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Objectives: We investigated the impact of our laboratory's reflex testing process for resolving ERBB2 (HER2) status on breast cancer samples that require additional workup after fluorescence in situ hybridization (FISH), per guideline recommendations published in 2018 by the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP)., Methods: In total, 500 breast cancer specimens with ERBB2 FISH results in groups 2 through 4 (all reported as immunohistochemistry [IHC] equivocal [2+] at external laboratories) were resubmitted for IHC testing in our laboratory. Per the ASCO/CAP guideline, FISH was rescored when internal IHC was also equivocal (2+), targeted to tumor areas demonstrating more intense IHC staining, if observed., Results: Reflex IHC/FISH testing changed the final reported ERBB2 status in 185 of 500 (37.0%) samples. Result changes included discordant IHC (n = 4 score 0, n = 132 score 1+, and n = 16 score 3+) and discordant FISH (n = 33). Numerical differences in FISH scores were comparable for targeted vs nontargeted FISH rescoring (P = .086 for ERBB2 copy number; P = .49 for ERBB2 ratio). Two cases showed larger differences in FISH scores, suggesting heterogeneity., Conclusions: Retesting of breast cancer samples with equivocal IHC frequently changes IHC results, but targeted reanalysis of borderline FISH results rarely identifies significant differences in ERBB2 copy number or ratio., (© American Society for Clinical Pathology, 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context.—: Guidelines for HER2 testing in breast cancer have changed over time, from the US Food and Drug Administration guideline to the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines published in 2007, 2013, and 2018., Objective.—: To investigate the change in assignment of HER2 status in breast cancers with equivocal (2+) immunohistochemistry (IHC) results by fluorescence in situ hybridization (FISH) following implementation of the ASCO/CAP 2018 guideline., Design.—: The study included 3556 invasive breast cancers that were HER2 equivocal (2+) by IHC and were submitted to our FISH laboratory after July 2018. Reflex testing (with repeat IHC staining) was performed on certain categories of FISH results known as groups 2, 3, and 4. Concomitant review of IHC and FISH was performed on these reflex cases per 2018 guideline recommendations. The FISH data were analyzed to compare US Food and Drug Administration and ASCO/CAP 2007, 2013, and 2018 interpretations., Results.—: Of 3548 invasive breast cancers with complete data available, the percentage agreement for FISH according to different guidelines was highest for ASCO/CAP 2018 versus US Food and Drug Administration (96.5%), followed by ASCO/CAP 2018 versus 2007 (93.8%), and lowest with ASCO/CAP 2018 versus 2013 (83.7%). Per the 2018 guideline, reflex IHC testing was performed on 633 breast cancers (17.8%); the majority of reflex testing results were negative (541 of 633; 85.5%). The overall distribution of HER2 FISH results (per the 2018 guideline) was 88.5% negative and 11.5% positive., Conclusions.—: By eliminating the equivocal FISH category, the 2018 ASCO/CAP guideline significantly reduced the HER2 FISH-positive rate in tumors with equivocal (2+) IHC results.

Background: The addition of lapatinib (L) to trastuzumab (T) was previously found to be synergistic in preclinical models and in the neoadjuvant setting. Prior to the results of the ALTTO trial, this study assessed the safety and feasibility of adding L to the standard adjuvant docetaxel, carboplatin, and trastuzumab (TCH) regimen in early-stage HER2-positive breast cancer (HER2+ BC)., Methods: In this single-arm, 2-stage, phase II study, patients with stages I-III HER2+ BC received TCH plus L at 1000 mg daily for a total of 12 months. The primary endpoint was the safety and tolerability, including the rate of diarrhea. Secondary endpoints included adverse event (AE) profile using the NCI CTCAE v3.0 and cardiac safety., Results: Thirty eligible patients were enrolled. Median follow-up is 5.3 years. Diarrhea was the most common AE with 50% Grade (G)1/2 and 43% G3 diarrhea. However, it was responsive to dose reduction of L (750 mg) and institution of anti-diarrheal medications. Cardiovascular AE were infrequent and no patients experienced congestive heart failure while on treatment., Conclusion: TCHL was a tolerable regimen at a starting L dose of 750 mg PO daily when given concurrently with chemotherapy.

Objectives: To develop and test an integrated approach to human epidermal growth factor receptor 2 (HER2) copy number analysis in breast cancer using in situ hybridization (ISH) and cytogenomic microarray (CMA)., Methods: CMA was performed on four clinical breast cancer samples with nonclassical patterns of HER2 ISH results. Integrated analysis was performed by correlating the data from pathology review, ISH, and CMA., Results: Integrated analysis provided a more comprehensive view of the genomic copy number landscape that informed HER2 copy number analysis, but ISH provided essential data in all cases., Conclusions: CMA can be helpful for clarifying HER2 amplification status in breast cancer. However, uncertainties over tumor percentage, clonal heterogeneity, and varying ploidy levels present challenges for genomic methods such as CMA. Accurate interpretation of HER2 copy number by CMA requires correlation with the pathology and ISH data., (© American Society for Clinical Pathology, 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Purpose: To develop a genomic signature that predicts benefit from trastuzumab in human epidermal growth factor receptor 2-positive breast cancer., Patients and Methods: DASL technology was used to quantify mRNA in samples from 1,282 patients enrolled onto the Combination Chemotherapy With or Without Trastuzumab in Treating Women With Breast Cancer (North Central Cancer Treatment Group N9831 [NCCTG-N9831]) adjuvant trastuzumab trial. Cox proportional hazard ratios (HRs), adjusted for significant clinicopathologic risk factors, were used to determine the association of each gene with relapse-free survival (RFS) for 433 patients who received chemotherapy alone (arm A) and 849 patients who received chemotherapy plus trastuzumab (arms B and C). Network and pathway analyses were used to identify key biologic processes linked to RFS. The signature was built by using a voting scheme., Results: Network and functional ontology analyses suggested that increased RFS was linked to a subset of immune function genes. A voting scheme model was used to define immune gene enrichment based on the expression of any nine or more of 14 immune function genes at or above the 0.40 quantile for the population. This model was used to identify immune gene-enriched tumors in arm A and arms B and C. Immune gene enrichment was linked to increased RFS in arms B and C (HR, 0.35; 95% CI, 0.22 to 0.55; P < .001), whereas arm B and C patients who did not exhibit immune gene enrichment did not benefit from trastuzumab (HR, 0.89; 95% CI, 0.62 to 1.28; P = .53). Enriched immune function gene expression as defined by our predictive signature was not associated with increased RFS in arm A (HR, 0.90; 95% CI, 0.60 to 1.37; P = .64)., Conclusion: Increased expression of a subset of immune function genes may provide a means of predicting benefit from adjuvant trastuzumab., (© 2015 by American Society of Clinical Oncology.)

Choice of therapy for breast cancer relies on human epidermal growth factor receptor-2 (HER2) and estrogen receptor α (ER) status. Before randomization in the phase III Adjuvant Lapatinib and/or Trastuzumab Treatment Optimisation (ALTTO) trial for HER2-positive disease, HER2 and ER were centrally reviewed by Mayo Clinic (Rochester, MN, and Scottsdale, AZ) for North America and by the European Institute of Oncology (IEO; Milan, Italy) for the rest of world (except China). Discordance rates (local vs. central review) differed between Mayo and IEO. Among locally HER2-positive cases, 5.8 % (Mayo) and 14.5 % (IEO) were centrally HER2 negative. Among locally ER-positive cases, 16.2 % (Mayo) and 4.2 % (IEO) were centrally ER-negative. Among locally ER-negative cases, 3.4 % (Mayo) and 21.4 % (IEO) were centrally ER-positive. We, therefore, performed a ring study to identify features contributing to these differing discordance rates. Mayo and IEO exchanged slides for 25 HER2 and 35 ER locally/centrally discordant cases. Both laboratories performed IHC and FISH for HER2 using the HercepTest(®) and PathVysion HER2 DNA probe kit/HER2/centromere 17 probe mixture. IHC for ER was tested centrally using the monoclonal ER 1D5 antibody (Mayo) or the DAKO cocktail of ER 1D5 and 2.123 antibodies (IEO). Mayo and IEO confirmed the central HER2-negative result in 100 % of 25 cases. Mayo and IEO confirmed the central ER result in 29 (85 %) of 34 evaluable cases. The five Mayo-negative/IEO-positive cases were ER-positive when retested at Mayo using the DAKO ER cocktail. In this ring study, ALTTO ineligibility did not change when HER2 testing was performed by either IEO or Mayo central laboratories. However, a dual antibody ER assay had fewer false-negative test results than an assay with a single antibody, and there was more discordance between the two ER reagents than has been previously reported. Using even slightly different assay methods yielded different results, even between experienced central laboratories.

Purpose: This study investigated the association between tumor MYC protein expression and disease-free survival (DFS) of patients randomized to receive chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C) in the N9831 (Alliance) adjuvant HER2(+) trastuzumab breast cancer trial., Experimental Design: This analysis included 1,736 patients randomized to Arms A, B, and C on N9831. Nuclear MYC protein expression was determined in tissue microarray sections containing three biopsies per patient or whole tissue sections using standard immunohistochemistry (clone 9E10). A tumor was considered positive for MYC protein overexpression (MYC(+)) if the nuclear 3+ staining percentage was more than 30%., Results: Five hundred and seventy-four (33%) tumors were MYC(+). MYC(+) was associated with hormone receptor positivity (χ(2), P = 0.006), tumors 2 cm or more (χ(2), P = 0.02), and a higher rate of nodal positivity (χ(2), P < 0.001). HRs for DFS (median follow-up: 6.1 years) for Arm C versus A were 0.52 (P = 0.006) and 0.65 (P = 0.006) for patients with MYC(+) and MYC(-) tumors, respectively (P(interaction) = 0.40). For Arm B versus A, HRs for patients with MYC(+) and MYC(-) tumors were 0.79 (P = 0.21) and 0.74 (P = 0.04), respectively (P(interaction) = 0.71). For Arm C versus B, HRs for patients with MYC(+) and MYC(-) tumors were 0.56 (P = 0.02) and 0.89 (P = 0.49), respectively (P(interaction) = 0.17)., Conclusions: Our data do not support an impact of tumor MYC protein expression on differential benefit from adjuvant trastuzumab., (©2013 AACR.)

Purpose: It has been suggested that PTEN, a negative regulator of PI3K/AKT signaling, is involved in tumor sensitivity to trastuzumab. We investigated the association between tumor PTEN protein expression and disease-free survival (DFS) of patients randomly assigned to receive chemotherapy alone (arm A) or chemotherapy with sequential (arm B) or concurrent trastuzumab (arm C) in the phase III early-stage human epidermal growth factor receptor 2 (HER2) -positive trial-North Central Cancer Treatment Group (NCCTG) N9831., Patients and Methods: The intensity and percentage of invasive cells with cytoplasmic PTEN staining were determined in tissue microarray sections containing three cores per block (n = 1,286) or in whole tissue sections (WS; n = 516) by using standard immunohistochemistry (138G6 monoclonal antibody). Tumors were considered positive for PTEN (PTEN-positive) if any core or WS had any invasive cells with ≥ 1+ staining. Median follow-up was 6.0 years., Results: Of 1,802 patients included in this analysis (of 3,505 patients registered to N9831), 1,342 (74%) had PTEN-positive tumors. PTEN positivity was associated with hormone receptor negativity (χ(2) P < .001) and nodal positivity (χ(2) P = .04). PTEN did not have an impact on DFS within the various arms. Comparing DFS of arm C to arm A, patients with PTEN-positive and PTEN-negative tumors had hazard ratios (HRs) of 0.65 (P = .003) and 0.47 (P = .005), respectively (interaction P = .16). For arm B versus arm A, patients with PTEN-positive and PTEN-negative tumors had HRs of 0.70 (P = .009) and 0.85 (P = .44), respectively (interaction P = .47)., Conclusion: In contrast to selected preclinical and limited clinical studies suggesting a decrease in trastuzumab sensitivity in patients with PTEN-negative tumors, our data show benefit of adjuvant trastuzumab for patients with HER2-positive breast cancer, independent of tumor PTEN status.

A comprehensive, blinded, pathology evaluation of HER2 testing in HER2-positive/negative breast cancers was performed among three central laboratories. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) analyses were performed on 389 tumor blocks from three large adjuvant trials: N9831, BCIRG-006, and BCIRG-005. In 123 cases, multiple blocks were examined. HER2 status was defined according to FDA-approved guidelines and was independently re-assessed at each site. Discordant cases were adjudicated at an on-site, face-to-face meeting. Results across three independent pathologists were concordant by IHC in 351/381 (92 %) and FISH in 343/373 (92 %) blocks. Upon adjudication, consensus was reached on 16/30 and 18/30 of discordant IHC and FISH cases, respectively, resulting in overall concordance rates of 96 and 97 %. Among 155 HER2-negative blocks, HER2 status was confirmed in 153 (99 %). In the subset of 102 HER2-positive patients from N9831/BCIRG-006, primary blocks from discordant cases were selected, especially those with discordant test between local and central laboratories. HER2 status was confirmed in 73 (72 %) of these cases. Among 118 and 113 cases with IHC and FISH results and >1 block evaluable, block-to-block variability/heterogeneity in HER2 results was seen in 10 and 5 %, respectively. IHC-/FISH- was confirmed for 57/59 (97 %) primary blocks from N9831 (locally positive, but centrally negative); however, 5/22 (23 %) secondary blocks showed HER2 positivity. Among 53 N9831 patients with HER2-normal disease adjudicated as IHC-/FISH-(although locally positive), there was a non-statistically significant improvement in disease-free survival with concurrent trastuzumab compared to chemotherapy alone (adjusted hazard ratio 0.34; 95 % CI, 0.11-1.05; p = 0.06). There were similar agreements for IHC and FISH among pathologists (92 % each). Agreement was improved at adjudication (96 %). HER2 tumor heterogeneity appears to partially explain discordant results in cases initially tested as positive and subsequently called negative.

Image analysis of the HER2 immunohistochemical (IHC) stain can help determine which breast cancer patients may benefit from HER2-targeted therapy. We studied the concordance of HER2 IHC and fluorescence in situ hybridization (FISH) as well as reproducibility of surgical pathologist (SP) and cytotechnologist (CT) interpretations using manual and image analysis methodologies on 154 IHC cases. Concordances with FISH were good for IHC negative (0, 1+) cases (range, 97%-100%) and positive (3+) cases (range, 87%-100%). Image analysis had fewer equivocal (2+) results (10.4%) than CT (14.9%) and SP (16.2%) manual methods, with higher concordances to FISH (31%, 26%, and 20% for image analysis, CT manual, and SP manual, respectively). CT manual (κ = 0.747) and image analysis (κ = 0.779) methods had better interobserver reproducibility than SP manual (κ = 0.697). CT image analysis had better intraobserver reproducibility (κ = 0.882) than CT (κ = 0.828) and SP (κ = 0.766) manual methods. HER2 IHC analysis performed by image analysis can produce accurate results with improved reproducibility.

The 2007 American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP) joint guidelines defined criteria for HER2 positivity of tumors that modified those of the US Food and Drug Administration (FDA), causing some confusion and uncertainty among clinicians. Using data from the HER2-positive breast cancer adjuvant trial N9831, we compared eligibility for patients who met both criteria, and disease-free survival (DFS) was assessed by Cox proportional hazards regression. The number of patients in the N9831 trial retrospectively eligible for trastuzumab therapy was decreased when ASCO/CAP criteria vs FDA criteria were applied to immunohistochemistry and/or fluorescence in situ hybridization results (107 [3.7%] of 2904 patients with immunohistochemistry results, 37 [1.3%] of 2809 patients with fluorescence in situ hybridization results, and 47 [1.7%] of 2809 patients with both results). Improvement in DFS was similar among patients treated with trastuzumab under either set of criteria (concurrent trastuzumab and chemotherapy compared with chemotherapy alone: by ASCO/CAP criteria, hazard ratio of DFS = 0.59, 95% confidence interval = 0.48 to 0.73; by FDA criteria but not ASCO/CAP criteria, hazard ratio = 0.60, 95% confidence interval = 0.12 to 3.13; number needed to treat to prevent one additional DFS event at 5 years: 10 and 11.2 patients, respectively). Following the 2007 ASCO/CAP criteria for HER2 positivity would negate the option of potentially life-saving trastuzumab therapy for a small but meaningful group of patients. We recommend using FDA-approved HER2 criteria for therapeutic decision making.

The accuracy and reliability of immunohistochemical analysis and in situ hybridization for the assessment of HER2 status remains a subject of debate. We developed a novel assay (HERmark Breast Cancer Assay, Monogram Biosciences, South San Francisco, CA) that provides precise quantification of total HER2 protein expression (H2T) and HER2 homodimers (H2D) in formalin-fixed, paraffin-embedded tissue specimens. H2T and H2D results of 237 breast cancers were compared with those of immunohistochemical studies and fluorescence in situ hybridization (FISH) centrally performed at the Mayo Clinic, Rochester, MN. H2T described a continuum across a wide dynamic range ( approximately 2.5 log). Excluding the equivocal cases, HERmark showed 98% concordance with immunohistochemical studies for positive and negative assay values. For the 94 immunohistochemically equivocal cases, 67% and 39% concordance values were observed between HERmark and FISH for positive and negative assay values, respectively. Polysomy 17 in the absence of HER2 gene amplification did not result in HER2 overexpression as evaluated quantitatively using the HERmark assay.

Accurate assessment of human epidermal growth factor receptor 2 is critical for the management of patients with breast cancer. We set out to study the impact of the 2007 American Society of Clinical Oncology/College of American Pathologists guidelines on the interpretation of human epidermal growth factor receptor 2 IHC results and its correlation with fluorescence in situ hybridization results. Invasive breast carcinomas with IHC HercepTest 3+ were retrieved from the archive of Mayo Clinic Rochester. The human epidermal growth factor receptor 2 slides were rereviewed, and results were recorded as percentage of invasive tumor cells with 3+, 2+, 1+, and 0 staining intensity. Human epidermal growth factor receptor 2 gene amplification by fluorescence in situ hybridization was performed on all tumors with 3+ staining in 70% or less of tumor cells. Of the 141 cases studied, 12 cases showed intense membrane staining in 11% to 30% of the invasive tumor cells and would have been scored as 2+ according to the new American Society of Clinical Oncology/College of American Pathologists guidelines. Of these 12 cases, 6 were positive for human epidermal growth factor receptor 2 gene amplification by fluorescence in situ hybridization (ratio >2.2), 4 cases were negative (HER2/CEP17 ratio of < 1.8), and 2 cases were equivocal (ratio of 1.8-2.2). One human epidermal growth factor receptor 2-positive case showed dramatic intratumoral heterogeneity with high-level amplification (ratio of 12.2) in the IHC 3+ area and no amplification (ratio of 1.0) in the IHC 1+/2+ areas. The 2007 American Society of Clinical Oncology/College of American Pathologists guidelines down-scored 2.8% of tumors from human epidermal growth factor receptor 2-positive (IHC 3+) to human epidermal growth factor receptor 2-negative (IHC 2+ equivocal and fluorescence in situ hybridization negative) in this study. Clinical studies are needed to determine whether the updated guidelines are better at predicting response to anti-human epidermal growth factor receptor 2 therapy.

Objective: Recent studies have validated the use of cytologic materials to determine HER-2/neu status. Good concordance has been shown between results obtained by immunocytochemistry (ICC), immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) on cytologic and surgical specimens. However, the utility of cytologic cell block material in determining HER-2/neu status has not been reported and is the subject of this study., Study Design: HER-2/neu status was determined in 25 cases of primary or metastatic breast carcinoma by IHC and FISH. All cases were formalin-fixed, paraffin-embedded (FPPE) cell block preparations. ICC was performed using monoclonal antibodies TAB250 (Zymed) and CB11 (Novacastra Laboratories). FISH analysis was performed using the PathVysion HER-2 probe kit (Vysis, Inc.). Results of ICC and FISH were compared in each case., Results: Of 25 cases studied, 17 showed no protein overexpression or amplification. Five cases showed protein overexpression and amplification. The remaining 3 cases showed 2+ staining intensity by ICC in 10, 20, and 50% of carcinoma cells, respectively, and all demonstrated lack of amplification., Conclusion: Immunocytochemistry performed on FFPE cell block material is a reliable method for determining HER-2/neu status in cytologic specimens. We recommend routine preparation of FFPE cell block material in instances of suspected primary of metastatic breast carcinoma.

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.

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.

Objective: The aim of this study is to examine Her-2/neu gene amplification and protein overexpression in a spectrum of ovarian neoplasms using both immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) techniques that are FDA approved. This study is focused on early stage tumors including both carcinomas and borderline tumors., Methods: FDA-approved IHC and FISH for Her-2/neu were performed on formalin-fixed, paraffin-embedded tissue from 79 ovarian neoplasms representing a broad spectrum of tumor types as well as four normal ovaries. All tumors were either stage I or stage II. Tumor and normal tissue were studied collectively using a tissue microarray (TMA). HercepTest (DAKO) and PathVysion Her-2/neu probe kit (Vysis Inc.) were used for IHC and FISH analysis., Results: FISH analysis of serous carcinomas demonstrated Her-2/neu gene amplification in 3 (18%) of 17 cases. Two of three cases showing Her-2/neu gene amplification were scored 1+ using IHC, while the remaining case was scored as 0. Analysis of endometrioid carcinomas demonstrated Her-2/neu amplification using FISH in 1 of 10 (10%) cases. IHC in this case was scored 2+ (positive). None of the remaining 44 tumors, including clear cell carcinoma (n = 12), transitional cell carcinoma (n = 1), mixed epithelial carcinoma (n = 7), carcinoma not otherwise specified (n = 1), and 31 borderline tumors (mucinous, n = 17; endometrioid, n = 7; serous, n = 7), showed Her-2/neu gene amplification or protein overexpression. Normal ovaries were negative as well., Conclusions: Amplification of Her-2/neu in early stage ovarian neoplasms is infrequent, 6.7% overall. Due to the limited number of informative cases, we were unable to determine the clinical significance of Her-2/neu amplification in this study. Her-2/neu amplification was restricted to carcinomas and was not encountered in ovarian borderline tumors.

We analyzed concordance between immunohistochemical 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 immunohistochemically, 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 immunohistochemical 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.

HER 2/neu is an important oncogene in breast cancer, but the prevalence and significance of HER 2/neu gene amplification in colon cancer have been poorly documented. We have evaluated HER 2/neu gene amplification and protein overexpression in a series of colon cancers to assess the frequency, concordance and clinical significance of these events. HER 2/neu gene copy number was measured in 154 primary colon tumors, 15 liver metastases and matched normal tissues using a quantitative PCR/ligase detection reaction (LDR) technique developed and validated in our laboratory. HER 2/neu copy number was confirmed by fluorescent in situ hybridization (FISH) in all tumors found to have gene amplification. In an independent and blinded fashion, HER 2/neu expression was assessed in paraffin sections from 139 of the tumor specimens using the HercepTest kit. HER 2/neu gene amplification was observed in 4 (2.4%) of the 169 tumor specimens and in none of the normal tissues. There was no apparent association with stage of disease, tumor grade or patient survival. Among 139 cases evaluated by immunohistochemistry (IHC), HER 2/neu overexpression was seen in 5 cases (3.6%). There was extremely high concordance (kappa = 0.852) between gene amplification and protein overexpression. The low prevalence of HER 2/neu gene amplification and protein overexpression suggests that this oncogene plays an infrequent role in the development and progression of colon cancer. These data indicate that the primary mechanism of dysregulated HER 2/neu expression in colon cancer, as in breast cancer, is gene amplification., (Copyright 2003 Wiley-Liss, Inc.)