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Dive into the research topics where Pasquale Pisapia is active.

Publication


Featured researches published by Pasquale Pisapia.


British Journal of Cancer | 2017

Development of a gene panel for next-generation sequencing of clinically relevant mutations in cell-free DNA from cancer patients

Umberto Malapelle; Clara Mayo de-las-Casas; Danilo Rocco; Mónica Garzón; Pasquale Pisapia; Nuria Jordana-Ariza; Maria Giovanna Russo; Roberta Sgariglia; Caterina De Luca; Francesco Pepe; Alejandro Martinez-Bueno; Daniela Morales-Espinosa; María González-Cao; Niki Karachaliou; Santiago Viteri Ramirez; Claudio Bellevicine; Miguel Angel Molina-Vila; Rafael Rosell; Giancarlo Troncone

Background:When tumour tissue is unavailable, cell-free DNA (cfDNA)can serve as a surrogate for genetic analyses. Because mutated alleles in cfDNA are usually below 1%, next-generation sequencing (NGS)must be narrowed to target only clinically relevant genes. In this proof-of-concept study, we developed a panel to use in ultra-deep sequencing to identify such mutations in cfDNA.Methods:Our panel (‘SiRe’) covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα)involved in non-small-cell lung cancer (NSCLC), gastrointestinal stromal tumour, colorectal carcinoma and melanoma. We evaluated the panel performance in three steps. First, we analysed its analytical sensitivity on cell line DNA and by using an artificial reference standard with multiple mutations in different genes. Second, we analysed cfDNA from cancer patients at presentation (n=42), treatment response (n=12) and tumour progression (n=11); all patients had paired tumour tissue and cfDNA previously genotyped with a Taqman-derived assay (TDA). Third, we tested blood samples prospectively collected from NSCLC patients (n=79) to assess the performance of SiRe in clinical practice.Results:SiRe had a high analytical performance and a 0.01% lower limit of detection. In the retrospective series, SiRe detected 40 EGFR, 11 KRAS, 1 NRAS and 5 BRAF mutations (96.8% concordance with TDA). In the baseline samples, SiRe had 100% specificity and 79% sensitivity relative to tumour tissue. Finally, in the prospective series, SiRe detected 8.7% (4/46) of EGFR mutations at baseline and 42.9% (9/21) of EGFR p.T790M in patients at tumour progression.Conclusions:SiRe is a feasible NGS panel for cfDNA analysis in clinical practice.


Journal of Clinical Pathology | 2016

Less frequently mutated genes in colorectal cancer: evidences from next-generation sequencing of 653 routine cases.

Umberto Malapelle; Pasquale Pisapia; Roberta Sgariglia; Elena Vigliar; Maria Biglietto; Chiara Carlomagno; Giuseppe Giuffrè; Claudio Bellevicine; Giancarlo Troncone

Aims The incidence of RAS/RAF/PI3KA and TP53 gene mutations in colorectal cancer (CRC) is well established. Less information, however, is available on other components of the CRC genomic landscape, which are potential CRC prognostic/predictive markers. Methods Following a previous validation study, ion-semiconductor next-generation sequencing (NGS) was employed to process 653 routine CRC samples by a multiplex PCR targeting 91 hotspot regions in 22 CRC significant genes. Results A total of 796 somatic mutations in 499 (76.4%) tumours were detected. Besides RAS/RAF/PI3KA and TP53, other 12 genes showed at least one mutation including FBXW7 (6%), PTEN (2.8%), SMAD4 (2.1%), EGFR (1.2%), CTNNB1 (1.1%), AKT1 (0.9%), STK11 (0.8%), ERBB2 (0.6%), ERBB4 (0.6%), ALK (0.2%), MAP2K1 (0.2%) and NOTCH1 (0.2%). Conclusions In a routine diagnostic setting, NGS had the potential to generate robust and comprehensive genetic information also including less frequently mutated genes potentially relevant for prognostic assessments or for actionable treatments.


Cancer Cytopathology | 2017

Consistency and reproducibility of next-generation sequencing and other multigene mutational assays: A worldwide ring trial study on quantitative cytological molecular reference specimens

Umberto Malapelle; Clara Mayo-de-las-Casas; Miguel Angel Molina-Vila; Rafael Rosell; Spasenija Savic; Michel P. Bihl; Lukas Bubendorf; Manuel Salto-Tellez; Dario de Biase; Giovanni Tallini; David H. Hwang; Lynette M. Sholl; Rajyalakshmi Luthra; Birgit Weynand; Sara Vander Borght; Edoardo Missiaglia; Massimo Bongiovanni; Daniel Stieber; Philippe Vielh; Fernando Schmitt; Alessandra Rappa; Massimo Barberis; Francesco Pepe; Pasquale Pisapia; Nicola Serra; Elena Vigliar; Claudio Bellevicine; Matteo Fassan; Massimo Rugge; Carlos E. de Andrea

Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v‐ras] oncogene homolog (NRAS), B‐Raf proto‐oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit α (PIK3CA). Next‐generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single‐institution studies rather than multicenter experiences.


Journal of Clinical Pathology | 2017

EGFR mutation detection on lung cancer cytological specimens by the novel fully automated PCR-based Idylla EGFR Mutation Assay

Caterina De Luca; Gianluca Gragnano; Pasquale Pisapia; Elena Vigliar; Umberto Malapelle; Claudio Bellevicine; Giancarlo Troncone

Aims In everyday practice, epidermal growth factor receptor (EGFR) testing is centralised in referral laboratories that receive paucicellular cytological specimens. Ideally, EGFR testing should be carried out in the centre where the patient is diagnosed such that the most cellular slide can be selected from in-house collected cytological material. However, available techniques are little standardised and difficult to be implemented in settings with little expertise in molecular testing. The Idylla EGFR prototype assay is a rapid and fully automated test which may easily be adopted by a wider number of pathological centres. This study assessed whether an Idylla EGFR prototype assay can be reliably applied to cytological lung cancer specimens. Methods The limit of detection (LOD) of the Idylla EGFR prototype assay was assessed by cell line dilution studies. A total of 10 ng was directly placed inside an Idylla EGFR prototype assay cartridge. Idylla results were compared with fragment length (exon 19 del) and Taqman assays. Results The Idylla EGFR prototype assay showed an LOD of 1% mutant allele and yielded valid results in 74/76 (97.3%) samples, detecting all the mutant cases (n=32) identified by standard techniques; in addition, Idylla detected two low abundance EGFR exon 19 deletions and two G719X exon 18 point mutations, not covered by our standard reference method. Conclusions Idylla EGFR prototype assay is sensitive on extracted DNA and can reliably be applied on cytological samples, enabling implementation of EGFR testing even in less experienced diagnostic units.


Expert Review of Molecular Diagnostics | 2017

Profile of the Roche cobas(®) EGFR mutation test v2 for non-small cell lung cancer.

Umberto Malapelle; Rafael Sirera; Eloisa Jantus-Lewintre; Pablo Reclusa; Silvia Calabuig-Fariñas; Ana Blasco; Pasquale Pisapia; Christian Rolfo; Carlos Camps

ABSTRACT Introduction: The discovery of driver mutations in non-small cell lung cancer (NSCLC) has led to the development of genome-based personalized medicine. Fifteen to 20% of adenocarcinomas harbor an epidermal growth factor receptor (EGFR) activating mutation associated with responses to EGFR tyrosine kinase inhibitors (TKIs). Individual laboratories’ expertise and the availability of appropriate equipment are valuable assets in predictive molecular pathology, although the choice of methods should be determined by the nature of the samples to be tested and whether the detection of only well-characterized EGFR mutations or rather, of all detectable mutations, is required. Areas covered: The EGFR mutation testing landscape is manifold and includes both screening and targeted methods, each with their own pros and cons. Here we review one of these companion tests, the Roche cobas® EGFR mutation test v2, from a methodological point of view, also exploring its liquid-biopsy applications. Expert commentary: The Roche cobas® EGFR mutation test v2, based on real time RT-PCR, is a reliable option for testing EGFR mutations in clinical practice, either using tissue-derived DNA or plasma-derived cfDNA. This application will be valuable for laboratories with whose purpose is purely diagnostic and lacking high-throughput technologies.


Cancer Cytopathology | 2017

ALK and ROS1 testing on lung cancer cytologic samples: Perspectives

Pasquale Pisapia; Maria D. Lozano; Elena Vigliar; Claudio Bellevicine; Francesco Pepe; Umberto Malapelle; Giancarlo Troncone

Cytologic sampling is the mainstay of diagnosing advanced lung cancer. Moreover, to select patients for personalized first‐line or second‐line treatment, epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) and c‐ros oncogene 1 (ROS1) rearrangements are tested on cytologic preparations. Commercially available fluorescence in situ hybridization (FISH) and immunocytochemistry (ICC) assays have primarily been used for the identification of cells harboring ALK or ROS1 gene fusions on histologic rather than cytologic preparations. However, it is now recognized that FISH and ICC also can be applied on cytologic samples provided the cytopathologist is aware that FISH and ICC results are not always concordant and that the performance of ICC largely depends on antibody clones, signal detection systems, and scoring systems. Notably, the routine clinical use of FISH and ICC may be replaced by emerging next‐generation sequencing and digital, color‐coded barcode technologies, which have the advantage of simultaneously evaluating ALK, ROS1, and EGFR alterations in a single analysis. Although their use in clinical cytologic practice remains to be fully established, it is conceivable that this technology will replace both FISH and ICC analyses in future diagnostic algorithms. Here, the authors review studies devoted to testing ALK and ROS1 on cytology specimens in an attempt to provide an update for the cytopathologist regarding current and evolving practice. Cancer Cytopathol 2017;125:817–30.


Cytopathology | 2016

Cytopathologists can reliably perform ultrasound-guided thyroid fine needle aspiration: a 1-year audit on 3715 consecutive cases.

Claudio Bellevicine; Elena Vigliar; Umberto Malapelle; Pasquale Pisapia; Giovanni Conzo; Bernadette Biondi; Antonio Vetrani; Giancarlo Troncone

In our Pathology Department, fine needle aspiration (FNA) of palpable thyroid nodules is performed by cytopathologists who ensure correct sample management and rapid on‐site evaluation (ROSE). Conversely, ultrasound (US)‐guided FNAs have traditionally been carried out by endocrinologists and radiologists in outside clinics, where the presence of a cytopathologist is not always feasible. To overcome this limitation, cytopathologists have started to perform US‐guided FNAs themselves. This study retrospectively evaluates 1 year of this novel practice.


Journal of Clinical Pathology | 2016

EGFR mutation detection on routine cytological smears of non-small cell lung cancer by digital PCR: a validation study

Umberto Malapelle; Caterina De Luca; Elena Vigliar; Francesca Ambrosio; Danilo Rocco; Pasquale Pisapia; Claudio Bellevicine; Giancarlo Troncone

Highly sensitive genotyping techniques are useful to detect epidermal growth factor receptor (EGFR) mutations on lung cancer cytological samples, when these specimens feature only few neoplastic cells. This study aimed to validate digital PCR (dPCR) methodology on cytological material. In plasmid model system, dPCR allowed for the detection of a minimal percentage (1%) of EGFR mutant alleles. Cytological samples (n=30), with neoplastic cell percentage ranging from 10% to 80% and yielding a quantity of extracted DNA ranging from 1.75 to 60 ng/µL were selected. Results previously generated by fragment length and TaqMan assays (n=8 exon 19 deletions, n=2 L858R mutations and n=20 wild-type DNA) were compared with those obtained by dPCR. Data were highly concordant (96.6%). However, dPCR detected an additional L858R mutation that had been missed by TaqMan assay on a paucicellular smear. This mutation was confirmed by cloning PCR products and sequencing. Thus, dPCR can reliably be used to increase EGFR mutation detection rate on scarcely cellular lung cancer smears.


Journal of Clinical Pathology | 2017

How to prepare cytological samples for molecular testing

Claudio Bellevicine; Umberto Malapelle; Elena Vigliar; Pasquale Pisapia; Giulia Vita; Giancarlo Troncone

This review is focused on the challenges in standardising and optimising molecular testing workflow in cytopathology. Although cytological samples yield optimal quality DNA, whose minimal amounts in most cases suffice even for multigene mutational profiling, the success of molecular testing is strongly dependent on standardised preanalytical protocols for maximising DNA yield and quality. Sample cytopreparation influences, even more, the quality of RNA and consequently the potential success of reverse transcription-PCR. Here, the educational and technical involvement of the cytopathologist as a relevant component of a multidisciplinary team, in the issues related to test request, specimen collection, fixation, processing, staining, tumour fraction enrichment, DNA quality/quantity assessment and storage conditions is discussed. In addition, the specific sample requirements related to more recent technological developments are examined, underlining the modern role of the cytopathologist, whose continuous education is crucial to meet the opportunities of molecular medicine.


CytoJournal | 2017

KRAS detection on archival cytological smears by the novel fully automated polymerase chain reaction-based Idylla mutation test.

Caterina De Luca; Elena Vigliar; Melania d'Anna; Pasquale Pisapia; Claudio Bellevicine; Umberto Malapelle; Giancarlo Troncone

Background: Molecular techniques are relevant to modern cytopathology, but their implementation is difficult without molecular expertise and infrastructure. The assessment of KRAS mutational status on cytological preparations may be useful either to refine uncertain diagnoses on pancreatic aspirates or to yield predictive information to plan targeted treatment of metastatic colorectal cancer (mCRC). The novel test Idylla™ enables fully automated KRAS genotyping in approximately 2 h, even in less experienced hands. Materials and Methods: This study aims to validate this methodology to detect KRAS mutations on archival cytological preparations of pancreatic cancer (n = 9) and mCRC (n = 9) by comparing the Idylla™ performance to that of standard real-time polymerase chain reaction. Results: The same 11 mutations (n = 4: p.G12D; n = 2: p.G12V; n = 2: p.A59E/G/T; n = 1: p.G12R; n = 1: p.G13D; n = 1: p.Q61H) were detected by both techniques. Conclusion: Even in less experienced laboratories, a cytopathologist may easily integrate morphological diagnostic report with accurate KRAS mutation detection, which is relevant for diagnostic and treatment decisions.

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Umberto Malapelle

Seconda Università degli Studi di Napoli

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Giancarlo Troncone

Seconda Università degli Studi di Napoli

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Claudio Bellevicine

University of Naples Federico II

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Francesco Pepe

University of Naples Federico II

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Elena Vigliar

University of Naples Federico II

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Caterina De Luca

University of Naples Federico II

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Roberta Sgariglia

University of Naples Federico II

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