Clinical application of molecular barcode sequencing for detection of low frequency variants in circulating tumor DNA of hepatocellular carcinoma

Abstract

간암은 한국인에게 높은 발병률을 나타내는 암 종으로서, 발병률과 사망률이 꾸준히 감소해오고 있음에도 불구하고, 여전히 여섯 번째로 많이 검진되는 암이며, 암으로 인한 사망률 중 네번째로 치명적이다. 특히 간세포암종의 경우, 50대와 60 대 남성에서 가장 많이 발생하며, 국내 간암의 주요원인은 B형과 C형 간염 바이러스가 대표적이다. 간암을 진단하기 위해서는 직접 암 조직을 획득하여 검사해야하는 조직학적 진단인 조직생체검사가 필수적이나, 바늘이나 내시경 등을 이용해 인체에 침습적으로 시행되어 환자에게 불안감을 초래할 수 있으며 반복적으로 조직 생검이 필요한 경우 부작용 및 환자나 의사 모두에게 부담스럽다는 단점이 있다. 또한 조직 생검을 통하여 얻은 종양 샘플의 경우, 종양의 비균질성으로 인하여 전체 암 조직의 특성을 파악하기가 힘들다. 이에 비해 혈액이나 복수 등 인체 내 체액을 이용한 액체 생검은 체액 내에 떠다니는 세포유리 DNA를 이용하여, 보다 덜 침습적이고 전반적인 암 조직의 특성을 잘 발견할 수 있다는 장점이 있으나, 체내에서의 반감기가 짧아서 세포유리 DNA의 양이 제한적이다. 이를 극복하기 위하여, 본 연구에서는 차세대 염기서열방식 (NGS)을 이용하여 간세포암종의 세포유리 DNA를 이용한 간세포암종의 체세포 돌연변이를 검출하고자 하였으며, 적은양의 세포유리 DNA로 낮은 대립유전자 빈도의 변이를 검출하기 위해 분자바코드가 포함되어 있는 어뎁터를 라이브러리에 부착하여 시퀀싱을 실시하였다. 총 149명의 환자의 혈장에서 세포유리 DNA를 추출하여 분자바코드를 이용한 NGS를 진행하였으며, 최소0.5%의 대립유전자 빈도를 가진 변이를 검출하였다. 또한 혈장에서 분리한 세포유리 DNA와 생체 종양 조직 DNA를 비교하여, 세포유리 DNA와 종양 조직 DNA의 동일한 변이를 검출할 수 있었다. 이를 근거로 하여, 두 검사결과를 비교하여 더욱 확실한 진단 결과를 담보할 수 있을 뿐만 아니라 액체 생검이 조직 생검의 대체적인 방법으로 제시될 수 있음을 시사한다. Liver cancer is predicted to be the sixth most commonly diagnosed cancer and the fourth leading cause of cancer-related death worldwide in 2018 and hepatocellular carcinoma accounts for seventy-five to eighty-five percent of total liver cancer cases. Similar to other solid tumor cancer, diagnosis of hepatocellular carcinoma is also required a tissue biopsy from a patient to determine malignancy of tumors. The tissue biopsy is, however, an invasive procedure in patients and cannot depict various genetic characteristics of the tumors as it collects a sample from a limited region in the tumors. To decrease the risk in patients and to overcome challenges associated with tumor heterogeneity, liquid biopsy, a less invasive procedure than the tissue biopsy, has been widely studied and applied in both research and diagnostic levels. Among various biomarkers of liquid biopsy, circulating tumor DNA, cell free DNA from tumors, is utilized as both diagnostic and prognostic biomarkers for cancer because it represents current status of tumors in molecular level as it is originated from cancer tumors. Comparing to isolation of other liquid biopsy biomarkers such as cell free RNA and exosome from blood, extraction protocols of cell free DNA are more straightforward and its concentrations in blood is higher than that of other liquid biopsy biomarkers. Next-generation sequencing has arisen in the past decade as an proficient technique for sequencing genetic materials and has been rapidly adopted in molecular diagnostic laboratories to detect genetic mutations in various diseases including cancers and rare diseases. Targeted sequencing, a type of next-generation sequencing, allows to detect variants with limit of detection as low as five percent of allele frequency under depth of coverage of five-hundred. To detect lower variant allelic frequency of variants from circulating cell free DNA by using next-generation sequencing, but still, increasing amount of genetic materials and greater depth of coverage are required. Unique molecular barcode sequencing has been developed to overcome the lower compatibility of input amount, higher error rate and less cost-effectiveness to detect rare and low allelic frequency variants in genetic materials. Total of one-hundred forty-nine patients were enrolled in this study and whole blood samples were collected in cell free DNA tubes for collection of plasma samples and twenty-seven of the total tissue samples were collected during surgery or tissue biopsy. Circulating tumor DNA from the plasma samples were extracted, libraries were constructed with unique molecular barcode embedded adapters and captured and, targeted sequencing was performed to detect somatic variants in the circulating tumor DNA. The limit of detection of molecular barcode sequencing was as low as 0.5 percent of allelic frequency and the molecular barcode sequencing of circulating tumor DNA demonstrated matched somatic variants in both plasma and tumor samples. It suggests that liquid biopsy using circulating tumor DNA could be an alternative method of tissue biopsy and could enable a study of low allelic frequency variants or rare variants in plasma from malignant diseases using molecular barcode sequencing.