Wilms tumour histology is determined by distinct types of precursor lesions and not epigenetic changes

Current models of Wilms tumour development propose that histological features of the tumours are programmed by the underlying molecular aberrations. For example, tumours associated with WT1 mutations arise from intralobar nephrogenic rests (ILNR), concur with CTNNB1 mutations and have distinct histology, whereas tumours with IGF2 loss of imprinting (LOI) often arise from perilobar nephrogenic rests (PLNR). Intriguingly, ILNR and PLNR are found simultaneously in Wilms tumours in children with overgrowth who have constitutional IGF2 LOI. We therefore examined whether the precursor lesions or early epigenetic changes are the primary determinant of Wilms tumour histology. We examined the histological features and gene expression profiles of IGF2 LOI tumours and WT1-mutant tumours which are associated with PLNR and/or ILNR. Two distinct types of IGF2 LOI tumours were identified: the first type had a blastemal-predominant histology associated with PLNR, while the second subtype had a myogenic histology, increased expression of mesenchymal lineage genes and an association with ILNR, similar to WT1-mutant tumours. These ILNR-associated IGF2 LOI tumours also showed signatures of activation of the WNT signalling pathway: differential expression of beta-catenin targets (MMP2, RARG, DKK1) and WNT antagonist genes (DKK1, WIF1, SFRP4). Unexpectedly, the majority of these tumours had CTNNB1 mutations, which are normally only seen in WT1-mutant tumours. The absence of WT1 mutations in tumours with IGF2 LOI indicated that CTNNB1 mutations occur predominantly in tumours arising from ILNR independent of the presence or absence of WT1 mutations. Thus, even though these two classes of tumours with IGF2 LOI have the same underlying predisposing epigenetic error, the tumour histology and the gene expression profiles are determined by the nature of the precursor cells within the nephrogenic rests and subsequent CTNNB1 mutations.     

Different CTNNB1 mutations as molecular genetic proof for the independent origin of four Wilms tumours in a patient with a novel germ line WT1 mutation

We describe a patient with a novel WT1 pS50X germ line mutation, who developed bilateral Wilms tumours, both with stromal-type histology. Both tumours showed loss of the wild type WT1 allele (loss of heterozygosity (LOH)) and a tumour specific mutation in catenin beta1 (CTNNB1), S45P in the left and Delta45S in the right tumour. Molecular analysis of microdissected cells from the left tumour revealed the same S45P CTNNB1 mutation in blastema, tubuli, stroma and muscle, and a different CTNNB1 mutation (T41A) in stromal cells isolated from another area of the same slide. Microdissection of two areas of muscle cells from the right tumour revealed the same Delta45S mutation and no CTNNB1 mutation nor LOH of WT1 in normal kidney cells. One year later, the patient developed a new set of bilateral tumours. Both tumours showed LOH of the wild type WT1 allele, but different CTNNB1 mutations as in the first tumours: S45C on the right and S45F on the left side, demonstrating that these developed independently and are not relapses. This case demonstrates the high risk for the development of Wilms tumours in patients with germ line truncation mutations.     

Wilms tumor genetics: mutations in WT1, WTX, and CTNNB1 account for only about one-third of tumors

Wilms tumor is genetically heterogeneous, and until recently only one Wilms tumor gene was known, WT1 at 11p13. However, WT1 is altered in only approximately 20% of Wilms tumors. Recently a novel gene, WTX at Xq11.1, was reported to be mutated in Wilms tumors. No overlap between tumors with mutations in WTX and WT1 was noted, suggesting that WT1 and WTX mutations could account for the genetic basis of roughly half of Wilms tumors. To assess the frequency of WTX mutations and their relationship to WT1 mutations in a larger (n = 125) panel of Wilms tumors which had been thoroughly assessed for mutations in WT1, we conducted a complete mutational analysis of WTX that included sequencing of the entire coding region and quantitative PCR to identify deletions of the WTX gene. Twenty-three (18.4%) tumors carried a total of 24 WTX mutations, a lower WTX mutation frequency than that previously observed. Surprisingly, we observed an equivalent frequency of WTX mutations in tumors with mutations in either or both WT1 and CTNNB1 (20.0%) and tumors with no mutation in either WT1 or CTNNB1 (17.5%). WTX has been reported to play a role in the WNT/beta-catenin signaling pathway, and, interestingly, WTX deletion/truncation mutations appeared to be rare in tumors carrying exon 3 mutations of CTNNB1, encoding beta-catenin. Our findings indicate that WT1 and WTX mutations occur with similar frequency, that they partially overlap in Wilms tumors, and that mutations in WT1, WTX, and CTNNB1 underlie the genetic basis of about one-third of Wilms tumors.     

Target genes of the WNT/beta-catenin pathway in Wilms tumors

The WNT/beta-catenin pathway is involved in numerous human cancers. Mutations of the CTNNB1 (beta-catenin) gene have also been detected in a subset of pediatric Wilms tumors, but the target genes of the deregulated WNT/beta-catenin pathway in these tumors have yet to be identified. To compare gene expression profiles of Wilms tumors with and without mutations of CTNNB1, we used 11.5-k cDNA microarrays. Most of the tumors (86%) had received preoperative chemotherapy as mandated by the European SIOP protocol. The comparison between Wilms tumors with and without CTNNB1 mutations revealed several target genes specifically deregulated in CTNNB1-mutated Wilms tumors. Among these, PITX2, APCDD1, and two members of the endothelin axis (EDN3 and EDNRA) are directly activated downstream targets of the WNT/beta-catenin pathway that may enhance proliferation of these tumor cells. In addition, several upstream inhibitors of WNT/beta-catenin signaling like WIF1 and PRDC were also strongly up-regulated in the CTNNB1-mutated Wilms tumors. This overexpression may be a negative feedback mechanism in tumors with uncontrolled WNT signaling. Moreover, we identified deregulated genes in both the retinoic acid and the RAS pathways, such as ATX/ENPP2 and RIS1, suggesting an association between these two pathways with that of WNT. In addition, the strong representation of muscle-related genes in the expression profile of CTNNB1-mutated Wilms tumors corresponded to histologically detectable areas of myomatous cells in these tumors that displayed intense and preferential nuclear beta-catenin antibody staining. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.     

Expression of beta-catenin in gastroenteropancreatic endocrine tumours: a study of 229 cases

AIMS: To clarify the role of beta-catenin in digestive endocrine carcinogenesis, a large and representative series of gastroenteropancreatic endocrine tumours was analysed in order to determine the incidence and pattern of beta-catenin changes and to analyse the clinical and histological characteristics of the tumours presenting immunohistochemically detectable changes in beta-catenin expression. METHODS: 229 cases of gastroenteropancreatic endocrine tumours (stomach, 11; duodenum and ampulla, 29; jejunum and ileum, 51; appendix, 13; colon and rectum, 17; and pancreas, 108) were studied by immunohistochemistry to assess the pattern of distribution of beta-catenin (membranous, cytoplasmic or nuclear). DNA was analysed to detect mutations in exon 3 of the CTNNB1 gene. RESULTS: The distribution of immunoreactive beta-catenin protein was membranous in 164 cases, cytoplasmic in 58 cases and nuclear in seven cases. No mutation was detected in exon 3 of the CTNNB1 gene in any case. The seven cases with nuclear accumulation of beta-catenin were large tumours (mean size 44 (standard deviation (SD) 18.5) mm) with metastases, including liver metastases in five cases, high Ki-67 index (mean 34% (SD 16.5%)) and cyclin D1 overexpression; p53 accumulation was detected in six cases. Five patients died of disease; the mean (SD) survival was 13.6 (4.8) months. CONCLUSIONS: Immunohistochemically detectable nuclear accumulation of beta-catenin is infrequent in gastroenteropancreatic endocrine tumours and is usually not associated with mutations in CNNTB1 exon 3. Changes in beta-catenin expression are late events in digestive endocrine carcinogenesis, associated with tumour progression and dissemination.     

Significance of beta-catenin and pRB pathway components in malignant ovarian germ cell tumours: INK4A promoter CpG island methylation is associated with cell proliferation

To clarify the mechanisms underlying cell cycle promotion in malignant germ cell tumours of the ovary (MGCTOs), beta-catenin and components of the pRB pathway, cyclin D1 and p16, were analysed in relation to cell proliferation. Immunohistochemically, p16 protein was not expressed in a number of MGCTOs (9 of 42 tumours: 21.4%) and was associated with p16 gene (INK4A) promoter 5'-CpG islands methylation. Amplification of the cyclin D1 gene (CCND1) was detected in a small number of MGCTOs (5 of 42 tumours: 13.5%). Reduced expression of p16 due to promoter methylation correlated significantly with increased cell proliferation as evidenced by Ki-67 labelling index (p < 0.001) and mitotic index (p < 0.01). In some tumour types, nuclear localization of beta-catenin has been reported to be associated with beta-catenin gene (CTNNB1) mutation, cyclin D1 overexpression, and increased cell proliferation. Nuclear localization of beta-catenin, which was observed in MGCTOs other than dysgerminoma, was not associated with cyclin D1 expression and increased cell proliferation, but appeared to be related to tumour differentiation. Furthermore, CTNNB1 mutations were not detected in any of the MGCTOs examined. Our results suggest that reduced expression of p16 due to INK4A promoter methylation is one of the principal factors that promote cell proliferation in MGCTOs. Thus, p16 may be a novel target for gene therapies to treat MGCTOs.     

Imprinting, expression, and localisation of DLK1 in Wilms tumours

BACKGROUND: Loss of imprinting (LOI) of the H19/IGF2 domain is a common feature of Wilms tumour. The GTL2/DLK1 domain is also imprinted and is structurally similar to H19/IGF2. The question arises as to whether DLK1 also undergoes LOI in Wilms tumour, or whether the LOI mechanism is restricted to the H19/IGF2 domain. AIM: To investigate the imprinting status of DLK1 in Wilms tumours with IGF2 LOI. The cellular localisation of DLK1 in the tumours was also examined. METHODS: DLK1 expression was measured by quantitative real time polymerase chain reaction (Q-PCR) in 30 Wilms tumours that had previously been classified according to whether they had IGF2 LOI, WT1 mutations, or 11p15.5 loss of heterozygosity. Allele specific expression of DLK1 was examined by direct sequencing using a DLK1 exon 5 polymorphism (rs1802710). Immunohistochemical analysis of DLK1 was performed on 13 tumours and two intralobar nephrogenic rests, in addition to two fetal kidneys and one fetal skeletal muscle sample. RESULTS: Ten of 30 tumours were heterozygous for rs1802710 and all tumours showed retention of imprinting of DLK1. Moderate to high expression of DLK1 was detected by Q-PCR in nine of 13 tumours with myogenic differentiation. Immunohistochemical expression of DLK1 was detected in the myogenic elements. CONCLUSION: LOI does not occur at the GTL2/DLK1 domain in Wilms tumour. This finding suggests that LOI at 11p15.5 does not reflect non-specific disruption of a shared imprinting mechanism. DLK1 expression in Wilms tumour might reflect the presence of myogenic differentiation, rather than an alteration of its imprinting status.     

Beta-catenin dysregulation in thyroid neoplasms: down-regulation, aberrant nuclear expression, and CTNNB1 exon 3 mutations are markers for aggressive tumor phenotypes and poor prognosis

beta-catenin has a role in cell adhesion and Wnt signaling. It is mutated or otherwise dysregulated in a variety of human cancers. In this study we assess beta-catenin alteration in 145 thyroid tumors samples from 127 patients. beta-catenin was localized using immunofluorescence and mutational analysis was performed by single-strand conformational polymorphism. Membrane beta-catenin expression was decreased in eight of 12 (66%) adenomas and in all 115 carcinomas (P: < 0.0001). Among carcinomas, reduced membrane beta-catenin was associated with progressive loss of tumor differentiation (P: < 0.0001). CTNNB1 exon 3 mutations and nuclear beta-catenin localization were restricted to poorly differentiated [7 of 28 (25%) and 6 of 28 cases (21.4%), respectively] or undifferentiated carcinomas [19 of 29 (65.5%) and 14 of 29 (48.3%) cases, respectively]. Poorly differentiated tumors always featured mutations involving Ser and Thr residues and were characterized by Thr to Ile amino acid substitutions (P: = 0.0283). The association between CTNNB1 exon 3 mutations and aberrant nuclear immunoreactivity (P: = 0.0020) is consistent with Wnt activation because of stabilizing beta-catenin mutations. Low membrane beta-catenin expression as well as its nuclear localization or CTNNB1 exon 3 mutations are significantly associated with poor prognosis, independent of conventional prognostic indicators for thyroid cancer but not of tumor differentiation. Analysis of beta-catenin dysregulation may be useful to objectively subtype thyroid neoplasms and more accurately predict outcomes.     

CTNNB1 mutations and beta-catenin expression in endometrial carcinomas

Mutations in the beta-catenin gene (CTNNB 1) with abnormal nuclear accumulation of beta-catenin have recently been identified in endometrial carcinoma (EC). Their relationship with microsatellite instability (MI) is unclear. It has been suggested that matrix metalloproteinase-7 (MMP-7) and cyclin D1 (cD) genes are targets for beta-catenin activation. DNA from 73 patients with EC was obtained from tumor and normal tissue (59 endometrioid and 14 nonendometrioid). CTNNB 1 mutations in exon 3 were assessed by single-strand conformation polymorphism and DNA sequencing. The results were correlated with immunostaining for beta-catenin, MMP-7, and cD. Three (CA)n repeats and mononucleotide tracts BAT 25 and BAT 26 had been previously used for MI analysis. CTNNB1 mutations were identified in 15 ECs (20.5%), all of them endometrioid carcinomas (15 of 59; 25.4%). They occurred in 6 of 19 MI-positive ECs (31.5%) and in 9 of 54 MI-negative ECs (16.6%). Eleven of the 15 CTNNB 1-mutated ECs showed beta-catenin nuclear immunostaining (P <.05). MMP-7 expression (>50% cells) was observed in 23 ECs, with 7 of these showing CTNNB 1 mutations. Significant expression of cD (>50% cells) was detected in 8 ECs, with 5 of these exhibiting CTNNB 1 mutations (P <.05). The results confirm that beta-catenin plays a role in endometrial carcinogenesis, particularly in endometrioid carcinomas. The results also suggest that MMP-7 and particularly cD may be targets of beta-catenin activation in ECs.     

Analysis of somatic APC mutations in rare extracolonic tumors of patients with familial adenomatous polyposis coli

Patients with familial adenomatous polyposis coli (FAP) carry heterozygous mutations of the APC gene. At a young age, these patients develop multiple colorectal adenomas that consistently display a second somatic mutation in the remaining APC wild-type allele. Inactivation of APC leads to impaired degradation of beta-catenin, thereby promoting continuous cell-cycle progression. The role of APC inactivation in rare extracolonic tumors of FAP patients has not been characterized sufficiently. Among tissue specimen from 174 patients with known APC germ-line mutations, we identified 8 tumors infrequently seen in FAP. To investigate the pathogenic role of APC pathway deregulation in these lesions, they were analyzed for second-hit somatic mutations in the mutational cluster region of the APC gene. Immunohistochemistry was performed to compare the expression pattern of beta-catenin to the mutational status of the APC gene. Exon 3 of the beta-catenin gene (CTNNB1) was analyzed for activating mutations to investigate alternative mechanisms of elevated beta-catenin concentration. Although CTNNB1 mutations were not observed, second somatic APC mutations were found in 4 of the 8 tumors: a uterine adenocarcinoma, a hepatocellular adenoma, an adrenocortical adenoma, and an epidermal cyst. These tumors showed an elevated concentration of beta-catenin. No APC mutations were seen in focal nodular hyperplasia of the liver, angiofibrolipoma, and seborrheic wart. This is the first study reporting second somatic APC mutations in FAP-associated uterine adenocarcinoma and epidermal cysts. Furthermore, our data strengthen a role for impaired APC function in the pathogenesis of adrenal and hepatic neoplasms in FAP patients.     

Cribriform-morular variant of papillary thyroid carcinoma: a pathological and molecular genetic study with evidence of frequent somatic mutations in exon 3 of the beta-catenin gene

The cribriform-morular variant (C-MV), an unusual and peculiar subtype of papillary thyroid carcinoma (PTC), has been observed frequently in familial adenomatous polyposis (FAP)-associated thyroid carcinoma and also in sporadic thyroid carcinoma. In this paper, five young women with the C-MV of PTC, aged 22-34 years at cancer diagnosis, are reported; two of them had attenuated FAP. Grossly, one FAP-associated tumour and one sporadic tumour were multicentric and the others were solitary. Histologically, the tumours were encapsulated and exhibited a combination of cribriform, follicular, trabecular, solid, and papillary patterns of growth, with morular areas. Immunohistochemically, the tumour cells showed cytoplasmic expression of thyroglobulin, neuron-specific enolase, epithelial membrane antigen, high- and low-molecular-weight cytokeratins, vimentin, and bcl-2 protein; nuclear expression of oestrogen and progesterone receptors, and retinoblastoma protein; and cytoplasmic and nuclear accumulation of beta-catenin. Germline mutations of the adenomatous polyposis coli (APC) gene were investigated using the protein truncation test in four subjects, including two FAP individuals. Germline APC mutation was identified in only one FAP patient with the multicentric C-MV of PTC, who had a thymidine deletion at codon 512, resulting in a frameshift leading to a premature stop codon. No loss of heterozygosity of loci close to the APC gene was detected in tumour tissues from these four patients. Somatic mutation analysis of exon 3 of the beta-catenin gene (CTNNB1) revealed alterations in seven tumours from all five individuals: one at a serine residue (codon 29), three at amino acids adjacent to serine or threonine residues (codons 22, 39, and 44), and three at other amino acids (codons 49, 54, and 56). Moreover, each of two different tumours examined from two patients with the multicentric C-MV of PTC, had different somatic mutations of the CTNNB1 gene. Taken together, these data suggest that accumulation of mutant beta-catenin contributes to the development of the C-MV of PTC.     

Do intronic mutations affecting splicing of WT1 exon 9 cause Frasier syndrome?

The WT1 gene, one of the genes responsible for Wilms tumour, is thought to play a crucial role in the development of the kidneys and gonads. This gene encodes four protein isoforms resulting from two alternative splicing sites, one of which involves inclusion or exclusion of lysine, threonine, and serine (KTS) between the third and fourth zinc finger domains. WT1 is virtually always mutationally inactivated in patients with Denys-Drash syndrome. We analysed WT1 in eight patients who had been diagnosed as having this syndrome, and identified five previously unknown mutations affecting splicing donor sites of intron 9. These mutations affect alternative splicing. The isoforms retaining KTS are not produced. The clinical features of the patients with these intronic mutations were consistent with those of Frasier syndrome, characterised by a more slowly progressive nephropathy than Denys-Drash syndrome, associated streak gonads, and no Wilms tumour development. Our results indicate that WT1 isoforms, including/excluding KTS, have different functions in tumorigenesis and organogenesis of the kidneys and gonads.     

Duplication of paternal IGF2 or loss of maternal IGF2 imprinting occurs in half of Wilms tumors with various structural WT1 abnormalities

The WT1 gene essential for the embryonic kidney development is mutated in 15-25% of Wilms tumors (WTs). To clarify whether genetic subtypes of WT1 abnormalities are correlated with IGF2 or CTNNB1 alterations or clinicopathological characteristics, we performed comprehensive WT1, IGF2, and CTNNB1 analyses of 36 WTs with WT1 abnormalities using single nucleotide polymorphism arrays, and methylation analysis of the IGF2-H19 differentially methylated region. The tumors were classified into three subtypes based on WT1 abnormalities: 13 with WT1 deletion, 12 with WT1 mutation, and 11 with both deletion and mutation. IGF2 alterations were found in 50% (18/36), paternal uniparental disomy (UPD) of 11p13-11p15 in 13 tumors, UPD limited to 11p15 in 3, and loss of IGF2 imprinting in 2. Quantitative RT-PCR analysis showed that tumors with IGF2 alteration had higher levels of IGF2 mRNA than tumors without IGF2 alteration (P = 0.02). WT1 mRNA levels were very low in six of eight WTs with WT1 deletion, whereas four of eight WTs with WT1 mutation or both deletion and mutation showed higher levels of WT1 mRNA than fetal kidneys. WTs with WT1 mutations occurred in younger patients (P < 0.01), and WTs with mutations or both deletion and mutation (12/23) were more frequent in syndromic patients than WTs (1/13) with the deletion (P = 0.02). WTs with WT1 mutations or both deletion and mutation had the triphasic histological-type (15/23; P = 0.03) and CTNNB1 mutation (17/23; P = 0.03) more frequently than WTs with the deletion (2/13 and 4/13). Thus, three WT1 subtypes were correlated with certain genetic and clinicopathological characteristics.     

Frequent nuclear beta-catenin accumulation and associated mutations in endometrioid-type endometrial and ovarian carcinomas with squamous differentiation

Focal squamous differentiation is a common feature of endometrioid endometrial and ovarian carcinomas (E-Em and E-Ova Cas). A close association between mutated beta-catenin accumulation and alteration in cellular morphology has recently been demonstrated in murine L cell lines. To clarify the possible role of beta-catenin abnormalities and changes in tumour morphology, 60 grade (G) 1 or G2 E-Em Cas with areas of squamous differentiation (SqD), including morules and squamous metaplastic (SqM) foci, as well as 32 G1 or G2 tumours without such lesions, were investigated and the results compared with findings for c-jun and wnt-1 expression. Twenty-three E-Ova Cas, with and without SqD lesions, were also examined. In E-Em Cas, frequent nuclear beta-catenin accumulation was observed in 22 (84.6%) of 26 tumours with morules and 15 (45.5%) of 33 with SqM foci, in contrast to 4 (12.5%) of 32 without such lesions. Similar findings were also noted for mutations in exon 3 of the beta-catenin gene, involving codons 32, 33, 34, 37, 41, and 45, the single nucleotide substitutions being identical between SqD and the surrounding carcinoma tissue in most informative cases. The mutations were positively related to nuclear immunopositivity, but inversely to membrane expression, while there was no association with the status of c-jun or wnt-1. These E-Ova Cas, nuclear beta-catenin accumulation and mutations were limited to tumours with SqD features, independent of c-jun and wnt-1 status. These data indicate that beta-catenin abnormalities are relatively common in E-Em and E-Ova Cas with SqD features, implying a role in the squamous differentiation of tumour cells, not necessarily related to c-jun and/or wnt-1 status.     

No difference in the occurrence of mismatch repair defects and APC and CTNNB1 genes mutation in a multi-racial colorectal carcinoma patient cohort

BACKGROUND AND AIMS: Colorectal cancers of different subtypes involve different pathogenic pathways like the Wnt and the mutator pathways. In this study, we screened 73 colorectal cancer cases from a multi-racial group for genetic and expression profile defects with the aim of correlating these with patients' clinicopathological characteristics. METHODS: Mutation screening of the entire coding region of APC and exon 3 of CTNNB1, loss of heterozygosity (LOH) of APC, and microsatellite instability (MSI) status were assessed for 44 patients with available paired frozen normal and tumour tissues. In addition, 29 cases with available paraffin embedded tumour blocks were screened for mutation in exon 3 of CTNNB1, the APC mutation cluster region (codon 1286-1513), and hMLH1, hMSH2, hMSH6 protein expressions by immunohistochemistry method. RESULTS: In our study, 15/73 cases showed APC mutations (20.5%), 1/73 cases had CTNNB1 mutation (1.4%), 5/32 cases had APC LOH (15.6%), and 16/70 (22.9%) cases revealed at least some form of mismatch repair (MMR) defect. Tumour grade (poor differentiation) was found to correlate significantly with right-sided tumour and mucinous histology (p = 0.01879 and 0.00320, respectively). Patients of younger age (below 45 years) more often had tumours of mucinous histology (p = 0.00014), while patients of older age (above 75 years) more often had tumours on the right side of the colon (p = 0.02448). Tumours of the mucinous histology subtype often had MMR defects (p = 0.02686). There was no difference in the occurrence of APC and CTNNB1 mutations and MMR defects found within our multi-racial colorectal cancer patient cohort. CONCLUSION: Our findings support the notion that racial factor may not be related to the occurrence of MMR defects and APC and CTNNB1 mutations in our multi-racial patient cohort.