Pre-Radiotherapy n-6/n-3 Fatty Acid Ratio Predicts Severe Oral Mucositis in Nasopharyngeal Carcinoma

Background and rationale. Nasopharyngeal carcinoma (NPC) is endemic in Southeast Asia and southern China, and most patients are diagnosed with locally advanced disease. Standard treatment for locally advanced NPC (LA-NPC) includes induction chemotherapy followed by concurrent chemoradiotherapy. Although effective, this approach causes substantial acute toxicity, and radiation-induced oral mucositis (RIOM) remains the most common severe complication. PD-1 inhibitors have enabled cisplatin-sparing strategies that maintain efficacy while reducing toxicity, but RIOM is still a major clinical challenge. Omega-6 and omega-3 polyunsaturated fatty acids (PUFAs) have opposing roles in inflammation. The erythrocyte membrane n-6/n-3 PUFA ratio reflects systemic fatty acid balance and inflammatory status over the preceding 2-3 months. This ratio has been linked to chronic disease risk and cancer progression, but its longitudinal changes and relationship with RIOM have not been prospectively examined in LA-NPC patients receiving PD-1 inhibitor-based, cisplatin-sparing regimens.

Study design and setting. This is a prospective, single-center, single-arm interventional study conducted at the Affiliated Hospital of Guangdong Medical University. The study enrolled treatment-naïve patients with LA-NPC (AJCC 9th edition stage II-III) and matched healthy controls. All patients received protocol-directed therapy and were followed prospectively. Healthy controls provided a single blood sample at enrollment. The study was approved by the Ethics Committee of the Affiliated Hospital of Guangdong Medical University (approval no. LY2025-10-025) and conducted in accordance with the Declaration of Helsinki. The study was prospectively registered at ClinicalTrials.gov.

Treatment protocol. All enrolled patients received a three-stage treatment regimen. First, patients received three 21-day cycles of induction chemoimmunotherapy. On day 1 of each cycle, they received intravenous toripalimab 240 mg, cisplatin 80 mg/m², and nab-paclitaxel 260 mg/m². After induction, patients received concurrent toripalimab and intensity-modulated radiotherapy (IMRT). Target volumes included the gross tumor volume of the nasopharynx (GTVnx), gross tumor volume of metastatic lymph nodes (GTVnd), high-risk clinical target volume (CTV1), and low-risk clinical target volume (CTV2). GTVnx and GTVnd received 69.96 Gy, CTV1 received 60 Gy, and CTV2 received 54 Gy, given once daily, five days per week, for 33 fractions. During radiotherapy, patients received three cycles of toripalimab 240 mg every 21 days. After radiotherapy, patients received six 3-weekly cycles of adjuvant toripalimab 240 mg intravenously.

Sample collection and laboratory assessments. Blood samples were collected at seven prespecified time points: T0 (baseline, within 1 week before treatment), T1-T3 (within 3 days after each induction cycle), T4 (pre-radiotherapy, within 1 week before radiotherapy), T5 (mid-radiotherapy, cumulative dose 40 Gy, 19 fractions), T6 (post-radiotherapy, within 3 days after completion), and T7 (1 month after radiotherapy). Healthy controls provided a single sample at enrollment. Erythrocyte membrane fatty acids were quantified by gas chromatography. Individually quantified omega-6 PUFAs were linoleic acid (LA), arachidonic acid (AA), and docosatetraenoic acid (DTA). Individually quantified omega-3 PUFAs were α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Total omega-6 and total omega-3 PUFAs were calculated as the sum of identified fatty acids. The n-6/n-3 ratio was defined as total omega-6 divided by total omega-3 PUFAs. The AA/EPA ratio and n-3 index (EPA + DHA as a percentage of total fatty acids) were also calculated. Plasma cytokines (IL-4, IL-6, IL-10, IL-12p70, IL-17, IFN-γ, and TNF-α) were measured by Luminex multiplex assay. Nutritional assessment included body weight, BMI, white blood cell count, total cholesterol, triglycerides, HDL, LDL, serum albumin, prealbumin, NRS 2002, and PG-SGA.

Data management and statistical analysis. Paper case report forms were double-checked, and two researchers independently entered data into an electronic database, followed by consistency verification and logical validation. A random 20% sample of key indicators was re-tested, and the intraclass correlation coefficient was computed for consistency. For variables with >5% missing data, multiple imputation using chained equations was applied. Statistical analyses were conducted using SPSS version 26.0. Categorical variables are presented as frequencies and percentages. Normally distributed continuous variables are presented as mean ± standard deviation, and non-normally distributed variables as median with interquartile range. Baseline comparisons used chi-square or Fisher's exact test and independent-samples t-test or Mann-Whitney U test. Longitudinal changes were evaluated by repeated-measures analysis of variance, with Greenhouse-Geisser correction when sphericity was violated. Trends in ordinal categorical variables were assessed using Cochran-Armitage and Cochran-Mantel-Haenszel tests. Spearman's rank correlation was used to examine associations between fatty acid parameters and toxicity severity. Univariate logistic regression and receiver operating characteristic (ROC) curve analysis were used to evaluate predictive performance, with the optimal cutoff determined by Youden's index. Calibration was assessed by the Hosmer-Lemeshow test. Because only 22 grade ≥3 RIOM events occurred, multivariable adjustment and internal validation were not performed to avoid overfitting, and predictive performance should be regarded as exploratory.

Sample size and limitations. A total of 105 patients with LA-NPC and 105 healthy controls were enrolled. All patients completed the protocol without loss to follow-up. This single-center, single-arm study in a homogeneous Chinese population may limit generalizability, and the non-randomized design precludes causal inference. The small number of grade ≥3 RIOM events led to wide confidence intervals and precluded extensive multivariable adjustment. No internal or external validation was performed; therefore, the predictive performance should be considered exploratory. Dietary records and dosimetric parameters were not collected. Residual confounding cannot be excluded.