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Abstract Prostate cancer (PCa) is the most common cancer amongst males and becomes incurable once it advances to the secondary site. The most common site of metastasis from PCa is the adipocyte-rich bone marrow. Adipocytes are metabolically active cells capable of secreting a variety of adipokines and free fatty acids, which can be utilized by tumor cells to enhance proliferation and cause resistance to chemotherapies. Our lab has shown that adipocytes promote PCa progression and therapy evasion through modulation of tumor metabolism and activation of pro-survival signaling; however, the molecular mechanisms behind tumor-promoting effects of fat cells are not understood. It is well known that high levels of iron within the tumor microenvironment help the growth of PCa cells. However, since iron overload can cause an increase in reactive oxygen species (ROS), which are harmful to cell structure, tumor cells have developed defense mechanisms to protect them from this oxidative damage. Intriguingly, our data show that the expression of ferritin, an iron storing protein, is reduced in PC3 and ARCaP(M) cells in co-culture with adipocytes. Since low ferritin levels have been associated with induction of ferroptosis, an iron and oxidative stress-regulated cell death process, we examined protein levels of ferroptosis markers GPX4 and NCOA4 in PCa cells. Surprisingly, our data show that interaction with adipocytes increases GPX4 and reduces NCOA4 expression in PCa cells suggesting an adipocyte-mediated defense mechanism against ferroptosis. The aim of the present study was to establish that adipocytes promote a defense mechanism against ferroptosis in PCa cells by the upregulation of GPX4 activity via the mTOR pathway. GPX4 is known to be an essential antioxidant enzyme that utilizes glutathione (GSH) to reduce lipid-peroxidation-induced ROS. We demonstrate that PCa cells exposed to adipocytes have reduced lipid-peroxidation induced ROS, which can be reversed with ferroptosis inducers. We also show that exposure of PC3 and ARCaP(M) cells to adipocytes leads to depletion of GSH, which may be indicative of higher GPX4 activity. It has been suggested that an increase in GPX4 can result from an interplay between ferroptosis and mTOR pathways, specifically mTORC1, which may mediate cysteine-induced GPX4 protein synthesis. Surprisingly, treatment of PCa cells co-cultured with adipocytes with mTORC1 inhibitor Everolimus (EVO) increases the protein and gene levels of GPX4. This coincides with reduced mRNA and protein expression of SLC7A11, transporter involved in the cysteine/glutamate antiporter system, which typically fuels GPX4 activity by bringing in the GSH precursor cysteine. Understanding the mechanisms of fat cell contribution to dysregulation of the mTOR pathway and escape from ferroptosis may have therapeutic implications for metastatic PCa. Citation Format: Alexis Wilson, Shane Mecca, Mackenzie Herroon, Laimar Garmo, Izabela Podgorski. Defense mechanism against oxidative damage in metastatic prostate cancer: Adipocyte-mediated modulation of mTOR and ferroptosis pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6319.

Abstract Prostate cancer (PCa) is the most common cancer amongst males and becomes incurable once it advances to the secondary site. The most common site of metastasis from PCa is the adipocyte-rich bone marrow. Adipocytes are metabolically active cells capable of secreting a variety of adipokines and free fatty acids, which can be utilized by tumor cells to enhance proliferation and cause resistance to chemotherapies. Our lab has shown that adipocytes promote PCa progression and therapy evasion through modulation of tumor metabolism and activation of pro-survival signaling; however, the molecular mechanisms behind tumor-promoting effects of fat cells are not understood. It is well known that high levels of iron within the tumor microenvironment help the growth of PCa cells. However, since iron overload can cause an increase in reactive oxygen species (ROS), which are harmful to cell structure, tumor cells have developed defense mechanisms to protect them from this oxidative damage. Intriguingly, our data show that the expression of ferritin, an iron storing protein, is reduced in PC3 and ARCaP(M) cells in co-culture with adipocytes. Since low ferritin levels have been associated with induction of ferroptosis, an iron and oxidative stress-regulated cell death process, we examined protein levels of ferroptosis markers GPX4 and NCOA4 in PCa cells. Surprisingly, our data show that interaction with adipocytes increases GPX4 and reduces NCOA4 expression in PCa cells suggesting an adipocyte-mediated defense mechanism against ferroptosis. The aim of the present study was to establish that adipocytes promote a defense mechanism against ferroptosis in PCa cells by the upregulation of GPX4 activity via the mTOR pathway. GPX4 is known to be an essential antioxidant enzyme that utilizes glutathione (GSH) to reduce lipid-peroxidation-induced ROS. We demonstrate that PCa cells exposed to adipocytes have reduced lipid-peroxidation induced ROS, which can be reversed with ferroptosis inducers. We also show that exposure of PC3 and ARCaP(M) cells to adipocytes leads to depletion of GSH, which may be indicative of higher GPX4 activity. It has been suggested that an increase in GPX4 can result from an interplay between ferroptosis and mTOR pathways, specifically mTORC1, which may mediate cysteine-induced GPX4 protein synthesis. Surprisingly, treatment of PCa cells co-cultured with adipocytes with mTORC1 inhibitor Everolimus (EVO) increases the protein and gene levels of GPX4. This coincides with reduced mRNA and protein expression of SLC7A11, transporter involved in the cysteine/glutamate antiporter system, which typically fuels GPX4 activity by bringing in the GSH precursor cysteine. Understanding the mechanisms of fat cell contribution to dysregulation of the mTOR pathway and escape from ferroptosis may have therapeutic implications for metastatic PCa. Citation Format: Alexis Wilson, Shane Mecca, Mackenzie Herroon, Laimar Garmo, Izabela Podgorski. Defense mechanism against oxidative damage in metastatic prostate cancer: Adipocyte-mediated modulation of mTOR and ferroptosis pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6319.

Abstract Background: Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer mortality. Acute adverse effects (AEs) from CRC treatments (surgery, chemotherapy, radiation therapy) may cause dose limitations and/or treatment discontinuation. Chronic AEs may include bowel symptoms, fatigue, anxiety, depression, and sarcopenic obesity. These acute and chronic AEs significantly impact quality of life (QoL). A comprehensive understanding of the pathophysiological mechanism(s) driving these AEs is lacking. Evidence supports the hypothesis that the gut microbiota may be an integrative point in the pathogenesis of several AEs. Dysbiosis alters the normal function of the gut and gut-brain-axis. CRC treatments can lead to dysbiosis and in turn may drive acute and chronic AEs. Our aims are to explore how CRC treatment affects the microbiota and the further path to recovery. Methods: A prospective feasibility study of n=35 participants in Calgary, Alberta of stage I-III CRC patients to evaluate: 1) The feasibility of collecting microbiota samples at diagnosis to one-year post diagnosis; 2) Longitudinal changes to microbiota over a 1-year period; and 3) Preliminary associations between changes in the microbiota and treatment completion, treatment AEs, clinical and tumor characteristics, and changes to patient reported outcomes (PROs). Inclusion: Newly diagnosed stage I-III CRC, aged ≥18, English speaking, and willing to provide 4 fecal samples. Exclusion: Inflammatory bowel disease, hereditary CRC syndromes, or stage IV. Convenience sampling will be used. Feasibility will include recruitment and retention rates, adherence to specimen collection protocols, specimen quality, and patient satisfaction. Microbiota will be evaluated using longitudinal fecal sampling for metabolomics, culture, and mechanistic studies to examine intra-individual differences in microbiota (α and b diversity). Shotgun sequencing libraries will be prepared to generate approximately 4M 150 bp read pairs/sample. Clinical data on tumor characteristics, treatments, and treatment AEs will be abstracted from medical records. Demographic data and a battery of PROs (diet, physical activity, depression, anxiety, QoL, CRC symptoms, cognitive function, and fatigue using validated questionnaires) will be collected. Results: This study will determine the feasibility of longitudinal prospective collection of biospecimen, clinical, and PROs in newly diagnosed stage I-III CRC patients. This study will also provide preliminary data on changes to the gut microbiota as a result of treatments and how these changes may in turn impact clinical and PROs. Conclusions: This novel investigation into dysbiosis as an integrative point driving CRC treatment AEs is timely and warranted given the persistence of debilitating problems post CRC treatment. Building on data from this project we plan to conduct a population-based cohort study. Our goal is to ultimately inform interventions to manage treatment AEs, improve clinical outcomes, and improve QoL for CRC survivors. Citation Format: Colleen Ann Cuthbert, Kathy McCoy, Anthony MacLean, Lin Yang, May Lynn Quan, Donald Buie. Investigating the effects of cancer treatment on gut microbiota in colorectal cancer patients: Study protocol [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B005.

Abstract Background: Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer mortality. Acute adverse effects (AEs) from CRC treatments (surgery, chemotherapy, radiation therapy) may cause dose limitations and/or treatment discontinuation. Chronic AEs may include bowel symptoms, fatigue, anxiety, depression, and sarcopenic obesity. These acute and chronic AEs significantly impact quality of life (QoL). A comprehensive understanding of the pathophysiological mechanism(s) driving these AEs is lacking. Evidence supports the hypothesis that the gut microbiota may be an integrative point in the pathogenesis of several AEs. Dysbiosis alters the normal function of the gut and gut-brain-axis. CRC treatments can lead to dysbiosis and in turn may drive acute and chronic AEs. Our aims are to explore how CRC treatment affects the microbiota and the further path to recovery. Methods: A prospective feasibility study of n=35 participants in Calgary, Alberta of stage I-III CRC patients to evaluate: 1) The feasibility of collecting microbiota samples at diagnosis to one-year post diagnosis; 2) Longitudinal changes to microbiota over a 1-year period; and 3) Preliminary associations between changes in the microbiota and treatment completion, treatment AEs, clinical and tumor characteristics, and changes to patient reported outcomes (PROs). Inclusion: Newly diagnosed stage I-III CRC, aged ≥18, English speaking, and willing to provide 4 fecal samples. Exclusion: Inflammatory bowel disease, hereditary CRC syndromes, or stage IV. Convenience sampling will be used. Feasibility will include recruitment and retention rates, adherence to specimen collection protocols, specimen quality, and patient satisfaction. Microbiota will be evaluated using longitudinal fecal sampling for metabolomics, culture, and mechanistic studies to examine intra-individual differences in microbiota (α and b diversity). Shotgun sequencing libraries will be prepared to generate approximately 4M 150 bp read pairs/sample. Clinical data on tumor characteristics, treatments, and treatment AEs will be abstracted from medical records. Demographic data and a battery of PROs (diet, physical activity, depression, anxiety, QoL, CRC symptoms, cognitive function, and fatigue using validated questionnaires) will be collected. Results: This study will determine the feasibility of longitudinal prospective collection of biospecimen, clinical, and PROs in newly diagnosed stage I-III CRC patients. This study will also provide preliminary data on changes to the gut microbiota as a result of treatments and how these changes may in turn impact clinical and PROs. Conclusions: This novel investigation into dysbiosis as an integrative point driving CRC treatment AEs is timely and warranted given the persistence of debilitating problems post CRC treatment. Building on data from this project we plan to conduct a population-based cohort study. Our goal is to ultimately inform interventions to manage treatment AEs, improve clinical outcomes, and improve QoL for CRC survivors. Citation Format: Colleen Ann Cuthbert, Kathy McCoy, Anthony MacLean, Lin Yang, May Lynn Quan, Donald Buie. Investigating the effects of cancer treatment on gut microbiota in colorectal cancer patients: Study protocol [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B005.

Abstract Adoptive cell therapy (ACT) using patient-derived tumor-specific T cells is a promising approach for cancer treatment especially for the treatment of hematological malignancies. However, therapeutic approaches using ACT in patients with solid tumors, such as hepatocellular carcinoma (HCC), has not shown substantially improved clinical outcomes in particular due to immunosuppressive tumor microenvironments. Therefore, strategies to enhance ACT T cell functionality in vivo and to overcome the immunosuppressive microenvironments in solid tumors are strongly needed. We developed a strategy combining nanomedicine with ACT, based on the chemical conjugation of cytokine-loaded nanoparticles (NPs) as synthetic “backpacks” to the surfaces of live lymphocytes for adoptive therapy. Novel protein nanoparticles were synthesized by crosslinking of single chain IL-12 (scIL-12)- or IL-15 superagonist through disulfide-containing crosslinkers, forming nanogels that when bound to the surface of T cells, release the supporting cytokines in response to antigen receptor signaling-mediated changes in cell surface redox state. This TCR-mediated drug release allowed drug delivery to the T cells to be focused in tumors and tumor-draining lymph nodes. Coupling of cytokine nanogels to T cell membranes induced significant cell activation in terms of interferon-γ production or proliferation in a concentration-dependent manner. Antigen-specific CD8+ T cells “backpacked” with scIL-12 or IL-15SA nanogels were capable of substantial tumor cell killing in a B16F10 melanoma cancer model in vivo, while avoiding lethal systemic toxicities triggered by systemically-administered immunomodulatory cytokines. Ongoing studies are seeking to apply this approach in HCC, and this approach bears the potential for the development of successful immunotherapeutic strategies against this and other solid tumors where effective treatment options are still lacking. Citation Format: Michael Fichter. T cell receptor signaling-responsive single chain IL-12 and IL-15 superagonist nanogel “backpacks” to enhance adoptive cell therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3565.

Abstract Adoptive cell therapy (ACT) using patient-derived tumor-specific T cells is a promising approach for cancer treatment especially for the treatment of hematological malignancies. However, therapeutic approaches using ACT in patients with solid tumors, such as hepatocellular carcinoma (HCC), has not shown substantially improved clinical outcomes in particular due to immunosuppressive tumor microenvironments. Therefore, strategies to enhance ACT T cell functionality in vivo and to overcome the immunosuppressive microenvironments in solid tumors are strongly needed. We developed a strategy combining nanomedicine with ACT, based on the chemical conjugation of cytokine-loaded nanoparticles (NPs) as synthetic “backpacks” to the surfaces of live lymphocytes for adoptive therapy. Novel protein nanoparticles were synthesized by crosslinking of single chain IL-12 (scIL-12)- or IL-15 superagonist through disulfide-containing crosslinkers, forming nanogels that when bound to the surface of T cells, release the supporting cytokines in response to antigen receptor signaling-mediated changes in cell surface redox state. This TCR-mediated drug release allowed drug delivery to the T cells to be focused in tumors and tumor-draining lymph nodes. Coupling of cytokine nanogels to T cell membranes induced significant cell activation in terms of interferon-γ production or proliferation in a concentration-dependent manner. Antigen-specific CD8+ T cells “backpacked” with scIL-12 or IL-15SA nanogels were capable of substantial tumor cell killing in a B16F10 melanoma cancer model in vivo, while avoiding lethal systemic toxicities triggered by systemically-administered immunomodulatory cytokines. Ongoing studies are seeking to apply this approach in HCC, and this approach bears the potential for the development of successful immunotherapeutic strategies against this and other solid tumors where effective treatment options are still lacking. Citation Format: Michael Fichter. T cell receptor signaling-responsive single chain IL-12 and IL-15 superagonist nanogel “backpacks” to enhance adoptive cell therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3565.

Abstract Fibrolamellar Carcinoma (FLC), a rare liver cancer, has yet no specific tools to evaluate its prognosis. This study aims, in partnership with the patient community, to search for associations between various demographic and clinical factors and overall survival of patients, to assess prognostic factors. We performed a retrospective analysis on a community-based patient registry (The Fibrolamellar Registry http://fibroregistry.org) to examine relations between survival and different variables. The variables examined were demographic (gender), clinical (age at diagnosis, tumor size, number of tumors inside the liver, presence of positive lymph nodes or distant metastasis) as well as treatment modalities (surgery or systemic therapy). Kaplan-Meier survival plots were created for the overall research population and for stratifying by unadjusted variables. Cox proportional hazards model was used for multivariable analysis of hazard ratio (HR) for death at any time among groups, adjusting for variables found significant in the univariate analysis. Among 150 patients, (89 females and 61 males), median overall survival time from diagnosis to death or loss of follow-up was 8 years and 2 months. Features with independent positive effect on survival were female gender, as well as being treated with surgery alone. In contrast, having 10 or more tumors inside the liver had a negative effect on survival. Positive lymph nodes at diagnosis, and distant metastasis at diagnosis, were both associated with poorer prognosis, though did not remain independent when adjusting for covariables. We could not find an effect of age at diagnosis or tumor size at its largest measurement, on survival. In conclusion, our study suggests gender is a significant prognostic factor for FLC patients, with females having a better outcome than males. Surgical removal of the tumor, without additional systemic therapy is a positive prognostic factor as well. Having a large number of tumors of 10 and above, despite all being inside the liver, is a negative prognostic factor. Citation Format: Amichai Berkovitz, Rachael D. Migler, Adam Qureshi, Roger Vaughan, Erin L. Marcotte, Sanford M. Simon. Clinical and demographic predictors of survival for fibrolamellar hepatocellular carcinoma patients - A patient-community registry-based study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2495.

Abstract Fibrolamellar Carcinoma (FLC), a rare liver cancer, has yet no specific tools to evaluate its prognosis. This study aims, in partnership with the patient community, to search for associations between various demographic and clinical factors and overall survival of patients, to assess prognostic factors. We performed a retrospective analysis on a community-based patient registry (The Fibrolamellar Registry http://fibroregistry.org) to examine relations between survival and different variables. The variables examined were demographic (gender), clinical (age at diagnosis, tumor size, number of tumors inside the liver, presence of positive lymph nodes or distant metastasis) as well as treatment modalities (surgery or systemic therapy). Kaplan-Meier survival plots were created for the overall research population and for stratifying by unadjusted variables. Cox proportional hazards model was used for multivariable analysis of hazard ratio (HR) for death at any time among groups, adjusting for variables found significant in the univariate analysis. Among 150 patients, (89 females and 61 males), median overall survival time from diagnosis to death or loss of follow-up was 8 years and 2 months. Features with independent positive effect on survival were female gender, as well as being treated with surgery alone. In contrast, having 10 or more tumors inside the liver had a negative effect on survival. Positive lymph nodes at diagnosis, and distant metastasis at diagnosis, were both associated with poorer prognosis, though did not remain independent when adjusting for covariables. We could not find an effect of age at diagnosis or tumor size at its largest measurement, on survival. In conclusion, our study suggests gender is a significant prognostic factor for FLC patients, with females having a better outcome than males. Surgical removal of the tumor, without additional systemic therapy is a positive prognostic factor as well. Having a large number of tumors of 10 and above, despite all being inside the liver, is a negative prognostic factor. Citation Format: Amichai Berkovitz, Rachael D. Migler, Adam Qureshi, Roger Vaughan, Erin L. Marcotte, Sanford M. Simon. Clinical and demographic predictors of survival for fibrolamellar hepatocellular carcinoma patients - A patient-community registry-based study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2495.

Abstract The adenomatoid odontogenic tumor (AOT) is a benign tumor of uncertain pathogenesis. Schimmelpenning syndrome is characterized by sebaceous nevi, which occurs often on the face, associated with variable ipsilateral abnormalities of the central nervous system, including ocular and skeletal abnormalities. It results from postzygotic autosomal dominant HRAS or KRAS lethal mutations that survive by somatic mosaicism. RAS genes mutations were previously reported in lesional tissue (including nevus sebaceous) of a patient, but not in normal skin or blood leukocytes, consistent with a somatic mosaic state. Interestingly, a case of multiple AOT was reported in a Schimmelpenning syndrome patient, which prompted us to evaluate RAS genes mutations, as well as 207 cancer genes mutations in a sample of one AOT from one Schimmelpenning syndrome patient having multiple tumors (index patient). We used the Ion AmpliSeqTM Cancer Hotspot Panel to interrogate these mutations by targeted next generation sequencing. We further included 3 sporadic AOT samples to assess if they shared similar mutations with the sample of the index patient. Additionally, molecular karyotyping analysis was performed in the index patient sample, as well as in one sporadic AOT sample by using a high-density whole genome array platform (Cytoscan® HD Array). The pathogenic KRAS G12V mutation was detected in the index patient sample of AOT, and in 2 out of 3 samples evaluated. No other pathogenic mutation was detected in the AOT samples. TaqMan® Mutation Detection probes specific to the c.35G>T KRAS gene substitution and/or Sanger sequencing were used to validate the mutation in all samples and in a panel of 4 additional sporadic AOT samples. A total of 7 out of 8 AOT samples showed the KRAS pathogenic mutation. We found a few copy number variations (CNVs), most of them common variations or alterations not encompassing genes. Loss of 7p15.3 encompassing the IGF2BP3 gene was detected in the sporadic AOT sample. In conclusion, we report for the first time the recurrent activating KRAS G12V mutation in a high proportion of investigated AOTs, while no other pathogenic mutation interrogated was detected. The importance of the 7p15.3 loss in the aetiopathogenesis of this tumor type remains to be established. Despite the benign nature of AOT, our results shed some light in future molecular targeted-therapy for the lesion. Supported by: CNPq, CAPES and FAPEMIG (Brazil). Citation Format: Carolina C. Gomes, Silvia F. Sousa, Grazielle F. Menezes, Thais S.F. Pereira, Rennan G. Moreira, Alessandra P. Duarte, Wagner H. Castro, Rolando A.R. Villacis, Silvia R. Rogatto, Marina G. Diniz, Ricardo S. Gomez. Recurrent KRAS G12V pathogenic mutation in adenomatoid odontogenic tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 88.

Abstract The adenomatoid odontogenic tumor (AOT) is a benign tumor of uncertain pathogenesis. Schimmelpenning syndrome is characterized by sebaceous nevi, which occurs often on the face, associated with variable ipsilateral abnormalities of the central nervous system, including ocular and skeletal abnormalities. It results from postzygotic autosomal dominant HRAS or KRAS lethal mutations that survive by somatic mosaicism. RAS genes mutations were previously reported in lesional tissue (including nevus sebaceous) of a patient, but not in normal skin or blood leukocytes, consistent with a somatic mosaic state. Interestingly, a case of multiple AOT was reported in a Schimmelpenning syndrome patient, which prompted us to evaluate RAS genes mutations, as well as 207 cancer genes mutations in a sample of one AOT from one Schimmelpenning syndrome patient having multiple tumors (index patient). We used the Ion AmpliSeqTM Cancer Hotspot Panel to interrogate these mutations by targeted next generation sequencing. We further included 3 sporadic AOT samples to assess if they shared similar mutations with the sample of the index patient. Additionally, molecular karyotyping analysis was performed in the index patient sample, as well as in one sporadic AOT sample by using a high-density whole genome array platform (Cytoscan® HD Array). The pathogenic KRAS G12V mutation was detected in the index patient sample of AOT, and in 2 out of 3 samples evaluated. No other pathogenic mutation was detected in the AOT samples. TaqMan® Mutation Detection probes specific to the c.35G>T KRAS gene substitution and/or Sanger sequencing were used to validate the mutation in all samples and in a panel of 4 additional sporadic AOT samples. A total of 7 out of 8 AOT samples showed the KRAS pathogenic mutation. We found a few copy number variations (CNVs), most of them common variations or alterations not encompassing genes. Loss of 7p15.3 encompassing the IGF2BP3 gene was detected in the sporadic AOT sample. In conclusion, we report for the first time the recurrent activating KRAS G12V mutation in a high proportion of investigated AOTs, while no other pathogenic mutation interrogated was detected. The importance of the 7p15.3 loss in the aetiopathogenesis of this tumor type remains to be established. Despite the benign nature of AOT, our results shed some light in future molecular targeted-therapy for the lesion. Supported by: CNPq, CAPES and FAPEMIG (Brazil). Citation Format: Carolina C. Gomes, Silvia F. Sousa, Grazielle F. Menezes, Thais S.F. Pereira, Rennan G. Moreira, Alessandra P. Duarte, Wagner H. Castro, Rolando A.R. Villacis, Silvia R. Rogatto, Marina G. Diniz, Ricardo S. Gomez. Recurrent KRAS G12V pathogenic mutation in adenomatoid odontogenic tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 88.

Abstract The most common form of genomic instability in sporadic colorectal cancer (CRC) is chromosomal instability, a phenotype characterized by a high frequency of DNA copy number gain (CNG) and loss (CNL). Given the fundamental role of chromosomal instability in the pathogenesis of CRC, this study aimed to investigate the concordance of DNA copy number variants (CNV) between primary and metastatic CRC to better understand how the genomic structure differs during tumor evolution. In this study, the SurePrint G3 Human Genome CGH+SNP 2x400K Microarray (Agilent, SC, California, USA) was used for genome-wide screening of CNV in 9 pairs of primary tumors and metachronous liver metastases obtained during surgical resection from patients diagnosed with advanced CRC. Data from the Microarray-based Comparative Genomic Hybridization was analyzed by COSMIC Cancer Gene Census (CGC) and an independent non-CGC panel to map hotspot regions harboring genes causally implicated in cancer in the course of the metastatic cascade. Also, unique CNV in metastases were further investigated to identify loci advantageous for tumor progression and those presumably associated with therapy resistance. Overall, CNG observed in primary tumors were highly concordant with those in liver metastases. The CNG hotspot regions for both tissues represented chr7 (p22.3-p13), chr8 (q21.13-q24.3), chr13 (q12.11-q13.3), chr20 (q11.1-q13.33) and chrX (q11.1-q28) loci, while the most frequent CNL was at chr8 (p21.3-p12) locus. All identified loci underwent CNV events with a higher frequency in metastases (67-100%) than in primary tumors (55%). The identified genomic loci with CNG encode DNA repair genes (such as PMS2, NBN, BRCA2, RAD54B, RAD21, RPA3,4, and TREX2), homeobox genes (HOXA9,11,13), genes encoding centrosome-associated proteins (CETN2, MPLKIP), proto-oncogenes (MYC), and also tumor suppressor (ATRX, ASXL1). Additionally, an analysis of unique CNV within distinct tissues revealed CNG at Chr6 (p25.1-p22.3) in 89% of metastatic samples and absent in all the primary tumor lesions. This particular genomic region contains proto-oncogene DEK. Although the analysis is still underway, we have identified several shared and unique regions of altered copy number in both, primary tumors and metastases. We conclude that despite the high inter- and intra-tumoral genomic heterogeneity, metachronous metastases “inherit” a similar profile of DNA CNV from their respective primary tumor tissues. We intend to further extend our studied group of patients and perform integrative analyses of CNV with gene expression. This study was supported by the Charles University (UNCE/MED/006) and the Grant Agency of the Czech Republic (21-04607X, 21-27902S) Citation Format: Michal Kroupa, Kristyna Tomasova, Katerina Saskova, Jana Drabova, Josef Horak, Ludmila Vodickova, Jachym Rosendorf, Vaclav Liska, Pavel Vodicka. Concordance of DNA copy number profiles between primary and metastatic colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 760.

Abstract The most common form of genomic instability in sporadic colorectal cancer (CRC) is chromosomal instability, a phenotype characterized by a high frequency of DNA copy number gain (CNG) and loss (CNL). Given the fundamental role of chromosomal instability in the pathogenesis of CRC, this study aimed to investigate the concordance of DNA copy number variants (CNV) between primary and metastatic CRC to better understand how the genomic structure differs during tumor evolution. In this study, the SurePrint G3 Human Genome CGH+SNP 2x400K Microarray (Agilent, SC, California, USA) was used for genome-wide screening of CNV in 9 pairs of primary tumors and metachronous liver metastases obtained during surgical resection from patients diagnosed with advanced CRC. Data from the Microarray-based Comparative Genomic Hybridization was analyzed by COSMIC Cancer Gene Census (CGC) and an independent non-CGC panel to map hotspot regions harboring genes causally implicated in cancer in the course of the metastatic cascade. Also, unique CNV in metastases were further investigated to identify loci advantageous for tumor progression and those presumably associated with therapy resistance. Overall, CNG observed in primary tumors were highly concordant with those in liver metastases. The CNG hotspot regions for both tissues represented chr7 (p22.3-p13), chr8 (q21.13-q24.3), chr13 (q12.11-q13.3), chr20 (q11.1-q13.33) and chrX (q11.1-q28) loci, while the most frequent CNL was at chr8 (p21.3-p12) locus. All identified loci underwent CNV events with a higher frequency in metastases (67-100%) than in primary tumors (55%). The identified genomic loci with CNG encode DNA repair genes (such as PMS2, NBN, BRCA2, RAD54B, RAD21, RPA3,4, and TREX2), homeobox genes (HOXA9,11,13), genes encoding centrosome-associated proteins (CETN2, MPLKIP), proto-oncogenes (MYC), and also tumor suppressor (ATRX, ASXL1). Additionally, an analysis of unique CNV within distinct tissues revealed CNG at Chr6 (p25.1-p22.3) in 89% of metastatic samples and absent in all the primary tumor lesions. This particular genomic region contains proto-oncogene DEK. Although the analysis is still underway, we have identified several shared and unique regions of altered copy number in both, primary tumors and metastases. We conclude that despite the high inter- and intra-tumoral genomic heterogeneity, metachronous metastases “inherit” a similar profile of DNA CNV from their respective primary tumor tissues. We intend to further extend our studied group of patients and perform integrative analyses of CNV with gene expression. This study was supported by the Charles University (UNCE/MED/006) and the Grant Agency of the Czech Republic (21-04607X, 21-27902S) Citation Format: Michal Kroupa, Kristyna Tomasova, Katerina Saskova, Jana Drabova, Josef Horak, Ludmila Vodickova, Jachym Rosendorf, Vaclav Liska, Pavel Vodicka. Concordance of DNA copy number profiles between primary and metastatic colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 760.

Abstract Innate immunity plays a vital role in detecting cytoplasmic nucleic acids resulting from viral infection or the presence of tumor cells. Cytosolic DNA is sensed by the cyclic-GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, and in the case of tumors, activation of this pathway has potential to both positively and negatively modulate cancer development. Activation of the STING pathway is a key prerequisite for type I IFN production that is needed for either endogenous or treatment-induced cancer immune responses, and reduced cGAS-STING expression is associated with poorer survival of patients with lung adenocarcinoma, invasive breast ductal carcinoma, and gastric cancer. Intrinsic activation of the cGAS-STING pathway in the tumor microenvironment (TME) promotes cancer cell-intrinsic senescence and recruitment of immune cells, resulting in antitumor effects. For cancers that are difficult to treat and characterized by immune tolerance, combination therapies of STING agonists with immune checkpoint inhibitors represents a new paradigm in disease management. To date, only two early clinical trials of the STING agonist ADU-S100 in combination with checkpoint inhibitors are underway (NCT02675439 and NCT03172936). To progress clinical trials more rapidly and with better safety outcomes for patients with advanced treatment-refractory metastatic solid tumors or lymphomas, being able to reliably screen a variety of STING agonists in different types of in vitro cancer models is of paramount importance. We have previously reported a novel functional assay that is capable of demonstrating the potency level of different STING agonists, based on the IFNβ response in THP-1 monocytic leukemia cells. We further investigated the role of newly developed STING agonists and clinical compounds in phenotypic in vitro cancer cell and TME models to inform on oncotherapeutic development. STING-expressing cancer cell lines, chosen based on microarray mRNA expression, were treated with agonists and assayed for cGAS-STING activation status using a fully automated platform for high-content imaging. In vitro analysis demonstrated high phospho-STING activation at 4 hours. In the BioMAP® Oncology Colorectal TME model, STING activation increased IL-6 release and the effect on other primary immune and tissue remodeling biomarkers will be discussed. Preclinical studies indicate that STING agonists, used as adjuvants in combination with other agents or radiation therapy, suppress tumor progression, reduce cellular toxicity, and eliminate metastases in breast and pancreatic cancer models. Phenotypic assays that provide human, translational data early in discovery are a valuable tool to accelerate progress in this area. Citation Format: Satheesh K. Sainathan, Justin H. Lipner, Jennifer I. Drake, Brogan A. Epkins, Brianna M. Roux, Alastair J. King. Measurement of cytosolic DNA sensing cGAS-STING pathway functional activity using in vitro phenotypic assay models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1877.

Abstract Innate immunity plays a vital role in detecting cytoplasmic nucleic acids resulting from viral infection or the presence of tumor cells. Cytosolic DNA is sensed by the cyclic-GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, and in the case of tumors, activation of this pathway has potential to both positively and negatively modulate cancer development. Activation of the STING pathway is a key prerequisite for type I IFN production that is needed for either endogenous or treatment-induced cancer immune responses, and reduced cGAS-STING expression is associated with poorer survival of patients with lung adenocarcinoma, invasive breast ductal carcinoma, and gastric cancer. Intrinsic activation of the cGAS-STING pathway in the tumor microenvironment (TME) promotes cancer cell-intrinsic senescence and recruitment of immune cells, resulting in antitumor effects. For cancers that are difficult to treat and characterized by immune tolerance, combination therapies of STING agonists with immune checkpoint inhibitors represents a new paradigm in disease management. To date, only two early clinical trials of the STING agonist ADU-S100 in combination with checkpoint inhibitors are underway (NCT02675439 and NCT03172936). To progress clinical trials more rapidly and with better safety outcomes for patients with advanced treatment-refractory metastatic solid tumors or lymphomas, being able to reliably screen a variety of STING agonists in different types of in vitro cancer models is of paramount importance. We have previously reported a novel functional assay that is capable of demonstrating the potency level of different STING agonists, based on the IFNβ response in THP-1 monocytic leukemia cells. We further investigated the role of newly developed STING agonists and clinical compounds in phenotypic in vitro cancer cell and TME models to inform on oncotherapeutic development. STING-expressing cancer cell lines, chosen based on microarray mRNA expression, were treated with agonists and assayed for cGAS-STING activation status using a fully automated platform for high-content imaging. In vitro analysis demonstrated high phospho-STING activation at 4 hours. In the BioMAP® Oncology Colorectal TME model, STING activation increased IL-6 release and the effect on other primary immune and tissue remodeling biomarkers will be discussed. Preclinical studies indicate that STING agonists, used as adjuvants in combination with other agents or radiation therapy, suppress tumor progression, reduce cellular toxicity, and eliminate metastases in breast and pancreatic cancer models. Phenotypic assays that provide human, translational data early in discovery are a valuable tool to accelerate progress in this area. Citation Format: Satheesh K. Sainathan, Justin H. Lipner, Jennifer I. Drake, Brogan A. Epkins, Brianna M. Roux, Alastair J. King. Measurement of cytosolic DNA sensing cGAS-STING pathway functional activity using in vitro phenotypic assay models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1877.