High Myc pathway activity and low stage of neuronal differentiation associate with poor outcome in neuroblastoma

Abstract

The childhood cancer neuroblastoma arises in the developing sympathetic nervous system and is a genotypically and phenotypically heterogeneous disease. Prognostic markers of poor survival probability include amplification of the MYCN oncogene and an undifferentiated morphology. Whereas these features discriminate high- from low-risk patients with precision, identification of poor outcome low- and intermediate-risk patients is more challenging. In this study, we analyze two large neuroblastoma microarray datasets using a priori-defined gene expression signatures. We show that differential overexpression of Myc transcriptional targets and low expression of genes involved in sympathetic neuronal differentiation predicts relapse and death from disease. This was evident not only for high-risk patients but was also robust in identifying groups of poor prognosis patients who were otherwise judged to be at low- or intermediate-risk for adverse outcome. These data suggest that pathway-specific gene expression profiling might be useful in the clinic to adjust treatment strategies for children with neuroblastoma.

Fig. 1.

Myc-pathway activation correlates to clinical stage, risk assessment, and patient outcome in neuroblastoma. (A and B) Pathway activity (PA) scores for a Myc gene expression signature (up- and down-regulation) in a microarray dataset of 251 neuroblastoma specimens (13) (Oberthuer) correlate to MYCN amplification (*, P < 0.001) (A), but not specifically to MYCN gene copy number (B). (C) High Myc pathway activity score correlates to advanced INSS stage (#, P < 0.001 vs. INSS 3, *, P < 0.01; **, P < 0.001 vs. INSS 4). (D) Myc signaling correlates to high COG risk (*, P < 0.001 vs. COG high risk tumors). (D). Data (A, C and D) are presented as means (open circle) ± two SEM. All P values relate to two-sided Student's t tests. (E and F) Categorization of tumors into groups, based on quartiles of Myc-up-regulated pathway activity scores only, shows that increasing strength of Myc signaling proportionally correlates to adverse patient outcome, when scoring either event-free survival (EFS) (P < 0.001, log-rank test) (E), or overall survival (OS) (P < 0.001, log-rank test) (F). These results were corroborated in an independent dataset of 101 tumors (15) (Wang) (EFS, P < 0.001, and OS, P < 0.001, log-rank tests) (G and H). (I) Cox's proportional hazards models (EFS) show for both datasets that Myc up-regulated signaling is a prognostic factor independently of MYCN amplification status.

Fig. 2.

Stage of differentiation correlates to neuroblastoma patient outcome. (A) Pathway activity (PA) scores, calculated over a gene expression signature for late sympathetic neuronal differentiation correlate to low INSS stage in a dataset representing 251 neuroblastoma cases (13) (Oberthuer) (#, P = 0.005; ##, P = 0.001 vs. INSS 3, *, P < 0.01; **, P < 0.001 vs. INSS 4). Presented as means (open circles) ± two SEM. (B) Late differentiation and Myc-up-regulated (Myc-up) signaling pathway activity scores correlate negatively. Blue circles: MYCN-non amplified tumor. Red triangles: MYCN-amplified tumor. (C and D) Categorization of tumors into groups, based on late differentiation pathway activity score quartiles, shows a correlation between decreasing stage of neuronal differentiation and adverse patient outcome scored as either event-free survival (EFS) (P < 0.001, log-rank test) (C) or overall survival (OS) (P < 0.001, log-rank test) (D). (E and F) This result was validated in an independent dataset of 101 tumors (15) (Wang) (EFS, P < 0.001, and OS, P < 0.001, log-rank tests).

Fig. 3.

Myc-signaling and stage of differentiation define patient subgroups with diverging clinical outcome. Pathway activity (PA) scores for Myc up-regulated signaling (Myc-up) and stage of neuronal differentiation (Late diff.) were used in hierarchical cluster analyses (Euclidian distance, average linkage clustering) of the Oberthuer (A–C) and Wang (D and E) datasets. Identified clusters were used for Kaplan–Meier analyses of event-free survival (EFS) and overall survival (OS) for the Oberthuer (B and C) and Wang (E and F) datasets respectively. Annotations are divided into INSS stage (1, 2, 3, 4, and 4S), MYCN amplification, COG risk (low, intermediate, high), outcome (diseased), and event (recurrence). Each tumor is represented by a filled black box when called into that annotation category. Missing values (Wang) are represented by a diagonal.

Fig. 4.

Myc-signaling and stage of differentiation correlate to outcome also for low- and intermediate-risk patients. Sample quartiles, based on the entire 251 patient Oberthuer cohort (13), for either Myc up-regulated signaling (distribution: 1^st quartile: n = 58; 2^nd 50; 3^rd 47; 4^th 22) (A and B) or late differentiation (distribution: 1^st quartile n = 20; 2^nd 42; 3^rd 59; 4^th 56) (C and D) were used for Kaplan–Meier analyses of the 177 low and intermediate risk tumors. (A) P < 0.001, (B) P < 0.001, (C) P = 0.02, (D) P = 0.09, log-rank tests. (E) Cox's proportional hazards models (EFS) by using high/low INSS stage, COG risk, MYCN amplification status, and either Myc-up-regulated or late differentiation quartile groups.

Fig. 5.

Myc signaling and stage of differentiation defines patient subgroups with diverging clinical outcome in the cohort of low- and intermediate-risk patients only. Pathway activity scores for Myc-induced signaling (Myc-up) and stage of neuronal differentiation (Late diff.) were used in hierarchical cluster analyses (Euclidian distance, average linkage clustering) of the Oberthuer low to intermediate risk tumors (n = 177) (A). Identified clusters were used for Kaplan–Meier analyses of event-free survival (EFS) and overall survival (OS) for the Oberthuer (B and C). Annotations are divided into INSS stage (1, 2, 3, 4, and 4S), MYCN amplification, COG risk (low, intermediate), outcome (diseased), and event (recurrence). Each tumor is represented by a filled black box when called into that annotation category.