Introduction: Clinical Aspects and Molecular Biology of Neuroblastoma

Neuroblastoma, a tumour derived from neural crest cells, represents the third most common pediatric cancer (accounting for approximately 7-10% of all childhood malignancies) and is the most common solid extracranial neoplasm of infancy and childhood, responsible for approximately 1-5% of all childhood cancer deaths. The clinical hallmark of neuroblastoma is heterogeneity, associated with a wide variety of likelihood of tumor progression. The most significant clinical predictors of outcome are age and stage, although an assessment of the patient's prognosis solely on the basis of clinical parameters is limited due to diverse biological tumor behavior and subsequent survival rates, even at distinct clinical stages.^1,2 Therefore, the heterogeneity of this tumor entity requires cellular and molecular markers in order to distinguish the different biological characteristics. Established molecular markers such as MYCN copy number and loss of heterozygosity for chromosome 1p3236 may help predicting poor outcome.³⁷ Based on the age-related prevalence and distinguishing genetic features of biologically favourable and unfavourable tumours, Brodeur et al⁸ proposed that NB are at least two or three diseases with distinct clinical and biological characteristics (Table 1). However, there is evidence that traditional prognostic parameters, such as analysis of MYCN amplification, which is considered an essential component of disease evaluation and treatment stratification, do not ensure completely accurate prognostic grouping.^9–11 Identifying new prognostic markers can increase the accuracy of risk assessment and can also identify biologically relevant targets for developing new therapies.

Table 1

Telomerase and Telomeres

A promising new marker is telomerase, a multicomponent ribonucleoprotein enzyme, which has been implicated in cell immortalization and tumorigenesis in almost all human tumors, including neuroblastomas.^12–17 Telomerase is a ribonucleoprotein polymerase which uses an internal RNA component as a template to synthesize telomeric DNA directly onto the ends of chromosomes.^18,19 These repetetive sequences are considered to be important in the protection and replication of chromosomes. Cells without telomerase activity display progressive shortening of telomeric repeats with each cell division, because lagging-strand DNA synthesis at the very end of linear chromosomes cannot be completed, a phenomenon generally referred to as the end-replication problem.^20–22 Telomere shortening contributing to genetic instability is believed to be the primary signal for senescence mediated by tumor suppressor genes, such as p53 and Rb.^23–26 Induction of telomerase activity in cells that have bypassed this control mechanism could give rise to clonal immortality by compensating for the loss of telomeric DNA and thus maintaining telomere length.^27,28 Telomere length itself has not proven to be a good indicator of malignancy, since both stabilization and extension caused by mechanisms other than telomerase activation have been reported.^29–34

Recently, the catalytic subunit hTERT (or hTRT, hEST2, TP2) of human telomerase was cloned and detection of hTERT expression by RT-PCR revealed a strong correlation with telomerase activity by the TRAP assay in the majority of tumors so far examined.^35–37 Other components of the the telomerase holoenzyme complex, such as human telomerase RNA (hTR) and telomerase protein 1 (TP1, TLP1, hTEP1) seem to be expressed in both normal and tumor tissues, and expression levels of these genes revealed no or only limited correlation with telomerase activity.^38–40

What Is the Role of Telomerase in Neuroblastomas?

Previous studies proposed that neuroblastomas with low telomerase activity might consist of cells that completely failed to repress telomerase activity during development, whereas tumors with high telomerase activity are likely to be derived from an immortalization event in a single cell.^15,29,41,42 This condition renders clinical implications, since high telomerase activity is supposed to be accompanied by several genetic alterations and poor prognosis, whereas low or even absent telomerase activity in neuroblastomas coincides with good prognosis, and eventually, spontaneous regression.^15,29 Exact prognostic implications on the basis of this two-entity model have so far been hampered by insufficient distinction of activity levels due to positivity for telomerase activity in the vast majority of neuroblastomas.^29,41

In our study, a total of 133 neuroblastomas of all stages were analyzed in blind-trial fashion by a modified nonradioactive and semiquantitative TRAP (telomeric repeat amplification protocol) assay. Telomerase activity (TA) was present in 39 (29%) tumor tissues including 4/24 (17%) stage 4S neuroblastomas, 25/41 (61%) stage 4 neuroblastomas, 8/23 (35%) stage 3 neuroblastomas, 0/13 (0%) stage 2 neuroblastomas, and 2/32 (6%) stage 1 neuroblastomas. Of the 104 tumors resected initially without cytotoxic pretreatment, 31 showed TA (29%). After cytotoxic pretreatment (either initially or after tumor recurrence), TA was present in 8/29 tumors (28%). For 128/133 tumors included in this study, MYCN analysis was available and relevant MYCN amplification >3 copies⁴³ could be observed in 21/128 (16%) samples. Data on LOH analysis of chromosome arm 1p were available for a subset of tumors (Table 2).

Table 2

Correlation of TRAP and Survival for all Stages Combined

The correlation of TA status determined by the TRAP assay with the clinical data of 133 patients included in the German Neuroblastoma Trial was established under simple-blind study conditions to prevent any subjective impact on the estimation of TA levels. Clinical outcome was significantly correlated with the presence of TA distinguishing between TA positive (without further discrimination of low and high grade TA) and negative tumors. For all tumors (n=133), 5-year event-free survival (EFS) for 94 telomerase-negative tumors was 0.86 ± 0.04 versus 0.25 ± 0.08 for 39 telomerase- positive tumors at p<0.0001. 5-year overall survival (OS) for telomerase negative tumors was 0.93 ± 0.03 versus 0.34 ± 0.09 for telomerase positive tumors at p<0.0001. For initial samples (n=104), 5-year EFS for 73 telomerase-negative tumors was 0.92 ± 0.03 versus 0.33 ± 0.10 for 31 telomerase-positive tumors at p<0.0001 (Fig. 1A). 5-year OS for telomerase-negative tumors was 0.97 ± 0.02 versus 0.42 ± 0.10 for telomerase-positive tumors at p<0.0001 (Fig. 1B). Numbers for EFS and OS for localized disease (stage 1,2,3), stage 4, and stage 4S are summarized in Table 2.

Figure 1

(A) Event-free survival (EFS) and (B) overall survival (Survival) in 104 initial neuroblastomas prior to chemotherapy. Telomerase activity (TA) negative, n=73; TA positive, n=31. p<0.0001.

Comparison of TA with MYCN Status

MYCN status was available for a total of 128 patients. The tumors of 107 patients had MYCN copy numbers £3. Patients with TA-positive tumors and MYCN copy numbers £3 in their tumors (n=22) taken before cytotoxic treatment were found to have a 5-year EFS of 0.31 ± 0.11, whereas those with TA-negative tumors (n=85) reached a 5-year EFS rate of 0.87 ± 0.04 (p<0.0001) (Fig. 2).

Figure 2

(A) Event-free survival (EFS) and overall survival (Survival) in relation to the MYCN status in 107 neuroblastomas with MYCN copy numbers £3. TA negative, n=85; TA positive, n=22. p<0.0001.

Catalytic Protein Subunit hTERT

From 75 neuroblastomas, frozen sections were made for extraction of both protein and RNA. For analysis, quantitative amounts of hTERT, hTR and hTEP1 gene expressions standardized on GAPDH expression were grouped on a score system ranging from 07 (see Material & Methods for details). On quantitative RT-PCR analysis on the LightCycler instrument, hTERT expression was found in 37 of 75 (49%) neuroblastomas. For correlating hTERT expression with TA in the TRAP assay, hTERT expression levels of each neuroblastoma sample was compared to hTERT expression of a dilution series (see Material & Methods) of a telomerase-positive (TRAP positive) Ewing tumor cell line. 22/75 (29.3%) neuroblastomas were positive for both telomerase activity and hTERT mRNA and 37/75 (49.3%) were negative for both TA and hTERT mRNA. One neuroblastoma (1.3%) was only positive for TA but not for hTERT mRNA, and 15 (20%) neuroblastomas were only positive for hTERT mRNA, but not for TA. Among the 22 neuroblastomas that were positive for both TA and hTERT mRNA, there was a significant statistical correlation (p<0.001) between quantitative expression levels of TA and hTERT mRNA. That is, 10 samples with intermediate or high TA levels (score 2 or 3 on a scale of 03) displayed intermediate or high hTERT mRNA levels (score 4 on a scale of 07) and, vice versa, 11 samples with low TA levels (score 1) displayed low hTERT mRNA levels (score 2 or 3). A single case of a stage 4 neuroblastoma after cytotoxic pretreatment revealed low TA (score 1) and high hTERT mRNA expression (score 5). Among the 15 samples which were positive for hTERT mRNA but negative for TA, almost all cases (n=14) had hTERT levels that were minimal (score 1). These minimal (score 1) hTERT levels always had values significantly lower than neuroblastomas with low TA and low hTERT. Furthermore, minimal hTERT levels were found in up to 50% of analyzed tumor-free tissues such as normal breast or normal liver (data not shown). However, there was a single case with no TA in TRAP assay which repeatedly displayed high hTERT mRNA levels by RT-PCR (score 4). This sample was from a stage 1 neuroblastoma of a 32-month-old (age at diagnosis) child with relapsefree clinical follow-up for 46 months. Further investigations revealed no Taq polymerase inhibitors in the TRAP assay and no MYCN amplification in this sample. Using cRNA sense and antisense probes for Northern Blots on RNA from a TA-positive Ewing's tumor and neuroblastoma cell line, the specificity of hTERT expression detected by our RT-PCR system was confirmed.

Based on 67 neuroblastomas without prior cytotoxic treatment, 5-year EFS for 37 hTERT negative tumors (score 0) was 0.85 ± 0.06, 1.0 ± 0.0 for 11 tumors with minimal (score 1) hTERT, 0.38 ± 0.17 for 9 tumors with low (score 23) hTERT, and 0.44 ± 0.17 for 10 tumors with intermediate to high (score 4) hTERT at p<0.0001. 5-year OS for 37 hTERT-negative tumors (score 0) was 0.93 ± 0.05, 1.0 ± 0.0 for 11 tumors with minimal (score 1) hTERT, 0.73 ± 0.17 for 9 tumors with low (score 23) hTERT, and 0.42 ± 0.17 for 10 tumors with intermediate to high (score 4) hTERT at p<0.0001.

RNA Component hTR

Positive hTR RNA expression at different levels was found in 74/75 neuroblastomas examined, and expression of hTR mRNA was also significantly correlated with TA (p<0.001). Based on 66 neuroblastomas without prior cytotoxic treatment, 5-year EFS for 34 tumors with negative (n=1) or low hTR expression (n=33) was 0.93 ± 0.05, 0.54 ± 0.11 for 24 tumors with intermediate hTR, and 0.54 ± 0.20 for 8 tumors with high hTR at p=0.0005. 5-year OS for 34 tumors with negative or low hTR was 0.96 ± 0.04, 0.75 ± 0.10 for 24 tumors with intermediate hTR, and 0.54 ± 0.20 for 8 tumors with high hTR at p=0.0012. From 4 of these 75 cases, slides were made from paraffin-embedded tissue for hTR in situ hybridization (Fig. 3), which revealed a strong correlation with both TA and hTR mRNA expression by RT-PCR. Interestingly, in a TA-negative and hTERT-negative (both by RTPCR and immunohistochemistry) neuroblastoma with intermixed differentiated ganglion cells, only the ganglion cells, but not the undifferentiated neuroblastic cells, displayed weak hTR in situ hybridization signals.

Figure 3

Telomerase activity (TRAP assay) and hTR RNA expression (in situ hybridization) in neuroblastomas. Lane A: intermediate telomerase activity; lane B: high telomerase activity; lane C: no telomerase activity.

Telomerase-Associated Protein hTEP1

Positive hTEP1 mRNA expression was found in 90% of the samples, but there was no correlation between TA and HTEP1 expression levels. High HTEP1 mRNA levels were also found in two-thirds of the TA-negative neuroblastomas, and at the same time low HTEP1 mRNA levels were also associated with some TA-positive neuroblastomas with high TA levels.

hTERT mRNA Expression in ParaffinEmbedded Tissues

hTERT expression by RT-PCR from fresh-frozen and paraffin-embedded tissues was compared in 16 selected cases from the archives. For assessing tissue sample preservation, RNA from these 16 formalin-fixed and paraffin-embedded neuroblastoma tissues was initially amplified by RT-PCR (TITAN system, Roche Diagnostics, Mannheim, Germany) for the GAPDH gene. In 11/16 cases, suitable RNA for RT-PCR was available. These 11 cases included five stage 4 neuroblastomas (with intermediate or high TA and hTERT mRNA expression in corresponding fresh-frozen tissue), three stage 3 neuroblastomas (with intermediate or high TA and hTERT mRNA expression in corresponding fresh-frozen tissue) and three stage 1 or 2 neuroblastoma (with neither TA nor hTERT mRNA expression in corresponding fresh-frozen tissue). Comparison of hTERT mRNA expression from the corresponding fresh-frozen tissues revealed similar expression patterns as with the paraffin-embedded tissue. hTERT mRNA expression in these two cases was correlated with fatal outcome, whereas the hTERT mRNA-negative stage 1 neuroblastoma case is relapse and disease free after 54 months of follow-up.

Conclusions: Telomerase Activity as New Molecular Marker for Treatment Stratification and Prognostic Grouping

Telomerase activity was demonstrated to be significantly correlated with poor prognosis in neuroblastoma. In our study according to stage classification, multivariate analysis revealed only TA and stage as independent prognostic factors, whereas serum LDH, MYCN status, and age were not. The high accuracy with which tumors prone to unfavorable outcome can be identified makes TA a reliable prognostic tool in neuroblastoma. Although it is not proven so far that TA will not change under cytotoxic pretreatment, our data suggest that TA analysis is suitable in all neuroblastomas irrespective of their pretreatment status: comparing the number of TA-positive tumors among all neuroblastomas in this study (n=133) vs. the subset of initial samples without pretreatment (n=104), we found no difference between these two groups (39/133 equals 29.3% of all neuroblastomas vs. 31/104 equals 29.8% of initial neuroblastomas).

One particular strength of TA as a prognostic marker is the differentiation of good and poor outcome in putative “favorable” clinical or biological subtypes of NB patients. These include, for example, patients in the age group of 01 years at diagnosis and patients with nonamplified MYCN in their tumors.⁴⁴ For all 63 patients < 1 year of age at diagnosis in this study combined, OS was ∼90% and EFS was ∼80%. However, for 10 of these patients with TA positive tumors, OS dropped down to ∼50%, and EFS down to ∼30%. The only favorable cases in this subset of TA-positive tumors were congenital neuroblastomas with minimal TA. These cases may represent NB derived from neuroblasts which retain the expression of fetal levels of TA because of a failure to repress telomerase, as suggested by Hiyama's NB development model.¹⁷ In the putative favorable group of patients with nonamplified MYCN, TA once again identifies good and poor prognostic subsets: for 86 initial samples without cytotoxic pretreatment, with MYCN < 3 copies, OS was ∼87% and EFS was ∼80%. However, for 20 of these patients with TA-positive tumors, OS dropped down to ∼40% and EFS down to ∼31%.

Above all, the significant correlation of clinical outcome with presence of telomerase activity strongly recommends that the analysis of telomerase activity in individual tumors should be accorded a priority at least equal to that of the determination of established markers, such as MYCN and LOH of chromosome 1p, thereby facilitating risk-directed therapy.⁴⁵

Acknowledgments

The authors thank Christina Scheel, Dr. KarlLudwig Schäfer and Dr. Bernhard Heine for their cooperation in several studies on telomerase activity in neuroblastomas.

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