Epstein-Barr Virus in Nasopharyngeal Carcinoma 內科部血液腫瘤科 徐正龍

(Cancer.Net)

Symptoms/signs Tumor mass in the nasopharynx (epistaxis, nasal obstruction, and discharge) Tumor mass in the nasopharynx (epistaxis, nasal obstruction, and discharge) Dysfunction of the eustachian tube, mass in paranasopharyngeal space ( tinnitus and deafness) Dysfunction of the eustachian tube, mass in paranasopharyngeal space ( tinnitus and deafness) Neck mass (upper neck) Neck mass (upper neck) Skull-base erosion and palsy of the V and VI cranial nerves and up extension of tumor (headache, diplopia, facial pain and numbness) Skull-base erosion and palsy of the V and VI cranial nerves and up extension of tumor (headache, diplopia, facial pain and numbness) Signs:Enlarged neck nodes (75%), cranial nerve palsy (20%) Signs:Enlarged neck nodes (75%), cranial nerve palsy (20%)

Trends Microbiol Aug;12(8):356-60

Epidemiology Male:Female = 2~3:1 Male:Female = 2~3:1 Chinese native > Chinese immigrant > North American native Chinese native > Chinese immigrant > North American native Both genetic and environmental factors Both genetic and environmental factors Environmental Environmental Viruses Viruses EBV- well documented viral “ fingerprints ” in tumor cells and also anti-EBV serologies with WHO type II and III NPC EBV- well documented viral “ fingerprints ” in tumor cells and also anti-EBV serologies with WHO type II and III NPC HPV - possible factor in WHO type I lesions HPV - possible factor in WHO type I lesions Nitrosamines - salted fish Nitrosamines - salted fish Others - polycyclic hydrocarbons, chronic nasal infection, poor hygiene, poor ventilation Others - polycyclic hydrocarbons, chronic nasal infection, poor hygiene, poor ventilation

Classification WHO classes (Based on light microscopy findings) WHO classes (Based on light microscopy findings) Type I - moderate to well differentiated squamous cell carcinoma Type I - moderate to well differentiated squamous cell carcinoma Type II - “ non-keratinizing ” carcinoma Type II - “ non-keratinizing ” carcinoma Type III - “ undifferentiated ” carcinoma Type III - “ undifferentiated ” carcinoma Differences between type I and types II & III Differences between type I and types II & III 5 year survival 5 year survival Type I - 10% Types II, III - 50% Type I - 10% Types II, III - 50% Long-term risk of recurrence for types II & III Long-term risk of recurrence for types II & III Viral associations Viral associations Type I - HPV Type I - HPV Types II, III - EBV Types II, III - EBV

Y S Chang, J Clin Microbiol November; 28(11): 2398–2402. Detection of Epstein-Barr virus DNA sequences in nasopharyngeal carcinoma cells by enzymatic DNA amplification

The Epstein–Barr virus genome (1)

Epstein – Barr virus(2) (Cell Research 2006)

Latent membrane proteins(3) (Cell Research 2006)

The EBV-encoded nuclear antigens (4)

Role of Epstein–Barr virus in the pathogenesis of NPC (5)

(2004)

Table 1. Major gene alterations in nasopharyngeal carcinoma Cancer Cell May;5(5): Role of Epstein–Barr virus in the pathogenesis of NPC (6)

Genetic background (1) three new susceptibility loci, three new susceptibility loci, TNFRSF19 on 13q12, EBV-LMP1, activate JNK pathway TNFRSF19 on 13q12, EBV-LMP1, activate JNK pathway MDS1-EVI1 on 3q26, EVI1, MDS1 and the fusion protein MDS1-EVI1, transcription factor, TGF-β signaling MDS1-EVI1 on 3q26, EVI1, MDS1 and the fusion protein MDS1-EVI1, transcription factor, TGF-β signaling CDKN2A-CDKN2B gene cluster on 9p21. tumor suppressor gene CDKN2A-CDKN2B gene cluster on 9p21. tumor suppressor gene leukemogenesis leukemogenesis HLA HLA (Nature Genetics 2010)

A gender-specific (male) association of CNV at 6p21.3 with NPC susceptibility A gender-specific (male) association of CNV at 6p21.3 with NPC susceptibility the CNV (Copy number variations, a major source of human genetic polymorphism ) located at chromosome 6p21.3, with single-copy deletion of the MICA and HCP5 genes, showed the highest association with NPC. the CNV (Copy number variations, a major source of human genetic polymorphism ) located at chromosome 6p21.3, with single-copy deletion of the MICA and HCP5 genes, showed the highest association with NPC. The MICA gene (11.7 kb) encodes a 383-amino-acid polypeptide and functions as a ligand for NKG2D on γ/δT cells, CD8+ α/β T cells and NK cells. (Immune function) The MICA gene (11.7 kb) encodes a 383-amino-acid polypeptide and functions as a ligand for NKG2D on γ/δT cells, CD8+ α/β T cells and NK cells. (Immune function) (Hum Mol Genet. 2011) Genetic background (2)

EBV gene (1) 1.Circulating free EBV DNA copies increased during the initial phase of radiotherapy after cell death The quantity of free plasma EBV DNA is related to the stage of the disease. The quantities of EBV DNA copies before and after treatment are significantly related to the rates of overall and disease-free survival When EBV DNA was used together with Anti-EBV VCA IgA, the sensitivity of early diagnosis of nasopharyngeal carcinoma increased Raised levels of EBV DNA were only detected in 67% of patients with locoregional recurrence, 116 and 122 although in those with distant metastasis levels of EBV DNA copies were heightened before the appearance of clinical abnormality

The Epstein–Barr virus genome (1)

Real-time Quantitative PCR

[Cancer Research 59, , March 15, 1999] Quantitative Analysis of Cell-free Epstein-Barr Virus DNA in Plasma of Patients with Nasopharyngeal Carcinoma1 Y. M. Dennis Lo, et al. The Chinese University of Hong Kong Y. M. Dennis Lo, et al. Cancer Research 1999

(NEJM, 2004) EBV gene (2-1)

EBV gene (2-2) (NEJM, 2004)

EBV gene (2-3) (NEJM, 2004)

Typing of EBV DNA from Plasma and Primary Tumor (2-4) (NEJM, 2004)

EBV DNA Kinetics T1/2(1-R/T) Surge at first week after R/T (therapy induced cancer cell death) Surge at first week after R/T (therapy induced cancer cell death) Median half life 3.8 days(2.4~4.4d) Median half life 3.8 days(2.4~4.4d) Cancer Res 2000

Median T1/2: 139min Median T1/2: 139min Median f/u 7 d, 8/11 undetectable, 1/8 & other 3 recur Median f/u 7 d, 8/11 undetectable, 1/8 & other 3 recur EBV DNA T1/2 (2-OP) Clin Can Res 2003

EBV DNA load correlate with tumor volume IJROBP 2006

Complement to TNM staging JCO 2006 Early stage (I+II) high load similar to III Early stage (I+II) high load similar to III

Pre-Tx EBV DNA level predict Meta/recur tx response and survival Cancer Feb Pre-tx (median) Median:2.33x105 Post-tx Detectable vs undetectable

EBV DNA in peripheral blood cells predict distant metastasis (JCO 2001)

EBV DNA T1/2 (OP) T1/2: 5.4 hrs T1/2: 5.4 hrs Local recur/ccrt Retrosternal LN

Recurrence-metastasis (1) Chemo:PUL 97-2 Lung meta

Gem+CDDP 94.4-T3N2M bone, liver meta Gem+cddp trial Recurrence-metastasis (2)

T1N1M0 Recurrence-metastasis (3) Bone meta

NPC patients (fresh) ~ , fresh: ~ , fresh:489 Male:Famale=361:121 (3:1) Male:Famale=361:121 (3:1) Age:13~84, Median:48 Age:13~84, Median:48 籍貫 :total:278, 閩 133 客 106 原 17 其他 22 籍貫 :total:278, 閩 133 客 106 原 17 其他 22 Path:scc:28, NKDC 63 NKUDC:398 Path:scc:28, NKDC 63 NKUDC:398 EBV DNA:0~3,948,614, posi:75.8%, median: 812 EBV DNA:0~3,948,614, posi:75.8%, median: 812 I:5(posi:40%, median: 145) I:5(posi:40%, median: 145) II:107(posi:67.2%:,median:257 ) II:107(posi:67.2%:,median:257 ) III:139(posi:68.3%,, median: 812) III:139(posi:68.3%,, median: 812) IVa+b:190 (posi:82.6%, median: 917) IVa+b:190 (posi:82.6%, median: 917) M1:40(posi:95%, median:9,643) M1:40(posi:95%, median:9,643)

Local Recurrence:61 AgeRange:19~72Median:48 SexMale:44(72.1%)Female:17(27.9%) EBV-DNA Positive:35 (57.3%) Range:0~58,632 Median:261 copies/mL Recurrence> 1 episode 8(13.1%) Metastasis7(11.4%) Tissue proof 52(85.2%) PET-CT48 6/48 negative MRI/CT61 1/61 negative Treatment Ccrt:31, r/t:9, Op:8,Op+ccrt:5 c/t:7, Supportive:1 Survival: Expire:18, Range:3~33m Median:9mSurvive:43,Follow-up>24m:20Median:32m

Metastasis (1) 73 metastatic patients 73 metastatic patients Male:female=5:1 Male:female=5:1 Response:CR:18(24.7%), PR:21(28.8%), SD:8(10.9%) Response:CR:18(24.7%), PR:21(28.8%), SD:8(10.9%) Median survival: 17m Median survival: 17m EBV DNA: EBV DNA: pretreatmen: 0~4,509,918 copies/ml, positive rate:70/73 (95%) median: 2,200 copies/ml

Metastasis(2)

Metastasis(3)

Summary Plasma EBV DNA is a novel tumor marker and independent prognostic factor in NPC Plasma EBV DNA is a novel tumor marker and independent prognostic factor in NPC Correlate with tumor burden, complement to TNM staging (especially in early stage with high viral load), and correlate with disease status. Correlate with tumor burden, complement to TNM staging (especially in early stage with high viral load), and correlate with disease status. More higher positive rate and viral load in metastasis than local recurrence More higher positive rate and viral load in metastasis than local recurrence Predict prognosis (treatment response and survival) in metastasis patients Predict prognosis (treatment response and survival) in metastasis patients

Acknowledgement 長庚醫院 長庚醫院醫研部內科部血液腫瘤科施麗雲教授王宏銘副教授 頭頸部腫瘤團隊 頭頸部腫瘤團隊 長庚大學 張玉生教授 黎欣白老師