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Table with Details of Patient Characteristics

Methods for quantitative immunofluorescence and IFN gamma treatment of PDXs

Supplementary Figures S1-S8

PDF file 103K, Cisplatin chemosensitivity in SK-LU-1 miR-34b/c transfected cells

PDF file115K, Suppl.Table S1. Patient characteristics. Suppl.Table S2. Lung AC lines with TP53 mutation. Suppl.Table S3. Primers used for qRT-PCR. Suppl.Table S4. Correlation between tumors miR-34b/c expression and clinical and molecular variables. Suppl.Table S5. Multivariate Cox model for DFS based upon miR-34b/c methylation (continuous). Suppl.Table S6. Multivariate Cox model for OS in 140 patients

Purpose: The microRNA-34b/c (miR-34b/c) is considered a tumor suppressor in different tumor types and a transcriptional target of TP53. The main objectives of this study were to investigate the clinical implications of miR-34b/c methylation in patients with early-stage lung adenocarcinoma and to determine the functional role of miR-34b/c re-expression in lung adenocarcinoma cell lines.Experimental Design: Aberrant methylation and expression of miR-34b/c were assessed in 15 lung adenocarcinoma cell lines and a cohort of 140 early-stage lung adenocarcinoma. Lung adenocarcinoma cell lines were transfected with miR-34b/c and the effects upon cell proliferation, migration, invasion, and apoptosis were investigated.Results: Aberrant methylation of miR-34b/c was detected in 6 (40%) of 15 lung adenocarcinoma cell lines and 64 of 140 (46%) primary lung adenocarcinoma. Expression of miR-34b/c was significantly reduced in all methylated cell lines and primary tumors, especially with TP53 mutations. Patients with increased miR-34b/c methylation had significantly shorter disease-free and overall survival as compared to patients with unmethylated or low level of miR-34b/c methylation. Ectopic expression of miR-34b/c in lung adenocarcinoma cell lines decreased cell proliferation, migration, and invasion.Conclusions: Epigenetic inactivation of miR-34b/c by DNA methylation has independent prognostic value in patients with early-stage lung adenocarcinoma. Reexpression of miR-34b/c leads to a less aggressive phenotype in lung adenocarcinoma cell lines. Clin Cancer Res; 19(24); 6842–52. ©2013 AACR.

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Purpose:EGFR exon 19 deletion (Ex19Del) mutations account for approximately 60% of lung cancer–associated EGFR mutations and include a heterogeneous group of mutations. Although they are associated with benefit from tyrosine kinase inhibitors (TKI), the relative inhibitor sensitivity of individual Ex19Del mutations is unknown.Experimental Design: We studied the TKI sensitivity and structural features of common Ex19Del mutations and the consequences for patient outcomes on TKI treatment.Results:We found that the L747-A750>P mutation, which represents about 4% of all Ex19Del mutations, displays unique inhibitor selectivity. L747-A750>P differs from other Ex19Del mutations in not being suppressed completely by erlotinib or osimertinib, yet is completely inhibited by low doses of afatinib. The HCC4006 cell line (with the L747-A750>P mutation) exhibited increased sensitivity to afatinib over erlotinib and osimertinib, and computational modeling suggests explanations for this sensitivity pattern. Clinically, patients with EGFR L747-A750>P mutant tumors showed inferior outcomes when treated with erlotinib than patients with E746-A750 mutant tumors.Conclusions:These results highlight important differences between specific Ex19Del mutations that may be relevant for optimizing TKI choice for patients.

Although most activating mutations of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancers (NSCLCs) are sensitive to available EGFR tyrosine kinase inhibitors (TKIs), a subset with alterations in exon 20 of EGFR and HER2 are intrinsically resistant and lack an effective therapy. We used in silico, in vitro, and in vivo testing to model structural alterations induced by exon 20 mutations and to identify effective inhibitors. 3D modeling indicated alterations restricted the size of the drug-binding pocket, limiting the binding of large, rigid inhibitors. We found that poziotinib, owing to its small size and flexibility, can circumvent these steric changes and is a potent inhibitor of the most common EGFR and HER2 exon 20 mutants. Poziotinib demonstrated greater activity than approved EGFR TKIs in vitro and in patient-derived xenograft models of EGFR or HER2 exon 20 mutant NSCLC and in genetically engineered mouse models of NSCLC. In a phase 2 trial, the first 11 patients with NSCLC with EGFR exon 20 mutations receiving poziotinib had a confirmed objective response rate of 64%. These data identify poziotinib as a potent, clinically active inhibitor of EGFR and HER2 exon 20 mutations and illuminate the molecular features of TKIs that may circumvent steric changes induced by these mutations.

Mechanisms of acquired resistance to immune checkpoint inhibitors (ICI) are poorly understood. We leveraged a collection of 14 ICI-resistant lung cancer samples to investigate whether alterations in genes encoding HLA Class I antigen processing and presentation machinery (APM) components or interferon signaling play a role in acquired resistance to PD-1 or PD-L1 antagonistic antibodies. Recurrent mutations or copy-number changes were not detected in our cohort. In one case, we found acquired homozygous loss of B2M that caused lack of cell-surface HLA Class I expression in the tumor and a matched patient-derived xenograft (PDX). Downregulation of B2M was also found in two additional PDXs established from ICI-resistant tumors. CRISPR-mediated knockout of B2m in an immunocompetent lung cancer mouse model conferred resistance to PD-1 blockade in vivo, proving its role in resistance to ICIs. These results indicate that HLA Class I APM disruption can mediate escape from ICIs in lung cancer. Significance: As programmed death 1 axis inhibitors are becoming more established in standard treatment algorithms for diverse malignancies, acquired resistance to these therapies is increasingly being encountered. Here, we found that defective antigen processing and presentation can serve as a mechanism of such resistance in lung cancer. Cancer Discov; 7(12); 1420–35. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1355

The emergence of drug resistance depends on the ability of the genome of cancer cells to constantly mutate and evolve under selective pressures. The generation of new mutations is accelerated when genes involved in DNA repair pathways are altered. Notably, although the emergence of new mutations fosters drug resistance, new variants can nevertheless become novel antigens that promote immune surveillance and even restrict cancer growth.

Preclinical studies have suggested that combining cytotoxic agents with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) to treat EGFR-mutated tumors may increase their inhibitory effect depending on the order of drug administration. The antitumor efficacy of different treatment sequences using vinorelbine (VNB) and gefitinib (GEF) was investigated both in vitro and in vivo in non-small cell lung cancer (NSCLC) cell lines with the rationale of potentially translating these findings into the clinical setting. The EGFR-wild-type A549 and the EGFR-mutated (exon 21 L858R/exon 20 T790M) H1975 cell lines were treated as follows: GEF followed by VNB, VNB followed by GEF and the two drugs applied individually or concurrently. Results in vitro demonstrated that the sequence of VNB followed by GEF was significantly more active than single-agent treatments. The expression of activated EGFR and its downstream pathway genes indicated that the increased cytotoxic effect of the VNB and GEF treatment sequence was accompanied by inhibition of EGFR, AKT and ERK1/2. Moreover, the increased inhibition of tumor growth after treatment with VNB followed by GEF was also confirmed in CD1-nude mice that were xenotransplanted with H1975 cells (p

Malignant pleural mesothelioma (MPM) is an aggressive human cancer and miRNAs can play a key role for this disease. In order to broaden the knowledge in this field, the miRNA expression was investigated in a large series of MPM to discover new pathways helpful in diagnosis, prognosis and therapy. We employed nanoString nCounter system for miRNA profiling on 105 MPM samples and 10 healthy pleura. The analysis was followed by the validation of the most significantly deregulated miRNAs by RT-qPCR in an independent sample set. We identified 63 miRNAs deregulated in a statistically significant way. MiR-185, miR-197, and miR-299 were confirmed differentially expressed, after validation study. In addition, the results of the microarray analysis corroborated previous findings concerning miR-15b-5p, miR-126-3p, and miR-145-5p. Kaplan-Meier curves were used to explore the association between miRNA expression and overall survival (OS) and identified a 2-miRNA prognostic signature (Let-7c-5p and miR-151a-5p) related to hypoxia and energy metabolism respectively. In silico analyses with DIANA-microT-CDS highlighted 5 putative targets in common between two miRNAs. With the present work we showed that the pattern of miRNAs expression is highly deregulated in MPM and that a 2-miRNA signature can be a new useful tool for prognosis in MPM.

Cell-free DNA (cfDNA) and circulating tumor cells (CTCs) are promising prognostic and predictive biomarkers in non-small cell lung cancer (NSCLC). In this study, we examined the prognostic role of cfDNA and CTCs, in separate and joint analyses, in NSCLC patients receiving first line chemotherapy. Seventy-three patients with advanced NSCLC were enrolled in this study. CfDNA and CTC were analyzed at baseline and after two cycles of chemotherapy. Plasma cfDNA quantification was performed by quantitative PCR (qPCR) whereas CTCs were isolated by the ScreenCell Cyto (ScreenCell, Paris, France) device and enumerated according to malignant features. Patients with baseline cfDNA higher than the median value (96.3 hTERT copy number) had a significantly worse overall survival (OS) and double the risk of death (hazard ratio (HR): 2.14; 95% confidence limits (CL) = 1.24–3.68; p-value = 0.006). Conversely, an inverse relationship between CTC median baseline number (6 CTC/3 mL of blood) and OS was observed. In addition, we found that in patients reporting stable disease (SD), the baseline cfDNA and CTCs were able to discriminate patients at high risk of poor survival. cfDNA demonstrated a more reliable biomarker than CTCs in the overall population. In the subgroup of SD patients, both biomarkers identified patients at high risk of poor prognosis who might deserve additional/alternative therapeutic interventions.

Originally formulated as an intravenous (i.v.) agent, vinorelbine is also currently available as an oral chemotherapeutic agent. Oral vinorelbine has demonstrated significant activity in different settings for NSCLC, including adjuvant treatment for resected disease, concurrent chemoradiation for locally advanced NSCLC and palliative chemotherapy for recurrent/metastatic NSCLC, as part of combination schedules or as a single-agent treatment.The authors explored the available data describing the use of oral vinorelbine in NSCLC. PubMed articles and abstracts presented at international conferences were analysed, and relevant trials were reported and discussed. Specific settings, including the treatment of elderly and unfit patients and metronomic schedules including oral vinorelbine, were evaluated. Available pharmacoeconomic data were also assessed.Oral vinorelbine is an appealing agent, particularly as part of combination regimens containing platinum derivatives, although it can have a role as a single-agent treatment as well. Its safety profile is generally favourable and its route of administration is generally preferred by patients receiving chemotherapy. Compared to i.v. vinorelbine and other antineoplastic agents, oral vinorelbine has been reported to be advantageous in terms of cost savings.

Introduction: Despite recent advances with new chemotherapeutic agents and target therapies, the prognosis of NSCLC remains poor. Recent results from clinical trials of immunotherapeutic agents, especially with immune checkpoint inhibitors, make this approach very exciting in NSCLC. Ipilimumab is a monoclonal antibody directed against cytotoxic T-lymphocyte antigen 4 that is able to stimulate the antitumour immune response by promoting T-cell activation. Areas covered: We have reviewed the literature and have described the most important results obtained with ipilimumab in NSCLC in recent trials with a specific focus on its peculiar toxicity profile and pattern of response. Trials ongoing with ipilimumab are also reported. Expert opinion: The results from clinical trials with ipilimumab are promising. Some important issues in the near future will be to identify prognostic and predictive biomarkers to select patients who could benefit from this drug. Further studies are warranted to understand how to combi...

Metastatic colorectal cancer (mCRC) is frequently characterized by the presence of mutations of the KRAS oncogene, which are generally associated with a poor response to treatment with anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibodies. With the methods currently used, a case is classified as KRAS-mutated when approximately 20% of the cells bear an activating KRAS mutation. These considerations raise the question of whether cells with a mutated KRAS can be found in mCRC cases classified as KRAS wild-type when more sensitive methods are used. In addition, the issue arises of whether these mCRC cases with low proportion of KRAS-mutated cells could account at least in part for the therapeutic failure of anti-EGFR therapies that occur in 40-60% of cases classified as KRAS wild type. In this study, we compared the classical assays with a very sensitive test, a locked nucleic acid (LNA) polymerase chain reaction (PCR), capable of detecting KRAS-mutated alleles at extremely low frequency (detection sensitivity limit 0.25% mutated DNA/wild-type DNA). By analyzing a cohort of 213 mCRC patients for KRAS mutations, we found a 20.6% discordance between the sequencing/TheraScreen methods and the LNA-PCR. Indeed, 44 mCRC patients initially considered KRAS wild type were reclassified as KRAS mutated by using the LNA-PCR test. These patients were more numerous among individuals displaying a clinical failure to anti-EGFR therapies. Failure to respond to these biological treatments occurred even in the absence of mutations in other EGFR pathway components such as BRAF.

Background Next Generation Sequencing (NGS) has become a valuable tool for molecular landscape characterization of cancer genomes, leading to a better understanding of tumor onset and progression, and opening new avenues in translational oncology. Formalin-fixed paraffin-embedded (FFPE) tissue is the method of choice for storage of clinical samples, however low quality of FFPE genomic DNA (gDNA) can limit its use for downstream applications. Methods To investigate the FFPE specimen suitability for NGS analysis and to establish the performance of two solution-based exome capture technologies, we compared the whole-exome sequencing (WES) data of gDNA extracted from 5 fresh frozen (FF) and 5 matched FFPE lung adenocarcinoma tissues using: SeqCap EZ Human Exome v.3.0 (Roche NimbleGen) and SureSelect XT Human All Exon v.5 (Agilent Technologies). Results Sequencing metrics on Illumina HiSeq were optimal for both exome systems and comparable among FFPE and FF samples, with a slight increase of PCR duplicates in FFPE, mainly in Roche NimbleGen libraries. Comparison of single nucleotide variants (SNVs) between FFPE-FF pairs reached overlapping values >90 % in both systems. Both WES showed high concordance with target re-sequencing data by Ion PGM™ in 22 lung-cancer genes, regardless the source of samples. Exon coverage of 623 cancer-related genes revealed high coverage efficiency of both kits, proposing WES as a valid alternative to target re-sequencing. Conclusions High-quality and reliable data can be successfully obtained from WES of FFPE samples starting from a relatively low amount of input gDNA, suggesting the inclusion of NGS-based tests into clinical contest. In conclusion, our analysis suggests that the WES approach could be extended to a translational research context as well as to the clinic (e.g. to study rare malignancies), where the simultaneous analysis of the whole coding region of the genome may help in the detection of cancer-linked variants. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2720-4) contains supplementary material, which is available to authorized users.

Supplemental Digital Content is available in the text, The presence of multiple primary tumors (MPT) in a single patient has been identified with an increasing frequency. A critical issue is to establish if the second tumor represents an independent primary cancer or a metastasis. Therefore, the assessment of MPT clonal origin might help understand the disease behavior and improve the management/prognosis of the patient. Herein, we report a 73-year-old male smoker who developed 2 primary lung cancers (adenocarcinoma and squamous cell carcinoma) and a malignant peritoneal mesothelioma (PM). Whole exome sequencing (WES) of the 3 tumors and of germline DNA was performed to determine the clonal origin and identify genetic cancer susceptibility. Both lung cancers were characterized by a high mutational rate with distinct mutational profiles and activation of tumor-specific pathways. Conversely, the PM harbored a relative low number of genetic variants and a novel mutation in the WT1 gene that might be involved in the carcinogenesis of nonasbestos-related mesothelioma. Finally, WES of the germinal DNA displayed several single nucleotide polymorphisms in DNA repair genes likely conferring higher cancer susceptibility. Overall, WES did not disclose any somatic genetic variant shared across the 3 tumors, suggesting their clonal independency; however, the carcinogenic effect of smoke combined with a deficiency in DNA repair genes and the patient advanced age might have been responsible for the MPT development. This case highlights the WES importance to define the clonal origin of MPT and susceptibility to cancer.

Lung cancer is still the leading cause of cancer related death worldwide. Fibroblast growth factor receptor (FGFR) is a tirosine-kinase receptor that is seen to be amplified or mutated in non-small cell lung cancer (NSCLC) and it plays a crucial role in tumour development and maintenance. The authors analyzed the state of the art of FGFR by reviewing the current literature. Fibroblast growth factor (FGF)-FGFR pathway and their aberrations are described, with the evaluation of their possible prognostic role in NSCLC and in particular in squamous cell carcinomas, in which FGFR is more often amplified. New therapeutic agents targeting FGFR signaling have been developed and are now in clinical evaluation. Dysregulation of FGF signaling in tumour cells is related to FGFR gene amplification or mutation, although it is still uncertain which of these aberrations represents a real predictor of response to specific inhibitors. However, recent evidence has questioned whether FGFR is a real target in squamous cell histology. The effectiveness of FGFR inhibitors is also still unclear since there are no clinical data on selected patients. Moreover, the management of specific side effects related to inhibition of the physiological role of FGF should be more thorough.

Immune checkpoint inhibitors (ICIs) including programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) antagonist antibodies for lung tumors mark a new era of cancer therapeutics harnessing a patient’s immune system to induce durable antitumor responses. Despite their impressive activity in some patients, challenges remain as most tumors exhibit primary or acquired resistance (AR) to ICIs, and the biologic mechanisms for resistance are poorly understood. Using a collection of 14 ICI-resistant lung cancer samples, we investigated whether alterations in genes encoding components of the HLA Class I antigen processing and presentation machinery or interferon signaling pathways play a role in AR to PD-1 or PD-L1 antagonistic antibodies. Although we did not detect any recurrent mutations or copy number changes in our AR cohort, we noted one case of an acquired homozygous loss of B2M that resulted in lack of HLA class I expression on the cell surface in both the patient’s tumor sample and the corresponding patient-derived xenograft (PDX). Downregulation of B2M was also found in two additional PDXs established from ICI-resistant tumors. To test if B2m expression confers sensitivity to ICIs in vivo, we used a CRISPR-mediated approach to knockout B2m in an ICI-sensitive, murine lung cancer cell line and transplanted the cells into syngeneic, immunocompetent mice. We found that the B2m knockout tumors were resistant to ICI therapy in contrast to the wild type tumors. Furthermore, RNA-seq analysis of a subset of the samples in the acquired resistance to ICI cohort revealed an inflammatory tumor microenvironment with significant upregulation of the inhibitory receptors LAG-3 and PD-1 in tumor-infiltrating T cells at ICI resistance. Overall, these findings provide a novel system for the evaluation and screening of candidate genes for their ability to mediate AR to ICIs in vivo. Moreover, they also provide evidence for the disruption of HLA Class I antigen processing and presentation as a mechanism for escape from ICIs in lung cancer and provide information on the immune microenvironment in ICI-resistant tumors. Citation Format: Katherine Hastings, Scott Gettinger, Choi Jungmin, Anna Truini, Ila Datar, Ryan Sowell, Anna Wurtz, Weilai Dong, Guoping Cai, Mary Ann Melnick, Joseph Schlessinger, Sarah Goldberg, Anne Chiang, Ignacio Melero, Jackeline Agorreta, Luis Montuenga, Richard Lifton, Soldano Ferrone, Paula Kavathas, David Rimm, Susan Kaech, Kurt Schalper, Roy Herbst, Katerina Politi. Impaired HLA Class I antigen processing and presentation as a mechanism of acquired Rrsistance to immune checkpoint inhibitors in lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A09.

9056Background: Exon 19 deletions (ex19 dels) account for ~50% of EGFR-mutant NSCLCs. The E746_A750 ex19 del accounts for 75% of these cases. Two frequent uncommon ex19 dels are L747_P753 > S and L...

Next-generation sequencing (NGS) is a cost-effective technology capable of screening several genes simultaneously, however, its application in a clinical context requires an established workflow to acquire reliable sequencing results. Here, we report an optimized NGS workflow analyzing 22 lung cancer-related genes to sequence critical samples such as DNA from formalin-fixed paraffin-embedded (FFPE) blocks and circulating free DNA (cfDNA). Snap frozen and matched FFPE gDNA from 12 non-small cell lung cancer (NSCLC) patients, whose gDNA fragmentation status was previously evaluated using a multiplex PCR-based quality control, were successfully sequenced with Ion Torrent PGM™. The robust bioinformatic pipeline allowed us to correctly call both Single Nucleotide Variants (SNVs) and indels with a detection limit of 5%, achieving 100% specificity and 96% sensitivity. This workflow was also validated in 13 FFPE NSCLC biopsies. Furthermore, a specific protocol for low input gDNA capable of producing good sequencing data with high coverage, high uniformity, and a low error rate was also optimized. In conclusion, we demonstrate the feasibility of obtaining gDNA from FFPE samples suitable for NGS by performing appropriate quality controls. The optimized workflow, capable of screening low input gDNA, highlights NGS as a potential tool in the detection, disease monitoring, and treatment of NSCLC.

The epidermal growth factor receptor (EGFR) signalling is one of the most deregulated pathways in non-small cell lung cancer (NSCLC). Recently, the development of novel irreversible tyrosine kinase inhibitors (TKI), such as afatinib, has significantly improved the survival of advanced NSCLC patients harbouring activated EGFR mutations. However, treatment with TKI is not always curative due to the development of resistance. In the present study, we investigated the sensitivity to afatinib in two NSCLC EGFR mutated cell lines (NCI-H1650 and NCI-H1975) by expression profile analysis of 92 genes involved in the EGF pathway. Thereafter, the established afatinib resistant clones were evaluated at different biological levels: genomic, by array comparative genomic hybridisation (aCGH) and deep sequencing; transcriptomic, by quantitative polymerase chain reaction (qPCR) and proteomic, by Western blot and immunofluorescence. The baseline gene expression of the two cell lines revealed that NCI-H1650, the less afatinib-responsive cell, showed activation of two main EGFR downstream pathways such as PI3K/AKT and PLCγ/PKC axes. Analysis of the afatinib-resistant cells showed PI3K/AKT and MAPK/ERK pathways activation together with a biological switch from an epithelial-to-mesenchymal phenotype might confer afatinib-resistant properties to this cell line. Our data suggest that the activation of EGFR-dependent downstream pathways might be involved in the occurrence of resistance to afatinib assuming that the EGFR mutational status should not be exclusively considered when selecting TKI treatments. In particular, the epithelial-to-mesenchymal transition might provide a new basis for understanding afatinib resistance.

Non-small cell lung cancer (NSCLC) is one of the most common causes of cancer-related death worldwide. Based on the patient's stage of disease, treatment options include surgery, radiotherapy, and chemotherapy. Although chemotherapy remains the main therapeutic approach for advanced NSCLC, targeted therapy represents a good chance of treatment for this subgroup of patients. Currently this approach is based on previous evaluation of clinically relevant mutations and the Sanger sequencing is the main approach to assign mutational status and to guide the appropriate treatment; however this tool is characterized by a low sensitivity. Recently, the advent of next-generation sequencing (NGS) has dramatically revolutionized the molecular knowledge of cancer by increasing the feasibility and possibility to sequence DNA ranging from large scale studies to targeted regions. This review reports an overview of different applications of the NGS as novel approach to study NSCLC, thereby providing information about mutational spectrum of this cancer in order to identify novel targetable mutations and to predict the emergence of drug resistance. All studies demonstrated several advantages of this approach over the traditional tools. In particular the NGS was also able to reveal mutations in low percentage, and to screen the mutational status of different critical samples such as biopsies, cytological samples and circulating plasma DNA, offering innovative diagnostic opportunities. Despite several problems have to be overcome toward the personalized therapy, the NGS represents a highly attractive system to identify mutations improving the outcome of patients with this deadly disease.

The epidemic increase of adenocarcinoma histology accounting for more than 50% of primary lung malignancies and the advent of effective molecular targeted-therapies against specific gene alterations characterizing this tumor type have led to the reconsideration of the pathologic classification of lung cancer. The new 2015 WHO classification provided the basis for a multidisciplinary approach emphasizing the close correlation among clinical, radiologic and molecular characteristics and histopathologic pattern of lung adenocarcinoma. The terms 'bronchioloalveolar carcinoma' and 'mixed adenocarcinoma' have been eliminated, introducing the concepts of 'adenocarcinoma in situ', 'minimally invasive adenocarcinoma' and the use of descriptive predominant patterns in invasive adenocarcinomas (lepidic, acinar, papillary, solid and micropapillary patterns). 'Invasive mucinous adenocarcinoma' is the new definition for mucinous bronchioloalveolar carcinoma, and some variants of invasive adenocarcinoma have been included, namely colloid, enteric and fetal-type adenocarcinomas. A concise update of the immunomorphologic, radiological and molecular characteristics of the different histologic patterns of lung adenocarcinoma is reported here.

Introduction: Tyrosine kinase inhibitors (TKI) are currently the most effective therapy for patients with NSCLC and common activating mutations of EGFR. Afatinib, an irreversible EGFR, HER2, and HER4 inhibitor, has recently been approved at the dose of 40 mg daily as front-line treatment for advanced NSCLC with common EGFR-activating mutations.Areas covered: The review analyzes the pharmacodynamics, pharmacokinetics, safety, and clinical efficacy of afatinb in NSCLC. This irreversible TKI was initially investigated in EGFR-unselected patients who progressed after TKIs, but the subsequent observation of its major activity against exon 19 mutant determined its indication in common EGFR mutations. Several Phase I to Phase III studies explored the role of afatinib in different settings of patients.Expert opinion: Afatinib showed efficacy as a front-line treatment in patients with EGFR common mutations. Currently, no statistically significant differences between afatinib, gefitinib, and erlotinib have been det...

Immunotherapy has become a promising approach for the treatment of NSCLC. In order to stimulate the host immune system against tumour antigens, several cancer vaccines have been generated and evaluated. Belagenpumatucel-L is a whole tumour cell vaccine expressing the antisense strand of the TGF-β2 gene.The purpose of this article is to review the most relevant findings of clinical trials testing belagenpumatucel-L in advanced NSCLC patients.Although the Phase III trial investigating belagenpumatucel-L in stage III/IV patients did not meet its primary end point, a survival benefit was observed in several subgroups of patients. Further studies are needed in order to select patients who may benefit from this vaccine.

Lung cancer is emerging as a paradigm for disease molecular subtyping, facilitating targeted therapy based on driving somatic alterations. Here we perform transcriptome analysis of 153 samples representing lung adenocarcinomas, squamous cell carcinomas, large cell lung cancer, adenoid cystic carcinomas and cell lines. By integrating our data with The Cancer Genome Atlas and published sources, we analyse 753 lung cancer samples for gene fusions and other transcriptomic alterations. We show that higher numbers of gene fusions is an independent prognostic factor for poor survival in lung cancer. Our analysis confirms the recently reported CD74-NRG1 fusion and suggests that NRG1, NF1 and Hippo pathway fusions may play important roles in tumours without known driver mutations. In addition, we observe exon-skipping events in c-MET, which are attributable to splice site mutations. These classes of genetic aberrations may play a significant role in the genesis of lung cancers lacking known driver mutations.

The aim of this study was to identify differentially-expressed miRNAs in the serum of non-small cell lung cancer (NSCLC) patients that might be a clinically-useful tool for lung cancer early detection. We performed miRNA expression profile analysis using TaqMan OpenArray Human panel in a discovery set of 70 serum samples obtained at lung tumor resection and 22 non-cancer subjects (NC). Selected serum miRNAs were then validated by quantitative PCR using an independent validation set of serum samples from LC patients (n = 84) and NC (n = 23). Sixty miRNAs were significantly up-regulated and 31 were down-regulated in the serum from NSCLC patients versus NC (adjusted p p p

Circulating tumor cells (CTCs) are cells spread from the primary tumor into the bloodstream that might represent an important biomarker in lung cancer. The prognosis of patients diagnosed with lung cancer is generally poor mainly due to late diagnosis. Recent evidences have reported that tumor aggressiveness is associated with the presence of CTCs in the blood stream; therefore, several studies have focused their attention on CTC isolation, characterization, and clinical significance. So far, the CellSearch(®) system is the only approach approved by FDA for metastatic breast, prostate, and colorectal cancer intended to detect CTCs of epithelial origin in whole blood and to assess prognosis. To date, no specific biomarkers have been validated in lung cancer and the identification of novel tumor markers such as CTCs might highly contribute to lung cancer prognosis and management. In the present review, the significance of CTC detection in lung cancer is examined through the analysis of the published studies in both non-small cell and small cell lung cancers; additionally the prognostic and the clinical role of CTC enumeration in treatment monitoring will be reported and discussed.

Introduction: Angiogenesis plays a crucial role in the proliferation and in the metastatic spread of tumour cells. Several agents with anti-angiogenic activity have been tested in advanced non-small-cell lung cancer (NSCLC) patients. Motesanib (AMG 706) is a promising anti-angiogenic multi-targeted tyrosine kinase inhibitor (TKI), which has been investigated as a monotherapy or in combination with chemotherapy, in several types of cancer.Areas covered: We have reviewed the literature, and we have presented the results of clinical trials that have investigated the administration of motesanib in advanced NSCLC patients.Expert opinion: Encouraging results have been described with the administration of motesanib as first-line treatment in combination with carboplatin and paclitaxel in Asian patients with non-squamous advanced NSCLC. Further studies are needed in order to identify the predictive biomarkers of response and to select patients who may benefit from this anti-angiogenic treatment.

Circulating tumor cells (CTCs) can be found in the bloodstream of patients with malignancies and are associated with disease recurrence and treatment response. Several studies have indicated an important role for CTCs in metastatic breast, colorectal and prostate tumors, as well as in non-small-cell lung cancer (NSCLC). The poor overall survival of NSCLC patients and the complex heterogeneity of the disease are significant challenges for therapeutic intervention. The detection of genetic abnormalities and the expression of biomarkers that can be therapeutically targeted in CTCs might yield new perspectives for the diagnosis and prognosis of NSCLC patients that are aimed at improving clinical outcomes.

The inhibition of the epidermal growth factor receptor (EGFR) through tyrosine kinase inhibitors (TKIs) represents an effective strategy for EGFR-mutated NSCLC. Afatinib is an irreversible erythroblastosis oncogene B (ErbB) family blocker, able to inhibit the kinase domains of EGFR, HER2 and HER4, and the transphosphorylation of ErbB3 that has recently been approved in the United States for the first-line treatment of EGFR-mutated NSCLC and in Europe and Japan for the treatment of EGFR-mutated TKI-naive patients.The authors analyzed the pharmacology and the clinical activity of afatinib in NSCLC through a review of the literature. Trials exploring different settings have been reported, including LUX-Lung 3 and LUX-Lung 6, where the drug achieved better outcomes in terms of response rate, progression-free survival and quality of life compared with chemotherapy. The main toxicities of afatinib are gastrointestinal and skin-related adverse events.Afatinib showed remarkable efficacy as a first-line treatment in the presence of common EGFR mutations. Afatinib showed some activity in NSCLC with acquired resistance to EGFR TKIs, although, currently, its efficacy after the failure of erlotinib or gefitinib has not been clearly stated. Direct clinical data comparing the activity and tolerability of different inhibitors are still needed.

ABSTRACT:After several decades of modest results with nonspecific immune stimulants, immunotherapy has become an exciting approach in the treatment of cancer. Although non-small-cell lung cancer has not been considered an immunogenic disease for very long, a better understanding of tumor immunology and the identification of new targets have led to the development of many clinical trials of immune-based therapies for this neoplasm. Promising results from many clinical trials suggest that immunotherapy could be an effective strategy in the management of advanced non-small-cell lung cancer. Further studies are required to help clinicians in the selection of patients who are more likely to benefit from immunotherapy strategies by the identification of biomarkers and to understand when the combination of immunotherapy with other agents should be recommended.

Malignant mesothelioma (MM) is an aggressive tumor, mainly derived from the pleura, which is predominantly associated with exposure to asbestos fibers. The prognosis of MM patients is particularly severe, with a median survival of approximately 9-12 months and latency between exposure and diagnosis ranging from 20-50 years (median 30 years). Emerging evidence has demonstrated that tumor aggressiveness is associated with genome and gene expression abnormalities; therefore, several studies have recently focused on the role of microRNAs (miRNAs) in MM tumorigenesis. miRNAs are small non-protein coding single-stranded RNAs (17-22 nucleotides) involved in numerous cellular processes that negatively regulate gene expression by modulating the expression of downstream target genes. miRNAs are often deregulated in cancer; in particular, the differential miRNA expression profiles of MM cells compared to unaffected mesothelial cells have suggested potential roles of miRNAs as either oncogenes or tumor suppressor genes in MM oncogenesis. In this review, the mechanism of MM carcinogenesis was evaluated through the analysis of the published miRNA expression data. The roles of miRNAs as diagnostic biomarkers and prognostic factors for potential therapeutic strategies will be presented and discussed.

Purpose: The microRNA-34b/c (miR-34b/c) is considered a tumor suppressor in different tumor types and a transcriptional target of TP53. The main objectives of this study were to investigate the clinical implications of miR-34b/c methylation in patients with early-stage lung adenocarcinoma and to determine the functional role of miR-34b/c re-expression in lung adenocarcinoma cell lines. Experimental Design: Aberrant methylation and expression of miR-34b/c were assessed in 15 lung adenocarcinoma cell lines and a cohort of 140 early-stage lung adenocarcinoma. Lung adenocarcinoma cell lines were transfected with miR-34b/c and the effects upon cell proliferation, migration, invasion, and apoptosis were investigated. Results: Aberrant methylation of miR-34b/c was detected in 6 (40%) of 15 lung adenocarcinoma cell lines and 64 of 140 (46%) primary lung adenocarcinoma. Expression of miR-34b/c was significantly reduced in all methylated cell lines and primary tumors, especially with TP53 mutations. Patients with increased miR-34b/c methylation had significantly shorter disease-free and overall survival as compared to patients with unmethylated or low level of miR-34b/c methylation. Ectopic expression of miR-34b/c in lung adenocarcinoma cell lines decreased cell proliferation, migration, and invasion. Conclusions: Epigenetic inactivation of miR-34b/c by DNA methylation has independent prognostic value in patients with early-stage lung adenocarcinoma. Reexpression of miR-34b/c leads to a less aggressive phenotype in lung adenocarcinoma cell lines. Clin Cancer Res; 19(24); 6842–52. ©2013 AACR.

Non-small cell lung cancer (NSCLC) is a major cause of cancer-related death worldwide. Although advanced NSCLC is still incurable, various anti-neoplastic agents have become available for the treatment of this disease. Pemetrexed , a multi-target folate antagonist, has improved the survival of non-squamous NSCLC patients. Currently, pemetrexed is approved for first-line treatment in combination with a platinum derivate, for second-line treatment as a single agent and, more recently, as maintenance treatment after first-line chemotherapy.The authors analyzed the state of the art of pemetrexed through a review of the literature. Clinical trials and meta-analyses involving pemetrexed in NSCLC were evaluated. Pemetrexed improved survival of non-squamous NSCLC in first-line, maintenance, and second-line treatments; this benefit is limited to non-squamous histology. Because pemetrexed has become part of the standard of care, current clinical trials are designed to compare it to other investigational combinations. Limited data on resectable disease are available, and additional clinical trials are being conducted.Pemetrexed has shown effectiveness and a favorable toxicity profile. Histology-driven indications and the relationship of pemetrexed with thymidylate synthase expression suggest that a more precise definition of predictive biomarkers could be further investigated.

Introduction: The current standard of care for advanced non-small cell lung cancer (NSCLC) is systemic chemotherapy or treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) for EGFR-mutated tumors. Preclinical studies suggest that the combination of cytotoxic agents and EGFR-TKIs may increase the inhibitory effect albeit influenced by the type of drug and the order of administration. Whether these combinations may exert similar effects in tumors poorly responsive to EGFR-TKIs is still unclear. The antitumor efficacy of different protocols with vinorelbine (VNB) and gefitinib (GEF) were investigated in two NSCLC cell lines, in vitro and in vivo, as a rationale to be potentially exploited in the clinical setting. Methods: The EGFR-wild-type A549 and the EGFR-mutated (exon 21 L858R/exon 20 T790M) H1975 cell lines were treated for 72 hrs with VNB and GEF according to different schemes and concentrations: GEF followed by VNB, VNB followed by GEF and the two drugs given individually or concurrently. The cytotoxicity was determined by MTT assay (short-term). In addition, the efficacy of repeated weekly VNB treatment along with sequential or continuous GEF administration was investigated by cell count (long-term). The expression levels of activated EGFR and downstream pathway molecules were investigated by western blot. The in vivo effects of single or combined treatments were studied in a CD1-Nude mouse model by subcutaneous injection of the H1975 cells. Cancer growth was determined by measurements of tumor diameters with a Vernier caliper, while lesion glucose consumption was estimated by means of a dynamic micro Positron Emission Tomography scans. To this purpose, a bolus of 3-6 MBq of 18F-fluorodeoxyglycose was injected through a tail vein during a list mode acquisition lasting one hour. After framing rate optimization, pixelwise maps of cancer glucose consumption (MRGlc) were created using dedicated software (PMOD) and applying Patlak graphical approach. Results: In vitro short and long-term studies demonstrated that the sequence of VNB followed by GEF was significantly more active than GEF followed by VNB or the concurrent administration of the two drugs. Western blot analyses indicated that the increased cytotoxic effect of the VNB and GEF sequence was accompanied by inhibition of p-EGFR, p-AKT and p-ERK1/2 expression levels, mainly in the EGFR-mutated H1975. Moreover, the increased antitumor efficacy of the sequence VNB followed by GEF was also confirmed in vivo by a significant inhibition of the H1975 tumor growth (p Conclusions: These preclinical findings in NSCLC cell lines, poorly responsive to EGFR-TKIs, showed that the sequential treatment with VNB followed by GEF induced a significant antitumor efficacy supporting the application to clinical trials. Citation Format: Angela Alama, Maria Giovanna Dal Bello, Irene Vanni, Anna Truini, Simona Coco, Erika Rijavec, Carlo Genova, Federica Biello, Cecilia Marini, Gianluca Bottoni, Gianmario Sambuceti, Francesco Grossi. In vitro and in vivo antitumor efficacy of sequentially combined vinorelbine and gefitinib in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2521. doi:10.1158/1538-7445.AM2015-2521