Genetic Variants in DNA Mismatch Repair Pathway predict prognosis of Lung Cancer patients with receiving Platinum-Based Chemotherapy

Objective: To investigate the relationships between genetic variants in DNA mismatch repair pathway genes and the prognosis of platinum-based chemotherapy in lung cancer patients. Methods: 346 lung cancer patients who received at least two cycles of platinum-based chemotherapy were recruited in this study. A total of 35 single nucleotide polymorphisms in 7 DNA mismatch repair genes were genotyped to investigate their associations with platinum-based chemotherapy prognosis. Result: The results revealed that patients carried MSH2 rs4608577 TT genotype had a significantly shorter progression free survival than patients with GG or GT genotypes (Additive model: P=0.003, OR =0.94, 95% CI =0.33-1.57). Patients with SAPCD1 rs707937 TT genotype had a significantly longer overall survival than patients with GG or GT genotypes (Additive model: P=0.0003, OR=0.75, 95% CI =0.35-1.14). Eight SNPs and fourteen SNPs were related to progression free survival and overall survival in subgroup analyses, respectively. Conclusion: Our findings suggest that the MSH2 rs4608577 and SAPCD1 rs707937 may be potential clinical biomarkers for predicting platinum-based chemotherapy prognosis in lung cancer patients.


Introduction
Lung cancer is the main causes of cancer-related mortality in the worldwide [1]. Two major pathologic types of lung cancer are non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), and NSCLC counted for about 85% of all cases [2,3]. Many new therapeutic agents such as gefitinib and erlotinib for target therapy were used for NSCLC patients, but it based on the genomic mutations of the patients. Thus, platinum-based chemotherapy regimens were still widely used for treatment of NSCLC in clinic [4][5][6][7]. However, the prognosis of the lung cancer patients is still dissatisfactory and obstacle the treatment [8,9]. The tumor microenvironment state and clinical stage are the traditional prognostic factors [10][11][12][13], and many factors including genetic polymorphism, histology type, clinical stage, gender, age, and so on have been reported to be associated with prognosis of platinum-based chemotherapy Ivyspring International Publisher [14][15][16][17][18][19][20]. It is important to discern some new biomarkers that can be used to predict the prognosis of platinum-based chemotherapy.
The DNA repair has four major repair pathways including base excision repair (BER), nucleotide excision repair (NER), double-strand break repair (DSBR) and DNA mismatch repair (MMR). DNA mismatch repair (MMR) is a very important repair system which actuates DNA damage response [21,22]. It can correct errors during DNA replication. MMR proteins have been collected together to form protein complexes that recognize and digest mismatch pieces of DNA and eventually fill in the mismatches [23][24][25][26][27]. Informally, MutLα plays a key role in the DNA mismatch repair system, and the endonuclease activity is related to DNA mismatch repair [28]. The complexes of MutLα and the MUTSα complex can guide an act on repairing the single-base and small-loop mismatches, and the MutSβ complex also guides the repair of small-to large-loop mismatches [29]. Therefore, MutSα and MutLα are important proteins in the MMR system and are used to cooperate the mismatch detection and repair events. In previous study, we found that MutSα and MutLα genes were associated with the toxicity of platinum-based chemotherapeutic in lung cancer [30,31].
Recent study showed that mutations in DNA repair genes are related to the prognosis of platinumbased chemotherapy in NSCLC patients [32,33]. MMR plays a key role in removing cisplatin-DNA adducts that the formation of platinum/DNA adducts blocks replication and transcription of DNA, and the mutations of MMR genes can decrease the response to platinum-based therapies and influence the prognosis through the highly conserved biological pathway [34][35][36][37]. The genetic polymorphisms of DNA mismatch repair genes were useful clinical biomarkers to predict chemotherapy response and prognosis in lung cancer patients [38].

Subjects and follow up
All patients were selected by the following inclusion criteria: (1) patients newly diagnosed with lung cancer by histological examination at the Affiliated Cancer Hospital or Xiangya Hospital of Central South University (Changsha, Hunan, China) between August 2009 and January 2013; (2) patients who received at least 2 cycles of platinum-based chemotherapy; (3) patients with no history of surgery before chemotherapy. All patients provided written informed consent before they participated in this study. The study performed in accordance with the principles of Declaration of Helsinki and was approved by the Ethics Committee of Xiangya School of Medicine, Central South University.
The terminate date for patient follow-up was July 15, 2019. Survival data were collected by telephone follow-up or residence registration. Overall survival (OS) time was defined as the period between the date diagnosed of lung cancer and the date of last follow-up or death. Progression-free survival (PFS) was calculated from the date diagnosed of lung cancer until the date of the first local recurrence or metastases in the last follow-up. Patients without progression were censored at the date of the last contact.

SNP selection, DNA extraction and genotyping
All of the common genetic variants in MLH1, MSH2, MSH3, MSH4, MSH5, SAPCD1 and MSH6 were selected by Haploview (Broad Institute, Cambridge, MA, USA) using pair-wise tagging with default settings (pairwise r 2 threshold = 0.8). SNPs with a minor allele frequency (MAF) ≥ 5% in the Han Chinese population were selected. Finally, Thirty-five SNPs were genotyped in the patients (Table 1). In our previous studies, the SNPs were investigated to be associated with platinum-based chemotherapy toxicity [30].
ALL blood samples were collected in the morning and stored in EDTA tube. Genomic DNA was isolated using a Genomic DNA Purification Kit (Promega, Madison, WI, USA) and stored at -20°C before use. Genotyping was analyzed using a Sequenom Mass ARRAY Genotyping Platform (Sequenom, San Diego, CA, USA) through polymerase chain reaction (PCR) system.

Statistical analysis
The covariates for logistic regression included sex, age, smoking status, histology and clinical stage. We applied three genetic models (dominant, additive, and recessive) to evaluate the associations between the single-nucleotide polymorphisms and prognosis of the patients. The cumulative PFS and OS were estimated by using the log-rank test or univariate COX regression. All analyses were performed using PLINK (version 1.9, http://pngu.mgh.harvard.edu/ purcell/plink/) and SPSS 18.0 software (SPSS Inc, Chicago, IL, USA). Odds ratios (OR) and their 95% confidence intervals (95% CI) were used to assess the associations between gene polymorphisms and prognosis. P<0.05 was considered statistically significant.

Demographic characteristics of patient characteristics
A total of 346 patients who received first-line platinum-based chemotherapy were collected for this study. As shown in Table 2, the median age was 52 years old (a range of 21 to 77 years old). Among them, 286 (82.7%) were males and 60 (17.3%) were females. There were 224 (64.7%) patients who had ever smoked, and the rest 122 (35.3%) patients never smoked. For histology, 234 (67.6%) patients were diagnosed with NSCLC, and 110 (28.9%) with NSCLC. 294 (85%) of the patients were in an advanced stage (stage III/IV/ED). The fundamental clinical information, outcomes of progression-free survival (PFS) and Overall survival (OS) of these lung cancer patients are summarized in Table 2.

Association of the MSH2 rs4608577 polymorphisms and PFS in lung cancer patients
In the univariate Cox regression analysis, we found that MSH2 rs4608577 was significantly associated with PFS of lung cancer patients received platinum-based chemotherapy.  Table 4). In additive analysis model, patients who carried the rs707937 GG genotype had a significantly longer OS than patients with CC genotype. In dominant analysis model, patients who carried rs707937 GG and GC genotypes had a significantly longer OS than patients with CC genotype. In recessive analysis model, patients who carried rs707937 GG genotype had a significantly longer OS than patients with CC and GC genotypes ( Figure 2).

Stratification analyses
Stratification analyses were used to investigate the associations of all 35 SNPs with PFS and OS in the subgroups stratified by age, smoking status, gender, histological type and stage. For PFS subgroup analyses, we found the SNPs were related to PFS of lung cancer patients received platinum-based chemotherapy as follows: MSH2 rs4608577 and FBXO11 rs2710163 in patients with age >55 years old; MSH3 rs6151914 in patients with age ≤55 years old; MSH2 rs4608577 and MSH6 rs3136329 in male patients; CLIC1 rs2293852 in patients with smoking; MSH2 rs4608577, MSH3 rs6151914, MSH4 rs3806162, CLIC1 rs2293852 and rs805304 in NSCLC patients; MSH3 rs1650665, rs26784 and rs6151670, and MLH1 rs1800734 in SCLC patients; MSH2 rs4608577, MSH6 rs3136329 and CLIC1 rs805304 in lung cancer patients with advanced or extensive stage (Table 5). For OS subgroup analyses, we found the SNPs were associated with OS of lung cancer patients received platinum-based chemotherapy in different subgroups as follows: MSH2 rs2302428, MSH3 rs6151914 and rs245346, MSH4 rs5745532 and SAPCD1 rs707937 in patients with ≤55 years old; MSH3 rs245346 in patients with >55 years old; MSH2 rs6544991, MSH3 rs245346 and rs6151892 and MSH6 rs2020910 in male patients; MSH3 rs6151892 and rs3816729, and MSH4 rs5745532 in patients with non-smoking; MSH3 rs26778, FBXO11 rs2710163 and SAPCD1 rs707937 in NSLCL patients; MSH2 rs2303428, MSH3 rs6151914, MSH5 rs707938 and rs31175772 in SCLC patients; MSH3 rs6151914, MSH4 rs3806162 and FBXO11 rs3732190 in patients with early or limited stage; MSH2 rs6544991 and rs13019654, and SAPCD1 rs707939 in patients with advanced or extensive stage ( Table 6).

Discussion
In this study, we emphasize to investigate whether polymorphisms of 7 MMR genes (MLH1, MSH2, MSH3, MSH4, MSH5, MSH6 and SAPCD1) were related to the prognosis of platinum-based chemotherapy in 346 lung cancer patients. Our results showed that MSH2 rs4608577 and SAPCD1 rs707937 were significantly related to the prognosis of lung cancer patients received platinum-based chemotherapy. Patients with GG genotype of MSH2 rs4608577 had better PFS, and patients with GG genotype of SAPCD1 rs707937 would have better OS.
In the subgroup analyses for PFS, MSH2 rs4608577, MSH3 rs1650665, rs26784, rs6151670, rs6151914; MSH4 rs3806162, MSH6 rs3136329, MLH1 rs1800734 were significantly associated with PFS in different subgroups. In the patients with older age (>55), we founded that the GG and GT genotype carriers in rs4608577 had significantly longer PFS when compared with TT genotype. For male patients, the GG and GT genotype also had significantly longer PFS. In adenocarcinoma subgroup, the hazard ratio for death was 1. 29  MST, median survival time; OR, odds ratio; CI, confidence interval; Additive model: comparison between minor allele subjects and major allele subjects. Dominant model: comparison between minor allele carriers and major homozygous subjects. Recessive model: comparison between major allele carriers and minor homozygous subjects. ** P < 0.01; *** P < 0.001.  In MMR system, the loss of functional MMR proteins such as hMutSα (MSH2-MSH6) and hMutLα (MLH1-PMS2) generate the tolerance with DNA alkylating agents. Apoptosis ensues directed by phosphorylation of p53 also related to these MMR repair gene functional in many cases [39]. The functions of DNA mismatch repair act on DNA replication fidelity, mutation avoidance and genome stability, all of which contribute to the viability of organs and cells. The association of DNA mismatch repair gene polymorphisms and platinum-based chemotherapy toxicity in NSCLC patients have been reported [40,41].
Gene variations in mutS homolog 2 (MSH2) have been founded to be related to tumor risk or survival in multiple kinds of tumors [42]. Polymorphism in ASCL1 target gene DDC is associated with clinical outcomes of small cell lung cancer patients [43,44]. Nonetheless, there is no report about the association between OS or PFS and polymorphisms in the platinum resistance related MSH2 and SAPCD1 in Chinese patients. In our research, carrying GG or GT genotype in MSH2 rs4608577 had better progressionfree survival in lung cancer patients. Therefore, molecular study was needed to elucidate the mechanism of rs4608577 in MSH2 that affects the MMR systems.
There are some limitations in this study. The samples were collected in the same region which also needed the multi-central study to overcome the analysis bias. Patients' sample size was not large enough to contribute to make results verification. The definite mechanism between MSH2 rs4608577, SAPCD1 rs707937 and the prognosis of platinumbased chemotherapy in lung cancer patients is still needed to be investigated. We hypothesized that rs4608577 and rs707937 were affect expression level of MSH2 and SAPCD1 respectively, and contributed to MMR activity.
In conclusion, our study showed that MSH2 rs4608577 was significantly related to progression-free survival and SAPCD1 rs707937 was significantly related to overall survival of platinum-based chemotherapy in lung cancer patients. MSH2 rs4608577 and SAPCD1 rs707937 perhaps could be biomarkers for predicting the prognosis in lung cancer patients with platinum-based chemotherapy.