Abstract 3153: A new circulating plasma/serum Epstein-Barr virus (EBV) DNA test by locked nucleic acid (LNA) PCR technique with enhanced sensitivity in diagnosis & monitoring of patients suffering from nasopharyngeal carcinoma (NPC) and other EBV associated malignancies
Authors: Yip Timothy T.C. , Kwok Hazel Y.Y. , You Sharon S.Y. , Kwong Dora L.W. , Fong Alvin H.W. , Cheng W.W. , Lai Christopher , Shek Anthony C.C. , Ma Victor W.S. , LiLung Maria , Ngan Roger K.C. 
Published: July 2016

Abstract
Circulating plasma/serum Epstein-Barr virus (EBV) test has been established as routine first line diagnostic test for Nasopharyngeal Carcinoma (NPC). This real time quantitative PCR test is useful in primary diagnosis of NPC as well as monitoring distant metastases of NPC to lung, liver and distant lymph nodes. However, it is still lacking sensitivity in diagnosis of early stage I/II NPC and local relapse in nasopharynx. Detection sensitivity of this PCR test relies on detecting small fragments of circulating EBV DNA shed into the blood stream from NPC tumor lesions which underwent apoptosis/necrosis. Previous literature already showed that the smaller the molecular size of the circulating EBV DNA targets detected, the higher the detection sensitivity. Using Locked Nucleic Acid (LNA) PCR technique, we have designed a new set of LNA primers for PCR amplification of a much smaller EBV gene target (BamH1-W DNA at 46 bp) than the conventional EBV gene target (BamH1-W at 76 bp). Using this LNA marker, we successfully achieved detection sensitivity of 95.7% in 46 NPC patients (44/46 positive) and detection specificity of 98.3% in 59 normal blood screening subjects (58/59 negative). 89.1% (i.e. 41/46) of the NPC patients had higher EBV DNA gene copies per mL in the LNA marker than the conventional marker. Folds of gene copies per mL of increase of LNA marker over those of conventional marker varied from 6 folds up to 3800 folds in 27/46 NPC patients. The results were confirmed in a validation set of 141 NPC patients (113/141) in a multicenter study under the umbrella of NPC Area of Excellence (AoE) program in Hong Kong with 79.6% (113/142) of NPC patients having higher gene copies per mL of LNA marker than the conventional marker initially tested. On longitudinal monitoring of the new LNA marker in 9 NPC patients for a period of 4.9 months to 5.6 months, higher sensitivity of detection was also achieved at disease onset, during the clinical course of disease and at distant metastases (2/9) than the conventional marker. Three patients with Lymphoepithelioma Like Carcinoma of Lung (LELC) and 3 patients with extra nodal nasal NK/T cell lymphoma were monitored from 3 months to 19.3 months. Detection sensitivity was also higher in the new LNA marker in 3/3 LELC and 1/2 NK/T lymphoma with disease progression whereas 1 NK/T lymphoma with remission had LNA marker remained negative. With such enlightening findings, we are expanding the scope of this new test to many more NPC patients as well as other EBV associated malignancies. We are also recruiting patients in embarking on a large scale prospective study.
Citation Format: Timothy T.C. Yip, Hazel Y.Y. Kwok, Sharon S.Y. You, Dora L.W. Kwong, Alvin H.W. Fong, W.W. Cheng, Christopher Lai, Anthony C.C. Shek, Victor W.S. Ma, Maria LiLung, Roger K.C. Ngan. A new circulating plasma/serum Epstein-Barr virus (EBV) DNA test by locked nucleic acid (LNA) PCR technique with enhanced sensitivity in diagnosis & monitoring of patients suffering from nasopharyngeal carcinoma (NPC) and other EBV associated malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3153.