Plasma EBV Research Articles

PO-0669: Adjuvant chemotherapy improves outcome in NPC patients with post-RT persistently detectable plasma EBV DNA

PO-0669: Adjuvant chemotherapy improves outcome in NPC patients with post-RT persistently detectable plasma EBV DNA

Free Circulating Epstein Barr Virus DNA Load In Indian Children With Hodgkin Lymphoma As a Biomarker Of EBV-Associated Disease

IntroductionEpstein-Barr virus (EBV) association has been described in a large majority of children with Hodgkin lymphoma (HL) in India. The significance of circulating plasma EBV load and its kinetics during HL therapy is largely unknown. This study aimed at assessing the value of circulating EBV-DNA as a biomarker of EBV-associated pediatric HL and of tumour burden, and the value of serial monitoring during therapy. MethodAll pediatric cases presenting with lymphadenopathy were prospectively recruited between 2007 and 2012. Lymph node biopsy was performed. Children with HL and controls with reactive nodal hyperplasia were enrolled in the study after prior informed consent. Untreated non-lymphoid malignancies and healthy controls were also included in the control group. Plasma EBV real-time quantitative-PCR (RQ-PCR) was assessed with LightCycler2.0, Roche. EBV-associated HL was defined by positive EBV latent membrane protein-1 immunohistochemistry on lymph-node biopsies. Risk-adapted ABVD chemotherapy was given to HL cases, and early treatment response was assessed. RQ-PCR was repeated after the first cycle, at the end of treatment and on follow-up. Kaplan-Meier survival analysis was done, including loss to follow-up as an event. ResultsThirty cases of HL (newly diagnosed–28, relapses–2) and 70 age/ sex-matched controls (benign lymphadenopathy–19, non-lymphoid malignancy–29, Burkitt lymphoma–5, healthy children–17) were included. HL stage distribution was stage I–4, II–9, III–4 and IV–13. EBV immunohistochemistry was positive in 16 (59.3%) out of 27 HL cases analyzed (14/19 MC, 1/6 NS, 0/2 LP, 1 unclassified). RQ-PCR was detectable in 19 (63.3%) out of 30 HL, with 87.5% accuracy (Kappa coefficient=0.69 [0.42-0.97]). All 70 controls were RQ-PCR negative (p<0.0001). RQ-PCR sensitivity and specificity in EBV-associated HL detection were 87.5% and 81.8% respectively. Three out of 4 cases with more than 10,000 EBV copies/mL had advanced stage disease (III-IV) and B symptoms. The highest viral load (430,000 copies/mL) was seen in a boy with stage IV-B disease and end-stage liver involvement. However, viral load was not significantly associated with tumor burden or with survival. All treated Q-PCR positive cases showed EBV clearance after the first cycle. One case, RQ-PCR negative 10 years after primary treatment of stage-I HL, relapsed locally and became RQ-PCR positive. Five-year overall survival (OS) and event-free survival (EFS) were 82.6±8.5% and 77.2±8.3% respectively. OS and EFS were not significantly different in EBV-positive and EBV-negative HL. ConclusionRQ-PCR detection of circulating EBV-DNA is a biomarker of EBV-associated HL, in contrast with pediatric non-lymphoid malignancies, sporadic Burkitt lymphoma and benign lymphadenopathies. EBV-DNA may be used as an early marker of response to therapy in EBV-associated HL and may increase in case of relapse. Disclosures:No relevant conflicts of interest to declare.

Characterizing The CpG Methylation Of Epstein-Barr Virus DNA In The Plasma Of Patients With Hodgkin Lymphoma and HIV-Associated Burkitt Lymphoma

BackgroundEpstein-Barr virus (EBV) is harbored as latent virus within the tumor cells of a subset of classical Hodgkin lymphoma (HL) and HIV-associated Burkitt lymphoma (BL). In HL, we and others have previously shown that elevated EBV DNA copies in plasma accurately distinguish EBV(+) tumors from EBV(-) tumors. However, EBV DNA may be present in plasma due to apoptosis of latently infected EBV(+) tumor cells (or latent EBV(+) benign lymphocytes), as a result of replicative lytic infection (particularly in immunocompromised hosts), or both. Thus, detecting elevated EBV DNA copies in plasma by does not always signify the presence of an EBV(+) tumor. However, a distinction is that the EBV DNA derived from latently infected cells is replicated by cellular DNA polymerase and is usually CpG methylated, whereas EBV DNA in lytic infection is replicated by the viral DNA polymerase and is never CpG methylated. MethodsTo improve upon plasma EBV DNA as a tumor-specific marker in EBV(+) lymphomas, we assessed the CpG methylation of EBV DNA in a subset of plasma specimens from immunocompetent patients with HL (enrolled in the Eastern Cooperative Oncology Group E2496 trial) and a subset from patients with HIV-associated BL (enrolled in the AIDS Malignancy Consortium 048 trial). The EBV tumor status was determined by in situ hybridization (ISH) on tissue for EBV RNAs (EBER). For the CpG methylation assay, methyl binding domain paramagnetic beads were used to first fractionate CpG methylated DNA from CpG unmethylated DNA in plasma. EBV DNA in each fraction were quantified by real time polymerase chain reaction (qPCR) to determine the percentage of methylated EBV DNA. Specimens with > 100 EBV DNA copies/mL plasma were analyzed. ResultsAmong patients with EBER(+) HL and elevated EBV DNA in plasma prior to therapy (n=8), EBV DNA was largely CpG methylated (median 95%, range 54%-99%). By contrast, in the rare cases where patients with EBER(-) HL tumors had high EBV DNA copy number in plasma prior to therapy (4 of 92, 4%), only small percentages of EBV DNA were methylated in 3 patients (5%, 9%, and 12%). In the fourth patient, the plasma EBV DNA was 99% CpG methylated. We suspect that patient 4 had an EBV-associated HL with either a technical failure of EBER-ISH or an instance of a tumor that harbors viral DNA but does not express EBERs. In patients with HIV-associated BL, plasma EBV DNA copies were elevated in 100% of patients with EBER(+) BL (n=4), but also in 4 of 9 (56%) patients with EBER(-) BL. This propensity to detect EBV DNA in the plasma of patients with HIV but without EBV(+) BL likely reflects replicative lytic infection in immunocompromised hosts, making plasma EBV DNA less reliable as a marker of EBV(+) lymphoma in this setting. Thus, we applied the methylation assay to specimens from patients with HIV and BL. Patients with HIV and EBER(+) BL tumors (n=4), EBV DNA was 28%, 46%, 47%, and 60% methylated, whereas a patient with HIV and EBER(-) BL had lower methylation at 8%. To better elucidate the meaning of plasma EBV DNA elevations during lymphoma treatment, we applied the methylation assay to specimens from patients with EBV(+) HL and elevated EBV DNA in plasma at month 6 of therapy (n=4). Dense CpG methylation (96%) of plasma EBV DNA was detected in 1 patient who went on to relapse at Month 15, while the plasma EBV DNA was largely unmethylated in 3 patients who remain in remission each with over 5 years of follow-up. ConclusionIn settings where EBV DNA is detected in plasma, the CpG methylation assay helps differentiate patients with EBV(+) HL or BL from those with EBV(-) tumors. In patients with HIV where lytic EBV replication can contribute greatly to elevations of EBV DNA in plasma, the fractionated quantification of plasma EBV DNA by the methylation assay aids in distinguishing EBV DNA that is tumor-derived from that which is the result of lytic replication. With growing interest in plasma EBV DNA as a serial marker of disease activity in EBV(+) lymphomas, the methylation assay is promising in its ability to differentiate patients with elevated EBV DNA in plasma but ongoing remission from those in whom plasma EBV DNA is tumor-derived and an early harbinger of relapse. Disclosures:No relevant conflicts of interest to declare.

Aberrantly hypermethylated Homeobox A2 derepresses metalloproteinase-9 through TBP and promotes invasion in Nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is notorious for its high invasiveness and metastatic ability. In this study, we identified a differential hypermethylated transcription repressor, Homeobox A2 (HOXA2), which may render NPC cells invasive and metastatic. Aberrant hypermethylation of HOXA2 led to low RNA expression in NPC tumors and cells. Addition of methylation inhibitor 5'Aza restored HOXA2 RNA expression in NPC cells. Methylated HOXA2 promoter reduces the binding affinity of the transcriptional co-activator p300, causing transcriptional repression of HOXA2. In NPC cells, re-expression of ectopic HOXA2 was correlated with decreased invasive ability and reduced metalloproteinase MMP-9 RNA and protein expression. Promoter, ChIP and DNA-pull down assays indicated that HOXA2 competes with the transcription activator, TATA-box binding protein (TBP) for a recognition sequence near the MMP-9 transcription start site, and suppresses MMP-9 transcription. Thus, HOXA2 acts as a suppressor or TBP-antagonist to inhibit MMP-9 expression; while methylation-mediated inactivation of HOXA2 in NPC derepresses MMP-9 production and increases invasion of NPC cells. In NPC plasma samples, increased plasma EBV copy number was correlated with increased in cell-free HOXA2 hypermethylation and elevated MMP-9 levels. Plasma EBV DNA and methylated cell-free HOXA2 can be used as biomarkers for monitoring NPC treatment.

Abstract B273: Multicenter Phase II study of MK-2206 in previously treated patients (pts) with recurrent and metastatic nasopharyngeal carcinoma (NPC): Mayo Clinic Phase II Consortium (Protocol: MC1079).

Abstract Background: NPC is endemic to Asia and over 40% of cases harbor PIK3CA amplification. MK2206 is an allosteric AKT inhibitor with activity in NPC in vitro. Methods: Pts who had progressed after palliative chemotherapy (chemo) for metastatic or recurrent NPC, received oral MK-2206 at 200 mg on Days 1, 8, 15 &amp; 22 of each 28-day cycle until disease progression. Plasma samples were collected at serial time points during cycle 1 for EBV DNA analysis, archived tumor samples were collected where feasible. The primary dual endpoints (2-stage design) consisted of RECIST-defined tumor response rate (RR) and 6-month (m) progression-free survival (PFS) rate. Secondary endpoints were overall survival (OS), PFS and adverse events (AEs). Results: Of the 21 pts enrolled, the median age was 47 years (range: 32-67), 91% were male, 81% had prior radiotherapy and 81% had &amp;gt; 1 prior line of palliative chemo. At the time of analysis, 20 out of 21 pts have ended treatment. The best responses were: 1 partial response (PR, 5%) lasting 4 ms; 10 stable disease (SD, 50%), 9 progressive disease (PD, 45%). The 6-m PFS rate was 38.9% (95% CI: 18.1-59.3%) and median PFS was 2.7 ms (95% CI: 0.9-7.2 ms). The 6-m OS rate was 67.9% (95% CI: 41.8-84.1%) and median survival has not been reached. In 7 pts who had SD more than or equal to 6 ms, the duration of treatment ranged from 6.4-13.9 ms. Of the 21 pts evaluable for AEs, 12 pts (57%) had at least one grade 3 AE [[Unable to Display Character: &amp;#8211;]] macular rash (6 pts - 29%), dysphagia (2 pts - 10%), hyperglycemia (2 pts - 10%) (see Table 1). The tumor samples of 7 pts were analyzed by FISH; 3 showed PIK3CA amplification, including 1 pt with chromosome 3 polysomy. Amongst these pts, 1 had SD more than 6ms, 1 had SD more than 12 ms, and 1 is still on treatment. Pts who had SD less than 6 m or PD did not have PIK3CA amplification. Conclusions: MK2206 is well tolerated and has signs of activity in unselected pts with NPC. Preliminary results suggest that PIK3CA amplification may be related to prolonged disease stabilization from MK2206, and analysis for other PIK3CA gene alterations in more samples will be undertaken. Result of the plasma EBV DNA result will be presented. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B273. Citation Format: Brigette B. Ma, Boon Cher Goh, Wan Teck Lim, Eng Huat Tan, Gilberto de Lima Lopes, Edwin P. Hui, Ann D. King, Kwok Wai Lo, Herbert Loong, Leung Li, Nathan Foster, Michael Kam, Sing Fai Leung, Charles Erlichman, Anthony TC Chan. Multicenter Phase II study of MK-2206 in previously treated patients (pts) with recurrent and metastatic nasopharyngeal carcinoma (NPC): Mayo Clinic Phase II Consortium (Protocol: MC1079). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B273.

FCGR2A and FCGR3A polymorphisms in classical Hodgkin lymphoma by Epstein–Barr virus status

FCGR2A and FCGR3A polymorphisms in classical Hodgkin lymphoma by Epstein–Barr virus status

Plasma Viral MicroRNA Profiles Reveal Potential Biomarkers for Chronic Active Epstein–Barr Virus Infection

Chronic active Epstein-Barr virus (CAEBV) infection has high mortality and morbidity, and biomarkers for disease severity and prognosis are required. MicroRNAs (miRNAs) are small noncoding RNAs, and EBV encodes multiple miRNAs. Because plasma contains sufficiently stable miRNAs, circulating EBV-associated miRNA profiles were investigated as novel biomarkers in CAEBV infection. Plasma miRNA expression was assessed for 12 miRNAs encoded within 2 EBV open reading frames (BART and BHRF). Expression levels were investigated in 19 patients with CAEBV infection, 14 patients with infectious mononucleosis, and 11 healthy controls. Relative expression levels of plasma miRNAs were determined by TaqMan probe-based quantitative assay. Plasma miR-BART1-5p, 2-5p, 5, and 22 levels in patients with CAEBV infection were significantly greater than those in patients with infectious mononucleosis and in controls. Plasma miR-BART2-5p, 4, 7, 13, 15, and 22 levels were significantly elevated in patients with CAEBV infection with systemic symptoms, compared with levels in patients with no systemic symptoms. The levels of miR-BART2-5p, 13, and 15 showed clinical cutoff values associated with specific clinical conditions, in contrast to plasma EBV loads. Levels of specific plasma EBV miRNAs were elevated differentially in patients with CAEBV infection. Several EBV miRNAs, particularly miR-BART2-5p, 13, and 15, are potentially biomarkers of disease severity or prognosis.

Plasma Epstein-Barr virus DNA predicts outcome in advanced Hodgkin lymphoma: correlative analysis from a large North American cooperative group trial

AbstractEpstein-Barr virus (EBV) is associated with Hodgkin lymphoma (HL) and can be detected by in situ hybridization (ISH) of viral nucleic acid (EBER) in tumor cells. We sought to determine whether plasma EBV-DNA could serve as a surrogate for EBER-ISH and to explore its prognostic utility in HL. Specimens from the Cancer Cooperative Intergroup Trial E2496 were used to compare pretreatment plasma EBV-DNA quantification with EBV tumor status by EBER-ISH. A cutoff of >60 viral copies/100 µL plasma yielded 96% concordance with EBER-ISH. Pretreatment and month 6 plasma specimens were designated EBV(-) or EBV(+) by this cutoff. Patients with pretreatment EBV(+) plasma (n = 54) had inferior failure-free survival (FFS) compared with those with pretreatment EBV(-) plasma (n = 274), log-rank P = .009. By contrast, no difference in FFS was observed when patients were stratified by EBER-ISH. Pretreatment plasma EBV positivity was an independent predictor of treatment failure on multivariate analyses. At month 6, plasma EBV(+) patients (n = 7) had inferior FFS compared with plasma EBV(-) patients (n = 125), log-rank P = .007. These results confirm that plasma EBV-DNA is highly concordant with EBER-ISH in HL and suggest that it may have prognostic utility both at baseline and after therapy. This trial was registered at www.clinicaltrials.gov as #NCT00003389.

Consistent high concentration of the viral microRNA BART17 in plasma samples from nasopharyngeal carcinoma patients - evidence of non-exosomal transport

BackgroundBecause latent Epstein Barr (EBV)-infection is a specific characteristic of malignant nasopharyngeal carcinoma (NPC), various molecules of viral origin are obvious candidate biomarkers in this disease. In a previous study, we could show in a few clinical samples that it was possible to detect a category of EBV microRNAs called miR-BARTs in the plasma of at least a fraction of NPC patients. The first aim of the present study was to investigate the status of circulating miR-BART17-5p (one of the miR-BARTs hereafter called miR-BART17) and EBV DNA in a larger series of NPC plasma samples. The second aim was to determine whether or not circulating miR-BART17 was carried by plasma exosomes.Patients and methodsPlasma samples were collected from 26 NPC patients and 10 control donors, including 9 patients with non-NPC Head and Neck squamous cell carcinoma and one healthy EBV carrier. Concentrations of miR-BART17 and two cellular microRNAs (hsa-miR-16 and -146a) were assessed by real-time quantitative PCR with spike-in normalization and absolute quantification. In addition, for 2 patients, exosome distributions of miR-BART17 and miR-16 were investigated following plasma lipoprotein fractionation by isopycnic density gradient ultrcentrifugation.ResultsThe miR-BART17 was significantly more abundant in plasma samples from NPC patients compared to non-NPC donors. Above a threshold of 506 copies/mL, detection of miR-BART17 was highly specific for NPC patients (ROC curve analysis: AUC=0.87 with true positive rate = 0.77, false positive rate = 0.10). In this relatively small series, the concentration of plasma miR-BART17 and the plasma EBV DNA load were not correlated. When plasma samples were fractionated, miR-BART17 co-purified with a protein-rich fraction but not with exosomes.ConclusionsDetection of high concentrations of plasma miR-BART17 is consistent in NPC patients. This parameter is, at least in part, independent of the viral DNA load. Circulating miR-BART17 does not co-purify with exosomes.

An International Collaboration to Harmonize the Quantitative Plasma Epstein-Barr Virus DNA Assay for Future Biomarker-Guided Trials in Nasopharyngeal Carcinoma

Persistently elevated posttreatment plasma EBV DNA is a robust predictor of relapse in nasopharyngeal carcinoma (NPC). However, assay standardization is necessary for use in biomarker-driven trials. We conducted a study to harmonize the method between four centers with expertise in EBV DNA quantitation. Plasma samples of 40 patients with NPC were distributed to four centers. DNA was extracted and EBV DNA copy number was determined by real-time quantitative PCR (BamHI-W primer/probe). Centers used the same protocol but generated their own calibrators. A harmonization study was then conducted using the same calibrators and PCR master mix and validated with ten pooled samples. The initial intraclass correlations (ICC) for the first 40 samples between each center and the index center were 0.62 [95% confidence interval (CI): 0.39-0.78], 0.70 (0.50-0.83), and 0.59 (0.35-0.76). The largest variability was the use of different PCR master mixes and calibrators. Standardization improved ICC to 0.83 (0.5-0.95), 0.95 (0.83-0.99) and 0.96 (0.86-0.99), respectively, for ten archival frozen samples. For fresh plasma with spiked-in EBV DNA, correlations were more than 0.99 between the centers. At 5 EBV DNA copies per reaction or above, the coefficient of variance (CV) was less than 10% for the cycle threshold (Ct) among all centers, suggesting this concentration can be reliably used as a cutoff for defining the presence of detectable EBV DNA. Quantitative PCR assays, even when conducted in experienced clinical labs, can yield large variability in plasma EBV DNA copy numbers without harmonization. The use of common calibrators and PCR master mix can help to reduce variability.

PO-0666: Plasma EBV DNA monitoring in advanced nasopharyngeal cancer patients receiving induction biochemotherapy and IMRT

PO-0666: Plasma EBV DNA monitoring in advanced nasopharyngeal cancer patients receiving induction biochemotherapy and IMRT

PD-0091: Plasma EBV DNA assay in post-treatment remission nasopharyngeal cancer patients- a prospective multi-center study

PD-0091: Plasma EBV DNA assay in post-treatment remission nasopharyngeal cancer patients- a prospective multi-center study

Clinical Significance of Quantitative Analysis of Plasma Epstein-Barr Virus DNA in Patients of Xinjiang Uygur Nationality with Hodgkin's Lymphoma

To investigate the relationship between plasma EBV-DNA concentration and clinicopathologic features of Hodgkin's lymphoma cases. At first, the positive rate of plasma EBV-DNA was determined with a nested-PCR method using 45 specimens from Uygur HL patients, as well as 110 healthy people sampled as normal controls. Secondly, using fluorescent quantitative nested-PCR, EBV viral load was assessed in the EBV-DNA positive plasma samples. Then, relationships between plasma EBV viral load and clinicopathologic features of HL patients were analyzed. The positive rate of plasma EBV-DNA of HL patients was significantly higher than that of normal controls (53.3%vs26.4%, P=0.001). There was no significant difference about plasma EBV viral load between EBV-associated HL and EBV-DNA positive normal people (P=0.490). Looking at patients' characteristics, plasma EBV viral load in 10-20 years EBV-associated HL was higher than in EBV cases which were less than 10 years or more than 35 years (P=0.025). Furthermore, in EBV-associated HL, concentration of plasma EBV-DNA was significantly higher in advanced stage disease (stages III-IV; P=0.013), and with B-symptoms (P=0.020). EBV-DNA levels were associated with part of clinicopathologic features of cases. It was of practical use to screen HL. Further etiological studies appear warranted.

Cell-free EBV DNA in Hodgkin lymphoma.

7 Background: EBV is associated with a subset of Hodgkin lymphoma (HL), identified by tissue-based techniques such as EBER in situ hybridization of tumor specimens. Recent evidence suggests that cell-free EBV DNA may be a surrogate for EBER and serve as a tumor marker in HL. Methods: EBV DNA copy number was quantitated by RT-PCR of plasma, serum, and peripheral blood mononuclear cells (PBMCs) from HL patients and compared using Pearson’s correlation. Receiver operator characteristic (ROC) curves determined thresholds for EBV DNA copy number to discriminate EBER status. Kaplan-Meier curves and Cox proportional hazards models were constructed to analyze failure-free survival (FFS). Results: Plasma and serum EBV DNA (n=30 paired samples) were correlated (r=0.96, p&lt;0.0001), while plasma and PBMC EBV DNA were not correlated (n= 25 paired samples, r=0.35, p=N.S.). Detectable plasma EBV DNA was 85% concordant with EBER status (n=34 paired samples) while detectable PBMC EBV DNA had a low concordance of 50% with EBER (n=78 paired samples). In a prospective clinical trial of HL patients treated with rituximab-ABVD, we confirmed the association between pre-treatment plasma EBV DNA and EBER (n=33 paired samples) and found a cut-off of &gt; 60 viral copies/100 µL plasma to have optimal test characteristics with 100% sensitivity, 93% specificity, and 94% concordance with EBER. We observed that EBV DNA was cleared from the plasma of patients with EBER(+) HL within one month of treatment, corresponding to a durable remission in each instance. In a larger prospective trial of HL (E2496), pre-treatment plasma EBV DNA was 95% concordant with EBER (n=121 paired samples), with a sensitivity of 92% and specificity of 96% at the same cut-off of 60 viral copies/100 µL plasma. Pre-treatment plasma EBV DNA positivity was found to be an independent predictor of inferior FFS with a hazard ratio of 2.0 (95% CI 1.2 – 3.5), as presented at ASCO 2012 (abstract #8003). Failure to clear the plasma of EBV with treatment corresponded to inferior 3-year FFS (44% vs 69%, log-rank p=0.03). Conclusions: These findings suggest that cell-free EBV DNA may be a useful tumor marker in HL, demonstrating concordance with EBER, prognostic significance prior to therapy, and utility as a marker of treatment response during therapy.

WS12-1 - Front-Line Concurrent Chemoradiotherapy (CCRT) Followed Consolidation Chemotherapy in Localized NASAL NK/T-Cell Lymphoma

ABSTRACT Background Extranodal, nature killer/T-cell lymphoma of nasal type, the nasal NKTCL, is known for its close association with EBV infection and poor clinical outcome after chemotherapy (C/T). The aims of the study are to evaluate the therapeutic efficacy of front-line concomitant chemoradiotherapy (CCRT) plus consolidation C/T and the impact of EBV viral status on the clinical outcomes in patients with localized nasal NKTCL. Methods Patients with newly diagnosed, measurable stage I/II nasal NKTCL were eligible. The CCRT was comprised of two cycles of dexamethasone and etoposide D1-3, plus cisplatin D1, the DEP regimen, q 4 weeks with concurrent 5040 cGy radiation in 28 fractions/5 weeks. Tumor assessment was carried out 4 weeks after completion of CCRT. Patients without disease progression after CCRT were subjected for two cycles of consolidation C/T consisted of dexamethasone, etoposide, ifosphamide, mesna and cisplatin D1-4, the DVIP regimen, q 4 weeks. Serial blood samples were collected for EBV viral titer and IFN-γ determination. Results A total of 33 patients were enrolled. The best tumor response was complete response (CR) in 23, partial response (PR) in 5, with an overall response rate and CR rate of 84.8% and 69.7%, respectively. The 2- and 5-year progression-free survival (PFS) rate of intent-to-treat population was 63.1% (95% CI, 46.5–79.7%) and 56.1% (95% CI, 36.5–75.7%), respectively; while the corresponding overall survival (OS) rate was 72.0% (95% CI, 56.5–87.65%) and 61.7% (95% CI, 42.7–80.8%), respectively. The most common treatment-related grade 3/4 toxic effects were leucopenia (84.9%), febrile neutropenia (39.4%), nausea/vomiting (33.4%), anemia (30.3%) and mucositis (30.3%). Detectable baseline plasma EBV DNA level was an adversely prognostic factor for both PFS (log-rank test, P = 0.04) and OS (log-rank test, P = 0.01) of CR/PR patients. Conclusions Front-line CCRT plus consolidation chemotherapy is feasible and active in localized nasal NKTCL.

1028P - Plasma EBV DNA Concentration Correlates with FDG Pet in Nasopharyngeal Carcinoma Treated with Induction Chemotherapy

ABSTRACT Background While local control rate in locally advanced NPC (LA-NPC) has improved with concurrent Cisplatin and IMRT, development of distant metastases remains common in these patients and limits survival. The role of induction chemotherapy in the treatment of LA- NPC is still a matter of debate. The aim of this retrospective review is to evaluate the effect of induction chemotherapy and to correlate between EBV-DNA concentration and tumor response by PET-CT. Methods Patients with stage III-IVB, WHO II/III received induction chemotherapy with 2 cycles of TPF : Docetaxel: 75 mg/m2, Cisplatin: 75 mg /m2, 5Fluorouracil: 750 mg / m2 CI for 96 hours followed 3-4 weeks later by concurrent weekly Cisplatin ( 40 mg /m2) and IMRT (GTV:70 Gy over 35 fractions). EBV-DNA quantification was performed at baseline and repeated before each cycle of TPF. PET scans were performed at baseline and repeated before IMRT. The max standard uptake values (SUV) were recorded in the primary tumors. Metabolic response was defined as a decrease in maximum SUV of 35% or more. Results 20 patients with LA-NPC (75%Stage IVA/IVB) were reviewed. All but one completed therapy. Objective response, according to RECIST criteria: CR: 4; PR: 14 NE: 2. Median concentration of EBV-DNA was 11,300 copies /ml (range: 1,184 - 43,000). Post TPF, reduction of EBV-DNA copies by >50% was observed in 83% pts and 66% achieved complete biochemical response. In the FDG-avid tumor pts, the median SUV at baseline was 12 (range 10.5 - 17.4); post TPF metabolic response was observed in 100% and was complete in 33%.All patients with complete biochemical response had also a complete metabolic response by PET. At 2-year loco-regional progression free rate is 95% and 2 year overall survival rate is 85%. No recurrence was seen in complete (biochemical / metabolic) responders. Conclusion A negative post induction FDG-PET and complete biochemical response after TPF are of significant value in LA-NPC and are useful determinant to predict outcome. Disclosure All authors have declared no conflicts of interest.

Role of plasma EBV DNA levels in predicting recurrence of nasopharyngeal carcinoma in a western population

BackgroundLoco-regionally advanced nasopharyngeal carcinomas can be cured by the combination of chemotherapy and radiotherapy. In Eastern countries, plasma levels of viral Epstein-Barr deoxyribonucleic acid (DNA) are accurate in predicting recurrence, but few data are available in Western populations. The aim of this prospective study was to evaluate the relationship between viral Epstein-Barr DNA copy numbers in plasma and the response rate, progression-free survival and overall survival in a cohort of Western patients with stage IIb-IVb nasopharyngeal cancer.MethodsWe evaluated plasma samples from 36 consecutive patients treated with induction chemotherapy followed by chemoradiation. EBV copy numbers were determined after DNA extraction using real-time quantitative polymerase chain reaction. Survival curves were estimated using the Kaplan–Meier method.ResultsCirculating Epstein-Barr virus DNA levels were measured before treatment, at the end of concomitant chemo- and radiotherapy, and during the follow-up period. Pre-treatment levels significantly correlated with the initial stage and probability of relapse. Their increase was 100% specific and 71.3% sensitive in detecting loco-regional or metastatic recurrence (an overall accuracy of 94.4%). Three-year progression-free and overall survival were respectively 78.2% and 97.1%.ConclusionsThe results of this study confirm that patients from a Western country affected by loco-regionally advanced nasopharyngeal carcinoma have high plasma Epstein-Barr virus DNA levels at diagnosis. The monitoring of plasma levels is sensitive and highly specific in detecting disease recurrence and metastases.

A multicenter randomized controlled trial (RCT) of adjuvant chemotherapy (CT) in nasopharyngeal carcinoma (NPC) with residual plasma EBV DNA (EBV DNA) following primary radiotherapy (RT) or chemoradiotherapy (CRT).

5511 Background: The benefit of adjuvant CT in NPC is unclear. Administering CT after full-dose CRT presents challenges in treatment compliance and toxicity. Post-RT EBV DNA predicts poor survival and may be a biomarker of subclinical residual disease. We conducted a biomarker driven RCT using EBV DNA to select high risk NPC patients (pts) for adjuvant CT while sparing low risk pts from unnecessary toxicity. Methods: Biopsy proven NPC, AJCC stage IIB-IVB, detectable EBV DNA at 6-8 wks post-RT, no persistent locoregional disease or distant metastasis, ECOG 0 or 1, adequate organ function. Randomised with stratification for primary therapy (RT Vs CRT) and tumor stage (II/III Vs IV) to arm A (adjuvant cisplatin 40 mg/m2 and gemcitabine 1000mg/m2, both given on D1+8 q3w x 6 cycles) or arm B (clinical follow-up). EBV DNA and PET scan were performed before and 6 months after randomization. Primary endpoint was relapse free survival and secondary endpoints included toxicity of adjuvant CT. With a hazard ratio of 2, 100 pts were required with a power of 0.8 and an alpha at 0.05. This safety analysis was approved by DMSC. Results: From 9/2006 to 12/2011, 514 pts consented for EBV DNA screening, 95 with detectable EBV DNA consented for adjuvant study. After work-up, 74 were eligible for randomization (37 to arm A; 37 to arm B). The two arms were well balanced in baseline characteristics. 80% received prior neoadjuvant and/or concurrent CT. Staging: IIB (36.5%), III (29.7%), IVA (18.9%), IVB (14.8%). Five pts refused adjuvant CT after randomization. Overall 65% and 57% completed 5 and 6 cycles respectively. Mean dose intensity (DI): 84% for cisplatin (22.5 mg/m2/wk, range 0.0-26.7), 92% for gemcitabine (612.8 mg/m2/wk, range 333.3-777.8). Treatment related adverse events above CTC G2 were summarized in Table. Conclusions: Delivery of 6 cycles of adjuvant CT is feasible with acceptable toxicity after full dose RT or CRT. [Table: see text]

Adjuvant chemotherapy in advanced nasopharyngeal carcinoma based on plasma EBV load

Nasopharyngeal carcinoma (NPC) has been shown to be a radio- and chemosensitive tumor and combined chemoradiotherapy is the standard of care for advanced-stage patients. Different chemotherapy strategies (neoadjuvant, concurrent, adjuvant) are incorporated with radiotherapy to enhance treatment outcome and each mode of combined therapy has advantages and disadvantages. There are many randomized clinical trials investigating the role of adding chemotherapy in NPC management. So far, concurrent and neoadjuvant chemotherapy are more frequently used for locoregionally advanced tumors. However, the current reality is that (1) distant failure is the most frequent site of relapse reported in all studies, (2) patients have already received neoadjuvant and/or concurrent chemotherapy, and (3) only some tumors recur after previous neoadjuvant/concurrent chemotherapy with radiotherapy. So, adjuvant chemotherapy is a reasonable timing to consolidate the treatment outcome for selected patients. Recently, several tumor markers can predict tumors with high probability of recurrence. Among these markers, plasma EBV DNA assay is a simple and reliable one in predicting prognosis. In this mini-review, I will focus on the role of adjuvant chemotherapy based on plasma EBV DNA load.

Abstract 729: Plasma EBV DNA concentration and clearance rate as good prognostic factors for metastatic nasopharyngeal carcinoma

Abstract Purpose: To investigate the pretreatment copy number and the clearance rate of plasma EBV DNA as novel prognostic outcome markers for metastatic nasopharyngeal carcinoma (NPC) Experimental Design: Nineteen newly diagnosed and fifty-four relapsed metastatic NPC patients were treated at outpatient department (i.v. or i.v. plus oral chemotherapy followed by local irradiation boost where applicable). Multiple blood samples were collected before and during the first 6 weeks of chemotherapy. Plasma EBV DNA concentrations and half-life values of plasma viral clearance rates, t1/2, were determined by a real-time quantitative PCR. The association between the copy number of plasma EBV DNA, t1/2, and clinical outcome were analyzed. Results: Treatment response evaluated after 3-6 months of treatment showed the best response of 18 complete responses (CR)(24.7%), 21 partial responses (PR)(28.8%) and overall response rate was 53.5%. The pretreatment plasma EBV DNA concentrations and the t1/2 of plasma EBV DNA clearance rates had significant effects on treatment response and overall survival prediction. In the chemotherapy regimen, gemcitabine plus cisplatin had better treatment outcome than cisplatin plus oral UFT and calcium folinate based regimen. Multivariate analysis revealed both pretreatment EBV DNA load and t1/2 of plasma EBV DNA clearance rates but not chemotherapy regimen had significant effects on overall survival. Conclusions: The pretreatment plasma EBV DNA copy number and their clearance rates are significant predictors for NPC treatment outcome. In low copy number group, the t1/2 in the first 6 weeks may provide the physician to choose a tailored regimen for the patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 729. doi:1538-7445.AM2012-729