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DFS by tumor stage stratified by genomic classifier

Supplementary Methods

Clinical information and 12 gene mutation status for cohorts C1-C3

Supplementary Tables S3-S13

Purpose:Patients with resected localized clear-cell renal cell carcinoma (ccRCC) remain at variable risk of recurrence. Incorporation of biomarkers may refine risk prediction and inform adjuvant treatment decisions. We explored the role of tumor genomics in this setting, leveraging the largest cohort to date of localized ccRCC tissues subjected to targeted gene sequencing.Experimental Design:The somatic mutation status of 12 genes was determined in 943 ccRCC cases from a multinational cohort of patients, and associations to outcomes were examined in a Discovery (n = 469) and Validation (n = 474) framework.Results:Tumors containing a von-Hippel Lindau (VHL) mutation alone were associated with significantly improved outcomes in comparison with tumors containing a VHL plus additional mutations. Within the Discovery cohort, those with VHL+0, VHL+1, VHL+2, and VHL+≥3 tumors had disease-free survival (DFS) rates of 90.8%, 80.1%, 68.2%, and 50.7% respectively, at 5 years. This trend was replicated in the Validation cohort. Notably, these genomically defined groups were independent of tumor mutational burden. Amongst patients eligible for adjuvant therapy, those with a VHL+0 tumor (29%) had a 5-year DFS rate of 79.3% and could, therefore, potentially be spared further treatment. Conversely, patients with VHL+2 and VHL+≥3 tumors (32%) had equivalent DFS rates of 45.6% and 35.3%, respectively, and should be prioritized for adjuvant therapy.Conclusions:Genomic characterization of ccRCC identified biologically distinct groups of patients with divergent relapse rates. These groups account for the ∼80% of cases with VHL mutations and could be used to personalize adjuvant treatment discussions with patients as well as inform future adjuvant trial design.

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.

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 incidence of renal cell carcinoma (RCC) is increasing worldwide, and its prevalence is particularly high in some parts of Central Europe. Here we undertake whole-genome and transcriptome sequencing of clear cell RCC (ccRCC), the most common form of the disease, in patients from four different European countries with contrasting disease incidence to explore the underlying genomic architecture of RCC. Our findings support previous reports on frequent aberrations in the epigenetic machinery and PI3K/mTOR signalling, and uncover novel pathways and genes affected by recurrent mutations and abnormal transcriptome patterns including focal adhesion, components of extracellular matrix (ECM) and genes encoding FAT cadherins. Furthermore, a large majority of patients from Romania have an unexpected high frequency of A:T>T:A transversions, consistent with exposure to aristolochic acid (AA). These results show that the processes underlying ccRCC tumorigenesis may vary in different populations and suggest that AA may be an important ccRCC carcinogen in Romania, a finding with major public health implications.

Context Marked unexplained national variations in incidence rates of kidney cancer have been observed for decades in Europe. Objective To investigate geographic variations at the regional level and identify European regions with high incidence rates of kidney cancer. Evidence acquisition Regional- and national-level incidence data were extracted from the Cancer Incidence in Five Continents databases, local cancer registry databases, and local published reports. World population age-standardised rates (ASRs) were calculated for the periods 2003–2007 and 1988–1992. Rates by period and sex were compared using map visualisation. Evidence synthesis During 2003–2007, the highest ASR was found in the Plzen region, Czech Republic (31.4/100 000 person-years in men). Other regions of the Czech Republic had ASRs of 18.6–27.5/100 000 in men, with a tendency for higher rates in regions south of Prague. Surrounding regions, including eastern Germany and regions of Slovakia and Austria, had medium-to-high incidence rates (13.0–16.8/100 000 in men). Three other areas in Europe showed higher incidence rates in men compared with the rest of the continent: Lithuania, Estonia, Latvia, and Belarus (15.0–17.6/100 000); Iceland (13.5/100 000), and northern Italy (up to 16.0/100 000). Similar regional differences were observed among women, with rates approximately half of those observed in men in the same region. In general, these regional geographic variations remained stable over the periods 1988–1992 and 2003–2007, although higher incidence rates were detected in the Baltic countries in 2003–2007. Conclusions Several European regions show particularly high rates of kidney cancer incidence. Large variations were observed within countries covered by national health-care systems, implying that overdetection is not the major factor. Patient summary We present regional geographic variations in kidney cancer incidence rates in Europe. We highlight several regions with high incidence rates where further studies should be conducted for cancer control and prevention.

Obtained from Haematologica/the Hematology Journal website http://www.haematologica.org, Peripheral T-cell lymphomas are very aggressive hematologic malignancies for which there is no targeted therapy. New, rational approaches are necessary to improve the very poor outcome in these patients. Phosphatidylinositol- 3-kinase is one of the most important pathways in cell survival and proliferation. We hypothesized that phosphatidylinositol- 3-kinase inhibitors could be rationally selected drugs for treating peripheral T-cell lymphomas. Several phosphatidylinositol-3-kinase isoforms were inhibited genetically (using small interfering RNA) and pharmacologically (with CAL-101 and GDC-0941 compounds) in a panel of six peripheral and cutaneous T-cell lymphoma cell lines. Cell viability was measured by intracellular ATP content; apoptosis and cell cycle changes were checked by flow cytometry. Pharmacodynamic biomarkers were assessed by western blot. The PIK3CD gene, which encodes the δ isoform of phosphatidylinositol-3-kinase, was overexpressed in cell lines and primary samples, and correlated with survival pathways. However, neither genetic nor specific pharmacological inhibition of phosphatidylinositol-3-kinase δ affected cell survival. In contrast, the pan-phosphatidylinositol-3-kinase inhibitor GDC-0941 arrested all T-cell lymphoma cell lines in the G1 phase and induced apoptosis in a subset of them. We identified phospho-GSK3b and phospho-p70S6K as potential biomarkers of phosphatidylinositol-3-kinase inhibitors. Interestingly, an increase in ERK phosphorylation was observed in some GDC-0941-treated T-cell lymphoma cell lines, suggesting the presence of a combination of phosphatidylinositol-3-kinase and MEK inhibitors. A highly synergistic effect was found between the two inhibitors, with the combination enhancing cell cycle arrest at G0/G1 in all T-cell lymphoma cell lines, and reducing cell viability in primary tumor T cells ex vivo. These results suggest that the combined treatment of pan-phosphatidylinositol-3-kinase + MEK inhibitors could be more effective than single phosphatidylinositol-3-kinase inhibitor treatment, and therefore, that this combination could be of therapeutic value for treating peripheral and cutaneous T-cell lymphomas., This work was supported by grants from the Asociación Española Contra el Cáncer, Fondo de Investigaciones Sanitarias (PI051623, PI052800 and PI080856), RTICC (RD06/0020/0107) and Ministerio de Ciencia e Innovación (SAF2008-0387-1). EMS is supported by a grant from the Department of Education, Universities and Research of the Basque Government (BFI08.207). MSB is supported by a Contract Miguel Servet from Fondo de Investigaciones Sanitarias (CP11/00018)

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. Using Illumina HumanHT-12 v3 Expression BeadChips, providing a genome-wide coverage of over 24,000 genes, we have analyzed gene expression variation in samples of 3 hepatocellular carcinomas (HCC) and 3 lung carcinomas (LC) cryopreserved at times up to 2 hours after resection. 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 -3.5%/hr (95% CI: -7.0%/hr to 0.1%/hr; p = 0.054). 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 genes (MT1G, MT1H, MT1E, MT1F, HABP2, SPINK1) was identified (FDR

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

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

Abstract 3494 The search of an efficient therapy for Peripheral T-cell lymphomas (PTCL) patients is still a challenge, in part due to the very little knowledge about the PTCL pathogenesis, and the absence of appropriate models. This heterogeneous group of very aggressive malignancies can not be cured with conventional therapies; therefore, new therapeutic strategies are needed to improve the poor outcome in these patients. PIM family is composed of 3 kinases (PIM1, PIM2 and PIM3) which play an essential role in cell proliferation and survival. They are mainly activated through JAK/STAT signaling pathway, and are frequently altered in human malignancies by amplification, mutation and overexpression. The aim of this study is to determine the efficiency and the mechanism of action of PIM inhibition in PTCL. Gene expression profiling of twenty two PTCL cases and seven reactive lymph nodes was performed. We observed a strong overexpression of the three PIM family genes in PTCL cases, especially PIM2. In addition, Gene Set Enrichment Analysis identified an overexpression of STAT3 and IL-2 pathways in PTCL cases, probably responsible for the strong expression of PIMs we found. Furthermore, PIM genes expression was confirmed by quantitative RT-PCR in 6 PTCL-derived cell lines compared to normal T cells from healthy donors, highlighting again the relevance of PIM2. Genetic inhibition was carried out using small interference RNA to specifically abolish the expression of each PIM1, PIM2 and PIM3 in a panel of 6 PTCL cell lines belonging to different PTCL subgroups. Additionally, pharmacological inhibition with one PIM inhibitor (ETP-39010) was performed. Surprisingly, genetic inhibition of each of the PIM gene alone did not show any cellular effect, neither cell cycle arrest nor apoptosis. But interestingly, we found that specific inhibition of each of the PIM genes caused an increased expression of the other PIM family members, probably leading to a compensatory mechanism among these kinases balancing the lack of one of them, avoiding pro-apoptotic effects and allowing cell survival. Accordingly, a simultaneous inhibition of PIM1, PIM2 and PIM3 using the pharmacological pan-PIM inhibitor produced a decrease in cell viability and a strong induction of apoptosis in all cell lines, without cell cycle arrest. Several PIM inhibitor biomarkers have been identified at the mRNA level, involving the DNA damage response signaling. In conclusion, our results indicate that PIM kinases inhibition could be an effective therapeutic approach for PTCL. Disclosures: No relevant conflicts of interest to declare.

Abstract 3493 Peripheral T-cell lymphomas (PTCL) are a heterogeneous group of very aggressive malignancies lacking efficient therapy. Unfortunately, there are neither animal models nor representative cell lines for most PTCL types, making functional and pharmacodynamic studies even more difficult. PI3K signaling is essential for cell proliferation and survival, is frequently altered in human cancer and seems to play a critical role in T-cell development and activation. The aim of this work is to determine the efficiency of PI3K inhibition in PTCL, looking for pharmacodynamic biomarkers, and to identify markers that could distinguish responders from non-responders. Twenty two PTCL cases and seven reactive lymph nodes were studied using gene expression profiling. We performed an in silico analysis using the Connectivity Map program to identify drugs that could potentially reverse the PTCL gene expression signature. Among them, several PI3K/mTOR inhibitors were found. Moreover, genomic studies using Gene Set Enrichment Analysis identified PIK3CD gene (encoding for the delta isoform of PI3K) to be the only one significantly correlated to the activation of CD40, NF-kB and TCR pathways. Quantitative RT-PCR confirmed the strong overexpression of PIK3CD in 6 PTCL-derived cell lines compared to normal T cells from healthy donors. Sequence analyses for the coding region of the PIK3CD gene identified a point mutation in one of these cell lines, described as activating in solid tumors. A panel of 6 PTCL cell lines belonging to different PTCL subgroups was treated with 3 PI3K inhibitors (LY294002, ETP-45658, GDC-0941). Moreover, genetic inhibition was also carried out using small interference RNA to specifically abolish the expression of alpha and delta isoforms of PI3K (PIK3CA and PIK3CD genes, respectively). In vitro studies showed very similar results with the three pharmacological PI3K inhibitors we used: they induced G1 cell cycle arrest in all cell lines, and apoptosis in some of them, in a time/dose-dependent manner. We also observed a decrease in the levels of pAKT(S473) in all cell lines, while pGSK3B(S9) and p-p70S6K(T389) were reduced after treatment only in sensitive cell lines. Our results indicate that genetic inhibition of PI3K delta isoform could induce apoptosis in those PTCL cell lines that were sensitive to PI3K inhibitors, but not in the resistant cell lines; while genetic inhibition of PI3K alpha isoform did not display such effects. Taken together these results could highlight the relevance of PI3K delta isoform in at least a subset of PTCL, indicating that PI3K inhibition, especially delta isoform, could be an effective therapeutic approach for PTCL and identifying potential markers for patients' stratification and pharmacodynamic assessment. Disclosures: No relevant conflicts of interest to declare.

Abstract 4917 T-cell lymphomas (TCL) are a heterogeneous group of aggressive malignancies lacking specific and efficient therapy. Unfortunately, there are neither animal models nor representative cell lines for most TCL types, making functional and pharmacogenomics studies even more difficult. PI3K and PIM are kinases involved in cell proliferation, frequently altered in human cancer that seems to play a critical role in T-cell development and activation. Genomic studies have identified PIK3CD subunit to be significantly associated with in activation of CD40, NF-kB and TCR-pathways. The aim of this project is to determine the efficiency of PI3K inhibitors (PI3Ki) and PIM inhibitors (PIMi) in TCL, looking for biomarkers of their mechanism of action and to identify markers that could identify responders from non-responders. Twenty PTCL and seven reactive lymph nodes were studied using gene expression microarrays. We performed an in silico analysis using the Connectivity Map program to identify drugs that could potentially reverse PTCL gene expression signature. Among them, several PI3K/mTOR inhibitors were found. A panel of 6 TCL cell lines belonging to different TCL subgroups were treated with 3 PI3Ki (LY294002, ETP-45658, GDC-0941) and one PIMi (ETP-39010). Functional studies were also done to establish the role of each of the targeted genes. In vitro studies showed that PI3Ki induced G1 cell cycle arrest in all cell lines, and apoptosis in a portion of them, in a time/dose-dependent manner. We also observed a decrease in the levels of pAKT(S473), pGSK3B(S9) and p-p70S6K(T389) after treatment. In addition, both the analysis of the PTCL gene expression signature as well as western blot studies on TCL cell lines has shown overexpression of PIM family genes, A decrease in cell viability, and a strong induction of apoptosis in all cell lines was seen after PIM inhibition, without cell cycle arrest. Several diagnostic and pharmacodynamic biomarkers of PIMi have been identified at the mRNA and protein level in both cell lines In conclusion, our results indicate that PI3Ki and PIMi are effective therapeutic approaches for TCLs, identifying potential markers for patient's stratification and pharmacodynamic assessment. Disclosures: No relevant conflicts of interest to declare.

Abstract 969 Introduction: Peripheral T-cell lymphoma (PTCL) has been the subject of a relatively limited number of studies to elucidate the molecular pathogenesis. As a result, molecular classification of PTCL is still to be developed, targeted drugs are in very early development and clinical outcome is dismal. Recently, new technologies in genomic analysis have offered the opportunity to improve the knowledge regarding microRNA and gene expression signatures in T-cell lymphoma as well as the potential of microRNAs as prognostic markers in this disease. Patients and methods: The study included a group of 22 patients with PTCL along with 7 reactive lymph nodes (LN) as controls. The global microRNA and gene expression profiles were examined using a commercially available Agilent platform. To identify microRNAs differentially expressed in PTCL versus LN samples and between different subgroups of PTCL, we used the significance analysis of microarrays (SAM) protocol and permutation tests (10,000 permutations). For analysis of pathways associated with PTCL pathogenesis, a gene set enrichment analysis (GSEA) was performed. Results were validated in an additional set of paraffin embedded samples. Results: A signature composed of 80 microRNAs was found to be differentially expressed in PTCL compared with LN, including the let-7 family, mir-10, mir-15, mir-16 and miR-101 (p Conclusions: Molecular analysis of PTCL, facilitated by the comparison with normal T-cell subpopulations, revealed the existence of a null-phenotype PTCL, characterized by aggressive behavior and expressing a microRNA oncogenic signature. This research suggests possible and novel roles for microRNAs in the diagnosis and pathogenesis of T-cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

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.

Primary cutaneous anaplastic large-cell lymphoma (C-ALCL) and cutaneous peripheral T-cell lymphoma not otherwise specified (C-PTL-NOS) are cutaneous T-cell lymphomas with distinct clinical behaviors. Whereas C-ALCL has a favorable prognosis with frequent spontaneous disease regression, C-PTL-NOS runs a more aggressive course. The molecular pathogenesis of these cutaneous T-cell lymphoma types has not yet been studied in detail. In this issue, van Kester et al. report new imbalances that could contribute to our understanding of the differences between these two lymphoma types.