Subdivision of de-novo metastatic nasopharyngeal carcinoma based on tumor burden and pretreatment EBV DNA for therapeutic guidance of locoregional radiotherapy

In total, 11,235 patients were newly diagnosed with NPC in the Sun Yat-sen University Cancer Center in China from November 2006 to October 2016. Of these, 498 patients were enrolled in this retrospective study (Fig. 1) according to the following inclusion criteria: (I) primary lesions in nasopharyngeal were diagnosed histologically and metastatic lesions of dmNPC were diagnosed radiologically or histologically; (II) age between 18 and 70 years; (III) no history of malignancy or synchronous cancer; (IV) treatment with cisplatin-based chemotherapy regimen; (V) normal hematopoietic function: white blood cell count ≥4 × 10⁹/L, platelets ≥100 × 10⁹/L, hemoglobin ≥90 g/L, and neutrophil granulocytes > 2.0 × 10/L; (VI) normal liver function test: aspartate aminotransferase and alanine aminotransferase < 2.5-fold of upper limit of normal (ULN), and total bilirubin < 2.0 × ULN; (VII) normal renal function test: creatinine clearance ≥60 ml/min or creatinine ≤1.5 × ULN; and (VIII) male or non-pregnant female. All patients were restaged based on the 8th edition of the American Joint Committee on Cancer/International Union Against Cancer staging system. This study was approved by the clinical research ethics committee of Sun-Yat sen university cancer center, and written informed consent was obtained from each patient.

During enrollment, all patients in the cohort received primary lesion biopsy under nasal endoscopy. Needle biopsy under CT guidance was performed for some metastatic organs such as liver and lung when the positions and volumes of metastatic lesions were suitable to perform it. Because it was difficult to perform biopsy for metastatic lesions of bones, biopsy on bones was not performed. General evaluation tests mainly included physical examination of the head and neck region (including nasopharynx and cervical lymph nodes), physical examination of the nervous system, EBV serologic tests, EBV DNA quantitative determination, nasal endoscopy, lesion biopsy, head and neck magnetic resonance imaging (MRI) scan, emission compared tomography (ECT) scan, and chest and abdominal CT scan. Positron emission tomography (PET-CT) was considered an optional evaluation test based on the patient’s financial burden.

Metastatic lesions and organs were evaluated based on radiological criteria. The number of metastatic bone lesions and metastatic lesions (excluding bones) were assessed by ECT (or PET-CT) and chest and abdominal CT (or PET-CT) scans, respectively. The number of metastatic organs was evaluated by both ECT and chest and abdominal CT (or PET-CT) scans.

Pretreatment plasma EBV DNA concentrations were measured using quantitative polymerase chain reaction. The detailed procedure is reported elsewhere [8].

All included patients received cisplatin-based induction chemotherapy (IC). The common PCT regimens were as follows: (I) triplet docetaxel–cisplatin–fluorouracil: 60 mg/m² of docetaxel and 60 mg/m² of cisplatin on day 1 plus 500–800 mg/m² of 5-fluorouracil for 120 h; (II) docetaxel–cisplatin: 75 mg/m² of docetaxel on day 1 plus 20–25 mg/m² of cisplatin on days 1–3; (III) cisplatin–fluorouracil: 20–25 mg/m² of cisplatin on days 1–3 plus 800–1000 mg/m² of 5- fluorouracil for 96 h; (IV) gemcitabine–cisplatin: 1000 mg/m² of gemcitabine on day 1 plus 20–25 mg/m² of cisplatin on days 1–3. Each regimen was administered intravenously every 3 weeks for a total of 4–6 courses. Among the 498 dmNPC patients, 311 received LRRT after IC with two-dimensional conventional radiotherapy (2DCRT) or IMRT techniques. There were 92 patients receiving 2DCRT and 219 patients receiving IMRT. The total radiation doses were 68–70 Gy for nasopharyngeal and neck lesions, with a frequency of five fractions per week divided in 1.8–2.2 Gy fractions [9].

All patients underwent follow-up examinations every 3 months for the first 3 years and every 6 months thereafter. The examinations included EBV DNA copy detection, nasopharyngoscopy, head and neck MRI scan, chest and abdominal CT scan, and ECT or PET/CT scans. The primary endpoint of this study was OS, which was measured from initial diagnosis to death from any cause or loss to follow-up.

The clinical characteristics of patients from different treatment groups were compared using the Pearson χ² test. The number of metastatic lesions and EBV DNA copies were transformed into dichotomous variables based on cutoff values defined by the receiver-operating characteristic (ROC) analysis. The survival outcomes of patients from different subgroups were analyzed using Kaplan-Meier curves and the log-rank test. The independent prognosis predictors were evaluated using the Cox proportional hazards regression model. All data analyses were performed using the Statistical Package for Social Sciences (SPSS for macOS, version 21.0, IBM Corp., Armonk, NY). A two-tailed p < 0.05 was considered statistically significant.

Among them, 375 (75.3%) and 123 (24.7%) patients had single and multiple metastatic organs, respectively. The median age was 47 years (range, 18–77 years) and most patients were male (83.1%, 414/498). Regarding metastatic lesions, 338 (67.9%) and 160 (32.1%) patients had five or less and more than five lesions, respectively. Based on ROC analysis, the EBV DNA copies cutoff value was set at 25,000 copies/ml and 284 (57.0%) patients had levels that surpassed this value. As shown in Table 1, statistical differences were found in the number of metastatic organs, number of metastatic lesions, and pretreatment EBV DNA copies between the different treatment groups.

All factors that may influence prognosis were included in the Cox proportional hazards regression model. As shown in Table 2, there was a higher mortality risk for patients who had metastasis in multiple organs (hazard ratio [HR], 1.897; 95% confidence interval [CI], 1.401–2.568; p < 0.001), more than five metastatic lesions (HR, 2.246; 95% CI, 1.670–3.020; p < 0.001), or pretreatment EBV DNA concentrations above 25,000 copies/ml (HR, 1.479; 95% CI, 1.132–1.930; p = 0.004), whereas patients who underwent LRRT had a lower risk of death (HR, 0.665; 95% CI, 0.511–0.864; p = 0.002). Thus, it could be concluded that multiple organs metastasis, over five metastatic lesions, and EBV DNA concentration above the cutoff value may represent risk factors. The addition of LRRT to PCT may reduce risk of death for dmNPC. The Kaplan-Meier survival curves also showed an association between LRRT and improved OS (3-year OS, 27% vs. 13%; p < 0.001) (Fig. 2a). However, compared with 2DCRT, IMRT might not improve OS of dmNPC (Fig. 3). Multivariable analysis (Table 3) also showed that the radiotherapy techniques were not an independent diagnostic factor. Obviously, patients with the aforementioned risk factors had shorter OS than other patients (p < 0.001 for all) (Fig. 2b-d).

According to the risk factors defined in the previous subsection, patients were divided into eight subgroups: group A, single organ metastasis, EBV DNA concentration ≤ 25,000 copies/ml, and 5 or fewer metastatic lesions; group B, single organ metastasis, EBV DNA concentration > 25,000 copies/ml, and 5 or fewer metastatic lesions; group C, multiple organs metastasis, EBV DNA concentration ≤ 25,000 copies/ml, and 5 or fewer metastatic lesions; group D, multiple organs metastasis, EBV DNA concentration > 25,000 copies/ml, and 5 or fewer metastatic lesions; group E, single organ metastasis, EBV DNA concentration ≤ 25,000 copies/ml, and more than 5 metastatic lesions; group F, single organ metastasis, EBV DNA concentration > 25,000 copies/ml, and more than 5 metastatic lesions; group G, multiple organs metastasis, EBV DNA concentration ≤ 25,000 copies/ml, and more than 5 metastatic lesions; and group H, multiple organs metastasis, EBV DNA concentration > 25,000 copies/ml, and more than 5 metastatic lesions.

The Kaplan-Meier survival curves showed that patients in groups C-H had shorter OS than those in groups A-B; moreover, the OS of group A was significantly longer than that of group B (p < 0.05 for all). However, further paired comparisons revealed no significant differences in OS among groups C-H (p > 0.05 for all) (Fig. 4a). Subsequently, group A was classified as a low-risk subgroup (single organ metastasis, EBV DNA concentration ≤ 25,000 copies/ml, and 5 or fewer metastatic lesions), group B was classified as an intermediate-risk subgroup (single organ metastasis, EBV DNA concentration > 25,000 copies/ml, and 5 or fewer metastatic lesions), and groups C-H were classified as a high-risk subgroup (multiple organs metastasis or more than 5 metastatic lesions or both). The survival curves of patients in different risk strata are displayed in Fig. 4b. According to the Pearson χ² test, the subgroups only differed in chemotherapy regimens (p < 0.001, p = 0.004 in low-risk and high-risk subgroups respectively) (Table 4) and no significant difference was found in other clinical characteristics.

The differences in OS between patients that did and did not receive LRRT in each classification of risk were further investigated in this study. Interestingly, not all patients benefited from LRRT. Statistical differences were found in OS among patients in the low-risk and intermediate-risk subgroups (p = 0.039 and p = 0.010, respectively), whereas no significant difference was found in the high-risk subgroup (p = 0.076) (Fig. 5). Subsequently, Cox proportional hazards regression model was performed for all subgroups (Table 5) and it was found that LRRT lowered the mortality risk for patients in the low-risk (HR, 0.490; 95% CI, 0.232–0.960; p = 0.042) and intermediate-risk subgroups (HR, 0.582; 95% CI, 0.357–0.947; p = 0.029); however, it did not affect high-risk patients (HR, 0.718; 95% CI, 0.499–1.033; p = 0.074). Regarding these patients, the mortality risk was higher for those who had multiple organs metastasis (HR, 1.518; 95% CI, 1.032–2.234; p = 0.034), whereas the presence of multiple (> 5) metastatic lesions or a pretreatment EBV DNA copies level above cutoff did not seem to worsen this risk (HR: 1.564, 95% CI: 0.955–2.562, p = 0.076; HR: 1.127, 95% CI: 0.745–1.707, p = 0.571, respectively).