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Open Access

Peer-reviewed

Research Article

The impact of MET, IGF-1, IGF1R expression and EGFR mutations on survival of patients with non-small-cell lung cancer

  • Samer Al-Saad ,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    samer.al-saad@unn.no

    Affiliations Institute of Medical Biology, UiT The Arctic University of Norway, Tromso, Norway, Department of Clinical Pathology, University Hospital of Northern Norway, Tromso, Norway

    ⨯
  • Elin Richardsen,

    Roles Conceptualization, Data curation, Writing – review & editing

    Affiliations Institute of Medical Biology, UiT The Arctic University of Norway, Tromso, Norway, Department of Clinical Pathology, University Hospital of Northern Norway, Tromso, Norway

    ⨯
  • Thomas K. Kilvaer,

    Roles Conceptualization, Data curation, Formal analysis, Methodology, Writing – review & editing

    Affiliations Institute of Clinical Medicine, UiT The Arctic University of Norway, Tromso, Norway, Department of Oncology, University Hospital of Northern Norway, Tromso, Norway

    ⨯
  • Tom Donnem,

    Roles Data curation, Investigation, Methodology, Visualization, Writing – review & editing

    Affiliations Institute of Clinical Medicine, UiT The Arctic University of Norway, Tromso, Norway, Department of Oncology, University Hospital of Northern Norway, Tromso, Norway

    ⨯
  • Sigve Andersen,

    Roles Data curation, Investigation, Visualization, Writing – review & editing

    Affiliations Institute of Clinical Medicine, UiT The Arctic University of Norway, Tromso, Norway, Department of Oncology, University Hospital of Northern Norway, Tromso, Norway

    ⨯
  • Mehrdad Khanehkenari,

    Roles Methodology, Validation, Writing – review & editing

    Affiliation Institute of Medical Biology, UiT The Arctic University of Norway, Tromso, Norway

    ⨯
  • Roy M. Bremnes,

    Roles Conceptualization, Data curation, Investigation, Methodology, Resources, Visualization

    Affiliations Institute of Clinical Medicine, UiT The Arctic University of Norway, Tromso, Norway, Department of Oncology, University Hospital of Northern Norway, Tromso, Norway

    ⨯
  • Lill-Tove Busund

    Roles Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing

    Affiliations Institute of Medical Biology, UiT The Arctic University of Norway, Tromso, Norway, Department of Clinical Pathology, University Hospital of Northern Norway, Tromso, Norway

    ⨯

Abstract

Introduction

To compare the efficacy of silver in situ hybridization (SISH) and immunohistochemistry (IHC) in detecting MET and IGF1R alterations and to investigate their prevalence and prognostic significance. A possible correlation between MET receptor expression, MET gene alterations and the two most frequent occurring EGFR gene mutations was also investigated.

Materials and methods

Stage I to IIIA tumors from 326 patients with NSCLC were immunohistochemically tested for protein expression of MET and IGF-1. Their cytoplasmic expression was compared with the gene copy number of the MET and IGF1Rgenes by SISH in paraffin-embedded, formalin-fixed material. Correlations were made with the immunohistochemical expression of two frequent EGFR mutations and clinicopathological variables. Univariate and multivariate survival analyses was used to evaluate the prognostic efficacy of the tested markers.

Results

In univariate analyses, high cytoplasmic MET expression showed a significant negative prognostic effect in adenocarcinoma patients (p = 0.026). MET gene to chromosome 7 ratio was a significant positive prognostic marker (p = 0.005), probably only due to the highly negative prognostic significance of chromosome 7 polysomy (p = 0.002). High IGF1R gene copy number was a negative prognostic marker for all NSCLC patients (p = 0.037). In the multivariate analysis, polysomy of chromosome 7 in tumor cells correlated significantly and independently with a poor prognosis (p = 0.011). In patients with adenocarcinoma, a high cytoplasmic MET expression was an independent negative prognostic marker (p = 0.013). In males a high IGF1R gene copy number to chromosome 15 count ratio was significantly and independently correlated to a poor prognosis (p = 0.018).

Conclusion

MET protein expression provides superior prognostic information compared with SISH. Polysomy of chromosome 7 is an independent negative prognostic factor in NSCLC patients. This finding has an important implication while examining genes located on chromosome 7 by means of SISH. High IGF1R gene copy number to chromosome 15 count ratio is an independent predictor of inferior survival in male patients with primary NSCLC.

Citation: Al-Saad S, Richardsen E, Kilvaer TK, Donnem T, Andersen S, Khanehkenari M, et al. (2017) The impact of MET, IGF-1, IGF1R expression and EGFR mutations on survival of patients with non-small-cell lung cancer. PLoS ONE 12(7): e0181527. https://doi.org/10.1371/journal.pone.0181527

Editor: Aamir Ahmad, University of South Alabama Mitchell Cancer Institute, UNITED STATES

Received: January 24, 2017; Accepted: July 3, 2017; Published: July 25, 2017

Copyright: © 2017 Al-Saad et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This research was funded by the Northern Norway Regional Health Authority (Helse Nord), Grant number SFP1032-12. The authors would like to acknowledge Roche Diagnostics Norway for providing our research laboratory with the two SISH antibodies MET gene and IGF1-R. Roche. The publication charges for this article have been funded by a grant from the publication fund of UiT The Arctic University of Norway. The Northern Norway Regional Health Authority, The Arctic University of Norway, and Roche Diagnostics Norway had no role in study design, data collection and analysis, results, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Lung cancer is the leading cause of cancer-related mortality in men and the second among women worldwide.[1] With annually about 1.3 million new registered non-small cell lung cancer (NSCLC) cases, every effort should be made towards finding more personalized cancer therapies[2].

MET (the hepatocyte growth factor receptor, HGFR, also known as c-Met, AUTS9; RCCP2; DFNB97, and as mesenchymal-epithelial transition factor) is activated by its ligand HGF and exerts broad biological effects associated with malignancy including cell proliferation, cell scattering and migration, induction of cell polarity, and angiogenesis.[3] MET is reported to regulate the morphogenesis of both epithelial and stromal cells [4], in addition to its role in the mesenchymal-epithelial transition of cells, and to play an essential role in tissue repair[5] (Fig 1).

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Fig 1. Simplified schema of the MET signaling adaptors and mediators.

Activation of the MET receptor by its ligand triggers tyrosines within the multifunctional docking site to become phosphorylated and recruit signaling effectors, including the adaptor protein growth factor receptor-bound protein 2 (GRB2). The MET pathway is modulated by cell surface molecules, including the HER family (HER1, HER2 and HER3) and IGF1R.MET can activate the effector molecule phosphatidylinositol 3-kinase (PI3K), and signals through the AKT/protein kinase B axis, which activates the mammalian target of rapamycin (mTOR) axis stimulating cell growth and protein synthesis. Moreover, the activation of either PI3K-AKT can activate NF-κB, which then can be released and stimulate gene transcription after being translocated to the nucleus.MET activation can result in the down-stream activation of the RAS-MAPK pathway. The nucleotide exchanger protein Son of Sevenless (SOS) activates the rat sarcoma viral oncogene homolog RAS by binding with the GRB2 (GRB2-SOS complex). This complex can activate the v-raf murine sarcoma viral oncogene homolog B1 (RAF) kinases, which successively activate MAPK effector kinase (MEK), and finally results in the activation of the mitogen-activated protein kinase (MAPK).Activation of MET cytoplasmic signalling cascades can additionally alter cell invasiveness, motility, and cytoskeleton, mediated through the RAS-related protein 1 (RAP1), the focal adhesion complex (FAC) as well as integrin connections. MET can additionally activate the downstream axis of the Signal transducer and activator of transcription 3 (STAT3) pathway. STAT3 can be activated through phosphorylation after binding directly to MET resulting in dimerization and translocation to the nucleus and consequently mediating cell proliferation, transformation, as well as tumorigenesis and invasion. [6–9].

https://doi.org/10.1371/journal.pone.0181527.g001

MET has recently also gained ground as an important target in the treatment of malignancy. Using the MET inhibitor onartuzumab plus erlotinib, an improved progression-free survival (PFS) and a better overall survival (OS) was seen in immunohistochemically MET-positive NSCLC patients, while a worse outcome was observed in MET-negative patients treated with onartuzumab.[10] However, recent published results by Spigel et al [11]from the III randomized trial of Onartuzumab plus Erlotinib versus Erlotinib trial failed to show similar results. Other trials are still ongoing[6, 12].

Herein, we conducted a study investigating the prevalence and the prognostic role of MET in NSCLC. We compared the expression of MET as detected by immunohistochemistry (IHC) with its gene amplification by means of silver in situ hybridization (SISH). Due to observations reporting MET activation and treatment resistance following anti EGFR treatment,[12, 13] we aimed to immunohistochemically study the prevalence and possible correlation between MET expression and two major forms of mutant EGFR; E746-A750deletion mutation of exon 19 (EGFRdel) and the single L858R deletion mutation of exon 21 (EGFRmut). In the light of studies indicating IGF1R as a putative coactivator of MET (Fig 1), we investigated the prevalence and the prognostic significance of IGF1R gene using SISH and the ligand IGF-1 using immunohistochemistry. Finally, we investigated the correlation between polysomy of chromosome 7, on which the MET gene is located, and disease-specific survival (DSS) in patients with NSCLC.

Materials and methods

Patients and clinical material

This retrospective study utilized primary tumor tissue from patients diagnosed with NSCLC stage I–IIIA; the tissue was surgically resected at the University Hospital of Northern Norway and Nordland Central Hospital between 1990 and 2004. Three hundred seventy-one patients were registered from the hospitals’ databases. The following exclusion criteria were employed: (1) radiotherapy or chemotherapy prior to surgery, (2) other malignancy within 5 years before the NSCLC diagnosis and (3) inadequate paraffin-embedded tissue blocks. Thirty-six patients fell into these three categories (criteria 1: n = 10; criteria 2: n = 13; criteria 3: n = 13) and were excluded from the study. Adjuvant chemotherapy had not yet been introduced as a therapeutic option in Norway during this time span (1990–2004). In total, 335 patients with complete medical records and adequate paraffin embedded tissue blocks were included in this study. The tumors were subtyped and histologically graded according to the recent World Health Organization (WHO) guidelines.[14] The patients were staged corresponding to the 7th edition of the UICC TNM classification, where 9 patients were regarded as having an in-situ disease regarding the new lung cancer classification resulting in a total of 326 patients eligible for this study.[15] The Regional Committee for Medical and Health Research Ethics, as well as the Norwegian Data Inspectorate, approved this study.

Microarray constructions

Two experienced pathologists (S.A.S. and K.A.S.) investigated all the lung cancer specimens thoroughly. Tissue microarray (TMA) blocks were constructed using a tissue-array instrument (Beecher Instruments, Silver Springs, MD, USA) as previously described [16].

Immunohistochemistry and silver in situ hybridization

The applied antibodies have been previously subjected to in-house validation by the manufacturer for IHC analysis of paraffin-embedded material. The antibodies used in this study were as follows: Phospho-MET Receptor (1:160; rabbit monoclonal, clone D26; #3077; Cell Signaling Technology,Danvers, MA, USA). EGF Receptor (E746-A750del Specific; 1:100; rabbit monoclonal, clone D6B6; #2085; Cell Signaling Technology, Danvers, MA, USA). EGF Receptor(L858R Mutant Specific; 1:100; rabbit monoclonal, clone 43B2; #3197; Cell Signaling Technology, Danvers, MA, USA).IGF1R gene and Chromosome 15 probe (prediluted by the manufacturer; INFORM IGF1R DNP Probe: 800–4458 and INFORM Chromosome 15 DIG Probe: 800–4459; Ventana Medical Systems, Illkirch, France). MET gene and chromosome 7 probe (prediluted by the manufacturer; INFORM MET DNA Probe: 800–4372; 05575311001 and INFORM Chromosome 7 Probe: 800–4342; 05278899001). IGF-I (1:100, rabbit polyclonal, clone H-70; #sc-9013; Santa Cruz Biotechnology incorporated, 10410 Finnell Street, Dallas, Texas 75220, USA). The detailed methodology for immunohistochemistry and silver in situ hybridization has been previously published[16, 17].

Scoring of immunohistochemistry (IHC) and silver in situ hybridization (SISH)

The tissue cores were scored by light microscopy to determine the degree of cytoplasmic and nuclear expression. Examples of various markers’ expressions are shown (Fig 2). Staining for MET and IGF1R genes resulted in signals as black dots on the corresponding chromosomes for both genes, while centromeres of chromosome 7 and 15 were stained as red dots. Regarding SISH scoring, uniform guidelines exist and were strictly followed for the interpretation of gene and chromosome signals.[18]Even though these guidelines were developed for breast cancer testing, we found similar staining results in NSCLC biopsies. Evaluation ofHER2sish is reported in breast cancer as the ratio of the average number of HER2 gene copies to the average number of chromosome 17 copies (HER2:chr17) per cell. Because no clear guidelines have been established for measuring MET or IGF1Rgene amplification in NSCLC, we also sought to determine whether the absolute number of MET and IGF1Rgene copies detected by SISH (i.e., the number of black dots observed in the nuclei of tumor cells) would add prognostic significance beyond that established by the gene copy number to chromosome count ratio. From each tumor, four cores were eligible for scoring. In each core, we counted genes and centromere signals in 20 cells at least in two cores, where one core was taken from the central part of the tumor and the other core was taken at the advancing edge of tumor. An overall average was taken for both gene and centromere count. The other two cores included for the most stromal tissue surrounding epithelial cells of NSCLC. Heterogeneity was not observed while scoring immunostains. Regarding SISH, in cases of heterogeneity hot spots with the highest gene or centromere count were scored. The number of gene copies was assessed according to the manufacturer’s protocols for INFORMHER2 DNA. Briefly, a discrete dot was counted as a single copy of MET, IGF1R, chromosome 7 or chromosome 15. Some nuclei showed multiple discrete copies. Clusters of dots representing many copies of the targetgenes were also apparent; a small cluster of multiple signals was counted as six copies and a large cluster was counted as 12 copies.

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Fig 2. Expression of investigated markers in NSCLC tissues.

A) High expression of MET in a patient suffering an adenocarcinoma. B) Tumor tissue with a high chromosome 7 count on which the MET gene is located. C) Tumor tissue with high MET gene copy number count. D) Balanced MET gene copy number to chromosome 7 count ratio. E) High IGF-1 IHC expression of a male patient with squamous cell carcinoma. F) Tumor tissue in a male patient with a high IGF1R gene copy number to chromosome 15 count. G) Tumor tissue in a male patient with a balanced IGF1R gene copy number to chromosome 15 count. H) An adenocarcinoma with positive IHC staining for the EGFR gene mutation E746-A750 deletion of exon 19. I) An adenocarcinoma with positive IHC staining for the EGFR gene single L858R deletion mutation of exon 21.

https://doi.org/10.1371/journal.pone.0181527.g002

Staining for MET, IGF-1, EGFRmut and EGFRdel resulted in a homogenous cytoplasmic staining (Fig 2). Due to homogenous staining, there was no need to score the density of positive cells. Scoring the intensity of staining was considered as representative to measure the protein expression by immunohistochemical staining. Tissue sections for the two first mentioned proteins were scored semi quantitatively for staining intensity in tumor epithelial cells as follows: 0 = negative, 1 = weak, 2 = intermediate, and 3 = strong. Regarding the immunohistochemical expression for IGF-1 and MET, tumors showing intermediate or strong positivity were regarded as the high expression group, while tumors with weak or negative staining were regarded as the low expression group. When evaluating EGFRmut and EGFRdel, only clear strong positive tumors where regarded as harboring gene mutations.

All of the anonymized samples were semi quantitatively and independently scored by two experienced pathologists (S.A.S. and E.R). In the event of disagreement, the slides were re-examined and a consensus was reached by the observers. When assessing one variable for a given core, the observers were blinded to the scores of the other variables and the outcome. The reproducibility of the IHC and SISH evaluation in randomly selected specimens is high. The IHC and SISH scores from each observer were compared for interobserver reliability using a 2-way random effects model with absolute agreement definition, yielding an intra-class correlation coefficient (reliability coefficient) and Cohen’s kappa. There was an excellent scoring agreement for two tested markers (MET SISH and EGFRdel) between the pathologists, with an intra-class correlation coefficient of 0.91 (P < 0.001) for MET SISH and 0.93 (P < 0.001) for EGFRdel.

Statistical analyses

The statistical analyses presented in this study were performed using the statistical package IBM SPSS, version 24 (SPSS Inc., Chicago, IL, USA).Chi-square and Fisher’s exact test were used to examine the correlation among different molecular markers and clinicopathological factors. The r-values represent Spearman’s rank correlation coefficients. The Kaplan–Meier method was used for drawing curves for univariate analysis of the association between marker expression and disease-specific survival (DSS). DSS was determined from the date of surgery until the time of lung cancer death. Statistical significance between the survival curves was assessed utilizing the log-rank test. Cut-offs were chosen by a minimal p-value approach, with regard to the association between markers and survival endpoints. The survival curves were terminated at 120 months as fewer than 10% of patients were at risk after this point. Statistically significant variables from the univariate analysis were included in the multivariate analysis, applying the Cox proportional hazards model. The data were run in a backward stepwise Cox regression with a probability for stepwise entry and a removal set at 0.05 and 0.10. The significance level was set at a P-value less than 0.05.

Results

Clinicopathological variables

We retrospectively examined a non-selected group of NSCLC patients, which was examined in previous works by our research group.[17]Clinical, demographic and histopathological variables are presented in Table 1.Of the 326 NSCLC patients, the majority were male (76%) and nearly all (96%) were previous or present smokers. The median age was 67 years (range 28–85 years) and the median follow-up of survivors was 105 months (range 73–234 months). Histologically subtypes presented as: (58.6%) 191 cases of squamous cell carcinoma (SCCs), (31.9%) 104 cases of adenocarcinomas (ACs) and (9.5%) 31 cases of large cell (anaplastic) carcinomas (LCCs).Fifty-nine patients (18%) were administered adjuvant radiotherapy due to nodal metastasis or non-radical surgical margins verified during surgery. The median follow-up of the survivors was 105 months (range 73–234 months).

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Table 1. Prognostic clinicopathologic variables as predictors for disease-specific survival in 326 NSCLC patients (univariate analyses; log-rank test).

https://doi.org/10.1371/journal.pone.0181527.t001

Biomarker expression and correlation in NSCLC tissue

Of 326 cases, all were immunohistochemically evaluable for the expression of MET and IGF-1, while 295 were evaluable for the silver in situ hybridization analysis of the MET gene copy number on chromosome 7, and 237 cases were eligible for the IGF1R gene copy number analysis on chromosome 15.

For the immunohistochemical analysis of EGFRgene mutations, 313 cases were eligible for EGFRmut, while 318 cases were eligible for the detection of the EGFRdel mutation.

MET, IGF-1, EGFRmut and EGFRdel showed a homogenous cytoplasmic staining pattern.

We found high (moderate to strong) cytoplasmic expression of MET and IGF-1 in 82.8% and 6.1% of valid tumor samples, respectively. The reported prevalence of phosphorylated MET in our material is consistent with results demonstrated by other investigators. [19–21]. However, a lower grade of prevalence has been observed by other reports [22–24]. Using various methods to detect the prevalence of phosphorylated MET in FFPE tissue, Dua et al[25] demonstrated that their c-MET FFPE assay could detect and quantify c-MET receptor levels in FFPE tumor specimens, and that these measurements would correlate well with measurements obtained by conventional methods.

A high chromosome 7 count, higher than 2 copies was found in 21.5% of valid tumor samples, while 6% of tumor samples showed polysomy, i.e. a chromosome 7 count higher than 3. A MET gene to chromosome 7 ratio higher than 1 was observed in 7.1% of valid tumors, while an IGF1R gene to chromosome 15 ratio higher than 1 was observed in 21.6% of valid tumors. In the male cohort population, 6.1% of valid tumors showed an IGF1R gene to chromosome 15 ratio higher than 1, p = 0.015, while this was observed in 15.5% of all females with NSCLC, p = 0.021. An EGFRdel mutated protein indicating a gene mutation was found in 6% of all valid patients (5.8% of males and 6.6% of women) while 5.4% showed an EGFRmut mutated protein, indicating a mutated gene (4.6% of males and 8% of women). Prevalence of investigated factors is shown (Table 2).The above-mentioned markers did not correlate with age, gender, smoking, WHO performance status, or vascular infiltration.

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Table 2. Prognostic Effect of MET, MET gene copy number, polysomy of chromosome 7, IGF1R gene copy number and IGF-1 Expression in Tumor Epithelial Cells of primary NSCLC in 326 patients (univariate analysis; log-rank test).

https://doi.org/10.1371/journal.pone.0181527.t002

Univariate analysis

Results from the univariate analysis regarding the clinical variables and their impact on DSS are presented in Table 2. T-stage (P<0.001), N-stage (P<0.001), pathological stage (P<0.001), WHO performance status (p = 0.016), histology (P = 0.028), vascular infiltration (P = 0.001), differentiation (p<0.001) and surgical procedure (p = 0.007) were significant prognosticators for the total patient population.

High cytoplasmic MET expression showed a significant negative prognostic effect only in patients with adenocarcinoma (p = 0.026), but not for the whole cohort (p = 0.411; Table 3 and Fig 3).MET gen copy count to chromosome 7 ratio>1 was a significant positive prognostic marker (p = 0.005). We sought to determine if different cut-off points for the MET gene/chromosome 7 ratio would provide an additional prognostic significance. A MET gene/chromosome 7 ratio >1.5 (p = 0.21) did not show a prognostic significance. The same applies for a MET gene/chromosome 7 ratio >2 (p = 0.43). However, the polysomy of chromosome 7 emerged as a highly specific (p = 0.002) negative prognosticator for all patients.IGF1Rgene copy number was a highly negative prognostic marker for all NSCLC patients (p = 0.037), but was even more significant in males (p = 0.015) than in females (p = 0.021).Finally, a high IGF-1 expression showed a trend as a negative prognostic marker in males (p = 0.053) but not for the whole cohort population. There was no significant correlation between DSS and tumor epithelial cell expression of the EGFR gene mutations EGFRmut (p = 0.628) and EGFRdel (p = 0.498).

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Fig 3. Kaplan–Meier curves of disease-specific survival.

Survival curves according to (A) MET immunohistochemical expression in patients with adenocarcinoma (B) Chromosome 7 copy number count in patients with NSCLC (C) IGF1R gene copy number to chromosome 15 ratio in NSCLC patients, and (D) IGF-1 immunohistochemical expression in patients with NSCLC.

https://doi.org/10.1371/journal.pone.0181527.g003

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Table 3. Results of Cox regression analysis summarizing significant independent prognostic factors for disease-specific survival.

https://doi.org/10.1371/journal.pone.0181527.t003

Multivariate Cox proportional hazards analysis

Significant clinic pathological and molecular variables from the univariate analyses were entered into the multivariate analysis. The statistically significant results are presented in Table 3.

Polysomy of chromosome 7 in tumor cells correlated significantly and independently with a poor prognosis (HR: 2.29; 95% CI: 1.21–4.35; p = 0.011). In patients with adenocarcinoma, a high cytoplasmic MET expression was an independent negative prognostic factor (HR: 2.85; 95% CI: 1.25–6.50; p = 0.013). In males with NSCLC a high IGF1R gene copy number to chromosome 15 count ratio higher than one, was significantly and independently correlated to a poor prognosis (HR: 2.67; 95% CI: 1.18–6.00; p = 0.018). For the whole cohort, the histologic subtypes did not reach a statistically prognostic significance, neither for the group of adenocarcinoma (p = 0.165), nor for the group of squamous cell carcinoma (p = 0.7) or for the large cell carcinoma group (p = 0.466).

Discussion

The chief aim of our study was to investigate the prognostic role of MET expression and the MET gene copy number gain in NSCLC. In addition to IHC we used SISH to investigate the METgene. While, in the adenocarcinoma patient population, independent of other clinicopathological variables, a high cytoplasmic MET expression was a significant negative prognosticator, as determined by IHC, a similar correlation was not found in patients with MET gene copy number gain, i.e. the absolute MET gene copy number in tumor cells did not affect the prognosis. Surprisingly, investigating the ratio of MET gene to chromosome 7, we found a trend of better survival in patients with a higher ratio in the whole cohort in the univariate analysis, but not in the multivariate analysis. However, a high MET gene to chromosome 7 ratio appeared to be a rather non-frequent event. We observed a MET gene to chromosome 7 ratio higher than 1 in 7.1% of our non-selected patients. This is in agreement with recent published data by Noonan et al[26], where–using fluorescence in situ hybridization in lung adenocarcinoma- they observed a MET gene to chromosome 7 ratio of 1.8 or higher only in 4.5% of adenocarcinoma patients. The rather confusing positive prognostic effect of a high MET gene to chromosome 7 ratio led us to investigate a possible role of chromosome 7 polysomy in NSCLC patients. Interestingly, we found a high chromosome 7 count, higher than three chromosome copies as a highly independent negative prognostic factor for the whole cohort population. We assumed that a dysfunction in the transcriptional or posttranscriptional controlling mechanisms could partly explain the discrepancies between the IHC and SISH results; still we wanted to explore other genes located on chromosome 7. Chromosome 7 is known to harbor genes whose alteration my play an important role in multiple diseases as cystic fibrosis[27], but also in tumorigenesis [28]with over than 1150 protein- coding genes, 605 of which have been validated by transcript sequences.[29] Nevertheless, three genes; EGFR, MET, and BRAF emerge as of special interest in NSCLC. Both MET and EGFR gene amplification are described to have a critical predictive role in NSCLC. [30]BRAF appears to have a role as a predictive marker in patients with advanced melanoma disease [31, 32], with a rather limited therapeutic effect followed by resistance development in NSCLC patients.[33] There are reports [34–36] proposing a mechanism for MET and EGFR axis regulation mediated by miRNAs. Additionally, MET protein activation has been associated with primary resistance to EGFR tyrosine kinase inhibitor (TKI) therapy in NSCLC patients (Fig 1).[13, 37] These observations have initiated a scientific debate about novel bispecific EGFR/MET inhibitors to obtain better therapeutic results. [10, 38, 39]In the light of this, we sought to determine any correlation between MET expression, MET gene copy number count (GCNC) and two of the most observed EGFR gene mutations in NSCLC; EGFRmut and EGFRdel [40, 41]in untreated patients. A critical issue in investigating EGFR mutations using immunohistochemistry was finding antibodies with an acceptable sensitivity and specificity. The specificity of the used antibodies has been certified by the manufacturer both by means of Western blot and flow cytometric analysis[42, 43] Additionally, Several groups[44–46] have concluded with an acceptable specificity and sensitivity of the aforementioned antibodies. Brevet et al. [47] reported that IHC using the EGFR L858R specific antibody showed a sensitivity of 95.2% and a specificity of 98.8%. They further found that the EGFR exon 19 mutant specific antibody would detect 100% of 15-bp (base pair) deletions with a high specificity, however, a significant lower sensitivity of about 48.6% in non-15-bp exon 19 deletions was observed. According to the COSMIC database, Non 15-bp exon 19 deletions account for about 35% of exon 19 deletions[48].

We did not find a statistic correlation between MET expression, MET GCNC and any of the examined EGFR gene mutations. Further, none of the investigated EGFR mutations had a prognostic significance, despite conflicting results by other groups[40, 49, 50].

The essential role of the HGF-MET cellular pathway has been further established following observations on MET-null mutant mice embryos, with malformation of liver, placenta,[7] melanocytes,[51] and testis.[52] Meanwhile, it has been observed that overexpression of MET can have an oncogenic potential by itself and can induce hepatocellular carcinoma in liver cells [53]. There is established evidence [54] that MET is sufficient for transformation of normal human osteoblasts causing an osteosarcoma-like disease in vivo. Furthermore, dysregulation of the HGF-MET pathway has been demonstrated in malignancies of epithelial cell origin, represented by carcinomas of the lung, mamma, hepatic cells, pancreas ovaries, papillary renal carcinoma, papillary thyroid carcinoma, and carcinomas of the colorectal system.[6]Dysregulation of the HGF-MET cellular axis may due to MET gene mutations, MET amplification, chromosomal rearrangement, MET transcriptional upregulation or changes in the autocrine or paracrine signaling. Several studies have investigated the prognostic role of the MET receptor and the MET gene alteration in NSCLC. While some groups investigated a rather small group of patients [55–57], other groups found a negative prognostic effect of high MET protein expression and MET gene copy number gain either independent of the histologic type[58–63] or only in patients with adenocarcinoma[64] or squamous cell carcinoma[65].

Exploring both the MET protein expression and the MET GCNC in 140 NSCLC patients, Dziadziuszko et al[66] concluded that neither was associated with prognosis. Meanwhile, Tran et al[67] observed that MET overexpression and MET high (GCNC) occur in a low proportion of primary NSCLCs and are associated with a good prognosis. Awad et al [68]reported MET exon 14 mutations to occur in rather older patients and that they may represent a clinically unique molecular subtype and a possible important therapeutical target in NSCLC. Preliminary findings from the PROFILE 1001 trial [69] show that crizotinib demonstrates a meaningful antitumor activity in patients with NSCLC harboring MET exon 14 alterations. Similar results were also recently published by Lu et al[70].

Recent studies have proposed a ligand-independent MET activation.[71, 72] Using prostate cancer cell lines, Varkaris et al,[73] proposed a full but rather delayed activation of MET through IGF1R (Fig 1). Consequently, we aimed to determine a possible correlation between the MET receptor expression, MET GCNC, and the IGF-1 axis.

MET receptor expression did not show a significant correlation with the MET GCNC (p = 0,77), the IGF1R GCNC (p = 0.64) or IGF-1 expression (p = 0.31).

However, investigating the prognostic significance of IGF-1 expression in epithelial NSCLC showed a trend towards worse survival. Furthermore, investigating the ratio of IGF1R GCNC to chromosome 15, this ratio immerged as a highly significant and independent negative prognostic indicator for disease specific survival in males, whereas it did not show prognostic influence in females. Gender differences in cancer tumorigenesis and survival are most likely associated with different sex hormone effects on various genes. While there are promising preliminary results among breast cancer and NSCLC patients treated with the IGF1R inhibitor Dalotuzumab,[74] according to our results, the subgroup of male patients with NSCLC appears to benefit the most of such a treatment. Promising preclinical trials investigating the role of IGF1R as a therapeutical target has resulted in the initiating of clinical trials on patients with multiple myeloma.[75]There are studies[76] suggesting IGF1R as a potential target in NSCLC treatment. While Tsuata et al[77] and Capuzzo et al[78]reported that the IGF1R expression did not represent a prognostic factor in resected NSCLC patients, other research groups[79–81] have either found a negative prognostic significance of high IGF1R expression or IGF1R GCNC.[82] However, future results of stratified treatment-trials among NSCLC patients will be needed to fortify these results.

Our current prognostic findings as detected by SISH for IGF1R and by immunohistochemistry for MET have a relevant practical implementation. While IGF1R SISH analyses seems to give additional information about the subgroup of NSCLC patients, who most likely would benefit of an anti-IGF1R therapy regimen, the MET SISH analyses seem to be biased when investigating the ratio of gene copy number to chromosome count due to the highly negative prognostic significance of chromosome 7 polysomy, higher than 3 chromosome copies. Our results would most probably apply to any SISH analysis investigating a gene to chromosome ratio located on chromosome 7. Even thought our findings would apply to small subgroups of patients, still finding therapeutic aid to these subgroups would be regarded as a significant step towards personalized NSCLC treatment. Until this, every effort should be made to find more specific and even more personalized potential molecular targets whose status in tumor samples might impact therapeutic responses.

Supporting information

S1 Table. Minimal data set.

Data set with scoring results for all the markers included in the study.

https://doi.org/10.1371/journal.pone.0181527.s001

(SAV)

Acknowledgments

The authors would like to acknowledge Roche Diagnostics Norway for providing our research laboratory with the two SISH antibodies MET gene and IGF1R. The publication charges for this article have been funded by a grant from the publication fund of UiT The Arctic University of Norway. Roche Diagnostics Norway and the publication fund of UiT had no role in study design, data collection and analysis, results, decision to publish, or preparation of the manuscript.

References

  1. 1. Torre LA, Siegel RL, Jemal A. Lung Cancer Statistics. Adv Exp Med Biol. 2016;893:1–19. pmid:26667336.
  2. 2. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87–108. pmid:25651787.
  3. 3. Sattler M, Reddy MM, Hasina R, Gangadhar T, Salgia R. The role of the c-Met pathway in lung cancer and the potential for targeted therapy. Ther Adv Med Oncol. 2011;3(4):171–84. pmid:21904579; PubMed Central PMCID: PMCPMC3150066.
  4. 4. Wang Y, Selden C, Farnaud S, Calnan D, Hodgson HJ. Hepatocyte growth factor (HGF/SF) is expressed in human epithelial cells during embryonic development; studies by in situ hybridisation and northern blot analysis. J Anat. 1994;185 (Pt 3):543–51. pmid:7649790; PubMed Central PMCID: PMCPMC1166661.
  5. 5. Xian CJ, Couper R, Howarth GS, Read LC, Kallincos NC. Increased expression of HGF and c-met in rat small intestine during recovery from methotrexate-induced mucositis. Br J Cancer. 2000;82(4):945–52. pmid:10732770; PubMed Central PMCID: PMCPMC2374397.
  6. 6. Ariyawutyakorn W, Saichaemchan S, Varella-Garcia M. Understanding and Targeting MET Signaling in Solid Tumors—Are We There Yet? J Cancer. 2016;7(6):633–49. pmid:27076844; PubMed Central PMCID: PMCPMC4829549.
  7. 7. Birchmeier C, Gherardi E. Developmental roles of HGF/SF and its receptor, the c-Met tyrosine kinase. Trends Cell Biol. 1998;8(10):404–10. pmid:9789329.
  8. 8. Peters S, Adjei AA. MET: a promising anticancer therapeutic target. Nature reviews Clinical oncology. 2012;9(6):314–26. Epub 2012/05/09. pmid:22566105.
  9. 9. Trusolino L, Comoglio PM. Scatter-factor and semaphorin receptors: cell signalling for invasive growth. Nature reviews Cancer. 2002;2(4):289–300. Epub 2002/05/11. pmid:12001990.
  10. 10. Spigel DR, Ervin TJ, Ramlau RA, Daniel DB, Goldschmidt JH, Blumenschein GR, et al. Randomized phase II trial of Onartuzumab in combination with erlotinib in patients with advanced non-small-cell lung cancer. J Clin Oncol. 2013;31(32):4105–14. pmid:24101053; PubMed Central PMCID: PMCPMC4878106.
  11. 11. Spigel DR, Edelman MJ, O'Byrne K, Paz-Ares L, Mocci S, Phan S, et al. Results From the Phase III Randomized Trial of Onartuzumab Plus Erlotinib Versus Erlotinib in Previously Treated Stage IIIB or IV Non-Small-Cell Lung Cancer: METLung. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2017;35(4):412–20. Epub 2016/12/13. pmid:27937096.
  12. 12. Ou S-HI, Kwak EL, Siwak-Tapp C, Dy J, Bergethon K, Clark JW, et al. Activity of crizotinib (PF02341066), a dual mesenchymal-epithelial transition (MET) and anaplastic lymphoma kinase (ALK) inhibitor, in a non-small cell lung cancer patient with de novo MET amplification. J Thorac Oncol. 2011;6(5):942–6. pmid:21623265.
  13. 13. Owusu BY, Bansal N, Venukadasula PKM, Ross LJ, Messick TE, Goel S, et al. Inhibition of pro-HGF activation by SRI31215, a novel approach to block oncogenic HGF/MET signaling. Oncotarget. 2016. pmid:27121052.
  14. 14. Travis WD, Brambilla E, Nicholson AG, Yatabe Y, Austin JH, Beasley MB, et al. The 2015 World Health Organization Classification of Lung Tumors: Impact of Genetic, Clinical and Radiologic Advances Since the 2004 Classification. J Thorac Oncol. 2015;10(9):1243–60. Epub 2015/08/21. pmid:26291008.
  15. 15. Rami-Porta R, Crowley JJ, Goldstraw P. The revised TNM staging system for lung cancer. Ann Thorac Cardiovasc Surg. 2009;15(1):4–9. Epub 2009/03/06. pmid:19262443.
  16. 16. Donnem T, Al-Saad S, Al-Shibli K, Delghandi MP, Persson M, Nilsen MN, et al. Inverse prognostic impact of angiogenic marker expression in tumor cells versus stromal cells in non small cell lung cancer. Clin Cancer Res. 2007;13(22 Pt 1):6649–57. pmid:18006765.
  17. 17. Al-Saad S, Al-Shibli K, Donnem T, Andersen S, Bremnes RM, Busund L-T. Clinical significance of epidermal growth factor receptors in non-small cell lung cancer and a prognostic role for HER2 gene copy number in female patients. J Thorac Oncol. 2010;5(10):1536–43. pmid:20802349.
  18. 18. Polónia A, Leitão D, Schmitt F. Application of the 2013 ASCO/CAP guideline and the SISH technique for HER2 testing of breast cancer selects more patients for anti-HER2 treatment. Virchows Arch. 2016;468(4):417–23. pmid:26754674.
  19. 19. Ichimura E, Maeshima A, Nakajima T, Nakamura T. Expression of c-met/HGF receptor in human non-small cell lung carcinomas in vitro and in vivo and its prognostic significance. Japanese journal of cancer research: Gann. 1996;87(10):1063–9. Epub 1996/10/01. pmid:8957065.
  20. 20. Levallet G, Vaisse-Lesteven M, Le Stang N, Ilg AG, Brochard P, Astoul P, et al. Plasma cell membrane localization of c-MET predicts longer survival in patients with malignant mesothelioma: a series of 157 cases from the MESOPATH Group. Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer. 2012;7(3):599–606. Epub 2012/01/17. pmid:22246193.
  21. 21. Sun W, Ai T, Gao Y, Zhang Y, Cui J, Song L. Expression and prognostic relevance of MET and phospho-BAD in non-small cell lung cancer. OncoTargets and therapy. 2013;6:1315–23. Epub 2013/10/05. pmid:24092988; PubMed Central PMCID: PMCPMC3787924.
  22. 22. Peng Z, Li Z, Gao J, Lu M, Gong J, Tang ET, et al. Tumor MET Expression and Gene Amplification in Chinese Patients with Locally Advanced or Metastatic Gastric or Gastroesophageal Junction Cancer. Molecular cancer therapeutics. 2015;14(11):2634–41. Epub 2015/09/04. pmid:26330547.
  23. 23. Lapere C, Cortot AB, Gregoire V, Cockenpot V, Tulasne D, Copin MC. Preferential Localization of MET Expression at the Invasion Front and in Spreading Cells Through Air Spaces in Non-Small Cell Lung Carcinomas. The American journal of surgical pathology. 2017;41(3):414–22. Epub 2017/01/19. pmid:28098570.
  24. 24. Tsuta K, Kozu Y, Mimae T, Yoshida A, Kohno T, Sekine I, et al. c-MET/phospho-MET protein expression and MET gene copy number in non-small cell lung carcinomas. Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer. 2012;7(2):331–9. Epub 2011/12/27. pmid:22198430.
  25. 25. Dua R, Zhang J, Parry G, Penuel E. Detection of hepatocyte growth factor (HGF) ligand-c-MET receptor activation in formalin-fixed paraffin embedded specimens by a novel proximity assay. PloS one. 2011;6(1):e15932. Epub 2011/02/02. pmid:21283737; PubMed Central PMCID: PMCPMC3024969.
  26. 26. Noonan SA, Berry L, Lu X, Gao D, Baron AE, Chesnut P, et al. Identifying the Appropriate FISH Criteria for Defining MET Copy Number-Driven Lung Adenocarcinoma through Oncogene Overlap Analysis. Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer. 2016;11(8):1293–304. Epub 2016/06/06. pmid:27262212.
  27. 27. Bazett M, Stefanov AN, Paun A, Paradis J, Haston CK. Strain-dependent airway hyperresponsiveness and a chromosome 7 locus of elevated lymphocyte numbers in cystic fibrosis transmembrane conductance regulator-deficient mice. J Immunol. 2012;188(5):2297–304. pmid:22287709.
  28. 28. Tsiambas E, Ragos V, Lefas AY, Georgiannos SN, Grapsa D, Grapsa D, et al. Chromosome 7 deregulation in non-small cell lung carcinoma molecular landscape. J BUON. 2015;20(6):1635–9. pmid:26854464.
  29. 29. Hillier LW, Fulton RS, Fulton LA, Graves TA, Pepin KH, Wagner-McPherson C, et al. The DNA sequence of human chromosome 7. Nature. 2003;424(6945):157–64. pmid:12853948.
  30. 30. Zhang Y, Wang W, Wang Y, Xu Y, Tian Y, Huang M, et al. Response to Crizotinib Observed in Lung Adenocarcinoma with MET Copy Number Gain but without a High-Level MET/CEP7 Ratio, MET Overexpression, or Exon 14 Splicing Mutations. J Thorac Oncol. 2016;11(5):e59–62. pmid:26724472.
  31. 31. Thompson JA. Major Changes in Systemic Therapy for Advanced Melanoma. J Natl Compr Canc Netw. 2016;14(5 Suppl):638–40. pmid:27226502.
  32. 32. Ribas A, Gonzalez R, Pavlick A, Hamid O, Gajewski TF, Daud A, et al. Combination of vemurafenib and cobimetinib in patients with advanced BRAF(V600)-mutated melanoma: a phase 1b study. Lancet Oncol. 2014;15(9):954–65. pmid:25037139.
  33. 33. Kim S-M, Kim H, Jang KW, Kim MH, Sohn J, Yun MR, et al. EGFR-mediated reactivation of MAPK signaling induces acquired resistance to GSK2118436 in BRAF V600E mutant NSCLC cell lines. Mol Cancer Ther. 2016. pmid:27196768.
  34. 34. Acunzo M, Romano G, Palmieri D, Laganá A, Garofalo M, Balatti V, et al. Cross-talk between MET and EGFR in non-small cell lung cancer involves miR-27a and Sprouty2. Proc Natl Acad Sci U S A. 2013;110(21):8573–8. pmid:23650389; PubMed Central PMCID: PMCPMC3666747.
  35. 35. Garofalo M, Romano G, Di Leva G, Nuovo G, Jeon Y-J, Ngankeu A, et al. EGFR and MET receptor tyrosine kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung cancers. Nat Med. 2012;18(1):74–82. pmid:22157681; PubMed Central PMCID: PMCPMC3467100.
  36. 36. Zhen Q, Liu J, Gao L, Liu J, Wang R, Chu W, et al. MicroRNA-200a Targets EGFR and c-Met to Inhibit Migration, Invasion, and Gefitinib Resistance in Non-Small Cell Lung Cancer. Cytogenet Genome Res. 2015;146(1):1–8. pmid:26184032.
  37. 37. van der Wekken AJ, Saber A, Hiltermann TJN, Kok K, van den Berg A, Groen HJM. Resistance mechanisms after tyrosine kinase inhibitors afatinib and crizotinib in non-small cell lung cancer, a review of the literature. Crit Rev Oncol Hematol. 2016;100:107–16. pmid:26852079.
  38. 38. Lee JM, Lee SH, Hwang J-W, Oh SJ, Kim B, Jung S, et al. Novel strategy for a bispecific antibody: induction of dual target internalization and degradation. Oncogene. 2016. pmid:26853467.
  39. 39. Castoldi R, Ecker V, Wiehle L, Majety M, Busl-Schuller R, Asmussen M, et al. A novel bispecific EGFR/Met antibody blocks tumor-promoting phenotypic effects induced by resistance to EGFR inhibition and has potent antitumor activity. Oncogene. 2013;32(50):5593–601. pmid:23812422; PubMed Central PMCID: PMCPMC3898114.
  40. 40. Rossi S, D'Argento E, Basso M, Strippoli A, Dadduzio V, Cerchiaro E, et al. Different EGFR Gene Mutations in Exon 18, 19 and 21 as Prognostic and Predictive Markers in NSCLC: A Single Institution Analysis. Mol Diagn Ther. 2016;20(1):55–63. pmid:26645830.
  41. 41. Boch C, Kollmeier J, Roth A, Stephan-Falkenau S, Misch D, Grüning W, et al. The frequency of EGFR and KRAS mutations in non-small cell lung cancer (NSCLC): routine screening data for central Europe from a cohort study. BMJ Open. 2013;3(4). pmid:23558737; PubMed Central PMCID: PMCPMC3641502.
  42. 42. Technology CS. EGF Receptor (L858R Mutant Specific) (43B2) Rabbit mAb #3197. 2017.
    • 43. Signaling C. EGF Receptor (E746-A750del Specific) (D6B6) XP® Rabbit mAb #2085. 2017.
      • 44. Kawahara A, Yamamoto C, Nakashima K, Azuma K, Hattori S, Kashihara M, et al. Molecular diagnosis of activating EGFR mutations in non-small cell lung cancer using mutation-specific antibodies for immunohistochemical analysis. Clinical cancer research: an official journal of the American Association for Cancer Research. 2010;16(12):3163–70. Epub 2010/04/29. pmid:20423982.
      • 45. Yu J, Kane S, Wu J, Benedettini E, Li D, Reeves C, et al. Mutation-specific antibodies for the detection of EGFR mutations in non-small-cell lung cancer. Clinical cancer research: an official journal of the American Association for Cancer Research. 2009;15(9):3023–8. Epub 2009/04/16. pmid:19366827.
      • 46. Riely GJ, Pao W, Pham D, Li AR, Rizvi N, Venkatraman ES, et al. Clinical course of patients with non-small cell lung cancer and epidermal growth factor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib. Clinical cancer research: an official journal of the American Association for Cancer Research. 2006;12(3 Pt 1):839–44. Epub 2006/02/10. pmid:16467097.
      • 47. Brevet M, Arcila M, Ladanyi M. Assessment of EGFR mutation status in lung adenocarcinoma by immunohistochemistry using antibodies specific to the two major forms of mutant EGFR. The Journal of molecular diagnostics: JMD. 2010;12(2):169–76. Epub 2010/01/23. pmid:20093391; PubMed Central PMCID: PMCPMC2871723.
      • 48. Forbes SA, Bindal N, Bamford S, Cole C, Kok CY, Beare D, et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic acids research. 2011;39(Database issue):D945–50. Epub 2010/10/19. pmid:20952405; PubMed Central PMCID: PMCPMC3013785.
      • 49. Hsu F, De Caluwe A, Anderson D, Nichol A, Toriumi T, Ho C. EGFR mutation status on brain metastases from non-small cell lung cancer. Lung Cancer. 2016;96:101–7. pmid:27133758.
      • 50. Li F, Du X, Zhang H, Ju T, Chen C, Qu Q, et al. Next Generation Sequencing of Chinese Stage IV Lung Cancer Patients Reveals an Association between EGFR Mutation Status and Survival Outcome. Clin Genet. 2016. pmid:27221040.
      • 51. Kos L, Aronzon A, Takayama H, Maina F, Ponzetto C, Merlino G, et al. Hepatocyte growth factor/scatter factor-MET signaling in neural crest-derived melanocyte development. Pigment Cell Res. 1999;12(1):13–21. pmid:10193678.
      • 52. Ricci G, Catizone A, Galdieri M. Expression and functional role of hepatocyte growth factor and its receptor (c-met) during fetal mouse testis development. J Endocrinol. 2006;191(3):559–70. pmid:17170214.
      • 53. Wang R, Ferrell LD, Faouzi S, Maher JJ, Bishop JM. Activation of the Met receptor by cell attachment induces and sustains hepatocellular carcinomas in transgenic mice. J Cell Biol. 2001;153(5):1023–34. pmid:11381087; PubMed Central PMCID: PMCPMC2174327.
      • 54. Patanè S, Avnet S, Coltella N, Costa B, Sponza S, Olivero M, et al. MET overexpression turns human primary osteoblasts into osteosarcomas. Cancer Res. 2006;66(9):4750–7. pmid:16651428.
      • 55. Kim IH, Lee IH, Lee JE, Hong SH, Kim TJ, Lee KY, et al. Prognostic Impact of Multiple Clinicopathologic Risk Factors and c-MET Overexpression in Patients Who Have Undergone Resection of Stage IB Non-Small-Cell Lung Cancer. Clinical lung cancer. 2016;17(5):e31–e43. Epub 2016/03/20. pmid:26992846.
      • 56. Casadevall D, Gimeno J, Clave S, Taus A, Pijuan L, Arumi M, et al. MET expression and copy number heterogeneity in nonsquamous non-small cell lung cancer (nsNSCLC). Oncotarget. 2015;6(18):16215–26. Epub 2015/06/05. pmid:26041880; PubMed Central PMCID: PMCPMC4599265.
      • 57. Masuya D, Huang C, Liu D, Nakashima T, Kameyama K, Haba R, et al. The tumour-stromal interaction between intratumoral c-Met and stromal hepatocyte growth factor associated with tumour growth and prognosis in non-small-cell lung cancer patients. British journal of cancer. 2004;90(8):1555–62. Epub 2004/04/15. pmid:15083185; PubMed Central PMCID: PMCPMC2409699.
      • 58. Cappuzzo F, Marchetti A, Skokan M, Rossi E, Gajapathy S, Felicioni L, et al. Increased MET gene copy number negatively affects survival of surgically resected non-small-cell lung cancer patients. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2009;27(10):1667–74. Epub 2009/03/04. pmid:19255323; PubMed Central PMCID: PMCPMC3341799.
      • 59. Guo B, Cen H, Tan X, Liu W, Ke Q. Prognostic value of MET gene copy number and protein expression in patients with surgically resected non-small cell lung cancer: a meta-analysis of published literatures. PloS one. 2014;9(6):e99399. Epub 2014/06/13. pmid:24922520; PubMed Central PMCID: PMCPMC4055667.
      • 60. Park S, Choi YL, Sung CO, An J, Seo J, Ahn MJ, et al. High MET copy number and MET overexpression: poor outcome in non-small cell lung cancer patients. Histology and histopathology. 2012;27(2):197–207. Epub 2011/12/31. pmid:22207554.
      • 61. Lim EH, Zhang SL, Li JL, Yap WS, Howe TC, Tan BP, et al. Using whole genome amplification (WGA) of low-volume biopsies to assess the prognostic role of EGFR, KRAS, p53, and CMET mutations in advanced-stage non-small cell lung cancer (NSCLC). Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer. 2009;4(1):12–21. Epub 2008/12/20. pmid:19096301.
      • 62. Beau-Faller M, Ruppert AM, Voegeli AC, Neuville A, Meyer N, Guerin E, et al. MET gene copy number in non-small cell lung cancer: molecular analysis in a targeted tyrosine kinase inhibitor naive cohort. Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer. 2008;3(4):331–9. Epub 2008/04/02. pmid:18379349.
      • 63. Siegfried JM, Weissfeld LA, Singh-Kaw P, Weyant RJ, Testa JR, Landreneau RJ. Association of immunoreactive hepatocyte growth factor with poor survival in resectable non-small cell lung cancer. Cancer research. 1997;57(3):433–9. Epub 1997/02/01. pmid:9012470.
      • 64. Dimou A, Non L, Chae YK, Tester WJ, Syrigos KN. MET gene copy number predicts worse overall survival in patients with non-small cell lung cancer (NSCLC); a systematic review and meta-analysis. PloS one. 2014;9(9):e107677. Epub 2014/09/19. pmid:25232729; PubMed Central PMCID: PMCPMC4169409.
      • 65. Go H, Jeon YK, Park HJ, Sung SW, Seo JW, Chung DH. High MET gene copy number leads to shorter survival in patients with non-small cell lung cancer. Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer. 2010;5(3):305–13. Epub 2010/01/29. pmid:20107422.
      • 66. Dziadziuszko R, Wynes MW, Singh S, Asuncion BR, Ranger-Moore J, Konopa K, et al. Correlation between MET gene copy number by silver in situ hybridization and protein expression by immunohistochemistry in non-small cell lung cancer. Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer. 2012;7(2):340–7. Epub 2012/01/13. pmid:22237262; PubMed Central PMCID: PMCPMC3358920.
      • 67. Tran TN, Selinger CI, Kohonen-Corish MR, McCaughan B, Kennedy C, O'Toole SA, et al. Alterations of MET Gene Copy Number and Protein Expression in Primary Non-Small-Cell Lung Cancer and Corresponding Nodal Metastases. Clinical lung cancer. 2016;17(1):30–8.e1. Epub 2015/09/24. pmid:26395411.
      • 68. Awad MM, Oxnard GR, Jackman DM, Savukoski DO, Hall D, Shivdasani P, et al. MET Exon 14 Mutations in Non-Small-Cell Lung Cancer Are Associated With Advanced Age and Stage-Dependent MET Genomic Amplification and c-Met Overexpression. J Clin Oncol. 2016;34(7):721–30. Epub 2016/01/06. pmid:26729443.
      • 69. AE D. Efficacy and safety of crizotinib in patients (pts) with advanced MET exon 14-altered non-small cell lung cancer (NSCLC). Journal of Clinical Ooncology. 2016;34(Suppl; abstr. 108).
      • 70. Lu X, Peled N, Greer J, Wu W, Choi P, Berger AH, et al. MET exon 14 mutation encodes an actionable therapeutic target in lung adenocarcinoma. Cancer Res. 2017. Epub 2017/05/20. pmid:28522754.
      • 71. Dulak AM, Gubish CT, Stabile LP, Henry C, Siegfried JM. HGF-independent potentiation of EGFR action by c-Met. Oncogene. 2011;30(33):3625–35. pmid:21423210; PubMed Central PMCID: PMCPMC3126872.
      • 72. Stabile LP, He G, Lui VWY, Thomas S, Henry C, Gubish CT, et al. c-Src activation mediates erlotinib resistance in head and neck cancer by stimulating c-Met. Clin Cancer Res. 2013;19(2):380–92. pmid:23213056; PubMed Central PMCID: PMCPMC3549019.
      • 73. Varkaris A, Gaur S, Parikh NU, Song JH, Dayyani F, Jin J-K, et al. Ligand-independent activation of MET through IGF-1/IGF-1R signaling. Int J Cancer. 2013;133(7):1536–46. pmid:23526299; PubMed Central PMCID: PMCPMC3713179.
      • 74. Scartozzi M, Bianconi M, Maccaroni E, Giampieri R, Berardi R, Cascinu S. Dalotuzumab, a recombinant humanized mAb targeted against IGFR1 for the treatment of cancer. Curr Opin Mol Ther. 2010;12(3):361–71. pmid:20521225.
      • 75. Menu E, van Valckenborgh E, van Camp B, Vanderkerken K. The role of the insulin-like growth factor 1 receptor axis in multiple myeloma. Archives of physiology and biochemistry. 2009;115(2):49–57. Epub 2009/02/24. pmid:19234898.
      • 76. Agullo-Ortuno MT, Diaz-Garcia CV, Agudo-Lopez A, Perez C, Cortijo A, Paz-Ares L, et al. Relevance of insulin-like growth factor 1 receptor gene expression as a prognostic factor in non-small-cell lung cancer. Journal of cancer research and clinical oncology. 2015;141(1):43–53. Epub 2014/08/02. pmid:25081930.
      • 77. Tsuta K, Mimae T, Nitta H, Yoshida A, Maeshima AM, Asamura H, et al. Insulin-like growth factor-1 receptor protein expression and gene copy number alterations in non-small cell lung carcinomas. Human pathology. 2013;44(6):975–82. Epub 2012/12/26. pmid:23266446.
      • 78. Cappuzzo F, Tallini G, Finocchiaro G, Wilson RS, Ligorio C, Giordano L, et al. Insulin-like growth factor receptor 1 (IGF1R) expression and survival in surgically resected non-small-cell lung cancer (NSCLC) patients. Annals of oncology: official journal of the European Society for Medical Oncology. 2010;21(3):562–7. Epub 2009/09/22. pmid:19767315; PubMed Central PMCID: PMCPMC3362271.
      • 79. Zhao S, Qiu Z, He J, Li L, Li W. Insulin-like growth factor receptor 1 (IGF1R) expression and survival in non-small cell lung cancer patients: a meta-analysis. International journal of clinical and experimental pathology. 2014;7(10):6694–704. Epub 2014/11/18. pmid:25400749; PubMed Central PMCID: PMCPMC4230063.
      • 80. Kim JS, Kim ES, Liu D, Lee JJ, Solis L, Behrens C, et al. Prognostic implications of tumoral expression of insulin like growth factors 1 and 2 in patients with non-small-cell lung cancer. Clinical lung cancer. 2014;15(3):213–21. Epub 2014/02/04. pmid:24485233; PubMed Central PMCID: PMCPMC4572464.
      • 81. Nakagawa M, Uramoto H, Oka S, Chikaishi Y, Iwanami T, Shimokawa H, et al. Clinical significance of IGF1R expression in non-small-cell lung cancer. Clinical lung cancer. 2012;13(2):136–42. Epub 2011/12/03. pmid:22133293.
      • 82. Dziadziuszko R, Merrick DT, Witta SE, Mendoza AD, Szostakiewicz B, Szymanowska A, et al. Insulin-like growth factor receptor 1 (IGF1R) gene copy number is associated with survival in operable non-small-cell lung cancer: a comparison between IGF1R fluorescent in situ hybridization, protein expression, and mRNA expression. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2010;28(13):2174–80. Epub 2010/03/31. pmid:20351332; PubMed Central PMCID: PMCPMC2860435.