Targeting MET Amplification: Opportunities and Obstacles in Therapeutic Approaches

Simple Summary The MET gene is crucial for cell growth and has shown promise as a cancer treatment target. However, distinguishing between focal amplification and polysomy, different types of gene multiplication, is challenging. Accurate differentiation requires techniques such as in situ hybridization (ISH) or next generation sequencing (NGS). As the effectiveness of MET inhibitors can vary, careful patient selection and defining the perfect amplification threshold are critical. Future studies should focus on determining optimal therapy combinations and innovating new treatments targeting MET amplification. Abstract The MET gene plays a vital role in cellular proliferation, earning it recognition as a principal oncogene. Therapies that target MET amplification have demonstrated promising results both in preclinical models and in specific clinical cases. A significant obstacle to these therapies is the ability to distinguish between focal amplification and polysomy, a task for which simple MET copy number measurement proves insufficient. To effectively differentiate between the two, it is crucial to utilize comparative measures, including in situ hybridization (ISH) with the centromere or next generation sequencing (NGS) with adjacent genes. Despite the promising potential of MET amplification treatment, the judicious selection of patients is paramount to maximize therapeutic efficacy. The effectiveness of MET inhibitors can fluctuate depending on the extent of MET amplification. Future research must seek to establish the ideal threshold value for MET amplification, identify the most efficacious combination therapies, and innovate new targeted treatments for patients exhibiting MET amplification.


Introduction
The MET gene, also referred to as c-MET, encodes a receptor tyrosine kinase (RTK) known as MET.This gene has garnered significant attention in the oncology field due to its pivotal role in tumorigenesis and metastasis [1].The aberrant activation of MET signaling has been implicated in the development and progression of several malignancies, including non-small cell lung cancer (NSCLC) [2], gastric cancer [3,4], colorectal cancer [5], papillary renal cell carcinoma (PRCC) [6], hepatocellular carcinoma [7], and breast cancer [8,9].
Multiple genetic alterations within MET have been identified as oncogenic drivers in cancer.One such alteration is exon 14 skipping, which results in an aberrant MET protein lacking a critical regulatory domain [10].The event leads to constitutive activation of the MET signaling pathway and contributes to tumorigenesis, particularly in NSCLC [11].Notably, studies have revealed that approximately 3% of NSCLC patients exhibit MET exon 14 skipping, highlighting its clinical relevance [10].
In addition to exon 14 skipping, gene amplification and single nucleotide variants have been implicated in MET-driven oncogenesis.MET amplification is characterized by an increased copy number of the MET gene, leading to elevated MET protein levels and hyperactivation of downstream signaling cascades, including the RAS-ERK/MAPK, PI3K-AKT-mTOR, or PLCgamma-PKC pathways [12][13][14][15].This phenomenon has been observed in various cancer types and holds promise as a therapeutic target [16,17].
The identification of specific MET alterations has paved the way for targeted therapies in cancer treatment.For instance, in the case of NSCLC patients with MET exon 14 skipping, the development of MET tyrosine kinase inhibitors (TKIs), such as capmatinib and tepotinib, has shown remarkable efficacy in clinical trials [18][19][20].These TKIs selectively inhibit the activated MET kinase, thereby suppressing aberrant MET signaling and impeding tumor growth.
Furthermore, MET amplification has emerged as an intriguing therapeutic target.Targeted therapies designed to counteract MET amplification have shown promise in preclinical studies and a subset of clinical studies [18,21].However, challenges remain, particularly regarding the development of effective inhibitors that can overcome resistance mechanisms associated with MET amplification [22][23][24][25][26].
In light of the significance of MET alterations in cancer, this review aims to comprehensively summarize the measurement, frequency, and therapeutic implications of MET amplification.We seek to elucidate the underlying mechanisms driving MET amplification and explore its potential as a therapeutic target.Additionally, we will discuss the challenges and prospects associated with targeting MET amplification, including the emergence of resistance mechanisms.

Gene Amplification and Protein Overexpression
Gene amplification is a prevalent genetic alteration observed in cancer [27][28][29].It usually causes protein overexpression by enhancing levels of the products encoded by the amplified gene [27].Although gene amplification and protein overexpression are distinct phenomena, they are often associated with each other [27,28].In general, protein overexpression of RTKs can have oncogenic effects by increasing local receptor concentration, leading to auto-dimerization of receptors, and subsequent hyperactivation of downstream signaling pathways [27].For instance, amplification of the HER2 (ERBB2) gene on chromosome 17 and the overexpression of HER2 protein play a crucial role in the pathogenesis of HER2-positive breast cancer [30].Fluorescent in situ hybridization (FISH) is a method that detects ERRB2 gene amplification, while HER2 immunochemistry (IHC) identifies overexpression of HER2 protein.These two assays are commonly used and exhibit a strong correlation, serving as biomarkers for anti-HER2 therapy [31,32].In the clinical setting of breast cancer, HER2 status determination often involves measuring HER2 protein expression using IHC initially, followed by FISH when the IHC result is equivocal (2+).Targeted therapies specially designed for HER2-positive breast cancer have demonstrated remarkable success, making them the most effective treatment in the realm of personalized medicine [33].
It has been observed that MET protein expression assessed by IHC does not always align with MET amplification [44][45][46].Therefore, it is crucial to recognize that the patient populations identified by MET amplification techniques such as FISH and next generation sequencing (NGS) may differ substantially from those identified using IHC for treatment selection.The underlying reasons for the discordance between MET protein expression and gene amplification are not yet fully understood, but intra-tumor heterogeneity has been suggested as a contributing factor [46,47].Previous studies investigating targeted therapies for MET based on IHC measurements have yielded inconsistent results [48][49][50].
Consequently, IHC may not be the appropriate approach to identifying the population that would benefit from MET-targeted therapy.

Focal Gene Amplification and Polysomy
Gene copy number gain in cancer can manifest as either focal gene amplification or polysomy (or aneuploidy of chromosome) [51].Focal amplification refers to the specific gain of gene copies in the specific gene, while polysomy involves an overall increase in chromosome copy number [51].In the case of HER2, FISH is employed to measure gene copy number (GCN) and the ratio of ERRB2 to centromere enumerator probe (CEP)17.However, it is crucial to consider the presence of polysomy when interpreting FISH results [52].Polysomy has been identified as the primary cause of equivocal HER2 FISH results, and its association with the efficacy or prognosis of trastuzumab treatment remains unclear [53,54].Consequently, the selection of appropriate candidates for anti-HER2 therapy requires careful consideration [55].
Similarly, in the context of MET, focal amplification of the MET gene entails a specific gain of gene copies, while polysomy involves an increase in chromosome 7 copy number, often accompanied by co-amplification of adjacent genes such as CDK6 and BRAF [56] (Figure 1).It has been observed that polysomy does not exhibit a favorable response to treatment with MET inhibitors alone [57].Currently, there is a lack of standardized methods, including the choice of diagnostic tools and the establishment of thresholds, to effectively define focal MET amplification as a therapeutic target [56,58].
therapies for MET based on IHC measurements have yielded inconsistent results [48][49][50].Consequently, IHC may not be the appropriate approach to identifying the population that would benefit from MET-targeted therapy.

Focal Gene Amplification and Polysomy
Gene copy number gain in cancer can manifest as either focal gene amplification or polysomy (or aneuploidy of chromosome) [51].Focal amplification refers to the specific gain of gene copies in the specific gene, while polysomy involves an overall increase in chromosome copy number [51].In the case of HER2, FISH is employed to measure gene copy number (GCN) and the ratio of ERRB2 to centromere enumerator probe (CEP)17.However, it is crucial to consider the presence of polysomy when interpreting FISH results [52].Polysomy has been identified as the primary cause of equivocal HER2 FISH results, and its association with the efficacy or prognosis of trastuzumab treatment remains unclear [53,54].Consequently, the selection of appropriate candidates for anti-HER2 therapy requires careful consideration [55].
Similarly, in the context of MET, focal amplification of the MET gene entails a specific gain of gene copies, while polysomy involves an increase in chromosome 7 copy number, often accompanied by co-amplification of adjacent genes such as CDK6 and BRAF [56] (Figure 1).It has been observed that polysomy does not exhibit a favorable response to treatment with MET inhibitors alone [57].Currently, there is a lack of standardized methods, including the choice of diagnostic tools and the establishment of thresholds, to effectively define focal MET amplification as a therapeutic target [56,58].

MET Amplification Detection by FISH
Various methods, such as FISH and NGS, have been employed to detect MET amplification [33].Among these methods, FISH has been regarded as the gold standard for measuring MET amplification [59][60][61].In previous studies, MET amplification was defined based on the MET/CEP7 ratio, using FISH analysis, and it was found to occur in approximately 5% of patients with NSCLC or gastric adenocarcinoma, with a cut-off value of MET/CEP > 2.2 [16].The correlation between FISH and IHC is under investigation.
However, a phase I study reported that patients with MET amplification, defined by a cut-off of MET/CEP ≥ 2.0, did not exhibit a favorable response to therapy with a MET inhibitor [62].This raises concerns regarding the accurate selection of the target population for MET amplification-directed treatment.Further investigations utilizing FISH measurements have revealed that only cases with high MET/CEP ratios, such as MET/CEP > 5, exhibit a favorable response to MET inhibitors [63][64][65].Guo  It is worth noting that the efficacy of MET inhibitors alone may be diminished when MET amplification coexists with other driver mutations.In such cases, combination therapies may be necessary to achieve optimal treatment outcomes.

MET Amplification Detection by NGS
MET amplification detection using NGS has gained popularity in clinical settings, offering a comprehensive analysis of genomic alterations.However, its efficacy as a tool for identifying optimal biomarkers to guide MET therapy remains a topic of debate.Several studies have indicated that NGS may not be as effective as FISH in predicting response to MET inhibitors [66,67].For instance, a comparative analysis between NGS-based gene copy number (GCN) assessment and FISH revealed that NGS alone is not sufficient for predicting MET inhibitor response [68].One potential limitation of NGS is its inability to accurately distinguish between polysomy and focal amplification, which may impact the identification of suitable therapeutic targets.This might stem from inaccurate interpretation of copy number gain result.Without recognizing the potential polysomy, the inaccurate interpretation of copy number gain might occur.Strategies to address this challenge are still under investigation, and a recent study proposed a method to differentiate polysomy from focal MET amplification by co-examining the amplification of genes on chromosome 7, such as BRAF and CDK6 [56].
Our group analyzed the data from 1025 patients with advanced solid tumors using a non-invasive cfDNA NGS panel known as Guardant360 [56].An algorithm to define focal amplification was developed.Focal amplification was defined as MET amplification without polysomy or an increase in the chromosome copy number itself.The Guardant360 test examined four genes located on chromosome 7: EGFR in 7p11.2,CDK6 in 7q21.2,MET in 7q31.2, and BRAF in 7q34.The MET gene was classified as focally amplified if there were no co-amplification of adjacent genes, such as CDK6 or BRAF.Conversely, MET non-focal amplification was defined as a MET copy number increase associated with polysomy, in which MET copy number increased together with either CDK6 and/or BRAF.
Several examples were given to illustrate this: a sample with only MET amplification would be classified as focal.If a sample had co-amplification of MET and EGFR without CDK6 amplification, it would be categorized as focal amplification, given that polysomy could not occur without increasing the copy numbers of all three genes together.The algorithm to describe focal amplification was defined as follows: (a) MET copy number ≥ 2.2.(b) MET is amplified without co-amplification of CDK6 and BRAF.Co-amplification status was defined as "increased together" when the copy number of the other gene (CDK6 or BRAF) ≥ 2.2, and the difference with MET amplification is within +/−0.5.(c) MET amplification that satisfies both (a) and (b) is defined as focal.
Results of analyzing patient cohort consisting with the testing cohort (291 patients), and validation cohort (734 patients) showed MET alterations related to abnormal signaling in approximately 10.7% (110 patients) of the entire patient population across nine different cancer types, most notably non-small cell and small cell lung cancers, gastroesophageal cancer, and prostate adenocarcinoma.MET alterations were found in 37 out of 291 patients.Among these, 24 had amplifications, 5 had exon 14 skipping, and 13 had single nucleotide variants (SNVs).Co-alterations, such as amplification, SNVs, were found in four samples.
Of the 24 MET amplifications, about 30% (7/24) were classified as focal amplification.The MET copy number was significantly higher with focal amplification compared to non-focal amplification (polysomy).In a validation cohort, focal MET amplification was detected in 4.2% of patients.Overall, the rate of focal amplification was 3.7% (=38/1025) across all patients.
This study showed that this approach can distinguish focal from non-focal MET amplification using comprehensive genomic profiling with NGS in patients with advanced cancer.The study also suggests that only 30% of all MET amplifications detected by the NGS are focal, and these are associated with a higher plasma MET copy number.
Nevertheless, it is pertinent to acknowledge a limitation inherent in this study, namely the selective focus on a restricted gene set localized within chromosome 7, encompassing MET, EGFR, BRAF, and CDK6.Inclusion of a broader array of genes or single nucleotide polymorphisms (SNPs) has the potential to substantially enhance the precision and fidelity of detecting focal amplification events.Furthermore, the absence of a well-defined correlation between EGFR and MET necessitates a more comprehensive exposition.
Although clinical investigations are warranted, NGS holds promise as a valuable tool for MET amplification analysis due to its widespread use in clinical practice and the potential to simultaneously examine other driver genes, such as EGFR.The integration of NGS with established methods such as FISH has been proposed in HER2-positive cases to capture those missed by the current IHC and FISH-based approaches [69].By leveraging the strengths of both techniques, NGS-based detection may enhance the sensitivity and accuracy of MET amplification identification, leading to improved patient stratification and personalized treatment decisions.
To fully exploit the potential of NGS in MET amplification assessment, further research is needed to establish standardized guidelines for interpreting NGS results, particularly in distinguishing between polysomy and focal amplification.Additionally, the development of bioinformatics algorithms and computational tools specific to MET amplification analysis will aid in the accurate classification of NGS data.Efforts to validate the clinical utility of NGS-based detection of focal amplification and its correlation with treatment response are crucial steps toward integrating NGS into routine clinical practice for precision cancer care.

MET Amplification as an Acquired Resistance Mechanism
MET amplification has emerged as a significant mechanism of acquired resistance in various targeted therapies, particularly in EGFR-mutant NSCLCs.MET amplification is known to stimulate signaling pathways such as RAS-ERK/MAPK, STAT, and PI3K/AKT downstream of EGFR, leading to resistance against EGFR TKIs [70,71].This resistance mechanism has been observed across all generations of EGFR TKIs [72][73][74].
Clinical studies have shed light on the prevalence of MET amplification as a resistance mechanism in NSCLC.Coleman et al. conducted a comprehensive analysis of key clinical trials in NSCLC and reported that MET amplification was identified as a mechanism of resistance in 7-15% of patients who experienced treatment failure with first-line osimertinib and in 10-22% following second-line osimertinib [26].These findings highlight the clinical relevance of MET amplification in acquired resistance scenarios.
Moreover, it has been recognized that MET amplification may contribute to treatment resistance not only in EGFR-positive NSCLCs but also in other oncogenic gene-positive NSCLCs, such as those with ALK fusion [75], RET-fusion [76,77], ROS1-fusion [78], NTRKfusion [79], or KRAS [80].Collectively, it is estimated that approximately 15% of NSCLC tumors harboring EGFR, KRAS, ALK fusion, and RET fusion alterations exhibit MET amplification [26].This underscores the importance of considering MET amplification as a potential resistance mechanism in the management of various oncogenic driver alterations.
Understanding the prevalence and clinical implications of MET amplification in acquired resistance scenarios is crucial for optimizing treatment strategies.Efforts are underway to develop effective therapeutic approaches targeting MET amplification to overcome acquired resistance.These include the investigation of combination therapies involving MET inhibitors with other targeted agents or immunotherapies to enhance treatment efficacy and prevent the emergence of resistance.Additionally, ongoing research aims to elucidate the underlying mechanisms and molecular interactions associated with MET amplification-mediated resistance, which will inform the development of novel therapeutic strategies for patients with MET-amplified NSCLC.
However, it is worth noting that the cutoff criteria for defining amplification varied across trials, and establishing standardized criteria for identifying optimal treatment targets remains a future challenge.

Combination Therapy
Combination therapy involving primary oncogene TKIs and MET TKIs has emerged as a rational strategy for addressing acquired resistance resulting from MET amplification.In recent years, significant advancements have been made in targeting both MET and EGFR, leading to the initiation of several clinical trials investigating the combination of MET inhibitors and EGFR inhibitors for acquired resistance in NSCLC.Table 2 provides an overview of combination therapy trials targeting MET amplification in EGFRmutant NSCLC.Capmatinib and tepotinib, both FDA-approved for NSCLC with MET exon14 skipping [18,19], have demonstrated positive outcomes when combined with gefitinib in pretreated NSCLC cases harboring both EGFR mutations and MET amplification [89,90].In a phase II study, the combination of capmatinib and gefitinib in patients with acquired resistance to EGFR-TKI and MET amplification (GCN ≥ 4 by FISH) or MET overexpression (IHC 3+) showed improved activity, particularly in the high MET amplification group (GCN ≥ 6; ORR 47% and mPFS 5.5 months) [89].Another phase II study, known as the INSIGHT study, investigated the combination of tepotinib and gefitinib in patients with acquired resistance to EGFR-TKI, MET amplification (GCN ≥ 5 or MET/CEP ≥ 2.0 by FISH), or MET overexpression (IHC 2+/3+).The ORR in this study was 67%, with a median PFS of 16.6 months compared to 4.2 months with chemotherapy (HR = 0.13, 90% CI = 0.04-0.43).Additionally, the overall survival was 37.3 months vs. 13.1 months with chemotherapy (HR = 0.08, 90% CI = 0.01-0.51)[90].
Savolitinib, a potent and selective MET TKI, in combination with osimertinib, was evaluated in the TATTON study for its efficacy in pretreated EGFR mutation-positive lung cancers with MET amplification [91].In patients who experienced resistance after first-or second-generation EGFR-TKIs with MET amplification (GCN ≥ 5 or MET/CEP ≥ 2.0 by FISH or GCN ≥ 5 by NGS), the ORR ranged from 64% to 67%.For patients with resistance after third-generation EGFR-TKIs and MET amplification, the objective response rate was 30%.A phase I study examining the combination of savolitinib and gefitinib in patients with acquired resistance to EGFR-TKI and MET amplification (GCN ≥ 5 or MET/CEP ≥ 2.0 by FISH) reported an objective response rate of 52% [92].It is important to note that the criteria for defining MET amplification varied across these studies.Furthermore, resistance-related amplification is often considered a subclonal population, which may warrant a lower threshold for defining the degree of amplification compared to de novo amplification [57,58].Nevertheless, studies have consistently demonstrated that higher degrees of amplification correspond to improved treatment efficacy [57].
Ongoing investigations are exploring the use of antibody-based therapies targeting MET.Telisotuzumab vedotin, an antibody-drug conjugate targeting MET, exhibited promising antitumor activity and acceptable toxicity in NSCLC cases with acquired resistance to EGFR-TKI and MET overexpression (H-score ≥ 150 by IHC).In a study involving patients with MET overexpression (including four patients with MET amplification out of a total of 28 patients), ORR was 32%, and mPFS was 5.9 months [50].

Ongoing Study
Several ongoing clinical trials are currently investigating treatment strategies targeting MET amplification.These trials aim to further evaluate the efficacy and safety of different therapeutic approaches.One notable trial is a phase III study evaluating the combination of savolitinib and osimertinib for acquired resistance in NSCLC (ClinicalTrials.govIdentifier: NCT05015608).This trial seeks to assess the potential benefits of combining a MET inhibitor (savolitinib) with a third-generation EGFR inhibitor (osimertinib) in patients who have developed resistance to previous treatments.
In addition, a phase II trial is underway to investigate the efficacy of savolitinib in combination with osimertinib for acquired resistance in NSCLC (ClinicalTrials.govIdentifier: NCT03778229).This study aims to provide further insights into the potential synergistic effects of these targeted therapies in overcoming resistance mechanisms, specifically in patients with MET amplification.
Furthermore, a phase II trial is evaluating the combination of savolitinib and durvalumab, an immune checkpoint inhibitor, for advanced gastric cancer (ClinicalTrials.govIdentifier: NCT05620628).This trial explores the potential of combining MET inhibition with immune checkpoint blockade to enhance therapeutic outcomes in this patient population.
Lastly, a phase I trial is investigating amivantamab, a human bispecific antibody targeting both EGFR and MET, in advanced NSCLC (ClinicalTrials.govIdentifier: NCT02609776).This study aims to assess the safety and efficacy of this novel antibody therapy in patients with advanced NSCLC harboring EGFR mutations and MET amplification.
These ongoing trials represent important steps in advancing our understanding of MET amplification as a therapeutic target and may contribute to the development of more effective treatment strategies for patients with acquired resistance in various cancer types.The results from these studies will provide valuable insights into the clinical utility of targeting MET amplification and may guide future treatment decisions in precision oncology.

Conclusions
The treatment of MET amplification represents a promising avenue, particularly in combination with EGFR-TKI therapy for pretreated NSCLC patients.However, the appropriate selection of patients is crucial for maximizing treatment efficacy.The underlying mechanisms contribute to the varying effectiveness of MET inhibitors based on the degree of MET amplification.Nevertheless, there remains a lack of consensus regarding the specific threshold or cut-off value for MET amplification across different studies.
Further research is needed to determine the optimal cutoff value for MET amplification, to identify the best combination therapies, and to develop new targeted therapies for patients with MET amplification.

Figure 1 .
Figure 1.Schematic representation of focal gene amplification and polysomy involving the MET gene and chromosome 7 copy number alterations.

Figure 1 .
Figure 1.Schematic representation of focal gene amplification and polysomy involving the MET gene and chromosome 7 copy number alterations.
et al. conducted a comprehensive analysis of the relationship between the definition of MET amplification by FISH and the response rate to targeted therapy in previous clinical trials.They consistently observed the most favorable outcomes in NSCLCs characterized by high-level MET amplification [57].
ORR; objective response rate, mPFS; median progression free survival.

Table 2 .
MET amplification combination therapy trials for EGFR-mutant NSCLC.