Fluorescence in situ hybridization method in isolated single nuclei extracted from paraffin-embedded hepatocellular carcinoma tissues.

BACKGROUND: Genetic analysis using the fluorescence in situ hybridization (FISH) method applied to intact tissue sections of formalin-fixed paraffin embedded (FFPE) tissue is well known to be relatively difficult. The frequent technical problems include unsuccessful hybridization as a result of poor probe penetration, excessive probe requirement, excessive background, auto-fluorescence, and overlapping or incomplete nuclei. These problems lead to absence or insufficiency of fluorescent signals, resulting in an inaccurate analysis. Formalin-fixed paraffin embedded tissue can be analyzed either as intact tissue sections or as a suspension of disaggregated, but intact, nuclei. Intact tissue sections have the advantage of preserved tissue architecture and morphology but have the intrinsic disadvantage of poor probe penetration, overlapping or incomplete nuclei and auto-fluorescence, accordingly reducing the accuracy of fluorescent signals evaluation. OBJECTIVE: To present the effective FISH method applied to isolated of single nuclei and the procedures for isolation of a single nuclei from formalin-fixed paraffin embedded tissues of hepatocellular carcinoma. MATERIAL AND METHOD: Ten paraffin-embedded blocks of hepatocellular carcinoma tissues from the department of pathology, Ramathibodi hospital, Thailand were studied. Isolated single nuclei were extracted from 10-microm sections of paraffin-embedded blocks of hepatocellular carcinoma tissue and hybridized with alpha-satellite centromeric DNA enumeration probes for chromosomes X (CEP X, spectrum green) and satellite III for chromosomes Y (CEP Y spectrum orange). The signal of at least, 200 interphase nuclei were counted from each specimen. RESULTS: The efficacy of this method has been evaluated in 10 formalin-fixed paraffin embedded tissue of hepatocellular carcinoma. The results showed bright, planar and an easy to score signal. CONCLUSION: FISH procedure described here is particularly suitable for retrospective studies of genetic aberration applied to formalin-fixed paraffin embedded tissues.

Preoperative chemoradiotherapy in locally advanced rectal cancer: Ramathibodi experience.

A retrospective study of the preoperative chemoradiotherapy in locally advanced rectal cancer performed at Ramathibodi Hospital. The median age of twelve patients was 52 years. The tumor locations (upper-, mid-, lower rectum) were 25%, 50% and 25%, respectively. Eleven patients had clinical stage 111 disease. All received concurrent 5-FU-based chemoradiotherapy followed by surgery (if resectable) and chemotherapy. The most common toxicity of preoperative treatment was gr. 1-2 diarrhea (58.3%). The response rate was 41.7%. Five patients (41.7%) underwent sphincter-sparing surgery. Four patients underwent AP resection. Twenty-five percent achieved pathological complete response. Pathological downstaging occurred in 33.3%. The remaining three patients had unresectable disease. With the median follow up of 13 months, five patients had progressive disease and one has expired. The local failure rate was 16.7%. The one-year recurrence-free survival was 75%. The authors conclude that preoperative chemoradiotherapy is an effective treatment with favorable outcome in locally advanced rectal cancer.