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YUICHI Saito



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    P02 - Diagnostics and Interventional Pulmonology (ID 110)

    • Event: WCLC 2020
    • Type: Posters
    • Track: Diagnostics and Interventional Pulmonology
    • Presentations: 1
    • Moderators:
    • Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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      P02.06 - A Novel Loop-Mediated Isothermal Amplification Method for Robust Detection of EGFR Mutations (ID 1137)

      00:00 - 00:00  |  Presenting Author(s): YUICHI Saito

      • Abstract
      • Slides

      Introduction

      Detection of EGFR mutation has been widely used for patients with pulmonary adenocarcinoma. Loop-mediated isothermal amplification (LAMP) has been used for the detection of bacteria, which amplifies DNA with high specificity and efficiency under isothermal conditions. Here we evaluated the usefulness of LAMP for detecting EGFR exon 19 deletion and L858Rmutations using cancer tissues in comparison to conventional PCR technique.

      Methods

      All tumor tissues were surgically resected from patients with pulmonary adenocarcinoma at the Saitama Cardiovascular and Respiratory Center between January 2019 and October 2019, and EGFR status of them were investigated by Therascreen EGFR PCR Kit as a conventional PCR. Among of them, only adenocarcinomas with EGFR exon 19 deletion, L858R mutations and EGFR wild type were enrolled into this study for comparison with EGFR status by LAMP method. A primer set for LAMP amplification of the partial sequence of the EGFR gene (NG_007726) was designed using Primer Explorer (primerexplorer.jp/e/). The primers were synthesized and purified by Eurofin Genomics. Block oligo and fluorophore‑labelled probes were synthesized and purified by Japan Bio Services Co., Ltd., or Gene Design, Inc. LAMP assays were performed using the 25 reaction volume with 30 mM KCl, 14 mM Tricine‑NaOH (pH 8.8), 0.5% Tween‑20, 1 mM DTT, 1.7 mM each dNTPs, 8.2 mM MgSO4, 1.6 µM each of the forward and backward inner primer, 0.8 µM each of the forward and backward loop primer, 0.2 µM forward and backward outer primer, 1.2 µM block oligo, 0.04 µM fluorophore‑labelled probe, and 12.3 units Bst-LF DNA polymerase. KCl, Trincine NaOH, Tween‑20, DTT, and MgSO4 were obtained from FUJIFILM Wako Pure Chemical Corporation (http://ffwk.fujifilm.co.jp /en/index.html). dNTPs were obtained from NIPPON GENE CO., Ltd. (https://www.nippongene.com/english/index.html). Bst-LF DNA polymerase was obtained from New England Biolabs Inc. (https://international.neb.com). The LAMP reaction was run for 120 min at 65˚C using a LightCycler 480 ® (Roche Diagnostics K.K.). Melting curve analysis was used to detect LAMP products. The LAMP amplicons were denatured at 95˚C for 5 min, followed by hybridization at 37˚C for 5 min. Subsequently, the temperature was gradually raised to 80˚C and the fluorescent intensity was measured 7 times per 1˚C increment. The obtained data were analyzed by the LightCycler 480 ® software (version 1.5.1.62; https://lifescience.roche.com/global_en/prod‑ucts/lightcycler14301‑480‑software‑version‑15.html) to calculate melting peak.

      Results

      Total 96 tumor samples (including EGFR wild type tumors) were analyzed by both Therascreen and LAMP assays. There were 20 tumors with exon 19 deletion, and 14 tumors with L858R EGFR mutations in Therascreen assay. LAMP method was performed for calculating LAMP value of all tumors. After standardization, we made each ROC curve in exon 19 deletion and L858R mutations. Sensitivity, specificity, and accuracy of LAMP method demonstrated 95.0%, 98.7%, and 97.9% in exon 19 deletion, on the other hand, 100%, 96.3%, and 96.8% in L858R mutations, respectively.

      Conclusion

      The sensitivity, specificity, and accuracy of detecting EGFR mutations in LAMP method were nearly equivalent to that in Therascreen method. This method may be a valuable alternative for the identification of EGFR mutations in pulmonary adenocarcinoma.

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    P04 - Early Stage/Localized Disease - Perioperative Therapy (Neoadjuvant Therapy, Surgery, Adjuvant Therapy) (ID 113)

    • Event: WCLC 2020
    • Type: Posters
    • Track: Early Stage/Localized Disease
    • Presentations: 1
    • Moderators:
    • Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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      P04.08 - Dynamics of Coagulation Factor XIII Activity After Thoracoscopic Lobectomy for Early-Stage Lung Cancer Patients (ID 2082)

      00:00 - 00:00  |  Author(s): YUICHI Saito

      • Abstract
      • Slides

      Introduction

      General thoracic surgeons generally tended to consider that VATS lobectomy must be less invasive than open lobectomy. However, these benefits are not as statistically robust in prospective randomized trial. In order to discuss the difference of surgical stress between VATS lobectomy and open lobectomy, we focused on the surgical trauma area of chest wall, as it is presumed that surgical stress would be correlated with the surgical trauma area of chest wall. From this viewpoint, we considered the invasiveness could be measured by the factors of wound-healing system. Coagulation factor XIII (FXIII) is not only a key factor that functions at the end of the coagulation cascade but also plays an important role in wound healing, making cross-linking fibrin monomers into stable polymers and counteracting fibrinolytic degradation. The purpose of the present study was to investigate the perioperative dynamics of Coagulation Factor XIII (FXIII) in non-small cell lung cancer patients undergoing Video-assisted thoracoscopic (VATS) lobectomy compared to open lobectomy and to investigate how it correlates with some other coagulation parameters.

      Methods

      Perioperative coagulation factors including FXIII were analyzed in 30 patients who underwent VATS lobectomy and 10 patients who underwent open lobectomy at Teikyo University Hospital between December 2017 and April 2019. All 40 patients were preoperatively diagnosed as NSCLC without any nodal metastasis by the institutional tumor-board. In our institution, all cases diagnosed as clinical N0 lung cancer were candidates for VATS lobectomy. However, when patients strongly desired to receive open lobectomy instead of VATS lobectomy, we have chosen open lobectomy preoperatively.

      Results

      VATS lobectomy group showed higher FXIII activity on POD5, compared to open lobectomy group (p=0.028). FXIII activity significantly reduced on POD3, POD5 and POD7 even in VATS lobectomy patients, compared to preoperative control and POD1 (p<0.001). No factors were discovered as the parameter which affects the maintenance of FXIII in VATS lobectomy group.

      Conclusion

      We found the difference of FXIII activity between VATS lobectomy patients and open lobectomy patients. From the characteristics of FXIII, FXIII activity may be a good marker for the invasiveness between VATS lobectomy and open lobectomy.

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