Clinical utility of circulating tumor DNA for colorectal cancer

Colorectal cancer (CRC) is currently the most common type of cancer in Japan, and its prognosis has improved because of development of diagnosis and advancement in treatments including surgery and chemotherapy. However, because of intratumor heterogeneity and clonal evolution, tumors often develop resistance to treatment. Genotyping tumor tissue in search of somatic genetic alterations for actionable information has become routine examination in clinical practice. However, the inherent molecular heterogeneity of metastatic tumors and the ability of cancer genomes to dynamically evolve are not properly captured by tissue specimens only. Circulating tumor DNA (ctDNA) carrying tumor‐specific genetic or epigenetic alterations is released into the circulation from tumor cells undergoing apoptosis or necrosis. Analysis of ctDNA has the potential to change clinical practice by exploiting blood rather than tissue, as a source of information. Here, we provide an overview of the characteristics of ctDNA and focus on detection methods for ctDNA, and the feasibility of use of ctDNA to monitor tumor dynamics for patients with colorectal cancer.     

循环肿瘤细胞和循环肿瘤内皮细胞计数对局限性肾癌术后复发转移监测的价值

目的:探讨局限性肾癌患者行根治或部分切除术后循环肿瘤细胞和循环肿瘤内皮细胞数量变化在预测肿瘤复发转移中的作用。方法:收集52例局限性肾癌患者术前和术后3个月7.5 mL外周血和20例健康者7.5 mL外周血。术后循环肿瘤细胞数量和循环肿瘤内皮细胞数量高于术前者纳入阳性组,反之纳入阴性组。分别对两组患者进行随访,终点事件为肿瘤复发转移,最长随访时间21个月。结果:CTC阳性组复发转移率为61.9%（13/21）,明显高于阴性组19.4%（6/31）,差异有统计学意义(P=0.003),CTEC阳性组复发转移率为57.1%（12／21）,阴性组复发转移率为22.6%（7/31）,差异有统计学意义（P=0.012）,双阳性组复发转移率为75.0%(12/16)。结论:术后3个月循环肿瘤细胞计数高于术前的肾癌患者易出现复发转移,若同时术后循环肿瘤内皮细胞计数高于术前,则提示患者复发转移率可能会更高。循环肿瘤细胞和循环肿瘤内皮细胞数量变化可作为较好的肾癌术后监测指标。     

Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients

Circulating tumor DNA (ctDNA) is a new circulating tumor biomarker which might be used as a prognostic biomarker in a way similar to circulating tumor cells (CTCs). Here, we used the high prevalence of TP53 mutations in triple negative breast cancer (TNBC) to compare ctDNA and CTC detection rates and prognostic value in metastatic TNBC patients. Forty patients were enrolled before starting a new line of treatment. TP53 mutations were characterized in archived tumor tissues and in plasma DNA using two next generation sequencing (NGS) platforms in parallel. Archived tumor tissue was sequenced successfully for 31/40 patients. TP53 mutations were found in 26/31 (84%) of tumor samples. The same mutation was detected in the matched plasma of 21/26 (81%) patients with an additional mutation found only in the plasma for one patient. Mutated allele fractions ranged from 2 to 70% (median 5%). The observed correlation between the two NGS approaches (R² = 0.903) suggested that ctDNA levels data were quantitative. Among the 27 patients with TP53 mutations, CTC count was ≥1 in 19 patients (70%) and ≥5 in 14 patients (52%). ctDNA levels had no prognostic impact on time to progression (TTP) or overall survival (OS), whereas CTC numbers were correlated with OS (p = 0.04) and marginally with TTP (p = 0.06). Performance status and elevated LDH also had significant prognostic impact. Here, absence of prognostic impact of baseline ctDNA level suggests that mechanisms of ctDNA release in metastatic TNBC may involve, beyond tumor burden, biological features that do not dramatically affect patient outcome.     

Analysis of colorectal cancer‐related mutations by liquid biopsy: Utility of circulating cell‐free DNA and circulating tumor cells

We recruited 56 colorectal cancer patients and compared the mutational spectrum of tumor tissue DNA, circulating cell‐free DNA (ccfDNA) and circulating tumor cell (CTC) DNA (ctcDNA) to evaluate the potential of liquid biopsy to detect heterogeneity of cancer. Tumor tissue DNA, ccfDNA, and ctcDNA were extracted from each patient and analyzed using next‐generation sequencing (NGS) and digital PCR. To maximize yields of CTC, three antibodies were used in the capture process. From 34 untreated patients, 53 mutations were detected in tumor tissue DNA using NGS. Forty‐seven mutations were detected in ccfDNA, including 20 not detected in tissues. Sixteen mutations were detected in ctcDNA, including five not detected in tissues. In 12 patients (35.3%), mutations not found in tumor tissues were detected by liquid biopsy: nine (26.5%) in ccfDNA only and three (8.8%) in ctcDNA only. Combination analysis of the two liquid biopsy samples increased the sensitivity to detect heterogeneity. From 22 stage IV patients with RAS mutations in their primary tumors, RAS mutations were detected in 14 (63.6%) ccfDNA and in eight (36.4%) ctcDNA using digital PCR. Mutations not detected in primary tumors can be identified in ccfDNA and in ctcDNA, indicating the potential of liquid biopsy in complementing gene analysis. Combination analysis improves sensitivity. Sensitivity to detect cancer‐specific mutations is higher in ccfDNA compared with ctcDNA.     

Detection of von Hippel-Lindau gene mutation in circulating cell-free DNA for clear cell renal cell carcinoma

The therapeutic landscape of metastatic clear cell renal cell carcinoma (ccRCC) has rapidly expanded, and there is an urgent need to develop noninvasive biomarkers that can select an optimal therapy or evaluate the response in real time. To evaluate the clinical utility of circulating tumor DNA (ctDNA) analysis in ccRCC, we established a highly sensitive assay to detect mutations in von Hippel-Lindau gene (VHL) using a combination of digital PCR and multiplex PCR–based targeted sequencing. The unique assay could detect VHL mutations with a variant allele frequency (VAF) <1.0%. Further, we profiled the mutation status of VHL in 76 cell-free DNA (cfDNA) and 50 tumor tissues from 56 patients with ccRCC using the assay. Thirteen VHL mutations were identified in cfDNA from 12 (21.4%) patients with a median VAF of 0.78% (range, 0.13%-4.20%). Of the 28 patients with VHL mutations in matched tumor tissues, eight (28.6%) also had VHL mutation in cfDNA with a median VAF of 0.47% (range, 0.13%-2.88%). In serial ctDNA analysis from one patient, we confirmed that the VAF of VHL mutation changed consistent with tumor size by radiographic imaging during systemic treatment. In conclusion, VHL mutation in cfDNA was detected only in a small number of patients even using the highly sensitive assay; nevertheless, we showed the potential of ctDNA analysis as a novel biomarker in ccRCC.     

Post-radiation circulating tumor DNA as a prognostic factor in locally advanced esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is a highly malignant and deadly tumor. Radiation therapy is one of the primary treatments for locally advanced ESCC. However, the biomarkers for prognosis of definitive radiation remain undefined. Peripheral blood circulating tumor (ct)DNA provides information of tumor genetic alterations and has been confirmed as a potential non-invasive biomarker for several types of cancer. The present study investigated the clinical implications of ctDNA detection in patients with ESCC and receiving definitive radiation therapy. Patients with locally advanced ESCC were retrospectively recruited. Plasma samples were collected before, during and following radiation therapy. Next-generation sequencing was performed to identify somatic mutations in 180 genes. A total of 69 baseline and post-radiation plasma samples were collected from 25 patients. A total of 59 non-silent single nucleotide variants were present in 33 genes. All pre-radiation and 58.3% (14/24) of post-radiation samples had at least one mutation. Patients with lymph node metastases (LNM) exhibited a higher number of pre-radiation mutations compared with those without LNM. The variables, progression-free survival (PFS) and overall survival (OS) of the patients with one baseline mutation were not significantly different compared with that in patients with more than one baseline mutation. Patients with initial ctDNA-positive post-radiation samples exhibited significantly reduced PFS (P=0.047) and OS (P=0.005) compared with that in patients with ctDNA-negative samples. The post-radiation plasma ctDNA status was an independent prognostic factor from univariate and multivariate analyses. Dynamic monitoring of ctDNA during follow-up was examined. The results indicated that ctDNA was a predictive and prognostic marker in patients with ESCC and receiving definitive radiation therapy, which may guide subsequent treatment.     

Current applications and challenges of circulating tumor DNA (ctDNA) in squamous cell carcinoma of the head and neck (SCCHN)

Liquid biopsies (LB) are emerging in the oncology field, with promising data as new diagnostic, prognostic and treatment-monitoring tools. Squamous cell carcinoma of the head and neck (SCCHN) is a heterogenous disease and many challenges remain to improve patient outcomes. Liquid biopsy could be of interest at different stages of SCCHN disease, including better screening to diagnose more patients at an early stage, early detection of relapse after curative treatment, and the implementation of precision medicine. As LB is very attractive by the ease of sampling, this field is moving fast. Therefore, it is important to be aware of the potential applications but also the limitations of these new tools in regards to technical aspects and interpretation of the data. In this review, we will first give an overview of potential clinical applications and technical challenges of circulating tumor DNA (ctDNA) and then focus on current available data of ctDNA in SCCHN. Although the literature on ctDNA analysis for SCCHN is scarce compared to other tumors, preliminary results seem to hold promise for the future, including the detection of minimal residual disease or the detection of potentially targetable events through liquid biopsy. Prospective liquid-biopsy driven clinical trials are needed to validate its clinical relevance.     

Serum cell-free DNA in renal cell carcinoma: a diagnostic and prognostic marker

BACKGROUND:

Currently, there are no established diagnostic and prognostic serum markers for renal cell carcinoma (RCC). The objective of this study was to evaluate the putative significance of serum cell‐free DNA.

METHODS:

Preoperative serum samples from 200 consecutive patients with sporadic, solid renal tumors were analyzed (157 patients with RCC and 43 patients with benign renal tumors). Quantitative real‐time polymerase chain reaction was used to assess total cell‐free DNA (ring finger protein 185 [RNF185]) and CpG island methylation of Ras association domain family member 1A (RASSF1A) von Hippel‐Lindau (VHL), prostaglandin‐endoperoxidase synthase 2 (PTGS2), and P16 (cyclin‐dependent kinase inhibitor 2A). Associations with RCC, pathologic variables, and disease‐specific survival were evaluated.

RESULTS:

Total cell‐free DNA levels and CpG island methylation of RASSF1A and VHL were highly diagnostic for RCC with an area under the receiver operating characteristic curve of 0.755, 0.705, and 0.694, respectively. VHL methylation was detected more frequently in patients with clear cell RCC than in those with other subtypes (P = .007). Total cell‐free DNA levels were higher in patients with metastatic RCC (P < .001) and necrotic RCC (P = .003) and were associated with poorer disease‐specific survival (P < .001). In multivariate analysis, the tumor stage, size, grade, and necrosis (SSIGN) score (P < .001) and categorized total cell‐free DNA levels (P = .028) were retained as independent prognostic factors.

CONCLUSIONS:

The current results indicated that cell‐free DNA represents a novel serum‐based diagnostic and prognostic biomarker for RCC. Total serum cell‐free DNA levels and CpG island methylation of RASSF1A and VHL may be useful diagnostic biomarkers for RCC. VHL methylation of cell‐free DNA is suggestive of clear cell RCC. Total serum cell‐free DNA may be a useful prognostic biomarker that may assist in tailoring postoperative surveillance and therapy. External prospective validation of these data will be required. Cancer 2012;. © 2011 American Cancer Society.     

循环肿瘤DNA在肾癌中的研究进展

基于循环肿瘤细胞(Circulating tumor cells,CTCs)和游离核酸的液体活检技术,在恶性肿瘤的诊断到随访的整个过程中都有潜在的应用价值。与传统组织活检相比,循环肿瘤DNA(Circulating tumor DNA,ctDNA)检测的优势在于风险小,可以进行连续检测,可以监测疾病的复发和随着时间推移对治疗的反应。ctDNA检测的灵敏度取决于使用的检测技术和遗传平台,还受肿瘤部位、分期、肿瘤异质性、肿瘤克隆性等的影响。ctDNA在肾细胞癌(Renal cell carcinoma,RCC)的治疗选择、耐药监测、对依维莫司的治疗反应预测等方面也得到很好的应用。本文主要对ctDNA在肾癌中的研究现状及挑战进行综述。     

代谢综合征与肾透明细胞癌相关性研究

目的 代谢综合征(metabolic syndrome,MS)是一组临床症候群,MS及其相关组分与癌症发生发展及病理特征具有密切关系.本研究旨在分析MS及其相关组分与肾透明细胞癌(clear cell cenal cell carcinoma,CCRCC)分期、分级及肿瘤大小的相关性.方法 回顾性分析2013-01-01-2015-12-30于山西医科大学第一医院就诊且病理诊断为CCRCC的375例患者的临床资料,包括年龄、性别、身高、体质量、血压、空腹血糖、生化结果、病理分期分级和肿瘤大小等.计数资料采用x2检验,计量资料以(x)±s表示,组间比较采用t检验,多因素分析采用Logistic回归分析.结果 MS组56例患者,其中男性患者32例,女性患者24例;非MS组319例患者,其中男性患者206例,女性患者113例.男女患者比较,差异无统计学意义,P=0.287.MS组与非MS组相比,年龄、吸烟、饮酒等差异无统计学意义,P值分别为0.100、0.691和0.269;而BMI指数、收缩压、空腹血糖、TG、HDL-C等差异均有统计学意义,均P＜0.001.在病理特点方面,MS与非MS相比,CCRCC病理分期(P=0.018)、分级(P=0.026)及肿瘤大小(P=0.026),差异均有统计学意义.MS相关疾病与CCRCC分期分析,糖尿病(P＜0.001)、高血压(P=0.015)、血脂紊乱(P=0.006)与CCRCC的分期有关.结论 CCRCC合并MS者病理分期较高、分级较低、肿瘤更大,糖尿病、高血压和血脂紊乱都可增加CCRCC的病理分期.     

Clinical significance of programmed death‐1 and programmed death‐ligand 1 expression in the tumor microenvironment of clear cell renal cell carcinoma

Recently, immunotherapy based on blocking immune checkpoints with programmed death‐1 (PD‐1) or PD‐ligand 1 (PD‐L1) Abs has been introduced for the treatment of advanced clear cell renal cell carcinoma (ccRCC), especially tumors resistant to vascular endothelial growth factor‐tyrosine kinase inhibitors (VEGF‐TKIs), but the significance of their expression in the tumor microenvironment is unclear. We investigated these immune checkpoint markers in tumor cells and tumor‐infiltrating immune cells (TIIC) in the tumor microenvironment of 100 untreated and 25 VEGF‐TKI‐treated primary ccRCC tissues. Upregulated expression of PD‐1 and PD‐L1 by TIIC, and PD‐L1 by tumor cells was associated with the histological grade and unfavorable prognosis of RCC patients. High PD‐1 and PD‐L1 expression by TIIC was associated with a poorer response to VEGF‐TKI, whereas PD‐L1 expression by tumor cells did not affect the efficacy of the treatment. Furthermore, increased PD‐1‐positive TIIC and PD‐L1‐positive TIIC were observed in tumors treated with VEGF‐TKIs compared with those in untreated tumors. Our data suggest that PD‐1 and PD‐L1 expression by TIIC in the tumor microenvironment is involved in treatment resistance, and that sequential therapy with immune checkpoint inhibitors could be a promising therapeutic strategy for ccRCC resistant to VEGF‐TKI treatment.     

Clinical implications of plasma circulating tumor DNA in gynecologic cancer patients

Molecular characterization of cancers is important in dictating prognostic factors and directing therapy. Next‐generation sequencing of plasma circulating tumor DNA (ctDNA) offers less invasive, more convenient collection, and a more real‐time representation of a tumor and its molecular heterogeneity than tissue. However, little is known about the clinical implications of ctDNA assessment in gynecologic cancer. We describe the molecular landscape identified on ctDNA, ctDNA concordance with tissue‐based analysis, and factors associated with overall survival (OS) in gynecologic cancer patients with ctDNA analysis. We reviewed clinicopathologic and genomic information for 105 consecutive gynecologic cancer patients with ctDNA analysis, including 78 with tissue‐based sequencing, enrolled in the Profile‐Related Evidence Determining Individualized Cancer Therapy ({"type":"clinical-trial","attrs":{"text":"NCT02478931","term_id":"NCT02478931"}}NCT02478931) trial at the University of California San Diego Moores Cancer Center starting July 2014. Tumors included ovarian (47.6%), uterine (35.2%), cervical (12.4%), vulvovaginal (2.9%), and unknown gynecologic primary (1.9%). Most ovarian and uterine cancers (86%) were high grade. 34% (N = 17) of ovarian cancers had BRCA alterations, and 22% (N = 11) were platinum sensitive. Patients received median 2 (range 0–13) lines of therapy prior to ctDNA collection. Most (75.2%) had at least one characterized alteration on ctDNA analysis, and the majority had unique genomic profiles on ctDNA. Most common alterations were TP53 (N = 59, 56.2% of patients), PIK3CA (N = 26, 24.8%), KRAS (N = 14, 13.3%), BRAF (N = 10, 9.5%), ERBB2 (N = 8, 7.6%), and MYC (N = 8, 7.6%). Higher ctDNA maximum mutation allele frequency was associated with worse OS [hazard ratio (HR): 1.91, P = 0.03], while therapy matched to ctDNA alterations (N = 33 patients) was independently associated with improved OS (HR: 0.34, P = 0.007) compared to unmatched therapy (N = 28 patients) in multivariate analysis. Tissue and ctDNA genomic results showed high concordance unaffected by temporal or spatial factors. This study provides evidence for the utility of ctDNA in determining outcome and individualizing cancer therapy in patients with gynecologic cancer.

Abbreviations

BMI

body mass index

BRCA

breast cancer susceptibility gene

CAP

College of American Pathologists

CI

confidence interval

CLIA

Clinical Laboratory Improvement Amendments

ctDNA

circulating tumor DNA

Del

deletion

HR

hazard ratio

ID

identification number

In/del

insertion/deletion

MAF

mutation allele frequency

NR

not reached

OS

overall survival

PREDICT

Profile‐Related Evidence Determining Individualized Cancer Therapy

SE

standard error

SNV

single nucleotide variant

UCSD

University of California San Diego

VUS

variants of unknown significance

     

肾细胞癌相关分子标志物研究现状及进展

近年,有关肾细胞癌病理学、遗传性及分子生物学方面的研究取得显著进展,这对提高肾细胞癌早期诊断、预后判断及治疗水平有重要意义。目前研究的多个肾细胞癌相关分子标志物(如CAⅨ、B7-H1等)对于肾细胞癌患者的预后判断有一定意义。本文总结了近年来与肾细胞癌患者有关的肿瘤标志物,分析其与肾细胞癌的诊断、治疗和预后方面关系。     

35例肾癌术后放疗的临床分析

目的：研究Ⅱ、Ⅲ期肾癌术后放疗的作用。方法：１９９２年１月－１９９５年１月，３５例Ⅱ和Ⅲ期肾癌行术后放疗。照射范围包括肾瘤床、肾蒂、主动脉旁及下腔静脉旁淋巴结区运用ＴＰＳ技术，采用直线加速器１８ＭＶＸ线照射，ＤＴ４５－５０Ｇｙ／５周。结果：失访２例按死亡处理，随访率９４．４％，全组无复发病例，三年及五年生存率各为５７％（２０／３５）和４５．７％（１６／３５）。三年生存率Ⅱ期为６９．２％（９／１３）     

The impact of tumor burden characteristics in patients with metastatic renal cell carcinoma treated with sunitinib

BACKGROUND:

An important goal of noncurative therapy for metastatic renal cell carcinoma (mRCC) is tumor burden (TB) control. However, to the authors' knowledge, the impact of TB characteristics on clinical outcome has not been studied in patients with mRCC who were treated with vascular endothelial growth factor‐targeted therapy.

METHODS:

Patients with clear cell mRCC who were treated with sunitinib between June 2004 and October 2007 were retrospectively identified. Computed tomography scans were re‐reviewed from baseline, at the time of maximal TB shrinkage (TS) while receiving sunitinib, and at the time of progressive disease (PD). Measurements were recorded as per Response Evaluation Criteria In Solid Tumors (RECIST).

RESULTS:

A total of 69 patients were identified. The majority (54%) were classified as being of favorable risk using Cleveland Clinic Foundation Tyrosine Kinase Inhibitor (CCF TKI) risk group criteria. All patients underwent prior nephrectomy and 77% received prior systemic therapy. There were a median of 8 metastatic deposits across all organs (range, 1‐27 deposits). The median TB at the initiation of therapy was 14.0 cm (range, 3.0 cm‐42.2 cm). On multivariable analysis, baseline characteristics of disease confined to above the diaphragm (P = .03) and a total TB <13 cm (P = .09) were found to be independent positive predictors of progression‐free survival. A+ baseline, total number of metastases <10 (P < .001) and TB above the diaphragm <6.5 cm (P = .05) were found to be independent positive predictors of overall survival (OS). Increased TS while receiving sunitinib was found to be significantly associated with OS (P < .001). At the time of PD, tumor location and pattern of disease progression were not found to be associated with survival as measured from the date of PD. However, total TB (P = .003) and total number of metastatic deposits (≤12 vs >12; P < .001) were found to be significant predictors of survival after PD.

CONCLUSIONS:

The results of the current study indicate that TB characteristics are associated with clinical outcome in patients with mRCC who are treated with sunitinib. Cancer 2011. © 2010 American Cancer Society.     

Expression and significance of tumor infiltrating dendritic cells in renal cell carcinoma

Dendritic cells(DCs) are considered to be the most effective antigen-presenting cells(APCs) that play a major role in initiating the antitumor immune response. Tumor infiltrating dendritic cells(TIDCs) are DCs that exist in microenviroment of tumors. Although the function and significance of TIDCs in the immune response to tumor have never been clearly demonstrated, their location suggests that they play a critical role in the presentation of tumor antigen to specific T cells[1]. For the…     

肾细胞癌的相关标志物

肾细胞癌是成年人肾脏最常见的恶性肿瘤,其自然过程极难预料,仅依靠分期和分级等尚不能提供足够的预后信息.寻找适合的肿瘤标志物有助于该病的诊断和监测,提供更有效的预后信息,确定发生复发和转移的风险,甚至可以作为肿瘤靶向治疗的标靶,并有助于深入了解肾细胞癌的疾病发展过程和揭示与之有关的分子生物学信息.本文回顾分析了近年来RCC相关性肿瘤标志物的最新研究进展情况.     

Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma

Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) have been recently investigated in several cancer types, but their respective clinical significance remains to be determined. In our prospective study, we compared the detection rate and the prognostic value of these two circulating biomarkers in patients with metastatic uveal melanoma. GNAQ/GNA11 mutations were characterized in archived tumor tissue. Using a highly sensitive and mutation‐specific bidirectional pyrophosphorolysis‐activated polymerization (bi‐PAP) technique, GNAQ c.626A>T, GNAQ c.626A>C and GNA11 c.626A>T copy numbers were quantified in plasma from 12 mL of blood. CTCs were detected at the same time in 7.5 mL of blood by the CellSearch® technique. Patient characteristics and outcome were prospectively collected. CTCs (≥1) were detected in 12 of the 40 included patients (30%, range 1–20). Among the 26 patients with known detectable mutations, ctDNA was detected and quantified in 22 (84%, range 4–11,421 copies/mL). CTC count and ctDNA levels were associated with the presence of miliary hepatic metastasis (p = 0.004 and 0.03, respectively), with metastasis volume (p = 0.005 and 0.004) and with each other (p < 0.0001). CTC count and ctDNA levels were both strongly associated with progression‐free survival (p = 0.003 and 0.001) and overall survival (p = 0.0009 and <0.0001). In multivariate analyses, ctDNA appeared to be a better prognostic marker than CTC. In conclusion, ctDNA and CTC are correlated and both have poor prognostic significance. CTC detection can be performed in every patient but, in patients with detectable mutations, ctDNA was more frequently detected than CTC and has possibly more prognostic value.     

Combination of RERG and ZNF671 methylation rates in circulating cell‐free DNA: A novel biomarker for screening of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia, hence, identifying easily detectable biomarkers for NPC screening is essential for better diagnosis and prognosis. Using genome‐wide and targeted analyses based on next‐generation sequencing approaches, we previously showed that gene promoters are hypermethylated in NPC tissues. To confirm whether DNA methylation rates of genes could be used as biomarkers for NPC screening, 79 histologically diagnosed NPC patients and 29 noncancer patients were recruited. A convenient quantitative analysis of DNA methylation using real‐time PCR (qAMP) was carried out, involving pretreatment of tissue DNA, and circulating cell‐free DNA (ccfDNA) from nonhemolytic plasma, with methylation‐sensitive and/or methylation‐dependent restriction enzymes. The qAMP analyses revealed that methylation rates of RERG, ZNF671, ITGA4, and SHISA3 were significantly higher in NPC primary tumor tissues compared to noncancerous tissues, with sufficient diagnostic accuracy of the area under receiver operating characteristic curves (AUC). Interestingly, higher methylation rates of RERG in ccfDNA were statistically significant and yielded a very good AUC; however, those of ZNF671, ITGA4, and SHISA3 were not significant. Furthermore, the combination of methylation rates of RERG and ZNF671 in ccfDNA showed higher diagnostic accuracy than either of them individually. In conclusion, the methylation rates of specific genes in ccfDNA can serve as novel biomarkers for early detection and screening of NPC.