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Background Prospective data on the risk of recurrence among women with hormone receptor-positive early breast cancer who temporarily discontinue endocrine therapy to attempt pregnancy are lacking. Methods We conducted a single-group trial in which we evaluated the temporary interruption of adjuvant endocrine therapy to attempt pregnancy in young women with previous breast cancer. Eligible women were 42 years of age or younger; had had stage I, II, or III disease; had received adjuvant endocrine therapy for 18 to 30 months; and desired pregnancy. The primary end point was the number of breast cancer events (defined as local, regional, or distant recurrence of invasive breast cancer or new contralateral invasive breast cancer) during follow-up. The primary analysis was planned to be performed after 1600 patient-years of follow-up. The prespecified safety threshold was the occurrence of 46 breast cancer events during this period. Breast cancer outcomes in this treatment-interruption group were compared with those in an external control cohort consisting of women who would have met the entry criteria for the current trial. Results Among 516 women, the median age was 37 years, the median time from breast cancer diagnosis to enrollment was 29 months, and 93.4% had stage I or II disease. Among 497 women who were followed for pregnancy status, 368 (74.0%) had at least one pregnancy and 317 (63.8%) had at least one live birth. In total, 365 babies were born. At 1638 patient-years of follow-up (median follow-up, 41 months), 44 patients had a breast cancer event, a result that did not exceed the safety threshold. The 3-year incidence of breast cancer events was 8.9% (95% confidence interval [CI], 6.3 to 11.6) in the treatment-interruption group and 9.2% (95% CI, 7.6 to 10.8) in the control cohort. Conclusions Among select women with previous hormone receptor-positive early breast cancer, temporary interruption of endocrine therapy to attempt pregnancy did not confer a greater short-term risk of breast cancer events, including distant recurrence, than that in the external control cohort. Further follow-up is critical to inform longer-term safety. (Funded by ETOP IBCSG Partners Foundation and others; POSITIVE ClinicalTrials.gov number, NCT02308085.).

Background: Pregnancy after breast cancer (BC) is of substantial importance for many young women at diagnosis and during follow-up. BC treatment including standard endocrine therapy (ET) (5-10 years) may reduce ovarian reserve and the chances of subsequent successful pregnancy, given conception is contraindicated during ET. A temporary interruption of ET to attempt and carry a pregnancy in this population has never been prospectively studied. Methods: POSITIVE is a single-arm, prospective, investigator-initiated, international trial evaluating the safety and pregnancy outcomes of interrupting ET for young women with early-stage hormone-receptor-positive (HR+) BC who desire pregnancy. The primary objective is to assess the risk of BC relapse associated with ET interruption for ~2 years to achieve pregnancy. Women ≤42 years with stage I-III HR+ BC who received adjuvant ET (SERM alone, GnRH analogue plus SERM or AI) for 18 to 30 months and wished to interrupt ET to attempt pregnancy were eligible. The primary endpoint is breast cancer free interval (BCFI) defined as the time from enrollment to the first BC event (local, regional, distant recurrence or a new invasive contralateral BC). Planned sample size was 500 patients. Three interim analyses of BCFI were reviewed by the Data Safety Monitoring Committee (DSMC) to assure a 95% chance of stopping the trial early if the annual BCFI event rate exceeded 4%; with primary analysis triggered after 1600 patient years of follow-up (pyfu) and no more than 46 BCFI events defined as the safety threshold. The DSMC recommended continuing the study following each interim analysis. We now report the primary results. Results: From 12/2014 to 12/2019, 518 women were enrolled. At enrollment, the median age of participants was 37 years (27-43 years); 75.0% were nulliparous, 93.4% had stage I/II disease, 66.3% node-negative. Median time from BC diagnosis to enrollment was 29 months (IQR: 25-32). Tamoxifen alone was the most prescribed ET (41.7%), followed by tamoxifen+ovarian function suppression (35.7%). 62.0% of participants had received neo/adjuvant chemotherapy. At a median follow-up of 41 months (1638 pyfu), 44 participants had experienced a BCFI event, not exceeding the pre-specified safety threshold of 46 events. The 3-year BCFI failure percent was 8.9% (95% CI: 6.3 to 11.6%), similar to the 9.2% (95% CI: 7.6 to 10.8%) calculated in the comparative external control cohort from the SOFT/TEXT trials (Sun et al, Breast 2020). Of 497 women followed for pregnancy status, 368 (74.0%) had at least one pregnancy, 317 (63.8%) had at least one live birth, with a total of 365 babies born. Based on competing risk analysis, 76.3% of patients resumed ET (half within 26 months), 8.3% had BCFI event/death before ET resumption, and 15.4% had not resumed ET yet. Conclusions: The POSITIVE trial demonstrates that for young women with early HR+ BC desiring pregnancy, temporary interruption of ET to attempt pregnancy does not confer a greater short-term risk of recurrence than that observed in a modern historical control group that did not interrupt ET. Most participants have had a live birth. Further follow-up is planned to confirm long-term safety. These results should be considered in counselling BC patients desiring future pregnancy. Citation Format: Ann Partridge, Olivia Pagani, Samuel M. Niman, Monica Ruggeri, Fedro Alessandro A. Peccatori, Hatem A. Azim, Marco Colleoni, Cristina Saura, Chikako Shimizu, Anna Saetersdal, Judith Kroep, Audrey Mailliez, Ellen Warner, Virginia F. Borges, Frédéric Amant, Andrea Gombos, Akemi Kataoka, Christine Rousset-Jablonski, Simona Borstnar, Junko Takei, Jeong Eon Lee, Janice Walshe, Manuel Ruiz Borrego, Halle Moore, Christobel Saunders, Vesna Bjelic-Radisic, Snezana Susnjar, Fatima Cardoso, Karen L. Smith, Teresa Ferreiro Vilarino, Karin Ribi, Kathryn Ruddy, Sarra El-Abed, Martine Piccart, Larissa A. Korde, Aron Goldhirsch, Richard D. Gelber. Pregnancy Outcome and Safety of Interrupting Therapy for women with endocrine responsIVE breast cancer: Primary Results from the POSITIVE Trial (IBCSG 48-14/BIG 8-13) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS4-09.

Background: Tumor and microenvironment features, including luminal phenotype as well as metabolic, immune and stroma activation, impact prognosis and treatment response in HER2-positive breast cancer. Here, we aimed to identify subgroups depicting biological processes associated with prognosis in patients receiving adjuvant trastuzumab in the phase III ALTTO trial, and to validate their biological and prognostic characteristics in the phase III neoadjuvant NeoALTTO trial. Methods: By applying a case-control approach (1:2), we selected from the ALTTO trastuzumab arm 134 and 268 patients with and without a distant relapse respectively, matched for clinicopathological characteristics. In ALTTO, RNA was obtained from FFPE tumor cores from surgical samples. RNA sequencing (RNAseq) data available from 254 frozen pre-treatment samples were used for validation in patients receiving trastuzumab and/or lapatinib in NeoALTTO. The Absolute Intrinsic Molecular Subtyping (AIMS) was used to compute PAM50 subtypes. Prognostic genes were identified using a multivariable Cox proportional hazard model (controlling for clinicopathological characteristics and PAM50 HER2-enriched [HER2-E] vs others) for distant relapse-free survival (DRFS). Clusters were identified using non-negative matrix factorization (NMF) and k-means clustering, and characterized with gene expression profiling and Gene Set Variation Analysis (GSVA, hallmark gene sets from MSigDB). Aiming at identifying the subgroups in external cohorts, we identified relevant genes to develop group-specific signatures with LASSO regression, and used the derived scores to build a multinomial classifier. Results: The case-control cohort includes a high-risk population, with higher proportions of >2cm, node positive and G3 tumors compared to the whole ALTTO trastuzumab arm. RNAseq data were generated for 386/402 patients. NMF and k-means clustering performed on genes associated with DRFS (false discovery rate < 0.05) identified 4 groups with distinct biological characteristics and prognosis: immune-enriched (IM, N = 69), proliferative/metabolic (P/M, N = 87, characterized by glycolysis, cholesterol homeostasis and proliferation pathways), mesenchymal/stroma-enriched (M/S, N = 76, characterized by epithelial-mesenchymal transition, angiogenesis and TGF-beta signaling), and hormone receptor positive-enriched (N = 154), further divided into PAM50 HER2-E (N = 91) and non-HER2-E (N = 63, LUM, mainly luminal A/B tumors). The IM and LUM groups presented better DRFS (91% and 87% 5-year DRFS, respectively) compared to the others (5-year DRFS of 72%, 58% and 51% for HER2-E, M/S and P/M, respectively). The gene expression-based classifier identified the same 5 groups in NeoALTTO. GSVA analysis and comparisons among the 5 groups showed similar results in the two studies. Of interest, we observed significant differences in event-free survival in NeoALTTO (P = 0.035 and P = 0.041 in the whole population and in the subgroup with residual disease, respectively), with IM and LUM presenting better outcomes compared to the other groups. Sensitivity to neoadjuvant treatment as described by pathological complete response (pCR, ypT0/is ypN0) differed across the groups, with LUM presenting lower pCR rates (8%) compared to HER2-E (40%), IM (35%), P/M (32%), and M/S (32%). Conclusions: Five biologically-driven HER2-positive breast cancer subgroups were identified in ALTTO, highlighting the heterogeneity of this disease. Of note, their biological features and clinical behavior were validated in the NeoALTTO population, suggesting the robustness of our findings. IM and LUM tumors could be considered for treatment de-escalation approaches. Additional validation in cohorts receiving standard (neo)adjuvant therapies is warranted. Citation Format: Mattia Rediti, David Venet, Andrea Joaquin Garcia, Dominique Agbor-Tarh, Marion Maetens, Delphine Vincent, Samira Majjaj, Sarra El-Abed, Takayuki Ueno, Serena Di Cosimo, Martine Piccart, Lajos Pusztai, Sherene Loi, Roberto Salgado, Giuseppe Viale, Françoise Rothé, Christos Sotiriou. Characterization and validation of biologically-driven HER2-positive breast cancer subgroups in the ALTTO and NeoALTTO clinical trials [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-09-05.

Background Despite huge efforts to identify biomarkers associated with long-term clinical outcomes in patients with early-stage HER2-positive breast cancer (HER2+ BC) treated with (neo)adjuvant anti-HER2 therapy, no reliable predictors have been identified so far. Fatty acid uptake, a process mediated by the transmembrane transporter CD36, has recently emerged as a potential determinant of resistance to anti-HER2 treatments in preclinical HER2+ BC models. Methods Here, we investigated the association between baseline intratumor CD36 gene expression and event-free survival in 180 patients enrolled in the phase III trial Neoadjuvant Lapatinib and/or Trastuzumab Treatment Optimization (NeoALTTO), which randomly assigned stage II-III HER2+ BC patients to receive neoadjuvant lapatinib, trastuzumab, or lapatinib-trastuzumab in combination with chemotherapy. To this aim, we selected NeoALTTO trial patients for whom pretreatment whole transcriptomic data were available. The main study results were validated in an independent cohort of patients enrolled in the neoadjuvant phase II trial NeoSphere. Results In 180 NeoALTTO patients, high intratumor CD36 expression was independently associated with worse event-free survival in patients treated with trastuzumab-based therapy (hazard ratio [HR] = 1.72, 95% confidence interval [CI] = 1.20 to 2.46), but not with lapatinib-based (HR = 1.02, 95% CI = 0.68 to 1.53) or trastuzumab-lapatinib–based (HR = 1.08, 95% CI = 0.60 to 1.94) therapy. Among 331 NeoSphere patients evaluated, high CD36 expression was independently associated with worse patient disease-free survival in both the whole study cohort (HR = 1.197, 95% CI = 1.002 to 1.428) and patients receiving trastuzumab-based neoadjuvant therapy (HR = 1.282, 95% CI = 1.049 to 1.568). Conclusions High CD36 expression predicts worse clinical outcomes in early-stage HER2+ BC treated with trastuzumab-based neoadjuvant therapy.

Purpose:The companion diagnostic test for trastuzumab has not changed much in the last 25 years. We used high-plex digital spatial profiling to identify biomarkers besides HER2 that can help predict response to trastuzumab in HER2-positive breast cancer.Experimental Design:Fifty-eight protein targets were measured in three different molecularly defined compartments by the NanoString GeoMx Digital Spatial Profiler (DSP) in a tissue microarray containing 151 patients with breast cancer that received adjuvant trastuzumab as part of the Hellenic Cooperative Oncology Group 10/05 clinical trial. Promising candidate biomarkers were orthogonally validated with quantitative immunofluorescence (QIF). RNA-sequencing data from the Neoadjuvant Lapatinib and/or Trastuzumab Treatment Optimisation Study (NeoALTTO) were accessed to provide independent cohort validation. Disease-free survival (DFS) was the main outcome assessed. Statistical analyses were performed using a two-sided test (α = 0.05) and multiple testing correction (Benjamini–Hochberg method, FDR < 0.1).Results:By DSP, high expression of alpha-smooth muscle actin (α-SMA), both in the leukocyte and stromal compartments, was associated with shorter DFS in univariate analysis (P = 0.002 and P = 0.023, respectively). High α-SMA expression in the stroma was validated by QIF after controlling for estrogen receptor and progesterone receptor status [HR, 3.12; 95% confidence interval (CI), 1.12–8.68; P = 0.029] showing recurrence on trastuzumab in the same cohort. In the NeoALTTO cohort, elevated levels of ACTA2 were predictive for shorter DFS in the multivariate analysis (HR, 3.21; 95% CI, 1.14–9.05; P = 0.027).Conclusions:This work identifies α-SMA as a novel, easy-to-implement biomarker of resistance to trastuzumab that may be valuable in settings where trastuzumab is combined with other therapies.

Signal to background ratio across different areas of interest for all targets

Table S5: Genes in the chr6q23-24 region, association with CN levels and pCR.

Supplementary Table and Supplementary Figure Legends

Purpose:The heterogeneity of response to anti-HER2 agents represents a major challenge in patients with HER2-positive breast cancer. To better understand the sensitivity and resistance to trastuzumab and lapatinib, we investigated the role of copy number aberrations (CNA) in predicting pathologic complete response (pCR) and survival outcomes in the NeoALTTO trial.Experimental Design:The neoadjuvant phase III NeoALTTO trial enrolled 455 patients with HER2-positive early-stage breast cancer. DNA samples from 269 patients were assessed for genome-wide copy number profiling. Recurrent CNAs were found with GISTIC2.0.Results:CNA estimates were obtained for 184 patients included in NeoALTTO. Among those, matched transcriptome and whole-exome data were available for 154 and 181 patients, respectively. A significant association between gene copy number and pCR was demonstrated for ERBB2 amplification. Nevertheless, ERBB2 amplification ceased to be predictive once ERBB2 expression level was considered. GISTIC2.0 analysis revealed 159 recurrent CNA regions. Lower copy number levels of the 6q23-24 locus predicted absence of pCR in the whole cohort and in the estrogen receptor–positive subgroup. 6q23-24 deletion was significantly more frequent in TP53 wild-type (WT) compared with TP53-mutated, resulting in copy number levels significantly associated with lack of pCR only in the TP53 WT subgroup. Interestingly, a gene-ontology analysis highlighted several immune processes correlated to 6q23-24 copy number.Conclusions:Our analysis identified ERBB2 copy number as well as 6q23-24 CNAs as predictors of response to anti–HER2-based treatment. ERBB2 expression outperformed ERBB2 amplification. The complexity of the 6q23-24 region warrants further investigation.

Table S1: Clinicopathological characteristics of the patients. Table S2: List of 49 genes associated with CNAs in breast cancer from the COSMIC Cancer Gene Census database. Table S3: Gene ontology analysis of the genes correlated with the genome instability index. Table S4: Gene ontology analysis of the genes anti-correlated with the genome instability index.

Background: The erythropoietin (EPO) receptor activation can stimulate tumor proliferation and may be implicated in resistance to anti-HER2 therapies. EPO administration can be used for the management of chemotherapy-induced anemia. We aim to assess if EPO intake during the adjuvant anti-HER2 treatment impacts the outcomes of HER2-positive early breast cancer (EBC) patients. Methods: For this post hoc analysis of the ALTTO trial (NCT00490139), we categorized two cohorts according to the use of EPO during adjuvant anti-HER2 treatment: i) at least one EPO dose (EPO-cohort) vs ii) no EPO (non-EPO-cohort). We compared their baseline characteristics to identify potential confounders. The primary endpoint disease-free-survival (DFS), and the secondary endpoint overall survival (OS) were compared between these two cohorts, with subgroup analysis according to menopausal status, tumor characteristics, and anti-HER2 treatment. Kaplan Meier method, log-rank test, and Cox regression model adjusted for confounders were performed for DFS and OS (removal criterion p2 cm (56.9%), positive nodal status (58.5%), positive hormone receptor (HR) status (61.8%), and poorly differentiated or undifferentiated histologic grade (64.2%). Baseline characteristics were similar in both cohorts, except for the timing of chemotherapy, for which 93.5% of patients from the EPO-cohort were enrolled in the concurrent designs (2/2B) vs 44.2% in the non-EPO-cohort (p Citation Format: Diogo Martins-Branco, Marie Kassapian, Véronique Debien, Rafael Caparica, Daniel Eiger, Urania Dafni, Charitini Andriakopoulou, Sarra El-Abed, Miguel Izquierdo, Malou Vicente, Saranya Chumsri, Martine Piccart-Gebhart, Alvaro Moreno-Aspitia, Ann Søegaard Knop, Janine Lombard, Evandro de Azambuja. The impact of erythropoietin administration concomitantly with adjuvant anti-HER2 treatment on the patients’ outcome: Sub-analysis of the ALTTO study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-14-05.

Background: Despite great efforts to identify tumor-related prognostic biomarkers in early-stage Human Epidermal growth factor Receptor 2 (HER2)-positive Breast Cancer (HER2+ BC), reliable determinants of long-term clinical outcomes are lacking. HER2+ BC is a lipogenic malignancy, and HER2-driven fatty acid (FA) biosynthesis plays a crucial role in sustaining cancer cell growth, proliferation, and metastatization. Recently, FA uptake, a process mediated by the transmembrane transporter CD36, has emerged as a new determinant of acquired resistance to anti-HER2 treatments in preclinical studies. Methods: The phase III NeoALTTO (NCT00553358) trial randomized 455 HER2+ BC patients to receive lapatinib, trastuzumab, or the combination of both drugs with weekly paclitaxel, followed by surgery, adjuvant fluorouracil, epirubicin, and cyclophosphamide and the same anti-HER2 therapy received before surgery. In the present analysis, we tested the hypothesis that higher intratumor CD36 mRNA levels could be associated with worse Event Free Survival (EFS) in a cohort of NeoALTTO patients for whom global gene expression analysis of baseline tumor samples through ClariomS platform was available. Results: A total of 180 patients were included (trastuzumab arm: n=66; lapatinib arm: n=65; combination arm: n=49). Higher baseline CD36 expression levels were associated with significantly worse EFS in patients treated with trastuzumab-based therapy, at both univariate (continuous scale, HR:1.73, 95% CI 1.20-2.49) and multivariable (HR:1.72, 95% CI 1.20-2.46) analysis, but not in patients treated with lapatinib- or trastuzumab plus lapatinib (HR:1.01; 95% CI: 0.69-1.47; p=0.98 and HR:1.02; 95% CI:0.54-1.95; p=0.94, respectively). CD36 levels did not predict pathological Complete Response (pCR) probability, while combining CD36 expression (based on median levels) with pCR status identified two subsets of patients with especially bad or good prognosis (7-year EFS rates in patients with high CD36/no pCR: 47%, 95% CIs: 0.26-0.66 vs. 79%, 95% CIs: 0.62-0.88 in patients with low CD36/any pCR status (Yes/No) OR high CD36/pCR; Log-Rank test p= 0.006). Conclusions: This is the first study to show that high CD36 expression is independently predictive of worse long-term clinical outcomes in HER2+ BC patients treated with neoadjuvant trastuzumab-based therapy. Along with recent preclinical data highlighting CD36 role in tumor resistance to anti-HER2 therapies, our findings point to CD36 as a novel promising target for HER2+ BC treatment. Citation Format: Francesca Ligorio, Serena Di Cosimo, Paolo Verderio, Chiara Maura Ciniselli, Sara Pizzamiglio, Lorenzo Castagnoli, Tiziana Triulzi, Elda Tagliabue, Sarra El-Abed, Miguel Izquierdo, Evandro de Azambuja, Paolo Nuciforo, Jens Huober, Luca Moscetti, Wolfgang Janni, M Coccia-Portugal, Paola Corsetto, Antonino Belfiore, Daniele Lorenzini, Maria Grazia Daidone, Andrea VIngiani, Serenella M Pupa, Giancarlo Pruneri, Claudio Vernieri. High CD36 expression predicts worse event free survival in HER2-positive breast cancer patients treated with neoadjuvant trastuzumab-based therapy: An exploratory analysis of the NeoALTTO study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-12.

Importance. Neoadjuvant therapy with dual HER2 blockade improved pathological complete response (pCR) rate. Nevertheless, it would be desirable to identify patients exquisitely responsive to single agent trastuzumab to minimize or avoid overtreatment. Objective. To evaluate the predictive and prognostic value of basal primary tumor miRNA expression profile within the trastuzumab arm of the NeoALTTO study.Design, Setting and Participants. RNA samples from baseline biopsies were randomized into training (n =45) and testing (n =47) sets. After normalization, miRNAs associated with Event-free survival (EFS) and pCR were identified by univariate analysis. Multivariate models were implemented to generate specific signatures which were first confirmed, and then analyzed according to other clinical and pathological variables.Main outcomes and measures. Association between miRNA expression and pCR and/or EFS.Results. We identified a prognostic signature including hsa-miR-153-3p (HR 1.831, 95%CI: 1.34-2.50) and hsa-miR-219a-5p (HR 0.629, 95%CI: 0.50 - 0.78). For two additional miRNAs (miR-215-5p and miR-30c-2-3p), we found a statistically significant interaction term with pCR (p.interaction: 0.017 and 0.038, respectively). Besides, a two-miRNA signature was predictive of pCR (hsa-miR-31-3p, OR 0.70, 95%CI: 0.53 - 0.92, and hsa-miR-382-3p, OR: 1.39, 95%CI: 1.01 -1.91). Notably, the performance of this predictive miRNA signature resembled that of the genomic classifiers PAM50 and TRAR, and did not improve when the extended models were fitted.Conclusions and relevance. Analysis of primary tumor tissue miRNAs holds the potential of a parsimonious tool to identify patients with differential clinical outcomes after trastuzumab based neoadjuvant therapy. Trial registration. ClinicalTrials.gov Identifier: NCT00553358. Table 1.MiRNAs associated with EFS. Results of the univariate Cox regression model- training set.miRNAsHR95% CIhsa-miR-12000.671(0.512; 0.879)ahsa-miR-1238-3p0.669(0.543; 0.826)ahsa-miR-12650.839(0.705; 0.997)ahsa-miR-129-5p0.785(0.628; 0.980)ahsa-miR-153-3p1.412(1.026; 1.943)ahsa-miR-15390.826(0.697; 0.979)ahsa-miR-1908-5p0.796(0.648; 0.978)ahsa-miR-205-5p0.737(0.553; 0.982)hsa-miR-219a-5p0.787(0.627; 0.989)ahsa-miR-25-5p0.566(0.391; 0.819)ahsa-miR-3000.581(0.399; 0.845)ahsa-miR-382-3p0.791(0.636; 0.985)ahsa-miR-4920.774(0.635; 0.944)ahsa-miR-519a-3p0.834(0.696; 0.998)ahsa-miR-551b-5p0.666(0.452; 0.981)ahsa-miR-5830.727(0.549; 0.963)ahsa-miR-6001.373(1.03; 1.829)ahsa-miR-6260.819(0.673; 0.998)ahsa-miR-651-5p0.611(0.382; 0.977)hsa-miR-7610.845(0.721; 0.991)ahsa-miR-891b0.658(0.479; 0.904)ahsa-miR-92a-2-5p0.787(0.637; 0.971)ahsa-miR-937-3p0.748(0.578; 0.967)aHR, Hazard Ratio; CI, Confidence Intervala microRNAs retained its statistically significance (at 10%) also when normalized for the overall mean. Table 2.MiRNAs associated with pCR. Results of the univariate logistic model - training set.miRNAsOR95% CIhsa-miR-132-3p0.410(0.169; 0.991)ahsa-miR-23b-5p0.520(0.316; 0.856)ahsa-miR-31-3p0.566(0.351; 0.912)ahsa-miR-31-5p0.508(0.291; 0.887)ahsa-miR-330-3p0.501(0.251; 0.999)hsa-miR-34b-3p0.673(0.474; 0.956)ahsa-miR-382-3p1.392(1.006; 1.925)ahsa-miR-548j-5p1.702(1.090; 2.658)aOR, Odds Ratio; CI, Confidence Interval. a microRNAs retained its statistically significance (at 10%) also when normalized for the overall mean. 2 Citation Format: Marilena Valeria Iorio, Sara Pizzamiglio, Giulia Cosentino, Chiara M Ciniselli, Loris De Cecco, Alessandra Cataldo, Ilaria Plantamura, Tiziana Triulzi, Sarra El-abed, Yingbo Wang, Mohammed Bajji, Paolo Nuciforo, Jens Huober, Susan Ellard, David Rimm, Andrea Gombos, Mariagrazia Daidone, Paolo Verderio, Elda Tagliabue, Serena Di Cosimo. The future of HER2-positive breast cancer patients might be written in miRNAs: An exploratory analysis from the NeoALTTO study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-26.

Purpose: The companion diagnostic test for trastuzumab has not changed much in the last 25 years. We used high-plex digital spatial profiling to identify biomarkers besides HER2 that can help predict response to trastuzumab in HER2-positive breast cancer. Experimental Design: Fifty-eight protein targets were measured in three different molecularly defined compartments by the NanoString GeoMx Digital Spatial Profiler (DSP) in a tissue microarray containing 151 patients with breast cancer that received adjuvant trastuzumab as part of the Hellenic Cooperative Oncology Group 10/05 clinical trial. Promising candidate biomarkers were orthogonally validated with quantitative immunofluorescence (QIF). RNA-sequencing data from the Neoadjuvant Lapatinib and/or Trastuzumab Treatment Optimisation Study (NeoALTTO) were accessed to provide independent cohort validation. Disease-free survival (DFS) was the main outcome assessed. Statistical analyses were performed using a two-sided test (α = 0.05) and multiple testing correction (Benjamini–Hochberg method, FDR < 0.1). Results: By DSP, high expression of alpha-smooth muscle actin (α-SMA), both in the leukocyte and stromal compartments, was associated with shorter DFS in univariate analysis (P = 0.002 and P = 0.023, respectively). High α-SMA expression in the stroma was validated by QIF after controlling for estrogen receptor and progesterone receptor status [HR, 3.12; 95% confidence interval (CI), 1.12–8.68; P = 0.029] showing recurrence on trastuzumab in the same cohort. In the NeoALTTO cohort, elevated levels of ACTA2 were predictive for shorter DFS in the multivariate analysis (HR, 3.21; 95% CI, 1.14–9.05; P = 0.027). Conclusions: This work identifies α-SMA as a novel, easy-to-implement biomarker of resistance to trastuzumab that may be valuable in settings where trastuzumab is combined with other therapies.

Purpose: The heterogeneity of response to anti-HER2 agents represents a major challenge in patients with HER2-positive breast cancer. To better understand the sensitivity and resistance to trastuzumab and lapatinib, we investigated the role of copy number aberrations (CNA) in predicting pathologic complete response (pCR) and survival outcomes in the NeoALTTO trial. Experimental Design: The neoadjuvant phase III NeoALTTO trial enrolled 455 patients with HER2-positive early-stage breast cancer. DNA samples from 269 patients were assessed for genome-wide copy number profiling. Recurrent CNAs were found with GISTIC2.0. Results: CNA estimates were obtained for 184 patients included in NeoALTTO. Among those, matched transcriptome and whole-exome data were available for 154 and 181 patients, respectively. A significant association between gene copy number and pCR was demonstrated for ERBB2 amplification. Nevertheless, ERBB2 amplification ceased to be predictive once ERBB2 expression level was considered. GISTIC2.0 analysis revealed 159 recurrent CNA regions. Lower copy number levels of the 6q23-24 locus predicted absence of pCR in the whole cohort and in the estrogen receptor–positive subgroup. 6q23-24 deletion was significantly more frequent in TP53 wild-type (WT) compared with TP53-mutated, resulting in copy number levels significantly associated with lack of pCR only in the TP53 WT subgroup. Interestingly, a gene-ontology analysis highlighted several immune processes correlated to 6q23-24 copy number. Conclusions: Our analysis identified ERBB2 copy number as well as 6q23-24 CNAs as predictors of response to anti–HER2-based treatment. ERBB2 expression outperformed ERBB2 amplification. The complexity of the 6q23-24 region warrants further investigation.

BackgroundNeutrophils kill antibody-opsonized tumor cells using trogocytosis, a unique mechanism of destruction of the target plasma. This previously unknown cytotoxic process of neutrophils is dependent on antibody opsonization, Fcγ receptors and CD11b/CD18 integrins. Here, we demonstrate that tumor cells can escape neutrophil-mediated cytotoxicity by calcium (Ca2+)-dependent and exocyst complex-dependent plasma membrane repair.MethodsWe knocked down EXOC7 or EXOC4, two exocyst components, to evaluate their involvement in tumor cell membrane repair after neutrophil-induced trogocytosis. We used live cell microscopy and flow cytometry for visualization of the host and tumor cell interaction and tumor cell membrane repair. Last, we reported the mRNA levels of exocyst in breast cancer tumors in correlation to the response in trastuzumab-treated patients.ResultsWe found that tumor cells can evade neutrophil antibody-dependent cellular cytotoxicity (ADCC) by Ca2+-dependent cell membrane repair, a process induced upon neutrophil trogocytosis. Absence of exocyst components EXOC7 or EXOC4 rendered tumor cells vulnerable to neutrophil-mediated ADCC (but not natural killer cell-mediated killing), while neutrophil trogocytosis remained unaltered. Finally, mRNA levels of exocyst components in trastuzumab-treated patients were inversely correlated to complete response to therapy.ConclusionsOur results support that neutrophil attack towards antibody-opsonized cancer cells by trogocytosis induces an active repair process by the exocyst complex in vitro. Our findings provide insight to the possible contribution of neutrophils in current antibody therapies and the tolerance mechanism of tumor cells and support further studies for potential use of the exocyst components as clinical biomarkers.

PURPOSE The PALLAS study investigated whether the addition of palbociclib, an oral CDK4/6 inhibitor, to adjuvant endocrine therapy (ET) improves invasive disease-free survival (iDFS) in early hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2–) breast cancer. In this analysis, we evaluated palbociclib exposure and discontinuation in PALLAS. METHODS Patients with stage II-III HR+, HER2– disease were randomly assigned to 2 years of palbociclib with adjuvant ET versus ET alone. The primary objective was to compare iDFS between arms. Continuous monitoring of toxicity, dose modifications, and early discontinuation was performed. Association of baseline covariates with time to palbociclib reduction and discontinuation was analyzed with multivariable competing risk models. Landmark and inverse probability weighted per-protocol analyses were performed to assess the impact of drug persistence and exposure on iDFS. RESULTS Of the 5,743 patient analysis population (2,840 initiating palbociclib), 1,199 (42.2%) stopped palbociclib before 2 years, the majority (772, 27.2%) for adverse effects, most commonly neutropenia and fatigue. Discontinuation of ET did not differ between arms. Discontinuations for non–protocol-defined reasons were greater in the first 3 months of palbociclib, and in the first calendar year of accrual, and declined over time. No significant relationship was seen between longer palbociclib duration or ≥ 70% exposure intensity and improved iDFS. In the weighted per-protocol analysis, no improvement in iDFS was observed in patients receiving palbociclib versus not (hazard ratio 0.89; 95% CI, 0.72 to 1.11). CONCLUSION Despite observed rates of discontinuation in PALLAS, analyses suggest that the lack of significant iDFS difference between arms was not directly related to inadequate palbociclib exposure. However, the discontinuation rate illustrates the challenge of introducing novel adjuvant treatments, and the need for interventions to improve persistence with oral cancer therapies.

Background: The potential gonadotoxicity of anti-HER2 agents remains largely unknown, and limited, conflicting evidence exists for taxanes. Antimüllerian hormone (AMH) is an established biomarker of ovarian reserve that may aid in quantifying anticancer treatment–induced gonadotoxicity. Patients and Methods: The present biomarker analysis of the randomized phase III neoadjuvant NeoALTTO trial included premenopausal women aged ≤45 years at diagnosis of HER2-positive early breast cancer with available frozen serum samples at baseline (ie, before anticancer treatments), at week 2 (ie, the “biological window” of anti-HER2 therapy alone), and/or at the time of surgery (ie, after completing paclitaxel + anti-HER2 therapy, before starting adjuvant chemotherapy). Results: The present analysis included 130 patients with a median age of 38 years (interquartile ratio [IQR], age 33–42 years). AMH values at the 3 time points differed significantly (PPPConclusions: This NeoALTTO biomarker analysis showed that anti-HER2 therapies alone had limited gonadotoxicity but that the addition of weekly paclitaxel resulted in marked AMH decline with possible negative implications for subsequent ovarian function and fertility.

The implementation of trastuzumab has revolutionized the clinical management of HER2 positive breast cancers. Unfortunately, 50% of patients are resistant to the treatment. Researchers have already designed alternative anti-HER2 agents, such as pertuzumab and lapatinib. Still, predicting which patients will benefit from the therapy would prevent overtreatment and avoid unnecessary risks of side effects. MiRNAs are small non-coding RNAs involved in post-transcriptional gene regulation, and participate in almost all biological processes, including cancer. Since aberrant miRNA levels can be detected both at tissue level and in the circulation, they are good candidates as predictive and prognostic biomarkers. This study had access to tumor tissue samples from the phase III NeoALTTO trial, aimed at evaluating the efficacy of a HER2 dual blockade with trastuzumab and Lapatinib vs single blocking, in concomitance with chemotherapy, in a pre-operative setting. The primary end-point of the study was pathologic complete response (pCR); the secondary end-point event-free survival (EFS). Focusing on the trastuzumab arm, we identified both a predictive signature (hsa-miR-31-3p, OR 0.70, 95%CI: 0.53-0.92 and hsa-miR-382-3p, OR 1.39, 95%CI: 1.01-1.91) with an AUC value of 0.73 (95%CI: 0.60-0.87), and a prognostic signature (miR-153-3p, HR 1.83, 95%CI: 1.34-2.50 and miR-219a-5p, HR 0.629, 95%CI: 0.51-0.79) leading to a C-statistics of 0.730 (95%CI: 0.63-0.83). Moreover, we identified 2 miRNAs (miR-215-5p and miR-30c-2-3p) associated to EFS with a statistically significant interaction term with pCR (p.interaction: 0.017 and 0.038, respectively). Aiming at deepening the understanding of resistance mechanism, we modulated the expression of miR-31-3p and miR-382-3p in vitro, upon trastuzumab treatment in HER2 positive breast cancer cell lines. Given that miR-31-3p negatively correlates with pCR, we transfected it in HER2-addicted SKBr3 cells to appreciate a possible gain of resistance to trastuzumab; conversely, miR-382-3p positively correlates with pCR and was transfected in HER2 non-addicted HCC1954 cells. Western blot analysis of HER2 signaling pathway highlighted that overexpression of miR-31-3p was able to counteract the reduction of phosphorylated HER2 levels induced by trastuzumab treatment in SKBR3 cells compared to control. Interestingly, miR-31-3p upmodulation increased the proliferation of both treated and non-treated SKBr3 cell in a 3D setting. Conversely, miR-382-3p overexpression in HCC1954 cells only slightly increased responsiveness to trastuzumab in the 3D setting, compared to control. Trastuzumab efficacy also relies on the immune system reaction, thus, in vivo experiments will likely provide further insights into the mechanism of action of these miRNAs. Citation Format: Giulia Cosentino, Sara Pizzamiglio, Chiara M. Ciniselli, Loris De Cecco, Alessandra Cataldo, Ilaria Plantamura, Tiziana Triulzi, Sarra El-abed, Yingbo Wang, Mohammed Bajji, Paolo Nuciforo, Jens Huober, Susan L. Ellard, David L. Rimm, Andrea Gombos, Mariagrazia Daidone, Paolo Verderio, Elda Tagliabue, Serena Di Cosimo, Marilena V. Iorio. miRNA and response to trastuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1229.

522 Background: Our preclinical findings suggest a role for the mevalonate pathway (MVA) in treatment resistance in HER2+ BC by providing alternative growth and survival signaling to bypass potent HER2 blockade, which could be overcome by the MVA inhibitors statins and nitrogen-containing bisphosphonates (NBs). Here we explored the effect of MVA inhibitors’ use on pts’ outcomes in the ALTTO trial (BIG2-06; NCT00490139). Methods: In the ALTTO trial, 8381 pts with HER2+ BC were randomized to 1 year of adjuvant lapatinib (L), trastuzumab (T), L+T, or T→L. All pts with documented treatment start with statins or NBs < 1 year after randomization were considered as MVA inhibitors users. Survival curves, with a median follow-up of 6.9 years, for disease-free survival (DFS), distant relapse-free interval (DRFI), BC-specific survival (BCSS), and overall survival (OS) according to MVA inhibitors use were estimated by the Kaplan Meier method and Log-rank test. All multivariate survival analyses employed a Cox proportional hazards regression model, adjusting for tumor size, nodal status, hormonal receptor (HoR), menopausal status, BMI, timing of chemo, and randomization arm. We considered interactions terms in Cox’s model between MVA inhibitors use and randomization arm, hormonal status, and BMI group. Results: Among the 8381 pts included in this study, 493 and 299 were statins or NBs users, respectively. Table 1 summarizes the significant differences in pts’ characteristics according to MVA inhibitors use ( P

509 Background: Several biologic features are implicated in the differences in response and survival to dual (trastuzumab and lapatinib [HL]) vs. single (trastuzumab [H]) HER2-blockade across neoadjuvant trials in early-stage HER2+ breast cancer. We evaluated the association of intrinsic subtypes and gene expression signatures with pathologic complete response (pCR) and event-free survival (EFS) in a pooled analysis of three independent phase III neoadjuvant studies with similar designs: CALGB 40601 (Alliance), NeoALTTO, and NSABP B-41. Methods: Gene expression profiling by RNA sequencing was assessed on 761 pre-treatment samples (264 from CALGB 40601, 249 from NeoALTTO, 248 from NSABP B-41). Intrinsic subtypes and 759 gene expression signatures were calculated. We studied the association of pCR and the benefit of dual (HL) vs. single (H) HER2-blockade by tumor intrinsic subtype in the pooled set. The ability of multiple gene expression signatures to predict pCR and EFS across the three studies was also tested by logistic and Cox regression analyses. Results: pCR status was associated with EFS only in HER2-Enriched (HR 0.45, 95% CI 0.29-0.71, p-value < 0.001) and Basal-like (HR 0.19, 95% CI 0.04-0.86, p-value 0.031) intrinsic subtypes, but not in Luminal and/or ER+ tumors. The EFS benefit of dual vs. single HER2-blockade was limited to HER2-Enriched tumors (HR 0.47, 95% CI 0.27-0.81, p-value 0.007). When evaluating the three clinical trials separately, we found 89/759 (11.7%) gene expression signatures in common for the prediction of pCR across the three clinical trials, including HER2-amplicon and immune activation signatures. Luminal-related signatures were associated with lower pCR rates but better EFS outcomes, especially in patients with residual disease. Stratified Cox regression models by study showed a significant and strong association of NK, B and plasma cells, as well as Ig-related signatures with a better EFS outcome, while vascular, proliferation, and metastasis signatures were associated with poor EFS. Conclusions: In early-stage HER2+ breast cancer, the relationship between pCR and EFS differs by tumor intrinsic subtype, and the benefit of dual vs. single HER2-blockade seems to be limited to HER2-Enriched subtype tumors. Immune signatures were associated with higher pCR rates and better EFS, luminal signatures were associated with lower pCR rates but good EFS outcomes, and vascular/proliferation/metastasis signatures were associated with poor EFS across the three clinical trials. Clinical trial identification: CALGB 40601: NCT00770809. (CALGB is part of the Alliance for Clinical Trials in Oncology). NeoALTTO: NCT00553358 NSABP B-41: NCT00486668

12084 Background: The potential gonadotoxicity of anti-HER2 agents remains largely unknown and limited conflicting evidence exists for taxanes. Anti-Mullerian hormone (AMH) is an established biomarker of ovarian reserve; its measurement during systemic therapies may aid in indicating gonadotoxicity, in the diagnosis and prediction of primary ovarian insufficiency (POI). The present analysis explored for the first time the impact of anti-HER2 therapy alone and then combined with weekly paclitaxel on ovarian reserve measured by AMH levels in breast cancer (BC) patients not previously exposed to anthracycline and cyclophosphamide. Methods: This biomarker analysis of the NeoALTTO (NCT00553358) randomized phase III neoadjuvant trial included premenopausal women aged ≤45 years at diagnosis of HER2-positive early BC with available frozen serum samples at baseline (i.e. before administering any anticancer treatment), at week 2 (i.e. “biological window” of anti-HER2 therapy alone) and/or at the time of surgery (i.e. after completion of paclitaxel plus anti-HER2 therapy and before starting adjuvant chemotherapy). Central AMH testing was performed with the Roche Elecsysâ AMH Plus assay (LoD = 0.01 ng/ml). AMH levels during anti-HER2 therapy alone and then combined with paclitaxel were assessed as a measure of treatment acute gonadotoxicity. The impact of different anti-HER2 agents, patients’ age, and baseline AMH levels on treatment gonadotoxicity were also investigated. Results: The present analysis included 130 patients with a median age of 38 years (IQR: 33-42 years). AMH values at the 3 time points differed significantly from each other (p < 0.001). At baseline, median AMH levels were 1.29 ng/mL (IQR 0.56 – 2.62 ng/mL). At week 2, a small but significant reduction in AMH levels was observed (median value: 1.10 ng/mL, IQR 0.45 – 2.09 ng/mL, p < 0.001). At surgery, there was a larger significant decline in AMH levels (median value: 0.01 ng/mL, IQR 0.01 – 0.03 ng/mL, p < 0.001). There was no significant difference between treatment arms (trastuzumab vs. lapatinib vs. trastuzumab plus lapatinib) in the degree of reduction in AMH levels at week 2 (p = 0.763) and at surgery (p = 0.700). Age and pre-treatment ovarian reserve had a major influence on treatment-induced gonadotoxicity risk, with older age and lower AMH levels at diagnosis being associated with a greater negative impact. Conclusions: This biomarker analysis of the NeoALTTO trial showed for the first time that anti-HER2 therapies alone had limited gonadotoxicity but the addition of weekly paclitaxel resulted in marked AMH decline which likely has implications for subsequent ovarian function and fertility. These data highlight the importance of oncofertility counselling among all premenopausal women with HER2-positive BC receiving systemic anticancer treatments. Clinical trial information: NCT00553358.

Background: Knowledge regarding risk of treatment-induced premature ovarian insufficiency (POI) is crucial for appropriate oncofertility counseling. While the gonadotoxicity of anthracycline- and cyclophosphamide-based chemotherapy (CT) in young breast cancer (BC) patients (pts) is well established, the impact of anti-HER2 agents remains largely unknown and limited conflicting evidence exists for taxanes. Anti-Mullerian hormone (AMH) is an established biomarker of ovarian reserve; its measurements during systemic therapies may aid in diagnosis and prediction of POI. The NeoALTTO trial represented a unique setting to explore the acute gonadotoxicity of anti-HER2 therapy alone and in combination with weekly paclitaxel in pts not previously exposed to any other potentially gonadotoxic anticancer treatment. Methods: NeoALTTO (NCT00553358) is a multicenter, open-label, randomized phase III neoadjuvant trial in HER2-positive early BC pts randomized to receive lapatinib (L), trastuzumab (T), or their combination (L+T) alone for 6 weeks followed by the addition of paclitaxel for 12 weeks before surgery.The present analysis included only premenopausal women aged ≤45 years with available frozen serum samples at baseline (i.e. before administering any anticancer treatment), at week 2 (i.e. “biological window” of anti-HER2 therapy alone) and/or at surgery (i.e. after completion of paclitaxel plus anti-HER2 therapy). Central AMH testing was performed at Erasme Hospital (Brussels, Belgium) with the Roche Elecsys® AMH Plus assay (LoD=0.010 ng/ml). AMH levels during anti-HER2 therapy alone and then combined with paclitaxel were assessed as a measure of treatment impact on ovarian reserve. The impact of different anti-HER2 agents (T and/or L), pts’ age and baseline AMH levels on treatment gonadotoxicity were also investigated. Results: The present analysis included 130 pts with a median age of 38 years (IQR: 33-42 years), of whom 86 (66.2%) were ≤40 years. At baseline, median AMH levels were 1.29 ng/mL (IQR 0.56 - 2.62 ng/mL). At week 2, a small but significant reduction in AMH levels was observed (median value: 1.10 ng/mL, IQR 0.45 - 2.09 ng/mL, p Citation Format: Matteo Lambertini, Marcello Ceppi, Richard A. Anderson, David A. Cameron, Marco Bruzzone, Maria Alice Franzoi, Claudia Massarotti, Sarra El-Abed, Yingbo Wang, Christophe Lecocq, Paolo Nuciforo, Rebecca Rolyance, Lajos Pusztai, Joohyuk Sohn, Jacopo Ligato, Maria Maddalena Latocca, Luca Arecco, Barbara Pistilli, Kathryn J. Ruddy, Alberto Ballestrero, Lucia Del Mastro, Fedro A. Peccatori, Ann H. Partridge, Cristina Saura, Michael Untch, Martine Piccart, Serena Di Cosimo, Evandro de Azambuja, Isabelle Demeestere. Impact of anti-HER2 therapy alone and in association with weekly paclitaxel on the ovarian reserve of young women with HER2-positive early breast cancer: Biomarker analysis of the NeoALTTO trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD5-05.

Aim To present the pre-specified analyses of >5-years follow-up of the Phase III ALTTO trial. Patients and methods 8381 patients with stage I-III HER2 positive breast cancer randomised to chemotherapy plus 1-year of trastuzumab (T), oral lapatinib (L; no longer evaluated), trastuzumab followed by lapatinib (T→L), and lapatinib + trastuzumab (L+T). The primary endpoint was disease-free survival (DFS). A secondary analysis examined DFS treatment effects by hormone receptor status, nodal status and chemotherapy timing; time to recurrence; overall survival (OS) and safety (overall and cardiac). Results At a median follow-up of 6.9 years, 705 DFS events for L+T versus T were observed. Hazard Ratio (HR) for DFS was 0.86 (95% CI, 0.74–1.00) for L+T versus T and 0.93 (95% CI, 0.81–1.08) for T→L versus T. The 6-year DFS were 85%, 84%, and 82% for L+T, T→L, and T, respectively. HR for OS was 0.86 (95% CI, 0.70–1.06) for L+T versus T and 0.88 (95% CI, 0.71–1.08) for T→L versus T. The 6-year OS were 93%, 92%, and 91% for L+T, T→L, and T, respectively. Subset analyses showed a numerically better HR for DFS in favour of L+T versus T for the hormone-receptor-negative [HR 0.80 (95% CI, 0.64–1.00; 6-yr DFS% = 84% versus 80%)] and the sequential chemotherapy [HR 0.83 (95% CI, 0.69–1.00; 6-yr DFS% = 83% versus79%)] subgroups. Conclusion T+L did not significantly improve DFS and OS over T alone, both with chemotherapy, and, therefore, cannot be recommended for adjuvant treatment of early-stage HER2-positive breast cancer. Trial Registration clinicaltrials.gov Identifier NCT00490139 .

524 Background: PREDICT+ is a widely used, free, online tool based on traditional clinico-pathological features, including HER2, developed to predict individual mortality of EBC pts and to aid clinical decision making for adjuvant therapy. However, its prognostic role in HER2+ EBC pts treated with chemotherapy (CT) and anti-HER2 therapies remains unclear. We aimed to investigate the prognostic performance of PREDICT+ in HER2+ EBC pts enrolled in the ALTTO trial. Methods: ALTTO is a phase III study evaluating adjuvant lapatinib (L) +/- trastuzumab (T) vs. T alone in pts with HER2+ EBC. Pts enrolled in the ALTTO trial and receiving T-based therapy started concurrently with CT were eligible for this analysis. We calculated PREDICT+ estimates using variables extracted from ALTTO database, blinded to pts outcomes. The prognostic performance of PREDICT+ was evaluated by assessing its calibration and discriminatory accuracy. For calibration, median predicted 5-year (5-yr) overall survival (OS) was compared to observed 5-yr OS. For discriminatory accuracy, the area under the receiver-operator characteristic (AUC under the ROC) curve and corresponding 95% confidence intervals (CI) for predicted 5-yr OS were calculated. Subgroup analyses were performed according to type of anti-HER2 therapy, type of CT, age, hormone receptor (HR) status, nodal status and tumor size. Results: This analysis included 2,794 pts. After a median follow-up of 6.0 years (IQR, 5.8-6.7), 182 deaths were observed. Overall, PREDICT+ underestimated 5-yr OS by 6.7% (95% CI, 5.8-7.6): observed 5-year OS was 94.7% vs. predicted 88.0%. The underestimation was consistent across all subgroups (Table). For discriminatory accuracy, AUC under the ROC curve was 73.7% (95%CI 69.7-77.8) in the overall population, ranging between 61.7% and 77.7% across the analysed subgroups. Conclusions: In HER2+ EBC pts enrolled in the ALTTO trial, the PREDICT+ score highly underestimated OS. The low performance of this prognostic tool was consistent across all pts subgroups. PREDICT+ should be used with caution to give prognostic estimation in HER2+ EBC pts treated in the modern era with effective chemotherapy and anti-HER2 targeted therapies.[Table: see text]

TPS597 Background: Early stage triple negative breast cancer (TNBC) is associated with a high risk of distant relapse. Because TNBC does not currently have specific targeted agents approved for use in the early setting, it is treated primarily with chemotherapy. TNBC may be more immunogenic than other subtypes of breast cancer. Atezolizumab (an anti–PD-L1 antibody), in combination with nab-paclitaxel has been approved in >70 countries for the treatment of PD-L1-positive unresectable locally advanced or metastatic TNBC based on the results of the randomized phase 3 IMpassion130 trial. The phase 3 IMpassion031 study, evaluating atezolizumab in combination with chemotherapy (nab-paclitaxel followed by doxorubicin and cyclophosphamide) in comparison to placebo plus chemotherapy as neoadjuvant treatment demonstrated a statistically significant and clinically meaningful improvement in pCR in both PD-L1 positive and PD-L1 negative tumors. ALEXANDRA/IMpassion030 is a global, prospective, randomized, open-label, phase 3 trial currently investigating the efficacy, safety and pharmacokinetic profile of adjuvant atezolizumab plus standard anthracycline/taxane adjuvant chemotherapy versus chemotherapy alone in early stage TNBC. Methods: ALEXANDRA/IMpassion030 will randomize 2300 patients with operable stage II-III TNBC, confirmed by central pathology review. Patients are stratified by type of surgery, nodal status, and centrally assessed PD-L1 status. Adjuvant chemotherapy consist of weekly paclitaxel 80 mg/m2 for 12 weeks followed by dose dense anthracycline (epirubicin 90 mg/m2 or doxorubicin 60 mg/m2) and cyclophosphamide 600 mg/m2 for 4 doses every 2 weeks or the same chemotherapy regimen (T-EC/AC) given concomitantly with atezolizumab 840 mg every 2 weeks followed by maintenance atezolizumab 1200 mg every 3 weeks until completion of 1 year of atezolizumab. The primary endpoint is invasive disease-free survival (iDFS) and secondary endpoints include, iDFS in the PD-L1 selected tumour status (IC1/2/3) and node-positive subpopulations, overall survival, safety, patient functioning and health related quality of life (HRQoL). Tumor tissue and blood samples will be collected for biomarker research. The first site was activated on May 4 2018, and approximately 373 sites in 30 countries are currently participating in this trial. This trial is sponsored by F. Hoffmann-La Roche Ltd and conducted in partnership with the Breast International Group, Frontier Science and Technology Research Foundation, Institute Jules Bordet and Alliance Foundation Trials. Clinical trial information: NCT03498716.