Impact of chrono-radiotherapy on the prognosis and treatment-related toxicity in patients with locally advanced nasopharyngeal carcinoma: A multicenter propensity-matched study

ABSTRACT The timing of radiotherapy (RT) delivery has been reported to affect both cancer survival and treatment toxicity. However, the association among the timing of RT delivery, survival, and toxicity in locally advanced nasopharyngeal carcinoma (LA-NPC) has not been investigated. We retrospectively reviewed patients diagnosed with LA-NPC who received definitive RT at multiple institutions. The median RT delivery daytime was categorized as morning (DAY) and night (NIGHT). Seasonal variations were classified into the darker half of the year (WINTER) and brighter half (SUMMER) according to the sunshine duration. Cohorts were balanced according to baseline characteristics using propensity score matching (PSM). Survival and toxicity outcomes were evaluated using Cox regression models. A total of 355 patients were included, with 194/161 in DAY/NIGHT and 187/168 in WINTER/SUMMER groups. RT delivered during the daytime prolonged the 5-year overall survival (OS) (90.6% vs. 80.0%, p = 0.009). However, the significance of the trend was lost after PSM (p = 0.068). After PSM analysis, the DAY cohort derived a greater benefit in 5-year progression-free survival (PFS) (85.6% vs. 73.4%, p = 0.021) and distant metastasis-free survival (DMFS) (89.2% vs. 80.8%, p = 0.051) in comparison with the NIGHT subgroup. Moreover, multivariate analysis showed that daytime RT was an independent prognostic factor for OS, PFS, and DMFS. Furthermore, daytime RT delivery was associated with an increase in the incidence of leukopenia and radiation dermatitis. RT delivery in SUMMER influenced only the OS significantly (before PSM: p = 0.051; after PSM: p = 0.034). There was no association between toxicity and the timing of RT delivery by season. In LA-NPC, the daytime of radical RT served as an independent prognostic factor. Furthermore, RT administered in the morning resulted in more severe toxic side effects than that at night, which needs to be confirmed in a future study.


Introduction
Nasopharyngeal carcinoma (NPC) is a cancer originating from the epithelial cells of the nasopharynx (Chen et al. 2019;Lo et al. 2012).Over 70% of NPC cases are initially detected in the locally advanced stages owing to the occult location of the tumor (Li et al. 2018).Unlike the treatment of head and neck tumors, which involves surgery, locally advanced NPC (LA-NPC) is predominantly treated with radiotherapy (RT) (Feng et al. 2010).Despite improved RT efficacy using intensity-modulated radiotherapy (IMRT), 10-20% of LA-NPC patients still experienced local recurrence due to radioresistance (Blanchard et al. 2015;Li et al. 2018).In addition, high-dose radical RT can cause increased toxicity and side effects.Therefore, efforts to improve the efficacy and safety of LA-NPC radiotherapy focus on modifying traditional RT approaches to reduce toxicity without compromising efficacy.Recently "chrono-radiotherapy" (chrono-RT), which refers to the time-of-day effects on RT, has been investigated to overcome the limitations of conventional RT.Studies have reported improved clinical response and alleviation of side effects with chrono-modulated RT in several cancers, including head and neck (Brolese et al. 2021;Elicin et al. 2021;Goyal et al. 2009), breast (Johnson et al. 2019;Noh et al. 2014), cervical (Shukla et al. 2010), rectal (Squire et al. 2017), and prostate (Hsu et al. 2016) cancer.The effects of chrono-RT on the prognosis and treatment-related toxicity of LA-NPC are yet to be elucidated.
The effectiveness of RT mainly relies on its ability to induce DNA damage.The cellular response to such damage is influenced by the circadian clock via its control of DNA repair enzymes across the cell cycle progression (Bermúdez-Guzmán et al. 2021;Gery et al. 2006).The G1-S phase cell-cycle arrest of NPC cells (Hou et al. 2020), rather than the radiosensitive G2-M phase (Pawlik and Keyomarsi 2004), indicates the possibility of radioresistance induction.The overexpression of period circadian regulator 2 (PER2), one of the circadian clock genes, has been reported to suppress tumor progression by regulating a checkpoint of the cell cycle (Cheng et al. 2015;Lu et al. 2015;Zhanfeng et al. 2016).Moreover, a preclinical study has demonstrated that PER2 overexpression can enhance chemotherapeutic efficacy by suppressing the proliferation, migration, and invasion of NPC cells, which further affirms its potential value in the chronotherapy of tumors (Hou et al. 2020).In this context, the likely interplay between RT delivery timings and its efficacy in NPC warrants further investigations.
Circadian and circannual variations have been shown to influence treatment-related toxicities in patients with NPC undergoing concurrent chemo-RT.Adverse effects commonly affect the hematologic system and gastrointestinal tract.Preclinical studies indicate that circadian and circannual rhythms can affect cell cycle and proliferation in intestinal and hematopoietic cells (Haus et al. 1984(Haus et al. , 1997;;Laerum et al. 1988;Smaaland et al. 2002).Limited research exists on the effects of circannual/ circadian variations in NPC.Exploring the implications of these variations is crucial to understand their influence on adverse symptoms and develop treatments to alleviate or prevent such symptoms.
This multi-centre retrospective study aimed to compare the toxicities and prognosis in terms of overall survival (OS), progression-free survival (PFS), locoregional recurrence-free survival (LRFS), and distant metastasis-free survival (DMFS) in patients with LA-NPC receiving RT at various timings: morning (DAY) vs. night (NIGHT) and darker half of the year (WINTER) vs. the brighter half (SUMMER).

Patients
We performed a retrospective review of patients with NPC who had received RT at the Affiliated Cancer Hospital, Institute of Guangzhou Medical University, Fujian Medical University Union Hospital, and The First Affiliated Hospital of Hainan Medical University between January 2013 and December 2015.The final follow-up was in December 2020.This research was approved by the institutional ethical committee of the participating hospitals.All patients underwent a pretreatment imaging evaluation and were restaged by two specialized radiation oncologists in accordance with the American Joint Committee for Cancer Staging System (8th Edition).Disagreements were resolved by mutual consensus.Patients or their guardians provided written informed consent for participation according to the tenets of the Declaration of Helsinki (World Medical Association 2013).The STROBE guidelines for cohort studies were followed for reporting (von Elm et al. 2008).
Patients with NPC were included in this study according to the following eligibility criteria: (a) 18-65 years of age, (b) confirmed to have non-keratinizing NPC via biopsy according to the current pathologic classification of the World Health Organization, (c) tumor -node -metastasis (TNM) stage (III or IV a), (d) definitive RT + concomitant chemotherapy with curative intent and a total delivered dose of 70 Gy in daily fractions of 2.0-2.5 Gy.The exclusion criteria were as follows: (a) RT delivery time was beyond the established range (09:00-13:00, 20:00-24:00), (b) RT delivery time was changed, for instance, at times in the morning and at times in the evening, (c) Karnofsky performance status (KPS) of < 70, (d) Targeted epidermal growth factor receptor therapy (nimotuzumab or cetuximab) was used concurrently with chemo-RT.Patient characteristics, RT details, and main outcomes were collected from the electronic database and retrospective chart review.RT timing data were collected from the Mosaiq® patient information system (Elekta, Stockholm, Sweden).

Time of RT
The RT delivered between 09:00 hours and 13:00 hours was termed as DAY, whereas that delivered during the dark hours (from 20:00 hours to 24:00 hours) was defined as NIGHT.The time point of the daily first therapeutic beam-on time was used to define each fraction of time.The limit for patients receiving RT twice a day to compensate for treatment interruptions was set at < 4 times for the entire treatment course (Elicin et al. 2021).March and September equinoxes were used to classify the year into SUMMER and WINTER seasons, and the median date of the RT course was used as the reference for the treatment season.

Outcomes
The co-primary endpoints of this study were to compare OS and PFS between two groups (DAY vs. NIGHT and WINTER vs. SUMMER).Secondary endpoints included the comparison of LRFS, DMFS, and the incidence of adverse events.OS was defined as the duration from the start of treatment to the date of death, while PFS was defined as the time from diagnosis to recurrence, metastasis, or death from any cause, or until the analysis was conducted.LRFS was defined as the period from diagnosis to the recurrence of the primary site and/or lymph nodes, and DMFS was defined as the time between the initial pathological diagnosis and the first detection of distant metastasis.Patients who were alive at the last follow-up were considered censored.
Toxic effects were assessed and graded based on CTCAE 4.0 (Common Terminology Criteria for Adverse Events, version 4.0).The study included the evaluation of eight common side effects associated with radiotherapy, including leukopenia, anemia, thrombocytopenia, dermatitis, dry mouth, oral mucositis, dysphagia, and vomiting.The highest grades of side effects observed during the radiotherapy period were recorded.

Statistical analysis
Patient characteristics were described using mean ± standard deviation for normally distributed continuous variables and median (range) for non-normally distributed variables.Comparisons between variables were conducted using t-test for normally distributed variables, Mann -Whitney U test for non-normally distributed variables, and chi-square test for categorical variables.Propensity score matching (PSM) was performed to balance variables, and a new datasheet was created for further analysis.Kaplan -Meier method and log -rank test was employed to estimate and compare cumulative survival rates (OS, PFS, LRFS, and DMFS).Cox proportional hazards model with the Forwards (LR) procedure was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs).Univariate and multivariate Cox proportional hazard models were applied to identify confounding factors for the endpoints, with p < 0.1 in univariate analyses considered for inclusion in multivariate analyses.Adverse events were compared using the rank-sum test.Statistical analyses were performed using SPSS (version 26.0) and R (version 4.0).Two-sided tests were conducted, and a p-value of <0.05 was considered statistically significant.

Patient characteristics
Of the 650 nasopharyngeal carcinoma (NPC) patients in the primary dataset, 355 met the inclusion criteria (Supplementary Figure S1).The baseline characteristics of the 355 eligible patients who were included in the study (194/161 in DAY/NIGHT and 187/168 in SUMMER/WINTER cohorts) are presented in Supplementary Table S1.
There was considerable variation in the baseline characteristics of the patients in both cohorts, including differences in NPC family history and hypertension between the DAY and NIGHT groups (Supplementary Table S2), as well as, differences in NPC family history and smoking status between the SUMMER and WINTER groups (Supplementary Table S3).After Propensity Score Matching (PSM) of patients in both cohorts, 141 patients in the DAY/NIGHT cohort and 146 patients in the SUMMER/WINTER cohort with characteristics wellbalanced between the groups (p > 0.05) (Supplementary Tables S2 and S3).
While no association was observed between timings of radiotherapy (RT) induction and 5-year Local Recurrence Free Survival (LRFS), RT delivered during the day significantly prolonged Distant Metastasis-Free Survival (DMFS), both before and after PSM (Supplementary Figures S1CD and S2CD).Timings of RT by seasons (WINTER/SUMMER) only significantly affected Overall Survival (OS) (before PSM: p = 0.051; after PSM: p = 0.034) (Figures 2 and Supplementary 3).Detailed long-term oncological outcomes and year-wise survival data for both chronotherapy comparative groups are provided in Tables 1 and 2.
Additionally, the SUMMER season was identified as an independent prognostic factor only for OS (HR = 0.528, 95% CI 0.292-0.955,p = 0.035).Furthermore, the clinical stage was an independent prognostic factor for OS, PFS, and DMFS.

Treatment-related toxicity
Two sets of data, after PSM processing, were analyzed to compare toxicity characteristics between the DAY/NIGHT and SUMMER/WINTER groups (Table 3 and Supplementary Table S8).Radiotherapy delivered at morning was significantly associated with a higher risk of leukopenia (Grade 0-2, p = 0.004; Grade 3-4, p = 0.006, Table 3) and radiation dermatitis (Grade 0-2, p = 0.007; Grade 3-4, p = 0.009, Table 3).However, no significant difference in toxicity was observed in the SUMMER/ WINTER group (Supplementary Table S8).Subgroup analysis was conducted to identify potential populations that may benefit from RT delivered during night-time and experience a lower incidence of Grade 3/4 toxicities (Table 4).RT during night-time was advantageous for patients ≤46 years of age, weighing >24 kg/m 2 , and having no family history of diabetes or hypertension.Patients with N3-4 stage, receiving adjuvant or neoadjuvant chemotherapy and IMRT, also showed a lower incidence of dermatitis with RT at evening time.Furthermore, non-smokers and patients with N3-4 stage receiving IMRT exhibited a significantly lower incidence of Grade 3-4 radiation dermatitis in the NIGHT group compared to the DAY group.In the subgroup analysis of the SUMMER/ WINTER group, no significant difference was observed between summer and winter RT at the subgroup level (Supplementary Table S9)

Discussion
Chronotherapy with regard to chemotherapy and radiotherapy has progressed enormously in various cancer types (Eriguchi et al. 2003;Gery et al. 2006;Sancar and Van Gelder 2021).Its role in NPC, which is mainly treated with radiotherapy, is highly valuable.Furthermore, there is a lack of studies examining the prognostic impact of chrono-RT on locally advanced NPC.This research offers a novel perspective on clinical RT delivery and chronotherapy in radiation oncology.
In this retrospective multicenter study, the daytime cohort of RT delivery was divided into two subgroups (DAY: 09:00-13:00 and NIGHT: 20:00-24:00) with a significant time interval, allowing for accurate assessment of circadian rhythm-related biological differences.The findings demonstrated that the timing of RT delivery, specifically DAY vs. NIGHT, independently influenced OS, PFS and DMFS in LA-NPC.This finding aligns with the growing body of evidence suggesting that the circadian rhythm significantly impacts cell cycle regulation, DNA repair mechanisms, and the body's response to oxidative stress, all of which are critical factors in the efficacy of radiotherapy.
Furthermore, RT administration during the daytime showed significantly improved outcomes in terms of leukopenia and radiation dermatitis compared to the NIGHT, indicating a potential modulation of tissue and blood system sensitivity to radiation-induced damage by circadian rhythms.Notably, RT delivery during the SUMMER season extended OS without affecting toxicity.Confounding factors were accounted for through PSM analysis.
The circadian clock gene PER2, whose expression in NPC is lower than that in the nasopharyngeal mucosa, has been demonstrated to act as a tumor suppressor by inhibiting the ERK/p38/MAPK signaling pathway to repair DNA damage (Hou et al. 2020).The inhibitory effect of PER2 DNA repair pathways suggests that they play a vital role in the development of radioresistance.An efficient and redundant DNA repair capacity is critical for radioresistance (Biau et al. 2019).Upregulation of PER2 results in enhanced radiosensitivity and vice versa.Although the tumor-intrinsic circadian rhythm of PER2 in NPC remains unknown, a high level of PER2 expression has been observed during the day in normal tissues (Corrà et al. 2017;Duong et al. 2011;Masri and Sassone-Corsi 2018).This finding may suggest a hypothesis for interpreting the significant difference in the efficiency of RT delivery between DAY and NIGHT.
Recent reports have highlighted the crucial role of the circadian rhythm in maintaining the balance of the immune system.Specifically, the mobilization and trafficking of leukocytes appear to be regulated in a manner that is dependent on the time of the day (Haspel et al. 2020;Wang et al. 2023).These findings may help explain the variations in treatment-related leukopenia resulting from daytime RT.Nathan et al. (2021) have recently demonstrated that the aging process is linked to a reduction in circadian outputs and a dysregulation of the immune system.The present study is consistent with these findings; a significant difference in the occurrence of leukopenia was noted following daytime RT in younger patients but not in older patients in the subgroup analysis.Nonetheless, this outcome must be interpreted cautiously due to the inability to verify the specific timings of blood collection given the retrospective nature of the study.
Consistent with our findings, prior research has demonstrated the remarkable superiority or a favorable trend of morning RT administration in other cancer types.For instance, a retrospective study by Hsu et al. investigated 409 patients with non-metastatic prostate adenocarcinoma, revealing a notable survival advantage for morning RT.The study reported a 6-year overall survival rate of 92% for the DAY group compared to 75% for the NIGHT group (p = 0.09), and a 5-year biochemical failure-free survival of 85% for the DAY group compared to 72% for the NIGHT group (p = 0.05) (Hsu et al. 2016).Two additional retrospective studies investigating brain metastasis in non-small cell lung cancer (NSCLC) have similarly shown that evening RT delivery positively affects oncologic survival (Badiyan et al. 2013;Rahn et al. 2011).Despite variations in the criteria used to define daytime in these studies, the results consistently indicated that the chronological effects of radiation influenced tumor responses.
Furthermore, circadian rhythms controlling the cell cycle tend to differ depending on the region of the body (Chan et al. 2017).Hence, the tumor's inherent circadian rhythm of the cell cycle in NPC could potentially account for this variation and influence treatment strategies.Previous research has indicated that the late afternoon and evening correspond to the most radiosensitive phase of the cell cycle (G2-M) in human oral mucosa, whereas the morning represents the G1-S phase.Consequently, radiation-induced oral mucositis tends to be more severe during the late afternoon and evening compared to the morning (Bjarnason et al. 2009;Goyal et al. 2009).However, this study did not find any differences in the effects of circadian rhythm on oral mucositis.The tumor's intrinsic circadian rhythm of NPC, which might be altered due to carcinogenesis, has not been reported.Nonetheless, research on the regulation of the NPC cell cycle to enhance radiosensitivity is on the rise (Break et al. 2021;He et al. 2022;Zhang et al. 2016).
The findings of the WINTER/SUMMER cohorts support the vitamin D hypothesis, suggesting that cancer survival is better in patients diagnosed in the and autumn seasons (SUMMER) compared to those diagnosed in the winter and spring seasons (WINTER).Following adjustment for patient characteristics using the PSM method, a significant improvement in overall survival was observed in NPC patients who underwent RT during the summer season.Additionally, there was a positive trend towards improved 5-year PFS in female patients within the SUMMER cohorts.The vitamin D hypothesis has been examined in both preclinical and clinical studies on head and neck squamous cell carcinoma and NSCLC (Elicin et al. 2021;Robsahm et al. 2004).However, RT is not the primary therapeutic management in these cancers.Recent preclinical studies assessing the effect of vitamin D on enhancing the radiosensitivity of cancer cells have yielded positive results (Gavrilov et al. 2010;Nuszkiewicz et al. 2020;Sharma et al. 2014;Wu et al. 2021;Yu et al. 2021Yu et al. , 2021)).However, there are no clinical studies to confirm this hypothesis.In contrast, Nino et al. have reported an inverse relationship between seasonality and vitamin D levels (Niino et al. 2021).Further studies are therefore needed to determine the effect of seasonality on RT delivery.
Based on these findings, we propose that the scheduling of RT in LA-NPC treatment could be optimized by aligning treatment times with patients' circadian rhythms to enhance therapeutic efficacy while minimizing toxicity.This chrono-therapeutic approach could involve scheduling RT sessions during windows of time when cancer cells are most susceptible to radiation but normal tissues are least likely to incur damage.Furthermore, our results advocate for the incorporation of circadian biology principles into the design of future clinical trials and treatment protocols for LA-NPC, and potentially other cancers as well.
This retrospective study has limitations including a small sample size, particularly in the female cohort during the exploratory analysis, which may introduce some bias.However, patient characteristics were well balanced in the DAY/NIGHT and WINTER/ SUMMER cohorts.Multivariate analysis effectively controlled for confounders, ensuring robust results in the initial cohort analysis.Certain clinical characteristics relevant to NPC, such as Epstein-Barr virus infection, smoking history, and alcohol and preserved food consumption, were insufficient for analysis in our cohort.Blood collection, influenced by the treatment schedule, lacked specific timing and should be interpreted with caution due to potential confounding bias.Additionally, factors related to chronotherapy, including patient chronotype, hormonal levels (cortisol, melatonin, etc.), and vitamin D levels throughout treatment, were not adequately detected and recorded.

Conclusions
To the best of our best knowledge, this is the first study to report the prognostic impact and toxicity of chronoradiotherapy in locally advanced NPC using PSM analysis.Significant improvement was observed in survival outcomes (OS, PFS, and DMFS for the DAY subgroup; OS for the SUMMER subgroup).Moreover, the daytime of RT delivery was identified to be an independent prognostic factor for locally advanced NPC.Furthermore, in comparison with RT administered in the NIGHT, RT delivery in the DAY resulted in a significant increase in the incidence of leukopenia and dermatitis.However, well-designed prospective studies should be conducted to confirm these findings.In addition, preclinical studies on the circadian rhythm of NPC are required to completely elucidate the underlying mechanisms.

Figure 1 .
Figure 1.Kaplan-Meier curves illustrate the survival of patients in DAY/NIGHT cohorts after PSM.(a) overall survival, (b) progressionfree survival, (c) locoregional recurrence-free survival and (d) distant metastasis-free survival.

Figure 2 .
Figure 2. Kaplan-Meier curves illustrate the survival of patients in SUMMER/WINTER cohorts after PSM.(a) overall survival, (b) progression-free survival, (c) locoregional recurrence-free survival and (d) distant metastasis-free survival.

Table 1 .
Comparisons of long-term oncological outcomes between DAY vs. NIGHT groups in the entire and PSM cohorts.

Table 2 .
Comparisons of long-term oncological outcomes between SUMMER vs. WINTER groups in the entire and PSM cohorts.

Table 3 .
Radiation toxicity profiles between DAY vs. NIGHT groups in the PSM cohorts.

Table 4 .
Effects of subgroups on 3-4 Grade toxicity underwent DAY versus NIGHT.