Yu QX, Fu PY, Zhang C, Li L, Huang WT. Mesenchymal-epithelial transition factor amplification correlates with adverse pathological features and poor clinical outcome in colorectal cancer. World J Gastrointest Surg 2024; 16(5): 1395-1406 [PMID: 38817281 DOI: 10.4240/wjgs.v16.i5.1395]
Corresponding Author of This Article
Wen-Ting Huang, MD, Chief, Chief Doctor, Professor, Department of Pathology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 113 Baohe Avenue, Longgang District, Shenzhen 518116, Guangdong Province, China. huangwt@cicams.ac.cn
Research Domain of This Article
Pathology
Article-Type of This Article
Basic Study
Open-Access Policy of This Article
This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Qiu-Xiao Yu, Ping-Ying Fu, Chi Zhang, Li Li, Wen-Ting Huang, Department of Pathology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Shenzhen 518116, Guangdong Province, China
Author contributions: Yu QX and Li L designed the research study; Yu QX performed the research; Fu PY and Zhang C contributed new reagents and analytic tools; Yu QX analyzed the data; Yu QX and Huang WT wrote the manuscript; and all authors have read and approve the final manuscript.
Supported bythe National Natural Science Foundation of China, No. 82002829.
Institutional review board statement: The study was reviewed and approved by the ethics committee of Shenzhen Cancer Hospital of Chinese Academy of Medical Sciences (Approval No. KYLX2023-107).
Conflict-of-interest statement: The authors have no relevant financial or non-financial interests to disclose.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Wen-Ting Huang, MD, Chief, Chief Doctor, Professor, Department of Pathology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 113 Baohe Avenue, Longgang District, Shenzhen 518116, Guangdong Province, China. huangwt@cicams.ac.cn
Received: January 10, 2024 Revised: February 22, 2024 Accepted: April 11, 2024 Published online: May 27, 2024 Processing time: 134 Days and 9.1 Hours
Abstract
BACKGROUND
Colorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer-related mortality worldwide. Mesenchymal-epithelial transition factor (MET) gene participates in multiple tumor biology and shows clinical potential for pharmacological manipulation in tumor treatment. MET amplification has been reported in CRC, but data are very limited. Investigating pathological values of MET in CRC may provide new therapeutic and genetic screening options in future clinical practice.
AIM
To determine the pathological significance of MET amplification in CRC and to propose a feasible screening strategy.
METHODS
A number of 205 newly diagnosed CRC patients undergoing surgical resection without any preoperative therapy at Shenzhen Cancer Hospital of Chinese Academy of Medical Sciences were recruited. All patients were without RAS/RAF mutation or microsatellite instability-high. MET amplification and c-MET protein expression were analyzed using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. Correlations between MET aberration and pathological features were detected using the chi-squared test. Progression free survival (PFS) during the two-year follow-up was detected using the Kaplan-Meier method and log rank test. The results of MET FISH and IHC were compared using one-way ANOVA.
RESULTS
Polysomy-induced MET amplification was observed in 14.4% of cases, and focal MET amplification was not detected. Polysomy-induced MET amplification was associated with a higher frequency of lymph node metastasis (LNM) (P < 0.001) and higher tumor budding grade (P = 0.02). In the survival analysis, significant difference was detected between patients with amplified- and non-amplified MET in a two-year follow-up after the first diagnosis (P = 0.001). C-MET scores of 0, 1+, 2+, and 3+ were observed in 1.4%, 24.9%, 54.7%, and 19.0% of tumors, respectively. C-MET overexpression correlated with higher frequency of LNM (P = 0.002), but no significant difference of PFS was detected between patients with different protein levels. In terms of concordance between MET FISH and IHC results, MET copy number showed no difference in c-MET IHC 0/1+ (3.35 ± 0.18), 2+ (3.29 ± 0.11) and 3+ (3.58 ± 0.22) cohorts, and the MET-to-CEP7 ratio showed no difference in three groups (1.09 ± 0.02, 1.10 ± 0.01, and 1.09 ± 0.03).
CONCLUSION
In CRC, focal MET amplification was a rare event. Polysomy-induced MET amplification correlated with adverse pathological characteristics and poor prognosis. IHC was a poor screening tool for MET amplification.
Core Tip: This study aimed to investigate pathological significance of mesenchymal-epithelial transition factor (MET) amplification in therapy-naïve colorectal cancer (CRC) and to propose a feasible screening strategy in clinical practice. In CRC harboring no RAS/RAF mutation or microsatellite instability, focal MET amplification was a rare event, while polysomy-caused MET amplification was observed in 14.4% of patients. Polysomy-caused MET amplification significantly correlated with frequent lymph node metastasis and higher tumor budding grade, which were two independent predictors of unfavorable CRC survival. Consistently, we discovered that MET amplification predicted poor outcome in a two-year follow-up. To our knowledge, this study firstly proved that c-MET immunohistochemistry (IHC) was not a suitable screening tool for MET amplification in CRC, and we recommend that tissue should be prioritized for fluorescence in situ hybridization over IHC to determine MET amplification.
Citation: Yu QX, Fu PY, Zhang C, Li L, Huang WT. Mesenchymal-epithelial transition factor amplification correlates with adverse pathological features and poor clinical outcome in colorectal cancer. World J Gastrointest Surg 2024; 16(5): 1395-1406
The mesenchymal-epithelial transition factor gene (MET) is a proto-oncogene located on chromosome 7q21-31 and encodes the receptor for hepatocyte growth factor[1,2]. The aberrant interaction between c-MET and HGF regulates cell survival, proliferation, migration, and differentiation by stimulating the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathways[2,3]. MET amplification is relevant to the clinical oncogenesis of diverse tumor types, including lung cancer, gastric cancer and papillary renal cell carcinoma[4,5]. The recognition of MET alterations has enhanced the development of MET tyrosine kinase inhibitors (TKIs), antibodies, and antibody-drug conjugates, which were actively investigated in numerous clinical trials and showed great potential[6]. Therefore, the identification of tumors that are oncogenically related to MET is crucial.
Colorectal cancer (CRC) ranks third in terms of incidence and second in mortality globally[7]. MET amplification has been identified in CRC, and the prevalence varies greatly from 2% to 18% due to different detection techniques and criteria applied[8,9]. Moreover, tumor status (therapy-naïve/therapy-resistant, primary/metastasis, mutated/wide-type) are important variables that must be considered. In CRC, the most commonly discussed function of MET amplification is an acquired feedback of the activation of PI3K/AKT and MAPK cascades in patients with resistance to anti-epidermal growth factor receptor (EGFR) therapies[10,11]. However, data on de novo MET amplification in therapy- naïve CRC biology are very limited. The incidence of de novo MET amplification and clinical values in newly diagnosed CRC patients need to be investigated.
In this study, we examined the incidence, pathological and prognostic significance of de novo MET amplification and c-MET expression in therapy- naïve CRC patients. We also investigated the correlation between MET fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) results to propose an efficient screening strategy for future clinical gene detection.
MATERIALS AND METHODS
Patients
From May 2020 to December 2021, 205 newly diagnosed CRC patients who underwent complete surgical resection without any preoperative therapy at Shenzhen Cancer Hospital of Chinese Academy of Medical Sciences were recruited. The criteria for selecting these patients were: (1) Diagnosed as having primary malignant CRC with relevant clinicopathological features; (2) being without other tumors diagnosed simultaneously; and (3) being RAS/RAF wild-type and microsatellite stable. The study was performed in accordance with the Declaration of Helsinki (as revised in 2013) and approved by the ethics committee of Shenzhen Cancer Hospital of Chinese Academy of Medical Sciences. Informed consent was obtained from all patients.
Assessment of histological features
The pathological profiles of patients were obtained from medical records, including gender, age, primary tumor site, histological tumor type, differentiation grade, tumor stage, presence, or absence of lymphovascular invasion (LVI), perineural invasion (PNI), extramural venous invasion (EMVI), lymph node metastasis (LNM), poorly differentiated clusters (PDC), and tumor budding (TB). All samples were sliced at 4 µm to obtain histological sections that were further stained with haematoxylin and eosin (H&E). All H&E slides were reviewed by two experienced pathologists. LVI was defined by the presence of tumor cells within endothelium-lined lymphatic or vascular channels[12]. PNI was defined as tumor cells found within the perineural space or the infiltration of cancer cells into the endoneurium[13]. EMVI was defined as the presence of tumor cells within an endothelium-lined space that was either surrounded by a rim of muscle or contains red blood cells[14]. PDC was defined as small group of more than five cancer cells in tumor stroma without gland formation[15]. TB was defined as the presence of cancer cells detached from the main tumor either as single cells or as clusters of fewer than five cells[16]. According to the three-tier system proposed by the ITBCC, all samples were divided into three categories: 0-4 buds (BD1), 5-9 buds (BD2) and 10 or more buds (BD3) based on the quantification of the buds at the hotspot (in a field measuring 0.785 mm2)[17].
FISH assay and interpretation
After reviewing H&E slides, representative formalin-fixed-paraffin-embedded (FFPE) tumor blocks were selected to determine MET amplification following the manufacturer’s protocol[18]. Briefly, the FFPE slides were pretreated using the Vysis paraffin pretreatment IV & post-hybridisation wash buffer kit (Abbott Molecular, Inc.; 01N31-005) after dewaxing and dehydration. A mixture of the MET (7q31) probe and centromere 7q (CEP7) probe (Abbott Molecular, Inc.; 06N05-020, 06J37-007) was added to the target tissue areas, and the slides were processed using the ThermoBrite Denaturation/Hybridization System (Abbott Molecular, Inc.). FISH signals were reviewed under a fluorescence microscope at a magnification of 600 × (Leica Microsystems GmbH). Two major methods were used to define MET amplification. A: MET amplification was classified as the presence of 5 or more copies of MET signal per cell [MET gene copy number (GCN) ≥ 5] using the Cappuzzo criteria[19]; B: Tumors with a MET/CEP7 ≥ 2 were defined as MET amplification using PathVysion criteria, which is also termed focal amplification[20].
IHC staining and evaluation
Same blocks were selected for c-MET IHC staining. The experiment was performed using the CONFIRM anti-total c-MET (SP44) rabbit monoclonal primary antibody (Ventana Medical Systems, Tucson, AZ; 790-4430) with a Ventana BenchMark XT automated slide processing system according to the manufacturer’s protocol. A pathologist without knowing the clinical information independently evaluated all stained slides. C-MET IHC was evaluated according to the scoring criteria established by Spigel et al[21] based on staining intensity (negative, weak, moderate, or strong) and the prevalence of these intensities in tumor cells. The following four subgroups were defined: 0 (no staining or < 50% of tumor cells with any intensity); 1+ (≥ 50% of tumor cells with weak or higher staining but < 50% with moderate or higher intensity); 2+ (≥ 50% of tumor cells with moderate or higher staining but < 50% with strong intensity); and 3+ (≥ 50% of tumor cells staining with strong intensity)[21].
Statistical analysis
The relationships between clinicopathological features and MET amplification or c-MET protein expression were detected using the chi-squared test in SPSS software version 15.0 (SPSS, Inc). Progression free survival (PFS) was determined using the Kaplan-Meier method, and survival curves were compared using log rank test. The comparison of MET GCN and MET/CEP7 between different c-MET IHC groups was performed using one-way ANOVA with GraphPad Prism software version 5.0 (GraphPad Software, Inc.). P values < 0.05 were considered significant.
RESULTS
General information
A total of 205 CRC patients were included in this study, and all patients had surgical resection tissue samples that were adequate for FISH and IHC analyses. The pathological characteristics of the patients are summarized in Table 1. The median age of disease onset was 60 years (range: 24 to 93 years), and the male-to-female ratio was 1.2. A total of 140 (68.3%) tumors occurred in the colon, and 65 (31.7%) occurred in the rectum. There were 151 (73.7%) tumors defined as exophytic type, and 54 (26.3%) as ulcerative type. The incidences of LVI, PNI, EMVI, LNM, and PDC in our cohort were 31.2%, 28.3%, 15.1%, 43.4%, and 74.1% respectively. In terms of TB, 117 (57.1%) were categorized into BD1, 40 (19.5%) were BD2, and 48 (23.4%) were BD3. Fifty-three (25.9%) CRC patients had poorly differentiated or mucinous carcinoma. According to the criteria of TNM staging after surgery, there were 34 (16.6%) patients with stage I disease, 72 (35.1%) patients with stage II, 80 (39.0%) patients with stage III, and 19 (9.3%) patients with stage IV.
Table 1 Histological and clinical characteristics of colorectal cancer patients, n (%).
Variable
Value
Gender
Male
112 (54.6)
Female
93 (45.4)
Age (yr)
≥ 60
106 (51.7)
< 60
99 (48.3)
Primary site
Colon
140 (68.3)
Rectum
65 (31.7)
Tumor type
Exophytic type
151 (73.7)
Ulcerative type
54 (26.3)
LVI
Present
64 (31.2)
Absent
141 (68.8)
PNI
Present
58 (28.3)
Absent
147 (71.7)
EMVI
Present
31 (15.1)
Absent
174 (84.9)
LNM
Present
89 (43.4)
Absent
116 (56.6)
PDC
Present
152 (74.1)
Absent
53 (25.9)
TB
BD1
117 (57.1)
BD2
40 (19.5)
BD3
48 (23.4)
Histologic differentiation
P/D or mucinous
53 (25.9)
M/D
142 (69.3)
W/D
10 (4.9)
Tumor stage
I
34 (16.6)
II
72 (35.1)
III
80 (39.0)
IV
19 (9.3)
c-MET immunohistochemistry
0
3 (1.4)
1+
51 (24.9)
2+
112 (54.7)
3+
39 (19.0)
MET FISH (Cappuzzo criteria)
MET GCN ≥ 5
28 (14.4)
MET GCN < 5
167 (85.6)
MET FISH (PathVysion criteria)
MET/CEP7 ≥ 2
0 (0.0)
MET/CEP7 < 2
195 (100.0)
MET amplification and c-MET expression in CRC patients
Of all the 205 cases detected, 195 showed interpretable FISH results. Following Cappuzzo criteria, 28 (14.4%) cases were defined as MET amplification (MET copy number ≥ 5) (Figure 1A and B). The PathVysion criteria indicated that the MET/CEP7 ratio was less than 2.0 in all 195 cases (Table 1). These data suggested that chromosome 7 polysomy, rather than focal gene amplification, was preferentially detected in primary lesions of therapy- naïve CRC patients. C-MET IHC was successfully performed in all 205 samples, and immunoreactivity was predominantly observed in the cytoplasm and membrane (Figure 1C-F). C-MET scores of 0, 1+, 2+, and 3+ were observed in 3 (1.4%), 51 (24.9%), 112 (54.7%), and 39 (19.0%) tumors, respectively (Table 1).
Figure 1 C-mesenchymal-epithelial transition factor immunohistochemistry (20 ×) and mesenchymal-epithelial transition factor fluorescence in situ hybridization (100 ×) results.
A: Mesenchymal-epithelial transition factor (MET) fluorescence in situ hybridization (FISH) positive (MET gene copy number ≥ 5 and MET/CEP7 < 2); B: MET FISH negative (MET gene copy number < 5 and MET/CEP7 < 2); C: Immunohistochemistry (IHC) 0; D: IHC 1+; E: IHC 2+; F: IHC 3+.
Correlation between MET amplification, c-MET expression, and clinicopathological features in CRC patients
Polysomy-induced MET amplification significantly correlated with frequent LNM (P < 0.001) and higher grade of TB (P = 0.02), while there were no significant differences regarding gender, age, tumor site, tumor type, LVI, PNI, EMVI, and PDC (P > 0.05) (Table 2).
Table 2 Correlations between polysomy-caused mesenchymal-epithelial transition factor amplification and clinicopathological characteristics of colorectal cancer patients, n (%).
Characteristic
MET FISH
P value
Positive MET GCN ≥ 5 (n = 28)
Negative MET GCN < 5 (n = 167)
Total (n = 195)
Gender
0.975
Male
15 (53.60)
90 (53.90)
105 (53.80)
Female
13 (12.90)
77 (46.21)
90 (46.20)
Age (yr)
0.085
≥ 60
19 (67.90)
84 (50.30)
103 (52.80)
< 60
9 (32.10)
83 (49.70)
92 (47.20)
Primary site
0.077
Colon
23 (82.10)
109 (65.30)
132 (67.70)
Rectum
5 (17.90)
58 (34.70)
63 (32.30)
Tumor type
0.187
Exophytic type
18 (64.30)
127 (76.00)
145 (74.40)
Ulcerative type
10 (35.70)
40 (24.00)
50 (25.60)
LVI
0.261
Present
11 (39.30)
48 (28.70)
59 (30.30)
Absent
17 (60.70)
119 (71.30)
136 (69.70)
PNI
0.377
Present
10 (35.70)
46 (27.50)
56 (28.70)
Absent
18 (64.30)
121 (72.50)
139 (71.30)
EMVI
0.500
Present
6 (21.40)
24 (14.40)
30 (15.40)
Absent
22 (78.60)
143 (85.60)
165 (84.60)
LNM
< 0.001
Present
21 (75.00)
60 (35.90)
81 (41.50)
Absent
7 (25.00)
107 (64.10)
114 (58.50)
PDC
0.153
Present
24 (85.70)
122 (73.10)
146 (74.90)
Absent
4 (14.30)
45 (26.90)
49 (25.10)
TB
0.020
BD1
10 (35.70)
99 (59.30)
109 (55.9)
BD2/3
18 (64.30)
68 (40.70)
86 (44.10)
Histologic differentiation
0.933
P/D or mucinous
7 (25.00)
43 (25.70)
50 (25.60)
W/D or M/D
21 (75.00)
124 (74.30)
145 (74.40)
In terms of c-MET expression, significant correlation was detected between high c-MET IHC scores and occurrence frequency of LNM (P = 0.002), while there was no significant difference regarding other pathological features (Table 3).
Table 3 Correlations between c-mesenchymal-epithelial transition factor protein expression and clinicopathological characteristics of colorectal cancer patients, n (%).
The significance of MET amplification and c-MET expression for patients’ survival
During a two-year follow-up after first diagnosis, 7 out of 28 (25%) CRC patients with de novo MET amplification experienced tumor recurrence or metastasis, while 11 patients were lost to follow up. In 167 patients with non-amplified MET, 13 (7.8%) had tumor recurrence or metastasis, and 58 were lost to follow up. In Kaplan-Meier analysis, significant difference in PFS was found between patients with positive-FISH and negative-FISH in our cohort (P = 0.003) (Figure 2A). The relationship between c-MET expression level and PFS was also investigated, and the recurrence/metastasis rates in group 0/1+, 2+ and 3+ were 11.8% (6/51), 10% (11/110), and 7.9% (3/38) respectively. No significant difference was detected between three IHC groups (P = 0.863) (Figure 2B).
Figure 2 Kaplan-Meier curve of progression free survival for colorectal cancer patients in a two-year follow-up.
A: Progression free survival (PFS) in colorectal cancer (CRC) patient with amplified-mesenchymal-epithelial transition factor (MET) and non-amplified MET; B: PFS in CRC patient with different c-MET protein levels. MET: Mesenchymal-epithelial transition factor; FISH: Fluorescence in situ hybridization; IHC: Immunohistochemistry.
C-MET IHC is a poor screening tool for MET amplification in CRC
Of the 28 (14.4%) patients with polysomy-induced MET amplification, 9 (32%), 10 (36%), and 9 (32%) patients had c-MET immunoscore of 0/1+, 2+ and 3+, respectively. MET FISH was negative in 167 (85.6%) patients, and 41 (24.0%), 98 (59.0%), and 28 (17.0%) were defined as c-MET IHC 0/1+, 2+ and 3+ (Figure 3A). The mean ± SEM of MET copy number showed no difference in the c-MET IHC 0/1+ (3.35 ± 0.18), 2+ (3.29 ± 0.11) and 3+ (3.58 ± 0.22) cohorts (Figure 3B). The MET-to-CEP7 ratio showed no difference in these three groups (1.09 ± 0.02, 1.10 ± 0.01, and 1.09 ± 0.03) (Figure 3C).
Figure 3 Comparison between mesenchymal-epithelial transition factor fluorescence in situ hybridization and immunohistochemistry results.
A: The c-mesenchymal-epithelial transition factor (MET) immunohistochemistry (IHC) status in MET fluorescence in situ hybridization positive and negative cases; B: The scatter plot representations of MET gene copy number in different c-MET IHC groups; C: The scatter plot representations of MET/CEP7 ratios in different c-MET IHC groups. MET: Mesenchymal-epithelial transition factor; GCN: Gene copy number; IHC: Immunohistochemistry.
DISCUSSION
Dysregulated MET signalling participates in tumor biology and may occur via various mechanisms, including protein overexpression, gene amplification and mutation[22]. In non-small cell lung cancer (NSCLC), de novo MET amplification occurs in 2%-4% of patients as primary oncogenic driver[4], and acquired MET amplification was detected in 10%-15% as a response to anti-EGFR drugs[23-25]. Various clinical guidelines recommended MET amplification as an actionable molecular target in the genetic profiling of NSCLC[26]. In CRC, MET amplification is less discussed. Cremolini et al[27] observed MET amplification in 8.5% of resistant CRC cases and suggested that acquired MET amplification might drive resistance to anti-EGFR antibodies in CRC patients. However, the role of de novo MET amplification in primary lesions of therapy-naïve CRC patients remains elusive, and validation of MET amplification in this cohort is critical to this field.
Our study detected MET amplification using FISH, the gold standard for gene copy detection. Currently, there is no consensus on the standardized cut-off value of MET amplification. In clinical trials, two major methods are commonly used. The first one by Cappuzzo et al[19] defines MET amplification as MET GCN ≥ 5, while other studies use GCN ≥ 6[28] or ≥ 15[29] as cut-offs. In our study, 14.4% of CRC had MET amplification (GCN range: 5.05-6.25) based on Cappuzzo criteria. Another research using PCR assay reported a MET amplification rate of 9% in primary CRC lesions[9]. This PCR-based method defined tumor as amplified when the GCN was ≥ 3 times the mean of the corresponding normal tissue. The data discrepancy between this study and ours may be explained by the different methodologies and interpretation criteria. One drawback of the GCN method is that focal amplification cannot be distinguished from polysomy. The second method calculates the ratio of MET-to-CEP7 and identifies focal MET amplification as MET/CEP7 ≥ 2.0[30,31]. Some clinical trials categorize amplified degree more accurately into three groups: Low ( ≥ 1.8 to ≤ 2.2); intermediate ( > 2.2 to < 5.0); and high ( ≥ 5.0)[32]. The MET/CEP7 ratio in our cohort ranged from 0.60 to 1.64, and no sample had focal amplification. Our data indicated that in primary CRC lesions exposed to no treatment, MET amplification occurred primarily via polysomy instead of focal gene duplication. Consistently with our data, Raghav et al[11] reported that the rate of focal MET amplification was 0% and 1.9% in resected liver metastases of two therapy-naïve CRC cohorts, indicating that focal MET amplification was a rare event in therapy-naïve CRC.
Our study demonstrated that polysomy-induced MET amplification significantly correlated with LNM and TB, which were two independent predictors of unfavorable survival in CRC patients[33]. Consistently, during the two-year follow up in our study, we found that the recurrence/metastasis rate in MET-amplified CRC was higher in MET-non-amplified CRC, and the Kaplan-Meier curve also showed significant difference between these two populations. Hence, we suggested that MET amplification was considered to predict a poor prognosis in CRC without RAS/RAF mutation or microsatellite instability-high (MSI-H). In terms of c-MET protein, our data revealed a correlation between c-MET overexpression and high frequency of LNM, but no significant difference was detected in patients’ outcome during the two-year follow-up. Currently, the prognostic value of c-MET still remains controversial. A systematic review of 11 retrospective studies found that high c-MET expression significantly predicted poor OS and PFS in 1895 CRC patients[34]. Another study conducted by Lai et al[35] demonstrated no significant difference in overall survival time between c-MET-high and c-MET-low tumors, which was similar to our finding. The contradiction between these studies may be attributed to different IHC scoring system applied and cohorts studied. In our study, we chose clinical score system established by Spigel et al[21], which was applied in multiple clinical trials investigating drug efficacy of MET inhibitors[36-38].
MET amplification may be an efficient indicator in future targeted therapies in CRC patients, therefore a feasible screening strategy should be established. IHC can be adopted as a screening tool in clinical practice for certain gene amplifications[39]. For example, HER-2 protein overexpression using IHC shows high concordance with gene amplification by FISH[40,41]. Therefore, we tried to figure out whether c-MET IHC was a predictive marker for amplification. No clear association between FISH and IHC was observed based on our data, indicating that IHC is not a screening tool for MET amplification in CRC. Similar conclusions were presented in lung cancer. A study of 81 lung sarcomatoid carcinomas showed that c-MET IHC correlated poorly with MET amplification (GCN ≥ 5 or MET/CEP7 ≥ 2)[42]. Another tri-institutional study tested 181 lung adenocarcinoma tissues and demonstrated that most c-MET IHC-positive cases were negative for MET amplification, and IHC missed patients with MET amplification, indicating that IHC was not a reliable screening tool for MET amplification in lung cancer[25]. The discordance between c-MET overexpression and amplification may be explained in two aspects: (1) MET amplification caused by low-level polysomy does not result in substantial protein expression; and (2) other than gene amplification, hypoxia or inflammation also induces c-MET protein expression by amplifying HGF signalling, including increasing autocrine or paracrine HGF, or stimulating an HGF activator or other epigenetic regulations[43-45].
Our study has some limitations. Firstly, patients were retrospectively recruited from single institution and the two years of follow-up was not long enough to fully assess the prognostic values of either MET amplification or c-MET overexpression. In addition, the pharmacological significance of polysomy-induced MET amplification in CRC patients still remains unclear. Therefore, future research directions could be: (1) The long-term prognostic significance of MET amplification and c-MET overexpression in CRC patients harboring no other classic gene mutations (RAS/RAF or MSI-H); and (2) the drug efficacy of MET-TKIs and other MET inhibitors in CRC patients with MET GCN ≥ 5.
CONCLUSION
Focal MET amplification was a rare event in primary lesions of therapy-naïve CRC, and polysomy-caused MET amplification was observed in 14.4% of this population. Both MET amplification and c-MET overexpression correlated with adverse clinicopathological features in CRC. MET amplification predicted poor clinical outcome. C-MET IHC was not a suitable tool for MET amplification screening, and we firstly recommend that tissue should be prioritised for FISH over IHC to determine MET amplification in CRC.
Footnotes
Provenance and peer review: Unsolicited article; Externally peer reviewed.
Zeng ZS, Weiser MR, Kuntz E, Chen CT, Khan SA, Forslund A, Nash GM, Gimbel M, Yamaguchi Y, Culliford AT 4th, D'Alessio M, Barany F, Paty PB. c-Met gene amplification is associated with advanced stage colorectal cancer and liver metastases.Cancer Lett. 2008;265:258-269.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 139][Cited by in F6Publishing: 150][Article Influence: 9.4][Reference Citation Analysis (0)]
Raghav K, Morris V, Tang C, Morelli P, Amin HM, Chen K, Manyam GC, Broom B, Overman MJ, Shaw K, Meric-Bernstam F, Maru D, Menter D, Ellis LM, Eng C, Hong D, Kopetz S. MET amplification in metastatic colorectal cancer: an acquired response to EGFR inhibition, not a de novo phenomenon.Oncotarget. 2016;7:54627-54631.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 43][Cited by in F6Publishing: 47][Article Influence: 7.8][Reference Citation Analysis (0)]
Lugli A, Kirsch R, Ajioka Y, Bosman F, Cathomas G, Dawson H, El Zimaity H, Fléjou JF, Hansen TP, Hartmann A, Kakar S, Langner C, Nagtegaal I, Puppa G, Riddell R, Ristimäki A, Sheahan K, Smyrk T, Sugihara K, Terris B, Ueno H, Vieth M, Zlobec I, Quirke P. Recommendations for reporting tumor budding in colorectal cancer based on the International Tumor Budding Consensus Conference (ITBCC) 2016.Mod Pathol. 2017;30:1299-1311.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 575][Cited by in F6Publishing: 636][Article Influence: 90.9][Reference Citation Analysis (0)]
Yu Q, Zhang C, Huang W, Li L. Heat-induced antigen retrieval in fluorescence in situ hybridization: An effective approach enhancing signal intensity in poor-quality FFPE sections.Exp Ther Med. 2021;22:1480.
[PubMed] [DOI][Cited in This Article: ][Reference Citation Analysis (0)]
Cappuzzo F, Marchetti A, Skokan M, Rossi E, Gajapathy S, Felicioni L, Del Grammastro M, Sciarrotta MG, Buttitta F, Incarbone M, Toschi L, Finocchiaro G, Destro A, Terracciano L, Roncalli M, Alloisio M, Santoro A, Varella-Garcia M. Increased MET gene copy number negatively affects survival of surgically resected non-small-cell lung cancer patients.J Clin Oncol. 2009;27:1667-1674.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 441][Cited by in F6Publishing: 468][Article Influence: 31.2][Reference Citation Analysis (0)]
Bean J, Brennan C, Shih JY, Riely G, Viale A, Wang L, Chitale D, Motoi N, Szoke J, Broderick S, Balak M, Chang WC, Yu CJ, Gazdar A, Pass H, Rusch V, Gerald W, Huang SF, Yang PC, Miller V, Ladanyi M, Yang CH, Pao W. MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib.Proc Natl Acad Sci U S A. 2007;104:20932-20937.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 1322][Cited by in F6Publishing: 1356][Article Influence: 79.8][Reference Citation Analysis (0)]
Guo R, Berry LD, Aisner DL, Sheren J, Boyle T, Bunn PA Jr, Johnson BE, Kwiatkowski DJ, Drilon A, Sholl LM, Kris MG. MET IHC Is a Poor Screen for MET Amplification or MET Exon 14 Mutations in Lung Adenocarcinomas: Data from a Tri-Institutional Cohort of the Lung Cancer Mutation Consortium.J Thorac Oncol. 2019;14:1666-1671.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 57][Cited by in F6Publishing: 117][Article Influence: 23.4][Reference Citation Analysis (0)]
Cremolini C, Morano F, Moretto R, Berenato R, Tamborini E, Perrone F, Rossini D, Gloghini A, Busico A, Zucchelli G, Baratelli C, Tamburini E, Tampellini M, Sensi E, Fucà G, Volpi C, Milione M, Di Maio M, Fontanini G, De Braud F, Falcone A, Pietrantonio F. Negative hyper-selection of metastatic colorectal cancer patients for anti-EGFR monoclonal antibodies: the PRESSING case-control study.Ann Oncol. 2017;28:3009-3014.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 54][Cited by in F6Publishing: 69][Article Influence: 11.5][Reference Citation Analysis (0)]
Yin X, Zhang T, Su X, Ji Y, Ye P, Fu H, Fan S, Shen Y, Gavine PR, Gu Y. Relationships between Chromosome 7 Gain, MET Gene Copy Number Increase and MET Protein Overexpression in Chinese Papillary Renal Cell Carcinoma Patients.PLoS One. 2015;10:e0143468.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 7][Cited by in F6Publishing: 12][Article Influence: 1.3][Reference Citation Analysis (0)]
Jurmeister P, Lenze D, Berg E, Mende S, Schäper F, Kellner U, Herbst H, Sers C, Budczies J, Dietel M, Hummel M, von Laffert M. Parallel screening for ALK, MET and ROS1 alterations in non-small cell lung cancer with implications for daily routine testing.Lung Cancer. 2015;87:122-129.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 42][Cited by in F6Publishing: 49][Article Influence: 4.9][Reference Citation Analysis (0)]
Noonan SA, Berry L, Lu X, Gao D, Barón AE, Chesnut P, Sheren J, Aisner DL, Merrick D, Doebele RC, Varella-Garcia M, Camidge DR. Identifying the Appropriate FISH Criteria for Defining MET Copy Number-Driven Lung Adenocarcinoma through Oncogene Overlap Analysis.J Thorac Oncol. 2016;11:1293-1304.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 101][Cited by in F6Publishing: 136][Article Influence: 17.0][Reference Citation Analysis (0)]
Hartmaier RJ, Markovets AA, Ahn MJ, Sequist LV, Han JY, Cho BC, Yu HA, Kim SW, Yang JC, Lee JS, Su WC, Kowalski DM, Orlov S, Ren S, Frewer P, Ou X, Cross DAE, Kurian N, Cantarini M, Jänne PA. Osimertinib + Savolitinib to Overcome Acquired MET-Mediated Resistance in Epidermal Growth Factor Receptor-Mutated, MET-Amplified Non-Small Cell Lung Cancer: TATTON.Cancer Discov. 2023;13:98-113.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 1][Cited by in F6Publishing: 57][Article Influence: 57.0][Reference Citation Analysis (0)]
Lu S, Wu L, Jian H, Cheng Y, Wang Q, Fang J, Wang Z, Hu Y, Han L, Sun M, Miao L, Ding C, Cui J, Wang K, Li B, Li X, Ye F, Liu A, Pan Y, Cang S, Zhou H, Sun X, Shen Y, Wang S, Zhang W, He Y. Sintilimab plus chemotherapy for patients with EGFR-mutated non-squamous non-small-cell lung cancer with disease progression after EGFR tyrosine-kinase inhibitor therapy (ORIENT-31): second interim analysis from a double-blind, randomised, placebo-controlled, phase 3 trial.Lancet Respir Med. 2023;11:624-636.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 14][Cited by in F6Publishing: 47][Article Influence: 47.0][Reference Citation Analysis (0)]
Wu YL, Zhang L, Kim DW, Liu X, Lee DH, Yang JC, Ahn MJ, Vansteenkiste JF, Su WC, Felip E, Chia V, Glaser S, Pultar P, Zhao S, Peng B, Akimov M, Tan DSW. Phase Ib/II Study of Capmatinib (INC280) Plus Gefitinib After Failure of Epidermal Growth Factor Receptor (EGFR) Inhibitor Therapy in Patients With EGFR-Mutated, MET Factor-Dysregulated Non-Small-Cell Lung Cancer.J Clin Oncol. 2018;36:3101-3109.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 162][Cited by in F6Publishing: 241][Article Influence: 40.2][Reference Citation Analysis (0)]
Couturier J, Vincent-Salomon A, Nicolas A, Beuzeboc P, Mouret E, Zafrani B, Sastre-Garau X. Strong correlation between results of fluorescent in situ hybridization and immunohistochemistry for the assessment of the ERBB2 (HER-2/neu) gene status in breast carcinoma.Mod Pathol. 2000;13:1238-1243.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 83][Cited by in F6Publishing: 79][Article Influence: 3.3][Reference Citation Analysis (0)]
Mignard X, Ruppert AM, Antoine M, Vasseur J, Girard N, Mazières J, Moro-Sibilot D, Fallet V, Rabbe N, Thivolet-Bejui F, Rouquette I, Lantuejoul S, Cortot A, Saffroy R, Cadranel J, Lemoine A, Wislez M. c-MET Overexpression as a Poor Predictor of MET Amplifications or Exon 14 Mutations in Lung Sarcomatoid Carcinomas.J Thorac Oncol. 2018;13:1962-1967.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 25][Cited by in F6Publishing: 46][Article Influence: 7.7][Reference Citation Analysis (0)]
