324

APC

BTPS2|DP2|DP2.5|DP3|GS|PPP1R46;adenomatous polyposis coli;APC;adenomatous polyposis coli

adenomatosis polyposis coli tumor suppressor|adenomatous polyposis coli protein|deleted in polyposis 2.5|protein phosphatase 1, regulatory subunit 46

5q21-q22

protein-coding

Hepatoblastoma (HB) is the most common malignant hepatic tumor during early childhood. Its molecular pathogenesis is still poorly understood. mutations of adenomatous polyposis coli (APC) gene have been identified in sporadic cases andin individuals associated with familial adenomatous polyposis syndrome. beta-catenin is a key element in the cadherin-mediated cell adhesion system and Wnt/wingless pathway, and is controlled by APC. APC affects the degradation of beta-catenin by its NH(2)-terminal phosphorylation on the serine/threonine residues of exon 3. mutations of these phosphorylation sites are primary targetsfor activating mutations in several types of human cancer and lead to nuclear accumulation of beta-catenin protein. In this study, we examined nine patients with HB using immunohistochemistry and direct DNA sequencing. ALL nine cases showed predominant nuclear expression of beta-catenin. Eight cases (89%) showed mutations involving exon 3 of the beta-catenin gene, including five with deletions and three with missense mutations. ALL five deletions were in-frame deletions without frameshift. The very high frequency of mutations in the beta-catenin gene suggests that beta-catenin mutations are crucial in the tumorigenesis of HB.

We describe the cytogenetic abnormalities in two cases of childhood hepatoblastoma. The first case was of fetal histology with squamous metaplasia, and cytogenetic study showed an add(5)(q31). Although an association between hepatoblastoma and familial adenomatous polyposis is recognized, the breakpoint in this case is distal to 5q21 and most probably does not involve the APC gene at that location. The second case was of macrotrabecular histology, and cytogeneticstudy showed an unbalanced translocation in the form of der(4)t(1;4)(q12;q34) ina hyperdiploid clone. Including our case, der(4)t(1;4)(q12;q34) has been recognized in four cases of hepatoblastoma, and it may be the first recurrent translocation in this tumor. Understanding the molecular mechanism and clinical significance of this translocation awaits analysis of more cases.

Brain malignancies represent the most common solid tumors in children, and they are responsible for significant mortality and morbidity. The molecular basis of the most common malignant pediatric brain tumor, medulloblastoma, is poorly understood. mutations in several genes including the human homologue of the Drosophila segment polarity gene, patched (PTCH), the adenomatous polyposis coligene (APC), beta-catenin, and p53 have been reported in subsets of hereditary and sporadic medulloblastoma. Inactivation of one Ptc allele in mice results in a 14% incidence of medulloblastoma. Here, we report a dramatic increase in the incidence (>95%) and accelerated development (prior to 12 weeks of age) of medulloblastoma in mice heterozygous for Ptc that lack p53. The acceleration of tumorigenesis in Ptc+/- mice is specific for loss of p53, because no change in tumor incidence was observed in Ptc+/- mice carrying a mutation in APC (Min+/-) or in Ptc+/- mice deficient in p19ARF. Thus, there is a specific interaction between p53 loss and heterozygosity of Ptc that results in medulloblastoma. Thismay be a consequence of increased genomic instability associated with loss of p53 function that may enhance the rate of acquisition of secondary mutations. Ptc+/-p53-/- mice provide a useful model for investigation of the molecular bases of medulloblastoma and for evaluation of the efficacy of therapeutic intervention strategies in a spontaneously arising endogenous brain tumor.

Hepatoblastoma (HBL) is the most common malignant liver tumor in young children.Recent reports have shown that the beta-catenin gene was frequently mutated or deleted in HBLS: To elucidate the role of beta-catenin abnormalities in HBLs, wesearched for mutations of beta-catenin and APC as well as expression of the target genes, cyclin D1, c-myc, and fibronectin, in 68 primary HBLS: The mutation analysis revealed that 44 (65%) tumors carried missense mutations or deletions of beta-catenin, ALL of which were somatic and targeted to the exon 3 encoding the amino acid residues involved in its degradation. However, no loss of function mutation of the APC gene was detected by the yeast functional assay. Of interest, beta-catenin mutation was significantly correlated with overexpression of the target genes, cyclin D1 and fibronectin, but not with that of c-myc in HBLs as measured by quantitative real-time reverse transcription-PCR. The immunohistochemical studies in 15 HBLs demonstrated that the nuclear/cytoplasmicaccumulation of beta-catenin was positive in 13 tumors, 9 of which had the deletion or mutation of the gene. The significant correlation between the beta-catenin gene abnormality and the positive staining of cyclin D1 was also confirmed. Furthermore, the nuclear accumulation of beta-catenin was strongly associated with the poorly differentiated tumor cell components as well as with the positive staining of cyclin D1 within the tumor. Thus, our present results suggested that the gain of function mutation of beta-catenin played a crucial role in the malignant progression of HBL in vivo.

Gardner syndrome (GS), caused by mutations in the adenomatous polyposis coli (APC) gene, is characterized by polyposis coli, osteomas, and various soft-tissue tumors. If undetected or untreated, virtually ALL patients develop colonic carcinoma at a young age. Early detection, while essential, can be difficult because of attenuated phenotypes or spontaneous mutations. We present the clinicopathologic features of 11 identical fibromatous lesions that we have termed Gardner-associated fibroma (GAF), which not only appear to be a part of the spectrum of lesions associated with GS but, in some cases, represent the sentinel event leading to its detection. The GAFs occurred in 11 patients (5 boys and 6 girls; age range, 3 months-14 years), were solitary (n = 7) or multiple (n= 4), and occurred in the superficial and deep soft tissues of the paraspinal region (n = 7), back (n = 3), face (n = 2), scalp (n = 2), chest wall (n = 2), thigh (n = 1), neck (n = 1), and flank (n = 1). Histologically, GAFs resemble nuchal-type fibromas (NFs), consisting of thick, haphazardly arranged collagen bundles between which are found occasional bland fibroblasts, and having marginsthat frequently engulf surrounding structures including adjacent fat, muscle andnerves. After surgical excision, four patients developed recurrences that were classic desmoid fibromatoses (DFs). In one patient with multiple GAFs, one lesion had the features of GAF and DF in the absence of surgical trauma. A family history of GS or polyposis (n = 6) or DF (n = 1) was known at the time of surgery in seven patients. In three patients, the diagnosis of GAF resulted in the diagnosis of unsuspected APC in older family members, with the detection of an occult colonic adenocarcinoma in one parent. In the family of the remaining patient, no stigmata of GS were present. Genetic analysis of this child was performed to investigate the presence of a spontaneous (new) mutation; however, no abnormalities were detected. The significance of GAF is that it serves as a sentinel event for identifying GS kindreds, including those with a high risk forthe development of DF, and it may potentially identify children with spontaneousmutations of the APC gene. Because NFs and GAFs resemble one another, we suggestthat a subset of NF occurring in multiple sites, unusual locations, or children may be GAF.

Gardner syndrome (GS), caused by mutations in the adenomatous polyposis coli (APC) gene, is characterized by polyposis coli, osteomas, and various soft-tissue tumors. If undetected or untreated, virtually ALL patients develop colonic carcinoma at a young age. Early detection, while essential, can be difficult because of attenuated phenotypes or spontaneous mutations. We present the clinicopathologic features of 11 identical fibromatous lesions that we have termed Gardner-associated fibroma (GAF), which not only appear to be a part of the spectrum of lesions associated with GS but, in some cases, represent the sentinel event leading to its detection. The GAFs occurred in 11 patients (5 boys and 6 girls; age range, 3 months-14 years), were solitary (n = 7) or multiple (n= 4), and occurred in the superficial and deep soft tissues of the paraspinal region (n = 7), back (n = 3), face (n = 2), scalp (n = 2), chest wall (n = 2), thigh (n = 1), neck (n = 1), and flank (n = 1). Histologically, GAFs resemble nuchal-type fibromas (NFs), consisting of thick, haphazardly arranged collagen bundles between which are found occasional bland fibroblasts, and having marginsthat frequently engulf surrounding structures including adjacent fat, muscle andnerves. After surgical excision, four patients developed recurrences that were classic desmoid fibromatoses (DFs). In one patient with multiple GAFs, one lesion had the features of GAF and DF in the absence of surgical trauma. A family history of GS or polyposis (n = 6) or DF (n = 1) was known at the time of surgery in seven patients. In three patients, the diagnosis of GAF resulted in the diagnosis of unsuspected APC in older family members, with the detection of an occult colonic adenocarcinoma in one parent. In the family of the remaining patient, no stigmata of GS were present. Genetic analysis of this child was performed to investigate the presence of a spontaneous (new) mutation; however, no abnormalities were detected. The significance of GAF is that it serves as a sentinel event for identifying GS kindreds, including those with a high risk forthe development of DF, and it may potentially identify children with spontaneousmutations of the APC gene. Because NFs and GAFs resemble one another, we suggestthat a subset of NF occurring in multiple sites, unusual locations, or children may be GAF.

Gardner syndrome (GS), caused by mutations in the adenomatous polyposis coli (APC) gene, is characterized by polyposis coli, osteomas, and various soft-tissue tumors. If undetected or untreated, virtually ALL patients develop colonic carcinoma at a young age. Early detection, while essential, can be difficult because of attenuated phenotypes or spontaneous mutations. We present the clinicopathologic features of 11 identical fibromatous lesions that we have termed Gardner-associated fibroma (GAF), which not only appear to be a part of the spectrum of lesions associated with GS but, in some cases, represent the sentinel event leading to its detection. The GAFs occurred in 11 patients (5 boys and 6 girls; age range, 3 months-14 years), were solitary (n = 7) or multiple (n= 4), and occurred in the superficial and deep soft tissues of the paraspinal region (n = 7), back (n = 3), face (n = 2), scalp (n = 2), chest wall (n = 2), thigh (n = 1), neck (n = 1), and flank (n = 1). Histologically, GAFs resemble nuchal-type fibromas (NFs), consisting of thick, haphazardly arranged collagen bundles between which are found occasional bland fibroblasts, and having marginsthat frequently engulf surrounding structures including adjacent fat, muscle andnerves. After surgical excision, four patients developed recurrences that were classic desmoid fibromatoses (DFs). In one patient with multiple GAFs, one lesion had the features of GAF and DF in the absence of surgical trauma. A family history of GS or polyposis (n = 6) or DF (n = 1) was known at the time of surgery in seven patients. In three patients, the diagnosis of GAF resulted in the diagnosis of unsuspected APC in older family members, with the detection of an occult colonic adenocarcinoma in one parent. In the family of the remaining patient, no stigmata of GS were present. Genetic analysis of this child was performed to investigate the presence of a spontaneous (new) mutation; however, no abnormalities were detected. The significance of GAF is that it serves as a sentinel event for identifying GS kindreds, including those with a high risk forthe development of DF, and it may potentially identify children with spontaneousmutations of the APC gene. Because NFs and GAFs resemble one another, we suggestthat a subset of NF occurring in multiple sites, unusual locations, or children may be GAF.

Pancreatoblastomas are unusual malignant neoplasms of the pediatric pancreas that may also rarely affect adults. The molecular pathogenesis of pancreatoblastomas is unknown. They are clinicopathologically distinct from adult pancreatic ductaladenocarcinomas, but their occasional occurrence in patients with Beckwith-Wiedemann syndrome and the case presented here of a pancreatoblastoma in an adult patient with familial adenomatous polyposis (FAP) suggests that they might bear a genetic similarity to other infantile embryonal tumors such as hepatoblastomas. We analyzed a series of nine pancreatoblastomas for mutations common to other embryonal malignancies including somatic alterations in the adenomatous polyposis coli (APC)/beta-catenin pathway and chromosome 11p, using immunohistochemistry for beta-catenin, 5q and 11p allelic loss assays, and direct DNA sequencing of exon 3 of the beta-catenin gene and the mutation cluster region of the APC gene. In addition, we analyzed the pancreatoblastomas for alterationsfound in adult-type pancreatic ductal adenocarcinomas including mutations in theK-ras oncogene and the p53 and DPC4 tumor suppressor genes, using direct DNA sequencing of exon 1 of K-ras and immunohistochemistry for p53 and Dpc4. Allelicloss on chromosome 11p was the most common genetic alteration in pancreatoblastomas, present in 86% (six of seven informative cases). Molecular alterations in the APC/beta-catenin pathway were detected in 67% (six of nine), including five neoplasms with activating mutations of the beta-catenin oncogene and the one FAP-associated tumor with biallelic APC inactivation (germline truncating mutation combined with loss of the wild-type allele); seven neoplasmsshowed abnormal nuclear accumulation of beta-catenin protein. In contrast, loss of Dpc4 protein expression was present in only two cases (one diffuse and one focal), and no alterations in the K-ras gene or p53 expression were detected. Our findings indicate that pancreatoblastomas are genetically distinct from the morecommon pancreatic ductal adenocarcinomas, but bear a close molecular pathogenesis to hepatoblastomas. In addition, pancreatoblastoma may represent an extracolonicmanifestation of FAP.

We identified patterns of differentially-expressed genes in cell lines derived from several pediatric solid tumors. Affymetrix Human cancer G110 Arrays, carrying 1,700 cancer-associated genes, were applied to a panel of 11 cell linesoriginating from Ewing tumors (ETs), neuroblastomas, and malignant melanoma of soft parts. Hierarchical clustering clearly differentiated these 3 entities and revealed groups of 75, 102, and 36 gene probe-sets exhibiting tumor-type specific up-regulation in these cell lines, respectively. Whereas ET lines demonstrated increased expression of microtubule-associated protein tau (MAPT), protein phosphatase 1 regulatory subunit 1A (PPP1R1A), NIMA (never in mitosis gene a)-related kinase 2 (NEK2), and cyclin D1 (CCND1), neuroblastoma samples exhibited high expression of wingless-type mouse mammary tumor virus integrationsite family member 11 (WNT11), Drosophila frizzled homolog 2 (FZD2), and adenomatous polyposis coli (APC) which are involved in regulating free beta-catenin levels. These genes likely maintain tumor-specific characteristics and participate in key downstream regulatory mechanisms. We also correlated the expression levels of up-regulated genes in ETs with their chromosomal localization and compared these data to the comparative genomic hybridization profiles of the cell lines. We demonstrate that gains of genetic material contribute essentially to differential gene expression.

Heterozygous mutations in one of the DNA mismatch repair genes cause hereditary nonpolyposis colorectal cancer (MIM114500). Turcot syndrome (MIM276300) has beendescribed as the association of central nervous system malignant tumors and familial colorectal cancer and has been reported to be both a dominant and recessive disorder. Homozygous and compound heterozygous mutations in APC, MLH1,MSH2, and PMS2 genes have been reported in five families. Here we describe a nonconsanguineous Pakistani family, including a son with lymphoma and colorectalcancer diagnosed at ages 5 and 8, respectively, and an 8-year-old daughter with glioblastoma multiforme. Both children had features of neurofibromatosis type 1 including atypical cafe au lait spots and axillary freckling without a family history consistent with neurofibromatosis type 1, familial adenomatous polyposis, or hereditary nonpolyposis colorectal cancer. mutational analysis was done for MLH1, MSH2, and MSH6 using denaturing high-performance liquid chromatography andsequencing of a blood sample from the daughter. A novel homozygous single base insertion mutation was identified (3634insT) resulting in a premature stop at codon 1,223 in exon 7 of the MSH6 gene. Both parents were found to be heterozygous for the 3634insT mutation. Microsatellite instability testing showed instability in the glioblastoma sample. We report here the first identification of a homozygous mutation in MSH6 in a family with childhood-onset brain tumor, lymphoma, colorectal cancer, and neurofibromatosis type 1 phenotype. Our findings support a role for MSH6 in Turcot syndrome and are consistent with an autosomal recessive mode of inheritance.

BACKGROUND: Hepatoblastoma (HB) is the most frequent liver tumor in childhood, occurring in the first few years of life. Surgery combined with chemotherapy hasresulted in dramatic improvements in prognosis. However, even today, about one quarter of affected children do not survive the disease. Compared to the generalpopulation, the risk of HB is 750-7,500 times higher in children predisposed to familial adenomatous polyposis (FAP), an autosomal-dominant cancer predispostionsyndrome caused by germline mutations in the tumor suppressor gene APC. Only limited data exist about the frequency of APC germline mutations in cases of apparently sporadic HB without a family history of FAP. PROCEDURE: In our sampleof 1,166 German FAP families, ALL known cases of HB were registered. In addition, 50 patients with apparently sporadic HB were examined for APC germline mutations. RESULTS: In the FAP families, seven unrelated cases of HB are documented; three had been detected at an advanced stage. In patients with apparently sporadic HB,germline mutations in the APC gene were identified in 10%. CONCLUSIONS: These data raise the issue of the appropriate screening for HB in children of FAP patients. To date, the efficiency of surveillance for HB is unclear. In Beckwith-Wiedemann syndrome (BWS), recent studies suggest an earlier detection of both Wilms tumor and HB by frequent screening. We discuss the rationale and implications of a screening program; besides the examination procedure itself, screening for HB in children of FAP patients would have important consequences for the policy of predictive testing in FAP. In a substantial fraction of sporadic HB, the disease is obviously the first manifestation of a de novo FAP. These patients should be identified by routine APC mutation screening and undergo colorectal surveillance thereafter.#CI- (c) 2005 Wiley-Liss, Inc.

The ependymoma is the second most common malignant brain tumor of childhood; however, its molecular basis is poorly understood. The formation of multiple ependymomas has been reported as an occasional feature of Turcot syndrome type 2(TS2), a familial cancer syndrome caused by inherited mutations of the APC tumorsuppressor gene, and characterised by the concurrence of a primary CNS tumor (predominantly medulloblastoma) and multiple colorectal adenomas. APC is a critical component of the Wnt/Wingless signaling pathway, which is disrupted in sporadic cancers (e.g., colorectal adenomas, hepatocellular carcinomas, and medulloblastomas) by somatic mutations affecting multiple genes encoding alternative pathway components, including APC and CTNNB1 (encoding beta-catenin). To investigate any role for genetic disruption of the Wnt/Wingless pathway in sporadic ependymomas, we performed mutation analysis of APC and CTNNB1 in 77 primary tumors. Two synonymous APC polymorphisms (PRO1442PRO; THR1493THR) were identified, which were detected at equivalent rates in ependymomas and control nonneoplastic DNA samples (n =50); however, no further APC or CTNNB1 sequence variations were found. In summary, although inherited APC mutations may be associated with ependymoma development in certain TS2 cases, these data indicatethat somatic mutations affecting APC and CTNNB1 do not play a major role in the pathogenesis of sporadic ependymomas.

Familial adenomatous polyposis (FAP) is an inherited condition causing numerous adenomatous colorectal polyps and a markedly elevated risk of colon cancer. FAP may be associated with various extracolonic manifestations such as desmoid fibromatosis and osteomas (termed Gardner's syndrome) and brain tumors, usually medulloblastoma or glioma [termed Brain tumor Polyposis (BTP) syndrome type 2]. We describe a pediatric patient who initially presented with prolactinoma and later was found to have Gardner's syndrome. A germline mutation of the APC (adenomatous polyposis coli) gene was identified. Our case illustrates the association between prolactinoma and FAP, which may represent a rare subtype of Gardner's and BTP syndromes.#CI- (c) 2007 Wiley-Liss, Inc.

tumors of the nervous system most often occur in both children and adults as sporadic events with no family history of the disease, but they are also among the clinical manifestations of a significant number of familial cancer syndromes, including familial retinoblastoma, neurofibromatosis 1 and 2, tuberous sclerosis, and Cowden, Turcot, Li-Fraumeni and nevoid basal cell carcinoma (Gorlin) syndromes. ALL of these syndromes involve transmissible genetic risk resulting from loss of a functional allele, or inheritance of a structurally defective allele, of a specific gene. These genes include RB1, NF1, NF2, TSC1, TSC2, TP53,PTEN, APC, hMLH1, hPSM2, and PTCH, most of which function as tumor suppressor genes. The same genes are also observed in mutated and inactive forms, or are deleted, in tumor cells in sporadic cases of the same tumors. The nature of the mutational events that give rise to these inactivated alleles suggests a possible role of environmental mutagens in their causation. However, only external ionizing radiation at high doses is clearly established as an environmental cause of brain, nerve and meningeal tumors in humans. Transplacental carcinogenesis studies in rodents and other species emphasize the extraordinary susceptibility of the developing mammalian nervous system to carcinogenesis, but the inverse relationship of latency to dose suggests that low transplacental exposures to genotoxicants are more likely to result in brain tumors late in life, rather than in childhood. While not ALL neurogenic tumor-related genes in humans have similar effects in experimental rodents, genetically engineered mice (GEM) increasingly provide useful insights into the combined effects of multiple tumor suppressor genes and of gene-environment interactions in the genesis of brain tumors, especially pediatric brain tumors such as medulloblastoma.

BACKGROUND AND AIMS: mutations in DNA mismatch repair genes are associated with high risk of digestive malignancies [hereditary non-polyposis colorectal cancer (HNPCC); Lynch syndrome]; mutations of APC and MYH are associated with classic and attenuated familial adenomatous polyposis (FAP). Although the early onset oftumors in both syndromes is characteristic of their genetic origin, pediatric malignancies remain rare. Certain reports have found familial colorectal cancer (CRC) occurring in very young patients associated with mutations in more than one gene. MATERIALS AND METHOD: A family corresponding to the Amsterdam criteria forHNPCC, including two cases of colorectal cancer before the age of 25 years, was analyzed for mutations in the MSH2 genes by sequencing. Because polyposis was observed in a patient who developed CRC at age 16, the APC gene was also sequenced. RESULTS: A truncating mutation in the MSH2 gene, c.258_259delTG, was carried by patients developing cancer of the colon (two patients), uterus, kidney, bladder, and/or small intestine at ages 16, 24, 43, 44, 45, and 57, respectively. A patient with CRC at age 16 was found to carry the APC c.3183_3187del5 mutation as well as the MSH2 mutation, and it is inferred that her father, deceased of CRC at age 24, was also a double heterozygote. INTERPRETATION: These results confirm that vigilance is required when interpreting molecular results for families with very young patients, as more than one gene may contribute to the genetic risk. cancer screening measures mustalso be adapted to the earlier and more penetrant risk to double heterozygotes.

Hepatoblastoma is the most common primary liver tumor in childhood and occurs more commonly in families with familial adenomatous polyposis. Germline mutations of the gene responsible for familial adenomatous polyposis--adenomatous polyposis coli (APC)--are described in patients with hepatoblastoma even without a family history. We investigated children presenting with apparently sporadic hepatoblastoma between 1991 and 2004. Blood samples were available from 29 children (18 boys) whose conditions were diagnosed at a median age of 22 months (range 6-119 months). No germline APC mutations were found, which does not support the need for routine screening in sporadic hepatoblastoma in the absenceof a suggestive family history of colorectal cancer or suspicion of familial adenomatous polyposis.

BACKGROUND & AIMS: Infantile and childhood liver tumors have been found in 0.42%of individuals with a germline mutation in the adenomatous polyposis coli (APC) gene. This study analyzed a hepatocellular adenoma of a 2-year-old child at riskfor familial adenomatous polyposis to identify genetic alterations in hepatic tumors initiated by APC germline mutations. METHODS: mutation screening was performed for the APC gene (protein truncation test and DNA sequence analysis), p53 gene (complementary DNA cloning and sequencing), and members of the Ras genefamily (complementary DNA sequence analysis). RESULTS: Both the mother and childhad a germinal CGA-->TGA transition at codon 1451 leading to an Arg1451Ter stop mutation in the APC gene. Loss of the wild-type APC allele as a second hit revealed hemizygosity of the inherited mutation in the tumor. Furthermore, a CGC-->CAC transition in the p53 gene of the adenoma resulted in an Arg-->His missense mutation in codon 175. No loss of heterozygosity was detected at the p53 locus. Ras gene mutations were not found. CONCLUSIONS: Biallelic inactivation ofAPC gene and p53 mutation are early events in hepatocellular tumorigenesis. Additional reports will confirm whether inherited APC gene mutations between codon 1444 and 1578 increase the risk for hepatic tumors.

Hepatoblastoma comprises only 1% of ALL cancers in childhood. Because of its lowfrequency, a small number of prognostic factors are described in hepatoblastoma and most of them are related to resectability. Microarray studies showed a largenumber of underexpressed genes in hepatoblastoma. Because aberrant DNA methylation has been recognized as an alternative mechanism for tumor suppressorgene inactivation, this could be involved with gene downregulation in these tumors. Despite the rarity of hepatoblastoma, this study evaluated the methylation pattern of 25 genes in 20 paraffin-embedded tumor specimens and fivenon-neoplastic liver samples (normal control) by quantitative methylation-specific PCR (QMSP). The examination of the methylation profile of hepatoblastoma samples and normal liver specimens revealed a high tumor-specificDNA hypermethylation in the promoter regions of five genes (APC, CDH1, MT1G, RASSF1A, and SOCS1). Furthermore, MT1G hypermethylation showed a significant correlation with poor prognosis of patients with hepatoblastoma. This study represents the first quantitative evaluation of promoter hypermethylation in hepatoblastoma and demonstrated that aberrant methylation is a frequent event inthis malignancy. Furthermore, our data provide evidence that MT1G hypermethylation may be useful as prognostic indicator for this disease and suggest that patients with hepatoblastoma may benefit from demethylating drug treatments.

BACKGROUND: Aberrant Wnt signaling due to deregulation of Wnt regulators is implicated in the development and progression of numerous embryonal tumors. Thisstudy addresses the questions if activation of Wnt signaling in germ cell tumors(GCTs) arising during childhood and adolescence is associated with aberrations of the tumor suppressor adenomatous polyposis coli (APC), and whether APC aberrations might be responsible for progression from benign teratoma to malignant yolk sac tumor (YST). PROCEDURE: Forty-eight GCTs were analyzed, including mature (n = 5) and immature (n = 7) teratomas, mixed malignant GCTs (n= 10), YSTs (n = 17) as well as dysgerminomas (n = 9). To screen APC for geneticaberrations, we conducted direct sequencing of the mutation cluster region (MCR), loss of heterozygosity analyses (LOH) and protein truncation test. Epigenetic analyses included methylation specific PCR and bisulfite genomic sequencing of the APC 1a promoter. Gene expression was determined by quantitative real-time PCR. RESULTS: Aberrant promoter methylation was detected in YSTs, teratomas and mixed malignant GCTs, with a pronounced hypermethylation exclusively in YSTs (11/13) while dysgerminomas were not methylated (0/9). Teratomas (2/2) and YSTs (4/5) show LOH at the APC locus. However, neither mutations within the MCR nor truncated protein were detected. APC expression did not significantly vary between the different histological subgroups. CONCLUSIONS: Methylation of APC and LOH 5q21-22 in YSTs and teratomas provide evidence for involvement of APC in theaccumulation of beta-catenin and activation of the WNT pathway. Our additional analyses suggest that APC is unlikely to be solely responsible for the formationand progression of childhood GCTs.#CI- Copyright (c) 2010 Wiley-Liss, Inc.

Desmoid fibromatosis is a rare, locally aggressive fibroblastic/myofibroblastic tumor that occasionally involves children. We examined a series of pediatric desmoids for CTNNB1 mutations, seen in sporadic tumors, and APC germline mutations, associated with familial adenomatous polyposis (FAP). Forty-four desmoids in pediatric patients were identified in the pathology files of 2 largereferral centers (1995-2009). Clinical charts were reviewed for history of FAP. Germline APC gene mutations were determined on blood samples from patients presenting with FAP. Immunohistochemistry for beta-catenin was performed. CTNNB1genotyping was done by Sanger sequencing on formalin-fixed paraffin-embedded tissue. CTNNB1 mutations were observed in 29 of 44 (66%) desmoids, with 3 mutations identified: T41A (64%), S45F (29%), and S45P (7%). Germline APC mutations were present in 7 (16%) desmoid patients. Eight (18%) patients had desmoids that were wild type for CTNNB1 and had no known clinical signs or family history suspicious for FAP at the time of testing or with extended follow up (n = 6). Beta-catenin nuclear labeling was observed in 38 of 41 (92%) tested cases, 34 (89%) of which showed mutations in either CTNNB1 (n = 29) or APC (n = 5). Nuclear localization of beta-catenin was seen in the majority of pediatric desmoids and was most often associated with somatic mutations in CTNNB1. However, a significant proportion of pediatric patients harbored germline mutations in APC.Given the implications, genetic counseling is recommended for children diagnosedwith desmoid tumors lacking CTNNB1 mutations because this population is enrichedfor FAP patients.

Infantile and childhood hepatoblastoma (HB) occurs more frequently in children with hereditary predisposition to familial adenomatous polyposis (FAP) than in the general population. The occurrence of HB in two infant siblings is reported.The sister died of the disease. The brother survived the HB and was later diagnosed with familial adenomatous polyposis and advanced rectal cancer. He wasfound to carry a germline mutation of the APC gene. Presuming that the HB in thetwo siblings was the first manifestation of FAP we performed APC mutation analysis in DNA from archived tumour tissue of his sister and in blood samples of both parents. Surprisingly, the mutation was neither found in both parents, nor in the tissue samples of the sister. We outline the impact of this finding for genetic counselling and review the literature on FAP and HB.

BACKGROUND: Approximately one-third of hepatoblastoma (HB) patients have associated congenital abnormalities, but familial recurrence is rare, except in association with familial adenomatous polyposis (FAP). This correlation may be missed if not actively sought, with implications for long-term outcome and management. METHODS: We retrospectively investigated 3 families with an HB-familial polyposis connection, from a cohort of 113 FAP families (1989 - 2010). Data were analysed to assess clinical problem, treatment, complications and management. Long-term morbidity and functional outcome were analysed to identify management difficulties. RESULTS: Three FAP families (2.65%) had an HB association. In one case, undiagnosed FAP at the time of HB diagnosis was only detected 5 years later, when the mother presented with advanced colorectal carcinoma. A chromosome 5 APC gene mutation (exon 15 codon 793 C-->T) was then identified. In a second case, a non-related male child presented with a stage 4 multifocal HB with lung metastases. Genetic studies identified an APC gene mutation (exon 6 codon 232 C-->T). Further family investigation showed >20 related FAP patients. A third HB-FAP association was identified in a known FAP family early in the study, prior to the availability of genetic testing. CONCLUSION: Although a rare association, a family history of FAP in HB patients is an important 'hidden connection'. Germline variation may be outside the usualFAP gene site. Identifying families with unknown HB/FAP is important due to long-term management implications and follow-up.

OBJECTIVES: We investigated four components of the Wnt signaling pathway in medulloblastomas. Medulloblastoma is the most common type of malignant pediatricbrain tumor, and the Wnt signaling pathway has been shown to be activated in this type of tumor. METHODS: Sixty-one medulloblastoma cases were analyzed for beta-catenin gene (CTNNB1) mutations, beta-catenin protein expression via immunostaining and Wnt signaling pathway-related gene expression. ALL data were correlated with histological subtypes and patient clinical information. RESULTS:CTNNB1 sequencing analysis revealed that 11 out of 61 medulloblastomas harbored missense mutations in residues 32, 33, 34 and 37, which are located in exon 3. These mutations alter the glycogen synthase kinase-3beta phosphorylation sites, which participate in beta-catenin degradation. No significant differences were observed between mutation status and histological medulloblastoma type, patient age and overall or progression-free survival times. Nuclear beta-catenin accumulation, which was observed in 27.9% of the cases, was not associated with the histological type, CTNNB1 mutation status or tumor cell dissemination. The relative expression levels of genes that code for proteins involved in the Wnt signaling pathway (CTNNB1, APC, AXIN1 and WNT1) were also analyzed, but no significant correlations were found. In addition, large-cell variant medulloblastomas presented lower relative CTNNB1 expression as compared to the other tumor variants. CONCLUSIONS: A small subset of medulloblastomas carry CTNNB1 mutations with consequent nuclear accumulation of beta-catenin. The Wnt signaling pathway plays a role in classic, desmoplastic and extensive nodularitymedulloblastoma variants but not in large-cell medulloblastomas.

BACKGROUND: Familial adenomatous polyposis (FAP) is a dominantly inherited disorder characterized by the presence of more than 100 adenomatous polyps in the colon and rectum starting in the second decade of life. FAP is associated with extra colonic manifestations, including desmoid tumors. METHODS: A 2-year-old girl presented with a rapidly enlarging tumor of the forehead and a family history of FAP. The tumor was cultured for cytogenetic studies. A DNA linkage study using flanking and intragenic polymorphisms of the adenomatous polyposis coli (APC) gene was performed to identify the allele loss in the tumor. Germlinemutation identification was by single strand conformation polymorphism analysis of exon 15 of the APC gene, with subsequent double stranded sequencing of fragments with conformational changes. A mutation-induced loss of a restriction site was used to confirm allele loss in the tumor. RESULTS: Microscopically, thetumor had desmoid features. Cytogenetic analysis of the tumor demonstrated loss of chromosome region 5(q21q22). A truncating adenomatous polyposis coli (APC) gene mutation was identified in the leukocyte DNA from the child and her affected father. Linked DNA markers suggested that the tumor had lost the maternal, wild-type allele. A mutation-induced restriction endonuclease site alteration demonstrated hemizygosity of the mutant sequence in the tumor DNA. CONCLUSIONS: These findings are compatible with the presence of a "second hit" inactivation of the APC gene and implicate this gene in the pathogenesis of desmoid tumors.

BACKGROUND: Hepatoblastoma is a rare, rapidly progressive, usually fatal childhood malignancy, which if confined to the liver can be cured by radical surgical resection. An association between hepatoblastoma and familial adenomatous polyposis (FAP), which is due to germline mutation of the APC (adenomatous polyposis coli) gene, has been confirmed, but correlation with siteof APC mutation has not been studied. AIM: To analyse the APC mutational spectrum in FAP families with hepatoblastoma as a possible basis to select kindreds for surveillance. PATIENTS: Eight patients with hepatoblastoma in seven FAP kindredswere compared with 97 families with identified APC gene mutation in a large Registry. METHODS: APC gene mutation was evaluated by RNase protection assay or in vitro synthesis protein assay. The chi 2 test and correlation were used for data analysis. RESULTS: APC gene mutation was identified in ALL seven FAP kindreds in which an at risk member developed hepatoblastoma. A male predominance was noted (six of eight), similar to literature cases (18 of 25, p < 0.01. mutations were restricted to codons 141 to 1230, but no significant difference in site of mutation between pedigrees with and without hepatoblastoma was identified. CONCLUSIONS: Hepatoblastoma occurs primarily in boys in FAP kindredsand is associated with germline APC mutation in the 5' end of the gene. However,the site of APC mutation cannot be used to predict occurrence of this extracolonic cancer in FAP pedigrees.

BACKGROUND & AIMS: Infantile and childhood liver tumors have been found in 0.42%of individuals with a germline mutation in the adenomatous polyposis coli (APC) gene. This study analyzed a hepatocellular adenoma of a 2-year-old child at riskfor familial adenomatous polyposis to identify genetic alterations in hepatic tumors initiated by APC germline mutations. METHODS: mutation screening was performed for the APC gene (protein truncation test and DNA sequence analysis), p53 gene (complementary DNA cloning and sequencing), and members of the Ras genefamily (complementary DNA sequence analysis). RESULTS: Both the mother and childhad a germinal CGA-->TGA transition at codon 1451 leading to an Arg1451Ter stop mutation in the APC gene. Loss of the wild-type APC allele as a second hit revealed hemizygosity of the inherited mutation in the tumor. Furthermore, a CGC-->CAC transition in the p53 gene of the adenoma resulted in an Arg-->His missense mutation in codon 175. No loss of heterozygosity was detected at the p53 locus. Ras gene mutations were not found. CONCLUSIONS: Biallelic inactivation ofAPC gene and p53 mutation are early events in hepatocellular tumorigenesis. Additional reports will confirm whether inherited APC gene mutations between codon 1444 and 1578 increase the risk for hepatic tumors.

PURPOSE: Gardner syndrome, a variant of familial adenomatous polyposis, is characterized by colonic polyps that undergo malignant change and benign and malignant extracolonic lesions. tumors frequently associated with Gardner syndrome include carcinoma of the ampulla of Vater, papillary carcinoma of the thyroid, and, in children, hepatoblastoma. The childhood malignancies often precede the appearance of other manifestations by several years. PATIENTS AND METHODS: Two patients are described. Gardner syndrome was diagnosed in a 15-year-old girl with fibrolamellar hepatocellular carcinoma after desmoid tumors and colonic polyposis developed. Classic hepatocellular carcinoma was also diagnosed in a 9 1/2-year-old boy with familial adenomatous polyposis. RESULTS: In patient 1, the diagnosis of fibrolamellar hepatocellular carcinoma preceded the diagnosis of Gardner syndrome by almost 2 years. The diagnosis was confirmedby identifying a germline mutation of the adenomatous polyposis coli (APC) gene.This is the first patient reported with fibrolamellar hepatocellular carcinoma associated with Gardner syndrome. Patient 2 had a strong family history of familial adenomatous polyposis but no manifestations of Gardner syndrome. He wasnot tested for the APC mutation. The current literature and previously reported cases of hepatocellular carcinoma in patients with Gardner syndrome or familial adenomatous polyposis are reviewed. CONCLUSIONS: Because hepatocellular carcinoma is uncommon in the pediatric and adolescent population, it is important to consider the possibility of Gardner syndrome or familial adenomatous polyposis in these patients.

We have extended the algebraic models for cancer initiation and progression developed by Nordling, Armitage-Doll and Knudson-Moolgavkar to include the effect of cell turnover rate in normal tissue, stochastic growth of preneoplastic adenomas, and the general case wherein a subfraction of the population is at risk. We have also gathered the mortality data available for the United States from 1900 to 1991 and categorically organized them by birth year cohorts and agespecific death rates for ages 0 to 104 in 5-year groupings. Using these data, wefirst explored the quantitative nature of the biases of underreporting or misdiagnosis as historical age-dependent functions. Then we used the extended algebraic model to calculate the parameters of subpopulation fraction at risk, mutation rates and adenoma growth rates. We observe that death rates for ALL cancers are low in childhood and early adulthood, rise in middle age in an approximately linear manner, reach a maximum in old age, and even after correction for reporting bias, decrease markedly in extreme old age. We represent this behavior as the natural result of a continuous process of cell division, death and mutation within a subpopulation at risk. This population at risk within any birth cohort is defined by the product of a constant inherited risk factor multiplied by a historically valuable environmental risk factor. Our formulationpermits explicit calculation of the fraction at risk of death from any cancer asa historical function. With regard to the algebraic description of the process of carcinogenesis, we use Nordling's concept that n genetic events in a cell population of constant cell number are required to initiate a colony capable of net cell growth or 'adenoma.' We adopt and extend Moolgavkar's use of the 'Gambler's Ruin' stochastic process to describe the probability of adenoma survival and the canonical expectation that a surviving adenoma will soon contain many initiated cells by virtue of stochastic distribution of surviving cells. Weconsider that within the growing adenoma, it is necessary for a cell to acquire m additional mutations in order to attain the carcinoma phenotype of cell growth rapid enough to kill in a short time. This would be irrespective of the need forany additional genetic events that may define the subsequent phenotypes of largelethal tumors, as these would be automatically acquired and be physiologically selected in any rapidly growing cell mass. It is evident that the steps of initiation and progression are dependent on both the rates of genetic change percell division and the cell kinetic rates of division and death. We have chosen to first examine colon cancer because the rates of cell division in normal colonic epithelium, dysplastic adenomas and small carcinomas have been directly observedas reported herein. For colon cancer, we calculate that about 65% of the US population is at risk for both males and females, and that this fraction has been constant for the earliest recorded birth cohorts of the mid-19th century to the beginning of the 20th century. The changes that have been observed in colon cancer mortality rates appear to arise from historical changes in death rates byunknown 'other causes of death', which share both genetic and environmental riskfactors with colon cancer and explicitly include undiagnosed deaths by colon cancer. Considering ALL possible values of n and m, we find the case of n=2 and m=1 to give the best concordance with present knowledge of mutations in the colon by the loss of two alleles of the APC gene and the observation that for m=1, a rate of genetic change approximately equal to that calculated for initiation mutation rates is obtained. Our estimates for the rate of initiation and progression mutation rates show no significant historical shifts and are approximately 1-2x10-7 events per cell division. (ABSTRACT TRUNCATED)