Evaluation of methylation degree from formalin-fixed paraffin-embedded DNA extract by field-amplified sample injection capillary electrophoresis with UV detection.

Alterations in global DNA methylation are implicated in several pathobiological processes. The tissues stored as paraffin blocks represent an important source of DNA for retrospective genetic and epigenetic analysis on a large scale. Therefore, we developed the first capillary electrophoresis method able to measure global methylation in formalin-fixed, paraffin-embedded (FFPE) DNA extracts. A field-amplified sample injection capillary electrophoresis method with UV detection for the separation and quantification of cytosine and 5-methylcytosine released following DNA hydrolysis by means of formic acid was employed. Analytes were baseline-separated within 8 min by using 300 mM tris(hydroxymethyl)aminomethane phosphate pH 3.75 as the running buffer. With use of electrokinetic injection the limit of detection (LOD) in real sample was 0.1 nM, thus improving by about 400-fold the LOD of the previously described methods based on capillary electrophoresis. Sample extraction and purification were optimized so that evaluation of the DNA methylation degree was possible starting from 0.5-1 µg of DNA with intra- and interassay relative standard deviations for the 5-methylcytosine to total cytosine ratio of 2.0 and 3.2%, respectively. Because of its high accuracy and throughput, our method will be useful for large-scale applications to determine the implications of genomic DNA methylation levels in tumorigenesis.

High sensitivity of reverse-hybridization methodology in the detection of KRAS mutations from formalin-fixed paraffin-embedded colorectal cancer samples.

Colorectal cancer is ranked the third most common cancer worldwide in terms of incidence and the second in terms of mortality. Recent advances in therapeutic approaches to colorectal cancer have identified a potential role of anti-epidermal growth factor receptor (EGFR) targeted therapies as adjuvant treatment in advanced disease. New evidences showed that patients harboring KRAS mutations on codons 12 and 13 are not responsive to anti-EGFR monoclonal antibodies. Therefore, new mutational screening tools have been proposed to select patients who will benefit from anti-EGFR targeted therapy, reducing inappropriate, expensive treatments and unwarranted side effects. We evaluated the performance of a reverse-hybridization-based assay in the identification of the most frequent KRAS mutations on a series of 50 formalin-fixed, paraffin-embedded, advanced colorectal cancer specimens, in comparison with the direct gene sequencing technique. Thirty-two of the 50 cases (64%) showed KRAS single point mutations by reverse-hybridization technique. In particular, 93.8% of the mutations were reported on codon 12, whereas 6.2% of the mutations were reported on codon 13. Direct gene sequencing showed KRAS mutations on 28 of the 50 cases (56%) with 96.4% of the mutations on codon 12 and 3.6% on codon 13. Concordance between the assays was observed in 92% of the cases. Both reverse hybridization and gene sequencing methods have been shown to be suitable tests in detecting KRAS mutations from formalin-fixed, paraffin-embedded tumor specimens. In our experience, reverse-hybridization technique has been shown to be an effective and more sensitive assay for the identification of the most common KRAS mutations.

S-100A1 is a reliable marker in distinguishing nephrogenic adenoma from prostatic adenocarcinoma.

Nephrogenic adenoma is a benign lesion that may occur at any site of the genitourinary tract, usually in association with previous urothelial injuries. Although its pathogenesis is still debated, recent studies seem to confirm its derivation from renal tubular epithelium, rather than from a metaplastic process of urothelium. In addition to its uncertain origin, there can be diagnostic difficulty in distinguishing nephrogenic adenoma from prostatic adenocarcinoma, particularly with lesions arising in the prostatic urethra. So far, immunohistochemical stains are often needed to make such a distinction, and several markers have been proposed, often with controversial results. S100A1 is a calcium binding protein that has been recently reported to be expressed in renal tubular cells and in a subset of renal cell neoplasms. Alpha-methylacyl-CoA racemase (AMACR), a recently identified prostate cancer marker, has also been found to be expressed in renal tubules and in some renal epithelial neoplasms. In this study, we investigated the expression of S100A1 and AMACR in 18 nephrogenic adenomas and in 100 prostatic adenocarcinomas. A strong and distinct cytoplasmic or nucleocytoplasmic staining of S100A1 was found in 17 out of 18 cases of nephrogenic adenoma (94%), but never in prostatic adenocarcinoma. In contrast, AMACR expression was detected in 14 of 18 nephrogenic adenomas (78%) and in 96 of 100 prostatic adenocarcinomas (96%). We conclude that (1) S100A1 is a specific and sensitive immunohistochemical marker to differentiate nephrogenic adenoma from prostatic adenocarcinoma; (2) AMACR immunostaining does not seem to be a useful marker in distinguishing between these 2 lesions; (3) given that both S100A1 and AMACR have been reported to be expressed in renal tubular cells and in a subset of renal cell neoplasms, our findings confirm the histogenetic relationship between nephrogenic adenoma and renal tubular epithelium.