Fresh Tumor Samples Research Articles

CTIM-22. NIVOLUMAB AND BEVACIZUMAB FOR RECURRENT GLIOBLASTOMA; T-CELL REACTIVITY AGAINST AUTOLOGOUS TUMOR CELLS

Abstract INTRODUCTION Glioblastoma is an aggressive brain tumor with a median survival of 14.6 months. We have no standard treatment for relapse and known options have limited effect. Novel treatments are necessary to improve survival and quality of life. METHODS We present our trial; phase II open label, two-armed translational study of Nivolumab and Bevacizumab for recurrent GBM, who have failed Stupp’s regimen. Patients are included in two arms depending on the possibility of salvage neurosurgical resection. Both arms receive Nivolumab and Bevacizumab administrated every second weekend, and the surgical arm also receive Nivolumab 7 days prior surgery. Forty-four patients were included by January 2021; 20 in each arm (four screen-failures). In the surgical arm, 20 fresh tumor samples as well as paired tissue from primary tumor were available. Tumor infiltrating lymphocytes (TILs) and tumor digest were produced in vitro from recurrent settings. Young TILs were expanded from fresh tumor fragments after minimal-culture, whereas rapidly expanded TILs (REP TILs) were obtained after massive expansion. By intracellular cytokine staining, we investigated the TIL reactivity after exposure to autologous tumor digest in order to evaluate whether the TILs were tumor-reactive, non-reactive or bystanders. RNA and whole exome sequencing were available before and after treatment. RESULTS Material from 19 patients was analyzed (one out of the 20 collected biopsies was limited in size, therefore no tumor digest could be produced). Four out of 19 TIL samples showed tumor reactivity after exposure to the autologous tumor digest. Tumor reactivity was ranged between 1,2 to 13,6 tox% in CD8+ TILs and between 2,8 to 10,9 tox% in CD4+ TILs. By flowcytometry we found, IgG4+ CD3+ TILS from tumor biopsies, meaning that Nivolumab were found in the brain. Currently controls are included to evaluate these results. CONCLUSIONS Updated results will be presented at SNO.

SURG-10. DEVELOPMENT OF NOVEL TOPICAL FLUORESCENT PROBE FOR INTRAOPERATIVE RAPID DETECTION OF GLIOMA

Abstract PURPOSE Fluorescence imaging is an important surgical adjunct in malignant glioma surgery. 5-aminolevulinic acid (5-ALA) has been proven effective for radical tumor resection and extended progression-free survival in a phase III randomized trial and therefore integrated into surgery for malignant glioma. Importantly, however, some limitations still exist in its use, which include false positivity and false negativity as well as inability of re-administration. In this study, we aimed to develop a novel, spray-type fluorescent probe using hydroxymethyl rhodamine green (HMRG) as a fluorescent scaffold. METHODS We have previously established a fluorescent probe library comprised of more than 320 kinds of HMRG probes. They have HMRG as a fluorescent scaffold with various types of dipeptides attached to it. Primary probe screening was performed using the homogenized tumor samples from patients with glioblastoma operated at our institution. Secondary screening followed using the selected probes and fresh tumor samples obtained from patients with glioblastoma operated from 2016 until 2018. Diced electrophoresis gel (DEG) assay, two-dimensional gel electrophoresis followed by a multi-well plate-based fluorometric assay, was performed to identify responsible enzymes for the selected probe. Further experiments with inhibitors, real-time PCR, immunohistochemistry, and western blotting were performed for confirmation. RESULTS Proline-arginine-HMRG (PR-HMRG) was selected as a candidate probe based upon the above two-step screenings. It achieved 79.4% accuracy in receiver operating characteristic curve analysis. Calpain-1 was found to be responsible to cleave PR-HMRG probe by DEG-proteome analysis. Calpain-1 protein was expressed at significantly higher level in tumors that were fluoresced by PR-HMRG than in those that were not. CONCLUSIONS Our innovative screening method was able to find PR-HMRG as a novel fluorescent probe effective for rapid detection of glioblastoma. A preclinical study is planned to assess the efficacy and safety of the selected probe.

Peptide vaccine targeting mutated GNAS: a potential novel treatment for pseudomyxoma peritonei

BackgroundPseudomyxoma peritonei (PMP) is a rare, slow-growing abdominal cancer with no efficacious treatment options in non-resectable and recurrent cases. Otherwise, rare activating mutations in the GNAS oncogene are remarkably frequent...

Tumor-Experienced Human NK Cells Express High Levels of PD-L1 and Inhibit CD8+ T Cell Proliferation.

Natural Killer (NK) cells play a key role in cancer immunosurveillance. However, NK cells from cancer patients display an altered phenotype and impaired effector functions. In addition, evidence of a regulatory role for NK cells is emerging in diverse models of viral infection, transplantation, and autoimmunity. Here, we analyzed clear cell renal cell carcinoma (ccRCC) datasets from The Cancer Genome Atlas (TCGA) and observed that a higher expression of NK cell signature genes is associated with reduced survival. Analysis of fresh tumor samples from ccRCC patients unraveled the presence of a high frequency of tumor-infiltrating PD-L1+ NK cells, suggesting that these NK cells might exhibit immunoregulatory functions. In vitro, PD-L1 expression was induced on NK cells from healthy donors (HD) upon direct tumor cell recognition through NKG2D and was further up-regulated by monocyte-derived IL-18. Moreover, in vitro generated PD-L1hi NK cells displayed an activated phenotype and enhanced effector functions compared to PD-L1- NK cells, but simultaneously, they directly inhibited CD8+ T cell proliferation in a PD-L1-dependent manner. Our results suggest that tumors might drive the development of PD-L1-expressing NK cells that acquire immunoregulatory functions in humans. Hence, rational manipulation of these regulatory cells emerges as a possibility that may lead to improved anti-tumor immunity in cancer patients.

Tumor-infiltrating lymphocytes predict survival outcomes in patients with cervical cancer treated with concurrent chemoradiotherapy

<h3>Objectives:</h3> The association between the prognosis of advanced cervical cancer treated with cisplatin-based concurrent chemoradiotherapy (CCRT) and the number of tumor-infiltrating lymphocytes (TILs) in biopsy samples collected before treatment was examined, to (i) identify correlations between selected immunogenic factors and clinicopathological characteristics, (ii) determine whether intra-tumoral abundance of various specific TILs is a prognostic indicator in women with advanced cervical cancer who undergo treatment with CCRT, and (iii) investigate subtypes of FOXP3+ T cells in cervical cancer samples. <h3>Methods:</h3> In this retrospective study, intratumoral lesions in colpo-scopic biopsies from 55 women with advanced cervical cancer who subsequently underwent CCRT were analyzed. The informed consent was obtained from all patients, and approval to conduct this study was granted from the institutional review board. The International Federation of Gynecology and Obstetrics clinical stages of these patients were as follows: Stage IIA1 (n=8), Stage IIA2 (n=6), Stage IIb (n=32), Stage IIIa (n=1), and Stage IIIb (n=8). Biopsy samples were subjected to automatic immunological staining using the following six antibodies: anti-CD3, anti-CD4, anti-CD8, anti-CD20, anti-CD206, and anti-FOXP3. Associations between the findings on automatic scoring of the number of each type of TIL in each specimen and clinicopathological characteristics or survial status were analyzed. Subtypes of FOXP3+ TILs in 15 additional fresh tumor samples were stained with CD45RA antibody and investigated by flow cytometry. <h3>Results:</h3> Correlations between density of TILs and clinicopathological characteristics were examined. Only weak infiltration of CD8+ lymphocytes was found to be associated with PLN metastasis. No other factors were found to be associated with immune cell infiltration. Better survival was found to be associated with high densities of CD3+, CD4+, CD8+, CD206+, and FOXP3+ T cells, while there were no statistically significant associations with CD20+ B cell density. The subtypes of FOXP3+ TILs were analyzed in 15 fresh tumor samples, and non-Tregs (FOXP3lowCD45RA–) which have anti-tumor activity, were detected in all cases. <h3>Conclusions:</h3> The abundance of various specific intratumoral TILs may be prognostic indicators in patients with advanced cervical cancer undergoing CCRT.

Optimizing Flow Cytometric Analysis of Immune Cells in Samples Requiring Cryopreservation from Tumor-Bearing Mice

Abstract Most shared resource flow cytometry facilities do not permit analysis of radioactive samples. We are investigating low-dose molecular targeted radionuclide therapy (MTRT) as an immunomodulator in combination with in situ tumor vaccines and need to analyze radioactive samples from MTRT-treated mice using flow cytometry. Further, the sudden shutdown of core facilities in response to the COVID-19 pandemic has created an unprecedented work stoppage. In these and other research settings, a robust and reliable means of cryopreservation of immune samples is required. We evaluated different fixation and cryopreservation protocols of disaggregated tumor cells with the aim of identifying a protocol for subsequent flow cytometry of the thawed sample, which most accurately reflects the flow cytometric analysis of the tumor immune microenvironment of a freshly disaggregated and analyzed sample. Cohorts of C57BL/6 mice bearing B78 melanoma tumors were evaluated using dual lymphoid and myeloid immunophenotyping panels involving fixation and cryopreservation at three distinct points during the workflow. Results demonstrate that freezing samples after all staining and fixation are completed most accurately matches the results from noncryopreserved equivalent samples. We observed that cryopreservation of living, unfixed cells introduces a nonuniform alteration to PD1 expression. We confirm the utility of our cryopreservation protocol by comparing tumors treated with in situ tumor vaccines, analyzing both fresh and cryopreserved tumor samples with similar results. Last, we use this cryopreservation protocol with radioactive specimens to demonstrate potentially beneficial effector cell changes to the tumor immune microenvironment following administration of a novel MTRT in a dose- and time-dependent manner.

Abstract 2649: An ex vivo 3D tumoroid model of fresh patient tissue (3D-EX) to assess the efficacy of anti-angiogenic compounds in renal cell carcinoma

Abstract Introduction: Renal Cell Carcinoma (RCC) is highly resistant to systemic chemotherapy; however, targeting pro-angiogenic pathways has shown significant clinical benefit. The development of targeted anti-angiogenetic agents has been hampered by the lack of clinically relevant models for screening. We have previously reported that the generation of 3D-tumoroids containing the unaltered tumor stroma could effectively be used to detect tumor response to small molecule inhibitors as well as antibody-based therapeutic agents. In this study we described a novel ex vivo platform to develop anti-angiogenic therapeutic strategies using fresh patient tumoroids of hepatocellular carcinoma and renal cell carcinoma. Materials and Methods: All fresh tumor samples were obtained with patient consent and relevant IRB approval. For the ex vivo assays, 3D tumoroids measuring 150 µm in size were treated with tyrosine kinase inhibitors (TKIs) sunitinib, sorafenib or axitinib. Treatment-mediated changes in endothelial cell viability was assessed by the confocal-based high-content real time imaging platform using fluorescent labeled anti-CD31 and anti-VEGFR2 antibodies, combined with custom image analysis algorithms. Additionally, treatment-mediated changes in tumor immune cell composition including CD4 and CD8 T-cells, Tregs, NK cells, macrophages, cell surface expression of checkpoint proteins as well as T-cell activation were evaluated by multiplex flow cytometry. Results: Our data shows that endothelial cells and capillary structures could clearly be visualized within3D tumoroids by confocal microscopy. Furthermore, we were able to quantify apoptotic cell death in endothelial cells induced by TKI inhibitors. Flow cytometry analysis demonstrated significant changes in T-cell activation upon treatment with TKIs that closely correlated with increased proinflammatory cytokine release. Observations found that the combination of anti-angiogenic treatments and immuno-modulatory agents significantly improved treatment responses. Conclusion: The ex vivo platform described in this study allowed assessment of the effect of anti-angiogenetic TKIs on tumor capillary endothelial cells as well as on tumor resident immune cell populations in intact RCC tumoroids of fresh patient tumor samples. We believe this clinically relevant approach can be used to identify the most effective anti-angiogenic and immunotherapeutic drugs and drug combinations in RCC and may improve personalized therapy for individual patients in clinical studies. Citation Format: Jared Ehrhart, Mibel M. Pabón, Stephen Iwanowycz, Zhisong Tong, Tina Pastoor, Soner Altiok. An ex vivo 3D tumoroid model of fresh patient tissue (3D-EX) to assess the efficacy of anti-angiogenic compounds in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2649.

Abstract 1566: Monitoring the impact of IL15 in combination with Sting agonists and nivolumab on activation and proliferation of tumor resident immune cells in 3D-EX platform of intact patient tumoroids

Abstract Background: The major goal of many immune-oncology (IO) therapeutics is improving the functionality of cytotoxic T cells through inhibiting checkpoints or through targeting stimulatory pathways. Tumor infiltrating myeloid cells are often educated within the tumor microenvironment (TME) to adopt regulatory phenotypes and become a major barrier to effective immune responses. Generation of an optimal immune response likely requires a combination of agents designed to promote anti-tumor T cell activation and to recondition the TME to support T cell functions. We generated patient derived 3D-tumoroids which retain the tumors stroma and suppressive immune landscape. This platform allowed for the screening of immune agonists targeting the IL15, Sting, or 41BB pathways alone or in combination with checkpoint inhibitors to identify the most effective combination in fresh patient tumoroid samples ex vivo. Methods: 3D-tumoroids approximately 150 µm in size were generated from fresh patient tumor samples which were obtained with informed consent and relevant IRB approval. Tumoroids were treated ex vivo with an IL15 agonist or Sting pathway agonists alone or in combination with nivolumab. Multi-parameter flow cytometry was used to analyze treatment-mediated changes inCD4/CD8 and NK cell activation. Additionally, we directly measured proliferation of T-cells, Tregs and myeloid cell populations in response to ex vivo treatments through EDU incorporation assay by flowcytometry. Immune cell function was further assessed by multiplex cytokine analysis and treatment-induced tumor cell killing was determined by high content confocal imaging. Results: Our data showed that IL15 treatment alone and select Sting agonists in combination and nivolumab had significant impact on activation and proliferation of tumor resident T-cells in their intact TME. Additionally, by using the 3D-EX platform, somewhat unexpectedly, we also observed meaningful changes in Tregs and multiple tumor infiltrating myeloid subsets including macrophages and dendritic cells upon ex vivo treatment with the IO drugs used in this study. Enhancement in pro-inflammatory cytokines production and immune cell functional phenotype were further correlated with tumor cell killing ex vivo. Conclusion: Our data reveals the power of the 3D-EX platform to identify combinations of immunotherapy agents capable of overcoming the unique suppressive environment developed by an individual tumor. Furthermore, the 3D-EX platform provides unique insight into the microenvironment to better identify compensatory mechanisms limiting the efficacy of IO therapeutics that may help to develop rational combination strategies. We believe the 3D-EX platform is clinically relevant and capable of significantly accelerating immuno-oncology drug discovery. Citation Format: Mibel M. Pabón, Jared Ehrhart, Stephen Iwanowycz, Zhisong Tong, Tina Pastoor, Soner Altiok. Monitoring the impact of IL15 in combination with Sting agonists and nivolumab on activation and proliferation of tumor resident immune cells in 3D-EX platform of intact patient tumoroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1566.

Abstract 1501: Employing 3D-ACT platform to assess the ability of stromal-targeting agents to improve penetration and efficacy of cell therapy

Abstract Background: Adoptive T cell therapy (ACT) has demonstrated great clinical success for the treatment of hematological malignancies. However, the unique challenges presented by solid tumors has significantly limited their utility. The primary characteristic of an effective ACT is the ability to selective identify tumor cells. In case of solid tumors, the ACT faces the additional hurdle requiring penetration into the tumor microenvironment (TME) while retaining function. Tumors develop highly immunosuppressive stroma which serves as a mechanical hinderance for the penetration and function of activated effector cells. In this study we used our 3D-ACT platform to investigate the ability of focal adhesion kinase (FAK) inhibitors to enhance the penetration of engineered cells into the intact patient tumor stroma and their efficacy within the tumor immune microenvironment. Methods: All tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids with intact TME measuring 150 µm in size were prepared from fresh tumor samples of renal cell carcinoma (RCC), and colorectal carcinoma (CRC) using proprietary technology developed at Nilogen Oncosystems. Engineered T cells were labeled with different cell tracker fluorescent dyes to monitor cell movements and locations within tumoroids by confocal analysis. 3D tumoroids were treated with vehicle only or FAK inhibitors and exposed to CAR-T cells at 10:1 E:T ratio. Cell penetration within tumoroids and tumor cell killing was measured by high-content confocal imaging combined with 3D-image analysis. ACT cell activation was monitored using multiparameter flow cytometry analysis and multiplex cytokine release assays to assess changes in the TME. Results: Our studies demonstrated that the confocal-based high-content real time imaging platform, combined with custom image analysis algorithms, allowed for monitoring of treatment-mediated tumor cell killing with structural analysis of engineered T-cells in the intact 3D tumoroids. Our data revealed that CAR-T cell penetration into tumoroids greatly varied between different tumor types and was heavily influenced by the tumor's stromal components. Treatment with FAK-inhibitors led to alterations within the TME, which subsequently impacted penetration and efficacy of CAR-T cells as assessed by flow cytometry and confocal imaging. Conclusion: The ex vivo model described here (3D-ACT) retains the TME and replicate the heterogeneity of tumor stroma to provide provides a clinically relevant platform to evaluate the influence of the tumor stromal components on engineered T cell functions. We believe the 3D-ACT platform can effectively be used to screen for drugs that can promote ACT penetration into the tumor stroma and survival within the tumor microenvironment. Citation Format: Zhisong Tong, Stephen Iwanowycz, Jared Ehrhart, Mibel M. Pabón, Tina Pastoor, Soner Altiok. Employing 3D-ACT platform to assess the ability of stromal-targeting agents to improve penetration and efficacy of cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1501.

Abstract 2996: Creating PDXs from continental African breast tumors: addressing racial inequity through science

Abstract To design interventions to reduce the burden of breast cancer (BC) in women with African ancestry, there is the need to understand the factors - heritable or non-heritable, modifiable or non-modifiable - that are associated with the disease. Patients and tumors from black women have been underrepresented in clinical research which are the main route to identifying new and improved treatments. Triple negative breast cancer (TNBC) is a clinically aggressive form of BC with propensity to metastasize and is enriched in patients with hereditary predisposition to BC. TNBC is most prevalent among black women despite lacking strong links with the known BC. This phenotype prevalence contributes to disparities in BC outcomes between blacks and other populations. There is an urgent need to represent black tumors in clinical research. Here we describe the process we used to transport fresh breast tumors from Ghana, west Africa to the USA to develop PDXs. Considering the shared ancestry between west Africans and diasporan African American, the results of this work will benefit both populations. Through an international collaboration between investigators at University of Michigan (UM) and Komfo Anokye Teaching Hospital (KATH) in Ghana, regular team site visits were initiated to generate surgical specimens for transport to the United States of America (USA). These specimens were used to create in vivo models. With informed consent, excess patient tumor tissue was taken from the operating room, at KATH. The tumors were cut into small pieces, and slow- frozen to -80°C in 10% DMSO/90%FBS. The samples were then transported overnight on dry ice in a commercial flight to UM. At UM, the tumor pieces were rapidly thawed and rinsed off with HBSS or PBS. The tumor sample was cut into smaller fragments (approximately 1-2 mm in diameter), mixed with matrigel (BD), and 25 μl of matrigel per injection site was injected into the mammary fat pad of female NOD/scid/IL2Rgnull (NSG) mice with an 18-gauge needle. From 2012 to 2017, we obtained fresh tumor samples from 49 Ghanaian patients. Of these, 23 (47%) were TNBC and 26 (53%) were other subtypes. Of the 23 TNBC samples, we implanted 18 into NSG mice and developed PDXs from 12. This gave us a success rate of 67%. We have fully characterized these tumors and their corresponding primary tumors. Our results demonstrate a maintenance of the histology and marker expression in the corresponding PDXs. Additionally, we obtained samples and developed PDXs from 17 Caucasian Americans, 13 African Americans, and 2 Asian patients from UM. 20 samples were TNBCs and 13 were non-TNBC. 18 were implanted in mice (12 TNBC, 4 ER+, and 2 Her2+). Of these, 11 developed into PDXs (9 TNBC, 2 ER+). Transporting African tumors to the USA is expensive, challenging, and limits large scale studies. These challenges can be overcome by enhancing African based research resources in order to conduct research in Africa. Citation Format: Evelyn M. Jiagge, Tahra K. Suhan, Jessica M. Bensenhaver, Michele Dziubinski, Joseph K. Oppong, Francis Aitpillah, Awuah Baffour, John D. Carpten, Lisa A. Newman, Sofia D. Merajver, Max S. Wicha. Creating PDXs from continental African breast tumors: addressing racial inequity through science [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2996.

Prognostic values of spindle checkpoint protein BUB1B in triple negative breast cancer

Objective: To identify important prognostic molecular markers of triple negative breast cancer (TNBC) using high throughput sequencing technology and to explore the correlation of spindle checkpoint protein BUB1B and clinicopathological features with patients' prognosis. Methods: The clinicopathological data and prognostic information of TNBC diagnosed at the West China Hospital of Sichuan University from 2009 to 2017 were collected. Forty-seven fresh tumor samples and 139 formalin fixed paraffin-embedded samples were selected. The fresh tumor samples were subject to RNA sequencing (RNA-seq). The enrichment analysis and protein-protein interaction (PPI) analysis were performed after intersection of difference analysis between RNAseq and GEO (Gene Expression Omnibus) datasets GSE38959 and GSE65194. Kaplan-Meier plotter database was used to analyze the relationship between expression of BUB1B and prognosis. Immunohistochemical staining was used to verify its expression in TNBC and correlation with clinicopathological features and prognosis. Results: Using edgeR to perform differential expression analysis between 47 TNBC tumor tissues and 12 normal tissues, 1 559 up-regulated genes and 1 376 down-regulated genes were identified, while only 131 differentially expressed genes were overlapping with those in GSE38959 and GSE65194. Enrichment analysis was mainly enriched in cell cycle, JAK-STAT signaling pathway and p53 signaling pathway. The top 10 genes ranked by degree of association were TOP2A, BUB1B, MKI67, PLK1, RRM2, PCNA, KPNA2, SMC4, PBK and IGF1. Kaplan-Meier plotter database analysis showed that the expression of BUB1B was significantly correlated with the prognosis of TNBC [overall survival, hazard ratio (HR)=0.52, 95%CI (0.35-0.77), P=0.001; distant metastasis-free, HR=0.72, 95%CI (0.52-0.98), P=0.038]. The immunohistochemical analyses of 139 formalin fixed paraffin-embedded samples showed that the low expression of BUB1B was correlated with poor prognosis in TNBC [HR=0.41, 95%CI (0.18-0.95), P=0.024]. Conclusions: The low expression of BUB1B protein is associated with poor prognosis in TNBC patients, and the molecular mechanism related with prognosis and potential therapeutic targets need to be further studied.

Anti-Angiogenic Treatment in Pseudomyxoma Peritonei—Still a Strong Preclinical Rationale

Simple SummaryPatients with pseudomyxoma peritonei that are not cured by the standard treatment (cytoreductive surgery and hyperthermic intraperitoneal chemotherapy) have no efficacious treatment options. Drugs that inhibit formation of new vessels (anti-angiogenic drugs) could be a therapeutic option for these patients. Using patient samples and animal models we show that angiogenesis is important in pseudomyxoma peritonei and that anti-angiogenic drugs may indeed have an effect. Our results support continued efforts to determine the role of anti-angiogenic treatment in pseudomyxoma peritonei.Pseudomyxoma peritonei (PMP) is a rare, slow-growing cancer characterized by progressive accumulation of intraperitoneal mucinous tumor deposits. Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) cures approximately 50% of patients, but in unresectable and recurrent cases, treatment options are limited. Anti-angiogenic treatment is being explored as a potential therapeutic option. Using PMP patient samples, microvessel densities (immunostaining for CD31 and CD105) and pro-angiogenic factors were analyzed, and the proliferative response upon incubation with human umbilical cord vascular endothelial cells (HUVEC) was determined. Growth inhibition by anti-angiogenic drugs was analyzed in patient-derived xenograft models of PMP. PMP tumor tissues were found to be highly vascularized and contained key pro-angiogenic factors, in particular related to vascular endothelial growth factor (VEGF) signaling, but interestingly, high levels of fibroblast growth factor 2 were also detected. HUVEC proliferation was stimulated upon incubation with fresh tumor samples and the observed proliferation could be inhibited by VEGF pathway inhibitor bevacizumab. In xenograft models the two VEGF pathway inhibitors, bevacizumab and aflibercept, inhibited tumor growth. This work reemphasizes the importance of angiogenesis as a major driver in PMP and strengthens the preclinical rationale for continued exploration of angiogenesis inhibition in the hope of providing novel treatment to a group of patients that have few other treatment options.

EX VIVO EXPANSION OF TUMOR INFILTRATING LYMPHOCYTES (TILS) AND CANCER STEM CELLS FROM MALIGNANT GLIOMAS

<h3>Background</h3> Gliomas are the most common type of primary malignant tumors of the Central Nervous System (CNS). Conventional therapies show low effectiveness and are frequently associated with important side effects. Glioblastoma (GBM) is the most lethal and frequent type of glioma, endowed with a subpopulation of cancer stem cells (CSC) responsible for resistance to conventional treatment and ensuing tumor relapse. Different forms of immunotherapy are increasingly recognized as promising therapeutic approach against aggressive tumors, including adoptive cell therapy with tumor infiltrating lymphocytes (TILs). However, studies accessing the potential of TILs in aggressive CNS tumors are limited. In this work, we describe the feasibility of isolating TILs and CSC from the same tumor specimen for ex vivo expansion and downstream immunotherapy application. <h3>Methods</h3> Fresh tumor samples were obtained from 10 patients (mean age: 60 years; 4 men and 6 women), diagnosed with malignant glioma and subjected to tumor resection in the Instituto do Câncer do Estado de São Paulo (ICESP), after informed consent approved by the Institutional Review Board. Brain tumor tissue was mechanically dissociated and enzymatically digested to generate a cell suspension. The CSC population was cultured in neurosphere formation media supplemented with EGF and FGF. TILs were expanded in culture medium supplemented with IL-2. <h3>Results</h3> Anatomopathological analyses of resected tumors confirmed diagnosis of 9 GBM and a grade I glioneural tumor. Tumorspheres could be generated in 8 out of 10 tumor samples, all of them GBM. No tumorspheres were formed in the respective cultures derived from the grade I glioneural tumor and from a GBM containing mutated ATRX. TILs could be expanded from all tumor samples tested, reaching a mean of 1,1 ± 0,7 × 10e6 total cells and mean cell viability of 60% ± 4%, after 5 days of in vitro expansion. <h3>Conclusion</h3> CSC could be expanded from all fresh glioma samples, except from a grade I glioma and a GBM with ATRX mutation, indicating a possible positive correlation with unfavorable clinical prognosis. Viable TILs could also be initially expanded from these glioma samples, indicating the possibility of using them to seed larger cultures subjected to typical rapid expansion protocols to attain clinical doses. Altogether, this procedure could help identify targetable CSC antigens, as well as screen for CSC-reactive TIL clones for possible adoptive cell therapy.

Combination of GC-MS based metabolomics analysis with mouse xenograft models reveals a panel of dysregulated circulating metabolites and potential therapeutic targets for colorectal cancer.

BackgroundColorectal cancer (CRC) is a common gastrointestinal tumor with subtle, often undetectable early symptoms, which means that upon diagnosis, patients often present in the middle or late stages of disease. Therefore, the need for an effective biomarker for the early diagnosis and development of novel therapeutic targets is urgent to prolong patient survival time and reduce mortality.MethodsTwenty mice were randomly divided into patient-derived xenograft (PDX) model (transplantation of fresh CRC tumor samples) and control groups (10 mice in each group). All the animals were euthanized using isoflurane at the end of the experiment. Gas chromatography-mass spectrometry (GC-MS)-based metabolomic profiling was performed to investigate the differential metabolites in the serum, and publicly available gene expression data (GSE106582) were analyzed to determine dysregulated metabolic pathways. Joint pathway analysis was used to identify potential metabolic targets. Immunohistochemistry analysis was performed to confirm the presence of the identified targets at the protein level.ResultsA total of 96 differential circulating metabolites were identified, which were predominantly involved in amino acid metabolism. In particular, the serum levels of amino acids such as phenylalanine and aspartic acid were significantly downregulated in the PDX group, suggesting an increased consumption of amino acids in CRC. Moreover, both the mRNA and protein levels of the amino acid transporters, SLC7A5 and SLC1A5, were found to be upregulated in CRC.ConclusionsBy combining GC-MS-based metabolomics profiling with a PDX model of CRC our study successfully identified potential diagnostic circulating metabolites. Dysregulated amino acid metabolism was found to be a significant feature of CRC. The amino acid transporters, SLC7A5 and SLC1A5, were identified as potential metabolic therapeutic targets. This study furthers the understanding of the metabolic processes involved in CRC.

Abstract PO026: Building the infrastructure required to research novel therapies for 35% of women with endometrial cancer who have rare subtypes

Abstract Whilst the majority of Endometrial Cancers (ECs) are common Type 1 endometrioid cancers, accounting for 75-80% of cases, with a good prognosis, Type 2 ECs include high-grade, clinically aggressive histologies, with poor response rates to hormonal therapies. Serous endometrial carcinoma is the second most common type, accounting for ~10 percent of cases, most with a p53 abnormality and a lesser proportion with HER2 overexpression/amplification/mutation. Clear cell endometrial carcinoma accounts for &amp;lt;5 % of EC, with the most aggressive cases also having p53 mutations. Mixed histologies and undifferentiated ECs are also aggressive. Carcinosarcoma (ECS) is a rare, aggressive, biphasic carcinoma that accounts for &amp;lt;5 percent of ECs, 90% with p53 abnormalities. Endometrial stromal sarcomas (ESS) can be low-grade or high-grade, or undifferentiated or resemble ovarian sex cord tumors. Uterine leiomyosarcoma (uLMS) are epithelioid or myxoid in type. Adenosarcoma involves a benign epithelial component mixed with a malignant stromal element. Whilst there have been considerable improvements in death rates for all cancers combined over the last 20 years, these improvements have not been seen for most rare cancers (RC). In order to ensure that those diagnosed with RC have access to research and novel therapies, we designed the Walter and Eliza Hall Institute of Medical Research (WEHI) Stafford Fox Rare Cancer Program (SFRCP). The WEHI-SFRCP has streamlined ethics, governance, consenting processes, including remote consent at home anywhere in Australia, and data collection protocols to allow the analysis of data and tissue of any type of RC. We have interconnected clinical and laboratory RC Databases within BioGrid Australia using the online REDCap platform. We have extensive laboratory processes in place for processing of tumour and blood samples, including the generation of PBMCs, DNA, RNA, to generate NGS including WGS; patient-derived xenografts (PDX), organoids, cell lines and other derivatives. We have high grade serous endometrial cancer (33 cases, including 4 PDX with 3 pending); uLMS (32 cases, including 2 PDX with 3 pending); endometrial carcinosarcoma (13 cases, including 3 PDX, 1 pending); rare (other, adenosarcoma, STUMP). We perform NGS testing on cases, depending on tumour purity, including Whole Genome Sequencing from fresh tumour samples; characterize PDX according to current chemotherapy and relevant novel therapeutics; study rare endometrial subtypes in specific projects and also provide information back to patients to guide therapy in the clinic. By integrating data sets with endometrial projects of similar depth we will drive forward the study of rare endometrial cancer subtypes. Citation Format: Clare L. Scott, Ratana Lim, Amandine Carmagnac, Cassandra Vandenberg, Gayanie Ratnayake, Genevieve Dall, Joshua Tram, Justin Bedo, Jocelyn Penington, Joep Vissers, Sean Grimmond, Matthew Wakefield, Anthony Papenfuss, Holly Barker. Building the infrastructure required to research novel therapies for 35% of women with endometrial cancer who have rare subtypes [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO026.

Abstract PO037: Identifying effective combinations of targeted therapies, using novel pre-clinical models, to improve treatment options for high-grade serous endometrial cancer

Abstract High-grade serous endometrial carcinoma (HGSEC) accounts for just 10% of endometrial cancer (EC) cases but is responsible for at least 40% of all EC-related deaths. It typically arises in post-menopausal women, with 70% of patients presenting with stage III or IV disease, does not respond to hormone therapy unlike the less aggressive forms of EC, and has a lower overall survival rate of just 18-27%, which has not improved over the past two decades. The primary treatment for HGSEC is surgery, followed by a combination of standard chemotherapies (platinum and taxane) with or without localised radiotherapy. However, recurrent HGSEC is less responsive to chemotherapy than are other subtypes of EC and even initial responses to chemotherapy are poor. Therefore, there is a great unmet clinical need to find better treatment options for women with this aggressive cancer. Apart from TP53 (mutated in up to 90% of cases), the other most frequently mutated genes in HGSEC are PPP2R1A (31%), PIK3CA (22%), FBXW7 (28%), CHD4 (17%) and BRCA2 (12%). Focal amplifications of the genes MYC, ERBB2, CCNE1, FGFR3 and SOX17 are also common. The presence of ERBB2 amplification and/or HER2 over-expression in around 30% of HGSEC suggests these patients may respond to HER2-targeting drugs, such as trastuzumab. However, only modest benefit has so far been seen for single-agent HER2-targeted therapies (ie trastuzumab or lapatinib) against HGSEC, suggesting resistance mechanisms are present. Another feature of HGSEC that could be exploited therapeutically is homologous recombination deficiency (HRD), which may be targeted with PARP inhibitors (PARPi). It is not clear what proportion of HGSEC are HRD and neither HER2-targeting drugs or PARPi have been approved for the treatment of HGSEC. Due to its rarity and a lack of pre-clinical models, HGSEC has so far been understudied, resulting in a lack of effective treatment options. We currently have 33 HGSEC patients consented to the WEHI-Stafford Fox Rare Cancer Program and have developed pre-clinical models from fresh patient tumour samples received (4 patient-derived xenograft (PDX) models validated, with 3 pending). Preliminary molecular analysis of whole-genome sequencing (5 samples, one of which gave rise to a PDX model), whole-exome sequencing (4 samples), and cancer panel sequencing (3 samples, 2 of which gave rise to PDX models; one harbouring ERBB2 amplification and one harbouring an AKT mutation) data from our HGSEC cohort has been performed. This has identified potential treatment targets, including ERBB2 amplifications and mutations in HR genes. I am using the PDX models for initial in vivo therapeutic characterization studies and to develop organoid models for use in high-throughput drug assays in vitro. This will guide subsequent novel drug combination testing in our PDX models. By combining specific targeted drugs I hope to overcome de novo resistance mechanisms and prevent acquired resistance. Results from this study will guide future decisions about therapeutic strategies to improve survival of women with HGSEC. Citation Format: Holly E. Barker, Ratana Lim, Amandine Carmagnac, Cassandra Vandenberg, Gayanie Ratnayake, Genevieve Dall, Briony Milesi, Angela Komiti, Emily O'Grady, Joshua Tram, Kym Pham Stewart, Justin Bedo, Jocelyn Penington, Joep Vissers, Sean Grimmond, Matthew Wakefield, Tony Papenfuss, Clare Scott. Identifying effective combinations of targeted therapies, using novel pre-clinical models, to improve treatment options for high-grade serous endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO037.

Visualization and quantification of pancreatic tumor stroma in fresh tissue via ultraviolet surface excitation.

.Significance: The study has confirmed the feasibility of using ultraviolet (UV) excitation to visualize and quantify desmoplasia in fresh tumor tissue of pancreatic adenocarcinoma (PDAC) in an orthotopic xenograft mouse model, which provides a useful imaging platform to evaluate acute therapeutic responses.Aim: Stromal network of collagen prominent in PDAC tumors is examined by imaging fresh tissue samples stained with histological dyes. Fluorescence signals are color-transferred to mimic Masson’s trichrome staining.Approach: Murine tumor samples were stained with Hoechst, eosin, and rhodamine B and excited at 275-nm. Fluorescence signals in the visible spectrum were captured by a CMOS color camera with high contrast and resolution at whole-tumor slice field of view.Results: Fluorescence imaging using UV excitation is capable of visualizing collagen deposition in PDAC tumors. Both fluorescence and histology data showed collagen content of up to 30%. The collagen modulation effect due to photodynamic priming treatment was observed showing 13% of collagen reduction. Necrosis area is visible and perfusion imaging using Texas Red dextran is feasible.Conclusions: The study demonstrates collagen visualization in fresh PDAC tumor samples using UV excitation. This imaging platform also provides quantitative stromal information from fiber analysis and visibility of necrosis and perfusion, suitable for therapeutic response assessment of photodynamic therapy.

Enhanced antitumor immunity by a novel small molecule HPK1 inhibitor

BackgroundHematopoietic progenitor kinase 1 (HPK1 or MAP4K1) has been demonstrated as a negative intracellular immune checkpoint in mediating antitumor immunity in studies with HPK1 knockout and kinase dead mice. Pharmacological...

TMOD-34. PATIENT-DERIVED XENOGRAFT AND CELL LINE MODELS FACILITATE NOVEL TREATMENT DISCOVERY IN CENTRAL NERVOUS SYSTEM LYMPHOMAS

Abstract Lymphomas of the central nervous system (CNSL) are rare tumors with few treatment options; however, recent genomic studies have uncovered several druggable targets. To facilitate novel treatment discovery, we sought to establish patient-derived xenograft (PDX) models and cell lines from patients with newly diagnosed or recurrent CNSL. PDX models were attempted from fresh tumor samples from 18 consented patients for whom rich clinical annotation was available. Orthotopic intracranial injections were performed via stereotactic injection. Cell line generation was attempted from both primary tumors (PDCL) and PDX samples (PDXCL). Characterization included histopathology, targeted exome sequencing, low-pass whole-genome sequencing for copy number evaluation, and expression profiling of matched patient tumors and PDXs. Drug response testing was performed using CellTiterGlo. From 18 samples attempted, 8 PDX models were successfully generated: 2 primary CNS diffuse large B-cell lymphoma (DLBCL), 5 secondary CNS DLBCL, and 1 secondary cutaneous T-cell lymphoma (CTCL). Mean time to moribund was 66 days. All 8 PDXs grew successfully as orthotopic models, and 4 also grew in subrenal capsule. Additionally, 6 PDCL and 2 PDXCL were established (&amp;gt; P3). Models faithfully represented primaries, showing nearly identical histopathology, immunoprofiles, and genomic signatures. To support expanded and rapid preclinical use of these models in drug testing we documented that PDX could be explanted to create short-term cell preparations or long-term cell lines with a 100% and 40% success rate, respectively. Proof of principle testing showed that explanted DLBCL PDX cells and cell lines showed sensitivity to copanlisib and venetoclax, which were not efficacious in the CTCL PDX. These findings showcase a diverse collection of CNSL models demonstrating high fidelity to primary tumors at the genomic and phenotypic levels, emphasizing their utility for preclinical studies and as patient avatars to rapidly determine sensitivities to existing and novel regimens prior to initiation of therapy.

Abstract PR07: A systems biology approach to reprogramming drug-resistant breast cancer stem-like cells

Abstract Breast cancer stem-like cells (CSCs) are hypothesized to be a cause of poor outcome in patients, and are drug-resistant in vitro and in PDX mice. We performed plate-based single-cell RNA sequencing (scRNA-seq) on flow sorted fresh tumor samples. These data were analyzed using our metaVIPER algorithm, which infers protein activity in a manner that is robust to the effects of noise and gene dropout. metaVIPER identified known breast CSC regulators, and analysis with our OncoTreat algorithm predicted several FDA approved drugs that could reprogram the breast CSCs into a state resembling that of differentiated tumor cells. We tested two of these predictions, ivermectin and albendazole, in TNBC PDX mice, and performed scRNA-seq on fresh tumor samples after 14 days of treatment. The results were exciting, as measured by single-cell entropy or lineage marker analysis. Breast CSCs were reprogrammed as predicted, while a tumor treated with a traditional chemotherapeutic (paclitaxel) had a dramatically higher proportion of CSCs. These results suggested a therapy in which cells are cyclically reprogrammed and treated with a conventional cytotoxic drug. This reprogram-kill-repeat therapy is being tested in TNBC PDX mice. We believe that the strategy developed here will prove to be a powerful tool for understanding and manipulating cell-state in heterogeneous populations. Citation Format: Jeremy Worley, Hongxu Ding, Heeju Noh, Evan Paull, Erin Bush, Piero Dalerba, Peter Sims, Fil Dela Cruz, Andrew Kung, Andrea Califano. A systems biology approach to reprogramming drug-resistant breast cancer stem-like cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PR07.