GENES, CHROMOSOMES & CANCER 53:1018–1032 (2014)
Comprehensive Exploration of Novel Chimeric Transcripts in Clear Cell Renal Cell Carcinomas Using Whole Transcriptome Analysis Masahiro Gotoh,1† Hitoshi Ichikawa,2† Eri Arai,1 Suenori Chiku,3 Hiromi Sakamoto,2 Hiroyuki Fujimoto,4 Masaki Hiramoto,5 Takao Nammo,5 Kazuki Yasuda,5 Teruhiko Yoshida,2 and Yae Kanai1* 1
Division of Molecular Pathology,National Cancer Center Research Institute,Tokyo,Japan Division of Genetics,National Cancer Center Research Institute,Tokyo,Japan 3 Science Solutions Division,Mizuho Information and Research Institute,Inc.,Tokyo,Japan 4 Department of Urology,National Cancer Center Hospital,Tokyo,Japan 5 Department of Metabolic Disorder,Diabetes Research Center,National Center for Global Health and Medicine,Tokyo,Japan 2
The aim of this study was to clarify the participation of expression of chimeric transcripts in renal carcinogenesis. Whole transcriptome analysis (RNA sequencing) and exploration of candidate chimeric transcripts using the deFuse program were performed on 68 specimens of cancerous tissue (T) and 11 specimens of non-cancerous renal cortex tissue (N) obtained from 68 patients with clear cell renal cell carcinomas (RCCs) in an initial cohort. As positive controls, two RCCs associated with Xp11.2 translocation were analyzed. After verification by reverse transcription (RT)-PCR and Sanger sequencing, 26 novel chimeric transcripts were identified in 17 (25%) of the 68 clear cell RCCs. Genomic breakpoints were determined in five of the chimeric transcripts. Quantitative RT-PCR analysis revealed that the mRNA expression levels for the MMACHC, PTER, EPC2, ATXN7, FHIT, KIFAP3, CPEB1, MINPP1, TEX264, FAM107A, UPF3A, CDC16, MCCC1, CPSF3, and ASAP2 genes, being partner genes involved in the chimeric transcripts in the initial cohort, were significantly reduced in 26 T samples relative to the corresponding 26 N samples in the second cohort. Moreover, the mRNA expression levels for the above partner genes in T samples were significantly correlated with tumor aggressiveness and poorer patient outcome, indicating that reduced expression of these genes may participate in malignant progression of RCCs. As is the case when their levels of expression are reduced, these partner genes also may not fully function when involved in chimeric transcripts. These data suggest that generation of chimeric transcripts may participate in renal carcinogenesis by inducing C 2014 The Authors. Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc. V dysfunction of tumor-related genes.
INTRODUCTION
Clear cell renal cell carcinoma (RCC) is the most common histological subtype of adult kidney cancer (Ljungberg et al., 2011). In general, RCCs at an early stage are curable by nephrectomy. However, some RCCs relapse and metastasize to distant organs. Even though molecular targeting agents have been developed for treatment of RCCs, their effectiveness for relapsed or metastasized RCCs after nephrectomy is very limited. To improve prognostication and the effectiveness of targeting therapy in patients with RCCs, the molecular background of renal carcinogenesis should be further elucidated. We and other groups have revealed both genetic and epigenetic events during renal carcinogenesis (Arai and Kanai, 2010). Especially, recent developments in high-throughput sequence capture methods and next-generation sequencing technologies have made exome sequencing technically feasible.
Such whole exome analyses have revealed that renal carcinogenesis involves inactivation of histone-modifying genes such as SETD2 (Dalgliesh et al., 2010), KDM5C (Dalgliesh et al., 2010), UTX (van Haaften et al., 2009), and PBRM1 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is nonommercial and no modifications or adaptations are made. Additional Supporting Information may be found in the online version of this article. Supported by: the Program for Promotion of Fundamental Studies in Health Sciences (10-42 and 10-43) of the National Institute of Biomedical Innovation (NiBio), Japan and the National Cancer Center Research and Development Fund (23-A-1), Japan. † Masahiro Gotoh and Hitoshi Ichikawa contributed equally to this work. *Correspondence to: Yae Kanai; Division of Molecular Pathology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. E-mail: [email protected] Received 17 April 2014; Accepted 7 August 2014 DOI 10.1002/gcc.22211 Published online 18 September 2014 in Wiley Online Library (wileyonlinelibrary.com).
C 2014 The Authors. Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc. V
1019
WHOLE TRANSCRIPTOME ANALYSIS IN RCCs
TABLE 1. The Clinicopathological Parameters of Clear Cell Renal Carcinomas Belonging to the Initial and Second Cohorts Clinicopathological parameters Age (mean 6 SD) Sex Male Female Tumor diameter (cm, mean 6 SD) Macroscopic configurationc Type 1 Type 2 Type 3 Predominant histological gradesd,e G1 G2 G3 G4 Highest histological gradesf G1 G2 G3 G4 Vascular involvementg Negative Positive Predominant growth patterne Expansive Infiltrative Most aggressive growth patternf Expansive Infiltrative Tumor necrosis Negative Positive Renal pelvic invasion Negative Positive Distant metastasis Negative Positive Pathological TNM stageh Stage I Stage II Stage III Stage IV
Initial cohort (n 5 68)
Second cohort (n 5 26)
P
62.25 6 11.00
57.12 6 10.80
0.078a
49 19 5.55 6 3.21
17 9 5.86 6 2.84
0.616b
25 17 26
13 5 8
0.562b
36 21 9 2
12 8 4 2
0.696b
5 32 16 15
1 6 11 8
0.105b
38 30
13 13
0.649b
61 7
24 2
1.000b
43 25
21 5
0.139b
51 17
16 10
0.212b
61 7
23 3
1.000b
58 10
24 2
0.500b
33 4 19 12
13 3 8 2
0.531b
0.407a
a
Mann–Whitney U test. Fisher’s exact test. No significant differences of clinicopathologcial parameters were observed between two cohorts. c Macroscopic configuration was evaluated on the basis of previously described criteria (Arai et al., 2006). d All the tumors were graded on the basis of previously described criteria (Fuhrman et al., 1982). e If the tumor showed heterogeneity, findings in the predominant area were described. f If the tumor showed heterogeneity, the most aggressive features of the tumor were described. g The presence or absence of vascular involvement was examined microscopically on slides stained with hematoxylin-eosin and elastica van Gieson. h All the tumors were classified according to the pathological Tumor-Node-Metastasis classification (Sobin et al., 2009). b
(Varela et al., 2011). Moreover, it is well known that clear cell RCCs are characterized by inactivation of the VHL tumor suppressor gene encoding a component of the protein complex that possesses ubiquitin ligase E3 activity (Baldewijns et al., 2010). Frequent mutation of a further component of the ubiquitin-mediated proteolysis pathway gene, BAP1 (Guo et al., 2012), and VHL-associated
transcription elongation factor, TCEB1 (Sato et al., 2013), has also been demonstrated on the basis of exome analyses. However, only a limited number of reports have described next-generation sequencing-based whole transcriptome analysis (RNA sequencing) of RCCs, and the molecular background of renal carcinogenesis has not been fully elucidated. Genes, Chromosomes & Cancer DOI 10.1002/gcc
Sample
Genes, Chromosomes & Cancer DOI 10.1002/gcc
K2
K3
K4
K5
K6
K7
K8
K9
K10
K11
K12
K13
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
(A) Initial cohort Clear K1
Subtype
NM_001193329 NM_001904 NM_001005862 NM_015506 NM_015506 NM_015153 NM_001001484
C9orf3
CTNNB1
ERBB2
MMACHC
MMACHC
PHF3
PTER
ASAP1
UPF3A
TEX264
RP11–322M19.1
SEMA6A
RIC8A
AC010724.1
POLR2G
RP1–45C12.1
ATXN7
EPC2
PLOD2
NM_001247996
NM_023011
NM_001129884
NM_020796
NM_021932
NM_002696
NM_000333
NM_015630
NM_000935
NM_003777
NM_001005417
B4GALT2
DNAH11
NM_032208
ANTXR1
Symbol
RNA accession no.
50 -partner gene
29 (ch 8, 131072825)
5 (ch 13, 115052104)
3 (ch 3, 51708578)
(ch 10, 89005872)
18 (ch 5, 115803279)
ADCY8
CDC16
FAM107A
MINPP1
CAMK4
RP11–34P13.7
CPEB1
(ch 15, 83207631) 1 (ch 11, 211349)
CYP1A2
KIFAP3
FHIT
RP11–715D1.1
CCNYL1
RAPGEF5
MSRB2
PTP4A1
BX004987.7
BX004987.7
LTBP4
PLAG1
PRUNE2
ZSWIM5
GKN1
Symbol
2 (ch 11, 62529376)
(ch 1, 171137243)
5 (ch 3, 63938159)
2 (ch 2, 149447942)
1 (ch 3, 145878668)
79 (ch 7, 21934617)
5 (ch 10, 16547159)
1 (ch 6, 64356700)
2 (ch 1, 45973222)
1 (ch 1, 45966085)
15 (ch 17, 37872192)
1 (ch 3, 41241161)
12 (ch 9, 97767898)
3 (ch 1, 44450537)
17 (ch 2, 69420547)
Exon (chromosome, position)
Exon boundariesb
NM_001115
NM_001078645
NM_001076778
NM_001178118
NM_001744
NM_001079533
NM_000761
NM_001204514
NM_001166243
NM_001142300
NM_012294
NM_012228
NM_003463
NM_001042545
NM_002655
NM_015225
NR_024270
NM_019617
RNA accession no
30 -partner gene
10 (ch 8, 131862049)
15 (ch 13, 115027362)
1 (ch 3, 58594984)
2 (ch 10, 89268093)
11 (ch 5, 110825288)
(ch 1, 92240)
8 (ch 15, 83218408)
7 (ch 15, 75047132)
21 (ch 1, 169890922)
2 (ch 3, 61186339)
(ch 3, 108828611)
4 (ch 2, 208602135)
7 (ch 7, 22306631)
4 (ch 10, 23399171)
1 (ch 6, 64237724)
(ch 1, 143529235)
(ch 1, 143529235)
12 (ch 19, 41116438)
3 (ch 8, 57083748)
19 (ch 9, 79229516)
1 (ch 1, 45769599)
2 (ch 2, 69204627)
Exon (chromosome, position)
789224
24742
6886406
262221
4977991
10777
1246321
2751820
372014
6852012
118976
97556013
97563150
18538382
1319062
215920
Distance (bp)
TCTGTGATGCGTCCACAGCCAGGAGACACG| ATCGTCTTTGCTGGACTTCTTGGAGTCTTT CTTGGCCCGGTTGAAGGTGTCCTCACCATG| GGTTTCGGAGTAAGGGGAGCGGCCCGGCGA CGCAGGGAGAAGGAAGAGGTGTTTGAAAAG| TATTGACTTGAAGCTGAAAGAAAAGCCTTA CCCGCGGCGGGAGGAGCCTGTTCCCCTGAG| ATTGGCCAAAATGGGAAGGATTGGATTCCA ACACTGCCAACCGGCCAGAGGACGAGTGTG| ATGTGGACGAGTGCCACCGCGTGCCGCCGC CCTTTTGGCTTCGAGGTTTACCCCTTCCAG| GTTATATGCAGTACTGAAGAGCAACTTCAG GTGGCCTACCATCTGGGCCGTGTTAGAGAG| GTTATATGCAGTACTGAAGAGCAACTTCAG ATCCCAATTTCCAGATGCCTTGTTCAACAG| AGACTTGCTTCAACACCAGGAAGAACATGA CATGCCTGATGATAACAAAAGAATTAGAAG| CCTTTCAGTGGGATCTACCTGAATAACAAG TTATCCTTCTACTTATGGCCTAGCCCAGTG| GTGCAGAGAGAGCTAGCAGCTGTTATTGCT CTCGGAGAAGCCCTCGAGCATCCCCACAG| TGTGACCTTAGCAATATATTACCACATAAAG AACAGCCAAAACAGTTCATTCATATTCAGC| GAGAGAGCTGCCTGCAGAGAGCGTGAGTCC TCAAACCGCAGGCATTTCAATCACATTATG| CTTTGAAGCTCAGGAAAGAAGAGAAATCCA AGATGACAATTCATGTTGTTTTCAGGAAAG| CCTTGGAATGGATGGCTTTGGCCAACCAGT CACCGAGGTGGAGGGGACCTGCACAGGGAA| AGAGCTGTGGGAGGACCCCTCTGAGTTCCG AGAGAAAGGAGAGACAATTATGTTCCTGAG| GTGCAGGTGATCCCCTGGGTATTAGCCGAC CTCATCTTCCTAGAGAAGCGTTTGCACAAG| ACCAAACCAATGCAGACCAAACCAATGCAG TGTCTTCCCATAATCACCAAGACAAGAAGG| AGGTCCACCAGGGCAAAATAGAAGCCTTGC CCTCAGCCTCCAAAGTAGCTGAGACTACAG| ATATGGAGTTTGGACCTCCAACAGTTAATG ATCGCTGTCTACTATGACAACCCCCACATG| AAAAAGAAATGAAGGCCCAGACACGTTACG AGATGGAGGCGAAGACAAGAGAGCTCATTG| GTAACAGTTGACAAATGGGAACCTTTGTTG CCCGTACCACTGCCCAGAAAAATCAATACG| AGTGATGCCAATGACCATCCAGTTCTCCAT
Flanking sequencesc
TABLE 2. The 33 Chimeric Transcriptsa from the 61 Genes Verified by Reverse Transcription-PCR and Sanger Sequencing
2
2
2
2
2
2
1
1
2
2
2
2
2
2
2
2
2
1
2
2
2
1
In-frame transcripts
1020 GOTOH ET AL.
K14
K15
K16
K17
Clear
Clear
Clear
Clear
K96
RBM10
NM_001204468
NM_006521
NM_014226
RAGE
TFE3
NM_003631
PARG
NM_001039350
NM_001145408
NONO
DPP6
NM_001961
NM_007030
NM_014244
NM_016207
NM_020640
EEF2
TPPP
ADAMTS2
CPSF3
DCUN1D1
Symbol
17 (ch X, 47041725)
4 (ch X, 48895722)
1 (ch 7, 153584819)
1 (ch 14, 102771299)
8 (ch 10, 51093249)
10 (ch X, 70517788)
1 (ch 19, 3985376)
2 (ch 5, 677865)
2 (ch 5, 178770768)
7 (ch 2, 9576490)
(ch 3, 182679014)
Exon (chromosome, position)
TFE3
RBM10
ACTR3B
EML1
BMS1
TFE3
ENHO
TERT
RP11–798K23.4
ASAP2
MCCC1
Symbol
NM_006521
NM_001204468
NM_020445
NM_001008707
NM_014753
NM_006521
NM_198573
NM_198253
NM_001135191
NM_020166
RNA accession no
30 -partner gene
5 (ch X, 48895639)
18 (ch X, 47044454)
4 (ch 7, 152498706)
2 (ch 14, 100317190)
6 (ch 10, 43287075)
6 (ch X, 48891766)
2 (ch 9, 34521854)
3 (ch 5, 1282739)
(ch 5, 178930741)
23 (ch 2, 9533611)
18 (ch 3, 182735125)
Exon (chromosome, position)
1853914
1851268
1086113
2454109
7806174
21626022
604874
159973
42879
56111
Distance (bp)
TGGGAGAATCCACCGCCATCCGCCACCATG| GCTCAGGACTGCAGGTAGACATCTCCACTG GAAGGATTCAAGGGAACCTTCCCTGATGCG| CTGCCTGTGTCAGGGAATCTGCTTGATGTG GCACTCTGTCTGCCAAATATTTGCACCCAG| GGTGTCAAGCTGTTCTACCTTTCTGGAATG TGTCACCGGCTTCCGCATCCAAGATGAAGA| ATGACAGCGCTTCTGCTGCAAGTAGCATGG AAGACCGCTAAGATGCAGGGGAACGTGATG| GTATACCAAGCTTGGCTACGCAGGCAACAC CTCACCATCGGGTCCAGCTCAGAGAAGGAG| ATTGCCAAGGACATGGAACGCTGGGCCCG GAGAAGCACAAGACCAAGACAGCTCAACAG| ATTGATGATGTCATTGATGAGATCATCAGC
TTGCAAAGAATCCAGGACAAAAAGGATTAG| GTGTTTGTCAAAGCTGGAGACAAAGTGAAA GAAGGGCTCAGGAGCTGCTCTTGATTCTAG| ATCCCCTGACCCCCACGCCGCCCCCACCCG TCTGTGGCGCTCAGCAACTGCGATGGGCTG| ACCTCTGAATAAGTCGTGGGAGCCCTCGGG TGACGTGGACATCGTCTTCAGCAAGATCAA| GGGTTGGCTGTGTTCCGGCCGCAGAGCACC
Flanking sequencesc
1
1
1
1
1
1
2
2
2
1
2
In-frame transcripts
b
Chimeric transcripts (33) included the transcripts (MMACHC-BX004987.7 and TFE3-RBM10) consisting of the same partner gene sets with a different exon boundary or different transcriptional direction, and the transcripts sharing a partner gene, TFE3. National Center for Biotechnology Information Database (Genome Build 37). c The exon boundaries are indicated by a vertical bar. Clear, clear cell renal cell carcinoma; Xp11.2, renal cell carcinoma associated with Xp11.2 translocation; ch, chromosome; 1, positive; 2, negative.
a
Xp11.2
(B) Positive controls Xp11.2 K69
Sample
Subtype
RNA accession no.
50 -partner gene
Exon boundariesb
TABLE 2. (Continued)
WHOLE TRANSCRIPTOME ANALYSIS IN RCCs
1021
Genes, Chromosomes & Cancer DOI 10.1002/gcc
1022
GOTOH ET AL.
TABLE 3. Genomic Breakpoints of Chimeric Transcripts Genomic breakpointsa 50 -partner gene
Sample
Symbol
30 -partner gene
Chr
Genomic position
Symbol
Chr
Genomic position
K6
POLR2G
11
62530558
CYP1A2
15
75045983
K7
AC010724.1
15
83207075
CPEB1
15
83219352
K9
SEMA6A
5
115796806
CAMK4
5
110823275
K13
ASAP1
8
131070249
ADCY8
8
131862252
K15
CPSF3
2
9578689
ASAP2
2
9532071
Flanking sequencesb TAGTCTCTCGGAAGATCTGGGTTGGGTTCT| GAGAATTGCTTGAACTCTGGAGGTAGAGGC GAGATTATTGAAGTAGATCCTGACACTAAG| GAAATTGGCTCCTCTCTTGTAACTTCTGCC CGTAAGAAATTTGGTACATAAGCTGGTATT| TTAATCCAATTCATCCAAATTATTCTATCG GGCAGACAACGATGACGAGCTCACATTCAT| TGCAAAGTTTCTCAATAGAGAGAGTGCTCT ACCCTGTCACCCAGGCTGGAGTGTGGTGGC| ACAATCATGGCTCACTGCAGCCTCCAACTC
a
National Center for Biotechnology Information Database (Genome Build 37). The genomic breakpoints are indicated by a vertical bar.
b
Human hematologic (Shima and Kitabayashi, 2011) and soft tissue malignancies (Cantile et al., 2013), prostatic adenocarcinoma (Tomlins et al., 2005), and distinct subtypes of lung adenocarcinoma (Soda et al., 2007; Kohno et al., 2012; Takeuchi et al., 2012) show “addiction” for gene fusion events. Although their incidence is low, fusion events involving the transcription factor TFE3 gene have been reported in RCCs: RCC associated with Xp11.2 translocation, which harbors TFE3 fusion, is considered to represent a distinct subtype according to the World Health Organization (WHO) classification (Eble et al., 2004). Moreover, fusion events including anaplastic lymphoma kinase (ALK), such as TMP3ALK, EML4-ALK, and VCL-ALK fusion, have been reported in a distinct group of RCCs, including socalled “unclassified RCC” and papillary RCC in adults (Sugawara et al., 2012) and pediatric RCCs associated with the sickle cell trait (Debelenko et al., 2011; Mari~ no-Enrıquez et al., 2011), based on fluorescence in situ hybridization (FISH) and immunohistochemistry. These findings have prompted us to perform comprehensive exploration of chimeric transcripts in the most common subtype, clear cell RCC, using next-generation sequencing technology. In the present study, to clarify the participation of expression of chimeric transcripts in renal carcinogenesis, whole transcriptome analysis was performed using tissue specimens of 68 clear cell RCCs in adults. MATERIALS AND METHODS Patients and Tissue Samples
The initial cohort subjected to whole transcriptome analysis comprised 68 samples of cancerous Genes, Chromosomes & Cancer DOI 10.1002/gcc
tissue (T) and 11 samples of non-cancerous renal cortex tissue (N) obtained from materials that had been surgically resected from 68 patients with primary clear cell RCCs. There were 49 men and 19 women with a mean (6standard deviation) age of 62.3 6 11.0 years (range, 36 to 85 years). All patients underwent nephrectomy at the National Cancer Center Hospital, Tokyo, and had not received any preoperative treatment. Two expert pathologists specializing in genitourinary pathology, E.A. and Y.K., examined all histological slides and performed histological diagnosis in accordance with the WHO classification (Eble et al., 2004). All the tumors were graded on the basis of previously described criteria (Fuhrman et al., 1982) and classified according to the macroscopic configuration (Arai et al., 2006) and the pathological Tumor-Node-Metastasis (TNM) classification (Sobin et al., 2009). As a positive control for chimeric transcript detection, two T samples showing histological findings compatible with Xp11.2 translocation RCC based on the WHO criteria were also subjected to whole transcriptome analysis. For comparison, three T samples of papillary RCCs diagnosed in accordance with the WHO criteria were also subjected to whole transcriptome analysis. The second cohort subjected to quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis comprised 26 paired T and N samples obtained from materials that had been surgically resected from 26 other patients with primary clear cell RCCs. These patients comprised 17 men and nine women with a mean (6standard
Figure 1. Levels of mRNA expression for the partner genes involved in chimeric transcripts in 26 paired samples of tumorous tissue (T) and non-cancerous renal cortex tissue (N) in the second cohort. mRNA expression was analyzed using custom TaqMan Gene Expression Assays on the 7500 Fast Real-Time PCR System (Life Technologies) employing the relative standard curve method. The probes and PCR primer sets used are summarized in Supporting Information Table S6. Experiments were performed in triplicate for each sample-
primer set, and the mean value for the three experiments was used as the CT value. All CT values were normalized to that of GAPDH in the same sample. Levels of mRNA expression for the MMACHC, PTER, EPC2, ATXN7, FHIT, KIFAP3, CPEB1, MINPP1, TEX264, FAM107A, UPF3A, CDC16, MCCC1, CPSF3, and ASAP2 genes were significantly reduced in T samples (shaded column) relative to N samples (white column). Bar, standard deviation.
1024
GOTOH ET AL.
TABLE 4. Correlations Between Levels of mRNA Expression for Each of the Partner Genes Involved in Chimeric Transcripts in Tumorous Tissue Samples and Clinicopathological Parameters Reflecting Tumor Aggressiveness in the Second Cohort MMACHC Clinicopathological parameters
Number of tumors
PTER
EPC2
ATXN7
Expressiona
P
Expressiona
P
Expressiona
P
Expressiona
P
0.0528 6 0.0226 0.0250 6 0.0116 0.0384 6 0.0202
1:6131022 c
0.558 6 0.557 0.324 6 0.400 0.277 6 0.248
1.88 3 1021c
0.114 6 0.065 0.0546 6 0.0480 0.0784 6 0.0596
1.23 3 1021c
0.125 6 0.099 0.0598 6 0.0374 0.128 6 0.099
2.12 3 1021c
0.086 0.0443 6 0.0222 0.0489 6 0.0233 0.0285 6 0.0105
5:0531022c
0.574 0.530 6 0.608 0.498 6 0.479 0.232 6 0.309
1.15 3 1021c
0.246 0.112 6 0.083 0.106 6 0.033 0.0373 6 0.0185
4:6531023c
0.228 0.134 6 0.144 0.124 6 0.082 0.0696 6 0.0311
1.64 3 1021c
0.0533 6 0.0254 0.0327 6 0.0132
2:5631022g
0.571 6 0.433 0.282 6 0.452
8:6031023g
0.125 6 0.067 0.0586 6 0.0383
7:2431023g
0.139 6 0.100 0.0876 6 0.078
5.01 3 1022g
0.046 6 0.0239 0.0306 6 0.0077
2:0031021g
0.482 6 0.492 0.194 6 0.129
1.57 3 1021g
0.0954 6 0.0659 0.0762 6 0.0541
7.53 3 1021g
0.121 6 0.100 0.0836 6 0.035
7.05 3 1021g
0.0503 6 0.0242 0.0314 6 0.0134
1:6931022g
0.548 6 0.513 0.232 6 0.277
1:4431022g
0.120 6 0.064 0.0472 6 0.0274
7:1331024g
0.142 6 0.106 0.0672 6 0.0281
2:6831022g
0.0451 6 0.0230 0.0270 6 0.0030
1:3431021g
0.469 6 0.471 0.101 6 0.018
8:4631023g
0.0973 6 0.0649 0.0487 6 0.0218
1.57 3 1021g
0.120 6 0.095 0.0620 6 0.0265
2.11 3 1021g
0.0450 6 0.0221 0.0185 6 0.0007
2:4631022g
0.413 6 0.460 0.589 6 0.572
4.98 3 1021g
0.0974 6 0.0625 0.0235 6 0.0021
5.54 3 1022g
0.120 6 0.093 0.0365 6 0.0106
3:6931022g
0.0430 6 0.0166 0.0277 6 0.0155 0.0549 6 0.0287 0.0185 6 0.0007
5:5431022c
0.561 6 0.573 0.204 6 0.232 0.251 6 0.163 0.589 6 0.572
4.69 3 1021c
0.104 6 0.056 0.0607 6 0.0503 0.100 6 0.078 0.0235 6 0.0021
1.78 3 1021c
0.112 6 0.095 0.112 6 0.099 0.135 6 0.097 0.0365 6 0.0106
1.87 3 1021c
b
Macroscopic configuration Type 1 13 Type 2 5 Type 3 8 d,e Histological grades G1 1 G2 6 G3 11 G4 8 Vascular involvementf Negative 13 Positive 13 e Growth pattern Expansive 21 Infiltrative 5 Tumor necrosis Negative 16 Positive 10 Renal pelvic invasion Negative 23 Positive 3 Distant metastasis Negative 24 Positive 2 h Pathological TNM stage Stage I 13 Stage II 3 Stage III 8 Stage IV 2
FHIT Clinicopathological Number of parameters Tumors
KIFAP3
Expressiona
P
CPEB1
Expressiona
Expressiona
P
TEX264 P
Expressiona
P
0.155 6 0.111 0.142 6 0.104 0.143 6 0.086
9.22 3 1021c
b
Macroscopic configuration Type 1 13 Type 2 5 Type 3 8 d,e Histological grades G1 1 G2 6 G3 11 G4 8 Vascular involvementf Negative 13 Positive 13 e Growth pattern Expansive 21 Infiltrative 5 Tumor necrosis Negative 16 Positive 10 Renal pelvic invasion Negative 23 Positive 3 Distant metastasis Negative 24 Positive 2 h Pathological TNM stage Stage I 13 Stage II 3 Stage III 8 Stage IV 2
0.0884 6 0.0432 3:8231022c 0.0362 6 0.0222 0.0675 6 0.0345
0.280 6:6231023c 0.211 6 0.172 0.130 6 0.044 0.0631 6 0.0308
0.118 1.18 3 1021c 0.004 4:2331022c 0.0862 6 0.0535 0.00383 6 0.00722 0.0718 6 0.0259 0.00173 6 0.00168 0.0556 6 0.0481 0.0123 6 0.0119
0.178 6 0.126 1:2031022g 0.0898 6 0.0503
0.0839 6 0.0384 5.68 3 1022g 0.00492 6 0.00742 1.00g 0.0599 6 0.0421 0.00615 6 0.00978
0.155 6 0.112 0.143 6 0.088
6.50 3 1021g
0.150 6 0.109 4:0931022g 0.0660 6 0.0410
0.0752 6 0.0446 5.69 3 1021g 0.00433 6 0.00664 1.05 3 1021g 0.0580 6 0.0207 0.0106 6 0.0139
0.158 6 0.104 0.112 6 0.066
3.40 3 1021g
0.174 6 0.114 5:5531024g 0.0699 6 0.0316
0.0820 6 0.0381 3:0931022g 0.0558 6 0.0431
Number of Tumors
Macroscopic configurationb Type 1 13
0.00275 6 0.00449 4:0831022g 0.0100 6 0.0115
0.148 9.75 3 1022c 0.233 6 0.154 0.142 6 0.068 0.0955 6 0.0427
0.183 6 0.110 6:0531023g 0.0943 6 0.0395
0.143 6 0.107 1.34 3 1021g 0.0763 6 0.0420 6.38 3 1022g 0.00461 6 0.00639 5.94 3 1021g 0.156 6 0.103 3.12 3 1021g 0.0643 6 0.0397 0.0380 6 0.0044 0.0127 6 0.0193 0.0922 6 0.0222 0.141 6 0.106 5.54 3 1022g 0.0755 6 0.0409 3:6931022g 0.0520 6 0.0014 0.0285 6 0.0078 0.161 6 0.124 1.40 3 1021c 0.0893 6 0.0302 0.127 6 0.090 0.0520 6 0.0014
FAM107A Clinicopathological parameters
0.00369 6 0.00572 3.31 3 1021c 0.0058 6 0.0102 0.0084 6 0.0113
0.177 6 0.125 4:7331022c 0.0782 6 0.0187 0.100 6 0.069
0.00458 6 0.00790 5.54 3 1022g 0.156 6 0.100 5.54 3 1022g 0.0170 6 0.0099 0.0638 6 0.0021
0.0730 6 0.0375 7.75 3 1022c 0.00300 6 0.00478 1.15 3 1021c 0.153 6 0.111 2.26 3 1021c 0.0400 6 0.0271 0.00100 6 0.00100 0.195 6 0.116 0.0930 6 0.0446 0.0085 6 0.0117 0.147 6 0.083 0.0285 6 0.0078 0.0170 6 0.0099 0.0638 6 0.0021
CDC16
CPSF3
ASAP2
Expressiona
P
Expressiona
P
Expressiona
P
Expressiona
P
0.312 6 0.184
5.51 3 1022c
0.113 6 0.054
1.35 3 1021c
0.0476 6 0.0255
2.78 3 1021c
0.0455 6 0.0346
1.85 3 1021c
Genes, Chromosomes & Cancer DOI 10.1002/gcc
1025
WHOLE TRANSCRIPTOME ANALYSIS IN RCCs
TABLE 4. (Continued) FAM107A Clinicopathological parameters
Number of Tumors
Type 2 Type 3 Histological gradesd,e G1 G2 G3 G4 Vascular involvementf Negative Positive Growth patterne Expansive Infiltrative Tumor necrosis Negative Positive Renal pelvic invasion Negative Positive Distant metastasis Negative Positive Pathological TNM stageh Stage I Stage II Stage III Stage IV
Expressiona
CDC16 P
Expressiona
CPSF3 P
0.0584 6 0.0377 0.0926 6 0.0753
Expressiona
ASAP2 Expressiona
P
0.0260 6 0.0151 0.0465 6 0.0286
P
5 8
0.0986 6 0.0779 0.203 6 0.242
0.0218 6 0.0191 0.0250 6 0.0227
1 6 11 8
0.685 0.209 6 0.140 0.313 6 0.197 0.100 6 0.129
2:1431022c
0.172 0.113 6 0.083 0.116 6 0.049 0.0475 6 0.0206
1:2831022c
0.0613 0.0575 6 0.0335 0.0464 6 0.0236 0.0256 6 0.0118
1.30 3 1021c
0.112 0.0399 6 0.0320 0.0411 6 0.0252 0.0122 6 0.0068
1:9331022c
13 13
0.258 6 0.182 0.217 6 0.226
3.11 3 1021g
0.123 6 0.063 0.0696 6 0.0458
2:5631022g
0.0518 6 0.0271 0.0344 6 0.0214
1.13 3 1021g
0.0508 6 0.030 0.0185 6 0.0203
7:9531024g
21 5
0.255 6 0.211 0.166 6 0.159
4.47 3 1021g
0.0975 6 0.0608 0.0914 6 0.0662
8.01 3 1021g
0.0437 6 0.0263 0.0409 6 0.0246
9.00 3 1021g
0.0372 6 0.0316 0.0240 6 0.0211
4.09 3 1021g
16 10
0.317 6 0.205 0.110 6 0.117
2:2431023g
0.121 6 0.063 0.0567 6 0.0278
5:0231023g
0.0535 6 0.0266 0.0266 6 0.0115
1:4431022g
0.0475 6 0.0317 0.0142 6 0.0086
2:7731023g
23 3
0.259 6 0.205 0.0726 6 0.0602
7.85 3 1022g
0.100 6 0.063 0.0650 6 0.0128
3.52 3 1021g
0.0453 6 0.0263 0.0263 6 0.0073
3.95 3 1021g
0.0368 6 0.0313 0.0179 6 0.0058
4.42 3 1021g
24 2
0.256 6 0.199 0.0165 6 0.0030
5.54 3 1022g
0.102 6 0.059 0.0245 6 0.0007
2:4631022g
0.0457 6 0.0249 0.0125 6 0.0019
1:2331022g
0.0363 6 0.0307 0.0151 6 0.0005
3.94 3 1021g
13 3 8 2
0.246 6 0.152 0.209 6 0.167 0.290 6 0.284 0.0165 6 0.0030
2.72 3 1021c
0.109 6 0.053 0.0513 6 0.0307 0.110 6 0.071 0.0245 6 0.0007
4:5931022c
0.0445 6 0.0279 0.0324 6 0.0170 0.0526 6 0.0220 0.0125 6 0.0019
9.67 3 1022c
0.0404 6 0.0279 0.0103 6 0.0082 0.0394 6 0.0376 0.0151 6 0.0005
1.86 3 1021c
a
Average mRNA levels/GAPDH 6 standard deviation. Macroscopic configuration was evaluated on the basis of previously described criteria (Arai et al., 2006). c Kruskal–Wallis test. P values of < 0.05 are underlined. d All the tumors were graded on the basis of previously described criteria (Fuhrman et al., 1982). e If the tumor showed heterogeneity, the most aggressive features of the tumor were described. f The presence or absence of vascular involvement was examined microscopically on slides stained with hematoxylin-eosin and elastica van Gieson. g Mann–Whitney U test. P values of
Comprehensive Exploration of Novel Chimeric Transcripts in Clear Cell Renal Cell Carcinomas Using Whole Transcriptome Analysis Masahiro Gotoh,1† Hitoshi Ichikawa,2† Eri Arai,1 Suenori Chiku,3 Hiromi Sakamoto,2 Hiroyuki Fujimoto,4 Masaki Hiramoto,5 Takao Nammo,5 Kazuki Yasuda,5 Teruhiko Yoshida,2 and Yae Kanai1* 1
Division of Molecular Pathology,National Cancer Center Research Institute,Tokyo,Japan Division of Genetics,National Cancer Center Research Institute,Tokyo,Japan 3 Science Solutions Division,Mizuho Information and Research Institute,Inc.,Tokyo,Japan 4 Department of Urology,National Cancer Center Hospital,Tokyo,Japan 5 Department of Metabolic Disorder,Diabetes Research Center,National Center for Global Health and Medicine,Tokyo,Japan 2
The aim of this study was to clarify the participation of expression of chimeric transcripts in renal carcinogenesis. Whole transcriptome analysis (RNA sequencing) and exploration of candidate chimeric transcripts using the deFuse program were performed on 68 specimens of cancerous tissue (T) and 11 specimens of non-cancerous renal cortex tissue (N) obtained from 68 patients with clear cell renal cell carcinomas (RCCs) in an initial cohort. As positive controls, two RCCs associated with Xp11.2 translocation were analyzed. After verification by reverse transcription (RT)-PCR and Sanger sequencing, 26 novel chimeric transcripts were identified in 17 (25%) of the 68 clear cell RCCs. Genomic breakpoints were determined in five of the chimeric transcripts. Quantitative RT-PCR analysis revealed that the mRNA expression levels for the MMACHC, PTER, EPC2, ATXN7, FHIT, KIFAP3, CPEB1, MINPP1, TEX264, FAM107A, UPF3A, CDC16, MCCC1, CPSF3, and ASAP2 genes, being partner genes involved in the chimeric transcripts in the initial cohort, were significantly reduced in 26 T samples relative to the corresponding 26 N samples in the second cohort. Moreover, the mRNA expression levels for the above partner genes in T samples were significantly correlated with tumor aggressiveness and poorer patient outcome, indicating that reduced expression of these genes may participate in malignant progression of RCCs. As is the case when their levels of expression are reduced, these partner genes also may not fully function when involved in chimeric transcripts. These data suggest that generation of chimeric transcripts may participate in renal carcinogenesis by inducing C 2014 The Authors. Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc. V dysfunction of tumor-related genes.
INTRODUCTION
Clear cell renal cell carcinoma (RCC) is the most common histological subtype of adult kidney cancer (Ljungberg et al., 2011). In general, RCCs at an early stage are curable by nephrectomy. However, some RCCs relapse and metastasize to distant organs. Even though molecular targeting agents have been developed for treatment of RCCs, their effectiveness for relapsed or metastasized RCCs after nephrectomy is very limited. To improve prognostication and the effectiveness of targeting therapy in patients with RCCs, the molecular background of renal carcinogenesis should be further elucidated. We and other groups have revealed both genetic and epigenetic events during renal carcinogenesis (Arai and Kanai, 2010). Especially, recent developments in high-throughput sequence capture methods and next-generation sequencing technologies have made exome sequencing technically feasible.
Such whole exome analyses have revealed that renal carcinogenesis involves inactivation of histone-modifying genes such as SETD2 (Dalgliesh et al., 2010), KDM5C (Dalgliesh et al., 2010), UTX (van Haaften et al., 2009), and PBRM1 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is nonommercial and no modifications or adaptations are made. Additional Supporting Information may be found in the online version of this article. Supported by: the Program for Promotion of Fundamental Studies in Health Sciences (10-42 and 10-43) of the National Institute of Biomedical Innovation (NiBio), Japan and the National Cancer Center Research and Development Fund (23-A-1), Japan. † Masahiro Gotoh and Hitoshi Ichikawa contributed equally to this work. *Correspondence to: Yae Kanai; Division of Molecular Pathology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. E-mail: [email protected] Received 17 April 2014; Accepted 7 August 2014 DOI 10.1002/gcc.22211 Published online 18 September 2014 in Wiley Online Library (wileyonlinelibrary.com).
C 2014 The Authors. Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc. V
1019
WHOLE TRANSCRIPTOME ANALYSIS IN RCCs
TABLE 1. The Clinicopathological Parameters of Clear Cell Renal Carcinomas Belonging to the Initial and Second Cohorts Clinicopathological parameters Age (mean 6 SD) Sex Male Female Tumor diameter (cm, mean 6 SD) Macroscopic configurationc Type 1 Type 2 Type 3 Predominant histological gradesd,e G1 G2 G3 G4 Highest histological gradesf G1 G2 G3 G4 Vascular involvementg Negative Positive Predominant growth patterne Expansive Infiltrative Most aggressive growth patternf Expansive Infiltrative Tumor necrosis Negative Positive Renal pelvic invasion Negative Positive Distant metastasis Negative Positive Pathological TNM stageh Stage I Stage II Stage III Stage IV
Initial cohort (n 5 68)
Second cohort (n 5 26)
P
62.25 6 11.00
57.12 6 10.80
0.078a
49 19 5.55 6 3.21
17 9 5.86 6 2.84
0.616b
25 17 26
13 5 8
0.562b
36 21 9 2
12 8 4 2
0.696b
5 32 16 15
1 6 11 8
0.105b
38 30
13 13
0.649b
61 7
24 2
1.000b
43 25
21 5
0.139b
51 17
16 10
0.212b
61 7
23 3
1.000b
58 10
24 2
0.500b
33 4 19 12
13 3 8 2
0.531b
0.407a
a
Mann–Whitney U test. Fisher’s exact test. No significant differences of clinicopathologcial parameters were observed between two cohorts. c Macroscopic configuration was evaluated on the basis of previously described criteria (Arai et al., 2006). d All the tumors were graded on the basis of previously described criteria (Fuhrman et al., 1982). e If the tumor showed heterogeneity, findings in the predominant area were described. f If the tumor showed heterogeneity, the most aggressive features of the tumor were described. g The presence or absence of vascular involvement was examined microscopically on slides stained with hematoxylin-eosin and elastica van Gieson. h All the tumors were classified according to the pathological Tumor-Node-Metastasis classification (Sobin et al., 2009). b
(Varela et al., 2011). Moreover, it is well known that clear cell RCCs are characterized by inactivation of the VHL tumor suppressor gene encoding a component of the protein complex that possesses ubiquitin ligase E3 activity (Baldewijns et al., 2010). Frequent mutation of a further component of the ubiquitin-mediated proteolysis pathway gene, BAP1 (Guo et al., 2012), and VHL-associated
transcription elongation factor, TCEB1 (Sato et al., 2013), has also been demonstrated on the basis of exome analyses. However, only a limited number of reports have described next-generation sequencing-based whole transcriptome analysis (RNA sequencing) of RCCs, and the molecular background of renal carcinogenesis has not been fully elucidated. Genes, Chromosomes & Cancer DOI 10.1002/gcc
Sample
Genes, Chromosomes & Cancer DOI 10.1002/gcc
K2
K3
K4
K5
K6
K7
K8
K9
K10
K11
K12
K13
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
Clear
(A) Initial cohort Clear K1
Subtype
NM_001193329 NM_001904 NM_001005862 NM_015506 NM_015506 NM_015153 NM_001001484
C9orf3
CTNNB1
ERBB2
MMACHC
MMACHC
PHF3
PTER
ASAP1
UPF3A
TEX264
RP11–322M19.1
SEMA6A
RIC8A
AC010724.1
POLR2G
RP1–45C12.1
ATXN7
EPC2
PLOD2
NM_001247996
NM_023011
NM_001129884
NM_020796
NM_021932
NM_002696
NM_000333
NM_015630
NM_000935
NM_003777
NM_001005417
B4GALT2
DNAH11
NM_032208
ANTXR1
Symbol
RNA accession no.
50 -partner gene
29 (ch 8, 131072825)
5 (ch 13, 115052104)
3 (ch 3, 51708578)
(ch 10, 89005872)
18 (ch 5, 115803279)
ADCY8
CDC16
FAM107A
MINPP1
CAMK4
RP11–34P13.7
CPEB1
(ch 15, 83207631) 1 (ch 11, 211349)
CYP1A2
KIFAP3
FHIT
RP11–715D1.1
CCNYL1
RAPGEF5
MSRB2
PTP4A1
BX004987.7
BX004987.7
LTBP4
PLAG1
PRUNE2
ZSWIM5
GKN1
Symbol
2 (ch 11, 62529376)
(ch 1, 171137243)
5 (ch 3, 63938159)
2 (ch 2, 149447942)
1 (ch 3, 145878668)
79 (ch 7, 21934617)
5 (ch 10, 16547159)
1 (ch 6, 64356700)
2 (ch 1, 45973222)
1 (ch 1, 45966085)
15 (ch 17, 37872192)
1 (ch 3, 41241161)
12 (ch 9, 97767898)
3 (ch 1, 44450537)
17 (ch 2, 69420547)
Exon (chromosome, position)
Exon boundariesb
NM_001115
NM_001078645
NM_001076778
NM_001178118
NM_001744
NM_001079533
NM_000761
NM_001204514
NM_001166243
NM_001142300
NM_012294
NM_012228
NM_003463
NM_001042545
NM_002655
NM_015225
NR_024270
NM_019617
RNA accession no
30 -partner gene
10 (ch 8, 131862049)
15 (ch 13, 115027362)
1 (ch 3, 58594984)
2 (ch 10, 89268093)
11 (ch 5, 110825288)
(ch 1, 92240)
8 (ch 15, 83218408)
7 (ch 15, 75047132)
21 (ch 1, 169890922)
2 (ch 3, 61186339)
(ch 3, 108828611)
4 (ch 2, 208602135)
7 (ch 7, 22306631)
4 (ch 10, 23399171)
1 (ch 6, 64237724)
(ch 1, 143529235)
(ch 1, 143529235)
12 (ch 19, 41116438)
3 (ch 8, 57083748)
19 (ch 9, 79229516)
1 (ch 1, 45769599)
2 (ch 2, 69204627)
Exon (chromosome, position)
789224
24742
6886406
262221
4977991
10777
1246321
2751820
372014
6852012
118976
97556013
97563150
18538382
1319062
215920
Distance (bp)
TCTGTGATGCGTCCACAGCCAGGAGACACG| ATCGTCTTTGCTGGACTTCTTGGAGTCTTT CTTGGCCCGGTTGAAGGTGTCCTCACCATG| GGTTTCGGAGTAAGGGGAGCGGCCCGGCGA CGCAGGGAGAAGGAAGAGGTGTTTGAAAAG| TATTGACTTGAAGCTGAAAGAAAAGCCTTA CCCGCGGCGGGAGGAGCCTGTTCCCCTGAG| ATTGGCCAAAATGGGAAGGATTGGATTCCA ACACTGCCAACCGGCCAGAGGACGAGTGTG| ATGTGGACGAGTGCCACCGCGTGCCGCCGC CCTTTTGGCTTCGAGGTTTACCCCTTCCAG| GTTATATGCAGTACTGAAGAGCAACTTCAG GTGGCCTACCATCTGGGCCGTGTTAGAGAG| GTTATATGCAGTACTGAAGAGCAACTTCAG ATCCCAATTTCCAGATGCCTTGTTCAACAG| AGACTTGCTTCAACACCAGGAAGAACATGA CATGCCTGATGATAACAAAAGAATTAGAAG| CCTTTCAGTGGGATCTACCTGAATAACAAG TTATCCTTCTACTTATGGCCTAGCCCAGTG| GTGCAGAGAGAGCTAGCAGCTGTTATTGCT CTCGGAGAAGCCCTCGAGCATCCCCACAG| TGTGACCTTAGCAATATATTACCACATAAAG AACAGCCAAAACAGTTCATTCATATTCAGC| GAGAGAGCTGCCTGCAGAGAGCGTGAGTCC TCAAACCGCAGGCATTTCAATCACATTATG| CTTTGAAGCTCAGGAAAGAAGAGAAATCCA AGATGACAATTCATGTTGTTTTCAGGAAAG| CCTTGGAATGGATGGCTTTGGCCAACCAGT CACCGAGGTGGAGGGGACCTGCACAGGGAA| AGAGCTGTGGGAGGACCCCTCTGAGTTCCG AGAGAAAGGAGAGACAATTATGTTCCTGAG| GTGCAGGTGATCCCCTGGGTATTAGCCGAC CTCATCTTCCTAGAGAAGCGTTTGCACAAG| ACCAAACCAATGCAGACCAAACCAATGCAG TGTCTTCCCATAATCACCAAGACAAGAAGG| AGGTCCACCAGGGCAAAATAGAAGCCTTGC CCTCAGCCTCCAAAGTAGCTGAGACTACAG| ATATGGAGTTTGGACCTCCAACAGTTAATG ATCGCTGTCTACTATGACAACCCCCACATG| AAAAAGAAATGAAGGCCCAGACACGTTACG AGATGGAGGCGAAGACAAGAGAGCTCATTG| GTAACAGTTGACAAATGGGAACCTTTGTTG CCCGTACCACTGCCCAGAAAAATCAATACG| AGTGATGCCAATGACCATCCAGTTCTCCAT
Flanking sequencesc
TABLE 2. The 33 Chimeric Transcriptsa from the 61 Genes Verified by Reverse Transcription-PCR and Sanger Sequencing
2
2
2
2
2
2
1
1
2
2
2
2
2
2
2
2
2
1
2
2
2
1
In-frame transcripts
1020 GOTOH ET AL.
K14
K15
K16
K17
Clear
Clear
Clear
Clear
K96
RBM10
NM_001204468
NM_006521
NM_014226
RAGE
TFE3
NM_003631
PARG
NM_001039350
NM_001145408
NONO
DPP6
NM_001961
NM_007030
NM_014244
NM_016207
NM_020640
EEF2
TPPP
ADAMTS2
CPSF3
DCUN1D1
Symbol
17 (ch X, 47041725)
4 (ch X, 48895722)
1 (ch 7, 153584819)
1 (ch 14, 102771299)
8 (ch 10, 51093249)
10 (ch X, 70517788)
1 (ch 19, 3985376)
2 (ch 5, 677865)
2 (ch 5, 178770768)
7 (ch 2, 9576490)
(ch 3, 182679014)
Exon (chromosome, position)
TFE3
RBM10
ACTR3B
EML1
BMS1
TFE3
ENHO
TERT
RP11–798K23.4
ASAP2
MCCC1
Symbol
NM_006521
NM_001204468
NM_020445
NM_001008707
NM_014753
NM_006521
NM_198573
NM_198253
NM_001135191
NM_020166
RNA accession no
30 -partner gene
5 (ch X, 48895639)
18 (ch X, 47044454)
4 (ch 7, 152498706)
2 (ch 14, 100317190)
6 (ch 10, 43287075)
6 (ch X, 48891766)
2 (ch 9, 34521854)
3 (ch 5, 1282739)
(ch 5, 178930741)
23 (ch 2, 9533611)
18 (ch 3, 182735125)
Exon (chromosome, position)
1853914
1851268
1086113
2454109
7806174
21626022
604874
159973
42879
56111
Distance (bp)
TGGGAGAATCCACCGCCATCCGCCACCATG| GCTCAGGACTGCAGGTAGACATCTCCACTG GAAGGATTCAAGGGAACCTTCCCTGATGCG| CTGCCTGTGTCAGGGAATCTGCTTGATGTG GCACTCTGTCTGCCAAATATTTGCACCCAG| GGTGTCAAGCTGTTCTACCTTTCTGGAATG TGTCACCGGCTTCCGCATCCAAGATGAAGA| ATGACAGCGCTTCTGCTGCAAGTAGCATGG AAGACCGCTAAGATGCAGGGGAACGTGATG| GTATACCAAGCTTGGCTACGCAGGCAACAC CTCACCATCGGGTCCAGCTCAGAGAAGGAG| ATTGCCAAGGACATGGAACGCTGGGCCCG GAGAAGCACAAGACCAAGACAGCTCAACAG| ATTGATGATGTCATTGATGAGATCATCAGC
TTGCAAAGAATCCAGGACAAAAAGGATTAG| GTGTTTGTCAAAGCTGGAGACAAAGTGAAA GAAGGGCTCAGGAGCTGCTCTTGATTCTAG| ATCCCCTGACCCCCACGCCGCCCCCACCCG TCTGTGGCGCTCAGCAACTGCGATGGGCTG| ACCTCTGAATAAGTCGTGGGAGCCCTCGGG TGACGTGGACATCGTCTTCAGCAAGATCAA| GGGTTGGCTGTGTTCCGGCCGCAGAGCACC
Flanking sequencesc
1
1
1
1
1
1
2
2
2
1
2
In-frame transcripts
b
Chimeric transcripts (33) included the transcripts (MMACHC-BX004987.7 and TFE3-RBM10) consisting of the same partner gene sets with a different exon boundary or different transcriptional direction, and the transcripts sharing a partner gene, TFE3. National Center for Biotechnology Information Database (Genome Build 37). c The exon boundaries are indicated by a vertical bar. Clear, clear cell renal cell carcinoma; Xp11.2, renal cell carcinoma associated with Xp11.2 translocation; ch, chromosome; 1, positive; 2, negative.
a
Xp11.2
(B) Positive controls Xp11.2 K69
Sample
Subtype
RNA accession no.
50 -partner gene
Exon boundariesb
TABLE 2. (Continued)
WHOLE TRANSCRIPTOME ANALYSIS IN RCCs
1021
Genes, Chromosomes & Cancer DOI 10.1002/gcc
1022
GOTOH ET AL.
TABLE 3. Genomic Breakpoints of Chimeric Transcripts Genomic breakpointsa 50 -partner gene
Sample
Symbol
30 -partner gene
Chr
Genomic position
Symbol
Chr
Genomic position
K6
POLR2G
11
62530558
CYP1A2
15
75045983
K7
AC010724.1
15
83207075
CPEB1
15
83219352
K9
SEMA6A
5
115796806
CAMK4
5
110823275
K13
ASAP1
8
131070249
ADCY8
8
131862252
K15
CPSF3
2
9578689
ASAP2
2
9532071
Flanking sequencesb TAGTCTCTCGGAAGATCTGGGTTGGGTTCT| GAGAATTGCTTGAACTCTGGAGGTAGAGGC GAGATTATTGAAGTAGATCCTGACACTAAG| GAAATTGGCTCCTCTCTTGTAACTTCTGCC CGTAAGAAATTTGGTACATAAGCTGGTATT| TTAATCCAATTCATCCAAATTATTCTATCG GGCAGACAACGATGACGAGCTCACATTCAT| TGCAAAGTTTCTCAATAGAGAGAGTGCTCT ACCCTGTCACCCAGGCTGGAGTGTGGTGGC| ACAATCATGGCTCACTGCAGCCTCCAACTC
a
National Center for Biotechnology Information Database (Genome Build 37). The genomic breakpoints are indicated by a vertical bar.
b
Human hematologic (Shima and Kitabayashi, 2011) and soft tissue malignancies (Cantile et al., 2013), prostatic adenocarcinoma (Tomlins et al., 2005), and distinct subtypes of lung adenocarcinoma (Soda et al., 2007; Kohno et al., 2012; Takeuchi et al., 2012) show “addiction” for gene fusion events. Although their incidence is low, fusion events involving the transcription factor TFE3 gene have been reported in RCCs: RCC associated with Xp11.2 translocation, which harbors TFE3 fusion, is considered to represent a distinct subtype according to the World Health Organization (WHO) classification (Eble et al., 2004). Moreover, fusion events including anaplastic lymphoma kinase (ALK), such as TMP3ALK, EML4-ALK, and VCL-ALK fusion, have been reported in a distinct group of RCCs, including socalled “unclassified RCC” and papillary RCC in adults (Sugawara et al., 2012) and pediatric RCCs associated with the sickle cell trait (Debelenko et al., 2011; Mari~ no-Enrıquez et al., 2011), based on fluorescence in situ hybridization (FISH) and immunohistochemistry. These findings have prompted us to perform comprehensive exploration of chimeric transcripts in the most common subtype, clear cell RCC, using next-generation sequencing technology. In the present study, to clarify the participation of expression of chimeric transcripts in renal carcinogenesis, whole transcriptome analysis was performed using tissue specimens of 68 clear cell RCCs in adults. MATERIALS AND METHODS Patients and Tissue Samples
The initial cohort subjected to whole transcriptome analysis comprised 68 samples of cancerous Genes, Chromosomes & Cancer DOI 10.1002/gcc
tissue (T) and 11 samples of non-cancerous renal cortex tissue (N) obtained from materials that had been surgically resected from 68 patients with primary clear cell RCCs. There were 49 men and 19 women with a mean (6standard deviation) age of 62.3 6 11.0 years (range, 36 to 85 years). All patients underwent nephrectomy at the National Cancer Center Hospital, Tokyo, and had not received any preoperative treatment. Two expert pathologists specializing in genitourinary pathology, E.A. and Y.K., examined all histological slides and performed histological diagnosis in accordance with the WHO classification (Eble et al., 2004). All the tumors were graded on the basis of previously described criteria (Fuhrman et al., 1982) and classified according to the macroscopic configuration (Arai et al., 2006) and the pathological Tumor-Node-Metastasis (TNM) classification (Sobin et al., 2009). As a positive control for chimeric transcript detection, two T samples showing histological findings compatible with Xp11.2 translocation RCC based on the WHO criteria were also subjected to whole transcriptome analysis. For comparison, three T samples of papillary RCCs diagnosed in accordance with the WHO criteria were also subjected to whole transcriptome analysis. The second cohort subjected to quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis comprised 26 paired T and N samples obtained from materials that had been surgically resected from 26 other patients with primary clear cell RCCs. These patients comprised 17 men and nine women with a mean (6standard
Figure 1. Levels of mRNA expression for the partner genes involved in chimeric transcripts in 26 paired samples of tumorous tissue (T) and non-cancerous renal cortex tissue (N) in the second cohort. mRNA expression was analyzed using custom TaqMan Gene Expression Assays on the 7500 Fast Real-Time PCR System (Life Technologies) employing the relative standard curve method. The probes and PCR primer sets used are summarized in Supporting Information Table S6. Experiments were performed in triplicate for each sample-
primer set, and the mean value for the three experiments was used as the CT value. All CT values were normalized to that of GAPDH in the same sample. Levels of mRNA expression for the MMACHC, PTER, EPC2, ATXN7, FHIT, KIFAP3, CPEB1, MINPP1, TEX264, FAM107A, UPF3A, CDC16, MCCC1, CPSF3, and ASAP2 genes were significantly reduced in T samples (shaded column) relative to N samples (white column). Bar, standard deviation.
1024
GOTOH ET AL.
TABLE 4. Correlations Between Levels of mRNA Expression for Each of the Partner Genes Involved in Chimeric Transcripts in Tumorous Tissue Samples and Clinicopathological Parameters Reflecting Tumor Aggressiveness in the Second Cohort MMACHC Clinicopathological parameters
Number of tumors
PTER
EPC2
ATXN7
Expressiona
P
Expressiona
P
Expressiona
P
Expressiona
P
0.0528 6 0.0226 0.0250 6 0.0116 0.0384 6 0.0202
1:6131022 c
0.558 6 0.557 0.324 6 0.400 0.277 6 0.248
1.88 3 1021c
0.114 6 0.065 0.0546 6 0.0480 0.0784 6 0.0596
1.23 3 1021c
0.125 6 0.099 0.0598 6 0.0374 0.128 6 0.099
2.12 3 1021c
0.086 0.0443 6 0.0222 0.0489 6 0.0233 0.0285 6 0.0105
5:0531022c
0.574 0.530 6 0.608 0.498 6 0.479 0.232 6 0.309
1.15 3 1021c
0.246 0.112 6 0.083 0.106 6 0.033 0.0373 6 0.0185
4:6531023c
0.228 0.134 6 0.144 0.124 6 0.082 0.0696 6 0.0311
1.64 3 1021c
0.0533 6 0.0254 0.0327 6 0.0132
2:5631022g
0.571 6 0.433 0.282 6 0.452
8:6031023g
0.125 6 0.067 0.0586 6 0.0383
7:2431023g
0.139 6 0.100 0.0876 6 0.078
5.01 3 1022g
0.046 6 0.0239 0.0306 6 0.0077
2:0031021g
0.482 6 0.492 0.194 6 0.129
1.57 3 1021g
0.0954 6 0.0659 0.0762 6 0.0541
7.53 3 1021g
0.121 6 0.100 0.0836 6 0.035
7.05 3 1021g
0.0503 6 0.0242 0.0314 6 0.0134
1:6931022g
0.548 6 0.513 0.232 6 0.277
1:4431022g
0.120 6 0.064 0.0472 6 0.0274
7:1331024g
0.142 6 0.106 0.0672 6 0.0281
2:6831022g
0.0451 6 0.0230 0.0270 6 0.0030
1:3431021g
0.469 6 0.471 0.101 6 0.018
8:4631023g
0.0973 6 0.0649 0.0487 6 0.0218
1.57 3 1021g
0.120 6 0.095 0.0620 6 0.0265
2.11 3 1021g
0.0450 6 0.0221 0.0185 6 0.0007
2:4631022g
0.413 6 0.460 0.589 6 0.572
4.98 3 1021g
0.0974 6 0.0625 0.0235 6 0.0021
5.54 3 1022g
0.120 6 0.093 0.0365 6 0.0106
3:6931022g
0.0430 6 0.0166 0.0277 6 0.0155 0.0549 6 0.0287 0.0185 6 0.0007
5:5431022c
0.561 6 0.573 0.204 6 0.232 0.251 6 0.163 0.589 6 0.572
4.69 3 1021c
0.104 6 0.056 0.0607 6 0.0503 0.100 6 0.078 0.0235 6 0.0021
1.78 3 1021c
0.112 6 0.095 0.112 6 0.099 0.135 6 0.097 0.0365 6 0.0106
1.87 3 1021c
b
Macroscopic configuration Type 1 13 Type 2 5 Type 3 8 d,e Histological grades G1 1 G2 6 G3 11 G4 8 Vascular involvementf Negative 13 Positive 13 e Growth pattern Expansive 21 Infiltrative 5 Tumor necrosis Negative 16 Positive 10 Renal pelvic invasion Negative 23 Positive 3 Distant metastasis Negative 24 Positive 2 h Pathological TNM stage Stage I 13 Stage II 3 Stage III 8 Stage IV 2
FHIT Clinicopathological Number of parameters Tumors
KIFAP3
Expressiona
P
CPEB1
Expressiona
Expressiona
P
TEX264 P
Expressiona
P
0.155 6 0.111 0.142 6 0.104 0.143 6 0.086
9.22 3 1021c
b
Macroscopic configuration Type 1 13 Type 2 5 Type 3 8 d,e Histological grades G1 1 G2 6 G3 11 G4 8 Vascular involvementf Negative 13 Positive 13 e Growth pattern Expansive 21 Infiltrative 5 Tumor necrosis Negative 16 Positive 10 Renal pelvic invasion Negative 23 Positive 3 Distant metastasis Negative 24 Positive 2 h Pathological TNM stage Stage I 13 Stage II 3 Stage III 8 Stage IV 2
0.0884 6 0.0432 3:8231022c 0.0362 6 0.0222 0.0675 6 0.0345
0.280 6:6231023c 0.211 6 0.172 0.130 6 0.044 0.0631 6 0.0308
0.118 1.18 3 1021c 0.004 4:2331022c 0.0862 6 0.0535 0.00383 6 0.00722 0.0718 6 0.0259 0.00173 6 0.00168 0.0556 6 0.0481 0.0123 6 0.0119
0.178 6 0.126 1:2031022g 0.0898 6 0.0503
0.0839 6 0.0384 5.68 3 1022g 0.00492 6 0.00742 1.00g 0.0599 6 0.0421 0.00615 6 0.00978
0.155 6 0.112 0.143 6 0.088
6.50 3 1021g
0.150 6 0.109 4:0931022g 0.0660 6 0.0410
0.0752 6 0.0446 5.69 3 1021g 0.00433 6 0.00664 1.05 3 1021g 0.0580 6 0.0207 0.0106 6 0.0139
0.158 6 0.104 0.112 6 0.066
3.40 3 1021g
0.174 6 0.114 5:5531024g 0.0699 6 0.0316
0.0820 6 0.0381 3:0931022g 0.0558 6 0.0431
Number of Tumors
Macroscopic configurationb Type 1 13
0.00275 6 0.00449 4:0831022g 0.0100 6 0.0115
0.148 9.75 3 1022c 0.233 6 0.154 0.142 6 0.068 0.0955 6 0.0427
0.183 6 0.110 6:0531023g 0.0943 6 0.0395
0.143 6 0.107 1.34 3 1021g 0.0763 6 0.0420 6.38 3 1022g 0.00461 6 0.00639 5.94 3 1021g 0.156 6 0.103 3.12 3 1021g 0.0643 6 0.0397 0.0380 6 0.0044 0.0127 6 0.0193 0.0922 6 0.0222 0.141 6 0.106 5.54 3 1022g 0.0755 6 0.0409 3:6931022g 0.0520 6 0.0014 0.0285 6 0.0078 0.161 6 0.124 1.40 3 1021c 0.0893 6 0.0302 0.127 6 0.090 0.0520 6 0.0014
FAM107A Clinicopathological parameters
0.00369 6 0.00572 3.31 3 1021c 0.0058 6 0.0102 0.0084 6 0.0113
0.177 6 0.125 4:7331022c 0.0782 6 0.0187 0.100 6 0.069
0.00458 6 0.00790 5.54 3 1022g 0.156 6 0.100 5.54 3 1022g 0.0170 6 0.0099 0.0638 6 0.0021
0.0730 6 0.0375 7.75 3 1022c 0.00300 6 0.00478 1.15 3 1021c 0.153 6 0.111 2.26 3 1021c 0.0400 6 0.0271 0.00100 6 0.00100 0.195 6 0.116 0.0930 6 0.0446 0.0085 6 0.0117 0.147 6 0.083 0.0285 6 0.0078 0.0170 6 0.0099 0.0638 6 0.0021
CDC16
CPSF3
ASAP2
Expressiona
P
Expressiona
P
Expressiona
P
Expressiona
P
0.312 6 0.184
5.51 3 1022c
0.113 6 0.054
1.35 3 1021c
0.0476 6 0.0255
2.78 3 1021c
0.0455 6 0.0346
1.85 3 1021c
Genes, Chromosomes & Cancer DOI 10.1002/gcc
1025
WHOLE TRANSCRIPTOME ANALYSIS IN RCCs
TABLE 4. (Continued) FAM107A Clinicopathological parameters
Number of Tumors
Type 2 Type 3 Histological gradesd,e G1 G2 G3 G4 Vascular involvementf Negative Positive Growth patterne Expansive Infiltrative Tumor necrosis Negative Positive Renal pelvic invasion Negative Positive Distant metastasis Negative Positive Pathological TNM stageh Stage I Stage II Stage III Stage IV
Expressiona
CDC16 P
Expressiona
CPSF3 P
0.0584 6 0.0377 0.0926 6 0.0753
Expressiona
ASAP2 Expressiona
P
0.0260 6 0.0151 0.0465 6 0.0286
P
5 8
0.0986 6 0.0779 0.203 6 0.242
0.0218 6 0.0191 0.0250 6 0.0227
1 6 11 8
0.685 0.209 6 0.140 0.313 6 0.197 0.100 6 0.129
2:1431022c
0.172 0.113 6 0.083 0.116 6 0.049 0.0475 6 0.0206
1:2831022c
0.0613 0.0575 6 0.0335 0.0464 6 0.0236 0.0256 6 0.0118
1.30 3 1021c
0.112 0.0399 6 0.0320 0.0411 6 0.0252 0.0122 6 0.0068
1:9331022c
13 13
0.258 6 0.182 0.217 6 0.226
3.11 3 1021g
0.123 6 0.063 0.0696 6 0.0458
2:5631022g
0.0518 6 0.0271 0.0344 6 0.0214
1.13 3 1021g
0.0508 6 0.030 0.0185 6 0.0203
7:9531024g
21 5
0.255 6 0.211 0.166 6 0.159
4.47 3 1021g
0.0975 6 0.0608 0.0914 6 0.0662
8.01 3 1021g
0.0437 6 0.0263 0.0409 6 0.0246
9.00 3 1021g
0.0372 6 0.0316 0.0240 6 0.0211
4.09 3 1021g
16 10
0.317 6 0.205 0.110 6 0.117
2:2431023g
0.121 6 0.063 0.0567 6 0.0278
5:0231023g
0.0535 6 0.0266 0.0266 6 0.0115
1:4431022g
0.0475 6 0.0317 0.0142 6 0.0086
2:7731023g
23 3
0.259 6 0.205 0.0726 6 0.0602
7.85 3 1022g
0.100 6 0.063 0.0650 6 0.0128
3.52 3 1021g
0.0453 6 0.0263 0.0263 6 0.0073
3.95 3 1021g
0.0368 6 0.0313 0.0179 6 0.0058
4.42 3 1021g
24 2
0.256 6 0.199 0.0165 6 0.0030
5.54 3 1022g
0.102 6 0.059 0.0245 6 0.0007
2:4631022g
0.0457 6 0.0249 0.0125 6 0.0019
1:2331022g
0.0363 6 0.0307 0.0151 6 0.0005
3.94 3 1021g
13 3 8 2
0.246 6 0.152 0.209 6 0.167 0.290 6 0.284 0.0165 6 0.0030
2.72 3 1021c
0.109 6 0.053 0.0513 6 0.0307 0.110 6 0.071 0.0245 6 0.0007
4:5931022c
0.0445 6 0.0279 0.0324 6 0.0170 0.0526 6 0.0220 0.0125 6 0.0019
9.67 3 1022c
0.0404 6 0.0279 0.0103 6 0.0082 0.0394 6 0.0376 0.0151 6 0.0005
1.86 3 1021c
a
Average mRNA levels/GAPDH 6 standard deviation. Macroscopic configuration was evaluated on the basis of previously described criteria (Arai et al., 2006). c Kruskal–Wallis test. P values of < 0.05 are underlined. d All the tumors were graded on the basis of previously described criteria (Fuhrman et al., 1982). e If the tumor showed heterogeneity, the most aggressive features of the tumor were described. f The presence or absence of vascular involvement was examined microscopically on slides stained with hematoxylin-eosin and elastica van Gieson. g Mann–Whitney U test. P values of
