Deciphering the Immune Landscape in Renal Cell Carcinoma and in Anti-PD-1 Therapy

Antigen recognition and T-cell mediated cytotoxicity are major tenets of cancer immunology that are not fully understood in clear-cell renal cell carcinoma (ccRCC). We evaluated multiregional treatment naïve nephrectomy samples from 27 patients as well as bloods samples from 21 and normal kidney tissue from 11 patients from the TRACERx Renal (TRAcking Cancer Evolution through therapy [Rx]) study via high dimensional flow cytometry. Results showed that the T cells in the tumour, normal kidney and blood have different phenotypes and differentiation patterns. A predominantly exhausted CD8 cell phenotype with expression of PD-1, TIM-3, Eomes, CD38 and CD39 was seen in the tumour immune microenvironment. ADAPTeR is a phase II study evaluating nivolumab (anti-PD1 antibody) in patients with treatment-naive metastatic ccRCC. Immunophenotyping by using high dimensional flow cytometry and multiplex immunofluorescence in addition to T cell receptor (TCR) sequencing was performed on 93 pre- and post-treatment, multi-region tumour and peripheral blood samples from 15 patients. We showed that an increased Granzyme B production in the CD8 cells and higher B cell infiltration at baseline were associated with response to Nivolumab. TCR sequencing analysis showed that maintenance of expanded TCR clones during the anti-PD1 treatment which were present pre-treatment and increased clustering of TCR clonotypes are associated with response to therapy. Comparing a responder patient with a non-responder by using single cell RNA Sequencing (SC RNA Seq) showed a more dysfunctional phenotype in the responder. In addition, Nivolumab bound CD8 cells in the responder also had higher Granzyme B and TCF7 expression suggesting a more cytotoxic and progenitor-like phenotype is associated with response. This study provides important data that needs to be validated in a bigger cohort to identify biomarkers of response to anti-PD-1 therapy in ccRCC

Determinants of anti-PD-1 response and resistance in clear cell renal cell carcinoma.

ADAPTeR is a prospective, phase II study of nivolumab (anti-PD-1) in 15 treatment-naive patients (115 multiregion tumor samples) with metastatic clear cell renal cell carcinoma (ccRCC) aiming to understand the mechanism underpinning therapeutic response. Genomic analyses show no correlation between tumor molecular features and response, whereas ccRCC-specific human endogenous retrovirus expression indirectly correlates with clinical response. T cell receptor (TCR) analysis reveals a significantly higher number of expanded TCR clones pre-treatment in responders suggesting pre-existing immunity. Maintenance of highly similar clusters of TCRs post-treatment predict response, suggesting ongoing antigen engagement and survival of families of T cells likely recognizing the same antigens. In responders, nivolumab-bound CD8+ T cells are expanded and express GZMK/B. Our data suggest nivolumab drives both maintenance and replacement of previously expanded T cell clones, but only maintenance correlates with response. We hypothesize that maintenance and boosting of a pre-existing response is a key element of anti-PD-1 mode of action

Higher COVID-19 pneumonia risk associated with anti-IFN-α than with anti-IFN-ω auto-Abs in children

We found that 19 (10.4%) of 183 unvaccinated children hospitalized for COVID-19 pneumonia had autoantibodies (auto-Abs) neutralizing type I IFNs (IFN-alpha 2 in 10 patients: IFN-alpha 2 only in three, IFN-alpha 2 plus IFN-omega in five, and IFN-alpha 2, IFN-omega plus IFN-beta in two; IFN-omega only in nine patients). Seven children (3.8%) had Abs neutralizing at least 10 ng/ml of one IFN, whereas the other 12 (6.6%) had Abs neutralizing only 100 pg/ml. The auto-Abs neutralized both unglycosylated and glycosylated IFNs. We also detected auto-Abs neutralizing 100 pg/ml IFN-alpha 2 in 4 of 2,267 uninfected children (0.2%) and auto-Abs neutralizing IFN-omega in 45 children (2%). The odds ratios (ORs) for life-threatening COVID-19 pneumonia were, therefore, higher for auto-Abs neutralizing IFN-alpha 2 only (OR [95% CI] = 67.6 [5.7-9,196.6]) than for auto-Abs neutralizing IFN-. only (OR [95% CI] = 2.6 [1.2-5.3]). ORs were also higher for auto-Abs neutralizing high concentrations (OR [95% CI] = 12.9 [4.6-35.9]) than for those neutralizing low concentrations (OR [95% CI] = 5.5 [3.1-9.6]) of IFN-omega and/or IFN-alpha 2

The role(s) of hypoxia-inducible factors (HIFs) in the development of renal cysts in mice deficient in fumarate hydratase (Fh1)

Fumarate hydratase (FH) encodes a Krebs cycle (also called TCA cycle) enzyme which converts fumarate to malate and also functions as a tumour suppressor gene. Germline mutations in FH predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). Affected individuals develop benign smooth muscle tumours (leiomyomas) of the skin and uterus and renal cysts and cancers; specifically type II papillary and collecting duct carcinomas. Kidney-specific deletion of Fh1 (the murine orthologue of FH) recapitulates HLRCC in part and mice develop renal cysts that are proliferative and express constitutively hypoxia inducible factors -1α and -2α (Hif-1α and Hif-2α). This phenomenon of pseudo-hypoxia (HIF expression in normal oxygen tension) is also evident in cultured cells deficient in FH and is proposed as a possible mechanism for initiation and/or progression of carcinogenesis. We have utilised our mouse model to investigate the role of Hif-1α and Hif-2α and Fh1 in renal cyst development and show remarkably that combined genetic inactivation of Hif-1α and Fh1 increased both cyst size and frequency. However, kidneys deficient for both Fh1 and Hif-2α were comparable to those deficient for Fh1. Immunohistochemical analyses show that Fh1 inactivation leads to increased proliferation, DNA damage and affects the cell cycle in the renal cysts. Hif-1α and Hif-2α inactivation is not shown to contribute to these. Through genome-wide mRNA expression analysis we have identified that Ccnd1 is significantly upregulated (or the most significantly upregulated gene) in Fh1/Hif-1α knockout kidneys compared to wild type and Fh1 knockout kidneys. Other upregulated genes in the double knockout kidneys include genes involved in cell cycle, proliferation and structural organization of the cell

Catheter-based renal denervation for treatment of resistant hypertension

Hypertension is a common disease associated with important cardiovascular complications. Persistent blood pressure of 140/90 or higher despite combined use of a reninangiotensin system blocker, calcium channel blocker and a diuretic at highest tolerated doses constitutes resistant hypertension. Excess sympathetic activity plays an important pathogenic role in resistant hypertension in addition to contributing to the development of metabolic problems, in particular diabetes. Reduction of renal sympathetic activity by percutaneous catheter-based radiofrequency ablation via the renal arteries has been shown in several studies to decrease blood pressure in patients with resistant hypertension, and importantly is largely free of significant complications. However, longer term follow-up is required to confirm both long-term safety and efficacy

The role(s) of hypoxia-inducible factors (HIFs) in the development of renal cysts in mice deficient in fumarate hydratase (Fh1)

Fumarate hydratase (FH) encodes a Krebs cycle (also called TCA cycle) enzyme which converts fumarate to malate and also functions as a tumour suppressor gene. Germline mutations in FH predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). Affected individuals develop benign smooth muscle tumours (leiomyomas) of the skin and uterus and renal cysts and cancers; specifically type II papillary and collecting duct carcinomas. Kidney-specific deletion of Fh1 (the murine orthologue of FH) recapitulates HLRCC in part and mice develop renal cysts that are proliferative and express constitutively hypoxia inducible factors -1α and -2α (Hif-1α and Hif-2α). This phenomenon of pseudo-hypoxia (HIF expression in normal oxygen tension) is also evident in cultured cells deficient in FH and is proposed as a possible mechanism for initiation and/or progression of carcinogenesis. We have utilised our mouse model to investigate the role of Hif-1α and Hif-2α and Fh1 in renal cyst development and show remarkably that combined genetic inactivation of Hif-1α and Fh1 increased both cyst size and frequency. However, kidneys deficient for both Fh1 and Hif-2α were comparable to those deficient for Fh1. Immunohistochemical analyses show that Fh1 inactivation leads to increased proliferation, DNA damage and affects the cell cycle in the renal cysts. Hif-1α and Hif-2α inactivation is not shown to contribute to these. Through genome-wide mRNA expression analysis we have identified that Ccnd1 is significantly upregulated (or the most significantly upregulated gene) in Fh1/Hif-1α knockout kidneys compared to wild type and Fh1 knockout kidneys. Other upregulated genes in the double knockout kidneys include genes involved in cell cycle, proliferation and structural organization of the cell.This thesis is not currently available in ORA

Effect of thermocycling on the amount of monomer released from bulk fill composite resins

Hatipoglu, Omer/0000-0002-4628-8551; Karadas, Muhammet/0000-0002-3357-6896WOS: 000510806600019PubMed: 31582597The goal of this study was to examine the effect of thermal cycling on the amount of monomer released from bulk fill composites. Five bulk fill composite resins were used in the study. Extraction solutions were obtained at the end of the time/thermal cycle periods: 0-1 day/0-1,500, 1-3 days/1,500-4,500 and 3-7 days/4,500-10,000. the monomers in the extractions samples taken at each time point were measured on an HPLC instrument. the obtained data were analyzed by repeated measures of variance analysis and tukey multiple comparison tests (p<0.05). the thermocycling increased the amount of monomer released from all composites at 0-1 day (p<0.05). At 0-1 and 1-3 days, Venus Bulk Fill and Filtek Bulk Fill composite resins were more affected. Polymer networks with high molecular weight monomers such as Bis-GMA and UDMA can be less affected by thermal changes compared to polymers with low molecular weight monomers.Recep Tayyip Erdogan University Research FundRecep Tayyip Erdogan University [2015.53001.111.03.01]The data presented in this study are a part of the doctoral thesis of Mr. Omer HATIPOGLU. This study was supported by Recep Tayyip Erdogan University Research Fund under Grant (Project code: 2015.53001.111.03.01)

Influence of different light-curing units on monomer elution from bulk fill composites

Hatipoglu, Omer/0000-0002-4628-8551; Karadas, Muhammet/0000-0002-3357-6896WOS: 000456292100008Objectives: This in vitro study assessed the effect of different light-curing units on the elution of monomers from bulk fill composites with different thicknesses. Methods: Five bulk fill composites (Filtek Bulk Fill Flowable, SonicFill 2, SDR, Tetric N-Ceram Bulk Fill, and Venus Bulk Fill) and one conventional composite (Filtek Z250) were selected for the study. the cylindrical specimens in thickness of 2 mm or 4 mm were prepared and photopolymerized for 20 s with a light-emitting diode (VALO Cordless) or a halogen (Monitex BlueLuxer) light-curing unit. the specimens in glass vials were covered with a 75% ethanol/water solution. Ethanol/water extraction solutions were collected for high-performance liquid chromatography analysis after 24 h, 3 days, and 7 days. the data were analyzed with repeated measures and three-way ANOVA (p < 0.05). Results: the total monomer amount was significantly influenced by light-curing source used and thickness. the highest levels of Bis-GMA and Bis-EMA were eluted from Tetric N-Ceram BulkFill and Venus Bulk Fill, respectively. SonicFill 2 released the highest level of TEGDMA at 4-mm thickness. the highest levels of UDMA release, from 4-mm-thick Filtek Bulk Fill Flowable, were attained using the halogen unit. Conclusions: Light-curing sources affected the number of monomers released by materials. the amount of eluted monomers declined over time. the increased ratio of released monomers to increased thickness is material dependent. the number of residual monomers is highly associated with the resin ratio and crosslinking network of the composites.Recep Tayyip Erdogan University Research FundRecep Tayyip Erdogan University [2015.53001.111.03.01]1 This study was supported by Recep Tayyip Erdogan University Research Fund under Grant (Project code: [2015.53001.111.03.01])