A total of 101 patients with NSCLC at peripheral stage I (T1N0M0) underwent surgical resection in the Unit of Thoracic Surgery in the Department of Surgical, Medical, Molecular Pathology and Critical Care at Pisa University (Pisa, Italy), and were retrospectively selected. Histological diagnoses were formulated by two pathologists independently (G.F. and A.S.), in accordance with the World Health Organization classification (20,21). Clinicopathological characteristics were noted in all cases. Written informed consent was obtained from each patient for tissue collection and molecular analysis.

Alignment of miRNAs with the TPL2 target gene was predicted using the microRNA target prediction programs microRNA.org (Harvard Medical School, Boston, MA, USA), MicroCosm Targets 5.0 (The European Bioinformatics Institute) and PITA (Weizmann Institute of Science).

Following standard deparaffinization and manual tumor macrodissection of the areas with prevalent adenocarcinomas, the miRNeasy formalin-fixed and paraffin-embedded (FFPE) kit (Qiagen GmbH, Hilden, Germany) was used to isolate total RNA, including miRNAs, from 5 µm sections of FFPE tissues, according to the manufacturer's protocol. A total of 600 ng of total RNA was used to synthesize cDNA using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a reaction volume of 20 µl.

Quantification of the mRNA expression of TPL2 was performed in triplicate using the Rotor Gene SYBR Green PCR kit (Qiagen GmbH) on a Rotor Gene 6000 (Qiagen GmbH). The following primers used for RT-qPCR are as follows: TPL2, forward: 5′-TAATCCACAAGCAAGCACCTC-3′ and reverse: 5′-TGATTGGGGTTTCTCTCCAG-3′); β-actin, forward: 5′-CCAACCGCGAGAAGATGA-3′ and reverse: 5′-CCAGAGGCGTACAGGGATAG-3′. Specific TaqMan^® MicroRNA assays (Applied Biosystems; Thermo Fisher Scientific, Inc.) were used to amplify miR-21 and RNU6B, according to the manufacturer's protocol. In addition, the threshold cycle and baselines were determined in an identical manner. The expression was calculated by relative quantification using β-actin and RNU6B as reference controls for TPL2 and miR-21, respectively. Fold expression changes were determined by the 2^−∆∆Cq method (22) using a pool of 12 non-cancerous tissues as a calibrator group. The analysis was performed using the DataAssist™ software (Applied Biosystems; Thermo Fisher Scientific, Inc.).

The protein expression of TPL2 was assessed in FFPE tissue samples using immunohistochemistry (IHC). The Cot (M-20) anti-TPL2 rabbit polyclonal antibody (dilution, 1:50; cat. no. sc-720, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was incubated at 37°C for 36 min to detect the protein. The avidin-biotin peroxidase method was used by developing the immunoreaction with diaminobenzidine. Simultaneous staining of a known TPL2-positive case was used as a positive control. Incubation of parallel slides omitting the first antibody was performed as a negative control. The number of TPL2 immunoreactive cells was determined by scoring a minimum of five high-power fields (magnification, ×40) and counting the number of immunoreactive cells out of the total epithelial cells analyzed in each field of view.

A proportion score (PS) was assigned by representing the estimated proportion of positively stained cells, and was divided into three groups: negative (score 0), 1–60% (score 1) and >60% (score 2). An intensity score (IS) was also assigned and divided into four classes: None (score 0), weak (score 1), intermediate (score 2) and strong (score 3). A total score was obtained from the sum of the PS and IS, and was graded into two classes: High (4–5, with a higher expression of TPL2) and low (0–3, with a lower or negative TPL2 expression).

One-way analysis of variance and chi-squared tests were used to determine the association between the expression of TPL2, miR-21 levels and the clinicopathological parameters. Survival analyses were performed using the Kaplan-Meier method, along with the log-rank test and the Cox proportional hazard model. Statistical analyses were performed using the JMP10 software (SAS Institute, Inc., Cary, NC, USA). P<0.05 was considered to indicate a statistically significant difference.

The present study was performed using 101 patients with early-stage NSCLC (71 males and 30 females), including 54 patients with adenocarcinoma (ADC) and 47 patients with squamous cell carcinoma (SCC). Regarding their histological classification, different histological subtypes of ADC were identified: Acinar (19/54, 35.2%), lepidic (18/54, 33.3%), mucinous (9/54, 16.7%), papillary (6/54, 11.1%) and solid (2/54, 3.7%). The median age at diagnosis was 67 years (range, 49–81; mean, 67.03 years), and the median follow-up was 63 months (range, 6–161 months). In terms of tumor grade, five tumors were G1 phase, 71 tumors were G2 phase and 25 were G3 phase. Disease progression and mortality from lung cancer were observed in 27 patients with ADC (26.7%) and 20 with SCC (19.8%) of the 101 patients with NSCLC, respectively. The median progression-free survival and the overall survival were 54 months (95% CI: 6–161) and 68 months (95% CI: 11–161), respectively. Regarding smoking habits of the entire patient group, there were 15 non-smokers, 44 former smokers and 42 current smokers (Table I).

TPL2 mRNA expression was quantified in 101 NSCLC tissues and in 12 non-cancerous tissues, and was normalized against the β-actin housekeeping gene, using RT-qPCR. The samples were divided into high and low expression groups based on the median fold-change value (0.815 for TPL2). TPL2 mRNA expression was low in 52/101 (51%) cases and high in 49/101 (49%) cases (data not shown). The present study determined whether TPL2 expression was correlated with the predominant clinicopathological characteristics. No statistically significant associations were observed between TPL2 mRNA expression and any of the predominant clinicopathological characteristics of the patients with NSCLC (Table I). However, focusing on the difference in TPL2 levels among the different subtypes of the 54 adenocarcinomas, it was identified that TPL2 mRNA expression was high in 6/19 (31.6%) acinar, 13/18 (72.3%) lepidic, 5/9 (55.6%) mucinous, 3/6 (50%) papillary and 1/2 (50%) solid subtypes, with a significantly higher level in lepidic when compared with all other subtypes (Chi-squared test, P=0.012; data not shown).

Using the 2-level (high/low) immunohistochemical scoring system, TPL2 protein expression was low in 45/101 (44%) and high in 56/101 (56%) cases (Fig. 1). Even if the correlation between the level of TPL2 mRNA and the score of its protein was not significant (chi-squared test, P=0.36), samples with a low TPL2 protein score expression typically exhibited lower mRNA levels (2.33-fold change value ±1.55) when compared with samples with high TPL2 score (4.0±1.2). In addition, a high score for TPL2 protein was identified in the lepidic adenocarcinoma subtype (P=0.003), as aforementioned for TPL2 mRNA expression.

Alignment of miRNAs with the TPL2 target mRNA was predicted by the miRNA target prediction programs miRBase, MicroCosm Targets and PITA. The present study identified the TPL2 gene as a potential target of miR-21 (Fig. 2). Firstly, the patients with NSCLC were divided into miR-21 high and low expression groups based on the median fold change values, which resulted as 1.7. Following this, the present study determined whether TPL2 expression was correlated with miR-21 levels, but no statistically significant associations were observed between groups (Chi-squared test, P=0.66).

To evaluate the potential association between TPL2 expression and the prognosis of the patients with NSCLC, a survival analysis by the Kaplan-Meier method using the disease-free interval (DFI) and the overall post-operative survival (OS) as endpoints was performed. It was noted that the tumors with a high TPL2 mRNA expression demonstrated a significantly longer mean DFI and OS compared with the patients with low expression of this mRNA (P=0.009 and P=0.024, respectively; Fig. 3 and Table II). In the multivariate analysis, TPL2 mRNA expression continued to be a good prognostic factor for DFI and OS (P=0.0005 and P=0.005; Table II). Tumor grading, a classical prognostic factor, was also associated with the DFI in the univariate (P=0.0019) and multivariate analyses (P=0.0008; Table II), as well as with the OS (P=0.0009 and P=0.0002, respectively). No significant differences were identified for other demographics or clinical characteristics (Table II). Furthermore, the median DFI and OS were similar in examined patients with either low or high miR-21 expression (data not shown).

Following this analysis, the survival difference between the histological cell types was investigated. No significant difference was observed between SCC and ADC (Table II). However, based on the difference of the TPL2 levels reported above, the NSCLCs were divided into three prognostic groups: Squamous cell carcinoma and the two predominant adenocarcinoma growth patterns (acinar and lepidic subtypes). The group with the most favorable prognosis was the lepidic predominant subtype, which was demonstrated earlier in the study to possess a higher level of TPL2, with a median DFI of 85 months and a median OS of 90 months. SCC and acinar ADC, both NSCLC tumors with lower TPL2 levels, presented a worse prognosis with a mean DFI of 53 and 68 months and with a mean OS of 65 and 68 months, respectively (P=0.02 and P=0.03; Fig. 4).