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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.

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.

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.