Correlation of Mammographic Calcifications with HER-2/neu Overexpression in Primary Breast Carcinomas

HER-2/neu is a valuable therapeutic and prognostic marker in primary breast carcinomas. The aim of this study was to evaluate the association between mammographic calcifications and HER-2/neu overexpression in primary breast carcinomas and assess its clinical perspective. A retrospective study of 152 preoperative mammograms in patients with breast carcinoma was performed. Expression of HER-2/neu was determined by immunohistochemical staining on 152 tissues that comprised specimens of 11 ductal carcinoma in situ (DCIS) and 141 primary invasive carcinomas. Mammographic calcifications were evaluated according to the Breast Imaging Reporting and Data System (BI-RADS), fourth edition. Calcifications were found in 73 (48.0%) out of 152 patients by mammography finding. Calcifications were more common in carcinomas with HER-2/neu overexpression (45:73, 61.6%) than in those without HER-2/neu overexpression (28:79, 35.4%; P = 0.001). Of the 73 carcinomas with calcifications on mammography, mass with spiculated margin as an associated finding of calcifications was more significantly frequent in carcinomas with HER-2/neu overexpression (15 of 45, 33.3%) than in those without HER-2/neu overexpression (2 of 28, 7.1%; P = 0.036). Fine linear morphology was more common in carcinomas with HER-2/neu overexpression (15:45, 33.3%) when compared with those without HER-2/neu overexpression (2:28, 7.1%; P = 0.036). Clustered distribution of calcifications was more common in carcinomas with HER-2/neu overexpression (26:45, 57.8%) compared with carcinomas without HER-2/neu overexpression (6:28, 21.4%; P = 0.048). Mammographic calcifications are correlated with HER-2/neu overexpression in primary breast carcinomas. Calcifications detected during screening mammography are not only of diagnostic value but of use in determining therapeutic options and prognosis.     

Differential gene expression patterns in HER2/neu-positive and -negative breast cancer cell lines and tissues

Overexpression of the oncogene HER2/neu (c-erbB-2) occurs in up to 30% of breast cancers and is correlated with reduced survival, especially in node-positive disease. The aim of this study was to identify genes associated with the aggressive phenotype of HER2/neu-positive breast cancer cells using cDNA microarrays. RNA was extracted from three HER2/neu-positive and three HER2/neu-negative breast cancer cell lines. Pooled RNA was hybridized in duplicate to the breast specific microarray filters from Research Genetics containing 5184 unique cDNAs. Subsequently, a similar comparison was performed for pooled RNAs from 10 node-positive, ER-positive invasive ductal carcinomas, half of which were HER2/neu overexpressers. In HER2/neu overexpressing breast cancer cell lines, 90 (1.7%) genes were up-regulated and 46 (0.9%) were down-regulated, compared to cell lines with low HER2/neu protein levels. In contrast, in HER2/neu overexpressing primary breast cancers, more genes were down-regulated (N = 132, 2.5%) than up-regulated (N = 19, 0.4%). Many of the differentially expressed genes have previously not been known to play a role in human neoplasia, and some of them may represent novel tumor suppressor or oncogenes. No genes were up-regulated, and only a small number of genes were down-regulated both in cell lines and in carcinomas with high HER2/neu protein levels. These included transforming acidic coiled-coil containing protein 1, glycogen phosphorylase BB, complement 1q and one EST. The differential expression of select genes was confirmed by Northern blotting (trefoil factor 3) or by immunocytochemistry (glycogen phosphorylase BB, vimentin, KAI1). In an extended validation study, 18 of 41 ER-negative, but none of 46 ER-positive, breast carcinomas were found to express vimentin, and all but one of the vimentin-positive tumors were confined to the HER2/neu-negative subgroup (P = 0.0019). Our findings support an important role of the mammary stroma in determining the clinical breast cancer phenotype.     

Expression of E-cadherin and c-erbB-2/HER-2/neu oncoprotein in high-grade breast cancer

E-cadherin (E-CD) is an epithelial-specific cell adhesion molecule, whose expression is lost in invasive lobular (ILC) but not in invasive ductal carcinoma (IDC) of the breast. This cell adhesion system can be disrupted by tyrosine kinase c-erbB-2/HER-2/neu. We examined 106 cases of high-grade invasive breast cancer, including 91 IDCs, 12 ILCs and 3 pleomorphic lobular carcinomas (PLCs). We determined Nottingham histological grade and performed immunohistochemistry for estrogen and progesterone receptors (ER/PR), Ki-67, E-CD and c-erbB-2/HER-2/neu with subsequent fluorescence in situ hybridization. Amplification of c-erbB-2/HER-2/neu gene was observed in 55/91 (60.4%) of IDCs, 3/12 (25%) of ILCs and 1/3 (33.3%) of PLCs, and associated with positive axillary lymph nodes. E-CD expression was lost in 14/91 (15.4%) of IDCs, 10/12 (83.3%) of ILCs and 2/3 (66.7%) of PLCs. The loss of E-CD immunoreactivity in IDCs appeared to be associated with c-erbB-2/HER-2/neu gene amplification, negative ER/PR status and positive lymph nodes, whereas E-CD-positive ILCs tended to be HER-2/neu-positive. The biological significance of E-CD expression seems to be different in high-grade IDC and ILC. Oncogenic pathway mediated by c-erbB-2/HER-2/neu may affect the E-CD expression in most invasive ductal breast carcinomas in vivo.     

Assessment of HER‐2/neu overexpression and/or gene amplification in breast carcinomas: should in situ hybridization be the method of choice?

AIMS: Since the release of Herceptin, pathology laboratories have been requested to test breast carcinomas for HER-2/neu overexpression and/or gene amplification. Standardized IHC and FISH are mandatory in order to get reliable results, but there are problems even with standardized procedures. We decided to evaluate the two methods to determine which, or possibly if both, should be the primary investigation method(s). METHODS AND RESULTS: The material consisted of 215 primary invasive breast carcinomas with complete clinical follow-up of 15 years. HER-2/neu protein expression was determined for all specimens, whereas FISH for assessing HER-2/neu gene signal number was done in 165 cases. IHC was double-checked with two or three different antibodies in 35 tumours, including all cases with discrepancies between IHC and FISH. Among these, there were discrepancies in a third. IHC overexpression of HER-2/neu was found in 13% and gene amplification in 18%. Discordance between IHC and FISH was found in 11 cases (8%). Five tumours were IHC+/FISH- and six were IHC-/FISH+. IHC and FISH positive cases as well as FISH only positive tumours had the same prognosis respecting survival. Tumours with >2 but 4 gene signals per nucleus. In contrast, cases with IHC overexpression without gene amplification had a prognosis similar to that of IHC-/FISH- tumours. CONCLUSIONS: From our data, it seems to be more important to assess HER-2/neu gene amplification than IHC overexpression. Failure to detect FISH-amplified (IHC-negative) cases would have an adverse effect on the survival of these patients. On the other hand, IHC overexpression tumours without gene amplification appear to belong to a better prognostic group, and failure to detect them would probably not have a negative effect on the survival of these women. Even though FISH is a more complex and expensive procedure, it should be considered the method of choice for primary assessment of HER-2/neu status in breast cancer patients.     

High-level SLP-2 expression and HER-2/neu protein expression are associated with decreased breast cancer patient survival

There is sufficient evidence that human stomatin-like protein 2 (SLP-2) is a novel cancer-related gene. Its protein is overexpressed in many human cancers. SLP-2 can contribute to the promotion of cell growth, cell adhesion, and tumorigenesis in esophageal squamous cell carcinoma and lymph node metastasis in laryngeal squamous cell carcinoma. Immunohistochemical detection of SLP-2, estrogen and progesterone receptors, and HER-2/neu were performed on 263 cases of primary invasive breast cancer with a tissue microarray. Of 263 cases, 138 (52.5%) showed high expression of SLP-2 protein, and 125 (47.5%) showed low or absent expression. In addition, there were significant positive associations between tumor stage and size (P = .020), lymph node metastasis (P < .001), clinical stage (P < .001), distant metastasis (P = .002), and HER-2/neu protein expression (P = .037) and high-level SLP-2 expression. High-level SLP-2 expression was associated with decreased overall survival (P = .011) and was more often found in patients with tumors larger than 20 mm, lymph node metastasis, advanced clinical stage, distant metastasis, and HER-2/neu protein-positive expression. More important, lymph node metastasis, HER-2/neu-positive expression, and high-level SLP-2 expression were associated with significantly decreased survival.     

Intratumoral heterogeneity of her-2/neu in invasive mammary carcinomas using fluorescence in-situ hybridization and tissue microarray

Fluorescence in-situ hybridization is increasingly being used to determine HER-2/neu status in patients with breast carcinoma. The possibility that intratumoral heterogeneity of HER-2/neu gene amplification may potentially contribute to inaccurate assessment of HER-2/neu status was investigated in routine cases of invasive mammary carcinomas. From 169 representative formalin-fixed, paraffin-embedded blocks of invasive duct mammary carcinomas with grade 3 architecture, 48 cases were analyzed by fluorescence in-situ hybridization and 59 analyses were performed. Intratumoral heterogeneity for HER-2/neu gene amplification was demonstrated in only 5 (16%) of 31 cases where morphologically similar areas of a single tumor were analyzed. We conclude from this study that intratumoral heterogeneity of HER-2/neu gene amplification is a demonstrable but relatively uncommon occurrence. For invasive mammary carcinomas, the accurate assessment of HER-2/neu status by fluorescence in-situ hybridization analysis is not significantly confounded by intratumoral heterogeneity of HER-2/neu gene amplification in individual tumors.     

Overexpression of HER-2/neu and its relationship with other prognostic factors change during the progression of in situ to invasive breast cancer.

Using permanent-section immunohistochemistry, we investigated the role of HER-2/neu in the development and progression of human breast cancer by measuring its overexpression in a series of hyperplastic (n = 30), dysplastic (n = 15), and malignant neoplastic (n = 708) lesions of ductal epithelium and by evaluating the relationships between overexpression and clinicopathologic features known to have prognostic significance in these lesions. The neoplasms included pure ductal carcinoma in situ (DCIS; n = 59) and infiltrating ductal carcinoma (IDC; n = 649). The latter were all node negative and stratified into IDC combined (n = 237) or not combined (n = 412) with a "significant amount" of DCIS (defined as DCIS greater than or equal to 10% of total tumor cellularity). Overexpression of HER-2/neu was not observed in any of the hyperplastic or dysplastic lesions. In contrast, it was present in 56% of pure DCIS and in 77% of the comedo subtype of this group. Only 15% of IDC overexpressed HER-2/neu. However, the rate of overexpression was significantly higher in the subset of IDC combined with DCIS compared with the subset of IDC not combined with DCIS (22% v 11%, respectively; P less than .0001). These results are consistent with the hypothesis that HER-2/neu plays a more important role in initiation than in progression of ductal carcinomas. They also suggest that overexpression decreases within individual tumors as they evolve from in situ to increasingly invasive lesions or, alternatively, that many invasive carcinomas arise de novo (ie, without progressing through a significant in situ stage) by mechanisms not involving HER-2/neu. In addition, overexpression of HER-2/neu was associated with several poor prognostic features (younger patient age, premenopause, negative estrogen receptor status, negative progesterone receptor status, and high nuclear grade) in the subset of IDC combined with DCIS. With one exception (negative estrogen receptor status) these associations were lost in IDC not combined with DCIS, also suggesting that the role of HER-2/neu changes during the progression of human breast cancer.     

Aneusomy 17 in breast cancer: its role in HER-2/neu protein expression and implication for clinical assessment of HER-2/neu status.

HER-2/neu protein overexpression in breast cancer is mostly caused by HER-2/neu gene amplification. However, it is unclear whether aneusomy 17 may also play a role. Using immunohistochemistry assay (IHC) with DAKO antibody and manual quantitation, 189 specimens were selected from archival invasive breast cancer specimens, including most IHC-positive and some IHC-negative cases (n = 158 and 31, respectively). They were then analyzed by PathVysion fluorescence in situ hybridization (FISH) assay (Vysis, Inc., Downers Grove, IL) and by an image analyzer (ACIS; ChromaVision Medical Systems, Inc., San Juan Capistrano, CA)-assisted IHC quantitation. Ninety-two cases contained disomy 17 (chromosome 17 centromere, 1.76-2.25 signals per cell) whereas 97 cases had aneusomy 17, including 82 with low polysomy (2.26-3.75 signals per cell), 10 with high polysomy (> or =3.76 signals per cell), and 5 with hypodisomy (> or =1.75 signals per cell). HER-2/neu protein expression had the highest correlation with HER-2/neu gene dosage (copy number; r =.826), followed by the HER-2/neu gene to chromosome 17 ratio (r =.733). The lowest correlation was with the chromosome 17 copy number (r =.307), on which the 10 cases with high polysomy 17 had a disproportionately high impact. The FISH assay using the PathVysion criterion for HER-2/neu gene amplification (HER-2/neu gene to chromosome 17 ratio, > or = 2.00) achieved higher concordance with ACIS IHC than did an alternative FISH criterion (absolute HER-2/neu gene copy number, > or = 4.00 signals per cell). Most ACIS IHC-PathVysion FISH-discordant cases contained disomy or low polysomy 17, whereas all 10 cases with high polysomy 17 had no such discordance. However, two cases with monosomy 17 had ACIS IHC-PathVysion FISH discordance, i.e., with gene amplification, but no protein overexpression. Both cases would have had no gene amplification if the alternative FISH criterion had been used. In conclusion, aneusomy 17 is common in breast cancer. Except in a certain subset of cases, aneusomy 17 probably is not a significant factor for HER-2/neu protein expression or for clinical assessment of HER-2/neu status.     

Pathobiologic findings in DCIS of the breast: morphologic features, angiogenesis, HER-2/neu and hormone receptors.

With the increasing incidence of ductal carcinoma in situ (DCIS) of the breast and its relationship to invasive breast carcinoma, it is important to understand the biology of this entity. We report on a hospital-based survey of 219 case subjects with DCIS of the breast without associated invasive carcinoma diagnosed between 1982 and 1994. The cases of DCIS were analyzed for architectural type, size, nuclear grade, necrosis, calcification, periductal fibrosis, neovascularization, estrogen receptor (ER), progesterone receptor (PR), and HER-2/neu expression. Periductal neovascularization was associated with tumor size, microcalcifications, periductal fibrosis, and HER-2/neu overexpression. Expression of ER and PR was observed in 60 and 62% of the cases, respectively, and HER-2/neu overexpression in 28% of the cases. ER and PR expression were both inversely associated with comedo histology and nuclear grade. HER-2/neu overexpression was positively associated with comedo histology, high nuclear grade, and periductal neovascularization and was inversely correlated with both ER and PR expression. High nuclear grade was positively associated with comedocarcinoma, necrosis, microcalcification, and periductal fibrosis. The role of these molecular/pathologic markers in the biology of DCIS and their potential clinical implications are discussed.     

Defining a test for HER-2/neu evaluation in breast cancer in the diagnostic setting.

In breast cancer amplification of the HER-2/neu oncogene and over-expression of the protein product is associated with poor prognosis, predicts response to some chemotherapeutic regimens and is the target for Herceptin treatment. To date there are several methods to assess the amplification/over-expression of HER-2/neu with each having advantages and disadvantages. We have studied amplification and over-expression of HER-2/neu in 250 consecutive cases of breast cancer (220 invasive and 30 in situ carcinomas) presenting to the Department of Pathology at Women's College Campus of Sunnybrook and Women's College Health Sciences Center. Thirty percent of the invasive carcinomas were node positive. HER-2/neu protein over-expression was assessed by immunohistochemistry (IH) using antibody CB11 and amplification of the gene by differential PCR. The percentage of tumor cells showing CB11 staining was determined and the most significant cut off point for positivity was > or =10% moderate or strong complete membranous staining. The gene was considered amplified if the density score of the product was > or =2. There was 94% concordance between the two methods (P value.0001). Both methods were positive in 16% of cases and negative in 78% of cases. Discrepant cases were examined by FISH which confirmed the IH results in 9/11 invasive carcinomas. These results show that there is excellent concordance between IH and PCR. However, immunohistochemistry is easier to perform and cheaper than PCR and could be used in routine assessment of HER-2/neu in breast cancer patients.     

Prognostic relevance of activated Akt kinase in node-negative breast cancer: a clinicopathological study of 99 cases.

Patients with lymphnode-negative breast cancer show a 10-year tumor recurrence rate of approximately 30%. Therefore, it is important to identify high-risk patients who would benefit from further adjuvant therapy. For this purpose, we examined the activation state of two kinases important in the regulation of cell proliferation and apoptosis in a series of 99 node-negative breast cancer cases with a mean follow-up of 10 years: Akt and extracellular regulated kinase (ERK1/2). The activation of Akt and ERK1/2 was investigated by immunohistochemistry using phospho-specific antibodies. The results were correlated with HER-2/neu expression, histological grading, receptor status, overall survival (OS) as well as with cell proliferation (Ki67 immunoreactivity, mitotic count) and tumor apoptosis assessed by TUNEL staining. Activation of Akt (pAkt) but not activation of ERK1/2 (pERK1/2) correlated with HER-2/neu overexpression (P<0.05) and was related to reduced tumor apoptosis (P<0.05). No association was found between pAkt or pERK1/2 with cell proliferation assessed by Ki67 and mitotic count (MC). Survival analysis of receptor status, HER2/neu expression, histological grading, MC and pAkt immunoexpression showed a significant correlation with decreased OS, but only pAkt reached statistical significance in the multivariate Cox regression analysis (P=0.015). Activation of Akt in node-negative breast cancer may indicate aggressive tumor behavior and may constitute an independent prognostic factor of OS. The determination of pAkt status may be of value in identifying high-risk patients, who would benefit from adjuvant therapy, and gives a rationale to investigate new therapy strategies by specific inhibition of the Akt signaling pathway in breast cancer.     

HER-2 status in breast cancer: correlation of gene amplification by FISH with immunohistochemistry expression using advanced cellular imaging system.

It has become important to accurately evaluate the status of HER-2/neu in invasive breast cancer, especially when one is considering the use of anti-HER-2 monoclonal antibody therapy (Trastuzumab). Almost one third of invasive breast carcinomas overexpress the HER-2/neu protein, so the use of the anti-HER-2/neu monoclonal antibody Herceptin (trastuzumab) to block the protein has become important in the management of and in prolonging the survival for patients with metastatic breast cancer. The effectiveness of this therapy is dependent on accurately evaluating the HER-2 status in these tumors, which can be done either by studying the expression of HER-2 protein by immunohistochemistry (IHC) or by evaluating HER-2 gene amplification by fluorescent in situ hybridization (FISH). Since interobserver variability may occur in manually grading HER-2 protein expression by IHC, the aim of this study was to compare the HER-2/neu expression by IHC using a computer-based image analysis system with that of the gene amplification by FISH. Formalin-fixed paraffin-embedded archival tissue from 108 primary infiltrating ductal carcinomas were immunostained using the HercepTest (DAKO). To reduce interobserver variability, membrane staining was evaluated using the Automated Cellular Imaging System (ACIS) by ChromaVision, and the cases were divided into four groups: group 1 (n=23) with HER-2/neu expression ACIS score less than or equal to 1.5; group 2 (n=17) with a score ranging from 1.6 to 1.9; group 3 (n=46) with a score 2.0 to 2.5; and group 4 (n=22) with a score greater than or equal to 2.6. FISH was performed on all of the 108 cases using the PathVysion HER-2/neu DNA probe kit from Vysis Inc. All cases were also manually reviewed and graded as negative, 1+, 2+, and 3+ according to the DAKO HercepTest grading scheme. Cases with negative and 1+immunostaining were considered as HER-2 not overexpressed, and cases with 2+ and 3+ staining were classified as showing HER-2 overexpression. In group 1, 1 of 23 (4%), in group 2, 2 of 17 (12%), in group 3, 5 of 46 (11%), and in group 4, 19 of 22 (86%) cases showed gene amplification by FISH. Furthermore, in group 4 all 15 (100%) cases with an ACIS score of 3 or greater were FISH positive. Correlation with manual IHC score and FISH showed that 2 of the 23 (9%) IHC negative (0 and 1+) cases and 25 of the 85 (29%) IHC positive (2+ and 3+) cases showed gene amplification by FISH. This study shows that the amplification of the HER-2/neu gene correlates better with overexpression of the HER-2/neu protein by IHC when the score is either less than 1.5 or greater than 2.6 by ACIS. Therefore, FISH may be useful to better evaluate HER-2/neu status in breast cancer in cases where the ACIS score by immunohistochemistry is 1.6 to 2.5, and since the correlation is so good, FISH may not be needed for HER-2 evaluation in cases with ACIS scores less than 1.5 and greater than 2.6.     

HER-2/neu protein expression in breast cancer evaluated by immunohistochemistry. A study of interlaboratory agreement

Immunohistochemistry (IHC) is used commonly for evaluating HER-2/neu protein expression in breast cancer. Given the potential clinical importance of HER-2/neu status in patient management, interlaboratory variability in HER-2/neu IHC results in a matter of legitimate concern. We compared the results from 2 laboratories for HER-2/neu determined by IHC on paraffin sections of the same 100 consecutive invasive breast cancers. Both laboratories used the same primary antibody; however, different methods for heat-induced epitope retrieval (microwave or steam) and immunostaining (automated equipment from different manufacturers) and different scoring systems (positive-negative and 0-4+) were used. Slides were read in a blinded fashion and the results from the 2 laboratories were compared. Of the 93 cases evaluable in both laboratories, 24% were scored as HER-2/neu-positive at 1 laboratory, and 23% were scored as positive at the other. Complete concordance in categorization of HER-2/neu status between the 2 laboratories was achieved in 90 of 93 cases. Excellent interlaboratory agreement for HER-2/neu IHC was attained using the same primary antibody to HER-2/neu, even without standardization of assay method or scoring criteria. However, standardization of these parameters remains an important objective to optimize interlaboratory agreement.     

Tyrosine kinase activation in breast carcinoma with correlation to HER-2/neu gene amplification and receptor overexpression.

The HER-2/neu oncogene encodes a transmembrane receptor with intrinsic tyrosine kinase activity. A pilot study was performed to investigate downstream effects of HER-2/neu (or related growth factor receptor) activation by identifying phosphorylated tyrosine. Fifty-four breast carcinomas were evaluated for HER-2/neu overexpression by the HercepTest (Dako, Carpinteria, CA) and the monoclonal CB11 antibody (Ventana, Tucson, AZ). Phosphotyrosine (an indication of tyrosine kinase activity) was detected by an antiphosphotyrosine mouse monoclonal antibody (Upstate Biotechnology, Lake Placid, NY). The gene amplification status was evaluated in 50 of the 54 cases by fluorescence in situ hybridization (FISH) using the Ventana gene probe. The HER-2/neu oncogene amplification was detected in 28% (14 of 50) of cases. Of the 14 cases showing oncogene amplification, tyrosine kinase activity was detected in 9 (64.2%) cases. There was moderate agreement between HER-2/neu gene amplification and tyrosine kinase activity (kappa = 0.43). Immunohistochemical staining of 3+ (with both HercepTest and CB11) showed better agreement with HER-2/neu oncogene amplification and increased tyrosine kinase activity than 2+ immunohistochemical staining. Overall, oncogene amplification and overexpression correlated with increased tyrosine kinase activity, supporting the mechanism of tyrosine kinase activation by HER-2/neu amplification and overexpression. However, 7 cases showing increased tyrosine kinase activity did not show gene amplification or 3+ receptor expression (by either HercepTest or CB11), raising the possibility of other growth factor receptors operating via the tyrosine kinase pathway. There was no apparent correlation between tyrosine kinase activity and hormone receptor status (estrogen or progesterone). Increased tyrosine kinase activity is more commonly associated with higher-grade tumors and thus may correlate with aggressive biologic behavior in breast carcinoma. The results of this pilot study suggest that a larger-scale investigation into downstream activation of tyrosine kinase and correlation to clinical outcome or response to Herceptin therapy may identify subsets of patients whose clinical response or outcome may be predicted by tyrosine kinase activation.     

Fluorescence in situ hybridization and immunohistochemical assays for HER-2/neu status determination: application to node-negative breast cancer

BACKGROUND: HER-2/neu (ERBB2) gene amplification and/or overexpression is a major event in human breast tumorigenesis. HER-2/neu gene alterations have been the most frequently assessed prognostic factors during the last 10 years in breast cancer and have recently emerged as a management decision tool and a therapeutic target. There is still controversy over the best method to determine whether a tumor is HER-2/neu positive. Because of the increasing demand for HER-2/neu gene status determination in clinical practice, we compared HER-2/neu gene alterations at the DNA level (gene amplification) and the protein level (overexpression) in a panel of patients with lymph node-negative breast cancer who had received local radiotherapy alone, with no adjuvant therapy. METHODS: We tested 100 excised lymph node-negative breast tumors, using fluorescence in situ hybridization (FISH) with a biotinylated HER-2/neu DNA probe and immunohistochemical assays (IHC) with 2 different antibodies. RESULTS: The FISH frequency of HER-2/neu gene amplification was 15%, and the IHC frequency of overexpression was 21%. CONCLUSION: Although HER-2/neu amplification by FISH and HER-2/neu overexpression by IHC correlated well in this panel of lymph node-negative breast carcinomas, there were a number of discordant cases, pointing to the important need for determining HER-2/neu alteration for the future management of HER-2/neu-based clinical applications.     

HER-2/neu testing in breast carcinoma: a combined immunohistochemical and fluorescence in situ hybridization approach.

We evaluated 750 consecutive invasive breast carcinomas for HER-2/neu utilizing a combination of immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) methodologies. IHC reactions of 3+ were considered HER-2/neu positive and 0 and 1+ IHC reactions were considered HER-2/neu negative. IHC reactions of 2+ were considered inconclusive and reflexed to FISH analysis. In addition, a 10% sampling and validation FISH analysis was performed on the positive and negative IHC tests. One hundred thirty-eight cases (18.4%) were HER-2/neu positive by IHC and/or FISH. One hundred twenty-three of the positive cases (89%) were 3+ IHC reactions and 14 positive cases were inconclusive by IHC and amplified by FISH. There was concordance with FISH in 77 of 78 (98.7%) of the positive or negative IHC cases that were tested (95% confidence interval [CI] = 93.1 to 100%). A single IHC-negative case showed HER-2/neu amplification by FISH. Thirty-nine cases were 2+ IHC (5.2%); 14 (36%) were amplified, 24 (62%) were not amplified, and one was not interpretable. HER-2/neu positivity was observed in 34% of grade 3 ductal carcinomas, 11.4% of grade 2 ductal carcinomas, 3.2% of grade 1 ductal carcinomas, and 3.2% of lobular carcinomas. Occasional cases with discordant IHC expression of HER-2/neu within the in situ and invasive carcinoma elements were also identified. IHC reliably characterized HER-2/neu in approximately 95% of the cases studied (95% CI = 93.0 to 96.2%) and was effective as a primary method for evaluating HER-2/neu status. In this study, 2+ IHC reactions were a heterogeneous group best regarded as indeterminate or inconclusive; in this series, only 36% were amplified by FISH analysis. Our findings suggest that a combination of IHC and FISH testing with FISH analysis performed reflexly on all 2+ IHC cases can optimize HER-2/neu testing.     

Predominance of the basal type and HER-2/neu type in brain metastasis from breast cancer.

Although breast cancer is the second most common cause of central nervous system (CNS) metastases with a notable increase of incidence, only few studies on brain-metastasizing breast cancer are available. In this immunohistochemical and fluorescence in situ hybridization (FISH) study, metastases to the CNS (n=85) and primary breast cancers, with known involvement of the CNS (n=44) including paired primary and metastasized tumours (n=23), were investigated retrospectively for the expression of oestrogen- (ER) and progesterone- (PR) hormone receptors, Her-2/neu, epidermal growth factor receptor (EGFR), Ki-67, and cytokeratins (CKs) 5/14. The majority of brain metastases were steroid hormone receptor negative (ER 66%, PR 82%) corresponding to the findings in primary tumours with known involvement of the CNS (68% ER-negative, 75% PR-negative). The frequency of HER-2/neu-overexpressing or -amplified cancers was increased in both groups (34 and 32%, respectively). EGFR expression was more frequent in metastases (41%) than in primary tumours (16%). The proportions of cases with a basal phenotype were 26 and 30%, respectively. In paired primary tumours and metastases to the CNS, constancy of Her-2/neu status was observed in 87% of cases with only one sample turning Her-2/neu-negative and two samples acquiring overexpression/amplification in brain metastases. In contrast, steroid hormone receptors exhibited more frequently a loss of expression (17%) than a gain (9%) with 74% revealing a constant phenotype. We conclude that brain-metastasizing breast cancer belongs predominantly to the basal type or Her-2/neu type. Primary and metastatic tumours differ from each other only in a minority of cases, leading rather to a loss of steroid hormone receptors and to a gain of EGFR and Her-2/neu.     

Change of HER-2/neu status in a subset of distant metastases from breast carcinomas

It is assumed that HER-2/neu status remains consistent in breast carcinoma during metastatic spread, but in most previous studies primary tumours were compared with concurrent regional lymph node metastases. The present study investigated 31 breast carcinomas and their corresponding lymph node and distant metastases for HER-2/neu status by immunohistochemistry (HercepTest) and fluorescence in situ hybridization (FISH) (PathVysion). In 14 cases, serum HER-2/neu (SHER-2) was measured sequentially using the Bayer Immuno 1 HER-2/neu assay. Comparing HER-2/neu immunohistochemistry of primary tumours and distant metastases case by case, increased HER-2/neu expression was found in the distant metastases in 15 cases (48.4%), three (9.7%) of which showed an increase from score 0 to score 3+. In contrast, lymph node metastases showed the same HER-2/neu expression as the primary tumours, confirmed by FISH. Two cases, which showed HER-2/neu score 3+ and HER-2/neu amplification in the primary tumours, revealed increased SHER-2 levels above 50 ng/ml at the first measurement. Five of the 14 cases (36%) showed an increase of SHER-2 above 50 ng/ml towards the end of the patients' life. On the basis of these results, there is evidence that in a subset of breast carcinomas, HER-2/neu amplification and overexpression occur de novo in distant metastases at a late disease stage.