Epithelial-Specific Methylation Marker: A Potential Plasma Biomarker in Advanced Non- small Cell Lung Cancer Chanida Vinayanuwattikun, MD, Virote Sriuranpong,

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Epithelial-Specific Methylation Marker: A Potential Plasma Biomarker in Advanced Non- small Cell Lung Cancer Chanida Vinayanuwattikun, MD, Virote Sriuranpong, MD, PhD, Suebpong Tanasanvimon, MD, Poonchavist Chantranuwat, MD, Apiwat Mutirangura, MD, PhD Journal of Thoracic Oncology Volume 6, Issue 11, Pages 1818-1825 (November 2011) DOI: 10.1097/JTO.0b013e318226b46f Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1. Comparisons of the absolute quantitative analysis of methylated alleles (AQAMA) and the conventional methylation-specific primer-based polymerase chain reaction (MSP-PCR). The results of AQAMA-PCR (solid line, filled triangle) indicated a near-perfect correlation with the known concentrations of the mixed samples compared with the semiquantitative measurement of MSP-PCR (solid line, filled square). Journal of Thoracic Oncology 2011 6, 1818-1825DOI: (10.1097/JTO.0b013e318226b46f) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3. Levels of plasma SHP-1 promoter 2 (SHP1P2) methylation were used to predict the survival rates of patients with advanced non-small cell lung cancer (NSCLC). The results of the Kaplan-Meier survival curve analysis for progression-free survival (PFS) (panel A) and overall survival (OS) (panel B) in patients with advanced NSCLC indicated that patients with low levels plasma of SHP1P2 methylation (<700 pg ml−1 [solid line, filled circle]) had improved PFS and OS rates compared with those with high levels of plasma SHP1P2 methylation (≥700 pg ml−1 [solid line, filled triangle]). Journal of Thoracic Oncology 2011 6, 1818-1825DOI: (10.1097/JTO.0b013e318226b46f) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2. The levels of SHP-1 promoter 2 (SHP1P2) methylation demonstrated enhanced diagnostic capability in patients with advanced non-small cell lung cancer (NSCLC) compared with the levels of total circulating DNA. Pretreatment plasma samples from 52 healthy controls and 38 patients with advanced NSCLC were collected. Both circulating DNA (panel A) and SHP1P2 methylation (panel B) in patients with advanced NSCLC were significantly higher than those of healthy controls. The median circulating DNA levels were 4512 pg ml−1(1339–49,888 pg ml−1) in patients with advanced NSCLC and 2077 pg ml−1 (34–26,955 pg ml−1) in healthy controls (p = 0.002). Nevertheless, levels of SHP1P2 methylation in controls were mostly undetectable, in contrast to the higher and broader range exhibited by patients with advanced NSCLC, with a median value of 770 pg ml−1 (0–26,500 pg ml−1) (p < 0.001). Journal of Thoracic Oncology 2011 6, 1818-1825DOI: (10.1097/JTO.0b013e318226b46f) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 4. Plasma levels of SHP1P2 methylation were analyzed in 20 patients with resectable stages I–III non-small cell lung cancer (NSCLC). The total quantity of circulating DNA (panel A) exhibited no significant difference between patients with early NSCLC and the healthy controls, with median levels of 1885 pg ml−1 (376–17,542 pg ml−1) and 2077 pg ml−1 (34–26,955 pg ml−1), respectively (p = 0.36). In contrast, the levels of SHP1P2 methylation (panel B) were higher and broader in patients with early stages of NSCLC compared with those of the healthy controls, with median values of 1249 pg ml−1 (358–10,488 pg ml−1) and undetectable, respectively (p < 0.001). Journal of Thoracic Oncology 2011 6, 1818-1825DOI: (10.1097/JTO.0b013e318226b46f) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions