SUMMARY:Synovial sarcomas (SS) are characterized by a chromosomal translocation t(X;18)(p11.2;q11.2) which usually fuses the SYT gene from chromosome 18 to SSX1 or SSX2 genes on chromosome X. Also, a variant SYT-SSX4 fusion gene has recently been shown in a single SS case. In addition to these cytogenetic changes, bcl-2 expression, as assessed by immunohistochemistry, has been reported to be an almost general constitutive alteration of SS. In the present work, we analyze a series of 36 SS surgical samples (from 34 patients) by RT-PCR for the presence of the SYT-SSX1 or the SYT-SSX2 fusion transcript. The analysis was extended to SYT-SSX4 on SYT-SSX1-negative and SYT-SSX2-negative cases only. Our results showed a significant correlation between the SYT-SSX2 fusion and the monophasic SS histologic subtype. SYT-SSX1 fusion transcripts were present in both monophasic and biphasic tumors. The SYT-SSX4 fusion type was detected in a single monophasic SS. In the same series of SS cases, we also confirmed and extended the previously reported constitutive expression of bcl-2 protein, by using both immunohistochemical and western blot analysis. Moreover, we demonstrated that the BCL-2 gene is not rearranged or amplified at genomic level, indicating that the high levels of bcl-2 expression observed in SS might result from transcriptional activation of the gene and/or protein stabilization. Finally, we show that bcl-2 is not phosphorylated in tumors from patients who had been preoperatively treated with radio/chemotherapy, in tumors from untreated patients, or in an SS cell line (CME-1) after in vitro treatment with cytotoxic concentrations of DNA-damaging agents or taxanes. These data indicate that SS cells are unable to activate an apoptosis pathway involving bcl-2 phosphorylation/inactivation and may provide a possible explanation for the limited effectiveness of conventional pharmacological treatments of this tumor type. (Lab Invest 2000, 80:805-813).

IntroductIon Single-Nucleotide Polymorphisms (snps) are the most common dna variants in the human genome, accounting for about 1/1000 bp changes. more or less, 65% of the substitutions are transitions, equally represented by a/g and c/t mutations, whereas 35% are transversions, and all the a/c, a/t, c/g, and g/t variants have the same frequency.1,2 today, snps are widely investigated in several fields-that is, as prognostic markers in pharmacogenetics, in studies on susceptibility to cancer and other complex diseases, as genetic markers (tagsnps) in genome-wide scans, and in many diagnostic protocols. associated with their high frequencies, the increasing popularity of snps is due to the availability of large-scale, high-throughput genotyping methods that have allowed the description of more than 7 million common variants worldwide. 2 to date, many real-time pcr-based approaches have been used for snp-typing in large-scale studies. one of the most commonly applied is the taqman ® method, also known as the 5′ exonuclease assay, which makes use of 2 fluorescently labeled probes for allelic discrimination. the presence of allele-specific probes guarantees a high sensitivity, offering appreciable advantages in terms of time and labor savings. other classical pcr-based genotyping techniques, such as the allele-specific oligonucleotide pcr (aso-pcr) assay or the restriction fragment length polymorphism pcr (rFlp-pcr) assay, still remain the most affordable for low-budget studies. high-resolution melting (hrm) analysis was first introduced in 1997 by ririe et al. 3 as the natural extension of a realtime monitoring process of pcr reactions. in brief, melting profiles of pcr fragments describe the amount of doublestranded dna that dissociates into single-stranded dna during incubation at increasing temperatures, monitored by measuring the fluorescence emitted by an intercalating dye. in particular, melting temperature (t m ) is defined as the temperature at which half of the dna amount is denatured. the quantity of information available from melting analyses has exponentially increased with the introduction of highly saturating intercalating dyes such as lcgreen (idaho technology, inc., salt lake city, ut) or evagreen (Biotium, inc., hayward, ca), which can be added at high concentration without inhibiting pcr reactions.during the past few years, hrm has been successfully applied to mutation detection, presequence scanning, methylation studies, and snp-typing. Because of the wide use of single-nucleotide polymorphisms (snps) as markers of genetic variation, several high-throughput genotyping methods have been developed and applied during the past decades. high-resolution melting (hrm) is a very attractive, advanced, fast, and cost-effective snp genotyping technology based on the analysis of the melting profile of pcr products, using intercalating fluorescent dyes to monitor the transition from unmelted to melted dna. the authors used hrm for genotyping 215 human dna samples for snps in the aBcB1, nQo1, and slc19a1 genes ...

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Point mutations of the K-RAS gene at codon 12 are found in about 40% of cases with colorectal cancer. The diagnostic implications of the detection of these mutations and their clinical utility are still unclear. The aim of this study was to test both the feasibility of the detection of the mutated K-RAS gene in serum and its potential role in colorectal cancer detection and monitoring. Codon 12 K-RAS mutations were examined in DNA extracted from the serum of 35 patients with colorectal cancer and were compared with the K-RAS status in the corresponding primary tumor. Molecular detection was performed by the mutant-enriched PCR (ME-PCR) assay, a sensitive method capable of distinguishing a small quantity of mutated DNA in the presence of abundant wild-type DNA. The occurrence of mutations was compared with clinicopathological parameters as well as CEA and CA19.9 serum levels. We found codon 12 K-RAS mutations in the tissue of 13/35 (37%) patients. Serum mutations were detected in 5/13 (38.5%) patients with mutated K-RAS in the tissue. 26/35 (74%) patients showed an identical K-RAS pattern in tissue and serum. No codon 12 K-RAS alterations were found in serum samples of 22 patients with benign gastrointestinal diseases. Elevated serum CEA levels were detected in 16 patients, four of whom also presented serum RAS mutations. Our results confirm that K-RAS mutations can be found in circulating DNA extracted from serum samples of patients with colorectal cancer and show that there is a correspondence between serum and tissue K-RAS patterns.

The replication-error positive (RER+) phenotype characterizes tumour cells with microsatellite instability. This 'mutator phenotype' is thought to induce spread mutations throughout the genome, thus increasing the risk of tumour development. Here we analyse spontaneously arising mutations at the tetranucleotide CCGG ( Msp I recognition site), at positions 14 067-14 070 of the p53 gene sequence, in three colon cancer cell lines, two with microsatellite instability and one without this characteristic. This restriction site covers hot-spot codon 248, which is often mutated in colon carcinomas. Using the Msp I RFLP-PCR assay we found that the mean mutation frequency at this site was not different among the cell lines considered. Taking the substitutions separately, none of the mutations involving codon 248 arose with significantly higher frequency in each of the RER+ cell lines (HCT116 and DLD1) compared with the RER-one (SW480). Only the CG transversion at nt 14 067 (codon 247) occurred with a slightly higher, but biologically insignificant, frequency in one of the RER+ cell lines (HCT116). Our in vitro data support the previously reported lack of correlation between microsatellite instability and p53 mutations in RER+ tumour specimens.