Lenvatinib for the treatment of renal cell carcinoma

Introduction: Renal cell carcinoma (RCC) accounts for 2-3% of all solid tumors. Expression of the receptor for the Vascular Endothelial Growth Factor (VEGF) is one of the most common features of RCC. Areas Covered: Lenvatinib is a novel multi-kinase inhibitor that has been studied in several solid tumors. It has shown promising results in the treatment of RCC, especially when combined with everolimus, In this review, we summarize the available data of lenvatinib for the treatment of advanced/metastatic renal cell carcinoma. Expert Opinion: Lenvatinib in combination with everolimus has provided encouraging results in both clinical and laboratory investigations showing that blocking angiogenesis and the mTOR signalling pathway could be a remarkable approach for treating RCC. As an additive to this type of approach it would be interesting in future clinical settings testing also the combination of lenvatinib and everolimus with immune-therapy.

A c c e p t e d M a n u s c r i p t Kidney cancer is the eight most common cancer-related cause of death in the world, with just in the USA an estimated 63,340 new diagnoses and 14,970 deaths in 2018 1 . The incidence of the disease is expected to increase by about 2% this year compared to last year 1,2 .
Renal cell carcinoma (RCC) originates in the renal cortex and it is responsible for approximately 90% of all primary renal neoplasms, with a prevalence twice as high in men compared to women.
Although different drugs are currently available for RCC, the prognosis of the metastatic stage of disease still remains poor [3][4][5] .
While conventional chemotherapy for RCC reported relatively poor outcomes, some innovative and biological drugs have been implemented for the treatment of RCC patients in the past ten years.

Such treatments, including small tyrosine kinase inhibitors of the Vascular Endotelial Growth
Factor Receptor (VEGFR) mTOR inhibitors and checkpoint inhibitors. In particular, after sunitinib, the Food and drug administration (FDA) approved other antiangiogenic drugs such as sorafenib, pazopanib, axitinib, and bevacizumab. Furthermore, the FDA approved mTOR inhibitors temsirolimus and everolimus and recently, the only two agents able to improve survival, cabozantinib and nivolumab 5 .
Motzer RJ et al. showed increased Progression Free Survival (PFS) and Overall Survival (OS) with the combination of lenvatinib plus everolimus in comparison to everolimus alone in a multi-centred randomized-to-control phase II study of RCC 9 . The aim of this mini-review is to summarize the first preliminary and clinically available data on lenvatinib in advanced/metastatic RCC. Finally, future directions will be discussed.

The molecular bases of tumor angiogenesis
A c c e p t e d M a n u s c r i p t It was not until relatively recent times that sporadic and hereditary mutations in the Von-Hippel-Lindau (VHL) gene were proven responsible for the development of RCC. The VHL gene was found on the human chromosome 3p25.5 for the first time in 1993 10 as a tumor suppressor gene.
Other than being implicated in the inherited von Hippel-Lindau disease, mutations of this gene are also found implicated in sporadic RCC. Mutations of the VHL gene have been associated with the majority of cases of sporadic RCC11. After these first discoveries, the pathway was considered as a potential candidate for targeted therapy of this disease. One of the most well-known targets of VHL gene is the hypoxia-inducible factor α (HIF-α), a transcription factor involved in the regulation of various angiogenic factors. In fact, in the absence of oxygen, HIF-α interacts with its heterodimer hypoxia-inducible β (HIF-β) to form a complex that leads to the transcription of hypoxia-inducible genes such as vascular endothelial growth factor (VEGF), platelet-derived growth factor β (PDGFβ), transforming growth factor α (TGF-α) and erythropoietin (EPO). In the presence of oxygen, however, the pVHL binds and hydroxylases two proline residues of HIF-α leading to its ubiquitination and subsequent degradation by proteasomes 10 . The presence of VHL gene mutations leads to inactivation of this oxygen-dependent degradation of HIF-α with an up-regulation of angiogenic factors.
In physiological conditions, and especially during early stages of life, angiogenesis is an important process by which new blood vessels are formed in order to deliver required oxygen and nutrients to peripheral tissues, maintaining the required levels of perfusion. Considered nowadays as a universal cancer hallmark, neo-angiogenesis in the context of a tumour provides new blood vessels that will ultimately result in enhanced tumor growth and increased metastatic potential. One of the most common features of cancer neo-angiogenesis is that it is highly regulated. It is mainly based on the interactions between tissue soluble VEGFs ligands and VEGFRs. These interactions work perfectly in healthy conditions, while they become deleterious during tumourigenesis and cancer progression.
Sub-variants of VEGF molecules in humans are VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PlGF) 12, 13 . The VEGF receptor family consists of three molecular subtypes A c c e p t e d M a n u s c r i p t (VEGFR-1, VEGFR-2 and VEGFR-3), which are type-II transmembrane tyrosine kinase (TK) proteins 12 . Among these receptor subtypes, the main one associated with the pathological formation of blood vessels in the context of solid tumors is VEGR-2 (Figure 1).
VEGFR-2 is usually expressed on the surface of vascular endothelial cells and circulating bone marrow-derived endothelial progenitor cells 13 . Upon binding of VEGF molecules to VEGFR-2, the receptor dimerizes and auto-phosphorylates at the carboxyl-terminal TK domain leading to activation of the signaling transduction pathway downstream. Consequently, various specific molecular pathways are activated at the same time (Figure 1): the PLCγ/PKC/MAPK pathway, related to endothelial cells migration; the PI3K/AKT/mTOR pathway, whose activation leads to increased survival of endothelial cells and increased vascular permeability; the Raf/MEK/Erk pathway, which promotes endothelial cells proliferation 14,15 . The VEGFR-2 has a significantly more robust kinase activity than VEGFR-1 even if the binding between VEGF-A and VEGFR-2 (KDR Flk-1 in mice) occurs with an affinity one order lower than that of VEGFR-1 (Flt-1 in mice) (Kd= 1-10 pM) 14 . The activation of the VEGFR-2 kinase activity results in a dramatic increase of blood vessels' formation, translating in an increased micro-vessel density and enhanced proliferation rate of vascular endothelial cells 13 . A larger study, enrolling 1091 RCC patients, showed that 79.6% of patients were positive for VEGF, 62.4% were positive for VEGFR2, 45% were positive for PDGF-B and 42.5% were positive for PDGFR-β 16 . On the basis of these data, VEGFR-2 is considered a key player in the process of tumor neo-angiogenesis in solid tumors and RCC. As a consequence, VEGFR-2 represents an attractive target for the development of novel anti-cancer therapies.
Sub-variants of fibroblast growth factor (FGF) molecules in humans are FGF1-10. The receptor family consists of four molecular subtypes (FGR-1, FGR-2, FGR-3 and FGR-4), which are highly conserved TK receptors. There are differences among subtypes in their ligand affinities and distribution across tissues [17][18][19] . Among the FGFR receptor-family, the most commonly amplified was the FGFR1, affected in 3.5% of the tumours 20 . Moreover, a study looking at 100 primary tumors and 40 metastatic lymph nodes obtained from 140 untreated RCC, showed that FGFR1 and A c c e p t e d M a n u s c r i p t FGFR2 were highly expressed in RCC by immunohistochemistry. Expression of FGFR1 was observed in 98% (98/100) of primary tumorous and in 82.5% (33/40) of lymph node metastases.
In normal kidneys FGFR1 expression was significantly lower (p=0.0001) than in RCC 21 .
There is strong evidence pointing out that fibroblast growth factor-2 (FGF2) has an important role in angiogenesis besides the VEGF. As a matter of fact FGF2 is a potent regulator of many cellular functions, such as proliferation, migration, survival, adhesion, motility, apoptosis and physiological functions like wound healing, tumorigenesis, angiogenesis, or blood vessel remodelling or embryonic growth [22][23][24] .

Lenvatinib: preclinical and phase I data
Authors performed also a post-hoc analysis to prove the robustness of the statistical analysis 31 . They used a blinded, independent radiological review (IRR) to assess the efficacy of their results.  A c c e p t e d M a n u s c r i p t mg of lenvatinib plus 5mg of everolimus (NCT03173560). Finally, a randomized, multi-centered, parallel assignment, open-label, phase III clinical trial made of 735 patients has been designed in order to test primarily PFS of first line lenvatinib in combination with everolimus (Arm A), or pembrolizumab (Arm B) compared to sunitinib (Arm C) in patients with mRCC. The study also aims to investigate OS and ORR as secondary outcomes (NCT02811861).This trial has been made to compare the efficacy and safety of the combination of these three arms as first-line treatment through the measurement of PFS. As major secondary outcome OS, ORR, AEs will be evaluated.

Expert Opinion
Lenvatinib in combination with everolimus has provided encouraging results in both clinical and A c c e p t e d M a n u s c r i p t (500g/mouse, twice in a week) had a substantial inhibition on tumour growth when compared to individual treatments. Moreover, a greater number of mice demonstrated complete response of tumour in the combination therapy group compared to single treatments 32,33 . Therefore, the combination of agents targeting VEGF and cellular immunotherapy-mediated pathways may block at the same time two critical signaling pathways activated in RCC, overcoming thereby some partial mechanisms crucial for the development of drug resistance in single agent therapy. In an era where immune-therapy is constantly progressing at a high pace, with always newer and more powerful molecules, the combination of lenvatinib with everolimus to some immune-modulation therapy in future randomized clinical trials could be also an interesting approach deserving some clinical validation.

Funding
This paper was not funded.

Declaration of Interest
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Peer reviewers on this manuscript have no relevant financial or other relationships to disclose. M a n u s c r i p t