Biomarkers in renal cell carcinoma: Towards a more selective immune checkpoint inhibition

Immune checkpoint inhibitors such as programmed death protein 1/programmed death-ligand 1 and cytotoxic T-lymphocyte–associated protein 4 inhibitors are already playing a central role in the treatment of metastatic renal cell carcinoma. However, they seem to be only effective in a subset of patients, with a high risk of innate and adaptive tumor resistance. Consequently, biomarkers capable of predicting immune treatment efficacy in advanced renal cancer are needed both in the clinical and the experimental setting. We hereby present a brief summary of evidence on the most studied biomarkers in metastatic renal cell carcinoma with a focus on the possible future place of T cell immunoglobulin and mucin domain-3 (TIM-3).     

Molecular signature of renal cell carcinoma by means of a multiplatform metabolomics analysis

Renal cell carcinoma (RCC) is a disease with no specific diagnostic method or treatment. Thus, the evaluation of novel diagnostic tools or treatment possibilities is essential. In this study, a multiplatform untargeted metabolomics analysis of urine was applied to search for a metabolic pattern specific for RCC, which could enable comprehensive assessment of its biochemical background. Thirty patients with diagnosed RCC and 29 healthy volunteers were involved in the first stage of the study. Initially, the utility of the application of the selected approach was checked for RCC with no differentiation for cancer subtypes. In the second stage, this approach was used to study clear cell renal cell carcinoma (ccRCC) in 38 ccRCC patients and 38 healthy volunteers. Three complementary analytical platforms were used: reversed-phase liquid chromatography coupled with time-of-flight mass spectrometry (RP-HPLC-TOF/MS), capillary electrophoresis coupled with time-of-flight mass spectrometry (CE-TOF/MS), and gas chromatography triple quadrupole mass spectrometry (GC-QqQ/MS). As a result of urine sample analyses, two panels of metabolites specific for RCC and ccRCC were selected. Disruptions in amino acid, lipid, purine, and pyrimidine metabolism, the TCA cycle and energetic processes were observed. The most interesting differences were observed for modified nucleosides. This is the first time that the levels of these compounds were found to be changed in RCC and ccRCC patients, providing a framework for further studies. Moreover, the application of the CE-MS technique enabled the determination of statistically significant changes in symmetric dimethylarginine (SDMA) in RCC.     

Prevention of renal carcinoma: the nutri-genetic approach

The development of renal cell carcinoma (RCC) has been associated with both genetic and environmental factors, with somatic and germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene and with tobacco smoking, obesity, long term exposure to some nutrients, pollutants, and industrial solvents such as trichloroethylene. Intra and interfamilial variability of expression of germline mutations in the VHL gene and variable susceptibility to carcinogens in the sporadic forms strongly suggest the involvement of conditional modifier genes. In order to identify sub groups of individuals at increased risk because of susceptibility genotypes, we have collected a series of 460 patients who developed an RCC and 79 families with the von Hippel Lindau disease. To collect clinical and mutational data for correlation analysis we have developed a unique tool the Universal Mutation Database. Comparison of the spectrum of germline and somatic mutations in the VHL gene showed that: 1) in sporadic RCC mutations lead more often to truncated proteins (83%), while the remaining mutations (17%), include 3/4 of transversions and 1/4 of transitions. This high proportion of transversions supports the involvement of carcinogens the impact of which is conditioned by the genetic variability of xenobiotic metabolizing enzymes; 2) whereas in familial cases missense mutations are more common; this difference allowed us to define a prognostic factor for the occurrence of RCC in a VHL context. In order to look for genotypes conferring a higher risk we genotyped the RCC patients for 8 different genes (50 genotypes). A significant relationship was observed for several combinations of alleles including CYP1A1 ("variant"), NAT2 and NAT1 (slow) and GSTM1 (null allele). Associations between specific mutational profiles and at risk genotypes at different tumoral stages should allow us to: 1) define more precisely the nature of specific patterns of mutations in relation with the deficiency or overexpression of such or such enzymes in presence of particular carcinogens; 2) demonstrate that certain combinations of genotypes confer a particular risk to develop a specific type of tumor in VHL patients. Thus tracking of potentially carcinogenic substances, through their footprints and through identification of conditionally detrimental genotypes of genes participating in their detoxification should permit a better prevention through an appropriate nutrition adapted to each individual.     

Risk factor for clear cell renal cell carcinoma in Chinese population: a case-control study

Background: Risk factors for clear cell renal cell carcinoma (ccRCC) differ among populations and remain controversial. We carried out a hospital-based case–control study to examine the effects of health status, lifestyle, and some genetic polymorphisms on ccRCC risk in Chinese subjects. Methods: Between 2007 and 2009, 250 newly diagnosed, histologically confirmed ccRCC cases and 299 sex-, age-matched healthy controls provided complete information including consumption of tea and alcohol, smoking, occupational exposure, body mass index (BMI), hypertension, diabetes, and urolithiasis by face-to-face interview in Shanghai. Genetic polymorphisms of cytochrome P450 mono-oxygenase (CYP1A1: 6235T>C, 4889A>G, and 4887C>A), glutathione S-transferase (GSTP1: 342A>G), and N-acetyltransferase (NAT2: 481C>T, 590G>A, and 857G>A) were identified by PCR-RFLP and DNA sequencing. Adjusted odds ratio (AOR) and 95% confidence interval (CI) were derived through multivariate logistic regression. Results: Green tea intake (≥500 ml/d) was inversely associated with ccRCC risk, with an AOR of 0.34 (95% CI 0.21–0.55). BMI (≥25 kg/m²), hypertension, and urolithiasis were independently associated with an increased risk of ccRCC, with AOR (95% CI) of 2.10 (1.32–3.34), 2.49 (1.57–3.93), and 3.33 (1.12–9.89), respectively. No association was observed between smoking, alcohol consumption, or occupational exposure with ccRCC risk. The polymorphisms and their interactions with the environmental exposures were mostly not associated with ccRCC risk. Conclusion: BMI (≥25 kg/m²), hypertension, and urolithiasis are independently associated with an increased risk, whereas green tea intake (≥500 ml/d) is independently associated with a decreased risk of ccRCC. The polymorphisms of the xenobiotic-metabolizing enzymes are weakly associated with ccRCC risk in Chinese subjects.     

Antioxidant enzymes in renal cell carcinoma

The aim of the study was to estimate the significance of oxidative/nitrosative damage and expression of antioxidant enzymes in renal cell carcinomas (RCC). For this we investigated immunohistochemically six antioxidant enzymes (AOEs) including MnSOD, ECSOD, thioredoxin, thioredoxin reductase, and gammaglutamyl cysteine synthetase heavy and light chain in 138 RCCs. As an indicator of oxidative/nitrosative damage, sections were stained with an antibody to nitrotyrosine. The extent of apoptosis was evaluated by TUNEL method and proliferation by immunohistochemistry to Ki67. Variable expression of all AOEs could be seen in RCC with expression of MnSOD being strongest. Nitrotyrosine was significantly associated with high grade tumors. MnSOD was associated with tumors of a lower stage. Cases showing ECSOD reactivity had higher and cases expressing thioredoxin lower apoptotic index than other tumors. No association with patient prognosis was observed. According to the results renal cell carcinomas show oxidative/nitrosative damage which, according to nitrotyrosine staining, was higher in high grade tumors. Of AOEs, MnSOD was more abundantly expressed in low stage tumors suggesting that its antioxidant function could play a main role to prevent development of oxidative damage leading to more aggressive tumors.     

Glutathione-S-transferase in human renal cell carcinoma

Homogenates of human renal cell carcinomas were tested for glutathione-S-transferase, an enzyme of normal proximal tubule cells. All tumors were positive; mean tumor fraction enzyme activity was 0.040 +/- 0.02 mumol/min/microgram protein. Glutathione-S-transferase activity in homogenates from normal kidney was 0.022 and 0.054 mumol/min/microgram protein. Finding similar levels of a major cytosolic enzyme in tumor and renal cortex confirms the origin of renal cell carcinoma in the proximal nephron. Glutathione-S-transferase, which binds carcinogens and steroids, may play a role in carcinogenesis and serve as a marker for this tumor.     

Candidate biomarkers in renal cell carcinoma

Although the human genome has been decoded, the knowledge about the pathogenesis of diseases including cancer is still limited. By focusing on renal cell carcinoma (RCC) we here summarize the data of various research groups analyzing the protein/peptide expression profiles of tumor lesions/cell lines or serum obtained from patients and respective controls. Different powerful approaches such as 2-DE, PROTEOMEX/SERPA/SPEARS, and T cell epitope discovery upon elution of MHC class I-bound peptides in combination with MS/LC-MS/MS revealed 500 differentially expressed proteins. The overlap in target recognition limits the pool to 299 unique protein identities, but only few thereof (12%) have been validated. The management, analysis, and interpretation of the distinct data sets derived from 27 publications required bioinformatic restructuring of the results. However, the comprehensive analysis of the results expands the knowledge about the pathophysiology of RCC in particular of the most prominent clear cell subtype by providing information on the differentially expressed proteins, their regulation status in RCC compared to normal kidney epithelium next to additional information on MHC-presented T cell epitopes and on serological targets. Despite the low number of validated differentially expressed proteins some of them might serve as candidate biomarkers for the diagnosis and/or as therapeutic targets.     

Immunosuppression in murine renal cell carcinoma

Summary Four cell-mediated immunological responses related to tumor elimination have been examined in mice injected with a transplantable renal cell carcinoma (Renca). Lymphokine-activated killer (LAK) cells generatedin vitro from spleen cells of normal mice were capable of attacking Renca, EL-4, P815 and YAC-1 targets, but those from mice bearing Renca for 3 weeks could not. Natural killer activity, stimulatedin vivo by administering poly(I) poly(C), was less than 50% of normal in Rencabearing hosts. In addition, development of cytotoxic T lymphocytes to allogeneic targets was markedly inhibited in mice possessing the renal tumor. Finally, the delayed hypersensitivity response to a dermally applied hapten was approximately 70% less than normal in tumor-bearing mice, no matter whether the tumor existed subcutaneously or intrarenally. A kinetic study of the development of nonresponsiveness using the LAK assay showed onset of poor response at 1 week, which became maximal within 3 weeks following receipt of tumor subcutaneously. The immunological depression was seen to be attributable in part to suppressor cells present among spleen cells but not bone marrow cells of tumor-bearing hosts. The suppressor cells preventedin vitro LAK generation by normal spleen cells and, when adoptively transferred to normal mice, they inhibited natural killer stimulation and delayed hypersensitivity generation. Another source of immunological downregulation was provided by Renca cells themselves. Incorporation of Renca cells that had been X-irradiated with 30000 rad into cultures of normal and Renca-derived splenic cells suppressed replication of both almost completely. Furthermore, the presence of X-irradiated Renca cells in cultures of normal spleen cells prevented development of LAK cells. Thus, the suppression seen in Renca-bearing mice derives from multiple sources and whether each is in any way related to the other has been discussed. Identification of the phenotypes of cells responsible for the lymphoid cell-mediated suppression and examination of its elimination are communicated in the companion paper.Offered in partial fulfillment of the requirements for the Ph. D. degree at Cleveland State University by SKG.     

Prognostic factors in renal cell carcinoma

We studied 569 cases of renal cell carcinoma in the files of the Department of Pathology of the Norwegian Radium Hospital from 1964 to 1974. A nephrectomy had been performed in all cases. Clinical information on sex, age, survival time and metastases was traced. The histological slides were examined and tumour growth pattern, cell type, cell shape, nuclear atypia, abnormal nucleoli, nuclear grade, vascular invasion and tumour demarcation were all evaluated. Besides well-known prognostic factors such as tumour stage, presence or absence of metastases and vascular invasion, nuclear grade was found to be a useful prognostic factor. Younger patients were found to do better than older, and women better than men. Smaller tumours carried a better prognosis than larger and clear cell tumours had a better prognosis than those composed of eosinophilic or basophilic cells. The presence of spindle cells was a bad prognostic omen.     

Occult renal cell carcinoma manifesting with epistaxis in a woman: a case report

Introduction

Metastatic disease in the sinonasal region occurs rarely and the primary site may be elusive. This case highlights the possibility of an occult renal tumor manifesting with nasal symptoms and the risk of severe bleeding following nasal biopsy.

Case presentation

We report the case of a 79-year-old Caucasian woman who presented with a six-week history of intermittent left-sided nosebleeds. She was fit, without previous surgery or anticoagulation. Nasal endoscopy and computed tomography showed a hemorrhagic mass occupying her left ethmoid cells and middle meatus. After a highly hemorrhagic biopsy, the lesion was histologically confirmed as clear cell carcinoma. Screening revealed a right kidney mass with widespread metastases. Palliative radiotherapy to the sinonasal metastasis and systemic treatment rendered her free of symptoms nine months after initial presentation.

Conclusions

General practitioners and ear, nose and throat (ENT) doctors are very often confronted with epistaxis. A small minority of patients with epistaxis show a primary or metastatic nasal mass. Detection of the origin of secondary sinonasal masses requires a high index of suspicion and examination of infraclavicular sites by a multidisciplinary team. Renal cell carcinoma metastases are prone to severe bleeding during any surgical intervention, therefore, preoperative embolization is recommended. Resection or radiotherapy to the sinonasal metastasis of renal origin is justified in order to prevent recurrent nosebleeds.

     

Increasing reactive oxygen species as a therapeutic approach to treat hereditary leiomyomatosis and renal cell carcinoma

Hereditary leiomyomatosis renal cell carcinoma (HLRCC)-associated renal tumors are aggressive and tend to metastasize early. There are currently no effective forms of therapy for patients with advanced HLRCC-associated kidney cancer. We have previously shown that HLRCC cells express a high level of reactive oxygen species (ROS). In the present study we investigated the cytotoxic-effects of increasing ROS level using bortezomib in combination with cisplatin on HLRCC cells in vitro and in an in vivo xenograft model. The cytotoxic effect of several ROS inducers on FH-deficient cells was assessed by synthetic lethality. ROS inducers had a pronounced impact on the viability of FH-deficient cells. Because of its high potency, the proteasome inhibitor bortezomib was further investigated. Bortezomib induced apoptosis in vitro in HLRCC cells and inhibited HLRCC tumor growth in vivo. Bortezomib-associated cytotoxicity was highly correlated with cellular ROS level: combining bortezomib with other ROS inducers enhanced cytotoxicity, while combining bortezomib with a ROS scavenger inhibited its cytotoxic effect. Finally, HLRCC murine xenografts were treated with bortezomib and cisplatin, another ROS inducer. This regimen induced HLRCC tumor regression in vivo. These findings suggest that increasing ROS level in HLRCC above a certain threshold can induce HLRCC-tumor cell death. Increasing tumor ROS with bortezomib in combination with cisplatin represents a novel targeted therapeutic approach to treat advanced HLRCC-associated renal tumors.Key words: HLRCC, bortezomib, cisplatin, ROS, kidney cancer, FH     

Immunosuppression in murine renal cell carcinoma

In our companion paper we have reported that cell-mediated immunity of mice bearing renal cell carcinoma is profoundly suppressed. The non-responsiveness of such animals was found to be attributable to Renca cells themselves and to splenic lymphoid cells that down-regulate other fully capable lymphoid cells. In this communication the lymphoid cell source of suppression within Renca-bearing mice has been explored with the aim of identifying phenotypes of the responsible cells, the manner by which suppression is mediated, and initial ways by which suppression may be eliminated. A plastic-adherent cell bearing the Thy1.2 surface marker as well as the Lyt1 and Lyt2 antigens has been found to operate, perhaps in conjunction with macrophages, to down-regulate lymphokine-activated killer (LAK) cell development for natural killer (NK) and non-NK targets that include Renca cells themselves. The splenic suppressor cells lost the capacity to suppress the NK response of normal recipient mice upon shallow irradiation (250 rad) prior to adoptive transfer. Spleen cells, presumably macrophages, from Renca-bearing mice were found to suppress the generation of LAK and NK cells in vitro by synthesizing prostaglandins. Indomethacin, a prostaglandin synthetase inhibitor, blocked the induction of suppression both in vitro and in vivo, suggesting the presence of endogenous prostaglandins in Renca-bearing mice. The suppression seen in Renca-bearing mice that derives from multiple sources and has been prevented by two separate methods has been discussed from the viewpoint of the inter-relatedness of the sources.     

Organic acid profile of isovaleric acidemia: a comprehensive metabolomics approach

Isovaleric acidemia (IVA, MIM 248600) can be a severe and potentially life-threatening disease in affected neonates, but with a positive prognosis on treatment for some phenotypes. This study presents the first application of metabolomics to evaluate the metabolite profiles derived from urine samples of untreated and treated IVA patients as well as of obligate heterozygotes. All IVA patients carried the same homozygous c.367 G > A nucleotide change in exon 4 of the IVD gene but manifested phenotypic diversity. Concurrent class analysis (CONCA) was used to compare all the metabolites from the original complete data set obtained from the three case and two control groups used in this investigation. This application of CONCA has not been reported previously, and is used here to compare four different modes of scaling of all metabolites. The variables important in discrimination from the CONCA thus enabled the recognition of different metabolic patterns encapsulated within the data sets that would not have been revealed by using only one mode of scaling. Application of multivariate and univariate analyses disclosed 11 important metabolites that distinguished untreated IVA from controls. These included well-established diagnostic biomarkers of IVA, endogenous detoxification markers, and 3-hydroxycaproic acid, an indicator of ketosis, but not reported previously for this disease. Nine metabolites were identified that reflected the effect of treatment of IVA. They included detoxification products and indicators related to the high carbohydrate and low protein diet which formed the hallmark of the treatment. This investigation also provides the first comparative metabolite profile for heterozygotes of this inherited metabolic disorder. The detection of informative metabolites in even very low concentrations in all three experimental groups highlights the potential advantage of the holistic mode of analysis of inherited metabolic diseases in a metabolomics investigation.     

Prognostic value of a gene signature in clear cell renal cell carcinoma

Renal cancer is a common urogenital system malignance. Novel biomarkers could provide more and more critical information on tumor features and patients’ prognosis. Here, we performed an integrated analysis on the discovery set and established a three-gene signature to predict the prognosis for clear cell renal cell carcinoma (ccRCC). By constructing a LASSO Cox regression model, a 3-messenger RNA (3-mRNA) signature was identified. Based on the 3-mRNA signature, we divided patients into high- and low-risk groups, and validated this by using three other data sets. In the discovery set, this signature could successfully distinguish between the high- and low-risk patients (hazard ratio (HR), 2.152; 95% confidence interval (CI),1.509–3.069; p < 0.0001). Analysis of internal and two external validation sets yielded consistent results (internal: HR, 2.824; 95% CI, 1.601–4.98; p < 0.001; GSE29609: HR, 3.002; 95% CI, 1.113–8.094; p = 0.031; E-MTAB-3267: HR, 2.357; 95% CI, 1.243–4.468; p = 0.006). Time-dependent receiver operating characteristic (ROC) analysis indicated that the area under the ROC curve at 5 years was 0.66 both in the discovery and internal validation set, while the two external validation sets also suggested good performance of the 3-mRNA signature. Besides that, a nomogram was built and the calibration plots and decision curve analysis indicated the good performance and clinical utility of the nomogram. In conclusion, this 3-mRNA classifier proved to be a useful tool for prognostic evaluation and could facilitate personalized management of ccRCC patients.     

Bananas as an energy source during exercise: a metabolomics approach

This study compared the acute effect of ingesting bananas (BAN) versus a 6% carbohydrate drink (CHO) on 75-km cycling performance and post-exercise inflammation, oxidative stress, and innate immune function using traditional and metabolomics-based profiling. Trained cyclists (N?=?14) completed two 75-km cycling time trials (randomized, crossover) while ingesting BAN or CHO (0.2 g/kg carbohydrate every 15 min). Pre-, post-, and 1-h-post-exercise blood samples were analyzed for glucose, granulocyte (GR) and monocyte (MO) phagocytosis (PHAG) and oxidative burst activity, nine cytokines, F?-isoprostanes, ferric reducing ability of plasma (FRAP), and metabolic profiles using gas chromatography-mass spectrometry. Blood glucose levels and performance did not differ between BAN and CHO (2.41±0.22, 2.36±0.19 h, P?=?0.258). F?-isoprostanes, FRAP, IL-10, IL-2, IL-6, IL-8, TNFα, GR-PHAG, and MO-PHAG increased with exercise, with no trial differences except for higher levels during BAN for IL-10, IL-8, and FRAP (interaction effects, P?=?0.003, 0.004, and 0.012). Of 103 metabolites detected, 56 had exercise time effects, and only one (dopamine) had a pattern of change that differed between BAN and CHO. Plots from the PLS-DA model visualized a distinct separation in global metabolic scores between time points [R2Y(cum)?=?0.869, Q2(cum)?=?0.766]. Of the top 15 metabolites, five were related to liver glutathione production, eight to carbohydrate, lipid, and amino acid metabolism, and two were tricarboxylic acid cycle intermediates. BAN and CHO ingestion during 75-km cycling resulted in similar performance, blood glucose, inflammation, oxidative stress, and innate immune levels. Aside from higher dopamine in BAN, shifts in metabolites following BAN and CHO 75-km cycling time trials indicated a similar pattern of heightened production of glutathione and utilization of fuel substrates in several pathways.     

Single cell metabolomics

Recent discoveries suggest that cells of a clonal population often display multiple metabolic phenotypes at the same time. Motivated by the success of mass spectrometry (MS) in the investigation of population-level metabolomics, the analytical community has initiated efforts towards MS-based single cell metabolomics to investigate metabolic phenomena that are buried under the population average. Here, we review the current approaches and illustrate their advantages and disadvantages. Because of significant advances in the field, different technologies are now at the verge of generating data that are useful for exploring and investigating metabolic heterogeneity.     

Angiogenetic pathway as a therapeutic target in renal cell carcinoma

For several decades, metastatic renal cell carcinoma (mRCC) was considered resistant to therapy and thus linked to poor prognosis. Recent years have seen a complete revolution of the therapeutic landscape, with the introduction of new target therapies that have radically changed treatment strategies in this setting. These new agents have proved to be effective if directed mainly against the angiogenic pathway. The vascular endothelial growth factor family is, in fact, the main target of a number of new drugs, such as monoclonal antibodies and different generations of tyrosine-kinase inhibitors that are currently approved or in different stages of clinical trial for the treatment of mRCC patients.