Personalized genomic medicine: non-small-cell lung cancer and anaplastic lymphoma kinase

Personalized genomic medicine: non-small-cell lung cancer and lymphoma kinase. Abstract Genomic medicine, that is to say, using genomic information about a patient in order to set the diagnostic path and to tailor therapy to his/her specific characteristics, is one of the cornerstones of modern precision medicine and forms an integral part of several fields, oncology first of all. Lung cancer is the leading cause of cancer mortality, causing more than 1.6 million deaths worldwide per year and non-small-cell lung cancer (NSCLC) accounts for approximately 85% of lung cancers. In a small subset of NSCLC (5%-8%), we can detect a genomic rearrangement on chromosome 2, between the Echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene, resulting in the chimeric protein EML4-ALK, that acts as an oncogene and that can be specifically targeted by an ALK-tyrosine kinase inhibitor (TKI) therapy. However, a major clinical challenge is represented by the fact that, after a first line ALK-TKI treatment, patients eventually develop acquired resistance to these agents, opening new scenarios for the right second-line drug choice.


INTRODUCTION
Targeted therapy is one of the greatest achievements in modern day precision oncology, allowing to specifically target molecular drivers, mainly responsible for cancer-cells development, proliferation and survival in several tumor subsets, anaplastic lymphoma kinase (ALK) rearranged non-small-cell lung cancer (NSCLC) included.
With reference to this particular cancer subtype, while treatment with ALK-tyrosine kinase inhibitor (TKI) gives raise to no concerns and is currently considered the standard-of-care by international guidelines, acquired resistance mechanisms to these agents represent a major clinical challenge. In fact, not only we do not fully understand the mechanism behind this phenomenon, but we are also struggling to develop effective drugs against the known resistance pathways.
Therefore, this paper aims to provide an up-to-date state-of-the-art review about ALK + NSCLC, genomics, epidemiology, diagnosis, treatment and acquired resistance mechanisms, jointly with an analysis about future developments and directions in this field.

NSCLC EPIDEMIOLOGY
Currently, lung cancer is both the most diagnosed and the deadliest cancer worldwide, and NSCLC accounts for 85% of all cases. However, NSCLC is not one single entity, in fact, it is subdivided into adenocarcinoma also known as lung adenocarcinoma (AC and LUAD respectively, 40% of all NSCLCs), lung squamous cell carcinoma (or LUSC, 20%-30% of all NSCLCs ), large cell carcinoma (2%-5% of all NSCLCs) or not otherwise specified (20% of all NSCLCs), according to the histological type [ Figure 1]; and in wild-type (without any known mutation) or mutated ("oncogene addicted"), if a mutation is present. Presently, we are only able to specifically target oncogenic mutations in the adenocarcinoma histological type, most notably epidermal growth factor receptor (EGFR, 15%-20% of all AC NSCLC) and ALK (4%-6% of all AC NSCLC, mainly younger and non smokers/light smokers patients) [1][2][3][4][5][6] .

THE ALK GENE
The ALK gene, located on the short arm of chromosome 2 (2p23), encodes for the homonymous receptor tyrosine kinase (ALK-RTK), consisting of an extracellular, a transmembrane and a catalytic cytoplasmic portion (that harbors the ATP binding cleft, responsible for starting phosphorylation and thus signal transduction) [7] .  Physiologically, ALK-RTK expression is limited to rare embryonic and adult brain tissue cells, suggesting a role in neural development and function [8,9] . However, in a small subset of NSCLC affected patients (4%-6%), typically following an inversion rearrangement in the short arm of chromosome 2 between the ALK gene and the Echinoderm microtubule-associated protein-like 4 (EML4) gene [inv2(p21;p23)], the resulting chimeric fusion protein (EML4-ALK) becomes aberrantly expressed, resulting in enhanced tyrosine kinase activity and constitutive activation of the signaling pathways mediated by mitogen-activated protein kinase, Janus kinase-signal transducer and activator of transcription proteins (JAK-STAT), Phosphatidylinositol-4,5-bisphosphate 3-kinase-RAC-alpha serine/threonine-protein kinase, leading to cell proliferation and anti-apoptosis and eventually to tumorigenesis [10][11][12] ; on a side note, ALK rearrangements with other genes (fusion partners) other than EML4 have also been reported (e.g., TFG, KLC1, PTPN3). However, the eventual outcome does not vary [13] .

DIAGNOSING ALK + NSCLC
Currently, we can use four different methods to detect ALK rearrangements: fluorescent in situ hybridization (FISH), immunohistochemistry (IHC), reverse transcriptase polymerase chain reaction (RT-PCR) and next generation sequencing (NGS); FISH and IHC are the only US Food and Drug Administration (FDA) approved techniques [16,17] .

FISH
To date, FISH is considered the gold standard assay for diagnosing ALK rearrangements. The rationale behind this method is represented by the fact that using two differently colored break-apart probes (green and red) specific to the inversion breakpoints of the ALK gene, we will obtain a single yellow signal in non rearranged cells, while two split signals (green and red) will be obtained in rearranged cells; however, although very reliable, this technique cannot be automated and requires trained personnel and equipment (fluorescence microscope and the devices for the hybridization probes) for results interpretation [16,17] .

IHC
Whereas ALK rearrangements are followed by EML4-ALK expression, IHC relies on highly ALK-specific monoclonal antibodies to detect the products of this inversion on formalin fixed tissue sections; while lowcost and easy to perform, IHC requires laboratory validation and standardization [16,17] .

RT-PCR
The use of this method is currently not recommended, in fact, it does not provide a good quality of RNA from the formalin-fixed paraffin embedded tissue and furthermore can only detect known fusion partners. However, with this approach subjectivity in assessment of the analysis can be completely ruled out, unlike IHC and FISH [16,17] .

NGS
Even though NGS is still not FDA-approved, it is an undoubtedly promising technique, in fact, it grants screening of both known and novel ALK gene rearrangements, alongside with screening for other NSCLCrelated gene mutations; nevertheless, trained personnel is required for results interpretation and costs are still prohibitive [16,18] .

ALK + NSCLC TREATMENT: ALK TKI
Being able to incorporate genomic information into the diagnostic and clinical pathways, shaping personalized precision cancer therapies, has been one of the biggest shift in modern day oncology, and ALK + NSCLC treatment is a perfect example. In fact, according to the most recent American Society of Clinical Oncology and the European Society for Medical Oncology guidelines, ALK-TKIs presently represent the standard of care in the treatment of this subgroup of patients, granting results far beyond chemotherapy; more specifically, three ALK-TKIs (alectinib, ceritinib and crizotinib) are FDA and European Medicines Agency approved for the clinical practice, while another TKI (brigatinib) is only FDA-approved for the clinical practice [19][20][21] . Furthermore, all of the ALK-TKIs share the same mechanism of action: specifically and competitively binding the ATP binding pocket they manage to block phosphorylation, thus inhibiting TKI signal transduction pathways and ultimately cell survival and proliferation [22] .
However, when compared to first-generation ALK-TKIs (crizotinib), second generation ones (ceritinib, alectinib and brigatinib) manage to grant superior clinical performances thanks to the improved chemical structure, specifically designed in order to be more selective and potent (and thus associated with lower IC50) and to be able to easily cross the blood-brain barrier, due to the high rates of brain metastases in ALK + NSCLCs [23,24] .

Crizotinib
Crizotinib was the first FDA-approved ALK-TKI, receiving accelerated approval in 2011 and regular approval in 2013 for ALK + NSCLC affected patients, based on the findings from the A8081007 study, a randomized (1:1) trial in which 347 ALK + NSCLC affected patients that had already received a previous platinumbased treatment were randomized to receive either crizotinib or standard of care chemotherapy (docetaxel/ pemetrexed). Results favored crizotinib over chemotherapy: progression free survival (PFS): 7.7 months vs.

Ceritinib
Ceritinib was the second ALK-TKI to receive FDA approval for the treatment of naive ALK + NSCLC patients in 2017, thanks to the results coming from the ASCEND 4 study, a phase III trial randomizing 376 patients (1:1) to receive first line ceritinib or standard of care chemotherapy (cisplatin/carboplatin + pemetrexed). The ceritinib regimen performed significantly better than the platinum-based one according to every endpoint: PFS: 16.6 months (ceritinib) vs. 8.1 months (chemotherapy), ORR: 73% (ceritinib) vs. 27% (chemotherapy); moreover, unlike crizotinib, ceritinib performed greatly also with respect to central nervous system (CNS) lesions, with an overall intracranial response rate of 57% vs. 22% (chemotherapy), and a median CNS response duration of 16.6 months [28] .

Alectinib
Alectinib was the third FDA-approved ALK-TKI (2017) for the treatment of naive NSCLC harboring ALK rearrangements in the first line setting and is currently considered the best upfront treatment available, due to the findings coming from the ALEX trial.

Second-line ALK-TKI
To date, ceritinib, alectinib and brigatinib are FDA-approved for the second-line treatment of ALK + NSCLC patients, after intolerance to or failure of a first line crizotinib treatment.

Ceritinib
In 2014, ceritinib was the first ALK-TKI to be granted FDA approval for the treatment of ALK + NSCLC patients who had progressed on or were intolerant to crizotinib treatment, thanks to a single-arm trial (163 patients) that showed an ORR of 44% and a DOR of 7.1 months [30] .

Alectinib
In 2015, alectinib was granted FDA-approval for the treatment of ALK + NSCLC patients, after intolerance to or failure of a first line crizotinib treatment, based on the findings from two different single-arm trials, that respectively showed an ORR of 38% and 44%, a DOR of 7.5 months and 11.2 months, alongside with a CNS ORR of 61% and a CNS DOR of 9.1 months [31] .

Brigatinib
In 2017, brigatinib was granted FDA approval for the treatment of ALK + NSCLC patients who had progressed on or were intolerant to crizotinib treatment, based on the results from the ALTA trial, a randomized phase II trial, evaluating two regimens of brigatinib 90 mg vs. 180 mg.

ACQUIRED RESISTANCE
Regarding ALK-TKI treatment, the main hurdle to overcome is represented by the cancer cells acquired mechanisms of resistance to therapy, that ultimately lead to the progression of disease; even though these mechanisms can consistently vary according to the administered ALK-TKI, clinical and pre-clinical models indicate that resistance can develop through 3 main mechanisms: activation of other oncogenic signals that allow the tumor to bypass the ALK signaling pathway, TKIs pharmacokinetic liabilities or secondary mutations that affect the kinase domain of ALK; nevertheless, secondary resistance mutations appear to be the major resistance mechanisms adopted by cancer cells and most importantly the only presently targetable one [33,34] .

Secondary resistance mutations
Considering one of the largest and most extensive systematic analysis addressing ALK inhibitors resistance published by Gainor et al. [33] in 2016, assessing 83 repeat biopsies -from as many ALK-positive patientsperformed from 2009 to 2016 following disease progression on first (crizotinib) and/or second (ceritinib, alectinib, brigatinib) generation ALK-TKIs (n = 51 patients received crizotinib, n = 24 patients received ceritinib, n = 17 patients received alectinib and n = 6 patients received brigatinib), ALK secondary resistance mutations were observed in just 20% of ALK + NSCLC affected patients progressing on crizotinib. On the other hand, the same mutations were detected in 56% of ALK-rearranged patients progressing on a secondgeneration ALK inhibitor (54% of patients progressing on ceritinib; 53% of patients progressing on alectinib and 71% of patients progressing on brigatinib). G1202R mutation was the most frequent one [33] .
Therefore, treatment with a second-generation ALK inhibitor seems to be directly related to a consistently higher frequency of ALK resistance mutations and to a different range of such ones.
Patients progressing on brigatinib ALK secondary resistance mutations were found in 5 of 7 (71%) specimens from patients progressing on brigatinib. Once again, just like patients progressing on alectinib or ceritinib, G1202R was the most frequently found ALK secondary resistance mutation, in fact, it was observed in three patients out of seven [33] .

Impact of ALK secondary resistance mutations
Therefore, taking these findings into account, while the main mechanism behind acquired resistance to crizotinib (first generation ALK-TKI) seems to be TKIs pharmacokinetic liability (i.e., resistance occurring due to continued daily therapy over a long period of time), the development of secondary resistance mutations (G1202R being the most prominent one) appears to be the primary mechanism accountable for resistance to and progression after second generation ALK-TKI (ceritinib, alectinib, brigatinib), seemingly due to the increased selectivity and potency of these drugs [35,36] .

Bypass signaling tracks
In contrast with secondary resistance mutations, the activation of bypass signaling pathways represents an "off target" resistance mechanism, meaning that tumor cells manage to exploit other RTKs and/or downstream molecules pathways to overcome ALK dependency in order to keep proliferating despite ALK inhibition. In fact, this activation can involve transmembrane receptors -most notably EGFR and human epidermal growth factor receptor family members -as well as downstream molecules: STAT3, TP53, PIK3CA mutations, MET (MET proto-oncogene, receptor tyrosine kinase) amplification or mitogen-activated protein kinase reactivation, for example [37][38][39] .
To date, none of these bypass pathways is targetable.

FUTURE DIRECTIONS
The correct employment of genomic information is key to develop even further personalized precision cancer therapies.
In the light of the latest ALK-TKI developments, it appears clear that in the near future these drugs will be administered according to a multi-step strategy, based on the information coming from genomic analyses, in order to choose the right drug for the right mutation at the right time, maximizing the benefits coming from the best possible therapeutic sequence.

Authors' contributions
All authors contributed equally to the article.

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