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702 Epigenomics of penile squamous cell carcinoma
Title
702
Epigenomics of penile squamous cell carcinoma Eur Urol Suppl 2015;14/2;e702
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Feber A. 1 , Arya M. 2 , De Winter P. 3 , Muhammad S. 4 , Nigam R.4 , Malone P.R. 4 , Tan W.S.3 , Rodney S. 3 , Lechner M. 1 , Freeman A. 5 , Jameson C.5 , Muneer A. 4 , Beck S. 1 , Kelly J.D. 3 1 UCL
Cancer Institute, Dept. of Cancer Biology, London, United Kingdom, 2 Queen Mary University of London, Barts Cancer Institute,
London, United Kingdom, 3 University College London, Dept. of Surgery & Interventional Science, London, United Kingdom, 4 University College London Hospital, Dept. of Urology, London, United Kingdom, 5 University College London Hospital, Dept. of Histopathology, London, United Kingdom INTRODUCTION & OBJECTIVES: The incidence of penile cancer (PeCa) in western nations is relatively low, 0.5-0.8/100,000 in the UK and USA. This contrasts with developing nations where incidence rates vary from 2.3-8/100,000. Other than HPV driven transformation little is known about the molecular genetic alterations defining the development of PeCa. We sought to identify the somatic alterations driving the tumourigenesis of PeCa, by performing whole exome sequencing (WES) of 27 PeCa samples. MATERIAL & METHODS: Sample Cohort. Informed consent was obtained from all subjects and ethical approval for this study was granted by the University College London (UCL) / University College London Hospital (UCLH) BioBank for studying Health and Human Disease (NC06.11). Whole-exome sequencing. Sequencing was performed using the Illumina TrueSeq or Nextrea exome capture kit according to the manufactures instructions and sequenced on a Hi-Seq2000 (Illumina). Reads were aligned to the human reference genome (build NCBI 37) using the Burrows-Wheeler Aligner (BWA) . Varscan and Annoar were used to identify and annotate somatic alterations. GPS1 mutations. Constructs expressing Flag-tagged wild-type GPS1 were a kind gift from Ning Wei (Yale University). Mutations in GPS1 were made using the Q5® Site-directed Mutagenesis kit (NEB). miRNA reporter assays. Dual-Luciferase® Reporter Assay system (Promega) was used to assess miRNA repression with both Let7 and mir100 reporter assays. RESULTS: WES revealed 810 genes containing somatic mutations among the 27 tumours, with a mean somatic mutation rate of 30 per sample, representing 1.78 non-silent mutations per megabase (range 0.72-7.5). There was no significant association between mutational burden and tumour stage, grade or age, high viral load tumours had a significantly lower mutational load (P<0.05) when compared to HPV negative. HPV high disease had significant enrichment TCW alterations, consistent with HPV induced APOBEC mutations. HPV low and negative disease had a significant enrichment of C>T alterations at CG dinucleotides, which correlates with a significant (P< 0.001) decrease in DNA methylation when compared to high viral load samples and matched normal tissue. Of the 810 mutated genes, 137 (17%) were recurrent alterations; 756(93%) have previously been identified as mutated in other tumour types. Of the recurrent mutations, TP53, FAT1 and the G-protein pathway suppressor CNS1(GPS1) were validated in an independent cohort of 70 PeCa samples. Of the novel genes identified, CSN1, was mutated in 11% and 14% of cases respectively. Recapitulation of CSN1 mutations resulted in the translocation of CSN1 to the cytoplasm and co-localization with AGO1 and AGO2-positive P-bodies and resulted in a significant inhibition of miRNA-mediated gene repression. CONCLUSIONS: This study represents the most comprehensive analysis of the genetic alterations in PeCa thus far. PeCa appears mutationally quiet, suggesting that penile cancer development is driven by an intricate pattern of alterations. The identification of a strong
file:///S|/IM/EURSUP/2015%20EAU%20Abstracts/content/data/702.html[19/02/2015 08:18:59]
Title CpG mutation signature in HPV negative tumours may suggest that changes to the epigenome (directly or indirectly) represent the major mechanism in the development in this disease. Furthermore, the identification of CSN1 mutation, and its role in aberrant miRNA processing, suggests aberrant miRNA regulation may play a key role in PeCa pathogenesis.
file:///S|/IM/EURSUP/2015%20EAU%20Abstracts/content/data/702.html[19/02/2015 08:18:59]
702
Epigenomics of penile squamous cell carcinoma Eur Urol Suppl 2015;14/2;e702
Print! Print!
Feber A. 1 , Arya M. 2 , De Winter P. 3 , Muhammad S. 4 , Nigam R.4 , Malone P.R. 4 , Tan W.S.3 , Rodney S. 3 , Lechner M. 1 , Freeman A. 5 , Jameson C.5 , Muneer A. 4 , Beck S. 1 , Kelly J.D. 3 1 UCL
Cancer Institute, Dept. of Cancer Biology, London, United Kingdom, 2 Queen Mary University of London, Barts Cancer Institute,
London, United Kingdom, 3 University College London, Dept. of Surgery & Interventional Science, London, United Kingdom, 4 University College London Hospital, Dept. of Urology, London, United Kingdom, 5 University College London Hospital, Dept. of Histopathology, London, United Kingdom INTRODUCTION & OBJECTIVES: The incidence of penile cancer (PeCa) in western nations is relatively low, 0.5-0.8/100,000 in the UK and USA. This contrasts with developing nations where incidence rates vary from 2.3-8/100,000. Other than HPV driven transformation little is known about the molecular genetic alterations defining the development of PeCa. We sought to identify the somatic alterations driving the tumourigenesis of PeCa, by performing whole exome sequencing (WES) of 27 PeCa samples. MATERIAL & METHODS: Sample Cohort. Informed consent was obtained from all subjects and ethical approval for this study was granted by the University College London (UCL) / University College London Hospital (UCLH) BioBank for studying Health and Human Disease (NC06.11). Whole-exome sequencing. Sequencing was performed using the Illumina TrueSeq or Nextrea exome capture kit according to the manufactures instructions and sequenced on a Hi-Seq2000 (Illumina). Reads were aligned to the human reference genome (build NCBI 37) using the Burrows-Wheeler Aligner (BWA) . Varscan and Annoar were used to identify and annotate somatic alterations. GPS1 mutations. Constructs expressing Flag-tagged wild-type GPS1 were a kind gift from Ning Wei (Yale University). Mutations in GPS1 were made using the Q5® Site-directed Mutagenesis kit (NEB). miRNA reporter assays. Dual-Luciferase® Reporter Assay system (Promega) was used to assess miRNA repression with both Let7 and mir100 reporter assays. RESULTS: WES revealed 810 genes containing somatic mutations among the 27 tumours, with a mean somatic mutation rate of 30 per sample, representing 1.78 non-silent mutations per megabase (range 0.72-7.5). There was no significant association between mutational burden and tumour stage, grade or age, high viral load tumours had a significantly lower mutational load (P<0.05) when compared to HPV negative. HPV high disease had significant enrichment TCW alterations, consistent with HPV induced APOBEC mutations. HPV low and negative disease had a significant enrichment of C>T alterations at CG dinucleotides, which correlates with a significant (P< 0.001) decrease in DNA methylation when compared to high viral load samples and matched normal tissue. Of the 810 mutated genes, 137 (17%) were recurrent alterations; 756(93%) have previously been identified as mutated in other tumour types. Of the recurrent mutations, TP53, FAT1 and the G-protein pathway suppressor CNS1(GPS1) were validated in an independent cohort of 70 PeCa samples. Of the novel genes identified, CSN1, was mutated in 11% and 14% of cases respectively. Recapitulation of CSN1 mutations resulted in the translocation of CSN1 to the cytoplasm and co-localization with AGO1 and AGO2-positive P-bodies and resulted in a significant inhibition of miRNA-mediated gene repression. CONCLUSIONS: This study represents the most comprehensive analysis of the genetic alterations in PeCa thus far. PeCa appears mutationally quiet, suggesting that penile cancer development is driven by an intricate pattern of alterations. The identification of a strong
file:///S|/IM/EURSUP/2015%20EAU%20Abstracts/content/data/702.html[19/02/2015 08:18:59]
Title CpG mutation signature in HPV negative tumours may suggest that changes to the epigenome (directly or indirectly) represent the major mechanism in the development in this disease. Furthermore, the identification of CSN1 mutation, and its role in aberrant miRNA processing, suggests aberrant miRNA regulation may play a key role in PeCa pathogenesis.
file:///S|/IM/EURSUP/2015%20EAU%20Abstracts/content/data/702.html[19/02/2015 08:18:59]