Expression of HER2, HER3, and TROP2 in primary tumors and brain metastases of breast cancer

Expression of HER2, HER3, and TROP2 in primary tumors and brain metastases of breast cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Expression of HER2, HER3, and TROP2 in primary tumors and brain metastases of breast cancer Shota Kusuhara, Takahiro Kogawa, Mototsugu Shimokawa, Chikako Funasaka, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7834157/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Mar, 2026 Read the published version in Breast Cancer → Version 1 posted 5 You are reading this latest preprint version Abstract Background Human epidermal growth factor receptor 2 (HER2)-directed antibody–drug conjugate (ADC) therapy has shown efficacy in HER2-positive breast cancer brain metastases. However, there is still an unmet medical need for further exploration of other suitable targets for ADCs in brain metastases (BMs). Methods The expression of HER2, HER3, and trophoblast surface antigen 2 (TROP2) was evaluated using immunohistochemistry (IHC) in pairs of primary tumors (PTs) and surgically resected BMs from 44 patients with breast cancer. Expression levels were classified as 0, 1+, 2+, or 3 + based on IHC intensity and the proportion of positive cells. Results The analysis revealed that HER3 and TROP2 expression (IHC ≥ 1+) was observed in all but one BM specimen. TROP2 was highly expressed (IHC ≥ 2+) in both BMs and PTs (86% and 70%, respectively; p = 0.11). High expression of HER3 was more frequent in BMs than in PTs (91% and PTs 59%, respectively; p < 0.01), regardless of breast cancer subtype. In individual paired samples, 95.5% (42/44) exhibited equal or higher HER3 expression in BMs than in PTs. In contrast, high HER2 expression (IHC ≥ 2+) was observed in similar proportions between BMs and PTs (43% and 41%, respectively; p = 1.00). Conclusions The observation of more frequent high-level expression of HER3 in BMs than in PTs, and high-level expression of TROP2 in both BMs and PTs suggests the potential of HER3- and TROP2-based ADC therapy for BMs from breast cancer. Further prospective studies are warranted to validate this hypothesis. Breast cancer brain metastasis HER2 HER3 TROP2 Figures Figure 1 Figure 2 Figure 3 Highlights HER3 and TROP2 were expressed at immunohistochemistry (IHC) 1+ or higher in all but one brain metastasis (BM) cases. High-level (IHC 2+ or 3+) HER3 expression was more frequent in BMs than in primary tumors (91% vs. 59% [p<0.01]). This study suggests the potential of HER3- and TROP2-targeting antibody–drug conjugates as drugs for breast cancer BMs. Introduction Brain metastases (BM) occur in one-third of breast cancer patients during their disease course. 1 Although local therapeutic interventions, such as whole brain radiation therapy (WBRT), stereotactic irradiation (SRI), and neurosurgery, are employed to manage BMs, 2 patient prognosis remains poor, with a median overall survival (OS) ranging from 2 to 16 months. 3 Once metastasis to the brain occurs, patients typically experience a significant decline in quality of life, culminating in organ failure and death. 4 Traditionally, the effectiveness of drug therapy for BMs has been considered low, 5 possibly because of the presence of the blood–brain barrier. However, in human epidermal growth factor receptor 2 (HER2)-positive (HER2+) breast cancer, new classes of drugs have been observed to challenge this notion. For instance, antibody–drug conjugates (ADCs), which comprise targeting antibodies that bind to cytotoxic payloads, can offer advantages over “naked” cytotoxic drugs. These benefits include a high therapeutic index, bystander killing through payload dispersion, and anti-tumor immune activity via antibody-dependent cytotoxicity. 6 One notable ADC is trastuzumab deruxtecan (T-DXd), which consists of a humanized HER2-directed monoclonal antibody and DXd, a topoisomerase inhibitor. T-DXd has shown promising clinical outcomes in trials involving HER2 + metastatic breast cancer patients with BMs. 7 – 10 Although active/progressing BM were excluded from the three pivotal studies DESTINY-Breast01, 02 and 03, a subsequent pooled analysis of patients with baseline BM in these trials demonstrated that T-DXd is effective in patients with stable BM and in active/untreated BM. In the recent Phase IIIb DESTINY-Breast12 study, which included a cohort with 263 patient at baseline BM, intra-clinical activity of T-DXd could be confirmed across all types of BM including active/progressing BM. 11 In addition, although T-DXd has demonstrated clinical activity against breast cancer with HER2-low expression, 12 its effectiveness against BMs from such HER2 non-overexpressing diseases remains uncertain. Therefore, other potential target proteins expressed in BMs should be explored. Recently, ADCs targeting cell surface proteins other than HER2, such as trophoblast surface antigen 2 (TROP2) 13 and HER3, 14 have emerged. TROP2 is a widely expressed glycoprotein belonging to the epithelial cell adhesion molecule family. 15 Sacituzumab govitecan (SG) is an ADC that combines SN-38, a topoisomerase inhibitor and active metabolite of irinotecan, with an anti-TROP2 monoclonal antibody. 16 SG has been approved by the Food and Drug Administration for treating advanced triple-negative breast cancer (TNBC) and hormone receptor-positive (HR+)/HER2-negative (HER2–) breast cancer. This approval was based on the results of phase III trials demonstrating the superiority of SG over physician’s choice treatments. 17 , 18 In these trials, SG achieved a 35% objective response rate (ORR) for extracranial lesions in patients with TNBC. 17 However, the intracranial response rate was modest, at only 3% (1/32). 19 HER3 is highly expressed in many solid tumors, including 30% to 50% of breast cancers. 20 – 22 High HER3 expression in breast cancer was reported to be associated with reduced OS. 21, 23 Patritumab deruxtecan (HER3-DXd) is a novel, first-in-class anti-HER3 ADC currently under clinical development for multiple indications such as non-small cell lung cancer (NSCLC) and breast cancer. A phase I/II study involving the use of HER3-DXd in patients with advanced breast cancer expressing HER3 also reported promising anti-tumor activity (ORRs of 30.1%, 22.6%, and 42.9% for patients with hormone receptor (HR)+/HER2– [n = 113], TNBC [n = 53], and HER2+ [n = 14], respectively). 14 Additionally, a phase II ICARUS-Breast 01 study evaluating HER3-DXd in patients with HR+/HER2– metastatic breast cancer who progressed on endocrine therapy and one line of chemotherapy also showed antitumor activity with ORR of 53.5% and a median progression free survival of 9.4 months. 24 In HERTHENA-Lung01, a phase II trial of HER3-DXd in advanced epidermal growth factor receptor-mutated NSCLC, the confirmed ORR was 29.8% and the intracranial ORR was 33.3% (10/30 patients; 95% CI, 17.3 to 52.8). 25 Despite the emerging potential of ADCs in treating BMs and their clinical applicability in targeting different proteins, evaluating target proteins for BMs is difficult in most cases owing to poor tumor accessibility. Tumors can alter their molecular profile during the development of BMs, 22 making it clinically relevant to understand the correlation between the expression levels of ADC target proteins, such as HER2, HER3, and TROP2 in primary tumors (PTs) and BMs in breast cancer. However, this area remains largely unexplored. Therefore, this study was undertaken to evaluate the expression patterns of HER2, HER3, and TROP2 in paired samples of surgically resected BMs and PTs from patients with breast cancer using immunohistochemistry (IHC). Methods Patients Patients with breast cancer who underwent surgical resection of BMs at the National Cancer Center Hospital in Japan were identified serially between January 2000 and December 2017. From this cohort, patients with available archival tissues from both resected BMs and PTs were selected. Tissue availability was assessed by pathologists (K.S., M.Y., and S.F.). Patient demographic information was collected through a retrospective chart review. IHC assays of PTs and BMs BM and PT tissues were subjected to IHC staining for estrogen receptor (ER), progesterone receptor (PgR), HER2, HER3, and TROP2 using an automated slide stainer (Ventana BenchMark ULTRA; Roche Diagnostics K.K., Basel, Switzerland). The Ventana UltraView Confirm ER (clone: SP1, Roche Diagnostics K.K.) and Ventana UltraView Confirm PGR (clone: 1E2, Roche Diagnostics K.K.) were used for ER and PgR IHC, respectively. The Ventana I-VIEW PATHWAY™ HER2 (clone: 4B5, Roche Diagnostics K.K.) and I-VIEW universal kit (Roche Diagnostics K.K.) were used for HER2 IHC. Anti-HER3 (clone: 7.3.8, Ventana Medical Systems, Inc., Oro Valley, US) and anti-TROP2 (clone: SP295, Abcam, Cambridge, UK) antibodies were used as primary antibodies for HER3 and TROP2 IHC, respectively. Heat-induced epitope retrieval for all targets was performed using ULTRA Cell Conditioning Solution #1 (Benchmark ULTRA CC1, Roche Diagnostics K.K.). The UltraView DAB IHC Detection Kit (Roche Diagnostics K.K.) was used to detect the chromogenic IHC signals of HER2, whereas the Ventana UltraView Universal DAB Detection Kit (Roche Diagnostics K.K.) was used to detect those of other targets. IHC for ER and PgR was evaluated by a pathologist (S.F.). ER and PgR statuses were classified as positive if ≥ 1% of tumor cells expressed the proteins. 26 Membranous IHC staining intensity for HER2, HER3, and TROP2 in tumor cells was scored visually by pathologists (R.N. and N.M.) using a microscope. HER2 IHC staining intensity (0, 1+, 2+, and 3+) was scored according to the updated 2018 American Society of Clinical Oncology/College of American Pathologists guidelines for HER2 testing in breast cancer. 23 In this study, PTs classified as HER2 2 + were considered HER2 + if there was a confirmation of either HER2 in situ hybridization (ISH) positivity or HER2 IHC 3 + in the pathology report documented in the medical record. Two HER2 + cases in the luminal group were considered HER2– because neither of them met the above definition. Three HER2 2 + cases were considered HER2 + because of ISH positivity (luminal-HER2, n = 1; pure HER2, n = 2). Breast cancer subtypes were classified according to the expression patterns of HR, ER, and PgR, and HER2 in PTs as follows: luminal (HR+/HER2–), TNBC (HR-/HER2–), luminal-HER2 (HR+/HER2+), and pure HER2 (HR–/HER2+). For HER3 and TROP2, IHC membranous staining intensity (0, 1+, 2+, and 3+) was scored using criteria generally applied to HER2 IHC scoring for gastric cancer: 24 0, no reactivity or membranous reactivity in < 10% of tumor cells; 1+, faint/barely perceptible membranous reactivity in ≥ 10% of tumor cells; 2+, weak to moderate complete, basolateral, or lateral membranous reactivity in ≥ 10% of tumor cells; 3+, strong complete, basolateral, or lateral membranous reactivity in ≥ 10% of tumor cells. An H-score (0–300) was also calculated based on membranous staining intensity using the following formula: H score = 3 × (percentage of strongly positive tumor cells) + 2 × (percentage of weakly to moderately positive tumor cells) + 1 × (percentage of faintly/barely perceptible positive tumor cells). 25 Representative expression patterns of HER2, HER3, and TROP2 are depicted in Fig. 1 . Statistics The proportions of high-level expression (IHC 2+/3+) for each protein were compared using McNemar’s test. A paired t-test was used to compare the H-scores of HER3 between PTs and BMs. OS, defined as the time from resection of BMs to death from any cause or the data cut-off date, was estimated using a log-rank test. The follow-up period was censored on December 31, 2017. OS was compared according to HER3 expression levels in BMs and changes in expression from PTs to BMs. Statistical analyses were performed using EZR software, version 1.62. Ethics This study was approved by the Institutional Review Board (IRB) of the National Cancer Center Hospital (IRB number: 2017 − 502). The IRB waived the requirement for written informed consent from the study participants. Results Patient characteristics A total of 44 patients were enrolled in this study. The clinical characteristics of the patients at the time of brain surgery are detailed in Table 1 . The median age was 53 (34–78) years, and most patients (88.6%) were under the age of 65 years. All but one patient had a performance status of 0 or 1; most patients had symptoms of BM. The median time from initial diagnosis of breast cancer to brain surgery was 2.4 years (range, 0.1–21.9) years. All but three patients had three or fewer BMs; however, a majority had at least one BM measuring ≥ 3 cm, potentially limiting initial SRI indications. BMs were the sole metastases in 11 (25%) patients. Sixteen patients (36.4%) had primary HER2 + disease, a proportion higher than that observed in the general population of breast cancer. Following surgical intervention, 36 (81.8%) and 5 (11.4%) patients underwent WBRT and SRI, respectively. Table 1 Patient characteristics at brain surgery Characteristics Patients (N = 44) Age Median, years (range) 53 (34–78) ≥ 65, n (%) 5 (11.4%) Sex Female 44 (100%) ECOG PS, n (%) 0 7 (15.9%) 1 36 (81.8%) 2 1 (2.3%) Symptoms from brain metastases Symptomatic 39 (88.6%) Asymptomatic 5 (11.4%) Time from diagnosis of metastatic disease to developing brain metastases < 6 months 14 (31.8%) 6 months ≤ 30 (68.2%) De novo stage IV Yes 9 (20.5%) No 35 (79.5%) Number of brain metastases 1 29 (65.9%) 2–3 12 (27.3%) more than 3 3 (6.8%) Maximum size of brain metastases < 3cm 6 (13.6%) 3cm ≤ 38 (86.4%) Concomitant extracranial metastases Present 33 (75.0%) lymph node 13 (29.5%) bone 13 (29.5%) liver 6 (13.6%) lung 14 (31.8%) Absent 11 (25.0%) Breast cancer subtype in primary tumor TNBC 8 (18.2%) Luminal 20 (45.5%) Pure HER2 4 (9.1%) Luminal-HER2 12 (27.3%) Treatment for brain metastases Surgery 44 (100%) Post-surgical STI 5 (11.4%) Post-surgical WBRT 36 (81.8%) Abbreviations: ECOG PS, Eastern Cooperative Oncology Group performance status; TNBC, triple-negative breast cancer; Luminal, hormone receptor-positive/HER2-negative; Pure HER2, hormone receptor-negative/HER2-positive; Luminal-HER2, hormone receptor-positive/HER2-positive; STI, stereotactic irradiation; WBRT, whole brain radiation therapy. HER3 was more frequently overexpressed BMs than in PTs All BM samples exhibited a certain level of HER3 expression (≥ IHC 1+; Table 2 and Figs. 2 and 3 ). The proportions of high-level expression (IHC 2+/3+) were higher in BMs than in PTs for HER3 but not for HER2 (HER3: 59% vs. 91% for PT and BMs, respectively (p < 0.01); HER2: 41% vs. 43% for PT and BMs, respectively (p = 1.00); Figs. 2 and 3 and Table 2 ). Table 2 Proportion of IHC2+/3 + of HER2, HER3, and TROP2 in PT and BM HER2 HER3 TROP2 PT BM PT BM PT BM All subtypes 18/44 (41%) 19/44 (43%) 26/44 (59%) 40/44 (91%) 31/44 (70%) 38/44 (86%) TNBC 0/8 (0%) 1/8 (13%) 4/8 (50%) 6/8 (75%) 7/8 (88%) 8/8 (100%) Luminal 2/20 (10%) 3/20 (15%) 12/20 (60%) 18/20 (90%) 12/20 (60%) 14/20 (70%) Luminal HER2 12/12 (100%) 11/12 (92%) 6/12 (50%) 12/12 (100%) 9/12 (75%) 12/12 (100%) Pure HER2 4/4 (100%) 4/4 (100%) 4/4 (100%) 4/4 (100%) 3/4 (75%) 4/4 (100%) Abbreviations: HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; TROP2, trophoblast surface antigen 2; BM, brain metastase; PT, primary tumor; TNBC, triple-negative breast cancer; Luminal, hormone receptor-positive/HER2-negative; Pure HER2, hormone receptor-negative/HER2-positive; Luminal-HER2, hormone receptor-positive/HER2-positive. The tendency for higher HER3 expression in BMs than in PTs was consistent across all breast cancer subtypes except the pure HER2 subtype. The proportions of IHC 2+/3 + in BM vs. PT were 75% vs. 50%, 90% vs. 60%, and 100% vs. 50% for TNBC (HR–/HER2–), luminal (HR+/HER2–), and luminal-HER2 (HR+/HER2+) subtypes, respectively (Table 2 ) (Fig. 3 ). For the pure HER2 subtype (HR–/HER2+), all patients showed IHC 2+/3 + HER3 expression in both BMs and PTs. In the HER2 + subtypes (luminal and pure HER2 subtypes), all 16 cases showed high HER3 expression in BMs (Table 2 ) (Fig. 3 ). On an individual patient basis, 80% (35/44) had higher membranous HER3 H-scores in BMs than in PTs, and the difference between BMs and PTs was statistically significant (Supplementary Fig. 1). For TROP2, all but one BM sample exhibited a certain level of expression (≥ IHC 1+, Table 2 and Fig. 3 ). TROP2 was highly expressed (IHC 2+/3+) in both PTs and BMs in the majority of cases (PTs 70% and BMs 86%, p = 0.11). HER3 expression in BM as a prognostic marker The impact of HER3 expression in BMs on patient survival was explored. No statistically significant difference in survival was observed between HER3 IHC scores in BMs (Supplementary Fig. 2). Similarly, no statistically significant difference in survival was observed between populations with decreased, equal, and increased BM HER3 expression compared with those with PT HER3 expression (Supplementary Fig. 2). Discussion This study demonstrated that HER3 and TROP2 were expressed at IHC 1 + or higher in all but one BM case, respectively. While TROP2 was highly expressed (IHC 2 + or 3+) in both PTs and BMs in the majority of cases, HER3 was more frequently expressed at high levels in BMs than in PTs (Figs. 2 and 3 and Table 2 ). In addition, in individual paired samples, 95.5% (42/44) of cases exhibited the same or higher HER3 expression in BMs than in PTs (Fig. 3 ). In contrast, HER2 exhibited high-level expression in similar proportions between BMs and PTs (Figs. 2 and 3 and Table 2 ). Our finding of more frequent high-level HER3 expression in BMs than in PTs is consistent with those of previous studies. A breast cancer study demonstrated HER3 positivity by IHC in 59% (22/37) of matched BMs and 30% (11/37) of PTs. 27 Another recent study indicated that HER3 was frequently expressed in BMs from breast cancer, with HER2 + and HER2-low BMs exhibiting significantly higher rates of HER3 co-expression than HER2-null BMs. 28 A study on NSCLC also demonstrated that HER3 was more abundantly expressed in BMs than in matched extracranial samples. 28 Considering that neuregulin 1, the ligand for HER3, is abundantly expressed in the brain and is released via various mechanisms, including hypoxia, 27 HER3 may play a role in the formation of BMs. In addition, HER3 overexpression has been suggested as a mechanism that confers resistance to anti-HER2 therapy. Therefore, the more frequent high-level HER3 expression in BMs than in PTs may result from clonal selection by anti-HER2 systemic treatment. Our finding that all HER2 + subtypes exhibited IHC 2+/3 + HER3 expression in BMs supports this hypothesis and is consistent with previous findings. 28 Although BMs have generally been considered resistant to drug treatment, ADCs, particularly those targeting HER2, have been observed to challenge this notion. Over the past decade, multiple ADCs have been developed for various types of cancer, including breast cancer, with promising clinical efficacy for BMs. Currently, the membranous expression level of the target protein is the most reliable predictor of response to ADCs. 7 The DAISY trial demonstrated that response rates to T-DXd decreased hierarchically with HER2 expression. 29 Additionally, a decrease in HER2 expression level has been suggested as one of the possible resistance mechanisms to T-DXd. These findings emphasize the importance of target protein levels for the efficacy of ADCs. Our finding that virtually all BMs expressed HER3 and TROP2 suggests that these proteins could be optimal targets for ADCs in patients with BMs from breast cancer. However, the intracranial response rate of SG has been reported to be only 3%. 19 It is currently unclear whether factors beyond target expression affect the intracranial activity of ADCs or if it depends more on the properties of the drugs. The TUXEDO-2 study is ongoing to evaluate the intracranial activity of datopotamab deruxutecan, another anti-TROP2 ADC, in patients with metastatic TNBC with active brain metastases (NCT05866432). Results from the first stage exploratory cohort were encouraging, three intracranial responses of seven evaluable patients, and the study has progressed to the second stage. 30 The DATO-BASE study (NCT06176261) is also ongoing to evaluate intracranial responses of Dato-DXd in patients with HER2– metastatic breast cancer. 31 Notably, a clinical trial specifically evaluating the clinical activity of HER3-DXd in breast cancer BMs is ongoing. 32 This study had several limitations. First, the sample size was relatively small, which may have limited the statistical power. Second, there were significant time gaps, up to 22 years, between the collection of PTs and BMs, raising concerns about differences in antigen preservation. However, considering that HER2 and TROP2 were expressed at similar levels and frequencies in both PTs and BMs (Table 2 ), the observed tendency for higher HER3 expression in BMs than in PTs is unlikely to be solely due to better antigen preservation in BMs. Third, only surgically removed BMs were included in this study, suggesting that these patients were diagnosed with oligometastases or oligoprogression of breast cancer. Patients with more advanced diseases, such as multiple BMs, might have different biological characteristics, potentially introducing bias in patient selection. In addition, this may explain why we did not observe a significant difference in survival by HER3 IHC scores in BM (Supplementary Fig. 2). In conclusion, this study demonstrated that HER3 was more frequently expressed at high levels in BMs than in PTs, and that TROP2 was highly expressed in both PTs and BMs. As multiple ADCs with various targets and payloads continue to be developed, HER3- and TROP2- ADCs may emerge as key drugs for BMs, regardless of the breast cancer subtype in the PT; however, this hypothesis warrants validation through clinical trials. Abbreviations ADCs, antibody–drug conjugates; BMs, brain metastases; Dato-DXd, Datopotamab deruxtecan; ER, estrogen receptor; HER2–, HER2-negative; HER2+, HER2-positive; HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; HER3-DXd, Patritumab deruxtecan; HR+, hormone receptor-positive; IHC, immunohistochemistry; IRB, Institutional Review Board; NSCLC, non-small cell lung cancer; ORRs, objective response rates; OS, overall survival; PgR, progesterone receptor; PTs, primary tumors; SG, sacituzumab govitecan; SRI, stereotactic irradiation; T-DXd, trastuzumab deruxtecan; TNBC, triple-negative breast cancer; TROP2, trophoblast surface antigen 2; WBRT, whole brain radiation therapy Declarations Acknowledgments Yuka Nakamura, Kokichi Honda, and Takanori Maejima performed IHC for ER and PgR, HER2, and TROP2, respectively. Ryoko Wanikawa selected appropriate slides of primary breast cancer and brain metastases. Editage provided language support and writing assistance. Funding This work was supported by JSPS KAKENHI [grant number 20K08973] and Daiichi Sankyo Co., Ltd. Author information Authors and Affiliations Department of Medical Oncology, National Cancer Center Hospital East, 6-5-1 Kashiwanoha, Kashiwa, Chiba, 277-8577, Japan Shota Kusuhara, Takahiro Kogawa, Chikako Funasaka, Chihiro Kondoh, Kenichi Harano, Nobuaki Matsubara, Yoichi Naito, Ako Hosono, Toru Mukohara Department of Advanced Medical Development, The Cancer Institute Hospital of Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo, 135-0063, Japan Takahiro Kogawa Department of Biostatistics, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamiogushi, Ube, Yamaguchi, 755-8505, Japan Mototsugu Shimokawa Department of Diagnostic Pathology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan Kaishi Satomi, Masayuki Yoshida Department of Molecular Pathology, Yokohama City University Hospital, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa, 236-0004, Japan Satoshi Fujii Department of Breast Surgery, National Cancer Center Hospital East, 6-5-1 Kashiwanoha, Kashiwa, Chiba, 277-8577, Japan Tatsuya Onishi Department of Breast Surgery, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan Akihiko Suto Department of Medical Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan Kan Yonemori Translational Research Laboratories, Daiichi Sankyo Co., Ltd, Shinagawa R&D Center, 1-2-58 Hiromachi, Shinagawa-ku,Tokyo, 140-0005, Japan Kumiko Koyama, Ryuichi Nakamura, Naoyuki Maeda Department of Neurosurgery and Neuro-Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan Yoshitaka Narita C ontributions S Kusuhara: data curation, formal analysis, writing—original draft, and writing—review. T Kogawa: Conceptualization, resources, data curation, formal analysis, supervision, investigation, writing—original draft, project administration, writing—review, and editing. T Mukohara: Supervision, investigation, writing— original draft, project administration, writing—review, and editing. All authors reviewed the manuscript. Corresponding author Correspondence to Takahiro Kogawa . Ethics declarations Co nflict of Interest Takahiro Kogawa received grants from Daiichi Sankyo, Eli Lilly, and AstraZeneca. Toru Mukohara received research funds from Sysmex, Eisai, MSD, Pfizer, AstraZeneca, Ono, Daiichi-Sankyo, and Gilead Sciences. Ethical approval This study was approved by the Institutional Review Board (IRB) of the National Cancer Center Hospital (IRB number: 2017-502). Informed consent Not Applicable. Registry and the registration no. of the study/trial Not Applicable. Animal studies Not Applicable. References Matsuo S, Watanabe J, Mitsuya K, Hayashi N, Nakasu Y, Hayashi M. 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Da Silva L, Simpson PT, Smart CE, Cocciardi S, Waddell N, Lane A, et al. HER3 and downstream pathways are involved in colonization of brain metastases from breast cancer. Breast Cancer Res. 2010;12:R46. https://doi.org/10.1186/bcr2603 . Tomasich E, Steindl A, Paiato C, Hatziioannou T, Kleinberger M, Berchtold L, et al. Frequent overexpression of HER3 in brain metastases from breast and lung cancer. Clin Cancer Res. 2023. https://doi.org/10.1158/1078-0432.Ccr-23-0020 . Mosele F, Deluche E, Lusque A, Le Bescond L, Filleron T, Pradat Y, et al. Trastuzumab deruxtecan in metastatic breast cancer with variable HER2 expression: the phase 2 DAISY trial. Nat Med. 2023;29:2110–20. https://doi.org/10.1038/s41591-023-02478-2 . Bartsch R, Berghoff AS, Furtner J, Marhold M, Starzer AM, Forstner H, et al. 187P Stage I results of a phase II study of datopotamab deruxtecan (DATO-DXd) in triple-negative breast cancer (TNBC) patients (pts) with active brain metastases (TUXEDO-2). ESMO Open. 2024;9:103209. https://doi.org/10.1016/j.esmoop.2024.103209 . DATO-BASE. a Phase 2 Trial of DATOpotamab-deruxtecan for Breast Cancer Brain MetAstaSEs [Available from: https://clinicaltrials.gov/study/NCT06176261 Bartsch R, Vaz Batista M, Berghoff AS, Furtner J, Marhold M, Oberndorfer F, et al. Patritumab deruxtecan (HER3-DXd) in active brain metastases from metastatic breast and non-small cell lung cancers, and leptomeningeal disease from advanced solid tumors: The TUXEDO-3 phase II trial. J Clin Oncol. 2024;42. https://doi.org/10.1200/JCO.2024.42.16_suppl.TPS2091 . TPS2091-TPS2091. Supplementary Files suppleFigure1.20251010.pdf Supplementary Figure 1: H-scores of membranous HER3 in the primary tumor and brain metastasis of each patient. Abbreviations: HER3, human epidermal growth factor receptor 3 suppleFigure220251010.pdf Supplementary Figure 2: Overall survival from resection of BMs. (A) Overall survival according to HER3 scores in BMs. (B) Overall survival according to the trajectory of HER3 from PTs to BMs: decreased, equal, or increased. Abbreviations: HER3, human epidermal growth factor receptor 3; BMs, brain metastases; PT, Cite Share Download PDF Status: Published Journal Publication published 13 Mar, 2026 Read the published version in Breast Cancer → Version 1 posted Editorial decision: Minor Revision 10 Jan, 2026 Reviewers agreed at journal 08 Nov, 2025 Reviewers invited by journal 06 Nov, 2025 Editor assigned by journal 14 Oct, 2025 First submitted to journal 13 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7834157","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":540881764,"identity":"c5e6daa9-58d3-4bec-a39d-8686bc423a27","order_by":0,"name":"Shota 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10:37:19","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127703,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7834157/v1/a50501ecc5316758bafaa8d5.html"},{"id":96075227,"identity":"bd706da9-031c-4838-8e7b-cd9b678556ad","added_by":"auto","created_at":"2025-11-17 10:37:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1177118,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative expression patterns of HER2, HER3, and TROP2.\u003c/p\u003e\n\u003cp\u003eAbbreviations: HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; TROP2, trophoblast surface antigen 2.\u003c/p\u003e","description":"","filename":"Figure120251010.png","url":"https://assets-eu.researchsquare.com/files/rs-7834157/v1/d6aaf6b6f50cf968b7e588fe.png"},{"id":96075225,"identity":"fef52f22-8698-4453-8c51-5030a2b162a7","added_by":"auto","created_at":"2025-11-17 10:37:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15173,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of IHC scores of HER2, HER3, and TROP2 in primary tumors and brain metastases.\u003c/p\u003e\n\u003cp\u003eAbbreviations: HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; TROP2, trophoblast surface antigen 2.\u003c/p\u003e","description":"","filename":"Figure220251010.png","url":"https://assets-eu.researchsquare.com/files/rs-7834157/v1/fe3bc57c535d4adb00b2a95d.png"},{"id":96075233,"identity":"2a89d9c6-7330-402a-beaf-e5d5c174cf46","added_by":"auto","created_at":"2025-11-17 10:37:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31928,"visible":true,"origin":"","legend":"\u003cp\u003eIHC scores of HER2, HER3, and TROP2 in the primary tumor and brain metastasis of each patient. Luminal, TNBC, pure HER2, and luminal HER2 indicates hormone receptor (HR)-positive/HER2–, HR–/HER2–, HR–/HER2+, and HR+/HER2+. UE indicates unevaluable.\u003c/p\u003e\n\u003cp\u003eAbbreviations: HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; HR, hormone receptor; TNBC, triple-negative breast cancer; TROP2, trophoblast surface antigen 2.\u003c/p\u003e","description":"","filename":"Figure320251010.png","url":"https://assets-eu.researchsquare.com/files/rs-7834157/v1/d20251dbf7093ef88fdddd9d.png"},{"id":104740393,"identity":"fbc8bc8b-7983-48f7-bfaa-8ad0c56a5ee6","added_by":"auto","created_at":"2026-03-16 16:17:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2791365,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7834157/v1/9caefab4-6176-4aff-bd92-f9612022a572.pdf"},{"id":96249299,"identity":"b942a5da-2e0f-428f-9398-e909ea9f754e","added_by":"auto","created_at":"2025-11-19 07:32:54","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":68313,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1\u003c/strong\u003e: H-scores of membranous HER3 in the primary tumor and brain metastasis of each patient. Abbreviations: HER3, human epidermal growth factor receptor 3\u003c/p\u003e","description":"","filename":"suppleFigure1.20251010.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7834157/v1/eda3a818bb73a97dbee3a535.pdf"},{"id":96246812,"identity":"10a8879b-b43a-4d6c-84de-300e4d233264","added_by":"auto","created_at":"2025-11-19 07:26:43","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":110178,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2:\u003c/strong\u003e Overall survival from resection of BMs. (A) Overall survival according to HER3 scores in BMs. (B) Overall survival according to the trajectory of HER3 from PTs to BMs: decreased, equal, or increased. Abbreviations: HER3, human epidermal growth factor receptor 3; BMs, brain metastases; PT,\u003c/p\u003e","description":"","filename":"suppleFigure220251010.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7834157/v1/ee5942647c1c0a07abb54ff8.pdf"}],"financialInterests":"","formattedTitle":"Expression of HER2, HER3, and TROP2 in primary tumors and brain metastases of breast cancer","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eHER3 and TROP2 were expressed at immunohistochemistry (IHC) 1+ or higher in all but one brain metastasis (BM) cases.\u003c/li\u003e\n \u003cli\u003eHigh-level (IHC 2+ or 3+) HER3 expression was more frequent in BMs than in primary tumors (91% vs. 59% [p\u0026lt;0.01]).\u003c/li\u003e\n \u003cli\u003eThis study suggests the potential of HER3- and TROP2-targeting antibody–drug conjugates as drugs for breast cancer BMs.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eBrain metastases (BM) occur in one-third of breast cancer patients during their disease course.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Although local therapeutic interventions, such as whole brain radiation therapy (WBRT), stereotactic irradiation (SRI), and neurosurgery, are employed to manage BMs,\u003csup\u003e2\u003c/sup\u003e patient prognosis remains poor, with a median overall survival (OS) ranging from 2 to 16 months.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Once metastasis to the brain occurs, patients typically experience a significant decline in quality of life, culminating in organ failure and death.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eTraditionally, the effectiveness of drug therapy for BMs has been considered low,\u003csup\u003e5\u003c/sup\u003e possibly because of the presence of the blood\u0026ndash;brain barrier. However, in human epidermal growth factor receptor 2 (HER2)-positive (HER2+) breast cancer, new classes of drugs have been observed to challenge this notion. For instance, antibody\u0026ndash;drug conjugates (ADCs), which comprise targeting antibodies that bind to cytotoxic payloads, can offer advantages over \u0026ldquo;naked\u0026rdquo; cytotoxic drugs. These benefits include a high therapeutic index, bystander killing through payload dispersion, and anti-tumor immune activity via antibody-dependent cytotoxicity.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e One notable ADC is trastuzumab deruxtecan (T-DXd), which consists of a humanized HER2-directed monoclonal antibody and DXd, a topoisomerase inhibitor. T-DXd has shown promising clinical outcomes in trials involving HER2\u0026thinsp;+\u0026thinsp;metastatic breast cancer patients with BMs.\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Although active/progressing BM were excluded from the three pivotal studies DESTINY-Breast01, 02 and 03, a subsequent pooled analysis of patients with baseline BM in these trials demonstrated that T-DXd is effective in patients with stable BM and in active/untreated BM. In the recent Phase IIIb DESTINY-Breast12 study, which included a cohort with 263 patient at baseline BM, intra-clinical activity of T-DXd could be confirmed across all types of BM including active/progressing BM.\u003csup\u003e11\u003c/sup\u003e In addition, although T-DXd has demonstrated clinical activity against breast cancer with HER2-low expression,\u003csup\u003e12\u003c/sup\u003e its effectiveness against BMs from such HER2 non-overexpressing diseases remains uncertain. Therefore, other potential target proteins expressed in BMs should be explored.\u003c/p\u003e\u003cp\u003eRecently, ADCs targeting cell surface proteins other than HER2, such as trophoblast surface antigen 2 (TROP2)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and HER3,\u003csup\u003e14\u003c/sup\u003e have emerged. TROP2 is a widely expressed glycoprotein belonging to the epithelial cell adhesion molecule family.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Sacituzumab govitecan (SG) is an ADC that combines SN-38, a topoisomerase inhibitor and active metabolite of irinotecan, with an anti-TROP2 monoclonal antibody.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e SG has been approved by the Food and Drug Administration for treating advanced triple-negative breast cancer (TNBC) and hormone receptor-positive (HR+)/HER2-negative (HER2\u0026ndash;) breast cancer. This approval was based on the results of phase III trials demonstrating the superiority of SG over physician\u0026rsquo;s choice treatments.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e In these trials, SG achieved a 35% objective response rate (ORR) for extracranial lesions in patients with TNBC.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e However, the intracranial response rate was modest, at only 3% (1/32).\u003csup\u003e19\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eHER3 is highly expressed in many solid tumors, including 30% to 50% of breast cancers.\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e High HER3 expression in breast cancer was reported to be associated with reduced OS.\u003csup\u003e21, 23\u003c/sup\u003e Patritumab deruxtecan (HER3-DXd) is a novel, first-in-class anti-HER3 ADC currently under clinical development for multiple indications such as non-small cell lung cancer (NSCLC) and breast cancer. A phase I/II study involving the use of HER3-DXd in patients with advanced breast cancer expressing HER3 also reported promising anti-tumor activity (ORRs of 30.1%, 22.6%, and 42.9% for patients with hormone receptor (HR)+/HER2\u0026ndash; [n\u0026thinsp;=\u0026thinsp;113], TNBC [n\u0026thinsp;=\u0026thinsp;53], and HER2+ [n\u0026thinsp;=\u0026thinsp;14], respectively).\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Additionally, a phase II ICARUS-Breast 01 study evaluating HER3-DXd in patients with HR+/HER2\u0026ndash; metastatic breast cancer who progressed on endocrine therapy and one line of chemotherapy also showed antitumor activity with ORR of 53.5% and a median progression free survival of 9.4 months.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e In HERTHENA-Lung01, a phase II trial of HER3-DXd in advanced epidermal growth factor receptor-mutated NSCLC, the confirmed ORR was 29.8% and the intracranial ORR was 33.3% (10/30 patients; 95% CI, 17.3 to 52.8).\u003csup\u003e25\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eDespite the emerging potential of ADCs in treating BMs and their clinical applicability in targeting different proteins, evaluating target proteins for BMs is difficult in most cases owing to poor tumor accessibility. Tumors can alter their molecular profile during the development of BMs,\u003csup\u003e22\u003c/sup\u003e making it clinically relevant to understand the correlation between the expression levels of ADC target proteins, such as HER2, HER3, and TROP2 in primary tumors (PTs) and BMs in breast cancer. However, this area remains largely unexplored. Therefore, this study was undertaken to evaluate the expression patterns of HER2, HER3, and TROP2 in paired samples of surgically resected BMs and PTs from patients with breast cancer using immunohistochemistry (IHC).\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePatients\u003c/h2\u003e\u003cp\u003ePatients with breast cancer who underwent surgical resection of BMs at the National Cancer Center Hospital in Japan were identified serially between January 2000 and December 2017. From this cohort, patients with available archival tissues from both resected BMs and PTs were selected. Tissue availability was assessed by pathologists (K.S., M.Y., and S.F.). Patient demographic information was collected through a retrospective chart review.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIHC assays of PTs and BMs\u003c/h3\u003e\n\u003cp\u003eBM and PT tissues were subjected to IHC staining for estrogen receptor (ER), progesterone receptor (PgR), HER2, HER3, and TROP2 using an automated slide stainer (Ventana BenchMark ULTRA; Roche Diagnostics K.K., Basel, Switzerland). The Ventana UltraView Confirm ER (clone: SP1, Roche Diagnostics K.K.) and Ventana UltraView Confirm PGR (clone: 1E2, Roche Diagnostics K.K.) were used for ER and PgR IHC, respectively. The Ventana I-VIEW PATHWAY\u0026trade; HER2 (clone: 4B5, Roche Diagnostics K.K.) and I-VIEW universal kit (Roche Diagnostics K.K.) were used for HER2 IHC. Anti-HER3 (clone: 7.3.8, Ventana Medical Systems, Inc., Oro Valley, US) and anti-TROP2 (clone: SP295, Abcam, Cambridge, UK) antibodies were used as primary antibodies for HER3 and TROP2 IHC, respectively. Heat-induced epitope retrieval for all targets was performed using ULTRA Cell Conditioning Solution #1 (Benchmark ULTRA CC1, Roche Diagnostics K.K.). The UltraView DAB IHC Detection Kit (Roche Diagnostics K.K.) was used to detect the chromogenic IHC signals of HER2, whereas the Ventana UltraView Universal DAB Detection Kit (Roche Diagnostics K.K.) was used to detect those of other targets.\u003c/p\u003e\u003cp\u003eIHC for ER and PgR was evaluated by a pathologist (S.F.). ER and PgR statuses were classified as positive if\u0026thinsp;\u0026ge;\u0026thinsp;1% of tumor cells expressed the proteins.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Membranous IHC staining intensity for HER2, HER3, and TROP2 in tumor cells was scored visually by pathologists (R.N. and N.M.) using a microscope. HER2 IHC staining intensity (0, 1+, 2+, and 3+) was scored according to the updated 2018 American Society of Clinical Oncology/College of American Pathologists guidelines for HER2 testing in breast cancer.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e In this study, PTs classified as HER2 2\u0026thinsp;+\u0026thinsp;were considered HER2\u0026thinsp;+\u0026thinsp;if there was a confirmation of either HER2 in situ hybridization (ISH) positivity or HER2 IHC 3\u0026thinsp;+\u0026thinsp;in the pathology report documented in the medical record. Two HER2\u0026thinsp;+\u0026thinsp;cases in the luminal group were considered HER2\u0026ndash; because neither of them met the above definition. Three HER2 2\u0026thinsp;+\u0026thinsp;cases were considered HER2\u0026thinsp;+\u0026thinsp;because of ISH positivity (luminal-HER2, n\u0026thinsp;=\u0026thinsp;1; pure HER2, n\u0026thinsp;=\u0026thinsp;2).\u003c/p\u003e\u003cp\u003eBreast cancer subtypes were classified according to the expression patterns of HR, ER, and PgR, and HER2 in PTs as follows: luminal (HR+/HER2\u0026ndash;), TNBC (HR-/HER2\u0026ndash;), luminal-HER2 (HR+/HER2+), and pure HER2 (HR\u0026ndash;/HER2+). For HER3 and TROP2, IHC membranous staining intensity (0, 1+, 2+, and 3+) was scored using criteria generally applied to HER2 IHC scoring for gastric cancer:\u003csup\u003e24\u003c/sup\u003e 0, no reactivity or membranous reactivity in \u0026lt;\u0026thinsp;10% of tumor cells; 1+, faint/barely perceptible membranous reactivity in \u0026ge;\u0026thinsp;10% of tumor cells; 2+, weak to moderate complete, basolateral, or lateral membranous reactivity in \u0026ge;\u0026thinsp;10% of tumor cells; 3+, strong complete, basolateral, or lateral membranous reactivity in \u0026ge;\u0026thinsp;10% of tumor cells.\u003c/p\u003e\u003cp\u003eAn H-score (0\u0026ndash;300) was also calculated based on membranous staining intensity using the following formula: H score\u0026thinsp;=\u0026thinsp;3 \u0026times; (percentage of strongly positive tumor cells)\u0026thinsp;+\u0026thinsp;2 \u0026times; (percentage of weakly to moderately positive tumor cells)\u0026thinsp;+\u0026thinsp;1 \u0026times; (percentage of faintly/barely perceptible positive tumor cells).\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Representative expression patterns of HER2, HER3, and TROP2 are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eStatistics\u003c/h3\u003e\n\u003cp\u003eThe proportions of high-level expression (IHC 2+/3+) for each protein were compared using McNemar\u0026rsquo;s test. A paired t-test was used to compare the H-scores of HER3 between PTs and BMs. OS, defined as the time from resection of BMs to death from any cause or the data cut-off date, was estimated using a log-rank test. The follow-up period was censored on December 31, 2017. OS was compared according to HER3 expression levels in BMs and changes in expression from PTs to BMs. Statistical analyses were performed using EZR software, version 1.62.\u003c/p\u003e\n\u003ch3\u003eEthics\u003c/h3\u003e\n\u003cp\u003e This study was approved by the Institutional Review Board (IRB) of the National Cancer Center Hospital (IRB number: 2017\u0026thinsp;\u0026minus;\u0026thinsp;502). The IRB waived the requirement for written informed consent from the study participants.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePatient characteristics\u003c/h2\u003e\u003cp\u003eA total of 44 patients were enrolled in this study. The clinical characteristics of the patients at the time of brain surgery are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The median age was 53 (34\u0026ndash;78) years, and most patients (88.6%) were under the age of 65 years. All but one patient had a performance status of 0 or 1; most patients had symptoms of BM. The median time from initial diagnosis of breast cancer to brain surgery was 2.4 years (range, 0.1\u0026ndash;21.9) years. All but three patients had three or fewer BMs; however, a majority had at least one BM measuring\u0026thinsp;\u0026ge;\u0026thinsp;3 cm, potentially limiting initial SRI indications. BMs were the sole metastases in 11 (25%) patients. Sixteen patients (36.4%) had primary HER2\u0026thinsp;+\u0026thinsp;disease, a proportion higher than that observed in the general population of breast cancer. Following surgical intervention, 36 (81.8%) and 5 (11.4%) patients underwent WBRT and SRI, respectively.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePatient characteristics at brain surgery\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCharacteristics\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePatients (N\u0026thinsp;=\u0026thinsp;44)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAge\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMedian, years (range)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e53 (34\u0026ndash;78)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026ge; 65, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5 (11.4%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSex\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e44 (100%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eECOG PS, n (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7 (15.9%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e36 (81.8%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 (2.3%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSymptoms from brain metastases\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSymptomatic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e39 (88.6%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAsymptomatic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5 (11.4%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTime from diagnosis of metastatic disease to developing brain metastases\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026lt; 6 months\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14 (31.8%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6 months \u0026le;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30 (68.2%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDe novo stage IV\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9 (20.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e35 (79.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNumber of brain metastases\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e29 (65.9%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u0026ndash;3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12 (27.3%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emore than 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3 (6.8%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMaximum size of brain metastases\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026lt; 3cm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6 (13.6%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3cm \u0026le;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e38 (86.4%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eConcomitant extracranial metastases\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePresent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e33 (75.0%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003elymph node\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13 (29.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ebone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13 (29.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eliver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6 (13.6%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003elung\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14 (31.8%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAbsent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11 (25.0%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBreast cancer subtype in primary tumor\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTNBC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8 (18.2%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLuminal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20 (45.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePure HER2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4 (9.1%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLuminal-HER2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12 (27.3%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTreatment for brain metastases\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSurgery\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e44 (100%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePost-surgical STI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5 (11.4%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePost-surgical WBRT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e36 (81.8%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"2\"\u003eAbbreviations: ECOG PS, Eastern Cooperative Oncology Group performance status; TNBC, triple-negative breast cancer; Luminal, hormone receptor-positive/HER2-negative; Pure HER2, hormone receptor-negative/HER2-positive; Luminal-HER2, hormone receptor-positive/HER2-positive; STI, stereotactic irradiation; WBRT, whole brain radiation therapy.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eHER3 was more frequently overexpressed BMs than in PTs\u003c/h3\u003e\n\u003cp\u003eAll BM samples exhibited a certain level of HER3 expression (\u0026ge;\u0026thinsp;IHC 1+; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The proportions of high-level expression (IHC 2+/3+) were higher in BMs than in PTs for HER3 but not for HER2 (HER3: 59% vs. 91% for PT and BMs, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01); HER2: 41% vs. 43% for PT and BMs, respectively (p\u0026thinsp;=\u0026thinsp;1.00); Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eProportion of IHC2+/3\u0026thinsp;+\u0026thinsp;of HER2, HER3, and TROP2 in PT and BM\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eHER2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eHER3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eTROP2\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eBM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eBM\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAll subtypes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18/44\u003c/p\u003e\u003cp\u003e(41%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19/44\u003c/p\u003e\u003cp\u003e(43%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26/44\u003c/p\u003e\u003cp\u003e(59%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e40/44\u003c/p\u003e\u003cp\u003e(91%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e31/44\u003c/p\u003e\u003cp\u003e(70%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e38/44\u003c/p\u003e\u003cp\u003e(86%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTNBC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0/8\u003c/p\u003e\u003cp\u003e(0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1/8\u003c/p\u003e\u003cp\u003e(13%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4/8\u003c/p\u003e\u003cp\u003e(50%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6/8\u003c/p\u003e\u003cp\u003e(75%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7/8\u003c/p\u003e\u003cp\u003e(88%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8/8\u003c/p\u003e\u003cp\u003e(100%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLuminal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2/20\u003c/p\u003e\u003cp\u003e(10%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3/20\u003c/p\u003e\u003cp\u003e(15%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12/20\u003c/p\u003e\u003cp\u003e(60%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e18/20\u003c/p\u003e\u003cp\u003e(90%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12/20\u003c/p\u003e\u003cp\u003e(60%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e14/20\u003c/p\u003e\u003cp\u003e(70%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLuminal HER2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12/12\u003c/p\u003e\u003cp\u003e(100%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11/12\u003c/p\u003e\u003cp\u003e(92%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6/12\u003c/p\u003e\u003cp\u003e(50%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12/12\u003c/p\u003e\u003cp\u003e(100%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9/12\u003c/p\u003e\u003cp\u003e(75%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e12/12\u003c/p\u003e\u003cp\u003e(100%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePure HER2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4/4\u003c/p\u003e\u003cp\u003e(100%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4/4\u003c/p\u003e\u003cp\u003e(100%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4/4\u003c/p\u003e\u003cp\u003e(100%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4/4\u003c/p\u003e\u003cp\u003e(100%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3/4\u003c/p\u003e\u003cp\u003e(75%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4/4\u003c/p\u003e\u003cp\u003e(100%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eAbbreviations: HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; TROP2, trophoblast surface antigen 2; BM, brain metastase; PT, primary tumor; TNBC, triple-negative breast cancer; Luminal, hormone receptor-positive/HER2-negative; Pure HER2, hormone receptor-negative/HER2-positive; Luminal-HER2, hormone receptor-positive/HER2-positive.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe tendency for higher HER3 expression in BMs than in PTs was consistent across all breast cancer subtypes except the pure HER2 subtype. The proportions of IHC 2+/3\u0026thinsp;+\u0026thinsp;in BM vs. PT were 75% vs. 50%, 90% vs. 60%, and 100% vs. 50% for TNBC (HR\u0026ndash;/HER2\u0026ndash;), luminal (HR+/HER2\u0026ndash;), and luminal-HER2 (HR+/HER2+) subtypes, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). For the pure HER2 subtype (HR\u0026ndash;/HER2+), all patients showed IHC 2+/3\u0026thinsp;+\u0026thinsp;HER3 expression in both BMs and PTs. In the HER2\u0026thinsp;+\u0026thinsp;subtypes (luminal and pure HER2 subtypes), all 16 cases showed high HER3 expression in BMs (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). On an individual patient basis, 80% (35/44) had higher membranous HER3 H-scores in BMs than in PTs, and the difference between BMs and PTs was statistically significant (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e\u003cp\u003eFor TROP2, all but one BM sample exhibited a certain level of expression (\u0026ge;\u0026thinsp;IHC 1+, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). TROP2 was highly expressed (IHC 2+/3+) in both PTs and BMs in the majority of cases (PTs 70% and BMs 86%, p\u0026thinsp;=\u0026thinsp;0.11).\u003c/p\u003e\n\u003ch3\u003eHER3 expression in BM as a prognostic marker\u003c/h3\u003e\n\u003cp\u003eThe impact of HER3 expression in BMs on patient survival was explored. No statistically significant difference in survival was observed between HER3 IHC scores in BMs (Supplementary Fig.\u0026nbsp;2). Similarly, no statistically significant difference in survival was observed between populations with decreased, equal, and increased BM HER3 expression compared with those with PT HER3 expression (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis study demonstrated that HER3 and TROP2 were expressed at IHC 1\u0026thinsp;+\u0026thinsp;or higher in all but one BM case, respectively. While TROP2 was highly expressed (IHC 2\u0026thinsp;+\u0026thinsp;or 3+) in both PTs and BMs in the majority of cases, HER3 was more frequently expressed at high levels in BMs than in PTs (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, in individual paired samples, 95.5% (42/44) of cases exhibited the same or higher HER3 expression in BMs than in PTs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, HER2 exhibited high-level expression in similar proportions between BMs and PTs (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eOur finding of more frequent high-level HER3 expression in BMs than in PTs is consistent with those of previous studies. A breast cancer study demonstrated HER3 positivity by IHC in 59% (22/37) of matched BMs and 30% (11/37) of PTs.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Another recent study indicated that HER3 was frequently expressed in BMs from breast cancer, with HER2\u0026thinsp;+\u0026thinsp;and HER2-low BMs exhibiting significantly higher rates of HER3 co-expression than HER2-null BMs.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e A study on NSCLC also demonstrated that HER3 was more abundantly expressed in BMs than in matched extracranial samples.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Considering that neuregulin 1, the ligand for HER3, is abundantly expressed in the brain and is released via various mechanisms, including hypoxia,\u003csup\u003e27\u003c/sup\u003e HER3 may play a role in the formation of BMs. In addition, HER3 overexpression has been suggested as a mechanism that confers resistance to anti-HER2 therapy. Therefore, the more frequent high-level HER3 expression in BMs than in PTs may result from clonal selection by anti-HER2 systemic treatment. Our finding that all HER2\u0026thinsp;+\u0026thinsp;subtypes exhibited IHC 2+/3\u0026thinsp;+\u0026thinsp;HER3 expression in BMs supports this hypothesis and is consistent with previous findings.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eAlthough BMs have generally been considered resistant to drug treatment, ADCs, particularly those targeting HER2, have been observed to challenge this notion. Over the past decade, multiple ADCs have been developed for various types of cancer, including breast cancer, with promising clinical efficacy for BMs. Currently, the membranous expression level of the target protein is the most reliable predictor of response to ADCs.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e The DAISY trial demonstrated that response rates to T-DXd decreased hierarchically with HER2 expression.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Additionally, a decrease in HER2 expression level has been suggested as one of the possible resistance mechanisms to T-DXd. These findings emphasize the importance of target protein levels for the efficacy of ADCs. Our finding that virtually all BMs expressed HER3 and TROP2 suggests that these proteins could be optimal targets for ADCs in patients with BMs from breast cancer. However, the intracranial response rate of SG has been reported to be only 3%.\u003csup\u003e19\u003c/sup\u003e It is currently unclear whether factors beyond target expression affect the intracranial activity of ADCs or if it depends more on the properties of the drugs. The TUXEDO-2 study is ongoing to evaluate the intracranial activity of datopotamab deruxutecan, another anti-TROP2 ADC, in patients with metastatic TNBC with active brain metastases (NCT05866432). Results from the first stage exploratory cohort were encouraging, three intracranial responses of seven evaluable patients, and the study has progressed to the second stage.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e The DATO-BASE study (NCT06176261) is also ongoing to evaluate intracranial responses of Dato-DXd in patients with HER2\u0026ndash; metastatic breast cancer.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Notably, a clinical trial specifically evaluating the clinical activity of HER3-DXd in breast cancer BMs is ongoing.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThis study had several limitations. First, the sample size was relatively small, which may have limited the statistical power. Second, there were significant time gaps, up to 22 years, between the collection of PTs and BMs, raising concerns about differences in antigen preservation. However, considering that HER2 and TROP2 were expressed at similar levels and frequencies in both PTs and BMs (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the observed tendency for higher HER3 expression in BMs than in PTs is unlikely to be solely due to better antigen preservation in BMs. Third, only surgically removed BMs were included in this study, suggesting that these patients were diagnosed with oligometastases or oligoprogression of breast cancer. Patients with more advanced diseases, such as multiple BMs, might have different biological characteristics, potentially introducing bias in patient selection. In addition, this may explain why we did not observe a significant difference in survival by HER3 IHC scores in BM (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e\u003cp\u003eIn conclusion, this study demonstrated that HER3 was more frequently expressed at high levels in BMs than in PTs, and that TROP2 was highly expressed in both PTs and BMs. As multiple ADCs with various targets and payloads continue to be developed, HER3- and TROP2- ADCs may emerge as key drugs for BMs, regardless of the breast cancer subtype in the PT; however, this hypothesis warrants validation through clinical trials.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eADCs, antibody–drug conjugates; BMs, brain metastases; Dato-DXd, Datopotamab deruxtecan; ER, estrogen receptor; HER2–, HER2-negative; HER2+, HER2-positive; HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; HER3-DXd, Patritumab deruxtecan; HR+, hormone receptor-positive; IHC, immunohistochemistry; IRB, Institutional Review Board; NSCLC, non-small cell lung cancer; ORRs, objective response rates; OS, overall survival; PgR, progesterone receptor; PTs, primary tumors; SG, sacituzumab govitecan; SRI, stereotactic irradiation; T-DXd, trastuzumab deruxtecan; TNBC, triple-negative breast cancer; TROP2, trophoblast surface antigen 2; WBRT, whole brain radiation therapy\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgments\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYuka Nakamura, Kokichi Honda, and Takanori Maejima performed IHC for ER and PgR, HER2, and TROP2, respectively. Ryoko Wanikawa selected appropriate slides of primary breast cancer and brain metastases. Editage provided language support and writing assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis\u0026nbsp;work was supported by JSPS KAKENHI [grant number 20K08973] and Daiichi Sankyo Co., Ltd.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor information\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors and Affiliations\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Medical Oncology, National Cancer Center Hospital East, 6-5-1 Kashiwanoha, Kashiwa, Chiba, 277-8577, Japan\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShota Kusuhara, Takahiro Kogawa, Chikako Funasaka, Chihiro Kondoh, Kenichi Harano, Nobuaki Matsubara, Yoichi Naito, Ako Hosono, Toru Mukohara\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Advanced Medical Development, The Cancer Institute Hospital of Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo, 135-0063, Japan\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTakahiro Kogawa\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Biostatistics, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamiogushi, Ube, Yamaguchi, 755-8505, Japan\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMototsugu Shimokawa\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Diagnostic Pathology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKaishi Satomi, Masayuki Yoshida\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Molecular Pathology, Yokohama City University Hospital, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa, 236-0004, Japan\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSatoshi Fujii\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Breast Surgery, National Cancer Center Hospital East, 6-5-1 Kashiwanoha, Kashiwa, Chiba, 277-8577, Japan\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTatsuya Onishi\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Breast Surgery, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAkihiko Suto\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Medical Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKan Yonemori\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranslational Research Laboratories, Daiichi Sankyo Co., Ltd, Shinagawa R\u0026amp;D Center, 1-2-58 Hiromachi, Shinagawa-ku,Tokyo, 140-0005, Japan\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKumiko Koyama, Ryuichi Nakamura, Naoyuki Maeda\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Neurosurgery and Neuro-Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYoshitaka Narita\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eC\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eontributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS Kusuhara: data curation, formal analysis, writing—original draft, and writing—review. T Kogawa: Conceptualization, resources, data curation, formal analysis, supervision, investigation, writing—original draft, project administration, writing—review, and editing. T Mukohara: Supervision, investigation, writing— original draft, project administration, writing—review, and editing. All authors reviewed the manuscript. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCorresponding author\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to \u003cstrong\u003e\u003cu\u003eTakahiro Kogawa\u003c/u\u003e\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics declarations\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003enflict of\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eInterest\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTakahiro Kogawa received grants from Daiichi Sankyo, Eli Lilly, and AstraZeneca. Toru Mukohara received research funds from Sysmex, Eisai, MSD, Pfizer, AstraZeneca, Ono, Daiichi-Sankyo, and Gilead Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical approval\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Review Board (IRB) of the National Cancer Center Hospital (IRB number: 2017-502).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInformed consent\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRegistry and the registration no. of the study/trial\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimal studies\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMatsuo S, Watanabe J, Mitsuya K, Hayashi N, Nakasu Y, Hayashi M. 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Patritumab deruxtecan (HER3-DXd) in active brain metastases from metastatic breast and non-small cell lung cancers, and leptomeningeal disease from advanced solid tumors: The TUXEDO-3 phase II trial. J Clin Oncol. 2024;42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1200/JCO.2024.42.16_suppl.TPS2091\u003c/span\u003e\u003cspan address=\"10.1200/JCO.2024.42.16_suppl.TPS2091\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. TPS2091-TPS2091.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"breast-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brca","sideBox":"Learn more about [Breast Cancer](http://link.springer.com/journal/12282)","snPcode":"12282","submissionUrl":"https://www.editorialmanager.com/brca/default2.aspx","title":"Breast Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Breast cancer, brain metastasis, HER2, HER3, TROP2","lastPublishedDoi":"10.21203/rs.3.rs-7834157/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7834157/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cdiv id=\"ASec1\" class=\"AbstractSection\"\u003e\u003cdiv class=\"Heading\"\u003eBackground\u003c/div\u003e\u003cp\u003eHuman epidermal growth factor receptor 2 (HER2)-directed antibody\u0026ndash;drug conjugate (ADC) therapy has shown efficacy in HER2-positive breast cancer brain metastases. However, there is still an unmet medical need for further exploration of other suitable targets for ADCs in brain metastases (BMs).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"ASec2\" class=\"AbstractSection\"\u003e\u003cdiv class=\"Heading\"\u003eMethods\u003c/div\u003e\u003cp\u003eThe expression of HER2, HER3, and trophoblast surface antigen 2 (TROP2) was evaluated using immunohistochemistry (IHC) in pairs of primary tumors (PTs) and surgically resected BMs from 44 patients with breast cancer. Expression levels were classified as 0, 1+, 2+, or 3\u0026thinsp;+\u0026thinsp;based on IHC intensity and the proportion of positive cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"ASec3\" class=\"AbstractSection\"\u003e\u003cdiv class=\"Heading\"\u003eResults\u003c/div\u003e\u003cp\u003eThe analysis revealed that HER3 and TROP2 expression (IHC\u0026thinsp;\u0026ge;\u0026thinsp;1+) was observed in all but one BM specimen. TROP2 was highly expressed (IHC\u0026thinsp;\u0026ge;\u0026thinsp;2+) in both BMs and PTs (86% and 70%, respectively; p\u0026thinsp;=\u0026thinsp;0.11). High expression of HER3 was more frequent in BMs than in PTs (91% and PTs 59%, respectively; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), regardless of breast cancer subtype. In individual paired samples, 95.5% (42/44) exhibited equal or higher HER3 expression in BMs than in PTs. In contrast, high HER2 expression (IHC\u0026thinsp;\u0026ge;\u0026thinsp;2+) was observed in similar proportions between BMs and PTs (43% and 41%, respectively; p\u0026thinsp;=\u0026thinsp;1.00).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"ASec4\" class=\"AbstractSection\"\u003e\u003cdiv class=\"Heading\"\u003eConclusions\u003c/div\u003e\u003cp\u003eThe observation of more frequent high-level expression of HER3 in BMs than in PTs, and high-level expression of TROP2 in both BMs and PTs suggests the potential of HER3- and TROP2-based ADC therapy for BMs from breast cancer. Further prospective studies are warranted to validate this hypothesis.\u003c/p\u003e\u003c/div\u003e","manuscriptTitle":"Expression of HER2, HER3, and TROP2 in primary tumors and brain metastases of breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-17 10:37:14","doi":"10.21203/rs.3.rs-7834157/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revision","date":"2026-01-10T17:25:36+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-11-08T10:06:33+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-06T09:30:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-14T08:58:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Breast Cancer","date":"2025-10-13T10:31:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"breast-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brca","sideBox":"Learn more about [Breast Cancer](http://link.springer.com/journal/12282)","snPcode":"12282","submissionUrl":"https://www.editorialmanager.com/brca/default2.aspx","title":"Breast Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e2d0fc70-0f91-455d-bdca-24f12c6fc943","owner":[],"postedDate":"November 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:14:57+00:00","versionOfRecord":{"articleIdentity":"rs-7834157","link":"https://doi.org/10.1007/s12282-026-01841-8","journal":{"identity":"breast-cancer","isVorOnly":false,"title":"Breast Cancer"},"publishedOn":"2026-03-13 15:58:54","publishedOnDateReadable":"March 13th, 2026"},"versionCreatedAt":"2025-11-17 10:37:14","video":"","vorDoi":"10.1007/s12282-026-01841-8","vorDoiUrl":"https://doi.org/10.1007/s12282-026-01841-8","workflowStages":[]},"version":"v1","identity":"rs-7834157","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7834157","identity":"rs-7834157","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}