R J M E Romanian Journal of Morphology & Embryology

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1 Rom J Morphol Embryol 2011, 52(3 Suppl): ORIGINAL PAPER R J M E Romanian Journal of Morphology & Embryology Conventional examination versus immunohistochemistry in the prediction of hormone profile of pituitary adenomas. An analysis on 142 cases B. BĂLINIŞTEANU 1), RALUCA AMALIA CEAUŞU 1), ANCA MARIA CÎMPEAN 1), IONELA BACIU 2), NICOLETA BĂCULESCU 2), M. COCULESCU 2), M. RAICA 1) 1) Department of Histology and Molecular Pathology, Victor Babeş University of Medicine and Pharmacy, Timisoara Angiogenesis Research Center, Timisoara 2) Department of Endocrinology, Carol Davila University of Medicine and Pharmacy, Bucharest Constantin I. Parhon Institute of Endocrinology, Bucharest Abstract The large majority of neoplasms located in the sella turcica are benign pituitary adenomas derived from cells of the adenohypophysis. Pituitary adenomas represent the third most common primary intracranial tumor in neurosurgical practice, outnumbered only by gliomas and meningiomas. Their biology of pituitary adenomas is complex and they can cause a variety of endocrine syndromes and disorders, based on hormone profile secreted by proliferating cells. The aim of this study was to evaluate the routine conventional methods and highly specific immunohistochemical methods in order to accurately predict the type of hormone secretion. Our study was investigated 142 cases admitted with pituitary adenomas and treated by open surgery. Sections from each case were stained with routine Hematoxylin and Eosin method for histopathologic evaluation. Immunohistochemistry was performed on additional slides in order to detect specific pituitary adenomas. Based on the immunohistochemical profile of pituitary adenomas we found following results: 37 growth hormone (GH)- secreting adenomas, 23 prolactin (PRL)-secreting adenomas, 15 mixed GH-cell/PRL-cell adenomas, three mammosomatotroph cell adenomas, two adrenocorticotrophic hormone (ACTH)-secreting adenomas, one thyrotrophic cell adenoma, 18 gonadotroph adenomas, 30 null cell adenomas and 13 plurihormonal adenomas. No correlation was found between conventional features and the immunohistochemical profile. The immunohistochemical profile of functional pituitary adenomas is mandatory for a correct diagnosis. We revealed that staining characteristics of the tumor cells, such as acidophilic, basophilic or chromophobe are nowadays outdated as main principle of classification, because they not identify specific adenoma types. Keywords: adenomas, immunohistochemistry, hormones, pituitary. Introduction The large majority of neoplasms located in the sella turcica are benign pituitary adenomas (75%) derived from cells of the adenohypophysis. Pituitary adenomas represent the third most common primary intracranial tumor in neurosurgical practice, outnumbered only by gliomas and meningiomas. They represent approximately 10 15% of all operated intracranial tumors and are encountered in 20 25% of autopsy-obtained pituitaries. Their prevalence increases with advancing age; both sexes are affected equally. Their biology is complex and they can cause a variety of endocrine syndromes and disorders [1]. Previously, pituitary adenomas were separated on the grounds of tinctorial characteristic of the cell cytoplasm into acidophilic, basophilic and chromophobic tumors. Based on their size, pituitary adenomas can be divided into microadenomas and macroadenomas. Modern pathologic classification is based on immunohistochemistry and ultrastructural methods. The development of immunohistochemistry permits the conclusive identification of the various cell types in the pituitary adenomas. The 2004 edition of the WHO histological typing of pituitary adenomas has illustrated a great progress in our understanding of these tumors. Correlation with clinical features and endocrine activity also became possible. The standard immunohistochemical battery includes the use of antibodies to GH, PRL, ACTH, TSH, FSH, LH, and the α-subunit of the glycoprotein hormones [2]. Even if the cellular hormonal immunoreactivity is common in pituitary tumors, this is not always correlated with serum levels of hormones or a clinical endocrine syndrome. These tumors are named clinically non-functioning adenomas (NFA). NFA have no clinical expression of produced hormones, either because they produce inactive molecule or synthesis but do not release hormones from cells in a sufficient amount raise the blood level and to determine an endocrine reaction. Another hypothesis includes a lysosomal catabolism of the hormone inside of cells [3]. Cytogenetic analysis of chromosomes and fluorescent in situ hybridization may provide important information regarding tumor development and progression [4].

2 1042 B. Bălinişteanu et al. Materials and Methods The study material included 142 pituitary adenomas. Resected tissue specimens were fixed in 10% buffered formalin for 48 hours and paraffin embedded. Five micrometers thick serial sections were performed from each paraffin block and sections were mounted on silanized slides. Sections from each case were stained with routine Hematoxylin and Eosin method for histopathologic evaluation. Paraffin sections of each tumor were immunostained using the primary antibodies against the following pituitary hormones: STH, PRL, ACTH, TSH, FSH, LH (DAKO, Carpinteria, California, USA). Visualization of the immune reactions was done by means of Streptavidin Biotin-peroxidase technique with use of 3,3 -diaminobenzidine as chromogen. All slides were immunostained automatically with the DAKO Autostainer. The presence of more than 10% of hormone immunopositive cells was considered secretory tumor. The immunostaining results for each patient were graded as being 0 (negative), 1+ (10 30% of cells), 2+ (30 50% of cells) or 3+ (over 50% of cells). The tumors with high co-expression GH/PRL immunoreactivity (>50% of cells) were considered mammosomatotrophic adenomas. Microscopic observation was performed by four independent observers using Nikon Eclipse 80i. The images were captured and processed with Lucia G software system (Nikon, Japan). Results Histologically, 105 tumors were acidophilic (Figure 1A), 14 basophilic (Figure 1B), seven chromophobes (Figure 1C), and 16 mixed. Figure 1 Types of pituitary adenomas according with cell features. Acidophilic pituitary adenoma (A HE staining, ob. 20), basophilic pituitary adenoma (B HE staining, ob. 20) and chromophobe pituitary adenoma (C HE staining, ob. 20). Morphological features of adenomas suggested following growth patterns: solid (85 cases) (Figure 2A), trabecular (22 cases) (Figure 2B), papillary (four cases) (Figure 2C), diffuse (one case) (Figure 2D), alveolar (one case) (Figure 2E) and mixed (29 cases). Figure 2 Pituitary adenoma classification based on histopathologic appearance: solid pattern (A HE staining, ob. 100); trabecular pattern (B HE staining, ob. 20); papillary pattern (C HE staining, ob. 20); diffuse pattern (D HE staining, ob. 10); alveolar pattern (E HE staining, ob. 10).

3 Conventional examination versus immunohistochemistry in the prediction of hormone profile of pituitary adenomas 1043 Based on the immunohistochemical profile of pituitary adenomas we found following results: 37 GHsecreting adenomas (26%), 23 PRL-secreting adenomas (16%), 15 mixed GH-cell/PRL-cell adenomas (10.5%), three mammosomatotroph cell adenomas (2.1%), two ACTH-secreting adenomas(1.4%), one TSH-secreting adenoma (0.8%), 18 gonadotroph (LH-FSH) adenomas (12%), 30 null cell adenomas (non-secreting) (21%) and 13 plurihormonal adenomas (9.1%), which represented by eight subtypes (Figure 3). The immunoreactivity patterns of pituitary adenomas presented following features: GH uniform intensity of cytoplasmic immunoreactivity and diffuse distribution (Figure 4A); PRL is typically paranuclear ( Golgi pattern ) (Figure 4B); TSH-diffuse cytoplasmic immunoreactivity (Figure 4C); ACTH variability of cytoplasmic immunoreactivity with diffuse distribution (Figure 4D); FSH/LH- cytoplasmic immunoreactivity with diffuse distribution or localized along cell membrane (Figure 4, E and F). In our study, we found three basophilic GHsecreting adenomas, four basophilic PRL-secreting adenomas, two ACTH-secreting adenomas and 14 acidophilic FSH/LH secreting adenomas. Moreover, seven of eight chromophobic adenomas had secretory activity. Figure 3 Pituitary adenoma distribution according with immunohistochemical expression of pituitary hormones. Figure 4 Pituitary hormones assessed by immunohistochemistry in pituitary adenomas: positive immunostaining for GH (A, ob. 40), PRL (B, ob. 40), TSH (C, ob. 40), ACTH (D, ob. 40), FSH (E, ob. 40) and LH (F, ob. 40).

4 1044 B. Bălinişteanu et al. Discussion Although several publications have discussed about pituitary adenomas, no large series analyzing morphologic and immunohistochemical features. Lack of clinicopathological correlations in the past classification of pituitary adenomas, which was based on the tinctorial properties of adenoma cells, limited the importance of histological diagnosis [5]. Acidophil adenomas were traditionally associated with acromegaly, basophilic adenomas with Cushing s syndrome, and chromophobe adenomas were thought to be nonfunctioning. Not all acidophilic tumors produce GH, nor are all GH-producing tumors acidophilic; some basophilic tumors do not cause Cushing s disease and more than half of chromophobic tumors are endocrinologically active, variously secreting GH, PRL, ACTH, TSH, LH/FSH. In our study, we found these differences, which are in contrast with conventional classification of pituitary adenomas. By using immunohistochemical methods, we will have new insight into the nature and pathogenesis of these tumors [6]. Immunohistochemistry can differentiate between different types of adenomas, but it does not correlate always with clinical features of patients. A large German study on 3489 pituitary adenomas collecting in 10 years of experience, revealed following immunohistochemical types of tumors: GH cell adenomas (15.5%), prolactin (PRL) cell adenomas (9.2%), mixed GH/PRL cell adenomas (5.2%), mammosomatotropic adenomas (1.1%), ACTH cell adenomas (15.1%), TSH cell adenomas (1.5%), FSH/LH cell adenomas (24.8%), null cell adenomas (25.1%) and plurihormonal adenomas (1.3%). The adenomas were classified according to the current WHO classification in the version of 2004 [7]. In comparison with our results, the differences can be observed to increased cases of ACTH adenomas and lower incidence of plurihormonal adenomas. With improved methods of immunostaining and the increased use of monoclonal antibodies, the incidence of plurihormonal adenomas is lower than expected from former studies with polyclonal antibodies [8]. According to the WHO recommendations, we did not consider tumors expressing both FSH and LH (gonadotropinomas) or somatoprolactinomas expressing both GH and prolactin as plurihormonal. Even with this limitation, plurihormonality was found to be a frequent finding in both hormonally active and clinically nonfunctioning pituitary adenomas. Pawlikowski M et al. were shown that over one-third (36.1%) of the investigated adenomas expressed more than one hormone. Plurihormonality, especially which is connected with co-expression of ACTH, seems to be more frequent in the recurrent tumors. Plurihormonal adenomas also possess higher Ki-67 indices, as compared to monohormonal tumors [9]. Plurihormonality is a common phenomenon in other endocrine tumors, such as pancreatic endocrine tumor and medullar carcinoma of the thyroid. Plurihormonal adenomas have been divided into monomorphous and plurimorphous tumors by ultrastructural studies [10]. In 2001, Ho DMT et al. studied 143 plurihormonal pituitary adenomas, which consisted of 26% of the surgically removed pituitary adenomas that we collected during a 12-year period. The staining results of each hormone subunit were recorded and the percentages of immunoreactive cells were graded as follows: (-), all cells negative; 1+, <5% cells positive; 2+, 5±20% cells positive; 3+, 20±50% cells positive; and 4+, >50% cells positive. Twenty-nine immunohistochemical subtypes of plurihormonal adenomas were recognized [11]. In our study, we found 13 plurihormonal adenomas (9.1%) and eight subtypes. This discrepancy may be due to different quantification methods of hormone immunoreactivity. Conclusions With the emergence of new methodology, it became all too clear that the tinctorial characteristics of the cell cytoplasm correlate poorly with reliable cell type recognition and secretory activity. Immunohistochemical staining of all the pituitary hormones is essential for diagnosis because the complexity of hormonal combinations in different subtypes and contribute to better characterization of secretory or non-secretory clinical behavior of the tumor. Moreover, the immunohistochemistry of pituitary adenomas can be used to identify the prognostic markers of these tumors, such as Ki67 LI, p53 expression or somatostatin receptors. Contribution Note All authors have an equal contribution to this work. References [1] Ezzat S, Asa SL, Couldwell WT, Barr CE, Dodge WE, Vance ML, McCutcheon IE, The prevalence of pituitary adenomas: a systematic review, Cancer, 2004, 101(3): [2] DeLellis RA, Lloyd RV, Heitz PU, Eng C, Pathology and genetics of tumours of endocrine organs, World Health Organization Classification of Tumours, IARC Press, Lyon, [3] Dumbrava M, Costinean S, Stancu C, Badiu C, Mind the gap in immunohistochemical techniques used in pathology of pituitary adenomas, Acta Endocrinol Buc, 2005, 1(4): [4] Kontogeorgos G, Predictive markers of pituitary adenoma behavior, Neuroendocrinology, 2006, 83(3 4): [5] Kontogeorgos G, Classification and pathology of pituitary tumors, Endocrine, 2005, 28(1): [6] Horvath E, Kovacs K, The adenohypophysis. In: Kovacs K, Asa SL (eds), Functional endocrine pathology, 2 nd edition, Blackwell Science, Malden, 1998, [7] Saeger W, Lüdecke DK, Buchfelder M, Fahlbusch R, Quabbe HJ, Petersenn S, Pathohistological classification of pituitary tumors: 10 years of experience with the German Pituitary Tumor Registry, Eur J Endocrinol, 2007, 156(2): [8] Saeger W, Wilczak W, Lüdecke DK, Buchfelder M, Fahlbusch R, Hormone markers in pituitary adenomas: changes within last decade resulting from improved method, Endocr Pathol, 2003, 14(1): [9] Pawlikowski M, Kunert-Radek J, Radek M, Plurihormonality of pituitary adenomas in light of immunohistochemical studies, Endokrynol Pol, 2010, 61(1):63 66.

5 Conventional examination versus immunohistochemistry in the prediction of hormone profile of pituitary adenomas 1045 [10] Kovacs K, Horvath E, Stefaneanu L, Bilbao J, Singer W, Muller PJ, Thapar K, Stone E, Pituitary adenoma producing growth hormone and adrenocorticotropin: a histological, immunocytochemical, electron microscopic, and in situ hybridization study. Case report, J Neurosurg, 1998, 88(6): [11] Ho DMT, Hsu CY, Ting LT, Chiang H, Plurihormonal pituitary adenomas: immunostaining of all pituitary hormones is mandatory for correct classification, Histopathology, 2001, 39(3): Corresponding author Anca Maria Cîmpean, Associate Professor, MD, PhD, Angiogenesis Research Center, Victor Babeş University of Medicine and Pharmacy, 2 Eftimie Murgu Square, Timişoara, Romania; Phone , ancacimpean1972@yahoo.com Received: August 13 th, 2011 Accepted: October 5 th, 2011