Ming-Yung Lee

Scoring mechanisms of p16INK4a immunohistochemistry based on either independent nucleic stain or mixed cytoplasmic with nucleic expression can significantly signal to distinguish between endocervical and endometrial adenocarcinomas in a tissue microarray study

BackgroundEndocervical adenocarcinomas (ECAs) and endometrial adenocarcinomas (EMAs) are malignancies that affect uterus; however, their biological behaviors are quite different. This distinction has clinical significance, because the appropriate therapy may depend on the site of tumor origin. The purpose of this study is to evaluate 3 different scoring mechanisms of p16INK4a immunohistochemical (IHC) staining in distinguishing between primary ECAs and EMAs.MethodsA tissue microarray (TMA) was constructed using formalin-fixed, paraffin-embedded tissue from hysterectomy specimens, including 14 ECAs and 24 EMAs. Tissue array sections were immunostained with a commercially available antibody of p16INK4a. Avidin-biotin complex (ABC) method was used for antigens visualization. The staining intensity and area extent of the IHC reactions was evaluated using the semi-quantitative scoring system. The 3 scoring methods were defined on the bases of the following: (1) independent cytoplasmic staining alone (Method C), (2) independent nucleic staining alone (Method N), and (3) mean of the sum of cytoplasmic score plus nucleic score (Method Mean of C plus N).ResultsOf the 3 scoring mechanisms for p16INK4a expression, Method N and Method Mean of C plus N showed significant (p-values < 0.05), but Method C showed non-significant (p = 0.245) frequency differences between ECAs and EMAs. In addition, Method Mean of C plus N had the highest overall accuracy rate (81.6%) for diagnostic distinction among these 3 scoring methods.ConclusionAccording to the data characteristics and test effectiveness in this study, Method N and Method Mean of C plus N can significantly signal to distinguish between ECAs and EMAs; while Method C cannot do. Method Mean of C plus N is the most promising and favorable means among the three scoring mechanisms.

Scoring of p16INK4a immunohistochemistry based on independent nuclear staining alone can sufficiently distinguish between endocervical and endometrial adenocarcinomas in a tissue microarray study

Endocervical adenocarcinomas and endometrial adenocarcinomas are malignancies that affect uterus; however, their biological behaviors are quite different. This distinction has clinical significance, because the appropriate therapy may depend on the site of tumor origin. The purpose of this study is to evaluate four different scoring methods of p16INK4a immunohistochemical staining in distinguishing between primary endocervical adenocarcinomas and endometrial adenocarcinomas from limited sizes of tissue specimens. A tissue microarray was constructed using formalin-fixed, paraffin-embedded tissue from hysterectomy specimens, including 14 endocervical adenocarcinomas and 21 endometrial adenocarcinomas. Tissue array sections were immunostained with a commercially available antibody of p16INK4a. Avidin–biotin complex method was used for antigens visualization. The staining intensity and area extent of the immunohistochemistry was evaluated using the semiquantitative scoring system. Of the four scoring methods for p16INK4a expression, Method Nucleus, Method Dominant Cytoplasm or Nucleus, and Method Mean of Cytoplasm plus Nucleus showed significant (P values <0.05), but Method Cytoplasm did not show significant (P=0.432), frequency distinction between endocervical adenocarcinomas and endometrial adenocarcinomas. In addition, Method Mean of Cytoplasm plus Nucleus had the highest overall accuracy rate (80%) for diagnostic distinction among these four score-counting methods. According to the data in this tissue microarray study, Method Nucleus is the most convenient and efficient method to distinguish between endocervical adenocarcinomas and endometrial adenocarcinomas. Although Method Dominant Cytoplasm or Nucleus as well as Method Mean of Cytoplasm plus Nucleus also revealed statistically significant results, they are relatively more inconvenient due to complicated score calculating means on the basis of mixed cytoplasmic and nuclear p16INK4a expressions. Method Cytoplasm is of no use in the diagnostic distinction between endocervical adenocarcinomas and endometrial adenocarcinomas.

Nuclear Receptor Interaction Protein (NRIP) expression assay using human tissue microarray and immunohistochemistry technology confirming nuclear localization

BackgroundA novel human nuclear receptor interaction protein (NRIP) has recently been discovered by Chen SL et al, which may play a role in enhancing the transcriptional activity of steroid nuclear receptors in prostate (LNCaP) and cervical (C33A) cancer cell lines. However, knowledge about the biological functions and clinical implications of NRIP, is still incomplete. Our aim was to determine the distribution of NRIP expression and to delineate the cell types that express NRIP in various malignant tumors and healthy non-pathological tissues. This information will significantly affect the exploration of its physiological roles in healthy and tumor cells.MethodsBy using tissue microarray (TMA) technology and an anti-NRIP monoclonal antibody immunohistochemical (IHC) survey, NRIP expression was examined in 48 types of tumors and in a control group of 48 matched or unmatched healthy non-neoplastic tissues.ResultsOur survey results showed that ten cases were revealed to express the NRIP in six malignancies (esophageal, colon, breast, ovarian, skin, and pancreatic cancers), but not all of these specific tumor types consistently showed positive NRIP expression. Moreover, malignant tumors of the stomach, prostate, liver, lung, kidney, uterine cervix, urinary bladder, lymph node, testis, and tongue revealed no NRIP expression. Among the control group of 48 matched and unmatched non-neoplastic tissues, all of them demonstrated IHC scores less than the cut-off threshold of 3. In addition, ten cores out of thirty-six carcinomatous tissues revealed positive NRIP expression, which indicated that NRIP expression increases significantly in carcinoma tissue cores, comparing to the matched controlled healthy tissues.ConclusionThis is the first study to use a human TMA and IHC to validate the nuclear localization for this newly identified NRIP expression. In considering the use of NRIP as a potential diagnostic tool for human malignancies survey, it is important to note that NRIP expression carries a sensitivity of only 23%, but has a specificity of 100%. There is also a significant difference in positive NRIP expression between primary carcinomatous tissues and matched controlled healthy tissues. Although further large-scale studies will merit to be conducted to evaluate its role as a potential adjunct for cancer diagnosis, data from this study provides valuable references for the future investigation of the biological functions of NRIP in humans.

True cytokeratin 8/18 immunohistochemistry is of no use in distinguishing between primary endocervical and endometrial adenocarcinomas in a tissue microarray study.

The choice of appropriate therapeutic plans for primary endocervical adenocarcinomas and endometrial adenocarcinomas depends on the site of origin of the tumor. The purpose of this study was to make clear whether the immunohistochemistry of the true cytokeratin 8/18 monoclonal antibody (Leica Microsystems, Newcastle, United Kingdom), instead of CAM 5.2 (Becton Dickinson Biosciences, San Jose, CA), has potential use in distinguishing between endocervical adenocarcinomas and endometrial adenocarcinomas. A tissue microarray was constructed using paraffin-embedded, formalin-fixed tissues from 34 hysterectomy specimens, including 14 endocervical adenocarcinomas and 20 endometrial adenocarcinomas. Using the Bond-Max autostainer (Leica Microsystems) and the associated Bond Refine Polymer Detection Kit, tissue array sections were immunostained with cytokeratin 8, 18, and 8/18 commercially available antibodies. The immunohistochemical expressions of all 3 markers, cytokeratin 8, 18, and 8/18 showed nonsignificant (P>0.05) frequency differences between the immunostaining results (positive vs. negative) in tumors of both gynecologic adenocarcinomas. Although CAM 5.2 has been reported to be helpful in distinguishing between primary endocervical adenocarcinomas and endometrial adenocarcinomas, we could not verify this point of view using the true cytokeratin 8/18 monoclonal antibody (Leica Microsystems). It has often been mistakenly cited that CAM 5.2 reacts with cytokeratin 8 and 18, and the results herein confer that there is a wrong impression that cytokeratin 8/18 is differentially expressed in these 2 gynecologic malignancies. In conclusion, the true cytokeratin 8/18 monoclonal antibody is of no use in distinguishing between primary endocervical adenocarcinomas and endometrial adenocarcinomas.

HER2 amplification and overexpression are significantly correlated in mucinous epithelial ovarian cancer

HER2 gene amplification and protein over-expression are important factors in predicting clinical sensitivity to anti-HER2 therapies in breast, gastric or gastroesophageal junction cancer patients. The aim of this study was to evaluate the correlation between HER2 gene copy numbers and HER2 protein expressions in mucinous epithelial ovarian cancer (EOC). Of the 49 tissue microarray samples of mucinous EOC, we applied 2010 ToGA trial (Trastuzumab for Gastric Cancer) surgical specimen scoring criteria to analyze the HER2 protein expression by an immunohistochemistry (IHC) test with Dako (Carpenteria, CA), c-erb-B2 antibody, and the HER2 gene amplification by the fluorescence in situ hybridization (FISH) test with Abbott/Vysis PathVysion HER2 DNA Probe Kit (Abbott Molecular Inc., Des Plaines, IA). We achieved a high overall concordance of 97.56% between nonequivocal HER2 results by IHC and FISH tests. In addition, HER2 gene copies before chromosome-17 correction increased significantly in a stepwise order through the negative, equivocal and positive IHC result categories (P<.001), as did the HER2 gene copies after chromosome-17 correction (P<.001). On the other hand, HER2 IHC results correlated significantly with both chromosome-17-uncorrected HER2 gene copy numbers (ρ=0.630, P<.001) and chromosome-17 corrected HER2 gene copy numbers (ρ=0.558, P<.001). We concluded that both chromosome-17 corrected and uncorrected HER2 gene copies correlated significantly with HER2 IHC results. Tests for the HER2 gene copies per tumor cell either before or after correction of chromosome-17 can be applied as a potentially valuable tool to analyze the HER2 status in mucinous EOC.

Assessing the HER2 status in mucinous epithelial ovarian cancer on the basis of the 2013 ASCO/CAP guideline update.

Her2 gene amplification and protein overexpression are important factors in predicting clinical sensitivity to anti-HER2 monoclonal antibody therapy in breast, gastric, or gastro-esophageal junction cancer patients. The purpose of this study was to evaluate the HER2 status in the mucinous epithelial ovarian cancer (EOC). Adopting the 2013 American Society for Clinical Oncology and the College of American Pathologists guideline update for HER2 testing, 49 tissue microarray samples of mucinous EOC were analyzed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) tests. The prevalence of HER2 positivity in Asian mucinous EOC was 9 of 49 Asian women (18.37%). The overall concordance was 100% between IHC and FISH results. Her2 gene copies before chromosome-17 correction increased significantly in a stepwise order through the negative, equivocal, and positive IHC result categories (P<0.001), as did the Her2 gene copies after chromosome-17 correction (P<0.001). Of the Taiwanese cohort (n=21), HER2 heterogeneity was 4.76% (1/21) in all but 14.26% (1/7) in HER2-positive cancer. In conclusion, we demonstrated that the prevalence of HER2 positivity in both Asian and white women was comparable; complete HER2 concordance existed between IHC and FISH tests for the Her2 gene copies per tumor cell either before or after correction of chromosome-17, and this can be applied as a potentially valuable tool to analyze the HER2 status. Polysomy-17 was absent under the CEP17 cutoff ≥3. The existence of HER2 heterogeneity can be discerned in certain HER2-expressed primary mucinous EOC in Taiwanese women.

Progesterone receptor does not improve the performance and test effectiveness of the conventional 3-marker panel, consisting of estrogen receptor, vimentin and carcinoembryonic antigen in distinguishing between primary endocervical and endometrial adenocarcinomas in a tissue microarray extension study.

ObjectiveEndocervical adenocarcinomas (ECA) and endometrial adenocarcinomas (EMA) are uterine malignancies that have differing biological behaviors. The choice of an appropriate therapeutic plan rests on the tumors site of origin. In this study, we propose to evaluate whether PR adds value to the performance and test effectiveness of the conventional 3-marker (ER/Vim/CEA) panel in distinguishing between primary ECA and EMA.MethodsA tissue microarray was constructed using paraffin-embedded, formalin-fixed tissues from 38 hysterectomy specimens, including 14 ECA and 24 EMA. Tissue microarray (TMA) sections were immunostained with 4 antibodies, using the avidin-biotin complex (ABC) method for antigen visualization. The staining intensity and extent of the immunohistochemical (IHC) reactions were appraised using a semi-quantitative scoring system.ResultsThe three markers (ER, Vim and CEA) and their respective panel expressions showed statistically significant (p < 0.05) frequency differences between ECA and EMA tumors. Although the additional ancillary PR-marker also revealed a significant frequency difference (p < 0.05) between ECA and EMA tumors, it did not demonstrate any supplementary benefit to the 3-marker panel.ConclusionAccording to our data, when histomorphological and clinical doubt exists as to the primary site of origin, we recommend that the conventional 3-marker (ER/Vim/CEA) panel is easier, sufficient and appropriate to use in distinguishing between primary ECA and EMA. Although the 4-marker panel containing PR also reveals statistically significant results, the PR-marker offers no supplemental benefit to the pre-existing 3-marker (ER/Vim/CEA) panel in the diagnostic distinction between ECA and EMA.

HER2 gene amplification in primary mucinous ovarian cancer: a potential therapeutic target

Sir: New cancer therapies can be directed at specific signalling pathways of tumour cells. Trastuzumab (Herceptin), a humanized monoclonal antibody that binds selectively to the HER2 protein, is currently being used in breast and some other types of tumours. The purpose of this study was to investigate whether HER2 could be a potential therapeutic target for the treatment of primary mucinous and non-mucinous epithelial ovarian cancer (EOC). Twenty-seven epithelial ovarian cancers, four mucinous and 23 non-mucinous, were analysed in a tissue microarray (TMA) study. In addition to its usefulness in surveys of specific biomarkers, TMA has become a popular tool for tissue-based research, because it allows for the massive acceleration of studies correlating molecular in-situ findings with clinicopathological information. However, the limitation of TMA seems to be its insufficiency in demonstrating tumour heterogeneity, because of the small sample size used. In 2007, the American Society of Clinical Oncology ⁄ College of American Pathologists (ASCO ⁄ CAP) established guidelines for the HER2 scoring algorithm for breast cancers. Although there are no valid data so far that correlate this HER2 scoring mechanism with clinical outcomes in ovarian cancers, we tried to apply the current ASCO ⁄ CAP guideline recommendations to interpret the results of HER2 HercepTest immunohistochemical (IHC) assays and PathVysion fluorescence in-situ hybridization (FISH) tests in EOC. The IHC results were evaluated by three pathologists (C.P.H., J.D.H. and H.C.) who did not know the patients’ clinical and FISH data. All of the FISH ⁄ IHC paired results confirmed that 19 of the 27 specimens gave concordant negative results and three gave concordant positive results. Of the remaining five specimens, all gave equivocal IHC results, whereas one gave FISH-positive and four gave FISHnegative results (Table 1). Surprisingly, we found that the overall rate of HER2 gene amplification was high in four of four (100%) mucinous EOC, but low in 0 out of 23 (0%) non-mucinous EOC. Figure 1A,B shows typical IHC and FISH results for one representative sample. The genetic basis for the oncogenic process of each histological subtype of EOC is very complex. For example, amplification and ⁄ or altered expression of oncogenes and tumour suppressor genes can play an important role. In 2003, Bookman et al. reported both a low frequency of HER2 overexpression (11.3%) and low response rates (7.3%) for single-agent trastuzumab therapy in patients with persistent or recurrent EOC in a phase II clinical trial. However, they applied oldfashioned semiquantitative IHC scoring scales, with a cut-off of more than 10% of tumour cells with total membranous rimmed staining. Additionally, they did not provide FISH data to confirm or validate the eligible cases, and there was no histological breakdown of their tested cases. Accordingly, we think that definitive HER2 data still remain inadequate for tumours originating from the ovarian surface epithelium. Until now, HER2 gene amplification has been the only established mechanism for the pathological HER-2 protein overexpression seen in human breast cancers. So far, no other common ⁄ fatal cancers have been studied as thoroughly or as comprehensively as breast cancer. Thus, adopting the more stringent ASCO ⁄ CAP guideline recommendations for breast cancer in this study, we investigated and evaluated the true state of HER2 overexpression in both mucinous and non-mucinous EOC. With careful exclusion of borderline and metastatic lesions, primary mucinous EOCs might be substantially less common than previously reported, and appear to constitute <3% of EOC. In this study, all four cases of mucinous EOC were strictly selected as primary ovarian tumours after careful review of the patients’ clinical histories of tumour staging, size and laterality, as well as vigilant performance of the appropriate immunohistochemical panel. Our results conclusively reveal that Table 1. Result categories of immunohistochemical (IHC) and fluorescence in-situ hybridization (FISH) tests obtained from 27 epithelial ovarian carcinomas

Assessing the impact of polysomy-17 on HER2 status and the correlations of HER2 status with prognostic variables (ER, PR, p53, Ki-67) in epithelial ovarian cancer: a tissue microarray study using immunohistochemistry and fluorescent in situ hybridization.

Although HER2 overexpression and Her2 amplification have been noted in breast and a variety of human cancers, we report here for the first time the impact of polysomy-17 on HER2 status and the correlations between HER2 status and other prognostic factors in patients with epithelial ovarian cancers (EOC).We analyzed HER2, estrogen receptor (ER), progesterone receptor (PR), p53, and Ki-67 protein overexpressions by immunohistochemistry (IHC) and determined Her2 gene amplification by fluorescence in situ hybridization (FISH) in 27 tissue microarray samples from EOC patients.We achieved 100% positive concordance (3/3) and 100% negative concordance (19/19) between HER2 testing by IHC and FISH. Both the total Her2 gene copies and FISH scores increased significantly in a stepwise order through the negative, equivocal, and positive HER2 IHC result categories in all 27 cases (P=0.001, P=0.001), and still increased significantly in 18 nonpolysomy-17 cases (P=0.007 and 0.013) after the exclusion of 9 polysomy-17 cases. HER2 protein expression is inversely correlated with both ER (P=0.002) and PR expressions (P=0.046). Her2 gene amplification is inversely correlated with ER expression (P=0.007) but not with PR expression (P=0.106).This study showed extremely high positive and negative concordances between Her2 FISH and HER2 IHC assays. Polysomy-17 is insufficient for causing a significant impact on the relationship between HER2 testing by IHC and FISH in EOC. ER and PR expressions were inversely correlated with HER2 protein expression. In addition, ER but not PR expression is inversely correlated with Her2 gene amplification.

p16INK4 and CEA can be mutually exchanged with confidence between both relevant three-marker panels (ER/Vim/CEA and ER/Vim/p16INK4) in distinguishing primary endometrial adenocarcinomas from endocervical adenocarcinomas in a tissue microarray study

The accurate distinction between primary endocervical adenocarcinomas (ECA) and endometrial adenocarcinomas (EMA) may require the use of multiple ancillary monoclonal antibodies in panels of immunohistochemistry stains. In addition to reappraising the expressions of four commonly used individual monoclonal antibodies [estrogen receptor (ER), Vimentin (Vim), carcinoembryonic antigen (CEA), and p16INK4], this study was designed to investigate whether CEA and p16INK4 can be effectively exchanged between two relevant three-marker panels (ER/Vim/CEA vs. ER/Vim/p16INK4) in distinguishing ECA from EMA. A tissue microarray was constructed using paraffin-embedded, formalin-fixed tissues from 35 hysterectomy specimens, including 14 ECA and 21 EMA. Utilizing the avidin–biotin technique, tissue array sections were immunostained with the four aforementioned individual markers (ER, Vim, CEA, and p16INK4). In addition to the four individual monoclonal antibodies, both their respective three-marker panels, proposed here, showed statistically significant (p < 0.05) frequency differences between these two gynecologic tumors (ECA vs. EMA). The panel performance and test effectiveness revealed that both three-marker panels are promising and very helpful. According to our data, when histomorphological and clinical doubt exists as to the primary site of origin, we recommend using either of these two conventional three-marker panels, which consist of ER/Vim/CEA and ER/Vim/p16INK4. CEA and p16INK4 can be interchanged with confidence without significantly influencing the panel presentations and efficiencies in distinguishing between adenocarcinomas of endocervical and endometrial origin.