Kyeongmee Park

HPV integration begins in the tonsillar crypt and leads to the alteration of p16, EGFR and c-myc during tumor formation

The prevalence of human papillomavirus (HPV) infection is high in the oropharyngeal mucosal regions, of which the tonsil is the most commonly affected. There may be a link between HPV and the pathogenesis of tonsillar cancer (TC), because of common anatomical characteristics between cervical and tonsillar cancer. We aimed to clarify whether HPV directly affects the oncogenesis and biologic behavior of TC by making a comparison between infection prevalence, physical status and viral loading numbers, and clinicopathologic prognostic factors. To compare HPV‐related molecules between TC and tonsillitis (CFT), p16, survivin, HIF‐1α, skp‐1, cyclin A, cyclin B1, c‐myc and EGFR were investigated. We observed a significant difference in HPV prevalence between 52 TCs and 69 CFTs (73.1% vs. 11.6%), and most of the HPVs were type 16 (87.2%) and nonepisomal (94.1%). Most TCs associated with HPV arose from the tonsillar crypts, and tended to be inverted and poorly differentiated. Compared with HPV‐negative TC, HPV‐positive TC showed a strong association with p16 overexpression (p < 0.0001), and an inverse association with EGFR amplification (p = 0.0478). HPV‐16 integration status was strongly associated with c‐myc amplification (p = 0.034) and HIF‐1α overexpression (p = 0.022). HPV‐16 integration could be directly related to tonsillar carcinogenesis initially in tonsillar crypts, followed by cell cycle aberration such as p16 overexpression related to the G1‐S phase.

Topoisomerase II-α (topoII) and HER2 amplification in breast cancers and response to preoperative doxorubicin chemotherapy

A significant proportion of breast cancers with HER2 amplification have simultaneous amplification of topoisomerase II-alpha (topoII). Amplification of HER2 and topoII was assayed using a novel chromogenic in situ hybridisation (CISH) method. HER2 and topoII amplification status and the response to preoperative doxorubicin chemotherapy were analysed in 67 locally advanced breast cancer patients. Response to chemotherapy was increased in the cases with coamplification of HER2 and topoII (18/19), whereas the response rate was significantly decreased in the cases without HER2 and topoII amplification (17/36). The 12 cases with HER2 amplification alone showed an intermediate response rate (9/12). The findings of the current study indicate that topoII amplification may play a role in determining chemosensitivity of breast cancers to doxorubicin chemotherapy.

E2F1 Expression is Related with the Poor Survival of Lymph Node-positive Breast Cancer Patients Treated with Fluorouracil, Doxorubicin and Cyclophosphamide

The expressions of E2F1 and retinoblastoma protein (pRB) were analyzed in 165 lymph node-positive breast cancers. All patients underwent adjuvant chemotherapy with fluorouracil, doxorubicin and cyclophosphamide (FAC). E2F1 was expressed in 43.6% and pRB was expressed in 46.1%. E2F1 expression was significantly increased in pRB-expressing tumors and was associated with an S-phase fraction. By univariate survival analyses, E2F1 expression and ER were identified as significant prognostic factors for disease recurrence and patient survival. E2F1 was the only significant prognostic factor of patient outcome after FAC chemotherapy by multivariate analysis.

Comparing Fluorescence In Situ Hybridization and Chromogenic In Situ Hybridization Methods to Determine the HER2/neu Status in Primary Breast Carcinoma using Tissue Microarray

Identification of HER2/neu status is important for predicting response to specific chemotherapy in breast carcinoma. Chromogenic in situ hybridization was performed using tissue microarray technology on 188 primary breast carcinomas. To validate the reliability of novel chromogenic in situ hybridization technology, the results of chromogenic in situ hybridization were correlated with the results of two-color fluorescence in situ hybridization done with the same tumors. On tissue microarray panels containing 188 breast carcinoma tissues, fluorescence in situ hybridization and chromogenic in situ hybridization were conducted simultaneously. HER2/neu amplification was detected in 46 tumors (24.5%) by fluorescence in situ hybridization and in 43 tumors (22.9%) by chromogenic in situ hybridization. Results of each method agreed with each other in 177 tumors (concordance: 94.1%). HER2/neu amplification by fluorescence in situ hybridization was associated with nuclear pleomorphism (P = .021), and HER2/neu amplification by chromogenic in situ hybridization was associated with poor nuclear grade (P = .037). High concordance between fluorescence in situ hybridization and chromogenic in situ hybridization indicated that chromogenic in situ hybridization can be a tempting alternative to fluorescence in situ hybridization for the detection of HER2/neu amplification in breast carcinoma because of its accuracy and relative low cost. HER2/neu appeared to have a prognostic implication because its amplification was associated with aggressive biologic features of the breast carcinoma. Integration of tissue microarray technology enabled high-throughput determination of HER2/neu amplification profile with rapidity and accuracy in large cohorts of the breast carcinoma.

Multiple gastrointestinal stromal tumors with a germline c-kit mutation

Familial gastrointestinal stromal tumor (GIST) is a rare autosomal dominant genetic disorder; thus far, only seven families have been reported with c‐kit germline mutations. Presented herein is a case of multiple intestinal GIST in a 38‐year‐old man with a germline mutation of the c‐kit gene. Operative specimens of the jejunal segment and multiple wedge resection specimens included approximately 30 masses, ranging in size from 1.0 to 6.0 cm. Microscopically, the tumors were composed of CD117‐positive spindle/epitheloid cells with variable numbers of mitotic counts, a characteristic of GIST. The mitotic rate increased to more than 5/50 high‐power fields. Interestingly, marked hypertrophy of the myenteric plexus with CD117‐positive cells was identified in the intestinal segment. By polymerase chain reaction–single‐strand conformation polymorphism analysis and direct DNA sequencing, a heterozygous c‐kit missense mutation at nucleotide 1676 of codon 559 (T → C, Val → Ala), part of the juxtamembrane domain, was detected in the normal tissue. The same mutation was homozygous in the tumor samples. The present case is the first proven case of multiple GIST with a c‐kit germline mutation in Korea and is distinguishable from other reported germ‐line c‐kit mutations because the same 1676 T → C missense mutation occurs in the normal allele as well as the affected allele, although the significance of the identical mutations remains to be investigated.

Chromosome 1p and 19q status and p53 and p16 expression patterns as prognostic indicators of oligodendroglial tumors: a clinicopathological study using fluorescence in situ hybridization.

To verify the prognostic implications of the statuses of chromosome 1p and 19q and the expressions of p53, p16 and GFAP in oligodendrogliomas, we investigated these parameters and correlated the results with patient outcome. Twenty‐seven cases of low‐grade oligodendroglioma (LO) and 29 cases of anaplastic oligodendroglioma (AO) were analyzed by FISH for 1p and 19q status and by immunohistochemistry for p53, p16, and GFAP expression using a tissue microarray. Direct sequencing of the p53 gene was also performed. 1p deletion was observed in 39 of 56 patients (69.9%), and 19q deletion in 41 of 56 (73.2%). Combined loss of 1p and 19q was found in 38 of 56 (67.9%) and exhibited distinct concomitant deletion (P = 0.000). p53 overexpression was observed in 17 cases (30.3%), GFAP expression in 18 cases (32.1%), and p16 loss in 40 cases (74%) of oligodendrogliomas. The expressions of p53 and GFAP were more frequent in AO than in LO (P = 0.015 and 0.001). In contrast, p53 expression was more common in oligodendrogliomas with an intact 19q (P = 0.029), or an intact 1p (P = 0.071). Only five of 14 patients with p53 expression showed TP53 mutation, which was inversely correlated with 1p deletion (P = 0.036). Patients with combined loss of 1p and 19q exhibited better overall survival (P = 0.045). Patients with p53 expression without combined 1p and 19q loss showed poor overall survival (P < 0.000). However, TP53 mutation along with 1p and 19q status could not predict patient outcome. Patients with p16 loss without combined 1p and 9q loss showed poor overall survival (P = 0.011). Therefore, in oligodendrogliomas, the absence of the combined deletion of 1p and 19q and the aberrant expression of p53 or loss of p16 could be used as poor prognostic markers.

Reduced expression of p27Kip1 protein is associated with poor clinical outcome of breast cancer patients treated with systemic chemotherapy and is linked to cell proliferation and differentiation

The cyclin‐dependent kinase inhibitor p27Kip1 is a negative regulator of cell proliferation. Its expression is known to be altered in a proteasome‐dependent manner without changes in DNA level. Reduced expression of p27Kip1 is associated with aggressive behavior in a variety of human cancers. We investigated expression of p27Kip1 protein in human breast cancer using immunohistochemistry to assess its biologic implication along with cell‐cycle analysis by flow cytometry. A total of 68 patients with invasive ductal cancer received adjuvant chemotherapy with cyclophosphamide, methotrexate, and 5‐FU every 3 weeks for six cycles. In epithelial cells of normal and benign breast disease, expression of p27Kip1 was well preserved while its expression markedly decreased in breast cancer (45 of 68). Expression of p27Kip1 is significantly reduced in poorly differentiated cancers and in the advanced stage of the disease. Levels of p27Kip1 expression correlated with cell populations in G0/G1 phase of the cell cycle. In survival analysis, p27Kip1 was useful to predict disease free survival but not overall survival of the patients after adjuvant chemotherapy. In summary, p27Kip1 seems to have a role in the cell proliferation and differentiation process during carcinogenesis of breast cancer. The results of the present study suggest that p27Kip1 can be used in predicting response to systemic chemotherapy in a subset of patients with breast cancer.

Heterogeneity of ERBB2 in gastric carcinomas: a study of tissue microarray and matched primary and metastatic carcinomas

Trastuzumab in association with systemic cytotoxic chemotherapy is a therapeutic option for patients with advanced or metastatic ERBB2+ gastric carcinoma. The status of the ERBB2 overexpression or gene amplification is an important predictive marker in gastric cancer. However, it is controversial whether the primary tumor is representative of distant metastases in terms of ERBB2 status. Quadruplicated tissue microarrays from formalin-fixed paraffin-embedded tissues from 498 advanced primary gastric carcinomas and 97 matched metastatic lymph nodes were investigated by immunohistochemistry with HercepTest and silver in situ hybridization. For further comparison, another set of 41 paired primary and distant metastatic gastric carcinomas were also tested. Intratumoral heterogeneity was defined as different results between tissue microarray cores. ERBB2-positivity was observed in 52 gastric carcinomas (10%) and was not associated with recurrence of disease or survival of patients. In ERBB2-positive primary gastric carcinomas, heterogeneous ERBB2 overexpression was observed in 21/63 (33%) gastric carcinomas and heterogeneous ERBB2 gene amplification in 14/62 (23%) cases. Repeated immunohistochemistry and silver in situ hybridization in representative paraffin tumor blocks confirmed focal ERBB2 overexpression and ERBB2 gene amplification and did not change the final results. Discrepancies in ERBB2 results between primary and paired metastatic lymph nodes were observed in 11% of cases by immunohistochemistry and 7% by silver in situ hybridization. Out of the 41 paired primary and distant metastases, 5 (12%) cases were ERBB2-positive, and discrepancy was observed in one case. Intratumoral heterogeneity and discrepant ERBB2 results in primary and metastatic tumor are not uncommon in gastric carcinoma. Results of silver in situ hybridization showed less frequent heterogeneity compared with immunohistochemistry. Wherever possible, ERBB2 immunohistochemistry testing should be performed in both primary and distant metastatic sites.

P63 and EGFR as prognostic predictors in stage IIB radiation-treated cervical squamous cell carcinoma

OBJECTIVES The purpose of this study was to determine the relation between p63, p53-related gene, epidermal growth factor receptor (EGFR), and spontaneous apoptosis in relation to radiotherapy in patients with FIGO stage IIB cervical carcinoma, who had undergone radiation and concurrent chemotherapy, retrospectively. METHODS Eighty-four patients with FIGO stage IIB squamous cell carcinoma (SCC) of the uterine cervix, who were treated with radiotherapy and concurrent chemotherapy between 1991 and 1996, were included in the present study. The clinicopathologic features, patterns of treatment failure, and survival data were compared with the expressions of p63 and EGFR, which were determined by immunohistochemistry and with apoptosis by TUNEL on tissue-arrayed slides. Univariate and multivariate analyses were performed to determine the prognostic factors that influence patient survival. RESULTS Overall the indices of the expressions of p63 and EGFR in stage IIB cervical carcinoma were 18.7 and 26.6%, respectively, and these were found to be correlated. EGFR expression was significantly associated with extrapelvic failure (P = 0.03), whereas p63 was associated with locoregional failure (P = 0.03). The spontaneous apoptotic index showed no prognostic value, but the immunoreactivities of p63 and EGFR were associated with a worse prognosis by both univariate (P = 0.01 and 0.04, respectively) and multivariate analysis (95% CI:2.0-4.4, RR:3.2 and 95% CI:4.9-8.7, RR:6.7, respectively). CONCLUSIONS The expression of p63 gene is associated with poor survival and locoregional failure, whereas EGFR expression was found to be a prognostic predictor of extrapelvic failure. Both molecules were found to be potent molecular risk factors in patients with FIGO stage IIB SCC of the uterine cervix, who had received radiotherapy and concurrent chemotherapy.

P16INK4a protein expression is associated with poor survival of the breast cancer patients after CMF chemotherapy.

Immunohistochemical assay for p16 protein expession was performed in 192 breast carcinoma patients treated with adjuvant chemotherapy. p16 expression was observed in 78 cases (40.6%). The frequency of p16 expression significantly decreased in moderately differentiated (histologic grade II) cancers, 20 (19.6%) of 102. In poorly differentiated cancers (histologic grade III), p16 expression was not observed in all 16 cases. p16 expression was significantly associated with histologic grade of the breast carcinomas (p < 0.001). The proliferative index (PI: S + G2/M) of individual tumors was measured by DNA flow cytometry. In 114 tumors with PI less than 20%, p16 expression was observed in 59 tumors (49.1%). In the tumors with PI equal or more than 20%, p16 expression was observed in 22 (28.2%) of 78 cases. p16 expression was significantly decreased in the tumor with higher PI (p = 0.003). For the other clinicopathologic variables, no significant association was found with p16 expression status. Immunohistochemical assay for p53 protein expression was performed on the same breast carcinomas. There was no significant association between p16 and p53 expression in breast carcinomas. During median follow-up period of 52 months (range: 40–72 months), 46 patients (25.8%) had recurrent disease and 32 patients (18.91%) died of recurrent disease. p16 expression was observed in 20 (43.5%) of 46 patients with recurrent disease, while its expression was observed in 58 patients (39.7%) of 146 patients who were free of recurrence during the study period. p16 expression had no significant impact on predicting recurrence of breast carcinoma. Fourteen patients (12.2%) of 114 patients whose tumors did not show p16 expression died of recurrent breast carcinoma, whereas 18 patients (23.1%) of 78 patients with p16 expressing tumor died during the follow-up period. There was a significant difference of patient survival according to p16 expression status (p = 0.039). These results indicate that p16 expression is useful in predicting response to chemotherapy in breast cancer patients. p16 protein seems to have a role in tumor growth and differentiation of the breast carcinoma.