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Renal cell carcinomas (RCC) are characterized by their heterogenous clinical outcomes, and due to their indeterminate behavior and the absence of routine biomarkers, it is difficult to identify patients who are at high-risk for relapse after curative nephrectomy. To identify genomic biomarkers for clear cell RCC (ccRCC) risk-stratification we interrogated somatic mutation status of 12 RCC-relevant genes using next-generation sequencing (NGS) in tumor-normal pairs from 943 patients with matched follow up data from the Cancer Genomics of the Kidney (CAGEKID) study. We examined associations between genomically-defined patient groups, explained below, and disease-free as well as RCC-specific survival independently in two cohorts of patients (N=469 for cohort 1; 474 for cohort 2). We used the Kaplan-Meier method with log-rank tests to compare survival functions, and Cox proportional hazards models to stratify for patient stage and age to estimate association of each group with survival. RCC-specific survival was assessed with a competing-risks method to include deaths from other causes. Within these cohorts, 76.4% of patients harbored somatic mutations in VHL, the most common driver gene in ccRCC. The most commonly mutated genes within VHL-mutated tumors were PBRM1 (39.7%), SETD2 (19%), BAP1 (14.3%), and KDM5C (8.3%). Less frequently mutated genes included ATM, COL11A1, DMD, TP53, and TRRAP (~3-5%).Among VHL-driven tumors, we identified a new genomic classifier on the basis of the number of mutations in additional RCC driver genes in the panel examined. Patients were classified based on the presence of mutations only in VHL (VHL+0), those with mutations in VHL and one other driver gene (VHL+1), two other driver genes (VHL+2), and 3 or more other driver genes (VHL≥3). We observed within both cohorts that both the risk of disease recurrence as well as RCC-specific death were associated with an increased number of mutations within this classification. When stratified for patient stage and age, the hazard-ratio for 5-year disease-free survival for VHL≥3 patients was 6.69 (p=0.000212), 4.31 for VHL+2 (p=0.000862), and 2.43 for VHL+1 (p=0.035662), compared to patients with only mutations in VHL. These observations were replicated in the second patient cohort, with hazards ratios of 4.55, 2.49, and 1.40, for VHL≥3, VHL+2, and VHL+1 classified patients respectively, indicating that risk of disease recurrence increases with the number of driver mutations. Notably, tumor mutational burden (TMB) was not significantly different between the aforementioned groups, demonstrating that our classifier is independent of TMB. We created a model based on a set of 12 RCC-relevant genes, which can predict risk of relapse for the ~80% of patients with ccRCC that are VHL-driven. This classification can be defined based on a small panel of genes, making it easily applicable to the clinic, in the context of tumor or liquid biopsy analysis. Citation Format: Kate I. Glennon, Naveen S. Vasudev, Ghislaine Scelo, Michelle Wilson, Louis Letourneau, Robert Eveleigh, Nazanin Nourbehesht, Madeleine Arseneault, Antoine Paccard, Lars Egevad, Juris Viksna, Edgars Celms, Sharon M. Jackson, Behnoush Abedi-Ardekani, Anne Y. Warren, Peter J. Selby, Sebastian Trainor, Michael Kimuli, Naeem Soomro, Adebanji Adeyoju, Poulam Patel, Magdalena B. Wozniak, Ivana Holcatova, Antonin Brisuda, Vladimir Janout, Estelle Chanudet, David Zaridze, Anush Moukeria, Oxana Shangina, Lenka Foretova, Marie Navratilova, Dana Mates, Viorel Jinga, Ljiljana Bogdanovic, Bozidar Kovacevic, Anne Cambon-Thomsen, Guillaume Bourque, Alvis Brazma, Jörg Tost, Paul Brennan, Mark Lathrop, Yasser Riazalhosseini, Rosamonde E. Banks. Genomic classification to refine prognosis in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB113.

Circulating miRNAs have shown great promises as noninvasive diagnostic and predictive biomarkers in several solid tumors. While the miRNA profiles of renal tumors have been extensively explored, knowledge of their circulating counterparts is limited. Our study aimed to provide a large-scale genome-wide profiling of plasma circulating miRNA in clear-cell renal cell carcinoma (ccRCC). Plasma samples from 94 ccRCC cases and 100 controls were screened for 754 circulating micro-RNAs (miRNA) by TaqMan arrays. Analyses including known risk factors for renal cancer-namely, age, sex, hypertension, obesity, diabetes, tobacco smoking and alcohol consumption-highlighted that circulating miRNA profiles were tightly correlated with the stage of the disease. Advanced tumors, characterized as stage III and IV, were associated with specific miRNA signatures that significantly differ from both controls and earlier stage ccRCC cases. Molecular pathway enrichment analyses of their gene targets showed high similarities with alterations observed in renal tumors. Plasma circulating levels of miR-150 were significantly associated with RCC-specific survival and could marginally improve the predictive accuracy of clinical parameters in our series, including age at diagnosis, sex and conventional staging. In summary, our results suggest that circulating miRNAs may provide insights into renal cell carcinoma progression.

The utility of KRAS mutations in plasma circulating cell-free DNA (cfDNA) samples as non-invasive biomarkers for the detection of pancreatic cancer has never been evaluated in a large case-control series. We applied a KRAS amplicon-based deep sequencing strategy combined with analytical pipeline specifically designed for the detection of low-abundance mutations to screen plasma samples of 437 pancreatic cancer cases, 141 chronic pancreatitis subjects, and 394 healthy controls. We detected mutations in 21.1% (N=92) of cases, of whom 82 (89.1%) carried at least one mutation at hotspot codons 12, 13 or 61, with mutant allelic fractions from 0.08% to 79%. Advanced stages were associated with an increased proportion of detection, with KRAS cfDNA mutations detected in 10.3%, 17,5% and 33.3% of cases with local, regional and systemic stages, respectively. We also detected KRAS cfDNA mutations in 3.7% (N=14) of healthy controls and in 4.3% (N=6) of subjects with chronic pancreatitis, but at significantly lower allelic fractions than in cases. Combining cfDNA KRAS mutations and CA19-9 plasma levels on a limited set of case-control samples did not improve the overall performance of the biomarkers as compared to CA19-9 alone. Whether the limited sensitivity and specificity observed in our series of KRAS mutations in plasma cfDNA as biomarkers for pancreatic cancer detection are attributable to methodological limitations or to the biology of cfDNA should be further assessed in large case-control series.

Epidemiologic studies have reported that moderate alcohol consumption is inversely associated with the risk of renal cancer. However, there is no information available on the associations in renal cancer subsites. From 1992 through to 2010, 477,325 men and women in the European Prospective Investigation into Cancer and Nutrition cohort were followed for incident renal cancers (n=931). Baseline and lifetime alcohol consumption was assessed by country-specific, validated dietary questionnaires. Information on past alcohol consumption was collected by lifestyle questionnaires. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated from Cox proportional hazard models. In multivariate analysis, total alcohol consumption at baseline was inversely associated with renal cancer; the HR and 95% CI for the increasing categories of total alcohol consumption at recruitment versus the light drinkers category were 0.78 (0.62-0.99), 0.82 (0.64-1.04), 0.70 (0.55-0.90), 0.91 (0.63-1.30), respectively, (p(trend)=0.001). A similar relationship was observed for average lifetime alcohol consumption and for all renal cancer subsites combined or for renal parenchyma subsite. The trend was not observed in hypertensive individuals and not significant in smokers. In conclusion, moderate alcohol consumption was associated with a decreased risk of renal cancer. What's new? Previous studies have indicated that environmental or lifestyle factors may be involved in the etiology of renal cancer, and that moderate alcohol consumption may reduce the risk of this type of cancer. In this very large European study (nearly 500,000 subjects), the authors found that, indeed, total alcohol consumption was inversely associated with renal cancer overall (for all subsites combined), and also with cancers of the renal parenchyma.

Findings from epidemiological studies examining physical activity in relation to pancreatic cancer risk have suggested decreased risks for physical activity; however, the results are inconsistent. The association between occupational and leisure-time physical activity and risk of pancreatic cancer was examined among 826 pancreatic cancer cases and 930 age-, sex- and center-matched controls from a large multicenter central European study in Czech Republic and Slovakia recruited between 2004 and 2012. Data on physical activity including type and dose (frequency, intensity, and duration) were examined using multivariable-adjusted logistic regression models. Occupational physical activity was not significantly associated with risk of pancreatic cancer [odds ratio (OR) 0.90, 95 % confidence interval (CI) 0.71–1.15]. A 35 % decrease in risk of pancreatic cancer was observed for regular leisure-time physical activity (OR 0.65, 95 % CI 0.52–0.87). The risk estimates were significant for low and moderate intensity of activity with the strongest protective effect among individuals who exercised during more than 40 weeks per year. The results for cumulated leisure-time physical activity assessed 1 year prior to diagnosis achieved the same level of risk reduction. In addition, stronger risk estimates for leisure-time physical activity were observed among women (men: OR 0.74, 95 % CI 0.54–1.01; women: OR 0.53, 95 % CI 0.37–0.75). The findings for female participants were stronger for intensity and frequency of leisure-time physical activity, in particular for light and moderate activity (OR 0.43, 95 % CI 0.25–0.75; and OR 0.57, 95 % CI 0.37–0.88, respectively). These results provide evidence for a decreased risk of pancreatic cancer associated with regular leisure-time physical activity.

Background Detection of lung cancer at an early stage by sensitive screening tests could be an important strategy to improving prognosis. Our objective was to identify a panel of circulating microRNAs in plasma that will contribute to early detection of lung cancer. Material and Methods Plasma samples from 100 early stage (I to IIIA) non–small-cell lung cancer (NSCLC) patients and 100 non-cancer controls were screened for 754 circulating microRNAs via qRT-PCR, using TaqMan MicroRNA Arrays. Logistic regression with a lasso penalty was used to select a panel of microRNAs that discriminate between cases and controls. Internal validation of model discrimination was conducted by calculating the bootstrap optimism-corrected AUC for the selected model. Results We identified a panel of 24 microRNAs with optimum classification performance. The combination of these 24 microRNAs alone could discriminate lung cancer cases from non-cancer controls with an AUC of 0.92 (95% CI: 0.87-0.95). This classification improved to an AUC of 0.94 (95% CI: 0.90-0.97) following addition of sex, age and smoking status to the model. Internal validation of the model suggests that the discriminatory power of the panel will be high when applied to independent samples with a corrected AUC of 0.78 for the 24-miRNA panel alone. Conclusion Our 24-microRNA predictor improves lung cancer prediction beyond that of known risk factors.

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by hotspot mutations in the KRAS gene (codons 12, 13 or 61) in 85-90% of cases. Codon 12 KRAS mutations have been detected in pancreatic juice, blood and stool samples from pancreatic cancer cases and represent promising biomarkers for early detection. However, the proportion of tumor-derived KRAS mutations in cell-free DNA fragments (cfDNA) has shown large variations, probably because of the heterogeneity in biosamples and assays tested. Deep sequencing technologies (NGS) allow the identification of low-abundance somatic variants, but have not previously been applied to the detection of KRAS hotspot mutations in cfDNA of PDAC cases. Moreover, variant calling methods have rarely been tested against cancer-free individuals so the proportion of false positives is unknown. We investigated whether deep sequencing of KRAS mutations at codons 12, 13 and 61 in plasma samples could represent a robust assay to distinguish pancreatic cancer from chronic pancreatitis and healthy controls. Methods: We developed an Ion Torrent-based NGS KRAS assay (partial exons 2 and 3, totalling 208bp) to screen cfDNA from plasma samples of 461 PDAC cases, 154 individuals with chronic pancreatitis and 421 healthy controls. cfDNA extraction (>4ng) and sequencing (>1000x coverage on average, and absence of systematic high sequencing error rates on the 208bp) performed well on 431 (93%) PDAC cases, 138 (90%) chronic pancreatitis, and 388 (95%) controls. We fit a robust negative-binomial regression to estimate the distribution of the sequencing errors at each DNA bp position and identified outlying samples, which were considered as KRAS positive when q-value Results: Sequencing of the serial dilutions of KRAS p.G12V mutated DNA indicated a detection threshold at a minor allele frequency of 0.2%. KRAS mutations in cfDNA were detected in 83 (19.3%) PDAC cases (73 on codon 12; 8 on codon 61; 1 on codon 13; and 1 multiple codons, i.e., similar in proportions as reported in tumor tissue from the International Cancer Genomic Consortium); 3 (2.2%) chronic pancreatitis (all on codon 12); and 8 (2.1%) healthy controls (4 on codon 12 and 4 on codon 61). Stage was significantly associated with the proportion of detected mutations in cancer cases (chi-squared p = 0.0005): the proportions of cases with detectable KRAS mutations in plasma were 7.9%, 14.9%, and 31.1% for local, regional, and advanced stages, respectively. Conclusions: The NGS-based KRAS mutation screening is a sensitive approach to detect low allelic fraction in plasma cfDNA, although its utility for early detection is still limited. However, it has the capacity to identify specific KRAS mutations, which could be useful in a panel of other non-invasive biomarkers. Citation Format: Florence Le Calvez-Kelm, Matthieu Foll, Magdalena B. Wozniak, Geoffroy Durand, Priscilia Chopard, Maroulio Pertesi, Tiffany Delhomme, Ivana Holcatova, Lenka Foretova, Vladimir Janout, Eleonora Fabianova, Maxime P. Vallée, Paul Brennan, James D. McKay, Graham Byrnes, Ghislaine Scélo. NGS-based detection of KRAS hotspot mutations in plasma cell-free DNA of pancreatic cancer cases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3137.

Lung cancer is the leading cause of cancer deaths worldwide. In 2008, 1.61 million new cases, and 1.38 million deaths due to lung cancer were recorded. This high mortality rate is mainly due to the late stage at which lung cancer is diagnosed. While early diagnosis has been successfully implemented through tomography-based population screenings in high-risk individuals, there is a need for simpler, non-invasive and more accessible methodologies for effective early cancer detection programs. Circulating microRNA (miRNA) profiles have been suggested as promising diagnostic and prognostic biomarkers for cancer, including lung cancer. However, the results have so far been inconsistent between studies. The objective of this study was to explore the potential of circulating miRNAs in plasma for early detection of lung cancer using global profiling approach. Plasma samples were collected from 100 early stage (I to IIIA) non-small-cell lung cancer patients (35 lung adenocarcinoma and 65 squamous cell carcinoma patients) and 100 age- and gender-matched healthy controls. 754 circulating miRNAs were analyzed via quantitative RT-PCR using TaqMan Low Density Arrays. Data were quantile normalized and limma analysis with adjustment for multiple testing to control for false discovery rate (FDR, Benjamini-Hochberg method) was performed to identify differentially regulated miRNA between cases and controls. Penalized Lasso logistic regression model (with penalty parameter tuning conducted by 10-fold cross-validation) was used to compute the least redundant panel of miRNAs for discriminating between cases and controls. The area under the receiver operating characteristic curve (AUC) was calculated to assess the discriminatory power of the model. Internal validation was conducted by calculating the bootstrap optimism-corrected AUC for the selected model. Sixty one plasma miRNAs were found to be significantly differentially expressed between lung cancer cases and controls including 33 upregulated and 28 downregulated miRNAs (p-value < 0.05). Penalized Lasso logistic regression identified a panel of 24 miRNA having the optimum classification performance. Multiple logistic regression analysis showed that combination of the 24 miRNAs alone could discriminate lung cancer cases from healthy controls (AUC of 0.92). This classification improved to AUC of 0.94 following addition of gender, age at interview and smoking status to the model. Internal validation of the model using the bootstrap optimism corrected AUC suggests that the discriminatory power of the panel will be high when applied to independent samples (corrected AUC of 0.86 and for the 24-miRNA panel alone, 0.87 for the model including gender, age and smoking status). These results highlight the potential of circulating miRNA as biomarkers for lung cancer. Independent prospective validation of the clinical potential of the panel within a cohort with pre-diagnostic samples is warranted. Citation Format: Magdalena B. Wozniak, Ghislaine Scelo, David Muller, Anush Moukeria, David Zaridze, Paul Brennan. Circulating microRNAs as non-invasive biomarkers for early detection of lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 884. doi:10.1158/1538-7445.AM2014-884

Background: The quality of tissue samples and extracted mRNA is a major source of variability in tumor transcriptome analysis using genome-wide expression microarrays. During and immediately after surgical tumor resection, tissues are exposed to metabolic, biochemical and physical stresses characterized as “warm ischemia”. Current practice advocates cryopreservation of biosamples within 30 minutes of resection, but this recommendation has not been systematically validated by measurements of mRNA decay over time. Methods: Using Illumina HumanHT-12 v3 Expression BeadChips, providing a genome-wide coverage of over 24,000 genes, we have analysed gene expression variation in samples of 3 hepatocellular carcinomas (HCC) and 3 lung carcinomas (LC) cryopreserved at times up to 2 hours after resection. Results: RNA Integrity Numbers (RIN) revealed no significant deterioration of mRNA up to 2 hours after resection. Genome-wide transcriptome analysis detected non-significant gene expression variations of -2.4%/hr (95% CI: -5.5%/hr to 0.6%/hr; p=0.12). In LC, no consistent gene expression pattern was detected in relation with warm ischemia. In HCC, a signature of 6 up-regulated genes (CYP2E1, IGLL1, CABYR, CLDN2, NQO1, SCL13A5) and 6 down-regulated (MT1G, MT1H, MT1E, MT1F, HABP2, SPINK1) was identified (FDR Conclusions: Overall, our observations support current recommendation of time to cryopreservation of up to 30 minutes and emphasize the need for identifying tissue-specific genes deregulated following resection to avoid misinterpreting expression changes induced by warm ischemia as pathologically significant changes. Citation Format: Florence Le Calvez-Kelm, Elodie Caboux, Maria Paciencia, Geoffroy Durand, Nivonirina Robinot, Magdalena Wozniak, Francoise Galateau-Salle, Graham Byrnes, Pierre Hainaut. Impact of delay to cryopreservation on RNA integrity and genome-wide expression profiles in resected tumor samples. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2000. doi:10.1158/1538-7445.AM2013-2000

Gene expression microarray and next generation sequencing efforts on conventional, clear cell renal cell carcinoma (ccRCC) have been mostly performed in North American and Western European populations, while the highest incidence rates are found in Central/Eastern Europe. We conducted whole-genome expression profiling on 101 pairs of ccRCC tumours and adjacent non-tumour renal tissue from Czech patients recruited within the “K2 Study”, using the Illumina HumanHT-12 v4 Expression BeadChips to explore the molecular variations underlying the biological and clinical heterogeneity of this cancer. Differential expression analysis identified 1650 significant probes (fold change ≥2 and false discovery rate

Renal cell carcinoma (RCC) is a poorly documented cancer, although time trends and geographical variations in incidence are intriguing. In addition to the frequent VHL mutation, recent studies have identified novel somatic alterations involved in RCC pathogenesis. Microarray and next generation sequencing efforts have been mostly performed on North American and Western European populations, while the highest incidence rates are found in Central/Eastern Europe. Our project has focused on the delineation of gene expression patterns specific to the Czech Republic population, where the incidence rates are the highest in the world. As a feasibility study, we performed whole genome expression profiling on 12 pairs of fresh frozen conventional clear cell RCC (ccRCC) tumors and adjacent non-tumor renal tissue using the Illumina HumanHT-12 v4 Expression BeadChips. To identify genes differentially expressed in ccRCC tumor versus normal tissue samples, the Significance Analysis of Microarrays (SAM) was used. Gene set enrichment analysis (GSEA) software was used for analysis of differentially expressed BioCarta categories and pathways associated with ccRCC pathogenesis. Following initial analyses a signature composed of 994 downregulated and 872 upregulated genes was found in ccRCC compared with normal tissue (FDR Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5103. doi:1538-7445.AM2012-5103

Mycosis fungoides (MF) is a low-grade cutaneous T-cell lymphoma, in which malignant T cell clones (mostly CD4+) arise in the skin from the early disease stages. IFN-α is widely used in the treatment of MF and when used in combination with PUVA has been reported as an effective treatment, with overall response rates of 30%–80%, and complete response rates of 14%–25%. However, up to date there is no information available on prognostic factors that could help to predict response to IFN-α /PUVA in MF. The purpose of the study was to find the molecular signature associated with IFN-α /PUVA resistance, or lack of remission after IFN-α /PUVA treatment. The gene expression profile of the pre-treatment samples from 30 MF patients enrolled in a random clinical trial with IFN-α and/or PUVA has been analyzed by use of cDNA microarrays. Following the treatment outcome, the patients have been divided into good responders (23 patients that have achieved complete remission in the time of 24 weeks or less) and bad responders (7 patients that have not reached completed remission or have shown progression of the disease during treatment). The genes associated with good vs. bad response have been identified. Four genes associated with cell cycle regulation and tumour microenviroment have been identified to predict good response by the significance analysis of microarrays (SAM) correlating expression data with survival time. Moreover, 38 genes involved in T cell receptor signaling pathway, NF-kB activation and Jak-Stat signaling pathway have been found to be associated with unfavorable response to treatment by use of SAM analysis. This was validated using other bioinformatics tools based on t-statistics and Cox-model, applying False Discovery Rate for multiple testing. Furthermore, using a web-based tool (Signs) that uses a combination of gene filtering, clustering and survival model building, a 2-gene model has been obtained. This model could distinguish two groups of MF patients with probability of remission at 24 weeks of 15%, and 60% (log-rank test, p:0.007). This confirms that TCR-signaling plays a key role in cutaneous T-cell lymphoma cell survival; and could potentially be used for stratifying MF patients treated with IFN-α /PUVA into different risk-groups, if confirmed in additional studies.