Non-invasive diagnosis of early-stage lung cancer using high-throughput targeted DNA methylation sequencing of circulating tumor DNA (ctDNA)

Abstract

Rational: LDCT screening can identify early-stage lung cancers yet introduces excessive false positives and it remains a great challenge to differentiate malignant tumors from benign solitary pulmonary nodules, which calls for better non-invasive diagnostic tools. Methods: We performed DNA methylation profiling by high throughput DNA bisulfite sequencing in tissue samples (nodule size < 3 cm in diameter) to learn methylation patterns that differentiate cancerous tumors from benign lesions. Then we filtered out methylation patterns exhibiting high background in circulating tumor DNA (ctDNA) and built an assay for plasma sample classification. Results: We first performed methylation profiling of 230 tissue samples to learn cancer-specific methylation patterns which achieved a sensitivity of 92.7% (88.3% - 97.1%) and a specificity of 92.8% (89.3% - 96.3%). These tissue-derived DNA methylation markers were further filtered using a training set of 66 plasma samples and 9 markers were selected to build a diagnostic prediction model. From an independent validation set of additional 66 plasma samples, this model obtained a sensitivity of 79.5% (63.5% - 90.7%) and a specificity of 85.2% (66.3% - 95.8%) for differentiating patients with malignant tumor (n = 39) from patients with benign lesions (n = 27). Additionally, when tested on gender and age matched asymptomatic normal individuals (n = 118), our model achieved a specificity of 93.2% (89.0% - 98.3%). Specifically, our assay is highly sensitive towards early‐stage lung cancer, with a sensitivity of 75.0% (55.0%-90.0%) in 20 stage Ia lung cancer patients and 85.7% (57.1%-100.0%) in 7 stage Ib lung cancer patients. Conclusions: We have developed a novel sensitive blood based non‐invasive diagnostic assay for detecting early stage lung cancer as well as differentiating lung cancers from benign pulmonary nodules.

Integrated epigenetic biomarkers in circulating cell-free DNA as a robust classifier for pancreatic cancer

“…Liquid biopsies, a well-known noninvasive method, have aroused public attention as diagnostic materials for cancer, particularly circulating tumor DNA (ctDNA) in plasma. Taking advantage of technical advances, both genetic and epigenetic aberrations of cell-free DNA (cfDNA) can be detected [2] and have shown promising performance in clinical practice, including diagnosis [3][4][5][6][7][8], prognosis [9][10][11][12], and drug resistance [13,14].…”

Circulating Tumor DNA Predicts the Response and Prognosis in Patients With Early Breast Cancer Receiving Neoadjuvant Chemotherapy

“…Advances in next-generation sequencing (NGS) 18 technology have enabled the rapid identification and broad coverage of tumor-specific genomic alterations in cell-free DNA (cfDNA) of individual patients. 19 - 21 The NGS assay provides an opportunity to screen more genes at a single time point and avoid missing mutations absent in biopsy because of intratumor heterogenicity. Here, using a large NGS panel that covers 1,021 cancer-related genes, we compared the change of ctDNA and imaging during NAC to determine whether ctDNA is superior to imaging in predicting tumor response during NAC and prognosis in patients with early breast cancer.…”

Ultrasensitive DNA hypermethylation detection using plasma for early detection of NSCLC: a study in Chinese patients with very small nodules

“…On the other hand, in the subgroup with tumor size 0-1 cm, the gene panel CDO1, TAC1, and SOX17 (sensitivity 71%, specificity 82%, and AUC 0.81, respectively) was slightly better than gene panel CDO1, SOX17, and HOXA7 (64%, 82%, and 0.75, respectively), indicating this panel may have better sensitivity and diagnostic accuracy in the detection of very small lung cancer lesions (Tables 5, 6 and 7). While the sensitivity for tumors < 1 cm is slightly lower than larger tumors, previous studies have not been able to detect this tumors even this well, and in most cases did not attempt to detect such earlystage lung cancer [29,35,36].…”

“…Capitalizing on the strengths of highly prevalent DNA methylation biomarkers and ultra-sensitive techniques to detect DNA methylation could facilitate early diagnosis of lung cancer with indeterminate screen-detected pulmonary nodules [27,28]. Recently, Liang and colleagues reported the high diagnostic accuracy by using highthroughput DNA bisulfite sequencing in tissue and plasma samples from patients with lung cancer [29]. Other studies have also sought to differentiate lung cancers from benign lung nodules by investigating ctDNA markers [30][31][32].…”