Jonathan F. Anker

Genomic landscape of DNA repair genes in cancer

DNA repair genes are frequently mutated in cancer, yet limited data exist regarding the overall genomic landscape and functional implications of these alterations in their entirety. We created comprehensive lists of DNA repair genes and indirect caretakers. Mutation, copy number variation (CNV), and expression frequencies of these genes were analyzed in COSMIC. Mutation co-occurrence, clinical outcomes, and mutation burden were analyzed in TCGA. We report the 20 genes most frequently with mutations (n > 19,689 tumor samples for each gene), CNVs (n > 1,556), or up- or down-regulated (n = 7,998). Mutual exclusivity was observed as no genes displayed both high CNV gain and loss or high up- and down-regulation, and CNV gain and loss positively correlated with up- and down-regulation, respectively. Co-occurrence of mutations differed between cancers, and mutations in many DNA repair genes were associated with higher total mutation burden. Mutation and CNV frequencies offer insights into which genes may play tumor suppressive or oncogenic roles, such as NEIL2 and RRM2B, respectively. Mutual exclusivities within CNV and expression frequencies, and correlations between CNV and expression, support the functionality of these genomic alterations. This study provides comprehensive lists of candidate genes as potential biomarkers for genomic instability, novel therapeutic targets, or predictors of immunotherapy efficacy.

Mutations in DNA repair genes are associated with increased neo-antigen load and activated T cell infiltration in lung adenocarcinoma

Mutations in DNA repair genes lead to increased genomic instability and mutation frequency. These mutations represent potential biomarkers for cancer immunotherapy efficacy, as high tumor mutational burden has been associated with increased neo-antigens and tumor infiltrating lymphocytes. While mismatch repair mutations have successfully predicted response to anti-PD-1 therapy in colorectal and other cancers, they have not yet been tested for lung cancer, and few have investigated genes from other DNA repair pathways. We utilized TCGA samples to comprehensively immunophenotype lung tumors and analyze the links between DNA repair mutations, neo-antigen and total mutational burden, and tumor immune infiltration. Overall, 73% of lung tumors contained infiltration by at least one T cell subset, with high mutational burden tumors containing significantly increased infiltration by activated CD4 and CD8 T cells. Further, mutations in mismatch repair genes, homologous recombination genes, or POLE accurately predicted increased tumor mutational burden, neo-antigen load, and T cell infiltration. Finally, neo-antigen load correlated with expression of M1-polarized macrophage genes, PD-1, PD-L1, IFNγ, GZMB, and FASLG, among other immune-related genes. Overall, after defining the immune infiltrate in lung tumors, we demonstrate the potential value of utilizing gene mutations from multiple DNA repair pathways as biomarkers for lung cancer immunotherapy.

Epithelial-mesenchymal transition (EMT) signature is inversely associated with T-cell infiltration in non-small cell lung cancer (NSCLC)

Epithelial-mesenchymal transition (EMT) is able to drive metastasis during progression of multiple cancer types, including non-small cell lung cancer (NSCLC). As resistance to immunotherapy has been associated with EMT and immune exclusion in melanoma, it is important to understand alterations to T-cell infiltration and the tumor microenvironment during EMT in lung adenocarcinoma and squamous cell carcinoma. We conducted an integrated analysis of the immune landscape in NSCLCs through EMT scores derived from a previously established 16 gene signature of canonical EMT markers. EMT was associated with exclusion of immune cells critical in the immune response to cancer, with significantly lower infiltration of CD4 T-cells in lung adenocarcinoma and CD4/CD8 T-cells in squamous cell carcinoma. EMT was also associated with increased expression of multiple immunosuppressive cytokines, including IL-10 and TGF-β. Furthermore, overexpression of targetable immune checkpoints, such as CTLA-4 and TIM-3 were associated with EMT in both NSCLCs. An association may exist between immune exclusion and EMT in NSCLC. Further investigation is merited as its mechanism is not completely understood and a better understanding of this association could lead to the development of biomarkers that could accurately predict response to immunotherapy.

Cancer immunotherapy in a neglected population: The current use and future of T-cell-mediated checkpoint inhibitors in organ transplant patients

Although the indications for immune checkpoint inhibitors continue to grow, organ transplant recipients with advanced malignancies have been largely excluded from clinical trials testing the safety and efficacy of these therapies given their need for chronic immunosuppression and the risk of allograft rejection. With the rapid growth of transplant medicine and the increased risk of malignancy associated with chronic immunosuppression, it is critical that we systematically analyze the available data describing immune checkpoint blockade in the organ transplant population. Herein we provide a current and comprehensive review of cases in which immune checkpoint blockade was used on organ transplant recipients. Furthermore, we discuss the differences in efficacy and risk of allograft rejection between CTLA-4 and PD-1 inhibitors and make recommendations based on the limited available clinical data. We also discuss the future of immune checkpoint blockade in this subpopulation and explore the emerging data of promising combination therapies with mTOR, BRAF/MEK, and BTK/ITK inhibitors. Further clinical experience and larger clinical trials involving immune checkpoint inhibitors, whether as monotherapies or combinatorial therapies, will help develop regimens that optimize anti-tumor response and minimize the risk of allograft rejection in organ transplant patients.

Role of gram-positive bacteria in chronic pelvic pain syndrome (CPPS)

Chronic pelvic pain syndrome (CPPS) is a complex disorder that affects a large proportion of all men. A limited understanding of its etiology and pathogenesis is reflected by the absence of effective therapies. Although CPPS is deemed clinically non‐infectious with no well‐defined etiological role for microbes, bacteria is readily isolated from both healthy and patient prostate secretion and urine samples. Our laboratory has previously demonstrated that a specific gram‐negative bacterial isolate can induce CPPS‐like symptoms in mice. Here we aimed to expand on these findings examining the role of gram‐positive patient‐derived bacteria in CPPS.

Overexpression of adhesion molecules and barrier molecules is associated with differential infiltration of immune cells in non-small cell lung cancer

Immunotherapy is emerging as a promising option for lung cancer treatment. Various endothelial adhesion molecules, such as integrin and selectin, as well as various cellular barrier molecules such as desmosome and tight junctions, regulate T-cell infiltration in the tumor microenvironment. However, little is known regarding how these molecules affect immune cells in patients with lung cancer. We demonstrated for the first time that overexpression of endothelial adhesion molecules and cellular barrier molecule genes was linked to differential infiltration of particular immune cells in non-small cell lung cancer. Overexpression of endothelial adhesion molecule genes is associated with significantly lower infiltration of activated CD4 and CD8 T-cells, but higher infiltration of activated B-cells and regulatory T-cells. In contrast, overexpression of desmosome genes was correlated with significantly higher infiltration of activated CD4 and CD8 T-cells, but lower infiltration of activated B-cells and regulatory T-cells in lung adenocarcinoma. This inverse relation of immune cells aligns with previous studies of tumor-infiltrating B-cells inhibiting T-cell activation. Although overexpression of endothelial adhesion molecule or cellular barrier molecule genes alone was not predictive of overall survival in our sample, these genetic signatures may serve as biomarkers of immune exclusion, or resistance to T-cell mediated immunotherapy.

Multi-faceted immunomodulatory and tissue-tropic clinical bacterial isolate potentiates prostate cancer immunotherapy

Immune checkpoint inhibitors have not been effective for immunologically “cold” tumors, such as prostate cancer, which contain scarce tumor infiltrating lymphocytes. We hypothesized that select tissue-specific and immunostimulatory bacteria can potentiate these immunotherapies. Here we show that a patient-derived prostate-specific microbe, CP1, in combination with anti-PD-1 immunotherapy, increases survival and decreases tumor burden in orthotopic MYC- and PTEN-mutant prostate cancer models. CP1 administered intra-urethrally specifically homes to and colonizes tumors without causing any systemic toxicities. CP1 increases immunogenic cell death of cancer cells, T cell cytotoxicity, and tumor infiltration by activated CD8 T cells, Th17 T cells, mature dendritic cells, M1 macrophages, and NK cells. CP1 also decreases intra-tumoral regulatory T cells and VEGF. Mechanistically, blocking CP1-recruited T cells from infiltrating the tumor inhibits its therapeutic efficacy. CP1 is an immunotherapeutic tool demonstrating how a tissue-specific microbe can increase tumor immunogenicity and sensitize an otherwise resistant cancer type to immunotherapy.CP1 is an uropathogenic Escherichia coli previously shown to promote inflammation and progression to prostate cancer. Here the authors show that in the context of a fully developed prostate cancer, CP1 promotes T cell infiltration into the tumour and increases the efficacy of anti-PD1 immunotherapy.

A bioluminescent and fluorescent orthotopic syngeneic murine model of androgen-dependent and castration-resistant prostate cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

MP11-07 CLINICALLY ISOLATED GRAM-POSITIVE PROSTATE BACTERIA INDUCE CHRONIC PELVIC PAIN.

maintained at day7 after LPS injection. Comprehensive cytokine assay (ELISA) showed that more than 2 folds of down-regulation of both inflammatory promoting cytokines (IL-1 alpha, IL-6) and chemokines (CCL3, CXCL1) were observed in epididymitis model of IDO KO mice compared with WT mice. On the other hand, more than 1.5 folds of upregulation of inflammatory inhibiting cytokines (IL-4, IL-10) were observed in epididymitis model of IDO KO mice. The peak expression of IL-1 alpha, IL-6, CCL3 and CXCL1 were at day1 and that of IL-4 was at day3. The expression of IL-10 increased in time dependent manner. Same results were introduced from separate quantitative analysis and immunohistochemical staining. After treatment of IDO inhibition and LPS, IL-1 alpha, IL-6, CCL3 and CXCL1 were significantly downregulated anytime in time series compared with WT mice using ELISA method (p<0.05). IL-4 and IL-10 were significantly up-regulated anytime in time series compared with WT mice (p<0.05). In the group of IDO inhibition, epididymal ductal structure was maintained at day7 after LPS injection and little invasion of inflammatory cells were observed anytime in time series. CONCLUSIONS: IDO should be involved in epididymal immunological reaction via cytokines. To inhibit IDO would contribute to protection of epididymis tissue when inflammation occurs in epididymis. Therefore, IDO might be a novel target for the therapy of the epididymitis in addition to antibodiotics.

MP11-08 REASSESMENT OF NON-TRADITIONAL UROPATHOGENS IN CHRONIC PELVIC PAIN SYNDROME (CP/CPPS)

maintained at day7 after LPS injection. Comprehensive cytokine assay (ELISA) showed that more than 2 folds of down-regulation of both inflammatory promoting cytokines (IL-1 alpha, IL-6) and chemokines (CCL3, CXCL1) were observed in epididymitis model of IDO KO mice compared with WT mice. On the other hand, more than 1.5 folds of upregulation of inflammatory inhibiting cytokines (IL-4, IL-10) were observed in epididymitis model of IDO KO mice. The peak expression of IL-1 alpha, IL-6, CCL3 and CXCL1 were at day1 and that of IL-4 was at day3. The expression of IL-10 increased in time dependent manner. Same results were introduced from separate quantitative analysis and immunohistochemical staining. After treatment of IDO inhibition and LPS, IL-1 alpha, IL-6, CCL3 and CXCL1 were significantly downregulated anytime in time series compared with WT mice using ELISA method (p<0.05). IL-4 and IL-10 were significantly up-regulated anytime in time series compared with WT mice (p<0.05). In the group of IDO inhibition, epididymal ductal structure was maintained at day7 after LPS injection and little invasion of inflammatory cells were observed anytime in time series. CONCLUSIONS: IDO should be involved in epididymal immunological reaction via cytokines. To inhibit IDO would contribute to protection of epididymis tissue when inflammation occurs in epididymis. Therefore, IDO might be a novel target for the therapy of the epididymitis in addition to antibodiotics.