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H. Torigoe



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    P1.02 - Poster Session with Presenters Present (ID 454)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Biology/Pathology
    • Presentations: 1
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      P1.02-071 - Detection of Multiple Low-Frequency Mutations by Molecular-Barcode Sequencing (ID 4554)

      14:30 - 15:45  |  Author(s): H. Torigoe

      • Abstract

      Background:
      Recent advantage of next-generation sequencing (NGS) in the field of cancer genome research has revealed extreme levels of genetic heterogeneity, suggesting the major roles of subclonal mutations in cancer relapse and in rapid emergence of acquired resistance. Unlike inherited mutations, somatic variants often occur at low allele frequencies that require sensitive methods for detection. Based on the results using another highly sensitive method, such as digital PCR, the study of cancer subclones requires to detect mutations that are present in <1% frequency, however, such a level of resolution cannot be obtained by conventional NGS approaches. In the current study, we performed molecular-barcode sequencing for primary lung adenocarcinoma cases in order to analyze mutations with <1% low frequency.

      Methods:
      Fresh frozen tumor samples from 58 primary lung adenocarcinoma patients whose tumors previously tested positive for EGFR by conventional method (the PNA-LNA PCR clamp method) were collected. All samples were obtained from surgically resected specimen. We used HaloPlex HS method, which is a high sensitivity amplicon-based targeted sequencing method incorporating molecular barcodes in the DNA library, allowing for the identification of duplicate reads to significantly improve the base calling accuracy even at low allelic frequencies compared to conventional NGS methods. We used a panel designed for 47 cancer-related genes including EGFR, and sequenced data was obtained by using Illumina Miseq Reagent v3 (600 Cycle). Normal tissue samples were also sequenced for threshold adjustment.

      Results:
      Out of 58 samples, EGFR ‘major’ mutation (L858R, Exon19 deletion, G719A/S, T790M) profiles of 57 samples by molecular-barcode sequencing corresponded to those by clinical method (98.3%). The mean coverage of EGFR major mutation was 3078 (243-8285), and the minimal detectable frequency was 0.45%. Of note, we could detect the minor frequency of T790M mutation in addition to the major frequency of L858R mutation, at the allele frequency of 1.92% (20/1042) for T790M and 10.62% (155/1459) for L858R mutation, respectively. Minor genetic alterations, except major mutation, in EGFR were detected in 82.8% (48/58) cases, and the mean number was 2.3 (0-11). These results suggest the clinical applicability of this method.

      Conclusion:
      We could demonstrate good concordance rate between clinical examination and molecular barcode sequencing. Minor genetic alterations in EGFR were detected in 82.8% (48/58) cases. Further investigations are warranted to establish the confident detection of subclonal mutations with low frequency.

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    P2.02 - Poster Session with Presenters Present (ID 462)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Locally Advanced NSCLC
    • Presentations: 1
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      P2.02-009 - Clinical Outcomes of Induction Chemoradiotherapy with High Dose Chest Radiation for Locally Advanced Non-Small Cell Lung Cancer Patients (ID 4362)

      14:30 - 15:45  |  Author(s): H. Torigoe

      • Abstract

      Background:
      In the treatment of patients with locally-advanced non-small cell lung cancer (LA-NSCLC), we usually apply chemoradiotheraphy (CRT) consisted of docetaxel and cisplatin with concurrent 40-60 Gy radiation therapy. The radiation dose of 60 Gy is generally planned in the case of definitive CRT. On the other hand, the radiation dose of 46 Gy is planned in the case of induction CRT, considering the safety of surgery. In the induction CRT, if the treatment response is poor and complete resection is supposed to be difficult, additional radiation is performed. In this study, we examined the safety and clinical outcome of lung resection after induction CRT using high-dose radiation in patients with LA-NSCLC.

      Methods:
      One hundred and eighteen patients with LA-NSCLC who underwent induction CRT followed by surgery between March 1999 and December 2014 in our hospital were reviewed. We categorized those patients into low-dose radiation group who received less than 60 Gy of radiation (n=105) and high-dose radiation group who received more than 60 Gy of radiation (n=13). We compared postoperative outcomes between these two groups applying match-paired analysis with using propensity score.

      Results:
      One hundred and eighteen cases consisted of 91 males and 27 females, and the average age was 60 years. Eleven patients had stage IIB disease, 73 patients had stage IIIA disease, and 34 patients had stage IIIB disease before CRT. The background between low-dose group and high-dose group was similar. There were no significant differences in the mortality (0.8% vs 0% in low-dose group and high-dose groups), the incidence of postoperative complication (57% vs 77%), and post-operative hospital days (median 22 vs 28 days) between each group. In addition, there were no significant differences in the 5-year OS rates (73% vs 77% in low-dose group and high-dose groups, p =0.66), and the 5-year DFS rates (56% vs 77%, p =0.11) between each group, even when we applied matched-paier analyses.

      Conclusion:
      This study showed that lung resection after induction CRT using high-dose radiation for LA-NSCLC patients had been performed safely with equivalent prognosis compared with that using low-dose radiation.

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    P3.02b - Poster Session with Presenters Present (ID 494)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Advanced NSCLC
    • Presentations: 1
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      P3.02b-121 - Targeting miR-200c/LIN28B Axis in Acquired EGFR-TKI Resistance Non-Small Cell Lung Cancer Cells Harboring EMT Features (ID 5677)

      14:30 - 15:45  |  Author(s): H. Torigoe

      • Abstract

      Background:
      MicroRNA (miR)-200 family members (miR-200s) are frequently silenced in advanced cancer and have been implicated in the process of epithelial-to-mesenchymal transition (EMT). We previously reported that miR-200s were silenced through promoter methylation in acquired EGFR-tyrosine kinase inhibitor (TKI) resistant non-small cell lung cancer (NSCLC) cells harboring EMT features. In this study, we examined the functional role of miR-200s in NSCLC cells and investigated the novel approach overcoming acquired EGFR-TKI resistance.

      Methods:
      We examined miR-200s expressions and promoter methylation statuses in 34 NSCLC cell lines. Then, we analyzed the altered pathway molecules associated with miR-200c statuses using publicly accessible database. Finally, we examined the antitumor effect targeting the molecules related to miR-200s expression in EGFR-TKI sensitive HCC4006 cells and acquired resistance cell line with EMT features HCC4006-GR cells.

      Results:
      In the analysis of NSCLC cell lines, miR-200s expression silenced cell lines showed each promoter methylation. There were significant correlations between miR-200c silencing and several oncogenic pathway alterations, including EMT-change and LIN28B overexpression, in database analysis. In addition, EGFR-wild type cell lines showed lower miR-200s expressions compared to EGFR-mutant cell lines. Introduction of miR-200c by using pre-miR-200c caused LIN28B suppression in HCC4006-GR cells. Interestingly, both introduction of miR-200c and knockdown of LIN28B showed antitumor effect in HCC4006-GR cells, whereas they were not effective in parental HCC4006.

      Conclusion:
      MiR-200c/LIN28B axis plays an important role in acquired resistance to EGFR-TKI and can be a therapeutic target overcoming drug resistance.