Droplet Digital PCR (ddPCR) as a Novel Technology in Detecting CTNNB1 Mutations in Desmoid Fibromatosis.

Identification of Familial Adenomatous Polyposis carriers among children with desmoid tumours

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 large referral 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. CTNNB1 genotyping 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 diagnosed with desmoid tumors lacking CTNNB1 mutations because this population is enriched for FAP patients.

Desmoid-type fibromatosis (DF) is a locally aggressive neoplasm characterized by mutations in the CTNNB1 gene, which encodes the β-catenin protein. We reviewed 85 cases of DF and performed Sanger sequencing for detecting mutations in CTNNB1 and immunostaining for detecting β-catenin localization. We included 70 DF samples, of which 56 cases demonstrated nuclear β-catenin localization and 43 cases harboured CTNNB1 mutations. CTNNB1-mutant DF samples consistently displayed nuclear β-catenin expression and were derived from larger-sized tumours compared to samples with wild-type CTNNB1. When we further classified DF cases into 2 subgroups based on the type of specimen, excised specimens with nuclear β-catenin expression frequently displayed CTNNB1 mutation and no statistical correlation between nuclear β-catenin expression and CTNNB1 mutation was observed in biopsies. When we classified CTNNB1 mutation cases into 2 subgroups (DF with T41A or T41I, and DF with S45F or S45P), T41A or T41I mutations were observed more frequently in males than in females. Additionally, DF tumours harbouring S45F or S45P mutations were located more frequently in the abdominal wall than tumours with T41A or T41I mutations. In conclusion, CTNNB1 mutation correlates with nuclear β-catenin expression in larger or excised DF tumours, and DF harbouring CTNNB1 mutations manifest variable clinical presentations.

Sinonasal hemangiopericytoma (HPC) is a tumor showing pericytic myoid differentiation and which arises in the nasal cavity and paranasal sinuses. CTNNB1 mutations appear to be a consistent aberration in sinonasal HPC, and nuclear expression of β-catenin has been reported. Our aim was to evaluate the frequency of β-catenin expression in sinonasal HPC and its histologic mimics in the upper aerodigestive tract. Cases were retrieved from the surgical pathology and consultation files. Immunohistochemical staining for β-catenin was performed on 50 soft tissue tumors arising in the sinonasal tract or oral cavity, and nuclear staining was recorded semiquantitatively by extent and intensity. Nuclear reactivity for β-catenin was present in 19/20 cases of sinonasal HPC; 17 showed moderate-to-strong multifocal or diffuse staining, and 2 had moderate focal nuclear reactivity. All solitary fibrous tumors (SFT) (10/10) showed focal-to-multifocal nuclear staining, varying from weak to strong in intensity. Most cases of synovial sarcoma (9/10) showed nuclear β-catenin expression in the spindle cell component, ranging from focal-weak to strong-multifocal. No cases of myopericytoma (0/10) showed any nuclear β-catenin expression. β-catenin expression is prevalent in sinonasal HPC, but is also frequent in SFT and synovial sarcoma. Our findings indicate that β-catenin is not a useful diagnostic tool in the evaluation of spindle cell tumors with a prominent hemangiopericytoma-like vasculature in the sinonasal tract and oral cavity, and that definitive diagnosis relies on the use of a broader immunohistochemical panel.

This study aimed to elucidate the genetic landscape of biliary papillary neoplasms. Of 28 cases examined, 7 underwent whole exome sequencing, while the remaining 21 were used for validation studies with targeted sequencing. In the whole exome sequencing study, 4/7 cases had mutations in either APC or CTNNB1, both of which belong to the Wnt/β-catenin pathway. Somatic mutations were also identified in genes involved in RAS signaling (KRAS, BRAF), a cell cycle regulator (CDC27), histone methyltransferase (KMT2C, KMT2D), and DNA mismatch repair (MSH3, MSH6, PMS1). Combined with discovery and validation cohorts, mutations in APC or CTNNB1 were observed in 6/28 subjects (21%) and were mutually exclusive. When the cases were classified into intraductal papillary neoplasms of the bile duct (IPNBs, n=14) and papillary cholangiocarcinomas (n=14) based on the recently proposed classification criteria, mutations in APC and CTNNB1 appeared to be entirely restricted to IPNBs with 6/14 cases (43%) harboring mutations in either gene. These genetic alterations were detected across the 3 nonintestinal histologic types. In immunohistochemistry, the aberrant cytoplasmic and/or nuclear expression of β-catenin was found in not only 5/6 IPNBs with APC or CTNNB1 mutations, but also 6/8 cases with wild-type APC and CTNNB1 (total 79%). In addition, APC and CTNNB1 alterations were exceptional in nonpapillary cholangiocarcinomas (n=29) with a single case harboring CTNNB1 mutation (3%). This study demonstrated recurrent mutations in APC and CTNNB1 in nonintestinal-type IPNBs, suggesting that activation of the Wnt/β-catenin signaling pathway is relevant to the development and progression of IPNBs.

Recent advances in genomics have improved the molecular classification of cutaneous melanocytic tumors. Among them, deep penetrating nevi (DPN) and plexiform nevi have been linked to joint activation of the MAP kinase and dysregulation of the β-catenin pathways. Immunohistochemical studies have confirmed cytoplasmic and nuclear expression of β-catenin and its downstream effector cyclin D1 in these tumors. We assessed nuclear β-catenin immunohistochemical expression in a large group of DPN as well as in the four most frequent differential diagnoses of DPN: "blue" melanocytic tumors, Spitz tumors, nevoid and SSM melanomas, and pigmented epithelioid melanocytomas (PEM). Nuclear β-catenin expression was positive in 98/100 DPN and 2/16 of melanomas (one SSM and one nevoid melanoma with a plexiform clone) and was negative in all 30 Spitz, 26 blue, and 6 PEM lesions. In 41% DPN, β-catenin expression was positive in more than 30% nuclei. No differences were observed in cytoplasmic and nuclear cyclin D1 expression between these tumor groups, suggesting alternate, β-catenin-independent, activation pathways. We have subsequently studied nuclear β-catenin expression in a set of 13 tumors with an ambiguous diagnosis, for which DPN was part of the differential diagnosis. The three out of four patients showing canonical DPN mutation profiles were the only β-catenin-positive cases. We conclude that nuclear β-catenin expression, independently from CCND1 expression, in a dermal melanocytic tumor is an argument for its classification as DPN. In ambiguous cases and in early combined DPN lesions, this antibody can be helpful as a screening tool. β-Catenin is also potentially expressed in a subset of malignant melanomas with CTNNB1 mutations.

The Wnt signaling pathway is pivotal in the adenoma-carcinoma sequence; however, its role in small bowel adenocarcinoma (SBA) remains insufficiently characterized. We analyzed the clinicopathological significance of Wnt pathway-related gene mutations and the expression of downstream or associated proteins in SBA. Immunohistochemical staining for β-catenin, cyclin D1, c-Myc, E-cadherin, and Wnt5a was performed in 75 primary SBA surgical specimens. Targeted next-generation sequencing was conducted in 48 of these cases. The genomic alterations in the Wnt pathway were identified as APC (14.6%) and CTNNB1 (8.3%), with no overlap between the two mutations. Aberrant (reduced membranous and/or nuclear) expression of β-catenin was observed in 37% of cases. Cyclin D1 and c-Myc were expressed in 60% and 41% of cases, respectively. Aberrant expression of β-catenin and/or Wnt5a was present in 60% of cases and was correlated with cyclin D1 and c-Myc expression. Mutations in APC and CTNNB1 were found in intestinal- and gastrointestinal-type SBAs, but were absent in gastric-type SBA. In intestinal-type SBA, the mutation frequency of APC and CTNNB1 was 39%, closely aligning with the 45% aberrant expression of β-catenin. Aberrant expression of β-catenin and/or Wnt5a, a ligand of the noncanonical Wnt pathway, was detected in 60% of cases and showed a correlation with both cyclin D1 and c-Myc expression. These findings suggest that both canonical and noncanonical Wnt pathway-related proteins are involved in SBA carcinogenesis and progression. Notably, the canonical Wnt pathway appears to play a predominant role in intestinal-type SBA.

ColoSeq Provides Comprehensive Lynch and Polyposis Syndrome Mutational Analysis Using Massively Parallel Sequencing

Loss of Membranous Expression of β-Catenin Is Associated with Tumor Progression in Cutaneous Melanoma and Rarely Caused by Exon 3 Mutations

11547 Background: Recently, first-in-class FDA approval was granted in the U.S. for use of the gamma secretase inhibitor (GSI), nirogacestat, for adults with progressive desmoid fibromatosis. In tandem, the unpredictable clinical behavior of desmoids, which ranges from local aggression to regression, raises consideration of whether diagnostic and molecular variability underlie their varying biological potential. With an aim to understand both, and to explore the potential for biomarkers for GSI therapy selection, prediction, and prognosis, we performed a retrospective review of comprehensive genomic profiling and histology of desmoids and other soft tissue tumors harboring CTNNB1 or APC mutations. Methods: Using real-world reference laboratory database of tumors submitted for clinical genomic assessment (Caris Life Sciences), we queried for samples with a diagnosis of desmoid fibromatosis, or for other neoplasms of soft tissue origin harboring CTNNB1 or APC mutations. Samples underwent next-gen sequencing of (whole exome) to identify gene variants/copy number alterations and of RNA (whole transcriptome) for expression and fusion profiling. Findings were correlated with available clinical data and whole slide image histologic review. Results: We identified 74 tumors submitted as desmoid fibromatosis, of which 80% harbored CTNNB1 and 15% harbored APC pathogenic or likely pathogenic variants. CTNNB1 variants included codon 41 (58%), codon 45 (41%), and ubiquitin motif codon 36 (1%), while 91% of APC variants detected were in exon 16. Recurrent co-alterations were rare, involving MUTYH (heterozygous G396D) in 2 samples, and TMB-High (≥10 mutations/Mb) present in 3 . Notably, 4 “desmoids” (5%) lacked characteristic mutations, one of which harbored COL1A1::USP6 fusion, reclassified as nodular fasciitis. Among 76 soft tissue tumors diagnosed as other entities at analysis but found to harbor CTNNB1/APC mutations, 6 (all limited core biopsies), could be confidently reclassified as desmoids. The remaining 70 CTNNB1/APC mutant neoplasms were diverse, including synovial sarcoma (11%) and rhabdomyosarcoma (10%). Conclusions: Correlation of genomics and histopathology may allow identification of other tumor types misclassified as desmoid fibromatosis. Conversely, genomic correlation facilitated recognition of additional desmoids among tumors submitted with other diagnoses. The striking lack of secondary mutations seen in this large cohort with comprehensive DNA sequencing implies that other mechanisms explain and could predict their variable behavior, for which we are exploring paired transcriptome profiling data. Finally, subsets of diverse, other soft tissue neoplasms harbor CTNNB1 or APC mutations, which may have implications for the design of future biomarker-selected Phase II basket trials.

Sinonasal myxoma (SNM) is a rare, benign mesenchymal neoplasm with distinct clinicopathologic features and aberrant nuclear localization of β-catenin by immunohistochemistry. The molecular underpinnings have been linked to that of a "myxoid variant" of desmoid fibromatosis. Herein, we describe a series of 8 cases of SNM and propose clinical and biologic differences compared with desmoid fibromatosis. Our patient cohort is comprised of 5 males and 3 females (age range: 10mo to 12y), 6 of whom are aged less than or equal to 24 months. All presented with facial swelling, reflecting lesions involving the maxillary bone, and all underwent resection. All tumors were variably cellular and comprised of bland spindled to stellate cells in a profusely myxoid background with diffuse nuclear β-catenin expression. All cases of SNM were analyzed by next-generation sequencing using the Oncopanel assay. Three cases failed sequencing, 2 of 5 successful cases exhibited exon 3 CTNNB1 alterations involving the ubiquitin recognition motif, and 3 had adenomatous polyposis coli ( APC ) deletions. One patient had APC germline testing which was negative. No germline testing was available for the remaining 7 patients. Follow-up data over a range of 1 month to 23 years was available for 7 of the 8 SNMs. One case patient had local recurrence, and all were alive without evidence of disease. This is in contrast to the high recurrence rate typically seen in desmoid fibromatosis, particularly after resection. Our findings expand the spectrum of tumors with underlying WNT/β-catenin pathway and highlight the histologic, clinical, and genetic differences of SNM compared with desmoid fibromatosis. APC deletion raises the possibility of underlying germline alteration and familial adenomatous polyposis.

Fibromatoses encompass a broad group of histopathologically similar fibroblastic/myofibroblastic proliferations with divergent clinical manifestations and behavior. Deep (desmoid-type) fibromatoses are typically large, rapidly growing, and locally aggressive tumors that occur in the abdominal wall, mesentery, and extra-abdominal soft tissue, principally the musculature of the trunk and extremities. Most sporadic cases of desmoid fibromatosis harbor inactivating mutations in CTNNB1, the gene encoding beta-catenin. Tumors occurring in the context of familial adenomatous polyposis and Gardner syndrome bear inactivating mutations in APC. By contrast, mutations in CTNNB1 or APC have not been identified in cases of superficial fibromatosis. Cutaneous involvement by desmoid fibromatosis is exceedingly rare. Here we present a 78-year-old male with desmoid-type fibromatosis arising in the dermis of the right medial calf with a pathogenic mutation in CTNNB1 and a variant of unknown significance in APC.

Wnt/β-catenin signal alteration and its diagnostic utility in basal cell adenoma and histologically similar tumors of the salivary gland

Desmoid fibromatosis (DSM) is a rare, locally aggressive mesenchymal tumor genetically characterized by mutations in the CTNNB1 gene. A local control rate of up to 65‒80% for DSM is achieved with multiple modality treatments, including watchful monitoring, radiation therapy, chemotherapy, and surgery. However, several variables, such as age < 30years, extremity tumor location, and tumor size of > 10cm in diameter, are associated with poor local control rates in patients with DSM. The definitive treatments for DSM have not been established. Therefore, it is necessary to develop novel treatments for DSM. Moreover, although patient-derived tumor cell lines are potent tools for preclinical research, no DSM cell lines have been reported. Therefore, this study aimed to establish and characterize a novel DSM cell line for preclinical studies on DSM. Herein, we established the first cell line derived from a patient with DSM exhibiting poor prognostic factors (27-year-old male patient with a DSM tumor of > 10cm in diameter located at the lower extremity) and named it NCC-DSM1-C1. NCC-DSM1-C1 cells had a T41A mutation in CTNNB1 and exhibited constant proliferation, spheroid formation, and invasion capability in vitro. Screening of antitumor agents in NCC-DSM1-C1 cells showed that bortezomib and romidepsin are effective against DSM. In conclusion, we report the first officially characterized DSM cell line derived from a patient with DSM exhibiting factors associated with poor prognosis. We believe that NCC-DSM1-C1 cell line is a useful tool for developing novel treatments for DSM.

Desmoid fibromatosis is a rare, nonmetastatic neoplasm marked by local invasiveness and relentless recurrence. Molecular determinants of desmoid recurrence remain obscure. beta-Catenin deregulation has been commonly identified in sporadic desmoids although the incidence of CTNNB1 (the gene encoding beta-catenin) mutations is uncertain. Consequently, we evaluated the prevalence of CTNNB1 mutations in a large cohort of sporadic desmoids and examined whether mutation type was relevant to desmoid outcome. Desmoid specimens (195 tumors from 160 patients, 1985 to 2005) and control dermal scars were assembled into a clinical data-linked tissue microarray. CTNNB1 genotyping was performed on a 138-sporadic desmoid subset. Immunohistochemical scoring was performed per standard criteria and data were analyzed using Kaplan-Meier and other indicated methods. CTNNB1 mutations were observed in 117 of 138 (85%) of desmoids. Three discrete mutations in two codons of CTNNB1 exon 3 were identified: 41A (59%), 45F (33%), and 45P (8%, excluded from further analysis because of rarity). Five-year recurrence-free survival was significantly poorer in 45F-mutated desmoids (23%, P < 0.0001) versus either 41A (57%) or nonmutated tumors (65%). Nuclear beta-catenin expression was observed in 98% of specimens and intensity was inversely correlated with incidence of desmoid recurrence (P < 0.01). In conclusion, CTNNB1 mutations are highly common in desmoid tumors. Furthermore, patients harboring CTNNB1 (45F) mutations are at particular risk for recurrence and therefore may especially benefit from adjuvant therapeutic approaches.