972 Cranial Desmoid Tumor Associated with Homozygous Inactivation of the Adenomatous Polyposis Coli Gene in a 2-Year-Old Girl with Familial Adenomatous Poly posis Deepthi C. de Silva, M.R.c.P.(uK)’ Morag F. Wright, B.SC? David A. J. Stevenson, B.SC.’ Caroline Clark, MSC.’ Elizabeth s. Gray, M.R.C.Path.3 John D. Holmes, M.Chir., F.R.c.s.~ John c. s. Dean, F.R.C.P.(Edin)’ Neva E. Haites, Ph.D.’ Malcolm G. Dunlop, M.D? ’ Department of Medical Genetics, Medical School, Foresterhill Aberdeen, Scotland. MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland. Department of Pathology, Medical School, Foresterhill, Aberdeen, Scotland. Department of Plastic Surgery, Royal Aberdeen Children’s Hospital, Aberdeen, Scotland. BACKGROUND. Familial adenomatous polyposis (FAP) is a dominantly inherited disorder characterized by the presence of more than 100 adenomatous polyps in the colon and rectum starting in the second decade of life. FAP is associated with extra colonic manifestations, including desmoid tumors. METHODS. A 2-year-old girl presented with a rapidly enlarging tumor of the forehead and a family history of FAP. The tumor was cultured for cytogenetic studies. A DNA linkage study using flanking and intragenic polymorphisms of the adenomatous polyposis coli (APC) gene was performed to identify allele loss in the tumor. Germline mutation identification was by single strand conformation polymorphism analysis of exon 15 of the APC gene, with subsequent double stranded sequencing of fragments with conformational changes. A mutation-induced loss of a restriction site was used to confirm allele loss in the tumor. RESULTS. Microscopically, the tumor had desmoid features. Cytogenetic analysis of the tumor demonstrated loss of chromosome region 5(q21q22). A truncating adenomatous polyposis coli (APC) gene mutation was identified in the leukocyte DNA from the child and her affected father. Linked DNA markers suggested that the tumor had lost the maternal, wild-type allele, A mutation-induced restriction endonuclease site alteration demonstrated hemizygosity of the mutant sequence in the tumor DNA. CONCLUSIONS. These findings are compatible with the presence of a “second hit” inactivation of the APC gene and implicate this gene in the pathogenesis of desmoid tumors. Cancer 1996; 77:972-6. 0 1996 American Cancer Society. KEYWORDS: desmoid tumor, familial adenomatous polyposis (FAP), chromosome deletion, adenomatous polyposis coli gene (APC gene). D. de Silva was funded by an ACTR Research Fellowship from the Aberdeen Royal Hospitals NHS Trust. APC mutation analysis and allele dosage studies were funded by Scottish Home and Health Department (SHHD) grant KIMRSI 50/C1837 to M.G. Dunlop. Address for reprints: Deepthi C. de Silva, Department of Medical Genetics, Medical School, Polwarth Building, Foresterhill, Aberdeen, AB9 220 Scotland. Received August 8, 1995; revision received November 6, 1995; accepted November 6, 1995. 0 1996 American Cancer Society F amilial adenomatous polyposis (FAP) is a dominantly inherited disorder characterized by the presence of over 100 adenomatous polyps in the rectum and colon developing from the second decade of life, which, if untreated, progress to carcinoma. Extracolonic manifestations include duodenal adenomatous polyps, epidermal and sebaceous cysts, fibromata, osteomata, and desmoid tumors. Congenital hypertrophy of the retinal pigment epithelium (CHRPE) is present in a characteristic distribution in a high proportion of affected individuals and is a useful indicator of the family members who have inherited the predisposing gene. The association between adenomatous polyps in the colon and other ectodermal and mesodermal tumors has been eponymously termed Gardner syndrome. The incidence of desmoid tumors in FAP is approximately lo%.’ The identification of the FAP gene locus on chromosome 5q222,3and the subsequent cloningz5of APC Gene Inactivation in a Cranial Desmoid TumorlDeSilva et al. 973 forehead lesion, the proband presented with a history of recurrent left limb paralysis lasting about 4 hours. There were no symptoms preceding the episode and five further episodes, occurring approximately fortnightly, were reported. On examination, the arm was flaccid, with weak biceps and absent triceps and supinator reflexes. Routine investigations, including full blood count, biochemistry, coagulation, immunoglobulins, and autoantibody and virology screens were all normal. X-ray examination of the skull and cervical spine revealed no abnormalities. Magnetic resonance imaging similarly revealed no evidence of a desmoid affecting the brain, thorax, or brachial plexus. Her recent symptoms remain unexplained, although a recurrence of the desmoid remains a possibility, despite the normal scans. Her father, who has FAP and had undergone regular colonoscopic screening, was initially noted to have polyps in the distal colon at age 26 years. He had a colectomy and ileorectal anastomosis performed when he was 28 years old. He has CHRPE but has not developed desmoid tumors. Gastric fundal polyps have been noted in the father, but his duodenum and periampullary region are normal. He also suffers from insulin-dependent diabetes. The proband’s paternal grandmother had died at the age of 29 years from a rectal carcinoma associated with multiple colonic polyps. There is no record of this woman having extracolonic tumors or abnormal fundoscopic findings. FIGURE 1. Proband, age 2 years. Desmoid tumor of left forehead. this gene (termed APC) have confirmed that these two conditions a.re allelic and that identical APC gene germline mutations can result in FAP or Gardner syndrome. CASE REPORT The proband was born at term following an uncomplicated pregnancy and a vaginal delivery. She has reached normal developmental milestones and was a healthy child prior to the present illness. When she was 2 years and 1 month old, her parents reported a rapidly enlarging mass on1 the left side of her forehead. At the surgical clinic 10 weeks later, it measured 7 cm x 6 cm x 2.5 cm (Fig. 1).The inass was nontender and nonpulsatile, had a firm texture, and was fixed to the deeper tissue. Results of her general examination were otherwise normal. Fundoscopy confirmed the presence of congenital hypertrophy of the retinal pigment epithelium (CHRPE). No rectal polyps were noted on sigmoidoscopy. Jaw X-rays were not performed. The tumor was resected with excellent cosmetic results, and 18 months following surgery no recurrence has been noted. One year after the initial resection of the MATERIALS AND METHODS The cytogenetic study was performed by culturing the resected cranial tumor using standard tissue culture techniques, and G banding of chromosomes was performed before analysis. DNA was purified to standard protocols from blood leukocytes and fresh tumor. The DNA linkage study was performed using the flanking, polymorphic microsatellite markers DP1,6YN 5.64,7and intragenic polymorphisms at nucleotide 1458 (exon 11)8and 5037 (exon 15)*and 37A/B (3’ untranslated region). Initial APC mutation screening was by single stranded conformation polymorphism (SSCP) analysis of leukocyte DNA from the affected father of the proband. SSCP screening was confined to exon 15, fragments A to M, by polymerase chain reaction (PCR) amplification using primer sequences described by Groden et aL5 PCR products were heat denatured and run on 6% polyacrylamide gels with 5% or 10% glycerol at room temperature under nondenaturing condition^.""^'^ Gels were dried and autoradiography was performed for 6-24 hours. PCR fragments showing any evidence of conformational changes were then reamplified by PCR from leukocyte DNA, electrophoresed on ultrapure low-melting-point agarose, and lightly ethidium stained. Each PCR fragment was cut from the gel with a fresh scalpel and gene cleaned (Stratech Scien- 974 CANCER March 1,1996 / Volume 77 / Number 5 FIGURE 3. Partial karyotype of tumor demonstrating interstitial deletion of chromosome 5jq21q22). FIGURE 2. Desmoid tumor of the scalp. Sweeping bundles of slender, elongated cells in a rather mucoid matrix (H and E; x120). tific). Gel purification was then subjected to double stranded sequencing as previously described."," DESMOID TUMOR ALLELE DOSAGE ANALYSIS After identification of the causative germline mutation, a search was made for a suitable mutation-induced restriction site alteration. The mutation was found to generate an Msel site. However, the Msel site in the wild-type sequence being in close proximity to the mutation site confounded the use of this alteration. Hence, a primer was made that removed this wild-type sequence Msel site by base substitution in the primer sequence itself. The new forward primer (GTTCTAATCATGGAATCTAATCA) was paired with primer E-reverse.5 PCR amplification with primer pair ElMse/E-reverse produced a 165 bp fragment with no Msel site for the wild-type sequence, but the mutation-induced Msel site generated 138 bp and 27 bp fragments after digestion. PCR amplification was then performed using desmoid and leukocyte DNA from the proband, leukocyte DNA from her affected father, and control DNA from a healthy individual, each incorporating 0.25 p.Ci of 3'P-CTP. The radiolabelled PCR fragments were digested to completion with Msel overnight at 37 "C in the manufacture's buffer and electrophoresed on 6% nondenaturing acrylamide gels. This analysis was repeated three times to ensure reproducibility. Gels were dried and analyzed on a phosphor imager using ImageQuant software. The relative allele dosages of mutant and wild-type bands were compared using peak pixel counts. FIGURE 4. Allele loss at DP1 locus in the desrnoid tumor DNA demonstrated by a reduction in intensity of the maternal band (marked by arrow) in the tumor DNA compared with the leukocyte DNA from the proband. Lane a, leukocyte DNA from affected father; lane b, desmoid tumor DNA; lane c, leukocyte DNA from proband; lane d, leukocyte DNA from unaffected mother. longer fascicles, more uniform fibroblasts, and evenly spaced small round vessels was more compatible with the diagnosis of a desmoid (Fig. 2). Cytogenetic studies showed an interstitial deletion of chromosome 5, de1(5)(q21q22),in three of five cells examined (Fig. 3), the normal karyotype in two of five cells presumably being due to contaminating normal tissue or leukocytes. There was a reduction in intensity of the maternal marker demonstrated for DP1 (Fig. 4) compatible with loss of the wild-type allele in the tumor. The loss is incomplete, possibly owing to contamination with normal tissue. The intragenic markers were uninformative in this family. No allele loss was apparent with markers YN 5.64. RESULTS Microscopic examination of the tumor revealed some features of cranial fasciitis, including myxoid change and occasional extravasated erythrocytes, but the presence of Identification of the Germline APC Mutation In leukocyte DNA from the father of the proband, an SSCP variant was identified in segment E of exon 15, and the APC Gene Inactivation in a Cranial Desmoid Tumor/DeSilva et al. 975 DISCUSSION FIGURE 5. Msel-digested and undigested radiolabelled PCR products (EliMse lorward and E-reverse) from blood and desmoid tumor DNA from proband (patient 956). with leukocyte DNA from her father (patient 603) and from a control healthy individual electrophoresed on 6% acrylaniide. Lanes A and B. undigested and Msel -digested products, respectively. Allele dosage and naked-eye assessment show that blood DNA from the proband and her father both show a mutant sequence band (138 bp), which is of approximately 50% the intensity of the wild-type band (165 bp) owing to the relative size and proportion of cytosine bases of each of the fragments. However, this pattern is clearly reversed in the desrnoid tumor. with 70% underrepresentation of the wild-type sequence on allele dose analysis. relevant product was reaniplified, gene cleaned, and sequenced as described. A frame shift mutation due to an insertion of thyrnidine a t nucleotide 3372 (codon 1124), resulting in a downstream premature stop codon, was identified (data not shown). APC sequencing was then repeated for leukocyte DNA from the proband, which confinned that she was also a carrier of the mutation. Sequencing of desmoid tumor DNA strongly suggested that the mutant sequence was overrepresented compared with the wild type, and quantification of the relative contribution of the mutant and wild-type alleles was performed using allele dosage analysis. Allele Dosage Analysis Phosphor imager analysis of the dried gels show that the wild-type sequence, after digestion with Mse1, is more intense than the smaller mutant due to the relative size and proportion of the cytosine bases of each of the fragments (Fig. 5). However, this pattern is dramatically reversed in the desmoid tumor even o n naked-eye assessment. ImageQuant software dosimetry on a number of separate analyses of peak pixel count of each band indicated a mean ratio of 2.06 for wild-typemutant alleles. Ilowever, this ratio was only 0.62 in the desnioid tumor DNA, indicating that thc wild-type sequence was only 30% of that expected if the mutation was heterozygous. The 2-year-old patient described herein was shown to have inherited the germline mutation of the APC from her affected father, although she has not yet manifested signs of colonic polyps. I ler presentation with a desmoid tumor is unusual for two reasons: her age and the site of the lesion. 1)esmoid tumors are commonly intraabdominal or on the abdominal wall. McAdam and Goligher’:’ reviewing 89 cases of desmoid tumors associated with FAP, reported that 44 were localized to the abdominal wall and 26 were intraabdominal, and in 19 the desmoids were present in both. Jarvinen14 in his review of 19 of 168 FAP patients with desmoids in the Finnish polyposis register reported three patients with extraabdominal desmoids, including one with a desmoid on the buttock and two with thoracic lesions. No cases of a cranial desmoid associated with a family history of FAP have been reported. In Jarvinen’s’”report, the average age at diagnosis of the desnioid tumor was 28.5 years (range 8-41 years). In McAdam and Goligher’s’‘’series, the average age of the affected females was 28 years and that of males 38 years. Childhood onset of desmoids is rare in association with FAP. Jarvinen” has reported an 8-month-old infant with an intraabdominal desmoid, and Kratzer et al“ have reported a family with FAP in which a 1-year-old infant who died of a hepatoma was also noted to have a desmoid near the umbilicus at post mortem. Cytogenetically visible abnormalities of chromosome 5q21 have been demonstrated in two desmoid tumors of patients with Gardner syndrome.16 These were an interstitial deletion of 5q14-21 and a random telomeric fusion of 5q. Non-Gardner syndrome patients with desmoids were also noted to have abnormalities of chromosome 5q21. Another study” of 10 desnioid tumors analyzed after short term culture and in situ hybridization demonstrated trisomy 8 in 4 of 10 tumors, and the authors refer to the fact that this trisomy was identified using conventional karyotype analysis in only one case of the series, indicating the need for more detailed cytogenetic studies in these tumors. We have attempted to identitji the parental origin of the deleted chromosome. The loss of the maternal 5q21 region was demonstrable at DP1, estimated to bc 1% distal to the A N : gene, but this does not extend to the proximal marker 5.64. The intragenic polymorphisnis were uninformative in this family. 1,oss of both APC gene copies has previously been reported for desnioid tuniors affecting the abdominal wall and mesentery. In a study of eight desmoids and corresponding normal tissue from seven patients with FAP using PCR, SSCP, and direct sequencing, Miyaki et all” demonstrated that all eight tumors had APC gene mutations. Seven of eight were exon 15 mutations leading to the formation of a stop codon and one of eight had loss of the normal allele. In one of the tumors, 976 CANCER March 1,1996 / Volume 77 I Number 5 loss of the wild-type allele was demonstrated by linkage studies in addition to the sequencing evidence. In the remainder, the presence of a germline mutation was assumed if this was present in both the normal surrounding tissue and the tumor. Our studies were complicated by the presence of normal tissue in the resected tumor specimen, and this normal tissue included infiltrating lymphocytes and normal stroma. The contamination with normal tissue was noted in both pathological and molecular investigations. The dosage protocol was specially designed to demonstrate dosage difference between the wild-type (maternal) and mutant (paternal) alleles in the tumor. 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