Bromodomain 4 inhibition leads to MYCN downregulation in Wilms tumor.

BACKGROUND: Wilms tumor is the most common childhood kidney cancer. Two distinct histological subtypes of Wilms tumor have been described: tumors lacking anaplasia (the favorable subtype) and tumors displaying anaplastic features (the unfavorable subtype). Children with favorable disease generally have a very good prognosis, whereas those with anaplasia are oftentimes refractory to standard treatments and suffer poor outcomes, leading to an unmet clinical need. MYCN dysregulation has been associated with a number of pediatric cancers including Wilms tumor. PROCEDURES: In this context, we undertook a functional genomics approach to uncover novel therapeutic strategies for those patients with anaplastic Wilms tumor. Genomic analysis and in vitro experimentation demonstrate that cell growth can be reduced by modulating MYCN overexpression via bromodomain 4 (BRD4) inhibition in both anaplastic and nonanaplastic Wilms tumor models. RESULTS: We observed a time-dependent reduction of MYCN and MYCC protein levels upon BRD4 inhibition in Wilms tumor cell lines, which led to cell death and proliferation suppression. BRD4 inhibition significantly reduced tumor volumes in Wilms tumor patient-derived xenograft (PDX) mouse models. CONCLUSIONS: We suggest that AZD5153, a novel dual-BRD4 inhibitor, can reduce MYCN levels in both anaplastic and nonanaplastic Wilms tumor cell lines, reduces tumor volume in Wilms tumor PDXs, and should be further explored for its therapeutic potential.

WT1-Mutant Wilms Tumor Progression Is Associated With Diverting Clonal Mutations of CTNNB1.

WT1-mutant Wilms tumors exhibit a high rate of concomitant CTNNB1 mutations, associated with activated Wnt signaling. Here, we show by laser and manual microdissection of different histologic cell types from 6 WT1-mutant tumor samples that 1 patient's tumor can contain up to 4 distinct mutations in CTNNB1 and/or WTX. Consecutive sections may also harbor different CTNNB1 mutations. The variability of activating CTNNB1 mutations demonstrates the multifocal nature of WT1-mutant Wilms tumors. As multiple independent tumors can occur in patients with constitutional WT1 mutations, they need to be surveyed more closely for tumor development.

Genomic and transcriptomic heterogeneity of colorectal tumours arising in Lynch syndrome.

Colorectal cancer (CRC) arising in Lynch syndrome (LS) comprises tumours with constitutional mutations in DNA mismatch repair genes. There is still a lack of whole-genome and transcriptome studies of LS-CRC to address questions about similarities and differences in mutation and gene expression characteristics between LS-CRC and sporadic CRC, about the molecular heterogeneity of LS-CRC, and about specific mechanisms of LS-CRC genesis linked to dysfunctional mismatch repair in LS colonic mucosa and the possible role of immune editing. Here, we provide a first molecular characterization of LS tumours and of matched tumour-distant reference colonic mucosa based on whole-genome DNA-sequencing and RNA-sequencing analyses. Our data support two subgroups of LS-CRCs, G1 and G2, whereby G1 tumours show a higher number of somatic mutations, a higher amount of microsatellite slippage, and a different mutation spectrum. The gene expression phenotypes support this difference. Reference mucosa of G1 shows a strong immune response associated with the expression of HLA and immune checkpoint genes and the invasion of CD4+ T cells. Such an immune response is not observed in LS tumours, G2 reference and normal (non-Lynch) mucosa, and sporadic CRC. We hypothesize that G1 tumours are edited for escape from a highly immunogenic microenvironment via loss of HLA presentation and T-cell exhaustion. In contrast, G2 tumours seem to develop in a less immunogenic microenvironment where tumour-promoting inflammation parallels tumourigenesis. Larger studies on non-neoplastic mucosa tissue of mutation carriers are required to better understand the early phases of emerging tumours. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Reduction of the tumorigenic potential of human retinoblastoma cell lines by TFF1 overexpression involves p53/caspase signaling and miR-18a regulation.

Trefoil factor family (TFF) peptides have been shown to play a pivotal role in oncogenic transformation, tumorigenesis and metastasis by changing cell proliferation, apoptosis, migration and invasion behavior of various cancer cell lines. In the study presented, we investigated the effect of TFF1 overexpression on cell growth, viability, migration and tumorigenicity of different retinoblastoma (RB) cell lines. Transient TFF1 overexpression significantly increases RB cell apoptosis levels. Stable, lentiviral TFF1 overexpression likewise decreases RB cell viability, proliferation and growth and significantly increases apoptosis as revealed by WST-1 assays, BrdU and DAPI cell counts. TFF1-induced apoptosis is executed via cleaved caspase-3 activation as revealed by caspase blockage experiments and caspase-3 immunocytochemistry. Results from pG13-luciferase reporter assays and Western blot analyses indicate that TFF1-induced apoptosis is mediated through transcriptional activity of p53 with concurrently downregulated miR-18a expression. In ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF1 overexpression significantly decreases the size of tumors forming from Y79 and RB355 cells and reduces the migration potential of RB355 cells. Differentially expressed genes and pathways involved in cancer progression were identified after TFF1 overexpression in Y79 cells by gene expression array analysis, underlining the effects on reduced tumorigenicity. TFF1 knockdown in RBL30 cells revealed caspase-3/7-independent apoptosis induction, but no changes on cell proliferation level. In summary, the in vitro and in vivo data demonstrate for the first time a tumor suppressor function of TFF1 in RB cells which is at least partly mediated by p53 activation and miR-18a downregulation.

Classification of a frameshift/extended and a stop mutation in WT1 as gain-of-function mutations that activate cell cycle genes and promote Wilms tumour cell proliferation.

The WT1 gene encodes a zinc finger transcription factor important for normal kidney development. WT1 is a suppressor for Wilms tumour development and an oncogene for diverse malignant tumours. We recently established cell lines from primary Wilms tumours with different WT1 mutations. To investigate the function of mutant WT1 proteins, we performed WT1 knockdown experiments in cell lines with a frameshift/extension (p.V432fsX87 = Wilms3) and a stop mutation (p.P362X = Wilms2) of WT1, followed by genome-wide gene expression analysis. We also expressed wild-type and mutant WT1 proteins in human mesenchymal stem cells and established gene expression profiles. A detailed analysis of gene expression data enabled us to classify the WT1 mutations as gain-of-function mutations. The mutant WT1(Wilms2) and WT1(Wilms3) proteins acquired an ability to modulate the expression of a highly significant number of genes from the G2/M phase of the cell cycle, and WT1 knockdown experiments showed that they are required for Wilms tumour cell proliferation. p53 negatively regulates the activity of a large number of these genes that are also part of a core proliferation cluster in diverse human cancers. Our data strongly suggest that mutant WT1 proteins facilitate expression of these cell cycle genes by antagonizing transcriptional repression mediated by p53. We show that mutant WT1 can physically interact with p53. Together the findings show for the first time that mutant WT1 proteins have a gain-of-function and act as oncogenes for Wilms tumour development by regulating Wilms tumour cell proliferation.

Genome-wide analysis associates familial colorectal cancer with increases in copy number variations and a rare structural variation at 12p12.3.

Colorectal cancer (CRC) is one of the most common cancer worldwide. However, a large number of genetic risk factors involved in CRC have not been understood. Copy number variations (CNVs) might partly contribute to the 'missing heritability' of CRC. An increased overall burden of CNV has been identified in several complex diseases, whereas the association between the overall CNV burden and CRC risk is largely unknown. We performed a genome-wide investigation of CNVs on genomic DNA from 384 familial CRC cases and 1285 healthy controls by the Affymetrix 6.0 array. An increase of overall CNV burden was observed in familial CRC patients compared with healthy controls, especially for CNVs larger than 50kb (case/control ratio = 1.66, P = 0.025). In addition, we discovered for the first time a novel structural variation at 12p12.3 and determined the breakpoints by strategic PCR and sequencing. This 12p12.3 structural variation was found in four of 2862 CRC cases but not in 6243 healthy controls (P = 0.0098). RERGL gene (RERG/RAS-like), the only gene influenced by the 12p12.3 structural variation, sharing most of the conserved regions with its close family member RERG tumor suppressor gene (RAS-like, estrogen-regulated, growth inhibitor), might be a novel CRC-related gene. In conclusion, this is the first study to reveal the contribution of the overall burden of CNVs to familial CRC risk and identify a novel rare structural variation at 12p12.3 containing RERGL gene to be associated with CRC.

Evaluating the performance of clinical criteria for predicting mismatch repair gene mutations in Lynch syndrome: a comprehensive analysis of 3,671 families.

Carriers of mismatch repair (MMR) gene mutations have a high lifetime risk for colorectal and endometrial cancers, as well as other malignancies. As mutation analysis to detect these patients is expensive and time-consuming, clinical criteria and tumor-tissue analysis are widely used as pre-screening methods. The aim of our study was to evaluate the performance of commonly applied clinical criteria (the Amsterdam I and II Criteria, and the original and revised Bethesda Guidelines) and the results of tumor-tissue analysis in predicting MMR gene mutations. We analyzed 3,671 families from the German HNPCC Registry and divided them into nine mutually exclusive groups with different clinical criteria. A total of 680 families (18.5%) were found to have a pathogenic MMR gene mutation. Among all 1,284 families with microsatellite instability-high (MSI-H) colorectal cancer, the overall mutation detection rate was 53.0%. Mutation frequencies and their distribution between the four MMR genes differed significantly between clinical groups (p < 0.001). The highest frequencies were found in families fulfilling the Amsterdam Criteria (46.4%). Families with loss of MSH2 expression had higher mutation detection rates (69.5%) than families with loss of MLH1 expression (43.1%). MMR mutations were found significantly more often in families with at least one MSI-H small-bowel cancer (p < 0.001). No MMR mutations were found among patients under 40-years-old with only colorectal adenoma. Familial clustering of Lynch syndrome-related tumors, early age of onset, and familial occurrence of small-bowel cancer were clinically relevant predictors for Lynch syndrome.

A pooled analysis of the outcome of prospective colonoscopic surveillance for familial colorectal cancer.

Surveillance guidelines for the management of familial colorectal cancer (FCC), a dominant family history of colorectal cancer in which the polyposis syndromes and Lynch syndrome have been excluded, are not firmly established. The outcome of colonoscopic surveillance is studied using data from six centers. DNA mismatch repair deficiency was excluded by genetic testing. Families were classified as FCC type X if they fulfilled the original Amsterdam criteria (AC) and late onset (LOFCC) if they fulfilled the AC apart from not having a cancer aged under 50. The most advanced findings on colonoscopy were analyzed. One thousand five hundred eighty-five individuals (median age 47.3, 44% male) from 530 FCC families (349 FCC type X) underwent a total of 4,992 colonoscopies with 7,904 patient-years of follow-up. Results for FCC type X and LOFCC were very similar. At baseline, 22 prevalent asymptomatic colorectal cancers were diagnosed, 120 (7.6%) individuals had high-risk adenomas and 225 (14.2%) simple adenomas. One thousand eighty-eight individuals had a further colonoscopy (median follow-up of 6.2 years). Of nine individuals diagnosed with cancer, eight had a previous history of at least one polyp/adenoma. High-risk adenomas were detected in 92 (8.7%) and multiple adenomas were detected in 20 (1.9%) individuals. Both FCC type X and LOFCC have a high prevalence of colorectal cancers and on follow-up develop high-risk adenomas (including multiple adenomas), but infrequent interval cancers. They should be managed similarly with five-yearly colonoscopies undertaken from between 30 and 40 with more intensive surveillance in individuals developing multiple or high-risk adenomas.

Evaluation of chromosome 11p imbalances in aniridia and Wilms tumor patients.

Newborn sporadic aniridia patients with an 11p13 deletion including the WT1 gene have an increased risk to develop Wilms tumor. At present a risk for Wilms tumor cannot be estimated in patients with deletions not extending into, but ending close to WT1. Therefore, it is important to determine the distance of deletion endpoints from the WT1 gene and survey these patients for a longer follow-up time to obtain a more defined risk estimation. Using molecular methods, such as Multiplex Ligation-dependent Probe Amplification (MLPA), deletion endpoints can be mapped more accurately than with FISH. We describe here the analysis of six aniridia patients, in two of these the deletions extend close to the 3' end of WT1. At the ages of 3.8 and 4 years they have not developed a Wilms tumor, suggesting a low tumor risk in such patients. In addition we have studied 24 non-AN cases with a higher likelihood for WT1 alterations with MLPA and found no deletions. In conclusion newborns with aniridia should be studied with molecular methods that can determine deletion endpoints in 11p13 exactly. For a better Wilms tumor risk estimation cases with deletion endpoints close to WT1 should be followed for at least 4-5 years. Furthermore germ line intragenic deletions affecting WT1 in patients with a higher likelihood for a WT1 association, for example, bilateral tumors, genitourinary aberrations, or nephrotic syndrome, were not found in this study, suggesting that deletions are rare events.

A novel inverted 17p13.3 microduplication disrupting PAFAH1B1 (LIS1) in a girl with syndromic lissencephaly.

We describe a female patient with mild lissencephaly (pachygyria), severe intellectual disability, and facial dysmorphisms with an inverted 1.4 Mb microduplication of chromosome 17p13.3. The 17p13.3 microduplication syndrome is associated with mild intellectual disabiltiy and contains, among others, the PAFAH1B1 (LIS1) gene, whereas microdeletions of the same segment cause Miller-Dieker syndrome (MDS) with severe to profound retardation. The duplication identified in our patient encompasses 29 genes, including CRK and YWHAE. The proximal breakpoint of the duplication is located in the first intron of the PAFAH1B1 gene. Analysis of total RNA showed that only one PAFAH1B1 allele is expressed. Therefore, this patient has a unique alteration: a duplication including YWHAE and CRK and haploinsufficiency of PAFAH1B1. Overexpression of YWHAE is associated with macrosomia, mild developmental delay, autism and facial dysmorphisms, and deletion of PAFAH1B1 alone leads to isolated lissencephaly (ILS). The patient described here shares features with MDS, but she is affected to a lesser degree. Her facial features are similar to MDS, and she has manifestations seen in other cases with YWHAE duplication.

Genetics of pediatric renal tumors.

Wilms tumor (WT) accounts for approximately 95 % of all pediatric renal tumors, with a peak incidence between 2 and 3 years of age. It occurs in sporadic and congenital forms, the latter often occurring before 1 year of age. Incidence declines with age, and WT rarely is observed in adults. WT is an embryonal tumor of the kidney caused by aberrant proliferation of early metanephric kidney cells. It can arise from more than one developmental error and therefore several subtypes can be defined. WT1, a zinc-finger transcription factor, was identified as the first WT gene. Other genes frequently altered somatically in subsets of WT are CTNNB1 and WTX; both genes influence the Wnt signalling pathway. Imprinting alterations of genes in 11p15 are also observed in a subset of WTs. Other pediatric renal tumors occur less often, e.g. malignant rhabdoid tumor of the kidney, clear-cell sarcoma, desmoplastic small-round-cell tumors, congenital mesoblastic nephroma, renal cell carcinoma of childhood, renal primitive neuroectodermal tumors, renal medullary carcinoma, and synovial sarcoma of the kidney. In most of these, characteristic genetic alterations have been identified that help in the unequivocal diagnosis of these childhood renal cancers that are often difficult to distinguish.

Gene expression studies of WT1 mutant Wilms tumor cell lines in the frame work of published kidney development data reveals their early kidney stem cell origin.

In order to get a better insight into the timing of WT1 mutant Wilms tumor development, we compared the gene expression profiles of nine established WT1 mutant Wilms tumor cell lines with published data from different kidney cell types during development. Publications describing genes expressed in nephrogenic precursor cells, ureteric bud cells, more mature nephrogenic epithelial cells and interstitial cell types were used. These studies uncovered that the WT1 mutant Wilms tumor cells lines express genes from the earliest nephrogenic progenitor cells, as well as from more differentiated nephron cells with the highest expression from the stromal/interstitial compartment. The expression of genes from all cell compartments points to an early developmental origin of the tumor in a common stem cell. Although variability of the expression of specific genes was evident between the cell lines the overall expression pattern was very similar. This is likely dependent on their different genetic backgrounds with distinct WT1 mutations and the absence/presence of mutant CTNNB1.

Pathological assessment of mismatch repair gene variants in Lynch syndrome: past, present, and future.

Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes and is the most prevalent hereditary colorectal cancer syndrome. A significant proportion of variants identified in MMR and other common cancer susceptibility genes are missense or noncoding changes whose consequences for pathogenicity cannot be easily interpreted. Such variants are designated as "variants of uncertain significance" (VUS). Management of LS can be significantly improved by identifying individuals who carry a pathogenic variant and thus benefit from screening, preventive, and therapeutic measures. Also, identifying family members that do not carry the variant is important so they can be released from the intensive surveillance. Determining which genetic variants are pathogenic and which are neutral is a major challenge in clinical genetics. The profound mechanistic knowledge on the genetics and biochemistry of MMR enables the development and use of targeted assays to evaluate the pathogenicity of variants found in suspected patients with LS. We describe different approaches for the functional analysis of MMR gene VUS and propose development of a validated diagnostic framework. Furthermore, we call attention to common misconceptions about functional assays and endorse development of an integrated approach comprising validated assays for diagnosis of VUS in patients suspected of LS.

Wilms tumor cells with WT1 mutations have characteristic features of mesenchymal stem cells and express molecular markers of paraxial mesoderm.

Wilms tumors (WTs) are genetically heterogeneous kidney tumors whose cells of origin are unknown. Tumors with WT1 mutations and concomitant loss of the wild-type allele represent a distinct subgroup, frequently associated with mutations in CTNNB1. Here, we describe the establishment and characterization of long-term cell cultures derived from five individual WTs with WT1 mutations. Three of these tumor cell lines also had CTNNB1 mutations and an activated canonical Wnt signaling pathway as measured by beta-catenin/T cell-specific transcription factor (TCF) transcriptional activity. Four of the five Wilms cell lines had a stable normal karyotype for at least 25 passages, and four lines showed loss of heterozygosity of chromosome 11p due to mitotic recombination in 11p11. Gene expression profiling revealed that the WT cell lines are highly similar to human mesenchymal stem cells (MSCs) and FACS analysis demonstrated the expression of MSC-specific surface proteins CD105, CD90 and CD73. The stem cell like nature of the WT cells is further supported by their adipogenic, chondrogenic, osteogenic and myogenic differentiation potentials. By generating multipotent mesenchymal precursors from paraxial mesoderm (PAM) in tissue culture using embryonal stem cells, gene expression profiles of PAM and MSCs were described. Using these published gene sets, we found coexpression of a large number of genes in WT cell lines, PAM and MSCs. Lineage plasticity is indicated by the simultaneous expression of genes from the mesendodermal and neuroectodermal lineages. We conclude that WTs with WT1 mutations have specific traits of PAM, which is the source of kidney stromal cells.

Biallelic MLH1 SNP cDNA expression or constitutional promoter methylation can hide genomic rearrangements causing Lynch syndrome.

BACKGROUND: A positive family history, germline mutations in DNA mismatch repair genes, tumours with high microsatellite instability, and loss of mismatch repair protein expression are the hallmarks of hereditary non-polyposis colorectal cancer (Lynch syndrome). However, in ~10-15% of cases of suspected Lynch syndrome, no disease-causing mechanism can be detected. METHODS: Oligo array analysis was performed to search for genomic imbalances in patients with suspected mutation-negative Lynch syndrome with MLH1 deficiency in their colorectal tumours. RESULTS AND CONCLUSION: A deletion in the LRRFIP2 (leucine-rich repeat flightless-interacting protein 2) gene flanking the MLH1 gene was detected, which turned out to be a paracentric inversion on chromosome 3p22.2 creating two new stable fusion transcripts between MLH1 and LRRFIP2. A single-nucleotide polymorphism in MLH1 exon 8 was expressed from both alleles, initially pointing to appropriate MLH1 function at least in peripheral cells. In a second case, an inherited duplication of the MLH1 gene region resulted in constitutional MLH1 promoter methylation. Constitutional MLH1 promoter methylation may therefore in rare cases be a heritable disease mechanism and should not be overlooked in seemingly sporadic patients.

Frequent long-range epigenetic silencing of protocadherin gene clusters on chromosome 5q31 in Wilms' tumor.

Wilms' tumour (WT) is a pediatric tumor of the kidney that arises via failure of the fetal developmental program. The absence of identifiable mutations in the majority of WTs suggests the frequent involvement of epigenetic aberrations in WT. We therefore conducted a genome-wide analysis of promoter hypermethylation in WTs and identified hypermethylation at chromosome 5q31 spanning 800 kilobases (kb) and more than 50 genes. The methylated genes all belong to alpha-, beta-, and gamma-protocadherin (PCDH) gene clusters (Human Genome Organization nomenclature PCDHA@, PCDHB@, and PCDHG@, respectively). This demonstrates that long-range epigenetic silencing (LRES) occurs in developmental tumors as well as in adult tumors. Bisulfite polymerase chain reaction analysis showed that PCDH hypermethylation is a frequent event found in all Wilms' tumor subtypes. Hypermethylation is concordant with reduced PCDH expression in tumors. WT precursor lesions showed no PCDH hypermethylation, suggesting that de novo PCDH hypermethylation occurs during malignant progression. Discrete boundaries of the PCDH domain are delimited by abrupt changes in histone modifications; unmethylated genes flanking the LRES are associated with permissive marks which are absent from methylated genes within the domain. Silenced genes are marked with non-permissive histone 3 lysine 9 dimethylation. Expression analysis of embryonic murine kidney and differentiating rat metanephric mesenchymal cells demonstrates that Pcdh expression is developmentally regulated and that Pcdhg@ genes are expressed in blastemal cells. Importantly, we show that PCDHs negatively regulate canonical Wnt signalling, as short-interfering RNA-induced reduction of PCDHG@ encoded proteins leads to elevated beta-catenin protein, increased beta-catenin/T-cell factor (TCF) reporter activity, and induction of Wnt target genes. Conversely, over-expression of PCDHs suppresses beta-catenin/TCF-reporter activity and also inhibits colony formation and growth of cancer cells in soft agar. Thus PCDHs are candidate tumor suppressors that modulate regulatory pathways critical in development and disease, such as canonical Wnt signaling.

WT1-dependent sulfatase expression maintains the normal glomerular filtration barrier.

Paracrine signaling between podocytes and glomerular endothelial cells through vascular endothelial growth factor A (VEGFA) maintains a functional glomerular filtration barrier. Heparan sulfate proteoglycans (HSPGs), located on the cell surface or in the extracellular matrix, bind signaling molecules such as VEGFA and affect their local concentrations, but whether modulation of these moieties promotes normal crosstalk between podocytes and endothelial cells is unknown. Here, we found that the transcription factor Wilms' Tumor 1 (WT1) modulates VEGFA and FGF2 signaling by increasing the expression of the 6-O-endosulfatases Sulf1 and Sulf2, which remodel the heparan sulfate 6-O-sulfation pattern in the extracellular matrix. Mice deficient in both Sulf1 and Sulf2 developed age-dependent proteinuria as a result of ultrastructural abnormalities in podocytes and endothelial cells, a phenotype similar to that observed in children with WT1 mutations and in Wt1(+/-) mice. These kidney defects associated with a decreased distribution of VEGFA in the glomerular basement membrane and on endothelial cells. Collectively, these data suggest that WT1-dependent sulfatase expression plays a critical role in maintaining the glomerular filtration barrier by modulating the bioavailability of growth factors, thereby promoting normal crosstalk between podocytes and endothelial cells.

Comprehensive Biology and Genetics Compendium of Wilms Tumor Cell Lines with Different WT1 Mutations.

PURPOSE: WT1 mutant Wilms tumors represent a distinct subgroup, frequently associated with CTNNB1 mutations. The genetic basis for the development of this subtype is currently not fully understood. METHODS: Live WT1 mutant Wilms tumors were collected during surgery of patients and cell cultures established in mesenchymal stem cell medium. They were studied for mutations in WT1 and CTNNB1, their differentiation capacity and protein activation status. Four cell lines were immortalized with a triple mutant ts SV40 largeT antigen and Telomerase. RESULTS: 11 cell lines were established from Wilms tumors of nine patients, including a left and right tumor from the same patient and a primary and second tumor from another patient. Six patients had germ line and three were tumor specific mutations. All cell lines harbored only mutant or deleted WT1 genes. CTNNB1 was wild type in three, all others carried mutations affecting amino acid S45. They had variable and limited capacities for mesenchymal differentiation, a high migratory capacity and a low invasive potential. All cells showed an activation of multiple receptor tyrosine kinases and downstream signaling pathways. CONCLUSIONS: These cell lines represent an important new tool to study WT1 mutant Wilms tumors, potentially leading to new treatment approaches.

No association between MUTYH and MSH6 germline mutations in 64 HNPCC patients.

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant tumour predisposition syndrome caused by germline mutations in mismatch repair (MMR) genes. In contrast to MLH1 and MSH2, germline mutations in MSH6 are associated with a milder and particularly variable phenotype. Based on the reported interaction of the MMR complex and the base excision repair protein MUTYH, it was hypothesised that MUTYH mutations serve as phenotypical modifiers in HNPCC families. Recently, a significantly higher frequency of heterozygosity for MUTYH mutations among MSH6 mutation carriers was reported. We examined 64 MSH6 mutation carriers (42 truncating mutations, 19 missense mutations and 3 silent mutations) of the German HNPCC Consortium for MUTYH mutations by sequencing the whole coding region of the gene. Monoallelic MUTYH mutations were identified in 2 of the 64 patients (3.1%), no biallelic MUTYH mutation carrier was found. The frequency of MUTYH mutations was not significantly higher than that in healthy controls, neither in the whole patient group (P=0.30) nor in different subgroups regarding mutation type. Our results do not support the association between MSH6 mutations and heterozygosity for MUTYH mutations.