Yasuhiko Kitayama

Alteration of immunoreactivity by hydrated autoclaving, microwave treatment, and simple heating of paraffin‐embedded tissue sections

The effects of treatment in a hydrated autoclave (121 °C, 2 atm for 20 min), microwave oven (in water), and simple heating (60 °C overnight in distilled water or 90 °C for 10 min in ZnSO4) on the stainability of 56 antigens by commercially available antibodies in formalin‐fixed paraffin‐embedded tissue sections were evaluated. The detectability of nuclear antigens, glycoprotein, lymphocytic surface markers, and chromogranin A was significantly and reproducibly improved by these treatments, whereas the detectability of viral antigens and peptide hormones was attenuated or unchanged. This enhancement includes not only the distinctiveness of the positive staining, but also the number of positive cells, as revealed by comparing serial sections. Among these four heating procedures, microwave heating and autoclaving were more effective than the others on p53, c‐erbB‐2, and CA125, whereas simple heating was best for smooth‐muscle actin (HHF35 and CGA7). Generally the effects of the heating procedures for these antigens were consistent among the cases, but the effects on GFAP varied with the case. The alterations we observed could significantly influence the interpretation of immunohistochemical staining of currently popular tumor markers such as p53 in terms of their prevalence (28%vs 64% in gastric cancer; 36%vs 82% in metastatic liver cancer) and other diagnostically important markers.

Human Sgo1 downregulation leads to chromosomal instability in colorectal cancer

Background and aims: Chromosomal instability (CIN) is recognised as a hallmark of cancer and is caused by a spindle assembly checkpoint disorder or chromosome mis-segregation during mitosis. Although the recent identification of human shugoshin (hSgo1), an important player in proper chromosome segregation, has suggested the involvement of hSgo1 in colorectal tumourigenesis, little is known about how it is involved. The aim of this study was to obtain information about the status of hSgo1 in human colorectal cancer. Method and results: Among the 46 colorectal cancer cases, hSgo1 mRNA expression was decreased in the tumour tissue in comparison with the corresponding normal tissue (p = 0.032). Human Sgo1-downregulated tumours (tumour to normal mucosa ratio<0.5) had preferential location on the left side large bowel rather than on the right side (p = 0.012), and a higher variation of centromere numbers revealed by fluorescence in situ hybridisation (FISH). To assess the effects of hSgo1 downregulation, hSgo1 knockdown was performed by transfecting the diploid HCT116 cell line with a short hairpin RNA expression vector. hSgo1 knockdown cells proliferated slowly because of both G2/M arrest and apoptosis (p<0.001), and markers of CIN in the form of aneuploidy (p<0.001) and micronuclei (p<0.005) were later observed in hSgo1 knockdown cells. Increased centrosome amplification (p<0.05), the presence of binucleated cells and mitotic catastrophes were also noted in hSgo1 knockdown cells. Conclusions: These findings suggest that hSgo1-downregulated colorectal cancers have a clinicopathological character of CIN, and hSgo1 downregulation leads to CIN in colorectal cancer cells.

Initial Intermittent Microwave Irradiation for Fluorescence In Situ Hybridization Analysis in Paraffin-Embedded Tissue Sections of Gastrointestinal Neoplasia

Initial Intermittent Microwave Irradiation for Fluorescence In Situ Hybridization Analysis in Paraffin-Embedded Tissue Sections of Gastrointestinal Neoplasia

Nonrandom Chromosomal Numerical Abnormality Predicting Prognosis of Gastric Cancer: A Retrospective Study of 51 Cases Using Pathology Archives

Chromosomal or centromerical numerical abnormality (CNA) is a well-known characteristic of human cancer, but the extensive and specific documentation of CNA in gastric cancer is still sparse, partly because of difficulty in obtaining cytogenetic information. Taking advantage of a recently developed fluorescence in situ hybridization protocol for formalin-fixed paraffin-embedded tissues, we investigated CNA of 51 gastric cancer cases with a panel of 18 chromosome-specific α-satellite probes (for chromosomes 1–4, 6–12, 15-18, 20, X and Y) and region specific probes (c-myc and p53) to enumerate respective chromosome numbers in interphase nuclei. The involved chromosomes exhibiting CNA were nonrandom in gastric cancer. Aberrations of chromosomes 1, 8, 17, 20, and X were frequent regardless of histologic types, whereas aberrations chromosomes 10, 15, and 18 occurred less often (p < 0.001). From a histopathologic standpoint, the mucocellular type had stable CNA in comparison with the tubular type (mucocellular type vs tubular type carcinoma: 21.0 ± 10.63% vs 62.8 ± 12.79%, p < 0.001). Interestingly, there was less extensive CNA in women (men vs women: 54.3 ± 9.49% versus 24.9 ± 12.23%, p < 0.001). A dramatic difference in the outcome was detected according to the involvement of chromosomes 3, 10, 11, 12, 17, and Y; that is, the cases with CNA of these chromosomes had worse prognosis.

Microsatellite instability of papillary subtype of human gastric adenocarcinoma and hMLH1 promoter hypermethylation in the surrounding mucosa

Gastric cancer has striking heterogeneity in histological pattern, cellular phenotype, genotype, biomarkers, and biological behavior. We focused on the specific morphological papillary phenotype of gastric adenocarcinoma and attempted to identify its distinct molecular characteristics. In our comparative study, early stage papillary (papillary‐dominant) gastric cancer showed a significantly higher and more widespread high‐frequency microsatellite instability (MSI‐H) than other morphological types. Analysis of mutations in a panel of five putative microsatellite instability (MSI)‐associated genes in the MSI‐H cases revealed that papillary or papillary‐dominant cancer displays a unique profile of mutations compared to profiles previously reported in gastric cancer. Immunohistochemical staining and methylation analysis revealed that silencing of hMLH1 by methylation in its promoter region was responsible for the failure of mismatch repair in papillary‐type gastric cancer, whereas aberrant promoter methylation of hMLH1 was not found in any cases without the unique mutator phenotype. Promoter hypermethylation of the hMLH1 genes was found to a lesser degree in the adjacent non‐tumor mucosa in four of the 10 cases with tumor having the mutator phenotype. Microsatellite instability itself could not be detected in the adjacent non‐tumor mucosa. Inactivation of hMLH1 expression by promoter hypermethylation may be an early event in carcinogenesis of this type of gastric cancer, preceding the development of the clear MSI phenotype of papillary carcinoma.

Fluorescence in situ hybridization analysis with a tissue microarray: 'FISH and chips' analysis of pathology archives

Practicing pathologists expect major somatic genetic changes in cancers, because the morphological deviations in the cancers they diagnose are so great that the somatic genetic changes to direct these phenotypes of tumors are supposed to be correspondingly tremendous. Several lines of evidence, especially lines generated by high‐throughput genomic sequencing and genome‐wide analyses of cancer DNAs are verifying their preoccupations. This article reviews a comprehensive morphological approach to pathology archives that consists of fluorescence in situ hybridization with bacterial artificial chromosome (BAC) probes and screening with tissue microarrays to detect structural changes in chromosomes (copy number alterations and rearrangements) in specimens of human solid tumors. The potential of this approach in the attempt to provide individually tailored medical practice, especially in terms of cancer therapy, is discussed.

Metastases from gastric adenocarcinoma presenting as multiple colonic polyps: Report of a case

A 53-year-old woman presented with symptoms of weight loss, diarrhea, and melena. A barium enema with endoscopy revealed multiple colonic polyps which were shown histologically to be metastatic deposits of poorly differentiated adenocarcinoma. The primary tumor, a poorly differentiated gastric adenocarcinoma, had been resected 11 years earlier. This appears to be only the second published report of polypoid colonic metastases from gastric adenocarcinoma.

Intratumor Heterogeneity of Centromere Numerical Abnormality in Multiple Primary Gastric Cancers: Application of Fluorescence in situ Hybridization with Intermittent Microwave Irradiation on Paraffin-embedded Tissue

Our recent success in retrieving distinct fluorescence signals in response to centromere specific probing of paraffin‐embedded tissues after intermittent microwave (MW) treatment provided the opportunity to analyze chromosome numbers or centromere abnormality in situ in human tumors in various clinicopathological settings. In this study, centromere numerical abnormality (CNA) was investigated by fluorescence in situ hybridization (FISH) in a case of multiple gastric cancer having intratumor histological heterogeneity. The different profiles as determined using a total of 20 specific probes on 4 multifocal lesions in the stomach confirmed the multi‐clonality of these tumors. FISH with probes specific for chromosomes 10, 11, 16 and 18 revealed intratumor heterogeneity of the CNA, which corresponded to the histological heterogeneity. Our report clearly demonstrates, for the first time, intratumor heterogeneity of CNA and its association with the histological picture, and substantiates the applicability of the MW‐assisted FISH protocol to paraffin‐embedded pathological specimens.

Primary osteosarcoma arising from cirrhotic liver

An autopsy case of a 67 year old man with primary osteosarcoma arising in cirrhotic liver is reported. His son had von Recklinghausen disease and he had had a history of hepatitis C virus infection for 10 years. A large tumor, about lO cm in diameter, was found in the right liver lobe. This tumor showed marked central necrosis and hemorrhage, and his‐tologically diffuse sarcomatous cell proliferation associated with extensive osteoid formation and calcification of the periphery. Examination of the whole tumor and the cirrhotic liver (155 tissue blocks) showed that the tumor consisted of sarcoma cells mixed with osteoid with no region resembling hepatocellular carcinoma or hepatoblastoma. Minute hepatocellular carcinomas were found in the cirrhotic liver distant from the sarcomatous area. On immunohistochemical examination, the main tumor gave a distinct positive reaction for vimentin, but not for keratin or other epithelial markers. These findings indicate that the tumor was a true primary osteosarcoma, not an osteoid metaplasia of hepatocellular carcinoma.

Chromosomal numerical abnormalities in early stage lung adenocarcinoma

Chromosomal numerical abnormalities (CNA) are ubiquitous in human cancers. However, the question of when a CNA occurs in the course of tumor generation and progression, is controversial. Recent radiological scrutiny has enabled the identification of small peripheral lesions in the lung. A chromosome‐wide investigation encompassing almost all the chromosomal centromeres was performed using modified fluorescence in situ hybridization on the archived pathological samples of 16 atypical adenomatous hyperplasia (AAH) and 30 lung adenocarcioma (AdCa) specimens including those smaller than 1 cm in size. The prevalence of the gain was more extensive in male than in female patients, and in non‐smokers than in smokers. It tended to be greater in poorly differentiated AdCa, in moderately differentiated AdCa, and in well‐differentiated AdCa cases, in that order. Most AAH had non‐specific gains affecting all the examined chromosomes. The prevalence of the gain differed significantly between AAH and bronchioloalveolar carcinoma (BAC) ≤ 1 cm, but not between BAC < 1 cm and well‐differentiated AdCa > 1 cm. It is proposed that the CNA is a distinct phenomenon occurring in the early or premalignant stage of lung AdCa, and that the CNA itself may not be a sequel in the carcinogenetic process, but a driving factor in carcinogenesis.