Comparative analysis of expression of mutant and wild-type alleles is essential for reliable PCR-based detection of MET exon 14 skipping

Mitiushkina, Natalia V.; Kholmatov, Maksim; Tiurin, Vladislav I.; Romanko, Alexandr A.; Yatsuk, Olga S.; Соколова, Татяна; Ivantsov, Alexandr O.; Kuligina, E.; Stepanov, Ilya; Belyaеv, Alexey M.; Togo, Alexandr V.; Imyanitov, Evgeny N.

doi:10.1016/j.biochi.2019.08.014

Cited by 11 publications

(14 citation statements)

References 30 publications

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“…All PCR reactions were run on the Bio-Rad CFX96 instrument. MET exon 14 skipping (MET ex14Δ) mutations were tested as described by Mitiushkina et al 15 Protocols for KRAS, NRAS, and BRAF mutational analysis were reported previously. 16,17 Next-generation sequencing (NGS) was performed using QIAseq-Targeted RNAscan Panel (Qiagen, Germany) with custom primers, which covered selected exons of the ALK, ROS1, RET, EGFR, KRAS, BRAF, and several other genes (Table S5).…”

Section: Methodsmentioning

confidence: 99%

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Comprehensive evaluation of the test for 5′‐/3′‐end mRNA unbalanced expression as a screening tool for ALK and ROS1 fusions in lung cancer

et al. 2022

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Background Despite the progress in the development of next‐generation sequencing (NGS), diagnostic PCR assays remain to be utilized in clinical routine due to their simplicity and low cost. Tests for 5′‐/3′‐end mRNA unbalanced expression can be used for variant‐independent detection of translocations, however, many technical aspects of this methodology require additional investigations. Methods Known ALK/ROS1 fusions and 5′‐/3′‐end unbalanced expression were analyzed in 2009 EGFR mutation‐negative non‐small cell lung cancer (NSCLC) samples with RT‐PCR tests, which were optimized for the use with FFPE‐derived RNA. Results Variant‐specific PCR tests for 4 common ALK and 15 common ROS1 translocations detected 115 (5.7%) and 44 (2.2%) rearrangements, respectively. Virtually all samples with common ALK fusions demonstrated some level of 5′/3′ mRNA ends unbalanced expression, and 8 additional NSCLCs with rare ALK fusions were further identified by PCR or NGS among 48 cases selected based on ALK expression measurements. Interestingly, NSCLCs with unbalanced 5′‐/3′‐end ALK expression but without identified ALK translocations had elevated frequency of RAS mutations (21/40, 53%) suggesting the role of RAS activation in the alternative splicing of ALK gene. In contrast to ALK, only a minority of ROS1 translocation‐positive cases demonstrated unbalanced gene expression, with both 5′‐ and 3′‐end mRNA expression being elevated in most of the samples with translocations. Surprisingly, high ROS1 expression level was also found to be characteristic for NSCLCs with activating mutations in other tyrosine kinases such as EGFR, ALK, or MET. Conclusions Comprehensive ALK analysis can be performed by the test for 5′‐/3′‐end unbalanced expression with minimal risk of missing an ALK rearrangement. In contrast, the use of the test for 5′‐/3′‐end unbalanced expression for the detection of ROS1 fusions is complicated; hence, the utilization of variant‐specific PCR assays for ROS1 testing is preferable.

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Section: Methodsmentioning

confidence: 99%

“…All PCR reactions were run on the Bio‐Rad CFX96 instrument. MET exon 14 skipping (MET ex14Δ) mutations were tested as described by Mitiushkina et al 15 Protocols for KRAS , NRAS , and BRAF mutational analysis were reported previously. 16 , 17 …”

Section: Methodsmentioning

confidence: 99%

Comprehensive evaluation of the test for 5′‐/3′‐end mRNA unbalanced expression as a screening tool for ALK and ROS1 fusions in lung cancer

et al. 2022

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“…The detection of ALK, ROS, RET, and NTRK1-3 rearrangements may be based on the immunohistochemistry (IHC) guided detection of the overexpression of the kinase portion of the corresponding protein or on the break-apart FISH assay (3). The methodology of MET testing remains to be standardized (4). PD-L1 expression analysis is apparently the most complicated assay for the time being.…”

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confidence: 99%

“…The latter approach allows for identification of all druggable gene fusions irrespective of the translocation variant ( 28 ). PCR analysis is relatively non-expensive and is more flexible for the incorporation of new predictive tests, as exemplified by the development of the assay for detection of MET exon 14 skipping mutations ( 4 ).…”

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Harmonization of Molecular Testing for Non-Small Cell Lung Cancer: Emphasis on PD-L1

2020

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Comprehensive molecular testing plays a critical role in the choice of treatment for non-small lung cell cancer (NSCLC). The analysis of druggable alterations in EGFR, BRAF, MET, KRAS, ALK, ROS1, RET and NTRK1/2/3 genes is more or less standardized and can be achieved using a single diagnostic platform, e.g., next generation sequencing (NGS) or polymerase chain reaction (PCR). In contrast to above targets, PD-L1 testing requires the use of immunohistochemistry (IHC). There are multiple PD-L1 IHC assays, which utilize distinct antibodies and detection systems. These PD-L1 tests are tailored to distinct drugs, often rely on different thresholds and scoring guidelines, and are characterized by incomplete inter-laboratory and inter-observer reproducibility. Several studies evaluated the performance of PD-L1 RNA expression tests, as PCR-based RNA analysis is compatible with other NSCLC molecular testing platforms, can be performed in a semi-automated manner, and has a potential for proper standardization. These investigations revealed a correlation between PD-L1 protein and RNA expression; however, there were NSCLCs demonstrating decent amounts of PD-L1 transcript in the absence of PD-L1 IHC staining. Clinical studies are required to evaluate, which of the two PD-L1 testing approaches, i.e., RNA or protein expression measurement, has a better predictive value.

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“…The answer likely lies buried in the details of the highly complex signaling and regulatory network that the MET/ HGF axis embodies. Mitiushkina et al developed a simple TaqMan polymerase chain reaction (PCR) assay specifically aimed at precise quantitation of mutant versus wild-type allele expression (20). Their assay identified 35 METex14 mutations out of 1,415 EGFR-negative lung adenocarcinoma samples (2.5%) which were confirmed by Sanger sequencing.…”

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Devil in the detail: MET overexpression fails as surrogate marker for MET exon 14 splice site mutations in NSCLC

Strickland¹

2020

Ann Transl Med

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Comparative analysis of expression of mutant and wild-type alleles is essential for reliable PCR-based detection of MET exon 14 skipping

Cited by 11 publications

References 30 publications

Comprehensive evaluation of the test for 5′‐/3′‐end mRNA unbalanced expression as a screening tool for ALK and ROS1 fusions in lung cancer

Comprehensive evaluation of the test for 5′‐/3′‐end mRNA unbalanced expression as a screening tool for ALK and ROS1 fusions in lung cancer

Harmonization of Molecular Testing for Non-Small Cell Lung Cancer: Emphasis on PD-L1

Devil in the detail: MET overexpression fails as surrogate marker for MET exon 14 splice site mutations in NSCLC

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