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Intratumoral diversity of telomere length in individual neuroblastoma tumors.

Pezzolo A, Pistorio A, Gambini C, Haupt R, Ferraro M, Erminio G, De Bernardi B, Garaventa A, Pistoia V - Oncotarget (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio di Oncologia, Istituto Giannina Gaslini, Genova, Italy.

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.

No MeSH data available.


Related in: MedlinePlus

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 sectionThe 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.
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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 sectionThe 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.

Mentions: The IQ-FISH procedure on tissue sections is a suitable approach for the assessment of TL in relation to cell type, and in the context of tissue architecture [30-36]. TL was measured in 102 primary NB tumors by a modified IQ-FISH assay with ploidy correction that showed high sensitivity (0.1 kb of telomere repeats) and accuracy (99%) (Fig 1 A-C). Seventy NB cases (68.6%) displayed homogeneous TL, of which 42 were short, 25 long, and 3 normal. In the remaining 32 NB cases (31.4%), single cell analysis revealed the coexistence in the same tumor of two cancer cell subpopulations with differing TL, and namely i) normal and short telomeres, or ii) long and short telomeres, or iii) normal and long telomeres. These 32 NB cases displaying heterogeneous TL showed stage and age distribution, risk group, favorable or unfavorable histology, frequency of MNA and ploidy comparable to cases with homogeneous TL (Table S1).


Intratumoral diversity of telomere length in individual neuroblastoma tumors.

Pezzolo A, Pistorio A, Gambini C, Haupt R, Ferraro M, Erminio G, De Bernardi B, Garaventa A, Pistoia V - Oncotarget (2015)

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 sectionThe 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480695&req=5

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 sectionThe 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.
Mentions: The IQ-FISH procedure on tissue sections is a suitable approach for the assessment of TL in relation to cell type, and in the context of tissue architecture [30-36]. TL was measured in 102 primary NB tumors by a modified IQ-FISH assay with ploidy correction that showed high sensitivity (0.1 kb of telomere repeats) and accuracy (99%) (Fig 1 A-C). Seventy NB cases (68.6%) displayed homogeneous TL, of which 42 were short, 25 long, and 3 normal. In the remaining 32 NB cases (31.4%), single cell analysis revealed the coexistence in the same tumor of two cancer cell subpopulations with differing TL, and namely i) normal and short telomeres, or ii) long and short telomeres, or iii) normal and long telomeres. These 32 NB cases displaying heterogeneous TL showed stage and age distribution, risk group, favorable or unfavorable histology, frequency of MNA and ploidy comparable to cases with homogeneous TL (Table S1).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio di Oncologia, Istituto Giannina Gaslini, Genova, Italy.

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.

No MeSH data available.


Related in: MedlinePlus