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

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.

Array-based dynamic allele specific hybridization (Array-DASH): Optimization-free microarray processing for multiple simultaneous genomic assays.

We report proof-of-principle experiments regarding a dynamic microarray protocol enabling accurate and semi-quantitative DNA analysis for re-sequencing, fingerprinting and genotyping. Single-stranded target molecules hybridise to surface-bound probes during initial gradual cooling with high-fidelity. Real-time tracking of target denaturation (via fluorescence) during a 'dynamic' gradual heating phase permits 'melt-curve' analysis. The probe most closely matching the target sequence is identified based on the highest melting temperature. We demonstrated a >99% re-sequencing accuracy and a potential detection rate of 1% for SNPs. Experiments employing Hypericum ribosomal ITS regions and HIV genomes illustrated a reliable detection level of 5% plus simultaneous re-sequencing and genotyping. Such performance suggests a range of potential real-world applications involving rapid sequence interrogation, for example, in the Covid-19 pandemic. Guidance is offered towards the development of a commercial platform and dedicated software required to bring this technique into mainstream science.

Expression and function of phosphoinositide 3-kinase delta in mesenchymal stromal cells from normal and leukaemic bone marrow.

Within lymphoid tissues, chronic lymphocytic leukaemia (CLL) cells interact with mesenchymal stromal cells (MSC). Inhibitors of phosphoinositide 3-kinase delta (PI3Kδ) cause release of CLL cells from lymphoid tissues into blood. PI3Kδ inhibitors are thought to target only CLL and other immune cells because PI3Kδ expression is restricted to haematopoietic cells. We found that PI3Kδ is unexpectedly expressed in primary MSC derived from CLL patients and healthy donors. PI3Kδ inhibition in MSC using idelalisib or duvelisib significantly reduced their ability to support CLL migration and adhesion. These observations provide the first evidence that PI3Kδ is expressed and functional in CLL MSC.

Associations between statin use and risk of non-Hodgkin lymphomas by subtype.

Non-Hodgkin lymphomas (NHL) are a group of cancers with highly heterogeneous biology and clinical features. Statins are increasingly prescribed to prevent cardiovascular diseases. Early evidence shows a preventive effect of statins for some cancers, but their effect on NHL risk is unclear. We conducted a population-based nested case-control study involving 5,541 NHL cases and 27,315 controls matched for gender, age, place of residence and length of period of available prescription drug data. We assessed the use of statins prior to diagnosis (excluding the 12 months prior to the index date). We used conditional logistic regression models to estimate odds ratio (OR) and 95% confidence interval (CI) for use of any statin, adjusting for medical conditions, number of family physician visits for 5 years prior to index date, healthcare utilization, income and use of other medications. Over one-quarter of cases and controls were prescribed statins. Ever-use of any statin was associated with lower risk of Total NHL (OR = 0.82, 95% CI 0.76-0.89) and of certain subtypes including diffuse large B-cell lymphomas (DLBCL, OR = 0.77, 95% CI 0.65-0.92), plasma cell neoplasms (PCN, OR = 0.76, 95% CI 0.63-0.91) and other B-cell NHL (0.75, 0.59-0.95). Analysis by statin type suggested that the association was limited to high potency statin and lipophilic statin users. No clear duration or dose-response relationships were observed. Our findings provide evidence that statin use can reduce the risk of DLBCL and plasma cell lymphomas, but not other NHL types. Further studies are warranted to verify these associations and to examine the biological mechanisms.

Negative regulators of cell death pathways in cancer: perspective on biomarkers and targeted therapies.

Cancer is a primary cause of human fatality and conventional cancer therapies, e.g., chemotherapy, are often associated with adverse side-effects, tumor drug-resistance, and recurrence. Molecularly targeted therapy, composed of small-molecule inhibitors and immunotherapy (e.g., monoclonal antibody and cancer vaccines), is a less harmful alternative being more effective against cancer cells whilst preserving healthy tissues. Drug-resistance, however, caused by negative regulation of cell death signaling pathways, is still a challenge. Circumvention of negative regulators of cell death pathways or development of predictive and response biomarkers is, therefore, quintessential. This review critically discusses the current state of knowledge on targeting negative regulators of cell death signaling pathways including apoptosis, ferroptosis, necroptosis, autophagy, and anoikis and evaluates the recent advances in clinical and preclinical research on biomarkers of negative regulators. It aims to provide a comprehensive platform for designing efficacious polytherapies including novel agents for restoring cell death signaling pathways or targeting alternative resistance pathways to improve the chances for antitumor responses. Overall, it is concluded that nonapoptotic cell death pathways are a potential research arena for drug discovery, development of novel biomarkers and targeted therapies.

Identification and Characterization of Novel Receptor-Interacting Serine/Threonine-Protein Kinase 2 Inhibitors Using Structural Similarity Analysis.

Receptor-interacting protein kinase 2 (RIP2 or RICK, herein referred to as RIPK2) is linked to the pathogen pathway that activates nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) and autophagic activation. Using molecular modeling (docking) and chemoinformatics analyses, we used the RIPK2/ponatinib crystal structure and searched in chemical databases for small molecules exerting binding interactions similar to those exerted by ponatinib. The identified RIPK2 inhibitors potently inhibited the proliferation of cancer cells by > 70% and also inhibited NFκB activity. More importantly, in vivo inhibition of intestinal and lung inflammation rodent models suggests effectiveness to resolve inflammation with low toxicity to the animals. Thus, our identified RIPK2 inhibitor may offer possible therapeutic control of inflammation in diseases such as inflammatory bowel disease, asthma, cystic fibrosis, primary sclerosing cholangitis, and pancreatitis.

Phosphatidylinositol-3,4-Bisphosphate and Its Binding Protein Lamellipodin Regulate Chemotaxis of Malignant B Lymphocytes.

Cell migration is controlled by PI3Ks, which generate lipid messengers phosphatidylinositol-3,4,5-trisphosphate and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and consequently recruit pleckstrin homology (PH) domain-containing signaling proteins. PI3K inhibition impairs migration of normal and transformed B cells, an effect thought to partly underlie the therapeutic efficacy of PI3K inhibitors in treatment of B cell malignancies such as chronic lymphocytic leukemia. Although a number of studies have implicated phosphatidylinositol-3,4,5-trisphosphate in cell migration, it remains unknown whether PI(3,4)P2 plays a distinct role. Using the PI(3,4)P2-specific phosphatase inositol polyphosphate 4-phosphatase, we investigate the impact of depleting PI(3,4)P2 on migration behavior of malignant B cells. We find that cells expressing wild-type, but not phosphatase dead, inositol polyphosphate 4-phosphatase show impaired SDF-induced PI(3,4)P2 responses and reduced migration in Transwell chamber assays. Moreover, PI(3,4)P2 depletion in primary chronic lymphocytic leukemia cells significantly impaired their migration capacity. PI(3,4)P2 depletion reduced both overall motility and migration directionality in the presence of a stable chemokine gradient. Within chemotaxing B cells, the PI(3,4)P2-binding cytoskeletal regulator lamellipodin (Lpd) was found to colocalize with PI(3,4)P2 on the plasma membrane via its PH domain. Overexpression and knockdown studies indicated that Lpd levels significantly impact migration capacity. Moreover, the ability of Lpd to promote directional migration of B cells in an SDF-1 gradient was dependent on its PI(3,4)P2-binding PH domain. These results demonstrate that PI(3,4)P2 plays a significant role in cell migration via binding to specific cytoskeletal regulators such as Lpd, and they suggest that impairment of PI(3,4)P2-dependent processes may contribute to the therapeutic efficacy of PI3K inhibitors in B cell malignancies.

Poly(ADP-ribose) polymerase-1 causes mitochondrial damage and neuron death mediated by Bnip3.

Excessive pathophysiological activity of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP1) causes neuron death in brain hypoxia/ischemia by inducing mitochondrial permeability transition and nuclear translocation of apoptosis-inducing factor (AIF). Bcl-2/adenovirus E1B 19 kDa-interacting protein (Bnip3) is a prodeath BH3-only Bcl-2 protein family member that is induced in hypoxia, and has effects on mitochondrial permeability and neuronal survival similar to those caused by PARP1 activation. We hypothesized that Bnip3 is a critical mediator of PARP1-induced mitochondrial dysfunction and neuron death. Hypoxic death of mouse cortical neuron cultures was mitigated by deletion of either PARP1 or Bnip3, indicating that both factors are involved. Direct normoxic PARP1 activation by a DNA alkylating agent enhanced Bnip3 expression, and caused Bnip3-dependent mitochondrial membrane permeability, AIF translocation, and neuron death. Hypoxia produced PARP1-dependent depletion of nicotinamide adenine dinucleotide (NAD(+)) and inhibition of the NAD(+)-dependent class III histone deactelyase (HDAC) sirtuin-1 (SIRT1). This, in turn, led to hyperacetylation and nuclear localization of the forkhead box (Fox) protein FoxO3a, followed by enhanced association of FoxO3a with the Bnip3 upstream promoter region, increased levels of Bnip3 transcript, and elevated mitochondrial Bnip3 immunoreactivity. Finally, FoxO3a silencing using a lentiviral short hairpin RNA approach significantly reduced hypoxic Bnip3 expression, mitochondrial damage, and neuron death. Together, these data illustrate a direct PARP1-mediated hypoxic signaling pathway involving NAD(+) depletion, SIRT1 inhibition, FoxO3a-driven Bnip3 generation, and mitochondrial AIF release.

Differential cellular responses induced by dorsomorphin and LDN-193189 in chemotherapy-sensitive and chemotherapy-resistant human epithelial ovarian cancer cells.

Inherent or acquired drug resistance is a major contributor to epithelial ovarian cancer (EOC) mortality. Novel drugs or drug combinations that produce EOC cell death or resensitize drug resistant cells to standard chemotherapy may improve patient treatment. After conducting drug tolerability studies for the multikinase inhibitors dorsomorphin (DM) and it is structural analogue LDN-193189 (LDN), these drugs were tested in a mouse intraperitoneal xenograft model of EOC. DM significantly increased survival, whereas LDN showed a trend toward increased survival. In vitro experiments using cisplatin (CP)-resistant EOC cell lines, A2780-cp or SKOV3, we determined that pretreatment or cotreatment with DM or LDN resensitized cells to the killing effect of CP or carboplatin (CB). DM was capable of blocking EOC cell cycle and migration, whereas LDN produced a less pronounced effect on cell cycle and no effect on migration. Subsequent analyses using primary human EOC cell samples or additional established EOC cells lines showed that DM or LDN induced a dose-dependent autophagic or cell death response, respectively. DM induced a characteristic morphological change with the appearance of numerous LC3B-containing acidic vacuoles and an increase in LC3BII levels. This was coincident with a decrease in cell growth and the altered cell cycle consistent with DM-induced cytostasis. By contrast, LDN produced a caspase 3-independent, reactive oxygen species-dependent cell death. Overall, DM and LDN possess drug characteristics suitable for adjuvant agents used to treat chemotherapy-sensitive and -resistant EOC.

Valproic acid enhances fludarabine-induced apoptosis mediated by ROS and involving decreased AKT and ATM activation in B-cell-lymphoid neoplastic cells.

Histone deacetylase (HDAC) inhibitors have been shown synergize with a number of cytotoxic drugs in leukemic cells. In chronic lymphocytic leukemia (CLL), the first line therapy is based on the combination of fludarabine, a nucleoside analogue, and rituximab, an anti-CD20 monoclonal antibody, and there are presently no HDAC inhibitors are used to manage CLL. In the present study, we found that the addition of valproic acid (VPA), a HDAC inhibitor, increases cell death in B-cell-neoplasm-derived cell lines, BJAB, NALM-6 and I-83. This increased apoptosis caused release of mitochondrial cytochrome c, activation of caspases, and increased reactive oxygen species (ROS). The addition of a ROS scavenger inhibited cell death induced by the VPA-fludarabine combination. In contrast, blocking the death receptor pathway failed to inhibit VPA increased fludarabine induced apoptosis. Combination of VPA and fludarabine treatment decreased both total and phosphorylated levels of AKT, an important anti-apoptotic protein, and ATM, a pivotal protein in DNA damage response. Chemical inhibition of AKT or ATM was sufficient to enhance fludarabine-induced apoptosis. We next examined patient samples from a local clinical trial where relapsed CLL patients were treated with VPA and examined the effects of VPA on AKT and ATM in vivo. After 30 days, there was a reduction in ATM levels in three out of the four patients treated, while AKT phosphorylation was reduced only in one patient. Taken together, VPA reduces ATM levels, thereby increasing ROS-dependent cell death via the mitochondrial apoptotic pathway when combined with fludarabine.

The creation of an adaptable informed consent form for research purposes to overcome national and institutional bottlenecks in ethics review: experience from rare disease registries.

Background: The lack of harmonization of evaluation criteria by Ethics Committees in the European Union (EU) has led to inconsistent ethics reviews received by research sites participating in multicenter non-interventional studies. The European General Data Protection Regulation (GDPR) appears to be implemented at national level with a substantial degree of variance in interpretation. The European Reference Networks (ERNs) were struggling in setting an Informed Consent Form (ICF) for registries, allowing reuse of data for research purposes. The aim of this work is to develop an adaptable ICF for research purposes to be used in ERN registries. Methods: To work on this challenge, a team was established within the European Joint Programme on Rare Diseases (EJP RD) to develop a patients' registry ICF template allowing easy adaptation to ERNs, country, and site-level specificities. ERN and patients' representatives validated the choice of developing a GDPR-compliant template for research purposes. The feedback received from 34 Ethics Committees on the Clinical Patient Management System ICF, including the submission of patients' data to the ERN registries and the EU consent regulatory framework were analyzed along with existing ontologies for data access and reuse. An adaptable ICF was developed following iterative cycles of consultation and review by clinicians, research experts, ethics and regulatory advisors, and patients' representatives. The development of pediatric material for minor participants was also undertaken. Results and Conclusion: Research oriented ICF templates for adults and for parents/legal representatives of patients were released in 26 national languages. This adaptable ICF aims to foster, according to patients' preferences, the reuse of registries data for research purposes in compliance with the applicable laws and standards. Pediatric material is being finalized to collect minors' assent. ICF machine-readability is also progressing to enhance data discovery and facilitate its access and reuse conditions.

Lysosome-Disrupting Agents in Combination with Venetoclax Increase Apoptotic Response in Primary Chronic Lymphocytic Leukemia (CLL) Cells Mediated by Lysosomal Cathepsin D Release and Inhibition of Autophagy.

Venetoclax and obinutuzumab are becoming frontline therapies for chronic lymphocytic leukemia (CLL) patients. Unfortunately, drug resistance still occurs, and the combination could be immunosuppressive. Lysosomes have previously been identified as a target for obinutuzumab cytotoxicity in CLL cells, but the mechanism remains unclear. In addition, studies have shown that lysosomotropic agents can cause synergistic cell death in vitro when combined with the BTK inhibitor, ibrutinib, in primary CLL cells. This indicates that targeting lysosomes could be a treatment strategy for CLL. In this study, we have shown that obinutuzumab induces lysosome membrane permeabilization (LMP) and cathepsin D release in CLL cells. Inhibition of cathepsins reduced obinutuzumab-induced cell death in CLL cells. We further determined that the lysosomotropic agent siramesine in combination with venetoclax increased cell death in primary CLL cells through an increase in reactive oxygen species (ROS) and cathepsin release. Siramesine treatment also induced synergistic cytotoxicity when combined with venetoclax. Microenvironmental factors IL4 and CD40L or incubation with HS-5 stromal cells failed to significantly protect CLL cells from siramesine- and venetoclax-induced apoptosis. We also found that siramesine treatment inhibited autophagy through reduced autolysosomes. Finally, the autophagy inhibitor chloroquine failed to further increase siramesine-induced cell death. Taken together, lysosome-targeting drugs could be an effective strategy in combination with venetoclax to overcome drug resistance in CLL.

Breast Tumor Metastasis and Its Microenvironment: It Takes Both Seed and Soil to Grow a Tumor and Target It for Treatment.

Metastasis remains a major challenge in treating breast cancer. Breast tumors metastasize to organ-specific locations such as the brain, lungs, and bone, but why some organs are favored over others remains unclear. Breast tumors also show heterogeneity, plasticity, and distinct microenvironments. This contributes to treatment failure and relapse. The interaction of breast cancer cells with their metastatic microenvironment has led to the concept that primary breast cancer cells act as seeds, whereas the metastatic tissue microenvironment (TME) is the soil. Improving our understanding of this interaction could lead to better treatment strategies for metastatic breast cancer. Targeted treatments for different subtypes of breast cancers have improved overall patient survival, even with metastasis. However, these targeted treatments are based upon the biology of the primary tumor and often these patients' relapse, after therapy, with metastatic tumors. The advent of immunotherapy allowed the immune system to target metastatic tumors. Unfortunately, immunotherapy has not been as effective in metastatic breast cancer relative to other cancers with metastases, such as melanoma. This review will describe the heterogeneic nature of breast cancer cells and their microenvironments. The distinct properties of metastatic breast cancer cells and their microenvironments that allow interactions, especially in bone and brain metastasis, will also be described. Finally, we will review immunotherapy approaches to treat metastatic breast tumors and discuss future therapeutic approaches to improve treatments for metastatic breast cancer.

Common conditions of use elements. Atomic concepts for consistent and effective information governance.

Myriad policy, ethical and legal considerations underpin the sharing of biological resources, implying the need for standardised and yet flexible ways to digitally represent diverse 'use conditions'. We report a core lexicon of terms that are atomic, non-directional 'concepts of use', called Common Conditions of use Elements. This work engaged biobanks and registries relevant to the European Joint Programme for Rare Diseases and aimed to produce a lexicon that would have generalised utility. Seventy-six concepts were initially identified from diverse real-world settings, and via iterative rounds of deliberation and user-testing these were optimised and condensed down to 20 items. To validate utility, support software and training information was provided to biobanks and registries who were asked to create Sharing Policy Profiles. This succeeded and involved adding standardised directionality and scope annotations to the employed terms. The addition of free-text parameters was also explored. The approach is now being adopted by several real-world projects, enabling this standard to evolve progressively into a universal basis for representing and managing conditions of use.

Getting your DUCs in a row - standardising the representation of Digital Use Conditions.

Improving patient care and advancing scientific discovery requires responsible sharing of research data, healthcare records, biosamples, and biomedical resources that must also respect applicable use conditions. Defining a standard to structure and manage these use conditions is a complex and challenging task. This is exemplified by a near unlimited range of asset types, a high variability of applicable conditions, and differing applications at the individual or collective level. Furthermore, the specifics and granularity required are likely to vary depending on the ultimate contexts of use. All these factors confound alignment of institutional missions, funding objectives, regulatory and technical requirements to facilitate effective sharing. The presented work highlights the complexity and diversity of the problem, reviews the current state of the art, and emphasises the need for a flexible and adaptable approach. We propose Digital Use Conditions (DUC) as a framework that addresses these needs by leveraging existing standards, striking a balance between expressiveness versus ambiguity, and considering the breadth of applicable information with their context of use.

Autophagy in clear cell ovarian cancer, a potential marker for hypoxia and poor prognosis?(#).

Autophagy contributes to cell survival and is up-regulated under hypoxia in many different cancers. Ovarian cancer has a poor prognosis and is generally resistant to chemotherapy. Through genetic profiling, it has becoming evident that ovarian cancer has distinct subtypes but the significance of these subtypes in ovarian cancer remains unclear. In this issue, Dr Lum and colleagues have presented evidence that autophagy as measured by LC3A staining occurs in a clear cell ovarian cancer that is correlated with hypoxic regions and poor overall survival. In addition, autophagy under hypoxia appears to be higher in clear cell ovarian cancer cells compared to other subtypes. This indicates that autophagy could be a factor in drug resistance and poor survival in clear cell ovarian cancer patients. This insight could lead to a better understanding of the role of autophagy under hypoxia in human ovarian cancer and could be a valuable biomarker for the development of better therapies for clear cell ovarian cancers.

A novel method to investigate drug resistance in the chronic lymphocytic leukemia (CLL) microenvironment: Analysis of CLL Cellular Environment and Response (ACCER).

Microenvironments such as lymph nodes allow chronic lymphocytic leukemia (CLL) cells to survive and become drug resistant. There are limited methods to study the to study the contribution of the stromal microenvironment. We have adapted a solid tumor microenvironment cell culture system that provides elements of the CLL microenvironment called Analysis of CLL Cellular Environment and Response (ACCER). We optimized the cell number for patient's primary CLL cells and HS-5 human bone marrow stromal cell line that will give sufficient cell number and viability with the ACCER. We then determined the amount of collagen type 1 to give the best extracellular matrix to seed CLL cells to the membrane. Finally, we determined that ACCER provide CLL cell protection against cell death following treatment with fludarabine and ibrutinib compared to co-culture conditions. This describes novel microenvironment model to investigate factors that promote drug resistance in CLL.

Elevated expression of interleukin 16 in chronic lymphocytic leukemia is associated with disease burden and abnormal immune microenvironment.

Interleukin-16 (IL-16) is a novel biomarker that has been implicated in many cancers as well as inflammatory diseases. In this study, we examined plasma levels of 30 cytokines and chemokines in chronic lymphocytic leukemia (CLL) and monoclonal B cell lymphocytosis (MBL) patients, and examined their association with disease stage, CLL biomarkers and T cell subsets. Interleukin 16 (IL-16) was identified as a relatively uncharacterized cytokine significantly elevated in CLL patients compared to healthy controls and MBL patients. Plasma levels of IL-16 were significantly elevated by Rai stage 0, increased by Rai stage 3-4, correlated strongly with lymphocyte count and were decreased after Ibrutinib treatment. CLL cells expressed IL-16 mRNA and spontaneously secreted IL-16 in vitro. CLL cells express IL-16 mRNA at significantly higher levels in lymphoid tissues than blood, and we observed that IL-16 release was increased in co-cultures of CLL and autologous CD4 + T cells. Elevated plasma IL-16 levels were associated with abnormalities in the immune microenvironment including multiple inflammatory cytokines and chemokines and expansion of type 1 follicular helper T cells. Taken together, our results identify IL-16 as a novel biomarker in CLL with potential functional roles in cellular interactions between CLL cells and T cells.

Clinical Outcomes in a Large Canadian Centralized CLL Clinic Based on Treatment and Molecular Factors over a Decade.

FISH cytogenetics, TP53 sequencing, and IGHV mutational status are increasingly used as prognostic and predictive markers in chronic lymphocytic leukemia (CLL), particularly as components of the CLL International Prognostic Index (CLL-IPI) and in directing therapy with novel agents. However, testing outside of clinical trials is not routinely available in Canada. As a centralized CLL clinic at CancerCare Manitoba, we are the first Canadian province to evaluate clinical outcomes and survivorship over a long period of time, incorporating the impact of molecular testing and the CLL-IPI score. We performed a retrospective analysis on 1315 patients diagnosed between 1960 and 2018, followed over a 12-year period, where 411 patients had molecular testing and 233 patients had a known CLL-IPI score at the time of treatment. Overall, 40.3% (n = 530) of patients received treatment, and 47.5% (n = 252) of patients received multiple lines of therapy. High-risk FISH and CLL-IPI (4-10) were associated with higher mortality (HR 2.03, p = 0.001; HR 2.64, p = 0.002), consistent with other studies. Over time, there was an increase in the use of targeted agents in treated patients. The use of Bruton's tyrosine kinase inhibitors improved survival in patients with unmutated IGHV and/or TP53 aberrations (HR 2.20, p = 0.001). The major cause of death in patients who received treatment was treatment/disease-related (32%, n = 42) and secondary malignancies (57%, n = 53) in those who were treatment-naïve. Our data demonstrate the importance of molecular testing in determining survivorship in CLL and underpinning the likely immune differences in outcomes for those treated for CLL.

Truncated forms of BNIP3 act as dominant negatives inhibiting hypoxia-induced cell death.

BNIP3 (Bcl-2/adenovirus E1B Nineteen Kilodalton Interacting Protein) is a pro-cell death member of the Bcl-2 family of proteins. Its expression is induced by the transcription factor Hypoxia Inducible Factor-1 (HIF-1) under conditions of low oxygen (hypoxia) and is found over expressed in hypoxic regions of many tumors. When over expressed, BNIP3 induces cell death through induction of mitochondrial dysfunction that is dependent on the presence of BNIP3's TM domain. Herein, we have determined that the SkOv3 ovarian cancer cell line expresses a truncated BNIP3 protein, which results in the elimination of the transmembrane domain. Truncation that eliminates all four domains of BNIP3 protein also inhibits hypoxia-induced cell death in SkOv3, HEK293, U251 and MCF-7 cells. Three different mutations in a BNIP3 expression vector that lead to a truncated BNIP3 protein, lacking TM domain only, or lacking CD, BH3, and TM domains resulted in inhibition of hypoxia-induced cell death when transfected into HEK293 cells. We found that truncated BNIP3 failed to associate with the mitochondria and the truncated BNIP3 lacking all four domains can bind to wild type BNIP3. Taken together, truncation of BNIP3 could be a novel mechanism for cancer cells to avoid hypoxia-induced cell death mediated by BNIP3 over expression.