β‐catenin mutation in ovarian solid pseudopapillary neoplasm

Primaly solid pseudopapillary neoplasm (SPN) of the ovary is a rare tumor; recently 6 cases have been reported. Its pathogenesis, however, remains largely unclear. We report an additional case of primary ovarian SPN of an 18‐year‐old girl. The aim of this study is to define the difference between pancreatic and ovarian SPN by histological and molecular examination. Microscopically the tumor predominantly showed a solid pattern and focally a pseudopapillary pattern. The tumor cells showed two patterns of abundant eosinophilic cytoplasm and intracytoplasmic vacuoles. Immunohistochemistry of the tumor was positive for β‐catenin (nuclear and cytoplasmic reactivity), α1‐antitrypsin, vimentin, CD56, synaptophysin (focal weak), CD10. Mutation analyses revealed a point mutation, c.110C >T, in exon 3 of the the β‐catenin gene (CTNNB1), which causes the replacement of serine with phenylalanine at codon 37. A Ser37 point mutation is known to be one of the oncogenic somatic mutations in pancreatic SPN and the major oncogenic β‐catenin mutation. Ovarian SPN of our case was similar to pancreatic SPN histologicaly and had the same genomic characteristics. We expected that both ovarian and pancreatic SPNs shared the same oncogenesis related to Wnt/β‐catenin pathway for tumorgenesis.     

Transcription factors involved in pancreas development are expressed in paediatric solid pseudopapillary tumours

Aims:  Solid pseudopapillary tumours (SPT) are rare pancreatic tumours, especially in children. The origin of this benign tumour remains unknown. Mutations of β-catenin , a gene essential for pancreatic development, are constantly found, leading to delocalization of immunohistochemical signals from the cytoplasm to the nuclei of tumour cells. The aim was to report clinical and histological data of eight children with SPT and explore the immunohistochemical expression of pancreatic duodenal homeobox (PDX) 1 and Sox9, known to be crucial for pancreatic development and linked to the β-catenin cascade.
Methods and results:  Eight children with features suggestive of SPT underwent surgical resection. Tumours displayed typical histological appearances. One was incompletely resected and recurred. Immunolabelling revealed nuclear location of β-catenin in all cases and strong cytoplasmic but no nuclear expression of PDX1 or Sox9 in all but one case.
Conclusions:  The clinical behaviour of SPT in the paediatric population is similar to its adult counterpart. Complete surgical resection is essential. PDX1 and Sox9 proteins are exclusively expressed in the cytoplasmic compartment in SPT, suggesting overexpression of the corresponding genes linked to β-catenin mutations. These findings favour the hypothesis that SPT originates from transformation of normally quiescent pancreatic stem cells.     

Neuropilin-2 is a novel marker expressed in pancreatic islet cells and endocrine pancreatic tumours

Neuropilin-2 (NP-2) is a cell surface transmembrane protein originally characterized as a receptor for the type 3 semaphorins, and more recently for a number of vascular endothelial growth factor (VEGF) isoforms. NP-2 expression has been recently localized to a subset of neuroendocrine cells in the gastrointestinal tract. The aim of this study was to define the expression pattern of NP-2 in normal pancreatic islets and to determine the utility of NP-2 expression as a diagnostic marker of pancreatic endocrine tumours. Paraffin-embedded tissue sections from 30 endocrine pancreatic tumours (EPTs) and from normal pancreas were immunostained with a rabbit polyclonal antibody generated towards NP-2. Nineteen of the tumours were hormonally functional (nine insulinomas, nine gastrinomas, and one glucagonoma). The NP-2 staining pattern was correlated with islet cell hormone expression. In addition, NP-2 expression was evaluated in other normal neuroendocrine tissues and neuroendocrine neoplasms. In normal pancreas, NP-2 stained a distinct subset of islet cells situated primarily at the islet periphery. Double immunohistochemical staining revealed co-localization with glucagon-expressing cells. Moderate to strong NP-2 staining was present in 27 of 30 EPTs. Serial staining of the pancreatic tumours with insulin, gastrin, glucagon, pancreatic polypeptide (PP) or somatostatin did not reveal a distinct pattern of co-localization. NP-2 expression was not detected in neuroendocrine cells outside the gastroenteropancreatic system, or in their corresponding neoplasms, except for focal staining in one bronchial carcinoid tumour. In conclusion, the vast majority of EPTs examined expressed NP-2, suggesting its utility as a diagnostic marker for these tumours. The function of NP-2 in islet cell biology or tumourigenesis remains to be elucidated.     

Insulin-like growth factor-II in endocrine pancreatic tumours. Immunohistochemical, biochemical and in situ hybridization findings

In earlier studies a high-molecular-weight (HMW) insulin-like growth factor-II (IGF-II) peptide was identified in adult human pancreas and localized to the insulin-producing B-cells. This peptide has now been investigated in neoplastic insulin cells. Forty endocrine pancreatic tumours and 17 pancreatic adenocarcinomas of ductal type were included in the study. All cases were investigated with immunohistochemical techniques using antibodies to IGF-II, insulin, pro-insulin, glucagon, somatostatin, pancreatic polypeptide, gastrin and vasoactive intestinal peptide (VIP). Frozen tissue from nine tumours and two normal pancreatic glands was extracted, gel separated, and quantified using radioimmunoassay. The tumours were also investigated by in situ hybridization. IGF-II-immunoreactive cells were found in nearly all the 18 insulin-producing tumours (16/18), in a minority of the other endocrine tumours, but not in pancreatic adenocarcinomas. All extracts from the endocrine tumours showed varying amounts of IGF-II and had different molecular-weight forms. The immunohistochemical and radioimmunoassay findings are both based on immunological binding and were further confirmed by Northern blot and in situ hybridization. These results show that IGF-II is expressed in insulin-producing tumours as well as in pancreatic tumours producing other peptides, in contrast to normal pancreatic islets where IGF-II is found exclusively in insulin-producing cells.     

p62 protein is expressed in pancreatic beta cells

p62 is a cellular protein that plays an adapter role in signal transduction pathways involved in such diverse biological functions as proliferation, differentiation, reaction to oxidative stress and immune response. Furthermore, p62 has recently been detected as a component of intracytoplasmic protein aggregates (inclusion bodies), which are hallmarks of a variety of chronic degenerative disorders, such as Parkinson's disease and Alzheimer's disease, but also of steatohepatitis. Here we report that p62 and insulin are co-expressed in a diffuse fashion in beta cells in normal human pancreas as well as in primary chronic pancreatitis and in normal pancreas from mouse and swine. In contrast, p62 protein is absent from, or only focally and very weakly expressed in, insulinomas, glucagonomas or non-functioning pancreatic neuroendocrine tumours or carcinomas that express insulin or other pancreatic as well as extrapancreatic hormones. Although the biological function of p62 in beta cells is unknown, the co-expression of p62 and insulin in non-neoplastic beta cells suggests that, in the beta cell, p62 may play a role in specific insulin-related signalling. Since p62 may also be involved in pro-apototic signal transduction, the loss of p62 expression in neuroendocrine neoplasms of the pancreas may render the tumour cells less sensitive to pro-apototic signals. Further research is necessary to elucidate the role of p62 in beta cell-specific signal transduction.     

Deletion at 3p25.3-p23 is frequently encountered in endocrine pancreatic tumours and is associated with metastatic progression

For several reasons, chromosome 3p is thought to be involved in the pathogenesis of sporadic endocrine pancreatic tumours (EPTs): von Hippel-Lindau's disease (VHL gene at 3p25.5) is associated with EPTs; 3p is frequently involved in solid human tumours; and comparative genomic hybridization has identified frequent losses at 3p in EPTs. This study investigated 99 benign and malignant tumours, including 20 metastases, from 82 patients, by microsatellite loss of heterozygosity (LOH) analysis and fluorescence in situ hybridization (FISH) in order to evaluate the importance of chromosome 3p deletions in the molecular pathogenesis and biological behaviour of EPTs, to elaborate a common region of deletion, and to narrow down putative tumour suppressor gene loci. Allelic losses of 3p were found in 58/99 (58.6%) of tumours in 45/82 (54.9%) patients; analysis of seven microsatellite markers (3p26-p21) revealed a common region of LOH at 3p25.3-p23. The LOH frequency was significantly higher in malignant than in benign neoplasms (70.2% versus 28.0%; p=0.001). In addition, a strong correlation was found between the loss of alleles on chromosome 3p and clinically metastatic disease (LOH of 73.7% in metastasizing versus 41.5% in non-metastasizing tumours; p=0.008). EPTs from these patients showed a tendency towards losing large parts or the entire short arm of chromosome 3 with tumour progression. Furthermore, FISH analysis revealed complete loss of chromosome 3 in ten out of 37 EPTs (27%). These results indicate that a putative tumour suppressor gene at 3p25.3-p23 may play a role in the oncogenesis of sporadic EPTs and that losses of larger centromeric regions are associated with metastatic progression.     

Prevalence of CD99 protein expression in pancreatic endocrine tumours (PETs)

AIMS: To determine the prevalence of CD99 expression in pancreatic endocrine tumours (PETs). We evaluated CD99 expression and analysed Ki67 labelling by immunohistochemistry in PETs. METHODS AND RESULTS: Thirty-eight PETs from 33 patients were analysed. CD99 immunoreactivity was consistently observed in normal islets of the pancreas, regardless of the cell type. Tumours comprising more than 30% CD99+ cells were defined as positively immunoreactive for CD99. CD99 expression was observed in 20 of the 38 PETs examined, but not in any of the pancreatic tumours of other histological subtypes (10 ductal adenocarcinomas, five intraductal papillary-mucinous tumours, and two acinar cell tumours). Loss of CD99 expression was related to markers of worse prognosis for PET, including gross local invasion, metastasis to the lymph nodes or other organs, lymphatic or blood vessel invasion, and neuroendocrine carcinoma (NEC). Thus, CD99 expression may have an efficiency comparable to that of high Ki67 labelling index (5% or more) for prognostication. CONCLUSIONS: CD99 expression was observed frequently and exclusively in PETs, and loss of CD99 expression in PETs was found to be associated with ominous prognostic indicators.     

Increased expression of histone deacetylase 2 is found in human gastric cancer

Accumulated evidence has established that aberrant regulation of histone deacetylases (HDACs) is one of the major causes of the development of human malignancies. Among different iso-enzymes of HDAC and sirtuins grouped as the HDAC super family, little is known as to how histone deacetylase 2 (HDAC2) causes carcinogenesis in solid tumors. Here, in order to investigate the possible role of HDAC2 in gastric carcinogenesis, we analyzed the expression of HDAC2 in 71 gastric adenocarcinomas by immunohistochemistry. Moderate to strong expression of HDAC2 was found in 44 (62%) out of a total of 71 tumors. The majority of positive tumors, which were detected in the nucleus but not in normal gastric epithelium, did not express HDAC2 or showed only weak positive staining. Interestingly, we also noted that HDAC2 expression appeared to be associated with tumor aggressiveness as HDAC2 expression was observed to be statistically significant in advanced gastric cancer (P=0.0023, Chi-square test) and in positive lymph node metastasis (P=0.0713, Chi-square test). Taken together, these results suggest that HDAC2 may play an important role in the aggressiveness of gastric cancer.     

'Difficult to diagnose' desmoid tumours: a potential role for CTNNB1 mutational analysis

Colombo C, Bolshakov S, Hajibashi S, Lopez‐Terrada L, Wang W‐L, Rao P, Benjamin R S, Lazar A J & Lev D
(2011) Histopathology 59 , 336–340 ‘Difficult to diagnose’ desmoid tumours: a potential role for CTNNB1 mutational analysis Aims: The utility of CTNNB1 (encoding β‐catenin) genotyping for diagnosing sporadic desmoid tumours (DT) when traditional clinicopathological parameters were inconclusive was evaluated. Methods and results: Cases included were: (i) new primary lesions where initial DT diagnosis was inconclusive; and (ii) possible recurrent DT versus scar. Formalin‐fixed paraffin‐embedded (FFPE) tissues were obtained via needle biopsy or a surgical excision (57 specimens) as part of initial assessment. DNA extraction, CTNNB1 exon 3 amplification and sequencing were conducted in a Clinical Laboratory Improvement Amendments of 1988 (CLIA)‐approved molecular diagnostics laboratory. For patients with no previous DT history (n = 47) sequencing identified mutations in 30 (64%), substantiating DT diagnosis. In biopsies with non‐mutated (NM) CTNNB1 (n = 17) the test was inconclusive; in seven of these, a diagnosis of DT was strongly favoured in the subsequent surgical resection specimen. Ten patients with previously resected DT were evaluated; mutation was identified in six cases (60%), indicating DT over scar. In two (20%) with primary tumours harbouring CTNNB1 mutation no mutation was found, favouring scar over DT; the other two NM‐CTNNB1 cases (20%) were inconclusive. Conclusions: CTNNB1 genotyping can be very useful in ‘difficult to diagnose’ lesions when the differential diagnosis includes DT. Recognizing inherent test limitations, the presence of CTNNB1 mutation can inform the therapeutic approach.     

beta-catenin nuclear expression correlates with cyclin D1 overexpression in sporadic desmoid tumours

The immunohistochemical expression of beta-catenin, cyclin D1, Ki-67 and PCNA was Examined in 38 cases of sporadic extra-abdominal or abdominal-wall desmoid tumours without familial adenomatous polyposis (FAP), to evaluate the hypothesis that the accumulated beta-catenin within the nuclei could affect the regulation of the cyclin D1 gene. There was a statistically significant correlation between beta-catenin accumulation and cyclin D1 overexpression (p=0.029). Each group with beta-catenin accumulation or cyclin D1 overexpression showed a higher PCNA-LI than those without, the difference being statistically significant (p=0.007, p=0.004, respectively). Differential PCR was also performed to detect amplification of the cyclin D1 gene and mutational analysis was undertaken for exon 3 of the beta-catenin gene. Amplification of the cyclin D1 gene was observed in 13 out of 22 cases (59.1%). There were nine-point mutations in 7 out of 18 cases (38.9%). The distribution of beta-catenin mutation fell within a wide range, from codon 21 to codon 67. In conclusion, beta-catenin nuclear expression correlated with cyclin D1 overexpression in sporadic desmoid tumours, which could be an in vivo model system for the APC-beta-catenin-Tcf pathway. In addition, beta-catenin mutations in desmoid tumours occurred at an unusually wide range of sites within the gene.     

High frequency of beta-catenin mutations in borderline endometrioid tumours of the ovary

Some low-grade endometrioid carcinomas arise from a background of endometrioid tumours of borderline malignancy. To determine the molecular mechanisms involved in the initiation of endometrioid carcinoma, the present study investigated whether the genetic alterations reported in these tumours (mutations in PTEN, KRAS, and beta-catenin genes, and microsatellite instability) are already present in endometrioid tumours of borderline malignancy. Eight endometrioid tumours of borderline malignancy were studied. By immunohistochemistry, beta-catenin was expressed in the nuclei of all tumours, suggesting the presence of stabilizing beta-catenin mutations. By mutational analysis, five different beta-catenin mutations were found in seven of eight cases (90%), affecting codons 32, 33, and 37. In contrast, only one tumour harboured a PTEN mutation, which affected codon 130. Neither KRAS mutations nor microsatellite instability was detected. A review of the literature indicated that beta-catenin mutations are characteristic of well-differentiated endometrioid carcinomas, since they were present in nearly 60% of grade I but in less of 3% of grade III tumours. In conclusion, the present study identifies beta-catenin mutation as a nearly constant molecular alteration in borderline endometrioid tumours, whereas PTEN and KRAS mutations and microsatellite instability are very infrequent. The findings in the present study, and previously reported data, strongly suggest that beta-catenin mutation is an early event in endometrioid ovarian carcinogenesis, and that it is involved in the development of low-grade endometrioid tumours.     

AMACR is highly expressed in gastric adenomas and intestinal-type carcinomas

Alpha-methylacyl-CoA racemase (AMACR) is a novel tumor biomarker expressed in a number of neoplasms, including colorectal and prostatic adenocarcinomas. However, AMACR expression has not been investigated in preneoplastic and neoplastic lesions of the stomach. Using immunohistochemistry we studied the expression of AMACR in normal gastric mucosa (n=32), intestinal metaplasia (n=26), adenomas (n=29) and adenocarcinomas (n=132) of the stomach from 135 patients. Synchronous adenocarcinomas arising in the background of adenomas were observed in 26 cases. AMACR immunoreactivity was not observed in all normal gastric mucosa. Tissue from intestinal metaplasia, adenomas, and adenocarcinomas was positive in 7.7% (2/26), 79.3% (23/29), and 62.9% (83/132) of cases, respectively. The difference in AMACR expression between adenomas or adenocarcinomas and non-neoplastic mucosa was statistically significant (p=0.0001). Moreover, intestinal-type carcinomas showed significantly higher expression of AMACR (69.8%) compared to diffuse-type carcinomas (47.2%) (p=0.02). Our results indicate that as well as being an additional diagnostic tool, altered AMACR expression in gastric adenomas and intestinal-type carcinomas suggests that AMACR may be involved early in the development of intestinal-type gastric carcinomas.     

IGFBP-1 is expressed specifically in ovarian clear cell adenocarcinoma

Sugita S, Morishita Y, Kano J, Furuya S, Shiba‐Ishii A & Noguchi M
(2011) Histopathology 58 , 729–738
IGFBP‐1 is expressed specifically in ovarian clear cell adenocarcinoma Aims: To investigate the specific expression of insulin‐like growth factor binding protein‐1 (IGFBP‐1) in ovarian clear cell adenocarcinoma (CCA). Methods and results: Immunohistochemistry and in situ hybridization for IGFBP‐1 were performed in normal endometrium, placenta, and 100 surgically resected cases of ovarian cancer including 31 CCAs and 69 non‐CCAs. Immunohistochemistry for hepatocyte nuclear factor‐1 (HNF‐1β) was also examined in all cases. Specific expression of IGFBP‐1 was confirmed in secretory endometrium, decidua of placenta and Arias‐Stella glands of miscarriage material. Among ovarian cancers, almost all cases of CCA showed expression of both IGFBP‐1 protein and mRNA, but non‐CCA hardly expressed IGFBP‐1. There was a significant difference between CCA and non‐CCA in the expression of IGFBP‐1 protein and mRNA. No correlation was found between the rate of IGFBP‐1 expression and pathological T and N factors of the tumour–node–metastasis (TNM) classification. All CCA cases except for one exhibited expression of HNF‐1β protein, whereas only 15.9% of non‐CCAs did so. Conclusion: The expression of IGFBP‐1 in CCA is more specific than that of HNF‐1β. IGFBP‐1 shows expression by decidual endometrium and Arias‐Stella glands, and CCA also exhibits characteristic expression. These results indicate that IGFBP‐1 is a immunohistochemical marker for CCA.     

Involvement of α‐ and β‐catenins and E‐cadherin in the development of mammary phyllodes tumours

Tsang J Y S, Mendoza P, Lam C C F, Yu A M C, Putti T C, Karim R Z, Scolyer R A, Lee C S, Tan P H & Tse G M
(2012) Histopathology  61, 667–674 Involvement of α‐ and β‐catenins and E‐cadherin in the development of mammary phyllodes tumours Aims: Phyllodes tumours (PT) are rare but clinically important fibroepithelial tumours of the breast. β‐Catenin, a key component in Wnt signalling, has been shown to be important in the development of PT. It also functions as a component of the cadherin complex, which may therefore be implicated in PT pathogenesis. By assessing stromal α‐catenin, β‐catenin and E‐cadherin expression in 158 PT cases using immunohistochemistry and examining associations with clinicopathological features, we aimed to determine the role of these proteins in PT pathogenesis. Methods and results: Cytoplasmic β‐catenin correlated with α‐catenin expression. A significantly higher expression of both markers was observed in borderline than in benign PT (P = 0.003 and <0.001, respectively), but a lower level was found in malignant PT. Cytoplasmic E‐cadherin expression was significantly higher in borderline and malignant than in benign PT (P = 0.001 and 0.012, respectively), but was not correlated with other markers. Both E‐cadherin and α‐catenin showed stronger correlations with histological parameters than β‐catenin. α‐Catenin showed a significant correlation with recurrence (P = 0.005 and 0.016, respectively). Conclusions: α‐ and β‐catenins may be important in the early stages of PT development, while E‐cadherin may be required for malignant development. The correlation of α‐catenin expression with tumour recurrence may be relevant in predicting PT behaviour.