Clinical biomarkers for thyroid immune-related adverse events in patients with stage III and IV gastrointestinal tumors

Background Thyroid immune-related adverse events (irAEs) associated with immune checkpoint inhibitor (ICI) treatment appear to correlate with a better prognosis. We aimed to investigate clinical biomarkers associated with thyroid irAEs. Methods We retrospectively analyzed data from 129 patients receiving programmed cell death protein 1 (PD-1) inhibitors for stage III and IV gastrointestinal tumors. Patients were divided into two groups: “thyroid irAEs” group and “no thyroid irAEs” group. We compared continuous variables using Mann–Whitney U and Kruskal–Wallis tests and categorical variables using Pearson’s chi–square test. Survival curves were generated using the Kaplan–Meier method, and associations between clinical features and thyroid irAEs were assessed using univariate and multivariate logistic regression models. Associations for thyroid irAEs and outcomes [progression-free survival (PFS), overall survival (OS)] of the patients were performed with a Cox proportional hazard model. Results A total of 129 patients, including 66 gastric cancer, 30 esophageal squamous cell carcinoma, and 33 hepatocellular carcinoma (HCC), were involved in this analysis with 47 cases of thyroid irAEs occurrence. The Cox proportional hazard model analysis confirmed the extended PFS [hazard rate (HR) = 0.447, 95% confidence interval (CI): 0.215 to 0.931, p = 0.031] and OS (HR = 0.424, 95% CI: 0.201 to 0.893, p = 0.024) for thyroid irAEs group when compared with those of the no thyroid irAEs group. Association between thyroid irAEs and clinical characteristics at baseline was analyzed subsequently by univariate analysis. Higher body mass index (p = 0.005), increased eosinophil count (p = 0.014), increased lactate dehydrogenase (p = 0.008), higher baseline thyroid stimulating hormone (TSH) (p = 0.001), HCC (p = 0.001) and increased adenosine deaminase (ADA) (p = 0.001) were linked with thyroid irAEs occurrence. The multivariable logistic regression model indicated that ADA [odds rate (OR) = 4.756, 95% CI: 1.147 to 19.729, p = 0.032] was independently associated with thyroid irAEs occurrence. Conclusions Increased baseline level of ADA was associated with thyroid irAEs occurrence in patients with advanced gastrointestinal tumors who received ICI treatment. In the case of abnormal ADA, attention should be paid to the risk of thyroid irAEs.


Introduction
The global incidence and mortality rates of gastrointestinal tumors in China have reached approximately 50% (1), indicating the necessity for increased attention toward their prevention, diagnosis, and treatment.Since 2011, immune checkpoint inhibitors (ICIs), which promote the anti-tumor immune response of activated T cells by blocking the inhibitory signal, including cytotoxic T-lymphocyte associated protein 4 inhibitors, programmed cell death protein 1 (PD-1) inhibitors, and programmed cell death ligand 1 inhibitors, have been used as a treatment method for unresectable or metastatic melanoma, lung cancer, breast cancer, and gastrointestinal tumors with monotherapy or combined therapy (2).Immune-related adverse events (irAEs) occur owing to the overactivation of the immune system following ICI treatment.This overactivation triggers enhanced T cell activity towards antigens found in both tumor and normal tissues, inducing an inflammatory response and alterations in the autoimmune environment, thereby initiating the incidence of irAEs (3,4).IrAEs appear to be associated with a favorable prognosis for ICI treatment, however, severe adverse reactions may necessitate discontinuation of medication and can even be life-threatening (2).
IrAEs can affect various organs and systems, causing dysfunction; thyroid dysfunction is a common endocrine irAE with a widely range of incidence rates in different reports (5)(6)(7).The mean incidence rate in phase III clinical trials is 10.8%, especially in patients using anti-PD1 drugs (8).Thyroid dysfunction includes primary hyperthyroidism, primary hypothyroidism, central hypothyroidism, and biphasic thyroiditis that transitions from transient hyperthyroidism to hypothyroidism with severe grade in rare cases (6).Thyroid dysfunction as an irAE has been associated with prolonged survival in various cancers, including caseds of non-small cell lung carcinoma (NSCLC), malignant melanoma, renal cell carcinoma, head and neck cancer, and gastric cancer (4)(5)(6).However, thyroid dysfunction can lead to metabolic abnormalities affecting the overall treatment effectiveness.
Few studies have focused on clinical biomarkers for the occurrence and timely monitoring of thyroid dysfunction as an irAE (5).High baseline levels of thyroid stimulating hormone (TSH) and positive thyroid related antibodies were associated with the occurrence of thyroid irAEs, but others biomarkers didn't reach consensus.This study is aimed to investigate clinical biomarkers for earlier diagnosis and follow-up of thyroid irAEs.Adenosine deaminase (ADA) is associated with the immune status in patients with solid tumors, with alterations observed during thyroid dysfunction occurred.ADA is a key enzyme regulating purine metabolism by degrading adenosine, thereby reducing its inhibitory effects on the immune system.Currently, no research has focused on the role of ADA in the occurrence of irAEs.In this study, we aimed to identify clinical biomarkers for the occurrence of thyroid irAEs.

Patients
Patients diagnosed with stage III and IV gastrointestinal tumors and undergoing treatment with anti-PD-1 antibody monotherapy or combined therapy (chemotherapy or targeted therapy) at The Fourth Hospital of Hebei Medical University from November 2018 and November 2021 were included in this study.The inclusion criteria were: (1)  All procedures were approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University.This retrospective study analyzed existing data, and the requirement for informed consent was waived.

Treatment and assessment
Patients received standard anti-PD-1 antibodies (sintilimab, camrelizumab, pembrolizumab, toripalimab, and tisleizumab) every 3 weeks as monotherapy or combined therapy (targeted therapy/chemotherapy) until disease progression, clinical worsening, unacceptable toxicity, or patient refusal.No cytotoxic T lymphocyte-associated antigen-4 inhibitor was used in these patients.Clinical and laboratory tests were performed during each cycle before dosing.Body computed tomography or magnetic resonance imaging scans were performed every 2-3 cycles.Progression-free survival (PFS) was defined as the time from the first administration of anti-PD-1 therapy to disease progression, death, or the study cutoff; overall survival (OS) was defined as the time from the commencement of anti-PD-1 therapy to death or the study cutoff values.PFS and OS were evaluated using the Response Evaluation Criteria in Solid Tumors version 1.1 as the study endpoints.

Statistical analysis
Continuous variables were compared using Mann-Whitney U and Kruskal-Wallis tests, while categorical variables were analyzed using Pearson's chi-square test.Survival curves were drawn using the Kaplan-Meier method.The associations between clinical features and thyroid irAEs were assessed using univariate and multivariate logistic regression models.The association between the clinical features and survival was analyzed using the log-rank test.The associations between thyroid irAEs and outcomes (PFS, OS) of the patients were performed with a COX proportional hazard model.The receiver operating characteristic (ROC) curve analysis determined the best cutoff value.All results were considered statistically significant at p <0.05.Statistical analysis was performed using SPSS version 25.0 software (IBM Corp, Armonk, NY, USA), and figures were generated using GraphPad Prism version 9 (GraphPad Software, San Diego, CA, USA).

Clinical characteristics analysis for thyroid irAEs and no-irAEs groups
In the cohort, 129 inpatients including 60 females and 69 males were involved in this study, with a median age of 63 years [interquartile range (IQR) 56-69 years], a median PFS of 146 days [95% confidence interval (CI): 181.44-241.21days] and a median OS of 239 days (95% CI: 260.78-322.32 days).Gastric cancer was the most common type of tumor (n = 66, 51.2%), while 33 patients had hepatocellular carcinoma (HCC), and 30 patients had esophageal squamous cell carcinoma.Thyroid irAEs were observed in 47 patients (11 thyrotoxicosis, 27 hypothyroidism, 9 biphasic thyroiditis), with a median of 3 (IQR 2-5) cycles of treatment.Patients with HCC were more prone to thyroid irAEs compared with other cancer types.The baseline information is shown in Table 1.

Thyroid irAEs associated with disease control rate, PFS and OS
As shown in Table 1, BMI, tumor type, ADA level, lactate dehydrogenase (LDH) level, and baseline TSH level differed between the thyroid irAEs and no thyroid irAEs groups, demonstrating that the baseline status of these indicators affected the occurrence of thyroid irAEs.The distribution frequency of thyroid irAEs incidence was not different for these 5 antibodies (p = 0.867).In Table 2, the disease control rate (DCR) between the two groups was significantly different (p= 0.002), which was observed 85.1% (95%CI:74.9%to 95.3%) in the thyroid irAEs group and 61.0%(95%CI:50.4% to 71.5%) in the no thyroid irAEs group.

Discussion
Thyroid irAEs were the most prevalent PD-1 inhibitors related irAEs including hypothyroidism, hyperthyroidism, biphasic thyroiditis, and central hypothyroidism, with hypothyroidism comprises the majority (9).Our study revealed thyroid irAEs in 47 out of 129 patients (36.4%), which is comparable to previous reports (42%) (4).Despite the occurrence of thyroid irAEs, most patients exhibited a low fatality rate and manifested symptoms, such as arrhythmia, myxedema, and marasmus (10).Timely monitoring of thyroid function is crucial to prevent thyroid dysfunction in cancer patients receiving the ICI treatment and maintain sustained therapeutic effects.Patients with thyroid irAEs tended to a longer OS and a trend of longer PFS than those without thyroid dysfunction, which was also comparable to the previous reports (6).
Patients with HCC were more prone to thyroid thyroid irAEs compared with other cancer types, although this conclusion needs to be validated in a larger population.The liver is the site for thyroid hormone synthesis and metabolism, HCC can initiate more synthesis and secretion of thyroxine binding globulin, which contributes to the occurrence of hypothyroidism by augmented thyroxine binding globulin-T4 binding (11, 12).In addition, signaling pathways, such  as TGF-b, Wnt, and Hedgehog, midiate inflammation and fibrosis of HCC, which can regulate the metabolism of thyroid hormones.Liver abnormalities are more likely to induce the abnormalities of thyroid hormone metabolism, which may be one of the reasons why patients with HCC are more prone to thyroid irAEs (13).
Our investigation identified a higher baseline level of ADA (>14.05U/L) as an independent biomarker associated with thyroid irAEs.ADA, a key enzyme involved in purine metabolism and immune system regulation, has been associated with thyroid autoimmune diseases at the transcriptional level (14).It can activate T lymphocytes (15) as well as alleviate immunosuppressive effects by degrading adenosine (16).The adenosine receptors on the dendritic cells (DCs) can interact with CD26 on the surface of T cells to mediate the CD4+ T cell differentiation (17), DCs deliver antigens to CD4+ T cells so as to stimulate the production of thyroid related antibodies, thereby destroying the thyroid follicular   We identified BMI (>21.06 kg/m 2 ), ECOG PS (≤1) and eosinophil count (>0.20 × 10 9 /L) as associated factors for thyroid irAEs.Consistent report demonstrated that a higher BMI is associated with increased incidence of irAEs and improved outcomes in various cancers such as NSCLC, melanoma, renal cell carcinoma, head and neck tumors, and epithelial urothelial carcinoma (20,21).Additionally, a study involving approximately 19% of patients with gastrointestinal tumors suggested a correlation between higher BMI and increased risk of overt thyroid dysfunction in ICI-treated cancers (21).Our study corroborated these findings, demonstrating that patients with gastrointestinal tumors and higher BMI are more susceptible to thyroid irAEs.One plausible explanation for this association is that obesity induces systemic inflammation and compromises immune response through modulation of leptin secretion, which is known to correlate with PD-1 expression in CD8+ T cells (22).
In our previous study, we observed that a lower ECOG PS was associated with a better prognosis (23).However, the relationship between ECOG PS and irAEs remains contentious due to the limited data.While ECOG PS (≤1) was identified as an independent risk factor for irAEs in patients with NSCLC who received ICI treatment (24), however, another study found no significant association between ECOG PS and irAEs for patients with metastatic renal cell carcinoma who received ICI treatment (25).This potential association between ECOG PS and thyroid irAEs should be re-evaluated in a larger population.

Conclusions
Increased baseline level of ADA was associated with thyroid irAEs occurrence in patients with advanced gastrointestinal tumors who received ICI treatment.In the case of abnormal ADA, attention should be paid to the risk of thyroid irAEs.

TABLE 1
Characteristics of patients with and without thyroid immune-related adverse events (irAEs).

TABLE 2
Effect of thyroid immune-related adverse events (irAEs) on immune checkpoint inhibitor (ICI) efficacy.
SD, stable disease; PR, partial response; PD, progressive disease; CR, complete response; ORR, objective response rate; DCR, disease control rate.

TABLE 3
Multivariate analysis of PFS and OS with COX regression models.

TABLE 4
Univariate and multivariate analysis of thyroid immune-related adverse events (irAEs) with Logistic regression models.