Quantitative real-time PCR analysis and microarray-based RNA expression of HER2 in relation to outcome.

BACKGROUND: Our aim was to use quantitative real-time PCR (Q-PCR) and RNA expression profiles (RNA-EPs) to investigate HER2 status in relation to outcome. PATIENTS AND METHODS: Cut-off levels for Q-PCR and RNA-EP were established in relation to immunohistochemistry (IHC) validated by FISH in a test set of frozen tissue samples from 40 primary breast cancers. The HER2 status was subsequently studied in another validation set of 306 tumors, where Q-PCR and RNA-EP results were compared with previously carried out IHC that we had validated by chromogenic in situ hybridization (CISH). RESULTS: Q-PCR and RNA-EP offered similar sensitivity (90% versus 77%), specificity (93% versus 95%), and negative (99% versus 98%) and positive (63% versus 61%) predictive values for HER2 determinations. Analyses of relapse-free survival (RFS) and overall survival on the basis of 5 and 10 years of follow-up indicated equivalent hazard ratios for all three techniques. In contrast to IHC/CISH, both Q-PCR and RNA-EP analyses of HER2 also gave statistically significant results regarding RFS and breast cancer-corrected survival after 10 years of follow-up. CONCLUSION: The use of RNA-EP and Q-PCR to analyze HER2 in frozen and formalin-fixed breast cancer samples may be an alternate approach to IHC in combination with FISH/CISH.

A single nucleotide polymorphism in the 3'untranslated region of the CDKN2A gene is common in sporadic primary melanomas but mutations in the CDKN2B, CDKN2C, CDK4 and p53 genes are rare.

In this report we present the results of mutational analysis of the CDKN2B, CDKN2C, CDK4, p53 genes and 5'UTR of the CDKN2A gene in a set of 44 sporadic primary melanomas, which had been earlier analysed for mutations in the CDKN2A (p16/p14(ARF)) gene. No tumour-associated mutations were detected except in 1 melanoma where we found a CC>T* deletion-mutation in the codon 151-152 (exon 5) of the p53 gene. On the basis of our preliminary results, we did extended genotyping of the 500 C>G and 540 C>T polymorphisms in the 3'UTR of the CDKN2A gene in 229 melanoma cases and 235 controls. The T-allele frequency (for 540 C>T polymorphism) in melanomas was significantly higher than in controls (0.14 vs. 0.08; chi(2) = 5.95, p = 0.01; OR = 1.71, 95%CI = 1.11-2.66). The heterozygote frequency for this polymorphism was 0.26 (59/229) in melanomas compared to 0.13 (30/235) in healthy controls (chi(2) = 11.4; p = 0.0007; OR = 2.34, 95% CI = 1.40-3.92). The frequency of the 500 C>G polymorphism in the 3'UTR in the CDKN2A gene was not significantly higher in melanomas compared to healthy controls. The 500 C>G polymorphism, however, was in linkage disequilibrium with approximately 50 kb apart the C>A intronic polymorphism in the CDKN2B gene (determined in 44 melanomas and 90 controls; Fisher exact test, p<0.0001). Finally, the sequence analysis of genomic DNA isolated from T cell lymphocytes of healthy individuals exhibited that the codon reported as last of exon 2 of the CDKN2C gene is rather the first codon of exon 3.

Polymorphic insertion of additional repeat within an area of direct 8 bp tandem repeats in the 5'-untranslated region of the p53R2 gene and cancer risk.

p53R2 is a recently cloned gene that functions in p53-induced DNA repair. In the 5'-untranslated region of the p53R2 gene two direct tandem 8 bp repeats are located. Within the region of these 8 bp direct repeats we have detected the insertion of an additional repeat. In order to determine a possible association of this novel polymorphism with any cancer or population, we carried out genotyping of 843 European and Asian controls and patients with various cancer types. In addition, 26 cancer cell lines were included in the study. No significant difference in polymorphic frequency could be demonstrated for any of the cancer types, although the allelic frequency in melanoma patients was lower than in controls (chi(2) = 3.28; P = 0.07; OR = 0.32; 95% CI 0.07-1.26). A significantly higher frequency of the polymorphism was detected in the compiled Caucasian individuals compared with Asians (chi(2) = 9.19; P = 0.002; OR = 3.13; 95% CI 1.39-7.43). In one tumour cell line we observed two extra inserted copies of the 8 bp repeat. The functional effect of the insertion polymorphism on the p53R2 gene transcription remains to be determined.

Analysis of G(1)/S checkpoint regulators in metastatic melanoma.

We have analyzed the expression of the CDKN1A (p21(CIP1)), CDKN1B (p27(Kip1)), TP53, RB1 and MDM2 proteins and tumor cell proliferation by immunohistochemical staining in 59 cases of metastatic melanoma. The genomic status of the CDKN2A (INK4-ARF, p16/p14(ARF)), CDKN2B (p15) and CDKN2C (p18) genes was determined by PCR-SSCP (single-strand conformation polymorphism) in 46 of these cases. These results were correlated with various clinico-pathological parameters, including the outcome of combined chemoimmunotherapy. We found positive correlations between the expression of CDKN1A and MDM2 (r = 0.5063, P = 0.001), between the expression of CDKN1B and RB1 (r = 0.5026, P = 0.001), and between RB1 expression and tumor cell proliferation (0.5564, P<0.001). Two mutations in the CDKN2A (p16) gene were detected, including a novel base change AAC-->ATC (Asn to Ile) at codon 71, that also changes the codon 85 of the alternative reading frame gene p14(ARF) from CAA to CAT (Gln to His). Homozygous deletion at exon 2 of the CDKN2A (INK4-ARF) gene was detected in six cases. In seven cases, the 540C-->G polymorphism in the 3'UTR of the CDKN2A (p16) gene was found in linkage disequilibrium with the 74C-->A polymorphism in intron 1 of the CDKN2B gene (P < 0.0001). These cases had significantly lower expression of the TP53 protein (P = 0.0032). Both 540C-->G and 580C-->T polymorphisms in the 3'UTR of the CDKN2A (p16) gene were associated with significantly shorter progression time from primary to metastatic disease (P = 0.0071). We conclude, that although none of the analyzed cell cycle regulators could be singled out as a major prognostic factor, G(1)/S checkpoint abnormalities remain one of the most significant factors in the development of malignant melanoma.

Increased frequency of LOH on chromosome 9 in sporadic primary melanomas is associated with increased patient age at diagnosis.

We carried out statistical analysis of the frequency of loss of heterozygosity (LOH) at 10 microsatellite markers on chromosome 9. In 44 microdissected sporadic primary melanomas a comparison of LOH frequency data with other patient data, like age at diagnosis and tumour thickness, showed an interesting correlation between patient age at diagnosis and frequency of LOH on chromosome 9. The patient group with age >72 years at diagnosis (n = 22, mean age 82.3 +/- 6.0 years, mean LOH 3.4 +/- 2.3) showed significantly increased LOH frequency (OR 3.1, 95% CI 1.8-5.3; chi(2) test, P < 0.0001) compared with age group

Loss of heterozygosity at chromosome 9p21 (INK4-p14ARF locus): homozygous deletions and mutations in the p16 and p14ARF genes in sporadic primary melanomas.

Loss of heterozygosity (LOH) was determined in 45 sporadic primary melanomas at six polymorphic microsatellite markers that flank the INK4a (p16-p14ARF) locus on chromosome 9p21. We also determined allelic loss at two markers on chromosome 9q and two markers at the Rb locus on chromosome 13. Homozygous deletion of the p16 and p14ARF genes was determined by a fluorescent-based quantitative multiplex polymerase chain reaction method. LOH at one or more polymorphic microsatellite markers on locus 9p21 was found in 32 of the melanomas (71%). The highest proportion of LOH was found at markers D9S736 and D9S104, which are telomeric and centromeric to the INK4 locus, respectively. Five melanomas showed LOH at all the analysed markers located on chromosome 9p21. LOH at markers D9S942 and D9S974, which are located close to the p16 and p14ARF genes, was found in 39% and 46% of melanomas, respectively. Analysis of the marker D9S257 on 9q22.1 showed LOH in 13 melanomas (44% of the informative cases). A subset of melanomas with LOH at the INK4 locus also carried inactivating mutations within the p16 coding sequence. Four melanomas carried homozygous deletions at the p16-p14ARF locus. Our results suggest, besides the involvement of the INK4 locus in sporadic melanomas, the possibility of the existence of additional tumour suppressor loci on chromosome 9.