Background
MBL is the most common malignant brain tumour of childhood, accounting for approximately 15-20% of central nervous system (CNS) malignancies [
1]. Despite multimodal therapy, 20% to 30% of MBL recur [
2]. Large cell/anaplastic (LC/A) variant is most commonly associated with metastatic disease [
3]. Brain and spine secondary localizations are frequent, while extraneural metastases (ENM) are rare (<10% of cases) and occur mostly in bone, lymph nodes, lung and liver [
4]. Subcutaneous ENM have been described in very few cases [
5,
6].
In recent years, high-throughput studies [
7‐
14] allowed to classify MBL into four subgroups (WNT, SHH-Sonic Hedgehog, Group 3 and Group 4) and revealed the existence of gene mutations and of expression patterns linked to a worse prognosis.
Here we report the case of a child affected by LC/A MBL who developed subcutaneous metastases after a ventriculo-peritoneal shunting procedure. We performed histological and molecular analysis to characterize in details this aggressive tumour, particularly focusing on the stem-like cell population.
Methods
Histology: Paraffin-embedded 3-μm-thick sections from MBL tumour sample were stained with haematoxylin and eosin (H&E). Histology was reviewed by 2 neuropathologists (F.G. and M.A.) and diagnosis was centralized to minimize inter-observer variability.
Immunohistochemistry (IHC): Monoclonal antibody to p53 (DO-7; Dako, Carpinteria, CA, USA; dilution 1:300), and polyclonal anti-Beta catenin antibody (BD Transduction Laboratories, San Jose, CA, USA; dilution 1:100) were used.
Fluorescence in situ hybridization (FISH): Tumour tissue sections were deparaffinised and pre-treated with pepsin before hybridization with c-myc and control (centromere of chromosome 8) probes (Abnova Corporation, Taipei, Taiwan).
Stem-like cells (SLC) culture: Cells were isolated as previously reported [
15]. Briefly, fresh tumour was dissociated to single cell suspension and cultured in DMEM/F12 medium supplemented with 0.6% glucose, 25 mg/ml insulin, 60 mg/ml N-acetyl-L-cystein, 2 mg/ml heparin, 20 ng/ml EGF, 20 ng/ml bFGF, 1× penicillin-streptomycin and B27 supplement without vitamin A.
Immunofluorescence: Immunofluerescence was performed as previously described [
15]. Primary antibodies used were anti-Sox2 (MAB4343 Millipore) and anti-Nestin (ab6142; Abcam).
RNA extraction, reverse transcription and gene expression analysis: RNA extraction, reverse transcription and gene expression analysis were performed as previously described [
16]. RNA from normal cerebella were purchased from Life Technologies. TaqMan Low Density Array was custom designed with TaqMan assays for genes of interest [
17,
18]. 1 μg RNA was reverse transcribed using High capacity cDNA reverse transcription Kit (Ambion- Life Technologies Corporation, Carlsbad, CA, USA). Gene expression analysis on samples was performed employing an ABI Prism 7900 HT sequence detection system (Applied Biosystems- Life Technologies Corporation, Carlsbad, CA, USA) according to manufacturer’s instructions. Transcripts quantification was expressed in arbitrary units as the ratio of the sample quantity to the calibrator or to the mean values of control samples. All values were normalized to the 4 endogenous gene controls: GAPDH, ß- ACTIN, ß2-MICROGLOBULIN and HPRT. Heat maps were generated employing SpotFire software according to Delta Ct values, as previously described [
19].
Cytofluorimetry: Samples were dissociated into single cells and incubated with APC-conjugated anti-CD133 or with isotype control (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to manufacturer’s instructions.
Ethics Committee of Bambino Gesù Children Hospital approved the case study.
Conclusions
MBL is a heterogeneous disease with survival rates ranging from 85% for average risk patients to lower rates (<65%) in the presence of risk factors [
21,
22]. The identification of MBL histological variant is critical for patient stratification: the presence of desmoplastic or of extensive nodular histology is a strong predictor for low risk disease in early childhood, while LC/A MBL has been associated with high risk and lower survival rates [
3]. Indeed, LC/A MBL has been defined as a separate entity in the current WHO classification of CNS tumours [
23].
Recently, Von Hoff suggested a better outcome for children with severe anaplastic histology without additional clinical and molecular risk factors (e.g. c-myc amplification, large cell histology, young age and metastases) [
3]. Amplification of the proto-oncogene c-myc, a well-known negative prognostic marker for MBL, has been associated to LC/A variant and could contribute to its aggressive behaviour [
24,
25]. Indeed, the frequency of c-myc amplification has been reported in about 5% of MBL in mixed cohorts [
26], while a higher percentage has been reported in LC/A MBL [
27]. Among the prognostic markers investigated to predict disease recurrence, only four have been confirmed as related to a poor outcome in a reproducible manner: p53, survivin, erbB-2 expression and amplification of myc genes [
2]. In detail, p53 immunopositivity is an adverse prognostic marker [
2,
28] and somatic TP53 mutations are associated with chemo- and radiotherapy resistance [
2,
29]. A recent study showed that somatic TP53 mutations are enriched almost exclusively among SHH and WNT subgroup and are highly predictive of extremely poor survival in SHH MBL [
30]. Presence of gross anaplasia and c-erbB-2 overexpression status are the most important predictors of recurrence rates [
31].
Our patient showed adverse prognostic markers: severe anaplastic histology, c-myc amplification and p53 immunoreactivity. According to the subgroup classification [
32], our patient was diagnosed as a group 3 (both for the primary tumour and the metastatic lesions), as expected from LC/A histology with c-myc amplification and with negative nuclear staining for Beta-Catenin. These histopathological features and biological properties might explain the occurrence of secondary localizations, including the early developed ENM.
Subcutaneous metastases have been reported in very few cases [
5,
6]. Our clinical report is the first in a well-documented LC/A MBL. Differently from what previously described, our patient showed subcutaneous infiltration in the posterior neck region, far from the path of the ventriculo-peritoneal shunt. Moreover, in the final stages of the disease, a cobblestone appearance of the scalp suggested further dissemination of tumour.
Presence of VP shunt has been suggested as a risk factor for MBL metastases [
33]; however, the general concept that systemic metastases are more frequent in patients with CSF shunts is not fully accepted [
34].
Interestingly, stem cell signatures have been associated with a poor prognosis in tumours, raising the concept that the stem cell population may indeed contribute to the aggressive behaviour [
35,
36]. Very recently, it has been reported that clonal genetic events observed in metastases can be demonstrated in a restricted sub-clone of the primary tumour, suggesting that only rare cells have the ability to metastasize [
37].
The opportunity to compare primary tumour with its metastases and SLC allowed us to highlight novel molecular findings. Our results are in agreement with the disease model based on different compartments: metastases were very similar among each other, while showing some differences versus the primary MBL. Differences were more evident between tumour samples and stem like cells.
Neoangiogenesis and cell invasion molecules were highly expressed in tumour bulk and even more in SLC. The same pattern was observed for the expression levels of molecules involved in EGF signalling, that may be linked to the occurrence of subcutaneous seeding.
In the era of molecular characterization of tumours, the identification of biological mechanisms of aggressiveness might well contribute to develop the most appropriate therapeutic strategies. In the present clinical case, we have shown that both primary tumour cells and SLC express ERBB family members, supporting a possible use of anti-ERBB specific therapies, which are already available [
38,
39]. In addition, our case expressed EZH2, a molecule that has been recently reported as a critical regulator in MBL growth, thus representing a novel potential therapeutic target [
40].
In conclusion, we report a rare case of subcutaneous metastatic LC/A MBL, which was analysed in details through a multidisciplinary approach. The molecular characterization of these aggressive tumours might improve the understanding of their pathogenesis and provide the rationale for targeted therapeutic strategies.
Consent
Written informed consent was obtained from the patient’s parents for publication of this Case report and of any accompanying images.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
EM, AP, GPS, RF, AG and EF carried out molecular studies. AM, FdB, GSC, AC and FL clinically followed the patient. MA, FRB, FG carried out pathologic assessments. All authors read and approved the final manuscript.