Analysis of 1p/19q-codeletion

Array CGH

DNA was extracted from fresh frozen tumor material by phenol-chloroform extraction according to standard protocols. Array CGH was performed on Agilent’s SurePrint G3 Human CGH Microarray Kit 2x400K (Design ID 021850, Agilent, Santa Clara, CA, USA) in keeping with the manufacturer’s protocol, but inversely using dyes assigned for sample and reference (Hackmann et al. 2012; doi: 10.1038/ejhg.2012.97.). Scanning was carried out on an Agilent microarray scanner and raw data were processed by Feature Extraction 9.5. Deleted and amplified regions were determined on Agilent’s Genomic Workbench Standard Edition 5.0.14.

Fluorescence In Situ Hybridization (FISH)

FISH analyses for identification of 1p/19q-codeletion were carried out on 10 µm sections of fresh frozen tumor tissue using dual color probes for 1p36/1q25 and 19q13/19p13 (ZytoVision GmbH, Bremerhaven, Germany) as previously described (Klink et al. 2010, Anal Cell Pathol (Amst). 2010;33(1):37-54). Locus specific probes on 1q25 and 19p13 were used as ploidy control. Signal enumeration, scoring and cutoff level determination were done as previously described (Klink et al. 2010, Anal Cell Pathol (Amst). 2010;33(1):37-54).

Results:

Since 1p/19q-codeletion is a frequent and important prognostic marker particularly in oligodendrogliomas and mixed oligoastrocytomas, we investigated the 1p/19q-status only in histologically proofed tumors with oligodendroglial component.

In our population, 32 patients initially had oligoastrocytomas WHO grade II; three patients had oligodendrogliomas WHO grade II, whereas the remaining 64 patients had diffuse astrocytomas.The 1p/19q-status of our patients with tumors harboring oligodendroglial components is shown in Table 5.

Histology / Gender f/m / IDH mutation / 1p19q codeletion / Methylated MGMT promoter
Pat 1 / OA / m / yes / yes / yes
Pat 2 / OA / f / yes / yes / yes
Pat 3 / OA / f / yes / yes / yes
Pat 4 / OD / f / yes / yes / yes
Pat 5 / OA / f / yes / yes / yes
Pat 6 / OA / m / yes / yes / no
Pat 7 / OA / m / yes / yes / no
Pat 8 / OD / m / yes / yes / yes
Pat 9 / OA / m / yes / yes / no
Pat 10 / OA / m / yes / no / yes
Pat 11 / OA / f / yes / yes / no
Pat 12 / OA / m / yes / yes / yes
Pat 13 / OD / m / yes / yes / yes
Pat 14 / OA / m / yes / yes / yes
Pat 15 / OA / m / yes / yes / yes
Pat 16 / OA / f / yes / no / yes
Pat 17 / OA / m / yes / yes / yes
Pat 18 / OA / m / yes / yes / yes
Pat 19 / OA / m / yes / no / yes
Pat 20 / OA / m / yes / yes / yes
Pat 21 / OA / f / yes / no / yes
Pat 22 / OA / f / yes / yes / yes
Pat 23 / OA / m / yes / no / no
Pat 24 / OA / m / yes / yes / yes
Pat 25 / OA / m / yes / no / no
Pat 26 / OA / m / no / yes / no
Pat 27 / OA / m / no / yes / yes
Pat 28 / OA / f / yes / yes / yes
Pat 29 / OA / f / no / no / no
Pat 30 / OA / m / yes / yes / yes
Pat 31 / OA / m / no / yes / no
Pat 32 / OA / f / yes / no / no
Pat 33 / OA / m / no / yes / no
Pat 34 / OA / f / yes / yes / no
Pat 35 / OA / m / yes / no / yes

Table S1: Characteristics of patients with tumors harboring oligodendroglial components in relation to their IDH mutation-, 1p/19q-codelation- and MGMT-promoter status.

All patients
n= 99 / Methylated MGMT promoter
n=58 / Unmethylated MGMT promoter
n= 41 / p-value
Histology precursor LGG WHO II[No. (%)]
Diffuse astrocytoma / 64 / (64.7%) / 34 / (53.1%) / 30 / (46.8%) / .129
Oligoastrocytomas/oligodendrogliomas / 35 / (35.3%) / 24 / (68.6%) / 11 / (31.4%)
Gender[No. (%)]
male / 54 / (55.7%) / 34 / (63.0%) / 20 / (37.0%) / .535
Female / 43 / (44.3%) / 24 / (55.8%) / 19 / (44.2%)
Course of malignization[No. (%)]
Anaplastic gliomas / 48 / (49.5%) / 31 / (64.6%) / 17 / (35.4%) / .109
Anaplastic gliomas /Glioblastoma / 25 / (25.8%) / 17 / (68.0%) / 8 / (32.0%)
Secondary Glioblastoma / 24 / (24.7%) / 10 / (41.7%) / 14 / (58.3%)
Secondary Glioblastoma
Age at malignancyyears [Median (Range)] / 44 / (23-77) / 45 / (23-71) / 42 / (30-77) / .843
Anaplastic gliomas / 45 / (26-71) / 46 / (26-71) / 39 / (30-70)
Anaplastic gliomas /Glioblastoma / 38 / (26-70) / 40 / (26-70) / 37 / (34-55)
Secondary Glioblastoma / 47 / (23-77) / 42 / (23-69) / 47 / (31-77)
Time to malignancyyears [Median (Range)] / 3.7 / (0.6-18.6) / 4.4 / (0.6-18.6) / 3.3 / (0.6-17.4) / .036
Anaplastic gliomas / 3.7 / (0.7-18.6) / 4.8 / (0.7-18.6) / 2.1 / (0.6-8.5)
Anaplastic gliomas /Glioblastoma / 4.1 / (2.2-12.6) / 4.3 / (2.2-12.6) / 3.7 / (2.3-7.9)
Secondary Glioblastoma / 3.4 / (0.6-17.4) / 3.6 / (0.6-11.6) / 3.3 / (0.7-17.4)
No. of OP since malignancy[No. (%)]
one / 77 / (79.4%) / 46 / (59.7%) / 31 / (40.3%) / 1.0
>1 / 20 / (20.6%) / 12 / (60.0%) / 8 / (40.0%)
Anaplastic gliomas / 0 / (0%) / <.001
Anaplastic gliomas /Glioblastoma / 16 / (64%)
Secondary Glioblastoma / 4 / (16.7%)
IDH-status[No. (%)]
mutated / 74 / (76.3%) / 47 / (63.5%) / 27 / (36.5%) / .226
Non-mutated / 23 / (23.7%) / 11 / (47.8%) / 12 / (52.2%)
Therapy after malignancy[No. (%)]
None / 4 / (4.1%) / 2 / 2 / .882
Radiotherapy and chemotherapy / 49 / (50.5%) / 31 / (63.3%) / 18 / (36.7%)
chemotherapy / 22 / (22.7%) / 13 / (59.1%) / 9 / (40.9%)
Radiotherapy / 22 / (22.7%) / 12 / (54.5%) / 10 / (45.5%)

Table S2: Comparison between the two groups of patients with secondary high-grade gliomas according to their MGMT promoter status in relation to clinical and molecular parameters. OP: operation. LGG: low-grade gliomas