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G.V. Scagliotti



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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): G.V. Scagliotti

      • 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): G.V. Scagliotti

      • 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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    P1.10 - Poster Session 1 - Chemotherapy (ID 204)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
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      P1.10-056 - The Elderly Patient Individualized Chemotherapy Trial (EPIC): A Randomized Phase III Multicenter Trial of Customized Chemotherapy versus Standard of Care for 1st Line Treatment of Elderly Patients with Advanced Non-Small-Cell Lung Cancer (ID 1117)

      09:30 - 09:30  |  Author(s): G.V. Scagliotti

      • Abstract

      Background
      This is an ongoing phase III multicenter randomized trial comparing first line pharmacogenomic-driven chemotherapy based on Excision-Repair-Cross-Complementing-1 (ERCC1), Ribonucleotide Reductase subunit M1 (RRM1) and Thymidylate Synthase (TS) gene expression, versus standard first line treatment in elderly patients (pts) with advanced non-small-cell lung cancer (NSCLC). Chemotherapy selection based on an individual patient’s molecular profile is a potentially promising approach to optimize efficacy with the already available cytotoxic drugs. In older pts this is particularly relevant owing to their rapid deterioration of symptoms and their increased propensity to suffer therapy-induced toxicity.

      Methods
      Pts aged >70 years, with ECOG Performance Status (PS) 0 or 1, previously untreated for stage IV NSCLC will be evaluated. In a 2:1 fashion, pts will be randomized to experimental arm (A) or standard arm (B). They must have measurable disease and EGFR negative mutational status. In arm A, treatment with single or dual-agent chemotherapy will be based on histology, ERCC1 (E), RRM1 (R) and TS (T) expression at the mRNA level. Expression of E, R and T is assessed by qRT-PCR on paraffin-embedded tumor specimens in a central laboratory. The cut off for high or low expression have been previously defined. Pts with squamous NSCLC who are: E low/R high will be treated with single agent carboplatin, E high/R low with single agent gemcitabine, E low/R low with carboplatin and gemcitabine and E high/R high with docetaxel or vinorelbine. In non-squamous NSCLC pts: E low/T high will be treated with carboplatin, E high/T low with pemetrexed, E low/T low with carboplatin and pemetrexed, E high/T high/R low with gemcitabine and E high/T high/R high with docetaxel or vinorelbine. In arm B treatment will be standard of care at the discretion of the care provider. The primary endpoint is overall survival (OS). The secondary endpoints are progression-free survival (PFS), disease response according to RECIST 1.1 and tolerability (using CTCAE version 4.0). Feasibility of treatment selection based on pharmacogenomic parameters will also be assessed. Treatment will continue to a maximum of 6 cycles if tolerated or until disease progression. Switch maintenance treatment is not allowed in either arm. Continuation maintenance (one or more of the agents used in the initial regimen) is allowed at the discretion of the investigator. Treatment upon progression is at the discretion of the care provider. Assuming an exponential survival distribution for both treatment arms and a median survival time of 8 months in the control arm we anticipate to detect an improvement of 3 months in the median survival time in the experimental arm. To have 90% power to detect a three-month improvement in median survival at a significance level of 5% (2-sided) and assuming a 10% failure rate in gene analyses or loss to follow up rate, a sample size of 567 patients is planned to be enrolled.

      Results
      Not Applicable

      Conclusion
      We hypothesize that such tailored approach will improve survival decreasing the exposure to ineffective toxic agents in advanced NSCLC elderly pts. To our knowledge this is the first pharmacogenomic-driven randomized trial in this population.

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    P2.18 - Poster Session 2 - Pathology (ID 176)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Pathology
    • Presentations: 1
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      P2.18-014 - Impact of Non Small Cell Lung Cancer (NSCLC) immunophenotyping in chemotherapy response. (ID 2679)

      09:30 - 09:30  |  Author(s): G.V. Scagliotti

      • Abstract

      Background
      The vast majority of non small cell lung cancers (NSCLC) presents as advanced disease and histological diagnosis is widely based on small biopsy or cytological samples. Recently, the adoption of new pharmaceutical agents targeting individual histotypes requires a precise subtyping of NSCLC. This task is often difficult according to morphological criteria only and the use of immunohistochemistry (IHC) is recommended for small samples or undifferentiated tumors to define the most probable histotype. However, the real impact of IHC characterization of NSCLC-Not otherwise Specified (NOS) in terms of response to therapy and outcome (compared to cases classified by morphology, only) is not well established.

      Methods
      A large series of 224 advanced "non-squamous" NSCLC diagnosed from year 2005 to 2010 on small biopsy or cytological samples and homogeneously treated, was retrospectively selected, all with adequate follow-up data available. All diagnoses were reviewed resulting in two groups of adenocarcinoma (ADC) and NSCLC-NOS. The latter were further characterized by IHC to identify the most probable differentiation lineage. Disease Control Rate (DCR) and Response Rate (RR) were calculated and Overall Survival (OS) curves were analyzed by Kaplan Meier.

      Results
      After review 120/224 (53.6%) cases were ADC based on morphological examination, only (“ADC morphology”) and 104/224 (46.4%) remained NSCLC-NOS. In terms of response to therapy no significant difference was found between the two groups (“ADC morphology” had DCR= 0.66 and RR=0.31; NSCLC-NOS had DCR=0.64 and RR=0.35; Chi-Square p=0.83). The NSCLC-NOS cases that underwent IHC profiling resulted in 66/104 (63.5%) cases that had an ADC phenotype (“NSCLC favor ADC”) and 38/104 (36.5%) cases that lack ADC features (including 5 “NSCLC favor squamous carcinoma” and 33 “NSCLC null phenotype”). The “NSCLC favor-ADC” had DCR and RR similar to “ADC morphology” group (Chi-Square p=0.23), while the “non-ADC” NSCLC group had significantly different both DCR=0.47 and RR=0.29 (Chi-Square p=0.006). Survival curves confirmed no difference in terms of survival between the “ADC morphology” and the “NSCLC favor-ADC” groups, while showed a significantly poorer survival for the “non-ADC” NSCLC group with respect the other two groups (median survival: 8.5 vs 12.3 months, respectively; HR=0.5999; p=0.018).

      Conclusion
      These preliminary findings indicate that the practice of minimizing the NSCLC-NOS diagnoses by means of IHC has an impact on chemotherapy response. Stratifying such tumors by IHC, cases having an ADC immunoprofile had response rates comparable to those of morphologically diagnosed ADC, thus supporting the value of IHC to maximize lung cancer histological typing in the perspective of obtaining the best response to therapy.

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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): G.V. Scagliotti

      • 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): G.V. Scagliotti

      • 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.

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    P3.12 - Poster Session 3 - NSCLC Early Stage (ID 206)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
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      P3.12-023 - International Tailored Chemotherapy Adjuvant Trial: ITACA Trial (ID 1452)

      09:30 - 09:30  |  Author(s): G.V. Scagliotti

      • Abstract

      Background
      This is an ongoing phase III multicenter randomized trial comparing adjuvant pharmacogenomic-driven chemotherapy, based on thymidilate synthase (TS) and excision-repair cross-complementing-1 (ERCC1) gene expression versus standard adjuvant chemotherapy in completely resected Stage II-IIIA non-small cell lung cancer (EudraCT #: 2008-001764-36).

      Methods
      For all the registered patients (pts) the expression of ERCC1 and TS is assessed by qRT-PCR on paraffin-embedded tumor specimens in a central laboratory. Randomization is stratified by stage and smoking status. Trial was emended on Feb, 2011 with the 7th staging system. Primary end point is overall survival; secondary end points include recurrence-free survival, therapeutic compliance, toxicity profile and comparative evaluation of ERCC1 and TS mRNA versus protein expression. It is assumed that the 5-year survival rate in the control arm is 45% and the hazard reduction associated to the experimental treatment is 30%. With a power of 90% to detect the estimated effect with log-rank test, a significant level of 5% (2 tails), 336 events have to be observed; the expected total number of pts is 700. The final statistical analysis will compare all pts in the control arms versus all those treated in the tailored chemotherapies groups. Efficacy analysis will be done on an intent-to-treat basis. Cox proportional hazard model will be used for estimating hazard ratios after adjusting for relevant variables. Within 45 days post-surgery, pts in each genetic profile are randomized to receive either a standard chemotherapy selected by the investigator (cisplatin/vinorelbine, cisplatin/docetaxel or cisplatin/gemcitabine) or an experimental treatment (tailored arms) selected as follows: 1) high ERCC1 and high TS 4 cycles of single agent paclitaxel 2) high ERCC1 and low TS 4 cycles of single agent pemetrexed 3) low ERCC1 and high TS 4 cycles of cisplatin/gemcitabine 4) low ERCC1 and low TS 4 cycles of cisplatin/pemetrexed. All chemotherapy regimens are administered for a total of 4 cycles on a 3-weekly basis.

      Results
      Not applicable

      Conclusion
      Currently, 558 pts have been randomized from 26 institutions mainly located in Italy and Germany (average enrolment: 19 patients/month). This is one of the pharmacogenomic-driven trial in the adjuvant setting in the European scenario.

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    P3.18 - Poster Session 3 - Pathology (ID 177)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Pathology
    • Presentations: 1
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      P3.18-014 - Implementing systematic histological and genotypic re-evaluation of Non Small Cell Lung Cancer (NSCLC) into routine clinical practice: a monocentric study (ID 2691)

      09:30 - 09:30  |  Author(s): G.V. Scagliotti

      • Abstract

      Background
      Cancer is a multistep process in which tumor cells progressively harbour genetic alterations that confer growth and spread advantages. These observations are also confirmed in NSCLCs where specific genetic abnormalities are sensitive to the action of targeted drugs, even if secondary mutations could develop conferring drug resistance. Furthermore, in recent studies, histologic and immunophenotypic changes have been described in patients (pts) with a specific molecular alteration re-biopsied after receiving a targeted therapy. This finding suggest the possibility of a "clonal resistance mechanism" in which genetic-similar cell populations had or acquire selective survival features thus escaping the inhibitory drug effect. In the present study histological examination and wide genetic analyses have been performed in tumor tissue sampled both before and after treatment in an unselected NSCLC patient population in order to elucidate progressive clinic-pathological and genetic abnormalities.

      Methods
      NSCLC pts with adequate tissue samples before and after at least one treatment have been evaluated from July 2006 to March 2013, at Department of Oncology of San Luigi Hospital. All had ECOG Performance Status of 0, median age of 51,5 years and IIIA-IV stage at diagnosis. After histological examination, mutational analyses for EGFR, K-RAS, PIK3CA, B-RAF, and HER2 genes were performed using pyrosequencing or RealtimePCR. ALK and c-MET genomic rearrangement were tested by FISH.

      Results
      A total of 24 (12 males and 12 females) pts were collected. Histological diagnoses were re-confirmed in all but one (4%) case in which morphological and immunophenotypical histology of neuroendocrine small cell lung cancer (SCLC) was found. All samples were adequate for molecular analyses. At the first biopsy EGFR activating mutations were 10/24 (42%) and 3/24 (12,5%) were the exon 20 EGFR p.T790M mutation, 2/3 (66%) associated to EGFR activating mutations. At the second biopsy 6/10 (60%) EGFR activating mutations were maintained and no acquired mutations in EGFR wild type pts at first were found at second biopsy. On the other hand, 6/24 (25%) p.T790M mutation were detected at second biopsy, 5/6 (83%) de novo acquired, 1/6 (17%) maintained and only 1/5 (20%) associated to EGFR activating mutations. The patient who acquired SCLC histology, maintained the L858R EGFR activating mutation after treatment with no other acquired mutation. K-RAS mutations were found in 4/24 (16.7%) first biopsies, while 5/24 (21%) were found at the second biopsies. No mutations were found in BRAF, HER2 and PIK3CA genes. ALK rearrangement was assessed in 5/24 (20,8%) patients; otherwise MET amplification was seen in 3/24 (12.5%) cases only 1/3 (30%) in EGFR mutant cases.

      Conclusion
      These preliminary data showed a complex scenario of basal and acquired alterations on tumor tissue before and after treatment highlighting the need of repeated histological and genotypic assessments to guide at the best treatment decisions.