Prognostic Significance of Chromosome 9p Deletion in Clear Cell Renal Cell Carcinoma

Abstract

The prognostic role of chromosomal copy number aberration in renal cell carcinoma (RCC) has been explored in several studies. Chromosome 9p deletion was reported as an independent prognostic factor in clear cell subtype (ccRCC). The findings from these studies initially appeared promising however they were of limited clinical applicability due to lack of standardisation of molecular techniques employed and short periods of follow-up. In addition these studies did not integrate 9p loss in well established, externally validated prognostic models to assess its impact on survival.

The current research aimed to assess the impact of somatic copy number deletion of chromosome 9p using I-FISH to allow clinical and analytical validation of previous studies.

In the current study, a variety of cytogenetic and molecular techniques was employed to assess copy number variation and zygosity status at chromosome 9p on two cohorts of patients with a minimum of eight years follow-up. A comprehensive protocol was designed for I-FISH scoring using a dual colour probe that hybridises to the CDKN2A region to facilitate further validation in future studies. There was a good degree of inter-observer agreement in the current study. Somatic copy number loss of chromosome 9p was associated with adverse histopathological features in ccRCC. It was also associated with a higher risk of recurrence and cancer-specific mortality. The integration of chromosome 9p loss improved the predictive accuracy of validated prognostic models for recurrence and cancer-specific survival respectively.

The findings from microsatellites analysis on the same cohort of cases confirmed that loss of heterozygosity involving chromosome 9p21 in particular LOH at the coding region of CDKN2A portends a worse prognosis in ccRCC in long term follow up and also validate the findings from I-FISH-based analysis of 9p deletion. Sixty percent of cases with LOH detected by microsatellites were copy number neutral (CNNLOH). A lack of sensitivity of microsatellites analysis in reference to FISH to detect copy number loss LOH was noted. Therefore, the current study has highlighted the importance of both techniques being considered complimentary, to assess copy number aberrations on chromosome 9p. The integration of the combination of copy number loss and LOH analysis at CDKN2A to prognostic models enhanced further their predictive accuracies expressed by concordance Index C.

Immunohistochemistry was employed on the same cohort to assess if copy number loss or LOH was associated with reduced protein expression by certain genes on chromosome 9p. The genes examined were those reported to be implicated in cancer progression. There was no association between copy number aberrations on chromosome 9p and levels of expression. However, the higher expression levels of ADFP and CAIX and lower levels of p14, p15 and p16 in ccRCC were in keeping with the more aggressive nature of this subtype compared to the other less common subtypes.

The array-based analysis within a different small cohort has demonstrated that copy number loss of the whole chromosome 9p is a common event in ccRCC and is more common than a segmental loss. It also validated the findings from the first cohort showing a survival advantage in patients without 9p loss.

The current research not only validates previous reports on chromosome 9p in ccRCC but also had a more robust study design, in a large cohort with the longest follow-up known worldwide to date. It highlights the role of CNV in predicting outcomes and opens the way for further studies to explore the underlying mechanisms responsible for ccRCC progression. It also justifies the need for further prospective studies assessing CNVs with zygosity status in addition to epigenetic changes and their effects on gene expression and the outcomes of surgically treated renal cell carcinoma.

Date of Award	2015
Original language	English
Supervisor	Ghulam Nabi (Supervisor) & Stewart Fleming (Supervisor)