Adamantinomatous and papillary craniopharyngiomas are characterized by distinct epigenomic as well as mutational and transcriptomic profiles

Craniopharyngiomas (CPs) are defined as histologically benign epithelial tumors of the sellar region [27, 34]. Reported world-wide incidence rates of 1.86 (1.60–2.14) new cases per million per year for all ages and 2.14 (1.53–2.92) for children under 15 years demonstrate that they represent rare lesions [15, 37, 39]. Although the growth pattern of CP is often locally aggressive and treatment is challenging, histological signs of malignancy are missing-thus defining them as grade I tumors according to the World Health Organization (WHO) classification [31]. There are two different subtypes of CP, papillary (papCP) and adamantinomatous (adaCP). Although clear histomorphological differences between both variants exist, a correct diagnosis is sometimes difficult to obtain, especially in small and/or fragmented specimens. Furthermore, the existence of mixed forms and the cell of origin of these tumors are areas of ongoing scientific debate [40, 41].

The papCP variant occurs almost exclusively in adults, at an age of 40–55 years [12]. They are composed of compact, monomorphic sheets of well-differentiated squamous epithelium and typically lack regressive changes like cholesterol clefts, calcifications, wet keratin and inflammation. Ciliated epithelium and PAS+ goblet cells are sometimes encountered, and histological morphology resembles that of Rathke’s cleft cysts with squamous metaplasia.

The adaCP variant is the most common non-neuroepithelial intracerebral neoplasm in children, accounting for 5–11 % of intracranial tumors in this age group [19, 42]. A second peak occurs in adulthood between 50–74 years [7]. Histopathological hallmarks of adaCP are the formation of differentiated epithelium disposed in cords, lobules, nodular whorls and irregular trabeculae bordered by palisaded columnar epithelium. Cystic cavities containing cell debris and fibrosis are lined by flattened epithelium. Pale nodules containing anucleate “ghost cells”/”wet keratin”, large areas of regressive changes i.e. inflammation and calcifications are representative. Further characteristic features of adaCP are finger-like tumor protrusions and the formation of a tumor-specific cellular environment in the surrounding brain tissue [8, 22]. This is accompanied by serious endocrinological and visual disturbances and significant long term morbidity and mortality rates, and makes treatment quite a challenge [26, 36].

Recent data has provided important insights into the molecular pathogenesis and origin of CP, and may provide distinguishing features for both subtypes associated with new molecular drug targets. Today it is common knowledge that the Wnt signaling pathway is strongly implicated in the pathogenesis of adaCP. Genetic analyses have shown that up to 95 % of the tumors harbor activating mutations in exon 3 of the CTNNB1 gene encoding β-catenin [5, 10, 28, 45]. Genetic alterations within the degradation targeting box of β-catenin lead to activation of the pathway in adaCP, indicated by aberrant nuclear accumulation of the protein and respective Wnt target gene activation [24]. This is described to be exclusive for adaCP [5, 30] and aberrant (nuclear) immunohistochemical staining for β-catenin in whirl-like cell clusters and single cells represent the most reliable marker for adaCP in the differential diagnosis of space occupying lesions in the sellar region [21]. Activated Wnt signaling influences tumor cell migration and the tumor initiating strength of this alteration was recently confirmed in vivo [18, 22, 48]. Furthermore, it was shown that EGFR- and SHH signaling pathways are also up-regulated in adaCP and associated with tumor cell migration [1, 2, 20, 23].

The molecular background of papCP initiation was largely unknown until exome sequencing studies revealed BRAF mutations (BRAF p.Val600Glu) in up to 95 % of these tumors [5]. This result was confirmed in smaller series of samples, where BRAF V600E mutations could be reliably detected by immunohistochemistry [29, 30, 44]. Interestingly, Brastianos and colleagues postulated that CTNNB1 and BRAF mutations were exclusive and clonal in each CP subtype, and they detected no other recurrent mutations or genomic aberrations in either subtype [5, 30]. In contrast, Larkin et al. claimed that BRAF mutations may coexist with CTNNB1 mutations in adamantinomatous tumors [30].

BRAF mutations were described in several other neoplasms and potent drugs have already shown a robust clinical response in melanomas, hairy cell leukemias, as well as brain tumors with BRAF p.Val600Glu such as pilocytic astrocytoma, pleomorphic xanthoastrocytoma and ganglioglioma [13, 14, 16, 33, 43]. Recently, two case reports of BRAF inhibitor treatment (Vemurafenib and Dabrafenib in combination with Trametinib) in patients with residual or recurrent papCP after surgery have been published, showing favorable short-term effects. However, the Vemurafenib-treated tumor displayed tumor regrowth after a drug holiday, indicating that further long-term studies are necessary [3, 4].

In order to verify and strengthen the above summarized molecular data and to reveal distinctive molecular characteristics that facilitate the diagnosis of both CP subtypes, we obtained CTNNB1 and BRAF mutational analysis as well as array-based gene expression and methylation profiling in one of the largest cohorts of human CP tissue samples published to date.

Two different subtypes of craniopharyngiomas (CP) have to be distinguished according to the current version of the WHO classification of tumors of the central nervous system [31]. Both variants, the adamantinomatous CP (adaCP) and the papillary CP (papCP), differ in their histomorphology, age distribution and clinical course. Whereas papCP occur almost exclusively in adults, adaCP have a bimodal age distribution with incidence peaks in children and adults aged 45–60 years [32]. The most significant factor associated with recurrence is the extent of surgical resection which depends on tumor size and localization. Nowadays there is a trend towards less radical extirpation in order to avoid hypothalamic injury [38]. To prevent a higher recurrence rate after incomplete surgical resection, additional radiotherapy is widely used. Histological evidence of brain invasion through the building of finger-like tumor protrusion, which is more frequently documented in the adaCP type than in the papCP type, seems not to correlate with higher recurrence rates in cases with gross surgical resection [49]. Discrimination of CP variants is often challenging in only small and/or fragmented surgical specimens, and the existence of CP with a mixed histological pattern was specified and promoted in several reports [40, 41]. Newly described molecular markers may help to solve this problem, and innovative approaches with large numbers and well characterized tumor samples are required in order to determine important implications for the differential diagnosis and treatment of CP. Our results, obtained in one of the largest cohorts to date, indicate that both variants can clearly be distinguished using genetic and epigenetic profiling. Targeted genotyping revealed activating mutations and small deletions in exon 3 of CTNNB1 (β-catenin) exclusively in adaCP cases. Using immunohistochemistry we were able to detect tumor cells with nuclear β-catenin accumulation only in the adaCP specimens tested. In contrast, BRAF mutations were detectable in all of the papCP tumor samples. Results of SSCP analyses could be verified using pyrosequencing and VE1 immunohistochemistry, with all mutations being of the hotspot V600E type. Our results are in line with a previous study published by Brastianos et al., showing CTNNB1 alterations in 92–96 % of adaCP and BRAFV600E mutations in a frequency of 95–100 % of papCP, depending on the detection method and the tumor purity [5]. The assumption that both mutations were mutually exclusive was recently questioned, however. In a small cohort of adaCP two tumors with CTNNB1 alterations in codon 41 (T41I) and additional BRAF V600E mutations were described [30]. To assess this specific correlation we included 11 adaCP with the described CTNNB1 gene mutation (codon 41, T41I) in our analysis. However, we were not able to detect additional alterations in BRAF in any of the adaCP samples studied. We also analyzed one patient showing a tumor relapse occurring 14 years after first surgery, but did not detect additional genomic alterations. Both the primary tumor and recurrence showed the same CTNNB1 mutation. Our work validates the hypothesis that these mutations are mutually exclusive in the CP subtypes and represent valuable molecular markers to differentiate papCP and adaCP. Several other studies have verified the benefit of CTNNB1 and BRAFV600E in the differential diagnosis of sellar lesions and we support this statement, with the caveat that V600E mutations may also occur for example in sellar pilocytic astrocytomas, and thus supportive histology is also required [21, 29, 44].

The apparent mutual exclusivity of CTNNB1 and BRAFV600E mutations in the CP variants indicates that both subtypes have a different molecular origin. This suggestion is supported by previous studies providing important insights into the molecular and cellular pathogenesis of these tumors [2, 8, 17, 25]. Several pathways have been described to be activated in adaCP cell clusters with nuclear β-catenin accumulations, including the Wnt pathway, the epidermal growth factor receptor pathway and the sonic hedgehog pathway [1, 18, 20, 23, 24, 46]. A novel tumor stem cell niche (CD133 and CD44), a specific cellular environment at the brain invasion border (TNC and MAP2) and a novel role for pituitary stem cells in the pathogenesis of adaCP has been proposed [2, 8, 17, 25]. Using array-based gene expression profiling we were able to confirm these associations in terms of transcriptional differences. Comparing papCP and adaCP tumor samples, the latter showed significant up-regulation of direct targets of the Wnt/β-catenin signaling pathway (LEF1 and AXIN2) as well as important components of the hedgehog signaling pathway (GLI2, PTCH1 and SHH). Furthermore, CLDN1 (a recently introduced marker to differentiate between CP subtypes) showed significantly higher expression levels in papCP [47]. Detection of tumor-specific signaling pathway activation enables the possibility of target-oriented intervention. Testing of novel pharmacological treatments for both CP variants is on the way, and will hopefully provide additional therapeutic options in the future, with BRAF V600E inhibition being a particularly attractive approach.

Reports regarding epigenomic differences in CP are missing so far. Therefore, we conducted DNA methylation profiling and were able to clearly distinguish the groups of adaCP and papCP. This was confirmed by gene expression analysis, demonstrating that the two subtypes are clearly molecularly distinct on both the epigenetic and transcriptional level.

One small biopsy of a histologically challenging case (pap11) was integrated in the analysis and grouped clearly into the papCP cluster, highlighting the utility of this approach for classification. It is of note, that differential methylation analysis revealed no significant difference between adaCP in children and adults. However, differential methylation and gene expression analysis emphasize an epigenetic impact on the Wnt- and Hedgehog signaling in adaCP as a whole. Further studies are currently underway to verify and specifically analyse additional differentially methylated genes and to correlate this data with gene expression profiles and clinical data.

This study clearly supports the assumption that adaCP and papCP are two distinct entities with different genetic and epigenetic backgrounds. They can be clearly diagnosed in most cases by using BRAF V600E and CTNNB1 mutation analysis but also by their methylation profile.

Innovative approaches studying large and well characterized tumor samples may help to further understand the pathogenesis of both variants and may lead to find new treatment strategies.