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The role of HER2 as a therapeutic biomarker in gynaecological malignancy: potential for use beyond uterine serous carcinoma

Published:December 08, 2022DOI:https://doi.org/10.1016/j.pathol.2022.11.004

      Summary

      Human epidermal growth factor receptor 2 (HER2) is a prognostic biomarker and therapeutic target in carcinomas of the breast, stomach and colon. In 2018, clinical trial data confirmed the prognostic and predictive role of HER2 in uterine serous carcinoma, with a demonstrated survival benefit from combined chemotherapy and anti-HER2 targeted therapy in patients with advanced or recurrent disease. Approximately one-third of uterine serous carcinomas demonstrate HER2 protein overexpression and/or gene amplification and HER2 immunohistochemistry, supplemented by in situ hybridisation in equivocal cases, is fast becoming a reflex ancillary test at time of diagnosis. The potential role of HER2 in gynaecological tumours other than uterine serous carcinoma is yet to be firmly established. With the advent of personalised medicine, routine tumour sequencing and pursuit of targeted therapies, this is a field currently under active investigation. Emerging data suggest triaging endometrial carcinomas for HER2 analysis based on molecular classification may be superior to histotype-based testing, with copy-number high/p53 mutant tumours enriched for HER2 overexpression or amplification. Accordingly, many carcinosarcomas and a subset of clear cell and high-grade endometrioid carcinomas may be eligible for HER2 targeted therapy, although any clinical benefit in this context is currently undefined. For ovarian carcinomas, combined data support the role of HER2 as a prognostic biomarker, however its use as a therapeutic target is yet to be elucidated through clinical trials. In the cervix, reported rates of HER2 overexpression vary and are generally low, and currently there is insufficient evidence to justify routine HER2 testing in this context. Limited data suggest HER2 holds promise as a prognostic and predictive biomarker in vulvar Paget disease. Future clinical trials, with pathologist input to develop and refine site-specific scoring criteria, are required to establish what role HER2 might play more broadly in gynaecological cancer care.

      Key words

      Introduction

      In an era of personalised medicine, molecular sequencing of rare or recurrent tumours is commonly performed in pursuit of therapeutic options other than conventional chemoradiation. The aim is to identify targetable genetic alterations, often in tumours of different histotype or location to those for which the therapeutic agent was originally developed. Some genetic alterations have reliable surrogate immunohistochemical markers, which may be performed more efficiently and economically than sequencing, offering rapid prognostic and theranostic information. An example is immunohistochemistry (IHC) for mismatch repair (MMR) proteins MLH1, PMS2, MSH2 and MSH6, with loss of MMR staining in tumour tissue a reliable surrogate for microsatellite instability and a predictor of response to immunotherapy.
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      Efficacy of pembrolizumab in patients with noncolorectal high microsatellite instability/mismatch repair-deficient cancer: results from the Phase II KEYNOTE-158 Study.
      Another is immunohistochemical overexpression of human epidermal growth-factor receptor 2 (HER2), a surrogate for HER2 (also known as ERBB2) gene amplification, a biomarker with prognostic and predictive value in carcinomas of the breast, stomach/gastro-oesophageal junction (GOJ) and colon. Recently, the utility of HER2 has also been established in uterine serous carcinoma, triggering interest in its potential application to other female genital tumours.
      The HER2 oncogene was first implicated in the pathogenesis of breast cancer in 1987,
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      Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.
      and in the decades since, accumulated literature has established the link between HER2 gene amplification and the pathogenesis of a range of malignancies, particularly breast, gastric/GOJ and colorectal cancer. HER2 is a transmembrane tyrosine kinase receptor encoded by the ERBB2 gene located on chromosome 17q12. It is one of a family of proteins [along with HER1/ERBB1 (also known as epidermal growth factor receptor/EGFR), HER3/ERBB3 and HER4/ERBB4] with important functions as mediators of cellular proliferation, tumourigenesis and apoptosis.
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      EGFR and HER2 exon 20 insertions in solid tumours: from biology to treatment.
      Ligand binding to the extracellular domain of HER1/EGFR or HER3/4 proteins initiates protein dimerisation, transphosphorylation of their intracellular domains and docking of numerous intracellular signalling molecules at these phosphorylated tyrosine residues. This triggers activation of myriad downstream intracellular and transmembrane signalling pathways.
      • Moasser M.M.
      The oncogene HER2: its signaling and transforming functions and its role in human cancer pathogenesis.
      HER2 has no known ligand and its signalling function is initiated by heterodimerisation with other HER family proteins, with signalling activity strongest when HER family proteins form heterodimers rather than homodimers;
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      The oncogene HER2: its signaling and transforming functions and its role in human cancer pathogenesis.
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      Activating HER2 mutations as emerging targets in multiple solid cancers.
      of all HER family proteins, HER2 has the most catalytic kinase activity and HER2-containing heterodimers the strongest signalling function.
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      The oncogene HER2: its signaling and transforming functions and its role in human cancer pathogenesis.
      HER2 manifests its tumourigenic potential via overexpression of the wild-type form, thought to be an early event and result of gene amplification or transcriptional deregulation.
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      The oncogene HER2: its signaling and transforming functions and its role in human cancer pathogenesis.
      More recently, activating somatic mutations in the HER2 kinase domain have also been implicated in tumourigenesis,
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      Activating HER2 mutations as emerging targets in multiple solid cancers.
      ,
      • Oh D.-Y.
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      HER2-targeted therapies - a role beyond breast cancer.
      and although genomic profiling has demonstrated recurrent somatic mutations in ERBB2 in human cancers, most of these are not associated with HER2 overexpression,
      • Connell C.M.
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      Activating HER2 mutations as emerging targets in multiple solid cancers.
      ,
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      Lung cancer: intragenic ERBB2 kinase mutations in tumours.
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      Abstract LB-103: landscape of somatic ERBB2 mutations: findings from AACR GENIE and comparison to ongoing ERBB2 mutant basket study.
      and occur at low frequency across all cancers, with no single mutant allele predominating.
      • Connell C.M.
      • Doherty G.J.
      Activating HER2 mutations as emerging targets in multiple solid cancers.
      ,
      • Oh D.-Y.
      • Bang Y.-J.
      HER2-targeted therapies - a role beyond breast cancer.
      The most frequent variants include S210F, HER2 exon 20 insertions and V777L missense mutations,
      • Oh D.-Y.
      • Bang Y.-J.
      HER2-targeted therapies - a role beyond breast cancer.
      and while these mutations may result in deregulation of HER2 signalling, they do not necessarily confer the same sensitivity to HER2 targeted therapies as strong HER2 overexpression or amplification,
      • Friedlaender A.
      • Subbiah V.
      • Russo A.
      • et al.
      EGFR and HER2 exon 20 insertions in solid tumours: from biology to treatment.
      • Connell C.M.
      • Doherty G.J.
      Activating HER2 mutations as emerging targets in multiple solid cancers.
      • Oh D.-Y.
      • Bang Y.-J.
      HER2-targeted therapies - a role beyond breast cancer.
      Tumours exhibiting overexpression of HER2 by IHC or gene amplification via fluorescent in situ hybridisation (FISH) are amenable to targeted therapy with monoclonal anti-HER2 antibodies such as trastuzumab, with reported improved patient survival.
      • Dawood S.
      • Broglio K.
      • Buzdar A.U.
      • Hortobagyi G.N.
      • Giordano S.H.
      Prognosis of women with metastatic breast cancer by HER2 status and trastuzumab treatment: an institutional-based review.
      • Romond E.H.
      • Perez E.A.
      • Bryant J.
      • et al.
      Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer.
      • Bang Y.-J.
      • Van Cutsem E.
      • Feyereislova A.
      • et al.
      Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial.
      This advance in cancer treatment has prompted enquiry into the potential role of this biomarker and application of HER2-targeted therapies in gynaecological malignancies. In the female genital tract, recent data indicate HER2 is overexpressed in roughly one-third of uterine serous carcinomas (USC),
      • Buza N.
      • English D.P.
      • Santin A.D.
      • Hui P.
      Toward standard HER2 testing of endometrial serous carcinoma: 4-year experience at a large academic center and recommendations for clinical practice.
      and in well-resourced settings it is increasingly standard that HER2 IHC is performed on all USC at time of diagnosis or tumour recurrence. As data emerge documenting HER2 overexpression and/or amplification in tumours other than USC, there is growing interest in applying HER2 analysis to other gynaecological malignancies. Proponents of this approach see it as analogous to MMR IHC, which is commonly performed universally on all carcinomas of the endometrium and ovary, not only to screen for Lynch syndrome, but also to explore therapeutic options. Currently the potential role of HER2 in female genital tumours beyond USC has not been established, although it is under investigation. Herein, we review the current literature regarding HER2 status, and potential therapeutic implications, in female genital tumours other than USC. We also provide a historical overview of the emergence of this biomarker in gynaecological pathology, its application in USC, and highlight important technical considerations when applying HER2 assays in routine practice.

      HER2 in uterine serous carcinoma

      With elucidation of the role of HER2 gene amplification in carcinogenesis, in 1998 the recombinant humanised monoclonal anti-HER2 antibody trastuzumab was approved by the United States Food and Drug Administration (FDA) for use as a targeted therapy for HER2-positive metastatic breast cancer.
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      • Leo C.
      • Szucs T.D.
      Breast cancer drug approvals by the US FDA from 1949 to 2018.
      Following release of clinical trial data in 2005,
      • Romond E.H.
      • Perez E.A.
      • Bryant J.
      • et al.
      Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer.
      ,
      • Piccart-Gebhart M.J.
      • Procter M.
      • Leyland-Jones B.
      • et al.
      Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer.
      the drug was approved by the European Commission, France, and some Canadian provinces, and its use subsidised by the Australian government from 2006.
      • Mackenzie R.
      • Chapman S.
      • Salkeld G.
      • Holding S.
      Media influence on Herceptin subsidization in Australia: application of the rule of rescue?.
      In 2010, the FDA approved its use for gastric cancer
      • Bang Y.-J.
      • Van Cutsem E.
      • Feyereislova A.
      • et al.
      Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial.
      and more recent trials show promise for use in treatment of colorectal cancer.
      • Siena S.
      • Di Bartolomeo M.
      • Raghav K.
      • et al.
      Trastuzumab deruxtecan (DS-8201) in patients with HER2-expressing metastatic colorectal cancer (DESTINY-CRC01): a multicentre, open-label, phase 2 trial.
      ,
      • Sartore-Bianchi A.
      • Trusolino L.
      • Martino C.
      • et al.
      Dual-targeted therapy with trastuzumab and lapatinib in treatment-refractory, KRAS codon 12/13 wild-type, HER2-positive metastatic colorectal cancer (HERACLES): a proof-of-concept, multicentre, open-label, phase 2 trial.
      HER2 protein expression and gene amplification in endometrial cancers has been studied since the late 1990s, with early reports drawing varied conclusions due to limitations in study methodology such as small sample size, inclusion of tumours of varied histotype and use of different scoring protocols measuring HER2 status. Inclusion of patients from different racial backgrounds also impacted upon data, with increased rates of expression in African American versus Caucasian patients; for example, one study examined 27 USC and reported 70% (7/10) of African American women exhibited HER2 IHC overexpression versus 24% (4/17) in Caucasian patients.
      • Santin A.D.
      • Bellone S.
      • Siegel E.R.
      • et al.
      Racial differences in the overexpression of epidermal growth factor type II receptor (HER2/neu): a major prognostic indicator in uterine serous papillary cancer.
      In early studies, HER2 amplification was found to be more common in high-grade tumours, with a trend to overexpression in USC
      • Grushko T.A.
      • Filiaci V.L.
      • Mundt A.J.
      • et al.
      An exploratory analysis of HER-2 amplification and overexpression in advanced endometrial carcinoma: a Gynecologic Oncology Group study.
      • Halperin R.
      • Zehavi S.
      • Habler L.
      • Hadas E.
      • Bukovsky I.
      • Schneider D.
      Comparative immunohistochemical study of endometrioid and serous papillary carcinoma of endometrium.
      • Fleming G.F.
      • Sill M.W.
      • Darcy K.M.
      • et al.
      Phase II trial of trastuzumab in women with advanced or recurrent, HER2-positive endometrial carcinoma: a Gynecologic Oncology Group study.
      but with mixed results. Reported rates of IHC overexpression ranged between 14% and 80%, with HER2 amplification by FISH ranging from 21% to 47%.
      • Buza N.
      • Roque D.M.
      • Santin A.D.
      HER2/neu in endometrial cancer: a promising therapeutic target with diagnostic challenges.
      ,
      • Xu M.
      • Schwartz P.
      • Rutherford T.
      • et al.
      HER-2/neu receptor gene status in endometrial carcinomas: a tissue microarray study.
      In response to these varied data, a comprehensive tissue microarray study assessed HER2 status in 75 pure USC, together with an additional 13 cases (independent of the microarray) from African American patients, using standard FDA-approved IHC (HercepTest) and FISH (PathVysion) test kits, with real time reverse transcriptase-polymerase chain reaction applied to cases with discordant IHC and FISH results. Of the combined 88 pure USC, 21.6% had elevated HER2 protein expression and/or gene amplification; 50% of the cohort of African American patients exhibited overexpression.
      • Xu M.
      • Schwartz P.
      • Rutherford T.
      • et al.
      HER-2/neu receptor gene status in endometrial carcinomas: a tissue microarray study.
      Subsequently, in 2013, the largest study to comprehensively evaluate HER2 expression in USC (discussed below) found 35% of 108 cases exhibited HER2 overexpression and/or gene amplification, also reporting heterogeneous protein expression in 53% of HER2-positive tumours,
      • Buza N.
      • English D.P.
      • Santin A.D.
      • Hui P.
      Toward standard HER2 testing of endometrial serous carcinoma: 4-year experience at a large academic center and recommendations for clinical practice.
      a phenomenon which likely produced an artificially lower rate of expression in the previous microarray study.
      Several case reports in the early 2000s documented partial or complete therapeutic response using trastuzumab in USC patients
      • Santin A.D.
      • Bellone S.
      • Roman J.J.
      • McKenney J.K.
      • Pecorelli S.
      Trastuzumab treatment in patients with advanced or recurrent endometrial carcinoma overexpressing HER2/neu.
      • Villella J.A.
      • Cohen S.
      • Smith D.H.
      • Hibshoosh H.
      • Hershman D.
      HER-2/neu overexpression in uterine papillary serous cancers and its possible therapeutic implications.
      • Jewell E.
      • Secord A.A.
      • Brotherton T.
      • Berchuck A.
      Use of trastuzumab in the treatment of metastatic endometrial cancer.
      and a further study demonstrated high in vitro sensitivity of USC cell lines to trastuzumab-mediated antibody-dependent cellular cytotoxicity and inhibition of proliferation.
      • Santin A.D.
      • Bellone S.
      • Gokden M.
      • et al.
      Overexpression of HER-2/neu in uterine serous papillary cancer.
      In 2010, the first Phase II Gynecologic Oncology Group clinical trial using single agent trastuzumab in advanced or recurrent HER2-positive endometrial carcinomas (of mixed histotypes) failed to demonstrate any therapeutic benefit
      • Fleming G.F.
      • Sill M.W.
      • Darcy K.M.
      • et al.
      Phase II trial of trastuzumab in women with advanced or recurrent, HER2-positive endometrial carcinoma: a Gynecologic Oncology Group study.
      and it was not until 2018 that the value of trastuzumab in this context was confirmed by a large multi-institutional trial. This Phase II randomized trial of women with advanced or recurrent HER2-positive USC showed improved progression-free and overall survival in 58 patients treated with combined trastuzumab and carboplatin-paclitaxel chemotherapy as compared to chemotherapy alone.
      • Fader A.N.
      • Roque D.M.
      • Siegel E.
      • et al.
      Randomized Phase II trial of carboplatin-paclitaxel versus carboplatin-paclitaxel-trastuzumab in uterine serous carcinomas that overexpress human epidermal growth factor receptor 2/neu.
      ,
      • Fader A.N.
      • Roque D.M.
      • Siegel E.
      • et al.
      Randomized Phase II trial of carboplatin-paclitaxel compared with carboplatin-paclitaxel-trastuzumab in advanced (stage III-IV) or recurrent uterine serous carcinomas that overexpress Her 2/Neu (NCT01367002): updated overall survival analysis.
      As discussed below, in this trial HER-2 status was scored using the 2007 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines, with specific modifications reported in detail in subsequent publications.
      • Buza N.
      HER2 testing in endometrial serous carcinoma: time for standardized pathology practice to meet the clinical demand.
      ,
      • Buza N.
      HER2 testing and reporting in endometrial serous carcinoma: practical recommendations for HER2 immunohistochemistry and fluorescent in situ hybridization: proceedings of the ISGyP Companion Society Session at the 2020 USCAP Annual Meeting.
      Based on the trial results, targeted anti-HER2 therapy is now endorsed in the US by the National Comprehensive Cancer Network and the Society of Gynecological Oncology.
      • Ferriss J.S.
      • Erickson B.K.
      • Shih I.-M.
      • Fader A.N.
      Uterine serous carcinoma: key advances and novel treatment approaches.
      • Koh W.-J.
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      • Bean S.
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      Uterine neoplasms, version 1.2018, NCCN clinical practice guidelines in Oncology.
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      Uterine serous carcinoma: molecular features, clinical management, and new and future therapies.

      HER2 scoring guidelines

      Disease-specific HER2 scoring guidelines have been developed for breast and gastric/GOJ carcinomas and recently advocated for in colorectal cancer, aimed at predicting clinical response to HER2-targeted therapy.
      • Bang Y.-J.
      • Van Cutsem E.
      • Feyereislova A.
      • et al.
      Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial.
      ,
      • Wolff A.C.
      • Hammond M.E.H.
      • Schwartz J.N.
      • et al.
      American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer.
      • Wolff A.C.
      • Hammond M.E.H.
      • Hicks D.G.
      • et al.
      Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update.
      • Wolff A.C.
      • Hammond M.E.H.
      • Allison K.H.
      • et al.
      Human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline focused update.
      • Bartley A.N.
      • Washington M.K.
      • Ventura C.B.
      • et al.
      HER2 testing and clinical decision making in gastroesophageal adenocarcinoma: guideline from the College of American Pathologists, American Society for Clinical Pathology, and American Society of Clinical Oncology.
      • Hofmann M.
      • Stoss O.
      • Shi D.
      • et al.
      Assessment of a HER2 scoring system for gastric cancer: results from a validation study.
      • Valtorta E.
      • Martino C.
      • Sartore-Bianchi A.
      • et al.
      Assessment of a HER2 scoring system for colorectal cancer: results from a validation study.
      These are summarised in Table 1. For USC, standardised scoring guidelines have only recently been proposed, based on the criteria used to select patients for the aforementioned 2018 clinical trial.
      • Buza N.
      • English D.P.
      • Santin A.D.
      • Hui P.
      Toward standard HER2 testing of endometrial serous carcinoma: 4-year experience at a large academic center and recommendations for clinical practice.
      ,
      • Fader A.N.
      • Roque D.M.
      • Siegel E.
      • et al.
      Randomized Phase II trial of carboplatin-paclitaxel versus carboplatin-paclitaxel-trastuzumab in uterine serous carcinomas that overexpress human epidermal growth factor receptor 2/neu.
      ,
      • Fader A.N.
      • Roque D.M.
      • Siegel E.
      • et al.
      Randomized Phase II trial of carboplatin-paclitaxel compared with carboplatin-paclitaxel-trastuzumab in advanced (stage III-IV) or recurrent uterine serous carcinomas that overexpress Her 2/Neu (NCT01367002): updated overall survival analysis.
      These criteria were underpinned by findings from the then largest study of HER2 expression in endometrial carcinomas (mentioned above), which examined a cohort of 85 serous and 23 mixed serous and endometrioid carcinomas.
      • Buza N.
      • English D.P.
      • Santin A.D.
      • Hui P.
      Toward standard HER2 testing of endometrial serous carcinoma: 4-year experience at a large academic center and recommendations for clinical practice.
      Notably, this study found that the 2007 ASCO/CAP breast scoring system (score as 3+ if uniform strong membrane staining in >30% of tumour cells) yielded the highest concordance between IHC and FISH for HER2 expression, also observing a lateral or basolateral pattern of staining, due to absent apical membrane staining, in 74% of HER2-positive USCs, particularly those with glandular differentiation. Both the stronger concordance between IHC and FISH results using the more stringent, 30% scoring cut-off, and similar rates of apical staining loss have since been reported by others.
      • Halle M.K.
      • Tangen I.L.
      • Berg H.F.
      • et al.
      HER2 expression patterns in paired primary and metastatic endometrial cancer lesions.
      ,
      • Ross D.S.
      • Devereaux K.A.
      • Jin C.
      • et al.
      Histopathologic features and molecular genetic landscape of HER2-amplified endometrial carcinomas.
      Based on the observation of heterogeneous staining in over 50% of HER2-positive tumours, this study also recommended the selection of blocks containing large areas of tumour for IHC or staining of multiple sections; heterogeneous immunohistochemical expression has been shown to correlate closely with heterogeneous gene amplification by FISH.
      • Ross D.S.
      • Devereaux K.A.
      • Jin C.
      • et al.
      Histopathologic features and molecular genetic landscape of HER2-amplified endometrial carcinomas.
      ,
      • Buza N.
      • Hui P.
      Marked heterogeneity of HER2/NEU gene amplification in endometrial serous carcinoma.
      The resultant criteria, representing a modification of the 2007 ASCO/CAP breast scoring system (modified to include tumours with basolateral/lateral staining and a HER2/CEP17 ratio of 2.0 or greater as positive), guided selection of patients for the 2018 clinical trial. Consequently, it is recommended that these criteria form the basis for future USC scoring in order to maximise therapeutic response.
      • Buza N.
      HER2 testing and reporting in endometrial serous carcinoma: practical recommendations for HER2 immunohistochemistry and fluorescent in situ hybridization: proceedings of the ISGyP Companion Society Session at the 2020 USCAP Annual Meeting.
      These criteria have since been applied in other retrospective clinicopathological studies,
      • Erickson B.K.
      • Najjar O.
      • Damast S.
      • et al.
      Human epidermal growth factor 2 (HER2) in early stage uterine serous carcinoma: a multi-institutional cohort study.
      • Rottmann D.
      • Assem H.
      • Matsumoto N.
      • Wong S.
      • Hui P.
      • Buza N.
      Does specimen type have an impact on HER2 status in endometrial serous carcinoma? Discordant HER2 status of paired endometrial biopsy and hysterectomy specimens in the presence of frequent intratumoral heterogeneity.
      • Rottmann D.
      • Snir O.L.
      • Wu X.
      • et al.
      HER2 testing of gynecologic carcinosarcomas: tumor stratification for potential targeted therapy.
      and have also been found to be reproducible among gynaecological pathologists, with moderate to substantial rates of interobserver agreement.
      • Buza N.
      • Euscher E.D.
      • Matias-Guiu X.
      • et al.
      Reproducibility of scoring criteria for HER2 immunohistochemistry in endometrial serous carcinoma: a multi-institutional interobserver agreement study.
      Table 1Current criteria (approved or proposed) for HER2 positivity by IHC and FISH in different tumour types
      HER2 IHC 3+HER2 FISH amplification
      Breast (ASCO/CAP 2018)>10% circumferential, strong, completeHER2/CEP17 ratio ≥2.0 and HER2 signal ≥4.0/nucleus

      OR ratio <2.0 and HER2 signal ≥6.0/nucleus (if IHC score 2+ or 3+)
      Gastric/gastro-oesophageal junction (ASCO/CAP 2016)≥10%, strong complete, or basolateral/lateralHER2/CEP17 ratio ≥2.0

      OR ratio <2.0 and HER2 signal >6.0/nucleus
      Colorectal (HERACLES trial)≥50% strong complete, or basolateral/lateralHER2/CEP17 ratio ≥2.0 in ≥50% of cells
      ASCO, American Society of Clinical Oncology; CAP, College of American Pathologists; FISH, fluorescent in situ hybridisation; IHC, immunohistochemistry.
      In summary, the proposed algorithm (depicted in Table 2) defines positive HER2 staining as an IHC 3+ score or a 2+ score with HER2 gene amplification by FISH. IHC score 3+ is assigned when more than 30% of tumour cells demonstrate strong complete or lateral/basolateral membranous staining and 2+ when strong complete or lateral/basolateral membrane staining is present in ≤30%, or weak to moderate staining is present in ≥10% of tumour cells. FISH is performed only on tumours with a 2+ IHC score, on a tumour area measuring approximately 1 cm2, correlating this with the HER2 IHC slide. A HER2/CEP17 ratio of ≥2 is considered amplified, as is a HER2/CEP17 ratio <2.0 with an average HER2 copy number ≥6.0 per nucleus. A recent study of 94 USC, aimed at refining the specific characteristics of HER2 gene amplification in USC by FISH, found 99% concordance with current gastric and breast HER2 FISH interpretations, endorsing these criteria.
      • Buza N.
      • Hui P.
      Characteristics of HER2 gene amplification by fluorescence in situ hybridization in endometrial serous carcinoma.
      The key points of difference between the proposed uterine serous versus breast and gastric scoring criteria lies in the more stringent percentage cut-off required for 3+ staining (30% for USC versus 10% for breast and gastric), with the inclusion of basolateral/lateral membrane staining as positive in USC and gastric but not in breast carcinoma algorithms. As stated, the key rationale for using this higher threshold in USC lies in the greater IHC-FISH concordance achieved using the 30% cut-off.
      • Buza N.
      • English D.P.
      • Santin A.D.
      • Hui P.
      Toward standard HER2 testing of endometrial serous carcinoma: 4-year experience at a large academic center and recommendations for clinical practice.
      The various HER2 staining patterns are depicted in Fig. 1, Fig. 2.
      Table 2Proposed HER2 IHC and FISH scoring criteria in endometrial serous carcinoma
      ScoreCriteria
      IHC
       0No staining in tumour cells
       1+Faint/barely perceptible, incomplete membrane staining in any proportion, or weak complete staining in <10% of tumour cells
       2+Strong complete or basolateral/lateral membrane staining in ≤30%, or weak to moderate complete or basolateral/lateral staining in ≥10% of tumour cells
       3+Strong complete or basolateral/lateral membrane staining in >30% of tumour cells
      FISH
       NegativeFISH HER2/CEP17 ratio <2.0 and average HER2 copy number <6/nucleus
       PositiveHER2/CEP17 ratio ≥2.0 or <2.0 with average HER2 copy number ≥6/nucleus
      ASCO, American Society of Clinical Oncology; CAP, College of American Pathologists; FISH, fluorescent in situ hybridisation; IHC, immunohistochemistry.
      Fig. 1
      Fig. 1HER2 protein expression in uterine serous carcinoma using the recently proposed endometrial carcinoma-specific scoring criteria: (A) HER2 3+: strong, complete and basolateral membranous staining in >30% of tumour cells; (B) HER2 2+: weak to moderate complete and basolateral membranous staining in ≥10% of tumour cells; (C) HER2 1+: faint, barely perceptible incomplete membranous staining; and (D) HER2 0: no staining in tumour cells.
      Fig. 2
      Fig. 2HER2 protein expression in uterine serous carcinoma frequently shows intratumoural heterogeneity (A) and lack of apical membrane staining resulting in a lateral/basolateral staining pattern (B).

      Prognostic and predictive value of HER2 in uterine serous cancer

      Numerous studies in the 1990s and early 2000s showed HER2 protein overexpression or gene amplification to be an independent predictor of diminished patient outcome with USC,
      • Slomovitz B.M.
      • Broaddus R.R.
      • Burke T.W.
      • et al.
      Her-2/neu overexpression and amplification in uterine papillary serous carcinoma.
      • Rolitsky C.D.
      • Theil K.S.
      • McGaughy V.R.
      • Copeland L.J.
      • Niemann T.H.
      HER-2/neu amplification and overexpression in endometrial carcinoma.
      • Togami S.
      • Sasajima Y.
      • Oi T.
      • et al.
      Clinicopathological and prognostic impact of human epidermal growth factor receptor type 2 (HER2) and hormone receptor expression in uterine papillary serous carcinoma.
      • Saffari B.
      • Jones L.A.
      • el-Naggar A.
      • Felix J.C.
      • George J.
      • Press M.F.
      Amplification and overexpression of HER-2/neu (c-erbB2) in endometrial cancers: correlation with overall survival.
      • Díaz-Montes T.P.
      • Ji H.
      • Smith Sehdev A.E.
      • et al.
      Clinical significance of Her-2/neu overexpression in uterine serous carcinoma.
      although occasional studies found no such association.
      • Grushko T.A.
      • Filiaci V.L.
      • Mundt A.J.
      • et al.
      An exploratory analysis of HER-2 amplification and overexpression in advanced endometrial carcinoma: a Gynecologic Oncology Group study.
      HER2 is now considered a prognostic biomarker in USC, with high HER2 protein expression significantly associated with aggressive disease, with one study showing that HER2 IHC staining proved to be a better predictor of survival than FISH.
      • Halle M.K.
      • Tangen I.L.
      • Berg H.F.
      • et al.
      HER2 expression patterns in paired primary and metastatic endometrial cancer lesions.
      Additionally, a recent multicentre retrospective cohort study in patients with stage I USC showed that amongst HER2-positive tumours, there were more recurrences, more lymphovascular space invasion and significantly worse progression-free and overall survival.
      • Erickson B.K.
      • Najjar O.
      • Damast S.
      • et al.
      Human epidermal growth factor 2 (HER2) in early stage uterine serous carcinoma: a multi-institutional cohort study.
      Other studies have correlated HER2 positivity in USC with advanced stage at presentation,
      • Santin A.D.
      • Bellone S.
      • Siegel E.R.
      • et al.
      Racial differences in the overexpression of epidermal growth factor type II receptor (HER2/neu): a major prognostic indicator in uterine serous papillary cancer.
      ,
      • Mentrikoski M.J.
      • Stoler M.H.
      HER2 immunohistochemistry significantly overestimates HER2 amplification in uterine papillary serous carcinomas.
      a variable associated with poor outcome.
      Regarding treatment response, heterogeneous HER2 protein expression or gene amplification has been found to impact the clinical response to targeted therapy in breast and gastric cancers, with tumours exhibiting homogeneous HER2 expression demonstrating a superior treatment response and improved progression-free and overall survival compared to those with heterogeneous HER2 protein expression. In addition, heterogeneous HER2 expression has been shown to be an independent predictor of poor prognosis in gastric cancer.
      • Muller K.E.
      • Marotti J.D.
      • Tafe L.J.
      Pathologic features and clinical implications of breast cancer with HER2 intratumoral genetic heterogeneity.
      ,
      • Kaito A.
      • Kuwata T.
      • Tokunaga M.
      • et al.
      HER2 heterogeneity is a poor prognosticator for HER2-positive gastric cancer.
      However, the potential prognostic impact of HER2 heterogeneity has not been studied in USC. As the 2018 clinical trial patients were selected based on IHC,
      • Fader A.N.
      • Roque D.M.
      • Siegel E.
      • et al.
      Randomized Phase II trial of carboplatin-paclitaxel versus carboplatin-paclitaxel-trastuzumab in uterine serous carcinomas that overexpress human epidermal growth factor receptor 2/neu.
      ,
      • Fader A.N.
      • Roque D.M.
      • Siegel E.
      • et al.
      Randomized Phase II trial of carboplatin-paclitaxel compared with carboplatin-paclitaxel-trastuzumab in advanced (stage III-IV) or recurrent uterine serous carcinomas that overexpress Her 2/Neu (NCT01367002): updated overall survival analysis.
      elucidating the correlation between clinical response and HER2 gene amplification also requires further study, as the best predictor of response to therapy, protein overexpression versus gene amplification, remains to be determined. Importantly, the response of patients with HER2 gene amplifications without HER2 protein overexpression merits further investigation. Interestingly, in breast cancer both ISH and FISH are equally predictive of clinical response to targeted therapy, while in gastric cancer HER2 protein expression shows the strongest association with treatment response. Consequently, while either IHC or FISH may be applied in breast cancer with reflex application of the other test in equivocal cases, the primary testing modality in gastric cancer should be IHC.
      • Wolff A.C.
      • Hammond M.E.H.
      • Allison K.H.
      • et al.
      Human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline focused update.
      ,
      • Bartley A.N.
      • Washington M.K.
      • Ventura C.B.
      • et al.
      HER2 testing and clinical decision making in gastroesophageal adenocarcinoma: guideline from the College of American Pathologists, American Society for Clinical Pathology, and American Society of Clinical Oncology.
      Another recent development in breast cancer is recognition of ‘HER2-low’ tumours. These tumours, traditionally classified as HER2 negative, exhibit IHC scores of 1+ or 2+ in the absence of HER2 gene amplification by ISH, yet have shown response to novel anti-HER2-antibody drug conjugates in patient with advanced or metastatic breast cancer refractory to standard therapies in early-phase clinical trials.
      • Modi S.
      • Park H.
      • Murthy R.K.
      • et al.
      Antitumor activity and safety of trastuzumab deruxtecan in patients with HER2-low-expressing advanced breast cancer: results from a Phase Ib study.
      ,
      • Banerji U.
      • van Herpen C.M.L.
      • Saura C.
      • et al.
      Trastuzumab duocarmazine in locally advanced and metastatic solid tumours and HER2-expressing breast cancer: a phase 1 dose-escalation and dose-expansion study.
      Two further clinical trials are currently ongoing in breast cancer,
      • Miglietta F.
      • Griguolo G.
      • Bottosso M.
      • et al.
      HER2-low-positive breast cancer: evolution from primary tumor to residual disease after neoadjuvant treatment.
      with this development challenging the traditional dichotomous approach to the allocation of anti-HER2 targeted therapies.

      Overview of technical aspects of HER2 testing

      Optimisation of testing

      In a single patient, HER2 results may vary depending on the nature of the specimen tested, such as biopsy versus definitive resection or primary versus metastasis/recurrence, with heterogeneity of expression often cited to explain this discordance. Tumour heterogeneity, while relatively uncommon in breast carcinoma,
      • Hanna W.
      • Nofech-Mozes S.
      • Kahn H.J.
      Intratumoral heterogeneity of HER2/neu in breast cancer--a rare event.
      ,
      • Greer L.T.
      • Rosman M.
      • Mylander W.C.
      • et al.
      Does breast tumor heterogeneity necessitate further immunohistochemical staining on surgical specimens?.
      is more frequently described at other sites, including the GOJ
      • Kunz P.L.
      • Mojtahed A.
      • Fisher G.A.
      • et al.
      HER2 expression in gastric and gastroesophageal junction adenocarcinoma in a US population: clinicopathologic analysis with proposed approach to HER2 assessment.
      and in USC, where heterogeneity has been defined as at least a two-fold change in the intensity of HER2 expression involving at least 5% of the tumour.
      • Buza N.
      • Hui P.
      Marked heterogeneity of HER2/NEU gene amplification in endometrial serous carcinoma.
      The proportion of USC that demonstrate heterogeneity ranges widely from 31% to 97%,
      • Buza N.
      • Hui P.
      Marked heterogeneity of HER2/NEU gene amplification in endometrial serous carcinoma.
      ,
      • Rottmann D.
      • Assem H.
      • Matsumoto N.
      • Wong S.
      • Hui P.
      • Buza N.
      Does specimen type have an impact on HER2 status in endometrial serous carcinoma? Discordant HER2 status of paired endometrial biopsy and hysterectomy specimens in the presence of frequent intratumoral heterogeneity.
      ,
      • Banet N.
      • Shahi M.
      • Batista D.
      • et al.
      HER-2 amplification in uterine serous carcinoma and serous endometrial intraepithelial carcinoma.
      likely reflective of different scoring systems used in these studies as well as different specimen types. To address the question of heterogeneity of HER2 status between paired endometrial biopsy and hysterectomy specimens, a recent study found 84% (31/37) exhibited concordant HER2 expression, with the most common pattern of discordance being positivity in the biopsy but not in the hysterectomy (4/6 discordant cases).
      • Rottmann D.
      • Assem H.
      • Matsumoto N.
      • Wong S.
      • Hui P.
      • Buza N.
      Does specimen type have an impact on HER2 status in endometrial serous carcinoma? Discordant HER2 status of paired endometrial biopsy and hysterectomy specimens in the presence of frequent intratumoral heterogeneity.
      Lack of uniform formalin fixation is postulated as one reason for this discordance,
      • Rottmann D.
      • Assem H.
      • Matsumoto N.
      • Wong S.
      • Hui P.
      • Buza N.
      Does specimen type have an impact on HER2 status in endometrial serous carcinoma? Discordant HER2 status of paired endometrial biopsy and hysterectomy specimens in the presence of frequent intratumoral heterogeneity.
      with possible false negative results in excision specimens due to less uniform cold ischaemic and formalin fixation intervals. Additionally, endometrial biopsies and curettings may provide a more spatially heterogeneous, random sample from different areas of the endometrial cavity compared to a more spatially limited area represented in tissue blocks from a hysterectomy specimen.
      In another study, Halle et al. demonstrated discordant HER2 expression between paired primary and metastatic tumours, with only 45% (9/20) of USC retaining staining in metastatic lesions. Conversely, in three of 15 patients with primary HER2 0–2+ tumours, HER2 overexpression was detected in the metastasis. Discordant HER2 expression between different metastases in the same patient was also observed.
      • Halle M.K.
      • Tangen I.L.
      • Berg H.F.
      • et al.
      HER2 expression patterns in paired primary and metastatic endometrial cancer lesions.
      A further study examined 68 cases of USC, serous endometrial intraepithelial carcinoma (SEIC) and synchronous SEIC and USC, finding discordant HER2 status in 47% (7/15) of SEIC and USC, also reporting high rates of intratumoural HER2 heterogeneity by IHC, identified in 97% (66/68) of cases.
      • Banet N.
      • Shahi M.
      • Batista D.
      • et al.
      HER-2 amplification in uterine serous carcinoma and serous endometrial intraepithelial carcinoma.
      These authors advise caution when evaluating HER2 status in small biopsies, advocating testing of both biopsy and excision specimens, and it has been demonstrated in gastric and GOJ adenocarcinomas that this approach, in addition to testing tumour recurrences/metastases, may increase detection of HER2 overexpression.
      • Huber A.R.
      • Buscaglia B.
      • Koltz B.R.
      • et al.
      Impact of specimen type and specimen number on HER2 status in gastroesophageal junction and gastric adenocarcinoma.
      In light of these data, repeat testing of the hysterectomy in cases where HER2 is negative on the initial biopsy and, similarly, testing of metastases when HER2 is negative on the initial biopsy or hysterectomy, could be considered. The findings of Halle et al. also suggest that testing of multiple metastatic sites in a single patient may further optimise results.
      • Halle M.K.
      • Tangen I.L.
      • Berg H.F.
      • et al.
      HER2 expression patterns in paired primary and metastatic endometrial cancer lesions.
      As previously stated, testing entire histological blocks with the largest amount of viable tumour is recommended, and when FISH is performed this should be interpreted in direct correlation with the strongest HER2 protein expression on the IHC slide. Time from tissue acquisition to fixation should be as short as possible, ideally less than 1 hour, with tissue fixed in 10% neutral buffered formalin for 6–72 hours before processing.
      • Wolff A.C.
      • Hammond M.E.H.
      • Allison K.H.
      • et al.
      Human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline focused update.
      ,
      • Bartley A.N.
      • Washington M.K.
      • Ventura C.B.
      • et al.
      HER2 testing and clinical decision making in gastroesophageal adenocarcinoma: guideline from the College of American Pathologists, American Society for Clinical Pathology, and American Society of Clinical Oncology.
      Finally, factors which may impact interobserver reproducibility when evaluating HER2 IHC include the experience and training of the pathologist, the antibody clone used and tumour histotype.
      • Buza N.
      • Euscher E.D.
      • Matias-Guiu X.
      • et al.
      Reproducibility of scoring criteria for HER2 immunohistochemistry in endometrial serous carcinoma: a multi-institutional interobserver agreement study.
      Given the increased routine diagnostic application of HER2 assays, quality assurance programs should consider including endometrial carcinoma in future HER2 IHC laboratory proficiency testing, in an effort to increase interlaboratory and pathologist reproducibility.

      Correlation between IHC, FISH and next generation sequencing

      The ASCO/CAP guidelines for HER2 testing of breast cancer state that a concordance level between IHC and FISH of 95% is desirable, and when equivocal results are excluded, concordance rates approach this in breast cancer.
      • Wolff A.C.
      • Hammond M.E.H.
      • Hicks D.G.
      • et al.
      Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update.
      Variable concordance is reported in USC. In 2005, Santin et al. found 100% agreement between HER2 IHC 3+ expression (using a 10% cut-off) and FISH gene amplification in nine USC and, similarly, no gene amplification in 10 USC scored by IHC as 0/1+.
      • Santin A.D.
      • Bellone S.
      • Van Stedum S.
      • et al.
      Determination of HER2/neu status in uterine serous papillary carcinoma: comparative analysis of immunohistochemistry and fluorescence in situ hybridization.
      In this study, 29% (2/7) of cases with IHC 2+ staining were amplified by FISH. By comparison, Slomovitz et al. found only 16% (2/12) of USC with HER2 IHC 2 or 3+ expression demonstrated FISH amplification; both cases with gene amplification were aneuploid with increased copies of both HER-2/neu and CEP 17.
      • Slomovitz B.M.
      • Broaddus R.R.
      • Burke T.W.
      • et al.
      Her-2/neu overexpression and amplification in uterine papillary serous carcinoma.
      Subsequently, in a microarray study (mentioned earlier),
      • Xu M.
      • Schwartz P.
      • Rutherford T.
      • et al.
      HER-2/neu receptor gene status in endometrial carcinomas: a tissue microarray study.
      Xu et al. demonstrated concordant HER2 IHC 3+ staining and FISH amplification in eight of 10 cases of USC. Similarly, a more recent study with larger case numbers (n=68) found 75% concordance between IHC and FISH for USC showing IHC 0/1+ or 3+ staining, using either the ASCO/CAP 2013 breast or ASCO/ASCP/CAP 2016 gastric scoring systems.
      • Banet N.
      • Shahi M.
      • Batista D.
      • et al.
      HER-2 amplification in uterine serous carcinoma and serous endometrial intraepithelial carcinoma.
      Notably, 50% of cases scored by IHC as HER2 3+ were non-amplified by FISH and 13–14% of IHC 0/1+ cases were FISH amplified. This study also evaluated HER2 status in SEIC and found a greater concordance between IHC and FISH in cases of SEIC alone, and when USC and SEIC cases were grouped together; in this context the concordance rates between HER2 IHC and FISH were 89% and 88% using breast and gastric criteria, respectively. The seminal study on which the USC scoring criteria are based, and which selected patients for the 2018 clinical trial, found that IHC concordance with FISH (for 0/1+ and 3+) was 78% (28/36) using the original FDA (HercepTest) breast criteria and 86% (25/29) using ASCO/CAP 2007 breast criteria.
      • Buza N.
      • English D.P.
      • Santin A.D.
      • Hui P.
      Toward standard HER2 testing of endometrial serous carcinoma: 4-year experience at a large academic center and recommendations for clinical practice.
      Tumours with equivocal (2+) IHC scoring showed gene amplification in 31% (5/16) and 26% (6/23) of cases when using the FDA HercepTest and ASCO/CAP scoring systems, respectively (10% vs 30% strong membranous staining cut-off). All of these results fall below the desired concordance rate recommended by the ASCO/CAP guidelines
      • Wolff A.C.
      • Hammond M.E.H.
      • Hicks D.G.
      • et al.
      Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update.
      which states that both IHC and FISH should be performed until testing concordance approaches 95%. Consequently, it has been suggested that until clinical trial data definitively establish the relationship of clinical response to HER2 status, as measured by IHC and FISH, both testing modalities should be routinely performed.
      • Banet N.
      • Shahi M.
      • Batista D.
      • et al.
      HER-2 amplification in uterine serous carcinoma and serous endometrial intraepithelial carcinoma.
      Finally, discordance between IHC and FISH has been reported in metastatic endometrial carcinoma (of varied histotypes), suggesting both should be performed in this context; Halle et al. reported that in endometrial carcinoma metastases, 12% (18/144) of tumours scored as IHC 3+ using HercepTest criteria lacked gene amplification.
      • Halle M.K.
      • Tangen I.L.
      • Berg H.F.
      • et al.
      HER2 expression patterns in paired primary and metastatic endometrial cancer lesions.
      Heterogeneity of HER2 protein expression and gene amplification may contribute to some of the observed discordance between IHC and FISH. In 2013, Buza and Hui investigated heterogeneity of gene amplification in a series of 17 USC with heterogeneous protein expression. In 10 of 17 cases, when a large area of tumour was examined, a heterogeneous ‘cluster amplification’ pattern was observed (defined as amplification of a cluster of 20 or more cells), with a further four cases showing diffuse amplification.
      • Buza N.
      • Hui P.
      Marked heterogeneity of HER2/NEU gene amplification in endometrial serous carcinoma.
      Direct correlation at a cellular level between the heterogeneity on IHC slides and FISH was also noted. These findings again highlight the importance of testing of large tumour samples from multiple specimens, ideally including the resection specimen, with direct targeting of regions of tumour for FISH analysis guided by the IHC slides. Other reasons offered to explain IHC overexpression in the absence of gene amplification include genetic aneuploidy, with chromosome 17 polysomy identified in cases of breast cancer that were IHC-positive but lacked gene amplification.
      • Lebeau A.
      • Deimling D.
      • Kaltz C.
      • et al.
      HER-2/neu analysis in archival tissue samples of human breast cancer: comparison of immunohistochemistry and fluorescence in situ hybridization.
      Conversely, a lack of IHC expression with FISH amplification may result from loss of antigenicity due to use of use of old archived sections, inadequate tumour fixation or expression of truncated variants of the HER2 protein due to shedding of the extracellular domain of HER2, a phenomenon which is seen more frequently in USC than in breast cancer, possibly resulting in failure to detect protein overexpression by IHC.
      • Robinson C.L.
      • Harrison B.T.
      • Ligon A.H.
      • et al.
      Detection of ERBB2 amplification in uterine serous carcinoma by next-generation sequencing: an approach highly concordant with standard assays.
      • Growdon W.B.
      • Groeneweg J.
      • Byron V.
      • et al.
      HER2 over-expressing high grade endometrial cancer expresses high levels of p95HER2 variant.
      • Carvajal-Hausdorf D.E.
      • Schalper K.A.
      • Bai Y.
      • Black J.
      • Santin A.D.
      • Rimm D.L.
      Objective, domain-specific HER2 measurement in uterine and ovarian serous carcinomas and its clinical significance.
      A recent study comprehensively evaluated HER2 FISH characteristics in 94 USC, the majority (90%, 85/94) of which had an IHC score of 2+, and 34 of them showed gene amplification by FISH. Comparing four sets of criteria for FISH scoring (2018 trial criteria, 2016 ASCO/CAP gastric, and ASCO/CAP 2013 and 2018 breast criteria) yielded almost identical results.
      • Buza N.
      • Hui P.
      Characteristics of HER2 gene amplification by fluorescence in situ hybridization in endometrial serous carcinoma.
      Polysomy of chromosome 17 occurred in 16% (15/94) and monosomy 17 in just 2% (2/94) cases.
      • Buza N.
      • Hui P.
      Characteristics of HER2 gene amplification by fluorescence in situ hybridization in endometrial serous carcinoma.
      Detection of HER2 amplification by next generation sequencing (NGS) has been shown to be highly concordant with IHC and FISH in USC.
      • Robinson C.L.
      • Harrison B.T.
      • Ligon A.H.
      • et al.
      Detection of ERBB2 amplification in uterine serous carcinoma by next-generation sequencing: an approach highly concordant with standard assays.
      Robinson et al. compared HER2 expression by IHC and FISH with targeted NGS in 93 USC using the 2018 ASCO/CAP breast guidelines. Of eight cases (9%) classified by IHC and FISH as HER2 overexpressed and/or amplified, there was 100% concordance with results of NGS.
      • Robinson C.L.
      • Harrison B.T.
      • Ligon A.H.
      • et al.
      Detection of ERBB2 amplification in uterine serous carcinoma by next-generation sequencing: an approach highly concordant with standard assays.
      Ross et al. performed targeted sequencing on 2042 endometrial carcinomas of various histological subtypes, finding HER2 amplification in 3.8% (77 tumours), with a HER2 IHC score of 2+ or 3+ reported in all evaluable cases (n=61).
      • Ross D.S.
      • Devereaux K.A.
      • Jin C.
      • et al.
      Histopathologic features and molecular genetic landscape of HER2-amplified endometrial carcinomas.
      Of 47 cases that underwent both NGS and FISH, 44 were positive for HER2 amplification in both analyses. The three discordant cases were amplified on NGS but were considered non-amplified by FISH, due to heterogeneous, focal HER2 amplification in single cells or small cell clusters. An overall low frequency of HER2 amplification by NGS, detected in only 8% (29/361)
      • Ross D.S.
      • Devereaux K.A.
      • Jin C.
      • et al.
      Histopathologic features and molecular genetic landscape of HER2-amplified endometrial carcinomas.
      and 9% (8/93)
      • Robinson C.L.
      • Harrison B.T.
      • Ligon A.H.
      • et al.
      Detection of ERBB2 amplification in uterine serous carcinoma by next-generation sequencing: an approach highly concordant with standard assays.
      of serous carcinomas in these series, was postulated to relate to high thresholds set to avoid false positives, low tumour purity and intratumoural heterogeneity, with NGS potentially not detecting gene amplification when present only within a minor subpopulation of cells.
      • Ross D.S.
      • Devereaux K.A.
      • Jin C.
      • et al.
      Histopathologic features and molecular genetic landscape of HER2-amplified endometrial carcinomas.
      Selection bias likely impacted results in the series by Robinson et al., which was enriched for tumours testing negative for HER2 by IHC and FISH.
      • Robinson C.L.
      • Harrison B.T.
      • Ligon A.H.
      • et al.
      Detection of ERBB2 amplification in uterine serous carcinoma by next-generation sequencing: an approach highly concordant with standard assays.
      These findings highlight the advantage of IHC and FISH over NGS, with gene amplification and heterogeneity visualised at a cellular level using the former two assays, whereas NGS provides an aggregated analysis of a larger tumour area. The lower cost and efficiency of IHC and FISH also support these modalities as the preferred method of testing.

      HER2 status in tumours other than UTERINE serous carcinoma

      Non-serous endometrial carcinoma

      The emergence of anti-HER2 targeted therapy for advanced and/or recurrent HER2-positive USC has prompted efforts to characterise HER2 expression in non-serous uterine tumours, with a view to extending this therapeutic option to more patients. While reported rates of HER2 protein overexpression and gene amplification vary widely in non-serous tumours,
      • Fleming G.F.
      • Sill M.W.
      • Darcy K.M.
      • et al.
      Phase II trial of trastuzumab in women with advanced or recurrent, HER2-positive endometrial carcinoma: a Gynecologic Oncology Group study.
      • Buza N.
      • Roque D.M.
      • Santin A.D.
      HER2/neu in endometrial cancer: a promising therapeutic target with diagnostic challenges.
      • Xu M.
      • Schwartz P.
      • Rutherford T.
      • et al.
      HER-2/neu receptor gene status in endometrial carcinomas: a tissue microarray study.
      ,
      • Rolitsky C.D.
      • Theil K.S.
      • McGaughy V.R.
      • Copeland L.J.
      • Niemann T.H.
      HER-2/neu amplification and overexpression in endometrial carcinoma.
      ,
      • Saffari B.
      • Jones L.A.
      • el-Naggar A.
      • Felix J.C.
      • George J.
      • Press M.F.
      Amplification and overexpression of HER-2/neu (c-erbB2) in endometrial cancers: correlation with overall survival.
      an emerging trend is the tendency for HER2 amplified tumours to have co-existing TP53 mutation and high-grade histology.
      • Halle M.K.
      • Tangen I.L.
      • Berg H.F.
      • et al.
      HER2 expression patterns in paired primary and metastatic endometrial cancer lesions.
      ,
      • Ross D.S.
      • Devereaux K.A.
      • Jin C.
      • et al.
      Histopathologic features and molecular genetic landscape of HER2-amplified endometrial carcinomas.
      ,
      • Rottmann D.
      • Snir O.L.
      • Wu X.
      • et al.
      HER2 testing of gynecologic carcinosarcomas: tumor stratification for potential targeted therapy.
      ,
      • Vermij L.
      • Horeweg N.
      • Leon-Castillo A.
      • et al.
      HER2 status in high-risk endometrial cancers (PORTEC-3): relationship with histotype, molecular classification, and clinical outcomes.
      ,
      • Jenkins T.M.
      • Cantrell L.A.
      • Stoler M.H.
      • Mills A.M.
      HER2 overexpression and amplification in uterine carcinosarcomas with serous morphology.
      A study of 790 endometrial carcinomas of varied histotype found that after USC, clear cell carcinoma, carcinosarcoma and high-grade endometrioid carcinoma exhibited the greatest rates of HER2 IHC overexpression, seen in 30%, 23% and 21% of cases, respectively, compared with just 11% of low-grade endometrioid tumours.
      • Halle M.K.
      • Tangen I.L.
      • Berg H.F.
      • et al.
      HER2 expression patterns in paired primary and metastatic endometrial cancer lesions.
      Another recent study investigated HER2 status in the PORTEC-3 trial population, comprising molecularly classified high-risk endometrial carcinomas.
      • Vermij L.
      • Horeweg N.
      • Leon-Castillo A.
      • et al.
      HER2 status in high-risk endometrial cancers (PORTEC-3): relationship with histotype, molecular classification, and clinical outcomes.
      Using IHC and FISH, 5.9% (24/407) of tumours of varied histotype showed HER2 overexpression or gene amplification. These comprised serous, endometrioid and clear cell carcinoma. HER2 positivity was strongly associated with the p53 abnormal subgroup, with 23 of 24 HER2-positive cases also exhibiting aberrant p53 expression. The correlation between HER2 and p53-abnormal status was significantly stronger than that between HER2 status and serous histology and HER2 status did not have independent prognostic value after correcting for the molecular subgroup.
      • Vermij L.
      • Horeweg N.
      • Leon-Castillo A.
      • et al.
      HER2 status in high-risk endometrial cancers (PORTEC-3): relationship with histotype, molecular classification, and clinical outcomes.
      A further retrospective analysis of 2042 unselected endometrial carcinomas subjected to molecular sequencing identified 77 cases (3.8%) with HER2 amplification, enriched for USC (38.5% of HER2 amplified cases), carcinosarcomas and high-grade endometrial carcinomas with ambiguous features (close to 30% of HER2 amplified cases), also identifying a co-existing TP53 mutation in 94% (72/77) of cases.
      • Ross D.S.
      • Devereaux K.A.
      • Jin C.
      • et al.
      Histopathologic features and molecular genetic landscape of HER2-amplified endometrial carcinomas.
      Given the propensity for carcinosarcomas to harbour a serous carcinoma component and TP53 mutation, HER2 expression in carcinosarcomas is of particular interest. In 2019, Rottmann et al. evaluated HER2 expression/amplification in 80 carcinosarcomas (65 uterine, 15 tubo-ovarian) finding 16% to be HER2-positive in the carcinoma component (13 cases, 12 uterine, 1 ovarian) by either IHC or FISH using 2013 ASCO/CAP breast scoring criteria (10% cut-off for 3+ staining) versus 13% positive (10 cases, 9 uterine, 1 ovarian) using 2007 criteria (30% cut-off for 3+ staining); interestingly, two cases had their HER2 status changed from negative (2007) to positive (2013) using different scoring systems.
      • Rottmann D.
      • Snir O.L.
      • Wu X.
      • et al.
      HER2 testing of gynecologic carcinosarcomas: tumor stratification for potential targeted therapy.
      All positive cases had a serous carcinoma component. Heterogeneity of protein expression was observed in 38% of HER2-positive tumours and lack of apical staining was frequent. The sarcomatous component was present on the HER2 stained slide in only 44 cases and none showed 3+ staining; five exhibited 2+ protein expression, with one of these showing equivocal (2007) and positive (2013) amplification by FISH. The authors concluded that adding evaluation of the sarcomatous component did not change the overall HER2 status and advocated use of the proposed USC scoring criteria in carcinosarcoma.
      • Rottmann D.
      • Snir O.L.
      • Wu X.
      • et al.
      HER2 testing of gynecologic carcinosarcomas: tumor stratification for potential targeted therapy.
      Jenkins et al. recently found HER2 expression and/or amplification in 15% (7/48) of carcinosarcomas, with all positive cases exhibiting serous morphology and aberrant p53 expression.
      • Jenkins T.M.
      • Cantrell L.A.
      • Stoler M.H.
      • Mills A.M.
      HER2 overexpression and amplification in uterine carcinosarcomas with serous morphology.
      This study also showed only minimal programmed cell death-ligand 1 (PD-L1) expression and no MMR loss in the HER2-positive cases, suggesting that HER2-positive carcinosarcomas may be less responsive to immune checkpoint inhibition. Similar findings have been reported by others with rates of HER2 overexpression or amplification in uterine carcinosarcoma ranging from 6% to 25%.
      • Rottmann D.
      • Snir O.L.
      • Wu X.
      • et al.
      HER2 testing of gynecologic carcinosarcomas: tumor stratification for potential targeted therapy.
      ,
      • Yoshida H.
      • Nishikawa T.
      • Matsumoto K.
      • et al.
      Histopathological features of HER2 overexpression in uterine carcinosarcoma: proposal for requirements in HER2 testing for targeted therapy.
      • Crane E.
      • Naumann W.
      • Tait D.
      • Higgins R.
      • Herzog T.
      • Brown J.
      Molecular variations in uterine carcinosarcomas identify therapeutic opportunities.
      • Cimbaluk D.
      • Rotmensch J.
      • Scudiere J.
      • Gown A.
      • Bitterman P.
      Uterine carcinosarcoma: immunohistochemical studies on tissue microarrays with focus on potential therapeutic targets.
      • Livasy C.A.
      • Reading F.C.
      • Moore D.T.
      • Boggess J.F.
      • Lininger R.A.
      EGFR expression and HER2/neu overexpression/amplification in endometrial carcinosarcoma.
      Commonly reported findings in carcinosarcomas are intratumoural heterogeneity of HER2 expression/amplification and lower HER2 expression in the sarcoma compared to the carcinoma component.
      • Ross D.S.
      • Devereaux K.A.
      • Jin C.
      • et al.
      Histopathologic features and molecular genetic landscape of HER2-amplified endometrial carcinomas.
      ,
      • Rottmann D.
      • Snir O.L.
      • Wu X.
      • et al.
      HER2 testing of gynecologic carcinosarcomas: tumor stratification for potential targeted therapy.
      ,
      • Jenkins T.M.
      • Cantrell L.A.
      • Stoler M.H.
      • Mills A.M.
      HER2 overexpression and amplification in uterine carcinosarcomas with serous morphology.
      ,
      • Yoshida H.
      • Nishikawa T.
      • Matsumoto K.
      • et al.
      Histopathological features of HER2 overexpression in uterine carcinosarcoma: proposal for requirements in HER2 testing for targeted therapy.
      ,
      • Livasy C.A.
      • Reading F.C.
      • Moore D.T.
      • Boggess J.F.
      • Lininger R.A.
      EGFR expression and HER2/neu overexpression/amplification in endometrial carcinosarcoma.
      It is recognised that tumours diagnosed as uterine clear cell carcinoma (CCC) have substantial morphological, immunohistochemical and molecular overlap with both USC and endometrioid carcinomas.
      • Fadare O.
      • Parkash V.
      • Dupont W.D.
      • et al.
      The diagnosis of endometrial carcinomas with clear cells by gynecologic pathologists: an assessment of interobserver variability and associated morphologic features.
      • DeLair D.F.
      • Burke K.A.
      • Selenica P.
      • et al.
      The genetic landscape of endometrial clear cell carcinomas.
      • Le Gallo M.
      • Rudd M.L.
      • Urick M.E.
      • et al.
      Somatic mutation profiles of clear cell endometrial tumors revealed by whole exome and targeted gene sequencing.
      Cagaanan et al. assessed HER2 status in 229 cases of CCC, endometrioid carcinoma and USC, and found HER2 to be positive in 48% of CCC, 19% of USC and 0% of endometrioid carcinoma. Detailed morphological and immunohistochemical review by a panel of gynaecological pathologists resulted in diagnostic disagreement in eight of 11 HER2-positive CCCs, with these cases showing overlap with USC.
      • Cagaanan A.
      • Stelter B.
      • Vu N.
      • et al.
      HER2 expression in endometrial cancers diagnosed as clear cell carcinoma.
      Aberrant p53 expression was present in 88% (22/25) of HER2-positive cases and all were MMR proficient. The small group of HER2-positive p53 wild-type tumours all lacked staining with oestrogen receptor (ER). These findings highlight the shortcoming of limiting HER2 testing to tumours diagnosed as USC, suggesting that testing of tumours with ambiguous morphology overlapping with USC should also be considered. Another series reported HER2 overexpression or amplification in two of six uterine CCC, with these authors concluding HER2 analysis should be performed on all uterine CCC.
      • Woo J.S.
      • Apple S.K.
      • Sullivan P.S.
      • Rao J.-Y.
      • Ostrzega N.
      • Moatamed N.A.
      Systematic assessment of HER2/neu in gynecologic neoplasms, an institutional experience.
      In a series of 33 undifferentiated endometrial carcinomas, HER2 IHC was negative,
      • Ramalingam P.
      • Masand R.P.
      • Euscher E.D.
      • Malpica A.
      Undifferentiated carcinoma of the endometrium: an expanded immunohistochemical analysis including PAX-8 and basal-like carcinoma surrogate markers.
      although rare reports of positive staining exist in the literature.
      • Woo J.S.
      • Apple S.K.
      • Sullivan P.S.
      • Rao J.-Y.
      • Ostrzega N.
      • Moatamed N.A.
      Systematic assessment of HER2/neu in gynecologic neoplasms, an institutional experience.
      HER2 testing has been applied to a series of 25 mesonephric-like adenocarcinoma of the uterine corpus. Five cases (20%) exhibited 2+ staining by IHC but all were negative by FISH.
      • Kim H.
      • Na K.
      • Bae G.E.
      • Kim H.-S.
      Mesonephric-like adenocarcinoma of the uterine corpus: comprehensive immunohistochemical analyses using markers for mesonephric, endometrioid and serous tumors.
      A conclusion drawn from these studies is the targeting of tumours for HER2 testing based on molecular classification, triaging all p53-abnormal ‘serous-like’ tumours for HER2 analysis, may be superior to histotype-directed testing.
      • Ross D.S.
      • Devereaux K.A.
      • Jin C.
      • et al.
      Histopathologic features and molecular genetic landscape of HER2-amplified endometrial carcinomas.
      ,
      • Vermij L.
      • Horeweg N.
      • Leon-Castillo A.
      • et al.
      HER2 status in high-risk endometrial cancers (PORTEC-3): relationship with histotype, molecular classification, and clinical outcomes.
      ,
      • Jenkins T.M.
      • Cantrell L.A.
      • Stoler M.H.
      • Mills A.M.
      HER2 overexpression and amplification in uterine carcinosarcomas with serous morphology.
      ,
      • Cagaanan A.
      • Stelter B.
      • Vu N.
      • et al.
      HER2 expression in endometrial cancers diagnosed as clear cell carcinoma.
      It is recognised that interobserver agreement, even among expert gynaecological pathologists, is poor when evaluating high-grade endometrial carcinomas.
      • Gilks C.B.
      • Oliva E.
      • Soslow R.A.
      Poor interobserver reproducibility in the diagnosis of high-grade endometrial carcinoma.
      On this basis, histotype-based selection for HER2 targeted therapy, purely on identification of serous morphology, may potentially leave some eligible patients unselected for HER2 analysis, and immunohistochemistry-based algorithms that include aberrant p53 may offer more objective and exhaustive selection of eligible patients. The findings of Cagaanan et al., where a small group of HER2-positive patients were ER negative/p53 wild-type, suggest adding ER to this triage algorithm may detect further patients eligible for HER2 targeted therapy.
      • Cagaanan A.
      • Stelter B.
      • Vu N.
      • et al.
      HER2 expression in endometrial cancers diagnosed as clear cell carcinoma.
      The application of targeted therapy in HER2 amplified non-serous tumours is yet to be evaluated by large clinical trials and such patients should be included in future studies.

      Ovarian carcinoma

      Various tumours of the ovary have been investigated with HER2 in search of a targetable biomarker. In ovarian mucinous tumours, HER2 overexpression has been noted in a subset of tumours and correlated with HER2 amplification.
      • Lin W.-L.
      • Kuo W.-H.
      • Chen F.-L.
      • et al.
      Identification of the coexisting HER2 gene amplification and novel mutations in the HER2 protein-overexpressed mucinous epithelial ovarian cancer.
      • Chao W.-R.
      • Lee M.-Y.
      • Lin W.-L.
      • Koo C.-L.
      • Sheu G.-T.
      • Han C.-P.
      Assessing the HER2 status in mucinous epithelial ovarian cancer on the basis of the 2013 ASCO/CAP guideline update.
      • McAlpine J.N.
      • Wiegand K.C.
      • Vang R.
      • et al.
      HER2 overexpression and amplification is present in a subset of ovarian mucinous carcinomas and can be targeted with trastuzumab therapy.
      Most studies did not designate the type of mucinous tumour, but the use of ‘carcinoma’ favours that they were mucinous carcinomas,
      • Lin W.-L.
      • Kuo W.-H.
      • Chen F.-L.
      • et al.
      Identification of the coexisting HER2 gene amplification and novel mutations in the HER2 protein-overexpressed mucinous epithelial ovarian cancer.
      ,
      • Chao W.-R.
      • Lee M.-Y.
      • Lin W.-L.
      • Koo C.-L.
      • Sheu G.-T.
      • Han C.-P.
      Assessing the HER2 status in mucinous epithelial ovarian cancer on the basis of the 2013 ASCO/CAP guideline update.
      while a single study determined that six of 33 (18%) of carcinomas and three of 16 (18%) borderline mucinous tumours showed amplification. Overall, amplification is reported in 12–26% of mucinous tumours, often in cases which also harbour TP53 mutations.
      • Mohammed R.A.A.
      • Makboul R.
      • Elsers D.A.H.
      • Elsaba T.M.A.M.
      • Thalab A.M.A.B.
      • Shaaban O.M.
      Pattern of HER-2 gene amplification and protein expression in benign, borderline, and malignant ovarian serous and mucinous neoplasms.
      ,
      • Cheasley D.
      • Wakefield M.J.
      • Ryland G.L.
      • et al.
      The molecular origin and taxonomy of mucinous ovarian carcinoma.
      Limited data are available on these patients in terms of response to targeted therapy.
      • McAlpine J.N.
      • Wiegand K.C.
      • Vang R.
      • et al.
      HER2 overexpression and amplification is present in a subset of ovarian mucinous carcinomas and can be targeted with trastuzumab therapy.
      High-grade serous carcinoma (HGSC) has been examined with particular interest given the promising findings in USC, as both tumours share TP53 as a driver mutation. However, most studies have demonstrated low numbers (between 2–4%) of tumours with amplification or overexpression of HER2,
      • Mohammed R.A.A.
      • Makboul R.
      • Elsers D.A.H.
      • Elsaba T.M.A.M.
      • Thalab A.M.A.B.
      • Shaaban O.M.
      Pattern of HER-2 gene amplification and protein expression in benign, borderline, and malignant ovarian serous and mucinous neoplasms.
      ,
      • Bookman M.A.
      • Darcy K.M.
      • Clarke-Pearson D.
      • Boothby R.A.
      • Horowitz I.R.
      Evaluation of monoclonal humanized anti-HER2 antibody, trastuzumab, in patients with recurrent or refractory ovarian or primary peritoneal carcinoma with overexpression of HER2: a phase II trial of the Gynecologic Oncology Group.
      ,
      • Ersoy E.
      • Cao Q.J.
      • Otis C.N.
      HER2 protein overexpression and gene amplification in tubo-ovarian high-grade serous carcinomas.
      with low response rates noted in studies where patients were treated with targeted therapies.
      • Bookman M.A.
      • Darcy K.M.
      • Clarke-Pearson D.
      • Boothby R.A.
      • Horowitz I.R.
      Evaluation of monoclonal humanized anti-HER2 antibody, trastuzumab, in patients with recurrent or refractory ovarian or primary peritoneal carcinoma with overexpression of HER2: a phase II trial of the Gynecologic Oncology Group.
      ,
      • Teplinsky E.
      • Muggia F.
      Targeting HER2 in ovarian and uterine cancers: challenges and future directions.
      A preclinical study which examined antitumour activity in 10 epithelial ovarian cancer cell lines, six serous and four CCC, showed treatment effect in the four tumours exhibiting HER2 overexpression (comprising 2 serous carcinomas and 2 CCC).
      • Menderes G.
      • Bonazzoli E.
      • Bellone S.
      • et al.
      Superior in vitro and in vivo activity of trastuzumab-emtansine (T-DM1) in comparison to trastuzumab, pertuzumab and their combination in epithelial ovarian carcinoma with high HER2/neu expression.
      However, guidelines for testing and treatment in ovarian HGSC are currently not available for routine practice.
      Ovarian carcinosarcoma, like its counterpart in the uterine corpus, can harbour a component of serous carcinoma which may show HER2 overexpression and/or amplification, although in the limited studies of this relatively rare tumour, low positivity (7%) has been reported
      • Rottmann D.
      • Snir O.L.
      • Wu X.
      • et al.
      HER2 testing of gynecologic carcinosarcomas: tumor stratification for potential targeted therapy.
      while 25–28% positivity rate was observed in cell lines in three studies.
      • Menderes G.
      • Bonazzoli E.
      • Bellone S.
      • et al.
      SYD985, a novel duocarmycin-based HER2-targeting antibody-drug conjugate, shows antitumor activity in uterine and ovarian carcinosarcoma with HER2/Neu expression.
      • Schwab C.L.
      • English D.P.
      • Black J.
      • et al.
      Neratinib shows efficacy in the treatment of HER2 amplified carcinosarcoma in vitro and in vivo.
      • Guzzo F.
      • Bellone S.
      • Buza N.
      • et al.
      HER2/neu as a potential target for immunotherapy in gynecologic carcinosarcomas.
      Efficacy in this tumour type is also difficult to ascertain, with available data only pertaining to activity in cell lines.
      • Menderes G.
      • Bonazzoli E.
      • Bellone S.
      • et al.
      SYD985, a novel duocarmycin-based HER2-targeting antibody-drug conjugate, shows antitumor activity in uterine and ovarian carcinosarcoma with HER2/Neu expression.
      • Schwab C.L.
      • English D.P.
      • Black J.
      • et al.
      Neratinib shows efficacy in the treatment of HER2 amplified carcinosarcoma in vitro and in vivo.
      • Guzzo F.
      • Bellone S.
      • Buza N.
      • et al.
      HER2/neu as a potential target for immunotherapy in gynecologic carcinosarcomas.
      A meta-analysis examining the association between HER2 expression and prognosis in over 5000 ovarian cancers determined that HER2 expression correlated with reduced overall and progression-free survival, supporting its use as a prognostic biomarker in this setting.
      • Luo H.
      • Xu X.
      • Ye M.
      • Sheng B.
      • Zhu X.
      The prognostic value of HER2 in ovarian cancer: a meta-analysis of observational studies.
      Further study is warranted to establish the role of HER2 analysis, scoring guidelines and potential therapeutic value of HER2 targeted therapy in ovarian tumours. In the absence of established guidelines for testing and treatment in this setting, HER2 analysis could be considered in an individualised fashion, at the discretion of the clinical and pathology team.

      Cervical carcinoma

      Attempts to characterise HER2 expression in cervical carcinomas have yielded varied results, and to date the prognostic and predictive role of this biomarker in the cervix remains ill-defined. A meta-analysis of 26 studies referencing standardised ASCO/CAP breast or gastric guidelines when assessing HER2 status in cervical cancer, found the estimated pooled prevalence of HER2 overexpression by IHC to be 5.7%.
      • Itkin B.
      • Garcia A.
      • Straminsky S.
      • et al.
      Prevalence of HER2 overexpression and amplification in cervical cancer: a systematic review and meta-analysis.
      Several immunohistochemical studies have reported disparate findings in the cervix. Ueda et al. found 46% (20/43) of adenocarcinomas of varied histotype (HPV-associated and independent) were positive for both HER2 (2+ or 3+ staining, 10% cut-off) and epidermal growth factor receptor (EGFR), with this finding significantly associated with lymph node metastasis, tumour stage and shorter relapse-free survival, suggesting that the co-expression of these biomarkers has prognostic value in cervical adenocarcinoma.
      • Ueda A.
      • Takasawa A.
      • Akimoto T.
      • et al.
      Prognostic significance of the co-expression of EGFR and HER2 in adenocarcinoma of the uterine cervix.
      A larger immunohistochemical study of 297 cervical adenocarcinomas of varied histotype found 3.8% of HPV-independent-gastric-type adenocarcinomas and 12.5% of HPV-associated mucinous carcinomas displayed HER2 overexpression.
      • Stolnicu S.
      • Barsan I.
      • Hoang L.
      • et al.
      Diagnostic algorithmic proposal based on comprehensive immunohistochemical evaluation of 297 invasive endocervical adenocarcinomas.
      Given the efficacy of HER2 targeted therapy in gastric cancer, there has been interest in investigating HER2 status in gastric type adenocarcinomas of the cervix. In one study, HER2 IHC staining was equivocal in six of 13 cases, with HER2 amplification identified in only one case.
      • Nakamura A.
      • Yamaguchi K.
      • Minamiguchi S.
      • et al.
      Mucinous adenocarcinoma, gastric type of the uterine cervix: clinical features and HER2 amplification.
      Most of these data suggest that only a small subset of cervical cancer patients might benefit from the routine application of HER2 analysis.
      Numerous molecular analyses of cervical carcinoma have been performed. The Cancer Genome Atlas report of 228 cervical cancers found that 17% harboured ERBB2 amplifications, with these more often seen in adenocarcinoma than squamous cell carcinoma.
      The Cancer Genome Atlas Research Network
      Integrated genomic and molecular characterization of cervical cancer.
      In another molecular study of 1015 cervical carcinomas of varied histotype, ERBB2 mutations were detected at rates of 2% for squamous cell carcinoma, 4.5% for adenocarcinoma and 10% for neuroendocrine carcinoma.
      • Xiang L.
      • Jiang W.
      • Ye S.
      • et al.
      ERBB2 mutation: a promising target in non-squamous cervical cancer.
      In this study, patients with ERBB2 mutation had worse prognosis than those without. Concurrent PIK3CA or KRAS mutations were detected in a significant proportion of these patients (19%); notably, mutations in the PIK3CA/PTEN pathway are known to be associated with resistance to HER2 targeted therapies.
      • Rimawi M.F.
      • De Angelis C.
      • Contreras A.
      • et al.
      Low PTEN levels and PIK3CA mutations predict resistance to neoadjuvant lapatinib and trastuzumab without chemotherapy in patients with HER2 over-expressing breast cancer.
      Zammataro et al. applied whole exome sequencing to 69 cervical squamous cell carcinomas and adenocarcinomas, most HPV-associated, and found 5.8% harboured ERBB2 mutations, also demonstrating response to combined anti-HER2 and PIK3CA inhibitors in experimental cervical tumour xenografts harbouring derangements in the ERBB2/PI3K/APT/mTOR pathway.
      • Zammataro L.
      • Lopez S.
      • Bellone S.
      • et al.
      Whole-exome sequencing of cervical carcinomas identifies activating ERBB2 and PIK3CA mutations as targets for combination therapy.
      Finally, a molecular analysis of 68 gastric-type adenocarcinomas of the cervix and vagina found that 8% harboured ERBB2 and 10% ERBB3 mutations.
      • Selenica P.
      • Alemar B.
      • Matrai C.
      • et al.
      Massively parallel sequencing analysis of 68 gastric-type cervical adenocarcinomas reveals mutations in cell cycle-related genes and potentially targetable mutations.
      The correlation between ERBB2 mutation and HER2 overexpression and/or amplification and treatment response remain to be established in this context.
      In terms of clinical application of HER2 targeted therapy, isolated case reports document sustained disease response in patients with metastatic HER2-positive cervical cancer
      • Fortman D.
      • Issa R.
      • Stanbery L.
      • Albrethsen M.
      • Nemunaitis J.
      • Kasunic T.
      HER2-positive metastatic cervical cancer responsive to first and second-line treatment: a case report.
      and a Phase II basket trial investigating use of neratinib (a pan-HER tyrosine inhibitor) in solid tumours has reported clinical benefit in 50% cervical cancer patients, with three of 12 patients having a partial response and three stable disease.
      • Oaknin A.
      • Friedman C.F.
      • Roman L.D.
      • et al.
      Neratinib in patients with HER2-mutant, metastatic cervical cancer: findings from the phase 2 SUMMIT basket trial.
      In a recently published consensus paper, the International Society of Gynaecological Pathologists (ISGyP) reflect on these aforementioned limited data, concluding there is currently insufficient evidence guiding use of predictive biomarkers, including HER2, in cervical adenocarcinoma.
      • Bosse T.
      • Lax S.
      • Abu-Rustum N.
      • Matias-Guiu X.
      The role of predictive biomarkers in endocervical adenocarcinoma: recommendations from the International Society of Gynecological Pathologists.
      Guidance for scoring of HER2 in this context is also lacking and future pathology-based clinical trials are required to establish these scoring systems and elucidate the predictive value of HER2 overexpression or amplification in this context.

      Vulvar carcinoma

      Frequent gene alterations in ERBB2 have been described in extramammary Paget disease (PD), suggesting this may represent a driver mutation during disease development and progression.
      • Ishida Y.
      • Kakiuchi N.
      • Yoshida K.
      • et al.
      Unbiased detection of driver mutations in extramammary Paget disease.
      Overexpression of HER2 has been demonstrated in 23–80% of vulvar PD
      • Brummer O.
      • Stegner H.-E.
      • Böhmer G.
      • Kühnle H.
      • Petry K.-U.
      HER-2/neu expression in Paget disease of the vulva and the female breast.
      • Richter C.E.
      • Hui P.
      • Buza N.
      • et al.
      HER-2/NEU overexpression in vulvar Paget disease: the Yale experience.
      • Gatalica Z.
      • Vranic S.
      • Krušlin B.
      • et al.
      Comparison of the biomarkers for targeted therapies in primary extra-mammary and mammary Paget’s disease.
      with overexpression in extramammary PD shown to be more prevalent in invasive lesions and to correlate with lymph node metastasis.
      • Richter C.E.
      • Hui P.
      • Buza N.
      • et al.
      HER-2/NEU overexpression in vulvar Paget disease: the Yale experience.
      ,
      • Masuguchi S.
      • Jinnin M.
      • Fukushima S.
      • et al.
      The expression of HER-2 in extramammary Paget’s disease.
      ,
      • Tanaka R.
      • Sasajima Y.
      • Tsuda H.
      • et al.
      Human epidermal growth factor receptor 2 protein overexpression and gene amplification in extramammary Paget disease: HER2 in extramammary Paget disease.
      Isolated case reports and a small series have described the successful use of trastuzumab, often combined with paclitaxel, in patients with advanced HER2-positive vulvar PD.
      • Bartoletti M.
      • Mazzeo R.
      • De Scordilli M.
      • et al.
      Human epidermal growth factor receptor-2 (HER2) is a potential therapeutic target in extramammary Paget’s disease of the vulva.
      • Ichiyama T.
      • Gomi D.
      • Fukushima T.
      • et al.
      Successful and long-term response to trastuzumab plus paclitaxel combination therapy in human epidermal growth factor receptor 2-positive extramammary Paget’s disease: a case report and review of the literature.
      • Hanawa F.
      • Inozume T.
      • Harada K.
      • Kawamura T.
      • Shibagaki N.
      • Shimada S.
      A case of metastatic extramammary Paget’s disease responding to trastuzumab plus paclitaxel combination therapy.
      • Ishizuki S.
      • Nakamura Y.
      Extramammary Paget’s disease: diagnosis, pathogenesis, and treatment with focus on recent developments.
      A clinical trial to investigate trastuzumab in patients with HER2-positive vulvar PD (NCT01427244) was unfortunately withdrawn. Future clinical trials are required to establish scoring criteria and efficacy of HER2 targeted therapy in this context.
      A recent analysis of 125 cases of vulvar squamous cell carcinoma, 80 with lymph node metastasis, examined IHC expression of EGFR, HER2, HER3 and HER4 using tissue microarray.
      • Baiocchi G.
      • Freitas R.R.
      • Bovolim G.
      • Badiglian-Filho L.
      • Costa A.A.B.A.
      • De Brot L.
      Immunohistochemical expression of ErbB/HER family proteins in patients with vulvar cancer.
      Positive HER2 expression was found in 0.9% of cases and in none of the lymph node metastases. Rates of EGFR and HER3 were also low, however HER4 was expressed in 22.8% of cases and was significantly related to adverse prognostic factors such as histological grade and presence of lymphovascular and perineural invasion. Another study found HER2 overexpression in only two of 101 vulvar squamous cell carcinomas and in three of 45 nodal metastases.
      • Garganese G.
      • Inzani F.
      • Fragomeni S.M.
      • et al.
      The Vulvar Immunohistochemical Panel (VIP) project: molecular profiles of vulvar squamous cell carcinoma.
      Several recent case reports document mammary-like adenocarcinomas of the vulva with HER2 overexpression;
      • Grewal J.K.
      • Eirew P.
      • Jones M.
      • et al.
      Detection and genomic characterization of a mammary-like adenocarcinoma.
      • Niakan S.
      • Love H.
      • Cao Q.
      • Kawar N.
      Primary invasive lobular carcinoma arising in mammary-like glands of the vulva managed with neoadjuvant trastuzumab-based chemotherapy, excision, and sentinel lymph node biopsy.
      • Xie Y.
      • Mata D.G.M.M.
      • Huang W.-Y.
      • Djordjevic B.
      • Jerzak K.J.
      A growing vulvar mass in a post-menopausal woman.
      one of these included a summary of reported cases of vulvar mammary-like adenocarcinoma, with 14% (2/14) exhibiting HER2 overexpression.
      • Niakan S.
      • Love H.
      • Cao Q.
      • Kawar N.
      Primary invasive lobular carcinoma arising in mammary-like glands of the vulva managed with neoadjuvant trastuzumab-based chemotherapy, excision, and sentinel lymph node biopsy.
      Two cases report use of targeted HER2 therapy. One patient achieved complete pathological response on excision of the vulvar primary, complicated by brain recurrence, with stable disease on HER2 targeted therapy at 4 years post-initial diagnosis.
      • Niakan S.
      • Love H.
      • Cao Q.
      • Kawar N.
      Primary invasive lobular carcinoma arising in mammary-like glands of the vulva managed with neoadjuvant trastuzumab-based chemotherapy, excision, and sentinel lymph node biopsy.
      The second patient did not respond to targeted therapy and died 2 years and 5 months post-initial diagnosis.
      • Grewal J.K.
      • Eirew P.
      • Jones M.
      • et al.
      Detection and genomic characterization of a mammary-like adenocarcinoma.

      Conclusions and future directions

      The confirmation in 2018 that trastuzumab provides a progression-free and overall survival benefit in advanced and recurrent HER2-positive USC has prompted development of site-specific guidelines to correctly identify patients eligible for targeted therapy, and spurred interest in applying this therapeutic option to other female genital malignancies. Currently, justification for the universal application of HER2 analysis in all gynaecological malignancies is lacking and the potential role of HER2 beyond USC requires further study, including large scale clinical trials ideally with pathologist input, in an effort to devise site-specific HER2 scoring criteria. Despite this, case reports and small series suggest that individualised testing and treatment in selected tumours from other gynaecological sites may be appropriate and clinically beneficial.
      Many lines of investigation require future analysis. Clinical trial data are required to determine the optimal testing modality and the IHC expression/FISH amplification profiles that best predict response to anti-HER2 therapies. The question of whether tumours with 1+ or 2+ (ISH negative) IHC expression might respond to targeted therapy also warrants investigation, particularly in light of recent developments with HER2-low breast cancers. The potential tailoring of scoring guidelines to deal with small biopsies, where tumour heterogeneity may result in a false negative result, should be explored; this issue has been addressed in scoring of gastric cancer in biopsy specimens, where any strong membranous expression is regarded as 3+ regardless of the percentage.
      • Ross J.S.
      • Mulcahy M.
      HER2 testing in gastric/gastroesophageal junction adenocarcinomas: unique features of a familiar test.
      The prognostic and predictive implications of tumour heterogeneity for HER2 expression and/or amplification is another area requiring investigation. Established guidelines on when to test and retest specimens during the course of an individual patient's disease are also required. Finally, as somatic ERBB2 mutations are reported across multiple tumour types,
      • Connell C.M.
      • Doherty G.J.
      Activating HER2 mutations as emerging targets in multiple solid cancers.
      ,
      • Stephens P.
      • Hunter C.
      • Bignell G.
      • et al.
      Lung cancer: intragenic ERBB2 kinase mutations in tumours.
      ,
      • Schram A.
      • Won H.H.
      • Andre F.
      • et al.
      Abstract LB-103: landscape of somatic ERBB2 mutations: findings from AACR GENIE and comparison to ongoing ERBB2 mutant basket study.
      ,
      • Pahuja K.B.
      • Nguyen T.T.
      • Jaiswal B.S.
      • et al.
      Actionable activating oncogenic ERBB2/HER2 transmembrane and juxtamembrane domain mutations.
      the potential role of HER2 targeted therapy in HER2 mutant cancers is being evaluated in pan-cancer trials.
      • Connell C.M.
      • Doherty G.J.
      Activating HER2 mutations as emerging targets in multiple solid cancers.
      ,
      • Oh D.-Y.
      • Bang Y.-J.
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      ,
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      Although preclinical data suggest that HER2 mutation may confer sensitivity to HER2 targeted therapies in these tumours, significant variation in response exists between different tumour types,
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      • Doherty G.J.
      Activating HER2 mutations as emerging targets in multiple solid cancers.
      reinforcing the need for ongoing research in this field.

      Conflicts of interest and sources of funding

      The authors state that there are no conflicts of interest to disclose.

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