Towards more powerful antibodies for fibroblast growth factor receptors in breast cancer

: Breast cancer (BC) is the most frequent form of malignancy and the second only to lung cancer as common cause of cancer-causing deaths in women. Notwithstanding many progresses in the field, metastatic BC has a very poor prognosis. As therapies are becoming more personalized to meet patients ‘ needs, a better knowledge of the molecular biology leading to the disease unfolds the possibility to project more precise compounds or antibodies targeting definite alteration at the molecular level expressed in cancer cells of patients or as antigens on the surface of cell membranes. Fibroblast growth factor receptor (FGFR) is a druggable target -which is activated by its own ligands -namely the Fibroblast Growth Factors (FGFs). This pathway provides a vast range of interesting molecular targets pursued at different levels of clinical investigation. Herein we provide an update on the knowledge on genetic alterations of the receptors in breast cancer, their role in tumorigenesis and the most recent drugs against this particular receptor to treat the disease. checkpoint inhibitors- both survival and quality of life of breast cancer patients new and more precise strategy of fighting cancer, focusing on checkpoints or drugs targeting driver mutations by cancer on


Biochemistry of the Receptor
As to the epidemiology of the disease, Breast Cancer (BC) is a devastating cancer for females. In the planet it is the first one of all types of tumors in terms of frequency and second in terms of mortality after lung cancer. Only in the United States 271270 patients were diagnosed with BC and the people who died in the continent were 41488 in the year 2019 1 . As personalized medicine advances, stratification of patients based on molecular targets is becoming the standard of care. FGFRs harbor cancer-driver mutations that can be targeted with antibodies for the treatment of BC.
The structure of the receptor has been fully described in our previously published review 2 . Briefly it is a receptor tyrosine kinase member, encompassing the cellular membrane in a single region. The FGFR family, in humans, consists of six receptors (FGFR1-6), bound by ligands. The FGFR1 gene is located on chromosome 18p11.23, while -2 is in chromosome 10q26.13, -3 is in chromosome 4p16.3, -4 is in chromosome 5q35.2, -5 is in chromosome 4p16.3, and finally -6 is located in chromosome 6p21.33 (also called Fibroblast Growth factor like-1). FGFR 1, 2 and 3 alternative splicing may encode alternative isoforms with different ligand binding specificities 3 . In addition to its involvement in cancer, like many other oncogenic drivers the receptor is also important for the development of the skeletal system 4,5 , the metabolism and embryogenesis 6,7 . Interestingly, Fibroblast Growth Factors (FGFs) engage with many cofactor, such as heparin or heparan sulfate proteoglycan (HSPGs) at the cell surface, increasing the affinity binding to cellular FGFR [8][9][10] . Indeed, FGFs after being secreted are almost immediately taken up by HSPGs. HSPGs, in turn, stabilizing the interaction between FGF ligand and FGFR 11 by safeguarding FGFs from degradation by proteases 12 . Ligands have different specificity in binding to FGFRs; some of them bind to different receptors, such as FGF1, while others, such as FGF6, bind only to one receptor isoform 13 .

FGFR Signalling
RTKs were discovered around fifty years ago. Since then, their signal transduction has been explained through the canonical or diffusion model 14 . It is known that cell membrane receptors are responding to a signal that is been transmitted from outside the cell through molecules that bind to them. Once bound the receptor becomes activated and it triggers a downstream series of events that activate other molecules. RTKs are the largest class of such receptors showing such capability. The mechanism leading to the FGFR signaling has been fully described in our previously published review 2 . Briefly, ligand-binding causes RTKs monomers to form dimers and this tethers tyrosine residues of the monomers close to each other, which thereby cross-phosphorylate and, as a direct consequence, activating each other 2,15 . It is through this mechanism of dimerization and cross-phosphorylation nearby could be tethered and cytoplasmic proteins phosphorylated. This ultimately activates signaling cascades 16,17 . FGFR substrate 2 (FRS2) is one of these adaptors. After FGF binding the FGFRs form dimers and FRS2 adaptor binds to the complex, by which a series of downstream signaling cascades occur leading to the activation of important tumorigenic pathways. Among such activated tumour-leading pathways are the phoshoinositide 3 kinase Protein Kinase A (PI3K-AKT) 18 and the Mitogen-Activated Protein Kinase (MAPK) 19 . In addition, FGFR on its own is connected to phospholipase C-gamma (PLC-γ), in mechanism that is FRS2 independent and it is also capable of activating Protein Kinase C (PKC) 20 . Notable, PKC can phosphorylate RAF, making the process of MAPK pathway activation to occur more effectively 21 .
The downstream molecules of the FGFR pathway, described in more detailed in our previous review 2 , constitute actionable targets that are captivating attention for the development of novel antibodies and/or small compounds against cancer-driver mutations in FGFRs and associated signaling molecules, to develop innovative anti-cancer drugs 22 (Figure 1).

The balancing of FGFR cascade
In order to have a balanced FGFR cascade, first the FGF signaling should be properly regulated. However, this process is poorly known and may vary based on the cell type 3 . Nevertheless, ubiquitination mediated internalization 14,23 , negative regulation through Spred, Sef and Spry genes 15,24,25 , are important negative feedback mechanisms balancing the FGFR/FGF axis cascade. Receptor auto-inhibition is another mechanism of control 26,27 . Moreover, Heparane Sulfate (HS)-binding site and the FGFR's acid box binding leads to a receptor binding closed conformation, an auto-inhibition mechanism 7,27 . This mechanism blocks the binding of FGF to FGFR. FGFs would therefore bind to other RTKs 28 .

FGFRs as Oncogenic Drivers
FGFRs' signaling pathways deregulation can work as cancer driving oncogenes, as evidenced by large series of experimental results gathered from experiments conducted with several types of tumors 12,13 . Therby, deregulation of FGFR's cascade leads to a blockade of apoptosis, an increase of mitogenesis and fosters epithelial-to-mesenchymal transitions 29 . Mechanisms of deregulation are the following ones: (i) expression of fusion proteins with FGFR resulting from gene-translocations that constitutively activate the kinase activity of FGFR 30 ; (ii) overregulation of genes and post-transcriptional events, ultimately increasing protein FGFR levels 31 ; (iii) high expression levels of FGF outside the cellular matrix, inside the stromal and tumour cells,, activating the paracrine/autocrine pathway 32 ; (iv) FGFR alternative splicing, change of its isoform, processes that ultimately change the ligand-to-receptor specificity, , increasing therefore the range of FGFs that are capable of inducing cellular growth 33 ; and (v) constitutively activating mutations of FGFR . Sarabipour et al. proved that the mutations constitutively activating the receptors give them the capacity to dimerize without ligands triggering them at physiological conditions, as described in (v) 34 . The stabilization of such unbound dimers takes place by a connection between the transmembrane and intracellular domains 35 . It is important to note that the phosphorylation of FGFRs that did not bind is retained, explaining thereby how the high expression level of FGFR leads to cancer formation 10,[36][37][38] . However, changes in the structure induced by the binding of the ligand to receptors that had dimerized inside the intracellular region of the membrane can change the transmembrane structure, which will switch its structural conformation into a specific one 39 . Based on dimers' transmembrane configuration the activity of the receptor is concerted 40 . Quite different are the FGF1 and FGF2 ligandsinduced conformational changes. Those ligands modify the intracellular domains distances changing therefore the amount dimers become phosphorylated 41 . For this reason FGFRs are found in different configurational states even after being bound by the ligand, some of them form an active state while others are intentionally inactivate 34 . Such series of mechanisms inside the cells lead to a fine tuning of the FGFR signaling cascade of events controlling apoptosis, mitogenesis, proliferation, epithelial-tomesenchymal-transitions, and oxygen deliver vessels formation in embryogenesis or tumorigenesis. There are different FGFR germline mutations in humans. Mainly they are GOFs in genes that give rise to hereditary diseases, such as osteochondrodysplasias, and tumorigenesis 42 . Fascinatingly, ther are mutationsin all the gene encoding for FGFR, and are not limited to the kinase region 43 . A peculiar aspect of the signaling pathway involving this receptor is the dependency on the type of tissue 44 ; each tumoor type hasdifferent aberrations in different FGFRs [45][46][47] . Here we focus on breast cancer fibroblast growth factors genes aberrations.

FGFR Genetic Alterations in Breast Cancer
In 1991 Adnane et al discovered for the first time amplifications of the genes encoding for the fibroblast growth factor receptors in the context of BCs 48 . Since then, several studies confirmed this data proving that the receptors have a role as oncogenes and they were further able to explain -until an extentthrough which molecular these molecules that sit on the cell membranes achieve this purpose. Additionally, point mutations (single nucleotide polymorphisms), high expression of ligands and FGFRs were also identified in previous studies, paving the way for an hypothesis where multiple molecular mechanisms could be leading to an overactivation of the receptors 49 . Deregulation of the fibroblast growth factor molecular cascade in human cancers is frequent. Interestingly, the most common alteration for these receptors is related to the first receptor -FGFR1 (located on the 8p11-12 region of the chromosomal)-while the other 5 receptors are barely found overamplified 10,50 .

FGFRs gene amplification
Around 15% of breast cancers have FGFR1 gene mutated 38,43,51 . The high expression levels of the FGFR1 gene and/or of the entire region of the 11q12-14 chromosome, (which entangles FGF3-4, and CCND1), was observed in 27% of patients positive for Human Epidermal Growth Factor Receptor 2 (HER2), as well as in 23% of patients positive for hormone receptor (HR+) and in 7% of patients who were Triple Negative, (TNBC) meaning they do not express any of the hormone receptors (progesterone and estrogen) nor HER2. The amplifications could be used as patients prognostic markers since they were found associated with early relapse and lower survival rates [52][53][54][55][56] . Andrè et al., using cell lines proved that cells with FGFR1 amplified have to induce overexpression of the gene to support B-cell line's survival, which is an oncogenic signature. The authors used a couple of commercially available cell lines with FGFR1 or FGFR2 amplified, MDA-MB-134 and SUM52, respectively. They proved in both cells that dovitinib (also known as TKI 1258), which is an anti-FGFR1 antibody, was capable to reduce downstream signalling by a down modulation of pFRS2 and pERK/MAPK as shown by western blotting, and in vivo it reduced tumours in primary breast cancer xenografted HBCx-2 mice 57 . The half maximal inhibitory concentration (IC50) measures the potency of a pharmaceutical construct by an equation standing for the amount of a specific drug required to inhibit in vitro to inhibit a chemical or biological function. To attain such value for TKI 1258 in the two cell lines, SUM52 and MDA-MB-134, 180 nmol/L and 190 nmol/L were needed, respectively. As expected, in control cells that did not express none of the two receptors (FGFR1 or FGFR2) the IC50 values were higher and above 2000 nmol/L. The authors showed that the drug (50 mg/kg) given to the HBCx-3 mice brought to a tumour regression vs empty control (p < 0.001) 57 . Moreover resistance to hormone therapies can be driven by FGFR1 amplifications. As a matter of fact, Turner et al., demonstrated in MDA-MB-134 and SUM44 BC cell lines, overexpressing FGFR1, a resistance to the endrocrine treatment, namely the 4-hydroxytamoxifen (4-OHT). The authors used cell TiterGlo assay to prove survival of the cells transfected with or without small interfering RNA targeting FGFR1 (siFGFR1), to silence the gene 38 . Treatment with siFGFR1 reversed such resistance. As a conclusion the authors evinced that FGFR1 is an important factor driving sensitivity to 4-OHT therapy. Another experiment adding to such proof-of-concept-elucidating that among the FGFRs specifically FGFR1 is responsible for the sensitivity to the drug-is the one where it was shown that the addition of FGFR2 to siFGFR1-treated cell lines was not capable to achieve the same results. The cells in the latter scenario were still resistant to 4-OHT. Therefore the resistance was FGFR1-dependent. Furthermore, FGFR1 inhibitor (PD173074) triggered a loss of colony formation ability of the cell lines, suggesting that FGFR1 confers transformative capabilities to the cells. This is in line with the evidences proving the oncogenic role of FGFR1 in breast cancer 38 .
In MONALEESA-2 based on the combination of letrozole with ribociclib (a Cyclin Dependent Kinase 4/6, CDK4/6, inhibitor), FGFR1 amplification was related to a lower PFS respect to wild type patients for the FGFR1 gene. In in vitro studies focused on estrogen receptor positive BC patients with amplification of FGFR1, it has been shown that there is correlation between FGFR1 amplification and resistance to CDK4/6 inhibitors ribociclib or palbociclib. Such resistance-to-treatment was overcome by the combination of anti-FGFR drugs, such as lucitanib. Moreover the combination of CDK4/6 inhibitor palbociclib and FDAapproved FGFR inhibitor erdafitinib showed a complete in vivo response in SCID/beige mice xenografted with estrogen receptor positive and FGFR1 amplified breast cancers. Moreover they showed that circulating tumor DNA from 34 patients progressing to the CDK4/6 drugs had amplifications or activating mutations of FGFR1 and FGFR2 was as high as 41% in the patients 58 . Hence, combining treatments acting on both pathways, namely the FGFR/FGF and the CDK4/6 pathways, could prove a great option to overcome resistance to molecules acting against the CDK4/6 pathway.
Different investigations have proven that amplifications of the FGFR2 (occurring in less than 5% of triple negative breast cancers), and other mutations activating the FGFRs have been related with maintenance of tumor-initiating cells and a high sensitivity to FGFR inhibitors 59 .

FGFRs Activating Mutations
Besides the oncogenic role that amplifications of FGFR gene have, a least common form of alterations causing FGFR-driven BCare mutations that constitutively activate the receptor 10 . There are different mechanisms by which activating FGFR mutations could lead to aberrant FGFR signaling. These ones include the following ones: (i) dimerization of FGFRs that become bound in a form that will not be reversible; (ii) receptor kinase domain over activation; and (iii) alterations of binding affinity between FGFR to FGF. In fact, the most frequently occurring oncogenic FGFR aberrations in BC are FGFR1 translocation 60 and FGFR1 amplification leading to activating mutations (10-15%) 38,52-56 . These two genetic aberrations can modify the phenotype of BC cells transforming them onto a phenotype exhibiting more sensitivity to FGFR inhibitors 55 . They can also drive the endocrine resistance, as explained in the previous chapter 38 . The translocation of the FGFR2 gene leading to activating phenotypes, can also bring to cellular transformation and make the cells more anti-FGFR sensitive 60 . In addition, FGFR2 amplification, present in less than 5% of BC cases, can make cancer cells more resistant to FGFR inhibitors, as seen in pre-clinical models [57][58][59]

Genome-wide studies
Genome-Wide-Association-Studies (GWAS) has brought evidence about the potential of risk prediction for the development of BC in individuals with Single Nucleotide Polymorphisms (SNPs) in the second intron of FGFR2 gene 70-73 . Easton et al., for example, in a cohort of 4,398 breast cancers vs 4,316 healthy individuals used GWAS to investigate common SNPs to find risk-associated factors 70 . Five new loci have been found significantly associated with BC (p value < 10 −7 ). FGFR2 is one of these loci, thereby corroborating the potential oncogenic role of alterations in this gene in BC 70 . According to well-powered GWAS, conducted by Stacey et al., rs4415084 and rs1094179 SNPs (located on chromosome 5p12) were associated with increased risk of encountering breast cancer. The difference reached the highest statistical significance ER + BC (p value = 1.3 × 10 −17 ) 71 . Meyer et al. proved with microarray gene expression analysis that FGFR2 is expressed at higher levels in rare homozygotes 72 . The authors then confirmed the data by Real-Time PCR (RT-PCR). They showed that patients with the rare homozygous had higher levels of FGFR2 as compared to that of normal homozygotes (Wilcoxon p-value = 0.028). They also showed that  59 . In fact, the authors demonstrated in BC, that CD29 high CD24 + TICs expressed significantly higher levels of GABRA4, FGFR2 and FOXA1 mRNA expression. Additionally, down-regulation of FGFR2 by short hairpin RNA (shRNA), which is a short molecule of RNA engineered to silence target genes (in this case FGFR2) via RNA interference, in mouse models substantially reduced (64 -70%) the CSCs subpopulation CD29 high CD24 + . Interestingly, the non-TIC cells (CD29 low CD24 − ) subpopulation was significantly increased (65 -67%) after using shFGFR.2. Therefore a down modulation of FGFR2 could cause a non-TICs increase and a TICs decrease 59 . Furthermore, they proved that in mice treated with shFGFR2 there is an increased of bipotent precursor-like cell population (K18+K14+). The generation of bipotent populations generated by FGFR2 knockdown could be overcome by FGFR2. Therefore, a valid strategy may consist in the inhibition FGFR2 in order to decrease those BC CSCs . Kim et al. additionally proved that tumour growth could be inhibited by treatment with FGFR2-inhibitor (TKI258) using NOD/SCID mouse models xenografted with breast cancer tumours overexpressing FGFR2. Such inhibition of tumour growth was followed by a significant reduction of protein FGFR2 phosphorylation together with Erk1/2 activation. This further proved that the inhibition was FGFR2 dependent 59 . Guagnano et al. through a screening that included BC cell lines with FGFR alterations studied cell sensitivity to NVP-BGJ398, which is an anti-FGFR inhibitor. The focused on 9 types of FGFR genetic alterations' well known from literature: FGFR1-FGFR2 chromosomal translocations; FGFR1-3 activating mutations; FGFR1-4 copy number gains. The drug was evinced as a powerful multi-kinase inhibitory molecule against Vascular Endothelial Growth Factor Receptor (VEGFR) 2, besides FGFR1-4 . Finally these researchers experiments demonstrated a predictive role for such alterations in the FGFR genes for response to NVP-BGJ398 therapy 60 . In a small study of 13 lobular BC, Reis-Filho et al. proved the presence of high copy number levels on 8p12-p11.2 the chromosomal location of six patients (46% of cases) 55 . Moreover, using small-interfering molecules against FGFR1 in SU5402 cell line, the authors were able to demonstrate that FGFR1 inhibitor can block strop breast cancer survival of ductal breast adenocarcinoma cell line MDA-MB-134 55 . In summary different research analyses of various research groups support that using drugs blocking the FGFR/FGF signaling pathway is a good approach that is worth experimenting at later stages of clinical development involving randomized-to-control patients. Accordingly to Next Generation Sequencing (NGS) investigating FGFR levels in breast cancer, were low levels of FGFR3 and FGFR4 were detected . As a matter of fact, the NGS study of Helsten et al. investigated their expression levels in 4853 solid tumors, with 522 breast cancer. In this experiment they showed a very low level of amplification of FGFR3 and FGFR4, lower than 1% and 2%, respectively 43 . Conversely, in a RT-PCR investigation of 103 breast-tumor samples and 10 tumor cell lines FGFR3 was not detected at all and FGFR4 was present in a maximum of 32% of the total BC population 75 .

Anti-FGFR Therapies
There is a growing interest in FGFR/FGF inhibitors to block the formation and progression of BC in developing new targeted therapies against this pathway 76 . Clinical evaluations have been conducted over small FGFR inhibitors, selective or nonselective, even though many are early clinical trials 57 . Novel drug development should be focused in the attainment of an increased selectivity to the FGFRs ATP-binding domain located in the intracellular region in order to reduce to the maximum extent the toxicity 45 . BGJ398 (infigratinib) is a pan-FGFR inhibitory molecule that has been evaluated on its own (NCT01004224) -to establish its maximum tolerated dose (MTD) for primary outcome and ORR together with pharmacokinetics and pharmacodynamics for secondary outcome measures 77 , respectively-was recently completed. In the 67 enrolled patients, ORR was 25.4% and DCR 64.2% 78 . Additionally, MTD for BGJ398 with BYL719 was investigated in another clinical investigation on phase I (NCT01928459) FGFR 1-3 and PIK3CA mutations bearing solid cancers, which was recently completed and whose results have not been posted yet. AZD4547 is an additional tyrosine kinase inhibitor, whose activity had been previously shown to be strong for FGFR-3. On the other its activity against FGFR4 was very low. A phase I is currently investigating safety and efficacy of this compound in endocrine progressing BC patients bearing polisomy or amplification of the FGFR1 gene (NCT01791985), which was recently completed and whose results are eagerly awaited. Another phase I study (NCT03238196) has been evaluating in ER+ HER2-MBC patients the FGFR inhibitor, called Erdafitinib, together with palbociclib and hormone therapy fulvestrant. . In preclinical studies multi-kinase inhibitors, capable of targeting also FGFRs together with other tyrosine kinases, have been showing promising results 7 . Phase I trial investigating such inhibitors have shown great positive as to safety and tolerability of this drug type. Dovitinib (TKI258) showed its effectiveness in targeting FGFR1-3, PDGFR and VEGFR1-3 79 , and has been experimentally used for the treatment of HER2-negative MBC in combination with fulvestrant. However this clinical trial was too slow and had to be terminated (NCT01528345).
E3810 (lucitanib), against colony stimulating factor 1 receptor (CSF1R), -3, FGFR1, FGFR2 and VEGFR1 has been studied on its own in two phase II clinical trials involving MBC patients with the presence or absence of FGFR1 amplification; one of them is a phase II study (NCT02202746), which was terminated by the sponsor of this clinical trial. Recently, the safety and tolerability of a triple kinase inhibitor (FGFR, PDGFR and VEGFR) was investigated in a phase I study made of 19 estrogen receptor positive MBC postmenopausal women (NCT02619162) . At PR2D (nintedanib with letrozole) there was a 55% mean increase in the plasma levels of FGF23 and there were no detectable levels of 17-B-estradio in the plasma of patients 78 .
Other undergoing strategies inhibiting FGFR/FGF inhibitors together other signaling pathways exist 80 , but these were not the main focus of this review. For a reference to such combinatorial strategies refer to our previous publication 2 . Antibodies against FGFR isoforms represent a valid therapeutic strategy to intervene in BC. As a matter of fact, GP369 recognizes FGFR-IIIb isoform and has exhibited good results in blocking breast cancer cell line proliferation 81 . Such positive preliminary results warrant further research. Lastly, another approach against the FGFR/FGF axis concerns the use of inhibitors of FGF ligands. Long pentraxin-3 (PTX3) is an inhibitor of various FGFR ligands, among them FGF2 and FGF8b, which have both been found to be implicated in breast cancer development 82 . FP-1039 is a recently developed ligandtrap in which a ligand-binding domain of FGFR1 is fused to an Ig-Fc domain. This compound showed promising activity in vitro and passed a phase I clinical trial (NCT00687505) for solid tumors, including breast cancer 83 . Table 1 outlines all the ongoing clinical investigations of anti-FGFRs therapies. . Although such results are crucial for the anti-FGFR therapy development, more knowledge of the molecular mechanisms by which FGFR function and lead to breast cancer in correlation with other well-known molecular pathways are also eagerly awaited and important to best design new treatments and to best give the most effective ones to each patient.

Discussion
The established BC oncogenic driver FGFR has been found involved in various tumour-related roles, leading to angiogenesis, tumor growth and apoptosis avoidance. There are different FGFR variations correlating with breast cancer. Therefore many different strategies have been design in order to block the FGFR/FGF axis. For this purpose phase I/II clinical investigations have been investigating several therapies targeting the FGF/FGFR axis. Among such molecules, some examples are futibatinib (TAS-120), nindetanib, rogaratinib (BAY-1163877), erdafitinib, an antipan-FGFR molecule infigratinib (BGJ-398), dovitinib and FGFR1-3 inhibitor AZD4547. Noteworthy, FGFR1 genomic aberrations are the most common ones, while gene amplifications in the FGFR2-6 and mutations constitutively activating the FGFR are uncommon. Thus, among the different targets for future therapies against FGFRs, FGFR1 should the considered as the primary one to be further pursued. A combination of anti-FGFR therapies should be experimented with other drugs targeting downstream pathways of the FGFRs/FGFs axis, mutation-bearing antigens and other Tyrosine Kinase cell membrane receptors such as AXL, CCK, EGF, HGF, PDGF, LMR, RET, TIE, RYK, ROS, and VEGF. As personalized medicine is marching with its great advancements in the field of oncology, stratification of patients -based on definite molecular modifications -and the discovery of always more precise biomarkers predictive both disease occurrence and treatment efficacy are leading to the development of accurate molecular-based methods aiding clinicians to choose the right therapy, or combination of therapies, for each individual patient. Furthermore, as immunotherapy is today standing at the front-line of innovation of anti-cancer treatments, it would be curious to test anti-FGFR or anti-ligand FGF drugs together with immunotherapeutic agentslike PD-1/PD-L1 or CTLA4-CD20 checkpoint inhibitors-to improve both survival and quality of life of breast cancer patients through new and more precise strategy of fighting cancer, focusing on checkpoints or drugs targeting driver mutations presented by cancer cells on their surfaces' membrane and beyond.
Author Contributions: N.S. contributed to the implementation of the work. He also contributed in reviewing the literature and editing the manuscript; P.O.F.V contributed to reviewing the literature, writing and editing the manuscript; C.F. contributed to reviewing the literature, writing and editing the manuscript; D.G. contributed with his opinion as an oncologist; Y.L. contributed to the original idea of the work. By giving his expert opinion he also contributed in the writing and editing of the manuscript.

Conflicts of interests:
The authors declare no conflicting interests.