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E.H.R. Olivieri



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    MA 01 - SCLC: Research Perspectives (ID 650)

    • Event: WCLC 2017
    • Type: Mini Oral
    • Track: SCLC/Neuroendocrine Tumors
    • Presentations: 1
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      MA 01.01 - Metastatic Behavior of Pulmonary Neuroendocrine Carcinomas Is Associated with Epithelial to Mesenchymal Transition Gene Profile (ID 9362)

      11:00 - 12:30  |  Author(s): E.H.R. Olivieri

      • Abstract
      • Presentation
      • Slides

      Background:
      The new 2015 WHO classification broadly divided pulmonary neuroendocrine tumor (NET) of the lung in low-grade typical carcinoid (TC) and atypical carcinoid (AC), to the high-grade large-cell neuroendocrine carcinoma (LCNEC) and the small-cell carcinoma (SCLC). The molecular alterations underlying the pathogenesis of these tumors have been studied showing two blocks of entities with independent cellular mechanisms. Many of the differences between the two NETs blocks can be ascribed to tobacco consumption, which induces epithelial to mesenchymal transition activation, responsible for invasive and metastatic behavior. These correlations further highlight the difference in OS for patients with low and high grade metastatic NETs. Therefore, epithelial to mesenchymal transition (EMT) genes profile emerge promise as indicator of invasion and metastasis in NETs.

      Method:
      Fresh frozen tissue from SCLC (n = 10), LCNEC (n = 4), AC (n = 5), TC (n = 5) and matched normal tissue samples were collected for qRT-PCR analysis carried out on StepOnePlus™ Real-Time PCR System (Applied Biosystems) with RT[2] Profiler PCR Array System for the EMT pathway with 84 target genes (Qiagen, Dusseldorf, Germany). Linear regression was done to evaluate association between gene expressions. Clinical variable such as age, gender, tobacco history, lymph node metastasis and histologic types were associated with gene expression. Differences were regarded as statistically significant at P < 0.05.

      Result:
      High expression of membrane receptor EGFR (p = 0.003), protein of the matrix metalloproteinase MMP3 (p = 0.044), transcriptional factor TCF3 (p = 0.022) and signaling pathway factor WNT5A (p = 0.013) were observed in patients with tobacco history. Metastatic LCNEC and SCLC presented significant lower expression of JAG1 gene and higher level of EGFR (p<0.01), transmembrane protein DSP (p = 0.03), TCF3 (p = 0.01), TGF-B3 (p = 0.04) and WNT5A (p = 0.01) compared to TC and AC. In addition to these genes, AKT1 and MAP1B were equally high expressed in metastatic NE carcinomas. Importantly, increased expression of these genes added of MMP2 gene was significantly associated with poor OS of the patients.

      Conclusion:
      A panel of 84 EMT genes was tested and the best biomarkers included EGFR, MMP2, MMP3, TCF3, WNT5A, JAG1, TGFB3, AKT1 and MAP1B with impact on unfavorable prognostic and overall survival of patients, highlight that EMT play a fundamental role in pathogenetic pathway of metastasis in NETs. Supported by CNPq project 301411/2016-6; FAPESP 2013/10113-7.

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    OA 07 - Biomarker for Lung Cancer (ID 659)

    • Event: WCLC 2017
    • Type: Oral
    • Track: Biology/Pathology
    • Presentations: 1
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      OA 07.08 - Clinical Potential of Sputum in Detecting Driver Mutations in Patients with Non-Small Cell Lung Cancer: A Preliminary Study (ID 7540)

      15:45 - 17:30  |  Author(s): E.H.R. Olivieri

      • Abstract
      • Presentation
      • Slides

      Background:
      The incidence of lung cancer has significantly increased over the last century and remains the most common cause of cancer deaths worldwide. Our better understanding of the tumor microenvironment and the systemic actions of tumors, combined with the recent advent of the liquid biopsy, may allow molecular diagnostics to be done with non-invasive method for detection and monitoring of patient tumors. Sputum has been the target for the discovery of non-invasive biomarkers for lung cancer because it contains airway epithelial cells, and molecular alterations identified in sputum are most likely to reflect tumor-associated changes. Since January of 2017, sputum samples have been prospectively collected at the time of diagnosis for future evaluation of actionable mutations in EGFR, KRAS, BRAF and NRAS in patients with non-small cell lung cancer in our center. Currently, from 20 sputum samples already collected, 5 are confirmed for driver mutations (one in KRAS and 4 in EGFR) in tissue biopsy, with 2 of the samples being positive for T790M in circulating tumor DNA (ctDNA) isolated from plasma. Our aim is to evaluate whether sputum may be representative in the detection of these mutations.

      Method:
      DNA was extracted from sputum using QIAamp DNA midi kit (Qiagen). Tumor somatic mutations were investigated by target-sequencing using a custom Ion Ampliseq™ Panel (ThermoFisher Scientific), containing hotspot regions of 14 genes frequently mutated in solid tumors (including EGFR). Multiplex amplification was performed with 10 ng of DNA using Multiplex PCR Master Mix (Qiagen) and high-throughput sequencing was performed using Ion Proton platform. Somatic mutations were considered if the variant allele was present in more than 0.5% of the reads, considering a minimum coverage depth of 20,000X. A medium coverage of 172,524X was obtained in the five samples.

      Result:
      We detected mutations in 3 out of 5 sputum samples of patients with previously known driver mutations (two exon 19 deletions and one exon 18 G719A in EGFR). The highest frequency was detected in the only patient with spontaneous sputum collection (23%).The other two mutations were detected in low frequencies (0.5 and 0.6%) in samples derived from sputum induction. We found T790M in one patient positive for T790M in ctDNA isolated from plasma.

      Conclusion:
      These preliminary findings indicate that driver mutations can be identified in sputum routinely obtained from sputum samples. Thus, the ability to examine sputum might provide a convenient source of sampling and may be adapted for future large-scale screening.

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    P3.15 - SCLC/Neuroendocrine Tumors (ID 731)

    • Event: WCLC 2017
    • Type: Poster Session with Presenters Present
    • Track: SCLC/Neuroendocrine Tumors
    • Presentations: 1
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      P3.15-006 - Comprehensive Analysis of EMT Gene Signature in Primary and Metastatic Small Cell and Non-Small Cell Carcinomas of the Lung (ID 8182)

      09:30 - 16:00  |  Author(s): E.H.R. Olivieri

      • Abstract
      • Slides

      Background:
      Metastasis are responsible for the death of 90% of patients with lung cancer (LC) indicating the necessity to know the multiple signaling pathways involved. Among them, high-grade neuroendocrine lung carcinomas (NELC) invade and metastasize rapidly. Therefore, biomarkers of aggressiveness in LC remain to be determined, especially in NELC. Epithelial to mesenchymal transition (EMT) genes profile emerge promise as indicator of invasion and metastasis. The aim was to investigate the expression of EMT markers and assessed their relationship with the clinicopathological features and prognosis.

      Method:
      Fresh frozen tissue from SCLC (n=15) and NSCLC (ADc n=23 and SqCC n=10) and matched normal tissue samples were collected for qRT-PCR analysis carried out on StepOnePlus™ Real-Time PCR with RT[2] Profiler PCR Array System for the EMT pathway with 84 target genes (Qiagen, Dusseldorf, Germany). Gene expression was correlated with clinicopathological variables in the SCLC and NSCLC groups. Survival curves were calculated using the Kaplan-Meier method and risk factors determined by multivariate Cox regression model. Differences were regarded as statistically significant at P<0.05.

      Result:
      Female patients presented significant higher expression of EGFR (p=0.03), ILK (p=0.05), JAG1 (p=0.01), MMP2 (p=0.04) and SNAI2 (p=0.04) genes. Tobacco history was associated with increased expression of EGFR (p<0.01), ITGAV (p=0.05), SPP1 (p<0.01) and WNT5A (p=0.02). NSCLC presented similar levels of EMT genes evaluated. Tumors from SCLC and NSCLC in advanced N and M stage expressed significant high levels of genes related to cellular membrane [EGFR (p=0.03), ILK (p<0.01), FR11 (p=0.05), ITGAV (p=0.02), ITGB1 (p<0.01), DSP (p=0.04)], extracellular matrix [COL5A2 (p=0.04), COL1A2 (p=0.04)], cytoplasm [GSK3B (p=0.01), VPS13A (p=0.02), MAP1B (p=0.01)] and nucleus SNAI2 (p=0.04). Interestingly, SCLC tumors expressed higher levels of FR11 (p=0.02), GSK3B (p=0.04), ILK (p<0.01), ITGB1 (p=0.01), JAG1 (p<0.01) and MAP1B (p=0.01) indicating more aggressiveness than NSCLC. A mathematical model controlled for N and M stage, histologic type and the gene expression showed that patients with SCLC expressing high levels of MMP2 and SPARC presented significant high risk of death (OR 5.41 and 4.94, respectively) compared to those with lower expression. Patients with NSCLC with low levels of ILK, SPP1, COL1A2, ITGB1 presented a low risk of death (OR -7.02, -0.4, -1.3 and -3.02, respectively).

      Conclusion:
      Different expression of EMT genes in SCLC and NSCLC, its relationship with histologic types, advanced stage, lymph node metastasis and death suggest a possible role of these markers in their malignancy, but more importantly provide a potential biomolecular marker to predict outcome.

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