The pathological reports of patients who underwent surgical resection for lung cancer between April, 2004 and May, 2012 at the Okayama University Hospital (Okayama, Japan) were reviewed. Of the 674 patients diagnosed with PA, 28 were found to have MPC. The ratio of MPC varied widely (3–80%) among these 28 patients. A total of 138 resected PAs without MPC were randomly selected in the same period to serve as age-, gender- and smoking status-matched controls to the PA-MPC cases (Table I). Our institutional review board approved this study's protocol and informed consent was obtained from all the patients.

Genomic DNA was obtained from primary tumors by standard phenol-chloroform (1:1) extraction followed by ethanol precipitation, or by using the DNeasy Tissue kit (Qiagen, Valencia, CA, USA). Total RNA was extracted from primary tumors using the RNeasy Mini kit (Qiagen) according to the manufacturer's protocol. Oligo(dT)-primed cDNA was synthesized using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) with DNase treatment.

Using DNA derived from frozen tumor specimens, genotyping was performed by SNaPshot, a targeted mutational analysis assay designed by Su et al (15). The platform involves two methods: a screen (SNaPshot) based on multiplex polymerase chain reaction (PCR), primer extension and capillary electrophoresis that was designed to assess 38 somatic mutations in 8 genes [(AKT1, BRAF, EGFR, KRAS, mitogen-activated protein kinase kinase 1 (MEK1), neuroblastoma RAS viral oncogene homolog (NRAS), phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α (PIK3CA) and phosphatase and tensin homolog (PTEN)]; and a PCR-based sizing assay that assesses EGFR exon 19 (deletions), EGFR exon 20 (insertions) and human epidermal growth factor receptor 2 (HER2) exon 20 (insertions).

PCR-based assays or direct sequencing were performed to confirm the results if mutations of EGFR and KRAS were detected by the SNaPshot assay.

The EGFR mutational status was determined using a PCR-based length polymorphism and restriction fragment length polymorphism assay, as previously reported (16). Briefly, the common deletions of exon 19 were distinguished from the wild-type based on PCR product length polymorphisms using 12% polyacrylamide gel electrophoresis (PAGE) via ethidium bromide staining. For the exon 21 L858R mutation, Sau96I digestion, which specifically digests the mutant type, was performed prior to 12% PAGE.

The KRAS mutations in codons 12 and 13 were examined using PCR-based direct sequencing on an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems), as previously reported (17–19).

EML4-ALK fusion was screened by ALK immunohistochemistry (IHC) and confirmed by reverse transcription (RT)-PCR assay and fluorescence in situ hybridization (FISH) analysis.

Unstained paraffin-embedded sections were deparaffinised in xylene, hydrated through, and rinsed in distilled water. Heat-induced epitope retrieval was performed with EnVision FLEX Target Retrieval Solution, High pH (Dako, Carpinteria, California, USA). The slides were then incubated at room temperature with mouse anti-ALK monoclonal antibody (clone 5A4; dilution, 1:100; cat. no. ab17127; Abcam) for 30 min. The slides were incubated at room temperature with EnVision FLEX+Mouse Linker (Dako) for 15 min. The immune complexes were then detected with the dextran polymer reagent (Fig. 2A) (20,21).

The primers used to identify the EML4-ALK fusion transcript were selected to enable the detection of all possible in-frame fusions of EML4 to exon 20 of ALK, in which the kinase domain of ALK would be preserved. The forward primers used were EML4 72F (5′-GTCAGCTCTTGAGTCACGAGTT-3′) and fusion-RT-S (5′-GTGCAGTGTTTAGCATTCTTGGGG-3′); the reverse primer was ALK 3078RR (5′-ATCCAGTTCGTCCTGTTCAGAGC-3′) (22). PCR was performed for EML4-ALK under the following conditions: 94°C for 10 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 64°C for 1 min and polymerization at 72°C for 1 min, with a final extension step at 72°C for 7 min. RT-PCR for GAPDH expression as an internal control was performed under the same conditions in each tumor sample.

FISH was performed on formalin-fixed, paraffin-embedded tumor tissues using a break-apart probe to the ALK gene (Vysis LSI ALK Dual Color, Break Apart Rearrangement Probe; Abbott Molecular, Abbott Park, IL, USA) as per the manufacturer's instructions. cases were defined as FISH-positive when there was a >15% split signal in the tumor cells (Fig. 2B) (23,24).

Differences in statistical significance among the categorized groups were compared using the Chi-square test or the Student's t-test. An analysis of overall survival and disease-free survival was performed using the Kaplan-Meier method with the log-rank test. The data were analyzed using SPSS v22.0 software (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference for each analysis.

Regarding genetic alterations, 13 (46.4%) of the 28 PA-MPCs harbored mutually exclusive mutations: 9 (32.1%) EGFR mutations, 1 (3.6%) KRAS mutation and 3 (10.7%) EML4-ALK fusion genes. PAs without MPC harbored 42 (30.4%) EGFR mutations, 17 (12.3%) KRAS mutations, 1 (0.7%) PIK3CA mutation and 3 (2.2%) EML4-ALK fusion genes in a mutually exclusive manner, except that 1 case had EGFR G719A and L861Q mutations. There were no mutations in the AKT1, BRAF, MEK1, NRAS, PTEN or HER2 genes in either group. EML4-ALK fusion genes appeared to occur significantly more frequently in PA-MPCs compared with PA without MPC (P=0.027) (Table II). Regarding the EGFR and KRAS mutational status, the results of the SNaPshot assay were consistent with those of PCR-based assays or direct sequencing.

To confirm the clinical outcome of PA-MPC, 11 cases with pathological stage IA PA-MPC were compared with 65 cases with pathological stage IA PA without MPC (Table III). As of December, 2013, 13 (17.1%) patients had succumbed to the disease, with a median follow-up period of 62.0 months; 2 (18.2%) of the patients with PA-MPC had succumbed to PA during follow-up; 11 (16.9%) patients with PA without MPC had succumbed (7 to PA and 4 to other causes). The 5-year overall survival rates of all pathological stage IA patients (n=76), patients with PA-MPC (n=11), and patients with PA without MPC (n=65) were 82.0, 87.5 and 81.0%, respectively. A total of 13 (17.1%) patients developed disease relapse (4 patients with PA-MPC and 7 patients with PA without MPC). The 5-year disease-free survival rates of all pathological stage IA patients, patients with PA-MPC, and patients with PA without MPC were 80.7, 58.4 and 84.2%, respectively. Patients with PA-MPC exhibited a significantly poorer disease-free survival rate compared with those with PA without MPC (log-rank test, P=0.04) (Fig. 3).