Discordance of HER2 Expression and/or Amplification on Repeat Testing

This article is featured in Highlights of This Issue, p. 901

HER2, or HER2/Neu, is a member of the EGFR family and encoded by the ERBB2 (1). Overexpression and/or amplification of HER2 leads to increased downstream activation of oncogenic pathways, resulting in increased cellular proliferation and tumorigenesis (1–3). HER2 has been recognized as a compelling target for cancer therapy and HER2-targeted agents have dramatically improved outcomes for patients with HER2-driven malignancies (4–7). As the use of next-generation sequencing (NGS) has become routine in modern oncology practice, it is understood that several tumor types beyond breast and gastric cancer are also driven by HER2 and may derive benefit from HER2-directed therapeutics (8–12).

A putative challenge with utilizing HER2-targeted therapies is the accurate characterization of HER2 status, which has implications for therapeutic decision-making both in the standard-of-care and investigational settings (13–15). There is some evidence to suggest that HER2 is an evolving biomarker, as several studies have demonstrated heterogeneity of HER2 expression within a tumor, or evolution of HER2 status when tumors are assessed at different time points (16, 17). Studies have also reported changes in HER2 status between primary and metastatic lesions, and furthermore, that this effect may be associated with exposure to HER2-targeted therapies (18–22). While the HER2 testing guidelines have standardized the interpretation of HER2 testing for breast and gastric cancer, many believe conversion or evolution in HER2 status remains a challenge (23–25). Interpretation of this literature is also complicated by the use of different HER2 assays for HER2 status assessments (26). As the landscape of HER2-targeted therapies is evolving, therapeutic indications may expand to patients who were previously declared “HER2-negative”, but are 1+ or 2+ by IHC and ERBB2 non-amplified by FISH (27). Thus, accurately determining the HER2 status of a tumor is critical for decision-making to ensure that the appropriate patients who may benefit from treatment are being considered.

To address the potential of HER2 discordance between testing, we studied a cohort of patients with metastatic HER2-amplified/expressing solid tumors who were being evaluated for a phase I clinical trial with a HER2-targeted therapy zanidatamab (NCT02892123) (28). Patients underwent reassessment of HER2 status by IHC and FISH, performed on either a new biopsy or submission of archival tissue, by a central testing laboratory for the purpose of pretrial screening. We sought to assess differences in HER2 status between local and central laboratory results and to determine if any factors were associated with discordance in HER2 status.

This study was approved by the Institutional Review Board at the University of Texas MD Anderson Cancer Center. Patients were 18 years of age or older, had advanced/metastatic solid tumors who progressed on standard-of-care therapy, and had documentation of HER2 expression (1+, 2+, or 3+ by IHC) and/or HER2 amplification (FISH positive or ERBB2-amplified by NGS) on local testing. As part of a pretrial screening for an investigational agent, patients underwent a reevaluation of HER2 status by central laboratory testing using tissue from either a new image-guided biopsy or from archival tissue (Fig. 1) after written informed consent. Tissues were formalin-fixed, paraffin embedded for assessment of HER2 status by IHC and FISH. Local testing included historical reports available from the electronic medical record and central testing included reports generated from either a new, fresh tissue biopsy or archival samples which were submitted at the time of pretrial screening. Demographics, clinical, and treatment data were retrospectively abstracted from a prospectively maintained clinical genomic database at our institution.

HER2 expression in the central laboratory was assessed by IHC using the Dako HercepTest assay (Agilent, Santa Clara, CA). For breast cancers, a score of 3+ was defined as a strong complete membrane staining observed in >10% of tumor cells, a score of 2+ was defined as a weak to moderate complete membrane staining is observed in >10% of tumor cells, a score of 1+ was defined as a faint/barely perceptible membrane staining detected in >10% of tumor cells, and a score of 0 was defined as no staining observed, or membrane staining is observed in <10% of the tumor cells (23). For gastric and other non-breast cancers, a score of 3+ was defined as tumor cell cluster with a strong complete, basolateral or lateral membranous reactivity irrespective of percentage of tumor cells stained, a score of 2+ was defined as a tumor cell cluster with a weak to moderate complete, basolateral or lateral membranous reactivity irrespective of percentage of tumor cells stained, a score of 1+ was defined as a tumor cell cluster with a faint/barely perceptible membranous reactivity irrespective of percentage of tumor cells stained, and a score of 0 was defined as no reactivity or no membranous reactivity in any tumor cell (25, 29, 30).

Amplification of ERBB2 was assessed by FISH using PathVysion HER-2 DNA Probe Kit (Abbott Laboratories, Chicago, IL). A positive FISH result for amplification was defined as FISH ratio [HER2 signal to centromere 17 (CEP17) signal] ≥ 2.0 or HER2 copy number ≥ 6.0 signals/cell, and a negative result was defined as FISH ratio < 2.0 or HER2 copy number < 4.0 signals/cell (23, 30). Tissue samples underwent review at an independent central testing laboratory where the assays were performed and interpreted independently by a pathologist (M.F. Press) without access to outside information.

For comparison of HER2 status between local and central testing, patients were stratified on the basis of local HER2 status and classified as: (i) HER2 overexpressing (3+ IHC) and/or HER2-amplified (FISH positive, ERBB2 amplification on NGS) or (ii) HER2 low-expressing (1/2+ IHC) and non-amplified (FISH negative or no ERBB2 amplification on NGS). Patients who were HER2 overexpressing and/or amplified on local testing were then reviewed to determine if HER2 status was discordant between local and central testing results. Discordant cases were defined as patients who were evaluated as HER2 overexpressing and/or HER2-amplified on local testing who were not overexpressing and not amplified on central HER2 assessment (IHC 0, 1+, 2+, and FISH negative).

Statistical analyses were performed using SPSS Statistics Version 24 (IBM, Armonk, NY), and figures were generated using GraphPad Prism version 9 (GraphPad, San Diego, CA) or JSFiddle Console for Sankey Diagram visualization (https://jsfiddle.net/gh/get/library/pure/highcharts/highcharts/tree/master/samples/highcharts/demo/sankey-diagram).

The relevant data to the study are included in the article and the datasets used for this study are available from the corresponding author upon reasonable request.

We first assessed the demographics and clinical characteristics of our cohort (Table 1). A total of 70 patients with HER2 expression (by IHC) and/or amplification (by FISH/NGS) on local testing underwent HER2 reassessment in the central laboratory, which was performed on either archival tissue sample [N = 13 (18.6%)] or on a new image-guided biopsy [N = 57 (81.4%)]. Patients were predominately white or Caucasian (64.3%), with a similar distribution of black (8.6%), Asian (12.9%), and Hispanic (11.4%) patients. Median age was 60 (range 24–75) years and there were 37 (52.9%) females. A broad range of cancers were represented, including 17 (24.3%) gastric/gastroesophageal junction (GEJ), 14 (20.0%) breast, 9 (12.9%) colorectal, 8 (11.4%) esophageal, and 7 (10.0%) biliary tract cancers, among others. For patients with archival tissues submitted for central testing, local testing was performed on 9 (69.2%) primary tumors and 4 (30.8%) from metastatic sites. For patients who underwent a new biopsy for central testing, local testing was performed predominantly on primary tumors (66.7%) followed by metastatic (29.8%) and liquid (3.5%) biopsies. Most samples analyzed in the central laboratory were from biopsies of metastatic sites (96.5%).

Baseline HER2 status was evaluated by reviewing reports of local IHC, FISH, and NGS that had already been done as standard of care. On local HER2 IHC, there were 30 (42.9%) patients who were IHC 3+, 15 (21.4%) who were IHC 2+, 1 (1.4%) patient who was IHC 1+, 2 (2.9%) who were IHC 0, and 22 (31.4%) patients who did not have IHC performed. For patients who had local HER2 FISH, there were 23 (32.9%) amplified, 2 (2.9%) equivocal, 7 (10.0%) non-amplified, and 38 (54.3%) who did not have FISH performed. In addition, there were 24 (34.3%) patients with ERBB2 amplification on NGS and 46 (65.7%) patients without local NGS results. On central HER2 IHC, there were 36 (51.4%) patients who were 3+, 15 (21.4%) who were 2+, 14 (20.0%) patients who were 1+, and 5 (7.1%%) who had 0 HER2 expression. In the central laboratory the carcinomas from 48 (68.6%) patients were ERBB2 amplified, while 21 (30.0%) were ERBB2 non-amplified, and 1 (1.4%) for whom the HER2 FISH assay was not successful (Supplementary Table S1).

Among 30 patients whose cancers were IHC 3+ by in a local laboratory, 25 (83.3%) had cancers that were ERBB2 amplified and 5 (16.7%) had cancers that were ERBB2 non-amplified by FISH in the central laboratory. 14 of 23 cancers with ERBB2 amplification on local FISH also demonstrated HER2 protein overexpression by IHC 3+ (52.2%) or equivocal (IHC 2+) expression (8.7%), while 9 had low HER2 protein immunostaining by IHC (IHC 1+, IHC 0; 39.1%%) on central testing. 21 (70.0%) were 3+, 5 (16.7%) were 2+, 2 (6.7%) were 1+, and 2 (6.7%) had 0 HER2 expression on central IHC. In 15 patients who were 2+ on local IHC, 5 (33.3%) were amplified on central FISH, and 2 (13.3%) were 3+, 5 (33.3%) were 2+, 7 (46.7%) were 1+, and 1 (6.7%) had 0 HER2 expression on central IHC. One patient who was 1+ on local IHC was 2+ and amplified on central testing, and neither of the 2 patients who were 0 on local IHC were amplified on central testing. Distribution of local versus central HER2 IHC and FISH results for patients had both local and central tests can be seen in Fig. 2A and B and flow diagram representing the directionality of HER2 IHC changes can be seen in Fig. 2C–E.

In 24 patients with ERBB2 amplification on local NGS, 17 (70.8%) were 3+, 4 (16.7%) were 2+, 2 (8.3%) were 1+, and 1 was 0 on central IHC, and 22 (91.7%) were amplified by FISH (2 patients were non-amplified). Patients had local NGS performed on several different panels, including 13 (54.2%) by Oncomine, 5 (20.8%) by FoundationOne (or CDx), 4 (16.7%) by AmpliSeq, 1 by Caris MI Profile, and 1 by an internal liquid biopsy panel.

Results were then compared in patients who were HER2 overexpressing (3+ by IHC) and/or amplified (FISH/NGS) on local testing who underwent central testing (Table 2). In the 8 patients who had central HER2 assessment from the same archival block used for local testing, none of patients were characterized as discordant.

Discordance was identified in 16 of 52 (30.8%) of patients who underwent a new biopsy for central testing (Fig. 3A). Of these, discordance in HER2 status was observed in 5 of 12 (41.7%) gastric/GEJ, 2 of 9 (22.2%) breast, and 2 of 7 (28.6%) colorectal tumors (Fig. 3B). The median days between tests was 614 (range 9–4,631) for patients with concordant HER2 results and 521 (range 249–3,130) for patients with discordant results (Fig. 3C). Discordant HER2 status was observed in 10 of 30 (33.3%) patients who received intervening HER2 therapy and in 6 of 22 (27.3%) patients who did not (Fig. 3D). Of the 16 patients with HER2 discordance, 8 were potentially eligible for trial enrollment based on local testing but ineligible based on central HER2 testing. Of note, there was also 1 patient identified with breast cancer who was IHC 2+/FISH- on local testing but IHC 2+/FISH+ on repeat central testing on a hepatic metastasis; this patient was considered ineligible for trial enrollment as patient had not gotten standard of care HER2-targeted therapy.

To better understand etiology of discordance in patients who had fresh biopsies for central testing, the characteristics of the cases with HER2 discordance are listed in Supplementary Table S2. Of these 16 cases, there were 12 patients who had local IHC performed and 5 with detailed reports which were available for review at our institution. Three of these cases were scored as 3+ with strong HER2 staining on pathology reports. One discordant case which was scored 2+ described a cancer cell cluster with a weak to moderate complete, basolateral, or lateral membranous reactivity irrespective of percentage of cancer cells positive, and another scored as 2+ reported 15% HER2 staining.

One patient who had a discordance had colon adenocarcinoma that was 3+ by IHC, FISH+, and ERBB2 amplified on local testing. However, the patient's tumor was found the be HER2 0+ on IHC and non-amplified on FISH on central testing. This led to central reassessment of the archival sample that was HER2-positive on local testing, and this analysis demonstrated intratumoral heterogeneity in the primary tumor, with HER2 amplification by FISH observed in superficial portion but no HER2 amplification observed in the deep invasive portion. (Fig. 4A–F).

Assessment of HER2 status is paramount for clinical decision-making in both standard-of-care and investigational settings. Here, we present a study of patients with metastatic solid tumors with HER2-expression/amplification on local testing as part of standard of care, who then underwent central reassessment of HER2 status by a subsequent image-guided biopsy or reassessment of archival samples. In this cohort of 12 different HER2-expressing cancers, discordance was found in nearly one-third of patients who were HER2 overexpressing and/or amplified on local testing who had a new biopsy performed for central HER2 reevaluation. There were 30% of patients who were HER2 3+ on local testing who were not 3+ on central reevaluation, and over half of patients with HER2 2+ expression on local IHC were 1+ or 0 expression on central review. In addition, HER2 discordance was not limited to those with intervening HER2-targeted therapy. These findings suggest that discordance of HER2 status is common and biomarker reassessment may help to inform therapeutic selection.

As the population of patients being considered for HER2-targeted therapies is growing, accurate characterization of HER2 status is essential for treatment decision-making. Accurate assessment is critical not only to help limit unnecessary treatments in HER2-negative patients that may not derive benefit from anti-HER2 therapy, but also to ensure all patients that may benefit from HER2-targeted therapy are offered therapy (17). For example, a patient in our study with IHC 2+/FISH− breast cancer on local testing but was IHC 2+/FISH+ on central testing of a fresh biopsy, which made her eligible for already approved HER2-targeted therapies. Likewise, this is of utmost importance in the evolving landscape of HER2-targeted therapies as “HER2 low” (IHC 1+/2+, FISH−) patients may become eligible for HER2-targeted therapies in the future (27, 31, 32).

Several prior studies have evaluated the evolution of HER2 status between primary and recurrent/metastatic samples, and have reported HER2 discordance between samples tested (20, 21, 33, 34). Loss of HER2 status has been observed following anti-HER2 therapy, suggesting that modulation of HER2 expression may be a mechanism of acquired resistance to HER2-targeted therapies (18, 19, 35, 36). A study by Griggs and colleagues investigated the discordance of HER2 status in stage I–III breast cancers between original and central laboratory testing, and found that HER2 discordance by IHC was 26%, but substantially lower (6%) when incorporating FISH results for patients who were 2+ by IHC (17). Notably, another study by Press and colleagues identified that of 1995 patients screened for enrollment to trial of lapatinib (N = 322 patients) for patients with HER2-amplified upper gastrointestinal adenocarcinomas, there were 29 patients (1.5%) with HER2 genomic heterogeneity (37).

This is one of the first studies to report on HER2 reassessment in such a diverse population of HER2-expressing cancers, which is notable as tumor agnostic basket trials have demonstrated activity of anti-HER2 therapy in several other malignancies in addition to breast and gastric cancers (4, 9, 10, 38–40). We found that HER2 discordance was observed in not only breast and gastric cancers, but also several other HER2-expressing tumor types. Interestingly, in 1 patient with colorectal cancer, we identified intratumoral heterogeneity, with HER2 expression/amplification observed in the superficial portions of the tumor but not in the deeper invasive portions. Several studies of HER2-positive gastrointestinal tumors have found discordance between primary and metastatic (21, 41). A study by Janjigian et. al. found that over 30% of patients with esophagogastric cancers had HER2 loss post-trastuzumab, suggesting that repeat HER2 evaluation may be warranted in this setting (42). As the present study was conducted in the context of pretrial screening, HER2 reassessment did impact patient selection, as 8 patients who had a new biopsy for repeat HER2 testing were not enrolled on the basis of their central HER2 status.

NGS is commonly used in the management of advanced/solid tumors. There have been genomic-biomarker-linked drug approvals by the FDA for over a dozen cancer types (43). Further, tumor-agnostic FDA approvals for any cancer with high tumor mutation burden, mismatch repair deficiency, or NTRK fusions provide a rationale for genomic testing for all advanced/metastatic solid tumors (43). Broad utilization of NGS creates an opportunity to detect ERBB2 amplification in diseases beyond those with current approval for HER2-targeted therapy. However, there have been questions about actionability of ERRB2 amplification detected by NGS, with concern that these patients may not necessarily have HER2 protein overexpression. In our series, in 24 patients with ERBB2 amplification on local NGS, almost all (91.7%) were ERBB2 amplified by FISH on central analysis. These findings suggest that HER2 discordances in ERBB2 amplified patients are relatively infrequent.

HER2 discordance can arise from several reasons, including evolution of the same tumor as determined by samples taken temporally at different time points, differences between primary and metastasis or in between metastasis, or due to differences due to age and processing of sample. HER2 discordance can also be seen due to assay used, such as the specific HER2 antibody or HER2 scoring used; such discordances can be best assessed by evaluating the discordance between test results of the same sample in different labs. In our study we sought to determine how often a patient with local HER2 results done as standard of care would have discordant results. either if they have a new biopsy, or re-testing of an existing tumor sample. In the small series where the same sample was retested, we observed no discordance, suggesting that tumor heterogeneity may be an important contributor to the discordance we observed. Indeed, 11 of the 16 discordant cases had a primary tumor tested initially, with a metastasis biopsied subsequently. However, as discordance of HER2 status is multifactorial, the cause of discordance cannot be definitively determined in a prospective clinical trial designed to screen patients rather than to formally study tumor evolution and interlab test concordance.

There are several limitations to the present study. As local HER2 results were assessed retrospectively, factors such as HER2 antibody used and interpretation of results were not able to be standardized. While this limitation is notable due to differences in HER2 IHC methodologies and staining intensity, this lack of standardization reflects a real-world clinical scenario. Although we did not see discordance when the same archival samples was assessed locally and centrally, this data was limited to only 8 patients. Our study only focuses on patients who were HER2-expressing or amplified by local testing. Therefore it does not assess the frequency of patients who may have been HER2-negative by local testing and may become HER2+ on repeat testing. Lastly, these results should be taken in the context of the population studied, which is a cohort of patients with metastatic cancers at a single institution, and additional research may be warranted to determine if these findings can be generalizable to a larger population.

Discordance in HER2 status on repeat biopsy is common in patients with tumors previously identified as HER2-expressing and/or amplified. In addition, HER2 discordance is not limited to patients with prior HER2-targeted therapy and could have therapeutic implications. Biomarker reassessment with a new biopsy may have value when considering HER2-targeted therapies. However, while there may be benefit derived from repeat HER2 testing with a new biopsy, this must be balanced with the risk of an additional invasive procedure and the potential delay in treatment initiation. Further study is warranted to determine if non-invasive methods (such as liquid biopsy or molecular imaging) have a role in reassessment of HER2 status.

D.D. Karp reports grants from NCI during the conduct of the study. M.F. Press reports personal fees from Biocartis, Cepheid, Zymeworks, Eli Lilly, AstraZeneca, Merck, Novartis; and other support from TORL Biotherapeutics outside the submitted work. S.A. Piha-Paul reports other support from AbbVie, ABM Therapeutics, Acepodia, Alkermes, Aminex Therapeutics, Amphivena Therapeutics, BioMarin Pharmaceutical, Boehringer Ingelheim, Bristol-Myers Squib, Cerulean Pharma, Chugai Pharmaceutical Co. Ltd., Curis, Cyclacel Pharmaceuticals, Daiichi Sankyo, Eli Lilly, ENB Therapeutics, Epigenetix Inc., Five Prime Therapeutics, F-Star Beta Limited, F-Star Therapeutics, Gene Quantum, Genmab A/S, Gilead Sciences, GlaxoSmithKline, Helix BioPharma Corp, Hengrui Pharmaceuticals Co. Ltd., HiberCell, Immorna Biotherapeutics, Immunomedics, Incyte Corp, Jacobio Pharmaceuticals Co. Ltd., Jiangsu Simcere Pharmaceutical Co. Ltd., Lytix Biopharma AS, Medimmune, Medivation, Merck Sharp and Dohme Corp, Nectin Therapeutics Ltd., Novartis Pharmaceuticals, Pieris Pharmaceuticals, Pfizer, Phanes Therapeutics, Principia Biopharma, Puma Biotechnology, Purinomia Biotech, Rapt Therapeutics, Replimune, Seattle Genetics, Silverback Therapeutics, Synlogic Therapeutics, Taiho Oncology, Tesaro, TransThera Bio, ZielBio; and grants from NCI/NIH; P30CA016672 – Core Grant (CCSG Shared Resources) outside the submitted work; and has worked as a consultant for CRC Oncology. E.E. Dumbrava reports grants from Bayer HealthCare Pharmaceuticals Inc, Immunocore LTD, Amgen, Aileron Therapeutics, Compugen Ltd., TRACON Pharmaceuticals, Unum Therapeutics, Gilead Immunomedics, BOLT Therapeutics, Aprea Therapeutics, Bellicum Pharmaceuticals, PMV Pharma, Triumvira, Seagen, Mereo BioPharma 5, Sanofi, Rain Oncology, Astex Therapeutics, Sotio, Poseida, Mersana Therapeutics, Genentech, Boehringer Ingelheim; other support from BOLT Therapeutics, Mersana Therapeutics, Orum Therapeutics, Summit Therapeutics, PMV Pharma, ASCO, LFSA Association, Rain Oncology, Banner MD Anderson Cancer Center; and other support from Catamaran Bio during the conduct of the study. F. Meric-Bernstam reports personal fees from Zymeworks during the conduct of the study; personal fees from AbbVie, Aduro BioTech, Alkermes, AstraZeneca, Daiichi Sankyo Co. Ltd., Calibr (a division of Scripps Research), DebioPharm, Ecor1 Capital, eFFECTOR Therapeutics, F. Hoffman-La Roche Ltd., GT Apeiron, Genentech, Harbinger Health, IBM Watson, Infinity Pharmaceuticals, Jackson Laboratory, Kolon Life Science, Lengo Therapeutics, Menarini Group, OrigiMed, PACT Pharma, Parexel International, Pfizer, Protai Bio Ltd., Samsung Bioepis, Seattle Genetics, Tallac Therapeutics, Tyra Biosciences, Xencor, Black Diamond, Biovica, Eisai, FogPharma, Immunomedics, Inflection Biosciences, Karyopharm Therapeutics, Loxo Oncology, Mersana Therapeutics, OnCusp Therapeutics, Puma Biotechnology, Seattle Genetics, Sanofi, Silverback Therapeutics, Spectrum Pharmaceuticals, Theratechnologies, Zentalis; grants from Aileron Therapeutics, AstraZeneca, Bayer HealthCare Pharmaceuticals, Calithera Biosciences, Curis, CytomX Therapeutics, Daiichi Sankyo Co. Ltd., Debiopharm International, eFFECTOR Therapeutics, Genentech, Guardant Health, Klus Pharma, Takeda Pharmaceutical, Novartis, Puma Biotechnology, Taiho Pharmaceutical Co.; and other support from European Organisation for Research and Treatment of Cancer (EORTC), European Society for Medical Oncology (ESMO), Cholangiocarcinoma Foundation outside the submitted work. No disclosures were reported by the other authors.

T.P. DiPeri: Data curation, formal analysis, investigation, visualization, writing–original draft, writing–review and editing. K. Kong: Resources, data curation, writing–review and editing. K. Varadarajan: Data curation, visualization, writing–review and editing. D.D. Karp: Resources, funding acquisition, writing–review and editing. J.A. Ajani: Resources, investigation, writing–review and editing. S. Pant: Resources, investigation, writing–review and editing. M.F. Press: Formal analysis, investigation, methodology, writing–original draft, writing–review and editing. S.A. Piha-Paul: Resources, investigation, writing–review and editing. E.E. Dumbrava: Resources, methodology, writing–review and editing. F. Meric-Bernstam: Conceptualization, resources, data curation, software, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing.

The authors would like to acknowledge Ivonne Villalobos, Angela Santiago, and Roberta Guzman who provided the administrative organization and technical support for this study in the USC central laboratory.

This work was supported by the following: NIH Training of Academic Surgical Oncologists (Award #: 5T32CA009599–32) (T. Diperi; PI: F. Meric-Bernstam), Center for Clinical and Translational Science (Award #: 5UL1TR003167–03; to F. Meric-Bernstam), and the MD Anderson Cancer Center support grant (Award #: P30 CA016672; to F. Meric-Bernstam), Tower Cancer Research Foundation (006886–0001; to M.F. Press), Breast Cancer Research Foundation (BCRF-21–132; to M.F. Press), and Dr. Miriam & Sheldon G. Adelson Medical Research Foundation (to M.F. Press). The ZW25–101 clinical trial, including central laboratory confirmation of HER2 status, was supported by Zymeworks.

The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.