Somatic but Not Germline Mutation of the APC Gene in a Case of ...

Anatomic Pathology / CRIBRIFORM-MORULAR VARIANT OF PAPILLARY THYROID CARCINOMA

Somatic but Not Germline Mutation of the APC Gene in a Case of Cribriform-Morular Variant of Papillary Thyroid Carcinoma José Cameselle-Teijeiro, MD, PhD,1 Clara Ruiz-Ponte, PhD,2 Lourdes Loidi, PhD,2 José Suarez-Peñaranda, MD, PhD,1 Javier Baltar, MD, PhD,3 and Manuel Sobrinho-Simoes, MD, PhD4 Key Words: Cribriform-morular variant; Familial adenomatous polyposis; APC gene; Polymerase chain reaction; Cytology; Papillary carcinoma; Thyroid cancer

Abstract We report a case of cribriform-morular variant (C-MV) of papillary thyroid carcinoma (PTC) in a 27year-old woman. In addition to conventional cytologic features of typical PTC, the fine-needle aspirate showed numerous epithelial cells with abundant, eosinophilic, very elongated cytoplasm. Microscopically, the tumor was encapsulated and highly cellular and exhibited a mixture of cribriform, follicular, papillary, trabecular, solid, and spindle cell patterns of growth, with morular foci showing peculiar nuclear clearing (biotin-rich nuclei). The cells were cuboidal or tall, with frequent nuclear pseudostratification and abundant eosinophilic cytoplasm. The nuclei were usually hyperchromatic, with grooving, pallor, and pseudoinclusions. Angioinvasion and foci of capsular invasion were observed. Immunohistochemically, the neoplastic cells showed reactivity for thyroglobulin, epithelial membrane antigen, low- and high-molecular-weight cytokeratins, vimentin, neuron-specific enolase, CD15, estrogen and progesterone receptors, and bcl-2 protein. Molecular genetic analysis of the APC gene revealed a mutation in exon 15 at codon 1309 in tumoral tissue but not in peripheral lymphocytes. These findings support a relationship between the morphologic pattern of the C-MV of PTC and the APC gene and the existence of this variant as a sporadic counterpart of familial adenomatous polyposis–associated thyroid carcinoma.

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Thyroid carcinoma has been reported in association with a variety of colorectal abnormalities, including familial adenomatous polyposis (FAP). FAP is an inherited condition caused by germline mutations in the adenomatous polyposis coli (APC) gene that is located in the 5q21 region.1 In 1994, Harach et al2 characterized the thyroid carcinoma developing in patients with FAP as a distinct tumor type in view of its histologic differences from papillary and follicular carcinomas. They also suggested that these unusual histologic features in a thyroid tumor, especially if multicentric, should alert the clinician to the possibility of FAP with its implications for family screening. Other studies3-6 confirmed the results of Harach et al.2 On the other hand, ret/PTC1 activation, a finding unique to papillary thyroid tumors, was found in 2 thyroid carcinomas of 3 patients with FAP reported by Cetta et al,7 strongly suggesting that these tumors are papillary carcinomas. More recently, Cameselle-Teijeiro and Chan8 reported 4 cases of thyroid carcinoma with histologic features similar to those of the cases described by Harach et al2 arising in patients without a family history or evidence of FAP and reviewed 5 similar cases reported in the literature. Because of the distinctive histologic features, they proposed naming this tumor the cribriform-morular variant (C-MV) of papillary thyroid carcinoma (PTC), and suggested that it could represent the sporadic counterpart of the FAP-associated thyroid carcinoma.8 In the present study, we report the clinical, cytologic, histologic, immunohistochemical, and molecular findings of a new case of C-MV of PTC. To the best of our knowledge, this is the first case of C-MV of PTC with somatic but not germline mutation in the APC gene. © American Society of Clinical Pathologists

Anatomic Pathology / CASE REPORT

Case Report A 27-year-old Spanish white woman was admitted to the hospital with a mass of 14 months’ duration in the right side of her neck. She had a history of diabetes mellitus secondary to relapsing acute pancreatitis induced by chronic alcoholism. There was no history of radiation exposure. No family history of FAP, goiter, or other endocrine disorders was noted. During the physical examination, a nodule was palpated in the right lobe of the thyroid without enlarged cervical lymph nodes. Ultrasonic examination revealed a solid tumor measuring 21 mm in diameter. Routine laboratory test results were within normal limits. Total thyroidectomy was performed after fine-needle aspiration biopsy (FNAB) yielded a diagnosis of PTC. After surgery, ophthalmoscopic analysis did not disclose congenital hypertrophy of the retinal pigment epithelium (CHRPE) or retinopathy. The patient was well and had no evidence of FAP 14 months after the diagnosis.

Material and Methods Cytologic, Histologic, and Immunohistochemical Examinations FNAB was performed, and smears were stained by the Diff-Quik (Merck, Darmstadt, Germany) method. The thyroidectomy specimen was fixed in buffered formaldehyde and embedded routinely in paraffin. The 5-µm-thick sections were stained with H&E, alcian blue (pH 2.5), and Mayer mucicarmine stain. Immunohistochemical studies were performed on paraffin sections using a panel of antibodies ❚Table 1❚ ; the reaction was detected by the indirect, non–avidin-biotin detection system known as EnVision (EnVision System, DAKO, Glostrup, Denmark). To examine the affinity of the biotin for streptavidin, a section was incubated without any primary antibody and the reaction evaluated by the streptavidin-biotin complex technique (StreptABComplex/HRP, DAKO). Nonimmune mouse and rabbit serum samples were substituted for the primary antibodies as negative controls. Appropriate positive controls were run concurrently for all antibodies tested. Molecular Genetic Analysis of Somatic and Germline APC Mutations Genomic DNA was isolated from paraffin-embedded tumoral tissues and peripheral lymphocytes by standard procedures. Seven sets of primers were used to amplify, by polymerase chain reaction (PCR) analysis, the regions of the APC gene where germline mutations in FAP with associated thyroid carcinomas were described ❚Table 2❚. © American Society of Clinical Pathologists

Amplification of fragments 1, 2, and 3 was performed by PCR: an initial denaturation at 94°C for 5 minutes followed by 3 cycles at 94°C for 30 seconds, 60°C for 40 seconds, and 72°C for 30 seconds; 3 cycles at 94°C for 30 seconds, 58°C for 40 seconds, and 72°C for 30 seconds; and 25 cycles at 94°C for 30 seconds, 55°C for 40 seconds, and 72°C for 30 seconds and a final extension at 72°C for 7 minutes. PCR conditions for fragments 4 to 7 were as follows: an initial denaturation at 94°C for 5 minutes, followed by 35 cycles at 94°C for 60 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, and, finally, an extension at 72°C for 7 minutes. Fragment Analysis To confirm amplification, PCR products were heat denatured before being loaded onto a standard 6% polyacrylamide denaturing gel in a semiautomated fluorescent system (A.L.F. express, Amersham-Pharmacia-Biotech, APB, Uppsala, Sweden). Electrophoresis was carried out at 55°C for 150 minutes (1,500 V, 60 mA, 30 W). Single-Strand Conformation Polymorphism Analysis PCR products were denatured and electrophoresed on homogeneous 20% polyacrylamide gels (Phast gel, APB) in nondenaturing conditions, using an electrophoretic system (Phast system, APB). The running temperature was 15°C (270 V, 7.2 mA, 2.0 W for 300 V per hour). DNA Sequencing PCR products of the normal sample and blood and tumor samples of the patient were sequenced directly. The aberrant bands obtained on single-strand conformation polymorphism (SSCP) analysis were cut directly from the dried gels. They were eluted and reamplified by the aforementioned conditions. These PCR products also were sequenced. Sequences were done by the dideoxy-chain-termination method of Sanger (Femtomol Sequencing Kit, Promega, Madison, WI). Samples were run on a 6% polyacrylamide gel in a semiautomatic sequencer (A.L.F. express, APB).

Results Cytologic, Gross, and Histologic Findings Cytologic smears showed a highly cellular sample composed of branching papillary fragments admixed with cells arranged in flat monolayers with a clean background. The individual cells were tall and columnar and held abundant, dense cytoplasm that was frequently spindly and very elongated ❚Image 1❚. The nuclei showed a fine chromatin pattern and an irregular contour, with frequent creases, Am J Clin Pathol 2001;115:486-493

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❚Table 1❚ Antibodies Used for Immunohistochemical Analysis and the Results Antigen

Antibody (Clone and Source)

Thyroglobulin Calcitonin Chromogranin A Synaptophysin Neuron-specific enolase Epithelial membrane antigen Keratins 8 and 18 Keratins 1, 2, 10, 11, 14, 15, 16, and 19 Keratins 1, 5, 10, and 14 Vimentin Carcinoembryonic antigen CA-19.9 CA-125 CD15 Estrogen receptor Progesterone receptor Ki-67 p53 protein bcl-2 protein

Tg6 (DAKO, Glostrup, Denmark) Polyclonal (BioGenex, San Ramon, CA) LK2H10 (BioGenex) SY38 (BioGenex) BBS/NC/VI-H14 (DAKO) E29 (DAKO) CAM5.2 (Becton Dickinson, Mountainview, CA) AE1/AE3 (Concepta Biosystems, Barcelona, Spain) 34beta E12 (Enzo, Farmingdale, NY) V9 (Concepta Biosystems) Polyclonal (DAKO) C241:5:1:4 (Novocastra, Newcastle upon Tyne, England) Ov185:1 (Novocastra) Leu-M1 (Becton Dickinson) 6F11 (Novocastra) 1A6 (Novocastra) MIB-1 (Immunotech, Marseille, France) DO-7 (DAKO) 124 (DAKO)

Dilution

Antigen Retrieval

Nonmorular Neoplastic Cells

Morular Cells

1:20 1:2,000 1:5 1:100 1:1,000 1:50 1:5

None None C/M C/M C/M C/M P, C/M

+ – – – + + +

+ – – – + – +

1:50

P, C/M

+

+

1:10 1:500 1:2,000 1:200

P, C/M C/M None C/M

+ + – –*

– – – +

1:200 1:100 1:10 1:5 1:20 1:10 1:5

C/M C/PC C/PC O, C/M P, C/M C/M O, C/M

– +† + + 3% – +

– NE – – NE – +

C, citrate; CA, carbohydrate antigen; M, microwave; NE, not evaluable; O, overnight (4°C); P, protease; PC, pressure cooker; +, positive; –, negative. * A few positive cells. † Focal positivity.

❚Table 2❚ Oligonucleotide Sequences of Primers Used for Polymerase Chain Reaction* Fragment 1 2 3 4 5 6 7

Oligonucleotides 5’-GACCCAAGTGGACTTTTCAGG-3’ 5’-ACAATAAACTGGAGTACACAAGG-3’ 5’-AGTCGTAATTTTGTTTCTAAACTC-3’ 5’-TTTGAAACATGCACTACGAT-3’ 5’-AGATGACCCATATTCTGTTTC-3’ 5’-CTTTAAAAGTAATATAAAACTC-3’ 5’-GAGAACAACTGTCTACAAAC-3’ 5’-GCTGCAGCACTTCCCATAGC-3’ 5’-CTACAGTGTTACCCAGCT-3’ 5’-GTTGCTGGATGGTAGTTG-3’ 5’-CAGATGAGCAGTTGAACTC-3’ 5’-TGTGGTTGGAACTTGAGGTG-3’ 5’-ACAGGAAGCAGATTCTGC-3’ 5’-GATTTGGTTCTAGGGTGC-3’

Codons

Codon of APC Gene Mutation

Size (bp)

Exon

420

3

74-141

1406

456

9

312-438

3136

275

14

582-653

5936

203

15

658-725

6986

118

15

828-867

8487

181

15

1032-1092

10617

123

15

1293-1334

13097

bp, base pairs. * Codons of the APC gene with the germline mutations already described in patients with thyroid carcinomas associated with familial adenomatous polyposis (FAP) are indicated. Two somatic mutations (codons 886 and 1061) were reported recently by Iwama et al5 in thyroid carcinomas from patients with FAP and germline mutations at codons 175 and 1110. Codons 778, 976, 993, and 1219, previously described by Soravia et al,6 were not analyzed in the present study. Only somatic APC mutation at codon 1309 was found in the present case.

grooves, and cytoplasmic invaginations (pseudoinclusions) (Image 1B). Nucleoli were not prominent. Colloid was scant, and there were isolated macrophages. Nuclear pleomorphism, mitotic figures, psammoma bodies, lymphocytes, and necrotic debris were not observed. The thyroidectomy specimen weighed 25 g. Transection revealed a well-circumscribed, white to tan mass measuring 21 mm in its largest dimension ❚Image 2❚. 488

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Microscopic examination revealed the tumor was cellular, encapsulated, and partially divided into lobules by a sclerotic septum. The neoplasm was composed of a combination of cribriform, follicular, papillary, solid, trabecular, spindle cell, and morular patterns of growth that merged intricately with one another ❚Image 3❚. The cribriform structures were formed by anastomosing bars and arches of a thickness of 1 or 2 cells with little or no fibrovascular © American Society of Clinical Pathologists

Anatomic Pathology / CASE REPORT

A

B

❚Image 1❚ A and B, Groups of tall columnar cells with abundant cytoplasm that is spindly and very elongated. B, Note an intranuclear cytoplasmic pseudoinclusion (rapid Romanowsky, original magnification ×400).

stroma; their lumina were usually round and empty, but some contained histiocytes. The cribriform areas merged with follicular structures usually devoid of colloid, as well as with nonarborizing papillary and pseudopapillary processes. The cells were mostly tall or cuboidal, with abundant oxyphilic cytoplasm and frequent nuclear pseudostratification (Image 3C). The tumor cell nuclei were usually hyperchromatic, grooved, and pale, with small nucleoli and cytoplasmic inclusions (Image 3D). There were 1 to 2 mitotic figures per 10 high-power fields. In the solid areas, the tumor cells changed from oval to more plump and spindly with nodular whorls (morules) ❚Image 4❚ . These morules resembled

❚Image 2❚ Right thyroid lobe transection showing a fleshy, encapsulated mass in the lower third.

© American Society of Clinical Pathologists

squamous metaplasia, but they lacked keratinization and intercellular bridges and showed a peculiar nuclear clearing reminiscent of viral inclusion bodies (Image 3D). No psammoma bodies, necrosis, or mucosubstances were found in the tumor. An additional oxyphilic adenoma measuring 9 mm was found in the same lobe of the gland. Immunohistologic Findings The results of the immunohistochemical study are summarized in Table 1. The tumor cells showed reactivity for thyroglobulin, epithelial membrane antigen, low- and highmolecular-weight cytokeratins, vimentin, neuron-specific enolase, CD15, estrogen and progesterone receptors, and bcl2 protein. In contrast, morules were negative for epithelial membrane antigen, high-molecular-weight cytokeratins, vimentin, and estrogen and progesterone receptors and positive for carbohydrate antigen (CA)-19.9 (Image 4). In the control section for biotin, false-positive nuclear staining was observed in the peculiar nuclear clearing. The proliferative index in the neoplasia, evaluated by MIB-1, was 3%. Molecular Genetic Analysis SSCP results showed an aberrantly migrating band in the amplified fragment 7, corresponding to the partial exon 15 APC gene tissue sample ❚Image 5A❚. Sequence data from this aberrant band revealed the presence of a known mutation in exon 15 of APC at codon 1309 (3927-3931 del AAAGA), resulting in a frameshift leading to a stop codon (TAG) at nucleotides 3939 to 3941 (codon 1314) ❚Image 5B❚. This frameshift gives rise to a truncated APC protein of 1309 amino acids in length (wild-type protein = 2,843 amino acids). The mutation was confirmed by sequence determination of Am J Clin Pathol 2001;115:486-493

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A

B

C

D

❚Image 3❚ The tumor showed a combination of cribriform, follicular, solid, morular, trabecular (A), and papillary (B) patterns of growth merging with one another (H&E, original magnification ×100). C, The cells were mostly tall with abundant cytoplasm and frequent pseudostratification (H&E, original magnification ×200). D, Morules with peculiar nuclear clearing reminiscent of viral inclusion bodies (top) also were found (H&E, original magnification ×400).

both strands. This mutation was not detected in the DNA blood sample of the patient.

Discussion FAP,6 Cowden syndrome,9 and a form of multinodular goiter and PTC10 are 3 conditions associated with hereditary predisposition to nonmedullary thyroid tumors, in which the APC, PTEN, and MNG1 genes are involved, respectively. Most recently, a new gene related to familial thyroid tumors with cell oxyphilia has been identified and called TCO.11 The C-MV of PTC is the term coined8 for a sporadic type of PTC morphologically indistinguishable from most of 490

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the thyroid carcinomas that arise in the setting of FAP.2 Thyroid tumors of both series2,8 had a female predilection and occurred in a younger age group (usually