Identification of Neoantigen-Reactive Tumor-Infiltrating Lymphocytes in Primary Bladder Cancer

Immune checkpoint inhibitors (ICIs) are effective in treating a variety of malignancies, including metastatic bladder cancer. A generally accepted hypothesis suggests that ICIs induce tumor regressions by reactivating a population of endogenous tumor-infiltrating lymphocytes (TILs) that recognize cancer neoantigens. Although previous studies have identified neoantigen-reactive TILs from several types of cancer, no study to date has shown whether or not neoantigen-reactive TILs can be found in bladder tumors. To address this, we generated TIL cultures from patients with primary bladder cancer and tested their ability to recognize tumor-specific mutations. We found that CD4+ TILs from one patient recognized mutated C-terminal binding protein 1 (CTBP1Q277R) in an MHC class II-restricted manner. This finding suggests that neoantigen-reactive TILs reside in bladder cancer, which may help explain the effectiveness of immune checkpoint blockade in this disease, and also provides a rationale for the future use of adoptive T-cell therapy targeting neoantigens in bladder cancer

Enterocyte-specific inactivation of SIRT1 reduces tumor load in the APC(+/min) mouse model.

SIRT1 is a mammalian NAD(+)-dependent histone deacetylase implicated in metabolism, development, aging and tumorigenesis. Prior studies that examined the effect of enterocyte-specific overexpression and global deletion of SIRT1 on polyp formation in the intestines of APC(+/min) mice, a commonly used model for intestinal tumorigenesis, yielded conflicting results, supporting either tumor-suppressive or tumor-promoting roles for SIRT1, respectively. In order to resolve the controversy emerging from these prior in vivo studies, in the present report we examined the effect of SIRT1 deficiency confined to the intestines, avoiding the systemic perturbations such as growth retardation seen with global SIRT1 deletion. We crossed APC(+/min) mice with mice bearing enterocyte-specific inactivation of SIRT1 and examined polyp development in the progeny. We found that SIRT1-inactivation reduced total polyp surface (9.3 mm(2) vs. 23.3 mm(2), p = 0.01), average polyp size (0.24 mm(2) vs. 0.51 mm(2), p = 0.005) and the number of polyps >0.5 mm in diameter (14 vs. 23, p = 0.04), indicating that SIRT1 affects both the number and size of tumors. Additionally, tumors in SIRT1-deficient mice exhibited markedly increased numbers of cells undergoing apoptosis, suggesting that SIRT1 contributes to tumor growth by enabling survival of tumor cells. Our results indicate that SIRT1 acts as a tumor promoter in the APC(+/min) mouse model of intestinal tumorigenesis

Enterocyte-specific SIRT1 deletion increases the rate of apoptosis in the intestinal tumors of APC^+/min mice.

<p>(A) A representative western blot showing expression of the wild-type SIRT1 protein in the intestinal epithelium of APC^+/min SIRT1^+/+ mice (first lane) and a truncated version in the epithelium of APC^+/min SIRT1^−/− mice (second lane). Liver cells of the APC^+/min SIRT1^−/− mice (third lane), as well as other tissues (not shown) express the wild-type protein. Pan-actin immunostaining served as a loading control. (B) Representative photographs of unfixed small intestines (distal segments) showing similar polyp number for APC^+/min SIRT1^+/+ (right) and APC^+/min SIRT1^−/− (left) mice. Scale bar indicates 5 mm length. (C) Representative photomicrographs of typical polyps from the two groups of mice, stained with hematoxylin and eosin. Scale bar indicates 100 µm length. (D) Representative photomicrographs and a bar graph showing Ki-67 immunohistochemical staining of polyp sections from APC^+/min SIRT1^+/+ (left) and APC^+/min SIRT1^−/− (middle) mice. Scale bar indicates 100 µm length. Proliferation index for polyps from APC^+/min SIRT1^+/+ and APC^+/min SIRT1^−/− mice (right), expressed as a fraction of Ki-67 positive cells within each polyp. Bars represent means ± SEM, n = 20 polyps per group. No statistically significant difference was observed. (E) Representative photomicrographs and a bar graph showing activated (cleaved) caspase-3 immunohistochemical staining of polyp sections from APC^+/min SIRT1^+/+ (left) and APC^+/min SIRT1^−/− (middle) mice. Scale bar indicates 100 µm length. Absolute numbers of apoptotic (caspase-3 positive) cells per high power field (400 x) for polyps from SIRT1^+/+ and SIRT1^−/− mice (right). Bars represent means ± SEM, n = 25 polyps per group. ***p<0.001.</p

SIRT1 inactivation inhibits Wnt and promotes p53 a pathway.

<p><b>A)</b> Representative photomicrographs showing β-catenin immunohistochemical staining of intestinal sections from APC^+/min SIRT1^+/+ and APC^+/min SIRT1^−/− mice. Scale bar indicates 50 µm length. Polyps from both groups demonstrate intense cytoplasmic and nuclear staining for β-catenin. B) Bar graph with frequencies of crypts with indicated number of cells with nuclear β-catenin staining in normal appearing mucosa of APC^+/min SIRT1^+/+ and APC^+/min SIRT1^−/− mice. The average number of β-catenin positive cells per crypt is reduced in APC^+/min SIRT1^−/− mice compared to APC^+/min SIRT1^+/+ animals 2.1±1.2 vs 3.2±1.3 (p = 1.5×10⁻⁵, at least 100 crypts per genotype were scored). C) Representative photomicrographs of normal appearing mucosa from APC^+/min SIRT1^+/+ and APC^+/min SIRT1^−/− mice demonstrating a reduced number of basal cells with nuclear β-catenin in APC^+/min SIRT1^−/− animals. Arrows are directed toward representative basal cells with nuclear β-catenin. Scale bar indicates 25 µm length. D) Inhibition of SIRT1 with EX527 (2 µM) and cambinol (50 µM) reduces activity of the TCF/LEF driven firefly luciferase reporter (TOP FLASH) transiently transfected into SW480 cells. TOP FLASH luciferase reporter contains minimal promoter along with three TCF binding sites, which have been mutated in FOP FLASH reporters. Bars represent means ± SEM of relative firefly luciferase activity normalized to renilla luciferase activity from the thymidine kinase promoter-driven renilla reporter that was co-transfected with TOP FLASH and FOP FLASH reporters. Each transfection is carried out in quadruplicate. **p<0.01. E) SW480 cells with shRNA-mediated downregulation of SIRT1 (top: western blot for SIRT1 and actin) exhibit reduced activity of the transiently transfected TCF/LEF driven firefly luciferase reporter (TOP FLASH). Bars represent means ± SEM of relative firefly luciferase activity normalized to renilla luciferase activity from the thymidine kinase promoter-driven renilla reporter that was co-transfected with TOP FLASH and FOP FLASH reporters. Each transfection is carried out in quadruplicate. *p<0.05. F) Immunoblot for acetyl-p53, p53 and actin from cells treated with etoposide, SIRT1 inhibitor EX527 or the combination of the two drugs. Inhibition of SIRT1 leads to p53 hyperacetylation in Hct116 colon cancer cell line. Hyperacetylation of p53 is observed in cells treated with EX527 and a combination of EX527 and etoposide. Etoposide alone modestly induces p53 acetylation.</p

Specific recognition of an FGFR2 fusion by tumor infiltrating lymphocytes from a patient with metastatic cholangiocarcinoma

Background Metastatic cholangiocarcinoma (CC), a form of gastrointestinal cancer that originates from the bile ducts, cannot be cured by currently available therapies, and is associated with dismal prognosis. In a previous case report, adoptive transfer of autologous tumor infiltrating lymphocytes (TILs), the majority of which recognized a tumor-specific point mutation, led to a profound and durable cancer regression in a patient with metastatic CC. Thus, more effective treatment for patients with this disease may be developed by using TILs that target cancer-specific mutations, but also other genetic aberrations such as gene fusions. In this context, fusions that involve fibroblast growth factor receptor 2 (FGFR2) and function as oncogenes in a subset of patients with intrahepatic CC (ICC) represent particularly attractive targets for adoptive cell therapy. However, no study to date has explored whether FGFR2 fusions can be recognized by patients’ T cells.Method To address whether FGFR2 fusions can be recognized by patients’ T cells, we tested TILs from four patients with FGFR2 fusion-positive ICC for recognition of peptides and minigenes that represented the breakpoint regions of these fusions, which were unique to each of the four patients.Results We found that CD4+ TILs from one patient specifically recognized the breakpoint region of a unique FGFR2-TDRD1 (tudor domain-containing 1) fusion, and we isolated a T-cell receptor responsible for its recognition.Conclusions This finding suggests that FGFR2 fusion-reactive TILs can be isolated from some patients with metastatic ICC, and thus provides a rationale for future exploration of T cell-based therapy targeting FGFR2 fusions in patients with cancer. Furthermore, it augments the rationale for extending such efforts to other types of solid tumors hallmarked by oncogenic gene fusions

Neoantigen Identification and Response to Adoptive Cell Transfer in anti PD-1 Naïve and Experienced Patients with Metastatic Melanoma

Immune checkpoint blockade (ICB) agents and adoptive cell transfer (ACT) of tumor infiltrating lymphocytes (TIL) are prominent immunotherapies used for the treatment of advanced melanoma. Both therapies rely on activation of lymphocytes that target shared tumor antigens or neoantigens. Recent analysis of patients with metastatic melanoma who underwent treatment with TIL ACT at the National Cancer Institute (NCI) demonstrated decreased responses in patients previously treated with anti PD-1 agents. We aimed to find a basis for the difference in response rates between anti PD-1 naïve and experienced patients. We examined the tumor mutational burden (TMB) of resected tumors and the repertoire of neoantigens targeted by autologous TIL in a cohort of 112 anti PD-1 naïve and 69 anti PD-1 experienced patients. Anti PD-1 naïve patients were found to have higher TMBs (352.0 vs. 213.5, p = 0.005) and more neoantigens (2 vs. 1, p = 0.003) compared to anti PD-1 experienced patients. Among patients treated with TIL ACT, TMB and number of neoantigens identified were higher in ACT responders than ACT non-responders in both anti PD-1naïve and experienced patients. Among patients with comparable TMBs and predicted neoantigen loads, patients naïve to anti PD-1 therapy were more likely to have identifiable neoantigens and tumor specific lymphocytes in their treatment products (2.5 vs. 1, p = 0.02). These results indicate that decreases in TMB and targeted neoantigens only partially account for the difference in response to ACT and that additional factors likely influence responses in these patients