Multistaining Optimization for Epstein-Barr Virus–Encoded RNA In Situ Hybridization and Immunohistochemistry of Formalin-Fixed Paraffin-Embedded Tissues Using an Automated Immunostainer

BACKGROUND: Single staining is commonly performed for practical pathologic diagnoses. However, this method is limited in its ability to specify cellular morphology and immunophenotype and often requires consumption of limited tissue. This study aimed to describe an optimized protocol for multiple in situ hybridization (ISH) and immunohistochemistry (IHC). METHODS: The quality of multistaining was evaluated by carefully changing each step of ISH and IHC in an angioimmunoblastic T-cell lymphoma (AITL) case on a Ventana BenchMark XT automated immunostainer. The optimized protocols were also performed using another immunostainer and in 15 cases of five Epstein-Barr virus (EBV)–associated malignancies using formalin-fixed paraffin-embedded tissue. RESULTS: The quality of various ISH-IHC staining protocols was semi-quantitatively evaluated. The best EBV-encoded RNA (EBER)-ISH/double IHC staining quality, equivalent to single staining, was obtained using the following considerations: initial EBER-ISH application, use of protease and antigen retrieval reagent (cell conditioning 1 [CC1] treatment time was minimized due to impact on tissue quality), additional baking/deparaffinization not needed, and reduced dilution ratio and increased reaction time for primary antibody compared with single immunostaining. Furthermore, shorter second CC1 treatment time yielded better results. Multiple staining was the best quality in another immunostainer and for different types of EBV-associated malignancies when it was performed in the same manner as for the Ventana BenchMark XT as determined for AITL. CONCLUSIONS: EBER-ISH and double IHC could be easily used in clinical practice with currently available automated immunostainers and adjustment of reagent treatment time, dilution ratio, and antibody reaction time.

Overexpression of C-reactive Protein as a Poor Prognostic Marker of Resectable Hepatocellular Carcinomas

BACKGROUND: C-reactive protein (CRP) is an acute phase reactant synthesized in the liver. CRP immunoreactivity is a feature of inflammatory hepatocellular adenomas with a higher risk of malignant transformation. A high serum CRP level denotes poor prognosis in hepatocellular carcinoma (HCC) patients. This study was conducted to determine whether CRP is produced in HCC and to assess the clinicopathologic significance of CRP expression in cancer cells. METHODS: CRP immunoreactivity was examined in treatment-naive HCCs (n=224) using tissue microarrays and was correlated with clinicopathologic parameters. The expression of CRP mRNA and protein was also assessed in 12 HCC cases by quantitative real-time polymerase chain reaction and immunoblotting. Hep3B and SNU-449 HCC cell lines were used for the analysis of CRP mRNA regulation by interleukin 6 (IL-6). RESULTS: CRP was expressed in 133 of 224 HCCs (59.4%) with a variable degree of immunoreactivity (grade 1 in 25.9%; grade 2 in 20.1%; grade 3 in 13.4%). There was an inverse relationship between grade 3 CRP immunoreactivity and cancer-specific survival (p=.0047), while no associations were found with other parameters, including recurrence-free survival. The CRP mRNA expression level was significantly higher in CRP immunopositive cases than in immunonegative cases (p<.05). CRP mRNA expression was increased in Hep3B cells, but was not detected in SNU-449 cells even after IL-6 treatment. CONCLUSIONS: We report the expression of CRP in HCC for the first time. CRP expression was associated with poor cancer-specific survival in patients with resectable HCC.

A Case of Leiomyoma of the Ovary / 대한산부인과학회지

Leiomyoma arising primarily in the ovary is a rare tumor, accounting for only 1% of benign ovarian neoplasms. About 50 cases have been reported in the literature to date. Most cases are asymptomatic and this benign neoplasm is usually found incidentally on routine pelvic examination, at surgery, or at autopsy. We present a case of ovarian leiomyoma in 39-year-old woman which has been experienced in our hospital with brief review of literature.

A Case of Squamous Cell Carcinoma of Bartholin's Gland / 대한산부인과학회잡지

Carcinomas of Bartholin's gland are rare tumors that account for less than 1% of all gynecological malignancies. Two major histological types, squamous cell carcinoma and adenocarcinoma, account for 80% to 90% of primary cases. The remainders are adenosquamous carcinoma, adenoid cystic carcinoma and so on. We experienced a case of squamous cell carcinoma of the Bartholin's gland managed by wide local excision and chemotherapy with cisplatin and 5-fluorouracil (5-FU). We present this case with a brief review of the literatures.