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1.  Which is worse: uterine papillary serous carcinomas or carcinosarcomas? 
It is clear that uterine carcinosarcomas and uterine papillary serous carcinomas (UPSC) have an adverse impact on outcome, but whether carcinosarcomas are worse than UPSC is unclear. The purpose of this study is to compare the pathology, survival, and disease recurrence of patients with carcinosarcomas to patients with UPSC.
The medical records of patients diagnosed with carcinosarcomas and UPSC between 1996 and 2009 at Samsung Medical Center were retrospectively analyzed. Information from pathology reports, site of relapse, time to recurrence, and death was obtained. The survival analysis was performed using the Kaplan-Meier method.
Thirty seven patients with carcinosarcomas and 38 patients with UPSC were identified during the study period. There was no significant difference in clinical characteristics including age, body mass index, proportion with advanced stage disease, rate of optimal debulking, and adjuvant treatment used. In addition, the pathology showed no significant difference in tumor size, myometrial involvement, lymphovascular invasion, peritoneal cytology, cervical invasion, and lymph node involvement. Patients with carcinosarcomas had similar patterns of relapse as the patients with UPSC. There was no difference in the progression-free and overall survival between the carcinosarcomas and UPSC patients (p=0.804 and p=0.651, respectively).
Patients with carcinosarcomas had similar clinicopathological features compared to the patients with UPSC.
PMCID: PMC3152760  PMID: 21860733
Uterine carcinosarcomas; Uterine papillary serous carcinomas; Epithelial endometrial carcinomas
2.  Her-2 neu (Cerb-B2) expression in fine needle aspiration samples of breast carcinoma: A pilot study comparing FISH, CISH and immunocytochemistry 
Breast cancers with Her-2 neu gene amplification are recognized as important markers for aggressive disease and targets which respond to therapy with trastuzumab. Her-2 neu testing on histological sections is routinely performed to select patients who may benefit from anti- Her-2 neu therapy. Few reports are available which document Her-2 neu status on fine needle aspirates (FNA).
This pilot study is to document expression of Her-2 neu (Cerb-B2) on cytospin smears from FNA of patients with breast carcinoma.
Materials and Methods:
Twenty samples of FNA already collected for diagnostic purposes from patients with primary breast carcinoma were studied for demonstration of Her-2 neu expression by immunohistochemistry (IHC), Fluorescent in-situ hybridization (FISH) and chromogenic in-situ hybridization (CISH) on cytospin smears from FNA. Their expression was compared with tissue sections where possible.
Good correlation was observed between Her-2 neu protein expression and gene amplification in cytospin smears. Three of five (60%) breast carcinomas cases with 2+ and 3+ staining on IHC showed gene amplification by FISH and CISH. Three of 7 (43%) and 5 of 7 (71%) cases negative/1+ staining on IHC did not show gene amplification by FISH and CISH respectively. Tissue sections from 10 cases with 2+ and 3+ staining for Her2neu by IHC showed gene amplification in 8 cases.
Demonstration of Her-2 neu by IHC, FISH or CISH in FNA is possible and may play a role in the management of patients with advanced breast cancer or those cases where surgical resection is not advisable.
PMCID: PMC3111708  PMID: 21713147
Fine needle breast aspirates; FISH; Her-2 neu; Immunohistochemistry
3.  Comparison of Chromogenic In Situ Hybridisation with Fluorescence In Situ Hybridisation and Immunohistochemistry for the Assessment of Her-2/neu Oncogene in Archival Material of Breast Carcinoma 
The successful treatment of breast cancer is dependent upon a number of complex factors. Her-2/neu gene amplification is known to be one of the most common genetic alterations associated with breast cancer and its accurate determination has become necessary for the selection of patients for trastuzumab therapy.
The aim of this study was to prove the consistency of chromogenic in situ hybridisation (CISH) technique after analyzing the overexpression of the Her-2/neu proto-oncogene in 100 invasive breast carcinomas and by comparing CISH results with immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). Moreover, it was done to evaluate the possible correlation of estrogen (ERs) and progesterone receptors (PRs), the proliferation marker Ki67 and the tumour suppressor gene p53 with HER-2/neu status of these breast carcinomas.
Of the 100 breast carcinomas that were analysed, 22 cases showed HER-2/neu amplification, 66 cases showed no amplification, whereas 12 cases were non-interpretable in both assays (FISH and CISH). Consequently, the overall concordance between FISH and CISH was 100%. Additionally, it was observed that when HER-2/neu gene was overexpressed, there was an association with negative PRs and ERs status, negative p53 protein expression and high Ki67 labelling index.
It is concluded that patients with tumours scoring 2+ with the CBE356 antibody (borderline immunohistochemistry-tested cases) would also benefit from CISH as it is shown to be highly accurate, practical and can be easily integrated into routine testing in any histopathology laboratory. Finally, CISH represents an important addition to the HER2 testing algorithm.
PMCID: PMC2447915  PMID: 18636112
breast cancer; CISH; FISH; HER-2/neu
4.  Lack of prognostic relevance of Her-2/neu, topoisomerase IIα and EGFR in advanced ovarian carcinoma 
Patients with advanced ovarian cancer (FIGO stage III) have a poor clinical prognosis. However, these patients show distinct differences in their survival time, possibly due to differing responses to chemotherapy and differing tumor biology. In contrast to histological subtype, grading and staging, which are known to affect a patient’s prognosis, the impact of the human epidermal growth factor receptor 2 (Her-2/neu), topoisomerase IIα and epidermal growth factor receptor (EGFR) on survival remain inconclusive. Therefore, the aim of this study was to assess their impact on survival in a group of advanced ovarian cancer patients. Tissue microarrays were constructed from specimens of 243 patients. Gene copy and chromosome numbers were evaluated by fluorescence in situ hybridization (FISH) and protein expression by immunohistochemistry (IHC). Scoring for the latter was calculated by considering the percentage of positive tumor cells and the relative staining intensity. FISH results were evaluated by previously published recommendations and correlated with overall survival. Using IHC, 1.6% of the cases that were tested for Her-2/neu and topoisomerase IIα were strongly positive, and 12.3% were positive for EGFR. Using FISH, 4.4% amplifications and 2.1% polysomies for Her-2/neu were identified; topoisomerase IIα showed 2.2% amplifications, 0.4% deletions and 3.5% polysomies. We observed 10.8% high polysomies, but no amplification for EGFR. None of the results obtained by IHC or FISH correlated with overall survival. In general, Her-2/neu, topoisomerase IIα and EGFR may be prognostic factors in ovarian carcinomas. However, within this group of FIGO stage III patients, differences in gene aberration or protein expression were not able to predict differences in survival.
PMCID: PMC3438672  PMID: 22969977
advanced ovarian cancer; Her-2/neu; topoisomerase IIα; epidermal growth factor receptor; co-amplification; prognosis; fluorescence in situ hybridization
5.  Quantitation of HER2/neu Expression in Primary Gastroesophageal Adenocarcinomas Using Conventional Light Microscopy and Quantitative Image Analysis 
Human epidermal growth factor receptor 2 (HER2/neu) is overexpressed in a proportion of gastroesophageal (GE) adenocarcinomas, and trastuzumab treatment results in significant improvement in overall survival in patients with HER2/neu-overexpressing GE tumors. Grading of HER2/neu expression in GE tumors and its clinical application is different from that of breast cancer. HER2/neu immunohistochemistry (IHC) image analysis (IA), widely used in breast cancer, has not been studied in GE tumors.
To evaluate the correlation between manual HER2/neu IHC scoring and HER2/neu IHC image analysis in GE adenocarcinomas with characterization of associated clinicopathologic features.
Tumor grade, growth pattern, and stage were evaluated in 116 cases of primary GE adenocarcinoma biopsy and resection specimens. Using anti-HER2/neu antibody and the proposed HER2/neu scoring system for gastric cancer, HER2/neu IHC expression was recorded after manual scoring and automated IA interpretation.
HER2/neu overexpression (IHC 3+) was detected in 19% (10 of 54) of gastric tumors, and overall correlation between manual HER2/neu IHC interpretation and IA interpretation was 78% (42 of 54). HER2/neu overexpression (IHC 3+) was detected in 26% (16 of 62) of GE junction tumors, and the overall correlation between manual HER2/neu IHC interpretation and IA interpretation was 84% (52 of 62).
The HER2/neu IHC scoring system for GE adenocarcinomas differs from that of breast carcinoma. Automated IA, validated for scoring of HER2/neu IHC in breast cancer, has a low correlation between HER2/neu IHC 2+ and IHC 3+ cases scored by conventional light microscopy and cannot be reliably used in the interpretation of HER2/neu IHC expression in GE adenocarcinomas.
PMCID: PMC3806979  PMID: 22646266
6.  Clinical significance of Akt and HER2/neu overexpression in African-American and Latina women with breast cancer 
Breast cancer patients with HER2/neu overexpression have poor outcomes with a decrease in disease-free survival (DFS) and overall survival. The biology of HER2/neu overexpression in breast tumors in African-American and Latina women is poorly understood. The purpose of this study is to understand the clinical significance of activated Akt (phospho-Akt or pAkt) expression in breast tumors from African-American and Latina patients with corresponding tissue HER2/neu overexpression. Cellular and molecular studies have shown that activation of the cell signaling phosphatidylinositol-3-kinase/Akt cascade via the HER2/neu and other receptor tyrosine kinases induces cell proliferation.
A total of 234 African-American and Latina patients were selected retrospectively. From this group, 141 tumor tissue samples were analyzed for tissue pAkt by immunohistochemistry (IHC). This cohort consisted of 46 HER2/neu-positive (3+ by IHC) and 95 HER2/neu-negative tumors. The prognostic value of activated tissue Akt in relation to HER2/neu overexpression for DFS was determined.
Patients with low pAkt and HER2-negative tumors had the best DFS. As expected, HER2/neu-overexpressing tumors with low pAkt had a decrease in DFS. Similarly, those with high pAkt and HER2-negative tumors also had poor DFS. However, those with an increase in both HER2 and pAkt had the worst DFS. An increase in pAkt was significantly associated with HER2/neu-positive and lymph node-positive breast tumors. Tumors with high HER2 and high pAkt were metastatic. Multivariate analysis demonstrated that, in addition to the common risk factors such as larger tumor size, lymph node involvement, estrogen receptor/progesterone receptor-negative tumors, and HER2/neu-positive tumors, overexpression of pAkt significantly was associated with a decrease in 5-year DFS. A decrease in DFS with an increase in pAkt was observed in both HER2/neu-positive and -negative groups. However, the DFS was similar between HER2/neu-positive/pAkt-negative and HER2/neu-negative/pAkt-positive groups.
Our data suggest that there may be differences in tumor phenotypes within the HER2/neu-overexpressing breast cancer patients. The overexpression of pAkt may be a powerful prognostic marker for predicting DFS and overall survival of breast cancer patients.
PMCID: PMC2374954  PMID: 18184439
7.  Chromogenic in situ hybridization (CISH): a novel alternative in screening archival breast cancer tissue samples for HER-2/neu status 
Breast Cancer Research  2004;6(5):R593-R600.
Chromogenic in situ hybridization (CISH) is emerging as a practical, cost-effective, and valid alternative to fluorescent in situ hybridization in testing for gene alteration, especially in centers primarily working with immunohistochemistry (IHC).
We assessed Her-2/neu alteration using CISH on formalin-fixed paraffin-embedded primary invasive ductal carcinoma tumors in which IHC (CB11 antibody) had previously been performed, and we compared the results with IHC. The 160 selected cases were equally stratified randomly into the four IHC categories (scores of 0, 1+, 2+, and 3+). We also compared age at diagnosis and tumor histologic grade with IHC and CISH Her-2/neu.
We were able to perform and evaluate CISH successfully on all cases. The agreement between 3+ IHC and CISH-amplified cases as well as between all IHC and CISH Her-2/neu negative cases was 100%, and the concordance on all positive cases was 72.50%, with an overall agreement of 86.25%. All the discordant cases had 2+ IHC scores. Although we noted Her-2/neu positivity more in premenopausal women, the age at diagnosis was not significantly associated with IHC or CISH results. Similarly, although the small group of well-differentiated tumors was apparently Her-2/neu negative in both tests, no significant association was noted between any tumor histologic grade and either IHC or CISH results.
CISH is easily integrated into routine testing in our laboratory. It is a necessary adjunct in determining the subset of non-amplified IHC-positive invasive tumors that will not benefit from trastuzumab therapy. Those cases with 2+ IHC results will be triaged and subjected to CISH. Her-2/neu testing should be done on all breast cancer cases regardless of age at presentation and tumor histologic grade.
PMCID: PMC549176  PMID: 15318940
breast cancer; chromogenic in situ hybridization; fluorescence in situ hybridization; Her-2/neu; immunohistochemistry
8.  HER2/neu-directed therapy for biliary tract cancer 
Biliary cancers are highly aggressive tumors that are often diagnosed an advanced disease stage and have a poor outcome with systemic therapy. Recent efforts towards molecular characterization have identified a subset of biliary patients that have HER2/neu amplification or mutation. HER2/neu amplification is associated with response to HER2/neu-directed therapy in breast and gastric cancers. However, the efficacy of HER2/neu-targeted therapy in biliary cancers is unknown.
Patients and methods
We retrospectively reviewed cases of advanced gallbladder cancer and cholangiocarcinoma with HER2/neu genetic aberrations or protein overexpression who received HER2/neu-directed therapy between 2007 and 2014. Clinical data were retrieved from medical records, and imaging studies were independently reviewed.
Nine patients with gallbladder cancer and five patients with cholangiocarcinoma had received HER2/neu-directed therapy (trastuzumab, lapatinib, or pertuzumab) during the study period. In the gallbladder cancer group, HER2/neu gene amplification or overexpression was detected in eight cases. These patients experienced disease stability (n = 3), partial response (n = 4), or complete response (n = 1) with HER2/neu-directed therapy. One patient had HER2/neu mutation and experienced a mixed response after lapatinib therapy. The duration of response varied from 8+ to 168 weeks (median 40 weeks), and three patients are still on therapy. One patient developed HER2/neu amplification as a secondary event after FGFR-directed therapy for FGF3-TACC3 gene fusion. The cholangiocarcinoma cases treated in this series had a higher proportion of HER2/neu mutations, and no radiological responses were seen in these patients despite HER2/neu-directed therapy.
HER2/neu blockade is a promising treatment strategy for gallbladder cancer patients with gene amplification and deserves further exploration in a multi-center study.
PMCID: PMC4469402  PMID: 26022204
Receptor; ErbB-2; Gallbladder neoplasms; Cancer of the biliary tract
9.  HER-2/neu Oncogene Amplification by Chromogenic in situ Hybridization in 130 Breast Cancers Using Tissue Microarray and Clinical Follow-up Studies 
Journal of Korean Medical Science  2004;19(3):390-396.
Determining of HER-2/neu oncogene amplification has become clinically important for managing breast cancer. Fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) are currently regarded as the standard methods. Chromogenic in situ hybridization (CISH) was investigated as a new modification with an accurate, sensitive technique. From 1998 to 2002, using CISH and IHC, the amplification and protein expression of the HER-2/neu oncogene were examined using paraffin sections in 130 breast carcinomas and to determine the prognostic role of HER-2/neu for outcome after a follow-up of 24-64 months. Amplifications by CISH and overexpression by IHC were observed in 28 (22%) and 27 cases (20.8%), respectively. Of the 104 patients, 20 patients (19.2%) with amplification had a shorter disease-free interval (34.9 months vs. 38.0 months in controls) (p=0.372). 15 patients (14.4%) had a disease recurrence, but there is no significant difference between 3 patients amplifying the oncogene and 12 patients without oncogene (20.6 months vs. 19.6 months) (p=0.862). 6 patients (5.8%) of these died. CISH is a useful alternative, particularly for confirming the IHC results. There is no relationship between the early recurrence and the HER-2/neu positive group, but lymph node status was statistically significant.
PMCID: PMC2816840  PMID: 15201505
Breast Neoplasms; Receptor, erbB-2; In Situ Hybridization; Immunohistochemistry; Pro-tein Array Analysis
10.  Overexpression/amplification of HER-2/neu is uncommon in hepatocellular carcinoma 
Journal of Clinical Pathology  2005;58(5):500-503.
Background: Hepatocellular carcinoma (HCC) is one of the most prevalent fatal cancers in the world. Despite advances in early diagnosis and improvements in surgical techniques, the survival of patients with HCC even after resection is poor because of the high incidence of recurrences. Therefore, the identification of prognostic factors may be helpful in the development of new treatment protocols.
Aims: To investigate HER-2/neu status in HCC by immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH), and to explore the possibility of using trastuzumab in the treatment of HCC.
Methods: Eight hundred and sixty eight surgical samples from patients with primary HCC were examined for their HER-2/neu status. IHC for HER-2/neu was performed with the HercepTest kit; FISH analysis was performed with the PathVysion HER-2 DNA probe kit. The correlations between HER-2/neu overexpression and clinicopathological characteristics were analysed statistically.
Results: HER-2/neu overexpression was detected in 21 (2.42%) of the 868 primary HCCs. Only one specimen showed HER-2/neu gene amplification by FISH. No significant associations were found between HER-2/neu overexpression and the clinicopathological parameters.
Conclusions: There is a low frequency of HER-2/neu overexpression/amplification in HCC. There appears to be no role for HER-2/neu as a prognostic marker and no benefit of anti-HER-2/neu trastuzumab treatment in patients with HCC.
PMCID: PMC1770655  PMID: 15858121
HER-2/neu; hepatocellular carcinoma; fluorescence in situ hybridisation; immunohistochemistry
11.  Clinicopathological and prognostic significance of HER-2/neu and VEGF expression in colon carcinomas 
BMC Cancer  2011;11:277.
HER-2/neu and VEGF expression is correlated with disease behaviors in various cancers. However, evidence for their expression in colon cancer is rather contradictory both for the protein expression status and prognostic value. HER-2/neu is found to participate in VEGF regulation, and has known correlation with VEGF expression in some tumors. In this study, we investigated HER-2/neu and VEGF expression in Chinese colon patients and explored whether there was any correlation between their expression patterns.
HER-2/neu and VEGF were investigated immunohistochemically using tumor samples obtained from 317 colon cancer patients with all tumor stages. Correlation of the degree of staining with clinicopathological parameters and survival was investigated.
Positive expression rates of HER-2/neu and VEGF in colon cancer were 15.5% and 55.5% respectively. HER-2/neu expression was significantly correlated with tumor size and distant metastases (P < 0.05), but was not an independent prognostic marker of survival (P > 0.05). Expression of VEGF was significantly correlated with tumor size, tumor stage, lymph node metastases, and distant metastases (P < 0.05). The 5-year survival rate in patients with negative and positive VEGF expression was 70.2% and 61.9% respectively; the difference was not statistically significant (P = 0.146). No correlation between HER-2/neu and VEGF expression was detected (P = 0.151).
HER-2/neu and VEGF are not important prognostic markers of colon cancer. The present results do not support any association between HER2/neu and VEGF expression in this setting.
PMCID: PMC3144457  PMID: 21708009
12.  Correlation of human epidermal growth factor receptor 2 expression with clinicopathological characteristics and prognosis in gastric cancer 
AIM: To investigate human epidermal growth factor receptor 2 (HER2) gene amplification and protein expression in Chinese patients with resectable gastric cancer and the association with clinicopathological characteristics and survival.
METHODS: One hundred and ninety-seven gastric cancer patients who underwent curative surgery procedures were enrolled into this study. HER2 gene amplification and protein expression were examined using fluorescence in-situ hybridization (FISH) and immunohistochemistry (IHC) analysis on formalin-fixed paraffin-embedded gastric cancer samples from all patients. For scoring, Hofmann’s HER2 gastric cancer scoring system was adopted. All cases showing IHC3+ or FISH positivity were defined as HER2 positive. Patient clinicopathological data and survival information were collected. Finally, χ2 statistical analysis was performed to analyze the HER2 positivity rate amongst the subgroups with different clinicopathological characteristics including; gender, age, tumor location, Lauren classification, differentiation, TNM staging, depth of invasion, lymph node metastases and distant metastasis. The probability of survival for different subgroups with different clinicopathological characteristics was calculated using the Kaplan-Meier method and survival curves plotted using log rank inspection.
RESULTS: According to Hofmann’s HER2 gastric cancer scoring criteria, 31 cases (15.74%) were identified as HER2 gene amplified and 19 cases (9.64%) were scored as strongly positive for HER2 membrane staining (3+), 25 cases (12.69%) were moderately positive (2+) and 153 cases (77.66%) were HER2 negative (0/1+). The concordance rate between IHC and FISH analyses was 88.83% (175/197). Thirty-six cases were defined as positive for HER2 gene amplification and/or protein expression, with 24 of these cases being eligible for Herceptin treatment according to United States recommendations, and 29 of these cases eligible according to EU recommendations. Highly consistent results were detected between IHC3+, IHC0/1 and FISH (73.68% and 95.42%), but low consistency was observed between IHC2+ and FISH (40.00%). The positivity rates in intestinal type and well-differentiated gastric cancer were higher than those in diffuse/mixed type and poorly-differentiated gastric cancer respectively (28.57% vs 13.43%, P = 0.0103; 37.25% vs 11.64%, P < 0.0001), but were not correlated with gender, age, tumor location or TNM stage, depth of invasion, lymph node metastases and distant metastasis. In poorly-differentiated gastric cancer patients, those without lymph node metastasis showed a higher HER2 positivity rate than those with lymph node metastasis (26.47% vs 7.14%, P = 0.0021). This association was not present in those patients with well-differentiated gastric cancer (28.57% vs 43.33%, P = 0.2832). Within our patient cohort, 26 cases were lost to follow-up. The median survival time for the remaining 171 patients was 18 mo. The median survival times of the HER2 positive and negative groups were 17 and 18.5 mo respectively. Overall survival was not significantly different between HER2-positive and negative groups (χ2 = 0.9157, P = 0.3386), but in patients presenting well-differentiated tumors, the overall survival of the HER2-positive group was significantly worse than that of the HER2-negative group (P = 0.0123). In contrast, patients with poorly differentiated and diffuse/mixed subtype gastric cancers showed no significant differences in overall survival associated with HER2. Furthermore, the median survival time of the HER2 positive group did not show any statistically significant differences when compared to the subgroups of gender, age, tumor location, TNM classification, lymph node metastases and distant metastasis.
CONCLUSION: Patients with intestinal type gastric cancer (GC), well-differentiated GC and poorly-differentiated GC without lymph node metastasis, may all represent suitable candidates for targeted therapy using Herceptin.
PMCID: PMC3627881  PMID: 23599643
Gastric cancer; Human epidermal growth factor receptor 2; Gene amplification; Protein expression; Clinicopathological characteristics
13.  Prognosis of Women With Metastatic Breast Cancer by HER2 Status and Trastuzumab Treatment: An Institutional-Based Review 
Journal of Clinical Oncology  2009;28(1):92-98.
The purpose of this study was to determine whether trastuzumab improves prognosis of women with metastatic human epidermal growth factor receptor 2 (HER2)/neu –positive breast cancer beyond that of women with HER2/neu-negative disease.
Patients and Methods
Two thousand ninety-one women with metastatic breast cancer diagnosed from 1991 to 2007, with known HER2/neu status and who had not received trastuzumab in the adjuvant setting, were identified. Disease was classified into the following three groups: HER2/neu negative, HER2/neu positive without first-line trastuzumab treatment, and HER2/neu positive with first-line trastuzumab treatment. Overall survival (OS) was estimated using the Kaplan-Meier product-limit method and compared between groups with the log-rank test. Cox proportional hazards models were used to determine associations between OS and HER2/neu status after controlling for patient characteristics.
One hundred eighteen patients (5.6%) had HER2/neu-positive disease without trastuzumab treatment, 191 (9.1%) had HER2/neu-positive disease and received trastuzumab treatment, and 1,782 (85.3%) had HER2/neu-negative disease. Median-follow-up was 16.9 months. One-year survival rates among patients with HER2/neu-negative disease, HER2/neu-positive disease and trastuzumab treatment, and HER2/neu-positive disease and no trastuzumab treatment were 75.1% (95% CI, 72.9% to 77.2%), 86.6% (95% CI, 80.8% to 90.8%), and 70.2% (95% CI, 60.3% to 78.1%), respectively. In a multivariable model, women with HER2/neu-positive disease who received trastuzumab had a 44% reduction in the risk of death compared with women with HER2/neu-negative disease (hazard ratio [HR] = 0.56; 95% CI, 0.45 to 0.69; P < .0001). This HR varied with time and was significant for the first 24 months and not significant after 24 months.
Our results show that women with HER2/neu-positive disease who received trastuzumab had improved prognosis compared with women with HER2/neu-negative disease.
PMCID: PMC2799236  PMID: 19933921
14.  Evaluation of gene amplification and protein expression of HER-2/neu in esophageal squamous cell carcinoma using Fluorescence in situ Hybridization (FISH) and immunohistochemistry 
BMC Cancer  2009;9:6.
Esophageal squamous cell carcinoma (ESCC) is the sixth most frequent neoplasia in Brazil. It is usually associated with a poor prognosis because it is often at an advanced stage when diagnosed and there is a high frequency of lymph node metastases. It is important to know what prognostic factors can facilitate diagnosis, optimize therapeutic decisions, and improve the survival of these patients. A member of the epidermal growth factor receptor (EGFR) family, c-erbB-2, has received much attention because of its therapeutic implications; however, few studies involving fluorescence in situ hybridization (FISH) analysis of HER-2/neu gene amplification and protein expression in ESCC have been conducted. The aim of this study was to verify the presence of HER-2/neu gene amplification using FISH, and to correlate the results with immunohistochemical expression and clinical-pathological findings.
One hundred and ninety-nine ESCC cases were evaluated using the Tissue Microarray (TMA) technique. A polyclonal antibody against c-erbB-2 was used for immunohistochemistry. Analyses were based on the membrane staining pattern. The results were classified according to the Herceptest criteria (DAKO): negative (0/1+), potential positive (2+) and positive (3+). The FISH reactions were performed according to the FISH HER2 PharmDx (DAKO) protocol. In each case, 100 tumor nuclei were evaluated. Cases showing a gene/CEN17 fluorescence ratio ≥ 2 were considered positive for gene amplification.
The c-erbB-2 expression was negative in 117/185 cases (63.2%) and positive in 68 (36.8%), of which 56 (30.3%) were 2+ and 12 (6.5%) were 3+. No significant associations were found among protein expression, clinicopathological data and overall survival. Among the 47 cases analyzed, 38 (80.9%) showed no gene amplification while 9 (19.1%) showed amplification, as demonstrated by FISH. Cases that were negative (0/1+) and potential positive (2+) for c-erbB-2 expression by immunohistochemistry showed no gene amplification. However, all cases with gene amplification were positive (3+) by immunohistochemistry. According to univariate analysis, there was a significant difference (p = 0.003) in survival rates when cases with and without HER-2/neu amplification were compared.
Our data demonstrate the correspondence between gene amplification and protein expression of HER-2/neu. Gene amplification is an indicator of poor prognosis in ESCC.
PMCID: PMC2648997  PMID: 19128465
15.  Simultaneous detection of expression and gene mutations of HER2/neu in Chinese patients with gastric cancer 
Oncology Letters  2010;1(3):559-563.
HER2/neu is one of the few identified oncogenes in tumorigenesis. Attention has been focused on the potential effect of HER2/neu mutations in the tyrosine kinase domain on carcinoma-targeted therapy. However, data concerning HER2/neu mutations in Chinese patients with gastric cancer (GC) are limited. This study aimed to detect the expression and somatic mutations of HER2/neu in Chinese patients with GC. Immunohistochemical staining for HER2/neu was performed on 72 formalin-fixed, paraffin-embedded specimens of GC (40 intestinal and 32 diffuse type). The correlation between the overexpression of HER2/neu and clinicopathological parameters was statistically analyzed. Somatic mutations in the tyrosine kinase domain of HER2/neu in the 72 patients were detected by direct sequencing. In the GC group, overexpression of HER2/neu was detected in 13 of the 72 GC patients and in 4 of the 72 adjacent tissues in the non-tumorous group (18.1 vs. 5.6%, P<0.05). Furthermore, the intestinal type of GC exhibited a higher rate of HER2/neu overexpression than the diffuse type (29.7 vs. 5.7%, P<0.05). The rate of HER2/neu overexpression in stage III-IV (TNM stage) GC cases was significantly higher than that in stage I-II (28.2 vs. 6.6%, P<0.05). HER2/neu overexpression correlated with a significantly less favorable patient survival (P=0.046). No somatic mutations in the tyrosine kinase domain of HER2/neu were detected in tumor tissues or the corresponding non-tumorous ones in the specimens obtained from the 72 Chinese GC patients. Results suggest that overexpression of HER2/neu is a frequent molecular event strongly associated with a poor patient prognosis, whereas the incidence of somatic mutations of the HER2/neu kinase domain is more likely a low-frequency event in Chinese GC patients.
PMCID: PMC3436403  PMID: 22966343
HER2/neu; gastric cancer; gene mutation; overexpression
16.  HER Receptor Family: Novel Candidate for Targeted Therapy for Gallbladder and Extrahepatic Bile Duct Cancer 
Biliary tract cancers (BTC) are uncommon in the United States, but are endemic in parts of South America and Asia. BTCs are aggressive tumors associated with poor survival. Activation of HER-2/neu (erbB2) and/or epidermal growth factor receptor (EGFR) are important in breast, colon, and lung cancers. Tumor specimens from patients from the United States and Chile were examined for expression of HER-2/neu, EGFR, and their activated forms (p-erbB2, p-EGFR).
Materials and Methods
Specimens from 77 gallbladder cancers (GBC), 16 extrahepatic bile duct cancers (EHBDC), 21 intrahepatic bile duct cancers (IHBDC), 11 cases of cholecystitis (CHOLE), and 8 normal gallbladders (NGB) were examined for HER-2/neu, p-erbB2, EGFR, and p-EGFR expression by immunohistochemistry (IHC), with scores of 2+ or 3+ defined as positive. HER-2/neu gene amplification was analyzed by double color HER-2/neu gene/chromosome 17 centromere (CEP17) fluorescence in situ hybridization (FISH) assays
HER-2/neu-positive IHC staining was found in 31.2% of GBC, 31.3%, of EHBDC, and 33.3% of IHBDC; 12.5% of CHOLE specimens showed 2+ staining and the remaining CHOLE and NGB were negative. HER-2/neu gene amplification was detected in 20.9% of GBC, 21.4% of EHBDC, and none of IHBDC. There was a significant correlation between IHC 2+ and 3+ and gene amplification (P =.0001).
HER-2/neu amplification was identified in more than 20% of GB and EHBDC. There was strong correlation between HER-2/neu IHC and FISH positivity. These findings indicate a role for HER-2/neu in some subsets of BTC, and provide a rationale for study of HER-2/neu-directed therapies in this setting.
PMCID: PMC2631213  PMID: 19262900
17.  IHC for Her2 with CBE356 antibody is a more accurate predictor of Her2 gene amplification by FISH than HercepTest™ in breast carcinoma 
Journal of Clinical Pathology  2005;58(10):1086-1090.
Background: Her2 (c-erbB-2/neu) overexpression in breast carcinoma predicts response to the anti-Her2 monoclonal antibody, trastuzumab, and is associated with a poor prognosis. When considering patients for trastuzumab treatment, Her2 protein expression is measured by imunohistochemistry (IHC) and, where staining is equivocal, by fluorescence in situ hybridisation (FISH) detection of Her2 gene amplification.
Aims: To compare IHC using CBE356 with IHC using the Food and Drug Administration approved HercepTest™.
Methods: CBE356 and HercepTest were analysed using 167 FISH characterised breast carcinomas. Immunohistochemical expression of Her2 was measured semiquantitatively. Sensitivity, specificity, predictive values, and overall accuracy were calculated for both IHC methods using gene amplification by FISH as the end point, and IHC and FISH assays were tested in Kaplan–Meier survival analysis.
Results: The accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of CBE356 positive (2+ and 3+) cases were 94%, 89%, 95%, 84%, and 97%, respectively, and of HercepTest positive (2+ and 3+) cases were 91%, 66%, 98%, 92%, and 91%, respectively. A positive result with CBE356, HercepTest, or FISH was associated with significantly decreased overall survival (log rank p = 0.005, p = 0.0017, and p = 0.0005, respectively).
Conclusions: Positive IHC staining for Her2 using CBE356 is 3% more accurate and 23% more sensitive at predicting Her2 gene amplification by FISH than positive staining with HercepTest. Negative IHC using CBE356 antibody is 6% more likely to represent a truly negative result than negative staining with HercepTest. Overall, CBE356 was a more accurate predictor of Her2 gene amplification by FISH than HercepTest.
PMCID: PMC1770743  PMID: 16189156
CBE356; immunohistochemistry; Her expression; breast carcinoma
18.  Estrogen receptor, progesterone receptor, and Her 2 Neu positivity and its association with tumour characteristics and menopausal status in a breast cancer cohort from northern Pakistan 
ecancermedicalscience  2012;6:283.
To measure the frequency of estrogen receptor (ER), progesterone receptor (PR), and Her 2 Neu positivity and to study their association with tumour characteristics and menopausal status in a breast cancer cohort from Northern Pakistan.
Patients attending NORI with histopathologically proven diagnosis of invasive ductal carcinoma of the breast were included after providing informed consent. Age, menopausal status, and tumour size were recorded. The presence or absence of nodal involvement (including site and number), distant metastases, sites of metastases, skin involvement, chest wall involvement, recurrent disease, and bilateral breast cancer were noted along with ER, PR, and Her 2 Neu status. Mean ± standard deviations were calculated for continuous variables like age. Frequency and percentage were calculated for categorical variables like ER, PR, and Her 2 Neu status. Association of ER, PR, and Her 2 Neu status with menopausal status and tumour-related characteristics were sought employing t test, x2 square test, and ANOVA wherever appropriate. P value >0.05 was considered significant.
There were 1226 patients included in the study. Mean patient age was 48.04 ± 11.97 years, and 743 (60.6%) patients were premenopausal. ER, PR, and Her 2 Neu were found positive in 763 (62.2%), 738 (60.1%), and 478 (38.9%) patients, respectively. Significant association (P < 0.05) was found between ER, PR positivity, and Her 2 Neu over expression with menopausal status, tumour size, involvement of skin, chest wall and lymph nodes and the presence of distant metastases. However, no significant association was detected between ER, PR, Her 2 Neu and recurrent disease.
The frequency of expression of hormonal receptors in breast cancer patients from Northern Pakistan is the same as reported in the literature although overexpression of Her 2 Neu is a little higher in our population. There is an inverse relationship between hormonal receptors expression and Her 2 Neu expression. Postmenopausal women have a higher incidence of ER and PR positivity and Her 2 Neu negativity. ER and PR negativity and Her 2 Neu positivity are associated with more advanced disease and poor outcome.
PMCID: PMC3530377  PMID: 23304239
estrogen receptor; progesterone receptor; Her 2 Neu oncogene; postmenopausal; overall survival
19.  Prognosis significance of HER-2/neu overexpression/amplification in Chinese patients with curatively resected gastric cancer after the ToGA clinical trial 
HER-2/neu-targeted therapy has been successfully used in advanced gastric cancer, but the role of HER-2/neu in the prognosis of gastric cancer is not yet clear. In this study, we investigated the correlation between HER-2/neu expression and amplification as well as their association with clinic outcomes in patients with curatively resected gastric cancer.
We constructed tissue microarray blocks containing >70% of gastric cancer tissue and matched adjacent normal gastric tissue for 227 patients. Expression of the HER-2/neu protein in these specimens was analyzed using immunohistochemical staining. Amplification of HER-2/neu was also analyzed for the same samples using fluorescence in situ hybridization. Data on clinicopathological features and relevant prognostic factors in these patients were analyzed.
Of the 227 gastric cancer samples, 11.89% were positive for HER-2/neu overexpression/amplification under the new scoring system. HER-2/neu overexpression/amplification was closely correlated to the Lauren type, degree of differentiation, tumor size and lymph node metastasis. HER-2/neu overexpression/amplification predicted poor survival in univariate analysis but not in a Cox proportional hazards model.
HER-2/neu overexpression/amplification was not an independent predictor for survival in patients with curatively resected gastric cancer.
PMCID: PMC3579675  PMID: 23249720
FISH; Gastric cancer; HER-2/neu; IHC; Prognosis
20.  HER-2/neu and CD117 (c-kit) overexpression in patients with pesticide exposure and extensive stage small cell lung carcinoma (ESSCLC) 
The rate of detection of HER-2/neu and CD117 (c-kit) overexpression in small cell lung cancer (SCLC) has varied widely; between 5–35% and 21–70% respectively.
To evaluate the relationship between pesticide exposure and HER-2/neu and CD117 overexpression in extensive stage SCLC (ESSCLC), we identified patients with ESSCLC and assessed pesticide exposure using a predetermined questionnaire. An exposure index (hours/day × days/year × years) ≥ 2400 hours was considered as 'exposed.' HER-2/neu overexpression was evaluated on archival tissue using the DAKO Hercep test, and CD117 testing was performed using immunohistochemistry (A4052 polyclonal antibody).
193 ESSCLC patients were identified. Pesticide exposure data could be obtained on 174 patients (84 females and 109 males) with a mean age of 68.5 years. 53/174 (30.4%) revealed HER-2/neu overexpression. 54/174 (31.03%) specimens showed CD117 overexpression by IHC. On multivariate analysis, HER-2/neu overexpression was associated with diminished survival (p < 0.001). In comparison, CD117 expression did not have an adverse prognostic value (p = 0.025). 41/53 (77.4%) patients with HER-2/neu overexpression and 47/121 (38.8%) patients without overexpression had exposure to pesticides (odds ratio: 5.38; p < 0.01). Among the cohort tested for CD117, 29/54 (53.7%) patients with CD117 overexpression and 59/120 (49.2%) patients without CD117 overexpression had pesticide exposure (odds ratio: 1.18; p = 0.12).
Pesticide exposure affects HER-2/neu but not CD117 overexpression. Future studies are needed to determine specific pesticide(s)/pesticide components that are responsible for HER-2/neu overexpression in ESSCLC, and to validate our findings in other solid tumors that overexpress HER-2/neu.
PMCID: PMC1180461  PMID: 15946380
Pesticide exposure; HER-2/neu overexpression; CD117 overexpression; c-kit overexpression; extensive stage small cell lung cancer; survival; epidemiology.
21.  Low frequency of HER2 amplification and overexpression in early onset gastric cancer 
Cellular Oncology (Dordrecht)  2011;34(2):89-95.
The recent ToGA trial results indicated that trastuzumab is a new, effective, and well-tolerated treatment for HER2-positive gastric cancer (GC). Although GC mainly affects older patients, fewer than 10% of GC patients are considered early-onset (EOGC) (presenting at the age of 45 years or younger). These EOGC show different clinicopathological and molecular profiles compared to late onset GC suggesting that they represent a separate entity within gastric carcinogenesis. In light of potential trastuzumab benefit, subpopulations of GC such as EOGC (versus late onset) should be evaluated for their frequency of amplification and overexpression using currently available techniques.
Tissue microarray (TMA) blocks of 108 early onset GC and 91 late onset GC were stained by immunohistochemistry (IHC, Hercep test, DAKO) and chromogenic in situ hybridization (CISH, SPoT-Light, Invitrogen).
Overall, we found only 5% HER2 high level amplification and 3% HER2 3+ overexpression (6/199). In addition, 8 patients (4%) showed a low level CISH amplification and 9 patients (4.5%) showed a 2+ IHC score. IHC and CISH showed 92% concordance and CISH showed less heterogeneity than IHC. In 2/199 cases (1%), IHC showed clinically relevant heterogeneity between TMA cores, but all cases with focal IHC 3+ expression were uniformly CISH high level amplified. Early onset GCs showed a significantly lower frequency of HER2 amplification (2%) and overexpression (0%) than late onset GCs (8% and 7% respectively) (p = 0.085 and p = 0.008 respectively). Proximal GC had more HER2 amplification (9% versus 3%) and overexpression (7% versus 2%) than distal tumours although this difference was not significant (p = 0.181 and p = 0.182 respectively). HER2 CISH showed more high level amplification in the intestinal type (7%, 16% if low-level included) compared to the mixed (5%, 5% if low-level included) and diffuse type (3%, 4% if low-level included) GCs (p = 0.029). A similar association was seen for HER2 IHC and histologic type (p = 0.008). Logistic regression indicated a significant association between HER2 expression and age, which remained significant when adjusted for both location and histological type.
Even focal HER2 overexpression in GC points to uniform HER2 amplification by CISH. We show for the first time that early onset GC has a lower frequency of HER2 amplification and overexpression than late onset GC, and confirm that intestinal type GC shows the highest rate of HER2 amplification and overexpression.
PMCID: PMC3063579  PMID: 21394646
HER2; Gastric cancer; Immunohistochemistry; In situ hybridization
22.  PAM50 Breast Cancer Subtyping by RT-qPCR and Concordance with Standard Clinical Molecular Markers 
BMC Medical Genomics  2012;5:44.
Many methodologies have been used in research to identify the “intrinsic” subtypes of breast cancer commonly known as Luminal A, Luminal B, HER2-Enriched (HER2-E) and Basal-like. The PAM50 gene set is often used for gene expression-based subtyping; however, surrogate subtyping using panels of immunohistochemical (IHC) markers are still widely used clinically. Discrepancies between these methods may lead to different treatment decisions.
We used the PAM50 RT-qPCR assay to expression profile 814 tumors from the GEICAM/9906 phase III clinical trial that enrolled women with locally advanced primary invasive breast cancer. All samples were scored at a single site by IHC for estrogen receptor (ER), progesterone receptor (PR), and Her2/neu (HER2) protein expression. Equivocal HER2 cases were confirmed by chromogenic in situ hybridization (CISH). Single gene scores by IHC/CISH were compared with RT-qPCR continuous gene expression values and “intrinsic” subtype assignment by the PAM50. High, medium, and low expression for ESR1, PGR, ERBB2, and proliferation were selected using quartile cut-points from the continuous RT-qPCR data across the PAM50 subtype assignments.
ESR1, PGR, and ERBB2 gene expression had high agreement with established binary IHC cut-points (area under the curve (AUC) ≥ 0.9). Estrogen receptor positivity by IHC was strongly associated with Luminal (A and B) subtypes (92%), but only 75% of ER negative tumors were classified into the HER2-E and Basal-like subtypes. Luminal A tumors more frequently expressed PR than Luminal B (94% vs 74%) and Luminal A tumors were less likely to have high proliferation (11% vs 77%). Seventy-seven percent (30/39) of ER-/HER2+ tumors by IHC were classified as the HER2-E subtype. Triple negative tumors were mainly comprised of Basal-like (57%) and HER2-E (30%) subtypes. Single gene scoring for ESR1, PGR, and ERBB2 was more prognostic than the corresponding IHC markers as shown in a multivariate analysis.
The standard immunohistochemical panel for breast cancer (ER, PR, and HER2) does not adequately identify the PAM50 gene expression subtypes. Although there is high agreement between biomarker scoring by protein immunohistochemistry and gene expression, the gene expression determinations for ESR1 and ERBB2 status was more prognostic.
PMCID: PMC3487945  PMID: 23035882
23.  Clinicopathologic significance of HER-2/neu protein expression and gene amplification in gastric carcinoma 
AIM: To study the HER-2/neu protein expression and gene amplification in gastric carcinoma and their relation.
METHODS: One hundred and forty-five formalin-fixed and paraffin- embedded tumor tissue samples from Chinese gastric carcinoma patients were studied with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) methods. Clinicopathologic data about all patients were collected.
RESULTS: The levels of HER-2 3+, HER-2 2+ and HER2 1+ were measurable in 6.9%, 8.3% and 17.2% of the samples, respectively. No HER-2 was stained in 67.6% of the samples. FISH showed that HER-2 gene was amplified in 18 samples, 10 HER-2 3+ samples, 5 HER-2 2+ samples, and 3 HER-2 1+ samples with IHC staining. HER-2 status was not correlated with the sex and age of patients, and tumor size, location or differentiation, but with the depth of invasion, TNM stage, lymph node and distant metastasis as well as histopathological classification of gastric cancer (P < 0.05).
CONCLUSION: All samples with IHC as HER-2 expression should be analyzed with FISH. Detection of HER-2 gene amplification can assess the malignant biological behaviors and prognosis of gastric cancer.
PMCID: PMC3070026  PMID: 21472111
Gastric carcinoma; HER-2; Clinocopathologic significance; Immunohistochemistry; Fluorescence in situ hybridization
24.  An analysis of current treatment practice in uterine papillary serous and clear cell carcinoma at two high volume cancer centers 
Despite the rarity of uterine papillary serous carcinoma (UPSC) and uterine clear cell carcinoma (UCCC), they contribute disproportionately to endometrial cancer deaths. Sufficient clinical information regarding treatment and prognosis is lacking. The aim of this study is to evaluate treatment outcomes in a rare cancer cohort based on the experience at two tertiary care cancer centers.
Clinicopathologic data were retrospectively collected on 279 patients with UPSC and UCCC treated between 1995 to 2011. Mode of surgery, use of adjuvant treatment, and dissection of paraaoritc lymph nodes were evaluated for their association with overall survival (OS) and progression-free survival (PFS).
40.9% of patients presented with stage I disease, 6.8% of patients presented with stage II disease and 52.3% of patients presented with stages III and IV. Median follow-up was 31 months (range, 1 to 194 months). OS and PFS at 5 years were 63.0% and 51.9%, respectively. OS and PFS were not affected by mode of surgery (open vs. robotic approach; OS: hazard ratio [HR], 0.68; 95% confidence interval [CI], 0.28 to 1.62; PFS: HR, 0.78; 95% CI, 0.40 to 1.56). Adjuvant treatment was associated with improved OS in stages IB-II (HR, 0.14; 95% CI, 0.02 to 0.78; p=0.026) but not in stage IA disease. There was no difference in OS or PFS based on the performance of a paraaoritc lymph node dissection.
Minimally invasive surgical staging appears a reasonable strategy for patients with non-bulky UPSC and UCCC and was not associated with diminished survival. Adjuvant treatment improved 5-year survival in stages IB-II disease.
PMCID: PMC4302281  PMID: 25376917
Adenocarcinoma, Clear Cell; Disease-free Survival; Endometrial Neoplasms; Lymph Node Excision; Retrospective Studies
25.  Human epidermal growth factor receptor-2 gene amplification in gastric cancer using tissue microarray technology 
AIM: To assess human epidermal growth factor receptor-2 (HER2)-status in gastric cancer and matched lymph node metastases by immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH).
METHODS: 120 cases of primary gastric carcinomas and 45 matched lymph node metastases from patients with full clinicopathological features were mounted onto multiple-punch and single-punch tissue microarrays, respectively, and examined for HER2 overexpression and gene amplification by IHC and CISH.
RESULTS: Twenty-four tumors (20%) expressed HER2 immunohistochemically. An IHC score of ≥ 2+ was observed in 20 tumors (16.6%). HER2 amplification was detected by CISH in 19 tumors (15.8%) and in their matched lymph node metastases. A high concordance rate was found between HER2 positivity (as detected by IHC) and HER2 gene amplification (as detected by CISH), since 19 of the 20 IHC positive cases were amplified (95%). All amplified cases had 2+ or 3+ IHC results. Amplification was associated with intestinal phenotype (P < 0.05). No association with grading, staging or survival was found.
CONCLUSION: In gastric cancer, HER2 amplification is the main mechanism for HER2 protein overexpression and is preserved in lymph node metastases.
PMCID: PMC3257442  PMID: 22253521
Human epidermal growth factor receptor-2; Immunohistochemistry; Chromogenic in situ hybridization; Gastric cancer

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