Can Next Generation Sequencing Be the Standard of Care for all Patients With Metastatic Non-Squamous Non-Small Cell Lung Cancer

The genetic alterations of most lung cancers are enormous. There are approximately 20,667 genes in the human genome comprised of over 3 billion base pairs. Smokers with lung adenocarcinoma have, on average, 8-10 mutations per 1 million bases (base pairs), which equates to over 50,000 single nucleotides [1]. The current standard of care in the treatment of stage IV nonsquamous non-small cell lung cancer (NSCLC) is based on tailoring treatment on just a handful of actionable genetic drivers. Specifically, the International Association for the Study of Lung Cancer (IASLC), the College of American Pathologists (CAP), and the Association for Molecular Pathology (AMP) issued a joint evidence-based guideline establishing recommendations for molecular diagnostic testing in lung cancer. In summation, their recommendations were to test all patients with lung adenocarcinoma for EGFR and ALK abnormalities regardless of clinical variables such as smoking history, gender, or ethnicity [2]. These guidelines are dated, and the landscape of actionable targets, as well as the availability and cost of technology, have changed dramatically since the recommendation. In light of the explosion of newly discovered gene targets, advances in sequencing technology, and development of novel pharmaceuticals, it is time to reexamine these guidelines and recommend every tumor be sequenced.


Introduction
The genetic alterations of most lung cancers are enormous.
There are approximately 20,667 genes in the human genome comprised of over 3 billion base pairs. Smokers with lung adenocarcinoma have, on average, 8-10 mutations per 1 million bases (base pairs), which equates to over 50,000 single nucleotides [1]. The current standard of care in the treatment of stage IV nonsquamous non-small cell lung cancer (NSCLC) is based on tailoring treatment on just a handful of actionable genetic drivers. Specifically, the International Association for the Study of Lung Cancer (IASLC), the College of American Pathologists (CAP), and the Association for Molecular Pathology (AMP) issued a joint evidence-based guideline establishing recommendations for molecular diagnostic testing in lung cancer. In summation, their recommendations were to test all patients with lung adenocarcinoma for EGFR and ALK abnormalities regardless of clinical variables such as smoking history, gender, or ethnicity [2]. These guidelines are dated, and the landscape of actionable targets, as well as the availability and cost of technology, have changed dramatically since the recommendation. In light of the explosion of newly discovered gene targets, advances in sequencing technology, and development of novel pharmaceuticals, it is time to reexamine these guidelines and recommend every tumor be sequenced.
The current standard of care for metastatic NSCLC is dependent on if the tumor has EGFR, ALK, ROS-1, BRAF V600, or PDL-1 gene alterations. If the tumor harbors an activating mutation in EGFR exon 19 or 21, an EGFR tyrosine kinase inhibitor is preferred as the first-line therapy [3]. In tumors harboring a ROS-1 or similarly ALK gene alterations, Crizotinib is preferred first-line therapy [4]. In carcinomas with BRAF V600E mutations, the FDA recently approved the combination of Dabrafenib plus Trametinib [5]. If a tumor is PDL-1 positive with a tumor proportion score of ≥50%, single-agent Pembrolizumab is preferred, but only if the tumor does not contain EGFR, ALK, or ROS1 alterations [6]. In the scenario that a tumor has none of the above actionable biomarkers, standard of care is treatment with carboplatin plus pemetrexed or carboplatin plus paclitaxel with or without bevacizumab [7].
Recently, mutations in the MET exon 14 and have been described as a potential target. The MET oncogene has been shown to be a sufficient cause of carcinoma exclusive of other oncogenic drivers including EGFR, KRAS, and BRAF mutations in addition to being exclusive to ALK ROS-1 and RET gene rearrangements [8]. Uniquely, MET mutations are not related to smoking history [8]. Treatment with MET Tki Crizotinib in MET exon 14 positive tumors have shown very high response rates. A single-arm study showing 34 patients with metastatic NSCLC with a MET mutation had median overall survival of 8.1 months, whereas those treated with a MET inhibitor had mean overall survival of 24.6 months [9].
While more evidence is needed, one should consider using a MET Tki as first or second line in patients with MET exon 14 skipping mutations. Despite 3% of lung adenocarcinomas being driven by MET exon 14 skip mutations, a target with known pharmacologic agents, the current standard of care does not currently require be markers of increased immunotherapy effectiveness [11]. With the simultaneous rise of advanced genomics and an increase in specialized pharmaceuticals, there has been an explosion of newly designed target therapies. When and if a clinician will use these targeted therapies, however, hinges on profiling the carcinoma genome for both breadth and depth of mutations, this is a task most suited for next genome sequencing (NGS). Therefore, as more molecular and gene targets emerge, is it now time to perform NGS on all patients with NSCLC rather than current standard single molecular target testing? (Table 1). ALK translocations 1st/Yes Alectinib [13], Crizotinib [4], Brigatinib [14] and Certinib [15] Brafv600e mutations 1st/Yes Dabrafenib and Trametinib [5] PD-L1 (tumor proportion score >50%) 1st/Yes Pembrolizumab [16] Met mutations or amplifications 2nd/No Crizotinib [17]and Cabozantinib [18] Her2 mutations 2nd/No Afatinib, Trastuzumab, and Ado-trastuzumab emtansine [19] RET mutation or translocation 2nd/No Cabozantinib [20], Vandetani [21] and Alectinib [22] LMNA-NTRK1 fusion 2nd/No LOXO-101 [23] From a clinical perspective, acting on this widespread menu of new actionable targets is only possible if those targets are pursued. Traditional DNA analysis is done with Sanger sequencing (SS), pyrosequencing, or traditional single-gene PCR assays [24].
The major issue with these technologies is only a few genes can be sequenced per run leading to exorbitant cost when looking at more than handful of genes [25]. Additionally, these technologies fail to incorporate intratumor heterogeneity with low sensitivities in identifying oncogenic drivers present at low frequency [26,27].
NGS is a technology in which the entire genome can be analyzed simultaneously, making it an ideal tool when the list of actionable targets is substantial [28].AdditionallyNGS is characterized by high coverage also called "deep sequencing," which allows for the characterization and identification of intratumor heterogeneity of low-frequency variants [29].
The first task for a clinician using NGS is with obtaining a sample. Analyzing samples can currently originate from FFPE tumor blocks, a diagnostic biopsy sample, or, more recently a simple blood draw (liquid biopsy). FFPE tumor blocks are common and allow the sample to undergo further pathological staining later.
Storage of FFPE is also commonplace making it easier for further testing in the future.
With liquid biopsy, one obstacle is yield of sufficient tumor.
Currently, this process yields, on average, one cancer cell in 106-

cells, which is insufficient genetic material without undergoing
an additional enrichment step [30].
This Enrichment step adds another layer of manageable complexity, which is yet another factor a physician needs to consider when interpreting the results. Important aspects to consider when tissue-based testing is not possible due to risk of biopsy location [33]. Advances in bioinformatic analysis are now at the point where as long as the sample has adequate coverage, liquid NGS is as sensitive as traditional based PCR in identifying mutations [34].
Additional factors include the availability and access to an NGS platform and the amount of time a clinician must wait for sample processing. While ten years ago, NGS was limited to large, wealthy,  Advances in treatment for NSCLC are being driven by an everdeepening and expanding knowledge of the biology of tumor cells on an individual level. Current guidelines are based on the treatment of just a few genes, but one must wonder why not all genes? Every passing year has brought numerous discoveries in gene targets and subsequent successes with novel pharmaceuticals. For instance, this past year saw the identification of pan tumor aberrations, such as NTRK, while extraordinary rare was able to be detected and targeted [44]. The breadth, ease, and cost of testing for these mutations, which can have profound impact on management, needs to be commonplace. Furthermore, making precision genetics part of the standard of care will further drive down costs, and the data generated from NGS will be a boon to knowledge, providing even more insight into other yet undiscovered genes. We believe we are at the point now both from a medical and economic standpoint to recommend that panel testing be routine for patients with NSCLC.