American Association For Cancer Research Research Articles

Abstract 500: Discovering epigenetic regulators of cells states in triple negative breast cancers

Abstract The implementation of targeted therapies have been instrumental in achieving clinical responses for ER, PR, and HER2 receptor expressing breast cancers. However, triple negative breast cancers in themselves do not respond to such therapies and are highly heterogeneous diseases with significantly worse progression and survival. Our preliminary data and literature have also shown that in TNBC different cancer cell subpopulations are present in any given tumor, each with its own unique chemo-response and resistance. To discover novel targets regulating cancer cell states in TNBCs, we have created a reporter cell line using CRISPR/Cas9 mediated knock-in of fluorescent reporter proteins after the endogenous loci of major cell type markers in SUM149. Using this reporter line allows for the direct visualization of ALDH1A3+/- and CD24+/- cells, facilities tracking of distinct cell state transitions in a given cell, and allows for lineage tracing following cell divisions. These reporter cells were subjected to HTS-FACS analysis after exposure to a siRNA library of known epigenetic regulators. Following secondary screening, three siRNA targets are able to uniquely inhibit distinct subpopulations of cells within the cell line DCAF1 (ALDH-/CD24- cells), MLL2 (ALDH-/CD24+ cells), and TRIM24 (ALDH+ cells). In combination, these siRNA's proportionally eliminated all cell types. Due to the inherent heterogeneity of TNBCs we then sought to determine if efficacy was consistent or unique across different cell lines with varying percentages of CD44, CD24, and Aldefluor+ cell populations. Most notably, in MDA-MB-468 which contains similar cell population percentages to SUM149 each siRNA was capable of reducing cell proliferation. Similarly, TRIM24 was also capable of reducing cellular proliferation in BT20, MLL2 in MDA-MB-231, and DCAF1 in HCC1937. Knockdown of TRIM24 consistently reduced Aldefluor+ cells in MDA-MB-468 and BT20 cells. Finally, coupled with sc-Omics technologies we utilized this efficacy data identify a subset of TNBC patients likely to respond to therapies which will target these genes in the future. Citation Format: Joseph Patrick Burnett, Garrett Johnson, Nathan Truchan, Michael Brooks, Max S. Wicha, Duxin Sun. Discovering epigenetic regulators of cells states in triple negative breast cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 500.

Abstract 3155: Interferon-gamma-induced melanoma plasticity and response to PD-1 blockade therapy

Abstract Melanoma is a cancer of melanocytes, which develop from neural crest cells during embryogenesis. One of the ways melanoma can resist targeted therapy or adoptive T cell transfer therapy (ACT) is by dedifferentiation, whereby the tumor becomes less melanocytic and more neural crest-like. ACT-induced dedifferentiation and therapy resistance was a result of tumor necrosis factor (TNF) from antitumor T cells (Landsberg, Nature 2012), which can serve as a positive control for inflammatory cytokine-induced melanoma plasticity. To study whether immune checkpoint blockade induces melanoma dedifferentiation, we analyzed RNA-seq data from paired biopsies of 68 patients before and during anti-PD-1 therapy. Surprisingly, the tumors of patients with complete or partial response dedifferentiate following therapy (paired p= 0.00015), but those of stable or progressive disease do not (paired p= 0.31 and 0.32, respectively). As the presence of interferon-gamma signatures in biopsies is best correlated with response to anti-PD-1 therapy, we hypothesized that interferon-gamma mediates dedifferentiation. We dedifferentiated four human melanoma cell lines by in vitro exposure to long-term (three to five weeks) interferon-gamma and compared it to three days of TNF as positive control. Although TNF and long-term interferon-gamma induced similar phenotypic changes based on flow cytometry, ATAC-seq showed that they each drive different global chromatin remodeling. Motif enrichment analyses revealed STAT6, TFAP2C, and SOX proteins are key transcription factors involved in interferon-gamma-induced dedifferentiation. Inferred protein activity analysis of the matching RNA-seq data showed that these regulators, as well as IRF3 and HMGA1, alter in activity selectively with dedifferentiation. In conclusion, dedifferentiation may be a marker of positive response to anti-PD-1 therapy, mediated by chronic exposure to T cells producing interferon-gamma. Citation Format: Yeon Joo Kim, Katherine M. Sheu, Jennifer Tsoi, Gabriel Abril-Rodriguez, Catherine Grasso, Alexander Hoffmann, Stephen T. Smale, Thomas G. Graeber, Cristina Puig-Saus, Antoni Ribas. Interferon-gamma-induced melanoma plasticity and response to PD-1 blockade therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3155.

Abstract 5795: Long-term diabetes risk among ovarian cancer survivors in a population-based cohort study

Abstract Objective. In the United States, ovarian cancer is the eleventh most common female cancer and the fifth most common cause of cancer death among women. Although incidence and death rates have been declining over the last 10 years, the 5-year survival rate is 47.6%. Diabetes was the seventh leading cause of death in the United States and is associated with an increased mortality rate among cancer patients. While previous studies have examined the association between diabetes and the risk of ovarian cancer, there are very few studies investigating the risk of diabetes after an ovarian cancer diagnosis. We previously reported increased risks of type I and II diabetes among endometrial cancer survivors, but there is no previous study, to our knowledge, that examined the risk of diabetes among ovarian cancer survivors. The aim of this study was to investigate long-term diabetes risk among ovarian cancer survivors compared with a general population of women. Methods. Ovarian cancer patients diagnosed from 1996 to 2012 in the Utah Cancer Registry were matched to up to 5 cancer-free women on birth state and birth year using the Utah Population Database. Diabetes diagnoses were identified with ICD-9 codes from statewide ambulatory surgery records, statewide inpatient databases and electronic medical records. We estimated hazard ratios with Cox Proportional Hazards models to: (1) investigate risk for diabetes after cancer diagnosis among ovarian cancer survivors compared with the general population, and (2) examine risk factors for type II diabetes among ovarian cancer survivors. Results. A total 1,520 ovarian cancer patients and 5,709 women from the general population were included in the final cohort. A slightly higher proportion of ovarian cancer patients were obese at baseline (16.3%) than the general population cohort (14.9%). Ovarian cancer survivors had a higher risk of type II diabetes in the first year after cancer diagnosis (HR=3.09, 95%CI=2.09, 4.56) and overall (HR=1.52, 95%CI=1.23, 1.88) compared with the general population. However, associations between ovarian cancer and type II diabetes were not observed in the later follow up periods. High BMI was a significant risk factor for type II diabetes among ovarian cancer survivors, with a dose-response relation (overweight; HR=1.81, 95%CI=1.21, 2.71, obese; HR=2.48, 95%CI=1.61, 3.83, P-trend <0.001). Clinical factors such as cancer treatment, stage, and histology did not contribute to an increased risk of type II diabetes among ovarian cancer patients. Other factors such as race and baseline comorbidities were also not associated with type II diabetes risk among ovarian cancer patients. Conclusion. Ovarian cancer survivors had an increased risk of type II diabetes compared with women in the general population. Risk was elevated only within the first year of cancer diagnosis, thus surveillance bias cannot be ruled out. Our findings suggest that the increased risk of type II diabetes does not persist beyond the first year after cancer diagnosis. Citation Format: Seungmin Kim, Kerry Rowe, John Snyder, Alison Fraser, Ken Smith, Vikrant G. Deshmukh, Michael Newman, Kimberley Herget, Chun-Pin Chang, Dominik Ose, Mary C. Playdon, David Gaffney, Mia Hashibe. Long-term diabetes risk among ovarian cancer survivors in a population-based cohort study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5795.

Abstract 190: Prognostic significance of esterase gene expression in multiple myeloma

Abstract Introduction: The esterase enzyme family is a subclass of the hydrolase enzyme superfamily that functions to split ester bonds. Several esterases have been identified, which differ in substrate specificity and biological function, and the expression of specific esterases may be dysregulated in cancer. Limited information is available regarding the expression of esterases in multiple myeloma (MM), despite potential for exploitation in novel therapeutic approaches such as peptide-drug conjugates (e.g. melflufen). We evaluated the biological significance of esterase enzyme expression in MM. Methods: For gene expression profiling, bone marrow aspirates were obtained from MM patients (pts) or healthy donors after obtaining written informed consent and following approved protocols in compliance with the Declaration of Helsinki. CD138+ plasma cells were enriched and used for RNA preparation. Illumina-compatible RNA sequencing libraries were prepared and sequenced. A cut-off value of >1 Reads Per Kilobase of transcript per Million mapped reads (RPKM) was used to filter expressed genes. The Welch two-sample t-test was used to identify significant variation in gene expression. Contribution of esterase gene expression to survival outcome was estimated by Kaplan-Meier analysis; significance between two groups was deduced using a Mantel-Cox log rank test. Results: Of 123 pts with MM, bone marrow aspirates were collected from 41 pts with newly diagnosed (ND) MM, 81 pts with relapsed/refractory (RR) MM, and 1 pt with stable disease. Samples were also obtained from two healthy donors. The most abundant esterases in MM samples were OVCA2, PAFAH1B2, NXPE3, UCHL3, LIPA, ABHD10, UCHL5, CASD1, ABHD13, and USP4 (median log2[RPKM] range: 3.9 to 2.1). The least abundant esterases were NLGN1, NLGN2, PON1, CEL, PON3, NLGN4Y, IL17A, AADAC, CES5A, and ASPG (median log2[RPKM]: -5.1 to -10.7). The expression levels of OVCA2, PAFAH1B2, NXPE3, UCHL5, SIAE, ESD, PNPLA6, and PAFAH1B3 were significantly higher (P<0.05), and of PCED1B significantly lower (P<0.05), in RRMM versus NDMM. The expression level of all these genes was comparatively higher in the two healthy donor samples. High expression of OVCA2, PAFAH1B3, CES4A, or PNPLA6 was associated with poorer prognosis (overall survival [OS]) versus low expression samples (median OS for high vs low expression [months]: OVCA2: 55 vs 122; PAFAH1B3: 55 vs 124; CES4A: 55 vs not reached; PNPLA6: 89 vs 127; all P≤0.01), whereas low expression of NXPE4 or GZMA was associated with poorer prognosis versus high expression samples (median OS for high vs low expression [months]: NXPE4: 120 vs 55; GZMA: 120 vs 74; both P≤0.03). Conclusion: Esterase expression levels in MM change on progression from NDMM to RRMM. High expression of OVCA2, PAFAH1B3, CES4A, and PNPLA6, and low expression of NXPE4 and GZMA, were identified as poor prognostic markers in pts with MM, suggesting a role for these esterases in myeloma biology. Citation Format: Romika Kumari, M. Mamun Majumder, Juha Lievonen, Raija Silvennoinen, Pekka Anttila, Nina N. Nupponen, Fredrik Lehmann, Caroline A. Heckman. Prognostic significance of esterase gene expression in multiple myeloma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 190.

Abstract 4306: Retrospective analysis of docetaxel in combination with ramucirumab for previously treated non-small cell lung cancer patients

Abstract BACKGROUND: Current clinical trials demonstrated that combination regimens comprising chemotherapy and immunotherapy lead to better patient outcomes compared to chemotherapy alone as the first line of treatment for non-small cell lung cancer (NSCLC). In addition, the combination therapy of docetaxel (Doc) and ramucirumab (Ram) was considered one of the standard treatments for advanced or relapsed NSCLC patients. However, little is known about the therapeutic responders of this combination therapy among previously treated NSCLC patients. In the present study, we aimed to identify predictive factors for therapeutic response, including programmed death-ligand 1 (PD-L1) expression in tumors, for Doc treatment in combination with Ram. METHODS: We retrospectively analyzed a total of 135 advanced or relapsed NSCLC patients who were refractory to platinum-based chemotherapy at eleven institutions in Japan between July 2016 and November 2018. RESULTS: Our observations showed that PD-L1 expression in tumors is not associated with the efficacy of combined therapy of Doc and Ram in previously treated NSCLC patients. Analysis of the patient clinical profiles indicated that prior treatment with immune checkpoint inhibitors (ICIs) is a reliable predictor for the good progression-free survival (PFS) to this combination therapy (P=0.041). CONCLUSIONS: Our retrospective study indicated that combination regimens comprising chemotherapy and ICIs followed by Doc and Ram could be an optimal therapeutic option for NSCLC patients regardless of the PD-L1 status of tumors. Further investigations are required to strengthen clinical evidence demonstrating the effectiveness of the combination therapy of Doc plus Ram in previously treated NSCLC patients. Citation Format: Rui Kitadai, Akihiro Yoshimura, Tadaaki Yamada, Yusuke Okuma, Takayuki Takeda, Takanori Kanematsu, Junji Uchino, Yasuhiko Nishioka, Koichi Takayama. Retrospective analysis of docetaxel in combination with ramucirumab for previously treated non-small cell lung cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4306.

Abstract 1674: Correlation of the mutational pattern and gene expression data of patient-derived Non-Hodgkin lymphoma xenografts (PDX) with the response to targeted therapies

Abstract Non-Hodgkin lymphomas (NHL) are lymphoid malignant neoplasms that represent a very heterogeneous group and still pose an important clinical challenge. In particular, the frequent development of resistance to the treatment with standard-of-care (SoC) drugs is associated with a high incidence of disease recurrence. Recent progress in molecular high-throughput profiling has helped to identify genetic drivers for many subtypes of B-cell lymphomas as well as T-cell lymphomas. Target validation and drug development depend on corresponding preclinical models representing the different subtypes. Therefore, we established and characterized a panel of patient-derived NHL xenografts. All lymphoma PDX were derived from peripheral blood, lymph node extirpations or core needle biopsies, respectively, and were routinely implanted subcutaneously into immunodeficient mice. For further characterization, established lymphoma PDX models were treated with SoC (e.g., cyclophosphamide, doxorubicine, vincristine, etoposide) and investigational drugs. To gain a deeper insight into the molecular biology of the lymphoma models, RNA sequencing was performed. More than 20 PDX models from different NHL entities have been successfully established and characterized. We observed heterogeneous individual responses to the SoC treatments as well as to target treatments such as rituximab or ibrutinib. Explorative analysis of RNA sequencing data confirmed the representation of the clinical lymphoma subgroups (e.g. DLBCL, Burkitt, AITL) in our panel of lymphoma models. Based on the RNA sequencing data, the individual Human Leukocyte Antigen (HLA) type of each lymphoma PDX was determined with the aim to enable personalized, HLA type-specific studies. Our lymphoma PDX portfolio provides an exceptional platform for the identification and validation of new targets and allows the preclinical screening of new combinations in translational research projects. Citation Format: Bernadette Brzezicha, Theresia Conrad, Michael Becker, Aitomi Bittner, Martin Janz, Clemens A. Schmitt, Ulrich Keilholz, Jens Hoffmann. Correlation of the mutational pattern and gene expression data of patient-derived Non-Hodgkin lymphoma xenografts (PDX) with the response to targeted therapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1674.

Abstract 6309: Development of protein conformational array ELISA for p53 and SHP2 allosteric regulator screening and analysis

Abstract P53 and SHP2 plays important role during cancer development. For p53, more than 50% of cancers were shown to have gene mutation in this tumor suppressor, however how these gene mutations impact the p53 protein conformation and function have not be studies in much detail. A Protein Conformational Array ELISA was developed with 17 polyclonal antibodies covering the whole p53 molecule, by measuring the linear epitope distribution on the surface of the p53 protein, the conformational (Higher Order Structure or HOS) can be described quantitively. Initial studies have shown that in lung cancer cells, the level of p53 is significantly increased and more interestingly, the p53 HOS has changed at its N-terminal as compared with the wild type. For SHP2, a protein phosphatase, a 27-antibody panel was developed to characterize the HOS changes. Because of the generic nature of the phosphate binding site, it has been very challenging to develop phosphatase inhibitors, however studies have shown that allosteric regulators can be identified with excellent anti-tumor activities. Currently, some of the allosteric regulators identified are being tested in the clinic. The SHP2 Conformational Array ELISA will provide a robust platform for the screening and testing of potential allosteric regulators with its molecular resolution and high-throughput capability. This presentation will describe the development of the two conformational arrays and their applications in the study of p53 and SHP2 from different cancer cell lines and the characterization of the allosteric regulators identified. Citation Format: Xing Wang. Development of protein conformational array ELISA for p53 and SHP2 allosteric regulator screening and analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6309.

Abstract 2087: Bringing advanced analytics experts to the data: Report of an interdisciplinary machine learning workshop

Abstract Gaining insights from medical and molecular omics data with advanced statistical learning methods requires the collaboration of experts with foundational knowledge of the data contents and context as well as computational and statistical background. The purpose of the workshop was to derive the key components that are necessary to apply machine learning methods to non-public real-world clinical, genomic and transcriptomic data from heterogeneous sources in a cross-institutional collaborative effort. Machine learning experts were invited to the premises of the institution that held a comprehensive collection of patient data. During the preparation phase, regular virtual meetings took place in order to discuss decisions regarding the research questions to analyze, transformations of the raw file formats and the technical infrastructure. The interdisciplinary group of experts decided to focus on models that would assign patients matching therapy classes based on their molecular variation profile. During the workshop phase, the experts spent several days at the same location working solely on the analysis in question. Here we present the lessons learned from conducting such an interdisciplinary workshop. The key components can be divided into a preparatory and a workshop phase. The first phase lays the foundation for the collaboration, including the formalities, discussion of the data and the applicable methods. It is especially important to involve all domains in decisions regarding the data preprocessing steps. As an example, a method might have particular requirements considering the data dimensions, which is information that the data experts need to know in order to aid with the meaningful reduction of the degrees of freedom, by e.g. aggregation or filtering. Sharing anonymized sample files that represent the data format in a realistic manner and sample scripts for testing the infrastructure helps all parties to set up the analysis pipelines efficiently. In the second phase, the intense working atmosphere allowed for quick iterations of feature engineering, model training and result evaluation. In the common case that a dataset is not yet publicly sharable, we demonstrate that it is a feasible option to invite analysts to the institution holding the data. Additionally, the focused atmosphere that is created by a limited-time workshop setting is advantageous to the motivation of the collaborators. Citation Format: Sophia Stahl-Toyota, Katrin Glocker, Analie Pascoe Perez, Alexander Knurr, Alexander Denker, Frank Ückert. Bringing advanced analytics experts to the data: Report of an interdisciplinary machine learning workshop [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2087.

Abstract 2450: Methyl CpG Binding Protein 2 suppresses Myc targeting miRNAs to promote context dependent tumor proliferation

Abstract Background: Epigenetic reader proteins maintain an imbalance between differentiation and self-renewal in cancer. Genetic alterations in the DNA methylation machinery (e.g., TET, DNMTs) and chromatin remodelers (e.g., KDMs, EZH1/2) are a hallmark of cancer. Methyl CpG binding domain protein 2 (MBD2) is an epigenetic reader protein which modulates regional gene transcription by recruiting co-repressor complexes to sites of CpG methylation. Our goal is to develop a novel class of anti-neoplastic epigenetic therapies targeting MBD2 to selectively reprogram tumor cells towards a terminally differentiated state and sensitize them to chemotherapy, radiation, and immunotherapy. Methods: To explore conserved mediators of MBD2 function in cancer, we used MBD2 targeting shRNA lentivirus to stably knockdown MBD2 in prostate cancer (PC3, DU145, LnCap), leukemia (SigM5, TET2WTK562 or TET2KOK562) and triple negative breast cancer (MDA-MB231-nanog-GFP) cell lines. Inhibition of MBD2 expression was confirmed by qRT-PCR and Western Blot. Proliferative potential was determined by cell counting and clonogenic potential was determined by methylcellulose-based colony forming assays. RNAseq analysis was performed on prostate cancer and leukemia cell lines. miRNA expression was analyzed using miScript miRNA PCR (Qiagen). In vivo tumor initiation capacity was analyzed using orthotropic and heterotopic xenografts in athymic NSG mice. Results: Our results show that MBD2 is required for the proliferation of triple negative breast cancer (TNBC), prostate cancer (PCa), and TET mutant leukemias (TML), in vitro. The functional mediators of MBD2's growth promoting effects were tissue/tumor context dependent. In leukemias, MBD2's growth promoting and tumor initiating effects were most pronounced in TET2 null cells (which accumulate 5mC). In TNBC, nanog-GFP reporter positive cells were more sensitive to MBD2 knockdown than reporter negative cells. In PCa and TNBC, RNAseq analysis revealed that knockdown of MBD2 led to downregulation of Myc pathway genes and increased the expression of Myc targeting microRNAs, miR33-5, miR34a, miR148a and miR363. Western blot analysis confirmed that MBD2 knockdown coordinately downregulated cMyc expression and activated p27 expression. We further demonstrated that inhibition of MBD2 diminished the tumor initiating capacity of TNBC and PCa in xenograft models and the in vivo engraftment rate of patient derived TET-/- AML. Conclusions: MBD2 knockdown diminished the proliferative capacity of PCa, TNBC and primary TET2 mutant leukemia cells in a genetic (TET2) and phenotypic (nanog+ stem/progenitor) context dependent manner. Delayed peak effect and altered differentiation markers after MBD2 inhibition suggest epigenetic reprogramming as the mechanism of growth suppression. MBD2 targets miRNA's upstream of cMyc in PCa and TNBC. MBD2 murine knockout models are developmentally normal, suggesting an acquired function in cancer with a favorable therapeutic window for targeting. Citation Format: Aysegul Balyimez, Yihong Guan, Emily Esakov, Shinjini Ganguly, Ofer Reizes, Daniel J. Lindner, Jaroslaw Maciejewski, Babal Jha, Omar Y. Mian. Methyl CpG Binding Protein 2 suppresses Myc targeting miRNAs to promote context dependent tumor proliferation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2450.

Abstract 4380: Positive expression of estrogen receptor alpha correlates with poor prognosis and metastasis in colorectal cancer

Abstract Background: The estrogen receptor beta (ERβ) is the predominant estrogen receptor in the normal colon mucosa, and its expression is downregulated in colorectal cancer (CRC). On the other hand, ER alpha (ERα) is very little expressed in the normal colon tissue. An increased ERα/β ratio has been shown in colon carcinomas. However, the role of ERα in CRC development and progression needs further evaluation. Aim: To investigate the role of ERα expression in CRC prognosis. Methods: A tumor tissue microarray of primary CRCs from 351 patients was stained for the expression of ERα. The nuclear staining intensity was evaluated using the Immuno-Reactive Score (IRS). Cox regression analysis and Kaplan-Meier curves were used to evaluate overall (OS) and disease-free survival (DFS). Colon cancer (CC) cells lines, SW-480, HCT-116 and HT-29 were used for in-vitro investigations. Two CC mouse models were used to investigate the expression of ERα. The zebrafish xenograft model was used to study CC cell metastasis. Untreated and ERα specific agonist (PPT, 40 nM) treated colon cancer cells were injected into the zebrafish perivitelline space. After 48 h the tale was checked for the presence of the metastasized cells. Results: ERα nuclear expression was negative in 232 and positive in 119 CRC patients, with a significantly higher expression in cancer tissue compared to normal tissue. Patients with positive ERα expression had higher TNM stage and more lymph node and distant metastasis. Positive ERα expression was independently associated with worse OS (HR=0.42; 95% CI: 0.26-0.51) and DFS (HR=0.32; 95% CI: 0.18-0.56), after adjustment for age and TNM stage. Moreover, patients with positive ERα expression had higher frequency of activated K-RAS mutation and higher expression levels of cysteinyl leukotriene receptor 1 (CysLT1R) and nuclear β-catenin, both with potent pro-tumorigenic properties. In the colon tissue from the colitis-associated colon cancer (CAC) mouse model with the Cysltr1 gene disruption, ERα expression was significantly decreased compared to wild-type (WT) mice, while in the colon of ApcMin/+ mice the ERα expression was significantly increased. In-vitro, PPT treatment significantly increased cell migration and wound closure compared to untreated cells. The zebrafish xenograft model showed that fish injected with PPT stimulated CC cells had significantly more tail metastasis compared with the control group (80% versus 67% respectively). Conclusion: Positive ERα expression correlates with poor prognosis and metastasis in colorectal cancer. Our results suggest that ERα-selective antagonists might be beneficial to combine therapeutic opportunities for improving the prognosis of CRC patients. Citation Format: Geriolda Topi, Shakti R Satapathy, Roy Roy Ehrnström, Marie-Louise Lydrup, Anita Sjölander. Positive expression of estrogen receptor alpha correlates with poor prognosis and metastasis in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4380.

Abstract 206: The landscape of oncogenic gene fusions in Chinese colorectal cancer patients

Abstract Background: Oncogenic gene fusions have been reported in patients with colorectal cancer (CRC) as promising actionable targets, but recognition of rare molecular subgroups is a challenge for precision oncology. We aimed to comprehensively characterize the clinical, pathological, and molecular landscape of Chinese CRC patients with oncogenic gene fusions. Methods: A total of 1226 patients with CRC underwent genomic testing between Aug. 2017 and Sep. 2019 using a tissue-based next-generation sequencing (NGS) assay. All the coding exons of 450 cancer-related genes and selected introns were captured by the custom hybridization capture panel. Microsatellite instability (MSI) and tumor mutational burden (TMB) were assessed in all patients. Results: Oncogenic gene fusions were found in 1.6% (20/1226) of Chinese CRC tumors, which was higher than that in the western population (0.68%, R. Madison, ESMO 2018 Congress). The most frequently detected fusions were NTRK1 (N = 5, 25%), BRAF (N = 5, 25%), ALK (N = 3, 15%), NTRK3 (N = 2, 10%), and RET (N = 2, 10%). FGFR1, ROS1, and EGFR fusions each occurred in one patient. Compared with patients without fusions, those with fusions were older (median age 69 vs 59 years, respectively, P=0.009), had right-sided CRC (75.0% vs 27.4%, respectively, P<0.001), MSI-high (45.0% vs 8.7%, respectively, P<0.001), and a higher TMB (median TMB of 9.8 vs 5.6 muts/mb, respectively). All MSI-high patients with fusions were TMB-high with a median value of 73.5 muts/Mb (range 51.8-180.3). The most frequently co-mutated genes in fusion cases were TP53 (70%), APC (60%), KMT2D (50%), ACVR2A (45%), RNF43 (45%), and TGFBR2 (40%). KRAS, NRAS, BRAF, POLE, and mismatch repair gene mutations were all mutually exclusive with oncogenic gene fusions. One patient harboring an EGFR-SEPT14 fusion, who had been heavily treated for right colon cancer metastasis, achieved a partial response to erlotinib. Conclusions: This study revealed that oncogenic gene fusions were found in 1.6% of Chinese CRC patients by using a tissue-based NGS assay. As oncogenic gene fusions are mutually exclusive with common oncogenic mutations, using NGS to detect fusions as actionable targets or resistance mechanisms has notable implications for precision therapy. Citation Format: Zhenhai Lu, Rongfeng Song, Kunsong Li, Ying Cheng, Wenjing Wang, Weifeng Wang. The landscape of oncogenic gene fusions in Chinese colorectal cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 206.

Abstract 2034: The young-onset colorectal cancer community speaks out about financial and emotional health

Abstract Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the United States and the second leading cause of cancer deaths. The 5-year survival rate of advanced CRC is low, at 14%. As CRC continues to rise in young people (20-49), we and others have shown that these patients are diagnosed at advanced stages of the disease. As a result, many of these individuals are subjected to aggressive treatments. Young-onset (YO) CRC patients and caregivers face unique clinical challenges including fear and stress around the disruption of family and career developmental tasks and goals suggesting a need for additional psychosocial and financial support. A cross-sectional study, conducted in the form of an online survey, was launched to better understand the experiences around employment and the resulting financial and psychological stress of YO-CRC patients and caregivers. YO-CRC patients and survivors (N=884) and caregivers (N=203) who were diagnosed between the ages of 20 to 50 years old completed an online questionnaire that was based on established instruments including PROMIS, EORTC-QOL-30, and EORTC-CR-29.Our survey found that 75% of patients and survivors were employed full-time at the time of diagnosis and only half of them were working at the time of the survey. Similarly, two-thirds of the respondents who were working part-time at diagnosis were working at the time of the survey. This suggests that a significant number of YO-CRC patients are unable to work after diagnosis. Interestingly, a quarter of them have been without evidence of disease for 2-10 years. Not surprisingly, more than half of the patient and survivor respondents indicated that they feel financially stressed (56%), worry about the loss of their family's financial stability because of the cost of cancer care (54%), and worry about the financial problems they will have in the future due to their illness/treatments (66%). Of those who feel financially stressed, 35% reported significant feelings of sudden panic/anxiety. Similarly, 40% of caregivers reported feeling financially stressed. This could be connected to 40% of them reporting taking a leave of absence, quitting a job, or leaving school because of the patient's diagnosis.These survey results indicate a need for the YO-CRC community to have access to resources that reduce the burden of loss of employment and resulting financial and psychological stress. Given the reality that 75% of our respondents had children, we hypothesize that employment and financial stress are potentially correlated to compounding parental responsibilities. Further studies will investigate financial and psychological well-being specific to the impact of child-rearing during the cancer-related loss of employment. Programs tailored at the YO-CRC community should consider the unique challenges presented by potential employment changes, parenting roles, and the likelihood of financial and psychological stress. Citation Format: Danielle Peterson, Tamara Springer, Kimberly Newcomer, Never2Young Board, Ronit Yarden. The young-onset colorectal cancer community speaks out about financial and emotional health [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2034.

Abstract 3766: Development of new FLT3/AXL dual inhibitor JRF104 with potent FLT3-TKD inhibition

Abstract Background: More than 30% of de novo AML patients were identified with FLT3 mutations. Two kinds of mutations, internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations, account for 23% and 7% of all AML cases, respectively. FLT3 inhibitors, which have been recommended by both NCCN and ELN guidelines, are often used as single or combo agent to treat FLT3 m+ AML. However, primary and secondary acquired resistance to FLT3 inhibitors is a remarkable challenge. It has been revealed that AXL can activate FLT3, resulting in reduction of response to FLT3 inhibitors. Furthermore, TKD mutations can also lead to development of drug resistance. Therefore, FLT3/AXL dual inhibitor with potent FLT3-TKD inhibition may provide better efficacy as novel AML treatment option. Results: JRF104 is a potent FLT3/AXL dual inhibitor with a kinase inhibition IC50 value of 0.42 nM and 1.22 nM, respectively. In vitro studies suggested that JRF104 could potently inhibit the proliferation of MV4-11 and Molm-13 cells, which endogenously express FLT3-ITD mutation. JRF104 shows wide spectrum in vitro antiproliferation activity against all FLT3 mutation types, including the “gate-keeper” F691L mutation. JRF104 can effectively reduce MDA-MB-231 cell migration in vitro, accomplished by blocking tumor migration pathway regulated by AXL. In MV4-11 xenograft model, JRF104 inhibited tumor growth at the dose of 1 mg/kg, QD (TGI: 65%); significantly reduce tumor volume at 5 mg/kg, QD (TGI: 118%). In Molm-13 xenograft model, tumor volume was well controlled at 15 mg/kg, QD (TGI: 94%), and all tumor volume reduced to zero at 50 mg/kg, QD (TGI: 104%). In CT-26 syngeneic model, JRF104 enhanced anti-tumor growth effect when combined with anti-PD-1 antibody and suppressed pERK and pAKT in tumor tissues. Additionally, JRF104 demonstrated high oral bioavailability in mouse, rat, and dog. In 28 days pre-clinical toxicology studies, JRF104 was well tolerated in all tested rats. HNSTD and NOEAL were determined to be 20 mg/kg QD and 3 mg/kg QD, respectively. All results support that JRF104 with high efficacy and safety. Conclusion: JRF104 demonstrated consistently potent inhibition of FLT3 and strong antitumor efficacy against FLT3-driven tumors in preclinical studies. Based on these datas, JRF104 may overcome resistance caused by AXL and FLT3-TKD mutation, representing a novel promising clinical antitumor agent for treating AML patients with FLT3 mutations. Citation Format: yuquan wei, Xiawei Wei, Yanzhong Zhang, Wanru Qin, Xiangyu Fu, Lihong Hu, Yuanfeng Xia, Jian Zuo, Zhong Xiong, Shanshan Yin, Shaohua Qin, Ge Dong, Ying Chen, Charles Ding, Chichung Chan, Shuhui Chen. Development of new FLT3/AXL dual inhibitor JRF104 with potent FLT3-TKD inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3766.

Abstract LB-021: Focal adhesive kinase inhibitor IN10018 sensitizes KRAS mutant cancer and overcomes drug resistance of KRAS G12C inhibition

Abstract KRAS mutation is found in around 20-30% of human cancer. Previously, lack of targeting therapy limited the treatment outcomes. Recently, KRAS G12C inhibitors like AMG510, MRTX849, and MRTX1257 were developed and exhibited promising therapeutic outcomes targeting the mutation. However, a considerable amount of cases showed less sensitivity, indicating either intrinsic or adaptive resistance mechanisms. Other reports and our own data exhibited that Focal Adhesive Kinase (FAK) inhibition showed good treatment effects to KRAS mutant cancer. Interestingly, TCGA data analysis reveal that FAK mRNA expression level is significantly higher in KRAS mutant tumors. Considering that FAK is one predominant target for cancer treatment resistance, we hypothesize that FAK is one of mediator leading to resistance mechanism of KRAS G12C inhibition. To explore our hypothesis, we employed high potent FAK inhibitor IN10018, which is being tested in phase I clinical trial for different cancer types. First, we found that IN10018 is active in a series of KRAS mutated cancer xenograft models. The compound sharply decreased the level of phosphoFAK Y397, a direct marker for FAK activity. Then we investigated whether combination of IN10018 with KRAS G12C inhibitors can show synergic effects in vitro and in vivo. IN10018 in combination with either AMG510 or MRTX1257 showed better effects in killing KRAS G12C cancer cells than each single treatment. More importantly a significant synergy between IN10018 with KRAS G12C inhibitors against KRAS mutated cancer cell lines was observed in vivo. Further evidence showed that KRAS G12C inhibition can significantly increase the expression of phosphoFAK Y397, suggesting a possible underlying mechanism of resistance to KRAS G12C inhibitors. IN10018 significantly decreased the upregulated FAK Y397 phosphorylation in the combination treatments, underlying the mechanism of synergy between the two inhibitors. Our results indicate that KRAS G12C cancer patients can be better treated by the combination of IN10018 with KRAS G12C inhibitor. Citation Format: Baoyuan Zhang, Yan Zhang, Jiangwei Zhang, Ping Liu, Bo Jiao, Zaiqi Wang, Ruibao Ren. Focal adhesive kinase inhibitor IN10018 sensitizes KRAS mutant cancer and overcomes drug resistance of KRAS G12C inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-021.

Abstract 2037: Association of molecular heterogeneity and actionable mutations with survival in lung cancer

Abstract Introduction: Today, the use of new technologies such as electronic medical records and testing methods are standard throughout healthcare centers. These new tools deliver an abundance of information to physicians, researchers, and the medical community. Harnessing the potential of this data can be an influential instrument to assist physicians in offering personalized medicine to patients and conduct complex research with ease. Nevertheless, the development, infrastructure, and implementation of distinct disease-specific registries for the collection, storage, and utilization of this data is crucial to benefit from this vital clinical and research information. Patients and Methods: In this study, we utilized an institutional thoracic patient registry complete with clinical and genomic data to carry out an in-depth analysis on lung adenocarcinoma patients (N=415). The patients selected and evaluated for analysis were seen at City of Hope from 2008 to 2016. The data was collected between 2016 and 2018 through retrospective chart review and compiled within the Thoracic Oncology Registry. This study was approved by the Institutional Review Board at COH under IRB 18217 and was conducted according to the Declaration of Helsinki. Data was de-identified and analyzed anonymously. Results: The lung adenocarcinoma median age was 64 (range, 22-92) years old, and the median OS was 33.29 months (95% CI, 29.77-39.48). The majority of patients were female (245/415; 59%). All the patients had metastatic disease, while the bulk of the patients (369/415; 89%) presented as Stage IV at the time of initial diagnosis. Oncogenes with the most commonly occurring mutations were found in EGFR (207/415; 50%), KRAS (97/352; 28%), and ALK rearrangement (28/377; 7%), while most frequent alterations in tumor suppressor genes were in TP53 (140/283; 49%), LRP1B (63/228; 28%), and STK11 (39/278; 14%). The top three actionable mutations identified include EGFR (177/415; 42.7%), ALK (28/377; 7.4%), and BRAF V600E (7/288; 2.4%). When survival was compared between the number of patients with actionable mutations (222/415; 53.5%) and wild-type patients (193/415; 46.5%), there was a substantial difference in median OS (39.8 months v 26.0 months, P<0.001). Comparing the TCGA and AACR Project GENIE dataset to our database, there was a surprising difference in the percentage of actionable mutations (15.0% v 22.3% v 53.5%). Conclusion: Our results showed an increased median overall survival in patients with lung adenocarcinoma that harbor actionable mutations compared to wild-type patients. Exploiting the appropriate use of clinical and genomic data collected and maintained in patient registries may better account for patient populations seen in the academic and community settings to improve cohort selection for clinical trials and survival outcomes. Citation Format: Jeremy Fricke, Isa Mambetsariev, Rebecca Pharaon, Yingyu Wang, Chen Chen, Fang Qiu, Lynette Smith, Surinder K. Batra, Wasim M. Nasser, Erminia Massarelli, Marianna Koczywas, Karen Reckamp, Ravi Salgia. Association of molecular heterogeneity and actionable mutations with survival in lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2037.

Abstract 5439: Distinct genomic profile in H. pylori associated gastric cancer

Abstract Background: Gastric cancer is one of the most common and deadly cancer types. Currently 4 subtypes have been identified with unique molecular alterations: Epstein-Barr virus (EBV)-positive, microsatellite instability (MSI), chromosomal instability (CIN), and genomic stable (GS) tumors. However, many gastric tumors are associated with the bacterium Helicobacter pylori but the genomic landscape of this subgroup of tumors remains largely unknown. Methods: Targeted-sequencing covering 425 genes were performed retrospectively on 1759 gastric tumor tissues and matched normal blood samples. The mean sequencing depth of tumors was 1159X. Nonsynonymous mutations, copy-number variation (CNV) and MSI status were called from human DNA reads; non-human DNA reads were mapped to NCBI microbial reference genome using Kraken and significant species were identified. Results: Overall, 37 (2.67%) samples were EBV-positive, 200 (11.37%) samples were H. pylori-positive, and 10 (0.57%) samples were positive for both. Other notable species identified in tumor samples were E. coli and C. acnes. No bacteria or viral reads were found in normal blood. Among the rest, 59 (3.35%) samples were MSI, 380 (21.6%) were CIN, and 1017 (57.82%) were GS. H. pylori-positive samples tend to be GS (85.5%, p < 0.001) and microsatellite stable (95%, p = 0.04). The most common mutations identified in H. pylori-positive tumors were TP53 (47%), CDH1 (12%) and ARID1A (8.5%). The top CNV events were amplification in MCL1 (14.5%), TERC (9%), CCNE1 (8.5%), PTPRD (8.5%), and MYC (7.5%). Compared to other GS tumors, mutation in AKT3 (p = 0.002) and amplification of TERC (p = 0.008) as well as MCL1 (p = 0.0.029) were significantly enriched in H. pylori-positive tumors. There was no difference in patients' sex or age among subtypes (p >= 0.05). Conclusion: This study characterized the genomic landscape of H. pylori positive gastric tumors using targeted-sequencing, which allowed direct comparison with other subtypes of gastric cancer. Identification of DNA reads from infectious agents in the tumor samples indicates that deep sequencing platform has promising utility in uncovering the characteristics of microbial environment of tumor. Co-infection between H. pylori and EBV occurred at a low frequency. H. pylori-positive tumors were GS and microsatellite stable with unique molecular features. Future study examining difference in treatment response among these subtypes of gastric cancer are warranted. Citation Format: Ao Wang, Hua Bao, Pengfei Hu, Yong Wu, Jinfeng Zhang, Xue Wu, Xiaonan Wang, Yang Shao. Distinct genomic profile in H. pylori associated gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5439.

Abstract CT266: Safety and efficacy of nimotuzumab in combination with chemotherapy in patients with recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC): A phase II study

Abstract Introduction: The prognosis od R/M HNSCC patients is poor and the cisplatin-based chemotherapy is currently the standard care of those patients. Nimotuzumab is a humanized EGFR monoclonal antibody which has been proven to be effective in locally-advanced head and neck squamous cell carcinoma. However, its efficacy in recurrent/metastatic disease remains unclear. Methods: R/M HNSCC patients (excluding nasopharyngeal carcinoma patients) who were not suitable for radical radiation therapy or operation were enrolled. No treatment after the diagnosis of recurrence and metastasis was allowed. Patients were given docetaxel(75mg/m2) +cisplatin (75mg/m2) (DP) or gemcitabine (1000mg/m2 d1,8) +cisplatin(75mg/m2) (GP) and nimotuzumab(200mg) every 3 weeks. Chemotherapy was given at most 6 cycles, and nimotuzumab was given until disease progression or intolerable adverse effects. The primary end point is progression free survival (PFS). Secondary endpoints were overall survival (OS), disease control rate (DCR),and objective response rate (ORR) according to RECIST v1.1,. Results: 25 eligible patients were enrolled (17 M, 8 F;median age 57) between 2018.10-2019.10. Sinuses was most involved region followed by tongue, oral cavity and hypopharynx. At a median follow-up of 9.5 months, median PFS was 5.8 months and median OS was 11.2 months. At 12 weeks after completion of chemotherapy, objective response rate (complete response (CR) + partial response (PR)) was 40%. The DCR (complete response (CR) + partial response (PR)+ stable disease (SD)) was 88%. Grade 3 or worse all-cause adverse events occurred in 8 (32%) of 25 treated patients. Grade III neutropenia was seen in 16% of the patients, and grade IV neutropenia was seen in 4% of the patients. Grade III thrombocytopenia was seen in 8% of the patients, and no grade IV thrombocytopenia was seen. Grade III anemia was seen in 8% of the patients, and no grade IV anemia was seen. Grade III vomiting was seen in 4% of the patients. And no other grade III-IV side effect was seen. Conclusion: Nimotuzumab + chemotherapy is a safe and effective regime for R/M HNSCC patients, which could be a considerable treatment choice worth further evaluation. Table 1Baseline Characteristics of the patientsVariableSex Male17(68%) Female8(32%)Age Median57 <6522(88%) ≥653(12%)ECOG 015(60%) 110(40%)Primary tumor site Sinuses7(28%) tongue6(24%) Oral cavity5(20%) Hypopharynx5(20%) Larynx2(8%)Extent of disease Locoregional recurrence12(48%) Metastasis with or without locoregional recurrence13(52%) Citation Format: Tao Hou, Min Lu, Xiayan Zhao, Yan He, Chunhong Hu, Ping Liu. Safety and efficacy of nimotuzumab in combination with chemotherapy in patients with recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC): A phase II study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT266.

Abstract 5180: Nuclear receptor tyrosine kinase c-MET restrains efficacy of PARP inhibitor in pancreatic cancer cells

Abstract Pancreatic tumors commonly have intra-tumor hypoxia and high reactive oxygen species (ROS) production and have become the fourth leading cause of death in the United States. Although ROS leads to oxidation of macromolecules such as DNA, protein, and lipids, pancreatic cancer cells are capable of surviving from the ROS-induced stress at relatively high tolerable levels. Therefore, identifying potential molecular mechanisms for pancreatic cancer cells to overcome ROS-induced cellular stress is important for develop therapeutic strategies in treating pancreatic cancer patients. While poly (ADP-ribose) polymerase 1 (PARP1) is a key protein for repairing ROS-induced DNA damages, we intended to include PARP inhibitors in targeted therapy strategy. Recent clinical trial showed that PARP inhibitor benefits pancreatic patients with BRCA mutation. However, our goal is to expand PARP inhibitor targeted therapy to pancreatic cancer patients without BRCA mutation with PARP inhibitor using strategies of combining kinase inhibitors as we demonstrated in breast cancer in our previous publications. Here, we used hydrogen peroxide (H2O2) as ROS stimuli and demonstrated that H2O2 induces nuclear transport of the receptor tyrosine kinase c-MET in AsPC-1 and BxPC-3 pancreatic cancer cells. We further showed that nuclear-located c-MET interacts with PARP1, and the interaction can be partially interrupted by crizotinib, a kinase inhibitor targeting c-MET. Moreover, combination of crizotinib and PARP inhibitor, olaparib, has synergistic anti-tumor effect in eliminating pancreatic cancer cells. Our data suggest that the intra-tumor hypoxia micro-environment of pancreatic cancer may promote nuclear localization of c-MET, which results in resistance to PARP inhibitor therapy. We further proposed a new therapeutic strategy of combining c-MET inhibitors and PARP inhibitors for advanced pancreatic cancer patients. Citation Format: Yuan Gao, Mei-Kuang Chen, Dihua Yu, Mien-Chie Hung. Nuclear receptor tyrosine kinase c-MET restrains efficacy of PARP inhibitor in pancreatic cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5180.

Abstract 5189: Neoadjuvant and adjuvant anti-PD-1 based combination immunotherapy with a novel AXL inhibitor, SKI-G-801 in syngeneic tumor model: A combined analysis of immune profiling

Abstract Introduction: Neoadjuvant therapy suggests the systemic treatment of cancer prior to surgical therapy. The main purpose of neoadjuvant therapy is to improve surgical outcomes in patients for whom a primary surgical approach is difficult to practice. Recently, Anti-PD-1 has been applied to neoadjuvant and adjuvant therapy, but the anti-cancer responses are not sufficient to prevent tumor recurrence. According to our previous study, a novel AXL inhibitor, SKI-G-801 strongly inhibits metastasis of syngeneic tumor with immune responses. The primary goal of this study is to overcome limitation of anti-PD-1 based neoadjuvant and adjuvant therapy with SKI-G-801. Methods: 4T1 cancer bearing mouse models were established for neoadjuvant or adjuvant treatment. Mice were treated with single anti-PD-1 (BIW), SKI-G-801 (QD) or a combination for 6 days, followed by the surgical dissection of tumor tissue in a subcutaneous xenograft model. In five mice per group, their survival rates were monitored for 50 days. We used flow cytometry and immunohistochemistry for comprehensive immune profiling and tumor-infiltrating lymphocytes (TILs) from three mice per group surgically removed tumor tissues. Three tumors per group were stained, and CD3-positive cells per unit area (mm2) were analyzed using Vectra Polaris. Results: According to results, the median survival of control group (only surgical operation without treatment) was 16 days (N=5), whereas, the longest survival group was neoadjuvant-combination (median survival = 37 days). Two out of five mice were survived until end of experiment (D-50). Most of the groups had similar outcomes excluding neoadjuvant-combination group. The median survival of adjuvant-anti-PD-1 group was 30 days, and adjuvant-SKI-G-801 and neoadjuvant-anti-PD-1 group were 25 days. The Neoadjuvant-SKI-G-801 was recorded 23.5 days. Statistically, survival rate of neoadjuvant-combination therapy was significantly higher than of neoadjuvant-anti-PD-1 and neoadjuvant-SKI-G-801 (log-rank test, p <0.01).The reason for neoadjuvant therapy is to improve immune responses before surgical operation. We investigated the effects of SKI-G-801 or anti-PD-1 alone and combination therapy on neoadjuvant treatment in the tumor microenvironment. IHC and flow cytometry were performed to elucidate tumor infiltrating immune cell populations with the tumor obtained by surgery. In IHC result, CD3+ cells population was significantly increased in SKI-G-801 alone and combination of neoadjuvant therapy compared to control group (p<0.05, p < 0.001, respectively), however, anti-PD-1 group was not significant. IFN-γ+ Tc cells, CD3+CD44+ memory Tc, and PD-1+Tc cells were significantly infiltrated into the tumor in the combination of neoadjuvant therapy (p < 0.01). Conclusion: This study confirmed that a potential combination with aPD-1 and SKI-G-801 applies to neoadjuvant therapy, as evidenced by increased T cell infiltration and function. Our findings provide a rationale for further clinical investigations. Keywords: Axl inhibitor, Neoadjuvant therapy, Adjuvant therapy, Immunotherapy, Anti-PD-1 Citation Format: Ha Ni Jo, Wongeun Lee, Chun-Bong Synn, Hee Kyu Lee, Jae-Hwan Kim, Yeongseon Byeon, Sung Eun Kim, Ji Min Lee, Do Hee Kim, Jung-Ho Kim, Beung-Chul Ahn, Min Hee Hong, Hye Ryun Kim, Kyoung-Ho Pyo, Byoung Chul Cho. Neoadjuvant and adjuvant anti-PD-1 based combination immunotherapy with a novel AXL inhibitor, SKI-G-801 in syngeneic tumor model: A combined analysis of immune profiling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5189.

Abstract 4428: Characterizing the tumor-infiltrating T-cell repertoire of breast cancer in indigenous African patients from Southwest Nigeria

Abstract Background: Women of African ancestry have a higher risk of young onset breast cancer than their European ancestry counterparts, with often more aggressive molecular tumor subtypes: these patients have fewer treatment options available to them, leading to higher mortality rates. Immunotherapy has recently garnered significant interest as a possible treatment for aggressive tumor subtypes (such as triple-negative breast cancer), but our lack of understanding of the tumor immune micro-environment has made it difficult to find more adaptive treatments for such cancers. Methods: We studied 96 invasive breast cancers collected from Nigerian women (mean age at diagnosis, 51.6±12.4 years), of which 62 (64.6%) were hormone receptor negative (HR-) by IHC, and 31 (32.3%) were basal-like subtype by PAM50 classification. Paired-end reads from bulk transcriptome sequencing were aligned to the human reference genome hg19 using STAR, and two algorithms (TRUST and MiXCR) were used to characterize the T-cell diversity in the tumors. To overcome the difficulty of extracting T-cell receptor (TCR) sequence information due to V(D)J recombination during T-cell maturation, we built a pipeline that incorporated data from public TCR databases (VDJdb and McPAS-TCR) to ensure a more accurate characterization of TCR diversity. Each potential TCR was first truncated to the appropriate start and end amino acids. Fragments of TCRs were discarded, and pairwise alignment of each output sequence was used to remove any duplicates. Results: Despite the many differences between TRUST and MiXCR, there was a 48.5% overlap in TCR sequences between the two methods, providing confirmation of the validity of these two algorithms. The intersection of the results from the two algorithms was then used for downstream analysis, which allowed for an expansive characterization of the tumor immune micro-environment, with 3,127 TCR sequences extracted over all 96 samples. We observed higher TCR diversity in basal-like tumors as compared to luminal-A/B tumors (p=0.011), whereas we saw no significance between HER2-enriched vs. luminal-A/B tumors (p=0.23). This is in accordance with other studies, which have shown that the most inflamed tumors are triple-negative/basal-like. Conclusion: Overall, this novel pipeline harnesses the power of two algorithms to effectively study the diversity of T-cells in the tumor, which, combined with further analyses of the tumor micro-environment, would allow for a more detailed understanding of the tumor immune infiltrates as potential therapeutic targets. Citation Format: Jean-Baptiste Reynier, Toshio F. Yoshimatsu, WABCS Working Group, Yonglan Zheng, Olufunmilayo I. Olopade. Characterizing the tumor-infiltrating T-cell repertoire of breast cancer in indigenous African patients from Southwest Nigeria [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4428.