The University of Southampton
University of Southampton Institutional Repository

Genetic alterations in potential precancerous skin conditions

Genetic alterations in potential precancerous skin conditions
Genetic alterations in potential precancerous skin conditions
Non melanoma skin cancer (NMSC) is the most common cancer worldwide. Exposure to ultraviolet radiation (UVR) causes DNA damage in keratinocytes leading to development of NMSCs and precancerous skin conditions such as actinic keratoses (AKs), Bowen’s disease (BD) and the clinically invisible p53 mutant patches/p53 immunopositive patches (PIPs). Based on published studies, the rate of progression from these precancerous skin conditions to cutaneous squamous cell carcinoma (cSCC) is variable and the highest rate is seen in AKs (up to 16%) followed by BD (3 - 5%) and the lowest rate of progression is seen in PIPs (1 cSCC for each 8,300 - 40,000 PIPs). The progression from precancerous lesions to cancer is usually accompanied by gain of molecular alterations that affect different genes. Molecular analyses of mutations in lesional DNA, using whole exome sequencing (WES) and targeted enriched sequencing, has demonstrated cSCCs harbour a high mutation burden (33 - 50 mutations/megabase (Mb)). The aim of the current work is to study the mutational status within the genome of precancerous skin conditions, namely AKs, BD and PIPs.This study was conducted in two stages. In the first stage, AKs were analysed for genetic changes (base pair alterations) using WES. Sections from formalin-fixed paraffinembedded (FFPE) AKs (N = 69) were initially characterised using histology (for dysplastic area) and immunohistochemistry (p53, beta catenin, CD4, CD8 and FOXP3 protein). DNA from the dysplastic and adjacent normal skin were isolated from 5 AK samples for WES analysis using SureSelect v5 kit (Agilent) and HiSeq 2000 sequencing system (Illumina). WES of the AKs showed that the median mutation burden within AKs was 34.5 mutations/Mb of DNA, with a median number of 1,275 (range 194 – 1,688) mutated genes per AK. In the second stage, 18 genes were prioritised for target enriched sequencing on a wider range of samples including AKs, BDs, cSCCs and PIPs based on the analysis of the WES/AK results, previous published cSCC WES data and genes mutated in other cancers in Catalogue Of Somatic Mutations In Cancer (COSMIC) database. The samples included 32 additional AKs (including 7 AK/cSCCs where a cSCC was adjoining an AK in the same histological section and considered to have arisen from the adjacent AK) and 37 BDs (including 8 BD/cSCCs where the cSCC was adjoining BD and deemed to have developed from BD), 23 PIPs, as well as corresponding non lesional skin. The target enriched sequencing was conducted using the Truseq custom amplicon kit and MiSeq sequencing system (Illumina), and demonstrated non-silent mutations in PIPs as well as in AKs, BDs and cSCCs. The more commonly mutated genes included TP53, NOTCH1, MLL2 and HMCN1. Additionally, the results showed that the genes mutated in PIPs were also mutated in the AKs and BDs, as well as in cSCCs with some identical mutations observed in the different types of lesions. Moreover, analysis of clonality showed some clonal mutations of CDKN2A, CACNA1C, MLL2 and GPR98 genes in PIPs, however, all TP53 mutations were subclonal. This suggests that in each case the TP53 mutation occurred after the initial genetic hit which initiated development of the PIP.Thus, this study has demonstrated that multiple genetic mutations within cancer related genes are present in precancerous skin lesions, including in clinically invisible PIPs that most of them are < 3,000 cells in size, and provides strong support for the “Big Bang” model of cancer in which it is estimated that subclonal evolution begins at an early stage of cancer development.
University of Southampton
Albibas, Amel Abdulsalam M.
0dccf7a0-80e6-4fd6-bad4-6d45131d6037
Albibas, Amel Abdulsalam M.
0dccf7a0-80e6-4fd6-bad4-6d45131d6037
Healy, Eugene
400fc04d-f81a-474a-ae25-7ff894be0ebd
Holloway, Judith
f22f45f3-6fc8-4a4c-bc6c-24add507037c

Albibas, Amel Abdulsalam M. (2016) Genetic alterations in potential precancerous skin conditions. University of Southampton, Doctoral Thesis, 280pp.

Record type: Thesis (Doctoral)

Abstract

Non melanoma skin cancer (NMSC) is the most common cancer worldwide. Exposure to ultraviolet radiation (UVR) causes DNA damage in keratinocytes leading to development of NMSCs and precancerous skin conditions such as actinic keratoses (AKs), Bowen’s disease (BD) and the clinically invisible p53 mutant patches/p53 immunopositive patches (PIPs). Based on published studies, the rate of progression from these precancerous skin conditions to cutaneous squamous cell carcinoma (cSCC) is variable and the highest rate is seen in AKs (up to 16%) followed by BD (3 - 5%) and the lowest rate of progression is seen in PIPs (1 cSCC for each 8,300 - 40,000 PIPs). The progression from precancerous lesions to cancer is usually accompanied by gain of molecular alterations that affect different genes. Molecular analyses of mutations in lesional DNA, using whole exome sequencing (WES) and targeted enriched sequencing, has demonstrated cSCCs harbour a high mutation burden (33 - 50 mutations/megabase (Mb)). The aim of the current work is to study the mutational status within the genome of precancerous skin conditions, namely AKs, BD and PIPs.This study was conducted in two stages. In the first stage, AKs were analysed for genetic changes (base pair alterations) using WES. Sections from formalin-fixed paraffinembedded (FFPE) AKs (N = 69) were initially characterised using histology (for dysplastic area) and immunohistochemistry (p53, beta catenin, CD4, CD8 and FOXP3 protein). DNA from the dysplastic and adjacent normal skin were isolated from 5 AK samples for WES analysis using SureSelect v5 kit (Agilent) and HiSeq 2000 sequencing system (Illumina). WES of the AKs showed that the median mutation burden within AKs was 34.5 mutations/Mb of DNA, with a median number of 1,275 (range 194 – 1,688) mutated genes per AK. In the second stage, 18 genes were prioritised for target enriched sequencing on a wider range of samples including AKs, BDs, cSCCs and PIPs based on the analysis of the WES/AK results, previous published cSCC WES data and genes mutated in other cancers in Catalogue Of Somatic Mutations In Cancer (COSMIC) database. The samples included 32 additional AKs (including 7 AK/cSCCs where a cSCC was adjoining an AK in the same histological section and considered to have arisen from the adjacent AK) and 37 BDs (including 8 BD/cSCCs where the cSCC was adjoining BD and deemed to have developed from BD), 23 PIPs, as well as corresponding non lesional skin. The target enriched sequencing was conducted using the Truseq custom amplicon kit and MiSeq sequencing system (Illumina), and demonstrated non-silent mutations in PIPs as well as in AKs, BDs and cSCCs. The more commonly mutated genes included TP53, NOTCH1, MLL2 and HMCN1. Additionally, the results showed that the genes mutated in PIPs were also mutated in the AKs and BDs, as well as in cSCCs with some identical mutations observed in the different types of lesions. Moreover, analysis of clonality showed some clonal mutations of CDKN2A, CACNA1C, MLL2 and GPR98 genes in PIPs, however, all TP53 mutations were subclonal. This suggests that in each case the TP53 mutation occurred after the initial genetic hit which initiated development of the PIP.Thus, this study has demonstrated that multiple genetic mutations within cancer related genes are present in precancerous skin lesions, including in clinically invisible PIPs that most of them are < 3,000 cells in size, and provides strong support for the “Big Bang” model of cancer in which it is estimated that subclonal evolution begins at an early stage of cancer development.

Text
PhD thesis for submission to the graduate school - Version of Record
Available under License University of Southampton Thesis Licence.
Download (10MB)

More information

Published date: November 2016

Identifiers

Local EPrints ID: 427141
URI: https://eprints.soton.ac.uk/id/eprint/427141
PURE UUID: 85f1c8ab-6867-42d1-bd45-5eb91a5fbffc
ORCID for Judith Holloway: ORCID iD orcid.org/0000-0002-2268-3071

Catalogue record

Date deposited: 03 Jan 2019 17:30
Last modified: 14 Mar 2019 01:47

Export record

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of https://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×