Delayed hypersensitivity reactions of cancer patients to antigens on lymphoid cell lines.

Four-hundred and fifty nine cancer patients were skin tested with extracts from five lymphoid cell lines. More than 50% of patients with lymphoma had positive skin tests with the extracts prepared from the cell line derived from Burkitt's lymphoma (BL) and more than 50% of nasopharyngeal carcinoma (NPC) patients reacted to the NPC-derived cell line extracts. Although the significant association between patient diagnosis and orgin of cell lines suggested that tumor-associated antigens were responsible for the pattern of delayed hypersensitivity, problems in standardization of antigen potency and non-specificity need to be resolved before this in vivo assay achieves its full potential.     

In vivo cell-mediated immunity in Chinese patients with nasopharyngeal carcinoma.

Delayed hypersensitivity to antigens derived from four EBV-related (KHLY 28, Raji, F265 and NC37) and one EBV-unrelated (Molt) T-lymphoid cell lines and to standard antigens (Trichophyton, Candida albicans and streptokinase/streptodornase) was measured in 104 NPC patients and 24 patients with other cancers. Of the NPC patients with non-disseminated disease, 55/86 (63.2%) had a positive skin reaction to HKLY 28 extract, compared with only 1/18 (5.6%) OC patients with non-disseminated disease. The frequencies of these NPC patients with positive skin reactions to the other cell-line extracts were significantly lower (1.7-5.4%). From the preliminary results of a longitudinal study, skin testing with this particular crude membrane extract from HKLY 28 cells appears to be of little practical value in monitoring the clinical evolution of NPC, although a significant association between clinical evolution and certain patterns of skin reactivity response to the extract was found in about two-thirds of the cases analysed.     

Comparative studies on immunity to EBV-associated antigens in NPC patients in North America, Tunisia, France and Hong Kong.

This study compared the relative antibody titers to EBV-related antigens in patients with nasopharyngeal carcinoma (NPC) and controls from a high-incidence (Hong Kong), an intermediate incidence (Tunisia), and two low-incidence (France, North America) areas to determine which of several EBV antibodies best differentiated NPC patients from controls. Antibodies measured include anti-virus capsid antigen (VCA), anti-early antigen (EA), anti-soluble antigen by complement-fixation (CF) and antibody-dependent lymphocyte cytotoxicity (ADLC). A matched pair analysis showed that significantly more NPC patients had higher VCA and EA but not CF or ADLC antibody titers than their matched cancer controls. The comparison of geometric mean titers between NPC cases and controls was more than seven-fold (816 vs 11.5) for EA antibody and more than three-fold (359.7 vs 95.4) for VCA anti-body (p less than 0.01). A two-fold difference was seen for CF antibody to soluble antigens (27.3 vs 12.9, p less than 0.01) and a three-fold difference (2657.7 vs 870.9, p less than 0.05) was observed for ADLC. Our finding of significant differences between NPC patients from four countries and their matched controls suggest that if EBV is the etiological agent of NPC in Chinese, it is quite likely to cause the majority of NPC cases in other ethnic groups living in other countries as well.     

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.     

Constitutive expression of HLA class II antigens on EBV positive malignant cells from nasopharyngeal carcinoma: possible involvement in T cell infiltration

Undifferentiated nasopharyngeal carcinoma (NPC) was originally referred to as "lymphoepithelial carcinoma." Two main cell types are constantly associated within these tumors: the malignant epithelial cells which harbor the Epstein-Barr virus (EBV) genome, and the nonmalignant lymphocytes mostly of the T lineage. Three characteristic features of malignant NPC cells might explain T cell migration within NPC tumor tissue: HLA class II molecule expression, IL-1 production, and presence of EBV antigens. Homogeneous suspensions of malignant NPC cells were derived from a nude-mouse transplantable tumor in order to specify the status of HLA class II molecules in these tumors. These suspensions were stained for HLA class II antigens and analyzed by flow cytometry in comparison with other carcinoma cell lines. Three characteristics of transplantable malignant NPC cells were demonstrated: constitutive and coordinate expression of DR, DP, and DQ molecules, and concomitant expression with the CDw40 antigen ("B lymphocyte carcinoma cross-reacting antigen").     

Correlation between nasopharyngeal carcinoma (NPC) and HLA in Hunan Province

24 patients with NPC and 49 normal individuals in Hunan Province were typed for their HLA-A and -B antigens. The results showed that the antigen frequencies of HLA-A1 and HLA-Bw63 were significantly higher in NPC patient group than in the normal group (P = 0.0098 and P = 0.0028, respectively) and the antigen frequency of HLA-A11 was significantly lower in NPC patient group than in the normal group (P = 0.0075). In addition, the joint occurrence of A2 and B17 was increased in NPC patient group (P = 0.0350). The results suggested that HLA-A1 be a regional antigen associated with NPC in Hunan or Southern China, and that HLA-Bw63 be a new kind of antigen associated with NPC.     

EB病毒VCA/IgA、Rta/IgG及EBNA1/IgA抗体水平在鼻咽癌高发区不同人群中的分布

目的 观察分析鼻咽癌高发区中的鼻咽癌患者、非鼻咽癌头颈部相似疾病患者和健康体检人群中EB病毒VCA/IgA、Rta/IgG及EBNA1/IgA的抗体水平分布情况。方法 收集211例未经治疗的鼻咽癌患者、203例头颈部相似症状患者和210例健康体检者的血清,采用免疫酶法检测VCA/IgA,采用酶联免疫吸附法（ELISA）检测Rta/IgG和EBNA1/IgA。应用秩和检验、受试者工作特征(ROC)曲线、多分类logistic回归模型等方法对结果进行分析评价。结果 鼻咽癌组的VCA/IgA、Rta/IgG及EBNA1/IgA抗体水平均显著高于头颈部相似疾病组和健康对照组(P<0.001)。头颈部相似疾病组的Rta/IgG及VCA/IgA抗体水平也明显高于健康对照组(P<0.001)。以头颈部相似疾病组和健康体检组为分析人群,分别作相关抗体的ROC曲线,VCA/IgA 的ROC曲线下面积为0.565,Rta/IgG抗体的ROC曲线下面积为0.604,具有统计学意义(P<0.05)。综合年龄、性别和3种EB病毒抗体等因素的多分类logistic回归分析显示,鼻咽癌、头颈部相似疾病和健康体检者的预测准确率分别为95.3％、70.9％和55.2％。结论 在鼻咽癌高发区EB病毒VCA/IgA及Rta/IgG抗体水平在头颈部相似疾病人群和健康人群中存在一定差异,在鼻咽癌的人群筛查和临床诊断中可根据具体情况设定不同的抗体阳性临界值。     

Immunofluorescent localization of associated antigen of anti-human nasopharyngeal carcinoma cell monoclonal antibody

The immunofluorescent localization of associated antigen of anti-human nasopharyngeal carcinoma (NPC) cell monoclonal antibody (McAb) was performed in different tissues (NPC, non-NPC tumor, chronic inflammation of nasopharyngeal mucosa, adult normal tissue and embryo tissue) with 5 control experiments. McAb (CN-1, Cs-C1) was produced by Department of Microbiology of our college and all the specimens were confirmed by pathology. The results revealed that Cs-C1 antigen was mainly found on the surface of NPC cells with a positive rate of 80.3%. Majority of the positive cells showed yellow-greenish linear fluorescence surrounding the cell membrane while some cells manifested granular fluorescence. In addition, Cs-C1 antigen was also found in a few embryo tissues, epithelial cells of the normal gastric mucosa and cells of the gastric cancer. It is suggested that Cs-C1 antigen be a kind of molecular structure, being gradually produced or increased in quantity during carcinogenesis, and belong to the tumor associated antigen. Cs-C1 antigen could be important in the study of carcinogenic mechanism of NPC cells and valuable to clinical immunodiagnosis.     

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.     

Discordant Epstein-Barr virus nuclear antigen (EBNA) antibody patterns in nasopharyngeal carcinoma

Epstein-Barr virus nuclear antigen (EBNA) preparations from three sources were tested with sera from normal individuals and patients with Hodgkin's disease, breast carcinoma, Burkitt's lymphoma, and American and Chinese nasopharyngeal carcinoma. Individual sera with discordant antibody patterns were noted in all groups. Sera from both NPC groups gave significantly higher anti-EBNA titers on cell lines converted with P3HR-1 or B95-8 virus compared with anti-EBNA titers on Raji cells. Anti-EBNA titers of Chinese NPC sera showed no correlation among the three EBNA sources, while all other groups had highly correlated titers. Cross-absorption experiments present evidence for more than one antigenic determinant on EBNA. These results suggest an additional parameter for distinguishing Chinese NPC from other EBV-related disorders.     

EB virus-associated antibodies in Caucasian patients with carcinoma of the nasopharynx and in long-term survivors after treatment

Sera from 30 Swedish patients with nasopharyngeal carcinoma (NPC) collected before or shortly after commencement of therapy and from 24 long-term survivors (LTS) were tested for antibodies to Epstein-Barr virus (EBV) capsid antigens (VCA), to the O and R components of the EBV-induced early antigen (EA) complex, and to EBV-determined cell membrane antigens (MA). In the tumor-bearing patients, all serologic reactivities increased overall from stage I to later stages of the disease: that is, with an increase in total tumor burden. In the LTS, who had remained apparently tumor-free for 3 to 29 years, all reactivities were substantially lower than in the tumor-bearing patients. While the data agreed in principle with those reported previously for Chinese NPC patients, there were considerable differences with respect to the incidence and titres of antibodies to the D component which, as discussed, might reflect a reduced responsiveness of Caucasian NPC patients or a difference in the distribution of various types of the disease among ethnic groups.     

THE HIGH-LEVEL EXPRESSION OF nm23(NDP)GENE IN NPC

WE have studied the expression of nm23(NDP)in 50cases nasopharyngeal biopsies with anti-nm23(NDP)antibodies. As a result, the NDP positive rate innasopharyngeal carcinoma(NPC)(95.54%) markedlyincreased (P<0.05), as compared with that in the normalnasopharyngeal epithelia (50.00-60.00%) and lympho-cytes (52.00%). There were cytoplasmic type, nucleustype and mixed cytoplasmonucleus type according to NDP,location in a cell Their positive rates were 64.44%,15.56%, and 20.00%, respectively in nasopharyngealcarcinoma. The expression of NDP had no relation withcervical lymphometastases in NPC, and the NDP positiverates had no significance between bilateral cervicallymphometastases and unilateral(P<0.05). But the NDPexpression had most relation with the NPC staging. Theexpression rate and the intensity in Ⅲ or Ⅳ stagepatients were markedly higher than that in Ⅱ stage. Itpoints out that the high-level expression of NDP hadrelation with the rapid cellular proliferation in NPC, andit may indicate the bad prognos     

血浆EB病毒DNA数量与鼻咽癌细胞凋亡的相关性分析

目的探讨血浆EBV-DNA数量与鼻咽癌细胞凋亡的关系。方法采用巢式荧光定量PCR,检测鼻咽癌患者血清EBV-DNA的拷贝数,TUNEL法检测鼻咽癌组织中癌细胞凋亡情况,分析EBV-DNA拷贝数与凋亡癌细胞的相关性。结果鼻咽癌患者血浆EBV-DNA检出率为96.4%（27/28）,中位数为1 800 copies/μl（0～22 100 copies/μl）。28例鼻咽癌活检组织中癌细胞平均凋亡指数为30.0±18.0。EBV-DNA拷贝数与鼻咽癌细胞凋亡呈正相关（γ=0.927,P〈0.05）。结论血浆EBV-DNA水平与鼻咽癌细胞凋亡呈正相关,鼻咽癌患者血浆EBV-DNA可能部分来源于凋亡的癌细胞。     

The 30-bp deletion variant: a polymorphism of latent membrane protein 1 prevalent in endemic and non-endemic areas of nasopharyngeal carcinomas in China.

Development of nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. However, NPC occurs with a marked geographic and racial distribution, whereas EBV infection is ubiquitous in the world. This leads to a question whether certain subtypes of EBV have a greater potential to induce cell transformation. Latent membrane protein 1 (LMP1) is an EBV-encoded oncogenic protein and its 30-bp deleted variant (del-LMP1) has been reported to be predominant in biopsies of NPC. We have assessed the polymorphism of LMP1 in 47 biopsies of NPC, 107 cases of throat washings (TWs) from NPC patients, and 106 cases of TWs from non-NPC patients in Guangzhou, an endemic area of NPC in southern China, as well as 103 cases of TWs from healthy donors in Haerbin, a non-endemic area of NPC in northern China. Our results found a similar extent of the LMP1 polymorphism between NPC patients and non-NPC patients in Guangzhou, with the del-LMP1 being predominant in both Guangzhou and Haerbin. Sequence analyses showed identical substitutions in other coding regions of the del-LMP1 isolated from Guangzhou and Haerbin. These results indicate that del-LMP1 represents a geographic or race-associated polymorphism rather than an NPC disease phenotype-associated polymorphism.     

Nasopharyngeal carcinoma (NPC). I. Types of cultures derived from tumour biopsies and non-tumorous tissues of Chinese patients with special reference to lymphoblastoid transformation

Biopsy specimens of nasopharyngeal carcinoma (NPC) from Chinese patients were sent from Hong Kong to Lyons and cultured in vitro. Four different types of culture were obtained: 1) epithelial growth of short duration (4 to 12 weeks) and limited extent around the explants; 2) early lymphocytic production (ELP) which lasted for 1 to 3 weeks; 3) fibroblastic cultures (either as primary or secondary to the epithelial growth); 4) “lymphoblastoid transformation” which occurred in 28% of the cultures and resulted in the establishment of long-term free-floating cell-lines. A few of these lines were intermittently dependent on the presence of fibroblasts during crises, while most of them were entirely independent. Biopsy specimens from head and neck tumours other than NPC, from hypertrophied and inflamed tonsils removed from children, and from apparently tumour-free nasopharyngeal mucosa gave rise to a similar spectrum of cultures, including the establishment of long-term lymphoblastoid cultures. The various lines thus obtained exhibited the presence of a herpes-type virus (HTV). It is proposed that the establishment of permanent lymphoblastoid lines is caused by the presence, in the original material, of an HTV having “transforming” properties. The significance of these results and the nature of the serological association between NPC and HTV are discussed.     

Detection of Epstein-Barr virus antigens with enzyme-conjugated antibody.

Monospecific conjugated (fluorescein isothiocynate and horseradish peroxidase) goat antisera, prepared against three human immunoglobulin classes, IM (mu), IgG (gamma) and IgA (alpha), were compared for their ability to detect human Ig classes possessing specificity for Epstein-Barr virus (EBV) viral capsid antigens (VCA) in a chronically infected human lymphoid cell line, P3J-HRIK. It was determined that the enzyme system was significantly more sensitive than immunofluorescence in detecting most of the immunoglobulins in sera from cancer patients. Some patients with nasopharyngeal carcinomas (NPC) had extremely high levels of EBV-specific IgA, whereas cancers other than NPC may have lower EBV-specific IgA titers.     

DNA content of nasopharyngeal carcinoma: an independent prognostic indicator

The purpose of this study was to examine whether tumor DNA content correlated with prognosis in nasopharyngeal carcinoma (NPC). DNA flow-cytometric analysis in fresh specimens of nasopharyngeal biopsy from 123 patients with clinical suspicion of NPC was collected initially. Histopathologic study and successful flow-cytometric analysis had 28 lymphoid hyperplasias and 87 NPCs. Seventeen NPC patients were treated elsewhere and were excluded. A total of 98 patients, including 28 lymphoid hyperplasias and 70 NPCs, formed the materials of this study. There were 34 (49%) diploid and 36 (51%) aneuploid in NPC patients. No lymphoid hyperplasias were aneuploid. The mean of S-phase fraction was higher in NPC than in lymphoid hyperplasia (P < .001), indicating higher cellular activity in NPC. DNA content failed to associate with age, gender, pathology, distant metastasis, and stage, indicating that DNA content was an independent prognostic indicator and possibly a clinical parameter. The log-rank test of overall survival curves was significant for stage (P = .002) and DNA ploidy (P = .042); it was almost significant for S-phase fraction (P = .057). Because the follow-up duration was not long enough, univariate and multivariate analysis were not significant for stage, ploidy, and S-phase fraction, except for distant metastasis. It is also most likely colinearity of clinical stage and distant metastasis that explained why clinical stage could not show significance in prognosis. Interestingly, the DNA content appeared to be a potential prognostic parameter in overall survival, although it was not statistically significant (P = .052). Our data suggested that NPC patients with aneuploid DNA and high S-phase fraction tend to have poor prognosis and should be treated more aggressively, even in the early stage of the disease.     

Epstein-Barr virus and nasopharyngeal carcinoma: is there an etiologic relationship?

Antibody titers to all the Epstein-Barr virus (EBV)-related antigens were significant elevated (P less than 0.001) in patients with nasopharyngeal carcinoma (NPC) compared with those of controls. EBV, early antigen, and anti-diffuse titers of Chinese-American and black patients with NPC were higher than those of white patients with NPC. Epstein-Barr viral capsid antigen antibody titers were elevated in white patients with squamous cell carcinoma of the pharynx, regardless of subsite. These results raise doubts about the specificity of the association between EBV and NPC.     

Increased expression of SHP-1 is associated with local recurrence after radiotherapy in patients with nasopharyngeal carcinoma

Background

Nasopharyngeal carcinoma (NPC) is a major cancer in southern China. Src homology phosphatase-1 (SHP-1) is a tyrosine phosphatase that regulates growth, differentiation, cell cycle progression, and oncogenesis. We determined the clinical significance of SHP-1 expression in the tumours of NPC patients from southern China who were treated with radiotherapy.

Patients and methods.

SHP-1 expression was determined by real-time polymerase chain reaction (PCR) and western blotting of NPC tissue samples of 50 patients and nasopharyngeal tissues of 50 non-NPC patients who had chronic nasopharyngeal inflammation. SHP-1 expression was measured in NPC tissue samples of 206 patients by immunohistochemistry and survival analysis was performed.

Results

The tumours of NPC patients had significantly increased expression of SHP-1 at mRNA and protein levels relative to patients with chronic nasopharyngeal inflammation. Survival analysis of NPC patients indicated that SHP-1 expression was significantly associated with poor local recurrence-free survival (p = 0.008), but not with nodal recurrence-free survival, distant metastasis-free survival, or overall survival.

Conclusions

SHP-1 appears to be associated with radiation resistance of NPC cells and can be considered as a candidate marker for prognosis and/or therapeutic target in patients with this type of cancer.