Home Translating Report News Physicians Diseases Body Sites Lab tests Search
Home Diseases and Health Information


HER2/neu protein has garnered a great deal of interest in the popular media.  However, it has long been known amongst pathologists and oncologists for its potential role as a tumor and prognostic marker. This protein exists on the surface of epithelial cells and functions in the normal cell as a receptor for a cellular growth factor. There is a protion of the protein that is clipped off and is present in the serum or plasma. In some cancerous cells, this protein loses its usual response to other regulatory proteins and leads to unregulated growth of cells forming a cancer.

To determine a patient's HER2/neu status, several diagnostic options are available to the pathologist. The first utilizes immunohistochemistry or special stains on the original tissue sections. The second utilizes a recent innovation in genetic probes called fluorescent in-situ hybridization or FISH. Genetic probes tagged with a fluorescent label are incubated with the chromosomes. If the gene is amplified or has more copies, it will be identified by the probe. The final method relies on detection of the protein in the patient's serum. An enzymatically linked test known as ELISA utilizes a monoclonal antibody tagged with a marker protein. This complex can bind to any HER2/neu protein present in serum. All three methods analyze slightly different aspects of the protein. The current controversy is which technique or combination of techniques is the best. Currently most pathologists utilize the first two techniques. Serum measurement may play a role in monitoring treatment. The final answer is not out yet but we will continue to keep you up to date on the developments. Meanwhile, the abstracts below highlight some of the current controversies and approaches. The most recent articles looking at both technologies finds both are valid but for values of 2+ on the her2-neu assay, additional follow up examination with FISH may be helpful.

There is a recent development utilizing Automated Cellular Imaging System (ACIS) which detects subtle immunohistochemical staining pattern differences on tissue slides. The abstracts below indicate a favorable comparison to tests such as FISH.


Comparison of Methodologies  
Reference Methods and Pathogenesis  
Clinical Utility  
Interfering Diseases or Substances that Alter Levels  
Commonly Used Terms  
Internet Links  

Chromogenic in-situ hybridization: a viable alternative to fluorescence in-situ hybridization in the HER2 testing algorithm.

Hanna WM, Kwok K.

1Sunnybrook and Women's College Health Sciences Centre, Toronto, ON, Canada.

Mod Pathol. 2006 Apr;19(4):481-7. Abstract quote  

Assessment of human epidermal growth factor receptor-2 status is standard practice in women with breast cancer. Most laboratories use immunohistochemistry as a screening test, with equivocal results confirmed by fluorescence in-situ hybridization (FISH). Chromogenic in-situ hybridization (CISH) is a relatively new method for detection of gene amplification using a peroxidase reaction, which can be viewed using a standard light microscope.

This study was undertaken to validate CISH as a method for assessing human epidermal growth factor receptor-2 gene amplification. The gene amplification status of human epidermal growth factor receptor-2 immunohistochemistry negative (0/1+, n=69; Group 1), immunohistochemistry positive (3+, n=50; Group 2) and equivocal tumor samples (2+, n=135; Group 3) was evaluated by FISH and CISH, and the concordance between FISH and CISH results calculated. In Group 1, 67/69 cases did not show amplification by CISH and 69/69 showed no amplification by FISH. Two cases were discordant; therefore, fluorescence/CISH concordance was 97%. In Group 2, 46/50 cases were amplified by FISH and 47/50 cases were amplified by CISH; three cases were not amplified by either method (immunohistochemistry false-positives). Only one case showed discordant FISH and CISH results, making the fluorescence/CISH concordance 98%. In Group 3, 89/135 cases were not amplified and 37/135 were amplified by both methods. Nine cases were discordant, giving a fluorescence/CISH concordance of 93%. The discordant cases were those with very low or borderline amplification with FISH.

The high level of concordance between FISH and CISH seen in this study suggests that CISH may be a viable alternative to FISH for use in the human epidermal growth factor receptor-2 testing algorithm.
Analytical Validation and Interobserver Reproducibility of EnzMet GenePro: A Second-Generation Bright-Field Metallography Assay for Concomitant Detection of HER2 Gene Status and Protein Expression in Invasive Carcinoma of the Breast.

Downs-Kelly E, Pettay J, Hicks D, Skacel M, Yoder B, Rybicki L, Myles J, Sreenan J, Roche P, Powell R, Hainfeld J, Grogan T, Tubbs R.

From the *Departments of Anatomical and Clinical Pathology, daggerBiostatistics and Epidemiology, Cleveland Clinic Foundation and the Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Cleveland, OH; double daggerSt. Rita's Medical Center, Lima, OH; section signVentana Medical Systems International, Tucson, AZ; and parallelNanoprobes Incorporated, Yaphank, NY.

Am J Surg Pathol. 2005 Nov;29(11):1505-1511. Abstract quote  

Fluorescence in situ hybridization (FISH) has both excellent sensitivity and specificity in detecting HER2 gene amplification in invasive breast carcinoma. FISH has not been widely implemented in clinical practice because of reagent costs and the special instrumentation and expertise required to perform and integrate the assay.

Immunohistochemistry (IHC) for HER2 protein is widely used, but false-positive and false-negative results are problematic. We developed a bright-field assay to visualize HER2 gene amplification and concomitant HER2 protein expression (EnzMet GenePro). This assay detects HER2 gene amplification via deposition of metallic silver by enzyme metallographytrade mark (EnzMettrade mark, Nanoprobes, Yaphank, NY) combined with HER2 protein detection by IHC using alkaline phosphatase and fast red K substrate visualization (CB11;Ventana, Tucson, AZ). The assay was performed on 94 invasive breast carcinomas, for which FISH (PathVysiontrade mark, Vysis, Downer's Grove, IL), conventional IHC (CB11), and enzyme metallography (EnzMettrade mark) results were known. The EnzMettrade mark component of the assay was scored as either HER2 gene amplified, polysomic, or nonamplified. The IHC component was scored using the conventional FDA scale of 0 to 3+. Concordance of the EnzMet component of the assay versus FISH was assessed and showed an excellent correlation (Pearson coefficient of 0.95; P < 0.001). The combination of gene and protein detection (EnzMet GenePro) displayed a specificity of 100% and an accuracy of 92.6% (95% confidence interval 85.3-97.0), facilitated recognition of gene/protein discordances, and allowed for efficient interpretation of the slide by conventional light microscopy.

The interobserver kappa for each component was excellent (IHC, kappa = 0.94; and EnzMettrade mark, kappa = 0.96). EnzMet is the first bright-field ISH assay in our experience that routinely and nonambiguously detects endogenous HER2 signals, essential for a reliable clinical HER2 assay, and in combination with HER2 protein enables improved diagnosis in borderline cases.
Reliability of chromogenic in situ hybridization for detecting HER-2 gene status in breast cancer: comparison with fluorescence in situ hybridization and assessment of interobserver reproducibility.

Gong Y, Gilcrease M, Sneige N.

1Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Mod Pathol. 2005 Aug;18(8):1015-21. Abstract quote  

Accurate determination of HER-2 status is important in the management of patients with breast cancer, especially in determining their eligibility for trastuzumab therapy. Fluorescence in situ hybridization (FISH) has been regarded as the gold standard method for detecting HER-2 gene amplification. Recently, chromogenic in situ hybridization (CISH), in which HER-2 is detected by a peroxidase reaction and the gene copies are determined by regular bright-field microscopy, has emerged as a potential alternative to FISH. However, this method requires validation before it can be adopted into clinical practice.

In this study, we evaluated 80 cases of invasive breast carcinoma by CISH, compared the results with those obtained by FISH, and assessed interobserver reproducibility among three observers. We found that agreement among the three pathologists on the CISH-determined HER-2 status was achieved in 73 cases (91%), all of which had results matching the corresponding FISH results: 54 nonamplified and 19 amplified. |

Of the 19 amplified cases, 13 were scored unanimously as high-level amplification; six had a minor scoring discrepancy (ie, low-level vs high-level amplification). A major scoring discrepancy (ie, nonamplification vs amplification) was found in the remaining seven cases, three of which were amplified and four of which were nonamplified by FISH. Two of the latter cases had a polysomy of chromosome 17. The cases that caused scoring difficulty were those with an equivocal or borderline signal number against a high background. Overall, there was nearly perfect agreement between the CISH and corresponding FISH results, and interpretation of CISH results were highly reproducible among the three pathologists.

We conclude that, in general, HER-2 status can be reliably assessed by CISH. Confirmatory FISH is recommended in cases with equivocal or borderline CISH copy numbers.
Analysis of Intratumoral Heterogeneity and Amplification Status in Breast Carcinomas With Equivocal (2+) HER-2 Immunostaining.

Lewis JT, Ketterling RP, Halling KC, Reynolds C, Jenkins RB, Visscher DW.

Division of Anatomic Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.

Am J Clin Pathol. 2005 Aug;124(2):273-81. Abstract quote  

Fluorescence in situ hybridization (FISH) and immunohistochemical analysis for assessment of HER-2 status in breast carcinomas are discordant in a significant proportion of cases with equivocal (2+) immunostaining.

To evaluate the role of intratumoral heterogeneity and degree of amplification, we performed additional HER-2 immunostains and FISH on tumor-bearing blocks from 20 invasive breast carcinomas with immunohistochemical scores of 2+ with gene amplification and in 18 cases without amplification.

Of the amplified cases, 11 (55%) had a 3+ immunohistochemical score on at least 1 additional slide, 8 (40%) remained 2+, and 1 (5%) had a slide scored 1+. All cases rescored 3+ showed high-level amplification in original and repeated FISH; cases remaining 2+ had a heterogeneous FISH profile (low-level amplification or a mosaic mixture of high-level amplified and nonamplified cells) in original and repeated FISH. Of nonamplified cases, 13 (72%) had a 1+ score on at least 1 additional slide, 4 (22%) remained 2+, and 1 (6%) had 1 slide scored 3+. In the nonamplified cases, 17 (94%) showed no amplification in repeated FISH.

Significant intratumoral heterogeneity and minimal (low-level) HER-2 amplification account for many breast cancers with 2+ HER-2 protein expression.
erb-b2 Amplification by Fluorescence In Situ Hybridization in Breast Cancer Specimens Read as 2+ in Immunohistochemical Analysis

Chieh Lan, MS, etal.
Am J Clin Pathol 2005;124:97-102 Abstract quote

We conducted this study to ascertain the prevalence of erb-b2 gene amplification in breast cancer specimens read as 2+ in immunohistochemical analysis. Slides from patients with metastatic or recurrent breast cancer were eligible for fluorescent in situ hybridization (FISH) study if they were read as 2+ immunohisto-chemically for erb-b2 by a certified pathologist.

The PathVysion kit (Vysis, Downers Grove, IL) was used for FISH studies. Amplification of the erb-b2 gene was defined as an erb-b2/CEP17 (chromosome 17 centromere) ratio of 2 or more in 30 tumor cells counted. From May 2003 to June 2004, 221 slides were submitted from 24 hospitals around the island. Of 216 successful hybridizations, 96 (44.4%) were determined to be erb-b2 amplified. In addition, the topoisomerase IIa gene was coamplified in 11 (21%) of 53 and deleted in 8 (15%) of 53 erb-b2 amplified cases.

The erb-b2 gene amplification rate was very high in cases determined to be 2+ by immunohistochemical analysis; therefore, determination of erb-b2 status by FISH in cases scored 2+ immunohistochemically is strongly recommended.
Comparison of Immunohistochemical and Fluorescence In Situ Hybridization Assessment of HER-2 Status in Routine Practice

Michelle Dolan, MD and Dale Snover, MD
Am J Clin Pathol 2005;123:766-770 Abstract quote

Because HER-2 expression in invasive carcinoma of the breast has well-documented ramifications for treatment and prognosis, accurate assessment of HER-2 status is critical. Comparative studies have shown high concordance rates between immunohistochemical analysis and fluorescence in situ hybridization (FISH) in cases with immunohistochemical scores of 0 or 1+ (negative) and 3+ (strongly positive) and low concordance rates among cases with immunohistochemical scores of 2+.

The present study was performed to determine concordance rates in a setting more representative of routine clinical practice, in which multiple pathologists submit specimens to a single cytogenetics referral laboratory. We found a higher rate of discordance between immunohistochemical analysis and FISH (approximately 92%) in the groups with immunohistochemical scores of 2+ than reported in other studies.

These results strongly support the practice of performing FISH in all cases with immunohistochemical scores of 2+, particularly in routine practice, in which interobserver variability in immunohistochemical scoring among multiple pathologists is likely to be high.
HER-2/neu Detection in Fine-Needle Aspirates of Breast Cancer
Fluorescence In Situ Hybridization and Immunocytochemical Analysis

Barbara G. Beatty, PhD, etal.
Am J Clin Pathol 2004;122:246-255 Abstract quote

We evaluated HER-2 receptor status by immunocytochemical and immunohistochemical analyses and fluorescence in situ hybridization (FISH) in 51 fine-needle aspiration (FNA) specimens together with the corresponding formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained from surgically resected breast cancers.

Three fixation methods were compared: ethanol, formalin, and CytoLyt-ThinPrep (Cytyc, Boxborough, MA). HER-2 was overexpressed and amplified in 8 (16%) of 51 FFPE specimens. Of the 8 cases, gene amplification was observed in 8 FNA specimens (100%) and overexpression in 2 (25%) ethanol-, 4 (50%) CytoLyt-, and 5 (63%) formalin-fixed FNA specimens. Strong pairwise k association between FISH results performed on FNA specimens and FFPE tissue samples (ethanol fixation, k = 0.848; ThinPrep, k = 0.918) and moderate (ThinPrep, k = 0.692; formalin fixation, k = 0.667) to poor (ethanol, k = 0.300) pairwise k agreement between tissue immunohistochemical and FNA immunocytochemical results was demonstrated.

We conclude that HER-2 protein expression on cytologic preparations was insufficiently reliable for clinical use, whereas HER-2 gene amplification determined by FISH demonstrated strong and consistent correlation with HER-2 status of FFPE tissue samples.
Comparative assays for the HER-2/neu oncogene status in breast cancer.

Vera-Roman JM, Rubio-Martinez LA.

Department of Pathology, Hospital General de Castellon, Castellon de la Plana, Spain
Arch Pathol Lab Med. 2004 Jun;128(6):627-33. Abstract quote  

CONTEXT: Tumor marker assays, especially those used to indicate the right therapy, should be standardized.

OBJECTIVE: To analyze the current methods for the HER-2/neu (h2n) oncogene status by immunohistochemical (IHC) analysis, fluorescence in situ hybridization (FISH), and chromogenic in situ hybridization (CISH) and compare those results with the chromosome 17 copy number and the status of the topoisomerase II alpha (TPIIalpha) gene.

DESIGN: We tested 50 infiltrating ductal breast carcinomas (pTNM status varied from pT1 N0 to pT4 N1) using the Food and Drug Administration (FDA)-approved methods HercepTest and Pathway for overexpression of h2n. We also used FISH and CISH to test for h2n amplification and CISH to test for chromosome 17 (c17) and TPIIalpha. The p53 and Ki-67 factors were also evaluated by IHC analysis.

RESULTS: h2n overexpression (3+) and amplification were observed in only 6 (12%) of 50 cases by IHC analysis, FISH, and CISH. Three cases that initially scored 3+ and 2+ had 4 to 5.95 signals (equivocal) by FISH but when corrected by the h2n/c17 ratio were nonamplified. TPIIalpha isomerase was amplified in only 2 (4%) of the 50 cases. Nineteen (38%) of the 50 cases were aneuploidic. All h2n amplified cases had high proliferative activity, but only 2 of 6 had p53 protein alterations.

CONCLUSIONS: The HercepTest and Pathway IHC assay h2n were fully concordant for the 3+ cases. The 3+ cases had to be confirmed in 75% of the tumor area examined. These 2 IHC assays were fully concordant with FISH and CISH. The 2 in situ hybridization (ISH) assays were 94% concordant for the 50 cases. The cutoff signal points for both ISH assays should be 6 or more. Thus, there is no need for the c17 ratio correction. Tumor heterogeneity appears not be a major problem, but our percentage of amplified cases is lower than previously reported. The FDA-approved IHC and ISH assays should give relatively uniform results when used following our recommendations.
HER-2 Testing in Breast Cancer Using Immunohistochemical Analysis and Fluorescence In Situ Hybridization
A Single-Institution Experience of 2,279 Cases and Comparison of Dual-Color and Single-Color Scoring

Priti Lal, MD, Paulo A. Salazar, Clifford A. Hudis, MD, Marc Ladanyi, MD, and Beiyun Chen, MD, PhD
Am J Clin Pathol 2004;121:631-636 Abstract quote

We analyzed concordance between immunohisto-chemical analysis and fluorescence in situ hybridization (FISH) in HER-2 status and studied the effect of dual-color (D-FISH) vs single-color FISH (S-FISH) scoring on the assignment of tumors to amplified or nonamplified categories. The assays were performed on formalin-fixed, paraffin-embedded sections of 2,279 invasive breast carcinomas. Immunohistochemical results were interpreted as negative (0, 1+) or positive (2+, 3+). For FISH analyses, a ratio for HER-2/chromosome 17 of 2.0 or more (D-FISH) or an absolute HER-2 copy number per nucleus of more than 4.0 (S-FISH) were interpreted as positive gene amplification.

We found 547 (24.0%) cases positive immunohisto-chemically, 326 (14.3%) by D-FISH, and 351 (15.4%) by S-FISH. Overall concordance in HER-2 status with immunohistochemical analysis was 87% for D-FISH and 86% for S-FISH. Excellent concordance was found among groups scored immunohistochemically as 0, 1+, and 3+ (with D-FISH , 97%; with S-FISH, 96%).

The most discordant category was the group scored 2+ immunohistochemically, in which only a quarter of the 2+ tumors were FISH(+). D-FISH and S-FISH scoring results were discordant in 89 tumors (4%), of which 8 (9%) had 3+ immunohistochemical staining and none showed high-level HER-2 amplification.

Among all FISH(+) tumors, 10% were negative by immunohisto-chemical analysis, and notably almost half (47%) showed borderline to low HER-2 amplification (D-FISH score, 2.0-3.9); the clinical significance of these findings warrants further investigation.
Enhanced Accuracy and Reliability of HER-2/neu Immunohistochemical Scoring Using Digital Microscopy

Kenneth Bloom, MD and Douglas Harrington, MD
Am J Clin Pathol 2004;121:620-630 Abstract quote

We evaluated the HER-2/neu status of 129 invasive breast cancer specimens for gene amplification by fluorescence in situ hybridization (FISH) and protein overexpression by immunohistochemical analysis. Each immunohistochemically stained slide was interpreted on a standard microscope independently by 10 pathologists. Separately, each pathologist reviewed the same slide set with the assistance of digital microscopy.

A total of 1,258 manual immunohistochemical scores and 1,269 digital microscopy immunohistochemical scores were completed. When the same 10 pathologists scored the same immunohistochemical slides with the assistance of digital microscopy, each reviewer improved concordance with FISH, and overall concordance with immunohistochemical analysis improved significantly, to 93% (P < .001). The interrater k was used to compare interobserver agreement in HER-2 immunohistochemical scoring for manual and digital microscopy interpretation. Significant improvement in interobserver agreement (k = 0.51 vs 0.86; P < .001) was achieved when HER-2 immunohistochemical analysis was scored with the assistance of the digital microscope.

The assistance of digital microscopy improves the accuracy and reliability of HER-2 immunohistochemical analysis. These data suggest that documented discrepancies between HER-2 immunohistochemical analysis and FISH reflect predominantly errors in manual immunohistochemical interpretation as opposed to immunohistochemical reagent limitations.

HER-2 testing in breast cancer using parallel tissue-based methods.

Yaziji H, Goldstein LC, Barry TS, Werling R, Hwang H, Ellis GK, Gralow JR, Livingston RB, Gown AM.

PhenoPath Laboratories, Seattle, Wash 98103, USA.
JAMA. 2004 Apr 28;291(16):1972-7. Abstract quote  

CONTEXT: Testing for HER-2 oncogene in breast cancer has increased because of its role as a prognostic and predictive factor. Some advocate gene testing by fluorescence in situ hybridization (FISH) vs protein testing by immunohistochemistry as the method which most accurately evaluates and predicts response to the anti-HER-2 antibody, trastuzumab. However, critical examination of FISH on a screening basis has yet to be performed.

OBJECTIVES: To determine the correlation between FISH and immunohistochemistry results by determining HER-2/neu gene status on tumor sections with indeterminate immunohistochemistry results (2+ score), confirm gene amplification on tumor sections with positive results (3+ score), and verify gene status on tumor sections with negative results (0 or 1+ score).

DESIGN, SETTING, AND PATIENTS: A quality control and quality assurance program for HER-2 testing by FISH, which used tumor specimens from 2963 patients (median age, 56 years) with breast cancer received from 135 hospitals and cancer centers in 29 states, was performed at a reference laboratory from January 1, 1999, to May 15, 2003. Every specimen evaluated by FISH was parallel tested with immunohistochemistry tests.

MAIN OUTCOME MEASURES: With FISH as the presumed standard testing method, the positive and negative predictive values and sensitivity and specificity of immunohistochemistry were calculated. RESULTS: A total of 3260 clinical HER-2 tests by FISH were performed on 2963 serially referred breast cancer specimens. Of these, 2933 tests were successful and 2913 breast cancer specimens had both FISH and immunohistochemistry results available. With FISH as the standard testing method, the positive predictive value of positive immunohistochemistry score (3+) was 91.6%, and the negative predictive value of negative immunohistochemistry score (0 or 1+) was 97.2%. The sensitivity of immunohistochemistry tests, including tumor sections with scores of 2+ or 3+, was 92.6% and the specificity of immunohistochemistry tests with scores of 3+ was 98.8%. The FISH test had a significantly higher failure rate (5% vs 0.08%) and reagent cost (140 dollars vs 10 dollars), and longer testing (36 hours vs 4 hours) and interpretation times (7 minutes vs 45 seconds) vs immunohistochemistry tests.

CONCLUSIONS: A testing algorithm for HER-2 determination is most efficient by using immunohistochemistry as the method of choice, with FISH performed for cancers with indeterminate results (2+ score). Successful quality control and quality assurance programs are a prerequisite for such approaches.

Assessment of methods for tissue-based detection of the HER-2/neu alteration in human breast cancer: a direct comparison of fluorescence in situ hybridization and immunohistochemistry.

Pauletti G, Dandekar S, Rong H, Ramos L, Peng H, Seshadri R, Slamon DJ.

Department of Medicine, Division of Hematology-Oncology, University of California at Los Angeles, UCLA School of Medicine, Los Angeles, CA, USA.

J Clin Oncol 2000 Nov 1;18(21):3651-64 Abstract quote

PURPOSE: To compare the efficacy of fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in detecting the HER-2/neu alteration in human breast cancer.

PATIENTS AND METHODS: Unselected stage I, II, and III breast cancer patients (N = 900) were tested for HER-2/neu gene amplification by FISH in paraffin-embedded, formalin-fixed archival material. Of these samples, 856 were tested for HER-2/neu overexpression by non-antigen-retrieval IHC with the polyclonal antibody R60, the sensitivity and specificity of which was preliminarily compared with the United States Food and Drug Administration-approved HercepTest (Dako Corp, Carpinteria, CA). Patient survival was analyzed in relation to the presence of the HER-2/neu alteration as determined by these two methodologies.

RESULTS: A total of 189 (21%) of 900 patients were positive by FISH and 147 (17.2%) of 856 were positive by IHC. This discrepancy is consistent with expected loss of IHC sensitivity associated with tissue fixation/embedding. The HercepTest did not improve sensitivity and introduced false positives. Comparison of R60-based IHC with FISH demonstrates that patient survival is associated progressively to gene amplification level as determined by FISH, whereas for IHC an association is found only in the highest (3+) immunostaining group. Among FISH-negative tumors, 45 (6.6%) of 678 were IHC-positive, with a survival probability similar to that of FISH-negative/IHC-negative cases; FISH-positive/IHC-negative patients have a survival probability similar to that of FISH-positive/IHC-positive cases.

CONCLUSION: IHC does not consistently discriminate patients likely to have a poor prognosis, whereas FISH provides superior prognostic information in segregating high-risk from lower-risk beast cancers. HER-2/neu protein overexpression in the absence of gene amplification occurs infrequently in breast cancer, in which case, patient outcome is similar to that of patients without the alteration.

Comparison of HER2/neu Status Assessed by Quantitative Polymerase Chain Reaction and Immunohistochemistry

Frances P. O'Malley, etal.

Am J Clin Pathol 2001;115:504-511 Abstract quote

We prospectively evaluated a series of 254 breast cancers by quantitative polymerase chain reaction (PCR) and immunohistochemistry using 3 antibodies: HercepTest, CB11, and TAB250.

DNA was extracted from a 10-µm tumor section for PCR, and 4-µm serial sections were taken from the same block for immunohistochemistry. The immunohistochemical results were scored using a semiquantitative immunohistochemical system. A positive tumor by immunohistochemistry had a score of 5 or more. The manufacturer's recommended scoring system was used for the HercepTest. Tumors were positive for gene amplification if the ratio of the HER2/neu gene to control gene after normalization was 2 or more.

Of 254 cases, 61 showed gene amplification. For immunohistochemistry, 23% of tumors were positive with CB11, 27% with TAB250, and 37% with the HercepTest. Results for each antibody were compared with PCR results.

The overall concordance for the HercepTest was 82%, which was significantly lower than that for CB11 (88%) or TAB250 (87%). The specificity for the HercepTest was 80% compared with 90% for TAB250 and 93% for CB11, while the positive predictive value for the HercepTest was 57% compared with 71% and 76% for TAB250 and CB11, respectively.

HER-2/neu in Breast Cancer: Interobserver Variability and Performance of Immunohistochemistry with 4 Antibodies Compared with Fluorescent In Situ Hybridization

Thomas A. Thomson, M.D., Malcolm M. Hayes, M.B.Ch.B., John J. Spinelli, Ph.D., Ernie Hilland, B.Sc., Christina Sawrenko, B.Sc., R.T., Don Phillips, R.T., Beverley Dupuis, R.T. and Robin L Parker, M.D.

BC Cancer Agency (TT, MH, JS, EH, CS, DP), Vancouver Hospital (BD), and University of British Columbia (RP), Vancouver, British Columbia, Canada

Mod Pathol 2001;14:1079-1086 Abstract quote

The immunohistochemistry (IHC) performance of 4 anti-HER-2/neu antibodies was compared with fluorescent in situ hybridization (FISH) analysis of HER-2/neu gene expression in breast cancer patients considered for Herceptin (Trastuzumab) therapy.

Interobserver variability in IHC interpretation was measured. Formalin-fixed tissue was received from 24 provincial hospital laboratories. The following anti-Her-2 antibodies were used: DAKO A0485 (polyclonal), Novacastra CB11 (monoclonal), Zymed TAB250 (monoclonal), and DAKO HercepTest (polyclonal). Additional sections were analyzed by FISH (Vysis).

Three pathologists blinded to FISH results independently interpreted invasive tumor cell membranous staining on a scale of 0 to +3. The HER-2/neu gene was considered amplified when the FISH signal ratio of HER-2/CEP-17 was 2.0. Blocks from all hospitals and of all ages were suitable for IHC and FISH analysis. No interlaboratory analysis variability was noted. The interobserver agreement (kappa) for stain intensity for each antibody was good for 0 and +3 but poor for +1 and +2. Reasonable concordance between IHC and FISH was found with three of the four antibodies. TAB250 was the most sensitive antibody. For the three pathologists, the IHC sensitivities and specificities compared with FISH using 0/+1 as negative and +2/+3 as positive were as follows: A0485, 63–84/95–98; CB11, 63–66/97–98; TAB-250, 82–100/94–95; HercepTest, 59–77/91–93. The positive and negative predictive values varied by stain intensity. Stain scores of 0 and +3 were highly predictive of gene status. Stain scores of +1 and +2 were not sufficiently predictive to classify cases as amplified versus nonamplified. IHC is a reasonable first test to assess HER-2/neu status in patients with breast cancer.

For most cases, DAKO A0485, TAB250, and HercepTest adequately predicted gene status. In cases with stain intensity of +1 or +2, the interobserver agreement is poor, and the predictive value is unsatisfactory for clinical use. Additional testing, preferably with FISH, is recommended.

Protein overexpression and gene amplification of c-erbB-2 in breast carcinomas: A comparative study of immunohistochemistry and fluorescence in situ hybridization of formalin-fixed, paraffin-embedded tissues

Masako Kobayashi, MD
Akishi Ooi, MD
Yoshio Oda, MD
Isao Nakanishi, MD

Hum Pathol 2002;33:21-28 Abstract quote

We evaluated 173 consecutive breast carcinomas for c-erbB-2 using a combination of immunohistochemistry (IHC) with a commercial polyclonal antibody (Nitirei) and dual-color fluorescence in situ hybridization (FISH) using the c-erbB-2–specific probe and the chromosome 17 centromere–specific probe from Vysis (Downers Grove, IL) and compared the results with the histologic characteristics of intraductal spread, cancer invasion, and intratumoral heterogeneity.

With correction for chromosome 17 copy number, c-erbB-2 amplification was observed in 26 tumors (13.5%): high-level amplification in 23 tumors, and low-level amplification in 3. The gene amplification was positively correlated with c-erbB-2 protein overexpression, defined as 2+ or 3+ immunostaining, on a case-by-case basis (P < .000001). All 3+ immunostaining tumors (19 tumors) showed high-level amplification, although gene amplification was found in only 5 of 27 2+ immunostaining tumors. Although the rates of overexpression and gene amplification did not differ in ductal carcinomas in situ and invasive carcinomas (P = .46 and .53, respectively), they were significantly higher in invasive carcinomas with intraductal spreading (P < .0001). Intratumoral heterogeneity of c-erbB-2 amplification was found in only 1 case; however, in 17 invasive carcinomas, intraductal components expressed c-erbB-2 more intensely than invasive components.

We conclude that in breast carcinomas, c-erbB-2 overexpression occurs mostly in tumors with high-level gene amplification, and such overexpression appears to endow carcinoma cells with the capacity for intraductal spreading. The best method for detecting breast carcinomas with c-erbB-2 aberrations using archival tissues is to screen cases by IHC; however, follow-up FISH assays are indispensable for excluding false-positive results.

HER2 Assessment by Immunohistochemical Analysis and Fluorescence In Situ Hybridization
Comparison of HercepTest and PathVysion Commercial Assays

Stanley R. McCormick, MD
Tamera J. Lillemoe, MD
Janet Beneke, MD
John Schrauth, MT(ASCP)
and John Reinartz, MD

Am J Clin Pathol 2002;117:935-943 Abstract quote

We determined HER2 protein overexpression by immunohistochemical analysis and HER2 gene amplification by fluorescence in situ hybridization (FISH) in 215 formalin-fixed, paraffin-embedded breast tumors.

Pathologist concordance for immunohisto-chemical scoring, and HER2 status concordance, as determined by immunohistochemistry and FISH, were high for immunohistochemical 3+, 1+, and 0 tumors but poor for 2+ tumors. Consensus immunohistochemical scores correlated with absolute and chromosome 17 (CEP17)–corrected HER2 gene copy number. Among HER2-nonamplified tumors, the immunohistochemical score and mean absolute chromosome 17 (CEP17) copy number were weakly correlated. Seventeen tumors were HER2-amplified using absolute HER2 gene criteria but nonamplified when corrected for chromosome 17 polysomy (8 of these were immnuohistochemical 2+). Assessment of benign epithelium within the immuno-histochemical slides revealed either no staining or basolateral membrane staining, suggesting normal HER2 protein expression. Twenty tumors showing similar basolateral HER2 immunostaining were all low-moderate grade, tubule-forming, and HER2-nonamplifed (17) or borderline amplified (3). Additional studies relating changes in HER2 gene content due to amplification or chromosome 17 polysomy and HER2 protein expression may be helpful to pathologists who interpret HER2 immnuohistochemical slides.

Breast tumors scored at 2+ should be analyzed by FISH, preferably using a dual-probe FISH assay capable of distinguishing HER2 gene amplification from chromosome 17 polysomy.

Determination of HER2 Gene Amplification by Chromogenic In Situ Hybridization (CISH) in Archival Breast Carcinoma.

Zhao J, Wu R, Au A, Marquez A, Yu Y, Shi Z.

Center for Biomedical Laboratory Science (JZ, ZS), San Francisco State University, San Francisco, California.

Mod Pathol 2002 Jun;15(6):657-65 Abstract quote

Purpose: To compare the efficacy of chromogenic in situ hybridization (CISH(TM)) with fluorescence in situ (FISH) hybridization and immunohistochemistry (IHC) in determination of the HER2 status in human breast cancer.

Materials and Methods: HER2 gene amplification was determined on formalin-fixed paraffin-embedded (FFPE) sections of 62 invasive breast cancers by FISH and followed by CISH using a digoxigenin (DIG)-labeled HER2 DNA probe generated by Subtraction Probe Technology (SPT(TM)), and a biotin-labeled chromosome 17 centromeric (chr.17cen) probe. The sections were heat treated and enzyme digested. After in situ hybridization, the HER2 probe was detected with fluorescein (FITC)-anti-DIG for FISH, followed by peroxidase-anti-FITC and diaminobenzidine (DAB) for CISH. The chr.17cen probe was detected with peroxidase-streptavidin and DAB. For CISH application, HER2 gene copies or chromosome 17 centromeres and morphology of cells were easily visualized simultaneously with a 40x objective under bright-field microscope in hematoxylin-counterstained sections. IHC study of HER2 overexpression was performed on adjacent sections using a panel of three HER2 antibodies (TAB 250, CB11, A0485), and staining was scored according to the criteria specified in the HercepTest.

Results: HER2 gene amplification detected by CISH was visualized typically as large DAB-stained clusters or by many dots in the nucleus. FISH and CISH identified HER2 gene amplification in 19% of the tumors. Chromosome 17 polysomy was detected in 31% of the tumors. HER2 overexpression was demonstrated in 19% (TAB 250), 23% (CB11), and 36% (A0485) of the tumors. Complete concordance between the results of CISH with FISH, TAB 250, CB11, and A0485 was seen in 100%, 97%, 94%, and 84% of the cases, respectively.

Conclusion: By permitting observation of morphology using a bright-field microscope, CISH is an accurate, practical, and economical approach to screen HER2 status in breast cancers. It is a useful methodology for confirming ambiguous IHC results.

Evaluation of HER-2/neu immunohistochemical assay sensitivity and scoring on formalin-fixed and paraffin-processed cell lines and breast tumors: a comparative study involving results from laboratories in 21 countries.

Rhodes A, Jasani B, Anderson E, Dodson AR, Balaton AJ.

UK NEQAS-ICC, The Department of Histopathology, University College London Medical School, England.

Am J Clin Pathol 2002 Sep;118(3):408-17 Abstract quote

Variation in assay sensitivity was studied in more than 90 laboratories that assayed 4formalin-fixed, paraffin-processed breast and ovarian carcinoma cell lines with graded levels of HER-2/neu protein overexpression and known levels of HER-2/neu gene amplification, in addition to breast carcinomas fixed and processed in the laboratories.

Main methods were the HercepTest (DAKO, Ely, England) and individualized protocols using a polyclonal antibody and the CB11 clone. While the proportion of laboratories achieving appropriate results with the HercepTest was significantly higher than for participants using other assays, laboratories using other assays showed significant improvement in the second assessment run. The level of agreement in evaluations by 26 laboratories using the HercepTest was excellent on cell lines and tumors and was significantly greater than that achieved by the remaining 41 laboratories using other immunohistochemical methods.

While laboratories using the DAKO HercepTest had the highest level of reproducibility in assay sensitivity and evaluation, the significant improvement in results by laboratories using other antibodies in the second assessment run suggests that stringent quality control and an ongoing quality assurance program using a standard reference material have the potential to improve the reliability of immunohistochemical assays for HER-2/neu, regardless of the antibody used.

Interobserver Reproducibility of Her-2/neu Protein Overexpression in Invasive Breast Carcinoma Using the DAKO HercepTest

Chih-Yi Hsu, MD, MHA, Donald Ming-Tak Ho, MD, FRCPC, FCAP, Ching-Fen Yang, MD, Chiung-Ru Lai, MD, I-Ting Yu, MD, and Hung Chiang, MD


Am J Clin Pathol 118:693-698 Abstract quote

Although there are criteria for interpretation of the staining results for Her-2/neu in the DAKO HercepTest, the determination of staining intensity and percentage of complete membrane staining is subjective.

We studied 46 cases of invasive breast carcinoma to evaluate interobserver reproducibility among 5 pathologists. Complete agreement was achieved in 22 (48%) of 46 cases. Generalized kappa values indicated substantial agreement (0.80). Discrepancies between negative (0, 1+) and positive (2+, 3+) results occurred in 2 cases (kappa = 0.96). One was because of a tangential cut of the basal part of the tumor that mimicked complete membranous staining, and the other was a borderline case that revealed focal (5%-15%) complete membranous staining. Distinguishing weakly (2+) from strongly (3+) positive results showed agreement in only 13 (59%) of 22 positive cases (kappa = 0.38). If more than 50% of tumor cells revealing strong complete membrane staining were regarded as strongly positive, agreement would be improved (kappa = 0.78).

While there was a high percentage (70%-80%) of negative cases during routine evaluation, the good interobserver agreement and high negative predictive value made immunohistochemical analysis an effective screening test to exclude negative cases.


Evaluation of HER-2/neu oncogene status in breast tumors on tissue microarrays.

Zhang D, Salto-Tellez M, Do E, Putti TC, Koay ES.

Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospital and Department of Pathology, National University of Singapore, Singapore.


Hum Pathol 2003 Apr;34(4):362-8 Abstract quote

The amplification and/or overexpression of the HER-2/neu oncogene and its encoded receptor protein are increasingly used for prognostication and prediction of therapeutic response to Herceptin in breast cancer. However, large-scale examination of archival tumor blocks by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) is prohibitively laborious and technically challenging.

The tissue microarray (TMA) technique enables hundreds of tumors to be studied simultaneously in a single experiment. To evaluate the HER-2/neu status of a selection of the breast tumors in our tumor bank, we constructed a TMA from 97 breast tumors, with a single 0.6-mm core per specimen. HER-2/neu gene amplification by FISH was found in 20 of the 87 interpretable cases (23%): in 14 of 14 IHC 3+ cases (100%), 5 of 8 IHC 2+ cases (62.5%) and 1 of 65 IHC 0/1+ cases (1.5%). Three of the 67 cases with no evidence of HER-2/neu gene amplification by FISH were moderately positive (2+) by IHC.

A close relationship was observed between these 2 assays as applied to the TMA (95.4% concordance: 95% CI, -2.2% to 6.8%; P <0.0001), and both HER-2/neu gene amplification and protein overexpression were strongly associated with tumor grade, estrogen receptor status, and progesterone receptor status. Gene amplification was found in most of the tumors with high-level overexpression (IHC 3+) and not in the unequivocal IHC-negative cases.

Complementary analysis by IHC and FISH are, however, recommended for tumors graded as 2+ by IHC, the group with the most result discrepancy. Hum Pathol 34:362-368. Copyright 2003 Elsevier Inc. All rights reserved.


Current diagnostic methods of HER-2/neu detection in breast cancer with special regard to real-time PCR.

Merkelbach-Bruse S, Wardelmann E, Behrens P, Losen I, Buettner R, Friedrichs N.

Institute of Pathology, University of Bonn, Germany.
Am J Surg Pathol. 2003 Dec;27(12):1565-70. Abstract quote  

In this study we compare different diagnostic methods to measure HER-2/neu gene amplification in breast cancer with special regard to real-time polymerase chain reaction. Fifty specimens of breast cancer were analyzed, and the use of laser-assisted microdissection prior to PCR was investigated.

A total of 38 of 50 cases showed HER-2/neu overexpression in immunohistochemistry. In the 2+ scored group, 2 of 23 cases turned out to be amplified after FISH analysis and 14 of 15 cases in the 3+ group were amplified. Of the 16 amplified cases, 3 initially were measured as nonamplified by real-time PCR but showed amplification after laser capture microdissection. One case showed amplification by PCR but turned out to have only one copy of chromosome 17 by FISH. All 0 or 1+ scored cases were measured as nonamplified both by FISH and PCR. Initial concordance rate between FISH and PCR was 92% and could be increased to 98% using laser-assisted microdissection. FISH and PCR showed high diagnostic accuracy and concordance while immunohistochemistry overestimates amplification within the 2+ scored group.

Therefore, either FISH or PCR should be applied in cases scored 2+ by immunohistochemistry. Diagnostic accuracy of PCR can be increased using laser-assisted microdissection.

Evaluation of her-2/neu status in carcinomas with amplified chromosome 17 centromere locus.

Departments of Pathology, Oregon Health & Science University, Portland.


Am J Clin Pathol. 2006 Nov;126(5):709-16 Abstract quote

Accurate assessment of Her-2/neu (erb-b2) status in breast carcinoma is essential for therapy planning. Clinical assays are targeted at protein overexpression (immunohistochemical analysis) or gene amplification (fluorescence in situ hybridization [FISH]). Cases with aberrant FISH signal patterns are problematic and may lead to underreporting of Her-2/neu amplification.

We performed FISH with additional chromosome 17 probes, SMS (Smith-Magenis syndrome critical region) and RARA (retinoic acid receptor), on 7 cases with unusual Her-2/CEP17 (chromosome 17 centromere control probe) results to assess whether different measurements of chromosome 17 copy number might clarify the Her-2/neu amplicon status. Although the Her-2/CEP17 ratio scores were within normal range (<2.0), the Her-2/SMS or Her-2/RARA ratio revealed amplification of Her-2/neu in 5 of 7 cases. Immunohistochemical analysis demonstrated Her-2/neu protein overexpression in the same 5 cases only. We describe novel application of SMS/RARA FISH probes for assessing cases with complex Her-2/CEP17 FISH patterns.

Such additional data, correlated with immunohistochemical analysis, may help guide therapy in patients with breast carcinoma.
Determination of HER-2 Status and Chromosome 17 Polysomy in Breast Carcinomas Comparing HercepTest and PathVysion FISH Assay

Dolly Varshney, MD, Yvonne Y. Zhou, PhD, Stephen A. Geller, MD, and Randa Alsabeh, MD
Am J Clin Pathol 2004;121:70-77 Abstract quote

We evaluated and compared 2 HER-2 tests (immunohistochemical analysis [HercepTest, DAKO , Carpinteria, CA] and fluorescence in situ hybridization [FISH]) and assessed chromosome 17 polysomy status in relation to these tests. HER-2 status was obtained in 690 cases.

The rinse step in the HercepTest before and after addition of the visualization reagent was 2 minutes in 188 cases and was increased to 5 minutes in 600 cases. HercepTest with both rinse steps was performed on duplicate slides in 98 cases. Chromosome 17 ploidy status based on FISH results was determined in 687 cases. Weak overexpression (2+) of HER-2 protein was not due to gene amplification in a majority of cases (67/76 [88%]). A small subset of breast carcinomas (19/687 [2.8%]) strongly overexpressed (3+) HER-2 protein without gene amplification. The aneuploidy rate was similar in negative and 2+ cases (60/141 [42.5%] and 12/26 [46%]), compared with 86% (18/21) in 3+ cases. The incidence of polysomy 17 in 2+ nonamplified cases (3/67 [4%]) was similar to that seen in negative cases (5.5%), in contrast with 47% (9/19) of 3+ nonamplified cases. Adding a longer rinse step to the HercepTest converted a subset (3/10 [30%]) of weakly positive cases to negative cases.

Weak overexpression of HER-2 protein in a majority of cases seems to represent an artifactual staining pattern. Chromosome 17 polysomy is a major factor in strong HER-2 protein overexpression in 3+ nonamplified cases.
Aneusomy 17 in Breast Cancer: Its Role in HER-2/neu Protein Expression and Implication for Clinical Assessment of HER-2/neu Status

Sijian Wang, M.D., Ph.D., M. Hossein Saboorian, M.D., Eugene P. Frenkel, M.D., Barbara B. Haley, M.D., Momin T. Siddiqui, M.D., Sefik Gokaslan, M.D., Linda Hynan, Ph.D. and Raheela Ashfaq, M.D.

Department of Pathology (SW, MHS, MTS, SG, RA), Department of Internal Medicine (EPF, BBH), and Academic Computing Services (LH), The University of Texas Southwestern Medical Center, Dallas, Texas

Mod Pathol 2002;15:137-145 Abstract quote

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.

Her-2/ neu Gene Amplification in Familial vs Sporadic Breast Cancer Impact on the Behavior of the Disease

Ana B. Espinosa, PhD, Maria D. Tabernero, MD, etal.
Am J Clin Pathol 2003;120:917-927 Abstract quote

We compared the incidence of Her-2/ neu amplification in patients with and without a family history of breast cancer and correlated gene status with clinicobiologic and prognostic features in sporadic and familial cases. Of 108 patients, 28.7% had gene amplification. Among 96 cases with family history information available, 28 had an affected first-degree relative.

The gene was amplified more frequently in familial than in sporadic cases (13/28 [46%] vs 14/68 [21%]; P = .01). Among familial cases, amplification was associated with adverse clinicobiologic features (poorly differentiated tumors [ P = .05], larger tumors [ P = .05], more lymph nodes involved [ P = .04], and DNA aneuploid [ P = .02] and highly proliferative tumors [ P = .005]), and the relapse ( P = .02) and disease-related death ( P = .05) rates were higher than in cases without amplification. Among sporadic cases, amplification was not associated with significantly different disease features, except for a higher incidence of DNA aneuploid tumors ( P = .01), percentage of S-phase tumor cells ( P = .006), and lower proportion of estrogen ( P = .001) and progesterone ( P = .002) receptors.

Her-2/neu amplification was observed more frequently among patients with a family history of breast cancer, in whom it was associated with adverse clinicobiologic features and a worse clinical outcome.


Amplification of Her-2/neu Gene in Her-2/neu-Overexpressing and -Nonexpressing Breast Carcinomas and Their Synchronous Benign, Premalignant, and Metastatic Lesions Detected by FISH in Archival Material

Ruliang Xu, M.D., Mary Ann Perle, Ph.D., Giorgio Inghirami, M.D., Wai Chan, B.S., Yara Delgado, M.D. and Helen Feiner, M.D.
New York University School of Medicine, Department of Pathology, New York, New York (RX, MAP, GI, WC, YD); and Quest Diagnostics, Anatomic Pathology, Teterboro, New Jersey (HF)

Correspondence: Address reprint requests to: Helen Feiner, M.D., Quest Diagnostics, Department of Anatomic Pathology, One Malcolm Avenue, Teterboro, NJ 07608.

Mod Pathol 2002;15:116-124 Abstract quote

Amplification of Her-2/neu in breast carcinoma is associated with poor prognosis, short disease-free interval, and short survival time in both node-negative and -positive patients. Little is known about the starting point of amplification of Her-2/neu and how it progresses from benign to malignant breast lesions.

We attempted to address these questions by evaluating amplification of Her-2/neu in benign, premalignant, and malignant lesions using fluorescence in situ hybridization (FISH). Twenty-six patients with Her-2/neu–overexpressing invasive ductal carcinomas (as judged by strong immunoreactivity with Her-2/neu antibody) and coexisting lesions of ductal hyperplasia (DH), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) in the vicinity of the invasive tumor (as judged by review of the hematoxylin and eosin–stained sections), as well as metastatic carcinoma in axillary lymph nodes (mets) were selected for this study.

In the primary carcinomas, a close relationship was present between overexpression as detected by immunohistochemistry (IHC) and amplification as demonstrated by FISH (85% concordance). Among these patients, amplification of Her-2/neu in ADH was demonstrated in 7 of 13 cases with ADH, and in DCIS, in 21 of 22 cases with DCIS. There was no amplification in DH or normal ductal epithelium. Significantly, in all 12 patients with synchronous positive axillary lymph nodes, there was concordant amplification of Her-2/neu in the primary and metastatic carcinoma. Amplification was consistent in multifocal metastases, despite morphological heterogeneity in some patients. Amplification ratios increased from ADH to DCIS to invasive carcinoma (P < .01, ADH versus DCIS; P < .05, DCIS versus invasive cancer), but there was no difference in amplification ratios between primary cancers and synchronous axillary metastases (P > .05). We also evaluated Her-2/neu amplification in 21 patients without Her-2/neu overexpression in their primary carcinomas (as judged by absent immunoreactivity with Her-2/neu antibody). Three showed amplification in both primary and metastatic lesions, with a low amplification ratio (approximately 2). One patient had amplification in the primary tumor but not in an axillary metastasis. Two patients exhibited slight amplification in the metastatic carcinoma (ratios 1.6 and 2), but not in their primary cancers.

This FISH study indicates that amplification of Her-2/neu can emerge de novo in any stage of the disease process, from ADH to metastatic lesions, but most often appears first in ADH or DCIS. The degree of Her-2/neu amplification increases with progression to invasive carcinoma, there being no further increase in synchronous metastasis. Our data suggest that amplification of Her-2/neu appears to be mainly involved in initiation of breast oncogenesis and that its role in progression of breast cancers is uncertain.

The Role of HER2/neu Overexpression/Amplification in the Progression of Ductal Carcinoma In Situ to Invasive Carcinoma of the Breast

E. K. Latta, M.D., S. Tjan, M.L.T., R. K. Parkes, M.Sc. and F. P. O’Malley, M.B.

Department of Pathology and Laboratory Medicine, Mount Sinai Hospital (EKL, ST, FPO’M), Toronto, Canada; Division of Epidemiology and Biostatistics, Samuel Lunenfeld Research Institute (RKP), Toronto, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto (EKL, FPO’M), Toronto, Canada

Modern Pathology 15:1318-1325 Abstract quote

HER2/neu overexpression/amplification is seen more frequently in ductal carcinoma in situ, particularly high-grade ductal carcinoma in situ (50–60%), than in invasive ductal carcinoma of the breast (25–30%). To date, however, the role of HER2/neu in the progression of in situ to invasive disease has not been clarified.

Two hundred fifty-one breast tumors were retrieved from the pathology files at Mount Sinai Hospital. These included 91 cases of ductal carcinoma in situ, 136 cases of invasive ductal carcinomas with associated ductal carcinoma in situ, and 24 cases of pure invasive carcinomas. All cases were reviewed and stained with two monoclonal antibodies to HER2/neu (CB11 and TAB250).

Immunohistochemical staining was recorded using a semiquantitative scoring system (1). Representative cases were also investigated using fluorescence in situ hybridization. HER2/neu protein overexpression (defined as immunohistochemical staining with score of 5) was seen in 34% of cases of pure ductal carcinoma in situ, 17% of invasive carcinomas with associated ductal carcinoma in situ, and 12.5% of pure invasive carcinomas (P = .01). Sixty percent of cases of high-grade ductal carcinoma in situ showed HER2/neu protein overexpression, versus 29% of high-grade invasive carcinomas with associated ductal carcinoma in situ and 22% of high-grade pure invasive ductal carcinomas (P = .02).

The concordance between the immunohistochemical staining in the in situ and invasive components of individual tumors was 90%. Thirty-three cases were also evaluated by fluorescence in situ hybridization and showed concordance between the immunohistochemical results and the degree of gene amplification in 91% of cases, whereas 3 of 33 cases showed HER2/neu gene amplification (HER2/CEP17 = 2.3–3.7) by fluorescence in situ hybridization in the absence of positive immunohistochemical staining. One case showed HER2/neu gene amplification in the associated ductal carcinoma in situ (HER2/CEP17 ratio = 6.5), with no evidence of gene amplification in the invasive tumor (HER2/CEP17 ratio = 1.14).

Multiple genetic events are required for the development of an invasive phenotype. The findings from this study suggest that the genetic event of HER2/neu gene amplification/protein overexpression may not play a key role in the progression of ductal carcinoma in situ to invasive carcinoma and that other molecular alterations may be more important in the initiation of invasion in ductal carcinoma of the breast.

c-myc amplification is associated with HER2 amplification and closely linked with cell proliferation in tissue microarray of nonselected breast cancers.

Park K, Kwak K, Kim J, Lim S, Han S.

Hum Pathol. 2005 Jun;36(6):634-9. Abstract quote  

Summary c- myc and HER2 amplification were analyzed on 214 consecutive breast cancers by fluorescence in situ hybridization using tissue microarray technology.

The frequencies of amplification were 15.4% (33/214) and 23.3% (49/210), respectively. c- myc amplification was significantly associated with HER2 amplification ( P < .001) and closely linked with cell proliferative activity, measured by Ki67 labeling index ( P = .010). In univariate survival analysis, lymph node status, tumor size, and histological grade were significant prognostic factors, but in multivariate analysis, lymph node status was the only significant factor. Patient survival did not differ according to c- myc amplification status, and c- myc amplification showed no significant correlation with clinicopathologic features of the tumors.

A strong correlation between c- myc and HER2 amplification and proliferative activity indicates a biological link between these genes in breast cancer cell.

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

Rohit Bhargava, MD
Rizwan Naeem, MD
Sharon Marconi, BS
Jason Luszcz, MS
Jane Garb, MS
Robert Gasparini, MS |
Christopher N. Otis, MD

Hum Pathol 2002;32:1344-1350. Abstract quote

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 ( = 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.


The Equivocally Amplified HER2 FISH Result on Breast Core Biopsy: Indications for Further Sampling Do Affect Patient Management.

The Departments of Pathology, Magee-Womens Hospital, Pittsburgh, PA.


Am J Clin Pathol. 2008 Mar;129(3):383-90. Abstract quote

To our knowledge, there are no universally accepted, evidence-based guidelines for how to resolve the HER2 status of tumors demonstrating equivocal amplification.

The present study was based on 17 breast core biopsy specimens demonstrating invasive carcinoma with equivocal HER2 amplification, defined as an HER2/chromosome 17 centromere ratio of 1.8 to 2.2. Each case had a corresponding resection specimen, on which HER2 immunohistochemical and repeated fluorescence in situ hybridization analyses were performed. A definitive change in HER2 status based on the resection specimen occurred in 10 (59%) of 17 cases, with 4 patients (24%) becoming eligible for trastuzumab therapy and 6 (35%) triaged as ineligible.

These results suggest that genetic and protein expression heterogeneity exists in tumors that show low-level HER2 gene copy numbers. For the purposes of uniform clinical management, HER2 status should be evaluated on a larger tumor sample if the core biopsy specimen demonstrates an equivocal result.
These results support the recent American Society of Clinical Oncology/College of American Pathologists recommendations for further testing in cases with equivocal HER2 results.
American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer.

American Society of Clinical Oncology, Alexandria, VA, USA.

J Clin Oncol. 2007 Jan 1;25(1):118-45.

Do we need HER-2/neu testing for all patients with primary breast carcinoma?

Taucher S, Rudas M, Mader RM, Gnant M, Dubsky P, Bachleitner T, Roka S, Fitzal F, Kandioler D, Sporn E, Friedl J, Mittlbock M, Jakesz R.

Department of Surgery, Vienna University Medical School, Vienna, Austria.
Cancer. 2003 Dec 15;98(12):2547-53 Abstract quote.  

BACKGROUND: HER-2/neu is a valuable prognostic marker in primary breast carcinoma. Controversy surrounds the correlation between HER-2/neu expression and other prognostic markers, as has been discussed in preclinical and clinical studies. The objective of the current study was to investigate the probability, calculated using parameters that are assessed routinely in clinical practice, that patients with breast carcinoma had positive HER-2/neu status.

METHODS: The authors evaluated HER-2/neu status in 923 consecutive patients with breast carcinoma by immunohistochemical methods. Correlations involving HER-2/neu status, estrogen receptor (ER) and progesterone receptor (PR) status, tumor grade, patient age, lymph node involvement, and tumor size were evaluated using the Mantel-Haenszel chi-square test and the Spearman correlation. The authors created a simple scoring system (i.e., the diagnostic instrument for validation of HER-2/neu score) to define subgroups of patients with breast carcinoma and to determine the likelihood of HER-2/neu positivity.

RESULTS: HER-2/neu overexpression was correlated significantly with negative ER (P = 0.0001) and PR status (P = 0.0001), Grade 3 (G3) lesions (P = 0.0001), and young age (P = 0.006). The likelihood of HER-2/neu positivity in a patient with positive ER and PR status and G1/G2 disease was approximately 6.1%.

CONCLUSIONS: The authors demonstrated in a large patient series that HER-2/neu overexpression was associated with negative hormone receptor status, G3, and young age. In a subgroup of patients presenting with hormone-responsive and G1/G2 tumors, the likelihood of HER-2/neu overexpression was very small. Therefore, the assessment of HER-2/neu status in this subgroup of patients with breast carcinoma may be considered unnecessary, unless the role of HER-2/neu status in adjuvant treatment has been proven.

Immunohistochemical detection of HER2/neu in patients with axillary lymph node negative breast carcinoma. A study of epidemiologic risk factors, histologic features, and prognosis.

Rosen PP, Lesser ML, Arroyo CD, Cranor M, Borgen P, Norton L.

Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.

Cancer 1995 Mar 15;75(6):1320-6 Abstract quote

BACKGROUND. Numerous studies have examined the prognostic significance of HER2/neu (HER) expression in patients with axillary lymph node negative breast carcinoma. Although some investigations suggest that the presence of the altered expression of HER is prognostically unfavorable, the subject remains controversial. This study explores the interaction of HER with three aspects of axillary lymph node negative breast carcinoma: epidemiologic risk factors, tumor histopathology, and prognosis.

METHODS. Immunohistochemical staining for HER was performed on 10% formalin fixed paraffin embedded primary carcinomas from 440 patients with negative axillary lymph nodes with a median follow-up of 119 months.

RESULTS. The immunohistochemical expression, or lack thereof, of HER did not prove to be prognostically significant in this group of patients with axillary lymph node negative breast carcinoma. There was also no consistent association with epidemiologic risk factors. The most striking results concerned the relationship of HER to histopathologic features of the carcinomas. Medullary carcinoma differed from other tumor types because it was HER(+) substantially less often (10%) than were other ductal (49%) or lobular (43%) carcinomas.

CONCLUSION. The results obtained in this study suggest that the immunohistochemical demonstration of HER is not a reliable prognostic indicator for patients with axillary lymph node negative breast carcinoma. This marker was not associated with major epidemiologic risk factors; however, there was a significant correlation between HER and the phenotypic features of breast carcinoma because medullary carcinoma is rarely HER(+). Although the associations may not be a strong enough basis for refining the classification of breast carcinoma, they could be useful for diagnosing individual patients. The changes in HER that are detectable by the immunohistochemical methods used in this study probably do not occur in the earliest stages of mammary carcinogenesis.

c-erbB-2 oncoprotein, CEA, and CA 15.3 in patients with breast cancer: prognostic value.

Molina R, Jo J, Filella X, Zanon G, Pahisa J, Mu noz M, Farrus B, Latre ML, Escriche C, Estape J, Ballesta AM.

Laboratory of Clinical Biochemistry, Hospital Clinic, Medical School, Barcelona, Spain.

Breast Cancer Res Treat 1998 Sep;51(2):109-19 Abstract quote

The diagnostic value of a new tumor marker, c-erbB-2, was studied in the sera of 50 healthy subjects, 58 patients with benign breast diseases, and 413 patients with breast cancer (186 locoregional, 185 with advanced disease, and 42 with no evidence of disease).

Using 15 U/ml as the cut-off, no healthy subjects or patients with benign diseases and only 2.4% of no evidence of disease patients had elevated serum levels. Abnormal c-erbB-2 levels were found in 29% (101/370) of the patients with breast carcinoma (locoregional 9%, metastases 45.4%). CEA (cut-off 5 U/ml) and CA 15.3 (cut-off 35 U/ml) sensitivity was 18% and 16% in patients with locoregional disease and 61% and 70% in those patients with advanced disease, respectively. A trend toward higher serum levels of all three tumor markers in patients with nodal involvement or greater tumor size was found, but was statistically significant only with CEA (p < 0.01). By contrast, c-erbB-2 was related to steroid receptors, in both locoregional and metastatic tumors.

When the prognostic value of these markers was evaluated, patients with abnormally high presurgical CEA and c-erbB-2 had a worse prognosis than those patients with normal values, in both node-negative (p < 0.05 and p < 0.001, respectively) and node-positive patients (p < 0.556 and p < 0.001, respectively).

By contrast, no relationship was found between CA 15.3 values and prognosis. Multivariate analysis showed that CEA and c-erbB-2 were also prognostic factors. The correlation between serum and tissue levels of c-erbB-2 was studied in the tumors of 161 patients. Significantly higher c-erbB-2 serum levels were found in patients with overexpression in tissue by immunohistochemistry, in both locoregional and advanced disease (p = 0.0001). Serum concentrations in patients with advanced disease were related to the site of recurrence, with significantly higher values in patients with metastases (mainly in those with liver metastases) than in those with locoregional recurrence.

In summary, c-erbB-2 serum levels seem to be a useful tumor marker in the prognosis of patients with breast cancer. Using all three tumor markers, sensitivity was 35% in patients with locoregional breast cancer and 88% in patients with recurrence.

C-erbB-2, CEA and CA 15.3 serum levels in the early diagnosis of recurrence of breast cancer patients.

Molina R, Jo J, Filella X, Zanon G, Farrus B, Munoz M, Latre ML, Pahisa J, Velasco M, Fernandez P, Estape J, Ballesta AM.

Laboratory of Biochemistry (Unit for Cancer Research), Hospital Clinic, School of Medicine, Barcelona, Spain.

Anticancer Res 1999 Jul-Aug;19(4A):2551-5 Abstract quote

C-erbB-2, CEA and CA 15.3 serial serum determinations were performed in 250 patients (follow-up: 1-4 years, mean 2.5 years) with primary breast cancer and no evidence of residual disease (NED) after radical treatment (radical mastectomy or simple mastectomy and radiotherapy). Ninety-five patients developed metastases during follow-up.

RESULTS: Abnormal c-erbB-2, CEA and CA 15.3 serum levels (> 20 U/ml, > 10 ng/ml or > 60 U/ml, respectively) prior to diagnosis were found in 28.4%, 31.6% and 46.3% of the 95 patients with recurrence, with a lead time of 4.2 +/- 2.4, 5.0 +/- 2.5 and 4.6 +/- 2.7 months, respectively. One of the tumor markers was the first sign of recurrence in 69.5% of the patients. Tumor marker specificity was 100% with levels lower than the cut-point in all 155 patients without recurrence. Tumor marker sensitivity was clearly related to the site of recurrence, with the lowest sensitivity found in locoregional relapse and the highest in patients with liver or bone metastases. C-erbB-2 sensitivity in early diagnosis was significantly higher in patients with c-erbB-2 overexpression in tissue (10/12, 83.3%) than in those without overexpression (1/34, 2.9%) (p = 0.0001). Likewise, higher levels of both, c-erbB-2 and CA 15.3 at diagnosis of recurrence, higher sensitivity in early diagnosis of relapse and a higher lead time were found in PgR+ patients (CA 15.3) or in PgR- patients (C-erbB-2) (p < 0.015).

In conclusion, tumor markers are useful tools for the early diagnosis of metastases, being the first sign of recurrence in 69.5% of patients with relapse (76.3% in patients with metastases).

HER2/neu Amplification in Breast Cancer
Stratification by Tumor Type and Grade

Elise R. Hoff, MD, Raymond R. Tubbs, DO, Jonathan L. Myles, MD, and Gary W. Procop, MD

Am J Clin Pathol 2002;117:916-921 Abstract quote

The presence of HER2/neu gene amplification is prognostically and therapeutically significant for patients with breast cancer.

We sought to determine whether a relationship exists between HER2/neu gene amplification and the histologic type and grade of tumor. The histologic features and corresponding HER2/neu amplification results of 401 cases of invasive breast carcinoma were reviewed. Lobular carcinomas were less likely than ductal carcinomas to have HER2/neu amplification. Amplification was less frequent in Scarff-Bloom-Richardson grade 1 ductal carcinomas than in grades 2 and 3. Metastatic carcinomas frequently displayed HER2/neu amplification (6/20 [30%]). Our results support a correlation between HER2/neu amplification and the histologic type and grade of breast cancer.

We suggest reexamination of tumors diagnosed as Scarff-Bloom-Richardson grade 1 invasive ductal carcinomas or lobular carcinomas if the lesion displays HER2/neu amplification to assure the exclusion of a higher grade of lesion or of missed ductal components.

Clinical laboratory assays for HER-2/neu amplification and overexpression: quality assurance, standardization, and proficiency testing.

Cell Markers And Cytogenetics Committees College Of American Pathologists.

Arch Pathol Lab Med 2002 Jul;126(7):803-8 Abstract quote

OBJECTIVE: To present and contrast the results of immunohistochemistry and fluorescence in situ hybridization (FISH) proficiency testing surveys for HER-2/neu, as conducted by the Cell Markers and Cytogenetics Committees of the College of American Pathologists.

DESIGN: During the past 2 years, unstained sections from invasive breast carcinomas have been used for both immunohistochemistry and interphase FISH proficiency surveys. In most instances, the same cases were used for both the Cell Markers and Cytogenetics surveys. Additional data regarding interpretative variability for immunohistochemistry surveys have also been captured.

RESULTS: The number of laboratories performing FISH for HER-2/neu testing doubled during the 2-year period. The results of FISH testing have been remarkably concordant for laboratories submitting results. In contrast, the results of immunohistochemistry testing continue to highlight substantial variability among laboratories evaluating the same cases. The data show that this variability is at least in part due to variability in interpretation among observers, as well as inherent differences between immunohistochemistry and FISH techniques.

CONCLUSIONS: College of American Pathologists proficiency survey programs provide useful information about clinical testing for HER-2/neu amplification/overexpression.

HER2 protein overexpression in estrogen receptor-positive ductal carcinoma in situ of the breast: frequency and implications for tamoxifen therapy.

Collins LC, Schnitt SJ.

1Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA.
Mod Pathol. 2005;18:615-620 Abstract quote  

Recent clinical data have suggested that the efficacy of tamoxifen in reducing the risk of local recurrence following lumpectomy and radiation therapy in patients with ductal carcinoma in situ (DCIS) is limited to patients with estrogen receptor (ER)-positive lesions. However, it is currently not known if HER2 protein overexpression might be associated with reduced tamoxifen benefit in patients with ER-positive DCIS, as has been suggested in patients with ER-positive invasive breast cancer and in preclinical models. Moreover, the frequency of HER2 overexpression in ER-positive ductal carcinoma in situ has not been previously evaluated in detail.

To address this issue, we studied ER expression and HER2 overexpression in 148 cases of DCIS using a sensitive double immunostaining technique and assessed the frequency of ER expression and HER2 overexpression in relation to each other and in relation to DCIS grade. Overall, ER expression was seen in 114 cases (77%) and HER2 protein overexpression was seen in 42 cases (28%). Of 114 ER-positive ductal carcinoma in situ, 14 (12%) showed concurrent HER2 protein overexpression, and all 14 of these DCIS lesions were of high nuclear grade. In addition, in all 14 ER-positive DCIS cases that showed HER2 overexpression, double immunostaining demonstrated that ER and HER2 protein were coexpressed by the same neoplastic cells.

We conclude that a subset of ER-positive DCIS show concomitant overexpression of HER2 protein. Whether or not HER2 overexpression is associated with a diminished response to tamoxifen in patients with ER-positive DCIS will require investigation in clinical outcome studies.

Assessment of HER-2/neu Status in Breast Cancer Automated Cellular Imaging System (ACIS)-Assisted Quantitation of Immunohistochemical Assay Achieves High Accuracy in Comparison With Fluorescence In Situ Hybridization Assay as the Standard

Sijian Wang, MD, PhD
M. Hossein Saboorian, MD
Eugene P. Frenkel, MD
Barbara B. Haley, MD
Momin T. Siddiqui, MD
Sefik Gokaslan, MD
Frank H. Wians, Jr, PhD
Linda Hynan, PhD
Raheela Ashfaq, MD

Am J Clin Pathol 2001;116:495-503 Abstract quote

This retrospective study of formalin-fixed infiltrating breast cancer specimens compared manual immunohistochemical assay with a new image analyzer–assisted immunohistochemical quantitation method, using fluorescence in situ hybridization assay (FISH) as the standard.

Following the manual immunohistochemical assay, 189 cases, including most manual immunohistochemically positive and some random negative cases, were analyzed by FISH assay for Her-2/neu gene amplification and by the Automated Cellular Imaging System (ACIS) for immunohistochemical staining. Using the FISH standard, the ACIS immunohistochemical assay attained a higher concordance rate and sensitivity than the manual immunohistochemical assay (91.0% and 88% vs 85.7% and 71%, respectively), with only a slight decrease in specificity (93% vs 96%, respectively). In particular, the ACIS immunohistochemical assay resulted in a higher correlation with the FISH assay in the manual immunohistochemical assay 2+ cases. The ACIS immunohistochemical assay achieved higher accuracy than the manual method according to receiver operating characteristic curve analysis.

The ACIS method represents a substantial improvement over the manual method for objective evaluation of the HER-2/neu status.

The Use of Cell Line Standards to Reduce HER-2/neu Assay Variation in Multiple European Cancer Centers and the Potential of Automated Image Analysis to Provide for More Accurate Cut Points for Predicting Clinical Response to Trastuzumab

Anthony Rhodes, PhD, Duncan Borthwick, PhD , etal.
Am J Clin Pathol 2004;122:51-60 Abstract quote

Immunohistochemical analysis, the most efficient way of assaying for HER-2/neu overexpression in patients with invasive breast cancer, is subject to variation in sensitivity and evaluation when used in multiple laboratories.

Cell lines with differing but constant levels of HER-2/neu expression have been advocated as standard material against which assay sensitivity can be gauged. Automated image analysis could provide more precise linear measurements of HER-2/neu expression than the subjective and categorical scoring system originally designed for the HER-2/neu clinical trials. Multiple European laboratories (range, 92-126) stained 7 cell line standards on 6 successive occasions using a variety of immunohistochemical assays for HER-2/neu.

During the 2-year study period, a trend toward a standard sensitivity level was observed, with significant improvement in numbers of laboratories achieving appropriate results. Image analysis gave reproducible and significantly different linear values for a total of 621 HER-2/neu results on cell lines previously categorized manually as 3+, 2+, 1+, or 0.

The use of cell line standards and image analysis have the potential to assist in standardizing immunohistochemical results for predictive markers and provide more accurate and quantifiable cut points for predicting clinical response to therapy, respectively.

A Formalin-Fixed, Paraffin-Processed Cell Line Standard for Quality Control of Immunohistochemical Assay of HER-2/neu Expression in Breast Cancer

Anthony Rhodes, PhD
Bharat Jasani, PhD
Jérôme Couturier, MD
Mark J. McKinley DipRCPath
John M. Morgan, PhD
Andrew R. Dodson, FIBMS
Hossein Navabi, MSc
Keith D. Miller, FIBMS
and André J. Balaton, MD

Am J Clin Pathol 2002;117:81-89 Abstract quote

To ensure the accuracy and reproducibility of immunohistochemical assays for determining HER-2/neu status of patients with breast cancer, a reliable standard for monitoring assay sensitivity is necessary.

We optimally fixed and paraffin processed human ovarian and breast carcinoma cell lines SKOV-3, MDA-MB-453, BT-20, and MCF-7 in quantities sufficient to meet the needs of a laboratory for the foreseeable future. The material was tested, alongside HercepTest kit cell lines (DAKO, Carpinteria, CA), by 7 breast cancer centers in the United Kingdom and France with different immunohistochemical assays and markers.

The cell lines also were analyzed by fluorescence in situ hybridization (FISH) by 2 centers using HER-2/neu kits. FISH produced 100% agreement between the 2 centers: SKOV-3 and MDA-MB-453 showed HER-2/neu amplification and BT-20 and MCF-7 did not. Immunohistochemical analysis and a common evaluation method produced 100% agreement that SKOV-3 and MCF-7 showed 3+ and zero HER-2/neu overexpression, respectively. For MDA-MB-453, there was 71% (5/7) concordance of 2+ immunohistochemical staining and 86% (6/7) concordance of zero or 1+ staining for BT-20.

The cell lines provide a valuable standard for gauging HER-2/neu assay sensitivity irrespective of the antibody, antigen retrieval system, detection system, or method of evaluation used.

Reliability of chromogenic in situ hybridization for detecting HER-2 gene status in breast cancer: comparison with fluorescence in situ hybridization and assessment of interobserver reproducibility.

Gong Y, Gilcrease M, Sneige N.

1Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Mod Pathol. 2005 Aug;18(8):1015-21. Abstract quote  

Accurate determination of HER-2 status is important in the management of patients with breast cancer, especially in determining their eligibility for trastuzumab therapy. Fluorescence in situ hybridization (FISH) has been regarded as the gold standard method for detecting HER-2 gene amplification. Recently, chromogenic in situ hybridization (CISH), in which HER-2 is detected by a peroxidase reaction and the gene copies are determined by regular bright-field microscopy, has emerged as a potential alternative to FISH. However, this method requires validation before it can be adopted into clinical practice.

In this study, we evaluated 80 cases of invasive breast carcinoma by CISH, compared the results with those obtained by FISH, and assessed interobserver reproducibility among three observers. We found that agreement among the three pathologists on the CISH-determined HER-2 status was achieved in 73 cases (91%), all of which had results matching the corresponding FISH results: 54 nonamplified and 19 amplified. |

Of the 19 amplified cases, 13 were scored unanimously as high-level amplification; six had a minor scoring discrepancy (ie, low-level vs high-level amplification). A major scoring discrepancy (ie, nonamplification vs amplification) was found in the remaining seven cases, three of which were amplified and four of which were nonamplified by FISH. Two of the latter cases had a polysomy of chromosome 17. The cases that caused scoring difficulty were those with an equivocal or borderline signal number against a high background. Overall, there was nearly perfect agreement between the CISH and corresponding FISH results, and interpretation of CISH results were highly reproducible among the three pathologists.

We conclude that, in general, HER-2 status can be reliably assessed by CISH. Confirmatory FISH is recommended in cases with equivocal or borderline CISH copy numbers.
HER-2/neu and Topoisomerase IIa Gene Amplification and Protein Expression in Invasive Breast Carcinomas
Chromogenic In Situ Hybridization and Immunohistochemical Analyses

Rohit Bhargava, MD, Priti Lal, MD, and Beiyun Chen, MD, PhD
Am J Clin Pathol 2005;123:889-895 Abstract quote

We studied HER-2/neu (HER-2) and topoisomerase IIa (topo2a) amplification (using chromogenic in situ hybridization) and overexpression (immunohistochemical analysis) in 113 invasive breast carcinomas. A gene copy number/chromosome 17 copy number ratio of 2.0 or higher indicated amplification. A topo2a/chromosome 17 ratio of less than 0.8 indicated gene deletion. HER-2 overexpression was scored according to standard HercepTest guidelines (DAKO, Carpinteria, CA).

Overexpression of topo2a was identified when nuclear staining was found in more than 5% of tumor cells. Of 113 tumors, 104 were analyzed successfully for HER-2 and topo2a amplification. Of the 104, 64 showed HER-2 amplification; 25 of these (39%) also showed topo2a amplification. No amplification was found in 40 tumors. Deletion of topo2a was seen in 7 (11%) of 64 HER-2– amplified tumors and 2 (5%) of 40 nonamplified tumors. Of 25 tumors with topo2a amplification, 18 (72%) overexpressed topo2a. Only 3 (4%) of 79 tumors without topo2a amplification overexpressed topo2a. Amplification of topo2a is associated with HER-2 amplification but not vice versa.

Amplification of topo2a resulted in protein overexpression in 72% of tumors, but topo2a overexpression rarely occurred without gene amplification. Identification of topo2a and HER-2 status might have therapeutic and prognostic implications.
Chromogenic In Situ Hybridization for the Detection of HER-2/neu Gene Amplification in Breast Cancer With an Emphasis on Tumors With Borderline and Low-Level Amplification
Does It Measure Up to Fluorescence In Situ Hybridization?

Rohit Bhargava, MD, Priti Lal, MD, and Beiyun Chen, MD, PhD
Am J Clin Pathol 2005;123:237-243 Abstract quote

We compared chromogenic in situ hybridization (CISH) with fluorescence in situ hybridization (FISH) for assessing HER-2/neu gene amplification using tissue microarrays (TMAs) made from formalin-fixed, paraffin-embedded tissue blocks from 113 cases of invasive breast carcinoma. TMAs were created using 0.6-mm tissue cores with 4 sampled cores per tumor. For both assays, a HER-2/chromosome 17 signal ratio of 2.0 or more was considered positive for gene amplification.

The average ratio of cores from the same tumor was used for determination of gene amplification status of that particular tumor. Of 113 cases, 102 were tested successfully by both assays. The results were concordant in 100.0% of cases (63 amplified; 39 nonamplified). All 22 cases of borderline (ratio, 2.0-2.5) or low-level (ratio, 2.6-3.9) amplification by FISH also showed HER-2 gene amplification by CISH. CISH is as sensitive as FISH in detecting borderline and low-level HER-2 amplification. Reliable recognition of the invasive carcinoma area by light microscopy and preservation of the test slides are added advantages of CISH.

CISH performs as well as FISH in the analysis of HER-2 gene amplification in breast cancer and might have advantages in certain situations.
Assessment of the HER2 status in breast cancer by fluorescence in situ hybridization: A technical review with interpretive guidelines.

Hicks DG, Tubbs RR.

Hum Pathol. 2005 Mar;36(3):250-61. Abstract quote  

Summary Diagnostic assays for HER2 in breast cancer provide important prognostic information and independently help guide management by identifying patients who are the most likely to benefit from Herceptin-targeted therapy. The biological events underlying HER2 -driven breast cancer that can be assessed in routine clinical specimens include the evaluation of gene amplification by fluorescence in situ hybridization (FISH), enhanced messenger RNA expression by real-time polymerase chain reaction, and the assessment of protein overexpression at the tumor cell membrane by immunohistochemistry (IHC).

Immunohistochemistry and FISH methodologies have the advantage of being morphologically driven, allowing for correlations between HER2 expression and morphologic features. However, each has important advantages and disadvantages, which are discussed in detail. Although immunohistochemistry is familiar and readily accommodated in most surgical pathology laboratories, increasing demands for FISH testing in the clinical setting will require greater familiarity with the technical aspects of FISH assays and their interpretation by the greater laboratory community. In this review, we provide an overview of FISH testing for HER2 in breast cancer, with an emphasis on technical considerations, interpretive guidelines, scoring criteria, and quality control.

The development of automated platforms for hybridization, image analysis for signal enumeration, and experience with FISH interpretation should broaden the availability of this technology for clinical diagnostic testing.
Detection and Quantitation of HER-2 Gene Amplification and Protein Expression in Breast Carcinoma

Anna M. Bofin, MD, etal.
Am J Clin Pathol 2004;122:110-119 Abstract quote

We compared fluorescence in situ hybridization (FISH), immunohistochemical analysis, immunocytochemical analysis, and relative quantification assays using polymerase chain reaction (PCR) as methods for estimating HER-2 gene amplification and protein overexpression in fine-needle aspirate (FNA) specimens and paraffin-embedded tissue samples from 49 cases of breast cancer.

FISH can be performed successfully on FNA smears. Immunohistochemical and immunocytochemical staining intensity of 3+ corresponds to a FISH ratio of more than 2.5. Immunohistochemical and immunocytochemical staining of 2+ and 1+ are not necessarily associated with gene amplification. Increased DNA PCR ratios might be seen without amplification, reflecting polysomy. HER-2 messenger RNA relative quantitation scores correlate well with HER-2 gene amplification.
Owing to the ease with which it can be performed and interpreted, we conclude that FISH is the test of choice for HER-2 estimation and, when possible, should be performed on whole nuclei, which are readily available in FNA smears or imprint cytology.

FISH may be used primarily or to confirm immunohistochemical, immunocytochemical, and PCR results.

Strong HER-2/neu protein overexpression by immunohistochemistry often does not predict oncogene amplification by fluorescence in situ hybridization.

Hammock L, Lewis M, Phillips C, Cohen C.
Hum Pathol. 2003 Oct;34(10):1043-7 Abstract quote

Breast cancer patients with HER-2/neu oncogene amplification by fluorescence in situ hybridization (FISH) have been shown to have a better response to trastuzumab (Herceptin) therapy than those showing HER-2/neu protein overexpression only. Many centers currently perform FISH only on tumors showing 2+ HER-2/neu positivity by immunohistochemistry (IHC), with the assumption that 3+ positivity virtually equates with amplification. Results of FISH performed on 102 breast cancer cases over a 12-month period were correlated with HER-2/neu IHC results.

FISH was performed using a ratio of HER-2/neu and chromosome 17 centromere signal counts (PathVysion; Vysis, Downers Grove, IL). Immunohistochemical expression of HER-2/neu was evaluated according to the published scoring guidelines of the HercepTest (Dako, Carpinteria, CA).

Only 22 of 45 tumors with 3+ positivity (49%) showed amplification by FISH. Only 2 of 25 cases with 2+ staining by IHC (6%) showed gene amplification, and 1 of 25 cases with negative IHC staining (4%) showed weak amplification. Of the 25 cases showing oncogene amplification, 22 (88%) showed 3+ IHC positivity, 2 (8%) showed 2+ positivity, and 1 (4%) was negative by IHC. More than 50% of breast tumors showing strong 3+ HER-2/neu staining do not show oncogene amplification by FISH. Most tumors with 2+ and negative IHC also fail to amplify.

In our experience, FISH studies should be performed on all 3+ and 2+ staining tumors to avoid inappropriate and toxic treatment. The decision to perform FISH on IHC-negative tumors should be guided by additional parameters, including tumor grade and estrogen receptor status.

Her-2/neu status in breast cancer metastases to the central nervous system.

Lear-Kaul KC, Yoon HR, Kleinschmidt-DeMasters BK, McGavran L, Singh M.

Department of Pathology, University of Colorado Health Sciences Center, Denver 80262, USA.
Arch Pathol Lab Med. 2003 Nov;127(11):1451-7. Abstract quote  

CONTEXT: Breast cancer is lethal when it metastasizes; one frequent site for spread is the central nervous system (CNS). Approximately 15% to 30% of breast cancers overexpress the protein HER-2/neu at the primary site, but there are few data on whether metastases from these tumors overexpress HER-2/neu and might be responsive to the potentially toxic anti-HER-2/neu immunotherapy (trastuzumab [Herceptin]) used in patients with disseminated disease.

OBJECTIVE: To assess CNS breast cancer metastases for HER-2/neu protein overexpression by immunohistochemistry and gene amplification by fluorescence in situ hybridization (FISH) and to compare the status in primary and metastatic sites in the same patient, whenever possible.

DESIGN: Central nervous system breast cancer metastases (n = 53) from 33 patients and corresponding primary breast cancer specimens in a subset of these patients (n = 12) were retrospectively identified in surgical pathology and autopsy databases. Fluorescence in situ hybridization analysis using PathVysion probes for HER-2/neu and chromosome enumeration probe 17 (CEP 17) and immunohistochemistry using the c-Erb-B2 antibody (Dako A0485) were compared. Immunohistochemical sections were evaluated by both visual and image analysis techniques.

RESULTS: Of 31 cases assessable by FISH, 26% showed gene amplification. One hundred percent concordance for HER-2/neu status was detected between the primary and CNS metastatic lesions in 10 of 10 patients analyzed by FISH; lesser concordance was noted in 12 cases compared by immunohistochemistry. In 9 patients with multiple CNS metastases, FISH showed concordance among different lesions within the same patient.

CONCLUSIONS: When FISH is the detection method, CNS metastases accurately reflect the HER-2/neu status of the primary tumor. Central nervous system metastases from breast cancer received as surgical specimens can therefore be used to assess HER-2/neu status in patients in whom the primary tumor is unavailable for analysis.


Interobserver interpretative reproducibility of GOLDFISH, a first generation gold-facilitated autometallographic bright field in situ hybridization assay for HER-2/neu amplification in invasive mammary carcinoma.

Tubbs R, Skacel M, Pettay J, Powell R, Myles J, Hicks D, Sreenan J, Roche P, Stoler MH, Hainfeld J.

Department Clinical Pathology, Cleveland Clinic Foundation, Ohio 44195-5131, USA

Am J Surg Pathol 2002 Jul;26(7):908-13 Abstract quote

Clinical laboratory testing for HER-2/neu gene amplification by fluorescence in situ hybridization is not widely used in diagnostic pathology laboratories. A bright field alternative permitting direct visualization of gene amplification using conventional microscopy may be more readily incorporated into routine diagnostic pathology practice. Interobserver reproducibility represents an important component of the validation of such an assay.

We tested the hypothesis that a first-generation bright field alternative to fluorescence in situ hybridization, a Nanogold (Nanoprobes, Inc, Yaphank, NY, USA) (or gold-label)/autometallographic assay for HER-2/neu gene amplification in breast carcinoma, can be reproducibly interpreted by pathologists. Reference standard was direct fluorescence in situ hybridization supplemented by RNA/RNA in situ hybridization. Reproducibility of selected conventional histologic parameters was captured based on a hematoxylin and eosin slide accompanying the GOLDFISH preparation (gold-facilitated autometallographic in situ hybridization) as an indication of comparative reproducibility. The average kappa among GOLDFISH observers was 0.84, which was at least or concordant of observers scoring nuclear grade (kappa = 0.50) and the presence of in situ carcinoma (kappa = 0.57) by conventional histopathology.

The GOLDFISH assay was specifically designed for qualitative interpretation, thus obviating the need for oil immersion microscopy and signal enumeration, and its interpretation was highly reproducible among five pathologists.

Correlation of HER-2 Status With Estrogen and Progesterone Receptors and Histologic Features in 3,655 Invasive Breast Carcinomas

Priti Lal, MD, Lee K. Tan, MD, and Beiyun Chen, MD, PhD
Am J Clin Pathol 2005;123:541-546 Abstract quote

We studied the inverse relationship between HER-2 and estrogen (ER) and progesterone (PR) receptors using HER-2 testing and correlated HER-2 status with histologic features in 3,655 unselected invasive breast carcinomas. Immunohistochemical analysis for ER, PR, and HER-2 and fluorescence in situ hybridization for HER-2 were performed. ER and PR expression were decreased significantly in HER-2+ tumors compared with HER-2– tumors (ER, 49.1% vs 78.17%; PR, 24.3% vs 53.13%). Even among HER-2+ tumors, the rate of ER or PR expression in high-grade tumors was significantly decreased compared with intermediate-grade tumors.

HER-2 was positive in 10.87% of grade 2 and 27.84% of grade 3 ductal carcinomas and negative in all grade 1 ductal carcinomas. HER-2 overexpression or amplification essentially was limited to grades 2 and 3 ductal carcinomas and correlated inversely with ER or PR expression.

Although ER or PR expression is decreased in HER-2+ tumors, a substantial proportion of them still express ER or PR.

Defining a Test for HER-2/neu Evaluation in Breast Cancer in the Diagnostic Setting

Wedad M. Hanna, M.D., Harriette J. Kahn, M.D., Margaret Pienkowska, Ph.D., John Blondal, M.D., Arun Seth, Ph.D and Alexander Marks, Ph.D

Sunnybrook and Women’s College Health Sciences CentreDepartments of Pathology (WMH, HJK, MP, AS), Medicine (JB), and Banting and Best Departments of Medical Research (AM), University of Toronto, Toronto, Ontario, Canada

Mod Pathol 2001;14:677-685 Abstract quote

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 [>=] 10% moderate or strong complete membranous staining. The gene was considered amplified if the density score of the product was [>=] 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.



Discrepancies in clinical laboratory testing of eligibility for trastuzumab therapy: apparent immunohistochemical false-positives do not get the message.

Tubbs RR, Pettay JD, Roche PC, Stoler MH, Jenkins RB, Grogan TM.

Department of Clinical Pathology, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.

J Clin Oncol 2001 May 15;19(10):2714-21 Abstract quote

BACKGROUND: Several studies have reported what seem to be false-positive results using the Food and Drug Administration (FDA)-approved HercepTest (Dako Corp, Carpinteria, CA) to profile Her-2/neu amplification and overproduction in breast carcinoma. False-positive status has been based on comparisons with gene copy enumeration by fluorescence in situ hybridization (FISH) and with comparisons to immunohistochemistry (IMH) results using a monoclonal antibody. However, simple overexpression by tumor cells that have normal gene copy has not been evaluated by profiling mRNA expression, ie, such cases could simply represent true-positive, transcriptionally upregulated overexpression.

MATERIALS AND METHODS: Four hundred infiltrating ductal carcinomas of breast were evaluated by IMH using monoclonal (CB11; Ventana Medical Systems, Inc, Tucson, AZ) and polyclonal (HercepTest; Dako) antibodies after antigen retrieval (AR). A polyclonal antibody sans AR (PCA/SAR) was also used. All IMH stains were evaluated and scored according to the guidelines for the FDA-approved HercepTest. A total of 145 of 400 carcinomas were subsequently evaluated by direct and digoxigenin-labeled (Dig) FISH, and 144 of 400 were evaluated by detection of mRNA overexpression via autoradiographic RNA:RNA in situ hybridization.

RESULTS: Overall HercepTest/CB11 IMH discordance was 12%. Expression of mRNA was highly concordant with FISH and DigFISH amplification and with CB11 and PCA/SAR immunohistology. IMH false-positive cases (no Her-2/neu gene amplification) occurred with both HercepTest (23%) and CB11 (17%), and the majority of false-positive results (34 of 44) were scored as 2+. All 2+ false-positive cases were mRNA-negative. Combined results of HercepTest and CB11 showed that 79% (38 of 48) of 3+ cases were Her-2/neu gene amplified, but only 17% (seven of 41) of 2+ cases had increased gene copy.

CONCLUSION: Discordant HercepTest/FISH results, and to a lesser extent discordance with CB11 IMH, are most commonly false-positive results with a score of 2+. The 2+ score as defined in the guidelines for the FDA-approved HercepTest should not be used as a criterion for trastuzumab therapy unless confirmed by FISH. Determination of Her-2 gene copy number by FISH may be a more accurate and reliable method for selecting patients eligible for trastuzumab therapy.

Laboratory testing for her2/neu in breast carcinoma: an evolving strategy to predict response to targeted therapy.

Diaz NM.

Interdisciplinary Oncology Program, H. Lee Moffitt Cancer Center & Research Institute at the University of South Florida, Tampa 33612, USA.

Cancer Control 2001 Sep-Oct;8(5):415-8 Abstract quote

BACKGROUND: Laboratory testing of HER2/neu in breast carcinoma has become vital to patient care following the approval of trastuzumab as the first therapy to target the HER2/neu oncoprotein. Initial clinical trials used immunohistochemistry (IHC) to test for HER2/neu overexpression in order to select patients for therapy. Fluorescence in situ hybridization (FISH), which tests for gene amplification, is more specific and sensitive than IHC when either assay is compared with HER2/neu overexpression as determined by Northern or Western blot analysis. Many weak overexpressors on IHC testing are not gene amplified on FISH analysis. Such weak overexpressors may be considered false-positives and raise the question of how best to test for HER2/neu.

METHODS: The literature was surveyed regarding testing for HER2/neu overexpression in breast carcinomas and alternative testing strategies.

RESULTS: False-positive results are a significant problem when IHC is exclusively used to test for HER2/neu overexpression. The false-positives are overwhelmingly confined to the group of 2-plus positives and do not respond to targeted therapy. In contrast, concordance between IHC and FISH is high when immunostaining is interpreted as either negative or strongly positive (3-plus). Whereas some recent studies have suggested that FISH may better predict response to anti-HER2/neu therapy than IHC, others have indicated that IHC is as effective a predictor as FISH. IHC is less technically demanding and costly than FISH.

CONCLUSIONS: IHC analysis of HER2/neu in breast carcinoma is a useful predictor of response to therapy with trastuzumab when strongly positive. Negative immunostaining is highly concordant with a lack of gene amplification by FISH. Most weakly positive overexpressors are false-positives on testing with FISH. Thus, screening of breast carcinomas with IHC and confirmation of weakly positive IHC results by FISH is an effective evolving strategy for testing HER2/neu as a predictor of response to targeted therapy.

Macpherson and Pincus. Clinical Diagnosis and Management by Laboratory Methods. Twentyfirst Edition. WB Saunders. 2006.
Rosai J. Ackerman's Surgical Pathology. Ninth Edition. Mosby 2004.
Sternberg S. Diagnostic Surgical Pathology. Fourth Edition. Lipincott Williams and Wilkins 2004.
Robbins Pathologic Basis of Disease. Seventh Edition. WB Saunders 2005.
DeMay RM. The Art and Science of Cytopathology. Volume 1 and 2. ASCP Press. 1996.
Weedon D. Weedon's Skin Pathology Second Edition. Churchill Livingstone. 2002
Fitzpatrick's Dermatology in General Medicine. 6th Edition. McGraw-Hill. 2003.
Weiss SW and Goldblum JR. Enzinger and Weiss's Soft Tissue Tumors. Fifth Edition. Mosby Elesevier 2008

Commonly Used Terms

Breast Cancer

Tumor Markers

Basic Principles of Disease
Learn the basic disease classifications of cancers, infections, and inflammation

Commonly Used Terms
This is a glossary of terms often found in a pathology report.

Diagnostic Process
Learn how a pathologist makes a diagnosis using a microscope

Surgical Pathology Report
Examine an actual biopsy report to understand what each section means

Special Stains
Understand the tools the pathologist utilizes to aid in the diagnosis

How Accurate is My Report?
Pathologists actively oversee every area of the laboratory to ensure your report is accurate

Got Path?
Recent teaching cases and lectures presented in conferences

Internet Links

Last Updated March 4, 2008

Send mail to The Doctor's Doctor with questions or comments about this web site.
Read the Medical Disclaimer.

Copyright © The Doctor's Doctor