Int J Clin Exp Med 2019;12(12):13741-13748 www.ijcem.com /ISSN:1940-5901/IJCEM0076963 Original Article FREM2 is an independent predictor of poor survival in clear cell renal cell carcinoma-evidence from the cancer genome atlas (TCGA) Weiping Huang, Yongyong Lu, Xixi Huang, Feng Wang, Zhixian Yu Department of Urology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325035, Zhejiang Province, China Received February 26, 2018; Accepted October 7, 2018; Epub December 15, 2019; Published December 30, 2019 Abstract: Fraser syndrome protein 1 (FRAS1) and FRAS1 related extracellular matrix protein 1 and 2 (FREM1, FREM2) are a novel group of basement membrane proteins. The relationship between the three gene (FRAS1, FREM1, FREM2) and renal clear cell carcinoma is completely unclear. Thus, in this research, we used the mRNA sequencing data derived from TCGA kidney renal clear cell carcinoma cohort to assess the association of FRAS1, FREM1 and FREM2 with different clinical features. FRAS1, FREM1 and FREM2 mRNA levels were downregulated in KIRC (kidney renal clear cell carcinoma) tissues than normal tissues (FRAS1, P < 0.0001; FREM1, P < 0.0001, FREM2, P = 0.0001), respectively. FRAS1, FREM1 and FREM2 were significantly different in histologic grade, patho- logic stage and pathologic T (all P < 0.001). Low FRAS1, FREM1 and FREM2 expression were correlated to worsen overall survival (all P < 0.01), and Low FREM1 and FREM2 expression had worse relapse-free survival (FREM1, P = 0.0113; FREM2, P = 0.0424). Multivariate Cox regression analysis revealed that FREM2 was an independent prog- nostic factor for overall survival. Taken together, FREM2 expression is an independent predictor of poor survival in renal clear cell carcinoma and is positively associated with advanced stage, high histologic grade. Keywords: FREM2, FRAS1, renal cell carcinoma, KIRC, TCGA Introduction sion of RCC [6]. With the further study of the molecular mechanism of RCC, finding a diag- Renal cell carcinoma (RCC), the third most nostic and prognostic molecular biomarker or malignant tumors of urinary system, account- an effective treatment will be a vital future ing for 63990 newly diagnosed cancer cases direction. and 14400 cancer deaths in the United States in 2017 [1, 2]. In China, an estimated 66800 Fraser syndrome protein 1 (FRAS1) and FRAS1 patients newly diagnosed with RCC and 23400 related extracellular matrix protein 1 and 2 patients died of RCC have occurred in 2015 [3]. (FREM1, FREM2) are a novel group of base- Clear cell RCC (ccRCC), the most common RCC ment membrane proteins [7]. Many research- histological subtype, consist of 70%-80% renal es have revealed that FRAS1/FREM proteins cancer [1, 4]. Surgery treatment is the first were mutated in patients with Fraser syndrome choice for patients with early stage RCC [4]. As [8, 9]. The deficiency of FRAS1/FREM proteins for patients with late stage RCC orrecurrent or could lead to several diseases, such as renal metastatic renal carcinoma, no effective treat- agenesis, congenital diaphragmatic hernia [10- ment was accessible, so the 3 and 5-year can- 12]. Furthermore, FRAS1 could mediates both cer-specific survival rates still remain poor [5]. the initiation of the mammalian kidney and the The occurrence and development of RCC is a integrity of renal glomeruli [7]. However, there multifactor, multistep and multistage complex are few studies about the functions of FRAS1/ biological process. Amounts of genes were FREM proteins in different cancers. So, the re- involved in the complex biological process, con- lationship between the three gene (FRAS1, tributing to tumorigenesis and tumor progres- FREM1, FREM2) and renal clear cell carcinoma FREM2 is an independent predictor of OS in ccRCC Table 1. Characteristics of KIRC patients in and the data of clinicopathological characteris- TCGA database tics for patients with kidney cancer were do- Variables Case, n (%) wnloaded from UCSC Xena (http://xena.ucsc. edu/). Sample type Primary Tumor 533 (87.95%) FRAS1, FREM1 and FREM2 expression in Solid Tissue Normal 72 (11.88%) KIRC patients Additional-New Primary 1 (0.17%) Age at diagnosis Compared to normal controls, RSEM (RNA-Seq by Expectation-Maximization) expression val- ≤ 60 264 (49.53%) ues of FRAS1, FREM1 and FREM2 were used > 60 269 (50.47%) for statistical analysis. We further analyzed the Sex association of FRAS1, FREM1 and FREM2 with Female 188 (35.27%) different clinical features, including age at ini- Male 345 (64.73%) tial pathologic diagnosis, gender, size (longest Tumor size dimension), histologic grade, pathologic stage, < 2 cm 305 (57.22%) pathologic T, pathologic N, pathologic M. ≥ 2 cm 121 (22.7%) Prognosis analysis NA 107 (20.08%) Histologic grade Differences in overall survival (OS) and rela- G1 + G2 243 (45.59%) pse-free survival (RFS) between “high” and G3 + G4 282 (52.91%) “low” expression groups were compared using GX 5 (0.94%) Kaplan-Meier curves, with p-values calculated NA 3 (0.56%) via log-rank test. Univariate and multivariate Cox regression models were used to identify Pathologic stage the prognostic effects of clinical features and I + II 324 (60.79%) the three genes. III + IV 207 (38.84%) NA 2 (0.38%) Statistical analysis Pathologic T All data were analyzed by using SPSS 23.0 T1 + T2 342 (64.17%) (SPSS, Inc., Chicago, IL, USA). The Mann-Whit- T3 + T4 191 (35.83%) ney U test was applied to compare the expres- Pathologic N sion of the three genes in terms of differ- N0 240 (45.03%) ent clinicopathological characteristics includ- N1-2 16 (3%) ing age at initial pathologic diagnosis, gender, NX 277 (51.97%) size (longest dimension), histologic grade, Pathologic M pathologic stage, pathologic T, pathologic N, M0 422 (79.17%) pathologic M. RSEM (RNA-Seq by Expectation- M1 79 (14.82%) Maximization) expression values were used for MX 30 (5.63%) statistical analysis. Univariate logistic regres- NA 2 (0.38%) sion analysis of the three genes expression as a categorical dependent variable was used, with a median expression of the three genes, in is completely unclear. Thus, in this research, we association with clinicopathologic characteris- used the mRNA sequencing data derived from tics. Differences in overall survival between TCGA kidney renal clear cell carcinoma cohort “high” and “low” expression groups (defined by to assess the association of FRAS1, FREM1 median value of the three genes expression) and FREM2 with different clinical features. were compared using Kaplan-Meier curves, with p-values calculated via log-rank test. Uni- Materials and methods variate Cox regression analysis was used to Data of RNA-Seq expression and clinicopatho- estimate survival based on the three genes logical characteristics from TCGA database expression and clinicopathologic factors. Mul- tivariate Cox analysis was used to compare The mRNA sequencing data (combining level 3 the influence of the three genes expression data from Illumina GA and HiSeq platforms) on survival along with clinicopathologic charac- 13742 Int J Clin Exp Med 2019;12(12):13741-13748 FREM2 is an independent predictor of OS in ccRCC Figure 1. FRAS1, FREM1 and FREM2 levels in KIRC and normal controls. A. FRAS1 (P < 0.0001); B. FREM1 (P < 0.0001); C. FREM2 (P < 0.0001). Table 2. Association between FRAS1 expression and logical and the three gene expression clinical characteristics in KIRC patients data were analyzed in January 2018. FRAS1 The median age at diagnosis was 61 Variables Numbers U value P value expression years (range, 26-90 years). All of the Age at diagnosis patients were evaluated according to the system for TNM described in the ≤ 60 264 32342 9.49 ± 1.55 0.75 AJCC cancer staging manual. The medi- > 60 269 9.27 ± 1.67 an of overall survival (OS) was 39.23 Sex months (range, 0.06-151.23 months) Female 188 31091.5 9.47 ± 1.54 0.431 and the median of relapse-free survival Male 345 9.33 ± 1.65 (RFS) was 40.27 months (range, 0.1- Tumor size 151.23 months). The clinicopathologi- < 2 cm 305 17499 9.45 ± 1.62 0.405 cal characteristics of the TCGA data- ≥ 2 cm 121 9.34 ± 1.58 base are summarized in Table 1. Histologic grade FRAS1, FREM1 and FREM2 expression G1 + G2 243 29124.5 9.62 ± 1.46 0.003 and association with clinicopathologi- G3 + G4 282 9.22 ± 1.67 cal characteristics Pathologic stage I + II 324 25507.5 9.59 ± 1.57 < 0.001 A total of 533 KIRC samples with the III + IV 207 9.04 ± 1.62 three genes expression and clinicopa- thological data were analyzed from Pathologic T TCGA. Solid normal tissue samples T1 + T2 342 25595.5 9.56 ± 1.57 < 0.001 were excluded. As shown in Figure 1, T3 + T4 191 9.06 ± 1.64 FRAS1, FREM1 and FREM2 mRNA lev- Pathologic N els were downregulated in KIRC tis- N0 240 1068 9.34 ± 1.56 0.003 sues than normal tissues (FRAS1, P < N1-2 16 8.25 ± 1.90 0.0001; FREM1, P < 0.0001, FREM2, P Pathologic M = 0.0001), respectively. Further, the M0 422 14379 9.42 ± 1.57 0.052 associations between FRAS1, FREM1 M1 79 9.03 ± 1.78 and FREM2 expression and clinicopa- thological characteristics in KIRC pati- ents were explored. FRAS1 was signifi- teristics. A P-value of less than 0.05 was con- cantly different in histologic grade (P = 0.003), sidered statistically significant. pathologic stage (P < 0.001), pathologic T (P < 0.001), pathologic N (P = 0.003) (Table 2). Results FREM1 expression was found to be significantly Patient characteristics from TCGA database different in sex (P < 0.001), histologic grade (P < 0.001), pathologic stage (P < 0.001), patho- From the TCGA KIRC data, 533 primary tumors logic T (P < 0.001), pathologic M (P = 0.001) and 72 normal controls with both clinicopatho- (Table 3).