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M. Lo Iacono



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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): M. Lo Iacono

      • 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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    P1.05 - Poster Session 1 - Preclinical Models of Therapeutics/Imaging (ID 156)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.05-015 - Assessment of the activity of Pemetrexed and Dasatinib as single agents and in combination in three malignant pleural mesothelioma cell lines (ID 2339)

      09:30 - 09:30  |  Author(s): M. Lo Iacono

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM), an asbestos exposure related disease, is a highly aggressive tumor. Pemetrexed is a third-generation multitargeted antifolate approved as single agent or in combination with cisplatin as standard of care in first/second line treatment of unresectable MPM. Thymidylate synthase (TS), a key enzyme in the de novo synthesis of thymidine, is the main target of Pemetrexed and its overexpression has been related to Pemetrexed-resistance. Experimental data suggest that c-SRC tyrosine kinase hyperactivation has a key role in MPM. The effect of c-SRC pharmacological inhibition in correlation with TS expression levels and Pemetrexed resistance, has been investigated in three MPM cell lines.

      Methods
      Cell growth inhibitory effects of both Pemetrexed and Dasatinib (10nM-100μM) were evaluated by MTS proliferation assay in epithelial (MPP89, REN) and biphasic (MSTO-211) MPM cell lines. Apoptosis was detected by AnnexinV-propidium iodide method using a FACScan, while drug-mediated changes in invasive ability were tested using “wound healing” scratch assay. Real-Time PCR and Western blot were assessed to identify drugs-associated genes and/or proteins modulation.

      Results
      The cell lines assayed displayed different sensitivity to both Pemetrexed and Dasatinib treatments. Among the three cell lines, MSTO-211 was the most sensitive to Pemetrexed (IC~50 ~0.5 μM); on the contrary REN was the most resistant (IC~50 ~5 μM). A similar trend was observed upon Dasatinib treatment with IC50 values ranging from 1 to 5 μM. The synergistic effect of Dasatinib and Pemetrexed was also evaluated, being significantly relevant in MPP89 and REN after 72h treatment while, in MSTO-211, was already detectable after 48h. Early and late apoptosis assessment confirmed, for both drugs, the ability to induce apoptosis, being MPP89 the most Dasatinib-sensitive and MSTO-211 the most Pemetrexed-sensitive cell line. In MPP89 and REN cells co-administration of Pemetrexed/Dasatinib significantly increased the apoptotic rate of 16 folds and this behaviour was enhanced in MSTO-211 (up to 27 folds). Both TS, gene and protein levels were higher in REN compared to MPP89 and MSTO-211 cells. Pemetrexed administration increased TS levels over time, in those cells most sensitive to the drug. Interestingly, in REN cells Pemetrexed treatment did not affect the high baseline TS levels but Dasatinib administration suppressed TS protein and, to a lesser extent, mRNA expression, thus increasing sensitivity to Pemetrexed. In addition, in REN cells the pretreatment with Dasatinib (5 μM) enhanced Pemetrexed sensitivity leading to a strong cell viability reduction. In all 3 cell lines, SRC was expressed (mRNA and protein), decreasing its levels from MSTO and MPP89 to REN, and activated. Dasatinib impaired also cell migration, as observed by wound-healing assay.

      Conclusion
      In vitro data suggest that inhibition of both TS and SRC might represent a potential therapeutic strategy in MPM. The evidence indicates that Dasatinib plays a role by inhibiting cell motility and, more surprisingly, by down-regulating TS. Dasatinib-mediated TS expression impairment suggests a cross-talk between SRC and TS pathways thus leading to hypothesize a therapeutic use of Dasatinib to sensitize those Pemetrexed-resistant MPM patients’ cohort.

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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): M. Lo Iacono

      • 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.

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    P3.02 - Poster Session 3 - Novel Cancer Genes and Pathways (ID 149)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.02-013 - ATF2 contributes to Platinum resistance in Non Small Cell Lung Cancer and cJUN/ATF2 Celastrol mediated modulation restores Platinum sensitization. (ID 2275)

      09:30 - 09:30  |  Author(s): M. Lo Iacono

      • Abstract

      Background
      ATF2 is a member of the basic helix-loop-helix (b-ZIP) transcription factor family playing important roles in Stress and DNA damage. Several studies reported correlation between ATF2/JNK-mediated activation and resistance to damaging agents. Celastrol is an ATF2 inhibitor extracted from a Chinese plant known as “Tunder of God Vine”. Celastrol is used in traditional Chinese medicine for anti-inflammatory properties. In addition, Celastrol is a triterpene with promising anticancer activity in several cancer models both in vivo and in vitro. The purpose of the present study was to investigate whether ATF2 might play a role in inducing drug resistance in Non-Small Cell Lung Cancer (NSCLC).

      Methods
      In NSCLC cell lines ATF2 expression levels were evaluated by quantitative PCR (qPCR) and Western Blot (WB) and correlated to cisplatin (CDDP) resistance. Celastrol mediated ATF2/cJUN activity was assessed by Luminometry, qPCR and western blotting (WB). Furthermore, matched tumors and corresponding normal tissues of 88 surgically resected NSCLC specimens, were collected and both qPCR and immunohistochemical analyses for ATF2 were performed.

      Results
      NSCLC cell lines CDDP-resistant (H522 and H1395) expressed high levels of ATF2 protein. Moreover, CDDP treatment increased ATF2 phosphorylation levels leading to an enrichment within the nuclear cell compartment. In our study, Celastrol reduced ATF2 activity by decreasing the production of ATF2 mRNA and blocking the CDDP-mediated phosphorylation/mRNA expression of cJUN, a main ATF2 partner. Furthermore, ATF2/cJUN functional inhibition mediated by Celastrol restored the response to CDDP in resistant lung cell Lines. ATF2, at both protein and mRNA level, was significantly up-modulated in NSCLC tumor samples compared to the paired normal lung tissue (mRNA: p<<0.01, mean Log2(FC)=+4.7). Moreover, high expression of ATF2 mRNA was correlated with the smoking status of the patients. Relevantly, smoker or former smoker patients expressed significantly high ATF2 mRNA levels compared to non-smokers (p=0.02 and p=0.04, respectively).

      Conclusion
      This study shows that in NSCLC cell lines a correlation between ATF2 protein expression and CDDP resistance occurs. Furthermore, our results suggest a potential increase of CDDP sensitivity, following the Celastrol-mediated ATF2/cJUN inhibition. For the first time it has been shown in NSCLC an up-regulation of ATF2 mRNA/protein levels compared to normal tissues and consistent with that detected in CDDP resistant cell lines. This data suggest a possible involvement of ATF2 in NSCLC CDDP-resistance.

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    P3.06 - Poster Session 3 - Prognostic and Predictive Biomarkers (ID 178)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.06-026 - Thymidylate synthase (TS) mRNA and protein expression in advanced non-small cell lung cancer (NSCLC) patients treated with pemetrexed-based therapy. (ID 2355)

      09:30 - 09:30  |  Author(s): M. Lo Iacono

      • Abstract

      Background
      In NSCLC, higher Thymidylate Synthase (TS) levels have been reported in both squamous and large cell carcinomas compared to adenocarcinoma. In clinical practice, Pemetrexed, a potent antifolate inhibitor of TS, showed a selective benefit in patients with "non-squamous" NSCLC. Two retrospective studies have shown that low TS protein levels are associated with better clinical outcome in NSCLC patients treated with pemetrexed. Aim of this study was to explore, in a series of advanced stage IV patients receiving pemetrexed-based regimens in first line of treatment, the association between TS mRNA and protein expression with overall survival (OS) and therapeutic response.

      Methods
      Two series of histologically confirmed non squamous-NSCLC, assessed in formalin-fixed and paraffin embedded specimens from patients treated with pemetrexed-based regimens were collected: the first series at San Luigi Hospital (n=64), the second series at Regina Elena National Cancer Institute (n=8). Due to the limited amounts of tissue specimens available, total RNA extraction was possible in 52 out of 72 cases. TS protein expression was performed using immunohistochemistry (mouse monoclonal TS106 antibody) and scored through H-SCORE method, considering both staining intensity (0 no staining; +1 weak; +2 moderate; or +3 strong) and percentage of tumor cells stained, resulting in semiquantitative H-scores ranging from 0 to 300. TS nuclear and cytoplasmic staining, respectively, were separately scored. Statistical analyses were performed using the STATISTICA10 software.

      Results
      The differential H-SCORE assessment showed a strong importance of TS localisation for clinical outcome prediction: in Cox regression analysis, a statistically significant association was observed between nuclear TS expression and OS (p < 0.009) indicating that lower nuclear TS expression levels were associated with longer OS. In addition, lower nuclear TS levels were significantly associated with a better response to therapy (p<0.001). On the contrary, TS cytoplasmic staining did not affect patients’ survival or clinical response (p>0.05). Four subgroups of patients, based on the dichotomized low/high TS expression in both nucleus and cytoplasm, were obtained: both high, both low, nucleus high/cytoplasm low and nucleus low/cytoplasm high. Significant differences in overall survival among these four groups were detected (p=0.017), confirming the strong and selective influence of nuclear TS, as compared to cytoplasmic TS, expression in clinical outcome. Moreover, Chi[2] test revealed a significant association between low nuclear TS and partial response to pemetrexed treatment, independently of cytoplasmic TS expression (p<0.001). No correlation between TS protein expression data and clinico-pathological data (age, gender) were identified. TS gene expression analyses are ongoing.

      Conclusion
      This retrospective study suggests that TS protein expression, selectively assessed at nuclear level, has a potential predictive role in advanced stage IV patients, receiving pemetrexed in first line of treatment. Patients with low nuclear TS expression showed prolonged overall survival and better response to therapy. Such preliminary results define TS assessment as a potential tool which may select the most appropriate group of patients to be treated with pemetrexed.