Correlations between histological characterizations and methylation statuses of tumour suppressor genes in Wilms' tumours

Yen‐Chein Lai; Meng‐Yao Lu; Wen‐Chung Wang; Tai‐Cheng Hou; Chen‐Yun Kuo

doi:10.1111/iep.12442

. 2022 Apr 18;103(3):121–128. doi: 10.1111/iep.12442

Correlations between histological characterizations and methylation statuses of tumour suppressor genes in Wilms' tumours

Yen‐Chein Lai ^1,^2,^✉, Meng‐Yao Lu ³, Wen‐Chung Wang ⁴, Tai‐Cheng Hou ⁵, Chen‐Yun Kuo ⁵

PMCID: PMC9107610 PMID: 35436013

Abstract

Wilms' tumour is a solid tumour that frequently occurs in children. Genetic changes in WT1 and epigenetic aberrations that affect imprinted control region 1 in WT2 loci are implicated in its aetiology. Moreover, tumour suppressor genes are frequently silenced by methylation in this tumour. In the present study, we analysed the methylation statuses of promoter regions of 24 tumour suppressor genes using a methylation‐specific multiplex ligation‐dependent probe amplification (MS‐MLPA)‐based approach in 6 Wilms' tumours. Methylation of RASSF1 was specific to all 6 Wilms' tumours and was not observed in normal tissues. Moreover, methylated HIC1 was identified in stromal‐type Wilms' tumours and methylated BRCA1 was identified in epithelial‐type Wilms' tumours. Unmethylated CASP8, RARB, MLH1_167, APC and CDKN2A were found only in blastemal predominant‐type Wilms' tumour. Our results indicated that methylation of RASSF1 may be a vital event in the tumorigenesis of Wilms' tumour, which informs its clinical and therapeutic management. In addition, mixed‐type Wilms' tumours may be classified according to epithelial, stromal and blastemal components via MS‐MLPA‐based approach.

Keywords: methylation detection, molecular pathology, multiplex ligation‐dependent probe amplification, nephroblastoma, tumour suppressor gene, Wilms' tumour

1. INTRODUCTION

Wilms' tumour, also known as nephroblastoma, is an embryonal malignant tumour of the kidney. It is the most commonly occurring solid tumour in children, excluding brain tumours. ¹ , ² , ³ The aetiology of Wilms' tumour is still poorly understood, although a number of associated genes and loci have been identified. ⁴ , ⁵ The genetic changes in Wilms' tumours are diverse and involve approximately 40 genes. ⁶ Genes that have been previously implicated include WT1, CTNNB1, FAM123B, DROSHA, DGCR8, XPO5, DICER1, SIX1, SIX2, MLLT1, MYCN, and TP53. ⁶ Whole‐genome sequencing and whole‐exome sequencing have led to the addition of BCOR, BCORL1, NONO, MAX, COL6A3, ASXL1, MAP3K4 and ARID1A. ⁷ Recently, 4 new predisposing genes have been identified—TRIM28, FBXW7, NYNRIN and KDM3B. ⁵

Hypermethylation of CpG islands upstream of tumour suppressor genes has been reported with alteration of methylation status in 1 or more types of tumours. ⁸ , ⁹ Wilms' tumour may develop mainly through alterations in epigenetic regulation triggered by dedifferentiation. ¹⁰ However, the risk of Wilms' tumour conferred by epigenetic changes associated with tumour suppressor genes is poorly characterized. In the present study, we investigated the methylation statuses of promoter regions of 24 tumour suppressor genes in 6 Wilms' tumours using a methylation‐specific multiplex ligation‐dependent probe amplification (MS‐MLPA)‐based approach.

2. MATERIALS AND METHODS

2.1. Study subjects

The 6 subjects have been described elsewhere. ¹¹ , ¹² , ¹³ Their paraffin‐embedded Wilms' tumour tissue samples were provided by the Department of Pediatrics of National Taiwan University Hospital. These patients had no history of the Denys–Drash syndrome, Frasier syndrome, or Beckwith–Wiedemann syndrome. The study procedures were approved by the Institutional Review Board of Chung Shan Medical University Hospital (reference number CS2‐16003). All procedures that involved human participants were conducted in accordance with the ethical standards of the institutional and/or national research committee and the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.

2.2. Histological examination

Tumour tissue samples (W7 to W12) were embedded in paraffin wax and cut into 5‐um‐thick slices. These sections were then stained with haematoxylin and eosin and reviewed by 2 of the authors (T‐C Hou and C‐Y Kuo) under a microscope to confirm their diagnostic classifications. Typing of Wilms' tumour has been described in detail elsewhere. ¹¹ , ¹³ W7 is of blastemal type, and W8 is of epithelial type. In addition, W10 and W11 are of stromal type. W9 is a triphasic Wilms' tumour comprised of 3 components: blastema, stroma and epithelium, while W12 is of mixed blastemal and epithelial type.

2.3. DNA extraction

Genomic DNA was purified from paraffin‐embedded tissues with the DNA FFPE Tissue Kit (Qiagen GmbH), according to the manufacturer's instructions, and dissolved in 100 μl of TE buffer (10 mM Tris‐HCl, pH 8.0, and 1 mM EDTA) as previously described. ¹¹ , ¹³ UV‐Vis measurements of DNA concentration of each sample were obtained using NanoDrop UV‐Vis Spectrophotometer (Thermo Scientific NanoDrop 2000c).

2.4. Methylation analysis

Methylation‐specific multiplex ligation‐dependent probe amplification analysis was performed using Salsa MS‐MLPA Kit ME001‐C2 Tumor Suppressor‐1 (MRC‐Holland) according to the manufacturer's instructions. Samples were subjected to capillary electrophoresis on an ABI PRISM 3130XL (Applied Biosystems). Twenty‐six MS‐MLPA probes were used to detect the methylation statuses of promoter regions of 24 tumour suppressor genes by HhaI digestion (Table 1). In addition, there were 15 reference probes that were unaffected by HhaI digestion. Upon digestion, peak signal was absent in unmethylated samples. In contrast, these probes generated a signal in methylated DNA. MLPA results were analysed using GeneMarker version 3.2.1 (SoftGenetics, LLC) to determine copy numbers and methylation statuses of the HhaI sites. The ‘internal methylation ratio’ was obtained by comparison of the HhaI‐digested aliquot (Figure 1B) with the paired undigested aliquot (Figure 1A) from each sample with intrasample data normalization according to the manufacturer's instructions. ¹⁴ Methylation (compared with normal reference) was assessed by comparing the probe methylation percentages (internal methylation ratio) of the test sample with the percentages of 5 normal reference blood samples from healthy individuals. Age‐matched controls were unavailable as Wilms' tumour tissue samples were delinked and age of subjects was unknown. Copy‐number ratio of 1.0 and methylation ratio of 0 were expected in most genes on normal reference. If a tumour suppressor gene was silenced by methylation, ‘methylation compared to normal reference’ was unlimited (∞). If methylation ratios of test and normal reference samples were appropriate, methylation compared with normal reference was around 1.0.

TABLE 1.

Chromosomal locations and methylation data of the 41 probes in ME001‐C2 Tumor Suppressor‐1

Size (NT)	Gene	MLPA probe	HhaI site	Cytoband	Methylation reference	W7	W8	W9	W10	W11	W12
142	TIMP3	02255‐L03752	+	22q12.3	‐	0/0	0.04/∞	0.05/∞	0/0	0/0	0/0
148	APC	01905‐L01968	+	5q22.2	‐	0/0	0.06/∞	0.15/∞	0.09/∞	0.08/∞	0.15/∞
161	CDKN2A	01524‐L01744	+	9p21.3	‐	0/0	0.05/∞	0.06/∞	0.09/∞	0.04/∞	0.07/∞
167	MLH1	01686‐L01266	+	3p22.2	‐	0/0	0.03/2.82	0.09/4.62	0.06/3.86	0.03/2.79	0.05/2.96
184	ATM	04044‐L03849	+	11q22.3	‐	0/0	0.04/∞	0.10/∞	0.10/∞	0.05/∞	0.18/∞
193	RARB	04040‐L01698	+	3p24.2	‐	0/0	0.05/9.28	0.10/11.75	0.12/23.95	0.06/8.18	0.09/10.25
211	CDKN2B	00607‐L00591	+	9p21.3	‐	0.18/3.72	0.54/8.85	0.15/2.58	0.13/3.15	0.26/5.40	0.07/1.05
220	HIC1	03804‐L00949	+	17p13.3	‐	0/0	0/0	0.05/∞	0.06/∞	0.04/∞	0/0
238	CHFR	03813‐L03753	+	12q24.33	‐	0/0	0/0	0/0	0/0	0/0	0/0
246	BRCA1	05162‐L04543	+	17q21.31	‐	0/0	0.04/∞	0.03/∞	0/0	0/0	0.03/∞
265	CASP8	02761‐L02210	+	2q33.1	‐	0/0	1.01/∞	0.79/∞	0.58/∞	0.55/∞	0.79/∞
274	CDKN1B	07949‐L07730	+	12p13.1	‐	0/0	0.04/∞	0/0	0/0	0/0	0/0
292	KLLN	02203‐L08261	+	10q23.3	‐	0.13/∞	0.07/∞	0.07/∞	0/0	0.05/∞	0/0
301	BRCA2	04042‐L03755	+	13q12.3	‐	0/0	0/0	0/0	0/0	0/0	0/0
319	CD44	03817‐L01731	+	11p13	‐	0.34/∞	0/0	0/0	0.07/∞	0/0	0/0
328	RASSF1	02248‐L01734	+	3p21.31	‐	0.84/∞	0.17/∞	0.74/∞	0.74/∞	0.28/∞	0.63/∞
346	DAPK1	01677‐L01257	+	9q21.33	‐	0/0	0.041/∞	0/0	0/0	0/0	0/0
353	VHL	03810‐L01211	+	3p25.3	‐	0/0	0/0	0/0	0/0	0/0	0/0
373	ESR1	02202‐L01700	+	6q25.1	‐	0/0	0.08/∞	0/0	0.13/∞	0/0	0/0
382	RASSF1	03807‐L02159	+	3p21.31	‐	0.94/∞	0.22/∞	0.74/∞	0.71/∞	0.33/∞	0.70/∞
400	TP73	04050‐L01263	+	1p36.32	‐	0/0	0.09/∞	0.12/∞	0.13/∞	0/0	0.10/∞
409	FHIT	02201‐L01699	+	3p14.2	‐	0/0	0/0	0/0	0/0	0/0	0/0
427	CADM1	03819‐L03848	+	11q23.3	‐	0/0	0.06/∞	0/0	0/0	0/0	0/0
436	CDH13	07946‐L07727	+	16q23.3	‐	0/0	0.08/∞	0.09/∞	0.11/∞	0/0	0.10/∞
454	GSTP1	01638‐L01176	+	11q13.2	‐	0/0	0.07/∞	0.09/∞	0.09/∞	0/0	0.09/∞
463	MLH1	02260‐L01747	+	3p22.2	‐	0/0	0/0	0/0	0/0	0/0	0/0
136	CREM	00981‐L00566	‐	10p12.1	Yes	1.00/1.15	1.17/1.00	1.11/1.05	1.00/0.72	1.15/1.30	0.89/0.97
154	PARK2	03366‐L02750	‐	6q26	Yes	0.97/1.16	1.32/1.00	1.26/0.58	0.98/0.98	1.36/1.07	1.20/0.72
175	TNFRSF1A	00554‐L13516	‐	12p13	Yes	0.99/1.09	1.10/1.55	0.99/1.45	1.02/1.62	1.12/1.36	0.80/1.36
202	MLH3	01245‐L00793	‐	14q24.3	Yes	0.99/1.04	1.09/1.04	1.11/1.04	0.93/1.07	1.18/1.16	0.87/0.84
229	PAH	02334‐L01820	‐	12q23.2	Yes	1.03/1.01	1.22/0.70	0.98/0.73	0.88/0.80	1.17/0.79	0.91/1.06
256	BCL2	00587‐L00382	‐	18q21.33	Yes	1.07/0.96	1.24/0.93	0.97/1.00	1.05/0.87	1.15/0.80	0.89/0.94
281	TSC2	01832‐L01397	‐	16p13.3	Yes	1.06/0.76	1.25/0.72	0.97/0.95	0.98/0.82	1.06/0.61	0.88/0.87
310	CDK6	03184‐L02523	‐	7q21.3	Yes	0.88/0.91	1.25/0.94	0.92/0.84	1.03/0.79	1.12/0.85	0.90/1.20
337	CDH1	02416‐L01862	‐	16q22.1	Yes	1.01/0.96	1.13/1.15	1.03/1.40	2.54/2.90	1.05/1.12	0.91/0.85
364	LOC254312	01234‐L00781	‐	10p14	Yes	0.95/1.03	1.32/1.06	1.17/0.84	1.01/0.74	1.14/1.05	0.82/0.88
390	KLK3	00713‐L00108	‐	19q13.33	Yes	1.11/1.00	1.21/064	0.99/1.02	0.93/0.78	1.03/0.77	0.75/1.01
418	BRCA2	01617‐L01199	‐	13q13.1	Yes	0.93/1.03	1.11/1.16	1.06/0.67	1.08/0.95	1.06/1.17	0.80/0.71
444	CD27	00678‐L00124	‐	12p13.31	Yes	0.95/0.84	1.22/1.45	0.93/1.50	0.98/1.27	0.99/1.17	0.83/1.94
475	CTNNB1	03984‐L03251	‐	3p22.1	Yes	0.90/1.18	1.20/0.98	0.91/0.68	1.07/0.87	0.92/0.98	0.82/0.83
483	CASR	02683‐L02148	‐	3q21.1	Yes	0.90/0.93	1.23/0.86	1.13/0.99	1.01/1.45	1.16/0.90	0.87/1.12

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Data are presented as internal methylation ratio/methylation compared with normal reference.

Detection of the methylation statuses of 24 tumour suppressor genes in W8 by MS‐MLPA. (A) Capillary electrophoresis pattern from undigested DNA. Red arrows indicate methylation of *RASSF1* and *CDKN2B* in all 6 Wilms' tumours. (B) Capillary electrophoresis pattern from the same sample but digested with *Hha*I site. Red arrows indicate that all DNA is unmethylated. Therefore, after MS‐MLPA, no signal was generated from the MS‐MLPA probes

2.5. Immunohistochemistry

Sections from 4‐micrometre‐thick, formalin‐fixed, and paraffin‐embedded Wilms' tumours were used for immunohistochemical analysis (IHC). IHC of RASSF1 was performed using a mouse monoclonal antibody (1:150 dilution; clone OTI2B11; Thermo Fisher Scientific). Tumour sections with reduction in RASSF1 protein expression were visualized via light microscopy.

3. RESULTS

The methylation statuses of the HhaI sites in 24 tumour suppressor genes were determined by 26 MS‐MLPA probes on MS‐MLPA analysis (Figure 1, Table 1). If the target site was unmethylated, the DNA–probe complex was digested to prevent exponential amplification. No signal was detected after fragment analysis (Figure 1B). The internal methylation ratio was 0 due to the absence of peak signal in unmethylated samples. In contrast, the internal methylation ratio was a number other than 0 in methylated DNA as a signal was generated. There were 2 MLPA probes for RASSF1 gene with different product sizes: hybridization to MV location 03‐050.353347 (reference hg18) resulted in product length of 382, and hybridization to MV location 03‐050.353298 resulted in product length of 328 (Table 1). There were also 2 MLPA probes for MLH1 gene with different product sizes: hybridization to MV location 03‐037.009621 (reference hg18) resulted in product length of 167, and hybridization to MV location 03‐37.010000 resulted in product length of 463 (Table 1).

3.1. Common features of all 6 paraffin‐embedded tumour specimens

Methylated RASSF1 and CDKN2B were found in all 6 Wilms' tumours (Figure 1A, Table 2). Methylation was observed in both probes in RASSF1 gene, and methylation of RASSF1 was specific to tumours but not normal tissues. Internal methylation ratio was not 0, and methylation compared with normal reference was unlimited (∞). Figure 2 shows examples of Wilms' tumours with RASSF1 protein expression.

TABLE 2.

Clinicopathological data and methylation statuses of six Wilms’ tumours

Variables	Gene	Size (NT)	W7	W8	W9	W10	W11	W12
Sex			Male	Male	Female	Male	Male	Female
Stroma			‐	‐	+	+	+	‐
Epithelium			‐	+	+	‐	‐	+
Blastema			+	‐	+ ^a	‐	‐	+
All	RASSF1	328	0.84/∞	0.17/∞	0.74/∞	0.74/∞	0.28/∞	0.63/∞
All	RASSF1	382	0.94/∞	0.22/∞	0.74/∞	0.71/∞	0.33/∞	0.70/∞
All	CDKN2B	211	0.18/3.72	0.54/8.85	0.15/2.58	0.13/3.15	0.26/5.40	0.073/1.05
None	VHL	353	0/0	0/0	0/0	0/0	0/0	0/0
None	MLH1	463	0/0	0/0	0/0	0/0	0/0	0/0
None	FHIT	409	0/0	0/0	0/0	0/0	0/0	0/0
None	CHFR	238	0/0	0/0	0/0	0/0	0/0	0/0
None	BRCA2	301	0/0	0/0	0/0	0/0	0/0	0/0
Stroma	HIC1	220	0/0	0/0	0.05/∞	0.06/∞	0.04/∞	0/0
Epithelium	BRCA1	246	0/0	0.04/∞	0.03/∞	0/0	0/0	0.03/∞
Blastema	CASP8	265	0/0	1.01/∞	0.79/∞	0.58/∞	0.55/∞	0.79/∞
Blastema	RARB	193	0/0	0.05/9.28	0.10/11.75	0.12/23.95	0.06/8.18	0.09/10.25
Blastema	MLH1	167	0/0	0.03/2.82	0.09/4.62	0.06/3.863	0.03/2.79	0.05/2.96
Blastema	APC	148	0/0	0.06/∞	0.15/∞	0.09/∞	0.08/∞	0.15/∞
Blastema	CDKN2A	161	0/0	0.05/∞	0.06/∞	0.09/∞	0.04/∞	0.07/∞
Others	ATM	184	0/0	0.04/∞	0.10/∞	0.10/∞	0.05/∞	0.18/∞
Others	TP73	400	0/0	0.09/∞	0.12/∞	0.13/∞	0/0	0.10/∞
Others	GSTP1	454	0/0	0.07/∞	0.09/∞	0.09/∞	0/0	0.09/∞
Others	KLLN	292	0.13/∞	0.07/∞	0.07/∞	0/0	0.05/∞	0/0
Others	CDH13	436	0/0	0.08/∞	0.09/∞	0.11/∞	0/0	0.10/∞
Others	ESR1	373	0/0	0.08/∞	0/0	0.13/∞	0/0	0/0
Others	CD44	319	0.34/∞	0/0	0/0	0.07/∞	0/0	0/0
Others	TIMP3	142	0/0	0.04/∞	0.05/∞	0/0	0/0	0/0
Others	DAPK1	346	0/0	0.041/∞	0/0	0/0	0/0	0/0
Others	CADM1	427	0/0	0.06/∞	0/0	0/0	0/0	0/0
Others	CDKN1B	274	0/0	0.04/∞	0/0	0/0	0/0	0/0

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Data are presented as internal methylation ratio/methylation compared with normal reference.

^{^a}

W9 revealed mixed pattern of three types of cellular components. However, more than two‐thirds were blastemal and epithelial.

Immunohistochemical analysis of RASSF1 staining in Wilms' tumours (200X). W9 (A) is mixed stromal, blastemal, and epithelial. W10 (B) and W11 (C) are stromal. Normal tissue (D) obtained from the kidney of the same patient (W11) without tumour invasion under pathological inspection

CDKN2B gene was methylated in normal reference and hypermethylated in 5 of the 6 tumours. In comparison with normal reference, methylation was not unlimited (∞). W7 to W11 methylation was 3.72‐, 8.85‐, 2.58‐, 3.15‐ and 5.40‐fold that of normal reference respectively. However, W12 methylation was only 1.05‐fold that of normal reference. Moreover, VHL, MLH1_463, FHIT1, CHFR and BRCA2 were unmethylated in all 6 Wilms' tumours (Figure 1B, Table 2).

3.2. Typing of Wilms' tumours by methylation statuses of tumour suppressor genes

There were methylation of HIC1 in the Wilms' tumours of stromal type (W9, W10 and W11) and methylation of BRCA1 in the Wilms' tumours of epithelial type (W8, W9 and W12). CASP8, RARB, MLH1_167, APC and CDKN2A were unmethylated only in W7, which was of blastemal predominant type (Table 2). In other words, there was methylation of CASP8, RARB, MLH1_167, APC and CDKN2A in stromal‐ and epithelial‐type Wilms' tumours but not in blastemal‐type Wilms' tumour.

3.3. Other findings

In addition to these 13 tumour suppressor genes (15 MS‐MLPA probes), there were differential methylation patterns for the other 11 tumour suppressor genes among the 6 Wilms' tumours (Table 2). Only two of these 11 tumour suppressor genes, KLLN and CD44, were methylated in W7. Only 1 of these 11 tumour suppressor genes, CD44, was unmethylated in W8. The 6 Wilms' tumours listed in order from minimum number to maximum number of these methylated genes are W7 and W11 (2), W12 (4), W9 and W10 (6), and W8 (10).

4. DISCUSSION

Traditional methods of methylation detection involve modifying DNA by methylation‐specific restriction enzymes or sodium sulphite, combined with Sanger sequencing, PCR or hybridization analysis. This process is cumbersome and difficult, with low reproducibility and insufficient sensitivity. MLPA is a PCR amplification reaction, while ME001 simultaneously detects changes in the degree of methylation of up to 24 different genes in a single reaction tube. Due to MLPA's easy operation, low cost and wide range of applications, our team has published several studies based on this method. ¹³ , ¹⁵ , ¹⁶ However, MLPA analysis of tumour samples only provides information regarding the ‘average’ situation in the cells from which the DNA samples were purified. ¹⁴ A potential limitation of this study is whether the methylation extent of blood sample is equal to that of normal kidney.

RASSF1 gene on 3p21.31 encodes a cytosolic RASSF1A protein similar to Ras effector proteins. ¹⁷ There were 2 MLPA probes for RASSF1 gene with different product sizes: product length 382 (shown in red in Figure 3) and product length 328 (shown in purple in Figure 3). Methylation was observed in both probes for RASSF1 gene and was specific to tumours. The results of this study regarding the methylation of RASSF1 gene are consistent with the findings of previous studies. ¹⁸ , ¹⁹ Moreover, methylation studies performed by Harada et al have shown that RASSF1 methylation is tumour‐specific and absent in adjacent non‐malignant tissues. ¹⁹ De novo methylation of the RASSF1 promoter is one of the most frequent epigenetic inactivation events in human cancer and leads to silencing of RASSF1 expression. ²⁰ , ²¹ Association of RASSF1 promoter methylation with Wilms' tumour has been reported, ²² and the methylation status of RASSF1 might be a novel biomarker for predicting outcome of Wilms' tumour patients. ²³ Moreover, methylation of RASSF1 may be an essential event in the tumorigenesis of Wilms' tumour, which informs its diagnostic, clinical and therapeutic management. CDKN2B gene hypermethylation was observed in W7 to W11, but not in W12 (shown in pink in Figure 3). CDKN2B gene on 9p21.3 encodes the p15^INK4B protein that binds to CDK4 or CDK6 and inhibits its activation. ²⁴ Hypermethylation of CDKN2B CpG islands has been found to occur in the majority of leukaemia patients. ²⁵

Summary of the methylation statuses of 24 tumour suppressor genes in Wilms' tumours by MS‐MLPA. A, Word cloud artwork illustrates the extent of methylation of 24 tumour suppressor genes. B, Methylation statuses of 24 tumour suppressor genes in different types of Wilms’ tumours. W12, W10, W8 and W7 histological patterns were selected for all, stromal, epithelial and blastemal types of Wilms’ tumours respectively

There were methylation of HIC1 in the Wilms' tumours of stromal type (W9, W10 and W11, shown in blue in Figure 3) and methylation of BRCA1 in the Wilms' tumours of epithelial type (W8, W9 and W12, shown in light blue in Figure 3). Hypermethylation at CpG islands in the 5′ ends of tumour suppressor genes is controversial and difficult to interpret. HIC1 gene on 17p13.3 encodes a transcriptional repressor for p21. ²⁶ Hypermethylation of HIC1 gene is found in 3% of Wilms' tumours. ²⁷ BRCA1 gene on 17q21.31 encodes a nuclear phosphoprotein that plays a role in maintaining genomic stability. ³³ However, BRCA1, which demonstrates promoter hypomethylation, is overexpressed in Wilms' tumour. ³⁴ RARB2 gene on 3p24.2 encodes retinoic acid receptor beta that is a type of nuclear receptor activated by all‐trans retinoic acid and 9‐cis retinoic acid. ³⁵ Our results were inconsistent with those of a study by Morris et al in which promoter methylation was absent at RARB2. ²²

CASP8, RARB, MLH1_167, APC and CDKN2A were unmethylated in blastemal‐type Wilms' tumour (shown in green, blue‐green in Figure 3). CASP8 gene on 2q33.1 encodes caspase‐8, an apoptosis‐related cysteine peptidase. ²⁸ In 43% of Wilms' tumours, there is methylation at CASP8. ²² MLH1 gene on 3p22.2 encodes proteins that detect and repair DNA mismatches. ²⁹ A small proportion of Wilms' tumours might be associated with the presence of microsatellite instability. ³⁰ CDKN2A gene on 9p21.3 encodes two proteins that regulate 2 critical cell cycle regulatory pathways, the p53 pathway and the RB1 pathway. ³¹ Arcellana‐Panlilio et al demonstrated methylation of the CpG island in the 5′ region of CDKN2A (p16) in 7 of 7 Wilms' tumours exhibiting decreased CDKN2A expression. ³² This is inconsistent with the finding of negligible methylation of the 5′ CpG island of CDKN2A by Erlich et al. ²¹ APC gene on 5q22.2 encodes a 312‐kDa protein that acts as an antagonist of the Wnt signalling pathway. ³⁶ Schweigert et al noted that activation of the Wnt/β‐catenin pathway is common in Wilms' tumour, but rarely through β‐catenin mutation and APC promoter methylation. ³⁷ However, Koesters et al demonstrated that β‐catenin mutation from CTNNB1 occurs in about 15% of Wilms' tumour and is associated with non‐anaplastic histology. ³⁸ In addition, based on the results of this study, there is notable APC promoter methylation in Wilms' tumour. An important finding is the possibility to further classify mixed‐type Wilms' tumours using genetic results of epithelial, stromal and blastemal components based on MS‐MLPA‐based approach. In particular, the methylation statuses of the above‐mentioned genes make them candidate molecular markers for the diagnosis, stratification and therapy of Wilms' tumours.

In Wilms' tumour, differentiation arrest of renal progenitor cells is incomplete, allowing for maturing lineages of varying proportions. ⁷ The outcome for stromal and epithelial predominant Wilms' tumours is generally excellent. ³⁹ Histological classification of Wilms' tumour is not always possible based on morphology alone. ⁴⁰ From our results, methylation of HIC1 in stroma, BRCA1 in epithelium, and CASP8, RARB, MLH1_167, APC and CDKN2A in either stroma or epithelium can be used to identify stromal predominant and epithelial predominant Wilms' tumours, which are associated with a good outcome. ³⁹ In the situation that the subtype of Wilms' tumour is difficult to diagnose, unmethylated CASP8, RARB, MLH1_167, APC and CDKN2A are potential diagnostic markers for blastemal‐type Wilms' tumours, which are associated with less favourable or poorer outcome. ⁴¹ Stratification of Wilms' tumour by epigenetic analysis of these genes highlights the benefits of methylation status analysis of important tumour suppressor genes. Is it possible that epigenetic modifications in Wilms' tumour provide potential therapeutic options? To answer this question, additional studies based on a larger number of cases and tissue microdissection are necessary.

In summary (Figure 3), all 6 Wilms' tumours showed methylation of RASSF1 specific to tumours, not normal tissues. Moreover, methylation of HIC1 was identified in the Wilms' tumours of stromal type and methylation of BRCA1 was identified in the Wilms' tumours of epithelial type. Unmethylated CASP8, RARB, MLH1_167, APC and CDKN2A were found only in the Wilms' tumour that was of blastemal predominant type. Our results indicated that methylation of RASSF1 may be a vital event in the tumorigenesis of Wilms' tumour, which informs its clinical and therapeutic management. In addition, mixed‐type Wilms' tumours may be classified using genetic results of epithelial, stromal and blastemal components based on MS‐MLPA‐based approach. However, a larger number of cases are necessary to further refine the molecular classification and pathogenesis of Wilms' tumours.

CONFLICT OF INTEREST

The authors declare that they have no competing interests.

ACKNOWLEDGEMENTS

The authors would like to thank the GenePhile Bioscience Laboratory of Ko's Obstetrics and Gynecology Clinic for help with acquisition of data.

Lai Y‐C, Lu M‐Y, Wang W‐C, Hou T‐C, Kuo C‐Y. Correlations between histological characterizations and methylation statuses of tumour suppressor genes in Wilms' tumours. Int J Exp Path. 2022;103:121–128. doi: 10.1111/iep.12442

Funding information

This work was supported by a grant from the Chung Shan Medical University (grant number CSMU‐INT‐110‐16). The funding body had no role in the study design, study setting, analysis or writing of the manuscript.

REFERENCES

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24. Roussel MF. The INK4 family of cell cycle inhibitors in cancer. Oncogene. 1999;18(38):5311‐5317. [DOI] [PubMed] [Google Scholar]
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26. Dehennaut V, Loison I, Boulay G, et al. Identification of p21 (CIP1/WAF1) as a direct target gene of HIC1 (Hypermethylated In Cancer 1). Biochem Biophys Res Commun. 2013;430(1):49‐53. [DOI] [PubMed] [Google Scholar]
27. Rathi A, Virmani AK, Harada K, et al. Aberrant methylation of the HIC1 promoter is a frequent event in specific pediatric neoplasms. Clin Cancer Res. 2003;9(10 Pt 1):3674‐3678. [PubMed] [Google Scholar]
28. Kruidering M, Evan GI. Caspase‐8 in apoptosis: the beginning of "the end"? IUBMB Life. 2000;50(2):85‐90. [DOI] [PubMed] [Google Scholar]
29. Hsieh P, Yamane K. DNA mismatch repair: molecular mechanism, cancer, and ageing. Mech Ageing Dev. 2008;129(7–8):391‐407. [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Diniz G, Aktas S, Cubuk C, et al. Tissue expression of MLH1, PMS2, MSH2, and MSH6 proteins and prognostic value of microsatellite instability in Wilms tumor: experience of 45 cases. Pediatr Hematol Oncol. 2013;30(4):273‐284. [DOI] [PubMed] [Google Scholar]
31. Møller MB, Ino Y, Gerdes AM, et al. Aberrations of the p53 pathway components p53, MDM2 and CDKN2A appear independent in diffuse large B cell lymphoma. Leukemia. 1999;13(3):453‐459. [DOI] [PubMed] [Google Scholar]
32. Arcellana‐Panlilio MY, Egeler RM, Ujack E, et al. Decreased expression of the INK4 family of cyclin‐dependent kinase inhibitors in Wilms tumor. Genes Chromosomes Cancer. 2000;29(1):63‐69. [DOI] [PubMed] [Google Scholar]
33. Roy R, Chun J, Powell SN. BRCA1 and BRCA2: different roles in a common pathway of genome protection. Nat Rev Cancer. 2011;12(1):68‐78. [DOI] [PMC free article] [PubMed] [Google Scholar]
34. Guerra J, Pereira BMS, Jgvd C, et al. Genes controlled by DNA methylation are involved in Wilms tumor progression. Cells. 2019;8(8):921. [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Allenby G, Bocquel MT, Saunders M, et al. Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids. Proc Natl Acad Sci USA. 1993;90(1):30‐34. [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene. 2017;36(11):1461‐1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
37. Schweigert A, Fischer C, Mayr D, et al. Activation of the Wnt/beta‐catenin pathway is common in wilms tumor, but rarely through beta‐catenin mutation and APC promoter methylation. Pediatr Surg Int. 2016;32(12):1141‐1146. [DOI] [PubMed] [Google Scholar]
38. Koesters R, Ridder R, Kopp‐Schneider A, et al. Mutational activation of the beta‐catenin proto‐oncogene is a common event in the development of Wilms' tumors. Cancer Res. 1999;59(16):3880‐3882. [PubMed] [Google Scholar]
39. Verschuur AC, Vujanic GM, Van Tinteren H, et al. Stromal and epithelial predominant Wilms tumours have an excellent outcome: the SIOP 93 01 experience. Pediatr Blood Cancer. 2010;55(2):233‐238. [DOI] [PubMed] [Google Scholar]
40. Popov SD, Sebire NJ, Vujanic GM. Wilms' Tumour ‐ Histology and Differential Diagnosis. In: van den Heuvel‐Eibrink MM, ed. Wilms Tumor. Codon Publication; 2016:3‐21. [PubMed] [Google Scholar]
41. Vujanic GM, Sandstedt B, Harms D, et al. Revised international society of paediatric oncology (SIOP) working classification of renal tumors of childhood. Med Pediatr Oncol. 2002;38(2):79‐82. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0001] 1. Brown KW, Malik KT. The molecular biology of Wilms tumour. Expert Rev Mol Med. 2001;2001:1‐16. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0002] 2. Pastore G, Znaor A, Spreafico F, et al. Malignant renal tumours incidence and survival in European children (1978–1997): report from the automated childhood cancer information system project. Eur J Cancer. 2006;42(13):2103‐2114. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0003] 3. Steliarova‐Foucher E, Colombet M, Ries LAG, et al. International incidence of childhood cancer, 2001–10: a population‐based registry study. Lancet Oncol. 2017;18(6):719‐731. [DOI] [PMC free article] [PubMed] [Google Scholar]

[iep12442-bib-0004] 4. de Almeida C, Cardoso L, Rodriguez‐Laguna L, et al. Array CGH analysis of paired blood and tumor samples from patients with sporadic Wilms tumor. PLoS One. 2015;10(8):e0136812. [DOI] [PMC free article] [PubMed] [Google Scholar]

[iep12442-bib-0005] 5. Mahamdallie S, Yost S, Poyastro‐Pearson E, et al. Identification of new Wilms tumour predisposition genes: an exome sequencing study. Lancet Child Adolesc Health. 2019;3(5):322‐331. [DOI] [PMC free article] [PubMed] [Google Scholar]

[iep12442-bib-0006] 6. Treger TD, Chowdhury T, Pritchard‐Jones K, et al. The genetic changes of Wilms tumour. Nat Rev Nephrol. 2019;15(4):240‐251. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0007] 7. Gadd S, Huff V, Walz AL, et al. A children's oncology group and TARGET initiative exploring the genetic landscape of Wilms tumor. Nat Genet. 2017;49(10):1487‐1494. [DOI] [PMC free article] [PubMed] [Google Scholar]

[iep12442-bib-0008] 8. Baylin SB. DNA methylation and gene silencing in cancer. Nat Clin Pract Oncol. 2005;2(Suppl 1):S4‐S11. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0009] 9. Lettini AA, Guidoboni M, Fonsatti E, et al. Epigenetic remodelling of DNA in cancer. Histol Histopathol. 2007;22(12):1413‐1424. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0010] 10. Yamada Y, Yamada Y. The causal relationship between epigenetic abnormality and cancer development: in vivo reprogramming and its future application. Proc Jpn Acad Ser B Phys Biol Sci. 2018;94(6):235‐247. [DOI] [PMC free article] [PubMed] [Google Scholar]

[iep12442-bib-0011] 11. Chiang MR, Kuo CW, Wang WC, et al. Correlations between histological and array comparative genomic hybridization characterizations of Wilms tumor. Pathol Oncol Res. 2019;25(3):1199‐1206. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0012] 12. Lu MY, Wang WC, Lin CW, et al. Identification of a constitutional mutation in the WT1 gene in Taiwanese patients with Wilms tumor. Adv Biosci Biotechnol. 2014;5:230‐234. [Google Scholar]

[iep12442-bib-0013] 13. Lu MY, Wang WC, Hou TC, et al. Methylation statuses of H19DMR and KvDMR at WT2 in Wilms tumors in Taiwan. Pathol Oncol Res. 2020;26(4):2153‐2159. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0014] 14. Homig‐Holzel C, Savola S. Multiplex ligation‐dependent probe amplification (MLPA) in tumor diagnostics and prognostics. Diagn Mol Pathol. 2012;21(4):189‐206. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0015] 15. Wang WC, Lai YC. Genetic analysis results of mature cystic teratomas of the ovary in Taiwan disagree with the previous origin theory of this tumor. Hum Pathol. 2016;52:128‐135. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0016] 16. Wang WC, Lai YC. Evidence of metachronous development of ovarian teratomas: a case report of bilateral mature cystic teratomas of the ovaries and systematic literature review. J Ovarian Res. 2017;10(1):17. [DOI] [PMC free article] [PubMed] [Google Scholar]

[iep12442-bib-0017] 17. Dubois F, Bergot E, Zalcman G, et al. RASSF1A, puppeteer of cellular homeostasis, fights tumorigenesis, and metastasis‐an updated review. Cell Death Dis. 2019;10(12):928. [DOI] [PMC free article] [PubMed] [Google Scholar]

[iep12442-bib-0018] 18. Wagner KJ, Cooper WN, Grundy RG, et al. Frequent RASSF1A tumour suppressor gene promoter methylation in Wilms' tumour and colorectal cancer. Oncogene. 2002;21(47):7277‐7282. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0019] 19. Harada K, Toyooka S, Maitra A, et al. Aberrant promoter methylation and silencing of the RASSF1A gene in pediatric tumors and cell lines. Oncogene. 2002;21(27):4345‐4349. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0020] 20. Pfeifer GP, Dammann R. Methylation of the tumor suppressor gene RASSF1A in human tumors. Biochemistry (Mosc). 2005;70(5):576‐583. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0021] 21. Ehrlich M, Jiang G, Fiala E, et al. Hypomethylation and hypermethylation of DNA in Wilms tumors. Oncogene. 2002;21(43):6694‐6702. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0022] 22. Morris MR, Hesson LB, Wagner KJ, et al. Multigene methylation analysis of Wilms' tumour and adult renal cell carcinoma. Oncogene. 2003;22(43):6794‐6801. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0023] 23. Ohshima J, Haruta M, Fujiwara Y, et al. Methylation of the RASSF1A promoter is predictive of poor outcome among patients with Wilms tumor. Pediatr Blood Cancer. 2012;59(3):499‐505. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0024] 24. Roussel MF. The INK4 family of cell cycle inhibitors in cancer. Oncogene. 1999;18(38):5311‐5317. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0025] 25. Takeuchi S, Matsushita M, Zimmermann M, et al. Clinical significance of aberrant DNA methylation in childhood acute lymphoblastic leukemia. Leuk Res. 2011;35(10):1345‐1349. [DOI] [PMC free article] [PubMed] [Google Scholar]

[iep12442-bib-0026] 26. Dehennaut V, Loison I, Boulay G, et al. Identification of p21 (CIP1/WAF1) as a direct target gene of HIC1 (Hypermethylated In Cancer 1). Biochem Biophys Res Commun. 2013;430(1):49‐53. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0027] 27. Rathi A, Virmani AK, Harada K, et al. Aberrant methylation of the HIC1 promoter is a frequent event in specific pediatric neoplasms. Clin Cancer Res. 2003;9(10 Pt 1):3674‐3678. [PubMed] [Google Scholar]

[iep12442-bib-0028] 28. Kruidering M, Evan GI. Caspase‐8 in apoptosis: the beginning of "the end"? IUBMB Life. 2000;50(2):85‐90. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0029] 29. Hsieh P, Yamane K. DNA mismatch repair: molecular mechanism, cancer, and ageing. Mech Ageing Dev. 2008;129(7–8):391‐407. [DOI] [PMC free article] [PubMed] [Google Scholar]

[iep12442-bib-0030] 30. Diniz G, Aktas S, Cubuk C, et al. Tissue expression of MLH1, PMS2, MSH2, and MSH6 proteins and prognostic value of microsatellite instability in Wilms tumor: experience of 45 cases. Pediatr Hematol Oncol. 2013;30(4):273‐284. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0031] 31. Møller MB, Ino Y, Gerdes AM, et al. Aberrations of the p53 pathway components p53, MDM2 and CDKN2A appear independent in diffuse large B cell lymphoma. Leukemia. 1999;13(3):453‐459. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0032] 32. Arcellana‐Panlilio MY, Egeler RM, Ujack E, et al. Decreased expression of the INK4 family of cyclin‐dependent kinase inhibitors in Wilms tumor. Genes Chromosomes Cancer. 2000;29(1):63‐69. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0033] 33. Roy R, Chun J, Powell SN. BRCA1 and BRCA2: different roles in a common pathway of genome protection. Nat Rev Cancer. 2011;12(1):68‐78. [DOI] [PMC free article] [PubMed] [Google Scholar]

[iep12442-bib-0034] 34. Guerra J, Pereira BMS, Jgvd C, et al. Genes controlled by DNA methylation are involved in Wilms tumor progression. Cells. 2019;8(8):921. [DOI] [PMC free article] [PubMed] [Google Scholar]

[iep12442-bib-0035] 35. Allenby G, Bocquel MT, Saunders M, et al. Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids. Proc Natl Acad Sci USA. 1993;90(1):30‐34. [DOI] [PMC free article] [PubMed] [Google Scholar]

[iep12442-bib-0036] 36. Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene. 2017;36(11):1461‐1473. [DOI] [PMC free article] [PubMed] [Google Scholar]

[iep12442-bib-0037] 37. Schweigert A, Fischer C, Mayr D, et al. Activation of the Wnt/beta‐catenin pathway is common in wilms tumor, but rarely through beta‐catenin mutation and APC promoter methylation. Pediatr Surg Int. 2016;32(12):1141‐1146. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0038] 38. Koesters R, Ridder R, Kopp‐Schneider A, et al. Mutational activation of the beta‐catenin proto‐oncogene is a common event in the development of Wilms' tumors. Cancer Res. 1999;59(16):3880‐3882. [PubMed] [Google Scholar]

[iep12442-bib-0039] 39. Verschuur AC, Vujanic GM, Van Tinteren H, et al. Stromal and epithelial predominant Wilms tumours have an excellent outcome: the SIOP 93 01 experience. Pediatr Blood Cancer. 2010;55(2):233‐238. [DOI] [PubMed] [Google Scholar]

[iep12442-bib-0040] 40. Popov SD, Sebire NJ, Vujanic GM. Wilms' Tumour ‐ Histology and Differential Diagnosis. In: van den Heuvel‐Eibrink MM, ed. Wilms Tumor. Codon Publication; 2016:3‐21. [PubMed] [Google Scholar]

[iep12442-bib-0041] 41. Vujanic GM, Sandstedt B, Harms D, et al. Revised international society of paediatric oncology (SIOP) working classification of renal tumors of childhood. Med Pediatr Oncol. 2002;38(2):79‐82. [DOI] [PubMed] [Google Scholar]

PERMALINK

Correlations between histological characterizations and methylation statuses of tumour suppressor genes in Wilms' tumours

Yen‐Chein Lai

Meng‐Yao Lu

Wen‐Chung Wang

Tai‐Cheng Hou

Chen‐Yun Kuo

Abstract

1. INTRODUCTION