Diminished Immune Surveillance during Histologic Progression of Intraductal Papillary Mucinous Neoplasms Offers a Therapeutic Opportunity for Cancer Interception.

PURPOSE: Intraductal papillary mucinous neoplasms (IPMN) are bona fide precursors to pancreatic ductal adenocarcinoma (PDAC). While genomic alterations during multistep IPMN progression have been well cataloged, the accompanying changes within the tumor immune microenvironment (TIME) have not been comprehensively studied. Herein, we investigated TIME-related alterations during IPMN progression, using multiplex immunofluorescence (mIF) coupled with high-resolution image analyses. EXPERIMENTAL DESIGN: Two sets of formalin-fixed, paraffin-embedded tissue samples from surgically resected IPMNs were analyzed. The training set of 30 samples consisted of 11 low-grade IPMN (LG-IPMN), 17 high-grade IPMN (HG-IPMN), and 2 IPMN with PDAC, while a validation set of 93 samples comprised of 55 LG-IPMN and 38 HG-IPMN. The training set was analyzed with two panels of immuno-oncology-related biomarkers, while the validation set was analyzed with a subset of markers found significantly altered in the training set. RESULTS: Cell types indicative of enhanced immune surveillance, including cytotoxic and memory T cells, and antigen-experienced T cells and B cells, were all found at higher densities within isolated LG-IPMNs compared with HG-IPMNs. Notably, the TIME of LG-IPMNs that had progressed at the time of surgical resection (progressor LGD) resembled that of the synchronous HG-IPMNs, underscoring that attenuated immune surveillance occurs even in LG-IPMNs destined for progression. CONCLUSIONS: Our findings provide a basis for interception of cystic neoplasia to PDAC, through maintenance of sustained immune surveillance using vaccines and other prevention approaches.

SRGN-Triggered Aggressive and Immunosuppressive Phenotype in a Subset of TTF-1-Negative Lung Adenocarcinomas.

BACKGROUND: Approximately 20% of lung adenocarcinoma (LUAD) is negative for the lineage-specific oncogene Thyroid transcription factor 1 (TTF-1) and exhibits worse clinical outcome with a low frequency of actionable genomic alterations. To identify molecular features associated with TTF-1-negative LUAD, we compared the transcriptomic and proteomic profiles of LUAD cell lines. SRGN , a chondroitin sulfate proteoglycan Serglycin, was identified as a markedly overexpressed gene in TTF-1-negative LUAD. We therefore investigated the roles and regulation of SRGN in TTF-1-negative LUAD. METHODS: Proteomic and metabolomic analyses of 41 LUAD cell lines were done using mass spectrometry. The function of SRGN was investigated in 3 TTF-1-negative and 4 TTF-1-positive LUAD cell lines and in a syngeneic mouse model (n = 5 to 8 mice per group). Expression of SRGN was evaluated in 94 and 105 surgically resected LUAD tumor specimens using immunohistochemistry. All statistical tests were 2-sided. RESULTS: SRGN was markedly overexpressed at mRNA and protein levels in TTF-1-negative LUAD cell lines (P < .001 for both mRNA and protein levels). Expression of SRGN in LUAD tumor tissue was associated with poor outcome (hazard ratio = 4.22, 95% confidence interval = 1.12 to 15.86, likelihood ratio test, P = .03), and with higher expression of Programmed cell death 1 ligand 1 (PD-L1) in tumor cells and higher infiltration of Programmed cell death protein 1-positive lymphocytes. SRGN regulated expression of PD-L1 as well as proinflammatory cytokines, including Interleukin-6, Interleukin-8, and C-X-C motif chemokine 1 in LUAD cell lines; increased migratory and invasive properties of LUAD cells and fibroblasts; and enhanced angiogenesis. SRGN was induced by DNA demethylation resulting from Nicotinamide N-methyltransferase-mediated impairment of methionine metabolism. CONCLUSIONS: Our findings suggest that SRGN plays a pivotal role in tumor-stromal interaction and reprogramming into an aggressive and immunosuppressive tumor microenvironment in TTF-1-negative LUAD.

CD8+ T cells inhibit metastasis and CXCL4 regulates its function.

BACKGROUND: The mechanism by which immune cells regulate metastasis is unclear. Understanding the role of immune cells in metastasis will guide the development of treatments improving patient survival. METHODS: We used syngeneic orthotopic mouse tumour models (wild-type, NOD/scid and Nude), employed knockout (CD8 and CD4) models and administered CXCL4. Tumours and lungs were analysed for cancer cells by bioluminescence, and circulating tumour cells were isolated from blood. Immunohistochemistry on the mouse tumours was performed to confirm cell type, and on a tissue microarray with 180 TNBCs for human relevance. TCGA data from over 10,000 patients were analysed as well. RESULTS: We reveal that intratumoral immune infiltration differs between metastatic and non-metastatic tumours. The non-metastatic tumours harbour high levels of CD8+ T cells and low levels of platelets, which is reverse in metastatic tumours. During tumour progression, platelets and CXCL4 induce differentiation of monocytes into myeloid-derived suppressor cells (MDSCs), which inhibit CD8+ T-cell function. TCGA pan-cancer data confirmed that CD8lowPlatelethigh patients have a significantly lower survival probability compared to CD8highPlateletlow. CONCLUSIONS: CD8+ T cells inhibit metastasis. When the balance between CD8+ T cells and platelets is disrupted, platelets produce CXCL4, which induces MDSCs thereby inhibiting the CD8+ T-cell function.

Contextual cues from cancer cells govern cancer-associated fibroblast heterogeneity.

Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis.

CD73 expression defines immune, molecular, and clinicopathological subgroups of lung adenocarcinoma.

INTRODUCTION: CD73 is a membrane-bound enzyme crucial in adenosine generation. The adenosinergic pathway plays a critical role in immunosuppression and in anti-tumor effects of immune checkpoint inhibitors (ICI). Here, we interrogated CD73 expression in a richly annotated cohort of human lung adenocarcinoma (LUAD) and its association with clinicopathological, immune, and molecular features to better understand the role of this immune marker in LUAD pathobiology. MATERIALS AND METHODS: Protein expression of CD73 was evaluated by immunohistochemistry in 106 archived LUADs from patients that underwent surgical treatment without neoadjuvant therapy. Total CD73 (T +) was calculated as the average of luminal (L +) and basolateral (BL +) percentage membrane expression scores for each LUAD and was used to classify tumors into three groups based on the extent of T CD73 expression (high, low, and negative). RESULTS: CD73 expression was significantly and progressively increased across normal-appearing lung tissue, adenomatous atypical hyperplasia, adenocarcinoma in situ, minimally invasive adenocarcinoma, and LUAD. In LUAD, BL CD73 expression was associated with an increase in PD-L1 expression in tumor cells and increase of tumor-associated immune cells. Stratification of LUADs based on T CD73 extent also revealed that tumors with high expression of this enzyme overall exhibited significantly elevated immune infiltration and PD-L1 protein expression. Immune profiling demonstrated that T-cell inflammation and adenosine signatures were significantly higher in CD73-expressing lung adenocarcinomas relative to those lacking CD73. CONCLUSION: Our study suggests that higher CD73 expression is associated with an overall augmented host immune response, suggesting potential implications in the immune pathobiology of early stage lung adenocarcinoma. Our findings warrant further studies to explore the role of CD73 in immunotherapeutic response of LUAD.

Female Gender Predicts Augmented Immune Infiltration in Lung Adenocarcinoma.

INTRODUCTION: Immune infiltration in lung adenocarcinomas (LUADs) has been associated with response to immune checkpoint inhibitors. Clinical features underlying differential responses of patients with LUADs to immunotherapy are not well understood. Here, we analyzed the association between LUAD immune infiltration and clinicopathologic variables. MATERIALS AND METHODS: Intratumoral CD3, CD8, and CD68 cell densities (tumor-associated immune cells [TAICs]) were immunohistochemically assessed in 146 surgically resected LUADs. LUADs were classified into 2 groups, low and high TAICs, based on the median values of cell densities for CD3, CD8, and CD68. Somatic mutation burden and driver gene mutation status were analyzed in a subset of the cases (n = 92). We statistically analyzed the association between the TAIC groups and various clinicopathologic and molecular variables by using the χ2/Fisher and Wilcoxon sum tests and multivariable logistic regression models. RESULTS: Patient gender, tumor size, and STK11 mutations were significantly associated with TAIC levels in LUAD. Female patients exhibited significantly elevated TAIC levels (P = .005) compared with male patients. Tumor size was inversely associated with TAIC levels (P = .012). STK11 mutated tumors were associated with lower TAICs (P = .008). Higher TAICs were consistently observed in female patients with LUADs after adjusting for stage, tumor size, and age. Multivariable regression models confirmed female gender as an independent variable associated with TAIC levels in LUAD (P = .0141). CONCLUSION: Immune infiltration in LUADs was significantly higher in female patients, warranting further exploration into the association between this clinical variable and immunotherapeutic response in LUAD.

An in vivo functional genomics screen of nuclear receptors and their co-regulators identifies FOXA1 as an essential gene in lung tumorigenesis.

Using a mini-library of 1062 lentiviral shRNAs targeting 40 nuclear hormone receptors and 70 of their co-regulators, we searched for potential therapeutic targets that would be important during in vivo tumor growth using a parallel in vitro and in vivo shRNA screening strategy in the non-small cell lung cancer (NSCLC) line NCI-H1819. We identified 21 genes essential for in vitro growth, and nine genes specifically required for tumor survival in vivo, but not in vitro: NCOR2, FOXA1, HDAC1, RXRA, RORB, RARB, MTA2, ETV4, and NR1H2. We focused on FOXA1, since it lies within the most frequently amplified genomic region in lung adenocarcinomas. We found that 14q-amplification in NSCLC cell lines was a biomarker for FOXA1 dependency for both in vivo xenograft growth and colony formation, but not mass culture growth in vitro. FOXA1 knockdown identified genes involved in electron transport among the most differentially regulated, indicating FOXA1 loss may lead to a decrease in cellular respiration. In support of this, FOXA1 amplification was correlated with increased sensitivity to the complex I inhibitor phenformin. Integrative ChipSeq analyses reveal that FOXA1 functions in this genetic context may be at least partially independent of NKX2-1. Our findings are consistent with a neomorphic function for amplified FOXA1, driving an oncogenic transcriptional program. These data provide new insight into the functional consequences of FOXA1 amplification in lung adenocarcinomas, and identify new transcriptional networks for exploration of therapeutic vulnerabilities in this patient population.

Stromal Hedgehog pathway activation by IHH suppresses lung adenocarcinoma growth and metastasis by limiting reactive oxygen species.

Activation of the Hedgehog (Hh) signaling pathway by mutations within its components drives the growth of several cancers. However, the role of Hh pathway activation in lung cancers has been controversial. Here, we demonstrate that the canonical Hh signaling pathway is activated in lung stroma by Hh ligands secreted from transformed lung epithelia. Genetic deletion of Shh, the primary Hh ligand expressed in the lung, in KrasG12D/+;Trp53fl/fl autochthonous murine lung adenocarcinoma had no effect on survival. Early abrogation of the pathway by an anti-SHH/IHH antibody 5E1 led to significantly worse survival with increased tumor and metastatic burden. Loss of IHH, another Hh ligand, by in vivo CRISPR led to more aggressive tumor growth suggesting that IHH, rather than SHH, activates the pathway in stroma to drive its tumor suppressive effects-a novel role for IHH in the lung. Tumors from mice treated with 5E1 had decreased blood vessel density and increased DNA damage suggestive of reactive oxygen species (ROS) activity. Treatment of KrasG12D/+;Trp53fl/fl mice with 5E1 and N-acetylcysteine, as a ROS scavenger, decreased tumor DNA damage, inhibited tumor growth and prolonged mouse survival. Thus, IHH induces stromal activation of the canonical Hh signaling pathway to suppress tumor growth and metastases, in part, by limiting ROS activity.

PI4KIIIß is a therapeutic target in chromosome 1q-amplified lung adenocarcinoma.

Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIß (PI4KIIIß). Molecular, biochemical, and cell biological studies show that PI4KIIIß-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIß-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIß antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIß-dependent secretion for cancer cell survival and tumor progression.

Procedural Requirements and Recommendations for Multiplex Immunofluorescence Tyramide Signal Amplification Assays to Support Translational Oncology Studies.

In the development of a multiplex immunofluorescence (IF) platform and the optimization and validation of new multiplex IF panels using a tyramide signal amplification system, several technical requirements are important for high-quality staining, analysis, and results. The aim of this review is to discuss the basic requirements for performing multiplex IF tyramide signal amplification (TSA) in formalin-fixed, paraffin-embedded cancer tissues to support translational oncology research. Our laboratory has stained approximately 4000 formalin-fixed, paraffin-embedded tumor samples using the multiplex IF TSA system for immune profiling of several labeled biomarkers in a single slide to elucidate cancer biology at a protein level and identify therapeutic targets and biomarkers. By analyzing several proteins in thousands of cells on a single slide, this technique provides a systems-level view of various processes in various tumor tissues. Although this technology shows high flexibility in cancer studies, it presents several challenges when applied to study different histology cancers. Our experience shows that adequate antibody validation, staining optimization, analysis strategies, and data generation are important steps for generating quality results. Tissue management, fixation procedures, storage, and cutting can also affect the results of the assay and must be standardized. Overall, this method is reliable for supporting translational research given a precise, step-by-step approach.

A novel patient-derived orthotopic xenograft model of esophageal adenocarcinoma provides a platform for translational discoveries.

Mouse models of gastroesophageal junction (GEJ) cancer strive to recapitulate the intratumoral heterogeneity and cellular crosstalk within patient tumors to improve clinical translation. GEJ cancers remain a therapeutic challenge due to the lack of a reliable mouse model for preclinical drug testing. In this study, a novel patient-derived orthotopic xenograft (PDOX) was established from GEJ cancer via transabdominal surgical implantation. Patient tumor was compared to subcutaneously implanted patient-derived tumor xenograft (PDX) and PDOX by Hematoxylin and Eosin staining, immunohistochemistry and next-generation sequencing. Treatment efficacy studies of radiotherapy were performed. We observed that mechanical abrasion of mouse GEJ prior to surgical implantation of a patient-derived tumor in situ promotes tumor engraftment (100%, n=6). Complete PDOX engraftment was observed with rapid intra- and extraluminal tumor growth, as evidenced by magnetic resonance imaging. PDOXs contain fibroblasts, tumor-associated macrophages, immune and inflammatory cells, vascular and lymphatic vessels. Stromal hallmarks of aggressive GEJ cancers are recapitulated in a GEJ PDOX mouse model. PDOXs demonstrate tumor invasion into vasculature and perineural space. Next-generation sequencing revealed loss of heterozygosity with very high allelic frequency in NOTCH3, TGFB1, EZH2 and KMT2C in the patient tumor, the subcutaneous PDX and the PDOX. Immunohistochemical analysis of Her2/neu (also known as ERBB2), p53 (also known as TP53) and p16 (also known as CDKN2A) in PDX and PDOX revealed maintenance of expression of proteins found in patient tumors, but membranous EGFR overexpression in patient tumor cells was absent in both xenografts. Targeted radiotherapy in this model suggested a decrease in size by 61% according to Response Evaluation Criteria in Solid Tumors (RECIST), indicating a partial response to radiation therapy. Our GEJ PDOX model exhibits remarkable fidelity to human disease and captures the precise tissue microenvironment present within the local GEJ architecture, providing a novel tool for translating findings from studies on human GEJ cancer. This model can be applied to study metastatic progression and to develop novel therapeutic approaches for the treatment of GEJ cancer.This article has an associated First Person interview with the first author of the paper.

Targeting CDK9 and MCL-1 by a new CDK9/p-TEFb inhibitor with and without 5-fluorouracil in esophageal adenocarcinoma.

BACKGROUND: CDK9 inhibitors are antitumorigenic against solid tumors, including esophageal adenocarcinoma (EAC). However, efficacy of a CDK9 inhibitor combined with 5-fluorouracil (5-FU) and target proteins that are targeted by these agents in EAC are unknown. METHODS: The anti-EAC efficacy of a new CDK9 inhibitor, BAY1143572, with and without 5-FU was assessed in vitro and in xenograft models in athymic nu/nu mice. Synergy between BAY1143572 and 5-FU in inhibiting cell proliferation was analyzed by calculating the combination index using CompuSyn software. Potential targets of BAY1143572 and 5-FU were identified by reverse-phase protein array. The effects of BAY1143572 and 5-FU on MCL-1 in vitro were analyzed by Western blotting, quantitative real-time polymerase chain reaction, and chromatin immunoprecipitation assay. MCL-1 protein expression in tumors from patients with locoregional EAC treated with chemoradiation and surgery was assessed by immunohistochemistry. RESULTS: BAY1143572 had dose-dependent antiproliferative and proapoptotic effects and demonstrated synergy with 5-FU against EAC in vitro. The median volumes of FLO-1 and ESO-26 xenografts treated with 5-FU plus BAY114352 were significantly smaller than those of xenografts treated with either agent alone (p < 0.05). BAY1143572 downregulated MCL-1 by inhibiting HIF-1α binding to the MCL-1 promoter. 5-FU enhanced BAY1143572-induced MCL-1 downregulation and stable MCL-1 overexpression reduced the apoptosis induced by BAY1143572 and 5-FU in vitro. High patients' tumor MCL-1 expression was correlated with shorter overall and recurrence-free survival. CONCLUSIONS: BAY1143572 and 5-FU have synergistic antitumorigenic effects against EAC. MCL-1 is a downstream target of CDK9 inhibitors and a predictor of response to neoadjuvant chemoradiation in EAC.

Targeting cyclin-dependent kinase 9 by a novel inhibitor enhances radiosensitization and identifies Axl as a novel downstream target in esophageal adenocarcinoma.

Cyclin-dependent kinase 9 (CDK9) transcriptionally regulates several proteins and cellular pathways central to radiation induced tissue injury. We investigated a role of BAY1143572, a new highly specific CDK9 inhibitor, as a sensitizer to radiation in esophageal adenocarcinoma. In vitro synergy between the CDK9 inhibitor and radiation was evaluated by clonogenic assay. In vivo synergy between the CDK9 inhibitor and radiation was assessed in multiple xenograft models including a patient's tumor derived xenograft (PDX). Reverse phase protein array (RPPA), western blotting, immunohistochemistry, and qPCR were utilized to identify and validate targets of the CDK9 inhibitor. The CDK9 inhibitor plus radiation significantly reduced growth of FLO-1, SKGT4, OE33, and radiation resistant OE33R xenografts and PDXs as compared to the cohorts treated with either single agent CDK9 inhibitor or radiation alone. RPPA identified Axl as a candidate target of CDK9 inhibition. Western blot and qPCR demonstrated reduced Axl mRNA (p = 0.02) and protein levels after treatment with CDK9 inhibitor with or without radiation in FLO-1 and SKGT4 cells. Axl protein expression in FLO-1 xenografts treated with combination of CDK9 inhibitor and radiation was significantly lower than the xenografts treated with radiation alone (p = 0.003). Clonogenic assay performed after overexpression of Axl in FLO-1 and SKGT4 cells enhanced radiosensitization by the CDK9 inhibitor, suggesting dependency of radiosensitization effects of the CDK9 inhibitor on Axl. In conclusion, these findings indicate that targeting CDK9 by BAY1143572 significantly enhances the effects of radiation and Axl is a novel downstream target of CDK9 in esophageal adenocarcinoma.

Tyrosine Threonine Kinase Inhibition Eliminates Lung Cancers by Augmenting Apoptosis and Polyploidy.

The spindle assembly checkpoint maintains genomic integrity. A key component is tyrosine threonine kinase (TTK, also known as Mps1). TTK antagonism is hypothesized to cause genomic instability and cell death. Interrogating The Cancer Genome Atlas revealed high TTK expression in lung adenocarcinomas and squamous cell cancers versus the normal lung (P < 0.001). This correlated with an unfavorable prognosis in examined lung adenocarcinoma cases (P = 0.007). TTK expression profiles in lung tumors were independently assessed by RNA in situ hybridization. CFI-402257 is a highly selective TTK inhibitor. Its potent antineoplastic effects are reported here against a panel of well-characterized murine and human lung cancer cell lines. Significant antitumorigenic activity followed independent treatments of athymic mice bearing human lung cancer xenografts (6.5 mg/kg, P < 0.05; 8.5 mg/kg, P < 0.01) and immunocompetent mice with syngeneic lung cancers (P < 0.001). CFI-402257 antineoplastic mechanisms were explored. CFI-402257 triggered aneuploidy and apoptotic death of lung cancer cells without changing centrosome number. Reverse phase protein arrays (RPPA) of vehicle versus CFI-402257-treated lung cancers were examined using more than 300 critical growth-regulatory proteins. RPPA bioinformatic analyses discovered CFI-402257 enhanced MAPK signaling, implicating MAPK antagonism in augmenting TTK inhibitory effects. This was independently confirmed using genetic and pharmacologic repression of MAPK that promoted CFI-402257 anticancer actions. TTK antagonism exerted marked antineoplastic effects against lung cancers and MAPK inhibition cooperated. Future work should determine whether CFI-402257 treatment alone or with a MAPK inhibitor is active in the lung cancer clinic.

ZEB1 suppression sensitizes KRAS mutant cancers to MEK inhibition by an IL17RD-dependent mechanism.

Mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitors have failed to show clinical benefit in Kirsten rat sarcoma (KRAS) mutant lung cancer due to various resistance mechanisms. To identify differential therapeutic sensitivities between epithelial and mesenchymal lung tumors, we performed in vivo small hairpin RNA screens, proteomic profiling, and analysis of patient tumor datasets, which revealed an inverse correlation between mitogen-activated protein kinase (MAPK) signaling dependency and a zinc finger E-box binding homeobox 1 (ZEB1)-regulated epithelial-to-mesenchymal transition. Mechanistic studies determined that MAPK signaling dependency in epithelial lung cancer cells is due to the scaffold protein interleukin-17 receptor D (IL17RD), which is directly repressed by ZEB1. Lung tumors in multiple Kras mutant murine models with increased ZEB1 displayed low IL17RD expression, accompanied by MAPK-independent tumor growth and therapeutic resistance to MEK inhibition. Suppression of ZEB1 function with miR-200 expression or the histone deacetylase inhibitor mocetinostat sensitized resistant cancer cells to MEK inhibition and markedly reduced in vivo tumor growth, showing a promising combinatorial treatment strategy for KRAS mutant cancers. In human lung tumor samples, high ZEB1 and low IL17RD expression correlated with low MAPK signaling, presenting potential markers that predict patient response to MEK inhibitors.

B7-H3 Expression in Merkel Cell Carcinoma-Associated Endothelial Cells Correlates with Locally Aggressive Primary Tumor Features and Increased Vascular Density.

PURPOSE: Merkel cell carcinoma (MCC) is an aggressive cutaneous malignancy whose pathogenesis and prognosis are related to the integrity of the host immune system. Despite promising clinical responses to immune-checkpoint blockade, response and resistance remain unpredictable, underscoring a critical need to delineate novel prognostic biomarkers and/or therapeutic targets for this disease.Experimental Design: Expression of immune-regulatory markers (PD-L2, B7-H3, B7-H4, IDO-1, ICOS, TIM3, LAG3, VISTA, and OX-40) was assessed using singlet chromogenic IHC in 10 primary MCCs. Multiplex immunofluorescence quantified CD31 and B7-H3 expression in 52 primary and 25 metastatic MCCs. B7-H3 and CD31 expressions were tabulated as a series of independent (X,Y) cell centroids. A spatial G-function, calculated based on the distribution of distances of B7-H3+ (X,Y) cell centroids around the CD31+ (X,Y) cell centroids, was used to estimate a colocalization index equivalent to the percentage of CD31-positive cell centroids that overlap with a B7-H3-positive cell centroid. RESULTS: Primary and metastatic MCCs exhibit a dynamic range of colocalized CD31 and B7-H3 expression. Increasing colocalized expression of B7-H3 with CD31 significantly associated with increased tumor size (P = 0.0060), greater depth of invasion (P = 0.0110), presence of lymphovascular invasion (P = 0.0453), and invasion beyond skin (P = 0.0428) in primary MCC. Consistent with these findings, increasing colocalized expression of B7-H3 and CD31 correlated with increasing vascular density in primary MCC, but not metastatic MCC. CONCLUSIONS: Our results demonstrate that colocalized expression of B7-H3/CD31 is a poor prognostic indicator and suggest therapies targeting B7-H3 may represent an effective approach to augmenting immune-activating therapies for MCC.

Concomitant targeting of the mTOR/MAPK pathways: novel therapeutic strategy in subsets of RICTOR/KRAS-altered non-small cell lung cancer.

Despite a therapeutic paradigm shift into targeted-driven medicinal approaches, resistance to therapy remains a hallmark of lung cancer, driven by biological and molecular diversity. Using genomic and expression data from advanced non-small cell lung cancer (NSCLC) patients enrolled in the BATTLE-2 clinical trial, we identified RICTOR alterations in a subset of lung adenocarcinomas and found RICTOR expression to carry worse overall survival. RICTOR-altered cohort was significantly enriched in KRAS/MAPK axis mutations, suggesting a co-oncogenic driver role in these molecular settings. Using NSCLC cell lines, we showed that, distinctly in KRAS mutant backgrounds, RICTOR blockade impairs malignant properties and generates a compensatory enhanced activation of the MAPK pathway, exposing a unique therapeutic vulnerability. In vitro and in vivo concomitant pharmacologic inhibition of mTORC1/2 and MEK1/2 resulted in synergistic responses of anti-tumor effects. Our study provides evidence of a distinctive therapeutic opportunity in a subset of NSCLC carrying concomitant RICTOR/KRAS alterations.

Immunohistochemical and Image Analysis-Based Study Shows That Several Immune Checkpoints are Co-expressed in Non-Small Cell Lung Carcinoma Tumors.

INTRODUCTION: The understanding of immune checkpoint molecules' co-expression in non-small cell lung carcinoma (NCLC) is important to potentially design combinatorial immunotherapy approaches. METHODS: We studied 225 formalin-fixed, paraffin-embedded tumor tissues from stage I-III NCLCs - 142 adenocarcinomas (ADCs) and 83 squamous cell carcinomas (SCCs) - placed in tissue microarrays. Nine immune checkpoint markers were evaluated; four (programmed death ligand 1 [PD-L1], B7-H3, B7-H4, and indoleamine 2,3-dioxygenase 1 [IDO-1]) expressed predominantly in malignant cells (MCs) and five (inducible T cell costimulator, V-set immunoregulatory receptor, T-cell immunoglobulin mucin family member 3, lymphocyte activating 3, and OX40) expressed mostly in stromal tumor-associated inflammatory cells (TAICs). All markers were examined using a quantitative image analysis and correlated with clinicopathologic features, TAICs, and molecular characteristics. RESULTS: Using above the median value as positive expression in MCs and high density of TAICs expressing those markers, we identified higher expression of immune checkpoints in SCC than ADC. Common simultaneous expression by MCs was PD-L1 + B7-H3 + IDO-1 in ADC and PD-L1 + B7-H3, or B7-H3 + B7-H4, in SCC. TAICs expressing checkpoint were significantly higher in current smokers than in never smokers. Almost all the immune checkpoint markers showed positive correlation with TAICs expressing inflammatory cell markers. KRAS-mutant ADC specimens showed higher expression of PD-L1 in MCs and of B7-H3, T-cell immunoglobulin mucin family member 3, and IDO-1 in TAICs than wild type. Kaplan-Meier survival curves showed worse prognosis in ADC patients with higher B7-H4 expression by MCs. CONCLUSIONS: We found frequent immunohistochemical co-expression of immune checkpoints in surgically resected NCLC tumors and correlated with tumor histology, smoking history, tumor size, and the density of inflammatory cells and tumor mutational status.