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  Table of Contents    
Year : 2020  |  Volume : 63  |  Issue : 1  |  Page : 86-89
Cellular mesenchymal epithelial transition (C-MET) gene copy number variation in gastric adenocarcinoma: A pilot search for new marker for targeted therapy in HER-2/neu resistance

1 Department of Pathology, University College of Medical Sciences and GTB Hospital, Delhi, India
2 Department of Surgical Disciplines, All India Institute of Medical Sciences, Delhi, India
3 Department of Reproductive Biology, All India Institute of Medical Sciences, Delhi, India

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Date of Web Publication31-Jan-2020


Increasing HER-2/neu resistance in gastric carcinoma has encouraged search for new biomarkers for targeted therapy. Cellular mesenchymal epithelial transition (C-MET) is one such tyrosine kinase inhibitor proposed for personalized salvage treatment. We determined frequency of C-MET gene copy number variation (CNV) by Fluorescent in-situ hybridization (FISH) in gastric adenocarcinoma (GAC) and sought its correlation with conventional clinicopathologic parameters. Dual-coloured FISH was done on 32 GAC cases. C-MET gene and centromere 7 signals were counted under fluorescent microscope and ratio was calculated for each case. Correlation between C-MET CNV and conventional clinic-pathologic parameters was done by Fischer exact test. CNV was identified in the form of amplification and polysomy (3.1% each) and associated with poorer prognostic parameters. Our pilot study highlights limited subset of patients that may benefit from anti-C-MET-targeted therapy and thus could be a novel biomarker for targeted intervention in GAC.

Keywords: Cellular mesenchymal epithelial transition, copy number variation, fluorescent in-situ hybridization, HER-2/neu, gastric adenocarcinoma

How to cite this article:
Jain P, Wadhwa N, Joshi MK, Jain M, Halder A, Mishra K. Cellular mesenchymal epithelial transition (C-MET) gene copy number variation in gastric adenocarcinoma: A pilot search for new marker for targeted therapy in HER-2/neu resistance. Indian J Pathol Microbiol 2020;63:86-9

How to cite this URL:
Jain P, Wadhwa N, Joshi MK, Jain M, Halder A, Mishra K. Cellular mesenchymal epithelial transition (C-MET) gene copy number variation in gastric adenocarcinoma: A pilot search for new marker for targeted therapy in HER-2/neu resistance. Indian J Pathol Microbiol [serial online] 2020 [cited 2022 Jan 24];63:86-9. Available from: https://www.ijpmonline.org/text.asp?2020/63/1/86/277381

   Introduction Top

Gastric carcinoma is the fourth most common malignancy worldwide and ranks second among digestive tract malignancy after oesophagus in both sexes in India.[1],[2] Adenocarcinoma is the commonest histologic type. Majority patients with gastric adenocarcinoma (GAC) present in advanced stage resulting in higher mortality. Targeted molecular treatment is realistic potential therapy for such patients. Directed molecular therapy against HER-2/neu positive advanced GAC in transtuzumab for gastric cancer (TOGA) trial yielded encouraging results. Since HER-2/neu amplification is found only in 15%-20% cases and resistance known in some of them, there is increasing search for new molecular markers.[3] C-MET (cellular mesenchymal epithelial transition) is an emerging novel marker implicated in gastric carcinogenesis. This proto-oncogene located on 7q21-31, induces cellular proliferation and local invasion. Amplification of C-MET gene is commonest mechanism leading to protein overexpression and also implicated in mediating resistance to HER-2/neu directed therapy in GAC. There is paucity of literature on C-MET copy number variation (CNV) from India. We undertook this pilot study to evaluate frequency of C-MET CNV in GAC and seek its correlation if any with conventional clinic-pathologic parameters.

   Methodology Top

This analytic study was performed in Department(s) of Pathology and Surgery of tertiary care centre. Forty-five histologically proven cases of GAC between 2006 and 2015 were retrieved from our records. Fluorescent in-situ hybridization (FISH) was done in 32 cases with well-preserved tumour morphology, high tumour cell density and minimal stromal tissue. The limited number of cases on which FISH was done was mainly due to logistic reason of limited amount of probe and loss of tissue during FISH in three cases. Other histological subtypes and cases which had received prior chemotherapy/targeted therapy were excluded from the study. Clinicopathological data were entered on pre-structured proforma according to CAP (College of American Pathologists) guidelines. FISH with dual-coloured probe against C-MET gene and centromere was performed using conventional 2-day procedure.[4] Patient anonymity and confidentiality was maintained. Institutional ethical clearance was taken prior to commencement of the study.

C-MET gene (green colour) and centromere 7 (orange colour) signals were counted under fluorescent microscope (Nikon NiU) in 20 non-overlapping interphase nuclei. Gene to centromere 7 ratio was derived for each case. Case was considered to be amplified when either C-MET/centromere 7 ratio ≥2.2 or ≥15 copies of C-MET gene in ≥10% of analysed cells or presence of tight C-MET gene clusters.[5],[6],[7] Polysomy was defined as C-MET/centromere 7 ratio: 1.8-2.2 (with presence of ≥3 copies of both gene and centromere 7 signals).[8],[9]

Statistical analysis

Results obtained were expressed qualitatively and correlation with conventional clinico-pathologic parameters was done by Fischer exact test. P value <0.05 was considered as significant.

   Results Top

We reviewed 45 cases of GAC and analysed C-MET gene CNV by FISH in 32 cases. Age of presentation ranged from 24 to 76 years with mean age of 56 ± 11.2 years and male preponderance (M:F = 1.6:1). Detailed clinicopathological characteristics of cases are mentioned in [Table 1]. Ten cases had associated Helicobacterpylori infection. Intestinal metaplasia was found in five cases. C-MET gene expression status was assessed only in GAC; associated/putative precursor lesions could not be studied due to cost constraints. There was no known family history of gastric cancer or any other malignancy.
Table 1: Clinicopathological details of the cases

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C-MET CNV (amplification and polysomy) was found in 2/32 (6.2%) cases of GAC. There was one case with C-MET amplification having C-MET/centromere 7 ratio of 2.3 [Figure 1]a [Figure 1]b [Figure 1]c]. This result was arrived at after counting of additional 20 nuclei by another pathologist. Since the ratio was near the cut off, evaluation was done by two pathologists in 20 additional nuclei. One case was polysomic showing multiple copies of C-MET gene and ratio being 2.0 [Figure 2]a [Figure 2]b [Figure 2]c]. Rest cases had disomy.
Figure 1: Dual-coloured FISH photomicrographs showing C-MET gene amplification in GAC (400 ×). (a) FITC photomicrograph showing C-MET gene signals (green dots). (b) Rhodamine photomicrograph shows orange centromere 7 signals (orange dots). (c) Composite image

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Figure 2: Dual-coloured FISH photomicrographs show C-MET gene polysomy in GAC (200×). (a) FITC photomicrograph shows C-MET gene signals (green dots). (b) Rhodamine photomicrographs show centromere signals (orange dots). (c) Composite image

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Statistical analysis was done on 32 cases on which FISH was performed. Cases with C-MET CNV showed association with several adverse clinico-pathologic factors. Both CNV were found in elderly males having large tumours (>5 cm), moderately differentiated to poorly cohesive adenocarcinomas invading beyond muscularis propria (≥pT2), having nodal metastasis (pN3) and advanced AJCC stage (≥III). Both showed lymphovascular and perineural invasion. Comparison of adverse prognostic factors in cases with C-MET CNV and cases with disomy is shown in [Table 2]. Despite evident clustering of adverse factors in these cases, statistical association of aberrant C-MET gene expression with the above-mentioned clinic-pathologic parameters could not be achieved owing to small group size.
Table 2: Comparison of adverse prognostic factors in cases with C-MET CNV and cases with disomy in 32 cases

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   Discussion Top

C-MET is an emerging novel marker implicated in pathogenesis of various malignancies such as lung, breast, colorectal, kidney, thyroid and stomach.[8] Recently, MET activation is found to induce resistance to targeted therapy in HER-2/neu amplified gastric cancer.[10] Most studies on role and clinical relevance of C-MET in gastric carcinoma are from south-east Asian countries where gastric carcinoma is common. We undertook this study to evaluate C-MET gene expression status in Indian patients with GAC and correlate it with conventional prognostic factors.

Frequency of amplification in our study (3.1%) is comparable to study by Hara et al. (3.9%), Lee et al. (3.4%) and Shah et al. (4.5%).[11],[12],[13] A few authors have observed even lower frequency of gene amplification ranging from 0% to –2%.[5],[7],[9] The variability in the frequency of amplification causes for geographical differences, tissue heterogeneity, tumour biology, different techniques used for assessment and different scoring criteria. Higher frequency of gene amplification is seen in south-east Asian countries and lower is observed from western population. Lee et al. conducted their study in Korean subset of patients and observed 3.4% cases to be amplified.[12] However, Janjigian et al. did not find amplification among Caucasian population which is a very low-prevalence area for gastric carcinoma.[9] Prevalence of gastric carcinoma in India is higher than western world and frequency of gene amplification is near comparable to Korean population.

Lack of uniform scoring criteria is another factor leading to discordant results among various studies. An et al. in their study used a FISH to asses C-MET CNV in metastatic/recurrent gastric cancer. They used a more liberal criteria of MET gene to centromere ratio ≥2 for defining amplification and found higher frequency of amplification of 8.3%.[14] Lower frequency of 2% was found by Lennerz et al. who applied criteria of C-MET/centromere 7 ratio ≥2.2.[5] We used more stringent criteria for CNV assessment and our frequency is comparable to the above study.

We observed C-MET polysomy in 1/32 (3%) patients. Polysomy is defined as ≥3 copies of centromere due to presence of additional chromosome. It can influence the FISH interpretation especially in those cases with concurrent CNV of gene and is important cause for equivocal FISH result. Janjigian et al. observed C-MET polysomy to be commoner than amplification in GAC in western population (33% vs. 0%). They concluded that polysomy was not likely to result in MET-driven cellular pathways. Despite increase in total C-MET protein expression (63.8%) they observed suboptimal activated protein expression phosphomet protein (2/38).[9] Even in breast carcinoma, HER-2/neu polysomy cases are said to behave like HER-2/neu negative and be unresponsive to targeted therapy.[8] Polysomy may not drive carcinogenesis unlike gene amplification which does so more often, hence differentiation using dual-coloured probes is essential. More studies are needed to study its role in gastric carcinoma.

We found C-MET CNV in old-aged males (>50 years), grade ≥G2, ≥T2 depth of invasion, >6 perigastric lymph nodes involvement, advanced stage, perineural invasion, however, with different histology. Amplified case was moderately differentiated intestinal type (Lauren's classification) while polysomic was diffuse infiltrative adenocarcinoma. Divergent observations are made by various authors regarding prognostic role of C-MET and its comparison with conventional clinicopathologic parameters. Lennerz et al. who observed MET amplification predominantly in intestinal type accounting for 80% (P value = 0.34) but found positive correlation with poor differentiation (P value = 0.02).[5] Nakajima et al. studied prognostic role of C-MET and observed ≥T2 invasion in 12/13 cases but could not establish any significance. They observed significant correlation of C-MET amplification with lymph node metastasis (P value = 0.04).[15] Both cases with C-MET CNV had peri-neural invasion; we found no previous reports on this association.

We noted trend of C-MET CNV toward poorer clinicopathological variable and outcome; this need to be ascertained in larger studies. Small sample size, lack of follow-up and economic constraints limiting number of cases for FISH are limitations of our study. Hence, we would like to refrain from drawing major conclusions from our study. Our study addresses C-MET expression in GAC in India, which has not been reported before.

   Conclusion Top

Our findings reiterate that C-MET gene amplification is a rare event in GAC. Prognostic significance of C-MET CNV and polysomy of chromosome 7 need to be ascertained by larger studies.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69-90.  Back to cited text no. 1
Mohandas KM, Jagannath P. Epidemiology of digestive tract cancers in India. VI. Projected burden in the new millennium and the need for primary prevention. Indian J Gastroenterol 2000;19:74-8.  Back to cited text no. 2
Dicken BJ, Bigam DL, Cass C, Mackey JR, Joy AA, Hamilton SM. Gastric adenocarcinoma: Review and considerations for future directions. Ann Surg 2005;241:27-39.  Back to cited text no. 3
ZytoLight SPEC MET/CEN 7 Dual Color Probe. Available from: http://www.zytovision.com/index.php/zytolight/17-zytolight-fish -products/209-z-2087. [Last accessed on 2015 Apr 11].  Back to cited text no. 4
Lennerz JK, Kwak EL, Ackerman A, Michael M, Fox SB, Bergethon K, et al. MET amplification identifies a small and aggressive subgroup of esophagogastric adenocarcinoma with evidence of responsiveness to crizotinib. J Clin Oncol 2011;29:4803-10.  Back to cited text no. 5
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. 6
Nagatsuma AK, Aizawa M, Kuwata T, Doi T, Ohtsu A, Ochiai A. Expression profiles of HER2, EGFR, MET and FGFR2 in a large cohort of patients with gastric adenocarcinoma. Gastric Cancer 2015;18:227-38.  Back to cited text no. 7
Liu Y, Ma L, Liu D, Yang Z, Yang C, Hu Z, et al. Impact of polysomy 17 on HER2 testing of invasive breast cancer patients. Int J Clin Exp Pathol 2013;7:163-73.  Back to cited text no. 8
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 Biomarks Prev 2011;20:1021-7.  Back to cited text no. 9
Chen C-T, Kim H, Liska D, Gao S, Christensen JG, Weiser MR. MET activation mediates resistance to lapatinib inhibition of HER2-amplified gastric cancer cells. Mol Cancer Ther 2012;11:660-9.  Back to cited text no. 10
Hara T, Ooi A, Kobayashi M, Mai M, Yanagihara K, Nakanishi I. Amplification of c-myc, K-sam, and c-met in gastric cancers: Detection by fluorescence in situ hybridization. Lab Investig 1998;78:1143-53.  Back to cited text no. 11
Lee HE, Kim MA, Lee HS, Jung E-J, 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. 12
Shah MA, Wainberg ZA, Catenacci DVT, Hochster HS, Ford J, Kunz P, et al. Phase II study evaluating 2 dosing schedules of oral foretinib (GSK1363089), cMET/VEGFR2 inhibitor, in patients with metastatic gastric cancer. PloS One 2013;8:e54014.  Back to cited text no. 13
An X, Wang F, Shao Q, Wang FH, Wang ZQ, Wang ZQ, et al. MET amplification is not rare and predicts unfavorable clinical outcomes in patients with recurrent/metastatic gastric cancer after chemotherapy. Cancer 2014;120:675-82.  Back to cited text no. 14
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. 15

Correspondence Address:
Pragya Jain
Department of Pathology, GB Pant Hospital, New Delhi
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/IJPM.IJPM_396_19

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