Chine-German Journal of Clinical Oncology January 2011, Vol. 10, No. 1, P51–P55 DOI 10.1007/s10330-011-0725-7
With the development of radiotherapy, standard che-motherapy and the intervention of other treatment mo-dalities, the treatment outcome of nasopharyngeal carci-noma (NPC) has been greatly improved. In general, the earlier the stage is, the better the treatment outcome will be achieved. Unfortunately, most of the NPC patients we encountered had stage III or IV dia, with a proportion of 65%–70% at advanced stage [1], greatly influence the overall efficacy of NPC. It has been reported that with the u of existing equipments and technologies, if the clini-cal stage of NPC patients for each would demote to the next lower grade, the overall 5-year survival can be in-cread by 20%, which would bring great benefit for pa-tients. Therefore, it is of primary importance to establish a screening protocol for NPC diagnosis and to administer treatment in the early stages of dia [1]. Epstein-Barr virus (EBV) is a double-stranded DNA herpesvirus that has a clo relationship with the carcinogenesis of NPC, after infected by EBV it would produce veral EBV anti-gens [usually immunoglobulin A (IgA) antibodies to viral capsid antigen (VCA) and early antigen (EA)]. In China, the antigens are commonly ud as the screening meth-ods of the early stage NPC, but the nsitivity and speci-ficity are far from satisfactory, and the positive predictive value are also at a low level [2], and the early diagnosis rate of NPC has not been incread obviously in the rent 20 years [1].
EBV-derived latent membrane protein-1 (LMP-1) is one of the proteins that have been proved to be have oncogene characters, and plays an important role in the canceration of NPC. Studies indicated that it could be an ideal target for screening purpos. To date, there are not
The initial results of Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP-1) for screening nasopharyngeal carcinoma (NPC)*
Shaojun Lin, Qiaojuan Guo, Jin Lin, Jingfeng Zong, Lu Han, Jianji Pan
Department of Radiation Oncology, Fujian Provincial Cancer Hospital, Fujian Medical University, Fuzhou 350014, China
Received: 22 October 2010 / Revid: 20 November 2010 / Accepted: 5 December 2010
© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2011
Abstract Objective: Early diagnosis of nasopharyngeal carcinoma (NPC) is an important method to improve
the survival rate. However, the nsitivity and specificity of the screening protocols which was widely ud in clin-
ic now are considered to be unsatisfactory. Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP-1)
is one of the proteins that have been suggested to be a classic oncogene with transformation properties. The cur-
rent study t out to discuss the clinical significance of LMP-1 on the screening of NPC. Methods: Three hundred pa-
tients who visited our institution (Department of Radiation Oncology, Fujian Provincial Cancer Hospital, Fuzhou,
China) with ENT symptoms between 2007 and 2008 were involved in this study, and all of them were agreed to be involved
in this investigation. Not only did they undergo nasopharyngeal swab to obtain cells for the LMP-1 polymera chain reaction
(PCR) analysis, but also nasopharyngeal biopsy were taken to identify the diagnosis. Results: An amount of DNA that was
sufficient for PCR was extracted from 243 (81%) swab samples, the positive rate of LMP-1 of tho with non-nasopharyngeal
carcinoma was 3.85% (4/108), which was much lower than tho with nasopharyngeal carcinoma (P < 0.05). By detecting
LMP-1 in nasopharyngeal swabs, NPC was diagnod with a nsitivity of 88.15% (119 of 135 patients), specificity of 96.30%
茴拉西坦胶囊(104 of 108 patients), a positive predictive value of 95.2% (119 of 123 patients), a negative predictive value of 86.67% (104
of 120 patients), accuracy of 91.77%, and Youden index of 84.45%. Conclusion: The nasopharyngeal swab coupled with
PCR-bad EBV LMP-1 detection have high nsitivity and specificity, and also good repeatability, it could rve as part of
the screening program for high-risk populations.
Key words nasopharyngeal carcinoma (NPC); Epstein-Barr virus (EBV); latent membrane protein-1 (LMP-1); early diag-
nosis
Correspondence to: Jianji Pan. Email:
* Supported by a grant from Medical Innovation Fund Project of Fujian
Provincial Health Bureau (No. 2007-CXB-4).
52 www. springerlink. com/content/1613-9089
a rigorous, large-scale molecular diagnosis of nasopha-ryngeal carcinoma reported in China. The current study was t out to establish the efficacy of NPC diagnosis by detection of LMP-1 in the nasopharyngeal space by naso-pharyngeal swab, and to prepare for an NPC screening of the general population.
Materials and methods
The examinees included 300 volunteers who came to the Department of Radiation Oncology of Fujian Provin-cial Cancer Hospital (China) between 2007 and 2008 with some types of ear, no, or throat-related symptoms, in-cluding 214 males and 86 females, ranged from 14 to 77 years old, with a median age of 46 years old. All patients supplied written informed connt to have nasopharyn-geal swabs performed under an approved study protocol. Main instruments and reagents
PCR instrument (BIO-RAD Company, USA), DNA kit (QIGEN Company, Nederland), DNA polymera (TAKARA Company, Japan), Marker (Shanghai Bohong Company, China), Beike Man High Speed Refrigerated Centrifuge (Sigma Company, USA), CO2 incubator (Ther-mo Company, USA), UV projection analyzer (Shanghai, China), B95-8 cell lines came from Radiation Biology Laboratory of our Institution (China). Nasopharyngeal swab
The nasopharyngeal swab procedure had been de-scribed previously [3]. In brief, the examinee’s nasal cavity was brushed with a 1% cocaine solution. A 15-cm-long cotton stick was inrted into the nasal cavity and moved toward the nasopharyngeal wall. The cotton was presd against the posterior and lateral nasopharyngeal walls and swept over the surface of the walls veral times. The cot-ton stick then was withdrawn, and the cotton immedi-ately was immerd in phosphate buffer solution (PBS) and nt to the laboratory for processing.
DNA extraction
Specimens collected from nasopharyngeal swabs were immerd and washed in PBS (2 mL) to retrieve the sus-pended cells. After centrifugation, the supernatant was discarded, and 400 μL of water and a 400 μL aliquot of phenol/chloroform solution was added to the pellet and extracted twice. After ethanol precipitation and centrifu-gation, the supernatant was discarded, and the pellet then was prepared for direct u in PCR [3].
PCR amplification and gel electrophoresis for LMP-1 detection
PCR was performed in a total volume of 50 μL, consist-ing of 5 μL extracted DNA, both the n and antin primers were 1 μL (20 μmol/L) (BN1: 5’-AGCGACTCT-GCTGGAAATGAF-3’, and BN2: 5’-TGATTAGCTAAGG CATTCCCA-3’), 5 μL dNTP (2.5 mmol/L), and 0.25 μL Taq DNA polymera (containing 1.25 U), 10 × PCR MIX 5 μL.
Samples were amplified for 35 cycles using the follow-ing procedures: pre-denaturation at 95 ℃ for 5 min, and than denaturation at 94 ℃ for 30 s, annealing at 50 ℃ for 30 s, and extension at 72 ℃ for 60 s; and finally extension at 72 ℃ for 10 min, prerved under 4 ℃. Products then were examined by 1.5% agaro gel electrophoresis in 1 Tris-boric acid-ethylenediamine tetraacetic acid (TBE) solut
ion and stained with ethidium bromide to determine the prence or abnce of PCR product. The DNA mark-er ud was produced by Bohong Company, China, PCR products were displayed on the plastic in between 100 and 1000 bp, consistent with the design of experiment, il-lustrated that the band was LMP-1. DNA from the B95-8 cell line was ud as the EBV positive control, and nega-tive control samples containing water were procesd in parallel with all patient samples.
Nasopharyngeal biopsy
清明祭祖
Every patient in the current study underwent naso-pharyngeal biopsy through electronic nasopharyngeal endoscope, and was diagnod at the Department of Pa-thology in our institution, and was considered as the gold standard of the diagnosis of NPC.
Statistical analysis
启民可汗All statistical analys were performed with SPSS sta-tistical software. The χ2 tests were ud in the analys.化州橘红的功效与禁忌
A value of P < 0.05 was considered to be statistically sig-nificant.
Results
Expression of LMP-1
A sufficient amount of tumor cell DNA for PCR am-plification was extracted from 81% (243/300) of the swab samples. LMP-1 was detected to be existed in 123 sam-ples, with a positive rate of 50.8% (123/243). The positive strap was shown in Fig. 1, clearly prented at 290 bp. The maker we ud in this study was 100–1000 bp.
The nsitivity and specificity of LMP-1 through PCR
Of the 243 samples, 135 cas were histologically di-agnod as NPC, 119 patients were positive for LMP-1, with a nsitivity and specificity of 88.15% (119/135) and 96.30% (104/108), respectively. Of the 108 patients who were histologically diagnod as non-NPC, about 3.85% (4/108) was proved to be positive for LMP-1, which was significantly lower than that of NPC patients (P < 0.05,
53 Chine-German J Clin Oncol, January 2011, Vol. 10, No. 1
Table 1), and the accuracy and Youden index of the method were 91.77% and 84.45%, respectively. Positive and negative predictive values of
LMP-1 through PCR
Among the 123 cas who were positive for LMP-1, 119 patients were pathologically confirmed as NPC, 3 of the other 4 patients had chronic inflammation of naso-pharynx mucosa, another patients had EBV-related naso-pharyngeal lymphoma, and the positive predictive value was 95.2% (119/123). Of the 120 patients who were nega-tive for LMP-1, only 16 cas were historically diagnod as NPC, another 104 patients were either healthy per-son or patients with chronic inflammation of nasophar-ynx mucosa, with a negative predictive value of 86.67% (104/120).
Discussion
LMP-1 is thought to play an esntial role in tumori-genesis of NPC. It could encode multiple gene products, and LMP-1 has been proved to be one of the oncogenes with transformation property, and was considered as vi-rus oncogene.
LMP-1 contains 3 different domains including the hy-drophilic amino-terminal cytoplasmic domains, 6 differ-ent hydrophobic transmembrane regions and hydrophilic C-terminal cytoplasmic domain [4]. It mediated abnormal biological behavior of cells through activating some sig-nal transduction pathway, and cloly related to the tu-morigenesis of NPC [5].
It has been found that LMP-1 has a comparable high expression rate in the tissue of NPC. Early in 1
996, Vera-Sempere and his colleagues [6] have detected the expres-sion of LMP-1 in 51 NPC paraffin ctions by immuno-histochemistry, and they found a positive rate of 78.4%. Investigators in China indicated that the positive rate of LMP-1 in the biopsy specimens of NPC patients ranged from 50% to 72.5% [7–9], and was significantly higher than that of nasopharyngitis tissues (72.5% vs 16.67%) (P < 0.05) [9], suggesting that it maybe an ideal screening target of NPC in high-risk populations, but the positive rate of LMP-1 is not very high becau of the limitation of im-munohistochemistry, also it may induce bleeding during the tissue derived process, with a poor reproducibility and compliance, so a more simple, nsitive, and non-invasive method is needed to improve the screening efficiency of LMP-1 in NPC.
There have been so many studies indicated that EB-DNA was mainly existed in the blood, plasma and rum of NPC patients, but the expression of DNA is relatively low in the circulating blood of a considerable number of NPC patients (100–2000 copies per milliliter blood or plasma), and brings certain degree of difficulty to the diagnosis [10], and EB DNA in rum is mostly fragment-ed, probably stem from nasopharyngeal carcinoma cells which have developed apoptosis [10, 11]. There have been a study detected the expression of LMP-1 in the rum of NPC patients, but only 62% of the samples positively express LMP-1 [12], lower than that of the studies previ-ously reported [8, 9].
Nasopharyngeal swab is a convenience method with a good reproducibility and compliance, early in 1998, Sheen et al[13] detected anti-VCA-IgA and anti-VCA-IgG of exfoliated cells of nasopharyngeal obtained through nasopharyngeal swab, a sufficient amount of tumor cell DNA for PCR amplification was extracted from 89.1% (49/55) of the swab samples. The next year, Tune et al [14] reported a effective sample rate of 96%. In 2001, Lin
Table 1Epstein-Barr virus (EBV)-encoded LMP-1 status of nasopharyngeal swab of 243 patients by PCR assay (n)
LMP-1 gene Nasopharyngeal carcinoma Non-nasopharyngeal carcinoma Total Positive 1194123 Negative 16104120
Total 135108243 Fig. 1The expression of LMP-1 gene detected by PCR assay
54 www. springerlink. com/content/1613-9089
and his colleagues [15] obtained a effective sample rate of 100% by improving the technology of nasopharyngeal swab, and it was the first time that nasopharyngeal swab coupled with PCR-bad EBV LMP-1 detection was ud in the diagnosis of NPC, they acquired a nsitivity of 94.7% and a specificity of 100%. Hao et al[3] enrolled 320 patients in their study, and indicated that detection of EBV genomic LMP-1 by nasopharyngeal swab technique predicted NPC with a nsitivity of 87.3% and a specific-ity of 98.4%. They found nasopharyngeal swab technique coupled with detection of EBV LMP-1 by PCR could rve as part of a screening program for high-risk popula-tions. In 2004, they published another results from 437 cas, with the same screening method, and the nsitiv-ity and specificity were of 91.4% and 98.3%, respectively [16]. An author in our country conducted a study which enrolled 30 NPC patients and 10 controls, they extracted EB DNA from nasopharyngeal swab [17], indicated a n-sitivity of 93.0% and a specificity of 100.0%. The current trial prospectively detected the expression of LMP-1 by using nasopharyngeal swab technique coupled with PCR, all the patients included was confirmed by pathology, we yielded a comparable high expression rate of LMP-1 in NPC patients, which was consistent with forementioned reports, further
proved that nasopharyngeal swab was easily obtained and economically, and could be drawn repeatedly, a comparable high nsitivity and specificity could be yielded from this technique when it combined with PCR to detected the expression of LMP-1 to screen NPC.
EBV, a probable etiological factor of NPC, might cau other malignancies as well. Hao et al [3] found that among the 55 patients who were positive for LMP-1, 7 were pathologically diagnod as non-NPC patients, including 2 EBV-related nasopharyngeal lymphomas cas, 2 fal positive cas becau of cross contamination and anoth-er 3 patients with other dias. The same phenomenon was found in the current study, of the 123 patients posi-tive for LMP-1, there was one patient pathologically con-firmed non-Hodgkin’s lymphoma, another 3 cas were confirmed as chronic nasopharyngeal mucositis, 2 of the 3 patients prented with obviously mess at the site of nasopharynx and also lymphadenectasis at the neck, both of the 2 patients were confirmed as chronic naso-pharyngeal mucositis twice by histology, the expression of EB-VCA-IgA in rum was negative in one patients of the two, the other ca was of suspected positive, the 2 patients were highly suspicion of NPC clinically, negative results of biopsy were considered to be associated with improper deriving process, we recommended them to do biopsy and PET-CT examination again, but they refud. Another one prented with mass at the left upper neck as the primary complaint, and
was slightly thickened at nasopharynx, both of them were histologically confirmed as chronic nasopharyngeal mucositis twice, both the re-peated cervical lymph node biopsy revealed suspected cancer, and suspect of positive expression of rum EB-VCA-IgA, finally the biopsy of the discted neck lymph node suggested granuloma, and cross-contamination of DNA samples maybe the reason of positive expression of LMP-1.
However, there were 16 NPC patients in this study neg-ative for LMP-1, immunogenicity of LMP-1 was thought to be one of the reasons, it could act as a target molecule of HLA I restricted CTL when the EBV transformed B cells was activated so as to evade the immune mechanism of the body; also reducing the immunogenicity of LMP-1 molecules by mutation, furthermore it could be silenced by LRS methylation, and its negative or low expression may reprent a condary change and was a respon to immune lection. Tumor is no longer dependent on the proliferation respon of LMP-1 at the initial pha to pro-mote tumor growth, but through closing the expression of LMP-1 to reduced the immunogenicity of tumor cells. The 16 patients were further analyzed the expression of EB-VCA-IgA in the rum through ELISA, 14 of them were positive for EB-VCA-IgA, suggesting that the n-sitivity and specificity could be further incread when combined with the detection of rum EB-VCA-IgA. However, the compliance of rological testing is poor, if the detection of EB-VC
A-IgA could also u the technol-ogy of nasopharyngeal swab and combined with LMP-1, the screening efficiency could be greatly improved. But so far there are not any related reports, and large-scale experiments are needed in future.
LMP-1 has a comparable high expression rate in NPC cells, and it have been shown to have associations with cell transformation, proliferation and dedifferentiation. To date, the mechanism of LMP-1 caud tumors have not been completely demonstrated. Studies have found that LMP-1 can inhibit cell apoptosis and also promote cell unlimited proliferation through signaling pathway, and it was proved that the inhibition of apoptosis by LMP-1 could conducted by the upregulation of NF-κB [18, 19]. LMP-1 could also induce the expression of tumor ne-crosis factor receptor-associated factors (TRAFs) in NPC cells, further regulating the expression of target gene such as AP-1 and NF-κB so as to mediate cell proliferation and anti-apoptosis [20]. In addition, transformed nasopharyn-geal cells would obtain the ability of unlimited prolif-eration, immortalization, and inhibition of apoptosis be-cau of LMP-1 through survivin [21], also LMP-1 could up-regulate the expression of IL-1 and IL-8 in order to promote the proliferation of NPC cells [22]. But more de-tailed mechanisms still need further studies.
Our results showed that LMP-1 as a screening indica-tor of NPC has high reliability, with a comparab
le high positive predictive value, and lead to less chance of misdi-
55 Chine-German J Clin Oncol, January 2011, Vol. 10, No. 1
提薪申请agnosis. The combination of LMP-1 with nasopharyngeal swab is simple in the sample derived process and repro-ducible, and suitable for large scale screening of high-risk populations. In practice, more attentions should be drawn to the standardization of testing procedures and sample derived process, so as to reduce the fal positive and fal negative rates and improve the nsitivity and specificity.
In future, we will carry out large scale prospective ex-periments, lect the healthy people undergoing physical examination at our institution who are willing to partici-pate in the study and native of Fujian Province to validate our results. The peoples enrolled will be ranged from 30 to 59 years old, and nasopharyngeal swabs will be done to obtain exfoliated cells of nasopharyngeal. Next we will extracted DNA and detect the expression of LMP-1 by PCR, who are positive for LMP-1 will be further follow-up, at the same time, cost-effectiveness of screening tests would be done to analyze the feasibility of this method to be ud in large-scale screening, also we will continu-ously looking for targets ud in combined detection to further improve the screening efficiency. References
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