Current Laboratory Testing Practices for Assessment of ERBB2/HER2 in Endometrial Serous Carcinoma and Colorectal Carcinoma Open Access

A list of 14 questions was developed by the CAP Molecular Oncology Committee and included as a supplemental questionnaire (SQ) in the 2021-A HER2 Immunohistochemistry Program distributed to laboratories subscribing to this CAP proficiency testing program (mailed March 15, 2021, and due back April 6, 2021). The full questionnaire is available in the supplemental digital content at https://meridian.allenpress.com/aplm in the October 2023 table of contents.

To annotate the SQ data, we extracted respondent institution type from CAP’s demographics database. For 69 laboratories not in the database, the missing type was determined by online institution searches. “Physician office laboratory/clinic” and “nonhospital laboratories” were combined into an “other, nonhospital laboratory” group.

Data validity adjustments were applied to responses that did not follow the skip-sequence directions in the SQ. Missingness checks were conducted to address incomplete and nonrandom response patterns. Responses relating to testing practices were excluded for laboratories that indicated that they performed the test at a reference laboratory. The “other” response code was populated for laboratories that wrote text in the open comment field without choosing any specific item.

Multivariate logistic regression models were used to test for laboratory characteristics associated with HER2 testing practices. The models were fit with 2 factors: institution location and institution type. Institution location was defined as a 2-level factor that classified laboratories as domestic (United States) or international. Institution type included 4 levels: independent/commercial reference laboratory, academic hospital/medical center laboratory, nonacademic hospital/medical center laboratory, and other, nonhospital laboratory. For any model that did not meet the convergence criterion, a second model was fit with the location factor. Multiple pairwise testing for the institution type differences was adjusted with a Bonferroni correction, and the adjusted P values are reported. Analyses were performed with SAS 9.4 (SAS Institute, Cary, North Carolina). A significance level of .05 was used for the statistical testing.

The survey was completed by 1229 of 1548 laboratories that received it (79.4%). Thirty-four (2.2%) of these were excluded because of survey duplication (3) or missing responses (31), resulting in the inclusion of 1195 questionnaires (77.2%) for analysis. Responding laboratories included 998 United States facilities (83.5%) and 197 international facilities (16.5%) from 37 countries. In the total group, 506 (42.3%) were nonacademic hospitals/medical centers, 399 (33.4%) were academic, and 192 (16.1%) were independent/commercial reference laboratories.

Among the 1195 included laboratories, 287 (24.0%) reported that they performed testing for HER2 in ESC, 94 (7.9%) planned to start offering the testing in 2021, 95 (7.9%) planned to start offering it after 2021, and the remaining 719 (60.2%) had no plans to start offering it (Figure 1). Domestic laboratories were more likely to perform in-house HER2 testing for ESC compared with international laboratories (25.9% [258 of 998] versus 14.7% [29 of 197]; P = .01) (Figure 2, A). Academic laboratories were also significantly more likely to perform testing (35.6%; 142 of 399) than the other institution types: nonacademic hospital laboratories (20.2%; 102 of 506; P < .001), commercial reference laboratories (20.8%; 40 of 192; P= .006), and other, nonhospital laboratories (3.1%; 3 of 98; P < .001).

Almost half of laboratories (141 of 287; 49.1%) reported performing this test on fewer than 10 specimens in 2020, 61 (21.3%) had a volume of 10 to 20 tests, and 32 (11.1%) had a volume of more than 20 specimens (Table 1).

With regard to indications for testing, 135 of 287 laboratories (47.0%) reported routinely performing the test only at the clinician’s request, 127 (44.3%) reported performing it reflexively in all cases, and 22 (7.7%) performed it reflexively for advanced-stage cases (International Federation of Gynecology and Obstetrics stages III and IV) (Table 1). Interestingly, international laboratories were statistically more likely to perform HER2 testing only at the clinician’s request (82.8% of international laboratories [24 of 29] versus 43.0% of domestic laboratories [111 of 258], P = .001).

Laboratories were asked which type of specimen was routinely selected if more than one was available. The most frequent response was primary tumor from the hysterectomy (117 of 286; 40.9%). The next most frequent response was “no selection made; our laboratory routinely tests any available tumor material” (77; 26.9%). Other laboratories routinely tested the biopsy/curettage or metastatic tumor (Table 1).

The most common testing method was IHC with reflex to ISH for equivocal results (254 of 286; 88.8%). Twenty laboratories (7.0%) reported simultaneous IHC and ISH testing. Three laboratories performed initial ISH with or without reflex to IHC (1.0%). Of laboratories that reported performing ISH in-house, 187 of 213 (87.8%) used FISH and 20 (9.4%) used CISH (Table 2).

The most commonly reported scoring criteria was the ASCO/CAP 2018 guideline for breast carcinoma¹  (195 of 281; 69.4%). Nine laboratories reported using the 2013 version³  or the 2007 version⁴  of the guideline for breast carcinoma (total of 3.2%); 45 (16.0%) reported using the Fader et al¹⁷ 2018 clinical trial guidelines, but the survey did not ask respondents to clarify their understanding of what those guidelines were. Ten laboratories (3.6%) stated that they gave an “overall” assessment of positive versus negative result, 14 (5.0%) used the original US Food and Drug Administration scoring criteria (HercepTest package insert) for breast carcinoma, and 8 (2.9%) reported “other,” including the gastric cancer criteria⁷  (4 respondents) and an in-house guideline (Table 2).

Similar to the results for ESC, 239 of 1185 respondents (20.2%) reported performing HER2 testing in this setting. Among the remaining 946 laboratories, 100 (8.4%) planned to start testing in 2021, 85 (7.2%) planned to start after 2021, and 761 (64.2%) had no plans to start testing (Figure 1). Testing was not more common in domestic versus international laboratories, but academic hospital laboratories (25.9%; 102 of 394) were more likely to offer it than either nonacademic hospitals (90 of 501; 18.0%; P = .02), commercial reference laboratories (33 of 192; 17.2%; P = .02), or other, nonhospital sites (14 of 98; 14.3%; P = .04) (Figure 2, B).

Among laboratories performing the testing, most (111 of 239; 46.4%) reported a volume of fewer than 10 specimens in 2020, and 41 (17.2%) reported more than 20 (Table 3). Some respondents (39) did not know their laboratory’s specimen volume.

Of the 239 laboratories that reported information about clinical indications for testing, 196 (82.0%) stated that the test was performed only at the clinician’s request, 20 (8.4%) indicated that they performed it for all metastatic cases, and 8 (3.3%) performed it in all cases (Table 3).

A question about specimen type (primary tumor from colectomy, primary tumor from a biopsy, metastatic tumor, etc) revealed no strong pattern of preference (Table 3).

The most common testing method was IHC with reflex to ISH for equivocal results (195 of 237; 82.3%) (Table 4); 18 laboratories (7.6%) reported performing both tests, 5 (2.1%) performed ISH with reflex to IHC, 7 (2.9%) performed IHC only with no reflex, and 4 (1.7%) performed ISH only. Most respondents (211 of 234; 90.2%) stated that they did not require the tumor to be RAS wild-type to obtain HER2 testing, 17 (7.3%) required the tumor to be RAS wild-type only if RAS status was available, and 6 (2.6%) absolutely required known RAS wild-type status before performing HER2 testing.

Half of the laboratories (115 of 233; 49.4%) indicated that they applied the CAP/American Society for Clinical Pathology/ASCO guideline for GC⁷  when scoring colorectal cancer, 70 (30.0%) used the scoring system validated for the HERACLES trial,²⁵  and 38 (16.3%) reported using the 2018 ASCO/CAP guideline for breast cancer.¹ 

Cross-tabulation of laboratories performing testing in ESC and CRC showed that 162 of 1195 respondents (13.6%) performed both types of testing and 824 (69.0%) performed neither type, whereas some offered HER2 testing in only one or the other tumor type. Our data do not indicate the reasons for these practices.

This study provides the first systematic survey of laboratory testing practices for HER2 in 2 tumor types, ESC and CRC, since the clinical adoption of HER2-targeted testing/therapies in these entities. Findings include that a minority of laboratories are performing the testing, and most of the rest do not plan to initiate testing in 2022. We assume that all respondents have the technical capability to offer HER2 IHC, because they subscribe to CAP HER2 IHC proficiency testing. It is not possible to determine from these data if laboratories with no plans to offer testing intend to obtain it via send-out to a reference laboratory or if, instead, they do not anticipate clinician requests for such testing.

Given that a minority of respondents perform the test or plan to do so, it might be expected that testing would be concentrated in a few reference laboratories; however, most laboratories performing this testing reported a low volume (<10 cases/y). This finding has implications for dissemination of guidelines for HER2 testing, because it indicates that both large and small stakeholders must be reached.

Our survey results show that most laboratories are using IHC as the primary test for both ESC (88.8%) and CRC (82.3%), with reflex to ISH for equivocal results. Therefore, the criteria used for IHC scoring are of great relevance to test performance. Our survey was designed to determine which criteria are in current use. In drafting the survey questions and responses, we assumed that most laboratories performing HER2 testing in ESC would either use criteria previously adopted for breast cancer or attempt to implement criteria specific for ESC. Similarly, we assumed that laboratories performing HER2 testing in CRC would use either breast cancer criteria or guidelines published for other gastrointestinal sites. The survey results support this assumption, as only 4 laboratories stated that they used GC criteria for ESC, and no laboratory reported using ESC criteria for CRC.

A recently published study demonstrated reasonable interobserver agreement rates among gynecologic pathologists when clear criteria for scoring HER2 IHC are used.²⁸  However, a barrier to consistent and widespread adoption of HER2 testing for ESC and CRC in clinical practice is the lack of consensus (or, rather, lack of clear guidance) on the optimal scoring criteria.

Comparing the published guidelines used for HER2 IHC interpretation in ESC shows the potential for both overcalling and undercalling HER2 overexpression by using breast carcinoma guidelines (Table 5) compared with the criteria that were used in the phase 2 trial¹⁷  but only comprehensively articulated in a separate and subsequent publication by Buza.²³  Most laboratories (69.4%) reported using ASCO 2018 criteria, which will lead to overcalling HER2 overexpression relative to the Buza criteria because cases with 10% to 29% strong circumferential membranous staining will be scored as positive in the former but not the latter. Using breast criteria (any version) will also lead to undercalling HER2 overexpression by not accepting basolateral staining as positive, whereas this pattern is accepted in Buza et al.²⁸  It should be noted that there is limited evidence to determine an actual gold standard set of criteria because there has not, to our knowledge, been a systematic study of borderline cases that would be assigned differently under different guidelines. This will be exceedingly difficult in ESC because these patterns are rare and ESC is itself a rare entity. Even in breast cancer, uncommon and unusual patterns of HER2 ISH results are interpreted based on expert opinion in the face of limited evidence.¹ 

Further guidance is needed to address HER2 interpretation when intratumoral heterogeneity is present, which is rare in breast cancer²⁹  and CRC,²⁵  but common in ESC^28,30  and GC.^7,31  The 2018 ASCO/CAP breast criteria¹  specify that staining for a 3+ score has to be “within a homogeneous and contiguous invasive cell population.” In tumor types where HER2 heterogeneity is common, this requirement would tend to move cases into the HER2-equivocal group. It will be helpful to obtain data (ideally with a clinical endpoint) to indicate whether HER2-heterogeneous ESC can be considered positive by IHC. When cases with intrasample heterogeneity do undergo reflex ISH testing, ESC²³  and GC⁷  guidelines recommend correlating with the IHC so that the area with highest HER2 expression can be analyzed.

With regard to intersample heterogeneity in ESC, our data showed a lack of agreement among laboratories as to whether the endometrial biopsy or hysterectomy should be tested (Table 1). This issue is important because recently published data showed that only 84% of ESCs had concordant HER2 on an endometrial biopsy/curettage and paired hysterectomy.³²  The discrepancies were attributed to both heterogeneity of HER2 expression and sampling issues.

Along similar lines, more than half of the laboratories (56.6%) reported that they tested the primary tumor from either biopsy or hysterectomy specimens (pooled data from Table 1), whereas 34.9% of laboratories tested either metastatic or primary tumor without specific preference, and only 5.6% tested metastatic tumor specifically, if available. We conclude that laboratories do not have a consistent approach to addressing differences between primary and metastatic sites. Change in HER2 status between the primary tumor and metastasis has been reported for breast and gastric/gastroesophageal cancer^33–36  and has been reported in ESC as well. In one report, only 45.0% of ESCs with HER2 overexpression at the primary site (3+ by HercepTest) were also 3+ at any metastatic site, and the metastatic sites sometimes were divergent from one another.³⁷  Given the clinical applicability of HER2 testing inadvanced-stage and/or recurrent ESC,¹⁹  it would be advisable for future studies and guidelines to acknowledge spatiotemporal heterogeneity and provide clear guidance on how to address it.

Like those performing HER2 testing in ESC, laboratories performing HER2 testing in CRC reported using a variety of guidelines (Table 4). The HERACLES investigators found that CRC HER2 results depended on the IHC assay that was used, with virtually all positive specimens (by gold standard ISH) having 50% or higher positivity with the Ventana 4B5 clone, whereas more than half had 50% or lower positivity by HercepTest.²⁵  The Ventana clone was ultimately incorporated into the HERACLES diagnostic criteria. Applying HERACLES criteria to tests performed with other antibodies could result in false-negative results (Table 6).

Responses also indicated that only 2.6% of laboratories required proof of RAS wild-type status before performing HER2 testing in CRC. RAS activation is common in CRC and predicts lack of response to receptor tyrosine kinase inhibition, presumably because it signals downstream from EGFR family members.³⁸  Although National Comprehensive Cancer Network guidelines recommend that all CRC be tested for RAS mutations,³⁹  there can be challenges in integrating results that may be obtained at different times, on different test platforms, and often in different laboratories without full information interchange.

Similar to IHC, there are also significant differences in criteria for HER2 ISH among different tumor types. Most breast cases are assigned based on the HER2/CEP17 ratio when performing dual-probe ISH, but the ASCO/CAP breast guidelines have additional clauses to address chromosome 17 aneuploidy,^1,3  which is not addressed in the Buza²³  guidelines for ESC or the HERACLES guidelines for CRC.²⁵  Further, the HERACLES criteria require CRC to have 50% of cells or more with HER2/CEP17 2.0 or higher for a positive result, whereas ASCO/CAP breast criteria are indifferent to the proportion of cells with amplification, but rather consider the average ratio across scored cells. Thus, for ISH as for IHC, guidance is needed to promote uniform HER2 interpretation for a given tumor type and to validate the recommended criteria.

Strengths of the current study include that it samples a large number and wide variety of laboratories, including those that, because they already offer HER2 IHC testing for breast cancer, are likely to have the technical resources to offer it for other tumor types. The high response rate of 77.2% means that the results are likely to be generalizable. Limitations are that the survey did not reach laboratories that do not participate in CAP IHC proficiency testing for HER2 because either they do not offer the test or they perform another type of proficiency testing. We did not gather data on the specific antibody clone or immunostaining platform used for IHC or the probe set used for ISH. These parameters affect the performance of a test system and must be specified as part of any clinical assay validation.

Prompt adoption of new clinical practices as trial data become available has been improving the care of cancer patients. In the rapidly evolving field of clinical oncology, timely communication among those involved in clinical trials and the pathology community is increasingly important to ensure adequate biomarker testing support. Practical implementation of biomarker assessment based on the knowledge of the practices in the field should be considered early in clinical trial design. Moreover, the diversity of testing platforms, instrumentation, and reagents calls for efforts to harmonize biomarker testing and reporting algorithms.

Laboratories performing HER2 testing and seeking to maintain CAP accreditation are required to perform annual proficiency testing, which is monitored as part of the CAP Laboratory Accreditation Program. Specific requirements pertaining to HER2 testing are described in the CAP Anatomic Pathology Checklist and Molecular Pathology Checklist. Based on the results of this survey study, it may be advisable to implement proficiency testing programs for ESC and CRC to promote quality and standardization of clinical testing for these disease types.

To gain a better understanding of current clinical HER2 testing practices for ESC and CRC, we surveyed laboratories participating in the CAP HER2 IHC proficiency testing. The survey revealed a lack of consensus as to the criteria to be used for HER2 interpretation in these disease types. Moreover, most laboratories report using interpretive criteria that are not specific to the disease type. Stakeholders, including CAP, should consider providing clear guidance for standardization of HER2 testing and interpretation in these diseases.

The authors thank Ellen Lazarus, MD, for providing medical editing support.