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Epigenetic mechanisms such as aberrant DNA methylation (DNAme) are known to drive esophageal squamous cell carcinoma (ESCC), yet they remain poorly understood. Here, we studied tumor-specific DNAme in ESCC cases from nine high-incidence countries of Africa, Asia, and South America. Infinium MethylationEPIC array was performed on 108 tumors and 51 normal tissues adjacent to the tumors (NAT) in the discovery phase, and targeted pyrosequencing was performed on 132 tumors and 36 NAT in the replication phase. Top genes for replication were prioritized by weighting methylation results using RNA-sequencing data from The Cancer Genome Atlas and GTEx and validated by qPCR. Methylome analysis comparing tumor and NAT identified 6,796 differentially methylated positions (DMP) and 866 differential methylated regions (DMR), with a 30% methylation (Δβ) difference. The majority of identified DMPs and DMRs were hypermethylated in tumors, particularly in promoters and gene-body regions of genes involved in transcription activation. The top three prioritized genes for replication, PAX9, SIM2, and THSD4, had similar methylation differences in the discovery and replication sets. These genes were exclusively expressed in normal esophageal tissues in GTEx and downregulated in tumors. The specificity and sensitivity of these DNAme events in discriminating tumors from NAT were assessed. Our study identified novel, robust, and crucial tumor-specific DNAme events in ESCC tumors across several high-incidence populations of the world. Methylome changes identified in this study may serve as potential targets for biomarker discovery and warrant further functional characterization.Significance:This largest genome-wide DNA methylation study on ESCC from high-incidence populations of the world identifies functionally relevant and robust DNAme events that could serve as potential tumor-specific markers.

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PDF file - 67KB, Antibodies to type-specific HPV proteins and the risk of lung cancer.

Human papillomavirus (HPV) infections have been implicated in lung carcinogenesis, but causal associations remain uncertain. We evaluated a potential causal role for HPV infections in lung cancer through an analysis involving serology, tumor DNA, RNA, and p16 protein expression. Association between type-specific HPV antibodies and risk of lung cancer was examined among 3,083 cases and 4,328 controls in two case–control studies (retrospective) and one nested case–control study (prospective design). Three hundred and thirty-four available tumors were subjected to pathologic evaluation and subsequent HPV genotyping following stringent conditions to detect all high-risk and two low-risk HPV types. All HPV DNA-positive tumors were further tested for the expression of p16 protein and type-specific HPV mRNA. On the basis of the consistency of the results, although HPV11 and HPV31 E6 antibodies were associated with lung cancer risk in the retrospective study, no association was observed in the prospective design. Presence of type-specific antibodies correlated poorly with the presence of the corresponding HPV DNA in the tumor. Although nearly 10% of the lung tumors were positive for any HPV DNA (7% for HPV16 DNA), none expressed the viral oncogenes. No association was observed between HPV antibodies or DNA and lung cancer survival. In conclusion, we found no supportive evidence for the hypothesized causal association between HPV infections and lung cancer. Cancer Res; 74(13); 3525–34. ©2014 AACR.

PDF file - 55KB, Association between type-specific HPV antibodies and risk of lung cancer by seropositivity threshold.

PDF file - 52KB, Comparison of HPV16 DNA positivity by sex, smoking status and lung histology.

Summary Background The contribution of alcohol to the large burden of oesophageal squamous cell carcinoma (ESCC) in east Africa remains uncertain and difficult to assess owing to complex consumption patterns of traditional and commercial drinks. We aimed to assess whether alcohol drinking, overall and at specific intake levels, contributes to ESCC risk in east Africa. Methods We did a hospital-based case-control study in Kenya, Tanzania, and Malawi, which included comprehensive assessment of a variety of locally consumed alcohol that we used to classify drinkers as exclusively low alcohol-by-volume (ABV

Genetic and epigenetic alterations of the telomere maintenance machinery like telomere length and telomerase reverse transcriptase (encoded by TERT gene) are reported in several human malignancies. However, there is limited knowledge on the status of the telomere machinery in periampullary carcinomas (PAC) which are rare and heterogeneous groups of cancers arising from different anatomic sites around the ampulla of Vater. In the current study, we investigated the relative telomere length (RTL) and the most frequent genetic and epigenetic alterations in the TERT promoter in PAC and compared it with tumor-adjacent nonpathological duodenum (NDu). We found shorter RTLs (1.27 vs 1.33, P=0.01) and lower TERT protein expression (p=0.04) in PAC tissues as compared to the NDu. Although we did not find any mutation at two reactivating hotspot mutation sites of the TERT promoter, we detected polymorphism in 45% (9/20) of the cases at rs2853669 (T>C). Also, we found a hypermethylated region in the TERT promoter of PACs consisting of four CpGs (cg10896616 with Deltabeta 7% ; cg02545192 with Deltabeta 9% ; cg03323598 with Deltabeta 19% ; and cg07285213 with Deltabeta 15%). In conclusion, we identified shorter telomeres with DNA hypermethylation in the TERT promoter region and lower TERT protein expression in PAC tissues. These results could be used further to investigate molecular pathology and develop theranostics for PAC.

Esophageal squamous cell carcinoma (ESCC) shows remarkable variation in incidence that is not fully explained by known lifestyle and environmental risk factors. It has been speculated that an unknown exogenous exposure(s) could be responsible. Here we combine the fields of mutational signature analysis with cancer epidemiology to study 552 ESCC genomes from eight countries with varying incidence rates. Mutational profiles were similar across all countries studied. Associations between specific mutational signatures and ESCC risk factors were identified for tobacco, alcohol, opium and germline variants, with modest impacts on mutation burden. We find no evidence of a mutational signature indicative of an exogenous exposure capable of explaining differences in ESCC incidence. Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC)-associated mutational signatures single-base substitution (SBS)2 and SBS13 were present in 88% and 91% of cases, respectively, and accounted for 25% of the mutation burden on average, indicating that APOBEC activation is a crucial step in ESCC tumor development.

Background Vulvar squamous cell carcinoma (VSCC) is a rare malignancy of the female genital tract. We aimed to determine the mucosal high-risk human papillomavirus (HPV)-attributable fraction of VSCCs from Italian women using multiple markers of viral infections. Methods VSCCs and 8 metastatic lymph node samples from 107 Italian women were analyzed by a highly type-specific multiplex genotyping assay for the presence of DNA from 119 different HPVs. Tissues were further analyzed for HPV RNA and for upregulation of the cellular protein p16INK4a. Results The rate of mucosal HPV-related tumors defined by viral DNA and RNA positivity was low (7.8%). HPV16 was the most prevalent, followed by 53, 56, and 58. Only five (4.9%) p16INK4a-positive tumors were also positive for both viral DNA and RNA. One (14.3%) metastatic lymph node sample was positive for all three markers. DNA of cutaneous HPVs was detected in only two VSCCs, i.e. genus beta types 5 and 110. Conclusion A small proportion of Italian VSCCs is putatively HPV-related, i.e. positive for both viral DNA and RNA of the same type, thus reinforcing the importance of HPV vaccination. Moreover, this study suggests that a direct role of HPV from genus beta and gamma in vulvar carcinogenesis is unlikely.

Genetic and epigenetic alterations of the telomere maintenance machinery like telomere length and telomerase reverse transcriptase (encoded by TERT gene) are reported in several human malignancies. However, there is limited knowledge on the status of the telomere machinery in periampullary carcinomas (PAC) that are rare and heterogeneous groups of cancers arising from different anatomic sites around the ampulla of Vater. In the current study, we investigated the relative telomere length (RTL) and the most frequent genetic and epigenetic alterations in the TERT promoter in PAC (n=20) and compared with tumor-adjacent nonpathological duodenum (NDu; n=16). We found shorter RTLs (1.27 vs 1.33, P=0.01) and lower TERT protein expression (p=0.04) in PAC tissues as compared to the NDu. Although we did not find any mutation at two reactivating hotspot mutation sites of the TERT promoter, we detected polymorphism in 55% (11/20) of the cases at rs2853669 (T>C). Also, we found a hypermethylated region in the TERT promoter of PACs consisting of four CpGs (cg10896616 with ∆β 7%; cg02545192 with ∆β 9%; cg03323598 with ∆β 19%; and cg07285213 with ∆β 15%). In conclusion, we identified shorter telomeres with DNA hypermethylation in the TERT promoter region and lower TERT protein expression in PAC tissues. Further studies with a larger sample size are necessary to substantiate these results.

Background: DNA isolation from formalin-fixed paraffin-embedded (FFPE) tissues for molecular analysis has become a frequent procedure in cancer research. However, the yield or quality of the isolated DNA is often compromised, and commercial kits are used to overcome this to some extent. Methods: We developed a new protocol (IARCp) to improve the quality and yield of DNA from FFPE tissues without using any commercial kit. To evaluate the IARCp's performance, we compared the quality and yield of DNA with two commercial kits, namely NucleoSpin® DNA FFPE XS (MN) and QIAamp DNA Micro (QG) isolation kit. Results: Total DNA yield for QG ranged from 120.0 to 282.0 ng (mean 216.5 ng), for MN: 213.6-394.2 ng (mean 319.1 ng), and with IARCp the yield was much higher ranging from 775.5 to 1896.9 ng (mean 1517.8 ng). Moreover, IARCp has also performed well in qualitative assessments by spectrophotometer, fluorometer, and real-time PCR assay. Conclusion: Overall, IARCp represents a novel approach to DNA isolation from FFPE which results in good quality and significant amounts of DNA suitable for many downstream genome-wide and targeted molecular analyses. This protocol does not require the use of any commercial kits or phenol for isolating DNA from FFPE tissues, making it suitable to implement in low-resource settings such as low and middle- income countries.

There are unexplained geographical variations in the incidence of kidney cancer with the high rates reported in Baltic countries, as well as eastern and central Europe. Having access to a large and well-annotated collection of “tumor/non-tumor” pairs of kidney cancer patients from the Czech Republic, Romania, Serbia, UK, and Russia, we aimed to analyze the morphology of non-neoplastic renal tissue in nephrectomy specimens. By applying digital pathology, we performed a microscopic examination of 1012 frozen non-neoplastic kidney tissues from patients with renal cell carcinoma. Four components of renal parenchyma were evaluated and scored for the intensity of interstitial inflammation and fibrosis, tubular atrophy, glomerulosclerosis, and arterial wall thickening, globally called chronic renal parenchymal changes. Moderate or severe changes were observed in 54 (5.3%) of patients with predominance of occurrence in Romania (OR = 2.67, CI 1.07–6.67) and Serbia (OR = 4.37, CI 1.20–15.96) in reference to those from Russia. Further adjustment for comorbidities, tumor characteristics, and stage did not change risk estimates. In multinomial regression model, relative probability of non-glomerular changes was 5.22 times higher for Romania and Serbia compared to Russia. Our findings show that the frequency of chronic renal parenchymal changes, with the predominance of chronic interstitial nephritis pattern, in kidney cancer patients varies by country, significantly more frequent in countries located in central and southeastern Europe where the incidence of kidney cancer has been reported to be moderate to high. The observed association between these pathological features and living in certain geographic areas requires a larger population-based study to confirm this association on a large scale. Supplementary Information The online version contains supplementary material available at 10.1007/s00428-020-02986-3.

Esophageal squamous cell carcinoma (ESCC) shows a remarkable variation in incidence which is not fully explained by known lifestyle and environmental risk factors. It has been speculated that an unknown exogenous exposure(s) could be responsible. Here we combine the fields of mutational signature analysis with cancer epidemiology to study 552 ESCC genomes from eight countries with varying incidence rates. The mutational profiles of ESCC were similar across all countries studied. Associations between specific mutational signatures and ESCC risk factors were identified for tobacco, alcohol, opium and germline variants, with modest impacts on mutation burden. We find no evidence of a mutational signature indicative of an exogenous exposure capable of explaining the differences in ESCC incidence. APOBEC associated mutational signatures SBS2 and SBS13 were present in 88% and 91% of cases respectively and accounted for a quarter of the mutation burden on average, indicating that activation of APOBEC is a crucial step in ESCC tumor development.

BACKGROUND: Recurrent mutations in the promoter of the telomerase reverse transcriptase (TERT) gene (C228T and C250T) detected in tumours and cells shed into urine of urothelial cancer (UC) patients are putative biomarkers for UC detection and monitoring. However, the possibility of detecting these mutations in cell-free circulating DNA (cfDNA) in blood and urine, or DNA from urinary exfoliated cells (cellDNA) with a single-gene sensitive assay has never been tested in a case-control setting. METHODS: We developed a single-plex assay (UroMuTERT) for the detection of low-abundance TERT promoter mutations. We tested 93 primary and recurrent UC cases and 94 controls recruited in France (blood, urine samples and tumours for the cases), and 50 primary UC cases and 50 controls recruited in Portugal (urinary exfoliated cell samples). We compared our assay with urine cytology. FINDINGS: In the French series, C228T or C250T were detected in urinary cfDNA or cellDNA in 81 cases (87·1%; 95% CI 78·6–93·2), and five controls (Specificity 94·7%; 95%CI 88·0–98·3), with 98·6% (95% CI 92·5–99·96) concordance in matched tumours. Detection rate in plasma cfDNA among cases was 7·1%. The UroMuTERT sensitivity was (i) highest for urinary cfDNA and cellDNA combined, (ii) consistent across primary and recurrent cases, tumour stages and grades, (iii) higher for low-risk non-muscle invasive UC (86·1%) than urine cytology (23·0%) (P

Epigenetic mechanisms such as aberrant DNA methylation (DNAme) are known to drive esophageal squamous cell carcinoma (ESCC), yet they remain poorly understood. Here, we studied tumor-specific DNAme in ESCC cases from nine high-incidence countries of Africa, Asia, and South America. Infinium MethylationEPIC array was performed on 108 tumors and 51 normal tissues adjacent to the tumors (NAT) in the discovery phase, and targeted pyrosequencing was performed on 132 tumors and 36 NAT in the replication phase. Top genes for replication were prioritized by weighting methylation results using RNA-sequencing data from The Cancer Genome Atlas and GTEx and validated by qPCR. Methylome analysis comparing tumor and NAT identified 6,796 differentially methylated positions (DMP) and 866 differential methylated regions (DMR), with a 30% methylation (Δβ) difference. The majority of identified DMPs and DMRs were hypermethylated in tumors, particularly in promoters and gene-body regions of genes involved in transcription activation. The top three prioritized genes for replication, PAX9, SIM2, and THSD4, had similar methylation differences in the discovery and replication sets. These genes were exclusively expressed in normal esophageal tissues in GTEx and downregulated in tumors. The specificity and sensitivity of these DNAme events in discriminating tumors from NAT were assessed. Our study identified novel, robust, and crucial tumor-specific DNAme events in ESCC tumors across several high-incidence populations of the world. Methylome changes identified in this study may serve as potential targets for biomarker discovery and warrant further functional characterization. Significance: This largest genome-wide DNA methylation study on ESCC from high-incidence populations of the world identifies functionally relevant and robust DNAme events that could serve as potential tumor-specific markers.

Background There are unexplained geographical variations in the incidence of kidney cancer with the high rates reported in Baltic countries, as well as eastern and central Europe. Analysis of non-neoplastic tissues is a way to better understand the carcinogenesis. Methods Having access to a rich, well-annotated collection of “tumor/non-tumor” pairs of kidney cancer patients from Czech Republic, Romania, Serbia, United Kingdom, and Russia for studying genomics of kidney cancer, we aimed to analyze morphology of non-neoplastic renal tissue. By applying digital pathology, we performed microscopic examination of 1012 frozen non-neoplastic kidney tissues from patients with renal cell carcinoma. Renal parenchyma was evaluated and scored for the interstitial inflammation and fibrosis, tubular atrophy, glomerulosclerosis and arterial wall thickening, globally called chronic renal parenchymal changes. Results Moderate or severe changes was observed in 54 (5.3%) of patients with predominance of occurrence in Romania (OR = 2.67, CI 1.07–6.67) and Serbia (OR = 4.37, CI 1.20-15.96) in reference to those from Russia. Further adjustment for comorbidities, tumor characterstics and stage did not change risk estimates. In multinomial regression model, relative probability of non-glomerular changes were 5.22 times higher for Romania and Serbia compered to Russia. Conclusion Our findings show that the frequency of chronic renal parenchymal changes in kidney cancer patients varies by country, significantly more frequent in countries located in central and southeastern Europe where the incidence of kidney cancer has been reported to be high. We suggest that these parenchymal changes, possibly linked to environmental exposures, may be relevant to renal carcinogenesis in these countries.

Background: The main risk factors for head and neck cancer (HNC) are tobacco and alcohol use. However, an important fraction of oropharyngeal cancer (OPC) is caused by human papillomaviruses (HPV), a subgroup with increasing incidence in several western countries. Methods: As part of the HPV-AHEAD study, we assessed the role of HPV infection in 772 archived tissue specimens of Belgian HNC patients: 455 laryngeal (LC), 106 oral cavity (OCC), 99 OPC, 76 hypopharyngeal (HC), and 36 unspecified parts of the head and neck. All specimens were tested for HPV DNA (21 genotypes); whereof all HPV DNA-positives, all HPV DNA-negative OPCs and a random subset of HPV DNA-negatives of the other HNC sites were tested for the presence of type-specific HPV RNA and p16(INK4a) over-expression. Results: The highest HPV DNA prevalence was observed in OPC (36.4 %), and was significantly lower (p < 0.001) in the other HNCs (OCC:7.5 %, LC:6.6 %). HPV16 was the most common HPV-genotype in all HNCs. Approximately 83.0 % of the HPV DNA-positive OPCs tested HPV RNA or p16-positive, compared to about 37.5 % and 44.0 % in OCC and LC, respectively. Estimation of the attributable fraction of an HPV infection in HNC was very similar for HPV RNA or p16 in addition to DNA-positivity; with 30 % for OPC, and 3 % for OCC and LC. Conclusion: Our study confirms the heterogeneity of HPV DNA prevalence across anatomical sites in HNC, with a predominance of HPV16 in all sites. The estimated proportion of HPV-driven HNC in Belgium, during the period 1980-2014, was 10 times higher in OPC compared to OCC and LC.

Human papillomavirus (HPV) causes oropharyngeal squamous cell carcinoma (OPSCC), although strongly divergent results have been reported regarding the prevalence of HPV16 in different countries, whether this represents important differences in etiology remains unclear. Applying rigorous protocols for sample processing, we centrally evaluated 1420 head and neck tumors (533 oropharynx, 395 oral cavity and 482 larynx) from studies conducted in the US, Europe and Brazil for mucosal HPV DNA and p16INK4a expression to evaluate regional heterogeneity in the proportion of HPV16-associated OPSCC and other head and neck cancer, and to assess covariates associated with the risk of HPV16-positive OPSCC. While majority of OPSCC in the US (60%) were HPV16-positive, this proportion was 31% in Europe and only 4% in Brazil (p

Role of human papillomavirus (HPV) in laryngeal carcinoma remains controversial. The aim of this study is to evaluate the role of HPV in laryngeal squamous cell carcinoma by determining presence of markers of viral infection. HPV DNA and E6*I mRNA status was determined by type-specific E7 PCR bead-based multiplex genotyping and RT-PCR assays in laryngeal squamous cell carcinoma biopsy samples. p16INK4a and COX-2 expression was determined by immunohistochemistry. Four cases out of 32 (13%) were HPV DNA+: HPV 11 (n = 1), HPV 31 (n = 3), HPV 59 (n = 1). One double infection: HPV 11 and HPV 31. p16INK4a was overexpressed in three cases (9%) and COX-2 in 17 cases (53%). Two of four HPV DNA+ samples had E6*I mRNA for HPV 31 and overexpressed p16INK4a and COX-2. HPV appears to play an active role in a small subset of laryngeal squamous cell carcinoma. p16INK4a can be used as a surrogate marker of transcriptionally active HPV infection; COX-2 expression had no correlation with HPV DNA and/or RNA positivity. J. Med. Virol. 86:642–646, 2014. © 2013 Wiley Periodicals, Inc.

Mutations of the MEN1 tumour suppressor gene predispose patients to the development of multiple endocrine neoplasia type 1 (MEN1) syndrome, which is characterized by multiple endocrine tumours, including prolactinomas. The recent findings of the interaction between menin, encoded by the MEN1 gene, and the oestrogen receptor, as well as the observation of rare cases of mammary carcinomas in our heterozygous Men1 mutant mice, led us to investigate a putative tumour suppressor function of the Men1 gene in mouse mammary cells by disrupting the gene in luminal epithelial cells. A significantly higher incidence of mammary intraepithelial neoplasia (MIN) was observed in mutant WapCre-Men1(F/F) mice (51.5%) than in WapCre-Men1(+/+) (0%) or Men1(F/F) (7.1%) control mice. The majority of MIN observed in the mutant mice displayed complete menin inactivation. Because of the leakage of WapCre transgene expression, prolactinomas were observed in 83.3% of mutant mice, leading to premature death. As there was no correlation between MIN development and elevated serum prolactin levels, and phospho-STAT5 expression was decreased in mammary lesions, the increased incidence of MIN lesions was most likely due to Men1 disruption rather than to prolactinoma development. Interestingly, in MIN lesions, we found a decrease in membrane-associated E-cadherin and beta-catenin expression, the latter of which is a menin partner. Finally, reduced menin expression was found in a large proportion of two independent cohorts of patients with breast carcinomas. Taken together, the current work indicates a role of Men1 inactivation in the development of mammary pre-cancerous lesions in mice and a potential role in human mammary cancer.

HPV16-positive cervical cancer lesions contain NFκB–ERα nuclear complexes to repress the TLR9 promoter., Human papillomavirus type 16 (HPV16) and other oncogenic viruses have been reported to deregulate immunity by suppressing the function of the double-stranded DNA innate sensor TLR9. However, the mechanisms leading to these events remain to be elucidated. We show that infection of human epithelial cells with HPV16 promotes the formation of an inhibitory transcriptional complex containing NF-κBp50–p65 and ERα induced by the E7 oncoprotein. The E7-mediated transcriptional complex also recruited the histone demethylase JARID1B and histone deacetylase HDAC1. The entire complex bound to a specific region on the TLR9 promoter, which resulted in decreased methylation and acetylation of histones upstream of the TLR9 transcriptional start site. The involvement of NF-κB and ERα in the TLR9 down-regulation by HPV16 E7 was fully confirmed in cervical tissues from human patients. Importantly, we present evidence that the HPV16-induced TLR9 down-regulation affects the interferon response which negatively regulates viral infection. Our studies highlight a novel HPV16-mediated mechanism that combines epigenetic and transcriptional events to suppress a key innate immune sensor.

Cervical cancer is the most common cancer among women in many areas of India which contributes for a fifth of the global burden of disease. Persistent infection with one of the high-risk human papillomaviruses (HPV) has been established as the cause for cervical cancer and the documentation of the prevalence of HPV types in cervical cancer in different regions of India is useful for a prevention program combining both screening and vaccination. In this study, the HPV type distribution and the frequency of p16(INK4a) immunoexpression have been determined in 125 cases of inflammatory lesions or grade 1 cervical intraepithelial neoplasia, 74 cases of grade 2, 72 cases of grade 3, and 113 cervical cancer cases diagnosed among women from rural Solapur and Osmanabad districts, Maharashtra. The overall prevalence of high-risk HPV was 37.6% in inflammatory lesions or grade 1 cervical intraepithelial neoplasia, 63.5% in grade 2, 97.2% in grade 3 and 92% in cervical cancer cases. HPV 16 and HPV 18 were detected in 80.6% of grade 3 cervical intraepithelial neoplasia and 86.5% of cervical cancer cases. 94.7% of the cervical cancer and 84.4% of the high grade lesions with a strong and full thickness staining for p16(INK4a) were positive for HPV infection; p16(INK4a) immunoexpression increased with worsening grade of cervical intraepithelial neoplasia. The HPV genotyping data showing a high HPV 16 and 18 prevalence in cancer specimens indicate that prophylactic HPV 16/18 vaccination would have a significant impact on the prevention of cervical cancer in India.

ΔNp73α, a dominant-negative inhibitor of p53 and p73, exhibits antiapoptotic and transforming activity in in vitro models and is often found to be upregulated in human cancers. The mechanisms involved in the regulation of ΔNp73α protein levels in normal and cancer cells are poorly characterized. Here, we show that that IκB kinase beta (IKKβ) increases ΔNp73α protein stability independently of its ability to activate NF-κB. IKKβ associates with and phosphorylates ΔNp73α at serine 422 (S422), leading to its accumulation in the nucleus, where it binds and represses several p53-regulated genes. S422A mutation in ΔNp73α abolished IKKβ-mediated stabilization and inhibition of p53-regulated gene expression. Inhibition of IKKβ activity by chemical inhibitors, overexpression of dominant-negative mutants, or gene silencing by siRNA also resulted in ΔNp73α destabilization, which under these conditions was rapidly translocated into the cytoplasm and degraded by a calpain-mediated mechanism. We also present evidence for the IKKβ and ΔNp73α cross talk in cancer-derived cell lines and primary cancers. Our data unveil a new mechanism involved in the regulation of the p73 and p53 network.

Detection and localization of Hepatitis C Virus (HCV) in liver tissue is useful for diagnostic purposes as well as to elucidate the mechanisms by which the virus participates in hepatocarcinogenesis. However, so far, a sensitive method for HCV detection at the cellular level is lacking. We describe here the application of a novel antibody, D4.12.9, developed against serum-derived HCV RNA-positive particles, for the detection of E2 proteins by immunohistochemistry in fixed, archived specimens including liver biopsies of HCV-infected patients and surgical specimens of hepatocellular carcinoma. We demonstrate that D4.12.9 is a powerful tool for sensitive and specific detection of HCV, independently of viral genotype. This approach has applications to diagnosis as well as exploratory pathological studies.

Exposure to aristolochic acid (AA, IARC Group 1 carcinogen) present in some traditional herbal medicines leads to aristolochic acid nephropathy (AAN), often complicated by development of multiple urothelial carcinomas of sequential onset. We used genome-scale mutational signature analysis of multiple urinary tract tumors of AAN cases from a unique patient group to determine the relationships of the patients’ late-onset second cancers to the AA exposure as well as to the first cancers. Aristolactam-DNA adduct-positive AAN patients (n=4) who developed cancer within 8 years following the initial exposure to AA were chosen for analysis of their first cancers (upper tract urothelial carcinomas, UTUC) and second cancers of delayed onset (1-9 years after first-cancer diagnosis, involving the bladder or ureteral meatus). All patients had received a kidney transplant before developing the second cancers and had a functional renal graft prior to prophylactic nephroureterectomy. Genomic DNAs were isolated from FFPE sections of the renal cortex and from the upper and lower tract tumors of each patient using laser capture micro-dissection or macro-dissection of the tumor areas. Low-coverage (~15x) exome 100-bp paired-end sequencing was performed using Illumina HiSeq2500. Customized variant calling identified somatic variants absent in non-tumor tissues. Non-negative matrix factorization was applied to extract mutational signatures in the tumor tissues. In all cases, we established the mutational signature of AA (the COSMIC signature 22) in the first UTUC as well as second cancers involving the bladder or lower ureter (meatus). Additionally, the first and second cancers harbored considerable overlaps in exposure-specific (A>T) somatic mutations. This finding provides evidence that the delayed onset of bladder urothelial carcinomas in AAN patients is likely due to distal seeding of cancer cells originating from the primary UTUC tumors. Our first-of-its-kind study addresses the risk as well as mechanistic factors leading to the second, late-onset bladder urothelial carcinomas following kidney transplantation and primary UTUC development. Our results underline the importance of long-term bladder follow-up in high-risk populations with established or suspected AA exposure. Funding: IARC; NYU Genome Technology Center is partially supported by the NIH/NCI (P30CA016087) grant. Citation Format: Xavier Castells, Maude Ardin, Sandrine Rorive, Nilufer Broeders, Yan Song, Stephanie Villar, Christine Carreira, Pierre-Paul Bringuier, Adriana Heguy, Thierry Quackels, Thierry Roumeguere, Joelle Nortier, Jiri Zavadil. Tracking the genetic relationship between first and late-onset second urothelial cancers by mutational signature analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5738. doi:10.1158/1538-7445.AM2017-5738

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.

Background The possible role of viruses in breast cancer etiology remains an unresolved question. We hypothesized that if some viruses are involved, it may be in a subgroup of breast cancers only. Epidemiological arguments drove our interest in breast cancer subgroups that are more frequent in Africa, namely inflammatory breast cancer (IBC) and triple-negative breast cancer. We tested whether viral prevalence was significantly higher in these subgroups. Materials and Methods One hundred fifty-five paraffin-embedded malignant breast tumors were randomly selected at the pathology laboratory of the University Hospital of Annaba (Algeria) to include one third of IBC and two thirds of non-IBC. They were tested for the presence of DNA from 61 viral agents (46 human papillomaviruses, 10 polyomaviruses, and 5 herpesviruses) using type-specific multiplex genotyping assays, which combine multiplex PCR and bead-based Luminex technology. Results Viral DNA was found in 22 (17.9%) of 123 tumors. The most prevalent viruses were EBV1 and HPV16. IBC tumors carried significantly more viruses (any type) than non-IBC tumors (30% vs. 13%, p

Human papillomavirus (HPV) infections have been implicated in lung carcinogenesis, but causal associations remain uncertain. We evaluated a potential causal role for HPV infections in lung cancer through an analysis involving serology, tumor DNA, RNA, and p16 protein expression. Association between type-specific HPV antibodies and risk of lung cancer was examined among 3,083 cases and 4,328 controls in two case–control studies (retrospective) and one nested case–control study (prospective design). Three hundred and thirty-four available tumors were subjected to pathologic evaluation and subsequent HPV genotyping following stringent conditions to detect all high-risk and two low-risk HPV types. All HPV DNA-positive tumors were further tested for the expression of p16 protein and type-specific HPV mRNA. On the basis of the consistency of the results, although HPV11 and HPV31 E6 antibodies were associated with lung cancer risk in the retrospective study, no association was observed in the prospective design. Presence of type-specific antibodies correlated poorly with the presence of the corresponding HPV DNA in the tumor. Although nearly 10% of the lung tumors were positive for any HPV DNA (7% for HPV16 DNA), none expressed the viral oncogenes. No association was observed between HPV antibodies or DNA and lung cancer survival. In conclusion, we found no supportive evidence for the hypothesized causal association between HPV infections and lung cancer. Cancer Res; 74(13); 3525–34. ©2014 AACR.

Role of human papillomavirus (HPV) in laryngeal carcinoma remains controversial. The aim of this study is to evaluate the role of HPV in laryngeal squamous cell carcinoma by determining presence of markers of viral infection. HPV DNA and E6*I mRNA status was determined by type-specific E7 PCR bead-based multiplex genotyping and RT-PCR assays in laryngeal squamous cell carcinoma biopsy samples. p16(INK4a) and COX-2 expression was determined by immunohistochemistry. Four cases out of 32 (13%) were HPV DNA+: HPV 11 (n = 1), HPV 31 (n = 3), HPV 59 (n = 1). One double infection: HPV 11 and HPV 31. p16(INK4a) was overexpressed in three cases (9%) and COX-2 in 17 cases (53%). Two of four HPV DNA+ samples had E6*I mRNA for HPV 31 and overexpressed p16(INK4a) and COX-2. HPV appears to play an active role in a small subset of laryngeal squamous cell carcinoma. p16(INK4a) can be used as a surrogate marker of transcriptionally active HPV infection; COX-2 expression had no correlation with HPV DNA and/or RNA positivity.

Human papillomavirus (HPV) is causally implicated in a subset of cancers of the upper aero-digestive tract (UADT). Methods Associations between type-specific HPV antibodies were examined among 1496 UADT cancer case subjects and 1425 control subjects by estimating odds ratios (ORs) in logistic regression analyses adjusted for potential confounders. The agreement between serology and tumor markers of HPV infection, including presence of HPV DNA and p16 expression, were examined in a subset of tumors. Results HPV16 L1 seropositivity was associated with increased risk of oral cavity and oropharyngeal cancer (OR = 1.94, 95% confidence interval [CI] = 1.03 to 3.65; OR = 8.60, 95% CI = 5.21 to 14.20, respectively). HPV16 E6 antibodies were present in 30.2% of oropharyngeal case subjects and only 0.8% of control subjects (OR = 132.0, 95% CI = 65.29 to 266.86). Combined seropositivity to HPV16 E6 and E7 was rare (n = 1 of 1425 control subjects). An agreement of 67% was observed between HPV16 E6 serology and the corresponding presence of an HPV-related cancer: four of six HPV DNA-positive/p16-overexpressing tumors were HPV16 E6 antibody positive. An HPV16 independent association was observed for HPV18 and oropharyngeal cancer (OR = 8.14, 95% CI = 2.21 to 29.99 for HPV18 E6 seropositivity) and HPV6 and laryngeal cancer (OR = 3.25, 95% CI = 1.46 to 7.24 for HPV6 E7 seropositivity). Conclusions These results confirm an important role for HPV16 infection in oropharyngeal cancer. HPV16 E6 antibodies are strongly associated with HPV16-related oropharyngeal cancers. Continuing efforts are needed to consider both HPV serology and p16 staining as biomarkers relevant to the etiology and natural history of HPV16-related oropharyngeal tumors. These results also support a marginal role for HPV18 in oropharyngeal cancer and HPV6 in laryngeal cancer

Mutations of the MEN1 tumour suppressor gene predispose patients to the development of multiple endocrine neoplasia type 1 (MEN1) syndrome, which is characterized by multiple endocrine tumours, including prolactinomas. The recent findings of the interaction between menin, encoded by the MEN1 gene, and the oestrogen receptor, as well as the observation of rare cases of mammary carcinomas in our heterozygous Men1 mutant mice, led us to investigate a putative tumour suppressor function of the Men1 gene in mouse mammary cells by disrupting the gene in luminal epithelial cells. A significantly higher incidence of mammary intraepithelial neoplasia (MIN) was observed in mutant WapCre-Men1(F/F) mice (51.5%) than in WapCre-Men1(+/+) (0%) or Men1(F/F) (7.1%) control mice. The majority of MIN observed in the mutant mice displayed complete menin inactivation. Because of the leakage of WapCre transgene expression, prolactinomas were observed in 83.3% of mutant mice, leading to premature death. As there was no correlation between MIN development and elevated serum prolactin levels, and phospho-STAT5 expression was decreased in mammary lesions, the increased incidence of MIN lesions was most likely due to Men1 disruption rather than to prolactinoma development. Interestingly, in MIN lesions, we found a decrease in membrane-associated E-cadherin and beta-catenin expression, the latter of which is a menin partner. Finally, reduced menin expression was found in a large proportion of two independent cohorts of patients with breast carcinomas. Taken together, the current work indicates a role of Men1 inactivation in the development of mammary pre-cancerous lesions in mice and a potential role in human mammary cancer.

Mutations of the multiple endocrine neoplasia type 1 (MEN1) gene predispose patients to MEN1 that affects mainly endocrine tissues, suggesting important physiological functions of the gene in adult endocrine cells. Homozygous disruption of Men1 in mice causes embryonic lethality, whereas the eventual involvement of the gene in embryonic development of the endocrine cells remains unknown. Here, we show that homozygous Men1 knockout mice demonstrate a reduced number of glucagon-positive cells in the E12.5 pancreatic bud associated with apoptosis, whereas the exocrine pancreas development in these mice is not affected. Our data suggest that menin is involved in the survival of the early pancreatic endocrine cells during the first developmental transition. Furthermore, chimerism assay revealed that menin has an autonomous and specific effect on the development of islet cells. In addition, using pancreatic bud culture mimicking the differentiation of α- and β-cells during the second transition, we show that loss of menin leads to the failure of endocrine cell development, altered pancreatic structure and a markedly decreased number of cells expressing neurogenin 3, indicating that menin is also required at this stage of the endocrine pancreas development. Taken together, our results suggest that menin plays an indispensable role in the development of the pancreatic endocrine cells.

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

Background Mutations of the MEN1 gene predispose to multiple endocrine neoplasia type 1 (MEN1) syndrome. Our group and others have shown that Men1 disruption in mice recapitulates MEN1 pathology. Intriguingly, rare lesions in hormone-dependent tissues, such as prostate and mammary glands, were also observed in the Men1 mutant mice. Methods To study the occurrence of prostate lesions, we followed a male mouse cohort of 47 Men1 +/- mice and 23 age-matched control littermates, starting at 18 months of age, and analysed the prostate glands from the cohort. Results Six Men1 +/- mice (12.8%) developed prostate cancer, including two adenocarcinomas and four in situ carcinomas, while none of the control mice developed cancerous lesions. The expression of menin encoded by the Men1 gene was found to be drastically reduced in all carcinomas, and partial LOH of the wild-type Men1 allele was detected in three of the five analysed lesions. Using immunostaining for the androgen receptor and p63, a basal epithelial cell marker, we demonstrated that the menin-negative prostate cancer cells did not display p63 expression and that the androgen receptor was expressed but more heterogeneous in these lesions. Furthermore, our data showed that the expression of the cyclin-dependent kinase inhibitor CDKN1B (p27), a Men1 target gene known to be inactivated during prostate cell tumorigenesis, was notably decreased in the prostate cancers that developed in the mutant mice. Conclusion Our work suggests the possible involvement of Men1 inactivation in the tumorigenesis of the prostate gland.