SDHB and SDHD Mutation Analysis in Renal Oncocytomas and Chromophobe Renal Cell Carcinomas

1. SDHB and SDHD Mutation Analysis in Renal Oncocytomas and Chromophobe Renal Cell Carcinomas Joanne Ramsay CMGS Spring Conference 2010

2. Renal Cell Cancer • Renal cell cancer (RCC) is a heterogeneous disease that can be classified into several subtypes • Around 2-3% of all RCC cases are familial • There are several genes reported to be associated with familial RCCs including • VHL • FLCN (Birt Hogg Dube) • FH (HLRCC) • and more recently SDHB (no specific subtype)

3. Renal Oncocytoma • Oncocytoma can occur in the thyroid gland, salivary gland, adrenal gland and the kidney • Renal oncocytomas (ROs) are benign epithelial tumours that arise from the intercalated cells of the renal collecting duct • The main characteristics of ROs is the accumulation of mitochondria in the cell cytoplasm and their deficiency in electron transport chain complex I • Loss of chromosomes 1 and 14 common but ROs are not associated with mutations in any one gene

4. Chromophobe RCC • Chromophobe RCC (CRCC) and RO have overlapping morphologies • CRCC can be malignant but low mortality • Hybrid tumours have been described • More significant chromosome loses • Both CRCC and RO have been reported in patients with SDHB mutations Renal Oncocytoma Chromophobe RCC

5. Succinate Dehydrogenase • SDH is a nuclear encoded mitochondrial enzyme • 4 subunits, A B C D • SDH acts in Kreb’s cycle to convert succinate to fumarate • Also acts as complex II in the electron transport chain • SDHB/SDHC/SDHD have tumour suppressor properties • Biallelic SDHA mutations cause the genetically heterogeneous Leigh syndrome

6. SDH Associated Phenotypes • Phaeochromocytoma (PC) and Paraganglioma (PGL) • PGL extra adrenal neuroendocrine tumours • PCs are essentially PGLs of the adrenal medulla • Mutations observed in sporadic and familial cases • SDHD associated PGLs/PCs show paternal inheritance • Renal Cancer – often with PGL • No genotype/phenotype correlation to explain why some get RCC • GIST • usually in combination with PGL • Cowden Syndrome – PTEN mutations • Renal/thyroid cancer in ~5% SDH mutation carriers

7. Aims Patient with germline SDHB mutation – later developed a RO LOH of normal allele observed in RO tissue SDHB associated ROs described in literature have not been confirmed by LOH analysis of tumour tissue This is the largest cohort of sporadic RO samples screened for SDHB/SDHD mutations Hoped to establish genotype/phenotype correlations Could then offer SDHB/SDHD mutation screening to patients with a certain RCC type Offer predictive testing to at-risk family members

8. Methods • DNA from 28 FFPE RO samples and 4 CRCC samples • From sporadic cases – no FH • Normal tissue supplied for comparison • Use of these tissues for our research was approved by the Ethical Committee of the Tayside Tissue Bank • Extraction by Pathology (lysis and column clean-up) • Screened SDHB and SDHD in 13 PCR fragments using direct bi-directional sequencing • 3 markers at each locus to identify LOH – compared tumour tissue to normal tissue

9. Results - Sequencing • No mutations identified in SDHB or SDHD • Several novel “polymorphisms” identified • All deeply intronic and unlikely to be disease causing • SDHD p.His50Arg unclassified variant detected in 2 samples • Had problems with DNA quality and quantity • PCR and sequencing failures • Increased cycles and amount of template DNA • Scored some fragments as ?No mutation detected • Identified skewed polymorphism ratios that indicated LOH

10. SDHB c.-37T>C • Mutation Surveyor DNA Quantification tool detected the C peak at 71.34% • Sample was later comfirmed to have LOH

11. SDHD – p.His50Arg • Identified in both tumour and normal tissues • Unclassified variant– based on literature and in silico evidence (PolyPhen predicts damaging, others suggest benign) • Recent study (Ni et al 2008) suggests Cowden Syndrome association & did not observe variant in 700 control chromosomes • Family studies would provide best info • Maternal inheritance goes against pathogenicity • Not possible in the context of this project • Freq. of around 1% in normal controls (Spanish population) • We aim to screen 500 Scottish controls (from Generation Scotland DNA bank) • Ethnic origin of the patients in our cohort are unknown

12. Summary of Variants Detected

13. Results - LOH • No LOH observed at SDHD locus • Chromosome 11 loss not common in ROs • About 50% samples had LOH at SDHB locus • Marker D1S170 was unstable in 4 samples • In MFAP2 gene (expressed in kidney) • Instability may contribute to tumourigenesis

14. Additional work • To confirm LOH, aCGH analysis was carried out on 2 samples • To characterise loss of chromosome 1 • To show that the technique works well on FFPE tissue • Identified contamination in one sample – introduced by pathology! • Loss of 1p36 confirmed in the other sample • In process of screening Generation Scotland DNA controls for the p.H50R SDHD variant • Using TaqMan allelic discrimination protocol • Looked at 92 samples so far – p.H50R not detected • Plan to examine at least another 350 samples

15. Conclusions • This is the largest cohort of ROs that have been screened for mutations in SDHB and SDHD • No mutations identified indicating that mutations in SDHB and SDHD are not associated with sporadic ROs • Confirmed that the SDHB region 1p36 is lost in about 50% of ROs • Should examine all RCC tumour from SDH mutation carriers for LOH • Requires good communication between clinicians, pathology and genetics

16. Acknowledgements • Thanks to Michelle, Dave and everyone else in molecular • Also to the Stewart Fleming, Leslie Christie and the rest of the pathology departments in Ninewells and Kirkcaldy • And to Cytogenetics especially Gordon and Jasbani for help with aCGH work