Liu, Minetta C.

3010 Background: PATHFINDER (NCT04241796) is an interventional, prospective study evaluating implementation of a blood-based multi-cancer early detection (MCED) test that uses targeted methylation-based cfDNA analysis to detect multiple cancer types and simultaneously predict cancer signal origin (CSO). We present a prespecified interim analysis of PATHFINDER evaluating an MCED test in a clinical setting. Methods: Participants (pts; ≥50y) were enrolled into 2 risk cohorts: non-elevated and elevated (smoking history, prior cancer [ > 3y post treatment], or genetic predisposition). MCED test results (cancer signal detected/not detected) were returned to investigators; pts with a signal detected also received a CSO prediction and underwent further diagnostic testing by their medical team. The primary objective was to assess the extent of diagnostic testing needed to achieve diagnostic resolution (eg, time to resolution, number/type of tests). Secondary endpoints included positive predictive value (PPV) and a measure of test satisfaction (following diagnostic resolution [signal detected] and post test [signal not detected]). Results: PATHFINDER consented 6796 pts before closing accrual on 12/4/20; as of October 6, 2020, 4086 consented, 4047 enrolled, and 4033 analyzable pts were included in the interim analysis (62.4% female, 92.1% white). Two study-related adverse events (anxiety of mild severity) were reported. Cancer signal was detected in 1.5% (62/4033) of pts; 40/62 reached diagnostic resolution to date. Kaplan-Meier estimate of median time to resolution was 78 (95% CI, 54-151) days. Among 40 pts that reached diagnostic resolution, ≥1 imaging test was performed in 93% (37/40); ≥1 invasive procedure was performed in 72% (13/18) versus 18% (4/22) of pts with diagnostic resolution of cancer versus no cancer, respectively. Based on results to date, PPV was 45% (95% CI, 30.7-60.2%; 18/40). Of 18 cancer diagnoses, 11 were solid tumors (3 stage IV, 6 stages I-III, 1 metastatic recurrence, 1 missing stage), and 7 were hematologic malignancies (1 stage IV, 4 stages I-III, 2 without AJCC stage). Accuracy of the top CSO prediction in true positives was 82.4% (95% CI, 59.0-93.8%; 14/17). Most pts were satisfied with the test (43.7% extremely satisfied, 30.7% very satisfied, 14.6% satisfied). Signal detection rate and test satisfaction were similar in the 2 risk cohorts; PPV tended to be higher in the elevated risk cohort, as expected. Conclusions: An interim analysis of this return of results study demonstrated promising MCED test results. Of 40 pts achieving diagnostic resolution, nearly half had a diagnostic workup confirming cancer; CSO was predicted with high accuracy for detected cancers. Taken together with the rarity of adverse events and high test satisfaction, these results support the feasibility of clinical implementation. Full enrollment cohort data will be available at the meeting. Clinical trial information: NCT04241796.

TPS1111 Background: Chest wall recurrence is a subtype of breast cancer that is challenging to treat, and associated with a short duration of response to treatment and an increased risk of development of distant metastases. Given the inflammatory nature of this disease and the association of chest wall disease with lymphovascular invasion, which is correlated with higher programmed cell death 1 (PD-1) expression, we hypothesized that immunotherapy may be beneficial as treatment. Combinations of immunotherapy and chemotherapy have a synergistic effect and demonstrated efficacy in the treatment of metastatic triple negative breast cancer (TNBC). This study is evaluating the efficacy of pembrolizumab, an anti-PD-1 antibody, in combination with carboplatin, in patients with chest wall infiltration from breast cancer. This drug combination has shown efficacy in advanced lung cancer. Methods: This is a multicenter, 2:1 randomized phase II study of pembrolizumab/carboplatin (Arm A, 56 patients) vs. carboplatin (Arm B, 28 patients) in 84 patients with chest wall disease from breast cancer, with or without distant metastases. Patients may have TNBC, hormone receptor positive/HER2 negative (following receipt of 2 prior hormone therapies), or HER2 positive breast cancer (with option to continue trastuzumab on study). Pembrolizumab is administered as 200 mg IV every 3 weeks, and carboplatin as AUC 5 IV every 3 weeks. Patients on Arm A may continue pembrolizumab +/- carboplatin (Arm Ax) after completion of 6 cycles of treatment, while patients in Arm B can cross-over to pembrolizumab (+/- carboplatin) on progression (Arm Bx). Patients must have adequate organ function, performance status ≤ 2, and may have received any number of lines of prior chemotherapy. Patients undergo serial chest wall photography and imaging (CT chest, abdomen, and pelvis, and bone scan) at baseline and every 6 weeks, as well as blood collection for correlative studies and chest wall biopsies at baseline and after 2 cycles of treatment. The primary endpoint is disease control rate (RECIST 1.1) at 18 weeks of treatment, and the study is powered to determine a 20% difference in disease control rates between arms (HR 0.52, a = 0.10, ß = 0.20). An interim analysis will occur for Arm B after 18 patients are enrolled, with a stopping rule for futility. Secondary endpoints include progression-free survival, toxicity (NCI CTCAE), and response based on irRECIST and tumor programmed death ligand 1 (PD-L1) expression. Exploratory objectives include evaluating changes in soluble PD-L1, tumor and peripheral blood immune composition, circulating tumor cells and cell-free DNA, and MYC oncogene expression. This study (NCT03095352) is open at 7 sites in the Translational Breast Cancer Research Consortium (TBCRC). 52 patients are enrolled. Grant funding is provided by Merck and UCSF. Clinical trial information: NCT03095352 .

TPS1110 Background: PARP inhibitors are approved for the treatment of HER2 negative metastatic breast cancer (MBC) with germline BRCA1/2 mutations, based on phase III studies demonstrating an improvement in progression-free survival (PFS) compared to chemotherapy in this population and better patient reported outcomes (Robson, NEJM, 2017; Litton, NEJM, 2018). However, germline BRCA1/2 mutations account for only 5-10% of breast cancer, limiting the current clinical applicability of PARP inhibitors. Somatic BRCA1/2 mutations are detectable in circulating cell-free DNA (cfDNA) in ̃13.5% of patients with MBC; in pre-clinical models, pathogenic somatic BRCA1/2 mutations have been shown to respond to PARP inhibition (Vidula, CCR, 2020). The purpose of this study is to evaluate the efficacy of talazoparib, a PARP inhibitor, in patients with MBC who have somatic BRCA1/2 mutations detectable in cfDNA, in the absence of a germline BRCA1/2 mutation, which we hypothesize will be effective in this setting. This study may help expand the population of patients with MBC who benefit from PARP inhibitors. Methods: This is an investigator initiated multicenter, single arm, phase II clinical trial studying the efficacy of talazoparib in 30 patients with MBC who have pathogenic somatic BRCA1/2 mutations detected in cfDNA. Patients with MBC who are found to have pathogenic somatic BRCA1/2 mutations detected in cfDNA in the absence of a germline BRCA1/2 mutation are eligible. Patients may have triple negative (with ≥ 1 prior chemotherapy), or hormone receptor positive/HER2 negative breast cancer (with ≥ 1 prior hormone therapy). Patients may have received any number of prior lines of chemotherapy, including a prior platinum (in the absence of progression). They must have adequate organ function and ECOG performance status ≤2, and should not have previously received a PARP inhibitor. Patients are treated with talazoparib 1 mg daily until disease progression or intolerability, with serial imaging using CT chest/abdomen/pelvis and bone scan performed at baseline and every 12 weeks, and cfDNA collection every 4 weeks. Primary endpoint is PFS by RECIST 1.1. Patients are being enrolled in a two-stage design with 80% power to demonstrate that the treatment is associated with “success” (PFS > 12 weeks) in ≥53% patients (4% alpha). Secondary endpoints include objective response rate and safety (NCI CTCAE v 5.0). Exploratory analyses include studying serial changes in cfDNA BRCA1/2 mutant allelic frequency and comparing pre-and post-treatment cfDNA for the emergence of BRCA1/2 reversion and resistance mutations. This study is activated and open at Massachusetts General Hospital, where 2 patients are completing screening. It is also opening soon at 6 other academic centers (NCT03990896). Grant support includes a Pfizer ASPIRE award and 2020 Conquer Cancer Foundation of ASCO – Breast Cancer Research Foundation – Career Development Award. Clinical trial information: NCT03990896 .

548 Background: sTILs are a well-established prognostic and predictive biomarker in patients with operable TNBC receiving pre or postoperative systemic therapy. We1 and others2,3 have also shown that sTILs are prognostic in patients who did not receive adjuvant chemotherapy. Here, we detail the outcomes of systemically untreated patients with N0 TNBC according to sTIL score. We focused on the N0 subset as a group of patients who may be candidates for future prospective therapy de-escalation trials. Methods: From a clinically annotated cohort of 605 patients with centrally confirmed TNBC (ER/PR < 1% and HER2 negative) with long-term outcomes data, we identified 182 patients treated with locoregional therapy only (breast surgery +/- radiation therapy and no chemotherapy). The clinicopathological characteristics of this cohort have previously been published1. In this analysis, we report the 5- and 10-year invasive disease-free survival (iDFS) and overall survival (OS) rates of patients with N0 TNBC according to sTIL levels. IDFS and OS were defined as per the STEEP classification and estimated using the Kaplan–Meier method. Comparisons of the survival distributions between groups were assessed by the log-rank test. sTILs were assessed as a continuous parameter according to the International TIL Working Group guidelines. For comparisons of outcomes between groups, tumors were classified as lymphocyte-predominant TNBC (defined as containing ≥50% sTILs) vs non-lymphocyte-predominant ( < 50% sTILs). Results: Of 182 systemically untreated patients, 149 (82%) were N0 and most (78%) were post-menopausal. T stage distribution was T1: 68%, T2: 28%, T3/4: 4%. Among N0 patients, 31 (21%) had lymphocyte-predominant TNBC, and in this group the 5-year iDFS and OS were 89% (95% CI 76-100) and 96% (95% CI 89-100), while the 10-year iDFS and OS were 89% (95% CI 76-100) and 87% (95% CI 73-100), respectively. In contrast, outcomes for patients with non-lymphocyte predominant TNBC were significantly worse. For this group, 5-year iDFS and OS were 62% (95% CI 53-73) and 78% (95% CI 71-86) while the 10-year iDFS and OS were 45% (95% CI 36-58) and 66% (95% CI 68-76), respectively ( log-rank p = 0.02 for iDFS and log-rank p = 0.03 for OS). Conclusions: sTIL quantification identifies a subset of patients with early-stage N0 TNBC with an exceedingly good prognosis, even in the absence of adjuvant chemotherapy. These data provide support for the evaluation of sTILs as part of prospective investigation of systemic therapy de-escalation strategies in N0 TNBC. References:1Leon-Ferre et al, Breast Cancer Res Treat (2018) 167:89-99 2Park et al, Ann Oncol (2019) 12:1941-1949 3De Jong et al, ESMO 2020

PURPOSE Predictive biomarkers to identify patients with human epidermal growth factor receptor 2 (HER2)–positive breast cancer who may benefit from targeted therapy alone are required. We hypothesized that early measurements of tumor maximum standardized uptake value corrected for lean body mass (SULmax) on 18 F-labeled fluorodeoxyglucose positron emission tomography-computed tomography (PET-CT) would predict pathologic complete response (pCR) to pertuzumab and trastuzumab (PT). PATIENTS AND METHODS Patients with stage II or III, estrogen receptor–negative, HER2-positive breast cancer received four cycles of neoadjuvant PT. 18 F-labeled fluorodeoxyglucose positron emission tomography-computed tomography was performed at baseline and 15 days after PT initiation (C1D15). Eighty evaluable patients were required to test the null hypothesis that the area under the curve of percent change in SULmax by C1D15 predicting pCR is ≤ 0.65, with a one-sided type I error rate of 10%. RESULTS Eighty-eight women were enrolled (83 evaluable), and 85% (75 of 88) completed all four cycles of PT. pCR after PT alone was 22%. Receiver operator characteristic analysis of percent change in SULmax by C1D15 yielded an area under the curve of 0.72 (80% CI, 0.64 to 0.80; one-sided P = .12), which did not reject the null hypothesis. However, between patients who obtained pCR and who did not, a significant difference in median percent reduction in SULmax by C1D15 was observed (63.8% v 41.8%; P = .004) and SULmax reduction ≥ 40% was more prevalent (83% v 52%; P = .03; positive predictive value, 31%). Participants not obtaining a 40% reduction in SULmax by C1D15 were unlikely to obtain pCR (negative predictive value, 91%). CONCLUSION Although the primary objective was not met, early changes in SULmax predict response to PT in estrogen receptor–negative and HER2-positive breast cancer. Once optimized, this quantitative imaging strategy may facilitate tailoring of therapy in this setting.

AbstractObjectivesInhibitors to the checkpoint proteins cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) and programmed cell death protein 1 (PD‐1) are becoming widely used in cancer treatment. However, a lack of understanding of the patient response to treatment limits accurate identification of potential responders to immunotherapy.MethodsIn this study, we assessed the expression of PD‐1 and CTLA‐4 on 19 leucocyte populations in the peripheral blood of 74 cancer patients. A reference data set for PD‐1 and CTLA‐4 was established for 40 healthy volunteers to determine the normal expression patterns for these checkpoint proteins.ResultsUnsupervised hierarchical clustering found four immune profiles shared across the solid tumor types, while chronic lymphocytic leukaemia patients had an immune profile largely unique to them. Furthermore, we measured these leucocyte populations on an additional cohort of 16 cancer patients receiving the PD‐1 inhibitor pembrolizumab in order to identify differences between responders and non‐responders, as well as compared to healthy volunteers (n = 20). We observed that cancer patients had pre‐treatment PD‐1 and CTLA‐4 expression on their leucocyte populations at different levels compared to healthy volunteers and identified two leucocyte populations positive for CTLA‐4 that had not been previously described. We found higher levels of PD‐1+ CD3+ CD4− CD8− cells in patients with progressive disease and have identified it as a potential biomarker of response, as well as identifying other significant differences in phenotypes between responders and non‐responders.ConclusionThese results are suggestive that categorisation of patients based on immune profiles may differentiate responders from non‐responders to immunotherapy for solid tumors.

Abstract Background: Growing data established the pivotal role of preexisting immune response in triple negative breast cancer (TNBC). Conventionally, preexisting immune response can be evaluated by quantifying tumor infiltrating lymphocytes mainly in the stroma or gene expression analysis from the whole tumor section. Due to technical challenges with these conventional methods, limited data regarding specific subtypes and spatial distribution of these immune infiltrates are currently available. Methods: NanoString IO360 gene expression analysis and Digital Spatial Profiling (DSP) were used. DSP was used to quantify 29 immune-related proteins in stromal and tumor-enriched segments from 44 TNBC samples from the FinXX trial (NCT00114816) and 335 samples from the Mayo Clinic (MC) cohort of centrally reviewed TNBC (Leon-Ferre BCRT 2018). In FinXX trial, 22 patients with recurrence and 22 patients without recurrence were included. In MC cohort, 217/335 patients received adjuvant chemotherapy while 118 patients had surgery only without adjuvant chemotherapy. Regions were segmented based on pancytokeratin staining. The general linear model was used for statistical analysis of differential expression with recurrence free survival (RFS) as a categorical variable (recur yes or no). Kaplan-Meier (KM) estimates and Cox regression models were also used for analysis. Results: In the FinXX trial, there were 12 out of 29 proteins in tumor epithelial segments (intraepithelial) which were significantly expressed at higher levels among patients who were free of recurrence. These proteins include Beta-2 microglobulin, CD11c, CD20, CD40, CD56, CD8, Granzyme B, HLA-DR, ICOS, PD-L1, PD-L2, and TGFB1. In contrast, merely 5 out of 29 proteins in stromal segments were significantly differentially expressed in these 2 groups of patients. Granzyme B, IDO1, PD-L1, and PD-L2 in stroma were significantly higher and SMA was significantly lower in patients without recurrence. Using Cox regression models, intraepithelial CD56, CD40, and HLA-DR were significantly associated with outcome. When comparing between highest and lowest intraepithelial protein expression by tertile, intraepithelial CD56 (HR 0.12, 95%CI 0.03-0.39, p &lt; 0.001), CD40 (HR 0.13, 95%CI 0.04-0.46, p = 0.002), and HLA-DR (HR 0.24, 95%CI 0.06-0.89, p = 0.032) were significantly associated with improved outcome. However, expression of these same proteins in stroma was not associated with outcome. Using KM estimates, intraepithelial CD56 (p &lt; 0.0001), CD40 (p = 0.0006), and HLA-DR (p = 0.013) were also significantly associated with improved outcome. Nonetheless, RNA expression of these proteins by IO360 from whole tumor sections were not significantly associated with outcome (CD56 p = 0.27, CD40 p = 0.21, HLA-DR p = 0.48). Similar findings with DSP were observed in MC TNBC cohort. Comparing between the highest and lowest quartiles, there were significantly fewer patients who developed recurrence with high protein expression of intraepithelial CD56 (p &lt; 0.001), CD40 (p = 0.002), and HLA-DR (p = 0.006). Conclusions: Using an in-depth analysis with spatially defined context, we identify that there were numerically more intraepithelial immune-related proteins associated with outcome compared to proteins in stroma. Specifically, intraepithelial CD56, CD40, and HLA-DR were significantly associated with improved outcome in both FinXX and MC TNBC cohorts. However, neither expression of these proteins in stroma nor RNA expression from whole tumor were associated with outcome. Our study highlights the impact of spatial biology and the importance of evaluating each potential biomarker in a spatially defined manner. Support: W81XWH-15-1-0292, BCRF 19-161, P50CA116201-9, P50CA015083 Citation Format: Saranya Chumsri, Jodi M. Carter, Yaohua Ma, Douglas Hinerfeld, Heather Ann Brauer, Sarah Warren, Torsten O. Nielsen, Karama Asleh, Heikki Joensuu, Edith A. Perez, Roberto A. Leon-Ferre, David W. Hillman, Judy C. Boughey, Minetta C. Liu, James N. Ingle, Krishna R. Kalari, Fergus J. Couch, Keith L. Knutson, Matthew P. Goetz, E. A. Thompson. Spatially defined immune-related proteins and outcome in triple negative breast cancer in the FinXX trial and Mayo Clinic cohort [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 PS6-02.

Abstract Background: Cyclin dependent 4/6 kinase inhibitors (CDK4/6i) and endocrine therapy (ET) have improved progression-free survival (PFS) and overall survival in hormone-receptor (HR)-positive metastatic breast cancer (MBC), but progression of disease is inevitable. Serum thymidine kinase-1 (TK1) is a secreted marker of proliferation that is prognostic in patients (pts) with HR-positive, HER2-negative MBC and may be predictive of ET and CDK 4/6i response. PROMISE (NCT0281902) is a prospective study enrolling women with HR-positive MBC starting palbociclib (Pb) + letrozole (L) (1st line) or Pb + fulvestrant (2nd line). We undertook a comprehensive “omic” assessment of blood, tumor, urine and the fecal microbiome in order to identify novel genomic variants and pathways associated with an early decline in TK1 (measured after 2 months) and PFS. Additionally, patient derived xenografts/organoids were generated at baseline and progression to test new therapeutic approaches to overcome resistance to CDK4/6i and ET. We report the initial association between the baseline genomic landscape and baseline TK1 levels. Methods: FFPE tumor biopsies were obtained for DNA/RNA sequencing (TempusTM) and blood samples for TK1 (Divitum® assay, Biovica) were collected pretreatment (pre-Pb) and after 2 cycles of Pb + ET (post-Pb2). Both whole-exome (exome capture) sequencing (WES) and RNA-Seq used the Integrated DNA Technologies xGen Exome Research Panel v1.0 capture kit. TK1+ disease was defined as &gt; 200 Du/L and TK1- disease as below limit of detection up to 200 Du/L. We tested the association between genomic and transcriptomic characteristics with baseline TK1 data in pretreatment samples where both WES and RNA-seq and TK1 was available. The data were analyzed using bioinformatics pipelines for somatic and germline mutations and copy number alterations. The current analysis focuses on baseline 1st-line pre-Pb omics data in conjunction with baseline TK1 levels. The database was locked for analysis on 5/29/2020. Results: Thirty-three pts (median age: 59 yrs.) were evaluable, with paired samples for TK1 in 32. Six pts had TK1+ disease pre-Pb and post-Pb2. Twenty-two pts had TK1- disease pre-Pb and post-Pb2. Four pts had a decrease in TK1 after 2 cycles of treatment that altered the classification from TK1+ to TK1-. Both baseline RNA seq and serum TK1 (n=16) were available for 4 TK1+ and 12 TK1- pts. In this group, 476 genes were differentially regulated (398 upregulated; 78 downregulated). Pathway analysis demonstrated enrichment in complement and coagulation cascade pathway, PPAR signaling pathway, and metabolism-related pathways related to up-regulation of CYP and UGT gene families. Further testing for the association of WES data with baseline TK1+ (n=8) and TK1- (n=16) disease demonstrated somatic copy number variations on chromosomes 6, 11, 12 and 15. CDK4 copy number gains were observed in 3/8 TK1+ pts and 0/16 TK1- pts. We also observed that somatic mutations (LOH, copy number and/or SNV/INDELs) were more prevalent in the TK1+ compared to the TK1- pre-Pb group in several cancer-associated genes (FAS [p=0.06] PTEN, PIK3CB, NAB2, SOX9 and FAT1 [p=0.08], TP53, and MAP2K4 [p=0.22]). Conversely, we also noted that 6/7 pts with GATA3 mutations had TK1- disease (p=0.23). Conclusions: Using a comprehensive “omics” approach, our data suggest that a secreted biomarker of proliferation (TK1) obtained prior to initiating CDK4/6i and ET for the first line treatment of HR+ MBC is associated with established and novel genes and pathways associated with prognosis of pts receiving ET and CDK 4/6i. Analysis of on-treatment (after 2 cycles) tumor RNA seq and its association with change in TK1 as well as data from the 2nd-line cohort will be presented at the meeting. Citation Format: Ciara C O Sullivan, Krishna R Kalari, Vera J Suman, Peter T Vedell, Ann Moyer, Abraham D Eyman Casey, Jason Sinnwell, Xiaojia Tang, Kevin Thompson, Alvaro Moreno-Aspitia, Donald W Northfelt, Minetta C Liu, Tufia C Haddad, Saranya Chumsri, Prema Peethambaram, Kathryn J Ruddy, Karthik V Giridhar, Roberto A Leon-Ferre, Adrian Nordmark, Mattias Bergqvist, Brendan P McMenomy, Richard M Weinshilboum, Liewei Wang, Matthew P Goetz. Novel genomic variants and pathways associated with baseline serum thymidine kinase 1 levels in HR-positive HER2-negative MBC patients commencing palbociclib and letrozole [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 PS5-24.

Abstract 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 &gt;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.

Abstract Introduction: Stromal tumor-infiltrating lymphocytes (sTILs) have established prognostic and predictive significance in triple-negative breast cancer (TNBC). However, the roles of other immune cells in TNBC are less well-established. We performed high-plex quantitative spatial profiling in a cohort of early-stage TNBC to 1) apply spatial context to tumoral immune landscapes and 2) identify immune proteins associated with clinical outcomes, independently of TILs and other established prognostic clinicopathologic variables, in patients (pts) treated with or without adjuvant chemotherapy (CTX). Methods: The Mayo TNBC cohort comprises pts with centrally-verified, CTX-naive tumors resected from 1985-2012. Using a cohort-based TMA, with Nanostring GeoMX DSP, we quantitated 58 proteins within spatially-distinct intra-epithelial, cytokeratin-positive tumor segments and adjacent cytokeratin-negative/nuclei-positive stromal segments. Differentially-expressed (DE) proteins were identified using a negative binomial generalized linear model (SNR&gt;2, p&lt; 0.05) and a target DE protein set was dichotomized (80th percentile). After adjusting for prognostic clinicopathologic variables, proteins associated with recurrence-free survival (RFS, defined as time from surgery to either local, regional, and distant recurrence, or death by any cause) were identified by performing variable selection using the Akaike Information Criterion (AIC) obtained from fitting all possible Cox proportional hazards regression models (performed separately for intra-epithelial/stromal segments, and in groups +/- adjuvant CTX. Results: From the TNBC TMA, DSP data (N=250 tumors) included 169 pts who received adjuvant CTX+ and 81 who did not (CTX-). Overall, 85/250 developed recurrent disease. In the CTX+ group, intra-epithelial tumor segments from pts without recurrent disease were enriched in 10 immune proteins, including CD8, markers involved in antigen presentation/dendritic cells (CD11c, CD40, HLA-DR) or NK cells (CD56) (FC: 1.4-2.1, p&lt;0.05); CD14 was increased in stroma (FC: 1.5, p&lt;0.05). In contrast, in the CTX- group, both the intra-epithelial tumor and stromal segments from pts without recurrences were enriched in immune proteins (N= 12 and 15 respectively; FC 1.6-5.5, p&lt; 0.05) most markedly CD40, IDO1 and HLA-DR (FC: 3.2-5.5, p&lt; 0.05). Overall, CD3, CD4, CD27, CD44, and ICOS among others were enriched only in the CTX- group; CD14 and CD56 were enriched only in the CTX+ group. Based on these spatial data, biologic function and DSP data from another set of TNBC (FinXX trial), CD11c, CD14, CD27, CD40, CD56, and IDO1 were selected for RFS analysis. After applying our model selection criterion and adjusting for pt age at surgery, tumor size, lymph node status, and sTILs, intra-epithelial CD56 was independently associated with improved RFS in the CTX+ group (HR: 0.31[0.12, 0.81]). In the CTX- group, intra-epithelial CD11c was independently associated with improved RFS (0.10 [0.01, 0.81]). Conclusion: In this early-stage TNBC cohort, spatially-distinct tumor immune landscapes were associated with RFS but differed according to receipt of CTX after surgical resection. In the patients who received CTX, the intra-epithelial compartment, rather than stromal compartment, was immune-enriched in pts without recurrences. Among a targeted protein set, intra-epithelial CD56 remained associated with improved outcomes, independent of sTILs and other clinicopathologic features. In the CTX- group, spatial landscapes were more balanced, and intra-epithelial CD11c was independently associated with improved outcomes. These data provide insight into the spatial context of intrinsic immune landscapes in TNBC, and identify candidate prognostic immune biomarkers which may inform therapeutic strategies. Citation Format: Jodi M Carter, Saranya Chumsri, David W Hillman, David M Zahrieh, Yaohua Ma, Xue Wang, Jennifer M Kachergus, Judy C Boughey, Minetta C Liu, Krishna R Kalari, JC Villasboas, Roberto A Leon Ferre, Fergus J Couch, Matthew P Goetz, E. Aubrey Thompson. Intra-epithelial tumor immune landscapes are associated with clinical outcomes in early-stage triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-01.