Identification of genomic predictors of non-melanoma skin cancer in solid organ transplant recipients

ARTICLE

Auteur(s) : Charis Kepron¹, Patricia Reis², Rikki Bharadwaj², James Shaw³, Suzanne Kamel-Reid^1,2,4, Danny Ghazarian^1,4

¹Department of Laboratory Medicine and Pathobiology, University of Toronto, Room 110, 100 College Street, Toronto, Ontario M5G 1L5, Canada
²Department of Cellular and Molecular Biology, University of Toronto, Princess Margaret Hospital, Room 9-620, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada
³Department of Medicine, Division of Dermatology, University of Toronto, Women’s College Hospital, 10^th floor Dermatology Clinic, 76 Grenville Street, Toronto, Ontario M5S 1B2, Canada
⁴Department of Pathology, University Health Network, Toronto General Hospital, Room 11E205, 200 Elizabeth Avenue, Toronto, Ontario M5G 2C4, Canada

With advances in transplant medicine, the long-term survival of solid-organ transplant recipients (SOTR) is increasing [1]. The drawback to this improved survival is a corresponding increase in the rates of complications such as post-transplant malignancy [1]. The most common tumors to occur in these patients are non-melanoma skin cancers (NMSC) which have a more aggressive clinical course than those seen in non-transplant patients, with increased risks of recurrence, metastases and death [1]. Immunosuppressive drugs appear to contribute to the development of tumors, however genetics also play a role since not all SOTRs will go on to develop NMSC. Although prior studies have examined the mechanisms underlying the formation of NMSC [2], no studies have yet focused on the molecular genetics of cancers in SOTRs.

With informed consent obtained according to the guidelines laid out by the hospital Research Ethics Board, skin tumors were obtained at the time of resection from the University Health Network Division of Dermatology, Toronto, Canada. Tissue was snap-frozen and stored at -80°C until RNA extraction and microarray analysis. Nodular BCCs from 8 SOTRs and 4 non-transplant patients (NTPs) were compared using the Human Genome U133A 2.0 plus GeneChip^® arrays (Affymetrix, Santa Clara, USA), which contain 14,500 unique genes. Non-tumorous “normal” skin was also sampled from areas adjacent to the tumours in order to control for changes in gene expression secondary to immunosuppressive medications. Data analysis using unsupervised hierarchical clustering showed distinct clustering of tumors from transplanted and non-transplanted patients. Differentially expressed genes between tumors from non-transplanted compared to transplanted patients were identified using Significance Analysis of Microarrays (SAM) and then validated using quantitative real-time PCR (QRT-PCR) as per previously published protocols [3].

SAM identified 123 under- and 29 over-expressed genes between the distinct sample clusters. Following pathways analysis and PCR validation, four genes were found to have significant differences in expression levels between BCCs and normal skin from SOTRs; GAL, EBP, MMP3, and cEBPα. The GAL gene was also found to be significantly overexpressed in tumours from SOTRs as compared to NTPs (table 1). While cEBPα showed a decrease in expression in tumors from SOTRs as compared to NTPs, this difference did not reach statistical significance.

In this preliminary analysis, three of the four genes found to have significant differences in gene expression between normal skin and BCCs in SOTRs are involved in cholesterol biosynthesis (EBP), inhibition of cell cycle progression (cEBPα), and the breakdown of extracellular matrix (MMP3). In addition, expression of the gene GAL, coding for the neuropeptide galanin, was significantly decreased in BCCs from SOTRs as compared to normal skin, but was increased in BCCs from SOTRs as compared to those from NTPs. Decreased expression of galanin in BCC as compared to normal skin has been previously reported [4], however the role played by this peptide in skin tumours has yet to be defined. MMP3 is known to be overexpressed in BCCs [5], a finding confirmed in this study, however alterations in the expression levels of EBP and cEBPα have not yet been described in BCCs. In order to further elucidate genetic alterations in skin tumours from SOTRs, additional samples are being collected which will allow for further microarray analyses as well as PCR validation of other genes identified by Pathways analysis. In addition to BCCs, squamous cell carcinomas from our two patient groups will also be investigated. Similar to basic science models in other areas of dermatologic research that have led to direct clinical applications [6], it is our hope that validated genes will lead to a better understanding of the mechanism underlying the formation of NMSC and ultimately identify biomarkers for diagnosis and gene targets for therapy.

Acknowledgements

References

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