Familial Nasopharyngeal Carcinoma 6

Prognostic Factors in Nasopharyngeal Cancer 107was assayed by a nested PCR using primer specific toEBV nuclear Antigen 1 (EBNA-1). The characteristicsof NPC patients with EBNA-1 positive and EBNA-1negative showed no significant difference. After amedian follow-up of 38 months, 29 of 88 EBNA-1 positivepatients developed distant metastasis and onlyone distant metastasis was observed in the patients ofthe EBNA-1 negative group (p = 0.00015). Overall survival,metastasis-free survival, and progression-freesurvival were all significantly lower for the patients inthe EBNA-1 positive group. Multivariate Cox analysisalso confirmed the same results. The authors concludedthat the presence of EBNA-1 DNA in peripheralblood cells of NPC patients was a novel biomarkerin predicting a significantly higher distant failure anda lower survival rate. Consistent sequence variation ofEBNA-1 in primary tumors and peripheral blood cellsin addition to the results of clinical observations dosupport that the EBV DNA that appeared in peripheralblood cells of NPC patients may have arisen fromthe disseminated cancer cells (Wang et al. 2002).Table 9.4 lists prognostic effects of peripheral bloodcells expressing tumor-associated genes in NPC.9.4.3Plasma/Serum EBV DNAWith recent advances in molecular biology, the PCRbasedtechnique makes it possible to detect a verysmall amount of biomolecules in a wide array of biologicalsamples. Cell-free circulating DNA could bedetected in plasma and serum. A previous reportindicated that the cancer patients had significantlyhigher mean free DNA in serum than did healthy controls(Leon et al. 1977). Studies on the characteristicsof DNA found in the plasma and serum of cancerpatients highly suggested that much of the circulatingDNA originated from the tumor cells and exhibitedtumor-associated alterations. The mechanisms underlyingthe release of cell-free DNA from the tumor tissuesinto the circulation have not yet been completelyelucidated. Some studies indicated that such cell-freeDNA was largely accountable through the release ofapoptotic and necrotic cancer cells (Fournie et al.1995; Jahr et al. 2001). As a consequence, circulatingcell-free DNA may serve as an attractive alternativefor protein-based tumor markers.Besides peripheral blood cells, plasma/serum sampleswere used for EBV DNA detection in NPC(Mutirangura et al. 1998; Shotelersuk et al. 2000;Hsiao et al. 2002). Using EBNA-2 as a target gene and40 cycles PCR, Mutirangura et al. (1998) demonstratedthat 13 of 42 NPC patients (31%) had detectableEBV DNA in their sera but not in all 82 normalcontrols. In a subsequent study, the detection rate ofserum/plasma EBV DNA increased to 58.7% in 167NPC patients studied by using nested PCR, but falsepositive was found in 10 of 77 (13%) normal blooddonors (Shotelersuk et al. 2000). Using EBNA-1 as atarget and conventional PCR of 35 and 50 cycles,Hsiao et al. (2002) reported that 50-cycle PCR assayenhanced detection sensitivity from 38.9% to 75% forpatients with newly untreated patients, from 27.8% to88.9% for patients with locoregional recurrence, andfrom 71.4% to 100% for patients with distant metasta-Table 9.4. Prognostic effects of peripheral blood cells expressing tumor-associated genes in nasopharyngeal carcinomaSeriesNo. ofcasesstage Treatment Timing andsampleAssay Target Results 3-y OS 3-y MFS 3-y PFSLin et al.(2001)124 I–IV RT ± Ch Pretreatment,PBCnestedPCREBNA-1 positive 65.5% 64.6% 59.2%negative 97.2% 97.1% 94.3%p = 0.001 p = 0.0004 p = 0.0004Lin et al.(2002)57 II–IV RT ± Ch Pretreatment,PBCnestedrt- PCRCK19mRNApositive – 49.9% –negative – 85.9% –p = 0.00893-y 3-year; OS overall survival; MFS metastasis-free survival; PFS progression-free survival; RT radiotherapy; Ch chemotherapy;PBC peripheral blood cells; PCR polymerase chain reaction; EBNA-1 Epstein–Barr Virus nuclear antigen 1; rt reverse transcriptase;CK19 cytokeratin 19; – not available