Chronic Lymphocytic Leukemia Cases Research Articles

CXCR4 as a prognostic marker in Egyptian chronic lymphocytic leukemia patients

Chronic lymphocytic leukemia (CLL) has variable clinical presentations, and molecular and biological prognostic markers. The C-X-C chemokine receptor 4 (CXCR4) and its ligand stromal cell-derived factor-1 (SDF-1) play an important role in trafficking of lymphocytes and monocytes. The aim was to study lymphocyte expression of CXCR4 and its prognostic value in CLL. A case control study was carried out on 30 newly diagnosed CLL cases and 30 healthy controls. Fludarabine, cyclophosphamide, and rituximab (FCR) was the standard treatment. Flowcytometric measurement of CXCR4 expression on lymphocytes was done. CXCR4 was significantly higher in patients than controls (81.67 ± 17.95 vs. 11.78 ± 2.78; P< 0.001). CXCR4 was significantly higher (P<0.001) in high risk CLL (93.63 ± 6.78) vs. intermediate risk (82.50 ± 7.13) and low risk (75.84 ± 12.23). CXCR4 was significantly higher (P<0.001) in non-responders (91.63 ± 6.98) vs. partial responders (83.11 ± 5.55) and complete responders (70.11 ± 4.44). CXCR4 was significantly lower in survivors vs. non-survivors (80.89 ± 5.09 vs. 85.43 ± 5.51; P< 0.001. CXCR4 had significant positive correlation with WBCs (r=0.45, P=0.01) and lymphocytes (r=0.40, P=0.01) measured at diagnosis. In conclusions, expression of CXCR4 in newly diagnosed CLL is significantly high. CXCR4 increased expression is associated with poor prognosis and resistance to the therapy.so it can be used as prognostic tool.

Assessment of Interleukin_10 level in Sudanese Patients with Chronic Lymphocytic Leukemia in Khartoum State

Background: Chronic lymphocytic leukemia (CLL) is the most predominant leukemia among adults. Interleukin‑10 (IL‑10) secretion has potent immunosuppressive effects and it has a strong stimulating impact on B-lymphocytes, inducing proliferation and differentiation. This study was conducted to investigate the serum levels of IL-10 in patients with CLL (cases) and comparing them with apparently healthy age and gender match volunteers as control group. Method: A total of 88 blood samples were randomly collected. 44 blood samples were withdrawn from cases, while the rest were obtained from control. Serum samples after centrifugation of blood were obtained for IL-10 level evaluation. Human IL-10 ELISA max Deluxe kit was used to determine IL-10 in each sample following manufacture instructions. The Data was analyzed through statistical package for the social science (SPSS) version 20. Results: Our results showed that the mean of IL_10 level among CLL patients (22.7±5.34 pg\ml) was significantly higher (P-value: 0.002) than the mean of IL_10 level among control group (4.99±0.55 pg\ml). Among case groups females showed IL-10 mean of 29.09±10.85 pg/ml that was significantly higher than male’s 18.22±5.04 pg/ml with p-value 0.023. Our result finds no significant difference between cases below 55years (23.62±8.03 pg/ml) and above55 years (22.07±7.19 pg/ml) in the level of IL-10 with P-value 0.893. Conclusion: In conclusion, this study determined higher serum IL-10 levels in CLL patients compared with healthy controls. Gender and duration of disease have significant effect on serum IL-10 level among CLL cases. However, sample size in this study was small and larger scale research is needed in this field with the inclusion of several factors such as disease, grade and outcome.

Chronic lymphocytic leukaemia trends and features at a tertiary hospital in South Africa (2011–2016)

Background: Chronic lymphocytic leukaemia (CLL) is a common lymphoproliferative disorder in developed countries. However, this condition is rare in Africa and there is a paucity of information on CLL, specifically on the continent.Aim: This study described, retrospectively, the frequency, demographics and laboratory features of CLL cases diagnosed from 2011 to 2016.Setting: Department of Pathology, National Health Laboratory Service, Tygerberg Academic Hospital, Cape Town.Methods: A retrospective analysis was performed for all CLL diagnoses made between 01 January 2011 and 31 December 2016.Results: Eighty CLL cases were diagnosed between 2011 and 2016. Men and women presented with the disease equally (48.8% vs. 51.2%, p 0.05). The mean age at diagnosis was 66.79 years (range of 37–95 years) and the modal age range (36.3%) was 60–69 years. Men presented with the disease at a significantly younger age than women (mean = 64 years vs. mean = 69.5 years, p 0.05). There were three (3.75%) human immunodeficiency virus (HIV)-positive patients (age range 43–50 years). Chromosome 13q14 deletion was found in 6 out of 19 patients (31.6%). Trisomy 12 and deletion 11q22 were found in 5 out of 21 (24%) and 7 out of 21 (33.3%) patients, respectively. Deletions 13q34 and 17p were negative for 6 and 20 patients, respectively.Conclusion: Chronic lymphocytic leukaemia at our facility presented equally in men and women. Men presented with the disease at a younger age than women. Additionally, our findings suggested that HIV is uncommon amongst CLL patients tested for HIV.

Regulation and Impact of BCR Induced Autophagy in Chronic Lymphocytic Leukemia

Introduction: Chronic lymphocytic leukaemia (CLL) is characterised by the accumulation of malignant B-cells within the peripheral blood and secondary lymphoid organs. CLL patients can be broadly divided into two groups depending on the mutational status of the immunoglobulin variable region genes (IGHV), with unmutated and mutated V- genes associated with progressive and indolent disease respectively. B cell receptor (BCR) expression and signalling capacity is pivotal to CLL pathogenesis and whilst drug which targeting this pathway are revolutionising the treatment of this disease. Resistance is already occurring and therefore a better understanding of this pathway is urgently required if we are to identify novel therapeutic strategies to treat this largely incurable disease. Autophagy primarily acts as a protective mechanism against cellular stress induced by starvation, cytokine removal, hypoxia, reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress and is important for normal B-cell development. However autophagy can also promote tumorigenesis and resistance to therapy. The role of autophagy in CLL cases in response to therapy has previously been investigated, however its role in CLL biology and how it is regulated has not previously been evaluated.Results: We investigated basal expression of a number of autophagy markers between CLL cases and normal donor B cells (NDB). We demonstrated that basal expression of LC3B-II p=0.0005, GABARAPL2 p=0.0113, ATG3 p=0.0005 and ATG7 p=0.034 was greater in CLL samples compared to NDB. Moreover even greater LC3B-II expression was observed in U-CLL cases compared to M-CLL (p=0.02). Using a pre-determined cut-off, LC3B-II expression identified a subset that had a shorter time to first treatment from first lymphocytosis (p=.017) and identified samples particularly within the M-CLL subset which were more likely to progress. Next using tissue extracted from CLL lymph nodes we showed greater LC3B expression in areas with higher levels of Ki67 by immunohistochemistry, indicating an association between proliferation centers and autophagy. This suggested that autophagy might be induced following BCR signalling. Indeed basal expression of LC3B-II was greater in samples with increased BCR signalling capacity. Therefore we investigated the role of the BCR in regulating autophagy. Activation of the CLL BCR with bead immobilised (BI) anti-IgM significantly induced expression of LC3B-II (p<.0001) at 2, 4 and 24 hours, with greater levels induced in U-CLL compared to M-CLL cases. Similar changes were observed with anti-IgD but at reduced levels compared to anti-IgM treatment. Interestingly using confocal microscopy and transmission electron microscopy we demonstrated that the beads coated with anti-IgM were being endocytosed by the CLL cells and the autophagosomes formed around the beads. To confirm that the BI anti-IgM induced autophagy was not solely a result of bead endocytosis we also treated CLL and NDB with soluble anti-IgM and anti-IgD. Soluble anti-IgM significantly induced LC3B-II (p=0.006) and GABARAPL2 (p=0.04) expression compared to untreated controls in CLL cases, but at slightly reduced levels compared to BI anti-IgM. In contrast soluble anti-IgD induced minimal changes or no change at all. Similar changes were also observed in NDB. Next we examined autophagic flux to determine whether BI anti-IgM increased RNA and protein expression or simply inhibited autophagosome breakdown. CLL samples treated with BI anti-IgM increased LC3B and GABARAP RNA expression within 2h (p=.0017 & p=.022 respectively) whilst treatment with BI anti-IgM plus HCQ significantly increased LC3B-II protein expression up to 24 hours compared to HCQ or anti-IgM alone (p<.0001). Confirming that activation of the BCR increased autophagic flux. To confirm the role of the BCR in the regulation of autophagy we treated CLL cells with ibrutinib or fostamatinib to inhibit BCR signalling capacity or treated cells with IL-4 to increase sIgM expression and signalling as we have previously demonstrated. As expected BCR kinase inhibitors prevented anti-IgM induced autophagy. In contrast IL-4 increased sIgM and signalling and consequently induced autophagy highlighting the importance of the BCR in regulating autophagy in CLL. Together these data suggest that the autophagy pathway might be therapeutically targeted for the treatment of CLL. DisclosuresStrefford:Roche: Research Funding. Forconi:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Janssen-Cilag: Speakers Bureau. Steele:Portola Pharmaceuticals: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria.

Daratumumab Decreases Treg-Mediated Immunosuppression and Potentiates CD8+ T-Cell-Induced Killing of Chronic Lymphocytic Leukemia (CLL) Cells Ex Vivo

Introduction: CD38 is expressed on the plasma membrane of lymphoid cells and in chronic lymphocytic leukemia (CLL) pts. increased CD38 expression is associated with a poor clinical outcome. While its role as a prognostic marker has been well established, the approval of daratumumab (Dara, first-in-class anti-CD38 mAb) offers a new role for CD38 as a potential therapeutic target in CLL. Matas-Cespedes et al have reported that Dara induces CLL cell death through antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP). While CD38 is expressed on CLL cells, it is also expressed on T-cell subsets, such as Tregs. We hypothesized that in addition to its direct immune-mediated activity, Dara modulates the immune cellular environment; skewing T-cell populations from an immunosuppressive towards immune-reactive milieu, thus promoting immune reconstitution for enhanced anti-CLL response.Methods: CD19+ cells were isolated from PBMCs of CLL pts. (n=30). Pts. with ≥30% CD38+ CLL cells were classified as CD38+ (n=22) and pts. with <30% CD38+ cells as CD38- (n=8). Dara-induced ADCC was assessed in Calcein AM labelled CLL cells cocultured with healthy donor PBMCs (E:T ratio, 50:1); CDC using 10% serum from a healthy donor and ADCP using human macrophages (E:T ratio, 2:1). Apoptosis was determined by annexin-V/PI staining followed by flow cytometry. Regulatory T-cells (Treg; CD3+CD4+CD25+CD127loFoxP3+) and cytotoxic effector T-cells (cTL, CD3+CD8+) were identified and isolated by FACS. Data are represented as mean±SEM.Results: Dara (0.1ug/mL) induced significantly greater ADCC in CD38+ (24.63±3.46%) vs. CD38- cells (14.11±1.11%) (p<0.001), whereas CDC (18.22±2.41 vs. 13.58±0.95) and ADCP (9.37±0.77% vs. 8.79±0.92%) were marginally, but insignificantly, increased in CD38+ vs. CD38- CLL cases. We also observed significantly greater apoptosis in CD38+ (36.41±3.34%) vs. CD38- CLL cells (23.39±1.70%) (p<0.005) (Fig 1A). Immune repertoire analysis revealed lower CD3+ T-cell numbers in CLL pts. (11.45±3.25%) compared to those in healthy donor PBMCs (23.64±7.36%); but with a higher percentage of Tregs (10.71±2.47 in CLL pts. vs. 1.96±0.60 in healthy donors). To determine whether Dara could decrease CLL-Tregs, we treated CLL-PBMCs withDara (10µg/ml; 5 days) ex vivo, and noted significant decrease in Treg cells from 12.75±1.33 (Isotype control) to 7.17±0.91, (n=5, p<0.001). As downregulation in Tregs can consequentially lead to a rise in other T-cell subtypes, we observed increased effector cTLs: from 50.08±5.41 to 64.53±3.19 (p<0.02) (Fig 1B). Further analysis on CFSE-labeled CLL-cTLs (primed or unprimed with Dara), showed cTLs proliferated significantly more so when co-cultured with Dara-treated apoptotic CLL (51.75±1.16) cells vs. ibrutinib (31.44±0.36) or rituximab-treated apoptotic CLL cells (36.25±3.77) or live CLL cells (7.04±3.34) (Fig 1C, representative CLL pt.). To determine if Dara-primed cTLs had enhanced cytolytic aptitude towards autologous CLL cells, we cocultured primed cTLs with CLL cells. Compared to unprimed cTLs, specific lysis of target CLL cells was greater in the primed cTL-CLL coculture (12.61±1.75% vs. 28.27±0.82%, respectively). And this effect was noted to be patient-specific as cTLs cultured with JVM13 (CD38+) cells showed lower specific lysis (Fig 1D, E).Conclusion: Here we demonstrate for the first time that Dara modulates CLL-Treg levels and increases effector cTL proliferation. Moreover, Dara-primed cTLs display heightened CLL-specific cytolytic activity than compared to Dara-naïve cTLs. Dara also induces direct apoptosis of CLL cells and exposure of Dara-killed CLL cells to cTLs increases cTL proliferation significantly more so than cTL exposure to CLL cells apoptosed using ibrutinib or rituximab. Altogether, our findings highlight the multiple mechanisms by which Dara (and potentially other anti-CD38 agents, currently being investigated by us) can induce CLL cell death and potentially revert dysfunctional cTL activity from that of a pro-CLL to an anti-CLL composure. [Display omitted] DisclosuresParikh:AstraZeneca: Honoraria; Pharmacyclics: Research Funding; Pharmacyclics: Honoraria. Ding:Merck: Research Funding. Sher:LAM Therapeutics, Inc: Research Funding. Malavasi:Jaansen Pharmaceuticals: Research Funding. Ailawadhi:Amgen: Consultancy, Honoraria; Pharmacyclics: Research Funding; Takeda: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Rare Germline Variant in NFATC4 Associated with Familial Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) has one of the highest familial risks of any cancer, with up to eightfold increased risk in first degree relatives of CLL patients. Genome-wide association studies (GWAS) have identified multiple common single nucleotide polymorphisms (SNPs) associated with risk, but these have modest effect. Similarly, identified germline variants associated with disease account for just a small minority of CLL patients, making familial CLL an attractive target for research, that could provide direct insight into pathobiology.We used whole exome sequencing (WES; Illumina) to search for rare germline variants (<1% Minor Allele Frequency) in 36 patients from 17 families with Mendelian-like CLL inheritance, where at least two affected family members were sequenced. Median age at diagnosis was 56.4 (range 41-72), 61% were male, 10/23 (43%) were IGHV -unmutated, and 5/24 (21%) had del17p and/or TP53 mutation. We found 3819 rare variants shared within all affected members of a given family. Of those, 71 non-synonymous variants were shared across at least 2 families, and 39 of these were predicted to be pathogenic by at least two commonly-used phenotype prediction tools. None of these variants are within known somatic CLL driver genes, nor were any found in genes near known CLL GWAS SNPs. In order to prioritize these variants, we assessed whether they were enriched among CLL cases (n = 646) as compared to ethnically matched normal population controls (n=8,920), a dataset we have recently reported (Tiao, Improgo et al, Leukemia). Using Fisher's exact test with Benjamini-Hochberg (BH) correction for multiple tests, we found that 27 of the 71 shared variants across multiple families were also enriched in unrelated CLL patients compared to controls. Among those, a rare variant of nuclear factor of activated T-cells 4 (NFATC4), rs200328372, was the most highly enriched in CLL (OR 20.8, Fisher-q-BH 0.026). This variant affects a splice acceptor site predicted to have a deleterious effect on the protein (FATHMM for non-coding variants). In a pathway analysis (MSigDB, DAVID) of genes carrying shared variants across families, NFATC4 together with SOS1 and PTPN7 were associated with the T-cell receptor, B-cell receptor, and MAP-kinase pathways, suggesting a possible role in CLL pathogenesis.The NFATC4 protein, also known as NFAT3, is part of a DNA-binding transcriptional complex that translocates to the nucleus after activation through dephosphorylation by calcineurin. This complex is involved in the inducible expression of cytokine genes in T cells, including interleukin (IL)-2 and IL-4. While NFATC1 has been implicated in CLL pathogenesis and was associated with more aggressive disease in a mouse model of CLL, the role of NFATC4 has not been elucidated. To further investigate this as well as its association with familial CLL, we compared gene expression profiling (GEP; Affymetrix U133 plus 2.0 array) data between familial and sporadic CLL patients and normal B-cells. While NFATC4 expression did not differ between normal B-cells (n=16) and CLL (n=143), it was significantly down-regulated in familial (n=47) as compared to sporadic (n=96) CLL cases (Mann-Whitney, p=0.005). In the 4 cases that harbor the shared risk variant, GEP results showed additional significant down-regulation of NFATC4 expression (Kruskal-Wallis, p=0.003 for all comparisons) (Figure 1).RNA sequencing (Illumina) demonstrated NFATC4 down-regulation in all CLL samples (not specifically in familial CLL as with GEP; n=100) versus normal B-cells (n=6; Mann-Whitney, p=-0.00024), suggesting a putative role for NFATC4 as a tumor suppressor. Since NFATC4 has over 30 alternatively spliced transcripts potentially affected by the rs200328372 variant, we used Cufflinks software to look at isoform-level RNA sequencing data, available on two relatives who carried this variant, compared to CLL cases and controls. Both relatives had reduced expression of all isoforms (Figure 2), suggesting that this mutation may reduce NFATC4 expression altogether. Studies of protein expression are ongoing.In conclusion, through rare germline variant analysis in familial CLL, we found a potentially deleterious variant of NFATC4 that is enriched in CLL. Subsequently, we used RNA sequencing data to show, for the first time, that NFATC4 expression is decreased in CLL versus normal B-cells, revealing a potential tumor suppressor mechanism in CLL. [Display omitted] DisclosuresBrown:Sun BioPharma: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Pharmacyclics: Consultancy; Redx: Consultancy; AstraZeneca: Consultancy; Pfizer: Consultancy; Janssen Oncology: Honoraria; AbbVie: Consultancy, Honoraria; Roche/Genentech: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Infinity Pharmaceuticals: Consultancy; Astellas Pharma: Consultancy.

Invariant NKT Are a More Effective and Versatile Platform Than T Cells for CAR Immunotherapy of CD1d-Expressing B Lineage Malignancies: Cellular and Molecular Mechanisms

Anti-CD19 chimeric antigen receptor T cell (CAR19T) immunotherapy has shown promising clinical potential in relapsed/refractory mature B cell malignancies. However, only about half of patients benefit, highlighting the need for more effective CAR-based strategies.iNKT cells are rare but powerful immunoregulatory and cytotoxic T lymphocytes, playing a pivotal anti-tumor role. iNKT cells are restricted by CD1d, a non-polymorphic, glycolipid-presenting HLA I-like molecule. We previously showed that CD1d, as well as on normal B cells, is also expressed on malignant CD19+ B cells in mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL) cells, while in up to 50% of patients with chronic lymphocytic leukemia (CLL) CD1d expression is very low or negative.We tested the hypotheses that a) bi-specific CAR iNKT 19 cells, targeting simultaneously CD19 and CD1d via the CD19-specific CAR and their natural invariant TCR respectively, would be more effective than CART19 cells against CD19+CD1d+ B cell malignancies and b) transcriptional enhancement of CD1d expression would increase the CAR19iNKT cytotoxic effect.We optimized a novel protocol for manufacturing CAR19iNKT cells. Their in vitro reactivity was assessed in cytotoxicity (flow cytometry), cytokine and cytotoxic granule intracellular staining and cytokine release assays (flow cytometry and Luminex technology) as well as real-time Incucyte proliferation assays. In vivo reactivity was assessed in systemic NSG xenograft assays with growth of the C1RCD1d B cells monitored by bioluminescence and magnetic resonance imaging. Transcription factor binding and histone mark enrichment at the promoter of CD1d was assessed by ChiP- and ChIP-re-ChIP-qPCR assays.We manufactured clinical scale 2nd and 3rd generation CAR19iNKT cells using a highly optimized protocol (>75% transduction efficiency, n=8). In vitro validation, using singly- or dual-positive CD1d and CD19 targets, demonstrated that CAR19iNKT cells are CD19-specific, retain their natural CD1d-restricted reactivity and exert additive dual-specific cytotoxicity against CD1d+CD19+ targets. Compared to same donor CAR19T, CAR19iNKT cells display a significantly higher (P<0.0001) expandability and proliferative potential and are equally or more effective in killing CD19+CD1d+ lymphoid cell lines (C1RCD1d and Farage) and consistently more effective against primary MCL, MZL and CLL cells.Since CD1d expression is low/negative in most cases of CLL, we sought to restore CD1d expression on leukemic cells for the purpose of CAR19iNKT cell immunotherapy. We found that surface CD1d expression can be restored by clinically relevant concentrations of ATRA in a time-dependent manner. Mechanistic dissection of CD1d transcriptional regulation in the CD1d-negative B lineage cell line U266 demonstrated the co-existence of bivalent chromatin marks at the promoter of CD1d commensurate with enriched binding and association of EZH2 with RARα. Notably, an EZH2 inhibitor exerted a synergistic effect along with ATRA in restoring CD1d transcription and surface expression and, in line with our hypothesis, 2nd and 3rd generation CAR19iNKT but not CAR19T cells displayed higher cytotoxic activity against ATRA-treated CLL cells.Finally, in an NSG xenograft model of lymphoma, while survival of T and iNKT cell-treated animals was the same as that of untreated animals (n=7, P>0.05), both CAR19T and CAR19iNKT cell-treated animals (n=19) had significantly and comparably improved overall survival (OS; P<0.0001). However, compared to CAR19T, CAR19iNKT cell immunotherapy led to a significantly improved OS (P=0.01) and also better disease control including of CNS lymphoma, with earlier, more profound and sustained complete responses resulting in a significantly improved tumor-free survival (P=0.03).We conclude thatCARiNKT19 are more effective than CART19 cells against CD19+CD1d+ B cell malignancies in vitro and in vivo and ATRA-mediated restoration of CD1d expression enhances the anti-lymphoma effect of CAR19iNKT immunotherapy in vitro . These properties together with the previously demonstrated ability of donor iNKT cells to protect from aGVHD raise the prospect of developing effective ‘off-the-shelf’ CAR19iNKT immunotherapy of lymphomas, which can mitigate against CD19-negative immune escape upon CAR treatment and can be further enhanced by transcriptional and epigenetic treatment. DisclosuresPule:UCL: Patents & Royalties: UCL Business; Autolus Ltd: Employment, Equity Ownership, Research Funding. Maher:Leucid Bio: Other: Chief scientific officer. Karadimitris:GlaxoSmithKline: Research Funding.

Chronic Lymphocytic Leukemia Patient Reported Outcomes and Quality of Life: Findings from the Cancer Experience Registry

Background: Approximately 20,000 new cases of chronic lymphocytic leukemia (CLL) are expected in the US in 2017 (ACS 2017), yet understanding of the psychosocial consequences of CLL is inadequate. Many people with CLL undergo an active surveillance (“watch and wait”) treatment plan, which can pose unique challenges. The purpose of this exploratory study was to compare how CLL patients describe their quality of life compared to other US population groups and describe the extent to which CLL affects daily life, finances, and professional and family relationships.Methods: 134 individuals with CLL enrolled in the Cancer Support Community's online Cancer Experience Registry, provided sociodemographic information and clinical history, and completed surveys including the Patient-Reported Outcomes Measurement Information System (PROMIS-29v2.0), which assesses seven health-related quality of life domains. We used multiple linear regression analysis to examine the association between course of CLL (undergoing observation, currently receiving first treatment, currently receiving second or subsequent treatment, in remission or maintenance therapy) and PROMIS subscale T-scores adjusting for sociodemographic variables (age, gender, education, income).Results: The sample was 47% female, 96% White; mean age was 61 (SD=9) years; mean time since CLL diagnosis was 7 (SD=5) years. More than one-third (37%) were undergoing observation (“watch and wait”) before initiating any treatment, 6% were receiving their first treatment, 12% were in active second or subsequent therapy, and 29% in remission. 38% of CLL respondents described their overall health as very good or excellent, and 45% as good; 17% indicated fair or poor health. When considering individual responses, considerable proportions of CLL patients reported substantially worse quality of life (>1 SD) than U.S. population national averages for anxiety (21% of respondents), fatigue (21%), physical functioning (15%), social functioning (12%), depression (11%), sleep disturbance (9%) and pain interference (5%). Additionally, many respondents indicated that CLL “somewhat to very much” affected their views on life expectancy (62%), quality of life (41%), finances (40%), ability to work (34%), and relationships with friends and family (26%). All regression models predicting PROMIS subscales were significant (R2 = .13 to .20, ps < .05). With respect to CLL course, worse anxiety was significantly associated with active treatment, whether first line of therapy (B=8.0, p <.05) or subsequent therapy (B=6.2, p <.05). Currently receiving second or subsequent therapy was significantly associated with worse physical (B=−5.2, p <.05) and social functioning (B=−6.4, p <.05), and greater levels of fatigue (B=7.6, p <.01) and depression (B=5.1, p <.05). There was also a significant relationship between sleep disturbance and currently receiving second or subsequent treatment (B=5.7, p <.01). No significant associations were found between observation (“wait and wait”) status or disease remission and quality of life outcomes.Conclusion: Over one-fifth (21%) of CLL survivors are experiencing substantial levels of anxiety and fatigue, compared to the general U.S. population. Poorer health-related quality of life was associated with active treatment but not active surveillance (“watch and wait”) or when disease was in remission. It remains unclear the extent to which advancing disease versus treatment contributes to worse quality of life. Next steps include further examination of factors that put CLL patients at greater risk for poorer outcomes and the evaluation of interventions designed to address emotional distress and quality of life concerns, especially among people with progressing disease. DisclosuresBuzaglo:Takeda Oncology: Research Funding; Pharmacyclics, Inc: Research Funding; Pfizer Oncology: Research Funding; Novartis: Research Funding; Janssen Biotech, Inc: Research Funding; Genentech, Inc: Research Funding; Eli Lilly and Company: Research Funding; Celgene Corporation: Research Funding; Bristol-Myers Squibb: Research Funding; Boehringer Ingelheim: Research Funding; AbbVie: Research Funding; Amgen Corporation: Research Funding; Bayer: Research Funding. Flowers:Burroughs Welcome Fund: Research Funding; Janssen Pharmaceutical: Research Funding; Infinity: Research Funding; TG Therapeutics: Research Funding; V Foundation: Research Funding; Celgene: Consultancy, Research Funding; OptumRx: Consultancy; Educational Concepts: Research Funding; Prime Oncology: Research Funding; Millennium/Takeda: Research Funding; Genentech/Roche: Consultancy, Research Funding; Onyx: Research Funding; Research to Practice: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Bayer: Consultancy; National Institutes Of Health: Research Funding; Seattle Genetics: Consultancy; Gilead: Consultancy; Abbvie: Consultancy, Research Funding; Acerta: Research Funding; Eastern Cooperative Oncology Group: Research Funding; Clinical Care Options: Research Funding; National Cancer Institute: Research Funding; Spectrum: Consultancy.

PI3Kδ/AKT Inhibition Augments 2-Phenylethynesulfonamide Induced Apoptosis Via the Unfolded Protein Response in Chronic Lymphocytic Leukemia

Introduction: Chronic lymphocytic leukemia (CLL) is largely comprised of two major subsets, IGHV unmutated (U-CLL) and mutated (M-CLL) CLL, associated with progressive and indolent disease respectively. Approximately 15% patients harbour a 17p deletion and/or a TP53 mutation which is largely associated with the worst clinical outcome. The development of BCR signalosome inhibitors such as ibrutinib and idelalisib have been instrumental in the treatment of CLL, however these drugs are not curative, only supress the disease and resistance is already developing particularly in patients with a complex karyotype associated with 17p del/ TP53 mutation. Consequently continued development of novel therapies targeting pathways which enhance selective killing of tumor cells independently of p53 are urgently required. We previously demonstrated that 2-phenylacetylenesulfonamide (PAS), induced apoptosis of CLL cells in a p53-independent manner. PAS is suggested to inhibit heat shock protein (HSP) 70, a key protein involved in protein folding. Therefore in CLL cells, PAS would be predicted to cause cellular stress due to the accumulation of misfolded proteins resulting in the activation of the unfolded protein response (UPR). The UPR serves to protect cells from the consequences of protein misfolding and can function in part by activating pro-survival signaling, including the PI3K/AKT pathway. Under normal conditions the ER chaperone BiP inhibits activation of the UPR by binding the three ER transmembrane receptors; inositol-requiring enzyme 1 (IRE-1α), pancreatic ER kinase-like ER kinase (PERK) and activating transcription factor 6 (ATF6). Activation of the UPR leads to the upregulation of HSP family members including HSP70 in the cytosol and BiP/GRP78 in the endoplasmic reticulum (ER). These HSPs aid in refolding misfolded proteins or facilitate their degradation via the proteasome. If the stress is resolved, the UPR signalling pathway is switched off; however, if stress persists the UPR remains active and switches from an anti-apoptotic to a pro-apoptotic signal via p38MAPK.Aim: To determine the mechanism of PAS induced apoptosis in CLL cells.Methods: Fifty two CLL samples were treated with PAS (5-20μM) and cellular responses assessed using immunoblotting, flow cytometry and MTT assay.Results: In this study we demonstrated that the UPR is induced in CLL cells following the induction of reactive oxygen species by PAS treatment. This consequently led to the activation of both pro-apoptotic (Noxa, p<0.0001) and anti-apoptotic (AKT, p<0.05) signalling pathways. PAS treatment further increased expression of key stress proteins BiP (p=0.0005) and HSP70 (p=0.001). PAS treatment also resulted in activation of the PERK and IRE-1α arms of the UPR (cleaved XBP-1, phosphorylated eiF2α, phosphorylated p38MAPK) within 2h, which persisted up to 24h. This pathway was pivotal for CLL cell death since small molecule inhibition of either p38MAPK or PERK signalling significantly reduced PAS mediated apoptosis by approximately 50%. However PAS induced apoptosis was simultaneously antagonised by activation of the pro-survival AKT pathway. Consequently we hypothesised that PAS treatment in combination with a PI3Kδ (idelalisib) or AKT (AKTi) inhibitor may promote greater CLL cell apoptosis. Indeed our combination indices (CI) studies demonstrated that simultaneous treatment with an AKTi or the clinically approved PI3Kδ inhibitor idelalisib and PAS synergistically and significantly enhanced apoptosis of CLL cells compared with either agent alone (p<0.05).Conclusion: This data suggests that inhibiting the pro-survival arm of the UPR (PI3K/AKT), whilst simultaneously activating the pro-death arm (HSP70/p38MAPK), may be a useful strategy for the treatment of CLL and particularly samples with a 17p del or TP53 mutation and warrants further clinical investigation particularly in CLL cases that are resistant to BCR kinase or Bcl-2 family inhibitors. DisclosuresStrefford:Roche: Research Funding. Johnson:Novartis: Consultancy, Honoraria; Genmab: Consultancy, Honoraria; Carrick: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Research Funding; Celgene: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Karus: Consultancy, Honoraria; Boehringer Ingelheim: Consultancy, Honoraria; Zenyaku Kogyo: Consultancy, Honoraria; Epizyme: Research Funding. Forconi:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Janssen-Cilag: Speakers Bureau. Steele:Gilead: Consultancy, Honoraria; Portola Pharmaceuticals: Consultancy, Honoraria, Research Funding.

CLL Stereotyped Subset #4 Igs Acquire Binding to Viable B Lymphocyte Surfaces By Somatic Mutations, Isotype Class Switching, and with the Prerequisite of IG Self-Association

The selection and transformation of a normal B cell into the leukemic state in chronic lymphocytic leukemia (CLL) is mediated at least in part by the structure of the B cell receptor (BCR) immunoglobulin (IG) antigen binding site. A dramatic indication of the role played by BCR is demonstrated by those CLL cases expressing “stereotyped” BCR IGs, characterized by very similar VH CDR3 amino acid sequences often encoded by specific IGHV, IGHD, and IGHJ gene segments. CLL stereotyped subset #4 BCR IGs expressing IGHV4-34/IGHD5-18/IGHJ6 and IGKV2-30/IGKJ2 rearrangements are prime examples of this. Moreover, these clones are always IgG isotype class switched, and contain IGHV and IGLVκ somatic mutations. Moreover, some of these mutations are shared among patients, and are almost invariably similar physicochemically and located at specific positions in the antigen binding site. In the following study, we evaluated the effects of these unique, acquired structural changes on the (auto)antigenic specificities of subset #4, using 3 mAbs belonging to the subset.Gamma-irradiated Ramos B and Jurkat T cell line cells were incubated with subset #4 IGs under non-permeabilizing conditions, and binding to the B and T cell surfaces was detected by flow cytometry. Unlike most other CLL BCR IGs, the average binding percentage to apoptotic Ramos B cells of subset #4 mAbs was low. Conversely, each subset #4 IG bound live B cells well (CLL 183 - 44.8%; CLL 240 - 55.6%; CLL 342 - 52.0%). Moreover, all 3 IGs exhibited minimum binding to live and apoptotic Jurkat T cells. Next we exposed the subset #4 IGs to viable human B lymphocytes from tonsillectomy samples to confirm that binding was not limited to transformed B cells, and indeed the IGs bound well to CD19+ while minimally to CD3+ tonsil cells. When murine mAbs reactive with appropriate B-cell surface antigens were used to define subpopulations of normal B cells with which the subset #4 IGs interacted, we found that the subset #4 IGs and a non-subset #4 IGHV4-34-expressing CLL case (CLL 141) bound viable, naïve human B cells as expected from other studies. However, it became clear that the subset #4 IGs also interacted with viable memory B cells (CLL 183 - 28.1%; CLL 240 - 34.0%; CLL 342 - 42.7%; CLL 141 - 11.8%), a subpopulation that CLL 141 do not bind; the latter similar to IGHV4-34-expressing IGs in other disease states. Furthermore, although DNA and the “i” and “I” carbohydrate antigens are common targets of IGHV4-34 utilizing antibodies in systemic lupus erythematosus and cold agglutinin disease, respectively, the subset #4 Igs did not bind to either of these autoantigens when tested by ELISA or glycan binding arrays, respectively.Strikingly, subset #4 IG binding to viable Ramos B cells and memory B lymphocytes depended on an asparagine (N) to aspartic acid (D) mutation at position 66 of FR3 in the rearranged IGKV2-30 gene (N vs. D: 5.26% vs. 55.6%); this amino acid residue is shared by many other clones in this subset and comes about by somatic hypermutation (SHM). Interestingly and consistent with the previous finding, another focused mutation of this subset, glutamic acid (E) at position 28 of FR1 in the rearranged IGHV4-34 (G28E), was responsible for the loss of DNA binding and for the diminished I/i glycan binding.Finally, binding to viable Ramos B cells required the presence of the IgG isotype characteristic of this subset, since reactivity with Ramos B lymphocytes disappeared when the stereotyped antigen-binding site was linked to IgM (183, 240, 342 IgG vs. IgM: 44.8%, 55.6%, 51.2% vs. 24.5%, 1.85%, 7.02%, respectively). This requirement for IgG isotype binding to live Ramos B cells is likely due to the documented subset #4 self-association which requires the gamma isotype, since the change of a single amino acid at VH FR1 (E16HA), which disrupts self-association, eliminated binding to Ramos B lymphocytes.Together our findings indicate that distinct autoantigens mediate the positive and negative selection of the structural elements acquired by SHM and isotype class switching that are characteristic of this unique subset of stereotyped BCR IGs. Moreover, our data show that the reactivity of subset #4 IGs with viable B-cell surface requires the self-association of these IGs and, therefore, strongly suggests that viable B-cell surface binding is mediated by a non-conventional binding site created by self-associated surface membrane bound IGs. DisclosuresKolitz:Gilead, Novartis, and Seattle Genetics: Other: Travel Support; Boehringer Ingelheim, Cantex, Erytech, and Millennium: Research Funding; Gilead, Magellan, Novartis, Pharmacyclics, and Seattle Genetics: Consultancy; Gilead, Magellan, and Novartis: Honoraria; Celgene, Jazz: Equity Ownership. Stamatopoulos:Novartis SA: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding.

Impact of Infrequently Mutated Genes on Prognosis in 531 Untreated CLL Patients

Background: In a previous multicentre study, 55 recurrent aberrations were identified in 538 chronic lymphocytic leukemia (CLL) cases including 278 pre-treatment samples (Landau et al., Nature 2015). Some of these aberrations are frequent and their impact on prognosis in CLL is well analyzed. However limited data exist on the rarely mutated genes.Aim: Characterize 531 untreated CLL patients in terms of infrequently mutated genes and impact on prognosis.Patients and Methods: 531 CLL patients at diagnosis or without prior treatment were screened for gene mutations by a targeted amplicon sequencing approach: ASXL1, ATM, BIRC3, BRAF, BTK, EGR2, FBXW7, KRAS, MAP2K1, MYD88, NRAS, NFKBIE, NOTCH1, PLCG2, POT1, SAMHD1, SF3B1, SRSF2, TP53, U2AF1, XPO1, and ZRSR2 . The mean overall read depth for all genes was 2,496 (range: 305-13,078). IGHV mutational status was analyzed in all cases. IGHV unmutated status (IGHV-U) was defined by sequence identity ≥98%. Chromosome banding analysis and interphase FISH with probes for 17p13, 13q14, 11q22 and centromeric region of chromosome 12 were performed in all cases. Male:female ratio was 1.6:1 and median age was 66 years (range: 30 - 87 years). In all cases data on time to first treatment (TTT) and overall survival (OS) was available with a median follow-up of 6 years, each.Results: Overall 223 (42%) mutated cases comprising 343 mutations were detected in 19/22 analyzed genes. The frequencies of the gene mutations were as follows: NOTCH1 (12%), ATM (9%), SF3B1 (9%), TP53 (7%), NFKBIE (5%), XPO1 (4%), BIRC3 (3%), MYD88 (2%), POT1 (2%), EGR2 (2%), FBXW7 (2%), KRAS (2%), BRAF (1%), NRAS (1%), ASXL1 (<1%), MAP2K1 (<1%), SAMHD1 (<1%), U2AF1 (<1%), and ZRSR2 (<1%). No mutations were detected in SRSF2 and, as expected in untreated CLL cases, also not in BTK and PLCG2 .We restricted the statistical analyses on infrequently mutated genes (≤5%) found in at least 10 mutated cases. Most of the gene mutations were associated with IGHV-U: NFKBIE (10% vs. 2%, p<0.001), POT1 (6% vs. 1%, p<0.001), EGR2 (4% vs. 1%, p=0.005), and XPO1 (11% vs. 1%, p<0.001). MYD88 was found only in IGHV-M (3% vs. 0%, p=0.017) and was associated with del(13q) sole (3% vs. 1%, p=0.049). NOTCH1 mutated vs. wild-type (wt) cases showed significantly higher frequencies of mutations in NFKBIE (11% vs. 4%, p=0.029), XPO1 (12% vs. 3%, p=0.004) and KRAS (8% vs. 1%, p=0.002). Accordingly, following genes were highly mutated in cases with trisomy 12 cases vs. without: NFKBIE (10% vs. 4%, p=0.037), KRAS (9% vs. 1%, p<0.001), BIRC3 (10% vs. 2%, p=0.003), and FBXW7 (10% vs. 1%, p<0.001). In contrast, most of the mutations were rare in patients with del(13q) sole: NFKBIE (2% vs. 7%, p=0.008), FBXW7 (0% vs. 4%, p=0.014), and KRAS (0% vs. 3%, p=0.005). Interestingly, EGR2 mutations were associated with del(11q) (7% vs. 1%, p=0.016), XPO1 with ATM (13% vs. 4%, p=0.011) and NFKBIE mutations (15% vs. 4%, p=0.021), and BIRC3 with FBXW7 mutations (18% vs. 3%, p=0.045).In Kaplan-Meier analyses, the infrequently mutated genes NFKBIE (4 vs. 8 years, p=0.006), POT1 (3 vs. 8 years, p=0.012), EGR2 (1 vs. 8 years, p=0.020), and XPO1 (3 vs. 8 years, p<0.001) showed significant shorter TTT vs. wt cases, but no effect on OS. Whereas BIRC3 showed only impact on OS (5 year-OS: 61% vs. 88%, p=0.002). Cox regression analysis was performed with following covariates: genes with at least 2% mutation frequency, IGHV status, FISH and cytogenetic aberrations. In multivariate analysis for TTT following markers showed significant effect: IGHV-M (RR=relative risk: 0.3, p<0.001), del(11q) (RR: 2.1, p=0.001), trisomy 12 (RR: 2.1, p<0.001) and SF3B1 mutation (RR: 1.9; p=0.001). The rarely mutated genes NFKBIE, POT1, EGR2 and XPO1 that were significant in univariate analysis showed no independent impact. For OS BIRC3 (RR: 4.1, p<0.001) besides IGHV-M (RR: 0.6, p=0.031) and del(17p) (RR: 5.3, P=0.002) retained significant effect. None of the other rarely mutated genes showed statistical significance already in univariate analysis.Conclusion: In 42% of 531 untreated CLL patients, mutations can be found in 19 distinct genes. Patients with the rarely mutated genes NFKBIE, POT1, EGR2 and XPO1 show reduced time to treatment, whereas BIRC3 mutations are associated independently with shorter overall survival. Even though most of the rare gene mutations analyzed here have no strong impact on prognosis, some of them might be interesting for targeted therapy approaches in the near future. DisclosuresJeromin:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

CLLU1 Expression Is Not Confined to Chronic Lymphocytic Leukemia Cells; Putative Role of Gene-Body Hypomethylation in Regulating Splice Variant-Specific Transcriptional Dynamics

Background: Alternative splicing of the CLLU1 gene generates 6 transcript variants (accession numbers from the National Center for Biotechnology Information: AJ845162-AJ845167), two of which comprise a putative coding DNA sequence (AJ845165 and AJ845166, CDS transcripts). Previous studies showed that CLLU1 overexpression is highly confined to chronic lymphocytic leukemia (CLL) cells and that only the AJ845162 is expressed in both high- and low-risk CLL patients, while expression of the CDS-transcripts is confined to the high-risk subgroup.Pair-wise linear regression analysis revealed a powerful linear relation between the expression levels of all the splice variants, thereby it was deduced that CLLU1 transcription is orchestrated by a common promoter, which is located upstream of CLLU1 exon 1 (Buhl et al., Leukemia 2009). We recently documented (Papamichos et al., Haematologica 2016; s1) that this promoter was evolutionarily derived by an endogenous retrovirus sequence.Herein we argue that CLLU1 expression is not restricted to CLL cells while also we highlight that transcription of CDS transcripts is regulated by an alternative intronic promoter.Methods:CLLU1 high-throughput sequencing of polyadenylated RNA (RNA-Seq) data across 51 human tissues and 2 human cell lines, generated by the Genotype-Tissue Expression (GTEx) project of the US National Institutes of Health Common Fund (GTEx Analysis Release V6p), were analyzed via GTEx Portal. CLLU1 GeneChip expression data (AJ845164 transcript-specific probe set 1558185_at from Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array), generated by Genevestigator project, were downloaded from Genevisible portal.CLLU1 chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-Seq) on histone H3 protein subunit modified at lysine position 4 with trimethylation (H3K4me3) as well as transcription factor (TF) ChIP-Seq data were downloaded from the Encyclopedia of DNA Elements (ENCODE) Regulation supertrack, which is available in UCSC Genome Browser Database. Data on CLLU1 gene-body methylation status in 63 cell lines from ENCODE were downloaded from the HAIB Methyl450 ENCODE track.Results:CLLU1 GeneChip expression data show that the AJ845164 transcript (corresponding to the RP11-693J15.4 long non-coding RNA) is promiscuously expressed in various normal B-cell subpopulations andin mature B-cell neoplasms other than CLL, especially in mantle cell lymphoma.RNA-Seq data analysis reveals that AJ845162 transcript is expressed in normal somatic tissues such as skeletal muscle, salivary gland, and esophageal mucosa. Vice versa, expression of CLLU1 CDS transcripts is confined to transformed lymphocytes.Structure interrogation of CDS transcripts indicates that these variants initiate from an alternative transcriptional start site which is located 2429 nucleotides downstream from the canonical one. H3K4me3 ChIP-Seq data signify the presence of an alternative intronic promoter in this genomic segment. TF Chip-Seq data reveal that the alternative promoter region comprises high affinity binding sites for signal transducer and activator of transcription 3 (STAT3) and Activator protein 1 (AP-1). Analysis of CLLU1 -body methylation status in five lymphoblastoid cell lines reveals that the alternative promoter remains heavily methylated in these cell types. This finding is in accordance with previous studies reporting that CLLU1 gene body is hypermethylated in B-cells from healthy individuals and in IgVH-mutated (IgVH-M) CLL cases while appears hypomethylated in IgVH-unmutated (IgVH-UM) CLL cells.Conclusion: Not all CLLU1 transcripts are equal in discriminating CLL from other mature B-cell malignancies.Whilst of retroviral origin, CLLU1 upstream promoter is not subjected to stringent epigenetic repression but remains active in somatic cells, a tantalizing finding that warrants further investigation.The level of intragenic DNA methylation represents a decisive regulator of alternative promoter usage (Neri et al., Nature 2017). It would be tantalizing to speculate that CLLU1 -body hypomethylation, occuring exclusively in IgVH-unmutated CLL, is mechanistically linked to the epigenetic derepression of the alternative intronic CLLU1 promoter and the subsequent high expression of CLLU1 CDS transcripts in high-risk CLL. DisclosuresNo relevant conflicts of interest to declare.

Tumor Microenvironment (TME) Regulation of Programmed Death-1 (PD1) Receptor and Its Ligands PDL1 and PDL2 in Chronic Lymphocytic Leukemia (CLL)

The PD1 pathway is involved in the inactivation of immune effectors and is potentially reactive against neoplastic cells in the TME. T cells from CLL patients exhibit defective immunity, decreased proliferative capacity, and impaired effector function leading to T cell exhaustion. These functional defects coincide with higher CD244, CD160 and PD1 expression on T cells. In the Eμ-TCL1 transgenic CLL model, PDL1 checkpoint blockade restores immune dysfunction and impairs leukemia growth. PD1/PDL1 ligation also affects BCR signaling, and because PD1 is also expressed on CLL cells, PD1 interference might directly influence tumor growth and proliferation directly.We studied the 1) expression the PD1 receptor and its ligands and their correlation with TFTT; 2) role of the TME in controlling PD1 axis and miRNA expression; 3) role of the BCRi ibrutinib (IB) in the expression of the receptor and ligands.Early-stage CLL patients were prospectively enrolled at diagnosis (n=211, O-CLL1: clinicaltrial.govID:NCT00917540). Gene expression (GE) profiling was performed using the GeneChipVR Gene1.0 STArray (Affymetrix) and validated by qPCR (Thermofisher) and flow-cytometry (FC, BD Biosciences) in an independent subset of patients. CLL lymph node samples underwent in situ immunolocalization analyses. Autologous T-cells (AAT) were obtained by in vitro exposure of patient T-cells with anti CD3/CD28 Dynabeads (Thermofisher) and IL2 in co-culture with CLL cells. Cultures were monitored until substantial clumping was observed and tested for PD1 axis expression. HS5 stromal cells were used in co-culture experiments with B-CLL (ratio 1:100). MiR-424 expression was evaluated by qPCR using validated TaqMan® MicroRNA Assays (Thermofisher). In selected experiments IB was added to cell culture. PD1 GEP of circulating B-CLL cells was significantly higher than of ligands. No differences in GEP of PD1 or its ligands were observed stratifying cases based on CD38, ZAP-70, or FISH markers. Significantly higher PDL1 GE was detected in IGHV-M cases. GEP results were validated by qPCR on 27 independent CLL samples. The impact of GE of the PD1 axis on clinical outcomes in CLL cases (median follow-up 39 months, range 6−82 months) indicated TTFT was significantly shorter in cases with higher levels of PDL2, while no significant differences were detected based on higher or lower PD1 or PDL1 levels. Multivariate analysis indicated higher PDL2 gene expression retained an independent prognostic power (HR=1.9, 95%CI 1.1-3.4, P=.022) in predicting TTFT together with IGHV-UM status, B-lymphocytosis≥5000/mm3 (P=.020), and CD38 expression (P=.021). In situ immunolocalization of CLL tissue infiltrates showed variable expression of PD1 that characterized small lymphoid elements, while PDL1 and PDL2 (PDL2>PDL1) characterized larger medium-sized elements within proliferation centers. Co-localization studies revealed PD1 co-expression of CD20+CLL cells and in scattered CD3+ cells. Both PDL1 and PDL2 were variably expressed in CD20+CLL cells; few T-cells and macrophages showed expression of both ligands within CLL infiltrates. FC analysis of CLL cells after AAT co-culture showed a higher percentage expression of circulating PD1 axis members (n=27, PDL1>>PDL2>PD1) compared to baseline indicating that the changes in PD1 axis expression may be due to events triggered by activation of the TME. In contrast, T cell populations had higher expression of CD3+, CD4+ and CD8+ cells bearing both PD1 and PDL1, while no substantial increment for either CD3+, CD4+ or CD8+/PDL2+ cells following AAT co-culture. GE analysis also showed a similar increase in PDLs (n=15, PDL2>>PDL1>PD1) expression with respect to non-activated B-CLL cells. Interestingly, a 48h co-culture of B-CLL with HS5 stromal cells (n=6) showed a similar increase in PD1 axis GE pattern as AAT cultures. Expression of miR-424 also decreased significantly (<80%) after AAT co-cultures (n=6), indicating its potential involvement in PDL1 regulation. IB reduced PD1/PDL1 gene and protein expression on CLL B-cells; the IB inhibitory effect was also observed only on CD8+ cells.We indicate that PDL2 expression characterizes a subset of high-risk early stage CLL patients. PD1 axis expression is characteristic of the CLL TME whereby modulatory co-stimulatory signals may be counteracted by IB. Further studies will examine the role of miR-424 regulation with this cellular context. DisclosuresNo relevant conflicts of interest to declare.

Real-World Outcomes of Chemoimmunotherapy in Chronic Lymphocytic Leukemia (CLL) Clinical Practice: Results from a Decade of Practice in Helsinki 2005-2015

Introduction: Around 350 new CLL cases are diagnosed annually in Finland, with approximately 1800 individuals living with the disease. However, no comprehensive disease outcome studies from clinical practice have been conducted to-date. In the absence of direct head-to-head trials, robust historical control data have become requisite to complement available clinical trial data and estimate the potential effects of novel therapies in local practice. In addition, patients (pts) in routine clinical care are far more heterogeneous than those included in clinical trials. The Finnish Hematology Registry (FHR) was created to collect real-world practice outcomes in CLL and other hematological malignancies, with Finland's centralized nationwide healthcare system allowing reliable pt identification and complete follow-up. The objective of this registry study is to describe treatment and survival outcomes for CLL pts from routine practice settings.Methods: A non-interventional, retrospective study design was used to collect routine clinical practice data from the FHR. Eligible pts aged ≥18 years diagnosed with CLL and receiving 1 or more treatment lines during 2005-2015 were identified from the FHR, with groupings of pts into those treated in the early (2005-2007), middle (2008-2010) and late (2011-2015) cohort time periods. As the registry had limited CLL pt data at study start, clinical investigators accessed medical records to retrieve and validate data. We report interim results on 66 pts from the Helsinki University Hospital, Helsinki, Finland, a hospital region accounting for 30% of the total national CLL incidence. The completion and validation of the Helsinki cohort (~130 pts) is ongoing and results from the whole dataset will be presented at ASH.Results: Median age at diagnosis was 67 years and 23% had Binet stage C. Longitudinal follow-up was available from 1st to 7th or higher treatment lines. Across all treatment lines, fludarabine-cyclophosphamide-rituximab (FCR) (52%), bendamustine-rituximab (BR) (48%), fludarabine-cyclophosphamide (FC) (27%), and chlorambucil (chl) -based therapies (24%) were the most frequently used treatments. FCR (48%) and FC (27%) were frequently used in the 1st line, whereas BR (34%) was the primary choice in the 2nd line. In later lines a wide array of treatment regimens were used. Only 2 pts received the novel agent ibrutinib as part of a named patient program; no other novel agents were used in this cohort during this period.FC and FCR were initiated in equal yet constantly decreasing proportions through 2008, when FCR became most common (0% FC vs. 40% FCR). A year later in 2009, BR was introduced into clinical practice and its use increased steadily from 14% to 30% through 2014 when its use then declined. The peak year of chl monotherapy or chl-based combinations was in 2006 with 38% of pts receiving chl-based treatment. Since then chl use has decreased dramatically, to 0% in 2015.Median overall survival (OS) from the start of any given 1st line treatment was 81 months (m). Pts receiving FC (p=0,009) and FCR (p<0,001) had significantly longer median OS (79 m and 82 m respectively) compared to bendamustine/BR (62 m) when given in the 1st line. FC and FCR resulted in longer, though not statistically different, time-to-next-treatment (TTNT) compared to bendamustine/BR (77 m vs 44 m). Despite the observed practice changes over the years, there were no detectable outcome differences in 1st and 2nd line median OS between those treated in early, middle and late time periods, possibly due to limited follow-up in the later period (data not shown).Conclusions: We describe real-world treatment trends and outcomes from a decade of practice in the Helsinki region. Our results show that chemoimmunotherapies have been vigorously implemented in clinical practice. However, interim results do not show detectable improvements for OS and TTNT during this decade of treatment in the Helsinki hospital region. In the Nordics, real-world evidence has become essential when introducing novel targeted therapies into clinical practice settings, with comprehensive disease registries expanding to allow for more detailed follow-up. [Display omitted] DisclosuresJarvinen:Janssen-Cilag Oy: Employment. Mehtälä:EPID Research: Employment, Other: EPID Research is a contract research organization that performs commissioned pharmacoepidemiological studies and thus its employees have been and currently are working in collaboration with several pharmaceutical companies.. Khanfir:EPID Research: Employment, Other: EPID Research is a contract research organization that performs commissioned pharmacoepidemiological studies and thus its employees have been and currently are working in collaboration with several pharmaceutical companies.. Juuti:EPID Research: Employment, Other: EPID Research is a contract research organization that performs commissioned pharmacoepidemiological studies and thus its employees have been and currently are working in collaboration with several pharmaceutical companies.. Leval:Janssen-Cilag Ab: Employment.

The Mutation Landscape of Follicular Lymphoma and Chronic Lymphocytic Leukemia Reveals a Distinctive Frequency and Composition of Aid-Related Mutation Signatures

Introduction: Somatic mutations associated with distinctive types of cancer can be globally classified by the trinucleotide context of single nucleotide variants into mutational signatures. Distinct signatures can be attributed to underlying causative mechanisms such as UV irradiation or smoking. B-cell lymphomas have only been analyzed globally and carry a signature characterized by APOBEC activity (Alexandrov, 2014).The two most common indolent B-cell neoplasms, chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL), are characterized by distinctive pathogenic processes, but mutagenesis caused by the APOBEC enzyme activation-induced cytosine deaminase (AID) may be shared by both malignancies. AID deaminates deoxycytidine to deoxyuridine, which is processed by DNA repair enzymes into a larger spectrum of point mutations and also DNA double-strand breaks. AID activity and subsequent DNA repair create two different mutation signatures: the canonical signature characterized by C>T/G transitions at RCY motifs and the non-canonical defined by A>C transversions at WAN motifs. More recently a third AID signature characterized by C>T transitions at RCG motifs has been associated with AID-mediated CpG-methylation dependent mutagenesis.We here investigate the hypothesis that AID mutational signatures contribute to the pathogenesis of FL, a lymphoma characterized by constitutional expression of AID, but also of CLL since high AID expression levels may account for a more aggressive disease, and a subset of CLL B-cells with AID expression display dissociation between class switch recombination and somatic hypermutation.Methods: We analyzed the mutational landscape in whole exome sequencing data from 31 FL and 24 CLL cases. Somatic variants were called by comparison of tumor and germline DNA with an in-house developed pipeline. The relative contribution of mutations at canonical and non-canonical AID hotspot motifs was computed by direct quantification of variants occurring in known AID motifs. Student's t test was used for comparison between CLL and FL.Mutational signatures were defined according to the 96-base substitution model (Alexandrov, 2013) by an unsupervised machine learning approach, with a local implementation of the SomaticSignatures R package.Results: The frequency of C>T/G transitions at canonical AID hotspot was more prevalent in CLL (9.0%; 95% CI: 8.1-10.0%) than in FL (6.6%, 95% CI: 5.9-7.3%)(p<0.001). Likewise, A>C transversions at non-canonical AID sites were significantly more frequent in CLL (1.9%; 95% CI: 1.6-2.2%) as compared to FL (0.5%; 95% CI: 0.4-0.6%)(p<0.0001).In the unsupervised analysis of FL and CLL cases, 97.7% of the mutation spectrum variance was explained by only 5 signatures (Figure 1). In FL, the mutational landscape was dominated by a signature (S2) characterized by C>A transversions in the trinucleotide KCH context (40%, 95% CI: 33-36%). The second most frequent signature (S3; 22%, 95% CI: 15-30%) was characterized by C>T transitions in the context of a hybrid between the canonical AID and the CpG hotspot motifs (RCY/RCG).In contrast, the mutational landscape of CLL was dominated by a signature (S5; 42%, 95% CI: 34-49%) characterized by C>T transitions in the RCY motif and A>C transversions in the WAN context, consistent with an overlapping between the canonical and the non-canonical AID hotspots (Kasar, 2016).Conclusions: The mutational landscape of both CLL and FL seems to be strongly shaped by AID activity. In FL, AID-induced mutations are mainly restricted to canonical AID hotspots and CpG-methylation dependent mutagenesis sites. In strong contrast, both canonical and non-canonical AID motifs contributed to the mutational landscape of CLL. In addition, FL carries a high prevalence of C>A substitutions that are associated with exposure to polycyclic aromatic hydrocarbons that produce bulky DNA adducts followed by the nucleotide excision repair process. These compounds are present in several industrial processes (food products, beverages, tobacco) that have been suggested as occupational risk factors for follicular lymphomas (Mester, 2009).Overall, our data suggest a much stronger role of AID in CLL pathogenesis than hitherto appreciated. The differential contribution of canonical versus non-canonical AID activity points to different regulation of AID in CLL compared to FL and warrants further investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Factors Associated with Non-Melanoma Skin Cancer (NMSC) Among Chronic Lymphocytic Leukemia (CLL) Patients

Background: The incidence of NMSC in patients with CLL is significantly higher compared to age- and sex- matched controls. Herein, we evaluate factors associated with risk of NMSC post-CLL diagnosis, including CLL clinical and prognostic characteristics, history of UV radiation exposure, severe sunburn, and combined genetic score of single nucleotide polymorphisms (SNPs) associated with NMSC.Methods: CLL patients newly diagnosed from 2002-2015 were enrolled and systematically followed by the Mayo Clinic/University of Iowa Lymphoma SPORE. Clinical and prognostic CLL data were obtained from the Mayo Clinic CLL database. NMSCs were abstracted both from medical records and from patient self-report. Self-reported history of the extent of mid-day sun exposure (h/week) at various ages (<13, 13-21, 22-40, >40 yrs) and severe sunburn were obtained from a risk factor questionnaire. The CLL International Prognostic Index (CLL-IPI) was computed as established using a weighted average of five independent CLL prognostic factors (IGHV mutational status, serum b2-microglobulin, Rai stage, age at CLL diagnosis and FISH 17p deletion/ TP53 status). CLL treatment was classified as positive if a person received T-cell immunosuppressive treatments that are purine analogue and/or alemtuzumab based treatments. All other therapies were combined into an “other category”. We computed a polygenetic risk score (PRS), a weighted average of the number of risk alleles across 13 SNPs that are known to be associated with NMSC, with the weights being the log of the odds ratio reported for each SNP. To evaluate associations with risk of NMSC post-CLL diagnosis we used Cox regression to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). CLL treatment was considered a time-dependent covariate in the analysis.Results: Among 1269 CLL patients, the median age at diagnosis was 63 years (range 24-91), 68% were male, and 6% had Rai stage III-IV at diagnosis. Based on the CLL-IPI, 44% were low risk, 34% intermediate risk, and 22% high/very-high risk. 9% of the CLL cases had prior history of NMSC and 44% reported history of severe sun burn. At a median follow-up of 7 years from CLL diagnosis, 196 patients had ≥1 NMSCs post-CLL diagnosis. Among these patients, 45 also had NMSC before CLL diagnosis. The most frequent NMSC was squamous cell carcinoma (63%), followed by basal cell (55%), and Merkel cell (1%). 264 patients received T-cell immunosuppressive treatments prior to NMSC diagnosis or prior to last follow up. Significant associations of clinical and prognostic characteristics with risk of NMSC post-CLL diagnosis were observed for age (HR=1.42 per 10 year increase, CI=1.24-1.62), male sex (HR=1.43, CI=1.04-1.97), prior history of NMSC (HR=3.85, CI=2.75-5.39), and CLL-IPI (HR=1.36, CI=1.13-1.64, adjusted for sex and prior NMSC). Mid-day sun exposures for each of the age groups considered showed no evidence of association with risk of NMSC post-CLL diagnosis; however, history of severe sunburn was significantly associated (HR=1.58, CI=1.06-2.36, adjusted for sex, CLL-IPI and prior NMSC). We observed an association between T-cell immunosuppressive treatments with risk of NMSC post-CLL diagnosis (HR=1.65, CI=1.14-2.40, adjusted for age, sex and prior NMSC). PRS was also associated with the risk of NMSC post-CLL diagnosis (HR=1.82, CI=1.21-2.72, adjusted for sex, CLL-IPI and prior NMSC).Conclusion: Screening for NMSC is important in CLL patients, and our data suggest that it is particularly important among patients with aggressive CLL according to CLL-IPI score, those receiving T-cell immunosuppressive treatments that are purine analogue and/or alemtuzumab based treatments, those who have a prior history of NMSC, or history of severe sunburn. PRS was also a significant factor in the risk of NMSC among CLL patients. Future studies should develop prediction models that include these factors. [Display omitted] DisclosuresKay:Tolero Corporation: Research Funding; Gilead: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding. Shanafelt:Genentech, Celgene, Pharamacyclics, Jansen, GlaxoSmithKline, AbbVie, Hospira, and Cephalon: Research Funding. Cerhan:Janssen: Other: Multiple Myeloma Registry Steering ; Janssen: Other: Scientific Advisory Board (REMICADELYM4001).

Genome-Wide DNA Methylation Profiling of Chronic Lymphocytic Leukemia Subsets Carrying Stereotyped B Cell Receptors

In recent years, subsets of chronic lymphocytic leukemia (CLL) patients carrying quasi-identical or stereotyped B cell receptors (BcRs) have been identified that share clinicobiological features and disease outcome. While these stereotyped subsets show distinct gene expression and genomic profiles, the DNA methylation landscape remains largely unexplored. By applying high-resolution 450K methylation arrays, we investigated 176 CLL subset cases belonging to: (i) the clinically aggressive, IGHV-unmutated (U-CLL) subsets #1 (clan I genes/IGKV(D)1-39, n=37) and #8 (IGHV4-39/IGKV1(D)-39, n=21); (ii) the IGHV1-69-expressing U-CLL subsets #3 (n=12), #5 (n=9), #6 (n=22), and #7 (n=12); and, (iii) the indolent, IGHV-mutated (M-CLL) subset #4 (IGHV4-34/IGKV2-30, n=28). In addition, we included subset #2 cases (IGHV3-21/IGLV3-21, mixed mutation status, n=35) that have a poor outcome independent of IGHV mutation status. For comparative purposes, we included a cohort of CLL cases that do not express stereotyped BcRs (‘non-subset’, n=325). These patients were subgrouped according to the recently proposed epigenetic classification of CLL, i.e. poor-prognostic, naïve-like CLL (n-CLL, n=102), favorable-prognostic, memory-like CLL (m-CLL; n=176), broadly corresponding to U-CLL and M-CLL, respectively, and a third intermediate CLL subgroup (i-CLL; n=47), which express borderline mutated IGHV genes and have an intermediate prognosis. Finally, a series of sorted normal subpopulations spanning different stages of B-cell differentiation [precursors (n=22), naïve B cells (n=19) and germinal center/memory B-cells (n=33)] were also included in the analysis. Overall, unsupervised analysis of subset vs. non-subset CLL revealed that all U-CLL subsets clustered with n-CLL, subset #4 clustered with m-CLL, while subset #2 clustered separately with i-CLL (Figure 1). Supervised analysis revealed a limited number of CpG sites that were differentially methylated when comparing each U-CLL or M-CLL subset with non-subset cases. In contrast, almost all subset #2 cases clustered separately from i-CLL in supervised analysis, indicating that this subset might represent a distinct subgroup of i-CLL. We recently demonstrated that the number of epigenetic changes that a tumor acquires, compared to its cellular origin (i.e. ‘epigenetic burden‘), may be a powerful predictor of clinical aggressiveness (Queiros et al, Cancer Cell 2016). When adopting this approach in CLL, comparison of specific subsets vs. their non-subset cases matched by epigenetic subgroup, revealed significant differences in the epigenetic burden amongst the various groupings; for instance, in subset #1 vs. n-CLL (72K vs. 67K, p<0.05) and in subset #2 vs. i-CLL (76K vs. 68K, p=0.001), while no difference was observed between subset #4 vs. m-CLL (83K vs. 82K, p=not significant). Subset #2 cases frequently carry del(11q) and harbor SF3B1 mutations, however, neither the IGHV mutation status nor the presence of del(11q) or SF3B1 mutations had any impact on the epigenetic burden within subset #2. In conclusion, U-CLL and M-CLL subsets generally clustered with n-CLL and m-CLL categories, respectively, implying common cellular origins. In contrast, subset #2 emerged as the first defined member of the i-CLL group, which in turn alludes to a distinct cellular origin and/or pathogenetic process for subset #2 and i-CLL patients. [Display omitted] DisclosuresPapakonstantinou:Janssen Pharmaceuticals: Research Funding; Gilead: Research Funding. Smedby:Janssen: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Gaidano:Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Ghia:AbbVie: Consultancy; Adaptive: Consultancy; Gilead: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy; Roche: Consultancy; Novartis: Research Funding. Stamatopoulos:Novartis SA: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.

Predicting Mechanisms of Ibrutinib-Resistance in Chronic Lymphocytic Leukemia (CLL) Using Computational-Based Modeling for Personalized Therapy with Clinical Validation

Background: Bruton's tyrosine kinase (BTK), a mediator of B cell receptor signaling, has been effectively exploited as a therapeutic target in Chronic lymphocytic leukemia (CLL). Ibrutinib is a BTK inhibitor that has shown excellent efficacy in treatment-naïve, relapsed/refractory, and high-risk CLL. However, concerns regarding ibrutinib resistance have emerged and characterizing the underlying mechanisms may aid in identifying patients with increased risk of resistance/disease progression. Moreover, understanding the interplay between determinants of ibrutinib resistance may reveal alternate therapeutic targets. Using a computational biology modeling (CBM) simulation-based method, we reverse-engineered the salient molecular architecture of a CLL patient progressing on ibrutinib to identify potential drug combinations to overcome drug resistance and induce remission.Methods: For validation of the computational biological modeling (CBM) software (Cellworks group), we selected a cohort of 15 ibrutinib resistant CLL patients from previously published studies (Table 1). Genomic data derived from whole exome sequencing (WES), targeted next-generation sequencing (NGS), copy number variation (CNV), and/or karyotype data were used in this study. All available genomic data for each profile was entered into the CBM, which generates a disease-specific protein network map using PubMed and other online resources. Disease-biomarkers that are unique to each patient were identified within the protein network maps by using CBM technology. Ibrutinib was selected from a digital drug library and simulated on every relapsed CLL patient case. Drug impact was assessed by quantitatively measuring the drug's effect on a cell growth score, which is a composite of cell proliferation, viability, and apoptosis, along with the simulated impact on each disease-specific biomarker score. Each patient-specific map was digitally screened to identify any Ibrutinib resistance mechanism(s).Results: In our modeled patient set, 4 different resistance mechanisms were identified across 14/15 ibrutinib- refractory CLL cases. 1) BTK C481S mutation (n=4) impacts ibrutinib binding affinity; 2) PLCG2 mutations (n=4) lead to activation of PRKCB and AKT1 signaling; 3) CARD11 mutation (n=2) results in constitutive activation of BTK downstream effectors; and 4) decreased G-protein coupled receptor (GPCR) pathway signaling, an upstream activator of BTK (n=5), was found to be a novel resistance mechanism identified by CBM. Knockdown (KD) of ALOX5AP, ARRB1 and CYSLTR2 reduced GPCR pathway signaling, resulting in reduced BTK activity (Table 1). After validating the CBM algorithm, we rendered a digital avatar for a relapsed-refractory CLL patient (Del17p+); progressing on ibrutinib. The patient had rapidly escalating ALC and lymphadenopathy; with the largest node measuring ~16cm. In addition to p53 loss, simulation modeling accounted for BTK C481S mutation (BAF 0.69) expressed in the patients tumor cells. CBM predicted venetoclax would be the most effective therapeutic for the patient due to several upstream signaling pathways converging on Bcl-2. Indeed, addition of venetoclax to the ongoing ibrutinib monotherapy dramatically reduced the patients ALC (from 113 to 3.3 x 109/L) by day 4 post-treatment, accompanied by reduction in lymphadenopathy (left axillary node decreased to ~3cm). Ibrutinib was discontinued within 1 month of starting the combination, with venetoclax monotherapy continued per dosing guidelines thereafter. Notably, the patient maintained remission status on venetoclax monotherapy alone.Conclusions: Computational modeling was used to identify ibrutinib resistance mechanisms in CLL patients. Using this novel approach, we identified an effective therapy (venetoclax) for a high-risk CLL patient who was progressing on ibrutinib. CBM-based approaches can be used to predict alternative therapeutic regimens for drug-refractory patients, targeting the disease-specific biomarkers unique to each patient. Altogether, our study demonstrates proof-of-principle on the use of computational predictive algorithms to simulate multiple protein-interaction networks operational in a tumor cell to exploit individualized therapeutic vulnerabilities. [Display omitted] DisclosuresYsebaert:Janssen: Consultancy, Research Funding, Speakers Bureau. Kumar:Cellworks: Employment. Singh:Cellworks Research India Pvt. Ltd: Employment. Ullal:Cellworks Research India: Employment. Bhargav:Cellworks: Employment. Azam:cellworks: Employment. Abbasi:Cellworks Group Inc.: Employment. Vali:Cellworks Group Inc.: Employment. Cogle:Celgene: Other: Membership on Steering Committee for Connect MDS/AML Registry.

Genomic Landscape of Chronic Lymphocytic Leukemia in China By Targeted Gene Sequencing

IntroductionIn recent years, whole-exome sequencing (WES) and whole-genome sequencing (WGS) have identified over 60 recurrently mutated genes in both coding and non-coding regions in chronic lymphocytic leukemia (CLL) patients. However, these studies were restricted to Europeans, and the genomic landscape of CLL in Asians remains less-well defined. To better understand the genetic basis of CLL in Asians, we used target gene sequencing to define the mutational status of 63 recurrently mutated genes in CLL and other small B-cell lymphomas in a cohort of 93 Chinese patients with CLL.MethodsA total of 93 CLL patients were retrospectively enrolled. Genomic DNA was isolated from cryopreserved mononuclear cells or bone marrow aspirate specimens. Targeted gene sequencing was performed on HiSeq Sequencer (Illumina, Inc, San Diego, CA) following the manufacturer's standard sequencing protocols. The sequencing reads were aligned to hg19 using the Burrows-Wheeler Aligner (BWA). SNV calling was performed using both GATK and Varscan programs, and the called SNV data were then combined. The variants at a frequency of 0.1% or more in the 1000 genomes database or 1000g (CHB and CHS) (http://www.1000genomes.org), esp6500 (http://evs.gs.washington.edu/EVS) or ExAC03 database (http://exac.broadinstitute.org) were removed from subsequent analysis.ResultsA total of 195 driver mutations across 50 genes were identified in 80 patients (86.0%). 57 cases (61.3%) harbored mutations involving at least two genes. The most frequently mutated genes included IGLL5 (21/93, 22.6%), MYD88 (15/93, 16.1%), LRP1B (11/93, 11.8%), TP53 (9/93, 9.6%), NOTCH1 (8/93, 8.6%), ATM (7/93, 7.5%), CHD2 (7/93, 7.5%) and KMT2D (7/93, 7.5%). Only 4 cases with SF3B1 mutations (4/93, 4.3%) were identified in our cohort (Figure 1). By comparing the results from the study by Landau et al, our results showed that CLL in Chinese displayed significantly different mutational profiles from those in Caucasians (Table 1).Cases with complex karyotype (CK) had higher frequencies of TP53 (7/16 [44%] vs 2/65 [3%], p <0.0001) and EGR2 mutations (3/16 [19%] vs 0/65 [0%], p=0.0066), as compared to cases without CK . By Kaplan-Meier analysis, we found that shorter time-to-first-treatment (TTT) was significantly associated with TP53 (p <0.0001), EGR2 (p=0.0019), KMT2D (p=0.047) and DDX3X mutations (p=0.0232). Multivariate analysis of TTT revealed that only TP53 mutation (hazards ratio[HR]: 2.945, 95%CI: 1.065-8.147; p=0.037) and advanced Binet stage (HR:4.464, 95%CI:2.037-9.784; p=0.0002) independently predicted inferior TTT (Table 2). TP53 (p <0.0001), SF3B1 (p=0.017), NOTCH1 (p=0.012), EGR2 (p <0.0001) and DDX3X mutations (p=0.007) predicted reduced overall survival. Multivariate Cox analysis revealed that TP53 mutation (HR: 20.361, 95%CI: 3.220-128.753, p=0.001), EGR2 mutation (HR: 15.497, 95%CI: 1.639-146.514; p=0.017) and SF3B1 mutation (HR: 20.230, 95%CI: 2.337-175.085, p=0.006) independently predicted unfavorable overall survival.ConclusionsWe found that Chinese CLL displayed a distinct mutational landscape from that of Caucasians. Our findings suggest different mechanisms involved in the pathogenesis of Chinese CLL. Besides, our results support that targeted gene sequencing can provide important values in the prognostification of CLL. Targeted gene sequencing of a large cohort and whole exome/genome sequencing of CLL cases are needed to better define the genomic landscape of CLL in Chinese. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High IGF1R Expression Is Associated with Worse Prognosis in CLL and Impacts Response to PI3K-δ Inhibitor Treatment

High IGF1R Expression Is Associated with Worse Prognosis in CLL and Impacts Response to PI3K-δ Inhibitor Treatment