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Purpose: Nasopharyngeal carcinoma (NPC) has been proved to be an Epstein-Barr virus (EBV)-associated cancer. By use of nested polymerase chain reactions (PCRs), we examined whether the presence of EBV DNA in the peripheral-blood cells (PBC) can serve as a prognostic indicator for NPC., Patients and Methods: Peripheral blood from 124 patients with NPC who had no evidence of distant metastasis and 114 healthy volunteers with serologically positive findings for EBV infection was collected prospectively. Plasma and erythrocytes were separated. DNA was extracted from PBCs and analyzed by a nested PCR using primers specific to Epstein-Barr virus nuclear antigen 1 (EBNA-1). All patients were treated by radiotherapy with or without chemotherapy. Clinical parameters and status of EBNA-1 in PBCs were used for survival analysis using the Kaplan-Meier method and the Cox proportional hazards model., Results: Positive rates of EBNA-1 DNA in PBCs of NPC patients and healthy volunteers are 71% and 14%, respectively (P =.001). No significant difference was observed with regard to the clinical characteristics of patients who were EBNA-1-positive (n = 88) and those who were EBNA-1-negative (n = 36). After a median follow-up period of 38 months (range, 24 to 56 months), 29 of 88 EBNA-1-positive patients and only one of 36 EBNA-1-negative patients developed distant metastases (P =.00015). Kaplan-Meier estimates of overall survival (P =.0010), metastasis-free survival (P =.0004), and progression-free survival (P =.0004) were significantly lower for the patients in the EBNA-1-positive group than for those in the EBNA-1-negative group. Multivariate Cox analysis confirmed the same results., Conclusion: The presence of EBNA-1 DNA in PBCs is a novel, important risk factor for patients with NPC that indicates a significantly higher risk of developing distant metastasis as well as a lower survival rate.

Background: Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC). An EBV-encoded immediate-early antigen, BZLF-1 replication activator (ZEBRA) initiates EBV replication and expression in all NPC tumors. In this study, we investigated whether immunoglobulin A (IgA) against ZEBRA is present in the sera of patients with NPC, and whether it was able to be determined by enzyme-linked immunosorbent assay (ELISA) using a recombinant ZEBRA prepared from Escherichia coli., Methods: A polymerase chain reaction-amplified cDNA fragment of the ZEBRA gene was inserted into the expression vector of E coli under the control of an IpL promoter. E coli bacteria containing the CI857 gene served as host to overexpress the ZEBRA protein by heat induction. Recombinant ZEBRA was collected by mechanical disruption of the bacteria, purified by column chromatography, and analyzed by SDS-PAGE and Western blot assay using sera from NPC patients. The recombinant ZEBRA was used to develop the ELISA to detect IgA against ZEBRA., Results: The amount of ZEBRA produced comprised 30% of total E coli protein. Western blot assay confirmed that affinity of the recombinant ZEBRA to IgA antibody was preserved. IgA against ZEBRA was shown to be positive by ELISA in 36 of 40 NPC sera, but in only nine of 55 patients with other head and neck malignancies, and two of 35 normal individuals. For serologic diagnosis of NPC, the sensitivity of IgA/ZEBRA detected by ELISA was 90% and the specificity was 87.4%., Conclusions: A recombinant ZEBRA was produced at high levels in E coli and retained affinity to IgA against ZEBRA. The recombinant ZEBRA was successfully used to develop an ELISA for the detection of IgA against ZEBRA. The high sensitivity and specificity of IgA against ZEBRA show that the ELISA is feasible for serologic diagnosis of NPC.

This study assessed 11C-acetate turnover (clearance) in nasopharyngeal carcinoma (NPC). Data were acquired by dynamic PET after the intravenous injection of 4.625 MBq.kg-1 body weight of 11C-acetate for 30 min. Tomograms were reconstructed and evaluated visually. A time-activity curve of the nasopharynx and neck was generated and the clearance rate of 11C-acetate from the nasopharynx in the slow phase and from NPC was calculated using 0.693/T1/2. Ten patients with nasopharyngeal carcinoma and nine normal subjects were studied. The clearance of 11C-acetate from the normal nasopharynx was rapid and biexponential, in contrast to the rapid uptake followed by extremely slow clearance in patients with NPC. The clearance rate (mean +/- S.D.) was 0.0074 +/- 0.0042 in NPC and 0.0263 +/- 0.0152 in controls in the slow phase, being significantly different between the two groups with no overlap. All nasopharyngeal carcinomas were clearly visualized, in contrast to no obvious retention in the normal nasopharynx. Our initial results indicate that 11C-acetate clearance can be used to differentiate nasopharyngeal carcinoma from a normal nasopharynx. This finding may lead to new applications of 11C-acetate in oncology.

Background: Soluble interleukin-2 receptor alpha (sIL-2R alpha) is a well-known indicator of T-cell activation noted to be increasing in nasopharyngeal cancer. However, the significance of sIL-2R alpha in monitoring disease relapse is unclear. This study was initiated to address this issue., Methods: Serum of 56 patients with NPC, which underwent either primary, salvage, or palliative treatments, from 1992 to 1993 at the Cancer Center, Veterans General Hospital-Taipei, were collected from our serum bank. According to their disease status at the time of study, at least two years after last treatments, the 56 patients were divided into four groups. The remission group represented those in remission at the time of study (n = 24). The metastasis group represented those with distant metastasis present at the time of study (n = 17). The recurrence group represented those with locoregional recurrence present at the time of study (n = 11). The combined group represented those with locoregional recurrence as well as distant metastasis (n = 4). The seral sIL-2R alpha concentrations of the 56 NPC patients were determined with enzyme-linked immunoabsorbent assay. The combined group was excluded in our statistical analysis. We performed statistical analysis on the differences of paired serum sIL-2R alpha concentrations between different periods of the diseases. The first analysis was on the differences of sIL-2R alpha concentrations between diagnosis and post-radiotherapy periods for 13 out of 24 patients in the remission group and 7 out of 11 patients in the recurrence group. The second analysis was on the differences of sIL-2R alpha concentration between follow-up before detection-of-relapse and after detection-of-relapse for 5 out of 17 patients in the metastasis group and six out of 11 patients in the recurrence group., Results: The first statistical analysis revealed no significant differences of sIL-2R alpha concentrations for the remission group (P = 0.946) and the recurrence group (P = 0.156) between diagnosis and post-radiotherapy periods. The second statistical analysis revealed no significant differences of sIL-2R alpha concentrations between before and after detection-of-relapse for the recurrence group, neither (P = 0.438). The results for the metastasis group were different. The sIL-2R alpha concentrations were shown to increase after the detection of metastasis for the 5 paired samples from the metastasis group, although the Wilcoxon signed ranks test on the differences only showed borderline significance (P = 0.063)., Conclusions: Our findings show that sIL-2R alpha would be of no value in monitoring the development of locoregional recurrence but might be useful in monitoring distant metastasis. Although our current limited data did not provide strong support for the role of sIL-2R alpha in monitoring metastasis, it might be delineated in the future by collecting more data.

Elevated serum IgA to antigens of EBV is associated with nasopharyngeal carcinoma (NPC). We have tested 620 NPC sera by ELISA for the presence of antibodies to EBV-encoded DNA binding protein, EBV-specific DNA polymerase, early antigen-diffused (EA-D), EBV nuclear antigen 1 (EBNA-1), EBV-specific thymidine kinase, and BamHI Z fragment EBV replication antigen. Sensitivity of these proteins was in the range of 51.5-79.5% for IgA and 69.4-82.8% for IgG. The complementary use of EBNA-1 with EA-D, however, could increase the sensitivity significantly to 98.1%. Western blot analysis further showed that the combination of EBNA-1 and EA-D is most useful for the detection of NPC. This is the first report of using double biomarkers including EBV gene products from both latent and active infections. The results of this study suggest that EBV in NPC may not be latent alone and that the method may be valuable for the early detection, early treatment, and better survival rate of patients with NPC. Because the application of recombinant EBV protein in ELISA is cost-effective and feasible for mass screening, the method may be of worth for further clinical investigation.

This is a phase II study to evaluate the efficacy and toxicity of short-course carboplatin in advanced-stage nasopharyngeal carcinoma (NPC). Thirty-three previously untreated stage III-IV NPC patients were studied. Carboplatin was given as a rapid intravenous injection every 3 weeks. The dose of carboplatin was calculated according to the individual patient's creatinine clearance and desired platelet nadir of 75,000/microliter according to the Egorin formula. Response and toxicity were evaluated. Thirty-two patients were evaluated for response. The median age was 54 years, range 30-70 years. Twenty-four patients had local regional disease and 8 patients had metastatic disease. The median dose of carboplatin given was 415 mg/m2 (range 91-791 mg/m2). Fourteen (44%) patients had a partial response with a 95% confidence interval of 26-62%. Fifteen (47%) patients had stable disease and 3 (9%) progressive disease. The overall median survival rate was not reached at 43 months. Overall toxicity was tolerable. Grade III-IV myelosuppression occurred in 4 (12%) patients. There were no other major toxicity- or treatment-related deaths. We conclude that carboplatin has a significant anticancer effect in advanced NPC. Thus carboplatin combination chemotherapy for the treatment of NPC is worthy of future clinical investigations.

Purpose: A newly designed concomitant chemoradiotherapy was undertaken to assess the feasibility and efficacy for advanced nasopharyngeal carcinoma (NPC)., Methods and Materials: Sixty-three patients with biopsy-proven NPC were entered in this Phase II trial from March 1992 to November 1993. Most patients present with Stage IV disease (93.4%) and poorly differentiated epidermoid carcinoma or undifferentiated carcinoma were the major pathologic type. Radiotherapy was delivered using a telecobalt unit and 10 MV x-rays and by altered fractionation (72-74 Gy/45 fractions/6 weeks). Chemotherapy with cisplatin 75 mg/m2, 2 h infusion at day 1, followed by 5-FU 400 mg/m2/day, continously infused for 4 days was given concurrently during the first and fifth weeks of radiotherapy., Results: The major toxicity was mucositis (61% belong to Grade 3, 31% to Grade 2). Weight loss, leucopenia, and skin reaction were frequently encountered. Three patients withdrew from treatment at 15, 25, and 55.5 Gy, three patients interrupted the radiotherapy for 1-4.5 weeks, and two patients refused the second cycle of concomitant chemotherapy due to toxicities. The initial tumor response showed 100% overall response rate, with 90.5% complete response. After a median follow-up time of 38 months, five patients failed at the primary and/or neck (four recurrent and one persistent), and 14 patients developed distant metastases alone. The 3-year primary disease-free, regional disease-free, distant disease-free, and overall survival rates are 89.1, 92.8, 74.3, and 73.6%, respectively. The late complication rate is acceptable so far., Conclusions: Our data indicates that concurrent chemoradiotherapy for advanced NPC is both feasible and effective, with acceptable toxicities. Distant metastases are the major site of treatment failure. Postradiation adjuvant chemotherapy to eradicate subclinical distant metastasis should be further studied.

In vivo demonstration of hypoxia is of significance for tumour patient management. Fluorine-18 fluoromisonidazole ([18F]FMISO) is a proven hypoxic imaging agent. We developed an [18F]FMISO tumour to muscle retention ratio (TMRR) for the detection of tumour hypoxia in nasopharyngeal carcinoma (NPC). Data were acquired by positron emission tomography (PET) of the nasopharynx and neck after intravenous injection of 370 MBq of [18F]FMISO. Two imaging protocols were adopted: a long protocol for comprehensive dynamic information and a short protocol for a simple, clinically convenient imaging procedure. Tomograms were reconstructed and evaluated visually. ROI analysis on the basis of time-activity curve evaluation was performed to calculate the TMRR of NPC or cervical nodal metastases (CNMs) in relation to the suboccipital muscles at 2 h. The calculation of the TMRR was exactly the same for both the long and the short protocol as two 30-min composite frames had been created immediately after intravenous injection and 2 h after injection of [18F]FMISO in the long protocol. The normal tissue to muscle retention ratio (NTMRR) was derived similarly from the normal nasopharynx. The data of 12 controls and 24 patients with NPC were analysed. The long protocol was used in 15 patients, and the short protocol in nine. In controls, the mean NTMRR+/-1 SD was 0.96+/-0.14. The mean TMRRs for NPC and CNMs were 2.56+/-1.50 and 1.35+/-0.51, respectively; these values were significantly higher than the mean NTMRR for normal controls (P<0.005 in each case). At the retention threshold value of 1.24, tumour hypoxia occurred in 100% of the primary lesions of NPC and 58% of CNMs. The TMRR for undifferentiated carcinoma was significantly lower than that for non-keratinized carcinoma (P<0.05). The [18F]FMISO TMRR is a simple and clinically useful index for detecting tumour hypoxia in NPC.

Bone scintigraphy plays an important role in the early detection of bone metastases in patients with nasopharyngeal carcinoma and serial scans may aid the clinician to assess the therapeutic response. A superscan is a pattern described as abnormal bone scan, indicating extensive bony metastases associated with various neoplastic diseases. Bone scans from 407 patients with nasopharyngeal carcinoma were reviewed retrospectively. Only six superscans (1.5%) were found. The appearance of a superscan is frequently accompanied by an abnormal titer of serological markers IgG-VCA and IgA-VCA, liver metastases, and poor survival. Although a superscan rarely occurs in nasopharyngeal carcinoma, its appearance may represent a poor prognosis in these patients.

Background: Cisplatin, 5-flourouracil (5-FU), and leucovorin (PFL) chemotherapy has been reported to be effective in the treatment of cancers but severe mucositis or neutropenia are dose limiting toxicities. This Phase II study evaluated the anticancer effect and the toxicities of a new weekly 24-hour infusional PFL chemotherapy in patients with nasopharyngeal carcinoma (NPC)., Methods: Forty-two patients with stage IV NPC were studied. Cisplatin 25 mg/m2/d, 5-FU 2200 mg/m2/d, and leucovorin 120 mg/m2/d were adminstered weekly by 24-hour intravenous continuous infusion in an outpatient setting. Clinical response and toxicity were evaluated weekly., Results: The complete response rate (CR) was 30% and the partial response (PR) rate 60% in the localized previously untreated group. The CR rate was 22.7% and PR rate 45.5% in local recurrent/metastatic group. The overall response rate was 79%. Eighty-one percent of patients who had no previous chemotherapy and 67% of patients who had previous chemotherapy responded to weekly PFL. There were no dose limiting toxicities. No patient had grade 3 or 4 mucositis or neutropenia. Thirty-two patients (76%) had no oral mucositis. Seven patients (17%) had grade 1 mucositis and 3 patients (7%) had grade 2 mucositis., Conclusions: Elimination of dose limiting toxicities is possible using a weekly 24-hour infusion schedule of PFL chemotherapy while retaining significant anticancer activity as demonstrated in these patients with advanced NPC. To discover whether this schedule is superior to cisplatin and 5-FU or other PFL chemotherapy regimens requires further investigation.

The aims of this study were to determine the incidence of hypertrophic pulmonary osteoarthropathy (HPO) in nasopharyngeal carcinoma (NPC) and assess its clinical significance. Altogether, 407 NPC patients were reviewed retrospectively. HPO was identified by 99Tcm-methylene diphosphonate bone scans and related clinical and radiographic evidence. Pulmonary metastases, bony metastases and titre of anti-Epstein Barr virus (EBV) immunoglobulin were assessed in patients with and without HPO. The patients had a mean (+/- S.D.) age of 50.4 +/- 12.4 (range 17-73) years. HPO was found in 27 of the 407 (6.6%) NPC patients, among whom 13 (48%) had pulmonary metastases. HPO preceded lung metastases by 7-22 months (14.4 +/- 6 months) in 7 (52%) patients. Six patients had overt lung metastases at the time of the bone scan. No significant difference was found in anti-EBV immunoglobulins between the patients with or without HPO, nor in the incidence of bony metastases between these two groups of patients. HPO should be regarded as an early sign of pulmonary metastases.

Epstein-Barr virus (EBV) encoded DNA polymerase (POL) was cloned and over-expressed in Escherichia coli. Western blot analysis confirmed the presence of antibody to this POL protein in sera from nasopharyngeal carcinoma (NPC) patients. By Western blot analysis, moderate to high concentration of IgG POL-specific antibodies were present in 43 of 48 NPC sera and only 4 of 48 healthy, seropositive controls. The POL-specific IgG antibodies appear as early as stage I of NPC, suggesting that the recombinant POL protein can be a useful diagnostic marker for early diagnosis of the disease. It was also found that human sera containing high titer of cytomegalovirus (CMV) antibodies or herpes simplex virus type 1 (HSV-1) antibodies did not cross-react with the recombinant EBV POL, despite the homology shared by DNA polymerase proteins of these viruses.

Background: Systemic disease progression occurs in the majority of patients with locally advanced nasopharyngeal carcinoma (NPC). Although a variety of chemotherapeutic drugs have had tumoricidal activity, the roles of chemotherapy and optimal regimens must be further defined. Based on high response rates of Cisplatin, 5-Fluororacil and Leucovorin (PFL) in patients with advanced squamous cell cancers of the head and neck, we tested a new outpatient PFL chemotherapy program in patients with advanced NPC., Methods: Patients with NPC and 1) previously untreated, locally advanced disease; 2) local regional recurrence (LR) after radiotherapy; or 3) metastatic disease were eligible for study. Cisplatin 20 mg/m2/d, 5-FU 800 mg/m2/d and Leucovorin 90 mg/m2/d were administered simultaneously by continuous 96-hour intravenous infusion every three weeks. Patients were evaluated for response, survival, and toxicity., Results: Thirty-five patients were studied. The response rates of PFL therapy were 100% (15% complete response [CR], 85% partial response [PR]) in 20 patients with locally advanced or locally recurrent disease, and 80% (13.3% CR, 67.7% PR) in 15 patients with metastatic disease. The overall median survival was 20 months after therapy (range, 2-21). The median survival rate for previously untreated, locally advanced patients was not reached. The median survival rate for previously treated, local recurrence was 34 months and for metastatic patients was 14 months. Mucositis and leukopenia were the dose-limiting toxicities (20-23%, grade III) and occurred more frequently in patients previously irradiated. No treatment-related deaths occurred., Conclusions: Outpatient PFL chemotherapy is active, safe, and convenient for advanced stage nasopharyngeal carcinoma patients, and the overall toxicities are tolerable.

The pharmacokinetics and pharmacodynamics of mitoxantrone were studied in 15 patients with advanced nasopharyngeal carcinoma (NPC) after single intravenous rapid infusion (12 to 14 mg/m2). Mitoxantrone plasma concentrations and urinary excretion were measured specifically with the use of a high-performance liquid chromatographic method with ultraviolet detection at 242 and 658 nm. The pharmacokinetic parameters are described adequately by a three-compartment model with a terminal half-life of 71.5 +/- 40.1 hours and a volume of distribution of 5037 +/- 2377 l. The total plasma clearance was 743 +/- 462 ml/minute, and the renal clearance was 18.8 +/- 8.49 ml/minute. Within 72 hours, 1.8 +/- 0.6% of the administration dose was excreted in urine as mitoxantrone parent compound. From the urinary excretion rate data, glomerular filtration and possible tubular reabsorption were the mechanisms involved in the urinary excretion of mitoxantrone. The values for unbound fraction (%) in plasma at time 0 and 5 minutes were 2.88 +/- 0.91% and 3.25 +/- 1.19%, with an average of 3.04 +/- 1.01%. The degree of protein binding of mitoxantrone was not affected by concentration (P greater than 0.05) in Chinese patients with NPC. The response rate for mitoxantrone was poor in this study. Clinical studies have demonstrated that mitoxantrone was generally well tolerated. Only very low incidences of nausea, vomiting, and alopecia were observed. The mild and rapidly reversible dose-limiting hematologic toxic effects have proven leukopenia. Although the toxicities reported here were tolerated for most patients, other combination regimens including mitoxantrone or other administration routes may be considered and need to be evaluated carefully.

Five of 23 patients with recurrent nasopharyngeal carcinoma (NPC) were diagnosed to have bone marrow metastasis. They all had advanced local-regional disease, and were treated with neoadjuvant chemotherapy and definitive radiotherapy after the initial diagnosis. Bone marrow metastasis developed 4-24 months later. The clinical features were anemia (5 of 5), leukopenia (3 of 5), thrombocytopenia (4 of 5), sepsis (3 of 5), tenderness of the sternum (3 of 5), and fever (4 of 5). Patients frequently had elevation of serum lactic dehydrogenase (LDH), alkaline phosphatase (ALK-P), and IgG and IgA antibody titers to Epstein-Barr viral capsid antigen when bone marrow involvement was diagnosed. However, clinical manifestations and laboratory tests were not specific. It is important that three patients had normal bone scans. All five patients had a rapid downhill course; four patients died within 23 days, and the fifth 3 months after the diagnosis of bone marrow metastasis. We concluded that bone marrow was a common metastatic site in NPC patients. Bone marrow metastasis adversely affected patients' survival and required a high index of suspicion for diagnosis. We suggested that bone marrow biopsy should be considered as a routine staging procedure in NPC patients and indicated especially when patients presented with abnormal blood counts, sepsis, bone pain, or tenderness of the sternum. It may be positive in the face of a normal bone scan.

The plasma pharmacokinetic profile of 4'-epidoxorubicin (epirubicin) was investigated in 28 patients with nasopharyngeal carcinoma (NPC) after single i.v. rapid infusions. All patients had normal liver and renal functions. Plasma concentrations of the parent compound were specifically determined by a high-performance liquid chromatographic (HPLC) method, with UV detection at 254 nm. Plasma levels of the compound were fitted to a three-compartment open model; a triexponential decrease in plasma concentrations with a long terminal plasma half-life (44.8 +/- 21.2 h) was observed in 27 patients. The respective mean (+/- SD) serum concentration at 72 h and the AUC, plasma clearance, and terminal elimination rate constant in complete responders were 7.67 +/- 1.98 ng/ml, 4,002 +/- 3,080 ng.h/ml, 26.6 +/- 12.9 l/h.m2, and 0.009 +/- 0.007 l/h, whereas those in nonresponders were 4.96 +/- 1.8 ng/ml, 1.88 +/- 652.8 ng.h/ml, 44.4 +/- 15 l/h.m2, and 0.017 +/- 0.006 l/h, respectively; these differences were significant (P less than 0.05). Epirubicin produced a 52% response rate, including 6 patients with a complete response, 8 with a partial response, 11 with no change, and 2 with progressive disease. No relationship could be found between the various pharmacokinetic parameters and either leukopenia, age, or sex. These observations strongly suggest that plasma clearance may be one of the determining factors affecting the response or nonresponse of NPC patients to epirubicin, and a dose adjustment according to plasma clearance would probably increase the response rate.