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C. Rolfo



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    MINI 03 - PD1 Axis Inhibition and EGFR (ID 101)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Treatment of Advanced Diseases - NSCLC
    • Presentations: 1
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      MINI03.11 - Discussant for MINI03.09, MINI03.10 (ID 3307)

      17:45 - 17:55  |  Author(s): C. Rolfo

      • Abstract
      • Presentation

      Abstract not provided

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    MINI 34 - RNA and miRNA (ID 162)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      MINI34.01 - MicroRNA Profiling of a Familial Primary Pulmonary Enteric Adenocarcinoma (ID 397)

      18:30 - 18:35  |  Author(s): C. Rolfo

      • Abstract
      • Presentation
      • Slides

      Background:
      Primary pulmonary enteric adenocarcinoma (PEAC) is defined as a pulmonary adenocarcinoma with a predominant component (>50%) of intestinal differentiation and tumor cells positive for at least one intestinal marker. Due to its peculiarity and rarity, the optimal clinical management of PEAC patients remains unclear. A total of thirty cases have been described in literature to date, though familial aggregation has never been reported before. This is the first study describing the histological and molecular characterization of the PEAC from a patient with a family member affected by the same tumor.

      Methods:
      We evaluated the molecular characteristics of proband’s PEAC applying a previously validated 47-miRNA signature and applying the predictive method to estimate tissue-of-origin probabilities. Immunohistochemical (IHC) staining (TTF-1, Napsin A, CDX2, cytokeratonins, mucins) and mutational analyses (EGFR, K-RAS, ALK) were performed on formalin-fixed, paraffin-embedded tissue of a patient affected by PEAC.

      Results:
      The familial aggregation of PEAC was associated with similar clinicopathological features (age at diagnosis, smoking-habit, tumor localization, multiple colon polyps), histologic findings (IHC staining negative for TTF-1 and positive for CDX2) and genetic findings (K-RAS(Gly12Asp) mutation, but no EGFR/ALK aberrations). MiRNA profiling revealed main similarities with NSCLC (75.98%), and partial overlap with pancreatic cancer (PDAC, 23.34%), but not with colorectal cancer (less than 0.5%). Notably, this PEAC shares with PDAC key miRNAs associated with tumor aggressiveness (mir-31/-126/-506/-508-3p/-514). Figure 1



      Conclusion:
      In conclusion, we described, for the first time, PEACs in two members of the same family, associated with clinicopathological features. Moreover, miRNA profiling resembled mostly NSCLC, with a partial overlap with PDAC’s pattern that could explain the aggressive behavior compared to most NSCLC and guide future tailored-therapy approaches.

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    P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)

    • Event: WCLC 2015
    • Type: Poster
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 3
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      P2.04-027 - Erlotinib Induce miRNA Alterations in T790M EGFR Mutated NSCLC: Preclinical Study (ID 2483)

      09:30 - 09:30  |  Author(s): C. Rolfo

      • Abstract
      • Slides

      Background:
      Lung cancer is one of the leading causes of cancer-related deaths worldwide. In the most common type, non-small cell lung cancer (NSCLC), an array of oncogenic driver mutations affecting growth factor receptors and signaling pathways, such as EGFR, KRAS, c-Met or ALK translocation has driven the development of directed-drug therapies with tyrosine kinase inhibitors (TKIs) and monoclonal antibodies. Nevertheless, the appearance of resistance mechanisms, such as c-Met amplification or de novo mutations in EGFR, decreases the effectiveness of these therapies. Therefore, we analyzed the levels of miRNA which have proved to be involved in disease progression (miR-30b-5p,195-5p,221-3p) in NSCLC cells that are resistant (H1975) or sensitive (HCC827) against first generation TKI (erlotinib) treatment, to assess whether they are markers of response to the treatment.

      Methods:
      The H1975 cell line (EGFR mutations T790M/L858R) was cultured in RPMI 1640 medium, supplied with 10% FBS, 2 mM L-glutamine and 1% penicillin/streptomycin (Gibco). The sulforhodamine B assay was performed to assess cell proliferation. The cells were treated during 72h with 10µM erlotinib (SelleckChem) or DMSO. After collection, cells were lysed and RNA was extracted through commercial kit (RNA Mini Spin, GE Healthcare). The miRNAs profile analysis was performed through TaqMan Real-Time PCR and miRNA RNU-48 was used as endogenous control. Data was processed according to the formula 2[-ΔΔct]. Control values (H1975 + vehicle) are used as baseline and results are shown in logarithmic scale (Image).

      Results:
      Cells treated with 10µM erlotinib show an increased expression of miR-30b-5p, miR-195-5p compared to control values. On the other hand, we detected that the expression of the miR-221-3p was strongly down-regulated in respect the control values.

      Conclusion:
      H1975 carries the T790M mutation in EGFR, associated in NSCLC with appearance of resistance to TKIs treatment. However, we observed after treatment an increase of onco-suppressor miR-30b-5p and decrease of the oncogenic mir-221-3p, which are reported to correlate with good prognosis (Garofalo 2013, Zhong 2014). Moreover, mir-195-5p, another miRNA related with onco-suppression, is also upregulated, which has been reported to correlate with good response (Liu, 2015). Overall, our data suggests that erlotinib treatment alters miRNA in an EGFR mutated cell line. Further analysis of miRNA in exosomes produced by H1975, and comparison with HCC827 exosomal and cellular miRNA levels is currently undergoing in our group to confirm their value as response to treatment biomarkers.Figure 1



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      P2.04-092 - Sequencing of Actionable EGFR Mutations from PtDNA in NSCLC: A Feasibility Study of Non-Invasive Analysis of Sensitivity to TKI (ID 984)

      09:30 - 09:30  |  Author(s): C. Rolfo

      • Abstract

      Background:
      In ~10 % of Caucasian patients with non-small cell lung cancer (NSCLC), the presence of an activating epidermal growth factor receptor (EGFR)-mutation has resulted in a more favourable prognosis by its exquisite sensitivity to a targeted treatment with tyrosine kinase inhibitors (TKI’s). However, in up to 20% of those patients, the presence of "driver" alterations cannot reliably be established due to an inadequate diagnostic sample and/or impossibility to re-biopsy a patient. Activating EGFR-mutations can be accurately detected in plasma with a high concordance to matched tumour tissue with allele-specific PCR assays of plasma tumor DNA (ptDNA). This ‘liquid biopsy’ can replace response to treatment, allow detection of early relapse in the follow-up and estimate prognosis. The aim of this study is to assess the feasibility of detecting ptDNA in patients with EGFR mutations, to assess the concordance of ptDNA levels to mutations in matched tissue samples and to correlate ptDNA levels with clinical response or relapse after start of TKI-treatment.

      Methods:
      Tumour tissue samples of 20 patients (10 with/10 without activating EGFR-mutations), was assessed for the presence of the respective mutation in matched ptDNA, obtained either at presentation or during follow up. DNA was extracted from aliquots (1ml) of plasma with the use of QIamp circulating nucleic acid kit (Qiagen). The target DNA is amplified and detected on the cobas z 480 analyzer using the amplification and detection reagents provided in the cobas EGFR Mutation Test kit. The concordance was estimated with Bayesian variables.

      Results:
      26 tissue and 34 plasma samples were collected from 20 Caucasian patients with median age of 67 years (54 to 84), 60% female, 30% non-smokers, 100% adenocarcinomas and 95% histologically or cytologically confirmed stage IV NSCLC. The median number of samples per patient was 3 (2 - 5). In the tissue samples 6 patients had an exon 19 deletion, 1 had exon 18 mutation, 2 had exon 20 mutation and in 1 patient a simultaneous exon 19 deletion and T790M mutation. The Bayesian characteristics of ptDNA determination are as follows:

      A: at sample level ( n = 34) B: at study population level (n = 20)
      1. Prevalence of mutation 8.8% 10%
      2. Sensitivity 12.5% 20%
      3. Specificity 100% 100%
      4. PPV 100% 100%
      5. NPV 32.2% 55%
      6. Accuracy 38% 60%
      7. Concordance 11.5% 7.7%


      Conclusion:
      Detection of ptDNA in EGFR-positive patients is feasible. However, ptDNA mutation testing by cobas[R] 4800_Blood Test was not reliable due to the low analytical sensitivity and the heterogeneity of the patient population. Further studies with other methods as next-generation sequencing or digital droplet PCR are warranted.

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      P2.04-094 - Exosomal MiRNA Analysis of Non-Small Cell Lung Cancer (NSCLC) Liquid Biopsies. Mirror of the Disease Status?: Proof of Concept Study (ID 1395)

      09:30 - 09:30  |  Author(s): C. Rolfo

      • Abstract
      • Slides

      Background:
      The discovery of the alterations in EGFR, c-Met or ALK in NSCLC has driven the development of targeted-drug therapy using tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is needed. The exosomes extracted from blood samples could be non-invasive and regularly updated biomarkers. Here we analyze selected exosomal miRNAs to evaluate its biomarker potential in NSCLC.

      Methods:
      1 ml of serum/plasma sample from 11 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. Exosome were isolated through commercial-kit or D~2~O/sucrose density-gradient ultracentrifugation. After exosome characterization (Western-Blot, Transmission Electron Microscopy) a panel of miRNAs (30b-5p, 30c-5p, 103,195,221-3p,222-3p), correlated with NSCLC disease (Garofalo et al, 2013), was analyzed. The miRNAs profile analysis was performed through TaqMan Real-Time PCR and mir-1228-3p was used as endogenous control. The data was processed according to the formula 2[-ΔΔct]. Control values are used as baseline and results are shown in logarithmic scale (figure).

      Results:
      Patients without molecular alterations (WMA): The levels of miR-30b-5p/30c-5p/103/221-3p/222-3p were down-regulated relative to the healthy controls. The patient with docetaxel treatment (P2) has an increased down-regulation of these miRNAs compared to the non-treated patient (P1). Patient with BRAF G464V: We observed an increased up-regulation of miR-30b-5p/30c-5p/103/221-3p/222-3p just after stopping Erlotinib treatment (P4a) compared with one month after the treatment (P4b). Patients with C-Met 3+ over-expression: We detected an increase of expression of miR-30b-5p/30c-5p and a decrease of expression of mir221-3p/222-3p in a patient treated with Crizotinib (P6) compared to a non-treated patient (P5). Patients with EGFR (exon 19 del): We observed a decrease of expression of mir221-3p/222-3p in a patient treated with Afatinib beyond progression (P8) compared to a non-treated patient (P7). Patients with ALK t(2p23): We detected a decrease of expression of miR-30b-5p/30c-5p/103 compared to healthy controls. No differences between treated (P9-P11) and non-treated (P3) patients were observed. Nevertheless, mir221-3p/222-3p differs significantly between patients treated with Ceritinib one week (P10) and one month (P11) after the treatment was started.

      Conclusion:
      This panel of exosomal miRNAs derived from patients with varying mutations is responsive to different treatments. The down-regulation of miR-30b-5p/30c-5p in exosomes of patients with WMA and ALK t(2p23) mutations, mirrored the reported low levels of these miRNAs in NSCLC tissue (Zhong et al.,2014). Follow-up analysis to correlate clinical progression and exosome miRNAs profile is currently ongoing. Figure 1



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    P3.07 - Poster Session/ Small Cell Lung Cancer (ID 223)

    • Event: WCLC 2015
    • Type: Poster
    • Track: Small Cell Lung Cancer
    • Presentations: 1
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      P3.07-001 - Anaplastic Lymphoma Kinase (ALK) Gene Rearrangement in Small-Cell Lung Cancer (SCLC): A Single Center Experience (ID 924)

      09:30 - 09:30  |  Author(s): C. Rolfo

      • Abstract
      • Slides

      Background:
      In small-cell lung cancer little is known about harboring an ALK translocation [1, 2]. The aim of this study was to investigate the prevalence of ALK expression and rearrangement in patients with SCLC.

      Methods:
      In this retrospective series, archival tissue from 17 treatment naïve patients with SCLC and neuroendocrine tumors was analyzed to detect ALK expression by immunohistochemistry (IHC) in all samples. Cut-off value for positivity was similar as in non-small-cell lung cancer (NSCLC): focally strong staining of the tumor cells Fluorescent in-situ hybridization (FISH) with Vysis LSI ALK probe was performed in the IHC positive cases [3]. The ALK FISH positive cases were submitted for exome sequencing (NGS) (Illumina MiseQ).

      Results:
      Of the 17 patients 9 were male and 8 female. Of these 17 patients 12 had a SCLC, 3 a neuroendocrine tumor, and 2 a neuroendocrine carcinoma. Three specimens could not be analyzed. Six of 17 specimens were ALK IHC positive, of which one neuroendocrine tumor, one neuroendocrine carcinoma and 4 of the SCLC.

      Conclusion:
      The IHC expression of ALK suggests a possible role of ALK-TKI in the treatment of SCLC. Mature FISH and NGS data will be presented at the meeting.

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