The standard of treatment for non-small cell lung cancer (NSCLC) patients, excluding those with driver mutations, involves the use of immune checkpoint inhibitors. Especially for individuals with high programmed death-ligand 1 (PD-L1) expression, PD-1/PD-L1 inhibitor monotherapy or chemoimmunotherapy is the preferred treatment in most cases. Although both PD-1/PD-L1 inhibitor monotherapy and chemoimmunotherapy are effective first-line treatment regimens for PD-L1 high NSCLC patients, several systematic reviews have suggested that chemoimmunotherapy may be more effective under certain conditions (1, 2). In addition, for non-squamous NSCLC patients, there are also many options for chemoimmunotherapy, including regimens that incorporate pemetrexed and paclitaxel. As a result, determining the optimal treatment for each patient is challenging, and currently, there is no clear distinction between these options.

Thyroid transcription factor-1 (TTF-1), known as a lineage-specific transcription factor, is a gene-regulating protein found in the thyroid, lungs, and brain (3, 4). In normal lung tissues, it is expressed in type II alveolar epithelial and Clara cells and promotes the transcription of surfactant protein genes (5). TTF-1 has been reported to be associated with lung cancer development; hence, TTF-1 is expressed in 60–80% of the cases of non-squamous non-small cell lung cancer (non-Sq NSCLC) and is commonly used for the histological diagnosis of lung cancer (6).

TTF-1 expression has been reported as a potential prognostic factor in patients with NSCLC and has also been previously reported to be significantly associated with therapeutic response to cytotoxic anticancer agents, particularly pemetrexed, with favorable results in TTF-1 positive patients (7, 8). Similarly, several reports on immunotherapy alone or chemoimmunotherapy have indicated that TTF-1-positive non-squamous NSCLC patients tend to have a better therapeutic response (9–11). However, there has been no evaluation of the impact of TTF-1 expression on the therapeutic effect of immunotherapy alone or chemotherapy in patients with NSCLC with high PD-L1 expression who are strongly affected by immunotherapy. Therefore, we evaluated whether TTF-1 has a clinical impact on this population.

This study is one of the largest studies to explore the impact of TTF-1 expression in patients with NSCLC and high PD-L1 expression who have undergone immunotherapy. In this cohort, there were no significant differences in PFS and OS between TTF-1-positive and negative populations. Additionally, we assessed the treatment regimen efficacy and stratified TTF-1 expression (positive/negative). Our findings revealed that chemoimmunotherapy with pemetrexed showed a tendency toward prolonged PFS compared with other regimens, irrespective of TTF-1 expression. Regarding OS, TTF-1-positive patients who received chemotherapy with pemetrexed had prolonged OS compared to the other groups. However, no significant difference in OS was observed in TTF-1-negative patients.

In TTF-1-positive NSCLC, especially in TTF-1-positive lung adenocarcinomas, high differentiation is often observed. Conversely, TTF-1-negative NSCLC cells exhibit lower differentiation and coexist with squamous cell carcinomas derived from atypical mucous column epithelial cells (13). Indeed, in this study, the TTF-1-positive group exhibited a significantly higher proportion of adenocarcinomas and a smaller proportion of non-adenocarcinoma NSCLCs than the TTF-1-negative group. Additionally, non-squamous and non-adeno NSCLCs are associated with a poorer prognosis than adenocarcinoma, suggesting that TTF-1 negative NSCLC has a biologically inferior prognosis compared to TTF-1 positive NSCLC (14). In terms of response to systemic chemotherapy, several previous studies have also indicated that TTF-1-negative NSCLC exhibits a less favorable therapeutic response with pemetrexed-based chemotherapy than TTF-1-positive NSCLC (7, 15, 16). Recent studies have reported that this result also holds true for chemoimmunotherapy (9, 10, 17–20). Actually, our previous study similarly reported a significantly lower therapeutic response to chemoimmunotherapy in the TTF-1-negative population compared to the TTF-1-positive population (11). However, this study, which demonstrated that TTF-1 expression does not affect the therapeutic efficacy of immunotherapy and chemoimmunotherapy, contrasts with the outcomes of previous studies. One hypothesis that could explain these contradictory results is the relationship between TTF-1 and PD-L1 expression levels in advanced NSCLC. In our previous study, we reported that PD-L1 expression was significantly higher in the TTF-1-positive population than in the TTF-1-negative population (11). This association between PD-L1 and TTF-1 expression levels has been reported in several other studies with the same trend (21, 22). It is therefore possible that one of the reasons for the low response to ICI treatment in the overall TTF-1-negative population was influenced by the relatively low PD-L1 expression. Consequently, it is possible that in this study, which was limited to PD-L1-high expressing patients with high efficacy of immunotherapy, there was no difference in response to ICI monotherapy or chemoimmunotherapy, regardless of TTF-1 status.

In this study, regardless of TTF-1 expression, chemoimmunotherapy with pemetrexed tended to prolong PFS compared to chemoimmunotherapy without pemetrexed. This result contradicts previous reports suggesting that conventional pemetrexed-based chemotherapy without immunotherapy results in shorter PFS and OS in TTF-1-negative populations. Several factors may have contributed to this discrepancy. First, there may be differences in the anti-tumor efficacies of immune checkpoint inhibitors. In this study, pemetrexed regimens included platinum + pemetrexed + pembrolizumab, whereas pemetrexed-free chemoimmunotherapy included mostly atezolizumab-based regimens, with the rest being pembrolizumab-based. Multiple immune checkpoint inhibitors, each with different anti-tumor effects, have been reported in clinical trials (23, 24). Second, pemetrexed, a cytotoxic anticancer agent, may have adjunctive effects that enhance the efficacy of immunotherapy. Pemetrexed induces the transcriptional activation of PD-L1 (encoded by CD274) by deactivating thymidylate synthase (TS) in NSCLC cells (25). This in turn activates T lymphocytes when combined with anti-PD-1/PD-L1 therapy. Compared with other anticancer agents, pemetrexed has a more pronounced effect on PD-1/PD-L1 inhibition. Especially in populations more likely to benefit from immunotherapy, such as those with high PD-L1 expression, the adjunctive effect of activating immunotherapy may have a greater impact than the reduced therapeutic effect of pemetrexed owing to TTF-1 negativity. Further large-scale studies are required to confirm these hypotheses.

This study had several limitations. Firstly, this was a retrospective study conducted only in Japan, and various confounding factors may have been involved. Specifically, the comparison of treatment regimens for each TTF-1 expression group, especially for chemoimmunotherapy, involved a few patients, precluding a comprehensive multivariate analysis. Second, despite propensity score matching, unknown factors may have influenced the results, making it challenging to completely align patient backgrounds based on TTF-1 expression.

In patients with NSCLC and high PD-L1 expression, tumor TTF-1 expression did not affect the therapeutic efficacy of PD-1 inhibitor monotherapy or chemoimmunotherapy. Moreover, while previous reports have indicated significant variations in the therapeutic effect of pemetrexed based on TTF-1 expression, the current study suggests that chemoimmunotherapy, including pemetrexed, may exhibit superior therapeutic efficacy compared to other regimens, irrespective of TTF-1 expression.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by The Ethics Review Board of Kyoto Prefectural University of Medicine. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin because We implemented an opt-out approach, and therefore, direct informed consent was not conducted. Participants were given the option to decline participation.

NN: Writing – original draft, Conceptualization, Investigation, Methodology, Software, Validation, Formal analysis. HK: Data curation, Writing – review & editing, Resources. TY: Writing – review & editing, Formal analysis, Investigation, Methodology, Project administration, Validation. MT: Resources, Writing – review & editing. YN: Resources, Writing – review & editing. YG: Resources, Writing – review & editing. AN: Resources, Writing – review & editing. SS: Resources, Writing – review & editing. KKT: Resources, Writing – review & editing. TT: Resources, Writing – review & editing. AO: Resources, Writing – review & editing. TH: Resources, Writing – review & editing. KD: Resources, Writing – review & editing. YC: Resources, Writing – review & editing. IH: Resources, Writing – review & editing. NT: Resources, Writing – review & editing. TM: Resources, Writing – review & editing. NS: Resources, Writing – review & editing. MIs: Resources, Writing – review & editing. YK: Resources, Writing – review & editing. KM: Resources, Writing – review & editing. MIw: Resources, Writing – review & editing. ST: Resources, Writing – review & editing. TK: Resources, Supervision, Writing – review & editing. KCT: Resources, Supervision, Writing – review & editing.