Determination of loss of heterozygosity in frozen and paraffin embedded tumors by denaturating high-performance liquid chromatography (DHPLC)

Spontaneous renal cell carcinoma (RCC) occurs with a high frequency in Eker rats carrying a germline alteration of the tuberous sclerosis-2 (Tsc-2) tu mor suppressor gene. To determine the frequency with which the wild-type al lele of the Tsc-2 gene is lost in RCC and the ability of DHPLC to detect lo ss of heterozygosity (LOH) at this gene locus, fresh-frozen and paraffin-em bedded formalin-fixed tumors from heterozygous Eker rats (Tsc-2(E lambda/+) ) were examined for LOH at the Tsc-2 locus. LOH was determined by quantitat ion of peak areas of PCR products specific for the mutant and wild-type Tsc -2 alleles. For normal DNA isolated from heterozygous animals, the allele r atio (AR) of mutant to wild-type PCR products war empirically determined to be 1.5+/-0.3 (n = 30) and LOH was defined as > 2 standard deviations away from this mean, i.e. any AR > 2.1. Analysis of 15 spontaneous frozen RCC sa mples showed LOH in 10/15 samples (66%). Carcinogen-induced tumors exhibite d an even higher frequency of LOH, with 6/6 paraffin-embedded. formalin-fix ed tumors exhibiting LOH. 100% concordance was observed between the results obtained by DHPLC and traditional methodologies. Therefore, LOH appears to occur with a high frequency in both spontaneous and carcinogen-induced RCC in this animal model and DHPLC is a sensitive and high throughput methodol ogy for detecting this type of genetic alteration. (C) 2001 Published by El sevier Science B.V.