Pilot study of VHL mutation analysis to predict response to interleukin-2 in patients with metastatic renal cell carcinoma

Abstract

14539 Background: More than 300 different mutations have been described in von Hippel-Lindau (VHL) families and nearly 100 in sporadic RCC. In familial cases, there is an excess of mutations in exon 3 and exon 1, whereas mutations are evenly distributed in sporadic cases. Recent evidence suggests the importance of genotype/phenotype correlations with different VHL gene mutations contributing to a tumor’s unique biological behavior. We performed a pilot study to compare the incidence and type of VHL mutation in IL-2 complete responders (CR) and non-responders (NR). Methods: Genomic DNA was extracted from 20 frozen clear cell tumour samples (10 CR and 10 NR) using Qiamp DNA mini kits (Qiagen). Four amplimers covering the entire coding sequence and three exon/intron junctions of the VHL gene were synthesized by PCR. PCR products were subsequently purified and were sequenced using Big Dye Primer Cycle Sequencing kit (Perkin-Elmer Applied Biosystems) and an automated DNA sequencer (Perkin-Elmer Applied Biosystems). Point mutation detection was performed on DNA sequencing gels from colored peak chromatograms. Mutation bearing sequences were confirmed by a second round of PCR and sequencing. Results: In the CR patients, only 2 out of 10 samples (20%) had evidence of a VHL mutation. These VHL mutations included a false sense mutations in exon 2, comprised of a C to T transition at position 646 in the cDNA, creating a stop codon, and a frameshift mutation in exon 1 introducing in one allele an abnormal stop codon at the beginning of exon 3 potentially leading to a shorter protein product. In the 10 non-responder samples, there was also a 2 out of 10 (20%) incidence of VHL mutations. These mutations included a complex 12 nucleotide deletion in the third exon in the a domain of the protein, and a G deletion in the first exon introducing a premature stop codon. Conclusions: In this small pilot study of metastatic RCC patients, there were relatively few VHL gene mutations, and VHL mutations did not appear to correlate with response to IL-2 therapy. This study, however, does not assess inactivation of VHL genes due to hypermethylation. An expanded genetic profile to evaluate IL-2 responders versus non-responders using 500k SNP Chips is underway. No significant financial relationships to disclose.