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ORIGINAL ARTICLE  
Year : 2022  |  Volume : 65  |  Issue : 4  |  Page : 786-790
c-MET positivity and its relationship with histopathological findings in gastric carcinomas exhibiting HER2 gene expression


Department of Pathology, Faculty of Medicine, Manisa Celal Bayar University, Manisa, Turkey

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Date of Submission07-Jan-2021
Date of Acceptance03-Jun-2021
Date of Web Publication02-Jun-2022
 

   Abstract 


Context: Co-expressions of receptor tyrosine kinases such as c-MET and HER2 were reported in many studies. The concomitant expression is associated with more aggressive clinical course. Aims: In this study, it was intended to investigate the correlation of the positivity of c-MET and HER2 with histopathologic findings and their impacts on prognosis. Subjects and Methods: After the decision of the ethics committee, a total of 64 cases, whose HER 2 status was studied by dual silver in situ hybridization/immunohistochemistry method, were included in the study. Immunohistochemical staining for c-MET was performed to all cases and the evaluation was performed similarly to the criteria for HER2 evaluation, but cytoplasmic staining was also considered significant. Statistical Analysis Used: The data were analyzed using SPSS 20 for Windows. Results: c-MET positivity which is considered by the score of 2+ and 3+ was found only in 34.4% of HER2 positive cases while it was 59.3% in HER2 negative cases (P = 0.045). The sole histopathological feature associated with c-MET positivity was distal gastric localization (P = 0.016). Conclusions: Even though higher rates of c-MET positivity in HER2 positive cases were stated in the literature, contrary results were obtained in this study. Comparing the HER2+/c-MET + co-expression group with the other groups, no difference was found about age, sex, macroscopic and microscopic characteristics. The presence of c-MET positivity in cases with HER2 expression suggests that c-MET expression might be associated with the resistance to Trastuzumab.

Keywords: c-MET, dual SISH/IHC, gastric carcinoma, HER2, immunohistochemistry, in situ hybridization

How to cite this article:
Coskun F, Ayhan S, Tan A, Isisag A. c-MET positivity and its relationship with histopathological findings in gastric carcinomas exhibiting HER2 gene expression. Indian J Pathol Microbiol 2022;65:786-90

How to cite this URL:
Coskun F, Ayhan S, Tan A, Isisag A. c-MET positivity and its relationship with histopathological findings in gastric carcinomas exhibiting HER2 gene expression. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 Nov 30];65:786-90. Available from: https://www.ijpmonline.org/text.asp?2022/65/4/786/346512





   Introduction Top


Although the incidence and mortality of gastric cancer is decreased, it is still 5th in terms of incidence around the world and 3rd in cancer-related deaths worldwide.[1] There are many studies reporting that the overexpression of c-MET which is a member of the family of receptor tyrosine kinases (RTKs) like HER2 is associated with progression and survival in gastric carcinomas. Preclinical studies in animal models have shown that c-MET antibodies or small molecule c-MET inhibitors prevent the proliferation of tumor cells and tumor progression in gastric epithelial cells expressing c-MET amplification. It has also been stated in several publications that activation of c-MET pathway might be play a role in the development of resistance to Trastuzumab treatment.[2]

In point of view of recent approaches on gastric cancer therapy, we aimed to determine the frequency of c-MET expression in gastric cancers and the association of it with histopathological prognostic factors and HER2 expression.


   Subjects and Methods Top


This study was approved by the ethics committee of Manisa Celal Bayar University.

In our study, 203 gastrectomy specimens were evaluated which had been diagnosed as gastric cancer between 1996 and 2016 in our department. Thirty-two cases evaluated with dual silver in situ hybridization/immunohistochemistry (SISH/IHC) method as HER2 positive and randomly selected HER2 negative 32 cases were included in the study. The hematoxylin-eosin-stained preparations were re-evaluated and confirmed according to World Health Organization (WHO) classification and Lauren classification. The findings stated in the pathology reports such as age and gender, location and size of the tumor, histological type, depth of invasion, perineural invasion, lymphovascular invasion, lymph node metastasis, local invasion or distant metastasis were recorded. Proper tissue blocks which are the most representative of the tumor were selected.

All cases (n = 64) were stained immunohistochemically by anti-Total c-MET (SP44) clone rabbit monoclonal antibody (Ventana, Cat. No. 790-4430, Predilue form). Immunohistochemical staining of sections from the FFPE tissue samples was performed with Ventana BenchMark GX (Roche Diagnostics, Basel, Switzerland), using the Ventana ultraView Universal DAB Detection Kit; the staining procedure is based on the indirect biotin-free system. Protocols involving heat induction- or protease digestion-based antigen retrieval were performed as recommended by the manufacturer with some modifications. Membranous and cytoplasmic staining with c-MET (SP44 clone) were considered as positive. The scoring was evaluated according to the classification system based on the proportion of stained cells and is shown in [Table 1]. The cases with a score of +2 and +3 were considered as c-MET positive.
Table 1: c-MET IHC scoring system

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For re-evaluation of cases which were previously labelled as HER2 positive, dual SISH/IHC combined method was performed. In a relatively new method, anti-HER2/neu (4B5) clone rabbit monoclonal antibody for IHC staining (Ventana, Cat. No. 790-2991, Predilue form) and inform HER2 dual ISH DNA probe cocktail (Ventana, Catalog number 780-4422) were used for ISH. Using recommended procedure, slides were first stained immunohistochemically and consequently performed dual SISH. In addition, HybClear solution was added to prevent reaction during hybridization in the transition between IHC and dual-SISH methods. Ultraview SISH DNP Detection Kit was applied for HER-2 gene, black signal was obtained with silver deposits, Ultraview Red ISH DIG Detection Kit was applied for chromosome 17 and red signal was obtained with naphthol and Fast Red. At the end of the whole process, IHC staining of HER2 and two-color signals of dual SISH in which red signal was representative for chromosome 17 while black signal was representative for HER2 gene loci were marked on the same slide. The staining protocol was performed in accordance with staining protocols of Ventana, BenchMark-XT fully automated slide stainer.

The data were analyzed using SPSS 20 for Windows (IBM SPSS Statistics Desktop 20.0 Windows Multilingual eAssembly).


   Results Top


c-MET positivity was determined in 30 (46,8%) of 64 cases. While it was 34,48% in HER2 positive cases (n = 11), HER2 negative cases were expressing significantly more of c-MET positivity (59.3%) (n = 19) (P = 0.045). In the c-MET positive cases, HER2 was scored as IHC 3+ in 9 cases (30%) and IHC 2+ in 21 cases (70%). While it was scored as IHC 1+ in 20 cases (58.8%) of c-MET negative group, 14 cases (41.2%) of same group with no immunoreactivity was scored as IHC 0.

In the c-MET positive cases male/female ratio was 1:2 (10/20) and the number of cases with and over the age of 65 and under 65 was equal. The tumor was located on the anastomosis line in 1 case (3.4%), at corpus in 4 cases (13.3%), at antro-pyloric region in 19 cases (63.3%), at cardia in 3 cases (10%) and observed as linitis plastica in 3 cases (10%). The diameter of the tumor was <5 cm in 11 cases (36.6%) and ≥5 cm in 19 cases (63.4%) [Table 2].
Table 2: Comparison of c-MET (+) and c-MET (-) cases in terms of clinicopathologic features

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While 19 (63.4%) of the c-MET positive cases were intestinal type, 9 (30%) diffuse type and 2 (6.6%) mixed type according to the Lauren classification, evaluation of gastric tumors in accordance with WHO classification represented 15 (50%) tubuler adenocarcinomas, 10 (33.3%) poorly cohesive carcinomas, 1 (3.3%) mucinous adenocarcinoma, 3 (10%) tubulo-papillary adenocarcinomas and 1 (3.3%) mixed adenocarcinoma morphology.

In classification of the c-MET positive cases in relation with the depth of invasion, 2 cases (6.6%) were in the early stage (T1a and T1b) and 28 cases (93.3%) were advanced (T2-T4a/b). While no lymph node metastasis (N0) observed in 7 cases (23.3%), one or more lymph node metastasis (N1-N3a/b) detected in 23 cases (76.6%). Lymphovascular invasion was detected in 25 cases (83.3%) and perineural invasion in 22 cases (73.3%). In 6 of 9 cases (66.7%) reported to be clinically metastatic was detected c-MET positivity whereas 2 of 3 cases (66.7%) with c-MET positivity was observed adjacent organ invasion.

When c-MET positive and c-MET negative cases were compared according to age, gender, macroscopic and histopathologic features; there was no statistically significant differences in terms of age, sex, Borman classification, tumor diameter, pT stage, pN stage, lymphovascular invasion, perineural invasion, adjacent organ invasion and distant organ metastasis (P > 0.05) [Table 3].
Table 3: Comparison of c-MET (+) and c-MET (-) cases in terms of histopathological features

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However, categorizing the gastric carcinomas as proximal or distal has revealed an statistically significant difference for 80% (24/30) of c-MET positive cases with distal gastric location (P = 0.016). Even though it is not statistically significant, HER2 expression in tumors with intestinal type was more frequent whereas c-MET positivity was observed mostly in diffuse type tumors (75%) (9/12).

The all data obtained from IHC and dual SISH staining, 4 different groups of HER2+/c-MET+cases (17.2%) (n = 11), HER2+/c-MET-cases (32.8%) (n = 21), HER2-/c-MET+cases (29.7%) (n = 19) and HER2-/c-MET-cases (20.3%) (n = 13) were revealed in which there was no statistically significant difference [Figure 1] and [Figure 2].
Figure 1: (a) Weak, vague membranous staining, c-MET (1+) (x400), (b) Moderate membranous staining, c-MET (2+) (x200), (c) Strong cytoplasmic and membranous staining of poorly cohesive carcinoma, c-MET (3+) (x200), (d) Strong cytoplasmic and membranous staining of tubular adenocarcinoma, c-MET (3+) (x400)

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Figure 2: (a) The case of HER2 (2+) tubular adenocarcinoma amplified by dual SISH/IHC method (x400), (b) Dual SISH/IHC method HER2/chromosome 17 ≥2, HER2 (2+) immunohistochemically (x1000), (c) Dual SISH/IHC method HER2/chromosome 17 ≥2, HER2 (3+) immunohistochemically (x400), (d) Dual SISH/IHC method, HER2/chromosome 17 <2, HER2 (3+) immunohistochemically (x400)

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   Discussion Top


In this study that was intended to reveal the association between HER2 gene amplification and c-MET, another member of RTKs family, in gastric cancers, c-MET was found as positive in 30 of 64 patients (46.8%) and surprisingly in HER2 negative cases the expression of c-MET was much more (P = 0.04). In the literature dealing with c-MET positivity in patients with gastric cancer Lee et al.,[1] Drebber et al.,[3] and Nakajama et al.[4] reported the c-MET positivity as 23.7%, 73.7% and 46.1%, respectively. In a meta-analysis involving 37 different studies conducted by Pyo et al.[5] 8395 cases of gastric carcinoma were reviewed and they reported that c-MET positivity ranged between 1.8% and 82.5% by IHC and mean c-MET overexpression have been found in 31.5% (2641/8395) in these studies. Besides having varying results in these previous studies, it is considerable that there are no specific and standardized criteria for interpretation of c-MET positivity by immunohistochemically. Reviewing the literature including the ones mentioned above, it has been observed that solely cytoplasmic staining or solely membranous staining was taken into account in some of the studies whereas in some others both cytoplasmic and membranous staining was considered and the staining thresholds were set in different percentages such as 5%, 10%, 25% and 50% in different studies. Therefore, it would not be reasonable to compare the data in the determination of c-MET expression unless having a standard scoring criteria.

There are controversial reports in the literature concerning with the relation between c-MET expression and clinicopathological variables. Retterspitz and colleagues determined c-MET overexpression in 51% (45/88) of diffuse type carcinomas. However, their study mostly consisted of diffuse type gastric cancer and did not include intestinal type gastric cancer. Similarly, Ma et al. found a correlation between c-MET overexpression and diffuse type carcinomas which are typed according to Lauren classification. This study, metastasis-associated with colon cancer-1 (MACC-1) and c-MET were evaluated together and it was shown that MACC-1 regulates the HGF/MET signaling. Although having no statistically significant association, c-MET expression was determined much more in diffuse type in our study. However, in the most of the reports in the literature, c-MET positivity has shown in intestinal type carcinomas.[1],[6],[7],[8],[9],[10] Pyo et al.[5] reported that overexpression of c-MET is associated with male gender, poor differentiation, lymph node metastasis, higher TNM stage and stated the poor prognostic efficacy with regard to overall survival. While in some studies c-MET overexpression was similarly found to be an independent prognostic factor associated with depth of tumor invasion, lymph node metastasis, distant metastasis, recurrence, perineural invasion, no association with clinicopathologic factors was determined in some other studies. Interestingly, in addition with literature a significant relationship was found between c-MET positivity and distal gastric location in our study. Although c-MET positivity was more prevalent in morphologically diffuse gastric carcinomas in our cases, no statistically significant relation was found with any of the histopathological factors except distal location. The development of gastric cancer is a complex, multi-step process involving multiple genetic and epigenetic changes of oncogenes, tumor suppressor genes, DNA repair genes, cell cycle regulators, and signal molecules such as MACC-1. According to our opinion the absence of a standardized method in the evaluation of overexpression and amplification of c-MET may lead to variable interpretation of the results of advanced gastric carcinomas in the literature.

In general, co-expressions of different members of the RTKs family such as EGFR, c-MET, FGFR-2, HER2 can be seen in gastric carcinomas.[11],[12] Pyo et al.[5] reported the correlation of HER2 positivity with c-MET positive cases, while Yıldız et al.[13] determined c-MET positivity in 84.6% in HER2 positive group and in 56.2% in HER2 negative group. On the other hand, in the study of Jia and colleagues in which c-MET, FGFR-2, HER2 expression was investigated, HER2+/c-MET+ expression was found in only 7% of cases. They also stated that the co-expression of RTKs is substantially associated with poor clinical outcomes; and the worst survival rates were observed in triple positive gastric carcinomas.[12] Cases with Japanese origin HER2+/c-MET+ co-expression was detected in 8% in the study of Fuse and colleagues.[14] In our study in which c-MET positivity was found in 34.3% (11/32) in HER2 positive cases, it was significantly higher, contrarily to literature findings in HER2 negative cases with 59.3% (19/32) of c-MET positivity (P < 0.05). As stated previously, the probable reason of reporting in various percentages in the literature, might be arised from using different criteria in the evaluation of c-MET by IHC.

In the study of Fuse et al.[14] which is intended to search the relation between survival and the efficacy of RTKs in advanced stage gastric carcinomas, co-expression of HER2, c-MET and EGFR was found to have statistically no significant effect on overall survival. However, a statistically significant difference between the HER2+/c-MET+ co-expressing group and the HER2+/c-MET- expressing group (P = 0.043) was observed and based on this, c-MET positivity has been implicated as a poor prognostic factor. In another study by Ha et al.[11] microarray tissue samples from 1589 gastrectomy materials were stained IHC for HER2, c-MET and EGFR and subsequently confirmed by FISH. Eventually, triple positivity (HER2+/c-MET+/EGFR+) was detected in 15 of 169 HER2 positive cases (9%) and c-MET positivity in 37 cases (22%) and EGFR positivity in 29 cases (17%) accompanying to HER2 expression was observed while 88 cases have solely HER2 expression. Mean overall survival was 63 months in the triple positive group and 113 months in the triple negative group. Increased RTKs co-expression was found in association with younger age, larger tumor size, and intestinal morphology (P < 0.05). In addition, they also investigated the treatment response by using of tumor cells isolated from the pericardial effusion of a HER2+/c-MET+ gastric carcinoma patient. It was found in their study, the combination of HER2 inhibitor (Lapatinib)+MET inhibitor (PHA665752) provides a more severe inhibition of tumor cell proliferation rather than the sole use of HER2 inhibitor. In our study, no statistically significant difference was observed by comparing HER2+/c-MET+ co-expressing group with others (HER2+/c-MET-, HER2-/c-MET+ and HER2-/c-MET-) in terms of histopathological prognostic findings. This is likely to be that most our cases are diagnosed at an advanced stage and our groups are associated with a limited number of cases.

Proto-oncogene c-MET, a member of the RTK family, is a known hepatocyte growth factor (HGF) receptor that is encoded by the MET gene. MET has a primary single-chain precursor protein made of alpha and beta subunits, the latter of which contains a cytoplasmic kinase domain and a docking site. Binding of HGF to the extracellular domain activates the kinase activity that phosphorylates the tyrosines at the carboxy terminal docking site. Phosphorylated MET (p-MET) can recruit a variety of proteins, including growth factor receptor-bound protein 2 (GRB2), GRB2-associated binding protein 1 (GAB1), phospholipase C (PLC)-gamma, SRC, and SHP2, and activates downstream signaling molecules such as phosphatidylinositol-3- kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathways. Similar to other RTKs, MET plays key roles in tumor survival, growth, angiogenesis, and metastasis.[2]

HER2 overexpression is observed in a small proportion of gastric carcinomas, and only advanced cases can benefit from treatment with Trastuzumab. In the majority of cases resistance to the treatment develop initially or within the first year. Considering that RTKs use common intracellular signaling pathways, alternate activations of c-MET and other RTKs, or co-expressions, might cause the intracellular signal to be continuously active in cross-pathways. This may lead to the development of resistance in some of the targeted therapies with HER2 and evokes a need for new targeted therapies in gastric carcinomas in which survival rates are low despite of surgery and various chemotherapeutic treatment protocols. In our study, the substantial detection of c-MET positivity in 34.3% of HER2 positive cases gave rise to thought that c-MET positivity might be responsible for some of the resistance to Trastuzumab treatment in these cases. However, immunohistochemically, in order to better explain the association of c-MET with histopathologic prognostic factors and to develop treatment modalities targeting c-MET, further standardization of scoring criteria is a priority.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Lee HE, Kim MA, Lee HS, Jung EJ, Yang HK, Lee BL, et al. MET in gastric carcinomas: Comparison between protein expression and gene copy number and impact on clinical outcome. Br J Cancer 2012;107:325-33.  Back to cited text no. 1
    
2.
Inokuchi M, Otsuki S, Fujimori Y, Sato Y, Nakagawa M, Kojima K. Clinical significance of MET in gastric cancer. World J Gastrointest Oncol 2015;7:317-27.  Back to cited text no. 2
    
3.
Drebber U, Baldus SE, Nolden B, Grass G, Bollschweiler E, Dienes HP, et al. The overexpression of c-met as a prognostic indicator for gastric carcinoma compared to p53 and p21 nuclear accumulation. Oncol Rep 2008;19:1477-83.  Back to cited text no. 3
    
4.
Nakajima M, Sawada H, Yamada Y, Watanabe A, Tatsumi M, Yamashita J, et al. The prognostic significance of amplification and overexpression of c-met and c-erb B-2 in human gastric carcinomas. Cancer 1999;85:1894-902.  Back to cited text no. 4
    
5.
Pyo JS, Sohn JH, Kim WH. Concordance rate between HER2 immunohistochemistry and in situ hybridization in gastric carcinoma: Systematic review and meta-analysis. Int J Biol Markers 2016;31:e1-10. doi: 10.5301/jbm.5000171.  Back to cited text no. 5
    
6.
Sotoudeh K, Hashemi F, Madjd Z, Sadeghipour A, Molanaei S, Kalantary E. The clinicopathologic association of c-MET overexpression in Iranian gastric carcinomas; an immunohistochemical study of tissue microarrays. Diagn Pathol 2012;7:57.  Back to cited text no. 6
    
7.
Janjigian YY, Tang LH, Coit DG, Kelsen DP, Francone TD, Weiser MR, et al. MET expression and amplification in patients with localized gastric cancer. Cancer Epidemiol Biomarkers Prev 2011;20:1021-7.  Back to cited text no. 7
    
8.
Ma J, Ma J, Meng Q, Zhao ZS, Xu WJ. Prognostic value and clinical pathology of MACC-1 and c-MET expression in gastric carcinoma. Pathol Oncol Res 2013;19:821-32.  Back to cited text no. 8
    
9.
Ha SY, Lee J, Kang SY, Do IG, Ahn S, Park JO, et al. MET overexpression assessed by new interpretation method predicts gene amplification and poor survival in advanced gastric carcinomas. Mod Pathol 2013;26:1632-41.  Back to cited text no. 9
    
10.
Retterspitz MF, Mönig SP, Schreckenberg S, Schneider PM, Hölscher AH, Dienes HP, et al. Expression of {beta}-catenin, MUC1 and c-met in diffuse-type gastric carcinomas: Correlations with tumour progression and prognosis. Anticancer Res 2010;30:4635-41.  Back to cited text no. 10
    
11.
Ha SY, Lee J, Jang J, Hong JY, Do IG, Park SH, et al. HER2-positive gastric cancer with concomitant MET and/or EGFR overexpression: A distinct subset of patients for dual inhibition therapy. Int J Cancer 2015;136:1629-35.  Back to cited text no. 11
    
12.
Jia YX, Li TF, Zhang DD, Fan ZM, Fan HJ, Yan J, et al. The co-expression and prognostic significance of c-MET, fibroblast growth factor receptor 2, and human epidermal growth factor receptor 2 in resected gastric cancer: A retrospective study. Onco Targets Ther 2016;9:5919-29.  Back to cited text no. 12
    
13.
Yıldız Y, Sokmensuer C, Yalcin S. Evaluation of c-Met, HGF, and HER2 expressions in gastric carcinoma and their association with other clinicopathological factors. Onco Targets Ther 2016;9:5809-17.  Back to cited text no. 13
    
14.
Fuse N, Kuboki Y, Kuwata T, Nishina T, Kadowaki S, Shinozaki E, et al. Prognostic impact of HER2, EGFR, and c-MET status on overall survival of advanced gastric cancer patients. Gastric Cancer 2016;19:183-91.  Back to cited text no. 14
    

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Correspondence Address:
Ayca Tan
Department of Pathology, Faculty of Medicine, Manisa Celal Bayar University, Manisa – 45040
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpm.ijpm_23_21

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