DNA Ligase 4 as a Prognostic Marker in Nasopharyngeal Cancer Patients Treated with Radiotherapy

Nasopharyngeal (NP) carcinoma arises from the lining of the nasopharynx, the narrow passage behind the nasal cavity. Worldwide, there are about 80,000 incident cases and 50,000 deaths annually (Ferlay et al., 2010). The incidence of NP carcinoma demonstrates marked geographical variation. In the United States and Western Europe, it is rare with an incidence of 0.5 to 2 per 100,000 (Parkin et al., 2005; Chang et al., 2006). In contrast, NP carcinoma is endemic in southern China, including Hong Kong, where the incidence may reach 25 cases per 100,000 per year. Intermediate risk regions include Southeast Asia, North Africa and the Middle Ease, and the Arctic. In South Korea, the incidence of NP cancer is 0.7 per 100,000 according to the Korean National Cancer Registry Report 2005, which is lower than that of Hong Kong but still much higher than that of Western countries (Kim et al., 2011). Because of the anatomical location of the nasopharynx and its proximity to critical neurovascular structures, radiation therapy (RT), rather than surgery, is a mainstay treatment for early stage NP carcinoma. And for more advanced disease, concurrent chemoradiation therapy


Introduction
Nasopharyngeal (NP) carcinoma arises from the lining of the nasopharynx, the narrow passage behind the nasal cavity.Worldwide, there are about 80,000 incident cases and 50,000 deaths annually (Ferlay et al., 2010).The incidence of NP carcinoma demonstrates marked geographical variation.In the United States and Western Europe, it is rare with an incidence of 0.5 to 2 per 100,000 (Parkin et al., 2005;Chang et al., 2006).In contrast, NP carcinoma is endemic in southern China, including Hong Kong, where the incidence may reach 25 cases per 100,000 per year.Intermediate risk regions include Southeast Asia, North Africa and the Middle Ease, and the Arctic.In South Korea, the incidence of NP cancer is 0.7 per 100,000 according to the Korean National Cancer Registry Report 2005, which is lower than that of Hong Kong but still much higher than that of Western countries (Kim et al., 2011).
Because of the anatomical location of the nasopharynx and its proximity to critical neurovascular structures, radiation therapy (RT), rather than surgery, is a mainstay treatment for early stage NP carcinoma.And for more advanced disease, concurrent chemoradiation therapy (CCRT) reduces the rate of distant metastasis and improves local control and overall survival compared to RT alone (Teo et al., 1996;Chua et al., 2002;Kwong et al., 2004;Ma et al., 2008).Thus, radiosensitivity is one of the most important factors determining the prognosis of NP cancer patients.However, methods to predict the radiosensitivity of NP cancer have not yet been developed.Most radiobiologists currently think that radiation primarily causes cell death by double-strand DNA damage, and that the inability of cancer cells to repair such damage with fidelity results in their death (Dikomey et al., 1998).
Ionizing radiation induces DNA double-strand breaks (DSBs), highly cytotoxic and cytostatic forms of damage.The capability for DNA DSBs repair is crucial for inherent radiosensitivity of tumor and normal cells.Repair of DSBs presumably involves two main mechanisms, nonhomologous end joining (NHEJ) and homologous recombination (HR).NHEJ is likely to be the major mechanism of repair of radiation-induced DSBs in humans because it is dominant for the repair of somatic cells and proliferating cells in the G1 stage, whereas HR is important for early embryogenesis and repair of proliferation (Hoeijmakers, 2009;Sakata et al., 2001;Kim et al., 2011).In addition, Ku 70, DNA-PKcs, MRN complex, RAD50, XRCC4, and DNA ligase4 (LIG4) play a critical role in DNA DSB repair through NHEJ (Iliakis, 2009;Davis et al., 2013).These DNA repair proteins in tumor cells may be potential prognostic markers for the prediction of the RT outcome.It has been demonstrated that down-regulated expressions of these molecules significantly sensitize cancer cell lines to ionizing radiation.But some studies showed opposite results.In those studies, patients with higher level of DNA repair proteins had better prognosis than patients with lower level.
In this study, we aimed to determine the relationship between the expression of DNA DSBs repair proteins and the prognosis of NP cancer patients treated with RT.

Patients and tumors
Data collection was overseen by the institutional review board (DAUH-IRB-14-090).65 patients who were histologically confirmed nasophayngeal cancer and treated with radiotherapy with or without chemotherapy from April 2000 to March 2009 at Dong-A University Medical Center and Busan National University Hospital were included in this study.For histological diagnosis, specimens were obtained from nasopharyngeal and lymph node biopsy in 61 and 4 patients, respectively.They were staged according to the TNM classification as presented in the AJCC Cancer Staging Manual (Stephen BE, 2010).Clinical follow-up information was obtained by review of the patients' records and retrospectively analyzed.

Statistical analysis
Categorical variables were summarized by counts and relative frequencies; continuous variables, by their median and range.The correlation between each markers and categorical variables were estimated by Kruskal-Wallis test; the correlation between each markers and continuous variables by linear regression.The probability of survival was calculated using the Kaplan-Meier method.The logrank test was used to evaluate the prognostic factors of the survival rate.The OS was calculated from the first day of diagnosis to the date of death from any cause or date of last follow-up.The TTP was calculated from the day of diagnosis to the locoregional and/or distant relapse in completely responded patients or by progression of disease in partially responded patients.Statistical significance was established as P value less than 0.05.

Treatment delivery and response
We treated all 65 patients with RT.Twenty-eight patients received CCRT alone.8 patients received RT alone.Ten patients received RT after induction chemotherapy and 6 patients received CCRT after induction chemotherapy.10 patients were treated with induction chemotherapy, CCRT, and chemotherapy, respectively.Only 3 patients were treated with chemotherapy followed by CCRT.Mean radiation dose was 67.88 cGy (range, 52.50 -93.80 cGy), and the chemotherapy during CCRT was cisplatin alone (75mg/m 2 ) delivered every 3 weeks.All the chemotherapy regimens included cisplatin.29 patients were treated with cisplatin and 5-FU, 23 were cisplan and docetaxel, 1 was cisplatin with S-1, and 4 were cisplatin alone.As a result of treatment, there were complete response (CR) in 33 patients (50.8%), partial response (PR) in 23 patients (35.4%), stable disease in 5 patients (7.7%), and progressive disease in 4 patients (6.2%).

Correlation between clinical outcome and expression of LIG4 and RAD50
There were no correlations between the positive proportion of LIG4 cells and stage, pathologic type, age, and gender.Also, there was no significant correlation between the expression of LIG4 and CR rate.However, there was a significant correlation between OS and expression of LIG4.The 5-year OS rate of patients with LIG4 (±) were 90% and 67.9%, respectively (p=0.035).There was also a significant correlation between the expression of LIG4 and TTP.The 5-year TTP rate of patients with LIG4 (±) were 75.9%, 55.5%, respectively (p=0.039).Figures 4 and 5 illustrate the Kaplan-Meier estimated survival curves for OS and TTP in patients classified according to the expression level of LIG4.No significant association was found between the level of RAD50 expression and clinical outcome.LIG4 and RAD50 were no statistically significant factor for TTP and OS in multivarient analysis.

Discussion
There are several clinical prognostic factors for NPC such as classification, sex, and age.However, among the same-staged patients, treatment outcomes are variable (Chua et al., 2004).Although there are many other variables that affect the prognosis of NP carcinoma patients, predicting the radiosensitivity of normal and tumor tissues before treatment will provide the most important information to determine the best treatment method for each patient with NP cancer.For that reason, there have been several studies focused on the relationships between expression levels of DNA DSBs repair proteins and prognosis of cancer.
Wilson et al. reported that a higher proportion of Ku70 positive tumor cells is closely associated with poor response to RT and a high risk of locoregional recurrence.The 5-year locoregional control rate was significantly higher in the low Ku70 group (85%) than in the high Ku70 group (42%) (p=0.0042).However, there were no differences in the metastases-free survival rates between the 2 groups (Ku70(+),82%); Ku70(-),78%; p=0.8672) (Lee et al., 2005).Similar results have been reported in tumors at other sites (Komuro et al., 2002;Wilson et al., 2000;Zhao et al., 2000).Uterine cervical cancers with low levels of Ku70 expression in biopsy samples were radiosensitive and displayed a significantly better RT outcome (Wilson et al., 2000).
Bouchaert et al. reported that localized prostate cancer patients with a high tumor level of DNA-PKcs had cancer that was more resistant to RT (p=0.0002).Positive DNA-PKcs nuclear expression was closely associated with biochemical recurrence after RT (p=0.0002).Of the 65 tumors with nuclear DNA-PKcs expression, 34 (>50%) experienced biochemical recurrence.In contrast, only 12 of 67 cases negative for DNA-PKcs had a relapse after treatment (Bouchaert et al., 2012).
In another study, reduced expression of the MRN complex predicted a poor effect of RT in patients with early breast cancer.In this study, moderate/strong expression of the MRN complex was related to a better prognosis compared with negative/weak MRN expression (p=0.02)(Soderlund et al., 2007).Karin et al. explained that their results could be related to the down-regulation or loss of proteins in the MRN complex, impairing the ability to recognize DNA strand breaks and failure to induce DNA damage signaling, cell cycle arrest, and apoptosis, thereby leading to increased risk of relapse.Impaired ability to detect and signal DNA damage is likely to also reduce DNA repair, and this could possibly increase the radiosensitivity of tumor cells.However, there are studies that have had opposite results.MRN complex is a protein complex consisting of Mre11, Rad50 and Nbs1.And overexpression of Nbs1 was a marker of poor prognosis in advanced head-and-neck squamous cell carcinoma and uveal melanoma (Ehlers et al., 2005;Yang et al., 2006).Karin et al. suggested that this discrepancy was due to differences in tumor characteristics and differences in evaluation staining.
Most of these studies tended to associate high NHEJ protein levels with a lower response to RT.However, other studies showed opposite results.In our study, the higher the expression of LIG4, the better prognosis the patients had.Increased expression of LIG4 was a good prognostic marker of disease recurrence or progression and OS in NP cancer patients treated with RT.No significant association was found between the level of RAD50 expression and clinical outcome.These results suggest that the response to DNA damage involving the NHEJ system could be dependent to the types of cancer cells (Pavon et al., 2008).Another possible explanation is that LIG4 may have other functions besides repairing DSB, like the DSB recognition of Nbs1.
Since this is a retrospective study, a number of factors in terms of the patients and tumor characteristics could  doi.org/10.7314/APJCP.2014.15.24.10985DNA Ligase 4 as a Prognositc Marker in Nasopharyngeal Cancer not be controlled.Furthermore, this study has some limitations.First, the sample size was too small.Second, the treatment delivery methods were variable, and the sequence of treatment and regimens of chemotherapy were variable.In addition, even though all chemotherapy included cisplatin, we didn't evaluate ERCC1, which is a significant prognostic factor in patients treated with platinum-based chemotherapy.Third, the follow up duration was variable.Consequently, these limitations might cause discrepancies.Nevertheless, this is the first study that suggesting the possibility of forecasting the prognosis of NP cancer treated with RT by performing immunohistochemical analysis of LIG4.
In conclusion, our results suggest the possibility of predicting the intrinsic radiosensitivity of NP cancer by performing immunohistochemical analysis of LIG4.Further prospective studies are needed to confirm our results.

Figure
Figure 4. Overall Survival (Kaplan-Meier Estimated Survival Curves) in patients with Nasopharyngeal Carcinomas with Expression of LIG4

Figure 5 .
Figure 5.Time to Progression (Kaplan-Meier Estimated Survival Curves) in Patients with Nasopharyngeal Carcinomas with Expression of LIG4