Full-length telomerase reverse transcriptase messenger RNA is an independent prognostic factor in neuroblastoma

Abstract

Telomerase activity (TA) is the most recently recognized prognostic factor in neuroblastoma, and its outstanding predictive power was documented by several studies. However, TA measurements require fresh tumor tissue that is not always available in daily clinical practice. We previously described a reverse transcriptase-polymerase chain reaction assay that we used to investigate the possible prognostic relevance of the telomerase catalytic subunit, hTERT, at the mRNA level. Because hTERT mRNA undergoes alternative splicing as a regulatory mechanism of TA, we discriminated between truncated and full-length hTERT transcripts. In a retrospective study on 124 neuroblastomas, 56 (45.2%) tumors showed spliced hTERT transcripts, whereas 30 (24.2%) contained full-length hTERT transcripts. The presence of both spliced and full-length hTERT transcripts was significantly associated with MYCN amplification. hTERT in general showed no correlation to other prognostic factors, ie, International Neuroblastoma Staging System stage, International Neuroblastoma Pathology classification grade, or age at diagnosis, whereas the presence of full-length transcripts was significantly associated with higher stages. The presence of any hTERT transcripts carried no significant prognostic information, yet full-length hTERT transcripts were highly predictive of poor outcome (P < 0.0001). In a multivariate analysis, full-length hTERT transcripts and International Neuroblastoma Pathology classification grade emerged as the sole independent predictors of event-free survival, with relative risks of 10.0 and 3.9, respectively. The strong statistical correlation of full-length hTERT transcripts with clinical outcome in neuroblastoma suggests that the reverse transcriptase-polymerase chain reaction analysis of hTERT transcripts may be equatable to TA measurements. Because this assay is well suited for archival material, it could become a useful adjunct in evaluating the prognosis of individual neuroblastoma cases.

Figure 1.

RT-PCR results of L428 cells and two neuroblastomas (Nbl). Positive controls (EWS transcripts in lanes 1, 4, and 7). Lanes 2, 5, and 8 show general (145 bp) and lanes 3, 6, and 9 show full-length (seminested PCR-products, 128 bp) hTERT transcripts. Note the presence of general hTERT transcripts and the lack of full-length hTERT transcripts in neuroblastoma no. 2. M, molecular weight marker.

Figure 2.

Quantitation of hTERT transcripts using Light Cycler chemistry (top) and detection threshold for the assay used in our study (bottom). Two hundred ng of tonsil RNA produced a signal corresponding to 100 copies of hTERT. The other curves show amplification of different copy numbers of hTERT-RNA for calibration of the assay (from left to right: 5 × 10⁵, 4.5 × 10⁴, 4 × 10³, 4 × 10², 4 × 10¹ hTERT copies). Nested RT-PCR (bottom) detects 500 copies (lane 1), 50 copies (lane 2), and 5 copies (lane 3) of hTERT mRNA but yields no signal with one copy (lane 4) or 200 ng of genomic DNA (lane 5).

Figure 3.

Kaplan-Meier analysis of EFS in 124 neuroblastoma patients with respect to general hTERT transcripts (top) and the presence of full-length hTERT (hTERT fl) transcripts (bottom).

Figure 4.

Kaplan-Meier analysis of the impact of hTERT transcripts on EFS in subgroups of neuroblastoma patients defined by the MYCN status (A) and INSS stage (B). To obtain sufficient sample sizes, stages 1, 2, 3, and 4S were grouped and compared to stage 4. sc, single copy; amp, amplified; fl, full-length transcripts; nfl, no full-length transcripts. For A and B, P is <0.0001.