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 ident ify 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 17 HHE 2018 | hospitalhealthcare.com 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 There is a growing body of evidence that challenges FISH as the gold standard for ALK testing when compared with NGS patients harbour ALK rearrangement from sequencing 54 cancer-related genes in plasma cfDNA. 72 This strategy could potentially overcome serial tissue biopsy limitations. Conclusions 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.