Cerebrospinal fluid cytokines in metastatic group 3 and 4 medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumour in the paediatric population [1]. MB can be further classified into 4 molecular subtypes (WNT, sonic hedgehog SHH, groups 3 and 4) on the basis of gene expression profiling [2]. Amongst these subtypes, Group 3 and 4 MB have higher propensity to metastasize [3]. In congruency with most human cancers whereby more than 90% of tumour deaths are metastasis-related [4], metastatic MB portends an extremely grim prognosis. This subset of patients usually succumbs to the disseminated spread of MB tumour cells both within and beyond the central nervous system (CNS) [2, 5, 6].

Traditional teaching advocates that MB disseminates to the leptomeninges via primary tumour cell shedding into the cerebrospinal fluid (CSF) followed by distal implantation and growth [7]. However, exact mechanisms of metastasis in MB at this stage, are still incompletely elucidated. The CSF is considered to be a fluid envelope that is responsible for the hydromechanical protection of the CNS. It has an essential role in the homeostasis of the brain parenchymal interstitial fluid and regulation of neuronal functioning [8]. Under physiological circumstances, the continual, directed flow of CSF enables the delivery of various factors within the ventricular system and targeted CNS regions [9]. Separately, inflammation, the 7th hallmark of cancer has been implicated as a key player in tumour development and metastasis [10‐12]. In particular, cytokines and their inflammation-related partners are known to coordinate various pro-inflammatory responses within the tumour microenvironment, acting on autocrine and, or paracrine pathways on both malignant and non-malignant cells [13].

As a step towards exploring inflammation in metastatic MB, the authors aim to investigate the involvement of circulating cytokines in the CSF of Group 3 and 4 MB patients with, and without disseminated disease. The 2 main hypotheses for this study are firstly, selected CSF cytokines will be differentially expressed between metastatic Group 3 and 4 MB patients versus non-metastatic Group 3 and 4 patients. Next, there will be different cytokine expressions between metastatic Group 3 and 4 MB, on the basis that these MB subtypes are molecularly distinct from each other.

Metastatic medulloblastoma (MB) portends an extremely poor prognosis. The worst outcomes occur in the subset of patients affected by disseminated MB tumour cells both within and beyond the CNS. Extrapolating findings from other human cancers, we are now aware that human body’s response to cancer parallels inflammation, and inflammatory cells and cytokines present in tumours are more likely to contribute to tumour growth and progression [17].

One of these culprit cytokines is CCL2, a pro-inflammatory mediator and chemoattractant. It has a critical role in recruiting macrophages and hence, promoting tumour cell survival [18, 19]. CCL2 is a low molecular weight cytokine with chemoattractant activity. As a prototypic chemokine, CCL2 orchestrates immune cell recruitment to specific sites. It is expressed constitutively for homeostatic functions such as regulating lymphocyte trafficking from blood to lymph nodes. Also, CCL2 is induced during inflammatory responses when leukocytes are required for tissue defence and repair [20]. Recently, Garzia et al. implicates a chemokine signalling axis involving C-C motif ligand 2 (CCL2) and its receptor C-C chemokine receptor type 2 (CCR2) as key players in the haematogenous spread of MB tumour cells [21]. Their pre-clinical study further notes that MYC-amplified Group 3 MB allograft mouse tumours have a higher tendency to express CCL2 [21]. Separately, the MYC gene is a well-characterized oncogene that drives tumorigenesis and metastasis [22], and the mechanisms by which MYC regulates transcription factors involved in metastasis-related processes such as cell migration, and invasion are well documented [22‐24]. Despite their findings, we remain uncertain of the mechanisms driving metastases in MB and, CCL2 is not yet clinically demonstrated in patients with metastatic MB at the time of this writing.

At this point, the authors emphasize that the 3 cases of Group 3 metastatic MB are highlighted for the following reasons. Despite their tumours having the same molecular subtype, each of them demonstrates distinct differences in their clinical presentations. Firstly, we observe that higher expression of CCL2 at the time of diagnosis do not reflect the radiological burden of disease in Patient C, versus the rest of the cohort who already have neural metastases at presentation. Furthermore, his CSF cytology was negative. For him, the initial location of disease progression is in the bone marrow while the brain and spine remain radiologically clear of tumour. It is only later that subsequent imaging showed extensive intracranial and leptomeningeal spread. As we are aware, extraneural MB metastases are infrequent [5, 6, 25, 26] and this clinical scenario suggests a haematogenous route, followed by CNS involvement during disease relapse. Here, our study findings suggest that the expression of CCL2 in CSF parallels the existence of metastases (at diagnosis or delayed), and is neither dependent on molecular subtype (that is, Group 3 or 4) nor MYC-amplification (within Group 3 subtypes). In addition, CSF CCL2 expression does not correlate to CSF cytology for our patient cohort.

In corroboration with Garzia et al, our study observes higher CCL2 CSF expression in the metastatic MB patients, especially for the MYC-amplified Group 3 MB. [21]. Putting it all together, our observations potentially justify a role for firstly, further investigation of CCL2 in metastatic MB from a translational perspective; and next, mechanistic exploration of CCL2 in MYC-amplified Group 3 MB, a subset of MB patients with very poor survival despite good surgical cytoreduction and adjuvant treatment.

At the same time, concurrently increased expressions of other cytokines in the CSF results in our study cohort are demonstrated. For example, TIMP1 and TIMP2 expressions, especially the former, are shown to correlate with CCL2 in patients with metastatic Group 3 and 4 MB. Although TIMPs are originally characterized as inhibitors of MMPs, we are now aware that TIMPs have broader biological functions, including effects on cell migration, angiogenesis [27, 28] and cancer progression [29]. The high expression of TIMP1 has also been correlated with adverse prognosis in breast carcinoma [30]. In addition, for the 2 Group 3, MYC-amplified MB patients, interval expression changes in CXCL1 concurrently with IL6 and IL8 are seen. These 3 cytokines have previously been implicated in other tumours as negative prognostic markers [31‐34]. Despite these intriguing observations, whether these selected cytokines contribute specifically to the process of MB metastasis needs to be investigated further. With the exception of CCL2, the other cytokines show variable expression at different times of each patient’s disease. The authors postulate that such findings may be a reflection of intertumoral heterogeneity, even within the same molecular subtype.

In summary, we report higher expression of CCL2 in the CSF samples of Group 3 and 4 metastatic MB versus non-metastatic Group 3 and 4 MB in our study cohort. As far as we know, this is the first CSF biomarker to be clinically demonstrated in metastatic MB patients. Furthermore, we show that selected cytokines similarly demonstrate higher expression in the subset of Group 3 MB patients. The authors advocate in-depth studies in a larger population, and international collaborative efforts for better understanding of this devastating disease.