Laboratory compliance with the American Society of Clinical Oncology/College of American Pathologists guidelines for human epidermal growth factor receptor 2 testing: a College of American Pathologists survey of 757 laboratories.
Abstract: Context.--To ensure quality human epidermal growth receptor 2 (HER2) testing in breast cancer, the American Society of Clinical Oncology/College of American Pathologists guidelines were introduced with expected compliance by 2008.

Objective.--To assess the effect these guidelines have had on pathology laboratories and their ability to address key components.

Design.--In late 2008, a survey was distributed with the HER2 immunohistochemistry (IHC) proficiency testing program. It included questions regarding pathology practice characteristics and assay validation using fluorescence in situ hybridization or another IHC laboratory assay and assessed pathologist HER2 scoring competency.

Results.--Of the 907 surveys sent, 757 (83.5%) were returned. The median laboratory accessioned 15 000 cases and performed 190 HER2 tests annually. Quantitative computer image analysis was used by 33% of laboratories. In-house fluorescence in situ hybridization was performed in 23% of laboratories, and 60% of laboratories addressed the 6- to 48-hour tissue fixation requirement by embedding tissue on the weekend. HER2 testing was performed on the initial biopsy in 40%, on the resection specimen in 6%, and on either in 56% of laboratories. Testing was validated with only fluorescence in situ hybridization in 47% of laboratories, whereas 10% of laboratories used another IHC assay only; 13% used both assays, and 12% and 15% of laboratories had not validated their assays or chose "not applicable" on the survey question, respectively. The 90% concordance rate with fluorescence in situ hybridization results was achieved by 88% of laboratories for IHC-negative findings and by 81% of laboratories for IHC-positive cases. The 90% concordance rate for laboratories using another IHC assay was achieved by 80% for negative findings and 75% for positive cases. About 91% of laboratories had a pathologist competency assessment program.

Conclusions.--This survey demonstrates the extent and characteristics of HER2 testing. Although some American Society of Clinical Oncology/College of American Pathologists guidelines have been implemented, gaps remain in validation of HER2 IHC testing.

(Arch Pathol Lab Med. 2010;134:728-734)
Subject: Immunohistochemistry (Usage)
Breast cancer (Development and progression)
Breast cancer (Diagnosis)
Breast cancer (Care and treatment)
Epidermal growth factor (Physiological aspects)
Epidermal growth factor (Measurement)
Pathologists (Practice)
Authors: Nakhleh, Raouf E.
Grimm, Erin E.
Idowu, Michael O.
Souers, Rhona J.
Fitzgibbons, Patrick L.
Pub Date: 05/01/2010
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2010 College of American Pathologists ISSN: 1543-2165
Issue: Date: May, 2010 Source Volume: 134 Source Issue: 5
Topic: Event Code: 200 Management dynamics
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 230246558
Full Text: Human epidermal growth receptor 2 gene ERBB2 (commonly referred to as HER2) amplification status is an important prognostic marker in breast cancer and is a factor in determining patient therapy. (1-5) The American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) introduced guidelines for HER2 testing in December 2006, which were subsequently published in January 2007. (6) These guidelines were produced in response to studies that indicated that up to 20% of HER2 results may be discordant with another laboratory. (7,8) The ASCO/CAP publications identified possible sources of variations and gave guideline recommendations to promote test standardization in the hope of For editorial comment, see p 660. improving test accuracy and patient safety. This was coupled with efforts by the Laboratory Accreditation Program (LAP) of the CAP to strengthen its accreditation standards regarding immunohistochemistry (IHC) in general and HER2 testing specifically. (9) As of January 2008, the LAP mandates that laboratories be in compliance with the ASCO/CAP guidelines. In an effort to understand the effect of these guidelines on laboratories, pathologists and staff involved in the CAP proficiency testing program conducted a survey to identify areas in which laboratories may be having difficulty successfully implementing the guideline recommendations.

MATERIALS AND METHODS

In late 2008, a survey was distributed with the HER2 IHC proficiency testing program. (10) The survey was composed of 30 questions; most questions had predetermined, multiple-choice answers. Some questions required a numeric answer. Thirteen questions (43%) elicited demographic institutional and laboratory information. Eight questions (27%) related to specific HER2 practices. Four questions (13%) addressed HER2 validation with fluorescence in situ hybridization (FISH), 4 questions (13%) addressed HER2 validation with another IHC laboratory assay, and 1 question (3%) addressed pathologist competency.

A series of multinomial logistic regression models were used to determine whether any demographic factors were significantly associated with any practice characteristics. For continuous variables, a stepwise, multivariate linear regression was used. All analyses were run using SAS version 9.2 software (SAS Inc, Cary, North Carolina) with a significance level of P < .05.

RESULTS

Of the 907 surveys that were distributed, 757 (83%) were returned. Table 1 shows the questions and answers regarding various institutional demographics. Nearly half of the respondents serve private nonprofit hospitals, with 65% in small- to medium-sized (<450 beds) facilities. About one-quarter of the laboratories have a pathology residency program, and about two-thirds serve a cancer center. The CAP accredits 84% of the laboratories. Table 2 shows the distribution of surgical pathology and HER2 IHC case volumes. The median number of accessioned cases in 2007 was 15 184, and the median number of HER2 stains was 190. Table 3 shows the distribution of full-time equivalent (FTE) histology personnel and pathologists working in the laboratory and performing HER2 testing. The distribution of histology FTE personnel indicates that fewer individuals perform HER2 testing than work in histology or perform IHC testing in general. A similar pattern is seen with the distribution of pathologist FTEs: Not all pathologists who sign out surgical pathology cases also sign out HER2 IHC cases.

Table 4 shows the frequency of HER2 slides prepared in house (81%), the use of quantitative computer image analysis (33%), and the frequency of in-house HER2 FISH analysis (23%). Table 5 shows how participating laboratories have dealt with issues related to tissue fixation and processing. All but 14.2% have instituted some change in practice to address fixation time, with 60% of laboratories processing tissues on the weekend to avoid prolonged fixation. Table 5 also shows that fixation time is addressed in the pathology report in some form in about 70% of laboratories. HER2 IHC testing is typically performed on the biopsy specimen 40% of the time, on the resection specimen 6% of the time, and on either specimen type 54% of the time. Two-thirds of laboratories take into consideration background staining in healthy tissues when interpreting IHC HER2 stains.

Table 6 shows data on test validation among laboratories that compared the results of IHC assays with FISH (60% of laboratories). Only 81% and 73% of these laboratories were able to achieve the required 95% concordance for HER2-negative and HER2-positive findings, respectively. Almost 10% of these laboratories reported achieving no greater than 50% concordance with FISH for either HER2-negative or HER2-positive results. Table 7 shows the number of cases used for validation. Only 78% of laboratories used the required minimum of 25 cases in their validation study.

Table 8 shows data on test validation among laboratories that compared the results of IHC assays with an IHC test performed in another laboratory (24% of laboratories). Even fewer laboratories choosing this method of test validation were able to achieve 95% concordance: 28% and 32% failed to achieve 95% concordance with the other laboratory for HER2-negative and HER2-positive results, respectively. One in 6 laboratories (17%) using this method of assay validation had no more than 50% concordance for IHC score 3 (HER2-positive) findings. Table 9 shows the number of cases used for test validation. Only 56% of laboratories used the required minimum of 25 cases in their validation study.

Table 10 shows that 91% of laboratories (639 of 704) have an active pathologist competency assessment process, with most (61%; 392 of 639) performing periodic challenges using external material.

In attempting to correlate various factors, some associations were identified: FISH analysis is performed at a higher frequency in larger hospitals (34.3% and 44.6% for 451-600 beds and >600 beds, respectively) compared with smaller facilities (4.2% and 4% for <150 beds and 151-300 beds, respectively; P < .001). Analysis using FISH is performed at least twice as often at reference IHC laboratories (51.8%) versus all other categories of institutions (<25%). Institutions with residency programs are more likely to perform FISH analysis than institutions without residency programs (48.1% versus 14.3%; P < .001). By contrast, institutions with a residency program are less likely to use quantitative image analysis for HER2 intensity scoring (18.2% versus 37.5%; P = .03). Table 11 shows that institutions accredited by the CAP are more likely to include fixation time in reports (P < .001) and to validate their HER2 IHC assays by comparing them with FISH (P = .01). Table 12 shows the number of laboratories that validated HER2 IHC with FISH and/or with another IHC laboratory test. There was no association that could be identified with a higher rate of HER2 validation by FISH or by validation with another IHC test. Factors tested included all laboratory and institutional demographics, as well as in-house HER2 slide preparation and FISH testing and the use of computer image analysis.

COMMENT

We conducted this survey to better understand the effect of the ASCO/CAP HER2 testing guidelines on surgical pathology laboratories. In doing so, for the first time, we are able to describe some of the characteristics and practices of laboratories performing HER2 IHC. Participants in this survey represent a broad spectrum of laboratories that serve small to large hospitals and operate in multiple practice settings. Almost two-thirds of laboratories offering HER2 testing by IHC are in small-to medium-sized (<450 beds) hospitals and about one-quarter also perform HER2 testing by FISH. Nearly one-fifth of the laboratories in this survey interpret HER2 slides that are prepared at another institution. About one-third of laboratories use computer image analysis to assess HER2 IHC.

One of the stated goals of the ASCO/CAP guidelines is to standardize testing procedures so that more consistent results are obtained within and among laboratories. Three areas that were addressed in the guidelines were also addressed with the addition of new LAP checklist questions (ANP.22997, ANP.22998, and ANP.22999). (6,9) These standards require laboratories to (1) properly validate their HER2 assays, (2) have a documented procedure for ensuring appropriate fixation, and (3) use the ASCO/CAP scoring criteria.

The results of this survey suggest that most laboratories are making an effort to comply with the guideline recommendations and the corresponding LAP requirements. Laboratories accredited by LAP document specimen fixation in their reports and performed validation studies at a higher rate than others performed them. However, some laboratories have been unable to achieve full compliance.

The first issue is validation. HER2 testing is covered by the Clinical Laboratory Improvement Amendments of 1988 (CLIA) as high-complexity testing. (11) As specified by CLIA, all such tests must be validated before they are offered. It is likely that relatively few laboratories appropriately validated their HER2 assays before the ASCO/CAP guidelines. Our data indicate that as of the end of 2008, about 70% of laboratories attempted to formally validate their procedures. But about 11% stated that they have not attempted validation. Almost 20% of laboratories interpret IHC HER2 slides that are prepared at another laboratory. This group does not have to validate their procedures but needs assurance that the laboratory doing IHC staining has performed appropriate validation.

Achieving 95% concordance in validation studies has also been challenging for laboratories. Among laboratories that validated their assay by comparing the results with FISH, the number of cases used in the validation studies ranged from 5 to 334, with a median of 40 cases. The 10th percentile of laboratories validated 21 cases, which is fewer than the 25 to 100 recommended. In this group, 81% and 73% of laboratories were able to achieve the 95% concordance target stated in the guideline for negative and positive cases.

Laboratories choosing to validate their assay by comparing their results with the results of another IHC laboratory test did even less well than those comparing their results with FISH did. Of those that validated with another IHC laboratory, the number of cases used in the validation study ranged from 5 to 145, with a median of 27. These results suggest that about 40% of laboratories have not validated their assay with the minimum of 25 cases. Also, 72% and 67% of laboratories were able to achieve the 95% concordance target stated in the guideline for negative and positive case results.

Of the laboratories that validated their IHC assay with FISH, 77% included cases classified as IHC score 2 in their validation study, even though validating equivocal results is not required. Similarly, 66% of laboratories that performed validation with another IHC laboratory included score 2 cases in their validation study. Although including a few equivocal cases in a large validation study might provide some useful information, the relatively high proportion of equivocal cases that were included (40% of laboratories validating with FISH included >10) suggests that a significant number of laboratories may have misunderstood the validation requirement.

In fairness, the data in this report represent a survey and not a formal evaluation of these programs for accreditation. Also, 13% of laboratories attempted validation by both methods, so it may be that some of these results reflect haphazard initial attempts at validation.

Fixation time is another important issue addressed in the HER2 testing guidelines, with a recommendation for formalin fixation of at least 6 hours and not more than 48 hours. Laboratories are now expected to develop policies to address cases that would otherwise be held in fixative longer than 48 hours (eg, on weekends). This survey shows that the guidelines have led to changes in pathology practices in about 85% of laboratories. These changes deal with the issue of minimizing the frequency and effect of prolonged fixation, either by reducing the number of cases fixed longer than 48 hours or by retesting such specimens by FISH.

The survey results indicate that about 60% of laboratories address this requirement by processing tissue during the weekend, with most of the remainder adopting other measures. It is not clear from this survey how many laboratories changed their weekend processing procedures solely because of this guideline. Of note, 14% of laboratories (103 of 727) reported that they do not have a specific policy to address prolonged fixation time (another 10% (74 of 727) chose "other" as the answer to this question). The guidelines recommend, but do not mandate, that fixation time be documented in the report. Our survey demonstrates that 72% of respondents (519 of 725) address fixation time in their report. There are a variety of methods used to document fixation time, as listed in Table 5.

Another recommendation in the new guidelines is that laboratories assessing HER2 by IHC use the ASCO/CAP scoring criteria. Our survey indicates that 84% of laboratories (605 of 722) use the ASCO/CAP scoring criteria, whereas 13% (95 of 722) use the scoring guidelines included in the test kit. One possible source for the confusion could be that some HER2 IHC assays are approved by the US Food and Drug Administration, and these assays include specific scoring language in the product insert. The difference between the test scoring approved by the US Food and Drug Administration (eg, HercepTest; Dako, Carpinteria, California) and the ASCO/CAP scoring criteria is mostly related to defining an equivocal category and the percentage of cells needed to classify a case as score 3. The ASCO/CAP scoring criteria improve the specificity of the test.

To achieve consistent performance in the interpretation of HER2 slides, the guidelines included a recommendation for ongoing competency assessment of individuals performing HER2 testing. Our survey indicates that about 91% of laboratories (689 of 757) have an active program for competency assessment of pathologists. Our survey also shows that fewer pathologists interpret HER2 cases than perform surgical pathology in general, suggesting that laboratories may be limiting the number of individuals performing HER2 testing to achieve more consistent results. A similar pattern is seen with histology personnel, with fewer performing IHC testing than work in histology and even fewer performing HER2 IHC than those that perform other IHC tests. Subspecialization has been advocated before in pathology with the realization that individual aptitude is more conducive to particular types of specimens. (12) This approach also makes it easier for individuals in that field to keep current with the literature and to serve as a point person or reference to other individuals or pathologists working in the same group.

Although not addressed in the ASCO/CAP guidelines, about one-third of laboratories stated that they did not take into consideration whether background staining was present in normal tissue findings. This has been suggested as a potential pitfall that may lead to false-positive results by IHC tests. (13)

In summary, we report our finding from a survey of 757 laboratories demonstrating the current state of practice regarding HER2 testing. Although some laboratories have successfully implemented the ASCO/CAP guidelines, substantial gaps remain in assay validation. The ASCO/ CAP guidelines were published with the intent of improving laboratory performance in HER2 testing. The data indicate that publication of guidelines did not, in and of itself, ensure uniform compliance and that enforcement through accreditation may be necessary to ensure widespread adherence. This survey was conducted when only about 25% of laboratories had been inspected by the LAP with the new HER2 standards in place. A follow-up survey after a complete cycle of inspection will be necessary to confirm this premise.

References

(1.) Pritchard KI, Shepherd LE, O'Malley FP, et al. HER2 and responsiveness of breast cancer to adjuvant chemotherapy. N Engl J Med. 2006;354(20):2103-2111.

(2.) Villman K, Sjostrom J, Heikkila R, et al. TOP2A and HER2 gene amplification as predictors of response to anthracycline treatment in breast cancer. Acta Oncol. 2006;45(5):590-596.

(3.) Konecney GE, Thomssen C, Luck HJ, et al. Her-2/neu gene amplification and response to paclitaxel in patients with metastatic breast cancer. J Natl Cancer Inst. 2004;96(15):1141-1151.

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

(5.) Vogel CL, Cobleigh MA, Tripathy D, et al. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol. 2002;20(3):719- 726.

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

(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(11):853-854.

(8.) Roche PC, Suman VJ, Jenkins RB, et al. Concordance between local and central laboratory HER2 testing in the breast intergroup trial N9831. J Natl Cancer Inst. 2002;94(11):855-857.

(9.) Commission on Laboratory Accreditation. Standards ANP.22997,ANP.22998, ANP. 22999 of the anatomic checklist of the Laboratory Accreditation Program. Northfield, IL: College of American Pathologists. Adopted on: September 27, 2007.

(10.) Fitzgibbons PL, Murphy DA, Dorfman DM, Roche PC, Tubbs RR; for the Immunohistochemistry Committee, College of American Pathologists. Interlaboratory comparison of immunohistochemical testing for HER2: results of the 2004 and 2005 College of American Pathologists HER2 Immunohistochemistry Tissue Microarray Survey. Arch Pathol Lab Med. 2006;130(10):1440-1445.

(11.) Clinical Laboratory Improvement Amendments of 1988; final rule. Fed Reg. 1992;57:7001-7186. To be codified at 42 CFR 1405.

(12.) Black-Schaffer WS, Young RH, Harris NL. Subspecialization of surgical pathology at the Massachusetts General Hospital. Am J Clin Pathol. 1996; 106(4)(suppl 1):S33-S42.

(13.) Gown AM. Current issues in ER and HER2 testing by IHC in breast Cancer. Mod Pathol. 2008;21(suppl 2):S8-S15.

Raouf E. Nakhleh, MD; Erin E. Grimm, MD; Michael O. Idowu, MD; Rhona J. Souers, MS; Patrick L. Fitzgibbons, MD

Accepted for publication October 28, 2009.

From the Department of Pathology, Mayo Clinic Florida, Jacksonville (Dr Nakhleh); the Department of Pathology, University of Washington Medical Center, Seattle (Dr Grimm); the Department of Pathology, Virginia Commonwealth University Health System, Richmond (Dr Idowu); the Department of Statistics, College of American Pathologists, Northfield, Illinois (Ms Souers); and the Department of Pathology, St Jude Medical Center, Fullerton, California (Dr Fitzgibbons).

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

Presented in part at the annual meeting of the College of American Pathologists, National Harbor, Maryland, October 11-14, 2009.

Reprints: Raouf E. Nakhleh, MD, Department of Pathology, Mayo Clinic Florida, 4500 San Pablo Rd, Jacksonville, FL 32224 (e-mail: Nakhleh.roauf@mayo.edu).
Table 1. Demographic and Accreditation Information

                                                           Response,
Parameters                                                  No. (%)

What best describes the institution in which you
    perform IHC for HER2? (n = 748)
  Private, nonprofit hospital                             347 (46.4)
  State, county, or city hospital                         106 (14.2)
  Independent laboratory, multihospital practice           87 (11.6)
  Private, for profit hospital                             65 (8.7)
  Reference IHC laboratory                                 56 (7.5)
  Independent laboratory, primary outpatient focus         54 (7.2)
  Other                                                    33 (4.4)

For institutions affiliated with a hospital, how many
    beds is the hospital licensed to support?
    (n = 519)
  0-150                                                    48 (9.2)
  151-300                                                 151 (29.1)
  301-450                                                 137 (26.4)
  451-600                                                  71 (13.7)
  >600                                                    112 (21.6)

Does your institution have a pathology residency
    program? (n = 748)
  Yes                                                     186 (24.9)
  No                                                      562 (75.1)

Service is provided to a designated cancer center?
    (n = 688)
  Yes                                                     454 (66.0)
  No                                                      234 (34.0)

Type of cancer center (n = 384)
  American College of Surgeons Commission on
    Cancer accredited                                     251 (65.4)
  NIH designated                                           37 (9.6)
  Self-designated cancer center                            94 (24.5)
  Other                                                    19 (4.9)

Organization that accredits/certifies your laboratory
    (n = 733). Multiple responses allowed.
  CAP                                                     614 (83.8)
  State                                                    44 (6.0)
  Joint Commission                                         39 (5.3)
  Other                                                    36 (4.9)

Abbreviations: CAP, College of American Pathologists; HER2, human
epidermal growth receptor 2; IHC, immunohistochemistry; NIH,
National Institutes of Health.

Table 2. Distribution of Surgical Pathology and Human Epidermal Growth
Receptor 2 (HER2) Accessions in 2007

                                                         Percentile

                          No.      Mean      Minimum     10th    25th

Surgical pathology        704    24 067.6          0     4630    8553
  cases accessioned
HER2                      674       925.4          0        0      80
  immunohistochemistry
  stains

                          Percentile

                          Median     75th     90th      Maximum

Surgical pathology        15 184    28 535   50 000     300 000
  cases accessioned
HER2                         190      380       750     130 000
  immunohistochemistry
  stains

Table 3. Distribution of Full-Time Employees (FTEs)

                No. (%) of FTEs      No. (%) of histology
                 in histology          FTEs who perform
Parameter    laboratory (n = 700)        IHC (n = 700)

    0              10 (1.4)                70 (10.0)
   1-4            265 (37.9)              506 (72.3)
  5-10            264 (37.7)              110 (15.7)
  11-20           106 (15.1)                9 (1.3)
  21-30            29 (4.1)                 5 (0.7)
  31-50            17 (2.4)
   >50              9 (1.3)

             No. (%) of histology      No. (%) of pathologist
               FTEs who perform      FTEs who sign out surgical
Parameter     HER2 IHC (n = 700)     pathology cases (n = 717)

    0             122 (17.4)                  2 (0.3)
   1-4            481 (68.7)                306 (42.7)
  5-10             87 (12.4)                270 (37.7)
  11-20             6 (0.9)                 105 (14.6)
  21-30             4 (0.6)                  20 (2.8)
  31-50                                      10 (1.4)
   >50                                        4 (0.6)

              No. (%) of pathologist
              FTEs who sign out HER2
Parameter       IHC cases (n = 715)

    0                 1 (0.1)
   1-4              476 (66.6)
  5-10              197 (27.6)
  11-20              36 (5.0)
  21-30               5 (0.7)
  31-50
   >50

Abbreviations: HER2, human epidermal growth receptor 2; IHC,
immunohistochemistry.

Table 4. Rate of In-House Human Epidermal Growth
Receptor 2 (HER2) Testing by Immunohistochemistry
(IHC) and Fluorescence In Situ Hybridization (FISH)
and Computer Image Analysis

                                                       Response,
                     Parameter                          No. (%)

How is HER2 IHC testing done? (n = 735)
  Interpret slides that have been stained in-house     598 (81.4)
  Interpret slides that have been stained at an
    outside laboratory                                 137 (18.6)

Is quantitative computer image analysis performed
    for HER2 intensity score? (n = 739)

  Yes                                                  242 (32.7)
  No                                                   497 (67.3)

Is in-house FISH performed? (n = 746)

  Yes                                                  169 (22.7)
  No                                                   577 (77.3)

Table 5. Tissue Fixation and Specimen-
Handling Information

                                                       Response,
                     Parameter                          No. (%)

How do you ensure accurate HER2 testing of
    breast specimens that are received late
    Thursday and Friday? (n = 727)

  Process and embed tissues on weekends; no
    specimens fixed longer than 48 h                   435 (59.8)
  No special policies for such specimens               103 (14.2)
  Other                                                 74 (10.2)
  Do FISH on all cases fixed longer than 48 h            46 (6.3)
  FISH on all IHC-negative cases fixed longer
    than 48 h                                            46 (6.3)
  Breast surgeries no longer done late Thursday
    and Friday                                           23 (3.2)
How is the fixation time conveyed in your
    report? (n = 725)
  Time of fixation not included in the report          206 (28.4)
  No. of hours stated in report                        197 (27.2)
  Standard disclaimer added to all HER2 reports
    that tissue fixation was within recommended
    guidelines (6-48 h)                                175 (24.1)
  Hours approximated using one of several
    categories                                           68 (9.4)
  Other                                                  43 (5.9)
  A statement on time of fixation added only if it
    does not meet the guidelines (<6 or > 48 h)          36 (5.0)

HER2 IHC testing is typically performed on:
    (n = 727)

  Either specimen type depending on fixation
    time, amount of invasive tumor, and/or
    clinical consideration                             390 (53.6)
  Initial diagnostic biopsy specimens, including
    needle biopsies                                    293 (40.3)
  Resection specimens                                    44 (6.1)

If HER2 overexpression is noted in the
    background of normal ducts, is this taken
    into consideration when interpreting the
    HER2 score? (n = 694)

  Yes                                                  466 (67.1)
  No                                                   228 (32.9)
Which HER2 IHC scoring guidelines are
  currently used in your laboratory? (n = 722)

  Scoring guidelines included in the CAP/ASCO
    guidelines                                         605 (83.8)
  Scoring guidelines included with the test kit         95 (13.2)
  Other                                                  22 (3.0)

Abbreviations: ASCO, American Society of Clinical Oncology; CAP,
College of American Pathologists; FISH, fluorescence in situ
hybridization; HER2, human epidermal growth receptor 2; IHC,
immunohistochemistry.

Table 6. Immunohistochemistry (IHC) Test Validation
With Fluorescence In Situ Hybridization (FISH)

                                                Response,
                 Parameter                       No. (%)

Did you validate your HER2 IHC stain by
    studying concordance with FISH?
    (n = 711)
  Yes                                           431 (60.6)
  No                                            144 (20.3)
  Not applicable                                136 (19.1)

HER2 IHC-negative cases (scores 0 and
    1+ combined) that were also
    negative by FISH (n 5 326), %
  0-50                                           29 (8.9)
  51-89                                          10 (3.1)
  90-94                                          23 (7.1)
  95-99                                         264 (81.0)

HER2 IHC-positive cases (score 3) that were
    also positive by FISH (n 5 322), %
  0-50                                           30 (9.3)
  51-89                                          30 (9.3)
  90-94                                          26 (8.1)
  95-99                                         236 (73.3)

Abbreviation: HER2, human epidermal growth receptor 2.

Table 7. Distribution of Cases Included in Human Epidermal Growth
Receptor 2 (HER2) Test Validation With Fluorescence In Situ
Hybridization (FISH) (n = 301 Laboratories)

                                           Minimum    10th     Median

No. of cases in validation study              5        21        40
  per laboratory (mean, 59)
                                                     No. of cases

                                            1-25     26-50     51-75

Laboratories performing FISH                21.6      39.9      11.0
  validation, %
                                       No. of cases

                                  0        1-5      6-10

Laboratories that performed validation
  for each score versus no. of cases
  performed for that score, %

IHC score 0                       10.0      25.6      20.9
IHC score 1                       11.6      21.3      26.9
IHC score 2                       23.3      19.6      17.3
IHC score 3                        2.0      21.3      31.9

                                            90th     Maximum

No. of cases in validation study             119       334
  per laboratory (mean, 59)
                                              No. of cases

                                           76-100     >100

Laboratories performing FISH                12.3      14.6
  validation, %
                                       No. of cases

                                11-20     21-40      >40

Laboratories that performed validation
  for each score versus no. of cases
  performed for that score, %

IHC score 0                      24.3      10.3       9.0
IHC score 1                      22.3      12.0       6.0
IHC score 2                      15.0      11.3      13.6
IHC score 3                      23.6      16.9       4.3

Abbreviation: IHC, immunohistochemistry.

Table 8. Immunohistochemistry (IHC) Test Validation
With Another IHC Test
                                                    Response,
Parameter                                            No. (%)
    IHC stain validated by studying concordance
    with IHC results of another laboratory
    (n = 685)
  Yes                                              161 (23.5)
  No                                               368 (53.7)
  Not applicable                                   156 (22.8)

HER2 IHC-negative cases (scores 0 and
    1+ combined) that also had IHC-negative
    findings in the other laboratory
    (n = 101), %
  0-50                                              11 (10.9)
  51-89                                              9 (8.9)
  90-94                                              8 (7.9)
  95-99                                             73 (72.3)

HER2 IHC-positive cases (score 3) that also
    had IHC-positive findings in the other
    laboratory (n = 99), %
  0-50                                              17 (17.2)
  51-89                                              8 (8.1)
  90-94                                              7 (7.1)
  95-99                                             67 (67.7)

Abbreviation: HER2, human epidermal growth receptor 2.

Table 9. Distribution of Cases Included in Test Validation With
Another Immunohistochemistry (IHC) Test (n = 301 Laboratories)

                                                   Percentile

                                           Minimum    10th     Median

No. of cases in validation study per          5        15        27
laboratory (mean, 59)

                                                  No. of cases

                                            1-25      26-50     51-75

Laboratories performing validation with     44.3      38.1       6.2
another IHC test, %

                                       No. of cases

Cases, No.                         0        1-5      6-10

Laboratories that performed validation
    for each score versus  no. of cases
    performed for that score, %
  IHC score 0                      3.1      28.9      28.9
  IHC score 1                     10.3      26.8      36.1
  IHC score 2                     34.0      30.9      20.6
  IHC score 3                      3.1      29.9      32.0

                                               Percentile

                                            90th     Maximum

No. of cases in validation study per         80        145
laboratory (mean, 59)

                                              No. of cases

                                           76-100     >100

Laboratories performing validation with      9.3       2.1
another IHC test, %

                                       No. of cases

Cases, No.                       11-20     21-40      >40

Laboratories that performed validation
    for each score versus  no. of cases
    performed for that score, %
  IHC score 0                     23.7      12.4       3.1
  IHC score 1                     14.4      10.3       2.1
  IHC score 2                      8.2       6.2       0.0
  IHC score 3                     27.8       6.2       1.0

Abbreviation: HER2, human epidermal growth receptor 2.

Table 10. Competency Assessment

                                                            Response,
Parameter                                                    No. (%)

Is there an active pathologist competency
    assessment process  in place? (n = 704)
  Yes                                                      639 (90.8)
  No                                                        65 (9.2)
How are you achieving ongoing competency
    assessment for HER2 scoring of practicing
    pathologists? (n 5 639) (multiple responses allowed)
  Periodic challenges using internal materials
    that are graded and documented for
    individual pathologist                                 106 (16.6)
  Periodic challenges using external material
    with documentation for individual
    pathologists                                           392 (61.3)
  Periodic review of past cases with
    documentation of agreement                             128 (20.0)
  Ongoing HER2 second-pathologist assessment
    with documentation in the report                       118 (18.5)
  Periodic conferences reviewing scoring criteria          158 (24.7)
  Comparison with the results of computer
    image analysis                                         102 (16.0)
  Other                                                     59 (9.2)

Abbreviation: HER2, human epidermal growth receptor 2.

Table 11. Correlation of Organization That Accredits/Certifies
Laboratory and Compliance With Fixation Time Included in Report and
Validation of Human Epidermal Growth Receptor 2 (HER2)
Immunohistochemistry (IHC) Concordance With Fluorescence In Situ
Hybridization (FISH)

                            Fixation Time Is Included in Report
                                     (P < .001), %
    Organization That
  Accredits/ Certifies
       Laboratory            Yes (n = 513)     No (n = 195)

CAP (n = 595)                    76.3              23.7
State (n = 41)                   65.8              34.2
Joint Commission (n = 37)        56.8              43.2
Other (n = 35)                   31.4              68.6

                            Validate HER2 IHC Stain by Studying
                            Concordance With FISH (P = .01), %
    Organization That
  Accredits/ Certifies
       Laboratory            Yes (n = 426)     No (n = 134)

CAP (n = 595)                    78.8              21.2
State (n = 41)                   60.0              40.0
Joint Commission (n = 37)        69.4              30.6
Other (n = 35)                   51.9              48.1

Abbreviation: CAP, College of American Pathologists.

Table 12. Correlation of the Laboratories That
Validated Epidermal Growth Receptor 2 (HER2)
Immunohistochemistry (IHC) Concordance With
Fluorescence In Situ Hybridization (FISH) Versus Those
That Validated With Another Laboratory's IHC Assay

                           Validation Using Another
                           Laboratory's IHC Assay

                                                  Not
                      Yes           No        Applicable
     FISH         (n = 157),    (n = 366),    (n = 152),
  Validation          No.           No.           No.

 Yes (n = 402)        88            273           41
 No (n = 142)         55             78            9
Not applicable
   (n = 131)          14             15          102
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