Genomic correlations for clinical outcomes in HER2-positive advanced gastric cancers treated using trastuzumab-based therapy

Genomic correlations for clinical outcomes in HER2-positive advanced gastric cancers treated using trastuzumab-based therapy | 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 Genomic correlations for clinical outcomes in HER2-positive advanced gastric cancers treated using trastuzumab-based therapy Sun Young Lee, Jaewon Hyung, Hyung-Don Kim, Hyungeun Lee, Meesun Moon, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7267337/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Although trastuzumab-based chemotherapy improves survival in HER2-positive advanced gastric cancer, some patients demonstrate suboptimal efficacy and limited response durations. We examined the relationship between clinical outcomes and genomic features, including co-mutations and the length of the ERBB2 -amplified segment. Methods We retrospectively analyzed 151 patients who had received first-line trastuzumab-based chemotherapy. Targeted next-generation sequencing was employed to assess genomic alterations. Progression-free survival (PFS) was defined as time from treatment initiation to disease progression or death. Results The median patient age was 62 years, and 73.5% were male. The median follow-up period was 45.8 months, and the median PFS was 8.2 months (95% confidence interval (CI), 6.5–9.4). Patients with a focal amplification of ERBB2 (≤ 879 Kb) had significantly longer PFS compared to those with non-focal amplifications (> 879 Kb) (10.1 vs. 6.1 months; log-rank p = 0.01). NOTCH3 alterations were associated with shorter PFS (log-rank p = 0.002). Multivariate analysis confirmed that ERBB2 focal amplification is an independent prognostic factor associated with improved prognosis, whereas NOTCH3 alterations serve as an independent prognostic factor for poorer outcomes. Conclusions ERBB2 focal amplification is associated with improved outcomes in trastuzumab-treated patients with HER2-positive gastric cancer, whereas NOTCH3 alterations predict a poor prognosis. These genomic features may support risk stratification and therapeutic decisions. HER2 positive trastuzumab advanced gastric cancer ERBB2 copy number amplification focal ERBB2 amplification NOTCH3 Figures Figure 1 Figure 2 Figure 3 Introduction Gastric cancer (GC) is the most commonly diagnosed cancer globally and ranks as the fourth leading cause of cancer-related death worldwide (Siegel et al., 2021 ). Over the last decade, several phase III clinical trials have been conducted to develop molecularly targeted therapies and immunotherapies (Ishii et al., 2019 ). Human epidermal growth factor receptor 2 (HER2/ ERBB2 ), a proto-oncogene encoded by ERBB2 on chromosome 17, is involved in cell proliferation, metastasis, and poor outcomes in various cancer types, including advanced gastric cancer (AGC) and gastroesophageal junction cancer (GEJC), with an incidence rate of 15–20% (Abrahao-Machado & Scapulatempo-Neto, 2016 ; Boku, 2014 ). Trastuzumab is a monoclonal antibody that targets the juxtamembrane portion of HER2 and inhibits HER2-mediated signaling by preventing HER2 dimerization, inhibiting the shedding of the HER2 extracellular domain, enhancing the endocytic destruction system, and inducing antibody-dependent cytotoxicity (Hudis, 2007 ). The combination of trastuzumab and chemotherapy in the first-line setting has shown significant survival benefits for patients with HER2-positive (immunohistochemistry (IHC) 2+/in situ hybridization positive (ISH) positive or IHC 3+) AGC, and trastuzumab-based therapy is the current standard treatment (Bang et al., 2010 ; Franchi et al., 2020 ; Janjigian et al., 2023 ; Koo et al., 2021 ). Nevertheless, the overall response rate (ORR) was approximately 47% in the ToGA trial (Bang et al., 2010 ), and the long-term benefits of trastuzumab-based treatment were observed only in a small number of patients (Pietrantonio et al., 2023 ). Some studies have shown that patients with higher HER2 protein expression level (An et al., 2017 ; Catenacci et al., 2016 ) or increased ERBB2 copy number (CN) (Gomez-Martin et al., 2013 ; Pietrantonio et al., 2023 ; Zhang et al., 2022 ), as assessed by IHC or NGS, respectively, can derive long-term benefits from trastuzumab-based therapy. Regarding primary drug resistance, mutations and amplifications in EGFR/MET/KRAS/PI3K , known as the AMNESIA panel, have been evaluated as negative prognostic factors for trastuzumab-based therapy (Pietrantonio et al., 2018 ). However, it is speculated that the impact of AMNESIA positivity on survival outcomes may vary depending on the cohort (Pietrantonio et al., 2018 ; Pietrantonio et al., 2023 ). Given that copy number variants (CNVs) occur extensively with varying lengths over 100 kilobases (Kb) (Steele et al., 2022 ), ERBB2 gene amplification may impact not only the ERBB2 gene itself but also adjacent genes within the amplified segment. This study aimed to evaluate the co-mutational status and ERBB2 -ampified segment length as prognostic factors for patient selection to optimize trastuzumab-based therapy. Methods Study patients Patients aged > 18 years with histologically confirmed locally advanced, unresectable, recurrent, or initially metastatic HER2-positive GC who received trastuzumab-containing first-line chemotherapy at Asan Medical Center, South Korea, from January 2015 to December 2023 were enrolled in the study. Subsequently, the patients were classified into three risk groups (poor-, moderate-, and good-risk) according to the prognostic model for AGC described in our previous article (Koo et al., 2011 ). The protocol used in this study was approved by the Institutional Review Board (IRB) of Asan Medical Center, Korea (2014 − 0301), and the study was conducted according to the Helsinki Declaration. HER2 status IHC was performed for HER2 using an anti-HER2/neu (4B5) rabbit monoclonal primary antibody (Ventana Medical System, Tucson, AZ). HER2 protein expression was scored on a scale of 0 to 3 according to the GC consensus guidelines (Bang et al., 2022 ). HER2 positivity was defined as IHC 3+, or as IHC 2 + with HER2 gene amplification conducted by ISH. Targeted NGS and bioinformatics analysis Genomic DNA was extracted from archived formalin-fixed, paraffin-embedded (FFPE) tissue specimens. Targeted next-generation sequencing (NGS) was performed using the NextSeq platform (Illumina, San Diego, CA, USA) with OncoPanel AMC v3, v4, and v4.5 panels, as described previously (Kim et al., 2022 ). The OncoPanel AMC version 3 (OP AMC v3), version 4 (OP AMC v4), and version 4.5 (OP AMC v4.5) captured 382, 323 and 343 cancer-related genes, respectively (OP AMC v3, 199 genes for entire exons, 8 genes for partial introns, and 184 genes for hotspots; OP AMC v4, 225 genes for entire exons, 6 genes for partial introns, and 99 for hotspots; OP AMC v4.5, 244 genes for entire exons, 14 genes for partial introns, and 110 for hotspots). Sequenced reads were aligned to the human reference genome (GRCh37; hg19) using the Burrows-Wheeler Aligner and processed using the Genome Analysis Toolkit pipeline. The CN variation analysis was performed using a CNV kit with the default segmentation method (circular binary segmentation; CBS), and copy numbers of tumors were analyzed against a panel of unmatched normal samples. Genes with an estimated copy number ≥ 5 were classified as amplifications, whereas those with a copy number ≤ 0 were classified as losses. For the ERBB2 -amplified segment length analysis, only segments that covered the entire ERBB2 gene region were used for comparison, excluding segments that covered a partial region of ERBB2 or were not calculable ( ERBB2 -amplified by manual detection). The maftools package in R was used to analyze pathway enrichment and for mutation comparison (Mayakonda et al., 2018 ). Statistical analysis Progression-free survival (PFS) was defined as the time from the initiation of trastuzumab-based first-line chemotherapy to the time of disease progression or death, whichever occurred first. Overall survival (OS) was defined as the time from the initiation of trastuzumab-based first-line chemotherapy to the time of death from any cause. The Kaplan–Meier method was used to estimate PFS and OS. Survival curves were compared using the log-rank test. ERBB2 copy number segment size cutoff was determined using the maximally selected rank statistics from the maxstat R package. The Cox proportional hazards model was used to estimate the hazard ratio (HR) for survival outcomes. All statistical analyses were performed using the statistical software package R version 4.3.3. Results Baseline characteristics of study patients The baseline characteristics of the included HER2-positive patients (n = 151) are summarized in Table 1 . The median age of the patients was 62 years (range, 25–84), while 111 patients (73.5%) were male, and 104 patients (68.9%) had initially metastatic disease. All patients received trastuzumab plus fluoropyrimidine and platinum double chemotherapy combination as first-line treatment. Among 109 patients (72%) with a measurable disease status, 82 patients (75%) exhibited a best response of either a partial response or better, according to the Response Evaluation Criteria in Solid Tumors version 1.1. With a median follow-up duration of 45.8 months (range, 15.4–84.4), the median PFS and OS were 8.2 months (95% CI 6.5–9.4), and 18.2 months (95% CI 14.9–23.4), respectively. Risk group data, as defined in the previous study, were available for 148 patients (Koo et al., 2011 ). The good, moderate, and poor risk categories included 57 (38.5%), 64 (43.2%), and 27 patients (18.2%), respectively. The PFS and OS curves for three risk groups differed significantly (Supplementary Fig. 1). Table 1 Baseline characteristics (n = 151). Characteristics n (%) Age > 60 years 83 (55.0) Sex Female 40 (26.5) Male 111 (73.5) HER2 IHC scale 2+ 27 (17.9) 3+ 124 (82.1) Treatment setting Initially metastatic 104 (68.9) Recurrent 43 (28.5) Locally advanced unresectable 4 (2.6) Histology WD/MD 82 (54.3) PD/SRC 69 (45.7) MSI result* MSS 141 (100.0) MSI-high 0 (0.0) EBV result* Negative 142 (95.9) Positive 6 (4.1) ECOG PS* 0/1 130 (87.8) ≥2 18 (12.2) Gastrectomy Performed 48 (31.8) Not performed 103 (68.2) Metastasis Peritoneal metastasis 68 (45.0) Liver metastasis 61 (40.4) Lung metastasis 14 (9.3) Bone metastasis 10 (6.6) Lymph node metastasis 92 (60.9) Others 133 (88.1) ALP > 120 IU/L* 43 (29.1) Albumin 1.2 mg/dL* 10 (6.8) Risk group* Good 57 (38.5) Moderate 64 (43.2) Poor 27 (18.2) IHC, immunohistochemistry; MSI, microsatellite instability; MSS, microsatellite stable; EBV, Epstein-Barr virus; ECOG PS, Eastern Cooperative Oncology Group Performance Status; ALP, alkaline phosphatase * Patients for whom the tests were available were included in the analysis. Genomic alterations identified among HER2-positive AGC The landscape of molecular alterations for our cohort is shown in Fig. 1 (A). The most frequently altered gene in HER2-positive tumors was TP53 (84%), followed by ERBB2 (75%), CDK12 (58%), RARA (38%), CCNE1 (34%), MYC (28%), and LRP1B (26%). Meanwhile, point mutations were most common in TP53 , followed by LRP1B (24%), and ERBB2 (16%). Regarding copy number variants, ERBB2 amplifications were the most prevalent (74%), with CDK12 and RARA amplifications enriched specifically in patients with ERBB2 amplification. There was no significant association between CDK12 or RARA co-amplification and survival outcomes (Supplementary Fig. 2). We examined the prognostic value of ERBB2 CN status in 111 patients harboring ERBB2 -amplified tumors. Using the optimal cutoff value of 60, patients with ERBB2 CN-high (CN ≥ 60) status had a superior PFS (median 13.1 vs. 7.5; p = 0.039) and tended to have a better OS (median 21.5 vs. 18.2; p = 0.061) compared to those with ERBB2 CN-low (CN < 60) status (Fig. 1 B–C). We detected 13 ERBB2 mutations in 12 patients (7.9%) with HER2-positive AGC in this study. The mutation loci were distributed in the extracellular domain (ECD), transmembrane domain (TMD), juxtamembrane domain (JMD), tyrosine kinase domain (TKD), and intracellular domain (ICD). Co-occurrence of the ERBB2 mutation and CN amplification was detected in 11 patients (7.0%); no significant correlation was observed between the variant allele frequency (VAF) of the mutation and its copy number (Supplementary Table 1). Of the 12 patients with ERBB2 mutations, activating mutations annotated by OncoKB (Chakravarty et al., 2017 ; Suehnholz et al., 2024 ) were detected in 7 patients. Survival analysis revealed no significant differences in PFS or OS between patients with and without ERBB2 -activating mutations. However, patients harboring mutations within the tyrosine kinase domain of ERBB2 exhibited a trend toward shorter PFS (median 2.0 vs. 9.5 months; p = 0.121) and a significantly shorter OS (median 8.9 vs. 30.7 months; p = 0.007) compared to those with other activating mutations (Supplementary Fig. 3). Survival outcomes according to ERBB2-amplified segment length To investigate the prognostic value according to the size of the ERBB2 -amplified region, we explored the lengths of all ERBB2 -amplified segments detected by CBS methods using CNVKit. Of the 111 patients with ERBB2 amplifications, we excluded ERBB2 -amplified segments from three patients that were not entirely covered by the ERBB2 gene region and two patients with ERBB2 amplification manually detected by the pathologist. In a total of 106 ERBB2 -amplified cohorts, the length of the amplified segments of the ERBB2 gene varied in the range of approximately 160 Kb–21 Mb. We defined the ERBB2 -amplified segment length into focal amplification (≤ optimal cutoff) and non-focal amplification (> optimal cutoff) using an optimal cutoff value of 879 Kb, determined by maximally selected rank statistics. Patients with ERBB2 focal amplification had a superior PFS (median, 10.1 vs. 6.1 months; log-rank p = 0.01) and tended to have a better OS (Fig. 2 ). NOTCH3 alterations are associated with poor prognosis To further identify genomic alterations with prognostic significance, we performed pathway enrichment analysis across the three risk groups: 57 in the good-risk group, 64 in the moderate-risk group, and 27 in the poor-risk group. Pathway analysis revealed that gene sets associated with the NOTCH signaling pathway, including NOTCH3 and FBXW7 , were more frequently altered in the moderate- and poor-risk groups, compared to the good risk group (Supplementary Fig. 4). We then assessed survival outcomes according to alterations in NOTCH3 and FBXW7 (Fig. 3 ). Patients harboring NOTCH3 alterations had significantly shorter PFS (median 4.5 vs. 8.5 months; log-rank p = 0.002) and OS (median 16.1 vs. 20.4 months; log-rank p = 0.004) compared to those without alterations. Patients harboring FBXW7 alterations tended to have a worse PFS (median 4.2 vs. 8.3 months; p = 0.056), but not OS (median 17.6 vs. 18.3 months; p = 0.127), compared to those without alterations. Clinical and genomic prognostic factors Univariate and multivariate analyses were conducted to evaluate the prognostic significance of ERBB2 -amplified segment length and NOTCH3 alterations. In the multivariable Cox proportional hazards model, adjusted for age, sex, disease setting, and prognostic group, ERBB2 focal amplification was significantly associated with improved PFS compared to non-focal amplification. Moreover, ERBB2 focal amplification emerged as a strong and independent prognostic factor for PFS (hazard ratio (HR), 0.52; CI, 0.31–0.86; p = 0.001; Table 2 ). In addition, multivariate analysis identified NOTCH3 alterations as independent negative prognostic factors for both PFS (HR, 1.93; 95% CI, 1.00–3.73; p = 0.049) and OS (HR, 2.03; 95% CI, 1.04–3.96; p = 0.037). Table 2 Multivariate analysis of ERBB2 focal amplification and NOTCH3 alterations. Progression-free survival Overall survival Univariate Multivariate Univariate Multivariate Hazard ratio (95% CI) p -value Hazard ratio (95% CI) p -value Hazard ratio (95% CI) p -value Hazard ratio (95% CI) p -value Age > 60 0.96 (0.64–1.45) 0.847 1.23 (0.76–1.98) 0.396 0.94 (0.63–1.42) 0.776 1.11 (0.70–1.75) 0.662 Male sex 0.58 (0.37–0.91) 0.018 0.56 (0.34–0.93) 0.025 0.61 (0.40–0.96) 0.031 0.54 (0.33–0.89) 0.014 Disease setting Initially metastatic Reference Reference Recurrent or Locally advanced unresectable 0.98 (0.61–1.56) 0.932 0.95 (0.59–1.51) 0.815 Prognostic group Good Reference Reference Reference Reference Moderate 1.03 (0.64–1.66) 0.895 0.92 (0.56–1.51) 0.736 1.52 (0.94–2.45) 0.086 1.37 (0.83–2.28) 0.221 Poor 2.19 (1.26–3.79) 0.005 2.11 (1.14–3.91) 0.018 2.48 (1.40–4.39) 0.002 1.94 (1.04–3.65) 0.038 ERBB2 focal amplification 0.55 (0.35–0.86) 0.008 0.52 (0.31–0.86) 0.001 0.68 (0.44–1.07) 0.094 0.76 (0.46–1.27) 0.297 NOTCH3 alterations 2.59 (1.40–4.79) 0.002 1.93 (1.00–3.73) 0.049 2.30 (1.28–4.14) 0.005 2.03 (1.04–3.96) 0.037 CI, confidence interval Conclusions This study evaluated the association between survival outcomes and genomic alterations identified through the targeted panel sequencing in patients with HER2-positive AGC. The findings in our research aligned with recent retrospective studies identifying a high CN status of ERBB2 as an independent prognostic factor in GC (Hino et al., 2022 ; Ichikawa et al., 2024 ; Pietrantonio et al., 2023 ), distinct from HER2 IHC status. Although these studies have shown that a high CN status of ERBB2 is associated with a better survival prognosis when combined with trastuzumab and chemotherapy, the potential impact of amplified segment length on treatment outcomes has remained largely unexplored. Based on the varying lengths of the ERBB2 -amplified segments, we investigated the differences in survival outcomes according to segment lengths. A recent study demonstrated that ERBB2 focal amplification, defined as an amplified segment less than 3 megabases (Mb) in length, was associated with improved clinical outcomes in breast cancer patients treated with trastuzumab-based therapy (Kim et al., 2023). Although the study did not analyze segment length in detail, its results suggest that the therapeutic efficacy of trastuzumab-based therapy may differ depending on the ERBB2-amplified segment length. ERBB2 -amplified segments encode numerous oncogenes and genes associated with drug resistance. Thus, we hypothesized that the tumor with ERBB2 non-focal amplification may have a worse prognosis due to attenuated HER2 dependency or acquired drug resistance. We found that the patients with ERBB2 focal amplification had a superior PFS. Although ERBB2 focal amplification was only associated with a trend toward improved OS, the lack of statistical significance may be attributed to the effects of subsequent treatments. Moreover, multivariate analysis confirmed ERBB2 focal amplification as an independent prognostic factor for PFS. The CDK12 and RARA genes, located near the ERBB2 gene, are frequently co-amplified with ERBB2 and have been associated with a poor prognosis in various cancers (Garattini et al., 2024 ; Yanai et al., 2020 ). We evaluated the association between co-amplification of ERBB2 with either CDK12 or RARA and survival outcomes. No statistically significant differences were observed, suggesting that additional genes co-amplified with ERBB2 , beyond CDK12 and RARA , should be investigated. These data indicate that the length of the ERBB2 -amplified segment may represent a novel biomarker for predicting responses to anti-HER2 therapies, although the underlying molecular mechanisms remain to be elucidated. This study identified NOTCH3 alterations as a poor prognostic factor in AGC. Although alterations in several genes, including NOTCH3 , were enriched in the poor- and moderate-risk groups, only NOTCH3 alterations were also statistically significant in multivariate analysis. NOTCH3 has previously been reported to potentially play important roles in cancer progression, including stemness, metastasis, and angiogenesis (Xiu et al., 2021 ). In terms of clinical significance, the overexpression of NOTCH3 is associated with poor survival outcomes in various cancers (Xiu et al., 2021 ). Particularly in GC, a prior meta-analysis revealed that NOTCH3 was frequently overexpressed and significantly associated with a poor prognosis, supporting its oncogenic role (Xu et al., 2024 ). However, the association of clinical outcomes with genomic alterations, such as CN loss and point mutation, has not yet been elucidated in GC. NOTCH3 can also act as a tumor suppressor gene in a context-dependent manner (Xiu et al., 2021 ). As the NOTCH3 alterations identified in our cohort lack further detailed characterization, the interpretation of their clinical significance remains limited. The interpretation of our findings has several limitations. First, this study is a single center observational study. However, all patients received trastuzumab-based therapy as first-line treatment and showed a correlation with survival outcomes in the overall study population as well as across risk groups. Second, we focused on mutations and CNVs detected by targeted panel sequencing. For the ERBB2 -amplified segment, the limitation of targeted panel sequencing, which leaves gaps in genome coverage, has prevented us from clearly defining the segment criteria due to the use of an optimal cutoff. Although we considered the ERBB2 -amplified segment to be the amplified interval, including the entire ERBB2 gene, and evaluated the cutoff using the maximally selected rank statistical analysis, different algorithms vary in tools and procedures for CNV calling, including normalization, calculation of copy ratio, and segmentation. Third, this study was not validated in an external cohort. Hence, further studies with a large sample size, utilizing whole-genome sequencing, are necessary to refine the criteria for distinguishing between non-focal and focal amplification. In conclusion, our study highlights the significance of ERBB2 focal amplification as a potential biomarker for predicting favorable outcomes to trastuzumab-based therapy in HER2-positive AGC. Declarations Funding: This work was supported by NRF (National Research Foundation of Korea) Grant funded by the Korean Government (NRF-2015-Fostering Core Leaders of the Future Basic Science Program/Global Ph.D. Fellowship Program) [Project Number NRF-2015H1A2A1033652]. Competing Interests: Nothing directly related to this work. Out of this work, MHR received honoraria from DAEHWA Pharmaceutical, Bristol Myers Squibb, Lilly, Ono Pharmaceutical, MSD, Taiho Pharmaceutical, Novartis, Daiichi Sankyo, and AstraZeneca, and served as a consultant for DAEHWA Pharmaceutical, Bristol Myers Squibb, Lilly and Ono Pharmaceutical. HDK received research grants from Roche/Genentech, Amgen, and AstraZeneca and honoraria from AstraZeneca, Bristol Myers Squibb, Ono Pharmaceuticals, Boryung Pharmaceuticals, MSD, Daiichi Sankyo, Astellas, Boostimmune, DAEHWA Pharmaceutical, LG Chem,and MustBio. Author Contributions: All authors contributed to the study conception and design. Material preparation, data collection, data analysis and interpretation were performed by Sun Young Lee, Jaewon Hyung, Hyung-Don Kim, Hyungeun Lee, Meesun Moon, Young Soo Park and Min-Hee Ryu. The first draft of the manuscript was written by Sun Young Lee and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Ethics approval: This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted the Institutional Review Board (IRB) of Asan Medical Center, Korea (2014-0301). Consent to participate: Informed consent was obtained from all individual participants included in the study. Acknowledgments This work was supported by NRF (National Research Foundation of Korea) Grant funded by the Korean Government (NRF-2015-Fostering Core Leaders of the Future Basic Science Program/Global Ph.D. Fellowship Program) [Project Number NRF-2015H1A2A1033652]. References Abrahao-Machado, L. F., & Scapulatempo-Neto, C. (2016). HER2 testing in gastric cancer: An update. World J Gastroenterol , 22 (19), 4619-4625. https://doi.org/10.3748/wjg.v22.i19.4619 An, E., Ock, C. Y., Kim, T. Y., Lee, K. H., Han, S. W., Im, S. A., Kim, T. Y., Liao, W. L., Cecchi, F., Blackler, A., Thyparambil, S., Kim, W. 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Pembrolizumab plus trastuzumab and chemotherapy for HER2-positive gastric or gastro-oesophageal junction adenocarcinoma: interim analyses from the phase 3 KEYNOTE-811 randomised placebo-controlled trial. Lancet , 402 (10418), 2197-2208. https://doi.org/10.1016/S0140-6736(23)02033-0 Kim, H. D., Ryu, M. H., Park, Y. S., Lee, S. Y., Moon, M., & Kang, Y. K. (2022). Insertion-deletion rate is a qualitative aspect of the tumor mutation burden associated with the clinical outcomes of gastric cancer patients treated with nivolumab. Gastric Cancer , 25 (1), 226-234. https://doi.org/10.1007/s10120-021-01233-1 Koo, D. H., Ryoo, B. Y., Kim, H. J., Ryu, M. H., Lee, S. S., Moon, J. H., Chang, H. M., Lee, J. L., Kim, T. W., & Kang, Y. K. (2011). A prognostic model in patients who receive chemotherapy for metastatic or recurrent gastric cancer: validation and comparison with previous models. Cancer Chemother Pharmacol , 68 (4), 913-921. https://doi.org/10.1007/s00280-011-1561-8 Koo, D. H., Ryu, M. H., Lee, M. Y., Chae, H., Kim, E. J., Moon, M. S., & Kang, Y. K. (2021). Trends in Chemotherapy Patterns and Survival of Patients with Advanced Gastric Cancer over a 16-Year Period: Impact of Anti-HER2-Targeted Agent in the Real-World Setting. Cancer Res Treat , 53 (2), 436-444. https://doi.org/10.4143/crt.2020.725 Mayakonda, A., Lin, D. C., Assenov, Y., Plass, C., & Koeffler, H. P. (2018). Maftools: efficient and comprehensive analysis of somatic variants in cancer. Genome Res , 28 (11), 1747-1756. https://doi.org/10.1101/gr.239244.118 Pietrantonio, F., Fuca, G., Morano, F., Gloghini, A., Corso, S., Aprile, G., Perrone, F., De Vita, F., Tamborini, E., Tomasello, G., Gualeni, A. V., Ongaro, E., Busico, A., Giommoni, E., Volpi, C. C., Laterza, M. M., Corallo, S., Prisciandaro, M., Antista, M.,…Di Bartolomeo, M. (2018). Biomarkers of Primary Resistance to Trastuzumab in HER2-Positive Metastatic Gastric Cancer Patients: the AMNESIA Case-Control Study. Clin Cancer Res , 24 (5), 1082-1089. https://doi.org/10.1158/1078-0432.CCR-17-2781 Pietrantonio, F., Manca, P., Bellomo, S. E., Corso, S., Raimondi, A., Berrino, E., Morano, F., Migliore, C., Niger, M., Castagnoli, L., Pupa, S. M., Marchio, C., Di Bartolomeo, M., Restuccia, E., Lambertini, C., Tabernero, J., & Giordano, S. (2023). HER2 Copy Number and Resistance Mechanisms in Patients with HER2-positive Advanced Gastric Cancer Receiving Initial Trastuzumab-based Therapy in JACOB Trial. Clin Cancer Res , 29 (3), 571-580. https://doi.org/10.1158/1078-0432.CCR-22-2533 Siegel, R. L., Miller, K. D., Fuchs, H. E., & Jemal, A. (2021). Cancer Statistics, 2021. CA Cancer J Clin , 71 (1), 7-33. https://doi.org/10.3322/caac.21654 Steele, C. D., Abbasi, A., Islam, S. M. A., Bowes, A. L., Khandekar, A., Haase, K., Hames-Fathi, S., Ajayi, D., Verfaillie, A., Dhami, P., McLatchie, A., Lechner, M., Light, N., Shlien, A., Malkin, D., Feber, A., Proszek, P., Lesluyes, T., Mertens, F.,…Pillay, N. (2022). Signatures of copy number alterations in human cancer. Nature , 606 (7916), 984-991. https://doi.org/10.1038/s41586-022-04738-6 Suehnholz, S. P., Nissan, M. H., Zhang, H., Kundra, R., Nandakumar, S., Lu, C., Carrero, S., Dhaneshwar, A., Fernandez, N., Xu, B. W., Arcila, M. E., Zehir, A., Syed, A., Brannon, A. R., Rudolph, J. E., Paraiso, E., Sabbatini, P. J., Levine, R. L., Dogan, A.,…Chakravarty, D. (2024). Quantifying the Expanding Landscape of Clinical Actionability for Patients with Cancer. Cancer Discov , 14 (1), 49-65. https://doi.org/10.1158/2159-8290.CD-23-0467 Xiu, M., Wang, Y., Li, B., Wang, X., Xiao, F., Chen, S., Zhang, L., Zhou, B., & Hua, F. (2021). The Role of Notch3 Signaling in Cancer Stemness and Chemoresistance: Molecular Mechanisms and Targeting Strategies. Front Mol Biosci , 8 , 694141. https://doi.org/10.3389/fmolb.2021.694141 Xu, J., Jin, X. L., Shen, H., Chen, X. W., Chen, J., Huang, H., Xu, B., & Xu, J. (2024). NOTCH3 as a prognostic biomarker and its correlation with immune infiltration in gastrointestinal cancers. Sci Rep , 14 (1), 14327. https://doi.org/10.1038/s41598-024-65036-x Yanai, Y., Kosaka, T., Nakamura, K., Aimono, E., Matsumoto, K., Morita, S., Mikami, S., Nishihara, H., & Oya, M. (2020). CDK12 and HER2 coamplification in two urothelial carcinomas with rapid and aggressive clinical progression. Cancer Sci , 111 (12), 4652-4655. https://doi.org/10.1111/cas.14672 Zhang, L., Hamdani, O., Gjoerup, O., Cho-Phan, C., Snider, J., Castellanos, E., Nimeiri, H., Frampton, G., Venstrom, J. M., Oxnard, G., Klempner, S. J., & Schrock, A. B. (2022). ERBB2 Copy Number as a Quantitative Biomarker for Real-World Outcomes to Anti-Human Epidermal Growth Factor Receptor 2 Therapy in Advanced Gastroesophageal Adenocarcinoma. JCO Precis Oncol , 6 , e2100330. https://doi.org/10.1200/PO.21.00330 Additional Declarations No competing interests reported. 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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-7267337","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":496463106,"identity":"0cbf0200-44e5-4b91-8638-fbaf088d3554","order_by":0,"name":"Sun Young Lee","email":"","orcid":"","institution":"Asan Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Sun","middleName":"Young","lastName":"Lee","suffix":""},{"id":496463107,"identity":"73fefa12-fde9-48e6-8268-5e30037132b0","order_by":1,"name":"Jaewon Hyung","email":"","orcid":"","institution":"Asan Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Jaewon","middleName":"","lastName":"Hyung","suffix":""},{"id":496463108,"identity":"0326227b-1a8e-4037-9716-208af608330f","order_by":2,"name":"Hyung-Don Kim","email":"","orcid":"","institution":"Asan Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Hyung-Don","middleName":"","lastName":"Kim","suffix":""},{"id":496463109,"identity":"37bc60c6-4819-481a-88cc-e03457871204","order_by":3,"name":"Hyungeun Lee","email":"","orcid":"","institution":"Asan Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Hyungeun","middleName":"","lastName":"Lee","suffix":""},{"id":496463110,"identity":"846a4486-9685-44df-b163-94df80fc2e02","order_by":4,"name":"Meesun Moon","email":"","orcid":"","institution":"Asan Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Meesun","middleName":"","lastName":"Moon","suffix":""},{"id":496463111,"identity":"d89d4e98-f595-4da5-b77a-a2fff588d3b2","order_by":5,"name":"Young Soo Park","email":"","orcid":"","institution":"Asan Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Young","middleName":"Soo","lastName":"Park","suffix":""},{"id":496463112,"identity":"3ad4cc7e-a754-4beb-8a05-8b8365db276e","order_by":6,"name":"Min-Hee Ryu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYDACZgaGA0BKDi7Ax8BGnBZjuAAbQS1QkNhAtBbddt6DhyvbDqf3Tzv8gPFHhU1iGwNb2gd8WswO8yUcPNt2OHfG7TQDZp4zaSAth2fg18JjcLARqKXhdg4DM2PbYaAW9ma8DoNpSZcHamH8+Y8ELQkGQC0MvA2HwQ4jrKXhXLrhRqBfDvMcSzNuY2ZLxq/l/Bnjjw1l1vJyt5MfPvxRYyPbz95mjFcLGDBCY+IAmGQmrAEI/hClahSMglEwCkYqAACx3kitqh22LwAAAABJRU5ErkJggg==","orcid":"","institution":"Asan Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Min-Hee","middleName":"","lastName":"Ryu","suffix":""}],"badges":[],"createdAt":"2025-08-01 04:53:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7267337/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7267337/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88642236,"identity":"7763e369-7940-4554-a08c-3468a55251b0","added_by":"auto","created_at":"2025-08-08 16:11:49","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1086625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic landscape for 151 HER2-positive AGC patients and survival outcomes according to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eERBB2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genomic status. (A)\u003c/strong\u003e Only the top five genes with copy number alterations and the top seven genes with mutations are shown. Each column represents a patient, and each row represents a gene. Survival analysis of \u003cem\u003eERBB2\u003c/em\u003e copy number for patients with \u003cstrong\u003e(B)\u003c/strong\u003e PFS or \u003cstrong\u003e(C)\u003c/strong\u003eOS as the endpoint. CN, copy number.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7267337/v1/9241aaf40521df6ec2aa4343.jpeg"},{"id":88644225,"identity":"2b57c0f3-7392-4f77-a441-9f9cfa632c86","added_by":"auto","created_at":"2025-08-08 16:19:49","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1006359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvival outcomes in relation to the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eERBB2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-amplified segment length.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Distribution of \u003cem\u003eERBB2\u003c/em\u003e-amplified segments in this cohort. \u003cstrong\u003e(B)\u003c/strong\u003ePFS and \u003cstrong\u003e(C)\u003c/strong\u003e OS according to focal/non-focal amplification of \u003cem\u003eERBB2\u003c/em\u003e. PFS, progression-free survival; OS, overall survival; focal, \u003cem\u003eERBB2\u003c/em\u003e-amplified segment length ≤ optimal cutoff; non-focal, \u003cem\u003eERBB2\u003c/em\u003e-amplified segment length \u0026gt; optimal cutoff.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7267337/v1/c66fd0eb2873357cdd4c7910.jpeg"},{"id":88642237,"identity":"44fa3cf0-719d-4de1-81bc-06d405239163","added_by":"auto","created_at":"2025-08-08 16:11:49","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":768912,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvival outcomes according to\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ealterations in\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e FBXW7 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e NOTCH3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e PFS and OS in patients with \u003cstrong\u003e(A)\u003c/strong\u003e \u003cem\u003eFBXW7\u003c/em\u003e gene alterations and \u003cstrong\u003e(B)\u003c/strong\u003e \u003cem\u003eNOTCH3\u003c/em\u003e gene alterations.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7267337/v1/9a3850e6697f6e8d880f326a.jpeg"},{"id":88752289,"identity":"515a455a-cd8b-4da3-830a-c31edb9b0519","added_by":"auto","created_at":"2025-08-11 06:31:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3996649,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7267337/v1/6e68e662-d3f8-4663-a251-4f4d58b4086a.pdf"},{"id":88642246,"identity":"978c1e4a-0e10-4b36-8521-4ea2679a856d","added_by":"auto","created_at":"2025-08-08 16:11:49","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":7386371,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialJCRCO.docx","url":"https://assets-eu.researchsquare.com/files/rs-7267337/v1/6fbaf38ef36140abf4c3cda1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genomic correlations for clinical outcomes in HER2-positive advanced gastric cancers treated using trastuzumab-based therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003c/p\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eGastric cancer (GC) is the most commonly diagnosed cancer globally and ranks as the fourth leading cause of cancer-related death worldwide (Siegel et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Over the last decade, several phase III clinical trials have been conducted to develop molecularly targeted therapies and immunotherapies (Ishii et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Human epidermal growth factor receptor 2 (HER2/\u003cem\u003eERBB2\u003c/em\u003e), a proto-oncogene encoded by \u003cem\u003eERBB2\u003c/em\u003e on chromosome 17, is involved in cell proliferation, metastasis, and poor outcomes in various cancer types, including advanced gastric cancer (AGC) and gastroesophageal junction cancer (GEJC), with an incidence rate of 15–20% (Abrahao-Machado \u0026amp; Scapulatempo-Neto, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Boku, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTrastuzumab is a monoclonal antibody that targets the juxtamembrane portion of HER2 and inhibits HER2-mediated signaling by preventing HER2 dimerization, inhibiting the shedding of the HER2 extracellular domain, enhancing the endocytic destruction system, and inducing antibody-dependent cytotoxicity (Hudis, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The combination of trastuzumab and chemotherapy in the first-line setting has shown significant survival benefits for patients with HER2-positive (immunohistochemistry (IHC) 2+/in situ hybridization positive (ISH) positive or IHC 3+) AGC, and trastuzumab-based therapy is the current standard treatment (Bang et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Franchi et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Janjigian et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Koo et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNevertheless, the overall response rate (ORR) was approximately 47% in the ToGA trial (Bang et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and the long-term benefits of trastuzumab-based treatment were observed only in a small number of patients (Pietrantonio et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Some studies have shown that patients with higher HER2 protein expression level (An et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Catenacci et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) or increased \u003cem\u003eERBB2\u003c/em\u003e copy number (CN) (Gomez-Martin et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Pietrantonio et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), as assessed by IHC or NGS, respectively, can derive long-term benefits from trastuzumab-based therapy. Regarding primary drug resistance, mutations and amplifications in \u003cem\u003eEGFR/MET/KRAS/PI3K\u003c/em\u003e, known as the AMNESIA panel, have been evaluated as negative prognostic factors for trastuzumab-based therapy (Pietrantonio et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, it is speculated that the impact of AMNESIA positivity on survival outcomes may vary depending on the cohort (Pietrantonio et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Pietrantonio et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGiven that copy number variants (CNVs) occur extensively with varying lengths over 100 kilobases (Kb) (Steele et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), \u003cem\u003eERBB2\u003c/em\u003e gene amplification may impact not only the \u003cem\u003eERBB2\u003c/em\u003e gene itself but also adjacent genes within the amplified segment. This study aimed to evaluate the co-mutational status and \u003cem\u003eERBB2\u003c/em\u003e-ampified segment length as prognostic factors for patient selection to optimize trastuzumab-based therapy.\u003c/p\u003e\u003c/div\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eStudy patients\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePatients aged \u0026gt; 18 years with histologically confirmed locally advanced, unresectable, recurrent, or initially metastatic HER2-positive GC who received trastuzumab-containing first-line chemotherapy at Asan Medical Center, South Korea, from January 2015 to December 2023 were enrolled in the study. Subsequently, the patients were classified into three risk groups (poor-, moderate-, and good-risk) according to the prognostic model for AGC described in our previous article (Koo et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The protocol used in this study was approved by the Institutional Review Board (IRB) of Asan Medical Center, Korea (2014 − 0301), and the study was conducted according to the Helsinki Declaration.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHER2 status\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIHC was performed for HER2 using an anti-HER2/neu (4B5) rabbit monoclonal primary antibody (Ventana Medical System, Tucson, AZ). HER2 protein expression was scored on a scale of 0 to 3 according to the GC consensus guidelines (Bang et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). HER2 positivity was defined as IHC 3+, or as IHC 2 + with \u003cem\u003eHER2\u003c/em\u003e gene amplification conducted by ISH.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTargeted NGS and bioinformatics analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGenomic DNA was extracted from archived formalin-fixed, paraffin-embedded (FFPE) tissue specimens. Targeted next-generation sequencing (NGS) was performed using the NextSeq platform (Illumina, San Diego, CA, USA) with OncoPanel AMC v3, v4, and v4.5 panels, as described previously (Kim et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The OncoPanel AMC version 3 (OP AMC v3), version 4 (OP AMC v4), and version 4.5 (OP AMC v4.5) captured 382, 323 and 343 cancer-related genes, respectively (OP AMC v3, 199 genes for entire exons, 8 genes for partial introns, and 184 genes for hotspots; OP AMC v4, 225 genes for entire exons, 6 genes for partial introns, and 99 for hotspots; OP AMC v4.5, 244 genes for entire exons, 14 genes for partial introns, and 110 for hotspots). Sequenced reads were aligned to the human reference genome (GRCh37; hg19) using the Burrows-Wheeler Aligner and processed using the Genome Analysis Toolkit pipeline. The CN variation analysis was performed using a CNV kit with the default segmentation method (circular binary segmentation; CBS), and copy numbers of tumors were analyzed against a panel of unmatched normal samples. Genes with an estimated copy number ≥ 5 were classified as amplifications, whereas those with a copy number ≤ 0 were classified as losses.\u003c/p\u003e\u003cp\u003eFor the \u003cem\u003eERBB2\u003c/em\u003e-amplified segment length analysis, only segments that covered the entire \u003cem\u003eERBB2\u003c/em\u003e gene region were used for comparison, excluding segments that covered a partial region of \u003cem\u003eERBB2\u003c/em\u003e or were not calculable (\u003cem\u003eERBB2\u003c/em\u003e-amplified by manual detection).\u003c/p\u003e\u003cp\u003eThe maftools package in R was used to analyze pathway enrichment and for mutation comparison (Mayakonda et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eProgression-free survival (PFS) was defined as the time from the initiation of trastuzumab-based first-line chemotherapy to the time of disease progression or death, whichever occurred first. Overall survival (OS) was defined as the time from the initiation of trastuzumab-based first-line chemotherapy to the time of death from any cause. The Kaplan–Meier method was used to estimate PFS and OS. Survival curves were compared using the log-rank test.\u003c/p\u003e\u003cp\u003e\u003cem\u003eERBB2\u003c/em\u003e copy number segment size cutoff was determined using the maximally selected rank statistics from the maxstat R package. The Cox proportional hazards model was used to estimate the hazard ratio (HR) for survival outcomes. All statistical analyses were performed using the statistical software package R version 4.3.3.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eBaseline characteristics of study patients\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe baseline characteristics of the included HER2-positive patients (n\u0026thinsp;=\u0026thinsp;151) are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The median age of the patients was 62 years (range, 25\u0026ndash;84), while 111 patients (73.5%) were male, and 104 patients (68.9%) had initially metastatic disease. All patients received trastuzumab plus fluoropyrimidine and platinum double chemotherapy combination as first-line treatment. Among 109 patients (72%) with a measurable disease status, 82 patients (75%) exhibited a best response of either a partial response or better, according to the Response Evaluation Criteria in Solid Tumors version 1.1. With a median follow-up duration of 45.8 months (range, 15.4\u0026ndash;84.4), the median PFS and OS were 8.2 months (95% CI 6.5\u0026ndash;9.4), and 18.2 months (95% CI 14.9\u0026ndash;23.4), respectively. Risk group data, as defined in the previous study, were available for 148 patients (Koo et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The good, moderate, and poor risk categories included 57 (38.5%), 64 (43.2%), and 27 patients (18.2%), respectively. The PFS and OS curves for three risk groups differed significantly (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e\u003c/div\u003e\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\u003eBaseline characteristics (n\u0026thinsp;=\u0026thinsp;151).\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=\"char\" char=\".\" 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\u003en (%)\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\u0026thinsp;\u0026gt;\u0026thinsp;60 years\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e83 (55.0)\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=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e40 (26.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e111 (73.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHER2 IHC scale\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\u003e2+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e27 (17.9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e124 (82.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTreatment setting\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\u003eInitially metastatic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e104 (68.9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRecurrent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e43 (28.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLocally advanced unresectable\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4 (2.6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHistology\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\u003eWD/MD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e82 (54.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePD/SRC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e69 (45.7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMSI result*\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\u003eMSS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e141 (100.0)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMSI-high\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0 (0.0)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eEBV result*\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\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e142 (95.9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6 (4.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eECOG PS*\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\u003e0/1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e130 (87.8)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026ge;2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18 (12.2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGastrectomy\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\u003ePerformed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e48 (31.8)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNot performed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e103 (68.2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMetastasis\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\u003ePeritoneal metastasis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e68 (45.0)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLiver metastasis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e61 (40.4)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLung metastasis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14 (9.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBone metastasis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10 (6.6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLymph node metastasis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e92 (60.9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOthers\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e133 (88.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eALP\u0026thinsp;\u0026gt;\u0026thinsp;120 IU/L*\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e43 (29.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAlbumin\u0026thinsp;\u0026lt;\u0026thinsp;3.3 g/dL*\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e51 (34.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal bilirubin\u0026thinsp;\u0026gt;\u0026thinsp;1.2 mg/dL*\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10 (6.8)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eRisk group*\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\u003eGood\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e57 (38.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eModerate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e64 (43.2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e27 (18.2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIHC, immunohistochemistry; MSI, microsatellite instability; MSS, microsatellite stable; EBV, Epstein-Barr virus; ECOG PS, Eastern Cooperative Oncology Group Performance Status; ALP, alkaline phosphatase\u003c/p\u003e\u003cp\u003e\u003csup\u003e\u003cb\u003e*\u003c/b\u003e\u003c/sup\u003e Patients for whom the tests were available were included in the analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGenomic alterations identified among HER2-positive AGC\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe landscape of molecular alterations for our cohort is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(A). The most frequently altered gene in HER2-positive tumors was \u003cem\u003eTP53\u003c/em\u003e (84%), followed by \u003cem\u003eERBB2\u003c/em\u003e (75%), \u003cem\u003eCDK12\u003c/em\u003e (58%), \u003cem\u003eRARA\u003c/em\u003e (38%), \u003cem\u003eCCNE1\u003c/em\u003e (34%), \u003cem\u003eMYC\u003c/em\u003e (28%), and \u003cem\u003eLRP1B\u003c/em\u003e (26%). Meanwhile, point mutations were most common in \u003cem\u003eTP53\u003c/em\u003e, followed by \u003cem\u003eLRP1B\u003c/em\u003e (24%), and \u003cem\u003eERBB2\u003c/em\u003e (16%). Regarding copy number variants, \u003cem\u003eERBB2\u003c/em\u003e amplifications were the most prevalent (74%), with \u003cem\u003eCDK12\u003c/em\u003e and \u003cem\u003eRARA\u003c/em\u003e amplifications enriched specifically in patients with \u003cem\u003eERBB2\u003c/em\u003e amplification. There was no significant association between \u003cem\u003eCDK12\u003c/em\u003e or \u003cem\u003eRARA\u003c/em\u003e co-amplification and survival outcomes (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e\u003cp\u003eWe examined the prognostic value of \u003cem\u003eERBB2\u003c/em\u003e CN status in 111 patients harboring \u003cem\u003eERBB2\u003c/em\u003e-amplified tumors. Using the optimal cutoff value of 60, patients with \u003cem\u003eERBB2\u003c/em\u003e CN-high (CN\u0026thinsp;\u0026ge;\u0026thinsp;60) status had a superior PFS (median 13.1 vs. 7.5; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.039) and tended to have a better OS (median 21.5 vs. 18.2; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.061) compared to those with \u003cem\u003eERBB2\u003c/em\u003e CN-low (CN\u0026thinsp;\u0026lt;\u0026thinsp;60) status (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u0026ndash;C).\u003c/p\u003e\u003cp\u003eWe detected 13 \u003cem\u003eERBB2\u003c/em\u003e mutations in 12 patients (7.9%) with HER2-positive AGC in this study. The mutation loci were distributed in the extracellular domain (ECD), transmembrane domain (TMD), juxtamembrane domain (JMD), tyrosine kinase domain (TKD), and intracellular domain (ICD). Co-occurrence of the \u003cem\u003eERBB2\u003c/em\u003e mutation and CN amplification was detected in 11 patients (7.0%); no significant correlation was observed between the variant allele frequency (VAF) of the mutation and its copy number (Supplementary Table\u0026nbsp;1).\u003c/p\u003e\u003cp\u003eOf the 12 patients with \u003cem\u003eERBB2\u003c/em\u003e mutations, activating mutations annotated by OncoKB (Chakravarty et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Suehnholz et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) were detected in 7 patients. Survival analysis revealed no significant differences in PFS or OS between patients with and without \u003cem\u003eERBB2\u003c/em\u003e-activating mutations. However, patients harboring mutations within the tyrosine kinase domain of \u003cem\u003eERBB2\u003c/em\u003e exhibited a trend toward shorter PFS (median 2.0 vs. 9.5 months; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.121) and a significantly shorter OS (median 8.9 vs. 30.7 months; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007) compared to those with other activating mutations (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eSurvival outcomes according to ERBB2-amplified segment length\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the prognostic value according to the size of the \u003cem\u003eERBB2\u003c/em\u003e-amplified region, we explored the lengths of all \u003cem\u003eERBB2\u003c/em\u003e-amplified segments detected by CBS methods using CNVKit. Of the 111 patients with \u003cem\u003eERBB2\u003c/em\u003e amplifications, we excluded \u003cem\u003eERBB2\u003c/em\u003e-amplified segments from three patients that were not entirely covered by the \u003cem\u003eERBB2\u003c/em\u003e gene region and two patients with \u003cem\u003eERBB2\u003c/em\u003e amplification manually detected by the pathologist. In a total of 106 \u003cem\u003eERBB2\u003c/em\u003e-amplified cohorts, the length of the amplified segments of the \u003cem\u003eERBB2\u003c/em\u003e gene varied in the range of approximately 160 Kb\u0026ndash;21 Mb. We defined the \u003cem\u003eERBB2\u003c/em\u003e-amplified segment length into focal amplification (\u0026le;\u0026thinsp;optimal cutoff) and non-focal amplification (\u0026gt;\u0026thinsp;optimal cutoff) using an optimal cutoff value of 879 Kb, determined by maximally selected rank statistics. Patients with \u003cem\u003eERBB2\u003c/em\u003e focal amplification had a superior PFS (median, 10.1 vs. 6.1 months; log-rank \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) and tended to have a better OS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eNOTCH3 alterations are associated with poor prognosis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further identify genomic alterations with prognostic significance, we performed pathway enrichment analysis across the three risk groups: 57 in the good-risk group, 64 in the moderate-risk group, and 27 in the poor-risk group. Pathway analysis revealed that gene sets associated with the \u003cem\u003eNOTCH\u003c/em\u003e signaling pathway, including \u003cem\u003eNOTCH3\u003c/em\u003e and \u003cem\u003eFBXW7\u003c/em\u003e, were more frequently altered in the moderate- and poor-risk groups, compared to the good risk group (Supplementary Fig.\u0026nbsp;4). We then assessed survival outcomes according to alterations in \u003cem\u003eNOTCH3\u003c/em\u003e and \u003cem\u003eFBXW7\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Patients harboring \u003cem\u003eNOTCH3\u003c/em\u003e alterations had significantly shorter PFS (median 4.5 vs. 8.5 months; log-rank \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) and OS (median 16.1 vs. 20.4 months; log-rank \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004) compared to those without alterations. Patients harboring \u003cem\u003eFBXW7\u003c/em\u003e alterations tended to have a worse PFS (median 4.2 vs. 8.3 months; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.056), but not OS (median 17.6 vs. 18.3 months; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.127), compared to those without alterations.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eClinical and genomic prognostic factors\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eUnivariate and multivariate analyses were conducted to evaluate the prognostic significance of \u003cem\u003eERBB2\u003c/em\u003e-amplified segment length and \u003cem\u003eNOTCH3\u003c/em\u003e alterations. In the multivariable Cox proportional hazards model, adjusted for age, sex, disease setting, and prognostic group, \u003cem\u003eERBB2\u003c/em\u003e focal amplification was significantly associated with improved PFS compared to non-focal amplification. Moreover, \u003cem\u003eERBB2\u003c/em\u003e focal amplification emerged as a strong and independent prognostic factor for PFS (hazard ratio (HR), 0.52; CI, 0.31\u0026ndash;0.86; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, multivariate analysis identified \u003cem\u003eNOTCH3\u003c/em\u003e alterations as independent negative prognostic factors for both PFS (HR, 1.93; 95% CI, 1.00\u0026ndash;3.73; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.049) and OS (HR, 2.03; 95% CI, 1.04\u0026ndash;3.96; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037).\u003c/p\u003e\u003c/div\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\u003e\u003cb\u003eMultivariate analysis of\u003c/b\u003e \u003cb\u003eERBB2\u003c/b\u003e \u003cb\u003efocal amplification and\u003c/b\u003e \u003cb\u003eNOTCH3\u003c/b\u003e \u003cb\u003ealterations.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003eProgression-free survival\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c10\" namest=\"c7\"\u003e\u003cp\u003eOverall survival\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eUnivariate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eMultivariate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003eUnivariate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eMultivariate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHazard ratio\u003c/p\u003e\u003cp\u003e(95% CI)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHazard ratio\u003c/p\u003e\u003cp\u003e(95% CI)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHazard ratio\u003c/p\u003e\u003cp\u003e(95% CI)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eHazard ratio\u003c/p\u003e\u003cp\u003e(95% CI)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAge\u0026thinsp;\u0026gt;\u0026thinsp;60\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.96 (0.64\u0026ndash;1.45)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.847\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.23 (0.76\u0026ndash;1.98)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.396\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.94 (0.63\u0026ndash;1.42)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.776\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.11 (0.70\u0026ndash;1.75)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.662\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMale sex\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.58 (0.37\u0026ndash;0.91)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.56 (0.34\u0026ndash;0.93)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.61 (0.40\u0026ndash;0.96)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.031\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.54 (0.33\u0026ndash;0.89)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.014\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDisease setting\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eInitially metastatic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eRecurrent or \u003c/p\u003e\u003cp\u003eLocally advanced unresectable\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.98 (0.61\u0026ndash;1.56)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.932\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.95 (0.59\u0026ndash;1.51)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.815\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePrognostic group\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eGood\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eModerate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.03 (0.64\u0026ndash;1.66)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.895\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.92 (0.56\u0026ndash;1.51)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.736\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.52 (0.94\u0026ndash;2.45)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.086\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.37 (0.83\u0026ndash;2.28)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.221\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003ePoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.19 (1.26\u0026ndash;3.79)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.11 (1.14\u0026ndash;3.91)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.48 (1.40\u0026ndash;4.39)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.94 (1.04\u0026ndash;3.65)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.038\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eERBB2\u003c/b\u003e \u003cb\u003efocal amplification\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.55 (0.35\u0026ndash;0.86)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.52 (0.31\u0026ndash;0.86)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.68 (0.44\u0026ndash;1.07)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.094\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.76 (0.46\u0026ndash;1.27)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.297\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNOTCH3\u003c/b\u003e \u003cb\u003ealterations\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.59 (1.40\u0026ndash;4.79)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.93 (1.00\u0026ndash;3.73)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.049\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.30 (1.28\u0026ndash;4.14)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.03 (1.04\u0026ndash;3.96)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.037\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003eCI, confidence interval\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis study evaluated the association between survival outcomes and genomic alterations identified through the targeted panel sequencing in patients with HER2-positive AGC. The findings in our research aligned with recent retrospective studies identifying a high CN status of \u003cem\u003eERBB2\u003c/em\u003e as an independent prognostic factor in GC (Hino et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ichikawa et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Pietrantonio et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), distinct from HER2 IHC status. Although these studies have shown that a high CN status of \u003cem\u003eERBB2\u003c/em\u003e is associated with a better survival prognosis when combined with trastuzumab and chemotherapy, the potential impact of amplified segment length on treatment outcomes has remained largely unexplored.\u003c/p\u003e\u003cp\u003eBased on the varying lengths of the \u003cem\u003eERBB2\u003c/em\u003e-amplified segments, we investigated the differences in survival outcomes according to segment lengths. A recent study demonstrated that ERBB2 focal amplification, defined as an amplified segment less than 3 megabases (Mb) in length, was associated with improved clinical outcomes in breast cancer patients treated with trastuzumab-based therapy (Kim et al., 2023). Although the study did not analyze segment length in detail, its results suggest that the therapeutic efficacy of trastuzumab-based therapy may differ depending on the ERBB2-amplified segment length. \u003cem\u003eERBB2\u003c/em\u003e-amplified segments encode numerous oncogenes and genes associated with drug resistance. Thus, we hypothesized that the tumor with \u003cem\u003eERBB2\u003c/em\u003e non-focal amplification may have a worse prognosis due to attenuated HER2 dependency or acquired drug resistance.\u003c/p\u003e\u003cp\u003eWe found that the patients with \u003cem\u003eERBB2\u003c/em\u003e focal amplification had a superior PFS. Although \u003cem\u003eERBB2\u003c/em\u003e focal amplification was only associated with a trend toward improved OS, the lack of statistical significance may be attributed to the effects of subsequent treatments. Moreover, multivariate analysis confirmed \u003cem\u003eERBB2\u003c/em\u003e focal amplification as an independent prognostic factor for PFS. The \u003cem\u003eCDK12\u003c/em\u003e and \u003cem\u003eRARA\u003c/em\u003e genes, located near the \u003cem\u003eERBB2\u003c/em\u003e gene, are frequently co-amplified with \u003cem\u003eERBB2\u003c/em\u003e and have been associated with a poor prognosis in various cancers (Garattini et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yanai et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). We evaluated the association between co-amplification of \u003cem\u003eERBB2\u003c/em\u003e with either \u003cem\u003eCDK12\u003c/em\u003e or \u003cem\u003eRARA\u003c/em\u003e and survival outcomes. No statistically significant differences were observed, suggesting that additional genes co-amplified with \u003cem\u003eERBB2\u003c/em\u003e, beyond \u003cem\u003eCDK12\u003c/em\u003e and \u003cem\u003eRARA\u003c/em\u003e, should be investigated. These data indicate that the length of the \u003cem\u003eERBB2\u003c/em\u003e-amplified segment may represent a novel biomarker for predicting responses to anti-HER2 therapies, although the underlying molecular mechanisms remain to be elucidated.\u003c/p\u003e\u003cp\u003eThis study identified \u003cem\u003eNOTCH3\u003c/em\u003e alterations as a poor prognostic factor in AGC. Although alterations in several genes, including \u003cem\u003eNOTCH3\u003c/em\u003e, were enriched in the poor- and moderate-risk groups, only \u003cem\u003eNOTCH3\u003c/em\u003e alterations were also statistically significant in multivariate analysis. \u003cem\u003eNOTCH3\u003c/em\u003e has previously been reported to potentially play important roles in cancer progression, including stemness, metastasis, and angiogenesis (Xiu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In terms of clinical significance, the overexpression of \u003cem\u003eNOTCH3\u003c/em\u003e is associated with poor survival outcomes in various cancers (Xiu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Particularly in GC, a prior meta-analysis revealed that \u003cem\u003eNOTCH3\u003c/em\u003e was frequently overexpressed and significantly associated with a poor prognosis, supporting its oncogenic role (Xu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, the association of clinical outcomes with genomic alterations, such as CN loss and point mutation, has not yet been elucidated in GC. \u003cem\u003eNOTCH3\u003c/em\u003e can also act as a tumor suppressor gene in a context-dependent manner (Xiu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As the \u003cem\u003eNOTCH3\u003c/em\u003e alterations identified in our cohort lack further detailed characterization, the interpretation of their clinical significance remains limited.\u003c/p\u003e\u003cp\u003eThe interpretation of our findings has several limitations. First, this study is a single center observational study. However, all patients received trastuzumab-based therapy as first-line treatment and showed a correlation with survival outcomes in the overall study population as well as across risk groups. Second, we focused on mutations and CNVs detected by targeted panel sequencing. For the \u003cem\u003eERBB2\u003c/em\u003e-amplified segment, the limitation of targeted panel sequencing, which leaves gaps in genome coverage, has prevented us from clearly defining the segment criteria due to the use of an optimal cutoff. Although we considered the \u003cem\u003eERBB2\u003c/em\u003e-amplified segment to be the amplified interval, including the entire \u003cem\u003eERBB2\u003c/em\u003e gene, and evaluated the cutoff using the maximally selected rank statistical analysis, different algorithms vary in tools and procedures for CNV calling, including normalization, calculation of copy ratio, and segmentation. Third, this study was not validated in an external cohort. Hence, further studies with a large sample size, utilizing whole-genome sequencing, are necessary to refine the criteria for distinguishing between non-focal and focal amplification.\u003c/p\u003e\u003cp\u003eIn conclusion, our study highlights the significance of \u003cem\u003eERBB2\u003c/em\u003e focal amplification as a potential biomarker for predicting favorable outcomes to trastuzumab-based therapy in HER2-positive AGC.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by NRF (National Research Foundation of Korea) Grant funded by the Korean Government (NRF-2015-Fostering Core Leaders of the Future Basic Science Program/Global Ph.D. Fellowship Program) [Project Number NRF-2015H1A2A1033652].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003eNothing directly related to this work. Out of this work, MHR received honoraria from DAEHWA Pharmaceutical, Bristol Myers Squibb, Lilly, Ono Pharmaceutical, MSD, Taiho Pharmaceutical, Novartis, Daiichi Sankyo, and AstraZeneca, and served as a consultant for DAEHWA Pharmaceutical, Bristol Myers Squibb, Lilly and Ono Pharmaceutical. HDK received research grants from Roche/Genentech, Amgen, and AstraZeneca and honoraria from AstraZeneca, Bristol Myers Squibb, Ono Pharmaceuticals, Boryung Pharmaceuticals, MSD, Daiichi Sankyo, Astellas, Boostimmune, DAEHWA Pharmaceutical, LG Chem,and MustBio.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eAll authors contributed to the study conception and design. Material preparation, data collection, data analysis and interpretation were performed by Sun Young Lee, Jaewon Hyung, Hyung-Don Kim, Hyungeun Lee, Meesun Moon, Young Soo Park and Min-Hee Ryu. The first draft of the manuscript was written by Sun Young Lee and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted\u0026nbsp;the Institutional Review Board (IRB) of Asan Medical Center, Korea (2014-0301).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u0026nbsp;\u003c/strong\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by NRF (National Research Foundation of Korea) Grant funded by the Korean Government (NRF-2015-Fostering Core Leaders of the Future Basic Science Program/Global Ph.D. Fellowship Program) [Project Number NRF-2015H1A2A1033652].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbrahao-Machado, L. F., \u0026amp; Scapulatempo-Neto, C. (2016). 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ERBB2 Copy Number as a Quantitative Biomarker for Real-World Outcomes to Anti-Human Epidermal Growth Factor Receptor 2 Therapy in Advanced Gastroesophageal Adenocarcinoma. \u003cem\u003eJCO Precis Oncol\u003c/em\u003e,\u003cem\u003e 6\u003c/em\u003e, e2100330. https://doi.org/10.1200/PO.21.00330 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HER2 positive, trastuzumab, advanced gastric cancer, ERBB2 copy number amplification, focal ERBB2 amplification, NOTCH3","lastPublishedDoi":"10.21203/rs.3.rs-7267337/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7267337/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eAlthough trastuzumab-based chemotherapy improves survival in HER2-positive advanced gastric cancer, some patients demonstrate suboptimal efficacy and limited response durations. We examined the relationship between clinical outcomes and genomic features, including co-mutations and the length of the \u003cem\u003eERBB2\u003c/em\u003e-amplified segment.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe retrospectively analyzed 151 patients who had received first-line trastuzumab-based chemotherapy. Targeted next-generation sequencing was employed to assess genomic alterations. Progression-free survival (PFS) was defined as time from treatment initiation to disease progression or death.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe median patient age was 62 years, and 73.5% were male. The median follow-up period was 45.8 months, and the median PFS was 8.2 months (95% confidence interval (CI), 6.5\u0026ndash;9.4). Patients with a focal amplification of \u003cem\u003eERBB2\u003c/em\u003e (\u0026le;\u0026thinsp;879 Kb) had significantly longer PFS compared to those with non-focal amplifications (\u0026gt;\u0026thinsp;879 Kb) (10.1 vs. 6.1 months; log-rank \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01). \u003cem\u003eNOTCH3\u003c/em\u003e alterations were associated with shorter PFS (log-rank \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002). Multivariate analysis confirmed that \u003cem\u003eERBB2\u003c/em\u003e focal amplification is an independent prognostic factor associated with improved prognosis, whereas \u003cem\u003eNOTCH3\u003c/em\u003e alterations serve as an independent prognostic factor for poorer outcomes.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003e\u003cem\u003eERBB2\u003c/em\u003e focal amplification is associated with improved outcomes in trastuzumab-treated patients with HER2-positive gastric cancer, whereas \u003cem\u003eNOTCH3\u003c/em\u003e alterations predict a poor prognosis. These genomic features may support risk stratification and therapeutic decisions.\u003c/p\u003e","manuscriptTitle":"Genomic correlations for clinical outcomes in HER2-positive advanced gastric cancers treated using trastuzumab-based therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-08 16:11:44","doi":"10.21203/rs.3.rs-7267337/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3ea8a232-6a27-4e74-b112-3e0e64ad75af","owner":[],"postedDate":"August 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-11T06:23:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-08 16:11:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7267337","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7267337","identity":"rs-7267337","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}