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Immunotherapy has shown limited efficacy in patients with EGFR-mutated lung cancer. Efforts to enhance the immunogenicity of EGFR-mutated lung cancer have been unsuccessful to date. Here, we discover that MET amplification, the most common mechanism of resistance to third-generation EGFR tyrosine kinase inhibitors (TKI), activates tumor cell STING, an emerging determinant of cancer immunogenicity (1). However, STING activation was restrained by ectonucleosidase CD73, which is induced in MET-amplified, EGFR-TKI–resistant cells. Systematic genomic analyses and cell line studies confirmed upregulation of CD73 in MET-amplified and MET-activated lung cancer contexts, which depends on coinduction of FOSL1. Pemetrexed (PEM), which is commonly used following EGFR-TKI treatment failure, was identified as an effective potentiator of STING-dependent TBK1-IRF3-STAT1 signaling in MET-amplified, EGFR-TKI–resistant cells. However, PEM treatment also induced adenosine production, which inhibited T-cell responsiveness. In an allogenic humanized mouse model, CD73 deletion enhanced immunogenicity of MET-amplified, EGFR-TKI–resistant cells, and PEM treatment promoted robust responses regardless of CD73 status. Using a physiologic antigen recognition model, inactivation of CD73 significantly increased antigen-specific CD8+ T-cell immunogenicity following PEM treatment. These data reveal that combined PEM and CD73 inhibition can co-opt tumor cell STING induction in TKI-resistant EGFR-mutated lung cancers and promote immunogenicity. Significance: MET amplification upregulates CD73 to suppress tumor cell STING induction and T-cell responsiveness in TKI-resistant, EGFR-mutated lung cancer, identifying a strategy to enhance immunogenicity and improve treatment.

Immunotherapy has shown limited efficacy in patients with EGFR-mutated lung cancer. Efforts to enhance the immunogenicity of EGFR-mutated lung cancer have been unsuccessful to date. Here, we discover that MET amplification, the most common mechanism of resistance to third-generation EGFR tyrosine kinase inhibitors (TKI), activates tumor cell STING, an emerging determinant of cancer immunogenicity (1). However, STING activation was restrained by ectonucleosidase CD73, which is induced in MET-amplified, EGFR-TKI–resistant cells. Systematic genomic analyses and cell line studies confirmed upregulation of CD73 in MET-amplified and MET-activated lung cancer contexts, which depends on coinduction of FOSL1. Pemetrexed (PEM), which is commonly used following EGFR-TKI treatment failure, was identified as an effective potentiator of STING-dependent TBK1-IRF3-STAT1 signaling in MET-amplified, EGFR-TKI–resistant cells. However, PEM treatment also induced adenosine production, which inhibited T-cell responsiveness. In an allogenic humanized mouse model, CD73 deletion enhanced immunogenicity of MET-amplified, EGFR-TKI–resistant cells, and PEM treatment promoted robust responses regardless of CD73 status. Using a physiologic antigen recognition model, inactivation of CD73 significantly increased antigen-specific CD8+ T-cell immunogenicity following PEM treatment. These data reveal that combined PEM and CD73 inhibition can co-opt tumor cell STING induction in TKI-resistant EGFR-mutated lung cancers and promote immunogenicity.Significance:MET amplification upregulates CD73 to suppress tumor cell STING induction and T-cell responsiveness in TKI-resistant, EGFR-mutated lung cancer, identifying a strategy to enhance immunogenicity and improve treatment.

Purpose: The blockade of immune checkpoints such as PD-L1 and PD-1 is being exploited therapeutically in several types of malignancies. Here, we aimed to understand the contribution of the genetics of lung cancer to the ability of tumor cells to escape immunosurveillance checkpoints.Experimental Design: More than 150 primary non–small cell lung cancers, including pulmonary sarcomatoid carcinomas, were tested for levels of the HLA-I complex, PD-L1, tumor-infiltrating CD8+ lymphocytes, and alterations in main lung cancer genes. Correlations were validated in cancer cell lines using appropriate treatments to activate or inhibit selected pathways. We also performed RNA sequencing to assess changes in gene expression after these treatments.Results: MET-oncogenic activation tended to associate with positive PD-L1 immunostaining, whereas STK11 mutations were correlated with negative immunostaining. In MET-altered cancer cells, MET triggered a transcriptional increase of PD-L1 that was independent of the IFNγ-mediated JAK/STAT pathway. The activation of MET also upregulated other immunosuppressive genes (PDCD1LG2 and SOCS1) and transcripts involved in angiogenesis (VEGFA and NRP1) and in cell proliferation. We also report recurrent inactivating mutations in JAK2 that co-occur with alterations in MET and STK11, which prevented the induction of immunoresponse-related genes following treatment with IFNγ.Conclusions: We show that MET activation promotes the expression of several negative checkpoint regulators of the immunoresponse, including PD-L1. In addition, we report inactivation of JAK2 in lung cancer cells that prevented the response to IFNγ. These alterations are likely to facilitate tumor growth by enabling immune tolerance and may affect the response to immune checkpoint inhibitors. Clin Cancer Res; 24(18); 4579–87. ©2018 AACR.

Supplementary Data from MET-Induced CD73 Restrains STING-Mediated Immunogenicity of EGFR-Mutant Lung Cancer

Study design participants, supplementary figure legends and List of cancer-related differentially methylated CpGs

Purpose: Lung cancer remains as the leading cause of cancer-related death worldwide, mainly due to late diagnosis. Cytology is the gold-standard method for lung cancer diagnosis in minimally invasive respiratory samples, despite its low sensitivity. We aimed to identify epigenetic biomarkers with clinical utility for cancer diagnosis in minimally/noninvasive specimens to improve accuracy of current technologies.Experimental Design: The identification of novel epigenetic biomarkers in stage I lung tumors was accomplished using an integrative genome-wide restrictive analysis of two different large public databases. DNA methylation levels for the selected biomarkers were validated by pyrosequencing in paraffin-embedded tissues and minimally invasive and noninvasive respiratory samples in independent cohorts.Results: We identified nine cancer-specific hypermethylated genes in early-stage lung primary tumors. Four of these genes presented consistent CpG island hypermethylation compared with nonmalignant lung and were associated with transcriptional silencing. A diagnostic signature was built using multivariate logistic regression model based on the combination of four genes: BCAT1, CDO1, TRIM58, and ZNF177. Clinical diagnostic value was also validated in multiple independent cohorts and yielded a remarkable diagnostic accuracy in all cohorts tested. Calibrated and cross-validated epigenetic model predicts with high accuracy the probability to detect cancer in minimally and noninvasive samples. We demonstrated that this epigenetic signature achieved higher diagnostic efficacy in bronchial fluids as compared with conventional cytology for lung cancer diagnosis.Conclusions: Minimally invasive epigenetic biomarkers have emerged as promising tools for cancer diagnosis. The herein obtained epigenetic model in combination with current diagnostic protocols may improve early diagnosis and outcome of lung cancer patients. Clin Cancer Res; 22(13); 3361–71. ©2016 AACR.

Supplementary Fig. 1. A, Western blot showing ectopic and transient expression of the LKB1 protein in the indicated cell lines; Supplementary Fig. 2. A, Profile of JAK2 mutations in NSCLC primary tumors, from the cBioPortal for Cancer Genomics (www.cbioportal.org); Supplementary Fig. 3. Western blot depicting the levels of the indicated proteins and cell lines, upon treatment with IFNγ, at a concentration of 30 ng/μL for 6 h. ACTIN and TUBULIN, protein-loading controls; Supplementary Fig. 4. Enriched gene ontology classifications (P

Epigenetic signature in lung cancer and non-tumoral paraffins classified based on histological subtypes.

Flow chart indicating study design and biological and bioinformatic filters

Predictions of cancer using the epigenetic model for the 24 false negatives cytologies.

Heatmap representing the patients' methylation profile for the four genes involved in the prediction model and Nomogram for prediction of cancer risk, constructed using the coefficients of our combined logistic regression model as weights.

Epigenetic and expression analysis based on histological subtypes from primary tissues of the TCGA database.

Primers for pyrosequencing and priority list.

Legends to supplementary figures from 1 to 4

Supplementary Table 1. Characteristics of tumors and patients included in the analysis; Supplementary Table 2. List of antibodies and treatments used; Supplementary Table 3. Sequence of primers used for real-time quantitative PCR and size of amplified; Supplementary Table 4. Alterations at the indicated genes in lung cancer cell lines. cell carcinomas; SCLC, small Supplementary Table S5: RNA-sequencing report; Supplementary Table S6: List of genes from the MET-Activated (MET-Sign) and Interferon Treatment (IFN

Non-small cell lung cancers (NSCLC) are the most common type of lung cancer and can be classified according to the presence of mutually exclusive oncogenic drivers. The majority of NSCLC patients present a non-actionable oncogenic driver, and treatment resistance through the amplification of the MET proto-oncogene (MET) or the expression of programmed cell death protein 1 ligand (PD-L1) is common. Herein, we investigated the relation between MET gene amplification and PD-L1 expression in patients with advanced NSCLC and no other actionable oncogenic driver (i.e., EGFR, ALK, ROS1). Our retrospective observational study analyzed data from 48 patients (78% men, median age 66 years) admitted to the Germans Trias i Pujol Hospital, Spain, between July 2015 and February 2019. Patients presenting MET amplification showed a higher proportion of PD-L1 expression (93% vs. 39%; p < 0.001) and overexpression (64% vs. 27%; p = 0.020) than those with non-amplified MET. PD-L1 expression was not significantly different when analyzed by sex (p = 0.624), smoking history (p = 0.429), and Eastern Cooperative Oncology Group Performance Status (p = 0.597) Overall survival rates were not significantly affected by MET amplification (high and intermediate amplification vs low amplification and non-amplificated) (p = 0.252) nor PD-L1 expression (> vs =< 50%) (p = 0.893). In conclusion, a positive correlation was found between MET gene amplification and PD-L1 expression and highly expressed (above 50%) in patients with NSCLC and no other actionable oncogenic driver. It could be translated as new guided-treatment oportunities for these patients.

Elucidating the adaptive mechanisms that prevent host immune response in cancer will help predict efficacy of anti-programmed death-1 (PD1)/L1 therapies. Here, we study the cell-intrinsic response of lung cancer (LC) to interferon-γ (IFNγ), a cytokine that promotes immunoresponse and modulates programmed death-ligand 1 (PD-L1) levels. We report complete refractoriness to IFNγ in a subset of LCs as a result of JAK2 or IFNGR1 inactivation. A submaximal response affects another subset that shows constitutive low levels of IFNγ-stimulated genes (IγSGs) coupled with decreased H3K27ac (histone 3 acetylation at lysine 27) deposition and promoter hypermethylation and reduced IFN regulatory factor 1 (IRF1) recruitment to the DNA on IFNγ stimulation. Most of these are neuroendocrine small cell LCs (SCLCs) with oncogenic MYC/MYCL1/MYCN. The oncogenic activation of MYC in SCLC cells downregulates JAK2 and impairs IγSGs stimulation by IFNγ. MYC amplification tends to associate with a worse response to anti-PD1/L1 therapies. Hence alterations affecting the JAK/STAT pathway and MYC activation prevent stimulation by IFNγ and may predict anti-PD1/L1 efficacy in LC. The authors thank Isabel Bartolessis (Cancer Genetics Group) and Eva Mussolen (Cancer Epigenetics Group) at IJC for technical assistance. This work was supported by Spanish grants from the MINECO (PID2020-114541RB-I00 to M.S.-C.) and from the Fundación Científica Asociación Española Contra el Cáncer (GCB14142170MONT to M.S.-C.). P.L. was supported by a fellowship from the Formación de Personal Investigador program (BES-2015-072204) and J.M.M. from the FI program AGAUR (2022 FI_B1 00085M). M.S. was supported by a Juan Rodes contract from the Instituto de Salud Carlos III (JR20/00015). O.A.R. was supported by an AECC contract (INVES19045ROME). We thank the CERCA Program/Generalitat de Catalunya for institutional support.

Small-cell lung cancer (SCLC) is an aggressive malignancy characterized by a rapid progression and a high resistance to treatments. Unlike other solid tumors, there has been a scarce improvement in emerging treatments and survival during the last years. A better understanding of SCLC biology has allowed for the establishment of a molecular classification based on four transcription factors, and certain therapeutic vulnerabilities have been proposed. The universal inactivation of TP53 and RB1, along with the absence of mutations in known targetable oncogenes, has hampered the development of targeted therapies. On the other hand, the immunosuppressive microenvironment makes the success of immune checkpoint inhibitors (ICIs), which have achieved a modest improvement in overall survival in patients with extensive disease, difficult. Currently, atezolizumab or durvalumab, in combination with platinum–etoposide chemotherapy, is the standard of care in first-line setting. However, the magnitude of the benefit is scarce and no predictive biomarkers of response have yet been established. In this review, we describe SCLC biology and molecular classification, examine the SCLC tumor microenvironment and the challenges of predictive biomarkers of response to new treatments, and, finally, assess clinical and molecular characteristics of long-term survivor patients in order to identify possible prognostic factors and treatment vulnerabilities.

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboración, si le hubiere, y los autores pertenecientes a la UAM, Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations, Background There is a lack of useful diagnostic tools to identify EGFR mutated NSCLC patients with long-term survival. This study develops a prognostic model using real world data to assist clinicians to predict survival beyond 24 months. Methods EGFR mutated stage IIIB and IV NSCLC patients diagnosed between January 2009 and December 2017 included in the Spanish Lung Cancer Group (SLCG) thoracic tumor registry. Long-term survival was defined as being alive 24 months after diagnosis. A multivariable prognostic model was carried out using binary logistic regression and internal validation through bootstrapping. A nomogram was developed to facilitate the interpretation and applicability of the model. Results 505 of the 961 EGFR mutated patients identified in the registry were included, with a median survival of 27.73 months. Factors associated with overall survival longer than 24 months were: being a woman (OR 1.78); absence of the exon 20 insertion mutation (OR 2.77); functional status (ECOG 0–1) (OR 4.92); absence of central nervous system metastases (OR 2.22), absence of liver metastases (OR 1.90) or adrenal involvement (OR 2.35) and low number of metastatic sites (OR 1.22). The model had a good internal validation with a calibration slope equal to 0.781 and discrimination (optimism corrected C-index 0.680). Conclusions Survival greater than 24 months can be predicted from six pre-treatment clinicopathological variables. The model has a good discrimination ability. We hypothesized that this model could help the selection of the best treatment sequence in EGFR mutation NSCLC patients.

Background Primary cardiac tumors are extremely rare; most are myxomas with a benign prognosis. However, primary sarcomas are highly aggressive and treatment options are limited. Radical surgery is often not feasible and conventional therapies provide only modest results. Due to the rare nature of primary cardiac tumors, there are no proper randomized studies to guide treatment. Their complexity requires alternative approaches in order to improve treatment efficacy. Methods We isolated DNA from 5 primary cardiac sarcomas; the quality of DNA from 3 of them was sufficient to perform high-resolution single nucleotide polymorphism (SNP) array analysis. Results In the present study, molecular karyotyping revealed numerous segmental chromosomal alterations and amplifications affecting actionable genes that may be involved in disease initiation and/or progression. These include chromosomal break flanking AKT2 in undifferentiated pleomorphic rhabdomyosarcoma, chromosomal break in promoter of TERT, and gain of CDK4 and amplification of MDM2 in inflammatory myofibroblastic tumor. We detected segmental break flanking MOS in high-grade myxofibrosarcoma. In addition, the high number of chromosomal aberrations in high-grade myxofibrosarcoma may cause multiple tumor-specific epitopes, supporting the study of immunotherapy treatment in this type of aggressive tumor. Conclusion Our results provide a genetic rationale that supports an alternative, personalized therapeutic management of primary cardiac sarcomas.

The incorporation into clinical practice of immune-checkpoint inhibitors (ICIs), such as those targeting the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and the programmed cell death 1 (PD-1) and its ligand (PD-L1), has represented a major breakthrough in non-small cell lung cancer (NSCLC) treatment, especially in cases where the cancer has no druggable genetic alterations. Despite becoming the standard of care in certain clinical settings, either alone or in combination with chemotherapy, a proportion of patients do not respond while others actually progress during treatment. Therefore, there is a clinical need to identify accurate predictive biomarkers and to develop novel therapeutic strategies based on ICIs. Although they have limitations, the current markers evaluated to select which patients will undergo ICI treatment are the levels of PD-L1 and the tumor mutational burden. In this paper we describe what is currently known about the dynamic interaction between the cancer cell and the immune system during carcinogenesis, with a particular focus on the description of the functions and gene alterations that preclude the host immunoresponse in NSCLC. We emphasize the deleterious gene alterations in components of the major histocompatibility complex (HLA-I or B2M) and of the response to IFNγ (such as JAK2) which are mutually exclusive and can affect up to one fifth of the NSCLCs. The participation of other gene alterations, such as those of common oncogenes and tumor suppressors, and of the epigenetic alterations will also be discussed, in detail. Finally, we discuss the potential use of the tumor's genetic profile to predict sensitivity to ICIs.

There is no effective therapy for patients with malignant pleural mesothelioma (MPM) who progressed to platinum-based chemotherapy and immunotherapy. Here, we investigate the antitumor activity of CDK4/6 inhibitors usingin vitroandin vivopreclinical models of MPM. Based on publicly available transcriptomic data of MPM, patients withCDK4orCDK6overexpression had shorter overall survival. Treatment with abemaciclib or palbociclib at 100 nM significantly decreased cell proliferation in all cell models. Both CDK4/6 inhibitors significantly induced G1 cell cycle arrest thereby increasing cell senescence and increased the expression of interferon signaling pathway and tumor antigen presentation process in culture models of MPM.In vivopreclinical studies showed that palbociclib significantly reduced tumor growth and prolonged overall survival in a platinum-naïve and platinum resistant MPM mouse model. Treatment of MPM with CDK4/6 inhibitors decreased cell proliferation, mainly by promoting cell cycle arrest at G1 and by induction of cell senescence. Our preclinical studies provide evidence for evaluating CDK4/6 inhibitors in the clinic for the treatment of MPM.

Background:There is no effective therapy for patients with malignant pleural mesothelioma (MPM) who progressed to platinum-based chemotherapy and immunotherapy. Methods:We aimed to investigate the antitumor activity of CDK4/6 inhibitors using in vitro and in vivo preclinical models of MPM. Results:Based on publicly available transcriptomic data of MPM, patients with CDK4 or CDK6 overexpression had shorter overall survival. Treatment with abemaciclib or palbociclib at 100 nM significantly decreased cell proliferation in all cell models evaluated. Both CDK4/6 inhibitors significantly induced G1 cell cycle arrest, thereby increasing cell senescence and increased the expression of interferon signalling pathway and tumour antigen presentation process in culture models of MPM. In vivo preclinical studies showed that palbociclib significantly reduced tumour growth and prolonged overall survival using distinct xenograft models of MPM implanted in athymic mice. Conclusions:Treatment of MPM with CDK4/6 inhibitors decreased cell proliferation, mainly by promoting cell cycle arrest at G1 and by induction of cell senescence. Our preclinical studies provide evidence for evaluating CDK4/6 inhibitors in the clinic for the treatment of MPM.

SummaryDespite the genetic inactivation of SMARCA4, a core component of the SWI/SNF-complex commonly found in cancer, there are no therapies that effectively target SMARCA4-deficient tumours. Here, we show that, unlike the cells with activated MYC oncogene, cells with SMARCA4 inactivation are refractory to the histone deacetylase inhibitor, SAHA, leading to the aberrant accumulation of H3K27me3. This is associated with impaired transactivation and significantly reduced levels of the histone demethylases KDM6A/UTX and KDM6B/JMJD3, which confer a strong dependency on the KDM6s in the SMARCA4-mutant cells, so that its inhibition compromises cell viability. Administering the KDM6 inhibitor GSK-J4 to mice orthotopically implanted with SMARCA4-mutant lung cancer cells or primary small cell carcinoma of the hypercalcaemic type (SCCOHT) had strong anti-tumour effects. Our results highlight the vulnerability of KDM6 inhibitors as a characteristic that could be exploited for treating SMARCA4-mutant cancer patients.

Purpose: The blockade of immune checkpoints such as PD-L1 and PD-1 is being exploited therapeutically in several types of malignancies. Here, we aimed to understand the contribution of the genetics of lung cancer to the ability of tumor cells to escape immunosurveillance checkpoints. Experimental Design: More than 150 primary non–small cell lung cancers, including pulmonary sarcomatoid carcinomas, were tested for levels of the HLA-I complex, PD-L1, tumor-infiltrating CD8+ lymphocytes, and alterations in main lung cancer genes. Correlations were validated in cancer cell lines using appropriate treatments to activate or inhibit selected pathways. We also performed RNA sequencing to assess changes in gene expression after these treatments. Results: MET-oncogenic activation tended to associate with positive PD-L1 immunostaining, whereas STK11 mutations were correlated with negative immunostaining. In MET-altered cancer cells, MET triggered a transcriptional increase of PD-L1 that was independent of the IFNγ-mediated JAK/STAT pathway. The activation of MET also upregulated other immunosuppressive genes (PDCD1LG2 and SOCS1) and transcripts involved in angiogenesis (VEGFA and NRP1) and in cell proliferation. We also report recurrent inactivating mutations in JAK2 that co-occur with alterations in MET and STK11, which prevented the induction of immunoresponse-related genes following treatment with IFNγ. Conclusions: We show that MET activation promotes the expression of several negative checkpoint regulators of the immunoresponse, including PD-L1. In addition, we report inactivation of JAK2 in lung cancer cells that prevented the response to IFNγ. These alterations are likely to facilitate tumor growth by enabling immune tolerance and may affect the response to immune checkpoint inhibitors. Clin Cancer Res; 24(18); 4579–87. ©2018 AACR.

Background: With the approval of the vaccines against SARS-CoV-2, oncologic scientific societies have recommended cancer p to be prioritized for vaccination. Since cancer p have not participated in vaccine development studies, these recommendations arise some questions regarding their efficacy, safety and impact on survival. The aim of this prospective study is to evaluate the immune response to the SARS-CoV-2 vaccine in LC p. Secondary objectives include vaccine-related adverse events (AE), cancer treatment AE after vaccination, impact of the vaccine on survival, immune response, toxicity and survival outcomes in p>75 y, (re)infection after vaccination, complications and mortality. Methods: LCp who receive the vaccine against SARS-COV-2 are candidates to participate in this study. A pre-vaccination IgG determination will be performed to identify p with previous infection, but asymptomatic course. After vaccination, IgG will be repeated at 3, 6 and 12 months. Information on short and long term vaccine-related AEs will be collected, as well as, serological results, tumor and treatment-related data, and survival. Results: From March, 31 to April 15, 2021,106p have participated in the study. 58.5% were male, median age was 66 y (46- 83), 90.6% were Non-Small Cell LC, 83% has stage IV at diagnosis, Systemic therapy included EGFR/ALK/ROS1/RET/MET TKI (22.6%), immunotherapy (IT) (39.6%), chemotherapy (CT) (19.4%) and CTIT (14.1%). 4p were not receiving active therapy. 94.3% received Moderna® vaccine on behalf of the Hospital Vaccination Program. AES to 1st dose (1D) included local pain (16%), swelling (0.9%), fever (2.8%) and myalgia (0.9%). 5p had prior known COVID infection. No vaccine-related AE were reported in this group. 6p were admitted after vaccination due to cancer-related symptoms. No deaths were reported. Definitive data on baseline and 3-m serological data, as well as complete 1D and 2D related-AE and potential interactions with cancer therapy will be presented later. Conclusions: 1D of SARS-COV-2 vaccine appears to be safe irrespective of systemic therapy in our cohort of LCp. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: M. Saigi: Financial Interests, Institutional, Funding: Merck;Financial Interests, Personal, Other: BMS;Financial Interests, Personal, Other: Pfizer. E. Carcereny Costa: Financial Interests, Personal, Advisory Board: AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, MSD, Novartis, Roche, Takeda;Financial Interests, Personal, Invited Speaker: AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, MSD, Novartis, Pfizer, Roche, Takeda, Amgen;Financial Interests, Personal, Other: Bristol-Myers Squibb, Pfizer, Roche, Takeda. M. Domenech: Financial Interests, Personal, Invited Speaker: AstraZeneca, BMS. A. Estival: Financial Interests, Personal, Other: Lilly, Pharmamar, Bayer, MSD, BMS, Roche. Honoraria: Roche, MSD, AstraZeneca, Pharmamar. M. Romeo Marin: Non-Financial Interests, Personal, Advisory Board: MSD, GSK;Non-Financial Interests, Personal, Other: AZ. M.T. Moran Bueno: Financial Interests, Personal, Advisory Board: AstraZeneca Boehringer Ingelheim Roche BMS. All other authors have declared no conflicts of interest.

Aberrant activation of MET as a result of exon 14-skipping (METex14) mutations or gene amplification is an oncogenic mechanism in non-small cell lung carcinoma (NSCLC) and a potential therapeutic target. The purpose of this study was to characterize MET alterations in a cohort of NSCLC patients treated with surgery. 157 NSCLCs of various histopathologies, including pulmonary sarcomatoid carcinomas (PSC), were tested for MET alterations. METex14 mutations, MET copy number alterations and the levels of MET protein were determined by Sanger sequencing, fluorescence in situ hybridization and immunohistochemistry, respectively. Concurrent alterations of other important cancer genes and immunostaining of the downstream effector, phopho-S6, were also determined. METex14 mutations and MET amplification were detected in seven tumors. MET genetic alterations were found predominantly in the lung adenocarcinoma (ADC) and PSC histopathologies. High levels of MET protein were found in most MET-amplified tumors, but not in all METex14-mutated tumors. Strong phopho-S6 staining was observed in about half of the MET-activated tumors. One tumor with METex14 exhibited concurrent ERBB2 amplification. MET activation, by either METex14 mutations or amplification, is characteristic of a subset of early stage NSCLCs and may coexist with ERBB2 amplification. This may have potential therapeutic implications. The presence of METex14 mutations was associated with low levels of MET protein, which may limit the use of total MET immunostaining as a marker for preselecting patients for MET-targeted therapies.

Cetuximab is a monoclonal antibody against epidermal growth factor receptor useful in the treatment of patients with Head and Neck Squamous Cell Carcinoma combined with radiotherapy or chemotherapy. Its pharmacokinetics are not influenced by hepatic status and there are no specific warnings concerning its indication in patients with impaired hepatic function. Patients with a previous liver transplant are at risk for hepatic toxicity and use immunosupressants to avoid rejection that can interact with other drugs. We present two cases of patients with a previous liver transplant in which cetuximab was administered to treat head and neck cancer.

Cancer is a dynamic process of somatic evolution. Distinct cell populations that undergo mutations, result in positive or negative selection depending on whether or not these mutations confer an advantage over other cells. In current clinical practice, predictive biomarkers such as EGFR mutation and ALK rearrangement are used routinely for treatment making decisions in non-small cell lung cancer (NSCLC). However, pressure from targeted therapies increases positive selection of resistant clones, eventually resulting in resistance to the drug. Monitoring the mutation pattern during the disease's course may allow the most suitable therapeutic approach to be selected. Here we report two clinical cases with lung adenocarcinoma demonstrating discordance in oncogene driver mutations between primary site and subsequent metastasis, and how this has contributed to therapeutic decisions.

Background The capability of tumors to avoid immune surveillance has emerged as therapeutically approachable, especially through the blockade of immune checkpoints (ICB) such as PD-L1/PD-1. We previously reported that B2M inactivating mutations in lung cancer (LC) impair immunorecognition through the disruption of the MHC-I complex. On the other hand, the refractoriness to IFNγ has also emerged as a mechanism that promotes tumor's immunoescape. Our purpose is to identify novel gene alterations that contribute to immunoescape in LC. Methods A panel of 44 NSCLC cell lines (35 commercially available and 9 ex vivo, derived from LC patients with metastatic pleural effusions, PE) were tested for response to IFNγ. Whole exome sequencing and RNA-sequencing was performed in the PE or gathered from public databases (Sanger and Cancer Cell Line Encyclopaedia) in the commercially available LC cells. The response to IFNγ was evaluated by determining the activation of various downstream targets, including an increase in the expression of CD274 (PD-L1) or IRF1 among others, using WB or RT-qPCR. We evaluated other parameters, such as the levels and location of the HLA-1/B2M proteins (HLA-I complex) by immunofluorescence, in selected LC cell lines. Results We could distinguish three different categories in the response to IFNγ; good responders, 75% (readily increase the levels of all downstream targets); non responders, 9% (did not increase the levels of any of the targets tested) or partial responders, 16% (were able to up-regulate all the targets tested but CD274/PD-L1, upon IFNγ exposure). By immunofluorescence, we observed that none of the non responders were able to increase HLA-1/B2M proteins in the cell surface, after IFNγ exposure. We identified biallelic inactivating alterations at JAK2 in three non responders (intragenic homozygous deletion, p.R426X and p.S507X in the NCI-H2126, NCI-H1993 and NCI-H1573, respectively). These mutations were concurrent with genetic alterations of main oncodrivers in LC. In the remaining non responder (PE-1), we identified a truncating mutation in a candidate gene. We overexpressed the wild type version of this gene and the response to IFNγ was restored. A mutational screening of this gene and immunostaining information for the encoded protein is currently ongoing in a cohort of 100 primary LC samples. Conclusions Alterations in molecules that are related with immunorecognition (B2M and HLA-I) are associated to immunotolerance, in LC. Here, we report that genetic alterations in factors that mediate the response to IFNγ are also involved in mediating immunotolerance in LC. Functional characterization of alterations in these genes is crucial to understand the mechanisms that contribute to tumor's immunoescape. Loss of function mutations in our candidate genes are likely to facilitate tumor growth by enabling immune tolerance and may affect the response to ICB. Citation Format: Juan Jose Alburquerque-Bejar, Maria Saigi, Eva Pros, Octavio Romero, Montse Sanchez-Cespedes. Searching for genetic alterations that drive the capability of evading tumor immunosurveillance in lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 998.

PURPOSE: The 5' ectonucleotidase (NT5E) encodes for the CD73 membrane protein that converts the extracellular AMP into adenosine (Ado), an immunosuppressive nucleoside that generates anti-inflammatory responses by stimulating T cell anergy. We aimed to analyze CD73 expression across non-small cell lung cancer (NSCLC) and explore its role in immunomodulation and its predictive value to immunotherapy (ICI). METHODS: We analyzed CD73 (NT5E) expression across lung adenocarcinoma samples from The Cancer Genome Atlas (TCGA) and determined top co-expressed genes or correlations with alterations in lung cancer (LC) driver genes. Correlations were validated in specific cancer cell lines using appropriate treatments to activate or inhibit selected pathways. A CRISPR-Cas9 CD73-knockout cell line model was generated to explore direct regulation of extracellular Ado production, as well as impact on secreted cytokines. Finally, we performed staining of CD73 and other immune-related molecules (CD8+ TILs, PD-L1, STING) in a pilot cohort of NSCLC patients (n=35) treated with first line ICI +/- chemotherapy at the DFCI. RESULTS: TCGA analysis revealed strong correlation between MET and CD73 expression and identified MET altered tumors, either by MET exon 14 skipping mutations (METex14) or MET amplification, as being enriched for high CD73 expression (p=0.03). Functional assays using LC cell lines showed that MET activation increases CD73 expression at a transcriptional and protein level by using qPCR, western-blot and flow-cytometry. Additional adenosinergic-related molecules, including the adenosine receptor A2BR (ADORA2B), were co-regulated in parallel with CD73, defining a specific phenotype. In our pilot cohort of patients, CD73 high expression (2+) was found in 20% of lung tumors, mostly adenocarcinomas, whereas 50% were negative (0+) for CD73 expression. CD73 was positively associated with high CD8+ (p= 0.02). Clinical outcomes are being evaluated to elucidate the potential role of CD73 as a predictive marker to ICI. CONCLUSIONS: MET altered tumors and LC cell lines express high levels of CD73 and other adenosinergic-related molecules, representing a potential mechanism of immune evasion and target for immunotherapy. The potential predictive role of CD73 expression in a larger cohort of patients treated with immune checkpoint inhibition (ICI) will be performed. Citation Format: Maria Saigi, Ryohei Yoshida, Erik H Knelson, Navin R Mahadevan, Amir Vajdi, Israel Cañadas, Tran C Thai, Mark M. Awad, Montse Sánchez-Céspedes, David A Barbie. Determinants of immune evasion in MET driven lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1012.

Purpose: Lung cancer remains as the leading cause of cancer-related death worldwide, mainly due to late diagnosis. Cytology is the gold-standard method for lung cancer diagnosis in minimally invasive respiratory samples, despite its low sensitivity. We aimed to identify epigenetic biomarkers with clinical utility for cancer diagnosis in minimally/noninvasive specimens to improve accuracy of current technologies. Experimental Design: The identification of novel epigenetic biomarkers in stage I lung tumors was accomplished using an integrative genome-wide restrictive analysis of two different large public databases. DNA methylation levels for the selected biomarkers were validated by pyrosequencing in paraffin-embedded tissues and minimally invasive and noninvasive respiratory samples in independent cohorts. Results: We identified nine cancer-specific hypermethylated genes in early-stage lung primary tumors. Four of these genes presented consistent CpG island hypermethylation compared with nonmalignant lung and were associated with transcriptional silencing. A diagnostic signature was built using multivariate logistic regression model based on the combination of four genes: BCAT1, CDO1, TRIM58, and ZNF177. Clinical diagnostic value was also validated in multiple independent cohorts and yielded a remarkable diagnostic accuracy in all cohorts tested. Calibrated and cross-validated epigenetic model predicts with high accuracy the probability to detect cancer in minimally and noninvasive samples. We demonstrated that this epigenetic signature achieved higher diagnostic efficacy in bronchial fluids as compared with conventional cytology for lung cancer diagnosis. Conclusions: Minimally invasive epigenetic biomarkers have emerged as promising tools for cancer diagnosis. The herein obtained epigenetic model in combination with current diagnostic protocols may improve early diagnosis and outcome of lung cancer patients. Clin Cancer Res; 22(13); 3361–71. ©2016 AACR.

Purpose: To explore the correlation between the expression of immunological biomarkers in a subset of NSCLC tumors presenting cMET activation. Methods: We retrospectively reviewed 200 tumor samples from a cohort of patients diagnosed with NSCLC in 3 different hospitals, from Spain and France. cMET activation was defined by the presence of one or more of the following criteria: 1- cMET mutations inducing exon 14 skipping (METex14), 2- cMET amplification, assessed by FISH 3- cMET overexpression, assessed by immunohistochemistry (IHC). METex14 mutations were determined by genomic sequencing and RT-PCR. FISH was interpreted according to Capuzzo scoring system, considering 5 or more gene copies as the mean value for positive amplification. Concurrent genetic alterations in EGFR and ERBB2 tyrosine kinase receptors were also determined. PD-L1 expression and density of tumoral infiltrating CD8+ lymphocytes were assessed by IHC. Statistical correlation analysis was performed by Chi-square and Fisher’s exact test. Results: METex14, cMET amplification and cMET overexpression were detected in 2%, 7.8% and 31% of tumors, respectively. Histological subtypes included: 70% adenocarcinoma, 20% squamous cell carcinoma, 8% large cell, 2% pulmonary sarcomatoid. Curiously, one of the METex14 adenocarcinoma was concurrent with ERBB2 amplification. Those tumors harbouring METex14 or cMET amplification were correlated with positivity for PD-L1 expression (p = 0.02). However, this association was not observed in cMET overexpressed tumors. Conclusions: We observed a direct correlation between PD-L1 positivity and METex14 or cMET amplification. Functional analysis and a large cohort of tumors are required to better characterize the potential role of cMET pathway in the regulation of PD-L1 expression. Citation Format: Maria Saigi, Carolina Pereira, Eva Pros, Elisabeth Brambilla, Ernest Nadal, Montse Sánchez-Céspedes. Molecular and immunological characterization of non-small cell lung cancer harboring cMET alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1765. doi:10.1158/1538-7445.AM2017-1765

Introduction: Lung cancer is the leading cause worldwide, mainly due to late diagnosis. The aim of this work was to identify a panel of epigenetic biomarkers for improving early diagnosis of lung cancer patients using minimally and non-invasive biological fluids. Patients and Methods: DNA hypermethylated biomarkers were identified performing a Genome-wide DNA methylation analysis (Infinium 450K array) in Non-small cell lung cancer (NSCLC) primary tumors from two different public databases (Discovery cohorts): CURELUNG FP7 Consortium (237 stage I NSCLC, 25 non-tumoral lung samples) and The Cancer Genome Atlas (TCGA; 350 stage I NSCLC, 62 non-tumoral lung samples). DNA methylation levels of selected candidates were analyzed by pyrosequencing in non- or minimally invasive samples from three independent cohorts of stage I NSCLC patients and non-tumoral controls (Validation cohorts): bronchoalveolar aspirates (82 NSCLC; 29 controls), bronchoalveolar lavages (51 NSCLC; 29 controls) and sputum (72 NSCLC; 26 controls). Combined Receiver Operating Characteristic (ROC) curve was obtained to evaluate the diagnostic utility of the epigenetic signature. Results: We identified a panel of 4 cancer-specific genes (BCAT1, CDO1, TRIM58 and ZNF177) with CpG island hypermethylation-associated silencing in early stage NSCLC primary tumors. All these genes presented significantly higher mean levels of%methylation (M) in NSCLC primary tumors respect to non-tumoral controls: BCAT1 (NSCLC: M>50%; Controls: M40%; Controls: M50%; Controls: M40%; Controls : M Conclusions: The herein identified DNA methylation signature could improve, in combination with current diagnostic protocols, the early diagnosis and outcome of NSCLC patients. The high diagnostic accuracy of this signature obtained in liquid biopsy offers a minimally invasive and easy accessible tool for early lung cancer diagnosis. Citation Format: A Diaz-Lagares, J Mendez-Gonzalez, D Hervas, M Saigi, MJ Pajares, D Garcia, AB Crujeiras, R Lopez-Lopez, R Pio, LM Montuenga, JJ Zulueta, E Nadal, A Rosell, M Esteller, J Sandoval. Identification of a DNA methylation signature in liquid biopsy for early non-small cell lung cancer (NSCLC) diagnosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-155.