Oncogenic Drivers in NSCLC

 

    Introduction

    Introduction

    Globally, lung cancer is one of the most commonly reported cancers, with approximately 2.2 million new cases worldwide each year.1 Approximately 80% of lung cancers are non-small cell lung cancer (NSCLC).1 The prognosis for patients with lung cancer is poor; the 5-year survival rate for patients with NSCLC is approximately 17%.2 Existing therapies have limited therapeutic benefit in unselected NSCLC.3–5 Therefore, there is a need for new treatments which can improve outcomes for patients with advanced NSCLC.

    There are three main histological NSCLC subtypes:

    Alternatively, NSCLC can be divided into molecular subsets based on specific biomarkers or mutations. Data suggest that 56% of non-squamous cell lung tumours have a driver mutation which can be detected through molecular testing.6 Identifying the particular molecular drivers present in individual patients may allow a personalised treatment approach to be used. The first molecular driver identified in NSCLC was mutation of the epidermal growth factor receptor (EGFR) gene.7 Among Caucasians, approximately 15% of patients with NSCLC have tumours bearing EGFR mutations, making them sensitive to specific inhibitors.7 Other known molecular drivers in NSCLC are aberrant forms of the receptor tyrosine kinases (RTK) anaplastic lymphoma kinase (ALK) and ROS1.8,9 These gene rearrangements lead to abnormal expression of constitutively active ALK or ROS1 fusion proteins that act as oncogenic drivers.8,9 As demonstrated in clinical trials and recommended in international guidelines, personalised treatment with ALK or ROS1 tyrosine kinase inhibitors is indicated in these subsets of patients.9–13

    Figure: Evolution of knowledge of NSCLC.14,15
    Adapted from Pao W, Girard N. Lancet Oncol 2011;12:175–180 and Shaw AT, et al. N Engl J Med 2011;365:158–167. Reproduced with permission from Elsevier and Massachusetts Medical Society ©2011

    References

    1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin 2011;65:5–29
    2. American Cancer Society. Cancer Facts & Figures 2015. Atlanta: American Cancer Society; 2015
    3. Scagliotti G, DeMarinis F, Rinaldi M, et al. Phase II randomized trial comparing three platinum-based doublets in advanced non-small cell lung cancer. J Clin Oncol 2002;20:4285–4291
    4. Herbst RS, Prager D, Hermann R, et al. TRIBUTE: A phase III trial of erlotinib HCl (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer. J Clin Oncol 2005;23:5892–5899
    5. Schiller JH, Harrington D, Belani CP, et al. Comparison of four chemotherapy regimens for advanced non-small cell lung cancer. N Engl J Med 2002;346:92–98
    6. Kris MG, Arcila ME, Lau C, et al. Two year results of LC-MAP: an institutional program to routinely profile tumor specimens for the presence of mutations in targeted pathways in all patients with non-small cell lung cancers. Oral presentation at World Conference on Lung Cancer, Amsterdam, The Netherlands, July 3–7, 2011 (Abstract 016.05)
    7. Korpanty GJ, Graham DM, Vincent MD, et al. Biomarkers that currently affect clinical practice in lung cancer: EGFR, ALK, MET, ROS-1, and KRAS. Front Oncol 2014;4:204
    8. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small cell lung cancer. Nature 2007;448:561–567
    9. Bubendorf L, Büttner R, Al-Dayel F, et al. Testing for ROS1 in non-small cell lung cancer: a review with recommendations. Virchows Arch 2016;469:489–503
    10. Masters GA, Temin S, Azzoli CG, et al. Systemic therapy for stage IV non-small-cell lung cancer: American Society of Clinical Oncology Clinical Practice Guideline Update. J Clin Oncol 2015;33(30):3488–3515
    11. National Comprehensive Cancer Network. NCCN clinical practice guidelines in oncology: non-small cell lung cancer – Version 2.2017.
    12. Novello S, Barlesi F, Califano R, et al. Metastatic non-small-cell lung cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2016;27(Suppl. 5):v1–v27
    13. Solomon B, Mok T, Kim D, et al. First-line crizotinib versus chemotherapy in ALK-positive lung cancer. N Engl J Med 2014;371:2167–2177
    14. Pao W, Girard N. New driver mutations in non-small-cell lung cancer. Lancet Oncol 2011;12:175–180
    15. Shaw AT, Forcione DG, Digumarthy SR, Iafrate AJ. Case 21-2011: A 31-year-old man with ALK-positive adenocarcinoma of the lung. N Engl J Med 2011;365:158–167

    ALK REARRANGEMENT

    The Role of ALK

    The anaplastic lymphoma kinase (ALK) protein is a transmembrane receptor tyrosine kinase (RTK).1 Like other RTKs, it is composed of three parts:1–3

    • An extracellular ligand-binding domain
    • A single membrane-spanning domain
    • A catalytic tyrosine kinase domain within the cytoplasm.

    When a ligand binds to the extracellular portion of the molecule, two ALK proteins come together, or dimerise.4 Dimerisation allows the tyrosine kinase domains of the two ALK proteins to phosphorylate, and thus activate, each other.4 This activation stimulates downstream signalling pathways involved in key cellular processes, such as cell proliferation and survival.3

    Figure: Structure of the ALK receptor tyrosine kinase.3
    Reproduced with permission, from Palmer RH, et al. Biochem J 2009;420:345–361. ©The Biochemical Society
    G-rich, glycin-rich; hALK, human ALK; LDLa, low-density lipoprotein class A; MAM, meprin–A5 antigen–PTPmu; PTK, protein tyrosine kinase

    The normal physiological role of ALK

    The normal function of the ALK protein is not fully understood, but it is thought to play a role in nervous system development and function.1 This is because ALK expression is seen in the brains and spinal cord of rats and mice, and ALK protein expression is at its highest during the first few weeks of life.1 However, knockout mice have a normal appearance.5 ALK mRNA has also been shown to be expressed in the small intestine. Low levels of ALK mRNA have been observed in the brain, colon, prostate and testis.6

    Figure: Normal ALK signalling.

    References

    1. Iwahara T, Fujimoto J, Wen D, et al. Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system. Oncogene 1997;14:439–449
    2. Garber K. ALK, lung cancer, and personalized therapy: portent of the future? J Natl Cancer Inst 2010;102:672–675
    3. Palmer RH, Vernersson E, Grabbe C, Hallberg B. Anaplastic lymphoma kinase: signalling in development and disease. Biochem J 2009;420:345–361
    4. Pulford K, Morris SW, Turturro F. Anaplastic lymphoma kinase proteins in growth control and cancer. J Cell Physiol 2004;199:330–358
    5. Mossé YP, Wood A, Maris JM. Inhibition of ALK signalling for cancer therapy. Clin Cancer Res 2009;15:5609–5614
    6. Chiarle R, Voena C, Ambrogio C, et al. The anaplastic lymphoma kinase in the pathogenesis of cancer. Nat Rev Cancer 2008;8:11–23
    7. Morris SW, Kirstein MN, Valentine MB, et al. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin’s lymphoma. Science 1994;263:1281–1284

    ALK as an Oncogenic Driver

    The anaplastic lymphoma kinase (ALK) gene is located on chromosome 2p23 and encodes a protein comprising 1,620 amino acids.1,2 The timeline shows the evolution of knowledge about ALK gene rearrangements and their role in cancer.3,4 The ALK gene was first identified in 1994 in samples from patients with anaplastic large-cell lymphoma (ALCL).2 It was shown that a chromosomal translocation occurred, resulting in the fusion of the ALK gene with the nucleophosmin (NPM) gene. This leads to the production of a fusion protein in which the catalytic domain of ALK is linked to the NPM protein, resulting in the constitutive activation of ALK. Between 55% and 85% of patients with ALCL express a genetic fusion involving the ALK gene.5

    This discovery led to further study of the ALK gene over the years.4

    Figure adapted by permission from Macmillan Publishers Ltd. Nat Rev Cancer 2008;8(1):11–23 ©2008

    A range of different fusion proteins involving ALK have been identified in different types of cancer.1,5,6 One of these is a fusion protein involving echinoderm microtubule-associated protein-like 4 (EML4) and ALK found in some patients with non-small cell lung cancer (NSCLC).

    ALK fusion proteins in different cancer types1,5–8

    Cancer type

    ALK fusion

    Incidence

    Lung cancer

    EML4-ALK

    3–5%

    KIF5B-ALK

    Rare

    TFG-ALK

    Rare

    Anaplastic large-cell lymphoma (ALCL)

    NPM-ALK

    60–80%

    TPM3-ALK

    12–18%

    TFG-ALK

    Rare

    CLTC-ALK

    Rare

    ATIC-ALK

    Rare

    TPM4-ALK

    Rare

    MSN-ALK

    Rare

    ALO17-ALK

    Rare

    MYH9-ALK

    Rare

    Inflammatory myofibroblastic tumour (IMT)

    TPM3-ALK or TPM4-ALK

    30%

    CARS-ALK

    Rare

    RANBP2-ALK

    Rare

    CLTC-ALK

    Rare

    SEC31L1-ALK

    Rare

    Other ALK gene alterations that do not involve the formation of fusion proteins have been found in some types of cancer, including point mutations (in thyroid cancer9 and neuroblastoma7) and amplification (in neuroblastoma7), but the clinical relevance of these gene alterations is unknown. This website is focused on the detection of ALK gene rearrangements in patients with NSCLC.

    References

    1. Palmer RH, Vernersson E, Grabbe C, Hallberg B. Anaplastic lymphoma kinase: signalling in development and disease. Biochem J 2009;420:345–361
    2. Morris SW, Kirstein MN, Valentine MB, et al. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science 1994;263:1281–1284
    3. Garber K. ALK, lung cancer, and personalized therapy: portent of the future? J Natl Cancer Inst 2010;102:672–675
    4. Chiarle R, Voena C, Ambogio C, et al. The anaplastic lymphoma kinase in the pathogenesis of cancer. Nat Rev Cancer 2008;8:11–23
    5. Barreca A, Lasorsa E, Riera L, et al. Anaplastic lymphoma kinase in human cancer. J Mol Endocrinol 2011;47:R11–R23
    6. Takeuchi K, Choi YL, Togashi Y, et al. KIF5B-ALK, a novel fusion oncokinase identified by an immunohistochemistry-based diagnostic system for ALK-positive lung cancer. Clin Cancer Res 2009;15:3143–3149
    7. Grande E, Bolós M-V, Arriola E. Targeting oncogenic ALK: a promising strategy for cancer treatment. Mol Cancer Ther 2011;10:569–579
    8. Lawrence B, Perez-Atayade A, Hibbard MK. TPM3-ALK and TPM4-ALK oncogenes in inflammatory myofibroblastic tumors. Am J Pathol 2000;157:377–384
    9. Murugan AK, Xing M. Anaplastic thyroid cancers harbour novel oncogenic mutations of the ALK gene. Cancer Res 2011;71:4403–4411

    ALK Gene Rearrangements in NSCLC

    The fusion gene of echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) was first discovered in non-small cell lung cancer (NSCLC) specimens in 2007.1,2 The fusion gene is created by an inversion in chromosome 2p, which results in the fusion of the N-terminal portion of the EML4 gene with sequence coding for the kinase domain of the ALK gene.1

    Figure adapted by permission from Macmillan Publishers Ltd. Nature 2007;448(7153):561–566 ©2007

    Since then, several variants of the EML4-ALK gene have been identified.3,4 All of these fusions contain the cytoplasmic portion of the ALK gene, including its entire kinase domain.3 However, the specific portion of the EML4 gene that acts as a fusion partner varies.3,4 Additionally, other fusion partners for ALK have been identified, such as TFG and KIF5B.2,5

    Different variants of EML4-ALK and non-EML4 fusion partners

    Reprinted from Eur J Cancer 2010;46(10):1773–1780. Sasaki T, Rodig SJ, Chirieac LR and Jänne PA. The biology and treatment of EML4-ALK non-small cell lung cancer ©2010, with permission from Elsevier

    Patients whose tumours express ALK fusion proteins comprise a distinct molecular subset of patients with NSCLC4 for whom personalised treatment with ALK inhibitors is indicated.6

    EML4-ALK as an oncogenic driver

    Fusion of the ALK and EML4 genes places the ALK kinase under the control of the EML4 promoter, driving aberrant expression.7 Fusion of ALK and EML4 also results in replacement of the extracellular and transmembrane portion of the ALK protein with a portion of the EML4 protein; therefore, unlike the normal ALK protein that is located on the cell surface, the abnormal ALK protein produced by the EML4-ALK fusion gene is located in the cytoplasm.1 The coiled coil (CC) portion of EML4 allows aggregation of the fusion proteins leading to constitutive activation without ligand binding.8 The result is persistent mitogenic signalling.8 This unregulated signalling induces cancer progression through its impact on cell proliferation and survival.9

    Figure adapted from Mano H. Cancer Sci 2008;99:2349–2355. ©2008. Reproduced with permission of Blackwell Publishing Ltd

    Figure adapted with permission. Shaw and Solomon. Clin Cancer Res 2011;17:2081–2086

    Watch the video clip above to see how EML4-ALK fusions form. https://www.youtube.com/watch?v=lPstArexiNY

    Scientists have shown that EML4-ALK (and the fusion protein NPM-ALK seen in anaplastic large-cell lymphoma) induces tumour formation when injected into nude mice.1 The mice were injected with fibroblast cells that had been transfected to express EML4, ALK, or the fusion proteins EML4-ALK or NPM-ALK.1 Only those receiving cells transfected with the fusion proteins developed tumours.1 Furthermore, mice injected with kinase-dead mutants of the fusion proteins did not develop tumours. This indicates that EML4-ALK kinase activity has in-vivo transforming potential capable of inducing tumour formation.

    Watch the video clip above to see Professor Keith Kerr of the University of Aberdeen, Scotland, explain the formation and function of the EML4-ALK fusion. https://www.youtube.com/watch?v=SfxTQZ6RAVY

    Tumour formation in nude mice after subcutaneous injection of cells expressing EML4-ALK or NPM-ALK fusion proteins, but not EML4 or ALK alone

    Figure adapted by permission from Macmillan Publishers Ltd. Nature 2007;448(7153):561–566 ©2007

    References

    1. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007;448:561–567
    2. Rikova K, Guo A, Zeng Q, et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 2007;131:1190–1203
    3. Lin E, Li L, Guan Y, et al. Exon array profiling detects EML4-ALK fusion in breast, colorectal, and non-small cell lung cancers. Mol Cancer Res2009;7:1466–1476
    4. Horn L, Pao W. EML4-ALK: Honing in on a new target in non-small-cell lung cancer. J Clin Oncol 2009;27:4232–4235
    5. Sasaki T, Rodig SJ, Chirieac LR, et al. The biology and treatment of EML4-ALK non-small cell lung cancer. Eur J Cancer 2010;46:1773–1780
    6. Reck M, Popat S, Reinmuth N, et al. Metastatic non-small-cell lung cancer (NSCLC): ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Ann Oncol 2014;25(Suppl. 3):iii27–iii39
    7. Pulford K, Morris SW, Turturro F. Anaplastic lymphoma kinase proteins in growth control and cancer. J Cell Physiol 2004;199:330–358
    8. Mano H. Non-solid oncogenes in solid tumors: EML4-ALK fusion genes in lung cancer. Cancer Sci 2008;99:2349–2355
    9. Palmer RH, Vernersson E, Grabbe C, Hallberg B. Anaplastic lymphoma kinase: signalling in development and disease. Biochem J 2009;420:345–361
    10. Mossé YP, Wood A, Maris JM. Inhibition of ALK signaling for cancer therapy. Clin Cancer Res 2009;15:5609–5614

    Clinical and Molecular Profile of ALK+ NSCLC

    A number of studies have examined clinical or demographic factors associated with EML4-ALK gene rearrangements. Compared with patients who have ALK-negative disease, factors associated with ALK-positive NSCLC are:

    • Younger median age (<60 years)1–5
    • Adenocarcinoma or mixed histology1,3–7
    • Non-smoking or light smoking history1–5,7
    • A solid growth pattern with signet-ring cells2,3

    ALK-rearranged NSCLC showing distinct solid growth pattern

    Figure adapted with permission. Rodig SJ, et al. Clin Cancer Res 2009;15:5216–5223

    Data have suggested that tumours with EML4-ALK gene rearrangements generally do not have mutations in the genes for epidermal growth factor receptor (EGFR)1–4,6,7 or V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue (KRAS).3–7 However, cases of NSCLC with EGFR mutations or KRAS mutations and concomitant EML4-ALK rearrangements have also been reported.5,8,9

    Despite the correlations between these clinical or histological features and ALK status, it is possible for any patient with NSCLC to have ALK-positive disease. For example, the EML4-ALK fusion gene has been found in older patients with a smoking history.2 This suggests that clinical characteristics alone cannot detect all patients and that molecular testing is essential to determine ALK status.

    Currently, the exact prevalence of EML4-ALK in NSCLC is unknown (see table below), but it has been estimated that between 3% and 5% of North American patients with NSCLC have this genetic inversion.10 Data vary between studies because of differences in patient selection criteria and EML4-ALK detection methods.11

    Study

    Nationality of patients with NSCLC

    No. with
    EML4-ALK

    Percent with
    EML4-ALK (%)

    Analytic method

    Soda et al. 200712

    Japanese

    5/75

    6.7

    RT-PCR

    Inamura et al. 200813

    Japanese

    5/221

    2.2

    RT-PCR

    Takeuchi et al. 200814

    Japanese

    11/364

    3.0

    RT-PCR

    Perner et al. 200815

    Swiss and US

    16/603

    2.7

    FISH, RT-PCR

    Koivunen et al. 200816

    Korean and US

    8/305

    2.6

    RT-PCR

    Korean

    6/167

    3.6

    US

    2/138

    1.4

    Shinmura et al. 200817

    Japanese

    2/77

    2.6

    RT-PCR

    Rodig et al. 20092

    US

    20/358

    5.6

    FISH

    Martelli et al. 20098

    Italian and Spanish

    9/120

    7.5

    RT-PCR

    Wong et al. 20094

    Chinese

    13/266

    4.9

    RT-PCR

    Shaw et al. 20093

    US*

    19/141

    13.5

    FISH

    Boland et al. 20096

    US

    6/335

    1.8

    FISH, RT-PCR

    Sakairi et al. 20101

    Japanese

    7/109

    6.4

    FISH, RT-PCR

    Takahashi et al. 20107

    Japanese

    5/313

    1.6

    RT-PCR

    Zhang et al. 20105

    Chinese

    12/103

    11.6

    RACE-coupled PCR sequencing

    Salido et al. 201118

    Spanish

    2/107

    1.9

    FISH

    Prevalence of EML4-ALK gene rearrangements in various studies

    *These patients were selected for genetic screening because of their higher risk of EML4-ALK based on clinical/demographic characteristics
    FISH: fluorescence in-situ hybridisation; RACE: rapid amplification of cDNA ends; RT-PCR: reverse transcription-polymerase chain reaction

    References

    1. Sakairi Y, Nakajima T, Yasufuku K, et al. EML4-ALK fusion gene assessment using metastatic lymph node samples obtained by endobronchial ultrasound-guided transbronchial needle aspiration. Clin Cancer Res 2010;16:4938–4945
    2. Rodig SJ, Mino-Kenudson M, Dacic S, et al. Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the Western population. Clin Cancer Res 2009;15:5216–5223
    3. Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbour EML4-ALK. J Clin Oncol 2009;27:4247–4253
    4. Wong DW-S, Leung EL-H, So KK-T, et al. The EML4-ALK fusion gene is involved in various histologic types of lung cancers from non-smokers with wild-type EGFR and KRAS. Cancer 2009;115:1723–1733
    5. Zhang X, Zhang S, Yang X, et al. Fusion of EML4 and ALK is associated with development of lung adenocarcinomas lacking EGFR and KRAS mutations and is correlated with ALK expression. Mol Cancer 2010;9:188
    6. Boland JM, Erdogan S, Vasmatzis G, et al. Anaplastic lymphoma kinase immunoreactivity correlates with ALK gene rearrangement and transcriptional up-regulation in non-small cell lung carcinomas. Hum Pathol 2009;40:1152–1158
    7. Takahashi T, Sonobe M, Kobayashi M, et al. Clinicopathologic features of non-small-cell lung cancer with EML4-ALK fusion gene. Ann Surg Oncol 2010;17:889–897
    8. Martelli MP, Sozzi G, Hernandez L, et al. EML4-ALK rearrangement in non-small cell lung cancer and non-tumor lung tissues. Am J Pathol 2009;174:661–670
    9. Yang J, Zhang X, Su J, et al. Concomitant EGFR mutation and EML4-ALK gene fusion in non-small cell lung cancer. Poster presented at the American Society of Clinical Oncology conference, June 3–7 2011, Chicago, IL [Abstract number 10517]
    10.  Garber K. ALK, lung cancer, and personalized therapy: portent of the future? J Natl Cancer Inst 2010;102:672–675
    11. Horn L, Pao W. EML4-ALK: Honing in on a new target in non-small-cell lung cancer. J Clin Oncol 2009;27:4232–4235
    12. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007;448:561–567
    13. Inamura K, Takeuchi K, Togashi Y, et al. EML4-ALK fusion is linked to histological characteristics in a subset of lung cancers. J Thorac Oncol 2008;3:13–17
    14. Takeuchi K, Choi YL, Soda M, et al. Multiplex reverse transcription-PCR screening for EML4-ALK fusion transcripts. Clin Cancer Res 2008;14:6618–6624
    15. Perner S, Wagner PL, Demichelis F, et al. EML4-ALK fusion lung cancer: a rare acquired event. Neoplasia 2008;10:298–302
    16. Koivunen JP, Mermel C, Zejnullahu K, et al. EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. Clin Cancer Res 2008;14:4275–4283
    17. Shinmura K, Kageyama S, Tao H, et al. EML4-ALK fusion transcripts, but no NPM-, TPM3-, CTCL-, ATIC-, or TGF-ALK fusion transcripts, in non-small cell lung cancer. Lung Cancer 2008;61:163–169
    18. Salido M, Pijuan L, Martínez-Avilés L, et al. Increased ALK gene copy number and amplification are frequent in non-small cell lung cancer. J Thorac Oncol 2011;6:21–27

    Natural History of ALK+ NSCLC

    Several studies have attempted to determine if ALK gene rearrangement is a prognostic factor in non-small cell lung cancer (NSCLC), and three studies controlled for independent prognostic factors or with matched patients have suggested that patients with ALK-positive NSCLC have a shorter overall survival and/or progression-free survival compared with patients with ALK-negative disease.1–3 Epidemiology of ALK-positive NSCLC is evolving, and future comparative studies should control for clinical and patient characteristics that might influence prognosis.

    A number of small molecule inhibitors of ALK have been assessed in clinical trials,4–6 and two of these have received regulatory approval in the European Union for the treatment of patients with advanced ALK-positive NSCLC.7,8 Further clinical trials of ALK inhibitors are ongoing.

    References

    1. Lee JK, Park HS, Kim D-W, et al. Comparative analyses of overall survival in patients with anaplastic lymphoma kinase-positive and matched wild-type advanced nonsmall cell lung cancer. Cancer 2012;118:3579–3586
    2. Yang P, Kulig K, Boland JM, et al. Worse disease-free survival in never-smokers with ALK lung adenocarcinoma. J Thorac Oncol 2012;7:90–97
    3. Kim HR, Shim HS, Chung J-H, et al. Distinct clinical features and outcomes in never-smokers with nonsmall cell lung cancer who harbor EGFR or KRAS mutations or ALK rearrangement. Cancer 2012;118:729–739
    4. Shaw AT, Kim DW, Nakagawa K, et al. Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. N Engl J Med 2013;368:2385–2394
    5. Shaw AT, Kim DW, Mehra R, et al. Ceritinib in ALK-rearranged non-small-cell lung cancer. N Engl J Med 2014;370:1189–1197
    6. Gadgeel SM, Gandhi L, Riely GJ, et al. Safety and activity of alectinib against systemic disease and brain metastases in patients with crizotinib-resistant ALK-rearranged non-small-cell lung cancer (AF-002JG): results from the dose-finding portion of a phase 1/2 study. Lancet Oncol 2014;15:1119–1128
    7. Pfizer Inc. EU XALKORI® (crizotinib) Summary of Product Characteristics, 2017
    8. Novartis Pharmaceuticals. EU ZYKADIA® (ceritinib). Summary of Product Characteristics, 2017

    ROS1 REARRANGEMENT

    The role of ROS1

    ROS1 belongs to the human receptor tyrosine kinase (RTK) family. This transmembrane receptor comprises:1

    • A very large extracellular domain, spanning more than 1,800 amino acids
    • One transmembrane domain
    • Intracellular tyrosine kinase domain.

    The tyrosine kinase domain of ROS1 shares a high degree of homology with ALK,2 explaining in part why some ALK inhibitors also inhibit ROS1.

    Figure: Structure of the human ROS1 receptor tyrosine kinase.
    Reprinted from Biochim Biophys Acta, 1795, Acquaviva J, et al., The multifaceted roles of the receptor tyrosine kinase ROS in development and cancer, 37–52, Copyright (2009)FN, fibronectin; PTK, protein tyrosine kinase

    To date, no ligand for human ROS1 has been identified and consequently, the physiological function of this orphan receptor remains unclear.1 Normal ROS1 protein expression has been reported in adult humans in the kidney, cerebellum, peripheral neural tissue, stomach, small intestine and colon, with lower expression in several other tissues.3 ROS1 expression has also been observed in normal and hyperplastic lung tissue.1,4 Like many other RTKs, ROS1 signalling feeds into multiple downstream pathways such as the RAS/RAF/MEK or MAPK, JAK/STAT3 and PI3K/AKT/mTOR pathways.5,6

    References

    1. Acquaviva J, Wong R, Charest A. The multifaceted roles of the receptor tyrosine kinase ROS in development and cancer. Biochim Biophys Acta 2009;1795:37–52
    2. Robinson DR, Wu YM, Lin SF. The protein tyrosine kinase family of the human genome. Oncogene 2000;19:5548–5557
    3. Rimkunas VM, Crosby KE, Li D, et al. Analysis of receptor tyrosine kinase ROS1-positive tumors in non-small cell lung cancer: identification of a FIG-ROS1 fusion. Clin Cancer Res 2012;18:4449–4457
    4. Lira ME, Choi Y-L, Lim SM, et al. A single-tube multiplexed assay for detecting ALK, ROS1, and RET fusions in lung cancer. J Mol Diagn 2014;16:229–243
    5. Chan BA, Hughes BGM. Targeted therapy for non-small cell lung cancer: current standards and the promise of the future. Transl Lung Cancer Res 2015;4:36–54
    6. Davies KD, Doebele RC. Molecular pathways: ROS1 fusion proteins in cancer. Clin Cancer Res 2013;19:4040–4045

    ROS1 Gene Rearrangements in NSCLC

    The ROS1 gene is located on chromosome 6 (6q22).1 The first ROS1 gene rearrangement to be discovered was in a glioblastoma cell line, an intrachromosomal deletion on chromosome 6 fused the 5’ region of the gene FIG to the 3’ region of ROS1.2 Since then, many other ROS1 fusion partners have been identified in non-small cell lung cancer (NSCLC), including CD74, SLC34A2 and SDC4 and the list is growing.3,4

    Figure: ROS1 fusion partners in NSCLC.
    Reproduced from Gainor and Shaw. Oncologist 20134

    CD74-ROS1 is the most frequently detected ROS1 fusion in NSCLC patients, reported in approximately one-third of ROS1-rerranged tumours.3,4

    Figure: Occurrence of different ROS1 fusions in NSCLC.
    Reproduced from Bubendorf L, et al. Virchows Arch 20163

    ROS1 gene rearrangements are present in 1–2% of NSCLCs.3–8 These patients comprise a distinct molecular group for whom personalised treatment with ROS1-targeted therapy is now the standard of care.9

    ROS1 as an Oncogenic Driver

    ROS1 fusion proteins are potent oncogenic drivers. Unlike in ALK, where the fusion partner provides a dimerisation domain that induces constitutive activation of ALK, the mechanism by which ROS1 fusion proteins become constitutively active is not completely understood. However, the signalling pathways activated generally promote cell proliferation and survival.3,4,10–12

    Patients with ROS1-rearranged NSCLC are eligible for targeted therapy with specific inhibitors.9 The availability of a targeted agent for ROS1-rearranged NSCLC emphasises the importance of reflex, rapid and accurate molecular testing in order to identify patients who will benefit from this treatment.3,13

    References

    1. Acquaviva J, Wong R, Charest A. The multifaceted roles of the receptor tyrosine kinase ROS in development and cancer. Biochim Biophys Acta 2009;1795:37–52
    2. Birchmeier C, Sharma S, Wigler M. Expression and rearrangement of the ROS1 gene in human glioblastoma cells. Proc Natl Acad Sci U S A 1987;84:9270–9274
    3. Bubendorf L, Büttner R, Al-Dayel F, et al. Testing for ROS1 in non-small cell lung cancer: a review with recommendations. Virchows Arch 2016;469:489–503
    4. Gainor JF, Shaw AT. Novel targets in non-small cell lung cancer: ROS1 and RET fusions. Oncologist 2013;18:865–875
    5. Bergethon K, Shaw AT, Ou S-HI, et al. ROS1 rearrangements define a unique molecular class of lung cancers. J Clin Oncol 2012;30:863–870
    6. Rikova K, Guo A, Zeng Q, et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 2007;131:1190–1203
    7. Seo JS, Ju YS, Lee WC, et al. The transcriptional landscape and mutational profile of lung adenocarcinoma. Genome Res 2012;22:2109–2119
    8. Takeuchi K, Soda M, Togashi Y, et al. RET, ROS1 and ALK fusions in lung cancer. Nat Med 2012;18:378–381
    9. Masters GA, Temin S, Azzoli CG, et al. Systemic therapy for stage IV non-small-cell lung cancer: American Society of Clinical Oncology Clinical Practice Guideline Update. J Clin Oncol 2015;33:3488–3515
    10. Charest A, Kheifets V, Park J, et al. Oncogenic targeting of an activated tyrosine kinase to the Golgi apparatus in a glioblastoma. Proc Natl Acad Sci U S A 2003;100:916–921
    11. Jun HJ, Johnson H, Bronson RT, et al. The oncogenic lung cancer fusion kinase CD74-ROS activates a novel invasiveness pathway through E-Syt1 phosphorylation. Cancer Res 2012;72:3764–3774
    12. Rimkunas VM, Crosby KE, Li D, et al. Analysis of receptor tyrosine kinase ROS1-positive tumors in non-small cell lung cancer: identification of a FIG-ROS1 fusion. Clin Cancer Res 2012;18:4449–4457
    13. Kerr KM, Bubendorf L, Edelman MJ, et al. Second ESMO consensus conference on lung cancer: pathology and molecular biomarkers for non-small-cell lung cancer. Ann Oncol 2014;25:1681–1690

    Clinical and Molecular Profile of ROS1+ NSCLC

    ROS1 rearrangements are not thought to overlap with mutations in other oncogenic drivers such as ALK or EGFR.1-4 However, patients with ROS1 rearrangements are thought to share similar characteristics to those commonly associated with ALK-positive non-small cell lung cancer (NSCLC). The following are commonly seen in patients with ROS1-rearranged NSCLC:3–7

    • Non-smoking history
    • Younger age
    • Adenocarcinoma
    • Asian ethnicity.

    However, clinical characteristics alone cannot detect all patients with ROS1 rearrangement. For example, ROS1 rearrangements have been observed in large cell and squamous cell histology and in patients with a smoking history. Consequently, molecular testing is essential to determine ROS1 status.7

    References

    1. Li C, Fang R, Sun Y, et al. Spectrum of oncogenic driver mutations in lung adenocarcinomas from East Asian never smokers. PLoS One 2011;6:e28204
    2. Rikova K, Guo A, Zeng Q, et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 2007;131:1190–1203
    3. Rimkunas VM, Crosby KE, Li D, et al. Analysis of receptor tyrosine kinase ROS1-positive tumors in non-small cell lung cancer: identification of a FIG-ROS1 fusion. Clin Cancer Res 2012;18:4449–4457
    4. Bergethon K, Shaw AT, Ou SH, et al. ROS1 rearrangements define a unique molecular class of lung cancers. J Clin Oncol 2012;30:863–870
    5. Davies KD, Le AT, Theodoro MF, et al. Identifying and targeting ROS1 gene fusions in non-small cell lung cancer. Clin Cancer Res 2012;18:4570–4579
    6. Gainor JF, Shaw AT. Novel targets in non-small cell lung cancer: ROS1 and RET fusions. Oncologist 2013;18:865–875
    7. Bubendorf L, Büttner R, Al-Dayel F, et al. Testing for ROS1 in non-small cell lung cancer: a review with recommendations. Virchows Arch 2016;469:489–503

    Natural history of ROS1+ NSCLC

    Information regarding the natural history of ROS1-positive non-small cell lung cancer (NSCLC) is currently limited, but several retrospective analyses have investigated the prognostic impact of ROS1 rearrangement.

    In one report, overall survival was significantly better in 14 patients with ROS1-positive NSCLC compared to that of 115 patients with ROS1-negative NSCLC, although it should be noted that 5 of the 14 ROS1-positive patients received a targeted therapy which could have confounded the results.1 In contrast, another study reported significantly shorter overall survival for 8 ROS1-positive patients compared with 384 ROS1-negative patients.2 Six additional studies have reported no significant difference in overall survival ROS1-positive NSCLC patients and ROS1-negative NSCLC patients.3–8

    These retrospective studies are limited by small sample sizes and differences in patient characteristics, ROS1 testing methodologies and definitions of the ROS1-negative control group. Cumulatively, however, the findings suggest that ROS1 rearrangement is unlikely to be a favourable prognostic factor in NSCLC, similar to what has previously been shown in ALK-positive NSCLC.9,10

    A tyrosine kinase inhibitor is now approved in the European Union for the treatment of advanced ROS1-positive NSCLC.11

    References

    1. Scheffler M, Schultheis A, Teixido C, et al. ROS1 rearrangements in lung adenocarcinoma: prognostic impact, therapeutic options and genetic variability. Oncotarget 2015;6:10577–10585
    2. Cai W, Li X, Su C, et al. ROS1 fusions in Chinese patients with non-small-cell lung cancer. Ann Oncol 2013;24:1822–1827
    3. Yoshida A, Kohno T, Tsuta K, et al. ROS1-rearranged lung cancer: a clinicopathologic and molecular study of 15 surgical cases. Am J Surg Pathol 2013;37:554–562
    4. Bergethon K, Shaw AT, Ou SH, et al. ROS1 rearrangements define a unique molecular class of lung cancers. J Clin Oncol 2012;30:863–870
    5. Chen Z, Akbay E, Mikse O, et al. Co-clinical trials demonstrate superiority of crizotinib to chemotherapy in ALK-rearranged non-small cell lung cancer and predict strategies to overcome resistance. Clin Cancer Res 2014;20:1204–1211
    6. Fu S, Liang Y, Lin YB, et al. The frequency and clinical implication of ROS1 and RET rearrangements in sesected stage IIIA-N2 non-small cell lung cancer patients. PLoS One 2015;10:e0124354
    7. Lee HJ, Seol HS, Kim JY, et al. ROS1 receptor tyrosine kinase, a druggable target, is frequently overexpressed in non-small cell lung carcinomas via genetic and epigenetic mechanisms. Ann Surg Oncol 2013;20:200–208
    8. Jin Y, Sun PL, Kim H, et al. ROS1 gene rearrangement and copy number gain in non-small cell lung cancer. Virchows Arch 2015;466:45–52
    9. Shaw AT, Yeap BY, Solomon BJ, et al. Effect of crizotinib on overall survival in patients with advanced non-small-cell lung cancer harbouring ALK gene rearrangement: a retrospective analysis. Lancet Oncol 2011;12:1004–1012
    10. Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. J Clin Oncol 2009;27:4247–4253
    11. Pfizer Inc. EU XALKORI® (crizotinib) Summary of Product Characteristics, 2017

    EGFR mutation

    The role of EGFR

    Similar to anaplastic lymphoma kinase (ALK) and ROS1, the epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase (RTK) composed of three important regions.1-3

    • An extracellular ligand-binding domain (includes four sub-domains I–IV)
    • The EGFR transmembrane domain
    • A tyrosine-protein kinase catalytic domain

    The extracellular ligand-binding domain binds to EGFR ligands, of which EGF is the most prominently upregulated in lung cancer.1 Subsequently, the EGFR undergoes autodimerisation and heterodimerisation with another EGFR or other member of the HER/erbB family of tyrosine kinases.1,2,4 The dimeric form impedes the auto-inhibitory role of the intracellular tyrosine kinase domain, and promotes tyrosine phosphorylation and down-stream signalling,1 through RAS/RAF/MEK/MAPK, PI3K/AKT, and JAK/STAT pathways.4,5 Activation of the EGFR mediates an array of diverse cellular responses such as cell lineage determination, proliferation, survival, angiogenesis, and migration.2,4,5

    Figure: EGFR structure1

    References

    1. Liu TC, Jin X, Wang Y, et al. Role of epidermal growth factor receptor in lung cancer and targeted therapies. Am J Cancer Res 2017;7:187–202
    2. Pines G, Köstler WJ, Yarden Y. Oncogenic mutant forms of EGFR: lessons in signal transduction and targets for cancer therapy. FEBS Lett 2010;584:2699–2706
    3. Bianco R, Gelardi T, Damiano V, et al. Rational bases for the development of EGFR inhibitors for cancer treatment. Int J Biochem Cell Biol 2007;39:1416–2031
    4. Zandi R, Larsen AB, Andersen P, et al. Mechanisms for oncogenic activation of the epidermal growth factor receptor. Cell Signal 2007;19:2013–2023
    5. Roengvoraphoj M, Tsongalis GJ, Dragnev KH, et al. Epidermal growth factor receptor tyrosine kinase inhibitors as initial therapy for non-small cell lung cancer: focus on epidermal growth factor receptor mutation testing and mutation-positive patients. Cancer Treat Rev 2013;39:839–850

    EGFR as an Oncogenic Driver

    Mutant forms of EGFR have been found in several human tumour types,1-3 and its expression is associated with a poor outcome in patients.3 Alterations in the EGFR gene are clustered in specific areas, known as 'hot spots', that are linked to elements of special functional or regulatory importance.1

    • Extracellular mutations ‘Ectodomain hotspot’ – deletions and point mutations in the ligand-binding domain that bypass the need for a ligand.1Mutant
      • EGFRvIII, a deletion spanning exons 2–7, is abundant in gliomas, but is also frequently detected in lung, breast and ovarian cancers.1,3-4
    • Intracellular tyrosine kinase mutations ‘Kinase hotspot’ – tumours, including non-small cell lung cancer, harbouring point mutations, small deletions or insertions of this region render this domain constitutively active.1
    • Intracellular mutations ‘C-tail hotspot’ – a deletion mutant in this region, i.e. EGFRvIV in gliomas, results in loss of kinase auto-regulation, implying that it remains constitutively active.1

    References

    1. Pines G, Köstler WJ, Yarden Y. Oncogenic mutant forms of EGFR: lessons in signal transduction and targets for cancer therapy. FEBS Lett 2010;584:2699–2706
    2. Zandi R, Larsen AB, Andersen P, et al. Mechanisms for oncogenic activation of the epidermal growth factor receptor. Cell Signal 2007;19:2013–2023
    3. Bianco R, Gelardi T, Damiano V, et al. Rational bases for the development of EGFR inhibitors for cancer treatment. Int J Biochem Cell Biol 2007;39:1416–1431
    4. Normanno N, De Luca A, Bianco C, et al. Epidermal growth factor receptor (EGFR) signaling in cancer. Gene 2006;366:2–16

    EGFR Gene Mutations in NSCLC

    Sensitising EGFR mutations are the most common actionable driver mutations found in patients with non-small cell lung cancer (NSCLC),1 and are most common in patients with adenocarcinoma histology, women, never smokers, and those of Asian ethnicity.2

    NSCLCs often exhibit prototypical point mutations or small insertions/deletions in the kinase domain-coding region (exons 18–21) of the EGFR gene that are believed to destabilise the inactive kinase conformation, driving it to a more active state.3

    • Exons 18 and 19 encode for the phosphate-binding loop. Exon 19 is usually affected by deletions, which represent 44% of kinase domain mutations.3
    • Exon 20, which encodes for the α-C helix, harbours relatively rare (4%) insertion mutations.3
    • Exon 21, encoding the activation loop (A loop), encompasses point mutations that account for 41% of mutations, including the prototypical L858R mutation.3

    The vast majority (~90%) of patients with EGFR-mutated NSCLC will have either an exon 19 deletion or an L858R substitution in exon 21.1,2 Most mutations involving exons 18, 19, and 21 are considered predictive of sensitivity to EGFR tyrosine kinase inhibitor (TKI) therapy, whereas mutations in exon 20 are typically resistant.1 The T790M mutation in exon 20 is well characterised as the most common mechanism of acquired resistance to EGFR TKI therapy, and it has been identified as a de novo T790M mutation, a germline mutation, and in combination with other genetic aberrations.1,2

    Figure: Frequency of mutations in exons 18–21 of the EGFR gene and the association with responsiveness to EGFR TKIs4
    Reprinted by permission from Macmillan Publishers Ltd: Mod Pathol. Cheng L, et al. Molecular pathology of lung cancer: key to personalized medicine. Mod Pathol 2012;25:347–69. Copyright (2012).
     

    References

    1. Castellanos E, Feld E, Horn L. Driven by Mutations: The Predictive Value of Mutation Subtype in EGFR-Mutated Non-Small Cell Lung Cancer. J Thorac Oncol 2017;12:612–623 
    2. Roengvoraphoj M, Tsongalis GJ, Dragnev KH, et al. Epidermal growth factor receptor tyrosine kinase inhibitors as initial therapy for non-small cell lung cancer: focus on epidermal growth factor receptor mutation testing and mutation-positive patients. Cancer Treat Rev 2013;39:839–850
    3. Pines G, Köstler WJ, Yarden Y. Oncogenic mutant forms of EGFR: lessons in signal transduction and targets for cancer therapy. FEBS Lett 2010;584:2699–2706
    4. Cheng L, Alexander RE, Maclennan GT, et al. Molecular pathology of lung cancer: key to personalized medicine. Mod Pathol 2012;25:347–369
    Further information
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