https://orcid.org/0000-0002-5899-1738
ABSTRACT
Objective
Axitinib is an oral multi-target tyrosine kinase inhibitor used for the treatment of renal cell carcinoma (RCC). Because of the severe adverse events (AEs) associated with axitinib, patients often need dose reductions or discontinue its use, highlighting the need for effective biomarkers to assess efficacy and/or AEs. The aim of this study was to investigate the relationship between single nucleotide polymorphisms (SNPs) in genes involved in the pharmacodynamic action of axitinib and clinical prognosis and AEs in metastatic RCC (mRCC) patients.
Methods
This study included 80 mRCC patients treated with first-, second-, or third-line axitinib (5 mg orally twice daily). Clinical parameters and genetic polymorphisms were examined in 75 cases (53 males and 22 females). We assessed three SNPs in each of three candidate genes namely, angiotensin-converting enzyme (ACE), nitric oxide synthase 3 (NOS3), and angiotensin II receptor type 1 (AT1R), all of which are involved in axitinib effects on vascular endothelial function.
Results
Axitinib-treated patients carrying the ACE deletion allele suffered more frequently from hand-foot syndrome and a deterioration in kidney function (p = .045 and p = 0.005, respectively) whereas those carrying the NOS3 G allele suffered more frequently from proteinuria and multiple AEs (p = .025 and p = 0.036, respectively).
Conclusions
Our study found that the ACE deletion allele and the NOS3 G allele are associated with increased AEs.
Introduction
Axitinib is an extensively used tyrosine kinase inhibitor (TKI) that has extremely robust efficacy worldwide in patients with metastatic renal cell carcinoma (mRCC).Citation1,Citation2 However, both the profile of the toxicity of axitinib can vary and are difficult to predict before the initiation of treatment.Citation3,Citation4 Because anti-angiogenic drugs act on nonmalignant endothelial cells as well as on tumor cells, the genetic background of the patient may play a crucial role in determining the efficacy of these drugs.Citation5 Of those, single nucleotide polymorphisms (SNPs) may affect the efficacy of axitinib.Citation6 Several studies have addressed the clinical implication of SNPs for the efficacy of TKIs. The largest study of SNPs showed the vascular endothelial growth factor receptor (VEGF)-A rs699947 AA genotype was related to a longer overall survival (OS) in patients treated with axitinib.Citation7 However, the association of these three SNPs with TKI-related adverse events (AEs) remains unelucidated. We hypothesized that genetic polymorphisms in three downstream genes in the VEGF pathway (angiotensin-converting enzyme, ACE; angiotensin II receptor type 1, AT1R; and NO synthase 3, NOS3) might alter the clinical outcome of VEGFR inhibitors.Citation8,Citation9 The ACE deletion, AT1R, and NOS3 polymorphism are the well-known risk factors associated with the development of cardiovascular diseases,Citation10 vascular vulnerability,Citation11 and lower level of production of active NO.Citation12
In this study, we investigated the association of SNPs and AE profiles in Japanese patients treated with axitinib using a novel approach focusing on polymorphisms in downstream genes in the VEGF pathway.
Results
Patient characteristics
The patient characteristics are summarized in (). The median patient age was 67 years (range: 25–85 years). All patients were Japanese, consisting of 53 men (71%) and 22 women (29%). Sixty-one (81%) patients underwent radical nephrectomy prior to systemic therapies. Thirty-five (47%) patients were administered axitinib as a first-line systemic therapy, 27 (36%) as a second-line therapy, and 13 (17%) as a third-line therapy. In the patients who received second- and third-line therapies, 17 (23%) patients were previously treated with cytokines, 21 (28%) with TKIs, 2 (3%) with nivolumab, and 1 (1%) with an mTOR inhibitor. Forty-eight patients (80%) had a Karnofsky performance status (PS) score of 80 or higher and 27 patients (36%) had a score of less than 80. Six patients (8%) were classified as favorable risk based on the International Metastatic Renal cell Carcinoma Database Consortium (IMDC) risk classification system, 40 (60%) as intermediate risk, and 29 as poor risk (39%). Twenty-two (29%) patients had one metastatic site, 24 (32%) had two, and 29 (39%) had 3 or more. The sites of metastasis were the lungs (n = 54, 72%), lymph nodes (n = 39, 52%), bones (n = 24, 32%), liver (n = 13, 17%), contralateral kidney (n = 9, 12%), adrenal (n = 7, 4%), and others (n = 20, 27%)
Table 1. Patients characteristics.
Patient outcome and genotype analyses
The mean follow-up duration after axitinib administration was 13.4 (range, 3–60) months. At the time of analysis, 14 patients continued taking axitinib. Thirty-six patients stopped axitinib treatment owing to disease progression, whereas 21 stopped treatment owing to AEs. Thirty-five patients had died at the time of analysis. The estimated median time to treatment failure (TTF), progression-free survival (PFS), and OS were 8.7 (range, 1–56) months, 6.9 (range, 1–59) months, and 14.2 (range, 1–65) months, respectively. The best objective response (BOR) assessment data were available for 72 patients (). The three patients that did not reach the first evaluation (early discontinuation) were excluded. Three patients (4%) showed complete remission (CR), 27 (36%) showed a partial response (PR), 29 (39%) showed disease stabilization, and 13 (21%) showed disease progression. The frequent AEs were hypothyroidism in 50 (66.7%) patients, hypertension in 48 patients (64.0%), proteinuria in 36 patients (48.0%), anorexia in 27 patients (36.0%), fatigue in 26 patients (34.7%), a hand-foot syndrome in 16 patients (21.3%), diarrhea in 14 patients (18.7%), hoarseness in 10 patients (13.3%), and renal dysfunction in 7 patients (9.3%). Fifty-two (69.3%) of 75 patients developed AEs of Grade 3 or higher, and 21 (28.0%) of 75 patients discontinued axitinib because of AEs ().
SNPs were successfully genotyped in 75 patients. No significant association was observed between the genotypes of the three SNPs and TTF, PFS, and OS (). The distribution of SNPs in patients exhibiting CR + PR + stable disease (SD) vs progressive disease (PD) or CR + PR vs SD + PD was assessed. No associations were observed between the SNPs and the BOR (). There were also no significant associations between polymorphisms and axitinib discontinuation due to AEs or risk of AEs of Grade 3 or higher (). However, the incidences of hand-foot syndrome and renal dysfunction of Grade 2 or higher were significantly higher in patients carrying the ACE rs4362 Deletion allele than in those carrying other genotypes [odd’s ratio (OR), 4.19; 95% confidence interval (CI), 1.08–16.24; p = .045: OR, 4.17; 95% CI, 1.55–10.87; p = 0.005; respectively, ]. In addition, patients carrying the NOS3 rs1799983 G allele had a significantly higher risk of proteinuria and multiple AEs (OR, 9.17; 95% CI, 1.07–78.77; p = .025: OR, 7.65; 95% CI, 1.08–65.69; p = 0.036; respectively, )
Table 2. Association between outcome and pharmacokinetic-associated genetic polymorphisms.
Table 3. Distribution of SNP genotypes for the best response.
Table 4. Association between adverse events and vascular endothelial related genetic polymorphisms.
Discussion
The identification of biological markers for predicting the efficacy and safety of the increasing number of options available for the treatment of advanced kidney cancer is a highly desirable, but as yet unachieved, goal. Although many VEGF-targeting agents are available, definitive strategies for treatment, including the appropriate choice of agent, and the optimal initial dose based on an individual’s background, have not been developed.
In this study, we sought to identify drug-related SNPs that could predict clinical outcomes and AEs following axitinib treatment. Our analysis found that there was no significant relationship between SNPs in three drug-treatment-related genes (ACE, NOS3, and AT1R) and TTF, PFS, OS, and BOR. On the other hand, several AEs were significantly associated with the deletion allele in the ACE gene and the G allele in the NOS3 gene. These significant associations suggest that alternative administration schedules, dose modification, or alternative treatment approaches should be considered in patients carrying the alleles.
There are two main approaches that have been used to elucidate the role of genetic polymorphisms in the action of anti-tumor agents. The first one is to assess polymorphisms in genes that affect the pharmacokinetics of the drug of interest, such as in ATP-binding cassette (ABC) pumps, cytochromes P450 (CYP) family members, uridine diphosphate glucuronosyltransferase (UGT), and olfactory receptors.Citation13–15 The second one is to search for gene polymorphisms in genes that affect the pharmacodynamics of the drug, for instance in the case of VEGFR inhibitors polymorphisms in the VEGFR 1 to 3, platelet-derived growth factor receptor (PDGFR), c-receptor tyrosine kinase (c-KIT), and fms-like tyrosine kinase (FLT)-3 genes have been examined.Citation13,Citation15
In the first approach, pharmacokinetic-related SNPs could be candidates that predict the clinical outcome of axitinib treatment.Citation16 However, in our previous study, we found there to be no significant relationship between the SNPs in pharmacokinetic-related genes and clinical outcomes.Citation4 Another group has also reported a negative result with respect to SNPs and axitinib clinical outcomes.Citation17 These negative results might not be surprising because clinical outcomes and AEs are presumably affected by a number of factors including the patient’s health condition, disease status, drug concentration, and drug metabolites rather than simply pharmacokinetics. Yamamoto et al. showed the usefulness of a predictive model that used the area under the plasma concentration-time curve (AUC) of axitinib and its relationship with six SNPs from ABC, UGT, and OR.Citation3
The second approach, namely the pharmacodynamic approach, analyzes the susceptibility of the molecules targeted by anti-cancer drugs. Polymorphisms in well-known target genes have been investigated by several groups.Citation6,Citation16 The largest study of SNPs in genes involved in the pharmacodynamic action of axitinib, reported by Escudier et al., showed that the VEGF-A rs699947 AA genotype was related to longer OS.Citation7 VEGF-A rs699947 variants lead to high expression levels of VEGF-A and consequently higher plasma levels of VEGF-A.Citation18 However, a high level of VEGF-A protein is not associated with an optimal response in mRCC patients treated with sunitinib.Citation19 This contradictory finding might arise because of the lack of an appropriate validation study.
In this study, we focused on downstream molecules in the VEGFR pathway. The binding of VEGF to VEGFRs initiates the VEGFR signaling pathway which then modulates endothelial cell function. VEGFR inhibitors could encumber endothelial function through vascular rarefaction and the down-regulation of NO production.Citation8,Citation9 Accordingly, the efficacy of VEGFR inhibitors could be influenced by factors that regulate the renin-angiotensin-aldosterone (RAS) system and NO generation. It is known that the RAS system plays an important role in the vascular rarefaction seen in patients with chronic kidney disease.Citation20 Individual functional differences in both ACE and AT1R have been studied in various diseases.Citation10,Citation21 The most common functional SNPs in ACE are reported to be insertion/deletion. The DD genotype is associated with higher levels of circulating ACE than the ID and II genotypes and is significantly more frequent in patients with myocardial infarction. The ACE D allele has also been reported not only to be associated with a decline in kidney function but also with resistance to reno-protective therapies provided by ACE inhibitors and beta-blockers despite their expected protection of the glomeruli.Citation22 These findings also illustrate a potential mechanism behind the worse effect of axitinib in patients carrying the ACE D allele. Patients with this allele might suffer more from vasculopathyCitation23 leading to kidney dysfunction and hand-foot syndrome. NO is well known to be a vasodilation factor that plays a crucial role in the relaxation of vascular smooth muscle. In vascular endothelial cells, NO is synthesized from L-arginine by NOS. Among the three known NOS isozymes, NOS1 and NOS2 are not expressed in the vascular endothelium so NOS3 is the most important enzyme that regulates NO production. The Rs1799983 G894T polymorphism is a common variant in NOS3 resulting in the substitution of Glu with Asp at amino acid residue 298 in exon 7. This variant is thought to be associated with a coronary disease,Citation24 hypertension,Citation25 chronic kidney disease,Citation26 and metabolic syndrome.Citation27 In type 2 diabetic patients, the NOS3 T allele contributes to the deterioration in proteinuria from micro- to overt-one. Moreover, hypertensive patients with the NOS3 T allele have early vascular damage, such as increased carotid intima-media thickness, albuminuria, and left ventricular hypertrophy.Citation28 In patients with this NOS3 T allele polymorphism, there could be an enhancement of the multiple vascular damages caused by TKIs. For example, vatalanib, a TKI which mainly inhibits VEGFR, PDGFR, and c-KIT, inhibits the phosphorylation of NOS3 at Ser1177 reducing NO synthesis in an ex vivo mouse artery model.Citation29 This might lead to a synergistic effect between axitinib and NOS3 gene variants potentially explaining the increased level of AEs seen in mRCC patients treated with axitinib.
There are some potential limitations to this study. First, owing to its retrospective study design, the treatment schedule, dose modifications, and the timing of radiological assessments were not organized by a strict protocol. Second, the patients were included over 10 years, and supportive care and sequential therapies have changed year by year. This change may affect study results. Third, when taking into account the three SNPs analyzed and nine AEs, a p-value less than 0.006 was the threshold for significance according to Bonferroni correction for multi-testing. However, Holm’s correction assured the statistical significance of the ACE deletion allele for the deterioration of kidney function. Finally, the main limitation of this study is the lack of statistical power, which might lead to a type II error, owing to the small sample size. Further studies in larger cohorts are needed to validate these results.
In conclusion, our retrospective assessment of SNPs in Japanese mRCC patients suggested that the rs4362 deletion allele in ACE and the rs1799983 G allele in NOS3 may be associated with increased toxicity such as proteinuria, hand-foot syndrome, renal dysfunction, and multiple AEs. If this finding is validated in a larger-scale study, alternative treatment approaches based on the SNPs may be considered in the future.
Materials and methods
Patient population
This study included 75 mRCC patients who had been treated between December 2008 and October 2017 with first-, second-, or third-line, or later, axitinib (standard schedule: 5 mg orally twice daily). Treatments were administered without interruption between cycles unless disease progression or intolerable toxicities were observed. Dose reduction was allowed to 2 mg minimally, depending on the severity of the toxicities. Treatment was discontinued in patients who showed disease progression or severe toxicity. Each patient was classified according to the IMDC risk scoring system at the beginning of systemic therapy. The choice of first- or second-line systemic therapy was based on the PS, the extent of diseases, comorbidities, previous treatments, and individual preferences. Some patients underwent metastasectomies. The response to axitinib was evaluated according to Response Evaluation Criteria in Solid Tumor (RECIST) criteria version 1.1. The assessment interval for individual patients was scheduled by attending doctors. All AEs were graded according to the National Cancer Institute Common Toxicity Criteria (NCI-CTC) version 4.0 (Institute et al., 2011). We selected the nine most frequent AEs from medical records and analyzed the association between AEs of Grade 2 and higher and SNPs. Patients who had more than three AEs of Grade 2 or higher were considered to have multiple AEs. All AEs leading to dose reduction or termination of axitinib treatment were recorded. All patients gave informed consent for this study, and the ethics committee of the Akita University Graduate School of Medicine approved the study.
Genetic analysis
We examined three representative SNPs in three candidate genes involved in vascular rarefaction and NO down-regulation. The associations between these SNPs and clinical outcomes, including PFS, OS, TTF, BOR, and AEs, were evaluated. The SNPs analyzed were ACE rs4362, NOS3 r1799983, and AT1R rs776746. DNA was extracted from a peripheral blood sample using a QIAamp Blood kit (Qiagen, Hilden, Germany) and was stored at −20°C until analysis. Each SNP was genotyped using the PCR-restriction fragment length polymorphism method. Primer sequences and PCR conditions for the polymorphism analysis are reported elsewhere.Citation30–32
Statistical analysis
TTF was calculated as the time between the initiation of axitinib treatment and the stoppage of axitinib due to intolerance. PFS was defined as the time between the initiation of axitinib treatment and disease progression or death, as confirmed by radiological images or obvious clinical manifestations of PD. OS was defined as the time between axitinib initiation and death. The database was closed upon patient death or final follow-up. The data were expressed as the mean ± SD, and differences with a p-value less than 0.05 were considered statistically significant. The chi-square test was used to examine differences in categorical data. TTF, PFS, and OS were stratified using the Kaplan–Meier method and were tested with the log-rank test. The Cox proportional hazard regression model was used for the analysis of hazard ratios and 95% CI. The analysis was performed using SPSS version 24.0 statistical software (SPSS Japan Inc., Tokyo, Japan). To test the population homogeneity of the subjects, the genotype frequencies of each polymorphism were tested against the Hardy-Weinberg equilibrium using the chi-square test.
Authors’ contributions
KN designed the study, performed laboratory research, analyzed the data and wrote the manuscript. RI and MT analyzed the data and drafted the manuscript. TN, SK, MS, SN, and TI collected and described the clinical material. TN and MM performed laboratory research and critically reviewed the manuscript. TH reviewed the data and edited the manuscript.
Abbreviations
| TKI | = | tyrosine kinase inhibitor |
| mRCC | = | metastatic renal cell carcinoma |
| SNP | = | single nucleotide polymorphism |
| VEGFR | = | vascular endothelial growth factor receptor |
| OS | = | overall survival |
| AEs | = | adverse events |
| ACE | = | angiotensin-converting enzyme |
| AT1R | = | angiotensin II receptor type 1 |
| NOS | = | NO synthase |
| PS | = | performance status |
| IMDC | = | International Metastatic Renal Cell Carcinoma Database Consortium |
| TTF | = | time to treatment failure |
| PFS | = | progression-free survival |
| BOR | = | best objective response |
| CR | = | complete remission |
| PR | = | partial response |
| SD | = | stable disease |
| PD | = | progressive disease |
| OR | = | Odd’s ratio |
| CI | = | confidence interval |
| ABC | = | ATP-binding cassette |
| CYP | = | cytochromes P450 |
| UGT | = | uridine diphosphate glucuronosyltransferase |
| PDGFR | = | platelet-derived growth factor receptor |
| c-KIT | = | c-receptor tyrosine kinase |
| FLT | = | fms-like tyrosine kinase |
| AUC | = | area under the plasma concentration-time curve |
| RAS | = | renin-angiotensin-aldosterone |
Acknowledgments
We are grateful for the assistance of Ms. Yoko Mitobe in data collection.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Additional information
Funding
References
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