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Chromophobe Renal Cell Carcinoma with and Without Sarcomatoid Change: a Clinicopathological, Comparative Genomic Hybridization, and Whole-Exome Sequencing Study

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Am J Transl Res 2015;7(11):2482-2499 www.ajtr.org /ISSN:1943-8141/AJTR0014993 Original Article Chromophobe renal cell carcinoma with and without sarcomatoid change: a clinicopathological, comparative genomic hybridization, and whole-exome sequencing study Yuan Ren1*, Kunpeng Liu1*, Xueling Kang3, Lijuan Pang1, Yan Qi1, Zhenyan Hu1, Wei Jia1, Haijun Zhang1, Li Li1, Jianming Hu1, Weihua Liang1, Jin Zhao1, Hong Zou1*, Xianglin Yuan2, Feng Li1* 1Department of Pathology, School of Medicine, First Affiliated Hospital, Shihezi University, Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Ministry of Education of China, Shihezi, China; 2Tongji Hospital Cancer Center, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 3Department of Pathology, Shanghai General Hospital, Shanghai, China. *Equal contributors. Received August 24, 2015; Accepted October 13, 2015; Epub November 15, 2015; Published November 30, 2015 Abstract: Chromophobe renal cell carcinomas (CRCC) with and without sarcomatoid change have different out- comes; however, fewstudies have compared their genetic profiles. Therefore, we identified the genomic alteration- sin CRCC common type (CRCC C) (n=8) and CRCC with sarcomatoid change (CRCC S) (n=4) using comparative genomic hybridization (CGH) and whole-exome sequencing. The CGH profiles showed that the CRCC C group had more chromosomal losses (72 vs. 18) but fewer chromosomal gains (23 vs. 57) than the CRCC S group. Losses of chromosomes 1p, 8p21-23, 10p16-20, 10p12-ter, 13p20-30, and 17p13 and gains of chromosomes 1q11, 1q21-23, 1p13-15, 2p23-24, and 3p21-ter differed between the groups. Whole-exome sequencing showed that the mutational status of 270 genes differed between CRCC (n=12) and normal renal tissues (n=18). In the functional enrichment analysis, the missense-mutated genes were classified into 6 biological processes (38 functions) and 5 pathways. The biological processes included cell adhesion, cell motility, ATP metabolism, sensory perception, carbo- hydrate and lipid metabolism and transport. The pathways included ATP-binding cassette transporter, extracellular matrix-receptor interaction, olfactory transduction, chondroitin sulfate biosynthesis, and hypertrophic cardiomyopa- thy. Whole-exome sequencing analysis revealed that the missense mutation statuses of 49 genes differed between the CRCC C and CRCC S groups. Furthermore, genetic alterations in metastasis suppressor 1, serine peptidase in- hibitor Kazal type 8, transient receptor potential cation channel super family M member 6, Rh family B glycoprotein, and mannose receptor C type 1 were located in different chromosomal regions. These alterations may provide clues regarding CRCC tumorigenesis and provide a basis for future targeted therapies. Keywords: Chromophobe renal cell carcinoma, chromophobe renal cell carcinoma with sarcomatoid change, com- parative genomic hybridization, whole-exome sequencing, genetic alteration Introduction being diagnosed at TNM stages III and IV, and with a median survival time of only 19 months Renal cell carcinoma (RCC) accounts for 2-3% [3-5]. Sarcomatoid RCCs account for about of all adult malignancies and is the seventh and 1-8% of all renal neoplasms; however, it is not a twelfth most common malignancy in men and distinct histological entity, as it arises from all women, respectively [1]. Chromophobe renal subtypes of RCC [6, 7]. Molecular genetic test- cell carcinoma (CRCC) is the third most com- ing is of great importance for the CRCC and mon histological subtype of RCC and carries CRCC S diagnosis and prognosis and may help a better prognosis than conventional RCC guide treatment. However, little is currently [2]. However, CRCC with sarcomatoid change known about genetic alterations in CRCC S, and (CRCC S) is associated with poor clinical out- few molecular genetics studies have compared comes, with most cases of sarcomatoid RCCs CRCC and CRCC S. Furthermore, until recently, A comparative genetic study of CRCC with and without sarcomatoid change whole-genome sequencing of CRCC has been standard phenol/chloroform extraction meth- little investigated. Therefore, we here identified od. DNA quality was checked on a 1% agarose and compared the genomic alterations in CRCC gel, and the amount of extracted DNA was mea- common type (CRCC C; 8 cases) and CRCC S (4 sured spectrophotometrically at 260 nm (impu- cases) using comparative genomic hybridiza- rity and the DNA to non-DNA ratio were also tion (CGH) and whole-exome sequencing. crosschecked at 280 nm). Extractions were stored at -80°C until nick translation labeling. Material and methods Comparative genomic hybridization (CGH) Ethics statement CGH was performed using a commercially avail- Ethical approval was obtained from Institutional able kit according to the manufacturer’s proto- Ethics Review Board (IERB), The First Affiliated col (Vysis Inc., Downers Grove, IL, USA). Briefly, Hospital, Shihezi University School of Medicine tumor DNA was labeled with fluorescein isothio- and all participants provided written informed cyanate (FITC), while the normal reference DNA consent for themselves. was labeled with cyanine 3 (Cy3). The hybridiza- tion mixture consisted of approximately 200 ng Specimens Spectrum Green-labeled test DNA and 200 ng Spectrum Red-labeled total genomic reference Paraffin-embedded CRCC (n=12) and normal DNA coprecipitated with 10 μg human Cot-1 renal tissues (n=18) were obtained from the DNA (Invitrogen, Carlsbad, CA, USA) and was archives of the Department of Pathology, dissolved in hybridization buffer before hybrid- School of Medicine, Shihezi University. The clin- ization to metaphase chromosomes. The probe icopathological data of these cases were col- mixtures were denatured at 73°C for 5 min and lected from the patients’ medical records after then competitively hybridized to the denatured obtaining patient consent and approval by normal metaphase chromosomes in a 37°C the Institutional Research Ethics Committee. humidified chamber for 3 days. After washing, CRCCs were classified according to the 2004 the chromosomes were counterstained with World Health Organization Classification of 4’6-diamidino-2-phenylindoledihydrochloride Renal Tumors, 2010 American Joint Committee (DAPI) II (Vysis Inc.) and embedded in an an- on Cancer guidelines, and International Union ti-fading agent to reduce photobleaching. To against Cancer tumor/lymph node metastasis/ provide a control in case of artifacts, normal distal metastasis (TNM) classification system. female DNA (labeled green) was used as a neg- Immunohistochemistry ative control. DNA extracted from the MPE600 breast cancer cell line was used as a positive Paraffin-embedded CRCC tissue sections we- control (labeled green). Threshold levels of re stained using the 2-step Envision method 1.25 and 0.8 were used to score gains and (Dako, Glostrup, Denmark). The following pri- losses, respectively. High-level amplification mary antibodies were used: cluster of differ- was indicated by a ratio greater than 1.5. All entiation (CD) 10 (GT200410, 1:100), cytokera- centromeres, chromosome p35-36, and het- tin (CK) AE1/3 (AE1/AE3, 1:100), vimentin erochromatic regions of chromosomes Y, 16, (Vim3B4, 1:100), CD117 (104D2, 1:300), 19, and 22 were excluded from further analysis P504S (13H4, 1:100), and CK7 (OV-TL12/30, because these regions can yield unreliable 1:50) (all from Dako). Briefly, thetissue sections hybridization owing to incompletely suppressed were incubated with primary antibodies acco- repetitive DNA sequences. rding to the manufacturer’s instructions. Im- munodetection was performed using 3,3’-dia- Whole-exome sequencing minobenzidine as the chromogenic horseradish peroxidase substrate, followed by hematoxylin DNA (1 μg) extracted from CRCC tissues (n=12) counterstaining. and normal renal tissues (n=18) were labeled with Illumina reagents and hybridized to Human DNA extraction Exome BeadChips (Illumina, San Diego, CA, USA). Illumina Expression Console software Total DNA was extracted from CRCC (n=12) and was used to perform the quality assessment. normal renal (n=18) tissue samples using a The significance analysis of microarrays algo- 2483 Am J Transl Res 2015;7(11):2482-2499 A comparative genetic study of CRCC with and without sarcomatoid change Table 1. Clinical characteristic of the 12 CRCC cases Case Age (y)/Sex Diagnosis Stage (tumor diameter, comment) Follow-up 1 52/M CRCC C PT2M0N0 stage 2 (7.5 cm, primary) Alive 2 36/M CRCC C PT1M0N0 stage 1 (6 cm, primary) Alive 3 56/F CRCC C pT1M0N0 stage 1 (5 cm, primary) Alive 4 53/M CRCC C pT3M0N0 stage 3 (20 cm, primary) Died 5 years after the operation 5 55/F CRCC C pT1M0N0 stage 1 (6.5 cm, primary) Alive 6 25/M CRCC C pT2M0N0 stage 2 (8 cm, primary) Alive 7 64/M CRCC C pT1M0N0 stage 1 (5.5 cm primary) Alive 8 60/M CRCC C pT2M0N0 stage 2 (8.5 cm, primary) Alive 9 39/M CRCC S pT3M0N1 stage 3 (15 cm, primary, 3/4 lymph nodes positive) Developed brain metastasis and died 1 month after the operation 10 52/F CRCC S pT3M0N1 stage 3 (15 cm, primary, 1/4 lymph nodes positive) Died 3 years after the operation 11 36/F CRCC S pT2M1N0 stage 4 (9.5 cm, primary, lung metastasis) Died 8 months after the operation 12 72/F CRCC S pT2M1N1 stage 4 (8.2 cm, primary, bone metastasis, 1/5 lymph nodes positive) Died 1 year after the operation Abbreviations: CRCC, chromophobe renal cell carcinoma; CRCC C, CRCC common type; CRCC S, CRCC with sarcomatoid change; F, female; M, male. Table 3. Chromosome aberrations in the 12 cases of CRCC
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  • Supplementary Table 1

    Supplementary Table 1

    Supplementary Table 1. 492 genes are unique to 0 h post-heat timepoint. The name, p-value, fold change, location and family of each gene are indicated. Genes were filtered for an absolute value log2 ration 1.5 and a significance value of p ≤ 0.05. Symbol p-value Log Gene Name Location Family Ratio ABCA13 1.87E-02 3.292 ATP-binding cassette, sub-family unknown transporter A (ABC1), member 13 ABCB1 1.93E-02 −1.819 ATP-binding cassette, sub-family Plasma transporter B (MDR/TAP), member 1 Membrane ABCC3 2.83E-02 2.016 ATP-binding cassette, sub-family Plasma transporter C (CFTR/MRP), member 3 Membrane ABHD6 7.79E-03 −2.717 abhydrolase domain containing 6 Cytoplasm enzyme ACAT1 4.10E-02 3.009 acetyl-CoA acetyltransferase 1 Cytoplasm enzyme ACBD4 2.66E-03 1.722 acyl-CoA binding domain unknown other containing 4 ACSL5 1.86E-02 −2.876 acyl-CoA synthetase long-chain Cytoplasm enzyme family member 5 ADAM23 3.33E-02 −3.008 ADAM metallopeptidase domain Plasma peptidase 23 Membrane ADAM29 5.58E-03 3.463 ADAM metallopeptidase domain Plasma peptidase 29 Membrane ADAMTS17 2.67E-04 3.051 ADAM metallopeptidase with Extracellular other thrombospondin type 1 motif, 17 Space ADCYAP1R1 1.20E-02 1.848 adenylate cyclase activating Plasma G-protein polypeptide 1 (pituitary) receptor Membrane coupled type I receptor ADH6 (includes 4.02E-02 −1.845 alcohol dehydrogenase 6 (class Cytoplasm enzyme EG:130) V) AHSA2 1.54E-04 −1.6 AHA1, activator of heat shock unknown other 90kDa protein ATPase homolog 2 (yeast) AK5 3.32E-02 1.658 adenylate kinase 5 Cytoplasm kinase AK7