The Downregulation of PTGS2 Mediated by ncRNAs is Tightly Correlated with Systemic Sclerosis-Interstitial Lung Disease

Background: Interstitial lung disease in systemic sclerosis (SSc-ILD) is one of the most severe complications of systemic sclerosis (SSc) and is the main cause of mortality. In this study, we aimed to explore the key genes in SSc-ILD and analyze the relationship between key genes and immune cell infiltration as well as the key genes relevant to the hallmarks of cancer.Methods: Weighted gene co-expression network analysis (WGCNA) algorithm was implemented to explore hub genes in SSc-ILD samples from the Gene Expression Omnibus (GEO) database. Logistic regression analysis was performed to screen and verify the key gene related to SSc-ILD. CIBERSORT algorithms were utilized to analyze immune cell infiltration. Moreover, the correlation between the key genes and genes relevant to cancer was also evaluated. Furthermore, non-coding RNAs (ncRNAs) linking to PTGS2 were also explored.Results: In this study, we first performed WGCNA analysis for three GEO databases to find the potential hub genes in SSc-ILD. Subsequently, we determined PTGS2 was the key gene in SSC-ILD. Furthermore, in CIBERSORT analyses, PTGS2 were tightly correlated with immune cells such as regulatory T cells (Tregs) and was negatively correlated with CD20 expression. Moreover, PTGS2 was associated with tumor growth. Then, MALAT1, NEAT1, NORAD, XIST identified might be the most potential upstream lncRNAs, and LIMS1 and RANBP2 might be the two most potential upstream circRNAs.Conclusion: Collectively, our findings elucidated that ncRNAs-mediated downregulation of PTGS2, as a key gene in SSc-ILD, was positively related to the occurrence of SSc-ILD and abnormal immunocyte infiltration. It could be a promising factor for SSc-ILD progression to malignancy.

IKZF1deletions Coupled with CD20 Expression Represents a Novel High-Risk Subtype in Adult B-Cell Progenitor Acute Lymphoblastic Leukemia

Genetic deletions of IKZF1 are associated with poor prognosis in B-cell acute lymphoblastic leukemia (B-ALL). Here we investigated the effect of IKZF1 deletions (IKZF1 del) plus with immunotype in adult B-ALL in PDT-ALL-2016 cohort. This cohort study involved 161 patients with B-ALL from 2016 to 2019, with detailed information about IKZF1 del and CD20 expression. Validation cohort consists N= patients from TARGET cohort. IKZF1 del was detected in 36.0% of patients with 3-year event-free survival (EFS) of 37.2±6.7% and overall survival (OS) of 51.1±7.3%, compared to IKZF1 wild-type (IKZF1 wt) with EFS 55.4±5.1% (P<0.01) and OS 74.6±4.5% (P<0.05), respectively. CD20 expression was also associated with inferior EFS than CD20-negative group (P<0.05). Furthermore, IKZF1 del coupled with CD20 expression, termed as IKZF1 del/CD20+, comprised 12.4% of patients with 3-year EFS of 25.0±9.7% compared with IKZF1 wt (P<0.05 ) and IKZF1 del/CD20- (P<0.05 ) groups, respectively. Multivariable analyses demonstrated independence of IKZF1 del/CD20+ with highest hazard ratio for EFS and OS. Furthermore, the prognostic strength of IKZF1 del/CD20+ was confirmed in TARGET validation cohort. Eighty-one patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT). Notably, neither IKZF1 del(P=0.6288), CD20 (P=0.0705) or IKZF1 del/CD20 (P=0.3410) groups were identified as poor outcome in allo-HSCT cohort. Collectively, our data demonstrate that IKZF1 del/CD20+ represents a very high-risk subtype in adult B-ALL; and particularly, allo-HSCT could overcome the poor outcome of IKZF1 del and IKZF1 del/CD20+. Disclosures No relevant conflicts of interest to declare.

Lw-213 Synergizes with Rituximab to Inhibit Diffuse Large B-Cell Lymphomas By Upregulating CD20

Background：Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens.Up to one-third of diffuse large B cell lymphoma (DLBCL) patients eventually develop resistance to R-CHOP regimen, since the remaining therapeutic options are limited. LW-213, a derivative of wogonin, is reported to possess antineoplastic properties in a variety of cancers, but whether it has effects on DLBCL is n-ot known. Studies have reported that upregulation of CD20 expression b-y either HDACi or silenced SOX2 expression showed sensitizing potential in Rituximab-induced cell death in malignant B cells. Our study was to explore whether LW-213 could sensitize DLBCL to Rixutimab thus improve therapeutic efficacy. Methods： Two DLBCL cell lines, RI-1 (ABC subtype) and Su-DHL -8 (GCB subtype), were used in our study. RI-1 and Su-DHL-8 cells were treated with LW-213 at different doses and for different times, and their proliferation and viability were detected by Cell counting kit-8 (CCK8).Flow cytometry was used to determine surface CD20 expression. Western blotting and q-PCR were applied to examine the protein and mRNA levels of CD20, SOX2, Ace-H3 and Ace-H3K27. CDC assay was used to evaluate the synergistic effects of LW-213 and Rixutimab. Results：We showed that LW-213 inhibited the proliferation of human DLBCL cell lines (Su-DHL-8、RI-1 ) in dose-and time-dependent manners with IC 50 values at the low μmol/L levels, meanwhile it potently inhibited primary lymphoma cells derived from peripheral blood of B-cel-l lymphoma patients. Furthermore, LW-213 significantly increased CD20 surface expression and the acetylation level of histone in DLBCL cell li-nes. Inversely，the SOX2 expression level remarkably decreased. Finally,Combination with LW-213 significantly synergized Rituximab-induced cell death in vitro. Conclusion： The results demonstrate that LW-213 sensitizes DLBCL cells to Rituximab in vitro by upregulating CD20 expression and the SOX2/ace-H3K27/ace-H3 axis may plays a critical role in CD20 upregulation processing. Even though this strategy is important in vitro models,the upregulating CD20 expression therapy against DLBCL proposed in this study warrants further study in vivo and clinical trials . Keywords：CD20 DLBCL Rituximab SOX2 Histone Deacetylation Disclosures No relevant conflicts of interest to declare.

PDK4-Mediated Metabolic Reprogramming Promotes Rituximab Resistance in Diffuse Large B-Cell Lymphoma Via Negative Regulation of MS4A1/CD20

Background: Diffuse large B cell lymphoma (DLBCL) heterogeneity promotes the recurrence and anti-CD20-based therapeutic resistance. In previous studies, it has been demonstrated that the downregulation of MS4A1/CD20 expression after chemoimmunotherapy with rituximab can lead to rituximab resistance. However, the the mechanisms of CD20-loss remains unknown. Methods: The expression levels of PDK4 were investigated in DLBCL patients and cell lines by RNA-seq, qRT-PCR, western blotting and immunofluorescence analysis. Lentiviral infection was used to regulate the level of PDK4 in DLBCL cells. The effects of PDK4 on apoptosis, drug sensitivity and proliferation of DLBCL cells were evaluated by flow cytometry and cell-counting kit-8 (CCK-8) assay, as well as being assessed in a murine model. Cell metabolism was conducted by measurement of glucose consumption, lactate production, ATP levels, ECAR and OCR with corresponding assay kit. Results: Our data showed that PDK4 expression levels elevated significantly in DLBCL cells derived from both the patients and cell lines with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone regimen) resistant. We further found that the overexpression of PDK4 in DLBCL cells can lead to cell proliferation and rituximab resistance both in vitro and in vivo. Furthermore, the loss of PDK4 expression or treatment with the PDK4 inhibitor dichloroacetate is effective in increasing the rituximab-induced cell apoptosis in DLBCL cells. According to the mechanism studies, PDK4 mediated a metabolic shift that the main energy source was changed from OXPHOS to glycolysis. More importantly, with the knockdown or overexpression of PDK4 in DLBCL cells leads to a reverse MS4A1/CD20 expression. Conclusion: Our data identify a metabolic reprogramming role of PDK4 in rituximab resistance of DLBCL and highlight the unique function of PDK4 as an attractive therapeutic target.

Cluster of Differentiation 20 Expression and Rituximab Use Do Not Improve Outcome of Classical Hodgkin Lymphoma Patients

Introduction: Cluster of differentiation 20 (CD20) is expressed in Reed Sternberg (RS) cells of 11-35% of classical Hodgkin lymphoma (cHL) cases with very controversial prognostic significance. Rituximab is not considered part of standard therapy in cHL but has shown promising responses in the relapsed setting. Early metabolic response has emerged as a robust prognostic tool for early and advanced stage cHL. This analysis reports the prognostic significance of CD20 expression and the therapeutic effect of rituximab in 310 previously untreated cHL patients. Methods: After due approval, all newly diagnosed cHL cases were retrospectively identified. CD20 immunehistochemical (IHC) stain was applied to all cases and considered positive when 10% of the RS cells showed expression. Overall survival (OS) and progression free survival (PFS) were computed using Kaplan-Meir method with log ranks test. Multivariate analyses for PFS were computed using Cox regression modeling incorporating variables with a p value ≤ 0.15 from the univariable model in addition to CD20 expression and rituximab use. Results: A: General characteristics Among 310 patients, 171 (55%) were males, the median age was 27 (14-89) years. Stage of disease was advanced (including early unfavorable) in 278 (90%), 214 (70%) had constitutional symptoms and 58 (19%) had bulky disease. The most common subtype was cHL-nodular sclerosis (NS) at 185 (60%). CD20 was positive in 66 (22%) of the cases. Majority of patients received ABVD 234 (76%) and out of the 66 patients with CD20 expression, 27 (41 %) received rituximab with front line chemotherapy. B: Characteristics and outcome based on CD20 expression Stratified by CD20 expression, there was no significant difference in terms of age, gender, stage at presentation, presence of constitutional symptoms or bulky disease between CD20+ and CD20- cases. NS subtype was more common among CD20- cases (p = 0.0001). More patients were able to achieve interim complete metabolic response (CMR) among CD20+ compared to CD20- at 85% vs. 69%, respectively (p = 0.032); this is further detailed in table I. After a median follow up of 51.3 (1.3-213) months, the 3-year PFS was 75.1% for CD20+ and 70% for CD20- (p = 0.36); this is further illustrated in figure I. C: Impact of rituximab therapy on CD20 positive cHL Stratifying CD20+ cases by rituximab therapy, there was no significant difference in terms of age, gender, stage at presentation, presence of constitutional symptoms or bulky disease. First line chemotherapy used was comparable; however more patients received involved field radiotherapy (IFRT) in the no rituximab group (p = 0.05). The addition of rituximab to chemotherapy did not affect the result of interim PET/CT (p = 0.84). Among the CD20+ cases, 27 (41 %) received rituximab with their first line chemotherapy. After a median follow up of 42.6 (1.3-188) months for CD20+ and 55.2 (2.2-213) months for the CD20- group, the 3-year PFS was 75.1% for CD20+ and 70% for CD20-; the 3-year OS was 89.2% for CD20+and 92.8% for CD20- (p = 0.36). After a median follow up of 36 (6.8-116) months for rituximab group and 55.8 (1.3-188) months for the no rituximab group, the 3-year PFS was 72.6% and 67.8% (p = 0.23) while the 3-year OS was 92.6% and 86.6% (p = 0.47)), respectively. This is further illustrated in figure I. On multivariate analysis the presence of constitutional symptoms and interim PET/CT positivity was significantly associated with worse outcome at HR 3.2 (1.14-9.01; p = 0.028) and 4.3 (2.27-8.1; p < 0.0001) respectively. Conclusions: We observed that neither CD20 expression nor nor rituximab use has improved the outcome of cHL patients. The presence of constitutional symptoms and interim PET/CT positivity were associated with worse outcome. A large prospective study is needed to confirm these findings. ptoms and interim PET/CT positivity are associated with worse outcome. A large prospective study is needed to confirm these findings. Figure Disclosures No relevant conflicts of interest to declare.

Obinutuzumab-Retreatment in Combination with Bendamustine Is Effective for Obinutuzumab Resistant Models in Vitro and In Vivo

Background: Follicular lymphoma (FL) commonly recurs and is difficult to cure. Obinutuzumab (OBI) is a humanized type II anti-CD20 antibody. It's mode of action is mainly characterized by the induction of direct cell death and it shows stronger antibody-dependent cellular cytotoxicity (ADCC) than rituximab. While OBI is indicated for previously untreated or relapsed/refractory (r/r) FL, there is no evidence on the efficacy of retreatment with OBI in r/r FL after prior OBI-containing therapy. To demonstrate the effectiveness of OBI-retreatment in a non-clinical study, we established in vitro and in vivo OBI resistant models, consisting of human non-Hodgkin lymphoma (NHL) cells resistant to OBI-induced ADCC and OBI-resistant tumors established from NHL cells under xenotransplantation conditions, and investigated the combination efficacy of OBI and bendamustine (Benda) in these OBI-resistant models. Methods: OBI-induced ADCC resistant clones were established from an RL cell line by inducing ADCC three times with CD16-transfected NK-92 cells. The in vivo resistant model was established from a SU-DHL-4 xenograft model through repeated treatment with OBI and re-inoculation of the regrown tumors. CD20 expression was assessed by flow cytometry or immunohistochemistry (IHC). ADCC activity was evaluated using calcein-AM with CD16-transfected NK-92 cells. To examine antitumor activity, OBI, human immunoglobulin G (HuIgG), Benda, or vehicle were intravenously administered on Day 1, 8 and 15 (OBI and HuIgG) or Day 1 and 2 (Benda and vehicle), and tumor volume was measured. Intratumorally infiltrated NK cells were assessed by IHC of CD335. Cell surface CD107a expression was assessed by flow cytometry. Results: OBI-induced ADCC resistant clones derived from RL (RL-OR-8 and RL-OR-22) showed a reduction in ADCC sensitivity compared with RL. These resistant clones exhibited CD20 expression similar to RL. Pretreatment of the effector NK cells with Benda enhanced ADCC induction of OBI in RL-OR-8 and RL-OR-22. Treatment with OBI (30 mg/kg) in combination with Benda (13.3 mg/kg) also significantly increased antitumor activity compared with each single agent alone on Day 18 (when tumor volume reached euthanasia criteria (*) in control and OBI monotherapy groups) in the RL-OR-22 xenograft model (Table 1). An in vivo OBI resistant model derived from SU-DHL-4 xenografts (SU-DHL-4-OR-18-8) was also established. While SU-DHL-4 xenografted tumors disappeared in 6/6 mice after the third OBI treatment (6 mg/kg), SU-DHL-4-OR-18-8 tumors did not regress in 6/6 mice. CD20 expression in SU-DHL-4-OR-18-8 tumors did not decrease compared with SU-DHL-4 tumors. The ratio of CD335-positive cells to tumor cells after 6 mg/kg of OBI treatment (Day 4) in SU-DHL-4-OR-18-8 was significantly decreased compared with the ratio in SU-DHL-4 (0.31 ± 0.12% vs 0.93 ± 0.23%). The combination efficacy of OBI and Benda was also assessed in an SU-DHL-4-OR-18-8 xenograft model. OBI (6 mg/kg) in combination with Benda (25 mg/kg) significantly increased the antitumor activity compared with each single agent alone on Day 22 (*in control group) and 29 (Table 1). Finally, the expression of CD107a, an NK cell-degranulation marker, was detected to examine the effect of Benda on NK activity in vitro. Pretreating effector NK cells with Benda upregulated CD107a expression on NK cell surfaces after OBI treatment in RL-OR-8 and RL-OR-22. Conclusions: The decreased CD335-positive cell ratio observed in the in vivo SU-DHL-4-OR-18-8 resistant model suggests that the mechanism of resistance to OBI also involves the attenuation of ADCC as in the in vitro OBI-induced ADCC resistant clones. The combination treatment of OBI and Benda was effective in both RL-OR-22 and SU-DHL-4-OR-18-8 xenograft models. It is possible that activation of NK cells by Benda might be involved in this combination mechanism. Although the mechanisms need to be examined in more detail, these results indicate the possible effectiveness of OBI-retreatment after prior OBI-containing therapy. Disclosures Yamashita-Kashima: Nippon Shinyaku Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Current Employment. Kawasaki:Chugai Pharmaceutical Co., Ltd.: Current Employment; Nippon Shinyaku Co., Ltd.: Research Funding. Yorozu:Chugai Pharmaceutical Co., Ltd.: Current Employment; Nippon Shinyaku Co., Ltd.: Research Funding. Yoshiura:Chugai Pharmaceutical Co., Ltd.: Current Employment; Nippon Shinyaku Co., Ltd.: Research Funding. Fujimura:Chugai Pharmaceutical Co., Ltd.: Current Employment; Nippon Shinyaku Co., Ltd.: Research Funding. Kurasawa:Chugai Pharmaceutical Co., Ltd.: Current Employment; Nippon Shinyaku Co., Ltd.: Research Funding. Harada:Chugai Pharmaceutical Co., Ltd.: Current Employment; Nippon Shinyaku Co., Ltd.: Research Funding. Yoshimura:Nippon Shinyaku Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Current Employment.

525 KIT mutation with a low MS4A1/CD20 expression is associated with poor prognosis in melanoma

BackgroundMelanoma has high response rate to immune checkpoint inhibitors. KIT, a driver mutation in melanoma seen in ~10% of the patients.1 However, the role of the KIT mutation in immune microenvironment of melanoma is not well established yet. Here we report a case with KIT mutation and a likely impaired B cell activity with poor response to Immune-checkpoint inhibitor therapy (ICI). We also describe the overall survival of melanoma depending on KIT mutation and MS4A1/CD20 expression, which encodes CD20, B-lymphocyte-specific membrane protein that plays a role in the development, differentiation, and activation of B-lymphocytes.2MethodsA case with poor response to ICI with KIT mutation and monoclonal B cell lymphocytosis was identified. Clinical and molecular characteristics of melanoma in TCGA was analyzed using cBioPortal web page. TCGA data were analyzed to determine KIT mutation status and MS4A1/CD20 expression in melanoma cohort. Samples in the upper 33 percentile of MS4A1 expression were identified as high expression, and the lower 33 percentile were identified as low expression. Mantel-Cox method was used for overall survival (OS) comparison between the cohorts.Results69-year-old male with initial diagnosis of stage III-B melanoma of the left thumb with local recurrence in the resection site and then lung metastases. Patient was then started on nivolumab/ipilimumab with rapid progression on immunotherapy. He was found to have KIT mutation (exon 13K642EMT), and started on imatinib, but he continued to have progression. He was switched to temozolomide with no response. He also had history of leukopenia, pre-dating the metastatic melanoma and was diagnosed with monoclonal B cell lymphocytosis. With the hypothesis that the patient‘s dysfunctional B cells may have impaired ability of ICI and poor prognosis; we analyzed TCGA database for KIT mutation and MS4A1/CD20 expression- which was used as marker for B cell activity. KIT mutation was seen in 10 of 147 patients with high MS4A1/CD20 expression, and 10 of 135 patients with low MS4A1/CD20 expression. Overall survival was 15 months for the patients with KIT mutation and low MS4A1/CD20 expression, and significantly lower when compared with other groups despite low number of patients. (P<0.0001) (figure 1).Abstract 525 Figure 1KIT mutation and MS41A/CD20 expression - overall survivalLow MS41A/CD20 expression with concurrent KIT mutation is associated with poor overall survivalConclusionsB cells have significant role in immune response to tumor. Lower expression of MS4A1/CD20 is known to be associated with poor prognosis in melanoma and other solid tumors.3 We demonstrated that a concurrent KIT mutation in melanoma with lower expression of MS4A1/CD20 contributes to poor prognosis in melanoma. Therefore, this small subset of aggressive tumors may need combination strategies involving targeting driver pathways with a kinase and immune checkpoint inhibitor.Referencesde Mendonça UB, Cernea CR, Matos LL, de Araujo Lima RR. Analysis of KIT gene mutations in patients with melanoma of the head and neck mucosa: a retrospective clinical report. Oncotarget 2018 May 1;9(33):22886.Tedder TF, Boyd AW, Freedman AS, Nadler LM, Schlossman S. The B cell surface molecule B1 is functionally linked with B cell activation and differentiation. The Journal of Immunology 1985 Aug 1;135(2):973–9.Liu Y, Wang L, Lo KW, Lui VW. Omics-wide quantitative B-cell infiltration analyses identify GPR18 for human cancer prognosis with superiority over CD20. Communications biology 2020 May 12;3(1):1–1.