Morimasa Matsuta

Detection of numerical chromosomal aberrations in malignant melanomas using fluorescence in situ hybridization

To evaluate the numerical chromosomal aberration i malignant melanoma, we have applied fluorescence in situ hybridization (FISH) with repetitive DNA probes specific for chromosomes 1, 6, 7, 9, 10, and 17 on 24 fresh malignant melanomas (primary: 14, metastatic: 8).

Immunohistochemical detection of Ki-67 in epithelial skin tumors in formalin-fixed paraffin-embedded tissue sections using a new monoclonal antibody (MIB-1).

The expression of the Ki‐67 antigen was investigated in 44 epithelial skin tumors using an immunohistochemical technique on formalin‐fixed, paraffin‐embedded tissue sections. Microwave oven heating was employed for retrieval of the antigen in these tissue sections.

Interphase cytogenetics of melanocytic neoplasms: numerical aberrations of chromosomes can be detected in interphase nuclei using centromeric DNA probes

This study shows that fluorescence in situ hybridization (FISH) to thin sections cut from paraffin‐embedded material can lie used to distinguish between groups of melanocytic neoplasms and thus may be useful as an investigational and diagnostic tool. FISH with a probe for a repealed, alpha satellite sequence specific to chromosome 17 was used to investigate the chromosomal composition of dysplastic (or Clarks nevus) and Spitzs nevi and malignant melanomas. Hybridization was to thin (∼6 μm) sections cut from paraffin blocks. The number of signals per nucleus in normal diploid cells is expected to be less than 2 since the sections are thinner than one nuclear diameter. Keratinocytes and lymphocytes m these same sections showed 1–2 signals per nucleus with a mean of 1.2. Dysplastic nevi showed 1–4 hybridization signals per nucleus with a mean of 1.5. Spitzs nevi showed 1–2 signals per nucleus with a mean of 1.3. Melanomas showed 1–6 signals per nucleus with a mean of 2.1. We were thus able to use FISH to demonstrate differences in chromosome numbers between groups of benign and malignant melanocytic neoplasms. Technical improvements in the near future can be expected lo result in more precise estimates of chromosomal number.

Immunohistochemical detection of p21WAF1/CIP1 and p53 proteins in formalin-fixed paraffin-embedded tissue sections of squamous cell carcinoma of the skin

Thirty-two cases of squamous cell carcinoma (SCC) of the skin were investigated as to the expression of p53 and p21 (WAF1/CIP1) using an immunohistochemical method. These cases were surgically resected or biopsied, tissue samples were then fixed in formalin and embedded in paraffin in the conventional way. Microwave heating was used for antigen retrieval. The primary monoclonal anti-p21 antibody and the monoclonal antibody against p53 were employed. The labeled-streptoavidin-biotin-peroxidase method was used for immunohistochemical staining. Of these, 30 cases showed overexpression of p53 staining, but normal epidermal cells were free of stain. p21 positive cells were detected faintly in the middle layer cells of normal epidermis. Of these, 30 cases showed overexpression of p21 staining. The staining pattern of p53 and p21 showed intratumoral heterogeneity in SCC. In general, there was the inverse relationship between p21 and p53 staining in tumors, namely p53 positive cells were p21 negative and vice versa. However, some of the tumor cells expressed both genes simultaneously. This study supports a hypothesis that p21 expression is regulated by p53, and that it is also regulated by an additional pathway(s) in SCC.

Immunohistochemical and Electron Microscopic Studies on Hashimoto′s Thyroiditis

The thyroid glands of nine patients with Hashimotos thyroiditis were studied by immunoperoxidase method for immunoglobulin (Ig) and thyroid hormone, and of these four tissue specimens were further examined by electron microscope. Immunoperoxidase method for Ig revealed that about 63% of the infiltrating cells contained Ig, and that about 90% of such Ig‐containing cells had IgG. IgG‐containing cells seemed to secrete autoantibodies. Immunoperoxidase method for thyroid hormone and electron microscopy revealed that there was a good correlation between the morphological features of the thyroid follicle and its immunohistochemical staining pattern. In follicles composed of columnar cells, the colloid and cytoplasm of some epithelial cells were immunohistochemically stained. Dense deposits, regarded as immune complexes, were observed in the basement membrane of these follicles, and lymphocytes were seen between the adjacent cells. When similar deposits appeared in the basement membrane of such follicles, 4 or more lymphocytes per follicle could be seen among the epithelial cells. The present findings seem to indicate that antibody‐dependent cell‐mediated cytotoxicity plays an important role in the pathogenesis of Hashimotos thyroiditis.

Cervical cytology by means of fluorescence in situ hybridization with a set of chromosome-specific DNA probes.

Objective: To reveal the numerical aberration of chromosome 1 and chromosome 17 in cervical neoplasia.

Applications of DNA Flow Cytometry and Fluorescence In situ Hybridization Using a Chromosome‐specific DNA Probe on Paraffin‐embedded Tissue Sections of Primary Malignant Melanomas

We have applied DNA flow cytometric analysis to paraffin‐embedded tissue sections of primary malignant melanomas. Conventionally, flow cytometric analysis of paraffin‐embedded tissue sections has been done by the method of Hedley et al. We added ultrasound treatment to the method of Hedley et al. and a lower value of coefficient of variation was shown. Furthermore, a new technique, fluorescence in situ hybridization with a chromosome‐specific repetitive DNA probe, was used for the analysis of chromosomal numerical aberrations in the same paraffin‐embedded tissue sections. The DNA flow cytometric analysis showed that in 8 cases six primary malignant melanomas were of the aneuploid pattern and two cases of lentigo maligna (melamona in situ) were of the diploid pattern. By fluorescence in situ hybridization, the two cases with the diploid pattern had spots/nucleus of 1.28 and 1.12, and those with the aneuploid pattern had spots/nucleus from 2.01 to 2.27. Only one nodular melanoma in an aneuploid case showed spots/nucleus of 1.71. These data indicate that fluorescence in situ hybridization with chromosome‐specific repetitive DNA probes can serve as a cytogenetic tool for the analysis of interphase nuclei of solid human tumors and may be useful for the study of tumor cell heterogeneity.

Malignant fibrous histiocytoma of the vagina

Abstract We describe here the case of an 82-year-old woman presenting with a hemorrhagic tumor on the anterior vaginal wall. She was diagnosed with malignant fibrous histiocytoma (MFH) from the findings of cytological analysis of biopsied surface tissue, histopathologic analysis of biopsied tissue, immunohistochemical staining, and electron microscopy. Cytological analysis of the biopsy sample harvested from the tumor surface showed multinucleated giant cells and large atypical cells with rough, granular, chromatin, as well as notable nucleoli. A storiform pattern was observed histopathologically, and immunohistochemical staining confirmed positive reactions to α1-antichymotripsin (α1-ACT), vimentin, and lysosome, but negative reactions to epithelial membrane antigen (EMA), cytokeratin, and α-smooth muscle action (α-SMA). Electron microscopy showed histiocyte-derived cells with a segmented nucleus with a large groove, pseudopodic cytoplasmic projections, and lysosome-like structures. However, intercellular adhesion factors were not notable, and microvilli were absent. Based on the above findings, a diagnosis of MFH originating from the vaginal wall was made. Because of the patients advanced age, and, in accordance with her wishes, three courses of cancer chemotherapy, consisting of doxorubicin hydrochloride, fluorouracil, and cisplatin were carried out, without surgery. No reduction in the size of the tumor was seen at follow up, and despite the absence of metastasis and no exacerbation of her general condition, she died suddenly at home 2 years after being discharged.

A case of subungual amelanotic malignant melanoma : an electron microscopic study of aberrant melanosomes

A 68‐year‐old woman with amelanotic malignant melanoma (AMM) of her left thumbnail bed was reported. The tumor cells were positive with S‐100 protein. Electron microscopic findings revealed the presence of typical melanosomes and a variety of aberrant melanosomes within the tumor cells, obviously different from the results for common malignant melanoma.

In Situ Hybridization for DNA: Fluorescent Probe

Fluorescence in situ hybridization (FISH) with chromosomespecific probes is one of the non-isotopic in situ hybridization methods that opens up several new areas of cytogenetic investigation by allowing visual determination of the presence and normality of specific genetic sequences in single metaphase or interphase cells (Pinkel 1986a, 1986b). With this approach, the genetic loci to be analyzed are made microscopically visible in single cells using in situ hybridization with nucleic acid probes specific to these loci. The DNA in target cells is made as a single strand and incubated with single-stranded fluoro chromedirectly- labeled or chemically modified DNA probe under conditions where the probe will anneal only with DNA sequences to which it has high DNA sequence homology. The bound chemically modified probe is made visible by immunofluorescent detection methods. This technique is often called standard FISH.