Gene Fusion in NSCLC

C H A P T E R

31 Gene Fusion in NSCLC: ALK, ROS1, RET, and Related Treatments Raffaele Palmirotta, Davide Quaresmini, Domenica Lovero, Francesco Mannavola, Franco Dammacco and Franco Silvestris Department of Biomedical Sciences & Human Oncology, University of Bari “Aldo Moro”, Bari, Italy

INTRODUCTION Lung cancer is a highly aggressive cancer responsible for more than 1.6 million deaths per year worldwide (Chan & Hughes, 2015). Smoking is the main risk factor, with a close causal relationship between the development of lung cancer and the amount and duration of tobacco smoking. Based on both the ontogeny and the morphology of the lung tumor, a major classification provided by the World Health Organization distinguishes two main types of lung cancer: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The latter accounts for nearly 80% of lung carcinomas and includes three main subtypes: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma (Travis et al., 2015). The diagnostic and therapeutic approach to NSCLC has changed remarkably over the past decade, as knowledge of the molecular mechanisms underlying carcinogenesis, especially those of adenocarcinoma, has grown.

Oncogenomics DOI: https://doi.org/10.1016/B978-0-12-811785-9.00031-4

The identification of major genomic alterations, including sequence mutations of critical genes, gene amplifications, and chromosomal translocations, as well as epigenetic rearrangements has expanded basic information on NSCLC biology to include recognition of the role played by molecular abnormalities, especially those involving the “driver genes” that regulate cell proliferation and tumor progression. Many of these abnormalities are now being specifically targeted by newly developed “small molecules.” The results of this approach have been impressive in terms of prolonging survival and reducing chemotherapy-related toxicities, as shown by recent clinical studies in patients with advanced NSCLC. These novel drugs have found extensive applications in clinical practice, but they are efficacious only in selected cohorts of patients (Palmirotta et al., 2016). Although the major genetic derangements in NSCLC were initially thought to occur predominantly as gene mutations, including those

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31. GENE FUSION IN NSCLC: ALK, ROS1, RET, AND RELATED TREATMENTS

affecting EGFR, BRAF, and/or RAS, several fusion genes have also been identified as important oncogenic drivers (Fig. 31.1). These are hybrid genes originating from chromosomal rearrangements, and they lead to the formation of chimeric proteins with oncogenic functions. The first fusion gene, known as the Philadelphia chromosome, was discovered in 1973 in patients with chronic myeloid leukemia. It originates from the fusion of the breakpoint cluster region (BCR) gene with the second exon of the Abelson murine leukemia

viral oncogene homolog-1 (ABL1) gene and drives the formation of an oncogenic fusion protein that constitutively activates the ABL1 kinase domain (Rowley, 1973). With the introduction of modern sequencing technologies, many other fusion genes have been identified in a wide array of solid tumors, including lung cancer, sarcomas, and tumors of the central nervous system (CNS) (Parker & Zhang, 2013). The first fusion gene identified in NSCLC was described in 2007. It involves translocation of the echinoderm microtubule associated protein-like-4 (EML4) gene to

Lung cancer histotypes Large cell carcinoma 15%

Squamous cell carcinoma 25%

Other 5% Small cell lung cancer 15%

Adenocarcinoma 40%

Squamous cell carcinoma DDR2 3%

PTEN 10%

Adenocarcinoma

NF1 8% BRAF 7% KRAS 25%

MET 4%

PIK3CA 12%

ALK 4% Other /Wild Type 55%

FGFR1 20%

EGFR 16%

Other /Wild Type 21%

NRG1 3% DDR2 3% R RIT1 2 NT OS1 % HE RK1 2% RE R2 2% T 2% 1%

FIGURE 31.1 Schematic representation of lung cancer histological types and specific molecular drivers. More than 85% of cases of lung cancer are classified as NSCLC, including adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and other rare histologies. The remaining 15% of tumors include highly aggressive SCLC. The range and the frequency of specific genomic pathway alterations that differentiate adenocarcinoma from squamous cell carcinoma are shown in the pie chart. Fusion oncogene rearrangements involving ALK, ROS-1, NRG1, NTRK1, and RET occur in , 7% of cases (Rosell & Karachaliou, 2016a).

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NSCLC: PAST AND FUTURE

the anaplastic lymphoma receptor tyrosine kinase (ALK) gene, both on chromosome 2 (Soda et al., 2007). This highly transforming oncogene (EML4-ALK fusion gene) promotes chronic activation of the ALK-tyrosine kinase domain and thus the hyperproliferation of cancer cells. Other fusions have been reported in lung adenocarcinomas, including the kinesin family member 5B-rat proto-oncogene (KIF5B-RET), CCDC6-c-Ros oncogene 1 (ROS1), and FGFR2-citron (CIT) (Parker & Zhang, 2013). Notably, these rearrangements typically occur in young patients, either nonsmokers or light smokers, and rarely coexist with other genetic alterations, supporting their role as independent oncogenic drivers. Given the huge impact of targeted treatments in patients with selected gene rearrangements and the relatively low rate of disease recurrence, the identification of patients likely to benefit from this type of therapy should be accurate, comprehensive, cost-effective, time-efficient, and compatible with strategies identifying all actionable genetic alterations. Among the methodologies currently applied to identify candidate patients are immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and reverse transcription polymerase chain reaction (RTPCR). However, novel techniques based on next-generation sequencing (NGS) are being increasingly adopted as they offer the advantage of all-in-one testing platforms to analyze multiple oncogenic drivers (Kerr & Lo´pez-Rı´os, 2016). Here, we summarize the state-of-the-art knowledge regarding fusion genes in lung cancer, with particular attention granted to their role in tumor physiology and development. We also discuss the methods used to identify these aberrant genes and recent clinical trials in which tyrosine kinase inhibitors (TKIs) have been used to target ALK, ROS1, and RET translocations.

NSCLC: PAST AND FUTURE Before the advent of the “oncogenomic era,” the treatment of lung cancer predominantly

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relied on platinum- and taxane-based chemotherapy, but their efficacies were very modest and both were toxic (Mayekar & Bivona, 2017). However, during the past 15 years, there has been a progressive shift from the use of conventional cytotoxic drugs toward novel targeted therapy and immunotherapeutic agents. In fact, investigations of lung cancer genomics have yielded important insights into the critical mechanisms underlying the carcinogenic process that can be efficiently disabled by engineered drugs. “Driver mutations” are among the genetic alterations that promote the uncontrolled proliferation and clonal expansion of tumor cells (Palmirotta et al., 2016). Over 60% of lung adenocarcinomas are characterized by at least one driver mutation which seems to exclude alterations in other potential driver genes (Chan & Hughes, 2015). With the introduction of TKIs, these mutations, and specifically the abnormally activated intracellular kinases they encode, can be directly targeted in molecularly selected patients. Due to the complex mutagenic landscape characterizing NSCLC, for therapeutic purposes a “molecular” classification is required to cluster tumors whose morphologies are similar but whose prognosis and responsiveness differ. Deep sequencing of the tumor genome has revealed that prevalent driver mutations in NSCLC affect the MAPK pathway, particularly the KRAS, EGFR, BRAF, and MEK oncogenes, with a respective frequency of 25%, 16%, 7%, and 1% of cases. Other genetic alterations, although less common, involve mutations to the MET, PI3KA, and HER2 genes, and still others consist of chromosomal rearrangements of ALK, ROS1, and RET (Rosell & Karachaliou, 2016a, 2016b; The Cancer Genome Atlas Research Network, 2014). Emergent knowledge regarding the oncogenomics of translocated NSCLC suggests that also the kinases encoded by the respective fusion genes can be targeted by TKIs in select patients. The impressive results from phase I and II trials of the first-in-call

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31. GENE FUSION IN NSCLC: ALK, ROS1, RET, AND RELATED TREATMENTS

ALK-inhibitor crizotinib in patients with ALK rearranged NSCLC led to its prompt approval by the U.S. Food and Drug Administration (FDA) in 2011 (Camidge et al., 2012; Kwak et al., 2010). Moreover, patients with tumors harboring other fusion genes, such as the ROS1 and RET proto-oncogenes, also showed a dramatically favorable response to crizotinib (Shaw et al., 2014).

ALK, ROS1, and RET: Roles in Normal Physiology and Cancer Development These three fusion genes generate oncogenic proteins, which in turn upregulate cell proliferation. However, in the normal cell, they participate in different molecular events and are thus best understood when considered separately. 1. ALK—Anaplastic lymphoma kinase (ALK, OMIM *105590) is a membrane receptor kinase originally described in anaplastic large cell lymphoma (ALCL) but subsequently identified in other cancers, including a minority of NSCLC adenocarcinomas. The ALK gene maps to the 2p23.2-p23.1 locus and is organized in 29 exons (Mosse et al., 2008) encoding 1,620 amino acids. ALK contains an extracellular ligand-binding portion, a single transmembrane domain, and an intracellular kinase domain that is highly conserved within the same protein family (Fig. 31.2) (Lee et al., 2010; Roskoski, 2013; Shaw, Hsu, Awad, & Engelman, 2013). The ALK transcript is a member of the insulin receptor superfamily, which plays a role in CNS development. In mouse models, ALK mRNA levels are highest in the embryo, decrease rapidly after birth, and are strongly downregulated thereafter and continuing over the whole life span of the animal (Palmer, Vernersson, Grabbe, & Hallberg, 2009; Stoica et al., 2001, 2002). In humans, ALK is constitutively expressed in the CNS,

small and large intestine, prostate, and testicle but not in other tissues, including lung and lymphoid cells (Palmer et al., 2009). The only known ligands for ALK are two polypeptides (midkine and pleiothrophin) involved in neural development, cell migration, and angiogenesis (Stoica et al., 2001, 2002). However, heparin, FAM150A, and FAM150B may also act as ALK ligands (Guan et al., 2015; Murray et al., 2015; Reshetnyak et al., 2015). Currently, the normal physiological role of ALK-activated pathways is unknown because most of the available data derive from basic and clinical research in oncology. The most well-studied pathway in which ALK acts as an oncoprotein is in the setting of glioblastoma, in which the NPM-ALK translocation encodes a protein that activates JAK-STAT, RAS-ERK-MAPK, and PI3K-AKT-mTOR signals, thus deregulating cell proliferation, survival, and apoptosis and inducing morphologic as well as metabolic alterations in tumor cells (Gorczynski, Prelowska, Adam, Czapiewski, & Biernat, 2014; Hallberg & Palmer, 2013) (Fig. 31.3). ALK oncogene “addiction” is primarily driven by the expression of the ALK kinase domain, which promotes oligomerization of the oncoprotein and in the process the loss of the ALK inhibitory domain (Palmirotta, Quaresmini, Lovero, & Silvestris, 2017). In tissues physiologically lacking ALK, the fusion of ALK with a partner gene encoding the coiled-coil domain necessary for transcript oligomerization results in an oncoprotein that chronically activates the associated signal transduction pathway (Roskoski, 2013). Moreover, the presence of the ALK translocation is generally mutually exclusive with that of other driver genes, such as EGFR and ROS1 (Palmirotta et al., 2016). ALK translocations act as driver mutations in approximately 5% of NSCLC

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NSCLC: PAST AND FUTURE Transmembrane region

(A)

(aa 1039-1059)

Extracellular domain

Intracellular domain (aa 1060-1620)

(aa 19-1038) MAM

NH2

MAM

(aa 264-427)

Signal peptide

Glycine-rich segment

PK domain

(aa 816 940)

(aa 1122-1376)

(aa 480-626)

COOH

LDLa

EML4 ALK 2p21 2p23

Breakpoint

ALK

EML4

(B)

Breakpoint

inve

rsion

inv(2)(p21p23) EML4/ALK

FIGURE 31.2 Schematic structure of the ALK gene and its fusion partner EML4. (A) The domains highlighted in different colors are shown: At the N-terminal, the extracellular region includes two MAM domains (amino acids 264
427 and 480
626, blue), a low-density lipoprotein receptor domain class A (LDLA, amino acids 453
471, red), a glycine-rich domain (amino acids 816
940, green), the transmembrane domain (TM, amino acids 1039
1059, orange) that connects the extra- and intracellular domains, the intracellular domain with a juxtamembrane domain (amino acids 1058
1122), and the tyrosine kinase catalytic domain (amino acids 1122
1376; gray). (B) Schematic EML4 ALK fusion gene is generated by an inversion on the short arm of chromosome 2.

patients, particularly in young nonsmoking Asian women affected by the adenocarcinoma phenotype (Dagogo-Jack, Shaw, & Riely, 2017). In ALK-addicted NSCLCs, the distal portion of the ALK gene is translocated to other chromosomes, resulting in the fusion of ALK with a partner gene and regulation by the latter’s promoter region. The partner gene for ALK in NSCLC is ELM4, and the first oncogenic mutation to be described was the (E13; A20) variant, consisting of a juxtaposition of exons 1
13 from EML4 to intron 19 of ALK, thereby joining exons 20
29, which encode the kinase domain. Other variants and isoforms of this fusion have since been described, as

have other fusion partners, including those involved in vesicular trafficking, such as kinesin family member 5b (KIF5b), kinesiclike chain 1 (KLC1), TRK-fused gene 1 (TFG1), striatin calmodulin-binding protein (STRN), translocated promoter region (TPR), and Huntingtin-interacting protein 1 (HIP1) (Table 31.1). 2. ROS1—ROS1 (OMIM *165020) maps to locus 6q22.1. Like the ALK gene, it encodes a tyrosine kinase receptor belonging to the insulin receptor superfamily and comprises three domains: the extracellular and transmembrane domains and the intracellular tyrosine kinase motif (Fig. 31.4). The ROS1 sequence shares 49% homology with ALK

V. LUNG CANCER: ROLE OF GENOMICS IN CLINICAL PRACTICE

ALK

ROS1

RET GLFs

Cell membrane GRFα

(b) RAS-MAPK

(a) JAK-STAT

(c) PLCγ

(d) PI3k-mTOR

(e)

IRS1 GRB2 JAK2

JAK3

SH2

PLC γ

PI3K

SHC

SRC

SHH

SOS

STAT

AKT

IP3 Ca2+

DAG

JUNB

CRKL-C3G-RAP1 GTPase

RAS STAT

P P

STAT

RAF

PKC mTOR

SNT2FRS2

MEK Bcl-XL

Bcl-A2 IGF-1R EIF4EBP1

ERK FOXO3a

MCL1 cfos

cjun

CASP9

p27

BAD

NK-kB

CYCLIN D1

CELL GROWTH, CELL CYCLE PROGRESSION, DIFFERENTATION,

Extracellular domain

NH2

N glycosylation site (aa 28-1859)

FIGURE 31.3

ANTI-APOPTOTIC SIGNALS, SURVIVAL AND PROLIFERATION

Transmembrane region (aa 1860-1882)

TM

Intracellular domain

PK domain

COOH

(aa 1945-2215)

Main interconnected signaling pathways activated by the three transmembrane tyrosine kinase receptor: ALK, ROS, and RET. In particular, for RET via a cell surface, the homodimeric GFLs (glial cell line
derived neurotrophic factor family ligands) activate the transmembrane receptor by binding to different GPI-linked GFRα receptors with high affinity (coreceptor of the GDNF family receptor-α). Upon this binding, RET receptor becomes phosphorylated (P) on multiple intracellular tyrosine residues by facilitating direct interactions with signaling molecules such as SRC and PLCγ or with other adaptor proteins that lead to the activation of multiple downstream signaling pathways that can promote cell growth, proliferation, survival, or differentiation (Arighi et al., 2005; Mulligan, 2014). ROS and ALK engage multiple signaling pathways to exert their transformation activity. For example, PLCγ, MAP kinase, IRS1 (signaling molecule to PI3K), and JAK (janus kinase) proteins can interact with ROS1 and ALK, and phosphorylation of these proteins can lead to the activation of the respective oncogenic pathways. AKT, v-akt murine thymoma viral oncogene homolog 1; IRS1, insulin receptor substrate 1; MAP, mitogen-activated protein kinase; mTOR, mechanistic target of rapamycin (serine/threonine kinase); P13K, phosphoinositide-3 kinase; PLCγ, phospholipase C gamma; STATS, signal transducers and activators of transcription-3.

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NSCLC: PAST AND FUTURE

TABLE 31.1 Chromosomal Translocations Involving ALK Gene in Non-Small Cell Lung Cancer Gene Fusion

Partner Protein

Locus of the Fusion Partner

Chromosomal Rearrangement

EML4-ALK

Echinoderm microtubule-associated protein like-4

2p21

inv(2)(p21p23)

TFG-ALK

TRK-fused gene

3q21

t(2;3)(p23;q21)

KIF5B-ALK

Kinesin family member 5B

10p11

t(2;10)(p23;p11)

KLC1-ALK

Kinesin light-chain 1

14q32

t(2;14)(p23;q32.1)

PTPN3

Protein tyrosine phosphatase, nonreceptor type 3

9q31.3

t(2;9)(p23;q31)

STRN

Striatin, calmodulin-binding protein

2p22.2

—

TPR

Translocated promoter region

1q31.1

t(1;2)(q31.1;p23)

HIP1

Huntingtin-interacting protein 1

7q11.23

—

and 77% homology at the ATP-binding site. This similarity provides the rationale for using ALK inhibitors to treat ROS1-driven cancers (Shaw et al., 2014). ROS1 is physiologically expressed in renal, gastric, intestinal, epididymal, and neural tissue and in bone but not in normal lung tissue. To date, however, no ligands for ROS1 have been identified (Chen, Heller, Poon, Kang, & Wang, 1991), nor is the function of the protein known. ROS knockout mice develop normally except for abnormalities in the male reproductive system (Legare & Sullivan, 2004). The first evidence of a role for ROS1 in cancerogenesis was in glioblastoma, in which a fusion oncoprotein resulting from ROS1 translocation to the FIG gene, which encodes a protein of the Golgi apparatus, was detected. In NSCLC, ROS1 translocations occur in 1%
2% of adenocarcinoma histotypes and involve additional fusion partners (Bergethon et al., 2012). Like ALK, ROS1 translocations are mutually exclusive with mutations in other driver genes, although a recent report described the coexistence of ROS1 mutations with EGFR mutations in 0.5% and with KRAS mutations in 1.8% of all cancer

samples investigated (Lin & Shaw, 2017). Lung adenocarcinomas characterized by ROS1 mutations develop in nonsmokers and have an earlier onset. The tumors show a high-grade histology, and 30% of patients have stage IV disease at clinical diagnosis (Bergethon et al., 2012). However, in contrast to ALK-addicted NSCLCs, patients with ROS1-addicted tumors seem to have a lower rate of CNS metastasis at diagnosis and a lower incidence of brain metastases during tumor progression (Lin & Shaw, 2017), but the biological mechanisms for this behavior are undefined. From a molecular standpoint, ROS1 dysregulation results from gene fusion, ROS1 overexpression, or ROS1 mutation. In ROS1 translocations, none of the fusion partners express dimerization domains, another feature distinguishing these tumors from those that are ALK-driven (Chin, Soo, Soong, & Ou, 2012). In 2007, two translocations associated with NSCLC were identified: SLC34A2ROS1 t(4;6) and CD74-ROS1 t(5;6)(q32;q22) (Rikova et al., 2007). SLC34A2 encodes an Na/P membrane cotransporter mediating inorganic phosphate homeostasis in small intestine, lung, testicle, liver, and breast

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31. GENE FUSION IN NSCLC: ALK, ROS1, RET, AND RELATED TREATMENTS

Transmembrane region (aa 1860-1882)

Extracellular domain NH2

N glycosylation site

T M

(aa 28-1859)

Ex 32

Intracellular domain

PK Domain

COOH

(aa 1945-2215)

Ex 34 Ex 35

T M

PK Domain

CD74-ROS1 (exon 32)

CD74

T M

PK Domain

CD74-ROS1 (exon 34)

EZR

T M

PK Domain

EZR-ROS1

T M

PK Domain

SLC34A2-ROS1 (exon 32)

T M

PK Domain

SLC34A2-ROS1 (exon 34)

T M

PK Domain

SDC4-ROS1 (exon 32)

T M

PK Domain

SDC4-ROS1 (exon 34)

TIMP3

T M

PK Domain

TIMP3-ROS1

3%

FIG

T M

PK Domain

FIG-ROS1

3%

CCD6

T M

PK Domain

CCD6-ROS1

1%

T M

PK Domain

LRIG3-ROS1

1%

CD74

SLC34A2 SLC34A2 SDC4 SDC4

LRIG3

42%

15%

12%

7%

FIGURE 31.4 Schematic representation of ROS1 gene structure and its fusion partner in lung adenocarcinoma. The domains highlighted in different colors are shown: N-glycosylation site (amino acids 28
1859, pink), the transmembrane domain (TM, amino acids 1860
1882, orange), and tyrosine kinase domain (PK, amino acids 1945
2215, gray). The ROS1 breakpoints are located at exons 32 (e32), 34 (e34), and 35 (e35). For each fusion, partners (in light pink) are shown with their percentage of frequency: CD74 (CD74 antigen), EZR (ezrin), SLC34A2 (solute carrier family 34 [sodium/phosphate cotransporter], member 2), SDC4 (syndecan 4), TIMP3 (tissue inhibitor of metalloproteinase 3), FIG (Golgi-associated PDZ and coiled-coil domain-containing protein), CCDC6 (coiled-coil domain-containing protein 6), and LRIG3 (leucine-rich repeats and immunoglobulin-like domain-containing protein 3).

tissue (Rikova et al., 2007). CD74, the most frequent fusion partner in NSCLC, is a membrane protein involved in peptide presentation to CD41 T cells (Davies et al., 2012). In addition to these partners, other ROS1 fusion partners identified in NSCLC are SDC4, EZR, FIG, TPM3, LRIG3, KDELR2, CCRC6, MSN, TMEM106B, TPD52L1, CLTC, and LIMA1 (Lin & Shaw, 2017) (Fig. 31.4). Once expressed, the fusion oncoprotein dimerizes and activates the tyrosine phosphatases SHP-1 and SHP-2, which further activate the RAS/RAF/MAPK,

JAK/STAT, VAV3/RHO, and PLCγ pathways, thus triggering cell proliferation, transformation, migration, and aggressive tumor growth (Palmirotta et al., 2016) (Fig. 31.3). However, depending on the fusion partner, the transduction pathway and the characteristics of the tumor may slightly differ. For example, CD74-ROS1 results in the phosphorylation of extended synaptotagminlike protein-1 (ESyt1), which confers a more invasive phenotype (Jun et al., 2012). 3. RET—The rearranged during transfection (RET) proto-oncogene (OMIM 164761) was

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NSCLC: PAST AND FUTURE

first described in 1985 by Takahashi, Ritz, & Cooper (1985) et al., as an oncogene activated through DNA rearrangement in the NIH-3T3 cell model. Its involvement in lung adenocarcinoma was first reported in 2012, although it is detected in ,1% of these tumors (Bos, Gardizi, Schildhaus, Buettner, & Wolf, 2013; Ju et al., 2012; Kohno et al., 2012, 2015; Lipson et al., 2012; Takeuchi et al., 2012). The RET gene is located on chromosome 10q11.2; its 21 exons encode a tyrosine kinase receptor normally expressed in neuronal tissue (Ishizaka et al., 1989). Due to alternative splicing of the mRNA at the carboxylterminal cytoplasmic tail, there are three isoforms of RET: RET51, RET43, and RET9 (long, intermediate, and short, respectively), with the number indicating the amino acid that follows the point of divergence (Fig. 31.5). RET51 and RET9 are the two major isoforms, and both are highly conserved over a broad range of species (Arighi, Borrello, & Sariola, 2005; Bos et al., 2013). The mature form of the RET receptor has a molecular mass of 170 kDa and consists of an extracellular domain containing four domains for cadherin binding, a calciumbinding site, and a cysteine-rich region, a transmembrane region, and an intracellular motif containing two distinct tyrosine kinase domains with 12 autophosphorylation sites that act as signaling sites for intracellular proteins (Arighi et al. 2005; Bos et al., 2013; Ishizaka et al., 1989). RET activation occurs when the receptor binds to a multimeric protein complex composed of members of the GRF-alpha group, namely alpha coreceptors of the GDNF family; neurotrophic factor derived from the glial cell line and their ligand-binding coreceptors, the GLFs; or ligands belonging to the GDNF family of ligands, including neurturin, artemin, and persephin (Durbec et al., 1996). After ligand binding activates the RET receptor, a RET homodimer is

451

formed, and the kinase domain is activated, resulting in autophosphorylation of the intracellular domains (Airaksinen & Saarma, 2002; Bos et al., 2013; Mulligan, 2014). RET activation additionally triggers multiple downstream signaling pathways, including RAS/RAF/ERK, PI3K/AKT, and JNK (Airaksinen & Saarma, 2002; Mulligan, 2014; Phay & Shah, 2010) (Fig. 31.3). The first link between RET and human cancer was in papillary thyroid carcinoma (PTC), with the identification of point mutations and translocations in the receptor (Bongarzone et al., 1989; Grieco et al., 1990). As many as 13 different oncogenic RET fusion products (RET/PTC) have been identified in PTC. All of them are derived from chromosomal translocation events in which the RET tyrosine kinase domain fuses with the 50 region of the heterologous gene in thyrocytes (Bos et al., 2013; Phay & Shah, 2010; Prescott & Zeiger, 2015). Notably, germline activating point mutations of RET have been associated with multiple endocrine neoplasia type 2 (MEN 2) syndrome (Arighi et al., 2005; Bos et al., 2013) and its individual phenotypes: MEN 2A (medullary thyroid carcinoma [MTC], pheochromocytoma [PC], and hyperparathyroidism); MEN 2B (MTC and PC); and familial MTC, strongly associated with specific mutations within the RET gene. Moreover, somatic mutations in RET account for 50% of sporadic medullary thyroid cancers (Arighi et al., 2005; Jhiang, 2000). In 2012, a link between the oncogenic RET fusion and lung adenocarcinoma was independently discovered by several groups of investigators (Ju et al., 2012; Kohno et al., 2012, 2015; Lipson et al., 2012; Takeuchi et al., 2012) who screened a large number of lung cancer patients. Most of these patients were nonsmokers ,60 years of age. Their tumors were of poorly differentiated histological type and negative for other

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31. GENE FUSION IN NSCLC: ALK, ROS1, RET, AND RELATED TREATMENTS

Extracellular domain

Transmembrane region (aa 636-652)

RET 51

T M

PK Domain

PK Domain

RET 43 RET 9

CRR

CLR (aa 191-270)

Intracellular domain

Tyrosine Kinase domain (724-1005)

KIF5B (exons 15/16/22/23)MT

PK Domain

PK Domain

RET 51 RET 43 RET 9

T M

PK Domain

PK Domain

RET 51 RET 43 RET 9

T M

PK Domain

PK Domain

RET 51 RET 43 RET 9

CCDC6

T M

PK Domain

PK Domain

RET 51 RET 43 RET 9

NCOA

T M

PK Domain

PK Domain

RET 51 RET 43 RET 9

T M

PK Domain

PK Domain

RET 51 RET 43 RET 9

T M

PK Domain

PK Domain

RET 51 RET 43 RET 9

T M

PK Domain

PK Domain

RET 51 RET 43 RET 9

FRMD4A

T M

PK Domain

PK Domain

RET 51 RET 43 RET 9

KIAA1217

T M

PK Domain

PK Domain

RET 51 RET 43 RET 9

KIF5B (exons 15/24) KIF5B (exon 24)

TRIM33 CUX1 KIAA1468

FIGURE 31.5 Schematic representation of RET gene structure and its fusion partner in lung adenocarcinoma. The domains highlighted in different colors are shown: the four extracellular cadherin-like repeats (CLR, amino acids 191
270, pale blue), the cysteine-rich region (CRR, light red), the transmembrane domain (TM, amino acids 636
652, orange), and the split two tyrosine kinase domains (PK, amino acids 724
1005, gray). The three major isoforms—RET9, RET43, and RET51—are indicated. Fusion partner (in light pink): KIF5B (kinesin family member 5B), CCDC6 (coiled-coil domain-containing protein 6), NCOA (nuclear receptor coactivator), TRIM33 (tripartite motif-containing protein 33), CUX1 (cut-like homeobox1), KIAA1468, FRMD4A (ferm domain-containing protein 4A), and KIAA1217.

driver mutations (EGFR, KRAS, NRAS, BRAF, HER2, and ALK). Together, these studies evidenced that RET rearrangements occur in 1%
2% of patients with NSCLC and in roughly 16% of NSCLC tumors lacking other oncogenic drivers (Bos et al., 2013; Lee et al., 2015; Wang et al., 2012). In lung cancers, eight RET variants fused to partner genes have been identified thus far (KIF5B, CCDC6, NCOA, TRIM33, CUX1, KIAA1468, KIAA1217, and FRMD4A), and in

all of them the coiled-coil domains of the partner proteins promote the dimerization of RET fusion proteins, resulting in constitutive activation of the RET kinase, similar to oncogenic ALK fusions (Kohno et al., 2015; Lee et al., 2016; Tsuta et al., 2014; Velcheti et al., 2017; Wang et al., 2012). Among these variants, RET-KIF5B is the most frequent translocation. It derives from a pericentric inversion of chromosome 10 determining a fusion between the first 15

V. LUNG CANCER: ROLE OF GENOMICS IN CLINICAL PRACTICE

METHODS TO DETECT GENE FUSIONS

exons of the KIF5B gene and exons 12
20 of the RET gene. Exons 1
15 of KIF5B codify for a coiled-coil domain that mediates homodimerization of the fusion proteins, whereas exons 12
20 of the RET gene contain the RET kinase domain, allowing cellular proliferation, migration, and differentiation stimulation through the downstream signal pathways of PI3K/AKT and/or RAS/MAPK/ERK and phospholipase c-γ (Bos et al., 2013; Chao, Briesewitz, & Villalona-Calero, 2012; Lipson et al., 2012; Wang et al., 2012). Seven more variants of the RET-KIF5B fusion gene involving KIF5B exons 15, 16, and 22
24 and RET exons 8
12 have also been identified (K15/R12, K15/R11, K16/R12, K22/R12, K23/R12, K24/R8, and K24/R11) (Kohno et al., 2015) (Fig. 31.5). 4. Other fusion genes—Analyses of the lung cancer genome and transcriptome have led to the detection of other oncogenic gene fusions as novel targetable driver genes in a fraction of NSCLC. These fusions involve NTRK1, which encodes a nerve growth factor receptor; TRKA (Vaishnavi et al., 2013); NRG1 (neuregulin) (FernandezCuesta et al., 2014); and fibroblast growth factor receptor fusions (FGFR1/2/3) (Wang et al., 2014). Oncogenic fusions of the NTRK1 gene with the CD74 and MPRIP genes were detected in 3% of patients investigated in an American cohort but not in other studies (Kohno et al., 2015; Vaishnavi et al., 2013). Thus, the prevalence of NTRK1 fusion remains unclear, whereas amplification of the FGFR1 gene as a major oncogene aberration has been confirmed in 10% of patients with squamous lung cancer (Kohno et al., 2015) (Table 31.2). Another fusion in NSCLC is between the E2A and PBX1 genes (Mo et al., 2013). A recent study used RNAseq to investigate potential oncogenes arising from alternative splicing and fusion genes in 86 pairs of tissue

453

samples from NSCLC and normal lung tissue (Hong, Kim, Bang, Lee, & Oh, 2016). Several novel candidate fusion transcripts were identified: AL137145.2-PFKFB3, C4orf3-KLHL2, TPPPBRD9, and HNRNPA2B1-SKAP2. The AL137145.2-PFKFB3 fusion protein was the most frequently detected (4/86 samples, 4.7%) but its functional significance in the pathogenesis of lung cancer remains unclear (Hong et al., 2016).

METHODS TO DETECT GENE FUSIONS Current methods for the detection of ALK, ROS1, RET, and other gene rearrangements are FISH, IHC, and RT-PCR, each with its concurrent advantages and disadvantages (Palmirotta et al., 2017). FISH is currently the gold standard for the detection of gene fusions, including those involving ALK and ROS1, and has been used in multicenter research trials (Rolfo et al., 2014; Shaw et al., 2014). The color-based differential display of separate chromosomal structures reveals breakpoints in metaphase and interphase chromosomes with structural rearrangements (Mertens, Johansson, Fioretos, & Mitelman, 2015). However, FISH is timeconsuming, labor-intensive, as well as expensive, and it requires a defined technical expertise; it is thus not affordable for routine clinical practice. Furthermore, the most commonly used FISH probes are able to detect only the driver gene and not the fusion partner gene. This is problematic because there is evidence suggesting that the nature of the fusion partner gene influences therapeutic sensitivity (Beadling et al., 2016). Furthermore, RET analyses have shown FISH signal differences depending on whether translocation is inter- or intrachromosomal (Lee et al., 2015). In lung tissue, ALK wild-type protein levels are not detectable by IHC, whereas ALK overexpression as a result of translocation (Pisapia et al., 2017)

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31. GENE FUSION IN NSCLC: ALK, ROS1, RET, AND RELATED TREATMENTS

TABLE 31.2 Other Rare Fusion Proteins in Nonsmall Cell Lung Cancer Gene Fusion

Gene Name

Locus of Each Partner

Reference

NTRK1-MPRIP

Neurotrophic receptor tyrosine kinase 1

1q23.1

Vaishnavi (2013)

Myosin phosphatase Rho interacting protein

17p11.2

Neurotrophic receptor tyrosine kinase 1

1q23.1

CD74 molecule, major histocompatibility complex, class II invariant chain

5q33.1

Neuregulin 1

1q23.1

CD74 molecule, major histocompatibility complex, class II invariant chain

5q33.1

Fibroblast growth factor receptor 1

8p11.23

BCL2-associated athanogene 4

8p11.23

Fibroblast growth factor receptor 2

10q26.13

CCAR2 cell cycle and apoptosis regulator 2

8p21.3

Fibroblast growth factor receptor 2

10q26.13

Citron rho-interacting serine/threonine kinase

12q24.23

Fibroblast growth factor receptor 3

4p16.3

Transforming, acidic coiled-coil containing protein 3

4p16.3

AL137145.2PFKFB3

—

Chromosome 10 Hong (2016)

6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3

10p15.1

C4orf3-KLHL2

Chromosome 4 open reading frame 3

4q26

Kelch-like family member 2

4q32.3

Tubulin polymerization promoting protein

5p15.33

Bromodomain containing 9

5p15.33

HNRNPA2B1SKAP2

Heterogeneous nuclear ribonucleoprotein A2/B1

7p15.2

src kinase associated phosphoprotein 2

7p15.2

E2A-PBX1

(TCF3) E2A immunoglobulin enhancer binding factors E12/E47 19p13.3

NTRK1-CD74

NRG1-CD74

FGFR1-BAG4

FGFR2-KIAA1967

FGFR2-CIT

FGFR3-TACC3

TPPP-BRD9

pre-B-cell leukemia homeobox 1

is easily identified. Thus, IHC is used as a screening tool of high sensitivity and specificity (Dietel et al., 2016; Lee et al., 2015). However, it is a weak diagnostic tool for ROS1 and RET translocations because ,30% are detectable based on relative mRNA overexpression, and in several instances

Vaishnavi (2013)

Fernandez-Cuesta (2014)

Wang (2014)

Wang (2014)

Wang (2014)

Wang (2014)

Hong (2016)

Hong (2016)

Hong (2016)

Mo (2013)

1q23.3

wild-type ROS1 and RET show IHC staining with an intensity equivalent to that resulting from translocations (Pisapia et al., 2017). RT-PCR is a highly sensitive method with the advantage that it requires only small amounts of biological material (Rolfo et al., 2014). The

V. LUNG CANCER: ROLE OF GENOMICS IN CLINICAL PRACTICE

TARGET THERAPY AGAINST FUSED GENES IN NSCLC

drawback is that obtaining high-integrity RNA from formalin-fixed paraffin-embedded (FFPE) cell blocks is often challenging. Moreover, RT-PCR is not always informative, such that current guidelines do not recommend it as an alternative to FISH or IHC (Lindeman et al., 2013; Pisapia et al., 2017; Rolfo et al., 2014). In the past few years, many of the previously mentioned diagnostic problems have been overcome using NGS. This methodology, although requiring both the preparation of different templates and different sequencing chemistries, offers a fast automated approach to the analysis of not only sequence variants but also the whole exome or transcriptome, thereby providing information on gene copy numbers and rearrangements not obtainable by FISH or IHC (Mertens et al., 2015; Palmirotta et al., 2017). To date, commercially available NGS kits, supported by bioinformatics pipelines, have enabled the simultaneous identification of a large number of rearrangements from small amounts of nucleic acids extracted from FFPE tissue samples or circulating tumor cells (Palmirotta et al., 2017; Pisapia et al., 2017) (Fig. 31.6).

TARGET THERAPY AGAINST FUSED GENES IN NSCLC Since driver gene fusions underlie oncogenesis in NSCLC, they also guide its treatment, as targets of several next-generation inhibitory agents. New, tailored, molecularly driven drugs have been designed by innovatively making use of oncogenomic data in combination with precision medicine approaches. Targeted therapy is aimed at specific genomic subsets of certain cancers and has yielded results that are significantly better than those achieved with standard chemotherapy. For example, progression-free survival (PFS) and overall survival (OS) in patients receiving traditional platinum-based therapy for metastatic NSCLC are 5.3 and 10.3 months, respectively (Scagliotti

455

et al., 2008) compared with a PFS of 10.9 and an OS of 19.2 months in patients with ALK- or ROS1-driven metastatic NSCLC treated with crizotinib as first-line therapy (Shaw et al., 2014; Solomon et al., 2014). Preliminary results from other new agents currently under investigation have also been promising.

ALK Inhibitors 1. Crizotinib—This small molecule was the first TKI to inhibit ALK translocations, by acting as an ATP competitor at the catalytic domain of ALK (Medves & Demoulin, 2012). The striking efficacy of crizotinib in lowering the tumor burden in patients with ALK-translocated lung adenocarcinoma led to the accelerated approval of the drug by the FDA. A phase I trial testing crizotinib in patients with ROS1translocated tumors showed a response rate of 61% and a median PFS of 9.7 months (Camidge & Doebele, 2012). Subsequent phase III trials confirmed the efficacy of crizotinib as a second-line drug in patients with metastatic disease, based on a 65% response rate (RR) and a PFS of 7.7 months versus 20% and 3.0 months, respectively, compared to traditional platinum-based chemotherapy (Shaw et al., 2013). As a firstline drug, crizotinib produced an RR of 74% and a median PFS of 10.9 months versus 45% and 7.0 months when randomized against a conventional platinum derivative plus pemetrexed (Solomon et al., 2014). Based on these data, crizotinib has been approved as both a first- and a second-line drug for patients with NSCLC positive for the ALK translocation (Kim et al., 2012) (Table 31.3). However, despite the encouraging response in the extension of PFS, a significant percentage of crizotinib-treated patients were shown to suffer tumor progression, characterized by either ALK-dependent or -independent mechanisms, within a mean

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31. GENE FUSION IN NSCLC: ALK, ROS1, RET, AND RELATED TREATMENTS

1. Biopsy/FFPE tumors

3. Template preparation

5. Data analysis

RNA extraction

2. Library preparation

RNA Reverse transcription

cDNA Clonal amplification

4. Sequencing

6. Results

Primers annealing

Barcodes and adapters ligation

Integrative genomics viewer

FIGURE 31.6 Workflow of NGS fusion gene assay performed with the Ion Torrent PGM platform. The sequential phases of the procedure are illustrated: (1) RNA is extracted from formalin-fixed paraffin-embedded (FFPE) tumor specimens. (2) Library preparation: The RNA is reverse transcribed into cDNA that is subsequently amplified with a multiplex of primer targeting fusion and native control transcripts. Each amplicon is ligated with adaptors and barcodes, and thus barcoded libraries can be combined and loaded onto a single ion chip to minimize the sequencing run time and cost. (3) Template preparation: The resulting libraries are ready for downstream template preparation—that is, clonally amplified by an emulsion PCR on beads followed by sequencing (4). (5) Data analysis: In run, the summary shows the percentage of wells in 318 Ion Torrent Chip loaded with template beads to generate reads. (6) Results: After automated processing of the bioinformatics pipeline, fusion transcripts are identified and confirmed by examining aligned reads in IGV (integrative genomics viewer).

time of 10 months. The former occurred in approximately 50% of patients and included either the development of secondary point mutations or copy number gains in the ALK gene (13% of patients) or secondary mutations (7%) (Palmirotta et al., 2016). The most frequent secondary point mutation was the so-called gatekeeper mutation in the

EML4-ALK oncoprotein, in which the L1196 residue located in the enzymatic fold of the ATP-binding site is replaced by a bulky sidechain amino acid, thus interfering with the crizotinib binding site (Shaw et al., 2017). In the remaining 50% of patients, crizotinib resistance was ALK-independent and instead involved the activation of alternative

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457

TARGET THERAPY AGAINST FUSED GENES IN NSCLC

TABLE 31.3 Main Clinical Trials for Approved Anti-ALK TKIs in NSCLC ALK-Translocated NSCLC Trial

Phase

Drug

Compared Arm

Progression-Free Survival (Months)

Response Rate (%)

Reference

PROFILE1001

I

Crizotinib

—

9.7

61

Camidge (2012)

PROVILE 1005

II

Crizotinib

—

8.1

60

Kim (2012)

PROFILE1007

III

Crizotinib

Pemetrexed or docetaxel

7.7 vs. 3.0

65 vs. 20

Shaw (2013)

PROFILE1014

III

Crizotinib

Cis/carboplatin plus pemetrexed

10.9 vs. 7.0

74 vs. 45

Solomon et al. (2014)

ASCEND-1

I

Ceritinib

—

6.9

56

Kim (2016)

ASCEND-3

II

Ceritinib

—

11.1

63.7

Felip et al. (2015)

ASCEND-4

III

Ceritinib

Cis/carboplatin plus pemetrexed

16.6 vs. 8.1

72.5 vs. 26.7

Soria (2017)

ASCEND-5

III

Ceritinib

Pemetrexed or docetaxel

6.7 vs. 1.6

42.6 vs. 6

Shaw (2017)

AF001JP

I/II

Alectinib

—

28

93.5

Seto (2013)

ALEX

III

Alectinib

Crizotinib

Not reached

82.9 vs. 75.5

Peters (2017)

pathways (Camidge et al., 2012). These included EGFR and KRAS mutations, the phosphorylation of proteins belonging to the ErbB family, amplification of the KIT gene, IFG1R activation, and activation of the epithelium-to-mesenchymal transition induced by hypoxia or autophagy. Other mutations potentially associated with crizotinib resistance include CSMD3, CDKN2A, MAG11, CREBBP, DOT1K, PBX1, and PRKDC genes (Giri, Patel, & Mahadevan, 2014). 2. Second-generation ALK inhibitors—To overcome crizotinib resistance, novel and second-generation ALK inhibitors have been developed. The activities of these molecules are in some cases independent of the presence of secondary mutations and are greater for secondary lesions in the CNS. The ALK inhibitor ceritinib is 20-fold more potent than crizotinib and is highly active against some of the main secondary mutations

of ALK, particularly L1196M, G1269S, G1202R, and C1156Y, but also IGF1R and ROS1. These features provided the rationale for recent trials exploring ceritinib activity in vivo. In preliminary data from a phase I trial, a therapeutic response in all patients was demonstrated, with an ORR of 56% in crizotinib-refractory patients and as high as 72% in the crizotinib-naı¨ve cohort. PFS was 6.9 and 8.3 months, respectively (Kim et al., 2016). These data led to FDA approval of ceritinib in patients with metastatic NSCLC who experienced progression despite crizotinib therapy. The efficacy of ceritinib was also demonstrated in a comparison with traditional platinum-based chemotherapy in patients with progression on crizotinib (Shaw et al., 2017; Soria et al., 2017) (Table 31.3). Alectinib is a highly selective TKI that is 10fold more potent than crizotinib. It competes with ATP for the binding of the kinase domain of ALK and can easily pass the blood
brain barrier because it is not recognized by a specific active-transport system. It is effective in patients

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458

31. GENE FUSION IN NSCLC: ALK, ROS1, RET, AND RELATED TREATMENTS

with disease progression after crizotinib, even in those whose tumors express the gatekeeper mutation L1196M (Song, Wang, & Zhang, 2015). Alectinib was first studied in a phase I/II trial; the RR was 94%, and the median PFS was 28 months (Seto et al., 2013). Further studies demonstrated a superior response compared to crizotinib also in the first-line setting, as evidenced by a RR of 82.9% versus 75.5% and a median PFS not reached in a median follow-up of 18.6 months (Peters et al., 2017). Many other TKIs are under intensive investigation with the aim of expanding the therapeutic arsenal against ALK-driven NSCLCs, by targeting resistance mechanisms and improving the curative power of these drugs (Table 31.3).

ROS1 Inhibitors 1. Crizotinib—The inhibitory effect of crizotinib in ROS1-driven tumors was investigated first in cellular models that took advantage of the high homology in the tyrosine kinase domains of ROS1 and ALK and later in patients with ROS1-translocated stage IV lung adenocarcinoma (Mazieres et al., 2015). The PROFILE1001 trial was expanded to include patients with ROS1-translocated NSCLC. The striking clinical response of this cohort confirmed the efficacy of crizotinib, based on an ORR of 72% and a median PFS of 19.2 months (Shaw et al., 2014). Accordingly, the FDA subsequently approved crizotinib for the treatment of metastatic ROS1-translocated NSCLC. However, as discussed for ALK-addicted tumors, ROS1-translocated NSCLCs usually progress after treatment with crizotinib because the frequency of secondary point mutations in the ROS1 gene is at least 50%
60%, which is higher than the 25%
30% in the ALK gene. The most frequent mutation is the G2032R, followed by D2033N, S1986Y/F (Gainor et al., 2017), L2026M, L1951R

(McCoach et al., 2016), and others. Additional mechanisms of resistance demonstrated in cellular models include the emergence of activating EGFR or KRAS mutations (Cargnelutti et al., 2015; Song, Kim, et al., 2015; Song, Wang, et al., 2015) and the higher level expression of genes involved in the epithelial-to-mesenchymal transition (Song, Kim, et al., 2015; Song, Wang, et al., 2015). 2. Second-generation ROS1 inhibitors—Other ROS1 inhibitors aimed at overcoming resistance mechanisms are being intensively researched, including ceritinib, which has been the focus of recent clinical trials. In a phase II study, a clinical response was obtained in 32 patients; the ORR was 67%, and the median PFS in crizotinib-naı¨ve patients was 19.3 months versus 9.3 months for the entire cohort (Lim et al., 2017). However, in the population enrolled in the study, crizotinib-pretreated patients remained resistant, and a measurable efficacy of ceritinib was detected only in patients whose tumors carried the ROS1 L2026M mutation (Dziadziuszko et al., 2016). The drug was not effective for other mutational states (Lin & Shaw, 2017). Other clinical studies investigated brigatinib, which yielded results similar to those obtained with ceritinib. In a phase I/II study (Gettinger et al., 2016), efficacy was again limited to the copresence of the L2026M mutation (Lin & Shaw, 2017). By contrast, independent of the L2026 mutational state, better results were achieved in a phase I clinical trial of lorlatinib: The ORR was approximately 50%, and the median PFS was 7 months (Felip et al., 2017). Moreover, lorlatinib was also active on brain metastases, consistent with its high penetration of the blood
brain barrier. Molecular studies have shown that lorlatinib is also active in the presence of L2026M, S1986Y/F, and D2033N point mutations of ROS1 (Facchinetti et al., 2016).

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FUTURE PERSPECTIVES

TABLE 31.4 Main Clinical Trials for Approved Anti-ROS1 TKIs in NSCLC ROS1-Translocated NSCLC Trial

Phase

Drug

Compared Arm

Progression-Free Survival (Months)

Response Rate (%)

Reference

PROFILE1001

I

Crizotinib

—

19.2

72

Shaw (2014)

EUROS1

Retrospective

Crizotinib

—

9.1

80

Mazie`res et al. (2015)

Entrectinib, a further second-generation TKI, is inactive in patients with L2026M- and G2032R-mutated ROS1-translocated NSCLC (Chong et al., 2017). In a recent phase I study in crizotinib-naı¨ve patients, this small molecule induced an ORR of up to 86% and a median PFS of 19 months, with efficacy also in CNS metastases (Drilon et al., 2017). Cabozantinib is a multikinase inhibitor of MET, VEGFR2, RET, KIT, and ROS1, in addition to ROS1 point mutations, including G2032R and D2033N (Chong et al., 2017). It is being investigated as a therapeutic option after crizotinib in patients with resistant tumors carrying these mutations. However, cabozantinib and other multikinase TKI inhibitors also have toxic effects such that alternative ROS1 inhibitors are under development, including DS6051b, which is active in crizotinib-naı¨ve patients (Nosaki et al., 2017), and TPX-0005 (Cui et al., 2017) (Table 31.4).

RET Inhibitors In NSCLC adenocarcinoma characterized by RET translocations, the efficacy of vandetanib, a RET and EGFR inhibitor (Gautschi et al., 2013), was demonstrated in a phase II clinical trial (NCT01823068). Other phase II clinical trials evaluating the efficacy of RET inhibitors such as sunitinib (NCT01829217), lenvantinib (NCT01877083), apatinib (NCT02540824), ponatinib (NCT01813734 and NCT01935336), and alectinib (NCT02314481) are ongoing. Data

concerning their use in NSCLCs harboring RET fusion genes are currently unavailable (Bos et al., 2013; Rosell & Karachaliou, 2016a, 2016b).

FUTURE PERSPECTIVES The therapeutic approach to NSCLC has evolved and will continue to do so, given the intrinsic limitations of classical chemotherapy in terms of response and survival. Specific therapies targeting druggable molecular alterations have recently gained favor. Based on the results of clinical trials, TKIs are currently the gold standard in the treatment of NSCLCs harboring EGFR mutations or ALK /ROS1 rearrangements. The detection of a growing number of genetic alterations has led not only to a better understanding of the molecular dynamics of cancer but also to the identification of new, targetable mutations, either in known or in newly discovered genes. Further efforts are being aimed at understanding the dynamic adaptations of cancer cells to therapeutic pressure and have led to an appreciation of the importance of biopsydriven therapeutic decision-making at any step of tumor progression. Although the range of TKIs available to treat NSCLC driven by gene rearrangements continues to expand, many questions remain to be answered, including determination of the TKI optimal as a first, second, or further line of therapy in ALK-, ROS1-, or RET-rearranged tumors.

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31. GENE FUSION IN NSCLC: ALK, ROS1, RET, AND RELATED TREATMENTS

These clinical considerations are being debated, but with the adoption of nextgeneration inhibitors, resistance will be increasingly limited to ALK-independent processes, including the activation of bypass signaling pathways. For this reason, drugs aimed at other targets but compatible with ALK/ ROS1 inhibitors are currently under investigation, including inhibitors of the Hsp90, mTOR, CDK4/6, and ERK5 pathways (Moore et al., 2014; Umapathy et al., 2014). These strategies are meant to weaken ancillary downstream pathways in ALK/ROS1/RET-driven cancers and to amplify tumor growth inhibition, including by preventing feedback signaling. In the development of such combination regimens, a key consideration is to increase therapeutic efficacy while limiting additional toxicity. In conclusion, although the achievements obtained with the use of TKIs in the treatment of EGFR- and ALK/ROS1-positive NSCLC have surpassed the initial short-term goals, many significant challenges clearly remain to be met if patient survival and quality of life are to be significantly improved.

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