The potential for enhancing the clinical utility of tumor classifications by concurrent evaluation of prognostic or predictive molecular markers remains to be realized for medulloblastoma. However, recent progress in our understanding of medulloblastoma biology has provided real scope for the creation of such schemes [8
]. Of particular value would be the identification of WNT tumors, which have a relatively good outcome with standard therapies and may benefit from a reduction in the intensity of adjuvant therapy, and of SHH tumors, which represent potential targets for novel pathway inhibitors. Despite these developments, burgeoning molecular data-sets present a challenge to those involved in treating medulloblastoma: how to develop an optimal therapeutic stratification, involving clinical, pathological, and molecular variables, with improved cure rate and reduced long-term adverse effects as end-points. Rising to this challenge involves determining (1) which molecular markers are independent indicators of outcome when tested alongside clinical and pathological variables in trial cohorts of patients and (2) how best to assay selected molecular markers in diagnostic material, which is submitted in the form of FFPE tissue. Our study describes an immunohistochemical method for distinguishing three molecular subgroups of medulloblastomas, which accurately matched SHH, WNT, and non-SHH/WNT subgroups in a validation set of profiled tumors, and provides data on associations between clinical, pathological, and molecular variables.
WNT medulloblastomas are characterized by up-regulation of the canonical WNT signaling pathway, which results in translocation of β-catenin to the nucleus. About two thirds harbor a CTNNB1
mutation. Mutations in other pathway elements, such as APC
, have been recorded in the absence of a CTNNB1
mutation, but are much less frequent [4
]. A robust immunohistochemical method, suitable for use with FFPE tissue samples, exists for identifying β-catenin nucleopositive WNT pathway medulloblastomas. However, we have demonstrated previously that the WNT tumor immunophenotype is variable [9
]. Most WNT tumors demonstrate widespread nuclear immunoreactivity for β-catenin, present in practically all tumor cells, while some examples show patchy staining. The extent of β-catenin nuclear immunoreactivity in these WNT pathway medulloblastomas always amounts to more than a third of the total tumor area and is clearly different from the situation where very few scattered β-catenin nucleopositive cells, representing less than 2% of tumor cells, are evident. Assays for CTNNB1
mutation and monosomy 6, which occurs in nearly all WNT pathway medulloblastomas, have helped to establish the status of tumors in these immunohistochemical categories [3
In this study, we have extended our earlier clinicopathological and cytogenetic data on WNT pathway medulloblastomas, corroborating data from other researchers. WNT pathway medulloblastomas are almost all classic tumors, and most (81% in this study) present between the ages of 6 and 12 years. Our previous studies have demonstrated a good outcome for WNT tumors with standard adjuvant therapy, and recent data from patients on the CNS9102 trial have been included in the present study [9
]. There is a close association between a WNT pathway immunophenotype and CTNNB1
mutation or monosomy 6, predicating a good outcome for medulloblastomas with these genetic abnormalities [3
]. Assaying chromosome 6 CNAs could prove to be a useful ancillary test alongside immunohistochemistry for β-catenin, but the latter remains the optimal method for determining WNT pathway status. While monosomy 6 is frequently encountered in WNT pathway medulloblastomas, other cytogenetic abnormalities, such as MYC
amplification or copy number changes on chromosome 17, are rare [3
]. The idiosyncratic nature of the WNT tumor, as evinced by its restricted range of age at presentation, its pathological and cytogenetic associations, and its relatively good outcome, all align with recent evidence from our group that its histogenesis is distinct from that of other medulloblastomas [17
While nuclear immunoreactivity for β-catenin is established as a robust marker of WNT pathway activation in diagnostic (FFPE) biopsies, finding a reliable immunohistochemical marker of SHH pathway activation for the clinical laboratory has been more challenging. Antibodies to up-regulated components of the SHH pathway that work well enough in animal models tend to perform erratically on FFPE tissue sections of human tumors, and none is established in diagnostic practice. Faced with such difficulties, we have resorted to surrogate markers of this molecular subgroup of medulloblastomas, utilizing expression profiling data to select antibodies that work well on FFPE tissue and target the protein products of genes overexpressed in SHH pathway medulloblastomas. Validating potential antibodies on medulloblastomas from a series used to generate the expression data, we selected GAB1 as a specific marker of SHH pathway medulloblastomas, and filamin A as a marker of WNT and SHH tumors. In a separate study, YAP1 nuclear immunoreactivity had already been shown to identify WNT and SHH pathway medulloblastomas [13
GAB1 (GRB2-associated binding protein 1) is a member of the Gab/DOS (Daughter of Sevenless) family of adapter proteins and is involved in multiple cell processes, including morphogenesis, proliferation, and cell adhesion and motility [19
]. GAB1 is tyrosine-phosphorylated upon stimulation of receptors by various growth factors, e.g. PDGF, HGF, and by cytokines, e.g. IL-6, IL-3, and interacts with multiple signaling molecules, e.g. SHP-2, PI3K, in several cell systems. Its role in the SHH pathway has yet to be clarified; dysregulation of GAB1 metabolism in other tumors that involve abnormalities of the SHH pathway has not been reported. On the basis of its biology, its specificity in classifying medulloblastomas might appear surprising. However, we have further validated the use of an anti-GAB1 antibody in this setting on over 80 tumors from the SJMB03 cohort, for which expression data are available (Dr. Amar Gajjar, personal communication). Filamin A is a large actin-binding protein involved in cell motility [48
]. Nonsense mutations in the gene encoding filamin A (FLNA
) cause an X-linked brain malformation, periventricular nodular heterotopia [5
]. In neoplasia, filamin A plays a role in both angiogenesis and metastasis [47
]. YAP1 is a transcriptional co-activator and can promote proliferation and transformation. It is a downstream target of the Hippo pathway, which upon activation inhibits YAP1 through phosphorylation and cytoplasmic sequestration. Overexpression of YAP1 is a feature of several neoplastic processes, including medulloblastoma, in which it is also amplified in a small proportion of cases [13
SHH pathway medulloblastomas encompass all desmoplastic tumors, including the conventional D/N medulloblastoma, MBEN, and paucinodular medulloblastoma. All of these variants are characterized by reticulin-positive internodular desmoplasia and are distinct from the less common (7% in the present series) non-desmoplastic medulloblastoma that contains a variable number of nodules with an identical cytology and immunophenotype to those in desmoplastic tumors. Previously, we have demonstrated that desmoplastic medulloblastomas, when compared with non-desmoplastic nodular tumors, present at a younger age and have distinct cytogenetic associations [30
]. Data from this study show that all non-desmoplastic nodular tumors fall into the non-SHH/WNT subgroup. Just over half (54%) of SHH pathway medulloblastomas are desmoplastic tumors. Among remaining non-desmoplastic SHH tumors, the LC/A variant is relatively overrepresented; the LC/A:classic ratio among the three subgroups was: SHH = 0.57; WNT = 0.06; non-SHH/WNT = 0.09. Data from this study confirm that desmoplastic variants contribute most medulloblastomas in infancy and adulthood [1
]. In addition, nearly all tumors in these age groups belong to the SHH pathway subgroup, LC/A and classic tumors as well as desmoplastic; only 15% of medulloblastomas in infancy and adulthood are non-SHH/WNT tumors or WNT tumors (single adult case).
Our molecular cytogenetic data link loss at 9q22, encompassing the PTCH1
locus, with SHH pathway medulloblastomas and are consistent with previous reports of 9q22 loss or PTCH1
mutations in a proportion of SHH tumors that ranges from one quarter to two thirds [7
]. While we did not have sufficient data to complete a survival analysis of molecular subgroups among infants, the high frequency of SHH tumors in this age group (83%) suggests that molecular subgroup testing would not augment or refine the role already identified for pathological variant as the major determinant of outcome among this subset of patients [40
Data derived from the CNS9102 cohort of children aged 3–16 years show that SHH and non-SHH/WNT tumors do not have significantly different outcomes. However, PFS and OS curves generated for all patients in these two molecular subgroups may hide divergent outcomes influenced by other risk factors, particularly pathology in the case of SHH pathway medulloblastomas, with their relatively high frequency of LC/A tumors. To explore this further, we compared survival curves for children split according to established risk factors in this cohort (M status, pathological variant, and presence or absence of MYC amplification), clearly demonstrating their importance for patients with both SHH and non-SHH/WNT tumors.
Non-SHH/WNT tumors dominated our series of medulloblastomas. Gene expression profiling has split this molecular subgroup into 2–4 further categories [2
]. In a recent study by Northcott et al. [32
], two non-SHH/WNT molecular subgroups were designated ‘C’ and ‘D’ alongside groups of SHH and WNT tumors. Group ‘C’ patients tended to present at a younger age and to have a worse outcome than group ‘D’ patients. The highest frequency of metastatic disease at presentation was observed in group ‘C’. MYC
amplification was also most common in group ‘C’, while isochromosome (isodicentric) 17q was most common in group ‘D’. This study demonstrated a higher male:female ratio in the non-SHH/WNT category, when compared to that in the SHH and WNT subgroups, a result supported by our data. Another concordant result was that most tumors with CNAs on chromosome 17 in our series occurred in the non-SHH/WNT category.
Reflecting an increased understanding of the heterogeneity of medulloblastomas, our data contribute to a developing consensus on the prognostic or predictive utility of molecular markers in the therapeutic stratification of childhood medulloblastoma. In combination with accepted clinical and pathological outcome indicators, molecular markers would be expected to refine stratification of current therapies and to facilitate the use of targeted therapies, such as GDC-0449, a novel SHH pathway inhibitor. GDC-0449 could have particular utility in infant and adult disease, where SHH tumors predominate, although the drug’s adverse effects on bone growth could be a limiting factor for infant disease [23
WNT tumors have a good outcome with standard therapies and, in the absence of high-risk factors, could be regarded as low-risk disease, amenable to strategies that reduce adjuvant therapy without compromising cure rate (). High-risk disease can now be defined by clinical (M+ disease), pathological (LC/A variant), and molecular (MYC
amplification) factors [8
]. However, further refinements to such a scheme might be expected as the biological behaviors of small disease subgroups are increasingly understood. Medulloblastomas with chromosome 17 CNAs or MYCN
amplification fall into this category. Various types of chromosome 17 CNA, such as isodicentric 17q or loss of 17p, have been proposed as outcome indicators in medulloblastoma [28
]. Data from our recent study of patients from the CNS9102 trial indicate that, once good-outcome WNT tumors (rarely showing chromosome 17 CNAs) have been removed from survival analyses, chromosome 17 CNAs of any type are not significantly associated with outcome [9
]. The status of MYCN
amplification as an outcome indicator is controversial, but its biological relevance could depend on cellular context; although numbers are small, our data suggest that MYCN
-amplified SHH tumors have a poorer outcome than MYCN
-amplified non-SHH/WNT tumors.
Characteristics of low-risk and high-risk medulloblastomas
In summary, our studies provide details on (1) a novel immunohistochemical method based on gene expression profiling for identifying SHH, WNT, and non-SHH/WNT molecular subgroups of medulloblastoma and (2) clinicopathological data relating to these molecular subgroups from a large cohort of patients representing the entire spectrum of disease.