Genetic polymorphism of the OPG gene associated with breast cancer.

BACKGROUND: The receptor activator of NF-κB (RANK), its ligand (RANKL) and osteoprotegerin (OPG) have been reported to play a role in the pathophysiological bone turnover and in the pathogenesis of breast cancer. Based on this we investigated the role of single nucleotide polymorphisms (SNPs) within RANK, RANKL and OPG and their possible association to breast cancer risk. METHODS: Genomic DNA was obtained from Caucasian participants consisting of 307 female breast cancer patients and 396 gender-matched healthy controls. We studied seven SNPs in the genes of OPG (rs3102735, rs2073618), RANK (rs1805034, rs35211496) and RANKL (rs9533156, rs2277438, rs1054016) using TaqMan genotyping assays. Statistical analyses were performed using the χ2-tests for 2 x 2 and 2 x 3 tables. RESULTS: The allelic frequencies (OR: 1.508 CI: 1.127-2.018, p=0.006) and the genotype distribution (p=0.019) of the OPG SNP rs3102735 differed significantly between breast cancer patients and healthy controls. The minor allele C and the corresponding homo- and heterozygous genotypes are more common in breast cancer patients (minor allele C: 18.4% vs. 13.0%; genotype CC: 3.3% vs. 1.3%; genotype CT: 30.3% vs. 23.5%). No significantly changed risk was detected in the other investigated SNPs. Additional analysis showed significant differences when comparing patients with invasive vs. non-invasive tumors (OPG rs2073618) as well as in terms of tumor localization (RANK rs35211496) and body mass index (RANKL rs9533156 and rs1054016). CONCLUSIONS: This is the first study reporting a significant association of the SNP rs3102735 (OPG) with the susceptibility to develop breast cancer in the Caucasian population.

High-resolution melting analysis as a sensitive prescreening diagnostic tool to detect KRAS , BRAF , PIK3CA , and AKT1 mutations in formalin-fixed, paraffin-embedded tissues.

CONTEXT: As the availability of targeted therapies for several tumor types increases, the need for rapid and sensitive mutation screening is growing. KRAS mutations constitutively activate the RAS/RAF/mitogen-activated protein kinase (MAPK) pathway and therefore play an important role in anti-epidermal growth factor receptor therapy for patients with colorectal cancers. Mutationally activated PIK3CA and AKT1 genes are promising therapeutic targets in breast cancer. In 60% to 70% of malignant melanomas, a mutation in BRAF can be found. Thus, the blocking of the oncogenic signaling induced by this mutation is now used as treatment approach. OBJECTIVE: To establish high-resolution melting assays for routinely used predictive analyses of KRAS , AKT1 , PIK3CA , and BRAF mutations. DESIGN: High-resolution melting assays were developed by using specifically designed primers and genomic DNA isolated either from cell lines or formalin-fixed paraffin-embedded tissues, oligonucleotides, or plasmids. Melting curve analyses were performed on the LightCyler platform and mutation analyses were additionally confirmed by Sanger sequencing. RESULTS: We developed high-resolution melting assays by using genomic DNA containing the desired mutation, which enabled us to detect percentages of mutated DNA (3.1% to 12.5%) mixed in a wild-type background. Assays were evaluated by hybridization probes and/or Sanger sequencing to exclude pseudogene amplification. The high-resolution melting assays were validated with genomic DNA from different tumor entities. The concordance between Sanger sequencing and high-resolution melting was 99% for KRAS exon 2 and PIK3CA exon 20 and 100% for the remaining assays. CONCLUSIONS: High-resolution melting provides a valid and powerful tool for detecting genomic mutations efficiently.

Different apoptotic responses of RA synoviocytes depending on different genotypes of the mdm2 SNP T309G.

Mdm2 is an ubiquitin ligase, which binds p53 and blocks its function as a transcription factor in the pathway of apoptosis. Recently we have showed that the SNP mdm2 T309G has a protective effect of the minor allele in rheumatoid arthritis (RA). However, a functional impact on apoptosis by the different genotypes of the mdm2 SNP has not been investigated. Genomic DNA was obtained from 49 cell lines of synoviocytes derived from 49 patients with RA, and the mdm2 SNP309 was genotyped by polymerase chain reaction and restriction enzyme analysis. Seven cell lines were identified as homozygous for TT (major allele of mdm2 SNP309), and four as homozygous for GG (minor allele). All 11 cell lines were stimulated with 5 ng/ml of TNF alpha and 2.5 ng/ml of Il-1beta. After staining with propidium iodide (25 µg/ml) DNA fluorescence was measured with FACS; the rate of apoptosis was defined as the percentage of cells with a sub-2n DNA content (= less than haploid DNA-content). The cell-lines genotyped with mdm2 SNP 309TT showed a significantly different apoptosis rate in percent compared with GG for both conditions with stimulation (19.4 ± 3.6 vs. 26.9 ± 1.8; P = 0.02) and without stimulation by TNF alpha and Il-1beta (27.4 ± 8.8 vs. 43.9 ± 2.4; P = 0.0002). In the cell culture in vitro model RA synoviocytes homozygous for mdm2 SNP 309GG had a higher apoptotic activity compared to TT, possibly identifying a protective effect of the minor allele.

Podocalyxin-like protein 1 expression is useful to differentiate pancreatic ductal adenocarcinomas from adenocarcinomas of the biliary and gastrointestinal tracts.

Metastases of adenocarcinomas from the pancreas, liver, and gastrointestinal tract are difficult to distinguish from each other because of their similar morphological and immunohistochemical features. So far, no specific marker for pancreatic ductal adenocarcinomas has been described. Podocalyxin-like protein 1 (PODXL-1) is expressed on vascular endothelium, hematopoietic precursor cells, and renal podocytes. We found that 44% (71/160) of pancreatic ductal adenocarcinomas expressed PODXL-1 in a membranous pattern. There was no expression in intrahepatic cholangiocarcinomas (0/18, P < .001), rarely in adenocarcinomas of the extrahepatic bile ducts (1/13, P = .009), and none in duodenal adenocarcinomas (0/5, P = .070). PODXL-1 expression was seen in only 9% of hepatocellular carcinomas (5/56, P < .001), 9% (4/47, P < .001) of gastric carcinomas, 10% of esophageal adenocarcinomas (2/20, P = .003), and 6% of colonic adenocarcinomas (1/17, P = .001). When used as a differential diagnostic marker, ampullary carcinoma needs to be excluded, as 30% (6/20, P = .24) of ampullary carcinomas stain positive, especially those of the signet-ring type (3/3). Adenocarcinomas of the lung and prostate, and liver metastases of colorectal carcinomas lacked PODXL-1 expression. It is concluded that immunoreactivity for PODXL-1 favors a pancreatic origin if ampullary carcinoma is excluded.