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Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease, for which it is crucial to promptly detect actionable and prognostic alterations to drive specific therapeutic decisions, regardless of tumor resectability status. Endoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA) is of key importance for PDAC diagnosis and can contribute significantly to tumor molecular profiling.Comprehensive genomic profile by targeted next-generation sequencing (NGS) was performed on 2 independent PDAC patient cohorts. Cohort 1 consisted of 77 patients with resectable PDAC for whom the histologic sample at the time of resection was available; for 56 patients cytologic specimens at the time of diagnosis also were obtained by EUS-FNA. Cohort 2 consisted of 20 patients with unresectable PDAC, for whom only the EUS-FNA cytologic sample was available.In cohort 1, a complete concordant mutational profile between the cytologic sample at diagnosis and the corresponding histologic specimen after surgery was observed in 88% of the cases, proving the ability to detect potential clinically relevant alterations in cytologic samples by NGS analysis. Notably, clinically actionable mutations were identified in 20% of patients. In cohort 2, comprehensive mutational profiling was obtained successfully for all samples. Consistent with the findings of cohort 1, KRAS, TP53, CDKN2A, and SMAD4 were the most altered genes. Most importantly, 15% of the patients harbored actionable mutations.Our findings show the feasibility of an NGS approach using both surgical specimens and cytologic samples. The model proposed in this study can be included successfully in the clinical setting for comprehensive molecular profiling of all PDAC patients irrespective of their surgical eligibility.

Assessment of HRD status is now essential for ovarian cancer patient management. A relevant percentage of high-grade serous carcinoma (HGSC) is characterized by HRD, which is caused by genetic alterations in the homologous recombination repair (HRR) pathway. Recent trials have shown that not only patients with pathogenic/likely pathogenic BRCA variants, but also BRCAwt/HRD patients, are sensitive to PARPis and platinum therapy. The most common HRD test is Myriad MyChoice CDx, but there is a pressing need to offer an alternative to outsourcing analysis, which typically requires high costs and lengthy turnaround times. In order to set up a complete in-house workflow for HRD testing, we analyzed a small cohort of HGSC patients using the CE-IVD AmoyDx HRD Focus Panel and compared our results with Myriad’s. In addition, to further deepen the mechanisms behind HRD, we analyzed the study cohort by using both a custom NGS panel that analyzed 21 HRR-related genes and FISH analysis to determine the copy numbers of PTEN and EMSY. We found complete concordance in HRD status detected by the Amoy and the Myriad assays, supporting the feasibility of internal HRD testing.

Simple Summary The number of druggable tumor-specific molecular alterations in the treatment of non-small cell lung cancer (NSCLC) has grown significantly in the past decade. Emerging technologies such as liquid biopsy and single-cell methods allow for studying targetable drivers and develop personalized treatments. However, although new therapies confer prolonged disease control and high tumor response rates, most patients eventually progress on targeted treatments. Intratumoral heterogeneity is a frequent event in NSCLC, driving the tumor cells to develop adaptive or new resistance mechanisms within the drug environment. This review summarizes the current and upcoming research on the biological role of tumor heterogeneity, highlighting the link between early and acquired drug resistance and tumoral heterogeneity in targetable driver mutated NSCLC. Abstract Tailored therapies based on the identification of molecular targets currently represent a well-established therapeutic scenario in the treatment of non-small cell lung cancer (NSCLC) patients. However, while aiming to improve patients’ response to therapy, development of resistance is frequently observed in daily clinical practice. Intratumoral heterogeneity is a frequent event in NSCLC, responsible for several critical issues in patients’ diagnosis and treatment. Advances in single-cell sequencing technologies have allowed in-depth profiling of tumors and attributed intratumoral heterogeneity to genetic, epigenetic, and protein modification driven diversities within cancer cell populations. This review highlights current research on the biological role of tumor heterogeneity and its impact on the development of acquired resistance in NSCLC patients.

Lung cancer remains the first cause of cancer-related deaths worldwide. Thanks to the improvement in the knowledge of the biology of non-small cell lung cancer (NSCLC), patients’ survival has significantly improved. A growing number of targetable molecular alterations have been identified. Next-generation sequencing (NGS) has become one of the methodologies entered in clinical practice and was recently recommended by the European society for medical oncology (ESMO) to perform a comprehensive molecular characterization in patients with cancer. The current review provides an overview of the clinical trials that have explored the impact of NGS in patients with cancer, its limits, and advantages.

Recently, the term mixed neuroendocrine non-neuroendocrine neoplasms (MiNEN) has been proposed as an umbrella definition covering different possible combinations of mixed neuroendocrine-exocrine neoplasms. Among these, the adenoma plus neuroendocrine tumor (NET) combination is among the rarest and not formally recognized by the 2019 WHO Classification. In this setting, the debate between either collision tumors or true mixed neoplasms is still unsolved. In this report, a pancreatic intraductal papillary mucinous neoplasm (IPMN) plus a NET is described, and the molecular investigations showed the presence in both populations of the same KRAS, GNAS, and CDKN2A mutations and the amplification of the CCND1 gene. These data prove clonality and support a common origin of both components, therefore confirming the true mixed nature. For this reason, mixed neuroendocrine-exocrine neoplasms, in which the exocrine component is represented by a glandular precursor lesion (adenoma/IPMN) only, should be included into the MiNEN family.

Objectives Circulating cell-free tumor DNA (ctDNA) isolated from the peripheral blood of non-small-cell lung cancer (NSCLC) patients provides biomarkers for both therapeutic target selection, particularly when direct tumor biopsy is unfeasible, and also for drug resistance monitoring. This study evaluates the reliability and feasibility of ctDNA analysis in an in-house clinical molecular diagnostic workflow. Materials and methods Mutation profiling by both standard methods and Next-Generation sequencing (NGS) was carried out and compared on 2 independent lung cancer patient cohorts. Cohort 1 consisted of 50 EGFR-mutated NSCLC patients, established on tumour biopsy, for whom ctDNA was collected at disease progression after TKI-inhibitor treatment and could be used to monitor drug resistance. Cohort 2 consisted of 50 newly diagnosed lung cancer patients for whom tumour biopsy was not possible and only ctDNA was available, providing the possibility of biomarker identification. Results ctDNA analysis of Cohort 1 verified the persistence of the tumour-detected EGFR activating mutation at disease progression by both standard and NGS methods, in 84% and 92% of the cases respectively. The T790M EGFR resistance mutation was identified in 71% of the ctDNA EGFR mutated samples providing vital information for their disease management. In newly diagnosed Cohort 2 patients, EGFR activating mutations were detected in 16% of the patients by both standard and NGS analysis of ctDNA in peripheral blood, providing indication to targeted-therapy otherwise unavailable for this group of patients. Conclusion The presented study investigated lung cancer ctDNA analysis, comparing conventional methods versus NGS sequencing, and demonstrated the successful use of plasma ctDNA as a template for targeted NGS tumor gene panel in an in-house routine clinical practice. More importantly, these data underline the advantages of the clinical application of ctDNA NGS analysis for identification of therapeutic targets, real-time monitoring of therapy, and resistance mechanisms in lung cancer patients.

Background:Histiocytoses are disorders characterized by tissue infiltration by macrophages, dendritic cells, or monocyte-derived cells. These diseases are classified in five groups based on histologic, clinical, and molecular features: Langerhans-related, cutaneous/mucocutaneous and malignant histiocytoses, Rosai-Dorfman disease, and hemophagocytic lymphohistiocytosis (1). Langerhans-related histiocytoses comprise Langerhans cell histiocytosis and Erdheim-Chester disease, both inflammatory myeloid-driven diseases characterized by clonal activating mutations along the MAPK or related pathways, most commonlyBRAFV600E, and by severe tissue and systemic inflammation (2). Here, we describe and characterize a novel, related histiocytosis chiefly manifested with severe synovial involvement.Objectives:Here, we describe a novel histiocytosis whose histologic and clinical picture (severe synovial involvement, systemic inflammation, skin lesions, and diabetes insipidus) differed from known histiocytic disorder. In addition, we performed molecular studies aimed at identifying causative activating mutations. Finally, by means of a dynamic 3D tissue culture system we characterized immune-metabolic mechanisms underlying disease pathogenesis and clinical response to treatment.Methods:The mutational status of oncogenes was determined with a mass spectrometry multiplexed genotyping approach (PentaPanel). Biospy samples were cultured in RCCSTM bioreactor (Synthecon) in the presence/absence of a MEK inhibitor (GSK1120212, 1nM), and then either processed for immunohistochemical analyses, or lysed for western blot analysis. Culture supernatants were collected for cytokine, chemokine and metabolome determination. The Bio-Plex Multiple-Cytokine Assay (Bio-Rad) and the Ella assay (ProteinSimple) were used to determine cytokine concentrations in supernatants and serum, respectively. Metabolomic studies were performed as described (3).Results:We identified a causative mutation in the proto-oncogeneKRAS(KRASG12D, not previously reported in related histiocytoses). In addition, 3D culture studies of patient’s biopsies revealed KRAS-driven signaling, phenotypic, and immunometabolic features. These included constitutive ERK and AKT phosphorylation, up-regulated glucose metabolism with glycolysis and TCA cycle activation, and deregulated release of pro-inflammatory cytokines IL-1β, IL-6 and TNFα. All these features reverted upon pharmacologic inhibition of the MAPK pathway. Characterization of this novel condition instructed effective treatment of the patient with the MEK inhibitor cobimetinib.Conclusion:Genetic, clinical, and histopathology features differentiate this condition from known histiocytic disorders. Mechanistically,KRASG12Dcauses constitutive activation of the MAPK pathway in macrophages, which results in maladaptive changes in cell energy metabolism sustaining rampant production of pro-inflammatory cytokines. Besides instructing effective treatment of this patient, these studies revealed metabolic rewiring as key to pathologic inflammatory activation of macrophages in human disease.References:[1]Emile JF, et al. Revised classification of histiocytoses. Blood. 2016[2]Cavalli G, et al. The multifaceted clinical presentations and manifestations of Erdheim-Chester disease. Ann Rheum Dis. 2013[3]Cavalli G, et al. Interleukin 37 reverses the metabolic cost of inflammation, increases oxidative respiration, and improves exercise tolerance. Proc Natl Acad Sci U S A. 2017Disclosure of Interests:Giulio Cavalli Consultant of: SOBI, Pfizer, Sanofi, Novartis, Paid instructor for: SOBI, Novartis, Speakers bureau: SOBI, Novartis, Giacomo De Luca Speakers bureau: SOBI, Novartis, Celgene, Pfizer, MSD, Riccardo Biavasco Employee of: Bluebird, Travis Nemkov: None declared, Angelo D’Alessandro: None declared, Rob Arts: None declared, Antonello Villa: None declared, Daniela Belloni: None declared, Greta Grassini: None declared, Giulia Cangi: None declared, Claudio Doglioni: None declared, Elisabetta Ferrero: None declared, Marina Ferrarini: None declared, Lorenzo Dagna: None declared

// Gilda Magliacane 1, * , Greta Grassini 1, * , Paola Bartocci 2, * , Ilaria Francaviglia 1 , Elena Dal Cin 1 , Gianluca Barbieri 2 , Gianluigi Arrigoni 1 , Lorenza Pecciarini 1 , Claudio Doglioni 1 , Maria Giulia Cangi 1 1 Unit of Pathology, IRCCS San Raffaele Scientific Institute, Milano, Italy 2 Diatech Pharmacogenetics Company, Jesi, Italy * These authors have contributed equally to this work Correspondence to: Maria Giulia Cangi, e-mail: cangi.mariagiulia@hsr.it Claudio Doglioni, e-mail: doglioni.claudio@hsr.it Keywords: Pathology, molecular diagnostics, solid tumors, high throughput mass-spectrometry, target therapy, mutations Received: July 01, 2015 Accepted: August 14, 2015 Published: September 02, 2015 ABSTRACT Tumor genotyping is an essential step in routine clinical practice and pathology laboratories face a major challenge in being able to provide rapid, sensitive and updated molecular tests. We developed a novel mass spectrometry multiplexed genotyping platform named PentaPanel to concurrently assess single nucleotide polymorphisms in 56 hotspots of the 5 most clinically relevant cancer genes, KRAS, NRAS, BRAF , EGFR and PIK3CA for a total of 221 detectable mutations. To both evaluate and validate the PentaPanel performance, we investigated 1025 tumor specimens of 6 different cancer types (carcinomas of colon, lung, breast, pancreas, and biliary tract, and melanomas), systematically addressing sensitivity, specificity, and reproducibility of our platform. Sanger sequencing was also performed for all the study samples. Our data showed that PentaPanel is a high throughput and robust tool, allowing genotyping for targeted therapy selection of 10 patients in the same run, with a practical turnaround time of 2 working days. Importantly, it was successfully used to interrogate different DNAs isolated from routinely processed specimens (formalin-fixed paraffin embedded, frozen, and cytological samples), covering all the requirements of clinical tests. In conclusion, the PentaPanel platform can provide an immediate, accurate and cost effective multiplex approach for clinically relevant gene mutation analysis in many solid tumors and its utility across many diseases can be particularly relevant in multiple clinical trials, including the new basket trial approach, aiming to identify appropriate targeted drug combination strategies.

OBJECTIVES: Alterations in mucin (MUC) glycosylation and expression have been described in cancer. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) can provide material for molecular biology analysis. This study assessed the feasibility of evaluating MUC expression from material obtained by EUS-FNA and studied the profile of MUC expression in benign and malignant pancreatic lesions. METHODS: A total of 90 patients with solid or cystic pancreatic lesions underwent FNA. The aspirated material was used for cytological analysis and RNA extraction to assess the expression pattern of MUCs by reverse transcription-PCR with primers specific for the MUC1, MUC2, MUC3, MUC4, MUC5A, MUC5B, MUC6, and MUC7 genes. RESULTS: RNA extraction was successful in 81% of the biopsies. The prevalences of MUC1, MUC2, MUC4, and MUC7 in ductal adenocarcinoma were 57.7, 51.4, 18.9, and 73.0%, respectively. Fifty percent of benign lesions and neuroendocrine tumors (NETs), and 63% of intraductal papillary mucinous neoplasms (IPMNs) were positive for MUC1. Twenty-five percent of benign lesions, 86% of NETs, and 47% of IPMNs were positive for MUC2. Of NETs, 50% were positive for MUC1, and 14% were positive for MUC7. None of the benign lesions or NETs expressed MUC4. MUC7 expression was highly significant for adenocarcinoma (P = 0.007) and borderline for IPMN (P = 0.05). MUC7 was expressed in 37.5% of chronic pancreatitis cases. CONCLUSIONS: RNA can be extracted from samples obtained under EUS-FNA. MUC7 could serve as a potential biological marker to identify malignant lesions, especially pancreatic adenocarcinoma. ZR 0 ZS 0 Z8 2 "OBJECTIVES: Alterations in mucin (MUC) glycosylation and expression have been described in cancer. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) can provide material for molecular biology analysis. This study assessed the feasibility of evaluating MUC expression from material obtained by EUS-FNA and studied the profile of MUC expression in benign and malignant pancreatic lesions.. . METHODS: A total of 90 patients with solid or cystic pancreatic lesions underwent FNA. The aspirated material was used for cytological analysis and RNA extraction to assess the expression pattern of MUCs by reverse transcription-PCR with primers specific for the MUC1, MUC2, MUC3, MUC4, MUC5A, MUC5B, MUC6, and MUC7 genes.. . RESULTS: RNA extraction was successful in 81% of the biopsies. The prevalences of MUC1, MUC2, MUC4, and MUC7 in ductal adenocarcinoma were 57.7, 51.4, 18.9, and 73.0%, respectively. Fifty percent of benign lesions and neuroendocrine tumors (NETs), and 63% of intraductal papillary mucinous neoplasms (IPMNs) were positive for MUC1. Twenty-five percent of benign lesions, 86% of NETs, and 47% of IPMNs were positive for MUC2. Of NETs, 50% were positive for MUC1, and 14% were positive for MUC7. None of the benign lesions or NETs expressed MUC4. MUC7 expression was highly significant for adenocarcinoma (P=0.007) and borderline for IPMN (P=0.05). MUC7 was expressed in 37.5% of chronic pancreatitis cases.. . CONCLUSIONS: RNA can be extracted from samples obtained under EUS-FNA. MUC7 could serve as a potential biological marker to identify malignant lesions, especially pancreatic adenocarcinoma.. . "

Cancer genomics role in patients management underlines the importance of rigorous validation of molecular diagnostics approaches to guide treatment decisions. Nowadays only next generation sequencing (NGS) assays can satisfy the need to match a comprehensive tumor molecular profile to the right therapies. Nevertheless, given the complexity, cost, and outcome implications for patients, a clinically valid NGS assay requires careful consideration of gene panel size, inclusion of appropriate markers, ability to detect multiple genomic aberration types, performance with low quality/quantity of nucleic acids, and workflow feasibility. In this study we describe the introduction of the Oncomine Comprehensive Assay (OCA) into a Molecular Pathology diagnostic framework. The OCA analyzes 143 genes of which 73 oncogenes are interrogated for mutational hotspots and 26 tumor suppressor genes for all exons. In addition, the OCA detects copy number variations in 49 genes and fusion drivers in 22 genes. Multiplexed PCR-based DNA and RNA libraries were sequenced on Ion S5 and data analysis was performed by the Torrent Suite using the Oncomine pipeline. The OCA was clinically validated analyzing a cohort of 200 tumor samples of 4 solid tumor types (100 Non Small Cell Lung Cancers, 40 Colon Adenocarcinomas, 40 Pancreatic Adenocarcinomas, 20 Esophageal Adenocarcinomas), for which routine molecular analysis was available. We successfully identified relevant somatic point mutations, indels, and high-level CNAs: TP53, KRAS and EGFR were the genes most frequently mutated though alteration frequencies varied among different tumor types, as expected. OCA DNA profiling demonstrated 95% concordance with standard methods for detecting commonly targeted somatic variants with highly concordant observed variant allele frequencies for detection of KRAS, EGFR and BRAF mutations. Also high-level CNAs strongly correlated with available routine standard tests. The concordance of OCA analysis and standard tests at the RNA level was definitely lower (80%): a subset of 45 lung samples were tested for ALK, RET, ROS1 rearrangements by OCA RNA assay and we successfully detected 15 of the 24 expected fusions, indicating the feasibility of identifying them by this NGS panel, but also stressing the limits of a targeted approach to detect widely variable breakpoints in the analyzed gene fusions. In order to detect all the ALK, ROS1, and RET fusions variants, we perform the parallel validation of the Archer FusionPlex RNA-based ALK, RET, and ROS1 gene fusion assay in 8 lung adenocarcinomas. Results are under evaluation. We conclude that the OCA DNA analysis can be a valuable tool in solid tumor molecular diagnostics being able to identify both common and additional relevant variants beyond current routine practice, and represents a broadly applicable targeted NGS assay which allows advanced precision oncology for different solid tumors types. Citation Format: Greta Grassini, Valeria De Pascali, Ilaria Francaviglia, Gilda Magliacane, Elena Dal Cin, Anna Talarico, Chiara Iacona, Claudio Doglioni, Lorenza Pecciarini, Maria Giulia Cangi. Targeted next generation sequencing in molecular diagnostics of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 645.

The analysis of circulating cell-free tumor DNA (ctDNA), which can be obtained from plasma by non-invasive procedures, was proven useful to provide biomarkers in the management of non-small-cell lung cancer (NSCLC) patients. Several studies assessed ctDNA prognostic and predictive value as source of key data for therapeutic target selection and drug resistance in such patients. The purpose of the present study was to compare the assessment of NSCLC common hot spot mutations in ctDNA samples using the NGS Oncomine cfDNA Lung Cancer assay (ThermoFisher) to the standard clinical tests (i.e. real-time quantitative PCR and droplet digital PCR), performed in both FFPE tumor and ctDNA samples. In particular we aimed to evaluate the feasibility and accuracy of this assay for (1) the detection of EGFR/KRAS mutations in 50 patients with newly diagnosed lung cancer and no possibility to obtain tissue samples and (2) the identification of anti-EGFR treatment acquired resistance mutations in 50 NSCLC EGFR-mutated patients with disease progression. The Oncomine cfDNA Lung assay is a multiplexed sequencing test, designed to detect 150 hotspot mutations in 11 lung cancer-related genes (ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, and TP53) with a limit of detection as low as 0.1%. We analyzed a total of 100 ctDNAs which were sequenced using the Ion S5 system. Libraries were templated using the Ion Chef and multiplexed as 8 libraries on a 520 chip. Data analysis was performed in Torrent Suite using the variant Caller plugin. The total process time, from ctDNA isolation to result reporting, was as short as 4 days, supporting a workflow where blood samples are received early on day 1 and final variant calls are available on day 4. ctDNA NGS analysis for the newly diagnosed patients with no available tumor sample identified KRAS mutations (7%) and more importantly, targetable EGFR mutations (10%). For the patients with progression disease, EGFR acquired resistance mutations were found in 78% of the cases. Overall, the most frequently mutated genes were EGFR (85%), TP53 (26%), PIK3CA (18%), ALK (10%), and MET (8%). Taken the EGFR mutation detected by routine methods as the gold standard, the concordance with EGFR variants detected by NGS was 97%. Interestingly, the NGS approach detected the same EGFR mutations with the same allelic frequency of standard methods. These preliminary data confirm the potential of the Oncomine cfDNA lung assay for plasma genotyping which allows both noninvasive multiplexed detection of targetable genomic alterations and monitoring of acquired resistance mutations in lung cancer. Citation Format: Ilaria Francaviglia, Gilda Magliacane, Greta Grassini, Salvatore Girlando, Daniela Medicina, Chiara Lazzari, Alessandra Bulotta, Lorenza Pecciarini, Claudio Doglioni, Maria Giulia Cangi. Identification and monitoring of somatic mutations in circulating cell-free DNA by next-generation sequencing in patients with lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5580.

Efficient, high-level expression of multiple genes is often difficult to achieve in retroviral vectors, due to positional effects affecting transcription of adjacent sequences. Here we describe the comparative analysis of different strategies for co-expressing two model cDNA sequences in the context of a second generation lentiviral vector system. A first option was based on the generation of a polycistronic construct by subcloning an internal ribosome entry site (IRES) sequence between tandem cDNAs. IRES-dependent translation of the cDNA placed downstream (3') of the first transgene was poor, and the protein was barely detectable in transduced cells. A similar result was obtained when both transgenes were placed under the transcriptional control of two independent internal promoters. When these independent transcription units were separated by the 5'HS4 chromatin insulator of the chicken beta-globin locus, a marked increase of the expression of the downstream protein was observed. Similarly, insertion of a polyadenylation sequence between the tandem transcription units fully restored - in transfection experiments - the expression of the downstream sequence, whose protein pattern was identical to the single-gene control, suggesting that in this specific construct transcriptional interference was the likely cause of the observed positional effects. These results indicate that chromatin insulator sequences can be useful molecular tools to overcome positional effects in the context of lentiviral vectors.

Objectives Erdheim-Chester disease (ECD) is a rare form of histiocytosis characterised by uncontrolled chronic inflammation. The oncogenic BRAF V600E mutation has been reported in biopsies in 19 out of 37 patients with ECD from the largest published cohort, but never found in the patients’ peripheral blood. Also, the role of the mutation in the pathogenesis of the disease has not been elucidated yet. BRAF V600E has been associated with oncogene-induced senescence (OIS), a protective mechanism against oncogenic events, characterised by the induction of proinflammatory pathways. Methods We verified the BRAF status in biopsies and peripheral blood from 18 patients with ECD from our cohort and matched controls by means of immunohistochemistry and of an ultrasensitive assay, based on the combination of a locked nucleic acid PCR and pyrosequencing. Droplet digital PCR was used to confirm the findings. We also evaluated the presence of senescence markers in ECD histiocytes. Results BRAF V600E mutation was present in all the biopsy and peripheral blood samples from patients with ECD and in none of the controls. ECD histiocytes and a fraction of circulating monocytes from patients with ECD showed signs of a constitutive activation of the MAPK pathway. Moreover, BRAF-mutated histiocytes expressed markers of OIS. Conclusions The oncogenic BRAF V600E mutation is present in biopsies and in the peripheral blood from all patients with ECD who were evaluated and is associated with OIS. These findings have significant implications for the pathogenesis, diagnosis and treatment of ECD.

The analysis of circulating cell-free tumor DNA (ctDNA), which can be obtained from plasma by non-invasive procedures, is expected to provide useful biomarkers in the management of non-small-cell lung cancer (NSCLC) patients. Indeed, several studies have assessed ctDNA prognostic and predictive value as source of key data for therapeutic targets and drug resistance in carcinoma patients. The expanding number of targeted therapeutics for NSCLC always expects a real-time wider tumor genotyping, and the use of ctDNA as either a complement or an alternative to tumor tissue DNA (ttDNA) could be a valid option for Next-Generation Sequencing (NGS) of key cancer genes. However, isolation and enrichment of ctDNA is a big challenge because of its high degree of fragmentation and its low concentration against the normally occurring background of cell-free DNA derived from healthy cells. Therefore standardized methods for ctDNA extraction and analysis are crucial aspects in the setting-up of a molecular diagnostic approach. In this study we aimed to evaluate the ctDNA use for molecular profiling, also analyzing the impact of both pre-analytical and analytical variables on DNA yield and mutation detection. Matched ttDNA and ctDNA from 30 NSCLC patients were extracted, quantified and quality-controlled, and then investigated by different standard methods (real-time PCR, digital PCR, Mass Spectrometry genotyping) for EGFR, KRAS, BRAF, PIK3CA status. Mutational screening of ctDNA samples by IonTorrent NGS (Oncomine™ Lung cfDNA Assay, ThermoFisher) was also performed. Further, we extended ctDNA evaluation to additional 30 lung cancer patients with no available tumor sample. We found that cell free DNA concentration in plasma correlated with both stage and number of metastatic sites. Analyzing matched ttDNA and ctDNA by standard methods, we identified mutations in EGFR, KRAS, PIK3CA genes, with an overall concordance of 77%. Interestingly, Oncomine Lung cfDNA assay detected these same mutations with the same allelic frequency of standard methods; mutations in TP53 and ALK genes were also found. In the 30 patients with no available tumor sample, we found EGFR (10%) and KRAS (7%) mutations on ctDNA by standard methods; NGS analysis is under evaluation. This study evaluated the use of multiple different methods to detect mutations in NSCLC and showed that ctDNA can be a feasible option for clinical monitoring of lung cancers, including for those patients who cannot undergo invasive diagnostic procedures, due to either comorbidities or absence of biopsable tumor lesions. Citation Format: Gilda Magliacane, Greta Grassini, Ilaria Francaviglia, Elena DalCin, Chiara Lazzari, Alessandra Bulotta, Monika Ducceschi, Vanesa Gregorc, Lorenza Pecciarini, Claudio Doglioni, Maria Giulia Cangi. ctDNA use in molecular diagnostics [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 744. doi:10.1158/1538-7445.AM2017-744

Pancreatic neuroendocrine tumors (PanNETs) are the second most common malignancy of the pancreas, and the only curative option is represented by surgery. PanNETs can be classified as well- or poorly- differentiated, based on histology. Poorly-differentiated neuroendocrine carcinomas (NECs) represent less than 10% of cases, but they are highly malignant and associated with poor prognosis, and, most importantly, the inconsistency in their clinical management is due to the insufficient data on NEC genetic features. In order to widen our knowledge of PanNET genetic alterations, we performed RNASeq analysis (using Illumina NextSeq platform) of a group of 18 PanNETs and 2 NECs. TopHat-Fusion algorithm identified, among others, a fusion involving SMAD3 transcription factor and CCDC149; the alteration of SMAD3 in a poorly differentiated NEC was particularly interesting since SMAD3 is involved in TGF-b signaling, which has an oncogenic role in malignant cells. The SMAD3-CCDC149 fusion was confirmed in the index case by RT-PCR using primers spanning the breakpoint, followed by Sanger sequencing. The fusion was also detected by FISH analysis performed on both FFPE and frozen samples. By both FISH analysis and Sanger sequencing we identified two other poorly-differentiated NEC samples characterized by the presence of the SMAD3-CCDC149 translocation. Immunohistochemical (IHC) staining revealed that the 3 tumors, positive for the SMAD3-CCDC149 fusion, were characterized by nuclear localization of SMAD3. In order to test the hypothesis that the fusion generates an active form of SMAD3 that localizes exclusively within the nucleus, we transfected BON-1 cell line with plasmids containing either SMAD3 full length or SMAD3 truncated form (the latter mimics the fusion product) showing that only the truncated form of SMAD3 had an exclusively nuclear localization. Interestingly, FISH analysis performed on 3 well-differentiated PanNET samples did not detected the presence of the SMAD3-CDC149 translocation and IHC analysis showed cytoplasmic localization of SMAD3. Moreover, IHC analysis performed on a cohort of 40 cases of non-pancreatic neuroendocrine tumors (NETs) revealed that only poor-differentiated tumors showed a significant percentage of samples with nuclear localization of SMAD3. These data support the hypothesis that the translocation is a typical phenomenon of poorly-differentiated tumors and suggests that it could be involved also in non-pancreatic NETs. In this study we identified a previous unknown SMAD3 translocation in poor-differentiated NECs, which generates an active SMAD3 form. Deepening the knowledge of the fusion product will shed light on PanNET genetic alterations, opening the way to new therapeutic strategies. Citation Format: Emanuela Brunetto, Greta Grassini, Valeria De Pascali, Anna Talarico, Francesca Invernizzi, Michaela Bowden, Massimo Loda, Luca Albarello, Massimo Falconi, Lorenza Pecciarini, Maria G. Cangi, Claudio Doglioni. TGF-β pathway alteration in pancreatic neuroendocrine tumors: characterization of a novel SMAD3 translocation [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 3519. doi:10.1158/1538-7445.AM2017-3519

HER2 gene amplification/overexpression has been identified in 10-34% of invasive breast cancers and correlates with reduced disease-free and overall survival. The HER2-targeted therapy with trastuzumab decreases the risk of relapse and prolongs survival. Resistance to trastuzumab is a significant barrier to the effective HER2-positive breast cancer treatment: response rate ranges from 20-35%, underlining that HER2-positive breast cancer is hardly a homogeneous disease and, more importantly, that other HER2-related pathways need to be unveiled. Recent studies provided evidence that HER2 overexpression increases skin tumorigenesis through CDC25A maintenance: HER2 suppresses DNA damage-induced checkpoint activation through lack of CDC25A degradation. CDC25A phosphatase, an essential component of the cell cycle machinery, is also a key player in integrating the specific signals of checkpoint control in response to DNA damage. CDC25A misregulation contributes to genomic instability and cancer development. Oncogene activation during early stages of tumor development causes DNA replication stress resulting in the DNA damage response (DDR) induction and the selection of cells defecting in their DDR could lead to malignant progression. Accordingly, CDC25A overexpression in primary human breast cells promotes the DDR alteration, increasing the chances of tumorigenic conversion by enhancing genomic instability at common fragile sites. The aim of our work was to verify a relation between HER2 and CDC25A in human breast cancer. To this end HER2 amplification and CDC25A overexpression were determined in 220 breast cancer patients. We found a statistically significant correlation between HER2 gene status and CDC25A expression (p=0,000): 64,4% of HER2 amplified tumours show CDC25A overexpression and 75,5% of tumours with no HER2 amplification do not overexpress CDC25A. Furthermore HER2 status and CDC25A expression significantly correlate with prognosis (p=0,003 and p=0,047 respectively). A mortality risk stratification was identified when four different combinations of HER2 status and CDC25A expression were evaluated for their relative effect on survival: the group with the highest mortality was the one in which breast carcinomas showed both HER2 amplification and CDC25A overexpression (p=0,019). Our in vitro studies demonstrate that HER2 can stabilize CDC25A protein levels in breast cancer cell lines, through PI3K-AKT pathway: decreased HER2-mediated signaling by HER2 inhibitors resulted in CDC25A decreased expression; remarkably CDC25A inactivation by specific inhibitor can restore response to IR-induced DNA damage in HER2-positive, both trastuzumab sensitive and resistant, breast cancer cell lines. These findings suggest a CDC25A role for a more specific characterization of HER2-positive breast cancers, warranting further study of its potential as therapeutic target in combination with HER2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3147. doi:10.1158/1538-7445.AM2011-3147