Thyroid transcription factor-1 as a prognostic indicator for stage IV lung adenocarcinoma with and without EGFR-sensitizing mutations

Despite several advances in cancer diagnosis and treatment, the prognosis of advanced lung cancer remains poor. In previous studies, patient-related factors such as performance, age, and female sex were identified as independent prognostic indicators of lung cancer stage [1]. Although there are many studies on prognostic biomarkers, most of them did not adjust for previous prognostic factors. Similar to epidermal growth factor receptor (EGFR) mutations, some specific driving mutations for target treatment are not only predictive factors for outcomes but also play a prognostic role [2]. Targeted therapy is a standard treatment modality in lung adenocarcinoma even in elderly patients or those with a poor performance status [3]. As such, more objective independent prognostic biomarkers are needed in clinical practice and in studies on lung cancer; this is particularly true for lung adenocarcinoma because of its increasing incidence worldwide [4].

Thyroid transcription factor (TTF)-1 is a tissue-specific transcription factor that has a homeodomain protein fold and regulates the expression of select genes in the thyroid and lung for embryonic development and differentiation [5]. The importance of TTF-1 extends into adulthood as it plays a critical role in maintaining the normal function of terminal respiratory unit cells by controlling surfactant proteins [6]. TTF-1 is a lineage marker and has been used as a diagnostic marker for lung adenocarcinoma and small cell carcinoma [7]. A subsequent study showed that TTF-1 overexpression was a favorable prognostic marker among patients with lung adenocarcinoma [8]. Anagnostou et al. showed that TTF-1 expression positively impacted the survival of stage I lung adenocarcinoma patients [9]. Although several studies also demonstrated similar results in advanced stage lung adenocarcinoma, there were some limitations such as small sample sizes or an uncontrolled driving mutation status. Chung et al. showed that TTF-1 was an independent prognostic factor among patients treated with EGFR tyrosine kinase inhibitors (TKI). However, they did not control for treatment lines, and EGFR-TKI treatment was not based on the EGFR mutation status [10]. The prognostic significance of TTF-1 among patients with wild-type EGFR adenocarcinoma has not been studied adequately. Moreover, research on the prognosis of patients with TTF-1–negative, EGFR-positive adenocarcinoma is also limited. The good prognostic capability of TTF-1 expression might be due to EGFR-sensitizing mutations as demonstrated by the positive correlation between TTF-1 expression and EGFR mutations [10, 11]. The purpose of this study was to explore the prognostic impact of TTF-1 expression based on the EGFR-sensitizing mutation status in lung adenocarcinoma patients.

In this study, we demonstrated that TTF-1 expression was a good prognostic indicator for OS and PFS in patients with stage IV lung adenocarcinoma regardless of the presence or absence of EGFR mutations. We also confirmed that TTF-1 positivity was strongly correlated with EGFR mutations. However, it is also of note that EGFR mutation positivity and TTF-1 expression negativity did not guarantee a good response of EGFR-TKI.

TTF-1 is a homeodomain nuclear transcription protein of the NKX2 gene family. By binding to specific gene sequences, TTF-1 modulates the transcriptional activation of target genes [5]. TTF-1 is expressed in type II pneumocytes and Clara cells and it regulates the surfactant and Clara cell secretory protein gene expression to maintain normal lung functions [14]. However, the role of TTF-1 in lung cancer pathogenesis and biology is uncertain. Some data suggest that TTF-1 might promote carcinogenesis by enhancing cell proliferation, namely at least adenocarcinoma [15‐17]. The NKX2–1 locus, which encodes TTF-1, is frequently amplified in the lung cancer genome [18]. TTF-1 could be important for the survival of a subset of patients with lung adenocarcinomas expressing TTF-1 based on the lineage-specific dependency model [19]. TTF-1 knockdown via RNA interference in these adenocarcinoma cell lines substantially induced tumor growth inhibition and apoptosis [17, 19]. In contrast, the results of both previous studies and the present study indicate that TTF-1 was associated with prolonged survival in patients with lung adenocarcinoma. There is quite a bit of evidence suggesting that TTF-1 plays paradoxical tumor-suppressive roles. Myosin-binding protein H (MYBPH) is one of the transcriptional targets of TTF-1. MYBPH inhibits actomyosin organization, which in turn reduces single cell motility and increases collective cell migration. MYBPH activation eventually results in inhibition of cancer invasion and metastasis [20]. Winslow et al. showed that TTF-1 downregulation in adenocarcinoma was related to a loss of differentiation and increased metastatic potential [21]. In their mouse model, NKX2–1-negativity was pathognomonic of high-grade, poorly differentiated tumors. NKX2–1 knockdown allowed the formation of more liver nodules after intrasplenic injection and more lung nodules after intravenous transplantation. In lung adenocarcinoma, TTF-1 plays not only an oncogenic role, but also a suppressive role for progression to an invasive condition while maintaining a minimum degree of differentiation paradoxically.

EGFR sensitive mutations have been associated with similar clinical characteristics to TTF-1 positive expression in patients with lung adenocarcinoma, particularly among women and non-smokers. Our data showed a significant correlation between TTF-1 and EGFR mutations, which is consistent with previous findings. Sheffield et al. demonstrated that TTF-1 expression and EGFR mutations were strongly correlated (p < 0.001) [22]. Shanzhi et al. also showed that TTF-1 expression in patients with lung adenocarcinoma was correlated with EGFR mutations [11]. They also demonstrated that the EGFR exon 21 mutation was more significantly correlated with TTF-1 overexpression than the exon 19 mutation. However, our data did not show a significant difference in the correlation of TTF-1 overexpression with specific mutations. From the molecular aspect, Yamaguchi et al. showed that TTF-1 transcriptional activation enhanced and sustained the pro-survival EGFR downstream pathway by inducing receptor tyrosine kinases such as orphan receptor 1 (ROR1) and activating c-Src [23]. They also noted that ROR1 knockdown induced growth inhibition not only of the EGFR sensitive mutation cell line, but also of the first-generation EGFR-TKI resistance T790 M mutation cell line. Recently, Clarke et al. showed that EGFR knockdown led to NKX2–1 upregulation, suggesting a negative feedback loop [16]. In our study, all patients with the acquired T790 M mutation after EGFR-TKI treatment were TTF-1 positive. This could be interpreted that the secondary acquired driving EGFR mutant still needs the TTF-1 pathway signal. Collectively, these findings suggest that beyond the simple clinical correlation, TTF-1 might play an important role in EGFR-driven lung adenocarcinoma oncogenesis and it might be a biomarker of EGFR oncogenic addiction among patients with EGFR mutation-positive lung adenocarcinoma.

TTF-1 expression might be important among patients with an EGFR-sensitizing mutation. In our study, five patients had EGFR mutation-positive and TTF-1-negative adenocarcinoma (Additional file 1: Table S4). Interestingly, these patients were not responsive to EGFR-TKI and their survival prognosis was significantly reduced. They showed rapid progression upon EGFR-TKI treatment. Loss of the anti-metastatic effect of TTF-1 might explain these poor outcomes. Another explanation is that the EGFR mutation may not be an oncogenic driver but a bystander mutation or there might be intra-tumoral heterogeneity in the EGFR mutation status. TTF-1 positivity could be a surrogate for EGFR mutations in driving oncogenicity in lung cancer patients. This might explain why TTF-1 positivity has a more significant prognostic impact than the EGFR mutation status among patients with advanced lung adenocarcinoma in the EGFR-TKI era [24]. Compared with previously documented factors such as good performance status, EGFR mutations, age, sex, smoking status, and distant metastasis, TTF-1 positivity has the most significant prognostic impact in advanced lung adenocarcinoma.

Pemetrexed combination treatment showed better efficacy in patients with non-squamous cell carcinoma than squamous cell carcinoma [25]. However, the mechanisms for such phenomenon are not well documented, and there is no validated predictive marker for pemetrexed treatment outcomes other than the histologic type. One retrospective study demonstrated that TTF-1 expression was a good marker for higher response rates and prolonged PFS and OS in patients with non-squamous, non-small-cell lung cancer [26]. However, these results should be interpreted with caution because they included patients who underwent first-line EGFR-TKI treatment and only examined EGFR mutations in approximately 50% of the studied patients. Moreover, the chemotherapy regimens were not controlled among the patients. The first-line pemetrexed-based chemotherapy was administered to only 21% of patients. Our results did not indicate that TTF-1 was a predictive marker for treatment outcomes following first-line pemetrexed-based therapy among patients with EGFR-negative adenocarcinoma. Recently, Schilsky et al. also showed TTF-1 expression was not predictive of the clinical benefit from pemetrexed-based treatment in patients with adenocarcinoma [24]. Further well-designed prospective studies to evaluate whether TTF-1 is a good response marker for specific chemotherapy regimens should be conducted.

Our present study indicated that TTF-1 expression was a good prognostic indicator for OS and PFS in patients with stage IV lung adenocarcinoma regardless of the presence or absence of EGFR mutations. Moreover, even in EGFR mutation-positive cases, the EGFR mutation might not be the driving oncogene if TTF-1 expression is negative. EGFR-TKI should be used with caution in these patients. These results should be validated further in well-designed prospective studies, particularly molecular studies.