Distinctive Molecular Genetic Alterations in Sporadic and Familial Adenomatous Polyposis-Associated Pancreatoblastomas : Frequent Alterations in the APC/β-Catenin Pathway and Chromosome 11p

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 ductal adenocarcinomas, 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 alterations found in adult-type pancreatic ductal adenocarcinomas including mutations in the K-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. Allelic loss 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 neoplasms showed 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 more common pancreatic ductal adenocarcinomas, but bear a close molecular pathogenesis to hepatoblastomas. In addition, pancreatoblastoma may represent an extracolonic manifestation of FAP.     

Solid-pseudopapillary tumors of the pancreas are genetically distinct from pancreatic ductal adenocarcinomas and almost always harbor beta-catenin mutations

Solid-pseudopapillary tumors (SPTs) are unusual pancreatic neoplasms of low malignant potential that most frequently affect young women. Genetic events contributing to the development of SPTs are unknown. Whereas the more common ductal adenocarcinomas of the pancreas essentially never harbor beta-catenin or APC gene mutations, we have recently identified alterations of the APC/beta-catenin pathway in other nonductal pancreatic neoplasms including pancreatoblastomas and acinar cell carcinomas. We analyzed a series of 20 SPTs for somatic alterations of the APC/beta-catenin pathway using immunohistochemistry for beta-catenin protein accumulation, direct DNA sequencing of beta-catenin exon 3, and direct DNA sequencing of the mutation cluster region in exon 15 of the APC gene in those SPTs that did not harbor beta-catenin mutations. Immunohistochemical labeling for cyclin D1 was performed to evaluate the overexpression of this cell-cycle protein as one of the putative downstream effectors of beta-catenin dysregulation. In addition, we analyzed the SPTs for genetic alterations commonly found in pancreatic ductal adenocarcinomas, including mutations in the K-ras oncogene and p53 and DPC4 tumor suppressor genes, using direct DNA sequencing of K-ras and immunostaining for p53 and Dpc4. Almost all SPTs harbored alterations in the APC/beta-catenin pathway. Nuclear accumulation of beta-catenin protein was present in 95% (19 of 20), and activating beta-catenin oncogene mutations were identified in 90% (18 of 20) of the SPTs. Seventy-four percent (14 of 19) showed overexpression of cyclin D1, ranging from 10 to 70% of tumor nuclei. In contrast, no K-ras mutations were present in any of the 20 SPTs, and Dpc4 expression was intact in all 16 SPTs for which immunohistochemical labeling was successful. Overexpression of p53 was limited to only 3 of 19 (15.8%) SPTs. These results emphasize the two distinct, divergent genetic pathways of neoplastic progression in pancreatic ductal and nonductal neoplasms.     

Azinuszellkarzinome und Pankreatoblastome

Acinar cell carcinomas and pancreatoblastomas are malignant tumors of the pancreas, showing predominantly acinar differentiation characterized by the immunohistochemical expression of pancreatic enzymes. Histologically, they usually display acinar and/or solid patterns, but may occasionally also exhibit cystic structures. The key feature of pancreatoblastomas is the presence of squamoid corpuscles. Acinar cell carcinomas predominantly occur in adults, pancreatoblastomas in children. Both tumor types commonly show allelic losses on chromosome 11p and mutations in the APC/beta-catenin signaling pathway. Pancreatoblastomas, in contrast to acinar cell carcinomas, are potentially curable.     

Molecular and immunohistochemical analysis of intraductal papillary neoplasms of the biliary tract

Intraductal papillary neoplasms (IPNs) of the biliary tract are uncommon lesions that may be solitary or may spread extensively along the biliary tree. Some biliary IPNs are histologically and radiologically similar to intraductal papillary mucinous tumors (IPMNs) of the pancreas and present a risk for progression to invasive cholangiocarcinoma. Unlike pancreatic IPMNs, little is known about their molecular pathogenesis. We studied 14 biliary IPNs (including 5 cases with associated invasive cholangiocarcinoma) for genetic alterations in the APC/beta-catenin pathway, K-ras oncogene mutations, p53/chromosome 17p alterations, and Dpc4/18q alterations. Immunohistochemistry was performed for beta-catenin, p53, and Dpc4, and microdissected tissue was analyzed using direct DNA sequencing for exon 1 of K-ras and exon 3 of beta-catenin and allelic loss assays on chromosomes 5q, 17p, and 18q. Activating mutations in codon 12 of the K-ras oncogene were present in 4 of 14 (29%) biliary IPNs. Of these 4 cases, 2 patients had associated invasive cholangiocarcinoma, and identical K-ras mutations were present in both the intraductal and invasive components. Allelic loss on chromosome 18q was present in 4 of 13 informative cases (31%); however, no loss of normal Dpc4 expression was detected by immunohistochemistry. Nuclear accumulation of beta-catenin protein was demonstrated in 3 of 12 cases (25%); however, there were no beta-catenin gene mutations, and allelic loss on 5q was present in only 1 of 10 informative cases (10%). Both immunohistochemistry for p53 and 17p allelic loss assays were negative. Biliary IPNs therefore demonstrate a K-ras gene mutation frequency that is lower than that previously reported for pancreatic IPMNs, but similar to that reported for hepatic cholangiocarcinomas. The presence of K-ras mutations in 2 purely intraductal neoplasms, and identical K-ras mutations in 2 cases with both intraductal and invasive components, suggests that these mutations arise early in tumorigenesis. Finally, the frequency of allelic loss on 18q suggests that a locus on 18q is involved in the molecular pathogenesis of biliary IPNs, but this locus is not DPC4.     

Acinar cell carcinoma of the pancreas and related neoplasms: a review

Acinar cell carcinomas (ACCs) are rare tumours of the exocrine pancreas, accounting for up to 2% of all pancreatic neoplasms in adults and 15% of those in children. They are typically solid, cellular, stroma-poor tumours composed of sheets of relatively uniform cells. This sheet-like arrangement is usually punctuated by variable numbers of acinar structures. Variable amounts of neuroendocrine elements in the form of scattered individual cells are quite common, and some cases have more significant neuroendocrine or ductal elements (mixed acinar neuroendocrine carcinoma and mixed acinar ductal carcinoma).Demonstration of acinar differentiation, usually by immunohistochemistry, is necessary for the diagnosis. Among the antibodies recognizing various pancreatic enzymes, trypsin and chymotrypsin are the most useful. Molecular alterations characteristic of ductal adenocarcinomas such as mutation in the KRAS oncogene are absent in ACCs. However, allelic loss on chromosomes 11p and mutations in the APC/β-catenin pathway have been identified in about 50% and 25% of cases, respectively.ACCs are fairly aggressive tumours, although they are not as dismal prognostically as ductal adenocarcinomas. Those patients who present with localized disease have a much better prognosis than those who present with metastases (5-year survival rate of 25% vs. 50%). Unfortunately, metastases, usually involving the liver, are present in 50% of patients at the time of diagnosis.     

Expression of novel markers of pancreatic ductal adenocarcinoma in pancreatic nonductal neoplasms: additional evidence of different genetic pathways.

Solid pseudopapillary tumor, pancreatoblastoma, undifferentiated carcinoma with osteoclastic-like giant cells, and acinar cell carcinomas are rare pancreatic nonductal neoplasms. Compared to the significant advances in our understanding of the pathogenesis of pancreatic ductal adenocarcinomas in the last decades, the molecular mechanisms underlying pancreatic nonductal neoplasms are poorly understood. In order to elucidate their molecular pathogenesis, we constructed tissue microarrays to study the expression of some novel pancreatic ductal adenocarcinoma-associated tumor markers in these nonductal pancreatic neoplasms. We analyzed nine markers including tumor suppressor gene (14-3-3 sigma), proliferation marker (topoisomerase II alpha), epithelial markers (prostate stem cell antigen, mesothelin and cytokeratin 19), stromal markers (fascin, hsp47 and fibronectin), and gamma-synuclein whose function is not delineated. In addition, we included tumor suppressor gene DPC4 and oncogene Beta-catenin to further confirm their expression in pancreatic nonductal tumors. Our results showed that in contrast to pancreatic ductal adenocarcinomas that show loss of Dpc4 protein in 55% of cases, loss of Dpc4 expression is absent in pancreatic nonductal neoplasms. Expression of 14-3-3 sigma is frequently seen in both pancreatic nonductal neoplasms (25-100%) and ductal adenocarcinomas (89%). Aberrant nuclear expression of beta-catenin is common in pancreatic nonductal neoplasms, specifically in solid pseudopapillary tumors (88%) and pancreatoblastomas (100%) but is rarely seen in pancreatic ductal adenocarcinomas (<5%). Expression of topoisomerase II alpha is not seen in solid pseudopapillary tumors and undifferentiated carcinomas with osteoclastic-like giant cells but is focally seen in pancreatoblastomas (50%) and acinar cell carcinomas (85%). Expression of PSCA and mesothelin was observed in pancreatic nonductal neoplasms but their expression was seen less frequently (0-50%) and weaker than that in pancreatic ductal adenocarcinomas (60-100%). CK19, a marker of pancreatic ductal adenocarcinomas, is not expressed in pancreatic nonductal neoplasms. Expression of gamma-synuclein as well as stromal markers (fascin, hsp47 and fibronectin) is frequently seen in both. Our findings indicate pancreatic nonductal neoplasms have distinctive patterns of protein expression relative to pancreatic ductal adenocarcinomas and suggest that pancreatic nonductal neoplasms have different genetic pathways from the more common pancreatic ductal adenocarcinomas.     

Allelic loss is often the first hit in the biallelic inactivation of the p53 and DPC4 genes during pancreatic carcinogenesis

The presumed precursor lesions of pancreatic ductal adenocarcinoma were recently classified according to their increasing grade of dysplasia and were designated as pancreatic intraepithelial neoplasia (PanIN) 1 through 3. In this study, we tested whether molecular genetic alterations can be correlated with this classification and may help to further categorize the various PanIN grades. We determined the frequencies of allelic loss at chromosomal arms 9p, 17p, and 18q in 81 microdissected duct lesions of various PanIN grades, using a combination of whole genome amplification and microsatellite analysis. In addition we examined the p53 and Dpc4 protein expression patterns by immunohistochemical analysis. In PanIN-1, we did not detect allelic losses. In PanIN-2, allelic losses were found in increasing frequency, and were particularly high in those lesions with moderate-grade dysplasia (low grade, 20, 33, and 17%, loss at 9p, 17p, and 18q, respectively; moderate grade, 46, 77, and 58%). PanIN-3 and invasive carcinomas exhibited abundant losses. Abnormal p53 and Dpc4 protein expression was only rarely identified in PanIN-2 lesions, but occurred frequently in PanIN-3 lesions and invasive carcinomas. The combined genetic and protein expression data support a model in which allelic loss is the first hit in the biallelic inactivation of the p53 and DPC4 tumor suppressor genes. In addition, our data indicate that allelic loss analysis may be useful in separating PanIN-2 lesions with low-grade dysplasia from those PanIN-2 lesions with moderate-grade dysplasia, each potentially representing a distinct progression step toward invasive carcinoma.     

Sporadic fundic gland polyps with epithelial dysplasia : evidence for preferential targeting for mutations in the adenomatous polyposis coli gene

Gastric fundic gland polyps (FGPs) occur in two distinct clinicopathological scenarios: sporadic and familial adenomatous polyposis (FAP) associated. FAP-associated FGPs arise through somatic second hit alterations of the adenomatous polyposis coli (APC) gene and frequently demonstrate epithelial dysplasia (Am J Pathol 2000, 157:747-754). Sporadic FGPs, in contrast, tend to contain beta-catenin gene mutations and only infrequently show dysplasia (Am J Pathol 2001, 158:1005-1010). However, sporadic FGPs with dysplasia have not been previously investigated. We studied 13 sporadic FGPs with surface/foveolar low-grade dysplasia or changes indefinite for dysplasia for alterations in the APC/beta-catenin pathway, using chromosome 5q allelic loss assays and direct DNA sequencing of the mutation cluster region in exon 15 of APC and the phosphorylation region in exon 3 of beta-catenin. In addition, to evaluate for possible additional genetic alterations in FGPs, all cases were evaluated for microsatellite instability using fluorescent-based amplification of a standard panel of five microsatellite markers. Alterations in APC were present in seven (53.8%) FGPs, including two cases with bi-allelic APC inactivation (truncating intragenic mutation plus 5q allelic loss), two cases with APC mutation only, and three cases with 5q allelic loss only. In contrast, only two (15.4%) FGPs contained stabilizing beta-catenin mutations. All 13 FGPs were microsatellite stable. These results indicate that sporadic FGPs with dysplasia/indefinite for dysplasia are molecularly similar to FAP-associated FGPs, and are dissimilar to the more common sporadic nondysplastic FGPs. Mutations in APC and beta-catenin, despite occurring in the same genetic pathway, show differing biological properties, a phenomenon that has previously been demonstrated in colorectal neoplasms. The lack of microsatellite instability in FGPs in this study and of K-ras mutations in a previous study suggests that secondary genetic alterations are rare in both dysplastic and nondysplastic FGPs.     

Pathology and genetics of pancreatic neoplasms with acinar differentiation

Pancreatic neoplasms with acinar differentiation, including acinar cell carcinoma, pancreatoblastoma, and carcinomas with mixed differentiation, are distinctive pancreatic neoplasms with a poor prognosis. These neoplasms are clinically, pathologically, and genetically unique when compared to other more common pancreatic neoplasms. Most occur in adults, although pancreatoblastomas usually affect children under 10 years old. All of these neoplasms exhibit characteristic histologic features including a solid or acinar growth pattern, dense neoplastic cellularity, uniform nuclei with prominent nucleoli, and granular eosinophilic cytoplasm. Exocrine enzymes are detectable by immunohistochemistry and, for carcinomas with mixed differentiation, neuroendocrine or ductal lineage markers are also expressed. The genetic alterations of this family of neoplasms largely differ from conventional ductal adenocarcinomas, with only rare mutations in TP53, KRAS, and p16, but no single gene or neoplastic pathway is consistently altered in acinar neoplasms. Instead, there is striking genomic instability, and a subset of cases has mutations in the APC/β-catenin pathway, mutations in SMAD4, RAF gene family fusions, or microsatellite instability. Therapeutically targetable mutations are often present. This review summarizes the clinical and pathologic features of acinar neoplasms and reviews the current molecular data on these uncommon tumors.     

Loss of Dpc4 expression in colonic adenocarcinomas correlates with the presence of metastatic disease

DPC4 is a candidate tumor suppressor gene on chromosome 18q21, a region that shows high frequencies of allelic losses in pancreatic and colorectal adenocarcinomas. Biallelic inactivation of DPC4 has been reported in half of pancreatic cancers, but are relatively infrequent in other tumor types. The role of DPC4 inactivation in colorectal neoplasms has not been fully characterized. An immunohistochemical assay for Dpc4 protein expression has been recently developed and shown to be a sensitive and specific surrogate for alterations in the DPC4 gene. In this study we examined the expression of Dpc4 protein in formalin-fixed archival tissue from 83 colorectal lesions, including 19 adenomas and 64 sporadic adenocarcinomas (11 stage I, 13 stage II, 17 stage III, and 23 stage IV cancers). None of the adenomas or stage I adenocarcinomas showed loss of Dpc4 expression, whereas one of 13 (8%) stage II, one of 17 (6%) stage III, and five of 23 (22%) of stage IV cancers showed loss of Dpc4 expression. There was a borderline significant difference in loss of Dpc4 reactivity in colorectal tumors with distant metastasis at presentation (22%) versus primary tumors without distant metastasis (5%) (Fisher's exact test, P = 0.05; chi(2) = 0.04). Poorly differentiated histology or status of pericolonic lymph nodes did not affect Dpc4 expression. Alterations in DPC4 are involved in the progression of a subset of colorectal carcinomas, especially those that present with advanced disease. In the sequential pathogenesis of colorectal tumors, inactivation of DPC4 is likely to be a late event.     

Fibromatosis of the breast and mutations involving the APC/beta-catenin pathway

Fibromatoses of the breast are nonmetastasizing tumors, but can be infiltrative and locally recurrent. Breast fibromatoses are rare, and their specific genetic alterations have not been elucidated. However, their occasional occurrence in patients with familial adenomatous polyposis (FAP) and their morphologic identification with other deep fibromatoses (desmoid tumors) suggest that alterations of the APC/beta-catenin pathway might be involved in the pathogenesis of sporadic and FAP-associated breast fibromatoses. We analyzed somatic beta-catenin and APC gene mutations in 33 breast fibromatoses (32 sporadic and 1 FAP-associated) using immunohistochemistry for beta-catenin, 5q allelic loss assays, and direct DNA sequencing for exon 3 of the beta-catenin gene and the mutation cluster region of the APC gene. Nuclear accumulation of beta-catenin was present in the stromal tumor cells in most (82%) cases but not in normal stroma or mammary epithelial cells. Somatic alterations of the APC/beta-catenin pathway were detected in 79% of breast fibromatoses, including activating beta-catenin gene mutations in 15 cases and somatic APC alterations (mutation or 5q allelic loss or both) in 11. These findings indicate that alterations of the APC/beta-catenin pathway with resultant nuclear translocation of beta-catenin are important in the pathogenesis of both sporadic and FAP-associated breast fibromatosis. The spectrum of beta-catenin and APC alterations is similar to that described for desmoid tumors of the abdomen, paraspinal region, and extremities.     

Pancreatic intraepithelial neoplasia and infiltrating adenocarcinoma: analysis of progression and recurrence by DPC4 immunohistochemical labeling.

Pancreatic intraepithelial neoplasia (PanIN) is thought to be a precursor lesion of infiltrating pancreatic ductal adenocarcinoma (IPA). DPC4 is a tumor-suppressor gene on chromosome 18q21.1 and is inactivated in approximately 55% of IPAs. Recently, immunohistochemical labeling using a monoclonal antibody to the Dpc4 protein has been shown to mirror DPC4 genetic status in invasive adenocarcinomas of the pancreas. In the present study, we examined the role of Dpc4 loss in neoplastic progression and recurrence. Two cases in which a PanIN clinically progressed to an invasive adenocarcinoma and a third of a patient with IPA of the head of the pancreas who later developed invasive adenocarcinoma in the tail of the pancreas were studied using Dpc4 immunolabeling. The first patient underwent pancreatic resection, which revealed PanIN-3 that lacked Dpc4 expression, and the patient developed an invasive pancreatic ductal carcinoma 10 years later that shared this loss of expression. The second patient had a pancreaticoduodenectomy for recurrent pancreatitis, and the resected pancreas contained PanIN-3 with intact Dpc4 expression. Seventeen months later, the patient developed an invasive adenocarcinoma of the distal pancreas that also had intact Dpc4 expression. In the third case, the patient underwent pancreaticoduodenectomy for an invasive ductal adenocarcinoma with negative margins. This carcinoma lacked Dpc4 expression. Three years later, resection of the pancreatic tail showed a second invasive adenocarcinoma. The cancer in the tail of the gland showed intact Dpc4 expression, suggesting it represented a second primary tumor, not a recurrence. We conclude that Dpc4 expression in PanIN can be predictive of Dpc4 expression in the subsequent invasive ductal adenocarcinoma. Additionally, Dpc4 expression can be used to differentiate recurrent or persistent adenocarcinoma from a second primary adenocarcinoma.     

Role of the DPC4 tumor suppressor gene in adenocarcinoma of the ampulla of Vater: analysis of 140 cases.

The K-ras oncogene is activated in approximately 90% of pancreatic adenocarcinomas, and the DPC4 (MADH4/SMAD4) tumor suppressor gene is inactivated in approximately 55% of pancreatic adenocarcinomas. The contributions of these genetic alterations to the development of adenocarcinoma of the ampulla of Vater have not been fully established. One hundred forty surgically resected ampullary adenocarcinomas (76 with associated adenomas with high-grade dysplasia) were immunohistochemically labeled for the DPC4 gene product, and in 85 cases the results were correlated with the status of the K-ras oncogene from previously reported data. The results were correlated with clinical outcome and with other pathologic predictors of prognosis. Complete loss of Dpc4 labeling was identified in 34% (95% confidence interval [CI]: 26%, 43%) of the invasive carcinomas and in none (upper 95% CI: 6%) of the associated adenomas. Focal loss of Dpc4 was seen in three (4%; 95% CI: 1%, 14%) of the areas of high-grade dysplasia. Complete loss of Dpc4 expression was seen in 28/77 intestinal-type tumors, in 17/46 pancreaticobiliary-type tumors, and in 0/10 colloid carcinomas. Activating point mutations in the K-ras gene were identified in 40% of the invasive cancers. There was no correlation between K-ras gene mutations and Dpc4 expression and no correlation between these variables and survival. The overall 5-year survival rate was 38%. Lymph node metastases were associated with shorter survival (P =.03). Loss of Dpc4 expression occurs in approximately one third of invasive ampullary cancers but is not seen in adenomas; thus, loss of Dpc4 expression occurs late in ampullary carcinogenesis. Although ampullary and pancreatic adenocarcinomas share histologic and molecular features, ampullary carcinomas are less likely to show loss of Dpc4 expression or K-ras gene mutations.     

BCL10 as a useful marker for pancreatic acinar cell carcinoma,especially using endoscopic ultrasound cytology specimens

Acinar cell carcinomas (ACCs) of the pancreas are characterized by the histological and immunohistochemical features of acinar cell differentiation. Recently, BCL10, originally identified as a recurrent t(1;14)(p22;q32) translocation in MALT B‐cell lymphoma, was found to be immunohistochemically positive in some solid tumors, including ACC. To evaluate its diagnostic efficacy, we performed BCL10 immunohistochemistry and evaluated molecular markers correlated to pancreatic tumor lineages (neuroendocrine markers and a mutation analysis of KRAS and GNAS) using samples from 126 pancreatic tumors (17 ACCs, 24 pancreatic ductal adenocarcinomas, 4 adenosquamous carcinomas, 9 intraductal papillary mucinous neoplasms, 10 mucinous cystic neoplasms, 44 neuroendocrine tumors, 9 serous cystic tumors and 10 solid‐pseudopapillary neoplasms). BCL10 was exclusively expressed in normal acini. In pancreatic tumors, 14 of 17 (82%) ACCs and 2 of 4 (50%) adenosquamous carcinomas were positive, while the other subtypes were almost negative. We subsequently examined the diagnostic utility of BCL10 in endoscopic ultrasound‐guided fine‐needle aspiration (EUS‐FNA) specimens using 57 pancreatic tumors. BLC10 correctly identified ACCs (9/13) and adenosquamous carcinomas (2/4) but none of the other subtypes (n = 41). Therefore, we suggested that BCL10 expression is a useful marker for acinar cell differentiation, particularly in the diagnosis of EUS‐FNA specimens.     

Ampulla of vater cancers: T-stage and histological subtype but not Dpc4 expression predict prognosis

Loss of immunohistochemical expression of Dpc4 occurs in about 50% of pancreatic ductal cancers and its loss correlates with DPC4/Smad4 gene inactivation. Dpc4 expression was also lost in 6 of 16 (37%) ampulla of Vater cancers (AVCs) previously analyzed. Furthermore, chromosomal losses involving 18q, where DPC4 is located, have been observed in 34% of AVCs and are associated with decreased survival. To evaluate the possibility that expression of Dpc4 may be correlated with survival, we analyzed 89 AVCs for inactivation of DPC4 by immunohistochemical staining. Thirty-seven cases showed no expression of Dpc4 (41%). Multivariate survival analysis was performed including age, sex, tumor size, histological subtype (intestinal or pancreatobiliary), grade of differentiation, T-stage, lymph-node metastases and Dpc4 status. T-stage and histological subtype were selected as independent prognostic factors, while Dpc4 immunostaining was not significantly associated with any clinicopathological variable, including histological subtype. Although Dpc4 expression is of no clinical relevance, its involvement in AVC gives additional weight to the hypothesis that, among all pancreatic exocrine and endocrine tumors, only AVC and common ductal adenocarcinomas have similar molecular fingerprints. Moreover, comparison of the frequencies of allelic loss on chromosomal arm 18q and the loss of Dpc4 expression (34% and 41%, respectively) is highly suggestive that DPC4 is the major target of these losses.     

Early loss of heterozygosity on chromosome arm 16q in flat epithelial atypia of the breast. Detection by microsatellite analyses

With the improvement of breast carcinoma screening, pre-malignant cell lesions such as flat epithelial atypia (FEA) are detected more frequently. Several studies have demonstrated that FEA show features of a ductal neoplasia, but is it really a precursor lesion? We have started a comparative genetic analysis of a panel of nine microsatellite markers on six different chromosomal regions to investigate whether FEAs show the same characteristic genetic alterations as ductal carcinomas in situ (DCISs) and invasive carcinoma of the breast. FEAs, DCISs and invasive carcinomas of the same patients were microdissected using PALM micro laser technology. DNA was isolated using the QIAamp DNA Micro Kit (QIAGEN). We have investigated a set of the polymorphic microsatellite markers D7S522, D8S522, NEFL, D10S541 (PTEN), D13S153 (RB1), D16S400, D16S402, D16S422 and D17S855 (BRCA1) using multiplex PCR for the detection of allelic imbalances. Most of the investigated FEAs showed a lower frequency of loss of heterozygosity than associated DCISs or invasive carcinomas. However, we were able to detect the same alterations in FEAs as in DCISs or invasive carcinomas in a number of cases. Notably, the microsatellite marker on 16q showed more prevalent allelic imbalances in FEAs than the other investigated markers. One of the hallmarks in the pathogenesis of a large subgroup of invasive breast carcinomas is the early loss of chromosome arm 16q. In this study, we were able to detect frequent genetic alterations on chromosome 16q in FEAs, associated DCISs and invasive carcinomas. This suggests that FEA is a precursor lesion in the low-grade pathway.     

Sporadic childhood hepatoblastomas show activation of beta-catenin, mismatch repair defects and p53 mutations.

Hepatoblastoma, a rare embryonic tumor that may arise sporadically or in the context of hereditary syndromes (familial adenomatous polyposis and Beckwith-Wiedemann's) is the most frequent liver cancer of childhood. Deregulation of the APC/beta-catenin pathway occurs in a consistent fraction of hepatoblastomas, with mutations in the APC and beta-catenin genes implicated in familial adenomatous polyposis-associated and sporadic hepatoblastomas, respectively. Alterations in other cancer-related molecular pathways have not been reported. We investigated a series of 21 sporadic paraffin-embedded hepatoblastoma cases for mutations in the p53 (exons 5-8) and beta-catenin (exon 3) genes, loss of heterozygosity at APC, microsatellite instability and immunohistochemical expression of beta-catenin and of the two main mismatch repair proteins, MLH1 and MSH2. No loss of heterozygosity at APC was detected. We found mutations in beta-catenin and p53 in 4/21 (19%) and 5/21 (24%) cases respectively, beta-catenin protein accumulation in 14/21 cases (67%), microsatellite instability in 17/21 cases (81%), of which eight resulted positive for high-level of microsatellite instability (in four cases associated with loss of MLH1/MSH2 immunostaining). No correlations between involved molecular pathway(s) and hepatoblastoma histotype(s) emerged. This study confirms that beta-catenin deregulation is involved in sporadic hepatoblastoma and also suggests that mismatch repair defects and p53 mutations contribute to this rare liver cancer. Sporadic hepatoblastoma appears to be molecularly and phenotypically heterogeneous and may reflect different pathways of liver carcinogenesis.