TTF-1 and p40 co-expression defines a distinct subtype of non-small cell lung cancer with frequent TP53 mutations and FGFR pathway dysregulation

Introduction

Lung cancer (LC) is the leading cause of cancer-related deaths and the second most common malignancy worldwide (1). Non-small cell lung cancers (NSCLCs), which account for approximately 85% of all cases, are subdivided into several pathological types, with adenocarcinoma (ADC) and squamous cell carcinoma (SCC) being the most common (2-7). In recent years, detailed analyses of the molecular pathogenesis of NSCLC have led to the identification of specific molecular targets for therapeutic intervention. Consequently, routine diagnostic practices for NSCLC now include molecular testing for EGFR, KRAS, ALK, ROS1, BRAF, NTRK1–3, NRG1, ERBB2, and MET in ADC cases, along with assessment of PD-L1 expression in both subtypes (8). This approach enables the application of personalized treatment strategies (5,9-11).

According to World Health Organization (WHO) guidelines, the diagnosis of lung cancer is based on morphological evaluation supplemented by immunohistochemical (IHC) staining of tumor samples (Table 1) (11-16). Thyroid transcription factor-1 (TTF-1) and p40 protein have emerged as key markers for distinguishing ADC and SCC, respectively (11,17-19). In addition to diagnosing SCC based on the unequivocal presence of keratinization and/or intercellular bridges, SCC can also be diagnosed when p40 positivity and TTF-1 negativity are observed (18). Conversely, in the absence of glandular structures or mucin production characteristic of glandular differentiation, ADC can be diagnosed when tumor cells are positive for TTF-1 and negative for p40 expression (11,18).

Although the WHO classification describes TTF-1 and p40 expression as typically mutually exclusive in NSCLC, co-expression within individual tumor cells has been documented, presenting a significant diagnostic dilemma (11,20-28). Current guidelines recognize that double positivity may occur and usually favors a diagnosis of ADC, although the possibility of adenosquamous carcinoma should also be considered, but only if two distinct tumor components are identified. Nevertheless, recommendations on the interpretation of such findings remain vague, emphasizing the importance of careful evaluation of staining extent and intensity to avoid misclassification explicit recommendations for interpreting such findings remain limited, necessitating careful evaluation of the extent and intensity of staining to mitigate the risk of diagnostic misclassification (11,27,28). The biological and clinical significance of TTF-1/p40 co-expression remains unclear, and its implications for diagnosis and treatment have not been fully explored. Therefore, the aim of this study was to perform a comprehensive clinicopathological, immunophenotypic, and molecular characterization of NSCLC cases exhibiting dual expression of p40 and TTF-1. We present this article in accordance with the REMARK reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-690/rc).

Methods

Sample selection and study design

A retrospective search of the patient database at the Central Clinical Hospital of the Medical University of Łódź was conducted to identify lung cancer cases diagnosed between May 2021 and November 2022. The inclusion criteria were histopathological confirmation of NSCLC, treatment-naïve status; and availability of p40 and TTF-1 IHC staining performed during routine diagnostics or sufficient archival material to perform these analyses. Cases were excluded if the diagnosis was other than NSCLC, the diagnosis of adenosquamous carcinoma, insufficient tissue was available for additional analyses, or p40 and TTF-1 staining could not be performed due to specimen limitations (e.g., cytology cell blocks reserved for potential future clinical use). Detailed demographic, clinicopathological, and IHC data were collected for all patients. Pathological review and characterization were performed according to the 5th edition of the World Health Organization Classification of Thoracic Tumors (11). The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Bioethical Committee at the Collegium Medicum in Bydgoszcz, Nicolaus, Copernicus University in Toruń (KB 395/2024) and individual consent for this retrospective analysis was waived.

Immunohistochemistry (IHC) and digitalization

IHC analysis was performed as part of the routine diagnostic workflow to evaluate the expression of thyroid transcription factor-1 (TTF-1; clone 8G7G3, Dako/Agilent) and p40 (clone BC28, Dako/Agilent). Tumors were classified as double expressors if ≥5% of tumor cells demonstrated co-expression of TTF-1 and p40 within overlapping tumor regions, and were allocated to the subgroup of strong expressors if >10% of tumor cells were co-positive. Entrapped non-neoplastic cells were excluded based on morphological criteria. To ensure accurate identification of double-positive cells, slides stained for p40 and TTF-1 were digitally aligned to enable visual overlay and evaluation of marker co-localization within the same tumor cells. The threshold for positivity was selected to ensure methodological consistency and biological relevance, based on prior evidence, particularly for TTF-1 (21,27,28). IHC interpretation was independently performed by two investigators (A.I.M., J.S.) and verified by two board-certified pathologists (M.B., R.K.).

Additional IHC staining was conducted for p53 (clone DO-7, Dako/Agilent), p63 (clone DAK-p63, Dako/Agilent), PD-L1 (clone 22C3, Dako/Agilent), and FGFR1–4 using 4 µm formalin-fixed, paraffin-embedded (FFPE) tumor sections following standardized protocols. Primary antibodies used for FGFR detection were: FGFR1 (NB100-2080, Novus Biologicals, USA; pH 6.0, 1:100), FGFR2 (H00002263-M01, Abnova, Taiwan; pH 6.0, 1:600), FGFR3 (B-9: sc-13121, Santa Cruz Biotechnology; pH 6.0, 1:50), and FGFR4 (M00769-2, Boster Bio, USA; pH 6.0, 1:500). Gastric and lobular breast ADCs were used as positive controls, while lymph node tissue served as the negative control. FGFR1–4 expression was semi-quantitatively scored using the H-score method (range, 0–300) and categorized as low or high based on tercile distribution.

Slide scanning, image acquisition, IHC review, and figure preparation were performed using the PANNORAMIC 1000 FLASH DX scanner, SlideCenter, and SlideViewer software (3DHISTECH Ltd., Budapest, Hungary), and the MedLAN Slide Viewer (MSVv2.0b, MedLAN, Białystok, Poland).

Next-generation sequencing (NGS)

Tumor dissection from FFPE blocks was performed using the TMA Master II system (3DHISTECH Ltd., Budapest, Hungary). DNA and RNA extraction and purification were carried out with the Ion Torrent Genexus FFPE DNA and RNA Purification Kit (Thermo Fisher Scientific; Part No. A45539), automated on the GPI system across 14 runs, with integrated fluorometric quantification via the built-in Qubit device.

Samples yielding ≥5 ng of nucleic acid were transferred to the Ion Torrent Genexus Integrated Sequencer (Thermo Fisher Scientific) for targeted NGS using the Oncomine™ Precision Assay GX. This panel enables simultaneous DNA- and RNA-based detection of single nucleotide variants (SNVs), insertions, deletions, copy number variants (CNVs), and gene fusions, although it does not capture large rearrangements involving entire exons or multiple exons. The minimum variant allele frequency thresholds were 2.5% for SNVs and 2.0% for insertions/deletions, with a reported sensitivity of 99–100%.

Data processing was performed using the Genexus integrated bioinformatics pipeline. The assay covers alterations in: AKT1, AKT2, AKT3, ARAF, CDK4, CHEK2, CTNNB1, ERBB4, FGFR4, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, MAP2K1, MAP2K2, MTOR, NRAS, PDGFRA, RAF1, SMO, TP53, CD274, CDKN2A, ERBB2, ERBB3, KRAS, PIK3CA, PTEN, NTRK2, NTRK3, NUTM1, RET, ROS1, RSPO2, RSPO3, ALK, ESR1, FGFR1, FGFR2, FGFR3, NRG1, NTRK1, AR, BRAF, EGFR, and MET.

In-silico validation

To contextualize findings, in-silico analysis was performed using publicly available NSCLC datasets from The Cancer Genome Atlas (TCGA; https://portal.gdc.cancer.gov/). mRNA expression data were downloaded from cBioPortal (https://www.cbioportal.org/) and analyzed as z-scores relative to all samples (log RNA Seq V2 RSEM) (29).

Statistical analysis

Patient demographics and clinical characteristics were summarized as mean ± standard deviation (SD) for continuous variables and as frequencies with percentages for categorical variables. For clinical outcome analyses, continuous data were reported as median with interquartile range (IQR). Distribution normality was assessed using the Shapiro-Wilk test. Group comparisons for continuous variables were performed using the Mann-Whitney U test for non-normally distributed data or Student’s t-test for normally distributed data. Categorical variables were compared using Pearson’s Chi-squared test. Associations between TP53 mutation status and overall survival (OS) were evaluated using univariable Cox proportional hazards models. All statistical analyses were performed using STATISTICA 13.1 (Dell Inc., Round Rock, TX, USA). A P value of <0.05 was considered statistically significant.

Results

Study cohort overview

Following application of the inclusion and exclusion criteria, 94 patients were included in the final study cohort (Figure S1). Among these, 18 cases (19.1%) demonstrated co-expression of TTF-1 and p40 (Figure 1; Figures S2-S19), with 8 classified as strong co-expressors and 10 as weak co-expressors (available online: https://cdn.amegroups.cn/static/public/tlcr-2025-690-1.xlsx). The remaining 76 cases (80.9%) exhibited immunoreactivity for only one of the two markers.

Figure 1 Example of p40 and TTF-1 co-expression in the same tumor areas from patient No. 16. (A,B) p40 immunostaining at 100× and 400× magnification, respectively. (C,D) TTF-1 immunostaining at 100× and 400× magnification, respectively. TTF-1, thyroid transcription factor 1.

Clinicopathological features and follow-up of p40/TTF-1 double expressors compared to other NSCLC cases

The double-expressing group consisted of 18 patients with a median age of 67.5 years (IQR, 65–70 years). Twelve patients (66.7%) reported a history of smoking exceeding 20 pack-years. The cohort included 8 women (44.4%) and 10 men (55.6%). Histologically, 11 tumors (61.1%) were classified as ADC and 7 (38.9%) as SCC. The median primary tumor size (assessed macroscopically or radiologically when pathologic evaluation was not available) was 3.0 cm (IQR, 1.6–4.0 cm). Pleural invasion was identified in 5 cases [out of 14 with reported pleural invasion (PL) status, 35.7%], lymph node metastases in 6 [out of 14 with reported metastasis to lymph nodes (pN) status, 42.8%], and distant metastases in 4 (out of 18 with reported M status, 22.2%). The distribution of clinical stages was as follows: stage I in 7/15 patients (46.7%), stage II in 2/15 (13.3%), stage III in 2/15 (13.3%), and stage IV in 4/15 (26.7%).

A significantly higher prevalence of solid growth patterns was observed in the double-expressing group, particularly among ADCs, representing a distinctive histopathological feature of this subset (Table 2). No significant differences were identified for other clinicopathological parameters. OS did not differ significantly between double expressors and non-double expressors [hazard ratio (HR): 0.77; 95% confidence interval (CI): 0.32–1.84; P=0.56, log-rank test; Figure 2]. Subgroup analyses stratified by histological subtype similarly showed no OS difference: for ADC cases, HR 1.18 (95% CI: 0.43–3.24; P=0.75), and for SCC cases, HR 0.27 (95% CI: 0.04–2.07; P=0.18), with non-double expressors serving as the reference group.

Figure 2 Kaplan-Meier curve showing OS probability stratified by p40/TTF-1 expression status, comparing double expressor and non-double expressor non-small cell lung carcinoma cases. The P value was calculated using the log-rank test. OS, overall survival; TTF-1, thyroid transcription factor 1.

Morphological, immunohistochemical and molecular characterization of p40/TTF-1 double expressors

Morphologically, the double-expressing tumors included seven SCCs and eleven ADCs (Figures S2-S19). Among SCCs, four were non-keratinizing and three were keratinizing. Solid components were present in 10 of the 11 ADC cases (Table 2). In all ADC cases with a solid component, co-expression of p40 and TTF-1 was predominantly observed within the solid areas. In terms of immunoreactivity, TTF-1 expression predominated over p40 in 11 tumors, p40 predominated in five tumors, and equivalent expression was observed in two cases (available online: https://cdn.amegroups.cn/static/public/tlcr-2025-690-1.xlsx).

Extended IHC profiling—including p63 (full-length isoform), napsin A, and mucicarmine—demonstrated strong p63 positivity in 10 cases, including seven tumors classified histologically as ADCs. Napsin A showed partial or weak positivity in nine cases, including two SCCs, while mucicarmine staining was positive in three ADCs. Regarding PD-L1 expression, the median combined positive score (CPS) was 40 (IQR, 15–60), and the median tumor proportion score (TPS) was 30 (IQR, 10–40) (Figure S20) (30). CPS and TPS values were numerically higher among ADCs compared to SCCs, though differences did not reach statistical significance (P=0.17 and P=0.09, respectively).

NGS was successfully performed on 15 of the 18 cases and another two had prior NGS data from clinical care. In one case, sequencing failed due to low nucleic acid quality and could not be repeated due to insufficient material. Key pathogenic variants were detected in 15 cases. Recurrent alterations included mutations in TP53, KRAS, EGFR, and FGFR1–3. Four tumors harbored clinically actionable alterations: two classic EGFR mutations [exon 19 deletion (NM_005228.5; c.2235_2249del; p.Glu746_Ala750del) and exon 21 L858R mutation (NM_005228.5: c.2573T>G; p.Leu858Arg)], one KRAS G12C mutation (NM_004985.5: c.34G>T; p.Gly12Cys), and one MET exon 14 skipping mutation (31).

Pathogenic or likely pathogenic TP53 mutations were detected in 11 of 17 evaluable cases (64.7%), non-druggable KRAS mutations (p.Gly13Cys and p.Gly12Asp) in four (23.5%), and FGFR1–3 alterations in four (23.5%). Notably, two tumors exhibited FGFR1 amplification, one had a pathogenic FGFR3 mutation (NM_000142.5: c.743G>A; p.Arg248His), and one showed a FGFR2 splice site variant of unknown significance (c.2302-1G>T). No pathogenic mutations were detected in two SCC cases.

Notably, most of the reported features—aberrant p53 expression, strong p63 expression, TP53 mutations, and FGFR alterations—were enriched in the subgroup of strong double expressors (available online: https://cdn.amegroups.cn/static/public/tlcr-2025-690-1.xlsx). Based on these molecular findings, three aspects were prioritized for further analysis: strong p63 expression across the cohort, the high frequency of TP53 mutations, and the presence of FGFR1–3 alterations.

Impact of TP53 mutational status and p63 expression on survival of p40/TTF-1 double expressor NSCLC

Strong p63 expression was observed in 10 of 18 p40/TTF-1 double expressor tumors; however, it was not associated with differences in OS compared to cases with weak or absent p63 expression (P=0.85). To validate the findings, in silico analysis was performed using data from 995 NSCLC patients retrieved from TCGA database. Patients were stratified based on the co-expression of TTF-1 and TP63 (the gene encoding both p40 and p63 proteins) into two categories: high TTF-1/TP63 mRNA co-expression, defined as expression levels above the third tercile (n=152) and all remaining cases without high co-expression (n=843). Consistent with our findings, no significant difference in OS was observed between cases with high TTF-1/TP63 co-expression and the rest of the cohort (log-rank P=0.72).

Given the high prevalence of TP53 mutations, we hypothesized that TP53 alterations may contribute to the biology of p40/TTF-1 co-expressing tumors. However, TP53 mutational status was not significantly associated with OS within the cohort (P=0.12). IHC analysis of p53 protein expression revealed that three tumors exhibited a wild-type staining pattern, while 13 showed either complete absence (null) or strong overexpression patterns. However, p53 IHC status was not prognostic (log-rank P=0.99).

This hypothesis was further examined using TCGA data. Interestingly, this analysis revealed that in cases with high TTF-1/TP63 co-expression, TP53 mutations were associated with significantly poorer OS (log-rank P=0.004; HR =0.29, 95% CI: 0.12–0.68; Figure 3A). No such association was observed in cases without high TTF-1/TP63 co-expression (log-rank P=0.33; HR =0.90, 95% CI: 0.72–1.15; Figure 3B).

Figure 3 Kaplan-Meier curves for overall survival comparing the prognostic value of TP53 mutation status in: (A) the group with high TTF-1/TP63 co-expression (serving as a surrogate for p40/TTF-1 double expressors), and (B) the control group without high TTF-1/TP63 co-expression; P values were calculated using the log-rank test. TTF-1, thyroid transcription factor 1.

FGFR1–4 alterations and expression in p40/TTF-1 double expressor NSCLC

Given the frequency of FGFR1–3 alterations and the emerging role of FGF-FGFR signaling in NSCLC, we further evaluated FGFR1–4 protein expression by IHC. All FGFR family members showed higher expression levels in tumor tissue compared to adjacent non-neoplastic lung parenchyma (Figure 4). FGFR4 was most highly expressed, followed by FGFR1 and FGFR2, with FGFR3 demonstrating the lowest expression levels. Median H-scores (IQRs) were: FGFR1—106 [65–165]; FGFR2—77.5 [29–150]; FGFR3—15 [5–35]; FGFR4—210 [175–250].

Figure 4 Representative immunohistochemical images of FGFR1–4 protein expression in lung tissue (A,C,E,G) adjacent to NSCLC (B,D,F,H), shown at 200× magnification. FGFR1–4, fibroblast growth factor receptor 1–4; NSCLC, non-small cell lung cancer.

No significant differences in FGFR1–4 expression was observed between ADC and SCC cases, nor were FGFR expression levels associated with TP53 mutational status, p53 IHC patterns, or p63 expression (Mann-Whitney U test P>0.15 for all comparisons). Clinically, high FGFR1 expression was more frequently associated with lymph node metastases (6/10 FGFR1-high vs. 0/4 FGFR1-low cases; P=0.07). Additionally, FGFR3 expression positively correlated with PD-L1 CPS (R=0.49; P=0.03).

Patients with high FGFR1 expression and/or FGFR1 amplification showed a trend toward poorer OS compared to those without these alterations (P=0.09; Figure 5). No survival relationships were identified for FGFR2–4 alterations.

Figure 5 Kaplan-Meier curve showing OS probability among p40/TTF-1 double expressor NSCLC cases, comparing patients with FGFR1 overexpression and/or amplification (FGFR1alt) to those without these alterations (FGFR1wt). The P value was calculated using the log-rank test. FGFR, fibroblast growth factor receptor; NSCLC, non-small cell lung cancer; OS, overall survival; wt, wild-type.

Discussion

In this study, we conducted a comprehensive clinicopathological, IHC, and molecular analysis of consecutive NSCLC cases, with a specific focus on tumors co-expressing p40 and TTF-1. We identified 18 such cases, demonstrating that p40/TTF-1 co-expression, while uncommon, is not exceedingly rare in diagnostic practice. These tumors exhibited distinct histological and molecular features that may differentiate them from conventional NSCLC subtypes (11).

The relatively high number of double expressors identified in our cohort may reflect differences in diagnostic workflows. In routine practice, p40 and TTF-1 IHC are not systematically applied, particularly in cases with clear morphological features. In addition, low-level expression of one marker may be underrecognized when overshadowed by strong expression of the other (27,28). Furthermore, in our study, all tumors containing cancerous cells co-expressing both markers—including those with limited or subclonal co-expression—were included. Double staining was confirmed by demonstrating nuclear co-localization of p40 and TTF-1 using multiple approaches, including digital slide alignment and visual overlay. To account for the variability among double expressors, we stratified tumors into weak and strong expressors using criteria consistent with the 5th edition WHO classification (11).

Histologically, a significantly higher frequency of solid growth patterns was observed among double-expressing ADCs. This finding is consistent with previous reports describing such tumors as poorly differentiated carcinomas (20,21,23,24). Extended IHC profiling revealed strong p63 expression in the majority of double positive cases, including those classified as ADC, whereas napsin A and mucicarmine staining were frequently weak or absent. PD-L1 expression was variable, with numerically higher scores in ADC cases, though the differences were not statistically significant.

NGS analysis revealed that almost 90% of tumors with TTF-1/p40 co-expression harbor pathogenic genetic alterations, supporting the necessity of molecular testing in such cases, irrespective of histological subtype (Table S1). KRAS mutations were detected in 24% of cases, including p.G12C, p.G13C, p.G12D, and p.G12S variants. Interestingly, half of these mutations occurred in SCC cases, despite KRAS alterations being considered rare in SCC (32,33). EGFR mutations were identified in two cases (12%), both classified as ADC, involving the classical exon 19 deletion (E746_A750del) and L858R point mutation—both well-established oncogenic drivers, particularly in Western populations (31,32).

The most frequently altered gene was TP53, mutated in 11/17 tumors (65%). This finding aligns with previous study reporting TP53 mutations in 60–80% of SCCs and 40–50% of ADCs, often co-occurring with KRAS mutations in the latter (34). All seven cases of SCC in our cohort harbored TP53 mutations, and three of the four KRAS-mutant cases demonstrated concurrent TP53 alterations. IHC assessment of p53 supported these findings, with most tumors exhibiting abnormal staining patterns consistent with underlying mutations. Further in silico analysis using TCGA data revealed that TP53 mutations were associated with worse OS specifically among tumors with high TTF-1/TP63 co-expression, reinforcing the pathogenic significance of TP53 alterations in this subset.

At the mechanistic level, TP53 is known to negatively regulate ΔNp63, an isoform of TP63 encoding the p40 protein. Loss of p53 function may facilitate ΔNp63 overexpression, impairing wild-type p53 transcriptional activity (35). This distribution parallels the diffuse p63 immunoreactivity observed in our cases. Furthermore, TAp63 - another isoform of TP63 gene encoding p63 protein is expressed in germ cells, generally more widely compared to the shorter ΔNp63 isoform which is predominantly expressed in basal and stratifying epithelia (34), consistent with the diffuse p63 immunoreactivity observed in our series. TP53 mutations may also affect the tumor microenvironment. Loss of p53 function has been linked to M1-to-M2 macrophage polarization, a transition further facilitated by the loss of LXRα/β receptors (36). This microenvironmental shift has been implicated in the development of lung tumors co-expressing TTF-1 and p63.

FGFR1–4 expression and genetic alterations were more prevalent in the p40/TTF-1 double-expressing group compared to the largest published study (37). High FGFR1 expression was associated with lymph node metastases and showed a trend toward poorer OS, while FGFR3 expression correlated positively with PD-L1 CPS scores—findings indicative of adverse prognostic significance, consistent with prior study (37). FGFR alterations frequently co-occur with TP53 mutations, particularly in SCC (37,38), supporting a potential mechanistic relationship. Notably, FGFR1 activation has been implicated in the development of TTF-1/p40 co-expressing tumors, particularly when occurring alongside ADC-type genomic alterations. FGFR signaling can activate transcription factors regulating lineage-specific genes, including NKX2-1 (encoding TTF-1), potentially promoting ADC differentiation pathways (37,39).

Notably, the key molecular and immunohistochemical features in our cohort were enriched among strong double expressors. Combined with previous studies suggesting that NSCLCs co-expressing TTF-1 and p40 may arise from basal reserve cells of the terminal respiratory unit (22,40), our findings support a model in which TP53 mutations in such progenitor cells enable aberrant expression of both p63 and TTF-1. This hypothetical mechanism is illustrated in Figure 6.

Figure 6 Hypothetical mechanistic explanation of p40/TTF-1 co-expression in NSCLC. This schematic illustrates a proposed model in which NSCLCs with dual p40/TTF-1 expression may originate from basal reserve cells of the terminal respiratory unit. TP53 mutation leads to deregulation of p63, promoting p40 expression. Concurrently, FGFR signaling and mutated p53 may support aberrant TTF-1 expression. These combined alterations facilitate p40/TTF-1 co-expression within the same tumor cell population. The tumor microenvironment may contribute to this phenotype through macrophage polarization from M1 to M2 states, driven by the loss of LXRα/β receptors and further enhanced by FGFR signaling. This model highlights both intrinsic and microenvironmental factors that may underpin the biphenotypic differentiation observed in this rare NSCLC subset. Figure was created with BioRender.com (license to J.S.). FGFR, fibroblast growth factor receptor; NSCLC, non-small cell lung cancer; TTF-1, thyroid transcription factor 1.

This study has several limitations. It was a retrospective, single-center analysis with a limited number of identified cases, inherently limiting statistical power and increasing the potential for false-negative results. Case selection was based on the availability of p40 and TTF-1 staining or sufficient tissue for additional staining, introducing possible selection bias toward atypical or larger specimens. The retrospective design also limited control over confounding factors such as adjuvant therapies and baseline comorbidities. The definition of double expressors relied on a 5% cut-off, selected based on prior validation studies (27,28), but nonetheless arbitrary. To account for variability in expression levels, we introduced a stratified classification into weak and strong expressors to better reflect the biological spectrum of this phenomenon. To improve technical precision, all stained slides were digitally aligned to enable visual overlay and single-cell co-localization analysis, reducing the risk of false interpretation due to adjacent but non-overlapping staining. Additionally, the targeted NGS panel used may have missed rare genomic rearrangements, gene fusions outside the assay’s scope, or epigenetic events. Survival analysis was restricted to univariable modeling due to sample size limitations. Lastly, although external validation was attempted using TCGA RNA expression data, mRNA levels may not perfectly reflect protein co-expression patterns, and true double expressors could not be definitively confirmed. Nevertheless, to our knowledge, this represents the largest and most comprehensively characterized cohort of TTF-1/p40 double-positive NSCLC cases reported to date, providing novel insights into their clinicopathological and molecular features.

Conclusions

In conclusion, NSCLCs co-expressing p40 and TTF-1 are uncommon but likely underrecognized tumors that pose diagnostic and therapeutic challenges—particularly when accounting for cases with focal co-expression, which represents a key strength of this study. Rather than aiming to reclassify these tumors, our objective was to assess whether even limited co-expression correlates with specific molecular or clinicopathological features. The frequent presence of TP53 mutations, FGFR pathway alterations, and mixed immunophenotypic profiles supports their biological distinctiveness. From a diagnostic perspective, reporting this phenomenon—even when present in a small proportion of tumor cells—and classifying such cases as NSCLC, not otherwise specified, may be more appropriate in biopsy material than assigning them strictly as ADC or SCC. Recognition of this distinct subgroup may have important implications for diagnostic workflows and therapeutic strategies, particularly if future studies validate the proposed model of biphasic differentiation driven by TP53 mutations.

Acknowledgments

We are grateful Sysmex/3DHistech (M. Bobrowski, A. Najdowski) and MedLAN (W. Mytnik) for technical assistance.

Footnote

Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-690/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-690/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-690/prf

Funding: This study was supported by the Medical University of Łódź (grant No. 503/1-034-03/503-11-001, to M.B.) and by a scientific grant for the best students at the Medical University of Łódź (grant No. 564/1-000-00/564-20-082, to G.J.S.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-690/coif). J.J. serves as an unpaid editorial board member of Translational Lung Cancer Research from October 2025 to September 2027. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Bioethical Committee at the Collegium Medicum in Bydgoszcz, Nicolaus, Copernicus University in Toruń (KB 395/2024) and individual consent for this retrospective analysis was waived.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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