Residual Cancer Burden Research Articles

Abstract 6610: Increasing blood volumes to detect minimal residual disease in neoadjuvant-treated early breast cancer patients

Abstract Breast cancer (BC) is the most frequent neoplasia affecting women worldwide normally detected at early stages. In this regard, early diagnosis drastically decreases mortality, however, around 20% of these patients will later relapse. This is mainly caused by undetectable molecular residual disease (MRD) not eliminated by standard primary treatments. Therefore, it is crucial to detect the after-treatment MRD to stratify the patients by their risk of relapse. Liquid biopsies have emerged as non-invasive method to obtain information about tumors and improve clinical cancer management. Regarding this, much has been hypothesized about utilizing high blood volumes to overcome the necessity of complex and resource-intensive next generation sequencing (NGS) methodologies to detect highly diluted blood tumor components in localized cancers. Herein, we employed a combined analysis of circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) together with high blood volumes and single-assay droplet digital PCR (ddPCR) to detect MRD with ultra-high sensitivity. We prospectively assayed 124 samples extracted at baseline, post-neoadjuvant therapy (NAT), post-surgery and a follow-up on a six-monthly basis. A median of 76.40 mL of blood to detect CTCs and 40 mL of plasma to detect ctDNA per patient from 19 BC women were used in this study. ddPCR assays were performed with a median of 14 partitions per determination to detect ctDNA and 12 partitions for CTCs. Overall, ctDNA, CTCs and ctDNA and/or CTCs were detected in 84.21%, 66.66% and 89.47% respectively in the pre-treatment blood samples. MRD (ctDNA and/or CTCs) was detected in 73.68% of the after NAT blood samples. On the other hand, it was detected in 46.66% and 70.00% of the post-surgery and follow-up samples respectively. Post-NAT MRD was detected in 57.14% (4/7) and 83.33% (10/12) of patients with and without pathological complete response pCR respectively. To note, the discordant patients achieving pCR in tissue with detectable MRD in blood were high-risk BC. Importantly, in one of the two patients without pCR and no MRD detected, not enough sample were available to complete the analysis. The other discordant patient presented a localized disease with residual cancer burden value of 1 and no lymph nodes affected. In 1 out of 19 (5.26%) patient clinically relapsed with a positive MRD detection 6 months earlier. Applying this methodology, we observed a sensitivity of 0.004% in ctDNA detection and 0.224 CTCs per mL of blood. Overall, this novel methodology greatly improves sensitivity for ctDNA and CTCs detection in treatment-naïve early BC. In addition, MRD was successfully detected in post-treatment samples antedating clinical relapse by 6 months in one patient. This prospective study is potentially demonstrating that using high blood volumes and a single-assay ddPCR is a cost-effective strategy to monitor localized BC and predict relapses. Citation Format: Alfonso Alba-Bernal, Ana Godoy-Ortiz, María Emilia Domínguez-Recio, Begoña Jimenez-Rodriguez, María Elena Quirós-Ortega, Esperanza López-López, Guillermo Carbajosa-Antona, Jesús Peralta-Linero, Luis Vicioso, Estefanía Bellagarza-García, Guadalupe Dolores Garrido-Ruiz, Cynthia Robles-Podadera, Alicia Garrido-Aranda, María Dunia Roldán-Díaz, Jesús Velasco-Suelto, Rocío Lavado-Valenzuela, Martina Álvarez, Nuria Ribelles, Javier Pascual, Emilio Alba, Iñaki Comino-Méndez. Increasing blood volumes to detect minimal residual disease in neoadjuvant-treated early breast cancer patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6610.

Deep Learning-Based Segmentation of Locally Advanced Breast Cancer on MRI in Relation to Residual Cancer Burden: A Multiinstitutional Cohort Study.

While several methods have been proposed for automated assessment of breast-cancer response to neoadjuvant chemotherapy on breast MRI, limited information is available about their performance across multiple institutions. To assess the value and robustness of deep learning-derived volumes of locally advanced breast cancer (LABC) on MRI to infer the presence of residual disease after neoadjuvant chemotherapy. Retrospective. Training cohort: 102 consecutive female patients with LABC scheduled for neoadjuvant chemotherapy (NAC) from a single institution (age: 25-73 years). Independent testing cohort: 55 consecutive female patients with LABC from four institutions (age: 25-72 years). Training cohort: single vendor 1.5T or 3.0 T. Testing cohort: multivendor 3.0 T. Gradient echo dynamic contrast-enhanced sequences. A convolutional neural network (nnU-Net) was trained to segment LABC. Based on resulting tumor volumes, an extremely randomized tree model was trained to assess residual cancer burden (RCB)-0/I vs. RCB-II/III. An independent model was developed using functional tumor volume (FTV). Models were tested on an independent testing cohort and response assessment performance and robustness across multiple institutions were assessed. The receiver operating characteristic (ROC) was used to calculate the area under the ROC curve (AUC). DeLong's method was used to compare AUCs. Correlations were calculated using Pearson's method. P values<0.05 were considered significant. Automated segmentation resulted in a median (interquartile range [IQR]) Dice score of 0.87 (0.62-0.93), with similar volumetric measurements (R=0.95, P < 0.05). Automated volumetric measurements were significantly correlated with FTV (R=0.80). Tumor volume-derived from deep learning of DCE-MRI was associated with RCB, yielding an AUC of 0.76 to discriminate between RCB-0/I and RCB-II/III, performing similar to the FTV-based model (AUC=0.77, P=0.66). Performance was comparable across institutions (IQR AUC: 0.71-0.84). Deep learning-based segmentation estimates changes in tumor load on DCE-MRI that are associated with RCB after NAC and is robust against variations between institutions. 2. Stage 4.

CtDNA and residual cancer burden are prognostic in triple-negative breast cancer patients with residual disease

Triple-negative breast cancer (TNBC) patients with residual disease (RD) after neoadjuvant systemic therapy (NAST) are at high risk for recurrence. Biomarkers to risk-stratify patients with RD could help individualize adjuvant therapy and inform future adjuvant therapy trials. We aim to investigate the impact of circulating tumor DNA (ctDNA) status and residual cancer burden (RCB) class on outcomes in TNBC patients with RD. We analyze end-of-treatment ctDNA status in 80 TNBC patients with residual disease who are enrolled in a prospective multisite registry. Among 80 patients, 33% are ctDNA positive (ctDNA+) and RCB class distribution is RCB-I = 26%, RCB-II = 49%, RCB-III = 18% and 7% unknown. ctDNA status is associated with RCB status, with 14%, 31%, and 57% of patients within RCB-I, -II, and -III classes demonstrating ctDNA+ status (P = 0.028). ctDNA+ status is associated with inferior 3-year EFS (48% vs. 82%, P < 0.001) and OS (50% vs. 86%, P = 0.002). ctDNA+ status predicts inferior 3-year EFS among RCB-II patients (65% vs. 87%, P = 0.044) and shows a trend for inferior EFS among RCB-III patients (13% vs. 40%, P = 0.081). On multivariate analysis accounting for T stage and nodal status, RCB class and ctDNA status independently predict EFS (HR = 5.16, P = 0.016 for RCB class; HR = 3.71, P = 0.020 for ctDNA status). End-of-treatment ctDNA is detectable in one-third of TNBC patients with residual disease after NAST. ctDNA status and RCB are independently prognostic in this setting.

Good practice: The experiences with the utilization of residual cancer burden-A single institution study.

The use of neoadjuvant therapy (NAT) has been showing an incraesing tendency in the treatment of locally advanced breast cancer. The evaluation of residual cancer could be performed by Residual Cancer Burden (RCB) calculator. The prognostic system takes the two largest diameters of the tumor, the cellularity, the amount of in situ carcinoma, the number of metastatic lymph nodes, and the size of the largest metastatic deposit into account. The aim of our study was to examine the reproducibility of RCB in NAT treated patients. Patients who were treated with NAT and had resection specimens between 2018 and 2021 were selected. Histological examination was performed by five pathologists. After assessment of the examined variables, RCB points and RCB classes were defined. For statistical analysis, interclass correlation was used (SPSS Statistics V.22.0 software). Altogether 100 patients were included in our retrospective, cohort study (average age: 57 years). In two-thirds of the cases, third generation chemotherapy was used, and mastectomy was performed. Significant concordance was found in the two largest diameters of the tumor (coefficients, 0.984 and 0.973), the cellularity (coefficient, 0.970), and the largest metastatic deposit (coefficient, 0.998). Although the amount of in situ carcinoma proved to be the least reproducible factor, it resulted in almost 90% of agreement (coefficient, 0.873). Regarding RCB points and classes, similar results were observed (coefficients, 0.989 and 0.960). Significant agreement was observed between examiners based on almost all RCB parameters, points, and classes, reflecting the optimal reproducibility of RCB. Therefore, we recommend the use of the calculator in routine histopathological reports in NAT cases.

Abstract P6-01-19: Multiparametric MRI-based Longitudinal-radiomics Analysis for Early Prediction of Treatment Response of Breast Cancers to Neoadjuvant Chemotherapy: A Multicenter Study

Abstract P6-01-19: Multiparametric MRI-based Longitudinal-radiomics Analysis for Early Prediction of Treatment Response of Breast Cancers to Neoadjuvant Chemotherapy: A Multicenter Study

Abstract PD11-06: PD11-06 Circulating tumor DNA association with residual cancer burden after neoadjuvant therapy in triple negative breast cancer in TBCRC 030

Abstract Background. Patients (pts) with early triple negative breast cancer (eTNBC) are at increased risk of breast cancer recurrence and death. Recent studies have focused on escalation of therapy, with current treatment standard of at least five drugs – and associated toxicities - for eTNBC. Though presence of residual disease after neoadjuvant therapy (NAT) as measured by residual cancer burden (RCB) helps guide addition of adjuvant treatment, more effective tools to tailor therapy are limited. Persistence of circulating tumor DNA (ctDNA) in the setting of residual disease is associated with high risk of distant recurrence. However, more sensitive minimal residual disease (MRD) assays are needed to potentially guide optimization of systemic therapy. Methods. TBCRC 030 is a phase II randomized study of 12 weeks of NAT single agent cisplatin or paclitaxel for stage II-III TNBC, followed by surgery. The primary objective of the parent study was to correlate baseline biomarker for homologous recombination deficiency and RCB by study arm. From this group, responders (RCB 0/1) and non-responders (RCB 2/3) from both study arms who did not receive additional NAT prior to surgery were selected for analysis from the study cohort, matched on baseline nodal status and tumor size. As a post hoc study amendment, available pts were followed for event free survival (EFS). Plasma samples were collected prior to treatment initiation (W0), at three weeks (W3), and at twelve weeks, prior to surgery (W12). Whole genome sequencing (WGS) was performed on primary tumor tissue to identify somatic mutations and design for each pt a tumor-informed, ctDNA assay tracking up to 1000 mutations to detect MRD. Detection limit was computed for each tested sample as previously described. For each sample assayed, we report tumor fraction (TFx) when MRD was detected and the detection limit at 90% power when MRD was not detected. Results. Of 139 study pts, 68 had complete tissue and plasma samples and no receipt of additional NAT. Of these, 22 were responders. These responders, and 22 matched non-responders were identified for analysis. Data from 22 pts – 11 responders, 11 non-responders - are described here; full analysis on all 44 pts will be presented at the meeting. Personalized ctDNA assays were designed targeting 434 to 1000 variants (median 1000) and applied to 66 plasma samples. At W0, 100% (22/22) were positive for ctDNA; 73% (16/22) and 55% (12/22) were positive at W3, and W12, respectively. In pts with T1-T2 tumors median TFx was 4.1e-3(7.8e-6, 3.4e-2) and 4.7e-1(4.3e-2, 9.0e-1) in pts with T3-T4 tumors. TFx decreased from W0 to W3 and from W0 to W12 in responders (Table 1). By W12, ctDNA had cleared in 7/8 pts with RCB 0, 1/3 with RCB 1, 2/8 with RCB 2, and 0/3 with RCB 3. Overall, ctDNA levels were broad with median TFx of 1.5e-3 (range 2.9e-6 to 0.90). Detection limit at 90% power for all tested samples was a median of 8.8e-6 (range 9.9e-7 to 6.8e-3). To investigate whether ctDNA persistence after NAT was associated with BC recurrence, we analyzed a separate group of all 8 pts with known recurrence and with complete data and samples. All pts had persistent ctDNA at W12 (median TFx 6.8e-3, [2.9e-6 to 6.6e-2]). Conclusions. After 3 weeks of NAT for eTNBC, ctDNA TFx decreased, with a 3900-fold change in responders and 18-fold change in non-responders. By W3, TFx for most pts with RCB 0/1 were below the 1 in 10,000 limit of detection for many currently available assays, emphasizing the need for sensitive tests to potentially guide therapy. Additional studies will determine if ctDNA-guided approaches in eTNBC can improve pt outcomes. Table 1: Tumer Fraction and Tumer Fraction Fold Change by Response to Neoadjuvant Therapy Citation Format: Heather A. Parsons, Timothy Blewett, Xiangying Chu, Sainetra Sridhar, Katheryn Santos, Kan Xiong, Vandana Abramson, Ashka Patel, Ju Cheng, Adam M. Brufsky, Justin Rhoades, Jeremy Force, Ruolin Liu, Tiffany A. Traina, Lisa Carey, Mothaffar Rimawi, Ahmed Elkhanany, Vered Stearns, Jennifer M. Specht, Harold Burstein, Antonio C. Wolff, Eric Winer, Nabihah Tayob, Ian Krop, Todd Golub, Erica L. Mayer, Viktor Adalsteinsson. PD11-06 Circulating tumor DNA association with residual cancer burden after neoadjuvant therapy in triple negative breast cancer in TBCRC 030 [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 PD11-06.

Abstract PD16-04: To monitor or not to monitor? MRI monitoring of neoadjuvant chemotherapy effect and its benefit on breast cancer patients’ pathological complete response rate

Abstract Introduction: Neoadjuvant chemotherapy (NACT) represents an essential tool for the treatment of selected breast cancer patients aimed to reduce the tumor size for a more conservative resection and to gain early information about the sensitivity to applied treatment. An effective early response assessment helps both to change the therapeutic strategy for patients nonresponding to the treatment used and to avoid the toxicity related to ineffective treatment. We assumed that changing the chemotherapy regimen might avoid the residual cancer burden (RCB) III outcome and lead to the highest possible rate of pathological complete response (pCR) achieved. We examined the benefit of MRI as a preferred method for interim response assessment to NACT with a primary focus on the relation between the pattern of responses to NACT within the MRI- monitored and non-monitored patients and the pattern of final outcomes on the RCB scale. Methods We present a real-life data-based retrospective analysis of 124 female patients with locally advanced breast carcinoma. All patients received NACT (anthracycline-based regimen followed by taxane-based regimen/early switch to taxanes after the 2nd cycle in nonresponders, and antiHER2 therapy if indicated) prior to surgery (mastectomy/breast-conserving surgery, sentinel lymph node biopsy/axillary dissection). Postoperative histopathological analysis of tumor specimens categorised 120 patients according to the RCB scale 0 – III. Four patients had surgery ex muros and were lost to follow-up. Patients were divided into two cohorts. Group A covered monitored patients with pre-treatment MRI and follow-up MRI after the 2nd cycle of NACT. If no response was detected, another follow-up MRI was indicated after the 2nd cycle of a new treatment. Group B comprised patients with no/incomplete MRI monitoring and patients without the change of therapy even though no response was detected by MRI after the 2nd cycle of NACT. Association between categorical variables was tested using chi-square tests. Statistical analysis was performed using StatsDirect® 3.3.5 (StatsDirect Ltd., Cheshire, UK). The data analysis was supported by a grant from the Cultural and Educational Grant Agency of the Ministry of Education, Science, Research, and Sport of the Slovak Republic (KEGA 041UK-4/2020). Results MRI-monitored patients had two times the odds of being RBC 0 (OR = 2.02, P = 0.122) and almost three times the odds of being RCB 0 or RCB I (OR = 2.83, P = 0.0206) than patients with no monitoring. Changing the NACT after the 2nd cycle in the cohort of monitored patients with no response to initial therapy was significantly associated with better outcomes on the RCB scale (P = 0.0042). This result was confirmed by comparing the pattern of results in patients with no response within the group of incompletely monitored patients with known MRI results after the 2nd cycle (P = 0.0257). Changing the ineffective NACT after the 2nd cycle significantly increased the proportion of RCB 0-I by 23.4%, confirming the benefit of response monitoring by MRI. Conclusion Breast cancer constitutes a heterogeneity of tumor subtypes and it is acknowledged that their behaviour during NACT is highly variable and often unpredictable. MRI represents an effective tool for the assessment of tumor response to applied NACT. To achieve the most clinically meaningful impact of the selected treatment it is critical to monitor the tumor response within the first 3 cycles. Our data clearly confirmed that clinical decisions related to the detection of early response or no response (resulting in the change of NACT) lead to better treatment outcomes and less toxicity. The rate of pCR defines the applied treatment efficacy. Every single patient with results different from RCB III has a better prognosis, reduced need for further expensive treatment and also a more favourable quality of life. These key facts need to be carefully considered when discussing the cost and benefits of MRI monitoring. Citation Format: Lucia Vanovcanova, Iveta Waszulikova, Bibiana Vertakova Krakovska. To monitor or not to monitor? MRI monitoring of neoadjuvant chemotherapy effect and its benefit on breast cancer patients’ pathological complete response rate [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 PD16-04.

Abstract PD11-04: 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 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 &amp;lt; 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&amp;lt; 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.

Abstract P5-05-05: Monitoring for response and recurrence in neoadjuvant-treated hormone receptor-positive HER2-negative breast cancer by personalized circulating tumor DNA testing

Abstract Background: The detection of circulating tumor DNA (ctDNA) may serve as an early predictor of response and recurrence. In this study, we used a tumor-informed ctDNA test to monitor clinical outcomes in patients with high-risk hormone receptor-positive HER2-negative (HR+HER2-) tumors who received neoadjuvant chemotherapy (NAC) on the I-SPY 2 trial (NCT01042379). Methods: We collected blood samples at pretreatment, during (at 3 and 12 weeks after initiation of paclitaxel-based treatment with or without an investigational drug), after NAC prior to surgery, 4 weeks after surgery, and annually until clinical diagnosis of recurrence. Cell-free DNA was isolated from plasma (N=329 samples) and ctDNA was detected using a personalized, tumor-informed multiplex polymerase chain reaction next generation sequencing-based test (SignateraTM). All patients were at high risk for recurrence by MammaPrint. The response endpoints were pathologic complete response (pCR) and residual cancer burden (RCB), and the survival endpoint was event-free survival (EFS). Results: This analysis included 66 patients with HR+HER2- breast cancer who had blood samples collected before, during, after NAC and had at least one blood sample after surgery with sufficient plasma for analysis. 57.1% (32/56) had grade III disease; 72.4% (42/58) were node-positive; 36.2% (21/58) had T3/T4 disease; and 33.3% (22/66) were MammaPrint High 2. The percent ctDNA positivity rates at pretreatment, after NAC prior to surgery, and 4 weeks after surgery were 79.7% (47/59), 6.5% (4/62), and 2% (1/50), respectively. Significantly higher ctDNA positivity rates at pretreatment were observed in patients with larger tumors (95% in T3/T4 vs. 69% in T1/T2, Fisher’s exact p=0.0387), higher grade tumors (94% in Grade III vs. 67% in Grade I/II, p=0.0147) and by MammaPrint score (100% in High 2 vs. 71% in High 1, p=0.0052). In this high-risk HR+/HER2- cohort, 10/66 (15.2%) achieved pCR/RCB 0, who were all ctDNA-negative at surgery. 56/66 (84.8%) had no-PCR, with RCB I (limited residual cancer), II (moderate) and III (extensive) in 7 (10.6%), 31 (47.0%) and 18 (27.3%), respectively. ctDNA-positivity after paclitaxel-based treatment was significantly associated with RCB II/III status (Fisher’s exact p=0.01). All patients in this cohort with persistent ctDNA subsequently had RCB II or III at surgery. 47 patients had paired samples collected after NAC prior to surgery and at 4 weeks after surgery. Of the 47, 91.5% (43/47) were ctDNA-negative at both time points and 8.5% (4/47) were discordant; 1 was ctDNA-negative and later tested ctDNA-positive, while 3 were ctDNA-positive and later tested ctDNA-negative. 61/66 patients had EFS data with a median of 1.6 years of follow up (range: 0.6 to 5.6). 5 tested ctDNA-positive in at least one time point after surgery. Of these, 2 experienced a recurrence (one local relapse and one distant metastasis) and both tested positive at the time of recurrence. For the patient who developed a distant recurrence it was the only blood sample available at a follow-up time point; for the patient who developed a local recurrence, blood from two earlier follow-up time points had tested negative. To date, no recurrences have been observed in those whose test(s) after surgery were negative for ctDNA. Conclusions: The persistence of ctDNA during neoadjuvant therapy is associated with the extent of residual disease in a cohort of patients with HR+HER2- breast cancer in the I-SPY 2 trial and thus may be useful in identifying patients who are not having an optimal response to therapy. I-SPY 2.2 will test whether ctDNA has utility in redirecting therapy to improve surgical outcome and subsequent prognosis. Citation Format: Mark Jesus M. Magbanua, Hope Rugo, Lamorna A. Brown Swigart, Ziad Ahmed, Gillian L. Hirst, Denise M. Wolf, Ruixiao Lu, Ekaterina Kalashnikova, Derrick Renner, Angel Rodriguez, Minetta C. Liu, Christina Yau, Laura J. Esserman, Laura Van ’t Veer, Angela DeMichele. Monitoring for response and recurrence in neoadjuvant-treated hormone receptor-positive HER2-negative breast cancer by personalized circulating tumor DNA testing [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 P5-05-05.

Abstract P5-02-09: Quantitative analysis of fiber-level collagen features in H&amp;E whole-slide images predicts neoadjuvant therapy response in patients with HER2+ breast cancer

Abstract Background: Neoadjuvant treatment (NAT) combining chemotherapy and HER2-targeted agents is frequently administered to HER2-positive (HER2+) breast cancer (BC) patients, with some experiencing a pathological complete response (pCR) and others having residual disease measured by the residual cancer burden (RCB) score. Here, we use a physics-guided machine learning (ML)-based approach to extract fiber-level collagen features from hematoxylin and eosin (H&amp;E)-stained whole slide images (WSIs) and identify collagen-related associations with treatment response in HER2+ patients receiving NAT. Methods: Clinical data and specimens from stage II-III HER2+ BC patients enrolled on the De-escalation to Adjuvant Antibodies Post-pCR to Neoadjuvant THP (DAPHNe; NCT03716180) clinical trial and treated with neoadjuvant paclitaxel/trastuzumab/pertuzumab were analyzed. An ML-based model trained to identify regions of BC tissue as invasive carcinoma, ductal carcinoma in situ (DCIS), diffuse inflammatory infiltrate, stroma, necrosis, or normal tissue was deployed on WSIs of H&amp;E-stained diagnostic core needle biopsies (N=89) to generate tissue overlays. Additional tissue areas were computed from the tissue model predictions using heatmap transformation, including tumor nests (continuous regions predicted as invasive cancer epithelium or DCIS), tumor nest borders (stromal region boundaries 10 μm from tumor nests), and bulk tumor borders (stromal region boundaries 300 μm from aggregated tumor nests). A separate ML-based model trained to identify fiber-level collagen features in WSIs of H&amp;E-stained specimens was also deployed to generate collagen overlays. A fiber feature extraction pipeline was utilized to characterize properties of all identified collagen fibers in the WSI (on the order of hundreds of thousands per slide), including length, width, tortuosity, and angle. These fiber features were then assessed based on their position within the tumor (e.g. relative to the tumor nest border). Combinatorial features (e.g. angle of fibers with respect to tumor boundary) were then explored univariately for associations (N=609) with treatment response. Patients with pCR (RCB=0; N=53) were considered responders, while all other cases (RCBI-III; N=36) were designated non-responders. Due to the small size of the cohort analyzed here, raw p-values are reported. Results: Using estrogen receptor status as a clinical covariate, a logistic regression-based univariate analysis of 609 collagen-associated features revealed six features to strongly associate with pCR (p&amp;lt; 0.05, AUC≥0.75; Table 1). Notable feature themes were identified: 1) fiber tortuosity in tumor nest borders and tumor borders, 2) angle of fibers in tumor border with respect to tumor boundary, and 3) distribution patterns of fiber width in tumor nest borders. The presence of fibers perpendicular to tumor boundary tangents was negatively associated with pCR, as was higher fiber tortuosity and thickness in tumor nest borders. Conclusions: Improved prediction of response to NAT in patients with BC is needed to determine appropriate treatment strategies for each patient. Here, using ML-based models to identify tissue features and collagen fibers, we identify collagen-associated features, measured directly from WSIs of H&amp;E-stained diagnostic BC biopsies, that negatively correlate with pCR. Additional development of this strategy, including the addition of cell identification models and known clinical information, is underway to further refine this novel predictive model. Citation Format: Tan H. Nguyen, Mohammad Mirzadeh, Aaditya Prakash, Emma L. Krause, Jun Zhang, Michael Pyle, Esther R. Ogayo, Harry C. Cramer, Busem Binboga Kurt, Jacqueline Brosnan-Cashman, Michael G. Drage, Stuart Schnitt, Andrew H. Beck, Michael Montalto, Ilan Wapinski, Laura Chambre, Sara Tolaney, Adrienne Waks, Justin Lee, Elizabeth A. Mittendorf. Quantitative analysis of fiber-level collagen features in H&amp;E whole-slide images predicts neoadjuvant therapy response in patients with HER2+ breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-09.

Abstract P4-03-32: Association between androgen receptor expression and recurrence free survival among locally advanced triple negative breast. Real world data from a single Institution in Mexico City

Abstract Background The androgen receptor (AR) is expressed in 12-41% of the TNBC, contributes with differentiation, proliferation, apoptosis, and angiogenesis; its expression is associated with better recurrence free survival (RFS) and overall survival (OS). Objective The aim is to determine the association between AR and pathologic complete response (PCR), recurrence free survival (RFS) in TNBC patients treated with neoadjuvant chemotherapy (neoCT). Patients and methods Cross-sectional, retrospective cohort trial. Adult women with stage II-III TNBC, treated with neoCT from 2014 to 2021 were included. Incomplete medical record, AR not-determined, secondary malignant neoplasm were excluded. AR was determined with the VENTANA assay system, &amp;gt; 1% were considered as positive. Statistical analyses included frequencies and range, comparison was done with X2, cox regression model was made for the prognostic association with a two-sided significance level of 0.05 95% CI. RFS were evaluated with Kaplan Meier method and log-rank test. Results 309 patients were analyzed; AR prevalence was 14.6%. Median age was 49 years (21-87), tumor size was 6.3 cm (0.1-22), ductal histology (95.1%) and nuclear-grade 3 (79%), these findings were not different between the AR+ vs AR-. Median Ki67 was 52% within AR+ vs 65% AR- (p=0.05). All patients were treated with neoCT, 14.9% had disease progression, PCR rates were similar among two groups (48.5% vs 42.2%) p=0.78. At a mean follow up of 19 months, 7 recurrences presented in AR+ (15.5%) vs 67 in the AR- (25.4%) p=NS. Median RFS was 54.9 (IC 95% 45.2-64-6) and 65.5 months (IC 95% 60.4-70.7), p=0.3. We analyze the prognostic factors, for RFS we found statistically significant the locally advanced stage OR 5.6 (1.4-22.9), residual cancer burden (RCB) OR 2.8 (2.1-3.9), N stage OR 1.4 (1.1-1.8) and progression during neoCT OR 2.8 (1.5-5.1); and for OS the RCB OR 2.8 (1.9-4.1) and progression during neoCT OR 3.1 (1.5-6.9). Conclusions In this trial the AR expression did not show significant association neither with PCR and RFS; main reasons are the low prevalence of AR expression, high rate of disease progression and high residual cancer burden after neoCT. We found association with other known factors (RCB, N stage, progression during neoCT and locally advanced stage), which are more important factors that determine the bad prognosis of TNBC. Citation Format: Eder Araiza Alvarado, Claudia Haydee Arce Salinas. Association between androgen receptor expression and recurrence free survival among locally advanced triple negative breast. Real world data from a single Institution in Mexico City [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 P4-03-32.

Abstract P5-12-03: Intratumor heterogeneity and intrinsic immune activation are associated with response to chemotherapy in BRCA-related breast cancers

Abstract Background Breast cancers in women with a germline BRCA1/2 mutation (gBRCAm) have homologous recombination deficiency (HRD) and are sensitive to therapies causing double-strand DNA breaks. In TBCRC 031 (INFORM), both neoadjuvant cisplatin and doxorubicin/cyclophosphamide (“AC”) in gBRCAm carriers resulted in a complete pathologic response in 18% and 26% respectively in patients with newly diagnosed HER2–negative breast cancer. Herein, we describe molecular features from tumor whole exome (WES) and transcriptome sequencing (RNAseq) associated with response. Methods TBCRC 031 (the INFORM Trial - NCT01670500) was a randomized phase II neoadjuvant trial comparing the efficacy of cisplatin versus AC in gBRCAm carriers with stage I-III HER2–negative breast cancer. Of 118 patients enrolled, 92 patients provided fresh frozen research biopsies, collected prior to chemotherapy initiation, which were subjected to WES and RNAseq. Variants were called using GATK best practices and intratumoral heterogeneity was inferred from mutations and variant allele frequencies using Mutant Allele Tumor Heterogeneity (MATH) scores. Mutational Signature 3 (Sig3) and Genomic Instability Score (GIS) were calculated with SigMA and scarHRD, respectively. RNAseq data were utilized to perform differential gene expression and functional analyses, while cellular deconvolution was performed with CIBERSORTx trained against breast cancer single cell data. Patients were grouped according to their residual cancer burden (RCB) as responders (RCB-0 or 1), or non-responders (RCB-2 or 3). P-values ≤ 0.05 were considered statistically significant and when appropriate, adjustment for multiple testing was performed using a false discovery rate ≤ 5%. Results Of the 92 patients with gBRCAm, 59 (64%) had triple-negative and 33 (36%) were hormone-receptor positive HER2-negative breast cancer, 40 (43%) were classified as responders and 52 (57%) as non-responders. WES analysis revealed that indices of HRD were high (Sig3 exposure predominant and GIS ≥ 42) across most samples irrespective of receptor status and not significantly different among responders and non-responders. In contrast, responders exhibited lower levels of intratumor heterogeneity than non-responders (median MATH 42.9 vs. 33.5, p = 0.01). 223 genes were differentially expressed between responders and non-responders following control for tumor hormone receptor status, BRCA1/2 mutation, and menopausal status. Pathways identified as significantly enriched in upregulated genes were indicative of intrinsic immune activation in responders (e.g., T-cell activation, immune response-signaling, and regulation of leukocyte activation). Cellular deconvolution of the tumor microenvironment confirmed that responders presented a higher proportion of T-cells (p = 0.025) and myeloid cells (p = 0.003) in the tumor samples, while perivascular-like cells were enriched in non-responders (p = 0.034). Conclusion In this analysis of the largest cohort of treatment-naïve gBRCAm breast cancer to date, we found that response to cytotoxic chemotherapy is associated with a transcriptional program of intrinsic immune activation and an increased population of intratumoral T-cells and myeloid cells. Lower levels of intratumor heterogeneity and higher immune activation were associated with response to chemotherapy gBRCAm carriers while HRD scores were not. Citation Format: Felipe Batalini, Doga C. Gulhan, Xanthi Lida Katopodi, Nikolas Kalavros, Antuan Tran, Dimitra Karagkouni, Emily Stern-Gatof, Stuart Schnitt, Judy E. Garber, Gerburg M. Wulf, Peter J. Park, Ioannis Vlachos, Nadine Tung. Intratumor heterogeneity and intrinsic immune activation are associated with response to chemotherapy in BRCA-related breast cancers [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 P5-12-03.

Abstract OT2-18-02: OlympiaN: a phase 2, multicenter, open-label study to assess the efficacy and safety of neoadjuvant olaparib monotherapy and olaparib plus durvalumab in patients with BRCA mutations and early-stage HER2-negative breast cancer

Abstract Background: In early breast cancer (BC), neoadjuvant therapy can promote de-escalation of surgery or treat micrometastases. Pathological complete response (pCR) is a standard primary endpoint in neoadjuvant BC studies, and correlates with long-term outcomes such as event-free survival (EFS). The poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and talazoparib are approved in the metastatic setting for patients with BC and a germline BRCA1 and/or BRCA2 pathogenic/likely pathogenic mutation (gBRCAm). Following the phase 3 OlympiA trial primary analysis (data cut-off [DCO] Mar 2020), the PARP inhibitor olaparib was FDA approved for the adjuvant treatment of patients with gBRCAm and HER2-negative, high-risk early BC who have been treated with neoadjuvant/adjuvant chemotherapy. At the second pre-specified OlympiA analysis (DCO Jul 2021), olaparib treatment sustained improvements in invasive disease-free survival (IDFS HR 0.63, 95% CI 0.50–0.78) and distant disease-free survival (DDFS HR 0.61, 95% CI 0.48–0.77), and also significantly reduced the risk of death (overall survival [OS] HR 0.68, 98.5% CI 0.47–0.97) vs placebo. PARP inhibitors have also shown efficacy in the neoadjuvant setting in smaller studies. Durvalumab, a programmed death-ligand 1 inhibitor, has been studied as neoadjuvant therapy in triple negative BC and showed improved IDFS, DDFS and OS vs placebo (GeparNuevo). For BRCAm carriers at low risk of recurrence, olaparib monotherapy may provide adequate neoadjuvant treatment, allowing de-escalation or omission of chemotherapy. For those at high risk of recurrence, it is hypothesized that addition of the immune checkpoint inhibitor durvalumab will enhance immunogenicity provided through cell death following PARP inhibition. Feasibility for a larger study will be assessed. Methods: OlympiaN is a phase 2, international, multicenter, open-label trial examining the efficacy and safety of neoadjuvant olaparib monotherapy and olaparib plus durvalumab in adults with deleterious/suspected deleterious BRCAm and operable, early-stage, HER2-negative, ER-negative or ER-low BC (immunohistochemistry nuclear staining &amp;lt; 10%). Patients will be enrolled in two cohorts: (A) patients at lower risk of recurrence: tumor size &amp;gt;5 to &amp;lt; 20 mm and nodal status N0 will receive olaparib 300 mg orally twice daily continuously in 28-day cycles; (B) patients at higher risk of recurrence: tumor size &amp;gt;20 to ≤50 mm and N0, or T1 and N1 will receive olaparib 300 mg orally twice daily continuously in 28-day cycles plus durvalumab 1500 mg IV infusion every four weeks. After four to six cycles, patients will undergo definitive surgery, then systemic and radiation therapy in accordance with local standard of care. Patients who achieve pCR at surgery will be able to receive adjuvant olaparib monotherapy in lieu of standard adjuvant systemic therapy for a total of twelve 28-day cycles of neoadjuvant and adjuvant olaparib therapy. The primary endpoint is the pCR rate after completion of neoadjuvant systemic therapy, assessed by central pathology review. Secondary endpoints include the pCR rate assessed by local pathology review, residual cancer burden, tumor volumetric analysis, EFS, safety and tolerability. Tumor tissue will be collected pre-treatment and at surgery to evaluate the mechanism of action of therapy. Baseline and longitudinal assessment of ctDNA and its association with clinical outcomes will also be performed. The primary pCR analysis will occur ~6 months after last participant enrollment; the final DCO will be 3 years after last participant dosed or once all participants have had an EFS event. No formal statistical analyses are planned, data will be summarized descriptively. Enrollment is planned to commence by late 2022, across ~65 sites in 10 countries; the target accrual is ~25 patients per cohort to ensure adequate precision in the estimated pCR rate. Funding: This study is funded by AstraZeneca UK Plc. Citation Format: Judith Balmaña, Mike Dymond, Elizabeth S. Lowe, Natalia Lukashchuk, Maria Winter, Nadine Tung. OlympiaN: a phase 2, multicenter, open-label study to assess the efficacy and safety of neoadjuvant olaparib monotherapy and olaparib plus durvalumab in patients with BRCA mutations and early-stage HER2-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-18-02.

Abstract P3-05-25: Clinical Factors associated with Survival Outcomes in Patients with Metaplastic Breast Cancer

Abstract Background Metaplastic breast cancer (MBC) is a rare and aggressive subtype of breast cancer (BC) defined by presence of both epithelial and mesenchymal components. Most are triple negative but are often chemo-refractory and associated with poor survival outcomes compared to non-metaplastic triple negative BC. Advances in therapy have led to improvements in survival outcomes of patients (pts) with BC over the last decade. Our multicenter retrospective study aims to assess (1) progression free survival (PFS) and overall survival (OS) (2) factors predictive of survival outcomes in MBC pts Methods We performed a retrospective analysis of pts diagnosed with MBC from 1997-2021 at Mayo Clinic (Florida, Arizona and Rochester) under IRB approval. Kaplan-Meier method was used to estimate OS and PFS at 1, 3 and 5 years. Cox regression models were used to examine the association between risk factors and survival outcomes. All tests were two-sided with p value &amp;lt; 0.05 considered statistically significant Results We evaluated 158 pts with MBC. Median age and tumor size was 61 (range: 20-91) years and 2.8 (range: 0.5-21) cm, respectively, with 80% of pts being postmenopausal. At diagnosis, 14.6% of pts had clinical T3/T4 disease, 16.4% were clinically node-positive, and 6.3% (10 pts) had distant metastases (DM). Spindle cell histology was seen in 36 pts. Most MBC pts were triple-negative (68.3%), high grade (77.2%) and high Ki-67 (36/47; 76.5%). Of the 61 pts tested for germline mutation, 8 (13.1%) were positive, with BRCA1/2, PTEN, NBN, CHEK2, and BARD1 mutations. Most pts had lumpectomy (53.8%), followed by simple mastectomy (38.5), and modified radical mastectomy (7.7%). Majority of pts had sentinel lymph node biopsy (71.8%). Adjuvant radiation therapy was performed in 65.6% of pts. Pathologic complete response (pCR) was noted in 4/38 (10.5%) evaluable pts who received neoadjuvant chemotherapy (NACT). Residual cancer burden (RCB) scores of 2 and 3 were seen in 76.2% of evaluable pts. Median follow-up time was 2.2 years (range: 6 days-24.6 years). Overall, 1-, 3-, and 5-year OS was 93.3%, 81.7%, and 76.0%, while PFS was 80.8%, 67.9%, and 60.9%, respectively. The presence of DM at diagnosis [HR 38.55 (11.18, 132.93), p &amp;lt; 0.001] and spindle cell histology (SC) [HR 2.57 (1.19, 5.53), p = 0.02] predicted worse OS in multivariable analysis. Inferior PFS was predicted by DM [HR 18.84 (6.53, 54.35), p &amp;lt; 0.001], SC [HR 2.46 (1.25, 4.86), p = 0.009], and node-positivity at diagnosis [HR 3.65 (1.5, 8.89), p = 0.004]. The 5-year OS and PFS were 22.2% and 0% for DM pts versus 80.0% and 65.6% for non-DM pts. The 5-year OS and PFS were 71.5% and 54.7% for SC pts versus 81% and 66.6% for non-SC pts. 5-year OS and PFS for NACT pts was 67.3% and 52.6% for NACT pts versus 78.2% and 62.8% for non-NACT pts. Age at diagnosis, menopausal status, family history of BC, grade, stage, tumor size, hormone-receptor and HER2 status, and use of NACT were not found to be significantly associated with OS or PFS in multivariate analysis. Conclusion This study is one of the largest and most recent review of institutional experiences with MBC. Overall, OS at 5 years was improved compared to prior older studies of MBC but still remains very low for those with DM, representing an area of unmet clinical need. SC and DM correlated with worse outcomes for both PFS and OS. Additionally, node positivity at diagnosis was a predictor of worse PFS. In contrast, no association was seen between survival and tumor size, stage, hormone receptor-positivity, HER2 receptor-positivity. The low pCR rates following NACT in our study are consistent with reported literature. Further, use of NACT does not impact survival, suggesting pts with resectable disease should proceed with surgery first. Citation Format: Siven Chinniah, Raza Zarrar, Zhuo Li, Kostandinos Sideras, Alvaro Moreno-Aspitia, Saranya Chumsri, Rohit Rao, Sarah McLaughlin, James Jakub, Emmanuel Gabriel, Sanjay Bagaria, Laura Vallow, Santo Maimone, Pooja Advani. Clinical Factors associated with Survival Outcomes in Patients with Metaplastic Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-25.

Abstract P6-01-14: Genomic characterization and oncology outcomes of a locally advanced ER+/HER2- breast cancer cohort treated with neoadjuvant chemotherapy

Abstract Background: Locally advanced ER+/HER2- breast cancer (LABC) is an aggressive condition often requiring multidisciplinary management. While early and metastatic breast cancer are well characterized, LABC is largely underrepresented in clinical trials and genomic studies. Herein we present comprehensive molecular profiling of an ER+/HER2- LABC cohort and their oncology outcomes. Method: The clinical records of locally advanced ER+/HER2- LABC (EC IIIA or higher) patients diagnosed and treated with neoadjuvant chemotherapy at Hospital de Base (Sao Jose do Rio Preto, Brazil) were reviewed. Comprehensive genomic profiling was performed on formalin-fixed paraffin-embedded (FFPE) tumor samples using capture-based hybrid next-generation sequencing (NGS) by targeting 425 cancer-related genes. The status of patients’ homologous recombination deficiency (HRD) and tumor mutation burden (TMB) were also measured. Survival outcomes were estimated using the Kaplan-Meier method. Univariable and multivariable analyses were performed to assess the association between oncology outcomes with clinicopathological and molecular characteristics. A p-value of 0.05 was considered statistically significant. FDR was utilized for multiple comparisons adjustment. Result: From May 2010 and December 2019, after inclusion and exclusion criteria, 90 patients were included. The median age of the cohort was 54 (24 – 88) years old. There were 21 (23%), 65 (72%), and 4 (5%) patients with stage IIIA, IIIB, and IIIC, respectively. A majority of the patients had tumors Grade 2 (72%, 65/90), with 10 (11%) Grade 1, 12 (13%) Grade 3, and the remaining 3 (4%) being undetermined. Most patients were postmenopause, 58% (52/90). All patients received chemotherapy-based neoadjuvant treatment, and 6 (7%) achieved pathological complete response (pCR). After a median follow-up of 63 months, the median recurrence-free survival (RFS) of the entire cohort was 80.4 months and the median overall survival (OS) was not reached yet. . A lower tumor grade was strongly associated with better RFS (p = 0.00058) and OS (p = 0.00028), while the pCR subgroup did not show significantly better RFS or OS. In terms of genomic profiling, PICK3CA (32/90, 35.6%) and TP53 (27/90, 30.0%) were the most frequently mutated genes. The median TMB was 4.1 muts/Mb, ranging from 0 to 29.7 muts/Mb. Altered NOTCH pathway was a negative prognostic factor (HR: 2.6; 95%CI: 1.0 - 6.5, p = 0.042) while NRF2 pathway aberrations demonstrated poorer RFS compared to their wildtype counterparts (HR: 3.1; 95%CI: 1.1 - 8.9, p = 0.035). Of note, mutated CUL3, a key player of the NRF2 pathway, was correlated with poor RFS (HR: 42.8; 95%CI: 7.0 - 262.5, adjusted p = 0.0004) and OS (HR: 48.4; 95%CI: 8.0 - 294.0, adjusted p = 0.0003) although the sample size was restricted. Furthermore, patients carrying NOTCH2 mutations (N = 2) showed significantly shorter RFS (HR: 14.9; 95%CI: 3.0 - 74.2, adjusted p = 0.004) and OS (HR: 28.8; 95%CI: 5.2 - 160.2, adjusted p = 0.0008). TMB was not a predictor of either pCR or survival. Eight patients carried BRCA1/2 pathogenic mutations (8.9%), and ten out of 44 HRD evaluable patients (22.7%) were HRD-high (HRD score ≥ 38). However, neither BRCA1/2 mutations nor HRD-positivity was associated with pCR, RFS, or OS. Conclusion: Comprehensive genomic profiling of ER+/HER2- LABC patients revealed that altered NOTCH and NFR2 pathway genes were associated with poor survival outcomes. An analysis involving residual cancer burden (RCB) is currently ongoing. Citation Format: Maira Abreu, Larissa Furlan, Yutong Ma, Hanlin Chen, Carla Ferreira, Aline Fares, Eduardo Constantino, Gustavo R. Nora, Gabriela Lucio, Tatiana Colombo, Rui Liu, Xue Wu, Qiuxiang Ou, Daniel V. Araujo. Genomic characterization and oncology outcomes of a locally advanced ER+/HER2- breast cancer cohort treated with neoadjuvant 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 P6-01-14.

Abstract P2-21-10: Tumour associated macrophages in Breast Cancer - Are they critical players in response to Neo Adjuvant Chemotherapy?

Abstract Background Breast Cancer (BC) patients who do not obtain pathologic complete response (pCR) after neoadjuvant chemotherapy (NACT) present higher rate of relapse and worse overall survival. Recent evidence suggests that chemotherapy (CT) efficacy relies on the capacity of chemotherapeutic agents to interact with the immune system. BC features a unique tumor microenvironment (TME) comprising of multiple immune cell types including Macrophages that share a double-edged relationship with cancer as they get polarised from M1 (anti-tumour) to M2 (Pro-tumour). Pro-tumour M2 macrophages are referred to as tumor-associated macrophages (TAMs) and are implicated extensively in angiogenesis, metastasis and therapy resistance. Establishing role of TAMs, facilitates the emergence of novel strategies that exploit them as theranostic targets/tools of interest for treating cancer. Utility of using Drugs like Zoledronic Acid, Trabectedin, Rebastinib in combination with 5-fu have shown promising anti-TAM activity and improved response to CT in clinical trials. In this study, we have characterized presence of M2-TAM and its correlation to NACT response in matched primary and residual tumours by examining expression of several biomarkers. Methods Treatment naïve primary and their matched residual tumour specimens from 45 women treated at a single center were accessed through IERB approved protocols. The study included locally advanced HR+HER2-ve and TNBC tumours treated with standard NACT regimes between 2018 to 2020. To determine the association of TAM population with response to NACT, expression levels of CD68 (pan macrophage marker) &amp; CD163 (marker of М2 macrophages), were detected by immunohistochemistry (IHC) and represented as combined H score. We also performed gene expression of chemokines, inflammatory cytokines and interleukins involved in M1-M2 polarization by q-RT-PCR. Residual Cancer Burden Scoring was used to assess response and patients were divided into three groups (complete responders, partial responders and non-responders). Univariate and multi variate analysis were performed between gene expression groups and IHC groups with clinicopathological parameters. Findings from this study was validated on public data bases like TCGA, METABRIC and GEO. Results: 20% of all patients included in the study were complete responders. We arrived at a Macrophage Polarisation Score (MPS) by Gene expression and also a combined H score by IHC. MPS and H-score had a positive correlation (p=0.083) overall. Interestingly, Combined analysis of H-Score and MPS with response to treatment showed a greater and statistically significant correlation with residual tumours as compared to treatment naïve tumours (p=0.009). We also observed that high MPS and high combined H score in residual tumours were associated with increased tumour size and LVI (p=0.055 and p=0.03). Other clinicopathological parameters like receptor status, grade and stage at diagnosis were not significantly associated with H score or MPS. Taken together we found that 11% of patients who exhibited high TAM score by both H sascore and MPS fell into the non-responders category. We therefore report TAMs in residual tumours being more indicative for response to therapy compared to primary tumours. Conclusion Although primary tumours are useful for building predictive models to therapy response, we have demonstrated that there is utility in examining residual tumours as well for choice to adjuvant chemotherapy, since the tumour is constantly evolving through the NACT period. More work to arrive at a “TAM score” that could aid in choice of additional adjuvant treatment strategies is underway. We believe our work is helping us to move one step closer to Precision Medicine in a low and middle income country like India that has a higher burden of locally advanced disease. Our analysis also lends itself to becoming a clinical test since it is performed on an FFPE specimen Citation Format: Lohita Krishna, Aruna Korlimarla, B S Srinath, Anugnya Ranjolkar, Sudipta Nascar, Hari PS, Durga Devi, Nidhi l, Rekha V Kumar. Tumour associated macrophages in Breast Cancer - Are they critical players in response to Neo Adjuvant 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 P2-21-10.

Abstract OT3-15-01: TBCRC-053: P-RAD: A Randomized Study of Preoperative Chemotherapy, Pembrolizumab and No, Low or High Dose RADiation in Node-Positive, HER2-Negative Breast Cancer

Abstract Background: The introduction of immune checkpoint inhibitors (ICI) to standard neoadjuvant chemotherapy regimens has been shown to significantly improve outcomes in patients with triple negative breast cancer and is being investigated for high-risk hormone receptor-positive (HR+)/human epidermal growth factor-2 negative (HER2-) breast cancer. Preclinical evidence suggests radiation therapy (RT) can stimulate intra-tumoral T cell infiltration and enhance the expression and immune detection of tumor-specific neoantigens. This phase II pilot randomized study (NCT04443348) aims to evaluate the safety and efficacy of two different doses of preoperative primary tumor RT boost when combined with neoadjuvant pembrolizumab, then followed by standard neoadjuvant chemotherapy. Dual co-primary endpoints include determining the pathologic complete response (pCR) rate in the non-irradiated and pathologically confirmed metastatic axillary lymph node(s) in each treatment arm and quantifying tumor-infiltrating T lymphocytes in on-treatment (C1D14) tumor biopsies. We hypothesize that high-dose RT will increase the proportion of tumors with high T cell infiltration (i.e., top quartile) from 25% to 55%. Secondary endpoints include measuring residual cancer burden, evaluating tolerability of the regimen, and assessing quality of life. Exploratory endpoints include evaluation of treatment-associated changes in the tumor immune microenvironment, circulating immune cell analyses, and circulating tumor DNA kinetics. Methods: The study plans to enroll 128 participants with either triple negative (n=80) or high-risk HR=/HER2- (n=48) breast cancer who will be randomized to receive no, low (9 Gy), or high (24 Gy) dose of preoperative RT boost, after which 24 participants of either breast cancer subtype will be enrolled to an exploratory high dose proton therapy boost cohort. The eligibility criteria include patients who have biopsy-proven, axillary lymph node-positive breast cancer that is either triple negative (defined as ER&amp;lt; 10%, PR&amp;lt; 10%, and HER2-negative) or high-risk HR+/HER2- (grade III or having a high-risk genomic assay score). Study treatment is given in 6-week cycles, with 400 mg Pembrolizumab given on day 1 of each cycle. For those participants randomized to receive a preoperative RT boost, treatment is delivered in 3 fractions (3 × 3 Gy or 3 × 8 Gy) over consecutive business days, where one of the fractions is given on the same day as C1D1 Pembrolizumab. Standard neoadjuvant chemotherapy begins on C1D15 with paclitaxel (plus carboplatin for triple negative) administered weekly for 12 weeks, and then starting on C3D15, dose-dense doxorubicin/cyclophosphamide is administered every 2 weeks for 8 weeks. Following neoadjuvant treatment, participants will receive standard breast surgery (including removal of the pathologically confirmed metastatic lymph node) followed by adjuvant pembrolizumab, radiation therapy, and standard-of-care systemic therapy as clinically indicated. Tissue samples from the primary tumor and biopsy-proven lymph node are taken at baseline, C1D14, and at the time of surgery. There are eleven blood collection timepoints throughout the neoadjuvant and adjuvant settings. Participants will be followed for 2 years after surgery to assess safety and durability of responses. Results: This study has accrued 12 participants to date, including 10 with triple negative breast cancer and 2 with high-risk HR+/HER2- breast cancer. Formal results for this study are forthcoming, as the trial is actively accruing at 6 institutions, with plans to open at 3 more within the year. For persons with a specific interest in this trial, please contact Joseph Connolly, Multi-Center Coordinator, at jconnolly28@mgh.harvard.edu. Citation Format: Joseph J. Connolly, Laura M. Spring, Alphonse G. Taghian, Michele Gadd, Laura Warren, Ana C. Garrido-Castro, Tari King, Elizabeth A. Mittendorf, Jose P. Leone, Dana L. Casey, Lisa Carey, Tiffany A. Traina, Yara Abdou, Atif Khan, George Plitas, Jean Wright, Cesar Augusto Santa-Maria, Lisa Jacobs, Rachel Blitzblau, E Shelley Hwang, Carey Anders, Ian Krop, Antonio C. Wolff, Alastair M. Thompson, Elyssa Denault, Gaorav Gupta, Alice Ho. TBCRC-053: P-RAD: A Randomized Study of Preoperative Chemotherapy, Pembrolizumab and No, Low or High Dose RADiation in Node-Positive, HER2-Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-15-01.

Abstract OT3-25-01: Neoadjuvant HER2-targeted Therapy +/- Immunotherapy with Pembrolizumab (neoHIP): An Open Label Randomized Phase II Trial

Abstract Background: Immune checkpoint inhibition (ICI) is synergistic with HER2-directed therapy in pre-clinical models. Clinically, pembrolizumab (K)-mediated ICI plus HER2-directed therapy with trastuzumab (H) was safe and demonstrated modest activity in H-resistant HER2-positive (HER2+) metastatic breast cancer. Because ICI may confer more robust activity when administered earlier in the course of disease, H and K administered in the curative-intent, treatment-naive setting may allow for de-escalation of cytotoxic chemotherapy; confer life-long, tumor-specific immunity; and ultimately, improve cure rates. Moreover, the synergy of H and K with paclitaxel (T) may overcome the need for dual HER2-blockade with H plus pertuzumab (P). In this randomized, multicenter, phase II, open-label, multi-center trial the efficacy and safety of neoadjuvant THP vs THP-K vs TH-K are explored. Methods: 174 patients (pts) ≥18y with previously untreated, stage II-III, HER2+ breast cancer will be randomized and stratified by clinical nodal status (positive vs. negative) and hormone receptor status (positive vs. negative). In arm A, pts receive T at 80mg/m2 weekly for 12 weeks, H at 8mg/Kg (loading dose) and then 6mg/Kg every 3 weeks x 3 doses, P at 840 mg (loading dose) and then 420mg/Kg every 3 weeks x 3 doses (THP). In arm B, pts receive THP plus K at 200mg every 3 weeks x 4 doses (THP-K). In arm C, pts receive TH-K; however, in a preplanned interim analysis, arm C did not meet the pre-defined efficacy threshold and this arm was subsequently closed. Enrollment to arms A and B continue. Definitive surgery is 3-6 weeks after the last dose. After surgery, pts are treated per the treating physician’s discretion including radiotherapy per local clinical standard. Pts whose tumors are hormone-receptor positive will receive hormone therapy per local standard-of-care. The primary end point is pathologic complete response (pCR) rate in the breast and axilla (ypT0/Tis ypN0). Secondary end points include pCR rate by ypT0ypN0 and ypT0/Tis, residual cancer burden index, event free survival, breast conserving surgery rate, safety and overall survival. Exploratory correlative studies will characterize potential immune biomarkers predictive of efficacy and/or toxicity. Funding sources: BCRF, Merck NCT03747120 Citation Format: Heather McArthur, Jorge Henrique Santos Leal, Christina DiLauro Abaya, Sangeetha Reddy, Meredith Carter, Reva Basho, Michelle Phillips, David Chan, Hugo Hool, Dorothy Park, Mary El-Masry, Philomena McAndrew, Swati Sikaria, Laura M. Spring, Aditya Bardia, Mourad Tighiouart, Farnaz Dadmanesh, Armando Giuliano, Stephen Shiao, David B. Page. Neoadjuvant HER2-targeted Therapy +/- Immunotherapy with Pembrolizumab (neoHIP): An Open Label Randomized Phase II 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 OT3-25-01.

Abstract P6-11-14: Mitochondrial structure and function adaptation in residual triple negative breast cancer cells surviving chemotherapy treatment

Abstract Background: Neoadjuvant chemotherapy (NACT) used for triple-negative breast cancer (TNBC) eradicates tumors in only 45% of patients. TNBC patients with substantial residual cancer burden have poor metastasis-free and overall survival rates. Our previous studies demonstrated mitochondrial oxidative phosphorylation (OXPHOS) was elevated, suggesting a unique therapeutic dependency of residual tumor cells that survived after NACT. However, mechanisms underlying this enhanced reliance on OXPHOS are yet unknown. Mitochondria are morphologically plastic organelles that cycle between fission and fusion to maintain mitochondrial integrity and metabolic homeostasis. Methods: We modeled residual disease in human TNBC cells by treating with chemotherapeutic agents at the IC50 of cell killing, then evaluating surviving cells after 48 hours of treatment. We modeled residual TNBC in orthotopic patient-derived xenograft (PDX) model (PIM001p) by treating with standard front-line NACT (Adriamycin + cyclophosphamide; AC), then longitudinally harvesting tumors prior to treatment, residual, and upon regrowth. We analyzed mitochondrial morphology, mtDNA content and integrity, mitochondrial oxygen consumption rate, and metabolomic flux. We developed a U-Net based deep learning model that automatically detects and quantifies mitochondrial features in transmission electron micrographs. To test the functional dependency of mitochondrial structure in TNBC, we perturbed mitochondrial fusion genetically (by knocking down the fusion-driving protein Optic Atrophy 1, OPA1) and pharmacologically (using the first-in-class small molecule OPA1 inhibitor, MYLS22). Results: Pharmacologic or genetic disruption of mitochondrial fusion and fission resulted in decreased or increased OXPHOS rate, respectively, in TNBC cells, revealing for the first time that mitochondria morphology regulates OXPHOS in TNBC. Upon comparing mitochondrial effects of conventional chemotherapies, we found that DNA-damaging agents (adriamycin, carboplatin) increased mitochondrial elongation, mitochondrial content, flux of glucose through the TCA cycle, and OXPHOS, whereas taxanes (paclitaxel, docetaxel) instead decreased mitochondrial elongation and OXPHOS rate. Increased levels of the short protein isoform of OPA1 were observed in residual cells that not killed by DNA-damaging chemotherapy treatment. Treatment of cells with adriamycin followed by MYLS22 or given concurrently with MYLS22 drastically decreased cell growth. Conversely, cells treated with adriamycin, inducing fusion, followed by the DRP1 inhibitor Mdivi-1, further inducing fusion, were less sensitive to adriamycin than were vehicle-treated cells. Further, we observed heightened OXPHOS, OPA1 protein levels, and mitochondrial elongation in residual tumors of the PDX model following AC treatment. We found that sequential treatment first with AC, thus inducing mitochondrial fusion and OXPHOS, followed by MYLS22 to inhibit OPA1 in residual tumors, was able to suppress mitochondrial fusion and OXPHOS and significantly inhibited residual tumor regrowth. Our deep-learning algorithm identified distinct changes in mitochondrial phenotypes in residual tumors of multiple PDX models. Treatment of non-chemotherapy-treated mice with the OPA1 inhibitor MYLS22 as a single agent had no effect on tumor growth, revealing that post-AC residual tumors have an enhanced dependency on mitochondrial fusion compared to treatment-naïve tumors. Taken together, our findings establish a functional role for mitochondrial structure in chemotherapeutic response and metabolic reprogramming, which may confer survival advantage to TNBC cells. These results suggest that pharmacologic perturbation of mitochondrial structure can overcome chemoresistance in TNBC cells when administered rationally based on our understanding of chemotherapy-induced mitochondrial adaptations. Citation Format: Lily Baek, Junegoo Lee, Katherine E. Pendleton, Mariah J. Berner, Emily Goff, Lin Tan, Sara Martinez, Iqbal Mahmud, Argenis Arriojas, Alexander Zhurkevich, Tao Wang, Matthew Meyer, Bora Lim, James P. Barrish, Weston Porter, Kourosh Zarringhalam, Philip L. Lorenzi, Gloria V. Echeverria. Mitochondrial structure and function adaptation in residual triple negative breast cancer cells surviving chemotherapy treatment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-14.

Abstract P6-04-08: Machine learning-based characterization of the breast cancer tumor microenvironment for assessment of neoadjuvant-treatment response

Abstract Background: Neoadjuvant treatment of breast cancer has been shown to potentially reduce the extent and morbidity of subsequent surgery. Response to neoadjuvant therapy may also be prognostic; complete pathologic response (pCR) following neoadjuvant treatment is associated with improved long-term outcomes. pCR, defined as the absence of residual invasive cancer, is determined by evaluation of H&amp;E-stained breast resections and regional lymph nodes following neoadjuvant treatment; however, pathologist assessment is subject to intra- and inter-reader variability. Here we report machine learning (ML)-based models to identify tissue regions and cell types in the tumor microenvironment (TME) of H&amp;E-stained breast cancer specimens. Model predictions were used to derive tumor bed area, a key component of the residual cancer burden score (RCB) used to assess neoadjuvant-treatment pathological response. Methods: Convolutional neural network (CNN) models were trained using digitized H&amp;E-stained whole slide images (WSIs) of 2700 neoadjuvant-treated breast cancer specimens (resections and biopsies) from 4 sources, and an additional 1100 breast cancer primary resections from TCGA. 229,901 pathologist annotations were used to train CNN models to segment tissue regions (cancer epithelium, stroma, diffuse inflammatory infiltrate, ductal carcinoma in situ, lymph nodes and necrosis) and cell types (cancer epithelial cells, fibroblasts, lymphocytes, macrophages, foamy macrophages and plasma cells) at single-pixel resolution. These tissue region segmentations were then used to derive tumor bed area using a convex hull algorithm. Each model was evaluated by board certified pathologists for performance. Model predictions of tumor bed area were evaluated in comparison to mean measurements from 3 pathologists for each of 22 held-out test slides. To further assess cell model performance, 5 pathologists exhaustively annotated 120 frames (300 x 300 pixels) on test samples from a dataset not used in model development (N=536; resections and biopsies) to produce consensus ground truth cell labels. Model predictions were compared with pathologist annotations in these frames using Pearson correlation, precision, recall, and F1 metrics. Only those classes with greater than 50 consensus cells identified were evaluated. Results: CNN predictions of tissue and cell classes within H&amp;E breast cancer WSIs showed concordance with manual pathologist consensus labels. The weighted average Pearson correlation (across the relevant cell types) between the model and consensus was 0.75, comparable to the correlation of 0.81 between pathologists and consensus. Classification metrics for each cell class are reported in Table 1. Reduced performance of the model relative to the average pathologist performance may be due to heterogeneous slide characteristics and infrequency of some cell types in the data. For prediction of tumor bed area, CNN model predictions showed moderate correlation with pathologist consensus (Pearson r=0.65, 95% CI: 0.38-0.81). Conclusions: CNN model classification of cell types and tissue regions across entire H&amp;E breast cancer WSIs shows concordance with pathologist consensus. Model predictions of tumor bed area also show concordance with pathologist assessment and can be used to derive the RCB score. These models can be reproducibly applied to quantify diverse histological features in large datasets, potentially enabling improved standardization and efficiency of pathologist evaluation of the breast cancer TME and neoadjuvant response. Classification Metrics for Individual Cell Classes Citation Format: Christian Kirkup, Sanjana Vasudevan, Filip Kos, Benjamin Trotter, Murray Resnick, Andrew H. Beck, Michael Montalto, Ilan Wapinski, Ben Glass, Mary Lin, Stephanie Hennek, Archit Khosla, Michael G. Drage, Laura Chambre. Machine learning-based characterization of the breast cancer tumor microenvironment for assessment of neoadjuvant-treatment response [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-04-08.