Association of genetic polymorphisms with laryngeal carcinoma prognosis in a Chinese population

We analyzed the effects of single-nucleotide polymorphisms (SNPs) on laryngeal carcinoma (LC) risk and overall survival (OS) in 170 Chinese male LC patients followed for 10 years. After assessment of clinical characteristics (age, laryngectomy, neck dissection, tumor differentiation, TNM status), the patients were genotyped for 24 SNPs associated with risk in multiple cancers. LC risk was assessed using log-rank test and Cox proportional hazard models. The median OS time was 48 months. By the follow-up deadline, OS was 41.2%. Kaplan-Meier analysis indicated 1-, 3-, and 5-year survival rates to be 84.7%, 57.2%, and 47.1%, respectively. Five LC clinicopathological characteristics, namely total laryngectomy (TL), low differentiation (LD), T3-T4, N1-N2, and clinical stage III-IV were associated with worse OS (HR: 2.35, p < 0.001; HR: 2.39, p = 0.02; HR: 2.17, p < 0.001; HR: 2.39, p < 0.001; and HR: 3.29, p < 0.001, respectively). Univariate cox regression analysis indicated that four SNPs were associated (p < 0.05) with LC OS in the codominant genetic model compared to patients with the homozygous wild-type genotype: rs10088262 G/A (HR = 1.57), rs1665650 A/G (HR = 0.65); rs3802842 C/C (HR = 2.18), and rs59336 T/A and T/T (HR = 0.61 and 2.61, respectively).


INTRODUCTION
Laryngeal carcinoma (LC) is a common tumor of the head and neck. It accounts for 1% -5% of all malignant tumors, comprises 3.3% -8.1% of all the head and neck malignant tumors, and among these its incidence is only lower than that of nasopharyngeal carcinoma [1]. According to GLOBOCAN 2012 [2], 156,877 individuals were diagnosed with LC worldwide, while 20,014 cases were diagnosed in China. In this country, the age-adjusted incidence was 1.1 per 100,000, and the age-adjusted mortality rate was 0.7 per 100,000 for both sexes. LC generally affects people aged 50-70 years, especially males. In China, the ratio of men to women diagnosed with LC was 10.5:1 [2,3]. In recent years, the incidence of LC has increased steadily because of multiple carcinogenic factors [4,5], and in China is predicted to reach 55,900 new cases/year in the next 5 years. As with other cancers, the pathogenesis of laryngeal carcinoma involves the combined effects of environmental and genetic factors. To date, several genes have been implicated in the occurrence of LC and have been shown to affect its prognosis [6][7][8]. Recent studies have shown that single genes may be associated with many related cancers [9]. For example, the murine double minute 2 (MDM2) www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 6), pp: 10255-10263 Research Paper gene has been proposed to contribute to the emergence and development of many tumors, especially digestive carcinomas [10] and LC [1].
The association of single nucleotide polymorphisms (SNP), the most common form of genetic variation, with multiple cancers, has also been highlighted [11,12]. While extensive studies have evaluated the relevance of clinicopathological parameters such as surgical treatment modalities, age, tumor stage, differentiation, and lymph node metastasis, as prognostic factors in head and neck cancers [13], only a few basic studies have revealed an essential role of specific genes in digestive tract cancers. Moreover, the association between SNPs within or near these genes and LC prognosis has not been fully investigated. In the present study, we analyzed the association of 24 SNPs related to digestive tract cancers with LC prognosis in 170 Han Chinese male patients, and identified four SNPs significantly associated with OS. These results shed light on the genetic component of LC and may prove useful to guide further studies addressing its pathogenesis.

Overall survival analysis
At the median follow-up period of 38 months (range, 3 to 122 months), the mean and median survival times were 62.13 and 48 months, respectively. By the follow-up time deadline, OS was 41.2% (100 dead and 70 survivors). Kaplan-Meier statistical analysis indicated that 1-, 3-, and 5-year survival rates were 84.7%, 57.2% and 47.1%, respectively [14].

Multivariate Cox proportional hazards analysis
We performed multivariate Cox regression analysis by including the five clinical predictors that showed statistical significance in univariate analyses. Results indicated that two SNPs (rs10088262 and rs3802842) correlated significantly with OS in LC patients: compared with "GG" in rs10088262, the risk rate of genotype "G/A" was 1.969 (95% CI: 1.26-3.09, p = 0.003); on the other hand, the risk rate of rs3802842 (C/C vs. A/A) was 1.839 (95% CI: 1.10-3.08, p = 0.021) ( Table 4).

DISCUSSION
Our study analyzed, during a ten-year follow-up, the prognostic association of clinical parameters and multiple cancer-related SNPs in 170 male LC patients from northwest China. We found that five clinical characteristics [laryngectomy, tumor differentiation, tumor status (T), regional lymph node status (N), and clinical (TNM) stage] were correlated with survival. Specifically, clinical outcome was affected by laryngectomy variants, as TL had poorer prognosis than PL, with a median survival of 30 months, compared with 73 months for PL. This conclusion, however, may be confounded by the fact that TL is more often performed to remove larger malignant tumors, which carry higher risk. The associations found for other clinical features were consistent with the findings of previous studies, as low tumor differentiation, and higher T, N, and clinical stages all correlated with worse prognoses [15,16].
Of the 24 SNPs analyzed, four showed an association with LC prognosis in univariate analyses. After adjusting for significant clinical parameters, multivariate regression analysis revealed that two SNP genotypes, i.e. "G/A" of rs10088262 (HR: 1.969) and "C/C" of rs3802842 (HR: 1.839) were significantly associated with LC prognosis. The SNPs analyzed here have been shown to be associated with risk for diverse     A/B stands for minor/major alleles on the sample frequencies.
types of cancer. Rs10088262 is an intergenic variant located on chromosome 8q24.13; its minor allele "A" has been correlated with reduced risk of esophageal cancer [12]. However, in the present study the genotype "G/A" of this SNP indicated poor prognosis, with a median survival time of 32 months, compared with 66 months for the "G/G" genotype.
Carrying the "C" allele at rs3802842 has been associated with a lower risk for rectal tumors [17]. Another study, however, suggested that the "CC" genotype of rs3802842 may significantly increase colorectal cancer (CRC) risk in the recessive model [18]. Our analysis revealed that the median survival of genotype "C/C" of the rs3802842 polymorphism was only 26 months, significantly less than the homozygous wild genotype "A/A" (48 months). Rs3802842 maps to a region between "colorectal cancer associated 1" and "colorectal cancer associated 2" (C11orf92/COLCA1-C11orf93/COLCA2) genes, located on 11q23.1, which are arranged on opposite strands and share a regulatory region that contains genetic variants that are in high linkage disequilibrium with rs3802842. Expression levels of COLCA1 and COLCA2 transcripts correlate with rs3802842 genotypes. Genetic, expression and immunohistochemical data implicate COLCA1 and COLCA2 in the pathogenesis of colon cancer, whereas histologic analyses indicate the involvement of immune pathways [19].

Study population
170 male patients, aged 32-82 years (average age, 60.7 years), were enrolled at the First Affiliated Hospital of Xi'an Jiao Tong University from January 30, 2002 to April 7, 2003; patient follow-up ended on April 7, 2013. Patients received neither radiotherapy nor chemotherapy before enrollment, and in all cases LC diagnosis was confirmed by two pathologists. All participants were unrelated Han Chinese and had no other malignancy histories.

Patient demographics and blood collection
A standardized epidemiological questionnaire including residential region, age, smoking status, alcohol use, ethnicity, education status, and family history of cancer was used to collect personal information through in-person interviews. Related information was collected through a consultation with the treating physicians or from medical chart reviews. LC staging relies on the TNM system designed jointly by the Union International Cancer Control Version 7.0 (UICC 7.0). Venous blood samples (5 ml) and signed informed consent were obtained from each participant. All blood samples were quickly frozen in liquid nitrogen and stored at -80°C. This study was approved by the ethics committee of the First Affiliated Hospital of Xi'an Jiao Tong University.

SNP selection and genotyping
Using the HapMap database, 24 candidate SNPs with minor allele frequencies > 5% in the Asian population and previously published associations with other cancers were selected from chromosomes 8, 9, 10, 11, and 12. Basic information about the 24 SNPs is listed in Table 5. Genomic DNA was extracted from peripheral blood using phenol-chloroform, and its concentration was measured using a DU530 UV/VIS spectrophotometer (Beckman Instruments, Fullerton, CA, USA) according to the manufacturer's protocol. MassARRAY Assay Design 3.0 Software (Sequenom, San Diego, California, USA) was used to design Multiplex SNP MassEXTEND assays [20]. Genotyping was performed using the Sequenom MassARRAY RS1000 following a standard protocol recommended by the manufacturer [20], and data were analyzed using Sequenom Typer 4.0 Software (Sequenom, San Diego, CA, USA) [20,21].

Statistical analysis
Patients' baseline characteristics, disease stage, and treatment modalities were summarized using descriptive statistics. The overall survival (OS) time was defined as the period from diagnosis until death of any cause or until the date of the last follow-up, at which data point was censored. All summary statistics on time-to-event variables were estimated according to the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate Cox proportional hazards regression models were used to calculate the hazard ratios (HR), and 95% confidence intervals (95% CI) of the effect of clinical variables and SNPs, respectively, on the overall survival (OS) of LC patients. SPSS software (version 21.0) was used for statistical analysis. A p value < 0.05 was considered significant.

CONCLUSION
We found that five clinicopathological characteristics, namely total laryngectomy, low differentiation, T3-T4, N1-N2, and clinical stage III-IV, were associated with survival in LC patients. Although four SNP were found to be significantly associated with OS in univariate cox regression analysis, multivariate analysis showed that two SNPs (rs10088262 and rs3802842) were associated with LC prognosis after adjustment for clinical factors. Combined with previous research, our study suggests an association for these SNPs with multiple cancers. Further larger studies are required to validate our findings and to assess the molecular mechanisms underlying the observed associations.