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The new kid on the block

Lung cancer continues to be the major cause of cancer-related death globally.1 More than two-thirds of lung cancer patients present with advanced disease,2,3 which excludes the option of potentially curative treatments. Another important reason accounting for the high mortality rate is excessive mutational load in patients with smoking history, a phenomenon central to the pathogenesis of lung cancer progression, compared with patients with age-related cancers.4 Five-year survival of all patients with lung cancer is only 18%.5 Recently, great advances have been made in terms of screening, minimally invasive techniques for diagnosis and new treatments.6–9
The recognition of genetic driver mutations in NSCLC has paved the way for the development of targeted therapies, which often provides outstanding responses in patients harbouring specific genetic mutations.10,11 Approximately two thirds of lung adenocarcinomas contain actionable driver mutations, which can be detected using comprehensive molecular profiling.11–13 ALK gene rearrangement in NSCLC was first discovered by Japanese researchers a decade ago.14 This gene rearrangement, which precipitates expression of oncogenic fusion proteins, is found in approximately 3–7% of patients with metastatic lung carcinoma based on early studies using reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridisation (FISH).14,15
ALK gene fusion with echinoderm microtubule- associated protein like 4 (EML 4) represents the most frequent rearrangement among the ALK alterations.12,16 Other fusion partners have also been reported such as TPR, HIP 1, FAM 179 A and COL25A1.17–20

Drug discovery

The discovery of ALK rearrangement led to advent of crizotinib, a tyrosine kinase inhibitor (TKI) with powerful activity against ALK. Crizotinib showed a response rate of 74% with progression-free survival (PFS) of 10.9 months compared with a response rate of 45% with PFS of 7 months in standard platinum doublet chemotherapy (either carboplatin or cisplatin plus pemetrexed).21 This superior outcome compared with standard platinum doublet chemotherapy laid the foundation for targeted therapy as the first-line treatment for ALK-positive NSCLC.21
Second-generation ALK inhibitors such as ceritinib and alectinib have not only been shown to be effective in the first-line treatment setting but are also effective in patients who develop crizotinib resistance.22–24 In 2013, FDA granted the approval of crizotinib for treatment of metastatic ALK-positive NSCLC with FISH as companion diagnostic based on efficacy and safety data of Phase II and III studies.21,25 

Benchmark technique

FISH is currently the benchmark technique for diagnosis of ALK rearrangements; however, meticulous preparation and skillful interpretation according to guidelines is necessary for achieving accurate results. Thus, it is expensive, labour-intensive and requires a high level of pathology expertise.26–29 On rare occasions, FISH may produce equivocal results because in 5–10% of NSCLC, the rate of rearrangement of positive cells falls within the range of 10–20%; however, the current accepted cut-off for positive cells is 15% or more.30,31
Immunohistochemistry (IHC) is another method that can be used for ALK diagnosis in lung cancer. An IHC companion diagnostic assay was approved in 2015 based on its ability to accurately identify patients with ALK-rearranged NSCLC.32,33 Although IHC has been extensively used in laboratories due to the cost effectiveness, its interpretation requires experience and rarely protein expression may be absent in cases with atypical ALK rearrangement.34,35 Despite these limitations, ALK IHC is gaining momentum in Europe as the primary test usually in a two-step approach with FISH being performed only to confirm positive or equivocal IHC results.36–38
However, a few studies have reported false negative results using IHC, which potentially risk excluding patients from receiving standard of care treatment.39,40,41 Molecular diagnosis could surpass the limitations of both FISH and IHC either as a stand alone assay or in concert with either FISH or IHC. Next-generation sequencing (NGS) has emerged as a promising molecular diagnostic technique for clinical practice due to its accuracy in detecting most genomic alterations by allowing parallel sequencing in a single assay.42,43
NGS is the blanket term used to describe a number of different second- and third- generation sequencing technologies, which are more efficient and show higher throughput than Sanger sequencing, a first-generation sequencing technology. Platforms for NGS from Illumina and Thermo Fisher are used widely.44
NGS can be applied in the form of large-scale sequencing to detect genetic alterations such as gene mutation and amplification by sequencing the whole genome, exome or transcriptome. By contrast, NGS can also be applied in the form of targeted sequencing to detect and validate genome alterations related to cancer genes by performing deep sequencing on genomic regions of interest.45
It has been acknowledged that molecular approaches improve the accuracy of ALK fusion detection, by resolving discordant or borderline cases.46–48 However, one of the most valuable advantages of NGS should be attributed to its high negative predictive value compared with FISH testing. Ali et al reported that 35% of ALK-positive cases detected by NGS were negative in ALK FISH, where only 20 of the 31 ALK-positive cases were concordant for ALK rearrangement and the remaining 11 cases were only NGS-positive.49
Importantly, the majority of ALK NGS-positive, FISH-negative patients responded to crizotinib, with a median response of 17 months. This reflects the sensitivity of NGS and the potential of denying life-prolonging treatment for this patient cohort due to false negative ALK FISH results.49
Interestingly, Ali et al also identified one out of the two NGS-positive but ALK FISH-negative cases (which did not respond to crizotinib) actually contained a TSC2 alteration. TSC2 alteration is well known to be associated with acquired resistance to targeted therapy, which could explain the de novo resistance in this patient.49,50  Drug resistance in patients is one of the most important reasons for treatment failure. 
Primary resistance occurs prior to treatment due to the presence of gene alterations conveying resistance, whereas secondary resistance occurs when initial useful treatment loses its effectiveness after initial success. NGS can be used to detect resistance genes and also predict resistance at a genetic level to guide treatment choices.51,52
The majority of ALK-positive patients on crizotinib will develop resistance approximately one year from start of treatment. This is due to resistance mutations in ALK or amplification of the ALK fusion gene or activation of other ALK-related signalling pathways such as c-Kit pathway through c-Kit gene amplification and other potential bypass mechanisms of resistance, including activating mutation of KRAS and EGFR.53–55
The beauty of NGS in this setting is that it can detect all of these resistance mutations in one test. Furthermore, the opportunistic testing of a panel of mutations would help to identify patients for clinical trials. It is crucial for the oncologist to evaluate all treatment options available for patients to ensure best standard of care is offered.
EGFR and ALK testing were first recommended in 2013; however, the College of American Pathologists (CAP), International Association for the Study of Lung Cancer (IASLC), Association for Molecular Pathology (AMP) and National Comprehensive Cancer Network (NCCN) guidelines have endorsed testing for ROS1, MET, RET, ERBB2, and BRAF, which further sets the foundation of precision medicine driven landscape in management of lung cancer that may significantly improve cancer mortality.11,41,56–58
However, this can be extremely challenging in terms of tissue sample conservation when multiple single-gene molecular assays are performed. Furthermore, diagnosis is usually made with small biopsy and cytology samples by employing minimal invasive technique such as endobronchial ultrasound (EBUS)-guided transbronchial needle aspiration.59,60 Thus, NGS technology could maximise available information from a single biopsy by identifying a select panel of clinically relevant mutations.61,62 NGS can be used in small biopsy specimens as well as cytological specimens.63

Overcoming limitations

Occasionally, a tumour biopsy or cytology sample would be insufficient for molecular testing because most lung cancers are metastatic or unresectable and diagnosis frequently relies on relatively small core or fine needle aspiration.64,65 Up to 23% of lung biopsies had insufficient material for pathological or cytological diagnosis, let alone molecular diagnosis, based on the report from UK National Lung Cancer Audit Report.66  After initial pathology diagnosis, only approximately 57% of biopsies had sufficient tissue for genomic analysis.67,68 Furthermore, serial biopsies may be warranted for patients who developed resistance to treatment in order to delineate the mechanism of resistance and tailor subsequent treatment. 
However, this is clinically challenging due to the invasive nature of the biopsy procedure with potential serious adverse risk for patients.69,70 Recently, NGS has been used to test genomic changes in liquid-based biopsy, specifically the cell-free DNA (cfDNA) from patients.71,72 cfDNA isolated from blood samples was shown to contain genetic changes, which were in concordance with primary tumour tissue DNA.69 In a study, Schwaederle et al established 10% of patients harbour ALK rearrangement from sequencing 54 cancer-related genes in plasma cfDNA.72 This strategy could potentially overcome serial tissue biopsy limitations.


In summary, it is critical to accurately identify ALK rearrangements in a patient sample, owing to the fact that false negative results would deny patients from receiving effective targeted therapies but false positive results would be equally deleterious as patients would be subjected to ineffective treatments. There is a growing body of evidence that challenges FISH as the gold standard for ALK testing when compared with NGS. This novel testing strategy is practical and reliable to use on tissue specimens and potentially on liquid biopsies, which will potentially further revolutionise the diagnostic landscape of lung cancer. 
By contrast, comprehensive molecular profiling with NGS remains controversial at present because clinical data to support its use are incomplete. The MOSCATO trial, which evaluates the clinical benefit of massive parallel sequencing approach showed that only 7% of successfully screened patients benefited with the use of this new technology.73 Dalton et al also investigated the role of this approach where the group reported only 6% of patients had benefited clinically.74
In addition, European Thoracic Oncology Platform Lungscape Consortium (ETOP) has appraised the efficacy of NGS and RT-PCR techniques in comparison to established diagnostic assays for diagnosis of ALK rearrangement in a large study of 96 IHC selected NSCLC cases. This working group reported similar sensitivity and specificity for NGS, RT-PCR and FISH in confirming ALK status, which was contrary to earlier studies.49,75,76 Thus, further investigation on the role of NGS and its magnitude of clinical impact is required; however, this ‘new kid’ is likely to become the mainstay technology for screening for targetable aberrations in lung cancer.


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