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Pathology – Research and Practice 204 (2008) 191–195

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Thyroid adenomatous nodule with bizarre nuclei: A case report and

mutation analysis of the p53 gene

Katsuaki Satoa,�, Yuzo Shimodeb, Mitsuyoshi Hirokawac,Yoshimichi Uedaa, Shogo Katsudaa

aDepartment of Pathophysiological and Experimental Pathology, Kanazawa Medical University, 1-1 Daigaku,

Uchinada, Kahoku, Ishikawa 920-0293, JapanbDepartment of Otorhinolaryngology, Kanazawa Medical University, Ishikawa, JapancDepartment of Diagnostic Pathology and Cytology, Kuma Hospital, Hyougo, Japan

Received 4 September 2007; accepted 10 October 2007

Abstract

We present a rare case of adenomatous nodule with bizarre nuclei. The patient was incidentally found to have anodule in the left lobe of the thyroid gland by ultrasonographic examination. Papillary thyroid carcinoma wassuspected by fine needle aspiration cytology, and hemithyroidectomy was performed. The demarcated 1.5-cm nodulehad a multinodular appearance with various features, including micro- and macrofollicular components, cysticdegeneration, a hyalinized area, and a papillary structure. Hyperchromatic bizarre nuclei with cytoplasmic inclusionswere restrictively observed in the microfollicular area. The bizarre nuclei demonstrated diffuse p53 proteinimmmunoreactivity, but no mutation in exons 5–9 of the p53 gene was detected. The bizarre nuclei were reactive foranti-5-methyl-20-deoxycytidine antibody, indicating the enclosure of presumably inactive methylated DNA. Theintranuclear cytoplasmic inclusions (ICIs) were proven to contain vimentin and b-catenin by immunohistochemistry.In this case, a degenerative process is involved in the formation of bizarre nuclei because of the compression bysurrounding micronodules, unidentifiable mitotic figures, and a quite low proliferative activity. This case suggests thatbizarre nuclei and ICIs, which might be identical to those of papillary carcinomas, can be seen in benign thyroidlesions, and overdiagnosis should be avoided regardless of immunohistochemical overexpression of p53.r 2007 Elsevier GmbH. All rights reserved.

Keywords: Adenomatous nodule; Bizarre nuclei; Intranuclear cytoplasmic inclusion; p53 gene; Thyroid

Introduction

Bizarre nuclei are large and hyperchromatic withmarked atypia, and are characteristically present in‘‘atypical adenomas’’ or ‘‘follicular adenomas withbizarre nuclei’’ in the thyroid gland. Tzen et al. [21]previously described that bizarre cells of atypical

e front matter r 2007 Elsevier GmbH. All rights reserved.

p.2007.10.003

ng author. Tel.: +8176 2188119; fax: +8176 2862484.

ss: sato-k@kanazawa-med.ac.jp (K. Sato).

adenomas showed a mutation of the p53 gene, andcould be precursors of anaplastic carcinomas. Anadenomatous nodule is a solitary benign hyperplasticlesion similar to a nodule of the multinodular goiter.Frable [8] mentioned a case of nodular colloid goiterwith regressive cell atypia in figures. Magro et al. [12]reported a case of nodular goiter with metaplasticbizarre spindle cells, however, examination for p53 wasnot performed. Here, we report a case of thyroidadenomatous nodule with bizarre nuclei presenting

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p53 protein overexpression by immunohistochemistry,but with no mutation of the p53 gene.

Clinical history

A 59-year-old man was incidentally found to have anodule in the left lobe of the thyroid gland byultrasonographic examination. He attended a hospital,and obtained medications for hypertension, hyper-lipidemia, and fatty liver, but had never receivedtreatment for the thyroid. His family history wasunremarkable. Fine needle aspiration cytology sus-pected papillary thyroid carcinoma, and hemithyroi-dectomy was performed. A follicular nodule withbizarre nuclei, probably a benign lesion, was suggestedby intraoperative frozen section diagnosis. There was noevidence of local recurrence or metastasis 15 monthsafter surgery.

Materials and methods

Surgically resected specimens were fixed in 10%neutral-buffered formalin and embedded in paraffin.Paraffin sections (4 mm) were used for hematoxylin andeosin staining and immunohistochemical examination.Primary antibodies used in this study were cytokeratin19 (RCK108, Dako, Glostrup, Denmark; 1:50), galec-tin-3 (B2C10, BD Biosciences Pharmingen, San Jose,CA, USA; 1:10), HBME-1 (HBME-1, Dako; 1:50),vimentin (V9, Dako; 1:100), b-catenin (17C2, Novocas-tra, Newcastle Upon Tyne, UK; 1:200), p53 (Bp53-12,Immuno-Biological Laboratories, Gunma, Japan;1:5,000), Ki-67 (MIB-1, Dako; 1:50), and anti-5-methyl-20-deoxycytidine antibody (kindly provided byYasuhiro Kagawa and Kazuaki Watanabe, BiologicalScience Labs, Toray Research Center, Inc., Kanagawa,Japan; 1:200). Antigen retrieval was performed usingappropriate methods for each antibody according to themanufacturer’s instructions. Detection was achievedwith an automated immunostainer (Ventana MedicalSystem, Tucson, AZ, USA).

DNA extracted from formalin-fixed and paraffin-embedded tissues was used for mutation analysis of thep53 gene by direct sequencing using specific primers forexon 5 (forward, 50-TGTTCACTTGTGCCCTGACT-30;reverse, 50-CAGCCCTGTCGTCTCTCCAG-30), exon 6(forward, 50-CCCAGGCCTCTGATTCCTCA-30; reverse,50-CAACCACCCTTAACCCCTCC-30), exon 7 (forward,50-ACTGGCCTCATCTTGGGCCT-30; reverse, 50-TG-TGCAGGGTGGCAAGTGGC-30), exon 8 (forward, 50-TCCTTACTGCCTCTTGCTTC-30; reverse, 50-TCTCC-TCCACCGCTTCTTGTC-30), and exon 9 (forward, 50-CACTAAGCGTGGTAAGCAAG-30; reverse, 50-CGG-CATTTTGAGTGTTAGAC-30).

Results

Macroscopically, surgically resected specimens re-vealed a demarcated 1.5-cm nodule in the lower portionof the left thyroid lobe. The nodule was gray-white incolor, and had a small cystic area. Microscopically, thenodule was surrounded by irregular fibrous tissues andhad a multinodular appearance with various features,including micro- and macrofollicular components, cysticdegeneration, a hyalinized region, and a papillarystructure (Fig. 1(A)). No compressive appearanceagainst the surrounding normal thyroid tissues ornecrosis was found. The areas containing bizarre cellsappeared to be compressed by the micronodule, andthe follicular structures were indistinct in these areas(Figs. 1(B) and (C)). Bizarre nuclei were commonlyobserved in the microfollicular area. Bizarre nucleifrequently contained intranuclear cytoplasmic inclusions(ICIs) (Fig. 1(D)). Mitotic figures were not identifiable.There was no capsular or vascular invasion. No othernodules or lymphocyte aggregates suggestive of chronicthyroiditis were present in the background of thethyroid.

Immunohistochemically, the bizarre cells diffuselyexpressed p53 protein, but the follicular cells werenegative (Fig. 2(A)). The bizarre nuclei were moreintensely and diffusely reactive for anti-5-methyl-20-deoxycytidine antibody than nuclei of follicular cells(Fig. 2(B)). The ICIs of the bizarre nuclei were re-active for vimentin (Fig. 2(C)). Expression of b-cate-nin was maintained on the bizarre cell membrane,and interspersed in the ICIs (Fig. 2(D)). Galectin-3,HBME-1, and cytokeratin 19 were not present inthe lesion. Ki-67 (MIB-1) labeling index was lessthan 0.1%.

No mutations were detected in exons 5-9 of thep53 gene.

Discussion

Bizarre nuclei are observed in neoplastic lesions of thethyroid, ‘‘atypical adenomas’’ or ‘‘follicular adenomaswith bizarre nuclei’’; however, hyperplastic lesionsincluding adenomatous goiter or nodules demonstratingbizarre cells are uncommon [8,12]. External radiationand 131I treatment may cause bizarre nuclear atypia infollicular cells, leading to misdiagnosis of anaplasticcarcinoma or papillary carcinoma in fine needle aspira-tion cytology [9]. The patient presented no past medicalhistory for the thyroid and had never received radiationtherapy. The solitary well-circumscribed nodule was afollicular lesion consisting of various histologicalcomponents: micro- and macrofollicular elements, focalcystic degeneration, hyalinized area, and papillarystructure, which were all different from the monotonous

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Fig. 1. Pathological findings of the nodule (HE staining): (A) A panoramic view of the nodule shows micro- and macrofollicular

components, cystic degeneration, a hyalinized area, and a papillary structure. Scale bar: 5mm. (B) The areas containing bizarre cells

are compressed by the surrounding micronodule, 40� . (C) Follicular structures are indistinct in the area presenting bizarre nuclei,

100� . (D) Bizarre nuclei are large, irregular, hyperchromatic, and frequently present intranuclear cytoplasmic inclusions, 400� .

K. Sato et al. / Pathology – Research and Practice 204 (2008) 191–195 193

follicular architecture of follicular adenomas or carci-nomas. The follicular cells in the papillary structurepresented blunt atypia, and showed no nuclear featureof papillary carcinoma such as ICIs, fine chromatin, andapparent nucleoli. No immunoreactivity for galectin-3,HBME-1, and cytokeratin 19, which were proposedthyroid markers of malignancy [2,3,14,16], was detected.Mitotic figures were not observed, and the proliferationrate estimated from Ki-67 labeling index was minimal.Accordingly, we diagnosed this lesion as an adenoma-tous nodule, quite benign lesion.

In the thyroid, p53 gene mutations are frequentlyidentified in anaplastic or poorly differentiated carcino-mas, but are rare or absent in well-differentiatedcarcinomas [5,20]. Tzen et al. [21] reported a case ofatypical follicular adenoma demonstrating p53 genemutation in bizarre cells, and suggested that the lesion

possibly represented an early stage of anaplasticcarcinoma. Bizarre cells with high-grade nuclear atypiaare also observed in benign lesions of other organs:atypical (bizarre) leiomyoma [4,6,11,13,18], ancientschwannoma [1], ancient melanocytic nevi [10], andbizarre cell pleomorphic adenoma [19], indicatingdegenerative changes. A case of atypical leiomyoma inthe larynx presented p53 overexpression [13], however,no p53-positive cells were found in bizarre cellpleomorphic adenoma [19] or bizarre leiomyoma ofthe scrotum [11]. There are occasionally significantcontroversial results between the immunohistochemicaloverexpression of p53 protein and p53 gene mutation.Overexpression of p53 protein may occur without p53

gene mutation. Not all mutations of the p53 gene causeoverexpression of p53 protein because truncated pro-teins that originated from non-sense mutations cannot

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Fig. 2. Immunohistochemical findings of the nodule: (A) P53 protein is diffusely overexpressed in bizarre nuclei but not in follicular

cells, 200� . (B) Bizarre nuclei are more intensely and diffusely reactive for anti-5-methyl-20-deoxycytidine antibody than nuclei of

follicular cells, 200� . (C) Intranuclear cytoplasmic inclusions express vimentin, 400� . (D) Membrane expression of b-catenin is

retained in the bizarre cells. Intranuclear cytoplasmic inclusions are occasionally reactive for b-catenin, 400� .

K. Sato et al. / Pathology – Research and Practice 204 (2008) 191–195194

be detected by immunohistochemistry. It is likely thatthe bizarre nuclei in our case might not be derived frommalignant transformation but from a degenerativeprocess, since the bizarre nuclei showed p53 proteinoverexpression by immunohistochemistry, but no muta-tion of exons 5–9 of the p53 gene was detected.

Bizarre nuclei are presumed to contain abundantheterochromatin, which relates to inactivated DNAbecause of hyperchromatic appearance. MethylatedDNA is known to be inactivated, and should beassociated with heterochromatin. Ernst et al. [7]reported that mean global levels of DNA methylationin symplastic leiomyomas were significantly higher thanthose in conventional leiomyomas, and striking nuclearatypia might be associated with excessive DNA methy-lation. Since bizarre nuclei were diffusely reactive foranti-5-methyl-20-deoxycytidine antibody in our case, we

suggest that DNA methylation is also related tohyperchromatic nuclear atypia in thyroid cases.

ICIs are occasionally observed in bizarre nuclei ofatypical leiomyomas [4,6,18]. Slone and O’Connor [18]suggested that the inclusions comprised aggregates ofintermediate filaments similar to hyaline globules in thecytoplasm of rhabdoid cells, but this was not confirmedby immunohistochemical or ultrastructural studies. Inthe thyroid, ICIs of papillary carcinomas were reportedto include intermediate filaments by electronmicro-graphs [15] and b-catenin immunostaining [17]. ICIs ofbizarre cells in the present case were also expressingimmunoreactivity for vimentin and b-catenin. Intra-nuclear invagination of the cytoplasm containing inter-mediate filaments might be a common process of bizarrenuclei in benign lesions and nuclei of thyroid papillarycarcinoma cells.

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Acknowledgment

The authors would like to thank Takayuki Nakata(Department of Pathology, Kanazawa Medical Uni-versity Hospital, Ishikawa, Japan) for immunohisto-chemistry.

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