Epigenetic markers for noninvasive early detection of nasopharyngeal carcinoma by methylation‐sensitive high resolution melting

Nasopharyngeal carcinoma (NPC) is a human malignancy that is closely associated with Epstein‐Barr Virus (EBV). Early diagnosis of NPC will greatly improve the overall survival. However, current EBV DNA marker detection still lacks the predictive value to perform well in high‐risk populations for early detection of NPC. Since aberrant promoter hypermethylation of tumor suppressor genes (TSGs) is widely considered to be an important epigenetic change in early carcinogenesis, this study identified a panel of methylation markers for early detection of NPC and also assessed the clinical usefulness of these markers with noninvasive plasma specimens instead of biopsies. MS‐HRM assays were carried out to assess the methylation status of a selected panel of four TSGs (RASSF1A, WIF1, DAPK1 and RARβ2) in biopsies, NP brushings and cell‐free plasma from NPC patients. High‐risk and cancer‐free groups were used as controls. DNA methylation panel showed higher sensitivity and specificity than EBV DNA marker in cell‐free plasma from NPC patients at early Stages (I and II) and in addition to the EBV DNA marker, MS‐HRM test for plasma and NP brushing DNA methylation significantly increased the detection rate at all NPC stages as well as local recurrence, using this selected four‐gene panel (p < 0.05). MS‐HRM assay on a selected gene panel has great potential to become a noninvasive and complementary test for NPC early and recurrent detection in combination with the EBV DNA test to increase the sensitivity for NPC detection at an early stage.     

Frequent hypermethylation of RASSF1A and TSLC1, and high viral load of Epstein-Barr Virus DNA in nasopharyngeal carcinoma and matched tumor-adjacent tissues

We examined the promoter hypermethylation of tumor-suppressor genes RASSF1A and TSLC1, quantitated EBV DNA load in nasopharyngeal carcinoma (NPC) tissues (T tissues), and matched tumor-adjacent tissues outside 0.5 cm (P tissues) and outside 1.0 cm (Z tissues) to evaluate the role of promoter hypermethylation of RASSF1A and TSLC1 as well as viral load in the pathogenesis of NPC. Methylation-specific polymerase chain reaction (PCR) for RASSF1A and TSLC1 and quantitative real-time PCR analysis of EBV DNA were performed on matched T, P, and Z tissues (n = 28) as well as chronic nasopharyngitis tissues (n = 8). Hypermethylated RASSF1A was frequently detected in the T (82%) and P tissues (75%), but less frequently in Z tissues (46%). he average quantities of EBV DNA (copies/microg DNA) in matched T, P, and Z tissues were 673,000, 90,000, and 7000. The differences of promoter hypermethylation of RASSF1A and EBV viral load among T, P, and Z tissues were statistically significant, with more frequent methylation and higher viral load detected when tissues examined were nearer to the NPC tissues. Our results suggest that aberrant hypermethylation of RASSF1A and high EBV load might be important events in NPC pathogenesis, and they may be useful molecular diagnostic markers for this cancer.     

High frequency of promoter hypermethylation of the death-associated protein-kinase gene in nasopharyngeal carcinoma and its detection in the peripheral blood of patients.

PURPOSE: Death-associated protein (DAP)-kinase gene is frequently inactivated by promoter hypermethylation in cancer. The aim of this study was to evaluate the promoter methylation status of the DAP-kinase gene in nasopharyngeal carcinoma (NPC). EXPERIMENTAL DESIGN: The methylation status was evaluated by methylation-specific PCR (MSP). Thirty-two NPC biopsy specimens, plasma and buffy coat of 12 patients, 5 NPC cell lines, 3 normal nasopharyngeal biopsy tissues, and 2 normal nasopharyngeal epithelial primary cultures were included in this study. RESULTS: There was no promoter hypermethylation in all 3 normal nasopharyngeal tissues and 2 normal nasopharyngeal primary cultures. Hypermethylation was found in 24 (75%) NPC primary tumor biopsies and 4 (80%) NPC cell lines. Of the 24 patients with hypermethylation of DAP-kinase promoter in the primary tumors, 12 patients had their plasma and buffy coat DNA available for MSP study. Hypermethylated DAP-kinase promoter was detectable in 5 patients in the plasma but not in the buffy coat, 2 patients in the buffy coat but not in the plasma, and 1 patient in both plasma and buffy coat. Four patients had no detectable hypermethylated DAP-kinase promoter in both plasma and buffy coat. Hypermethylation of DAP-kinase promoter was found in both early- and late-stage NPC. CONCLUSIONS: Our results show that hypermethylation of the DAP-kinase promoter is a common early event in NPC. The high frequency of identification of hypermethylated DAP-kinase promoter in plasma and buffy coat of NPC patients illustrates its potential clinical application as tumor marker for the diagnosis and monitoring of treatment result.     

Quantification of Epstein–Barr virus DNA load in nasopharyngeal brushing samples in the diagnosis of nasopharyngeal carcinoma in southern China

Nasopharyngeal carcinoma (NPC) is highly incident in southern China, where 40% of world's new cases arise each year. Detection of Epstein–Barr virus (EBV) DNA load in nasopharyngeal (NP) brush/swab samples has gradually been established as a method for diagnosis of NPC. However, its applicable value in NPC diagnosis has never been investigated in southern China. It is important to explore whether such a test could be applicable to our local population. A total of 245 consecutive participants undergoing NP brushing examination were recruited to obtain the NP brushing samples in this study. Quantitative PCR assays were used to obtain the EBV DNA load. Mann–Whitney, ANOVA and receiver operating characteristic tests were used to analyze its diagnostic value. NP brushing samples from NPC patients showed extremely high levels of EBV DNA load (mean = 46360 copy/ng DNA) compared to its expression from non‐NPC control (mean = 28 copy/ng DNA) and high‐risk control (mean = 50 copy/ng DNA) groups. It produced 96% sensitivity and 97% specificity, at the COV = 225 copy/ng DNA. Furthermore, EBV DNA load could reflect disease progress. Our data showed a better performance of EBV DNA load in NP brushing samples compared with an initial biopsy, immunoglobulin A (IgA) antibody titers to viral capsid antigen in serum and EBV DNA load in plasma. Detection of EBV DNA load in NP brushing samples could be an effective supplement for NPC diagnosis. Being minimally invasive and low cost, NP brush sampling combined with EBV DNA detection demonstrates great potential for screening high‐risk populations for NPC.     

Quantitative detection of Epstein-Barr virus DNA methylation in the diagnosis of nasopharyngeal carcinoma by blind brush sampling

Detecting EBV DNA load in nasopharyngeal (NP) brushing samples for the diagnosis of nasopharyngeal carcinoma (NPC) has attracted widespread attentions. Currently, NP brush sampling mostly relies on endoscopic guidance, and there are few reports on diagnostic markers suitable for nonguided conditions (blind brush sampling), which is of great significance for extending its application. One hundred seventy nasopharyngeal brushing samples were taken from 98 NPC patients and 72 non-NPC controls under the guidance of endoscope, and 305 blind brushing samples were taken without endoscopic guidance from 164 NPC patients and 141 non-NPC controls (divided into discovery and validation sets). Among these, 38 cases of NPC underwent both endoscopy-guided NP brushing and blind brushing. EBV DNA load targeting BamHI-W region and EBV DNA methylation targeting 11029 bp CpG site located at Cp-promoter region were detected by quantitative polymerase chain reaction (q-PCR). EBV DNA load showed good classification accuracy for NPC in endoscopy-guided brushing samples (AUC = 0.984). However, in blind bushing samples, the diagnostic performance was greatly reduced (AUC = 0.865). Unlike EBV DNA load, the accuracy of EBV DNA methylation was less affected by brush sampling methods, whether in endoscopy-guided brushing (AUC = 0.923) or blind brushing (AUC = 0.928 in discovery set and AUC = 0.902 in validation set). Importantly, EBV DNA methylation achieved a better diagnostic accuracy than EBV DNA load in blind brushing samples. Overall, detection of EBV DNA methylation with blind brush sampling shows great potential in the diagnosis of NPC and may facilitate its use in nonclinical screening of NPC.     

Noninvasive diagnosis of nasopharyngeal carcinoma: nasopharyngeal brushings reveal high Epstein-Barr virus DNA load and carcinoma-specific viral BARF1 mRNA

Nasopharyngeal carcinoma (NPC) is the most prevalent ENT-tumour in Indonesia. We investigated the primary diagnostic value of Epstein-Barr virus (EBV) DNA load and mRNA detection in noninvasive nasopharyngeal (NP) brushings, obtained prospectively from consecutive Indonesian ENT-patients with suspected NPC (N=106) and controls. A subsequent routine NP biopsy was taken for pathological examination and EBER-RISH, yielding 85 confirmed NPC and 21 non-NPC tumour patients. EBV DNA and human DNA load were quantified by real-time PCR. NP brushings from NPC patients contained extremely high EBV DNA loads compared to the 88 non-NPC controls (p<0.0001). Using mean EBV DNA load in controls plus 3 SD as cut-off value, specificity, sensitivity, positive and negative predictive values were 98, 90, 97 and 91%, respectively. Epstein-Barr nuclear antigen 1 (EBNA1) and the carcinoma-specific BARF1 mRNA were detected by nucleic acid sequence based amplification and found in 86 and 74% of NP brushings, confirming NPC tumour cell presence. EBV RNA positivity was even higher in fresh samples stored at -80 degrees C until RNA expression analyses (88% for both EBNA1 and BARF1). EBV RNA-negative NP brushings from proven NPC cases had the lowest EBV DNA loads, indicating erroneous sampling. No EBV mRNA was detected in NP brushings from healthy donors and non-NPC patients. In conclusion, EBV DNA load measurement combined with detection of BARF1 mRNA in simple NP brushings allows noninvasive NPC diagnosis. It reflects carcinoma-specific EBV involvement at the anatomical site of tumour development and reduces the need for invasive biopsies. This procedure may be useful for confirmatory diagnosis in large serological NPC screening programs and has potential as prognostic tool.     

Hypermethylation of epithelial-cadherin gene promoter is associated with Epstein-Barr virus in nasopharyngeal carcinoma

Background: Epstein-Barr virus (EBV) is documented as the important etiologic agent of nasopharyngeal carcinoma (NPC) but the mechanism of development and pathogenesis induced by EBV is presently unclear. Hypermethylation of epithelial-cadherin (E-cadherin) promoter has been shown to be induced in NPC cell line by EBV LMP1 via DNA methyltransferase activation. EBV genomes and hypermethylation of E-cadherin promoter were investigated in NPC tissues to evaluate the role of EBV in the hypermethylation and pathogenesis of NPC. Methods: Methylation-specific polymerase chain reaction (MSP) was performed to detect E-cadherin promoter hypermethylation in paraffin embedded tissues from patients with NPC and normal nasopharyngeal tissues. EBV genomes were detected by PCR in the tissue samples. Results: Hypermethylation of E-cadherin promoter and EBV were predominantly detected in undifferentiated and non-keratinizing NPC compared to those in squamous cell NPC. Hypermethylation of E-cadherin was found in 28 of 38 (73.7%) patient samples. EBV was detected in 22 of the 28 (78.6%) NPC samples demonstrating E-cadherin hypermethylation. EBV genomes and hypermethylation were not detected in normal nasopharyngeal tissues. Significant association was found between E-cadherin hypermethylation and EBV genomes (p < 0.001; Fisher's exact test). Hypermethylation of E-cadherin was more frequently detected in advanced stages compared to those in early stages of NPC (p = 0.036; Fisher's exact test). Conclusions: The high incidence of EBV with the consistency of E-cadherin hypermethylation, particularly in undifferentiated and non-keratinizing NPC suggests the role of EBV in the hypermethylation. EBV exists at early stage of NPC that induces the hypermethylation and contributes to progression of the disease to the advanced stage of NPC.     

Hypermethylation of epithelial-cadherin gene promoter is associated with Epstein-Barr virus in nasopharyngeal carcinoma

Background: Epstein-Barr virus (EBV) is documented as the important etiologic agent of nasopharyngeal carcinoma (NPC) but the mechanism of development and pathogenesis induced by EBV is presently unclear. Hypermethylation of epithelial-cadherin (E-cadherin) promoter has been shown to be induced in NPC cell line by EBV LMP1 via DNA methyltransferase activation. EBV genomes and hypermethylation of E-cadherin promoter were investigated in NPC tissues to evaluate the role of EBV in the hypermethylation and pathogenesis of NPC. Methods: Methylation-specific polymerase chain reaction (MSP) was performed to detect E-cadherin promoter hypermethylation in paraffin embedded tissues from patients with NPC and normal nasopharyngeal tissues. EBV genomes were detected by PCR in the tissue samples. Results: Hypermethylation of E-cadherin promoter and EBV were predominantly detected in undifferentiated and non-keratinizing NPC compared to those in squamous cell NPC. Hypermethylation of E-cadherin was found in 28 of 38 (73.7%) patient samples. EBV was detected in 22 of the 28 (78.6%) NPC samples demonstrating E-cadherin hypermethylation. EBV genomes and hypermethylation were not detected in normal nasopharyngeal tissues. Significant association was found between E-cadherin hypermethylation and EBV genomes (p < 0.001; Fisher's exact test). Hypermethylation of E-cadherin was more frequently detected in advanced stages compared to those in early stages of NPC (p = 0.036; Fisher's exact test). Conclusions: The high incidence of EBV with the consistency of E-cadherin hypermethylation, particularly in undifferentiated and non-keratinizing NPC suggests the role of EBV in the hypermethylation. EBV exists at early stage of NPC that induces the hypermethylation and contributes to progression of the disease to the advanced stage of NPC.     

Promoter hypermethylation of multiple genes in nasopharyngeal carcinoma.

PURPOSE: The methylation profile of nasopharyngeal carcinoma (NPC) has been investigated by a candidate gene approach. EXPERIMENTAL DESIGN: Four NPC cell lines, 4 NPC xenografts, 33 NPC primary tumors, and 6 samples of normal nasopharyngeal epithelium were subjected to methylation-specific PCR for analysis of promoter methylation of eight cancer-related genes. These eight genes were RASSF1A, RARbeta2, DAP-kinase, p16, p15, p14, MGMT, and GSTP1. The correlation between methylation status of these genes and clinical features such as stage, local-regional recurrence, distant metastasis, and survival has been analyzed. RESULTS: The incidence of promoter methylation in NPC samples was 84% for RASSF1A, 80% for RARbeta2, 76% for DAP-kinase, 46% for p16, 17% for p15, 20% for p14, 20% for MGMT, and 3% for GSTP1. No methylation of these genes was detected in the six normal nasopharyngeal epithelium samples. All NPC tumor samples in this study displayed aberrant methylation in at least one of these eight genes. No significant correlation between methylation status of these genes and clinical parameters of the patients was found. CONCLUSIONS: A high frequency of aberrant methylation of the 5' CpG island of the RASSF1A, RARbeta2, DAP-kinase, and p16 genes in the present study was noted. Our findings suggest that methylation of the genes in the critical pathways is common in NPC.     

Predicting hepatocellular carcinoma by detection of aberrant promoter methylation in serum DNA.

PURPOSE: Most hepatocellular carcinomas (HCC) are diagnosed at an advanced stage. Hypermethylation of CpG islands in promoter regions is now recognized as an important early event in carcinogenesis and detection of methylated DNA has been suggested as a potential biomarker for early detection of cancer. There are no studies on epigenetic changes in samples from HCC patients before diagnosis. We explored the possible diagnostic value of aberrant promoter hypermethylation of three tumor suppressor genes in serum DNA for early detection of HCC. EXPERIMENTAL DESIGN: Aberrant promoter hypermethylation was investigated in DNA isolated from the serum of 50 HCC patients who provided repeated blood samples before diagnosis and 50 controls enrolled in a cancer screen program in Taiwan. Methylation-specific PCR was used to determine the methylation status of p16, p15, and ras association domain family 1A (RASSF1A). RESULTS: Among cases, aberrant methylation was found in serum DNA 1 to 9 years before clinical HCC diagnosis. RASSF1A had the highest frequency of hypermethylation with 35 (70%) cases having at least one positive sample compared with 22 (44%) for p16 and 12 (22%) for p15. Six subjects were hypermethylation negative for all three genes. For the 50 controls, promoter hypermethylation was found in three and two subjects for RASSF1A and p16, respectively; none had methylation of p15. A receiver operating characteristic curve that included clinical risk factors (age, HBsAg status, anti-hepatitis C virus status, smoking, and alcohol status) and hypermethylation biomarkers gave an overall predictive accuracy of 89% with sensitivity and specificity 84% and 94%, respectively. CONCLUSIONS: The analysis of epigenetic changes on RASSF1A, p16, and p15 tumor suppressor genes in serum DNA may be a valuable biomarkers for early detection in populations at high risk of HCC.     

Comparison of plasma Epstein-Barr virus (EBV) DNA levels and serum EBV immunoglobulin A/virus capsid antigen antibody titers in patients with nasopharyngeal carcinoma

BACKGROUND: Serologic measurement of antibodies to Epstein-Barr virus (EBV) immunoglobulin A/viral capsid antigen (IgA/VCA) and early antigen (IgA/EA) has been used widely to screen for nasopharyngeal carcinoma (NPC) in China. Recently, it was found that plasma EBV DNA concentration is an indicator for the staging and prognosis of patients with NPC. To determine whether there is a correlation between plasma EBV DNA levels and serum levels of IgA/VCA, the authors measured both in patients with NPC and in a control group. METHODS: Real-time polymerase chain reaction was used for quantitative analysis of plasma EBV DNA concentration, and enzyme-linked immunoadsorbent assay was used to measure EBV VCA/IgA in patients with primary NPC (n = 120 patients), locally recurrent NPC (n = 8 patients), and distant metastatic NPC (n = 21 patients) among 76 patients with NPC after the completion of radiotherapy, in 60 patients with NPC in clinical remission, in 38 patients with non-NPC tumors, and in 47 control individuals. RESULTS: The median plasma EBV DNA levels were 6200 copies/mL, 9200 copies/mL, and 2050 copies/mL in patients with primary, locally recurrent, and distant metastatic NPC, respectively, but declined to 0 copies/mL in patients with clinically remissive NPC, in patients who completed radiotherapy, in patients with non-NPC tumors, and in the control group. In contrast, EBV VCA/IgA titers and detection rates remained high in all NPC groups. Plasma EBV DNA levels were significantly higher in patients who had serum VCA/IgA titers > or = 1:640 (median, 83,450 copies/mL) compared with the levels in patients who had titers < or = 1:320 (median, 17,200 copies/mL). Patients with NPC who had advanced TNM stage (Stages III and IV; median, 8530 copies/mL) and T classification (T3 and T4 tumors; median, 8530 copies/mL) had significantly higher plasma EBV DNA levels compared with patients who had early TNM stage (Stages I and II; median, 930 copies/mL) and T classification (T1 and T2 tumors; median, 3700 copies). Patients who had advanced TNM stage NPC had significantly higher mean VCA/IgA titers (1:424) compared with patients who had early TNM stage NPC (1:246), but there was no correlation between IgA/VCA titer and T or N classification of NPC. CONCLUSIONS: The results suggest that plasma EBV DNA detection is a more sensitive and specific marker than the serum IgA/VCA titer for the diagnosis and monitoring of patients with NPC. These findings provide convincing evidence for the use of plasma EBV DNA measurements for the early diagnosis and staging of NPC as well as for monitoring recurrence and metastasis of this tumor.     

Epstein-Barr virus DNA in serum/plasma as a tumor marker for nasopharyngeal cancer.

Nasopharyngeal cancer (NPC) constitutes a type of carcinoma encountered frequently in Southern China, among Eskimos of the Arctic region, and to a lesser extent in Southeast Asia. Because EBV DNA present in plasma or serum of NPC patients has proven to represent a promising noninvasive tumor marker, the present study was designed to determine the incidence of serum/plasma EBV DNA by nested PCR during various disease management stages. By this method, we could detect EBV DNA in plasma/serum of 98 of 167 NPC patients prior to treatment, compared with 10 of 77 samples derived from healthy blood donors serving as controls, with a similar prevalence observed in plasma versus serum. Investigation of 13 patients subjected to radiotherapy revealed plasma EBV DNA to persist in the plasma of one case, whereas among the remaining patients, it had vanished during the early phase of treatment. Finally, with 52 samples derived from 37 NPC patients during follow-up, we established 100% specificity and 0% false-positive rate for plasma DNA detection by nested PCR. Moreover, we subjected 24 known EBV DNA-positive serum samples to DNase digestion prior to DNA extraction and amplification to differentiate between free and encapsulated viral DNA, which demonstrated complete absence of the human beta-globin genomic DNA in contrast to EBV DNA detectable in 14 samples. In conclusion, applying this noninvasive method, serum/plasma EBV DNA constitutes a reliable tumor marker prior to, during, and after treatment of NPC.     

Laboratory markers of tumor burden in nasopharyngeal carcinoma: a comparison of viral load and serologic tests for Epstein-Barr virus

Epstein-Barr virus (EBV) is present within the tumor cells of most cases of nasopharyngeal carcinoma (NPC). Recent studies suggest that tumor burden is proportional to the level of EBV DNA in blood and that rapid blood testing can be used to guide therapeutic intervention. The relative utility of viral load vs. serology has been insufficiently studied. In our study, EBV viral load was measured by quantitative PCR using either real-time or end-point detection systems in serum samples from 124 NPC patients (93 pretreatment, 13 relapsed, 18 in remission) and 40 controls. Serologic titers against EBV early antigen were measured in the same serum samples. EBV DNA was detectable in 64 of 93 untreated NPC patients (69%; mean viral load 11,211 copies/ml), 11 of 13 relapsed NPC patients (85%; mean 53,039 copies/ml) and 0 of 18 remission patients. EBV DNA was detectable in only 1 of 40 non-NPC controls (3%). In 34 instances where paired plasma and serum samples were available for testing, both were effective sample types, and there was no significant difference between end-point and real-time methods for measuring viral load. Early antigen (EA) IgA and IgG titers were elevated in most NPC patients regardless of whether their disease was active or in remission. EBV viral load was more informative than was EA serology for distinguishing remission from relapsed disease. EBV DNA measurement appears to be a noninvasive way to monitor tumor burden after therapy.     

Quantitative analysis of the transrenal excretion of circulating EBV DNA in nasopharyngeal carcinoma patients

PURPOSE: The existence of transrenal clearance of circulating cell-free DNA is controversial. In this study, we used NPC as a model to investigate if circulating EBV DNA can be excreted into urine and to quantify the contribution of renal excretion to the clearance of plasma EBV DNA. EXPERIMENTAL DESIGN: Quantitative analysis of urine EBV DNA was done for 74 NPC patients using real-time PCR with two different amplicon sizes. The urine concentration of EBV DNA was expressed as copies per millimole of creatinine (copies/mmol Cr) to minimize the effects of interindividual variations in hydration status. RESULTS: EBV DNA was detectable in the urine of 56% NPC patients using a 59-bp real-time PCR assay. The median urine EBV DNA concentrations measured by the 59- and 76-bp assays were 7,040 and 290 copies/mmol Cr, respectively. Patients with detectable urine EBV DNA had significantly higher plasma concentrations, with a positive correlation between the plasma and urine concentrations of EBV DNA. The fraction of plasma EBV DNA excreted into the urine was 0.0026% of that for creatinine. CONCLUSIONS: We have shown that circulating EBV DNA can be excreted transrenally into urine in NPC patient and the fraction of excretion is negatively associated with the size of the DNA molecules. Because there is a positive correlation between plasma and urine EBV DNA concentration, urine EBV DNA analysis may potentially be applicable as an ultra-noninvasive test for the monitoring and prognostication of NPC patients.     

Promoter hypermethylation of tumor-related genes in gastric intestinal metaplasia of patients with and without gastric cancer

Promoter hypermethylation is an alternative mechanism of gene silencing in human cancers including gastric cancer. While intestinal metaplasia (IM) is generally regarded as a precancerous lesion of the stomach, our study examines the presence of gene promoter hypermethylation in IM of patients with and without gastric cancer. We examined 31 samples of gastric cancer, 36 gastric IM (21 associated with gastric cancer and 15 from noncancer patients) and 10 normal gastric biopsies. Tissues containing foci of IM were carefully microdissected from paraffin-embedded section. Bisulfite-modified DNA was examined for gene promoter hypermethylation in DAP-kinase, E-cadherin, GSTP1, p14, p15, p16, RASSF1A and hMLH1 by methylation-specific-PCR. None of the control gastric tissues had hypermethylation detected, but gene promoter hypermethylation was frequently detected in gastric cancer and IM. The mean number of methylated genes in cancer and IM was 3.0 and 1.4, respectively (p < 0.0001). Methylation in IM from cancer patients was all associated with concurrent methylation in the corresponding tumor samples. The numbers of methylated genes were similar in IM obtained from cancer and noncancer patients. By examining the methylation patterns of these genes, 3 differential methylation patterns were recognized: hypermethylation was more frequent in cancer than in IM (DAP-kinase, p14, p15 and p16); comparable frequencies of methylation in cancer and IM (E-cadherin and hMLH1); and no methylation (GSTP1). Aberrant methylation in tumor-related genes is frequently detected in gastric IM of both cancer and noncancer patients, suggesting their early involvement in the multistep progression of gastric carcinogenesis.     

The RASSF1A tumor suppressor gene is commonly inactivated in adenocarcinoma of the uterine cervix.

PURPOSE: Development of adenocarcinoma (AC) of the uterine cervix, as well as squamous cell carcinoma (SCC), is strongly linked to infection by high-risk human papillomavirus (HPV) types. Human HPV E6 and E7 proteins inactivate the tumor suppressor genes p53 and retinoblastoma, respectively. However, additional genetic alterations may be required to maintain a malignant phenotype. Allelic loss at the short arm of chromosome 3 is one of the most frequent genetic changes found in cervical cancer and various other types of human cancer, including lung, breast, and ovarian cancer. This implies that a resident tumor-suppressor gene in this region is involved in the genesis of these tumors. RASSF1A, which is located at 3p21.3, is rarely inactivated by mutations but has been suggested as a target tumor suppressor gene on the basis of its frequent inactivation through promoter hypermethylation and loss of heterozygosity in a variety of primary human cancers. In the present study, we sought to determine whether epigenetic silencing of RASSF1A caused by hypermethylation of the promoter region plays a role in the development of uterine cervical cancer. Experimental Design: We studied 51 uterine cervical carcinoma samples. These 51 cases were comprised of 31 SCCs and 20 ACs. Real-time methylation-specific PCR system was used for the detection and quantitation of the bisulfite-converted methylated version of the RASSF1A promoter region. The 20 cases of cervical AC were also analyzed for the presence of oncogenic HPV 16 DNA using a PCR-based method. RESULTS: We found complete methylation of the RASSF1A promoter in 45% (9 of 20 samples) of AC cases. There was no promoter methylation observed in any of the 31 cases of SCC. We also correlated RASSF1A promoter hypermethylation to oncogenic HPV 16 infection. HPV 16 DNA was found in 3 of 9 (33%) AC tumors with RASSF1A promoter hypermethylation and 5 of 11 (45%) AC tumors without RASSF1A promoter hypermethylation. We could not demonstrate an inverse correlation between RASSF1A methylation and HPV 16 infection in AC of the uterine cervix. CONCLUSIONS: Hypermethylation of the RASSF1A promoter region is common in AC of the uterine cervix and rare in squamous carcinoma of uterine cervix. HPV infection does not correlate with RASSF1A methylation status in AC of the uterine cervix, but the absence of RASSF1A methylation in SCC of the uterine cervix coupled with the high incidence of HPV 16 infection in this subtype is in accord with previous reports. Our results suggest that epigenetic silencing of RASSF1A may play a role in the development of AC of the uterine cervix.     

Quantitative analysis of cell-free Epstein-Barr virus DNA in plasma of patients with nasopharyngeal carcinoma

Using real-time quantitative PCR, cell-free EBV DNA was detectable in the plasma of 96% (55 of 57) of nasopharyngeal carcinoma (NPC) patients (median concentration, 21058 copies/ml) and 7% (3 of 43) of controls (median concentration, 0 copies/ml). Advanced-stage NPC patients had higher plasma EBV DNA levels than those with early-stage disease. At 1 month after completion of radiotherapy, plasma EBV DNA was undetectable in 7 of 15 subjects (47%) but remained high in the remaining 8 subjects (53%). Clinical examination revealed that all of the former seven subjects had complete tumor regression, whereas six of the eight latter subjects exhibited evidence of disease persistence or had developed distant metastases. These results suggest that quantitative analysis of plasma EBV DNA may be a useful clinical and research tool in the screening and monitoring of NPC patients.     

血浆EB病毒DNA浓度预测鼻咽癌远处转移的研究

背景与目的：鼻咽癌治疗失败的主要原因之一是远处转移,近年多项放化疗综合治疗的临床研究未显示出明显的远处转移率的降低和远期生存的获益,综合治疗适宜个体及最佳模式尚未确立。血浆EB病毒DNA（EBV DNA）浓度是一项能反映鼻咽癌分期、治疗反应、预后的灵敏、特异的分子生物学指标。我们设计此前瞻性研究。探讨通过鼻咽癌患者血浆EBV DNA浓度预测远处转移的发生．为个体化的综合治疗模式的选择提供分子学指标。方法：69例初治鼻咽癌患者分别在治疗前和治疗结束时采用荧光定量PCR方法检测血浆EBV DNA浓度。所有患者按计划随访。进行远期疗效及生存的评价。Kaplan-Meier法计算无转移生存率及总生存率,多因素分析用Cox回归模型。结果：远处转移患者治疗前血浆EBV DNA中位浓度（27 000copies／m1）及治疗后EBVDNA检出率（55．56％）均高于持续缓解者（4 000 copies／ml,5．56％）及局部复发者（3 850 copies／ml,0％）（P值分别为0．039,0．001）。以治疗前20 000 copies／ml、治疗后0 copies／ml为界值点．EBV DNA低浓度患者的无复发生存率、无转移生存率及总生存率均高于高浓度患者,差异有显著性。Cox回归分析显示治疗前EBV DNA浓度（P=0．050,RR=3．95）、治疗后EBVDNA浓度（P=0．001,RR=11．74）均是影响无转移生存的危险因素。进一步将患者治疗前后EBVDNA浓度变化综合进行生存分析,结果表明治疗后EBV DNA浓度能否降到0是影响无转移生存最重要的预后因素（P=0．000）。结论：鼻咽癌患者治疗前、后血浆EBV DNA浓度,尤其是治疗后能否降到0．能预测远处转移的发生,有望为放化综合治疗筛选高危患者,指导放化结合模式的选择。