Limits...
Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma.

Shao H, Chung J, Lee K, Balaj L, Min C, Carter BS, Hochberg FH, Breakefield XO, Lee H, Weissleder R - Nat Commun (2015)

Bottom Line: MGMT (O(6)-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy.We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients.We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, Massachusetts 02114, USA.

ABSTRACT
Real-time monitoring of drug efficacy in glioblastoma multiforme (GBM) is a major clinical problem as serial re-biopsy of primary tumours is often not a clinical option. MGMT (O(6)-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy. Yet, serial clinical analysis remains difficult, and, when done, primarily relies on promoter methylation studies of tumour biopsy material at the time of initial surgery. Here we present a microfluidic chip to analyse mRNA levels of MGMT and APNG in enriched tumour exosomes obtained from blood. We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients. We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.

No MeSH data available.


Related in: MedlinePlus

Analysis of clinical samples.(a) Measurement of mRNA for EPHA2, EGFR, PDPN in serum samples. GBM patients (n=17) generally showed higher levels of markers compared with healthy controls (n=15). (b) Correlation of exosomal MGMT mRNA against tumour tissue methylation status. Tissue methylation correlates inversely with exosomal MGMT copy number. The mRNA levels were significantly higher (*P<0.001; Tukey's multiple comparison test) in GBM patients with negative tissue MGMT methylation, than that of patients with positive methylation or healthy controls. The latter categories were non-distinguishable (NS) from each other. (c) Longitudinal exosomal MGMT and APNG mRNA analyses were performed in seven GBM patients; two representative examples are shown (see Supplementary Fig. 15 for other patients). Clinical assessments (NR, S, R) were based on radiological findings, clinical examination and lab values. All changes were plotted as mean±s.d. (d) Sequential exosomal mRNA changes between two time points were analysed in GBM patients (n=7) undergoing TMZ treatment. All changes were normalized to their initial values and plotted according to clinical evaluation at the end of the assessment period (the later time point). All changes were independent of initial tissue MGMT methylation status.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4430127&req=5

f5: Analysis of clinical samples.(a) Measurement of mRNA for EPHA2, EGFR, PDPN in serum samples. GBM patients (n=17) generally showed higher levels of markers compared with healthy controls (n=15). (b) Correlation of exosomal MGMT mRNA against tumour tissue methylation status. Tissue methylation correlates inversely with exosomal MGMT copy number. The mRNA levels were significantly higher (*P<0.001; Tukey's multiple comparison test) in GBM patients with negative tissue MGMT methylation, than that of patients with positive methylation or healthy controls. The latter categories were non-distinguishable (NS) from each other. (c) Longitudinal exosomal MGMT and APNG mRNA analyses were performed in seven GBM patients; two representative examples are shown (see Supplementary Fig. 15 for other patients). Clinical assessments (NR, S, R) were based on radiological findings, clinical examination and lab values. All changes were plotted as mean±s.d. (d) Sequential exosomal mRNA changes between two time points were analysed in GBM patients (n=7) undergoing TMZ treatment. All changes were normalized to their initial values and plotted according to clinical evaluation at the end of the assessment period (the later time point). All changes were independent of initial tissue MGMT methylation status.

Mentions: The exosomal levels of gene product diagnostic markers (EPHA2, EGFR and PDPN) were heterogeneous among clinical blood samples (Fig. 5a). No detectable EGFR mRNA was found in some GBM patients (n=7) despite the fact that we used EGFR protein for GBM enrichment (see Supplementary Fig. 13); such discrepancies between exosomal mRNA and protein levels have been observed in other tumour studies as well14. The average levels of EPHA2 and EGFR were significantly higher (P<0.05; two-tailed t-test) in GBM patients (n=17) than in healthy controls (n=15); PDPN level was similar in both groups (P=0.15; Supplementary Fig. 14a). Using a single marker alone, the iMER assay correctly identified GBM cases at the accuracies of 84.4% (EPHA2) and 78.1% (EGFR). The accuracy increased to 90% when the diagnosis was based on combined markers (Supplementary Fig. 14b).


Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma.

Shao H, Chung J, Lee K, Balaj L, Min C, Carter BS, Hochberg FH, Breakefield XO, Lee H, Weissleder R - Nat Commun (2015)

Analysis of clinical samples.(a) Measurement of mRNA for EPHA2, EGFR, PDPN in serum samples. GBM patients (n=17) generally showed higher levels of markers compared with healthy controls (n=15). (b) Correlation of exosomal MGMT mRNA against tumour tissue methylation status. Tissue methylation correlates inversely with exosomal MGMT copy number. The mRNA levels were significantly higher (*P<0.001; Tukey's multiple comparison test) in GBM patients with negative tissue MGMT methylation, than that of patients with positive methylation or healthy controls. The latter categories were non-distinguishable (NS) from each other. (c) Longitudinal exosomal MGMT and APNG mRNA analyses were performed in seven GBM patients; two representative examples are shown (see Supplementary Fig. 15 for other patients). Clinical assessments (NR, S, R) were based on radiological findings, clinical examination and lab values. All changes were plotted as mean±s.d. (d) Sequential exosomal mRNA changes between two time points were analysed in GBM patients (n=7) undergoing TMZ treatment. All changes were normalized to their initial values and plotted according to clinical evaluation at the end of the assessment period (the later time point). All changes were independent of initial tissue MGMT methylation status.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4430127&req=5

f5: Analysis of clinical samples.(a) Measurement of mRNA for EPHA2, EGFR, PDPN in serum samples. GBM patients (n=17) generally showed higher levels of markers compared with healthy controls (n=15). (b) Correlation of exosomal MGMT mRNA against tumour tissue methylation status. Tissue methylation correlates inversely with exosomal MGMT copy number. The mRNA levels were significantly higher (*P<0.001; Tukey's multiple comparison test) in GBM patients with negative tissue MGMT methylation, than that of patients with positive methylation or healthy controls. The latter categories were non-distinguishable (NS) from each other. (c) Longitudinal exosomal MGMT and APNG mRNA analyses were performed in seven GBM patients; two representative examples are shown (see Supplementary Fig. 15 for other patients). Clinical assessments (NR, S, R) were based on radiological findings, clinical examination and lab values. All changes were plotted as mean±s.d. (d) Sequential exosomal mRNA changes between two time points were analysed in GBM patients (n=7) undergoing TMZ treatment. All changes were normalized to their initial values and plotted according to clinical evaluation at the end of the assessment period (the later time point). All changes were independent of initial tissue MGMT methylation status.
Mentions: The exosomal levels of gene product diagnostic markers (EPHA2, EGFR and PDPN) were heterogeneous among clinical blood samples (Fig. 5a). No detectable EGFR mRNA was found in some GBM patients (n=7) despite the fact that we used EGFR protein for GBM enrichment (see Supplementary Fig. 13); such discrepancies between exosomal mRNA and protein levels have been observed in other tumour studies as well14. The average levels of EPHA2 and EGFR were significantly higher (P<0.05; two-tailed t-test) in GBM patients (n=17) than in healthy controls (n=15); PDPN level was similar in both groups (P=0.15; Supplementary Fig. 14a). Using a single marker alone, the iMER assay correctly identified GBM cases at the accuracies of 84.4% (EPHA2) and 78.1% (EGFR). The accuracy increased to 90% when the diagnosis was based on combined markers (Supplementary Fig. 14b).

Bottom Line: MGMT (O(6)-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy.We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients.We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, Massachusetts 02114, USA.

ABSTRACT
Real-time monitoring of drug efficacy in glioblastoma multiforme (GBM) is a major clinical problem as serial re-biopsy of primary tumours is often not a clinical option. MGMT (O(6)-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy. Yet, serial clinical analysis remains difficult, and, when done, primarily relies on promoter methylation studies of tumour biopsy material at the time of initial surgery. Here we present a microfluidic chip to analyse mRNA levels of MGMT and APNG in enriched tumour exosomes obtained from blood. We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients. We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.

No MeSH data available.


Related in: MedlinePlus