IFN-alpha-stimulated genes and Epstein-Barr virus gene expression distinguish WHO type II and III nasopharyngeal carcinomas

Nonkeratinizing nasopharyngeal carcinoma (NPC) is 100% associated with Epstein-Barr Virus (EBV) and divided into two subtypes (WHO types II and III) based on histology. We tested whether these subtypes can be distinguished at the molecular genetic level using an algorithm that analyzes sets of related genes (gene set enrichment analysis). We found that a class of IFN-stimulated genes (ISG), frequently associated with the antiviral response, was significantly activated in type III versus type II NPC. Consistent with this, replication of the endogenous EBV was suppressed in type III. A strong association was also seen with a subset of ISGs previously identified in systemic lupus erythematosus, another disease in which 'normal' EBV biology is deregulated, suggesting that this pattern of ISG expression may be linked to the increased EBV activity in both diseases. In contrast, unsupervised hierarchical clustering of the complete expression profiles failed to distinguish the two subsets. These results suggest that type II and III NPC have not originated from obviously distinct epithelial precursors; rather, the histologic differences may be a consequence of a differential antiviral response, involving IFNs, to chronic EBV infection.     

Nasopharyngeal brush biopsies and detection of nasopharyngeal cancer in a high-risk population.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an important tumor in many countries. Ethnic and regional factors strongly influence disease risk. NPC is usually diagnosed late in disease development, and 10-year survival rates are as low as 10%. Epstein-Barr virus (EBV), a possibly causative agent, is present in all cells of essentially all undifferentiated NPCs. We wished to determine the following: 1) whether an ambulatory nasopharyngeal brush biopsy could provide sufficient tumor cell DNA for the detection of EBV and 2) whether the detection of EBV in this locale reflects the presence of tumor cells or simply EBV carrier status. METHODS: We collected nasopharyngeal tissue via ambulatory brush biopsies from 21 patients with newly diagnosed NPC and from 157 subjects with other otolaryngologic complaints. The majority of study subjects were from high-risk populations. Sample DNA was analyzed for the presence of EBV genomic sequences by use of the polymerase chain reaction (PCR). RESULTS: Ninety-six percent of samples yielded sufficient DNA for PCR amplification. Nineteen of 21 patients with NPC brushed positive for EBV DNA, while all but two (1.3%) of 149 informative control subjects were negative for EBV (two-sided P<.0001). One of the EBV-positive control subjects had an EBV-positive inverted sinonasal papilloma; the other EBV-positive control subject exhibited no overt clinical disease. CONCLUSION: Demonstration of EBV DNA in nasopharyngeal brush biopsy specimens detects NPC with a sensitivity of at least 90% (95% confidence interval = 89.63%-91.32%) and a specificity of approximately 99% (95% confidence interval = 98.64%-98.68%). This technique merits further testing as a possible ambulatory screening strategy in high-risk populations.     

Serum EBV DNA as a biomarker in primary nasopharyngeal carcinoma of Indian origin

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a unique tumor due to its etiology and endemic distribution. Ethnic and regional factors are found to strongly influence the risk of disease; however, there have been no well-conducted studies on Indian patients. The present study assesses the relationship between Epstein-Barr Virus (EBV) and sporadic Indian NPC and the role of serum EBV DNA in NPC detection. METHODS: Primers directed against non-polymorphic Epstein-Barr nuclear antigen-1 (EBNA-1) gene were used to detect the presence of EBV DNA from fresh tissue and serum in NPC, using PCR. RESULTS: EBV DNA was detected in 69% of the biopsies and 58% of the serum of the NPC patients. With respect to histology, WHO Type III NPC, WHO Type II tumors and WHO I tumors showed 100%, 72.2% and 33% EBV positivity, respectively. EBV positivity was also observed in 23% (6/26) of benign samples. All biopsies of patients with positive serum samples were positive for EBV DNA. CONCLUSION: EBV infection was found in sporadic NPC of South Indian origin, which confirms the etiological role of EBV in NPC. Detection of EBNA-1 in the serum and corresponding tissues of NPC patients suggests that the serum EBV DNA originates from NPC and also indicates the benefit of circulating viral DNA as an early marker in the diagnosis of NPC. Serum DNA-PCR methods can be extrapolated to follow-up studies involving tumor regression or to assess the response to various therapies.     

血浆 EB病毒游离 DNA检测对监测鼻咽癌患者预后的意义

背景与目的:有报道 , 测定血浆中的 EB病毒游离 DNA( EBV-DNA)的拷贝数可作为诊断及监测鼻咽癌患者病情变化的手段之一.本研究旨在评价血浆 EBV-DNA检测在鼻咽癌患者预后监测上的价值, 并进一步与 VCA/IgA、 EA/IgA进行比较.方法:比较鼻咽癌放疗后 30例远处转移患者、 22例局部复发患者、 24例无 瘤生存者血浆中 EBV-DNA、 VCA/IgA、 EA/IgA水平.分别应用荧光定量 PCR方法检测血浆 EBV-DNA水平,免疫酶法检测 VCA/IgA、 EA/IgA;前瞻性观察 20例初诊鼻咽癌患者放疗前、放疗剂量达 40 Gy时及放疗结束时上述指标的变化. 结果:放疗后各组不同预后患者的血浆 EBV-DNA含量的中位数有显著性差异, 远处转移组为 135 100 copies/ml(四分线区域 5 525～ 1 003 750 copies/ml) >局部复发组的 20 500(四分线区域 0～ 58 500 copies/ml) > 无瘤生存组的 0 copy/ml(四分线区域 0～ 0 copy/ml), P均 < 0.05. 远处转移组的血浆 EBV-DNA水平高者较多, 当阳性标准为 1 000 000 copies/ml时,诊断远处转移组的敏感性为 27.3%,而诊断局部复发组的敏感性为 0.0%,特异性均为 100.0%.在初诊患者放疗前、放疗剂量达 40 Gy时及放疗结束时, EBV-DNA水平逐渐降低,平均含量分别为 32 050 copies/ml(四分线区域 3 880～ 317 750 copies/ml)、 0 copy/ml(四分线区域 0～ 14 375 copies/ml)、 0 copy/ml(四分线区域 0～ 2 940 copies/ml), P均 < 0.05, 而 VCA/IgA、 EA/IgA的水平未见明显变化. 结论: 血浆 EBV-DNA检测可用于监测鼻咽癌患者预后,其价值明显优于 VCA/IgA、 EA/IgA.     

Detection of LMP-1 gene in middle ear effusion of NPC

Nasopharyngeal carcinoma (NPC) is closely related with Epstein-Barr virus (EBV). Almost every NPC tumor cells carries clonal EBV genomes. Detection of EBV derived latent membrane protein-1 gene (LMP-1) indicate the presence of NPC. Middle ear effusion (MEE) is a frequent sign of NPC. There have been no reports on LMP-1 in MEE. Tympanocentesis of 88 ears with MEE of 66 patients were done in three groups of patients, group (I) NPC, 31 patients, 50 ears, (II) other head and neck cancers, five patients, six ears and (III) no cancer history, 30 patients, 32 ears. The middle ear aspirate and nasopharyngeal swab specimen were collected to detect LMP-1 with a PCR-based method. Sixty aspirates (68%) out of 88 ears with MEE had enough DNA for PCR amplification. LMP-1 was detected in six middle ear aspirate specimen from three patients in group I who had petrous apex invasion. LMP-1 was detected in 30 swab specimen (93.8%) out of 32 nasopharyngeal swabs in group I. LMP-1 was not detected in middle ear aspirates or nasopharyngeal swab in group II and III patients. LMP-1 was not detected in MEE in patients without NPC. In NPC patients, the detection of LMP-1 may indicate petrous apex invasion.     

Detection of cell free Epstein-Barr virus DNA in sera from patients with nasopharyngeal carcinoma

BACKGROUND: The detection of tumor-derived DNA within the circulation of patients with malignant disease using polymerase chain reaction (PCR)-based strategies has opened a new avenue for the diagnosis and monitoring of these patients. Because of the universal association of Epstein-Barr virus (EBV) with the nonsquamous type of nasopharyngeal carcinoma (NPC; World Health Organization types II and III), the detection of cell free EBV DNA in sera from patients with NPC may be a valuable tool for monitoring the progress of tumors or to provide advanced warning of tumor recurrence. METHODS: Serum samples were obtained from different patients, and cell free EBV DNA was detected with a conventional PCR approach. A total of 134 patients were sampled, including 36 patients with primary NPC, 28 control patients, 18 patients suffering from locoregional recurrence, 7 patients with distant metastasis, and 45 patients with NPC in clinical remission. A conventional PCR approach employing standard 35-cycle and 50-cycle reactions was used to detect cell free EBV genomes. Results from the two PCR cycles were compared to provide a semiquantitative picture of the relative quantity of EBV genome in each serum sample. RESULTS: The EBV DNA detection rates, i.e., the rates of positive detection, for 35-cycle and 50-cycle PCR analyses, respectively, were 38.9% and 75% for patients with primary NPC, 3.5% and 10.7% for control patients, 27.8% and 88.9% for patients with locoregional disease recurrence, 71.4% and 100% for patients with distant metastasis, and 7.1% and 36.5% for patients with disease in clinical remission. The rates of positive detection among patients with active disease all appeared to be significantly greater compared with the rates among patients with disease in clinical remission. Longitudinal data for six patients with recurrent tumors revealed a close correlation between the relative quantity of circulating cell free EBV genomes and the disease course of these patients. The sensitivity, specificity, positive predictive value, and negative predictive value of the 50-cycle PCR analysis for detecting recurrent disease were 92%, 63.5%, 42.6%, and 96.4%, respectively. CONCLUSIONS: This study demonstrated that, by using a 50-cycle PCR-based approach, high sensitivity and high negative predictive value for detecting recurrent disease can be obtained from the detection of the cell free EBV genome in sera from patients with NPC. The 50-cycle PCR analysis, therefore, may provide a simple, clinically useful adjuvant method for monitoring patients with NPC.     

鼻咽癌前期病变中的EB病毒感染

背景与目的：鼻咽癌中的浸润性癌细胞均感染了EB病毒（Epstein-Barr virus．EBV）。前期病变可见于早期鼻咽癌癌旁上皮。本研究旨在通过检测前期病变中的EB病毒,探讨EB病毒感染存鼻咽癌变过程中的作用,及其基因型在鼻咽癌变过程中发生的宿主内演变。方法：采用核酸原位杂交检测15例早期鼻咽癌活检组织中的EB病毒编码RNA（EBV—encoded RNA,EBER）。采用巢式PCR法检测前期病变和癌巢中的EB病毒类型和潜伏膜蛋白1（latent membrane protein 1,LMPI）EB病毒株。具有代表性的LMPI基因羧基末端PCR产物采用四色荧光终止序列技术进行DNA序列分析。结果：所有15例早期鼻咽癌中的绝大多数浸润性癌细胞均呈EBER阳性。在15例的期病变中．14例可检测到EBER阳性的异常上皮细胞和／或浸润性淋巴细胞。单个A型EB病毒可在9例癌巢（11例适用）及9例前期病变（10例通用）的DNA样本中检测到。EB病毒LMP1基因羧基末端在15例癌巢DNA样本中均可检测到,其中14例是30bp缺失型LMP1 EB病毒株,1例是野生型和30bp缺失型LMP1株的混合感染。在11例适合做EB病毒LMP1基因羧基末端扩增的前期病变的DNA样本中,5例呈野生型和30bp缺失型LMP1 EB病毒株的混合感染,4例是单个缺大型LMP1 EB病毒株感染,1例呈单个野生型LMP1 EB病毒株感染,1例呈阴性反应。野生型LMP1基因羧基末端的DNA序列与B95—8细胞的DNA序列完全一致;30bp缺大型LMP1基因羧基末端的DNA序列却其有30bp缺失（密码子：346～355）和4个错义点突变（密码子：334、335、338和366）。结论：鼻咽上皮细胞的EB病毒感染是癌变过程中侵袭前的事件;而在鼻咽癌变过程中,EB病毒基因型会产生宿主内的演变。     

Relationship between the Epstein-Barr virus and undifferentiated nasopharyngeal carcinoma: correlated nucleic acid hybridization and histopathological examination.

In order to examine critically the closeness of association between Epstein-Barr virus (EBV) DNA and nasopharyngeal carcinoma (NPC) a correlated histopathological and nucleic acid hybridization study was performed on 51 undifferentiated NPC, 4 NPC with some signs of squamous differentiation, 7 nasophayngeal tumors of other histological types and 14 head and neck carcinomas located outside the nasopharynx. All 51 undifferentiated NPCs contained significant numbers of EBV-genome copies per cell. Two of the somewhat differentiated NPCs were also EBV-DNA-positive, whereas 2 were negative. Of the 7 other nasopharyngeal tumors, 1 was EBV-DNA-positive. Histological examination, however, showed that this was a typical Burkitt lymphoma. The other 6 tumors were all EBV-DNA-negative lymphoproliferative malignancies. All 14 had head and neck carcinomas located outside the nasopharynx were EBV-DNA-negative. The sera of undifferentiated NPC patients had elevated antibody titers against the EBV-determined antigens, the EA (D) componet in particular. These findings confirm that there is a regular association between EBV-DNA and undifferentiated NPC.     

鼻咽癌中EB病毒LMP1基因N端Xho I酶切位点的丢失

背景与目的：众所周知,EB病毒LMP1基因在鼻咽癌变过程起着一定的作用。本研究通过检测广东地区鼻咽癌组织EB病毒LMP1基因N-末端区Xho I酶切位点的丢失,探讨LMP1基因变异在鼻咽癌发生发展中的作用。方法：收集中山大学肿瘤防治中心鼻咽癌患者鼻咽新鲜活检标本63例。收集EB病毒健康携带者外周血单个核细胞(PBMCs)10例作为对照。采用QIAamp DNA Mini Kit和QIAamp DNA Blood Mini Kit分别抽取组织和外周血单个核细胞的DNA,应用巢式PCR扩增EB病毒LMP1基因的N-末端区,并用Xho I对扩增产物进行酶切。采用四色荧光末端终止法对扩增产物进行序列分析。结果：10例健康携带者外周血单个核细胞的EB病毒LMP1基因N-末端区均未见Xho I酶切位点的丢失。63例鼻咽癌组织中有50例(79．37％)出现Xho I酶切位点的丢失(Xho I—loss),还有4例(6．34％,)为Xho I酶切位点部分丢失,只有9例(14．29％)未见Xho I酶切位点的丢失(wt-Xho I)。除了Xho I酶切位点的丢失(nt：169423～169428;GAGCTC→GA□TCTC)外,还发现四个错义点突变。结论：本研究所检测的广东地区EB病毒健康携带者外周血单个核细胞所携带的：EB病毒LMP1基因为wt—Xho I,而在鼻咽癌组织中主要为Xho I-loss。因此,我们认为EB病毒LMP1基因N-末端区Xho I酶切位点的丢失和其他的错义点突变可能是在鼻咽癌的发生发展过程中产生的。     

Screening nasopharyngeal carcinoma by detection of the latent membrane protein 1 (LMP-1) gene with nasopharyngeal swabs

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a common head and neck cancer in Taiwan. The goals of the current study were to investigate whether a nasopharyngeal swab technique could provide enough DNA for polymerase chain reaction (PCR) analysis of the Epstein-Barr virus (EBV)-derived latent membrane protein 1 (LMP-1) gene and to determine the feasibility and reliability of diagnosing NPC by detection of LMP-1 in the nasopharynx. METHODS: 320 adults underwent nasopharyngoscopy and nasopharyngeal swab to obtain cells for the LMP-1 PCR assay; some patients also underwent nasopharyngeal biopsy. RESULTS: An amount of DNA that was sufficient for PCR was extracted from 96.3% of the swab samples. By detecting LMP-1 in nasopharyngeal swabs, NPC was diagnosed with a false positive rate of 12.7% (7 of 55 patients), a false negative rate of 1.6% (4 of 253 patients), sensitivity of 87.3% (48 of 55 patients), specificity of 98.4% (249 of 253 patients), a positive predictive value of 92.3% (48 of 52 patients), and a negative predictive value of 97.3% (249/256 patients). NPC was diagnosed by nasopharyngoscopy with a false positive rate of 38% (30 of 79 patients), a false negative rate of 0.4% (1 of 241 patients), sensitivity of 62% (49 of 79 patients), specificity of 99.6% (240 of 241 patients), a positive predictive value of 98% (49 of 50 patients), and a negative predictive value of 88.9% (240 of 270 patients). Only 7 (0.2%) of 256 patients with a diagnosis other than NPC had LMP-1 detected in the nasopharyngeal space. CONCLUSIONS: Detecting EBV genomic LMP-1 by nasopharyngeal swab diagnosed NPC with 87.3% sensitivity and 98.4% specificity. EBV genomic DNA usually is not detected by PCR-based methods in the nasopharyngeal space. Its incidence is estimated to be as low as 0.2% among the general population. The nasopharyngeal swab coupled with PCR-based EBV LMP-1 detection could serve as part of a screening program for high-risk populations.     

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.     

Epstein-Barr virus in nasopharyngeal and salivary gland carcinomas of Greenland Eskimoes

Biopsy specimens from nasopharyngeal carcinomas (NPC) or salivary-gland carcinomas (SGC) in Greenland Eskimoes were examined for the presence of Epstein-Barr virus (EBV) DNA and sera from the patients were tested for EBV-specific antibody titres. Six out of 7 NPCs and one from an undifferentiated SGG were positive for EBV DNA. The EBV-specific antibody spectra and titres of the patients with NPC or undifferentiated SGG conformed to the results of earlier studies in other high-incidence areas.     

Histopathological types of nasopharyngeal carcinoma in a low-risk area: Japan.

NPC in Japan was studied histopathologically by examining 816 NPCs from among 3 338 biopsies from patients with tumours of the nasopharynx and other parts of the upper respiratory system. In addition, a comparative study on NPC among Taiwanese and an immunoserological study were carried out. When comparing histological types of malignant tumours of the upper respiratory tract, poorly-differentiated squamous-cell carcinoma predominated only in those of the nasopharynx. Malignant nasopharyngeal tumours in Japanese patients were characterized by two histological features: predominance of squamous-cell carcinoma, especially of the poorly-differentiated type, and a relatively high frequenty of malignant lymphoma. An analysis of 731 cases of NPC showed that a vast majority (86.7%) were poorly-differentiated and a minority (13.3%) well-differentiated squamous-cell carcinomas. The former included 45.8% spindle-polygonal-cell carcinomas, 26.6% transitional-cell carcinomas ana 14.3% lymphoepitheliomas. Although the transitional-cell carcinomas and lymphoepitheliomas showed a peculiar morphology, it was confirmed that they are of a squamous nature. A comparative study of the histology of NPCs in a high-risk area, Taiwan, and in a low-risk area, Japan, revealed considerable differences between the two groups. Well-differentiated carcinomas were infrequent in both groups but were more frequent in Japanese than in Taiwanese, while the frequency of poorly-differentiated carcinomas, especially transitional-cell carcinomas and lymphoepitheliomas, was much higher among Taiwanese. A seroepidemiological study on the relation of anti-VCA antibody titres to histological type of tumour in 84 Japanese NPC patients revealed that the rate of positivity and the geometric mean of the titres were considerably higher in patients with lymphoepitheliomas. Althougropathological study along this line would appear to be important, since the results obtained in Japanese NPC cases may suggest that EBV genomes in NPC cells vary with the grade of differentiation of NPC.     

定量分析鼻咽癌患者外周血游离EBV

目的:分析外周血游离Epstein-Barr病毒(EBV)是否特异地出现于鼻咽癌患者。方法:收集鼻咽癌患者和非鼻咽癌肿瘤患者血浆各50例,正常人血浆30例,提取DNA,荧光定量PCR检测EBV拷贝数,比较游离EBV在上述人群的差异。结果:在25例(50%)鼻咽癌患者血浆中检测到游离EBV,其拷贝数波动于1×103拷贝/ml血浆～2.6×106拷贝/ml血浆,中位数为5.3×105拷贝/ml血浆。在50例非鼻咽癌肿瘤患者和30例正常人血浆中均未检测到游离EBV。结论:游离EBV出现于鼻咽癌患者外周血,可能是一个较特异的鼻咽癌标志物。     

Biological response of nasopharyngeal carcinoma to radiation therapy: a pilot study using serial 18F-FDG PET/CT scans

We used serial (18)F-fluorodeoxyglucose positron emission tomography/computed tomography ((18)F-FDG PET/CT) to evaluate tumors' maximum standardized uptake value (SUV(max)) before, during, and after radiotherapy to explore the biological behavior of and response to radiation therapy in various subtypes of nasopharyngeal carcinoma (NPC). Sixty-one patients with pathologically diagnosed NPC were prospectively enrolled into the study. WHO type II(B) disease had a higher initial SUV(max) and more significant biological response at the primary site as compared with type II(A) subtype. The two subtypes of WHO type II NPC may significantly differ in their biological behavior and response to radiotherapy.     

Quantitative Epstein-Barr virus DNA analysis and detection of gene promoter hypermethylation in nasopharyngeal (NP) brushing samples from patients with NP carcinoma.

PURPOSE: Nasopharyngeal carcinoma (NPC) is highly prevalent in southern China and characterized by a strong association with EBV. We aimed to detect EBV DNA and cancer-related gene promoter hypermethylation in nasopharyngeal (NP) brushing samples and provide a novel noninvasive approach for NPC detection. EXPERIMENTAL DESIGN: Twenty-eight NPC cases and 26 noncancerous subjects were prospectively recruited. NP brushing samples were subjected to quantitative real-time PCR analysis of EBV DNA and methylation-specific PCR analysis of the DAP-kinase, RASSF1A, and p16 genes. RESULTS: EBV DNA quantity in NP brushing samples from NPC patients (median, 8.94 copies/actin) was significantly higher than that of controls (median, 0 copies/actin; P < 0.0001). Twenty-seven of 28 NPC patients had detectable EBV DNA in NP brushes, whereas 25 of 26 controls had undetectable or very low levels of EBV DNA. Elevated EBV DNA level in brushing samples as a tumor marker had a sensitivity of 96.4% and a specificity of 96.2% for NPC detection. Moreover, T(1) disease had a significantly lower EBV DNA level as compared with locally more advanced disease (P = 0.037). In brushing samples of NPC patients, the frequencies of DAP-kinase, RASSF1A, and p16 promoter hypermethylation were 50.0%, 39.3%, and 46.4%, respectively. Seventy-eight percent of cases showed methylation of at least one gene. No aberrant hypermethylation was detected in control samples. CONCLUSIONS: Our study demonstrated the feasibility of detecting multiple molecular tumor markers in NP brushing samples with a high sensitivity and specificity for NPC detection. It offers a powerful yet noninvasive approach for the diagnosis of NPC in high-risk populations.     

鼻咽癌患者血浆EBV DNA水平的研究进展

EB病毒与鼻咽癌的发生有密切关系。近年来,随着PCR技术的发展,在鼻咽癌患者血浆中可定量检测EBVDNA水平。大量研究表明,鼻咽癌患者血浆EBVDNA的检出率及拷贝数明显高于正常人群。放疗后转移、复发患者其EBVDNA的阳性率及拷贝数高于持续缓解患者。血浆EBVDNA水平在鼻咽癌的早期诊断、临床分期、预后判断及监测治疗后转移、复发中均有重要临床意义。     

鼻咽癌中EB病毒LMP1基因N端XhoⅠ酶切位点的丢失

背景与目的:众所周知, EB病毒 LMP1基因在鼻咽癌变过程起着一定的作用.本研究通过检测广东地区鼻咽癌组织 EB病毒 LMP1基因 N-末端区 XhoⅠ酶切位点的丢失,探讨 LMP1基因变异在鼻咽癌发生发展中的作用.方法 :收集中山大学肿瘤防治中心鼻咽癌患者鼻咽新鲜活检标本 63例.收集 EB病毒健康携带者外周血单个核细胞 (PBMCs) 10例作为对照.采用 QIAamp DNA Mini Kit和 QIAamp DNA Blood Mini Kit分别抽取组织和外周血单个核细胞的 DNA,应用巢式 PCR扩增 EB病毒 LMP1基因的 N-末端区,并用 XhoⅠ对扩增产物进行酶切.采用四色荧光 末端终止法对扩增产物进行序列分析.结果: 10例健康携带者外周血单个核细胞的 EB病毒 LMP1基因 N-末端区均未见 XhoⅠ酶切位点的丢失.63例鼻咽癌组织中有 50例(79.37%)出现 XhoⅠ酶切位点的丢失(XhoⅠ-loss),还有 4例(6.34%)为 XhoⅠ酶切位点部分丢失,只有 9例(14.29%)未见 XhoⅠ酶切位点的丢失(wt-XhoⅠ ).除了 XhoⅠ酶切位点的丢失 (nt: 169423～169428; GAGCTC→ GATCTC)外,还发现四个错义点突变.结论:本研究所检测的广东地区 EB病毒健康携带者外周血单个核细胞所携带的 EB病毒 LMP1基因为 wt-XhoⅠ,而在鼻咽癌组织中主要为 XhoⅠ-loss.因此,我们认为 EB病毒 LMP1基因 N-末端区 XhoⅠ酶切位点的丢失和其他的错义点突变可能是在鼻咽癌的发生发展过程中产生的.     

The association between circulating tumor cells and Epstein-Barr virus activation in patients with nasopharyngeal carcinoma

Background: Circulating tumor cells (CTCs) and microemboli (CTM) are attracting increasing attention in medical biology and clinical practice. However, the clinical relevance of CTCs in nasopharyngeal carcinoma (NPC) has not yet been ascertained, and no study has focused on the influence of Epstein-Barr virus (EBV) status on CTCs in NPC patients. These issues were therefore examined. Methods: Peripheral blood samples were prospectively obtained from 33 NPC patients before treatment. CTCs and CTM were captured using the Isolation by Size of Epithelial Tumor (ISET) method. Immunohistochemistry on CK5/6 (cytokeratin5/6) and P63, as well as in situ hybridization of EBERs (EBV-encoded RNAs) were used to validate the harvested tumor cells. Results: Out of 33 NPC patients, CTCs were detected in 22 cases (66.7%), and CTM were observed in 2 cases (6.1%). CTCs were presented in all stages of NPC patients but had no association with the TNM stages (all P > 0.05). The presence of CTCs appeared to correlate with EBV activation status. Among the total NPC patients, the EBV VCA-IgA levels in CTC-positive cases were higher than that in CTC-negative cases (negative vs. positive: 3.88 vs. 4.86, P = 0.016). A similar result was observed in the patients without distant metastasis (negative vs. positive: 3.76 vs. 4.95, P = 0.009). When the number of CTCs was considered, CTC counts were found to correlate with EBV VCA-IgA (R = 0.382, P = 0.041) and EBV DNA load (R = 0.396, P = 0.033) for all NPC patients as well as NPC patients without distant metastases. Conclusions: These findings strongly suggested detectable CTCs/CTM in all stages of NPC patients and support a positive correlation between CTCs and EBV activation in NPC patients.