Journal articles: 'Kosciuszko House (Philadelphia, Pa.)' – Grafiati (2024)

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Abstract Background: RNF43, a single transmembrane circular E3 ubiquitin ligase, has been identified as a tumor suppressor for a variety of cancers, including ovarian cancer, gastric cancer, pancreatic cancer, endometrial cancer and colorectal cancer. It is even considered to be carcinogenic driver mutations in cholangiocarcinoma, ovarian cancer, gastric cancer, endometrial cancer and colorectal cancer (CRC). However, the correlation between RNF43 mutation and CRC immunotherapy has not been reported yet. Methods: We evaluated the role of RNF43 in CRC using publicly available data from The Cancer Genome Atlas (TCGA) and Memorial Sloan Kettering Cancer Center (MSKCC). An in-house Chinese CRC cohort (n=2290) was used for the analysis of immune-related markers. The relationship between clinical pathologic features and RNF43 were analyzed with using the two-sided chi-squared test or the Fisher exact test. Clinicopathologic characteristics associated with overall survival using Cox regression and the Kaplan-Meier method. Results: In the MSKCC cohort, a total of 108 CRC patients receiving immunotherapy were enrolled, of which 38 (34.9%) were over 60-year-old and 61 (56.0%) were male. RNF43 mutation was significantly associated with high TMB and high MSI score (all p <0.05). Besides these, RNF43 mutation was discovered to be enriched in MSI instable. Kaplan-Meier survival analysis showed that patients with RNF43 mutation obtained better OS compared to RNF43 wild-type (not reach vs. 13 months, HR, 0.12; 95% CI 0.03-0.49; p=0.0034). In the TCGA cohort, CRC patients of stage I-IV were 16.6%, 38.5%, 28.2% and 14.2%, respectively. All of these patients received radiation therapy. No association was observed between RNF43 and OS (HR, 1.83; 95% CI 0.66-5.07; p=0.2479). In the in-house Chinese CRC cohorts, 210 (9.2%) patients carried RNF 43 mutations, of which 123 patients (58.6%) were male. RNF43 mutations were significantly associated with higher TMB levels (p<0.0001) and PD-L1 TPS> 1% (p=0.0110) in the in-house Chinese RCC cohorts. Citation Format: Changjun Yu, Weibiao Kang, Yu Yuan, Junling Zhang, Mengli Huang, Yanan Chen, Xihua Xia, Yuezong Bai. RNF43 mutation as a predictor of immunotherapeutic efficacy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5089.

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Abstract Hematopoietic progenitor kinase 1 (HPK1), a hematopoietic cell-restricted serine/threonine protein kinase, has been reported to serve as a critical negative feedback regulator of T lymphocytes by phosphorylating the adaptor protein SLP76 in the TCR complex. The negative feedback role of HPK1 in TCR signaling makes it a promising target for immuno-oncology therapy. It has been recently demonstrated that the kinase activity of HPK1 is essential for its antitumor activity, and HPK1 inhibitors can be potentially combined with immune checkpoint inhibitor therapy (such as PD-1 antibody) for effective cancer treatment. Here, we report a potent and selective HPK1 inhibitor BGB-15025 which has been identified through in-house high throughput screening and structure-guided drug design. BGB-15025 potently inhibits HPK1 kinase activity in biochemical assay, with a 50% inhibitory concentration (IC50) of 1.04 nM under Km concentration of ATP. Consistently, BGB-15025 can potently reduce SLP76 phosphorylation and increase downstream ERK phosphorylation in a concentration dependent manner in T cells-based assay. As a consequence, BGB-15025 induces TCR activation and IL-2 production in T cells. Based on in-house profiling, BGB-15025 shows good selectivity profile among MAP4K family and does not affect ZAP70 phosphorylation up to 1 μM. Oral administration of BGB-15025 demonstrates dose-dependent pSLP76 inhibition in splenic T cells and induces serum IL-2 in mouse model. In efficacy studies, BGB-15025 exhibits anti-tumor activity in GL261 tumor model as single agent. Moreover, BGB-15025 demonstrated combination effect with anti-PD-1 antibody in CT26 and EMT-6 syngeneic tumor models. BGB-15025 is currently in phase I clinical trial to treat patients with advanced solid tumor, both as a single agent and in combination with anti PD-1 antibody tislelizumab. Citation Format: Ye Liu, Jing Li, Zhuo Li, Qin Wang, Xing Zhou, Xiaoxin Liu, Bo Zhang, Xitao Wang, Sanjia Xu, Hanzi Sun, Xiaomin Song, Xi Yuan, Zhiwei Wang, Xuesong Liu. BGB-15025, a potent and selective HPK1 inhibitor, is efficacious as a single agent or in combination with PD-1 antibody in multiple tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5541.

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Abstract Background: The von Hippel-Lindau (VHL) gene is a tumor suppressor gene. Although VHL gene alterations play a key role in the pathogenesis of renal cell carcinoma (RCC), and several therapies targeting this molecular pathway have been studied, such as sunitinib, pazopanib and axitinib. However, it is not clear whether VHL mutation is a prognostic factor for immunotherapy in renal cell carcinoma. Methods: The impact of VHL mutations on survival outcomes in RCC patients received immunotherapy or non-immunotherapy were verified in the Memorial Sloan Kettering Cancer Center (MSKCC) trials and The Cancer Genome Atlas (TCGA) RCC cohort, respectively. An in-house Chinese RCC cohort (n=950) was used for the analysis of immune-related markers. The relationship between clinical pathologic features and VHL were analyzed with using the two-sided chi-squared test or the Fisher exact test. Clinicopathologic characteristics associated with overall survival using Cox regression and the Kaplan-Meier method. Results: In the MSKCC cohort, a total of 143 RCC patients receiving immunotherapy were enrolled, of which 117 (81.8%) were clear cell RCC and 143 (100%) microsatellite-stable RCC. VHL mutation was significantly associated with high TMB score (P =0.017). Kaplan-Meier survival analysis showed that patients with VHL mutation obtained better OS compared to VHL wild-type (50 months vs. 23 months, HR, 0.36; 95% CI 0.21-0.64; P<0.001). In the TCGA cohort, all RCC patients received non- immunotherapy. No association was observed between VHL and OS (HR, 1.00; 95% CI 0.73-1.38; P=0.981). VHL mutations were significantly associated with higher TMB levels (P=0.006) and MSI-H (P=0.004) in the in-house Chinese RCC cohorts. Conclusions: Our results suggest that VHL mutation may be associated with better OS in RCC patients receiving immunotherapy. The exact mechanisms underlying VHL are needed to be further evaluated. Citation Format: Dong Shen, Yufeng Huang, Zhiwei Wu, Junling Zhang, Mengli Huang, Yanan Chen, Xihua Xia, Yuezong Bai. Prognosis of renal cell carcinoma patients with VHL mutations to immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5090.

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Abstract Background: NOTCH, as an oncogene, has been shown to be closely associated with radiation and chemotherapy, hormone therapy, and other molecular targeted therapies (such as anti-EGFR and anti-PI3K molecular targeted therapies) in a variety of tumors, and even in drug resistance mechanisms. However, the correlation between NOTCH mutations and immunotherapy in colorectal cancer (CRC) has not been reported. Methods: We retrospectively identified NOTCH mutation and treatment outcomes. The relationship between clinical pathologic features and NOTCH were analyzed with using the two-sided chi-squared test or the Fisher exact test. Clinicopathologic characteristics associated with overall survival using Cox regression and the Kaplan-Meier method. Results: We retrospectively analyzed 108 CRC patients who received immunotherapy, including 31 patients with NOTCH mutation (MUT) and 78 patients with NOTCH wide-type (WT). NOTCH mutation was discovered to be enriched in MSI instable and associated with significant better overall survival (HR, 0.4; 95% CI, 018-0.9; P=0.0272). Although compared with NOTCH WT, no association was observed between NOTCH1/2/4 MUT and overall survival, Kaplan-Meier survival analysis showed that patients with NOTCH3 MUT obtained better OS (not reach vs. 14 months, HR, 0.17; 95% CI 0.04-0.74; P=0.0176). In addition, in an in-house Chinese CRC cohort (n=1968), we found 322 (16.4%) patients with NOTCH mutations, including NOTCH1/2/3/4 mutation frequency were 43.5%, 32.9%, 46.9% and 12.1%, respectively. 206 (60.4%) patients had one NOTCH mutation, and only 20 (6.2%) patients had more than four mutations. NOTCH mutations were significantly associated with higher TMB levels (P<0.0001) and PD-L1 positive (P=0.0011) in the in-house Chinese CRC cohorts. Conclusions: Our results suggest that NOTCH1/2/3/4 mutation may be a potential predictor to favorable ICI response in CRC patients. The exact mechanisms underlying NOTCH mutations are needed to be further evaluated. Citation Format: Xiaoyang Xia, DongShan You, Jing Cao, JingHui Huang, Junling Zhang, Mengli Huang, Yanan Chen, Xihua Xia, Yuezong Bai. Identification of NOTCH mutation as novel predictor to efficacious immunotherapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5088.

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Abstract Expression of the epithelial cytokine, thymic stromal lymphopoietin (TSLP), has been observed across several barrier surfaces and plays a critical role in both physiological and pathological processes. Via T helper (Th) 2 cytokine production, TSLP has been implicated in asthma, atopic dermatitis, inflammatory bowel disease, multiple sclerosis, and chronic obstructive pulmonary disease. This range in which TSLP exerts its effects on inflammatory diseases makes the TSLP/TSLPR axis an attractive therapeutic target. At Biocytogen, we generated a novel TSLP/TSLPR double humanized mouse model that enables in vivo assessment of human anti-TSLP and anti-TSLPR antibodies. In our B-hTSLP/hTSLPR mice, the murine Tslp and Tslpr genes are replaced with the human TSLP and TSLPR genes by hom*ologous recombination. ELISA analysis showed that human TSLP (after induction with calcipotriol) was exclusively detectable in hom*ozygous B-hTSLP/hTSLPR mice compared to wild type mice. Similarly, flow cytometry analysis indicated TSLPR expression was detectable in monocytes, neutrophils, macrophages, total DCs, cDC1, cDC2, Mo-DC and Pre-DC. Percentages of T cells, B cells, NK cells, DCs, granulocytes, monocytes, macrophages, CD8+ T cells, CD4+ T cells and Tregs in hom*ozygous B-hTSLP/hTSLPR mice were comparable to those in the wild type mice, demonstrating that introduction of human TSLP and TSLPR in place of their mouse counterparts did not impair the overall development, differentiation, or distribution of these cell types. Furthermore, using an asthma mouse model via induction with ovalbumin (OVA), we showed that an anti-TSLP antibody (Tezepelumab, synthesized in house) was efficacious in controlling asthma progression in B-hTSLP/hTSLPR mice. Altogether, B-hTSLP/hTSLPR mice are a promising model for preclinical in vivo pharmacodynamic assessment of TSLP antibodies. Citation Format: Shujin Zhang, Yanhui Nie, Jiahui Chang, Veronika Chromikova, Luke (Zhaoxue) Yu, Mari Kuraguchi. Evaluating in vivo efficacy of anti-TSLP antibodies in humanized B-hTSLP/hTSLPR mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2089.

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Abstract Extracellular vesicles (EV), facilitated with cancer-targeted antibody or peptide, have been demonstrated as an effective delivery vehicle of chemotherapy and gene therapy. This study aimed to develop and evaluate a targeted EV to deliver a newly identified natural cytotoxic marine compound verrucarin A (Ver-A) or combination with highly potent microtubule polymerization inhibitor mertansine (DM1). The tumor models, glioblastoma (GBM), triple-negative breast cancer (TNBC) and others, were used to validate the developed targeted therapy. First, the stirred-tank bioreactor-based EV biomanufacturing was optimized by developing a two-step purification procedure using size exclusion (300 kDa) and affinity (CD63) columns in liquid chromatography system. Second, both single and dual tumor-targeting monoclonal antibodies (mAbs), such as anti-SSTR2, CD276, EGFR and/or CD47 mAb either developed in house or used in clinics, were tagged on the surface of PEGylated EV to construct mAb-EV-drugs. The flow cytometry analysis, live-cell confocal images and IC50 assay revealed the effective surface binding, intracellular drug delivery and high anti-cancer cytotoxicity of mAb-EV-drugs in cancer cell lines, and the biodistribution study confirmed the tumor targeting in xenograft mouse models. Furthermore, the maximum tolerated dose study showed minimal systemic toxicity, as confirmed by hematoxylin and eosin staining of multiple important organs. Finally, the anti-tumor efficacy study demonstrated significant inhibition of tumor growth by mAb-EV-Ver-A or Ver-A/DM1 in cell line-derived xenograft models, patient-derived xenograft models, and/or immunocompetent xenograft models. This study suggested that the targeted EV is an efficient platform to deliver combined potent chemotherapies to treat cancer or tumor. Citation Format: Kai Chen, Yingnan Si, Seulhee Kim, Zhuoxin Zhou, Lufang Zhou, X. Margaret Liu. Targeted extracellular vesicle to deliver combined chemotherapies to treat cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5326.

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Abstract V-set and immunoglobulin domain containing 4 (VSIG4) protein is an immune checkpoint molecule that is related to the B7 family of immune regulatory proteins. B7 family members have been implicated in both positive and negative regulation of T cell activation, particularly VSIG4 is a known negative regulator of T cell proliferation and IL-2 production. Pathological consequences of VSIG4 expression are associated with inflammatory disorders, lung cancer development, high-grade glioma, and multiple myeloma, suggesting VSIG4 is a potential target for drug development. Therefore, Biocytogen developed humanized VSIG4 mice (B-hVSIG4 mice) by replacing the murine Vsig4 gene with the human VSIG4 gene through hom*ologous recombination. Flow cytometry analysis showed human VSIG4 was detectable in peritoneal exudate macrophages (PEMs) in hom*ozygous B-hVSIG4 mice compared to wild type mice. Additionally, percentages of T cells, B cells, NK cells, DCs, granulocytes, monocytes, macrophages, CD8+ T cells, CD4+ T cells and Tregs in hom*ozygous B-hVSIG4 mice were similar to those in wild type mice, demonstrating that introduction of human VSIG4 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types. For in vivo efficacy evaluation of an anti-human VSIG4 antibody (Ab-A, synthesized in house) using B-hVSIG4 mice, mouse colon adenocarcinoma MC38 cells were subcutaneously implanted into hom*ozygous B-hVSIG4 mice (female, 7-week-old, n=5). Mice were grouped and treated when the tumor volume reached approximately 100 mm3. Results showed that an anti-hVSIG4 antibody was efficient at inhibiting tumor growth in B-hVSIG4 mice. In conclusion, B-hVSIG4 mice are as a promising model for preclinical in vivo studies to assess pharmacodynamic of anti-hVSIG4 antibodies. Keywords: VSIG4, humanized mice, immune checkpoint molecule Citation Format: Ruili Lyu, Chengzhang Shang, Leila Kokabee, Yanhui Nie, Yi Yang. Evaluating in vivo efficacy of anti-VSIG4 antibodies in humanized B-hVSIG4 mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1642.

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Abstract Contributions of the microenvironment to soft tissue sarcoma progression are relatively undefined, representing a major impediment to identifying essential regulatory networks in sarcomagenesis. Furthermore, genetic and molecular characteristics that distinguish precancerous versus cancerous microenvironments are not well known across human cancer types. While animal models have the potential to reveal these complex processes, significant impediments to such inquiries include (1) the difficulty in distinguishing microenvironmental cells from precancerous or cancer cells in tissue specimens; and (2) the challenge in defining a discrete tissue with known cancer predilection that represents a precancerous microenvironment. We developed a unique zebrafish model that allows segregation of microenvironmental, precancerous, and cancerous cell populations by fluorescence-activated cell sorting. This model exhibits high predilection for malignant peripheral nerve sheath tumor (MPNST), a type of soft tissue sarcoma with a particularly poor prognosis due to aggressive growth, limited response to conventional treatment, and ineffective targeted therapy options. Using RNA-seq, we profiled the transcriptomes of microenvironmental cells from our zebrafish MPNST model and determined that the precancerous and cancerous microenvironments exhibit broad activation of inflammatory and immune-associated signaling networks. Markers for both M1 and M2 macrophage polarization were upregulated in precancerous and cancerous microenvironments, suggesting the presence of a mixed macrophage population during sarcomagenesis. Patterns of ligand and receptor expression based on a previously defined human ligand-receptor network identified significant upregulation of multiple tumor-promoting ligands in both precancerous and cancerous microenvironments. We also identified specific ligand-receptor pairs that may mediate key signaling events during sarcoma initiation and progression. Together this work provide new insight into distinguishing characteristics of the cancer-prone cellular microenvironment that may promote MPNST initiation and progression in vertebrates. Citation Format: Heather R. Shive, John S. House, Jordan L. Ferguson, Dereje D. Jima, Aubrie A. Selmek, Dillon T. Lloyd. Characterization of the precancerous and cancer microenvironment in a zebrafish sarcoma model [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR011.

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Abstract Introduction: T cell activation is critical in the initiation and potentiation of anti-tumor immune responses. Hematopoietic Progenitor Kinase 1 (HPK1/MAP4K1) is a member of the MAP4K family whose activity has been demonstrated to restrain T cell activation through phosphorylation of SLP-76 at Serine 376 leading to TCR disassembly. Mediators generated in the tumor microenvironment (TME) such as adenosine, PGE2 and TGFβ can dampen T cell activity and present a significant barrier to cancer therapy. HPK1 inhibition may allow for enhanced T cell activation under such suppressive conditions. Herein, we describe studies to assess the effects of HPK1 inhibition on T cell activation and in alleviating suppression. We also test the ability of HPK1 inhibition in combination with adenosine receptor antagonism to further amplify T cell activation. Methods: CD8+ T cells were isolated from human blood and RNA was isolated for qPCR analysis of MAP4K1-7 expression. Cells were activated using CD3/28 stimulation and supernatants were assayed for IL-2, IFNɣ and TNFα using CBA. CRISPR knockout of HPK1 in CD8+ T cells and reference or in-house HPK1 inhibitors used to characterize HPK1 function. We used a flow cytometry-based assay to quantify activation markers CD69 and CD25, and phospho-SLP-76 in isolated CD8+ T cells and in human and mouse whole blood. Results: Using publicly available gene expression databases, we identified that HPK1 is primarily expressed in immune cells, with highest expression in T cells, B cells, and dendritic cells. In-house gene expression analyses demonstrated that HPK1 is the predominant MAP4K family member in human T cells. CRISPR knockout in primary human CD8+ T cells was used to demonstrate that HPK1 but not MAP4K3 and MAP4K4 negatively regulates T cell activation. Consistent with this, reference and in-house HPK1 kinase inhibitors reduced phosphorylation of SLP-76 in both isolated T cells and human and mouse whole blood. In human T cells, HPK1 inhibition dose-dependently increased IL-2, IFNɣ and TNFα secretion, highlighting the negative role of HPK1 in T cell activation. Using adenosine, PGE2 and TGFβ, expression of CD69 and T cell cytokine secretion is diminished. HPK1 inhibition restored T cell activation to unsuppressed levels. Consistent with these results, HPK1KO CD8+ T cells displayed significant resistance to adenosine, PGE2 and TGFβ-induced immunosuppression. Finally, upon suppression using adenosine, combining HPK1 inhibition with adenosine receptor antagonism further elevated T cell activation above individual inhibitor treatments alone. Conclusion: These data demonstrate that HPK1 plays a significant role in dampening T cell activation and provide a strong therapeutic rationale for targeting HPK1 to relieve T cell immunosuppression in the TME and amplify anti-tumor immune responses. Citation Format: Rajesh K. Singh, Rameshwari Rayaji, Bindi Patel, Kristen Zhang, Sachie Marubayashi, Soonweng Cho, Stefan Garrido-Shaqfeh, Joseph Kulusich, Cesar Meleza, Nidhi Tibrewal, Joice Thomas, Pradeep Nareddy, Ehesan Sharif, Sharon Zhao, David Green, Manmohan R. Leleti, Jay P. Powers, Daniel DiRenzo, Matthew J. Walters. HPK1 inhibition enhances T cell activation and relieves the immunosuppressive phenotype of inhibitory signals found in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1367.

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Abstract Introduction: Nasopharyngeal carcinoma (NPC) is a malignancy characterized by distinct geographic, ethnic, and genetic differences. It presents a major threat to human health, particularly in East Asia and Southeast Asia. NPC commonly invades adjacent tissues and organs, resulting in complex and difficult to treat clinical presentations. NPC commonly remains largely asymptomatic during early disease stages due to its small primary foci. Hence, NPC patients are diagnosed mostly at advanced stage. Currently, no accurate and effective methods are available for the early diagnosis, evaluation, or prognosis for NPC. While Epstein-Barr virus (EBV) DNA and EBV serology tests have been advocated for early diagnosis and screening for NPC, the sensitivities of these two tests vary. due to the fact that only a small proportion of individuals infected with EBV ultimately develop NPC. The ability to detect neoplasms at early stages provides useful information for early intervention. We have recently demonstrated that interrogating the serum glycoproteome, using a proprietary platform that couples artificial intelligence (AI) to targeted liquid chromatography-massspectrometry (LC-MS) yields highly informative biomarkers in a range of neoplastic diseases. Experimental procedures in this proof-of-principle study, a total of 106 plasma samples (1:1 ratio of histologically confirmed cases of NPC and cancer-free controls), which were matched for age (average 49 years) were used. All subjects were male, from a multi-ethnic patient population in Malaysia, including three ethnic minority groups (Bidayuh, Iban and Kadaza). To identify diagnostic glycoprotein profiles for NPC we applied an in-house developed targeted glycoproteomics workflow, whereby plasma proteins were first digested with trypsin/LysC, followed by targeted analysis using LC-MS. The LC-MS generated chromatographic peaks were integrated using an in-house AI-based software for high throughput analysis and further subjected to multivariate statistical analysis. Summary of new data among the 579 glycopeptides interrogated in this study we found 62 glycopeptides statistically significantly abundant in NPC cases compared to healthy controls (at FDR <0.1). From these, we built a predictive algorithm that differentiates NPC cases from controls with high sensitivity and selectivity (AUC of 0.955). Six glycopeptide markers were found as associated with ethnicity. Conclusions: In summary, our results indicate that LC-MS-based glycopeptide profiling provides a highly accurate means of for differentiating NPC cases from healthy controls. Follow-up studies with larger sample size are currently in progress. Citation Format: Klaus Lindpaintner, Thi Thin Aye, Gege Xu, Rachel Rice, Alan Mtchell, Daniel Serie, A Malek, LH Low, Lp Tan, Ct Low, Cy Wong, H Sawali, Yy Yapp, As Khoo. Peripheral blood glycoproteomic biomarkers as a powerful new tool for the detection of nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2799.

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Rickettsial diseases (RDs) are major under-diagnosed causes of arthropod borne acute febrile illness (AFI) presenting with a range of symptoms from mild self-limiting fever to fatal sepsis. The spotted fever group (SFG) and typhus group (TG) are major RDs, which are commonly caused by Rickettsia conorii and Rickettsia typhi, respectively. The limited availability and role of serological tests in the acute phase of illness warrants rapid reliable molecular methods for diagnosis and epidemiological studies. Two hundred patients with AFI in whom the routine fever diagnostics were negative, were enrolled over a period of two months (April 2019 to May 2019). DNA was extracted and in-house nested PCR using primers specific for both SPG and TG pathogens was used. The positive amplified products were sequenced for species identification and phylogenetic analysis was performed using MEGA 7.0.14 software (iGEM, Temple University, Philadelphia, PA 19122, USA). The demographic details of the RD cases were documented. The prevalence of RD among AFI cases was 7% (14/200); SFG and TG were identified as the cause in 4% and 3% of AFI cases, respectively. The median age of the RD cases was 22 years (range 2–65). The median duration of fever was 3 days (range 1–12). The RD cases presented with respiratory symptoms or signs (44.44%), jaundice (22.22%), abdominal pain (22.22%), diarrhea (22.22), vesicular rash (11.11%), vomiting (11.11%), loss of appetite (11.11%), headache (11.11%), leukocytosis (88.88% with mean count 22,750/mm3), and thrombocytopenia (33.33%). The cases were treated empirically with piperacillin-tazobactam (66.66%), clindamycin (44.44%), cefotaxime (33.33%), meropenem (33.33%), metronidazole (33.33%), doxycycline (22.22%), azithromycin (22.22%), ceftriaxone (11.11%), and amoxicillin-clavulanic acid (11.11%). The mortality among the RD cases was 11.11%. The present pilot study shows that RD is not an uncommon cause of AFI in north India. The febrile episodes are usually transient, not severe and associated with heterogenous clinical presentation without documented history of tick exposure in the hospitalized patients. The transient, non-severe, febrile illness could be due to transient rickettsemia resulting from empirical antimicrobial therapy as the rickettsial organisms are expected to be more susceptible to higher doses of β-lactam antibiotics. The study emphasizes the molecular method as a useful tool to identify rickettsial etiology in AFI.

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Abstract Epithelial ovarian cancer (EOC) remains the fifth leading cause of cancer related death in women worldwide, partly due to the survival of chemoresistant, stem-like tumor-initiating cells (TICs) that promote disease relapse. Previous studies described the role of the NF-kB pathway in promoting TIC chemoresistance and survival through the action of key transcription factors (TFs) like RelA and RelB. These TFs serve as key master regulators of genes important for the inflammatory response, which also overlap with the regulation of targets associated with cancer promotion. We previously showed that RelA and RelB exert different effects in ovarian cancer cells, with RelB relatively more important for tumor initiation. In this study, we hypothesize that NF-kB signaling regulates the expression of several microRNAs (miRNAs) differentially through RelA and RelB to promote TIC survival and proliferation. miRNAs comprise a subset of small, noncoding RNAs that regulate gene expression and present potential therapeutic targets for clinical use. Inducible shRNA stably was expressed in OV90 EOC cells to knockdown RelA or RelB; cells were subsequently analyzed by miR-seq to identify differentially expressed miRNAs in cells grown in TIC vs adherent (adh) conditions. To validate the miR-seq findings, we performed qPCR assays of the candidate miRNAs differentially expressed in TIC or adh conditions with RelA or RelB knocked down. We confirmed the decreased expression of oncogenic miRNAs hsa-miR-105-5p and hsa-miR-452-5p, while also confirming the increased expression of tumor suppressive miRNA hsa-miR-34a-5p observed in the miR-seq using OV90 cells. Ongoing work will validate these expression changes in additional cell lines and conduct functional assays to characterize the effect of mimicking or inhibiting these miRNAs of interest. The identification of miRNAs differentially expressed in TICs under the control of RelA or RelB will provide key insights in designing treatments against them to prevent disease relapse in patients with EOC. Citation Format: Rahul D. Kamdar, Brittney S. Harrington, Soumya Korrapati, Nathan Wong, Carrie D. House, Christina M. Annunziata. NF-κB signaling regulates miRNA expression in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5820.

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Abstract Purpose: Intratumoral heterogeneity emerges from accumulating genetic and epigenetic changes during tumorigenesis, which may contribute to therapeutic failure and drug resistance. We aimed to develop a tool that can evaluate the intratumoral epigenetic heterogeneity using the DNA methylation profiles from bulk tumors. Methods: Ggenomic DNA of three laser micro-dissected tumor regions, including digestive tract surface (DTS), central bulk (CB), and invasive margin (IM), was extracted from formalin-fixed paraffin-embedded (FFPE) sections of 98 colorectal cancer patients. The genome-wide methylation profiles were generated with methylation array. The most variable methylated probes were selected to construct a DNA methylation-based heterogeneity (MeHEG) estimation tool that can deconvolve the proportion of each reference tumor region with the support vector machine model-based method. A PCR-based assay for quantitative analysis of DNA methylation (QASM) was developed to specifically determine the methylation status of each CpG in MeHEG assay at single-base resolution to realize fast evaluation of epigenetic heterogeneity. Results: In the discovery set with 79 patients, the differentially methylated CpGs among the three tumor regions were found. The 7 most representative CpGs were identified by random forest analysis and subsequently selected to develop the MeHEG algorithm. We validated its performance of deconvolution of tumor regions in an independent cohort with 19 patients. In addition, we showed the significant association of MeHEG-based epigenetic heterogeneity with the genomic heterogeneity in mutation and copy number variation in our in-house and TCGA cohorts. Finally, we found that the patients with higher MeHEG score had worse disease-free and overall survival outcomes. Conclusion: By constructing a 7-loci panel with a machine learning approach and QASM assay to facilitate its use in a PCR manner, we developed a valuable method to evaluate the intratumoral epigenetic heterogeneity. The MeHEG algorithm provides innovative insights into the intratumoral heterogeneity from the epigenetic perspective that may add valuable sources to current knowledge about tumor heterogeneity. Citation Format: Yumo Xie, Yu Zhang, Guannan Tang, Xiaolin Wang, Meijin Huang, Yanxin Luo, Huichuan Yu. Spatial deconvolution from bulk DNA methylation profiles determines intratumoral epigenetic heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6079.

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Abstract Purpose: Breast cancer is a heterogeneously complex disease. A number of molecular subtypes with distinct biological features lead to different treatment responses and clinical outcomes. Traditionally, breast cancer is classified into subtypes based on gene expression profiles; these subtypes, namely intrinsic subtypes, include luminal A, luminal B, basal-like and HER2-enriched breast cancer. This molecular taxonomy, however, could only be appraised through transcriptome analyses. We aim to classify breast cancer intrinsic subtypes using un-annotated approach using deep learning models.Methods: 388 pathological whole slide images (WSIs) from TCGA-BRCA dataset were downloaded from TCGA-GDC portal. These WSIs underwent patches generation and normalization using PyHIST tool and Macenko algorithm, respectively. Laplacian algorithm was applied to remove patches with blurry areas and pixelated. The remaining patches (n = 1,833,889) were divided into 3 parts for training (70%), testing (5%) and validation (25%). We applied a 2-step transfer learning with 2 pre-trained models, namely ResNet50, ResNet101, Xception and VGG16, which have been trained on another in-house breast cancer pathological image dataset. Results: these four models shown promising classification results of 4 breast cancer intrinsic subtypes with accuracy ranged from 0.68 (ResNet50 model) to 0.78 (ResNet101 model) in both testing and validation sets. The average AUC score for these models were from 0.88 (ResNet50 model) to 0.94 (ResNet101 model), whereas ResNet101_imgnet with “imagenet” weight archived an accuracy of 0.73 and AUC of 0.92. The overall accuracy of patient-wise prediction even shown a higher average accuracy of 0.914. These models’ prediction visualization was also used to demonstrate that the process of model learning was based on pathological cells’ clusters. Conclusion: Our study demonstrated the feasibility and capability of the deep learning model in classifying breast cancer intrinsic subtypes without region of interest annotation, which wound facilitate the clinical applicability of proposed models. Citation Format: Chi-Cheng Huang, Nam Nhut Phan, Ling-Ming Tseng. Predicting molecular subtypes of breast cancer using [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-02-15.

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Abstract Background Cytomegalovirus (CMV) Polymerase chain reaction (PCR) test is a valuable tool for diagnosis and therapeutic monitoring of CMV infection. Academic medical centers with a high volume of organ transplant and immunosuppressed patients often utilize CMV quantitative and qualitative PCR testing. CMV qualitative PCR typically does not alter clinical management, and positive CMV qualitative test often needs follow up quantitative test. Despite its lack of usefulness, CMV qualitative PCR is often over-ordered and may unnecessarily raise the cost of hospitalization. We evaluated utility of CMV PCR testing at a tertiary care medical center. Methods We reviewed CMV PCR testing done from June 2015-November 2016 at Hahnemann University Hospital in Philadelphia, PA. CMV qualitative test was performed at LabCorp and CMV quantitative test was done at Focus Labs. Data collected included demographics, length of stay, and immunosuppression. Selected patients had either CMV qualitative PCR positive without follow up quantitative PCR, or negative CMV qualitative PCR with unnecessary CMV quantitative PCR ordered. Results We evaluated 226 CMV PCR test results including 162 qualitative and 64 quantitative CMV PCR in 139 patients. 39 (28%) patients had superfluous CMV testing. Mean age was 52.6 years, 61% were male, 46% were African American. Mean length of stay was 24 days. A half (N = 19,49%) were immunocompromised. 28 (17.2%) results were positive for CMV qualitative PCR, 7 (25%) of whom follow up CMV quantitative PCR were not sent. Thirty-two patients had negative CMV qualitative testing yet had CMV quantitative PCR sent. Six had redundant CMV quantitative PCR tests within 7 days likely resulting from delayed result report from send out. After performing cost analysis, these unnecessary tests would have saved $3930. Conclusion In our cohort, significant unnecessary CMV testing resulted in increased health care cost and patient discomfort. Positive Qualitative CMV PCR without Quantitative testing impairs diagnosis and treatment follow up. Given complicated testing algorithm and limited value of CMV qualitative PCR testing in the adult population, we plan to simplify CMV testing to quantitative only and perform in house testing to shorten result time. Disclosures All authors: No reported disclosures.

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Abstract Background: Liposarcoma (LPS) is an understudied form of soft tissue sarcoma (STS). The well-differentiated (WD) and de-differentiated (DD) subtypes of LPS are associated with indolent and aggressive disease courses, respectively, but this histologic stratification fails to fully capture disease heterogeneity. Molecular approaches may help refine prognostication and inform treatment intensification. Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3 or IMP3), is an RNA-binding protein that regulates gene expression by controlling mRNA stability and has been implicated in tumorigenesis and poor prognosis in many cancers. We hypothesized IGF2BP3 would refine the prognostication of LPS beyond its WD/DD histologic status. Methods: We examined the association between IGF2BP3 gene or protein expression and clinical data in four datasets: (1) patients with STS subtypes (n=206) in the cancer genome atlas (TCGA) database, (2) an in-house gene microarray of lipomatous tumors (n=71), LPS cell lines (n=3) and patient-derived xenografts (PDX, n=3), (3) an in-house tissue microarray (TMA) of lipomatous tumors (n=115), LPS cell lines (n=3) and PDXs (n=3) and (4) an in-house TMA of WD/DD LPS (n=71). IGF2BP3 protein expression in TMAs was quantified by immunohistochemistry (IHC). IGF2BP3 gene and protein expression values from identical samples were compared by Pearson correlation (n=43). The Kaplan-Meier method and log-rank test were used to compare survival outcomes. Results: In the TCGA cohort, which does not include WD LPS, IGF2BP3 expression was a poor prognostic factor solely in DD LPS (n=50, median overall survival (mOS): 1.6 vs 5.0 years, p=0.009). Among gene microarray samples, IGF2BP3 expression was highest in DD LPS (n=18) compared to WD LPS (n=29) and lipoma (n=7) by paired t-tests (p=0.03 and 0.002, respectively) and IGF2BP3 expression was associated with worse survival in WD/DD LPS (mOS 7.7 vs 21.5 years, p=0.02). In both TMAs, IGF2BP3 expression (>25% cell positivity/core) portended worse survival in WD/DD LPS (mOS (3): 3.7 vs 13.8 years, p<0.001 and mOS (4): 2.7 vs 14.9 years, p<0.001). IGF2BP3 was not expressed in myxoid LPS (n=21) or lipoma (n=8) samples. Gene and protein expression of IGF2BP3 were positively correlated in WD/DD LPS (r2 = 0.69). IGF2BP3 expression was more strongly associated with survival than LPS differentiation status (mOS: 7.0 (DD) vs 15.2 years (WD), p=0.02). Furthermore, all LPS cell lines and PDXs demonstrated high gene and protein expression of IGF2BP3. Conclusion: IGF2BP3 is highly expressed in a subset of LPS. Across independent datasets, IGF2BP3 is also a biomarker of disease progression and worse survival, and may stratify patients more effectively than histologic differentiation. Mechanistic studies of IGF2BP3 in LPS tumorigenesis and progression using aforementioned LPS cell lines and PDX models are ongoing. Citation Format: Kyle D. Klingbeil, Jack Pengfei Tang, Sarah M. Dry, Fritz C. Eilber, Dinesh S. Rao, Brian E. Kadera, Anusha Kalbasi. IGF2BP3 (IMP3) expression is associated with worse survival in well-differentiated/dedifferentiated (WD/DD) liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3490.

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Abstract Glial buffering of K+ prevents seizures, hence gliomas in patients on anticonvulsants may harbor potassium related anomalies. Cell membrane potentials are very sensitive to K+ and may link to tumor acidosis when they become more positive than the equilibrium potential for H+ ions (Vaupel, P, et al., Cancer Res, 1989). Kir5.1(KCNJ16) heterodimerizes with Kir4.1(KCNJ10), a major glial potassium channel, to stop K+ conductance in acidosis. Trek1&2(KCNK2&10) are also pH sensitive K+ channels. Glioblastomas (more malignant and invasive) versus oligodendrogliomas showed decreased KCNJ16 in patients on anticonvulsants (Beckner, ME, Proc AACR, A2319, 2021). However, loss of genomic integrity in tumors and potential variation between assays prompted house keeping gene (HKG) normalization to lessen confounding factors. REMBRANDT (12/31/2020, Georgetown Database of Cancer) had 220 glioblastomas and 67 oligodendrogliomas with microarray gene expression via medians of each reporter (1-8 per gene) in Adobe Flash readouts (ended 1/1/21). A subgroup, 75 glioblastomas and 24 oligodendroglioma patients, took anticonvulsants. Comparison of genes of interest (GOI) initially had t tests between types of gliomas in the subgroup. Here, differences between glioma types for each GOI reporter were compared to differences for HKGs (PPIA, RPLP0, YWHAZ, and B2M). In gliomas with anticonvulsants, paired 2-tailed t tests for a null difference of median reporters between tumor types were less sensitive than new comparisons of oligodendroglioma minus glioblastoma (OMG) differences for GOIs versus OMG differences in HKGs. For KCNJ16, the t test, p = 0.015, for a decrease in glioblastomas (versus oligodendrogliomas) in initial studies improved to p ≤ 0.0001 by using HKGs in comparisons. The KCNK2&10 decrease in glioblastomas became significant, p = 0.047. Among other glycolytic and acidosis related genes, CA12, GLO1&2, PFKFB1&3, showed stronger trends, with p values from 0.08 to 0.13 (decreases and increase) in comparisons using HKGs. Conclusion: HKG normalization presumably corrects for loss of genomic integrity, assay variations, etc. in comparisons using microarrays of different tumors. The pH-sensitive K+ channel genes studied here were expressed less in glioblastomas. The resulting pH-insensitive K+ distributions in glioblastomas putatively shift their cell membrane potentials to less negative values which are sufficient to allow passive H+ efflux that causes extracellular acidosis with accompanying activation of invasion. Acknowledgement: The data utilized in this study were provided by the Georgetown Database of Cancer (G-DOC), a project of the Georgetown Lombardi Comprehensive Cancer Center designed to provide translational research tools to the scientific community. Citation Format: Marie E. Beckner. Decreased pH-sensitive potassium channel gene expressions in glioblastomas compared to oligodendrogliomas detected with house keeping genes may shift glioblastoma membrane potentials towards proton efflux to the microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3039.

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Abstract Ovarian cancer is the deadliest gynecological cancer with over 13,000 deaths estimated in the United States this year. While there is an initial high response rate to cytotoxic chemotherapy, over 80% of advanced cases relapse within 24 months, presenting an urgent need to better understand the molecular mechanisms permitting relapse. Current research suggests that cancer recurrence can be attributed to a small subpopulation of cancer stem-like tumor-initiating cells (TICs) that possess the ability to both resist chemotherapy and initiate tumorigenesis. Our lab previously demonstrated an essential role for alternative NF-kB signaling in maintaining ovarian TICs, yet it is not fully understood how this pathway becomes activated in ovarian cancer. To begin to address this, we used an NF-kB reporter system to screen soluble factors known to be in the ovarian tumor microenvironment (TME) to identify the strongest inducers of NF-kB activation in ovarian cancer cells. We found that tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a strong inducer of alternative NF-kB signaling. In vitro experiments demonstrate enhanced expression of stemness genes including SOX2, OCT4 and NANOG and increased spheroid formation ability after stimulation with TWEAK. Moreover, flow cytometry analysis of several TIC markers shows a significant enrichment of TICs when chemotherapy was combined with TWEAK, relative to chemotherapy alone. Stimulation of CD117+ cells led to higher expression of stemness genes relative to stimulation of CD117- cells, suggesting TWEAK activity may be specific to TICs and enhances their stem-like phenotype. In support of this, we found that CD117+ cells have higher expression of the TWEAK receptor FN14. We investigated the role of TWEAK in aiding relapse in vivo using an intraperitoneal xenograft mouse model and found that administration of a small molecule inhibitor of TWEAK-FN14 signaling as a maintenance treatment following chemotherapy significantly prolonged survival. These findings highlight a potential mechanism of TIC enrichment in response to chemotherapy that contributes to relapse. Citation Format: Denay N. Stevenson, Ryne M. Holmberg, Samuel F. Gilbert, Mikella Robinson, Carrie D. House. TWEAK cytokine supports ovarian cancer tumor-initiating cell (TIC) phenotypes important for relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3129.

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Abstract Immune checkpoint inhibitors have become an important tool for the treatment of cancer. However, the majority of patients does not respond to the currently employed antibody-based treatments, indicating a need for additional targets and new treatment modalities. The invention of 3rd generation chemistries like locked nucleic acids (LNAs) has allowed the development of highly specific and efficacious antisense oligonucleotides (ASOs) that allow target knockdown in vivo without the need for complex delivery formulations. The membrane-bound protein neuropilin-1 (NRP1) was initially identified as a factor involved in cell migration, -survival, and neoangiogenesis making it an attractive target for cancer therapy. It was subsequently shown that it has additional immune-mediated pro-tumorigenic roles. It can suppress anti-tumor immune activity via regulation of trafficking of tumor-associated macrophages, phenotypic stability of Tregs, and exhaustion of effector T cells. Considering that these effects are mediated by multiple domains of NRP1, there is a conceptual advantage to down-regulate the expression of the whole protein over the functional or steric blockade of individual domains. We used our in-house Oligofyer™ bioinformatics system to design both human NRP1 specific LNAplus™ ASOs, as well as murine surrogates, which both achieved target knock-down of more than 85% in vitro. Furthermore, the murine surrogate down regulated NRP1 in tumor mouse models. Systemic application of mouse-specific ASOs without additional carriers or adjuvants has led to a strong delay of tumor growth or complete eradication in syngeneic mouse models. This effect was almost completely abrogated in immune-compromised NSG mice. Furthermore, re-challenge of immune-competent mice after tumor eradication did not result in new tumor growth, suggesting induction of a long-lasting immunity by LNAplus™ ASO-mediated knockdown of NRP1. Taken together, these encouraging results indicate that the ASO-mediated down-regulation of NRP1 has the potential to become a promising treatment option for patients in need. We are currently performing further experiments to fully elucidate the mechanisms that underlie the observed anti-tumor efficacy. Citation Format: Andre Maaske, Nicole Kirchhammer, Julia Festag, Laura Fernandez Rodriguez, Mélanie Buchi, Monika Schell, Stefanie Raith, Sven Michel, Richard Klar, Alfred Zippelius, Frank Jaschinski. Knock-down of Neuropilin-1 by locked nucleic acid antisense oligonucleotides facilitates cancer immune control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4149.

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Abstract Lenvatinib is a multiple receptor tyrosine kinase inhibitor and an angiogenesis inhibitor targeting mainly vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) receptors. In our previous study, we reported that human xenograft tumors sensitive to lenvatinib showed higher microvessel density (“MVD (IHC)”) than relatively resistant tumors in immunohistochemistry (IHC). Although MVD (IHC) is a potential indicator to predict antitumor activity of lenvatinib, available IHC data are limited. Here, we investigated a novel gene expression signature (MVD gene score) calculated from RNA-sequencing (RNA-seq) data that reflects MVD (IHC) and antitumor effect of lenvatinib. Tumor tissues were collected from 12 mouse syngeneic tumor models followed by RNA-seq. We also stained CD31 in the same tumors and calculated MVD (IHC) score by dividing the number of CD31 positive blood vessels by tumor area. Genes that met the following criteria were selected for MVD gene score calculation: (1) genes that are specifically expressed on endothelial cell cluster in in-house single cell RNA-seq data using Hepa 1-6 mouse hepatocellular carcinoma tumor tissues; (2) well-known angiogenesis markers from literature or genes whose expression show significant correlation with MVD (IHC) in mouse syngeneic tumor models. MVD gene score was calculated as the average of logarithm transformed gene expression values. In order to examine applicability of MVD gene score, we compared MVD (IHC) and MVD gene score to lenvatinib antitumor activities on mouse syngeneic tumors. We measured MVD (IHC) and computed MVD gene score from RNA-seq data using matched tumor tissues of 12 mouse syngeneic models. MVD gene score was correlated with MVD (IHC). As reported, lenvatinib sensitive tumor models showed higher MVD (IHC) score. They also showed higher MVD gene score calculated from RNA-seq data. Our newly developed MVD gene score has a potential to predict the antitumor activity of angiogenic inhibitors such as lenvatinib using RNA-seq data in tumor samples. Further validation study is ongoing using various human tumor samples. Citation Format: Megumi Kuronishi, Yoichi Ozawa, Takayuki Kimura, Yu Kato. Development of a novel gene expression signature that correlates with intratumor microvessel density and antitumor activity of lenvatinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5136.

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Abstract Epithelial ovarian cancer (EOC) is a global health burden, with the poorest five-year survival rate of the gynecological malignancies due to diagnosis at advanced stage and high recurrence rate. Recurrence in EOC is driven by the survival of chemoresistant, stem-like tumor-initiating cells (TICs) that are supported by a complex extracellular matrix (ECM) and immunosuppressive microenvironment. To target TICs to prevent recurrence, we identified genes critical for TIC viability from a whole genome siRNA screen. A top hit was the cancer-associated, ECM synthesis enzyme UDP-glucose dehydrogenase (UGDH). Immunohistochemistry was used to delineate UGDH expression in histological and molecular subtypes of EOC. Clustering analysis was performed to characterize EOC cell lines to aligned to the molecular subtypes observed in tumors. UGDH expression was modulated in the cell lines using inducible shRNA, and the effects on TICs and on the cells in the supporting microenvironment were examined. High UGDH expression was observed in the majority of high-grade serous ovarian cancers and variable expression was observed in clear cell, mucinous and endometrioid histotypes. A distinctive survival prognostic for UGDH expression was revealed when serous cancers were stratified by molecular subtype, where high UGDH was associated with a poor prognosis in the mesenchymal subtype. Using OV90 as a representative cell line for the mesenchymal subtype, we examined the effect of UGDH knockdown on the tumor cells alone, and on mesothelial support cells in co-culture with the tumor cells. Knock down of UGDH in the OV90 cells reduced the viability, sphere-formation and colony formation capacity of TICs and reduced extracellular hyaluronan production. Knocking down UGDH in the tumors affected the mesothelial cells in co-culture by significantly reducing the expression of ECM signaling and remodeling proteins Versican, Vitronectin, Laminin and matrix metalloprotease 1. These data show that modulation of UGDH expression in tumors influences cells in the microenvironment and reveal a distinct role for UGDH in the mesenchymal molecular subtype of EOC. UGDH is a strong prospective therapeutic target in TICs, for the treatment of metastatic and recurrent EOC, particularly in patients with the mesenchymal molecular subtype. Citation Format: Brittney S. Harrington, Rahul Kamdar, Nathan Wong, Carrie D. House, Christina M. Annunziata. Modulating UGDH expression in ovarian cancer tumor-initiating cells alters the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3188.

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Abstract Around 80 percent of high-grade serous ovarian cancer patients will relapse with chemotherapy resistant disease. Despite extensive research and developments in immunotherapy, there are limited improvements in overall survival for these patients. Recent research has found that the tumor microenvironment (TME) plays a crucial role in tumor initiation, therapy resistance, and tumor relapse. The TME, which consists of immune cells, blood vessels, and an extracellular matrix (ECM) built primarily by fibroblasts, may enhance the survival and growth of tumor-initiating cells (TICs), chemoresistant stem-like cells thought to be responsible for tumor relapse. Cytotoxic chemotherapies have been shown to modify the TME and this modulation may favor the survival of TICs. For example, carboplatin can activate stromal cells such as fibroblasts to secrete an altered ECM profile, including increases in collagen 1a1 and fibronectin production, which can bind specific integrins upregulated in TICs. Using ex-vivo decellularized peritoneum tissues from mice exposed to chemotherapy or vehicle treatment, we observed a 2-fold increase in growth in peritoneums pre-exposed to chemotherapy relative to those pre-exposed to vehicle. We hypothesize that alterations in the ECM following chemotherapy treatment permit TIC adhesion and growth. To investigate this, mouse embryonic fibroblasts (3T3s) were exposed to chemotherapy during ECM production, decellularized, and reseeded with a panel of ovarian cancer cell lines. Our results suggest a significant increase in cell survival in matrices pre-exposed to chemotherapy compared to control matrices pre-exposed to vehicle. Using this model, we are examining the features of cells that preferentially survive on these matrices. In addition to drug resistance, we are using flow cytometry to assess TIC markers, such as CD117, and CD133, and qRT-PCR to measure stemness genes such as SOX2, OCT4, and NANOG. These studies highlight the need for further investigation into the systemic effects of chemotherapies on the TME and how these changes may be fostering cancer cell adhesion, growth, and survival. Understanding these mechanisms will allow us to develop therapies to achieve better clinical outcomes. Citation Format: Omar Lujano-Olazaba, Mikella Robinson, Samuel F. Gilbert, Emily Kogan, Carrie D. House. The role of chemotherapy-induced fibrosis in the maintenance of tumor initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3833.

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Abstract Background: Cyclin-dependent kinases (CDK) are a class of enzymes that, along with their regulatory cyclin binding partners, drive cell cycle progression. Cell cycle dysregulation is a hallmark of cancer and targeting its genetic drivers can confer therapeutic benefits. Cyclin E1 (CCNE1) gene alterations are common in patients with ovarian cancer and can be found in ~20% of high-grade serous ovarian cancer cases, which tend to be platinum therapy resistant and therefore represent a high medical need. Cyclin E1 is the canonical binding partner of CDK2, which becomes constitutively active when CCNE1 is amplified and overexpressed. Selectively inhibiting CDK2 is an attractive therapeutic option for CCNE1-amplified tumors and may limit off-target CDK-driven toxicities. Here we report preclinical validation studies leading to the development of an orally available CDK2 inhibitor, BLU-222, for the treatment of ovarian cancer harboring a CCNE1 amplification. Methods: BLU-222 selectivity was measured by enzyme assays, cellular target engagement assays (NanoBRET), and proliferation assays in a panel of ovarian cancer cell lines. In vitro cellular potency was assessed by phospho-Rb levels. In vivo antitumor activity of BLU-222 as a single agent or in combination with carboplatin was measured in an OVCAR-3 cell line-derived xenograft (CDX) tumor model harboring a CCNE1 amplification. Results: BLU-222 demonstrated selectivity, both biochemically and in cells, with low nanomolar potency for CDK2 vs other CDK family members (CDK1, -4, -6, -7, and -9). In a panel of ovarian cancer cell lines, those with CCNE1 amplifications were highly sensitive to BLU-222. Consistent with the in vitro profiling, BLU-222 exhibited significant antitumor activity in the OVCAR-3 CDX model. While administration of single-agent carboplatin led to stasis in vivo, the combination of BLU-222 + carboplatin induced durable tumor regression even after treatment cessation. Conclusions: These data provide a strong rationale for advancing BLU-222 towards clinical development in patients with CCNE1-amplified ovarian cancer. Citation Format: Victoria Brown, Phil Ramsden, Nealia House, Richard Vargas, Jian Guo, Ruduan Wang, Riadh Lobbardi, Maxine Chen, Douglas Wilson, Joseph Kim, Neil Bifulco, Michelle Maynard, Emanuele Perola, Dean Zhang, Steve Wenglowsky, Yoon Jong Choi. BLU-222, an investigational, potent, and selective CDK2 inhibitor, demonstrated robust antitumor activity in CCNE1-amplified ovarian cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2306.

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Abstract Gastric cancer (GC) disproportionally affects people of Latino ancestry and represent a leading cause of incidence and mortality disparities in this minority populations. When compared to Non-Latino Whites, Latinos are ~2-fold more likely to be diagnosed with and to die of GC. The goal of our research program is to contribute to GC research in patients of Latino ancestry and advance cancer health disparity research. One of the research projects aims at identifying germline variants associated with GC risk. To do so, we are identifying novel gastric cancer genes and detecting mutations in previously reported GC genes, using whole-exome sequencing, in 500 patients who have been diagnosed with early-onset GC and/or who had GC and family history of cancer. Whole exome sequence data collection was recently completed and is being analyzed with the primary goals of estimating the prevalence and penetrance in known cancer genes as well as the identification of new GC genes. Using in-house exome data and publicly available genetic data, we will identify variants with predicted impact in the phenotype to design a panel that will be used for their detection in gastric preneoplasia biopsies. These variants will be tested using duplex sequencing, a highly sensitive method that can detect very rare mutations with ultra-depth sequencing. We will then select the variants with likely functional consequences for tumor initiation and/or progression to generate isogenic gastric organoids to investigate their effect in proliferation, differentiation, and gene expression. We aim to generate a body of work that can be used for risk assessment, prevention, early diagnostic, and that will lead to better outcomes for GC in this important U.S. minority. Dr. Ana Estrada-Florez is the recipient of the AACR Cancer Health Disparities Fellowship. Citation Format: Ana Patricia Estrada-Florez, Paul Lott, Ted Toal, Nicole Halmai, Elizabeth Quino, Alma Poceros-Coba, Alix Guevara, Fabian Castro, Jhon Suarez, John Mcpherson, Rasika Venkatesh, Hongyong Zhang, Apri Vang, Guadalupe Polanco, Joana Guerra, Graciela Molina, Carol Parra, Adriana Della Valle, Esteban Castelao, Manuel Teixeira, Mabel Bohorquez, Javier Torres, Alejandro Carvajal Corvalan, Luis G. Carvajal-Carmona. Identification of predisposition and progression gastric cancer biomarkers in Latinos [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2274.

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Abstract BACKGROUND: The incidence of brain metastases (BM) is tenfold higher than that of primary brain tumors. BM predominantly originate from primary lung, breast, and melanoma tumors with a 90% mortality rate within one year of diagnosis, posing a large unmet clinical need to identify novel therapies against BM. The goal of this work is to uncover the molecular factors that drive the formation of BM and investigate whether we can slow down and ultimately block BM formation. METHODS: The Singh lab has generated a large in-house biobank of patient-derived BM cell lines that are established from BM patient tumor samples. We use these BM cell lines to generate murine orthotopic xenograft models of BM and interrogate the biological processes that lead to BM. These models have successfully recapitulated all the stages of their respective BM cascades and additionally captured a “pre-metastatic” population of BM cells that have just seeded the brains of mice before forming mature, clinically detectable tumors. Pre-metastatic cell populations are impossible to detect in human patients but represent a therapeutic window wherein metastasizing cells can be targeted and eradicated before establishing clinically detectable and difficult to treat brain tumors. RESULTS: RNA sequencing of pre-metastatic BM cells revealed a unique deregulated transcriptomic profile that is specific to pre-metastatic cells despite the tumor of origin. Subsequent Connectivity Map analysis revealed compounds that we biologically characterized in vitro for selective anti-BMIC phenotypes. This effort led to a lead compound that exhibits anti-BM activity in vitro, while remaining ineffective against normal brain cell controls. Preliminary in vivo work has shown that following both orthotopic and intracardiac injection of BM cells, treatment with this lead compound reduces the tumor burden compared to mice being treated by a vehicle control, while providing a significant survival advantage. Ongoing mechanistic investigations aim to delineate the protein target of this compound in the context of the observed selective anti-BM phenotype. CONCLUSION: Identification of novel small molecules that target premetastatic BM cells could slow or prevent the formation of BM and dramatically improve the prognosis of at-risk cancer patients. Citation Format: Agata Kieliszek, Blessing Bassey-Archibong, Chitra Venugopal, Sheila Singh. Interrogating the pre-metastastic gene signature to block brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 976.

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Abstract Cancers acquire somatic mitochondrial DNA (mtDNA) mutations undetected in surrounding tissues. Cancer-specific mtDNA mutations primarily constitute variants of unknown significance whose oncogenic consequences are poorly understood. Analysis of large-scale sequencing datasets suggests that human cancers generally select against severe mtDNA mutations; however, the presence of selective pressure on the mtDNA genome has yet to be experimentally demonstrated. Here we optimized a FACS-based protocol to reconstitute the highly metastatic human melanoma cell line A375 with exogenous mtDNA, thereby generating nuclear-isogenic cytoplasmic hybrid (cybrid) lines. hom*oplasmic cybrids were generated exclusively harboring either exogenous wildtype mtDNA, mtDNA with partial loss of OXHPHOS function (SNPs in mt.ND1 (of Complex I) or mt.ATP6 (of Complex V)), or mtDNA with complete loss of OXPHOS function (derived from a human melanoma harboring a truncating frameshift mutation in mt.CO1 (of Complex IV)). Compared to wildtype cybrids, subcutaneous xenografts of ND1 and ATP6 cybrids into NOD–SCID Il2rg−/− (NSG) mice had elevated primary tumor ROS levels and similar primary tumor growth rates. Xenografts of our most dysfunctional OXPHOS cybrid, CO1, formed tumors albeit with lowered growth rates. Uniquely, the ND1 and CO1 xenografts respectively exhibited diminished metastatic burden and no metastatic burden. Our results suggest that OXPHOS impairing mtDNA mutations are tolerable during primary tumor growth but reduce the severity and incidence of metastasis. Lastly, we generated heteroplasmic A375 cybrids, harboring both wild-type and mutant alleles, and developed a ddPCR method to characterize allelic frequency with single-cell precision. Extended in vitro passage of the heteroplasmic cybrids failed to significantly shift the heteroplasmic mtDNA allelic frequency. However, following subcutaneous xenografting of the heteroplasmic cybrids, primary tumors significantly shifted their variant allele frequency to favor the wildtype allele. In summary, these results indicate that a functional mtDNA haplotype is not absolutely required for tumorigenesis but contributes to metastatic efficiency. In addition, we experimentally demonstrated the existence of selective pressure in favor of functional mtDNA during tumorigenesis. Citation Format: Spencer Shelton, Sara House, Vijayashree Ramesh, Zhengkang Chen, Claire Llamas, Alpaslan Tasdogan, Ralph DeBerardinis, Sean Morrison, Prashant Mishra. OXPHOS impairing mitochondrial DNA mutations suppress melanoma growth and metastatic progression [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR003.

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-Leslie G. Desmangles, Joan Dayan, Haiti, history, and the Gods. Berkeley: University of California Press, 1995. xxiii + 339 pp.-Barry Chevannes, James T. Houk, Spirits, blood, and drums: The Orisha religion in Trinidad. Philadelphia: Temple University Press, 1995. xvi + 238 pp.-Barry Chevannes, Walter F. Pitts, Jr., Old ship of Zion: The Afro-Baptist ritual in the African Diaspora. New York: Oxford University Press, 1993. xvi + 199 pp.-Robert J. Stewart, Lewin L. Williams, Caribbean theology. New York: Peter Lang, 1994. xiii + 231 pp.-Robert J. Stewart, Barry Chevannes, Rastafari and other African-Caribbean worldviews. London: Macmillan, 1995. xxv + 282 pp.-Michael Aceto, Maureen Warner-Lewis, Yoruba songs of Trinidad. London: Karnak House, 1994. 158 pp.''Trinidad Yoruba: From mother tongue to memory. Tuscaloosa: University of Alabama Press, 1996. xviii + 279 pp.-Erika Bourguignon, Nicola H. Götz, Obeah - Hexerei in der Karibik - zwischen Macht und Ohnmacht. Frankfurt am Main: Peter Lang, 1995. 256 pp.-John Murphy, Hernando Calvo Ospina, Salsa! Havana heat: Bronx Beat. London: Latin America Bureau, 1995. viii + 151 pp.-Donald R. Hill, Stephen Stuempfle, The steelband movement: The forging of a national art in Trinidad and Tobago. Philadelphia: University of Pennsylvania Press, 1995. xx + 289 pp.-Hilary McD. Beckles, Jay R. Mandle ,Caribbean Hoops: The development of West Indian basketball. Langhorne PA: Gordon and Breach, 1994. ix + 121 pp., Joan D. Mandle (eds)-Edmund Burke, III, Lewis R. Gordon ,Fanon: A critical reader. Oxford: Blackwell, 1996. xxi + 344 pp., T. Denean Sharpley-Whiting, Renée T. White (eds)-Keith Alan Sprouse, Ikenna Dieke, The primordial image: African, Afro-American, and Caribbean Mythopoetic text. New York: Peter Lang, 1993. xiv + 434 pp.-Keith Alan Sprouse, Wimal Dissanayake ,Self and colonial desire: Travel writings of V.S. Naipaul. New York : Peter Lang, 1993. vii + 160 pp., Carmen Wickramagamage (eds)-Yannick Tarrieu, Moira Ferguson, Jamaica Kincaid: Where the land meets the body: Charlottesville: University Press of Virginia, 1994. xiii + 205 pp.-Neil L. Whitehead, Vera Lawrence Hyatt ,Race, discourse, and the origin of the Americas: A new world view. Washington DC: Smithsonian Institution Press, 1995. xiii + 302 pp., Rex Nettleford (eds)-Neil L. Whitehead, Patricia Seed, Ceremonies of possession in Europe's conquest of the new world, 1492-1640. Cambridge: Cambridge University Press, 1995. viii + 199 pp.-Livio Sansone, Michiel Baud ,Etnicidad como estrategia en America Latina y en el Caribe. Arij Ouweneel & Patricio Silva. Quito: Ediciones Abya-Yala, 1996. 214 pp., Kees Koonings, Gert Oostindie (eds)-D.C. Griffith, Linda Basch ,Nations unbound: Transnational projects, postcolonial predicaments, and deterritorialized nation-states. Langhorne PA: Gordon and Breach, 1994. vii + 344 pp., Nina Glick Schiller, Cristina Szanton Blanc (eds)-John Stiles, Richard D.E. Burton ,French and West Indian: Martinique, Guadeloupe and French Guiana today. Charlottesville: University Press of Virginia; London: Macmillan Caribbean, 1995. xii + 202 pp., Fred Réno (eds)-Frank F. Taylor, Dennis J. Gayle ,Tourism marketing and management in the Caribbean. New York: Routledge, 1993. xxvi + 270 pp., Jonathan N. Goodrich (eds)-Ivelaw L. Griffith, John La Guerre, Structural adjustment: Public policy and administration in the Caribbean. St. Augustine: School of continuing studies, University of the West Indies, 1994. vii + 258 pp.-Luis Martínez-Fernández, Kelvin A. Santiago-Valles, 'Subject People' and colonial discourses: Economic transformation and social disorder in Puerto Rico, 1898-1947. Albany: State University of New York Press, 1994. xiii + 304 pp.-Alicia Pousada, Bonnie Urciuoli, Exposing prejudice: Puerto Rican experiences of language, race, and class. Boulder: Westview Press, 1996. xiv + 222 pp.-David A.B. Murray, Ian Lumsden, Machos, Maricones, and Gays: Cuba and hom*osexuality. Philadelphia: Temple University Press, 1996. xxvii + 263 pp.-Robert Fatton, Jr., Georges A. Fauriol, Haitian frustrations: Dilemmas for U.S. policy. Washington DC: Center for strategic & international studies, 1995. xii + 236 pp.-Leni Ashmore Sorensen, David Barry Gaspar ,More than Chattel: Black women and slavery in the Americas. Bloomington: Indiana University Press, 1996. xi + 341 pp., Darlene Clark Hine (eds)-A. Lynn Bolles, Verene Shepherd ,Engendering history: Caribbean women in historical perspective. Kingston: Ian Randle; London: James Currey, 1995. xxii + 406 pp., Bridget Brereton, Barbara Bailey (eds)-Bridget Brereton, Mary Turner, From chattel slaves to wage slaves: The dynamics of labour bargaining in the Americas. Kingston: Ian Randle; Bloomington: Indiana University Press; London: James Currey, 1995. x + 310 pp.-Carl E. Swanson, Duncan Crewe, Yellow Jack and the worm: British Naval administration in the West Indies, 1739-1748. Liverpool: Liverpool University Press, 1993. x + 321 pp.-Jerome Egger, Wim Hoogbergen, Het Kamp van Broos en Kaliko: De geschiedenis van een Afro-Surinaamse familie. Amsterdam: Prometheus, 1996. 213 pp.-Ellen Klinkers, Lila Gobardhan-Rambocus ,De erfenis van de slavernij. Paramaribo: Anton de Kom Universiteit, 1995. 297 pp., Maurits S. Hassankhan, Jerry L. Egger (eds)-Kevin K. Birth, Sylvia Moodie-Kublalsingh, The Cocoa Panyols of Trinidad: An oral record. London & New York: British Academic Press, 1994. xiii + 242 pp.-David R. Watters, C.N. Dubelaar, The Petroglyphs of the Lesser Antilles, the Virgin Islands and Trinidad. Amsterdam: Foundation for scientific research in the Caribbean region, 1995. vii + 492 pp.-Suzannah England, Mitchell W. Marken, Pottery from Spanish shipwrecks, 1500-1800. Gainesville: University Press of Florida, 1994. xvi + 264 pp.

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Abstract Hypoxia and the development of spatially heterogeneous hypoxic and/or necrotic regions in non-small cell lung cancer (NSCLC) and other solid tumors is associated with chemotherapy and radiation resistance. One mechanism by which this could occur is through modification of the cell-cell interactions (or games) between the resistant and naïve cells within the tumor. Cell-cell interactions can modify the fitness of cells and thereby change the evolutionary dynamics of a tumor. Hypoxia may, in this way, change the game dynamics between cells and directly support maintenance of a population of resistant cells. To understand the mechanism by which hypoxic heterogeneity modulates evolutionary dynamics and therapy response, we combine computational modeling and in vitro experiments under a spatial hypoxia gradient. We utilize in-house game assay protocols to probe the evolutionary games between cell types. The experimental system is adapted from Carmona-Fontaine's MEMIC plate. This system allows us to create stable oxygen and nutrient gradients for 12 evolutionary replicates of NSCLC PC-9 cells. Fluorescence labeled gefitinib-resistant PC-9 cells and naïve parental PC-9 cells are co-cultured in a closed chamber with limited availability to oxygen and nutrient supply at one end. A green hypoxia probe is used to verify the establishment of a hypoxia gradient and a red Annexin V probe monitors apoptosis. Once microenvironmental gradients are established due to cellular consumption of oxygen and nutrients, images of cells are taken every 1.5 hours for 5 days. We carry out multiple experiments in which we vary the initial proportions of parental and resistant cells. Computationally, green and red fluorescence from the images captured by the microscopy system is quantified by an in-house image processing pipeline. We apply a random forest machine learning classifier, illumination correction, and object identification to each image to gather fluorescence counts for each well. These counts are used to compute the background rate of cell death and absolute vs. relative oxygen levels. The subsequent cell counts over time were used to calculate the growth rate (fitness) of the cell types in different environments and therefore the strength of environmental and frequency dependent interactions between the cell types. This framework allows for tightly coupled computational and in vitro experiments aimed at understanding and predicting the clinical implications of hypoxia resistance across a range of tumor types and microenvironments. We have measured the effect of hypoxia upon game interactions between gefinib-resistant and susceptible NSCLC cells. The evolutionary game changes with changing levels of hypoxia–and for the first time we are able to write down a continuous equation representing the game as a function of oxygen. Citation Format: Mina Dinh, Bezhou Feng, Jeff Maltas, Emily Dolson, Masahiro Hitomi, Steph Owen, Jacob Scott. Exploring the effect of hypoxia and spatial interactions on the dynamics between gefitinib resistant and naïve NSCLC cell lines [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A027.

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Abstract Duocarmycins are natural DNA alkylators which have potent toxicity with no established mechanism of resistance. Prodrug versions need to be developed to ensure a sufficient therapeutic window for cancer treatment (1). A series of duocarmycin bioprecursors have been developed in-house which lack a key hydroxyl trigger group, required for spirocyclisation and subsequent activation. This hydroxyl group can be regioselectivity restored by specific Cytochrome P450 enzymes (CYPs), with a primary focus on cancer associated isoforms CYP1A1 and CYP2W1 (2). An exploratory library of analogues of our lead compound, ICT2700 (CYP1A1 and CYP2W1 activated), has been synthesized and elucidated aspects of the structure-activity relationship (SAR) between the compound and subsequent toxicity in CYP transfected and mock cell lines, using MTT assays. The data shows the importance of having an indole in the DNA recognition subunit (the non-alkylating subunit) for CYP1A1 and CYP2W1 activation. Further, a compound with thus far unexplained activity was identified. ICT11038 has a benzofuran instead of an indole in the recognition subunit and subsequently demonstrates inherent nanomolar toxicity consistently in all cell lines tested, including a range of cancerous and non-cancerous cells, which is not dependent on cytochrome expression. CYP1A1 has been shown to enhance the toxicity of ICT11038 further but is not required for nanomolar toxicity. Further SAR exploration and western blots confirm that the inherent toxicity is achieved through DNA damage despite lacking the key trigger group, suggesting a novel activation pathway. Additionally, our SAR studies show that replacing the indole of the DNA alkylation subunit with a benzofuran ablates CYP dependent activity, with one exception, ICT2724. ICT2724 is non-toxic in mock transfected cells but is toxic in CYP1A1 transfected cells, albeit it at higher concentration than ICT2700. ICT2724 is analogous to ICT2700, except for the direct indole to benzofuran substitution. Other compounds with benzofurans in the alkylating subunit compounds are not metabolized to a toxic compound by CYP1A1 or CYP2W1 despite their indole equivalents being good prodrug candidates. To conclude, the introduction of the benzofuran functional group into the CYP-activated duocarmycin bioprecursor pharmacophores produces an unexplained pattern of CYP activation and, in one example, an intrinsically toxic DNA damaging agent (ZYP-11038). References (1) Z. Jukes et al, Drug Discov Today, 2020, 26(2), 577-584, https://doi.org/10.1016/j.drudis.2020.11.020 (2) H. Sheldrake et al, J. Med. Chem, 2013, 56(16); 6273-6277, dx.doi.org/10.1021/jm4000209 Citation Format: Zoe H. Jukes. Effect of benzofuran pharmacophores on CYP-activated duocarmycin bioprecursors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4050.

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Abstract Ovarian cancer is the most lethal gynecological malignancy in the United States, with a known predilection for metastasizing to the omentum, a sheet of fatty tissue that encloses the abdomen. While it has been shown that ovarian cancer cells invade towards mature adipocytes in vitro, the contribution of stromal cells that reside in the omentum is not well understood. We hypothesize that preadipocytes, the precursor cells that give rise to omental adipocytes, are important mediators of cancer progression in the omental tumor microenvironment as they have a pro-inflammatory secretome that is distinct from that of mature adipocytes. To evaluate this hypothesis, we tested whether preadipocyte secreted factors altered tumorgenicity of ovarian cancer cells using colony formation and cell viability in vitro and a limiting dilution in vivo in a subcutaneous mouse model. The mouse studies showed that low dilutions of cancer cells require preadipocytes for engraftment and tumor formation. A transplantation assay showed that cancer cells require the presence of preadipocytes for sustained tumor formation capacity. In vitro co-culture assays revealed that preadipocytes secrete factors that increase clonogenicity and extend cell viability in serum-free conditions. To identify signaling pathways induced by preadipocytes we co-cultured cancer cells with either primary human omental preadipocytes or differentiated mature adipocytes derived from the same female donor and performed RNA sequencing and gene set enrichment analysis (GSEA). We identified differentially expressed genes in cancer cells that were unique to preadipocyte co-culture, including DCN, MMP2, COL6A2 and COL12A1, genes involved in extracellular matrix organization. The most significantly upregulated gene was insulin-like growth factor binding protein 5 (IGFBP5), which has functions in insulin-like growth factor signaling and ECM interactions. Ongoing studies using CRISPR edited cancer cells suggest IGFBP5 mediates the enhanced tumorigenesis of cancer cells in the presence of preadipocytes. Our findings highlight the role of preadipocytes in the ovarian tumor microenvironment and implicate IGFBP5 as a preadipocyte-mediated gene. Future studies will determine if there is a link between the upregulation of IGFBP5 and modulation of extracellular matrix proteins that promote cancer progression. Understanding these processes will enable the design of more effective therapies for treating ovarian cancer. Citation Format: Jennifer A. Waters, Mikella Robinson, Samuel F. Gilbert, Ixchel Urbano, Carrie D. House. Omental preadipocytes support ovarian cancer tumorigenesis by mediating genes important for extracellular matrix reorganization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2535.

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Abstract Background: Extracellular vesicles (EVs) are secreted by all cells into extracellular fluids and are found in most body fluids, including blood and urine. EVs contain many bioactive molecules, including proteins, RNA, DNA, and phospholipids. EVs have been reported to be associated with various diseases, including cancer metastasis and progression, suggesting that they play an important role in cell-to-cell communication. EVs are a promising modality for liquid biopsy and their clinical applications are desired, but methods for EVs isolation and quantification are still controversial. Traditionally used methods include ultracentrifugation (UC), polymer-based precipitation, and immunoprecipitation (IP) against surface marker antigens, but they are often inadequate in recovery efficiency and purity and are not standardized. In addition, it is very difficult to separate and recover EVs from body fluids that contain many contaminants. Therefore, we developed the original IP method (EViSTEPTM) in which a chelating agent-based reagent combined with in-house antibodies (CD9 and CD63) separate EVs with high purity and high recovery from body fluids such as serum and plasma for clinical applications. In this study, we evaluated whether EViSTEPTM in combination with expression levels of a newly identified long non-coding RNA (lncRNA), LOC100507412 (HEVEPA), could be used for stratification of pancreatic ductal adenocarcinoma (PDAC). Methods: EVs were isolated from serum of PDAC (22 cases), intraductal papillary mucinous neoplasm (IPMN) (23 cases), and healthy controls (HC) (21 cases) using EViSTEPTM, and HEVEPA expression analysis was performed by digital droplet PCR. Results: In the analysis of HC, IPMN, and PDAC samples, HEVEPA was significantly more highly expressed in PDAC compared to HC and IPMN. In ROC analysis between PDAC versus non-PDAC (IMPN and HC), the AUC of HEVEPA (0.80) was comparable to other tumor makers such as CEA (0.48) and CA19-9 (0.89). Furthermore, the AUC improved to 0.99 when HEVEPA was combined with CA19-9 but was not significantly different from that of CA19-9 alone. Conclusion: EViSTEPTM enables us to analyze HEVEPA in EVs, and the combination of HEVEPA and CA19-9 is expected to improve the diagnostic performance of PDAC. The combination of the EViSTEPTM method of EVs isolation and HEVEPA expression levels was suggested to be useful for the stratification of PDAC. Citation Format: Yuta Shimizu, Kenji Takahashi, Fumi Asai, Tatsutoshi Inuzuka. Development of the diagnostic method for pancreatic cancer using novel isolation technology of extracellular vesicles. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P048.

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Abstract The stromal tumor microenvironment plays a central role in the development, progression and therapy resistance of prostate cancer (PCa), the second leading cause of cancer-related death in males in western nations. Functional and single cell RNA sequencing (scRNA-seq) studies unequivocally demonstrate the coexistence of functionally and spatially distinct fibroblast and mural cell states within the tumor microenvironment of various non-prostate tumors. Stromal cellular heterogeneity in PCa however remains poorly understood. This study therefore aimed to (1) comprehensively analyze stromal cell states within the PCa microenvironment and (2) to identify and characterize onco-supportive fibromuscular entities in respect to their potential clinical exploitation. Thus, we performed bulk tissue scRNA-seq on biopsy cores from benign and cancerous prostate tissue of 4 different patients. Analyses presented here focused on the fibroblast and mural cell clusters, which each delineated further into 2 subclusters. One fibroblast subcluster showed strong similarity to published signatures of inflammatory cancer-associated fibroblasts (iCAF). A closely related but distinct fibroblast subcluster exhibited hallmarks of benign interstitial fibroblasts with enrichment of GO terms such as antigen processing and presentation. Based on expression signatures and spatial localization via immunohistochemistry, the 2 mural cell subclusters (MCAM+) were identified as vascular smooth muscle cells (vSMCs) and dedifferentiated prostate SMCs/myofibroblasts. Notably, the gene signature of this latter subcluster correlated with increasing Gleason score and decreased disease-specific survival. On the basis of scRNA-seq data from this and independent datasets, panels of subcluster-specific markers were selected for validation studies via FACS, multiplex immunohistochemistry and quantitative real time PCR. Thus far, immunohistochemical studies validated not only the in vivo presence of the 4 distinct cellular states but also revealed their distinct spatial localization. We will present molecular and functional data from these ongoing analyses in (i) immortalized fibroblast and smooth muscle cell lines, (ii) heterogeneous ex vivo outgrowth cultures of primary PCa fibroblasts/myofibroblasts and (iii) primary cells from single cell suspensions of PCa biopsies enzymatically digested using an in-house approach optimized for maximal recovery of fibromuscular cells. These studies aim to evaluate the specificity, robustness and suitability of the chosen markers to delineate distinct tumor-relevant stromal cell subtypes enabling their functional contribution to PCa pathophysiology and clinical relevance to be assessed in future experiments. Citation Format: Elisabeth Damisch, Elena Brunner, Sieghart Sopper, Isabel Heidegger, Andreas Pircher, Georgios Fotakis, Zlatko Trajanoski, Sandra Theresa Deichsler, Georg Schäfer, Natalie Sampson. Functional heterogeneity of cancer-associated stromal subtypes in the prostate cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4013.

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Abstract Background: Human conventional dendritic cells (cDC) are essential for the anti-tumor immune response. Their immunogenic and tolerogenic states in cancer remain controversial. Our objective was to define the range of DC activation states in vitro and to determine their relevance ex vivo in cancer. Methods: First, in vitro, we activated healthy donor cDC with 16 different stimuli and measured cDC and T cells cytokine and chemokine secretion upon co-culture (n = 130 individual experiments) to define new maturation archetypes. Second, we established the relevance of these archetypes in cancer, ex vivo in human. We performed in depth analysis of cDC infiltrating head and neck cancer (HNSCC) by flow cytometry (n = 22 patients), transcriptomic analysis (n = 6 patients), and cDC-enriched single-cell transcriptomics (ScRNAseq) (n = 10503 cells from 2 patients). Then we performed a merged analysis of cDC from in house and public ScRNAseq datasets (n = 36 tumor samples from HNSCC, Lung, and Breast cancer, and, as comparator, n = 33 blood and juxtatumor samples, n = 19 healthy donor samples, and n = 23 samples from inflammatory diseases). Results: In vitro, we identified two archetypes of cDC activation: (i) PD-L1highICOSLlow cDC that produce large amounts of inflammatory cytokines and chemokines, labeled Secretory cDC; (ii)PD-L1lowICOSLhigh cDC that induce a broad range of Th cytokines, labeled Helper cDC. This functional dichotomy of cDC was mutually exclusive and not receptor specific. In cancer, we identified Secretory cDC, and these cells aligned with mature LAMP3+cDC. Helper cDC were not found ex vivo. Secretory cDC simultaneously expressed stimulatory (CD40, TNFRSF9 (4-1BB)), inhibitory (CD274, CD200, IDO1), and mixed (PVR) checkpoints. Secretory cDC were associated with T cell inflammation in HNSCC, triple-negative breast cancer (TNBC), and melanoma (all p < 1.10-15), with improved overall survival in HNSCC and TNBC (all p < 0.01), and with response to checkpoint blockade in 2 melanoma cohorts (all p < 0.01). Conclusion: We identify and characterize Secretory cDC in several solid cancers. This novel phenotypic and functional dichotomy of human cDC activation states has broad implications for all types of immunotherapies and provides rational for drug combinations. Citation Format: Caroline Hoffmann, Floriane Noël, Maximilien Grandclaudon, Lucile Massenet-Regad, Paula Michea, Philemon Sirven, Lilith Faucheux, Aurore Surun, Olivier Lantz, Mylene Bohec, Jian Ye, Weihua Guo, Juliette Rochefort, Jerzy Klijanienko, Sylvain Baulande, Charlotte Lecerf, Maud Kamal, Christophe Le Tourneau, Maude Guillot-Delost, Vassili Soumelis. PD-L1 high ICOSL low Secretory dendritic cells infiltrate human solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2105.

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Abstract Ovarian cancer is the most lethal gynecologic malignancy in the US. Although high-grade serous ovarian cancer (HGSOC) patients initially respond to chemotherapy, over 70% relapse in two years. This paradigm can be understood through ovarian cancer’s pronounced heterogeneity whereby a majority of cancer cells are highly proliferative and chemosensitive and a minority of cells, termed tumor-initiating cells (TICs), are relatively quiescent and chemoresistant stem-like cells. Both are presumed to be important for tumor repopulation. Our lab previously demonstrated that TICs exhibit an upregulation of stem cell genes and NF-κB signaling. RNA sequencing of cells grown in TIC enhancing, non-adherent conditions shows significantly increased expression of NF-kB genes NFKB2, RELA, and RELB, as well as stem cell genes SOX2 and ALDH1A2. In this study, we are investigating the mechanism through which the NF-κB transcription factors RelA and RelB support TICs to promote relapse in ovarian cancer. RNA sequencing of shRNA knockdowns of RELA or RELB from cells grown in TIC conditions shows that RELA knockdown impacts 1415 unique genes, RELB knockdown impacts 2016 unique genes, and RELA or RELB knockdown decreases expression of 1912 shared genes. Gene ontology analysis suggests RELA regulates genes in growth and differentiation pathways while RELB regulates genes in metabolic pathways. Specifically, RELA knockdown significantly decreased expression of NF-kB pathway genes (RELA, RELB, NFKB2) as well as stem cell genes CD117 and ALDH1A2. RELB knockdown significantly decreased expression of NF-kB pathway genes (RELA, RELB) as well as stem cell genes CD117, CD133, ALDH1A1, and ALDH1A2. To expand on these findings we performed ChIP-sequencing of RelA and RelB in OV90 cells cultured in TIC enhancing or adherent monolayer conditions. Our results show that both RelA and RelB bind at promoter sites for NFKBIA and NUAK1 in both conditions. In monolayer cultures RelA uniquely binds 14 different genes, including lncRNAs, miRNAs, and Cyclin L1, important in G0-G1 cell cycle progression. In TIC conditions, RelB uniquely binds 16 different genes, including lncRNAs, miRNAs, and WNT10A, important in stem cell self-renewal. Experiments are underway to validate our top hits in ovarian cancer, using siRNA knockdowns to corroborate the genes and pathways through which RelB supports self-renewal and RelA supports cell cycle progression. The ultimate goal of this project is to identify downstream pathways regulated by NF-kB that can be targeted to overcome relapse in HGSOC. Citation Format: Emily M. Mu, Samuel F. Gilbert, Mikella Robinson, Jacqueline Lara, Omar Lujano-Olazaba, Christina M. Annunziata, Carrie D. House. Using ChIP-seq to identify genes regulated by RelA or RelB that support ovarian cancer tumor-initiating cell (TIC) characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5727.

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Abstract Sialoglycans have emerged as a critical glyco-immune checkpoint that impairs antitumor response by inhibiting innate and adaptive immunity. We have previously reported that Bi-Sialidase - an engineered human sialidase-Fc fusion - potentiates antitumor immune response by cleaving terminal sialic acids from sialoglycans (desialylating) on tumor cells and immune cells. Furthermore, we have shown that a tumor-targeted sialidase, a heterodimeric molecule consisting of one chain of sialidase-Fc and a second chain of a HER2-targeting antibody (trastuzumab), leads to more efficient desialylation of tumor cells than the non-targeted Bi-Sialidase and demonstrates antitumor activity in trastuzumab-resistant and low HER2-expressing tumor models. To evaluate the potential of targeted desialylation of both tumor cells and immune cells, we designed and characterized a PD-L1-targeted sialidase in preclinical models, since PD-L1 is expressed on both tumor cells and immune cells. Furthermore, the design of the PD-L1-targeted sialidase enables the combinatorial blockade of two orthogonal immune checkpoint pathways, inhibiting both the PD-1/PD-L1 axis and immunosuppressive sialoglycans. We generated a humanized anti-human PD-L1 antibody with comparable PD-1/PD-L1 blockade potency to the existing anti-PD-L1 drugs, atezolizumab and avelumab. Subsequently, we constructed a bifunctional heterodimeric molecule, consisting of one chain of sialidase-Fc of a third generation of engineered human sialidase (Neu 2) and a second chain of the in-house generated anti-PD-L1 antibody. The PD-L1-targeted sialidase maintained its potency for inhibiting the PD-1/PD-L1 axis as compared to its parental anti-PD-L1 antibody, and demonstrated improved desialylation of PD-L1-expressing tumor cells and immune cells in vitro. We tested the PD-L1-targeted sialidase in transgenic mouse tumor models that express human PD-1 and PD-L1 replacing their murine counterparts, since the parental PD-L1 antibody doesn’t cross-react with the mouse antigen. In the transgenic human PD-L1-expressing mouse colon carcinoma CT26 subcutaneous tumor model, the PD-L1-targeted sialidase exhibited enhanced efficacy compared to the Bi-Sialidase or the anti-PD-L1 antibody. Furthermore, the PD-L1-targeted sialidase demonstrated a dose-dependent tumor growth inhibition and modulation of immune cell infiltration. In conclusion, these results suggested that PD-L1-targeted sialidase offers a promising cancer immunotherapeutic approach, which simultaneously inhibits immunosuppressive sialoglycans and the PD-1/PD-L1 axis through targeted delivery of engineered human sialidase to PD-L1-expressing tumor cells and immune cells. Citation Format: Jenny Che, Lihui Xu, Wayne Gatlin, Robert LeBlanc, Lizhi Cao, James Broderick, Li Peng. Development of PD-L1-targeted sialidase as a novel cancer immunotherapeutic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB221.

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Abstract Background: Resistance mechanisms to anti-PD1 therapy remain poorly understood. Studies have implicated that efficient anti-PD1 therapy relies specifically on intact CD28/B7 signaling. We have previously shown that membranal CD28 is actively shed by MMP-2 and MMP-13 upon T cell stimulation in humans, and that soluble CD28 can counteract the efficacy of anti-PD1 blocking antibodies in-vitro. Here we describe that CD28-shedding process is a potential novel resistance mechanism to PD-1 therapies in cancer patients, by using several ex-vivo and in-vivo methodologies. The prominent unfavorable effect of CD28-shedding on PD-1 blockade prompted the generation of BND-67, an agent that can selectively and efficiently block CD28 shedding. Methods: Soluble CD28 detection in plasma of cancer patients and in the culture media of stimulated T cells was achieved using a proprietary in-house ELISA. Cancer patient processed tumors and PBMC were used ex-vivo to identify membrane/soluble CD28 by ELISA and flow-cytometry. MC-38 challenged human CD28 transgenic mice (CD28-shedding permissive model), and WT mice (non-permissive CD28-shedding model) were compared for efficacy to anti-PD1 blocking antibody. BND-67 was isolated by screening a VHH library for binding to the CD28-shedding prone region and blocking of CD28 shedding. The lead clone was further affinity-matured and reformatted into a half-life extended construct. Results: CD28 receptor density was found to be lower on tumor-resident T cells then their matched counterparts in the periphery. Evaluation of plasma samples from patients undergoing anti-PD-1 therapy demonstrated an association of diseases relapse with elevated plasma levels of soluble CD28. Correspondingly, relapses from response to anti-PD-1 therapy were significantly higher in CD28-shedding permissive mice vs non-permissive mice. To overcome this novel regulatory mechanism, we generated BND-67, which is an affinity-matured VHH that can effectively bind CD28, block CD28 shedding and has no agonistic activity. In addition, an optimal approach for increasing the half-life of the agent was identified by fusing the VHH to human Fc. Conclusions: We report here the discovery of a potential novel resistance mechanism to anti-PD1 blockade involving the shedding of the CD28 receptor. This process occurs in the setting of cancer and was further reinforced by results observed in transgenic mice. Biond has generated BND-67, a therapeutic drug candidate for the inhibition of this novel regulatory mechanism. Citation Format: Motti Hakim, Anna Fridman Dror, Tal Shahar Gabay, Dror Alishekevitz, Edna Meilin, Ilana Mandel, Yair Sapir, Avidor Shulman, Tehila Ben Moshe. CD28 shedding is a novel resistance mechanism to anti PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 654.

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Abstract Background: Glioblastoma is the most prevalent and severe type of malignant brain tumor in adults. Although the genetic make-up initiating glioblastoma is increasingly better understood, a better understanding in the mechanisms that drive its evolution, heterogeneity and therapy resistance may reveal new directions for therapy development. To get better insights into glioblastoma evolution, we analyzed and de-convoluted transcriptomes of primary and recurrent glioblastoma resections. Material and Methods: Matching primary and secondary resections from n=185 glioblastoma patients were collected as part of EORTC Study 1542. The study was extended with tumor pairs from n=51 patients from the international GLASS study. The datasets were subjected to differential and deconvolution analysis using in-house algorithms. Results: When mapping the tumor samples into a reduced Glioblastoma Intrinsic Transcriptional Subtype space, we visualized subtype traversal, indicating that the CL subtype most often switches. As we found no more transitions from MES to other subtypes than to be expected by chance, we concluded that MES is an end-state. On average, tumor cell percentages decreased from ~67% to ~50% mostly due to an increase in TAM/microglia. Differential expression analysis was performed with correction for tumor cell percentages. While expression of most known oncogenes did not change considerably over time, marker genes of TAM/microglia, neurons and oligodendrocytes were up-regulated whereas endothelial cell markers were down-regulated over time. Furthermore, a cluster of ~30 extracellular matrix-associated genes increase significantly over time. A signature representing the gene-set was significantly associated with poor survival; high signatures were in particular associated to survival in secondary resections (P = 6.613e-06, Kaplan-Meier estimator). This suggests that the increase of extracellular matrix expression fulfils an important role in glioblastoma evolution. Conclusion: Using a large cohort, we interrogated changes in the glioblastoma transcriptome over time and found that in particular the composition of the tumor and its environment changes. The tumor cell percentage drops, suggesting more invasion or recruitment of non-malignant cells or a combination of both. This change is independent of an increase in the prognostic increase in extracellular matrix expression. Citation Format: Youri Hoogstrate, Kaspar Draaisma, Santoesha A. Ghisai, Iris de Heer, Levi van Hijfte, Wouter Coppieters, Melissa Kerkhof, Astrid Weyerbrock, Marc Sanson, Ann Hoeben, Slávka Lukacova, Giuseppe Lombardi, Sieger Leenstra, Monique Hanse, Ruth Fleischeuer, Colin Watts, Joseph McAbee, Nicos Angelopoulos, Thierry Gorlia, Vassilis Golfinopoulos, Johan M. Kros, Vincent Bours, Martin J. van den Bent, Pierre A. Robe, Pim J. French. Transcriptional evolution of glioblastoma reveals changes in bulk composition, mesenchymal sub-type as end-state, and a prognostic association with increased extracellular matrix gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6140.

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Abstract Introduction: Orthotopic tumour mouse models are often preferred to subcutaneous models due to their increased clinical relevance and pathophysiological features, as the tumour microenvironment can influence stromal interaction, immune response, angiogenesis, tumour growth and progression as well as response to treatment. Orthotopic implantation of liver cancer cell lines can be time consuming and inefficient due to the surgical procedures involved to establish the tumour. This study assesses the benefits of replacing traditional invasive surgical orthotopic implantation of two hepatic tumour cell lines with a minimally invasive ultrasound guided approach. Tumour growth of both implantation methods were assessed using ultrasound and bioluminescence, as well as duration of surgery and animal recovery time. In addition, the immune landscape of both implantation methods was assessed using flow cytometry. Methods: Hep3B and Hepa 1-6 cells were transfected with luciferase for bioluminescence imaging. Hep3B-luc or Hepa 1-6-luc cells were orthotopically injected into the left liver lobe of C57BL/6 mice via either conventional surgery or ultrasound guided injection. Tumour growth was measured using both Ultrasound (Fujifilm Vevo 3100 imaging system, Visualsonics) and bioluminescence (IVIS® Spectrum in vivo imaging system, Perkin Elmer). Surgery times were recorded as the duration each animal was under anaesthesia. Recovery times were reported as the time animals were held in recovery before returning to their cage. An in house validated flow cytometry T cell panel was used to assess T cell infiltration in the Hepa1-6 syngeneic model. Results: A comparison of tumour volume versus tumour associated bioluminescence demonstrated good correlation between both methods for each cell line. The duration of surgery and post implantation recovery times were significantly reduced for mice that underwent ultrasound-guided implantation compared to surgery, as well as fewer post-operative clinical observations. No significant difference in body weight was observed throughout the study. T cell infiltration by flow cytometry assessment enabled further evaluation of potential differences in the immune landscape of Hepa 1-6 tumours. Conclusion: Ultrasound guided implantation of orthotopic hepatic tumours is a minimally invasive technique that leads to improved recovery times for animals, which adheres to the principles of the 3R’s. The time taken for ultrasound guided implantation compared to traditional surgical methods was also significantly reduced, enabling a higher throughput of animals on study. Tumour growth kinetics in both Hep3B and Hepa 1-6 models were comparable between the 2 methods. Citation Format: Chris Payne, Yasmin Amer, Ruth Storer, Ganisha Hutchinson, Lucy Harris, Amanda Miles, Yinfei Yin. Comparison between surgical and ultrasound-guided orthotopic implantation of hepatocellular carcinoma models in mice using ultrasound and bioluminescence imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1660.

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Abstract OBJECTIVE. A clinical challenge of ductal carcinoma in situ (DCIS) is to accurately predict individual risk for recurrence. Prior studies have highlighted the importance of stromal collagen surrounding breast ducts in breast cancer aggression. We studied the collagen structure surrounding DCIS in a matched case-control cohort of women with DCIS using second-harmonic generation (SHG) and hematoxylin and eosin (H&E) images to determine whether stromal collagen could provide additional risk information. METHODS. A cohort of 122 patients with DCIS (61 cases with recurrence matched with 61 controls) were retrospectively analyzed. Whole-side SHG and H&E images were obtained and regions-of-interest (ROI) of stromal collagen adjacent to DCIS was automatically segmented using an in-house trained deep learning algorithm. A quantitative histology image analysis pipeline (termed “histomics”) was exploited to extract a total of 123 histomics features (intensity, statistical and textural) from the segmented stromal region from both SHG and H&E ROI images. Clinical features previously shown to associate with DCIS recurrence including tumor size, margins and treatment were also collected. Conditional logistic regression model with lasso penalty was used to assess the performance of these features to stratify patients with and without recurrence. RESULTS. We found the combination of SHG, H&E and clinical features reached the highest concordance of 0.93 and the combination of only SHG and H&E features reached a concordance of 0.91. In contrast, clinical features alone attained a concordance index of 0.86. Model comparison by the Akaike information criterion (AIC​) shows that the higher concordance of the combination of SHG, H&E and clinical features may indicate better discrimination, whereas the higher concordance of combination of only SHG and H&E features was merely the result of including the larger number of features. Various textural features were found to be statistically significant (p<0.05) in both the SHG and H&E images, while the intensity or statistical features did not reach statistical significance. CONCLUSION. Textural stromal features extracted from SHG and H&E image-based histomics indicate a significant association of some features with DCIS recurrence. Combination of these histomics features with conventional clinical features are a promising quantitative tool to predict recurrence in patients with DCIS. Citation Format: Taman Upadhaya, Mary-Kate Hayward, Mi-Ok Kim, Ronald Balassanian, Valerie Weaver, Olivier Morin, Catherine Park. Prediction of breast ductal carcinoma in situ recurrence using histomics analysis of stromal collagen from second-harmonic generation and hematoxylin and eosin stain-based images [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-02-16.

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Abstract This poster describes logistical issues related to implementing a randomized, double- blinded, placebo-controlled Phase III interventional trial on the nine-valent HPV vaccine (9vHPV) among cisgender men and transgender women living with HIV, at the Mexico site. The trial seeks to demonstrate that 9vHPV reduces the incidence of persistent oral HPV infection (a surrogate for HPV-associated oropharyngeal cancer) with the 9 vaccine types. Five-hundred participants will be randomized in a 1:1 allocation to receive 9vHPV or placebo, stratified based on clinical site (Brazil, Mexico, Puerto Rico) and age. The team invites potential participants through local community organizations and public HIV clinics. People may be invited when waiting in line in the morning to get laboratory testing done, when they have an appointment for HIV care or through their treating physician or a community organizer. Initially, we worked at a single HIV clinic, although we did distribute study flyers to treating physicians at other clinics. As of mid-2022 we began enrolling at two additional clinics. Participants are prescreened when initially invited or by phone to prevent unnecessary trips for those ineligible. Once prescreened, participants are given an appointment for their first study visit; reminders about their first or other study visits are sent by text message 2-3 times before the appointment. Participants are provided with financial compensation in cash at each visit. We have implemented both study-wide mechanisms and additional locally-designed strategies and forms to guarantee quality control. For example, registering participant issues, study agent trail and persons invited, pre-screened and enrolled (including reasons for exclusion). Data is registered on paper forms and in a bespoke data base program (DatStat, designed at Moffitt Cancer Center). DatStat carries out the randomization (only the Mexico site pharmacist is unblinded) and requires a wireless internet connection, which can sometimes fail even though a router is installed by the study at each clinic. Vaccine and syringe importation can be time consuming and cause enrollment delays, given the need to acquire permissions for importation or problems getting the shipment out of Customs. Making sure shipments go through an airport with better functioning Customs offices is also important. Citation Format: Betania Allen-Leigh, Alejandra Portillo-Romero, Manuel Quiterio, Maribel Acosta, Abraham Rivera- Ramirez, Guillermina Sanchez, Aurelio Cruz, Tonatiuh Barrientos, Carlos Magis, Kimberly Isaacs-Soriano, Martha E. Abrahamsen, Margaret House, Emma Brofsky, Vikrant Sahasrabuddhe, Timothy Wilkin, Anna Giuliano, Luisa Villa, Eduardo Lazcano-Ponce. Logistical issues in implementing a clinical trial on oral cancer prevention through HPV vaccination: Implementation of Ulacnet201 in Mexico [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A002.

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Abstract TMB is a biomarker with potential for predicting positive patient response to immune checkpoint inhibitors. TMB measurements can be determined using genomic DNA extracted from FFPE-preserved tissue biopsy samples. However, assessment of TMB from a liquid biopsy, or blood TMB (bTMB), is an attractive alternative clinical diagnostic tool that would allow clinicians and patients to avoid the invasive challenge of tissue biopsies. Accurately measuring bTMB with targeted gene panels has been problematic, thus we have developed the new Seraseq® bTMB reference materials at various mutational burden levels from genomic DNA extracted from tumor-derived and SNP-matched normal cell lines. Somatic variants were identified in each cell line by whole exome sequencing (WES). TMB assessments were made by an in-house developed TMB bioinformatics pipeline based on recommendations by the Friends of Cancer Research (FoCR) TMB Harmonization Consortium. DNA from tumor and normal cell lines were blended at 0%, 0.5%, and 2% tumor fractions and fragmented to approximate the size of cell free DNA (cfDNA). The resulting bTMB mixes were enriched with the TruSight™ Oncology 500 ctDNA Assay (2x151 bp), in triplicate, and sequenced on a NovaSeq™ 6000. Blood TMB scores were determined using the DRAGEN™ TruSight Oncology 500 ctDNA Analysis Software v1.1. The observed bTMB scores for the tumor-containing materials were slightly lower in the TF 0.5% mix versus the TF 2% mix due to variants moving below the limit of detection of the TSO500 ctDNA assay (Table 1). As is the case with patient samples and clonal hematopoiesis, there are background variants in 0% tumor content material that may elevate apparent bTMB scores, thus adjusted bTMB scores are shown. The Seraseq Blood TMB Score reference materials provide a tumor-normal matched blood TMB control to aid development, validation and QC of cfDNA NGS assays for accurate determination of bTMB Scores. Table 1. Average bTMB scores and adjusted bTMB scores for each mix. Mix Average bTMB Score (mutations/megabase ± 1 SD) Adjusted bTMB Score (mutations/megabase ± 1 SD) Score 7 0% 7.5 ± 1.7 0 Score 7 0.5% 13.1 ± 2.6 5.6 ± 3.1 Score 7 2% 17.9 ± 1.3 10.4 ± 2.1 Score 13 0% 4.6 ± 0.5 0 Score 13 0.5% 18.7 ± 2.1 14.1 ± 2.2 Score 13 2% 24.6 ± 0.8 20.0 ± 0.9 Score 20 0% 7.5 ± 1.4 0 Score 20 0.5% 26.0 ± 2.3 18.5 ± 2.7 Score 20 2% 35.6 ± 1.0 28.1 ± 1.7 Score 26 0% 6.0 ± 0.5 0 Score 26 0.5% 20.7 ± 5.5 14.7 ± 5.5 Score 26 2% 30.4 ± 1.5 24.4 ± 1.9 Citation Format: Dana Ruminski Lowe, Benedicta Forson, Matthew G. Butler, Yves Konigshofer, Catherine Huang, Omoshile Clement, Russell K. Garlick, Bharathi Anekella. Development of blood TMB (bTMB) reference materials for validation of cfDNA-based targeted NGS assays that measure tumor mutational burden (TMB) in patient blood samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 537.

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Abstract Background: For breast cancer patients undergoing lumpectomy, margin status remains an important risk factor for local recurrence. Positive margins mandate re-excision, and some patients may require multiple resections and mastectomy to obtain negative margins. In this study, association of various clinicopathologic factors with positive surgical margins for primary invasive breast cancer was assessed using both public and in-house datasets. Method: The datasets were obtained from The Cancer Genome Atlas-Breast Cancer (TCGA-BC) and Clinical Breast Care Project (CBCP). The proportion of positive to negative margins in both TCGA-BC (3.86%, 38/984) and CBCP (3.78%, 15/397) was similar. The role of each clinicopathologic factor for margin prediction was evaluated using logistic regression, Fisher’s exact test and Cox proportional hazards models. Results: The univariable and multivariable logistic regression using both TCGA-BC and CBCP data sets showed that higher tumor stage, tumor size and positive lymph nodes significantly contributed (p < 0.05) to positive surgical margins. In agreement with the results of logistic regression, Fisher’s exact test also showed the association of tumor stage with margin status. Furthermore, PAM50 subtype normal-like was significantly associated (p = 0.044) with positive margins while Her2 subtype was close to significance (p= 0.062) in TCGA-BC. However, they were not significant in the multivariable model. In addition, young patients also showed close significance to positive margins (p = 0.054) in the multivariable model. The bivariable cox hazards model demonstrated that margin status along with PAM50 subtype significantly associated with progression of the disease in TCGA-BC. On the other hand, margin status along with stage or TNM (T:Tumor size, N: Lymph Node Status, M: Metastasis) was insignificant. This shows that margin status was surrogate to tumor stage or TNM. Conclusion: This characterization study showed that chances of obtaining positive margins increases with higher tumor stage, tumor size and positive lymph nodes. Subtype and age also trended to affect margin status. If validated in a dataset of a larger number of cases with positive margin, our results may provide an additional perspective of how the margin status is associated with the clinical outcome of breast cancer patients which may further impact care provider’s decision making for re-excisions. Disclaimer: The contents of this publication are the sole responsibility of the author(s) and do not necessarily reflect the views, opinions or policies of USUHS, HJF, the DoD or the Departments of the Army, Navy or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsem*nt by the U.S. Government. Citation Format: Anupama Praveen Kumar, Diego Vicente, Praveen Kumar Raj Kumar, Jianfang Liu, Brenda Deyarmin, Brad Mostoller, Albert J. Kovatich, Jeffrey A. Hooke, Leigh Fantacone-Campbell, Xiaoying Lin, Craig D. Shriver, Hai Hu. Clinicopathologic factors associated with surgical margins in primary invasive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3363.

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Abstract Background: Epidermal growth factor receptor (EGFR) inhibitors, including monoclonal antibodies and tyrosine kinase inhibitors, have limited efficacy in head and neck squamous cell carcinoma (HNSCC) patients. In the randomized phase II PREDICTOR trial, we aimed at identifying predictive and pharmacodynamics biomarkers of 2-4 weeks afatinib (an irreversible pan-HER inhibitor) versus no treatment in the pre-operative setting (NCT01415674). We previously reported a 59% metabolic response rate on PET imaging in the afatinib arm. We report here the evaluation of predictive genomic and transcriptomic biomarkers of afatinib efficacy. Patients and Methods: All patients (41 in the afatinib arm and 20 in the no treatment arm) underwent a pre-treatment biopsy. We performed targeted DNA sequencing using an in-house NGS panel of 571 genes on baseline biopsies from 56 patients, and RNA-sequencing (RNAseq) in 54 patients. In the afatinib arm, 26 patients had paired pre- and post-treatment tumor samples. DNA and RNA alterations were correlated with metabolic response to afatinib using PET imaging, as well as overall survival (OS). Results: Most frequent molecular alterations, including known activating mutations and/or focal amplifications for oncogenes or hom*ozygous deletions and inactivating mutations for tumor suppressor genes, involved genome integrity (TP53 [70%]), cell cycle (CCND1 [38%], CDKN2A [32%], CDKN2B [14%]), senescence (TERT [23%]), Wnt signaling (NOTCH1 [16%]), and the PI3K pathway (PIK3CA [14%]). In the afatinib arm, metabolic response was observed in 1 out of 7 patients (14%) and in 19 out of 28 patients (68%) in the Wnt altered and unaltered groups (p = 0.03, fisher exact test), respectively. In the whole cohort of patients, hom*ozygous deletions of both CDKN2A and CDKN2B correlated with shorter OS, with 6-year survival of 22% in the CDKN2A/B altered group and 70% in the CDKN2A/B wild-type group (p = 0.004; log-rank test). In the afatinib treated patients, using a generalized linear mixed model with a patient as random effect and a quasi-binomial family, the ratio of B cells expression levels in the post-treated versus pre-treated samples was significantly higher in responder as compared to non-responder patients (p = 0.001). Conclusions: Wnt signaling pathway alterations and treatment-related dynamic changes in B cells proportions were identified as predictive and pharmacodynamics biomarkers of afatinib efficacy. CDKN2A/B hom*ozygous deletions were associated with a poor prognosis in HNSCC patients treated with upfront surgery. Citation Format: Grégoire Marret, Maud Kamal, Jocelyn Gal, Stéphane Temam, Jerzy Klijianenko, Jean-Pierre Delord, Caroline Hoffmann, Gilles Dolivet, Olivier Malard, Jerôme Fayette, Olivier Capitain, Caroline Even, Sébastien Vergez, Lionel Geoffrois, Frédéric Rolland, Philippe Zrounba, Laurent Laccourreye, Joël Guigay, Nicolas Aide, Valérie Bénavent, Constance Lamy, Elodie Girard, Marta Jimenez, Ivan Bièche, Christophe Le Tourneau. Randomized phase II trial of pre-operative afatinib in non-metastatic head and neck squamous cell carcinoma patients: Identification of predictive biomarkers of response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1237.

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Abstract Introduction: Analytical methods of extracellular vesicles (EVs) is becoming an increasingly promising field of study due to them being an effective biomarker for cancers. EVs are present in various types of body fluids, which can be easily used for diagnostic purpose. Prior research has explored numerous techniques for isolating and analyzing these EVs based on their physical and biochemical properties, however, the complexity of biological samples makes conventional EV isolation difficult to exclusively extract EVs of a certain type of cell. Current downstream analysis methods lack the ability to differentiate exosomes of different origins in a sample. Recent studies suggested the presence of certain proteins in cancer exosomes that facilitates preferential uptake of the exosomes by organ-specific cancer cells, called organotropism. Using this unique property, we devised a microfluidic platform to examine the uptake of specific exosomes onto their respective progenitor cell lines, thus aiming to use the interaction for cancer diagnosis purposes. Methods: The CellExoChip was prepared following the standard PDMS-based soft lithography method. The device consists of an inlet and outlet, with dimensions of 22x22 mm with a cell capture area of about 50 mm2. The microfluidic device was functionalized by Streptavidin and the cancer cells were biotinylated with EZ-Link-NHS-Biotin to create an intense affinity between the cells and the device. We further injected dyed exosomes with different origins through the device and evaluated their specific uptake using fluorescence microscope. Results: The prepared CellExoChip successfully immobilized over 1,500 cells onto the surface and viability evaluation demonstrated that only 6.79% of the initial cells were sacrificed during the biotinylation and on-chip binding process. The average on-chip cell viability showed 75.47±7.68%. The uptake of lung cancer cell exosomes into three different cancer cell lines (lung, bladder and breast) was measured on chip. The relative uptake of lung cell exosomes by the respective lung cells was 100% compared to the bladder cells and breast cell which were 15.87% and 40.31%, respectively. We extended this specific uptake evaluation to other LungCell-LungExo combinations using H1650, A549, and in-house CTC cell line, CTCR5, and the results demonstrated the organotropism nature of the exosomes in lung cancer. Discussion and conclusion: We present a novel screening method to accurately characterize specific cancer-derived EVs using a microfluidic platform. This process bypasses the requirement of analyzing and profiling these embedded proteins prior to EV isolation. Our microfluidic device facilitates this interaction between cells and exosomes through the diagnosis process of liquid biopsy. Citation Format: Kruthi Srinivasa Raju, Zeqi Niu, Joseph Marvar, Shawn Fortna, Nna-Emeka Onukwugha, Yoon-Tae Kang, Sunitha Nagrath. On-chip evaluation of cancer cell-extracellular vesicle interactions using a novel microfluidic microsystem (CellExoChip) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2792.

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Abstract Background: Personalized neoantigen cancer vaccines have recently showed marked therapeutic potential in both preclinical and early phase clinical studies. However, significant challenges remain in the accurate prediction of neoepitopes and the efficient delivery of the vaccine components for eliciting potent antitumor T cell responses. To overcome these challenges, we have developed novel vaccine platform designated BP1209, which is characterized by being composed of neoantigen peptides that contain a high affinity binding motif for human antibodies. Methods: We developed vaccine with enhanced efficacy designed to form a complex with checkpoint antibodies. The vaccine instantly assembles a divalent peptide-antibody complex by simply mixing with therapeutic antibodies in physiological condition. We evaluated the efficacy of BP1209 vaccine in combination with DC-targeting antibodies in vivo. Next, we generated a series of neoantigen peptides in both murine and human origins using in-house bioinformatic algorithms and evaluated the advantages of BP1209 vaccine. Results: BP1209 vaccine showed robust T-cell response and anti-tumor activity when combined with anti-CD40 agonistic antibody or atezolizumab compared with a control peptide vaccine or the antibody alone. Treatment of BP1209 murine neoantigen vaccine to syngeneic tumor bearing mice exhibited robust anti-tumor response resulting in complete tumor regression in therapeutic setting. Preclinical evaluation of neoantigen vaccines from murine and human tumors showed notably increased CD8+ T cell response even for epitopes that do not respond with a conventional long peptide vaccine. Further analysis of DCs and T cells induced by BP1209 suggested that peptide delivery was mediated by increased cross-presentation of epitope peptides on DCs supported by DC maturation by the checkpoint antibody and increased vaccine peptide uptake by the antibody mediating peptide delivery. TCR repertoire analysis of the tumor infiltrating lymphocytes (TILs) of vaccinated mice revealed remarkable accumulation of clonal CTLs in the BP1209 vaccinated group. Conclusions: BP1209 cancer vaccine dramatically improves T-cell response and anti-tumor activity over conventional long-peptide vaccines. We demonstrated that the enhancement of vaccine potency was achieved not only by the function of the antibodies on DCs but mainly by the increased antigen delivery to DCs. Enhanced T-cell response was more remarkable in CD8+ T cells, especially in clonal expansion of the CTL in TILs, suggesting that BP1209 vaccine facilitates antigen cross-presentation and inducing functional CTLs. Analysis using a number of predicted neoantigen peptides showed that the BP1209 vaccine enhances the vaccine efficacy independent of epitope sequences. Thus, our new platform of cancer vaccines provides a critical solution to enhance personalized neoantigen vaccines. Citation Format: Yuji Mishima, Fumiko Isoda, Noriko Matsumoto, Noriko Watanabe, Kazushi Hiranuka, Takashi Yamada, Norihiro Fujinami, Manami Shimomura, Toshihiro Suzuki, Tetsuya Nakatsura, Norihiro Nakamura. Personalized neoantigen cancer vaccine assembled on DC targeting antibody improves cancer immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3552.

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Abstract Through somatic mutations, cancer cells accumulate cancer-specific mutated peptides, commonly referred to as neoepitopes or neoantigens, that can be recognized by T cells. Due to their cancer-specific nature, neoantigens are a highly attractive area of study for developing cancer immunotherapies. By comparing whole-exome sequencing of patient tumor tissue and matched normal tissue, researchers have been able to identify cancer-specific neoepitopes. However, the filters that can accurately delineate immunogenic neoepitopes from non-immunogenic remain elusive. Current methods, such as the pipeline developed by the Tumor Neoantigen Selection Alliance (TESLA), typically select short peptides based on hypothesized rules of immunogenicity. Importantly, only a small percentage of these epitopes have been reported to be recognized by T cells through experimental validation assays. In this study, we systematically tested the neoantigen prediction capabilities of our in-house pipeline, Identify-Prioritize-Validate (IPV), and the TESLA pipeline. Whole-exome sequencing from 11 head and neck squamous cell carcinoma patients was used to identify somatic variants. With IPV, variants were filtered and ranked primarily based on variant allele frequencies in RNA and DNA and 20mer peptides were generated from each mutation. In parallel, the TESLA pipeline was used to rank 8- 12-mer peptides based on a combination of HLA binding and stability predictions as well as expression levels of source antigens. Pools of the top 10 peptides from both methods were generated. To enable the direct comparison of TESLA and IPV while accounting for the disparate peptide lengths produced by the pipelines, two additional pools were generated: one pool of 20mers from the TESLA pipeline and one pool of 8-12mers from IPV. The four peptide pools were individually cultured in vitro with patient-matched PBMCs and immune recognition was measured through IFNg and IL5 using fluorospot assays. The IPV pipeline consistently outperformed the TESLA pipeline in predicting neoantigens that elicited an immune response. 5 of the 11 patients showed significant immune recognition of the IPV 20mer peptide pool, only 2 patients responded to the 20mer TESLA pool, while no patients responded to the TESLA and IPV 8-12mer pools. Through this data, we prove the efficacy of IPV, and its filters, and highlight the importance of utilizing 20mer peptides over shorter peptides. Finally, we deconvoluted the positive IPV responses and were able to provide the sequences of novel immunogenic neoepitopes. Our work underscores the improvement in the predictive ability of IPV in comparison to typical pipelines that output short peptides based on parameters derived from a priori knowledge and demonstrates the ability of IPV to be utilized as a tool to identify novel neoantigens. Citation Format: Leila Yasmine Chihab, Zeynep Kosaloglu-Yalcin, Jason Greenbaum, Aaron Miller, Stephen Schoenberger, Bjoern Peters. Experimental comparison of two pipelines to identify neo-epitopes in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2719.

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Abstract Introduction: The FDA has approved 11 therapies for non-small cell lung cancer (NSCLC) patients diagnosed with gene fusions. Importantly, these life-saving targeted therapies remain underutilized due to a lack of cost-effective, comprehensive, and simple-to-use technologies for gene fusion detection. Our previous work using ASPYRE technology (allele-specific pyrophosphorolysis reaction; Silva et al, 2020) demonstrated detection of somatic variants at single-molecule level from DNA extracted from tumor tissue. However, DNA-based fusion detection requires tiling across large introns, which limits sensitivity particularly if breakpoints are located in repetitive regions of the genome. Here, we show that ASPYRE technology can also be utilized for highly sensitive detection of RNA fusions, scaling to over 30 fusion targets covering the main 3’ fusion partners (ALK, ROS1, RET, NTRK1, NTRK3) and from a single reverse transcription PCR (RT-PCR) amplification reaction. Methods: The ASPYRE assay consists of four sequential enzymatic stages: RT-PCR; enzymatic clean-up; combined exonuclease digestion, hybridization, pyrophosphorolysis and ligation; rolling circle isothermal amplification and detection. All targets are detected across two reaction wells using four channels on a standard real-time PCR instrument. A panel of synthetic RNA oligonucleotides comprising gene fusion boundaries was spiked into a background pool of lung-RNA, quantified using digital PCR (dPCR) and compared against a commercial reference standard to validate its suitability for assessing assay performance. RNA was extracted from formalin-fixed paraffin-embedded (FFPE) lung tissue from healthy donors to determine assay specificity, and from FFPE lung tissue from NSCLC patients with known fusions to exemplify end-to-end sample analysis. Results: We demonstrate equivalence of our in-house reference samples to commercial reference standards. Using serial dilutions of input RNA standards, we show that detection by ASPYRE is consistent with detection of single molecules as input for common ROS1, ALK, RET and NTRK3 fusions. In addition, ASPYRE was able to detect previously confirmed ROS1 and ALK fusions and MET exon skipping variants in FFPE samples. ASPYRE had high specificity, correctly confirming the absence of RNA fusions and exon skipping variants in healthy control FFPE samples. Conclusions: ASPYRE approached single-molecule sensitivity for over 30 RNA fusion targets in a single reaction. The assay required only 1 ng RNA input, with a turn-around time of <4 hours from extracted RNA to result, opening the door to routine detection of RNA fusions from limited specimens. The simplicity and ease of use of ASPYRE, combined with its rapid turnaround time and analytical performance characteristics, provides a new tool for rapid and comprehensive treatment selection in NSCLC. Citation Format: Eleanor Gray, Justyna Mordaka, Efthimia Christoforou, Kristine von Bargen, Nicola Potts, Rebecca Palmer, Barnaby Balmforth. Low-cost, simple and rapid assay for single-molecule detection of gene fusions from RNA with ASPYRE technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4104.

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Abstract Background: Cancer progression is generally accompanied by loss of differentiation and gain of stemness features. In prostate cancer (PCa), androgen receptor (AR) signaling at the transcription level has been reported to decline in high-grade vs. low-grade tumors. Also, loss of AR expression promotes a stem-like cell phenotype, implicating inverse relationship between AR signaling and stemness in PCa. Here, we aim to understand the dynamic changes in stem-like features and AR signaling activity (AR-A) at the global transcriptomic level, and the relationship between the AR-A and stemness, across the spectrum of PCa development and progression. Methods: We adapted and developed bioinformatic pipelines to assess the degree of cancer stemness (i.e., aggressiveness) and the AR-A during PCa development and progression. And the analysis was performed in published and in-house RNA-seq datasets encompassing normal prostate, primary PCa (TCGA), metastatic castration-resistant PCa (mCRPC), PCa with long-term or neo-adjuvant androgen-deprivation therapy (ADT), and castration-resistant xenografts models. Results: In the normal prostate, significantly higher AR-A and lower stemness were observed in luminal cells compared to basal cells, consistent with the knowledge that luminal cells are differentiated cells regulated by AR signaling whereas the basal cell compartment harbors stem cell activity. In PCa, we have observed that: 1). Surprisingly, both stemness and AR-A are significantly increased in primary PCa compared to benign prostates; 2). In untreated primary PCa, tumor progression is accompanied by significantly reduced AR-A and an increasing trend of stemness; 3). In mCRPC, the AR-A inversely correlates with stemness; 4). Interestingly, while long-term ADT leads to reduced AR-A and increased stemness, short-term ADT during neo-adjuvant therapy (i.e., before the surgery) results in decreased stemness; 5). Our xenograft CRPC models have confirmed the AR-A is significantly reduced in CRPC. Conclusions: PCa development and early growth are accompanied by increased AR-A as well as the stemness. In contrast, the progression of treatment-naïve PCa is accompanied by decreasing AR-A and increasing stemness. In treatment-failed mCRPC, the AR-A shows an inverse correlation with the stemness. Duration of ADT influences the stemness, and stemness functions as a factor of treatment-induced reprogramming. Significance: Developing gene signature scores to assess stemness and AR signaling has allowed us to track the global differentiation status of PCa, and to better understand the cancer cell heterogeneity and plasticity along the course of PCa development, progression and treatment resistance. Our results suggest that increased stemness represents a ‘universal’ feature of PCa progression and aggressiveness and should be therapeutically targeted. Citation Format: Xiaozhuo Liu, Eduardo Cortes Gomez, Yibing Ji, Wanjun Song, Jun Yong, Jianmin Wang, Qiang Hu, Song Liu, Dean Tang. Bioinformatics approaches to dissecting androgen receptor activity and cancer stemness during prostate cancer development and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2703.