Neu Gene Copy Number Research Articles

Assessment of HER-2/neu, с-MYC andCCNE1 gene copy number variations andprotein expression in endometrial carcinomas.

To analyze copy number variations of HER-2/neu, c-MYC and CCNE1oncogenes and their protein expression in endometrioid endometrial carcinomas in relation to the degree of tumor progression and presence of a family history of cancer in cancer patients. The study was conducted on endometrial cancer (EC) samples from 68patients with I-II FIGO stages of disease. Copy number analysis of HER-2/neu, c-MYC and CCNE1genes was performed by quantitative PCR. Protein expression was analyzed using immunohistochemistry. Assessment of copy number variations of HER-2/neu, c-MYC and CCNE1genes revealed their amplification in the tumors of 18.8, 25.0and 14.3% of EC patients, respectively. High expression of corresponding proteins was detected in 14.6, 23.5and 65.6% of patients, respectively. It was established that HER-2/neu gene amplification is more common in the group of tumors of low differentiation grade than in moderate grade EC (35.7and 5.5% of cases, respectively, p < 0.05). Also, high expression of c-Myc protein was more frequently observed in low differentiated tumors compared to the moderately differentiated EC (36.6and 13.2% of cases, respectively, p < 0.05). Expression of HER-2/neu and cyclin E proteins was found to be dependent on the depth of tumor invasion into the myometrium. High expression of HER-2/neu protein was observed in 25.0and 4.1% of EC patients with tumor invasion > ½ and < ½ of the myometrium, respectively, and cyclin E- in 86.7and 46.6% of cases, respectively, p < 0.05. It was shown that among patients with a family history of cancer, a larger proportion of cases with high expression of c-Myc protein was observed compared to the group of patients with sporadic tumors (43.8and 17.3%, respectively; p < 0.05). Amplification of HER-2/neu gene, along with high expression of c-Myc, HER-2/neu and cyclin E proteins, are associated with such indices of tumor progression as a low differentiation grade and deep myometrial invasion, suggesting the potential possibility of including these markers in the panel for determining the molecular EC subtype associated with an aggressive course of the disease. In a certain category of EC patients, there is a relationship between a family history of cancer and high expression of c-Myc protein.

The identification of a small but significant subset of patients still targetable with anti-HER2 inhibitors when affected by triple negative breast carcinoma

Triple (ER-, PR-, HER2-) negative breast carcinoma lack targeted therapies, making this group of tumors difficult to treat. By definition, the lack of HER2 expression means a case scoring 0 or 1+ after immunophenotypical analysis and makes the patients avoiding therapeutical chances with anti-HER2 inhibitors. We sought to recruit from a group of triple negative breast carcinoma, patients eligible for effective personalized targeted therapy with anti-HER therapies on the basis of their HER2 gene status. 135 patients diagnosed with IHC triple negative breast carcinoma were studied. Whole tissue sections were used for in situ hybridization analysis. 8/100 (8%) of ductal-type triple negative breast carcinoma presented Her-2/neu gene amplification versus 2/35 (5.7%) non-ductal triple negative breast carcinoma. Three cases showed a ratio 2.5. One case showed Her-2/neu heterogeneous gene amplification, ratio 2.3. The other six showed from 7 to 8 absolute Her-2/neu gene copy number. Two cases staged pT1c, and eight cases staged pT2. Eight cases graded G3 and two cases G2. (1) Eight percentage of ductal and 5.7% non-ductal-type triple negative breast carcinoma present Her-2/neu gene amplification, (2) the standard diagnostic flowchart "do not FISH in 0-1+ (HER2-) breast carcinoma" should be replaced by "do FISH in triple (ER-, PR-, HER2-) negative breast carcinoma," to avoid loss of therapeutical chances in a cohort of such a patients, (3) we demonstrated the identification of a small but significant subset of patients targetable with anti-HER2 inhibitors, giving patients affected by (ex)triple negative breast carcinoma new personalized therapeutical chances.

Correlation Between Quantitative HER-2 Protein Expression and Risk for Brain Metastases in HER-2+ Advanced Breast Cancer Patients Receiving Trastuzumab-Containing Therapy

Patients with human epidermal growth factor receptor (HER)-2+ breast cancer are at particularly high risk for brain metastases; however, the biological basis is not fully understood. Using a novel HER-2 assay, we investigated the correlation between quantitative HER-2 expression in primary breast cancers and the time to brain metastasis (TTBM) in HER-2+ advanced breast cancer patients treated with trastuzumab. The study group included 142 consecutive patients who were administered trastuzumab-based therapy for HER-2+ metastatic breast cancer. HER-2/neu gene copy number was quantified as the HER-2/centromeric probe for chromosome 17 (CEP17) ratio by central laboratory fluorescence in situ hybridization (FISH). HER-2 protein was quantified as total HER-2 protein expression (H2T) by the HERmark® assay (Monogram Biosciences, Inc., South San Francisco, CA) in formalin-fixed, paraffin-embedded tumor samples. HER-2 variables were correlated with clinical features and TTBM was measured from the initiation of trastuzumab-containing therapy. A higher H2T level (continuous variable) was correlated with shorter TTBM, whereas HER-2 amplification by FISH and a continuous HER-2/CEP17 ratio were not predictive (p = .013, .28, and .25, respectively). In the subset of patients that was centrally determined by FISH to be HER-2+, an above-the-median H2T level was significantly associated with a shorter TTBM (hazard ratio, [HR], 2.4; p = .005), whereas this was not true for the median HER-2/CEP17 ratio by FISH (p = .4). Correlation between a continuous H2T level and TTBM was confirmed on multivariate analysis (HR, 3.3; p = .024). These data reveal a strong relationship between the quantitative HER-2 protein expression level and the risk for brain relapse in HER-2+ advanced breast cancer patients. Consequently, quantitative assessment of HER-2 protein expression may inform and facilitate refinements in therapeutic treatment strategies for selected subpopulations of patients in this group.

P2-12-05: Correlation between Quantitative HER2 Protein Expression and Risk of Brain Metastases in HER2−Positive Advanced Breast Cancer Patients Receiving Trastuzumab-Containing Therapy.

Abstract Background. Patients with HER2−positive breast cancer are at particularly high risk for brain metastases; however, the biological basis is not fully understood. Within HER2−positive breast cancer tumors, it is possible to resolve a ∼1.5-log range of HER2 protein expression using a novel quantitative HER2 assay (HERmark®). We investigated the correlation between quantitative HER2 protein expression in primary breast cancers and the time to brain metastases (TTBM) in HER2−positive advanced breast cancer patients treated with trastuzumab. Methods. The study group included 142 consecutive patients who were administered trastuzumab-based therapy for HER2−positive metastatic breast cancer, defined as 3+ categorical staining by immunohistochemistry (IHC). HER-2/neu gene copy number was subsequently quantified as HER2/CEP17 ratio by central laboratory fluorescence in situ hybridization (FISH). HER2 protein was quantified as total HER2 protein expression (H2T) by the HERmark assay in formalin-fixed, paraffin-embedded primary tumor samples. HER2 variables were correlated with clinical features and TTBM measured from the initiation of trastuzumab-containing therapy. Results. H2T level (continuous variable) was correlated with shorter TTBM (HR=2.3; p=0.013), whereas HER2 gene amplification by FISH (p=0.28) and continuous HER2/CEP17 ratio (p=0.25) had no significant prognostic impact. The correlation between continuous H2T level and TTBM was confirmed in a multivariate analysis (HR=3.2; p=0.021). Controlling for the competing risk of death from causes other than brain metastases, continuous H2T remained a strong correlate of TTBM (HR=2.7; p=0.0009). In the subset of patients that was centrally-determined HER2 positive by FISH (117 patients), above-median H2T level was significantly associated with shorter TTBM (HR=2.4; p=0.005), whereas this was not true for median FISH/CEP17 ratio (p=0.4). In a multivariate analysis of this subset, continuous H2T (p=0.021) and a time dependent covariate capturing time to non-brain metastases (p=0.0044) were prognostic for TTBM, whereas FISH/CEP17, ER, PgR and grade were not. Conclusions. These data reveal a strong relationship between quantitative HER2 protein expression levels and the risk of brain relapse in HER2−positive advanced breast cancer patients. Consequently, quantitative assessment of HER2 protein expression may inform and facilitate refinements in therapeutic treatment strategies for selected subpopulations of patients in this group. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-05.

Adjuvant Therapy with Trastuzumab for HER-2/neu-Positive Breast Cancer

Breast cancer is the most common cancer in women in the U.S. and western Europe. Amplification of the her-2/neu gene occurs in approximately 25% of invasive ductal carcinomas of the breast. In experimental models, transfection of the her-2/neu gene results in transformation of mammary epithelial cells. In human breast cancer, amplification of the her-2/neu gene results in protein over expression and poor prognosis. Patients whose tumors have her-2/neu gene amplification have a shorter disease-free survival time than patients whose tumors exhibit a normal her-2/neu gene copy number. her-2/ neu gene amplification identifies a biologically unique subset of aggressive breast tumors that are sensitive to growth inhibition and apoptosis induced by anti-HER-2/neu-targeted therapies. The first HER-2/neu-targeted approach to reach the clinic was trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of the HER-2/neu protein. Trastuzumab therapy prolongs the survival of patients with metastatic HER-2/neu-overexpressing breast cancer when combined with chemotherapy and has recently been demonstrated to lead to dramatic improvements in disease-free survival when used in the adjuvant therapy setting in combination with or following chemotherapy. However, potential cardiotoxicity requires careful patient selection. Here, we review the recently completed clinical trials of adjuvant trastuzumab in the adjuvant setting. HER-2/neu testing, patient selection, cardiotoxicity, duration of therapy, and directions for future research are discussed.

AQUA and FISH analysis of HER‐2/neu expression and amplification in a small cell lung carcinoma tissue microarray

Most small cell lung carcinoma (SCLC) patients have metastatic disease at the time of diagnosis and are faced with poor prognosis and limited treatment options. Reports of HER-2/neu gene amplification and overexpression in this malignancy have raised the possibility of applying targeted immunotherapy with trastuzumab, the monoclonal antibody used to treat metastatic breast cancer. However, a review of the studies measuring HER-2/neu gene amplification and protein expression in SCLC reveals discordant results. The aim of the present study was to re-examine HER-2/neu expression in SCLC in relation to gene copy number using the new, highly sensitive, immunofluorescence automated quantitative analysis (AQUA) technology. Fluorescence in situ hybridization (FISH) was used to measure HER-2/neu gene copy number and amplification status and AQUA was used to measure protein expression in a series of 23 SCLC tumours on a tissue microarray. None of the 17 SCLC specimens assessable by FISH exhibited HER-2/neu gene amplification as defined by a HER-2/neu/chromosome 17 ratio = or > 2. Twelve of 17 (70.1%) SCLC samples were polysomic for chromosome 17 with corresponding increases in HER-2/neu gene copy numbers. Intermediate levels of protein expression corresponding to AQUA scores in the range of 4-24 were detected in all 23 specimens. High protein expression levels corresponding to AQUA scores up to 83, observed previously in association with gene amplification and poor prognosis in breast cancer cases, were not detected in the present study. No statistically significant association was observed between absolute chromosome 17 or HER-2/neu gene copy numbers and protein expression levels in tumour cells (P > 0.45). The lack of gene amplification and robust HER-2/neu protein expression in SCLC tumour cells in this series does not suggest a prominent role for the HER-2/neu gene in SCLC tumour progression and does not support the general applicability of targeted immunotherapy with trastuzumab to this malignancy.

Pathology of borderline HER-2/neu breast carcinoma: a biologically distinct phenotype

The significance of HER-2/neu results obtained by immunohistochemical analyses (IHC) which are neither negative nor strongly positive is controversial. The incidence of fluorescence in situ hybridization (FISH) positivity in these tumors is small and the implication is that these borderline results represent laboratory misclassification. We analyzed the tumor characteristics of these HER-2/neu borderline tumors to determine if they represent a unique tumor type. HER-2/neu status was determined by image analysis (IA) of IHC sections in 669 cases of invasive breast cancer. Borderline cases were reflexed to FISH to determine gene status. HER-2/neu results were compared to tumor morphology and other tumor markers. HER-2/neu was negative, borderline and positive in 69.5, 15.8, and 14.6% of cases, respectively. HER-2/neu amplification was present in 17.3% of borderline cases. The borderline group is significantly different from the HER-2/neu positive group for all parameters studied except Ki-67. Compared to the HER-2/neu negative group, the borderline group showed a significantly higher HER-2/neu gene copy number and a trend towards lower progesterone receptor expression (p=0.058). Compared to the HER-2/neu negative group, the HER-2/neu borderline/FISH-negative group showed significantly lower PR expression. Compared to the HER-2/neu positive group, the HER-2/neu borderline/FISH positive group showed significant differences with multiple parameters. Borderline HER-2/neu tumors are a unique tumor type and do not represent laboratory imprecision. Hormone receptor alterations are associated with early changes in HER-2/neu expression. While IA is capable of detecting these changes, current FISH methodology is not.

Polysomy 17 in HER-2/neu Status Elaboration in Breast Cancer: Effect on Daily Practice

To assess the effect of chromosome 17 copy number on HER-2/neu status determination in breast cancers. HER-2/neu gene copy and chromosome 17 centromere numbers were evaluated on 893 breast carcinomas using double color fluorescence in situ hybridization (FISH). The net and chromosome 17 corrected (ratio) HER-2/neu copy numbers were compared and related to immunohistochemistry done according to the Food and Drug Administration (FDA)-approved scoring system (0, 1+, 2+, and 3+) as a first screening step in 584 cases. When a ratio > or = 2 was considered as criterion for FISH positivity, 49.3% (440 of 893) of cases showed amplification versus 56.2% (502 of 893) by using a net HER-2/neu gene copy number >4 as a alternative criterion; 14.8% (67 of 453) of cases having a ratio <2 had a net HER-2/neu gene copy number >4 and 1.1% (5 of 440) with a ratio > or = 2 had a net HER-2/neu gene copy number <4. Among discordant cases, 88.8% (64 of 72) were polysomic (>2.25 chromosomes 17/cell) and among polysomic cases, 12.8% (40 of 312) of the low polysomic (2.26-3.75 chromosomes 17/cell) and 36.9% (24 of 65) of the highly polysomic (>3.75 chromosomes 17/cell) cases showed discordance. In cases with a ratio <2, polysomy 17 incidences were 85.7% (6 of 7) in IHC 3+, 42.4% (79 of 186) in IHC 2+, 33.3% (15 of 45) in IHC 1+, and 29.1% (16 of 55) in IHC 0. A net increase in HER-2/neu gene copy number consecutive to polysomy 17 in the absence of specific gene amplification might lead to a strong protein overexpression in a small subset of breast carcinomas. HER-2/neu status determination by FISH is dependent on the criterion considered for positivity in clinical practice.

Analysis of HER-2/neu amplification in endometrial carcinoma by chromogenic in situ hybridization. Correlation with fluorescence in situ hybridization, HER-2/neu, p53 and Ki-67 protein expression, and outcome

Fluorescence in situ hybridization (FISH) is the most widely used technique to detect HER-2/neu gene amplification; however, it is only available in some institutions. In contrast, chromogenic in situ hybridization (CISH) can be evaluated by routine light microscopy. In endometrial carcinoma there are few data concerning HER-2/neu status and prognosis. Therefore, we determined HER-2/neu gene status by CISH using a digoxigenin-labelled probe on 60 formalin-fixed paraffin-embedded endometrial carcinomas. The data were compared with the immunohistochemistry of HER-2/neu (A0485, TAB250), p53, Ki-67, clinicopathological factors, and survival. By conventional light microscopy, HER-2/neu amplification (⩾6 copies >50% cancer cells) was detected in 14% (8/59) tumours, HER-2/neu overexpression (>10% cells moderate/strong complete membrane staining) in 22% (13/60) for A0485, and 18% (11/60) for TAB250, p53 (>10% +cells) in 61% (36/59), and Ki-67 (>50% +cells) in 50% (30/60). Discordant cases for CISH and immunohistochemistry, as well as all (2+) were further analysed by FISH (Vysis). Among 10 cases (2+) and not amplified by CISH, two showed low-level amplification by FISH. Significant correlation was found between amplification and protein overexpression (P⩽0.001), and a trend with nonendometrioid type, higher grade, and older age. A better outcome (Kaplan–Meier) was observed for patients with nonamplified (1–5 copies per nucleus) or low-level (6–10 copies) amplification tumours, low Ki-67 expression, age <50 years, endometrioid type, low FIGO (International Federation of Obstetrics and Gynaecology) grade and stage, superficial myometrial infiltration, and no lymph–vascular invasion (P⩽0.036), but only as a trend for HER-2/neu protein negative (P=0.13). Cox analysis revealed age, FIGO grade and stage, myometrial infiltration, and lymph–vascular invasion to be independent prognostic factors (P⩽0.05), and a trend for HER-2/neu gene copy number (0.18). In endometrial carcinoma, HER-2/neu gene status can be readily assessed by CISH in routine clinical practice, and it gives more prognostic information than HER-2/neu by immunohistochemistry. FISH analysis in (2+) cases but negative by CISH may detect additional tumours with low-level amplification.

HER-2/neu Gene Copy Number Quantified by Real-Time PCR: Comparison of Gene Amplification, Heterozygosity, and Immunohistochemical Status in Breast Cancer Tissue

Amplification of the oncogene HER-2/neu influences breast cancer pathogenesis, and therapy and prognosis may be affected by the degree of amplification. The extent of amplification or protein overexpression typically is analyzed by fluorescence in situ hybridization or immunohistochemistry (IHC), but quantitative PCR techniques have been described that may provide alternatives to these methods. We developed a rapid-cycle, real-time PCR assay for quantification of HER-2/neu gene status. We compared results obtained with this assay with short tandem repeat findings by capillary electrophoresis (CE) and with protein overexpression assessments by IHC. Accuracy and linearity were tested on cell lines and with simulation experiments. We analyzed the amplification of HER-2/neu in 51 clinical tissue samples from patients with suspected breast cancer. The intra- and interrun CVs for HER-2/neu quantification by real-time PCR were 12% and 18%, and the CV for different simulated amplification and deletion experiments was <7%. The results for HER-2/neu gene status in cell lines matched the values reported in literature. We detected HER-2/neu amplification by real-time PCR in 11 samples, all from patients with invasive ductal carcinoma. Allelic imbalances were found by CE analyses in three samples and by protein overexpression in six samples; five of these were also detected by real-time PCR. Comparison of the quantification results with known prognostic indices yielded results similar to those reported in several other published studies. The assay is suitable for accurate and precise quantification of HER-2/neu copy numbers in tumor tissue samples obtained in routine clinical practice.

Quantitative association between HER-2/neu and steroid hormone receptors in hormone receptor-positive primary breast cancer.

HER-2/neu, which encodes a receptor tyrosine kinase, is amplified and overexpressed in 20%-25% of human breast cancers. Such tumors are often resistant to hormone therapy. Despite a general inverse association between HER-2/neu amplification/overexpression and estrogen receptor (ER) and/or progesterone receptor (PR) expression, a fraction of patients are both HER-2/neu- and hormone receptor (HR)-positive. The efficacy of hormone therapy in this group is currently a matter of debate. To better understand the relationship between HER-2/neu positivity and HR expression, we analyzed HER-2/neu, ER, and PR as continuous variables in breast cancer cell lines and two cohorts of primary breast cancer patients. HER-2/neu and ER/PR expression was analyzed by enzyme-linked immunosorbent assay (ELISA) and enzyme immunoassay (EIA), respectively, in 14 human breast cancer cell lines, some of which had been transfected with the HER-2/neu gene. For the clinical study population, HER-2/neu protein levels were assessed by ELISA (cohort A, n = 665), and HER-2/neu gene copy number was determined using fluorescence in situ hybridization (cohort B, n = 894). ER/PR expression was analyzed by EIA (cohort A) or radioligand binding (cohort B). Associations between HER-2/neu and ER/PR expression were analyzed using Spearman's rho correlation and the chi-square test, and absolute levels were compared using the Mann-Whitney U test. All statistical tests were two-sided. HR-positive human breast cancer cell lines transfected with the HER-2/neu gene expressed statistically significantly lower levels of ER and PR than parental lines. In the clinical cohorts, levels of HER-2/neu overexpression and gene amplification were inversely correlated with ER/PR levels (Cohort A [n = 112]: for ER, r = -0.34, P<.001; for PR, r = -0.24, P =.010. Cohort B [n = 188]: for ER, r = -0.39, P<.001; for PR, r = -0.26, P<.001). Among patients with HR-positive tumors, HER-2/neu-positive tumors had statistically significantly lower ER/PR levels than HER-2/neu-negative ones (Cohort A: for ER, median = 25 fmol/mg [interquartile range [IQR] = 13-78] versus median = 38.5 fmol/mg [IQR = 17-99] and P =.031; for PR, median = 35 fmol/mg [IQR = 12-119] versus median = 88.5 fmol/mg [IQR = 22-236] and P<.001. Cohort B: for ER, median = 44 fmol/mg [IQR = 13-156] versus median = 92 fmol/mg [IQR = 35-235] and P<.001; for PR, median = 36 fmol/mg [IQR = 13-108] versus median = 84 fmol/mg [IQR = 24-250] and P<.001). Patients with higher levels of HER-2/neu overexpression or amplification had statistically significantly lower levels of ER/PR than patients with lower levels of HER-2/neu overexpression or amplification. Because absolute HR levels are strongly related to response to hormone therapy in primary and advanced breast cancer, reduced ER/PR expression may be one mechanism to explain the relative resistance of HER-2/neu-positive:HR-positive tumors to hormone therapy.

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 (≥3.76 signals per cell), and 5 with hypodisomy (≤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, ≥2.00) achieved higher concordance with ACIS IHC than did an alternative FISH criterion (absolute HER-2/neu gene copy number, ≥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.

Evaluation of c-erbB-2 overexpression and Her-2/neu gene copy number heterogeneity in Barrett's adenocarcinoma.

Amplifiction of the Her-2/neu gene is accompanied by overexpression of its cell surface receptor product, c‐erbB‐2 protein. To investigate the degree of intratumoural heterogeneity we applied immunohistochemistry in primary Barrett’s adenocarcinoma (BCA) (n = 6) and dysplasia adjacent to the carcinoma (n = 4). In addition, fluorescence in situ hybridisation (FISH) was performed in primary BCA (n=5) and dysplastic areas (n=4). For an objective evaluation digital image analysis and laser scanning microscopy were used. Five of six BCA showed a marked intratumoural heterogeneous staining pattern ranging from areas in which the tumour cells were negative or faintly positive to tumour areas with a strong staining of the entire membrane. Among the two dysplastic areas also a heterogenous staining pattern was observed. FISH analysis revealed marked heterogeneity of intratumoural gene copy number changes in all BCA showing populations with different fractions of cells with polysomy, low level amplification and high level amplification. One dysplasia showed a minor population with Her‐2/neu signal clusters. In conclusion, we observed marked intratumoural heterogeneity of c‐erbB‐2 protein overexpression and Her‐2/neu gene copy number in the majority of the primary BCA analyzed. Digital image analysis and laser scanning microscopy were helpful in quantifying the variations in protein expression and DNA copy number in individual tumour cells. The observed heterogeneity could hamper the exact diagnostic determination of the c‐erbB‐2 status in small biopsies and possibly influence the effectiveness of a potential c‐erbB‐2 targeting therapy.Figures on http://www.esacp.org/acp/2000/20‐1/walch.htm.

Prognostic impact of HER-2/neu gene copy number determination by fluorescence in situ hybridisation (FISH) versus immunohistochemically detected overexpression in node positive primary breast cancer

Prognostic impact of HER-2/neu gene copy number determination by fluorescence in situ hybridisation (FISH) versus immunohistochemically detected overexpression in node positive primary breast cancer

Improved Quantitation of HER-2/neuGene Copy Number in Breast Tumor-Derived DNA Samples

A Southern blot-based assay is presented that increases the simplicity and accuracy of the HER-2/neu gene copy number assignment in breast cancer DNA. Genomic DNA was hybridized simultaneously with probes corresponding to portions of HER-2/neu and a single-copy gene, myeloperoxidase. A unique restriction fragment was detected for each gene. The use of DNA probes of similar mass and equal specific activities resulted in a ratio of band intensities on the resultant autoradiograph that reported the ratio of gene copy numbers directly. Patient samples containing amplified levels of the HER-2/neu gene were identified by simple visual inspection of a single autoradiograph. Analysis of breast cancer samples alongside the cell line DNAs, representing a range of HER-2/neu gene copy numbers, permits visual quantitation of the tumors' gene copy numbers. The authors show that the HER-2/neu gene copy number can be determined accurately in marginally degraded DNA, a feature of some clinical samples.

Neu-protein overexpression in breast cancer. Association with comedo-type ductal carcinoma in situ and limited prognostic value in stage II breast cancer.

Amplification of the neu proto-oncogene in breast cancer has been reported to correlate with the presence of lymph-node metastases and with a poor prognosis. We describe a method for the immunohistochemical detection of overexpression of neu protein on formalin-fixed paraffin-embedded tissue, with the use of two different monoclonal antibodies. In a group of tumors with a known neu-gene copy number, intense membrane staining of tumor cells was present in all tumors with neu-gene amplification. Of 189 tumors from patients with Stage II breast cancer, 27 (14 percent) had neu-membrane staining. Neu overexpression was associated with larger tumor size (P = 0.006) but not with lymph-node involvement. Neu-protein expression in lymph-node metastases was the same as its expression in primary tumors. Among the patients with neu overexpression (median follow-up, 37 months), disease-free survival was not significantly shorter; overall survival was reduced significantly in these patients (P = 0.042), but this reduction did not remain significant after adjustment for tumor size. Of 45 ductal carcinomas in situ, 19 (42 percent) had neu-membrane staining. These 19 were all of the large-cell, comedo growth type. None of 16 ductal carcinomas in situ of small-cell, papillary, or cribriform growth type had neu overexpression. We conclude that neu overexpression may be an early step in the development of a distinct histologic type of carcinoma of the breast, but we could find no association of overexpression with lymph-node status or tumor recurrence.