Evaluating the Urinary Exosome microRNA Profile of von Hippel Lindau Syndrome Patients with Clear Cell Renal Cell Carcinoma.

INTRODUCTION: Renal cell carcinoma is one of the ten more common malignant tumors worldwide, with a high incidence and mortality rate. Kidney cancer frequently presents at an advanced stage, and it is almost invariably fatal. Much progress has been made in identifying molecular targets for therapy in the hope of improving survival rates, but still, we have no good markers for early detection or progression of the disease. Von Hippel Lindau syndrome (VHL) is an autosomal dominant cancer hereditary syndrome in which affected individuals are at risk of developing bilateral and multifocal renal cell carcinomas (RCC) as well as other tumors. These patients provide an ideal platform to investigate the potential of urinary exosomal miRNA biomarkers in the early development of ccRCC, as these patients are regularly imaged and tumors are actively monitored until the tumor reaches 3 cm before surgical excision. This allows for pre- and post-surgical urine collection and comparison to excised tumor tissues. Studying different biomarkers in urine can provide comprehensive molecular profiling available to patients and physicians and can be a great source of additional tumor genetic information. METHODS: Pre- and postoperative urine samples were obtained from a cohort of VHL patients undergoing surveillance and surgical excision of ccRCCs, and exosomes were extracted. MicroRNA-Seq analysis was performed on miRNA extracted from both urine-derived exosomes and FFPE material from excised ccRCCs. RESULTS: MicroRNA-Seq analysis highlighted a significant difference in the urinary exosome-derived miRNA expression profiles between VHL patients and normal control individuals. This included decreased expression of the miR-320 family, such as miR-320a, known to be decreased in sporadic ccRCC and suppressed by the HIF1α transcription factor activated by the loss of the VHL gene. MiR-542-5p represented a potential marker of VHL-associated ccRCC that was lowly expressed in normal control urinary exosomes, significantly increased in the preoperative urinary exosomes of tumor-bearing VHL patients, and subsequently reduced to normal levels of expression after tumor excision. In concordance with this, the expression of miR-542-5p was increased in the VHL-associated ccRCC in comparison to the normal kidney. CONCLUSIONS: This study shows the potential for miRNA profiling of exosomes from readily available biofluids to both distinguish VHL patient urine from normal control urine microRNAs and to provide biomarkers for the presence of VHL syndrome-associated ccRCC. Further validation studies are necessary to demonstrate the utility of urinary exosome-derived miRNAs as biomarkers in kidney cancer.

Efficacy, safety, and biomarker analyses of bintrafusp alfa, a bifunctional fusion protein targeting TGF-ß and PD-L1, in patients with advanced non-small cell lung cancer.

BACKGROUND: Bintrafusp alfa, a first-in-class bifunctional fusion protein targeting transforming growth factor-ß (TGF-ß) and programmed cell death ligand 1, has demonstrated encouraging efficacy as second-line treatment in patients with non-small cell lung cancer (NSCLC) in a dose expansion cohort of the phase 1, open-label clinical trial (NCT02517398). Here, we report the safety, efficacy, and biomarker analysis of bintrafusp alfa in a second expansion cohort of the same trial (biomarker cohort). METHODS: Patients with stage IIIb/IV NSCLC who were either immune checkpoint inhibitor (ICI)-naïve (n=18) or ICI-experienced (n=23) were enrolled. The primary endpoint was the best overall response. Paired biopsies (n=9/41) and peripheral blood (n=14/41) pretreatment and on-treatment were studied to determine the immunological effects of treatment and for associations with clinical activity. RESULTS: Per independent review committee assessment, objective responses were observed in the ICI-naïve group (overall response rate, 27.8%). No new or unexpected safety signals were identified. Circulating TGF-ß levels were reduced (>97%; p<0.001) 2 weeks after initiation of treatment with bintrafusp alfa and remained reduced up to 12 weeks. Increases in lymphocytes and tumor-associated macrophages (TAMs) were observed in on-treatment biospies, with an increase in the M2 (tumor trophic TAMs)/M1 (inflammatory TAMs) ratio associated with poor outcomes. Specific peripheral immune analytes at baseline and early changes after treatment were associated with clinical response. CONCLUSIONS: Bintrafusp alfa was observed to have modest clinical activity and manageable safety, and was associated with notable immunologic changes involving modulation of the tumor immune microenvironment in patients with advanced NSCLC.

GNRH2 Polymorphism in Men With Prostate Cancer Treated With Androgen Deprivation Therapy.

BACKGROUND/AIM: Gonadotropin-releasing hormone 2 (GNRH2) is a poorly-studied peptide hormone that is widely distributed in the central nervous system and expressed in peripheral tissues of mammals. The non-synonymous rs6051545 variant in GNRH2 (A16V) has been linked to higher serum testosterone concentrations. This study investigated whether the A16V variant is associated with altered androgen-deprivation therapy (ADT) progression-free survival (PFS) and overall survival (OS). PATIENTS AND METHODS: We examined the expression of GNRH2 in prostate tissue microarrays comprising normal tissue, prostatic hyperplasia, and prostate cancer using immunofluorescence. We also evaluated the GNRH2 genotype in 131 patients with prostate cancer who received ADT and compared PFS and OS between the variant and wild-type genotypes. RESULTS: GNRH2 was detected in all prostate tissues, although expression did not vary with Gleason grade or disease stage (p=0.71). The GNRH2 A16V genotype was not associated with PFS or OS; however, univariate and multivariate analyses revealed Gleason score and definitive local therapy were each associated with PFS (p≤0.0074), whereas age and Gleason score were associated with OS (p≤0.0046). CONCLUSION: GNRH2 is expressed in normal, hyperplastic, and neoplastic prostate tissues; the A16V variant is not related to treatment outcome or survival.

Neoadjuvant PROSTVAC prior to radical prostatectomy enhances T-cell infiltration into the tumor immune microenvironment in men with prostate cancer.

BACKGROUND: Clinical trials have shown the ability of therapeutic vaccines to generate immune responses to tumor-associated antigens (TAAs). What is relatively less known is if this translates into immune-cell (IC) infiltration into the tumor microenvironment. This study examined whether neoadjuvant prostate-specific antigen (PSA)-targeted vaccination with PROSTVAC could induce T-cell immunity, particularly at the tumor site. METHODS: An open-label, phase II study of neoadjuvant PROSTVAC vaccine enrolled 27 patients with localized prostate cancer awaiting radical prostatectomy (RP). We evaluated increases in CD4 and CD8 T-cell infiltrates (RP tissue vs baseline biopsies) using a six-color multiplex immunofluorescence Opal method. Antigen-specific responses were assessed by intracellular cytokine staining after in vitro stimulation of peripheral blood mononuclear cells with overlapping 15-mer peptide pools encoding the TAAs PSA, brachyury and MUC-1. RESULTS: Of 27 vaccinated patients, 26 had matched prevaccination (biopsy) and postvaccination (RP) prostate samples available for non-compartmentalized analysis (NCA) and compartmentalized analysis (CA). Tumor CD4 T-cell infiltrates were significantly increased in postvaccination RP specimens compared with baseline biopsies by NCA (median 176/mm² vs 152/mm²; IQR 136-317/mm² vs 69-284/mm²; p=0.0249; median ratio 1.20; IQR 0.64-2.25). By CA, an increase in both CD4 T-cell infiltrates at the tumor infiltrative margin (median 198/mm² vs 151/mm²; IQR 123-500/mm² vs 85-256/mm²; p=0.042; median ratio 1.44; IQR 0.59-4.17) and in CD8 T-cell infiltrates at the tumor core (median 140/mm² vs 105/mm²; IQR 91-175/mm² vs 83-163/mm²; p=0.036; median ratio 1.25; IQR 0.88-2.09) were noted in postvaccination RP specimens compared with baseline biopsies. A total of 13/25 patients (52%) developed peripheral T-cell responses to any of the three tested TAAs (non-neoantigens); five of these had responses to more than one antigen of the three evaluated. CONCLUSION: Neoadjuvant PROSTVAC can induce both tumor immune response and peripheral immune response. TRIAL REGISTRATION NUMBER: NCT02153918.

Expression and mutational status of c-kit in thymic epithelial tumors.

BACKGROUND: Overexpression of c-kit, a tyrosine kinase receptor protein encoded by the protooncogene kit, has been previously reported in thymic epithelial tumors and in other neoplasms such as gastrointestinal stromal tumors, myeloproliferative disorders, melanoma, and seminoma. Mutations in the kit gene have been related to response to imatinib in gastrointestinal stromal tumor and one case report of thymic carcinoma. We studied expression of c-kit in a large retrospective series of thymic epithelial malignancies and sequenced the whole gene in a subset of patients. METHODS: Thymic epithelial tumors from 120 patients (13 thymic carcinomas and 107 thymomas) were examined. Immunohistochemical staining with an antic-kit polyclonal antibody was performed on a tissue microarray. Mutation analyses of exons 1 to 20 were conducted by direct DNA sequencing of polymerase chain reaction products in eight thymic carcinomas, five thymomas, and one thymic carcinoma cell line. RESULTS: The percentage of c-kit positive cells was significantly higher in thymic carcinoma (46%) than in thymoma (4%). Decreased disease-related survival and progression-free survival were observed in c-kit positive tumors. No mutations were detected. CONCLUSION: c-kit expression is strongly but not exclusively related to thymic carcinoma histotype, and it is of prognostic value. Mutations are very rare.