{"id":36352,"date":"2020-12-23T20:05:59","date_gmt":"2020-12-23T20:05:59","guid":{"rendered":"http:\/\/touchoncology.com\/?p=36352"},"modified":"2022-10-28T15:50:56","modified_gmt":"2022-10-28T14:50:56","slug":"met-exon-14-skipping-alterations-in-nsclc-current-understanding-and-therapeutic-advances","status":"publish","type":"post","link":"https:\/\/touchoncology.com\/lung-cancer\/journal-articles\/met-exon-14-skipping-alterations-in-nsclc-current-understanding-and-therapeutic-advances\/","title":{"rendered":"MET Exon 14 Skipping Alterations in NSCLC: Current Understanding and Therapeutic Advances"},"content":{"rendered":"

Advanced non-small cell lung carcinoma (NSCLC) treatment paradigms have evolved during the past decade. Identification of tumor-specific molecular alteration in cancer driver genes has led to the development of targeted therapies.1\u20133<\/span>\u00a0Most of the tumors harboring such alterations are sensitive to tyrosine kinase inhibitor (TKI) drugs, making such oncogenic drivers promising targets for the development of antitumor therapeutics.4,5<\/span><\/p>\n

MET<\/span><\/em>\u00a0is a proto-oncogene that can act as an oncogenic driver after certain genomic alterations. It is expressed in many epithelial as well as mesenchymal cells, including hepatocytes, hematopoietic cells, and neuronal cells, and is essential for important biological processes, such as embryonic development and organogenesis.6,7<\/span>\u00a0However, mutations and its aberrant activation can promote tumor development and cancer progression by dysregulating downstream signaling pathways.8,9\u00a0<\/span>Initially, abnormal MET signaling was believed to be the mechanism of resistance acquired by NSCLC tumor cells against certain therapeutics.10\u201312<\/span>\u00a0Further reports demonstrated the role of\u00a0MET<\/span><\/em>\u00a0alterations in sustained\u00a0MET<\/span>\u00a0pathway dysregulation, leading to oncogenesis.13\u201315<\/span>\u00a0Clinically, NSCLCs with\u00a0MET<\/span><\/em>\u00a0alterations are associated with poor prognosis, and these alterations have been recognized as an important therapeutic target in various cancers, including NSCLC.16\u201318<\/span><\/p>\n

In this review, we discuss the current understanding of the implications of aberrant\u00a0MET<\/span><\/em>\u00a0activation in NSCLC harboring\u00a0MET<\/span><\/em>\u00a0exon 14 (METex14<\/span><\/em>) skipping alteration, available diagnostic options, potential therapies in the pipeline, and the future clinical landscape.<\/p>\n

Structure and function of the MET receptor<\/h2>\n

MET<\/span><\/em>\u00a0was first identified in a chemically treated human-osteosarcoma-derived cell line as a transforming gene from a fusion of\u00a0TPR-MET<\/span>.19<\/span>\u00a0The\u00a0MET<\/span><\/em>\u00a0gene is located on chromosome 7q31 in the human genome, which spans about 125 kb DNA and contains 21 exons and 20 introns.20<\/span>\u00a0MET is encoded as a precursor, which is modified into a mature protein by proteolytic cleavage between its\u00a0a<\/span>\u00a0and\u00a0b<\/span>\u00a0subunits.21<\/span>\u00a0A mature MET protein is composed of a small\u00a0a<\/span>\u00a0subunit (50 kDa) and a larger\u00a0b<\/span>\u00a0(145 kDa) subunit linked together by a disulfide bridge.8<\/span>\u00a0The\u00a0a<\/span>\u00a0subunit and a portion of\u00a0b<\/span>\u00a0subunit together form the extracellular region of the heterodimer protein, while the remainder of the\u00a0b<\/span>\u00a0subunit comprise the transmembrane and intracellular regions (Figure 1A<\/span><\/em>).<\/p>\n

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The extracellular component of MET contains three domains. N-terminal Sema (Sema-phorin) is the largest domain comprising 500 residues, which encompasses the\u00a0a<\/span>\u00a0and a part of\u00a0b<\/span>\u00a0subunits. The domain is essential for the ligand binding,22<\/span>\u00a0dimerization, and activation of MET.23,24<\/span>\u00a0The Sema domain is followed by the plexins-semaphorins-integrins (PSI) domain, containing four disulphide bonds, which are essential for the proper orientation of the receptor for ligand binding.25<\/span>\u00a0The PSI domain is connected to the transmembrane helix of MET through the immunoglobulin-plexins-transcription factor domain. The intracellular portion of the receptor includes a juxtamembrane (JM) domain, a tyrosine kinase (TK) catalytic domain, and a C-terminal multifunctional docking site.22<\/span>\u00a0Binding of its ligand, hepatocyte growth factor (HGF), which is also known as scatter factor, is essential for the activation of the kinase activity.26,27<\/span>\u00a0HGF is the only MET receptor ligand known so far and binds to the receptor with high affinity.22,28<\/span><\/p>\n

MET signaling and its dysregulation in NSCLC<\/h2>\n

HGF binding to MET causes dimerization of the receptor leading to the autophosphorylation of intracellular residues Y1234 and Y1235 in the kinase domain followed by phosphorylation of two additional tyrosine residues, Y1349 and Y1356, in the C-terminal outside of the kinase domain (Figure 1B<\/span><\/em>). Phosphorylation of the C-terminal residues leads to the formation of the docking site, which is necessary for the engagement of signaling partners.29<\/span>\u00a0Subsequently, adapter and effector proteins, such as GRB2 (growth factor receptor bound protein 2), GAB1 (GRB2 associated binding protein 1) and SHC (Src homology 2 domain-containing), bind to the docking site triggering downstream signaling.30\u201336<\/span>\u00a0MET signaling plays a crucial role in executing various cellular functions.37\u201339<\/span>\u00a0To maintain functional balance and cellular integrity, MET activity is regulated through various mechanisms. The active MET receptor can phosphorylate at residue Y1003 in the JM domain, a site for the recruitment of E3-ligase Casitas B-lineage lymphoma (CBL), and subsequently undergo ubiquitin-mediated lysosomal degradation, leading to the downregulation of\u00a0MET<\/span>\u00a0(Figure 2A<\/span><\/em>).40\u201342<\/span>\u00a0Additionally, it has been shown that phosphorylation of S985 at JM domain acts as a counterbalance to receptor activation, by negatively regulating its activity, even in the presence of HGF.43,44<\/span>\u00a0Furthermore, proteolytic cleavage of MET by ADAMs (a disintegrin and metalloproteinase) and gamma-secretase may also contribute to the downregulation of MET receptor activity.45,46<\/span><\/p>\n

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Alterations in\u00a0MET<\/span><\/em>\u00a0can result in the dysregulation of\u00a0MET<\/span>\u00a0<\/em>signaling, which is present in various solid tumors including NSCLC and is associated with tumor progression and metastasis.47\u201349<\/span>\u00a0Gene amplification, rearrangement, and skipping alterations, which lead to the overexpression and impaired degradation of MET, are the major underlying factors of aberrant MET activation.50,51<\/span>\u00a0Alteration or deletion of crucial residues in regulatory domain interfere with mechanisms that help to maintain MET receptor turnover leading to its accumulation and hyperactivation.52\u201354<\/span><\/p>\n

METex14<\/span>\u00a0<\/span><\/em>skipping alteration in NSCLC<\/h2>\n

Skipping of\u00a0METex14<\/span><\/em>\u00a0in NSCLC was first reported in 2005.55<\/span>\u00a0Substitutions or deletions at 3\u2032<\/span>\u00a0splice site in intron 13 or the 5\u2032<\/span>\u00a0end splice site of intron 14 results in\u00a0METex14<\/span><\/em>\u00a0skipping.56,57<\/span>\u00a0This somatic alteration, at or around the splice junction of\u00a0METex14<\/span><\/em>, leads to the loss of exon 14 in the transcript and synthesis of the MET protein with an in-frame deletion of 47 amino acids in the JM domain (including residue Y1003) ablating the CBL-mediating ubiquitination and degradation of the receptor (Figure 2B<\/span><\/em>).24,58<\/span>\u00a0Consequently,\u00a0METex14<\/span><\/em>\u00a0skipping results in increased levels of MET protein, which can drive activation of downstream signaling pathways that promote tumor development.57,59<\/span><\/p>\n

Splicing occurs through two sequential steps involving various parts of the intron. The splice donor site and the splice acceptor site are present at the 5\u2032<\/span>\u00a0and 3\u2032<\/span>\u00a0ends, respectively. The splice acceptor site is flanked upstream by the branch point and poly-pyrimidine tract sites (Figure 3A<\/span><\/em>). First, the branch point nucleotide performs a nucleophilic attack on the first nucleotide of the intron at the splice donor site. This forms an intermediate loop or lariat. Subsequently, the 3\u2032<\/span>\u00a0end of the released exon performs a similar nucleophilic attack on the last nucleotide of the SA thereby fusing the exons and releasing the intron lariat.60,61<\/span>\u00a0Most\u00a0METex14<\/span><\/em>\u00a0skipping alterations involve the branch point, poly-pyrimidine tract or splice acceptor site in intron 13 or the splice donor site in intron 14. As shown in\u00a0Figure 3B<\/span><\/em>, these alterations interfere with the splicing mechanism leading to exon 14 skipping.<\/p>\n

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Interestingly,\u00a0METex14<\/span><\/em>\u00a0skipping alterations are primary oncogenic drivers in NSCLC, as these alterations are most likely to be mutually exclusive to other known oncogenic drivers, such as\u00a0KRAS<\/span>,\u00a0EGFR<\/span>,\u00a0ALK<\/span>,\u00a0ROS1<\/span>\u00a0<\/em>or\u00a0RET<\/span><\/em>.57,62,63\u00a0<\/span>Approximately 3\u20134% of NSCLCs harbor the\u00a0METex14<\/em>\u00a0<\/span>alteration (Table 1<\/span><\/em>).18,57,63\u201368<\/span>\u00a0They are associated with some histologic subtypes of NSCLC but are not related to tumor stage. Among the histological subtypes,\u00a0METex14<\/span>\u00a0<\/em>skipping alteration is commonly found in sarcomatoid carcinoma (4.9\u201331%),69\u201372<\/span>\u00a0adenosquamous carcinoma (4\u20138%),18,73,74<\/span>\u00a0adenocarcinoma (3\u20134%),1,18,57,65,75,76<\/span>\u00a0and squamous cell carcinoma (2%).74,77<\/span>\u00a0Also, among adenocarcinomas, the predominant subtypes are acinar (35\u201352.9%) or solid subtypes (35.3\u201353%).64,70,73,74,77<\/span>\u00a0Clinically,\u00a0METex14<\/span><\/em>\u00a0skipping abnormality is found mostly in patients of advanced age.18,63,68,70,73,74,77,<\/a>78<\/span><\/p>\n

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Detection of\u00a0METex14<\/span>\u00a0<\/span><\/em>skipping alteration<\/h2>\n

Immunohistochemical analysis is a routine practice for the detection of\u00a0MET<\/span><\/em>\u00a0overexpression. However, this technique on its own cannot specifically confirm\u00a0METex14<\/span><\/em>\u00a0skipping or an underlying alteration. Therefore, DNA- and RNA-based molecular assays are preferred methods for the detection of\u00a0METex14<\/span><\/em>\u00a0alteration. DNA-based sequencing assay can detect\u00a0MET\u00a0<\/span><\/em>alterations such as insertions, deletions, point mutations, or duplications in splice sites, which may cause exon 14 skipping. Identification of such mutational hotspots leading to\u00a0METex14<\/span>\u00a0skipping alteration are used to predict the possible skipping event. However,\u00a0METex14<\/span><\/em>\u00a0skipping is associated with more than 120 reported sequence variants in splice sites, which makes it challenging to detect these mutations using only DNA-based assays.57,63<\/span>\u00a0Therefore, analysis of RNA transcripts allows for the verification of fusion between exons 13 and 15.83<\/span>\u00a0In ideal cases, both DNA- and RNA-based assays are used to complement each other for reliable detection of\u00a0METex14<\/span>\u00a0<\/em>alterations (Table 1<\/span><\/em>).1,18,63<\/span>\u2013<\/span>82<\/span><\/p>\n

Reverse transcription polymerase chain reaction (RT-PCR), quantitative real time RT-PCR, and Sanger sequencing are the routine approaches used for the analysis of mutations and\u00a0METex<\/span>14<\/em> alteration.18,73<\/span>\u00a0mRNA transcript can be reverse transcribed using RT-PCR and corresponding complementary DNA is sequenced using Sanger techniques to verify exon 14 skipping from the sample. However, efficiency of the method relies on the quality of RNA, which is often derived from formalin-fixed paraffin-embedded or frozen tissue.83<\/span>\u00a0Sanger sequencing of\u00a0METex14<\/span>\u00a0<\/em>and its splice sites is still in routine practice for small scale analysis covering a portion of genomic region, which is performed using the PCR amplicon from genomic DNA covering exon 13 and exon 15, or cDNA from\u00a0MET<\/span>\u00a0transcript. However, the European Society for Medical Oncology (ESMO) guidelines has proposed next-generation sequencing (NGS) and RNA sequencing, if possible, to detect\u00a0METex14<\/em>\u00a0<\/span>alteration in its updated guidelines on September 2020.84,85<\/span><\/p>\n

Recently, NGS has become a common diagnostic method to identify\u00a0METex<\/span>14<\/em> alterations. This high throughput method allows the large-scale analysis of multiple samples in a short time with comprehensive genomic coverage.1,64,86,87<\/span>\u00a0The two most popular NGS sequencing panels used for targeted sequence profiling are hybridization capture and amplicon-based sequencing panels. The hybridization capture panel allows more comprehensive profiling for all alteration types, whereas the amplicon-based panel is ideal for analysing single nucleotide variants and indels (insertions and deletions). NGS analysis also can simultaneously detect other mutations or translocations (such as\u00a0ALK<\/span>,\u00a0ROS1<\/span>,\u00a0RET<\/span>,\u00a0NTRK1,<\/span>\u00a0and\u00a0NRG1<\/span>\u00a0fusions) in a single assay.83<\/span>\u00a0Due to the inherent difficulties in acquiring sufficient RNA material for testing, DNA-based NGS panels are used more frequently to identify\u00a0METex14<\/em>\u00a0<\/span>skipping alterations. Recently, the US Food and Drug Administration (FDA) approved FoundationOne\u00ae<\/span>\u00a0CDx (Foundation Medicine, Cambridge, MA, USA) as a companion diagnostic test for this indication.88<\/span>\u00a0Circulating tumor DNA (CtDNA) or RNA from plasma\/blood samples (liquid biopsy) can also be used to identify\u00a0METex14<\/span><\/em>\u00a0alterations using NGS technologies. A clinical trial (VISION; ClinicalTrials.gov Identifier: NCTO2864992) aiming to test\u00a0<\/span>METex14<\/span><\/em>\u00a0skipping alterations in circulating free DNA using plasma liquid biopsy is ongoing.89<\/span>\u00a0Some of the commercially available targeted NGS assays that are used to detect these alterations are compared in\u00a0Table 2<\/span><\/em>.90<\/span>\u2013<\/span>101<\/span><\/p>\n

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Therapeutic intervention of NSCLC with\u00a0METex14<\/em>\u00a0<\/span>skipping alteration<\/h2>\n

NSCLC characterized with\u00a0METex14\u00a0<\/span>skipping alterations is targetable.57<\/span>\u00a0Although many\u00a0METex14<\/span><\/em>\u00a0skipping tumors were found to express programmed death-ligand-1 (PD-L1), the overall response rate to PD-1\/PD-L1-directed immune checkpoint inhibitors has been found to be low, and median progression-free survival (mPFS) was found to be short in patients with NSCLC.102,103<\/span>\u00a0It should be noted that the mutation burden is generally low in such tumors. There are three therapeutic approaches to target tumors harboring\u00a0METex14<\/em><\/span>\u00a0skipping alteration:<\/p>\n