Potential evolution of neurosurgical treatment paradigms for craniopharyngioma based on genomic and transcriptomic characteristics

The recent genomic and transcriptomic characterization of human craniopharyngiomas has provided important insights into the pathogenesis of these tumors and supports that these tumor types are distinct entities. Critically, the insights provided by these data offer the potential for the introduction of novel therapies and surgical treatment paradigms for these tumors, which are associated with high morbidity rates and morbid conditions. Mutations in the CTNNB1 gene are primary drivers of adamantinomatous craniopharyngioma (ACP) and lead to the accumulation of β-catenin protein in a subset of the nuclei within the neoplastic epithelium of these tumors. Dysregulation of epidermal growth factor receptor (EGFR) and of sonic hedgehog (SHH) signaling in ACP suggest that paracrine oncogenic mechanisms may underlie ACP growth and implicate these signaling pathways as potential targets for therapeutic intervention using directed therapies. Recent work shows that ACP cells have primary cilia, further supporting the potential importance of SHH signaling in the pathogenesis of these tumors. While further preclinical data are needed, directed therapies could defer, or replace, the need for radiation therapy and/or allow for less aggressive surgical interventions. Furthermore, the prospect for reliable control of cystic disease without the need for surgery now exists. Studies of papillary craniopharyngioma (PCP) are more clinically advanced than those for ACP. The vast majority of PCPs harbor the BRAFv600e mutation. There are now 2 reports of patients with PCP that had dramatic therapeutic responses to targeted agents. Ongoing clinical and research studies promise to not only advance our understanding of these challenging tumors but to offer new approaches for patient management.

The recent genomic and transcriptomic characterization of human craniopharyngiomas has provided important insights into the pathogenesis of these tumors and supports that these tumor types are distinct entities.Critically, the insights provided by these data offer the potential for the introduction of novel therapies and surgical treatment paradigms for these tumors, which are associated with high morbidity rates and morbid conditions.Mutations in the CTNNB1 gene are primary drivers of adamantinomatous craniopharyngioma (ACP) and lead to the accumulation of b-catenin protein in a subset of the nuclei within the neoplastic epithelium of these tumors.Dysregulation of epidermal growth factor receptor (EGFR) and of sonic hedgehog (SHH) signaling in ACP suggest that paracrine oncogenic mechanisms may underlie ACP growth and implicate these signaling pathways as potential targets for therapeutic intervention using directed therapies.Recent work shows that ACP cells have primary cilia, further supporting the potential importance of SHH signaling in the pathogenesis of these tumors.While further preclinical data are needed, directed therapies could defer, or replace, the need for radiation therapy and/or allow for less aggressive surgical interventions.Furthermore, the prospect for reliable control of cystic disease without the need for surgery now exists.Studies of papillary craniopharyngioma (PCP) are more clinically advanced than those for ACP.The vast majority of PCPs harbor the BRAF v600e mutation.There are now 2 reports of patients with PCP that had dramatic therapeutic responses to targeted agents.Ongoing clinical and research studies promise to not only advance our understanding of these challenging tumors but to offer new approaches for patient management.
key members of the canonical WNT signaling pathway is b-catenin, which is encoded by the CTNNB1 gene. 10,32his protein plays a critical role in development, cellular proliferation, differentiation, and cell migration. 10,11,16,41A destruction complex restrains b-catenin within the cytosol and facilitates its ubiquitination and proteasomal degradation. 31The Wnt protein family includes approximately 20 different proteins that bind to the Frizzled (Fz) family of receptors.When Wnt proteins bind the Fz receptors, the Wnt pathway is activated, 31 leading to an intracellular signaling cascade that ultimately prevents formation of the b-catenin destruction complex. 9,11,16,22,31Without the destruction complex, b-catenin protein accumulates and ultimately translocates to the nucleus, where it facilitates transcription of b-catenin target genes and stimulates cellular proliferation and other Wnt regulated cellular processes. 8,9,11,16,22,31ncreased migratory capacity and invasiveness have been observed in neoplastic cells with elevated b-catenin levels and activated target genes. 22In ACP, b-catenin accumulation can be found within cell clusters localized at invasive protrusions of tumor into normal brain 22 (Fig. 1A).It is possible that this increased mobility and invasive capacity are related to the overexpression of fascin-1 (fascin), a b-catenin/T-cell factor (TCF) signaling target gene that is commonly involved with reorganization of the actin cytoskeleton, which is required for cellular motility. 22,23b-catenin can regulate fascin expression by binding to a TCF-binding domain on the promoter region for fascin. 31Other cancers, such as colorectal, have increased fascin expression, particularly at the invasive edges. 22-catenin binding of fascin promotes reallocation of bcatenin, changes cell adhesion properties, and reduces bcatenin destruction.22 As b-catenin accumulates it can reduce E-cadherin expression, which reduces cell adhesion and likely leads to cells that are more motile and, consequently, more invasive.22 Inhibition of fascin or b-catenin expression can decrease the migratory capacity of ACP tumor cells in culture.22

CTNNB1 Mutation
Nuclear accumulation of b-catenin results from mutations within exon 3 of the CTNNB1 gene.This exon encodes a degradation targeting motif, mutation of which confers resistance to the b-catenin destruction complex. 9,41While mutations have been identified at a number of different codons, these all impact the binding of GSK3b. 9,11,12,24,28,38s a result of this mechanism, nuclear accumulation of b-catenin is a histological hallmark of CTNNB1 mutation. 10,12,16,26,41In the case of craniopharyngiomas, mutation of CTNNB1 has been described in 69%-100% 9,20,28 of ACP specimens, while it is not routinely observed in PCP or other parasellar tumors. 9,28In the most detailed study to date, CTNNB1 mutation was identified in 95% of ACP specimens, using massively parallel sequencing and targeted genotyping. 5In the same study, Brastianos and Santagata determined that CTNNB1 mutation was not present in PCP. 5 Other groups have identified CTNNB1 mutation in 70%-80% of ACP specimens. 9,12,19,31Sampling error or the significant intratumoral heterogeneity present in these tumors, which often have scant neoplastic epithelium, may explain the variability in the frequency with which CTNNB1 mutation is detected.Presently, however, despite the presence of b-catenin dysregulation in multiple cancer cell types, no targeted therapies are available.As such, direct intervention regarding CTNNB1 mutation is unlikely to impact neurosurgical treatment algorithms in the short term.
Nevertheless, insights related to the mutation affecting the GSK3b binding domain of CTNNB1 have implications regarding the origins of ACP.A clearer understanding of these may facilitate efforts to identify directed therapies.For example, definitive evidence of pituitary origin would indicate that ACP is not protected by the blood-brain barrier, thereby suggesting that a wider range of systemically delivered antitumor therapies could be effective.While similar mutations are observed in other lesions that share histological features with ACP, such as pilomatricoma and calcifying cystic odontogenic tumor, 21 multiple potential origins for ACP remain to be explored.As mentioned above, it has been postulated that ACP arises from undifferentiated anterior pituitary stem cells. 17,20,23,39An additional potential mechanism employs the paracrine tumorigenesis theory, in which small clusters of CTNNB1 mutated cells, characterized by nucleocytoplasmic b-catenin, leverage paracrine/autocrine signaling to promote tumorigenic activity in a much larger nonmutated cell population. 7,35e Sonic Hedgehog Pathway Sonic hedgehog (SHH) plays an integral role in the maintenance of adult stem cells 2 and in the normal development of several organs, including the pituitary gland and Rathke's pouch. 18,31Pathological upregulation of SHH signaling has been demonstrated in a multitude of cancers, including meningioma, basal cell carcinoma, and medulloblastoma. 1,2,4,14,29,40With regard to ACP, SHH overexpression has been described in tumors in humans and in a novel murine model, in which it colocalizes in cells with nuclear accumulation of b-catenin (Fig. 1B). 2 It is hypothesized that both autocrine and paracrine SHH signaling may contribute to ACP tumorigenesis.Two studies of the human ACP transcriptome have shown an upregulation of SHH pathway genes 18,20 (Fig. 2).The therapeutic relevance of this finding was solidified by the demonstration that ACP uniquely expressed the active isoform of SHH protein, when compared with other pediatric brain tumor types and to normal brain. 20As such, preclinical animal studies of the smoothened inhibitor vismodegib are ongoing (personal communication, J. P. Martinez-Barbera, 2016).
An additional application of novel insights regarding the SHH pathway in craniopharyngioma lies in the differentiation of ACP from PCP. Hölsken and colleagues 25 demonstrated a much higher expression of SHH pathway genes in the transcriptomes of ACP compared with PCP.These findings correlated with distinct methylation patterns between the 2 tumor types.This again suggests that ACP and PCP are biologically distinct tumors.Interestingly, among ACP specimens, the authors identified no difference in the methylation pattern of tumors based on the patient's age at presentation.This implies that therapies identified through the study of pediatric tumors may also have relevance in the adult setting.The distinction between ACP and PCP was further supported by a recent study showing different patterns of primary cilia between these lesions. 15Using immunohistochemistry for ARL13B, Coy and colleagues showed that ACPs have primary cilia throughout their neoplastic epithelium (Fig. 1C), whereas PCPs have primary cilia only in the basal cells of the neoplastic epithelium.Because neoplasms that are driven by oncogenic activation of the hedgehog pathway, such as basal cell carcinoma, and a subset of medulloblastomas often retain primary cilia and require these organelles for hedgehog signaling, the identification of primary cilia throughout the epithelium of ACP further supports the potential importance of SHH signaling and perhaps other cilia-dependent signaling pathways in the pathogenesis of ACP. 15

Epidermal Growth Factor Receptor
There does not appear to be genome-level dysregulation of the epidermal growth factor (EGF) pathway in ACP.However, downstream upregulation of this pathway may contribute to ACP cell growth and infiltration, and offers the prospect of effective targeted therapies that could influence the role of neurosurgical intervention for patients with ACP.The EGF receptor (EGFR) is a transmembrane glycoprotein that can be activated by multiple growth factors, resulting in the initiation of intracellular cascades involved in the regulation of cell proliferation, differentiation, apoptosis, and motility. 23There is evidence that, when activated and phosphorylated, EGFR and b-catenin colocalize to the nucleus, along with fascin.As described above, this occurs in cell clusters at the infiltrating borders of the tumor, in which the canonical Wnt pathway is upregulated. 23,31,33Correlation between increased EGFR pathway activity and ACP cell growth and migration has been seen in laboratory data 16,20 and cell culture models. 23dditional evidence supporting the role of EGFR in tumorigenesis of ACP includes high levels of EGF pathway genes in the ACP transcriptome, 20 the involvement of EGFR in the regulation of the expression of stem cell markers in ACP, and the presence of the activated EGFR pathway in b-catenin accumulating cells. 26As with therapies directed at the SHH pathway, those targeting the EGF pathway require further clarification through preclinical study before they could enter clinical use and impact neurosurgical care.

Additional Relevant Pathways
In addition to SHH and EGFR pathways, other molecules and pathways are uniquely expressed in craniopharyngioma, but are not yet as well described.The ACP transcriptome is characterized by high levels of certain matrix metallopeptidases, 19,20 including MMPs 2, 3, 7, 9, and 12. Elevated levels of p63 have been found in both ACP and PCP, with elevated expression also identified in the ACP transcriptome. 20P63 is a gene homolog of the p53 tumor suppressor family. 12,16Impaired p63 expression has been suggested to influence neoplastic cell transformation. 12In addition, transcriptome analysis identified significant overexpression-in ACP-of LCK, EPHA, and SRC, each of which is a target of the tyrosine kinase inhib-itor dasatinib. 20While still in the preclinical stages, these unique characteristics of ACP may at some point allow for effective therapeutic intervention without radical surgery.

Neurosurgical Implications of ACP
While current therapy for ACP relies upon surgery, radiation, or cyst-directed intervention, the biological characteristics described above hold promise for the introduction of targeted antitumor treatments.The details of therapy delivery, such as the timing and route (e.g., sys-temic, intrathecal), and therefore the precise role of operative intervention, remain to be determined.Presently, preclinical studies to identify the paradigms with the highest likelihood of success are ongoing.

Papillary Craniopharyngioma Distinction From ACP
As described above, ACP and PCP demonstrate distinct genetic aberrations.Different transcriptome profiles and differences in genome-wide methylation patterns support the view that these 2 tumors are biologically distinct entities with different mechanisms of pathogenesis.This distinction has substantial implications for neurosurgeons, as case reports of successful directed therapy for PCP, derived through experience with more common tumors, have been published.Successful clinical trials of these treatments could alter the role of the neurosurgeon in the management of PCP.Genomic studies support the findings from histological studies that show nuclear and cytoplasmic accumulation of b-catenin in ACP.In PCP, b-catenin localizes to the cell membrane, similar to the pattern of localization in other CTNNB1 wild-type tumors of the sellar region 9,12,16,31 and throughout the body.

brAF
Mutation of the BRAF proto-oncogene is well characterized in the context of multiple human cancers, making it the most clinically relevant genome-level aberration that has been identified in human craniopharyngioma.BRAF mutation upregulates MAP kinase signaling, thereby driving cell division.Although multiple mutations of BRAF have been described in human cancers, the most common is a substitution of valine by glutamate at codon number 600, termed the BRAF v600e mutation.The work of Brastianos and colleagues, discussed above, 7 identified BRAF v600e mutation in 92.8% of PCP specimens.This mutation was not found in any of the ACP specimens examined.This raised the potential for effective targeted therapy using medications that were developed in the context of other tumors that harbor BRAF mutation, most notably cutaneous melanoma.In this report, however, whole-exome sequencing was undertaken in only 3 PCP specimens.As such, the existence of additional lower frequency recurrent genomic events remains a possibility.Nevertheless, multiple studies demonstrate the presence of BRAF v600e mutation in nearly every specimen of confirmed PCP. 30,36,37An additional intriguing report suggests that a small subset of human craniopharyngioma is characterized by coexisting mutations in both CTNNB1 and BRAF. 32This could be of substantial clinical interest and merits further exploration, but presently does not impact neurosurgical treatment strategies.
Leveraging the presence of the BRAF v600e mutation, 2 recent case reports demonstrated clinically relevant responses of PCP to directed therapies.Aylwin and colleagues 3 described a near-total radiographic response of a BRAF v600e mutant PCP following treatment with single agent vemurafenib, a mutation-specific BRAF inhibitor.Importantly, after therapy was halted for a CSF leak associated with tumor shrinkage, tumor recurrence occurred within 6 weeks and long-term control was not achieved after therapy was restarted.In a 39-year-old patient with multiply recurrent PCP, who had required 4 craniotomies and an endonasal resection for multiple cystic recurrences, Brastianos and collegues 6 reported an excellent initial response to combination therapy using the RAF inhibitor dabrafenib and MEK inhibitor trametinib (Fig. 3).Furthermore, the authors identified BRAF v600e mutant DNA in the patient's peripheral blood during therapy, thus presenting the prospect of noninvasive diagnosis for patients with suspected PCP.These reports highlight the facts that multiple directed therapies are potentially available for patients with PCP and that dual or multiagent therapy is likely to be required to reliably obtain durable tumor control.As such, a multi-institutional trial of combined BRAF and MEK inhibition for patients with PCP is forthcoming. 5n addition to offering therapeutic potential, the high prevalence of the BRAF v600e mutation in PCP may simplify diagnosis of these tumors.A mutation-specific antibody (clone VE1) recognizes the BRAF v600e mutant protein but not the wild-type protein. 13This is now being employed by pathologists for immunohistochemical identification, thereby distinguishing PCP from other masses of the sellar region, including pituitary adenomas, ACP, and Rathke's cleft cysts with squamous metaplasia. 27,30,37argeted genotyping of the BRAF v600e mutant allele is also used for diagnosis and other biomarkers have been proposed as well. 34

Neurosurgical implications of PCP
For neurosurgeons, the potential impact of recent advances in our biological understanding of PCP, as well as noninvasive identification of BRAF mutated cells, is considerable.Neoadjuvant systemic therapy that does not necessitate aggressive (or any) surgical intervention could assume a prominent position in the treatment paradigm for patients with PCP.While PCP represents the minority of craniopharyngiomas, and is almost exclusively limited to the adult population, this represents dramatic progress in the clinical management of this tumor associated with high morbidity.

Conclusions
Prospects for effective novel therapies against craniopharyngiomas in humans have improved considerably with recent advances in our understanding of the biological basis of this disease.Genomic and transcriptomic analyses indicate that ACP and PCP will require different therapeutic approaches.Promising options for PCP have emerged from previous experience with more common tumors that harbor the BRAF v600e mutation, and human clinical trials will further investigate these advances.These trials may allow neurosurgeons to reserve aggressive intervention, with the associated morbidity, for the minority of cases that do not demonstrate a durable response to directed therapies.Although we currently lack therapies that target the b-catenin mutation that characterizes ACP, multiple additional targets, such as the SHH and EGFR pathways, have been described.Further preclinical investigation of these prospects is ongoing and may ultimately, as in the case of PCP, influence neurosurgical management paradigms.

Fig. 1 .
Fig. 1.A: Immunohistochemistry for β-catenin demonstrates nuclear and cytoplasmic staining in clusters of tumor cells in human ACP.b: In situ hybridization shows a high level of expression of SHH mRNA in areas with nuclear β-catenin.C: Immunohistochemistry for the cilia marker ARL13B shows primary cilia in the majority of tumor cells in ACP.

Fig. 3 .
Fig. 3. Effect of dual therapy using dabrafenib and trametinib on multiply recurrent PCP.H & E-stained specimens prior to (A) and after (b) treatment.Immunohistochemistry that recognizes BRAF V600E mutant protein specifically (VE1 clone) shows that the BRAF mutation is present in the tumor epithelium in both the pretreatment (C) and posttreatment (D) specimens, thereby demonstrating that the treatment did not entirely eradicate tumor cells.Treatment with dabrafenib and trametinib results in a marked decrease in the number of cycling cells (MIB-1 immunohistochemistry on pretreatment [e] and posttreatment [F] samples).