ALBERT

Description: 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-FcγR 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.

Description: Background Cancer associated fibroblasts (CAFs) are one of the major components constituting the tumor microenvironment and known to be deeply involved in the growth and metastasis of solid tumors. We previously reported that CAFs supported survival of primary lymphoma cells via the increased glycolytic metabolism (Aoki et al. Oncotarget 2017). Recent findings indicate that exosomes secreted from tumor cells play an important role of the survival and metastasis. Although the information about exosomes secreted from tumor cells has been accumulated, the role of them secreted from cells constituting the tumor microenvironment has been largely unexplored. Purpose To uncover the role of exosomes in lymphoma microenvironment, we investigated the function of them derived from CAFs those were isolated from primary lymphoma samples. Methods CAFs were successfully isolated from primary lymph node samples of various types of non-Hodgkin lymphoma including follicular lymphoma, diffuse large B-cell lymphoma, angioimmunoblastic T-cell lymphoma, mantle cell lymphoma, and T lymphoblastic lymphoma (N=20), and subsequently exosomes secreted from the representative 4 CAFs were obtained by a standard procedure using ultracentrifugation. Exosomes were confirmed by immunoblotting, electron microscope, and the nano tracking analysis. The functional role of CAFs and exosomes in an interaction between lymphoma cells and its microenvironment were investigated. Written informed consent for the experimental use of patient lymph node samples was obtained, and all experimental procedures were approved by the institutional review board of Nagoya University Hospital. Results We isolated CAFs from different types of non-Hodgkin lymphoma samples, and studied whether the survival of patient lymphoma cells could be supported in co-culture with CAFs. The survival of cells differed depending on CAFs indicating the diversity of the ability of CAFs to support lymphoma cells. Then we analyzed cellular glycolysis and ATP production of lymphoma cells in co-culture with CAFs. As expected, the increase of glycolysis and production of ATP differed among 4 CAFs probably due to the extent of the Warburg effect. Next, we investigated whether CAFs secreted extracellular vesicles into culture supernatant. We found that vesicles with CD9 positive and matched exosomes in size were accumulated in culture media (Figure A, B), and the amount of released exosomes differed among CAFs in line with the ability to support the survival of lymphoma cells. Exosomes displayed survival support of lymphoma cells in a dose-dependent manner(Figure C), and cellular glycolysis and ATP production were increased in the presence of exosomes as well as CAFs, which indicated that the ability of CAFs to support lymphoma cells was, at least in part, elicited by exosomes. While exosomes secreted from CAFs with the strong ability to support lymphoma cells displayed the support of the survival, the survival of lymphoma cells was not observed in the presence of exosomes derived from CAFs with the weak support of lymphoma cells even in higher dose of exosomes, indicating that not only the quantity but also the quality of exosomes could be a determinant of the survival effect. Next, we focused on exosomes secretion-related proteins of nSMase2 and Rab27b to uncover the underlying mechanism of secretion of exosomes. NSMase2 required for exosomes formation and Rab27b involved in the migration of multivesicular body from the pericule to the membrane were strongly expressed in CAFs with higher ability to support lymphoma cells. Using nSMase2- and Rab27b- specific siRNA, the amount of exosomes secretion was reduced resulting in the decreased survival support. Finally, we studied the presence of CAFs or exosomes associated with the drug resistance. As expected, primary lymphoma cells demonstrated resistance to gemcitabine and cytarabine in the presence of CAFs or exosomes, and the resistance was restored in the presence of CAFs transduced with Rab27b-specific siRNA. Conclusion Our results suggest that CAFs and exosomes secreted from them are involved in the survival and drug resistance of patient lymphoma cells and play a pivotal role in the microenvironment of non-Hodgkin lymphoma. Exosomes would be a novel attractive therapeutic target. Disclosures Kiyoi: Sanofi K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Celgene Corporation: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Novartis Pharma K.K.: Research Funding; Astellas Pharma Inc.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria.

Description: Background and Objective Primary mediastinal large B-cell lymphoma (PMBL) accounts for 2 to 4% of non-Hodgkin lymphomas and is characterized by distinct clinical, pathological and genetic features. Although the utility of DA-EPOCH-R without radiotherapy (RT) and a PET-guided RT approach were recently reported, a standard therapy has not yet been established, mostly due to the lack of data from prospective randomized studies. In addition, the prognosis for patients (pts) with relapsed PMBL is not well understood. Therefore, we conducted a multicenter, cooperative retrospective study to evaluate the clinical outcome of pts with PMBL. Patients and Methods We analyzed a total of 345 pts with newly diagnosed PMBL from 65 institutes between May 1986 and September 2012 in Japan. Pts were treated according to each institutional protocol or physicians' decisions. In pts treated with R-CHOP, the role of PET before RT was analyzed. In addition, we analyzed prognostic factors for PMBL pts and constructed a novel prognostic model using data from patients treated with R-CHOP. Results The median age was 32 (range, 17-83) years, and female pts were predominant (58%) among the patient population. Median tumor diameter was 10 cm. Stage I/II, low-risk by IPI, and PS 0/1 were also predominant (68%, 52% and 75%, respectively). The presence of pleural or pericardial effusion, elevated lactate dehydrogenase level and extra-nodal lesions were observed in 46%, 80% and 44% of pts, respectively. With a median follow-up of 48 months in surviving pts, overall survival (OS) and progression-free survival (PFS) at 4 years were 87% and 70%, respectively. The OS and PFS were improved in pts treated with rituximab(R)-containing chemotherapy (n = 267) (4-year OS: 91% vs. 77%, P 〈 0.001; 4-year PFS: 75% vs. 54%, P 〈 0.001, respectively). The OS at 4 years for patients treated with CHOP (n = 44), R-CHOP (n = 187), DA-EPOCH-R (n = 9), second- or third-generation regimens (n = 45; 28 with R and 17 without R), and chemotherapy followed by ASCT (n = 57; 43 with R and 14 without R) were 67%, 90%, 100%, 91% and 92%, respectively (P 〈 0.001). The PFS at 4 years were 40%, 71%, 100%, 83% and 76%, respectively (P 〈 0.001) (Figure 1). Consolidative RT was given to 42% of the patient population. A total of 119 of 187 pts treated with R-CHOP were assessed by PET/CT after the completion of R-CHOP, and 64 pts received consolidative RT after R-CHOP. In pts with negative PET after R-CHOP (n = 84), the OS (100% vs. 100%, P 〉 0.99) and PFS (92% vs. 72%, P = 0.36) at 4 years were similar when comparing pts treated with (n =25) or without RT (n =59). However, in pts with positive PET after R-CHOP (n = 28), both OS (100% vs. 60%, P = 0.010) and PFS (80% vs. 17%, P 〈 0.001) at 4 years were superior in pts who received RT (n = 21) than in pts who did not receive RT (n = 7). A total of 97 pts (28%) relapsed or progressed after first-line therapy. Of these, 67 (19%) and 11 pts (3%) relapsed in the mediastinum and CNS, respectively. Median time from initial diagnosis to relapse or progression was 9 months. Median OS after relapse or progression was 16 months and was higher in patients treated with stem-cell transplantation (SCT) (n = 58; 44 ASCT, 14 allogeneic SCT) than in patients who did not undergo SCT (4-year OS: 67% vs. 31%, P 〈 0.001). The IPI was predictive for OS (P = 0.002) and PFS (P 〈 0.001) in pts with PMBL treated with R-CHOP. Moreover, multivariate analysis showed that the presence of pleural or pericardial effusion was a significant and independent prognostic factor for PFS. We constructed a novel prognostic model (PMBL prognostic index; PMBIPI) and classified pts treated with R-CHOP into three different risk groups using these two factors (the presence of pleural or pericardial effusion, and IPI high/intermediate-risk or high-risk). For 93 pts (51%) classified as the low-risk group (0 factor), OS and PFS at 4 years were 97% and 89%, respectively. For 61 pts (34%) classified as the intermediate-risk group (1 factor), OS and PFS at 4 years were 85% and 59%, respectively. For 27 pts (15%) classified as the high-risk group (2 factors), OS and PFS at 4 years were 72% (P = 0.001) and 44% (P 〈 0.001), respectively (Figure 2). Conclusions The combination of R and chemotherapy improved outcomes for patients with PMBL. In addition, PET could predict the necessity for RT in pts with PMBL treated with R-CHOP. PMBIPI is a promising tool for risk-stratification of pts with PMBL. These findings require further validation in prospective studies. Disclosures: No relevant conflicts of interest to declare.