Comparative Outcome of Thai Pediatric Osteosarcoma Treated with Two Protocols: the Role of High-Dose Methotrexate (HDMTX) in a Single Institute Experience

Osteosarcoma is the most common primary bone tumor in children and adults. Approximately 60% of all incidences of osteosarcoma occur in patients aged less than 20 years (Fletcher et al., 2006; Stiller et al., 2006; Mirabello et al., 2009; Mirabello et al., 2009). In Thailand, the annual incidence of pediatric osteosarcoma was approximately 1.9 cases per million children (Wiangnon et al., 2011). After the introduction of neoadjuvant chemotherapy with radical surgery, the disease-free survival rate of patients with non-metastatic osteosarcoma has increased dramatically from below 20%, with surgery only (Coventry and Dahlin, 1957; Dahlin DC, 1978;


Introduction
Osteosarcoma is the most common primary bone tumor in children and adults. Approximately 60% of all incidences of osteosarcoma occur in patients aged less than 20 years (Fletcher et al., 2006;Stiller et al., 2006;. In Thailand, the annual incidence of pediatric osteosarcoma was approximately 1.9 cases per million children (Wiangnon et al., 2011). After the introduction of neoadjuvant chemotherapy with radical surgery, the disease-free survival rate of patients with non-metastatic osteosarcoma has increased dramatically from below 20%, with surgery only (Coventry and Dahlin, 1957;Dahlin DC, 1978;Marcove et al., 1970;Gaffney et al., 2006), to 60-76% much more recently (Delepine et. al. 1996;Ferrari et al., 2001;Lewis et al., 2007). High-dose methotrexate (HDMTX) with leucovorin rescue is recognized as an efficient component of therapy in childhood osteosarcoma when used in combination with other drugs such as cisplatin (CDDP), carboplatin (CBDCA), doxorubicin (ADR) and ifosfamide (IFO) (Jaffe et al., 1983;Jaffe et al., 1985;Winkler et al., 1990;Rosen et al., 1993;Goorin et al., 2003;Meyers et al., 2008). Since then, several schemes of multiagent adjuvant chemotherapy have been assessed, but recent meta-analysis had still shown no consensus in the addition of HDMTX to treatment of pediatric osteosarcoma. This lack of consensus results from the lack of an appropriate randomized-and case-controlled trial comparing the anti-tumor efficacy of HDMTX between treatment groups (Van Dalen et al., 2011).
The purpose of this retrospective study was to assess the therapeutic efficacy in two consecutive eras in order to demonstrate the role of HDMTX-containing chemotherapeutic protocol (MTX(+) protocol) in the treatment of pediatric osteosarcoma at our university hospital. We also aimed to demonstrate the incidence and the epidemiological data, and to identify the prognostic significance influencing the treatment outcome of pediatric osteosarcoma. This approach might provide the legacy of treatment regimen and identify the patient at-risk of refractory disease, as well as who may benefit from other investigational or escalation of treatment.

Patients
As a single-institution retrospective study, we enrolled pediatric patients ≤18 years of age with a diagnosis of osteosarcoma between January 1999 and June 2014, and followed up with treatment until the 30th of June 2014 at Ramathibodi Hospital, the tertiary-care referral and research university hospital in Bangkok, Thailand. Clinical data collected for each patient included clinical characteristics (age at diagnosis, sex, medical background and genetic predisposition to cancer), tumor characteristics (location, size, metastatic status at diagnosis), treatment (preoperative chemotherapy, surgery, postoperative treatment, adverse effects), outcome (remission, relapse, survival) and laboratory investigations (complete blood count (CBC), alkaline phosphatase (ALP)). All patients were assessed for the extension of primary tumor by a magnetic resonance imaging (MRI) scan of involved anatomical sites and the metastatic status at diagnosis by chest roentgenogram, bone scintigraphy and computerized tomography (CT) of the chest.

Chemotherapy Regimens and Administration
The chemotherapeutic protocols for pediatric osteosarcoma are shown in Table 1. Patient characteristics are given in Table 2. After the histological diagnosis of osteosarcoma by tumor biopsy, chemotherapy was administered to all patients according to chemotherapeutic protocol regardless of patients' characteristics, size of primary tumor, osteosarcoma subtypes and metastatic status at diagnosis. For patients diagnosed between 1999 and early 2007, we used 3-4 cycles of CDP/ADM preoperatively and CBDCA/IFO alternately with CDP/ ADM postoperatively (MTX(-) protocol) in intervals of 3 -4 weeks. G-CSF (5 mcg/kg/dose) was administered daily during a period of 8 -10 days as primary prophylaxis of febrile neutropenia. For patients diagnosed between late 2007 and 2014, we used MTX(+) protocol, consisting mainly of cycles of 2 courses of CDP/ADM and 4 courses of HDMTX preoperatively, and alternating cycles of CDP/ADM, IFO/etoposide (VP-16) and HDMTX postoperatively, as shown in Table 1. Hydration (2,500 ml/m 2 /day) and urine alkalinization with intravenous sodium bicarbonate were performed for 24 hours prior to MTX infusion to maintain urine pH 7 -8. 12gm/m 2 MTX was infused over 4 hours, and then folinic acid rescue was given intravenously at 24 hours from the beginning of MTX infusion, at a dose of 10 mg/m 2 every 6 hours, through a total of 11 doses. The dosage of folinic acid was adjusted according to a plasma MTX concentration at 24, 48 and 72 hours starting from the beginning of MTX infusion by a nomogram for pharmacokinetically guided leucovorin rescue after high-dose methotrexate administration (Bleyer WA. 1981). Plasma MTX concentration was collected every additional 24 hours until the plasma concentrations were <0.1umol/L following MTX infusion. G-CSF was also administered daily for 8 -10 days as primary prophylaxis of febrile neutropenia in each cycle of CDP/ADR and IFO/VP-16. Complete blood count, blood chemistries, liver function test (LFT) and kidney function were monitored regularly before every cycle of chemotherapy.
After preoperative chemotherapy for 8-10 weeks, all patients were radiologically evaluated for the feasibility of surgical procedure and obtaining patients' informed consent for surgery. The decision of radical surgical procedures, such as wide resection with intraoperative radiotherapy, limb-salvage surgery, rotatoplasty or amputation, was based on orthopedic surgeons' opinion, patients' consent and the volume of primary tumor and neurovascular involvement by MRI before and after Ifosfamide 2800 mg/m 2 /day, day 1-5; E: Etoposide 100 mg/m 2 /day, day 1-5; G-CSF (5 mcg/kg/day) was administered daily after completion of chemotherapy chemotherapy. The therapeutic effect was evaluated using clinical (size of primary tumor), radiographic (bone scintigraphy, CT of chest, MRI of primary tumor) and pathological parameters (surgical margin, percentage of tumor necrosis; Huvos score). Progressive disease (PD) was defined by the evidence of subsequently new or worsening metastatic lesions, including pulmonary nodule(s) by CT scan, nuclear uptake lesion(s) by bone scintigraphy or size of primary tumor by physical examination and/or imaging.

Statistical analysis
Survival analysis was done according to the Kaplan-Meier method and was compared by means of the log-rank test based on demographic data, pathological diagnosis and treatment protocol. A value of p<0.05 was accepted as statistically significant. Overall survival (OS) rate was determined as the time from the date of diagnosis to death from any cause or by the last follow-up examination. Disease-free survival (DFS) rate was calculated from the date of diagnosis to the first documentation of an event (disease progression, death from any cause or the most recent follow-up examination). The statistically significant parameters in survival analysis were entered into a multivariate analysis by stepwise Cox regression model.

Results
For the entire cohort, a total of seventy-six patients with pediatric osteosarcoma were identified by reviewing their medical records at the Registry of Division of Pediatric Hematology-Oncology, Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University. Six patients excluded from our study were those who declined treatment after diagnosis and those who were referred out to receive treatment at a regional hospital by Thailand's Universal Health Coverage scheme. Only seventy patients were eligible for our study. Of these patients, the majority were male (65.7%) with a median and mean age at diagnosis of 11.60 years (SD=2.11) and 11.41years (5.67-15.31 years), respectively. 32 patients (45.7%) had initial pulmonary metastasis at the time of diagnosis, while only 2 of them had combined pulmonary and distant bone metastasis. No patient had isolated bone metastasis at diagnosis. Primary tumor site was an extremity in 68 patients (97.1%), the scapula in 1 patient (1.4%) and iliac wing in 1 patient (1.4%). The two most common primary sites were at distal femur (35.7%) and proximal tibia (27.1%). The most frequent presenting symptoms were pain with or without minor trauma (65.7%) and abnormal mass at primary site (64.3%). The median time from initial symptom to diagnosis was 2.45 months (0.5-5.25). After tumor biopsy, chemotherapy could not be given in four patients because of severe osteomyelitis and surgical wound infection. All of them subsequently died with tumor progression locally and metastatically. 7 patients who were declined surgery died, despite intensive chemotherapy. Overall limb-salvage and amputation rates were 12.9% and 77.7%, respectively. Two patients developed acute kidney injury as documented by a rising serum creatinine of more than 1.5 times of the upper normal limit for their ages and by elevated transaminase enzymes, which included total bilirubin after administration of HDMTX. They were treated and fully recovered with leucovorin rescue and force diuresis and also tolerated well a subsequent HDMTX administration. None of the patients had treatment-related toxic death or other serious toxicities, including infectious complications in our study.
According to chemotherapeutic protocols, the presence of good histological response (GHR; percent tumor necrosis of primary tumor >90%) occurred at a significantly higher frequency in patients who received the MTX(+) protocol than those with MTX(-) protocol (42.1% and 0%, respectively, p=0.002). There was no statistically significant difference between groups of treatment protocol in terms of clinical characteristics of patients that included each patient's sex, age at diagnosis, metastatic status at diagnosis, primary sites of tumor, histological subtypes (conventional/variant) and type of surgical procedures (limb-sparing/amputation). All patients who underwent surgery had adequate surgical margin histologically. No correlation was found between age and histological subtypes. Up to 50% of the data regarding primary tumor volume were missing because of poor documentation and missing sections.
Regardless of treatment protocols, the median survival time was 45.0 months (SD=14.20) with a median and mean follow-up time of 29.65 months (SD=40.83) and 43.66 months (3.33-176.30), respectively. The Kaplan-Meier analysis for 3, 5-year DFS and 3, 5-year    Figure 1A. Patients with GHR had a statistically significant higher 3-year DFS and OS than those with poor histological response (PHR) (p=0.008 and p=0.016, log rank test) [100%, 100% for GHR and 56.1±9.5%, 41.0±9.6% for PHR, respectively]. For localized osteosarcoma, the 3-and 5-year OS were 65.1±6.9% and 54.4±8.7%, respectively. The 3-year DFS and OS were higher significantly for MTX(+) protocol as compared to MTX(-) protocol (p=0.010 and p=0.009, log rank test) [69.8±10.5%, 79.8±9.1% for MTX(+) and 31.1±6.9%, 42.2±7.4% for MTX(-) protocol, respectively]. In subgroup analysis, patients with metastatic osteosarcoma treated with MTX(+) protocol also had a statistically significant higher 3-year DFS and OS than those treated with MTX(-) protocol (66.7±13.6%, and 15.0±8.0% for 3-year DFS, p=0.010, 73.3±13.2% and 20±8.9% for 3-year OS, p=0.006, respectively). For patients with localized osteosarcoma who were treated with MTX(+) protocol, the 5-year OS was up to 88.9±10.5%. The presence of initial pulmonary metastasis, with an age at diagnosis ≤11.6 years, had a statistically significant association with lower 3-year DFS (p=0.043 and p=0.035, respectively), but had no significant association with 3-year OS (p=0.058 and p=0.209, respectively). According to the protocol, patients with initial pulmonary metastasis tended to have higher 3 and 5-year OS than those with localized disease for MTX(+) protocol. For MTX(-) protocol, patients with initial pulmonary metastasis had statistically significant higher 3-and 5-year OS than those without metastasis, as shown in Figure 2. However, age at diagnosis, sex, histological subtypes and types of surgery were not found to be statistically significant factors determining DFS and OS.
Two parameters with statistically significant associations with higher 3-year DFS and OS, histological response and treatment protocol, were included in the backward stepwise Cox regression model for a multivariate analysis. The multivariate analysis identified only the treatment with MTX(-) protocol as the only independent predictor of inferior OS rate with a hazard ratio (HR) of 3.53 (95% confidence interval of 1.2-10.41, p=0.022).

Discussion
Osteosarcoma is the most common primary bone tumor in childhood and adolescents. The introduction of neoadjuvant chemotherapy has dramatically improved the outcome in osteosarcoma and has thus minimized the micro-metastasis of the disease. It has also increased the chance of radical resection of primary tumor including feasibility of limb-salvage surgery and has also reduced the risk of local recurrence after limb-salvage surgery as compared to that for amputation (Merkel et al., 1991). The effective chemotherapeutic agents against osteosarcoma are CDDP (Ochs et al., 1978;Baum et al., 1979), CBDCA (Choeyprasert et al., 2014), ADR (Cores et al., 1972;Choeyprasert et al., 2014) and IFO (Harris et al., 1995;Harris et al., 1998) andHDMTX (Jaffe et al., 1983;Rosen G, 1993;Delepine et al., 1996;Goorin et al., 2003;Lewis et al., 2007). Recently, HDMTX seems to be one of the most active agents against osteosarcoma; however, there is still no consensus on a standard chemotherapy approach, including even the HDMTX-based protocol (Jaffe et al., 1985;van Dalen et al., 2011).
Recently, there were few literatures regarding the epidemiologic data of pediatric osteosarcoma in Thailand (Wiromrat et al., 2012;Choeyprasert et al., 2014). Only one report from the Northern region of Thailand demonstrated the outcome of treatment in pediatric osteosarcoma by the combination of CBDCA and ADR (Choeyprasert et al., 2014). Like the previous literature, males were affected approximately 1.91 times more frequent than females. The age at diagnosis was comparable to previous studies worldwide with a peak age incidence in adolescents Smith et al., 2010;Wiromrat et al., 2012). Almost all of patients had a primary tumor at extremities, especially around the knee joint (62.8%). Approximately half of the patients had distant metastasis at diagnosis, especially at lungs, which was higher than those of Western countries (Link et al., 1986;Eilber et al., 1987). However, our finding was correlated with a previous study from the Northern region of Thailand (Choeyprasert et al., 2014). For our study, patients with osteosarcoma were managed with a similar chemotherapeutic protocol and with surgical approaches that were aimed at complete resection of primary tumor regardless of metastatic status at diagnosis.
For the entire cohort, the 3-and 5-year OS rate during 1999-2014 for both localized and metastatic osteosarcoma were 53.2±6.1%, 44.8±6.4%, respectively. Our OS rates for patients with localized osteosarcoma were slightly inferior to those with the international data, which show a 5-year OS up to 70% (Bielack et al., 2002;Meyers et al., 2005;Ferrari et al., 2005). In an earlier era, we described a 3-year DFS of 44±9.9% for localized osteosarcoma treated with MTX(-) protocol as compared to 46% from the study of The European Osteosarcoma Intergroup (Lewis et al., 2000), which also treated localized osteosarcoma with CDDP and ADM. The 3-year overall survival for pediatric osteosarcoma treated with MTX (+) protocol were 88.9±10.5% for those without initial pulmonary metastasis and 73.3±13.2% for those with initial pulmonary metastasis (p=0.387) and B) Kaplan-Meier analysis for estimated 3-year overall survival for pediatric osteosarcoma treated with MTX (-) protocol were 60±9.8% for those without initial pulmonary metastasis and 20±8.9% for those with initial pulmonary metastasis (p=0.008) treatment regimens for pediatric osteosarcoma evolved over time. We adapted the HDMTX-based protocol from the European and American Osteosarcoma Study Group, EURAMOS-1 protocol (Marina et al., 2009), for our institute. Apparently, the MTX(+) protocol statistically improved outcomes for patients with both localized and metastatic osteosarcoma as compared to the MTX(-) protocol. For MTX(+) protocol, patients with localized osteosarcoma had a 3-year OS of 88.9±10.5 %, which was consistent with previous literatures, in which an HDMTXbased regimen was used by such study groups as the Intergroup Study-0133 (Meyers et al., 2005;Meyers et al., 2008;Smeland et al., 2011) and the Scandinavian Sarcoma Group (SSG) XIV protocol (Hegyi et al., 2011). Our study showed a superior outcome compared to those from previous studies from Asia using HDMTX-based regimens (Lin et al., 2011;Han et al., 2012). As regards histological response to chemotherapy, GHR (tumor necrosis ≥90%) induced by preoperative MTX(+) protocol demonstrated a significant correlation with outcomes of pediatric osteosarcoma, which were the same as in previous studies (Hudson et al., 1990;Bramwell et al., 1992;Bramwell et al., 1997;Souhami et al., 1997) We received an unsatisfactory response by the MTX(-) protocol, since no GHR was observed in the MTX(-) subgroup. Types of surgical procedures were not a statistically significant prognostic factor for osteosarcoma in our study, possibly because of the adequacy of surgery in our institute and the efficacy of preoperative chemotherapy making total resection feasible. A younger age at diagnosis was also a good prognostic factor determining DFS, consistent with previous studies (Mankin et al., 2004;Lee et al., 2009;Hagleitner et al., 2011), but not for OS in our study. However, the influence of age on outcome in pediatric osteosarcoma is still inconclusive because of the variability of treatment and metastatic status at diagnosis in each study.
Multivariate analyses showed that the treatment with MTX(-) protocol (HR 3.26, p=0.015) was an independent predictor of OS. The pulmonary metastasis was not the significant prognostic factor in our study; however, patients who had initial metastasis tended to have inferior outcomes, as shown in Figure 2. These results must be interpreted carefully because the small population, especially in MTX(+) arm, might render the subgroup analysis erroneous. The additional limitations were the missing data on the size of primary tumor, type of histology and histological response, especially in early era of treatment in our institute.
In conclusion, this study showed that HDMTXbased regimen has a superior efficacy against pediatric osteosarcoma as compared to other alternatives. Although the high incidence of initial pulmonary metastasis in this study was higher than those of Western countries, treatment outcome was still comparable. The HDMTXbased regimen should be conducted in larger populations in order to determine its efficacy and antitumor activity, especially in those with initial pulmonary metastasis and of an age >11.6 years. Furthermore, the refinement in risk, treatment stratification and dose intensification for pediatric osteosarcoma is a future challenge for researchers to improve outcomes, especially in metastatic patients who may need a more intensive regimen.