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P. Bironzo



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    O22 - Mesothelioma III (ID 122)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Mesothelioma
    • Presentations: 1
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      O22.01 - Next generation sequencing in malignant pleural mesothelioma: preliminary data from a retrospective cohort of 123 patients (ID 2290)

      16:15 - 16:25  |  Author(s): P. Bironzo

      • Abstract
      • Presentation
      • Slides

      Background
      The median survival of patients with advanced stage malignant pleural mesothelioma (MPM) ranges between 9 and 12 months after diagnosis, regardless of the recent achievements with systemic therapies combining cisplatin and antifolates such as pemetrexed or raltitrexed. Since MPM is a relatively rare malignancy and early pre-neoplastic lesions are clinically difficult to be identified, the understanding of molecular pathogenesis including sequential accumulation of genetic/epigenetic alterations for MPM development has lagged behind other common malignancies. According to the COSMIC database the most frequently mutated genes in MPM include CDKN2A, NF2 and BAP1, followed by other 12 genes having been found mutated in a fraction of MPM cases (c-MET, VHL,WT1 among others). Clearly, a better and more systematic understanding of the role of genomic alterations in MPM is needed. In this retrospective study, a consecutive series of 123 formalin-fixed, paraffin embedded (FFPE) MPM tissue samples with clinical annotates, collected at two institutions, was retrospectively analyzed through Next-Generation Sequencing (NGS) technology to enhance knowledge about tumor-specific genomic profiling.

      Methods
      Genomic DNA was extracted by tumour microdissected FFPE samples for all 123 patients. Amplicons NGS libraries for 50 Oncogene included in Ion AmpliSeq™ Cancer Hotspot Panel (CHP) v.2 were generated as indicated by manufacturer, and sequenced in Personal Genome Machine IonTorrent. Variant Caller included in Torrent Suite Software was utilised to identify mutations in the samples, annotation was performed with Annovar software. Genomic analysis for BAP1 and NF2 (not included in the CHP) is separately ongoing.

      Results
      Of 123 advanced stage MPM patients, all treated with pemetrexed-based chemotherapy, 70% were males, current smokers 50%, median age 66.5 (range 36-82) years and histological subtypes were 96/22/5 epithelioid/biphasic/sarcomatous. With a cut off for allele frequency(AF)>=10% a total of 966 non-synonymous, 8 del-ins, 62 nonsense, 637 intronic, 204 regulatory and 1140 synonymous somatic sequence variations were detected in 107 patients already screened. Excluding synonymous mutations and irrespective of AF, the five most frequently altered genes were CSF1R (mut:154, pts:80), KDR (mut:148, pts:73), FLT3 (mut:132, pts:99), PIK3CA (mut:126, pts:60), TP53 (mut:111, pts:66). Evaluating mutations identified at least once, a correlation between HRAS and PIK3CA mutations and patient status (dead or alive) was observed (p=0.017 and p=0.039, respectively). Specifically, HRAS silent mutation p.H27H (rs12628) was responsible for the association (p=0.021) and occurred in 54% of 107 MPM compared to 30% of reported AF in available databases. PIK3CA p.I391M missense mutation (rs2230461; AF 24% in this series) was significantly associated to progression-disease (p=0.003). Among the other SNPs reported in at least 15 pts there are rs3729674(PIK3CA), rs1800863(RET), rs3822214(KIT), rs10006115(KDR), rs75580865(FLT3) and rs5030613(SMARCB1).

      Conclusion
      These extremely preliminary data indicate that NGS technology is feasible in FFPE MPM tissues and some of the detected genetic mutations are novel observations of potential prognostic and therapeutic interest.

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    P2.06 - Poster Session 2 - Prognostic and Predictive Biomarkers (ID 165)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.06-013 - Genetic profiling of lung cancer in young adults patients: early data assessment using Next-Generation Sequencing. (ID 1189)

      09:30 - 09:30  |  Author(s): P. Bironzo

      • Abstract

      Background
      In 3% of cases, lung cancer is diagnosed in patients younger than 45 years. The epidemiology, biology and clinical history of young lung cancer patients is generally different from the adult counterpart: the higher percentage of mutations has the potential to influence both tolerability and response to treatment with consequent impact on quality of life and survival. The biomolecular characterization of the disease in this subgroup will allow the design of clinical studies dedicated to young patients, that will lead to the identification of specific items that are not deducible from trials opened to the general adult population. In this study, Next-Generation Sequencing (NGS) technology has been applied to archival tissue samples to enhance tumor-specific genomic profile knowledge in this selected cohort of young patients (pts).

      Methods
      A retrospective analysis has been performed at the Thoracic Unit of San Luigi Hospital from January 2007 to March 2013, collecting 13 lung cancer-diagnosed pts (10 completely sequenced; in 3 cases the analysis is ongoing), aged between 15-39 years. Genomic DNA was extracted by microdissected formalin-fixed and paraffin embedded (FFPE) tumor samples of all pts and by lymphocytes of three healthy controls (ctrl). Amplicons NGS libraries for 46 oncogenes included in the Ion Torrent Cancer Panel were generated, following manufacture guidelines, and sequenced in Personal Genome Machine (PGM) Ion Torrent instrument. Variant Caller included in Torrent Suite Software was used to identify mutations.

      Results
      Twenty-two non-synonymous, 3 frameshifts, 3 stop-gain and 55 synonymous somatic sequence variations were found in 10 young adult patients (allelic frequency ≥ 10%). Excluding synonymous mutations, the most frequently altered genes in patients were TP53 (7 mutations; 25%), followed by EGFR and KDR (5 mutations; 18%), PI3K (3 mutations; 11%), KIT (7 mutations; 14%), FGFR3-ABL1-MET-ATM-RB1-SMO (1 mutation; 3.6%). Furthermore, 14 of these mutations are annotated in SIFT or in PolyPhen databases as “mutations that could damage affected protein”. Overall, we identified 28 mutations annotated in COSMIC database, among which the most frequent were COSM149673, COSM28026 and COSM6223-COSM22413 with 5,4 and 2 counts, respectively. We also found 93 SNPs in our cohort, including the most frequent rs7688609, rs1873778 and rs1050171 with 13 counts (10 pts; 3 ctrl) followed by rs1800861 (9 pts; 3ctrl); rs41115 (8 pts; 2ctrl); rs1870377 (4 pts; 1ctrl); rs3822214 (2 pts; 2ctrl); rs3729687 (2 pts; 1ctrl); rs2228230 (2 pts) and rs3730358-rs3135898 (1 pt; 1ctrl).

      Conclusion
      The development of new biological techniques, such as the next-generation sequencing, could allow to collect a wide number of mutations. From these preliminary results, some interesting data have been discovered concerning SNPs or mutations. This pivotal retrospective analysis is the basis for the ongoing prospective collection. A better definition of molecular-genetic pattern in this selected young population of patients could increase the knowledge about the lung cancer etiology and suggest age-related new trials design.