Real-world safety and tolerability of rapid infusion obinutuzumab in chronic lymphocytic leukemia, small lymphocytic lymphoma and non-Hodgkin lymphoma: a provincial review

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Interaction of TAPP adapters with the phosphoinositide PI(3,4)P2 regulates B cell activation and differentiation

Phosphoinositide 3-kinase is a family of lipid kinases that function by phosphorylating the D3 position of phosphoinositide (PI) lipids generating PI(3)P, PI(3,4)P2 and PI(3,4,5)P3. These D3 phosphoinositides regulate various cellular processes through the recruitment of effector proteins containing lipid specific pleckstrin homology (PH) domains. PI phosphatases such as PTEN and SHIP function to restrain PI3K signaling by limiting the amount of D3 PI available for binding. Deletion of either PTEN or SHIP significantly alters B cell function and humoral immune responses. TAPP1 and TAPP2 are dual PH domain containing adaptors which selectively bind the phosphoinositide PI(3,4)P2 via their C-terminal PH domains. PI(3,4)P2 is a lipid messenger generated by PI3K and through the inositol phosphatase activity of SHIP. The function of PI(3,4)P2 remains incompletely understood. To identify the functional role of TAPP-PI(3,4)P2 interactions, we utilized a knock-in (KI) mouse bearing mutations within the PI-binding pocket of both TAPPs. Our study assessed the effect of PI3K dependent KI mutation on B lymphocyte development, activation and antibody production. Flow cytometry analyses of lymphoid tissues found that TAPP KI mice develop relatively normal frequencies of mature B cell populations with the exception of peritoneal B1 cells, which are increased by approximately 50%. Strikingly, TAPP KI mice developed substantially elevated serum antibody levels. TAPP KI mice were able to generate high affinity antigen-binding antibodies upon immunization with NP-OVA in alum adjuvant; however, total immunoglobulin production was markedly increased under this immunization condition. We further assessed the germinal centre (GC) response, which are known to require PI3K signaling and a hallmark of T cell dependent (TD) antibody responses. TAPP KI mice generated larger germinal centers (GC) upon immunization, which was associated with increased GC B cell survival. We further assessed whether uncoupling of TAPPs from PI(3,4)P2 alters B cell signaling and functional responses in vitro. B cells purified from TAPP KI mice were found to have altered functional responses in vitro, with significantly increased survival and cell division following antigen receptor cross-linking. Consistent with increased cell survival, TAPP KI B cells show increased Akt phosphorylation on Ser473 and Thr308 after antigen receptor cross-linking. However, reconstitution of B cell deficient mice with either WT or TAPP KI B cells was found to generate similar GC responses, suggesting that activation of other cells may contribute to the enhanced in vivo responses. Consistently, when we examined the CD4+ T follicular helper cells, a subset providing critical cues to GC responses, we found increased expression of ICOS activation marker. Our results indicate the interactions of TAPP adapters with PI(3,4)P2 serve to restrain lymphocyte activation and limit antibody production, providing the first in vivo evidence that this interaction is important for immune function.February 201

Interaction of TAPP adapter proteins with phosphatidylinositol (3,4)-bisphosphate regulates germinal centre response and development of autoimmunity (P1107)

Abstract Activation of PI3-kinase enzymes is essential for lymphocyte development and function. Active PI3Ks generate several types of D3 phosphoinositides, including PI(3,4,5)P3 and PI(3,4)P2. Tandem PH domain containing proteins (TAPPs) are adaptor molecules which bind specifically to PI(3,4)P2, but their functions in immune cells are largely unknown. We have performed studies on TAPP mutant mice, in which a mutation was introduced to the C terminal domain of both TAPP1 and TAPP2, to determine the effect of uncoupling TAPPs from PI(3,4)P2. We found that these TAPP KI mice exhibit elevated serum antibody levels and also develop autoantibodies and kidney pathology resembling that of lupus. TAPP KI mice generated elevated germinal center (GC) responses upon immunization with T-dependent antigen, suggesting an inhibitory role associated with TAPPs in lymphocytes. The GC B cell population was increased in frequency and showed reduced expression of apoptosis markers, and expansion of follicular helper T cell subset. We hypothesize that TAPP KI mice have disruption in a regulatory mechanism within the GC leading to sustained B cell activation and survival, and contributing to development of autoimmunity. TAPP KI B cells were found to be hyper-responsive to BCR cross-linking in vitro, and T cell responses are currently being assessed. Our goal is to define the B cell and T cell intrinsic signaling mechanisms enhanced as a result of disrupted inhibitory signaling mediated by TAPPs.</jats:p

The landscape of T-cell engagers for the treatment of follicular lymphoma

Follicular lymphoma (FL), the second most common subtype of non-Hodgkin lymphoma, relies on interactions with immune elements in the tumor microenvironment, including T-follicular helper cells and follicular dendritic cells, for its survival and progression. Despite its initial responsiveness to chemoimmunotherapy, FL is generally considered incurable. Strategies to improve immune-mediated control of FL could significantly benefit this population, particularly as it includes many elderly and comorbid patients. Immune cell engagers, especially bispecific antibodies (BsAbs), are crucial in targeting FL by bridging tumor and effector cells, thereby triggering T-cell activation and cytotoxic killing. CD3 × CD20 BsAbs have shown the most promise in clinical development for B-NHL patients, with structural variations affecting their target affinity and potency. This review summarizes the current clinical trials of BsAbs for relapsed/refractory FL, highlighting the approval of some agents, their role in first-line treatment or combination therapies, their toxicity profiles, and the future of this therapeutic approach compared to other immune cell therapies

Interaction of TAPP adapter proteins with phosphatidylinositol (3,4)-bisphosphate regulates B cell activation and autoantibody production (159.5)

Abstract Activation of PI3-kinase enzymes through the BCR is essential for B cell development and function. Active PI3Ks generate D3 phosphoinositide PI(3,4)P2 that bind to signaling molecules such as TAPP (Tandem PH domain containing protein) adaptor via their C terminal PH domain. This interaction leads to translocation of TAPPs to the plasma membrane. However, the role of TAPP adaptors in regulating PI3K signaling in B cell is not completely understood. We have performed studies on TAPP mutant mice, where a mutation was introduced to the C terminal domain of both TAPP1 and TAPP2 to determine the effect of uncoupling TAPPs from PI(3,4)P2. The studies showed an enhanced B cell activation and elevated serum antibody levels in these mice, suggesting an inhibitory role associated with TAPPs in B cells. To test whether the aberrant responses observed in B cells leads to autoimmunity in these animals, we analyzed their serum and observed a progressive increase in anti-dsDNA antibodies in the aging cohort. This along with other observed characteristics such as the presence of anti-nuclear antibodies within the serum, progressive development of lymphopenia and glomerulonephritis in aged mice is reminiscent of lupus. We are currently working to define the cellular interactions involved in this autoimmune phenotype and the signaling mechanism associated with the inhibitory role of TAPPs.</jats:p

TAPP adaptors control Akt-dependent metabolic activation of germinal center B cells and suppress autoimmunity via binding to the SHIP product PI(3,4)P2

Abstract The phosphoinositide phosphatase SHIP regulates B cell activation by converting the PI 3-kinase product PI(3,4,5)P3 to PI(3,4)P2. While this activity of SHIP appears critical for generation of effective adaptive immunity while avoiding autoimmunity, the function of PI(3,4)P2 in B cells remains relatively unexplored. Tandem PH domain containing proteins (TAPPs) are adaptor proteins that specifically bind to PI(3,4)P2 via their C-terminal PH domains, targeting them to the plasma membrane. Mice bearing inactivating mutations in the PH domains of both TAPP1 and TAPP2, uncoupling them from PI(3,4)P2, exhibit hypergammaglobulinemia and develop lupus-like characteristics including anti-DNA antibodies and deposition of immune complexes in kidneys. Here we find that TAPP KI mice develop chronic germinal centres (GCs) and age-associated increases in B cell expression of activation, exhaustion and memory markers, all of which are B cell intrinsic and dependent on the co-stimulatory molecule ICOS. TAPP KI B cells exhibit elevated phosphorylation of Akt and its target GSK3, and increased survival. Strikingly, TAPP KI B cells exhibit increased metabolic activity associated with elevated oxygen consumption rate, increased extracellular acidification rate, increased expression of glucose transporter Glut1 and increased glucose transport rate. Together our findings suggest that TAPP-PI(3,4)P2 interaction is important for regulating signalling via Akt, B cell metabolism and development of autoimmunity.</jats:p

Risk Factors for the Development of Skin Cancers in Patients with Chronic Lymphocytic Leukemia: A Retrospective Cohort Study

Introduction: Individuals with chronic lymphocytic leukemia (CLL) are often immunosuppressed and at increased risk of infection and secondary malignancies. The primary aim of this study was to determine the incidence of skin cancer amongst the CLL population at Kingston Health Sciences Centre (KHSC), Ontario, Canada. Secondly, we sought to identify the risk factors associated with the development of skin cancer in CLL patients. Methods: Consecutive patients seen at KHSC with a diagnosis of CLL between 2014 January 1 and 2019 December 31 formed the primary study cohort. KHSC serves a region of ~ 550,000 including a high proportion of older individuals and those living in rural areas. Approval was provided by Queen's University Research Ethics Board. Four independent reviewers conducted retrospective electronic chart review, initially in duplicate with review of any areas of discrepancy to ensure a standardized approach. Data collected included age, sex, CLL date of diagnosis, stage, genetics and treatment; histological diagnoses of other cancers (skin and other), smoking status (ever/never) and date of last follow up or death. The primary outcome was the development of the first skin cancer (squamous cell carcinoma, basal cell carcinoma, melanoma, or sarcoma) confirmed via pathology reports that are available in our local institution. All statistical analysis was performed using SAS Enterprise v. 7.15. Categorical variables were compared using chi-square or Fisher exact tests, medians using Wilcoxon and Mann-Whitney tests, and continuous variables using t-tests. Risk factors for development of skin cancer were assessed using multivarable Cox-proportional hazard models. Results: Of the total cohort of 377 individuals with CLL, 251 (67%) were male. Median age at diagnosis of CLL was 65 years (range 36 - 93 years of age). Median follow-up from the time of CLL diagnosis was 6.5 years (range 0.27 - 30.98). Of these, 80 individuals (21.2%) developed at least one skin cancer after their diagnosis of CLL, with an age-adjusted incidence of 16.96/1000 patient years (95% CI 12.5 - 23). Among the 297 who did not develop skin cancer post-CLL diagnosis, 13 individuals who had documented skin cancer pre-CLL diagnosis only, are included in the non-skin cancer group (Figure 1). Females had a lower incidence of skin cancer compared with males (63 males versus 17 females, p=0.009). There was no difference between the groups based on Rai stage at diagnosis, smoking history, or IGHV /p53 status. Individuals treated with ibrutinib had a lower incidence of skin cancer (3 versus 49, p=0.002). Development of skin cancer was associated with development of other invasive tumours including CLL transformation (p=0.001) (Table 1). Conclusion: Limitations of this study include its small size, and single institution setting. Our data likely reflect a conservative estimate as skin cancers may be diagnosed outside of the institution, and not all CLL is treated at tertiary care centres. Consistent with other studies we found that males with CLL are at increased risk of developing skin cancer, as compared to females. Individuals with CLL and skin cancer were more likely to develop another malignancy or Richter's transformation. The finding of lower incidence of skin cancer with ibrutinib treatment is novel; further investigation in larger populations is needed to determine if it may offer a protective effect. Disclosures Asai: Sanofi Canada: Honoraria, Research Funding; AllerGen NCE: Research Funding; Pfizer: Honoraria, Research Funding; Janssen: Honoraria; Leo Pharma: Honoraria; Eli Lilly: Honoraria; Novartis: Honoraria; Abbvie: Honoraria, Research Funding; Canadian Institutes of Health Research: Research Funding. Hay:Roche: Research Funding; Janssen: Research Funding. </jats:sec

Real world risk of infusion reactions and effectiveness of front-line obinutuzumab plus chlorambucil compared with other frontline treatments for chronic lymphocytic leukemia

Abstract Background Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in North America. Previous studies have shown improved progression free survival (PFS) and response rates in unfit patients treated with obinutuzumab compared to other regimens. The aim of this study was to evaluate the obinutuzumab-chlorambucil regimen in the context of historical treatments and first-dose infusion reactions at CancerCare Manitoba (CCMB). Methods A retrospective chart review was conducted for patients treated with obinutuzumab from January 1, 2014 to December 31, 2017 at CCMB. A minimum data set was extracted for patients treated with other front-line therapies. Descriptive statistics were used to evaluate patient demographics, toxicity, duration and dosing of obinutuzumab treatment. Kaplan–Meier curves were used to evaluate time-to-next-treatment (TTNT), overall survival (OS) and PFS for patients treated with obinutuzumab. A multivariable logistic regression model was used to investigate associations between infusion related reactions (IRRs) and age at treatment, pre-treatment lymphocyte count, cumulative illness rating scale (CIRS) and receipt of prior chemotherapy. Results Forty seven percent of patients receiving frontline therapy received chlorambucil and obinutuzumab. Sixty-seven patients were treated with obinutuzumab and consisted of 36 males (53.7%) and 31 females (46.3%) with 29 patients (43.3%) over age 75 years. Rates of grade 3 and 4 obinutuzumab IRRs were lower (6%) compared to the CLL11 clinical trial (20%) due to local practices including slower infusion rates and using chlorambucil before starting obinutuzumab treatment. Many patients had difficulty tolerating the full dosage of chlorambucil. Only 26 patients (38.8%) had their dose of chlorambucil escalated to the full dose of 0.5 mg/kg. In addition, only 18 patients (26.9%) received all doses of obinutuzumab and all 12 doses of chlorambucil. Conclusions In summary, first dose infusion reactions with obinutuzumab can be markedly reduced by using chlorambucil to decrease the lymphocyte count before obinutuzumab and by using a very slow initial obinutuzumab infusion rate. Modifications in chlorambucil dosing and obinutuzumab administration can improve tolerance without significant loss in efficacy. </jats:sec