Int J Clin Exp Pathol 2015;8(7):8311-8335 www.ijcep.com /ISSN:1936-2625/IJCEP0010165 Original Article Papillary renal cell carcinoma: a clinicopathological and whole-genome exon sequencing study Kunpeng Liu1*, Yuan Ren1*, Lijuan Pang1, Yan Qi1, Wei Jia1, Lin Tao1, Zhengyan Hu1, Jin Zhao1, Haijun Zhang2, Li Li2, Haifeng Yue3, Juan Han3, Weihua Liang4, Jianming Hu1, Hong Zou1*, Xianglin Yuan3, Feng Li1* 1Department of Pathology, School of Medicine, Shihezi University, Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Ministry of Education of China; 2Department of Pathology, First Affiliated Hospital of Medical School, Shihezi University, China; 3Community hospital of Shihezi University, Shihezi City, Xin Jiang, China; 4Tongji Hospital Cancer Center, Tongji Medical College, Huazhong University of Science and Technology, China. *Equal contribu- tors. Received May 12, 2015; Accepted June 26, 2015; Epub July 1, 2015; Published July 15, 2015 Abstract: Papillary renal cell carcinoma (PRCC) represents the second most common histological subtype of RCC, and comprises 2 subtypes. Prognosis for type 1 PRCC is relatively good, whereas type 2 PRCC is associated with poor clinical outcomes. The aim of the present study was to evaluate the clinicopathological and mutations charac- teristics of PRCC. Hence, we reported on 13 cases of PRCC analyzed using whole-exome sequencing. Histologically, type 2 PRCC showed a higher nuclear grade and lymphovascular invasion rate versus type 1 PRCC (P < 0.05). Immunostaining revealed type 1 PRCC had higher CK7 and lower Top IIα expression rates (P < 0.05). Whole-exome sequencing data analysis revealed that the mutational statuses of 373 genes (287 missense, 69 silent, 6 nonsense, and 11 synonymous mutations) differed significantly between PRCC and normal renal tissues (P < 0.05). Functional enrichment analysis was used to classify the 287 missense-mutated genes into 11 biological process clusters (comprised of 61 biological processes) and 5 pathways, involved in cell adhesion, microtubule-based movement, the cell cycle, polysaccharide biosynthesis, muscle cell development and differentiation, cell death, and negative regulation. Associated pathways included the ATP-binding cassette transporter, extracellular matrix-receptor inter- action, lysosome, complement and coagulation cascades, and glyoxylate and dicarboxylate metabolism pathways. The missense mutation status of 19 genes differed significantly between the groups P( < 0.05), and alterations in the EEF1D, RFNG, GPR142, and RAB37 genes were located in different chromosomal regions in type 1 and 2 PRCC. These mutations may contribute to future studies on pathogenic mechanisms and targeted therapy of PRCC. Keywords: Papillary renal cell carcinoma, whole-exome sequencing, gene mutation Introduction divided into two subtypes, types 1 and 2 [2]. Type 1 PRCC is generally considered to have a Renal cell carcinoma (RCC) accounts for better prognosis than type 2 PRCC, although no approximately 90% of all renal malignancies. consensus regarding the standard treatment Papillary RCC (PRCC), the second most com- for metastatic PRCC exists [3-7]. mon RCC subtype, accounting for approximate- ly 10% of all cases, is a renal parenchyma Molecular genetic studies are highly important malignant tumor with papillary or tubulopapil- in diagnosis and prognosis evaluation, and may lary architecture that presents as type 1 or 2 provide treatment directions. MET locates at PRCC; type 1 PRCC is composed of single lay- 7q, and its mutation relates to susceptibility to ered small cell and scanty cytoplasm, type 2 PRCC [8]. Mutations of MET have been identi- PRCC is characterized by pseudostratified large fied to cause hereditary PRCC, and occur in a cells and eosinophilic cytoplasm is a renal small proportion of sporadic PRCC and a great- parenchyma malignant tumor with papillary or er number show somatic copy number gains tubulopapillary architecture [1]. Based on the involving chromosome 7q [8, 9]. In addition, cytologic and histologic features, PRCC can be leucine-rich repeat kinase 2 (LRRK2) is overex- PRCC pressed and amplified in PRCC. MET and samples by using a standard phenol/chloro- LRRK2 have a synergistic effect during tumor form extraction method. The quantity of DNA growth via the mTOR and STAT3 pathway [10]. was measured by reading A260/280 ratios by The exome BeadChip can not only identify gene spectrophotometer. When A260/280 ratios mutations, but also identify diagnostic and located range 1.8 to 2.0, DNA was available. therapeutic oncogenes and tumor suppressor Extractions were stored at -80°C until they genes. Although the pathologic and immuno- were labeled by nick translation. phenotypic of PRCC have been investigated, whole-genome exon sequencing reports are Whole-exome sequencing limited. Therefore, we here examined the clini- copathological and gene mutation characteris- A total of 1 μg of DNA from each of the 13 PRCC tics of PRCC by a combination of immunohisto- tissues and 18 normal kidney tissues were chemistry and exon chip analyses. labeled with Illumina reagents and hybridized to Human Exome BeadChips (Illumina, USA). Materials and methods The quality assessment was performed by Illumina Expression Console software. Com- Specimens pared with normal renal tissues, the mutative genes were identified the mutated genes by The study contained 13 paraffin-embedded significance analysis of microarrays (SAM) algo- PRCCs and 18 normal kidney tissues. 13 rithm in PRCC tissues. The mutative genes tumors consisted of 6 case of type 1 and 7 associated with cell cycle regulation and other case of type 2 PRCC. All tissues were obtained biological functions were determined by Gene from the archives of the Department of Ontology biological process (Gene Ontology BP) Pathology, School of Medicine, Shihezi enrichment of the classification analysis. The University. After asked for the view of the pathways associated with PRCC were con- patients and the Institutional Research Ethics firmed by the Kyoto Encyclopedia of Genes and Committee, we make a collection of the clinico- Genomes database (KEGG). pathological data for these cases in the patients’ medical records. All specimens were Statistical analysis observed by two independent pathologists. Nuclear grading was done according to the All statistical calculations were done using Fuhrman nuclear grade system. Tumor stages SPSS 17.0. Difference of measurement data were according to the 2010 TNM (T = Tumor, N was compared with single factor analysis of = Node, M = Metastases) classification of the variance. Count data were analyzed using American Joint Committee on Cancer. Fisher’s exact test. Classification enrichment of gene function and pathway were used to ana- Immunohistochemistry (IHC) lyze gene function (Gene Ontology of Biological Processes, Molecular function) by DAVID data- IHC staining was performed on 4 μm thick for- base and KEGG Database. P value < 0.05 was malin-fixed, paraffin-embedded tissue sections a difference in statistics. by the 2-step Envision technique (Dako, Denmark). The primary antibodies included Results cluster of differentiation (CD) 10 (GT200410, 1:100), cytokeratin (CK) (AE1/AE3, 1:100), Clinical features vimentin (Vim3B4, 1:100), CD117 (1:300), alpha-methylacyl-CoA racemase (AMACR), Top The clinical characteristics of type 1 and type 2 IIα, MDM2, p53, (13H4, 1:100), and CK7 (OV- PRCC are summarized in Table 1. In this cohort, TL12/30, 1:50), and purchased from Dako 7 patients were men and 6 were women (1.2 company. Negative or positive control was set male/female ratio); mean age was 53.9 (range up on the basis of antibodies. from 26 to 74); the average age of the patients was 61.5 (range from 48 to 74) with type 1 DNA extraction PRCC, 47.4 (range from 26 to 63) with type 2 PRCC. The male-to-female ratio, the mean age Total DNA was isolated from the 13 cases of of the patients, and metastasis were not signifi- PRCC and 18 cases of normal kidney tissue cantly different between the two groups. In the 8312 Int J Clin Exp Pathol 2015;8(7):8311-8335 PRCC Table 1. Distribution of analyzed clinicopathologic features and under a microscope. Type 2 outcome of type 1 and type 2 PRCC PRCC had higher nuclear grade P (P = 0.049) and Lymphova- Characteristics Type 1 Type 2 value scular invasion in relative to No. of patients 6 7 type 1 (P = 0.049). Age of patients Mean ± SD 61.5 ± 11.8 47.4 ± 13.7 0.075 Immunohistochemistry Range 48-74 26-63 Sex of patients Male 3 4 Results of immunohistochemi- Female 3 3 0.617 cal staining were summarized Metastasis Positive 0 1 0.538 in Table 2. All PRCC expressed Tumor size (cm) Mean ± SD 6.92 ± 3.06 7.27 ± 3.10 0.840 AMACR (Figure 1C), CK7 posi- tive expression rate of type 1 Range 3.5-11 3.9-13 PRCC (6/6) was higher in com- Fuhrman grade Low (1-2) 6 2 pared with type 2 (2/7) (P = High (3) 0 5 0.049 0.016). In contrast, Top IIα Lymphovascular invasion Negative 6 3 immunoreactivity was negative Positive 0 4 0.049 (0/6) in type 1 PRCC, while the stage I-II 6 4 majority of type 2 PRCC (4/7) III -IV 0 3 0.122 were positive for Top IIα (P = Outcome Dead 5 (5/6) 0.049). P value: type 1 PRCC vs. type 2 PRCC; Fisher’s exact test. Whole-exome sequencing 13 cases, 3 were asymptomatic, 6 were pre- In the whole-exome sequencing data analysis, sented with osphyalgia, and 4 were presented the mutational status of 373 genes was found with hematuria. Ultrasonic examination and to be significantly different P( < 0.05) between Computed tomography (CT) showed inhomoge- PRCC and normal renal tissues. In PRCC tis- neous mass, as the tumor mass often had sues, 287 missense, 69 silent, 6 nonsense, hemorrhage, necrosis, or cystic degeneration. and 11 synonymous mutations were detected Follow-up found the tumor related survival rate (Table 3).