The anti-CD20 mAb rituximab has substantially improved the clinical outcome of patients with a wide range of B-cell malignancies. However, many patients relapse or fail to respond to rituximab, and thus there is intense investigation into the development of novel anti-CD20 mAbs with improved therapeutic efficacy. Although Fc-Fc␥R interactions appear to underlie much of the therapeutic success with rituximab, certain type II anti-CD20 mAbs efficiently induce programmed cell death (PCD), whereas rituximab-like type I anti-CD20 mAbs do not. Here, we show that the humanized, glycoengineered anti-CD20 mAb GA101 and derivatives harboring non-glycoengineered Fc regions are type II mAb that trigger nonapoptotic PCD in a range of B-lymphoma cell lines and primary B-cell malignancies. We demonstrate that GA101-induced cell death is dependent on actin reorganization, can be abrogated by inhibitors of actin polymerization, and is independent of BCL-2 overexpression and caspase activation. GA101-induced PCD is executed by lysosomes which disperse their contents into the cytoplasm and surrounding environment. Taken together, these findings reveal that GA101 is able to potently elicit actin-dependent, lysosomal cell death, which may potentially lead to improved clearance of B-cell malignancies in vivo. IntroductionThe addition of the anti-CD20 mAb rituximab to chemotherapy has substantially improved the clinical outcome for many patients with a wide range of B-cell malignancies. 1-3 However, despite the unprecedented success of rituximab, a substantial proportion of patients with CD20-positive B-cell malignancies fail to achieve a complete remission or relapse after receiving rituximab-containing immunochemotherapy. 4 These areas of unmet clinical need highlight the requirement to develop improved treatments for these patients. Given both the success with rituximab and the rapid development of mAb engineering technology, there is currently intense investigation into the development of novel anti-CD20 mAbs aimed at improving therapeutic efficacy. Central to this challenge, is an enhanced understanding of the mechanism of action of anti-CD20 mAbs.Anti-CD20 mAbs can activate a range of potential tumor cell killing pathways (reviewed in Lim et al 5 ) including Fc-Fc␥ receptor (Fc␥R) interactions (namely Ab-dependent cellular cytotoxicity [ADCC] and phagocytosis mediated by Fc␥R-expressing immune effector cells such as macrophages and/or NK cells), complement-dependent cytotoxicity (CDC), or the direct induction of programmed cell death (PCD). Although it is well established that Fc-Fc␥R interactions are critical for the in vivo efficacy of anti-CD20 mAbs, 6-8 the role of complement remains disputed as to whether it is beneficial, 9,10 inconsequential, 7,11,12 or even detrimental to anti-CD20 mAb efficacy. 13,14 However, the potential importance of PCD in enhancing anti-CD20 mAb potency remains largely underinvestigated, perhaps because it does not appear to play a major role in the therapeutic efficacy of rituximab. 15 We have c...

Lynch syndrome or hereditary non-polyposis colorectal cancer is caused by mutations of DNA mismatch repair (MMR) genes. The extracolonic tumour spectrum includes endometrial, ovarian, gastric, small bowel, pancreatic, hepatobiliary, brain, and urothelial neoplasms. Families were referred on the basis of clinical criteria. Tumour immunohistochemistry and microsatellite testing were performed. Appropriate patients underwent sequencing of relevant exons of the MMR genes. Proven and obligate mutation carriers and first-degree relatives (FDRs) with a Lynch syndrome spectrum cancer were considered mutation carriers, as were a proportion of untested, unaffected FDRs based on the proportion of unaffected relatives testing positive in each age group. Kaplan-Meier analysis of risk to 70 years was calculated. One hundred and eighty-four Lynch syndrome spectrum extracolonic cancers in 839 proven, obligate, or assumed mutation carriers were analysed. Cumulative risk for females of an extracolonic tumour is 47.4% (95% CI 43.9-50.8). The risk to males is 26.5% (95% CI 22.6-30.4). There was no reduction in gynaecological malignancies due to gynaecological screening (examination, transvaginal ultrasound scan, hysteroscopy and endometrial biopsy). Males have a higher risk of gastric cancer than females (p = 0.0003). Gastric cancer risk in those born after 1935 does not justify surveillance. These penetrance estimates have been corrected for ascertainment bias and are appropriate for those referred to a high-risk clinic.

mAbs are becoming increasingly utilized in the treatment of lymphoid disorders. Although Fc-FcγR interactions are thought to account for much of their therapeutic effect, this does not explain why certain mAb specificities are more potent than others. An additional effector mechanism underlying the action of some mAbs is the direct induction of cell death. Previously, we demonstrated that certain CD20-specific mAbs (which we termed type II mAbs) evoke a nonapoptotic mode of cell death that appears to be linked with the induction of homotypic adhesion. Here, we reveal that peripheral relocalization of actin is critical for the adhesion and cell death induced by both the type II CD20-specific mAb tositumomab and an HLA-DRspecific mAb in both human lymphoma cell lines and primary chronic lymphocytic leukemia cells. The cell death elicited was rapid, nonapoptotic, nonautophagic, and dependent on the integrity of plasma membrane cholesterol and activation of the V-type ATPase. This cytoplasmic cell death involved lysosomes, which swelled and then dispersed their contents, including cathepsin B, into the cytoplasm and surrounding environment. The resulting loss of plasma membrane integrity occurred independently of caspases and was not controlled by Bcl-2. These experiments provide what we believe to be new insights into the mechanisms by which 2 clinically relevant mAbs elicit cell death and show that this homotypic adhesion-related cell death occurs through a lysosome-dependent pathway.Introduction mAbs are becoming increasingly utilized in the treatment of lymphoid disorders (1, 2). In particular, mAb directed to cell-surface antigens on malignant B cells has proven the most clinically effective, with the anti-CD20 mAb, rituximab, being the first to be approved by the US FDA for the treatment of cancer. Rituximab has substantially improved outcome for patients with many different types of non-Hodgkin lymphoma and has now been administered to over 1 million patients in the decade since its approval. Despite such success, treatment is not curative and there is intense preclinical and clinical investigation of many other engineered mAbs directed to both CD20 and a host of other cell-surface antigens (2).

The anti-CD20 monoclonal antibody (mAb) rituximab has revolutionized the treatment of B-cell malignancies. This unprecedented success has not only substantially changed the mindset of the clinical community about the ability of mAb to improve outcomes but has catalyzed the interest in the pharmaceutical industry to develop the next generation of anti-CD20 mAbs. Since the introduction of rituximab 15 years ago, we have learned much about the potential mechanisms underlying the therapeutic efficacy of anti-CD20 mAbs. In parallel, many novel anti-CD20 mAbs have entered the clinic, each designed with modifications to structure aimed at further improving efficacy. On review of the newer generation of anti-CD20 mAbs entering clinical trials, it appears that the link between the novel mechanistic insights and the development of these next-generation anti-CD20 mAbs is unclear. As we move into an era of personalized medicine, it will become increasingly important for us to develop closer links between the emerging mechanistic insights and the clinical development, to further enhance the potency of anti-CD20 mAbs beyond that achieved with rituximab. (Blood. 2011;117(11): 2993-3001) IntroductionThe advent of monoclonal antibody (mAb) technology after the Nobel Prize-winning scientific contribution of Kohler and Milstein 1 led to a great expectation that mAbs would provide effective targeted therapy for cancer. After early promise came a period of despondency in the late 1980s and early 1990s with largely disappointing early phase clinical trial results, with the notable exception of anti-idiotype antibodies in follicular lymphoma (FL). 2,3 More than 20 years elapsed before mAbs began to fulfill their early promise as effective anticancer therapeutics, and many lessons were learned from these clinical trial failures. When the first successes were seen in hematologic malignances, the importance of the antigen target specificity and developing "humanized" mAbs was recognized. In hematologic malignancies, the major success of mAb therapy to date has been seen with anti-CD20 mAbs. Although the first B cell-specific antibody B1 (renamed tositumomab), which targets the B cell-specific antigen that we now know as CD20, was discovered as long ago as 1981, 4 it was not until 1997 that the anti-CD20 rituximab became the first mAb to be approved by the U.S. Food and Drug Administration (FDA) for use in relapsed indolent lymphoma. 5 mAbs are now routinely delivered in a wide range of hematologic malignancies, and rituximab is widely accepted to be the single most important factor leading to improved outcome in a range of B-cell lymphomas 6-10 and more recently in B-cell chronic lymphocytic leukemia (B-CLL). 11,12 As we reflect on the development of rituximab in the late 1990s, there were considerable challenges and uncertainties for those early pioneers to overcome in integrating rituximab into treatment protocols. Given those difficulties, much of what has now become standard practice was the result of serendipity and pragmatism ...

Monoclonal antibodies (mAbs) have revolutionized the treatment of B-cell malignancies. Although Fc-dependent mechanisms of mAb-mediated tumor clearance have been extensively studied, the ability of mAbs to directly evoke programmed cell death (PCD) in the target cell and the underlying mechanisms involved remain under-investigated. We recently demonstrated that certain mAbs (type II anti-CD20 and anti-HLA DR mAbs) potently evoked PCD through an actin-dependent, lysosome-mediated process. Here, we reveal that the induction of PCD by these mAbs, including the type II anti-CD20 mAb GA101 (obinutuzumab), directly correlates with their ability to produce reactive oxygen species (ROS) in human Blymphoma cell lines and primary B-cell chronic lymphocytic leukemia cells. ROS scavengers abrogated mAb-induced PCD indicating that ROS are required for the execution of cell death. ROS were generated downstream of mAb-induced actin cytoskeletal reorganization and lysosome membrane permeabilization. ROS production was independent of mitochondria and unaffected by BCL-2 overexpression. IntroductionMonoclonal antibodies (mAbs) have revolutionized the treatment of cancer. The first mAb approved for this purpose, rituximab, which targets the CD20 antigen on B-lymphocytes, has substantially improved the clinical outcome of patients with B-cell malignancies in combination with chemotherapy. 1,2 However, a substantial proportion of patients still relapse and acquire resistance to rituximab. 3 In an attempt to further improve therapeutic outcomes and develop novel therapies for rituximabrefractory patients, many next-generation mAbs directed against CD20 or various other cell surface antigens have been developed by the pharmaceutical industry. An enhanced understanding of the mechanisms underlying mAb-induced tumor clearance is therefore pivotal for improving the therapeutic performance of mAbs.In addition to classic Fc-dependent mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), antibodydependent cellular phagocytosis (ADCP), and complementdependent cytotoxicity (CDC), certain mAbs can eliminate B cells by triggering intracellular signaling on antigen ligation to directly induce programmed cell death (PCD). Although Fc-dependent mechanisms of mAb-induced tumor clearance have been extensively studied, and many novel mAbs with optimized Fc properties are currently being developed, the role of direct PCD and its underlying mechanisms remain largely under-investigated. A significant advantage of exploiting the direct induction of PCD to augment mAb efficacy is that, unlike Fc-dependent mechanisms, it does not rely on host immune effector mechanisms which are amenable to saturation or exhaustion, such as in patients with high tumor burden, or immune effector cell failure secondary to depletion by chemotherapy regimes. 4 Further investigation of the mechanism of mAb-induced PCD therefore provides the potential for improved therapeutic efficacy through the development of optimized next-generation mAbs and novel, mechanism-b...

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant condition caused by inactivating mutations of DNA mismatch repair (MMR) genes. An accurate estimation of colorectal cancer risk for mutation carriers is essential for counselling and rationalizing screening programmes. Families were referred on the basis of clinical criteria. Tumour immunohistochemistry and microsatellite testing were performed. Appropriate patients underwent sequencing of all relevant exons of the MMR genes. Proven and obligate mutation carriers and first-degree relatives (FDRs) with an HNPCC spectrum cancer were considered mutation carriers, as were a proportion of untested, unaffected FDRs based on the proportion of unaffected relatives testing positive in each age group. The cumulative lifetime risk was calculated by Kaplan-Meier analysis. Three hundred and forty-one colorectal cancers in 839 proven, obligate, or assumed mutation carriers were analysed. The cumulative risk to age 70 years for all mutation carriers combined was 50.4% (95% CI 47.8-52.9). The cumulative risk in males was 54.3% (95% CI 50.7-57.8), which was significantly higher than in females (log rank p = 0.02) who had a risk of 46.3% (95% CI 42.8-49.9). These penetrance estimates from HNPCC families attending high-risk clinics have been corrected for ascertainment bias and are appropriate risks for those referred to a high-risk clinic. Current colonoscopic screening guidelines are appropriate.

Molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) underlies the variable outcomes achieved with immunochemotherapy. However, outcomes of gene expression profiling (GEP)-defined molecular subgroups in a real-world DLBCL population remain unknown. Here we examined the prevalence and outcomes of molecular subgroups in an unselected population of 1149 patients with de novo DLBCL in British Columbia, Canada. Evaluable biopsies were profiled by fluorescence in situ hybridization (FISH), immunohistochemistry and digital GEP to assign cell-of-origin (COO) and the so-called "double-hit signature" (DHITsig) - a signature originally described as being characteristic for high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). DHITsig was expressed in 21% of 431 germinal center B-cell-like (GCB)-DLBCL and all 55 Burkitt lymphomas examined. Reflecting this latter finding, DHITsig has been renamed the "dark zone signature" (DZsig). DZsigpos-DLBCL, non-DZsigpos GCB-DLBCL and activated B-cell-like (ABC)-DLBCL were associated with a 2-year overall survival of 57%, 89% and 71%, respectively. 62% of DZsigpos tumors were negative for HGBCL-DH-BCL2 by FISH, but were associated with outcomes similar to HGBCL-DH-BCL2. A small group of HGBCL-DH-BCL2 that lacked DZsig expression had different molecular features compared to DZsig-expressing HGBCL-DH-BCL2, and were associated with favorable outcomes comparable DLBCL, not otherwise specified. DZsigpos and ABC-DLBCL had shorter diagnosis-to-treatment interval (DTI) than GCB-DLBCL, with this metric being associated with outcome. In conclusion, DZsig expression extends beyond HGBCL-DH-BCL2 and captures a poor-prognosis DLBCL subgroup with short DTI, including patients unidentifiable by routine FISH testing, that should be considered for treatment intensification or novel therapies in prospective trials.

Background ABO blood groups have been linked to susceptibility to infection with certain microorganisms, including coronaviruses. We examined the relationship between blood group and clinical outcomes in individuals infected with severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) and compared their blood group distribution with the general population. Methods At the inception of the pandemic, all individuals testing positive for SARS‐CoV‐2 in Kuwait were admitted to one designated coronavirus disease 2019 (COVID‐19) hospital and enrolled in a prospective registry. Patients admitted from February 24 to May 27, 2020, were stratified according to blood group. As a control, blood groups of 3,730,027 anonymized individuals representing almost Kuwait's entire population were obtained from a national database. Results Of 3305 SARS‐CoV‐2–positive patients, 37.1%, 25.5%, 28.9%, and 8.5% were groups O, A, B, and AB, respectively. Univariate analysis revealed no significant differences in severe clinical outcomes or death among the blood groups. However, multivariable analysis demonstrated that group A individuals had higher odds of developing pneumonia compared with non–group A (adjusted odds ratio 1.32, 95% confidence interval 1.02–1.72, p < .036). Compared with the general population, the COVID‐19 cohort had a lower frequency of group O, equivalent frequency of A, and higher frequency of B and AB. No significant difference in the RhD group was found. Conclusion This study supports potential involvement of the ABO blood group system in predisposing to infection with SARS‐CoV‐2 in an unselected population. Examination of the mechanistic link between blood group and COVID‐19 and its implications on controlling the current pandemic is warranted.