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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.

Advanced breast cancer represents a challenge for patients and for physicians due its dynamic genomic changes yielding to a resistance to treatments. The main goal is to improve quality of live and survival of the patients through the most appropriate subsequent therapies based on the knowledge of the natural history of the disease. In these guidelines, we summarize current evidence and available therapies for the medical management of advanced breast cancer.

Kaplan-Meier Curves for Progression-Free Survival and Overall Survival by high TMB {greater than or equal to}7 mutations/Mb and PTEN alterations in Chemotherapy-Treated mTNBC. (A) Progression-free survival and (C) overall survival by tumor mutational burden

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CONSORT DIAGRAM

Purpose:Few patients with metastatic triple-negative breast cancer (mTNBC) benefit from immune checkpoint inhibitors (ICI). On the basis of immunotherapy response correlates in other cancers, we evaluated whether high tumor mutational burden (TMB) ≥10 nonsynonymous mutations/megabase and PTEN alterations, defined as nonsynonymous mutations or 1 or 2 copy deletions, were associated with clinical benefit to anti-PD-1/L1 therapy in mTNBC.Experimental Design:We identified patients with mTNBC, who consented to targeted DNA sequencing and were treated with ICIs on clinical trials between April 2014 and January 2019 at Dana-Farber Cancer Institute (Boston, MA). Objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) were correlated with tumor genomic features.Results:Sixty-two women received anti-PD-1/L1 inhibitors alone (23%) or combined with targeted therapy (19%) or chemotherapy (58%). High TMB (18%) was associated with significantly longer PFS (12.5 vs. 3.7 months; P = 0.04), while PTEN alterations (29%) were associated with significantly lower ORR (6% vs. 48%; P = 0.01), shorter PFS (2.3 vs. 6.1 months; P = 0.01), and shorter OS (9.7 vs. 20.5 months; P = 0.02). Multivariate analyses confirmed that these associations were independent of performance status, prior lines of therapy, therapy regimen, and visceral metastases. The survival associations were additionally independent of PD-L1 in patients with known PD-L1 and were not found in mTNBC cohorts treated with chemotherapy (n = 90) and non-ICI regimens (n = 169).Conclusions:Among patients with mTNBC treated with anti-PD-1/L1 therapies, high TMB and PTEN alterations were associated with longer and shorter survival, respectively. These observations warrant validation in larger datasets.

Purpose: We previously detected promising efficacy of neoadjuvant nintedanib (a multityrosine kinase inhibitor, TKI) in early HER2-negative breast cancer. In a preclinical study, we monitored stromal hypoxia with 18F-fluoromisonidazole-positron emission tomography (18F-FMISO-PET); we found that reoxygenation of tumors (or lack of it) during a window-of-opportunity (WoO) treatment with TKIs correlated with the benefit (or lack of it) from TKI-plus-chemotherapy combinations. We studied the predictive role of 18F-FMISO-PET for the TKI nintedanib in the neoadjuvant setting in a phase II WoO randomized trial.Experimental Design: Patients were randomized to a 14-day WoO of nintedanib preceded and followed by an 18F-FMISO-PET, followed by nintedanib plus weekly paclitaxel (Arm A) or an 18F-FMISO-PET followed by weekly paclitaxel (Arm B) before surgery. The endpoint was residual cancer burden (RCB). The objective was to detect the patients with no response (RCB-III) on the basis of the baseline or evolutive 18F-FMISO-PET values/changes.Results: One-hundred and thirty HER2-negative patients were randomized. Seventeen (27.9%), 34 (55.7%), and 8 (13.1%) patients had an RCB of III, II, and I/0, respectively, in Arm A. In this arm, baseline hypoxic tumors had a 4.4-fold higher chance of experiencing RCB = 3 (P = 0.036) compared with baseline normoxic tumors. Nintedanib WoO induced tumor reoxygenation in 24.5% of the patients; those not reoxygenating showed a trend toward higher chance of experiencing RCB-III (6.4-fold; P = 0.09). In Arm B, 18F-FMISO-PET lacked predictive/prognostic value.Conclusions: Baseline hypoxic tumors (measured with 18F-FMISO-PET) do not benefit from neoadjuvant nintedanib. Clin Cancer Res; 23(6); 1432–41. ©2016 AACR.

Extended methods and results, including e-references

Purpose:We do not yet have validated biomarkers to predict response and outcome within hormone receptor–positive/HER2-positive (HR+/HER2+) breast cancer. The PAM50-based chemo-endocrine score (CES) predicts chemo-endocrine sensitivity in hormone receptor–positive/HER2-negative (HR+/HER2−) breast cancer. Here, we evaluate the relationship of CES with response and survival in HR+/HER2+ breast cancer.Experimental Design:Intrinsic subtype and clinicopathologic data were obtained from seven studies in which patients were treated with HER2-targeted therapy either with endocrine therapy (ET) or with chemotherapy (CTX). CES was evaluated as a continuous variable and categorically from low to high scores [CES-C (chemo-sensitive), CES-U (uncertain), and CES-E (endocrine-sensitive)]. We first analyzed each dataset individually, and then all combined. Multivariable analyses were used to test CES association with pathologic complete response (pCR) and disease-free survival (DFS).Results:A total of 457 patients were included (112 with ET and 345 with CTX). In the combined cohort, CES-C, CES-U, and CES-E were identified in 60%, 23%, and 17% of the patients, respectively. High CES (i.e., CES-E) was associated with a lower probability of achieving pCR independently of clinical characteristics, therapy, intrinsic subtype, and study (adjusted OR = 0.42; P = 0.016). A total of 295 patients were analyzed for DFS with a median follow-up of 66 months. High CES was also associated with better DFS (adjusted HR, 0.174; P = 0.003) independently of pCR, clinical characteristics and intrinsic subtype. In patients with residual disease, the adjusted DFS HR of CES was 0.160 (P = 0.012).Conclusions:In HER2+/HR+ breast cancer, CES is useful for predicting chemo-endocrine sensitivity and provides additional prognostication beyond intrinsic subtype and clinicopathologic characteristics.

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.

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Hypoxic volumes

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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.

Introduction: Tumor-Infiltrating Lymphocytes (TILS) is a well-known predictor of response to primary chemotherapy1,2 and a prognostic factor for improved survival in different breast cancer subtypes3. We present data on the association of the level of TILs and morphologic characteristics of such infiltrate with pCR. Material and methods: A series of 477 breast cancer patients (479 tumors) treated with primary chemotherapy at Catalan Institut of Oncolgy - H.U. Bellvitge between January 2009 and December 2016. Chemotherapy consisted on anthracyclines and taxanes (plus trastuzumab if Her-2 positive disease). Levels of percentage of TILs using hematoxylin-eosin-stained sections of diagnostic core-needle biopsy were evaluated according to international guidelines in a prospectively defined retrospective analysis. Characterization of TILS consisted on identification of plasma cells, intraepithelial infiltrate vs stromal infiltrate and homogeneous vs heterogeneous infiltrate. A sub-classification using levels of TILS and heterogeneity was done for statistical purposes. Levels of TILS and their morphological characteristics were examined for their associations with pCR adjusted for predictive clinic-pathological factors, by univariate and multivariate logistic regression, statistical significance set at 0.05. A ROC curve was performed to look for a cut-point of TILs to predict PCR. Results: The mean value of TILs was 23.79% (SD, 24%). TILs were significantly higher in ductal carcinomas (39.1% vs 0%, p=0.007), grade 3 (55.2 vs 17.7%p30 (48.8% vs 24.5%, p 20% (15.2% vs 41.5%, OR: 3.96 [95%CI, 2.57-6.10]; P < 0.001); plasma cells (OR 6.61 [95%CI, 1.51-28.8]; P=0.01), intraepithelial TILS (OR: 10.34 [95%CI, 2.22-48.01]; P =0.003), homogeneous high infiltrate (OR: 13.6 [95%CI, 3.04-60.77]; P =0.001). In luminal tumors, TILs over 20% predicted pCR (OR 12.3 [95%CI, 4.0-37.7]; P < 0.001) as well as in TNBC (OR 4.32 [95%CI, 1.77-10.53]; P=0 .001) but not in those cases with HER2 positive tumors (luminalB HER2 + HER2) (OR 1.65 [95%CI, 0.88-3.07]; P=0.118). In the multivariate analyses, levels of TILs > 20% were associated with higher pCR rates (adjusted odds ratio, 2.44 [95%CI, 1.48-4.01]; P < .001). Conclusions: The presence of TILs over 20% at diagnosis is an independent, positive, predictive marker of pCR in early breast cancer treated with neoadjuvant chemotherapy. Interestingly, the predictive information added by TILs >20% was higher in luminal and triple negative tumors compared to HER2 positive cases. Careful morphological characterization of TILS may add valuable predictive information and can be done in current pathologic laboratories with a well-trained breast cancer pathologist. References: 1.J Clin Oncol 2009; 28:105-113. doi: 10.1200/JCO.2009.23.73702.JAMA Oncol. 2015;1(4):448-454. doi:10.1001/jamaoncol.2015.08303.Lancet Oncol 2018: 19: 40-50 http://dx.doi.org/10.1016/S1470-2045(17)30904-X. N%Age years (mean, sd)Grade*I326.7II19540.7III15152.6Ki 67≤3020442.6>3027557.4Molecular subtype*Luminal A469.6Luminal B HER2 -14329.9Luminal B HER2+9219.2HER+ enriched7615.9Triple negative12225.5Pathologic responseNon-pCR35874.7pCR12125.3 Citation Format: Catalina Falo, Juan Azcarate, Ana Petit, Andrea Vethencourt, Sergi Fernandez Gonzalez, Amparo Garcia-Tejedor, Silvia Vazquez, Hector Perez, Maria Laplana, Charo Taco, Esther Guerra, Anna Guma, Raul Ortega, Agostina Stradella, Sabela Recalde, Adela Fernandez-Ortega, Rafael Villanueva, F Javier Perez, M Jesus Pla, Miriam Campos, Diana Perez, Eulalia Fernandez-Montoliu, Veronica Obadia, Monica Cejuela, Miguel Gil-Gil, Sonia Pernas, Mar Varela, Teresa Soler-Monzo. Morphologic characterization of tumor-infiltrating lymphocytes and its relation with pathological response in a series of breast cancer patients treated with primary chemotherapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-26.

Background: Novel therapies are needed upon progression to first-line CDK4/6 inhibitor (CDKi) plus endocrine therapy in advanced HR+ breast cancer. One frequent alteration driving resistance to CDKi plus hormones is FGFR1/2 amplification/overexpression. We have demonstrated that a combined assay of RNAScope and FISH, detecting RNA overexpression and genomic amplification of FGFR1/2, captures more FGFR-aberrant cases than either test alone. When these alterations exist, the triple estrogen receptor (ER), CDK4/6 and FGFR1/2 blockade is able to revert acquired resistance to CDKi (Breast Cancer Res; 23:21-37; 2021). Rogaratinib is a selective FGFR1-4 kinase inhibitor with significant activity in FGFR-aberrant malignancies. In this trial we will test the safety of the triple blockade combining fulvestrant, palbociclib and rogaratinib. We will also study its role as a resistant-reversion combo for FGFR1/2-positive, HR+ patients progressing to a CDK4/6 inhibitor plus aromatase inhibitor (AI) in the first-line metastatic setting. Trial design: Single-arm, prospective, multicentric, open-label, phase I dose-escalation trial. Patients will be pre-screened for amplification and/or overexpression of FGFR1/2 by FISH and RNAScope, during their first-line treatment. Positive patients will start treatment: on day 1, patients will receive rogaratinib q12 hours. On day 2, patients will continue on rogaratinib, receive fulvestrant (500 ug IM) and start on palbociclib (100 mg/day on days 1-21). A second fulvestrant dose (500 ug) will be administered in day +15. The first cycle will be 29 days, followed by 28-day cycles (continuous rogaratinib q12 hours days 1-28, fulvestrant 500 ug day 1, and palbociclib 100 mg/day days 1-21). Rogaratinib will be escalated, starting on 400 mg/bid in 200 mg/bid increments, following a classic 3+3 schedule. Patients experiencing ≤ grade 1 tolerable side effects as the greatest toxicity will be allowed to escalate palbociclib to 125 in cycle 2. A full pharmacokinetic profile and pharmacodynamics (plasma FGF23 and phosphate levels) will be performed on days 1 and 15 of cycle 1. The impact of the triple combination in immunodynamics will be assessed on PBMCs on days 2 and 15. Ten additional HR+, FGFR1/2 + patients will be accrued at the RP2D in an expansion cohort to study efficacy. RECIST 1.1 and NCI CTC AE V 5.0 criteria will be used for assessing disease control (q8 weeks) and toxicity. Eligibility criteria: Pre-screening: Women >18 year old diagnosed with advanced, non-curable HR+ breast cancer; 2) who are receiving first-line treatment with an AI and any oral CDKi; 3) Adequate organ function. Full screening: 4) Positivity for amplification (FISH ratio>2.2) and/or overexpression (RNAScope score of 3+ or 4+) of either FGFR1/2. 5) PD to first-line AI + CDKi within the last 28 days. 6) Recovery from previous toxicity to < tolerable G2. Specific aims: Primary: 1) To determine RP2D of the triple combination of fulvestrant, palbociclib and rogaratinib. 2) To assess the safety and toxicity of the former combination 3) To study the pharmacokinetics, pharmacodynamics and immunodynamics of the triplet. Secondary: 1) To determine the PFS time and 6-month PFS rate of the triplet in FGFR1/2 amplified/overexpressed patients AI + CDKi doublet. Statistical methods: We will follow a 3+3 escalation schedule; thus, the minimum and maximum number of patients for the dose-escalation phase will range between 6 and 18 (no escalation planned beyond 800 mg/bid rogaratinib). Ten additional patients will be accrued in the expansion cohort (N=28). The anticipated rate of positive screening is 40%; thus, we will screen approximately 70 patients. . Present accrual/target accrual: 55/70 screened; 16 FGFR1/2-positive (of 28 planned); 2 accrued and treated. Citation Format: Sonia Pernas, Cristina Hernando, Begoña Bermejo, Noelia Martinez-Jañez, Jose A García Saenz, Serafin Morales, Luis Manso, Jorge Silva, Juan A Guerra, Diego Malon, Silvana Mouron, Eduardo Caleiras, Miguel Quintela-Fandino. Rogaratinib, palbociclib and fulvestrant in advanced hormone receptor-positive (HR+), FGFR1/2-positive breast cancer: Phase I trial plus an expansion cohort [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 OT2-20-01.

Background: Most breast cancers (BC) exhibit low immune infiltration and are unresponsive to immunotherapy. Inhibitors of the Receptor Activator of NFkB (RANK) pathway, such as denosumab, used for the treatment of bone metastasis, have been shown to prevent BC, reducing tumor cell proliferation and survival. Our recent data supports that RANK pathway inhibition in BC cells enhances anti-tumor immune response. However, the population of BC patients who may benefit from denosumab remains to be identified. The aim of the study is to evaluate the antiproliferative, proapoptotic and/or immunomodulatory activity of denosumab in early BC and to identify biomarkers of response. Methods: Patients with early stage HER2-negative BC, candidates to tumor excision as first therapeutic approach were included. Patients were randomized 2: 1 to denosumab (two doses of 120mg/Kg subcutaneous denosumab, days 1 and 8 plus oral calcium 1000 mg daily for a month) and control arm (no treatment). Blood and tumor samples were collected at baseline and at surgery (2-4 weeks later). Putative changes in tumor cell proliferation by Ki67 immunohistochemistry (IHC), survival by Cleaved caspase-3 IHC and tumor infiltrating lymphocytes (TILs) quantified in H&E were evaluated between initial biopsy and surgery. RANK and RANKL expression will be analysed by IHC and the infiltrating immune populations will be characterized by specific antibodies (CD3, CD4, CD8, CD20, FoxP3, CD68, PDL1, PD1). We will analyse denosumab driven gene expression changes in tumor samples and tools such as CIBERSORT will be used to characterize the immune infiltrate. To assess the increase in TILs and the variation in Ki 67 and Cleaved caspase-3, the T-test for paired samples was used. A value of p less than 0.05 was defined as statistically significant. Result: We present results from the initial 45 patients enrolled out of 60, 31 cases in the experimental arm and 13 in the control arm. Mean age was 55,97 (range 37-88) years. 38 women with luminal breast cancer were analysed and 7 cases of triple negative BC (5 in experimental arm and 2 in control arm). Clinical and tumor characteristics were well balanced between both groups. No relevant toxicities were reported. There was no reduction of Ki67 in either of the two arms and no changes in Cleaved caspase-3 were observed. Interestingly, a statistically significant increase in TILs was observed in the denosumab treated group (p=0.0092, Paired t test) but not in the control group (p=0.68). 29.03% of patients treated with denosumab showed a ≥10% increase in TILs vs 7.14% in the control group (p=0.13). Denosumab was associated with an effective systemic inhibition of RANKL. No relationship was found between serum RANKL levels at baseline and response to denosumab. RNAseq analysis and immunophenotyping is still in progress. Conclusion: Short term neoadjuvant denosumab induces an immunomodulatory effect with an increase in stromal TILs in early BC. ClinicalTrials.gov Identifier: NCT03691311 Table 1.ResultsBiopsySurgeryKi 67 (mean frequency) Control24.5729.57Ki 67 (mean frequency) Experimental22.3927.90Cleaved caspase 3 (mean area) Control0.260.58Cleaved caspase 3 (mean area) Experimental0.350.42TILs (mean percentage) Control9.3410.29TILs (mean percentage) Experimental8.2913.54TILs incrementControl p=0.68 vs. Experimental p=0.009 Citation Format: Andrea Vethencourt, Eva M Trinidad, Anna Petit, María T Soler-Monsó, Clara Gómez Aleza, Ander Urruticochea, Amparo García-Tejedor, Anna Gumà Martinez, Veronica Obadia, Silvia Vazquez, Rafael Villanueva, Adela Fernánez, Monica Cejuela, Sabela Recalde Penabad, Agostina Stradella, Miguel Gil-Gil, Sonia Pernas, Eva Gonzalez-Suarez, Catalina Falo. First results of the randomized window of opportunity clinical trial D-Biomark: Immunomodulatory effect of denosumab in early breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-10.

Background: Overexpression of human epidermal growth factor receptor 2 (HER2) occurs in 15-20% of breast cancers (BC). At the same time, HER2-low tumors (immunohistochemistry 1+ or 2+ with no gene amplification) comprise ~50% of all BC. Trastuzumab Deruxtecan (T-DXd) is an anti-HER2 antibody drug conjugate that has shown impressive and durable response rates not only in HER2+ and HER2low BC, but also in other cancer subtypes expressing the HER2 receptor. However, not all patients respond or benefit to the same extent. Thus, there is a need to identify predictive biomarkers. Here, we hypothesize that by performing several molecular studies in both tissue and plasma samples of those patients participating in the DESTINY studies receiving T-DXd, we will shed more light on the molecular features of HER2 expressing BC and will better characterize the patient population that benefits benefit from this promising new anti-HER2 agent. Methods: HER2-PREDICT is a multi-center, observational study within the biomarker program of SOLTI group, which will include patients who received T-DXd-only while participating in DESTINY trials. Both HER2-positive and HER2-low unresectable and/or metastatic breast cancer may be included in SOLTI-1804 HER2-PREDICT study (NCT04257162). All patients need to consent to obtain a fresh tumor biopsy or donate an archival metastatic biopsy. Primary tumors may be allowed under SOLTI acceptance. Additionally, for patients included before initiating T-DXd treatment blood samples for biomarker analyses on Cycle 1 Day 1 (C1D1), C2D1 and end of treatment will be obtained. Collection of tumor biopsies is an essential part of this study. Pathological analyses will include hematoxylin and eosin (H&E) staining of formalin-fixed paraffin-embedded (FFPE) tissue, identification of areas with the greater amount of tumor cells and determination of their tumor cell percentage. RNA will be isolated and analyzed using the nCounter platform (Nanostring Technologies). Molecular intrinsic subtypes will be identified by a research-based version of PAM50. Furthermore, we aim to evaluate 771 additional genes (+5 housekeeping genes) that encompass genomic signatures and individual genes of importance in breast cancer by means of the nCounter®Breast Cancer 360 Panel. Somatic mutations in PIK3CA, TP53, and additional genes (e.g., GATA3 and ERBB2) will be identified using next-generation sequencing (NGS) in FFPE tumor blocks and plasma samples. Objectives: The primary objectives are (1) to identify an optimal ERBB2 mRNA cut-off point predictive of T-DXd response and (2) to evaluate the correlation of baseline ERBB2 mRNA levels (as a continuous variable) with overall response rate (ORR). Secondary objectives include: to evaluate the association of ERBB2 mRNA levels, PAM50 intrinsic subtypes and immune-related genes with ORR, progression-free survival (PFS) and overall survival (OS); to design a new gene expression signature predictive of clinical benefit; to evaluate the correlation in early changes in circulating tumor DNA (ctDNA) with ORR, PFS and OS; and to identify acquired somatic mutations of resistance upon progression in plasma samples. The study is active in 13 sites in Spain, and 39 patients have been included in the trial since December 2019 until June 2021. Acknowledgments: We thank Daiichi Sankyo Inc. for their financial support for the study. Citation Format: Aleix Prat, Cristina Saura, Josefina Cruz, Esteban Nogales Fernández, Javier Salvador Bofill, Joaquin Gavilá, Maria José Bermejo-Perez, Vanesa Quiroga, Serafin Morales, Sonia Servitja, Ruben de Toro, Pilar Zamora, Patricia Galván, Nuria Chic, Débora Martínez, Fara Brasó-Maristany, Jordi Canes, Laia Paré, Juan M Ferrero-Cafiero, Tomás Pascual, Sonia Pernas. Solti-1804 HER2-PREDICT: Translational study of tumor samples from breast cancer patients treated with trastuzumab deruxtecan in the metastatic setting [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 OT1-12-01.

INTRODUCTION: The Objective was to investigate the incidence of lymphedema after breast cancer treatment and to analyze the risk factors involved in a tertiary level hospital. METHODS: Prospective longitudinal observational study over 3 years post-breast surgery. 232 patients undergoing surgery for breast cancer at our institution between September 2013 and February 2018. Sentinel lymph node biopsy (SLNB) or axillary lymphadenectomy (ALND) were mandatory in this cohort. In total, 201 patients met the inclusion criteria and had a median follow-up of 31 months (range, 1–54 months). Lymphedema was diagnosed by circumferential measurements and truncated cone calculations. Patients and tumor characteristics, shoulder range of motion limitation and local and systemic therapies were analyzed as possible risk factors for lymphedema. RESULTS: Most cases of lymphedema appeared in the first 2 years. 13.9% of patients developed lymphedema: 31% after ALND and 4.6% after SLNB (p

To critically review the existing evidence on immune checkpoint inhibitors (ICIs) in early-stage and metastatic breast cancer and discuss emerging strategies in the different breast cancer subtypes.Immunotherapy has become one of the major milestones in contemporary oncology, revolutionizing the treatment of multiple solid tumors. ICI agents combined with chemotherapy have demonstrated significant efficacy in both early-stage and metastatic triple-negative breast cancer. However, only a subgroup of patients responds to those agents and some associated toxicities, although infrequent, can be life-disabling. Emerging data from immunotherapy studies in advanced hormone receptor-positive (HR +) breast cancer as well as HER2-positive disease are arising with mixed results. Although breast cancer has not classically been considered a hot tumor, ICIs have proven to be effective in a subset of breast cancer patients. However, much remains to be learned, and the identification of new biomarkers beyond PD-L1 expression is essential not only to improve the efficacy of ICI but also to identify patients who can avoid them, together with their toxicities and costs.

Background: Current trends in breast cancer treatment include the use of less aggressive surgeries to reduce morbidity, shorten hospital stays and improve cosmetic results. The aim of the study is to assess tumor cell viability after percutaneous ultrasound (US)-guided radiofrequency ablation (RFA) for small breast cancer by a combination of staining techniques. Methods: A prospective study was conducted at a single institution from 2013 to 2017. Twenty women with invasive ductal carcinoma of the breast measuring ≤ 20 mm were treated with US-guided RFA followed immediately by surgical resection. Tumor viability pre- and post-RFA was assessed with Hematoxylin and Eosin (H&E), Nicotinamide adenine dinucleotide (NADH), Succinate dehydrogenase (SDH), Cytochrome c oxidase (COX), Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Cytokeratin 18 and 19 (CK18/CK19) staining techniques. Outcomes and correlation with the different techniques were evaluated with principal component analysis Cronbach’s alpha. Results: Oxidative enzymes in frozen sections showed loss of SDH and NADH in 13 of the 16 tumors (81%) and COX in 11 of the 13 tumors (84%). In paraffin-embedded tissues, CK18 was negative or markedly reduced in 98% and CK19 in 100% of the cases. Lack of evidence of cell death was seen in 3 cases where the maximum temperature achieved at the center of the tumor was ≤ 70ºC. The reliability and internal consistency between the different staining techniques was high (Cronbach’s alpha, 0.8), with concordance between the staining results of the oxidative enzymes and of CK18/CK19. Conclusion: Loss of tumor viability in small breast tumors after US-guided percutaneous RFA could be assessed in our series with different staining methods. CK18 and CK19 could be used in paraffin-embedded tissues as surrogate markers of tumor cell viability after immediate RFA.

Introduction and objectives. Metaplastic breast cancer is an heterogenous and infrequent disease (0.5-2%). Histologically, it is defined by differentiation of neoplastic epithelium into squamous or mesenchymal-like elements. Although they are mainly triple negative tumors, they present a more aggressive course with less response to chemotherapy and worse prognosis. The aim of our study was to characterize the molecular profile and tumor evolution in matched (primary-relapse) samples of patients (pts) with metaplastic breast cancer. Material and Methods. Genomic profiling of tumor biopsies from different time points in patients with early-stage metaplastic breast cancer who had disease recurrence/progression, and were treated at our institution between 2010 and 2020. Tumor samples were analyzed by DNA- Next Generation Sequencing (NGS; Illumina 2x75bp) using the ActionOncoKitDX panel (Imegen-Health in Code group). It allowed the study of point mutations in 50 genes, CNVs, fusion genes among 8 genes and pharmacogenetic SNPs associated with treatment efficacy or toxicity. It also determined MSI through 110 markers. The results were classified following the recommendations of the ACMG (American College of Medical Genetics). Only pathogenic and likely pathogenic variants were considered for analysis and afterwards we categorized them following the ComPerMed (The Personalized Medicine Commission) criteria. Results. We have analyzed 21 matched diagnosis-relapse tumor samples (8 primary tumor samples, and 13 loco-regional or metastatic disease) from 8 patients. In 4 patients, genomic characterization was performed at 3 different time points of their tumor evolution. Pathogenic alterations identified were mutations in TP53 (in 100% of the samples), TERT promoter (29%), and MYC amplifications (24% of samples, of which 75% were relapse/progression samples). We didn’t find any mutation in PI3KCA, but PTEN was found to have variations in 38% samples (10% mutations and 38% deletions). Amplification of FGFR1 was identified in 13% of the pts (only in the primary tumor). No ERBB2 or EGFR gene amplifications were detected, neither high grade MSI. We did not find a significant increase in point mutations between primary and relapse/progression samples, although gene amplifications were found more frequently in the former ones. Sixty three percent of the cases were Tier I and II category alterations, which could have future implications in the management of this neoplasia. Conclusion. In our series of metaplastic carcinoma, the most frequent pathogenic alterations occurred in cycle regulation’s genes, including TP53 and TERT promoter mutations, and MYC amplifications. Relapse/progression samples were enriched in MYC amplification and inTP53 allelic frequency. Larger studies are required to better characterize these tumors, and identify new strategies to improve the prognosis of these patients. Citation Format: Mónica Cejuela, Agostina Stradella, Anna Petit, Pablo Gargallo, Jan Bosch, Paula Carbonell, Sabela Recalde, Andrea Vethencourt, Adela Fernández, Catalina Falo, Miguel Gil-Gil, Silvia Vazquez, Rafael Villanueva, Teresa Soler, Inés Calabria, Sonia Pernas. Genomic characterization and tumor evolution in matched(primary-relapse)samplesof patients with METAPLASTIC breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-07-06.