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

 

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 

 

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

 

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.

 

1 Ferlay J et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015;136:E359–86.

2 National Cancer Intelligence Network. Stage Breakdown by CCG 2014. London: NCIN; 2016. 

3 Northern Ireland Cancer Registry, Queens University Belfast, Incidence by stage 2010–2014. Belfast: NICR; 2016. 

4 Alexandrov LB et al. Signatures of mutational processes in human cancer. Nature 2013;500:415–21.

5 Howlader N et al. SEER Cancer Statistics Review, 1975–2014, based on November 2016 SEER data submission. Bethesda, MD: National Cancer Institute; 2017. https://seer.cancer.gov/csr/1975_2014/ (accessed May 2018). 

6 Johnson DH, Schiller JH, Bunn PA Jr. Recent clinical advances in lung cancer management. J Clin Oncol 2014;32:973–82. 

7 Reck M et al. Management of non-small-cell lung cancer: recent developments. Lancet 2013;382:709–19. 

8 Forde PM, Ettinger DS. Targeted therapy for non-small-cell lung cancer: past, present and future. Expert Rev Anticancer Ther 2013;13:745–8. 

9 Ettinger DS. Ten years of progress in non-small cell lung cancer. J Natl Compr Canc Netw 2012;10:292–5. 

10 Swanton C, Govindan R. Clinical implications of genomic discoveries in lung cancer. N Engl J Med 2016;374:1864–73.

11 Kris MG et al. Using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs. JAMA 2014;311:1998–2006

12 Kwak EL et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010;363:1693–703.

13 Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature 2014;511:543–50.

14 Soda M et al. Identification of the transforming EML4-ALK fusion gene in non small cell lung cancer. Nature 2007;448:561–6.

15 Takeuchi K et al. Multiplex reverse transcription-PCR screening for EML4-ALK fusion transcripts. Clin Cancer Res 2008;14:6618–24.

16 Hallberg B, Palmer RH. The role of the ALK receptor in cancer biology. Ann Oncol 2016;27(Suppl 3):iii4–iii15.

17 Choi YL et al. A novel fusion of TPR and ALK in lung adenocarcinoma. J Thorac Oncol 2014;9:563–6.

18 Fang DD et al. HIP1-ALK, a novel ALK fusion variant that responds to crizotinib. J Thorac Oncol 2014; 9:285–94.

19 Hong M et al. HIP1-ALK, a novel fusion protein identified in lung adenocarcinoma. J Thorac Oncol 2014;9:419–22.

20 Cui S et al. Use of capture-based next-generation sequencing to detect ALK fusion in plasma cell-free DNA of patients with non-small-cell lung cancer. Oncotarget 2017;8(2):2771–80. 

21 Solomon BJ et al. First-line crizotinib versus chemotherapy in ALK-positive lung cancer. N Engl J Med 2014;371:2167–77.

22 Seto T. et al. CH5424802 (RO5424802) for patients with ALK-rearranged advanced non-small-cell lung cancer (AF-001JP study): a single-arm, open-label, phase 1-2 study. Lancet Oncol 2013;14:590–8.

23 Peters S et al. Alectinib versus crizotinib in untreated ALK-positive non–small-cell lung cancer. N Engl J Med 2017;377(9):829–38.

24 Iwama E et al Development of anaplastic lymphoma kinase (ALK) inhibitors and molecular diagnosis in ALK rearrangement-positive lung cancer. Onco Targets Ther 2014;7:375–85.

25 Shaw AT, Kim DW, Nakagawa K. Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. N Engl J Med 2013;368:2385–94. 

26 Hunt J. Molecular pathology in anatomic pathology practice:a review of basic principles. Arch Pathol Lab Med 2007;132(2):

248–60.

27 Babic A et al. The impact of preanalytical processing on staining quality for H&E, dual hapten, dual color in situ hybridization and fluorescent in situ hybridization assays. Methods.2010;52(4):287–300.

28 Minca E et al. ALK status testing in nonsmall cell lung carcinoma: correlation between ultrasensitive IHC and FISH. J Mol Diagn 2013;15(3):341–6.

29 Sholl L et al. Combined use of ALK immunohistochemistry and FISH for optimal detection of ALK-rearranged lung adenocarcinomas. J Thorac Oncol 2013;8(3):322–8.

30 Camidge DR et al. Native and rearranged ALK copy number and rearranged cell count in non-small cell lung cancer. Implications for ALK inhibitor therapy. Cancer 2013;119(22):3968–75.

31 Selinger C et al. Testing for ALK rearrangement in lung adenocarcinoma: a multicenter comparison of immunohistochemistry and fluorescent in situ hybridization. Mod Pathol 2013;26(12):1545–53.

32 Conde E et al. Accurate identification of ALK positive lung carcinoma patients: novel FDA-cleared automated fluorescence in situ hybridization scanning system and ultrasensitive immunohistochemistry. PLoS One 2014;9:e107200.

33 Ying J et al. Diagnostic value of a novel fully automated immunochemistry assay for detection of ALK rearrangement in primary lung adenocarcinoma. Ann Oncol 2013;24:2589–93.

34 Kim H et al. Discordance between anaplastic lymphoma kinase status in primary non-small-cell lung cancers and their corresponding metastases. Histopathology 2013;62(2):305–14.

35 Salido M et al. Increased ALK gene copy number and amplification are frequent in non-small cell lung cancer. J Thorac Oncol 2011;6(1):21–7.

36 Yatabe Y. ALK FISH and IHC: you cannot have one without the other. J Thorac Oncol 2015;10:548–50.

37 Savic S et al. Screening for ALK in non-small cell lung carcinomas: 5A4 and D5F3 antibodies perform equally well, but combined use with FISH is recommended. Lung Cancer 2015; 89:104–9.

38 Leighl NB et al. Molecular testing for selection of patients with lung cancer for epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinase inhibitors: American Society of Clinical Oncology endorsement of the College of American Pathologists/International Association for the study of lung cancer/association for molecular pathology guideline. J Clin Oncol 2014;32:3673–9.

39 Cabillic F et al. Parallel FISH and immunohistochemical studies of ALK status in 3244 non-small-cell lung cancers reveal major discordances. J Thorac Oncol 2014;9:295–306.

40 Ilie MI et al. Discrepancies between FISH and immunohistochemistry for assessment of the ALK status are associated with ALK ‘borderline’-positive rearrangements or a high copy number: a potential major issue for anti-ALK therapeutic strategies. Ann Oncol 2015;26:238–44.

41 Antonescu CR et al. Molecular characterization of inflammatory myofibroblastic tumors with frequent ALK and ROS1 gene fusions and rare novel RET rearrangement. Am J Surg Pathol 2015;39:957–67.

42 Roychowdhury S et al. Personalized oncology through integrative high-throughput sequencing: a pilot study. Sci Transl Med 2011;3:111ra121.

43 Frampton GM et al. Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing. Nat Biotechnol 2013;31:1023–31. 

44 Kamps R et al. Next-generation sequencing in oncology: Genetic diagnosis, risk prediction and cancer classification. Int J Med Sci 2017;18(2):308.

45 Wu K et al. Next-generation sequencing for lung cancer. Future Oncol 2013;9:1323–36.

46 Demidova I et al. Detection of ALK rearrangements in 4002 Russian patients: The utility of different diagnostic approaches. Lung Cancer 2017;103:17–23.

47 Li W et al. Combinational analysis of FISH and immunohistochemistry reveals rare genomic events in ALK fusion patterns in NSCLC that responds to crizotinib treatment. J Thorac Oncol 2017;12:94–101.

48 Pekar-Zlotin M et al. Fluorescence in situ hybridization, immunohistochemistry, and next-generation sequencing for detection of EML4-ALK rearrangement in lung cancer. Oncologist 2015;20:316–22.

49 Ali SM et al. Comprehensive genomic profiling identifies a subset of crizotinibresponsive ALK-rearranged non-small cell lung cancer not detected by fluorescence in situ hybridization. Oncologist 2016;6:762–70.

50 Pirazzoli V et al. Acquired resistance of EGFR-mutant lung adenocarcinomas to afatinib plus cetuximab is associated with activation of mTORC1. Cell Rep 2014;7:999–1008.

51 Coco S, Truini A, Vanni I. Next generation sequencing in non-small cell lung cancer: new avenues toward the personalized medicine. Curr Drug Targets 2015;16:47–59.

52 Oxnard GR, Arcila ME, Sima CS. Acquired resistance to EGFR tyrosine kinase inhibitors in EGFR-mutant lung cancer: distinct natural history of patients with tumors harboring the T790M mutation. Clin Cancer Res 2011;17:1616–22.

53 Choi YL, Soda M, Yamashita Y. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N Engl J Med 2010;363:1734–9.

54 Doebele RC, Pilling AB, Aisner DL. Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer. Clin Cancer Res 2012;18:1472–82.

55 Sasaki T., Koivunen J., Ogino A. A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors. Cancer Res 2011;71:6051–60.

56 Ettinger DS et al. Non-small cell lung cancer, Version 6.2015. J Natl Compr Canc Network 2015;13(5):515–24.

57 Lee CK et al. Impact of EGFR inhibitor in non-small cell lung cancer on progression-free and overall survival: A meta-analysis. J Natl Cancer Inst 2013;105:595–605. 

58 Lindeman N et al. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors. J Mol Diagn 2018;20:129–59.

59 Bulman W, Saqi A, Powell CA. Acquisition and processing of endobronchial ultrasound-guided transbronchial needle aspiration specimens in the era of targeted lung cancer chemotherapy. Am J Respir Crit Care Med 2012;185:606–11. 

60 Jurado J et al. The efficacy of EBUS-guided transbronchial needle aspiration for molecular testing in lung adenocarcinoma. Ann Thorac Surg 2013;96:1196–202.

61 Sequist LV et al. Implementing multiplexed genotyping of non-small-cell lung cancers into routine clinical practice. Ann Oncol 2011;22:2616–24. 

62 Li T et al. Genotyping and genomic profiling of non-small-cell lung cancer: Implications for current and future therapies. J Clin Oncol 2013;31:1039–49.

63 Endris V et al. Molecular diagnostic profiling of lung cancer specimens with a semiconductor-based massive parallel sequencing approach: feasibility, costs, and performance compared with conventional sequencing. J Mol Diagn 2013;15:765e775.

64 Sorber L et al. Circulating cell-free nucleic acids and platelets as a liquid biopsy in the provision of personalized therapy for lung cancer patients. Lung Cancer 2017;107:100–7.

65 Travis WD et al. Diagnosis of lung adenocarcinoma in resected specimens: implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification. Arch Pathol Lab Med 2013;137:685–705. 

66 National Lung Cancer Audit Report 2012 [Internet]. 2012. www.hqip.org.uk/assets/NCAPOP-Library/NCAPOP-2012-13/Lung-Cancer-Nationa… (accessed May 2018).

67 Rosen S. World Market for Cancer Diagnostics, 5th Edition. 2013. 

68 Travis WD et al. International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society: International multidisciplinary classification of lung adenocarcinoma: executive summary. Proc Am Thorac Soc 2011;8:381–5.  

69 Xu Set al. Circulating tumor DNA identified by targeted sequencing in advanced-stage non-small cell lung cancer patients. Cancer Lett 2016;370:324–31.

70 Ross K et al. The potential diagnostic power of circulating tumor cell analysis for non-small-cell lung cancer. Expert Rev Mol Diagn.2015;15:1605–29.

71 Lanman RB et al. Analytical and clinical validation of a digital sequencing panel for quantitative, highly accurate evaluation of cell-free circulating tumor DNA. PLOS One 2015;10(10): e0140712. 

72 Schwaederle M et al. Detection rate of actionable mutations in diverse cancers using a biopsy-free (blood) circulating tumor cell DNA assay. Oncotarget 2016;7(9):9707–17.

73 Massard C et al. High-throughput genomics and clinical outcome in hard-to-treat advanced cancers: Results of the MOSCATO 01 Trial. Cancer Discov.2017;7:586–95.

74 Dalton WB et al. Personalized medicine in the oncology clinic: Implementation and outcomes of the Johns Hopkins Molecular Tumor Board. JCO Precision Oncology 2017; 31 May [Epub ahead of print].

75 Rogers T-M et al. Multiplexed transcriptome analysis to detect ALK, ROS1 and RET rearrangements in lung cancer. Sci Rep 2017;7:42259.

76 Letovanec I et al. Evaluation of NGS and RT-PCR methods for ALK rearrangement in European NSCLC patients: Results from the ETOP Lungscape Project. J Thorac Oncol 2018;13(3):413–25.