Cancer and ALK, RET, ROS1 gene fusions

Lung cancer causes over 1.5 million deaths annually, making it the leading cause of cancer-related deaths worldwide (2, 3). While there are multiple types of the disease, non-small cell lung cancer (NSCLC) comprises 85% of all lung cancers (4). Cigarette smoking is the number 1 cause of NSCLC, but genetics and exposure to radon, asbestos or air pollution are also risk factors (5, 6). NSCLC is an aggressive disease with high metastatic potential. Indeed, the majority of NSCLC are locally advanced or metastatic disease. Most new diagnoses survive less than a year, and among all NSCLC cases, the 5-year survival rate is 16% (7).

Cancer is caused by mutations in specific genes. Many of these genes, including ALK, RET and ROS1, are called driver oncogenes because they are critical to the survival, growth and proliferation of cancer cells (8). Driver oncogene profiles are unique for each cancer type. Even histologically similar cancers can be stratified into molecularly distinct subsets of a given type of cancer (9). Many of these driver oncogenes, including ALK, RET and ROS1, encode tyrosine kinases and researchers have identified tyrosine kinase inhibitors (TKIs) that selectively inhibit the activity of some oncogene translocations(9).

Anaplastic lymphoma kinase (ALK)

ALK is a gene located on chromosome 2p23 that spans 29 exons. The gene encodes a 22-kDa protein, a receptor tyrosine kinase belonging to the classical insulin superfamily. The protein consists of an extracellular ligand-binding domain, a transmembrane domain and an intracellular tyrosine kinase domain (10). ALK is expressed during embryogenesis and plays a role in neuronal development and differentiation (7). Although the actual physiological function of ALK is uncertain, studies have shown that pleiotrophin and midkine are activating ligands. ALK is activated by dimerization and autophosphoyrlation in trans, resulting in the activation of downstream targets including Janus kinase, mammalian target of rapamycin (mTOR), phosphoinisitode-3-kinase (PI3K) and hypoxia-inducible factor 1 alpha (HIF-1 alpha) (10). ALK signaling also involves microRNAs downstream of activation (miR-135b, miR-29a and miR-16) (10).

ALK rearrangements

ALK was first discovered in 1994 in anaplastic non-Hodgkin’s lymphoma as a translocation that was fused to nucleophosmin, resulting in constitutively active ALK activity (11). Since then, ALK activating mutations have been detected in many types of cancer, as shown in the ALK gene fusion table below.

ALK is activated through 3 main mechanisms: overexpression, activating point mutations and fusion protein expression. ALK has 22 known fusion partners, with the partners regulating expression and localization and the conserved ALK kinase domain controlling ALK activity (10). ALK was first discovered in NSCLC in 2007 as a fusion with echinoderm microtubule-associated protein-like 4 (EML4). Multiple EML4-ALK fusion variants have since been discovered that modify protein size, frequency in NSCLC and sensitivity to inhibitors (7, 12, 13).

While multiple driver mutations can occur in a single cancer incidence, ALK rearrangements are almost always mutually exclusive. ALK rearrangements occur in 3-5% of patients with lung cancer. Given that lung cancer has the highest world-wide cancer incidence, NSCLC patients positive for ALK fusions make up the largest population of ALK-positive patients (7, 14).

ALK fusion detection

Fluorescence in situ hybridization (FISH) is one method for detecting ALK rearrangements in NSCLC samples. While current detection panels using green and red probes provide accurate visual determination, there are several limitations to this method. Limitations include: high cost, technically challenging sample preparation and interpretation, long turn-around time and the inability to identify the type of translocation.

Immunohistochemistry (IHC) measures the level of ALK fusion protein expressed in a tissue section. For cancers such as anaplastic large cell lymphoma (ALCL), this low cost, straightforward test works well because the ALK expression level is high enough to make an accurate determination (15, 16). However, ALK expression levels in NSCLC sections are not high enough to enable accurate determination. Furthermore, the tissue preparation, poor antibody choice and the lack of a standard scoring system make IHC inadequate for ALK fusion determination in NSCLC (17, 18).

Reverse transcription polymerase chain reaction (RT-PCR) is another method to detect ALK fusions. This approach is less expensive than FISH; however, the nature of the test requires specific primers for both fusion partners, making unknown fusion partners undetectable by RT-PCR. Cross-sample contamination and RNA degradation can also prevent accurate fusion gene detection.

ALK tyrosine kinase inhibitors

Crizotinib is a TKI that effectively targets EML4-ALK fusion activity. However, ALK expression can change based on the stage of the disease. For example, one study reported ALK expression in 11.9% of primary NSCLC and 25% of metastatic disease. Studies have shown point mutations can occur in the fusion sequence that increase ALK fusion gene copy number and the outgrowth of other driver mutations, resulting in resistance to the inhibitor (1, 19-23). Research is currently investigating ALK inhibitors that overcome resistance.

Rearranged during transfection (RET)

RET is a proto-oncogene located on chromosome 10q11.2 that encodes a receptor tyrosine kinase (24). RET is expressed in testis germ cells, urogenital tract cells, renal medullary cells, neurons and ganglia and is involved in embryogenesis, including renal organogenesis and enteric nervous system development (25-29). Similar to ALK, the protein consists of an extracellular ligand-binding domain, a transmembrane domain and an intracellular tyrosine kinase domain. The receptor binds to the family of glial cell line-derived neurotrophic factor (GDNF) ligands, and activation requires formation of a multimeric complex with the GDNF family receptor (GFR) alpha coreceptor (30-33). RET is autophosphorylated and activates the RAS/mitogen-activated protein kinase/extracellular-regulated kinase (RAS-MAPK-ERK) and PI3K-AKT pathways along with phospholipase C (PLC)-gamma to stimulate cell proliferation, migration and differentiation (34).

RET rearrangements

RET gene fusions were first detected in thyroid cancer in 1990 and have since been discovered in various carcinomas including NSCLC, as shown in the RET gene fusion table below (35). RET gene fusions are found in 1-2% of NSCLC, and these rearrangements are predominantly intrachromosomal, meaning that the RET fusion partners are also located on chromosome 10. As with ALK, these gene fusions encode transcripts comprised of the 3’ kinase domain of RET fused to a partner that conveys ligand-independent dimerization of the kinase domain, resulting in constitutive RET activity (36).

RET fusion detection

There is no gold-standard technique to detect RET gene fusions, and most studies use multiple techniques for detection and validation. For example, RET gene fusions were first detected in NSCLC in 2011-2013 by multiple groups using multiple techniques, including whole-genome screening (WGS), RNA sequencing (RNA-Seq), RT-PCR with confirmatory FISH and FISH with confirmatory RT-PCR (37-40). Although normal lung tissue shows low RET expression, IHC is not a reliable method to detect overexpressed RET because staining can vary (resulting in false-positive results) and the immunoreactivity of available antibodies is weak (37, 41, 42).

ROS1

ROS1 is located on chromosome 6 and encodes an orphan receptor tyrosine kinase with no clearly identified ligand. Although the native protein function is undefined, it has been reported to play a role in epithelial-mesenchymal transition in intestine, heart, lung, kidney and testis (36, 43-45). Rearrangements in the gene were first reported in NSCLC in the same paper showing ALK rearrangements in NSCLC and have been found in various cancers (see ROS1 gene fusion table below) (12).

ROS1 rearrangements

Approximately 1-2% of NSCLC carry ROS1 rearrangements (36). Similar to ALK and RET, ROS1 fusions are diverse but have conserved breakpoints that preserve ROS1 kinase activity; however, the exact mechanism of how ROS1 is a driver oncogene is unknown. Unlike ALK and RET, most ROS1 fusion partner proteins lack dimerization domains that facilitate ligand-independent homodimerization to catalyze ROS1 kinase activity (39). Activation is thought to stimulate signal transduction leading to SHP-1 and -2 upregulation and activation of the PI3K-AKT-mTOR, JAK-STAT and MAPK-ERK pathways leading to survival and proliferation (46).

ROS1 fusion detection

ROS1 rearrangements can be detected by FISH; however, similar to ALK, dual-probe FISH is limited when the fusion partner is unknown or in close proximity to ROS1. There is limited data on using RT-PCR alone to detect rearrangements, but IHC is reported to be an effective method to detect ROS1 rearrangements thanks to an effective antibody and unique staining patterns (47).

Detecting fusions using Archer® NGS tests

Archer® FusionPlex® next-generation sequencing (NGS) tests rapidly detect translocations from total nucleic acid isolated from tumor samples—including FFPE preserved specimens. Archer®’s proprietary Anchored Multiplex PCR (AMP™) chemistry allows for rapid preparation of highly multiplexed NGS libraries for targeted capture of mRNAs produced from fusion genes. The Archer® technology permits the simultaneous detection of both known recurrent fusions as well as previously unidentified fusions at key breakpoints in target genes. Archer®’s FusionPlex® tests offer a complete fusion detection solution, from library preparation through data analysis, for both the Illumina® or Ion Torrent™ platforms.

Want to learn more about how Archer® FusionPlex®NGS tests detect ALK, RET and ROS1 mutations? Click here.

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