Matilde Lombardero

MGMT immunoexpression predicts responsiveness of pituitary tumors to temozolomide therapy

Temozolomide, an alkylating imidazol tetrazine derivative, inhibits DNA replication and is used successfully in the management of patients with gliomas and other malignancies [5, 12]. Recent studies showed the drug to be eVective in patients with pituitary carcinoma and aggressive pituitary adenoma [1, 2, 7, 10, 14]. We reported the case of a 46-year-old man with an aggressive, prolactin (PRL) secreting pituitary tumor [4, 13]. Temozolomide treatment caused marked clinical improvement, reduction in blood PRL levels, and tumor shrinkage [13]. Morphologically, the treated tumor showed necrosis, hemorrhage, Wbrosis and neuronal transformation. Mitotic activity and Ki-67 labeling were signiWcantly decreased [4]. Based on these dramatic clinical, laboratory, imaging and morphologic changes, temozolomide was given to a 41-year-old man with an aggressive silent subtype 2 corticotroph pituitary adenoma. Neither clinical improvement nor tumor shrinkage was observed. No morphologic diVerences were seen between the preand posttreatment tumors. The cytotoxic eVect of the temozolomide depends upon methylation of guanine at the O-6 position in DNA. O-6 methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme, which removes the alkyl group adducts from the O-6 position and induces resistance to temozolomide [6, 9, 11]. Previous immunohistochemical studies showed that gliomas with low MGMT levels respond, whereas those with high levels resist temozolomide eVect [6, 9, 11]. This suggests that MGMT immunostaining may serve as an indicator of treatment responsiveness. Based on the results of previous studies, we investigated MGMT immunopositivity in the above noted cases and in an additional three pituitary adenomas. The immunohistochemical method was described in previous publication [3, 8]. Similar to the results obtained in gliomas, our study showed lack of MGMT staining in the responsive tumor (Fig. 1) and high expression in the resistant tumor (Fig. 2) as well as in the three other adenomas studied. We suggest that MGMT expression should be immunohistochemically assessed before embarking upon temozolomide treatment of aggressive pituitary tumors. If MGMT expression is K. Kovacs Department of Pathology, St. Michael’s Hospital, University of Toronto, Toronto, ON, Canada

Erythropoietin: a hormone with multiple functions.

Erythropoietin (EPO), the main hemopoietic hormone synthesized by the kidney as well as by the liver in fetal life, is implicated in mammalian erythropoiesis. Production and secretion of EPO and the expression of its receptor (EPO-R) are regulated by tissue oxygenation. EPO and EPO-R, expressed in several tissues, exert pleiotropic activities and have different effects on nonhemopoietic cells. EPO is a cytokine with antiapoptotic activity and plays a potential neuroprotective and cardioprotective role against ischemia. EPO is also involved in angiogenesis, neurogenesis, and the immune response. EPO can prevent metabolic alterations, neuronal and vascular degeneration, and inflammatory cell activation. Consequently, EPO may be of therapeutic use for a variety of disorders. Many tumors express EPO and/or EPO-R, but the action of EPO on tumor cells remains controversial. It has been suggested that EPO promotes the proliferation and survival of cancer cells expressing EPO-R. On the other hand, other reports have concluded that EPO-R plays no role in tumor progression. This review provides a detailed insight into the nonhemopoietic role of EPO and its mechanism(s) of action which may lead to a better understanding of its potential therapeutic value in diverse clinical settings.

A descriptive and comparative lectin histochemical study of the vomeronasal system in pigs and sheep.

The accessory olfactory bulb (AOB) is the primary target of the sensory epithelium of the vomeronasal organ (VNO), and thus constitutes a fundamental component of the accessory olfactory system, which is involved in responses to behaviour‐related olfactory stimuli. In this study we investigated the characteristics of the AOB, VNO, vomeronasal nerves (VNNs) and caudal nasal nerve (CdNN) in pigs and sheep, species in which olfaction plays a key behavioural role both in the neonatal period and in adulthood. The patterns of staining of the AOB by the Bandeiraea simplicifolia and Lycopersicon esculentum lectins were the same in the 2 species, whereas the Ulex europeus and Dolichos biflorus lectins gave different patterns. In both species, lectin staining of the AOB was consistent with that of the VNNs, while the CdNN did not label any of the structures studied. The entire sensory epithelium of the pig was labelled by Ulex europeus and Lycopersicum esculentum lectins, and all 4 lectins used labelled the mucomicrovillar surface of the sensory epithelium in sheep.

Clinicopathologic correlates of giant pituitary adenomas.

Giant adenomas comprise a clinical/therapeutic subset of pituitary adenomas that pose a surgical challenge. The study population consisted of 28 patients who had giant pituitary adenomas, which are defined as tumors with a diameter greater than 5cm. Clinically, five tumors (18%) were endocrinologically functional and 23 (82%) were not. During surgery, one tumor was radically excised, four were subtotally excised, 12 were partially excised, and 11 were biopsied. All of the tumors showed typical histological features of pituitary adenoma. Of the 23 clinically non-functional adenomas, 18 were gonadotrophic tumors, four were null cell adenomas and one was a silent corticotroph adenoma. The MIB-1 labeling indices ranged from 0.1% to 2.0%. The mean topoisomerase labeling index was 0.75%. Microvessel density ranged from 0.42% to 5.55%, and there was moderately intense immunostaining for vascular endothelial growth factor. The present study found giant adenomas to be invasive but slow growing, histologically benign and often gonadotrophic in subtype.

Multiple endocrine neoplasia type 1–associated thyrotropin-producing pituitary carcinoma: report of a probable de novo example

Pituitary carcinomas are exceedingly rare. At present, the sole diagnostic criterion is metastatic spread, either craniospinal or systemic. There is no agreement on a histologic, immunohistochemical, and/or ultrastructural definition. We report a clinically and morphologically well-documented example of pituitary thyrotropin cell carcinoma in a man with multiple endocrine neoplasia type 1 syndrome. The tumor produced thyrotropin, alpha-subunit, and prolactin and, through electron microscopy, was found to consist solely of Thyrotroph cells. Over a protracted course, craniospinal and systemic metastases were noted. The primary and metastatic deposits of this aggressive tumor were studied. To our knowledge, this tumor is the first reported case of thyrotropin cell carcinoma occurring in association with the multiple endocrine neoplasia type 1 syndrome. The literature regarding thyrotropin carcinomas is reviewed. Based on the study of several biopsies during disease progression, we believe that the carcinoma originated de novo without an intermediary adenoma phase.

An easy method for the removal of Epon resin from semi-thin sections. Application of the avidin-biotin technique

SummaryA simple procedure is described for removing Epon resin from semi-thin 1 μm sections, which permits excellent postembedding immunohistochemical staining (avidin-biotin complex technique). The procedure was developed for the detection of growth hormone and prolactin in bovine adenohypophysis fixed with 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 m sodium cacodylate buffer pH 7.4–7.6. The results indicate that the removal of the epoxy embedding medium prior to the application of the immunohistochemical reagents was essential for the successful localization of the antigenic determinants of the two hormones. The immunocytochemical reactivity was obtained only after treating the sections with a solution of potassium hydroxide in a mixture of absolute methyl alcohol and propylene oxide (Maxwells solution). An enhanced immunoreactivity was obtained when this treatment was followed by an additional treatment with either 4% hydrogen peroxide or a saturated aqueous solution of sodium metaperiodate. Because of the easy preparation of the Epon removal solution and the good structural preservation without damage to the antigenic determinants, Maxwells solution is suggested as a good etching agent which can be used in immunohistochemical studies on semi-thin sections with excellent results.

Distribution of the arterial supply to the vomeronasal organ in the cat

The main goal of this work was to investigate the general distribution of arterial blood around and inside the vomeronasal organ (VNO) of the cat.

Effect of estrogen on the blood supply of pituitary autografts in rats.

Estrogens are known to cause pituitary enlargement and lactotroph proliferation. They also modulate pituitary angiogenesis and induce tumor formation. Pituitary grafts, due to the loss of hypothalamic dopamine, also show lactotroph hyperplasia. We investigated the role of estrogen on rat pituitary autograft vascularization by light and transmission electron microscopy, and assessed prolactin (PRL) blood levels, microvessel density (MVD) and cell proliferation using the BrdU labeling index. All adenohypophysial cell types were identified by immunohistochemistry (streptavidin‐biotin‐peroxidase complex method). The proangiogenic factors, vascular endothelial growth factor (VEGF), its receptor Flk‐1, and hypoxia inducible factor‐1α (HIF‐1α) were similarly demonstrated. The prevalence of lactotrophs, as well as more intense staining for VEGF, Flk‐1 and HIF‐1α, was noted in those grafts exposed to estrogen, mainly in the area surrounding the central necrotic core. Immunostaining showed Flk‐1 expression increased in endothelial cells of the estrogen‐exposed grafts as compared with those unexposed. In contrast to the grafts not exposed to estrogen, in the estrogen‐exposed grafts, only fenestrated endothelium could be demonstrated, suggesting that estrogen induces fenestration of newly formed capillaries. There was an increase in blood PRL levels in the estrogen‐treated groups as compared with controls. Both MVD and BrdU labeling indices were higher in grafts exposed to estrogen, especially after 4 weeks. Our results suggest that estrogen administration not only enhances the expression of proangiogenic factors in the pituitary grafts but also induces their expression at earlier stages, leading to rapid neoformation of purely fenestrated capillaries.

Vascularization of rat pituitary autografts

Pituitary autotransplantation eliminates direct vascular contact between the hypothalamus and the adenohypophysis, and enables us to study the role of the hypothalamus in regulating adenohypophysial endocrine activity. The aim of this study was to investigate vascularization of the pituitary autografts. Three‐month‐old male Wistar rats were hypophysectomized, and their adenohypophyses were autotransplanted under the renal capsule. The animals were killed 3 weeks after autotransplantation. The grafts were removed and studied by using histology, immunohistochemistry and transmission electron microscopy. In the central portion of the grafts, organizing necrosis was apparent. The peripheral portion of the graft contained all adenohypophysial cell types, with a predominance of lactotrophs. Vascular endothelial growth factor and hypoxia‐inducible factor were expressed in the graft mainly in the perinecrotic areas. Several capillaries inside the grafts were lined by continuous unfenestrated epithelium, while others were lined by fenestrated endothelium, suggesting that neovascularization is the result of two processes: ingrowths of capillaries from the renal capsule to the graft, and neoformation of capillaries from pre‐existing adenohypophysial vessels. In conclusion, hypoxia seems to be an important factor in the vascularization of pituitary autografts. Mediated via hypoxia‐inducible factor, hypoxia stimulates vascular endothelial growth factor secretion, which plays a crucial role in angiogenesis.

Hormonal and morphological study of the pituitaries in reeler mice

Reelin is a neuronal glycoprotein that plays a crucial role in brain layer formation during prenatal development. The reeler mutant mouse lacks Reelin, leading to abnormalities in the neuronal layering of cerebral cortex and cerebellum, producing ataxia, tremor and abnormal locomotion. Reeler mice are reported to have growth retardation and most of them are sterile or unable to bring up their newborns. Since the brain is one of the main regulator of pituitary hormone secretion and no information was reported regarding pituitary function and structure in these mutant mice, we studied pituitary endocrine activity and morphology in reeler mice. Mice were classified in three groups as reeler homozygote (RHM), reeler heterozygote (RHT) or control (CO). Pituitary hormone blood levels were assessed by enzyme immunoassay (EIA) and immunoradiometric assay (IRMA). Animals and their pituitaries were weighted and pituitaries were studied by histology, immunohistochemistry and electron microscopy. Results showed statistically significant differences in body weight and in adrenocorticotropic hormone (ACTH) and luteinizing hormone (LH) blood levels between the three groups. In contrast, growth hormone (GH) blood levels showed a high individual variation and no decrease in reeler groups compared with CO. Morphological studies revealed no differences in pituitary cell types except that somatotrophs appeared to be slightly smaller in RHM and RHT. Although it seems that pituitary hypofunction is not responsible for growth retardation, more studies are needed to obtain a deeper insight into the endocrine status of these mutant mice to elucidate the cause of their low body weight and reproductive behaviour.