Cell Membrane and Cytoplasmic Staining for MIB-1
in Hyalinizing Trabecular Adenoma of the
Thyroid Gland
Mitsuyoshi Hirokaw...
Sections were stained with monoclonal antibody MIB-1
(1:50 dilution; Immunotech, Marseilles, France). Lymph
node germinal ...
Non-neoplastic Thyroid Follicles
The non-neoplastic cells lining the follicles surround-
ing the tumors were generally neg...
Acknowledgment
The authors thank Dr. T. Manabe for access to cases at the
Department of Pathology, Kawasaki Medical School...
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Cell membrane and_cytoplasmic_staining_for_mib-1_in_hyalinizing_trabecular_adenoma_of_the_thyroid_gland

  1. 1. Cell Membrane and Cytoplasmic Staining for MIB-1 in Hyalinizing Trabecular Adenoma of the Thyroid Gland Mitsuyoshi Hirokawa, M.D., Ph.D., and J. Aidan Carney, M.D., Ph.D. The monoclonal MIB-1 antibody reacts with the nuclei of cells in the late G1, S, G2, and M phases of the cell cycle. Previ- ously, we found two cases of hyalinizing trabecular adenoma that showed cell membrane and cytoplasmic immunopositivity for the antibody. The purpose of this investigation was to con- firm this exceptional reactive pattern of MIB-1 in hyalinizing trabecular adenoma. For the study, we collected 13 additional hyalinizing trabecular adenomas and stained a total of 15 tu- mors using MIB-1 antibody. Ten cases of papillary thyroid carcinoma were studied similarly. All hyalinizing trabecular adenomas showed strong positivity for the antibody in 90% or more of the tumor cells, localized especially to the cell mem- brane and also to the cytoplasm. There was no cell membrane or cytoplasmic MIB-1 positivity among the 10 papillary carci- nomas. Luminal border of normal extratumoral thyroid follicles rarely showed faint immunopositivity. Our findings indicate that strong cell membrane and cytoplasmic immunoreactivity for MIB-1 is a characteristic of the hyalinizing trabecular ad- enoma. Staining for MIB-1 will be useful in differentiating hyalinizing trabecular adenoma from papillary carcinoma, which shares a number of cytologic and histologic findings with hyalinizing trabecular adenoma. Key Words: Thyroid—Hyalinizing trabecular adenoma— MIB-1—Ki67—Immunohistochemistry—Papillary carcinoma. Am J Surg Pathol 24(4): 575–578, 2000. Hyalinizing trabecular adenoma of the thyroid gland is a subtype of follicular adenoma described by Carney et al.1 in 1987. This benign tumor shares several histologic features with papillary carcinoma, including intranuclear cytoplasmic invaginations, nuclear grooves, and psam- moma bodylike formations. It coexists occasionally with papillary carcinoma.1,8,10 These and other findings have led to the suggestion that hyalinizing trabecular adenoma and papillary carcinoma are closely related tumors.2 In 1995, we reported two cases of hyalinizing trabec- ular adenoma in which the cytoplasm of the tumor cells was reactive for MIB-1.3 Because MIB-1 reacts with cell nuclei in the late G1, S, G2, and M phases of the cell cycle, the cytoplasmic immunoreactivity of MIB-1 in hyalinizing trabecular adenoma was unexpected and un- explained. We have now examined 13 additional cases of hyalinizing trabecular adenoma for MIB-1 immunoreac- tivity and found the same intense cytoplasmic positivity for the antigen in all cases. Herein, we confirm the ex- ceptional reactive pattern of MIB-1 in hyalinizing tra- becular adenoma and point out its diagnostic usefulness in differentiating the tumor from papillary carcinoma. MATERIALS AND METHODS We collected 15 cases of hyalinizing trabecular ade- noma from the surgical pathology files of Kawasaki Medical School, the University of Tokushima School of Medicine, and the consultation files of the authors. He- matoxylin– and eosin-stained slides of the tumors were reviewed, and the diagnosis of hyalinizing trabecular ad- enoma was confirmed. The patients included 13 women and two men whose age ranged from 25 to 72 years (median, 46.2 years). The tumors ranged in diameter from 1.0 to 4.0 cm. Juxtaposed hyalinizing trabecular adenoma and papillary carcinoma were present in one patient. Routinely processed, formalin-fixed, paraffin-embedded tumor blocks were sectioned at 3 to 5 mm. After depa- raffinization and rehydration, the tissue sections were placed in 10 mol per liter citrate buffer (pH 6.0) and heated for three periods of 5 minutes each in a micro- wave oven at 500 W. The immunohistochemical proce- dure was based on the avidin–biotin–peroxidase method. From the Department of Pathology (M.H.), University of Tokushima School of Medicine, Japan; and the Department of Laboratory Medicine and Pathology (J.A.C.), Mayo Clinic and Mayo Foundation, Rochester, MN, U.S.A. Address correspondence and reprint requests to Mitsuyoshi Hirokawa, MD, PhD, Department of Pathology, University of Tokushima School of Medicine, 3-18-15 Kuramoto-cho, Tokushima, 770-8503, Japan. The American Journal of Surgical Pathology 24(4): 575–578, 2000 © 2000 Lippincott Williams & Wilkins, Inc., Philadelphia 575
  2. 2. Sections were stained with monoclonal antibody MIB-1 (1:50 dilution; Immunotech, Marseilles, France). Lymph node germinal center centrocytic nuclei were used as positive controls. Negative control sections were stained in parallel, the specific antibody having been replaced with phosphate-buffered saline. The nuclear MIB-1- positive rate was calculated by counting the number of stained and unstained nuclei in randomly selected fields, using a light microscope equipped with a 100× objective lens and a 10× ocular lens. The result was expressed as positive (labeled) nuclei per 1,000 nuclei counted. Ten cases of conventional papillary thyroid carcinoma from the surgical pathology files of Kawasaki Medical School were studied similarly. These patients included eight women and two men whose age ranged from 32 to 64 years (median, 48.8 years). RESULTS Hyalinizing Trabecular Adenoma The limiting membrane of almost all of the tumor cells was uniformly and strongly positive for MIB-1 (Fig. 1). The reactivity was particularly intense in cell membrane facing intratrabecular spaces or lumina, and sometimes was located in discrete, round, oval, and elongated zones. The cytoplasm was also positive but to a lesser degree, with its peripheral portion showing slightly stronger im- munoreactivity than the central (perinuclear) zone. The occasional intranuclear cytoplasmic invaginations were also weakly positive for MIB-1. Bipolar staining concen- trated at the luminal and basal aspects of the cytoplasm was present in one case. Nuclear positivity was observed in a few tumor cells in two cases; the positive rate was not estimated because nuclear staining was overwhelmed by the cytoplasmic positivity. Negative controls showed no reactivity. Papillary Carcinoma None of the papillary carcinomas showed cell mem- brane or cytoplasmic positivity for MIB-1. Occasional nuclei were stained (positive rate range, 0.78–3.14%; Fig. 2). FIG. 1. Hyalinizing trabecular adenoma. Tumor cells ex- hibited strong cell membrane and less strong cytoplasmic staining for MIB-1. FIG. 2. Papillary carcinoma and hyalinizing trabecular ad- enoma. Papillary carcinoma (top) showed nuclear immu- noreactivity for MIB-1, compared with cell membrane and cytoplasmic staining in hyalinizing trabecular adenoma (bottom). M. HIROKAWA AND J. A. CARNEY576 Am J Surg Pathol, Vol. 24, No. 4, 2000
  3. 3. Non-neoplastic Thyroid Follicles The non-neoplastic cells lining the follicles surround- ing the tumors were generally negative for MIB-1. An occasional luminal border revealed weak positivity (Fig. 3). Rare nuclei of follicular epithelial cells also revealed positivity (positive rate range, 0–0.58%). DISCUSSION The monoclonal antibody MIB-1 (Ki67) reacts with antigens associated with human nuclear cell prolifera- tion. These antigens are expressed during the G1, S, G2, and M phases of the cell cycle and are absent in the G0 phase. An epitope of Ki67 is destroyed by formalin fixa- tion and routine processing so that it can be studied only in frozen sections. In contrast, the MIB-1 epitope is fixa- tion resistant, permitting its use with formalin-fixed tis- sues in conjunction with an antigen retrieval procedure. These antibodies have been used in numerous studies designed to predict the biologic behavior of tu- mors.6,7,9,11 During these investigations, the antibodies have reacted invariably with nuclei. High positive rates for nuclear staining have been associated generally with aggressive tumor behavior. In 1995, we reported two cases of hyalinizing trabec- ular adenoma that had intense cytoplasmic immunoreac- tivity for MIB-1.3 In the current study, undertaken to investigate further this phenomenon in a larger number of cases, we report the MIB-1 findings in 13 additional cases of the tumor. The results revealed the same pattern of cell membrane and cytoplasmic positivity among the new cases as was observed in our original two cases,3 thus confirming our earlier results. A few intranuclear cytoplasmic invaginations were also positive for MIB-1. Kaleem and Davila4 studied Ki67 immunoreactivity in a case of hyalinizing trabecular adenoma and found an incidence of nuclear positivity of 0.6%. The second case in their report does not appear to be a hyalinizing tra- becular adenoma because the hyalinization depicted in the case illustration is intertrabecular and stromal, and not intratrabecular and cytoplasmic. Rare nuclear posi- tivity was observed in only two of our 15 cases of hya- linizing trabecular adenoma. We could not determine the positive rate precisely because cytoplasmic positivity overwhelmed the nuclear staining. To the best of our knowledge, the MIB-1 cell mem- brane and cytoplasmic staining pattern we describe in hyalinizing trabecular adenoma has not been reported in other thyroid (or nonthyroid) tumors. Nor has there been reference to the patchy positive staining of the luminal cell membrane in rare normal thyroid follicles that we observed. Katoh et al.5 studied nuclear MIB- 1 reactivity in a large series of neoplastic and non- neoplastic thyroid conditions. Their material included adenomas, normo/macrofollicular (n ‫ס‬ 14), tubular (n ‫ס‬ 18), solid/trabecular (n ‫ס‬ 6), and oxyphilic (n ‫ס‬ 5), and carcinomas, papillary (n ‫ס‬ 60) and follicular (n ‫ס‬ 14). They made no mention of finding cell mem- brane or cytoplasmic positivity in any of the tumors. It is improbable that the cytoplasm of the hyalinizing trabec- ular adenoma cells and that of normal thyroid follicles contains an epitope identical to the nuclear proliferation antigen. More likely, both cell types have a cytoplasmic antigen that cross-reacts with the anti-MIB-1 antibody. The MIB-1 staining results we obtained with hyalin- izing trabecular adenoma contrasted markedly with those of papillary carcinoma, in which cell membrane and cy- toplasmic staining was not present. Approximately 1% of papillary carcinoma nuclei were immunopositive. Our findings thus provide another morphologic difference be- tween hyalinizing trabecular adenoma and papillary car- cinoma, casting further doubt on the validity of the sug- gestion of Fonseca et al.2 that the hyalinizing trabecular adenoma is an encapsulated variant of papillary carci- noma. In practice, staining for MIB-1 should prove to be useful diagnostically in distinguishing between the two tumors. ᮀ FIG. 3. Normal thyroid tissue. Luminal border of follicular lining cells is weakly positive for MIB-1 (arrows). THYROID TRABECULAR ADENOMA 577 Am J Surg Pathol, Vol. 24, No. 4, 2000
  4. 4. Acknowledgment The authors thank Dr. T. Manabe for access to cases at the Department of Pathology, Kawasaki Medical School, Japan. REFERENCES 1. Carney JA, Ryan J, Goellner JR. Hyalinizing trabecular adenoma of the thyroid gland. Am J Surg Pathol 1987;11:583–91. 2. Fonseca E, Nesland JM, Sobrinho–Simo˜es M. Expression of strati- fied epithelial-type cytokeratins in hyalinizing trabecular adeno- mas supports their relationship with papillary carcinomas of the thyroid. Histopathology 1997;31:330–5. 3. Hirokawa M, Shimizu M, Manabe T, Kuroda M, Mizoguchi Y. Hyalinizing trabecular adenoma of the thyroid: its unusual cyto- plasmic immunopositivity for MIB 1. Pathol Int 1995;45:399–401. 4. Kaleem Z, Davila RM. Hyalinizing trabecular adenoma of the thyroid. A report of two cases with cytologic, histologic and im- munohistochemical findings. Acta Cytol 1997;41:883–8. 5. Kerns B-JM, Jordan PA, Faerman LL, Berchuck A, Bast RC, Layfield LJ. Determination of proliferation index with MIB-1 in advanced ovarian cancer using quantitative image analysis. Am J Clin Pathol 1994;101:192–7. 6. Katoh R, Bray CE, Suzuki K, et al. Growth activity in hyperplastic and neoplastic human thyroid determined by an immunohisto- chemical staining procedure using monoclonal antibody MIB-1. Hum Pathol 1995;26:139–46. 7. Lynch DAF, Clarke AMT, Jackson P, Axon ATR, Dixon MF, Quirke P. Comparison of labeling by bromodeoxyuridine, MIB-1, and proliferating cell nuclear antigen in gastric mucosal biopsy specimens. J Clin Pathol 1994;47:122–5. 8. Molberg K, Albores–Saavedra J. Hyalinizing trabecular carcinoma of the thyroid gland. Hum Pathol 1994;25:192–7. 9. Ozaki O, Ito K, Mimura T, Sugino K. Anaplastic transformation of papillary thyroid carcinoma in recurrent disease in regional lymph nodes: a histologic and immunohistochemical study. J Surg Oncol 1999;70:45–8. 10. Rosai J, Carcangiu ML, DeLellis RA. Tumors of the thyroid gland. In: Atlas of tumor pathology. 3rd series, fascicle 5. Washington, DC: Armed Forces Institute of Pathology, 1992:31–8. 11. Sugitani I, Yanagisawa A, Shimizu A, Kato M, Fujimoto Y. Clin- icopathologic and immunohistochemical studies of papillary thy- roid microcarcinoma presenting with cervical lymphadenopathy. World J Surg 1998;22:731–7. M. HIROKAWA AND J. A. CARNEY578 Am J Surg Pathol, Vol. 24, No. 4, 2000

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