Background
Methods
Patients and samples
DNA preparations and NGS analysis
Gene mutations in different renal carcinomas from cBioPortal for Cancer genomics database
Amplification refractory mutation system and sanger sequencing
Immunohistochemistry
Name of antibody | Protein target | Manufacturer | Catalog | Dilution used |
---|---|---|---|---|
CK7 | Cytokeratin 7 | Fuzhou Maixin Biotech | Kit-0021 | 1:1 |
P504S | alpha-methylacyl-CoA racemase (AMACR), P504S | Fuzhou Maixin Biotech | RMA-0546 | 1:1 |
WT-1 | Wilms tumor 1 | Fuzhou Maixin Biotech | MAB-0678 | 1:1 |
CD57 | human natural killer-1 | Fuzhou Maixin Biotech | MAB-0257 | 1:1 |
P53 | p53 | Fuzhou Maixin Biotech | MAB-0674 | 1:1 |
BCL2 | B-cell lymphoma 2 | Fuzhou Maixin Biotech | RMA-0660 | 1:1 |
CCND1 | Cyclin-D1 | Abcam | ab16663 | 1:200 |
P16 | P16INK4A | Fuzhou Maixin Biotech | MAB-0673 | 1:1 |
p-ERK | phosphorylated Thr202/Tyr204-p44/42 MAPK | Cell Signaling Tech | 4370 | 1:100 |
ERK | total p44/42 MAPK | Cell Signaling Tech | 4695 | 1:100 |
p-MEK | phosphorylated MEK1/2 (Ser221) | Cell Signaling Tech | 2338 | 1:100 |
MEK | total MEK1/2 | Cell Signaling Tech | 4694 | 1:100 |
DUSP4/MKP-2 | Dual specificity protein phosphatase 4/MAP kinase phosphatase-2 | Abcam | ab216576 | 1:50 |
DUSP6/MKP-3 | Dual specificity protein phosphatase 6/MAP kinase phosphatase-3 | Abcam | ab54940 | 1:50 |
Results
Clinicopathologic features of a retrospective MA cohort
Deep-sequencing reveals novel mutations in MA
Present study (n = 28) | Renal Clear Cell Carcinoma (n = 499) | Wilms Tumor (n = 102) | Papillary Renal Cell Carcinoma (n = 293) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Gene | Mutant | Freq | Gene | Mutant | Freq | Gene | Mutant | Freq | Gene | Mutant | Freq | |
1 | BRAF | 23 | 82% | VHL | 235 | 51% | TP53 | 15 | 15% | MET | 23 | 7% |
2 | NF1 | 6 | 21% | PBRM1 | 158 | 36% | HLA-DQB1 | 11 | 7% | KMT2C | 20 | 6% |
3 | NOTCH1 | 5 | 18% | MUC4 | 142 | 21% | CTNNB1 | 8 | 7% | MUC4 | 20 | 5% |
4 | SPEN | 5 | 18% | SETD2 | 60 | 13% | CDK11A | 6 | 6% | SETD2 | 19 | 6% |
5 | AKT2 | 4 | 14% | BAP1 | 44 | 10% | DROSHA | 7 | 6% | KIAA1109 | 17 | 6% |
6 | APC | 4 | 14% | KDM5C | 30 | 7% | TMPRSS13 | 5 | 4% | BAP1 | 16 | 5% |
7 | ATRX | 3 | 11% | MTOR | 23 | 5% | ADCK5 | 4 | 4% | AR | 16 | 5% |
8 | ETV4 | 3 | 11% | PABPC1 | 20 | 5% | WT1 | 4 | 4% | KMT2D | 15 | 5% |
9 | FANCD2 | 2 | 7% | AHNAK2 | 19 | 4% | SIX1 | 4 | 4% | PCLO | 14 | 4% |
10 | FAT3 | 2 | 7% | PTEN | 19 | 4% | MAP3K4 | 4 | 4% | FAT1 | 13 | 4% |
11 | KDM6A | 2 | 7% | ATM | 18 | 3% | ACTB | 3 | 3% | NEFH | 13 | 4% |
12 | KDR | 2 | 7% | KMT2C | 17 | 4% | ZNF880 | 3 | 3% | WDFY3 | 13 | 4% |
13 | NOTCH3 | 2 | 7% | MAGEC1 | 16 | 4% | ZNF595 | 3 | 3% | SYNE1 | 13 | 4% |
14 | TET2 | 2 | 7% | MUC6 | 16 | 3% | DGCR8 | 3 | 3% | CUL3 | 13 | 4% |
15 | TSC2 | 2 | 7% | LRP1B | 15 | 4% | AGRN | 3 | 3% | ZNF814 | 13 | 3% |
16 | PCLO | 15 | 4% | MADCAM1 | 4 | 3% | ALMS1 | 13 | 3% | |||
17 | SYNE1 | 15 | 4% | HYDIN | 3 | 3% | DNAH8 | 12 | 4% | |||
18 | ARID1A | 14 | 3% | CUBN | 12 | 4% | ||||||
19 | KMT2D | 14 | 3% | PBRM1 | 12 | 4% | ||||||
20 | MUC2 | 14 | 3% | PCF11 | 12 | 4% |
Gene | Number of mutation events (frequency) | Exon_Rank | Missense or stop-gained mutations | Base mutation | Synonymous or intronic mutations | Base mutation |
---|---|---|---|---|---|---|
BRAF | 23 | 15 | p.V600E, LOC100507389-BRAF, STARD9-BRAF, CUX1-BRAF | c.1799 T > A, chrom3:142634278_7:140492322, chrom15:42936216_7:140492968, chrom7:101809365_7:140492220 | \ | \ |
NF1 | 6 | 43, 46, 32 | p.G1403S, p.Y2285* | c.4207G > A, c.6855C > A | p.S2152= | c.6456 T > C |
NOTCH1 | 5 | 29, 32, 14 | p.D1808N, p.H2018Q, p.D740N | c.5422G > A, c.6054C > A, c.2218G > A | \ | \ |
SPEN | 5 | 11, 2 | p.R807H, p.R128C | c.2420G > A, c.382C > T | p.K926= | c.2778A > G |
AKT2 | 4 | NA | \ | cn_amp | \ | |
APC | 4 | 16 | p.L662I | c.1984C > A | p.P1634= | c.4902G > A |
ATRX | 3 | 9 | \ | \ | p.T899=, p.S651= | c.2697G > A, c.1953 T > G |
ETV4 | 3 | NA | \ | \ | cn_amp | \ |
FANCD2 | 2 | 25, 30 | p.N791S, p.T981I | c.2372A > G, c.2942C > T | \ | \ |
FAT3 | 2 | 20, 23 | p.R3849Q | c.11546G > A | p.Y4222= | c.12666C > T |
KDR | 2 | 10, 15 | p.W460 L, p.I753V | c.1379G > T, c.2257A > G | \ | \ |
NOTCH3 | 2 | 4, 24 | p.A198V | c.593C > T | p.P1331= | c.3993G > A |
TET2 | 2 | 11 | p.E1973K, p.N1765 K | c.5917G > A, c.5295C > G | \ | \ |
TSC2 | 2 | 30, 35, 5 | p.V1144 M, p.E1513K | c.3430G > A, c.4537G > A | \ | \ |
ABL1 | 1 | 4 | \ | \ | p.L217= | c.649 T > C |
ALOX12B | 1 | 12 | p.A525T | c.1573G > A | \ | \ |
AMER1 | 1 | 2 | p.G149_A156del | c.446_469delGAGCCACAGAGAAAGCTGTGGCTG | \ | \ |
ARID1A | 1 | 1 | \ | \ | p.A281= | c.843G > A |
ASXL1 | 1 | 12 | p.Q778* | c.2332C > T | \ | \ |
AURKB | 1 | 6 | p.G161R | c.481G > A | \ | \ |
AXL | 1 | 2 | p.R71Q | c.212G > A | \ | \ |
BCORL1 | 1 | 3 | p.P312S | c.934C > T | \ | \ |
CCND1 | 1 | NA | \ | \ | cn_amp | \ |
CD79A | 1 | 2 | \ | \ | p.T65= | c.195C > T |
CDH1 | 1 | 9 | p.D433N | c.1297G > A | \ | \ |
CDK12 | 1 | 13 | \ | \ | p.S1191= | c.3573A > G |
DIS3 | 1 | 11 | p.V509 L | c.1525G > C | \ | \ |
DOT1L | 1 | 24 | p.G1087S | c.3259G > A | \ | \ |
EP300 | 1 | 2 | \ | \ | p.Y207= | c.621C > T |
EPHA3 | 1 | 7 | p.A515T | c.1543G > A | \ | \ |
EPHA5 | 1 | 5 | \ | \ | p.Y437= | c.1311 T > C |
ERBB2 | 1 | 3 | \ | \ | p.P122= | c.366G > A |
ERBB4 | 1 | 27 | p.A1130S | c.3388G > T | \ | \ |
EZH2 | 1 | 2 | p.R18C | c.52C > T | \ | \ |
FGFR1 | 1 | NA | \ | \ | cn_amp | NA |
FGFR4 | 1 | 18 | p.D785H | c.2353G > C | \ | \ |
GRIN2A | 1 | 3 | p.V122I | c.364G > A | \ | \ |
HRAS | 1 | 4 | \ | \ | p.D108= | c.324C > T |
IKBKE | 1 | 6 | p.R134C | c.400C > T | \ | \ |
IL7R | 1 | 5 | p.A199G | c.596C > G | \ | \ |
INHBA | 1 | 2 | p.K45R | c.134A > G | \ | \ |
KAT6A | 1 | 15 | \ | \ | p.E993del | c.2977_2979delGAG |
KDM5A | 1 | 23 | \ | \ | p.G1200 fs | c.3597dupA |
KDM6A | 1 | 5 | p.Y143C | c.428A > G | \ | \ |
KMT2D | 1 | 10 | \ | \ | p.P556= | c.1668G > T |
MAP2K1 | 1 | 10 | p.Q354H | c.1062A > C | \ | \ |
MET | 1 | NA | \ | \ | cn_amp | NA |
MTOR | 1 | 28 | \ | \ | p.A1388= | c.4164C > T |
NCOR1 | 1 | 4 | \ | \ | p.L133= | c.399G > A |
NF2 | 1 | 5 | \ | \ | p.R160= | c.478C > A |
NPM1 | 1 | 7 | p.V192 M | c.574G > A | \ | \ |
NSD1 | 1 | 5 | \ | \ | p.K513= | c.1539G > A |
NTRK1 | 1 | 8 | p.N323S | c.968A > G | \ | \ |
PAK3 | 1 | 1 | p.S31G | c.91A > G | \ | \ |
PARP2 | 1 | 8 | p.E231K | c.691G > A | \ | \ |
PARP4 | 1 | 31 | \ | \ | p.R1332= | c.3996C > T |
PAX5 | 1 | 6 | \ | \ | p.L234= | c.700C > T |
PIK3C2G | 1 | 29 | p.R1316G | c.3946A > G | \ | \ |
PIK3CG | 1 | 8 | \ | \ | p.I879= | c.2637C > T |
PMS2 | 1 | 9 | p.R304T | c.911G > C | \ | \ |
PRDM1 | 1 | 5 | p.H409Q | c.1227C > A | \ | \ |
RAD52 | 1 | 9 | p.A248T | c.742G > A | \ | \ |
RET | 1 | 7 | \ | \ | p.G453= | c.1359G > C |
RPTOR | 1 | 14 | \ | \ | p.N513= | c.1539C > T |
SMARCA4 | 1 | 35 | p.R1633Q | c.4898G > A | \ | \ |
SOX10 | 1 | 4 | p.T240P | c.718A > C | \ | \ |
TSC1 | 1 | 13 | p.M425 V | c.1273A > G | \ | \ |
TSHR | 1 | 5 | p.G132R | c.394G > C | \ | \ |
n | Total | BRAF V600E | BRAF wild-type |
---|---|---|---|
27 | 22 | 5 | |
Gender | F:M = 9:18 | F:M = 5:17 | F:M = 1:4* |
Age range, year | 12~ 80 | 25~ 73 | 12~ 47** |
Median age, year | 39 | 40 | 29 |
Tumor size, cm | 2~ 7 | 2.5~ 7 | 2~ 5.5* |
Median size, cm | 3.1 | 3.2 | 2.2 |
BCL2, (negative/positive) | 5/22 | 2/20 | 3/2** |
p16, (negative/positive) | 4/23 | 1/21 | 3/2** |
p-MEK, (negative/positive) | 0/27 | 0/22 | 0/5 |
p-ERK, (negative/positive) | 15/12 | 13/9 | 2/3 |
DUSP6, (negative/positive) | 4/23 | 2/20 | 2/3 |
Immunohistochemical analysis
Patient Number | BRAF status | CK7 | AMACR | WT-1 | CD57 | P53 | BCL2 | CCND1 | P16 | p-ERK | ERK | p-MEK | MEK | DUSP6 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | BRAF V600E | – | – | 3+ | 2+ | – | 3+ | – | 3+ | – | 3+ | 2+ | 3+ | 2+ |
2 | BRAF wild-type | – | – | 3+ | 3+ | – | – | – | 2+ | – | 3+ | 2+ | 3+ | 1+ |
3 | BRAF V600E | – | – | 1+ | 2+ | – | – | – | 1+ | – | 2+ | 1+ | 2+ | 1+ |
4 | BRAF wild-type | – | – | 2+ | 2+ | – | – | – | 1+ | – | 2+ | 1+ | 3+ | – |
5 | BRAF V600E | – | – | 3+ | 3+ | – | 1+ | – | – | – | 3+ | 2+ | 3+ | 1+ |
6 | BRAF V600E | – | – | 3+ | 2+ | – | – | – | 1+ | – | 2+ | 1+ | 2+ | 1+ |
7 | BRAF V600E | – | – | 1+ | 2+ | – | 2+ | – | 2+ | 1+ | 1+ | 1+ | 2+ | 1+ |
8 | BRAF V600E | – | – | 2+ | 2+ | – | 2+ | – | 2+ | – | 2+ | 1+ | 3+ | – |
9 | BRAF V600E | – | – | 3+ | 3+ | – | 3+ | – | 2+ | 1+ | 2+ | 1+ | 3+ | 1+ |
10 | BRAF V600E | – | – | 1+ | 3+ | – | 2+ | – | 2+ | 1+ | 1+ | 1+ | 3+ | 2+ |
11 | BRAF V600E | – | – | 3+ | 2+ | – | 3+ | – | 3+ | 1+ | 2+ | 1+ | 2+ | 2+ |
12 | BRAF V600E | – | – | – | 3+ | – | 3+ | – | 3+ | – | 2+ | 2+ | 1+ | 2+ |
13 | BRAF V600E | – | – | 2+ | – | – | 3+ | – | 2+ | 1+ | 1+ | 1+ | 2+ | 1+ |
14 | BRAF V600E | – | – | 3+ | 2+ | 2+ | – | 1+ | 1+ | 1+ | 1+ | 2+ | 1+ | |
15 | BRAF V600E | – | – | – | 1+ | – | 3+ | – | 1+ | – | 2+ | 2+ | 1+ | 2+ |
16 | BRAF V600E | – | – | 1+ | 1+ | – | 2+ | – | 1+ | – | 1+ | 2+ | 3+ | – |
17 | BRAF wild-type | – | – | 1+ | 1+ | – | – | – | – | 1+ | 2+ | 1+ | 3+ | 1+ |
18 | BRAF V600E | – | – | 1+ | 1+ | – | 3+ | – | 3+ | – | 3+ | 1+ | 3+ | 1+ |
19 | BRAF V600E | – | – | – | 1+ | – | 3+ | – | 2+ | – | 3+ | 1+ | 1+ | 2+ |
20 | BRAF Intron8-STARD9/ CUX1/ LOC100507389 fusion | +/− | – | 1+ | 1+ | – | 3+ | – | 3+ | 1+ | 2+ | 1+ | 2+ | 1+ |
21 | BRAF V600E | – | – | 1+ | 1+ | – | 2+ | – | 1+ | – | 1+ | 1+ | 3+ | 1+ |
22 | BRAF V600E | – | – | 1+ | 1+ | – | 3+ | – | 1+ | 1 | 1+ | 1+ | 2+ | 1+ |
23 | BRAF V600E | +/− | 1+ | 1+ | 1+ | – | 3+ | – | 2+ | 1 | 1+ | 1+ | 2+ | 1+ |
24 | BRAF wild-type | – | – | 1+ | 1+ | – | 2+ | – | – | 1+ | 2+ | 1+ | 1+ | 1+ |
25 | BRAF wild-type | – | – | 1+ | 1+ | – | 3+ | – | – | 1+ | 3+ | 2+ | 2+ | – |
26 | BRAF V600E | – | – | 1+ | 1+ | – | 3+ | – | 1+ | – | 3+ | 1+ | 3+ | 2+ |
27 | BRAF V600E | – | – | 1+ | 1+ | – | 2+ | – | 2+ | – | 1+ | 1+ | 3+ | 1+ |
28 | BRAF V600E | – | – | 1+ | 1+ | – | 3+ | – | 1+ | 1+ | 1+ | 1+ | 1+ | 1+ |