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INTRODUCTION: Breast cancer (BC) is a highly prevalent and heterogeneous disease, entailing different so-called intrinsic subtypes (IS) according to gene expression, namely Luminal A, Luminal B, HER2-Enriched (HER2E) and Basal-like, as well as a normal breast-like group. The HER2E is still a poorly understood entity, which needs further biologic characterization to improve therapeutic management. MATERIAL AND METHODS: Patients (pts) treated at Hospital Clinic (Barcelona, Spain) over 18 years with a diagnosis of metastatic BC, with available matched primary and metastatic tumor samples for gene expression analyses were retrospectively recruited. Using the nCounter® Breast Cancer 360 panel, we studied the expression of 776 genes pertaining to different tumor and immune pathway-related signatures, with a focus on HER2E vs. non-HER2E tumors. Significant changes were considered at a false discovery rate (FDR) < 5%. Main clinicopathological features were also compared. IS changes from primary to metastatic disease were assessed. RESULTS: Ninety-one pts with paired tumor samples were included. Briefly, primary tumors were 67.0% hormone receptor-positive (HR+)/HER2-negative (HER2-), 14.8% were HER2-positive (HER2+) and 18.2% were triple-negative (TNBC). IS distribution in primary tumors was the following: 25 Luminal A (28%), 22 Luminal B (24%), 24 HER2E (26%), 12 Basal-like (13%), and 8 Normal-like (9%). In contrast, IS distribution in metastatic tumors was the following: 13 Luminal A (14%), 22 Luminal B (24%), 34 HER2-E (37%), 16 Basal-like (18%), and 6 Normal-like (7%). The HER2E disease proved to be a relatively stable subtype, with 16 (66.7%) tumors not changing IS in the transition to metastatic disease (p=0.078). Particularly, within HR+/HER2-, HER2+ and TNBC, 8/12 (66.7%), 8/10 (80.0%) and 1/2 (50.0%) tumors remained HER2E. Conversely, an overall significant switch from Luminal to non-Luminal tumors at the metastatic progression was observed (p=0.031), with 14/19 (73.7%) new non-Luminal BC being HER2E. When considering all primary and metastatic tumors, HER2E were observed to be less frequently estrogen receptor (ER) positive than non-HER2E tumors (55.8% vs. 78.7%; p=0.002), with lower mean progesterone receptor (PR) levels (15.3% vs. 25.8%; p=0.027). Compared to the other subtypes (as a group), the PAM50 risk of recurrence score (ROR-P) was higher for HER2E tumors (59.1 vs. 41.7; p < 0.001), which were also more frequently HER2+ (36.5% vs. 5.8%) and less likely HR+/HER2- (50.0% vs. 73.6%) and TNBC (13.5% vs. 20.7%), compared to non-HER2E (p < 0.001). No significant differences in grade, TILs, Ki67 and histotype were observed. Overall, 140/776 genes were significantly downregulated in HER2E vs. non-HER2E tumors, including genes related to cell adhesion, migration, DNA damage repair, apoptosis, estrogen signalling pathway, senescence. Conversely, 178/776 genes were significantly upregulated, including the tyrosine-kinase receptor FGFR4, genes involved in nuclear activity, DNA transcription regulation, MAP-kinase signaling pathways, proliferation, cytokine response, epithelial-to-mesenchymal transition (EMT), cell cycle progression, and immune-related genes such as CD274 (PD-L1 gene). DISCUSSION: A switch towards more aggressive IS from primary to advanced disease was observed, with an increase in HER2E prevalence. HER2E tumors, which tend to maintain their IS at the metastatic disease, showed upregulation of genes related to proliferation, survival and EMT, coherently with their well-known worse prognosis. FGFR4 and CD274 upregulation, along with downregulation of genes involved in DNA damage repair suggest these tumors might benefit from targeted therapeutic approaches. Genomic higher risk was not convoyed by consistent clinicopathological features, except for lower PR levels and HER2 overexpression at IHC. Further characterization according to primary/metastatic and HR status is ongoing. Intrinsic subtype distribution across primary and metastatic breast tumors Citation Format: Francisco Javier Muñoz-Carrillo, Laia Paré, Benedetta Conte, Adela Rodríguez, Patricia Galván, Esther Sanfeliu, Blanca González-Farré, Claudette Falato, Isabel Garcia-Fructuoso, Barbara Adamo, Nuria Chic, Olga Martínez-Sáez, Tomás Pascual, Reinaldo Moreno, Montserrat Muñoz, Fara Brasó-Maristany, Aleix Prat, Francesco Schettini, Maria Vidal. Clinico-Pathological and Molecular Characterization of HER2-Enriched Breast Tumors Independently of HER2 Status [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 P2-23-16.

Purpose:To assess palbociclib in combination with trastuzumab with or without endocrine therapy in patients with HER2-positive advanced breast cancer.Patients and Methods:PATRICIA is a prospective, open-label, multicenter phase II trial. Patients had received 2–4 prior lines of anti-HER2–based regimens. Treatment consisted of palbociclib 200 mg daily for 2 weeks and 1 week off plus trastuzumab. The study was based on a Simon two-stage design comprising three cohorts: estrogen receptor (ER)-negative (cohort A), ER-positive (cohort B1), and ER-positive with letrozole (cohort B2). ER-positive patients were randomized to cohorts B1 or B2. Primary endpoint was progression-free survival rate at 6 months (PFS6). Secondary objectives included safety and evaluation of the PAM50 intrinsic subtypes.Results:Seventy-one patients were recruited (n = 15 in cohort A and 28 in each cohort B). The PFS6 rate in cohorts A, B1, and B2 was 33.3% (5/15), 42.8% (12/28), and 46.4% (13/28), respectively. Regarding safety, grade 1–2 and 3–4 toxicities occurred in 97.7% and 84.4% of patients, respectively. The most common grade 3–4 toxicities were neutropenia (66.4%) and thrombocytopenia (11.3%). Regarding PAM50, 59 (83.1%) tumors were profiled. Luminal disease defined by PAM50 was found independently associated with longer PFS compared with non-luminal disease (10.6 vs. 4.2 months median PFS; adjusted hazard ratio = 0.40; P = 0.003).Conclusions:Palbociclib in combination with trastuzumab is safe and exhibits promising survival outcomes in trastuzumab pretreated ER-positive/HER2-positive advanced breast cancer with a PAM50 Luminal A or B subtype. The enrollment was stopped prematurely, and a new randomized cohort was opened in this population.

Supplementary Data

BACKGROUND: Metastatic breast cancer (mBC) remains an incurable disease and is the cause of nearly all deaths related to BC. Next-generation sequencing technologies are allowing the application of personalized targeted molecular therapies, thereby improving outcomes in breast cancer patients. However, they are not routinely used in the clinic and their cost could be a cause of disparity. One strategy to overcome the barriers of implementing NGS in the clinic is to promote the active participation of patients with mBC in the management of their disease and offering free access to these tests. With this in mind, we designed HOPE (SOLTI-1903), a Spanish real-world study where patients lead their inclusion, participation, and follow-up through a digital tool (DT) that guides them in every step of the journey. The ultimate objective of HOPE is to gather real-world data on the utilization of molecular information in the management of mBC and to empower these patients. TRIAL DESIGN: Patients diagnosed with locally advanced or mBC can be included. Basic demographic data, disease characteristics, treatment history and quality of life data are collected by patients through a DT. The study is complemented by a patient empowerment program including informative workshops and precision medicine video-tutorials. Patients are encouraged to involve their physicians in HOPE. A total of 600 patients will be included in Spain. PATIENT JOURNEY: Once patients request participation through the DT, a dedicated team in SOLTI assists them in the subsequent steps while validating that eligibility criteria are met according to patient-provided data. Then, patients receive instructions from SOLTI’s team to attend the nearest partner local laboratory, where they sign the study informed consent form. A metastatic (preferably) or primary archival tumor sample is requested from the patient’s reference hospital and analyzed by FoundationOne®CDx. Patients that are in progression or not receiving active systemic chemo- or radiotherapy undergo a blood draw to receive a Guardant360 analysis. These two NGS tests are offered to all patients, and the blood test is performed even if no tissue is available. The results from the molecular analyses are regularly reviewed by a Molecular Advisory Board (MAB). The MAB, based on its joint experience in clinical oncology, genomics, bioethics, and pathology, may add some advice to these reports via DT, making comments about detected molecular alterations and adding further recommendations for specific treatment options or available CT with targeted therapies and/or additional genetic tests such as germline validation of potentially significant findings. From that moment, patients are requested to record their disease evolution in the DT every 3 months for 2 years. The primary objective is to assess the real-world clinical practice integrating molecular profiling in the Standard of Care management of patients with mBC connected through a DT. Secondary objectives include to i) describe the genetic mutational profile of mBC, ii) estimate the enrollment rate in CT of patients engaged in a patient-centered strategy for molecular tumor assessment, iii) assessing Progression Free Survival, Overall Survival and Quality of Life status among patients enrolled in CT according to the tumor’s genomic profile and iv) evaluate the logistic feasibility of the study. Recruitment started on October 2020. By June 2021, 362 patients had been enrolled. ACKNOWLEDGEMENTS: This study is sponsored by SOLTI and financially supported by Novartis and three non-profit organizations: Asociación Cáncer de Mama Metastásico, Asociación Saray and Fundación Actitud frente al Cáncer. Roche and Guardant Health provide their tests for all patients. Citation Format: Ana Casas, Eva Ciruelos, Mafalda Oliveira, Cristina Saura, Meritxell Bellet, Sonia Pernas, Joaquín Gavilá, Montserrat Muñoz, Maria Vidal, Blanca González-Farré, Juan M. Cejalvo, Rafael López, Ana Vivancos, Marcos Malumbres, Javier Salvador Bofill, Isabel Blancas, Emilio Alba, Valentina Boni, Susana De la Cruz, Elena Galve, Antonia Perelló, Mireia Margelí, Meritxell Soler, Rubén Olivera-Salguero, Helena Masanas, Rosa Olmos, Marga Forns, Pilar Fernández Pascual, Elia Seguí, Tomas Pascual, Aleix Prat. Solti-1903 HOPE: Real-world clinical practice study to assess the impact of using genomic data on the next treatment decision making-choice in patients with locally advanced or metastatic breast cancer in Spain [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 OT2-06-01.

Background: Baseline HER3 protein or ERBB3 mRNA levels do not seem to predict efficacy from HER3-DXd in early-stage and advanced HR+/HER2- breast cancer (Prat et al. ESMO Breast 2022; Krop et al. ASCO 2022). Here, we evaluated potential baseline pre-treatment genetic determinants of efficacy to HER3-DXd. Methods: SOLTI TOT-HER3 (NCT04610528) is a window of opportunity, multicenter, pre-operative trial which enrolled, in part A, 77 evaluable patients with untreated HR+/HER2- operable (≥1 cm) breast cancer. Patients received a single dose of HER3-DXd (6.4 mg/kg). The primary objective was to evaluate the CelTIL score variation between pre- and post-treatment (day 21) samples. CelTIL combines % of tumor cellularity and % of tumor-infiltrating lymphocytes into a single score. DNA and RNA were purified from pre-treatment baseline FFPE tumor samples. Gene expression was evaluated using a custom 67-gene panel on the nCounter. NGS-based DNA-seq was performed using the VHIO-300 panel, which estimates tumor mutational burden (TMB), identifies copy-number aberrations (CNAs) across the entire genome and calls mutational status of >300 genes. From CNA data, 150 previously defined DNA-based signatures (Xia et al. Nat Comm 2019) trained to capture RNA- and protein-based phenotypes such as the PAM50-related biology were evaluated. Associations of each variable with efficacy (i.e., CelTIL relative changes, and tumor cellularity relative changes) were adjusted for multiple-testing (false discovery rate [FDR] < 5%). The area under the ROC Curve (AUC) was used to estimate the discrimination performance of each variable. Results: RNA and DNA data were obtained from 45 (58%) patients. Baseline characteristics in this subset of patients were generally similar to the original TOT-HER3 population. Among 228 variables (single mutation status, single gene expression, PAM50 signatures, TMB, and DNA CNA-based signatures), 139 (61%) were found significantly associated (FDR< 5%) with CelTIL changes at day 21. Among them, TP53 mutations (n=7) were found associated with higher CelTIL response compared to TP53 wild type (71% [95% CI=-5.4-17.8] vs. 24% [95% CI=15.9-55.4], FDR=2.1%). In addition, RNA-based genes tracking Basal-related biology (e.g., CCNE1, AUC=0.71) or immune expression (e.g., PDCD1, AUC=0.73, or CD68, AUC=0.62), together with RNA/DNA-based signatures tracking proliferation and/or basal-related biology (e.g., retinoblastoma loss-of-heterozygozity [RB-LOH], AUC=0.76), were associated with high CelTIL response. Conversely, RNA/DNA-based signatures tracking endocrine sensitivity/Luminal A-related biology (e.g., Scorr_IE_Correlation, AUC=0.76) were associated with low/lack of CelTIL response. PIK3CA somatic mutations (n=14, 31% of cases), and TMB (range 2.2-12.7) were not found associated with CelTIL response. Similar overall results were obtained when relative changes in tumor cellularity (instead of CelTIL) was evaluated as the efficacy endpoint. Conclusions: TP53 mutations, immune-related genes, and DNA/RNA-based phenotypic signatures tracking Basal- or Luminal A-related biology such as the DNA-based RB-LOH score or the endocrine sensitivity score (Scorr_IE_Correlation) are associated with CelTIL changes in response to HER3-DXd in HR+/HER2- breast cancer. Further RNA- and DNA-based analyses will be evaluated. Citation Format: Fara Brasó-Maristany, Claudette Falato, Olga Martínez-Sáez, Juan Miguel Cejalvo, Mireia Margelí, Pablo Tolosa, Francisco Javier Salvador Bofill, Josefina Cruz, Blanca González-Farré, Esther Sanfeliu, Míriam Arumí, Guillermo Villacampa, Eva Ciruelos, Martín Espinosa-Bravo, Yann Izarzuzaga, Patricia Galván, Judit Matito, Sonia Pernas, Anu Santhanagopal, Stephen Esker, Parul Patel, Pang-Dian Fan, Juan Manuel Ferrero-Cafiero, Ana Vivancos, Tomás Pascual, Aleix Prat, Mafalda Oliveira. Genetic determinants of response to patritumab deruxtecan (HER3-DXd) in hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer: a correlative analysis from SOLTI TOT-HER3 trial [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 P5-02-31.

Background: The HER2DX assay is a genomic test in early-stage HER2-positive (HER2+) breast cancer that provides prognostic and predictive information. HER2DX is a supervised learning algorithm incorporating tumor size, nodal staging, and 4 gene expression signature scores (immune/IGG, tumor cell proliferation, luminal differentiation and the expression of the HER2 amplicon). Among them, the IGG signature is associated with both overall survival and probability of achieving a pathologic complete response (pCR). Here, we studied the association of percentage (%) of tumor infiltrating lymphocytes (TILs) with HER2DX scores, immune genes and other breast cancer-related genes. Methods: HER2DX and %TILs were evaluated in 670 formalin-fixed paraffin-embedded (FFPE) samples of HER2+ breast cancer, including in 3 clinical studies SHORTHER (n=437), PAMELA (n=86), a cohort of patients treated with anti-HER2 therapy plus chemotherapy at Hospital Clínic of Barcelona (n=147). The %TILs were quantified by histological evaluation with hematoxylin eosin staining according to International TILs Working Group guidelines. The nCounter platform determined the expression of 192 genes and HER2DX scores. Pearson correlations (Cor) and Significance Analysis of Microarrays (SAM) with a false-discovery rate (FDR) < 5% assessed the association between %TILs and the expression of individual genes or HER2DX signature scores. Results: A moderate correlation was observed between %TILs and the immune IGG signature (Cor=0.56, p< 0.001). Of note, 171 (25.52%) cases had low %TILs (< 30%) and high IGG score, while 1 (0.15%) case had high %TILs (≥30%) and low IGG score. The %TILs were significantly associated with the expression of immune genes, ERBB2, IGG signature and HER2 amplicon score, and negatively associated with the expression of luminal genes (i.e., ESR1, PRG and BCL2). An unclear relationship between TILs and proliferation genes was observed. Finally, moderate correlations were observed between %TILs and HER2DX pCR score (Cor=0.48, p< 0.001) and between %TILs and HER2DX risk score (Cor=0.33, p< 0.001). Conclusions: Important differences exist between the %TILs and the HER2DX IGG signature in early-stage HER2+ breast cancer. The %TILs should not be used to predict the HER2DX scores. Biologically, a higher %TILs indirectly capture a higher ERBB2 expression and a lower expression of luminal genes, both associated with response to anti-HER2 treatment. Citation Format: Esther Sanfeliu, Fara Brasó-Maristany, Maria Vittoria Dieci, Mercedes Marín-Aguilera, Blanca González-Farré, Gaia Griguolo, Tomas Pascual, Patricia Galván, Laura Angelats, Oleguer Castillo, Paula Blasco, Valeria Sirenko, Pedro Jares, Joan Antón Puig-Butillé, Laia Paré, Antonio Martínez, Antonio Llombart-Cussac, Javier Cortés, Ana Vivancos, Patricia Villagrasa, Joel S Parker, Charles M Perou, Aleix Prat, PierFranco Conte, Valentina Guarneri. Association between tumor infiltrating lymphocytes (TILs) and the HER2DX assay in early-stage of HER2-positive (HER2+) breast cancer [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 P4-02-30.

BACKGROUND: In early-stage triple negative breast cancer (TNBC), the addition of carboplatin (CBDCA) to neoadjuvant chemotherapy (CT) increases pathologic complete response (pCR) and relapsed-free survival. However, it is unclear whether CBDCA improves overall survival (OS). In addition, the prognostic and/or predictive role of pathogenic germline variants (PGV) in BRCA1/2 genes and other cancer risk in this setting is not fully understood. Here, we assessed the efficacy of (neo)adjuvant CBDCA and the prognostic and predictive role of a panel of 14 genes in patients (pts) with eTNBC. METHODS: This is a retrospective study on 117 pts diagnosed with early-stage TNBC between 2000-2021 at Hospital Clinic of Barcelona. Eighty-one pts (69%) were candidates for PGV testing. Overall, 14 genes (PGV) (BRCA1, BRCA2, PALB2, BRIP1, CHEK2, TP53, ATM, RAD51C, RAD51D, BARD1, MLH1, MSH2, MSH6 and PMS2) were assessed using the TruSight hereditary cancer panel (Illumina MySeq platform) according to local guidelines. Univariable and multivariable logistic regression and Cox regression analyses were performed to identify clinical and molecular predictors of pCR and relapse-free survival (RFS), respectively. Chi-squared or Fisher’s exact tests were used to assess characteristics’ distribution as appropriate. RESULTS: Of 117 pts, 83 (71%) received CT in the neoadjuvant setting and 28 (24%) in the adjuvant setting. CBDCA was added to standard CT in 68 pts (82%) in the neoadjuvant cohort and 7 pts (6%) in the adjuvant cohort. Among pts with germline testing, 32/81 (39%) harbored PGV. BRCA1 was the most frequently mutated gene (18/32, 56%), followed by BRCA2 (5/32, 16%), PALB2 (4/32, 13%), BRIP1 (2/32, 6%), CHEK2, TP53 and PMS2 (3/32, 6%). Percentages of pts receiving CBDCA were similar between patients with and without PGV (14/16,87% and wild type (53/67,79%)(p=0.120). In the neoadjuvant cohort (n=83), CBDCA was the only variable significantly associated with pCR at both univariate (pCR rates of 58.5% with CBCDA and 14.3% without CBCDA; odds ratio [OR]=8.2 [95% CIs 2.0-55.7], p=0.008) and remained statistically significant after adjusting for PGV, tumor size and nodal status (OR=6.9 [95% CIs 1.4-53.0], p=0.028). pCR rates according to PGV are reported in Table 1. In terms of RFS, addition of CBDCA to neoadjuvant therapy (hazard ratio [HR]=0.2 [0.1-0.45], p< 0.001), PGV (HR=0.2 [0.05-0.9], p=0.048), pCR (HR=0.2 [0.1-0.6], p=0.004) and nodal status (HR=5.1 [1.7-15.2], p< 0.003) were significantly associated with RFS in univariate analyses. In a multivariable model, CBDCA remained an independent predictor of improved RFS along with pCR. When the neoadjuvant and adjuvant cohort were pooled together (n=117), platinum-based CT remained significantly associated with better RFS (HR=0.2 [0.14-0.86], p=0.021) regardless of time of administration (i.e., neoadjuvant or adjuvant). With a median follow-up of 5 years, CBCDA use was not found associated with OS (HR=0.7 [0.3-1.8], p= 0.560). CONCLUSIONS: The addition of CBDCA to standard CT was significantly associated with pCR and RFS but not OS, consistent with the phase III data. The benefit in terms of RFS was independent of the presence and the type of pathogenic germline alterations. Table 1. pCR rates according to PGV Citation Format: Adela Rodríguez Hernández, Benedetta Conte, Laia Fernández, Fara Brasó-Maristany, Belén Pastor, Miriam Potrony, Lorena Moreno, Elia Grau, Joan Antón Puig-Butillé, Aurora Sánchez, Blanca González-Farré, Esther Sanfeliu, Olga Martínez-Sáez, Claudette Falato, Maria Vidal, Nuria Chic, Tomás Pascual, Francesco Schettini, Montserrat Muñoz, Francesc Balaguer, Aleix Prat, Barbara Adamo. Efficacy of platinum-based chemotherapy and germline mutational status in early-stage triple-negative breast cancer: a unicenter retrospective analysis with long-term follow-up [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 P6-01-09.

Background Hormone receptor–positive/HER2-negative (HR+/HER2-) breast cancer (BC) is associated with low % of stromal tumor-infiltrating lymphocytes (sTIL) and immune gene expression and poor response to immune checkpoint inhibitors. Evaluating the effect of letrozole and ribociclib (L+R) on the immune microenvironment may suggest new opportunities for immunotherapy-based approaches for HR+/HER2- BCs. Here, we present an exploratory correlative analysis from CORALLEEN, a trial that evaluated the efficacy of L+R (vs. chemotherapy [CHT]) in patients with high-risk PAM50 Luminal B BC (Prat et al. Lancet Oncology. 2022). Methods CORALLEEN is a randomized exploratory study in postmenopausal women with operable stage I-III breast cancer, HR+/HER2- and Luminal B by Prosigna®. Patients were randomized 1:1 to receive either 6 cycles of ribociclib (600mg; 3-weeks-on/1-week-off) plus daily letrozole or CHT: 4 cycles of AC followed by 12 doses of weekly paclitaxel. The primary endpoint was the rate of PAM50 Risk of Relapse (ROR)-low score at surgery in each arm. Samples were prospectively collected at baseline, day 15, and surgery. sTILs score, ki67 IHC and gene expression analysis were determined in all available samples. Complete cell cycle arrest (CCCA) was defined as Ki67≤2.7%. Gene expression profiling by mRNA sequencing (RNAseq) was evaluated. We applied a collection of 194 immune- gene expression signatures (iGES), representing multiple biological pathways and cell types, including. Results 106 patients were randomly assigned to receive neoadjuvant L+R (n=52) or CTH (n=54). Overall, Ki67, sTILs and RNA-seq was available in 95.4%, 96.7% and 83.1% of the samples across the 3 time-points. In terms of cell-cycle inhibition, L+R achieved a significant decrease in Ki67 protein expression and led to higher rates of CCCA at 2 weeks (89.6% vs. 43.2%, p< 0.001) and surgery (45.9% vs 25.5%, P=0.054) compared with CHT. Interestingly, the 11-gene PAM50 proliferative score was significantly lower in tumors with CCAA than in those with non-CCCA (p< 0.001) after L+R, but not after CTH (p = 0.682). In contrast, tumors with CCCA after CHT had a significantly lower rate of tumor cellularity compared to tumors with non-CCAA (p = 0.002). This was not observed in the L+R arm (p=0.141). Compared to baseline, no clear and significant patterns in % of sTILs were observed at week 2 and surgery. However, % of TILs at surgery in tumors with CCCA after CHT was higher than in tumors with non-CCCA (median 15% versus 1%, p=0.017). This was not observed in the L+R arm (median 1% and 5%, p=0.584). Interestingly, this inverse relationship between immune infiltration and CCAA was further confirmed by RNA- CHT compared to tumors with non-CCCA, whereas 174 (89.7%) of iGES were upregulated (FDR< 5%) in tumors with non-CCCA after L+R compared to tumors with CCCA. Finally, L+R and CTH treatment at week 2 and surgery showed an increase in adaptive immune signatures indicative of activated T-cell and B-cell phenotypes; however, CTH was uniquely associated with increased cytokine signaling, enhanced antigen presentation, dendritic, granulocyte, macrophage and NK cells and decrease in Th17, Th2 and Treg cells. Conclusion In early-stage Luminal B breast cancer, L+R induce a potent anti-proliferative effect compared to CHT. Both treatments generally increased T- and B-cell immune infiltration; however, an inverse relationship between immune infiltration and anti-proliferative response at surgery exists according to treatment, where immune infiltration is increased in residual tumors with non-CCAA when treated with L+R, but the opposite is observed with CHT. The prognostic value of immune and anti-proliferative effects of L+R in residual tumors is currently being evaluated in the prospective RIBOLARIS phase II clinical trial (NCT05296746). Citation Format: Tomás Pascual, Nuria Chic, Aranzazu Fernandez-Martinez, Blanca González-Farré, Laia Paré, Cristina Saura, Cristina Hernando, Montserrat Muñoz, Míriam Arumí, Patricia Galván, Xavier Gonzalez-Farré, Mafalda Oliveira, Miguel Gil Gil, Eva Ciruelos, Patricia Villagrasa, Joaquin Gavila Gregori, Aleix Prat, Charles M. Perou. Cell-Cycle Inhibition and Immune Microenvironment in HR+/HER2- Breast Cancer During and After preoperative ribociclib and letrozole vesus chemotherapy: A correlative analysis of the 1402-SOLTI/CORALLEEN phase 2 trial [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 PD17-07.

Background: Different approaches for treatment de-escalation are being investigated; however, the current ongoing phase III adjuvant trials with CDK4/6 inhibitors are not addressing the question if these drugs can replace multi-agent chemotherapy in high-risk early breast cancer. Here, we present the primary results of the CORALLEEN phase 2 trial, which evaluates the efficacy of ribociclib plus endocrine therapy (ET) as neoadjuvant treatment in patients with high-risk Luminal B disease. Methods: CORALLEEN is a parallel, multicenter, two-arm, randomized exploratory study in postmenopausal women with primary operable hormone receptor-positive (HR+)/HER2-negative breast cancer, Luminal B by Prosigna®. Other eligibility criteria include stage I-III operable breast cancer and ECOG 0-1. Patients were randomized 1:1 to receive either six 28-days cycles of ribociclib (600mg; 3-weeks-on/1-week-off) plus daily letrozole (2.5mg) or chemotherapy (CT): 4 cycles of AC (doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2 every 21 days) followed by weekly paclitaxel (80 mg/m2) during 12 weeks. Baseline, Day 15 on-treatment, and surgical specimens were collected for molecular characterization and evaluation of response. The primary endpoint is the rate of PAM50 Risk of Relapse (ROR)-low disease at surgery in each arm. PAM50 ROR score integrates gene expression data, tumor size, and nodal status to define a low-risk group in the adjuvant setting (i.e. >90% distant relapse-free survival at 10 years). ROR-low was defined using the standard cutpoints as Citation Format: Joaquín Gavilá, Cristina Saura, Tomás Pascual, Cristina Hernando, Montserrat Muñoz, Laia Paré, Blanca González Farré, Pedro Fernandez, Patricia Galván, Xavier González Farré, Mafalda Oliveira, Miguel Gil Gil, Miriam Arumi, Neus Ferrer Tur, Alvaro Montaño, Yan Izarzugaza, Antonio Llombart Cussac, Raquel Bratos, Santiago González, Eduardo Martínez, Sergio Hoyos, Beatriz Rojas, Juan Antonio Virizuela, Vanesa Ortega, Rafael López, Pamela Céliz, Eva Ciruelos, Patricia Villagrasa, Aleix Prat. Primary results of SOLTI-1402/CORALLEEN phase 2 trial of neoadjuvant ribociclib plus letrozole versus chemotherapy in PAM50 Luminal B early breast cancer: An open-label, multicenter, two-arm, randomized study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS2-06.

Background: CDK4/6 inhibitors increase tumor immunogenicity in preclinical models of breast cancer and several trials combining CDK4/6 inhibitors and anti-PD1/PDL1 therapies are underway. However, immune response data in tumor samples from patients (pts) treated with CDK4/6 inhibitors are scarce. Here, we present exploratory results of the CORALLEEN trial, which evaluated the efficacy of ribociclib and endocrine therapy in patients with high-risk Luminal B disease (Gavilá et al. submitted to SABCS 2019). Methods: CORALLEEN is a randomized exploratory study in postmenopausal women with operable stage I-III breast cancer, hormone receptor positive (HR+)/HER2-negative and Luminal B by Prosigna®. Pts were randomized 1:1 to receive either 6 cycles of ribociclib (600mg; 3-weeks-on/1-week-off) plus daily letrozole (R+L) or chemotherapy (CHT): 4 cycles of AC followed by 12 doses of weekly paclitaxel. The primary endpoint is the rate of PAM50 Risk of Relapse (ROR)-low disease at surgery in each arm. Baseline and surgical specimens were also collected for stromal tumor infiltrating lymphocyte (TIL) determination and gene expression analysis. Expression of 770 genes and 31 biological signatures were determined using the Breast360TM panel (nCounter). In order to identify genes whose expression correlated with TIL, a significance of microarrays (SAM) quantitative analysis was used with a false discovery rate (FDR) Citation Format: Nuria Chic, Blanca González Farré, Laia Paré, Tomás Pascual, Cristina Saura, Cristina Hernando, Montserrat Muñoz, Pedro Fernandez, Patricia Galván, Xavier González Farré, Mafalda Oliveira, Miguel Gil Gil, Pamela Céliz, Eva Ciruelos, Patricia Villagrasa, Joaquín Gavilá, Aleix Prat. Immune response following neoadjuvant ribociclib plus letrozole versus chemotherapy in PAM50 Luminal B early breast cancer: A correlative analysis of the SOLTI-1402 CORALLEEN phase 2 randomized trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-11-01.

Summary Background In hormone receptor-positive, HER2-negative early stage breast cancer, cyclin-dependent kinases 4 and 6 (CDK4/6) inhibition in combination with endocrine therapy could represent an alternative to multiagent chemotherapy. We aimed to evaluate the biological and clinical activity of neoadjuvant ribociclib plus letrozole in the luminal B subtype of early stage breast cancer. Methods CORALLEEN is a parallel-arm, multicentre, randomised, open-label, phase 2 trial completed across 21 hospitals in Spain. We recruited postmenopausal women (≥18 years) with stage I–IIIA hormone receptor-positive, Eastern Cooperative Oncology Group Performance Status 0–1, HER2-negative breast cancer and luminal B by PAM50 with histologically confirmed, operable primary tumour size of at least 2 cm in diameter as measured by MRI. Patients were randomly assigned (1:1) using a web-based system and permuted blocks of 25 to receive either six 28-days cycles of ribociclib (oral 600 mg once daily for 3 weeks on, 1 week off) plus daily letrozole (oral 2·5 mg/day) or four cycles of doxorubicin (intravenous 60 mg/m2) and cyclophosphamide (intravenous 600 mg/m2) every 21 days followed by weekly paclitaxel (intravenous 80 mg/m2) for 12 weeks. The total duration of the neoadjuvant therapy was 24 weeks. Randomisation was stratified by tumour size and nodal involvement. Samples were prospectively collected at baseline (day 0), day 15, and surgery. The primary endpoint was to evaluate the proportion of patients with PAM50 low-risk-of-relapse (ROR) disease at surgery in the modified intention-to-treat population including all randomly assigned patients who received study drug and had a baseline and at least one post-baseline measurement of ROR score. The PAM50 ROR risk class integrated gene expression data, tumour size, and nodal status to define prognosis. This trial was registered at ClinicalTrials.gov , NCT03248427 . Findings Between July 27, 2017 to Dec 7, 2018, 106 patients were enrolled. At baseline, of the 106 patients, 92 (87%) patients had high ROR disease (44 [85%] of 52 in the ribociclib and letrozole group and 48 [89%] of 54 in the chemotherapy group) and 14 (13%) patients had intermediate-ROR disease (eight [15%] and six [11%]). Median follow-up was 200·0 days (IQR 191·2–206·0). At surgery, 23 (46·9%; 95% CI 32·5–61·7) of 49 patients in the ribociclib plus letrozole group and 24 (46·1%; 32·9–61·5) of 52 patients in the chemotherapy group were low-ROR. The most common grade 3–4 adverse events in the ribociclib plus letrozole group were neutropenia (22 [43%] of 51 patients) and elevated alanine aminotransferase concentrations (ten [20%]). The most common grade 3–4 adverse events in the chemotherapy group were neutropenia (31 [60%] of 52 patients) and febrile neutropenia (seven [13%]). No deaths were observed during the study in either group. Interpretation Our results suggest that some patients with high-risk, early stage, hormone receptor-positive, HER2-negative breast cancer could achieve molecular downstaging of their disease with CDK4/6 inhibitor and endocrine therapy. Funding Novartis, Nanostring, Breast Cancer Research Foundation-AACR Career Development Award.

Background: CDK4/6 inhibition combined with anti-HER2 therapy is currently being explored in HER2-positive (HER2+) breast cancer (BC). Here, we report the final efficacy and genomic analysis of cohort A and B of the PATRICIA phase II trial evaluating palbociclib in combination with trastuzumab in advanced HER2+ BC. Methods: PATRICIA is a prospective, open-label, multicenter phase II trial. Patients had received 2-4 prior lines of anti-HER2-based regimens. Treatment consisted of palbociclib 200 mg daily for 2 weeks and 1 week off plus trastuzumab. The study was based on a Simon two-stage design comprising three cohorts: estrogen receptor (ER)-negative (cohort A), ER-positive (cohort B1), and ER-positive with letrozole (cohort B2). Patients with ER-positive tumors were randomized to cohorts B1 or B2. Primary endpoint was progression-free survival (PFS) rate at 6 months (PFS6). Secondary objectives included PFS, overall survival (OS) and the association of the research-based PAM50 intrinsic subtyping with PFS and OS. PAM50 was performed from FFPE samples using the nCounter platform. For each sample we calculated the PAM50 signature scores (Basal-like, HER2-E, Luminal A and B, Normal-like), CES, ROR-Subtype, ROR-proliferation and the proliferation signature score. Multivariable Cox regression analyses evaluating PAM50 subtypes, age, performance status, treatment line, type of biopsy, and ER status. Results: Seventy-one patients were recruited (n = 15 in cohort A and 28 in each cohort B). Median follow-up was 42.3 months (IQR 34.7-54.8). The PFS6 rate in cohorts A, B1, and B2 was 33.3% (5/15), 42.8% (12/28), and 46.4% (13/28), respectively. Median PFS was 4.2 months (95% CI 0.7-6.7) in cohort A, 6.0 months (95% CI 4.0-10.6) in cohort B1 and 5.1 months (95% CI 3.7-9.1) in cohort B2. Regarding PAM50, 59 (83.1%) tumors samples (42.4% metastasis) were profiled. 49.2% of the tumor samples were identified as HER2-E, followed by Luminal B (22.0%), Luminal A (16.9%), normal-like (10.2%), and Basal-like (1.7%). Luminal disease defined by PAM50 was independently associated with longer PFS compared with non-luminal disease (10.6 vs. 4.2 months median PFS; adjusted hazard ratio [HR] = 0.34; P = 0.007). Median OS was 21.8 months (95% CI 13.8-32.2) in cohort A, 28.0 months (95% CI 14.2-48.8) in cohort B1 and 34.3 months (95% CI 20.6-47.6) in cohort B2. Luminal disease defined by PAM50 was not independently associated with OS compared with non-luminal disease (34.3 vs. 26.1 months; adjusted HR = 0.753; P = 0.365). Among the 9 PAM50 signatures, expression of 3 signatures were found significantly associated with OS: CES (HR = 0.50; p=0.021), Luminal A score (HR=0.33; p=0.022) and ROR-S (HR=1.018; p=0.027). Conclusion: Our analysis shows that the promising PFS previously reported in trastuzumab pretreated ER-positive/HER2+ advanced breast cancer with a PAM50 Luminal A or B subtype were maintained after a median of >3 years of follow-up. A longer OS was seen in patients with luminal tumors, but results were not statistically significant and could have been influenced by the low sample size. Cohort C of PATRICIA is currently randomizing patients with HR-positive/HER2+, PAM50 Luminal A or B tumors to palbociclib and endocrine therapy plus trastuzumab or treatment of physician’s choice (NCT02448420). Acknowledgements: This study is sponsored by SOLTI and financially supported by Pfizer Citation Format: Eva Ciruelos, Tomás Pascual, Mafalda Oliveira, Santiago Escrivá-de-Romaní, Sonia Pernas, Laia Paré, Barbara Adamo, Eduardo Martínez, Javier Cortés, Antonia Perelló, Maria Galan, Mireia Melé, Pablo Tolosa, Blanca González-Farré, Patricia Galván, Jordi Canes, Paolo Nuciforo, Xavier Gonzalez, Patricia Villagrasa, Aleix Prat. Palbociclib and trastuzumab for HER2-positive metastatic breast cancer (SOLTI-1303 PATRICIA): Final results from cohort A and B, prospective, open-label, multicenter phase II 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 PD8-03.

BACKGROUND: Metastatic breast cancer (mBC) remains an incurable disease and is the cause of nearly all deaths from breast cancer. Targeted molecular therapies and the evolving role of next-generation sequencing (NGS) technologies are increasing and may improve outcomes in breast cancer patients. However, they are not being routinely used in the clinic. One strategy to overcome the barriers of implementing NGS in the clinic is to promote the active participation of mBC patients in the management of their disease. With this in mind, we designed HOPE (SOLTI-1903), a national real-world study where patients lead their inclusion, participation and follow-up in the study through a digital tool that will guide them in every step of the journey. Our objective is empowering mBC patients and gather real-world data about the utilization of molecular information in the management of mBC. TRIAL DESIGN: Patients diagnosed with mBC who are receiving, have just received, or will receive standard treatment or treatment in a clinical trial (CT) can be included. Demographic data, disease characteristics, treatment history and quality of life data will be collected through a digital tool (DT) by the patient. Patients are encouraged to involve their physician`s in the study journey. The study is complemented by a patient empowerment program including informative workshops and precision medicine video-tutorials. A total of 600 patients will be included in Spain. Patient Journey Once patients request to participate in HOPE through the DT, a dedicated team from SOLTI will assist them in the following steps while validating that eligibility criteria are met according to data introduced by themselves. Then, patients will receive instructions via DT to go to the nearest partner local laboratory, where they will sign the study consent form. A metastatic (preferably) or primary archival tumor sample will be requested to the patient’s reference hospital and analyzed by FoundationOne®CDx. Also, a blood sample will be collected and analyzed by Guardant360. For all patients, two NGS tests will be offered (If no tissue available, only blood test will be performed). Both molecular analyses results will be reviewed during regular meetings by a Molecular Advisory Board (MAB). The MAB, based on their joint experience in clinical oncology, genomics, molecular biology, bioethics and pathology, may add some advice to these reports via DT, making comments about detected molecular alterations and adding further recommendations for specific treatment options or available CT with targeted therapies. From that moment, patients will record their disease evolution in the DT each 3 months for 2 years. The primary objective is to assess the real-world clinical practice integrating molecular profiling in the Standard of Care management of mBC patients connected through a DT. Secondary objectives include to describe genetic mutational profile of mBC, to evaluate the enrollment rate in CT of patients engaged in a patient-centered strategy for molecular tumor assessment, to asses Progression Free Survival, Overall Survival and Quality of life status among patients enrolled in CT according to the tumor’s genomic profile and those receiving standard treatment and to describe logistic feasibility of the study. This study is sponsored by SOLTI and financially supported by Novartis and two non-profit organizations: Asociación Cáncer de Mama Metastásico y Fundación Actitud frente al Cáncer. Roche and Guardant Health provide their test for all patients. Citation Format: Ana Casas, Eva Ciruelos, Mafalda Oliveira, Cristina Saura, Meritxell Bellet, Sonia Pernas, Joaquín Gavilá, Montserrat Muñoz, María Vidal, Blanca Gonzalez Farré, Juan M. Cejalvo, Rafael Lopez, Ana Vivancos, Maurizio Scaltriti, Javier S. Bofill, Isabel Blancas, Emilio Alba, Valentina Boni, Susana De la Cruz, Elena Galve, Antonia Perelló, Mireia Margelí, Jordi Canes, Pamela Céliz, Helena Masanas, Rosa Olmos, Marga Fonts, Pilar Fernandez-Pascual, Patricia Villagrasa, Aleix Prat. Solti-1903 hope: Real-world clinical practice study to assess the impact of using comprehensive genomic data on the next treatment decision making-choice in patients with locally advanced or metastatic breast cancer in Spain [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-17-01.