Course Faculty Response

The CAP/IASLC/AMP Guidelines published in 2013 (Lindeman, 2013) and endorsed by ASCO in 2014 (Leighl, 2014) recommend routine testing for EGFR mutations and ALK gene rearrangements in patients with advanced  adenocarcinoma, or mixed carcinoma with adenocarcinoma component including adenosquamous carcinoma. Importantly, clinical characteristics such as age, gender, race, and smoking history are not recommended as a selection criterion for molecular testing.  Exceptions to these rules are limited lung cancer specimens (biopsy, cytology) where an adenocarcinoma component cannot be excluded, and cases of squamous or small cell carcinomas occurring in patients with unusual clinical characteristics such as young age or lack of smoking history. There is a strong argument that more comprehensive testing that would include genes for emerging targeted agents should be considered.

The updated draft of the CAP/IASLC/AMP Guideline that was posted for public comments in 2016 recommended (in addition to ALK and EGFR) ROS1, RET, HER2, BRAF and MET to be included in a broad testing panel. We are currently awaiting the final guideline for these additional biomarkers to be published. Testing for genes other than ALK and EGFR is not recommended to be performed as stand-alone tests, but rather as a part of a multigene panel.  ROS1 and RET are translocations that can be detected by FISH, RT-PCR, IHC or NGS. HER2 mutations most frequently occur as insertions in exon 20 and can be detected by mutation detection techniques. They do not correlate with the gene amplification or protein expression and therefore HER2 FISH or IHC should not be performed on the lung cancer specimens. MET gene is more complex and variety of mechanisms of dysregulation of the MET pathway in lung cancer have been reported. Two most important partially overlapping mechanisms include MET exon 14 alterations and MET gene amplification.  Although individual assays (i.e. mutation assays for MET exon 14 mutations or FISH/CISH for MET amplification) can be used, NGS approach seems to be more time and cost-effective.
 
The NGS approach is more efficient than stand-alone sequential assays, particularly in a patient who is known to be negative for some of the more common alterations. Furthermore, some NGS platforms can simultaneously detect mutations, copy number changes and rearrangements. Detection of MET exon 14 alterations can be very challenging for diagnostic testing, because of their diversity (Cui, 2014) (Frampton, 2015). Currently, DNA based broad, hybrid capture NGS is the most frequently used approach. It is worth mentioning   that MET exon 14 variants may not be included in the every NGS platform used in the clinical laboratories.

Therefore, communication between oncologist and pathologists about testing platforms is essential. Although lung cancers with MET exon 14 alterations tend to show high expression of MET by IHC, there are some cases that will be IHC negative despite the presence of mutations (Awad, 2016). More studies are needed to confirm correlation between MET exon 14 alterations and protein overexpression before MET IHC can be used as a screening tool. MET exon 14 alterations are mutually exclusive with other lung cancer driver mutations such as EGFR or KRAS, and have not been associated with acquired resistance to EGFR TKI (Frampton, 2015). In contrast, MET exon 14 mutations can co-exist with MET and MDM2 amplifications. From morphological standpoint, MET exon 14 alterations occur in 3-4% of lung adenocarcinoma, and in 20-30% of sarcomatoid carcinomas (Tong, 2016) (Liu, 2016).

Course Faculty Response

For METexon 14 alterations testing see case 1.

De novo MET amplification occurs in 1-5% of lung adenocarcinoma and its prevalence depends on the assay and defined cutoff points. Amplification and polysomy are two main mechanisms that can result in copy number changes. Broad, hybrid capture NGS assays has been increasingly used for detection of gene amplifications (Cheng, 2015). It is essential to compare the sequence of targeted regions from the tumor sample to a diploid normal sample to distinguish between polysomy and gene amplification.  It is also important that NGS platforms are validated against tumor samples with known gene amplifications determined by other methods such as HER2 FISH. NGS approach has a main advantage of providing information about other concurrent genomic alterations that may guide treatment decisions and that could be obtained from the same, frequently limited sample.  Cutoff points vary significantly between the laboratories and platforms, and is something that needs to be standardized.  FISH has been traditionally used for detection of gene amplification in clinical practice.

Currently, there is no guideline for cutoff of MET positivity in lung cancer specimens. Several positivity criteria have been reported such as five or more MET signals per cell (Cappuzzo, 2009), a MET/CEP7 ratio =2 (PathVysion kit) and adopted HER2 criteria for breast cancer. Lack of standardized interpretation criteria complicates comparison of reported studies, particularly in respect to outcomes from therapy.

Furthermore, MET amplification can occur synchronously with other oncogenic mutations such as KRAS, EGFR, BRAF etc in 41 to 63% of the lung cancer cases (Noonan, 2016). However, it has been shown that cases with a high level of amplification defined as MET/CEP7 ratio =5 show no overlap with other oncogenic mutations suggesting that MET amplification is probably a true oncogenic driver (Noonan, 2016).

It is also worth mentioning that about 20% of lung adenocarcinomas with METexon14 mutations also have high-level MET amplification (Awad, 2016) (Paik,2015) (Tong, 2016). Therefore, the choice of assay used for MET TKIs remains to be determined. Cases with METexon 14 mutations without MET amplification show response to MET TKI; however, it is uncertain if MET amplification alone can be used as a predictive biomarker. The proposed CAP/IASLC/AMP guideline does not provide recommendation for MET testing in respect to assay. Therefore, based on available studies testing for both METexon14 mutations and MET amplification may be considered for all MET inhibitors.
 
In this case, liquid biopsy sample showed concordant results with the tissue sample. Liquid biopsy is an evolving approach to noninvasive capture of circulating tumor cells (CTCs) and nucleic acids including cell-free RNA, microRNA, and circulating cell-free DNA (cfDNA), of which a subset may represent circulating tumor DNA. In lung cancer so far, the FDA has approved the cobas EGFR mutation test v2, a blood-based companion diagnostic for Tarceva. No other blood tests for other gene targets have been approved mostly because of low sensitivity (Copin, 2016).

 Faculty Summary
1.    MET exon 14 alterations are mutually exclusive with other lung cancer driver mutations such as EGFR or KRAS and have not been associated with acquired resistance to EGFR TKI. MET exon 14 alterations most frequently occur in sarcomatoid carcinomas (20-30%).
2.    Detection of MET exon 14 alterations can be very challenging for diagnostic testing, because of their diversity. Currently, DNA-based broad, hybrid capture NGS is the most frequently used approach that allows simultaneous detection of gene mutations and copy number changes.  MET exon 14 variants may not be included in the every NGS platform used in the clinical laboratories. Therefore, it is essential to know what alterations your laboratory’s assay can detect.
3.    De novo MET amplification occurs in 1-5% of lung adenocarcinoma and its prevalence depends on the assay and defined cutoff points. About 20% of lung adenocarcinomas with METexon14 mutations also have high-level MET amplification (FISH MET/CEP7 ratio ≥5).