Intratumoral diversity of telomere length in individual neuroblastoma tumors

Abstract

The purpose of the work was to investigate telomere length (TL) and mechanisms involved in TL maintenance in individual neuroblastoma (NB) tumors. Primary NB tumors from 102 patients, ninety Italian and twelve Spanish, diagnosed from 2000 to 2008 were studied. TL was investigated by quantitative fluorescence in situ hybridization (IQ-FISH) that allows to analyze individual cells in paraffin-embedded tissues. Fluorescence intensity of chromosome 2 centromere was used as internal control to normalize TL values to ploidy. Human telomerase reverse transcriptase (hTERT) expression was detected by immunofluorescence in 99/102 NB specimens.The main findings are the following: 1) two intratumoral subpopulations of cancer cells displaying telomeres of different length were identified in 32/102 tumors belonging to all stages. 2) hTERT expression was detected in 99/102 tumors, of which 31 displayed high expression and 68 low expression. Alternative lengthening of telomeres (ALT)-mechanism was present in 60/102 tumors, 20 of which showed high hTERT expression. Neither ALT-mechanism nor hTERT expression correlated with heterogeneous TL. 3) High hTERT expression and ALT positivity were associated with significantly reduced Overall Survival. 4) High hTERT expression predicted relapse irrespective of patient age. Intratumoral diversity in TL represents a novel feature in NB.In conclusion, diversity of TL in individual NB tumors was strongly associated with disease progression and death, suggesting that these findings are of translational relevance. The combination of high hTERT expression and ALT positivity may represent a novel biomarker of poor prognosis that deserves further investigation.

Keywords: ALT mechanism; neuroblastoma; telomerase; telomere length.

Figure 1. A: Interphase Quantitative Fluorescence Hybridization (IQ-FISH) using pan-telomere (red) and chromosome 2 centromeric (green) peptide nucleic acid (PNA) probes in paraffin embedded NB tissue section

The nuclei are stained with DAPI (blue). Magnification x100. B: Calibration of IQ-FISH for TL measurements using four NB cell lines (IMR32, SHSY-5Y, GILIN and HTLA-230) and fetal adrenal medulla as long telomere controls, HeLa and MCF-7 cell lines as short telomere controls, peripheral blood mononuclear cells from adult healthy donors as well as adult adrenal medulla as normal telomere controls. We defined the minimum and maximum cut-off values of fluorescence ratio units (FRU) as 412 and 503, respectively. C: FRU values analyzed by two readers by means of the Bland and Altman's plot. Except for four data points (4/141; 2.8%) all values fell within 95% of the limits of agreement.

Figure 2. A: Kaplan-Meier Event-Free Survival curve for five patient groups based upon telomere length (TL)

B: Kaplan-Meier Overall Survival curve for five groups based upon TL.

Figure 3. A: ALT positive NB cells showing ALT-associated bright intra-nuclear foci of telomere FISH signals (red) (arrows)

Nuclear DNA was counterstained with DAPI (blue). B: Immunofluorescence nuclear labeling for the catalytic subunit of telomerase hTERT (green). C: Variance analysis of TL in relation to presence or absence of ALT and to high or low hTERT expression. D: Kaplan-Meier Event-Free Survival curve for long/normal TL and hTERT expression. E: Kaplan-Meier Event-Free Survival curve for short TL and hTERT expression.

Figure 4. A: Kaplan-Meier Overall Survival curve for ALT positivity/negativity and hTERT high/low expression

B: Kaplan-Meier Event-Free Survival curve for hTERT high/low expression and age at diagnosis (> or ≤18 months).