Circulating tumor DNA sequencing for colorectal cancers: A comparative analysis of colon cancer and rectal cancer data
Kaibin HuangHongyue QuXiaoni ZhangTanxiao HuangXiaoxia SunWan HeMingwei LiLiewen LinMingyan XuShifu ChenLigang Xia
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Abstract:
Circulating tumor DNA (ctDNA) has been recognized as a promising biomarker for colorectal cancer (CRC) early diagnosis and postoperative monitoring. However, we hypothesize that the clinical value of ctDNA sequencing may differ for colon cancer (CC) and rectal cancer (RC).Forty-three patients with primary CRC were prospectively enrolled. Tumor tissue samples, paired preoperative plasma samples and a series of postoperative plasma samples were obtained. Mutations in each sample were identified and compared.For 73.0% patients, at least one concordant mutation was detected in both tumor tissue DNA and paired preoperative ctDNA. The mutation concordance rate were higher in CC patients compared to RC patients (92.3% vs 45.5%; p= 0.004). For early stage patients, the mutation concordance rate was 72.7%. The recurrence rate was 33.3% for patients with postoperative ctDNA positive mutations, and 3.4% for patients with negative ctDNA (HR 10.767; 95% CI 1.1-103.8; p= 0.040).Liquid biopsy via ctDNA sequencing has great potential for the early detection and postoperative monitoring of CRC. The DNA of CC tissues is more likely to be released into blood than the DNA of RC tissues. This should be considered when diagnosing CRC patients with ctDNA sequencing technology.Keywords:
Concordance
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Circulating tumor DNA
Highly sensitive technologies are available for the molecular characterization of solid tumors, including digital PCR (dPCR). Liquid biopsy, based on the analysis of cell-free DNA (cfDNA), is often used to assess EGFR or RAS alterations in lung and colorectal cancers. Our study aimed to compare the results of two different dPCR platforms for the detection of mutations in cfDNA.Plasma samples from lung and colorectal cancer patients collected as per routine procedures have been tested. cfDNA Was extracted from plasma, and samples were screened on the droplet digital PCR (ddPCR, BioRad) and solid dPCR QIAcuity (Qiagen).A total of 42 samples were analyzed, obtained from 20 Non-Small Cell Lung Cancer (NSCLC) patients carrying an EGFR or a KRAS mutation on tissue at diagnosis, and from 22 samples of colorectal cancer (CRC) patients, 10 of which presenting a KRAS mutation. EGFR mutation detection was 58.8% for ddPCR and 100% for dPCR (κ = 0.54; 95% CI, 0.37-0.71), compared to tissue results. The detection rate for RAS mutations was 72.7% for ddPCR and 86.4% for dPCR (κ = 0.34; 95% CI, 0.01-0.68), compared to tissue results.This study showed moderate agreement between dPCR and ddPCR. Sampling effect or threshold settings may potentially explain the differences in the cfDNA data between the two different platforms.
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Abstract Background Mutations within the telomerase reverse transcriptase promoter (TERTprom) and isocitrate dehydrogenase (IDH) account for the most common genetic alterations in gliomas. Each of these mutations impact clinicopathologic diagnosis and course of diseases. While TERTprom mutations are frequently detected in glioblastoma, IHD mutations are assigned to astrocytoma of grade 2-4, thus mostly associated with better prognosis. In the era of precision oncology, molecular profiling and continuous monitoring of treatment response or relapse are of increasing importance. Accordingly, this study aims to detect TERTprom and IDH mutations in plasma-derived cell-free (cf)DNA of gliomas. The mutant allele frequencies (MAF) will be compared retrospectively to clinico-pathological parameters including extent of resection and tumor progression. Material and Methods Digital droplet PCR (ddPCR) analyses were performed using the QX200TM Digital Droplet System from BioRad. First, to evaluate probes for ddPCR, genomic DNA of several brain tumor cell models (n=6) and tumor tissue (n=1), as well as cfDNA of plasma (n=3) from samples with known TERTprom and IDH mutation status was investigated. For detection of IDH mutations, the unique assay ID dHSaMDV2010055 (IDH1p.R132H) and for TERTprom mutations the TaqMan dPCR Liquid Biopsy Assays for C228T (Hs000000092) and C250T (Hs000000093) were used. The results of ddPCR were analyzed with QuantaSoftTM software and the MAF was calculated Results To validate the detection method for IDH1R132H, we analyzed the MAF in one tissue and corresponding plasma sample of a confirmed IDH1-mutated astrocytoma. In addition, plasma from one astrocytoma grade 2-3 as well as from an IDH1-mutated glioblastoma was tested. Interestingly, both astrocytoma cases exhibited undetectable or very low MAF ranging from 0.1 to 1% in tissue as well as in plasma samples, while in plasma from the high-grade glioblastoma case, IDH1R132H was detected with a frequency of 1.9%. Due to the high GC content of the TERT promoter region, amplification steps are challenging. Accordingly, we first optimized ddPCR conditions for C228T and C250T probes by adding 7-deaza-2-deoxyguanosine-5-triphosphate (7-ddGTP) in varying concentrations to each ddPCR reaction. When using 4µM of 7-ddGTP per sample, a clear separation between mutant and wild-type droplets was reached, detecting MAF between 36-63% in DNA from cell culture models. Conclusion Within this pilot study we optimized the ddPCR method for the detection of IDH1R132H and TERTprom mutations in plasma and tissue samples. Subsequently, we hypothesize that these mutations are suitable liquid biomarkers correlating with extent of resection and tumor progression in gliomas.
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The "sample-to-answer" integration and automation of circulating tumor DNA (ctDNA)-based liquid biopsy using digital PCR (dPCR) has been hampered by the complicated operations of liquids with volumes ranging from milliliter samples to nanoliter droplets. On the basis of a "3D extensible" design paradigm proposed previously, an integrated droplet digital PCR (IddPCR) microdevice was successfully developed to automate the entire process of liquid biopsy, from the extraction of ctDNA in 2 mL of plasma using magnetic beads to the generation, amplification, and screening of over 30 000 droplets for detection. A series of reagent mixing structures, including macro-, meso-, and micromixers, was designed to enable efficient reagent handling and mixing at different volume scales. The volume thresholds of the microscale and macroscale in the IddPCR device were calculated to be 40 and 100 μL, respectively, based on the fluid dynamics and sizes of the device structures, so that different mixers can be selected according to the reagent volumes. The DNA extraction efficiency obtained on the device was determined to be ∼60%, and the on-chip ddPCR demonstrated a high correlation with an R2 of 0.9986 between the readouts and the estimations by a Poisson distribution. Finally, the IddPCR microdevice was able to detect rare tumor mutations (T790M) with an occurring frequency as low as ∼1% from 2 mL of human plasma in a "sample-to-answer" manner. This work offers a feasible solution for the automation of liquid biopsy and paves the way for its broad applications in clinics.
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Circulating tumor DNA (ctDNA) is a component of the "naked" DNA found in blood. It can be isolated from plasma and represents combined genetic material from the primary tumor and metastases. Quantitative and qualitative information about a cancer, including mutations, can be derived using digital polymerase chain reaction and other technologies. This "liquid biopsy" is quicker and more easily repeated than tissue biopsy, yields real-time information about the cancer, and may suggest therapeutic options. All stages of cancer therapy have the ability to benefit from ctDNA, starting with screening for cancer before it is clinically apparent. During treatment of metastatic disease, it is useful to predict response and monitor disease progression. Currently, ctDNA is used in the clinic to select patients who may benefit from epidermal growth factor receptor-targeted therapy in non-small cell lung cancer. In the future, ctDNA technology promises useful applications in every part of clinical oncology care.
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Opportunities and challenges in translational application of ctDNA along with recent developments in chip-based ctDNA detection technologies have been reviewed.
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Liquid biopsy allows the identification of targetable cancer mutations in a minimally invasive manner. In patients with advanced non-small cell lung cancer (NSCLC), droplet digital PCR (ddPCR) is increasingly used to genotype the epidermal growth factor receptor (EGFR) gene in circulating cell-free DNA (cfDNA). However, the sensitivity of this method is still under debate. The aim of this study was to implement and assess the performance of a ddPCR assay for detecting the EGFR T790M mutation in liquid biopsies.A ddPCR assay was optimized to detect the EGFR T790M mutation in plasma samples from 77 patients with NSCLC in progression.Our ddPCR assay enabled the detection and quantification of the EGFR T790M mutation at cfDNA allele frequency as low as 0.5%. The mutation was detected in 40 plasma samples, corresponding to a positivity rate of 52%. The number of mutant molecules per mL of plasma ranged from 1 to 6,000. A re-biopsy was analyzed for 12 patients that had a negative plasma test and the mutation was detected in 2 cases. A second liquid biopsy was performed for 6 patients and the mutation was detected in 3 cases.This study highlights the value of ddPCR to detect and quantify the EGFR T790M mutation in liquid biopsies in a real-world clinical setting. Our results suggest that repeated ddPCR tests in cfDNA may obviate tissue re-biopsy in patients unable to provide a tumor tissue sample suitable for molecular analysis.
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Circulating tumor DNA(ctDNA) is the DNA fragment released into blood by tumor cells.Wheather it presents or not and its plasma concentration are closely related to the prognosis of patients. The common detection methods of ctDNA include digital polymerase chain reaction,second-generation sequencing,methylation detection technology and so on. Detecting specific point mutations or methylation of ctDNA can not only assist in the diagnosis of pancreatic cancer,but also be expected to identify pancreatic cancer at an early stage. Detecting ctDNA after operation can help predicting tumor recurrence and metastasis effectively,so that patients with high recurrence and metastasis risks can be intervened in advance. Accordingly,this article intends to review detection technology of ctDNA and its clinical applications in the early diagnosis of pancreatic cancer,the prediction of tumor recurrence and metastasis after surgery,and the evaluation of patient prognosis.循环肿瘤DNA（ctDNA）是肿瘤细胞释放入血的DNA片段，其存在与否、血浆浓度的高低与患者预后息息相关。常见的检测方法有数字聚合酶链式反应技术、二代测序、甲基化检测等。针对ctDNA的特异性点突变或甲基化进行检测，不但能辅助诊断胰腺癌，还有望在早期阶段识别胰腺癌。针对术后ctDNA的检测能有效预测肿瘤复发转移，对患者进行风险分层，从而得以对高复发转移风险患者提前进行干预。据此，本文对ctDNA检测技术及ctDNA在胰腺癌早期诊断、手术后复发转移的预测和患者预后评估等方面的临床应用研究进行综述。.
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The use of liquid biopsy is of potential high importance for children with high grade (HGG) and diffuse midline gliomas (DMG), particularly where surgical procedures are limited, and invasive biopsy sampling not without risk. To date, however, the evidence that detection of cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA) could provide useful information for these patients has been limited, or contradictory.
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