Potentially functional variants of autophagy‐related genes are associated with the efficacy and toxicity of radiotherapy in patients with nasopharyngeal carcinoma

Abstract Background Nasopharyngeal carcinoma (NPC) is one of the major invasive malignant neoplasms of head and neck, while radiotherapy is the primary therapy for NPC. Genetic variants could affect the efficacy and toxicities of radiotherapy in NPC patients. Methods In the current study, we aimed to investigate 10 potentially functional SNPs of autophagy‐related genes (ATG) with the efficacy and toxicity of radiotherapy in 468 NPC patients. Results We found ATG10 rs10514231, rs1864183, and rs4703533 were significantly associated with worse efficacy of radiotherapy at both at the primary tumor and lymph node, while ATG16L2 rs10898880 was significantly associated with better efficacy of radiotherapy at both primary tumor and lymph node. Besides, we also found ATG10 rs10514231 and ATG16L2 rs10898880 were significantly associated with the occurrence of grade 3–4 oral mucositis (allelic model, for rs10514231: OR = 1.95, 95% CIs = 1.31–2.9, p = .001; for rs10898880: OR = 1.56, 95% CIs = 1.19–2.04, p = .001) and grade 3–4 myelosuppression (allelic model, for rs10514231: OR = 2.08, 95% CIs = 1.39–3.09, p < .001; for rs10898880: OR = 1.51, 95% CIs = 1.1–2.06, p = .010). Conclusions This should be the first report identifying ATG10 rs10514231, rs1864183, rs4703533, and ATG16L2 rs10898880 could contribute to the efficacy and toxicity of radiotherapy in NPC patients. Further investigation of the underlying molecular mechanisms and prospective clinical trials in NPC patients are needed to validate our results.


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YANG ANd LIU et al., 2012). While in southern China it is much greater, with annual rates between 15 and 30 NPC cases per 100,000 (Kamran, Riaz, & Lee, 2015). Radiotherapy alone or chemoradiotherapy, is an important component of the primary therapy of NPC for its highly radio-sensitivity (Miao et al., 2019;Zhan, Zhang, Wei, Fu, & Zheng, 2019). However, predictors of the efficacy and toxicity response to radiotherapy of NPC have not been yet fully identified (Chen et al., 2019;Kamran et al., 2015;Miao et al., 2019).
The discovery of suitable biomarkers is needed to predict efficacy and toxicity of radiotherapy in patients with NPC. Recently, single nucleotide polymorphisms (SNPs) of candidate genes have been to be associated with the outcomes and toxicity in patients accepting radiotherapy of many cancers, including lung cancer, NPC, prostate cancer, breast cancer, oropharyngeal cancer, thyroid cancer, and so on (Kerns et al., 2019;Lewin et al., 2019;Liu et al., 2018;Tao et al., 2018;Wang et al., 2017;Wen et al., 2018). Studies showed that autophagy played an important role in various stages of cancer development, progression, radio-sensitivity and toxicity, including NPC (Liang et al., 2018;Lin et al., 2014;Qin et al., 2013;Wen et al., 2018;K. Xie et al., 2016;Yang et al., 2018;Yuan et al., 2017;Zhu et al., 2018). Autophagy could selectively target dysfunctional organelles, intracellular microbes, and pathogenic proteins, and deficiencies in these processes might lead to occurrence of cancers (Levine & Kroemer, 2019). During this process, autophagy-related genes (ATG) play an essential role in autophagy, and directly or indirectly accelerate cancer development and progression (Levine & Kroemer, 2019;Tsuboyama et al., 2016). The ATG family is a big family, and only a small part of the family members are currently known in humans (Klionsky, 2007). Some potentially functional variants of ATGs have been identified to be associated with the development, progression, radio-sensitivity and toxicity of other cancers, like lung cancer, hepatocellular carcinoma, prostate cancer, bladder cancer, breast cancer, and so on (Budak Diler & Aybuga, 2018;Li et al., 2019;Nikseresht et al., 2018;Zhou et al., 2017). Inspired by these findings, the present study was conducted to establish the relationships, if any, between potentially functional variants of ATGs and the efficacy of radiotherapy, as well as radiation-induced toxicity reaction in NPC patients.

| Study populations
The current study totally recruited 468 pathological diagnosed NPC patients treated with radiotherapy. The inclusion criteria was a first-time diagnosis of NPC, no prior treatment of anticancer therapies, no severe disorders of lung, heart, liver, pancreas, or kidney diseases. At recruitment, each participant or family members signed the informed consent form and a 5 ml of blood sample from the patients was collected. Genetic DNA of all patients was extracted using Wizard Genomic DNA Purification Kit (Promega), and stored at −80°C for further evaluation. Questionnaires on patient demographics were collected prior to treatment. The present study's protocol was approved by the ethics committee of the Shengjing Hospital Affiliated to China Medical University.

| Treatment efficacies and toxic reactions
All the patients were treated with intensity modulated radiation-therapy (IMRT), with a tumoricidal radiation dose of 66-70 Gy in 30-33 fractions for nasopharyngeal primary focus and the positive lymph nodes. All the patients underwent fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) after treatment. Treatment efficacies at the primary tumor and lymph node were evaluated in line with the Response Criteria in Solid Tumors (PERCIST), which defined treatment efficacy as complete metabolic response (CMR). Radiation-induced toxic reactions, including. dermatitis, oral mucositis and myelosuppression, were evaluated according to the radiation toxicity grading criteria of the Radiation Therapy Oncology Group or European Organization for Research and Efficacy of Cancer (RTOG/ EORTC). Patients were defined as "non-sensitive or mildly radiosensitive" group (grade 0-2) and "highly radiosensitive" group (grade 3-4).

| Selection of SNPs and genotyping
The selection of candidate SNPs were mainly based on study previously published by Wen et al. (2018). Eight of the nine functional SNPs [ATG2B rs17784271 (3ʹUTR) and rs4900321 (3ʹUTR); ATG10 rs10514231 (intron 2), and rs4703533 (the promoter region); ATG12 rs26538 (the promoter region) and rs1058600 (3ʹUTR); ATG16L2 rs1126205 (the promoter region) and rs10898880 (the promoter region)], were included (MAF of rs6884232 in Chinese was 0). We also included two widely reported SNPs in ATG10 gene, rs1864182 and rs1864183. This means totally 10 SNPs were included in this study. The genotyping was performed using the TaqMan methodology and read with the Sequence Detection Software on an ABI-Prism 7,900 instrument according to the manufacturer's instructions (Applied Biosystems, Foster City, CA).

| Statistical analysis
All statistical tests were two-sided and a p value of .05 was considered significant, and all analyses were performed using SAS software version 9.2 (SAS Institute). Univariate logistic regression was performed to determine the association of the 10 SNPs with the efficacy at the primary tumor and lymph node, as well as the radiation-induced toxicity reaction in NPC patients adjustment for age, gender, BMI, smoking, drinking, family history of cancer, EBV-DNA, chemotherapy, and TNM stage.

| Associations between candidate
SNPs and the efficacy of radiotherapy Table 2 presents the associations between candidate SNPs and the efficacy of radiotherapy at the primary tumor and lymph node in NPC patients. We found ATG10 rs10514231, rs1864183, and rs4703533, were significantly associated with worse efficacy of radiotherapy at both at the primary

| DISCUSSION
In present study, we investigated the associations of 10 potentially functional SNPs in ATG2B, ATG10, ATG12, and ATG16L2 with the efficacy and toxicity of radiotherapy in 468 NPC patients. We found ATG10 rs10514231, rs1864183, and rs4703533 were significantly associated with worse efficacy of radiotherapy at both at the primary tumor and lymph node, while ATG16L2 rs10898880 was significantly associated with better efficacy of radiotherapy at both at the primary tumor and lymph node. Besides, we also found ATG10 rs10514231 and ATG16L2 rs10898880 were significantly associated with the occurrence of grade 3-4 oral mucositis and myelosuppression. These results suggest that potentially functional variants of ATGs might be useful biomarkers for predicting efficacy and toxicity of radiotherapy in NPC patients, once these results were validated by additional investigations.
With the rapid development of radio-genomics, many studies have presented significant associations between genetic variants of candidate gene with the efficacy and toxicity of radiotherapy in NPC patients Ma et al., 2017;Xie et al., 2014;Yu et al., 2016). Xie et al. (2014) found that the p53 codon 72 polymorphism could be an independent prognostic marker for locoregionally advanced NPC. Guo et al. (2017) reported that CDKN2A rs3088440 was significantly related with a poorer treatment efficacy on the primary tumor and cervical lymph node after radiotherapy, and also with a decreased risk of grade 3-4 acute radiation-induced myelosuppression. Ma et al. (2017) found that polymorphisms in angiogenesis related genes could contribute to clinical outcomes of radiotherapy in NPC patients. Yu et al. (2016) detected that CTNNB1 rs1880481 and rs3864004, and GSK3β rs3755557 were significantly associated with poorer efficacy of radiotherapy in NPC patients, while GSK3β rs375557 and APC rs454886 were correlated with acute grade 3-4 radiation-induced dermatitis and oral mucositis, respectively. These findings above revealed that genetic variants could potentially work as the indicator of efficacy and toxicity of radiotherapy in NPC patients.
Emerging evidence has revealed that autophagy process, which degrades intracellular components through the lysosomal machinery, plays an essential role in the process of cancer development and progression (Avalos et al., 2014; Levine, Cuervo, & Klionsky, 2008), while ATGs could control autophagic formation, and directly or indirectly accelerate cancer development and progression (Levine & Kroemer, 2008). Xie et al. (2016) identified that ATG10 rs10514231, rs1864182 and rs1864183 were associated with poor lung cancer survival and positively correlated with ATG10 expression. In current study, we also found ATG10 rs10514231, rs1864183, and rs4703533 were significantly associated with worse efficacy of radiotherapy at both primary tumor and lymph node. Qin et al. (2013) reported that ATG10 rs1864182 and rs10514231 were significantly associated with a decreased risk of breast cancer in Chinese population. Yuan et al. (2017) also revealed that genetic variations in ATGs were significantly associated with clinical outcomes of advanced lung adenocarcinoma treated with gefitinib. Recently, Wen et al. (2018) found ATG16L2 rs10898880 contributed to a better prognosis of patients with non-small cell lung cancer (NSCLC) after definitive radiotherapy, and a greater risk of developing severe radiation pneumonitis. This results were similar to the findings in current study, which revealed that ATG16L2 rs10898880 was significantly associated with better efficacy of radiotherapy at both at the primary tumor and lymph node, and had a greater risk of developing grade 3-4 oral mucositis and myelosuppression.

| CONCLUSIONS
Conclusively, we identified ATG10 rs10514231, rs1864183, rs4703533, and ATG16L2 rs10898880 could contribute to the efficacy and toxicity of radiotherapy in NPC patients. Further investigation of the underlying molecular mechanisms to explain how these polymorphisms affect response to radiotherapy and prospective clinical trials in NPC patients are needed to validate our results.

ACKNOWLEDGMENT
This study was funded by a grant from the National Natural Science Foundation of China (project No. 81470086).

CONFLICTS OF INTEREST
The authors declare that they have no conflict of interest.

AUTHOR CONTRIBUTIONS
L.Z. and Y.Z. conceived and designed the experiments. L.Z. and Y.Z. performed the experiments. L.Z. and Y.Z. analyzed the data and wrote the paper.