Immune checkpoint inhibitors (ICIs) have transformed the therapeutic paradigm for advanced non-small cell lung cancer (NSCLC), yielding substantial clinical benefits. However, durable responses are observed in only a minority of patients, underscoring the critical need for predictive biomarkers to optimize patient selection and therapeutic outcomes (1).

Skeletal muscle wasting (SMW) is a multifactorial condition influenced by aberrant interactions among cytokines, endocrine signaling pathways, metabolic alterations, nutritional deficits, and reduced physical activity (2). SMW, related to inflammation and metabolic dysfunction, may impair immune response and reduce immunotherapy efficacy in NSCLC (2). Moreover, in NSCLC and other malignancies, SMW has emerged as a negative prognostic factor and a potential negative biomarker of immunotherapy response (3–5).

Interestingly, type 2 and 3 iodothyronine deiodinases (DIO2 and DIO3) are present in skeletal muscle, where thyroid hormone signaling plays a crucial role in muscle development, function, and regeneration (6). In this light, the ratio of free triiodothyronine (fT3) to free thyroxine (fT4) is an easily assessable and reliable indicator of deiodinases regulating active thyroid hormones. Exploratory studies have linked a low fT3/fT4 ratio with reduced muscle mass and SMW in several non-cancer conditions (7, 8). Moreover, the fT3/fT4 ratio has been found to correlate with survival outcomes in patients with solid tumors (colorectal cancer and renal cell carcinoma) treated with tyrosine kinase inhibitors (9, 10) and in patients with urothelial carcinoma treated with immunotherapy (11). fT3/fT4 ratio, strictly related to muscle metabolism and systemic inflammation (6, 12), may reflect metabolic status and immune system function and thus serve as a useful predictor of immunotherapy response in NSCLC. Based on this evidence, we hypothesized that the fT3/fT4 ratio could serve as an easily assessable surrogate marker of skeletal muscle status and an indirect predictor for response to immunotherapy in patients with advanced NSCLC. We therefore conducted a study to explore the relationship between fT3/fT4 ratio, SMW and clinical outcome in NSCLC patients treated with upfront pembrolizumab.

We retrospectively analyzed a cohort of consecutive patients with advanced NSCLC treated with first-line pembrolizumab monotherapy between June 2018 and September 2021, at three Italian oncology centers (University of Verona, Gemelli University Hospital and Parma University Hospital). Inclusion criteria were: age >18 years; histologically confirmed diagnosis of advanced and measurable disease according to Response Evaluation Criteria In Solid Tumors (RECIST) 1.1 criteria; PD-L1 expression ≥ 50% assessed by immunohistochemistry (IHC); prior administration of more than one dose of pembrolizumab as first-line treatment, based on clinical judgment, aligned with established guidelines and best clinical practices; availability of pre-treatment abdominal computed tomography (CT) scans, performed within 3 months before starting immunotherapy. Patients with incomplete clinical data or lacking adequate imaging were excluded.

The study was approved by the local Ethical Committee (Prot. 2193CESC, 2022) and it was conducted according to the ethical standards of Declaration of Helsinki.

The following clinical-radiological and laboratory features were collected: age, gender, Eastern Cooperative Oncology Group Performance Status (ECOG PS), smoking history, comorbidities, disease stage, baseline thyroid hormones levels (fT3 and fT4), weight, height, Body Mass Index (BMI) and data on routine pre-immunotherapy CT based body composition.

Body composition assessment was performed by measuring the cross-sectional areas of skeletal muscle and fat tissue using a single axial CT slice taken at the level of the third lumbar vertebra (L3), a region known to reliably reflect total body tissue distribution (3).

Image analysis at the L3 level was carried out using a freehand segmentation approach, which closely aligned with the principles of the Alberta protocol (an established method based on tissue attenuation thresholds) utilizing built-in functionalities of SliceOmatic software version 5.0 (TomoVision, 3280 Chemin Milletta, Magog, J1X 0R4, Canada), distinguishing muscle from visceral, subcutaneous and intermuscular adipose tissues using anatomic knowledge and tissue-specific Hounsfield unit (HU) ranges. Skeletal muscle area (SMA) was identified within an attenuation range of −29 to +150 HU, visceral adipose tissue (VAT) was identified between -150 and -50 HU, subcutaneous adipose tissue (SAT) and intermuscular adipose tissue (IMAT) between -190 and -30 HU. The evaluated muscle groups included the psoas, erector spinae, quadratus lumborum, transversus abdominis, internal and external obliques, and rectus abdominis. To account for individual body size, the SMA was normalized to body surface area, resulting in the skeletal muscle index (SMI), expressed in cm²/m². This step was essential to precisely quantify muscle volume, which can vary significantly depending on an individual’s body composition. To categorize patients exhibiting skeletal muscle wasting (SMW), we employed sex-specific and BMI-adjusted SMI thresholds as outlined by Martin et al (13).

SMW was defined as SMI of <43 cm²/m² for men with BMI < 25 kg/m², SMI < 53 cm²/m² for men with BMI ≥ 25 kg/m², and SMI < 41 cm²/m² for women. The main objective of the study was the association of SMW with predictive factors (e.g., ECOG PS, fT3/fT4 ratio).

Continuous variables were expressed as medians and range, categorical variables as frequencies and percentages. Progression-free survival (PFS) and overall survival (OS) were estimated using Kaplan-Meier methods and compared using the log-rank test. A p-value < 0.05 was considered statistically significant. A multivariate logistic regression, including ECOG PS and fT3/fT4 ratio, was developed using stepwise regression (forward selection, enter limit and remove limit, p = 0.10 and p = 0.15, respectively), to identify independent predictors of outcome. Receiver operating characteristic (ROC) analysis was conducted to determine the optimal cut-off for the fT3/fT4 ratio in predicting SMW. Statistical evaluation was performed by use of the SPSS 29.0 (SPSS Inc., Chicago, IL, USA) for Windows.

Early identification, through clinical (and biological) biomarkers, of long-term responders to immunotherapy is essential for optimizing treatment strategies and improving clinical outcomes.

In our study, low fT3/fT4 ratio was significantly associated with a higher prevalence of SMW, even after adjusting for ECOG PS, preliminarily suggesting that fT3/fT4 ratio may serve as a proxy identifying SMW in patients with advanced NSCLC. Accordingly, recent findings in euthyroid individuals with type 2 diabetes mellitus demonstrated that a low fT3/fT4 ratio was significantly associated with sarcopenia, muscle mass, strength, and physical performance (7). Moreover, a significant inverse association between fT3/fT4 ratio and SMI (even after adjusting for age, metabolic factors, and comorbidities) was evaluated in a large retrospective study across Chinese subjects over 45 years old without cancer (8). In this light, fT3/fT4 ratio may reflect not only metabolic resilience but also systemic alterations associated with disease burden and catabolism, highlighting its potential role in identifying patients at increased risk of SMW and enabling earlier intervention and tailored supportive strategies in lung cancer.

Early detection of SMW is particularly relevant, representing one of the few ideally modifiable prognostic factors in several malignancies, including NSCLC (3). The baseline identification of these patients could facilitate their prompt referral to dedicated, multidisciplinary therapeutic interventions, such as tailored nutritional counselling, personalized exercise programs, and pharmacological approach, aimed at optimizing body composition and thus improving overall prognosis. Furthermore, while no statistically significant correlation was observed between the fT3/fT4 ratio and PFS (probably due to the small sample size), a significant association was identified with OS, suggesting a potential prognostic role for this parameter in immunotherapy-treated NSCLC.

However, our study has some limitations. Firstly, the small sample size may limit the statistical power and generalizability of our findings. Secondly, the exclusive inclusion of patients treated with pembrolizumab monotherapy may restrict the applicability of the results to broader NSCLC populations receiving other immune checkpoint inhibitors or chemoimmunotherapy combinations. Lastly, we assessed only baseline fT3/fT4 ratio values, without evaluating their longitudinal changes over the course of treatment, which could provide further insight into the dynamic relationship between thyroid function, treatment response and clinical outcomes. These limitations, along with our preliminary findings, have prompted us to design a prospective observational study to further investigate the role of the fT3/fT4 ratio in relation to body composition, as well as muscle strength and physical performance, and treatment response in advanced NSCLC. The study will specifically include patients undergoing immunotherapy, either alone or in combination with chemotherapy, to strengthen the clinical applicability of fT3/fT4 ratio. Furthermore, we will monitor longitudinal changes in fT3/fT4 ratio during treatment to evaluate its dynamic association with disease progression, therapeutic response, and muscle mass changes.

fT3/fT4 ratio may represent a simple but reliable proxy for early detection of SMW in NSCLC patients receiving upfront mono-immunotherapy. Given the prognostic relevance of SMW, this parameter could support baseline clinical risk stratification. Further prospective studies are warranted to validate its utility and explore its association with immunotherapy response and long-term outcomes.