Loss of NOTCH2 positively predicts survival in subgroups of human glial brain tumors

Abstract

The structural complexity of chromosome 1p centromeric region has been an obstacle for fine mapping of tumor suppressor genes in this area. Loss of heterozygosity (LOH) on chromosome 1p is associated with the longer survival of oligodendroglioma (OD) patients. To test the clinical relevance of 1p loss in glioblastomas (GBM) patients and identifiy the underlying tumor suppressor locus, we constructed a somatic deletion map on chromosome 1p in 26 OG and 118 GBM. Deletion hotspots at 4 microsatellite markers located at 1p36.3, 1p36.1, 1p22 and 1p11 defined 10 distinct haplotypes that were related to patient survival. We found that loss of 1p centromeric marker D1S2696 within NOTCH2 intron 12 was associated with favorable prognosis in OD (P = 0.0007) as well as in GBM (P = 0.0175), while 19q loss, concomitant with 1p LOH in OD, had no influence on GBM survival (P = 0.918). Assessment of the intra-chromosomal ratio between NOTCH2 and its 1q21 pericentric duplication N2N (N2/N2N-test) allowed delineation of a consistent centromeric breakpoint in OD that also contained a minimally lost area in GBM. OD and GBM showed distinct deletion patterns that converged to the NOTCH2 gene in both glioma subtypes. Moreover, the N2/N2N-test disclosed homozygous deletions of NOTCH2 in primary OD. The N2/N2N test distinguished OD from GBM with a specificity of 100% and a sensitivity of 97%. Combined assessment of NOTCH2 genetic markers D1S2696 and N2/N2N predicted 24-month survival with an accuracy (0.925) that is equivalent to histological classification combined with the D1S2696 status (0.954) and higher than current genetic evaluation by 1p/19q LOH (0.762). Our data propose NOTCH2 as a powerful new molecular test to detect prognostically favorable gliomas.

Figure 1. Glioma patients with centromeric 1p allelic loss show better survival.

(A) Deletion patterns on chromosome 1p in GBM and OD. Somatic deletion mapping in 118 GBM and 26 OD WHO grades II and III was performed using 43 microsatellite markers. Four markers at deletion hotspots (D1S2845 at 1p36.3, D1S507 at 1p36.1, D1S216 at 1p22 and D1S2696 at 1p11) were selected to define chromosome 1p haplotypes. In GBM, 10 haplotypes were grouped into tumors with centromeric (H8–H10), interstitial (H5–H7) and telomeric (H2–H4) deletion patterns. In OD, only haplotypes H1 and H10 were observed. Chromosomal positions of 1p markers are shown on the left and areas of allelic loss are shown in grey. (B) Kaplan-Meier cumulative survival curve of the haplotype groups.

Figure 2. Added value of chromosomes 1p and 19q molecular markers on glioma patient survival.

D1S2696 is the best chromosome 1p discriminator for better survival of GBM patients.

Figure 3. Refined somatic 1p deletion mapping in OD and GBM converge to the NOTCH2 gene.

(A) Schematic drawing of the chromosome 1 pericentric duplication and principle of the PCR-based N2/N2N test. NOTCH2 markers are in black and N2N markers are in grey. (B) The N2/N2N test distinguishes GBM (left) from OD (right). Electrophoretograms of GBM with (G10 and 091) or without (G49) 1p loss always show N2/N2N balance while OD have reduced NOTCH2 copy number relative to N2N. In GBM, NOTCH2 and N2N copy numbers indicated underneath are deduced from allelic retention (G10) or loss (091) at marker 210WF10. AO80 and AO84 are two recent OD with 1p/19q loss not included in the initial statistics. (C) Refined somatic deletion mapping deduced from the N2/N2N test. Chromosomal positions of markers and pericentric duplication are shown on the left. In GBM, minimally lost area is distally delimited by marker D1S514 (GBM G40 and G01) and proximally by NOTCH2 exon 4 (GBM G10). In OD, the minimal centromeric boundary of 1p loss is given by NOTCH2 exon 1 (all OD), while 2 homozygous losses target Notch2 exon 4 (OD AO80 and AO84). Conventional LOH data are shown by circles, data from the N2/N2N test are shown by squares. White: retention of both alleles; black: loss of heterozygosity; black on a black background: homozygous loss; grey: non informative. Areas of minimal allelic loss, highlighted in grey, are aligned with the targeted gene NOTCH2 shown with its sense of transcription.

Figure 4. The N2/N2N test provides the highest accuracy to predict glioma patient survival.

Receiver Operating Characteristics (ROC) curves indicating the specificities (or 1-specificities, respectively) and the corresponding sensitivities at continuously varying cut-off points for survival time. Thus, a given cut-off (particular survival time), the test result was determined for all individual tests (based on either molecular markers D19S589, D1S2845, D1S216, D1S2696, N2/N2N, or histology) as being true or false positive, or true or false negative, respectively. Based on these data, the specificities (or 1-specificities, respectively) and sensitivities calculated for each of the cut-off points.