In recent years, the advent of personalized medicine combined with comprehensive genomic profiling has revolutionized the therapeutic landscape of non-small cell lung cancer (NSCLC), leading to the development of targeted therapies that radically changed cancer care in molecular selected patients [1]. In addition to the well-known oncogene-addicted NSCLC subgroups, including EGFR (epidermal growth factor receptor) activating mutations, BRAF (B-Raf proto-oncogene) V600E mutations, ALK (anaplastic lymphoma kinase) and ROS1 (v-ros avian UR2 sarcoma virus oncogene homolog 1) gene rearrangements, several different drivers (RET rearrangements, HER2 amplification/mutation, KRAS G12C mutation, NTRK 1-3 translocations) were identified in the last decade expanding the list of potential actionable oncogenes [2]. RET chromosomal rearrangements were initially identified in 10%–20% of papillary thyroid cancers. By contrast, RET mutations are the major oncogenic alteration reported in sporadic medullary thyroid cancers (MTC) (50%) and the most frequent germline mutations found in multiple endocrine neoplasia type 2 (MEN2) ^[3][4][3,4]. The RET proto-oncogene was first identified in lung cancer in 2012 and RET fusions were found in 1–2% of NSCLC cases examined by four different research groups from United States, Korea and China [5]. Initial reports showed that similarly to other oncogene drivers, RET fusions were typically associated with younger age, female gender, non-smoker status, Asian ethnicity, advanced stage, and adenocarcinoma subtype. However retrospective analysis suggested that RET-positive NSCLC were poorly differentiated compared with other oncogene-addicted (e.g., EGFR, ALK) tumors [6].

Since the beginning, RET fusion genes have been considered mutually exclusive with other molecular alterations. However, a retrospective analysis showed the presence of concomitant genomic alterations in 4 of the 12 patients with RET-rearranged NSCLC analyzed, harboring EGFR, MAP2K1, CTNNB1, and AKT1 mutations [7]. Moreover, EGFR mutated patients, experiencing disease progression under EGFR tyrosine kinase inhibitors (TKIs) therapy, may present RET rearrangements as mechanism of resistance [8].

Following RET rearrangement identification, different targeted therapies have been investigated. Multikinase inhibitors (MKIs) have been initially evaluated. Due to their concomitant inhibition of other kinases as vascular endothelial growth factor receptor 2 (VEGFR2) and EGFR, they were, unfortunately, characterized by limited efficacy with significant off-target adverse events and negative impact on health related quality of life, leading to high rates of high grade toxicities and dose reductions in NSCLC patients [9]. These disappointing results have contributed to the further development of selective RET kinase inhibitors, characterized by promising activities and more favorable tolerability.

2. Molecular Pathway

The RET gene, located in the chromosome 10, is composed by the extracellular region, including four N-terminal cadherin-like domains (named CLD1 to CLD4) followed by a single cysteine-rich domain (CRD), the transmembrane region, and the intracellular region composed by a bipartite tyrosine kinase [10].

RET signaling is involved in the embryonic development of some organs, such as kidney, peripheral and central nervous systems, as well as in the Peyer’s patch organogenesis and spermatogenesis process. Furthermore, RET signaling plays a key role in regulating cell proliferation, survival and differentiation process across different neurons subpopulations. The RET ligands include four members of the glial cell line-derived neurotrophic factor (GDNF) family, GDNF, neurturin, artemin and persephin, leading to the autophosphorylation of intracellular tyrosine residues, with subsequent activation of multiple downstream pathways, such as RAS-MAPK, PI3K-AKT, JAK-STAT, PLCγ and PKC [11].

The architecture of RET extracellular domain (ECD) was revealed by small angle X-ray scattering (SAXS) and electron microscopy (EM). The EM structure for RET–GFL–GFRα complex has a 2:2:2 stoichiometry: a dimer of GDNF binds two co-receptor molecules that recruits two RET receptors, exhibiting positive cooperativity. This geometry, named ternary complex, reveals a composite ligand-binding site, characterized by a GFRα1-binding hotspot that contacts the CLD containing calcium sites regions, and couples the CRD region ligand recognition leading to the receptor homodimerization. The activation of the kinase domain depends from the intermolecular autophosphorylation of intracellular tyrosine residues, working as docking sites for downstream signaling proteins carrying SRC homology 2 (SH2) or phosphotyrosine-binding (PTB) domains ^[12][13][14][12,13,14].

RET proto-oncogene was discovered in 1985 by Takahashi et al. as a gene that REarranged during Trasfections (RET) of DNA extracted from human T-cell lymphoma into NIH-3T3 cells [15]. Grieco et al. showed that the rearrangements were detected in all of the transfectants and of the original tumor DNAs, but not in normal DNA of the same patients, indicating that this genetic lesion occurred in vivo and was specifically related to sporadic tumors [16]. The intracellular region contains a tyrosine kinase domain and tyrosine phosphorylation sites located next to the C terminal region, where two major isoforms, RET9 and RET51, are positioned due to alternative splicing. The latter isoform has stronger tumorigenic activity even if both are co-expressed across different tissues. Although Y1062 is the most important docking site of major pathways, autophosphorylation of certain docking sites specifically gives rise to separate downstream pathways: Y1096 to RAS/MAPK and PI3K/AKT pathways; Y1015 to PLCγ; Y752 and Y928 to JAK/STAT pathway; and Y687 and Y981 to Shp2 and Src kinases, respectively [17].

RET proto-oncogene may be aberrantly activated by point mutation, fusion, or rearrangement. Sporadic mutations and rearrangements have been mainly detected in papillary thyroid cancer and NSCLC, while germline mutations have been reported in MEN ^[17][18][17,18]. Recently, the applications of next-generation sequencing (NGS) technologies supported the identification of RET alterations in several other malignancies, including pancreatic cancer, salivary gland cancer, colorectal cancer, ovarian cancer, breast cancer, and Spitz tumors. ^{[19][20][21][22][23]}[19,20,21,22,23] To date, more than 35 different RET fusion genes partners have been described, leading to the RET kinase expression and aberrant activation in cell types where both RET and its co-receptors are not normally expressed. In-frame KIF5B (the kinesin family 5B gene)-RET fusion occurred predominantly in lung adenocarcinoma (70–90%), and is composed of 638 N-terminal amino acid residues of the KIF5B protein and 402 C terminal amino acid residues of the RET protein. Coiled-coil domain containing 6 (CCDC6)-RET (RET/PTC1) is the second most frequent fusion described in NSCLC samples, accounting for 10–25% of the overall RET fusions. Other uncommon RET fusion partners, currently identified in lung cancer patients, include NCOA4, TRIM33, ZNF477P, ERCC1, HTR4, CLIP1, FRMD4, and WAC ^[24][25][26][24,25,26]. As with other oncogenic fusions, such as ALK and ROS1, adenocarcinomas are the most frequent histology to carry out RET rearrangements, followed by adeno-squamous, squamous cell, and neuroendocrine cancers [27].

3. The Available Techniques to Detect RET Rearrangements

Since there is not yet a universally accepted standard approach to detect RET rearrangements, several methods may be used in the clinic, including Fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), reverse-transcriptase polymerase chain reaction (RT-PCR), and NGS.

In several pathology laboratories FISH still represents the main technique used for the detection of RET fusions in NSCLC. The break-apart FISH probe is designed to hybridize against the 3′ and 5′ sides of the 10q11.21 RET chromosome region. To date, RET FISH is strongly suggested as a sensitive method to detect RET locus aberrations; however, this technique does not provide any information about the RET fusion partner and is not characterized by high specificity. Indeed the diagnostic sensitivity of FISH for the detection of RET fusions in lung cancer patients was estimated to range between 85.8% and 100%, while the specificity was reported to range between 62.1 and 96.8%, although it may be underestimated given the positivity cutoff set at ≥10% tumor cells.

FISH presents some limitations, such as inadequate identification of small intrachromosomal rearrangements, since only large gene deletions or amplifications can be detected and quantified by the immunofluorescence probes. As a consequence, FISH may produce some false-positive results, considering that all rearrangements occurring within the RET locus are detected, regardless of whether these result or not in a functional oncogenic fusion [28].

Yang et al., tested FISH performance in RET-rearranged NSCLC, showing a high sensitivity for both KIF5B (95%) and CCDC6 (95%) fusion partners while reporting a lower percentage of RET-rearranged tumor cells for NCOA4 fusions, with sensitivity near 67% [29].

IHC can be used to measure RET protein expression, which may serve as a surrogate marker for RET fusions. Despite the growing number of diagnostic assays, the variability in their performance represents a significant challenge for harmonizing RET IHC testing. In previous studies, RET IHC has shown poor correlation with RET fusion status as determined by both FISH and RT-PCR, thus may not be considered a settled approach for the RET rearrangement detection [30]. Based on this data IHC is not currently recommended for RET fusion genes diagnostic purpose in clinical practice, while either FISH or NGS are needed.

Literature data suggest a wide range of RET-RNA expression levels in tumor samples by RT-q-PCR technology, considered an inadequate approach to detect either novel fusion partners or isoforms. Approximately 371 NSCLC patients, including 270 adenocarcinomas and 101 squamous cell carcinomas, were investigated to identify the clinical-pathological characteristics associated with the KIF5B/RET fusion. The RET fusion genes were detected only in three cases of adenocarcinomas analyzed by an RT-PCR-based assay while fusion partners were identified by direct sequencing [31].

In this scenario, NGS assays targeted-based approaches are able to identify either known or unknown mutations within gene panel reference range, ensuring higher diagnostic accuracy, faster turnaround time for low sample volumes, and lower costs. To date, several NGS panels for routine mutation analysis are commercially available enabling the simultaneous analysis of a plethora of clinically relevant hotspots in target genes, including RET. Targeted RNA sequencing (RNAseq) completes the DNA based one, allowing a more comprehensive approach for simultaneous detection of both gene fusions and somatic mutations in tumor samples. In detail, RNAseq assay approach allows the detection of chimeric RNA, the discrimination of splicing isoforms, and also the quantification of fusion transcripts. [32].

Most of the positive aspects of the RNAseq approach consists of its ability to allow an adequate detection of different RET fusion partners. Although researchers are conscious that this kind of information do not currently affect clinical decisions; however research data showed that specific fusion partners could predict different survival outcome in RET-rearranged NSCLC patients. For example KIF5B-RET fusions seem highly dependent from EGFR signaling to promote enhanced cell growth, as compared both CCDC6-RET and NCOA4-RET fusions, in preclinical models [33].

Rich et al. showed as non-KIF5B-RET fusions contributed to anti-EGFR therapy resistance and [34] the same authors also reported specific RET fusions as mechanisms of resistance following exposure to third generation EGFR TKIs [34].

Finally, RNA seq allows the simultaneous testing of multiple biomarker beyond RET, including ALK, ROS1, NTRK, NRG1, Met ex 14 skipping, as recently suggested/recommended by the international ESMO guidelines [35].

NGS allows the detection of copy number alterations, gene rearrangements, and somatic mutations with 99% specificity and >99% sensitivity for base substitutions at ≥5 mutant allele frequency and >95% sensitivity for copy number alterations [26].

Despite this evidence, recent clinical trials, LIBRETTO-001 and ARROW, leading to the regulatory approval of RET-TKIs in NSCLC, included patients who tested positive for RET fusion by the different methods used at each local facility (NGS, RT-PCR, or FISH), without requiring a central confirmatory NGS analysis. ^[32][36][32,36].

The European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group (TR and PM WG) recently presented the recommendations for the routine detection of targetable RET rearrangements and mutations for the implementation of a rational approach in solid tumors. In particular, in NSCLC patients, multigene NGS is recommended. If NGS is not available, FISH or RT-PCR is indicated, depending on local availability, cost and/or number of tumor cells. In the case of a negative test result, NGS is always recommended. If a tissue sample is not available or exhausted, liquid biopsy may be considered ^{[35][36][37][38]}[35,36,37,38]. Perhaps even more impactful is the ability for liquid biopsy to detect acquired RET rearrangements and/or mutations as resistance mechanisms alterations to targeted therapies in oncogene-addicted NSCLC ^[39][40][39,40].

An analysis of over 32,000 plasma samples collected from advanced cancer patients was performed to elucidate the co-occurring RET alterations oncogenic signaling pathways identified by liquid biopsy. This study was the largest cancer cohort with somatic activating RET alterations, reporting that non-KIF5B-RET fusions contributed to anti-EGFR therapy resistance [34]. However, the sensitivity of NGS analysis for the detection of RET fusions on plasma free-circulating nucleic acids is significantly lower as compared to tissue analysis, requiring further validation in dedicated studies. [41].