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Masaoki Ito
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FP12 - Tumor Biology and Systems Biology - Basic and Translational Science (ID 188)
- Event: WCLC 2020
- Type: Posters (Featured)
- Track: Tumor Biology and Systems Biology - Basic and Translational Science
- Presentations: 1
- Moderators:
- Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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FP12.03 - SRC-Homology 2 Domain-Containing Phosphatase 2 (SHP2) in Resected Lung Adenocarcinoma (LUAD) (ID 992)
00:00 - 00:00 | Presenting Author(s): Masaoki Ito
- Abstract
Introduction
Epidermal growth factor (EGFR)-mutant lung adenocarcinomas (LUADs) display impaired phosphorylation of extracellular signal-regulated kinase (ERK) and SRC-homology 2 domain-containing phosphatase 2 (SHP2) in comparison with EGFR wild-type LUADs. However, the function of SHP2 in early EGFR-mutant LUADs and EGFR wild-type LUADs has not been reported. We posit that SHP2 mRNA expression could be a predictive marker in resected EGFR-mutant LUADs versus EGFR wild-type patients (pts).
Methods
We examined 267 resected LUADs from Japan and Spain. mRNA expression levels of AXL, MET, CDCP1, STAT3, YAP1 and SHP2 were analyzed by quantitative reverse transcriptase polymerase chain reaction (PCR). EGFR mutant cell lines were investigated for their activity of SHP2.
Results
Among the 267 enrolled pts, 100 (37.3%) were EGFR-mutant LUADs. Five-year recurrence-free survival (RFS) and overall survival (OS) were lower for EGFR-mutant LUADs with high SHP2 mRNA levels (hazard ratio= 1.83 and 2.28, respectively. p= 0.03 and p=0.04). However, SHP2 was not associated with RFS nor OS in the 167 wild-type EGFR LUADs. In EGFR-mutant cells, RMC-4550 (SHP2 inhibitor) plus erlotinib showed synergism via inhibition of AKT (S473) and ERK1/2 (T202/Y204). While erlotinib displaces SHP2 into the nucleus, either RMC-4550 alone, or in combination with erlotinib, restores SHP2 into the cytoplasm membrane, limiting AKT and ERK activation.
Conclusion
High SHP2 mRNA is related to shorter RFS and OS in EGFR-mutant LUADs, but not in EGFR wild-type LUADs. The findings indicate that the addition of SHP2 inhibitors could improve adjuvant therapy in EGFR-mutant LUADs.
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MA09 - Prognosis and Staging (ID 187)
- Event: WCLC 2020
- Type: Mini Oral
- Track: Staging
- Presentations: 1
- Moderators:
- Coordinates: 1/31/2021, 09:15 - 10:15, Scientific Program Auditorium
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MA09.09 - Positive EGFR Mutation Status is a Risk of Recurrence in pN0–1 Lung Adenocarcinoma When Considering pStage and Histological Subtype (ID 836)
09:15 - 10:15 | Presenting Author(s): Masaoki Ito
- Abstract
Introduction
Recurrence risk of resected lung adenocarcinoma is represented by pathological stage (pStage), histological subtype, and potentially by EGFR mutation status. However, the relationship among these factors and their combined impact on prognosis are unclear. Using a multicenter database, we retrospectively investigated the recurrence risk of pN0–1M0 non-variant lung adenocarcinoma based on pStage, histological subtype, and EGFR mutation status.
Methods
Non-variant type adenocarcinoma less or equal to 5 cm in pathological size were involved. The prognostic impacts of pStage, histological subtype, and EGFR mutation status were analyzed in pN0–1M0 adenocarcinoma cases. For the histological malignancy grading, cases were divided into low (lepidic predominant adenocarcinoma), intermediate (papillary or acinar predominant adenocarcinoma), or high malignant subtype (solid or micropapillary predominant adenocarcinoma) according to histological features. Recurrence-free survival (RFS) and overall survival (OS) were used for prognostic evaluation.
Results
A total of 1155 pN0–1M0 non-variant adenocarcinoma cases ≤ 5cm in diameter (pStage 0–IIIA) were analyzed. The median follow-up term was 1080 days (range: 9-2785). In total, 50.6% cases harbored EGFR mutation. The clinicopathological features in which EGFR mutation was likely to be harbored were female (p < 0.001), never–smoker (p < 0.001), low SUV (p < 0.001), low malignant subtype (p < 0.001), no pleural invasion (p < 0.001), no lymphovascular invasion (p = 0.003), and pStage IA1–IB cases (p = 0.002). Regarding to histological subtype, EGFR mutations were likely to be harbored in adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), low or intermediate malignant subtype.
The majority of EGFR-mutant cases was AIS, MIA, and low malignant subtype. However, AIS and MIA showed no recurrence, and low malignant grade subtype and pStage IA1–IB intermediate malignant subtype showed high 5–year RFS regardless of EGFR mutation status (98.5% and 85.3%, respectively). Whereas, in pStage IA1–IB high malignant grade subtypes and pStage IIA1–IIIA cases, positive EGFR mutation cases showed significantly lower 5-year RFS than EGFR-negative cases (49.6% vs 75.6%, respectively; HR = 1.84; 95% CI = 1.38–7.42; p < 0.01). Multivariate analysis indicated positive EGFR mutation status, lymphovascular invasion, intrapulmonary metastasis, and lymph node metastasis were significantly related to recurrence (EGFR mutation: HR = 2.005, 95% CI = 1.029–3.906, p = 0.041).
OS showed similar tendency with RFS in considering 3 status. The significant difference was confirmed only between low malignant subtypes vs IA1–IB/intermediate/EGFR(±) plus IA1–IB/ high/EGFR(-), HR=2.01, 95% CI=2.92–44.3, p < 0.001.
Conclusion
Risk of recurrence changes by pStage and histological subtype in adenocarcinoma. Distribution of EGFR mutation also varies among pStage and histological subtypes. Risk of postoperative relapse should be discussed among cases with recurrent risks.
EGFR mutation status enables better stratification of recurrence risk when considering pStage and histological malignant subtype.
EGFR mutant cases showed worse RFS in pStageIA1–IB/high malignant subtype and pStgae IIA–IIIA. RFS stratified by pStage and histological subtype was more finely stratified by EGFR mutation status compared to classification by single status only.
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P61 - Tumor Biology and Systems Biology - Basic and Translational Science - KRAS (ID 199)
- Event: WCLC 2020
- Type: Posters
- Track: Tumor Biology and Systems Biology - Basic and Translational Science
- Presentations: 1
- Moderators:
- Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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P61.01 - Imipramine Blue (IP) plus MET Tyrosine Kinase Inhibitors (TKI) Suppress Lung Adenocarcinoma (LUAD) KRAS Mutation Tumor Growth (ID 1348)
00:00 - 00:00 | Author(s): Masaoki Ito
- Abstract
Introduction
KRAS mutations in LUAD co-occur with TP53 mutations and LKB1 non-synonymous mutations (nsm), portending a poor prognosis. MET amplification has not been considered. Previously, we identified OTSSP167 as a PAK1 kinase inhibitor with significant activity in A549 (KRASG12-C and LKB1nsm). OTSSP167 plus auranofin (PKCι) shows high synergism and inhibits tumor growth in mice in the H23 cell line (KRASG12-C, p53mut and LKB1nsm) (Ito et al, Cell Comm and Signaling 2019). In addition, OTSSP167 has a potent MELK inhibition effect. We hypothesize that MET expression could be upregulated in KRAS-mutant cell lines, based on the fact that the putative signaling pathway, MELK-forkhead box M1 (FOXM1)-MET, could be present in KRAS mutant cells. In the current study, we examined the combination of MET TKIs with imipramine blue (FOXM1 inhibitor).
Methods
Quantitative real time PCR of MET and FOXM1 was performed in 4 KRAS-mutant cell lines (A549, H23, H460 and Calu6). LKB1 mRNA was assessed in 32 advanced KRAS-mutant LUAD patients. Cell proliferation assays were performed in A549 and H23, and in the EBC1 (MET amplified lung cancer cell line). Synergy was defined by a combination index (CI) of < 0.75 by Chou-Talalay. Cell lines were treated with IP and MET TKIs (crizotinib, savolitinib and tepotinib).
Results
MET mRNA was elevated in A549 and H23 (which both carry LKB1nsm) but not in H460 and Calu6. FOXM1 mRNA was overexpressed in H23. Synergy (CI<0.75) was seen with IP plus tepotinib in the A549 and H23 cell lines, but not in H460 and Calu6. Synergy was also noted with IP plus crizotinib, but not with savolitinib. The CI of IP plus MET TKIs in the EBC1 cell line (which is only MET amplified) was >1. The median overall survival for KRAS-mutant LUAD patients with low LKB1 was 1.1 months versus 19.4 for those with high LKB1 (p=<0.005).
Conclusion
The bona fide of MET TKIs (tepotinib) plus IP in KRAS cell lines with LKB1 nsm, encourages the determination of clinical benefit of tepotinib plus IP in KRAS mutant LUAD patients. The liaison of MET and LKB1 nsm should be further investigated. LKB1 mRNA expression, together with MET and FOXM1 mRNA expression, warrants assessment in KRAS LUAD patients.
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P65 - Tumor Biology and Systems Biology - Basic and Translational Science - NC RNA (ID 204)
- Event: WCLC 2020
- Type: Posters
- Track: Tumor Biology and Systems Biology - Basic and Translational Science
- Presentations: 1
- Moderators:
- Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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P65.04 - Tracking circRNAs in Lung Adenocarcinoma Samples as Promising Biomarkers for Cancer Detection using the NanoString nCounter® (ID 3502)
00:00 - 00:00 | Author(s): Masaoki Ito
- Abstract
Introduction
Lung cancer is positioned as the foremost cause of cancer death at global scale. The lack of reliable biomarkers for early detection seems to be the main cause underlying this high mortality rate; therefore, the discovery of new candidates allowing timely diagnosis of the disease is rather imperative.
Circular RNAs (circRNAs) have strongly emerged as valuable tissue-specific biomarkers of different disorders, including lung cancer. The covalently linked 5´-3´ends provide them not only with a distinctive structure but also keeps them exempted from the exonuclease activity making them very stable. They have been described to be highly expressed in extracellular vesicles (EVs) when compared with mRNA; however, despite of the aforementioned properties, their potential as biomarkers has not been fully explored in lung cancer, yet very far to be implemented in the liquid biopsy settings.
Through this study we demonstrate the feasibility of using the NanoString nCounter® Flex platform for the study of circRNAs in different fresh and formalin-fixed paraffin embedded (FFPE) lung cancer material including EVs, hence paving the way for the development of new diagnosis platforms based on this technology and the different circRNA expression patterns.
Methods
Lung cancer cell lines were cultured under standard laboratory conditions until harvested for RNA extraction. EVs were isolated from the cell culture medium by ultracentrifugation.
FFPE tissue samples were retrospectively collected; samples were macro-dissected and total RNA was extracted.
Patient blood was separated into plasma and cellular fractions by centrifugation. Plasma-derived EVs were isolated using the miRCURY Exosome Serum/Plasma Kit.
RNA from EVs was extracted with TRI-Reagent.
For mRNA depleted samples, a treatment with RNase R was performed.
Gene expression analysis was carried out using the NanoString nCounter® platform with a customized panel harboring 85 circRNA related to the biology of the disease.
Results
First runs showed circRNA expression in lung cancer cell lines. A comparative analysis of total RNA versus mRNA-depleted RNA was performed resulting in an overall circRNA enrichment of the latter. An analogous experiment was performed in FFPE PC9 cells; however, circRNA enrichment was not achieved.
Likewise, FFPE lung cancer tissue samples were analyzed; Consequently, circRNA expression has been evident in all samples analyzed so far.
EV-derived RNA (exRNA) from cell lines was also tested in the nCounter®. Counts corresponding to different transcripts were evident in all samples without previous amplification step. Similar results were found in exRNA from one NSCLC patient. More patient samples are currently being collected to validate these results.
Conclusion
Through this project we have demonstrated the feasibility of using nCounter® for the study of circRNAs in different lung cancer materials. RNase R treatment proved to be beneficial for circRNA enrichment in fresh samples but not FFPE samples, probably due to the mechanical and chemical breakage they may experiment during purification, thus becoming susceptible to RNase R degradation.
CircRNAs were also expressed in EVs; however, more patient samples need to be tested. Pre-amplification steps are advisable in the future to explored the sensitivity of the technology before being implemented into the liquid biopsy settings.
