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Background Today, there is no clear therapeutic algorithm for patients with metastatic HER2-positive (HER2+) breast cancer (BC) who have progressed to trastuzumab, pertuzumab, tyrosine kinase inhibitors and antibody-drug conjugates (ADC). Among the emerging strategies, the use of immune checkpoint inhibitors in combination therapy is showing promising clinical benefit in the advanced setting of HER2+ BC by overcoming immune resistance and enhancing antitumor cellular immunity. The intrinsic subtypes Basal-like and HER2-enriched (i.e PAM50 non-luminal tumors) represent approximately the 60% of HER2+ BC and are associated with higher expression of immune-related genes, tumor-infiltrating lymphocytes (TILs) presence and high tumor mutational burden (TMB), compared to luminal subtypes. Additionally, immune infiltration and TMB in HER2+ BC are associated with chemo/antiHER2 responsiveness and with potential benefit from anti-PD-1/PD-L1 inhibitors. We hypothesize that combining atezolizumab with trastuzumab and vinorelbine may improve outcomes in HR- or PAM50 non-luminal/HR-positive (HR+) disease within HER2+ MBC. Methods ATREZZO is an open-label, single-arm, Simon 2-stage, multicenter phase II study. The trial will include 55 pre- or post-menopausal female or male patients with unresectable locally advanced or metastatic HR- or PAM50 non-luminal/HR+ HER2+ BC and progressed to trastuzumab-based chemotherapy and anti-HER2 ADC. Prior pertuzumab is allowed, but not required. Treatment consists of atezolizumab IV 1200 mg every 3 weeks combined with trastuzumab and vinorelbine. Patients with stable, progressing, or untreated brain metastasis not requiring immediate local therapy are eligible. The primary objective is to evaluate the Overall Response Rate (ORR) according to RECIST v 1.1 and secondary endpoints include ORR in patients with PD-L1 positive breast cancer, clinical benefit rate, overall survival and progression-free survival. The final recruited population will contain no more than 60 % of patients with PD-L1 negative tumors. Tumor assessments will be performed every 9 weeks. Incidence, duration and severity of adverse events, and further correlative molecular analyses will be also evaluated. An interim analysis will be conducted when 19 patients are evaluable for ORR and if the number of responses is ≥ 3, 36 additional patients will be included. As of July 15th, 2022, 48 patients were screened and 15 were included in sixteen Spanish sites. This study was funded by Roche Farma SA. Trial identification: NCT04759248 Citation Format: Eva Ciruelos, Antonia Perelló, Santiago González-Santiago, Ana López, Francisco Javier Salvador Bofill, Cinta Albacar, Juan Miguel Cejalvo, Santiago Escrivá-de-Romani, Isabel Blancas, Sonia Pernas, Olga Martínez-Sáez, Josefina Cruz, Jose Ponce, Sonia Servitja, Maria-Eva Perez-Lopez, Juan A Guerra, Esther Sanfeliu, Cesar A Rodríguez, Guillermo Villacampa, Lorea Villanueva, Pablo Tolosa, Tomás Pascual, Aleix Prat. SOLTI-1907 ATREZZO: Targeting hormonal receptor negative (HR-) or PAM50 non-luminal disease with atezolizumab in combination with trastuzumab and vinorelbine in HER2-positive metastatic breast cancer (MBC) [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 OT2-08-02.

Background: Residual disease (RD) following neoadjuvant chemotherapy (NAC) in early HER2-negative breast cancer (BC) remains an unmet medical need. However, no therapies to date have tested their activity directly in chemo-resistant RD. Here, we hypothesized that combining an oncolytic virus such as T-VEC with atezolizumab may offer clinical benefit in patients (pts) with RD after standard NAC. To our knowledge, PROMETEO is the first trial that examines the activity of immunotherapy in pts with RD prior to surgery. Methods: PROMETEO (NCT03802604) is a single-arm, open-label, multicenter phase II trial. Women with triple-negative BC (TNBC) or hormone receptor-positive/HER2-negative (HR+/HER2-) BC with baseline (i.e., before NAC) ki67 ≥ 20% were eligible. RD was confirmed with a magnetic resonance imaging (MRI) showing a tumor diameter ≥ 10 mm and a core-biopsy detecting the presence of invasive cells. Before surgery, T-VEC was administered intratumorally on week 1 (106 pfu/mL), then in week 4 and every 2 weeks thereafter (108 pfu/mL) for 4 injections. Atezolizumab (840 mg) was administered intravenously every 2 weeks for 4 infusions, starting at week 4. Surgery was performed in < 3 weeks after completing the treatment. The primary objective was to evaluate the efficacy of the combination, measured by the rate of residual cancer burden (RCB) class 0/1 at surgery. Tumor samples collected at 5 timepoints (before NAC, during screening period, after first dose of T-VEC, after first dose of T-VEC and atezolizumab and at surgery) were mandatory to assess gene expression, tumor-infiltrating lymphocytes (TILs), immune cells PD-L1 IHC (SP142), tumor mutational burden (TMB) by FoundationOne and other translational endpoints. Results: Between Dec 2018 to Feb 2022, 28 pts were enrolled: 20 pts with HR+/HER2- disease and 8 pts with TNBC. Median age was 47 (range 31-71) and 71% of pts were premenopausal. At diagnosis before NAC, clinical stage II disease represented 60.7%, cN+ 60.7%, median Ki-67 was 37.5% (range 20%-95%), high TILs (≥10%) 37%, median TMB was 3 (0-19) and only 1 of 27 pts (3.7%) had a PD-L1-positive tumor. After NAC, mean tumor size by MRI was 28.3 mm (10-93). Two pts discontinued from the trial (1 withdrawal of consent and 1 COVID infection). The completion of 5 cycles of treatment was achieved by 73% of pts. The overall RCB-0/1 rate was 25% (7 of 28, 95% IC 10.7 – 44.9%), all with RCB 0 (pathologic complete response [pCR]). The pCR rate was 30% in HR+/HER2- disease and 12.5 % in TNBC. Radiological response by MRI was achieved by 3 of 28 pts (10.7%). Interestingly, none of the 7 pts with a pCR had radiological response (stable disease n=5, progressive disease [PD] n=2). Six pts (21.4%) had radiological PD and had RCB 2/3. Overall, 27 (96%) patients had at least one treatment-emergent adverse event (TEAE) of any grade. Most common grade 1 or 2 AEs were fever (11 pts, 39.3%), ALT increased (9 pts, 32.1%), AST increased (8 pts, 28.6%), arthralgia (6 pts, 21.4%) and anemia (6 pts, 21.4%). Grade 3 reversible neutropenia occurred in 1 patient. Across all pts, significant increases (p< 0.001) in TILs, immune genes and immune PDL1+ cells were observed after 1 dose of TVEC, 1 dose of the combination and at surgery. Intrinsic subtype changes at surgery occurred in 73.1% of cases, mostly (46.1%) Luminal A/B converting to Normal-like. At surgery, 19 of 26 (73.1%) of tumors were PDL1+. Conclusions: Two months of T-VEC in combination with atezolizumab induced a pCR in a subgroup of pts with chemoresistant HER2- breast cancer. This effect is probably related to the immune activation provoked by the combined treatment. Interestingly, a high discrepancy was observed between the pre-surgical radiological imaging and the actual surgical pathological report. Pre-operative window-of-opportunity trials in this context might provide important clues regarding the activity of novel treatment strategies. Citation Format: Tomás Pascual, Maria Vidal, Juan Miguel Cejalvo, Estela Vega, Esther Sanfeliu, Sergi Ganau, Ana Julve, Esther Zamora, Ignacio Miranda, Ana Delgado, Begoña Bermejo, Luis de la Cruz-Merino, Manel Juan, Patricia Galván, Xavier Gonzalez-Farré, Juan Manuel Ferrero-Cafiero, Patricia Villagrasa, Mafalda Oliveira, Aleix Prat. PD11-04 Primary results of SOLTI-1503 PROMETEO phase 2 trial of Combination of Talimogene Laherparepvec (T-VEC) with Atezolizumab in patients with residual breast cancer after standard neoadjuvant multi-agent chemotherapy [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 PD11-04.

Background Trastuzumab deruxtecan (T-DXd) has shown durable antitumor activity in pretreated patients with HER2-positive advanced breast cancer (ABC), but its efficacy has not yet been evaluated in patients with active brain metastases (BMs). DEBBRAH aims to assess T-DXd in patients with HER2-positive or HER2-low ABC and central nervous system involvement. Methods This ongoing, five-cohort, phase II study (NCT04420598) enrolled patients with pretreated HER2-positive or HER2-low ABC with stable, untreated, or progressing BMs, and/or leptomeningeal carcinomatosis. Here, we report findings from HER2-positive ABC patients with non-progressing BMs after local therapy (n = 8; cohort 1), asymptomatic untreated BMs (n = 4; cohort 2), or progressing BMs after local therapy (n = 9; cohort 3). Patients received 5.4 mg/kg T-DXd intravenously once every 21 days. The primary endpoint was 16-week progression-free survival (PFS) for cohort 1 and intracranial objective response rate (ORR-IC) for cohorts 2 and 3. Results As of October 20, 2021, 21 patients received T-DXd. In cohort 1, 16-week PFS rate was 87.5% (95%CI, 47.3-99.7; P < .001). ORR-IC was 50.0% (95%CI, 6.7-93.2) in cohort 2 and 44.4% (95%CI, 13.7-78.8; P < .001) in cohort 3. Overall, the ORR-IC in patients with active BMs was 46.2% (95%CI, 19.2-74.9). Among patients with measurable intracranial or extracranial lesions at baseline, the ORR was 66.7% (12 out of 18 patients; 95%CI, 41.0-86.7), 80.0% (95%CI, 28.4-99.5) in cohort 1, 50.0% (95%CI, 6.7-93.2) in cohort 2, and 66.7% (95%CI, 29.9-92.5) in cohort 3. All responders had partial responses. The most common adverse events included fatigue (52.4%; 4.8% grade ≥3), nausea (42.9%; 0% grade ≥3), neutropenia (28.6%; 19% grade ≥3), and constipation (28.6%; 0% grade ≥3). Two (9.5%) patients suffered grade 1 interstitial lung disease/pneumonitis. Conclusions T-DXd showed intracranial activity with manageable toxicity and maintained the quality of life in pretreated HER2-positive ABC patients with stable, untreated, or progressing BMs. Further studies are needed to validate these results in larger cohorts.

Breast cancer; Heterodimerization Cáncer de mama; Heterodimerización Càncer de mama; Heterodimerització Anti-HER2 therapies have markedly improved prognosis of HER2-positive breast cancer. However, different mechanisms play a role in treatment resistance. Here, we identified AXL overexpression as an essential mechanism of trastuzumab resistance. AXL orchestrates epithelial-to-mesenchymal transition and heterodimerizes with HER2, leading to activation of PI3K/AKT and MAPK pathways in a ligand-independent manner. Genetic depletion and pharmacological inhibition of AXL restored trastuzumab response in vitro and in vivo. AXL inhibitor plus trastuzumab achieved complete regression in trastuzumab-resistant patient-derived xenograft models. Moreover, AXL expression in HER2-positive primary tumors was able to predict prognosis. Data from the PAMELA trial showed a change in AXL expression during neoadjuvant dual HER2 blockade, supporting its role in resistance. Therefore, our study highlights the importance of targeting AXL in combination with anti-HER2 drugs across HER2-amplified breast cancer patients with high AXL expression. Furthermore, it unveils the potential value of AXL as a druggable prognostic biomarker in HER2-positive breast cancer. A.A.-A., E.J.A., and F.B.-M. were supported by Asociación Española contra el Cáncer AECC (PRDVA18013LLUC to A.A.-A., POSTD211413AREN to E.J.A., and AECC_Postdoctoral17-1062 to F.B.-M.). A.M.-S. was funded by the Spanish Government (PFIS FI20/00188). J.Ar. is supported by Breast Cancer Research Foundation (BCRF-20-08), Instituto de Salud Carlos III Project reference number AC15/00062, and the EC under the framework of the ERA-NET TRANSCAN-2 initiative cofinanced by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181), and Asociación Española Contra el Cáncer (AECC). A.P. was supported by Instituto de Salud Carlos III—PI19/01846, Breast Cancer Now—2018NOVPCC1294. P.E. and A.L. were funded by Instituto de Salud Carlos III and cofinanced by FEDER (PI18/01219 to P.E. and CB16/12/00481 to A.L.). J.M.C. was funded by Sociedad Española de Oncología Médica (Rio Hortega-SEOM) and Compromiso ADAMED.

Supplementary Figure from mTOR Inhibition and T-DM1 in HER2-Positive Breast Cancer

Supplementary Figure S5 shows a forest plot of the exploratory categorical analysis of selected (meta)gene expression and their association with overall survival on the capecitabine arm vs. observation.

Purpose:Predictive biomarkers for capecitabine benefit in triple-negative breast cancer (TNBC) have been recently proposed using samples from phase III clinical trials, including non-basal phenotype and biomarkers related to angiogenesis, stroma, and capecitabine activation genes. We aimed to validate these findings on the larger phase III GEICAM/CIBOMA clinical trial.Experimental Design:Tumor tissues from patients with TNBC randomized to standard (neo)adjuvant chemotherapy followed by capecitabine versus observation were analyzed using a 164-gene NanoString custom nCounter codeset measuring mRNA expression. A prespecified statistical plan sought to verify the predictive capacity of PAM50 non-basal molecular subtype and tested the hypotheses that breast tumors with increased expression of (meta)genes for cytotoxic cells, mast cells, endothelial cells, PDL2, and 38 individual genes benefit from adjuvant capecitabine for distant recurrence-free survival (DRFS; primary endpoint) and overall survival.Results:Of the 876 women enrolled in the GEICAM/CIBOMA trial, 658 (75%) were evaluable for analysis (337 with capecitabine and 321 without). Of these cases, 553 (84%) were profiled as PAM50 basal-like whereas 105 (16%) were PAM50 non-basal. Non-basal subtype was the most significant predictor for capecitabine benefit [HRcapecitabine, 0.19; 95% confidence interval (CI), 0.07–0.54; P < 0.001] when compared with PAM50 basal-like (HRcapecitabine, 0.9; 95% CI, 0.63–1.28; P = 0.55; PinteractionP value = 0.01). Analysis of biological processes related to PAM50 non-basal subtype revealed its enrichment for mast cells, extracellular matrix, angiogenesis, and features of mesenchymal stem-like TNBC subtype.Conclusions:In this prespecified correlative analysis of the GEICAM/CIBOMA trial, PAM50 non-basal status identified patients with early-stage TNBC most likely to benefit from capecitabine.

Supplementary Table S1 shows the gene list for the NanoString custom nCounter codeset included in the study. Supplementary Table S2 shows patient characteristics in the GEICAM/CIBOMA translational study cohort versus the original trial. Supplementary Table S3 shows the distribution of PAM50 intrinsic subtypes in the GEICAM/CIBOMA translational study cohort. Supplementary Table S4 shows the differential gene expression analysis performed on the 164 genes included in the custom codeset comparing PAM50 non-basal vs. basal-like subtype and their biological processes. Supplementary Table S5 shows multivariate survival analysis and interaction tests for the four genes and metagenes included in the prespecified hypotheses testing their association with DFS. Supplementary Table S6 shows multivariate survival analysis and interaction tests for the continuous expression of the 38 individual genes included in the prespecified hypotheses testing their association with DRFS. Supplementary Table S7 shows multivariate survival analysis and interaction tests for the continuous expression of the 38 individual genes included in the prespecified hypotheses testing their association with OS. Supplementary Table S8 shows multivariate survival analysis and interaction tests for the continuous expression of the 38 individual genes included in the prespecified hypotheses testing their association with DFS. Supplementary Table S9 shows multivariate survival analysis and interaction tests restricted to the PAM50 non-basal subgroup. Supplementary Table S10 shows Exploratory analysis for the prognostic capacity of the continuous expression of genes and metagenes restricted to the PAM50 non-basal subgroup.

Supplementary Data S1 shows exploratory analysis for the predictive capacity of the continuous expression of additional genes and metagenes included in the codeset. Supplementary Data S2 shows exploratory analysis for the prognostic capacity of the continuous expression of the selected genes and metagenes

Primers obtained from PrimerBank database.

NRF2 expression analysed by Western Blot after subcellular fractionation.

Purpose:Despite the clinical advantage of the combination of trastuzumab and platinum-based chemotherapy in HER2-amplified tumors, resistance will eventually develop. The identification of molecular mechanisms related to primary and acquired resistance is needed.Experimental Design:We generated lapatinib- and trastuzumab-resistant clones deriving from two different HER2-amplified gastric cancer cell lines. Molecular changes such as protein expression and gene-expression profile were evaluated to detect alterations that could be related to resistance. Functional studies in vitro were corroborated in vivo. The translational relevance of our findings was verified in a patient cohort.Results:We found RPS6 activation and NRF2 to be related to anti-HER2 drug resistance. RPS6 or NRF2 inhibition with siRNA reduced viability and resistance to anti-HER2 drugs. In knockdown cells for RPS6, a decrease of NRF2 expression was demonstrated, suggesting a potential link between these two proteins. The use of a PI3K/TORC1/TORC2 inhibitor, tested in vitro and in vivo, inhibited pRPS6 and NRF2 expression and caused cell and tumor growth reduction, in anti–HER2-resistant models. In a cohort of HER2-amplified patients treated with trastuzumab and chemotherapy, a high level of NRF2 at baseline corresponds with worse progression-free survival.Conclusions:NRF2 through the PI3K/AKT/mTOR/RPS6 pathway could be a potential effector of resistance to anti-HER2 drugs in our models. RPS6 inhibition decreases NRF2 expression and restores sensitivity in HER2-amplified gastric cancer in vitro and in vivo. High NRF2 expression in gastric cancer patients predicts resistance to treatment. RPS6 and NRF2 inhibition could prevent resistance to anti-HER2 drugs.

Purpose: Predictive biomarkers for capecitabine benefit in triple-negative breast cancer (TNBC) have been recently proposed using samples from phase III clinical trials, including non-basal phenotype and biomarkers related to angiogenesis, stroma, and capecitabine activation genes. We aimed to validate these findings on the larger phase III GEICAM/CIBOMA clinical trial. Experimental Design: Tumor tissues from patients with TNBC randomized to standard (neo)adjuvant chemotherapy followed by capecitabine versus observation were analyzed using a 164-gene NanoString custom nCounter codeset measuring mRNA expression. A prespecified statistical plan sought to verify the predictive capacity of PAM50 non-basal molecular subtype and tested the hypotheses that breast tumors with increased expression of (meta)genes for cytotoxic cells, mast cells, endothelial cells, PDL2, and 38 individual genes benefit from adjuvant capecitabine for distant recurrence-free survival (DRFS; primary endpoint) and overall survival. Results: Of the 876 women enrolled in the GEICAM/CIBOMA trial, 658 (75%) were evaluable for analysis (337 with capecitabine and 321 without). Of these cases, 553 (84%) were profiled as PAM50 basal-like whereas 105 (16%) were PAM50 non-basal. Non-basal subtype was the most significant predictor for capecitabine benefit [HRcapecitabine, 0.19; 95% confidence interval (CI), 0.07–0.54; P < 0.001] when compared with PAM50 basal-like (HRcapecitabine, 0.9; 95% CI, 0.63–1.28; P = 0.55; Pinteraction Conclusions: In this prespecified correlative analysis of the GEICAM/CIBOMA trial, PAM50 non-basal status identified patients with early-stage TNBC most likely to benefit from capecitabine.

Improved selection of cancer patients who are most likely to respond to immune checkpoint inhibitors remains an unmet clinical need. Recently, a positive correlation between levels of

Background: A previous phase 2 study in metastatic breast cancer compared treatment with intravenously delivered oncolytic reovirus, pelareorep (pela), in combination with paclitaxel (PTX) versus PTX alone. This study demonstrated a statistically significant improvement in overall survival (OS), without differences in objective response or progression-free survival. We hypothesized that the OS benefit from pela + PTX may be attributed to an adaptive immune response triggered by pela. To test this hypothesis, and examine if pela can mediate the priming of an anti-tumor immune response, we designed a study called AWARE-1 (A window-of-opportunity study of pela in Early Breast Cancer), which is currently enrolling and for which initial translational research results are presented. Methods: AWARE-1 is evaluating the safety and effect of pela ± atezolizumab on the tumor microenvironment (TME) in 38 women with early breast cancer. Patients are treated with pela on days 1, 2, 8, and 9, while atezolizumab is administered on day 3. Tumor biopsies are collected at diagnosis, day 3, and day ~21. Five cohorts will be examined: Cohort 1: Hormone Receptor-positive/HER2-negative (HR+/HER2-neg) (10 patients), pelareorep + letrozole. Cohort 2: HR+/HER2-neg (10 patients), pelareorep + letrozole + atezolizumab. Cohort 3: Triple Negative Breast Cancer (TNBC) (6 patients), pelareorep + atezolizumab. Cohort 4: Hormone Receptor-positive/HER2-positive (HR+/HER2+) (6 patients), pelareorep + trastuzumab + atezolizumab. Cohort 5: Hormone Receptor-negative/HER2-positive (HR-/HER2+) (6 patients), pelareorep + trastuzumab + atezolizumab. The primary endpoint of the study is CelTIL score, a metric for quantifying the changes in tumor cellularity and infiltration of TILs, where an increase in CelTIL is associated with a favorable response to treatment. Tumor tissue was examined for pela replication, and changes to the TME were assessed by imaging mass cytometry (IMC), immunohistochemistry, and T cell receptor sequencing (TCR-seq). Peripheral blood was also examined by TCR-seq. Results: Detailed translational research results will be presented from patients in cohort 1, who received just pelareorep and letrozole. CelTIL score increased in 5/10 patients at day 3 biopsies and 6/10 patients at day 21 biopsies. Preliminary results show high levels of viral replication (>50% of tumor cells) while immunohistochemistry and IMC analysis revealed changes to the TME, with increases in CD8+ T cells and upregulation of PD-L1 at both day 3 and day 21 biopsies. Overall, preliminary data from cohort 1 of AWARE-1 demonstrate pela-mediated priming of an adaptive immune response. (NCT04102618) Citation Format: Luis Manso, Patricia Villagrasa, Nuria Chic, Begoña Bermejo, Juan Miguel Cejalvo, Yann Izarzugaza, Blanca Cantos, Salvador Blanch, Mireia Margeli, Jose Luis Alonso, Alejandro Martínez, Rafael Villanueva, Juan Antonio Guerra, Raquel Andrés, Pilar Zamora, Esteban Nogales, Manel Juan, Blanca González, Rita Laeufle, Gerard Nuovo, Grey Wilkinson, Matt Coffey, Azucena González, Débora Martínez, Laia Paré, Fernando Salvador, Xavier Gonzalez, Aleix Prat, Joaquín Gavilá. A window-of-opportunity study with atezolizumab and the oncolytic virus pelareorep in early breast cancer (REO-027, AWARE-1) [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 PS12-08.

Background: Breast cancer (BC) is one of the most serious health problems worldwide. The implementation of screening programs as well as advances in diagnosis and therapeutics have achieved an increase in survival. However, there are patients who do not benefit from screening programs: very young or older than 69 years old patients and interval cancers.The main aim of this breast cancer fast-track program (BCFP) is to reduce the time from the onset of breast cancer signs and symptoms until the beginning of treatment. · Methods: The programme began in June 2009; we hereby present data from the first ten years (June 2009-June 2019) in the Clinico-Malvarrosa Health Department in Valencia, Spain. Breast surgeons, primary care (PC) physicians, radiologists who are experts in breast cancer and oncology coordinators regularly met to discuss suspected cases of BC, and initially set guidelines with the criteria to be used by PC physicians in order to refer patients to BCFP. On the same day that the PC physician identified a patient with suspected BC, an index card was sent to the oncology coordinator, who reviewed these cases and referred those meeting previously defined criteria to either breast surgeons or breast radiologists. · Results: 1849 proposals were sent to the hospital from which 1778 patients came to the first visit at the specialist. 312 (16.9%) referred cases were diagnosed with BC. 206 (66%) women were diagnosed of BC in the age range in which screening with mammography is not recommended in our general population (between 45-69 years old). 296 patients had a localized BC (94%) and 16 (6%) an advanced BC. The median time from submission of a proposal until the specialist assessment was 13 days for diagnosed patients, 2 days from the first visit to histopathological diagnosis, and 29 days from the histopathological diagnosis until a treatment is initiated (either oncological or surgical). It took a median of 18 days to confirm the absence of BC in 1466 patients with initial suspicion of it. • Conclusions: Our data show that the time interval between patient referral by the PC physician to the specialist, diagnosis of breast cancer, and start of therapy can be reduced. We optimized existing resources with no additional costs associated with the implementation of this programme. Keywords: Breast Cancer, Clinical Guidelines, Early diagnosis Authors: M.T. Martinez1*, S. Moragon1*, S. Simón1, B. Ortega1, J.V. Montón1, J. Navarro2, A. Sanmartin2, A. Julve3, E. Buch4, O. Burgues5, C. Hernando1, J. M. Cejalvo1, A. Lluch1, B. Bermejo1, A. Cervantes1, I. Chirivella1.1Medical Oncology Department, INCLIVA Biomedical Research Institute, Valencia, Spain, 2Management department, Hospital Clinico Universitario de Valencia, Valencia, Spain3 Radiodiagnosis Department, Hospital Clinico Universitario de Valencia, Valencia, Spain4 Senology Department, Hospital Clinico Universitario de Valencia, Valencia, Spain5 Pathological Anatomy Department. Hospital Clinico Universitario de Valencia, Valencia, Spain Citation Format: Maria Teresa Martínez, Santiago Moragon, Soraya Simon, Belen Ortega, Jose Vicente Monton-Bueno, Jorge Navarro, Ana Sanmartin, Ana Julve, Elvira Buch, Octavio Burgués, Cristina Hernando, Juan Miguel Cejalvo, Ana Lluch, Begoña Bermejo, Andrés Cervantes, Isabel Chirivella. Breast cancer fast-track programme to shorten time between initial symptoms, diagnosis and initiation of treatment. 10 years update [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 PS7-71.

BACKGROUND: MicroRNAs have emerged as new diagnostic and therapeutic biomarkers for breast cancer. Herein, we analyzed miR-99a-5p expression levels in primary tumours and plasma of breast cancer patients to evaluate its usefulness as a minimally invasive diagnostic biomarker. METHODS: MiR-99a-5p expression levels were determined by quantitative real-time PCR in three indepent cohorts of patients: I) Discovery cohort: breast cancer tissues (n=103) and healthy breast tissues (n=26). II) Testing cohort: plasma samples from 105 patients and 98 healthy donors. III) Validation cohort: plasma samples from 89 patients and 85 healthy donors. Receiver operating characteristic (ROC) curve analyses were applied to evaluate the diagnostic potential of miR-99a-5p expression levels in tissue and plasma samples. RESULTS: MiR-99a-5p was significantly downregulated in breast cancer tissues compared to healthy breast tissues (p < 0.0001), being able to discriminate BC from healthy breast tissues with an AUC of 0.85, 87.38% sensitivity, 76.92% specificity, and 85.27% accuracy. Conversely, miR-99a-5p levels were significantly higher in breast cancer patients than in healthy controls in plasma samples from both testing and validation cohorts (p < 0.0001). ROC curve analysis revealed that miR-99a-5p has good diagnostic potential, with an AUC of 0.76, 63.81% sensitivity, 79.59% specificity, and 71.43% accuracy. The value of circulating miR-99a-5p levels as a breast cancer biomarker was further validated in an independent cohort, where it was able to identify breast cancer with 57.30% sensitivity, 67.06% specificity, and 62.07% accuracy. Besides, we also confirmed that circulating miR-99a-5p levels were able to discriminate early breast cancer from healthy controls with a 66.67% accuracy, 68.80% sensitivity, 65.28% specificity, and an AUC of 0.69 (p < 0.0001). CONCLUSION: MiR-99a-5p’s deregulated expression distinguished healthy individuals from breast cancer patients in two different types of samples (tissues and plasma). Interestingly, plasma expression levels were significantly lower in healthy controls than in early-stage breast cancer patients. Our findings suggest that circulating miR-99a-5p as a novel promising non-invasive biomarker for breast cancer detection. Citation Format: Iris Garrido-Cano, Vera Constâncio, Anna Adam-Artigues, Ana Lameirinhas, Soraya Simón, Belen Ortega, María Teresa Martínez, Cristina Hernando, Begoña Bermejo, Ana Lluch, Paula Lopes, Rui Henrique, Carmen Jerónimo, Juan Miguel Cejalvo, Pilar Eroles. Circulating miR-99a-5p expression in plasma: A potential biomarker for early diagnosis of breast cancer [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 PS2-31.

Mechanisms driving tumor progression from less aggressive subtypes to more aggressive states represent key targets for therapy. We identified a subset of luminal A primary breast tumors that give rise to HER2-enriched (HER2E) subtype metastases, but remain clinically HER2 negative (cHER2(–)). By testing the unique genetic and transcriptomic features of these cases, we developed the hypothesis that FGFR4 likely participates in this subtype switching. To evaluate this, we developed 2 FGFR4 genomic signatures using a patient-derived xenograft (PDX) model treated with an FGFR4 inhibitor, which inhibited PDX growth in vivo. Bulk tumor gene expression analysis and single-cell RNA sequencing demonstrated that the inhibition of FGFR4 signaling caused molecular switching. In the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohort, FGFR4-induced and FGFR4-repressed signatures each predicted overall survival. Additionally, the FGFR4-induced signature was an independent prognostic factor beyond subtype and stage. Supervised analysis of 77 primary tumors with paired metastases revealed that the FGFR4-induced signature was significantly higher in luminal/ER(+) tumor metastases compared with their primaries. Finally, multivariate analysis demonstrated that the FGFR4-induced signature also predicted site-specific metastasis for lung, liver, and brain, but not for bone or lymph nodes. These data identify a link between FGFR4-regulated genes and metastasis, suggesting treatment options for FGFR4-positive patients, whose high expression is not caused by mutation or amplification.

New treatment strategies such as immune checkpoint inhibitors and oncolytic viruses are opening new possibilities in cancer therapy. Preliminary results in melanoma and other tumors showed that the combination of talimogene laherparepvec with an anti-PD-1/PD-L1 or anti-CTLA4 has greater efficacy than either therapy alone, without additional safety concerns beyond those expected for each agent. The presence of residual cancer after neoadjuvant chemotherapy in early breast cancer patients is an unmet medical need. SOLTI-1503 PROMETEO is a window of opportunity trial, which evaluates the combination of talimogene laherparepvec in combination with atezolizumab in women with operable HER2-negative breast cancer who present residual disease after neoadjuvant chemotherapy. The primary end point is the rate of residual cancer burden 0/1.Clinical Trial Registration: NCT03802604

Understanding the biological aspects of immune response in HER2+ breast cancer is crucial to implementing new treatment strategies in these patients. It is well known that anti-HER2 therapy has improved survival in this population, yet a substantial percentage may relapse, creating a need within the scientific community to uncover resistance mechanisms and determine how to overcome them. This systematic review indicates the immunological mechanisms through which trastuzumab and other agents target cancer cells, also outlining the main trials studying immune checkpoint blockade. Finally, we report on anti-HER2 vaccines and include a figure exemplifying their mechanisms of action.

Trastuzumab treatment has significantly improved the prognosis of HER2-positive breast cancer patients. Despite this, resistance to therapy still remains the main clinical challenge. In order to evaluate the implication of microRNAs in the trastuzumab response, we performed a microRNA array in parental and acquired trastuzumab-resistant HER2-positive breast cancer cell lines. Our results identified miR-146a-5p as the main dysregulated microRNA. Interestingly, high miR-146a-5p expression in primary tumor tissue significantly correlated with shorter disease-free survival in HER2-positive breast cancer patients. The gain- and loss-of-function of miR-146a-5p modulated the response to trastuzumab. Furthermore, the overexpression of miR-146a-5p increased migration and angiogenesis, and promoted cell cycle progression by reducing CDKN1A expression. Exosomes from trastuzumab-resistant cells showed a high level of miR-146a-5p expression compared with the parental cells. In addition, the co-culture with resistant cells’ exosomes was able to decrease in sensitivity and increase the migration capacities in trastuzumab-sensitive cells, as well as angiogenesis in HUVEC-2 cells. Collectively, these data support the role of miR-146a-5p in resistance to trastuzumab, and demonstrate that it can be transferred by exosomes conferring resistance properties to other cells.

Background: The antibody-drug conjugate trastuzumab deruxtecan (T-DXd) significantly improved survival outcomes of HER2-low advanced breast cancer (ABC) patients (pts) compared to standard chemotherapy in the DESTINY-Breast04 trial. DEBBRAH is assessing the efficacy and safety of T-DXd in HER2[+] and HER2-low ABC pts with a history of brain metastases (BM) and/or leptomeningeal carcinomatosis (LMC); here, we report results of HER-low ABC pts. Methods: DEBBRAH (NCT04420598) is a multicenter, open-label, five-cohort, non-comparative, phase 2 study across 18 hospitals in 2 countries. A total of 39 pts aged ≥18 years with pretreated HER2[+] or HER2-low ABC with stable, progressing, or untreated BM and/or LMC, were enrolled in 5 cohorts: (1) HER2[+] ABC with non-progressing BM after radiotherapy and/or surgery; (2) HER2[+] or HER2-low ABC with asymptomatic untreated BM; (3) HER2[+] ABC with progressing BM after local treatment; (4) HER2-low ABC with progressing BM after local treatment; (5) HER2[+] or HER2-low ABC with LMC. Pts received 5.4 mg/kg T-DXd intravenously once every 21 days until disease progression, unacceptable toxicity, or consent withdrawal. Primary endpoint for cohorts 2 and 4 was intracranial overall response rate (ORR-IC) according to RANO-BM. Secondary endpoints included overall response (ORR) according to RECIST v1.1, progression-free survival (PFS), duration of response (DoR), clinical benefit rate (CBR); and safety and tolerability as per NCI-CTCAE v.5.0. Primary analysis is the estimation of ORR-IC (H0: ORR-IC ≤5%; H1: ORR-IC ≥40%) based on the one-sided binomial exact test. Sample size was planned to attain an 80% power at a nominal α level of 0.05 in each cohort. Results from cohort 2 should be considered descriptive since formal testing has to be performed in the whole cohort of pts with HER2[+] or HER2-low ABC and asymptomatic untreated BM. Results: From Oct 23, 2020, through Feb 15, 2022, 6 pts and 7 pts were allocated into cohorts 2 and 4, respectively. One patient with LMC included in cohort 4 was excluded from analysis. Median age was 54 (range 40–73) years. Median number of previous lines of therapy for advanced disease was 7 (range, 4-8) and 3 (range, 2-4) for cohorts 2 and 4, respectively. Median follow-up was 9.5 months (range, 1.6-15.7). At data cutoff (Apr 29, 2022), no patient of cohort 2 and 3 (50.0%) pts of cohort 4 remained on therapy. In cohort 2, ORR-IC was 66.7% (4 of 6 pts had intracranial partial response [PR]; 95% CI, 22.3–95.7). In cohort 4, ORR-IC was 33.3% meeting the primary endpoint (2 of 6 pts had intracranial PR; 95% CI, 4.3–77.7; P = .033). Overall, ORR-IC in all pts was 50% (6 of 12 pts; 95% CI, 21.1-78.9) and CBR was 66.7% (8 of 12 pts; 95% CI, 34.9-90.1). Combining pts with measurable intracranial or extracranial disease from cohorts 2 and 4, ORR, CBR and median DoR were 41.7% (5 of 12 pts; 95% CI, 15.2–72.3), 50.0% (6 of 12 pts; 95% CI, 21.1–78.9), and 7.2 months (95% CI, 2.5-16.4), respectively. Median PFS was 5.7 months (95% CI, 4.7-NA) among these pts. The most common treatment emergent adverse events (TEAEs) of any grade (G) were fatigue (58.3%; 8.3% G≥3) and nausea (50.0%; 0% G≥3). Two (16.7%; 0% G≥3) cases of interstitial lung disease/pneumonitis were reported. Serious unrelated TEAEs occurred in 2 (16.7%) of 12 pts; 1 case of general pain (G3) and 1 case of venous embolism (G5) that led to death. There were no drug-related deaths due to TEAEs. Conclusions: T-DXd showed a preliminary antitumor activity in pretreated HER2-low ABC pts with asymptomatic untreated or progressing BM after local treatment. The substantial response of BM to T-DXd in this setting is promising and warrants further investigation. Citation Format: José Manuel Pérez-García, Marta Vaz Batista, Patricia Cortez-Castedo, Manuel Ruiz Borrego, Juan Miguel Cejalvo, Juan de la Haba-Rodríguez, Laia Garrigós, Fabriccio Racca, Sonia Servitja, Salvador Blanch, Maria Gión, Mónica Nave, Adela Fernández, Alejandro Martínez-Bueno, Antonio Llombart-Cussac, Miguel Sampayo-Cordero, Andrea Malfettone, Javier Cortés, Sofía Braga. Trastuzumab Deruxtecan in patients with Unstable Central Nervous System Involvement from HER2-Low Advanced Breast Cancer: The DEBBRAH 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 PD7-02.

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: CD166 is broadly expressed in normal epithelium and overexpressed in many types of malignancies, including breast cancer. Probody® therapeutic candidates are masked antibodies, conditionally activated by tumor-associated proteases, which restricts their activity to the tumor microenvironment and minimizes ‘off-tumor’ toxicity. CX-2009 is a conditionally activated humanized anti-CD166 monoclonal antibody conjugated to DM4 that showed clinical activity in ABC patients in a phase 1 study (Boni et al. Clin Cancer Res. 2022). This phase 2 study (NCT04596150) evaluates CX-2009 as monotherapy in patients with advanced HR+/HER2− BC (Arm A) and TNBC (Arm B), and in combination with pacmilimab (a conditionally activated PD-L1) in TNBC (Arm C). Methods: Key eligibility criteria for all cohorts include: ECOG 0-1, acceptable end-organ function, measurable disease, willingness to receive ocular prophylaxis for DM-4 related toxicity, and available tumor tissue for CD166 evaluation. Eligibility criteria for HR+ BC include: 2-4 prior regimens (excluding single-agent hormonal therapy with up to 2 prior cytotoxic regimens) and a prior CDK 4/6 inhibitor in the metastatic setting; eligibility criteria for TNBC include CD166 by IHC >1% by central assessment, 1-3 prior regimens in the metastatic setting and prior taxane. All patients initially received 7 mg/kg Q3W; the protocol was subsequently amended to enroll patients at 6 mg/kg Q3W. The primary endpoint was overall response rate (ORR) using RECIST v1.1 assessed by central review. Other key endpoints include ORR by investigator assessment, clinical benefit rate at 24 weeks (CBR24; defined as any response, confirmed or unconfirmed, or SD for 24 weeks), duration of response, and progression-free survival by investigator. Archival tumor specimens and blood samples were collected for correlative research including genomic analyses. Results: As of 13 May 2022, 60 patients were enrolled in Arm A (all patients started at 7 mg/kg); 52 were evaluable for efficacy by investigator. Median duration of follow-up was 29.1 weeks (range: 3.6-60.7). Median age was 60.5 years (36, 83); pts received a median of 3.5 (1, 6) prior treatments for ABC. CD166 H-Score > 200 was reported in 53.3% of patients. Arm A met the primary efficacy endpoint with a confirmed ORR by central radiology of 14.9% (n=47); by investigator, ORR was similar at 15.4% (n=52); an additional 9 patients (17.3%) had an unconfirmed response. CBR24 was 40.4%; using only confirmed responses, CBR24 was 23.1%. Median PFS was 11.4 weeks (95% CI 9.0, 13.9). Common treatment-related all-grade adverse events (TRAEs) included blurred vision (42%), nausea (35%), fatigue (35%), diarrhea (25%), peripheral neuropathy (27%), infusion-related reaction (23%) and decreased appetite (20%). Grade ≥3 ocular and neuropathic TRAEs were 15% and 10%, respectively. AEs resulting in treatment discontinuation (AEDC) were 25%. For Arm B and C, 55 and 10 patients were enrolled (the majority received a starting dose of 6 mg/kg). For Arm B, the futility boundary was crossed (ORR < 10%). Grade ≥3 ocular and neuropathic TRAEs and AEDC at 7 mg/kg in Arm B were similar to Arm A (11%, 11% and 21% respectively); whereas at 6 mg/kg, they were reduced at 3%, 0% and 0%, respectively. Biomarker data and correlation with outcomes will be presented. Conclusions: Praluzatamab ravtansine demonstrated single-agent activity in unselected heavily pretreated patients with HR+/HER2- ABC. Time to event analyses, such as PFS, were confounded by higher-than-expected toxicity at a starting dose of 7 mg/kg. The toxicity profile was generally consistent with a DM4 payload. The lower dose of 6 mg/kg appears to be better tolerated. Additional clinical studies in HR+ABC, incorporating a starting dose of 6 mg/kg and potentially including a biomarker strategy, are warranted. Citation Format: Kathy Miller, Sara Tolaney, Leisha A. Emens, Sung-Bae Kim, Erika Hamilton, Cristina Saura, Lucia Sanz, Valentina Boni, Filipa Lynce, Juan Miguel Cejalvo, Jennifer Crozier, Shirley Wang, Hirdesh Uppal, Alison L. Hannah, Sara Hurvitz. Preliminary results from a phase 2 study of praluzatamab ravtansine (CX-2009) in patients with advanced breast cancer (ABC) [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 P4-01-15.

Improved selection of cancer patients who are most likely to respond to immune checkpoint inhibitors remains an unmet clinical need. Recently, a positive correlation between levels of PD1 mRNA and clinical outcome in response to PD1 blockade across diverse tumor histologies has been confirmed in several datasets. ACROPOLI is a parallel cohort, non-randomized, phase II study that aims to evaluate the efficacy of the anti-PD1 immune checkpoint inhibitor spartalizumab as monotherapy in metastatic patients with solid tumors that express high levels of PD1 (cohort 1; n = 111). An additional cohort of 30 patients with tumors expressing low levels of PD1, where PD1/PD-L1 antibodies in monotherapy are standard treatment, will also be included (cohort 2). Primary end point is overall response rate in cohort 1. Considering previous data, we hypothesized that anti-PD1 monotherapy is effective across multiple cancer types in patients with PD1-high mRNA expressing tumors. Given these observations, ACROPOLI is a phase II study designed to study spartalizumab in this population. This article describes the SOLTI-1904 ACROPOLI phase II trial, `Efficacy of spartalizumab across multiple cancertypes in patients with PD1-highmRNA expressing tumors defined by a single and pre-specified cutoff.' The primary objective is to determine overall response rate (ORR) in the PD1-high population. Secondary objectives include other measures of clinical efficacy and evaluation of safety and tolerability. Spartalizumab has shown to have a manageable safety profile and preliminary clinical efficacy according to several phase I/II clinical trials [1-3].

In patients with trastuzumab-resistant HER2-positive breast cancer, the combination of everolimus (mTORC1 inhibitor) with trastuzumab failed to show a clinically significant benefit. However, the combination of mTOR inhibition and the antibody-drug conjugate (ADC) trastuzumab-emtansine (T-DM1) remains unexplored. We tested T-DM1 plus everolimus in a broad panel of HER2-positive breast cancer cell lines. The combination was superior to T-DM1 alone in four cell lines (HCC1954, SKBR3, EFM192A, and MDA-MB-36) and in two cultures from primary tumor cells derived from HER2-positive patient-derived xenografts (PDX), but not in BT474 cells. In the trastuzumab-resistant HCC1954 cell line, we characterized the effects of the combination using TAK-228 (mTORC1 and -2 inhibitor) and knockdown of the different mTOR complex components. T-DM1 did not affect mTOR downstream signaling nor induct autophagy. Importantly, mTOR inhibition increased intracellular T-DM1levels, leading to increased lysosomal accumulation of the compound. The increased efficacy of mTOR inhibition plus T-DM1 was abrogated by lysosome inhibitors (chloroquine and bafilomycin A1). Our experiments suggest that BT474 are less sensitive to T-DM1 due to lack of optimal lysosomal processing and intrinsic resistance to the DM1 moiety. Finally, we performed several in vivo experiments that corroborated the superior activity of T-DM1 and everolimus in HCC1954 and PDXderived mouse models. In summary, everolimus in combination with T-DM1 showed strong antitumor effects in HER2-positive breast cancer, both in vitro and in vivo. This effect might be related, at least partially, to mTOR-dependent lysosomal processing of T-DM1, a finding that might apply to other ADCs that require lysosomal processing., This work was supported by ISCIII (CIBERONC CB16/12/00481, CB16/12/00241, PI18/00382, PI18/00006, PI18/01219), Generalitat de Catalunya (2017 SGR 507). MINECO through gBFU2015-71371-R grant and the CRIS Cancer Foundation supported work in AP lab. D. Casadevall was supported by ISCIII (Rio Hortega Research Contract CM16/00023 and Juan Rodés Research Contract JR18/00003). F.J. Sánchez-Martín and S. Menéndez were supported by Department de Salut Generalitat de Catalunya (PERIS SLT002/16/00008 and PERIS SLT006/17/00040). M. Qin received financial support from the China Scholarship Council (CSC) for her doctoral fellowship. Work carried out in our laboratories receives support from the European Community through the Regional Development Funding Program (FEDER).

Due to the lack of specific targets, cytotoxic chemotherapy still represents the common standard treatment for triple-negative breast patients. Despite the harmful effect of chemotherapy on tumor cells, there is evidence that treatment could modulate the tumor microenvironment in a way favoring the propagation of the tumor. In addition, the lymphangiogenesis process and its factors could be involved in this counter-therapeutic event. In our study, we have evaluated the expression of the main lymphangiogenic receptor VEGFR3 in two triple-negative breast cancer in vitro models, resistant or not to doxorubicin treatment. The expression of the receptor, at mRNA and protein levels, was higher in doxorubicin-resistant cells than in parental cells. In addition, we confirmed the upregulation of VEGFR3 levels after a short treatment with doxorubicin. Furthermore, VEGFR3 silencing reduced cell proliferation and migration capacities in both cell lines. Interestingly, high VEGFR3 expression was significantly positively correlated with worse survival in patients treated with chemotherapy. Furthermore, we have found that patients with high expression of VEGFR3 present shorter relapse-free survival than patients with low levels of the receptor. In conclusion, elevated VEGFR3 levels correlate with poor survival in patients and with reduced doxorubicin treatment efficacy in vitro. Our results suggest that the levels of this receptor could be a potential marker of meager doxorubicin response. Consequently, our results suggest that the combination of chemotherapy and VEGFR3 blockage could be a potentially useful therapeutic strategy for the treatment of triple-negative breast cancer.

Background: PIK3CA is the most frequently mutated oncogene in breast cancer (BC). These mutations are considered drivers, indeed, several novel PI3K inhibitors are under evaluation in clinical trials. Recently, alpelisib, a PI3Kα inhibitor, combined with hormonotherapy has improved progression-free survival in mutated advanced luminal BC. As a result, alpelisib has been FDA-approved for this setting. In some tumors, double PIK3CA mutations in cis demonstrated increasing oncogenicity and sensitivity to PI3Kα inhibitors compared to those tumors with single-hotspot mutations. We aim at assessing the proportion and distribution of PIK3CA double mutations of a monocentric genomic screening program to select patients for trials with experimental targeted agents. Methods: We assessed PIK3CA mutation in a cohort of 345 consecutive metastatic BC patients diagnosed at Hospital Clínico València-INCLIVA from Jan-13 to Apr-20. Molecular screening of hotspot mutations was performed in primary (36.2%, 125/345) and metastatic tissue (63.8%, 220/345), with either MassArray or Next-Generation Sequencing. Hotspots were selected according to public published databases. To be considered mutant, tumors needed to harbor at least 5% of mutant alleles. Our cohort is enriched in luminal BC, according to clinical trials developing PI3K inhibitors. Results: Patients with PIK3CA mutations represented 34.8% (120/345) of the whole cohort. The distribution of BC subtypes was: 80.5% Luminal, 12.0% HER2, and 7.5% triple-negative. The most frequent metastatic site was liver (32.8%) followed by nodes (21.4%), and bone (11.4%). We detected 27 different hotspots, of which three comprised 73% of all PIK3CA mutations: H1047R (32%), E545K (27%), and E542K (14%). Others hotspots were: E545D (4%), H1047L (3%), M1043I (2%), N345K (2%), C420R (1%), E545A (1%), R88Q (1%). Furthermore, 12% PIK3CA mutations were unique ( In our series, 19 patients (15.8%) of all mutant tumors presented a double PIK3CA mutation. In almost all double-PIK3CA mutated tumors (89%, 17/19), one of the hotspots was either a helical or kinase domain (E542, E545, and H1047). Interestingly, E545D was never detected among tumors with a single-mutations. Conclusions: In our cohort, a wide spectrum of PIK3CA mutations was found in patients with metastatic breast cancer, suggesting the clinical relevance of molecular screening. Double PIK3CA mutations were identified in 15.8% of mutant tumors and maybe related to higher sensitivity to PI3K inhibitors. E545D mutation was always associated with a second mutation suggesting a different biological behavior. Citation Format: Juan Miguel Cejalvo, Santiago Moragón, Valentina Gambardella, Belén Ortega, Cristina Hernando, Maria Teresa Martinez, Noelia Tarazona, Octavio Burgués, Soraya Simón, Jesús Poveda, Alba Viala, Jorge Martín, Pilar Rentero-Garrido, Sheila Zúñiga-Trejos, Ana Lluch, Begoña Bermejo, Desamparados Roda, Andrés Cervantes. Frequency and spectrum of doublePIK3CAsomatic mutations in metastatic breast cancer patients [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 PS18-20.

Purpose: Despite the clinical advantage of the combination of trastuzumab and platinum-based chemotherapy in HER2-amplified tumors, resistance will eventually develop. The identification of molecular mechanisms related to primary and acquired resistance is needed. Experimental Design: We generated lapatinib- and trastuzumab-resistant clones deriving from two different HER2-amplified gastric cancer cell lines. Molecular changes such as protein expression and gene-expression profile were evaluated to detect alterations that could be related to resistance. Functional studies in vitro were corroborated in vivo. The translational relevance of our findings was verified in a patient cohort. Results: We found RPS6 activation and NRF2 to be related to anti-HER2 drug resistance. RPS6 or NRF2 inhibition with siRNA reduced viability and resistance to anti-HER2 drugs. In knockdown cells for RPS6, a decrease of NRF2 expression was demonstrated, suggesting a potential link between these two proteins. The use of a PI3K/TORC1/TORC2 inhibitor, tested in vitro and in vivo, inhibited pRPS6 and NRF2 expression and caused cell and tumor growth reduction, in anti–HER2-resistant models. In a cohort of HER2-amplified patients treated with trastuzumab and chemotherapy, a high level of NRF2 at baseline corresponds with worse progression-free survival. Conclusions: NRF2 through the PI3K/AKT/mTOR/RPS6 pathway could be a potential effector of resistance to anti-HER2 drugs in our models. RPS6 inhibition decreases NRF2 expression and restores sensitivity in HER2-amplified gastric cancer in vitro and in vivo. High NRF2 expression in gastric cancer patients predicts resistance to treatment. RPS6 and NRF2 inhibition could prevent resistance to anti-HER2 drugs.

Estrogen receptor-positive (ER+) is the most common subtype of breast cancer. Endocrine therapy is the fundamental treatment against this entity, by directly or indirectly modifying estrogen production. Recent advances in novel compounds, such as cyclin-dependent kinase 4/6 inhibitors (CDK4/6i), or phosphoinositide 3-kinase (PI3K) inhibitors have improved progression-free survival and overall survival in these patients. However, some patients still develop endocrine resistance after or during endocrine treatment. Different underlying mechanisms have been identified as responsible for endocrine treatment resistance, where ESR1 gene mutations are one of the most studied, outstanding from others such as somatic alterations, microenvironment involvement and epigenetic changes. In this scenario, selective estrogen receptor degraders/downregulators (SERD) are one of the weapons currently in research and development against aromatase inhibitor- or tamoxifen-resistance. The first SERD to be developed and approved for ER+ breast cancer was fulvestrant, demonstrating also interesting activity in ESR1 mutated patients in the second line treatment setting. Recent investigational advances have allowed the development of new oral bioavailable SERDs. This review describes the evolution and ongoing studies in SERDs and new molecules against ER, with the hope that these novel drugs may improve our patients’ future landscape.

Circulating microRNAs have emerged as potential diagnostic biomarkers. The deregulation of the microRNA miR-99a-5p has been previously described as an effective biomarker of early breast cancer. Herein, we present a new nanoporous anodic alumina (NAA)-based biosensor that can detect plasma miR-99a-5p with high sensitivity and selectivity. NAA pores are loaded with rhodamine B and capped with a specific oligonucleotide that is able to block cargo release until the target is present. In the presence of miR-99a-5p, the capping oligonucleotide recognizes the miR-99a-5p sequence and displaces it allowing the release of the encapsulated dye. This method is able to successfully distinguish healthy controls from breast cancer patients, even at early stages with high efficiency, showing the presented system as a promising tool for breast cancer detection.

Ligand-dependent corepressor (LCOR) mediates normal and malignant breast stem cell differentiation. Cancer stem cells (CSCs) generate phenotypic heterogeneity and drive therapy resistance, yet their role in immunotherapy is poorly understood. Here we show that immune-checkpoint blockade (ICB) therapy selects for LCORlow CSCs with reduced antigen processing/presentation machinery (APM) driving immune escape and ICB resistance in triple-negative breast cancer (TNBC). We unveil an unexpected function of LCOR as a master transcriptional activator of APM genes binding to IFN-stimulated response elements (ISREs) in an IFN signaling-independent manner. Through genetic modification of LCOR expression, we demonstrate its central role in modulation of tumor immunogenicity and ICB responsiveness. In TNBC, LCOR associates with ICB clinical response. Importantly, extracellular vesicle (EV) Lcor-messenger RNA therapy in combination with anti-PD-L1 overcame resistance and eradicated breast cancer metastasis in preclinical models. Collectively, these data support LCOR as a promising target for enhancement of ICB efficacy in TNBC, by boosting of tumor APM independently of IFN. This work was supported by the Instituto de Salud Carlos III-FSE (nos. MS17/00037 and PI18/00014) and the Cancer Research Institute, Clinic and Laboratory Integration Program (grant no. CRI2477 to T.C.-T). We also thank the AECC LAB (grant no. LABAE19007CELI) and the FERO foundation (to T.C.-T). This work was also supported by ISCIII (CIBERONC nos. CB16/12/00481, CB16/12/00241, PI18/00006 and PI21/00002), Generalitat de Catalunya (no. 2017 SGR 507) and the European Community through the Regional Development Funding Program to J. Albanell. Y.K. is supported by the Brewster Foundation, the Breast Cancer Research Foundation, the Susan G. Komen Foundation and the American Cancer Society. J. Arribas is funded by the Breast Cancer Research Foundation (no. BCRF-20-08), Instituto de Salud Carlos III (no. PI19/01181), Asociación Española Contra el Cáncer (no. GCAEC19017ARRI) and Fundación BBVA (no. CAIMI VHIO-FBBVA 2018-2021). M.M.A. is funded by MICINN and the Spanish Government (no. PGC2018-094091-B-I00). D.C. was funded by Instituto de Salud Carlos III (no. JR1800003). S.M. is funded by PERIS (no. SLT006/17/00040). The wotk of R.R.G. is funded by MICINN, the Spanish Government (no. PID2019-104948RB-I00) and the BBVA Foundation. S.B and J.P. acknowledge support from the Spanish Ministry of Science, the EMBL partnership and the CESO and CERCA Program.

Introduction Molecular-matched therapies have revolutionized cancer treatment. We evaluated the improvement in clinical outcomes of applying an in-house customized Next Generation Sequencing panel in a single institution. Methods Patients with advanced solid tumors were molecularly selected to receive a molecular-matched treatment into early phase clinical trials versus best investigators choice, according to the evaluation of a multidisciplinary molecular tumor board. The primary endpoint was progression-free survival (PFS) assessed by the ratio of patients presenting 1.3-fold longer PFS on matched therapy (PFS2) than with prior therapy (PFS1). Results Of a total of 231 molecularly screened patients, 87 were eligible for analysis. Patients who received matched therapy had a higher median PFS2 (6.47 months; 95% CI, 2.24–14.43) compared to those who received standard therapy (2.76 months; 95% CI, 2.14–3.91, Log-rank p = 0.022). The proportion of patients with a PFS2/PFS1 ratio over 1.3 was significantly higher in the experimental arm (0.33 vs 0.08; p = 0.008). Discussion We demonstrate the pivotal role of the institutional molecular tumor board in evaluating the results of a customized NGS panel. This process optimizes the selection of available therapies, improving disease control. Prospective randomized trials are needed to confirm this approach and open the door to expanded drug access.

[EN] We report herein an evaluation of the effect of several mesoporous silica nanoparticles (MSNs) on the cellular uptake and in vitro cytotoxicity in human cells. Bare MSNs and MSNs functionalized with polyethylene glycol or hyaluronic acid are employed to evaluate uptake efficiency and mechanisms of endocytosis in cancer (MDA-MB-231) and non-cancer (MCF10A) cells. Moreover, changes in viability, cell cycle, oxidative stress, and mitochondrial membrane potential are evaluated. Our results confirm that MSNs are internalized efficiently by human cells and that uptake mechanisms differ for cell types and particles. We also confirm that MSNs are biocompatible materials that do not induce ROS/RNS production, nor changes on mitochondrial membrane potential or cell cycle., The authors want to thank the Spanish Government RTI2018-100910-B-C41 (MCUI/AEI/FEDER, UE) and PI18/01219 (ISCIII), the Generalitat Valenciana (PROMETEO/2018/024 and ACIF/2016/030), and CIBER-BBN (CB07/01/2012) and CIBER-ONC (CB16/12/00481) for support.

Background Recently, microRNAs have been demonstrated to be potential non-invasive biomarkers for diagnosis, prognosis assessment or prediction of response to treatment in cancer. In this study, we evaluate the potential of miR-30b-5p as a biomarker for early diagnosis of breast cancer (BC) in tissue and plasma. Methods Expression of miR-30b-5p was determined in a series of 112 BC and 40 normal breast tissues. Circulating miR-30b-5p levels in plasma samples were determined in a discovery cohort of 38 BC patients and 40 healthy donors and in a validation cohort of 83 BC patients and 83 healthy volunteers. miR-30b-5p expression was measured by quantitative real-time PCR and receiver operating characteristics curve analysis was carried out. Results The miR-30b-5p expression was significantly lower in BC tissue than in healthy breast samples. In contrast, circulating miR-30b-5p levels were significantly higher in BC patients compared with healthy donors. Furthermore, circulating miR-30b-5p levels were significantly higher in patients with positive axillary lymph node and de novo metastatic patients. Receiver operating characteristics curve analysis demonstrated a good diagnostic potential of miR-30b-5p to detect BC even at an early stage of the disease. Conclusion Thus, we highlight the potential of miR-30b-5p as a non-invasive, fast, reproducible and cost-effective diagnostic biomarker of BC., Highlights Our results show the following: • Low miR-30b-5p expression was detected in breast tumor tissue compared with healthy breast tissue. • High circulating miR-30b-5p levels in plasma were associated to breast cancer. • miR-30b-5p levels in plasma identify breast cancer of all subtypes and stages of the disease. • Circulating miR-30b-5p levels relate with patients' tumor burden. • Circulating miR-30b-5p is a potential non-invasive and cost-effective diagnostic BC biomarker.

Càncer de mama; Genòmica del càncer; Recerca translacional Cáncer de mama; Genómica del cáncer; Investigación traslacional Breast cancer; Cancer genomics; Translational research Novel antibody-drug conjugates against HER2 are showing high activity in HER2-negative breast cancer (BC) with low HER2 expression (i.e., 1+ or 2+ and lack of ERBB2 amplification). However, the clinical and molecular features of HER2-low BC are yet to be elucidated. Here, we collected retrospective clinicopathological and PAM50 data from 3,689 patients with HER2-negative disease and made the following observations. First, the proportion of HER2-low was higher in HR-positive disease (65.4%) than triple-negative BC (TNBC, 36.6%). Second, within HR-positive disease, ERBB2 and luminal-related genes were more expressed in HER2-low than HER2 0. In contrast, no gene was found differentially expressed in TNBC according to HER2 expression. Third, within HER2-low, ERBB2 levels were higher in HR-positive disease than TNBC. Fourth, HER2-low was not associated with overall survival in HR-positive disease and TNBC. Finally, the reproducibility of HER2-low among pathologists was suboptimal. This study emphasizes the large biological heterogeneity of HER2-low BC, and the need to implement reproducible and sensitive assays to measure low HER2 expression. This work was supported by the grants from the European Union’s Horizon 2020 Research and Innovation Programme under Grant agreement No. 847912 (to A.P.), the Instituto de Salud Carlos III-PI16/00904 (to A.P.), Pas a Pas (to A.P.), Save the Mama (to A.P.), Breast Cancer Now-2018NOVPCC1294 (to A.P.), Fundación Científica Asociación Española Contra el Cáncer-Ayuda Postdoctoral AECC 2017 (to F.B.-M.), Fundación SEOM, Becas FSEOM para Formación en Investigación en Centros de Referencia en el Extranjero 2018 (to T.P.) and PhD4MD - Departament de Salut expedient SLT008/18/00122 (to N.C.).

Background A previous phase 2 study in metastatic breast cancer demonstrated a statistically significant improvement in overall survival (OS) in patients treated with pelareorep (pela) in combination with paclitaxel (PTX) versus PTX alone.1 Given that pela is an intravenously delivered immuno-oncolytic reovirus, we hypothesized that the OS benefit from pela + PTX may be attributed to an adaptive T cell response triggered by pela. To examine if pela can mediate the priming of an anti-tumor immune response, we are conducting together with the SOLTI group the AWARE-1 study (a window-of-opportunity study of pela in early breast cancer), which is currently enrolling and for which initial translational research results are presented. Methods AWARE-1 is a window-of-opportunity study to evaluate the safety and effect of pela ± atezolizumab on the tumor microenvironment (TME) in 38 women with early breast cancer. Patients are treated with pela on days 1, 2, 8, and 9, while atezolizumab is administered on day 3. Tumor biopsies are collected at diagnosis, day 3, and day ~21. Five cohorts will be examined: Cohort 1: HR+/HER2-neg (10 patients) receiving pelareorep + letrozole; Cohort 2: HR+/HER2-neg (10 patients) receiving pelareorep + letrozole + atezolizumab; Cohort 3: TNBC (6 patients) receiving pelareorep + atezolizumab; Cohort 4: HER2+/HR+ (6 patients) receiving pelareorep + trastuzumab + atezolizumab; Cohort 5: HER2+/HR- (6 patients) receiving pelareorep + trastuzumab + atezolizumab. The primary endpoint of the study is CelTIL score [2], a metric for quantifying the changes in tumor cellularity and infiltration of TILs, where an increase in CelTIL is associated with a favorable response to treatment. Tumor tissue is being examined for pela replication, and changes to the TME are being assessed by immunohistochemistry and T cell receptor sequencing (TCR-seq). Peripheral blood is also being examined by TCR-seq. Results Detailed TCR-seq results from peripheral blood and tumor tissue are presented for the ten-patients enrolled into Cohort 1 who received pela and letrozole. In tumor tissue, T cell clonality increased in day 21 biopsies relative to baseline biopsies, with similar increases in T cell fraction (the number of T cells) in the majority of patients. In general, most of the tissue-expanded T cell clones were also seen in the peripheral blood. Conclusions Overall, these preliminary data from cohort 1 of AWARE-1 demonstrate that pela mediates priming of a T cell-based immune response that occurs both systemically and within breast cancer tissue. Trial Registration NCT04102618 Ethics Approval This study was approved by the Spanish Health Authority, protocol number 2018-003345-42. References Bernstein V, et al. A randomized phase II study of weekly paclitaxel with or without pelareorep in patients with metastatic breast cancer: final analysis of Canadian Cancer Trials Group IND.213. Breast Cancer Res Treat 2018;167(2):485-493. Nuciforo P, et al. A predictive model of pathologic response based on tumor cellularity and tumor-infiltrating lymphocytes (CelTIL) in HER2-positive breast cancer treated with chemo-free dual HER2 blockade. Ann Oncol 2018;29(1):170-177.