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Aim To present our innovative robotic approach for the treatment of rectus diastasis with concurrent primary or incisional ventral hernias. Material & Methods We performed 45 r-TARRD repairs for symptomatic rectus diastasis with concomitant associated ventral/incisional umbilical and/or epigastric hernias. Data on patient demographics, type of hernia, operative time, complications, recurrence rate and hospital stay were retrospectively analyzed. Follow-up was scheduled at 1 month, 6 months and 1 year after surgery. Results 45 patients (13 M, 32 F) underwent r-TARRD repair. Mean age was 54,8 years (range 31–68) and mean BMI was 26,74 Kg/m2 (range 21,1–31). Mean ASA was 2,2 (range 1–3). In all patients we used a polypropylene mesh 25×15 cm, properly shaped. Mean operative time was 192 min (range115–260). Mean hospital stay 4,2 days (range 2–7). No conversion to laparoscopy or open surgery and no major complications occurred. At 1 month follow up one mesh infection (2,22%) was observed and it was treated conservatively. Four recurrences (8,88%) were reported at 1 year follow up. Conclusions Robot-assisted TARRD repair is conceived as a novel alternative minimally invasive procedure for RD with concurrent midline defects ensuring a primary fascial defect closure and mesh implantation in a sublay position with a wide overlap. It is important to better evaluate the suture that should be used to perform the repair and multicenter studies with standardization of patient's demographics, RD characteristics and long-term follow-up outcomes are mandatory to assess the effectiveness and durability of r-TARDD repair.

Supplementary Table S1. Analyses of MSI, DNA methylation, KRAS, BRAF, and PIK3CA mutations, and neoantigen load. Supplementary Table S2. List of antibodies and fluorophores used in the immunofluorescence procedure. Supplementary Table S3. Statistical methods. Supplementary Table S4. Macrophage density, M1-like macrophage density, M2-like macrophage density, and M1:M2 macrophage density ratio in overall tissue regions and patient survival with inverse probability weighting (IPW). Supplementary Table S5. Densities of macrophage populations defined by positivity for single polarization marker in tumor intraepithelial and stromal regions and patient survival with inverse probability weighting (IPW). Supplementary Table S6. Clinical, pathological, and molecular characteristics of colorectal cancer cases according to year of diagnosis. Supplementary Table S7. Macrophage densities and M1:M2 macrophage density ratio in tumor intraepithelial and stromal regions and patient survival in strata of MSI status with inverse probability weighting (IPW). Supplementary Table S8. Macrophage densities and M1:M2 macrophage density ratio in tumor intraepithelial and stromal regions and patient survival in strata of year of diagnosis with inverse probability weighting (IPW). Figure S1. Comparison of staining patterns between multiplex immunofluorescence and standard immunohistochemistry. Figure S2. Flowchart of the cyclic immunofluorescence procedure. Figure S3. Analysis of immunofluorescence slides to quantify tumor intraepithelial and stromal macrophage densities. Figure S4. Macrophage densities across 10 TMAs. Figure S5. Fluorescence signal intensities across 10 TMAs. Figure S6. t-SNE analysis of a random sample of 0.5% of all cells based on fluorophore signal intensities shows no clear clustering according to the TMAs. Figure S7. Core-to-core correlation of macrophage densities in two randomly chosen cores of tumors with two or more cores. Figure S8. Inverse probability weighting-adjusted Kaplan-Meier survival curves of colorectal cancer survival according to the ordinal quartile categories (Q1-Q4) of intraepithelial (A) and stromal (B) macrophage densities. Figure S9. Forest plots of inverse probability weighting-adjusted Cox regression models of colorectal cancer specific survival according to the densities of intraepithelial and stromal M1-like polarized macrophages, with M1-like macrophages defined using different cut-offs (10-50%) of the M1-end of the macrophage polarization (M1:M2) index distribution. Figure S10. Forest plots of inverse probability weighting-adjusted Cox regression models of colorectal cancer specific survival according to the densities of intraepithelial and stromal M2-like polarized macrophages, with M2-like macrophages defined using different cut-offs (10-50%) of the M2-end of the macrophage polarization (M1:M2) index distribution. Figure S11. Forest plots of inverse probability weighting-adjusted Cox regression models of colorectal cancer specific survival according to the intraepithelial and stromal M1:M2-density ratio with M1-like and M2-like macrophages defined using different cut-offs (10-50%) of both tails of the macrophage polarization (M1:M2) index distribution. Figure S12. Densities of M1-like and M2-like macrophages in relation to histologic lymphocytic reaction patterns.

Macrophages are among the most common cells in the colorectal cancer microenvironment, but their prognostic significance is incompletely understood. Using multiplexed immunofluorescence for CD68, CD86, IRF5, MAF, MRC1 (CD206), and KRT (cytokeratins) combined with digital image analysis and machine learning, we assessed the polarization spectrum of tumor-associated macrophages in 931 colorectal carcinomas. We then applied Cox proportional hazards regression to assess prognostic survival associations of intraepithelial and stromal densities of M1-like and M2-like macrophages while controlling for potential confounders, including stage and microsatellite instability status. We found that high tumor stromal density of M2-like macrophages was associated with worse cancer-specific survival, whereas tumor stromal density of M1-like macrophages was not significantly associated with better cancer-specific survival. High M1:M2 density ratio in tumor stroma was associated with better cancer-specific survival. Overall macrophage densities in tumor intraepithelial or stromal regions were not prognostic. These findings suggested that macrophage polarization state, rather than their overall density, was associated with cancer-specific survival, with M1- and M2-like macrophage phenotypes exhibiting distinct prognostic roles. These results highlight the utility of a multimarker strategy to assess the macrophage polarization at single-cell resolution within the tumor microenvironment.

Copy number variation (CNV) of samples in TCGA

ORF overexpression screen data

Supplementary detailed methods

DEDD and MCL1 overexpression are not necessary for initiation of tumorigenesis.

Effective therapies for non–small cell lung cancer (NSCLC) remain challenging despite an increasingly comprehensive understanding of somatically altered oncogenic pathways. It is now clear that therapeutic agents with potential to impact the tumor immune microenvironment potentiate immune-orchestrated therapeutic benefit. Herein, we evaluated the immunoregulatory properties of histone deacetylase (HDAC) and bromodomain inhibitors, two classes of drugs that modulate the epigenome, with a focus on key cell subsets that are engaged in an immune response. By evaluating human peripheral blood and NSCLC tumors, we show that the selective HDAC6 inhibitor ricolinostat promotes phenotypic changes that support enhanced T-cell activation and improved function of antigen-presenting cells. The bromodomain inhibitor JQ1 attenuated CD4+FOXP3+ T regulatory cell suppressive function and synergized with ricolinostat to facilitate immune-mediated tumor growth arrest, leading to prolonged survival of mice with lung adenocarcinomas. Collectively, our findings highlight the immunomodulatory effects of two epigenetic modifiers that, together, promote T cell–mediated antitumor immunity and demonstrate their therapeutic potential for treatment of NSCLC.Significance: Selective inhibition of HDACs and bromodomain proteins modulates tumor-associated immune cells in a manner that favors improved T-cell function and reduced inhibitory cellular mechanisms. These effects facilitated robust antitumor responses in tumor-bearing mice, demonstrating the therapeutic potential of combining these epigenetic modulators for the treatment of NSCLC. Cancer Discov; 7(8); 852–67. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 783

Supplementary Table 1. HDAC inhibitors tested with healthy donor PBMCs and their biochemical potency (nM) across HDACs 1, 2, 3, and 6. Supplementary Table 2. Information for consented Non-small cell lung cancer (NSCLC) patients that underwent surgical resectioning as part of their treatment plan and whose tumor specimen and blood samples obtained after surgery were analyzed. Supplementary Figure 1. Reduced CD4+FOXP3+ Treg cells in healthy donor and NSCLC patient PBMC in the presence of ricolinostat. Supplementary Figure 2. Up-regulation of CD69 on T cells in healthy donor PBMC cultures in the presence of ricolinostat. Supplementary Figure 3. Viability of immune cells within dissociated tumor specimens cultured in the presence of ricolinostat or entinostat. Supplementary Figure 4. Effector function of T cells within 2-D cultures of disaggregated tumor specimens from NSCLC patients. Supplementary Figure 5. Increased expression of MHC class II and CD86 on monocytes in healthy donor PBMC in the presence of ricolinostat. Supplementary Figure 6. Phenotype of T cells infiltrating lung tumors of genetically engineered mice treated with ricolinostat. Supplementary Figure 7. Gene expression profile of Tumor-infiltrating T cells. Supplementary Figure 8. Ricolinostat promotes up-regulation of MHC class II and CD86 on tumor-associated macrophages. Supplementary figure 9. Immunohistochemical and flow cytometric analyses of acetylated α-tubulin in lung tumors of KP mice. Supplementary Figure 10. Kinetics of tumor growth in KP mice treated with ricolinostat. Supplementary Figure 11. Phenotype of tumor-infiltrating T cell subsets in lung tumors of KP mice treated with JQ1. Supplementary Figure 12. Immunohistochemical and flow cytometric analyses of phospho-STAT5 levels in lung tumors of KP mice. Supplementary Figure 13. Suppressive function of Tregs isolated from the spleen of lung tumor-bearing KP mice treated with JQ1. Supplementary Figure 14. Phenotype of Tregs in the spleen or lung tumors of genetically engineered mouse models (GEMM) of NSCLC. Supplementary Figure 15. Phenotype of Tregs within cultures of dissociated tumor specimen from NSCLC patients. Supplementary Figure 17. Histology and immunohistochemical staining of lung tumor sections from treated KP mice. Supplementary Figure 18. Kinetics of tumor growth in TL or wild-type mice. Supplementary Figure 19. Quantification of T cell subsets and TAMs in tumors of KP mice. Supplementary Figure 20. Phenotype of CD8+T cells infiltrating lung tumors of treated KP mice. Supplementary Figure 21. Gene expression profile of tumor-infiltrating macrophages (TAMs) in treated KP mice. Supplementary Figure 22. Gene expression profile of tumor-infiltrating macrophages T cells in treated KP mice. Supplementary Figure 23. Proportion of CD4+Foxp3+ Tregs present within tumor-infiltrating T cells utilized in gene expression studies. Supplementary Figure 24. Gating strategy. Supplementary Figure 25. Correlation plot for the expression of indicated genes in CD45+ leukocytes in KP tumors as evaluated by single cell RNA-Sequencing.

Supplementary Figure 1. In-vivo mouse model's schema; Supplementary Figure 2. Total BLI Flux of SCID.beige myeloma xenograft model and Kaplan-Meier survival analysis of the NSG xenograft model; Supplementary Figure 3. Elotuzumab does not affect myeloma cell proliferation and homing to the bone marrow.

Focal amplification of chromosome 1q23.3 in patients with advanced primary or relapsed urothelial carcinomas is associated with poor survival. We interrogated chromosome 1q23.3 and the nearby focal amplicon 1q21.3, as both are associated with increased lymph node disease in patients with urothelial carcinoma. Specifically, we assessed whether the oncogene MCL1 that resides in 1q21.3 and the genes that reside in the 1q23.3 amplicon were required for the proliferation or survival of urothelial carcinoma. We observed that suppressing MCL1 or the death effector domain–containing protein (DEDD) in the cells that harbor amplifications of 1q21.3 or 1q23.3, respectively, inhibited cell proliferation. We also found that overexpression of MCL1 or DEDD increased anchorage independence growth in vitro and increased experimental metastasis in vivo in the nonamplified urothelial carcinoma cell line, RT112. The expression of MCL1 confers resistance to a range of apoptosis inducers, while the expression of DEDD led to resistance to TNFα-induced apoptosis. These observations identify MCL1 and DEDD as genes that contribute to aggressive urothelial carcinoma.Implications:These studies identify MCL1 and DEDD as genes that contribute to aggressive urothelial carcinomas.

High-grade serous ovarian cancer (HGSOC) is often sensitive to initial treatment with platinum and taxane combination chemotherapy, but most patients relapse with chemotherapy-resistant disease. To systematically identify genes modulating chemotherapy response, we performed pooled functional genomic screens in HGSOC cell lines treated with cisplatin, paclitaxel, or cisplatin plus paclitaxel. Genes in the intrinsic pathway of apoptosis were among the top candidate resistance genes in both gain-of-function and loss-of-function screens. In an open reading frame overexpression screen, followed by a mini-pool secondary screen, anti-apoptotic genes including BCL2L1 (BCL-XL) and BCL2L2 (BCL-W) were associated with chemotherapy resistance. In a CRISPR-Cas9 knockout screen, loss of BCL2L1 decreased cell survival whereas loss of proapoptotic genes promoted resistance. To dissect the role of individual anti-apoptotic proteins in HGSOC chemotherapy response, we evaluated overexpression or inhibition of BCL-2, BCL-XL, BCL-W, and MCL1 in HGSOC cell lines. Overexpression of anti-apoptotic proteins decreased apoptosis and modestly increased cell viability upon cisplatin or paclitaxel treatment. Conversely, specific inhibitors of BCL-XL, MCL1, or BCL-XL/BCL-2, but not BCL-2 alone, enhanced cell death when combined with cisplatin or paclitaxel. Anti-apoptotic protein inhibitors also sensitized HGSOC cells to the poly (ADP-ribose) polymerase inhibitor olaparib. These unbiased screens highlight anti-apoptotic proteins as mediators of chemotherapy resistance in HGSOC, and support inhibition of BCL-XL and MCL1, alone or combined with chemotherapy or targeted agents, in treatment of primary and recurrent HGSOC.Implications:Anti-apoptotic proteins modulate drug resistance in ovarian cancer, and inhibitors of BCL-XL or MCL1 promote cell death in combination with chemotherapy.

Elotuzumab, a recently approved antibody for the treatment of multiple myeloma, has been shown to stimulate Fcγ receptor (FcγR)-mediated antibody-dependent cellular cytotoxicity by natural killer (NK) cells toward myeloma cells. The modulatory effects of elotuzumab on other effector cells in the tumor microenvironment, however, has not been fully explored. Antibody-dependent cellular phagocytosis (ADCP) is a mechanism by which macrophages contribute to antitumor potency of monoclonal antibodies. Herein, we studied the NK cell independent effect of elotuzumab on tumor-associated macrophages using a xenograft tumor model deficient in NK and adaptive immune cells. We demonstrate significant antitumor efficacy of single-agent elotuzumab in immunocompromised xenograft models of multiple myeloma, which is in part mediated by Fc–FcγR interaction of elotuzumab with macrophages. Elotuzumab is shown in this study to induce phenotypic activation of macrophages in vivo and mediates ADCP of myeloma cells though a FcγR-dependent manner in vitro. Together, these findings propose a novel immune-mediated mechanism by which elotuzumab exerts anti-myeloma activity and helps to provide rationale for combination therapies that can enhance macrophage activity. Mol Cancer Ther; 17(7); 1454–63. ©2018 AACR.

Supplementary Table 3

List of differentially present metabolites. The table lists fold change value of metabolites differentially detected in CAF co-cultures compared to separately cultured cells in at least one of the cell lines. Bold font indicates statistically significant (p

Supplementary Tables S1-5 and Supplementary Fig. S1

Using a three-dimensional coculture model, we identified significant subtype-specific changes in gene expression, metabolic, and therapeutic sensitivity profiles of breast cancer cells in contact with cancer-associated fibroblasts (CAF). CAF-induced gene expression signatures predicted clinical outcome and immune-related differences in the microenvironment. We found that fibroblasts strongly protect carcinoma cells from lapatinib, attributable to its reduced accumulation in carcinoma cells and an elevated apoptotic threshold. Fibroblasts from normal breast tissues and stromal cultures of brain metastases of breast cancer had similar effects as CAFs. Using synthetic lethality approaches, we identified molecular pathways whose inhibition sensitizes HER2+ breast cancer cells to lapatinib both in vitro and in vivo, including JAK2/STAT3 and hyaluronic acid. Neoadjuvant lapatinib therapy in HER2+ breast tumors lead to a significant increase of phospho-STAT3+ cancer cells and a decrease in the spatial proximity of proliferating (Ki67+) cells to CAFs impacting therapeutic responses. Our studies identify CAF-induced physiologically and clinically relevant changes in cancer cells and offer novel approaches for overcoming microenvironment-mediated therapeutic resistance. Cancer Res; 76(22); 6495–506. ©2016 AACR.

Supplemental methods, results, figures, tables, and references

Figure S1. REMARK diagram Figure S2. NanoString Tumor Inflammation Signature by breast cancer intrinsic subtype Figure S3. NanoString individual gene expression changes by cohort Table S1. Pre-treatment immune microenvironment and response to preoperative therapy Table S2. Tissue-based immune biomarkers in patients with pathologic complete response Table S3. References for classification of genes as M1-like or M2-like

Purpose:Hormone receptor–positive/HER2-negative (HR+/HER2−) breast cancer is associated with low levels of stromal tumor-infiltrating lymphocytes (sTIL) and PD-L1, and demonstrates poor responses to checkpoint inhibitor therapy. Evaluating the effect of standard chemotherapy on the immune microenvironment may suggest new opportunities for immunotherapy-based approaches to treating HR+/HER2− breast tumors.Experimental Design:HR+/HER2− breast tumors were analyzed before and after neoadjuvant chemotherapy. sTIL were assessed histologically; CD8+ cells, CD68+ cells, and PD-L1 staining were assessed immunohistochemically; whole transcriptome sequencing and panel RNA expression analysis (NanoString) were performed.Results:Ninety-six patients were analyzed from two cohorts (n = 55, Dana-Farber cohort; n = 41, MD Anderson cohort). sTIL, CD8, and PD-L1 on tumor cells were higher in tumors with basal PAM50 intrinsic subtype. Higher levels of tissue-based lymphocyte (sTIL, CD8, PD-L1) and macrophage (CD68) markers, as well as gene expression markers of lymphocyte or macrophage phenotypes (NanoString or CIBERSORT), correlated with favorable response to neoadjuvant chemotherapy, but not with improved distant metastasis-free survival in these cohorts or a large gene expression dataset (N = 302). In paired pre-/postchemotherapy samples, sTIL and CD8+ cells were significantly decreased after treatment, whereas expression analyses (NanoString) demonstrated significant increase of multiple myeloid signatures. Single gene expression implicated increased expression of immunosuppressive (M2-like) macrophage-specific genes after chemotherapy.Conclusions:The immune microenvironment of HR+/HER2− tumors differs according to tumor biology. This cohort of paired pre-/postchemotherapy samples suggests a critical role for immunosuppressive macrophage expansion in residual disease. The role of macrophages in chemoresistance should be explored, and further evaluation of macrophage-targeting therapy is warranted.

Purpose:Increasing tumor-infiltrating lymphocytes (TIL) is associated with higher rates of pathologic complete response (pCR) to neoadjuvant therapy (NAT) in patients with triple-negative breast cancer (TNBC). However, the presence of TILs does not consistently predict pCR, therefore, the current study was undertaken to more fully characterize the immune cell response and its association with pCR.Experimental Design:We obtained pretreatment core-needle biopsies from 105 patients with stage I–III TNBC enrolled in ARTEMIS (NCT02276443) who received NAT from Oct 22, 2015 through July 24, 2018. The tumor-immune microenvironment was comprehensively profiled by performing T-cell receptor (TCR) sequencing, programmed death-ligand 1 (PD-L1) IHC, multiplex immunofluorescence, and RNA sequencing on pretreatment tumor samples. The primary endpoint was pathologic response to NAT.Results:The pCR rate was 40% (42/105). Higher TCR clonality (median = 0.2 vs. 0.1, P = 0.03), PD-L1 positivity (OR: 2.91, P = 0.020), higher CD3+:CD68+ ratio (median = 14.70 vs. 8.20, P = 0.0128), and closer spatial proximity of T cells to tumor cells (median = 19.26 vs. 21.94 μm, P = 0.0169) were associated with pCR. In a multivariable model, closer spatial proximity of T cells to tumor cells and PD-L1 expression enhanced prediction of pCR when considered in conjunction with clinical stage.Conclusions:In patients receiving NAT for TNBC, deep immune profiling through detailed phenotypic characterization and spatial analysis can improve prediction of pCR in patients receiving NAT for TNBC when considered with traditional clinical parameters.

Purpose:We report results from a phase II study assessing the efficacy of the WEE1 inhibitor adavosertib with cisplatin in metastatic triple-negative breast cancer (mTNBC).Patients and Methods:Patients with mTNBC treated with 0–1 prior lines of chemotherapy received cisplatin 75 mg/m2 i.v. followed 21 days later by cisplatin plus adavosertib 200 mg oral twice daily for five doses every 21 days. The study had 90% power to detect the difference between null (20%) and alternative (40%) objective response rates (ORR) with a one-sided type I error of 0.1: an ORR >30% was predefined as making the regimen worthy of further study. RNA sequencing and multiplex cyclic immunofluorescence on pre- and post-adavosertib tumor biopsies, as well as targeted next-generation sequencing on archival tissue, were correlated with clinical benefit, defined as stable disease ≥6 months or complete or partial response.Results:A total of 34 patients initiated protocol therapy; median age was 56 years, 2 patients (6%) had BRCA2 mutations, and 14 (41%) had one prior chemotherapy. ORR was 26% [95% confidence interval (CI), 13–44], and median progression-free survival was 4.9 months (95% CI, 2.3–5.7). Treatment-related grade 3–5 adverse events occurred in 53% of patients, most commonly diarrhea in 21%. One death occurred because of sepsis, possibly related to study therapy. Tumors from patients with clinical benefit demonstrated enriched immune gene expression and T-cell infiltration.Conclusions:Among patients with mTNBC treated with 0–1 prior lines, adavosertib combined with cisplatin missed the prespecified ORR cutoff of >30%. The finding of immune-infiltrated tumors in patients with clinical benefit warrants validation.

Supplementary Figures, Detailed Methods, and Supplementary References

Supplementary Figure Legend from Chemotherapy Induces Tumor Clearance Independent of Apoptosis

Supplementary Figure 1 from Chemotherapy Induces Tumor Clearance Independent of Apoptosis

Recently developed inhibitors of polymerase theta (POLθ) have demonstrated synthetic lethality in BRCA-deficient tumor models. To examine the contribution of the immune microenvironment to antitumor efficacy, we characterized the effects of POLθ inhibition in immunocompetent models of BRCA1-deficient triple-negative breast cancer (TNBC) or BRCA2-deficient pancreatic ductal adenocarcinoma (PDAC). We demonstrate that genetic POLQ depletion or pharmacological POLθ inhibition induces both innate and adaptive immune responses in these models. POLθ inhibition resulted in increased micronuclei, cGAS/STING pathway activation, type I interferon gene expression, CD8+ T cell infiltration and activation, local paracrine activation of dendritic cells and upregulation of PD-L1 expression. Depletion of CD8+ T cells compromised the efficacy of POLθ inhibition, whereas antitumor effects were augmented in combination with anti-PD-1 immunotherapy. Collectively, our findings demonstrate that POLθ inhibition induces immune responses in a cGAS/STING-dependent manner and provide a rationale for combining POLθ inhibition with immune checkpoint blockade for the treatment of HR-deficient cancers.

Background: Platinum agents induce DNA crosslinking and cause accumulation of genotoxic stress, which leads to immune activation via IFN-γ signaling, making the combination with nivolumab (PD-1 antibody) an attractive strategy to enhance the benefit of either agent alone in metastatic triple-negative breast cancer (mTNBC). Methods: In this phase II open-label, investigator-initiated, multicenter trial, patients with unresectable locally advanced or mTNBC treated with 0-1 prior lines of chemotherapy in the metastatic setting were randomized 1:1 to carboplatin (AUC 6) with or without nivolumab (360 mg) IV every 3 weeks. Stratification factors included: germline BRCA (gBRCA) status, prior neo/adjuvant platinum, and number of prior lines of metastatic therapy. After approval of PD-L1 inhibition for mTNBC, the study was amended to include first-line mTNBC only and PD-L1 status was added as a stratification factor. Patients randomized to carboplatin alone were allowed to crossover at progression to receive nivolumab (+ nab-paclitaxel post-amendment). The primary objective was to compare progression-free survival (PFS) per RECIST 1.1 criteria of carboplatin with or without nivolumab in first-line mTNBC in the intent-to-treat (ITT) population. Key secondary objectives were objective response rate (ORR), overall survival (OS), clinical benefit rate, and duration and time to objective response. PD-L1 status was confirmed centrally using the SP142 Ventana assay (positive, ≥1% IC). Paired research biopsies at baseline, on-treatment and at progression were performed, if safely accessible. The trial closed to accrual prior to reaching target accrual due to approval of PD-1 inhibition in combination with platinum-based chemotherapy for PD-L1+ mTNBC. Results: Between 1/30/2018 and 12/9/2020, 78 patients enrolled. Three patients did not receive protocol treatment, and the safety analysis was conducted among the 75 that received any treatment; 37 received carboplatin + nivolumab (Arm A), 38 received carboplatin alone (Arm B). Median age was 59.1 yrs (range: 25.4-75.8). Four patients (5.3%) had a known gBRCA1/2 mutation. Sixty-two (82.7%) patients received 0 prior lines (ITT population) and 13 (17.3%) 1 prior line of metastatic therapy. Sixty-seven patients (89.3%) experienced any grade ≥2 treatment-related adverse event (AE). The most frequent AE were platelet count decrease (n=40; 53.3%), anemia (n=36; 48.0%), neutrophil count decrease (n=33; 44.0%) and fatigue (n=24; 32.0%). Grade 3/4 AE were observed in 46 (61.3%) patients, and there was one grade 5 AE (COVID19 pneumonia). Any grade ≥2 immune-related AE (irAE) were observed in 25 of the 37 (67.6%) patients treated with carboplatin + nivolumab. Grade 3/4 irAE were observed in 11 (29.7%) patients. In the ITT population (32 on Arm A; 30 on Arm B), median PFS was 4.2 months with carboplatin + nivolumab, and 5.5 months with carboplatin (stratified HR 0.98, 95% CI [0.51 - 1.88]; p=0.95). ORR was 25% vs. 23.3%, respectively. At a median follow-up of 23.5 months, median OS was 17.5 months vs. 10.7 months (stratified HR 0.63, 95% CI [0.32 - 1.24]; p=0.18). In patients with PD-L1+ mTNBC (13 on Arm A; 11 on Arm B), median PFS was 8.3 months and 4.7 months, respectively (stratified HR 0.63, 95% CI [0.21 - 1.89]; p=0.41). ORR was 23.1% vs. 27.3%, respectively. Median OS was 17.5 months vs. 9.6 months (stratified HR 0.59, 95% CI [0.20 - 1.75]; p=0.34). Conclusions: Addition of nivolumab to carboplatin in patients with previously untreated mTNBC, unselected by PD-L1 status, did not significantly improve PFS. A trend toward improved PFS and OS was observed in patients with PD-L1+ mTNBC. Tissue, blood and intestinal microbiome biomarker analyses are planned; bulk tumor and single-cell sequencing, and TCR sequencing in peripheral blood are ongoing. Clinical trial information: NCT03414684. Citation Format: Ana C Garrido-Castro, Noah Graham, Kevin Bi, Jihye Park, Jingxin Fu, Tanya Keenan, Edward Thomas Richardson, Ricardo Pastorello, Paulina Lange, Victoria Attaya, Robert Wesolowski, Natalie Sinclair, Zarah Lucas, Steve Lo, Nadine Tung, Meredith Faggen, Peter A Kaufman, Caroline C Block, Fred Briccetti, Madhavi Toke, Wendy Chen, Kai Wucherpfennig, Sascha Marx, Ye Tian, Judith Agudo, Jennifer L Guerriero, Stuart Schnitt, Nancy U Lin, Eric P Winer, Elizabeth A Mittendorf, Nabihah Tayob, Eliezer Van Allen, Sara M Tolaney. A randomized phase II trial of carboplatin with or without nivolumab in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-14-18.

Background: Although immune checkpoint inhibitors (ICI) are most widely used in PD-L1 positive, metastatic triple-negative breast cancer (TNBC), the clinical utility of ICI in other tumor subtypes and patient populations is not well defined. In this study, we measured infiltration of multiple immune cell populations in primary and metastatic HR+ tumors to infer potential benefit from ICI. We investigated how risk stratifying features such as pathologic grade and Oncotype Dx scores affect immune cell infiltration across subtypes. Lastly, we compared immune cell infiltration in breast tumors from younger versus older women, as our pre-clinical studies demonstrated differential enrichment of immune response pathways by age and association with improved response to ICI. Methods: ImmunoProfile, a multiplexed immunofluorescence (IF) assay, was performed on FFPE tissue from 167 archived breast cancer tumors from the Dana-Farber Cancer Institute (HR+/HER2- n=124, HER2+ n=21, TNBC n=22). Unmatched samples were obtained from either primary breast (HR+/HER2- n=104, HER2+ n=13, TNBC n=15) or metastatic core biopsies. IF staining signal was measured for T cell markers CD8 and co-localized CD8/PD-1, T regulatory cell marker FOXP3, PD-L1 and PD1 expression on tumor cells as well as PD-L1 expression on inflammatory cells. Inflammatory, tumoral (TPS) and combined inflammatory and tumor (CPS) pathologic PD-L1 scores were calculated. PD-L1 staining was also assessed by IHC (E1L3N) staining and compared to multiplexed IF stains. IF staining signal (Vectra analyzed, log2+1 transformed) was analyzed against clinical and pathologic variables, including tumor subtype, primary versus metastatic status, pathologic grade, and Oncotype Dx score, using non-parametric statistical comparisons. Given the decline in adaptive immunity that occurs with normal physiological aging and our own group’s previous reports, we also explored whether immune cell staining signal associated with age. Results: TPS, CPS, and inflammatory PD-L1 scores strongly correlated between IHC and IF staining methods (Spearman r = 0.660, 0.683, 0.638 respectively, p < 2.2e-16). When HR+/HER2- (n=124), HER2+ (n=21) and TNBC (n=22) cases were analyzed separately, there was no statistical difference between unmatched primary and metastatic cases in any of the analyzed immune cell population stains for any subtype. When all primary cases were analyzed, TNBC showed higher CD8, CD8/PD1, PD1 (all p < 0.05), and FOXP3 staining as well as IF CPS, TPS and inflammatory PD-L1 scores (all p < 0.005) than HR+ cases. HR+/HER2- cases from women < age 65 (n=56) showed higher intra-tumoral CD8 (p=0.039), PD1 (p=0.043), and CD8/PD1 (p=0.022) than cases from women > age 65 (n=38). There was a statistically significant step-wise increase in IF staining of all immune cell populations with increasing pathologic grade (I, II, III) as well as Oncotype Dx scores (< 11, 11-25, >25) among primary HR+/HER2- cases (n=74). Conclusions: Strong correlations between PD-L1 scores based on IF and IHC methods suggest clinical utility for multiplexed IF immune cell staining in therapeutic planning. ImmunoProfile demonstrated the previously known pattern that primary TNBC contains higher immune cell infiltration than HR+ or HER2+ tumors. Women < age 65 with HR+ cancer showed higher levels of CD8 and PD1 stained populations, suggesting there may be greater utility of ICI in younger patients. Lastly, the association of higher pathologic grade and Oncotype Dx score with increasing immune cell infiltration supports exploration of ICI in biologically unfavorable primary HR+ cancers. Citation Format: Tess A. O'Meara, Tanya E. Keenan, Adrienne G. Waks, Kristen D. Felt, Bijaya Sharma, Scott Rodig, Melissa Hughes, Nancy U. Lin, Judith Agudo, Jennifer L. Guerriero, Sandra S. McAllister, Elizabeth Mittendorf, Sara Tolaney. Multiplexed immunofluorescence staining of intra-tumoral immune cell populations and associations with immunohistochemical, clinical, and pathologic variables in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-05.

Background: Immunological differences between HER2+ breast tumors that are HR+ versus HR- have not been widely explored. DAPHNe was a single-arm prospective clinical trial enrolling patients with clinical anatomic stage II-III HER2+ breast cancer to neoadjuvant treatment with THP. Here, we examine differential expression of immune-related proteins between HR+/HER2+ and HR-/HER2+ tumors in pre-treatment biopsies from trial participants, and analyze correlates of pathologic response to THP, overall and by HR status. Methods: A baseline research biopsy was required in all participants prior to initiation of neoadjuvant therapy. All patients received 12 weeks of neoadjuvant THP; 5 patients also received neoadjuvant AC given incomplete clinical response to THP, and are excluded from residual cancer burden (RCB) analyses. The trial primary endpoint, feasibility of de-escalation to antibody doublet therapy only (HP) following pCR, was previously reported. Protein expression profiling of baseline research biopsies was performed on the NanoString GeoMx® platform using a 58-protein immune cell profiling panel. Regions of interest (ROI; up to 12 per slide) were identified by a pathologist based on tumor cell, immune cell, and/or T cell presence, and all ROI were segmented into tumor vs stroma. Protein data quality controls were normalized by isotypes. Linear mixed effect models were used for differential expression comparisons; p-values were estimated using Satterthwaite’s method. Results: 97 pre-treatment breast tumor specimens were analyzed: 64 patients had HR+ tumors, 32 HR-, 1 unknown; 67 patients had favorable (RCB 0/1) response to preop therapy, 30 patients had unfavorable response (RCB 2/3). Multiple significant differences were observed between protein expression in HR+ versus HR- tumors (Table 1). Multiple immune cell types, as well as checkpoint proteins PD-L1 and IDO1, were higher in HR- patients, whereas checkpoint proteins B7-H3 and TIM3 were higher in HR+ patients. In the overall population, higher HER2 and PD-L1 expression were significant predictors of favorable RCB response, while higher ER alpha and Bcl-2 expression were significant predictors of unfavorable RCB response (Table 2). Conclusions: As anticipated, the strength of HER2 expression and ER expression were the most significant predictors of favorable and unfavorable RCB response to neoadjuvant THP, respectively. This highly multiplexed protein expression analysis demonstrated significant differences between the immune microenvironment of HR+ and HR- HER2+ breast tumors, implying that distinct immunological targets should be explored in the treatment of HER2+ breast cancer according to HR status. Table 1.Differences between HR+/HER2+ and HR-/HER2+ patientsStroma compartmentTumor compartmentHigher in HR+ER alpha* (fold-change 2.25)ER alpha* (8.94)Fibronectin (1.68)Bcl-2* (1.98)B7-H3 (1.44)Fibronectin* (1.90)PR (1.69)TIM-3 (1.37)Higher in HR-CD27* (fold-change 1.36)IDO1 (1.50)IDO1 (1.77)S100B (1.44)CD45 (1.45)MART1 (1.33)CD3 (1.35)CD20 (1.34)STING (1.33)CD4 (1.30)ICOS (1.30)CD45RO (1.28)CD40 (1.28)CD127 (1.27)VISTA (1.25)PD-L1 (1.24)4-1BB (1.21)*FDR p-value < 0.05 (otherwise, p-value < 0.05). Table 2: Predictors of RCB response Overall populationHigher in RCB 0/1HER2* (fold-change 3.75)PD-L1 (1.25)Higher in RCB 2/3ER alpha* (2.44)Bcl-2 (1.46)HR+/HER2+ patientsStroma compartmentTumor compartmentHigher in RCB 0/1HER2* (fold-change 4.28)HER2* (4.21)CTLA4 (1.64)Higher in RCB 2/3Bcl-2* (2.18)*FDR p-value < 0.05 (otherwise, p-value < 0.05).HR-/HER2+ patients are not shown in Table 2 as only 2 HR- patients had unfavorable RCB response. Citation Format: Adrienne G. Waks, Ying Huang, Tyler Hether, Esther R Ogayo, Sapana Kadel, Jillian Alberti, Sara M Tolaney, Ian E Krop, Prajan Divakar, Otto Metzger, Madison L O'Donnell, Sarah Church, Eric P Winer, Jennifer L. Guerriero, Elizabeth A Mittendorf. Correlation of immune-related protein expression with hormone receptor (HR) status and pathologic response to neoadjuvant paclitaxel/trastuzumab/pertuzumab (THP) among patients with early-stage HER2+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-07-03.

Background: TOPACIO was a phase I/II study evaluating the PARP inhibitor (PARPi) niraparib in combination with the anti-PD-1 antibody pembrolizumab in patients with locally advanced and metastatic triple-negative breast cancer (TNBC, n=55) and ovarian cancer irrespective of BRCA mutation status. In the efficacy-evaluable population (n=47) the objective response rate (ORR) was 21% and disease control rate (DCR) 49%. Although activity was greater in patients with BRCA mutations (7/15, ORR=47% and 12/15, DCR=80%), durable clinical benefit was seen in patients with wild-type BRCA tumors (3/27, ORR=11% and 9/27, DCR=33%). In a limited cohort of 20 patients with durable clinical benefit, there were 8 BRCA wildtype patients, four of whom had mutations in genes associated with the homologous recombination repair and other DNA damage repair pathways. Pre-treatment tissues were collected and evaluated for tumor PD-L1 status. Patients with PD-L1 positive tumors (28/47, 60%) had a higher response rate (9/28, ORR=32%) than those with PD-L1 negative tumors (1/13, ORR=8%; 6 tumors had unknown PD-L1 status). It remains unstudied whether the tumor’s gene expression profile or immune status in baseline biospecimens is predictive of treatment response. In this study we conducted exploratory biomarker analyses to test the hypothesis that gene expression patterns and immune status are associated with treatment response. Methods: Transcriptional profiling of baseline samples was performed using the BC360 (n=41) and PanCancer IO360 (n=42) panels (Nanostring) and multigene signatures were used to measure tumor and immune activities as well as relative immune cell abundance. Transcriptional analysis was paired with high-dimensional, single-cell cyclic immunofluorescence (CyCIF) of samples that had adequate tissue for analysis (n=19) to characterize the composition and topology of the immune microenvironment at single-cell resolution. Results: Nanostring transcriptional analysis revealed that PAM50 genes stratified tumor samples into 4 subgroups with distinct histology as determined by CyCIF. Each subgroup was capable of responding to niraparib plus pembrolizumab. Multiple genes involved in WNT signaling (WNT5B, TANKS1, TANKS2, PARP4, and NET02) were associated with favorable clinical responses. Low neuropilin and tolloid-like protein 2 (NETO2) gene expression was strongly correlated with favorable progression free survival (PFS; R=-0.61, p=0.0008, Spearman’s correlation), suggesting it may be a predictive biomarker of therapeutic response. Nanostring gene expression signatures for tumor inflammation, apoptosis, and inflammatory chemokines also distinguished responders from non-responders (p Citation Format: Jennifer L Guerriero, Gregory J Baker, Jia-Ren Lin, Yu-An Chen, Ricardo Pastorello, Tuulia Vallius, Janae Davis, Clarence Yapp, Sarah E Church, Eric Miller, Anniina Färkkilä, Shaveta Vinayak, Melinda L Telli, Giulia Fulci, Alan D'Andrea, Geoffrey I Shapiro, Sara M Tolaney, Sandro Santagata, Peter K Sorger, Elizabeth A Mittendorf. High-dimensional, single-cell analysis and transcriptional profiling reveal novel correlatives of response to PARP inhibition plus PD-1 blockade in triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-07-13.

PURPOSE: Parents of neonates with seizures are at risk of mental health symptoms due to the impact of illness on family life, prognostic uncertainty, and the emotional toll of hospitalization. A family-centered approach is the preferred model to mitigate these challenges. We aimed to identify strategies to promote family-centered care through an analysis of parent-offered advice to clinicians caring for neonates with seizures. BASIC PROCEDURES: This prospective, observational, and multi-center (Neonatal Seizure Registry) study enrolled parents of neonates with acute symptomatic seizures. Parents completed surveys about family well-being at 12 months, 18 months, and 24 months corrected gestational age. Parents were asked open-ended questions eliciting their advice to clinicians caring for neonates with seizures. Responses were analyzed using a conventional content analysis approach. MAIN FINDINGS: Among the 310 parents who completed surveys, 118 (38%) shared advice for clinicians. These parents were predominantly mothers (n=103, 87%). Three overarching themes were identified. (1) Communicate information effectively: Parents appreciate when clinicians offer transparent and balanced information in an accessible way. (2) Understand and validate parent experience: Parents value clinicians who display empathy, compassion, and a commitment to parent-partnered clinical care. (3) Provide support and resources: Parents benefit from emotional support, education, connection with peers, and help navigating the healthcare system. PRINCIPAL CONCLUSIONS: Parents caring for neonates with seizures appreciate a family-centered approach in healthcare encounters, including skilled communication, understanding and validation of the parent experience, and provision of support and resources. Future interventions should focus on building structures to reinforce these priorities.

Immune checkpoint inhibitors (ICIs) have minimal therapeutic effect in hormone receptor-positive (HR+ ) breast cancer. We present final overall survival (OS) results (n = 88) from a randomized phase 2 trial of eribulin ± pembrolizumab for patients with metastatic HR+ breast cancer, computationally dissect genomic and/or transcriptomic data from pre-treatment tumors (n = 52) for molecular associations with efficacy, and identify cytokine changes differentiating response and ICI-related toxicity (n = 58). Despite no improvement in OS with combination therapy (hazard ratio 0.95, 95% CI 0.59–1.55, p = 0.84), immune infiltration and antigen presentation distinguished responding tumors, while tumor heterogeneity and estrogen signaling independently associated with resistance. Moreover, patients with ICI-related toxicity had lower levels of immunoregulatory cytokines. Broadly, we establish a framework for ICI response in HR+ breast cancer that warrants diagnostic and therapeutic validation. ClinicalTrials.gov Registration: NCT03051659., A randomized phase 2 clinical trial has recently shown no benefit of the combination eribulin and pembrolizumab over pembrolizumab alone in HR + metastatic breast cancer patients (NCT03051659). Here, the authors are reporting the final OS data and biomarker analyses on a subset of samples to analyze molecular correlates

Purpose: Increasing tumor-infiltrating lymphocytes (TIL) is associated with higher rates of pathologic complete response (pCR) to neoadjuvant therapy (NAT) in patients with triple-negative breast cancer (TNBC). However, the presence of TILs does not consistently predict pCR, therefore, the current study was undertaken to more fully characterize the immune cell response and its association with pCR. Experimental Design: We obtained pretreatment core-needle biopsies from 105 patients with stage I–III TNBC enrolled in ARTEMIS (NCT02276443) who received NAT from Oct 22, 2015 through July 24, 2018. The tumor-immune microenvironment was comprehensively profiled by performing T-cell receptor (TCR) sequencing, programmed death-ligand 1 (PD-L1) IHC, multiplex immunofluorescence, and RNA sequencing on pretreatment tumor samples. The primary endpoint was pathologic response to NAT. Results: The pCR rate was 40% (42/105). Higher TCR clonality (median = 0.2 vs. 0.1, P = 0.03), PD-L1 positivity (OR: 2.91, P = 0.020), higher CD3+:CD68+ ratio (median = 14.70 vs. 8.20, P = 0.0128), and closer spatial proximity of T cells to tumor cells (median = 19.26 vs. 21.94 μm, P = 0.0169) were associated with pCR. In a multivariable model, closer spatial proximity of T cells to tumor cells and PD-L1 expression enhanced prediction of pCR when considered in conjunction with clinical stage. Conclusions: In patients receiving NAT for TNBC, deep immune profiling through detailed phenotypic characterization and spatial analysis can improve prediction of pCR in patients receiving NAT for TNBC when considered with traditional clinical parameters.

Background: PARP inhibitors (PARPi) significantly extend progression-free survival (PFS) compared to chemotherapy in pts with BRCA1/2 mutated (BRCA1/2m) ABC, but responses are not durable. PARPi activate the cGAS-STING pathway leading to increased PD-L1 expression and cytotoxic T-cell recruitment, creating a tumor microenvironment (TME) that may be more vulnerable to immunotherapy. This study was conducted to evaluate the safety, efficacy and effects on the TME of the PARPi tala combined with the PD-L1 inhibitor avelumab in ABC, and to assess the impact of BRCA1/2 status on clinical outcomes. Methods: TALAVE was an open-label, multi-institutional trial (NCT03964532) for pts with HER2-negative ABC. Pts were enrolled in two cohorts: cohort 1 - BRCA1/2m and HER2-negative ABC; cohort 2 - BRCA1/2 wildtype TNBC. Pts received a 4-week induction of tala (1mg po daily D1-D28), followed by a combination of daily tala and avelumab (800mg IV D1, D15). The primary objective was the safety and tolerability of the combination. Secondary objectives included ORR, OS and PFS. Pts underwent serial biopsies to investigate molecular signatures associated with BRCA status or clinical benefit using multiple omics techniques: RNA profiling by NanoString PanCancer IO 360™ Panel, GeoMx® Digital Spatial Profiler (DSP) Whole Transcriptome Atlas (WTA) and protein spatial analysis by multiplex immunofluorescence (mIF) and Cyclic Immunofluorescence (CyCIF). Results: 12 pts were enrolled in each cohort. In cohort 1, 5 pts had gBRCA1, 6 had gBRCA2 and 1 had sBRCA2 mutation. The median age was 50 [IQR:43-59.5]; all pts were female, with median of 1 prior therapy for ABC [IQR: 0-2.5]. 42% pts had prior platinum. ORR was 42% (83% in cohort 1; 0% in cohort 2). There were 10 PRs, all in cohort 1. mPFS was 5.1 months (mo) (95% CI: 3.7-7.3 mo); 9.3mo in cohort 1 and 2.9mo in cohort 2. 5 out of 24 pts remain on treatment, all in cohort 1. Treatment related adverse events (TRAEs) included anemia 33%, neutropenia 25% (gr3+ 13%), thrombocytopenia 21% (gr3+ 13%), fatigue 33% and nausea 29%. Other TRAEs gr3+ included dyspnea (4%) and AST elevation (4%). There were no gr5 events. RNA analysis showed that in cohort 1, tala monotherapy disrupted MMEJ, induced antiproliferative effects and expression of genes in the cGAS-STING pathway, including TBK1-mediated IRF3 activation, with downstream induction of T-cell, dendritic cell and cytokine gene expression. These effects were not seen in biopsies post tala monotherapy in cohort 2. mIF analyses demonstrated T-cell and macrophage infiltration in BRCA1/2m tumors. Analyses of post-combination biopsies is ongoing. Conclusions: There were no new safety signals of PARPi combined with immunotherapy. Responses were limited to pts with BRCA1/2m. RNA and protein analyses indicate cGAS-STING activation and immune cell infiltration in BRCA1/2m tumors, validating murine preclinical findings. Citation Format: Filipa Lynce, Kenichi Shimada, Xue Geng, Edward T. Richardson, Candace Mainor, Mei Wei, Julie M. Collins, Paula P. Pohlmann, Arielle L. Heeke, Kelly F. Zheng, Madeline Townsend, Jane Staunton, Stuart J. Schnitt, Joan S. Brugge, Hongkun Wang, Claudine Isaacs, Geoffrey I. Shapiro, Jennifer L. Guerriero. TALAVE: Induction talazoparib (tala) followed by combined tala and avelumab in patients (pts) with advanced breast cancer (ABC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT142.

Modulation of immunosuppressive tumor microenvironments (TMEs) can enhance immunotherapy response rates in solid cancers. One of the key TME regulators is the macrophage migration inhibitory factor (MIF). In addition to its role in tumor cell proliferation, angiogenesis and metastasis, MIF induces polarization of macrophages to a suppressive M2-like subtype, suppresses cytotoxic T cells and correlates with poor response to immune checkpoint therapy. Therapeutic interventions are hampered due to MIF’s ubiquitous expression and non-pathological roles. In contrast, the disease-related structural isoform of MIF, termed oxMIF, is exclusively present in solid tumor tissue, can be specifically targeted by antibodies and oxMIF neutralization blocks protumorigenic activities attributed to MIF. We determined the antitumorigenic and TME-modifying potential of the new oxMIF-specific antibody ON203 in preclinical models including fresh tumoroids retaining an intact TME isolated from colorectal adenocarcinoma (CRC) patients. 3D tumoroids were treated with ON203 and tumor cell killing was analyzed by high-content 3D computational bioimaging. ON203-induced TME modulation was assessed by secretome analysis and flow cytometry. To understand ON203’s action on the TME and to determine factors influencing the response to ON203 we performed dimensionality reduction (UMAP) to comparatively visualize the composition of the tumoroids. To further characterize the immune population differences between conditions, we employed multi-parameter clustering (FlowSOM/XShift) and compared the abundance of each cluster across conditions. We also investigated functional marker expression differences across each of the conditions. ON203 demonstrated excellent tumor penetration and retention and reduced tumor cell proliferation in mouse xenograft models. oxMIF presence was confirmed in all freshly isolated CRC tumoroids and correlated with a “cold” phenotype with high IL-10 and TGF-β levels. Four out of five ON203-treated CRC tumoroids responded with significant tumor cell death. In the responding tumoroids ON203 activated NK and NKT cells (upregulation of Granzyme B and CD107a) and supported an anti-tumor M1-like polarization and macrophage activation (upregulation of CD16 and HLA-DR). Taken together, the anti-oxMIF antibody ON203 demonstrated antitumorigenic effects by reducing tumor cell proliferation and by modulating the TME towards immunosupportive functions. In the upcoming clinical Phase 1 trial ON203’s safety, tolerability, pharmacokinetics, and pharmacodynamics in patients with solid tumors will be analyzed to evaluate its potential as a standalone or combinatorial therapy with immune checkpoint inhibitors. Citation Format: Barbara Maurer, Eric R. Haas, Irina Mirkina, Julia Mayer, Alexander Schinagl, Jennifer L. Guerriero, Michael Thiele. Targeting the oxidized form of macrophage migration inhibitory factor (oxMIF) with antibody ON203 activates the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2974.

Despite objective responses to poly(ADP-ribose) polymerase (PARP) inhibition and improvements in progression-free survival (PFS) compared to standard chemotherapy in patients with BRCA-associated triple-negative breast cancer (TNBC), benefits are transitory. Using high-dimensional single-cell profiling of human TNBC, here we demonstrate that macrophages are the predominant infiltrating immune cell type in breast cancer susceptibility (BRCA)-associated TNBC. Macrophages are an innate immune cell that play a critical role in host defense and maintaining tissue homeostasis, however their infiltration into tumors has been associated with disease progression and resistance to therapy. Tumor associated macrophages (TAMs) represent a significant proportion of solid tumors, including breast cancer. TAMs play a major role in tumorigenesis as they can enhance tumor cell growth, angiogenesis and metastasis. In addition, TAMs can inhibit anti-tumor responses of T cells. Our recent work has shown that removal or conversion of TAMs to an anti-tumor phenotype enhances chemo- and immuno-therapy establishing TAMs as targets for anti-cancer therapy. Through multi-omics profiling, we show that PARP inhibitors enhance both anti- and pro-tumor features of macrophages through glucose and lipid metabolic reprogramming, driven by the sterol regulatory element-binding protein 1 (SREBF1, SREBP1) pathway. Combining PARP inhibitor therapy with colony-stimulating factor 1 receptor (CSF1R)-blocking antibodies significantly enhanced innate and adaptive antitumor immunity and extended survival in mice with BRCA-deficient tumors in vivo, and this was mediated by CD8+ T cells. Collectively, our results uncover macrophage-mediated immune suppression as a liability of PARP inhibitor treatment and demonstrate that combined PARP inhibition and macrophage-targeting therapy induces a durable reprogramming of the tumor microenvironment (TME), thus constituting a promising therapeutic strategy for TNBC. Therefore, targeting TAMs offers great potential to enhance both chemo- and immuno-therapy. Deep analysis of TAMs in solid tumors has revealed the complexity of TAMs and revealed major gaps in our knowledge of the functional and phenotypic characterization of TAM subsets associated with cancer, before and after treatment. Here we will discuss the complexity of TAMs in solid tumors including characterizing TAM subsets, location, and crosstalk with neighboring cells, as well as novel TAM-modulating strategies and combinations that are likely to enhance current therapies and overcome chemo- and immuno-therapy resistance. Citation Format: Jennifer L. Guerriero. The complexities of tumor associated macrophages and the key to effective targeting for anti-cancer therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr SY17-01.

Cancers deficient in homologous recombination (HR) repair secondary to mutations in genes such as BRCA1 or BRCA2, are dependent on alternative DNA damage response (DDR) pathways to maintain genomic integrity, rendering them susceptible to synthetic lethal targeting of these pathways. Recently, inhibitors of polymerase theta (POLθ, encoded by POLQ), the critical enzyme in microhomology-mediated end-joining (MMEJ), have been shown to be synthetic lethal with HR repair deficiency (Zhou et al. Nature Cancer 2021). Both HR and MMEJ require nucleolytic DNA end-resection to allow for DSB repair, and we have previously shown that MMEJ acts as a barrier to DNA end-resection at DSBs (Patterson-Fortin et al. Cancer Research 2022). Given the synthetic lethality between HR and MMEJ leads to unrestrained DNA end-resection generating chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, we hypothesized that POLθ inhibition in HR-deficient cancers would activate the cGAS/STING innate immune response pathway and facilitate immunotherapy. To investigate the interactions of POLθ inhibition with the immune microenvironment in HR-deficient cancers, we used human cell lines and genetically modified mouse models representative of BRCA1-deficient triple-negative breast cancer (TNBC) and BRCA2-deficient pancreatic ductal adenocarcinoma (PDAC). Genetic or pharmacological inhibition of POLθ using novobiocin, a first-in-class inhibitor of the POLθ ATPase domain, induced significantly increased cytosolic dsDNA contained in micronuclei. This free DNA was sensed by the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS), increasing synthesis of cGAMP, in HR-deficient tumor cells but not in HR-proficient tumor cells. Increased cGAMP bound to and activated stimulator of interferon genes (STING), triggering phosphorylation of TBK1 and ultimately of IRF3. Activation of the cGAS/STING pathway by POLθ inhibition drove the expression of type I interferon response elements, including PD-L1. Depletion of STING by siRNA or by CRISPR abrogated this pro-inflammatory signaling and abolished the anti-tumor efficacy of novobiocin-mediated POLθ inhibition. Pharmacologic inhibition of POLθ enhanced Granzyme B+ CD8+ T-cell tumor infiltration. Importantly, antibody-mediated depletion of CD8+ T-cell severely compromised the anti-tumor efficacy of novobiocin-mediated POLθ inhibition, whereas anti-tumor activity of POLθ inhibition was augmented with the addition of either anti-PD-1 or anti-CTLA-4 antibodies. These results demonstrate that POLθ inhibition in HR-deficient cancers mediates a pro-inflammatory response in HR-deficient TNBC or PDAC tumor microenvironments, and that immune checkpoint blockade inhibition enhances the therapeutic efficacy of POLθ inhibition. Citation Format: Jeffrey Patterson-Fortin, Heta Jadhav, Constantia Pantelidou, Tin Phan, Carter Grochala, Anita K. Mehta, Jennifer L. Guerriero, Gerburg M. Wulf, Brian M. Wolpin, Ben Z. Stanger, Andrew J. Aguirre, James M. Cleary, Alan D. D'Andrea, Geoffrey I. Shapiro. Polymerase theta inhibition activates the cGAS-STING pathway and cooperates with immune checkpoint blockade in BRCA-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6190.

Laparoscopic transabdominal preperitoneal (TAPP) is a valid option for bilateral primary groin hernia and recurrent cases. Robotic approach for inguinal hernia is still debated. The aim of this study is to investigate the potential role of robotic-assisted single site-TAPP (RASS-TAPP) reporting our experience. We performed 44 RASS TAPP in 32 patients from February 2016 to July 2018. Data on patient demographics, type of hernia, operative time, complications, recurrence rate and hospital stay were retrospectively analyzed. Follow-up was scheduled at 1 week, 4 months and 1 year after surgery. Forty-two hernias were treated in 32 patients (27 M). Mean age was 48.6 years (range 20–67), mean BMI was 26.49 kg/m2 (range 16–34.9). Mean operative time was 54.8 min (range 28–150). In two cases (6%) a conversion to laparoscopy was necessary. At 1 week, two scrotal hematomas and four seromas were observed and treated conservatively. At 4 months follow-up, one patient (3.1%) complained temporary pain. No patient had inguinal recurrence or incisional umbilical hernia and chronic pain at 1-year follow-up. RASS TAPP is feasible and safe with a high patient satisfaction. However, the surgeon experiences a technical discomfort due to the conflict of the instrumentation which influences negatively the choice of this approach, despite the better vision and augmented dexterity provided by the robot.

Reinforced prosthetic crural repair is particularly indicated for giant hiatal hernias. The rationale is to reduce the recurrence rate in the long term. The aim of our study is to evaluate the outcomes of laparoscopic giant hiatal hernia repair using a biosynthetic mesh. We retrospectively analyzed 44 patients who underwent laparoscopic mesh-reinforced hiatal closure and fundoplication using a biosynthetic material. Inclusion criterion was large hiatal defects (> 5 cm). Follow-up was scheduled at 6, 12 and 36 months after surgery. 44 patients (29F) with a mean age of 62 years (range 14–85) and mean of BMI 24.5 kg/m2 (range 21–29) underwent successful laparoscopic repair. Twenty-six (59.1%) patients had Nissen–Rossetti fundoplication, whereas 18 (40.9%) had Toupet fundoplication. Six-month questionnaire for the evaluation of symptoms was available for 43 patients (97.7%) and for 40 (90.9%) patients at 12 and 36 months. Mean preoperative symptoms score analysis was 1.68 ± 0.73. Mean scores at each follow-up time were significantly improved compared to baseline (p > 0.05). Barium swallow was available in 37 patients (84.1%) at 1 year after surgery. Radiologic recurrence was observed in two patients (4.5%). No patient had symptoms attributable to recurrence or required revisional surgery. There were no mesh-related complications at 3 years follow-up. The use of biosynthetic mesh for crural reinforcement is associated with a low incidence of mesh-related complications and with a reasonably low recurrence rate (4.5%) at 36 months. However, additional data with longer follow-up are needed to determine long-term safety and efficacy.

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.