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Despite revolutionizing cancer management, immunotherapies dysregulate the immune system, leading to immune-mediated adverse events. These common and potentially dangerous toxicities are often treated with corticosteroids, which are among the most prescribed drugs in oncology for a wide range of cancer and noncancer indications. While steroids exert several mechanisms to reduce immune activity, immunotherapies, such as immune checkpoint inhibitors (ICI), are designed to enhance the immune system's inherent antitumor activity. Because ICI requires an intact and robust immune response, the immunosuppressive properties of steroids have led to a widespread concern that they may interfere with antitumor responses. However, the existing data of the effect of systemic steroids on immunotherapy efficacy remain somewhat conflicted and unclear. To inform clinical decision-making and improve outcomes, we review the impact of steroids on antitumor immunity, recent advances in the knowledge of their impact on ICI efficacy in unique populations and settings, associated precautions, and steroid-sparing treatment approaches.

The tumor-associated vasculature imposes major structural and biochemical barriers to the infiltration of effector T cells and effective tumor control. Correlations between stimulator of interferon genes (STING) pathway activation and spontaneous T cell infiltration in human cancers led us to evaluate the effect of STING-activating nanoparticles (STANs), which are a polymersome-based platform for the delivery of a cyclic dinucleotide STING agonist, on the tumor vasculature and attendant effects on T cell infiltration and antitumor function. In multiple mouse tumor models, intravenous administration of STANs promoted vascular normalization, evidenced by improved vascular integrity, reduced tumor hypoxia, and increased endothelial cell expression of T cell adhesion molecules. STAN-mediated vascular reprogramming enhanced the infiltration, proliferation, and function of antitumor T cells and potentiated the response to immune checkpoint inhibitors and adoptive T cell therapy. We present STANs as a multimodal platform that activates and normalizes the tumor microenvironment to enhance T cell infiltration and function and augments responses to immunotherapy.

Supplementary Figure 1. Expression of immune related genes in the peripheral blood is associated with good outcome following NAC. Supplementary Figure 2. Monocytes are most abundant in blood of patients with good outcomes following NAC. Supplementary Figure 3. Monocytes are most abundant in blood of patients with good outcomes following NAC.

Differentially expressed genes between pCR and RD samples.

Immune checkpoint inhibitors (ICI) are now routinely used in multiple cancers but may induce autoimmune-like side effects known as immune-related adverse events (irAE). Although classical autoimmune diseases have well-known risk factors, including age, gender, and seasonality, the clinical factors that lead to irAEs are not well-defined. To explore these questions, we assessed 455 patients with advanced melanoma treated with ICI at our center and a large pharmacovigilance database (VigiBase). We found that younger age was associated with a similar rate of any irAEs but more frequent severe irAEs and more hospitalizations (OR, 0.97 per year). Paradoxically, however, older patients had more deaths and increased length of stay (LOS) when hospitalized. This was partially due to a distinct toxicity profile: Colitis and hepatitis were more common in younger patients, whereas myocarditis and pneumonitis had an older age distribution both in our center and in VigiBase. This pattern was particularly apparent with combination checkpoint blockade with ipilimumab and nivolumab. We did not find a link between gender or seasonality on development of irAEs in univariate or multivariate analyses, although winter hospitalizations were associated with marginally increased LOS. This study identifies age-specific associations of irAEs.

Number and percentage of reported cases of colitis, autoimmune hepatitis, pneumonitis, and myocarditis based on age group reported in Vigibase.

Legends for Supplementary Figures 1-3

Biomarkers of response are needed in breast cancer to stratify patients to appropriate therapies and avoid unnecessary toxicity. We used peripheral blood gene expression and cell-type abundance to identify biomarkers of response and recurrence in neoadjuvant chemotherapy–treated patients with breast cancer. We identified a signature of IFN and complement response that was higher in the blood of patients with pathologic complete response. This signature was preferentially expressed by monocytes in single-cell RNA sequencing. Monocytes are routinely measured clinically, enabling examination of clinically measured monocytes in multiple independent cohorts. We found that peripheral monocytes were higher in patients with good outcomes in four cohorts of patients with breast cancer. Blood gene expression and cell type abundance biomarkers may be useful for prognostication in breast cancer.Significance:Biomarkers are needed in breast cancer to identify patients at risk for recurrence. Blood is an attractive site for biomarker identification due to the relative ease of longitudinal sampling. Our study suggests that blood-based gene expression and cell-type abundance biomarkers may have clinical utility in breast cancer.

ICD and CPT codes used for synthetic derivative analyses.

PDF file 62K, MCL1 overexpression enhances docetaxel resistance in non-amplified TNBC cell lines

PDF file 77K, Association of gene alterations with gene expression

XLSX file 706K, This file contains 8 supplementary data tables which include patient demographics, genes and alterations assessed by NGS, Nanostring gene expression data, and bioinformatics analyses

XLS file - 44K

Therapeutic antibodies blocking programmed death-1 and its ligand (PD-1/PD-L1) induce durable responses in a substantial fraction of melanoma patients. We sought to determine whether the number and/or type of mutations identified using a next-generation sequencing (NGS) panel available in the clinic was correlated with response to anti–PD-1 in melanoma. Using archival melanoma samples from anti–PD-1/PD-L1-treated patients, we performed hybrid capture–based NGS on 236–315 genes and T-cell receptor (TCR) sequencing on initial and validation cohorts from two centers. Patients who responded to anti–PD-1/PD-L1 had higher mutational loads in an initial cohort (median, 45.6 vs. 3.9 mutations/MB; P = 0.003) and a validation cohort (37.1 vs. 12.8 mutations/MB; P = 0.002) compared with nonresponders. Response rate, progression-free survival, and overall survival were superior in the high, compared with intermediate and low, mutation load groups. Melanomas with NF1 mutations harbored high mutational loads (median, 62.7 mutations/MB) and high response rates (74%), whereas BRAF/NRAS/NF1 wild-type melanomas had a lower mutational load. In these archival samples, TCR clonality did not predict response. Mutation numbers in the 315 genes in the NGS platform strongly correlated with those detected by whole-exome sequencing in The Cancer Genome Atlas samples, but was not associated with survival. In conclusion, mutational load, as determined by an NGS platform available in the clinic, effectively stratified patients by likelihood of response. This approach may provide a clinically feasible predictor of response to anti–PD-1/PD-L1. Cancer Immunol Res; 4(11); 959–67. ©2016 AACR.

Immune checkpoint inhibitors (ICI) targeting CTLA4 or PD-1/PD-L1 have transformed cancer therapy but are associated with immune-related adverse events, including myocarditis. Here, we report a robust preclinical mouse model of ICI-associated myocarditis in which monoallelic loss of Ctla4 in the context of complete genetic absence of Pdcd1 leads to premature death in approximately half of mice. Premature death results from myocardial infiltration by T cells and macrophages and severe ECG abnormalities, closely recapitulating the clinical and pathologic hallmarks of ICI-associated myocarditis observed in patients. Using this model, we show that Ctla4 and Pdcd1 functionally interact in a gene dosage–dependent manner, providing a mechanism by which myocarditis arises with increased frequency in the setting of combination ICI therapy. We demonstrate that intervention with CTLA4–Ig (abatacept) is sufficient to ameliorate disease progression and additionally provide a case series of patients in which abatacept mitigates the fulminant course of ICI myocarditis.Significance:We provide a preclinical model of ICI-associated myocarditis which recapitulates this clinical syndrome. Using this model, we demonstrate that CTLA4 and PD-1 (ICI targets) functionally interact for myocarditis development and that intervention with CTLA4–Ig (abatacept) attenuates myocarditis, providing mechanistic rationale and preclinical support for therapeutic clinical studies.See related commentary by Young and Bluestone, p. 537.This article is highlighted in the In This Issue feature, p. 521

Supplementary Table 1: Results for samples obtained (less than or equal to) 12 months prior to therapy (optimal) vs. those obtained >12 months from treatment or shortly after starting therapy (non-optimal). Supplementary Table 2: Cox proportional hazards model of OS adjusting for baseline variables. Supplementary Table 3: Cox proportional hazards model of PFS adjusting for baseline variables. Figure S1: (A) Performance of mutational load across a range of potential thresholds. (B) Receiver Operating Curve (ROC) for mutational loads cutoffs of 3.3 mutations/MB (low mutational load group) and 23.1 mutations/MB (high mutational load group). Figure S2: (A) Mutational load in tumors with cutaneous/unknown primaries in responders vs. non-responders. (B) Mutational load in tumors with non-cutaneous primaries (acral, mucosal, uveal) in responders vs. non-responders. (C) Gene amplifications and deletions in responders vs. non-responders. Figure S3: (A) LRP1B mutations/variants of unknown significance in responders vs. non-responders. (B) Total number of mutations among melanomas with and without LRP1B mutations. (C) Association between number of LRP1B mutations and total mutations in the melanoma TCGA. Figure S4: (A) T cell receptor (TCR) clonality in responders vs. non-responders. (B) T cell fraction in responders vs. non-responders. (C) TCR clonality in responders vs. non-responders in ideal samples, defined as those obtained within 4 months of anti-PD-1/PD-L1 treatment without other prior therapies. (D) T cell fraction in these ideal samples. Figure S5: Mutation load correlated with (A) T cell receptor (TCR) clonality and (B) T cell fraction.

S3. Representative images of cells grown in 3D Matrigel for 14D and treated with ABT-263 (1.0 uM). S4. Whole cell lysates from cells treated with ABT-263 (1.0uM) for 0=24 hours.

An estimated 40,000 deaths will be attributed to breast cancer in 2016, underscoring the need for improved therapies. Evading cell death is a major hallmark of cancer, driving tumor progression and therapeutic resistance. To evade apoptosis, cancers use antiapoptotic Bcl-2 proteins to bind to and neutralize apoptotic activators, such as Bim. Investigation of antiapoptotic Bcl-2 family members in clinical breast cancer datasets revealed greater expression and more frequent gene amplification of MCL1 as compared with BCL2 or BCL2L1 (Bcl-xL) across three major molecular breast cancer subtypes, Luminal (A and B), HER2-enriched, and Basal-like. While Mcl-1 protein expression was elevated in estrogen receptor α (ERα)-positive and ERα-negative tumors as compared with normal breast, Mcl-1 staining was higher in ERα+ tumors. Targeted Mcl-1 blockade using RNAi increased caspase-mediated cell death in ERα+ breast cancer cells, resulting in sustained growth inhibition. In contrast, combined blockade of Bcl-2 and Bcl-xL only transiently induced apoptosis, as cells rapidly acclimated through Mcl-1 upregulation and enhanced Mcl-1 activity, as measured in situ using Mcl-1/Bim proximity ligation assays. Importantly, MCL1 gene expression levels correlated inversely with sensitivity to pharmacologic Bcl-2/Bcl-xL inhibition in luminal breast cancer cells, whereas no relationship was seen between the gene expression of BCL2 or BCL2L1 and sensitivity to Bcl-2/Bcl-xL inhibition. These results demonstrate that breast cancers rapidly deploy Mcl-1 to promote cell survival, particularly when challenged with blockade of other Bcl-2 family members, warranting the continued development of Mcl-1–selective inhibitors for targeted tumor cell killing.Implications: Mcl-1 levels predict breast cancer response to inhibitors targeting other Bcl-2 family members, and demonstrate the key role played by Mcl-1 in resistance to this drug class. Mol Cancer Res; 15(3); 259–68. ©2016 AACR.

PDF file 45K, Association of high Ki67 with basal-like status

PDF file 26K, Association of node status with RFS and OS, stratified by menopausal status

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PDF 118K, TSC1 truncation in the RD of a TNBC after NAC

PDF file 1474K, MEK activity is required for MYC-induced soft agar colony formation

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PDF file 64K, Legends for all supplementary data figures (1-7)

Background: Immune checkpoint inhibitors (ICI) have improved patient overall and progression-free survival in some cancer types but yielded limited success in breast cancer. Phase-III clinical trials in triple negative breast cancer (TNBC) patients, who harbor extensive tumor-infiltrating lymphocytes within tumor stroma, have demonstrated increased progression-free survival (IMpassion130) and pathologic complete response (KEYNOTE-522). Consequently, combinations of ICI and chemotherapy have been FDA-approved for metastatic TNBC patients, and potentially in the early breast cancer setting. Despite FDA-approval, the therapeutic benefit of ICI alone and the most efficacious chemotherapy combinations are poorly characterized. Objective: We sought to model ICI response in vivo to elucidate the mechanisms responsible for immunotherapy efficacy in breast cancer and ascertain the therapeutic benefits of different chemotherapeutic combinations with ICI. Methods: In this study, we used an immunocompetent EMT6 orthotopic mammary tumor model to investigate the efficacy of single-agent immunotherapy and in combination with standard-of-care chemotherapy (paclitaxel [PAC] or doxorubicin [DOX]). We used single-cell RNA sequencing to analyze the cellular landscape of the primary tumor in response to combinatorial therapeutic strategies. Additionally, we serially sampled and analyzed peripheral blood from mice with differential responses by bulk and T-cell receptor (TCR) sequencing to identify systemic genetic alterations and T-cell expansion. Results: Single-agent anti-PD-L1 robustly suppressed primary tumor growth (p =0.0046) and extended survival (p Citation Format: Ann Hanna, Xiaopeng Sun, Paula I. Gonzalez-Ericsson, Violeta M. Sanchez, Melinda E. Sanders, Justin M. Balko. Host myeloid response to tumor and immunotherapy is associated with heterogeneity in outcomes to anti-PDL1 [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 P1-04-03.

Purpose:The phase III ExteNET trial showed improved invasive disease-free survival in patients with HER2+ breast cancer treated with neratinib versus placebo after trastuzumab-based adjuvant therapy. The benefit from neratinib appeared to be greater in patients with ER+/HER2+ tumors. We thus sought to discover mechanisms that may explain the benefit from extended adjuvant therapy with neratinib.Experimental Design: Mice with established ER+/HER2+ MDA-MB-361 tumors were treated with paclitaxel plus trastuzumab ± pertuzumab for 4 weeks, and then randomized to fulvestrant ± neratinib treatment. The benefit from neratinib was evaluated by performing gene expression analysis for 196 ER targets, ER transcriptional reporter assays, and cell-cycle analyses.Results:Mice receiving “extended adjuvant” therapy with fulvestrant/neratinib maintained a complete response, whereas those treated with fulvestrant relapsed rapidly. In three ER+/HER2+ cell lines (MDA-MB-361, BT-474, UACC-893) but not in ER+/HER2− MCF7 cells, treatment with neratinib induced ER reporter transcriptional activity, whereas treatment with fulvestrant resulted in increased HER2 and EGFR phosphorylation, suggesting compensatory reciprocal crosstalk between the ER and ERBB RTK pathways. ER transcriptional reporter assays, gene expression, and immunoblot analyses showed that treatment with neratinib/fulvestrant, but not fulvestrant, potently inhibited growth and downregulated ER reporter activity, P-AKT, P-ERK, and cyclin D1 levels. Finally, similar to neratinib, genetic and pharmacologic inactivation of cyclin D1 enhanced fulvestrant action against ER+/HER2+ breast cancer cells.Conclusions:These data suggest that ER blockade leads to reactivation of ERBB RTKs and thus extended ERBB blockade is necessary to achieve durable clinical outcomes in patients with ER+/HER2+ breast cancer.

Supplementary Figure 1: Representative images of high and low sTILs.

Dataset 2: Metadata and clonotype data for TCRβ sequencing (Archer) in 4 patients (peripheral CD8+ PD-1HI and CD8+ PD-1NEG T cells and post-NAC tumor), before (n=3) and after (n=4) NAC.

Supplementary Tables - PDF file 148K, Table S1. Characteristics of HER2-negative breast cancers, separated by HR status, TCGA Table S2. Characteristics of triple-negative breast cancers with residual disease post neoadjuvant chemotherapy and univariate Table S3. Bimodal analysis of metabolism-related genes ranked by bimodality index (BI)

Purpose: We undertook this study to determine the prevalence of estrogen receptor (ER) α (ESR1) mutations throughout the natural history of hormone-dependent breast cancer and to delineate the functional roles of the most commonly detected alterations.Experimental Design: We studied a total of 249 tumor specimens from 208 patients. The specimens include 134 ER-positive (ER+/HER2−) and, as controls, 115 ER-negative (ER−) tumors. The ER+ samples consist of 58 primary breast cancers and 76 metastatic samples. All tumors were sequenced to high unique coverage using next-generation sequencing targeting the coding sequence of the estrogen receptor and an additional 182 cancer-related genes.Results: Recurring somatic mutations in codons 537 and 538 within the ligand-binding domain of ER were detected in ER+ metastatic disease. Overall, the frequency of these mutations was 12% [9/76; 95% confidence interval (CI), 6%–21%] in metastatic tumors and in a subgroup of patients who received an average of 7 lines of treatment the frequency was 20% (5/25; 95% CI, 7%–41%). These mutations were not detected in primary or treatment-naïve ER+ cancer or in any stage of ER− disease. Functional studies in cell line models demonstrate that these mutations render estrogen receptor constitutive activity and confer partial resistance to currently available endocrine treatments.Conclusions: In this study, we show evidence for the temporal selection of functional ESR1 mutations as potential drivers of endocrine resistance during the progression of ER+ breast cancer. Clin Cancer Res; 20(7); 1757–67. ©2014 AACR.

Purpose: Although most patients with estrogen receptor α (ER)-positive breast cancer initially respond to endocrine therapy, many ultimately develop resistance to antiestrogens. However, mechanisms of antiestrogen resistance and biomarkers predictive of such resistance are underdeveloped.Experimental Design: We adapted four ER+ human breast cancer cell lines to grow in an estrogen-depleted medium. A gene signature of estrogen independence was developed by comparing expression profiles of long-term estrogen-deprived (LTED) cells to their parental counterparts. We evaluated the ability of the LTED signature to predict tumor response to neoadjuvant therapy with an aromatase inhibitor and disease outcome following adjuvant tamoxifen. We utilized Gene Set Analysis (GSA) of LTED cell gene expression profiles and a loss-of-function approach to identify pathways causally associated with resistance to endocrine therapy.Results: The LTED gene expression signature was predictive of high tumor cell proliferation following neoadjuvant therapy with anastrozole and letrozole, each in different patient cohorts. This signature was also predictive of poor recurrence-free survival in two studies of patients treated with adjuvant tamoxifen. Bioinformatic interrogation of expression profiles in LTED cells revealed a signature of MYC activation. The MYC activation signature and high MYC protein levels were both predictive of poor outcome following tamoxifen therapy. Finally, knockdown of MYC inhibited LTED cell growth.Conclusions: A gene expression signature derived from ER+ breast cancer cells with acquired hormone independence predicted tumor response to aromatase inhibitors and associated with clinical markers of resistance to tamoxifen. Activation of the MYC pathway was associated with this resistance. Clin Cancer Res; 17(7); 2024–34. ©2011 AACR.

Supplementary Figure 5: The change in sTILs during NAC is not prognostic for outcome in TNBC patients with residual disease.

Dataset 1: Metadata and clonotype data for TCRβ sequencing (Adaptive) in 15 pairs of NAC-treated patients (tumor data).

Supplementary Figure 2: Pre-NAC sTILs have minimal prognostic value in breast cancer patients with residual disease.

Efferocytosis is the process by which apoptotic cells are cleared from tissue by phagocytic cells. The removal of apoptotic cells prevents them from undergoing secondary necrosis and releasing their inflammation-inducing intracellular contents. Efferocytosis also limits tissue damage by increasing immunosuppressive cytokines and leukocytes and maintains tissue homeostasis by promoting tolerance to antigens derived from apoptotic cells. Thus, tumor cell efferocytosis following cytotoxic cancer treatment could impart tolerance to tumor cells evading treatment-induced apoptosis with deleterious consequences in tumor residual disease. We report here that efferocytosis cleared apoptotic tumor cells in residual disease of lapatinib-treated HER2+ mammary tumors in MMTV-Neu mice, increased immunosuppressive cytokines, myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg). Blockade of efferocytosis induced secondary necrosis of apoptotic cells, but failed to prevent increased tumor MDSCs, Treg, and immunosuppressive cytokines. We found that efferocytosis stimulated expression of IFN-γ, which stimulated the expression of indoleamine-2,3-dioxegenase (IDO) 1, an immune regulator known for driving maternal-fetal antigen tolerance. Combined inhibition of efferocytosis and IDO1 in tumor residual disease decreased apoptotic cell- and necrotic cell-induced immunosuppressive phenotypes, blocked tumor metastasis, and caused tumor regression in 60% of MMTV-Neu mice. This suggests that apoptotic and necrotic tumor cells, via efferocytosis and IDO1, respectively, promote tumor ‘homeostasis’ and progression.Significance:These findings show in a model of HER2+ breast cancer that necrosis secondary to impaired efferocytosis activates IDO1 to drive immunosuppression and tumor progression.

Supplemental Figure S6. Tumors harvested on treatment d 7 were assessed by IHC for FoxP3 and Arg-1. Representative images are shown. Original magnification = 400X. Asterisks represent areas of acellular debris. Black arrows indicate tumor infiltrating lymphocytes (TILs). Yellow arrows indicate hyper-condensed nuclei characteristic of apoptotic bodies / apoptotic debris.

Supplementary Tables S1-S10.

Purpose:The recent approval of anti-programmed death-ligand 1 immunotherapy in combination with nab-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor immune microenvironment (TIME).Experimental Design:We examined immune-related gene expression patterns before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 TNBCs, from patients with residual disease (RD). Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). In addition, we sought to characterize the systemic effects of NAC through single-cell analysis (RNAseq and cytokine secretion) of programmed death-1–high (PD-1HI) CD8+ peripheral T cells and examination of a cytolytic gene signature in whole blood.Results:In non-TNBC, no change in expression of any single gene was associated with RFS or OS, while in TNBC upregulation of multiple immune-related genes and gene sets were associated with improved long-term outcome. High cytotoxic T-cell signatures present in the peripheral blood of patients with breast cancer at surgery were associated with persistent disease and recurrence, suggesting active antitumor immunity that may indicate ongoing disease burden.Conclusions:We have characterized the effects of NAC on the TIME, finding that TNBC is uniquely sensitive to the immunologic effects of NAC, and local increases in immune genes/sets are associated with improved outcomes. However, expression of cytotoxic genes in the peripheral blood, as opposed to the TIME, may be a minimally invasive biomarker of persistent micrometastatic disease ultimately leading to recurrence.

Supplementary Figures - PDF file 440K, Figure S1. Figure S1. Validation the IHC protocol for LDHB using FFPE MDAMB231 cells with stable knockdown of LDHA or LDHB. Reduced staining for LDHB was detected only in the LDHB knockdown line. Control cells were infected with a non-silencing shRNA. Figure S2. LDHB, when present, substantially contributed to the total LDH activity in cell lines. Figure S3. A, verification of LDHA or LDHB knockdown by LDH activity in stable isogenic cell lines, MDAMB231 and HCC1937. Figure S4. LDHB association with PAM50 intrinsic subtype in primary breast cancers. A, LDHB mRNA expression separated by intrinsic subtype: Perou GSE10893 (8) Figure S5. High LDHB was associated with triple-negative breast cancer. LDHB mRNA expression of HER2 (by IHC/FISH)-negative disease separated by clinical classification Figure S6. LDHB mRNA expression within PAM50 intrinsic subtypes for HR-positive/HER2-negative breast cancers. Figure S7. LDHB mRNA expression within PAM50 intrinsic subtypes for triple-negative breast cancers Figure S8. LDHB mRNA expression was highly associated with cell cycle proliferation marker, CCNB1, for basal-like cancers within the triple-negative group. Figure S9. Association between mRNA expression and copy number for LDHB for the breast invasive carcinoma cohort separated by (A) basal and (B) luminal A/B subtypes for TCGA "manuscript samples" within the cBio Cancer Genomics Portal