Trastuzumab, pertuzumab, and a taxane (THP) has been the standard first-line therapy for HER2+ advanced breast cancer for over a decade. With new regimens emerging, genomic tools like HER2DX may help identify patients who benefit durably from THP versus those requiring intensification. Here, baseline tumor tissue from 122 patients with HER2+ treated with THP in Poland was tested with HER2DX. A previously published Spanish real-world cohort (n = 93) was added to generate a combined cohort (n = 215). Univariable analyses were performed in the Polish cohort, and multivariable Cox and logistic regression models were applied to the combined cohort. A HER2DX metastatic prognostic score was trained on overall survival (OS) in the Spanish cohort and validated in the Polish cohort. In the Polish cohort, high ERBB2 mRNA scores were associated with significantly longer real-world progression-free survival (rwPFS) (33.8 vs. 17.9 months; hazard ratio [HR] 0.57; p = 0.022) and real-world overall survival (rwOS) (75.1 vs. 40.2; HR 0.48; p = 0.009). In the combined cohort, ERBB2 high-score tumors showed prolonged rwPFS (33.8 vs. 12.5; HR 0.50; p < 0.001) and rwOS (not reached vs. 37.1; HR 0.36; p < 0.001), and higher rwORR (84.4% vs. 52.0%; p < 0.001). Prognostic value was independent of clinical variables, including number of metastatic sites. Subgroup analyses showed particularly favorable outcomes in patients with <3 sites (median rwPFS 51.7 vs. 20.3 months). The HER2DX metastatic prognostic score outperformed ERBB2 alone in the validation cohort. In conclusion, the HER2DX ERBB2 mRNA score provides independent prognostic information in HER2+ advanced breast cancer treated with THP. The HER2DX metastatic prognostic score further improves prognostic accuracy.

PURPOSE:HER2DX is a validated genomic assay used to support treatment decisions in early-stage HER2-positive (HER2+) breast cancer. It provides three scores: relapse risk, likelihood of pathologic complete response (pCR), and ERBB2 mRNA expression. This study aimed to evaluate the association between HER2DX and histopathologic features and assess its relationship with pCR after neoadjuvant therapy. EXPERIMENTAL DESIGN:Patients with newly diagnosed stage I to III HER2+ breast cancer were analyzed based on available HER2DX results during routine care in Spain (January 2022-June 2025). Centralized HER2DX testing was performed on formalin-fixed, paraffin-embedded tumor samples. Histopathologic analysis included tumor grade, hormone receptor status, histologic subtype, Ki67 index, HER2 IHC score, stromal tumor-infiltrating lymphocytes (TIL), tertiary lymphoid structures, and spatial immune distribution. Univariate and multivariable logistic regression analyses were conducted to identify factors associated with pCR after neoadjuvant trastuzumab-based therapy. RESULTS:A total of 410 HER2+ tumors were analyzed, and 250 patients received neoadjuvant trastuzumab-based therapy with available surgical outcomes (36% achieved a pCR). HER2DX pCR scores were significantly associated with all eight histopathologic features, whereas relapse risk and ERBB2 scores were associated with five and two, respectively. TIL correlated with the immune/immunoglobulin signature (r = 0.59), and Ki67 with the proliferation signature (r = 0.50). The HER2DX pCR score remained the only independent predictor of pCR in multivariable analysis (OR, 1.77; 95% confidence interval, 1.08-2.97; P = 0.030). CONCLUSIONS:HER2DX reflects key biological and pathologic features of HER2+ breast cancer and independently predicts pCR, supporting its utility for individualized treatment decision-making.

Supplementary Figure S1. Clinical trial design of the ELIPSE trial.

Background: The HER2DX assay, a 27-gene test, was developed to provide prognostic information and predict treatment responses in patients with early-stage HER2+ breast cancer. This assay evaluates four gene expression signatures: immune/immunoglobulin (IGG), proliferation, luminal, and HER2, offering a comprehensive risk score, a likelihood estimates for pathological complete response (pCR) and ERBB2 expression levels. In this study, we evaluated the HER2DX assay in HER2+ DCIS, aiming to understand its biology and relationship with HER2+ invasive breast cancer. Methods: Standardized HER2DX genomic test was evaluated centrally on 28 formalin-fixed paraffin-embedded tumor samples of HER2+ DCIS across three hospitals in Spain. DCIS features such as nuclear grade, comedonecrosis, architectural pattern, tumor size and hormone receptor (HR) status were evaluated. Percentage (%) of stromal tumor infiltrating lymphocytes (TILs), their spatial distribution (i.e.: inflamed, desert or excluded), and presence of tertiary lymphoid structures (TLS, defined as spatially organized, non-encapsulated areas of immune cell aggregates with or without germinal center [GC]) were assessed on hematoxylin eosin slides. Descriptive statistics were used. Results: The HER2DX assay was evaluated in 28 cases of HER2+ DCIS. Most cases had a 3 nuclear grade (67.9%) and presented with comedonecrosis (60.7%). HR positivity (HR+) was found in 42.9% of cases, as determined by immunohistochemistry. The median tumor size was 30 mm, with a range from 5 mm to 90 mm. The median % of TILs was 20% (range 0.5-70%). TILs, as a continuous variable, showed a moderate correlation with the HER2DX IGG signature (Pearson correlation coefficient=0.43, p-value=0.023), and a tendency with the inflamed spatial distribution (p-value=0.063). TLS were identified in 82.1% of DCIS samples, and their presence was associated with higher expression of the IGG signature (52.2% TLS in IGG-high vs. 21.7% in IGG-low). Regarding the HER2DX luminal signature, 41.7% of cases were classified as luminal-high, associated with HR+ disease (p=0.025). For the HER2DX proliferation signature, 82.1% of cases were identified as proliferation-low. In HER2DX risk stratification, 100% of cases were categorized as low-risk, 46.4% were pCR-high, and 78.6% were ERBB2-high. Notably, only one (3.6%) HER2+ DCIS case was found to be ERBB2-low. Conclusions: The HER2DX assay revealed that all HER2+ DCIS cases were categorized as low-risk, with most cases showing high ERBB2 expression and a high predicted response to anti-HER2-based therapy. These findings underscore the underlying biology of HER2+ DCIS and its tumor immune microenvironment, indicating that HER2+ DCIS shares similar tumor biology with low-risk HER2+ breast cancer. Citation Format: Esther Sanfeliu, Anabel Martinez-Romero, Mercedes Marín-Aguilera, Vicente Marco, Felip Garcia, Vicente Peg, Blanca González-Farré, Ivonne Vazquez, Patricia Galván, Oleguer Castillo, Paula Blasco, Valeria Sirenko, Angela Aguirre, Laia Paré, Guillermo Villacampa, Antonio Martínez, Jesus Soberino, Aleix Prat, Fara Brasó-Maristany. HER2DX assay in HER2-positive (HER2+) breast ductal carcinoma in situ (DCIS) [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-07-18.

In advanced HER2-positive breast cancer, the standard taxane-trastuzumab-pertuzumab (THP) regimen faces competition from new therapies, emphasizing the need for biomarkers to guide treatment. This study evaluates the HER2DX ERBB2 mRNA score as a prognostic predictor, aiming to tailor treatment strategies. We retrospectively analyzed 94 patients treated with the THP regimen between 2010 and 2024. The HER2DX ERBB2 mRNA score was categorized as low (n = 14), medium (n = 20), or high (n = 60), and its correlation with progression-free survival (PFS) and overall survival (OS) was assessed using Cox regression models. The median follow-up was 31.5 months. Patients with ERBB2-high scores had significantly better median PFS (33.9 vs. 10.6 months, hazard ratio [HR] = 0.40, 95% CI: 0.24–0.69, p < 0.001) and OS (not reached vs. 30.8 months, HR = 0.26, 95% CI: 0.13–0.49, p < 0.001) compared to ERBB2-low patients. Based on these findings, further validation of this biomarker in tumor samples from the CLEOPATRA phase III trial is ongoing, which could help optimize treatment strategies in this population.

Supplementary Figure S3. PAM 50 subtype characterisation: A) PAM 50 subtype distribution at baseline and B) PAM50 subtype switching from baseline to post-treatment (day 28) tumor samples.

Introduction: In first-line HER2-positive (HER2+) metastatic breast cancer (MBC), the standard regimen of taxane-trastuzumab-pertuzumab (THP) faces increasing competition from new therapies. This shift highlights the need for precise biomarkers to guide treatment decisions. Two previous real-world evidence studies in HER2+ MBC have shown that, compared to HER2DX ERBB2-low/med groups, the HER2DX ERBB2-high group is associated with better progression-free survival (PFS) and overall survival (OS) following THP in the first-line setting (Bayona et al. ESMO Breast 2024) and following T-DM1 monotherapy in the second-line setting or beyond (Brasó-Maristany et al. JNCI 2022). Here, for the first time, we evaluated the ability of the HER2DX ERBB2 mRNA score to predict long-term outcomes in the pivotal trial that established THP in the first-line setting. Methods: We analyzed available formalin-fixed paraffin-embedded (FFPE) tumor RNA from participants in the CLEOPATRA phase III trial (Baselga et al., NEJM 2012; Swain et al., Lancet 2020; NCT00567190) who were randomized to THP (docetaxel, trastuzumab, and pertuzumab). Standardized HER2DX assay was conducted at Reveal Genomics’ central lab (Barcelona, Spain) blinded from clinical data. The HER2DX ERBB2 score, evaluated both as a continuous variable (1 to 99) and as pre-defined groups (low, medium, and high), was correlated with overall response rate (ORR), PFS, and OS in an exploratory analysis using logistic regression and Cox proportional hazards models. The significance level for all statistical analyses was set at a two-sided alpha of 0.05. Results: In this CLEOPATRA subpopulation, representing 53% of the original cohort (214 out of 402 patients randomized to THP), the median PFS was 18.7 months (95% confidence interval [CI]: 16.2-22.8) and the median OS was 59.5 months (95% CI: 49.3-83.5). These survival outcomes are similar to the original trial cohort. The assay success rate was 96.8% (214/221). The HER2DX ERBB2-low, -medium, and -high groups distribution was 13.5% (n=29), 8.9% (n=19), and 77.6% (n=166), respectively. The HER2DX ERBB2 score, analyzed as a continuous variable, demonstrated an association with PFS (hazard ratio [HR] per 10-unit increment=0.90; 95% CI: 0.83-0.97) and OS (HR per 10-unit increment=0.91; 95% CI: 0.83-1.00), after adjusting for IHC status (IHC 0+, 1+, 2+ versus 3+). Patients with ERBB2-high tumors showed superior median PFS (20.3 months, 95% CI: 18.2-26.4) compared to those with ERBB2-low (10.4 months, 95% CI: 8.4-12.7) and ERBB2-medium (18.7 months, 95% CI: 16.6-36.9) disease. Similarly, a better median OS was observed in patients with ERBB2-high tumors (72.7 months, 95% CI: 51.8-102.1) compared to those with ERBB2-low (40.3 months, 95% CI: 28.8-59.5) and ERBB2-medium (56.5 months, 95% CI: 37.1-59.5) disease. The ERBB2 score, as a continuous variable, was not associated with the ORR (odds ratio per 10-unit increment=1.11, 95% CI: 0.95-1.31). Conclusion: The HER2DX ERBB2 mRNA score is strongly associated with long-term survival outcomes in metastatic HER2+ breast cancer treated with first-line THP. Notably, HER2DX identified patients with ERBB2-low disease, as the subgroup with the poorest outcomes in both PFS and OS. Further analyses will be presented at the conference. Citation Format: Javier Cortés, Fara Brasó-Maristany, Guillermo Villacampa, Eva Ciruelos, Mercedes Marín-Aguilera, Patricia Galván, Sanne de Haas, Eleonora Restuccia, Mahesh Shivhare, Laia Paré, Wesley Buckingham, Joel S. Parker, Ana Vivancos, Charles M. Perou, Patricia Villagrasa, Julia Maués, Aleix Prat, and Sandra Swain. HER2DX ERBB2 mRNA Score in First-Line Metastatic HER2-Positive Breast Cancer Treated with Docetaxel, Trastuzumab and Pertuzumab: Correlative Analysis from CLEOPATRA Phase III Trial [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr PS4-06.

Abstract Purpose: Elacestrant has shown significantly prolonged progression-free survival compared with standard-of-care endocrine therapy in estrogen receptor–positive (ER-positive), HER2-negative metastatic breast cancer, whereas potential benefit in early-stage disease requires further exploration. The SOLTI-ELIPSE window-of-opportunity trial investigated the biological changes induced by a short course of preoperative elacestrant in postmenopausal women with early breast cancer. Patients and Methods: Eligible patients with untreated T1c (≥1.5 cm)-T3, N0, ER-positive/HER2-negative breast cancer with locally assessed Ki67 ≥10% received elacestrant at a daily dose of 345 mg for 4 weeks. The primary efficacy endpoint was complete cell cycle arrest, defined as Ki67 ≤2.7%, on day 28. Results: Overall, 22 patients were evaluable for the primary endpoint. Elacestrant was associated with a complete cell cycle arrest rate of 27.3% and a statistically significant Ki67 geometric mean change of −52.9% (P = 0.007; 95% confidence interval, −67.4 to −32.1). Notably, the treatment with elacestrant led to a shift toward a more endocrine-sensitive and less proliferative tumor phenotype based on PAM50-based gene signatures. Elacestrant increased the expression of immune-response genes (GZMB, CD4, and CD8A) and suppressed proliferation and estrogen-signaling genes (MKI67, ESR1, and AR). These biological changes were independent of the levels of Ki67 suppression on day 28. The most common adverse events were grade 1 anemia (21.7%), hot flushes (8.7%), constipation (8.7%), and abdominal pain (8.7%). One patient experienced a grade 3 cutaneous rash, leading to treatment discontinuation. No other serious adverse events were reported. Conclusions: Preoperative treatment with elacestrant in early breast cancer demonstrated relevant biological and molecular responses and exhibited a manageable safety profile. These findings support further investigation of elacestrant in the early setting.

Supplementary Table S2. Complete cell cycle arrest in the overall study population and in subgroups defined according to baseline Ki67 expression.

Complete lists of genes evaluated using the nCounter platform (plus five housekeeping genes: ACTB, MRPL19, PSMC4, RPLP0, SF3A1).

Methylation and gene expression changes in sensitive and resistant TNBC PDX tumors.

Clinical significance of methylation signatures of resistance

HER2-enriched molecular subtype: patients' characteristics according to PIK3CA gene status.

Baseline characteristics of the patients included (PIK3CA cohort) or not (no-PIK3CA cohort) in the PIK3CA analysis.

Pattern of PIK3CA mutations in the ShortHER trial (n=803 patients).

Additional prognostic value (DFS) of TILs, PIK3CA mutation, PDCD1 expression and MYBL2 expression to integrated prognostic models.

Clinical significance of methylation signatures of resistance

Supplementary Table S1. Models, point mutations and CNA Supplementary Table S2. Differentially expressed genes, pathways and co-occurrence