Fluorescence Lifetime Imaging Microscopy, a Novel Diagnostic Tool for Metastatic Cell Detection in the Cerebrospinal Fluid of Children with Medulloblastoma

Abstract

In pediatric brain tumours, dissemination of malignant cells within the central nervous system confers poor prognosis and determines treatment intensity, but is often undetectable by imaging or cytology. This study describes the use of fluorescence lifetime (FLT) imaging microscopy (FLIM), a novel diagnostic tool, for detection of metastatic spread. The study group included 15 children with medulloblastoma and 2 with atypical teratoid/rhabdoid tumour. Cells extracted from the tumour and the cerebrospinal fluid (CSF) 2 weeks postoperatively and repeatedly during chemo/radiotherapy were subjected to nuclear staining followed by FLT measurement and cytological study. Control CSF samples were collected from patients with infectious/inflammatory disease attending the same hospital. Median FLT was prolonged in tumour cells (4.27 ± 0.28 ns; P < 2.2*10^-16) and CSF metastatic cells obtained before chemo/radiotherapy (6.28 ± 0.22 ns; P < 2.2*10^-16); normal in inflammatory control cells (2.6 ± 0.04 ns) and cells from children without metastasis before chemo/radiotherapy (2.62 ± 0.23 ns; P = 0.858) and following treatment (2.62 ± 0.21 ns; P = 0.053); and short in CSF metastatic cells obtained after chemo/radiotherapy (2.40 ± 0.2 ns; P < 2.2*10^-16). FLIM is a simple test that can potentially identify CSF spread of brain tumours. FLT changes in accordance with treatment, with significant prolonged median values in tumours and metastases. More accurate detection of metastatic cells may guide personalised treatment and improve the therapeutic outcome.

Figure 1

(a) Distribution of the FLT (ns) measured for each slide. Patients were categorised according to stage of treatment. The control group contains slides from patient with inflammatory disease. (b) Distribution of the fluorescence lifetime (ns) in each source group. (c) Hierarchical clustering (average, 1-correlation) for each source group of patients. (Patient numbers and groups are keyed to Tables 1 and 2 respectively). An image of a cell produced by the FLIM system (d) from a group B patient with a FLT of 4.8 ± 0.43 ns, and (e) from a group C patient with a FLT of 1.17 ± 0.3 ns.

Figure 2

Fluorescence lifetime for each stage of therapy in 5 patients with medulloblastoma, (a) patient 3, (b) patient 6, (c) patient 7, (d) patient 8, (e) patient 13, and one patient with ATRT (f), patient 10. (Patient numbers are keyed to Tables 1 and 2).

Figure 3

Density plots of fluorescence lifetime findings in tumour cells and CSF cells from patients with (a) metastatic disease, (b) localised disease (c), before chemo/radiotherapy (d), after chemo/radiotherapy.