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Evaluation of A Liquid Biopsy Protocol Using Ultra

This study evaluates a liquid biopsy protocol using ultra-deep massive parallel sequencing to detect and quantify circulating tumor DNA in colorectal cancer patients. The assay demonstrated a 92% concordance in mutation detection between plasma and tissue samples, highlighting its potential as a non-invasive alternative to traditional tissue biopsies. The research aims to improve targeted therapy selection by accurately identifying actionable mutations in key driver genes such as KRAS, NRAS, and BRAF.

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13 views2 pages

Evaluation of A Liquid Biopsy Protocol Using Ultra

This study evaluates a liquid biopsy protocol using ultra-deep massive parallel sequencing to detect and quantify circulating tumor DNA in colorectal cancer patients. The assay demonstrated a 92% concordance in mutation detection between plasma and tissue samples, highlighting its potential as a non-invasive alternative to traditional tissue biopsies. The research aims to improve targeted therapy selection by accurately identifying actionable mutations in key driver genes such as KRAS, NRAS, and BRAF.

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Le Doan
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Evaluation of a liquid biopsy protocol using ultra-deep massive parallel sequencing for detecting and

quantifying circulation tumor DNA in colorectal cancer patients.


Huu Thinh Nguyen, Hoai-Nghia Nguyen et al.,

Abstract
The identification and quantification of actionable mutations are critical for guiding targeted therapy
and monitoring drug response in colorectal cancer. Liquid biopsy (LB) based on the plasma cell-free DNA
analysis has emerged as a non-invasive approach with many clinical advantages over conventional tissue
sampling. Here, we developed a LB protocol using ultra-deep massive parallel sequencing and validated
its clinical performance for detection and quantification of actionable mutations in three major driver
genes (KRAS, NRAS, and BRAF). The assay showed a 92% concordance for mutation detection between
plasma and paired tissues and great reliability in quantification of variant allele frequency.
Keywords: liquid biopsy, colorectal cancer, actionable mutation, ultra-deep sequencing, circulating
tumor DNA.

Introduction
Colorectal cancer (CRC) is the fifth and the third most common cancers in term of both incidence and
mortality in Vietnam and the world respectively, accounting for a million death per year worldwide.
Currently targeted therapy is one of the effective treatment for colorectal cancer patients.
Overexpression of EGFR was reported in 60-80% of CRC cases. Therefore, EGFR has become a
therapeutic target for small molecule inhibitors or monoclonal antibodies such as cetuximab and
panitumumab, which have been approved by the Food and Drug Administration. However, drug
resistance was observed in CRC patients who acquired somatic mutations in other oncogenes including
KRAS (30-40%), NRAS (2-3%) and BRAF (8-12%). As such, the American Society of Clinical Oncology
recommended simultaneous testing for KRAS, NRAS and BRAF mutation in order to select the optimal
treatment regimen, thereby improving the survival rate of advanced CRC patients.
Tissue biopsy is regarded as the gold standard for tumor genetic profiling. However, it is an invasive
approach and not feasible for repeated biopsy in patients with metastatic tumor. Furthermore, tissue
sampling might not represent the genetic heterogeneity of the entire tumor. Recently, liquid biopsy has
emerged as a non-invasive approach that better reflects the whole genetic complexity of tumors and
enables real-time monitoring of treatment response. This approach detects genetic alterations in
circulating tumor DNA (ctDNA) released into the blood circulation by tumor cells undergoing cell death.
However, the low abundance of ctDNA in the circulation requires as highly sensitive analytical technique
to accurately identify genetic alterations. Several methods have been developed to detuct such low
abundant mutation in plasma including amplification refractory mutation system (ARMS) and droplet
digital PCR (ddPCR). While ARMS has been shown to be not sensitive enough to detect mutations in
ctDNA, ddPCR exhibited good sensitivity for both detection and quantification in ctDNA. However,
ddPCR only allows analyzing a limited number of known mutation per reaction. Unlike ddPCR, massively
parallel sequencing (MPS) has the ability to simultaneously explore a complete mutation landscape of
multiple driver genes at lower cost per mutations. It has recently been reported that MPS combining
with unique molecular barcoding and ultra-deep sequencing could achieve high sensitivity for detection
and quantification of low frequent mutations in plasma. Hence, in this study, we evaluated the clinical
utility of MPS in detection and quantifying actionable KRAS, NRAS and BRAF mutations in plasma
samples collected from CRC patients.

Methods
Patient recruitment
A total of 56 patients diagnosed with CRC were recruited from the University Medical Center at Ho Chi
Minh city. Of those patients, 50 provided paired samples of tissue biopsies and plasma samples while
the remaining 6 provided only plasma samples (Table S1). Written informed consents were obtained
from all patients. Comprehensive patients’ clinical information were summarized Table S2. This study
was approved by the Ethic Committee of University of Medicine and Pharmacy at Ho Chi Minh City,
Vietnam (Ethic number: 027/DHYD-HD).
Clinical sample collection
Prior to tissues biopsy, 10 ml of peripheral blood was drawn in K2-EDTA tubes (BD Vacutainer, USA),
stored at room temperature for maximum of 8 hours before undergoing 2 rounds of centrifugation (2,00
x g for 10 min then 16,000 x g for 10 min) to separate plasma from blood cells. More than 95% of the
samples had a collection-to-spin time of less than 4 hours. The remaining samples had a collection-to-
spin time of 6-77 hours. With these samples, care was taken to enxure that no hemolysis was observed
after the first spin to avoid contamination of genomic DNA from white blood cells. The plasma fractions
(4-6ml) were then collected, aliquoted (2ml per aliquot) and stored at -80oC until cell free DNA
extraction. Tissue biopsied were collected, formalin-fixed and paraffin-embedded (FFPE) then the
tumor-rich areas of the FFPE tissues containing at least 50% of tumor cells identified by a hematoxylin
and eosin staining were micro-dissected and subjected to DNA extraction.

DNA isolation
Cell free DNA was extracted from an aliquot of 2ml of plasma using the MagMAX Cell-free DNA Isolation
kit (thermos fisher, USA0 following the manufacturer’s instructions. Tumor tissue-derived DNA was
extracted from FFPE samples using QIAamp DNA FFPE tissue kit (Qiagen, USA) following the
manufacturer’s instructions. Both cell free DNA from plasma and genomic DNA from FFPE (2µl of
sample) were quantified using the QuantiFlour dsDNA system (Promega, USA) and Quantus Flourometer
(Promega, USA).

Ultra-deep massively parallel sequencing (MPS) with unique molecular identifier tagging
Library preparation
For cell free DNA (cfDNA), library with unique molecular identifier tagging was prepared from 2ng of
cfDNA using the Accel-NGS 2S Plus DNA library kit (Swift Biosciences, USA) following the manufacturer’s
instructions. Library concentrations

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