Chronic impairment of ERK signaling in glutamatergic neurons of the forebrain does not affect spatial memory retention and LTP in the same manner as acute blockade of the ERK pathway

The ERK/MAPK signaling pathway has been extensively studied in the context of learning and memory. Defects in this pathway underlie genetic diseases associated with intellectual disability, including impaired learning and memory. Numerous studies have investigated the impact of acute ERK/MAPK inhibition on long-term potentiation and spatial memory. However, genetic knockouts of the ERKs have not been utilized to determine whether developmental perturbations of ERK/MAPK signaling affect LTP and memory formation in postnatal life. In this study, two different ERK2 conditional knockout mice were generated that restrict loss of ERK2 to excitatory neurons in the forebrain, but at different time-points (embryonically and post-natally). We found that embryonic loss of ERK2 had minimal effect on spatial memory retention and novel object recognition, while loss of ERK2 post-natally had more pronounced effects in these behaviors. Loss of ERK2 in both models showed intact LTP compared to control animals, while loss of both ERK1 and ERK2 impaired late phase LTP. These findings indicate that ERK2 is not necessary for LTP and spatial memory retention and provide new insights into the functional deficits associated with the chronic impairment of ERK signaling

Expression in Aromatase Inhibitor Responder vs. Non-Responder HR+ Breast Cancer Cohorts

Hormone receptor positive (HR+) breast cancer patients have an excellent prognosis with over 85% cure rates. However, 20,000 women with HR+ breast cancer die annually from tumor recurrence. Those at highest risk for recurrence are those women whose tumors continue to proliferate through estrogen blockade (Figure 4). Addition of chemotherapy for these patients failed to improve outcomes. We retrospectively identified women with HR+ breast cancer treated neoadjuvantly with aromatase inhibitors (AI), and selected matched patient cohorts for responders and non-responders determined by post-AI Ki67 o

Multiplex immunofluorescence to measure dynamic changes in tumor-infiltrating lymphocytes and PD-L1 in early-stage breast cancer.

BACKGROUND: The H&E stromal tumor-infiltrating lymphocyte (sTIL) score and programmed death ligand 1 (PD-L1) SP142 immunohistochemistry assay are prognostic and predictive in early-stage breast cancer, but are operator-dependent and may have insufficient precision to characterize dynamic changes in sTILs/PD-L1 in the context of clinical research. We illustrate how multiplex immunofluorescence (mIF) combined with statistical modeling can be used to precisely estimate dynamic changes in sTIL score, PD-L1 expression, and other immune variables from a single paraffin-embedded slide, thus enabling comprehensive characterization of activity of novel immunotherapy agents. METHODS: Serial tissue was obtained from a recent clinical trial evaluating loco-regional cytokine delivery as a strategy to promote immune cell infiltration and activation in breast tumors. Pre-treatment biopsies and post-treatment tumor resections were analyzed by mIF (PerkinElmer Vectra) using an antibody panel that characterized tumor cells (cytokeratin-positive), immune cells (CD3, CD8, CD163, FoxP3), and PD-L1 expression. mIF estimates of sTIL score and PD-L1 expression were compared to the H&E/SP142 clinical assays. Hierarchical linear modeling was utilized to compare pre- and post-treatment immune cell expression, account for correlation of time-dependent measurement, variation across high-powered magnification views within each subject, and variation between subjects. Simulation methods (Monte Carlo, bootstrapping) were used to evaluate the impact of model and tissue sample size on statistical power. RESULTS: mIF estimates of sTIL and PD-L1 expression were strongly correlated with their respective clinical assays (p \u3c .001). Hierarchical linear modeling resulted in more precise estimates of treatment-related increases in sTIL, PD-L1, and other metrics such as CD8+ tumor nest infiltration. Statistical precision was dependent on adequate tissue sampling, with at least 15 high-powered fields recommended per specimen. Compared to conventional t-testing of means, hierarchical linear modeling was associated with substantial reductions in enrollment size required (n = 25➔n = 13) to detect the observed increases in sTIL/PD-L1. CONCLUSION: mIF is useful for quantifying treatment-related dynamic changes in sTILs/PD-L1 and is concordant with clinical assays, but with greater precision. Hierarchical linear modeling can mitigate the effects of intratumoral heterogeneity on immune cell count estimations, allowing for more efficient detection of treatment-related pharmocodynamic effects in the context of clinical trials. TRIAL REGISTRATION: NCT02950259

Transcriptional and immunohistological assessment of immune infiltration in pancreatic cancer.

Pancreatic adenocarcinoma is characterized by a complex tumor environment with a wide diversity of infiltrating stromal and immune cell types that impact the tumor response to conventional treatments. However, even in this poorly responsive tumor the extent of T cell infiltration as determined by quantitative immunohistology is a candidate prognostic factor for patient outcome. As such, even more comprehensive immunophenotyping of the tumor environment, such as immune cell type deconvolution via inference models based on gene expression profiling, holds significant promise. We hypothesized that RNA-Seq can provide a comprehensive alternative to quantitative immunohistology for immunophenotyping pancreatic cancer. We performed RNA-Seq on a prospective cohort of pancreatic tumor specimens and compared multiple approaches for gene expression-based immunophenotyping analysis compared to quantitative immunohistology. Our analyses demonstrated that while gene expression analyses provide additional information on the complexity of the tumor immune environment, they are limited in sensitivity by the low overall immune infiltrate in pancreatic cancer. As an alternative approach, we identified a set of genes that were enriched in highly T cell infiltrated pancreatic tumors, and demonstrate that these can identify patients with improved outcome in a reference population. These data demonstrate that the poor immune infiltrate in pancreatic cancer can present problems for analyses that use gene expression-based tools; however, there remains enormous potential in using these approaches to understand the relationships between diverse patterns of infiltrating cells and their impact on patient treatment outcomes

Impact of chemotherapy (chemo) on t-cell maturation and clonal proliferation in early-stage and metastatic breast cancer

Background Adaptive immunity is initiated by T-cell receptor (TCR) engagement with cognate tumor antigen, leading to T-cell maturation (i.e. conversion from naïve to effector state), clonal proliferation, and potentially tumor elimination. Therefore, broad T-cell repertoire diversity could be an important determinant of anti-tumor immunity. In breast cancer, chemo may facilitate adaptive immunity, but conversely may also be lymphotoxic. We characterize the impacts of curative-intent chemo and palliative combination chemo/immune checkpoint inhibition (ICI) on lymphocyte quantity, maturation, and clonal proliferation. Methods Peripheral blood mononuclear cells (PBMCs) were collected at baseline and serially in stage IIII subjects receiving curative-intent chemo (dose-dense doxorubicin, cyclophosphamide, paclitaxel, n=20), and in stage IV subjects receiving palliative ICI (pembrolizumab) plus chemo (paclitaxel, n=15; or capecitabine, n=14). Flow cytometry was conducted on fresh PBMCs to minimize cellular losses related to cryopreservation. DNA was extracted for T-cell clonality analysis using the immunoSEQ Assay (Adaptive Biotechnologies) at deep resolution. T-cell richness, calculated using the iChao1 richness estimator was used to assess overall T-cell diversity. Results Both dose-dense chemo and palliative chemo/ICI were lymphodepleting, however dose-dense chemo was associated with greater reductions in CD4+ naïve T cell count (week 8 counts 34% vs. 96% of baseline, p\u3c.001) and T-cell richness (week 8 richness 57% vs 90% of baseline, p\u3c.001). These effects were durable, with reductions in lymphocyte subsets (ALC, CD4, CD8, naïve, effector, and central memory subsets) and T-cell richness (estimated rearrangements 4.37 vs 3.29 x 10 5/sample) sustained at metastatic relapse. T-cell richness correlated with CD4+ naïve cell proportion (R2=0.52), suggesting a major contribution of CD4+ naïve cells to overall T-cell diversity. In this dataset, acute memory T-cell expansions were observed following initiation of chemo, but only among younger patients (age\u3c60). Conclusions Curative-intent cytotoxic chemotherapy durably alters the quantity and diversity of peripheral T-cells, particularly of naïve cells, contributing to lymphopenia and reduced T-cell richness at metastatic relapse. Acute expansions of EM subsets were also observed but only in younger individuals. These contrasting immunologic effects highlight the potential importance of sequencing of ICI relative to chemo, and raise concerns regarding age-related thymic involution and immunosenescence. Ongoing research is warranted to investigate the predictive/prognostic utility of T-cell diversity/quantity in breast cancer, and whether thymic T-cell regeneration can be targeted therapeutically to enhance immunotherapy response, for example with cytokine therapies (IL-12 or IL-7, NCT04095689) or sex steroid inhibition (NCT03650894)

Pharmacological Inhibition of ERK Signaling Rescues Pathophysiology and Behavioral Phenotype Associated with 16p11.2 Chromosomal Deletion in Mice

The human 16p11.2 microdeletion is one of the most common gene copy number variations linked to autism, but the pathophysiology associated with this chromosomal abnormality is largely unknown. The 593 kb deletion contains the ERK1 gene and other genes that converge onto the ERK/MAP kinase pathway. Perturbations in ERK signaling are linked to a group of related neurodevelopmental disorders hallmarked by intellectual disability, including autism. We report that mice harboring the 16p11.2 deletion exhibit a paradoxical elevation of ERK activity, cortical cytoarchitecture abnormalities and behavioral deficits. Importantly, we show that treatment with a novel ERK pathway inhibitor during a critical period of brain development rescues the molecular, anatomical and behavioral deficits in the 16p11.2 deletion mice. The ERK inhibitor treatment administered to adult mice ameliorates a subset of these behavioral deficits. Our findings provide evidence for potential targeted therapeutic intervention in 16p11.2 deletion carriers. SIGNIFICANCE STATEMENT The ERK/MAPK pathway is genetically linked to autism spectrum disorders and other syndromes typified by intellectual disability. We provide direct evidence connecting the ERK/MAP kinases to the developmental abnormalities in neurogenesis and cortical cytoarchitecture associated with the 16p11.2 chromosomal deletion. Most importantly, we demonstrate that treatment with a novel ERK-specific inhibitor during development rescues aberrant cortical cytoarchitecture and restores normal levels of cell-cycle regulators during cortical neurogenesis. These treatments partially reverse the behavioral deficits observed in the 16p11.2del mouse model, including hyperactivity, memory as well as olfaction, and maternal behavior. We also report a rescue of a subset of these deficits upon treatment of adult 16p11.2del mice. These data provide a strong rationale for therapeutic approaches to this disorder