Title: Specific Mutations in the β-Catenin Gene (CTNNB1) Correlate with Local Recurrence in Sporadic Desmoid Tumors
Abstract: Desmoid fibromatosis is a rare, nonmetastatic neoplasm marked by local invasiveness and relentless recurrence. Molecular determinants of desmoid recurrence remain obscure. β-Catenin deregulation has been commonly identified in sporadic desmoids although the incidence of CTNNB1 (the gene encoding β-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 β-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. Desmoid fibromatosis is a rare, nonmetastatic neoplasm marked by local invasiveness and relentless recurrence. Molecular determinants of desmoid recurrence remain obscure. β-Catenin deregulation has been commonly identified in sporadic desmoids although the incidence of CTNNB1 (the gene encoding β-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 β-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. Desmoid fibromatosis is a rare soft tissue monoclonal neoplasm demonstrating fibroblastic to myofibroblastic differentiation. With an estimated incidence of two to four desmoid tumors arising per million population per year, ∼1000 new desmoid diagnoses are made annually in the US.1Reitamo JJ Hayry P Nykyri E Saxen E The desmoid tumor. I. Incidence, sex-, age- and anatomical distribution in the Finnish population.Am J Clin Pathol. 1982; 77: 665-673Crossref PubMed Scopus (439) Google Scholar Desmoids predominantly affect young adults, especially females, and primarily involve the extremities (including proximal points of attachment at the pelvic and shoulder girdles), the trunk, and the intestinal mesenteries.2Lev D Kotilingam D Wei C Ballo MT Zagars GK Pisters PW Lazar AA Patel SR Benjamin RS Pollock RE Optimizing treatment of desmoid tumors.J Clin Oncol. 2007; 25: 1785-1791Crossref PubMed Scopus (225) Google Scholar Desmoid tumors can infrequently occur as part of familial syndromes such as familial adenomatous polyposis (FAP) and familial infiltrative fibromatosis, both identified as caused by a germ-line mutation in the adenomatosis polyposis gene (APC).3Eccles DM van der Luijt R Breukel C Bullman H Bunyan D Fisher A Barber J du Boulay C Primrose J Burn J Fodde R Hereditary desmoid disease due to a frameshift mutation at codon 1924 of the APC gene.Am J Hum Genet. 1996; 59: 1193-1201PubMed Google Scholar, 4Kinzler KW Nilbert MC Su LK Vogelstein B Bryan TM Levy DB Smith KJ Preisinger AC Hedge P McKechnie D Finner R Markham A Groffen J Boguski MS Altschul SF Horii A Andoh H Miyoshi Y Miki Y Nishisho I Nakamura Y Identification of FAP locus genes from chromosome 5q21.Science. 1991; 253: 661-665Crossref PubMed Scopus (2007) Google Scholar, 5Giardiello FM Petersen GM Piantadosi S Gruber SB Traboulsi EI Offerhaus GJ Muro K Krush AJ Booker SV Luce MC Laken SJ Kinzler KW Vogelstein B Hamilton SR APC gene mutations and extraintestinal phenotype of familial adenomatous polyposis.Gut. 1997; 40: 521-525PubMed Google Scholar, 6O'Sullivan MJ McCarthy TV Doyle CT Familial adenomatous polyposis: from bedside to benchside.Am J Clin Pathol. 1998; 109: 521-526PubMed Google Scholar, 7Latchford A Volikos E Johnson V Rogers P Suraweera N Tomlinson I Phillips R Silver A APC mutations in FAP-associated desmoid tumours are non-random but not ‘just right’.Hum Mol Genet. 2007; 16: 78-82Crossref PubMed Scopus (48) Google Scholar, 8Miyaki M Yamaguchi T Iijima T Takahashi K Matsumoto H Yasutome M Funata N Mori T Difference in characteristics of APC mutations between colonic and extracolonic tumors of FAP patients: variations with phenotype.Int J Cancer. 2008; 122: 2491-2497Crossref PubMed Scopus (26) Google Scholar However, most desmoids are sporadic and there is a global lack of knowledge regarding risk factors associated with their etiology and development. Diagnosis is made on the basis of clinical, radiological, and histological parameters. However, distinguishing desmoid tumors from reactive processes such as scar or other benign fibroblastic neoplasms, and even differentiating from low-grade sarcomas can be difficult at times, especially when diagnosis is based on a needle biopsy.9Carlson JW Fletcher CD Immunohistochemistry for b-catenin in the differential diagnosis of spindle cell lesions: analysis of a series and review of the literature.Histopathology. 2007; 51: 509-514Crossref PubMed Scopus (212) Google Scholar, 10Montgomery E Folpe AL The diagnostic value of b-catenin immunohistochemistry.Adv Anat Pathol. 2005; 12: 350-356Crossref PubMed Scopus (46) Google Scholar Although unable to metastasize, these highly infiltrative and locally destructive lesions have a significant tendency to recur, with recurrence rates ranging from 19 to 38% in multiple studies.2Lev D Kotilingam D Wei C Ballo MT Zagars GK Pisters PW Lazar AA Patel SR Benjamin RS Pollock RE Optimizing treatment of desmoid tumors.J Clin Oncol. 2007; 25: 1785-1791Crossref PubMed Scopus (225) Google Scholar, 11Leibel SA Wara WM Hill DR Bovill Jr, EG de Lorimier AA Beckstead JH Phillips TL Desmoid tumors: local control and patterns of relapse following radiation therapy.Int J Radiat Oncol Biol Phys. 1983; 9: 1167-1171Abstract Full Text PDF PubMed Scopus (129) Google Scholar, 12Bataini JP Belloir C Mazabraud A Pilleron JP Cartigny A Jaulerry C Ghossein NA Desmoid tumors in adults: the role of radiotherapy in their management.Am J Surg. 1988; 155: 754-760Abstract Full Text PDF PubMed Scopus (87) Google Scholar, 13McKinnon JG Neifeld JP Kay S Parker GA Foster WC Lawrence Jr, W Management of desmoid tumors.Surg Gynecol Obstet. 1989; 169: 104-106PubMed Google Scholar, 14McCollough WM Parsons JT van der Griend R Enneking WF Heare T Radiation therapy for aggressive fibromatosis. The Experience at the University of Florida.J Bone Joint Surg Am. 1991; 73: 5717-5725Google Scholar, 15Acker JC Bossen EH Halperin EC The management of desmoid tumors.Int J Radiat Oncol Biol Phys. 1993; 26: 851-858Abstract Full Text PDF PubMed Scopus (107) Google Scholar, 16Catton CN O'Sullivan B Bell R Cummings B Fornasier V Panzarella T Aggressive fibromatosis: optimisation of local management with a retrospective failure analysis.Radiother Oncol. 1995; 34: 17-22Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 17Ballo MT Zagars GK Pollack A Pisters PW Pollack RA Desmoid tumor: prognostic factors and outcome after surgery, radiation therapy, or combined surgery and radiation therapy.J Clin Oncol. 1999; 17: 158-167PubMed Google Scholar, 18Jelinek JA Stelzer KJ Conrad E Bruckner J Kliot M Koh W Laramore GE The efficacy of radiotherapy as postoperative treatment for desmoid tumors.Int J Radiat Oncol Biol Phys. 2001; 50: 121-125Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 19Gronchi A Casali PG Mariani L Lo Vullo S Colecchia M Lozza L Bertulli R Fiore M Olmi P Santinami M Rosai J Quality of surgery and outcome in extra-abdominal aggressive fibromatosis: a series of patients surgically treated at a single institution.J Clin Oncol. 2003; 21: 1390-1397Crossref PubMed Scopus (293) Google Scholar, 20Abbas AE Deschamps C Cassivi SD Nichols III, FC Allen MS Schleck CD Pairolero PC Chest-wall desmoid tumors: results of surgical intervention.Ann Thorac Surg. 2004; 78: 1219-1223Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 21Duggal A Dickinson IC Sommerville S Gallie P The management of extra-abdominal desmoid tumours.Int Orthop. 2004; 28: 252-256Crossref PubMed Scopus (36) Google Scholar, 22Phillips SR A'Hern R Thomas JM Aggressive fibromatosis of the abdominal wall, limbs and limb girdles.Br J Surg. 2004; 91: 1624-1629Crossref PubMed Scopus (75) Google Scholar, 23Kotiligam D Lazar AJ Pollock RE Lev D Desmoid tumor: a disease opportune for molecular insights.Histol Histopathol. 2008; 23: 117-126PubMed Google Scholar Currently there are no reliable methods to identify those tumors with a higher risk for recurrence; the molecular determinants of desmoid behavior remain primarily unknown.23Kotiligam D Lazar AJ Pollock RE Lev D Desmoid tumor: a disease opportune for molecular insights.Histol Histopathol. 2008; 23: 117-126PubMed Google Scholar Surgical resection remains the mainstay of desmoid therapy; however, these tumors frequently require multiple and at times debilitating surgical interventions for their control, resulting in significant treatment-related morbidity such as amputation or loss of significant portions of foregut. Anti-inflammatory agents, hormonal blockade, and cytotoxic chemotherapy also may be useful in selected patients, but overall response rates to these modalities remains modest at best.23Kotiligam D Lazar AJ Pollock RE Lev D Desmoid tumor: a disease opportune for molecular insights.Histol Histopathol. 2008; 23: 117-126PubMed Google Scholar, 24Gega M Yanagi H Yoshikawa R Noda M Ikeuchi H Tsukamoto K Oshima T Fujiwara Y Gondo N Tamura K Utsunomiya J Hashimoto-Tamaoki T Yamamura T Successful chemotherapeutic modality of doxorubicin plus dacarbazine for the treatment of desmoid tumors in association with familial adenomatous polyposis.J Clin Oncol. 2006; 24: 102-105Crossref PubMed Scopus (151) Google Scholar, 25Patel SR Benjamin RS Desmoid tumors respond to chemotherapy: defying the dogma in oncology.J Clin Oncol. 2006; 24: 11-12Crossref PubMed Scopus (71) Google Scholar, 26Patel SR Evans HL Benjamin RS Combination chemotherapy in adult desmoid tumors.Cancer. 1993; 72: 3244-3247Crossref PubMed Scopus (210) Google Scholar Investigation of FAP-related desmoids has led to the identification of a potential role for β-catenin deregulation in desmoid tumorigenesis.27Alman BA Li C Pajerski ME Diaz-Cano S Wolfe HJ Increased b-catenin protein and somatic APC mutations in sporadic aggressive fibromatoses (desmoid tumors).Am J Pathol. 1997; 151: 329-334PubMed Google Scholar β-Catenin is a cadherin-binding protein involved in cell-cell adhesion, but also functions as a transcriptional activator when complexed in the nucleus with members of the T-cell factor/lymphocyte enhancer factor family of proteins.28Willert K Jones KA Wnt signaling: is the party in the nucleus?.Genes Dev. 2006; 20: 1394-1404Crossref PubMed Scopus (509) Google Scholar Control of cytosolic levels of β-catenin occurs via a multiprotein complex, in which APC, axin/conductin, and glycogen synthetase kinase-3β (GSK-3β) negatively regulate β-catenin expression. Once bound to this protein complex, β-catenin is sequentially phosphorylated on four critical amino acids (serines 45, 37, and 33 and threonine at 41), ultimately resulting in the targeting of β-catenin for ubiquitination and proteosomal degradation.29Xu W Kimelman D Mechanistic insights from structural studies of b-catenin and its binding partners.J Cell Sci. 2007; 120: 3337-3344Crossref PubMed Scopus (192) Google Scholar Mutations in the CTNNB1 gene coding for β-catenin have been described in a variety of epithelial-originating malignancies, in which deregulated β-catenin has been identified as contributing to their protumorigenic phenotype.30Clevers H Wnt/beta-catenin signaling in development and disease.Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4518) Google Scholar, 31Polakis P The oncogenic activation of b-catenin.Curr Opin Genet Dev. 1999; 9: 15-21Crossref PubMed Scopus (607) Google Scholar, 32Willert K Nusse R b-Catenin: a key mediator of Wnt signaling.Curr Opin Genet Dev. 1998; 8: 95-102Crossref PubMed Scopus (666) Google Scholar, 33Polakis P The many ways of Wnt in cancer.Curr Opin Genet Dev. 2007; 17: 45-51Crossref PubMed Scopus (757) Google Scholar, 34Lazar AJ Calonje E Grayson W Dei Tos AP Mihm Jr, MC Redston M McKee PH Pilomatrix carcinomas contain mutations in CTNNB1, the gene encoding beta-catenin.J Cutan Pathol. 2005; 32: 148-157Crossref PubMed Scopus (112) Google Scholar, 35Gavert N Ben-Ze'ev A Beta-catenin signaling in biological control and cancer.J Cell Biochem. 2007; 102: 820-828Crossref PubMed Scopus (146) Google Scholar Most CTNNB1 mutations occur within exon 3 of the gene in the region encoding for the β-catenin protein sequence that contains the serine/threonine residues described above. These mutations result in β-catenin stabilization, increased nuclear accumulation, and transcriptional activation of target genes such as c-MYC, c-JUN, and cyclin D.28Willert K Jones KA Wnt signaling: is the party in the nucleus?.Genes Dev. 2006; 20: 1394-1404Crossref PubMed Scopus (509) Google Scholar, 32Willert K Nusse R b-Catenin: a key mediator of Wnt signaling.Curr Opin Genet Dev. 1998; 8: 95-102Crossref PubMed Scopus (666) Google Scholar, 33Polakis P The many ways of Wnt in cancer.Curr Opin Genet Dev. 2007; 17: 45-51Crossref PubMed Scopus (757) Google Scholar Recently, mutations in exon 3 of the CTNNB1 gene have also been identified in sporadic desmoid tumors.27Alman BA Li C Pajerski ME Diaz-Cano S Wolfe HJ Increased b-catenin protein and somatic APC mutations in sporadic aggressive fibromatoses (desmoid tumors).Am J Pathol. 1997; 151: 329-334PubMed Google Scholar, 36Shitoh K Konishi F Iijima T Ohdaira T Sakai K Kanazawa K Miyaki M A novel case of a sporadic desmoid tumour with mutation of the b-catenin gene.J Clin Pathol. 1999; 52: 695-696Crossref PubMed Scopus (42) Google Scholar, 37Amary MF Pauwels P Meulemans E Roemen GM Islam L Idowu B Bousdras K Diss TC O'Donnell P Flanagan AM Detection of b-catenin mutations in paraffin-embedded sporadic desmoid-type fibromatosis by mutation-specific restriction enzyme digestion (MSRED): an ancillary diagnostic tool.Am J Surg Pathol. 2007; 31: 1299-1309Crossref PubMed Scopus (84) Google Scholar, 38Saito T Oda Y Kawaguchi K Tanaka K Matsuda S Tamiya S Iwamoto Y Tsuneyoshi M Possible association between higher b-catenin mRNA expression and mutated beta-catenin in sporadic desmoid tumors: real-time semiquantitative assay by TaqMan polymerase chain reaction.Lab Invest. 2002; 82: 97-103Crossref PubMed Scopus (44) Google Scholar, 39Tejpar S Nollet F Li C Wunder JS Michils G dal Cin P Van Cutsem E Bapat B van Roy F Cassiman JJ Alman BA Predominance of b-catenin mutations and b-catenin dysregulation in sporadic aggressive fibromatosis (desmoid tumor).Oncogene. 1999; 18: 6615-6620Crossref PubMed Scopus (286) Google Scholar, 40Miyoshi Y Iwao K Nawa G Yoshikawa H Ochi T Nakamura Y Frequent mutations in the b-catenin gene in desmoid tumors from patients without familial adenomatous polyposis.Oncol Res. 1998; 10: 591-594PubMed Google Scholar, 41Jilong Y Jian W Xiaoyan Z Xiaoqiu L Xiongzeng Z Analysis of APC/b-catenin genes mutations and Wnt signalling pathway in desmoid-type fibromatosis.Pathology. 2007; 39: 319-325Crossref PubMed Scopus (34) Google Scholar Summarizing all desmoid tumors analyzed in the several initial published series (n = 106 total desmoid specimens) suggests that CTNNB1 mutations occur in ∼50% of sporadic tumors. However, a recent publication evaluating the β-catenin status in the largest single cohort analyzed to date (n = 76) identified a mutational prevalence of 87%, potentially rendering sporadic desmoid tumors as a neoplasm possessing among the highest rate of β-catenin mutations yet described.37Amary MF Pauwels P Meulemans E Roemen GM Islam L Idowu B Bousdras K Diss TC O'Donnell P Flanagan AM Detection of b-catenin mutations in paraffin-embedded sporadic desmoid-type fibromatosis by mutation-specific restriction enzyme digestion (MSRED): an ancillary diagnostic tool.Am J Surg Pathol. 2007; 31: 1299-1309Crossref PubMed Scopus (84) Google Scholar This incidence discrepancy remains to be resolved. In the current study we attempted to evaluate the prevalence and spectra of β-catenin mutations in a large clinically annotated cohort of sporadic human desmoid tumor specimens. Furthermore, we correlated genotyping results with clinical and immunohistochemical features, including time to recurrence from the original tumor resection and β-catenin protein expression pattern and intensity. With institutional review board approval, formalin-fixed, paraffin-embedded desmoid tumor specimens accrued between 1985 and 2005 were retrieved from The University of Texas M.D. Anderson Cancer Center (UTMDACC) pathology archives amounting to 195 specimens from 160 patients. When available, matching normal tissue was also retrieved. For tissue microarray (TMA) construction, dermal scar tissue specimens (n = 18) to be used as controls were also retrieved. All specimens were further screened and evaluated by an experienced soft tissue pathologist (A.J.L.), and only those with confirmed desmoid tumor histology and also containing tissue adequate for analytic purposes were included. Normal tissue and scar specimens were likewise confirmed as per these criteria as well. Clinical information including demographic, therapeutic, tumor, and clinical outcome variables were retrieved from patient medical records and from the UTMDACC soft tissue tumor database and were tabulated for correlative analyses. For all patients, analysis of time to recurrence was calculated from initial surgery for the primary desmoids to the first recurrence, regardless of whether the surgery was performed at UTMDACC or elsewhere. Site was categorized as superficial trunk (to include the abdominal wall, chest wall, and back), extremity, deep trunk/mesentery, and head and neck. All patients were treated surgically with curative intent; this was a criteria for inclusion in the study. Postoperative radiotherapy was given to ∼20% of patients. Cases in which patients were diagnosed with FAP or had a family history of FAP were excluded; therefore, only sporadic desmoid tumors were ultimately evaluated. DNA was extracted from two 20-μm-thick formalin-fixed, paraffin-embedded tissue rolls cut from blocks with at least 70% tumor using the QIAamp DNA mini kit DNA isolation kit (n = 160, including 151 sporadic desmoids tumors; Qiagen Valencia, CA). Polymerase chain reaction (PCR) used primers (BCAT-DES-F: 5′-AGTCACTGGCAGCAACAGTC-3′ and BCAT-DES-R: 5′-TCTTCCTCAGGATTGCCTT-3′) and thermocycling conditions previously reported to amplify exon 3 of CTNNB1 (phosphorylation domain, codons 30 to 48) with appropriate controls.42Koch A Denkhaus D Albrecht S Leuschner I von Schweinitz D Pietsch T Childhood hepatoblastomas frequently carry a mutated degradation targeting box of the b-catenin gene.Cancer Res. 1999; 59: 269-273PubMed Google Scholar PCR products were detected by gel electrophoresis in 2% agarose, and amplicon bands were purified using the QIAquick gel extraction kit (Qiagen). Direct sequencing used the above primers (forward and reverse), ABI Prism dye terminator cycle sequencing ready reaction kit, and ABI Prism 3100-Avant genetic analyzer (Applied Biosystems, Foster City, CA). Both strands were analyzed by the NCBI Blast Alignment Tool (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) to identify mutations. For TMA construction, hematoxylin and eosin (H&E)-stained sections were reviewed to define areas of desmoid tumor; dermal scars served as controls. Using an automated TMA apparatus (ATA-27; Beecher Instruments, Sun Prairie, WI), 0.6-mm punch samples (two per case; 408 total) were obtained from the donor desmoid (n = 195) and scar (n = 18) blocks and formatted into three recipient blocks. H&E-staining of 4-μm TMA sections was used to verify all samples. The polymeric biotin-free horseradish peroxide method on a Microsystems Bond Max stainer (Beecher Instruments, Bannockburn, IL) was used for immunohistochemistry. Four-μm-thick sections were prepared from formalin-fixed paraffin-embedded tissue blocks and were dried in a 60-degree oven for 20 minutes. These sections were placed in the automated Bond Max stainer that pretreated the slides with enzyme-induced epitope retrieval for 2 minutes followed by incubation with antibodies against β-catenin (clone 14, dilution 1:500; BD Biosciences, San Jose, CA). Anti-mouse secondary antibody and the Refine polymer detection kit (Leica) was used for immunostaining, with 3,3-diaminobenzidine serving as chromagen. Positive and negative controls were run in parallel. Labeling intensity was graded by a soft tissue pathologist (A.J.L.) as none (=0), weak (=1), moderate (=2), or strong (=3) for both the cytosolic and nuclear compartments, and the percentage of positive tumor cells was estimated from the two paired TMA samples for each case. For nuclear staining, weak (=1) was defined functionally as having to view nuclei at ×400 to confirm nuclear accumulation. Moderate (=2) staining could be viewed at ×200 and the blue hematoxylin nuclear counterstain was apparent. In strong staining, the nuclear counterstain was no longer visible in the majority of nuclei. The staining on the entire array was performed in the UTMDACC clinical immunohistochemistry facility on two occasions and each was scored independently. Associations among the possible predictors of time from primary surgery to initial recurrence were examined using Fisher's exact test, Kruskal-Wallis test, or Spearman's correlation coefficient as appropriate. Variables considered included CTNNB1 genotype, tumor size, tumor site, gender, and age at initial diagnosis. Time-to-recurrence was examined using Cox proportional hazards regression and the Kaplan-Meier method. Strata were compared using the log-rank test. Multivariate models were constructed by introducing all variables simultaneously into the model and then eliminating variables using the backward selection method. P values were two-tailed and considered significant at α <0.05. Analyses were conducted using SAS for Windows (release 9.1; SAS Institute, Cary, NC). We first sought to evaluate the prevalence of CTNNB1 mutations in a relatively large cohort of clinically annotated sporadic desmoid tumors treated at a single institution. Initially, 160 tumors from as many patients were included. All cases had sufficient DNA extracted for CTNNB1 exon 3 genotyping; the results were definitive in 154 (96%) of cases. After excluding cases with a personal or family history of FAP (n = 9, none with CTNNB1 mutations) and seven cases lacking sufficient clinical information, a collection of 138 sporadic desmoid tumors remained and were included in this analysis (Table 1). Of these, 86 patients were female (62%) and 52 were male (38%). Median age was 32 years and ranged from 0.2 to 78 years. The following sites were involved: 54 in superficial trunk (39%), 53 in extremities (38%), 18 in deep trunk/mesentery (13%), and 13 in head and neck (9%). Size was known in 108 cases and ranged from 1.1 to 30 cm (median, 6 cm). The primary tumors were available for 89 patients and the clinical details of this subset are presented in Table 1. A mutation in exon 3 of the CTNNB1 gene was identified in 117 (85%) of the 138 cases. No mutations were identified in adjacent normal tissue in all cases in which this was tested (n = 25). Correlating the presence of CTNNB1 gene mutation with patient and tumor variables, female patients were much more likely than male patients to lack a mutation (22% versus 4%, P = 0.0032); associations with age at diagnosis, tumor sites, and desmoid size were not noted (Table 2).Table 1Desmoid Tumor Cohort DataTotal, n = 138Primary, n = 89n%n%Gender Female86625360 Male52383640Age, years (median, range)32 (0.2 to 78)33 (1 to 78)Site Superficial trunk54393337 Extremity53383135 Deep trunk/mesentery18131416 Head and neck13101112Size, cm (median, range) (total)6 (1.1 to 30)(n = 108)6.8 (1.1 to 30)(n = 76)Clinical data for the desmoid cohort. There were 138 desmoid patients with sufficient clinical and molecular data for analysis. The entire cohort of 138 was used for the analysis of clinical variables associated with CTNNB1 mutation genotype. The 89 patients with primary desmoid tumors on the array were used for the β-catenin immunohistochemical study. Open table in a new tab Table 2Patient and Tumor Properties with CTNNB1 GenotypeTotalWT41A45F45PVariablen%n%n%n%n%Gender Female866219223642252967 Male5238243363142736Age at diagnosis <30 years614412203151142347 >30 years77569123849253257Tumor site Superficial trunk5439101927501120611 Extremity5338592751203812 Deep trunk/viscera18133171161211211 Head and neck131032343164600Tumor size*Tumor size was available only for 108 of the total of 138 patients in this cohort. The total row gives the percentage of each tumor type in the population. WT, wild type (not mutated). <6 cm56*Tumor size was available only for 108 of the total of 138 patients in this cohort. The total row gives the percentage of each tumor type in the population. WT, wild type (not mutated).5211202443162959 >6 cm52*Tumor size was available only for 108 of the total of 138 patients in this cohort. The total row gives the percentage of each tumor type in the population. WT, wild type (not mutated).486113058142724Total13821156950392897Distribution of CTNNB1 mutation types tabulated with patient clinical and tumor data. Females were more likely to lack a mutation in CTNNB1 and males were more likely to have a 41A mutation. There was a trend toward association of the 45F mutation with tumors involving an extremity.* Tumor size was available only for 108 of the total of 138 patients in this cohort. The total row gives the percentage of each tumor type in the population. WT, wild type (not mutated). Open table in a new tab Clinical data for the desmoid cohort. There were 138 desmoid patients with sufficient clinical and molecular data for analysis. The entire cohort of 138 was used for the analysis of clinical variables associated with CTNNB1 mutation genotype. The 89 patients with primary desmoid tumors on the array were used for the β-catenin immunohistochemical study. Distribution of CTNNB1 mutation types tabulated with patient clinical and tumor data. Females were more likely to lack a mutation in CTNNB1 and males were more likely to have a 41A mutation. There was a trend toward association of the 45F mutation with tumors involving an extremity. Next, we determined the CTNNB1 mutational spectra. Interestingly, only three different point mutations in two different codons (41 and 45) could be identified in all mutated samples (n = 117 of 138 total) (Figure 1): ACC to GCC in codon 41 (41A), resulting in the replacement of threonine by alanine, was identified in 69 samples (59%); TCT to TTT in codon 45 (45F), resulting in the replacement of serine by phenylalanine, was identified in 39 cases (33%); and TCT to CCT in codon 45 (45P), resulting in the replacement of serine with proline, was identified in nine samples (8%). When analyzing the incidence of the specific CTNNB1 gene mutations as a correlate of patient and tumor variables, we identified that males have a higher incidence of the 41A mutation than females (63% versus 42%, P = 0.0109) and in terms of tumor site there was a trend toward tumors involving the extremities having a higher incidence of the 45F mutation (38%, P = 0.0640) versus all other sites. No other associations were noted. We sought to evaluate whether any specific CTNNB1 gene mutation correlated with clinical outcome. Time to first recurrence was calculated from date of the initial attempt at complete surgical resection irrespective of whether this occurred at UTMDACC or elsewhere. In that the study cohort was representative of the referral pattern to a large tertiary center, it therefore included a large proportion of patients (34%) presenting with recurrent desmoid tumors, and the 5-year recurrence rate from time of diagnosis of initial tumor was 49% (95