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) tumor suppressor gene. To determine the frequency with which the wild-type allele of the Tsc-2 gene is lost in RCC and the ability of DHPLC to detect loss of heterozygosity (LOH) at this gene locus, fresh-frozen and paraffin-embedded formalin-fixed tumors from heterozygous Eker rats (Tsc-2(Ek/+)) were examined for LOH at the Tsc-2 locus. LOH was determined by quantitation 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 ratio (AR) of mutant to wild-type PCR products was 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 samples showed LOH in 10/15 samples (66%). Carcinogen-induced tumors exhibited an even higher frequency of LOH, with 6/6 paraffin-embedded, formalin-fixed 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 methodology for detecting this type of genetic alteration.