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Lung cancer is a heterogeneous disease in which patient-specific treatments are desirable and the development of targeted therapies has been effective. Although mutations in KRAS are frequent in lung adenocarcinoma, there are currently no targeted agents against KRAS. Using a mouse lung adenocarcinoma cell line with a Kras mutation (CMT167), we previously showed that PPARγ activation in lung cancer cells inhibits cell growth in vitro yet promotes tumor progression when activated in myeloid cells of the tumor microenvironment. Here, we report that PPARγ activation in myeloid cells promotes the production of TGFβ1, which, in turn, acts on CMT167 cancer cells to increase migration and induce an epithelial–mesenchymal transition (EMT). Targeting TGFβ1 signaling in CMT167 cells prevented their growth and metastasis in vivo. Similarly, another mouse lung adenocarcinoma cell line with a Kras mutation, LLC, induced TGFβ1 in myeloid cells through PPARγ activation. However, LLC cells are more mesenchymal and did not undergo EMT in response to TGFβ1, nor did LLC require TGFβ1 signaling for metastasis in vivo. Converting CMT167 cells to a mesenchymal phenotype through overexpression of ZEB1 made them unresponsive to TGFβ1 receptor inhibition. The ability of TGFβ1 to induce EMT in lung tumors may represent a critical process in cancer progression. We propose that TGFβ receptor inhibition could provide an additional treatment option for KRAS-mutant epithelial lung tumors.Implications: This study suggests that TGFβ receptor inhibitors may be an effective therapy in a subset of KRAS-mutant patients with non–small cell lung cancer, which show an epithelial phenotype.

S1: Differentiation state of CMT-luc and LLC-luc cells. S2: Intratumoral myeloid cell populations do not change with PPARγ knockout or activation. S3: TGFβRII knockdown CMT-luc cells are less responsive to TGF-β1 S4: Loss of PPARγ in myeloid cells does not affect LLC-luc tumor progression. S5: LLC cell response to TGF-β1 treatment.

Terminology definitions as used by the Precision Oncology Decision Support (PODS) team at MD Anderson Cancer Center

Supplementary Table S1. FDA approved HER2-targeted therapies

This shows the efficiency of immuodepletion experiments

Prominent commercial vendors providing decision support services (as of November 1, 2017)

The complement cascade is a part of the innate immune system that acts primarily to remove pathogens and injured cells. However, complement activation is also peculiarly associated with tumor progression. Here we report mechanistic insights into this association in multiple immunocompetent orthotopic models of lung cancer. After tumor engraftment, we observed systemic activation of the complement cascade as reflected by elevated levels of the key regulator C3a. Notably, growth of primary tumors and metastases was both strongly inhibited in C3-deficient mice (C3−/− mice), with tumors undetectable in many subjects. Growth inhibition was associated with increased numbers of IFNγ+/TNFα+/IL10+ CD4+ and CD8+ T cells. Immunodepletion of CD4+ but not CD8+ T cells in tumor-bearing subjects reversed the inhibitory effects of C3 deletion. Similarly, antagonists of the C3a or C5a receptors inhibited tumor growth. Investigations using multiple tumor cell lines in the orthotopic model suggested the involvement of a C3/C3 receptor autocrine signaling loop in regulating tumor growth. Overall, our findings offer functional evidence that complement activation serves as a critical immunomodulator in lung cancer progression, acting to drive immune escape via a C3/C5–dependent pathway.Significance: This provocative study suggests that inhibiting complement activation may heighten immunotherapeutic responses in lung cancer, offering findings with immediate implications, given the existing clinical availability of complement antagonists. Cancer Res; 78(1); 143–56. ©2017 AACR.

With the increasing availability of genomics, routine analysis of advanced cancers is now feasible. Treatment selection is frequently guided by the molecular characteristics of a patient's tumor, and an increasing number of trials are genomically selected. Furthermore, multiple studies have demonstrated the benefit of therapies that are chosen based upon the molecular profile of a tumor. However, the rapid evolution of genomic testing platforms and emergence of new technologies make interpreting molecular testing reports more challenging. More sophisticated precision oncology decision support services are essential. This review outlines existing tools available for health care providers and precision oncology teams and highlights strategies for optimizing decision support. Specific attention is given to the assays currently available for molecular testing, as well as considerations for interpreting alteration information. This article also discusses strategies for identifying and matching patients to clinical trials, current challenges, and proposals for future development of precision oncology decision support. Clin Cancer Res; 24(12); 2719–31. ©2018 AACR.

These figures show experiments of tumor growth in Factor B-/- mice. They also show gating strategy for flow cytometry and changes in inflammatory cells. Data showing loss of CIITA in cancer cells alters the response to complement depletion.

Actionable gene list for the Precision Oncology Decision Support (PODS) team at MD Anderson Cancer Center (as of November 1, 2017)

A peptide sequencing scheme utilizing fluorescence microscopy and Edman degradation to determine the amino acid position in fluorophore-labeled peptides was recently reported, referred to as fluorosequencing. It was observed that multiple fluorophores covalently linked to a peptide scaffold resulted in a decrease in the anticipated fluorescence output and worsened the single-molecule fluorescence analysis. In this study, we report an improvement in the photophysical properties of fluorophore-labeled peptides by incorporating long and flexible (PEG)(10) linkers at the peptide attachment points. Long linkers to the fluorophores were installed using copper-catalyzed azide-alkyne cycloaddition conditions. The photophysical properties of these peptides were analyzed in solution and immobilized on a microscope slide at the single-molecule level under peptide fluorosequencing conditions. Solution-phase fluorescence analysis showed improvements in both quantum yield and fluorescence lifetime with the long linkers. While on the solid support, photometry measurements showed significant increases in fluorescence brightness and 20 to 60% improvements in the ability to determine the amino acid position with fluorosequencing. This spatial distancing strategy demonstrates improvements in the peptide sequencing platform and provides a general approach for improving the photophysical properties in fluorophore-labeled macromolecules.

Objective: Multiplex immunohistochemistry (mIHC) and multiplexed ion beam imaging (MIBI) images are usually phenotyped using a manual thresholding process. The thresholding is prone to biases, especially when examining multiple images with high cellularity. Results: Unsupervised cell phenotyping methods including PhenoGraph, flowMeans, and SamSPECTRAL, primarily used in flow cytometry data, often perform poorly or need elaborate tuning to perform well in the context of mIHC and MIBI data. We show that, instead, semi-supervised cell clustering using Random Forests, linear and quadratic discriminant analysis are superior. We test the performance of the methods on two mIHC datasets from the University of Colorado School of Medicine and a publicly available MIBI dataset. Each dataset contains numerous highly complex images.

PurposeLung cancers bearing oncogenic EML4-ALK fusions respond to targeted tyrosine kinase inhibitors (TKIs; e.g. alectinib), with variation in the degree of shrinkage and duration of treatment (DOT). We previously demonstrated a positive association of a TKI-induced interferon gamma (IFNγ) transcriptional response with DOT in EGFR-mutant lung cancers. Herein, we used murine models of EML4-ALK lung cancer to test a role for host immunity in the therapeutic response to alectinib.Experimental DesignThree murine EML4-ALK cell lines (EA1, EA2, EA3) were implanted orthotopically into the lungs of immunocompetent and immunodeficient mice and treated with alectinib. Tumor volumes were serially measured by μCT. Immune cell content was measured by flow cytometry, multispectral immunofluorescence and CyTOF. Transcriptional responses to alectinib were assessed by RNAseq and secreted chemokines were measured by ELISA.ResultsAll cell lines were sensitive to alectinib in vitro. EA1 and EA3 tumors retained residual disease that rapidly progressed upon termination of treatment while EA2 tumors were eliminated by TKI treatment. Alectinib induced inflammatory transcriptional programs and multiple chemokines in all cell lines while untreated tumors exhibited distinct baseline chemokine expression patterns and content of CD8+ T cells and myeloid subsets. When propagated in immune-deficient mice, all three cell line-derived lung tumor models exhibited significant shrinkage followed by prompt progression despite continuous alectinib treatment.ConclusionsThe findings support an hypothesis that host and TKI-stimulated production of chemokines by tumor cells promotes functional engagement of adaptive immune cells within the tumor microenvironment that enhances the durability and depth of TKI response.Statement of Translational RelevancePatients with metastatic lung cancer harboring ALK fusions are treated with targeted tyrosine kinase inhibitors (TKI) in the first line setting. Despite bearing the same driver oncogene, patients experience a range of tumor burden reduction and variable amounts of residual disease. Residual disease burden associates with patient survival and contributes to the emergence of drug resistance yielding treatment failure. The factors mediating this differential response to TKI and residual disease are incompletely understood. Our group has developed a panel of murine ALK driven lung cancer cell lines that reproducibly show differences in the depth and duration of response when implanted into immunocompetent mice. Data using this model indicate that the presence of CD8+ T cells is a major contributor to the depth and duration of response. These models will be critical in developing rational combination therapies to augment the immune microenvironment engagement along with TKIs to improve outcomes for these patients.

Introduction Symptomatic early onset pulmonary events (EOPEs) were observed in 3% to 6% of patients within 1 week of starting brigatinib at 90 mg daily for 7 days followed by 180 mg daily. We conducted a prospective observational cohort study to measure pulmonary function changes on initiating brigatinib. Methods Patients initiating brigatinib were eligible. Pulmonary function test (PFT) with diffusing capacity for carbon monoxide (DLCO), Borg dyspnea scale, six-minute walk test, and blood draw for cytometry by time-of-flight were performed at baseline, day 2, and day 8 plus or minus day 15 of brigatinib. The primary end point was the incidence of PFT-defined EOPEs, prespecified as greater than or equal to 20% DLCO reduction from baseline. An interim analysis was performed owing to a higher than expected incidence of DLCO reduction. Results A total of 90% (nine of 10) experienced DLCO reduction with the nadir occurring on day 2 or day 8. Median DLCO nadir was −13.33% from baseline (range: −34.44 to −5.00). Three participants met the PFT-defined EOPE criteria. All patients, including these three, were asymptomatic, none required brigatinib interruption or dose reduction, and all patients escalated to 180 mg without further issues. Despite continued dosing, by day 15, all assessed patients experienced DLCO recovery. Dyspnea and six-minute walk test results did not correlate with DLCO changes. Patients with a PFT-defined EOPE had significantly higher levels of activated neutrophils at baseline and day 8. Conclusions DLCO reduction occurred in 90% during the first 8 days of brigatinib dosing without any related symptoms. DLCO improved in all six patients assessed at day 15 despite continued dosing and dose escalation. Pretreatment levels of neutrophil activation should be explored as a biomarker for developing EOPEs.

Lung cancer is a heterogeneous disease in which patient-specific treatments are desirable and the development of targeted therapies has been effective. Although mutations in KRAS are frequent in lung adenocarcinoma, there are currently no targeted agents against KRAS. Using a mouse lung adenocarcinoma cell line with a Kras mutation (CMT167), we previously showed that PPARγ activation in lung cancer cells inhibits cell growth in vitro yet promotes tumor progression when activated in myeloid cells of the tumor microenvironment. Here, we report that PPARγ activation in myeloid cells promotes the production of TGFβ1, which, in turn, acts on CMT167 cancer cells to increase migration and induce an epithelial–mesenchymal transition (EMT). Targeting TGFβ1 signaling in CMT167 cells prevented their growth and metastasis in vivo. Similarly, another mouse lung adenocarcinoma cell line with a Kras mutation, LLC, induced TGFβ1 in myeloid cells through PPARγ activation. However, LLC cells are more mesenchymal and did not undergo EMT in response to TGFβ1, nor did LLC require TGFβ1 signaling for metastasis in vivo. Converting CMT167 cells to a mesenchymal phenotype through overexpression of ZEB1 made them unresponsive to TGFβ1 receptor inhibition. The ability of TGFβ1 to induce EMT in lung tumors may represent a critical process in cancer progression. We propose that TGFβ receptor inhibition could provide an additional treatment option for KRAS-mutant epithelial lung tumors. Implications: This study suggests that TGFβ receptor inhibitors may be an effective therapy in a subset of KRAS-mutant patients with non–small cell lung cancer, which show an epithelial phenotype.

Introduction In patients with NSCLC, the prognostic significance of the tumor microenvironment (TME) immune composition has been revealed using single- or dual-marker staining on sequential tissue sections. Although these studies reveal that relative abundance and localization of immune cells are important parameters, deeper analyses of the NSCLC TME are necessary to refine the potential application of these findings to clinical care. Currently, the complex spatial relationships between cells of the NSCLC TME and potential drivers contributing to its immunologic composition remain unknown. Methods We used multispectral quantitative imaging on the lung adenocarcinoma TME in 153 patients with resected tumors. On a single slide per patient, we evaluated the TME with markers for CD3, CD8, CD14, CD19, major histocompatibility complex II (MHCII), cytokeratin, and 4′,6-diamidino-2-phenylindole (DAPI). Image analysis, including tissue segmentation, phenotyping, and spatial localization, was performed. Results Specimens wherein greater than or equal to 5% of lung cancer cells expressed MHCII (MHCIIhi TME) had increased levels of CD4+ and CD8+ T cells and CD14+ cell infiltration. In the MHCIIhi TME, the immune infiltrate was closer to cancer cells and expressed an activated phenotype. Morphologic image analysis revealed cancer cells in the MHCIIhi TME more frequently interfaced with CD4+ and CD8+ T cells. Patients with an MHCIIhi TME experienced improved overall survival (p = 0.046). Conclusions Lung cancer cell-specific expression of MHCII associates with levels of immune cell infiltration, spatial localization, and activation status within the TME. This suggests that cancer cell-specific expression of MHCII may represent a biomarker for the immune system’s recognition and activation against the tumor.

Eicosanoids represent a family of active biolipids derived from arachidonic acid primarily through the action of cytosolic phospholipase A2-α. Three major downstream pathways have been defined: the cyclooxygenase (COX) pathway which produces prostaglandins and thromboxanes; the 5-lipoxygenase pathway (5-LO), which produces leukotrienes, lipoxins and hydroxyeicosatetraenoic acids, and the cytochrome P450 pathway which produces epoxygenated fatty acids. In general, these lipid mediators are released and act in an autocrine or paracrine fashion through binding to cell surface receptors. The pattern of eicosanoid production is cell specific, and is determined by cell-specific expression of downstream synthases. Increased eicosanoid production is associated with inflammation and a panel of specific inhibitors have been developed designated non-steroidal anti-inflammatory drugs. In cancer, eicosanoids are produced both by tumor cells as well as cells of the tumor microenvironment. Earlier studies demonstrated that prostaglandin E2, produced through the action of COX-2, promoted cancer cell proliferation and metastasis in multiple cancers. This resulted in the development of COX-2 inhibitors as potential therapeutic agents. However, cardiac toxicities associated with these agents limited their use as therapeutic agents. The advent of immunotherapy, especially the use of immune checkpoint inhibitors has revolutionized cancer treatment in multiple malignancies. However, the majority of patients do not respond to these agents as monotherapy, leading to intense investigation of other pathways mediating immunosuppression in order to develop rational combination therapies. Recent data have indicated that PGE2 has immunosuppressive activity, leading to renewed interest in targeting this pathway. However, little is known regarding the role of other eicosanoids in modulating the tumor microenvironment, and regulating anti-tumor immunity. This article reviews the role of eicosanoids in cancer, with a focus on their role in modulating the tumor microenvironment. While the role of PGE2 will be discussed, data implicating other eicosanoids, especially products produced through the lipoxygenase and cytochrome P450 pathway will be examined. The existence of small molecular inhibitors and activators of eicosanoid pathways such as specific receptor blockers make them attractive candidates for therapeutic trials, especially in combination with novel immunotherapies such as immune checkpoint inhibitors.

BackgroundProgrammed death 1/programmed death ligand 1 (PD-1/PD-L1) targeted immunotherapy affords clinical benefit in ~20% of unselected patients with lung cancer. The factor(s) that determine whether a tumor responds or fails to respond to immunotherapy remains an active area of investigation. We have previously defined divergent responsiveness of two KRAS-mutant cell lines to PD-1/PD-L1 blockade using an orthotopic, immunocompetent mouse model. Responsiveness to PD-1/PD-L1 checkpoint blockade correlates with an interferon gamma (IFNγ)-inducible gene signature and major histocompatibility complex class II (MHC II) expression by cancer cells. In the current study, we aim to identify therapeutic targets that can be manipulated in order to enhance cancer-cell-specific MHC II expression.MethodsResponsiveness to IFNγ and induction of MHC II expression was assessed after various treatment conditions in mouse and human non-small cell lung cancer (NSCLC) cell lines using mass cytometric and flow cytometric analysis.ResultsSingle-cell analysis using mass and flow cytometry demonstrated that IFNγ consistently induced PD-L1 and MHC class I (MHC I) across multiple murine and human NSCLC cell lines. In contrast, MHC II showed highly variable induction following IFNγ treatment both between lines and within lines. In mouse models of NSCLC, MHC II induction was inversely correlated with basal levels of phosphorylated extracellular signal-regulated kinase (ERK) 1/2, suggesting potential mitogen-activated protein (MAP) kinase-dependent antagonism of MHC II expression. To test this, cell lines were subjected to varying levels of stimulation with IFNγ, and assessed for MHC II expression in the presence or absence of mitogen-activated protein kinase kinase (MEK) inhibitors. IFNγ treatment in the presence of MEK inhibitors significantly enhanced MHC II induction across multiple lung cancer lines, with minimal impact on expression of either PD-L1 or MHC I. Inhibition of histone deacetylases (HDACs) also enhanced MHC II expression to a more modest extent. Combined MEK and HDAC inhibition led to greater MHC II expression than either treatment alone.ConclusionsThese studies emphasize the active inhibitory role that epigenetic and ERK signaling cascades have in restricting cancer cell-intrinsic MHC II expression in NSCLC, and suggest that combinatorial blockade of these pathways may engender new responsiveness to checkpoint therapies.

MHC class II (MHCII) expression is usually restricted to antigen presenting cells, but can be expressed by cancer cells. We examined the effect of cancer cell-intrinsic MHC class II (csMHCII) expression in lung adenocarcinoma on T cell recruitment to tumors and response to anti-PD-1 therapy. The functional significance of altering csMHCII expression was explored using two orthotopic immunocompetent murine models of non-small cell lung cancer: CMT167 (CMT) and Lewis Lung Carcinoma (LLC). We previously showed that CMT167 tumors are eradicated by anti-PD1 therapy, while LLC tumors are resistant. RNA-seq analysis of cancer cells recovered from tumors revealed that csMHCII correlated with response to anti-PD1 therapy, with immunotherapy-sensitive CMT167 cells being csMHCII positive, while resistant LLC cells were csMHCII negative. To test the functional effects of csMHCII, MHCII expression was altered on the cancer cells through loss- and gain-of-function of CIITA, a master regulator of the MHCII pathway. Loss of CIITA in CMT167 decreased csMHCII, and converted tumors from anti-PD-1-sensitive to anti-PD-1-resistant. This was associated with decreased T cell infiltration, lower levels of Th1 cytokines, increased B cell number and decreased macrophage recruitment. Conversely, overexpression of CIITA in LLC cells resulted in csMHCII in vitro and in vivo. Enforced expression of CIITA increased T cell infiltration and sensitized tumors to anti-PD-1 therapy. csMHCII expression was also examined in a subset of surgically resected human lung adenocarcinomas by multispectral imaging, provided a survival benefit and positively correlated with T cell infiltration. These studies demonstrate a functional role for csMHCII in regulating T cell infiltration and sensitivity to anti-PD-1.

The complement cascade is a part of the innate immune system that acts primarily to remove pathogens and injured cells. However, complement activation is also peculiarly associated with tumor progression. Here we report mechanistic insights into this association in multiple immunocompetent orthotopic models of lung cancer. After tumor engraftment, we observed systemic activation of the complement cascade as reflected by elevated levels of the key regulator C3a. Notably, growth of primary tumors and metastases was both strongly inhibited in C3-deficient mice (C3−/− mice), with tumors undetectable in many subjects. Growth inhibition was associated with increased numbers of IFNγ+/TNFα+/IL10+ CD4+ and CD8+ T cells. Immunodepletion of CD4+ but not CD8+ T cells in tumor-bearing subjects reversed the inhibitory effects of C3 deletion. Similarly, antagonists of the C3a or C5a receptors inhibited tumor growth. Investigations using multiple tumor cell lines in the orthotopic model suggested the involvement of a C3/C3 receptor autocrine signaling loop in regulating tumor growth. Overall, our findings offer functional evidence that complement activation serves as a critical immunomodulator in lung cancer progression, acting to drive immune escape via a C3/C5–dependent pathway. Significance: This provocative study suggests that inhibiting complement activation may heighten immunotherapeutic responses in lung cancer, offering findings with immediate implications, given the existing clinical availability of complement antagonists. Cancer Res; 78(1); 143–56. ©2017 AACR.

Congenital myasthenic syndromes (CMSs) are genetic disorders characterized by a faulty neuromuscular junction causing muscle weakness with or without respiratory and oculomotor involvement. Congenital myopathies (CMs) can present similarly to CMS but are secondary to muscle protein defects. CMSs cause muscular weakness, hypotonia, and ptosis and can respond to treatment1,2; CMs have no proven therapies. We present a patient with DOK7 CMS, previously diagnosed as CM for 25 years.

Targeting PD-1/ PD-L1 is only effective in ~20% of lung cancer patients, but determinants of this response are poorly defined. We previously observed differential responses of two murine K-Ras lung cancer cell lines to anti-PD-1 therapy: CMT167 tumors were eliminated while LLC tumors were resistant. The goal of this study was to define mechanism(s) mediating this difference. RNA-Seq analysis of cancer cells recovered from lung tumors revealed that CMT167 cells induced an IFNγ signature that was absent in LLC cells. Silencing Ifngr1 in CMT167 resulted in tumors resistant to IFNγ and anti-PD-1 therapy. Conversely, LLC cells had high basal expression of Socs1, an inhibitor of IFNγ. Silencing Socs1 increased response to IFNγ in vitro and sensitized tumors to anti-PD-1. This was associated with a reshaped TME, characterized by enhanced T cell infiltration and enrichment of PD-L1 high myeloid cells. These studies demonstrate that targeted enhancement of tumor-intrinsic IFNγ signaling can induce of cascade of changes associated with increased therapeutic vulnerability.SummaryMechanisms regulating response to anti-PD-1 therapy in lung cancer are not well defined. This study, using orthotopic immunocompetent mouse models of lung cancer, demonstrates that intrinsic sensitivity of cancer cells to IFNγ determines anti-PD-1 responsiveness through alterations in the tumor microenvironment.

Dietary specialists tend to be less susceptible to the effects of chemical defenses produced by their prey compared to generalist predators that feed upon a broader range of prey species. While many researchers have investigated the ability of insects to detoxify dietary allelochemicals, little research has been conducted in marine ecosystems. We investigated metabolic detoxification pathways in three species of butterflyfishes: the hard coral specialist feeder, Chaetodon multicinctus, and two generalist feeders, Chaetodon auriga and Chaetodon kleinii. Each species was fed tissue homogenate of the hard coral Porites lobata or the feeding deterrent compound homarine (found in the coral extract), and the expression and catalytic activity of cytochrome P450 (CYP) 3A-like and CYP2-like enzymes were examined after one-week of treatment. The P. lobata homogenate significantly induced content and catalytic activity of CYP2-like and CYP3A-like forms, by 2-3 fold and by 3-9 fold, respectively, in C. multicinctus. Homarine caused a significant decrease of CYP2-like and CYP3A-like proteins at the high dose in C. kleinii and 60-80% mortality in that species. Homarine also induced CYP3A-like content by 3-fold and catalytic activity by 2-fold in C. auriga, while causing non-monotonic increases in CYP2-like and CYP3A-like catalytic activity in C. multicinctus. Our results indicate that dietary exposure to coral homogenates and the feeding deterrent constituent within these homogenates caused species-specific modulation of detoxification enzymes consistent with the prey selection strategies of generalist and specialist butterflyfishes.

9538 Background: Phase I-III studies reported symptomatic pulmonary toxicity within the first week of initiating brigatinib in 6% patients post-crizotinib and 3% in TKI naive patients with standard dosing (90mg QD for 7 days then 180mg QD as tolerated). A prospective observational study of pulmonary function testing (PFT) on initiating brigatinib was conducted. Methods: Patients PS≤2, with resting O2 sats on RA ≥90% and Hg ≥10 g/dL, without significant heart/lung disease or steroid use initiating brigatinib 90 mg QD were eligible. PFT with DLCO, Borg dyspnea and 6-minute walk tests were performed at baseline (prior to brigatinib), and on day 2 (D2), 8 (D8), and 15 (D15) of brigatinib. D15 analyses were initially as clinically indicated but became mandatory if DLCO had not returned to baseline by D8. Peripheral blood was collected at baseline, D2 and D8 for CyTOF analysis. The primary endpoint was the incidence of Early Onset Pulmonary Events (EOPEs), defined as a DLCO reduction of ≥ 20% from baseline. An interim analysis was performed on the first 10 patients due to a higher than expected incidence of DLCO reduction. Results: D2 and D8 measurements were captured in all 10 patients, D15 in 7 patients. Ninety percent (9/10) of patients experienced DLCO reduction with nadir occurring on D2 in 4/9 and on D8 in 5/9 patients. Median DLCO nadir was −13.33% from baseline (range: −34.44 to −5.00). Three patients (30%) met EOPE criteria, all on D8, all without symptoms. Brigatinib was not held and all 10 patients escalated to 180mg on D8. Despite continued dosing, 4/9 patients recovered DLCO to baseline or above by D15 (2/3 EOPEs cases), 2/9 recovered above nadir but below baseline by D15 (1/3 EOPE case), and 3/9 did not have improvement from nadir values but no D15 assessment was performed. Dyspnea and 6-minute walk test did not correlate with DLCO changes. Patients who experienced an EOPE had significantly higher levels of activated neutrophils (pERKhi) at baseline. On the day of the EOPE event, patients who met EOPE criteria had significantly higher levels of activated neutrophils and fewer activated CD4+ effector memory T cells. Conclusions: Modest DLCO reduction occurred in 90% (9/10) patients during the first 8 days of brigatinib-dosing without associated symptoms. When rechecked on D15, DLCO improved in 100% patients (6/6) despite continued dosing and standard dose escalation at D8. Patients unlikely to tolerate even this modest, short-lived change should consider shallower step-up dosing or alternative drugs. CyTOF analysis suggests levels of pretreatment neutrophils may be a biomarker for developing EOPEs. Clinical trial information: NCT03389399 .

Immune checkpoint inhibitors targeting PD-1/PD-L1 interactions have shown clinical efficacy in the treatment of multiple cancer, including non-small cell lung cancer (NSCLC). However, the factors determining the extent and duration of therapy are incompletely understood. While antigen presentation via MHC class I interacting with CD8+ T cells has been characterized, the role of MHC class II (MHCII) interacting with CD4+ T cells is less well defined. MHCII expression is usually restricted to antigen-presenting cells, but can be expressed by cancer cells. We examined the effect of cancer cell-intrinsic MHC class II (csMHCII) expression in lung adenocarcinoma on T-cell recruitment to tumors and response to anti-PD-1 therapy. The functional significance of altering csMHCII expression was explored using two orthotopic immunocompetent murine models of non-small cell lung cancer: CMT167 (CMT) and Lewis Lung Carcinoma (LLC). We previously showed that CMT167 tumors are eradicated by anti-PD1 therapy, while LLC tumors are resistant. RNA-seq analysis of cancer cells recovered from tumors revealed that csMHCII correlated with response to anti-PD1 therapy, with immunotherapy-sensitive CMT167 cells being csMHCII positive, while resistant LLC cells were csMHCII negative. To test the functional effects of csMHCII, MHCII expression was altered on the cancer cells through loss and gain of function of CIITA, a master regulator of the MHCII pathway. Loss of CIITA in CMT167 decreased csMHCII and converted tumors from immunotherapy sensitive to immunotherapy resistant. This was associated with decreased T-cell infiltration and lower levels of Th1 cytokines. Conversely, overexpression of CIITA in LLC cells resulted in csMHCII in vitro and in vivo. Enforced expression of CIITA increased T-cell infiltration and sensitized tumors to anti-PD-1 therapy. csMHCII expression was also examined in a subset of surgically resected human lung adenocarcinomas by multispectral imaging, and positively correlated with T-cell infiltration. These studies demonstrate a functional role for csMHCII in regulating T-cell infiltration and sensitivity to anti-PD-1. Citation Format: Amber M. Johnson, Bonnie L. Bulock, Alexander J. Neuwelt, Erin L. Schenk, Eric T. Clambey, Raphael A. Nemenoff. Cancer cell-intrinsic expression of MHCII regulates the immune microenvironment and response to immune therapy in lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A20.

BACKGROUND The goal of this study was to determine a set of timing, shape, and statistical features available through noninvasive monitoring of maternal electrocardiogram and photoplethysmography that identifies preeclamptic patients. METHODS Pregnant women admitted to Labor and Delivery were monitored with pulse oximetry and electrocardiogram for 30 minutes. Photoplethysmogram features and heart rate variability were extracted from each data set and applied to a sequential feature selection algorithm to discriminate women with preeclampsia with severe features, from normotensive and hypertensive controls. The classification boundary was chosen to minimize the expected misclassification cost. The prior probabilities of the misclassification costs were assumed to be equal. RESULTS Thirty-seven patients with clinically diagnosed preeclampsia with severe features were compared with 43 normotensive controls; all were in early labor or beginning induction. Six variables were used in the final model. The area under the receiver operating characteristic curve was 0.907 (standard error [SE] = 0.004) (sensitivity 78.2% [SE = 0.3%], specificity 89.9% [SE = 0.1%]) with a positive predictive value of 0.883 (SE = 0.001). Twenty-eight subjects with chronic or gestational hypertension were compared with the same preeclampsia group, generating a model with 5 features with an area under the curve of 0.795 (SE = 0.007; sensitivity 79.0% [SE = 0.2%], specificity 68.7% [SE = 0.4%]), and a positive predictive value of 0.799 (SE = 0.002). CONCLUSIONS Vascular parameters, as assessed noninvasively by photoplethysmography and heart rate variability, may have a role in screening women suspected of having preeclampsia, particularly in areas with limited resources.

Single-protein molecules are identified in parallel through fluorescence monitoring of Edman degradation. The identification and quantification of proteins lags behind DNA-sequencing methods in scale, sensitivity, and dynamic range. Here, we show that sparse amino acid–sequence information can be obtained for individual protein molecules for thousands to millions of molecules in parallel. We demonstrate selective fluorescence labeling of cysteine and lysine residues in peptide samples, immobilization of labeled peptides on a glass surface, and imaging by total internal reflection microscopy to monitor decreases in each molecule's fluorescence after consecutive rounds of Edman degradation. The obtained sparse fluorescent sequence of each molecule was then assigned to its parent protein in a reference database. We tested the method on synthetic and naturally derived peptide molecules in zeptomole-scale quantities. We also fluorescently labeled phosphoserines and achieved single-molecule positional readout of the phosphorylated sites. We measured >93% efficiencies for dye labeling, survival, and cleavage; further improvements should enable studies of increasingly complex proteomic mixtures, with the high sensitivity and digital quantification offered by single-molecule sequencing.

Genomic testing is increasingly performed in oncology, but concerns remain regarding the clinician's ability to interpret results. In the current study, the authors sought to determine the agreement between physicians and genomic annotators from the Precision Oncology Decision Support (PODS) team at The University of Texas MD Anderson Cancer Center in Houston regarding actionability and the clinical use of test results.On a prospective protocol, patients underwent clinical genomic testing for hotspot mutations in 46 or 50 genes. Six months after sequencing, physicians received questionnaires for patients who demonstrated a variant in an actionable gene, investigating their perceptions regarding the actionability of alterations and clinical use of these findings. Genomic annotators independently classified these variants as actionable, potentially actionable, unknown, or not actionable.Physicians completed 250 of 288 questionnaires (87% response rate). Physicians considered 168 of 250 patients (67%) as having an actionable alteration; of these, 165 patients (98%) were considered to have an actionable alteration by the PODS team and 3 were of unknown significance. Physicians were aware of genotype-matched therapy available for 119 patients (71%) and 48 of these 119 patients (40%) received matched therapy. Approximately 46% of patients in whom physicians regarded alterations as not actionable (36 of 79 patients) were classified as having an actionable/potentially actionable mutation by the PODS team. However, many of these were only theoretically actionable due to limited trials and/or therapies (eg, KRAS).Physicians are aware of recurrent mutations in actionable genes on "hotspot" panels. As larger genomic panels are used, there may be a growing need for annotation of actionability. Decision support to increase awareness of genomically relevant trials and novel treatment options for recurrent mutations (eg, KRAS) also are needed. Cancer 2018;124:966-72. © 2017 American Cancer Society.

Macrocyclic peptides are an increasingly important class of biopharmaceuticals. A new method of macrocyclization is reported that involves reaction of the N-terminal amine and thiol side chain of cysteine with a Meldrum’s acid derived conjugate acceptor. This reaction, which utilizes naturally occurring amino acids and requires no orthogonal protection of side chains, can also be reversed to yield the original linear peptide as desired.

Using an immunocompetent mouse model of NSCLC, this study demonstrates that tumor-intrinsic response to IFNγ determines response to anti–PD-1 through alterations in the tumor microenvironment., Targeting PD-1/PD-L1 is only effective in ∼20% of lung cancer patients, but determinants of this response are poorly defined. We previously observed differential responses of two murine K-Ras–mutant lung cancer cell lines to anti–PD-1 therapy: CMT167 tumors were eliminated, whereas Lewis Lung Carcinoma (LLC) tumors were resistant. The goal of this study was to define mechanism(s) mediating this difference. RNA sequencing analysis of cancer cells recovered from lung tumors revealed that CMT167 cells induced an IFNγ signature that was blunted in LLC cells. Silencing Ifngr1 in CMT167 resulted in tumors resistant to IFNγ and anti–PD-1 therapy. Conversely, LLC cells had high basal expression of SOCS1, an inhibitor of IFNγ. Silencing Socs1 increased response to IFNγ in vitro and sensitized tumors to anti–PD-1. This was associated with a reshaped tumor microenvironment, characterized by enhanced T cell infiltration and enrichment of PD-L1hi myeloid cells. These studies demonstrate that targeted enhancement of tumor-intrinsic IFNγ signaling can induce a cascade of changes associated with increased therapeutic vulnerability.

Metal-selective self-assembly with rare-earth cations is possible with suitable rigid, symmetrical bis-tridentate ligands. Kinetically controlled formation is initially observed, with smaller cations preferentially incorporated. Over time, the more thermodynamically favorable complexes with larger metals are formed. This thermodynamic control is a cooperative supramolecular phenomenon and only occurs upon multiple-metal-based self-assembly: single-metal ML3 analogues do not show reversible selectivity. The selectivity is dependent on small variations in lanthanide ionic radius and occurs despite identical coordination-ligand coordination geometries and minor size differences in the rare-earth metals.

Reactive alcohol functionality has been incorporated into a self-assembled M2L3 mesocate. Post-synthetic modification of this complex with suitable isocyanates is not only possible, but is self-catalyzed by multiple internal hydrogen bonds from the self-assembly. As the metal-ligand coordination is reversible at elevated temperature, the isomeric distribution of product changes upon reaction, due to the different steric bulk conferred on the assembly after the modification.

Lung cancer is the leading cause of cancer-related deaths worldwide and the second most common cancer among both men and women in the United States. Immunotherapy regimens that disrupt the inhibitory PD-1/PD-L1 axis have recently emerged as promising treatments that can unleash anti-tumor immunity against a wide range of cancers, including a subset of lung cancers. How these therapies elicit potent anti-tumor immunity remains controversial, with markers for immunotherapy-sensitivity still an active area of investigation. To study determinants of lung cancer growth in vivo we studied two orthotopic mouse models of non-small cell lung cancer (NSCLC). These Kras-mutant tumor lines dramatically differ in their response to immunotherapy: CMT167 tumor cells are exquisitely susceptible to PD-1/PD-L1 targeted immunotherapy whereas LLC tumor cells are immunotherapy-resistant. Immunotherapy-sensitive cells showed induction of the MHC class II antigen processing and presentation pathway in vitro and in vivo, and were capable of stimulating CD4 T cells directly in ex vivo co-culture. To study the impact of tumor cell-intrinsic MHC class II induction, we used RNA interference to knockdown CIITA, a transcriptional inducer of the MHC class II pathway. CIITA knockdown in immunotherapy-sensitive cells impaired the induction of tumor cell-intrinsic MHC class II in vitro and abrogated CD4 T cell stimulation in co-culture. CIITA knockdown further converted tumors from immunotherapy-sensitive to immunotherapy-resistant. These studies identify tumor cell-intrinsic expression of MHC class II as a potential direct target for CD4 T cell recognition that may promote sensitivity to PD-1/PD-L1 blockade.

EnterotoxigenicEscherichia coli(ETEC) is a common cause of travelers' and postweaning diarrhea in humans and swine, respectively. The extent to which ETEC damages host cells is unclear. Experiments are presented that probe the ability of porcine ETEC isolates to induce apoptosis and cell death in porcine intestinal epithelial cells. Quantification of host phosphatidylserine exposure following ETEC infection suggested that ETEC induced changes in plasma membrane asymmetry, independent of the expression of the heat-labile enterotoxin. Significant host cell death was not observed. ETEC infection also caused a drastic inhibition of host esterase activity, as measured by calcein fluorescence. While ETEC infection resulted in activation of host caspase 3, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling of DNA double-strand breakage, indicative of late stages of apoptosis, was not observed. Camptothecin-induced apoptosis markedly increased subsequent ETEC adherence. Transfer of cell-free supernatants from apoptotic cells to bacterial inocula prior to infection of naïve cells increased the transcriptional activity of the regulatory region upstream of the K88ac operon and promoted subsequent adherence to host cells.