Introduction
According to the 2007 WHO classification of tumors of the central nervous system (CNS), CNS primitive neuroectodermal tumors (PNETs) can be further subdivided into CNS neuroblastoma/ganglioneuroblastoma, medulloepithelioma (MEPL), and ependymoblastoma (EBL) [
18]. In addition, “embryonal tumor with abundant neuropil and true rosettes” (ETANTR) has been discussed as a possibly unique variant of CNS PNET [
1,
2,
4,
6,
8,
10,
11,
19].
CNS neuroblastomas histologically and molecularly resemble subsets of medulloblastomas and peripheral neuroblastomas [
18]. They are characterized by the presence of Homer Wright (neuroblastic) rosettes, foci of neurocytic and/or ganglion cell maturation, intense synaptophysin expression, and
MYC/MYCN amplifications in almost 50 % of cases [
3,
18]. On the other hand, ETANTR, EBL, and MEPL are rare neoplasms characterized by the presence of similar histological patterns, namely multilayered and pseudo-stratified rosette-forming structures of variable shape and size. Both EBL and ETANTR include the so-called “ependymoblastic rosettes” harboring well-formed central round or slit-like lumina in the absence of an outer membrane [
4,
6,
11,
12,
14,
18]. MEPL is histologically characterized by papillary and tubular structures surrounded by an external limiting membrane, reminiscent of the developing neural tube [
4,
18]. These structures are sometimes also referred to as “medulloepithelial” rosettes. Moreover, some MEPL have also been reported to display ependymoblastic rosettes [
18]. These three variants of embryonal CNS tumors show a clinically uniform behavior, in that they predominantly affect infants under the age of 4 years and are associated with a highly aggressive course with reported survival times up to 24–36 months, but typically averaging 12 months [
1,
5,
9,
11,
15,
23].
Applying FISH analysis, we previously found amplifications at 19q13.42 involving the
C19MC cluster in 93 % of tumors diagnosed either as ETANTR, EBL, or MEPL with ETANTR features, but not in any other pediatric brain tumors [
15]. These results demonstrate that this genetic aberration is highly sensitive and specific to embryonal CNS tumors with multilayered rosettes irrespective of other features and that these subtypes are highly interrelated. Recently, Paulus and Kleihues therefore proposed to use the term “embryonal tumor with multilayered rosettes” (ETMR) as a general name for these tumors, a new entity, in part defined by the
C19MC amplification itself [
22].
To further test whether the three histological variants of ETMR represent a single entity, we performed clinicopathological and molecular analyses in 97 ETMR samples initially designated as ETANTR, EBL, or MEPL.
Materials and methods
Ninety-seven diagnostic specimens diagnosed histopathologically as either ETANTR, EBL, or MEPL were received for this study from various sources around the globe and collected during the last 5 years. Among these sources were Burdenko Neurosurgical Institute, Moscow, Russia; University of Bonn, Germany; Ludwig-Maximilians University, Munich, Germany; University of Münster, Germany; University of Tübingen, Germany; Università Sapienza, Rome, Italy; Necker Hospital, Paris, France; Academic Medical Center, Amsterdam, the Netherlands; University of Cambridge, Cambridge, UK; Institute of Neurology, Vienna, Austria; Hospital for Sick Children, Toronto, Canada; Memorial Sloan Kettering Cancer Center, New York, USA; and University of California, San Francisco, USA. A subset of these cases was previously published [
15,
16].
All cases were routinely formalin fixed and paraffin embedded. For diagnostic purposes, routine histopathological examination and immunohistochemical (IHC) analyses were performed in the different institutions participating in this study. Further centralized evaluation of all H&E slides was performed in the Heidelberg University Department of Neuropathology. In all 97 cases, IHC analysis applying a LIN28A polyclonal antibody and FISH analysis for the 19q13.42 locus were performed as previously described [
15,
16].
For samples for which sufficient DNA was available (
n = 41), we analyzed copy number aberrations (CNAs) using data generated with Illumina Human Methylation 450 k BeadChip arrays as described previously [
13,
26]. For the detection of amplifications, chromosomal gains and losses, automatic scoring was verified by manual assessment of the respective loci for each individual profile as previously described [
26].
To evaluate the molecular specificity of potential ETMR subtypes, we performed comparative cluster analysis of 450 k profiles generated for 41 ETMR together with 110 other primary pediatric brain tumors including pilocytic astrocytoma (PA; n = 10), ependymoma (EPN; n = 10), glioblastoma grade IV (GBM; n = 40), atypical teratoid rhabdoid tumor (AT/RT; n = 10), and medulloblastoma (MB; n = 40). Eight normal cerebellum (CBM) samples were also included. The following criteria were applied to filter the data: removal of probes targeting the X and Y chromosomes (n = 11,551), removal of probes containing a single-nucleotide polymorphism (dbSNP132 Common) within five base pairs of and including the targeted CpG-site (n = 24,536), and probes not mapping uniquely to the human reference genome (hg19) allowing for one mismatch (n = 9,993). In total, 438,370 probes were kept for analysis. For unsupervised hierarchical clustering of 41 ETMR samples, we selected the 4,756 most variably methylated probes across the dataset (s.d. >0.25). Samples were clustered using 1-Pearson correlation coefficient as the distance measure and average linkage (x-axis). Methylation probes were reordered by hierarchical clustering using euclidean distance and average linkage (y-axis). The heatmap illustration of 41 ETMR samples and 118 other pediatric brain tumor and control samples was generated by separately determining the 2,500 most variably methylated probes between the medulloblastoma subgroups, the K27-, G34-, IDH-, and wt (not H3.3 or IDH mutated) GBM subgroups, the non-ETMR tumor samples, and across the whole dataset. Probes were only used once (n = 6,540). Methylation probes were reordered by hierarchical clustering using euclidean distance and average linkage (y-axis).
Discussion
Currently, the verification of a distinct nosologic position for various human malignancies is complemented (or sometimes defined) by a comprehensive molecular work-up [
23,
26]. A number of refinements have recently been introduced into the current histological classification of pediatric CNS tumors. For example, the routine application of molecular diagnostics distinguishes AT/RTs from other PNETs [
18] and current studies strongly suggest incorporating four molecular medulloblastoma subgroups into the classification as separate tumor entities [
20]. These molecular data will help to further subdivide existing tumor entities by identifying disease variants with diverse clinical outcomes, distinct biology and clues regarding cell of origin. In contrast, molecular analyses of a representative cohort of ETANTR, EBL, and MEPL, three rare variants of embryonal CNS neoplasms, strongly suggests their integration into one single tumor entity called ETMR. All tumors were positive for LIN28A, a marker highly specific for ETMRs, and almost all cases displayed amplification of the
C19MC miRNA cluster at 19q13.42, as well as frequent trisomy 2. Furthermore, no significant differences in other CNAs were observed between these three histological variants and their DNA methylation patterns were highly concordant.
Morphologically, ETMR manifests uniformly with the presence of multilayered true rosettes: “ependymoblastic” and/or “medulloepithelial”, with variable shape and size. As already noted, it may frequently be difficult to distinguish between EBL and MEPL [
18]. Diagnostic differences between these tumor categories are descriptive and conceptual because they are, in general, based only on the absence of an outer collagen IV-positive membrane and apical cytoplasmic blebs in EBL structures. Moreover, similar clinical parameters and a highly aggressive course of disease for all ETMR histological variants (resistance to treatment, inevitable tumor recurrence and rapid death) also support our suggestion of commonality [
1,
8,
9,
11,
15,
16,
19].
Previously, molecular analysis of various ETMR subtypes has been hampered by limited cohort sizes and to date, only a few chromosomal imbalances were detected by conventional CGH analysis [
7,
25]. Recently, we described a focal unique amplicon at 19q13.42 spanning 0.89 Mb and covering the
C19MC genomic locus, the largest known cluster of human microRNA-coding genes [
24]. Further, the
C19MC locus was found to be amplified in 93 % of ETMR diagnosed previously on the basis of their characteristic morphology [
15], and these findings have been confirmed by others [
5,
17,
21,
23,
27,
28]. Thus,
C19MC amplification is a highly specific genetic ETMR hallmark, similar to loss of the
SMARCB1 locus in AT/RT or t(11;22) in Ewing sarcoma [
18]. This suggests that the
C19MC amplification could serve as a “driver” oncogenic event in ETMR. Comprehensive analysis of CNAs in our ETMR cohort revealed few other recurrent chromosomal gains and losses, and no subtle cytogenetic differences between the tumors with various histological features could be defined.
Common molecular signatures between the three histological variants of ETMR suggest that they may share a common origin, such as a primitive cell population in the subependymal region, with further evolution into a wide range of morphological appearances and mimics. In support of this suggestion, analysis of the 11 recurrent ETANTR samples revealed its frequent histological evolution into either EBL or MEPL phenotypes, while at the same time the molecular genetic make-up did not change between the initial and advanced stages of disease. Such stepwise morphologic transformations in the “classic” ETANTR appearance due to disease progression allow one to suppose that the three “histological variants” of ETMR may constitute either “polar ends” of a morphologic spectrum or varying differentiation stages of a single tumor entity rather than separate nosologic categories.
In conclusion, we identified uniform molecular signatures occurring in a representative subset of embryonal brain tumors with multilayered rosettes indicating that ETANTR, EBL, and MEPL comprise a single biological entity, which could potentially be designated in future WHO schemes as ETMR. For molecular diagnosis of this tumor category and to distinguish them from other embryonal tumors of the CNS, combined LIN28A IHC and FISH analysis of the 19q13.42 locus are recommended as routine diagnostic markers. Since misdiagnosis for controversial “poorly differentiated” embryonal CNS neoplasms is not uncommon, all tumors and especially CNS-PNETs harboring combined LIN28 expression and 19q13.42 amplification should be classified as ETMR, even in absence of the key histological patterns. As a next step, it will be important to understand the distinct biological significance of the prototypic molecular events in ETMR which may provide therapeutic targets for novel treatment strategies for these highly aggressive and therapy-resistant pediatric CNS malignances.