The Cumulative Cisplatin Dose Affects the Long-Term Survival Outcomes of Patients with Nasopharyngeal Carcinoma Receiving Concurrent Chemoradiotherapy

The prognostic value of the cumulative cisplatin dose (CCD) remains controversial for patients with nasopharyngeal carcinoma (NPC) receiving only concurrent chemoradiotherapy (CCRT). We retrospectively reviewed 549 consecutive patients with non-metastatic, histologically-proven NPC treated using intensity-modulated radiotherapy (IMRT) at Sun Yat-sen university cancer center. Patient survival between different CCD groups were compared. The cut-off value of pre-treatment plasma EBV DNA (pre-DNA) and CCD based on disease-free survival (DFS) were 1460 copies/ml (AUC, 0.691; sensitivity, 0.717; specificity, 0.635) and 240 mg/m2 (AUC, 0.506; sensitivity, 0.526; specificity, 0.538), respectively. Of the entire cohort, 92/549 (16.8%) patients received a CCD ≥ 240 mg/m2 and 457 (83.2%) patients, <240 mg/m2. For CCD ≥ 240 mg/m2 vs. < 240 mg/m2, the estimated 4-year DFS, overall survival (OS), locoregional-free survival (LRFFS) and distant metastasis-free survival (DMFS) rates were 89.1% vs. 81.3% (P = 0.097), 92.4% vs. 90.0% (P = 0.369), 95.6% vs. 91.2% (P = 0.156), and 91.3% vs. 88.4% (P = 0.375), respectively. For the whole cohort, multivariate analysis identified the CCD was an independent prognostic factor for DFS (HR, 0.515; 95% CI, 0.267–0.995; P = 0.048). However, CCD (≥240 mg/m2) had no prognostic value in subgroup analysis with stratification by the cut-off value of pre-DNA (P > 0.05 for all rates).

cisplatin was given every 3 weeks to reach a targeted cumulative dose of 300 mg/m 23,5 . However, a cumulative dose of 200 mg/m 2 cisplatin had been reported to be sufficient to yield a beneficial anti-tumor effect in other head and neck cancers 8 and NPC 9,10 . Most recently, Ou et al. 11 revealed a total cisplatin dose of 300 mg/m 2 was an independent prognostic factor for better distant metastasis and overall survival in local advanced NPC. This controversial results urgently need to be investigated further.
Therefore, we retrospectively assessed the relationship between the cumulative cisplatin dose (CCD) and the long-term outcomes of patients with NPC receiving concurrent chemoradiotherapy (CCRT) and intensity-modulated radiation therapy (IMRT). Moreover, the relationship between the pre-treatment Epstein-Barr virus DNA load (pre-DNA) and cumulative cisplatin dose was explored.

Methods and Materials
Patient Selection. We retrospectively reviewed 1811 patients with previously untreated, biopsy-proven NPC with no evidence of distant metastasis treated using IMRT between November 2009 and February 2012 at Sun Yat-sen University Cancer Center. The 549 (30.3%) patients receiving only cisplatin during CCRT were included in this investigation. All experimental protocols were approved by the Research Ethics Committee of Sun Yat-sen University Cancer Center, patient confidentiality was protected at all times, and informed consent was obtained from all patients. All the methods were carried out in accordance with the approved guidelines in this study.
Clinical Staging. Routine staging included a complete medical history, clinical examination of head and neck, direct fiber-optic nasopharyngoscopy, magnetic resonance imaging (MRI) of skull base and entire neck, chest radiography, whole-body bone scan, abdominal sonography and positron emission tomography (PET)-CT. The tumour-associated markers immunoglobulin A (IgA) antibodies to EBV viral capsid antigen (VCA) and EBV early antigen (EA) and plasma EBV DNA were quantified. All patients had a dental evaluation before radiotherapy and were restaged according to the 7 th edition of the International Union against Cancer/American Joint Committee on Cancer (UICC/AJCC) system 12 . All MRI materials and clinical records were reviewed to minimize heterogeneity in restaging. Two radiologists employed at our hospital separately evaluated all scans; disagreements were resolved by consensus.
Quantitation of Epstein-Barr Virus DNA. Plasma EBV DNA was quantified before treatment using a real-time quantitative PCR assay 13 that targets the BamHI-W region of the EBV genome using the primers 5′ -GCCAGAGGTAAGTGGACTTT-3′ and 5′ -TACCACCTCCTCTTCTTGCT-3′ and dual fluorescence-labeled oligomer probe 5′ -(FAM)CACACCCAGGCACACACTACACAT(TAMRA)-3′ . EBV genome sequence data were obtained from the GeneBank sequence database.

Treatment
Radiotherapy. All patients received definitive IMRT at our center while immobilized using a custom-made head-to-neck thermoplastic cast and neck support. A high-resolution planning computed tomography scan (Siemens, Plus 4) with contrast was taken from the vertex to 2 cm below the sternoclavicular joint (slice thickness, 3 mm). Target volumes were delineated slice-by-slice on treatment planning CT scans using an individualized delineation protocol that complies with International Commission on Radiation Units and Measurements reports 50 and 62. Prescribed doses were 66-72 Gy at 2.12-2.43 Gy/fraction to planning target volume (PTV) of primary gross tumour volume (GTVnx), 64-70 Gy to PTV of GTV of involved lymph nodes (GTVnd), 60-63 Gy to PTV of high-risk clinical target volume (CTV1), and 54-56 Gy to PTV of low-risk clinical target volume (CTV2). All targets were treated simultaneously using the simultaneous integrated boost technique.
Chemotherapy. According to institutional guidelines, we recommended radiotherapy alone for stage I disease, CCRT for stage II and CCRT + /− neoadjuvant/adjuvant chemotherapy for stage III-IVA-B. In addition, patients with stage I disease and high pre-DNA would also receive CCRT which was decided by clinicians. Neoadjuvant or adjuvant chemotherapy was cisplatin with 5-fluorouracil (PF), cisplatin with toxoids (TP), or cisplatin with both 5-fluorouracil and taxoids (TPF) (every three weeks; two or three cycles). CCRT was weekly cisplatin (30-40 mg/m 2 ) or 3-weekly cisplatin (80-100 mg/m 2 ) on weeks 1, 4 and 7 of radiotherapy.

Follow-Up and Statistical Analysis
Patient follow-up was measured from first day of therapy to last examination or death. Patients were examined at least every three months during first two years, with follow-up examinations every six months thereafter until death. End-points (time to first defining event) were disease-free survival (DFS), overall survival (OS), loco-regional relapse-free survival (LRRFS) and distant metastasis-free survival (DMFS). DFS was the primary end-point in our study.
Receiver operating characteristic (ROC) curves were used to calculate cut-off value for pre-DNA and CCD based on DFS. Serum lactate dehydrogenase (LDH) were classified as described previously 14,15 . The Chi-square test was used to compare clinical characteristics. Life-table estimation was performed using the Kaplan-Meier method and log-rank test. The multivariate Cox proportional hazards model was used to estimate hazard ratios (HR) and 95% confidence intervals (CI). All statistical tests were two-sided; P < 0.05 was considered significant. STATA statistical package (STATA 12; StataCorp LP, College Station, Texas, USA) was used for all analyses.

Discussion
This current study demonstrated a CCD ≥ 240 mg/m 2 is associated with significantly improved DFS among patients with NPC receiving only cisplatin during CCRT, which was consistent with the results of previous studies 9,11 . Moreover, no definitely prognostic association was found between CCD and pre-DNA.
Since group B had a higher percentage of stage T4 and IV disease, patients in that group therefore received a higher CCD compared with that of patients in group A. Moreover, the results of this current study revealed that a higher CCD had no benefit in terms of DMFS. The reasonable explanation is that cisplatin delivered during CCRT increases the effect of radiotherapy but does not reduce the risk of distant metastasis, which has been recommended by the NCCN guidelines. Therefore, the DFS benefit of a higher CCD may be attributed to increased regional control. Due to the relatively insufficient follow-up time, no significant difference in OS could be detected when the patients were stratified by CCD.
In the prospective NPC-9901 and NPC-9902 trials 17 , at least two cycles of cisplatin (100 mg/m 2 ) improved local-free survival (LFS) and OS compared with one cycle. Moreover, 200 mg/m 2 cisplatin was reported as an appropriate cumulative dose in other retrospective studies of NPC 9,10 and head and neck cancer (HNSCC) 8,18 . It seemed that 200 mg/m 2 has been used as the standard lowest cumulative dose in NPC and HNSCC. However, more recently, Ou et al. reported a total cisplatin dose > 300 mg/m 2 was an independent prognostic factor for OS, DFS and DMFS in local advanced NPC 11 . Notably, a proportion of patients in this study also received induction or adjuvant chemotherapy. However, several prospective clinical trials have proven patients with locoregionally advanced NPC do not benefit from induction [19][20][21][22][23] or adjuvant chemotherapy 24 . Therefore, the lowest effectively CCD in this study may have been inflated by delivering cisplatin-based induction or adjuvant chemotherapy 11 .
Notably, the CCD cutoff value of 240 mg/m 2 did not have prognostic value in subgroup analysis based on pre-DNA, indicating that this cutoff value may be not appropriate for patients with pre-DNA higher or lower than 1460 copies/ml. The effective CCD may be higher in patients with pre-DNA < 1460 copies/ml and lower for patients with pre-DNA > 1460 copies/ml, or no definite relationship may exist between CCD and pre-DNA. Since no related studies have investigated this relationship, we therefore could not make a conclusion about the relationship between CCD and pre-DNA only based on the negative results of this current study. Future prospective studies are warranted to investigate the relationship further.
The optimal cisplatin schedule for CCRT in NPC remains under debate: is the weekly or 3-weekly regimen more effective? A third planned dose of 100 mg/m 2 cisplatin was omitted in a substantial number of patients with HNSCC 8 . Moreover, in the NPC-9901 trial 5 , only 52% of patients received ≥ three cycles of 100 mg/m 2 cisplatin. This poor compliance may constrain the wide-spread use of 3-weekly cisplatin (100 mg/m 2 ). The weekly cisplatin (40 mg/m 2 ) regimen was first reported in 2002 by Chan et al. 25 . Loong et al. observed a significant association between > five concurrent weekly cycles of 40 mg/m 2 cisplatin and better OS in subgroup analysis of 142 patients with stage II-III NPC 10 , and other retrospective studies also support the weekly strategy [26][27][28] . This current study also observed no significant prognostic differences between of patients receiving the weekly or 3-weekly cisplatin regimens, which further prove the efficacy of weekly regimen.
The purpose of concurrent chemotherapy is to achieve prognosis improvement with minimal and acceptable toxicities. Therefore, it is of great importance to establish the optimal dose of cisplatin administer during RT to gain survival benefit and avoid great toxicities. We identified a cumulative cisplatin dose ≥ 240 mg/m 2 was an independent prognostic factor for DFS in patients with NPC receiving only cisplatin during CCRT. Therefore, in the clinic, at least six cycles of 40 mg/m 2 may be considered when delivering the weekly cisplatin regimen, whereas three cycles of 80 mg/m 2 may be adequate for the 3-weekly regimen.
This study is limited by its retrospective nature and a relatively short follow-up time, though we selected DFS as the major end-point to address these limitations. Moreover, the sample bias may existed because the data was from a single institution. However, it should not influence the results because we recruited all the eligible patients and the cohort was big. Further prospective studies of larger cohorts are warranted to confirm these results and further define the relationship between pre-DNA and the CCD to deliver more individualized therapeutic regimens in NPC.

Conclusions
In this current study, a cumulative cisplatin dose ≥ 240 mg/m 2 was identified as an independent prognostic factor for DFS in patients with NPC receiving single-agent cisplatin-based CCRT. Therefore, either weekly 40 mg/m 2 cisplatin or 3-weekly 80 mg/m 2 cisplatin could be appropriate standard regimens for patients with NPC receiving CCRT based on IMRT, during which a cumulative cisplatin dose of ≥ 240 mg/m 2 should be delivered.