Impact of pathologic response and individual prognosis after neoadjuvant treatment in patients with early HER2+ and triple-negative breast cancer.

1
Introduction
Breast cancer (BC) is the leading cause of cancer-related death in women around the world and is currently classified into at least three biological subtypes, each with prognostic and predictive relevance for clinical practice [1,2]. Human epidermal growth factor receptor 2-positive (HER2+) and triple-negative breast cancer (TNBC) account for approximately 15-20% and 10-15% of all breast tumors, respectively [3]. In these subtypes, neoadjuvant treatments (NATs) are increasingly utilized in curative settings as part of multimodal strategies [3].
From a surgical perspective, NAT can lead to less invasive procedures, improving the chances of breast conservation and lowering the likelihood of axillary lymph node dissection [4]. Additionally, this approach allows for the assessment of pathological complete response (pCR), which has prognostic value for individual patients, and supports treatment intensification for those with residual disease (RD) [5,6]. Specifically, in HER2+ BC treated with NAT, patients experiencing pCR typically complete one year of anti-HER2 therapy, with or without adjuvant endocrine treatment if estrogen receptor-positive (ER+). For those with RD, treatment is escalated by administering the antibody-drug conjugate (ADC) trastuzumab emtansine (T-DM1) for up to 14 cycles [7]. The KEYNOTE-522 regimen is the standard of care for stage II-III TNBC, combining neoadjuvant chemotherapy with pembrolizumab, which continues for nine cycles post-surgery [[8], [9], [10], [11]]. For patients with RD, adjuvant treatment may be escalated with 6-8 cycles of capecitabine [6]. Those with high-risk HER2-negative tumors and germline breast cancer susceptibility gene (BRCA)1/2 mutations may receive one year of olaparib [12,13].
While overall stage at presentation, including tumor size (cT) and nodal status (cN), is known to impact OS – with more advanced disease linked to poorer outcomes – pCR after NAT is believed to improve prognosis, regardless of initial stage. However, recent studies suggest that stage at diagnosis may still influence outcomes, even in patients who experience pCR [14,15]. Furthermore, KEYNOTE-522 data on event-free survival (EFS) by stage and pathological response indicated that patients who experienced pCR with immunotherapy had numerical better outcomes compared to those treated with neoadjuvant chemotherapy alone, across both stage II and III disease [11]. These findings suggest that the prognostic benefit of pCR may vary depending on stage at presentation and the treatment used to achieve it. Therefore, this study aims to assess how tumor size and nodal status at diagnosis influence recurrence-free survival (RFS) and OS in patients with HER2+ and TNBC treated with different NAT regimens.

2
Methods
2.1
Study design and patient cohort
This single-center retrospective study included patients diagnosed at Dana-Farber Cancer Institute with TNBC or HER2+ early-stage BC who underwent surgery following NAT. Patients with available data on pCR, RD, RFS, and OS were identified through the Clinical Outcomes Quality Database (COQD) from its inception in 2016. To ensure adequate follow-up and minimise selection bias, only patients who had surgery before June 2021 were included. For patients with multifocal or multicentric tumors, the most aggressive biology was considered. Bilateral synchronous BCs were excluded. A CONSORT flow diagram illustrating the steps taken to build the patient cohort for the single-center retrospective study is shown in Supplementary Fig. 1. All procedures followed the World Medical Association's Code of Ethics (Declaration of Helsinki) for human research and were approved by the institutional review board (IRB #24-026).
Consistent with American Society of Clinical Oncology-American Pathologists guidelines, ER and progesterone receptor (PR) statuses were evaluated by immunohistochemistry (IHC); ≥1% of stained tumor cells for ER and/or PR were considered hormone receptor-positive. HER2 status was evaluated by IHC and/or by in-situ hybridization (ISH) [16]: HER2- was defined as IHC 0 or 1+, or 2+ with fluorescence in situ hybridization (FISH)-negative or not amplified; HER2+ was defined as IHC 3+ or 2+ with FISH-positive or amplified. Clinical stage was defined by using the 7th edition of the American Joint Committee on Cancer BC staging system. Patients were classified according to their response to NATs, using pathological T and N categories from surgery, and were grouped as having either pCR (defined as ypT0/is, ypN0) or RD.

2.2
Statistical analysis and reporting
Clinicopathologic characteristics were compared using the Kruskal-Wallis test for numeric variables and Chi-square test for categorical variables. Kaplan-Meier (KM) methods were used to estimate time-to-event outcomes and to generate survival curves. Fixed-time survival probabilities (e.g., 3-year survival rates with 95% confidence intervals [CIs]) were reported when the median was not reached. Univariate associations of clinical tumor size (cT), nodal status (cN), and subtype with RFS and OS were evaluated in the overall cohort and in patients with pCR (ypT0/is, ypN0) or RD using Cox proportional hazard models, with results reported as hazard ratios (HRs). Multivariable analysis incorporating cT, cN, and subtype was conducted similarly, with adjusted HRs reported.
For analysis, cT1 and cT2 tumors were grouped together, as were cT3 and cT4. Nodal status was categorized as cN0 (negative) or cN+ (cN1, cN2, cN3). Tumor subtype was classified as hormone receptor-negative (HR-)/HER2+, hormone receptor-positive (HR+)/HER2+, or TNBC. RFS was defined as the time from surgery to the first recurrence (local, regional, or distant) or death from any cause, while OS was defined as the time from surgery to death from any cause.
Results were reported with 95% CI, and a p-value of < 0.05 was considered statistically significant. All analyses were performed using SAS9.4 and R software (Version 4.3.1, www.r-project.org). Study reporting followed the ESMO-Guidance for Reporting Oncology real-World Evidence, with a checklist available in Supplementary File 2.

3
Results
3.1
Patient demographics
A total of 863 patients underwent surgery at our institution between January 2016 and June 2021. The median age at surgery was 50.2 years (range: 21.0-85.4), with a median follow-up of 3.5 years. The demographic characteristics of the cohort are summarized in Table 1. The majority of patients were Caucasian (714, 82.7%), followed by African American (59, 6.8%), Asian, or Pacific Islander (44, 5.1%). Regarding disease stage, 80 patients (9.3%) were diagnosed at stage I, 602 (69.8%) at stage II, and 181 (21.0%) at stage III. 374 patients (43.3%) experienced pCR, while 489 patients (56.7%) had RD.

3.2
Clinicopathologic characteristics by tumor subtype
Among the 863 patients included, ductal histology was predominant (772, 89.5%) across all subtypes, with no significant differences observed (p = .125). Grade 3 tumors were most frequent (p < .001), particularly in TNBC (355, 91.7%). The majority of patients (655, 75.9%) had cT1-2 tumors, and 52.6% were cN0 (n = 454), with no significant differences in tumor size (p = .246) or nodal status (p = .063) between groups. pCR rates varied significantly (p < .001), with the highest frequency in HR-/HER2+ patients (130, 71.8%) and lowest in TNBC (125, 32.3%). NAT patterns differed by subtype; nearly all HER2+ patients received HER2-targeted therapy (179, 98.9% HR-/HER2+; 292, 99.0% HR+/HER2+), while 94.8% of TNBC patients were treated with chemotherapy alone (p < .001). Recurrence rates were highest in TNBC (74, 19.1%) compared to HER2+ subtypes (p < .001), with the brain (39, 4.5%), bone (37, 4.3%), and lung (33, 3.8%) being the most common sites of recurrence. Table 2 summarizes the detailed clinicopathologic characteristics of the patient population by subtype.

3.3
Recurrence-free survival
Median RFS was not reached, so median survival was not reported as the KM curves did not fall below 50%. Three-year RFS was reported, based on a median follow-up of 3.5 years. Three-year RFS for the entire cohort was 87% (95% CI, 85-89), with 80% (95% CI, 76-84) in TNBC and 93% (95% CI, 91-96) in HER2+ BC. Patients with pCR had a 3-year RFS of 98% (95% CI, 97-100) compared to 79% (95% CI, 75-82) in those with RD. In TNBC, 3-year RFS was 97% (95% CI, 95-100) for patients with pCR vs. 72% (95% CI, 66-77) for those with RD. In HER2+ BC, 3-year RFS was 99% (95% CI, 97-100) for patients with pCR compared to 87% (95% CI, 82-92) for those with RD. Specifically, in HR+/HER2+ BC, 3-year RFS was 99% (95% CI, 97-100) for patients with pCR compared to 88% (95% CI, 83-93) for those with RD; in HR-/HER2+ BC, 3-year RFS was 98% (95% CI, 96-100) for patients with pCR compared to 83% (95% CI, 72-96) for those with RD.
Higher cT (cT3-4 vs cT1-2, HR: 2.72; 95% CI, 1.88-3.94, p < .001), positive cN (cN+ vs cN0, HR: 2.43; 95% CI, 1.65-3.59, p < .001), TNBC subtype (TNBC vs. HR+/HER2+, HR: 2.57; 95% CI, 1.63-4.04, p < .001; HR-/HER2+ vs HR+/HER2+, HR: 0.94; 95% CI, 0.49-1.80, p = .8), and RD (pCR vs. RD, HR: 0.11; 95% CI, 0.06-0.22, p < .001) were all associated with poorer 3-year RFS.
In patients with pCR, 3-year RFS was numerically higher in cT1-2 (99%) compared to cT3-4 (95%) and in cN0 (99%) compared to cN+ (97%), though the differences were not significant (cT3-4 vs. cT1-2, HR: 2.97; 95% CI, 0.84-10.52, p = .09; cN+ vs cN0, HR: 2.85; 95% CI, 0.74-11.01, p = .1). Subtype stratification showed no associations (Fig. 1).
In patients with RD, higher cT (cT3-4 vs. cT1-2, HR: 2.26; 95% CI, 1.54-3.33, p < .001), positive cN (cN+ vs. cN0, HR: 2.31; 95% CI, 1.54-3.47, p < .001), and TNBC subtype (TNBC vs. HR+/HER2+, HR: 2.42; 95% CI, 1.51-3.86, p < .001) were associated with poorer 3-year RFS, while HR-/HER2+ showed no significant difference (HR: 1.66; 95% CI, 0.79-3.49, p = .2) (Fig. 1).
These results were consistent when using hormone receptor cutoff of <10% to define ER and PR negativity (Supplementary Fig. 2).
In the multivariate model for RFS, no associations were identified in patients with pCR. In those with RD, higher cT (cT3-4 vs. cT1-2, HR: 1.86; 95% CI, 1.24-2.80, p = .003), positive cN (cN+ vs. cN0, HR: 2.13; 95% CI, 1.39-3.28, p = .001), and TNBC subtype (TNBC vs. HR+/HER2+, HR: 2.60; 95% CI, 1.63-4.17, p < .001) remained associated with poorer 3-year RFS (Supplementary Fig. 3).

3.4
Overall survival
Median OS was not reached, so median survival was not reported as the KM curves did not fall below 50%. Three-year OS was reported, based on a median follow-up of 3.5 years. Three-year OS for the entire cohort was 93% (95% CI, 91-95), with 87% (95% CI, 83-98) in TNBC and 98% (95% CI, 97-99) in HER2+ BC. Patients with pCR had a 3-year OS of 100% (95% CI, 99-100), compared to 88% (95% CI, 85-91) in those with RD. In TNBC, 3-year OS was 99% (95% CI, 98-100) for patients with pCR vs. 81% (95 CI, 76-86) for those with RD. In HER2+ BC, 3-year OS was 100% (95% CI, 100-100) for patients with pCR compared to 96% (95% CI, 93-99) for those with RD. Specifically, in HR+/HER2+ BC, 3-year OS was 100% (95% CI, 100-100) for patients with pCR compared to 95% (95% CI, 92-99) for those with RD; in HR-/HER2+ BC, 3-year RFS was 100% (95% CI, 100-100) for patients with pCR compared to 98% (95% CI, 94-100) for those with RD.
Higher cT (cT3-4 vs cT1-2, HR: 2.94; 95% CI, 1.81-4.77, p < .001), positive cN (cN+ vs cN0, HR: 3.08; 95% CI, 1.79-5.30, p < .001), TNBC subtype (TNBC vs. HR+/HER2+, HR: 3.21; 95% CI, 1.75-5.92, p < .001; HR-/HER2+ vs HR+/HER2+, HR: 0.38; 95% CI, 0.11-1.34, p = .1), and RD (pCR vs. RD, HR: 0.08; 95% CI, 0.03-0.22, p < .001) were all associated with poorer 3-year OS.
In patients with pCR, 3-year OS was equally high in cT1-2 (100%) and cT3-4 (100%), as well as in cN0 (100%) and to cN+ (99%) (cT3-4 vs. cT1-2, HR: 1.29; 95% CI, 0.13-12.43, p = .8; cN+ vs cN0, HR: 1.14; 95% CI, 0.16-8.07, p = .9). Subtype stratification revealed no significant associations (Fig. 2).
In patients with RD, higher cT (cT3-4 vs. cT1-2, HR: 2.57; 95% CI, 1.56-4.23, p < .001), positive cN (cN+ vs. cN0, HR: 3.18; 95% CI, 1.80-5.63, p < .001), and TNBC subtype (TNBC vs. HR+/HER2+, HR: 3.00; 95% CI, 1.59-5.64, p < .001) were associated with poorer 3-year OS, while HR-/HER2+ showed no significant difference (HR: 0.59; 95% CI, 0.13-2.63, p = .5) (Fig. 2).
These results were consistent when using hormone receptor cutoff of <10% to define ER and PR negativity (Supplementary Fig. 4).
In the multivariate model for OS, no associations were identified in patients with pCR. In those with RD, higher cT (cT3-4 vs. cT1-2, HR: 2.00; 95% CI, 1.18-3.37, p = .01), positive cN (cN+ vs. cN0, HR: 2.99; 95% CI, 1.64-5.43, p < .001), and TNBC subtype (TNBC vs. HR+/HER2+, HR: 3.28; 95% CI, 1.74-6.18, p < .001) remained associated with poorer 3-year OS (Supplementary Fig. 5).

4
Discussion
The findings of this study align with existing literature, confirming that experiencing pCR predicts a favorable prognosis, particularly in aggressive subtypes such as TNBC and HER2+ BCs. Our data show that while tumor size, nodal status, and subtype influenced RFS and OS in patients with RD after NAT, these factors were not prognostic in patients who experienced pCR.
At first glance, our findings may appear to differ from those of two previous studies. One study, involving 3710 patients with HER2+ BC across 11 clinical trials, found that cT and cN were significant prognostic factors for EFS, and cT was a predictor of OS after a median follow-up of 61.2 months. In patients with RD, cT, cN, and hormone receptor status were independent predictors of both EFS and OS [14]. The second study analyzed 35,598 patients with stage I-III TNBC from the National Cancer Database, of whom 11,967 experienced pCR [15]. In this subgroup, 10-year OS was 88.5%, with the best outcomes in cT1-2, cN0 patients (90.0%) and the worst in cT3-4, cN2-3 patients (72.0%).
Our data, based on a smaller cohort with a median follow-up of 3.5 years, showed for patients experiencing a pCR a numerical trend towards worse 3-year RFS in TNBC, cT3-4 or cN+ disease, though differences were not statistically significant. OS differences were not observed, and median survival was not reached within the follow-up period.
These data altogether contribute to understanding the extent to which experiencing pCR influences individual patient prognosis. While pCR was once believed to equalize prognosis regardless of stage at presentation, the evidence discussed above suggests this may not be the case [14,15]. However, if significant effects are observed only in larger cohorts, it may indicate that while poor prognostic factors such as larger tumor size and nodal involvement remain relevant after pCR, experiencing a pCR substantially mitigates the adverse impact of higher disease stage at diagnosis. These findings confirm the importance of experiencing a pCR, supporting ongoing efforts to refine NAT regimens to maximize pCR rates in HER2+ BC and TNBC.
In patients with RD, our study found that higher cT, cN, and TNBC subtype at diagnosis was associated with poorer outcomes, consistent with existing literature. This underscores the need for tailored treatment escalation in this group and for continued research to develop novel therapies that can improve prognosis for patients with RD.
Our study has some limitations. First, as a retrospective study, it carries inherent biases typical of this study design, including potential selection bias and incomplete data collection. Second, the follow-up period of 3.5 years is relatively short, reflecting the date our institutional database was established, which also limited the number of patients receiving neoadjuvant immunotherapy (Table 2). Additionally, we were unable to evaluate EFS and focused on RFS instead. This is due to the design of our institutional dataset, which does not clearly capture progression of the primary cancer during preoperative therapy.
Furthermore, our study did not capture previously all post-neoadjuvant treatments that patients with residual disease may have received, which evolved over time since the database was established in 2016. HER2+ and TNBC are heterogeneous diseases, and we did not account for the biology of residual disease, which may influence prognosis but could not be assessed in this study. Lastly, we did not systematically document patients who discontinued NAT early due to side effects or poor tolerance. We recognize that this may reduce the likelihood of experiencing pCR, potentially affecting long-term outcomes.

5
Conclusion
This study confirms that patients experiencing pCR have better outcomes than those with RD. Three-year RFS and OS varied based on clinical tumor size, nodal status, and subtype at diagnosis. In patients with RD, cT, cN, and subtype remained independent prognostic factors. In contrast, no significant associations of baseline clinical-pathological characteristics were observed in patients with pCR, likely due to the low number of events.

CRediT authorship contribution statement
Chiara Corti: Writing – original draft, Methodology, Investigation, Conceptualization. Tianyu Li: Writing – review & editing, Visualization, Validation, Methodology, Formal analysis. Alyssa R. Martin: Writing – review & editing, Data curation. Melissa E. Hughes: Writing – review & editing, Project administration, Data curation. Tonia Parker: Writing – review & editing, Project administration, Data curation. Tyzaire S. Duporte: Writing – review & editing, Data curation. Giuseppe Curigliano: Writing – review & editing. Tari A. King: Writing – review & editing. Elizabeth A. Mittendorf: Writing – review & editing. Nancy U. Lin: Writing – review & editing. Nabihah Tayob: Writing – review & editing, Supervision. Sara M. Tolaney: Writing – review & editing, Supervision, Methodology, Investigation, Funding acquisition, Conceptualization.

Data sharing statement
The dataset generated and analyzed during the current study is available from the corresponding author, Sara M. Tolaney (sara_tolaney@dfci.harvard.edu), upon reasonable request. Data will be made available following publication. Access will be granted to researchers whose proposed use of the data has been approved. Investigator support will be provided for all data analyses.

Funding
This research was funded by the Elaine and Eduardo Saverin Foundation, OOFOS, and the Benderson Family Fund.

Declaration of competing interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
CCo reports reimbursement for travel and lodging by Veracyte (SABCS23); her research was supported by IEO-Monzino Foundation (2023-2024) and is currently supported by the Fondazione Gianni Bonadonna (FGB) and Associazione Italiana per la Ricerca contro il Cancro (AIRC) (2024-2027). All competing interests were outside the submitted work. GC reports honoraria for speaker's engagements for Roche, Seattle Genetics, Novartis, Lilly, Pfizer, Foundation Medicine, NanoString, Samsung, Celltrion, BMS, MSD; honoraria for providing consultancy from Roche, Seattle Genetics, NanoString; honoraria for participating in an Advisory Board from Roche, Lilly, Pfizer, Foundation Medicine, Samsung, Celltrion, Mylan; honoraria for writing engagements from Novartis, BMS; honoraria for participation in the Ellipsis Scientific Affairs Group; and institutional research funding for conducting phase I and II clinical trials from Pfizer, Roche, Novartis, Sanofi, Celgene, Servier, Orion, AstraZeneca, Seattle Genetics, AbbVie, Tesaro, BMS, Merck Serono, Merck Sharp & Dohme, Janssen-Cilag, Philogen, Bayer, Medivation, Medimmune. All competing interests were outside the submitted work. TAK reports speaker honoraria for Exact Sciences, compensated service on the FES Steering Committee for GE Healthcare, compensated service for advisory board role for Veracyte, and compensated service as faculty for PrecisCa cancer information service. EAM reports compensated service on scientific advisory boards for AstraZeneca, BioNTech, and Merck; uncompensated service on steering committees for Bristol Myers Squibb and Roche/Genentech; speakers honoraria and travel support from Merck Sharp & Dohme; and institutional research support from Roche/Genentech (via SU2C grant) and Gilead. EAM also reports research funding from Susan Komen for the Cure for which she serves as a Scientific Advisor, and uncompensated participation as a member of the American Society of Clinical Oncology Board of Directors. NUL reports institutional research support from Genentech, Pfizer, Merck, Seattle Genetics, Zion Pharmaceuticals, Olema Pharmaceuticals, AstraZeneca, Iksuda, and Stemline; consulting honoraria from Seattle Genetics, Daiichi-Sankyo, AstraZeneca, Olema Pharmaceuticals, Stemline/Menarini, Artera Inc., Eisai, Shorla Oncology, and Denali Therapeutics; royalties from Up to date (book); and travel support from Olema, AstraZeneca, and Daiichi Sankyo.
All competing interests were outside the submitted work. NT reports receiving honoraria for a Clinical Research Workshop at the San Antonio Breast Cancer Symposium outside the submitted work.
SMT reports consulting or advisory roles for Novartis, Pfizer/SeaGen, Merck, Eli Lilly, AstraZeneca, Genentech/Roche, Eisai, Bristol Myers Squibb/Systimmune, Daiichi Sankyo, Gilead, Blueprint Medicines, Reveal Genomics, Sumitovant Biopharma, Artios Pharma, Menarini/Stemline, Aadi Bio, Bayer, Jazz Pharmaceuticals, Natera, Tango Therapeutics, eFFECTOR, Hengrui USA, Cullinan Oncology, Circle Pharma, Arvinas, BioNTech, Launch Therapeutics, Zuellig Pharma, Johnson&Johnson/Ambrx, Bicycle Therapeutics, BeiGene Therapeutics, Mersana, Summit Therapeutics, Avenzo Therapeutics, Aktis Oncology, Celcuity, Boehringer Ingelheim, Samsung Bioepis, Olema Pharmaceuticals, Tempus, Boundless Bio, Denali Therapeutics, and Relay Therapeutics; research funding for Genentech/Roche, Merck, Exelixis, Pfizer, Lilly, Novartis, Bristol Myers Squibb, AstraZeneca, NanoString Technologies, Gilead, SeaGen, OncoPep, Daiichi Sankyo, Menarini/Stemline, Jazz Pharmaceuticals, and Olema Pharmaceuticals; and travel support from Lilly, Gilead, Jazz Pharmaceuticals, Pfizer, Arvinas, and Roche. The remaining authors declare no competing interests.