COLD-PCR技术的原理及应用

低变性温度下的复合PCR(COLD-PCR)技术是一种新开发的PCR方法,能够从大量野生型DNA中选择性扩增低水平未知突变。该技术显著地提高了低浓度突变的检测灵敏度,已经被应用于医药的很多领域,包括肿瘤检测、产前诊断以及传染性疾病检测等。同时COLD-PCR可以与很多分子生物学检测方法结合使用,在临床诊断上具有较好的应用前景。对COLD-PCR技术做基本的介绍,并结合其在医药领域的应用进行综述。     

循环肿瘤DNA的检测及研究现状

中国癌症发病率与世界水平相持平,死亡率要高于世界水平。虽然更多先进的技术已经运用到癌症治疗领域,但是癌症的死亡率依然居高不下。癌症之所以难克服,是因为肿瘤细胞具有异质性。“液体活检”技术的出现,使无创性治疗方法开始应用到肿瘤治疗领域,循环肿瘤DNA(circulating tumor DNA,ctDNA)则是“液体活检”中的一种重要素材。血液中游离的DNA被称为cfDNA(Cell free DNA),含有突变的cfDNA就被称作ctDNA。ctDNA所含有的突变一般是单个碱基替换所造成的,只有癌细胞才会有这种情况的发生,这使得ctDNA具有极高的特异性,可作为高灵敏的生物标记物。由于ctDNA存在于血液中,其无创性价值重大。     

一种检测kras基因突变的新型TB-ARMS qPCR方法的建立

在荧光定量PCR基础上建立一种简单有效并且高度灵敏的TB-ARMSkras基因突变检测方法,并对其检测性能进行评估,探讨其临床应用价值。针对kras基因8种常见的点突变类型,通过设计并优化突变特异性引物、野生型特异性封闭引物并综合应用突变富集扩增反应条件等多种手段,提高点突变检测的灵敏度和特异性,采用已知野生型基因组样品和构建的突变质粒作为标准品,进行方法学评价;通过对临床样本的检测及与现有商品化试剂盒的比较进行性能验证;通过对术前血浆和配对组织样品的对比检测,评估方法是否适用于血液样本的检测。建立了TB-ARMS kras突变检测的新方法,能检测的最低突变率可达到0.01%。通过综合采用野生型特异性封闭引物和突变富集扩增条件等方法证明了其0.01%的突变检测灵敏度。检测准确性优于现有商品化试剂盒,血浆DNA TB-ARMS qPCR检测结果与配对组织DNA测序结果相符合。因此,TB-ARMS kras基因突变检测方法具有广泛的临床应用价值,既适用于临床组织样品的检测,也可应用于液体活检。     

扩增子测序技术应用于乳腺癌循环游离DNA无创性检测的研究

目的:应用扩增子测序方法检测乳腺癌循环游离DNA肿瘤相关突变。方法:军事医学科学院附属医院的10例HER2阳性晚期乳腺癌患者血样入组,应用扩增子测序技术检测血细胞和血浆中的循环游离DNA(cfDNA),筛选出肿瘤相关基因突变,即循环肿瘤DNA(ctDNA)。结果:检测10例晚期乳腺癌患者外周循环血50个基因,共检出11个突变基因,其中PIK3CA、ERBB4的阳性检出率均达到100%,AKT、TP53的阳性检出率为90%。结论:检测覆盖度及深度均良好,结果提示11个基因的热点突变区域出现单核苷酸的多态性基因突变,突变基因与PI3K-AKT-m TOR通路、Ras-Raf-MEK-ERK通路明显相关。扩增子测序技术和筛选方法可以用于乳腺癌ctDNA的无创性检测。     

基于锁核酸及等位位点特异性扩增技术的KRAS基因突变检测荧光定量PCR方法研究

基于等位位点特异性扩增的原理,设计锁核酸修饰KRAS基因突变特异性扩增引物,结合封阻探针技术,建立检测KRAS基因突变的荧光定量PCR方法。结果发现,锁核酸修饰的引物及探针可显著提高等位位点特异性扩增技术用于复杂样本中的微量基因突变检测的敏感度,该技术检测KRAS基因突变的敏感性可达0.01%~0.1%。进一步用建立的荧光定量PCR方法检测52例结直肠癌患者血浆标本,并用DNA测序法作为对照,同时用健康人血浆标本建立阴性检测结果判读标准,以初步评价该方法应用于循环DNA中KRAS基因突变检测的可行性。结果发现结直肠癌患者KRAS基因突变主要是G12C、G12A和G12R,而且q PCR法的阳性检出率为46.15%,高于DNA测序法(13.46%),阴性结果与DNA测序法的符合率为100%。此外,结直肠癌患者外周血KRAS基因的突变检出率与文献报道组织标本中的突变检出率及常见突变类型基本相符。上述结果说明该方法检测循环肿瘤DNA(circulating tumor DNA,ct DNA)具有较高的可靠性,可以用于肿瘤患者循环血液中KRAS基因突变的检测。     

粪便DNA突变检测在快速筛选大肠癌中的应用

大肠癌是发病率和死亡率都很高的常见疾病, 对高危人群进行大规模的普查筛选, 可以降低大肠癌的发生率和死亡率。粪便DNA突变检测是新近发展的可用作大肠癌普查的技术, 与常规结肠镜检测和大便隐血实验相比, 具有特异性高、灵敏度高和易于被患者接受等优点。文章阐述了粪便DNA突变检测的相关基因、肿瘤特异性DNA提取方法和检测方法等, 并对其在快速筛选大肠癌中的应用进行了展望。大肠癌是发病率和死亡率都很高的常见疾病, 对高危人群进行大规模的普查筛选, 可以降低大肠癌的发生率和死亡率。粪便DNA突变检测是新近发展的可用作大肠癌普查的技术, 与常规结肠镜检测和大便隐血实验相比, 具有特异性高、灵敏度高和易于被患者接受等优点。文章阐述了粪便DNA突变检测的相关基因、肿瘤特异性DNA提取方法和检测方法等, 并对其在快速筛选大肠癌中的应用进行了展望。     

循环肿瘤DNA检测技术的研究进展

循环肿瘤DNA(circulating tumor DNA,ct DNA)检测,即所谓的"液体活检"。与传统的组织活检技术相比,"液体活检"能够更加全面地反映肿瘤的特征。随着ct DNA研究的逐渐深入,其检测技术的灵敏度和特异性都得到了很大的提升,而其中最具代表性的检测技术有数字PCR(digital PCR,d PCR)技术、流式技术的磁珠乳液扩增方法(bead,emulsion,amplification and magnetic,BEAMing)、标记扩增深度测序(tagged-amplicon deep sequencing,TAm-Seq)等,这些技术的发展有望开启精准医疗的新时代。     

Taqman多重实时荧光定量PCR检测裸鼠肿瘤转移模型中肿瘤转移率方法的建立

目的:建立基于多重荧光定量PCR技术的动物体内恶性肿瘤转移模型中肿瘤转移率高效检测的方法。方法:以人和小鼠的β2m基因为目标,设计相应的引物和Taqman探针;分别抽提人涎腺腺样囊性癌ACC细胞和小鼠Sp2/0细胞的基因组DNA作为模板,建立两物种双重荧光定量PCR检测方法,且分别以人和鼠定量基因检测为参照考察双重荧光定量PCR方法的特异性、灵敏性和精确性,并与传统转移灶称重计算肿瘤转移率的方法进行比较。结果:双重Taqman实时荧光定量PCR检测方法具有很好的特异性和较高的灵敏度(0.006ng/μl DNA),重复性较好(批间平均CV=0.036),有较强稳定性,比传统称重法更高效,更准确。结论:双重荧光定量PCR能够在同一反应体系下同时对动物组织中人肿瘤转移灶和周围组织进行快速准确的定量检测,计算出肿瘤转移率,具有经济、快速、特异性强等优点,在肿瘤动物模型肿瘤转移率检测方面具有很高的应用价值。     

大鼠冠状病毒和仙台病毒的双重PCR检测方法的建立与应用

目的建立快速检测实验大鼠冠状病毒和仙台病毒的双重PCR方法。方法根据大鼠冠状病毒N基因、仙台病毒L基因设计特异性引物;经过双重PCR优化,特异性和敏感性的检测,建立双重PCR体系。应用该PCR体系检测人工感染仙台病毒组织DNA样本和实验动物组织样本,并与ELISA方法比对。结果双重PCR扩增出大鼠冠状病毒(168 bp)和仙台病毒(262 bp)目的条带,PCR扩增产物测序结果利用核酸BLAST功能进行同源序列对比,仙台病毒和大鼠冠状病毒同源性分别为100%和99%。仙台病毒和大鼠冠状病毒的检测下限为1.56×10~2 copies/μL。特异性检测对小鼠肝炎病毒扩增,产生片段大小近似大鼠冠状病毒产物。应用建立的双重PCR体系检测人工感染仙台病毒组织DNA样本,30份DNA标本均被检出;检测94份实验动物肺组织样本,结果均阴性。结论建立的双重PCR方法操作简单、快速、特异性强、灵敏度高,能够实现对实验动物仙台病毒和大鼠冠状病毒病原体的快速检测。     

PCR-ELISA检测HBV DNA的研究

目的:建立一种敏感、特异的乙型肝炎病毒(HBV)DNA检测方法。方法:应用PCR扩增技术和核酸杂交技术结合酶促显色技术(即PCRELISA技术)来检测血清中的HBVDNA。结果:应用PCRELISA技术能够检出许多PCR琼脂糖凝胶电泳所检测不到的HBVDNA,大大地提高了检出率,而且,特异性强。结论:PCRELISA方法灵敏度高,特异性强,检测结果数据化,不受主观因素的影响 。     

Combined multimodal ctDNA analysis and radiological imaging for tumor surveillance in Non-small cell lung cancer

BackgroundRadiology is the current standard for monitoring treatment responses in lung cancer. Limited sensitivity, exposure to ionizing radiations and related sequelae constitute some of its major limitation. Non-invasive and highly sensitive methods for early detection of treatment failures and resistance-associated disease progression would have additional clinical utility.MethodsWe analyzed serially collected plasma and paired tumor samples from lung cancer patients (61 with stage IV, 48 with stages I-III disease) and 61 healthy samples by means of next-generation sequencing, radiological imaging and droplet digital polymerase chain reaction (ddPCR) mutation and methylation assays.ResultsA 62% variant concordance between tumor-reported and circulating-free DNA (cfDNA) sequencing was observed between baseline liquid and tissue biopsies in stage IV patients. Interestingly, ctDNA sequencing allowed for the identification of resistance-mediating p.T790M mutations in baseline plasma samples for which no such mutation was observed in the corresponding tissue. Serial circulating tumor DNA (ctDNA) mutation analysis by means of ddPCR revealed a general decrease in ctDNA loads between baseline and first reassessment. Additionally, serial ctDNA analyses only recapitulated computed tomography (CT) -monitored tumor dynamics of some, but not all lesions within the same patient. To complement ctDNA variant analysis we devised a ctDNA methylation assay (^methcfDNA) based on methylation-sensitive restriction enzymes. cfDNA methylation showed and area under the curve (AUC) of > 0.90 in early and late stage cases. A decrease in ^methcfDNA between baseline and first reassessment was reflected by a decrease in CT-derive tumor surface area, irrespective of tumor mutational status.ConclusionTaken together, our data support the use of cfDNA sequencing for unbiased characterization of the molecular tumor architecture, highlights the impact of tumor architectural heterogeneity on ctDNA-based tumor surveillance and the added value of complementary approaches such as cfDNA methylation for early detection and monitoring     

Detection of ctDNA with Personalized Molecular Barcode NGS and Its Clinical Significance in Patients with Early Breast Cancer

We attempted to detect circulating tumor DNA (ctDNA), taking advantage of molecular barcode next-generation sequencing (MB-NGS), which can be more easily customized to detect a variety of mutations with a high sensitivity than PCR-based methods. Sequencing with a gene panel consisting of the 13 most frequently mutated genes in breast tumors from stage I or II patients revealed 95 somatic mutations in the 12 genes in 62% (62/100) of tumors. Then, plasma DNA from each patient (n = 62) before surgery was analyzed via MB-NGS customized to each somatic mutation, resulting in the detection of ctDNA in 16.1% (10/62) of patients. ctDNA was significantly associated with biologically aggressive phenotypes, including large tumor size (P = .004), positive lymph node (P = .009), high histological grade (P < .001), negative ER (P = .018), negative PR (P = .017), and positive HER2 (P = .046). Furthermore, distant disease-free survival was significantly worse in patients with ctDNA (n = 10) than those without ctDNA (n = 52) (P < .001). Our results demonstrate that MB-NGS personalized to each mutation can detect ctDNA with a high sensitivity in early breast cancer patients at diagnosis, and it seems to have a potential to serve as a clinically useful tumor marker for predicting their prognosis.     

Detection of oncogenic mutations in paired circulating tumor DNA and circulating tumor cells in patients with hepatocellular carcinoma

Background and aimsCirculating tumor cells (CTCs) or circulating tumor DNA (ctDNA) may be used for diagnostic or prognostic purposes in patients with hepatocellular carcinoma (HCC). We aim to determine whether CTCs or ctDNA are suitable to determine oncogenic mutations in HCC patients.MethodsTwenty-six mostly advanced HCC patients were enrolled. 30 mL peripheral blood from each patient was obtained. CellSearch system was used for CTC detection. A sequencing panel covering 14 cancer-relevant genes was used to identify oncogenic mutations. TERT promoter C228T and C250T mutations were determined by droplet digital PCR.ResultsCTCs were detected in 27% (7/26) of subjects but at low numbers (median: 2 cells, range: 1–15 cells) and ctDNA in 77% (20/26) of patients. Mutations in ctDNA were identified in several genes: TERT promoter C228T (77%, 20/26), TP53 (23%, 6/26), CTNNB1 (12%, 3/26), PIK3CA (12%, 3/26) and NRAS (4%, 1/26). The TERT C228T mutation was present in all patients with one or more ctDNA mutations, or detectable CTCs. The TERT C228T and TP53 mutations detected in ctDNA were present at higher levels in matched primary HCC tumor tissue. The maximal variant allele frequency (VAF) of ctDNA was linearly correlated with largest tumor size and AFP level (Log10). CtDNA (or TERT C228T) positivity was associated with macrovascular invasion, and positivity of ctDNA (or TERT C228T) or CTCs (≥ 2) correlated with poor patient survival.ConclusionsOncogenic mutations could be detected in ctDNA from advanced HCC patients. CtDNA analysis may serve as a promising liquid biopsy to identify druggable mutations.     

Intercalation of manganese-mefenamic acid complex into double stranded of calf thymus DNA

Abstract

The interaction of the [Mn(mef)₂(phen)H₂O] complex in which mef is mefenamic acid drug and phen is 1,10 phenanthrolin ligand with calf thymus DNA (ct-DNA) was studied by using different spectroscopic methods, molecular docking and viscometery. The competitive fluorescence and UV–Vis absorption spectroscopy indicated that the complex interacted with ctDNA via intercalating binding mode with the binding constant of 1.16?×?10⁴ Lmol^?1. The thermodynamic studies showed that the reaction between the complex and ctDNA is exothermic. Furthermore, the complex induced changes in DNA viscosity. Circular dichroism spectroscopy (CD) was employed to measure the conformational changes of ctDNA in the presence of the complex and verified intercalation binding mode. The molecular modeling results illustrated that the complex interacted via intercalation by relative binding energy of ?28.45?kJ mol^?1.     

Utility of ctDNA in predicting response to neoadjuvant chemoradiotherapy and prognosis assessment in locally advanced rectal cancer: A prospective cohort study

BackgroundFor locally advanced rectal cancer (LARC) patients who receive neoadjuvant chemoradiotherapy (nCRT), there are no reliable indicators to accurately predict pathological complete response (pCR) before surgery. For patients with clinical complete response (cCR), a “Watch and Wait” (W&W) approach can be adopted to improve quality of life. However, W&W approach may increase the recurrence risk in patients who are judged to be cCR but have minimal residual disease (MRD). Magnetic resonance imaging (MRI) is a major tool to evaluate response to nCRT; however, its ability to predict pCR needs to be improved. In this prospective cohort study, we explored the value of circulating tumor DNA (ctDNA) in combination with MRI in the prediction of pCR before surgery and investigated the utility of ctDNA in risk stratification and prognostic prediction for patients undergoing nCRT and total mesorectal excision (TME).Methods and findingsWe recruited 119 Chinese LARC patients (cT3-4/N0-2/M0; median age of 57; 85 males) who were treated with nCRT plus TME at Fudan University Shanghai Cancer Center (China) from February 7, 2016 to October 31, 2017. Plasma samples at baseline, during nCRT, and after surgery were collected. A total of 531 plasma samples were collected and subjected to deep targeted panel sequencing of 422 cancer-related genes. The association among ctDNA status, treatment response, and prognosis was analyzed. The performance of ctDNA alone, MRI alone, and combining ctDNA with MRI was evaluated for their ability to predict pCR/non-pCR.Ranging from complete tumor regression (pathological tumor regression grade 0; pTRG0) to poor regression (pTRG3), the ctDNA clearance rate during nCRT showed a significant decreasing trend (95.7%, 77.8%, 71.1%, and 66.7% in pTRG 0, 1, 2, and 3 groups, respectively, P = 0.008), while the detection rate of acquired mutations in ctDNA showed an increasing trend (3.8%, 8.3%, 19.2%, and 23.1% in pTRG 0, 1, 2, and 3 groups, respectively, P = 0.02). Univariable logistic regression showed that ctDNA clearance was associated with a low probability of non-pCR (odds ratio = 0.11, 95% confidence interval [95% CI] = 0.01 to 0.6, P = 0.04). A risk score predictive model, which incorporated both ctDNA (i.e., features of baseline ctDNA, ctDNA clearance, and acquired mutation status) and MRI tumor regression grade (mrTRG), was developed and demonstrated improved performance in predicting pCR/non-pCR (area under the curve [AUC] = 0.886, 95% CI = 0.810 to 0.962) compared with models derived from only ctDNA (AUC = 0.818, 95% CI = 0.725 to 0.912) or only mrTRG (AUC = 0.729, 95% CI = 0.641 to 0.816). The detection of potential colorectal cancer (CRC) driver genes in ctDNA after nCRT indicated a significantly worse recurrence-free survival (RFS) (hazard ratio [HR] = 9.29, 95% CI = 3.74 to 23.10, P < 0.001). Patients with detectable driver mutations and positive high-risk feature (HR_feature) after surgery had the highest recurrence risk (HR = 90.29, 95% CI = 17.01 to 479.26, P < 0.001). Limitations include relatively small sample size, lack of independent external validation, no serial ctDNA testing after surgery, and a relatively short follow-up period.ConclusionsThe model combining ctDNA and MRI improved the predictive performance compared with the models derived from individual information, and combining ctDNA with HR_feature can stratify patients with a high risk of recurrence. Therefore, ctDNA can supplement MRI to better predict nCRT response, and it could potentially help patient selection for nonoperative management and guide the treatment strategy for those with different recurrence risks.

Zhen Zhang and colleagues conducted a cohort study to investigate the utility of circulating tumor DNA in predicting response to neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer in China.     

IS MEASUREMENT OF CIRCULATING TUMOR DNA OF DIAGNOSTIC USE IN PATIENTS WITH THYROID NODULES?

Objective: Circulating tumor DNA (ctDNA), a subset of cell-free DNA (cfDNA), is a potential biomarker for thyroid cancer. We determined the performance of a ctDNA panel for detecting thyroid malignancy in patients with thyroid nodules.Methods: Sixty-six patients with thyroid nodules without a prior history of cancer enrolled in a prospective, 1-year study in which blood was drawn for ctDNA analysis prior to undergoing fine-needle aspiration biopsy (FNAB) of thyroid nodules. The ctDNA panel consisted of 96-mutations in 9 cancer driver genes. The primary outcome measures were the sensitivity, specificity, and positive and negative predictive values (PPV, NPV) of our ctDNA panel for the diagnosis of thyroid malignancy as determined by pathologic and/or molecular tissue examination.Results: Results from 10 subjects could not be determined due to inadequate volume or technical issues. The final classifications of the thyroid nodules were 13 malignant and 43 benign lesions. A KRAS G12V mutation was detected in the plasma of 1 patient with stage IVA papillary carcinoma whose tissue contained the same mutation. Two of the 43 patients with benign lesions also had ctDNA detected, giving a sensitivity of 7.7%, specificity of 95.35%, PPV of 33.33%, and NPV of 77.35%. There were no significant differences between benign or malignant lesions in cfDNA levels.Conclusion: Neither cfDNA measurements nor our panel of ctDNA mutations are sensitive or specific enough to provide valuable information over FNAB. An expanded panel and the inclusion of proteomics may improve sensitivity and specificity for thyroid cancer detection.Abbreviations: cfDNA = cell-free DNA; ctDNA = circulating tumor DNA; FNAB = fine-needle aspiration biopsy; NIFTP = noninvasive follicular thyroid neoplasm with papillary-like nuclear features     

Detection of circulating tumor-derived material in peripheral blood of pediatric sarcoma patients: A systematic review

BackgroundDetection of circulating tumor-derived material (cTM) in the peripheral blood (PB) of cancer patients has been shown to be useful in early diagnosis, prediction of prognosis, and disease monitoring. However, it has not yet been thoroughly evaluated for pediatric sarcoma patients.MethodsWe searched the PubMed and EMBASE databases for studies reporting the detection of circulating tumor cells, circulating tumor DNA, and circulating RNA in PB of pediatric sarcoma patients. Data on performance in identifying cTM and its applicability in diagnosis, and evaluation of tumor characteristics, prognostic factors, and treatment response was extracted from publications.ResultsA total of 79 studies were assigned for the present systematic review, including detection of circulating tumor cells (116 patients), circulating tumor DNA (716 patients), and circulating RNA (2887 patients). Circulating tumor cells were detected in 76% of patients. Circulating DNA was detected in 63% by targeted NGS, 66% by shallow WGS, and 79% by digital droplet PCR. Circulating RNA was detected in 37% of patients.ConclusionOf the cTM from Ewing's sarcoma and rhabdomyosarcoma ctDNA proved to be the best target for clinical application including diagnosis, tumor characterization, prognosis, and monitoring of disease progression and treatment response. For osteosarcoma the most promising targets are copy number alterations or patient specific micro RNAs, however, further investigations are needed to obtain consensus on clinical utility.     

Groove binding between ferulic acid and calf thymus DNA: spectroscopic methodology combined with chemometrics and molecular docking studies

Abstract

Ferulic acid (FA), a dietary phenolic acid compound, is proved to possess numerous biological activities. Hence, this study was devoted to explore the interaction between FA and calf thymus DNA (ctDNA) by UV???vis absorption, fluorescence, circular dichroism (CD) spectroscopy combined with multivariate curve resolution-alternating least-squares (MCR???ALS) and molecular docking studies. The concentration curves and the pure spectra of compositions (FA, ctDNA and FA???ctDNA complex) were obtained by MCR???ALS approach to verify and monitor the interaction of FA with ctDNA. The groove binding mode between FA and ctDNA was confirmed by the results of melting analysis, viscosity measurements, single-stranded DNA experiments, and competitive studies. The binding constant of FA???ctDNA complex was 4.87?×?10⁴ L mol^?1 at 298?K. The values of enthalpy (ΔH°) and entropy (ΔS°) changes in the interaction were ?16.24?kJ mol^?1 and 35.02?J mol^?1 K^?1, respectively, indicating that the main binding forces were hydrogen bonds and hydrophobic interactions. The result of CD spectra suggested that a decrease in right-handed helicity of ctDNA was induced by FA and the DNA conformational transition from the B-form to the A-form. The results of docking indicated that FA binding with ctDNA in the minor groove. These findings may be conducive to understand the interaction mechanism of FA with ctDNA and the pharmacological effects of FA.

Communicated by Ramaswamy H. Sarma

     

Circulating tumor DNA dynamics and recurrence risk in patients undergoing curative intent resection of colorectal cancer liver metastases: A prospective cohort study

BackgroundIn patients with resectable colorectal liver metastases (CRLM), the role of pre- and postoperative systemic therapy continues to be debated. Previous studies have shown that circulating tumor DNA (ctDNA) analysis, as a marker of minimal residual disease, is a powerful prognostic factor in patients with nonmetastatic colorectal cancer (CRC). Serial analysis of ctDNA in patients with resectable CRLM could inform the optimal use of perioperative chemotherapy. Here, we performed a validation study to confirm the prognostic impact of postoperative ctDNA in resectable CRLM observed in a previous discovery study.Methods and findingsWe prospectively collected plasma samples from patients with resectable CRLM, including presurgical and postsurgical samples, serial samples during any pre- or postoperative chemotherapy, and serial samples in follow-up. Via targeted sequencing of 15 genes commonly mutated in CRC, we identified at least 1 somatic mutation in each patient’s tumor. We then designed a personalized assay to assess 1 mutation in plasma samples using the Safe-SeqS assay. A total of 380 plasma samples from 54 patients recruited from July 2011 to Dec 2014 were included in our analysis. Twenty-three (43%) patients received neoadjuvant chemotherapy, and 42 patients (78%) received adjuvant chemotherapy after surgery. Median follow-up was 51 months (interquartile range, 31 to 60 months). At least 1 somatic mutation was identified in all patients’ tumor tissue. ctDNA was detectable in 46/54 (85%) patients prior to any treatment and 12/49 (24%) patients after surgery. There was a median 40.93-fold (19.10 to 87.73, P < 0.001) decrease in ctDNA mutant allele fraction with neoadjuvant chemotherapy, but ctDNA clearance during neoadjuvant chemotherapy was not associated with a better recurrence-free survival (RFS). Patients with detectable postoperative ctDNA experienced a significantly lower RFS (HR 6.3; 95% CI 2.58 to 15.2; P < 0.001) and overall survival (HR 4.2; 95% CI 1.5 to 11.8; P < 0.001) compared to patients with undetectable ctDNA. For the 11 patients with detectable postoperative ctDNA who had serial ctDNA sampling during adjuvant chemotherapy, ctDNA clearance was observed in 3 patients, 2 of whom remained disease-free. All 8 patients with persistently detectable ctDNA after adjuvant chemotherapy have recurred. End-of-treatment (surgery +/− adjuvant chemotherapy) ctDNA detection was associated with a 5-year RFS of 0% compared to 75.6% for patients with an undetectable end-of-treatment ctDNA (HR 14.9; 95% CI 4.94 to 44.7; P < 0.001). Key limitations of the study include the small sample size and the potential for false-positive findings with multiple hypothesis testing.ConclusionsWe confirmed the prognostic impact of postsurgery and posttreatment ctDNA in patients with resected CRLM. The potential utility of serial ctDNA analysis during adjuvant chemotherapy as an early marker of treatment efficacy was also demonstrated. Further studies are required to define how to optimally integrate ctDNA analyses into decision-making regarding the use and timing of adjuvant therapy for resectable CRLM.Trial registrationACTRN12612000345886.     

Somatic TP53 Mutations Are Detectable in Circulating Tumor DNA from Children with Anaplastic Wilms Tumors

BACKGROUND: Diffuse anaplastic Wilms tumor (DAWT) is a rare, high-risk subtype that is often missed on diagnostic needle biopsy. Somatic mutations in TP53 are associated with the development of anaplasia and with poorer survival, particularly in advanced-stage disease. Early identification of DAWT harboring TP53 abnormalities could improve risk stratification of initial therapy and monitoring for recurrence. METHODS: Droplet digital polymerase chain reaction (ddPCR) was used to evaluate 21 samples from 4 patients with DAWT. For each patient, we assessed TP53 status in frozen tumor, matched germline DNA, and circulating tumor DNA (ctDNA) from plasma, serum, and urine collected throughout treatment. RESULTS: Mutant TP53 was detectable in ctDNA from plasma and serum in all patients. We did not detect variant TP53 in the same volume (200 μl) of urine. One patient displayed heterogeneity of TP53 in the tumor despite both histological sections displaying anaplasia. Concentration of ctDNA from plasma/serum taken prenephrectomy varied significantly between patients, ranging from 0.44 (0.05-0.90) to 125.25 (109.75-140.25) copies/μl. We observed variation in ctDNA throughout treatment, and in all but one patient, ctDNA levels fell significantly following nephrectomy. CONCLUSION: We demonstrate for the first time that ddPCR is an effective method for detection of mutant TP53 in ctDNA from children with DAWT even when there is intratumoral somatic heterogeneity. This should be further explored in a larger cohort of patients, as early detection of circulating variant TP53 may have significant clinical impact on future risk stratification and surveillance.