Chumsri, Saranya

Abstract Background: The intrinsic immune response in triple negative breast cancer (TNBC) has both prognostic and predictive utility, and immune-related biomarkers are now actionable targets in TNBC. Current modalities for immune profiling are limited by lack of spatial context and precise quantitation. We used quantitative digital spatial profiling (DSP) to 1) characterize the immune architecture of tumoral and stromal regions in TNBC and 2) identify prognostic immune signatures. Methods: From the FinXX trial of adjuvant capecitabine therapy, tumor sections (FFPE) matched for patient characteristics, treatment arm and RFS-based outcome (N=44) were assessed with Nanostring GeoMxTM DSP. Briefly, with 3-plex immunofluorescence (pan-cytokeratin, CD45, CD68 with SYTO-13 dye for nuclei), spatially-defined CD45-enriched or CD68-enriched stromal segments and adjacent tumor segments (N=950) were selected for digital quantitation (NanoString nCounter) of 39 immune proteins (e.g. immune cell profiling proteins: CD3, CD4, CD8, CD11c, CD20, CD56, CD68, HLA-DR, PD-1, PD-L1, Granzyme B), immune drug targets (e.g. VISTA, STING, IDO1), and activation status-related proteins (e.g. ICOS, PD-L2, CD40, CD40L, CD44). Normalized, differentially-expressed (DE) proteins were evaluated, and weighted gene co-expression network analysis (WGCNA) was performed to identify modules of highly co-expressing immune proteins, and association with RFS as a categorical variable was determined. Results: 20 DE proteins in tumor segments were associated with RFS. Among them, those with the highest RFS-associated fold changes (FC) included CD20, CD40, CD56, HLA-DR, Granzyme B, and PD-L2 (FC): 2.1-4.4) (p < 0.05). In contrast, only 4 DE proteins were associated with RFS in stromal segments (Granzyme B, IDO-1, PD-L1, and PD-L2 (FC: 1.5-3.3) (p < 0.05). By WGCNA, 2 tumor-based immune modules [IM1 (lymphocyte infiltration) and IM2 (lymphocyte activation)] were identified based on their co-expression. The top three co-expressed proteins in IM1 were CD4, CD40, and CD45 (r >0.9). The three top co-expressed proteins in IM2 were PD-L1, PD-L2, and Granzyme B (r > 0.85). Together with CD56 expression, IM1 and IM2 comprised a set of 3 intratumoral immune features that associated with RFS. For recurring tumors, all 3 immune features clustered with low scores . Among non-recurrent tumors, two distinct, non-overlapping clusters with inverse characteristics were identified: high IM2, low IM1, low CD56 vs. low IM2, high IM1, and high CD56. Overall, across all 3 immune features, PDL2, CD40 and CD56 expression most strongly associated with RFS. Conclusion: With spatially-defined, precise quantification of immune proteins, we identified 3 immune features, including two distinct immune modules, IM1 and IM2 that correlate with durable RFS. These immune modules of highly co-expressing proteins were associated with RFS only when present in the tumor compartment but not tumor-adjacent, immune cell-rich stroma. Citation Format: Jodi M. Carter, Saranya Chumsri, Yaohua Ma, Xue Wang, Jennifer M. Kachergus, Douglas Hinerfeld, Heather Ann Brauer, Sarah Warren, Heikki Joensuu, Edith A. Perez, Aubrey Thompson. Quantitative digital spatial profiling reveals novel prognostic intratumoral immune signatures in triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1004.

3535 Background: Peripheral blood allows for a simple non-invasive method for isolating various cancer associated circulating stromal cells (CStCs) which may predict for cancer presence. Cancer Associated Macrophage-Like cells (CAMLs), a specific CStC, are phagocytic myeloid cells that derive from an immunological response to cancer and emanate from primary tumors. Using a filtration platform we screened the peripheral blood of untreated newly diagnosed cancer patients (n = 308) for CAMLs. In parallel, we screened patients with newly diagnosed non-malignant diseases, i.e. lupus, benign cysts, etc. (n = 39), and healthy control samples (n = 76). We found that CAMLs are highly prevalent (87%) in the blood of cancer patients, but uncommon in non-malignant conditions (20%) & absent in healthy individuals (0%). Methods: Anonymized peripheral blood were taken from 308 cancer patients after confirmation of invasive malignancy [stage I (n = 76), stage II (n = 73), stage III (n = 72), stage IV (n = 65) and unstaged non-metastatic (n = 22)] with pathologically confirmed lung (n = 65), pancreas (n = 53), breast (n = 52), prostate (n = 40), esophageal (n = 30), renal cell (n = 18), hepatocellular (n = 15), neuroblastoma (n = 10), melanoma (n = 8), and other (n = 17). Further, anonymized blood was taken from patients with untreated non-malignant conditions including benign breast masses (n = 19), lupus (n = 11), liver cirrhosis (n = 5), benign prostatic hyperplasia (BPH) (n = 3), and viral infection (n = 1); or from healthy control volunteers (n = 76). CAMLs were isolated from whole peripheral blood by the CellSieve™ microfiltration technique and defined as enlarged, multinuclear cells with cytokeratin and/or CD45/CD14 positive. Results: CAMLs were found in 87% of all cancer patients regardless of stage, ~5.4 CAMLs/7.5mL blood. Specifically, CAMLs were found in 80% of Stage I, 90% Stage II, 89% Stage III, and 97% Stage IV patients. No CAMLs were found in any healthy controls, but were found in 26% of benign breast masses, 18% of lupus, 0% of BPH and 0% of cirrhosis. In total, CAML sensitivity in cancer vs healthy was 87% (CI95% 82-90%), specificity = 100% (CI95% 95-100%), PPV = 100% (CI95% 100%), NPV = 67% (CI95% 58-71%). CAML sensitivity in cancer vs benign was 87% (CI95% 82-90%), specificity = 80% (CI95% 64-91%), PPV = 97% (CI95% 95-98%), NPV = 43% (CI95% 35-51%). Conclusions: CAMLs, a Circulating Stromal Cell subtype, is a sensitive blood based biomarker found in all stages of cancer that is rare in non-malignant conditions and absent in healthy individuals.

529 Background: Multiple prognostic models exist to predict late relapse risk in early stage hormone receptor-positive (HR+) breast cancer (BC). The CTS5 is one such model that has been validated in HR+ HER2-negative BC. The value of this model in HR+ HER2+ has not been established. Here, we assessed CTS5 in patients (pts) with early stage HER2+ BC treated in the NCCTG N9831 (Alliance) trial. Methods: Pts with stage I-III HER2+ HR+ BC who survived ≥ 5 years were included. The online CTS5 calculator was used to determine CTS5 score and risk group (low, intermediate, and high) based on age, tumor size, grade, and number of involved nodes. Kaplan-Meier (KM) estimates, Cox regression models, and C index were used for analysis. Results: From 3,130 pts, 1,204 pts met the criteria and were included. Median age was 49 (22-79) years and median tumor size was 2.4 (0.1-12) cm. 63.6% had grade 3 tumors, 33.6% grade 2, and 2.8% grade 1. Median follow up was 10.89 (5.01-15.32) years. Based on CTS5, 821 (68.2%) pts were classified as high risk, 289 (24%) as intermediate risk, and 94 (7.8%) as low risk. Overall, using univariate Cox regression analysis, there was no statistically significant difference in recurrence free survival (RFS) among pts with intermediate vs. low (HR 0.47 95%CI 0.18-1.22, p = 0.12) and high vs. low (HR1.23 95%CI0.57-2.67, p = 0.6) with the C index of 0.58. Among pts who received concurrent trastuzumab (H) with HR+ BC, there was also no statistical difference in RFS between high vs. low (HR 0.68 95%CI0.24-1.97, p = 0.48) with the C index of 0.55. Paradoxically, pts with intermediate risk had better RFS than low risk (HR 0.18 95%CI0.03-0.97, p = 0.05). As a continuous variable, there is also no significant improvement in RFS per 1 unit increase in CTS5 score (HR 1.19 95%CI 0.73-1.96, p = 0.49) with the C index of 0.54. After 5 years, 7.06% (n = 30/425) of HR+ pts treated with concurrent H recurred. Conclusions: The CTS5 model is not prognostic in pts with early stage HR+ HER2+ BC receiving adjuvant H. While most HR+ HER2+ pts are classified as high risk by CTS5, the recurrence between years 5-10 was low in pts who received adjuvant H. This study highlights the need to develop a new predictive model for risk of late relapse in this specific group of pts to enable clinicians to determine which pts would benefit from extended adjuvant endocrine therapy. Support: BCRF-19-161, U10CA180821, Genentech. https://acknowledgments.alliancefound.org Clinical trial information: NCT00005970 .

510 Background: Several studies have established the critical role of preexisting immune response in triple negative breast cancer (TNBC). Most studies evaluated the tumor infiltrating lymphocytes in stroma. However, limited data are available with regards to the importance of specific subtypes and spatial distribution of these immune infiltrates. Methods: NanoString IO360 gene expression analysis and Digital Spatial Profiling (DSP) were used. DSP was used to quantify 39 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 estimates and Cox regression models were also used for analysis. Results: In the FinXX trial, using global gene expression analysis with IO360, there was no signature significantly associated with RFS. However, using DSP, high protein expression of CD56 in the tumor-enriched segments was associated with significant improvement in RFS (HR 0.26, 95%CI 0.09-0.78, p 0.01). Nevertheless, CD56 expression in the stroma (HR 0.66, 95%CI 0.29-1.53, p 0.33) and all segments (HR 0.53, 95%CI 0.23-1.25, p 0.14) was not significantly associated with improved outcome. We further validated these findings in the MC TNBC cohort where intratumoral CD56 expression was associated with a significant improvement in RFS (HR 0.23, p 0.002) but not stromal CD56 (p 0.79). Interestingly, when evaluating the MC TNBC cohort according to receipt of chemotherapy, intratumoral CD56 was associated with improved outcome only in patients who received chemotherapy (p 0.02 vs. 0.07). In both cohorts, higher expressions of intratumoral PD-L1, HLA-DR, and CD8 were associated with improved outcome. Conclusions: Using an in-depth analysis with spatially defined context, we identify that intratumoral CD56-positive NK cells are associated with improved outcome in TNBC. Our study highlights the potential role of NK cells in TNBC and future implications for biomarkers and therapeutic targets.Support: W81XWH-15-1-0292, P50CA116201-9, P50CA015083. Clinical trial information: NCT00114816 .

Increasing data support the importance of preexisting host immune response and neoantigen burden for determining response to immune checkpoint inhibitors (ICIs). In lung cancer and melanoma, tumor mutational burden (TMB) has emerged as an independent biomarker for ICI response. However, the significance of TMB in breast cancer, particularly in the context of PD-L1 negativity, remains unclear. This report describes a patient with HER2-negative breast cancer with high TMB and an apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) trinucleotide signature; her disease was refractory to multiple lines of treatments but achieved durable complete response using ICIs and capecitabine. Additional analysis of the tumor revealed a low amount of stromal tumor-infiltrating lymphocytes (sTILs) and PD-L1 negativity, reflecting a poor preexisting host immune response. In collaboration with Foundation Medicine, comprehensive genomic profiling from 14,867 patients with breast cancer with the FoundationOne test was evaluated. Using the cutoff of ≥10 mutations/megabase (mut/Mb) for high TMB, PD-L1 positivity and TMB-high populations were not significantly overlapping (odds ratio, 1.02;P=.87). Up to 79% of TMB-high tumors with >20 mut/Mb were PD-L1–negative. Our study highlights that despite having low TILs and PD-L1 negativity, some patients may still experience response to ICIs.

Abstract Background: Growing evidence supports the critical role of preexisting immune response in triple negative breast cancer (TNBC). However, there are limitations with current evaluation approaches: inability to functionally assess the type of immune infiltration with traditional pathologic evaluation and loss of spatial distribution in conventional high-plex gene or protein expression analyses from the whole tumor section. Here, we report the initial analysis of immune protein expression as a function of spatial distribution and clinical outcomes in TNBC samples. Methods: NanoString GeoMxTM Digital Spatial Profiling (DSP) was used to quantify 39 immune-related proteins in stromal and tumor segments from 44 TNBC samples from the FinXX trial. Samples were matched for patient characteristics, treatment arm (capecitabine vs. 5-fluorouracil), and outcome based on recurrence-free survival (RFS) with 22 samples from patients who recurred and 22 samples from patients with durable RFS. Regions of interest (ROIs) were selected based on expression of cytokeratin (tumor), CD45 (leukocytes), or CD68 (macrophages). Each ROI was segmented into tumor (pancytokeratin-positive area) and stroma (pancytokeratin-negative/nuclear SYTO13-positive area). The general linear model was used for statistical analysis of differential expression with RFS as a categorical variable (recur yes or no). Results: A total of 950 tumor and stroma segments were included in this initial analysis. In both tumor and stroma segments, over-expression of T cell activation markers (CD137, GITR) was associated with better outcome, whereas T cell markers (CD3, CD4, CD8) were not significantly associated with outcome. In tumor segments alone, improved outcome was significantly associated with increased protein expression [> 2-fold change (FC) at p<0.001] of CD56, PD-L2, HLA-DR, CD137, GITR, and CD40. In CD45-enriched stroma, improved outcome was associated with elevated expression (FC>2.0, p<0.001) of PD-L2; whereas durable RFS was associated with elevated PD-L2 and IDO1 expression in CD68-enriched stroma. In contrast, macrophage/dendritic cell markers CD68 and CD11c were not associated with outcome. In tumor cells adjacent to CD45-enriched stroma, durable RFS was associated with increased abundance (FC>2.0, p<0.001) of PD-L2, CD56, CD27, GITR, CD20, HLA-DR, and IDO1. Similarly, in tumor segment associated with CD68-enriched stroma, proteins associated with RFS included PD-L2, HLA-DR, CD56, GITR, and CD137. Among 39 immune function proteins, only elevated CTLA4 expression in CD68-enriched stroma was associated with recurrence (FC=0.55, p<0.001), whereas tumor-segment CTLA4 was associated with RFS. Conclusions: Using an in-depth analysis to precisely quantify the abundance of multiple immune function proteins in a spatially defined manner, we observed that PD-L2, IDO1, and T cell activation markers were robustly associated with durable RFS in both tumor and stromal segments. In contrast, MHC components (HLA-DR, beta-2-microglobulin), B cell markers (CD20), and NK cell markers (CD56) were strongly associated with favorable outcome in tumor but not in stromal segments. Our study highlights the conclusion that the immune landscape of TNBC is far too complex to be encompassed by any single molecular marker, and more detailed analyses of the DSP data reported here are ongoing with a view towards using quantitative multiplex analyses to refine our understanding of how therapeutic outcome is influenced by interactions among immune cells and between immune and tumor cells. Acknowledgements: Supported by the Breast Cancer Research Foundation (BCRF18-161), Bankhead Coley (6BC05 Florida Department of Health), 26.2 with Donna Foundation, US National Cancer Institute (CA15083), and the Canadian Cancer Center Citation Format: Saranya Chumsri, Douglas Hinerfeld, Jennifer M. Kachergus, Yaohua Ma, Heather A Brauer, Sarah Warren, Xue Wang, Torsten O. Nielsen, Karama Asleh, Heikki Joensuu, Edith A. Perez, E. A. Thompson. Digital spatial mapping of the immune landscape of triple negative breast cancer reveals novel features of immune-tumor cell interaction [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 PD5-06.

Abstract Background: Tumor-associated macrophages have been shown to play a critical role in metastasis, angiogenesis, and immune escape. Cancer associated macrophage-like cells (CAMLs) are circulating stromal cell with macrophage expression subtypes, detected in the peripheral blood of patients with solid tumors. We have previously shown that enlargement of CAML cells is an independent predictor of poorer overall survival and an indication of progression in patients with metastatic breast cancer (MBC). However changes in CAML sizes after induction of therapy has not been investigated. Here we evaluated the association between early changes in numbers and sizes of CAMLs as it relates to clinical response after induction of new therapy. Method: Peripheral blood samples from 29 patients with metastatic breast cancer who failed at least 2 prior lines of therapies were prospectively collected. Baseline (BL) blood samples were taken prior to initiation of a new therapy and the second samples (T1) were taken approximately 30 days after initiation of a new systemic therapy. Blood samples were filtered using CellSieveTM filtration. The quantity and size of CAMLs were measured then compared between the BL and T1 time points. Univariate analysis was used to determine the association of changes in CAML numbers and changes in CAML size, with objective response at the first follow up CT scan with progression free survival (PFS). Results: CAMLs were detected in 97% of BL samples (averaging 20.9 CAMLs/7.5ml blood) and 93% of T1 samples (averaging 18.5 CAMLs/7.5mL blood). At the time of the first follow up CT scan, 17 of 29 patients (58.6%) had progressive disease based on the RECIST criteria. Among those, 14 of 17 patients (82%) had an increase in CAML size (~85% overall enlargement) and 3/17 (18%) had a decrease CAML size (~60% overall shrinkage) in T1 samples compared to baseline. In contrast, 10 of 12 patients (83%) with clinical benefit, including stable disease and partial or complete response, had a 92% reduction in their CAML’s size and 2 of 12 patients (17%) had 61% enlargement in their CAML’s size after treatment. Overall, CAML size change after therapy induction was 83% accurate in predicting clinical benefit based on subsequent scans. Furthermore, patients with enlarging CAML size at T1 had significantly shorter PFS of 4 months compared to 10 months in patients with decreasing CAML size (HR 3.7; 95%CI1.5-10.1; p 0.020). Conclusion: CAMLs can be commonly found in most treatment-refractory metastatic breast cancer patients and their morphological size is clinically correlated to poorer survival. Our data suggests that monitoring the size changes of a patient’s CAMLs after 30 days of new treatment initiation may predict clinical benefit earlier than conventional scans and act as real time monitoring of MBCs response to new therapies. Additional studies are currently ongoing to validate these results. Citation Format: Saranya Chumsri, Markus D. Lacher, William V. Williams, R. K. Alpaugh, Stuart S. Martin, Rena Lapidus, Cha-Mei Tang, Massimo Cristofanilli, Daniel L Adams. Circulating cancer associated macrophage-like cells (CAMLs) are early predictors of response to new line therapies in metastatic breast cancer [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 P5-06-27.

Abstract Background: The combination of cyclin dependent 4/6 kinase inhibitors (CDK4/6i) with endocrine therapy (ET) has resulted in clinically significant improvements in progression-free survival (PFS) and overall survival (OS) in hormone-receptor (HR)-positive metastatic breast cancer (MBC). However, most patients’ disease ultimately progresses on CDK4/6i and ET. Therefore, further research is necessary to understand the mechanisms driving primary and secondary resistance. PROMISE is a multicenter prospective cohort study enrolling women with HR-positive MBC commencing treatment with palbociclib + letrozole (1st line) or palbociclib + fulvestrant (2nd Line). The study provides a comprehensive “omic” assessment of blood, tumor, urine and the fecal microbiome to identify molecular or cellular features associated with primary endocrine resistance (e.g. disease progression ≤ 12 months on treatment) and acquired resistance to CDK 4/6i. Additionally, patient derived xenografts and organoids are created to test new drug strategies designed to overcome resistance to CDK 4/6i and ET. Here, we present initial sequencing results from pretreatment biospecimens collected from PROMISE study participants. Methods: On-study tumor biopsies and blood samples were collected for DNA/RNA sequencing (TempusTM). The analyzed biospecimens were all obtained prior to initiation of palbociclib and ET. We correlated patient clinical characteristics (phenotypes) with molecular data and responses to protocol treatment. The data were analyzed using a series of cutting-edge bioinformatics pipelines for somatic and germline mutations in addition to copy number alterations (CNAs). The study database was locked for analysis on 06/20/2019. Results: We analyzed the somatic single nucleotide variants/INDELs (sSNV/INDEL) profiles across the tumor samples to determine the genes that were least likely to occur as a result of background mutation processes. Twenty-six patients had somatic copy number alterations (sCNA) and/or sSNV/INDEL in at least one of 18 genes with the most significant sSNV/INDEL profiles (p < 0.03) which included clinically and biologically relevant genes. The genes with the most statistically significant sSNV/INDEL mutation profiles were GATA3, PIK3CA, CDH1, and ESR1 (p < 0.0009). We observed a high percentage of tumors with somatic alterations in GATA3 (23% sSNV/INDEL, 15% sCNA), PIK3CA (38%, 12%), CDH1 (19%, 50%) and ESR1 (19%, 58%). ESR1 mutations were more frequent in patients receiving 2nd line treatment. Other frequently altered genes included TP53 (15%, 46%), MAP2K4 (8%, 50%), DNAAF1 (8%, 50%), and CDKN1B (8%, 35%). Further, ZNF317 and F3 were altered in 9 and 7 patients, respectively. Twenty-four samples had alterations in at least one of the CDK4/6 pathway genes (RB1, CCNE2, CCND1, CDK6, ESR1, CDKN2A, CCND3, CDK4, CDK2 and CCNE1). Four patients progressed on therapy; three of the four patients had mutations in PIK3CA, and one had a mutation in ESR1. Results of the RNA sequencing data (N=26) will be presented at the SABCS meeting. Conclusions: This is the first report of a prospective study designed to characterize the genomic landscape of ER+/HER2- MBC prior to palbociclib treatment. We observed high frequencies of known targetable alterations in PIK3CA and ESR1, including in patients that progress, which is consistent with previous reports. RNA sequencing data will be presented at the meeting. Citation Format: Ciara C O Sullivan, Krishna R Kalari, Vera J Suman, Peter T Vedell, Ann Moyer, Erin Carlson, Jason Sinnwell, Tejaswi Alaparthi, Xiaojia Tang, Kevin Thompson, Jaeyun Sung, Alvaro Moreno-Aspitia, Donald Northfelt, Minetta C Liu, Tufia C Haddad, Prema Peethambaram, Saranya Chumsri, Kathryn J Ruddy, Karthik V Giridhar, Roberto A Leon-Ferre, Paula Gill, Mohammad Ranginwala, Asad Javed, Sameer Batoo, Brendan P. McMenomy, Richard Weinshilboum, Liewei Wang, Matthew P Goetz. Comprehensive tumor sequencing to identify biomarkers of palbociclib response: First report of the PROMISE study [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 P2-11-07.

78 Background: Previous study demonstrated that activation of RAS/MAPK pathway is associated with reduced tumor infiltrating lymphocytes and poor response to neoadjuvant chemotherapy in triple negative breast cancer (TNBC). Further study showed that inhibition of MAPK pathway with a MEK inhibitor is synergistic with anti-PD1/PD-L1 therapies. Methods: Patients with unresectable locally advanced or metastatic TNBC with ≤ 3 prior lines of therapy without prior anti-PD-1/PD-L1/PD-L2 therapies were enrolled. Treatment was started with a 2-week run in period with single agent binimetinib. Dose level 0 was binimetinib at 45 mg oral twice daily continuously and dose level -1 was 30 mg twice daily. Pembrolizumab was given at a fixed dose of 200 mg every 3 weeks at both dose levels. Phase I study was based on the standard 3+3 design. Results: A total of 12 patients were enrolled and treated in the phase 1. Five patients were enrolled at dose level 0, 1 patient withdrew prior to treatment and 1 patient was not evaluable for dose limiting toxicity (DLT). Among 3 evaluable patients, 2 patients experienced DLT with grade 3 flank pain and ALT abnormality. Additional 8 patients were enrolled at dose level -1. Out of 6 evaluable patients for DLT, there was 1 DLT observed with grade 3 AST and ALT abnormality. However, this particular patient had liver metastasis with grade 1 AST and ALT abnormality at baseline and her liver function test (LFT) normalized in 3 weeks after treatment discontinuation and oral prednisone. Other grade 1-2 common AEs included rash, LFT increase, abdominal pain, mucositis, nausea, cardiac troponin T increase without EKG change. The efficacy data will be presented at the meeting after the phase II interim analysis. Conclusions: Pembrolizumab in combination with binimetinib at 30 mg twice daily appears to be safe based on the initial cohort. Phase II part is currently ongoing with binimetinib 30 mg twice daily and pembrolizumab 200 mg every 3 weeks with a total of 23 patients planned where the safety and efficacy of this combination will be further evaluated. Clinical trial information: NCT03106415.

Abstract Purpose: Recent studies have demonstrated a benefit of adjuvant capecitabine in early breast cancer, particularly in patients with triple-negative breast cancer (TNBC). However, TNBC is heterogeneous and more precise predictive biomarkers are needed. Experimental Design: Tumor tissues collected from TNBC patients in the FinXX trial, randomized to adjuvant anthracycline–taxane–based chemotherapy with or without capecitabine, were analyzed using a 770-gene panel targeting multiple biological mechanisms and additional 30-custom genes related to capecitabine metabolism. Hypothesis-generating exploratory analyses were performed to assess biomarker expression in relation to treatment effect using the Cox regression model and interaction tests adjusted for multiplicity. Results: One hundred eleven TNBC samples were evaluable (57 without capecitabine and 54 with capecitabine). The median follow-up was 10.2 years. Multivariate analysis showed significant improvement in recurrence-free survival (RFS) favoring capecitabine in four biologically important genes and metagenes, including cytotoxic cells [hazard ratio (HR) = 0.38; 95% confidence intervals (CI), 0.16–0.86, P-interaction = 0.01], endothelial (HR = 0.67; 95% CI, 0.20–2.22, P-interaction = 0.02), mast cells (HR = 0.78; 95% CI, 0.49–1.27, P-interaction = 0.04), and PDL2 (HR = 0.31; 95% CI, 0.12–0.81, P-interaction = 0.03). Furthermore, we identified 38 single genes that were significantly associated with capecitabine benefit, and these were dominated by immune response pathway and enzymes involved in activating capecitabine to fluorouracil, including TYMP. However, these results were not significant when adjusted for multiple testing. Conclusions: Genes and metagenes related to antitumor immunity, immune response, and capecitabine activation could identify TNBC patients who are more likely to benefit from adjuvant capecitabine. Given the reduced power to observe significant findings when correcting for multiplicity, our findings provide the basis for future hypothesis-testing validation studies on larger clinical trials.