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Pleural effusions are among the first clinical manifestations of malignant pleural mesothelioma (MPM) and often constitute the only available material for diagnosis. Although an MPM diagnosis can be reliable on cytology, the reported sensitivity is low (30% to 75%). Particularly, it can be hard to discriminate epithelioid MPM, the most common histotype, from reactive mesothelial hyperplasia (MH). Currently, BRCA1-associated protein 1 (BAP1) and CDKN2A (p16), evaluated by immunohistochemistry and fluorescent in situ hybridization, respectively, are the most valuable markers to discriminate MPM and MH. Both markers have a high specificity, but their sensitivity is not always satisfying, even when used together. We have recently developed a 117-gene expression panel, based on Nanostring technology, able to differentiate epithelioid MPM from MH pleural tissues better than BAP1 and p16. Herein, we evaluated the efficacy of the same panel on an independent retrospective cohort of 23 MPM and 11 MH pleural effusions (cell blocks and smears). The overall sensitivity and specificity of the panel were equal to 0.9565 and 1, respectively. Moreover, the panel performance was compared with BAP1 and p16 on 25 cell blocks. Sensitivity levels of gene panel, BAP1 alone, p16 alone, and BAP1 plus p16 were 1, 0.5882, 0.4706, and 0.7647, respectively. Specificity was always 1. Although further validation is needed, this gene panel could really facilitate patients' management, allowing a definitive MPM diagnosis directly on pleural effusions.

Pembrolizumab has been approved as first-line treatment for advanced Non-small cell lung cancer (NSCLC) patients with tumors expressing PD-L1 and in the absence of other targetable alterations. However, not all patients that meet these criteria have a durable benefit. In this monocentric study, we aimed at refining the selection of patients based on the expression of immune genes. Forty-six consecutive advanced NSCLC patients treated with pembrolizumab in first-line setting were enrolled. The expression levels of 770 genes involved in the regulation of the immune system was analysed by the nanoString system. PD-L1 expression was evaluated by immunohistochemistry. Patients with durable clinical benefit had a greater infiltration of cytotoxic cells, exhausted CD8, B-cells, CD45, T-cells, CD8 T-cells and NK cells. Immune cell scores such as CD8 T-cell and NK cell were good predictors of durable response with an AUC of 0.82. Among the immune cell markers, XCL1/2 showed the better performance in predicting durable benefit to pembrolizumab, with an AUC of 0.85. Additionally, CD8A, CD8B and EOMES showed a high specificity (&gt, 0.86) in identifying patients with a good response to treatment. In the same series, PD-L1 expression levels had an AUC of 0.61. The characterization of tumor microenvironment, even with the use of single markers, can improve patients’ selection for pembrolizumab treatment.

Resistance to EGFR tyrosin kinase inhibitors (TKI) inevitably occurs. Here it is reported the case of a young patient affected by lung adenocarcinoma harboring the L858R EGFR sensitive mutation. The patient developed multiple TKI resistance mechanisms: T790M EGFR resistance mutation, detected only on tumor cell-free DNA, squamous cell transformation and MET amplification, both detected on a tumor re-biopsy. The co-occurrence of squamous cell transformation and de novo MET amplification is an extremely rare event, and this case confirms how dynamic and heterogeneous can be the temporal and spatial tumor evolution under treatment pressure.

Gene fusions have a pivotal role in non-small cell lung cancer (NSCLC) precision medicine. Several techniques can be used, from fluorescence in situ hybridization and immunohistochemistry to next generation sequencing (NGS). Although several NGS panels are available, gene fusion testing presents more technical challenges than other variants. This is a PubMed-based narrative review aiming to summarize NGS approaches for gene fusion analysis and their performance on NSCLC clinical samples. The analysis can be performed at DNA or RNA levels, using different target enrichment (hybrid-capture or amplicon-based) and sequencing chemistries, with both custom and commercially available panels. DNA sequencing evaluates different alteration types simultaneously, but large introns and repetitive sequences can impact on the performance and it does not discriminate between expressed and unexpressed gene fusions. RNA-based targeted approach analyses and quantifies directly fusion transcripts and is more accurate than DNA panels on tumor tissue, but it can be limited by RNA quality and quantity. On liquid biopsy, satisfying data have been published on circulating tumor DNA hybrid-capture panels. There is not a perfect method for gene fusion analysis, but NGS approaches, though still needing a complete standardization and optimization, present several advantages for the clinical practice.

Objectives The separation of benign from malignant mesothelial proliferations and exact subclassification of mesothelioma subtypes is crucial to determining patient care and prognosis but morphologically can be very difficult. Methods This session of the 2018 IMIG meeting addressed these problems. Results A new immunohistochemical marker, methylthioadenosine phosphorylase, was shown to correlate well with CDKN2A FISH and is cheaper and faster to run. A 117 gene expression panel also provided good separation on both tissue biopsy and cytology samples. Review of a series of mesotheliomas thought to be biphasic produced only a moderate level of agreement among expert pathologists with some cases being classified as purely epithelioid or sarcomatoid; these classifications had prognostic significance. The entity called transitional mesothelioma was found to behave exactly like sarcomatoid mesothelioma. RNA-seq analysis of a large series of mesotheliomas from a public database showed that, genetically, the morphologic breakdown into epithelioid, sarcomatoid, or biphasic mesotheliomas is artificial because there is a continuous spectrum of genomic changes. There are now criteria for the diagnosis of mesothelioma in situ and this is potentially important, since such cases might be curable. Conclusions This session documented new morphological and molecular approaches to separating benign from malignant mesothelial proliferations and to subclassifying malignant mesoteheliomas in clinical relevant ways.

The disruption of the Hippo pathway occurs in many cancer types and is associated with cancer progression. Herein, we investigated the impact of 32 Hippo genes on overall survival (OS) of cancer patients, by both analysing data from The Cancer Genome Atlas (TCGA) and reviewing the related literature. mRNA and protein expression data of all solid tumors except pure sarcomas were downloaded from TCGA database. Thirty-two Hippo genes were considered; for each gene, patients were dichotomized based on median expression value. Survival analyses were performed to identify independent predictors, taking into account the main clinical-pathological features affecting OS. Finally, independent predictors were correlated with YAP1 oncoprotein expression. At least one of the Hippo genes is an independent prognostic factor in 12 out of 13 considered tumor datasets. mRNA levels of the independent predictors coherently correlate with YAP1 in glioma, kidney renal clear cell, head and neck, and bladder cancer. Moreover, literature data revealed the association between YAP1 levels and OS in gastric, colorectal, hepatocellular, pancreatic, and lung cancer. Herein, we identified cancers in which Hippo pathway affects OS; these cancers should be candidates for YAP1 inhibitors development and testing.

Malignant pleural mesothelioma (MPM), an asbestos-induced tumor, represents significant diagnostic challenges for pathologists. Its histological diagnosis is stepwise and should be based on morphological assessment, supported by clinical and radiological findings, and supplemented with immunohistochemistry (IHC) and, more recently, molecular tests. The main diagnostic dilemmas are the differential diagnoses with benign mesothelial proliferations and other pleural malignant tumors. The present review is an update regarding the morphological, immunohistochemical, and molecular features with respect to MPM diagnosis. Data sources include a survey of the biomedical literature from PubMed (http://www.ncbi. nlm.nih.gov/pubmed) and textbooks focusing on the pathological diagnosis of MPM and associated immunohistochemical and molecular markers. The histological findings of MPM could facilitate its diagnosis and provide important prognostic information. The immunohistochemical approach should rest on the application of a panel including positive (mesothelial-related) and negative markers with greater than 80% sensitivity and specificity, which need to be selected based on morphology and clinical information. Moreover, in challenging cases, fluorescent in situ hybridization (FISH) testing for the p16 deletion and IHC to evaluate the loss of BRCA1-associated protein 1 (BAP1) expression could be useful in distinguishing benign from malignant pleural proliferations.

The present case report describes the infrequent coexistence of squamous cell transformation and the epidermal growth factor receptor (EGFR) T790M mutation as resistance mechanisms to first line treatment with tyrosine kinase inhibitors. The patient was a 44-year-old female, diagnosed with a primitive advanced lung adenocarcinoma with bone metastases. The tumor was positive for the EGFR exon 19 deletion, therefore the patient was treated with afatinib (40 mg/day, orally) and radiotherapy for bone lesions. After 16 months, the patient developed resistance. Cytological examination of the pleural effusion confirmed an adenocarcinoma positive for the EGFR exon 19 deletion and the T790M mutation within exon 20, while a biopsy from the upper left bronchus revealed a keratinizing squamous cell carcinoma positive for the EGFR exon 19 deletion. In addition, the EGFR mutations were concomitantly detected in circulating cell-free tumour DNA. Due to the presence of the T790M mutation, the patient underwent osimertinib therapy (80 mg/day, orally), which resulted in a partial tumour regression at the 2-month follow-up, whereas the squamous lesions were treated with radiotherapy. The adenocarcinoma and squamous carcinoma components may share the same origin, according to the presence of the EGFR exon 19 deletion in both lesions. More accurate characterization of resistance mechanisms may lead to the development of improved treatment regimens.

Pulmonary sarcomatoid carcinomas (PSC) are a rare group of lung cancer with a median overall survival of 9–12 months. PSC are divided into five histotypes, challenging to diagnose and treat. The identification of PSC biomarkers is warranted, but PSC molecular profile remains to be defined. Herein, a targeted whole transcriptome analysis was performed on 14 PSC samples, evaluated also for the presence of the main oncogene mutations and rearrangements. PSC expression data were compared with transcriptome data of lung adenocarcinomas (LUAD) and squamous cell carcinomas (LUSC) from The Cancer Genome Atlas. Deregulated genes were used for pathway enrichment analysis; the most representative genes were tested by immunohistochemistry (IHC) in an independent cohort (30 PSC, 31 LUAD, 31 LUSC). All PSC cases were investigated for PD-L1 expression. Thirty-eight genes deregulated in PSC were identified, among these IGJ and SLMAP were confirmed by IHC. Moreover, Forkhead box signaling and Fanconi anemia pathways were specifically enriched in PSC. Finally, some PSC harboured alterations in genes targetable by tyrosine kinase inhibitors, as EGFR and MET. We provide a deep molecular characterization of PSC; the identification of specific molecular profiles, besides increasing our knowledge on PSC biology, might suggest new strategies to improve patients management.

Purpose: MET-deregulated NSCLC represents an urgent clinical need because of unfavorable prognosis and lack of specific therapies. Although recent studies have suggested a potential role for crizotinib in patients harboring MET amplification or exon 14 mutations, no conclusive data are currently available. This study aimed at investigating activity of crizotinib in patients harboring MET or ROS1 alterations. Patients and Methods: Patients with pretreated advanced NSCLC and evidence of ROS1 rearrangements (cohort A) or MET deregulation (amplification, ratio MET/CEP7 >2.2 or MET exon 14 mutations, cohort B) were treated with crizotinib 250 mg twice daily orally. The coprimary endpoint was objective response rate in the two cohorts. Results: From December 2014 to March 2017, 505 patients were screened and a total of 52 patients (26 patients per cohort) were enrolled onto the study. At data cutoff of September 2017, in cohort A, objective response rate was 65%, and median progression-free survival and overall survival were 22.8 months [95% confidence interval (CI) 15.2–30.3] and not reached, respectively. In cohort B, objective response rate was 27%, median progression-free survival was 4.4 months (95% CI 3.0–5.8), and overall survival was 5.4 months (95% CI, 4.2–6.5). No difference in any clinical endpoint was observed between MET-amplified and exon 14–mutated patients. No response was observed among the 5 patients with cooccurrence of a second gene alteration. No unexpected toxicity was observed in both cohorts. Conclusions: Crizotinib induces response in a fraction of MET-deregulated NSCLC. Additional studies and innovative therapies are urgently needed.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer, with most patients diagnosed at advanced stages. First-line treatment based on a combined chemotherapy (FOLFIRINOX or gemcitabine plus nab-paclitaxel) provides limited benefits. Olaparib, a PARP inhibitor, has been approved as maintenance for PDAC patients harboring germline BRCA1/2 pathogenic mutations and previously treated with a platinum-based chemotherapy. BRCA1/2 germline testing is recommended, but also somatic mutations could predict responses to PARP inhibitors. Analysis of tumor tissues can detect both germline and somatic mutations and potential resistance alterations. Few data are available about BRCA1/2 testing on pancreatic tumor tissues, which often include limited biological material. We performed BRCA1/2 testing, by an amplicon-based Next Generation Sequencing (NGS) panel, on 37 consecutive PDAC clinical samples: 86.5% of cases were adequate for NGS analysis, with a success rate of 81.2% (median DNA input: 10 nanograms). Three BRCA2 mutations were detected (11.5%). Failed samples were all from tissue macrosections, which had higher fragmented DNA than standard sections, biopsies and fine-needle aspirations, likely due to fixation procedures. BRCA1/2 testing on pancreatic tumor tissues can also be feasible on small biopsies, but more cases must be analyzed to define its role and value in the PDAC diagnostic algorithm.

We thank the authors for their interest in our study and for their comments. Wiesweg and colleagues correctly reported that the minor allele frequency of both p.T1010I and p.R988C MET variants is close to 1% in the European population, but these mutations were described also as somatic (T1010I

Context.— Patients with non–small cell lung cancer harboring ALK receptor tyrosine kinase (ALK), ROS proto-oncogene 1 (ROS1), and ret proto-oncogene (RET) gene rearrangements can benefit from specific kinase inhibitors. Detection of fusion genes is critical for determining the best treatment. Assessing rearrangements in non–small cell lung cancer remains challenging, particularly for lung cytology. Objective.— To examine the possible application of the multiplex, transcript-based NanoString system (NanoString Technologies, Seattle, Washington) in the evaluation of fusion genes in lung adenocarcinoma samples. Data Sources.— This study is a narrative literature review. Studies about NanoString, gene fusions, and lung adenocarcinoma were collected from PubMed (National Center for Biotechnology Information, Bethesda, Maryland). We found 7 articles about the application of the NanoString system to detect fusion genes on formalin-fixed, paraffin-embedded tumor tissues and one article evaluating the adequacy of lung cytologic specimens for NanoString gene expression analysis. Conclusions.— To maximize the yield of molecular tests on small lung biopsies, the NanoString nCounter system has been suggested to detect fusion genes. NanoString fusion gene assays have been successfully applied on formalin-fixed, paraffin-embedded tissues. Although there are only a few studies available, the application of NanoString assays may also be feasible in lung cytology. According to available data, the NanoString system could strengthen the routine molecular characterization of lung adenocarcinoma.

Malignant pleural mesothelioma (MPM) is an aggressive tumor associated with asbestos exposure. Histopathological analysis of pleural tissues is the gold standard for diagnosis; however, it can be difficult to differentiate malignant from benign pleural lesions. The purpose of this review is to describe the most important biomarkers and new diagnostic tools suggested for this differential diagnosis. There are many studies concerning the separation between MPM and benign pleural proliferations from both pleural tissues or effusions; most of them are based on the evaluation of one or few biomarkers by immunohistochemistry (IHC) or enzyme-linked immunosorbent assays (ELISAs), whereas others focused on the identification of MPM signatures given by microRNA (miRNA) or gene expression profiles as well as on the combination of molecular data and classification algorithms. None of the reported biomarkers showed adequate diagnostic accuracy, except for p16 [evaluated by fluorescent in situ hybridization (FISH)] and BAP1 (evaluated by IHC), both biomarkers are recommended by the International Mesothelioma Interest Group guidelines for histological and cytological diagnosis. BAP1 and p16 showed a specificity of 100% in discerning malignant from benign lesions because they are exclusively unexpressed or deleted in MPM. However, their sensitivity, even when used together, is not completely sufficient, and absence of their alterations cannot confirm the benign nature of the lesion. Recently, the availability of new techniques and increasing knowledge regarding MPM genetics led to the definition of some molecular panels, including genes or miRNAs specifically deregulated in MPM, that are extremely valuable for differential diagnosis. Moreover, the development of classification algorithms is facilitating the application of molecular data for clinical practice. Data regarding new diagnostic tools and MPM signatures are absolutely promising; however, before their application in clinical practice, a prospective validation is necessary, as these approaches could surely improve the differential diagnosis between malignant and benign pleural lesions.

Maternal uniparental disomy of chromosome 14 (upd(14)mat) or Temple syndrome is an imprinting disorder associated with a relatively mild phenotype. The absence of specific congenital malformations makes this condition underdiagnosed in clinical practice. A boy with a de novo robertsonian translocation 45,XY,rob(13;14)(q10;q10) is reported; a CGH/SNP array showed a loss of heterozygosity in 14q11.2q13.1. The final diagnosis of upd(14)mat was made by microsatellite analysis, which showed a combination of heterodisomy and isodisomy for different regions of chromosome 14. Obesity after initial failure to thrive developed, while compulsive eating habits were not present, which was helpful for the clinical differential diagnosis of Prader-Willi syndrome. In addition, the boy presented with many phenotypic features associated with upd(14)mat along with hypoesthesia to pain, previously unreported in this disorder, and bilateral cryptorchidism, also rarely described. These features, as well as other clinical manifestations (i.e., truncal obesity, altered pubertal timing), may suggest a hypothalamic-pituitary involvement. A detailed cytogenetic and molecular characterization of the genomic rearrangement is presented. Early genetic diagnosis permits a specific follow-up of children with upd(14)mat in order to optimize the long-term outcome.

Lung adenocarcinoma patients harboring anaplastic lymphoma kinase (ALK) gene rearrangements respond well to approved ALK inhibitors. However, to date, limited evidence is available regarding whether using circulating free tumor mRNA to identify aberrant ALK expression is possible, and its feasibility remains to be clearly addressed. The present study evaluated ALK expression by a one-step reverse transcription‑polymerase chain reaction (PCR) assay on the circulating free tumor mRNA from 12 lung adenocarcinoma patients. Additionally, the present study tested for ALK rearrangements by fluorescence in situ hybridization (FISH) and immunohistochemistry. A molecular genetic characterization was performed on tumor tissues and plasma samples. Aberrant ALK expression was detected in 2/12 patients using mRNA purified from plasma specimens and the results agreed with the FISH and immunohistochemistry findings of solid biopsy samples. The detection of aberrant ALK expression on circulating free tumor RNA may be feasible using a one‑step real‑time PCR assay and may be particularly helpful when a solid biopsy sample is not available.

e20656 Background: In lung cancer the evaluation of MET, a tyrosine kinase receptor involved in tumor growth and invasiveness, for copy number amplification and point mutations is relevant for prognosis and therapy. In lung adenocarcinoma (ADC) MET amplification is observed in 1-2% and MET mutations in 3-4% of cases. Mutations mainly occur in exon 14 (ex14), which encodes for the juxtamembrane negative regulatory domain, including splice site alterations and missense mutations within the exon.Commonly, MET ex14 alterations, in particular splice site ones, are mutually exclusive with other driver mutations, but co-occurrence with MET and MDM2 amplification and KRAS mutations was described. We evaluated METex14 mutation frequency and coexistence with additional driver alterations in a prospective cohort of Italian ADC patients. Methods: 315 ADC patients were tested (January-December 2016) for MET ex14 alterations by Sanger Sequencing on formalin-fixed paraffin-embedded tissues and cytological smears (tumor cells > 40%). MET positive cases were screened also for other oncogenes, among which KRAS, BRAF and PIK3CA, using a MALDI-TOF platform, and for ROS1 translocations and MET amplification by FISH. Results: 16 patients (5%) were MET positive: 7 splice site mutations (43%) and 9 (57%) other alterations within ex14. Among splice site mutations 1 co-occurred with ROS1 translocation and 1 with MET amplification. Among other mutations we found 5 T1010I missense mutations: 3 co-occurring with G12D/G13C/Q61H KRAS, 1 with G469A BRAF and 1 with a MET intron 13 point mutation; 2 R988C mutations: 1 with G13C KRAS and 1 with G13C KRAS plus ROS1 translocation; 1 P1026S mutation and 1 frameshift deletion (p.A991del;R992fs*999), both with G12C KRAS. Conclusions: Splice site mutations cause MET activation by exon skipping and increase sensitivity to tyrosine-kinase inhibitors, whereas how other ex14 mutations affect MET function is not fully understood. In our cohort we found a high rate of METex14 mutations together with alterations in other oncogenes, mostly KRAS. The interaction of MET with cancer signaling pathways deserve further investigation in order to better define the role of ex14 mutations in the context of target therapy.

Background and aims Increased concentrations of soluble mesothelin-related peptides (SMRP) have been found in sera of patients with malignant pleural mesothelioma (MPM) even if a relatively high rate of false positives has hampered their clinical use as a tumor marker. Individual SMRP levels could be affected by polymorphic elements. The aim of this study was to investigate the association between single nucleotide polymorphisms within the promoter-5'UTR regions and SMRP levels in healthy asbestos-exposed individuals and patients suffering from MPM. Methods The promoter-5'UTR regions of the mesothelin gene were genotyped in 59 healthy asbestos-exposed subjects and 27 MPM patients. SMRP levels were measured using a commercially available ELISA kit. Results Two novel polymorphisms, an A>C variant (called New1) and a C>T variant (called New2), were identified. In healthy subjects, high SMRP levels were associated with the C-variant of New1, with an average 1.62-fold increase compared with AA homozygotes (pConclusions New1-New2 genotypes could be employed as markers for setting individualized and appropriate thresholds of “normality” when SMRP is used in surveillance programs of asbestos-exposed people.