Abstract
Background: Organic cation transporter 6 (OCT6) encoded by solute carrier family 22 member 16 (SLC22A16) is involved in regulating cellular sensitivity and resistance to platinum derivatives. SLC22A16 has functional genetic variants but the association between these variants and the effectiveness of antitumor drugs remains unexplored. Patients and Methods: This study retrospectively analyzed data from 160 patients with advanced non-small cell lung cancer treated with platinum-based combination chemotherapy for first-line chemotherapy between October 2010 and May 2018. We investigated the association between the genetic variant of SLC22A16 and clinical outcomes. Results: Patients with the rs714368 GG genotype had a shorter progression-free survival than those with AA or AG. Gene polymorphism was not associated with adverse effects. The predictive effect of rs714368 was confirmed in multivariate analysis using a Cox proportional hazards model. Conclusion: A genetic variant of SLC22A16 is a potential predictive biomarker for response to platinum-based chemotherapy for non-small cell lung cancer.
Treatment of non-small-cell lung cancer (NSCLC) has changed dramatically because of the evolution of kinase inhibitors and the development of immune checkpoint inhibitors. Although selected patients benefit tremendously from the efficacy of these precision medicines, cytotoxic chemotherapy is still important for use in combination with immunotherapy (1-3) or in patients for whom molecular or immunotherapy fails. Platinum derivatives (cisplatin and carboplatin) and third-generation anticancer drugs are key agents for treating NSCLC. Pemetrexed is more effective than other drugs for non-squamous NSCLC (4). Therefore, the combination of platinum derivatives and pemetrexed is standard in such cases. The efficacy of cytotoxic chemotherapy is limited, however, and the mechanisms underlying resistance to it remain unclear. Multiple factors have been found to be associated with cisplatin resistance (5). One of the important mechanisms is the reduction of the intracellular platinum concentration caused by reduction of influx or promotion of efflux.
We previously reported that organic cation transporter 6 (OCT6) is a mediator of platinum influx into cancer cells, and down-regulation of OCT6 is one mechanism of acquired platinum resistance in lung cancer (6), as it is with other platinum derivatives (7). Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation. Polymorphisms of solute carrier family 22 member 16 (SLC22A16) are reported to influence doxorubicin pharmacokinetics (8) and are associated with neutropenia in doxorubicin-based chemotherapy (9). However, it is unknown whether SLC22A16 polymorphisms affect the efficacy of platinum-based cytotoxic chemotherapy. The aim in this study was to clarify the association between SNPs of SLC22A16 and clinical outcomes of patients with NSCLC treated with platinum-based cytotoxic chemotherapy.
Patient characteristics (n=160).
Patients and Methods
Patients and chemotherapies. We retrospectively reviewed data from patients with advanced NSCLC who received platinum-based first-line chemotherapy at Nagoya City University Hospital with any of the following regimens: Cisplatin (75 mg/m2) or carboplatin [area under the curve of 6 mg/ml/min (AUC 6)] plus pemetrexed (500 mg/m2), with or without bevacizumab (15 mg/kg); carboplatin (AUC 6) plus paclitaxel (200 mg/m2) or nab-paclitaxel (100 mg/m2); cisplatin (80 mg/m2) or carboplatin (AUC 5) plus gemcitabine (1000 mg/m2); or cisplatin (80 mg/m2) plus docetaxel (60 mg/m2). Patients who underwent surgery or curative chemoradiotherapy were excluded. We obtained peripheral blood samples from the patients before treatment in order to analyze SNPs. We recorded patient characteristics, clinical outcomes according to Response Evaluation Criteria in Solid Tumors version 1.1 and adverse effects (myelosuppression, liver function and creatinine) according to National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0 using data supplied by the attending physicians from medical records. All procedures involving human participants were in accordance with the ethical standards of the institutional research committee of Nagoya city university (no. 70-00-0095) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. We obtained written informed consent from all patients, and all medical data were anonymized.
The genotypic and allelic frequencies of solute carrier family 22-member 16 single nucleotide polymorphisms (SNPs) determined in this study.
Association between solute carrier family 22 member 16 rs714368 single nucleotide polymorphism and response to chemotherapy.
Genomic DNA extraction and detection of drug transporter polymorphisms. Genomic DNA was extracted from the blood samples of 160 patients with NSCLC using QIAamp DNA Mini Kit (Qiagen; Hilden, Germany) according to the manufacturer's instructions. SLC22A16 SNPs were detected using the StepOnePlus Real-Time PCR System (Applied Biosystems; Foster City, CA, USA) and TaqMan SNP Genotyping Assays (rs714368, NM_033125.4:c.146A>G, C___2256675_10; and rs12210538, NM_033125.4:c.1226T>C, C__22271871_10). We chose these SNPs based on a previous report (10). All assays were purchased from Applied Biosystems and used in accordance with the manufacturer's instructions.
Statistical analysis. The response rate (RR) was defined as the sum of the complete response (CR) and partial response (PR) rates, and the disease control rate (DCR) was defined as the sum of CR, PR and stable disease (SD) rates. RR and DCR were compared using Fisher's exact test, with p<0.05 considered statistically significant.
Association between solute carrier family 22 member 16 rs714368 single nucleotide polymorphism and survival.
Progression-free survival (PFS) was defined from the date chemotherapy was first begun to the date of disease progression, death, or last follow-up. Overall survival (OS) was defined from the date chemotherapy was first begun to the date of death or last follow-up. These outcomes were analyzed according to SNP genotype using the Kaplan–Meier method and log-rank test, with p<0.05 considered statistically significant. Correlations of the frequency of adverse effects (myelosuppression, liver function or renal function) with gene polymorphisms were compared using Fisher's exact test. Finally, we performed multivariate analysis using a Cox proportional hazards model to examine the relationship of clinical outcomes and adverse effects with SNP genotype and clinical characteristics. In this model, we used p=0.10 in the log-rank test as the threshold for inclusion of a covariate in the model, with p<0.05 subsequently considered statistically significant. All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria), which is a modified version of R commander designed to add statistical functions frequently used in biostatistics (11).
Results
Data from a total of 160 patients were assessed in this study (Table I). The median age was 67 years, 117 were men, and 116 were current or former smokers. Most patients had disease stage IV (83.8%). The histological diagnosis was lung adenocarcinoma in the majority of patients (68.1%). Thirty-six patients had driver mutations: epidermal growth factor receptor (EGFR) mutation in 31 patients (15 with exon 19 deletion, 14 with L858R mutation and two with L861Q mutation) and anaplastic lymphoma kinase fusion gene in five patients. Programmed cell death ligand 1 expression was positive in 15 patients but not evaluated in most (87.5%).
The RR was 43.8%, the DCR was 82.6%, the median PFS was 124 days, and the median OS was 533 days. Two patients were excluded from assessment of treatment response as the treatment efficacy was not evaluated.
The genotypic and allelic frequencies targeted SNPs are shown in Table II. We excluded rs12210538 as a covariate as all patients in the study had the major homoallele, a finding that concurred with the results of the 1000 Genomes Project data in the East Asian population. Outcomes according to SNPs are shown in Tables III and IV. Carriers of the rs714368 A allele had a significantly longer median PFS than those with the GG genotype (140 vs. 93 days, p=0.022) (Figure 1). RR, DCR and OS did not significantly differ by SNP genotype (RR=44.8% vs. 41.7%, p>0.99; DCR, 83.6% vs. 83.3%, p>0.99; median OS, 530 vs. 533 days, p=0.85).
Progression-free survival according to the dominant model of the solute carrier family 22 member 16 rs714368 single nucleotide polymorphism. These data were analyzed by Kaplan–Meier methods and compared using the log-rank test.
We performed univariate analysis based on patient background to determine factors associated with clinical outcomes (Table V). In addition to carriers of the rs714368 A allele, patients with stage III disease had a longer PFS than those with stage IV (median=172.5 vs. 121 days, p=0.042), and those with history of smoking tended to have a longer PFS than never smokers (median=126 vs. 108 days, p=0.076). Multivariate analysis was conducted to assess predictive factors significantly associated with PFS. Stage, smoking history, and rs714368 genotypes were used as covariates in this model. Factors predictive of a poor PFS were stage IV versus III disease [hazard ratio (HR)=1.76, 95% confidence interval (CI)=1.117-2.771; p=0.015] and rs714368 GG genotype versus AA or AG (HR=1.945, 95% CI=1.212-3.122; p=0.006) (Table VI).
Univariate analysis of survival according to clinical characteristics.
There were no associations between gene polymorphisms and adverse effects (myelosuppression, liver function or renal function) (Table VII).
Discussion
Cytotoxic chemotherapy for NSCLC yields paradoxical tumor responses from patient to patient, even among those with similar tumor pathology. For this reason, it is important to find biomarkers predictive of treatment response so as to give more patients greater treatment benefits. In this study, we investigated the association between treatment outcomes in advanced NSCLC treated with platinum-based cytotoxic chemotherapy and SNPs of SLC22A16. A novel finding was that A carriers of the SLC22A16 rs714368 had a longer PFS compared with those with GG genotype.
Platinum derivatives are key drugs for treating lung cancer, particularly when used in combination with immune checkpoint inhibitors (1-3). Additional cytotoxic chemotherapy yields tumor volume reduction, which helps prevent early treatment failure of immune-cytotoxic combination therapy. Platinum-based chemotherapy combined with EGFR tyrosine kinase inhibitors may provide better efficacy than sequential monotherapy (12). Given the importance of cytotoxic chemotherapy in combination with newer agents to treat NSCLC, predictive biomarkers of response or resistance are needed.
Understanding the mechanisms of resistance to cytotoxic chemotherapy is a key issue. Tumor cells become resistant to cisplatin by multiple pathways, including reduction of intake, inactivation by glutathione (13), increased DNA repair (14, 15), and overexpression of drug-efflux pumps (16-18). One of the major factors determining the chemotherapeutic activity of platinum is drug influx (19), as that directly affects the intracellular drug concentration and cell damage the drug causes. The OCT family is associated with the transport of platinum drugs (20). OCT2, expressed at the basolateral membrane of renal tubular epithelial cell, transports cisplatin and is associated with nephrotoxicity (21). Certain OCT2 SNPs have been shown to be associated with this adverse effect of platinum-based chemotherapy for NSCLC (22). Down-regulation of OCT3 in hepatocellular carcinoma leads to diminished cisplatin sensitivity (23). We previously reported that OCT6 contributes to platinum influx into tumor cells. Cisplatin-resistant cell lines have reduced OCT6 expression and lower intracellular platinum accumulation compared with their respective parental cell lines. Up-regulation of OCT6 results in intracellular platinum accumulation and tumor sensitivity to cisplatin (6). OCT6 affects transport into tumor cells not only of cisplatin but also of other platinum derivatives (7). In addition, cisplatin-resistant cell lines were also less sensitive to carboplatin or other platinum derivatives than parental cell lines (24). In summary, OCT6 appears to affect cancer cell sensitivity to platinum derivatives, and its down-regulation is associated with tumor platinum resistance.
Multivariate analysis of progression-free survival.
The frequency of adverse event about myelosuppression, liver function and renal function.
The SLC22A family of transporters have a similar membrane topology: 12 α-helical transmembrane domains, a large extracellular loop between domains 1 and 2, and a large intracellular loop between domains 6 and 7 (25). Among SLC22A16 polymorphisms, rs714368 is located at the large extracellular loop and rs12210538 at domain 8. Most reports of SLC22A16 SNPs have assessed adverse effect of doxorubicin rather than treatment outcome. Some indicated that the rs714368 G allele increased exposure to doxorubicin (8) and gastrointestinal events (26). Other studies showed that the rs714368 A allele increased the incidence of having to delay doses of doxorubicin because of myelosuppression (27), apparently resulting from overexposure to doxorubicin in patients with A-bearing genotypes. These findings suggest rs714368 may have an important role in modulating doxorubicin pharmacokinetics. In addition, SLC22A16 may have an especially large adverse influence on hematopoiesis by accumulation of doxorubicin, as the gene's expression is restricted to hematopoietic cells (28) and testis (29) in normal adult tissues. The large difference in the expression of each organ may be the cause of seemingly contradictory associations between SNPs and adverse effects. While the G allele increases drug concentration in the blood, the A allele may increase doxorubicin influx into target tissues such as tumor cells and hematopoietic cells. On the other hand, our study showed no association between rs714368 polymorphisms and adverse effects (Table VII). Although doxorubicin is mainly metabolized in the liver, platinum derivatives are excreted from the kidneys. The differences of drug excretion route may affect the findings regarding adverse effects. As previously mentioned, OCT2 is mainly expressed in renal cells and is associated with nephrotoxicity, and SLC22A16 polymorphisms may have no influence on adverse effects. the functional implications of differences in SLC22A16 SNPs are still unclear. However, it is reported that other transporters introducing different SNPs affected drug sensitivity of tumor cell lines without changing the transporter expression level (30). Without a change in expression level, SLC22A16 rs714368 may influence not only pharmacokinetics but also transporter function and influx of drugs, resulting in varied clinical outcomes because of differences in intracellular platinum concentrations.
Platinum derivatives are usually used with third-generation cytotoxic drugs including pemetrexed, which is a key drug in non-squamous NSCLC treatment strategy. Using multiple biomarkers associated with each cytotoxic drug to predict combination chemotherapy outcomes would be a reasonable idea. It is known that treatment outcomes in pemetrexed containing chemotherapy are influenced by gene polymorphisms of folylpoly-γ-glutamate synthase, an enzyme that metabolizes pemetrexed (31). Tumor markers are also potential predictors of response to chemotherapy or kinase inhibitors (32, 33). Combining several biomarkers may therefore be more informative for predicting treatment response rather than using just one.
This study may have selection bias, as it was a retrospective, single-institution study. However, investigations of how SLC22A16 gene polymorphisms affect OCT6 function are important in furthering our understanding of tumor response and resistance.
In conclusion, we demonstrated that a SLC22A16 SNP is a potential biomarker predictive of the effect of platinum-based chemotherapy for advanced NSCLC. A paradigm shift in NSCLC treatment strategies is occurring with the use of cytotoxic chemotherapy combined with immune checkpoint inhibitors or tyrosine kinase inhibitors. Identification of acquired treatment-resistance mechanisms for each particular regimen is important in individualizing treatment for this cancer.
Acknowledgements
The Authors would like to thank Enago (www.enago.jp) for the English language review.
Footnotes
Authors' Contributions
AT and TO designed the study and wrote the initial draft of the article. AT, SF, KS, and YK analyzed gene polymorphisms. AT, TO, SF, KS, TU, OT and KM contributed to the analysis and interpretation of data and assisted in the preparation of the article. TO, SF, KM, KF, YK, TT, HO, MT, YI and AN contributed to data collection and interpretation. All authors reviewed the article and approved the definitive version of the article and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Conflicts of Interest
TO received honorarium by Chugai Pharmaceutical and research funding by Boehringer Ingelheim.
Funding
This study was funded by JSPS KAKENHI (grant number JP15K08593).
- Received May 13, 2020.
- Revision received July 3, 2020.
- Accepted July 6, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
