β‐Catenin (CTNNB1) mutations and clinicopathological features of mesenteric desmoid‐type fibromatosis

Aims and methods: Desmoid‐type fibromatosis (desmoid) is a fibroblastic tumour that shows locally aggressive growth. Mesenteric desmoid is a rare lesion that shares morphological and biological features with fibromatoses occurring in the abdominal wall or in extraabdominal sites, but differs in terms of gross appearance and clinical presentation. We report on a series of 56 cases of mesenteric desmoids from our consultation files and compare them with cases of non‐mesenteric desmoids and retroperitoneal fibrosis. Results: Primary diagnosis of desmoid‐type fibromatosis was correct in 42%, and gastrointestinal stromal tumour was a common misdiagnosis. Nuclear expression of β‐catenin was detected in 91.6% of all desmoids. Mutational analysis of exon 3 of the β‐catenin gene (CTNNB1) revealed that mesenteric desmoids carried mutations significantly more often (51/56, 91.1%) than non‐mesenteric tumours (20/28; 71.4%; P = 0.027). p.T41A occurred significantly more frequently in mesenteric fibromatoses (80.4%) than in abdominal wall and extra‐abdominal fibromatoses (46.4%; P = 0.002). Two novel mutations (p.S45C and p.D32G) were found. In retroperitoneal fibrosis, mutations and nuclear β‐catenin expression were absent. β‐Catenin‐negative desmoids either carried a CTNNB1 mutation or were associated with Gardner syndrome. Conclusions:  Our study provides evidence that some clinical and genetic features of mesenteric desmoids differ from those of non‐mesenteric fibromatosis, and corroborates the usefulness of mutational analysis, especially in diagnosing β‐catenin‐negative mesenteric desmoids.     

Prevalence of the CTNNB1 mutation genotype in surgically resected fibromatosis of the breast

Kim T, Jung E A, Song J Y, Roh J H, Choi J S, Kwon J E, Kang S Y, Cho E Y, Shin J H, Nam S‐J, Yang J H & Choi Y‐L
(2012) Histopathology  60, 347–356
Prevalence of the CTNNB1 mutation genotype in surgically resected fibromatosis of the breast Aims: To investigate CTNNB1 mutation and β‐catenin expression in resected breast fibromatosis and to identify potential molecular markers of fibromatosis of the breast. Methods and results: We selected 12 patients with fibromatosis of the breast who underwent surgical resection and were confirmed by histological examination. Ultrasonography findings for 10 patients were reviewed and only two cases were suspicious for fibromatosis on imaging. On core needle biopsy for pre‐operative diagnoses, only three cases were histologically suspicious for fibromatosis. Mutations in exon 3 of CTNNB1 were detected by direct DNA sequencing in nine (75.0%) cases: all were c.121G>A (p.T41A), which was much more frequent in breast fibromatoses than in other soft tissue lesions. Nuclear β‐catenin expression was observed in all cases and the level of expression was higher in cases with mutation. In eight of nine cases, the matched biopsy specimen showed the same CTNNB1 mutation status as the pre‐operative specimen. Conclusions: In the majority of cases, clinical presentation and breast imaging are highly suspicious for carcinoma. Definitive pre‐operative pathological diagnosis by core needle biopsy is difficult. CTNNB1 mutation and nuclear β‐catenin expression are frequently detected in sporadic breast fibromatoses, suggesting their potential as a useful tool to distinguish breast fibromatoses from other neoplasms.     

Focal β‐catenin mutation identified on formalin‐fixed and paraffin‐embedded inflammatory hepatocellular adenomas

The identification of hepatocellular adenoma (HCA) with mutation in exon 3 of the CTNNB1 gene encoding for β‐catenin is clinically relevant due to a higher risk of malignant transformation. Inflammatory HCA (IHCA) can exhibit β‐catenin activation (β‐IHCA). We report two cases with multiple IHCA in which focal β‐catenin activation has been found in one of the IHCA. In both cases, the diagnosis of IHCA was confirmed on the resected nodules by routine stains, immunohistochemical detection of C‐reactive protein (CRP) and molecular biology on frozen material. An additional molecular analysis was performed on formalin‐fixed paraffin‐embedded (FFPE) material that showed focal glutamine synthetase (GS) staining, the surrogate marker of β‐catenin activation. In case 1, it was a 1.8‐cm area within the 7.5 cm IHCA, and in case 2 a small 0.3‐cm area within a 1.8 cm resected IHCA located close to a larger IHCA, negative for GS. In both cases, nuclear β‐catenin expression and decreased reticulin network were observed in the GS expressing foci, together with cholestasis and diffuse CD34 expression in case 1. Molecular analysis by pyrosequencing on FFPE material using the GS‐stained slides as reference to select areas with/without positive staining revealed a CTNNB1 exon 3 mutation restricted to the areas exhibiting both positive GS and CRP expression, whereas wild‐type CTNNB1 was found in areas showing only CRP staining. These two cases illustrate focal β‐catenin activation that can occur within IHCAs. Additional data are needed to determine if β‐catenin mutation is a secondary event in IHCA.     

A lesion categorized between ghost cell odontogenic carcinoma and dentinogenic ghost cell tumor with CTNNB1 mutation

Ghost cell odontogenic carcinoma (GCOC) is a rare malignant neoplasm characterized by the presence of ghost cells. It is considered to arise either de novo or from a preexisting benign precursor, calcifying odontogenic cyst (COC), or dentinogenic ghost cell tumor (DGCT). We report a case of a 44‐year‐old Japanese male with a left maxillary tumor. The patient received treatment to resect the left maxillary cyst 25 years prior; however, the details were uncertain. The tumor was resected with clear margins. Taken together with the results of histological and immunohistochemical examinations, the tumor was categorized between GCOC and DGCT, and we diagnosed the tumor as GCOC suggesting similarity to DGCT. Further, we focused on CTNNB1, which encodes β‐catenin and is frequently mutated in COCs. In this tumor, we identified CTNNB1 Ser33Cys, one of the mutations typically found in COCs. This finding suggests that CTNNB1 is a common target for the pathogenesis of tumors accompanied by ghost cells.     

Near universal detection of alterations in CTNNB1 and Wnt pathway regulators in desmoid‐type fibromatosis by whole‐exome sequencing and genomic analysis

CTNNB1 mutations or APC abnormalities have been observed in ～85% of desmoids examined by Sanger sequencing and are associated with Wnt/β‐catenin activation. We sought to identify molecular aberrations in “wild‐type” tumors (those without CTNNB1 or APC alteration) and to determine their prognostic relevance. CTNNB1 was examined by Sanger sequencing in 117 desmoids; a mutation was observed in 101 (86%) and 16 were wild type. Wild‐type status did not associate with tumor recurrence. Moreover, in unsupervised clustering based on U133A‐derived gene expression profiles, wild‐type and mutated tumors clustered together. Whole‐exome sequencing of eight of the wild‐type desmoids revealed that three had a CTNNB1 mutation that had been undetected by Sanger sequencing. The mutation was found in a mean 16% of reads (vs. 37% for mutations identified by Sanger). Of the other five wild‐type tumors sequenced, two had APC loss, two had chromosome 6 loss, and one had mutation of BMI1. The finding of low‐frequency CTNNB1 mutation or APC loss in wild‐type desmoids was validated in the remaining eight wild‐type desmoids; directed miSeq identified low‐frequency CTNNB1 mutation in four and comparative genomic hybridization identified APC loss in one. These results demonstrate that mutations affecting CTNNB1 or APC occur more frequently in desmoids than previously recognized (111 of 117; 95%), and designation of wild‐type genotype is largely determined by sensitivity of detection methods. Even true CTNNB1 wild‐type tumors (determined by next‐generation sequencing) may have genomic alterations associated with Wnt activation (chromosome 6 loss/BMI1 mutation), supporting Wnt/β‐catenin activation as the common pathway governing desmoid initiation. © 2015 Wiley Periodicals, Inc.     

Beta‐catenin status in paediatric medulloblastomas: correlation of immunohistochemical expression with mutational status,genetic profiles,and clinical characteristics

Medulloblastoma is the most frequent malignant paediatric brain tumour. The activation of the Wnt/β‐catenin pathway occurs in 10‐15% of medulloblastomas and has been recently described as a marker for favourable patient outcome. We report a series of 72 paediatric medulloblastomas evaluated for β‐catenin protein expression, CTNNB1 mutations, and comparative genomic hybridization. Gene expression profiles were also available in a subset of 40 cases. Immunostaining of β‐catenin showed extensive nuclear staining (>50% of the tumour cells) in six cases and focal nuclear staining (<10% of cells) in three cases. The other cases either exhibited a signal strictly limited to the cytoplasm (58 cases) or were negative (five cases). CTNNB1 mutations were detected in all β‐catenin extensively nucleopositive cases. The expression profiles of these cases documented strong activation of the Wnt/β‐catenin pathway. Remarkably, five out of these six tumours showed a complete loss of chromosome 6. In contrast, cases with focal nuclear β‐catenin staining, as well as tumours with negative or cytoplasmic staining, never demonstrated CTNNB1 mutation, Wnt/β‐catenin pathway activation or chromosome 6 loss. Patients with extensive nuclear staining were significantly older at diagnosis and were in continuous complete remission after a mean follow‐up of 75.7 months (range 27.5–121.2 months) from diagnosis. All three patients with focal nuclear staining of β‐catenin died within 36 months from diagnosis. Altogether, these data confirm and extend previous observations that CTNNB1‐mutated tumours represent a distinct molecular subgroup of medulloblastomas with favourable outcome, indicating that therapy de‐escalation should be considered. International consensus on the definition criteria of this distinct medulloblastoma subgroup should be achieved. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

'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.     

Multiple pilomatricomas with somatic CTNNB1 mutations in children with constitutive mismatch repair deficiency

Constitutional mismatch repair deficiency (CMMR‐D) due to biallelic germline mutations in one of four mismatch repair genes causes a childhood cancer syndrome characterized by a broad tumor spectrum including hematological malignancies, and brain and Lynch syndrome‐associated tumors. Herein, we report three children who had in addition to CMMR‐D‐associated malignancies multiple pilomatricomas. These are benign skin tumors of hair matrical differentiation frequently associated with somatic activating mutations in the ß‐catenin gene CTNNB1. In two of the children, the diagnosis of CMMR‐D was confirmed by the identification of biallelic germline PMS2 mutations. In the third individual, we only found a heterozygous germline PMS2 mutation. In all nine pilomatricomas with basophilic cells, we detected CTNNB1 mutations. Our findings indicate that CTNNB1 is a target for mutations when mismatch repair is impaired due to biallelic PMS2 mutations. An elevated number of activating CTNNB1 alterations in hair matrix cells may explain the development of multiple pilomatricomas in CMMR‐D patients. Of note, two of the children presented with multiple pilomatricomas and other nonmalignant features of CMMR‐D before they developed malignancies. To offer surveillance programs to CMMR‐D patients, it may be justified to suspect CMMR‐D syndrome in individuals fulfilling multiple nonmalignant features of CMMR‐D (including multiple pilomatricomas) and offer molecular testing in combination with interdisciplinary counseling. © 2013 Wiley Periodicals, Inc.     

Hepatoblastoma—An attempt of histological subtyping on fine‐needle aspiration material

Hepatoblastoma (HB) is classified into epithelial, mixed (epithelial/mesenchymal), and small‐cell (anaplastic) type. Wnt/β‐catenin pathway plays a key role in hepatic development, regeneration, and tumorigenesis, and HB is known to present β‐catenin mutations (50–90%). The present study was undertaken to delineate the cytomorphologic features of HB and to evaluate the feasibility of subtyping of HB on fine‐needle aspiration cytology (FNAC). The expression of β‐catenin in these tumors was also evaluated both of histopathologic sections and on the aspirated material. Thirty‐three cases with fine‐needle aspirates of HB were retrieved over a period of 12 years. Cytologic diagnosis was reviewed in the light of clinicoradiological data, response to therapy, and subsequent histopathology. Immunochemistry for β‐catenin was performed in 19 of 33 cases on histopathologic sections (n = 10)/cell blocks (n = 6)/cytosmears (n = 3). Based on the cytologic features, the cases were divided into fetal HB (n = 17), embryonal HB (n = 4), combined epithelial HB (n = 8), and mixed HB (n = 4). Four cases of histopathologically proven mixed HB were reported as pure epithelial HB on FNAC, as mesenchymal elements were not represented in the cytology smears. Cytoplasmic as well as nuclear staining for β‐catenin was noted in a total of 10 of 19 cases. FNAC can accurately categorize epithelial HB; however, in mixed type, the accuracy depends on number of areas sampled. Cell block can be of help to perform ancillary investigations especially β‐catenin for both diagnostic and therapeutic purposes. Cytopathol. 2013. © 2012 Wiley Periodicals, Inc.     

Novel intra‐genic large deletions of CTNNB1 gene identified in WT desmoid‐type fibromatosis

A wait and see approach for desmoid tumors (DT) has become part of the routine treatment strategy. However, predictive factors to select the risk of progressive disease are still lacking. A translational project was run in order to identify genomic signatures in patients enrolled within an Italian prospective observational study. Among 12 DT patients (10 CTNNB1‐mutated and 2 wild type) enrolled from our institution only two patients (17%) showed a progressive disease. Tumor biopsies were collected for whole exome sequencing. Overall, DT exhibited low somatic sequence mutation rate and no additional recurrent mutation was found. In the two wild type (WT) cases, two novel alterations were detected: a complex deletion of APC and a pathogenic mutation of LAMTOR2. Focusing on WT DT subtype, deep sequencing of CTNNB1, APC and LAMTOR2 was conducted on a retrospective series of 11 WT DT using a targeted approach. No other mutation of LAMTOR2 was detected, while APC was mutated in two cases. Low‐frequency (mean reads of 16%) CTNNB1 mutations were discovered in five samples (45%) and two novel intra‐genic deletions in CTNNB1 were detected in two cases. Both deletions and low frequency mutations of CTNNB1 were highly expressed. In conclusion, a minority of DT is WT for either CTNNB1, APC or any other gene involved in the WNT pathway. In this subgroup novel and hard to be detected molecular alterations in APC and CTNNB1 were discovered, contributing to explain a portion of the allegedly WT DT cases.     

Genetic alteration and immunohistochemical staining patterns of ovarian high‐grade serous adenocarcinoma with special emphasis on p53 immnnostaining pattern

We evaluated p53, KRAS, BRAF and CTNNB1 mutation and p53, WT1, p16 and beta‐catenin expression in 31 ovarian high‐grade serous adenocarcinoma. Twenty‐five (80.6%) tumors contained functional mutations of p53; three frameshift, four nonsense and 19 missense mutations. None of the tumors showed KRAS, BRAF or CTNNB1 mutation. In all 18 tumors with missense mutations, ≥60% of tumor cells were strongly positive for p53 immunostaining whereas all tumors with frameshift or nonsense mutations were completely negative. Missense mutation was correlated with diffuse and strong imunoreaction and frameshift/nonsense mutation was correlated with completely negative immunoreaction (P = 0.000). Tumors with wild‐type p53 revealed a wide range of immunostaining patterns. In 27 (87.1%) and 18 (58.1%) tumors, ≥50% of tumor cells were moderate to strongly positive for WT1 and p16, respectively. A considerable intratumoral heterogeneity for p16 expression was present. None of the tumors demonstrated nuclear beta‐catenin expression. p53 mutations appear to be a powerful molecular marker for ovarian high‐grade serous adenocarcinoma. Using p53 with an appropriate interpretation criteria together with WT1, p16 and beta‐catenin, most of the high‐grade serous adenocarcinoma could be distinguished from other ovarian tumors.     

Inactive Wnt/β‐catenin pathway in conventional high‐grade osteosarcoma

Osteosarcoma is the most common malignant bone tumour, with a peak incidence in children and young adolescents, suggesting a role of rapid bone growth in its pathogenesis. The Wnt/β‐catenin pathway plays a crucial role in skeletal development and is indispensable for osteoblasts' lineage determination. Previous studies suggesting an oncogenic role for the Wnt/β‐catenin pathway in osteosarcoma were based on cytoplasmic staining of β‐catenin or the detection of one component of this pathway. However, those approaches are inappropriate to address whether the Wnt/β‐catenin pathway is functionally active. Therefore, in this study, we examined nuclear β‐catenin expression in 52 human osteosarcoma biopsies, 15 osteoblastomas (benign bone tumours), and four human osteosarcoma cell lines by immunohistochemistry. Furthermore, we modulated Wnt/β‐catenin pathway activity using a GIN (GSK3β inhibitor) and evaluated its effect on cell growth and osteogenic differentiation. Absence of nuclear β‐catenin staining was found in 90% of the biopsies and all osteosarcoma cell lines, whereas strong nuclear β‐catenin staining was observed in all osteoblastomas. Wnt‐luciferase activity was comparable to the negative control in all osteosarcoma cell lines. GIN stimulated the Wnt/β‐catenin pathway, as shown by translocation of β‐catenin into the nucleus and increased Wnt‐luciferase activity as well as mRNA expression of AXIN2, a specific downstream target gene. Stimulation of the Wnt/β‐catenin pathway by GIN significantly reduced cell proliferation in the cell lines MG‐63 and U‐2‐OS and enhanced differentiation in the cell lines HOS and SJSA‐1, as shown by an increase in alkaline phosphatase (ALP) activity and mineralization. In contrast with the oncogenic role of the Wnt/β‐catenin pathway in osteosarcoma as previous studies suggested, here we demonstrate that this pathway is inactivated in osteosarcoma. Moreover, activation of the Wnt/β‐catenin pathway inhibits cell proliferation or promotes osteogenic differentiation in osteosarcoma cell lines. Our data suggest that loss of Wnt/β‐catenin pathway activity, which is required for osteoblast differentiation, may contribute to osteosarcoma development. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.