Evaluation of markers of immunity in different metastatic immune microenvironments suggests more suppression within breast to liver metastases in breast cancer.

PURPOSE: Programmed death receptor ligand-1 (PD-L1) expression and tumor mutational burden (TMB) are approved screening biomarkers for immune checkpoint inhibition (ICI) in advanced triple negative breast cancer. We examined these biomarkers along with characterization of the tumor microenvironment (TME) between breast tumors (BrTs), axillary metastases (AxMs), liver metastases (LvMs), non-axillary lymph node metastases, and non-liver metastases to determine differences related to site of metastatic disease. METHODS: 3076 unpaired biopsies from breast cancer patients were analyzed using whole transcriptome sequencing and NextGen DNA depicting TMB within tumor sites. The PD-L1 positivity was determined with VENTANA PD-L1 (SP142) assay. The immune cell fraction within the TME was calculated by QuantiSeq and MCP-counter. RESULTS: Compared to BrT, more LvM samples had a high TMB (≥ 10 mutations/Mb) and fewer LvM samples had PD-L1+ expression. Evaluation of the TME revealed that LvM sites harbored lower infiltration of adaptive immune cells, such as CD4+, CD8+, and regulatory T-cells compared with the BrT foci. We saw differences in innate immune cell infiltration in LvM compared to BrT, including neutrophils and NK cells. CONCLUSIONS: LvMs are less likely to express PD-L1+ tumor cells but more likely to harbor high TMB as compared to BrTs. Unlike AxMs, LvMs represent a more immunosuppressed TME and demonstrate lower gene expression associated with adaptive immunity compared to BrTs. These findings suggest biopsy site be considered when interpreting results that influence ICI use for treatment and further investigation of immune composition and biomarkers expression by metastatic site.

A comprehensive single-cell breast tumor atlas defines epithelial and immune heterogeneity and interactions predicting anti-PD-1 therapy response.

We present an integrated single-cell RNA sequencing atlas of the primary breast tumor microenvironment (TME) containing 236,363 cells from 119 biopsy samples across eight datasets. In this study, we leverage this resource for multiple analyses of immune and cancer epithelial cell heterogeneity. We define natural killer (NK) cell heterogeneity through six subsets in the breast TME. Because NK cell heterogeneity correlates with epithelial cell heterogeneity, we characterize epithelial cells at the level of single-gene expression, molecular subtype, and 10 categories reflecting intratumoral transcriptional heterogeneity. We develop InteractPrint, which considers how cancer epithelial cell heterogeneity influences cancer-immune interactions. We use T cell InteractPrint to predict response to immune checkpoint inhibition (ICI) in two breast cancer clinical trials testing neoadjuvant anti-PD-1 therapy. T cell InteractPrint was predictive of response in both trials versus PD-L1 (AUC = 0.82, 0.83 vs. 0.50, 0.72). This resource enables additional high-resolution investigations of the breast TME.

Entinostat, nivolumab and ipilimumab for women with advanced HER2-negative breast cancer: a phase Ib trial.

We report the results of 24 women, 50% (N = 12) with hormone receptor-positive breast cancer and 50% (N = 12) with advanced triple-negative breast cancer, treated with entinostat + nivolumab + ipilimumab from the dose escalation (N = 6) and expansion cohort (N = 18) of ETCTN-9844 ( NCT02453620 ). The primary endpoint was safety. Secondary endpoints were overall response rate, clinical benefit rate, progression-free survival and change in tumor CD8:FoxP3 ratio. There were no dose-limiting toxicities. Among evaluable participants (N = 20), the overall response rate was 25% (N = 5), with 40% (N = 4) in triple-negative breast cancer and 10% (N = 1) in hormone receptor-positive breast cancer. The clinical benefit rate was 40% (N = 8), and progression-free survival at 6 months was 50%. Exploratory analyses revealed that changes in myeloid cells may contribute to responses; however, no correlation was noted between changes in CD8:FoxP3 ratio, PD-L1 status and tumor mutational burden and response. These findings support further investigation of this treatment in a phase II trial.

Mimicking the breast metastatic microenvironment: characterization of a novel syngeneic model of HER2+ breast cancer.

Preclinical murine models in which primary tumors spontaneously metastasize to distant organs are valuable tools to study metastatic progression and novel cancer treatment combinations. Here, we characterize a novel syngeneic murine breast tumor cell line, NT2.5-lung metastasis (-LM), that provides a model of spontaneously metastatic neu-expressing breast cancer with quicker onset of widespread metastases after orthotopic mammary implantation in immune-competent NeuN mice. Within one week of orthotopic implantation of NT2.5-LM in NeuN mice, distant metastases can be observed in the lungs. Within four weeks, metastases are also observed in the bones, spleen, colon, and liver. Metastases are rapidly growing, proliferative, and responsive to HER2-directed therapy. We demonstrate altered expression of markers of epithelial-to-mesenchymal transition (EMT) and enrichment in EMT-regulating pathways, suggestive of their enhanced metastatic potential. The new NT2.5-LM model provides more rapid and spontaneous development of widespread metastases. Besides investigating mechanisms of metastatic progression, this new model may be used for the rationalized development of novel therapeutic interventions and assessment of therapeutic responses targeting distant visceral metastases.

Organoid generation from mouse mammary tumors captures the genetic heterogeneity of clinically relevant copy number alterations.

Breast cancer metastases exhibit many different genetic alterations, including copy number amplifications. Using publicly available datasets, we identify copy number amplifications in metastatic breast tumor samples and using our organoid-based metastasis assays, and we validate FGFR1 is amplified in collectively migrating organoids. Because the heterogeneity of breast tumors is increasingly becoming relevant to clinical practice, we demonstrate our organoid method captures genetic heterogeneity of individual tumors.

Myeloid-Derived Suppressor-Cell Dynamics Control Outcomes in the Metastatic Niche.

Myeloid-derived suppressor cells (MDSC) play a prominent role in the tumor microenvironment. A quantitative understanding of the tumor-MDSC interactions that influence disease progression is critical, and currently lacking. We developed a mathematical model of metastatic growth and progression in immune-rich tumor microenvironments. We modeled the tumor-immune dynamics with stochastic delay differential equations and studied the impact of delays in MDSC activation/recruitment on tumor growth outcomes. In the lung environment, when the circulating level of MDSCs was low, the MDSC delay had a pronounced impact on the probability of new metastatic establishment: blocking MDSC recruitment could reduce the probability of metastasis by as much as 50%. To predict patient-specific MDSC responses, we fit to the model individual tumors treated with immune checkpoint inhibitors via Bayesian parameter inference. We reveal that control of the inhibition rate of natural killer (NK) cells by MDSCs had a larger influence on tumor outcomes than controlling the tumor growth rate directly. Posterior classification of tumor outcomes demonstrates that incorporating knowledge of the MDSC responses improved predictive accuracy from 63% to 82%. Investigation of the MDSC dynamics in an environment low in NK cells and abundant in cytotoxic T cells revealed, in contrast, that small MDSC delays no longer impacted metastatic growth dynamics. Our results illustrate the importance of MDSC dynamics in the tumor microenvironment overall and predict interventions promoting shifts toward less immune-suppressed states. We propose that there is a pressing need to consider MDSCs more often in analyses of tumor microenvironments.

Breast cancer and neurotransmitters: emerging insights on mechanisms and therapeutic directions.

Exploring the relationship between various neurotransmitters and breast cancer cell growth has revealed their likely centrality to improving breast cancer treatment. Neurotransmitters play a key role in breast cancer biology through their effects on the cell cycle, epithelial mesenchymal transition, angiogenesis, inflammation, the tumor microenvironment and other pathways. Neurotransmitters and their receptors are vital to the initiation, progression and drug resistance of cancer and progress in our biological understanding may point the way to lower-cost and lower-risk antitumor therapeutic strategies. This review discusses multiple neurotransmitters in the context of breast cancer. It also discusses risk factors, repurposing of pharmaceuticals impacting neurotransmitter pathways, and the opportunity for better integrated models that encompass exercise, the intestinal microbiome, and other non-pharmacologic considerations. Neurotransmitters' role in breast cancer should no longer be ignored; it may appear to complicate the molecular picture but the ubiquity of neurotransmitters and their wide-ranging impacts provide an organizing framework upon which further understanding and progress against breast cancer can be based.

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging.

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

Neurotransmitter signaling: a new frontier in colorectal cancer biology and treatment.

The brain-gut axis, a bidirectional network between the central and enteric nervous system, plays a critical role in modulating the gastrointestinal tract function and homeostasis. Recently, increasing evidence suggests that neuronal signaling molecules can promote gastrointestinal cancers, however, the mechanisms remain unclear. Aberrant expression of neurotransmitter signaling genes in colorectal cancer supports the role of neurotransmitters to stimulate tumor growth and metastatic spread by promoting cell proliferation, migration, invasion, and angiogenesis. In addition, neurotransmitters can interact with immune and endothelial cells in the tumor microenvironment to promote inflammation and tumor progression. As such, pharmacological targeting of neurotransmitter signaling represent a promising novel anticancer approach. Here, we present an overview of the current evidence supporting the role of neurotransmitters in colorectal cancer biology and treatment.

Revival and Recharacterization of a Preclinical Model of Hormone-Dependent Breast Cancer to Study Immunotherapy.

Immune checkpoint inhibitors have yet to significantly improve outcomes for hormone-dependent estrogen/progesterone receptor-positive breast cancer. To address this issue, there is a need for murine models that more closely mimic hormone receptor-positive breast cancer. In this issue, Gil Del Alcazar and colleagues provide an in-depth characterization of a Nitroso-N-methylurea-induced mammary tumor model in outbred Sprague-Dawley rats that meets these needs as it mimics the heterogeneity for mutational profiles, estrogen receptor expression, and immune evasive mechanisms observed in human breast cancer. See related article by Gil Del Alcazar et al., p. 680 (1).

Entinostat Decreases Immune Suppression to Promote Antitumor Responses in a HER2+ Breast Tumor Microenvironment.

Therapeutic combinations to alter immunosuppressive, solid tumor microenvironments (TME), such as in breast cancer, are essential to improve responses to immune checkpoint inhibitors (ICI). Entinostat, an oral histone deacetylase inhibitor, has been shown to improve responses to ICIs in various tumor models with immunosuppressive TMEs. The precise and comprehensive alterations to the TME induced by entinostat remain unknown. Here, we employed single-cell RNA sequencing on HER2-overexpressing breast tumors from mice treated with entinostat and ICIs to fully characterize changes across multiple cell types within the TME. This analysis demonstrates that treatment with entinostat induced a shift from a protumor to an antitumor TME signature, characterized predominantly by changes in myeloid cells. We confirmed myeloid-derived suppressor cells (MDSC) within entinostat-treated tumors associated with a less suppressive granulocytic (G)-MDSC phenotype and exhibited altered suppressive signaling that involved the NFκB and STAT3 pathways. In addition to MDSCs, tumor-associated macrophages were epigenetically reprogrammed from a protumor M2-like phenotype toward an antitumor M1-like phenotype, which may be contributing to a more sensitized TME. Overall, our in-depth analysis suggests that entinostat-induced changes on multiple myeloid cell types reduce immunosuppression and increase antitumor responses, which, in turn, improve sensitivity to ICIs. Sensitization of the TME by entinostat could ultimately broaden the population of patients with breast cancer who could benefit from ICIs.

Evaluating the impact of age on immune checkpoint therapy biomarkers.

Both tumors and aging alter the immune landscape of tissues. These interactions may play an important role in tumor progression among elderly patients and may suggest considerations for patient care. We leverage large-scale genomic and clinical databases to perform comprehensive comparative analysis of molecular and cellular markers of immune checkpoint blockade (ICB) response with patient age. These analyses demonstrate that aging is associated with increased tumor mutational burden, increased expression and decreased promoter methylation of immune checkpoint genes, and increased interferon gamma signaling in older patients in many cancer types studied, all of which are expected to promote ICB efficacy. Concurrently, we observe age-related alterations that might be expected to reduce ICB efficacy, such as decreases in T cell receptor diversity. Altogether, these changes suggest the capacity for robust ICB response in many older patients, which may warrant large-scale prospective study on ICB therapies among patients of advanced age.

Phase I Study of Entinostat and Nivolumab with or without Ipilimumab in Advanced Solid Tumors (ETCTN-9844).

PURPOSE: Epigenetic modulators improve immune checkpoint inhibitor (ICI) efficacy and increase CD8+ effector:FoxP3+ regulatory T cell ratios in preclinical models. We conducted a multicenter phase I clinical trial combining the histone deacetylase inhibitor entinostat with nivolumab ± ipilimumab in advanced solid tumors. PATIENTS AND METHODS: Patients received an entinostat run-in (5 mg, weekly × 2) prior to the addition of ICIs. Dose escalation followed a modified 3+3 design [dose level (DL)1/2: entinostat + nivolumab; DL 3/4: entinostat + nivolumab + ipilimumab]. Blood and tissue samples were collected at baseline, after entinostat run-in, and after 8 weeks of combination therapy. Primary endpoints included safety and tolerability, and the recommended phase II dose (RP2D). Secondary endpoints included antitumor activity and change in tumor CD8/FoxP3 ratio pre- and post-therapy. RESULTS: Thirty-three patients were treated across four dose levels. Treatment-related adverse events (AE) included fatigue (65%), nausea (41%), anemia (38%), diarrhea (26%), and anorexia (26%). Grade 3/4 AEs included fatigue (n = 7, 21%), anemia (n = 9, 27%), and neutropenia (n = 4, 12%). The RP2D was 3 mg entinostat weekly, 3 mg/kg every 2 weeks nivolumab, and 1 mg/kg every 6 weeks ipilimumab (max four doses). The objective response rate by RECIST 1.1 was 16%, including a complete response in triple-negative breast cancer. A statistically significant increase in CD8/FoxP3 ratio was seen following the addition of ICIs to entinostat, but not post-entinostat alone. CONCLUSIONS: The combination of entinostat with nivolumab ± ipilimumab was safe and tolerable with expected rates of immune-related AEs. Preliminary evidence of both clinical efficacy and immune modulation supports further investigation.

Lymph Node Immune Profiles as Predictive Biomarkers for Immune Checkpoint Inhibitor Response.

The need for predictive biomarkers that can accurately predict patients who will respond to immune checkpoint inhibitor (ICI) immunotherapies remains a clinically unmet need. The majority of research efforts have focused on expression of immune-related markers on the tumour and its associated tumour microenvironment (TME). However, immune response to tumour neoantigens starts at the regional lymph nodes, where antigen presentation takes place and is regulated by multiple cell types and mechanisms. Knowledge of the immunological responses in bystander lymphoid organs following ICI therapies and their association with changes in the TME, could prove to be a valuable component in understanding the treatment response to these agents. Here, we review the emerging data on assessment of immunological responses within regional lymph nodes as predictive biomarkers for immunotherapies.

Clocking cancer: the circadian clock as a target in cancer therapy.

Disruption of the cellular pathway modulating endogenous 24-h rhythms, referred to as "the circadian clock", has been recently proven to be associated with cancer risk, development, and progression. This pathway operates through a complex network of transcription-translation feedback loops generated by a set of interplaying proteins. The expression of core circadian clock genes is frequently dysregulated in human tumors; however, the specific effects and underlying mechanisms seem to vary depending on the cancer types and are not fully understood. In addition, specific oncogenes may differentially induce the dysregulation of the circadian clock in tumors. Pharmacological modulation of clock components has been shown to result in specific lethality in certain types of cancer cells, and thus holds great promise as a novel anti-cancer therapeutic approach. Here we present an overview of the rationale and current evidence for targeting the clock in cancer treatment.

Adopting an alternative structure for clinical trials in immunotherapy.

Background: This evaluation emphasizes the main points of the original article 'Position paper: new insights into the immunobiology and dynamics of tumor-host interactions require adaptations of clinical studies' by Sprenger et al. and provides further justification for the use of an alternative approach in the design of human clinical trials for new investigational drugs in the field of immuno-oncology.Objective: Standard trial design utilizing the double blind placebo trial approach, while effective for drugs that directly treat tumors, is too costly, slow, and not effective for drugs and protocols that depend on activation of the immune system for killing of tumors.Methods/results: This paper has proposed through the use of detailed diagnostic profiling, small groups of patients with similar tumor microenvironment characteristics be grouped to determine the clinical benefit of immunological combinations that enter clinical trials. In addition, mega data from larger trials in which patients are subcategorized as above can provide the necessary data as a substitute for current double blind placebo trials which do not take into account the immune status of the host and tumor.Conclusion: There needs to be evolution of the clinical trial landscape so that it matches the exponential growth of the field of immunotherapy.

Regulation of the tumor immune microenvironment and vascular normalization in TNBC murine models by a novel peptide.

Triple-negative breast cancer (TNBC) is a highly metastatic and aggressive disease with limited treatment options. Recently, the combination of the immune checkpoint inhibitor (ICI) atezolizumab (anti-PD-L1) with nab-paclitaxel was approved following a clinical trial that showed response rates in at least 43% of patients. While this approval marks a major advance in the treatment of TNBC it may be possible to improve the efficacy of ICI therapies through further modulation of the suppressive tumor immune microenvironment (TIME). Several factors may limit immune response in TNBC including aberrant growth factor signaling, such as VEGFR2 and cMet signaling, inefficient vascularization, poor delivery of drugs and immune cells, and the skewing of immune cell populations toward immunosuppressive phenotypes. Here we investigate the immune-modulating properties of AXT201, a novel 20 amino-acid integrin-binding peptide in two syngeneic mouse TNBC models: 4T1-BALB/c and NT4-FVB. AXT201 treatment improved survival in the NT4 model by 20% and inhibited the growth of 4T1 tumors by 47% over 22 days post-inoculation. Subsequent immunohistochemical analyses of 4T1 tumors also showed a 53% reduction in vascular density and a 184% increase in pericyte coverage following peptide treatment. Flow cytometry analyses demonstrated evidence of a more favorable anti-tumor immune microenvironment following treatment with AXT201, including significant decreases in the populations of T regulatory cells, monocytic myeloid-derived suppressor cells, and PD-L1 expressing cells and increased expression of T cell functional markers. Together, these findings demonstrate immune-activating properties of AXT201 that could be developed in combination with other immunomodulatory agents in the treatment of TNBC.

Management of Breast Cancer During the COVID-19 Pandemic: A Stage- and Subtype-Specific Approach.

The COVID-19 pandemic has rapidly changed delivery of cancer care. Many nonurgent surgeries are delayed to preserve hospital resources, and patient visits to health care settings are limited to reduce exposure to SARS-CoV-2. Providers must carefully weigh risks and benefits of delivering immunosuppressive therapy during the pandemic. For breast cancer, a key difference is increased use of neoadjuvant systemic therapy due to deferral of many breast surgeries during the pandemic. In some cases, this necessitates increased use of genomic tumor profiling on core biopsy specimens to guide neoadjuvant therapy decisions. Breast cancer treatment during the pandemic requires multidisciplinary input and varies according to stage, tumor biology, comorbidities, age, patient preferences, and available hospital resources. We present here the Johns Hopkins Women's Malignancies Program approach to breast cancer management during the COVID-19 pandemic. We include algorithms based on tumor biology and extent of disease that guide management decisions during the pandemic. These algorithms emphasize medical oncology treatment decisions and demonstrate how we have operationalized the general treatment recommendations during the pandemic proposed by national groups, such as the COVID-19 Pandemic Breast Cancer Consortium. Our recommendations can be adapted by other institutions and medical oncology practices in accordance with local conditions and resources. Guidelines such as these will be important as we continue to balance treatment of breast cancer against risk of SARS-CoV-2 exposure and infection until approval of a vaccine.

Best Foot Forward: Neoadjuvant Systemic Therapy as Standard of Care in Triple-Negative and HER2-Positive Breast Cancer.

Neoadjuvant systemic treatment of early-stage breast cancer has been used to improve resectability and reduce the extent of breast and axillary surgery. More recently, several other merits of neoadjuvant systemic treatment have emerged, including the ability to tailor clinically available adjuvant systemic therapy options based on pathologic response and to serve as a platform for early assessment of novel agents and response biomarkers and as an avenue for treatment optimization investigations (local and systemic therapy escalation and de-escalation trials guided by pathologic response). Attainment of a pathologic complete response (pCR) is associated with excellent long-term outcomes; conversely, the presence of residual disease is associated with a high risk of recurrence for patients with HER2-positive breast cancer and triple-negative breast cancer (TNBC). Treatment strategies in early-stage HER2-positive breast cancer include regimens incorporating trastuzumab, pertuzumab, ado-trastuzumab emtansine, and neratinib, resulting in high pCR rates and overall excellent long-term outcomes. Currently available cytotoxic regimens yield pCR for 35% to 55% of patients with TNBC, and immune checkpoint inhibition is showing early promise for this subtype. New drug and predictive biomarker evaluations in the neoadjuvant setting aim to develop optimal treatment strategies for the individual patient, with the ultimate goal of maximizing efficacy and minimizing toxicity. Research efforts involving novel agents are being undertaken to address the high risk of recurrence for patients with residual disease. Omission of breast surgery following neoadjuvant chemotherapy requires further development of imaging and biopsy techniques to accurately assess the extent of residual disease before clinical application.