Katsuhide Ikeda

Diagnostic usefulness of EMA, IMP3, and GLUT‐1 for the immunocytochemical distinction of malignant cells from reactive mesothelial cells in effusion cytology using cytospin preparations

To differentiate reactive mesothelial cells (RMs) from metastatic carcinoma and malignant mesothelioma (MM) in effusion cytology is crucial for the cytologic diagnosis and the management of the patients. In the present study, the immunocytochemical staining profile of the epithelial membrane antigen (EMA), the insulin‐like growth factor‐II mRNA‐binding protein 3 (IMP3), and the glucose transporter‐1 (GLUT‐1) was examined to distinguish RMs from malignant cells. A total of 171 pleural (n = 87) and peritoneal (n = 84) effusion specimens, including 50 benign effusions with RMs, 11 MM effusions, and 110 metastatic malignant effusions, were evaluated for immunocytochemistry. EMA, IMP3, monoclonal GLUT‐1, and polyclonal GLUT‐1 immunoreactivity were observed in 26.0%, 6.0%, 20.0%, and 18.0% of RMs, respectively. In contrast to RMs, the immunoreactivity in MM was 100%, 36.4%, 100%, and 90.9%; adenocarcinoma (AC) was 100%, 80.8%, 81.7%, and 72.1%; squamous‐cell carcinoma was 83.3%, 83.3%, 83.3%, and 66.7%. EMA, IMP3, mGLUT‐1, and pGLUT‐1 expressions were observed in 98.4%, 65.6%, 88.5%, and 75.4% in the pleural effusion with malignant cells, and 100%, 88.3%, 78.3%, and 71.7% in ascites containing malignant cells, respectively. The findings of the present study indicate that the immunocytochemical staining for EMA, IMP3, and GLUT‐1 is a useful diagnostic tool for distinguishing effusions containing malignant cells from those that contain benign cells, and in particular, we suggest that the combination of mGLUT‐1 and EMA, and IMP3 and EMA are extremely useful in pleural effusion and in ascites, respectively. Diagn. Cytopathol. 2011;39:395–401.

Coordinate expression of cytokeratin 8 and cytokeratin 17 immunohistochemical staining in cervical intraepithelial neoplasia and cervical squamous cell carcinoma: An immunohistochemical analysis and review of the literature

OBJECTIVE Several Cytokeratin (CK) isoforms have been analyzed in cervical intraepithelial lesions. However, previously reported numbers of specimens have been too low to evaluate any correlation between CK and CIN. METHODS We examined the immunohistochemical staining of p16, CK8, and CK17 in 134 cervical tissues obtained by punch biopsy and graded as follows: CIN I (n=39), CIN II (n=31), CIN III (n=43), SCC (n=21). RESULTS p16 staining was identified in 74.4% of CIN I, 93.6% of CIN II, 97.7% of CIN III, and 100% of SCC cases. CK8 and CK17 staining were identified in 12.8% and 33.3% of CIN I, 22.6% and 58.1% of CIN II, 62.8% and 81.4% of CIN III, and 71.4% and 95.2% of SCC cases, respectively. Interestingly, positivity for CK8 and CK17 correlated with increasing lesion grade of the intraepithelial lesions and also correlated with p16 staining (p16, p=0.0008; CK8, p<0.0001, and CK17, p<0.0001), and a coordinate expression profile of CK8[+]/CK17[+] correlates with increasing CIN grade and carcinoma (likewise, a coordinate expression profile of CK8[-]/CK17[-] correlates with decreasing CIN grade and absence of carcinoma), but expression of just CK8 (CK8[+]/CK17[-]) or just CK17 (CK8[-]/CK17[+]) does not correlate with increasing CIN grade and carcinoma. CONCLUSIONS Results of the present study showed that p16, CK8, and CK17 immunostaining differed according to the degree of cervical intraepithelial lesions and SCC, and surprisingly, that staining was significantly correlated with increasing lesion grade of CIN and SCC.

IMP3/L523S, a novel immunocytochemical marker that distinguishes benign and malignant cells: the expression profiles of IMP3/L523S in effusion cytology.

Differentiating reactive mesothelial cells from metastatic carcinoma and malignant mesothelioma is critical in effusion cytology. Numerous immunohistochemical/cytochemical reports use various antibodies in effusion samples, and most antibodies differentiate metastatic adenocarcinoma from malignant mesothelioma, but no antibodies help distinguish malignant mesothelioma from reactive mesothelial cells. A mouse monoclonal antibody (IMP3/L523S) against KOC is a 580-amino acid oncofetal RNA-binding protein containing 4 K homology domains. IMP3/L523S has been identified in several human malignant tumors. The immunocytochemical staining profile of IMP3 was determined in 95% alcohol-fixed cytologic effusion specimens. A total of 229 cases of pleural and peritoneal effusion cytospecimens were evaluated for the study, including 39 benign effusions with reactive mesothelial cells and 190 metastatic malignant effusions. IMP3 immunoreactivity was observed in 2 (5.1%) of 39 cases of reactive mesothelial cells, 138 (72.6%) of 190 cases of malignant effusion, 4 (36.4%) of 11 cases of malignant mesothelioma, 106 (75.7%) of 140 cases of metastatic adenocarcinoma, and 8 (100%) of 8 cases of squamous cell carcinoma. The overall specificity for the diagnosis of malignancy was 94.9%, whereas the sensitivity was 72.6%. In the peritoneal effusions, the sensitivity for the diagnosis of metastatic adenocarcinoma to distinguish reactive mesothelial cells was 92.3%. In conclusion, IMP3 staining is present in many carcinomas and is not a useful marker for distinguishing between carcinomas arising in different organs. However, the IMP3 antibody is a highly specific marker for malignant lesions, and thus, IMP3 staining is useful for distinguishing neoplastic cells from reactive mesothelial cells in effusion samples.

Comparison of Immunocytochemical Sensitivity Between Formalin-Fixed and Alcohol-Fixed Specimens Reveals the Diagnostic Value of Alcohol-Fixed Cytocentrifuged Preparations in Malignant Effusion Cytology

The most commonly used fixative in effusion cytology is formalin. In the present study, the immunocytochemical properties of formalin-fixed and alcohol-fixed specimens were compared to evaluate the usefulness of alcohol-fixed cytocentrifuged preparations for routine cytologic diagnosis. A total of 269 effusion samples and 17 primary antibodies were used. The sensitivity of immunocytochemical studies in alcohol-fixed specimens was similar and correlated to that of formalin-fixed specimens, suggesting that alcohol-fixed cytocentrifuged preparations are useful in effusion cytology. Pretreatment with or without heat-induced antigen retrieval revealed that antigen retrieval was unnecessary for immunocytochemical studies with most primary antibodies in alcohol-fixed cytocentrifuged preparations. The present study describes the use of immunocytochemical studies with alcohol-fixed cytocentrifuged preparations for diagnosis in routine effusion cytology.

Cytomorphologic features of well-differentiated papillary mesothelioma in peritoneal effusion: a case report.

Well‐differentiated papillary mesothelioma (WDPM), a distinct subtype of diffuse malignant mesothelioma, usually occurs in the peritoneum and is seen most commonly in women of reproductive age. Histologic features of WDPM include papillary growth and stout fibrous cores surrounded by a single layer of tumor cells. We present the case of a 73‐year‐old woman without subjective symptoms who showed signs of peritoneal effusion during a routine examination and for whom cytologic examination of the ascitic fluid was performed. Many spherical clusters, with a smooth external surface composed of a single layer of uniform cuboidal cells, were observed. Within each cluster, a collagenous ball showed light green Papanicolaou staining. Immunohistochemistry of surgical specimens showed tumor cells positive for calretinin, D2‐40, and HBME‐1 staining. The histologic diagnosis was WDPM. The identification of a collagenous ball within these clusters is a useful cytologic finding for the diagnosis of WDPM. WDPM should be suspected when numerous collagenous balls are present by effusion cytology and isolated cells are not. Diagn. Cytopathol. 2008;36:512–515.

Clear cell adenocarcinoma arising from a giant cystic adenomyosis: a case report with immunohistochemical analysis of laminin-5 gamma2 chain and p53 overexpression.

We report a case of a clear cell adenocarcinoma arising from a giant cystic adenomyosis, with immunohistochemical analysis of p53 and laminin-5 gamma2 chain overexpression. Microscopically, not only clear cell adenocarcinoma showing myometrial invasion but also single-layered clear cell adenocarcinoma cells lining the cyst wall were observed. Transition from these single-layered tumor cells to papillary proliferative lesions of various degrees was recognized. Moreover, these tumor cells were continuous with minimal atypical cells. Although the tumor cells within the uterus showed a low positive cell ratio for p53, the metastatic foci showed a remarkable p53 overexpression. Laminin-5 gamma2 chain expression was low in papillary proliferation and high in myometrial invasion and metastatic foci. The single-layered tumor cells showing non-invasive proliferation also contained laminin-5 gamma2 chain-positive cells. When non-invasive tumor cells were considered to be at an early stage in tumor progression, some tumor cells had already acquired an invasive feature. p53 overexpression was not related to expression of the laminin-5 gamma2 chain.

Fine needle aspiration cytology of primary proximal-type epithelioid sarcoma of the perineum: A case report

BACKGROUND Epithelioid sarcoma is a malignant soft tissue tumor of unknown histogenesis. We describe the cytologic findings in a case of primary proximal-type epithelioid sarcoma of the perineum and results of an immunofluorescence analysis of rhabdoid cells from this tumor. To the best of our knowledge, the 3-color immunofluorescence features of proximal-type epithelioid sarcoma have never before been reported. CASE An 8-cm-diameter mass with a 2.5-cm ulcer was found in the perineum of a 36-year-old man. After excision of the tumor, histopathologic examination of the resected specimen suggested a diagnosis of proximal-type epithelioid sarcoma. Fine needle aspiration cytology showed numerous rhabdoid cells with globular intracytoplasmic inclusions. A few isolated cells and polygonal cells were also observed in the smears. Three-color immunofluorescence analysis indicated that the intracytoplasmic inclusions in the rhabdoid cells were positive for cytokeratin, vimentin and CD34. The cytoplasmic staining pattern differed between rhabdoid and epithelioid sarcoma cells. CONCLUSION Immunofluorescent staining of rhabdoid cells from a primary perineal proximal-type epithelioid sarcoma revealed an uneven distribution of cytokeratin in intracytoplasmic inclusions, with the highest concentration at the periphery of the inclusions.

Effusion cytomorphology and immunocytochemistry of malignant melanoma: Five cases of melanotic melanoma and one case of amelanotic melanoma

Effusion cytological analyses of amelanotic malignant melanoma (AMM) are very rare and no concise description of AMM related cytomorphologic features using effusion have been reported. Here, we report the cytomorphological, immunohistochemical, and immunocytochemical findings in the effusion cytology of six cases of malignant melanoma (MM), one case of AMM, and five cases of melanotic malignant melanoma.

Proximal-type epithelioid sarcoma in a 36-year-old man: Closer immunoelectron-microscopic resemblance of the tumor cells to epithelial cells than to mesenchymal cells

Proximal‐type epithelioid sarcoma (PES) is a rare neoplasm. We report a case of PES that arose in the perineal subcutis of a 36‐year‐old Japanese man who died within 4 months of the first clinical sign, probably due to massive pulmonary metastases. In the present study, we analyzed the tumor obtained at surgery, immunohistochemically, immunoelectron‐microscopically and genetically. Although the tumor cells in the patient expressed both cytokeratin and vimentin immunohistochemically, they showed epithelial characteristics immunoelectron‐microscopically because they had tonofilaments constructed of cytokeratin, not vimentin. In addition, the cytokeratins expressed on the tumor were glandular‐type keratins. These findings indicate that PES may be a form of carcinoma in soft tissue. To ascertain the possible origin of the tumor, we compared the tumor immunohistochemically with fetal tissues. Although notochord and fetal peritoneal mesothelium were similar to the tumor antigenically, we could not confirm the specific origin of the tumor. Furthermore, the p53‐WAF1 pathway did not contribute to tumorigenesis in the patient because the tumor had no mutation in exons 5–8 of the p53 gene and was immunohistochemically positive for WAF1.

Multiple immunoenzyme labeling using heat treatment combined with the polymer method: An analysis of the appropriate inactivation conditions of primary antibodies

Multiple immunoenzyme labeling is of considerable value to detect several antigens in the same specimen, although this technique is limited when the primary antibodies have been raised in the same animal species. Multiple immunoenzyme labeling using heat treatment is a simple, reliable and straightforward technique wherein the heat treatment prevents mixed labeling and cross-reaction. The present study determined the inactivation time for primary antibodies by heat treatment in order to apply this procedure to routine histopathological diagnosis and research, and found that the inactivation time differed among the primary antibodies. The secondary antibodies and the labeling enzyme were completely inactivated by heating for 10 min. Therefore, the inactivation of the primary antibodies is crucial to perform multiple immunoenzyme labeling using heat treatment. The sequential combination of the primary antibodies is also important; in the study presented here, an anti-thyroid transcription factor-1 (TTF-1) antibody should be used first and anti-cytokeratin AE1/AE3 antibody second, but not in the opposite sequence, to avoid a mixed-colour-labeling reaction. The present data provided the optimum combination of primary antibodies for multiple immunoenzyme labeling using heat treatment.