Original article
Pituitary adenomas and craniopharyngiomas are CDX2 negative neoplasms

Introduction

The evolutionary conserved caudal-related homeo-box transcription factor CDX2 (previously called CDX3 in humans) is important in axial development and differentiation of the intestine. It modulates proliferation, apoptosis and cell-adhesion [9]. While absence of CDX2 gene in knock-out mice is lethal, heterozygotic mice develop neoplastic changes in the colon suggesting that CDX2 plays a role as a tumour suppressor gene [5]. In vitro studies suggest that Cdx2 gene expression is regulated through NF-kappaB, PI3K/PTEN and OCT1 signalling pathways [15,16]. In humans, CDX2 is constantly expressed in normal colorectal mucosa and, to a lesser extent, in several other tissues such as pancreas [22]. Loss of CDX2 expression is associated with increasing tumour grade, tumour stage and lymph node metastasis in colorectal neoplasms [1]. However, there is a growing list of neoplasms that may also show an expression of CDX2 in neoplastic cells. These include ovarian [21], lung [21,23], endometrial [36], breast [23], thyroid [37] and prostate [11] cancer. Furthermore CDX2 has been implicated in lymphoma progression [10] and aberrant expression has been demonstrated in acute myeloid leukaemia [27] and acute lymphoblastic leukaemia [24,34]. These studies suggest a tissue-dependent role of CDX2 in carcinogenesis and indicate that possible involvement of CDX2 needs to be determined for each tumour type individually.
Tumours of the anterior pituitary lobe account for approximately 7.5 percent of all intracranial tumours with a reported annual incidence of 8.2-14.7 per 100 000 population [4]. The prevalence increases with age and shows a female predominance which has been attributed to the expression of sex steroid receptors by some authors [33]. While a significant correlation exists between proliferative activity and tumour invasiveness [14,19] a correlation with tumour recurrence has not been established. The current WHO classification lists pituitary adenomas with an “elevated” mitotic index and a MIB-1 labelling index greater than 3% as “atypical adenomas” [18], while pituitary carcinomas are exceptionally rare neoplasms.
Craniopharyngiomas are epithelial tumours that are assumed to arise from a neoplastic transformation of ectodermal-derived Rathke’s pouch cell remnants [25]. The tumours have bimodal peak incidences. While adamantinous cranioharyngiomas are seen in children and adults, the papillary subtype is almost restricted to adults [3]. Although slowly-growing benign neoplasms, they tend to infiltrate adjacent bone and brain structures and surgical resection might be a challenge [28]. The rate of recurrence is dependant on the radicality of tumour resection on the one hand, but on the other hand the p53 protein expression in the tumour plays a significant role [32].
Since CDX2 is expressed in normal neuroendocrine cells of the intestine and gastric fundus [2] as well as in many carcinoid tumours [6,22], and since genome-wide mRNA analysis revealed elevated expression le-vels for CDX2 in normal pituitary tissue that were above leukaemic cell lines [30], we hypothesized that CDX2 might play a role in the tumourigenesis of pituitary adenomas. Additionally, we examined adamantinous and papillary craniopharyngiomas since CDX2 expression has been recently reported in squamous epithelia [13,35,36], but to date craniopharyngiomas have not been examined for a possible CDX2 expression.

Material and methods

We examined paraffin-embedded samples from normal pituitary gland (n = 28), pituitary adenomas (n = 75) and craniopharyngiomas (n = 23) by immunohistochemistry.
Normal pituitary tissue (11 male, 17 female; age 13-86 yrs, mean 55.6 yrs) was taken from routine autopsy brains (n = 21) and from neurosurgical spe-cimen (n = 7) which contained normal pituitary tissue coincidentally. In the latter, surgery had been performed for Rathke’s cleft cysts or hypophysitis. From the autopsy samples two probes (1.0 mm in diameter, one centrally and one laterally) were taken from the anterior lobe (pars distalis) and aligned on a tissue micro array using a conventional tissue microarrayer (Beecher Instruments, Sun Prairie, Wisconsin, USA). For surgical cases the whole slides were analyzed.
Of the pituitary adenomas (39 male, 38 female; age 19-79 yrs, mean 51.3 yrs), seven were classified as atypical pituitary adenoma, 7 showed the aspect of obvious invasive growth in imaging studies according to grade 3 or 4 of parasellar growth [17], 11 were re-current tumours (up to 10 years after first operation). 34 were hormonally silent (null-cell-adenomas), 20 expressed growth hormone, 7 were corticotrophs, 9 were prolactinomas, 3 were mixed tumours, 1 FSHoma and 1 TSHoma.
The craniopharyngiomas were in 17 cases adamantinous (6 female, 11 male, age 4-74 yrs, mean 36.7) and in 6 papillary (3 female, 3 male, age 33-49, mean 41.8 yrs). Two representative probes (1.0 mm in diameter) were taken from each tumour and aligned on a tissue micro array.
Samples were immunostained using a monoclonal mouse IgG1 antibody directed against the full length human CDX2 protein (Zytomed Systems, Berlin, Germany; clone CDX2-88, dilution 1:25) using an automated immunohistochemistry staining system (BenchMark®, Ventana Medical Systems, Tucson, Arizona, USA). The protocol (extended cell conditioning pretreatment for 30 min) is based on an indirect biotin-avidin system and uses a universal biotinylated immunoglobulin secondary antibody and diaminobenzidine substrate as chromogen. Sections were finally counterstained with haematoxylin. In negative controls, the primary antibody was omitted. Normal human colon tissue served as positive control (Fig. 1A). This study was carried out according to the guidelines of the University’s ethics commission.

Results

CDX2 expression in normal adenohypophysis
All autopsy cases and surgical specimen examined lacked expression of CDX2 in the nuclei of epithelium, connective tissue stroma or blood vessels (Fig. 1B). Furthermore, CDX2 expression was also absent in neighbouring brain tissue, including ependymal cells and cyst epithelium (wall of Rathke’s cleft cysts).

CDX2 expression in pituitary adenomas and craniopharyngiomas
No nuclear CDX2 immunostaining was found in any of the pituitary adenomas, neither in classical adenomas (Fig. 1C) nor in atypical, invasive or recurrent adenomas.
Likewise, all adamantinous (Fig. 1D) and papillary craniopharyngiomas were immunonegative for CDX2.

Discussion

CDX2 is a nuclear transcription factor and commercially available antibodies are widely used to characterize colorectal adenocarcinomas [37]. How-ever, CDX2 is not specific to intestinal epithelium. Large studies have shown that CDX2 is also expressed in various other epithelial neoplasms, including tumours of ovaries, prostrate, breast, lung and uterus [2,22,37], but these studies did not include pituitary adenomas. Moskaluk et al. reported an absence of CDX2 in normal pituitary tissue, but did not give detailed particulars on the number of samples that he examined [22]. A study by Erickson et al. reporting CDX2 expression in neuroendocrine tumours also included 12 pituitary adenomas but did not specify the hormonal activity of these tumours [6].
Therefore, we included in our series samples from normal pituitary gland and pituitary adenomas putting emphasis on different hormonal expression as well as hormonally silent tumours (including recurrent and atypical adenomas). In all cases investigated normal and neoplastic adenohypophyseal tissue lacked expression of CDX2, indicating that CDX2 is not involved in differentiation of the ante-rior pituitary or in tumourigenesis of pituitary adenomas. Likewise, neuroendocrine tumours of parathyroid, paragangliomas and pheochromocytomas have been previously reported to be immunonegative for CDX2 [6].
Metastases to the pituitary are not uncommon [7,12,20,26,31] and need to be distinguished from primary pituitary tumours. The incidence of intracranial metastases of colorectal tumours varies in the literature from 2-16% of all carcinomas [29,31]. Strickland-Marmol and colleagues reported for CDX2 a specifi-city of 100%, and 83% sensitivity to determine gastrointestinal adenocarcinomas metastatic to the brain. Thus, the now confirmed negative immunoreactivity of pituitary adenomas for CDX2 might be useful for diagnostic purposes, especially in cases of metastatic neurodenocrine tumours, which also express synaptophysin, but in contrast to pituitary adenomas are known to express CDX2 [6].
An aberrant CDX2 expression has also been reported in epithelial lesions with squamous differentiation [13,35,36]. Interestingly, CDX2 expression is epigenetically silenced in squamous oesophageal cancer [8]. To our best knowledge, CDX2 expression in craniopharyngiomas has not been previously examined. The current work shows that the squamous tumour cells in craniopharyngiomas are constantly negative for CDX2.
In summary, we demonstrate that CDX2 is absent in normal pituitary gland as well as in pituitary adenomas and craniopharyngeomas. Thus, CDX2 may be helpful to distinguish metastatic carcinomas affecting the sellar region from primary pituitary tumours.

References


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