Treatment-naive Chronic Lymphocytic Leukemia Patients Research Articles

Reduced prognostic value of beta-2-microglobulin for time to first treatment in CLL patients with compromised kidney function

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults and characterized by a highly heterogeneous clinical course. The CLL-IPI and the OCLL-1 scores are among the best validated tools to predict time-to-first-treatment. In both models, elevated beta-2-microglobulin plasma level (B2M) is an independent prognostic factor. Yet, B2M is commonly increased in patients with chronic kidney disease (CKD) per se and both models were not adjusted for CKD. We analyzed the clinical outcomes of 297 treatment-naive CLL patients between 2000 and 2022. B2M was more frequently elevated in CKD patients and lost prognostic significance at the threshold > 2.5 mg/L. Both CLL-IPI and OCLL-1 failed to facilitate prognostic segregation in CKD patients. 22.2% of CKD patients were assigned to a higher CLL-IPI risk group due to elevated B2M. Our results suggest that both models overestimate the risk for disease progression and need to be interpreted with caution in CKD patients.

P66Shc deficiency in CLL cells enhances PD-L1 expression and suppresses immune synapse formation.

Introduction: Escape from immunosurveillance is a hallmark of chronic lymphocytic leukemia (CLL) cells. In the protective niche of lymphoid organs, leukemic cells suppress the ability of T lymphocytes to form the immune synapse (IS), thereby hampering T-cell mediated anti-tumoral activities. By binding its cognate receptor PD-1 at the surface of T lymphocytes, the inhibitory ligand PD-L1, which is overexpressed in CLL cells, mediates the T-cell suppressive activities of CLL cells. However, the molecular mechanism underlying PD-L1 overexpression in CLL cells remains unknown. We have previously reported a defective expression of the pro-apoptotic and pro-oxidant adaptor p66Shc in CLL cells, which is causally related to an impairment in intracellular reactive oxygen species (ROS) production and to the activation of the ROS-sensitive transcription factor NF-κB. The fact that PD-L1 expression is regulated by NF-κB suggests a mechanistic relationship between p66Shc deficiency and PD-L1 overexpression in CLL cells. Methods: 62 treatment-naive CLL patients and 43 healthy donors were included in this study. PD-L1 and p66Shc expression was quantified in B cells by flow cytometry and qRT-PCR. IS architecture and local signaling was assessed by flow cytometry and confocal microscopy. CD8+ cell killing activity was assessed by flow cytometry. Results: Here we show that residual p66Shc expression in leukemic cells isolated both from CLL patients and from the CLL mouse model Eμ-TCL1 inversely correlated with PD-L1 expression. We also show that the PD-L1 increase prevented leukemic cells from forming ISs with T lymphocytes. Reconstitution of p66Shc, but not of a ROS-defective mutant, in both CLL cells and the CLL-derived cell line MEC-1, enhanced intracellular ROS and decreased PD-L1 expression. Similar results were obtained following treatment of CLL cells with H2O2 as exogenous source of ROS, that normalized PD-L1 expression and recovered IS formation. Discussion: Our data provide direct evidence that the p66Shc-deficiency-related ROS depletion in CLL cells concurs to enhance PD-L1 expression and provides a mechanistic basis for the suppression of T cell-mediated anti-tumoral functions in the immunosuppressive lymphoid niche.

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.

Real-World Venetoclax-Obinutuzumab in 232 Treatment Naive CLL Patients: Feasibility and Tolerability

Fixed duration venetoclax-obinutuzumab (VO) is approved in treatment naïve (TN) chronic lymphocytic leukemia (CLL), based on CLL14 study addressed to patients (pts) with coexisting conditions. The combinations showed also to be effective and well tolerated in the younger population enrolled in the randomized CLL13 trial. The aim of this retrospective, multicenter Italian study is to evaluate the feasibility and tolerability of this combination in TN CLL in common clinical practice as well as reasons for choosing fixed-duration VO instead of continuous BTKi. Consecutive pts receiving first-line therapy after VO approval/reimbursement in Italy (May 2022) were considered. Reasons for choosing the fixed duration treatment were recorded. The impact of clinical, biological, and demographic characteristics on VO feasibility (definitive treatment discontinuation and/or schedule modifications due to toxicity) and tolerability were analyzed. Debulking (Deb) phase (D1-D56) was separately evaluated to better understand its influence on treatment management. From May 2022 to June 2023, 790 treatment naïve continuous pts were treated with targeted agents in 49 centers. Treatment choice consisted of: continuous BTKi in 558 pts and fixed-duration VO in 232 (the latter representing the population of our analysis). The main reasons given by physicians for choosing VO instead of BTKi were: disease biology 46% (mutated IgHV status), fixed duration 33%, comorbidities 15%, patient choice 6%. Pts' characteristics are shown in Table 1. When considering Deb-phase (232 pts), VO was regularly administered according to the investigator brochure in 150 (64.7%). In 22 cases (9.5%) the schedule was a priori modified by clinicians: 12 pts started with V led-in and postponed O infusion at C2D1; in 10 planned O D8 and D15 infusions were omitted. Overall, 12 pts (5.2%) permanently discontinued VO due to toxicity. 3 of them dying due to: COVID pneumonia, invasive aspergillosis, VO-unrelated cachexia. In the remaining 48 (20.7%) schedule was adjusted due to AEs. Treatment modifications, overlapping in some cases were: 42 extended the Deb-phase (median days to completion 63, range 59-130); 14, never reached the full V dose; 31 did not perform the planned 5 O infusions (median 3 infusions, range 1-4). No age, neither coexisting conditions influenced treatment feasibility. The only parameters having an impact on Deb-phase discontinuation or schedule modifications due to toxicity were: baseline ANC<1500/mmc (OR 3.1, p=.035), hypogammaglobulinemia (OR 2.2, p=.018), concomitant use of anticoagulants (3.13, p=.029). Overall 23 pts (9.9%), including 7 categorized as high risk TLS, were hospitalized for treatment initiation. During Deb-phase grade 3-4 AEs occurred in 69 pts (30%): neutropenia (18%); thrombocytopenia (14%); infections (5%); IRR (5%). Development of any severe AE was influenced by: upper GI disorder, baseline neutropenia or thrombocytopenia. TLS was recorded in 13 pts (6%): 11 O-related and 4 V-related. All 4 clinical TLS were considered as secondary to O. Only high CLL-CI risk was associated with TLS. None of the baseline factors nor type of steroids premedication predicted IRR. At the present follow-up of the 232 pts considered: 142 completed the 6 cycles of combinations treatment, 75 are still ongoing, 15 definitively discontinued treatment for AEs. Overall, 42 pts received VO with an adjusted schedule for toxicities: 22 are receiving V at reduced dose, 39 pts did not complete the planned O infusions (treatment modifications overlapping in some cases). G 3-4 toxicity developed in 78 pts being neutropenia and infections the most common reported. At univariate analysis factors affecting treatment feasibility were: presence of endocrine comorbidity; intermediate/high CLL-CI and hypogammaglobulinemia. To our knowledge this is the first real-world study on clinical factors that may influence VO feasibility. Despite this setting of unselected population, most clinicians were adherent to VO schedule. A low rate of pts permanently discontinued therapy during Deb-phase with no impact of age or comorbidities on feasibility. Furthermore, TLS and IRR were in line with clinical trials. Longer follow-up data will be reported at the meeting allowing by multivariate analysis to better clarify the role of comorbidities on treatment management and the reproducibility of VO tolerability in common clinical practice.

Advancements in the Treatment of CLL: The Rise of Zanubrutinib as a Preferred Therapeutic Option.

Ibrutinib, the first-in-class Bruton's tyrosine kinase inhibitor (BTKi), is a commonly deployed therapeutic option for previously untreated and relapsed/refractory (R/R) patients with chronic lymphocytic leukemia (CLL). The use of ibrutinib is, however, partially limited by off-target side effects. Zanubrutinib (zanu) is a second-generation BTKi with enhanced target selectivity and occupancy of the kinase binding site. The SEQUOIA study showed that zanu significantly prolonged progression-free survival (PFS) when compared to bendamustine-rituximab (BR) in treatment-naive CLL patients. More recently, data from the phase III ALPINE trial, which directly compared zanu with ibrutinib, demonstrated that zanu's advantages include an improved safety profile as well as enhanced clinical efficacy. Based on the results of the SEQUOIA and ALPINE pivotal trials, the Food and Drug Administration (FDA) and European Medicines Agency (EMA) licensed zanu for the treatment of patients with CLL or small lymphocytic lymphoma (SLL) in January 2023. The updated (v2.2023) National Comprehensive Cancer Network (NCCN) guidelines and the most recent German CLL algorithm suggest that zanu may replace first-generation BTKis as a preferred therapeutic option for patients with CLL/SLL due to its increased selectivity for the kinase binding site, improved therapeutic efficacy, and favorable toxicity profile. Some drug class-related characteristics such as drug resistance, low complete remission (CR) rates, and indefinite treatment duration still remain with zanu, and the results from recently completed and ongoing fixed-duration clinical trials, combining zanu with an anti-BCL2 agent, are eagerly awaited with the possible promise of a reduced treatment duration and lower financial burden.

Continuous Venetoclax in Previously Untreated Patients with Chronic Lymphocytic Leukemia and TP53 Abnormalities. a Study of the Italian Campus CLL

BACKGROUNG. Fixed duration venetoclax-based therapy, either in combination with BTK inhibitors or anti-CD20 monoclonal antibody, is a highly active front-line treatment in patients with chronic lymphocytic leukemia (CLL). However, patients with TP53 abnormalities may early relapse after the end of treatment. Continuous treatment with BTK inhibitor is a valid option in TP53 disrupted patients, counteracted by a relatively high rate of discontinuation due to adverse events. In the phase II study investigating venetoclax in TP53-disrupted CLL patients, only 5 patients were treatment naive (TN) and 4/5 were remission-free after a median follow-up of 70months (Stilgenbauer S, EHA2022). AIM. The aim of this retrospective study is to describe the efficacy and discontinuation rate of continuous venetoclax in TN CLL patients with TP53 disruption in the real-life setting. METHODS. Medical charts of CLL patients from 16 centers belonging to the Italian Campus CLL network were retrospectively reviewed to identify CLL with del17p13(cut-off 10%) and/or TP53 mutation (TP53m) treated front-line with continuous venetoclax. The primary endpoint was the rate of treatment discontinuation. Secondary endpoints were overall response rate (ORR), measurable residual disease (MRD) by flow-cytometry, progression-free survival (PFS), overall survival (OS) and comparison with a cohort of 100 TN patients treated with ibrutinib (updated from Visentin A, AJH2022). CLL diagnosis and response assessment were carried out according to the iwCLL2018 guidelines. Adverse events were classified according to the CTCAEv5.0 grading. Statistical analyses were performed with Prism7. RESULTS. Thirty-four TN CLL with TP53 abnormalities were recruited. The median age was 69 years (range 46-84), median CIRS score was 3.5 (range 0-12), 24% had a creatinine clearance <60ml/min. LDH and β2-microglobulin were increased in 38% and 48% of patients. Seventy-one % of patients were IGHV unmutated, 79% harbored del17p13 and 74% TP53m. According to the tumor lysis syndrome (TLS) risk score, 12% were classified at low-risk, 35% intermediate-risk and 53% at high-risk of TLS. Ninety-nine patients were hospitalized during the ramp-up phase while the remaining in the out-patient clinics. No patient developed clinical or biochemical TLS. Eighty-two % of patients were able to reach 400mg of venetoclax. The best ORR was 85%, including 50% complete responses (CR) and 35% partial responses (PR) (Fig. 1). The median time to CR was 14.9 months. MRD was assessed in the peripheral blood of 14 patients, achieving undetectable MRD4 in 6 (43%), detectable MRD4 in 6 (43%) and detectable MRD2 in 2 (14%) patients. After a median follow-up of 20 months, 9 patients decreased the venetoclax dose and 3 discontinued therapy (thrombocytopenia 1 and Richter transformation 2). The median PFS and OS were not reached. The 12 and 24-month PFS were 96% and 83%, respectively (Fig. 1). All patients were alive after 12 months and the estimated 24-month OS was 88%. Regarding safety, we recorded of grade ≥3 (G≥3) adverse events in 23 (67%) patients, including hematological toxicity in 14 (11 neutropenia, 2 anemia, 1 thrombocytopenia), second primary malignancies in 3, diarrhea in 3, infections in 2 and major bleeding in 2. Atrial fibrillation occurred in 2 patients. Compared to patients treated with ibrutinib (n=100 , comparable for age and comorbidities, median follow-up 24months), we observed a higher CR rate (50% vs 9%, p<0.0001), a trend for a longer PFS (p=0.10) and a lower incidence of discontinuation (24-month 14% vs 35%, p=0.0194) in patients receiving venetoclax (Fig. 1). We also observed a higher rate of all G≥3 events (67% vs 22%, p<0.0001) and severe neutropenia in patients treated with venetoclax (32% vs 4%, p<0.0001), but a lower rate of infections (6% vs 22%, p=0.0386) and cardiovascular events (6% vs 14%, p=0.3573). CONCLUSION. To our knowledge, we report the largest real-life study of continuous venetoclax in TN CLL patients with TP53 abnormalities, indicating a high efficacy of venetoclax in this subset of patients, not previously studied systematically in clinical trials. Only one patient discontinued venetoclax due to toxicity. The use of continuous venetoclax in TN CLL patients with TP53 disruption deserve further investigation, as it can be an effective alternative option beside BTK inhibitor. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Extrinsic interactions in the microenvironment in vivo activate an antiapoptotic multidrug-resistant phenotype in CLL

AbstractThe Bcl-2 inhibitor venetoclax has yielded exceptional clinical responses in chronic lymphocytic leukemia (CLL). However, de novo resistance can result in failure to achieve negative minimal residual disease and predicts poor treatment outcomes. Consequently, additional proapoptotic drugs, such as inhibitors of Mcl-1 and Bcl-xL, are in development. By profiling antiapoptotic proteins using flow cytometry, we find that leukemic B cells that recently emigrated from the lymph node (CD69+/CXCR4Low) in vivo are enriched for cell clusters simultaneously overexpressing multiple antiapoptotic proteins (Mcl-1High/Bcl-xLHigh/Bcl-2High) in both treated and treatment-naive CLL patients. These cells exhibited antiapoptotic resistance to multiple BH-domain antagonists, including inhibitors of Bcl-2, Mcl-1, and Bcl-xL, when tested as single agents in a flow cytometry–based functional assay. Antiapoptotic multidrug resistance declines ex vivo, consistent with resistance being generated in vivo by extrinsic microenvironmental interactions. Surviving “persister” cells in patients undergoing venetoclax treatment are enriched for CLL cells displaying the functional and molecular properties of microenvironmentally induced multidrug resistance. Overcoming this resistance required simultaneous inhibition of multiple antiapoptotic proteins, with potential for unwanted toxicities. Using a drug screen performed using patient peripheral blood mononuclear cells cultured in an ex vivo microenvironment model, we identify novel venetoclax drug combinations that induce selective cytotoxicity in multidrug-resistant CLL cells. Thus, we demonstrate that antiapoptotic multidrug-resistant CLL cells exist in patients de novo and show that these cells persist during proapoptotic treatment, such as venetoclax. We validate clinically actionable approaches to selectively deplete this reservoir in patients.

Use of Bendamustine Combined with Rituximab in Chronic Lymphocytic Leukemia Patients: A Single Center Experience

Background:There is no single standard treatment (tx) modality for symptomatic chronic lymphocytic leukemia (CLL) patients (pts). Fludarabine, cyclophosphamide, and rituximab (FCR) is the prefered regimen for healthy, young pts (<65 years). However, through the disease course almost all pts in this group will relapse and may become refractory to fludarabine-containing regimens. Additionally, for older adults (>65 years) and pts with comorbidities fludarabine is not a suitable agent. Bendamustine combined with rituximab (BR) is an acceptable alternative in these patient groups.Aim: To evaluate the efficacy and safety of BR in previously untreated or relapsed/refractory (R/R) CLL pts.Patients and Methods: Ptswho received BR as first- or further lines of tx were enrolled in the study. Data were analysed as of August 2017. The standard dose of bendamustine was 70 mg/m2 in D1 and D2 for R/R pts and 90 mg/m2 in D1 and D2 for tx naive pts. Rituximab dose was 375 mg/m2 in the first cycle and 500 mg/m2 in consecutive cycles. Pts' demographics, disease stages, biological and cytogenetic prognostic markers, comorbidities, previous treatments, bendamustine dose, cycle durations, use of G-CSF, adverse events (AEs), tx responses and follow-up periods were noted retrospectively. Evaluation of response was established according to NCI-WG guidelines [Hallek et al, 2008]. All toxicities were assessed according to the National Cancer Institute's Common Toxicity Criteria (NCI-CTC), version 4.Results: Nineteen ptswere enrolled in the study (Table 1). Thirteen pts were male and the median age at the time of BR treatment was 61 years (range, 41-80 years), with 9 pts (47%) older than 65 years. At the time of diagnosis, 7 pts (37%) were Rai stage IV. Del(13q), del(11q), and del(17p) were positive in 5, 1, and 2 pts, respectively. All pts had active disease as defined by the NCI-WG. Five pts received BR as first-line. Of the previously treated 14 pts, 71% had received fludarabine with either alkylating agents and/or rituximab. Median number of previous lines of tx was 2.Among comorbidities, hypertension (32%), asthma/COPD (32%) and type 2 diabetes mellitus (3%) were the three most common.Bendamustine dose ranged from 70 mg to 100 mg/m2. According to the physicians' decision, bendamustine was started as 70 mg in first cycle of tx and was escalated to the planned dose in all pts but three. All 6 intended courses were administered to 15 pts (79%), and in the remaining 4 pts, 3 died due to infections and one patient required early discontinuation due to prolonged neutropenia. In 8 pts (42%) tx cycle prolonged to 35 days and in 11 cases (58%) G-CSF were used during tx.After a median follow-up of 17 months (mos), five of 19 pts (26%) had progressed at the time of analysis, and median time to next treatment (TTNT) was 12 mos (range, 5-34 mos). Treatment responses are shown in Table 2. During the follow-up 5 pts (26%) died. Two had multiple comorbidities, three received bendamustine doses ≥90 mg/m2. Deaths were due to R/R disease and/or infections. Median OS after treatment was 13 mos (range, 5-34 mos).Overall, grade III-IV AEs occurred in 14 cases (74%): the most common toxicity was hematologic AEs (anemia in 2, neutropenia in 12, and thrombocytopenia in 2 cases), and serious infection occurred in six pts (2 bacterial pneumonia, 4 neutropenic fever). Non-hematologic toxicities were grade II rash in one, and grade II nausea in one patient.Conclusion: BR is effective and safe in treatment naive CLL patients and shows notable activity in R/R pts. Major toxic effect are myelosuppresion and infections. Higher doses of bendamustine should be avoided in patients with comorbidities and older age. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High Prevalence of Multiple B Cell Clones in Patients with Chronic Lymphocytic Leukemia

INTRODUCTIONStudies suggest that the first clonal events in chronic lymphocytic leukemia (CLL) may be acquired at hematopoietic stem cell stage. Consistently, it has been reported that CD34+cells from CLL patients engrafted in immunodeficient mice give rise to clonal B cell lymphoproliferation while CD34+ cells from healthy donors did not (Kikushige, Cancer Cell 2011). Furthermore, in rare patients with early CLL or monoclonal B cell lymphocytosis two clonal populations can be demonstrated. We hypothesized that immunofixation electrophoresis could serve as a sensitive tool to assess the prevalence of a second, clonally distinct, lymphoproliferative event in CLL patients. METHODSWe studied 262 treatment-naïve patients with a diagnosis of chronic lymphocytic leukemia (CLL) enrolled in our natural history study (NCT00923507). All patients had diagnostic peripheral blood flow cytometry testing (Laboratory of Pathology, NCI) and serum immunofixation electrophoresis (Department of Laboratory Medicine, Clinical Center, NIH) done. RESULTSOf the 262 patients, 148 (56.49%) were male and 114 (43.51%) were female. The median age of the study population was 65 years. Amongst the 262 patients, 243 (92.74%) were Caucasians, 8 (3.05%) were African Americans, 3 (1.14%) were Asians and 3 (1.14%) were multiracial. The race was undisclosed for 5 patients. Two of the 262 CLL patients had another B-cell malignancy on diagnostic workup, Waldenstrom's macroglobulinemia (Lymphoplasmacytic Lymphoma) and Hairy Cell Leukemia. Nine (3.44%) patients had two distinct clones of CLL by flow cytometry. In 4 both kappa and lambda clones were present. In 5, the two clones expressed the same light chains but had a distinct immunophenotype resulting in bi-phenotypic clones. By serum IFE, 62 (23.66%) of 262 patients had monoclonal gammopathy; in 44 (16.8%) patients one monoclonal band was detected, while 18 (6.9%) patients had 2 or more bands. Among patients with one monoclonal band 25 were kappa and 19 lambda light chain. In 19 of 25 patients with kappa clonal CLL by flow cytometry, the monoclonal band on IFE was kappa (concordant flow and IFE): IgM Kappa (in 8), IgG Kappa (10), and IgA Kappa (1); and in 6 patients with kappa clonal CLL the monoclonal band was lambda (discordant flow and IFE): IgM Lambda (in 2), and IgG Lambda (4). In 15 of 19 patients with lambda clonal CLL, flow cytometry and IFE were concordant: IgM Lambda (in 6), IgG Lambda (6), IgA Lambda (1), free lambda light chain (2), and discordant in 4: IgM Kappa (in 2), IgG Kappa (2). Amongst the 18 patients with 2 or more bands on IFE, only 5 patients had concordant flow cytometry and IFE, while 13 had discordant findings. In summary, using IFE we detected a second clonal event in 23 (8.8%) of 262 CLL patients. Considering the 11 patients with two distinct clones by flow cytometry, 34 (13%) of 262 treatment-naive CLL patients had at least one additional clonal B cell process. In addition, 18 patients with concordant flow and IFE findings had IgG or IgA type gammopathy, consistent with a concurrent plasma cell dyscrasia of the same light chain restriction as the CLL clone. CONCLUSIONIn 13% of CLL patients we found evidence for a second B cell lymphoproliferative process. The prevalence of biclonal CLL by flow cytometry at 3.4% is comparable to findings by others, reporting between 0.7% and 3.4%. In contrast, the prevalence of monoclonal gammopathy (23.7%) in our patients (median age 65) was much higher than reported in the general population: 3.2 % at age 50 and above, and 5.3% at age 70 and above (Kyle, NEJM 2006). Importantly, in 8.8% of all patients the monoclonal gammopathy was discordant with the CLL clone. These data support the existence of a genetic event in an early progenitor cell that predisposes to the development of clonal B cell expansions.This research was supported by the Intramural Research Program of NHLBI and NCI. DisclosuresWiestner:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding.

Ibrutinib Disrupts IL-4R - IL-4 Axis By Inhibiting IL-4R Signaling and Reversing Th2/Th1 Polarization through Diminished CLECL1 in CLL B Cells

The BTK inhibitor, ibrutinib, inhibits chronic lymphocytic leukemia (CLL) cell growth by blocking microenvironmental support in situ, releasing cells from tissues niches, and diminishing IL-4-producing T cells, all of which deprive leukemic cells of nurturing signals. However, little is known about the mechanisms whereby ibrutinib affects surface membrane (sm) levels of IL-4R and IL-4R signaling, Th2 polarization, particularly the relative sensitivities of U-CLL versus M-CLL to these effects of the drug, and how these parameters impact on those intraclonal CLL B cell fractions defined by time since last cell division. Hence we addressed these issues here.We studied 28 treatment-naive CLL patients receiving ibrutinib as single agent therapy. CLL B cells collected after ibrutinib treatment expressed lower densities of sm CD5, CD20, HLA-DR, CD25, and CD28; they also transiently displayed increased smIgM levels and persistently exhibited elevated but signaling-impeded smCXCR4. Moreover, ibrutinib treatment significantly lowered smIL-4R, without changing RNA or total protein content of the residual cells, suggesting that ibrutinib treatment may interrupt membrane trafficking. Finally, CLL B cells treated with ibrutinib exhibited significantly lower pSTAT6 and pJAK3 upon IL-4 stimulation, a here-to-fore unknown effect of BTK inhibition. Thus, ibrutinib impairs IL-4R sm expression and function.We next investigated if ibrutinib-impaired IL-4R signaling differentially affected the survival of U-CLL versus M-CLL cells. Supplementing leukemic B cell cultures with autologous T cells improved survival of U-CLL but not M-CLL cells. This T cell promoted survival was blocked by pretreatment of T cells with ibrutinib, or by co-culturing cells with IL-4 neutralizing antibody. Similarly, IL-4 improved the survival of U-CLL but not M-CLL cells in vitro, but only if the CLL B cells were tested before, but not after ibrutinib treatment in vivo . These findings were confirmed in a xenograft mouse model using primary treatment-naïve CLL patient cells; we observed greater inhibition of tissue-resident CLL B cell growth for U-CLL than M-CLL with ibrutinib therapy. Thus, IL-4R signaling is critical for CLL B cell survival, and U-CLL cells are more dependent on this than M-CLL.The CLL B cells remaining post-ibrutinib treatment were smaller, less viable and exhibited the resting phenotype described above; in almost all cases, ibrutinib eliminated the recently born, “proliferative fraction” (PF) of CLL cells, defined by a CXCR4DimCD5Bright phenotype. As suggested by our gene expression studies, we found that CLL PF cells display the highest levels of smIL-4R and smCLECL1, the latter is a receptor that induces Th2 polarization. Here we documented this by T cell polarization assays using autologous naïve CD4+ T cells co-cultured with CLL PF cells. However after ibrutinib treatment, PF cells taken from patients expressed lower smCLECL1 and failed to induce Th2 polarity. Thus, the Th1 polarization bias that occurs with ibrutinib treatment might result from direct targeting of ITK in T cells, as suggested by others, and by indirect targeting of CLECL1 on CLL cells as demonstrated here.Finally, PF cells from U-CLL patients have the highest levels of the anti-apoptotic protein BCL2. Ibrutinib treatment for 1 cycle significantly reduced BCL2 protein in U-CLL but not M-CLL cases. Notably, U-CLL cases with initial reduction of BCL2 that we followed to the end of cycle 3 treatment displayed increased BCL2 after cycles 2 and 3, suggesting that BTK inhibition initially selects clones expressing higher levels of BCL2. M-CLL cases however had invariant BCL2 protein during treatment, which might contribute to the better survival of M-CLL cells after the loss of environmental support.In summary, we report that ibrutinib interrupts the IL-4R - IL-4 axis by diminishing smIL-4R, smCLECL1, intracellular BCL2; and by promoting a non-IL-4 microenvironment thus favoring a Th1 cell bias. U-CLL B cells, in particular their PF, are more vulnerable to this action than M-CLL cells and therefore are more prone to die in tissue niches, consistent with M-CLL patients having greater and longer lymphocytosis when treated with ibrutinib. Thus our results provide a mechanistic understanding for the differences in primary ibrutinib sensitivity between U-CLL and M-CLL patients, supporting the rationale to combine inhibitors of BTK and BCL2 in CLL. DisclosuresBarrientos:AbbVie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Janssen: Consultancy. Burger:Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Novartis: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Gilead: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; TG Therapeutics: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding.

CLL-132: Mutational Status of IGVH in Correlation with Genetic Abnormalities of Patients with Chronic Lymphocytic Leukemia in Macedonia - A Single-Centre Experience

Context B-cell chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. One of the most important molecular predictors, the immunoglobulin VH gene mutational status, divides CLL into two prognostic groups, where unmutated U-CLL is associated with remarkably worse prognosis than mutated M-CLL. In many studies, multiple recurring chromosomal aberrations appear to be effective prognostic markers in CLL. Objective The aim of the study was first-time evaluation of rearrangement of IG genes and genetic abnormalities in Macedonian CLL patients. Design Prospective study of Macedonian patients with CLL at early and advanced stage of disease in a period of time from January 2011 to January 2019. The median follow-up was 48 months (1-96 months). Setting Mutational status and configuration of IGHV-IGHD-IGHJ rearrangements in 90 treatment-naive CLL patients were analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing methodology. We used multiplex ligation-dependent probe amplification (MLPA) for detection of most common genetic abnormalities. Interventions We introduced prognostic markers like cytogenetic aberrations, and mutational status of immunoglobulin heavy chain in prognostic stratification of Macedonian patients with CLL. Results Our evaluation has shown that 54.7% patients belonged to the U-CLL subset, whereas 45.3% belonged to the M-CLL subset. The most frequently expressed IGHV subgroup was IGHV3 (40%), followed by IGHV4 (29%), IGHV1(27%), and IGHV5 (3%). The most frequent subgroup with 8% were IGHV1-69*13 and IGHV4-34*0. In the IGHD and IGHJ sets, most frequently expressed genes were IGHD3 (59.7%) and IGHJ6(53.1%), respectively. We found that 80% of IGHV1, 65.6% of IGHD3, and 83.3% of IGHJ6 genes were U-CLL (p=0.0005). Frequency of genetic aberrations by MLPA were 13q14 deletion (20%) followed by 11q deletion (14%), then trisomy 12 (13%) and 17p deletion (6.6%), multiple mutations (6.7%), and NOTCH mutation (5.3%). Conclusions Our evaluation of mutational status on IGVH, IGDH, and IGJH genes in Macedonian CLL patients resulted with data that are consubstantial to those from the Mediterranean area. Novel mutations discovered in this study must be validated through large cohort studies and may offer clues to the mechanisms underlying the difference in CLL survival. In the future, therapeutic strategies targeting these genes should be evaluated and considered.

Frontline Therapy of Chronic Lymphocytic Leukemia: Changing Treatment Paradigm.

The treatment landscape of treatment-naive chronic lymphocytic leukemia (TN-CLL) is rapidly evolving. As more and more new drugs and combinations are becoming part of therapeutic armamentarium, it becomes highly pertinent to understand the evidence for each of the treatment options to select the right drug for the right patient. We summarize the recent data of the available frontline treatment options. The novel agents can overcome adverse biological attributes and provide long-term disease control. MRD may become a reliable surrogate for survival in the evaluation of future therapies. FCR still remains one of the best options in a young fit CLL with mutated IGVH. Long-term follow-up data of ibrutinib confirm its efficacy and safety in both high-risk and elderly TN-CLL patients. A combination of venetoclax with obinutuzumab has provided the hope of fixed-duration therapy and the potential for functional cure in TN-CLL. Several other trials testing the efficacy of other targeted agents and the optimal sequencing approaches are underway. Chemoimmunotherapy holds its ground as an effective treatment in the IGVH-mutated CLL. The targeted agents either singly or in combination have become standard of care in many subsets of TN-CLL.

Mitochondrial Genome-Derived Mc-COX2 with Novel Characteristics Predicts Prognosis of Patients with Chronic Lymphocytic Leukemia

Background Circular RNAs (circRNAs), a novel family of non-coding RNAs, have crucial roles in cancer progression. Conventional research is mainly focus on nuclear genome derived circRNAs (nu-circRNAs). The biological and clinical characteristics of mitochondrial genome derived-circRNAs (mt-circRNA) remains largely unknown, especially in chronic lymphocytic leukemia (CLL), the most prevalent incurable B-cell neoplasm in western countries. Lack of convenient and reliable clinical biomarkers is a hindrance in monitoring the progression of CLL. It is in urgent need of screening effective new biomarkers and exploring potential therapeutic targets associated with CLL initiation and progression. Recent studies have shown that circRNAs are enriched and stable in serum exosomes. However, the biological mechanisms of exosomal circRNAs remain unclear. In this study, we attempted to identify the novel characteristics of a mt-circRNA mc-COX2, which was abundant in plasma exosomes and could be involved in the progression of CLL. Since CLL patients have specific expression features of exosomal circRNAs in plasma, the function of the circRNAs and their clinical significance is urged to be explored. Methods Firstly, to unveil circRNAs expression profiles in CLL, plasma samples from five treatment-naive CLL patients and five age-/sex-matched healthy donors (HDs) were collected for circRNA microarray analyses. Northern blot and RNA- FISH were conducted to verify the existence of circRNAs in mitochondria. qPCR and other functional analysis such as RNase R, actinomycin D and RIP experiments were carried out to demonstrated the clinical and biological characteristics of mc-COX2, one of the mt-circRNAs. Cell apoptosis ability was determined by FCM. Moreover, electron microscope, partical size analysis, FCM and Western blot were used to explore the existence of exosomes and q-PCR analysis was performed to detect the expression of mc-COX2. Results Mt-circRNAs were highly expressed in CLL patients plasma (Figure A, B). Herein, we reported a novel circRNA named as mc-COX2 which was generated from the COX2 gene on mitochondrial genome by back-splicing and closely related to prognosis of CLL patients (Figure C). The enrichment of mc-COX2 in the mitochondria was further confirmed by RNA-FISH (Figure D). Northern blot was performed using head-to-tail probe of mc-COX2 and the results showed that mc-COX2 was detectable within the splice sites (Figure E). Notably, obviously different from nu-circRNAs, mc-COX2 showed lower stability with lower tolerance to RNase R and actinomycin D, but it was much more stable compared with linear RNAs (Figure G, F). And mc-COX2 cannot bind to AGO2 protein, suggesting that it probably function via other mechanisms instead of acting as ceRNA (Figure H). Furthermore, the up-regulated expression of mc-COX2 was positively associated with leukemogenesis and worse survival of CLL patients (Figure I, J). CLL patients with TP53 deletion rather than mutation displayed higher expression of mc-COX2 (Figure K). The endogenous reduction of mc-COX2 could induce cell apoptosis (Figure L). In addition, we indicated that mc-COX2 was highly enriched in exosomes, by which circRNAs could enter the circulation and be readily measured in the serum (Figure M). Moreover, the existence of mc-COX2 in plasma suggests that mc-COX2 may serve as a potentially novel prognosis biomarker for CLL patients, guiding targeted therapy in clinic. Conclusions In summary, we demonstrated the existence and clinical significance of mc-COX2, a novel class of circRNA species abundant in CLL plasma exosomes for the first time, which was distinct from nu-circRNAs. Furthermore, the specific high expression of mc-COX2 in CLL plasma which was strongly correlated with P53 deletion, can indicate worse prognosis of CLL patients. Taken together, our study not only identifies a novel circRNA which may serve as a new "liquid biopsy" biomarker for CLL prognosis but also expands the current knowledge regarding molecular mechanisms of circRNAs, providing potential diagnostic and therapeutic implications for CLL. It would be of great interest to explore the biogenesis of mt-circRNAs and their impact on mitochondrial function in future studies. Figure Disclosures No relevant conflicts of interest to declare.

Elevated Lactate Dehydrogenase Has Prognostic Relevance in Treatment-Naïve Patients Affected by Chronic Lymphocytic Leukemia with Trisomy 12.

Chronic Lymphocytic Leukemia (CLL) patients with +12 have been reported to have specific clinical and biologic features. We performed an analysis of the association between demographic; clinical; laboratory; biologic features and outcome in CLL patients with +12 to identify parameters predictive of disease progression; time to treatment; and survival. The study included 487 treatment-naive CLL patients with +12 from 15 academic centers; diagnosed between January 2000 and July 2016; and 816 treatment-naïve patients with absence of Fluorescence In Situ Hybridization (FISH) abnormalities. A cohort of 250 patients with +12 CLL followed at a single US institution was used for external validation. In patients with +12; parameters associated with worse prognosis in the multivariate model were high Lactate DeHydrogenase (LDH) and β-2-microglobulin and unmutated immunoglobulin heavy-chain variable region gene (IGHV). CLL patients with +12 and high LDH levels showed a shorter Progression-Free-Survival (PFS) (30 months vs. 65 months; p < 0.001), Treatment-Free-Survival (TFS) (33 months vs. 69 months; p < 0.001), Overall Survival (OS) (131 months vs. 181 months; p < 0.001) and greater CLL-related mortality (29% vs. 11% at 10 years; p < 0.001) when compared with +12 CLL patients with normal LDH levels. The same differences were observed in the validation cohort. These data suggest that serum LDH levels can predict PFS; TFS; OS and CLL-specific survival in CLL patients with +12.

CYP2B6 polymorphism and lipoprotein lipase expression in chronic lymphocytic leukemia: impact on the outcome of fludarabine–cyclophosphamide regimen

Background Chronic lymphocytic leukemia (CLL) is the most common type of leukemia and has a highly variable clinical course. Cyclophosphamide (CPA)-containing regimens are the standard of care for patients lacking the 17p deletion. CYP2B6 is a polymorphic cytochrome P450 isoform that converts CPA to its active form. Lipoprotein lipase (LPL) catalyzes the hydrolysis of triacylglycerol. Remarkably, a growing body of data emphasizes its role in the biology of different tumors. Objectives This study aimed to study CYP2B6 polymorphism and LPL expression in fludarabine cyclophosphamide (FC)-treated CLL patients lacking 17p deletion. Methods 46 treatment-naive CLL patients negative for 17p deletion and indicated to receive chemotherapy were enrolled. CYP2B6 genotyping and lipoprotein lipase mRNA expression were assayed by Realtime PCR. FC-protocol was given then treatment-related toxicities, response, and event free survival were traced. Results CYP2B6*6 allele was associated with lower rates of treatment-related anemia and hospital admission. The response to FC was affected only by CYP2B6 polymorphism. The event-free survival of responders was significantly higher in patients having low LPL expression. Conclusion CYP2B6*6 infers lower CPA efficacy with lower treatment-induced side effects and increased risk of nonresponding to FC chemotherapy in CLL. LPL expression is a predictor of outcome in CLL, indicating poor survival.

A phase 2 study of lenalidomide and dexamethasone in previously untreated patients with chronic lymphocytic leukemia (CLL)

Lenalidomide has anti-tumor activity in CLL but can be complicated by tumor lysis syndrome (TLS) and tumor flare (TF). In our previous study using low-dose lenalidomide in treatment-naive CLL, TLS was averted but TF remained frequent and complete responses (CR) were rare, despite treatment to progression. The addition of dexamethasone may mitigate TF and enable lenalidomide dose escalation, achieving durable response without long-term use. In this phase 2 trial, 31 treatment-naive CLL patients received lenalidomide (target 25mg daily) plus dexamethasone for a finite 18 cycles. No patients developed TLS and TF was infrequent. Overall responses were 74.2% (CR 9.7%) and median progression-free survival 27 months. Cereblon-binding proteins IKZF1 and IKZF3 were largely downregulated, with associated increased IRF4 levels. We therefore report that lenalidomide plus dexamethasone can achieve durable responses in a subset of patients without continuing therapy until progression. Upregulation of IRF4 may contribute to anti-CLL activity of immunomodulatory agents.This trial was registered at www.clinicaltrials.gov as NCT01133743.

Reduced Dose Ibrutinib Due to Financial Toxicity in CLL.

Ibrutinib is the only approved novel agent that is available for the treatment of relapsed-refractory and treatment-naive chronic lymphocytic leukemia patients with deletion 17p or TP53 mutation in India. The cost of ibrutinib is still prohibitive for most Indian CLL patients. We report here for the first time dose reductions due to the patient preference of financial toxicity. We prospectively followed up patients of CLL receiving ibrutinib at a tertiary referral center in India. The period of study was from April 2016 to April 2018. Reduced dose ibrutinib was defined as a sustained (≥ 12months) dosing at < 420mg/day, either at treatment initiation or within 3months from starting therapy. Progression free survival was compared using Kaplan-Meier analysis. There were a total of three patients on reduced dose and twelve patients on standard dose ibrutinib. Two patients discontinued standard dose ibrutinib due to adverse events. The patient age, cytogenetics, number of prior therapies and follow-up were not significantly different between the two groups. The rate of ≥ grade3 adverse events was significantly different between the two groups. The overall response rate and median PFS were also not significantly different between the two groups. In the limited patient numbers and follow-up period we show that outcomes of reduced dose ibrutinib are comparable to standard dose ibrutinib but with fewer adverse events. This study provides a proof of concept that a subset of patients might do well on reduced dose ibrutinib.

Front-line treatment of patients with chronic lymphocytic leukemia: a systematic review and network meta-analysis.

A systematic literature review and network meta-analysis were conducted to determine the relative efficacy and safety of interventions for treatment-naive chronic lymphocytic leukemia patients, as comparative evidence is scarce. Relative treatment effects of progression-free survival, overall survival and safety outcomes were estimated via network meta-analysis based on data identified via systematic literature review. Ibrutinib was superior in all pairwise comparisons for progression-free survival (probability to be better [P] range: overall population:69-100%; fludarabine-ineligible population: 69-100%) and overall survival (P range: overall: 89-100%; fludarabine-ineligible: 91-100%) and had the highest probability of being best for all outcomes. Ibrutinib provides superior benefit in survival and safety compared with other front-line treatments of chronic lymphocytic leukemia.

Characterization and Prognostic Impact of Multiple Productive IGHV Rearrangements in CLL

Background:In chronic lymphocytic leukemia (CLL) one of the strongest prognostic factors is IGHV mutational status. Infrequently, patients present not only with a single IGHV rearrangement but with multiple productive rearrangements. In about 2% of all CLL patients analyzed on cDNA level multiple rearrangements display the same mutational status and are categorized accordingly following ERIC recommendations. In another 1% rearrangements with discordant IGHV mutational status are detected and preclude a definite risk assignment. Only limited data exist on these rare subgroups.Aim:To characterize treatment-naive CLL patients with multiple productive IGHV rearrangements and determine the impact on prognosis.Patients and Methods:Out of 8,016 treatment-naive CLL patients between 2005 and 2015 and with data on IGHV mutational status we identified 204 (3%) with multiple productive rearrangements. IGHV mutational status was analyzed on cDNA and in all cases according to ERIC recommendations. IGHV mutated status (M) was defined by sequence identity <98% and unmutated status (U) by ≥98%. Chromosome banding analysis was available in 102 cases and interphase FISH with probes for 17p13, 13q14, 11q22 and centromeric region of chromosome 12 in 191. Male:female ratio was 3:1 and median age 68 years (range: 38-89). Additionally, data on SF3B1 and TP53 mutations was present in all cases. Follow-up data on time to first treatment (TTT) and overall survival (OS) was available in 105 cases with a median follow-up of 4 years. For statistical comparison we used a cohort of 1,262 untreated CLL patients with single IGHV rearrangement (median age: 67 years; range: 30-91, median follow-up: 6 years).Results:Out of 204 patients with multiple, productive rearrangements 199 (98%) presented with two and 5 patients (2%) with three IGHV rearrangements. Concordant IGHV mutated status (MM) was present in 120 cases (59%), whereas concordant unmutated status (UU) was seen in 34 patients (17%). In 50 cases (25%) a mixed IGHV status (UM) was detected.We analyzed frequencies of complex karyotype by CBA, biclonality according to immunophenotype (concurrent kappa restricted and lambda restricted subpopulations) and/or CBA, TP53 disruption (TP53mut and/or del(17p)), SF3B1mut, del(11q), trisomy 12, and del(13q). Overall, a higher frequency of biclonality was detected in patients with multiple vs. single IGHV rearrangements (16% vs. 1%, p<0.001). However, association to neither MM, UU nor UM existed. MM presented with molecular and cytogenetic characteristics similar to M. Correspondingly, UU showed similar frequencies of mutations and aberrations to U, except for higher frequency of trisomy 12 in UU vs. U (42% vs. 19%, p=0.003). Interestingly, UM presented with characteristics similar to U and UU. UM was associated with TP53 disruption vs. M (16% vs. 5%, p=0.003) and vs. MM (5%, p=0.035) as well as with SF3B1mut vs. M (16% vs. 5%, p=0.008). Furthermore, UM cases showed high frequency of del(11q) vs. M (29% vs. 3%, p<0.001) and vs. MM (1%, p<0.001) and less frequently del(13q) sole vs. M (41% vs. 60%, p=0.011) and MM (41% vs. 69%, p=0.001).No significantly differences in TTT were observed between MM and M (median: 13 vs. 14 years) and between UU and U (6 vs. 4 years), respectively. However, the difference between MM vs. UU (p=0.022) and M vs. U (p<0.001) was significant. The UM subgroup presented with a TTT (median: 4 years) similar to U and UU, whereas it was significantly shorter vs. M (p=0.003) and MM (p=0.006), respectively. A similar picture emerged for survival. 5-year OS of MM was not different vs. M (94% vs. 90%) but vs. U (78%, p=0.001). The statistical analysis of OS in UU was hampered by low case numbers. UM presented again with similar 5-year OS vs. U (81% vs. 78%, n.s.) and significantly worse OS vs. M (90%, p=0.049) and vs. MM (94%, p=0.014).Conclusions: (1) Patients with multiple productive IGHV rearrangements and concordant IGHV status show similar prognosis and characteristics to patients with single rearrangement with the respective IGHV status. (2) Cases with mixed IGHV status show similar prognosis to patients with IGHV unmutated status and accordingly are characterized by high frequencies of adverse prognostic factors like TP53 disruption, SF3B1mut, and del(11q), whereas del(13q) sole is less frequent. DisclosuresJeromin:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Dicker:Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

CC-122 Repairs T Cell Activation in Chronic Lymphocytic Leukemia That Results in a Concomitant Increase in PD-1:PD-L1 and CTLA-4 Immune Checkpoint Expression at the Immunological Synapse

Immunomodulatory drugs (IMiDs®) such as lenalidomide and immune checkpoint blockade (ICB) antibodies can enhance autologous anti-tumor T cell immunity and have the potential to elicit durable control of disease in B cell malignancies. These immunotherapies are likely to be most effective when employed in treatment combinations. Thus, the goal of pre-clinical research should be to reveal mechanisms of action (MOA) in the tumor microenvironment (TME) and identify biomarkers to guide development of combination therapy for patients. CC-122 is a novel first-in-class pleiotropic pathway modifier (PPM®) that has potent anti-proliferative, anti-angiogenic and immunomodulatory activities and is currently in Phase I clinical trials for lymphoma and chronic lymphocytic leukemia (CLL). Here, we have utilized the immunological synapse bioassay to examine T cell interactions with CLL tumor cells (modeling anti-tumor T cell responses in the TME) following CC-122 treatment and measure the expression of co-signaling complexes at the synapse.Conjugation assays and confocal imaging were used to visualize intercellular conjugate interactions and F-actin polymerization at the immune synapse between CD4+ and CD8+ T cells and autologous CLL tumor cells pulsed with superantigen (acting as antigen-presenting cells, APCs). Peripheral blood was obtained from treatment naive CLL patients (n=40) representative of disease heterogeneity. Treatment of both purified CLL cells and CD4+ or CD8+ T cells with CC-122 (0.01 - 1 μM for 24h) dramatically enhanced the number of T cells recognizing tumor cells (% conjugation) and increased the formation of F-actin immune synapses (area, μm2) compared to vehicle treated cells (P<.01). Notably, CC-122 treatment induced T cells to engage in multiple tumor cell synapse interactions that were more pronounced in restored CD8+ T cell lytic synapses. This immunomodulatory activity was detected across all CLL patient samples and drug concentrations tested. In addition, synapse strength as measured by total fluorescence intensity of F-actin per T cell:APC conjugate increased significantly with CC-122 (P<.01). A critical MOA of lenalidomide is activation of T cell immune synapse signaling. Here, our comparative studies revealed that CC-122 (0.1 - 1 μM) significantly enhanced autologous T cell synapse activity in CLL by 4 - 5 fold versus vehicle (P<.01), whereas lenalidomide (1 μM) enhanced activity by 3 fold vs vehicle. Moreover, CC-122 treatment resulted in increased expression and polarization of tyrosine-phosphorylated proteins at T cell synapses compared to lenalidomide and vehicle treatment (P<.01). This data provides evidence that CC-122 induces functional T cell synapses that control the assembly of signaling complexes between the T cell receptor (TCR) and the F-actin cytoskeletal layer. Following T cell recognition of APCs, co-signaling receptors co-localize at the immune synapse where they synergize with TCR signaling to promote (co-stimulatory receptors) or inhibit (co-inhibitory/'immune checkpoint' receptors) T cell activation. Quantitative image analysis studies revealed that restoration of T cell synapse activity with CC-122 was accompanied by an increased recruitment of inducible co-stimulator (ICOS) to the synapse that was dose-dependent (P<.01). CC-122 treatment also increased polarized expression of CTLA-4 and PD-1 immune checkpoint proteins at the synapse with PD-L1+ tumor cells. The observed up-regulation of co-inhibitory receptors led to combining CC-122 with anti-PD-L1, anti-PD-1 or anti-CTLA-4 blocking antibodies. Results show that these treatment combinations increased T cell synapse activity compared to using these immunotherapies alone (P<.01).In conclusion, our results demonstrate for the first time that CC-122 can activate T cell immune synapse signaling against autologous CLL tumor cells and this immunomodulatory capability is more potent than lenalidomide. We further show that CC-122 activation of T cells is associated with enhanced expression of the co-stimulatory receptor ICOS and co-inhibitory checkpoints CTLA-4 and PD-1 at the synapse site. Importantly, our pre-clinical data demonstrates that this regulatory feedback inhibition can be exploited by the addition of anti-PD-L1, anti-PD-1 or anti-CTLA-4 ICB to CC-122 to more optimally stimulate T cell activity against immunosuppressive tumor cells. DisclosuresHagner:Celgene: Employment, Equity Ownership. Pourdehnad:Celgene: Employment. Gandhi:Celgene: Employment, Equity Ownership. Ramsay:MedImmune: Research Funding; Celgene: Research Funding.