Introduction

The MET gene is a proto-oncogene, and it encodes the hepatocyte growth factor receptor (HGFR). The binding of its ligand, hepatocyte growth factor, induces dimerization and activation of the receptor. This activates the downstream RAS/ERK/MAPK, PI3K/AKT, and Wnt/b-catenin signaling pathways, which play a role in cellular survival, embryogenesis, cellular migration, invasion, angiogenesis, and the epithelial to mesenchymal transition (EMT) [1].

Abnormalities of the MET gene are one of the most frequently identified genetic disorders in neoplastic diseases. Germline mutations in the MET gene have been found in hereditary papillary renal carcinoma (HPRC). Somatic MET mutations have been observed in sporadic papillary renal cell carcinoma, head and neck squamous cell carcinoma, and childhood hepatocellular carcinoma. Amplification and overexpression of this gene are also associated with multiple human cancers. The MET gene is altered in 5% of non-small cell lung cancer (NSCLC) patients. Alterations in exon 14 of the MET gene are detected in 3–4% of lung adenocarcinoma patients. The prevalence of de novo MET amplification in NSCLC ranges from 1% to 5% of patients, depending on the assay and the positivity cut-point used. Several agents have been developed to target MET or HGF. They are divided into small molecule inhibitors and monoclonal antibodies. Currently, two tyrosine kinase inhibitors (TKIs), i.e. tepotinib and capmatinib, have been approved by the Food and Drug Administration (FDA) for the treatment of NSCLC patients with a splice site mutation in exon 14 of the MET gene. Response to therapy occurs in 46–68% of patients, depending on the mutation testing method (liquid biopsy vs. tissue) and the treatment line. However, it has also been shown that MET inhibitors (crizotinib, cabozantinib, and capmatinib) may be effective in NSCLC patients with MET gene amplification [2–5].

Splice site mutations in exon 14 of the MET gene are currently examined with the next-generation sequencing (NGS) technique. In turn, with the use of fluorescence in situ hybridization (FISH), the ratio of MET to the centromeric portion of chromosome 7 (CEP7) can be used to distinguish between chromosome polysomy and gene amplification. However, there are other cheaper, simpler, and faster methods for the examination of MET exon 14 mutations and the MET gene copy number, e.g. quantitative PCR (qPCR), including one using reverse transcription (RT-qPCR). However, PCR-based methods have numerous limitations, for example, low sensitivity and specificity. In this study, we attempted to detect selected abnormalities in the MET gene using various molecular techniques.

Material and methods

Patients

The study group consisted of 26 patients with lung adenocarcinoma (median age: 67.2 ± 8.5 years, 8 women, 18 men) diagnosed and treated in the Department of Pneumonology, Oncology, and Allergology from 2014 to 2019. All enrolled patients had a diagnosis of abnormalities in the EGFR gene (real-time PCR method), the ALK gene (IHC, FISH, and RT-qPCR methods), the ROS1 gene (FISH and RT-qPCR methods), the MET gene (FISH, qPCR, RT-qPCR methods), and the RET gene (qRT-PCR method), as well as assessment of PD-L1 protein expression (IHC method). We enrolled one patient with ALK gene rearrangement, one patient with RET gene rearrangement, and 6 patients with EGFR gene mutations (three with deletions in exon 19 and three with substitution Leu858Arg). Seven patients had PD-L1 expression on ≥ 50% of tumor cells. The ALK-positive patient was treated with crizotinib in first-line therapy and alectinib in second-line therapy. Three patients with EGFR gene mutations received erlotinib, and other 3 patients with these mutations were administered afatinib. In two patients with progression after erlotinib or afatinib, a Thr790Met mutation was detected and osimertinib was administered. Two EGFR-positive patients received chemotherapy with cipslatin and pemetrexed. Only one patient with PD-L1 expression on ≥ 50% of tumor cells was treated with pembrolizumab. The other patients received first-line platinum-based chemotherapy (13 patients — cisplatin plus pemetrexed, 4 patients — cisplatin plus vinorelbine, and one patient — cisplatin plus gemcitabine). In second-line therapy applied in chemotherapy-resistant patients, atezolizumab was used in 9 patients, nivolumab in 2 patients, and docetaxel plus nintedanib in one patient. The median overall survival of our patients was 48 months (95% CI: 16.3–48.0). The demographic and clinical-pathological characteristics of the patients are presented in Table 1.

All aspects of the work covered in this manuscript were approved by the Ethics Committee of the Medical University of Lublin, Poland (No. KE-0254/169/2014).

Routine diagnosis of predictive factors in adenocarcinoma patients

DNA was isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissues or cytological specimens (cell blocks). DNA was extracted using the QIAamp DNA FFPE Tissue Kit (CE-IVD marked, Qiagen, Germany). Isolation was performed according to the manufacturer’s instructions (the same isolated DNA was also used for examination of the MET gene copy number using the qPCR technique). The concentration and quality of isolated DNA were estimated by spectrophotometry. Mutations of the EGFR gene were identified using the EntroGen EGFR Mutations Analysis Kit (CE-IVD marked, EntroGen, Woodland Hills, Canada) in the Cobas Z 480 real-time PCR system (Roche Diagnostics, USA). We examined all the most common mutations in exons 18 to 21 [6].

Abnormal ALK protein and PD-L1 protein expression were examined using an immunohistochemistry test. ALK protein IHC staining was conducted on the Ventana Benchmark GX platform using CE-IVD approved anti-ALK Rabbit Monoclonal Primary Antibody (clone D5F3). The OptiView Amplification Kit and the OptiView DAB IHC Detection Kit were used as detection systems. Rabbit monoclonal negative control immunoglobulin was used as a negative control (Ventana Medical System, Tuscon, USA). CE-IVD approved Ventana SP263 antibody was used for PD-L1 protein IHC staining. The same equipment and detection systems were used for the examination of ALK expression. Rabbit monoclonal negative control immunoglobulin (Ventana Medical System, Tucson, AZ, USA) was used as a negative control. The slides were assessed by pathologists using an Olympus BX41 microscope [7–8].

All positive results of ALK expression obtained in IHC staining were re-evaluated with the FISH method to visualize the presence of ALK rearrangement using the Vysis ALK Break Apart FISH Probe Kit (Abbot Molecular, USA) and the paraffin-pretreatment IV and Post-Hybridization Wash Buffer Kit (Abbot Molecular, USA). In the diagnosis of ROS1 gene rearrangement, we used the ZytoLight SPEC ROS1 DualColor Break Apart Probe (ZytoVision, Germany) and the Vysis Paraffin Pretreatment and Post-hybridization Wash Buffer Kit (Abbott, USA). Fluorescence signals were assessed using an Axio Scope microscope (Zeiss, Germany). Interpretation of FISH results was conducted in accordance with the American Food and Drug Administration (FDA) and International Association for the Study of Lung Cancer (IASLC) guidelines [8].

Reverse transcriptase PCR analysis of ALK, ROS1, and RET gene rearrangements and MET gene skipping mutations

Total RNA was extracted from FFPE tissues with the miRNeasy FFPE Kit (Qiagen Inc., Germany) according to the manufacturer’s instructions. The RNA concentration was measured with Qubit 4 fluorometers (Invitrogen, Thermo Fisher Scientific, Waltham, USA). RNA samples were stored at –80oC until RT-qPCR (reverse transcriptase-quantitative PCR) was performed. The same isolated RNA was also used for the examination of the MET mRNA expression.

To detect ALK, ROS1, and RET gene fusions, as well as MET exon 14 skipping mutations, we used the Lung Cancer RNA Panel kit (EntroGen, Woodland Hills, Canada) according to the manufacturer’s instructions. There were 8 reactions of twenty microliters in the volume of one-step RT-qPCR for one patient. Every reaction mixture contained 10 µL of One-Step RT-qPCR Reaction Mix, 1 µL of RT Enzyme Mix, 4 µL of Reaction Detection Primer Mix (one from eight), and 5 µL of RNA (concentration 16 ng/µL). The RT-qPCR reaction was performed on the Illumina Eco real-time PCR platform (Illumina, San Diego, USA) in the following conditions: 55oC for 10 minutes, 95oC for 1 minute, and next 40 cycles: 95oC for 10 seconds and 60oC for 45 seconds. Ct values were obtained, and analysis was performed according to the manufacturer’s instructions.

MET gene amplification assessment with the FISH technique

The MET gene amplification status was assessed using the ZytoLight SPEC MET/CEN 7 Dual Color Probe (CE, ZytoLight, Germany). The Paraffin-Pretreatment and Post-Hybridization Wash Buffer Kit (Abbot Molecular, USA) was also used for the pre-staining procedure. Three to five μm thick paraffin sections were cut and mounted on the positively charged glass slides. All procedures were carried out according to the manufacturer’s procedure.

The SPEC MET/CEN 7 Dual Color Probe is a combination of a probe with an orange fluorochrome direct labeled specific for the alpha satellite centromeric region of chromosome 7 (D7Z1) and a probe with a green fluorochrome direct labeled targeted at the locus in the MET gene located at 7q31.2. In a normal interphase nucleus, two orange and two green signals are expected. In cells with amplification of the MET gene locus, multiple copies of the green signal or green signal clusters will be observed. The fluorescence signals were assessed using an Axio Scope microscope (Zeiss, Germany).

We classified the cases into two categories: MET-positive (with amplification) and MET-negative (without amplification). The cutoff of the MET/CEP7 ratio was 2.0. A sample was considered to have MET amplification if the mean MET/CEP7 ratio was ≥ 2.0 or if the MET/CEP7 ratio was < 2.0, but the MET-copy number was ≥ 5 copies per nucleus or MET signal clusters were seen in more than 10% of tumor cell nuclei. According to a different classification, MET amplification was also classified using the low ratio (≥ 1.8 to ≤ 2.2 MET/CEP7), intermediate ratio (> 2.2 to < 5 MET/CEP7), and high ratio (≥ 5 MET/CEP7) [9–10].

MET gene copy number assessment with the qPCR technique

The MET gene copy number was assessed using a TaqMan primer set and probe (TaqMan Copy Number Assays, Hs00305306_cn, Thermo Fisher Scientific, Waltham, Massachusetts, USA). RNazeP (TaqMan Copy Number Reference Assay, human, RNaseP, Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used as an internal control. The 10-microlitre qPCR reaction mixture contained 5 μL of Genotyping Master Mix, 0.5 μL of TaqMan Copy Number Assays or TaqMan Copy Number Reference Assay, and 4.5 μL of DNA (concentration 5 ng/μL). The real-time PCR was performed in Illumina Eco (Illumina Inc., San Diego, California, USA). The temperature conditions of qPCR were as follows: enzyme activation at 95°C for 10 minutes followed by 40 cycles of two-stage PCR at 95°C for 15 seconds and next at 62°C for 90 seconds. Method 2-DdCt was used for the calculations. The calibrator, which was a mixture of DNA obtained from lymphocytes of healthy subjects, was used for the calculations. According to the literature data, we assumed that more than 3 copies of the MET gene allowed us to find a high copy number of this gene.

MET mRNA expression analysis using RT-qPCR

RNA reverse transcription was performed using the High Capacity cDNA Reverse Transcription Kits (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s instructions. cDNA was stored at –20° C until qPCR was performed.

The expression of MET mRNA (cDNA) was assessed using primers and the TaqMan probe kit (TaqMan™ Gene Expression Assay Hs01565584_m1, Thermo Fisher Scientific, Waltham, Massachusetts, USA). GAPDH (Gene Expression Reference Assay Hs03929097_g1, Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used as an internal control. The composition of the reaction mixture (10 μL) was as follows: 5 μL of TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 0.5 μL of TaqMan Gene Expression Assay or GAPDH reference assay, 3.5 μL of nuclease-free water, and 1 μL of cDNA (RT-PCR reaction product). The real-time PCR was performed in Illumina Eco (Illumina Inc., San Diego, California, USA). The temperature conditions of the qPCR reaction were as follows: 50°C for 2 minutes (UNG, uracil-N-glycosylase activation), 95°C for 20 minutes (polymerase activation) followed by 40 cycles of two-stage PCR (95°C for 3 seconds then 62°C for 45 seconds). Method 2-DCt was used for the calculations.

Statistical analysis

The U-Mann Whitney test was used for testing the equality of population medians among groups differing in clinical and demographic factors. The Spearman test was used to calculate the correlation between countable variables. Data were expressed as a percentage (for the categorized variable), median, and standard deviation (for continuous variables). These tests were performed with Statistica v. 13.1 (Tibco Software, USA). Survival analyses were performed using the Kaplan-Meier estimation method in MedCalc 15.8 (MedCalc Software, Ostend, Belgium) with a calculation of the 95% confidence interval (CI). We considered p values below 0.05 to be statistically significant.

Results

In our patients, we did not find MET exon 14 skipping mutations with the use of the RT-qPCR technique. We also did not demonstrate the presence of MET gene amplification or chromosome 7 polysomy with the FISH technique. The median MET/CEP7 ratio was 1.04 with a standard deviation of 0.145, and the median number of chromosome 7 was 2.5 ± 0,788. In the FISH method, the median MET gene copy number was 2.6 ± 0.457. In contrast, the median MET gene copy number was 3.43 ± 1.539 in the qPCR study. Fifteen patients had more than 3 copies of the MET gene detected by the qPCR technique. According to the criteria adopted by other authors, we may conclude that 57.7% of the patients had a high MET gene copy number. The relative MET mRNA expression was low, and its median was 0.01 with a standard deviation of 0.045.

The age (division into two groups according to the median), stage of disease (IIIB versus IV), and smoking status did not affect the assessed parameters (MET/CEP7 ratio, CEP7 number, MET gene copy number in the FISH, and qPCR methods, and MET mRNA expression). However, the MET/CEP7 ratio and MET mRNA expression were slightly higher in the women than in the men (p = 0.054 and p = 0.055, respectively, Fig. 1 and 2). Moreover, the MET gene copy number in the FISH method was insignificantly higher in patients with genetic driver alterations (EGFR gene mutations, ALK and RET gene rearrangements) than in patients without these abnormalities (p = 0.077, Fig. 3).

Figure 1. Value of the MET/CEP7 ratio assessed with the FISH technique depending on the sex of the studied lung adenocarcinoma patients

Figure 2. Relative MET mRNA expression assessed with the RT-qPCR method depending on the sex of the studied lung adenocarcinoma patients

Figure 3. MET gene copy number assessed with the FISH method in patients with and without genetic driver alterations (EGFR gene mutations, ALK and RET gene rearrangements)

We found a significant positive correlation (R = +0.573, p = 0.0022) between the MET gene copy number assessed with the qPCR method and the relative MET mRNA expression (Fig. 4). We did not detect a correlation between the MET/CEP7 ratio and the MET gene copy number assessed with the qPCR method, as well as the MET mRNA expression. The MET gene copy number in the FISH technique did not significantly correlate with the MET gene copy number assessed with the qPCR technique and with the relative MET mRNA expression.

Figure 4. Significant positive correlation between the MET gene copy number assessed with the qPCR method and the relative MET mRNA expression in lung adenocarcinoma patients

Discussion

Most studies on the CNV of MET gene have been conducted in Asian populations. However, two studies on Caucasian NSCLC patients need to be mentioned. Capuzzo et al. [9], studied the CNV of the MET gene using the FISH technique in 435 Italian NSCLC patients, and Bubendorf et al. [11] studied abnormalities in the MET gene using the silver in situ hybridization (SISH) technique in the European population. Capuzzo et al. were the first to introduce a MET gene amplification evaluation system using the FISH technique. They found a high MET gene copy number (mean ≥ 5 copies/cell) in 48 cases (11.1%), including 18 cases with true gene amplification (4.1%). The high MET gene copy number was associated with an advanced stage (p = 0.01), a low grade of tumor differentiation (p = 0.016), and amplification of the EGFR gene in tumor cells (p < 0.0001). The authors found no relationship between patients’ sex and the MET gene copy number in the cancer cells. No patient with an EGFR activating mutation showed a high MET gene copy number. MET-positive patients had shorter survival than MET-negative patients (p = 0.005) [9]. These results differ from those presented in our study in which we showed that the women and patients with genetic driver alterations had a slightly higher MET gene copy number than the men and patients without other genetic abnormalities. On the other hand, we did not find patients with true MET gene amplification with an oncogenic character (responsible for tumor growth). This is likely to be related to the high percentage of patients with other genetic driver alterations included in our study. Driver abnormalities do not usually coexist in one patient.

Bubendorf et al. published results from the European Thoracic Oncology Platform (ETOP) Lungscape Project, which involved 1572 patients with surgically resected NSCLC. MET gene amplification was defined as a MET/CEP7 ratio ≥ 2 and a high MET gene copy number as ≥ 5, as well as high MET protein expression in the IHC test as ≥ 2 + intensity in ≥ 50% of tumor cells. One hundred and eighty-two patients with MET protein expression and without mutations in the EGFR and KRAS genes were analyzed for the MET exon 14 skipping mutation. The high expression of the MET protein was significantly associated with the female sex and small tumor size. MET amplification occurred in 4.6% of patients, and a high MET gene copy number was detected in 4.1% of patients. The MET gene abnormalities were not significantly associated with the clinical and demographic characteristics of the patients. The MET exon 14 skipping mutation was detected in 5 of the 182 (2.7%) patients, including 4 adenocarcinoma patients (4.5%). The authors emphasized that the large inter-laboratory variability in the MET status assessment highlights the challenge of these analyses in routine practice [11].

Dziadziuszko R et al. [12] assessed the MET gene copy number using the SISH technique in 140 Polish NSCLC patients. The median value of the MET gene copy number per cell was 3.12 (from 1.74 to 11.84). Three patients (2.1%) showed gene amplification (MET gene clusters) and 14 (10%) had tumors with 5 or more gene copies per nucleus. There was a significant correlation between the MET copy number and the protein expression. The authors found no association between the MET gene copy number and the demographic or clinical features, including sex (p = 0.54), disease stage (p = 0.21), tumor grade (p = 0.86), and histology (p = 0.84), or smoking status (p = 0.47). They showed no associations between the MET copy number and disease-free survival or overall survival. In our study, we found that the median MET gene copy number tested with the qPCR technique exceeded 3, which is consistent with the observations reported by Dziadziuszko et al. [12], who used the FISH technique. It is debatable whether 3 or more copies of the gene should be used as a cutoff point for recognition of a high MET gene copy number in NSCLC patients [12].

In another study on the Polish NSCLC population, Kowalczuk et al. [13] used the qPCR technique to study the MET gene copy number and mRNA MET expression. In total, 151 patients with paired surgical samples of tumor and tumor-distant normal lung tissues were enrolled in the study. A high MET gene copy number (more than 3.0 copies per cell) was found in 18.5% of patients and occurred more frequently in adenocarcinoma with an increased EGFR and HER2 gene copy number and with EGFR activating mutations (p = 0.051). The MET mRNA expression was 1.76-fold higher in the tumor compared to unaffected lung tissue, and it was associated significantly with the MET gene copy number. The results of this study are partially consistent with our results [13].

Aguado et al. [14] examined 422 NSCLC patients and identified 13 patients (3%) with MET exon 14 mutations and 15 patients (3.5%) with very high MET mRNA expression, which was analyzed using the quantitative transcript-based hybridization technology. These two subgroups of patients were mutually exclusive, displayed distinct phenotypes, and did not generally coexist with other genetic driver alterations. Ninety-two percent of patients with very high MET mRNA expression had MET gene amplification detected by FISH and/or NGS. However, FISH failed to identify three patients with very high MET mRNA expression, among whom one received MET tyrosine kinase inhibitors and obtained clinical benefit. These results indicated that MET mRNA expression assessment could improve the selection of patients for MET TKIs [14].

Kim JH et al. [15] performed a meta-analysis to evaluate the prognostic value of a high MET gene copy number in NSCLC patients. From 21 studies, 7647 patients were included in the pooled analysis of hazard ratios for disease-free survival or overall survival. Patients with a high MET gene copy number showed significantly worse survival than patients with a low MET gene copy number. The method used for MET CNV analysis included FISH, SISH, bright field in situ hybridization (BISH), and qPCR. The FISH technique was mostly used, but various cutoff criteria were adopted. A high MET gene copy number occurred in 1–38.9% of patients, depending on the technique used and the cut-point for positivity. Using the qPCR technique, more than 3 MET gene copies were considered a high MET gene copy number in most studies. A high MET gene copy number was detected in 5.6%, 18%, and 18.5% of NSCLC patients. In one study, the cutoff was the mean MET gene copy number, which was 1.31. In this study, a high MET gene copy number was found in 4.8% of patients. Surprisingly, there is a large discrepancy in the results and the fact that as many as 57.7% of patients in our study had over 3 MET gene copies detected by the qPCR test [15].

Conclusions

Our study has several inherent limitations that need to be discussed. The group of adenocarcinoma patients was very small. Moreover, the group was heterogeneous regarding the occurrence of somatic driver alterations. Therefore, we did not detect any rare abnormalities in the MET gene. The 3 gene copy number cutoff for our qPCR test appears to be understated. This produces incorrect high MET gene copy number results in several patients. Nevertheless, the agreement of the MET mRNA expression and FISH results, as well as the positive correlation between the MET mRNA expression and the MET gene copy number in the qPCT test, indicate that these methods could complement each other. Performance of these two tests simultaneously (e.g. determination of mRNA expression and gene copy number) increases the reliability of MET gene assessment. Such a tool allows correct qualification of our patients for molecularly targeted therapies.

Conflict of interest

Authors declare no conflict of interest.

References