases. Blinded histopathological examination of the spleens revealed 35 SMZL and 11 SDLU. Paired assessment of BMB and spleen specimens did not identify any discriminating pattern of BM infiltration and/or morphology between SMZL and SDLU; notably, the immunophenotypical profile of BMB and spleen specimens was identical when 14 different markers have been utilized (Table 1). In conclusion, our study documents the value of BMB histopathology in the diagnosis of PSMZLs and their differential diagnosis from other small B-cell lymphomas primarily or secondarily involving the spleen. Presently, it is not possible to distinguish SMZL from SDLU based on the only BMB histopathology. Finally, our results confirm and expand the considerable heterogeneity of PSMZL, which could reflect different activation status and/or different histogenesis of the clonogenic cells.Table 1.Histology and immunohistochemistry.Pattern of infiltrationPure intrasinusoidal6/108Interstitial19/108Nodular4/108Diffuse8/108Mixed intrasinusoidal-interstitial39/108Nodular combined with other types32/108MorphologySmall lymphocytes105/108plasmacytic differentiation (PC)13/105onocytoid differentiation (MC)20/105Pure MC population3/108ImmunohistochemistryCD20+ CD79á+BCL2+108/108CD43+BCL6+CD10+CyclinD1+0/108CD5+7/98CD23+4//91CD5+CD23+0/91TRAP+31/58DBA44+59/93CD27+44/70SIgD+30/61SIgD+CD27+18/24 Disclosures: No relevant conflicts of interest to declare.