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The Role of cDNA in Gastric Cancer
- Thread starter ginfreely
- Start date
Simple Summary
DNA release from tumour cells (call circulating tumour DNA) into the blood stream can be found in patients with gastric cancer through a blood test call a liquid biopsy. This less invasive test can assess the genetic make-up of tumours to provide important information on the mechanisms of cancer development, identify mutations which can be targeted with drugs and could be used to screen for patients with gastric cancer. This article will review the current and future uses of liquid biopsies in gastric cancer.
Abstract
Circulating tumour DNA (ctDNA) has potential applications in gastric cancer (GC) with respect to screening, the detection of minimal residual disease (MRD) following curative surgery, and in the advanced disease setting for treatment decision making and therapeutic monitoring. It can provide a less invasive and convenient method to capture the tumoural genomic landscape compared to tissue-based next-generation DNA sequencing (NGS). In addition, ctDNA can potentially overcome the challenges of tumour heterogeneity seen with tissue-based NGS. Although the evidence for ctDNA in GC is evolving, its potential utility is far reaching and may shape the management of this disease in the future. This article will review the current and future applications of ctDNA in GC.
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1. Introduction
Gastric cancer (GC) is the fifth most common cancer worldwide and accounts for almost 800,000 deaths each year [1]. Early-stage GC is potentially curable; however, it only accounts for approximately 30–40% of all GC diagnoses [2,3]. There are no GC-screening modalities with established evidence to improve GC-related mortality in large, randomised trials. Most GC cases are diagnosed as advanced disease [3]. The diagnosis is made using an invasive tissue biopsy usually through an upper gastrointestinal endoscopy after a symptomatic presentation or sometimes incidentally. For patients with an early-stage of disease, the standard treatment as per NCCN/ESMO guidelines is peri-operative combination chemotherapy and surgery [4,5]. In those who undergo surgical resection, there is no universally accepted programme to monitor recurrence. The treatment of metastatic gastric cancer (mGC) has changed over time, with incremental improvements in survival [6,7]. The optimal first line treatment in HER2-negative GC is combination chemotherapy with or without immunotherapy depending on the patient’s PDL1 status [8,9]. The addition of trastuzumab to combination chemotherapy improves survival in patients with HER2-amplified tumours [10]. However, many of the established prognostic and predictive biomarkers require invasive tissue-based testing including for HER2 and PDL1. The use of non-invasive, circulating, and reproducible methods to screen, diagnose, monitor, and molecularly characterise GC for both predictive and prognostic potential is needed. The circulating biomarkers of cancer are of interest given they are non-invasive and acceptable to patients. Circulating tumour cells (CTCs) and circulating free DNA (cfDNA), including the tumour fraction (i.e., circulating tumour DNA (ctDNA)), are examples of circulating biomarkers that can be detected in the blood. Other circulating biomarkers including exosomes and circulating RNA are being studied across many tumour types; however, they will not be covered in this review article. The advantages and challenges of tissue and liquid biopsies in gastric cancer are shown in Table 1.
Table 1
Advantages and challenges of tissue and liquid biopsies.Tissue Biopsy | Liquid Biopsy |
|---|---|
Requires invasive procedure | Minimally invasive |
Unable to capture tumour heterogeneity | Overcomes challenges of tumour heterogeneity |
Unable to assess temporal genomic changes | Real time genomic monitoring and cancer evolution monitoring |
Very low risk of false positives (CHIP) | Risk of false positives (CHIP) |
Risk of non-diagnostic sample | Variable detection rate (dependant on stage, site of metastases, type of cancer) |
Technical consideration for tissue processing required (storage of tissue, cutting, histopathological review) | Pre-analytical va |
1.1. Circulating Tumour Cells
Circulating tumours cells are cancer cells released into bloodstream originating from either the primary tumour or metastatic lesions. They can be detected and isolated in the blood of patients with cancer and play a role in metastasis [11]. However, CTCs form only a very small portion of the total blood cells and are more challenging to isolate and sequence compared to ctDNA [12]. In addition, the sensitivity to detect genomic aberrations is reduced when using CTCs compared with ctDNA [13,14,15]. ctDNA also provides an estimate of tumour burden through quantification methods to measure the fraction of ctDNA in the blood, which is not possible with CTCs.
1.2. cfDNA and ctDNA
cfDNA is extra-cellular DNA secreted from cells into the bloodstream. It can also be secreted into other body fluids, including CSF (cerebrospinal fluid) and urine. cfDNA may also be secreted from normal cells during physiological processes including necrosis and apoptosis, and elevated levels can be seen during pregnancy, renal failure, in auto-immune conditions, and following exercise [16,17,18,19,20,21]. Individuals with cancer often have elevated levels of cfDNA, and a small fraction of this is tumour-derived (i.e., ctDNA) [22]. Mutations in cfDNA may not always be tumour-derived. For example, genomic alterations found through the analysis of white blood cells (WBC) can signify the clonal haematopoiesis of indeterminant potential (CHIP). This somatic blood cell clonally derived variant represents a false positive result when using ctDNA-based sequencing [23]. Germline variants can also be detected using blood-based ctDNA analyses
1.3. ctDNA Sequencing Methods
ctDNA concentrations are higher in serum compared to plasma [24]. However, sera also contain DNA released from white blood cells; therefore, plasma-based assays have a higher sensitivity in isolating and analysing tumour-derived DNA [24]. Generally, the concentration of ctDNA in the blood is low and requires very sensitive tools to isolate and subsequently perform the sequencing of DNA.ctDNA analysis can detect multiple genomic aberrations, including point mutations (single nucleotide variants), insertions/deletions, amplifications (copy number variants), and gene fusions. Current technologies can isolate and sequence ctDNA at concentrations (i.e., variant allelic frequencies—VAF) as low as 0.01% [25,26]. Targeted sequencing techniques can analyse a single gene using droplet digital PCR (ddPCR) or BEAMing (beads, emulsion, amplification, and magnetics) PCR. Targeted sequencing can also be employed on a multi-gene basis, using next generation sequencing (NGS) assays encompassing hundreds of genes. ddPCR is a highly specific method used to detect known mutations at very low allelic frequencies. However, it can only detect one mutation per assay. BEAMing is a PCR-based sequencing method that uses primers to tag sequences prior to amplification. BEAMing and ddCR-targeted techniques are useful in detecting MRD and relapsed disease through a tumour-informed approach. NGS techniques can detect multiple tumour-specific genomic aberrations and can provide a broader molecular profile of tumours. This is useful in situations where the genomic profile of the tumour is unknown (i.e., a tumour agnostic approach) when using a plasma-only assay. Table 2 shows the different ctDNA technologies.
ctDNA-sequencing Technologies.
Technology | Example | Molecular Targets | Detection Limit | Limitations | Benefits |
|---|---|---|---|---|---|
Allele-Specific PCR Assay | Roche/Cobas | Known mutations | <0.01% | Only semi-quantitative; less sensitive compared with ddPCR | Highly specific with broad coverage |
Emulsion PCR Assays | ddPCR BEAMing | Known mutations | <0.01% | Less specific. Unable to detect CNV/fusions | Fully quantitative |
Targeted NGS Assays |
Amplicon-based | TAM-Seq | Hotspot SNV and CNV | <0.1% | Less sensitive and limited variant analysis compared to capture-based assay | Fast and cost effective |
Capture-based | Guardant360© | SNV, CNV, fusions | <0.1% | Lower specificity compared to amplicon-based assays, complex, and slower. | Higher sensitivity compared with amplicon sequencing |
Non-targeted NGS Assays | Whole-genome sequencing Whole-exome sequencing | All variants | <1% | Reduced sequencing depth compared with NGS, costly | Genome-wide analysis |
The methylation of DNA regulates gene expression in normal and cancer cells. There are distinct patterns of methylation seen in patients with cancer [27,28,29,30]. These abnormal epigenetic aberrations can be detected through analysing ctDNA. The employment of DNA methylation detection is useful as abnormal patterns occur early in cancer development, particularly in Barret’s oesophagus, making it useful in screening [31]. Several studies have shown that ctDNA methylation has reasonable sensitivity and specificity in the detection of early cancer, as outlined below [32].
This review article will focus on the current technology and utility of ctDNA in gastric cancer including in screening, the detection of MRD, and comprehensive genomic profiling in the advanced disease setting.
This review article will focus on the current technology and utility of ctDNA in gastric cancer including in screening, the detection of MRD, and comprehensive genomic profiling in the advanced disease setting.
2. ctDNA Detection in Gastric Cancer
ctDNA can be detected in individuals with early- and late-stage GC. A study using a plasma-based, whole-genome NGS panel in 44 patients with any stage gastric (n = 39) and oesophageal cancer (n = 5) revealed a ctDNA detection rate of approximately 39%, with a VAF range of 2.5–8%. Interestingly, the concordance with tissue was only 54% [33]. Another study of 29 patients with any stage GC showed a ctDNA detection rate of 91.3% using a targeted NGS panel, with a VAF ranging from 2.8% to 87.1% and an average of 5.4 somatic variants detected per patient. The tissue concordance was 47.8% in this cohort. Even small tumours such as T1-T2 shed ctDNA [34].
Wow very high detection rate 39% just by using blood vs 54% using tissue. Very good!
The GC somatic mutation landscape has been described previously using tissue-based sequencing, but also using ctDNA-based sequencing [34]. A large comprehensive genomic-profiling study using ctDNA to detect targetable genetic mutations in patients with advanced gastrointestinal (GI) cancers was performed in Japan [35]. The primary aim of this study was to identify patients for interventional clinical trials using targeted agents on a national scale comparing ctDNA-based (GOZILA Study) and tissue-based sequencing (GI-SCREEN). The GOZILA study included 1687 patients for ctDNA analysis, including 260 patients with oesophageal, junctional, and gastric adenocarcinomas. An NGS-based ctDNA analysis revealed genomic alterations, MSI prevalence, and germline BRCA mutations in 85%, 2.5%, and 1.5% of patients, respectively. In addition, the most common molecular alterations were a TP53 mutation (53%), a PIK3CA mutation/amplification (20%), a CCNE1 amplification (20%), an EGFR amplification (15%), and a HER2 amplification (12%) in OGA [35].
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