Implementation of American Society of Clinical Oncology/College of American Pathologists HER2 guideline recommendations in a tertiary care facility increases HER2 immunohistochemistry and fluorescence in situ hybridization concordance and decreases the number of inconclusive cases.
Abstract: Context.--The American Society of Clinical Oncology/ College of American Pathologists (ASCO/CAP) guideline recommendations from January 2007 identified many sources of immunohistochemistry (IHC) testing variation.

Objective.--In this current study, we implemented the guidelines and addressed our institution's preanalytic, analytic, and postanalytic variables relating to HER2 testing to improve clinical outcomes.

Design.--We evaluated core biopsies performed on breast lesions from 2006 through 2007. Prognostic/predictive markers obtained by IHC were correlated with HER2 fluorescence in situ hybridization (FISH). Preanalytic sources of biopsy testing variation were studied by collecting data on the number of biopsies that needed repeat testing because of inconclusive FISH results.

Results.--In the year preceding implementation of the guidelines, the HER2 IHC and FISH concordance was 98%. In an additional 10.8% of cases, the FISH results were in conclusive. When additional material became available to retest the inconclusive cases, the results were informative. Further evaluation of the inconclusive cases revealed that the core needle biopsies received, on average, 4 hours of formalin fixation. After implementation of a minimum 6 hours of fixation and the ASCO/CAP guideline recommendations, the HER2 IHC and FISH concordance was 98.5%. The number of FISH inconclusive cases decreased from 10.8% to 3.4% (a 64% reduction). Repeat estrogen-receptor IHC requests decreased by 40% from 38 in 2006 to 23 in 2007.

Conclusions.--We have shown that standardized fixation and adherence to the ASCO/CAP guidelines for HER2 testing has resulted in a greater HER2 IHC and HER2 FISH correlation, decreased numbers ofinconclusive FISH cases, decreased repeat estrogen-receptor requests, and financial savings to the Department of Pathology.
Article Type: Report
Subject: Immunohistochemistry (Standards)
In situ hybridization (Standards)
Health facilities (Management)
Authors: Middleton, Lavinia P.
Price, Kathy M.
Puig, Pamela
Heydon, Lori J.
Tarco, Emily
Sneige, Nour
Barr, Kaye
Deavers, Michael T.
Pub Date: 05/01/2009
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 College of American Pathologists ISSN: 1543-2165
Issue: Date: May, 2009 Source Volume: 133 Source Issue: 5
Topic: Event Code: 350 Product standards, safety, & recalls; 200 Management dynamics Computer Subject: Company business management
Product: Product Code: 8000300 Medical Facilities & Planning NAICS Code: 62 Health Care and Social Assistance
Organization: Organization: American Society of Clinical Oncology; College of American Pathologists
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 230151999
Full Text: The human epidermal growth factor receptor 2 gene, ERBB2 (HER2), located on chromosome band l7q21, is amplified in approximately 20% of all invasive breast cancers. (1-3) Immunohistochemical analysis of HER2 is a standard method of evaluating HER2 protein expression and is widely used by nearly all pathologists practicing in community and academic hospitals. As a prognostic marker, HER2 results help tailor individualized therapy in breast cancer. In newly diagnosed patients with breast cancer, findings of HER2 positivity are associated with higher rates of disease recurrence and mortality in select patients who do not receive any adjuvant systemic therapy. As a predictive marker, HER2 expression is predictive for several types of systemic therapies and sensitivity or resistance to different types of chemotherapeutic regimens. Numerous studies have shown that agents like trastuzumab that target the HER2 receptor are effective. Trastuzumab, a humanized monoclonal antibody, improves response rates, slows time to progression, and increases survival when used alone or in combination with standard chemotherapeutic regimens in patients with HER2-positive metastatic breast cancer. (4-6) Yet diagnostic test results may be inaccurate. Studies comparing HER2 immunohistochemistry (IHC) results obtained from the original pathology department with HER2 IHC results at a high-volume central laboratory have shown up to a 20% discordance rate. (7,8) It is important to have accurate results to ensure that every patient with breast cancer who may benefit from this therapy is identified, yet spare those who do not need this costly and potentially cardiotoxic therapy. (9)


The American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guideline recommendations published in January 2007 identified many sources of IHC testing variation. (9) In this current study, we implemented the guidelines during the course of a year and addressed our institution's preanalytic, analytic, and postanalytic variables relating to standardized testing of HER2. Specifically, we set out to achieve 100% accuracy of HER2 IHC testing of breast core biopsy specimens through standardization of preanalytic processes at M. D. Anderson Cancer Center (Houston, Texas) and, therefore, improve clinical outcomes in a 12-month period, as part of a clinical safety and effectiveness project.

In this study, HER2 IHC results were compared with fluorescence in situ hybridization (FISH) results for HER2 gene amplification.


Using the pathology database (PowerPath, IMPAC Medical Systems, Sunnyvale, California), we identified the number of core biopsies performed on breast lesions during the calendar years 2006 and 2007. We evaluated the prospectively reported prognostic/predictive markers. A program was designed to capture the HER2 IHC results and HER2 FISH results. The results for both years were correlated. We identified and set up a series of meetings with all those who were involved in obtaining, processing, interpreting, and reporting breast biopsies to assess the current status and identify improvement opportunities (Figure 1).

We studied our process of breast core needle biopsy (CNB) evaluation (Figure 2, A and B) and created a detailed process management chart to fully comprehend the flow of the information from the radiologist to the pathologist and ultimately to the patient's provider. We studied preanalytic sources of biopsy testing variation by collecting data on the number of biopsies that needed repeat testing because of inconclusive FISH results or tissue floating because those may have been caused by poor tissue preservation or fixation. We refined our methodology and standardized our tissue fixation and processing as it related to breast tissue specimens. We improved work flow in the histology laboratory to increase the efficiency of delivery of slides to the pathologist.


Analytic sources of HER2 testing variation were explored. For HER2 IHC, testing was performed on formalin-fixed, paraffinembedded tissue using a known, consistent level of antibody and subjecting each test to a similar test environment (AB8 Neomarkers, dilution 1:300, Labvision, Fremont, California). A previous study from our institution validated Neomarkers HER2 testing against the US Food and Drug Administration (FDA)-approved Dako HercepTest (Dako, Carpinteria, California). (10) We adopted the current guideline recommendations to reserve 3+ membranous staining to those cases where there was uniform intense membranous staining in more than 30% of the tumor cells. All tumors exhibiting 1 + ,2+ ,and3+ immunoreactivity were reflexively sent for FISH.

Fluorescence In Situ Hybridization

The PathVysion HER2 DNA probe kit (Vysis, Inc, Downers Grove, Illinois) containing 2 DNA probes that are directly labeled with spectrum orange fluorophore (HER2 gene locus on band 17q11.2-12), and spectrum green fluorophore-labeled chromosome 17 enumeration probe (CEP17; band 17p11.1-11.1) was used for all 1+ ,2+ ,and3+ scored HER2 immunoreactivity.

The slides were enumerated using an Olympus AX or BX61 (after August 2007) epifluorescence microscope (Olympus, Melville, New York) fitted with a Spectrum Orange, Spectrum Green, and 4',6-diamidino-2-phenylindole (DAPI; Vysis) triple-filter set. For each case, at least 60 tumor nuclei were counted, and the copy numbers of HER2 and CEP17 were recorded for each nucleus. HER2 was quantified using the ratio of HER2 to CEP17 signal counts. After January 2007, in accordance with the guideline recommendation, HER2 gene amplification was defined as a HER2: CEP17 signal ratio greater than 2.2. Tumor cells that could not be counted because hybridization signals were not uniform or the background was obscured were interpreted as inconclusive.

Postanalytic sources of HER2 variation include interpretation criteria, reporting elements, and quality assurance procedures. We shared cases in which the IHC and FISH results were discordant among our group of breast pathologists to refine our diagnostic ability. We modified our reports to include all recommended reporting elements, and we subscribed to the CAP general IHC survey, tissue microarray IHC survey, and CAP IHC


Q-Probes for HER2. For our daily laboratory quality assurance and quality control assessments, we ran a daily HER2 control with the cases and had one pathologist routinely screen the slide. We began the process of accumulating a repository of 1+ ,2+ , and 3+ formalin-fixed paraffin-embedded tissue blocks for controls.


Using the pathology database, we identified 1033 core biopsies performed on breast lesions during the calendar year 2006, before the publication of the guidelines. Two-thirds or 67% (687) were diagnosed as invasive breast cancer. This percentage is high because our institution is a tertiary cancer center where most patients are referred and outside diagnoses are confirmed (often with repeat biopsy at this institution). Prognostic/predictive markers performed to tailor therapy were evaluated. In the year preceding implementation of the guidelines, the HER2 IHC and FISH concordance was 98% for breast specimens obtained by CNB. Cases interpreted as equivocal were excluded from the correlation. In 2% of cases, the results were discordant.

In an additional 10.8% of cases from 2006, the FISH results were inconclusive. When additional material became available to retest the inconclusive cases (from subsequent excision of new tumors or receipt of additional pathology), the results were informative and no longer in conclusive. Further evaluation of the inconclusive cases revealed that the CNBs were obtained and submitted for evaluation after 2 PM. In 50% of the inconclusive cases, the patients had biopsies, and tissue was submitted to pathology after 3 PM. Time in formalin averaged 4 hours.

Figure 1 shows a cause-and-effect Ishakawa or "fishbone" diagram, outlining all potential issues with specimen collection, HER2 testing, and interpretation. We defined the current process, identified gaps between the current and the preferred state, and identified opportunities for improvement (Figure 2). After reviewing the data and determining that the majority of inconclusive cases had not been adequately fixed, we settled on the problem statement that there was a lack of standardization in biopsy specimen fixation resulting in less than optimal results.

Although not specifically recommended in the published guidelines, existing data support fixing all specimens, including biopsy material in formalin, for a minimum of 6 hours. (11) Working closely with the radiologists, our first intervention was to modify the existing specimen requisition form to include the time the specimen was placed in formalin. Secondly, we put all of the breast specimens, including CNBs, on a dedicated processor to ensure that a minimum fixation period of 6 hours was achieved.

Six-hour fixation was chosen because Goldstein et al (11) demonstrated that the maximum hormone receptor score occurs after 6 to 8 hours fixation. The small amount of tissue being fixed on CNB does not diminish the amount of time needed for fixation, as the same authors identified that the greatest correlation between core biopsy and excisional biopsy hormone receptor results occurs at a minimum of 6 hour fixation.11 Since implementation of standardized fixation times, our number of core biopsy cases that were inconclusive for FISH has decreased dramatically from 10.8% to 3.4%. In 2007, there were 1186 breast biopsies identified in the breast pathology database. Invasive breast cancer was diagnosed in 68% of cases. HER2 IHC and HER2 FISH results were concordant in 98.5% of all evaluated samples.


Figure 3 represents a statistical process control chart. Statistical process control charts allow you to tease out the variability inherent within a process to better understand whether interventions have the desired impact, and if so, whether the improvement is sustainable beyond the time period under study.12 The chart takes each individual point in time and calculates 3 SDs greater than and less than the mean. The red line is the upper control of the limit, and the blue line is the lower control of the limit. The green line is the mean. Figure 3 shows that after our third and final intervention, the percentage of cases that were inconclusive for analysis decreased from 10.8% to 3.8%, a 64% reduction. The variation has also decreased, reflecting that our improvements have been sustainable over time.

Although not an initial aim of our study, an unexpected benefit was that we found that the number of repeat estrogen receptor immunohistochemistry requests decreased significantly by 40%, from 38 in 2006 to 23 in 2007 after implementation of standardized fixation and the ASCO/CAP guideline recommendations (Figure 4). Currently, we run controls on every IHC batch and also evaluate staining in adjacent nonneoplastic breast parenchyma. Floating of tissue sections is much less of a problem, and there are fewer repeat requests for IHC tests on all breast CNB material.


Our study has shown that standardization of processing, with a 6 hour minimum fixation time for breast CNB specimens, has translated not only into greater HER2 IHC and FISH correlation but also resulted in a reduction of both FISH inconclusive cases and repeat estrogen receptor staining. We were able to increase our HER2 IHC and FISH concordance from 98% to 98.5%. Granted this is a modest increase of concordance beyond our baseline and represents concordance between 1+,2+,and 3+ immunoreactivity and HER2 gene amplification by FISH, but a clinical decision error of less than 1% has a tremendous implication for patient safety. As important, we were able to reduce the percentage of cases deemed inconclusive by FISH analysis by 64% and decrease repeat estrogen-receptor hormonal analysis requests to the laboratory by 40%. Whereas the minimum time of fixation is important, the maximum time that a specimen is fixed in formalin may not be as critical, and to date, that has not been scientifically proven. Study of the hour-by-hour effect of formalin fixation on HER2 testing during the range of fixation intervals seen in routine clinical practice has not been performed. (13)

Patients with HER2-positive breast cancers are eligible to be treated with trastuzumab, a drug that has been shown to increase survival and decrease disease recurrence. (4-6) The treatment is expensive and potentially cardiotoxic and should only be reserved for those patients with HER2-positive tumors. Our goal to achieve 100% accuracy of HER2/neu IHC testing of breast core biopsy specimens through standardization of preanalytic factors was lofty, but we were able to show a 0.5% improvement in the HER2 IHC and HER2 FISH correlation by standardizing fixation times and interpretation criteria. Moreover, by instituting standardized specimen processing, we were able to reduce the frequency of repeat FISH testing for inconclusive cases from a baseline rate of 10.8% to 3.4%.


Our interventions were simple and primarily aimed at preanalytic factors; we modified the pathology specimen request card to include the time the biopsy was placed in fixative. We educated our radiologists about why this information was needed and the importance of completing the forms. We placed all breast biopsies on a dedicated processing machine and ensured that all specimens received a minimum of 6 hours of fixation. We improved work flow in the histology laboratory to increase the efficiency of delivery of slides to the pathologist.

Concurrently, we established professional competencies in evaluating HER2 IHC. We followed the new recom mendation to reserve 3+ IHC scoring for those tumors with uniformly intense membrane staining in greater than 30% of tumor cells and followed the new guidelines for interpreting and reporting FISH. (9) We standardized our reporting elements.

The financial savings to the pathology department were significant. All inconclusive FISH tests need to be repeated. In 2006, all denials for duplicate services billed under the HER2 FISH professional billing code were appealed and paid. However, many more inconclusive tests were actually performed. Fluorescence in situ hybridization tests are always run twice when the result is inconclusive, although the patient is only billed once, resulting in an unreimbursed cost of $450 per case. This translates into a savings of $34000/y in cost avoidance for repeat FISH testing of patient biopsy material.

The inefficient use of pathology and billing resources and the inability of the patient to start on his or her appropriate course of treatment is substantial, but harder to quantify. Our findings directly related to our institution's strategic vision for 2005-2010, that is, to enhance the excellence, value, safety, and efficiency of our patient care and contain costs.

An additional benefit of the changes in our study was the improvement in estrogen-receptor staining results and the reduction of repeat staining for this biomarker. Al though not an aim of our study, we fortuitously found that the number of repeat estrogen-receptor requests on core biopsy material significantly decreased by 40% from 38 in 2006 to 23 in 2007 after implementation of standardized fixation and the ASCO/CAP guidelines. Additionally, fewer tissue sections floated after a minimum of 6 hour of fixation.


By standardizing the preanalytic factors related to time in formalin and processing of breast core biopsies, we have implemented processes that may be applicable to other specimens and other IHC tests.

In summary, we have shown that adherence to the ASCO/CAP guidelines for HER2 testing has resulted in a greater correlation between HER2 IHC and HER2 FISH, decreased the number of inconclusive FISH cases, decreased repeat estrogen-receptor hormone analysis, and resulted in financial savings to the department of pathology.


(1.) Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuireWL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/ neu oncogene. Science. 1987;235(4785):1 77-182.

(2.) Yaziji H, Goldstein LC, Barry TS, et al. HER-2 testing in breast cancer using parallel tissue-based methods. JAMA. 2004;291(1 6):1972-1977.

(3.) Owens MA, Horten BC, Da Silva MM. HER2 amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues. Clin Breast Cancer. 2004;5(1):63-69.

(4.) Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpress es HER2. N EnglJMed. 2001;344(11):783-792.

(5.) Cobleigh MA, Vogel CL, Tripathy D, et al. Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. JClin Oncol. 1999;17(9):2639-2648.

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(7.) Paik S, Bryant J, Tan-Chiu E, et al. Real-world performance of HER2 testing: National Surgical Adjuvant Breast and Bowel Project experience. J Natl Cancer Inst. 2002;94(1 1):852-854.

(8.) Roche PC, Suman VJ, Jenkins RB, et al. Concordance between local and central laboratory HER2 testing in the breastintergroup trial N9831. JNatlCancer Inst. 2002;94(1 1):855-857.

(9.) Wolf AC, Hammond EH, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med. 2007;131(1):18-43.

(10.) Hoang MP, Sahin AA, Ordonez NG, Sneige N. HER-2/neu gene amplification compared with HER-2/neu protein overexpression and interobserver reproducibility in invasive breast carcinoma. Am J Clin Pathol. 2000;1 13(6):852 859.

(11.) Goldstein NS, Ferkowicz M, Odish E, Mani A, Hastah F. Minimum formalin fixation time for consistent estrogen receptor immunohistochemical staining of invasive breast carcinoma. Am J Clin Pathol. 2003;120(1):86-92.

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(13.) Lombardo JF. American Society ofClinical Oncology/CollegeofAmerican Pathologists guidelines should be scientifically validated. Arch Pathol Lab Med. 2007;131(10):1510.

Lavinia P. Middleton, Kathy M. Price, Pamela Puig, Lori J. Heydon, Emily Tarco, Nour Sneige, Kaye Barr, Michael T. Deavers,

Accepted for publication September 12, 2008.

From the Departments of Pathology (Drs Middleton, Sneige, and Deavers and Mss Puig, Tarco, and Barr), Performance Improvement, (Ms Price), and Laboratory Informatics (Ms Heydon), The University of Texas M. D. Anderson Cancer Center, Houston.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Lavinia P. Middleton, MD, Department of Pathology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd--Unit 85, Houston, TX 77030 (e-mail: lpmiddleton@
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