IGHV Unmutated Patients Research Articles

Safety and Effectiveness in Treatment Naive CLL Patients with or without High Risk Features Treated with Veno Under Real-World Conditions in Austria, Germany, and Switzerland

Background In chronic lymphocytic leukemia (CLL), the association of genetic markers such as del(17p), TP53 and IGHV status with disease prognosis is well established. In clinical trials, treatment with Venetoclax in combination with Obinutzumab (VenO) has shown promising efficacy and good tolerability in all subgroups 1. However, there is limited real-world data in specific genetic subgroups. Aim Effectiveness and safety data were analyzed for therapy naive patients treated with VenO focusing on CLL patients with or without the genetic risk factors TP53 mut and/or del(17p) as well as IGHV status. Methods VeRVe is a non-interventional, observational, prospective study with 60 study sites in Germany, Austria, and Switzerland. CLL patients treated with VenO according to the local label 2 were included. Study documentation is possible at baseline, weekly during ramp-up, monthly until the end of month 6 and quarterly afterwards up to a maximum of 2 years. Response assessment was documented after ramp-up, 3, 12 and 24 months. Results Until April 12 th, 2023, 101 patients were enrolled and received at least one dose of Venetoclax, 42 % were IGHV unmutated. Baseline characteristics were balanced between the genetic risk groups. Patients with TP53 aberrations were older compared TP53 wildtype patients (71 vs. 66 a). 70% of patients with TP53 aberration, 68% of patients with wt TP53, 74% of patients with IGHVunmutated and 71% of patients with IGHVmut had at least 1 comorbidity. The best overall response rate after 12 months for 88 evaluable patients is shown in the figure. Estimated PFS-rate after 1 year was 93% in the total population, 100% in patients with TP53 aberrations, 91% in patients with wt TP53 95.1% of patients with IGHVunmutated and 82.5% of patients with IGHVmut. Estimated OS -rate after 1 year was identical to the estimated PFS. CTCAE grade AEs 3-5 were reported in 35% of the total population, in 21% in the TP53 aberrant, in 35% in the TP53 wt, in 33% in the IGHVunmutated and 10% in the IGHVmut population. A total of 3 patients had laboratory TLS. No new safety signals were detected. Conclusion In summary, both patients with or without TP53 aberration and with mutated or unmutated IGHV had high rates of best ORR, estimated PFS, and OS. Reassuringly, CR/CRi rates were high in patients with TP53 aberration or IGHV unmutated patients. Treatment was well tolerated. Disclosures DR has received honoraria or research grants from AbbVie, AstraZeneca, Gilead, Janssen, Verastem, Roche, Cellestia. IS has received honoraria from AbbVie, Amgen, AstraZeneca, BeiGene, Janssen, Roche, Servier. HH received speaker or consulting fees from AbbVie, AstraZeneca, Celgene, Janssen, Roche, and Beigene. CL received consulting fees from AbbVie, Amgen. TW received honaria or research funding from Novartis, Celgene, Roche, Bayer, Teva and Abbvie. CL and JH are employees of AbbVie and may own AbbVie stock. BS received speaker or consulting fees from AbbVie, Alexion, Amgen, AstraZeneca, BeiGene, BMS, Hexal, Janssen, Novartis, Pfizer, Sanofi-Aventis and SOBI. PP has received honoraria from Amgen, Gilead, Janssen, Sanofi-Aventis, Takeda. Honorarium paid by AbbVie was not related to authorship of this abstract. No honoraria or payments were made for authorship. AbbVie sponsored this study and contributed to the design, study conduct. AbbVie participated in the interpretation of data, review, and approval of the publication. All authors had access to all relevant data. Statistical support was provided by Daniela Giesder and Anna Eisen of GermanOncology GmbH, which was funded by AbbVie.

Obinutuzumab plus chlorambucil versus ibrutinib in previously untreated chronic lymphocytic leukemia patients without TP53 disruptions: A real-life CLL campus study.

One of the main issues in the treatment of patients with chronic lymphocytic leukemia (CLL) deals with the choice between continuous or fixed-duration therapy. Continuous ibrutinib (IB), the first-in-class BTK inhibitor, and obinutuzumab-chlorambucil (G-CHL) are commonly used therapies for elderly and/or comorbid patients. No head-to-head comparison has been carried out. Within the Italian campus CLL network, we performed a retrospective study on CLL patients without TP53 disruption treated with IB or G-CHL as first-line therapy. Patients in the G-CHL arm had a higher CIRS score and the worst renal function. The overall response rates between the G-CHL and IB arms were similar, but more complete remissions (CRs) were achieved with G-CHL (p = 0.0029). After a median follow-up of 30 months, the progression-free survival (PFS, p = 0.0061) and time to next treatment (TTNT, p = 0.0043), but not overall survival (OS, p = 0.6642), were better with IB than with G-CHL. Similar results were found after propensity score matching and multivariate analysis. While PFS and TTNT were longer with IB than with G-CHL in IGHV unmutated patients (p = 0.0190 and 0.0137), they were superimposable for IGHV mutated patients (p = 0.1900 and 0.1380). In the G-CHL arm, the depth of response (79% vs. 68% vs. 38% for CR, PR and SD/PD; p < 0.0001) and measurable residual disease (MRD) influenced PFS (78% vs. 53% for undetectable MRD vs. detectable MRD, p = 0.0203). Hematological toxicities were common in the G-CHL arm, while IB was associated with higher costs. Although continuous IB provides better disease control in CLL, IGHV mutated patients and those achieving an undetectable MRD show a marked clinical and economic benefit from a fixed-duration obinutuzumab-based treatment.

High Cell Death Rates at Start of Ibrutinib Therapy Predict for Deeper Remissions in Patients with Chronic Lymphocytic Leukemia (CLL)

Introduction: The BTK inhibitor ibrutinib blocks B-cell receptor and other signaling pathways in CLL cells and has become a standard CLL therapy. Isotopic labeling with deuterated water (2H2O) to measure the effects of ibrutinib on CLL cell proliferation and death directly in patients revealed that ibrutinib profoundly inhibits proliferation and induces high rates of leukemia cell death (registered at clinicaltrials.gov as NCT01752426; Burger et al., JCI Insight, 2017). Here, we report a long-term follow-up of this trial in which we analyzed long-term outcomes and correlated CLL cell birth and death rates with depth of response over time. Methods: Thirty patients with previously untreated CLL/SLL enrolled in the study to receive continuous long-term ibrutinib monotherapy, after one month of labeling with deuterated water. For our analyses, we stratified patients in groups based on high or low CLL cell death rates in the peripheral blood (PB) or lymphatic tissues (lymph nodes, spleen), i.e. death rates above or below the median, assessed over the first months on therapy, as detailed in the original publication. Responses were assessed according to 2008 IWCLL guidelines, with the exception that lymphocytosis was not used as a sole criterion for progression. PB MRD was quantified by flow cytometry with a sensitivity 10-4. Results: The median age of the participants was 64 years (range, 48 - 78); 63.3% were male. Thirteen patients (43%) had advanced stage disease, 57% had unmutated IGHV genes, and 10% carried 17p deletion (del17p). Twenty-two patients (73%) completed 5 years of protocol treatment and subsequently continued treatment with commercial supply ibrutinib. The patients were followed for a median time of 107 months (range, 6 - 111). The reasons for eventual treatment discontinuation (n=21) were adverse events (n=8, 38%), progressive disease (n=4, 19%), change to another treatment (n=5, 24%), death (n=2, 7%) and other reasons (n=2, 7%). To date, nine patients are still receiving ibrutinib. Five patients (17%) achieved a complete remission, 24 (80%) achieved partial remission, and one patient (3%) had stable disease, resulting in an overall response rate of 97%. Median progression-free (PFS) and overall survival (OS) were not reached at a median follow-up of 107 months. Five-year PFS was 80.6%, and OS was 90%. PB MRD was measured in 18 patients after 5 years of ibrutinib (54 to 60 cycles); all patients had detectable MRD (median 11.8%, range, 0.06 - 47.30%). We found that CLL cell death rates in PB and tissues after treatment initiation inversely correlated with the level of PB MRD after 5 years (r=-0.5872, P=0.0104 and r=-0.4407, P=0.0672). MRD levels were significantly lower in patients with high CLL cell death rates in PB (7.03% ± 3.68% vs. 29.46% ± 5.53%, mean ± SEM, P=0.0056) and in tissues (9.73% ± 5.49% vs. 28.89% ± 4.42%, P=0.0155) as compared with patients with lower CLL cell death rates (Figure 1). Tissue CLL cell death rates were significantly higher in patients with IGHV-unmutated CLL compared to mutated (28.88% ± 2.75% vs. 13.16% ± 2.82%, P=0.0005), and MRD levels trended lower (IGHV-unmutated: 10.71% ± 5.39% vs. IGHV-mutated: 28.36% ± 6.02%, P=0.0878). No statistically significant correlation was found between CLL cell birth rate at baseline or after start of ibrutinib and MRD levels after 5 years of treatment. Conclusion: CLL cell death rates during early treatment correlated with achieving lower MRD levels. We found that CLL cell death rates are higher in IGHV-unmutated patients suggesting a higher dependency of IGHV-unmutated clones on BCR signaling and, therefore, resulting in deeper responses. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BRAF and BIRC3 Mutations Stratify a Poor Prognostic Subgroup in FCR Treated Chronic Lymphocytic Leukemia

Abstract Background. The current shift of therapy of chronic lymphocytic leukemia (CLL) towards novel targeted agents mandates the recognition of molecular predictors to identify patients who can still benefit from chemoimmunotherapy and those who should instead be considered for novel targeted agents upfront. In the case of FCR (fludarabine, cyclophosphamide, rituximab), the IGHV mutation status and FISH karyotype stratify low-risk patients carrying mutated IGHV genes and devoid of both del11q and del17p who maximally benefit from such treatment; intermediate-risk patients harboring unmutated IGHV genes and/or del11q in the absence of del17p are a case mix of good and poor responders to FCR; while high-risk patients harboring del17p are unsuitable for chemoimmunotherapy. This model fails to consider the impact of recurrent CLL mutations of potential prognostic relevance, which may be improved by their inclusion. Purpose. We aimed at refining the genetic-based stratification of FCR-treated CLL patients by integrating the mutational profile in a prognostic model together with the IGHV mutation status and FISH karyotype. Methods. A multicenter cohort of 173 (162 with complete molecular data) untreated CLL receiving first-line therapy with FCR in the real-life clinical practice was evaluated by target resequencing. Tumor genomic DNA collected at the time of treatment was analyzed for mutations in the coding exons plus splice sites of CLL cancer driver genes (n=23). Deep next-generation-sequencing (NGS) of the gene panel was performed on the Illumina MiSeq platform (coverage >2000x in >90% of the target). Non-synonymous mutations represented in >10% of tumor allele were called by using VarScan2, and a stringent bioinformatic pipeline was developed to protect against the false call of polymorphisms and sequencing errors. Medical statistics was performed using SPSS version 24.0 and R version 3.3.2. Results. The cohort characteristics and mutational profile were consistent with those reported in CLL receiving FCR as initial treatment. After a median follow-up of 7.2 years, 114 patients progressed, accounting for a median PFS of 4.5 years. Among patients categorized as low-risk by IGHV and FISH status, gene mutations associated with poor prognosis were virtually absent (Fig. 1A). By univariate analysis, none of the cancer driver gene mutations significantly associated with PFS among low-risk patients. Consistently, by recursive partitioning, the proportion of low-risk patients failing FCR early was not explained by the co-occurrence of a cancer driver gene mutation. Among patients categorized as intermediate-risk by IGHV and FISH status, mutations of BIRC3 (HR: 6.452; 95% CI 2.467-16.875; p Download : Download high-res image (256KB) Download : Download full-size image Disclosures 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. Zaja: Novartis: Honoraria, Research Funding; Janssem: Honoraria; Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Abbvie: Honoraria; Takeda: Honoraria; Gilead: Honoraria. Rigolin: Celgene: Honoraria, Other: Advisory Board; Mundipharma: Other: Advisory Board; Gilead: Honoraria. Coscia: Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Tedeschi: AbbVie: Consultancy; Gilead: Consultancy, Other: travel expenses; Janssen: Consultancy, Other: travel expenses. Laurenti: Roche: Other: Advisory Board; Gilead: Other: Advisory Board; Janssen: Other: Advisory Board; Abbvie: Other: Advisory Board. Cuneo: Gilead: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; Abbvie: Honoraria, Other: Advisory Board; Roche: Honoraria, Other: Advisory Board. Foa: AbbVie: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Sandoz: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; janssen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau. Gaidano: Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

Ibrutinib Therapy Downregulates Toso, the Fcr for IgM, Expression in CLL Patients

Background: Toso, the FcR receptor (FcmR) for IgM, is a type I transmembrane protein belonging to the immunoglobulin gene superfamily. Toso is expressed in lymphocytes, and plays an important role in B cell development and survival. In normal B cells, Toso expression increases after B cell receptor (BCR) stimulation, and Toso enhances BCR signaling induced cell survival, NK-kB pathway activation, and BCL-xL expression. CLL B cells express high levels of Toso, especially in IgHV unmutated patients. Upon binding of IgM to Toso on CLL cells, the Toso-IgM complex is internalized and undergoes lysosomal degradation. Given the importance of BCR signaling in CLL pathogenesis and treatment, we performed a series of preclinical and correlative studies to examine the cross talk between Toso and BCR signaling, effects of Toso on CLL cell survival and how Toso is affected by BCR signaling inhibition with ibrutinib. Methods and Results: Toso gene expression (FAIM3) was analyzed in serial samples from eight CLL patients treated with ibrutinib plus rituximab using Affymetrics HG U133 plus 2.0 oligonucleotide arrays at baseline, and after 1 and 4 weeks of continuous ibrutinib-based therapy. We noted that the relative mean Toso gene expression declined during ibrutinib therapy (-0.26 ± 0.12 after 2 weeks and -0.3 ± 0.18 after 4 weeks). Accordingly, Toso surface expression on CLL cells, assessed by flow cytometry, also significantly decreased after 1 and 4 weeks of ibrutinib therapy (Figure 1). We next measured plasma levels of soluble Toso (sToso), which is encoded by an alternative spliced Toso transcript, in serial samples from 35 CLL ibrutinib treated patients using an enzyme-linked immunosorbent assay (ELISA). We noted that baseline sToso levels in CLL patients were significantly higher than in 6 normal controls, i.e. 70 ± 12.1 arbitrary units (AU, n=35) versus 18 ± 8 AU (n=6, p&lt;0.01). sToso levels significantly declined during ibrutinib therapy to 51.9 ± 7.5 AU (n=35, p&lt;0.01) after 1 week of treatment and further decrease in the subsequent time points to 11.9 ± 2.8 AU (p&lt;0.01) after 12 months of ibrutinib therapy. In addition, we found that patients with unmutated IgHV had higher sToso levels at baseline compared to patients with mutated IgHV (76.9 ± 10.8 AU (n=19) versus 45.4 ± 6.7 AU (mean ± SEM, n=11, p&lt;0.05)(Figure 1). Furthermore, time to normalization of sToso levels was longer in unmutated IgHV patients. We then interrogated effects of in vitro stimulation of Toso on CLL viability and BCR signaling-related chemokines CCL3 and CCL4, using mouse monoclonal antibodies (clone 2A5) or artificial human Fc Toso (hToso-Fc) for Toso triggering. After stimulation of CLL cells with 1 or 10 mg/mL of 2A5 antibody or hToso-FC for 24 hours, we noted significantly improved CLL cell survival and increased levels of CCL3 and CCL4 chemokine secretion, particularly in samples from IgHV unmutated patients. The mean relative CLL cell viability after Toso stimulation was 154.2 ± 23.6% compared to unstimulated controls for 2A5 and 140.9 ± 17.3% after hToso-Fc treatment (n=6, p&lt;0.05). Toso triggering induced very high levels of CCL3 and CCL4 secretion using 2A5 (3901 ± 464% of controls) or hToso-FC (6712 ± 1717% of controls, n=6, p&lt;0.05)(Figure 1). Conclusions: These studies demonstrate that ibrutinib therapy downregulates Toso (FAIM3) gene expression, as well as cell surface and soluble Toso expression. IgHV unmutated patients have higher levels of soluble Toso and the effect on Toso activation on viability and CCL3 and CCL4 secretion is more pronounced in this group of patients. These findings corroborate the close relation between BCR signaling and Toso in CLL. Disclosures Wierda: Cyclcel: Research Funding; Miragen: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Loxo Oncology Inc.: Research Funding; Janssen: Research Funding; Xencor: Research Funding; Acerta Pharma Inc: Research Funding; Pharmacyclics LLC: Research Funding; Genentech: Research Funding; AbbVie: Research Funding; GSK/Novartis: Research Funding; Gilead Sciences: Research Funding; Juno Therapeutics: Research Funding; KITE pharma: Research Funding; Sunesis: Research Funding. Burger:Janssen Pharmaceuticals: Consultancy, Honoraria; Aptose Biosciences, Inc: Research Funding; Gilead Sciences: Research Funding; Pharmacyclics, an AbbVie company: Research Funding; AstraZeneca: Honoraria; BeiGene: Research Funding.

Ibrutinib and Rituximab Provides Superior Clinical Outcome Compared to FCR in Younger Patients with Chronic Lymphocytic Leukemia (CLL): Extended Follow-up from the E1912 Trial

Shanafelt: Pharmacyclics: Research Funding; Patent: Patents & Royalties: US14/292,075 on green tea extract epigallocatechin gallate in combination with chemotherapy for chronic lymphocytic leukemia; Polyphenon E International: Research Funding; Merck: Research Funding; Abbvie: Research Funding; Genentech: Research Funding; Celgene: Research Funding; Glaxo-SmithKline: Research Funding; Hospira: Research Funding; Cephalon: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. O'Brien:Janssen: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Astellas: Consultancy; Aptose Biosciences, Inc: Consultancy; Amgen: Consultancy; Alexion: Consultancy; Acerta: Research Funding; Eisai: Consultancy; Gilead: Consultancy, Research Funding; GlaxoSmithKline: Consultancy; Verastem: Consultancy; Celgene: Consultancy; Sunesis: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Vaniam Group LLC: Consultancy; Regeneron: Research Funding; Kite: Research Funding. Barrientos:AbbVie: Consultancy, Research Funding; AstraZeneca: Consultancy; Genentech: Consultancy; Bayer: Consultancy; Gilead: Consultancy; Janssen: Honoraria; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Sandoz: Consultancy; Oncternal Therapeutics: Research Funding. Coutre:Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Astra Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Gilead: Research Funding; BeiGene: Other: Travel, Accommodations, Expenses & Data Safety Monitoring Committee; Genentech: Consultancy. Barr:Pharmacyclics LLC, an AbbVie company: Consultancy, Research Funding; Gilead: Consultancy; Verastem: Consultancy; Genentech: Consultancy; Seattle Genetics: Consultancy; Merck: Consultancy; Celgene: Consultancy; Janssen: Consultancy; AbbVie: Consultancy; Astra Zeneca: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding. Cashen:Celgene: Other: Speaker's Bureau; Seattle Genetics: Other: Speaker's Bureau; Novartis: Other: Speaker's Bureau. Mato:AstraZeneca: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Celgene: Consultancy; Johnson & Johnson: Consultancy, Research Funding; TG Therapeutics: Consultancy, Other: DSMB member , Research Funding; LOXO: Consultancy, Research Funding; DTRM Biopharma: Research Funding; Genentech: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Gilead: Research Funding; Acerta: Consultancy; Janssen: Consultancy. Erba:Amgen: Consultancy; MacroGenics: Consultancy, Other: Lecture fees, Research Funding; MacroGenics: Consultancy, Other: Lecture fees, Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Speakers Bureau; Agios: Consultancy, Speakers Bureau; Agios: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; ImmunoGen: Consultancy, Research Funding; Astellas Pharma: Consultancy; Astellas Pharma: Consultancy; Amgen: Consultancy; GlycoMimetics: Consultancy, Other: Chair, data and safety monitoring board, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Celgene: Consultancy, Other: chair, AML Registry Scientific Steering Committee, Speakers Bureau; ImmunoGen: Consultancy, Research Funding; AbbVie: Consultancy, Other: Chair, IRC for phase III studies, Research Funding; GlycoMimetics: Consultancy, Other: Chair, data and safety monitoring board, Research Funding; Covance: Other: Fees for serving as chair on an independent review board for AbbVie Phase III studies; Covance: Other: Fees for serving as chair on an independent review board for AbbVie Phase III studies; AbbVie: Consultancy, Other: Chair, IRC for phase III studies, Research Funding; Celgene: Consultancy, Other: chair, AML Registry Scientific Steering Committee, Speakers Bureau; Pfizer: Consultancy; Pfizer: Consultancy; Seattle Genetics: Consultancy; Seattle Genetics: Consultancy; Daiichi Sankyo: Consultancy, Research Funding. Stone:Arog Pharmaceuticals: Consultancy, Honoraria, Research Funding; Otsuka-Astex Pharmaceuticals: Consultancy; Fujifilm: Consultancy; AstraZeneca: Consultancy; Celator Pharmaceuticals: Consultancy; Abbvie: Consultancy, Research Funding; Ono Pharmaceutical-Theradex Oncology: Consultancy; Cornerstone Pharmaceuticals: Consultancy; Daiichi Sankyo: Consultancy; Takeda: Other: Fees for serving on a data and safety monitoring board ; Orsenix: Consultancy; MacroGenics: Consultancy; Jazz Pharmaceuticals: Consultancy; Agios: Consultancy, Research Funding; Stemline Therapeutics: Consultancy; Pfizer: Consultancy; Celgene: Consultancy, Other: Fees for serving on a steering committee, and fees for serving on a data and safety monitoring board; Novartis: Consultancy, Research Funding; Actinium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Astellas Pharma: Membership on an entity's Board of Directors or advisory committees; Argenx: Other: Fees for serving on a data and safety monitoring board ; Roche: Consultancy. Tallman:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Time-to-progression after front-line fludarabine, cyclophosphamide, and rituximab chemoimmunotherapy for chronic lymphocytic leukaemia: a retrospective, multicohort study.

Fludarabine, cyclophosphamide, and rituximab (FCR) has become a gold-standard chemoimmunotherapy regimen for patients with chronic lymphocytic leukaemia. However, the question remains of how to treat treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia. We therefore aimed to develop and validate a gene expression signature to identify which of these patients are likely to achieve durable remissions with FCR chemoimmunotherapy. We did a retrospective cohort study in two cohorts of treatment-naive patients (aged ≥18 years) with chronic lymphocytic leukaemia. The discovery and training cohort consisted of peripheral blood samples collected from patients treated at the University of Texas MD Anderson Cancer Center (Houston, TX, USA), who fulfilled the diagnostic criteria of the International Workshop on Chronic Lymphocytic Leukemia, had received at least three cycles of FCR chemoimmunotherapy, and had been treated between Oct 10, 2000, and Oct 26, 2006 (ie, the MDACC cohort). We did transcriptional profiling on samples obtained from the MDACC cohort to identify genes associated with time to progression. We did univariate Cox proportional hazards analyses and used significant genes to cluster IGHV-unmutated samples into two groups (intermediate prognosis and unfavourable prognosis). After using cross-validation to assess robustness, we applied the Lasso method to standardise the gene expression values to find a minimum gene signature. We validated this signature in an external cohort of treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia enrolled on the CLL8 trial of the German Chronic Lymphocytic Leukaemia Study Group who were treated between July 21, 2003, and April 4, 2006 (ie, the CLL8 cohort). The MDACC cohort consisted of 101 patients and the CLL8 cohort consisted of 109 patients. Using the MDACC cohort, we identified and developed a 17-gene expression signature that distinguished IGHV-unmutated patients who were likely to achieve a long-term remission following front-line FCR chemoimmunotherapy from those who might benefit from alternative front-line regimens (hazard ratio 3·83, 95% CI 1·94-7·59; p<0·0001). We validated this gene signature in the CLL8 cohort; patients with an unfavourable prognosis versus those with an intermediate prognosis had a cause-specific hazard ratio of 1·90 (95% CI 1·18-3·06; p=0·008). Median time to progression was 39 months (IQR 22-69) for those with an unfavourable prognosis compared with 59 months (28-84) for those with an intermediate prognosis. We have developed a robust, reproducible 17-gene signature that identifies a subset of treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia who might substantially benefit from treatment with FCR chemoimmunotherapy. We recommend testing the value of this gene signature in a prospective study that compares FCR treatment with newer alternative therapies as part of a randomised clinical trial. Chronic Lymphocytic Leukaemia Global Research Foundation and the National Institutes of Health/National Cancer Institute.

A Randomized Phase III Study of Ibrutinib (PCI-32765)-Based Therapy Vs. Standard Fludarabine, Cyclophosphamide, and Rituximab (FCR) Chemoimmunotherapy in Untreated Younger Patients with Chronic Lymphocytic Leukemia (CLL): A Trial of the ECOG-ACRIN Cancer Research Group (E1912)

A Randomized Phase III Study of Ibrutinib (PCI-32765)-Based Therapy Vs. Standard Fludarabine, Cyclophosphamide, and Rituximab (FCR) Chemoimmunotherapy in Untreated Younger Patients with Chronic Lymphocytic Leukemia (CLL): A Trial of the ECOG-ACRIN Cancer Research Group (E1912)

The Magnitude of Improvement in Progression-Free Survival with Targeted Therapy in Relapsed/Refractory Chronic Lymphocytic Leukemia Based on Genetic Risk: A Systematic Review and Meta-Analysis

Background Currently, only genetic testing, del(17p) or TP53, determines choices of therapy in chronic lymphocytic leukemia (CLL) whereas mutational status of IGHV and 11q deletion are not recognized as reliable parameters for treatment selection (with exception for fludarabine, cyclophosphamide and rituximab [FCR] in mutated IGHV).To increase understanding of the predictive value of prognostic parameters in the novel therapy era, we conducted a systematic review and meta-analysis assessing the magnitude of improvement in progression-free survival (PFS) with B-cell receptor (BCR) or BCL2 pathway inhibitors based on the presence or absence of 17p deletion/TP53 mutations, 11q deletion and IGHV mutational status in relapsed refractory (R/R) CLL patients.Methods Randomized trials comparing BCR or BCL2 inhibitors to other treatment regimens were considered eligible for this analysis. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement was utilized as a reporting guideline. The search strategy yielded 1184 records for screening with 97 full-text articles assessed for eligibility. Finally, seven randomized trials were included in the quantitative meta-analysis, all of which compared BCR or BCL2 inhibitors to a different comparator.Results Meta-analysis of seven randomized trials comprising 2415 patients with R/R CLL revealed that treatment with BCR or BCL2 pathway inhibitors improved OS compared with the combined comparator treatments [Hazard ratio (HR) 0.555;95% confidence interval (CI): 0.452-0.680;P=0.000]. Of note, the I2 statistic for heterogeneity (i.e.,8.8% [P=0.36] ) and Q values (i.e., 6.583) indicated a high level of homogeneity of results across studies. Similar results were obtained with PFS (HR, 0.196; 95% CI: 0.144-0.268; P=0.000) although significant statistical heterogeneity existed across the studies (I2 = 78.6%, P<0.00001;Q=28.08).Next, we evaluated the magnitude of improvement in PFS obtained with BCR or BCL2 pathway inhibitors in R/R CLL patients with high-risk genetic features (Fig 1). The risk of progression was lower for 17p deleted patients (n= 450) treated with BCR or BCL2 inhibitors in comparison to combined comparator treatments (HR, 0.206; 95% CI, 0.108-0.392; P = .000; I2 = 76%; P=0.001;Q=20.853). The same applied with the analysis of TP53 which included five studies enrolling 582 R/R CLL patients with mutated TP53 (HR, 0.231 (95% CI:0.1370.390;P=0.000;I2=75.8%,P=0.0002;Q=16.513).Data for PFS by 11q deletion status, restricted to 4 studies (3 of ibrutinib and one of venetoclax) including 433 patients , revealed a significantly higher risk of progression for 11q deleted patients treated with comparator treatments [HR,0.081; 95% CI: 0.054-0.121; I2= 0.0% (P=0.56), Q =2.06]. Finally, data for PFS by IGHV mutational status that relied on 6 studies including 1580 IGHV unmutated patients favoured treatment with BCR or BCL2 inhibitors ( HR, 0.172; 95% CI, 0.109-0.272; P=0.000; I2= 84.8%, P<0.0001; Q= 32.98).An additional pooled meta-analysis restricted to patients with low risk genetic features revealed that the benefit observed with BCR or BCL2 inhibitors in patients with high-risk genetic features may extend to former (data not shown). Of note, the interaction analysis comparing the magnitude of PFS improvement in patients with and without high risk genetic features, respectively, showed that the benefit was independent of mutational status of IGHV (HR,1.39; 95% CI,0.77-2.40;P=0.26), mutational status of TP53 (HR,1.06; 95% CI,0.56-2.00;P=0.43) and 17p deletion (HR,1.10;95% CI,1.10;95%CI, 0.52-2.35;P=0.40). Interestingly, R/R CLL patients who carried 11q deletion treated with BCR or BCL2 inhibitor fared significantly better in terms of the improvement of PFS than patients without 11q deletion (HR,0.39; 95% CI,0.25-0.62;P<0.0001) indicating the particular value of novel therapies in patients with this genetic defect.Conclusions The important information provided by present meta-analysis is that the improvement over traditional treatments observed with BCR or BCL2 pathway inhibitors is common to all patients with R/R CLL, including those patients with unfavorable and favourable prognostic parameters. BCR signal transduction and BCL2 inhibitors have broad utility and overcome many but not all of the traditional poor risk molecular features in CLL. DisclosuresMolica:Gilead: Other: Advisory board; AbbVie: Other: Advisory board; Roche: Other: Advisory board; Jansen: Other: Advisory board. Levato:Novartis: Other: Advisory board. Shanafelt:Genentech: Research Funding; GlaxoSmithKline: Research Funding; Jansen: Research Funding; Pharmacyclics: Research Funding.

Calcium Mobilization in Unfavorable-Prognosis Chronic Lymphocytic Leukemia Patients Mediates Focal Adhesion Kinase (FAK) Cleavage, Thereby Its Activation

INTRODUCTIONChronic Lymphocytic Leukemia (CLL) is the most common leukemia in the western world and is characterized by the accumulation of monoclonal B cells, due to both increased proliferation and apoptosis resistance. Although in the last years with the introduction of new kinase inhibitors blocking the pathways mediated by B-cell receptor (BCR) signaling we got an astonishing progress in the comprehension and treatment of this disease, CLL is still an incurable disease and many characteristics of its pathogenesis still remain unclear. Signaling events downstream the BCR engagement are central for the progression of CLL. Focal Adhesion Kinase (FAK), one of the primary enzyme involved in the engagement of integrins and assembly of focal adhesions, plays a major role in cellular adhesion and metastasis of various cancers, being regulated by Calcium (Ca2+) flux and by Src-kinases (e.g. Lyn) through a Calpain-dependent manner following BCR triggering. FAK has been demonstrated to be over-expressed in many human cancers but a down-modulation of its expression has also been reported. Studies concerning FAK expression in CLL are lacking in the literature. However, since an interaction of FAK with molecules implicated in BCR signal transduction, such as the Src-kinase Lyn, has been demonstrated we hypothesize that this kinase could have a key role in CLL pathogenesis.METHODSFAK expression was analyzed in B-lymphocytes from 107 CLL patients and 10 healthy subjects by Western blotting (WB) and the obtained expression data were correlated with the clinical features of the patients. In 25 out of 107 patients studied, surface IgM and IgD expression has been evaluated by flow cytometry (FC). For Ca2+ mobilization assessment, 1x107 cells were incubated with 4μM Fluo-4-AM at 37°C for 30min and then analyzed by FC; after 30s of baseline acquisition, α-IgM F(ab‘)2 and α-IgD F(ab‘)2 (10μg/ml) were added and fluorescence intensity was recorded for 5min. Ionomycin was added as positive control. Phosphorylation at Tyr397 was assessed with a specific antibody. Leukemic B cells from patients were treated in vitro with 5μM Defactinib (FAK inhibitor) and the apoptosis induction was evaluated by Annexin V/Propidium Iodide flow cytometry test and by the presence of cleaved PARP by WB.RESULTSBy WB analyses we demonstrated a slightly significant difference in FAK expression between patients and controls (p<0.05), the protein being particularly down-regulated in unmutated IGHV and del17p/del11q/12+ CLL patients. We observed that FAK down-modulation was limited to its whole form detected in WB at 125kDa, while bands related to FAK cleavage (92/94 and 84kDa) were detected also in those patients lacking full length-FAK. Cleaved-FAK is due to Calpain protease activity, when stimulated by the bond with Ca2+ ions. We then compared FAK expression with the capability of the cell to mobilize Ca2+ from intracellular stores, observing that patients with this capability had less amount of full length-FAK, which translated into a higher presence of cleaved/activated form of FAK. The cleavage bands infact were found phosphorylated at activatory Tyr397. Of note, only IGHV-unmutated patients showed these features. Lastly, we studied the effect of Defactinib, a specific FAK inhibitor, in CLL cells; this molecule was able to induce apoptosis in leukemic cell in a caspase-dependent way, as assessed by the presence of the cleaved PARP.CONCLUSIONSWe herein propose that full length-FAK down-modulation could be considered as a new marker of unfavorable prognosis. In our model, poor prognosis CLL patients (particularly IGHV unmutated ones) presenting Ca2+ mobilization, are more prone to activate Calpain, which in turn activates FAK. Together with data from the literature, our results suggest that CLL cells missing the full length-FAK, not only are unaffected by the lack of it, but they rather present a cleaved/activated form of FAK that could favor cell migration and metastatic invasion. Moreover, since Defactinib can induce apoptosis in CLL cells, should these data be confirmed by in vivo studies, this FAK inhibitor could represent a new therapeutic approach for CLL. DisclosuresTrentin:Gilead: Research Funding; Abbvie: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding.

Resistance to complement activation, cell membrane hypersialylation and relapses in chronic lymphocytic leukemia patients treated with rituximab and chemotherapy.

The anti-CD20-specific monoclonal antibody rituximab (RTX), in combination with chemotherapy, is commonly used for primary treatment in chronic lymphocytic leukemia (CLL). However, relapses remain important and activation of the complement pathway is one of the mechanisms by which RTX generates the destruction of B cells directly by complement-dependent cytotoxicity (CDC), or indirectly by antibody-dependent cellular phagocytosis. In this study, the RTX capacity to induce CDC was established in 69 untreated CLL patients, this cohort including 34 patients tested before the initiation of RTX-chemotherapy. In vitro CDC-resistance to RTX predicts lower response rates to RTX-chemotherapy and shorter treatment free survival. Furthermore, the predictive value of CDC-resistance was independent from the clinical, cytogenetic and FcγR3A V158F polymorphism status. In contrast, CLL cell resistance to CDC predominates in IGHV unmutated patients and was related to an important α2-6 sialyl transferase activity, which in turn increases cell surface α2-6 hypersialylation. Suspected factors associated with resistance to CDC (CD20, CD55, CD59, factor H, GM1, and sphingomyelin) were not differentially expressed or recruited between the two CLL groups. Altogether, results provide evidence that testing RTX capacity to induce CDC in vitro represents an independent predictive factor of therapeutic effects of RTX, and that α2-6 hypersialylation in CLL cells controls RTX response through the control of the complement pathway. At a time when CLL therapy is moving towards chemo-free treatments, further experiments are required to determine whether performing an initial in vitro assay to appreciate CLL CDC resistance might be useful to select patients.

Early Achievement of MRD-Negativity in IGHV-Mutated (IGHV-M) Patients Portends Highly Favorable Outcomes after First-Line Treatment of CLL with Fludarabine, Cyclophosphamide and Rituximab (FCR). Serial Monitoring for Minimal Residual Disease (MRD) in Blood after Achieving MRD-Negativity Predicts Subsequent Clinical Relapse

IntroductionAchieving MRD-negative remission after FCR therapy is an important predictor of longer progression-free (PFS) and overall survival (OS); however, with long-term follow-up, many MRD-negative patients subsequently relapse. The time course and predictive factors for relapse in initially MRD-negative patients have not been extensively characterized.MethodsWe prospectively analyzed 289 patients treated first-line with FCR for CLL at M.D. Anderson Cancer Center from 2008-15, to determine pre-treatment characteristics associated with post-treatment MRD negativity and the time course of relapse in MRD-negative patients. MRD analysis was performed in BM after course 3 (n=239) and at end of therapy (EOT) (n=231) using standardized 4-color flow cytometry (FLC). Ninety-five MRD-negative patients had 12-monthly serial MRD monitoring on blood.ResultsPretreatment characteristics are shown below (N=289).For the total cohort, the only pre-treatment characteristic significantly associated with PFS was IGHV-unmutated (IGHV-UM) (HR 3.0 [1.6-5.5], p<0.001). Analyzed separately, among IGHV-mutated (IGHV-M) patients, only ZAP70 positivity (38/92 [41%]) by immunohistochemistry was significantly associated with shorter PFS (HR 3.2 [1.3-8.2], p=0.01); among IGHV-UM patients only B2M ≥4.0mg/L (64/146 [44%]) was significantly associated with shorter PFS (HR 1.9 [1.2-3.0], p=0.005).Overall response rate was 96%. 46/239 patients (19%) after 3 courses and 120/231 (51%) at EOT achieved MRD negativity in BM. The following pre-treatment characteristics were significantly associated in univariableanalysis with achieving MRD-negativity at EOT in all patients: negative FISH (OR 3.1 [1.3-7.0], p=0.01), trisomy 12 (OR 2.7 [1.1-6.3], p=0.03, IGHV-M [OR 3.2 (1.8-5.9), p<0.001], β2-microglobulin <4.0mg/L [OR 1.85 (1.1-3.1), p=0.02] and ZAP70 negativity [OR 1.8 (1.03-3.2), p=0.04]. On multivariable analysis, only IGHV-M was significantly associated with MRD-negativity [OR 3.7 (1.7-8.2), p=0.001]. There were trends toward association between negative FISH [OR 5.4 (0.9-31.8), p=0.07] and trisomy 12 [OR 4.9 (0.8-29.8), p=0.08] with MRD-negativity. MRD negativity at EOT correlated with PFS, independent of IWCLL response category, and correlated with PFS in both IGHV-M and IGHV-UM patients (Figure A). In IGHV-M patients who were MRD-negative at EOT, there was a trend toward superior PFS if MRD-negativity was attained after 3 courses of FCR, even though 11/22 patients who were MRD-negative after 3 courses received no further therapy and only 3/22 received 6 courses (Figure B).Re-emergence of MRD in blood was detected in 45/95 MRD negative patients, at a median of 49 months post- treatment; this appeared less frequent in IGHV-M patients (Figure C). Re-emergence of MRD preceded clinical relapse by a median of 25 months. There were no significant differences in time-to-subsequent clinical relapse after re-emergence of MRD according to mutation status (Figure D). Among IGHV-M patients who were MRD-negative after 3 cycles, 4/22 had subsequent re-emergence of MRD.ConclusionsAmong patients with CLL receiving first-line FCR, the best pre-treatment predictor of achieving MRD-negativity and subsequent longer PFS was IGHV-M. Post-treatment MRD-negativity correlated with subsequent PFS; patients with IGHV-M who achieved MRD-negativity after 3 courses had a particularly favorable outcome, even after abbreviation of therapy. However, despite achieving MRD-negativity, many patients subsequently relapse; serial MRD monitoring in peripheral blood can herald relapse with a lead-time of approximately 2 years and potentially direct monitoring and/or early intervention strategies. [Display omitted] [Display omitted] DisclosuresThompson:Pharmacyclics: Consultancy, Honoraria. O'Brien:Janssen: Consultancy, Honoraria; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding. Jain:Incyte: Research Funding; Celgene: Research Funding; Seattle Genetics: Research Funding; Servier: Consultancy, Honoraria; Abbvie: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Infinity: Research Funding; Novimmune: Consultancy, Honoraria; BMS: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Genentech: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding. Kadia:Novartis: Honoraria; BMS: Research Funding. DiNardo:Novartis: Research Funding; Daiichi Sankyo: Research Funding; Agios: Research Funding; Abbvie: Research Funding; Celgene: Research Funding. Wierda:GSK/Novartis: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Genentech/Roche: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Emergent: Consultancy, Research Funding; Sanofi: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; Merck: Consultancy; Karyopharm: Research Funding; Acerta: Research Funding; Janssen: Research Funding; Juno Therapeutics: Research Funding; KITE Pharma: Research Funding.

Presence of a Translocation Is Associated with Short Time to Treatment from Diagnosis in IGHV Mutated Chronic Lymphocytic Leukemia (CLL) Patients

Chronic Lymphocytic Leukemia (CLL) has a varied clinical course; some patients experience a long survival and others succumb to disease in a short time. Clinical factors correlated with either time to first treatment (TFT) and/or overall survival include Rai stage, IGHV somatic hypermutation status, fluorescence in situ hybridization (FISH) abnormalities, especially del(17p), karyotypic complexity and the presence of a cytogenetic translocation. Previous studies have included patients both at diagnosis and at various times throughout their diseases, and many included limited numbers of patients, precluding extensive analyses of relationships between the prognostic factors and their relative impact on clinical outcome. We sought to identify which factors determined within a short time of diagnosis (i.e., 1 year) were prognostic for TFT in untreated CLL patients.We identified 329 untreated CLL patients who had stimulated karyotypic and FISH analyses within 1 year of diagnosis seen at The Ohio State University (OSU). Patient characteristics and outcome were obtained from patient records. The studies were approved by the OSU IRB and were conducted according to the Declaration of Helsinki. A complex karyotype was defined as ≥ 3 unrelated aberrations by karyotype. Patient characteristics are given in Table 1. Translocations occurred in 87 (26.4%) patients: 38 balanced and 49 unbalanced translocations. Initial statistical analyses showed no large difference in TFT between balanced and unbalanced translations, so they were combined for final analyses. 144 patients (49 with and 95 without a translocation) had unmutated IGHV, and 144 patients (22 with and 122 without a translocation) had mutated IGHV. IGHV data were not available for 41 patients.TFT was calculated from date of diagnosis to date of first treatment. Untreated patients were censored at last known untreated date. Kaplan-Meier curves estimated TFT probability, and proportional hazard models were used to examine the association between potential risk factors and TFT. Using backward selection, variables with statistical significance when adjusting for all other covariates were included in the final model. To evaluate potential effect modifications, pairwise interactions among all the variables in the final model were examined and retained if statistically significant. Stata 14 (College Station TX) was used, and all tests were two-sided with statistical significance set at p<0.05.Median follow-up for censored patients was 30 months (range 0.03-102 months). Median TFT for the entire cohort was 47 months (95% confidence interval (CI) 40-63 months). In a univariable model, the following factors were significant: presence of a translocation (hazard risk (HR) 2.69, CI 1.91-3.78, p<0.001), Rai stage III/IV (HR 3.73, CI 2.32-5.99, p<0.001), complexity (HR 2.92, CI 1.98-4.31, p<0.001), unmutated IGHV (HR 3.54, CI 2.42-5.17, p<0.0001), del17p (HR 2.10, CI 1.31-3.37, p=0.002), del11q (HR 2.91,CI 1.92-4.40, p<0.001). In the multivariable model, there was significant effect modification of IGVH status on the relationship between translocation and TFT (p<0.001). In IGHV mutated patients, those with a translocation had over 5 times the risk of starting treatment relative to those without a translocation (HR 5.30, CI 2.76-10.17); however, in IGHV unmutated patients, a translocation did not significantly increase the risk of starting treatment (HR 1.32, CI 0.86-2.03). Independent of IGHV and translocation, Rai Stage (HR 2.07, CI 1.24-3.45, p=0.01) and del11q (HR 1.68, CI 1.09-2.60, p=0.02) were the only variables that remained statistically significant. Notably, once these variables were accounted for in the model, complexity did not provide additional significant prognostic information (p=0.12), perhaps due to its strong association with a translocation (p<0.001). In summary, the presence of a translocation in IGHV mutated patients appeared to negate the improved prognosis associated with mutated IGHV, but the presence of a translocation did not have an effect on TFT in high-risk IGHV unmutated patients (Figure 1). [Display omitted] [Display omitted] DisclosuresJones:Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding. Andritsos:Hairy Cell Leukemia Foundation: Research Funding. Woyach:Morphosys: Research Funding; Acerta: Research Funding; Karyopharm: Research Funding. Awan:Pharmacyclics: Consultancy; Novartis Oncology: Consultancy; Innate Pharma: Research Funding.

Prognostic Impact of NOTCH1 Hotspot Mutation in TP53-Mutated Patients with Chronic Lymphocytic Leukemia

IntroductionA presence of activating mutations in NOTCH1 gene has been recently associated with reduced survival and chemo-immunotherapy resistance in chronic lymphocytic leukemia (CLL). However, a prognostic significance of the NOTCH1 mutations with respect to TP53mutation status has not been fully explained yet.MethodsAn examined cohort included 409 patients with CLL enriched for high risk cases; in 121 patients consecutive samples were investigated. To determine the TP53 mutation status, a functional analysis of separated alleles in yeast (FASAY, exons 4-10) combined with direct sequencing was performed; the ambiguous cases were retested using an ultra-deep next generation sequencing (MiSeq platform; Illumina). The presence of NOTCH1 hotspot mutation (c.7544_7545delCT) was analyzed using direct sequencing complemented by allele-specific PCR in the selected samples. In several patients harboring concurrent NOTCH1 and TP53 mutations, single separated cancer cells were examined using multiplex PCR followed by direct sequencing. A correlation between mutation presence and patient overall survival, time to first treatment and other molecular and cytogenetic prognostic markers was assessed using Log-rank (Mantel-cox) test and Fisher’s exact test, respectively.ResultsThe NOTCH1 and TP53 mutations were detected in 16% (65/409) and 27% (110/409) of the examined patients, respectively; a coexistence of these mutations in the same blood samples was observed in 11% (19/175) of the mutated patients. The detected increased mutation frequency attributes to more unfavorable profile of the analyzed cohort; in the TP53-mutated patients missense substitutions predominated (75% of TP53 mutations).As expected, a significantly reduced overall survival in comparison to the wild-type cases (147 months) was observed in the NOTCH1-mutated (115 months; P = 0.0018), TP53-mutated (79 months; P < 0.0001) and NOTCH1-TP53-mutated patients (101 months; P = 0.0282). Since both NOTCH1 and TP53 mutations were strongly associated with an unmutated IGHV gene status (P < 0.0001 and P = 0.0007), we reanalyzed the IGHV-unmutated patients only and interestingly, the impact of simultaneous NOTCH1 and TP53 mutation presence on patient survival was missed in this case (P = 0.1478).On the other hand, in the NOTCH1 and/or TP53-mutated patients significantly reduced time to first treatment was identified as compared to the wild-type cases (41 months vs. 25 months in NOTCH1-mutated, P = 0.0075; 17 months in TP53-mutated, P < 0.0001; and 18 months in NOTCH1-TP53-mutated patients, P = 0.0003). The similar results were observed also in the subgroup of the IGHV-unmutated patients, with the exception of patients carrying sole NOTCH1 mutation (P = 0.2969). Moreover, in the NOTCH1-TP53-mutated patients an increased frequency of del(17p)(13.1) was found in comparison to the TP53-mutated patients only (72% vs. 56%); this cytogenetic defect was not detected in the patients with sole NOTCH1 mutation.Our results might indicate, that NOTCH1 mutation could preferentially co-selected with particular, less prognostic negative type of TP53 defects. Notably, in our cohort the NOTCH1 mutation predominated in the patients harboring truncating TP53 mutations localized in a C-terminal part of the TP53 gene behind the DNA-binding domain (P = 0.0128). Moreover, in one of the NOTCH1-TP53-mutated patients the analysis of separated cancer cells revealed a simultaneous presence of NOTCH1 mutation and TP53 in-frame deletion in the same CLL cell. In contrast, in the other examined NOTCH1-TP53-mutated patient the concurrent NOTCH1 mutation and TP53 missense substitution (with presumed negative impact on patient prognosis) were found in different CLL cells.ConclusionsThe parallel presence of NOTCH1 hotspot mutation might be detected in a significant proportion of TP53-mutated patients and it seems to be associated with less prognostic unfavorable TP53 mutations. Nevertheless, these preliminary data should be further confirmed in a large cohort of patients.This study was supported by projects VaVPI MSMT CR CZ.1.05/1.1.00/02.0068 of CEITEC, IGA MZ CR NT13493-4/2012, NT13519-4/2012 and CZ.1.07/2.3.00/30.0009. DisclosuresBrychtova:Roche: Travel grants Other. Doubek:Roche: Travel grants Other.

Frequency analysis of HLA class I alleles in Iranian patients with progressive and non-progressive chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a malignant disorder of B cell origin, with low incidence in Asian populations. In this study we investigated the HLA-class I A and B allele frequencies in 87 Iranian CLL patients and 64 healthy controls using sequence specific primer-polymerase chain reaction (SSP-PCR) technique. Our results showed increased frequencies of HLA-A11:01 (p=0.02) and HLA-B35:01 (p=0.002) alleles and HLA-A11:01/B35:01 haplotype (p=0.036) and decreased frequencies of HLA-A01:01 (p=0.02), HLA-A26:01 (p=0.03), HLA-B65:01 (p=0.03) and HLA-B53:01 (p<0.00001) alleles in CLL patients compared to the control group. Classification of the patients into non-progressive and progressive groups did not reveal significant differences for the frequency of any of the HLA-A and -B alleles or haplotypes between these two subtypes. Comparison between patients with immunoglobulin heavy chain variable region genes (IGHV) mutated (n=56) and unmutated (n=31) subtypes showed a significant increase in HLA-A32:01 (p=0.05) and HLA-A33:01 (p=0.05) alleles in IGHV unmutated patients compared to IGHV mutated patients. Similarly, a higher frequency of HLA-B52:01 (p=0.037) alleles was observed in CD38+ compared with CD38− patients. Our results obtained from an Iranian population indicate that CLL is associated with distinct HLA class I alleles and haplotypes some of which are linked to disease prognostic factors.

Genomic Aberrations Dramatically Improve The Strong Prognostic Impact Of IGHV Mutational Status In Chronic Lymphocytic Leukemia (CLL)

▪CLL is a heterogeneous disease with patients (pts) experiencing rapid disease progression and others living for years without requiring treatment. Recently, next generation sequencing has revealed new molecular alterations, targeting the NOTCH1 and BIRC3 genes which occur in about 10% CLL at diagnosis and correlate with poor outcome. Given the possibility of targeting NOTCH1 and BIRC3 with drugs currently under development, the primary endpoints of our research were: 1) to determine overall survival (OS) upon IGHV, NOTCH1, TP53 and BIRC3 in univariate analysis; 2) to correlate these genomic aberrations with other biological or clinical prognostic factors, and finally 3) to confirm NOTCH1, BIRC3 and TP53 as independent prognostic factors. We investigated 475 pts with a median age of 65 years (range 33-89), whose 160 had low Rai stage, 301 intermediate stage and 14 high stage. NOTCH1 mutations (mut) were studied by ARMS PCR for c.7544-7545delCT and by Sanger sequencing of NOTCH1 exon 34. Mutations of TP53 were analysed by DNA direct sequencing, while BIRC3 disruption (disr) was studied by Sanger sequencing for mutations and by interphase FISH for deletions. All these alterations were studied at diagnosis or before any chemotherapeutic approach. NOTCH1mut and TP53mut pts were 52 (10.9%) and 36/475 (7.6%), respectively. Thirty four patients were BIRC3mut (7.2%) and 26 BIRC3 deleted (5.5%) for a total of 46 cases (9.7%) BIRC3disr. NOTCH1, TP53 and BIRC3 alterations were mutually exclusive. There were significant correlations between NOTCH1 (P<0.00001), TP53 (P=0.004), BIRC3 status (P=0.00004) and IGHV mutations. Concerning FISH cytogenetics (460 patients), a significant correlation (P<0.0001) was found between NOTCH1mut and trisomy 12 (20/62; 32%). TP53mut were strictly associated with del17p (15/25; 60%; P<0.0001), while BIRC3disr was found mainly within 11q22-q23 deletions subset (22/46;49%; P<0.0001). With regard to clinical outcome, 30 (83%) of 36 TP53mut pts (P=0.00009), 47 (90%) of 52 NOTCH1mut (P<0.00001) and 40 (87%) of 46 BIRC3disr pts had received chemotherapy at the time of analysis. Twenty nine NOTCH1mut (56%), 15 TP53mut (42%) and 18 BIRC3disr (39%) pts underwent at least two lines of treatment (P<0.0001). Noteworthy, shorter OS was observed in IGHV unmutated (UM) patients (12% vs 80% at 18 years, P<0.00001), in NOTCH1mut pts (12% vs 71% at 16 years, P<0.00001), in TP53mut pts (9% vs 76% at 14 years, P<0.00001) and in BIRC3disr pts (29% vs 65% at 16 years, P=0.00001). To further explore the prognostic impact of NOTCH1mut, TP53mut and BIRC3disr, we investigated them within the UM (153 pts) IGHV subset, notoriously at worst prognosis. As a matter of fact, NOTCH1mut (16% vs 45% at 14 years, P=0.012), TP53mut (0% vs 43% at 13 years, P=0.002) and BIRC3disr (0% vs 57% at 11 years, P=0.011) pts showed significant shorter OS [Figure]. Within the mutated IGHV subgroup we obtained similar results. In multivariate analysis of OS, TP53mut (HR 5.2, P<0.00001), age >60 years (HR 3.8, P=0.00002), IGHV UM status (HR 0.30, P=0.0001), intermediate/high Rai stages (HR 2.8, P=0.0002), NOTCH1mut (HR 2.6, P=0.001), and BIRC3disr (HR 2.5, P=0.005) were confirmed to be independent adverse prognostic factors. Noteworthy, here, we demonstrated that genomic aberrations are able to improve the historical prognostic ability of the IgHV mutational status. In conclusion, genomic aberrations, particularly TP53mut, NOTCH1mut and BIRC3disr should be considered as novel important prognostic parameters in CLL and therefore they have to be necessarily considered in updated scoring prognostic systems. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

IGHV Mutational Status Is Prognostic in Del(11q) and Del(17p) Chronic Lymphocytic Leukemia

Abstract 4598 IntroductionThe importance of interphase fluorescence in-situ hybridization (FISH) DNA analysis as a predictive tool for survival in patients with chronic lymphocytic leukemia (CLL) is well-established. However, even among cytogenetically defined groups, survival varies widely. In CLL patients who harbor del(13q), their IGHV mutational status predicts for Rai stage progression, treatment initiation and overall survival. Here we analyze, the influence of IGHV mutational status upon overall survival in CLL patients harboring the poor prognostic CLL FISH findings of a del(11q) or del(17p). MethodsAfter IRB approval, we retrospectively reviewed all patients who had IGHV sequence homology analysis performed who also had del(11q) or del (17p) detected by FISH evaluation during the clinical course of their disease. An IGHV non-homologous sequence of <2% was defined as unmutated. ResultsBetween 2006 and March 2011, IGHV mutational studies were performed on 159 CLL patients of whom 70 (44%) had a documented del(11q) or del(17p). Four patient's IGHV mutational status could not be resulted; they were excluded from further analysis. Of the remaining 66 patients: 39 (59%) had del(11q) and 27 (41%) had del(17p): no patient in this series harbored both del(11q) and del(17p). Fifty (71%), had an unmutated IGHV sequence and 16 (29%) were mutated.The median time from diagnosis to last follow-up for IGHV unmutated patients was 3.1 (range: 0.1–14.6) years and 3.1 (range: 0.6–19.4) years for the IGHV mutated patients (p=0.12). When comparing unmutated to mutated IGHV patients, the median age and Rai stage presentation were similar: in each cohort > 75% were Rai stage 0 or 1. While the majority of patients were treatment naïve at the time of this consultation, unmutated patients were more likely to have received treatment before to this evaluation: 34% vs. 6%, p=0.04. The median time between diagnosis and first FISH analysis was 2.0 (range: 0–13.2) years in the unmutated group and 0.7 (range: 0–18.9) years in the mutated group (p=0.3).No patient died without first receiving CLL treatment. The most common reasons for treatment initiation were either bulky adenopathy or progression to an advanced Rai stage. The 5 year cumulative probability of receiving treatment for the unmutated group was 76% compared to 57% for the mutated group (log rank p = 0.05). Eleven of 67 patients (16%) underwent allogeneic bone marrow transplantation. All eleven transplants were performed in the IGHV unmutated population (11/50; 22%).The median survival for the unmutated patients was 11.3 years with a 5 year OS probability of 68%. For mutated patients, the median survival was not reached and the 5 year OS probability was 89% (log rank p = 0.06). Of the 18 deaths, only one death was not a direct result of CLL; this death occurred four years after the CLL diagnosis in a patient with IGHV unmutated, del(17p) disease. Discussion:We recently reported del(13q) CLL patients with an unmutated IGHV sequence predictably suffer an aggressive disease phenotype. Here, we did not limit analysis to those who concurrently harbored the salutary del(13q). Again, we found, the IGHV mutational status to be the dominate predictor of clinical outcome: despite the presence of del(11q) and/or del(17p), the presence of a mutated IGHV sequence was associated with an indolent clinical course. These analyses suggest CLL FISH aberrations predict clinical outcome because they track with the IGHV mutational status: del (13q) predicts for indolent disease because it is often accompanied by a mutated IGHV sequence; del(11q) and del (17p) predict for aggressive disease as these aberrations are often accompanied by an unmutated IGHV sequence.Importantly, we report a high discordance in our high risk cytogenetic patients and their IGHV mutational risk assessment. Nearly a third of our del(17p)/del(11q) patients had a mutated IGVH sequence, offering an explanation for the variable clinical outcomes seen in high risk cytogenetic groups. Moreover, these findings question the prevailing recommendation of allogeneic bone marrow transplantation to all suitable high risk cytogenetic CLL patients in first response. Additional outcomes research needs to be performed to better inform and guide clinical care in CLL patients who harbor both high and low risk factors. Disclosures:No relevant conflicts of interest to declare.

Age at diagnosis and the utility of prognostic testing in patients with chronic lymphocytic leukemia

A study was undertaken to analyze the survival of chronic lymphocytic leukemia (CLL) patients relative to age-matched individuals in the general population and determine the age-stratified utility of prognostic testing. All 2487 patients diagnosed with CLL between January 1995 and June 2008 and cared for in the Mayo Clinic Division of Hematology were categorized by age at diagnosis and evaluated for differences in clinical characteristics, time to first treatment, and overall survival (OS). Among Rai stage 0 patients, survival was shorter than the age-matched general population for patients aged <55 years (P < .001), 55 to 64 years (P < .001), and 65 to 74 years (P < .001), but not those aged ≥75 years at diagnosis (P = not significant). CD38, IGHV mutation, and ZAP-70 each predicted time to first treatment independent of stage for all age groups (all P < .04), but had less value for predicting OS, particularly as age increased. IGHV and fluorescent in situ hybridization (FISH) predicted OS independent of stage for patients aged <55 years (P ≤ .001), 55 to 64 years (P ≤ .004), and 65 to 74 years (P ≤ .001), but not those aged ≥75 years. CD38 and ZAP-70 each predicted OS independent of stage for only 2 of 4 age categories. Among Rai 0 patients aged <75 years, survival was shorter than the age-matched population only for IGHV unmutated (P < .001) patients or those with unfavorable FISH (P < .001). Survival of CLL patients aged <75 years is shorter than the age-matched general population regardless of disease stage. Among patients aged <75 years, the simple combinations of stage and IGHV or stage and FISH identifies those with excess risk of death relative to the age-matched population. Although useful for predicting time to first treatment independent of stage for patients of all ages, prognostic testing had little utility for predicting OS independent of stage among patients aged ≥75 years.