Clinical Validity of Next-Generation Sequencing Multi-Gene Panel Testing for Detecting Pathogenic Variants in Patients With Hereditary Breast-Ovarian Cancer Syndrome
2020; 40(2): 148-154
Ann Lab Med 2021; 41(2): 198-206
Published online March 1, 2021 https://doi.org/10.3343/alm.2021.41.2.198
Copyright © Korean Society for Laboratory Medicine.
Hyojin Chae, M.D., Ph.D.1,2,* , Pil Soo Sung, M.D., Ph.D.3,* , Hayoung Choi, M.S.2 , Ahlm Kwon, M.S.2 , Dain Kang, B.S.E.2 , Yonggoo Kim, M.D., Ph.D.1,2 , Myungshin Kim, M.D., Ph.D.1,2,* , and Seung Kew Yoon, M.D., Ph.D.3,*
1Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea; 2Catholic Genetic Laboratory Center, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea; 3Department of Internal Medicine, Seoul St. Mary’s Hospital, The Catholic University Liver Research Center, College of Medicine, The Catholic University of Korea, Seoul, Korea
Correspondence to: Myungshin Kim, M.D., Ph.D.,
Department of Laboratory Medicine,
Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 06591, Korea
Seung Kew Yoon, M.D., Ph.D.,
Division of Hepatology, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 06591, Korea
* These authors contributed equally to this work.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Hepatocellular carcinoma (HCC) is the second-most-common cause of cancer-related deaths worldwide, and an accurate and non-invasive biomarker for the early detection and monitoring of HCC is required. We assessed pathogenic variants of HCC driver genes in cell-free DNA (cfDNA) from HCC patients who had not undergone systemic therapy.
Methods: Plasma cfDNA was collected from 20 HCC patients, and deep sequencing was performed using a customized cfDNA next-generation sequencing panel, targeting the major HCC driver genes (TP53, CTNNB1, TERT) that incorporates molecular barcoding.
Results: In 13/20 (65%) patients, we identified at least one pathogenic variant of two major HCC driver genes (TP53 and CTNNB1), including 16 variants of TP53 and nine variants of CTNNB1. The TP53 and CTNNB1 variants showed low allele frequencies, with median values of 0.17% (range: 0.06%–6.99%) and 0.07% (range: 0.05%–0.96%), respectively. However, the molecular coverage of variants was sufficient, with median values of 5,543 (range: 2,317–9,088) and 7,568 (range: 2,400–9,633) for TP53 and CTNNB1 variants, respectively.
Conclusions: Our targeted DNA sequencing successfully identified low-frequency pathogenic variants in the cfDNA from HCC patients by achieving high coverage of unique molecular families. Our results support the utility of cfDNA analysis to identify somatic gene variants in HCC patients.
Keywords: Hepatocellular carcinoma, Cell-free DNA, Next-generation sequencing, Molecular barcoding, Pathogenic variants, TP53, CTNNB1, TERT
Hepatocellular carcinoma (HCC) is the second-most-common cause of cancer-related deaths worldwide . In Korea, the mortality rate of HCC was 21.5 per 100,000 population in 2016, and HCC ranked as the second-leading cause of cancer-related deaths . The major risk factors of HCC include chronic hepatitis B virus (HBV) and hepatitis C virus infection, alcohol abuse, liver cirrhosis, and exposure to aflatoxin B1 . Alpha-fetoprotein (AFP) is the most widely used biomarker for early detection of HCC. However, AFP has poor reliability and low sensitivity for early-stage HCC detection . The lack of an early diagnostic marker for HCC has posed a major challenge to curative treatments, including liver resection and liver transplantation. In addition, early detection of recurrence during monitoring after curative therapy is associated with improved survival in HCC patients . Thus, for early detection of HCC as well as for recurrence monitoring after curative surgical resection, an accurate and non-invasive HCC biomarker is required .
Circulating cell-free DNA (cfDNA) has potential as a noninvasive biomarker for detecting and monitoring tumor cells. In addition, cfDNA has prognostic value and may be useful in strategies to select patients eligible for targeted therapy [7, 8]. Several studies have demonstrated significantly higher cfDNA levels in sera and plasma of patients with both early and advanced HCC [9, 10]. Recent studies have explored HCC-related genomic alterations and have identified frequent gene variants, including those in the
In this study, we aimed to assess pathogenic variants of HCC driver genes in cfDNA from advanced HCC patients who had not undergone systemic therapy. We used a customized targeted NGS panel that incorporates unique molecular identifiers (UMIs) to reduce PCR-based NGS errors and to distinguish reads amplified from the same original DNA molecule (on the basis of identical UMIs).
Twenty patients, including 17 men and 3 women with a median age of 60 years (range: 47–79 years), admitted at Seoul St. Mary’s Hospital, Seoul, Korea, between June 2018 and May 2019 were enrolled in this study. All patients were diagnosed as having HCC according to the guidelines from the American Association for the Study of Liver Diseases and the European Association for the Study of the Liver . Cirrhosis was present in all 20 (100%) patients, with viral hepatitis B being the main etiology for the underlying liver disease in 15/20 (75%) patients. Most patients had multiple nodules (17/20, 85%), and macrovascular invasion and metastasis were present in 9/20 (45%) and 14/20 (70%) patients, respectively (Table 1). All participants, including three healthy adults recruited as healthy controls, provided written informed consent. This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board/Ethics Committee of Seoul St. Mary’s Hospital (IRB No. K18TESI0295).
Peripheral blood samples (10 mL) were drawn in ethylenediaminetetraacetic acid (EDTA)-containing tubes, and plasma was separated within one hr of collection in two centrifugation steps: 2,000 × g at 4°C for 10 minutes, followed by 16,000 × g at 4°C for 10 minutes . Plasma samples were immediately aliquoted and stored at –80°C for up to nine months.
Circulating cfDNA was isolated from 4 mL of plasma using the MagMAX Cell-Free DNA Isolation Kit (Applied Biosystems, Waltham, MA, USA) and the KingFisher Duo Prime Magnetic Particle Processor (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The size of the purified plasma DNA was estimated using a 2,100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA, USA), and its concentration was determined using a Qubit fluorometer (Thermo Fisher Scientific) in combination with a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Seraseq ctDNA Reference Material v.2 (SeraCare Life Sciences, Milford, MA, USA) was used to validate the limit of detection. The reference material consisted of 40 cancer-relevant somatic variants spiked into a background of wild-type DNA (purified from a reference cell line, GM24385) at defined variant allele frequencies (VAFs) of 2%, 1%, 0.5%, 0.25%, 0.125%, and 0% . Seraseq ctDNA was extracted and analyzed in duplicate. Methods used for library preparation and sequencing were the same as those used for the participant samples.
We designed a custom 88-amplicon panel (mean read length: 107 bp) targeting three HCC driver genes, namely
We used the optimal amount of input cfDNA (20 ng in 8.3 µL) recommended by the manufacturer to generate libraries using an Ion AmpliSeq HD library kit (Thermo Fisher Scientific) and the Custom Ion AmpliSeq HD panel. Library quantification was performed using the TapeStation 2200 High Sensitivity D1000 Kit (Agilent Technologies). Clonal amplification of the libraries was performed by emulsion PCR on an Ion Chef System using an Ion 540 Kit-Chef (Thermo Fisher Scientific). Template-positive ion sphere particles were enriched, loaded on an Ion 540 Chip, and sequenced using an Ion S5 XL Sequencer (Thermo Fisher Scientific), according to the manufacturer’s instructions.
Sequence data were processed for primary and secondary analyses, using standard Ion Torrent Suite Software (Thermo Fisher Scientific) running on the Torrent Server (Thermo Fisher Scientific). Raw signal data were analyzed using Torrent Suite v. 5.10.1 (Thermo Fisher Scientific) and Ion Reporter (Thermo Fisher Scientific). The pipeline included signal processing, base calling, quality score assignment, adapter trimming, PCR duplicate removal, read alignment, mapping quality control, coverage analysis, and variant calling. The sequenced reads were aligned against the hg19 reference genome (Genome Reference Consortium GRCh37). Sequence variants were identified using the Ion Reporter software v. 5.10 (Thermo Fisher Scientific) and Ion AmpliSeq HD Workflow template for Liquid Biopsy-w2.1-DNA-Single Sample, and the coverage of each amplicon was determined using the Coverage Analysis Plugin Software v. 5.10.0 (Thermo Fisher Scientific). The application of UMIs enabled the grouping of reads into molecular families. Random errors generated during library construction and the sequencing process were removed automatically. At least three independent molecular families were required to identify and call a variant.
Categorical clinical variables between patients with and without pathogenic or likely pathogenic variants identified in the cfDNA were compared using Fisher’s exact test. Observed and designed VAFs were compared using Spearman’s rank correlation and Passing–Bablok regression. Statistical analyses were performed using MedCalc v. 17.2 (MedCalc Software, Ostend, Belgium).
The cfDNA output of each sample and the sequencing metrics are presented in Table 2. The median concentration of plasma cfDNA from all HCC patients was 4.1 ng/mL (range: 0.8-15.3 ng/mL). The library concentrations were 2,050–14,500 pM. The median sequencing coverage was 62,694 (range: 44,765–81,672), and all 20 samples had a median read coverage greater than 25,000, which is the median read coverage across targets specified by the manufacturer to ensure a 0.1% limit of detection.
The results of the analysis of the reference materials for six variants (one in
Targeted NGS using an in-house panel of three HCC driver genes identified at least one pathogenic variant in the plasma cfDNA of 13/20 patients (65%). These included 16 variants of
Of the 16 pathogenic variants of
In the present study, we identified 16 pathogenic variants of TP53 and nine pathogenic variants of
Two recent studies investigated the molecular landscape of cfDNA in HCC patients [22, 23] (Table 4). In the European study, 29 pathogenic or likely pathogenic variants in eight genes were detected in 18/51 (35%) patients. The median read depth was 486, and the median VAF was 0.12. In the study on 206 HCC patients from the USA, alterations, including amplifications, synonymous alterations, and variants of undetermined significance, in addition to pathogenic or likely pathogenic variants, were detected in 181/206 (87.8%) patients. The median VAF was 0.49%, and
Although these were larger scale studies than our study, to the best of our knowledge, our study is the first to use a customized cfDNA NGS panel targeting the major HCC driver genes, incorporating molecular barcoding. Using an in-house customized panel targeting three HCC driver genes implemented on a robust and standardized NGS platform, we detected at least one pathogenic variant in plasma cfDNA among 68% of the patients analyzed (13/19). These included 16 variants of
We acknowledge some limitations of our study. First, somatic variant profiles from synchronously collected tumor biopsies were not available for concordant analysis. However, a previous study indicated that, even without prior knowledge of the variant repertoire in an HCC biopsy, high-depth sequence analysis of plasma cfDNA can represent somatic variants in an HCC biopsy in a significant proportion of therapy-naive HCC patients . Second, as a pilot study, our study enrolled small number of patients. However, we found strong evidence that plasma cfDNA analysis using NGS can reliably detect pathogenic variants in HCC driver genes in HCC patients. Furthermore, as a proof-of-concept study, correlations between detected cfDNA variants and clinical characteristics were not the main focus of this study. These should be assessed in a larger patient cohort in future studies. Lastly, we did not confirm the very-low-frequency variants using other methods, such as digital droplet PCR.
In conclusion, by using targeted cfDNA NGS, we achieved a very high coverage of the entire coding regions of three major HCC driver genes, allowing the detection of low-frequency variants in a large number of unique molecular families. cfDNA could be used as a reliable biomarker to identify somatic variants in HCC, and our results support the utility of cfDNA analysis in a larger cohort of HCC patients.
We thank all patients who participated in this study, the referring clinicians, and The Catholic Genetic Laboratory Center for assisting us with this study.
PSS, YK, MK, and SKY conceived and designed the work; HC, AK, and DK contributed to the acquisition of data; HJC wrote the manuscript; PSS, YK, MK, and SKY revised the manuscript critically for important intellectual content.
No potential conflicts of interest relevant to this article were reported.
This research was partly supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) and funded by the Ministry of Science, ICT and Future Planning (2019R1I1A1A01059642, S.P.S).