Analysis of Cell-free Epstein-Barr Virus-associated RNA in the Plasma of Patients with Nasopharyngeal Carcinoma

Chen et al. (1) and Nawroz et al. (2) have reported that tumor-derived DNA is detectable in the plasma and serum of cancer patients and have opened up a new molecular approach for the early detection of malignancy. It is not known, however, whether circulating tumor-derived RNA is also present in plasma, because of the lability of RNA. To address this possibility, we used nasopharyngeal carcinoma (NPC) as a model system and attempted to detect Epstein-Barr virus (EBV)-latent gene transcripts in cell-free plasma samples from NPC patients. NPC constitutes one of the commonest cancers in Hong Kong and Southern China (3). Previous studies have indicated that EBV is consistently detected in all undifferentiated NPC cases and is present in all cancer cells (3). Latent EBV infection is an early event in the development of this cancer (4). These findings suggested that the EBV genome and latency products may serve as potential markers for the screening and diagnosis of this cancer. Among the EBV-latent genes, the small EBV-encoded RNAs (EBERs) are expressed in all NPC cases and are the most abundant latency-associated transcripts in NPC cells (∼105 to 106 copies per cell) and are widely used for the detection of EBV-associated human tumors, using in situ hybridization (5). We hypothesize that EBER RNA may also be detectable in the plasma of NPC patients. In this study, we used reverse transcription-PCR (RT-PCR) and oligonucleotide hybridization to analyze the presence of EBER-1 RNA in cell-free plasma samples of 26 NPC patients and 29 healthy subjects. The project was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong, and informed consent was obtained from all participants. Total RNA was extracted from 250 μL of fresh plasma sample from each individual, using a Trizol LS kit (Life Technologies) and following the manufacturer’s recommendations. The RNA was then dissolved in 5 μL of RNase-free water, and 2 μL of plasma RNA was subjected to 40 cycles of RT-PCR amplification, using an EZ rTth RNA PCR kit (PE Applied Biosystems). The Tth DNA polymerase had both reverse transcriptase and DNA polymerase activities (6) and thus was able to perform both reactions in a single tube. For each sample, duplicate aliquots were subjected to RT-PCR analysis. Primers specific to the EBER-1 gene were used as described previously (sense, 5′-AAAACATGCGGACCACCAGC-3′; antisense, 5′-AGGACCTACGCTGCCCT-AGA-3′) (7). The PCR products were analyzed using 3% agarose gel electrophoresis, and their identities were confirmed by Southern blotting and hybridization using an EBER-1-specific internal probe (5′-ACGGTGTCTGTGGTTGTCTT-3′) (7). A 167-bp RT-PCR product was detected in the plasma samples containing EBER-1 RNA (Fig. 1). Detection of EBER-1 RNA in the plasma of NPC patients (lanes 2–10) and healthy controls (lanes 13–19) by RT-PCR. Lanes 1 and 12, an EBV-transformed lymphoblastoid cell line (CB14022) as a positive control; lanes 11 and 20, reagent control without RNA. The 167-bp EBER-1 RT-PCR product (arrows) is indicated in the positive control lanes (lanes 1 and 12), plasma samples from NPC patients (lanes 2–4 and 6–10), and a non-NPC control (lane 16). Among the 26 NPC patients, EBER-1 RNA was detected in 23 of 26 (88.5%) plasma samples. Control reactions without reverse transcriptase were negative, confirming that the products were amplified from RNA. In the three negative cases, transcripts of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), were detectable, demonstrating the integrity of RNA in these samples. Among the healthy control subjects, 6 of 29 (20.7%) plasma samples showed detectable EBER-1 RNA. The difference in the proportion of cases in which EBER-1 RNA was detectable in NPC and non-NPC cases was statistically significant (χ2 test, P <0.001). The detection of EBER-1 RNA in the plasma of the healthy individuals is probably attributable to the presence of latent EBV-infected B lymphocytes in healthy carriers of the virus (3). This study shows for the first time that cell-free tumor-related RNA can be detected in the plasma of patients with NPC. The sensitivity and specificity of the plasma RNA assay for NPC were 88.5% and 79.3%, respectively. This novel approach may be useful for early detection of NPC and other EBV-associated malignancies. The specificity of the detection method could potentially be increased by testing other NPC-related genes. Mutirangura et al. (8) have evaluated the presence of cell-free EBV DNA in the serum samples of NPC patients. They reported that only 13 of 42 (31%) patients were positive for EBV DNA in their sera. Using real-time quantitative PCR, we have detected cell-free EBV DNA in the plasma of 96% of NPC patients (9). Real-time quantitative PCR can potentially be applied to the detection of EBV-associated RNA in the plasma of NPC patients. This approach may potentially enhance the clinical usefulness of EBV-associated RNA detection for NPC diagnosis because of improved discrimination between NPC subjects and EBV RNA-positive subjects without NPC. This development is possible because the latter group of subjects generally has a smaller amount of plasma EBV-associated RNA (Fig. 1 , lane 16, which shows a relatively weak RT-PCR signal) than those with NPC (Fig. 1 , lanes 2–4 and 6–10). A further advantage of real-time PCR for NPC diagnosis is that no post-PCR manipulation is necessary, which greatly increases throughput and reduces the risk of carryover contamination. Our data highlight the concept that the detection of tumor-associated RNA in plasma may be a promising new direction for cancer detection. Recent advances in the expression genetics of cancer have successfully identified large panels of differentially expressed genes in human malignancies (10) and could potentially provide numerous new markers for plasma RNA-based molecular analysis. This work was supported by Research Grants Council Earmarked Grants CUHK 261/96 M and CUHK 259/96 M.