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Background: Patients treated for early stage breast cancer (BC) have a 30% lifetime risk of developing metastatic disease. Numerous studies have demonstrated that dormant bone marrow disseminated tumor cells (DTCs) are independently associated with risk of recurrence and death, yet interventions targeting these cells are lacking. The PENN-SURMOUNT (Surveillance Markers of Utility for Recurrence after (Neo)adjuvant Therapy) Screening Study was launched in 2016 to screen high risk BC survivors for DTCs using bone marrow aspirate (BMA) and identify eligible DTC positive patients for clinical trials. Given the novelty of this approach, we concurrently developed and pilot tested a PRO measurement strategy to study how the screening method of BMA and disclosure of DTC results impacts early-stage BC patients. Methods: PENN-SURMOUNT is a single center prospective, longitudinal cohort study examining BM and blood biomarkers of MRD among patients within 5 years of BC diagnosis who have high risk criteria (positive axillary nodes, triple negative biology, ER+ with Oncotype Dx ≥ 25 and/or high risk Mammaprint, or pathologic residual disease after neoadjuvant chemotherapy). From May 2019 – August 2021, we recruited patients on SURMOUNT to complete PRO surveys at baseline (T0), after BMA (T1), and after disclosure of DTC results (T2). Surveys were administered in paper form initially, then electronic form starting Feb 2021. PRO survey instruments were selected through literature review, followed by consensus among multidisciplinary clinical and research experts and patient advocates. PRO measures assess recurrence distress (Quality of Life in Adult Cancer Survivors, QLACS), illness intrusiveness (Illness Intrusiveness Ratings Scale, IIRS), and decision making (Decision Regret Scale). Additional survey items assess tolerability of the BMA and patients’ risk perception and cognitive understanding after DTC results disclosure. Descriptive statistics summarize PRO survey compliance and responses at T0, T1, and T2 in the total population and the population who reported longitudinal data for T2. Results: 61 of 66 eligible patients on the SURMOUNT trial enrolled in the PRO pilot study and completed a baseline survey, of which 47 (77%) tested negative for DTCs. Mean completion rates were 0.92 at T0, 0.85 at T1, and 0.56 at T2. After electronic survey implementation, completion rates increased to 0.94 (T0), 0.97 (T1) and 0.81 (T2). At T0, 36 (59%) patients reported a high risk perception of developing BC recurrence at 5 years and 42 (69%) during their lifetime. Mean T0 recurrence distress using the QLACS subscale was 14.6 (SD 6.3) out of possible score 4-28, compared to an expected mean of 11.42 (SD 5.48) in a general survivorship population. Mean T0 illness intrusiveness was 27.3 (SD 13.9) out of possible score 13-91. At T1, approximately 85% of patients agreed that they correctly understood the purpose of the bone marrow procedure and what the procedure would entail. 44 (72%) of patients reported a maximum pain score Citation Format: Tara Kaufmann, Patrick Chang, Shoshana Rosenberg, Elizabeth Frank, Brian Hobbs, Lauren J. Bayne, Isoris Nivar, Brooke L. Goodspeed, Killian M. Rohn, Emily M. Kugler, Kevin Fox, Susan Domchek, Angela Bradbury, Payal Shah, Hayley Knollman, Rachel C. Jankowitz, Igor Makhlin, Amy S. Clark, Lewis A. Chodosh, Angela DeMichele, Katherine Goodfellow. Pilot study of a patient-reported outcome (PRO) measurement strategy to determine impact of screening for minimal residual disease (MRD) in high-risk breast cancer survivors [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 P5-08-01.

Background CDK4/6 inhibitors (CDK4/6i) in combination with endocrine therapy (ET) have a well-established role in the management of hormone receptor-positive (HR+)/HER2- metastatic breast cancer (MBC). The benefit of continuing CDK4/6i beyond progression in combination with a different ET has not been confirmed. Preclinical data suggest synergy between CDK4/6i and PD-L1 inhibition. The PACE trial prospectively evaluates whether continuation of the CKD4/6i palbociclib beyond progression on prior CDK4/6i and aromatase inhibitor (AI), with a change in ET to fulvestrant, improves outcomes beyond change to fulvestrant alone, as well as explores the activity of the palbociclib, fulvestrant, and avelumab triplet. Methods PACE is a multicenter randomized open-label investigator-initiated phase II trial, open at 11 U.S. sites. Eligible patients (pts) had HR+/HER2- evaluable MBC with prior progression on AI and any CDK4/6i after > 6 months (mo) of therapy in the MBC setting, or during/within 12 mo in the adjuvant setting, with no more than 1 prior line of chemotherapy for MBC. Pts were randomized 1:2:1 to fulvestrant alone (F); fulvestrant and palbociclib (F+P); or fulvestrant, palbociclib, avelumab (F+P+A), with tumor assessments every 8 weeks. Blood for circulating tumor DNA (ctDNA) analysis was collected at baseline, at times of tumor assessments, and at progression. The primary objective was to evaluate progression-free survival (PFS) with F+P vs F; secondary objectives included PFS with F+P+A vs F, objective response rate (ORR) in all arms, and safety. A sample size of 220 patients was planned to provide 80% power to detect an improvement in PFS with HR 0.6154 with F+P vs F (6.5 vs 4 mo; α(1)=0.05). Results A total of 220 pts were randomized from 9/2017-2/2022 (F: n=55, F+P: n=111, F+P+A: n=54); median age 57 years (range 25-83), 85% non-Hispanic (7.7% non-Hispanic black), 8.6% Hispanic, 6.4% unknown. 40% had de novo MBC, 60% had visceral disease, and 14% bone-only disease. 16% had 1 prior line of chemotherapy for MBC, 90% had received prior palbociclib, 4.5% ribociclib, 4.1% abemaciclib, 1.4% palbociclib and ribociclib. Pts entered the trial after a median 19 mo of prior CDK4/6i plus AI (interquartile range 12-31 mo). A total of 10 (5%) pts received protocol therapy as first line ET for MBC, 169 (77%) as second line, and 41 (17%) as beyond second line. 88% entered the trial directly after progression on CDK4/6i. After a median follow-up of 24 mo, 18 pts remained on protocol treatment. PFS was not improved with F+P vs F (median 4.6 vs 4.8 mo; HR=1.11, 90% CI 0.79-1.55; 2-sided p=0.62). Median PFS was 8.1 mo with F+P+A (HR=0.75 vs F, 90% CI 0.50-1.12; 2-sided p=0.23). ORR was 7.3% (90%CI 1.5-13.0) with F, 9.0% F+P (4.5-13.5%) and 13.0% F+P+A (5.4-20.5%). No new safety signals have been observed. Analysis of ctDNA panel sequencing encompassing 70 genes from 184 baseline samples, including correlation with known and hypothesized resistance genes, will be presented. Conclusions For ER+/HER2- breast cancer, combining palbociclib with fulvestrant beyond progression on prior CDK4/6i and AI did not significantly improve PFS compared with using fulvestrant alone. The observed longer PFS when a PD-L1 inhibitor was added to fulvestrant plus palbociclib is an intriguing signal in this ER+ population. Translational studies of blood and tumor tissue are ongoing and will be presented. Citation Format: Erica L. Mayer, Yue Ren, Nikhil Wagle, Reshma Mahtani, Cynthia Ma, Angela DeMichele, Massimo Cristofanilli, Jane Meisel, Kathy D. Miller, Trevor Jolly, Elizabeth Riley, Rubina Qamar, Priyanka Sharma, Sonya Reid, Natalie Sinclair, Meredith Faggen, Caroline Block, Naomi Ko, Ann Partridge, Wendy Y. Chen, Michelle K. DeMeo, Victoria Attaya, Amanda Okpoebo, Yuan Liu, Eric Gauthier, Harold Burstein, Meredith Regan, Sara Tolaney. GS3-06 Palbociclib After CDK4/6i and Endocrine Therapy (PACE): A Randomized Phase II Study of Fulvestrant, Palbociclib, and Avelumab for Endocrine Pre-treated ER+/HER2- Metastatic 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 GS3-06.

Background: Breast cancer with low HER2 expression (HER2-low) is of high clinical relevance because of new therapeutic options with antibody-drug conjugates. We have recently shown in a large cohort from neoadjuvant clinical trials that HER2-low breast cancer has different molecular characteristics as well as different clinical outcomes compared to HER2-zero. Considering the positive correlation between HER2-low expression and hormone receptor positivity observed consistently in many investigations, we have extended our analysis to HR+ tumors from the post-neoadjuvant PenelopeB trial. In PenelopeB, patients with HR+ breast cancer and residual disease after neoadjuvant chemotherapy (NACT) were randomized to post-neoadjuvant palbociclib versus placebo in addition to endocrine therapy. We evaluated the molecular phenotype and clinical outcomes of HER2-low compared to HER2-zero patients. Methods: A total of 1250 patients were randomized, HER2 status was available for 1151 tumors from pretherapeutic core biopsy, determined mainly by local pathology, and from 1213 tumors from the post-NACT sample, determined as part of central pathology. For 1119 patients a paired HER2-status was both available. HER2-zero was defined as IHC0 and HER2-low-positive was defined as IHC1+ or IHC2+/ISH-. Gene expression analysis of 2549 genes using the HTG oncology biomarker panel was performed in 620 pretherapeutic biopsies and 780 post-NACT residual tumor samples, with 539 paired gene expression samples. Breast cancer subtypes were determined using the AIMS approach. Results: In pretherapeutic biopsies, 695 tumors (60%) were HER2-low and 457 (40%) were HER2-zero. A HER2-low status in the biopsy was significantly linked to improved iDFS (HR 0.76 (0.60-0.96; p=0.02). In residual tumors, 632 tumors (60%) were HER2-low and 581 (40%) were HER2-zero, without any prognostic impact of HER2 low status. In addition, a shift of HER2-low-status comparing core biopsy and residual tumor was observed in 415 (37%) of 1119 tumors. 161 (14%) had a shift from HER2-zero to HER2-low and 254 (23%) shifted from HER2-low to HER2-zero. A shift from HER2-zero to HER2-low in the post-NACT samples was significantly linked to reduced iDFS (HR 1.43 [95%CI 1.01-2.01]), p=0.04), compared to HER2-low group, while a shift from HER2-low to HER2-zero was associated with better iDFS compared to HER2-zero group, although not statistically significant (p=0.17). We did not observe a significant correlation of HER2-low status and AIMS molecular subtypes. In particular, the HER2-enriched (HER2E) subtype was assigned to only 4.3% of HER2-zero and 3.1% of HER2-low tumors. Significant iDFS differences were observed for HER2-low-status in combination with AIMS subtypes (lumB/basal/HER2E vs. lumA/normL; overall p-value < 0.0001) for both pretherapeutic biopsies and residual tumor. Patients with post-NACT HER2-low tumors had an improved survival in the subgroups of aggressive AIMS subtypes (lumB/basal/HER2E), but not in the less aggressive AIMs subtypes (lumA/normL), with a positive test for interaction (p=0.02). For the pre-NACT samples a similar, but non-significant trend was observed. We evaluated a total of 620 core biopsies for differences in gene expression comparing HER2-low and HER2-zero tumors. A total of 417 genes were statistically significantly different, but in a hierarchical clustering there was no clear separation of HER2-low and HER2-zero tumors. Conclusions: In the PenelopeB cohort of HR+ tumors, a HER2-low status in pretherapeutic core biopsies is related to improved disease-free survival, especially for those tumors that have a more aggressive intrinsic subtype. A shift of HER2-low status was observed before and after chemotherapy, indicating an adaptation of the pathway activity to therapy-induced stress, which might become relevant for future diagnostic and therapeutic approaches. Citation Format: Carsten Denkert, Miguel Martín, Michael Untch, Hervé R. Bonnefoi, Erik S. Knudsen, Seock-Ah Im, Angela DeMichele, Agnieszka Witkiewicz, Laura Van ’t Veer, Sung-Bae Kim, Harry D. Bear, Nicole McCarthy, Karen Gelmon, Frederik Marmé, José Ángel García-Sáenz, Nicholas Turner, Federico Rojo, Martin Filipits, Lesley-Ann Martin, Peter A. Fasching, Christian Schem, Catherine M. Kelly, Toralf Reimer, Masakazu Toi, Hope Rugo, Michael Gnant, Andreas Makris, Yuan Liu, Karsten Weber, Sivaramakrishna Rachakonda, Sibylle Loibl. HER2-06 Outcome analysis of HER2-zero or HER2-low hormone receptor-positive (HR+) breast cancer patients - characterization of the molecular phenotype in combination with molecular subtyping [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 HER2-06.

PURPOSE To examine incremental values of estrogen receptor (ER) status, body mass index (BMI), menopausal status, and a previously reported multi-gene classifier over commonly used clinical factors (i.e. age, tumor grade, comedonecrosis, surgical margins, and treatment) in predicting risk of any ipsilateral recurrence (IR) event within five years after DCIS diagnosis. METHODS A derivation cohort consisted of participants in the Translational Breast Cancer Research Consortium (TBCRC) 038, a retrospective multicenter cohort study in women undergoing surgical resection for DCIS between 01/01/1998 and 02/29/2016 (n=216). The validation cohort, the Repository of Archival Human Breast Tissue (RAHBT) at Washington University School of Medicine, provided cases meeting the same eligibility criteria as TBCRC038 (n=97). Participants in both cohorts had RNA-seq data and either developed IR 1-5y after initial DCIS diagnosis or were free from subsequent breast events for at least five years. The previously reported 812-gene classifier had been developed from a subset of the TBCRC038 samples using a negative-binomial regression model to identify differentially expressed genes in the primary tumor associated with subsequent recurrence events. This classifier has been shown to be highly correlated with 5-year invasive, DCIS, and all breast cancer events, and validated in the RAHBT cohort. Cox proportional hazards regression was used to estimate hazard ratios (HRs) of IR in the TBCRC038 cohort (76 with IR). The clinical score was developed using clinical predictors (aforementioned clinical factors and ER) and their regression coefficients from the model with the maximum predictive accuracy (e.g. c-index) and the minimum number of predictors; the summary score integrated the clinical score and multi-gene classifier. Predictive performance of both clinical and summary scores was validated in the RAHBT cohort (20 with IR). RESULTS In the TBCRC cohort derivation set, we used a multivariable model based on clinical factors alone (clinical score) and found that ER status, but not BMI or menopausal status, was independently associated with a higher IR risk (HR=2.06, 95% CI 1.18-3.58). Adding the multi-gene classifier to the clinical factors-based model (summary score) in the TBCRC038 test set increased predictive accuracy (c-index 0.68 to 0.70), with the genomic classifier-adjusted HR of 14.96 (95% CI 8.64-25.91). The summary score had higher predictive performance for IR risk than clinical score alone (c-index 0.82 vs. 0.70). In the RAHBT validation samples, model performance was similarly improved using summary scores clinical factors-based model plus multigene classifier as compared to clinical scores along (c-index 0.74 vs. 0.58). CONCLUSION Combining clinical factors and a multigene classifier provided more accurate risk estimates of IR within five years after excision of DCIS than clinical factors alone. Figure 1. Observed and predicted recurrence-free survival in the first five years after initial DCIS diagnosis in the RAHBT validation cohort, by risk groups defined by clinical scores (left) and clinical score plus multigene classifier (right). Citation Format: Ying Liu, Siri H. Strand, Lorraine King, Bryan Harmon, Fergus J. Couch, Kristalyn Gallagher, Mark Kilgore, Shi Wei, Angela DeMichele, Tari King, Priscilla F. McAuliffe, Jeffrey Marks, Carlo Maley, Robert West, E Shelley Hwang, Graham A. Colditz. Using clinical characteristics and molecular markers to predict the risk of subsequent ipsilateral breast events after excision of DCIS [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 P1-07-02.

Purpose: To predict pathologic complete response (pCR) in breast cancer patients undergoing neoadjuvant chemotherapy (NAC), from baseline and early-treatment DCE-MRI scans, in the context of the ACRIN 6698/I-SPY 2 BMMR2 challenge. Materials and Methods: The BMMR2 dataset consists of 191 patients undergoing NAC for locally advanced breast cancer as part of the ACRIN 6698/I-SPY 2 trial. DCE-MRI was obtained at time points T0 (pre-NAC), T1 (3 weeks), and T2 (12 weeks). The BMMR2 challenge provided the MRI scans, tumor annotations, and limited clinical and demographic information. The data were split 60/40; using the 60% training data, the task was to develop models to predict pCR; the competition was for best area under the curve (AUC) when applied to the 40% unseen test data. Using the publicly available CaPTk software we calculated 3 types of radiomic features within the segmented tumor volume: 1) texture of the signal enhancement ratio (SER) kinetic map of T0 images; 2) texture of the difference between the T1 kinetic maps (PE, WIS, WOS, and SER) and corresponding T0 maps; 3) texture of the difference between the T1 kinetic maps and the corresponding T0 maps, with T1 scans deformably registered to T0 scans. ComBat harmonization was applied to the extracted features to account for MRI acquisition differences. We computed the tumor longest diameter, functional tumor volume (FTV), and clinical tumor size each at T0 and T1. We modeled pCR via logistic regression. Using the training data alone, with the criteria of performance in univariable modeling and low collinearity, we selected radiomic features and clinical, demographic, and size covariates. We then performed PCA on the combined set of selected radiomic features and size covariates. We evaluated multivariable models including the selected clinical covariates in combination with the first few PCs via cross-validated AUC (5-fold, 200 repetitions) on the training data. The best models were submitted for independent evaluation on the unseen test data of the BMMR2 challenge. Results: Of the available clinical covariates, only hormone receptor (HR)± and human epidermal growth factor receptor 2 (HER2)± had any association with pCR. We retained these in all models, and performed PCA on the set combining the best-performing features and the size variables FTV at T0, FTV at T1, and longest diameter at T1. Models based on the first few PCs, HR, and HER2, had training AUCs in 0.78–0.81. Our best-performing model had an AUC on test data of 0.84, using the covariates PCs 1–5, HR, and HER2 (Table 1). Conclusions: Our preliminary results suggest that radiomic phenotyping of changes in tumor heterogeneity can accurately predict pCR early in the course of NAC. Future analysis with larger samples from ISPY-2 could also examine the effect of different therapies, including targeted therapy and immunotherapy. Table 1: Performance of candidate logistic regression models on training and test data. AUC: Area under receiver operating characteristic curve. * Mean 5-fold cross-validated AUC across 200 replicates. † Competition best-performing predictions. Citation Format: Eric A. Cohen, Rhea D. Chitalia, Snekha Thakran, Walter C. Mankowski, Alex Anh-Tu Nguyen, Hannah Horng, Elizabeth S. McDonald, Michael Feldman, Angela DeMichele, Despina Kontos. Title: Characterizing Changes in Tumor Heterogeneity via Radiomic Phenotyping for Predicting Response to Neoadjuvant Chemotherapy for Locally Advanced Breast Cancer: Preliminary Results from the ACRIN 6698/I-SPY 2 trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD16-08.

Background: As the development and use of oral anticancer agents increases, it is critical to understand patient adherence to both standard and investigational agents. The open label, phase 3 multicenter PALLAS trial investigates whether adding 2 years of the CDK4/6 inhibitor palbociclib (P) to adjuvant endocrine therapy (ET) improves invasive disease-free survival (iDFS) over adjuvant ET alone in patients (pts) with HR- positive, HR2-negative, stage II-III breast cancer. Pts were randomly assigned to either Palbociclib (125 mg/day, 3 weeks on, 1 week off, in a 28-day cycle) plus ongoing provider/patient-choice adjuvant ET (P+ET) versus ET alone. We examined patient-reported adherence to ET +/- P during the first two years of study treatment. Methods: Adherence outcomes were measured in English-speaking pts in the U.S., UK, Ireland and Australia, Spanish-speaking pts in Spain and Mexico, and German-speaking pts in Germany and Austria. Adherence measures included drug diaries completed at each cycle, pill counts (for P only) collected at each study visit, and the Morisky Medication Adherence Scale-4 item and the McHorney Adherence Estimator questionnaires completed at cycles 2, 3, 6, 12, 18, and 24 (22 months). Mean adherence for each cycle was defined as the average proportion of prescribed pills taken (via drug diary) across all patients who initiated that cycle. Persistence was defined as the duration of drug initiation to treatment cessation (via drug diary). Generalized estimating equations were used to model the “most adherent” pts on the Morisky (score = 5 vs score 0) to compare the average difference between arms over time for ET, adjusting for baseline demographic and clinical variables. Results: 81% (N=4688) of PALLAS pts were included in this analysis. Median persistence to ET was 23.6 months in P + ET (n=2169), 23.7 months in ET alone (2136) and 20.4 months for P (n=2194). The number of pts who initiated each cycle for ET declined over time and was similar between arms; the decline was more marked for P (Table 1). Mean adherence range as measured by drug diary was 98.2-99.3% for ET in P+ET and 98.0 - 99.4% in ET alone; and for P, ranged from 93.4 - 98.8%. The adherence and persistence results were nearly identical whether measured by drug diary or pill count for P. The observed percent “most adherent” for P measured by the Morisky scale ranged from 71.9% - 79.6% and the percent “low risk” for adherence problems measured by the McHorney scale, 64.0% - 73.4%. The percent of pts “most adherent” and “low risk” for adherence problems to ET was higher, on average over time, in the P+ET group compared to ET alone (75% vs 68%, p Table 1.Adherence and persistence in PALLAS over the 24 month treatment periodPalbocicilb + ETETPalbociclibETETTreatment cycleMean Adherence (SD)N*Mean Adherence (SD)NMean Adherence (SD)N193.4 (13.8)229098.7 (6.1)230999.0 (5.2)2343294.7 (13.3)218198.8 (6.4)220398.4 (7.1)2206397.4 (10.1)211298.7 (7.3)214298.8 (6.3)2168497.6 (10.2)203598.6 (7.8)211099.0 (5.7)2154597.6 (11.1)197598.3 (7.9)209398.1 (8.3)2148698.1 (8.7)189998.7 (6.6)203798.5 (8.2)2116798.1 (9.3)185599.0 (6.3)201298.6 (7.3)2102898.0 (9.1)181098.2 (8.2)199798.1 (8.2)2092998.4 (7.4)173399.0 (6.4)196298.8 (6.8)20511098.3 (8.6)171499.2 (4.9)194699.0 (5.5)20381198.3 (8.0)169198.5 (7.3)193798.2 (8.2)20311298.7 (6.2)161299.1 (5.3)191098.7 (7.5)19861398.5 (7.7)159698.9 (6.4)189398.8 (7.1)19691497.9 (9.3)157698.4 (7.3)188298.0 (9.1)19611598.5 (6.9)151499.2 (4.6)185499.0 (6.0)19281698.2 (8.7)148699.1 (4.6)183599.1 (5.7)19131798.1 (9.8)147998.3 (7.8)182698.5 (6.4)19031898.7 (6.8)141699.3 (4.1)176899.2 (5.3)18601998.7 (7.3)140899.3 (3.8)175699.4 (3.1)18482098.4 (8.9)139798.9 (5.3)174298.7 (5.8)18382198.5 (7.5)130199.0 (6.1)167698.8 (7.1)17842297.8 (9.7)124698.8 (6.2)164799.2 (5.7)17692398.1 (8.8)118098.2 (8.5)161998.4 (8.0)17552498.8 (7.2)108699.0 (6.3)146898.9 (7.1)16192598.8 (6.7)99699.2 (5.2)143298.8 (7.1)15892697.9 (11.2)84599.1 (5.7)140998.6 (8.8)1578ET=Endocrine therapy * Number of pts who initiated the treatment cycle. Citation Format: Eileen Shinn, David Zahrieh, Angela DeMichele, Nick Zdenkowski, Julie Lemieux, Jun Mao, Vesna Bjelic-Radisic, Michelle Naughton, Georg Pfeiler, Karen Gelmon, Ingrid Mayer, Daniel Egle, Gabriele Zoppoli, Tiffany Traina, Miguel Martin Jiménez, Silvia Antolin Novoa, Tufia Haddad, Arlene Chan, Alistair Edward Ring, Antonio Wolff, Jose JuanPonce Lorenzo, Dhanusha Sabanathan, Hal Burstein, Zbigniew Ireneusz Nowecki, Gunda Pristauz-Telsnigg, Adam Brufsky, Meritxell Bellet-Ezquerra, Theodoros Foukakis, Yelena Novik, Gabor Rubovszky, Karoline Muehlbacher, Otto Metzger, Theodora Goulioti, Ernest Law, Ann Partridge, Lisa Carey, Alex Zoroufy, Dominik Hlauschek, Christian Fesl, Erica Mayer, Michael Gnant. Adherence with adjuvant endocrine therapy with or without Palbociclib in the PALLAS trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-12-01.

Background. DCIS consists of a molecularly heterogeneous group of premalignant lesions, with variable risk of invasive progression. Understanding biomarkers for invasive progression could help individualize treatment recommendations based upon tumor biology. As part of the NCI Human Tumor Atlas Network (HTAN), we conducted comprehensive genomic analyses on two large DCIS case-control cohorts. Methods. We performed smart3-seq and low-pass whole genome sequencing on two independent, retrospective, longitudinally sampled DCIS case-control cohorts. TBCRC 038 was a multicenter cohort diagnosed with DCIS between 1998 and 2016 at one of the Translational Breast Cancer Research sites; the RAHBT (Resource of Archival Human Breast Tissue) cohort included women identified through the St. Louis Breast Tissue Repository, and the Women’s Health Repository diagnosed between 1997 and 2001. We studied the spectrum of molecular changes present and sought genomic predictors of subsequent ipsilateral breast events (iBEs: DCIS recurrence or invasive progression) in both DCIS epithelium and stroma in formalin fixed paraffin embedded tissue. We generated de novo tumor and stroma-centric subtypes for DCIS that represents fundamental transcriptomic organization. Copy number analysis was performed using low-pass DNA sequencing. Non-negative matrix factorization (NMF) was applied to the RNA expression of all coding genes to identify clusters. A negative-binomial regression model was used to identify differentially expressed genes. Results. We analyzed 677 DCIS samples from 481 patients with 7.1 years median follow-up. In TBCRC samples, we identified three clusters via NMF in TBCRC referred to as ER low, quiescent, and ER high. The ER-low cluster had significantly higher levels of ERBB2 and lower levels of ESR1 compared to quiescent and ER-high clusters. Quiescent cluster lesions were less proliferative and less metabolically active than ER high and ER low subtypes. These findings were replicated in the RAHBT cohort. Focusing on the stromal component of DCIS from laser capture microdissection in RAHBT samples, we identified four distinct DCIS-associated stromal clusters. A “normal-like” stromal cluster with ECM organization and PI3K-AKT signaling; a “collagen-rich” stromal cluster; a “desmoplastic” stromal cluster with high fibroblast and total myeloid abundance, mostly associated with macrophages and myeloid dendritic cells (mDC); and an “immune-dense” stromal cluster. Further, we compared differentially expressed genes in patients with or without subsequent iBEs within 5 years of diagnosis. Hypothesizing that the resulting 812 DE genes (DESeq2) represent multiple routes to subsequent iBEs, we leveraged NMF to identify paths to progression. In both TBCRC and RAHBT cohorts, poor outcome groups exhibited increased ER, MYC signaling, and oxidative phosphorylation, supporting that these pathways are important for DCIS recurrence and progression. Conclusion. Comprehensive genomic profiling in two independent DCIS cohorts with longitudinal outcomes shows distinct DCIS stromal expression patterns and immune cell composition. RNA expression profiles reveal underlying tumor biology that is associated with later iBEs in both cohorts. These studies provide new insight into DCIS biology and will guide the design of diagnostic strategies to prevent invasive progression. Citation Format: Siri H Strand, Belén Rivero-Gutiérrez, Kathleen E Houlahan, Jose A Seoane, Lorraine M King, Tyler Risom, Lunden Simpson, Sujay Vennam, Aziz Khan, Timothy Hardman, Bryan E Harmon, Fergus J Couch, Kristalyn Gallagher, Mark Kilgore, Shi Wei, Angela DeMichele, Tari King, Priscilla F McAuliffe, Julie Nangia, Joanna Lee, Jennifer Tseng, Anna Maria Storniolo, Alastair Thompson, Gaorav Gupta, Robyn Burns, Deborah J Veis, Katherine DeSchryver, Chunfang Zhu, Magdalena Matusiak, Jason Wang, Shirley X Zhu, Jen Tappenden, Daisy Yi Ding, Dadong Zhang, Jingqin Luo, Shu Jiang, Sushama Varma, Cody Straub, Sucheta Srivastava, Christina Curtis, Rob Tibshirani, Robert Michael Angelo, Allison Hall, Kouros Owzar, Kornelia Polyak, Carlo Maley, Jeffrey R Marks, Graham A Colditz, E Shelley Hwang, Robert B West. The Breast PreCancer Atlas DCIS genomic signatures define biology and correlate with clinical outcomes: An analysis of TBCRC 038 and RAHBT cohorts [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 GS4-07.

Background: Circulating IL-6, an activator of JAK/STAT signaling, is associated with poor outcomes and aromatase inhibitor (AI) resistance in hormone-receptor positive (HR+) metastatic breast cancer (MBC). We previously presented clinical outcomes of JAKEE, a single arm, phase II, Simon two-stage clinical trial that tested Ruxolitinib (Rux), an oral selective inhibitor of JAK1/2, and exemestane (EXE) in 25 participants (pts) with HR+ MBC that relapsed/progressed on non-steroidal AI (NSAI); specifically, the primary endpoint of safety was met, but there were no complete or partial responses and 6/25 (24%) achieved stable disease (SD) for ≥6 cycles. We investigated whether host circulating inflammatory markers, IL-6 genotypes, and estrogen levels were associated with differential response to therapy.Methods: Responders (R) were defined as having achieved SD≥6 cycles. Flow cytometry was performed on baseline and on-treatment peripheral blood samples to assess downstream CD3+ T-cell phosphoSTAT3 inhibition by Rux. Serum concentrations of C-reactive protein (CRP), IL-6, serum amyloid A (SAA), Estrone (E1) and Estradiol (E2) were measured at baseline and serially on treatment. Sanger sequencing was performed to assess for three functional variants of the IL-6 promoter: −572G>C (rs1800796), −597G>A (rs1800797), and −174G>C (rs1800795), with high-risk polymorphisms being -597G/G and/or -174G/G. Non-parametric median testing was employed to test for differences in circulating markers by response groups given non-normal distribution, with a two-sided alpha of 0.05.Results: The cohort was heavily pre-treated: 28% received ≥2 lines of chemotherapy for MBC and 20% had CNS disease at enrollment. Among 17/25 pts with samples for pharmacodynamic assessment, Rux exhibited a 25% median inhibition (range 0-77%) of phosphoSTAT3. There was no differential effect in R vs non-responders (NR) (median inhibition 20% vs 29%, p=0.15). Frequency of high-risk IL-6 genotypes and distribution of baseline serum CRP, IL-6, SAA, E1 and E2 are depicted in the table. 15/25 (60%) harbored a high-risk IL-6 promoter polymorphism, with no significant difference in frequency between R and NR (50% vs 63%, p=0.65). 19 pts had samples for inflammatory biomarker analysis. 16/19 had baseline CRP≥10mg/L. While median levels of baseline CRP, SAA, and IL-6 were above upper limit of normal, there was no difference between R and NR (table). The proportion of pts with baseline undetectable E1 and E2 were similar between R and NR (E1: 36.9% vs 33.3%, p=1.0, E2: 52.6% vs 50%, p=1.0); notably, a significantly lower proportion with high-risk IL-6 genotype had undetectable baseline E1 (20% vs 60%, p=0.041), while no difference was noted for baseline E2, nor in the % change in E1 or E2 levels from baseline to cycle 4 by responder status.Conclusions: The JAKEE cohort represents an inflamed population with elevated circulating inflammatory markers and a high proportion with high-risk IL-6 genotypes. Examination of host inflammatory markers, IL-6 genotypes and estrogen levels did not reveal a differential response to the combination of Rux and EXE in patients with HR+ MBC that had progressed on prior NSAI. At tolerable dosing, Rux exhibited only a modest inhibition of phosphoSTAT3. Further work is needed to optimize strategies for targeting inflammation and JAK/STAT signaling in HR+ MBC. IL-6 GenotypeFrequencyFrequency by ResponderSignificanceHigh-Risk -174G/G and/or -597G/G15/25 (60%)Non-responder12/19 (63.2%)Responder3/6 (50%)p-value0.56Pretreatment Inflammatory Biomarkers [Upper limit of normal]Median (Range)Median Level by Responder GroupSignificanceCRP [8mg/L]SAA [10mg/L]IL-6 [2pg/mL]24.0 (0.2-146.8)12.8 (2.8-162.5)4.2 (1.8-11.5)Non-responder23.219.84.5Responder24.7 12.42.7p-value0.510.150.15Baseline Estrogen BiomarkersMedian (Range)Non-responderResponderp-valueEstrone (E1) (pg/mL)Estradiol (E2) (pg/mL)79.5.0 (0.2-1039.0)0.2 (0.2-44.1)79.8 0.247.8 4.20.410.91 Citation Format: Igor Makhlin, Nicholas McAndrew, E. Paul Wileyto, Amy Clark, Robin Holmes, Lisa N Bottalico, Grace R Jeschke, Kevin R Fox, Susan M Domcheck, Jennifer M Matro, Angela R Bradbury, Natalie Shih, Michael D Feldman, Elizabeth O Hexner, Jacqueline F Bromberg, Angela DeMichele. Analysis of host inflammatory and estrogen biomarkers in JAKEE: A phase II trial of the JAK inhibitor ruxolitinib in combination with exemestane for estrogen receptor-positive metastatic 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 P2-07-04.

Background: Quality of life (QOL) in breast cancer patients (pts) can be greatly impacted by initial treatment and ongoing therapy, particularly if side effects and symptoms are not well tolerated. The PALLAS trial investigated whether the addition of 2 years of palbociclib (palbo) to adjuvant endocrine therapy (ET) improved invasive disease-free survival (iDFS) over adjuvant ET alone. We report on the main patient-reported outcome (PRO) quality of life (QOL) and symptom severity results of this trial by treatment arm. Methods: PALLAS is an ongoing multicenter, open label phase 3 trial that enrolled hormone receptor positive, HER2-negative, stage II-III breast cancer patients at 406 cancer centers across 21 countries. Patients were randomly assigned (1:1) to either 2 years of palbo (125mg/day, 3 weeks on 1 week off) plus ongoing provider or patient-choice adjuvant ET (palbo+ET) versus ongoing ET alone. The primary study endpoint was iDFS. Treatment with palbo in all pts stopped at the time of the second interim analysis (5/2020) due to futility; all pts then moved to follow up. The PRO analyses were triggered for completion after awareness of the pre-specified 469 iDFS cases in November 2020. Eight PRO endpoints were measured serially (i.e., day 1 of each monthly cycle for the first 3 months, then every 3 months for the first 2 years, and once at year 3). The endpoints were the EORTC QLQ-C30 global health status/QOL score, the Brief Fatigue Inventory score, the modified Brief Pain Inventory severity and pain interference scores, the EORTC QLQ-BR23 alopecia score, and the Breast Cancer Prevention Trial hot flash symptoms, vaginal problems, and musculoskeletal pain scores. Linear mixed models compared the average difference between arms across time points during the initial 2-year treatment period adjusting for cycle 1 day 1 (C1D1) PRO scores, demographic and clinical variables. The average differences between arms (palbo+ET vs ET alone) and the two-sided (1-[0.05/8]) x 100% confidence intervals, adjusted for multiple comparisons, were calculated. Analysis of covariance compared the average between-arm differences by endpoint at 3 years. Results: The PRO intention to collect population included 4688 (81%) of the overall trial pts, and was clinically and demographically representative of the remaining 1073 pts. Each analysis population, with measures at C1D1 and at least 1 post-C1D1 assessment for each PRO endpoint, comprised ≥ 89% of the 4688 pts and the proportions were similar between arms. After adjustment for baseline covariates, on average, no clinically important differences between arms were observed for any of the eight endpoints over the 2 year treatment period (Table 1). All effect sizes were below the pre-specified 0.2 threshold. These PRO results were similar at the 3 year time point. Conclusions: No clinically significant differences in either patient-reported QOL or symptom severity were found, on average, between participants in the two PALLAS treatment arms while either taking palbo+ET or ET alone, or after study-wide termination of palbo. In general, the addition of palbociclib in the adjuvant breast cancer setting did not contribute to increased symptom burden within this survivorship population. Further analyses will examine the relationship between PROs and treatment discontinuation by arm and study time point. Support: AFT, Pfizer, https://acknowledgments.alliancefound.org Table 1.Results of the Patient-Reported QOL and Symptom Severity Analyses Between the Two Treatment Arms During the First 2 Years of PALLASPRO Endpoint *Palbo + ET Adjusted average score (95% CI)ET alone Adjusted average score (95% CI)Average Difference + (Palbo + ET vs ET alone)[99.38% CI] Clinically ++ Important DifferenceEORTC QLQ-C30 Global Health Status/QOL71.7 (71.2, 72.2)74.0 (73.5, 74.5)-2.3 (-3.3, -1.4)**NoBrief Fatigue Inventory Score2.3 (2.2, 2.3)2.1 (2.0, 2.1)0.2 (0.1, 0.3)NoModified Brief Pain Inventory - Severity Score2.3 (2.2, 2.3)2.4 (2.4, 2.5)-0.2 (-0.3, -0.1)NoModified Brief Pain Inventory - Interference Score1.7 (1.6, 1.7)1.7 (1.6, 1.7)0.0 (-0.1, 0.1)NoEORTC QLQ-BR23 Alopecia1.4 (1.4, 1.4)1.3 (1.3, 1.3)0.1 (0.1, 0.1)NoBreast Cancer Prevention Trial - Hot Flash Symptoms1.2 (1.2, 1.3)1.2 (1.2, 1.3)0.0 (-0.1, 0.1)NoBreast Cancer Prevention Trial - Vaginal Problems0.8 (0.8, 0.8)0.8 (0.7, 0.8)0.0 (0.0, 0.1)NoBreast Cancer Prevention Trial - Musculoskeletal Pain1.2 (1.2, 1.2)1.3 (1.3, 1.3)-0.1 (-0.2, 0.0)No* For the EORTC QLQ-C30 Global Health Status/QOL subscale, higher scores indicate better QOL. For all other PRO endpoints, higher scores indicate worse symptom levels. ** The lower bound of the one-sided CI (adjusted for multiple comparisons) was -3.3. Because the lower limit is greater than the pre-specified non-inferiority margin of -3.44, non-inferiority of palbo+ET relative to ET-alone was concluded. The non-inferiority margin corresponds to a 0.2 SD in the EORTC QLQ-C30 global health/QOL score. +The average difference was adjusted for the following baseline covariates: Cycle 1 day 1 score, region (if applicable), age category, first adjuvant ET, race, ethnicity, N-stage, T-stage, histological grade, PgR, prior chemotherapy, ECOG Performance Status. ++Based on each instrument’s published clinically relevant cut-offs, if available. After calculating Cohen’s d treatment effect sizes, (i.e. by dividing the average difference by the ET-alone arm standard deviation from cycle 1 day 1), all effect sizes were below the pre-specified 0.2 threshold, and would not be considered clinically important. Citation Format: Michelle Joy Naughton, David Zahrieh, Michael Gnant, Nicholas Zdenkowski, Julie Lemieux, Jun J Mao, Vesna Bjelic-Radisic, Eileen Shinn, Marija Balic, Christoph Thomssen, Jane Neisel, Manuel Ruiz-Echarri, Sibylle Loibl, Claudine Isaacs, David Cameron, Fernando Manuel Henao Carrasco, Matthew Goetz, Viktor Wette, Gustavo Werutsky, Hope Rugo, Marcus Vetter, Ling-Ming Tseng, Kathy Miller, Florian Fitzal, Juan Miguel Gil Gil, Haeseong Park, Barbro Linderholm, Emilio Bajetta, Zoneddy Dayao, Aleix Prat, Karin Ehrhardt, Otto Metzger, Amal Arahmani, Ernest Law, Ann Partridge, Lisa Carey, Alex Zoroufy, Amylou Dueck, Dominik Hlauschek, Angela DeMichele, Erica Mayer. Quality of life and symptom severity in the PALLAS randomized trial of palbociclib with adjuvant endocrine therapy in early breast cancer (AFT-05) [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 P4-10-01.

Background: Black patients with triple negative breast cancer (TNBC) have worse survival outcomes, even after adjusting for stage at diagnosis, income, insurance status and other socioeconomic factors. Little is known regarding anti-tumor immune responses in Black patients and how these differences affect responses to treatment in TNBC. Limited data exists regarding the stromal tumor infiltrating lymphocytes (sTILs, which are strongly prognostic in TNBC) distribution based on race and ethnicity. Here we evaluate the prevalence, distribution, and prognostic impact of sTILs in TNBC by race/ethnicity from 2 prospective clinical trials of adjuvant anthracycline/taxane-based chemotherapy (E2197 and E1199). Methods: Full-face hematoxylin and eosin-stained sections of 481 tumors from ECOG-ACRIN trials E2197 and E1199 were previously evaluated for density of sTILs and shown to be associated with disease-free survival (DFS), distant recurrence-free interval (DRFI), and overall survival (OS) (Adams, et al JCO 2014). Further analyses were undertaken to evaluate the impact of race/ethnicity. Results: The majority of the 481 TNBC were from White patients (82.3%, n=403); with 12.3% (n=59) Black patients, 1.6% (n=14) other (9 Hispanic, 3 Asian, 2 Other), and 0.5% (n=5) unknown race. Age distribution (mean 49.2 for White and 49.2 for Black) and node negative disease (White 68/403 (42%), Black 24/59 (41%)) were similar. However, tumor size ≤2cm was seen more commonly in White patients (34%, 137/403) compared with Black patients (20%, 12/59). Black patients had a higher proportion of high sTILs (≥30%) with 23.7% (14/59) compared to White patients (11.4%, 46/403). The association of continuous stromal TILs with DFS (hazard ratio for a 10-point difference) was 0.84 (95% CI 0.72, 0.98) for White patients and 0.94 (95% CI 0.73, 1.20) for Black patients [159 DFS events for Whites, 26 DFS events for Blacks]. Conclusions: This is the first dataset from prospective clinical trials evaluating sTILs in TNBC in Black patients. Prevalence of high sTILs was greater in Black patients compared to White patients. The association between increasing sTILs and improved invasive disease-free survival across racial/ethnic groups must be investigated in larger datasets. Table 1.Race/EthnicityTotal (n=481)White (n=403)Black (n=59)Other (n=19)Mean age49.049.249.245.6T1 (tumor 2 and Citation Format: Natalie Klar, Robert J Gray, Sylvia Adams, Joseph A Sparano, Lori J Goldstein, Angela M DeMichele, Antonio C Wolff, Nancy E Davidson, George W Sledge, Sunil S Badve. Stromal tumor infiltrating lymphocytes analysis by race and ethnicity in triple negative breast cancers from 2 phase III randomized adjuvant breast cancer trials: ECOG-ACRIN E2197 and E1199 [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 P1-08-35.

Background: In high-risk hormone-receptor (HR)+/HER2- BC patients germline (g) mutations can be found in approximately 14% in BRCA1/2 and in BRCA1/2 and other BC predisposition genes in 20% (Pohl-Rescigno E, et al. JAMA Oncol 2020). In metastatic BC CDK4/6 inhibitors may have greater activity in patients with a BRCA mutation detected in ctDNA (André F, et al. J Clin Oncol 2020). The PENELOPE-B trial did not to show an improved invasive disease-free survival (iDFS) by adding palbociclib to ET in high-risk HR+/HER2- BC (Loibl S, et al. J Clin Oncol 2021). Methods: Blood samples from 898 of 1250 PENELOPE-B patients were available. 445 patients were sampled following a case-cohort design (220 cases defined as patients with any event during follow-up and 225 randomly selected patients without any event [non-cases]) and analyzed for germline variants in BRCA1/2 and 16 non-BRCA1/2 cancer predisposition genes (ATM, BARD1, BRIP1, CDH1, CHEK2, FANCM, MRE11A, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53, XRCC2) by targeted next generation sequencing (NGS). The primary definition of mutational status was the prevalence of a pathogenic mutation (mt) in one or more analyzed BC predisposition genes. Statistical analyses for time-to-event endpoints (iDFS, distant disease-free survival [DDFS], and overall survival [OS]) were based on inverse probability weighting: weighted Cox proportional hazard models and Kaplan-Meier estimates were used. Results: 442 of 445 patients (placebo arm: 104 cases and 105 non-cases; palbociclib arm: 114 cases and 119 non-cases) were successfully analyzed for mutational status. A total of 42 (9.5%) patients (placebo arm: 9.1%; palbociclib arm: 9.9%) carried any mutation. 15 (3.4%) patients had a gBRCA1/2 mt (one of whom carried a gATM mt and one a gCHEK2 mt in addition to gBRCA2 mt) and 29 (6.6%) had mutations in one of the other BC predisposition genes (n=8 CHEK2, n=7 PALB2, n=5 ATM, n=2 RAD50, n=1 for BARD1, FANCM, MRE11A, RAD51C, RAD51D, TP53 and n=1 both RAD51D and BRIP1). The mutational status with respect to all genes analyzed showed no significant correlation to clinical baseline variables. With regard to gBRCA1 and gBRCA2 genes only, the mutational status significantly correlated with age but not with other clinical variables: all 15 (100%) gBRCA mt carriers were younger than 50 years compared to 238 (56%) wildtype (wt) patients (p=0.002). The iDFS rate after 3 years was 80.9% in patients with any mutation and 79.5% in patients without. Mutational status (mt vs. wt) based on all genes analyzed was not prognostic (iDFS: hazard ratio 1.015, 95%CI 0.558-1.784; DDFS: 0.970, 95%CI 0.521-1.758; OS: 0.768, 95%CI 0.274-1.615). Neither the mutated patients had a benefit from palbociclib treatment (palbociclib vs placebo; iDFS: hazard ratio 0.766, 95%CI 0.263-3.022; DDFS: 0.897, 95%CI 0.275-3.489; OS: 0.666, 95%CI 0.063-5.671) nor the wt patients (iDFS: hazard ratio 0.918, 95%CI 0.650-1.303; DDFS: 0.966, 95%CI 0.679-1.393; OS: 0.901, 95%CI 0.573-1.433); interaction tests for treatment arm/mutational status for all time-to-event endpoints were not statistically significant. Analysis in the subgroups of patients by gBRCA1/2 showed similar results but had less statistical power. Conclusions: In this case-cohort analysis of 442 patients enrolled in the PENELOPE-B trial, the detection of BC predisposition genes was lower than expected with 10%. This is probably due to the low rate of gBRCA1/2 carriers (3.4%), which could be influenced by the selection criteria of the trial. Patients with gBRCA1/2 or other BC disposition genes had a comparable outcome to non-carriers in the PENELOPE-B trial. Citation Format: Sibylle Loibl, Jan Hauke, Karen Gelmon, Frederik Marmé, Corinna Ernst, Miguel Martin, Michael Untch, Hervé Bonnefoi, Erik Knudsen, Seock-Ah Im, Angela DeMichele, Laura Van’t Veer, Sung-Bae Kim, Harry Bear, Nicole McCarthy, Nicholas Turner, Agnieszka Witkiewicz, Federico Rojo, Peter A Fasching, José A García-Sáenz, Catherine M Kelly, Toralf Reimer, Masakazu Toi, Hope S Rugo, Carsten Denkert, Michael Gnant, Andreas Makris, Yuan Liu, Olga Valota, Bärbel Felder, Karsten Weber, Valentina Nekljudova, Eric Hahnen. Germline BRCA1/2 and other predisposition genes in high-risk early-stage HR+/HER2- breast cancer (BC) patients treated with endocrine therapy (ET) with or without palbociclib: A secondary analysis from the PENELOPE-B study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-36.

Background: The CK12 expression signature consists of genes CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, CXCL10, CXCL11, CXCL13 and was previously shown to associate with the presence of T and B cell rich tertiary lymphoid structures in melanoma and other cancers, and with better patient survival independent of tumor staging and treatment. I-SPY 2 is a biomarker-rich, phase II neoadjuvant platform trial for high risk early stage breast cancer. Here we leverage the I-SPY 2 biomarker program to test the hypothesis that this signature associates with sensitivity to neoadjuvant immunotherapies and potentially other classes cancer therapeutics in breast cancer. Methods: Data from 1130 patients across 12 arms of I-SPY2 (control (ctr): 210; veliparib/carboplatin (VC): 71; neratinib (N): 114; MK2206: 93; ganitumab: 106; ganetespib: 93; AMG386: 134; TDM1/pertuzumab(P): 52; H/P: 44; pembrolizumab (pembro): 69; durvalumab/olaparib (durva/olap): 71; pembro/SD101: 72) were available for analysis. Pre-treatment FF (n=987) or FFPE (n=143) biopsies were assayed using Agilent gene expression arrays. Signature scores were calculated as the average expression level across the 12 genes, after z-score normalization. We used logistic modeling to assess association with pCR in each arm in a model adjusting for HR and HER2 (likelihood ratio test, p Citation Format: Hatem Soliman, Denise Wolf, Jo Chien, Christina Yau, Michael Campbell, Mark Magbanua, Ruixiao Lu, Nicholas O'Grady, Lamorna Brown-Swigart, Gillian Hirst, Beverly Parker, Laura Sit, Smita Asare, Doug Yee, Angie DeMichele, Rita Nanda, Lajos Pusztai, Don Berry, Laura Esserman, Laura Van't Veer. Chemokine12 (CK12) tertiary lymphoid gene expression signature as a predictor of response in 3 immunotherapy arms of the neoadjuvant ISPY 2 TRIAL - pembrolizumab with and without SD101, and durvalumab combined with olaparib - and in 9 other arms of the trial including platinum-based and dual-anti-HER2 therapies [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 PD10-07.

Background: Understanding health-related quality of life (HRQOL), including side effects, is critical to guide supportive care during chemotherapy. The EA1131 trial demonstrated that Platinum (Plat) was unlikely to improve outcomes compared to capecitabine (Cape) in patients with stage II-III triple-negative breast cancer (TNBC) of basal subtype and ≥1 cm residual disease after neoadjuvant chemotherapy (NAC), supporting Cape as the continued standard of care. Patient-reported outcomes (PRO) were administered as a sub-study to understand HRQOL and symptoms from the patient’s perspective. Methods: EA1131 was amended in 9/2017 to add PRO endpoints and all patients enrolled after this amendment were eligible for the PRO sub-study. The Functional Assessment of Cancer Therapy-Breast Cancer Symptom Index (FBSI) and the Functional Assessment of Cancer Therapy/Gynecologic Oncology Group Neurotoxicity Subscale (NtxS; Plat arm only) were administered at baseline (BL), cycle 3 day 1 (C3D1), and following treatment at 6 and 15 months. Due to early trial termination, the PRO sub-study target accrual (n=362) was not reached. It was hypothesized that HRQOL, assessed by the FBSI-Treatment Side Effect (TSE) subscale (range 0-16, higher score = less side effects, better HRQOL), would indicate fewer post-treatment side effects at 6 and 15 months following Plat compared to Cape. The Wilcoxon rank sum test was used to compare FBSI-TSE subscale scores and total FBSI scores (range 0-64, higher score = better overall HRQOL) between arms at BL, C3D1, 6 months and 15 months. Two-sample t-tests were used to compare change in FBSI-TSE subscale scores and total FBSI scores from BL to C3D1 between arms. Change in NtxS scores (range 0-44, higher score = less neurotoxicity) from BL to C3D1 was evaluated with the paired t-test. Analyses were exploratory and p-values 1.5 points) in 27% of patients on Cape and 23% of patients on Plat (p-value 0.51). Mean total FBSI scores did not differ between arms at any time (BL: Cape 50.6, Plat 49.7; C3D1: Cape 48.1, Plat 48.0; 6 months: Cape 49.9, Plat 51.1; 15 months: Cape 53.3, Plat 50.3; all p > 0.05). Mean change in total FBSI scores from BL to C3D1 did not differ between arms (Cape -2.20, Plat -1.83, p = 0.75). Mean (standard deviation) NtxS scores for the Plat arm were 38 (6.3), 36.1 (7.8), 36 (7.1) and 34.5 (7.9) at BL, C3D1, 6 months and 15 months, respectively. Mean NtxS score decreased (indicating worsening neurotoxicity) from BL to C3D1 (p-value 0.006). Conclusions: Despite more frequent severe toxicity by CTCAE criteria for Plat than Cape, patient-reported side effects worsened during treatment with Cape but not Plat. Overall, changes in HRQOL were small for both arms and resolved after therapy. However approximately one-fourth of patients had clinically meaningful worsening side effects on both arms. PRO-assessed neurotoxicity increased in the Plat arm. This PRO sub-study demonstrates that PROs capture toxicities beyond CTCAE criteria and provides novel data about patients’ experience during adjuvant chemotherapy following NAC for TNBC. Citation Format: Karen L Smith, Fengmin Zhao, Ingrid A Mayer, Amye J Tevaarwerk, Sofia F Garcia, Carlos L Arteaga, William F Symmans, Ben H Park, Brian L Burnette, Della F Makower, Margaret Block, Kimberly A Morley, Chirag R Jani, Craig Mescher, Shabana J Dewani, Ursa Brown-Glaberman, Lisa E Flaum, Erica L Mayer, William M Sikov, Eve T Rodler, Angela M DeMichele, Joseph A Sparano, Antonio C Wolff, Kathy D Miller, Lynne I Wagner. Patient-reported outcomes in EA1131: A randomized phase III trial of platinum vs. capecitabine in patients with residual triple-negative breast cancer after neoadjuvant 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 P4-10-02.

Background: Molecular plasticity of breast cancer is crucial for the development of therapy-resistant disease. In this investigation, we studied changes in molecular signatures between pretherapeutic (pre-Tx) and post-therapeutic (post-NACT) tumor samples from patients included in the PENELOPE-B (NCT01864746) trial. The phase III PENELOPE-B study did not show a significant benefit from palbociclib in women with centrally confirmed HR+, HER2- primary breast cancer without a pathological complete response after taxane-containing neoadjuvant chemotherapy (NACT) and at high-risk of relapse (CPS-EG score ≥3 or 2 and ypN+) (Loibl et al. JCO 2021). However, first translational investigations showed that a small number of patients with a luminal-B tumor subtype, based on absolute intrinsic molecular subtyping (AIMS, Paquet & Hallet, JNCI 2014) subtyping after NACT, had a numerical benefit from post-NACT palbociclib. We have therefore extended the analysis and included a cohort of paired pre-Tx and post-NACT samples. Methods: We investigated gene expression in pre-Tx (n=259) tumor tissue samples using the HTG EdgeSeq Oncology Biomarker Panel including 2549 genes (HTG Molecular Diagnostics Inc.); for the same patients the same panel on post-NACT residual tumor samples were available. The paired samples were selected based on a case-cohort approach. Based on 91 genes of this panel, the AIMS subtype was calculated. In addition, we performed exploratory biomarker analyses to identify genes and gene signatures with prognostic and predictive relevance. After completion of NACT, PENELOPE-B patients were randomized to palbociclib versus placebo in addition to standard endocrine therapy. Results: The prevalence of AIMS subtypes, in particular LumA vs LumB, changed in pre-Tx and post-NACT tumors. In the pre-Tx samples, 115 (44%) and 123 (47%) of tumors had LumA and LumB subtypes, respectively, as expected from a high-risk cohort. However, in the post-NACT samples, LumA tumors were predominant (n=183, 71%) over LumB (n=30; 12%). 78 (30%) and 6 (2%) tumors switched their subtype from LumB to LumA and LumA to LumB, respectively. For further analyses, we compared the groups of low proliferating (LumA and NormL) and high proliferating subtypes (LumB, BasalL and HER2E). In bivariable Cox regression analysis, the grouped pre-Tx and post-NACT AIMS subtypes were independent prognostic factors for iDFS: HR=1.85 (1.16-2.98, p=0.011) for pre-Tx LumB/BasalL/HER2E vs LumA/NormL and HR=2.18 (1.24-3.84, p=0.007) for post-NACT. Similar results were found when adjusted for prognostic clinical factors and for DDFS and OS endpoints although the pre-Tx subtype did not reach significance. These and further Cox models investigating interaction effects show that patients with tumors developing from high (pre-Tx) to low proliferation (post-NACT) had a higher iDFS risk compared to stable low proliferating tumors but a lower iDFS risk compared to stable high proliferating tumors. Neither in the pre-Tx LumB/BasalL/HER2E nor in the pre-Tx LumA/NormL subgroup a benefit from palbociclib was observed. Based on the results of the AIMS subtyping, we extended the exploratory analysis to identify genes that might be involved in the prognostic and predictive effects as well as genes driving the subtype switch. The analysis is ongoing and the relevant genes will be presented at the conference. Conclusions: Our findings show that the switch from high-risk molecular subtypes (in particular LumB) to low-risk subtypes (in particular LumA) is common in neoadjuvant therapy of luminal tumors. The adaptation of luminal high-risk tumors to therapy-induced stress is crucial for the clinical outcome and the results suggest that molecular defined tumor subtypes might not be as stable as originally thought. Citation Format: Carsten Denkert, Frederik Marmé, Miguel Martin, Michael Untch, Hervé Bonnefoi, Sung-Bae Kim, Harry Bear, Agnieszka Witkiewicz, Seock-Ah Im, Angela DeMichele, Laura Van’t Veer, Nicole McCarthy, Thorsten Stiewe, Karen A. Gelmon, José A. García-Sáenz, Catherine M. Kelly, Toralf Reimer, Erik Knudsen, Nicholas Turner, Federico Rojo, Peter A. Fasching, Julia Teply-Szymanski, Yuan Liu, Masakazu Toi, Hope S. Rugo, Michael Gnant, Andreas Makris, Bärbel Felder, Karsten Weber, Sibylle Loibl. Molecular plasticity of luminal breast cancer and response to CDK 4/6 inhibition - The biomarker program of the PENELOPE-B trial investigating post-neoadjuvant palbociclib [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 PD2-04.

Background: I-SPY2 is a multicenter, phase 2 trial using response-adaptive randomization within biomarker subtypes defined by hormone-receptor (HR), HER2, and MammaPrint (MP) status to evaluate novel agents as neoadjuvant therapy for high-risk breast cancer. The primary endpoint is pathologic complete response (pCR). Cemiplimab is an anti-PD-1 inhibitor approved for the treatment of NSCLC and cutaneous basal and squamous cell CA. Lymphocyte activation gene 3 (LAG-3) binds MHC class II leading to inhibition of T-cell proliferation and activation and is often co-expressed with PD-1. REGN3767 is a fully humanized mAb that binds to LAG-3 and blocks inhibitory T-cell signaling. Concurrent blockade of LAG-3 with an anti-PD-1 may enhance efficacy of an anti-PD-1. Methods: Women with tumors ≥ 2.5cm were eligible for screening. Only HER2 negative (HER2-) patients were eligible for this treatment; HR positive (HR+) patients had to be MP high risk. Treatment included Paclitaxel 80 mg/m2 IV weekly x 12 and Cemiplimab 350 mg and REGN3767 1600 mg both given q3weeks x 4, followed by doxorubicin/cyclophosphamide (AC) every 2 weeks x 4. The control arm was weekly paclitaxel x 12 followed by AC every 2-3 weeks x 4. Cemiplimab/REGN3767 was eligible to graduate in 3 of 10 pre-defined signatures: HER2-, HR-HER2-, and HR+HER2-. The statistical methods for evaluating I-SPY 2 agents has been previously described. To adapt to changing standard of care, we constructed “dynamic controls” comprising ‘best’ alternative therapies using I-SPY 2 and external data and estimated the probability of Cemiplimab/REGN3767 being superior to the dynamic control. Response predictive subtypes (Immune+ vs Immune-) were assessed using pre-treatment gene expression data and the ImPrint signature. Results: 73 HER2- patients (40 HR+ and 33 HR-) received Cemiplimab/REGN3767 treatment. The control group included [357 patients with HER2- tumors (201 HR+ and 156 HR-) enrolled since March 2010. Cemiplimab/REGN3767 graduated in both HR-/HER2- and HR+/HER2- groups; estimated pCR rates (as of June 2022) are summarized in the table. Safety events of note for Cemiplimab/REGN3767 include hypothyroidism 30.8%, adrenal insufficiency (AI) 19.2%, hyperthyroidism 14.1%, pneumonitis 1.3%, and hepatitis 3.8%. All were G1/2 except for 6 (7.7%) G3 AI and 3 (3.8%) G3 colitis. Rash occurred in 62.8%, 9% G3 and 2 pts (2.6%) had pulmonary embolism. X% of adrenal insufficiency cases required replacement therapy. 40 patients (11 HR+ and 29 HR-) in Cemiplimab/REGN3767 were predicted Immune+; 32 (29 HR+ and 3 HR-) were predicted Immune-. In the HR+ group pCR was achieved in 10/11 (91%) patients with Immune+ subtype compared with 8/29 (28%) with Immune- subtype. Additional biomarker analyses are ongoing and will be presented at the meeting. Conclusion: The I-SPY 2 study aims to assess the probability that investigational regimens will be successful in a phase 3 neoadjuvant trial. Dual immune blockade with a LAG-3 inhibitor and anti-PD1 therapy resulted in a high predicted pCR rate both in HR-/HER2- (60%) and HR+/HER2- (37%) disease. The novel Imprint signature identified a group of HR+ patients most likely to benefit from this active regimen. Table 1: Estimated pCR rates Citation Format: Claudine Isaacs, Rita Nanda, Jo Chien, Meghna S. Trivedi, Erica Stringer-Reasor, Christos Vaklavas, Judy C. Boughey, Amy Sanford, Anne Wallace, Amy S. Clark, Alexandra Thomas, Kathy S. Albain, Laura C. Kennedy, Tara B. Sanft, Kevin Kalinsky, Hyo S. Han, Nicole Williams, Mili Arora, Anthony Elias, Carla Falkson, Smita Asare, Ruixiao Lu, Maria Pitsouni, Amy Wilson, Jane Perlmutter, Hope Rugo, Richard Schwab, W. Fraser Symmans, Nola M. Hylton, Laura Van’t Veer, Douglas Yee, Angela DeMichele, Donald Berry, Laura J. Esserman, I-SPY Investigators. Evaluation of anti-PD-1 Cemiplimab plus anti-LAG-3 REGN3767 in early-stage, high-risk HER2-negative breast cancer: Results from the neoadjuvant I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS5-03.

Background: I-SPY2 is a multicenter, phase 2 trial using response-adaptive randomization within biomarker subtypes defined by hormone-receptor (HR), HER2, and MammaPrint (MP) status to evaluate novel agents as neoadjuvant therapy for high-risk breast cancer. The primary endpoint is pathologic complete response (pCR). Cemiplimab (Cemi) is a PD-1 inhibitor approved for the treatment of NSCLC, cutaneous basal, and squamous cell cancer. Here, we report current efficacy rates of Cemi in combination with paclitaxel followed by AC. Methods: Women with tumors ≥ 2.5cm were eligible for screening. Only HER2 negative (HER2-) patients were eligible for this treatment; HR positive (HR+) patients had to be MP high risk. Treatment included paclitaxel 80 mg/m2 IV weekly x 12 and Cemi 350 mg IV given q3weeks x 4, followed by doxorubicin/cyclophosphamide (AC) every 2 weeks x 4. The control arm was weekly paclitaxel x 12 followed by AC every 2-3 weeks x 4. All patients undergo serial MRI imaging; and imaging response (at 3 weeks, 12 weeks and prior to surgery) were used along with accumulating pCR data to continuously update and estimate pCR rates for trial arms. Analysis was modified intent to treat. Patients who switched to non-protocol therapy count as non-pCR. The goal is to identify (graduate) regimens with ≥85% Bayesian predictive probability of success (i.e. demonstrating superiority to control) in a future 300-patient phase 3 neoadjuvant trial with a pCR endpoint within responsive signatures. Cemi was eligible to graduate in 3 pre-defined signatures: HER2-, HR-HER2-, and HR+HER2-. To adapt to changing standard of care, we constructed “dynamic controls” comprising ‘best’ alternative therapies using I-SPY 2 and external data and estimated the probability of Cemi being superior to the dynamic control. Results: 60 HER2- patients (28 HR+ and 32 HR-) received Cemi arm treatment. The control group included 357 patients with HER2- tumors (201 HR+ and 156 HR-) enrolled since March 2010. Cemi graduated in HR-/HER2- signature. Estimated pCR rates (as of June 2022) are summarized in the table. Immune-related endocrine disorders include: hypothyroid (14.5%), adrenal insufficiency (10%), hyperthyroid (4.8%),) and thyroiditis (3.2%). Only one grade 3 adrenal insufficiency was observed. All immune related AE’s were manageable. Additional biomarker analyses are ongoing and will be presented at the meeting. Response predictive subtypes (Immune+ vs Immune-) and additional predictive biomarkers were assessed. Associations with pCR will be presented at SABCS. Conclusion: The I-SPY 2 study aims to assess the probability that investigational regimens will be successful in a phase 3 neoadjuvant trial. Anti-PD-1 therapy with Cemi resulted in a higher predicted pCR rate in HR-/HER2- 55 rate% disease compared to control at 29%. Immune-mediated AE’s were observed. This data is consistent with previously published data using check point inhibitors in early-stage HR-/HER2- breast cancer. Estimated pCR rates Citation Format: Erica Stringer-Reasor, Rebecca A. Shatsky, Jo Chien, Anne Wallace, Judy C. Boughey, Kathy S. Albain, Hyo S. Han, Rita Nanda, Claudine Isaacs, Kevin Kalinsky, Zahi Mitri, Amy S. Clark, Christos Vaklavas, Alexandra Thomas, Meghna S. Trivedi, Janice Lu, Smita Asare, Ruixiao Lu, Maria Pitsouni, Amy Wilson, Jane Perlmutter, Hope Rugo, Richard Schwab, W. Fraser Symmans, Nola M. Hylton, Laura Van ’t Veer, Douglas Yee, Angela DeMichele, Donald Berry, Laura J. Esserman, I-SPY Investigators. PD11-01 Evaluation of the PD-1 Inhibitor Cemiplimab in early-stage, high-risk HER2-negative breast cancer: Results from the neoadjuvant I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD11-01.

Background: The Penelope-B trial did not show improvement in invasive disease-free survival (iDFS) with the addition of palbociclib to endocrine therapy (ET) in patients with high-risk early breast cancer (BC) after neoadjuvant chemotherapy (NACT). Biomarkers may be able to identify subgroups of patients deriving benefit from Palbociclib and guide future studies. Estrogen-receptor (ER), progesterone-receptor (PgR) and Ki-67 might be helpful in identifying patients benefiting from palbociclib. Concordantly, tumors with elevated expression of Cyclin D1 and phosphorylated retinoblastoma protein (phospho-RB) may harbor more dependency on CDK4/6 and thus higher sensitivity to palbociclib. Methods: The percentage of positive ER and PgR cells and Ki-67 assessed in surgical specimens after NACT were combined to obtain the immunohistochemical score 3 (IHC3, Cuzick et al JCO 2011, low vs high based on the median IHC3 value). Cyclin D1 and phospho-RB Ser 807/811 immunoreactive (phospho-RB) scores were analyzed in residual tumors after NACT (range 0-12 each). Proportional hazard regression model was used to assess the predictive and prognostic value of IHC3 and treatment on iDFS. Subgroup analysis was performed according to BC intrinsic subtypes (luminal-A/normal-like, luminal-B/HER2-enriched/basal) and HER2-status (HER2 0, HER2 low). Cox/Fine-Gray regression was used to define the predictive and prognostic value of CyclinD1 (≤1, >1), phospho-RB (≤2, >2) as dichotomized and continuous variables on iDFS, distant DFS (DDFS), locoregional invasive recurrence-free interval (LRRFI) and overall survival (OS). Multivariate analyses (MVA) were adjusted for age (≤50 vs >50), Ki-67 (≤15 vs >15), region (non-Asian vs Asian), ypN (ypN0-1 vs ypN2-3), risk status (CPS-EG=2 ypN+ vs ≥3), cT (cT1-2 vs cT3-4), ypT (ypT0-2 vs ypT3-4), and grade (G1-2 vs G3). The MVA for IHC3 includes all the covariates above except Ki-67. p< 0.05 was defined as statistically significant. Results: Data for ER, PgR, Ki-67, HER2, Cyclin D1 and phospho-RB were available for 1250 patients. Overall, 98.9% of the patients had ER+ tumors, 75.0% PgR+, 52.2% had HER2 low, 25.5% Ki-67>15, 50% had IHC3 score higher than median, 93.9% had Cyclin D1 >1, 57.8% had phospho-RB >2. Patients with IHC3 score high had a worse iDFS compared to patients with IHC3 score low (MVA HR 2.28 95%CI (1.78-2.91), p< 0.0001). Patients with luminal-A/normal-like tumors and IHC3 low had an improved iDFS with the addition of palbociclib to ET (MVA HR 0.35 95%CI (0.14-0.90), test for interaction p=0.01). No difference was observed according to HER2 status. Cyclin D1>1 has no predictive value but is prognostic for better iDFS (MVA HR 0.62 95%CI (0.41-0.94), p=0.023), LRRFI (MVA HR 0.30 95%CI (0.15-0.63), p=0.001) and OS (MVA HR 0.50 95%CI (0.28-0.89), p=0.019). Similar results were obtained when Cyclin D1 was analysed as a continuous variable. Phospho-RB had neither predictive nor prognostic value. Phospho-RB highly correlates with Ki-67 (p< 0.001, Spearman correlation 0.248). Conclusions: Patients with high Cyclin D1 expression had a favorable prognosis independent of treatment arm, but patients with luminal-A/normal-like tumors and IHC3 low after NACT had an improved outcome when receiving palbociclib in addition to adjuvant ET. Theses exploratory studies suggest specific signatures/phenotypes could predict benefit from Palbociclib in high-risk early breast cancer. Citation Format: Erik S. Knudsen, Sivaramakrishna Rachakonda, Frederik Marmé, Miguel Martín, Michael Untch, Hervé R. Bonnefoi, Wolfgang D. Schmitt, Sung-Bae Kim, Harry D. Bear, Agnieszka Witkiewicz, Seock-Ah Im, Angela DeMichele, Laura Van’t Veer, Nicole McCarthy, Bruno V. Sinn, Karen Gelmon, José Ángel García-Sáenz, Catherine M. Kelly, Toralf Reimer, Nicholas Turner, Federico Rojo, Martin Filipits, Peter A. Fasching, Christian Schem, Lesley-Ann Martin, Yuan Liu, Masakazu Toi, Hope Rugo, Michael Gnant, Andreas Makris, Jenny Furlanetto, Karsten Weber, Carsten Denkert, Sibylle Loibl. Immunohistochemical markers and determinants of clinical response in the Penelope-B trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD17-06.

Background: In previous work we leveraged the I-SPY2 trial to create treatment response predictive subtypes (RPS) incorporating tumor biology beyond clinical HR/HER2, to better predict drug responses in an expanded treatment landscape that includes platinum agents, dual HER2-targeting regimens and immunotherapy [1]. We showed that best performing schemas incorporate Immune, DRD and HER2/Luminal phenotypes, and that treatment allocation based on these would increase the overall pCR rate to 63% from 51% using HR/HER2-based treatment selection. The RPS schema has been selected for prospective evaluation in I-SPY2. Using the RPS, one would prioritize platinum-based therapy for HER2-/Immune-/DRD+, immunotherapy for HER2-/Immune+, and dual-anti-HER2 for HER2+ that are not luminal. HER2+/Luminal patients have low response rates to dual-anti-HER2 therapy but may respond better to anti-AKT. However, there is still a considerable ‘biomarker-negative’ group of resistant cancers (HER2-/Immune-/DRD-) with very low pCR rates to all tested agents, that require a new therapeutic approach. Here we characterize the protein signaling architecture of these tumors to identify new target candidates. Methods: 987 I-SPY 2 patients from 10 arms of the trial were considered for this analysis. All have gene expression, pCR and RPS; 944 have distant recurrence free survival (DRFS) data; and 736 have reverse phase protein array (RPPA) data from laser capture microdissected tumor epithelium. These data – known collectively as the I-SPY2-990 mRNA/RPPA Data Resource - were recently made public on NCBI’s Gene Expression Omnibus [GEO: GSE196096]. We focus on HER2-/Immune-/DRD- tumors, applying Wilcoxon and t-tests to identify phosphoproteins that differ between HR+HER2-/Immune-/DRD- and other HR+HER2- tumors; and between TN/Immune-/DRD- and other TNs. The Benjamini-Hochberg (BH) method is used to adjust p-values for multiple hypothesis testing. In addition, the Kaplan-Meier method is used to estimate DRFS. Results: 201/736 I-SPY 2 patients with RPPA data are classified HER2-/Immune-/DRD- (HR+HER2-: n=138; TN: n=63). Of these, 8.5% (17/201) achieved pCR. Non-responding HER2-/Immune-DRD- had worse outcomes than responders (~75% vs. ~95% DRFS at 5 years). 60/139 phospho-proteins differ significantly between HR+HER2-/Immune-/DRD- and other HR+HER2- tumors (n=122). These tumors are relatively ‘cold’, in that 90% (54/60) of the phosphoprotein activities characterizing this group are at lower levels than in the overall HR+HER2- population; including immune (e.g. pPDL1, pJAK/STAT) and proliferation (e.g., Ki67, CyclinB1, pAURK) endpoints. Phosphoproteins showing higher levels in this subset include ERBB2 (BH p=1.7E-06), Cyclin D1 (BH p=1.4E-05), pAR (BH p=1.4E-05), and ER (BH p=3E-04). Within the TN subset, only 3/139 phospho-proteins differed significantly between TN/Immune-/DRD- and other TN tumors (n=189). These were all immune-related (pPDL1, pSTAT1, and HLA DR), with lower expression in the TN/Immune-/DRD- group. Conclusion: HR+HER2- and TN patients who are Immune-Low and DRD-Low have very low pCR rates to all tested therapeutics in I-SPY2 including standard chemotherapy, platinum, and immunotherapy. Senolytics (possibly targeting Cyclin D1), HER2low agents, and AR modulators may overcome resistance in HR+HER2-/Immune-/DRD-, whereas an immune activator beyond checkpoint inhibition is suggested for TN/Immune-/DRD- patients. [1] Wolf et. al., Redefining Breast Cancer Subtypes to Guide Treatment Prioritization and Maximize Response: Predictive Biomarkers across 10 Cancer Therapies. Cancer Cell 2022 Citation Format: Denise M. Wolf, Christina Yau, Julia Wulfkuhle, Rosa I. Gallagher, Lamorna A. Brown Swigart, Gillian L. Hirst, Jean-Philippe Coppe, Mark Jesus M. Magbanua, Rosalyn Sayaman, I-SPY2 Investigators, Laura Sit, Nola M. Hylton, Angela DeMichele, Donald A. Berry, Lajos Pusztai, Douglas Yee, Laura J. Esserman, Emanuel F. Petricoin, Laura Van ’t Veer. PD5-04 Characterizing the HER2-/Immune-/DNA repair (DRD-) response predictive breast cancer subtype: the hunt for new protein targets in a high-needs population with low response to all I-SPY2 agents [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 PD5-04.

Background: The PENELOPE-B (NCT01864746) and PALLAS (NCT02513394) trials are large prospective, randomized, phase III trials that evaluated adjuvant palbociclib (PAL) + endocrine treatment (ET) vs ET in patients with hormone receptor–positive/human epidermal growth factor receptor 2–negative (HER2–) early breast cancer (EBC). Both studies did not meet the primary endpoint of improving invasive disease-free survival (iDFS). We conducted biomarker analyses to identify patients who might benefit from PAL + ET in EBC. Methods: Resected tumor tissue was collected from consenting patients. Gene expression analyses were conducted using the HTG EdgeSeq Oncology Biomarker Panel including 2549 genes. Based on 91 genes from the HTG panel, the intrinsic molecular subtypes were calculated using Absolute Intrinsic Molecular Subtyping (AIMS). Potential predictive treatment biomarkers were established in PENELOPE-B (n=906 with resected tissue) as the development set using an outcome-oriented approach based on iDFS with a selection procedure that maximized the log-rank statistic to estimate a standard Z score–based optimal cutoff. Independent validation was conducted on PALLAS (n=2085; PENELOPE-B-like with resected tissue and HTG data). Hazard ratios and corresponding 95% CIs were calculated using the Cox proportional hazards model, and iDFS distributions between treatment arms were compared using the log-rank test. Interaction between treatment and biomarker status was assessed. Results: Patient baseline characteristics were well balanced, with no differences in iDFS between the intent-to-treat set and the biomarker set for both trials. Approximately 73% of patients (PENELOPE-B [n=663] and PALLAS [n=1516]) had luminal A subtypes whereas only 7.1 % (PENELOPE-B [n=64]) and 8.3 % (PALLAS [n=172]) had a luminal B subtype. AIMS subtypes showed overall similar prognostic patterns for iDFS between PENELOPE-B and PALLAS. The biomarker-defined subgroup found in PENELOPE-B with optimal cutoff demonstrated a preferential benefit from PAL + ET (n=364 [96 events]; hazard ratio [95% CI], 0.63 [0.42, 0.95]; P=0.025). Independent validation of the PALLAS subgroup using the pre-defined optimal cutoff confirmed a significant benefit from PAL + ET (n=916 [70 events]; 0.55 [0.34–0.90]; P=0.015) while not in the rest of the patients (interaction p=0.0025). Significant treatment effects remained (0.55 [0.34–0.89]; P=0.015) after adjusting for the randomization stratification factors of PALLAS. Conclusions: The composite predictive biomarker defined from PENELOPE-B was independently validated in a prospectively defined retrospective analysis of a subset of patients selected from PALLAS. The composite biomarker identified a subset of EBC patients deriving benefit from the addition of PAL to ET. This patient stratification approach can potentially be applied to future adjuvant clinical trials for treatment of hormone receptor–positive/HER2– EBC. Citation Format: Sibylle Loibl, Carsten Denkert, Yuan Liu, Erik S. Knudsen, Angela DeMichele, Zhe Zhang, Julia Teply-Szymanski, Martin Filipits, Peter A. Fasching, Michael Gnant, Shibing Deng, Marija Balic, Federico Rojo, Mark Watson, Chetan Deshpande, Nicholas Turner, Otto Metzger, Kathy Puyana Theall, Agnieszka Witkiewicz, Olga Valota, W. Fraser Symmans, Erica L. Mayer. Development and Validation of a Composite Biomarker Predictive of Palbociclib + Endocrine Treatment Benefit in Early Breast Cancer: PENELOPE-B and PALLAS Trials [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD17-05.

Background. Immunotherapy has emerged as an important component of neoadjuvant therapy for some patients with breast cancer (BC). As a result, immune-related adverse events (irAEs) are increasing and have effects on both short and long term symptoms significantly impacting patient quality of life. BC patients may develop new conditions including arthralgias, gastrointestinal issues, endocrinopathies, and fatigue during or after cancer therapy that may be acute or long-lasting in nature. Monitoring for early onset and severity of symptoms, and adjusting treatment and symptom management could optimize therapy for a particular patient, maximizing potential efficacy while mitigating toxicity. We sought to identify patient demographic characteristics and symptom patterns associated with risk for development of irAEs in the context of a randomized trial for patients with early-stage high-risk breast cancer. Methods. I-SPY2 is a multi-center, phase 2 trial using response-adaptive randomization for high-risk early-stage women with BC. The study population for this analysis includes enrolled patients receiving combinations of experimental immunotherapy and chemotherapy. Groups considered for statistical comparisons included those that developed an irAE versus those that did not develop an irAE up until the surgery timepoint. In I-SPY adverse events are documented through the Common Terminology Criteria for Adverse Events (CTCAEv5.0). Hypothyroidism, adrenal insufficiency, and pneumonitis were the irAEs considered in this study. A chi-square test was used to assess associations between race and ethnicity (White, Asian, Black, non-Hispanic) and irAEs. One-way ANOVA was used to evaluate the association between age (>50 vs < 50) and irAEs. 33 symptoms reported at CTCAE grade 2 or higher were included in the analyses and a symptom burden score was calculated using area under curve (AUC) which combined the duration of each symptom between baseline and week 6 of treatment, and grade of adverse event. Regularized regression using leave-one out cross validation was used to evaluate early symptoms (as quantified by the symptom burden score) as predictors, and irAEs as surrogate responses. Results. Out of 461 patients, percentages of patients wth irAEs of interest included hypothyroidism (13%), adrenal insufficiency (9%), and pneumonitis (4%). Demographic information was available for 333 patients, of which 270 (81%) were White, 23 (7%) were Asian, 37 (11%) were African American (AA) and 278 (17%) were non-Hispanic. There were proportionately higher number of white patients that developed hypothyroidism than non-white patients (35 of 265 (13%) vs 2 of 63 (3%), P < 0.04). Pneumonitis was more common in patients over 50 years old than under 50 years old (P < 0.02). Symptoms that were most commonly reported up to week 6 of treatment among patients who developed an irAE included: diarrhea (36%), fatigue (15%), dizziness (12%) and shortness of breath (SOB) (11%). Symptoms associated with the development of hypothyroidism included fatigue (15%, mean AUC=11.8 vs 5.8 for those that did not develop irAE), SOB (11%, 4.3 vs 2.8), and blurry vision (1%, 1.0 vs 0.12). Development of adrenal insufficiency was associated with early reports of diarrhea (36%, 19.0 vs 10.5), SOB (11%, 7.8 vs 2.6), joint pain (3%, 2.29 vs 0.58), decreased appetite (3%, 3.55 vs 0.91), and constipation (1%, 3.6 vs 0.02). No significant early symptoms emerged for pneumonitis due to a limited number of events. Conclusion. Our study utilizes an analysis framework that is aimed to determine symptom clusters that predict the development of irAEs. We describe specific symptoms presenting early with the development of hypothyroidism and adrenal insufficiency, in recognition of allowing physicians to be more diligent in active and post treatment monitoring. Citation Format: Amrita Basu, Saumya Umashankar, Michelle Melisko, Ruixiao Lu, Hongmei Yu, Christina Yau, Smita Asare, Maria Pitsouni, Rebecca A. Shatsky, Claudine Isaacs, Angela DeMichele, Dawn Hershman, Rita Nanda, Mi-Ok Kim, Laura J. Esserman, Hope Rugo. Identification of symptoms that are associated with irAEs in the I-SPY clinical 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 GS5-04.

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.

Introduction: One of the principal limiting factors to achieving cures in patients with cancer is drug resistance. Drug repositioning is the application of FDA-approved drug compounds for novel indications beyond the scope of the drug’s original intended use. This approach offers advantages over traditional drug development by reducing development costs and providing shorter paths to approval, as drug safety has already been established during the drug’s original regulatory process. One approach for computational drug repositioning involves generating a disease gene expression signature and then identifying a drug that can reverse this disease signature. In this study, we extracted drug resistance signatures from the I-SPY 2 TRIAL by comparing gene expression profiles of responder and non-responder patients stratified by treatment and molecular subtype. We then applied our drug repositioning pipeline to predict compounds that can reverse the gene expression profiles of these drug resistance signatures. We hypothesize that reversing these drug resistance signatures will resensitize tumors to treatment and improve patient outcome. Methods: We first generated the drug resistance signatures by performing differential expression between responders (RCB 0/I) and non-responders (RCB III) within treatment arms and molecular subtypes. An optimal log fold-change cutoff was selected for each signature by identifying the cutoff that best separates the responder and non-responder samples using k-means clustering. We then applied our drug repositioning pipeline to identify compounds that significantly reverse these signatures using the drug perturbation profiles generated in a breast cancer cell line in the Connectivity Map v2 dataset. Briefly, the pipeline uses a non- parametric, rank-based pattern-matching strategy based on the Kolmogorov-Smirnov (KS) statistic to assess the enrichment of resistance genes in a ranked drug gene expression list. Significance of each prediction is estimated from a null distribution of scores generated from random gene signatures. Results: We found that few individual genes are shared among the resistance signatures across the treatment arms and molecular subtypes, with the most common genes present in only 5/17 of the treatment arm and molecular subtype groups. At the pathway-level, however, we found that immune-related pathways are generally enriched among the responders and estrogen-response pathways are generally enriched among the non-responders. Although most of our drug predictions are unique to treatment arms and molecular subtypes, our drug repositioning pipeline identified the selective estrogen receptor degrader (SERD) fulvestrant as a compound that can potentially reverse resistance across a majority of the treatment arms and molecular subtypes. Conclusion: We applied our drug repositioning pipeline to identify novel agents to sensitize drug-resistant tumors in the I-SPY 2+ clinical trial and identified a SERD, fulvestrant, as a potential candidate for multiple molecular subtypes and treatment arms. Citation Format: Katharine Yu, Amrita Basu, Christina Yau, Denise Wolf, Gillian Hirst, Laura Sit, Nicholas O’Grady, Thelma Brown, I-SPY 2 TRIAL Investigators, Angela DeMichele, Don Berry, Nola Hylton, Doug Yee, Laura Esserman, Laura van 't Veer, Marina Sirota. Computational drug repositioning for the identification of new agents to sensitize drug-resistant breast tumors across treatment arms and molecular subtypes [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-04.

I-SPY2 is a neoadjuvant trial evaluating experimental therapies in combination with cytotoxic chemotherapy compared to chemotherapy alone with the primary endpoint of pathologic complete response (pCR). Abundant preclinical evidence suggested the type I insulin-like growth factor receptor (IGF-1R) regulated breast cancer growth, although multiple clinical trials did not show benefit. We were the first to report the results of a monoclonal IGF-1R antibody ganitumab (G) in combination with chemotherapy. PGM followed by doxorubicin/cyclophosphamide (AC) did not result in substantial increases in pCR when compared to P followed by AC. In this report, we examined several potential predictive biomarkers. IGF-1R inhibitors induce hyperglycemia and we examined hemoglobin A1C (HgbA1c) as a measure of glucose control in patients before and after PGM therapy. 106 patients received PGM and 104 patients had baseline HgbA1c with a median of 5.4%. However, 27% (28/104) had levels greater than 5.7% the upper limit of normal as defined by the NIDDK. 4 of 104 had HgbA1c greater than 6.5%, a level associated with type 2 diabetes. pCR rates are similar between patients with baseline HgbA1c ≤5.7% (21%) vs. >5.7% (25%) (Fisher test p=0.79). 72 of these patients had an additional HgbA1c during the course of PGM therapy. For patients with HgbA1c ≤5.7%, 27% (14/52) had subsequent elevation above 5.7% after PGM. For patients with a baseline HgbA1C >5.7%, all 20 patients continued to have elevated levels through PGM. We also examined pre-treatment tumor gene expression profiles derived from custom Agilent 44K full-genome microarrays. We studied 11 genes associated with the IGF-1R signaling (IGF1, IGF2, IGF1R, INSR, IGFBP2, IRS1, IRS2, IGFBP3, IGFBP4, IGFBP5, CDH1), the IGFBP5/IGFBP4 ratio, and two IGFR expression signatures (Creighton, et al. J Clin Oncol 26:4078 2008 PMID: 18757322; Mu, et al. Breast Cancer Res Treat 133:321 2012 PMID: 22297468). The 2 signatures evaluated: the IGF1 ligand score and the IGF1-R signature are anti-correlated (Rp= - 0.79). In the population as a whole, lower levels of IRS1 and IGFBP5 significantly associated with response to PGM (likelihood ratio test (LR) p< 0.05), as do lower levels of the IGF1 ligand score and higher levels of the IGF-1R signature. However, levels of IRS1 and the two expression signatures also trend toward or are significantly associated with response in the control arm; and treatment interactions for all four biomarkers are non-significant (LR p>0.05). Therefore, none of these biomarkers qualify as specific predictors of response to PGM. Similarly, high MammaPrint scores (MP2) were associated with higher pCR scores in both PGM and Control arms. Previous gene expression profiles were divided into tertiles (low, intermediate, high). Similar to the continuous case, IGF1Rsig-class associates with pCR in both the PGM and control arms (Fisher test p=0.033 and 0.044, respectively), and thus also fails as a specific predictor of response to PGM. We conclude that PGM therapy results in worsening of glucose control and likely increases serum insulin levels. While IGF gene expression profiling associated with treatment response, they were not specific for PGM. Further, biomarker analysis and strategies to control glucose will be needed to optimize anti-IGF-1R therapies. Citation Format: Douglas Yee, Paul Haluska, Denise M Wolf, Christina Yau, Amy Wilson, I-SPY2 TRIAL Consortium, Angela DeMichele, Claudine Isaacs, Jane Perlmutter, Joan Venticinque, Hope S Rugo, Richard Schwab, Nola M Hylton, W Fraser Symmans, Michelle E Melisko, Teresa L Helsten, Laura J van't Veer, Donald A Berry, Laura J Esserman. Biomarker analysis of paclitaxel, ganitumab, and metformin (PGM) therapy in the I-SPY2 neoadjuvant clinical trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-08.

Background: Disseminated tumor cells (DTCs) in bone marrow detected after treatment may represent occult residual disease. We enumerated DTCs after neoadjuvant chemotherapy (NACT) in patients (pts) diagnosed with high-risk early stage breast cancer and examined the relationship of these cells with response and survival. Methods: I-SPY SURMOUNT is a sub-study of the I-SPY 2 TRIAL (NCT01042379). Pts enrolled on I-SPY 2, who signed consent for this sub-study, had bone marrow aspirates (BMA) collected after NACT at the time of surgery. DTCs were isolated and enumerated from BMA using immunomagnetic enrichment/flow cytometry (IE/FC). DTCs were defined as EPCAM-positive and CD45-negative nucleated cells. Samples were considered positive using a predetermined threshold of >4 DTCs per mL (Magbanua et al, unpublished data). Pathologic response was assessed using the residual cancer burden (RCB) method at local sites, and pts underwent standard adjuvant therapy if indicated and follow up for recurrence events and death. Relationship of DTCs with clinicopathologic variables was examined using Chi-squared test. Group means were compared using t tests. The log-rank test was used to compare survival curves. Results: A total of 73 patients were enrolled, 51 of whom had successful DTC assessment. The median DTC per mL was 4 (interquartile range 1.2-11.6). 24/51 (47%) were DTC-positive. Clinical characteristics by DTC status are shown in the table. DTC-positive pts were significantly younger (p=0.02) and had larger pretreatment tumors (longest diameter by magnetic resonance imaging) compared to DTC-negative pts (p=0.032). DTCs were not associated with receptor subtype. Thirty pts (41%) achieved a pathologic complete response (pCR). DTCs were not associated with pCR (p= 0.166); however, DTC-positive patients were significantly more likely to have residual cancer (RCB-II/III) after NACT compared to DTC-negative patients (OR 3.3, p=0.037). Median follow up of this cohort was 2.8 years (range: 0.9-4.8). Interim survival analysis showed that DTCs were not significantly correlated with EFS (p=0.6) or DRFS (p=0.41). Conclusions: Detection of DTCs at surgery after NACT is significantly more common in young patients, those with larger tumors, and those with residual disease at surgery. While these associations suggest higher risk for later recurrence, larger studies and longer follow up are necessary to determine if DTCs add prognostic value over pathologic evaluation alone for pts receiving NACT. Citation Format: Mark Jesus M Magbanua, Laura van 't Veer, Amy Clark, A. Jo Chien, Judy Boughey, Heather Han, Anne Wallace, Heather Beckwith, Minetta Liu, Christina Yau, E. Paul Wileyto, Lamorna Brown Swigart, Jane Perlmutter, Lauren Bayne, Shannon Deluca, Stephanie Yee, Erica Carpenter, Laura Esserman, John Park, Lewis Chodosh, Angela DeMichele. Outcomes associated with disseminated tumor cells at surgery after neoadjuvant chemotherapy in high-risk early stage breast cancer: The I-SPY SURMOUNT study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-07.

Background: I-SPY 2 is a multicenter, phase 2 trial using response-adaptive randomization within molecular subtypes defined by receptor status and MammaPrint (MP) risk to evaluate novel agents as neoadjuvant therapy for women with high-risk stage II/III breast cancer. The primary endpoint is pathologic complete response (pCR, ypT0/Tis ypN0). SGN-LIV1A is an investigational antibody drug conjugate consisting of an antibody to LIV1A, a zinc transporter highly prevalent in breast cancer, and a highly potent microtubule inhibitor, monomethyl auristatin E. Retrospective IHC analysis of LIV1A expression levels amongst tumor samples from 100 previous ISPY-2 patients showed 88% of breast tumor samples with moderate to high expression of LIV1A (Yau, C. et al SABCS 2018).Methods: Women with tumors ≥ 2.5cm were eligible for screening. Only HER2 negative (HER2-) patients were eligible for this treatment, hormone-receptor positive (HR+) patients had to have MP high molecular profile. Treatment included SGN-LIV1A 2.5 mg/kg (max dose 250 mg) every 3 weeks x 4, followed by doxorubicin/cyclophosphamide (AC) every 2-3 weeks x 4. The control arm was weekly paclitaxel x 12 followed by AC every 2-3 weeks x 4. All patients undergo serial MR imaging where response at 3 & 12 weeks combined with accumulating pCR data are used to estimate, and continuously update, predicted pCR rate for the trial arm. Analysis set was modified intention to treat with patients who switched to non-protocol therapy counted as non-pCR and not as their pCR status at time of surgery. The goal is to identify/graduate regimens with ≥85% Bayesian predictive probability of success (i.e. demonstrating superiority to control) in a future 300-patient phase 3 neoadjuvant trial with a pCR endpoint within signatures defined by HR & HER2 status & MP result. This investigational arm was eligible for graduation in 3 of 10 pre-defined signatures: HER2-, HR+HER2- and HR-HER2-. Regimens may also leave the trial for futility (< 10% probability of success), maximum sample size accrual (10% < probability of success Results: Sixty patients were randomized and evaluable to SGN-LIV1A. The study arm was stopped due to reaching the predetermined time limit for patient accrual of 2 yrs. Final estimated pCR rates are below. The estimated pCR rates were similar between the SGN-LIV1A and control arms for any tumor subtype. Preliminary safety events for SGN-LIV1A include increased rates of transaminitis and hyperglycemia and reduced rates of peripheral neuropathy compared to control. One patient was removed from the analysis as she was determined to have angiosarcoma of the breast. Notably, this patient had a dramatic early response and subsequent pCR to SGN-LIV1A treatment. Conclusion: The value of I-SPY 2 is to give insight about the performance of an investigational agent’s likelihood of achieving pCR. SGN-LIV1A delivered every 3 weeks was comparable to paclitaxel for the primary endpoint of pCR in I-SPY2 and may have a similar side effect profile, however, with less peripheral neuropathy. Clinical trials evaluating weekly dosing of SGN-LIV1A are ongoing. A trial of SGN-LIV1A in the treatment of angiosarcoma is under consideration at this time. Final Estimated pCR Rates and Predictive ProbabilitiesEstimated pCR rate(95% prob interval)SignatureSGN-LIV1AControlProbability SGNLIV1A Superior to ControlPredictive Probability of Success in Phase 3HER2-0.16 (0.08-0.24) N= 600.20 (0.16-0.25) N= 3270.180.02HR-/HER2-0.25 (0.12-0.37) N=360.28 (0.21-0.35) N=1460.310.06HR+/HER2-0.09 (0-0.18) N=240.14 (0.09-0.19) N=1810.150.03 Citation Format: Heather Beckwith, Richard Schwab, Christina Yau, Erica Stringer-Reasor, Shi Wei, A. Jo Chien, Kathy S Albain, Kevin Kalinsky, Anne Wallace, Anthony Elias, Douglas Yee, Amy S Clark, Judy C Boughey, Heather Han, Rita Nanda, Claudine Isaacs, Zahi Mitri, Julie E Lang, Alexandra Thomas, Tara Sanft, Angela DeMichele, Jane Perlmutter, Hope S Rugo, Nola M Hylton, W. Fraser Symmans, Michelle E Melisko, Laura J van't Veer, I-SPY 2 Consortium, Amy Wilson, Smita M Asare, Ashish Sanil, Donald A Berry, Laura J Esserman. Evaluation of SGN-LIV1a followed by AC in high-risk HER2 negative stage II/III breast cancer: Results from the I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD1-10.

Introduction: Given excellent survival outcomes in breast cancer and new methods to predict treatment response, oncologists are interested in de-escalating the amount of chemotherapy delivered to patients. This is particularly important in the setting of COVID-19, where patient perspectives of de-escalation may be altered by perception of COVID-19 risk.Methods: This concurrent mixed methods study included (1) semi-structured interview data from patients with breast cancer treated at the University of Alabama at Birmingham and patient advocates from nationally representative advocacy organizations (10/2019-5/2020) and (2) cross-sectional survey data from a nationwide sample of women with breast cancer (11/19-12/2019). Questions evaluated interest in de-escalation study participation, perceived barriers/facilitators to participation, and language describing de-escalation. Participant perspectives surrounding COVID-19 impact on de-escalation were elicited in interviews post 3/2020.Results: Quantitative and qualitative findings were synergistic. Interviews were conducted with 40 female participants (24 patients, 16 patient advocates). Participant ages ranged from 33-79 years old; 30% were minorities; 35% didn’t have a college degree. Common barriers to acceptance of de-escalation included fear of recurrence, worry about decision regret, lack of clinical trial interest, and dislike for the focus on less treatment. Fear of recurrence was the most commonly expressed barrier, with one participant stating, “I’m just afraid it wouldn’t get it all”. Common facilitators included trust in the physician, toxicity avoidance, monitoring with the option of increasing treatment intensity, perception of good prognosis, and impact on daily life. Participants interviewed during the COVID-19 pandemic (n=16) expressed substantial virus-related fear, including fear of exposure, fear of infecting their personal contacts or health care team, fear of cancer-related complications, and fear about their immunocompromised state. These fears contributed to participants perspective on de-escalation, as highlighted by participants stating, “I wouldn't worry about getting the chemo as much as I would worry about getting the virus” and “Less is more for me right now”.Of 91 survey respondents (69% response rate), median age was 58 years (interquartile range [IQR] 48-69), 86% had early stage breast cancer. Many (43%) patients were not interested in participation in a study testing lower doses of chemotherapy than standard of care. Patients not interested in participating were more often unmarried (55% vs. 32%, V=.23), disabled (56% vs. 40%, V=.17), or diagnosed with early stage cancer (45% vs. 22%, V=.14). Barriers to participation included fear of cancer recurrence (85%) and regret about the decision to receive less chemotherapy if the cancer were to recur (79%). Few patients (19%) considered clinical trials themselves as a barrier. Patients were interested in participation due to lessened physical side effects of treatment (82%), lessened long-term problems related to treatment (76%), and lessened impact on daily life (72%). The most popular terminology describing chemotherapy de-escalation was “lowest effective chemotherapy dose” (53%); no patients preferred the term “de-escalation.” Conclusion: Fear of recurrence is a common barrier to de-escalation clinical trial participation in patients with breast cancer. Fears may be altered for patients considering treatment during the COVID-19 pandemic. Trust in the physician and use of patient-generated language, such as “customized” instead of “de-escalation”, are potential areas for future interventions engaging patients in trials. Citation Format: Gabrielle Rocque, Courtney P. Williams, Courtney J. Andrews, Kathleen Gallagher, Timothy C. Childers, Kimberly D. Wiseman, Alan Balch, Stacey A. Ingram, Thelma Brown, Tara Kaufman, Nadine Tung, Mary Lou Smith, Antonio C. Wolff, Angela DeMichele, Lynn Wagner. Patient perspectives on chemotherapy de-escalation: “Don’t de-escalate! I don’t want to die!” [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD3-10.

Background: Achieving a pathologic complete response (pCR) has been shown on the patient level to predict excellent long-term event-free survival outcomes. Residual cancer burden (RCB) quantifies the extent of residual disease for patients who did not achieve pCR. A high proportion of metastatic events to the central nervous system (CNS), a known chemotherapy sanctuary site, was previously observed among the small number of relapses in patients achieving a pCR (Symmans et al 2017), raising the possibility that these CNS events may be independent of response in the breast. I-SPY2 is an adaptively randomized, phase II, platform trial that evaluates new drugs and combinations in the neoadjuvant setting for women with high-risk primary breast cancer. In this study, we evaluated the type and sites of recurrences by RCB classes in the I-SPY 2 TRIAL. Methods: I-SPY 2 patients enrolled prior to 11/2016 across 9 experimental and control arms, with available RCB and event-free survival (EFS) data were included in this analysis. The median follow-up is 3.8 years. We summarized the EFS event type, further sub-dividing the distant recurrence events by their site of relapse (CNS-only, CNS and other sites, Non-CNS). We estimated the overall and site-specific distant recurrence incidence in each RCB class at 3 years using a competing risk (Fine-Gray) model. In addition, we assessed the association between RCB and distant recurrence free survival including all distant recurrences (DRFS), as well as excluding the CNS-only recurrences (non-CNS DRFS) using a Cox model. Our statistics do not adjust for multiplicities beyond variables evaluated in this study. Results: Among 938 subjects, there were 180 EFS events, including 28 (16%) local recurrences (without distant recurrence and/or death) and 152 DRFS events. Among the DRFS events, 25 patients died without a distant recurrence. 127 experienced distant recurrences, including 22 (17.3%) with CNS-only, 16 (12.6%) with CNS and other sites, 87 (68.5%) with non-CNS distant recurrence; 2 (1.6%) patients had missing recurrence site information. Incidence of CNS-only recurrences are low and are similar across RCB classes (pCR/RCB-0 (n=338): 1%, RCB-I (n=129): 3%, RCB-II (n=328): 2%, RCB-III (n=143): 2% at 3 years). In contrast, the incidence of non-CNS recurrences increase with increasing RCB (RCB-0: 2%, RCB-I: 4%, RCB-II: 11%, RCB-III: 19% at 3 years). DRFS of RCB-I patients do not significantly differ from those achieving a pCR/RCB-0 (DRFS at 3 years: 92% vs. 95%, hazard ratio: 1.77 (0.87-3.63)); the small numerical difference is further reduced when the CNS-only recurrences are excluded (non-CNS DRFS at 3 years: 95% vs. 96%, hazard ratio: 1.48 (0.61-3.58)). CNS recurrences among DRFS events are proportionally higher within the pCR (5/16 (31%)) and RCB-I (5/12 (42%)) than in the RCB-II (8/57 (14%)) and RCB-III (4/42 (9%)) groups largely because of the relative low frequency of non-CNS recurrence events. Conclusions: In our high-risk I-SPY 2 cohort, CNS-only recurrences are uncommon but appear similar across RCB groups, independent of response, suggesting that the CNS is a treatment sanctuary site. In contrast, non-CNS recurrence rates increase as RCB increases. These findings, if confirmed, support the use of RCB to identify patients with excellent outcomes beyond those achieving a pCR; and suggest that inclusion of CNS only recurrences as an outcome event may impact the association between neoadjuvant therapy response and long-term outcome. Citation Format: Christina Yau, Angela DeMichele, W. Fraser Symmans, Lajos Pusztai, Douglas Yee, Amy S. Clark, Christos Hatzis, Jeffrey B. Matthews, Jodi Carter, Yunn-Yi Chen, Kimberly Cole, Laila Khazai, Molly Klein, Dina Kokh, Gregor Krings, Sunati Sahoo, Kathy S. Albain, A. Jo Chien, Kirsten K. Edmiston, Anthony D. Elias, Erin D. Ellis, David M. Euhus, Heather S. Han, Claudine Isaacs, Qamar J. Khan, Julie E. Lang, Janice Lu, Jane L. Meisel, Zaha Mitri, Rita Nanda, Donald W. Northfelt, Tara Sanft, Erica Stringer-Reasor, Rebecca K. Viscusi, Anne M. Wallace, Rachel Yung, Nola M. Hylton, Judy C. Boughey, Michelle E. Melisko, Jane Perlmutter, Hope S. Rugo, Richard Schwab, Laura J. van' t Veer, Donald A. Berry, Laura J. Esserman. Site of recurrence after neoadjuvant therapy: Clues to biology and impact on endpoints [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-20-02.

Background: A variety of investigational HER2-inhibitor agents/combinations have been tested in I-SPY 2, including neratinib (N), TDM1 combined with pertuzumab (P) (TDM1/P), and trastuzumab (H) combined with pertuzumab (H/P; prior to this combination becoming standard of care), all with trastuzumab as control (Ctr). All three experimental arms graduated, showing improved efficacy over control in one or more receptor subsets (HR+HER2+, HR-HER2+, or/and HER2+). Here we assess 10 biomarkers in the HER2, ER/PR, and proliferation pathways on multiple levels of resolution (expression, protein, phospho-protein) as predictors of response in these four arms, hypothesizing that highly HER2-activated, proliferative tumors may be more sensitive to HER2-inhibition than those that are more luminal and quiescent. Methods: 192 HER2+ patients were considered in this analysis: (31 Ctr, 65 N, 52 TDM1/P, and 44 H/P). 10 biomarkers relating to HER2, ER, or proliferation were evaluated from pre-treatment biopsies: HER2 IHC (n=145), 3 expression signatures (n=192), BluePrint subtype (n=192), and 5 protein/phospho-protein analytes by RPPA (n=175). Each biomarker was tested for association with pCR in the whole population and within each arm using a logistic model. This analysis was adjusted for HR status and treatment arm as covariates, and performed within receptor subtypes. This analysis does not adjust for multiplicities of other biomarkers. Results: In the population as a whole, HER2 and HER2-signaling biomarkers, evaluated at multiple levels of resolution: IHC, total-/phospho-protein by RPPA, and mRNA (HER2 amplicon module) - are highly correlated (rho=0.8 [0.65-0.92]). Higher HER2 levels and activity are associated with response: HER2 IHC 3+ status (LR p=0.00032), total quantitative ERBB2 protein by RPPA (LR p=5.4E-09), ERBB2 activation levels (LR p=6.58E-06 (pERBB2 (Y1248)) and 9.95E-06 (pEGFR Y1173)), and the ERBB2 amplicon expression signature (LR p=2.38E-08). In contrast, higher average ER/PR expression associates with non-response to HER2-targeted therapy (LR p=4.28E-08). Both HER2 and ER/PR signaling phenotypes are captured by BluePrint subtyping; and consistent with the individual pathway markers, tumors classified Luminal-type had a lower pCR rate relative to those classified as Her2-type (or Basal-type) (LR p=4.84E-11). These associations all retain significance in a model adjusting for HR status and treatment arm, and in the HR+HER2+ subset. In addition, we quantitatively assessed proliferation markers at the total protein (RPPA: Ki67), phospho-protein (pAURKA) and mRNA (proliferation signature Module11_Prolif) levels. All three proliferation biomarkers predict response overall; but this association is strongest within the HR+HER2+ subset (LR p=0.0012 (Module11_prolif), 0.0036 (pAURK), and 0.045 (Ki67)). None of the biomarkers tested were associated with response in the HR-HER2+ subset. Numbers are small within individual arms. Within the HR+HER2+ subtype, higher HER2 and lower ER/PR is observed in responders in all experimental arms; but the proliferation markers Module11_Prolif (LR p=0.0031) and Ki67 total protein (LR p=0.0029) are associated with response to TDM1/P but not H/P or N. Conclusion: High HER2 signaling at the expression, protein, and phospho-protein levels, and low ER signaling, all predict response to HER2-inhibition across treatment arms. Proliferation markers may be useful for prioritizing therapies in the HR+HER2+ subset. Citation Format: Denise M Wolf, Christina Yau, Julia Wulfkuhle, Lamorna Brown-Swigart, Smita M. Asare, Gillian L. Hirst, Laura Sit, Jane Perlmutter, I-SPY 2 TRIAL Consortium, Minetta Liu, John Park, Angela DeMichele, Douglas Yee, Don Berry, Laura Esserman, Emanuel Petricoin, Laura van t' Veer. HER2 signaling, ER, and proliferation biomarkers predict response to multiple HER2-targeted agents/combinations plus standard neoadjuvant therapy in the I-SPY 2 trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-02.

Background: Patients (pts) treated for early stage breast cancer (BC) have a 30% lifetime risk of developing incurable, distant metastatic disease. Yet, standard monitoring after definitive therapy for primary disease is passive observation. Numerous studies have demonstrated that dormant bone marrow (BM) disseminated tumor cells (DTCs) are independently associated with recurrence, but assessment of DTCs is not performed in clinical practice, largely because of concerns about the acceptability and logistics of bone marrow aspiration (BMA) and lack of established therapies that target DTCs. As part of a large scale screening study for a clinical trial targeting DTCs, we examined pt attitudes about DTC screening and subsequently assessed feasibility and tolerability of BM DTC assessment. Methods: The PENN-SURMOUNT (Surveillance Markers of Utility for Recurrence after (Neo)adjuvant Therapy) Screening Study is a single center prospective, longitudinal cohort study examining BM and blood biomarkers of minimal residual disease (MRD) among pts within 5 years of BC diagnosis, who meet one of the following high risk criteria: positive axillary nodes, triple negative biology, ER+ with Oncotype Dx ≥ 25 and/or high risk Mammaprint, or residual disease (RD) after neoadjuvant chemotherapy (NACT). Consented pts undergo a baseline outpatient BMA; if negative, pts can repeat screening annually. During trial design, we surveyed 25 women with stage 2-3 BC at random from the breast clinic at the University of Pennsylvania to assess feasibility. On the SURMOUNT Study, we collected demographic and clinical characteristics of pts, and patient-centered survey data regarding feasibility and acceptability of the BMA that is administered within 48 hours of the procedure. Results: In the pre-trial feasibility survey, 21/25 (84%) pts indicated they were very/definitely interested in knowing if they harbored DTCs. Of those, 18 (86%) indicated moderate/definite interest in testing for DTCs with BMA after the BMA was described to them in detail. 20 (95%) of pts indicated moderate/definite interest in taking oral therapy to eradicate DTCs. 14 (67%) pts stated undergoing up to 3 additional BMA would not change their likelihood of undergoing the clinical trial; only 1 stated much less likely. In the subsequent SURMOUNT study, 361 pts have been referred to date; 167 were eligible, and 136 (81%) subsequently enrolled. 21 (13%) are still in screening. 130 pts have had at least 1 BMA with annual re-screens in 37 (year 1) and 8 (year 2). 39% traveled >50 miles to participate. Post-BMA symptoms were rare (bleeding 2%; redness 12%) though 59%/70% reported mild-moderate pain/tenderness. After BMA, 47%/29%/25% reported it was better/same/worse than expected. 30%/32%/22% reported minimal/moderate/high anxiety prior to the BMA. Afterward, only 20%/5%/4% reported minimal/moderate/high residual anxiety. In 128 pts with results, 38 (30%) have ≥ 1 DTC (30 initial, 8 on follow-up); by risk group: 20/77 (26%) node positive, 20/64 (31%) triple negative, 3/8 (38%) ER+/RS≥25, 7/29 (24%) with RD post-NACT. DTC+ pts were similar to DTC- in median age (50.4), race, distance traveled, menopausal status and BMI. 94% of DTC+ pts have entered the CLEVER clinical trial. Conclusion: A majority of BC survivors want to know DTC status; a majority of these are willing to have BMA and enroll on a clinical therapeutic trial, many are willing to travel to participate and are willing to undergo annual BMA assessment. The SURMOUNT study shows that screening for DTCs is feasible and effective in identifying pts for therapeutic intervention targeting MRD to reduce recurrence. Citation Format: Isoris Nivar, Tara Kauffman, Lauren Bayne, Paul Wiley, Brooke Goodspeed, Michael Feldman, Lewis Chodosh, Amy Clark, Angela DeMichele. Patient attitudes, experience and results of screening for minimal residual disease (MRD) for therapeutic intervention [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-12-01.

Background: Palbociclib (PB), the first clinically available oral CDK4/6 inhibitor, in combination with endocrine therapy has become standard of care for HR+/HER2- advanced/metastatic breast cancer (MBC). The phase III PALOMA-2 trial demonstrated significant progression-free survival benefit of PB plus letrozole (LE) vs LE + placebo (27.6 vs 14.5 months; HR=0.56, 0 Table 1. Patient characteristics and overall survival (OS)VariablePB+LELE alone(N=766)(N=622)Median age (IQR), years66.0 (58.0—73.0)70.0 (61.0—79.0)White (%)68.368.0Median number of metastatic sites (n)2.02.0Bone-only disease (%)27.731.2Visceral disease (%)52.949.4De novo MBC (%)41.539.4Median follow-up (95%CI), months 22.0 (20.5—23.7)19.0 (16.7—21.2)Percent of events censored (%)79.970.6Estimated OS rate (%)6 months95.988.912 months91.384.624 months81.270.836 months72.060.6PB+LE= Palbociclib plus letrozole; LE= Letrozole alone; CI = Confidence interval; IQR = interquartile range Citation Format: Angela DeMichele, Massimo Cristofanilli, Adam Brufsky, Xianchen Liu, Jack Mardekian, Lynn McRoy, Rachel M Layman, Hope S Rugo, Richard S Finn. Overall survival for first-line palbociclib plus letrozole vs letrozole alone for HR+/HER2- metastatic breast cancer patients in US real-world clinical practice [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-02.

Background: I-SPY2 is a multicenter, phase 2 trial using response-adaptive randomization within biomarker subtypes to evaluate novel agents as neoadjuvant therapy for high-risk breast cancer. The primary endpoint is pathologic complete response (pCR) at surgery. The goal is to identify (graduate) regimens with ≥ 85% Bayesian predictive probability of success (i.e., demonstrating superiority to control) in a future 300-patient phase 3 1:1 randomized neoadjuvant trial with pCR endpoint within signatures defined by hormone-receptor (HR), HER2, and MammaPrint (MP) status. Regimens may leave the trial for futility (< 10% probability of success), maximum sample size accrual (with probability of success ≥ 10% and < 85%), or safety concerns as recommended by the independent DSMB. For HER2+ patients, the I-SPY2 control arm was 12 weekly cycles of paclitaxel+trastuzumab (TH, control) followed by doxorubicin/cyclophosphamide (AC) q2-3 weeks x4 and surgery. Patritumab is a fully human monoclonal antibody that inhibits HER3. In this experimental arm for HER2+ patients, patritumab was given q3w x 4 cycles (18mg/kg loading dose followed by 9mg/kg/dose) concurrent with paclitaxel and trastuzumab q1w x 12 weeks (PTH, treatment), followed by AC q2-3w. Methods: Women with tumors ≥ 2.5cm were eligible for screening. MP low/HR+ tumors were ineligible. MRI scans (baseline, 3 weeks after start of therapy, prior to AC, and prior to surgery) were used in a longitudinal statistical model to predict pCR for individual patients. Analysis was intention to treat. Patients who switched to non-protocol therapy count as non-pCR. Patients on treatment arm therapy at the time of arm closure are non-evaluable. Graduation potential was in 3 of 10 pre-defined signatures: all HER2+, HR-/HER2+, and HR+/HER2+. Results: The PTH regimen was stopped at the recommendation of the Safety Working Group and DSMB based on a safety event (bilateral sensorineural hearing loss, Gr 3) observed in one patient. At the time of arm closure, N=31 patients had received PTH treatment; 4 patients receiving PTH were changed to non-protocol therapy and removed from the analysis. The final estimated pCR report will consider 27 PTH and 31 TH as evaluable patients. Accrual was insufficient to assess graduation, however, there appears to be good signal in the HER2+HR- but not HER2+HR+ signatures. I-SPY 2 TRIAL Est. pCR at time of arm closureSignaturesPTH (Treatment)N= 31TH (Control)N = 31All (HER2+)0.40 (0.22 - 0.59), n=310.23 (0.09 - 0.37), n=31HR-/HER2+0.64 (0.36 - 0.91), n=110.30 (0.12 - 0.47), n=12HR+/HER2+0.28 (0.08 - 0.48), n=200.20 (0.06 - 0.34), n=19 HR+/HER2+0.28 (0.08 - 0.48), n=200.20 (0.06 - 0.34), n=19The patient who developed Gr3 sensorineural hearing loss 6 days after the 2nd patritumab (and 4th paclitaxel/trastuzumab) treatment, did not recover her hearing after patritumab was stopped, and also reported Gr3 vulvovaginal pain, vulvitis, and vaginal inflammation. Other gynecological symptoms in the PTH arm include: 1 pt with Gr1 vaginal hemorrhage, and 1 pt with Gr2 dyspareunia. There was a higher frequency of Gr3 hypokalaemia (12.5% vs. 3.2%). One pt in the PTH arm reported Gr3 small intestinal obstruction which resolved with conservative management. Conclusion: The I-SPY 2 study aims to assess the probability that investigational regimens will be successful in a phase 3 neoadjuvant trial; PTH was stopped due to safety concerns, although there was activity in the HER2+ HR- signature. This is the first report of Gr3 hearing loss associated with patritumab/paclitaxel/trastuzumab, and thus attribution is uncertain. Citation Format: Teresa L Helsten, Shelly S Lo, Christina Yau, Kevin Kalinsky, Anthony D Elias, Anne M Wallace, A. Jo Chien, Janice Lu, Julie E Lang, Kathy S Albain, Erica Stringer-Reasor, Amy S Clark, Judy C Boughey, Erin D Ellis, Douglas Yee, Angela DeMichele, Claudine Isaacs, Jane Perlmutter, Hope S Rugo, Richard Schwab, Nola M. Hylton, W. Fraser Symmans, Michelle E Melisko, Laura J van't Veer, Amy Wilson, Ruby Singhrao, Smita M Asare, Ashish Sanil, Donald A Berry, Laura J Esserman. Evaluation of patritumab/paclitaxel/trastuzumab over standard paclitaxel/trastuzumab in early stage, high-risk HER2 positive breast cancer: Results from the neoadjuvant I-SPY 2 trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-02.

Background: I-SPY2 is a multicenter, response-adaptive randomization phase 2 trial to evaluate novel agents when added to standard neoadjuvant therapy for women with high-risk stage II/III breast cancer - weekly paclitaxel + investigational treatment x 12 wks followed by doxorubicin & cyclophosphamide(AC) q3 wks x 4 vs. weekly paclitaxel/AC (control). The primary endpoint is pathologic complete response (pCR). The goal for all investigational arms is to identify/graduate regimens with ≥85% Bayesian predictive probability of success (i.e. demonstrating superiority to control) in a future 300-patient phase 3 1:1 randomized neoadjuvant trial with a pCR endpoint within signatures defined by hormone-receptor (HR) & HER2 status & MammaPrint (MP). Findings from the graduated, previously reported Pembro4 arm (Nanda et al, ASCO 2017) supported investigation of de-escalating therapy, and determining if pembrolizumab (an anti-PD-1 antibody) alone q3 wks x 4 after weekly paclitaxel x 12 wks + pembrolizumab q3 wks x 4 was sufficient to sustain response without AC. Methods: Women with tumors ≥2.5cm were eligible for screening. MP low/HR+ were ineligible. MRI scans (at baseline, 3 wks, 12 wks, and prior to surgery) were used in a longitudinal statistical model to predict pCR for individual patients (pts). Pts who receive non-protocol therapy (e.g., carboplatin or AC for the Pembro8-noAC arm) count as non-pCR. Pembro8-noAC was open to HER2- pts for evaluation in 3 of 10 pre-defined signatures: HER2-, HR+/HER2-, and HR-/HER2-. Regimens exit the trial for futility ( Results: Pembro8-noAC was randomized to 73 pts, 3 of whom progressed while receiving pembrolizumab alone on study. Randomization to this arm continued after the first report because the rate of progression during AC over the course of the trial was estimated to be 6.5% based on serial MRI studies. However, notification of the third case prompted the study team to ask the DSMB for the summary response for this arm. Although it did not meet formal stopping rules for either graduation or futility, Pembro8-noAC was not near the target threshold pCR rates of 60% for HR-/HER2- and 30% for HR+/HER2+. As a result of this information, combined with the on-treatment progressions, assignment to Pembro8-noAC was discontinued. Treatment with pembrolizumab alone was no longer allowed due to the potential concern for progression, and investigators were given the option to administer AC with pembrolizumab or proceed with definitive surgery following the 12 weeks of paclitaxel + pembrolizumab. 34 pts had surgery results at the time the study was closed. Of the remaining 39 pts, 34 pts have on-therapy MRI assessments. Estimated pCR rates were based on all pts with information at the time (see table). Immune-related adverse events included grade 3 colitis (n=2), grade 3 pneumonitis (n=1), grade 3 transaminitis (n=1), grade 3 hypothyroidism (n=1), and grade 1-2 adrenal insufficiency (n=5). Conclusion: Although Pembro8-noAC is performing at least as well as standard paclitaxel/AC, the likelihood is very low that the regimen would be successful in a phase 3 trial. Pembrolizumab alone following 12 weeks of paclitaxel + pembrolizumab was not sufficient to sustain a response. This was quickly assessed with a small number of patients. Estimated pCR rateSignature(95% prob interval)Pembro8-noACControlHER2-0.210.2(0.09-0.32)(0.15-0.25)HR-/HER2-0.270.27(0.09-0.45)(0.19-0.35)HR+/HER2-0.150.15(0.01-0.29)(0.09-0.20) Citation Format: Minetta C. Liu, Patricia A Robinson, Christina Yau, Anne M Wallace, A. Jo Chien, Erica Stringer-Reasor, Rita Nanda, Douglas Yee, Kathy S Albain, Judy C Boughey, Heather S Han, Anthony D Elias, Kevin Kalinsky, Amy S Clark, Kathleen Kemmer, Claudine Isaacs, Julie E Lang, Janice Lu, Tara Sanft, Angela DeMichele, Nola M Hylton, Michelle E Melisko, Jane Perlmutter, Hope S Rugo, Richard Schwab, W. Fraser Symmans, Laura J van't Veer, Patricia K Haugen, Amy Wilson, Ruby Singhrao, Smita Asare, Ashish Sanil, Donald A Berry, Laura J Esserman. Evaluation of a novel agent plus standard neoadjuvant therapy in early stage, high-risk HER2 negative breast cancer: Results from the I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-09-02.

Importance: It is essential to refine the populations most likely to benefit from targeted chemotherapy combinations. Background: MINDACT showed that molecularly low risk patients, as assessed by the Amsterdam 70-gene profile MammaPrint® (Agendia) (MP), did not benefit from chemotherapy. In the I-SPY2 trial, MP low risk Hormone Receptor (HR) positive/HER2 negative patients are not eligible; however, HR negative or HER2 positive patients were considered high risk and included for treatment with chemotherapy plus novel agents. The neoadjuvant setting provides an opportunity to evaluate whether molecularly low risk HER2 positive patients are good candidates for neoadjuvant chemo/anti-HER2 therapy, where pathologic complete response (pCR) and 3-year outcomes are measured. Objective: To evaluate and further characterize the fraction of and outcomes for patients molecular low-risk disease in the I-SPY2 trial. Methods: The I-SPY2 platform trial is an ongoing multicenter phase 2 multi-arm study utilizing adaptive design and evaluating efficacy and tolerability of investigational agents in combination with standard-of-care chemotherapy for patients with high-risk anatomic stage II/III breast cancer. Patients with HR+HER2- MP low-risk tumors are not eligible. All enrolled patients undergo pretreatment biopsy for genomic evaluation of early recurrence risk with MP for risk stratification and with the 80-gene BluePrint® (Agendia) test for intrinsic subtype classification. Primary endpoint of the study is pCR in breast and lymph nodes (ypT0/is, ypN0). Secondary outcomes included event-free survival (EFS) and distant-recurrence free survival (DRFS). Following standard neoadjuvant treatment with or without a concomitant investigational agent, patients are followed for long-term outcomes. EFS and DRFS at 3 and 5 years in the pCR vs non-pCR groups within histologic and molecular subtypes are determined using the Kaplan Meier method. Results: Of the 1038 enrolled patients by November 2016, 24 patients (3.2%) had molecular low-risk disease as determined by MP. Of these, 21 (87.5%) had HR+HER2+, 1 (4.2%) had HR-HER2+, and 2 (8.4%) had HR-HER2-. BluePrint expression subtyping classified 17 as luminal-type, 6 HER2 enriched-type and 1 basal type. One patient withdrew consent for follow-up. Of the 23 remaining, none achieved pCR and only one (HR+/HER2+) had an EFS event (with median follow-up of 4.3 years). Overall, EFS and DRFS data were available for 950 of the 1038 patients (as of February 2019), with median follow-up of 3.8 years. Of the 173 HR+HER2+ in this cohort, 20 (11.6%) had molecular low-risk tumors. The EFS and DRFS for the 173 HER2+HR+ pCR group is 97% and 98% (non-pCR group 89% and 94%), respectively at 3 years. Removing the molecular low-risk HER2+ patients from the denominator reveals the worse outcome for patients with non pCR: EFS 86% and DRFS 92% at 3 years. The difference is more apparent at 5 years, changing from 74% to 66% for EFS and from 81% to 75% for DRFS. Intrinsic subtypes vary and do not predict early risk. Conclusions: I-SPY2 focuses on women with high clinical risk with only 3.2% of enrolled patients having low molecular risk tumors. However, 12% of HER+ breast cancer enrolled patients had molecular low risk tumors of which 95% were also HR+. In this group, early recurrence is very low despite absence of pCR and irrespective of intrinsic subtype. Refining the population using molecular high-risk classification of patients reveals a greater difference between the pCR and non-pCR groups within the HR+HER2+ subtype in the EFS and DRFS analysis. Clinical trial designs could address the opportunity to find more targeted and less toxic treatments for the HER2+ low molecular risk patient population. Citation Format: Paula R Pohlmann, Christina Yau, Angela DeMichele, Claudine Isaacs, Judy C Boughey, Nola Hylton, Michelle E Melisko, Jane Perlmutter, Hope S Rugo, W. Fraser Symmans, I-SPY 2 TRIAL Consortium, Donald A Berry, Laura Esserman. Amsterdam 70-gene profile (MammaPrint) low risk, even in the HER2 positive subset, identifies a population of women with lower early risk for recurrence despite low response rates to chemotherapy and to HER2 targeted therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-06.