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Supplementary Figure from Clinical Outcomes and Immune Markers by Race in a Phase I/II Clinical Trial of Durvalumab Concomitant with Neoadjuvant Chemotherapy in Early-Stage TNBC

expression compartment comparisons

methods table describing batches and antibodies

Supplementary Table from Clinical Outcomes and Immune Markers by Race in a Phase I/II Clinical Trial of Durvalumab Concomitant with Neoadjuvant Chemotherapy in Early-Stage TNBC

Purpose:The incidence of triple-negative breast cancer (TNBC) is higher among Black or African American (AA) women, yet they are underrepresented in clinical trials. To evaluate safety and efficacy of durvalumab concurrent with neoadjuvant chemotherapy for stage I–III TNBC by race, we enrolled additional AA patients to a Phase I/II clinical trial.Patients and Methods:Our study population included 67 patients. The primary efficacy endpoint was pathologic complete response (pCR; ypT0/is, N0) rate. χ2 tests were used to evaluate associations between race and baseline characteristics. Cox proportional hazards models were used to assess association between race and overall survival (OS) and event-free survival (EFS). Multivariate logistic regression analyses were used to evaluate associations between race and pCR, immune-related adverse events (irAE) and recurrence.Results:Twenty-one patients (31%) self-identified as AA. No significant associations between race and baseline tumor stage (P = 0.40), PD-L1 status (0.92), and stromal tumor–infiltrating lymphocyte (sTIL) count (P = 0.57) were observed. pCR rates were similar between AA (43%) and non-AA patients (48%; P = 0.71). Three-year EFS rates were 78.3% and 71.4% in non-AA and AA patients, respectively [HR, 1.451; 95% confidence interval (CI), 0.524–4.017; P = 0.474]; 3-year OS was 87% and 81%, respectively (HR, 1.72; 95% CI, 0.481–6.136; P = 0.405). The incidence of irAEs was similar between AA and non-AA patients and no significant associations were found between irAEs and pathologic response.Conclusions:pCR rates, 3-year OS and EFS after neoadjuvant immunotherapy and chemotherapy were similar in AA and non-AA patients. Toxicities, including the frequency of irAEs, were also similar.

reproducibility graphs

Ultimapper schematic and images

Univariate and multivariate analysis

Purpose:In both the IMpassion 130 trial in the metastatic setting and in Keynote 522 in the neoadjuvant setting, patients with triple-negative breast cancer (TNBC) showed benefit from PD-1 axis immunotherapy. Here, we assess PD-L1 expression on both tumor and immune cells using quantitative immunofluorescence to assess association with benefit from neoadjuvant durvalumab concurrent with chemotherapy in TNBC.Experimental Design:Pretreatment core needle biopsies (n = 69) were obtained from patients who participated in a phase I/II clinical trial (NCT02489448). The final analysis included 45 patients [pathologic complete response (pCR) = 18, non-pCR = 27] due to technical issues and insufficient tissue. Slides were stained using a previously validated Ultivue DNA-based Ultimapper kit (CD8, CD68, PD-L1, Cytokeratin/Sox10, and Hoechst counterstain). The PD-L1 expression was analyzed by molecular compartmentalization without segmentation using AQUA software (version 3.2.2.1) in three tissue compartments including tumor (cytokeratin-positive cells), CD68+ cells, and overall stroma.Results:In patients with pCR, PD-L1 expression was significantly higher in tumor cells, in CD68+ cells and in the stroma compared with patients non-pCR. There was no difference in the amount of CD68+ cells in the tumor or stromal compartments between cases with pCR and non-pCR.Conclusions:Expression of PD-L1 in tumor cells, immune cells in stroma, and colocalized with CD68+ cells is associated with higher rates of pCR to durvalumab and chemotherapy in TNBC.

Images of PD-L1 expression

Tumours use various strategies to evade immune surveillance1,2. Immunotherapies targeting tumour immune evasion such as immune checkpoint blockade have shown considerable efficacy on multiple cancers3,4 but are ineffective for most patients due to primary or acquired resistance5–7. Recent studies showed that some epigenetic regulators suppress anti-tumour immunity2,8–12, suggesting that epigenetic therapies could boost anti-tumour immune responses and overcome resistance to current immunotherapies. Here we show that, in mouse melanoma models, depletion of KDM5B—an H3K4 demethylase that is critical for melanoma maintenance and drug resistance13–15—induces robust adaptive immune responses and enhances responses to immune checkpoint blockade. Mechanistically, KDM5B recruits the H3K9 methyltransferase SETDB1 to repress endogenous retroelements such as MMVL30 in a demethylase-independent manner. Derepression of these retroelements activates cytosolic RNA-sensing and DNA-sensing pathways and the subsequent type-I interferon response, leading to tumour rejection and induction of immune memory. Our results demonstrate that KDM5B suppresses anti-tumour immunity by epigenetic silencing of retroelements. We therefore reveal roles of KDM5B in heterochromatin regulation and immune evasion in melanoma, opening new paths for the development of KDM5B-targeting and SETDB1-targeting therapies to enhance tumour immunogenicity and overcome immunotherapy resistance. KDM5B recruits SETDB1 to repress endogenous retroelements such as MMVL30, suppressing anti-tumour immunity, and the depletion of KDM5B induces a robust adaptive immune response and enhances the response to immune checkpoint blockade.

516 Background: The incidence of triple negative breast cancer (TNBC) is higher among Black or African American (AA) women, yet AA patients (pts) are underrepresented in clinical trials, exemplifying racial disparity in oncology. We conducted a phase I/II trial to assess the safety and efficacy of durvalumab concurrent with weekly nab-paclitaxel and dose dense doxorubicin/cyclophosphamide (ddAC) neoadjuvant therapy for stage I-III TNBC. The primary efficacy endpoint was pathologic complete response (pCR; ypT0/is,N0) rate. Given the unclear efficacy and safety of immunotherapy in AA pts with breast cancer, we extended our accrual to recruit AA pts, with the goal of evaluating the association between racial groups and PD-L1 expression, stromal tumor infiltrating lymphocytes (sTILs), toxicities, treatment response and survival. Methods: Our study population included 67 pts. PD-L1 immunohistochemistry results and sTIL counts were available on 59 and 60 pts, respectively. Chi-Squared test was used to evaluate associations between race and baseline characteristics. Cox proportional hazards model was used to assess association between AA race and overall survival (OS) and event free survival (EFS), adjusting for age, comorbidities and pCR status. Multivariate logistic regression analyses were used to evaluate the association between race and pCR, development of immune-related adverse events (irAEs) and breast cancer recurrence. Results: Twenty-one pts (31%) self-identified as AA. No significant associations between AA race and baseline body mass index (BMI; p=0.075), Charlson comorbidity index (p=0.32), tumor stage (p=0.40), grade (p=0.54), PD-L1 status (0.92) and sTIL count (p=0.57) were observed. pCR rates did not significantly differ between AA and non-AA pts: 9/21 (43%) AA vs. 22/48 (48%) non-AA (p=0.71). 3-yr OS was 87% in the non-AA versus 81% in the AA cohort (HR 1.72, 95% CI 0.481-6.136; p=0.405); 3 yr EFS were 78.3% and 71.4% in non-AA and AA pts respectively. (HR 1.451, 95% CI 0.524-4.017; p=0.474). Pts with pCR were more likely to remain event-free at 3 yrs, irrespective of race (HR 0.234, 95% CI 0.066-0.829; p=0.024). In multivariate logistic regression analyses, lack of pathologic response (OR for pCR 0.17, 95% CI 0.03-0.7; p=0.02) and node positive status (OR 4.13, 95% CI 1.05-19.88; p=0.05) were associated with recurrence. The incidence of irAEs was similar between AA and non-AA pts and no significant associations were found between irAEs and pathologic response. Conclusions: pCR rates after neoadjuvant immunotherapy and chemotherapy were similar in AA and non-AA pts. Stromal TILs, PD-L1 status, 3yr OS and EFS, and the frequency of irAEs were also similar. These results suggest that when patients receive identical treatment and are monitored closely, disparities in outcomes can be mitigated or abolished. Clinical trial information: NCT02489448.

Background: Several recent studies that compared small cohorts of metastatic and primary lesions, suggested substantial heterogeneity in tumor infiltrating lymphocyte count, immune gene expression and PD-L1 protein expression across different metastatic sites and between primary breast cancers and metastasis. Understanding the frequency of PD-L1 positivity rates across different tissue sites can indicate differences in the immune microenvironment and may also guide biopsy site selection. We compared PD-L1 positivity on immune cells and tumor cells in primary and metastatic triple negative breast cancer tumors (TNBC).Methods: A retrospective data analysis of the Foundation Medicine PD-L1 IHC database was conducted on 340 cases of TNBC. PD-L1 positivity was determined by IHC using SP142CDx. Results are reported as percent of PD-L1 stained immune cells (IC) in the tumor area. A tumor was considered PD-L1 positive if ≥ 1% IC stained positive with PD-L1. As an exploratory analysis, PD-L1 positivity of tumor cells (TC) was also assessed. PD-L1 percent positive staining results are reported as means with 95% CI. The proportion of PD-L1 positive and negative IC and TC in primary tumors vs metastatic sites was compared using Chi-Square test. Prism 8 was used for all data analysis.Results: All patients were female, with median age 56 years (range 26-89); 179 samples were from primary tumors and 161 from metastatic lesions, representing 15 different tissue sites. Overall, PD-L1 expression on immune cells was statistically significantly more frequent in primary tumors compared to metastatic sites (63.7% [n=114] vs 42.9% [n=69]), p Table 1: Sample Characteristics and % PD-L1 positivity on immune cellsSample typeTotal N (%)N PD-L1 positive (%, 95% CI)Primary Tumor179 (52.6)114 (63.7%, 56.2% - 70.7%)Metastatic Lesion161 (47.4)69 (42.9%, 35.1% - 50.9%)Sites of MetastasesN (% of metastatic samples)N PD-L1 positive (%, 95% CI)Lung16 (10.0)11 (68.8%, 41.3% - 90.0%)Soft Tissues23 (14.3)15 (65.2%, 42.7% - 83.6%)Lymph Nodes45 (28.0)23 (51.1%, 35.8% - 66.3%)Skin21 (13.0)5 (23.8%, 8.22% - 47.2%)Liver23 (14.3)4 (17.4%, 5.00% - 38.8%)Bone12 (7.5)2 (16.7%, 2.10% - 48.4%)Brain9 (5.6)5Mediastinum4 (2.5)1Pleura2 (1.2)0Muscle1 ( Table 2: Comparison of PD-L1 positivity in primary versus metastatic sitesTissuePDL1+ Immune CellPDL1- Immune CellP valuePDL1+ Tumor CellPDL- Tumor CellP valuePrimary114650.0001151640.1313Metastasis69927154 Citation Format: Mariya Rozenblit, Richard Huang, Natalie Danziger, Brian Alexander, Shakti Ramkissoon, Kim Blenman, Jeffrey Ross, David Rimm, Lajos Pusztai. Comparison of PD-L1 protein expression between primary tumors and metastatic lesions in triple negative breast cancers [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 PS5-08.

Objectives Checkpoint inhibitor (CPI)–associated colitis can limit therapy and has resemblance to inflammatory bowel disease (IBD). Studies exploring mechanistic similarities between these colitides are limited, yet therapeutic targets for either disorder could emerge from shared pathophysiology. Methods The morphology and inflammatory content of colonic biopsy specimens from anti–CTLA-4 and anti–PD-1/PD-L1 antibody-treated patients with CPI colitis were compared with initial biopsy specimens from patients with IBD. Predictors of the need for infliximab were sought in CPI patients. Results Biopsy specimens from CPI patients showed significantly lower chronicity scores and similar activity scores compared with patients with IBD. Anti–CTLA-4 and IBD groups showed equivalent CD8, CD4, PD-1, and PD-L1 expression, while FoxP3 scores were lower and CD68 scores were higher in anti–CTLA-4 compared with IBD biopsy specimens. Anti–PD-1/PD-L1 group had lower scores for CD8, CD4, and PD-1 and equivalent scores for FoxP3, PD-L1, and CD68 compared with IBD. Anti–CTLA-4 biopsy specimens had higher scores for CD8, PD-1, PD-L1, and CD68 than anti–PD-1/PD-L1 biopsy specimens. CD8/FoxP3 ratios and CD68 scores were higher among CPI patients requiring infliximab therapy for colitis compared with those responding to steroids. Conclusions The proinflammatory immune phenotype of anti–CTLA-4–associated colitis has significant overlap with IBD. CD8/FoxP3 ratios may predict therapeutic response in CPI-associated colitis.

The goal of this Phase I/II trial is to assess the safety and efficacy of administering durvalumab concurrent with weekly nab-paclitaxel and dose-dense doxorubicin/cyclophosphamide (ddAC) neoadjuvant therapy for stages I–III triple-negative breast cancer. The primary endpoint is pathologic complete response (pCR:ypT0/is, ypN0). The response was correlated with PDL1 expression and stromal tumor-infiltrating lymphocytes (sTILs). Two dose levels of durvalumab (3 and 10 mg/kg) were assessed. PD-L1 was assessed using the SP263 antibody; ≥1% immune and tumor cell staining was considered positive; sTILs were calculated as the area occupied by mononuclear inflammatory cells over the total intratumoral stromal area. 59 patients were evaluable for toxicity and 55 for efficacy in the Phase II study (10 mg/kg dose). No dose-limiting toxicities were observed in Phase I. In Phase II, pCR rate was 44% (95% CI: 30–57%); 18 patients (31%) experienced grade 3/4 treatment-related adverse events (AE), most frequently neutropenia (n = 4) and anemia (n = 4). Immune-related grade 3/4 AEs included Guillain–Barre syndrome (n = 1), colitis (n = 2), and hyperglycemia (n = 2). Of the 50 evaluable patients for PD-L1, 31 (62%) were PD-L1 positive. pCR rates were 55% (95% CI: 0.38–0.71) and 32% (95% CI: 0.12–0.56) in the PD-L1 positive and negative groups (p = 0.15), respectively. sTIL counts were available on 52 patients and were significantly higher in the pCR group (p = 0.0167). Concomitant administration of durvalumab with sequential weekly nab-paclitaxel and ddAC neoadjuvant chemotherapy resulted in a pCR rate of 44%; pCR rates were higher in sTIL-high cancers.

Purpose: In both the IMpassion 130 trial in the metastatic setting and in Keynote 522 in the neoadjuvant setting, patients with triple-negative breast cancer (TNBC) showed benefit from PD-1 axis immunotherapy. Here, we assess PD-L1 expression on both tumor and immune cells using quantitative immunofluorescence to assess association with benefit from neoadjuvant durvalumab concurrent with chemotherapy in TNBC. Experimental Design: Pretreatment core needle biopsies (n = 69) were obtained from patients who participated in a phase I/II clinical trial (NCT02489448). The final analysis included 45 patients [pathologic complete response (pCR) = 18, non-pCR = 27] due to technical issues and insufficient tissue. Slides were stained using a previously validated Ultivue DNA-based Ultimapper kit (CD8, CD68, PD-L1, Cytokeratin/Sox10, and Hoechst counterstain). The PD-L1 expression was analyzed by molecular compartmentalization without segmentation using AQUA software (version 3.2.2.1) in three tissue compartments including tumor (cytokeratin-positive cells), CD68+ cells, and overall stroma. Results: In patients with pCR, PD-L1 expression was significantly higher in tumor cells, in CD68+ cells and in the stroma compared with patients non-pCR. There was no difference in the amount of CD68+ cells in the tumor or stromal compartments between cases with pCR and non-pCR. Conclusions: Expression of PD-L1 in tumor cells, immune cells in stroma, and colocalized with CD68+ cells is associated with higher rates of pCR to durvalumab and chemotherapy in TNBC.

Purpose: Validation of artificial intelligence (AI) algorithms in digital pathology with a reference standard is necessary before widespread clinical use, but few examples focus on creating a reference standard based on pathologist annotations. This work assesses the results of a pilot study that collects density estimates of stromal tumor-infiltrating lymphocytes (sTILs) in breast cancer biopsy specimens. This work will inform the creation of a validation dataset for the evaluation of AI algorithms fit for a regulatory purpose. Approach: Collaborators and crowdsourced pathologists contributed glass slides, digital images, and annotations. Here, "annotations" refer to any marks, segmentations, measurements, or labels a pathologist adds to a report, image, region of interest (ROI), or biological feature. Pathologists estimated sTILs density in 640 ROIs from hematoxylin and eosin stained slides of 64 patients via two modalities: an optical light microscope and two digital image viewing platforms. Results: The pilot study generated 7373 sTILs density estimates from 29 pathologists. Analysis of annotations found the variability of density estimates per ROI increases with the mean; the root mean square differences were 4.46, 14.25, and 26.25 as the mean density ranged from 0% to 10%, 11% to 40%, and 41% to 100%, respectively. The pilot study informs three areas of improvement for future work: technical workflows, annotation platforms, and agreement analysis methods. Upgrades to the workflows and platforms will improve operability and increase annotation speed and consistency. Conclusions: Exploratory data analysis demonstrates the need to develop new statistical approaches for agreement. The pilot study dataset and analysis methods are publicly available to allow community feedback. The development and results of the validation dataset will be publicly available to serve as an instructive tool that can be replicated by developers and researchers. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License.

We assessed associations between PD-L1 protein expression, RS, tumor grade, and stromal tumor-infiltrating lymphocyte (TIL) count in early-stage ER + cancers.FFPE tissue blocks of 213 patients with RS in 2012-2017 were identified. PD-L1 immunohistochemistry was performed with SP142 assay, cases with ≥ 1% tumor-infiltrating immune cell positivity in the tumor area were considered PD-L1 + . TIL scores were determined following the international TIL counting guidelines. PD-L1 expression positivity rates were compared across RS ( 11, 11-25, 25) and TIL categories ( 10%, 10-29%, 30%), and tumor grade using Wilcoxon and Chi-square tests. Multivariate analysis was performed using logistic regression.PD-L1 and TIL results were available for 201 and 203 patients. Overall, 53% of cases were PD-L1 +. PD-L1 expression was higher among cases with RS 25, versus RS 11 (p = 0.00019) and RS 11-25 (p = 0.0017). PD-L1 positivity also correlated with TIL score, tumor grade, and tumor size. Among cancers with TIL 30%, 92% were PD-L1 + versus 44% PD-L1 + among TIL 10% (p = 2.8 × 10PD-L1 expression is significantly more frequent and higher in larger tumors (T2, T3), grade 3 cancers, and in cancers with RS 25. PD-L1 expression also correlates with TIL score.

Background: The RxPONDER and TAILORx trials demonstrated benefit from adjuvant chemotherapy in patients < 50 years with node-positive breast cancer and Recurrence Score (RS) 0-25, and with node-negative disease and RS 16-25, respectively. Neither trial showed benefit in older women with RS < 26. It is unclear what explains the interaction between age and adjuvant chemotherapy benefit. Methods: We analyzed transcriptomic and genomic data from n=4,507 ER+/HER2- breast cancers to compare differences in estrogen receptor (ER), proliferation, and immune-related gene expressions, and somatic mutation patterns and mutation burden between younger (< 50 years of age) and older (>55 years) patients. We restricted our analysis to patients in the lower 80% range of in silico RS distribution to mimic the RxPONDER and TAILORx populations. Results: Five data sets were analyzed independently to assess consistency of results (TCGA n=530; microarray cohort A n=865; Cohort B n=609, METABRIC n=867, SCAN-B n=1636). Older patients had significantly higher somatic mutation burden and more frequent copy number gain in ESR1, LATS1, ARID1B, SGK1, and MYB genes (odds ratio [OR] > 8.5, FDR< 0.05), but lower frequency of GATA3 mutations (12% versus 26%, P< 0.0001). Younger patients had higher rate of ESR1 copy number loss (OR: 0.45, FDR: 0.03). There was no difference in proliferation-related gene expression. ESR1 mRNA expression was significantly lower in younger women in all cohorts (P < 0.001). A regression model of ESR1 mRNA expression using age and ER IHC positivity indicated that lower ER expression in younger patients is primarily driven by lower ESR1 mRNA per cancer cell and not by fewer ER positive cells. We also assessed four gene signatures associated with endocrine therapy sensitivity including a 4-gene ERS, a 7-gene ERS-Lum, a 106-gene ERS-Pos signature, and a 59-gene ERS-Neg signature associated with endocrine resistance. In the TCGA and METABRIC cohorts, the ERS, ERS-Lum, and ERS-Pos signatures were all lower (FDR< 0.03) while the ERS-Neg signature was higher (FDR< 0.001) in younger patients. Similarly, in both microarray cohorts, and in the SCAN-B-cohort, the ERS-Pos signature was lower and the ERS-Neg signature was higher in younger patients (FDR< 0.002). Next, we assessed 4 different immune cell signatures that have been associated with response to chemotherapy. In the TCGA, B-cell, T-cell, Mast-cell, and TIS signatures were significantly higher (FDR Citation Format: Tao Qing, Thomas Karn, Mariya Rozenblit, Julia Foldi, Michal Marczyk, Naing Lin Shan, Kim Blenman, uwe Holtrich, Kevin Kalinsky, Funda Meric-Bernstam, Lajos Pusztai. Molecular differences between younger versus older estrogen receptor positive/human epidermal growth factor receptor-2 negative breast cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD9-09.

Background: In the IMpassion 130 trial, TNBC patients treated with Atezolizumab and nab-Paclitaxel that showed immune cell expression of PD-L1 showed better overall survival. The goal of this analysis was to assess association between quantitative PD-L1 expression, in various tissue compartments, and pathologic complete response (pCR: ypT0/N0) to neoadjuvant anti-PD-L1 therapy concurrent with nab-paclitaxel (100 mg/m2) x 12 followed by ddAC x 4 chemotherapy in stage I-III triple negative breast cancer (TNBC). Methods: Pre-treatment core needle biopsies (n=69) were obtained from patients who participated in a Phase I/II clinical trial (NCT02489448). Of these 24 patients samples were excluded due to a range of clinical and technical issues. The final analysis had 45 patients (pCR = 18, non-pCR = 27). The slides were stained using a previously validated Ultivue DNA-based Ultimapper® kit (CD8, CD68, PD-L1, Cytokeratin/Sox10 and Hoechst counterstain for nuclear staining). Briefly, mouse monoclonal antibodies with a short DNA strand attached to the Fc region were amplified and attached with a complimentary DNA strand attached to a fluorophore followed by an amplification procedure. We stained 8 batches of slides with 2 control slides for each batch to assess for variability between batches (CD8, CD68 and PD-L1). The images were analyzed by molecular compartmentalization without segmentation using AQUA software (version 3.2.2.1) for each marker in different compartments (tumor, stroma and CD68 for PD-L1). A column graph and Mann-Whitney U-test was used to analyses the results for statistical significance between pCR and non-pCR. P-values shown are not adjusted for multiple comparisons. Results: Reproducibility between batches was excellent (CD68; R2 = 0.86 to 0.94 and PD-L1; R2 = 0.87 to 0.98). PD-L1 expression was significantly higher in the tumor compartment, the stromal compartment, and in the CD68 compartment in pCR patients compared to non-pCR. However, comparative analysis between the pCR and non-pCR patients showed no difference in the overall presence of CD68 in any of the molecular compartments. Conclusion: Expression of PD-L1 in tumor cells and immune cells in stroma and the CD68 expression compartment (predominantly macrophages) is associated with response to Durvalumab and chemotherapy in this small neoadjuvant TNBC trial. This result, the association of PD-L1 expression in macrophages with outcome, has also been seen in melanoma (Toki et al CCR 2019) and lung cancer (Liu et al, in revision). Further study is required to validate this potential predictive biomarker. Citation Format: Fahad Shabbir Ahmed, John McGuire, Patricia Gaule, Kim Blenman, Lajos Pusztai, David L Rimm. Quantitative assessment of PD-L1 protein expression on macrophages and tumor cells as predictive markers of response to neoadjuvant durvalumab and chemotherapy in triple negative breast cancer (TNBC) [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 P4-10-14.

Background: The goal of this analysis was to identify immune cell types and mRNA expression signatures that are associated with pathologic complete response (pCR: ypT0/N0) to neoadjuvant durvalumab concurrent with weekly nab-paclitaxel (100 mg/m2) x 12 followed by ddAC x 4 chemotherapy in stage I-III triple negative breast cancer (TNBC). Methods: Pre-treatment core needle biopsies were obtained from 57 patients (n=25 pCR, n=32 residual disease (RD)) who participated in Phase I/II clinical trial (NCT02489448) for RNA sequencing and histology assessment. Formalin-fixed paraffin-embedded (FFPE) core biopsies were stained by immunofluorescence for CD8, CD68 and cytokeratin, images were acquired on the Vectra-Polaris system and analyzed using InForm software with the adaptive segmentation into three compartments: Tumor, Stroma, and All (Tumor + Stroma). Correlation between immune cell density in the tissue compartments and pCR was assessed. RNA was extracted from core biopsies collected into RNAlater and poly-A enriched mRNAs were sequenced on Illumina platform using NovaSeq paired-end, 100bp fragments, with a depth of 50 million reads. Six specimens failed QC due to insufficient tumor cells in the sample. Correlation between pCR and immune markers and previously published immune, proliferation and DNA damage response deficiency (DDRD) gene signatures were assessed using logistic regression. Due to high overlap of genes across signatures, no multiple testing correction was done. Differentially expressed genes were found using DESeq2 R package with Benjamini-Hochberg correction for multiple testing. Broadside, an interaction mining tool, was used to extract sets of combinations of genes associated with pathologic response. Results: In cases with pCR, we detected significantly higher overall CD8 cell density and a trend for higher CD8 in tumor and stroma compartments. There was no significant difference in CD68 cell density between response groups. At the RNA level, high expression of DDRD, IFNγ, T-cell, B-cell, dendritic cell, M1 macrophage signatures and the tumor inflammation gene signature were significantly associated with higher rate of pCR. High expression of epithelial mesenchymal transition and TGFβ and IL8/VEGF gene signatures were associated with higher rate of residual disease. Proliferation gene signatures were not associated with response in this TNBC population. At individual gene level, the highest pCR predictive value was observed for the STAT1 + PCF11 and LAMP3 + SDR39U1 doublets. Conclusion: High CD8 cell density, high expression of a broad range of immune gene expression signatures and DNA damage response deficiency are associated with greater sensitivity to neoadjuvant anti-PD-L1 and chemotherapy. Citation Format: Kim RM Blenman, Xiaotong Li, Michal Marczyk, Tess O'Meara, Vesal Yaghoobi, Vignesh Gunasekharan, Tristen Park, David Rimm, Lajos Pusztai. Predictive markers of response to durvalumab concurrent with nab-paclitaxel and dose dense doxorubicin cyclophosphamide (ddAC) neoadjuvant therapy for triple negative breast cancer (TNBC) [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 P3-09-05.

Purpose: Because durable response to programmed cell death 1 (PD-1) inhibition is limited to a subset of melanoma patients, new predictive biomarkers could have clinical utility. We hypothesize that pretreatment tumor-infiltrating lymphocyte (TIL) profiles could be associated with response. Experimental Design: Pretreatment whole tissue sections from 94 melanoma patients treated with anti–PD-1 therapy were profiled by multiplex immunofluorescence to perform TIL quantification (CD4, CD8, CD20) and assess TIL activation (CD3, GZMB, Ki67). Two independent image analysis technologies were used: inForm (PerkinElmer) to determine cell counts, and AQUA to measure protein by quantitative immunofluorescence (QIF). TIL parameters by both methodologies were correlated with objective response or disease control rate (ORR/DCR) by RECIST 1.1 and survival outcome. Results: Pretreatment lymphocytic infiltration, by cell counts or QIF, was significantly higher in complete or partial response than in stable or progressive disease, particularly for CD8 (P < 0.0001). Neither TIL activation nor dormancy was associated with outcome. CD8 associations with progression-free survival (HR > 3) were independently significant in multivariable analyses and accounted for similar CD3 associations in anti–PD-1-treated patients. CD8 was not associated with melanoma prognosis in the absence of immunotherapy. Predictive performance of CD8 cell count (and QIF) had an area under the ROC curve above 0.75 (ORR/DCR), which reached 0.83 for ipilimumab plus nivolumab. Conclusions: Pretreatment lymphocytic infiltration is associated with anti–PD-1 response in metastatic melanoma. Quantitative TIL analysis has potential for application in digital precision immuno-oncology as an “indicative” companion diagnostic.

Epigenetic therapies are emerging strategies to prime host immune system to immunotherapies. Here we show that depletion of H3K4 demethylase KDM5B in melanoma cells induces anti-tumor T cell immunity through up-regulation of retroelements, which activates cytosolic nucleic acid sensing pathways and subsequent type I interferon responses. Mechanistically, KDM5B recruits H3K9 methyltransferase SETDB1 to the chromatin to repress these retroelements. Ablation of KDM5B enhances responses of poorly immunogenic melanoma tumors to anti PD-1 treatment. Our studies suggest that therapies targeting KDM5B are a novel approach to enhance tumor immunogenicity and overcome immunotherapy resistance.

Programmed Death Ligand 1 (PD-L1) positivity rates differ between different metastatic sites and the primary tumor. Understanding PD-L1 expression characteristics could guide biopsy procedures and motivate research to better understand site-specific differences in the tumor microenvironment. The purpose of this study was to compare PD-L1 positivity on immune cells and tumor cells in primary and metastatic triple negative breast cancer (TNBC) tumors. Retrospective study utilizing the PD-L1 database of Foundation Medicine containing the SP142 companion diagnostic immunohistochemistry assay (SP142 CDx) and Food and Drug Administration guidelines for scoring. 340 TNBC cases (179 primary tumors and 161 unmatched metastatic lesions) were evaluated. The primary outcome measures were PD-L1 positivity rates in immune cells and tumor cells. χ2 test was used for comparisons. Spearman’s correlation coefficient was used for correlations. More primary tumors were positive for PD-L1 expression on immune cells than metastatic lesions (114 (63.7%) vs 68 (42.2%), p

Since the advent of microscopy imaging and flow cytometry, there has been an explosion in the number of probes, consisting of a component binding to an analyte and a detectable tag, to mark areas of interest in or on cells and tissue. Probe tags have been created to detect and/or visualize probes. Over time, these probe tags have increased in number. The expansion has resulted in arbitrarily created synonyms of probe tags used in publications and software. The synonyms are problematic for readability of publications, accuracy of text/data mining, and bridging data from multiple platforms, protocols, and databases for Big Data analysis. Development and implementation of a universal language for probe tags will ensure equivalent quality and level of data being reported or extracted for clinical/scientific evaluation as well as help connect data from many platforms. The International Society for Advancement of Cytometry Data Standards Task Force composed of academic scientists and industry hardware/software/reagent manufactures have developed recommendations for a standardized nomenclature for probe tags used in cytometry and microscopy imaging. These recommendations are shared in this technical note in the form of a Probe Tag Dictionary. © 2020 International Society for Advancement of Cytometry.

FCS 3.2 is a revision of the flow cytometry data standard based on a decade of suggested improvements from the community as well as industry needs to capture instrument conditions and measurement features more precisely. The unchanged goal of the standard is to provide a uniform file format that allows files created by one type of acquisition hardware and software to be analyzed by any other type. The standard retains the overall FCS file structure and most features of previous versions, but also contains a few changes that were required to support new types of data and use cases efficiently. These changes are incompatible with existing FCS file readers. Notably, FCS 3.2 supports mixed data types to, for example, allow FCS measurements that are intrinsically integers (e.g., indices or class assignments) or measurements that are commonly captured as integers (e.g., time ticks) to be more represented as integer values, while capturing other measurements as floating-point values in the same FCS data set. In addition, keywords explicitly specifying dyes, detectors, and analytes were added to avoid having to extract those heuristically and unreliably from measurement names. Types of measurements were formalized, several keywords added, others removed, or deprecated, and various aspects of the specification were clarified. A reference implementation of the cyclic redundancy check (CRC) calculation is provided in two programming languages since a correct CRC implementation was problematic for many vendors. © 2020 International Society for Advancement of Cytometry.

PURPOSE A subset of estrogen receptor–positive (ER-positive) breast cancer (BC) contains high levels of tumor-infiltrating lymphocytes (TILs), similar to triple-negative BC (TNBC). The majority of immuno-oncology trials target TNBCs because of the greater proportion of TIL-rich TNBCs. The extent to which the immune microenvironments of immune-rich ER-positive BC and TNBC differ is unknown. PATIENTS AND METHODS RNA sequencing data from The Cancer Genome Atlas (TCGA; n = 697 ER-positive BCs; n = 191 TNBCs) were used for discovery; microarray expression data from Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; n = 1,186 ER-positive BCs; n = 297 TNBCs) was used for validation. Patients in the top 25th percentile of a previously published total TIL metagene score distribution were considered immune rich. We compared expression of immune cell markers, immune function metagenes, and immuno-oncology therapeutic targets among immune-rich subtypes. RESULTS Relative fractions of resting mast cells (TCGA Padj = .009; METABRIC Padj = 4.09E-15), CD8+ T cells (TCGA Padj = .015; METABRIC Padj = 0.390), and M2-like macrophages (TCGA Padj= 4.68E-05; METABRIC Padj = .435) were higher in immune-rich ER-positive BCs, but M0-like macrophages (TCGA Padj = 0.015; METABRIC Padj = .004) and M1-like macrophages (TCGA Padj = 9.39E-08; METABRIC Padj = 6.24E-11) were higher in immune-rich TNBCs. Ninety-one immune-related genes (eg, CXCL14, CSF3R, TGF-B3, LRRC32/GARP, TGFB-R2) and a transforming growth factor β (TGF-β) response metagene were significantly overexpressed in immune-rich ER-positive BCs, whereas 41 immune-related genes (eg, IFNG, PD-L1, CTLA4, MAGEA4) were overexpressed in immune-rich TNBCs in both discovery and validation data sets. TGF-β pathway member genes correlated negatively with expression of immune activation markers ( IFNG, granzyme-B, perforin) and positively with M2-like macrophages ( IL4, IL10, and MMP9) and regulatory T-cell ( FOXP3) markers in both subtypes. CONCLUSION Different immunotherapy strategies may be optimal in immune-rich ER-positive BC and TNBC. Drugs targeting the TGF-β pathway and M2-like macrophages are promising strategies in immune-rich ER-positive BCs to augment antitumor immunity.

We evaluated the spontaneous and immunotherapy-induced histological changes in the tumor microenvironment of a mouse melanoma regression model consisting of immunocompetent C57BL/6J mice implanted with syngeneic YUMMER1.7 melanoma cells. We focused on tumor regression phenotypes and spatial relationships of melanoma cells with B cells and neutrophils since this was not previously described. We found common themes to the host response to cancer irrespective of the mode of tumor regression. In nonregression tumors, melanoma cells were epithelioid shaped and tightly packed. In regression tumors, melanoma cells were spindle shaped and discohesive. B cells including plasmablasts and plasma cells were numerous and were increased with immunotherapy. Neutrophils were in direct contact with dead or dying melanoma cells. Immunotherapy increased neutrophil counts and induced neutrophil extracellular traps (NETs)-like formations and geographic necrosis. Beyond tumor regression, the increase in the B cell and neutrophil response could play a role in immunotherapy induced adverse reactions.

Phenotyping immune cells and cell clusters in situ, including their activation state and function, can aid in interpretation of spatial relationships within the tissue microenvironment. Immune cell phenotypes require multiple biomarkers. However, conventional microscopy setups can only image up to four biomarkers at one time. In this report, we describe and give an example of a workflow to phenotype, quantitate, and visualize greater than four biomarkers in silico utilizing multiplexed fluorescence histology and the TissueFAXS quantitative imaging system with a conventional microscopy setup. Biomarkers were conjugated to Cy3 or Cy5. Multiplexed staining was performed on formalin-fixed paraffin-embedded tissue sections. We imaged the slides, inactivated the dyes, and repeated the process until all biomarkers were stained. Phenotype profiles were built based on in silico combinations of the biomarkers. We used algorithms that aligned all images to create a composite image, isolated each cell in the image, and identified biomarker positive cells in the image. The in silico phenotypes were quantitated and displayed through flow cytometry-like histograms and dot scatterplots in addition to backgating into the tissue images. The advantage of our workflow is that it provides visual verification of cell isolation and identification as well as highlight characteristics of cells and cell clusters. © 2018 International Society for Advancement of Cytometry.

Tumor invasion into draining lymph nodes, especially sentinel lymph nodes (SLNs), is a key determinant of prognosis and treatment in breast cancer as part of the TNM staging system. Using multicolor histology and quantitative image analysis, we quantified immune cells within SLNs from a discovery cohort of 76 breast cancer patients. We found statistically more in situ CD3+ T cells in tumor negative vs. tumor positive nodes (mean of 8878 vs. 6704, respectively, p = 0.006), but no statistical difference in CD20+ B cells or CD1a+ dendritic cells. In univariate analysis, a reduced hazard was seen with a unit increase in log CD3 with HR 0.49 (95% CI 0.30–0.80) and log CD20 with HR 0.37 (95% CI 0.22–0.62). In multivariate analysis, log CD20 remained significant with HR 0.42 (95% CI 0.25–0.69). When restricted to SLN tumor negative patients, increased log CD20 was still associated with improved DFS (HR = 0.26, 95% CI 0.08–0.90). The CD20 results were validated in a separate cohort of 21 patients (n = 11 good outcome, n = 10 poor outcome) with SLN negative triple-negative breast cancer (TNBC) (“good” mean of 7011 vs. “poor” mean of 4656, p = 0.002). Our study demonstrates that analysis of immune cells within SLNs, regardless of tumor invasion status, may provide additional prognostic information, and highlights B cells within SLNs as important in preventing future recurrence., Immunology: Lymph nodes full of B cells linked to better outcomes B cells within the tumor-draining lymph nodes may have an important biological role in preventing relapse of breast cancer. A team led by Peter Lee from City of Hope in Duarte, California, USA, quantified the levels of three populations of immune cells—T cells, B cells and dendritic cells—within sentinel lymph nodes biopsied from a cohort of 76 patients. They found that larger numbers of T cells and B cells were both linked to longer progression-free survival in the women. However, after statistically accounting for correlations between the two immune cell types, the researchers concluded that B cells had the dominant beneficial effect on survival times. They validated the finding that high B-cell counts are a prognostic indicator of better outcomes in a separate cohort of 21 women with triple-negative breast cancer.

DARC/ACKR1 erythrocyte expression, also known as the “Duffy blood group,” is understood to sequester pro-inflammatory chemokines and thereby regulate circulating gradients that direct immune cell infiltration. We hypothesize that this function also determines immune cell landscapes in the tumor microenvironment. Due to evolutionary selection pressures of malaria, individuals with sub-Saharan African ancestry typically carry the Duffy-null allele (rs4849887) and this lack of DARC/ACKR1 expression gives immunity to malaria while also allowing chronically high inflammation levels. Over 68% of African Americans (AA) have been found to have the Duffy-null genotype, compared to a rare 1-3% in European American individuals and we have shown it increases predisposition for Triple-Negative Breast Cancer (TNBC). In addition, we are currently studying if Duffy-null may contribute to higher breast cancer mortality that disproportionately affects AA women. This may be in part due to the role that low-DARC/ACKR1 expression plays in chronic inflammation, altering levels of several chemokines that modulate the migration and differentiation of specific immune cells. This role will impact tumor immune cell infiltration as well as the immune cell population composition overall, depending upon levels of DARC/ACKR1. Using RNA sequencing, our initial results indicated that for breast cancer tumors with high DARC/ACKR1 expression there was a higher estimated presence of CD8+ T cells, CD4+ T cells, regulatory T cells, follicular helper T cells, and memory B cells. Whereas with low DARC/ACKR1 expression, there was markedly less expression of resting dendritic cells and memory B cells. Therefore, in order to ascertain the influence DARC status has on spatial deposition and functional status of immune cell landscapes across the tumor microenvironment, we performed imaging mass cytometry on primary TNBC tumors. The panel contained tumor, structural, and immune markers, and was used to characterize the spatial differences between samples that had been verified to be DARC-high or DARC-low through immunohistochemistry. Our imaging analyses indicated that high DARC/ACKR1 expression correlates with infiltration of monocytes, macrophages, and cytotoxic T cells into the solid tumor microenvironment. Conversely, tumors with low DARC/ACKR1 expression showed monocytes and cytotoxic T cells contained in the tumor stroma. Using single-cell phenotyping, we were also able to identify distinct cell populations between DARC-high and -low. The tSNE analysis and heatmaps performed using Histology Topography Cytometry Analysis Toolbox (histoCAT), allowed us to visualize the spatial distribution of these cell populations, indicating an immune-suppressive tumor microenvironment in DARC-low tumors. These differences may be implicated in the causality of tumor progression as well as how to approach treatment given the cell heterogeneity of TNBC. This work provides greater context on the role that Duffy-null plays in chronic inflammation on the tumor microenvironment. Citation Format: Yanira Guerra, Rachel Martini, Jessica Bensenhaver, Yalei Chen, Joseph K. Oppong, Ishmael Kyei, Frances S. Aitpillah, Michael O. Adinku, Joseph K. Oppong, Ernest K. Adjei, Aisha Jibril, Baffour Awuah, Mahteme Bekele, Engida Abebe, Kwasi Ankomah, Ernest B. Osei-Bonsu, Kofi K. Gyan, Clayton Yates, Kim Blenman, Olivier Elemento, Lisa Newman, Melissa B. Davis. The role of African Duffy-null allele related inflammation on the tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-141.

Human melanomas exhibit relatively high somatic mutation burden compared to other malignancies. These somatic mutations may produce neoantigens that are recognized by the immune system, leading to an antitumor response. By irradiating a parental mouse melanoma cell line carrying three driver mutations with UVB and expanding a single-cell clone, we generated a mutagenized model that exhibits high somatic mutation burden. When inoculated at low cell numbers in immunocompetent C57BL/6J mice, YUMMER1.7 (Yale University Mouse Melanoma Exposed to Radiation) regresses after a brief period of growth. This regression phenotype is dependent on T cells as YUMMER1.7 tumors grow significantly faster in immunodeficient Rag1-/- mice and C57BL/6J mice depleted of CD4 and CD8 T cells. Interestingly, regression can be overcome by injecting higher cell numbers of YUMMER1.7, which results in tumors that grow without effective rejection. Mice that have previously rejected YUMMER1.7 tumors develop immunity against higher doses of YUMMER1.7 tumor challenge. In addition, escaping YUMMER1.7 tumors are sensitive to anti-CTLA-4 and anti-PD-1 therapy, establishing a new model for the evaluation of immune checkpoint inhibition and antitumor immune responses.

564 Background: What tumor biological differences, if any, contribute to the higher incidence and worse prognosis of triple negative breast cancer (TNBC) in African American (AA) compared to NonAA patients are unknown. We hypothesized that differences in the tumor immune microenvironment may contribute to the outcome disparities. The purpose of this study was to characterize and compare the immune microenvironment of TNBC between patients self-identified as NonAA or AA. Methods: Formalin fixed paraffin embedded surgically resected cancer and paired normal tissues collected before any systemic therapy and the corresponding clinical data were collected for NonAA (n = 56) and AA (n = 54) stage I-III TNBC treated at Yale Cancer Center between 2000-2017. The two cohorts were matched for clinical stage, age of diagnosis, and year of diagnosis. We performed somatic and germline whole exome sequencing (WES), bulk RNA sequencing, and immunohistochemistry to assess PD-L1 expression (SP142). Stromal tumor infiltrating lymphocytes (sTILs) were assessed on H&E slides. Mutation load, mutation frequencies, and gene expression differences were compared at gene and pathway level. Immune cell composition was estimated through gene expression deconvolution analyses (TIDE). Results: Tumor mutational burden was similar between the two cohorts. At gene level, few genes had significantly different somatic mutation frequencies, or differential mRNA expression between AA and NonAA samples. Pathway level alterations showed inflammation, immunity (adaptive; innate), antigen presentation, and allograft rejection pathways were more affected by somatic mutations in AA samples. The affected genes differed from cancer to cancer and were not recurrent and therefore were missed at gene level analysis. Gene set enrichment and co-expression analysis also showed higher immune related pathway expression in AA samples. Unsupervised co-expression cluster analysis confirmed coordinated overexpression of genes involved in immunity, inflammation, and cytokine/chemokine signaling in AA patients. Two immunotherapy response predictive signatures, immune inflamed and the IFNG as well as sTILs score and PD-L1 positivity were also higher in AA samples. These findings raise the possibility that immune checkpoint inhibitors might be particularly effective in AA patients. In NonAA samples, the EMT transition, angiogenesis, adipogenesis, myogenesis, fatty acid metabolism, TGFβ signaling, UV-response, and hypoxia pathways were overexpressed. TIDE analysis suggested higher levels of TAM M2, overall TIDE score, and the Immune Exclusion score in NonAA samples. Conclusions: TNBC in AA patients more frequently harbor somatic mutations in genes involved with immune functions and overexpress immune and inflammatory genes compared to NonAA patients.

Background: Cancer driver genes are characterized by frequently recurrent mutations, substantial functional impact on in vitro and in vivo cancer growth, and several of the represent clinically important therapeutic targets. Whole exome and genome sequencing studies identified mutations in at least one cancer driver gene in about 70% breast cancers. These studies also revealed that each cancer also harbors hundreds to thousands of additional non-recurrent mutations which follow a long-tail distribution. Historically, these long tail “passenger” mutations are considered random due to genomic instability and not expected to contribute to the biology of the disease. We hypothesize that “long-tail” mutations could also have functional importance and contribute to the unique biology of a given cancer. The goal of the current analysis was to identify the long-tail mutations in breast cancer, and estimate their overall functional importance. Methods: We obtained somatic mutations from the breast cancer TCGA cohort (N=1076) and calculated the somatic mutation frequency for each gene. The dNdScv algorithm was used to estimate significantly mutated genes. A gene was considered to be in the long tail of mutations if its somatic mutation frequency was 1-3%, and the dNdScv p-value was < 0.05. We performed pathway enrichment analysis with 21 cancer pathways assembled by NanoString Technologies (Seattle, WA) for the long-tail genes using Fisher’s exact test and obtained gene dependency scores (DS) from The Cancer Dependency Map (DepMap) project which performed genome-wide pooled loss of function screening for 17,634 human genes using and CRISPR-Cas9-mediated (CRISPR) gene editing to estimate tumor cell viability after gene silencing in 563 cell lines. The more negative (i.e. lower) the dependency score the more important the gene is to sustain cell viability. We compared the DS of the long tail genes with known cancer driver genes and other human genes excluding known breast cancer driver genes. We also compared the dependency score of genes with long-tail mutations between breast cancer and other cancer types using the Mann-Whitney U test. We estimated the trend of average dependency score across genesets using the Jonckheere Terpstra test and using Kendall's tau (τ) coefficient to show the increasing (positive value) or decreasing (negative value) trend. Results: Seventy percent of breast cancers (n=763) carried long-tail mutations in 115 different genes. The average number of long tail mutations was N=3 (range from 1 to 118). Genes with long-tail mutations were enriched in epithelial-mesenchymal transition, extracellular matrix, angiogenesis, adaptive immunity, MAPK, innate immunity, inflammation, and RAS pathways (FDR Citation Format: Tao Qing, Hussein Mohsen, Mariya Rozenblit, Michal Marczyk, Julia Foldi, Kim Blenman, Vignesh Gunasekharan, Lajos Pusztai. Functional importance of long-tail mutations in 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 PS19-05.

Background: The history of the race concept sheds the light on the lack of scientific basis of racial categories. Early attempts of using race in scientific practice (ca. 17th century) took place during times of colonial expansions and the transatlantic slave trade. The lenses through which scientists approached race categories changed over the next centuries, but legacies of using race categories in genetic studies are still embedded in some of today’s practices. Consequently, this engenders the need for novel approaches that transcend categories of or related to race in genomics to avoid unintended consequences and improve the study of disease. Methods: We introduce an approach that dynamically generates trait-specific cohorts based on genomic clustering at a subset of loci known to contribute to the disease under study. Reflecting the broad diversity of traits, the number of clusters and cluster memberships vary depending on the trait and its implicated genomic regions. Our method hence directs the lens of studying human biodiversity with respect to disease to the trait level rather than “population level.” Results: We test our approach on cancer and control whole genome sequencing datasets to partition each based on predisposition to 8 cancer types. Focusing on COSMIC genes known to have a germline contribution to cancer, we demonstrate that resulting clusterings vary across cancer types and transcend race, ethnicity—and ancestry categories, supporting our call to generate dynamic categorization sets at the trait level. Conclusion: Our suggested method highlights the importance of historical perspectives to co-shape today’s scientific practice. Experimental results resonate with previous joint calls from scholars in genetics and the humanities and social sciences to focus on the study racial and ethnic disparities in health as a result of rampant societal and environmental disparities—rather than genomic differences partly based on historical attempts to impose fixity on fluid social constructs. Citation Format: Hussein Mohsen, Kim Blenman, Lajos Pusztai. Dynamic clustering of cancer genomics datasets beyond pre-defined human categories [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PR12.

Introduction: Although there are many possible causes, it has been observed that Triple Negative Breast Cancer (TNBC) has a worse overall outcome in African American (AA) than white patients. Recently, there is also evidence that the patients response to the tumor, or the biological response expressed as the tumor immune microenvironment, impacts outcome. The goal of this study was to compare the Tumor Microenvironment (TME) of TNBC in the two mentioned groups in order to seek molecular parameters that are different in TNBC in AA patients as compared to whites. Methods: We selected N=50 AA and N=50 white TNBC samples from the Yale Pathology archives that were matched by diagnosis date. We measured CD45, CD3, CD8, CD20, CD14, CD68, PD-L1, THY1, aSMA and Fibroblast Activation Protein (FAP) expression in both the tumor and stromal compartments in whole slides from formalin fixed paraffin embedded (FFPE) tissues using multiplexed quantitative immunofluorescence (QIF). We also assessed the activation status of CD3-positive cells using Ki67 and Granzyme B markers. Median of each marker expression was calculated for each case by using quantitation from all Fields of View (FOV) that contained at least 10% tumor area. Results: AA tumors contained a significantly higher CD45-positive cells in overall, within tumor and stromal compartments (p = 0.0102, 0.0007 and 0.0280 respectively). Being more specific about CD45 marker, AA also showed to have a significantly higher level of overall lymphocyte population, measured by quantitating CD45+ CD14- cells (p =0.0081). AA tumors also had a significantly higher level of CD8 expression (p Conclusion: Significant differences are seen in the TME of AA vs. white TNBCs. Further characterization of macrophages, fibroblasts, regulatory T-cells and Extracellular Matrix are underway to more specifically define TME and determine the relationship between TME features and outcome. Citation Format: Vesal Yaghoobi, Vasiliki Pelekanou, Tess O'Meara, Andrea Silber, Lajos Pusztai, David Rimm, Kim Blenman. Comparison of tumor immune microenvironment in triple negative breast cancer (TNBC) of African American versus that of white patients [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 4973.

3117 Background: A 101-gene algorithm established as a molecular subtyping method for triple-negative breast cancer (TNBC) includes assignment of an immunomodulatory (IM) subtype based on genes active in immune cell processes. Recently, we isolated the IM concept to an independent 27-gene algorithm and its predictive ability for immunotherapy response was demonstrated in lung cancer. The objective of this study was to validate the predictive accuracy of the IM subtype as determined by the 27-gene algorithm for pathological complete response (pCR) compared with PD-L1 immunohistochemistry (IHC) staining in TNBC. Methods: We obtained RNA sequencing data from pretreatment core needle biopsies in 55 patients with stage I-III primary TNBC who received neoadjuvant immunotherapy (durvalumab with weekly nab-paclitaxel followed by ddAC) in phase I/II trial (NCT02489448). The 27-gene algorithm was used to determine IM positivity using a cutoff point previously validated from 71 lung cancer biopsy patients treated with immunotherapy. Results from the algorithm and PD-L1 IHC (antibody, SP263) were compared with pCR. Predictive accuracy of both methods was determined by diagnostic indicators. In cases positive for the IM subtype and pCR, we analyzed the immune microenvironment by deconvoluting the immune infiltration using a computational algorithm. Results: Of the 55 patients, 25 (45%) had pCR. Compared with previous subtyping methods, the 27-gene algorithm showed stronger predictive value (odds ratio, 4.125; 95% CI, 1.36-13.47; P < 0.015). For PD-L1 IHC, the odds ratio was 2.63 (95% CI, 0.82-9.21; P = 0.11). The positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio for PD-L1 IHC were 0.55, 0.68, 1.43, and 0.54, respectively. For the 27-gene algorithm, these metrics were 0.65, 0.69, 2.09, and 0.51, indicating its superior accuracy for predicting pCR. The computational algorithm showed that the IM subtype and pCR were negatively associated with a macrophage-enriched microenvironment. CD4+ T cells and dendritic cells were significantly increased among the baseline immune cell population in IM-positive patients. Conclusions: We conclude that 27-gene algorithm is a clinically applicable and a possible predictive marker for response to neoadjuvant immunochemotherapy for patients with primary TNBC. Our immune microenvironment results suggest that antigen-presenting immune cells have a crucial role in immunochemotherapy response.

Background A subset of estrogen receptor-positive (ER+) breast cancers have significant tumor infiltrating lymphocytes (TILs), similar to triple-negative breast cancer (TNBC). We examined differences in the immune microenvironment of immune-rich ER+ and immune-rich TNBC to find out if similar or different immunotherapy strategies are appropriate for these distinct disease types. Methods ER+/HER2-negative and TNBC cases were obtained from The Cancer Genome Atlas (TCGA) (n = 818 RNA Seq) and METABRIC (n = 1465 microarray). An immune gene expression score, as surrogate for TILs, was calculated for each case (Danaher et al). Signature scores were correlated with histologic TILs (R = 0.44, p Results In TCGA and METABRIC, CIBERSORT showed more M0 (p = 0.015, p = 0.0043) and M1 macrophages (p = 9.4e-08, p = 6.24e-11) in immune-rich TNBC compared to ER+. Mast cells (p = 0.0093, p = 4.09e-15) and M2 macrophages (p = 4.4e-05, p = 0.053) were more abundant in immune-rich ER+. QIF confirmed higher macrophage content in TNBC (p = 0.0001). In both datasets, 36 IO targets were higher expressed in TNBC and 15 in ER+ cases (Table). Notably, coordinated high expression of TGFb pathway members TGFb3, TGFb-R2, and LRRC32 was seen in ER+ cancers and correlated positively with M2 and negatively with M1 macrophage content across all cases. Table . 185PD Potential ER+ Immuno- Oncology Targets TNBC Mean Expression ER+ Mean Expression Fold-Change Mean Expression p-value FDR-adjusted IL6ST 2,717 11,815 4.35 5.13E-21 2.32E-19 TGF-b3 2,085 4,745 2.28 1.32E-24 8.97E-23 CXCR1 (IL-8 receptor A) 439 988 2.25 1.04E-09 7.46E-09 CSF3R 1,050 2,025 1.93 8.46E-12 7.67E-11 RORC 1,008 1,855 1.84 3.65E-08 2.26E-07 ADORA2A 2,163 3,442 1.59 6.18E-08 3.65E-07 LRRC32 (TGFb activator) 7,549 11,152 1.48 1.09E-07 5.31E-07 TLR3 880 1,300 1.48 1.65E-05 5.92E-05 CXCL12 1,055 1,535 1.45 8.44E-05 2.53E-04 CLEC14A 492 705 1.43 2.68E-05 9.11E-05 TGFb-R2 7,559 9,845 1.30 2.08E-04 5.90E-04 TNFSF14 799 872 1.09 2.94E-03 6.89E-03 MICA 349 374 1.07 7.54E-03 1.51E-29 NLRP3 3,015 3,145 1.04 3.12E-03 7.20E-03 JAK1 12,683 13,058 1.03 0.019 0.035 Conclusions IO drugs targeting TGFb, M2 macrophages and mast cells are attractive therapeutic candidates in immune-rich ER+ breast cancer based on the expression characteristics of these targets. Legal entity responsible for the study The authors. Funding Howard Hughes Medical Institute Medical Fellows Grant. Disclosure V. Pelenkanou: Full / Part-time employment: Sanofi, US. D. Rimm: Advisory / Consultancy: Amgen; Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Advisory / Consultancy: Agendia; Advisory / Consultancy: Biocept; Advisory / Consultancy: BMS; Advisory / Consultancy: Cell Signaling Technology; Advisory / Consultancy, Research grant / Funding (institution): Cepheid; Advisory / Consultancy: Daiichi Sankyo; Advisory / Consultancy: GSK; Advisory / Consultancy: InVicro/Konica/Minolta; Advisory / Consultancy: Merck; Advisory / Consultancy, Research grant / Funding (institution): NanoString; Advisory / Consultancy, Research grant / Funding (institution): Perkin Elmer; Advisory / Consultancy: PAIGE.AI; Advisory / Consultancy, Research grant / Funding (institution): Ventana; Advisory / Consultancy, Research grant / Funding (institution): Ultivue; Shareholder / Stockholder / Stock options: PixelGear; Research grant / Funding (institution): Navigate/Novartis; Research grant / Funding (institution): NextCure; Research grant / Funding (institution): Lilly. L. Pusztai: Honoraria (self), Advisory / Consultancy: Merck; Honoraria (self), Advisory / Consultancy: AstraZeneca; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (self), Advisory / Consultancy: Seattle Genetics; Honoraria (self), Advisory / Consultancy: Pfizer; Honoraria (self), Advisory / Consultancy: Almac. All other authors have declared no conflicts of interest.

e14180 Background: Tumor infiltrating lymphocytes (TILs) are powerful prognostic and predictive factors in TNBC. We hypothesized that survival differences in TNBC by race may be caused by differences in the tumor immune microenvironment. We assessed racial differences in the extent and composition of immune infiltration in TNBC and correlated these differences with clinical characteristics and disease-free survival (DFS). Methods: Formalin fixed paraffin embedded TNBC samples and clinical information were collected for n = 43 AA and n = 43 Caucasian cases, matched by diagnosis date and stage. Stromal TILs were assessed on H&E-stained slides. Multiplexed immunofluorescence was performed to quantify CD68 (macrophage), CD8 (cytotoxic T cell) and PD-L1 protein expression in the whole-section, tumor and stromal compartments. Average expression for each marker was calculated over all fields of view. Cox proportional hazards were used to assess associations between DFS, staining markers and clinical variables. Results: Characteristics of AA and Caucasian cases were not significantly different. There were 14 and 8 recurrences in the AA and Caucasian cohorts, respectively (median follow-up 8.7 vs 9.4 yrs). TIL counts (p = 0.031) and overall CD68 expression (p = 0.005) were higher in AA compared to Caucasian patients. 21% percent of AA cases had TIL predominant phenotype versus 3% of Caucasians, but CD8 expression was similar by race. PD-L1 expression was higher in stroma compared to tumor across all patients (median 401 vs 267 au, p = 0.002) and did not differ by race. Higher overall and stromal PD-L1 expression were associated with better DFS in the entire population (median 3501 vs 1895 days, p = 0.0009) and in each race separately. Higher CD68 expression was also associated with better DFS (median 3015 vs 2111 days, p = 0.0004). In multivariate analysis of DFS, stage at presentation remained significant (p = 0.002) in addition to PD-L1 and CD68 expression. Conclusions: AA TNBC had higher TIL counts and CD68 expression but similar CD8 and PD-L1 expression compared to Caucasians. High CD68 and PD-L1 expression were associated with better DFS in both race cohorts.

Background: Durable responses to immune checkpoint blockade against programmed cell death 1 (PD-1) are limited to a subset of metastatic melanoma patients, so there is a need for predictive biomarkers. Tumor-infiltrating lymphocytes (TILs) are the major cellular target of anti-PD-1 therapy, so we hypothesized that pretreatment TIL profiles would be associated with response. Methods: Pretreatment whole-tissue sections from 94 melanoma patients treated with anti-PD-1 therapy (pembrolizumab, nivolumab, or ipilimumab plus nivolumab) from 2011-17 were collected from Yale Pathology archives, and stained to visualize nuclei (DAPI) and melanoma cells (S100 & HMB45) in combination with two multiplex immunofluorescence panels to: (1) perform TIL quantitation of helper T cells by CD4 (SP35, Spring), cytotoxic T cells by CD8 (C8/144B, Dako), and B cells by CD20 (L26, Dako); and (2) assess TIL activation by identifying T cells by CD3 (SP7, Novus), cytolytic activity by GZMB (4E6, Abcam), and proliferation by Ki67 (MIB-1, Dako). Cell phenotyping and counting were performed using inForm software (PerkinElmer) and protein expression was measured by the AQUA method of quantitative immunofluorescence (QIF). Response Evaluation Criteria In Solid Tumors (RECIST) 1.1 were used to classify best overall response as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). Objective response rate (ORR; CR/PR), disease control rate (DCR; CR/PR/SD), and progression-free survival (PFS) were correlated with TIL parameters measured by both methodologies. Results: Pretreatment lymphocytic infiltration, by cell counts or QIF, was significantly higher in CR/PR than in SD/PD, particularly for CD3 and CD8 (P < 0.0001). Neither TIL activation (CD3 high, Ki67 and/or GZMB high) nor TIL dormancy (CD3 high, Ki67 and GZMB low) was significantly associated with outcome. Multivariable analyses revealed significant CD8 associations (HR > 3) with PFS independent of age, sex, mutation, stage, treatment, and prior immune checkpoint blockade, which accounted for similar CD3 PFS associations as expected. The favorable predictive performance of CD8 cell count (and QIF) had an area under the receiver operating characteristic (ROC) curve of 0.75 for ORR and 0.78 for DCR, which reached 0.83 (ORR or DCR) for dual therapy (ipilimumab plus nivolumab). Interestingly, in contrast to previous hypothetical classifications, there were a number of responders (CR/PR) in the CD3 or CD8 low (immune desert) category. Conclusions: Pretreatment lymphocytic infiltration, by cell counts or QIF, is associated with anti-PD-1 response in metastatic melanoma. Multiplex analysis of the tumor microenvironment has the potential to be used as a companion diagnostic test for precision immunotherapy. Citation Format: Pok Fai Wong, James W. Smithy, Kim R. Blenman, Harriet M. Kluger, David L. Rimm. Quantitative assessment of tumor-infiltrating lymphocytes and immunotherapy outcome in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3638.

Multiple studies have reported high levels of IL-10 in SLE patients and in murine models of lupus. IL-10 is a regulatory cytokine mainly produced by B cells, which use this cytokine to support their proliferation, and by myeloid cells, which use IL-10 to reduce proinflammatory responses. IL-10 is also produced by a subset of CD4+ T regulatory cells. Various manipulations of IL-10 levels with repeated administrations of anti-IL-10 neutralizing antibodies, genetic ablation or injections of recombinant cytokine have shown contradictory results, which is likely to reflect the opposite effects of this cytokine on the two major effector arms of lupus pathologenesis, namely B cells and inflammation. We have investigated the role of IL-10 in a novel congenic model of lupus, B6.Sle1.Sle2.Sle3 (B6.TC), which consists of the three NZM2410-derived SLE susceptibility loci combined on a C57BL/6 background. We first investigated in this model the source of elevated IL-10 and shown that it results from a larger number of CD4+ T cells producing the cytokine, and from a greatly increased B1-a cell pool, which is the main IL-10 producing compartment. We have then used AAV-mediated skeletal muscle gene delivery to overexpress IL-10 in young B6.TC mice and follow disease marker expression up to 7 months of age. We show here that continuous overexpression of low levels of IL-10 significantly delayed antinuclear auto-antibody production and decreased clinical nephritis. B cell phenotypes were largely unaffected, while T-cell activation was significantly reduced. This highlighted the pivotal role played by T-cell activation in this model, and suggested that this pathway could be effectively targeted for therapeutic interventions. These results also reinforce the notion that IL-10 exerts multiple functions and commend caution in equating high levels of IL-10 and increased pathogenesis in systemic autoimmunity.

The purpose of this study was to evaluate the ability to induce TNFalpha-dependent apoptosis in vivo in predisease lupus-prone NZM2410 and derived B6.NZM congenic mouse strains. An endotoxicosis model that utilizes LPS and d-galactosamine to induce mortality by TNFalpha/TNFR1-dependent hepatocyte apoptosis was used to assess TNFalpha production, apoptotic signaling, and effects on the production of IL-6 and IL-10. NZM2410 was found to be resistant to endotoxicosis and to produce significantly less TNFalpha-induced IL-6 and IL-10. At low doses of LPS, partial resistance was associated with the Tnfa(w) allele. At higher doses of LPS, partial resistance cosegregated with lupus-susceptibility loci and functionally mapped downstream of caspase 3. Additional partial resistance in NZM2410 was also found upstream of FADD. These results demonstrate the existence of multiple defects in the TNFalpha/TNFR1 signaling pathway in the NZM2410 mouse and their relevance to lupus pathogenesis is discussed.

INTRODUCTION: Talactoferrin Alfa (TLF) is a unique recombinant form of human lactoferrin. The hypothesized mechanism of action involves TLF binding to the intestinal endothelium, which induces dendritic cell maturation and cytokine release leading to infiltration of tumor with monocytes and T-lymphocytes and inhibition of tumor growth. Based on promising phase II trial results, this correlative study was undertaken to further examine immune mechanism of action of TLF in metastatic NSCLC patients. METHODS: Talactoferrin was administered orally at 1.5 gm bid weeks 1–12 with 2 weeks off on a 14-week cycle. Enrolled patients had a pathologic diagnosis of NSCLC and were previously treated with at least 2 lines of systemic treatment. Patients had a CT guided core biopsy of tumor before initiation of talactoferrin and at week 7 on TLF. Flow cytometry was performed and quantitative immunohistochemistry for immune correlates was performed on the biopsied specimens. RESULTS: Four patients with metastatic NSCLC were enrolled. The trial was halted prematurely in light of the negative phase III trial results with the compound as a single agent in NSCLC. For the 2 patients who had repeat on-treatment tumor biopsies, a consistent increase in monocytes as a percentage of total immune cells was observed. Otherwise, no clear trend of increase or decrease was observed in any other immune cell parameters compared to matched patient pre-treatment biopsies. CONCLUSION: Repeat biopsies for immune correlates by flow cytometry and quantitative immunohistochemistry in NSCLC patients are feasible. In the few patients sampled before trial closure, increased monocytes as a total percentage of the immune cell population within tumor was observed in response to TLF.