Supplementary Figure and Table Legends
Supplementary Figure 1. Oncoprint showing the clinical and molecular characteristics of 112
IDH-mutant, 1p19q-codeleted oligodendrogliomas.
Supplementary Figure 2. A. 38/F, frontal lobe IDH mutant, 1p19q co-deleted Grade 3
oligodendroglioma, with FISH for ALT. Arrows show microvascular proliferation. B. 61/M,
frontal lobe IDH mutant 1p19q co-deleted oligodendroglioma, Grade 2, with FISH for ALT.
C. 27/M, frontal lobe IDH mutant 1p19q co-deleted Grade 3 oligodendroglioma, with FISH forALT.

D. 41/M, frontal lobe IDH mutant 1p19q co-deleted Grade 2 oligodendroglioma, withFISH for ALT.

E. 39/M, periventricular IDH mutant 1p19q co-deleted Grade 2oligodendroglioma, with FISH for ALT.
Supplementary Table 1. Multivariate analysis of clinical and molecular features of IDHmutant, 1p19q-codeleted gliomas.
Supplementary Table 2. Correlation between ALT and molecular features in IDH-mutant,
1p19q-codeleted gliomas.
Supplementary Table 3. List of genes studied by target sequencing.
Supplementary Table 4. Frequency of mutations in 40 ALT-positive, IDH-mutant, 1p19qcodeleted gliomas.
Supplementary Table 5. Gene mutations and survival in 40 ALT-positive, IDH-mutant
oligodendrogliomas.
Supplementary Table 6. Clinical characteristics of 112 patients of IDH-mutant, 1p19qcodeleted gliomas.
Online Materials and Methods

1Supplementary Figure 1
Age

Sex

Location
GradeResection

Temozolomide

Radiotherapy

OS

ALT

TERT rearrangement

CDKN2A/B

TERTp

EGFR
PDGFRA

MYC

MYCN************************************************************************************************************************************************FUBP1
CIC************************************************************************NOTCH1************************************************************************

KMT2C************************************************************************

KMT2D************************************************************************PIK3CA************************************************************************

PIK3R1

unknown
Alive

Wildtype
ResectionTotal
Subtotal

ALT
Positive
Negative

CNVs
Loss
Amplification

Temozolomide/radiotherapy
YesNo

TERT rearrangement
Positive
Negative

Gene mutation
MutantWildtype
Grading23
See also online Supplementary Tables 4 and 5.

TERTp mutation
Mutant*OS status
DeadHemisphere
Non-hemisphereAge
55 or above<55

Tumor location

Sex
MaleFemaleSupplementary Figure 2A

100 µm5 µm

38/F, frontal lobe IDH mutant, 1p19q co-deleted Grade 3 oligodendroglioma, with FISH for ALT.
Arrows show microvascular proliferation.

Supplementary Figure 2B

100 µm5 µm61/M, frontal lobe IDH mutant 1p19q co-deleted oligodendroglioma, Grade 2, with FISH for ALT.

Supplementary Figure 2C

100 µm10 µm

27/M, frontal lobe IDH mutant 1p19q co-deleted Grade 3 oligodendroglioma, with FISH for ALT.

Supplementary Figure 2D

100 µm

10 µm41/M, frontal lobe IDH mutant 1p19q co-deleted Grade 2 oligodendroglioma, with FISH for ALT.

Supplementary Figure 2E

100 µm5 µm

39/M, periventricular IDH mutant 1p19q co-deleted Grade 2 oligodendroglioma, with FISH for ALT.

Supplementary Table 1. Multivariate analysis of clinical and molecular features of IDH-mutant, 1p19q-codeleted gliomas.
FeaturesPFSHR (95% CI)
p valuesAge<55 years old
10.991≥55 years old
0.995 (0.392-2.521)
Sex
MaleFemale

1
0.841 (0.408-1.734)

0.639

2
3

1
0.684 (0.291-1.608)

0.384

Hemisphere
Non-hemisphere10.671 (0.086-5.222)

0.703

Total resection
Non-total resection

13.343 (1.529-7.312)

0.003

YesNo

10.756 (0.357-1.603)

0.466

Yes
No

1
0.828 (0.377-1.818)

0.639

NegativePositive1
3.462 (1.598-7.498)

0.002

GradingLocation

Operation

Temozolomide

Radiotherapy

ALTSupplementary Table 2. Correlation between ALT and molecular features in IDH-mutant, 1p19q-codeleted gliomas.
FeaturesTERT rearrangement
Yes
No

Number of cases

ALT negative (n, %) ALT positive (n, %)p values

12 (10.7%)
100 (89.3%)

9 (8.0%)63 (56.3%)

3 (2.7%)37 (33.0%)

0.412

Homozygous deletion (HD)No HD

13 (11.6%)
99 (88.4%)

8 (7.1%)64 (57.1%)

5 (4.5%)35 (31.3%)

0.826

Amplification
No amplification

1 (0.9%)111 (99.1%)

1 (0.9%)71 (63.4%)

0 (0%)
40 (35.7%)

0.454

Amplification
No amplification

7 (6.3%)105 (93.8%)

1 (0.9%)71 (63.4%)

6 (5.4%)34 (30.4%)

0.004

Amplification
No amplification

12 (10.7%)
100 (89.3%)

7 (6.3%)65 (58.0%)

5 (4.5%)35 (31.3%)

0.649

CDKN2A/B

EGFR

MYC

PDGFRASupplementary Table 3. List of genes studied by target sequencing.
ABCB1

ABCC9ADAM29AKT1ATRXBCORBCORL1BRAF

CCND1CCND2CCND3CDH18

CDK4CDK6CDKN2A

CDKN2BCDKN2CCIC

COL1A2

CSF1R

CTNNB1DDX3X

DRD5EGFRERBB2ERBB3ERBB4FAT1

FGFR1

FGFR2

FGFR3FGFR4FUBP1GABRA6

H3F3A

HDAC9HIST1H3B

HIST1H3C

HMCN1HRAS

IDH1IDH2KDRKELKITKLF4KMT2B

KMT2C

KMT2D

KRAS

LZTR1MDM2

MDM4MET

MLH1MSH2MSH6MTOR

MYC

MYCN

NF1NF2NLRP5NOTCH1NRASPBRM1PDGFRAPDGFRBPIK3CAPIK3CGPIK3R1PIK3R2PMS2

POLE

PPM1D

PTCH1

PTEN

PTPN11

PTPRD

RB1

ROS1

SCN9A

SEMA3C

SEMG1

SETD2

SMARCAL1

SMO

SPTA1

STAG2

TCF12

TCHH

TP53

Supplementary Table 4. Frequency of mutations in 40 ALT-positive, IDH-mutant, 1p19q-codeleted gliomas.
GradeGenes

Total cases (n, %)2 (n, %)

3 (n,%)

p value

ATRX

1 (2.5%)

1 (2.5%)0 (0%)

0.641

CIC

27 (67.5%)

24 (60.0%)

3 (7.5%)

0.125

FUBP1

12 (30.0%)

9 (22.5%)

3 (7.5%)0.414

NOTCH1

11 (27.5%)

9 (22.5%)2 (5.0%)0.944

PIK3CA6 (15.0%)

4 (10.0%)

2 (5.0%)0.268

PIK3R17 (17.5%)

5 (12.5%)2 (5.0%)

0.396

ROS1

5 (12.5%)

4 (10.0%)

1 (2.5%)

0.875

TCF12

2 (5.0%)2 (5.0%)0 (0%)

0.504

TP53

0 (0%)

0 (0%)

0 (0%)NA

Supplementary Table 5. Gene mutations and survival in 40 ALT-positive, IDH-mutant oligodendrogliomas.
p value
Genes
Total (n)
PFS
OSCIC

27 (67.5%)

0.308

0.364

FUBP1

12 (30.0%)

0.377

0.497

NOTCH1

11 (27.5%)

0.704

0.465

PIK3CA

6 (15.0%)

0.3840.871

PIK3R17 (17.5%)

0.141

0.299

* Only 2 TCF12 mutations were found.

Supplementary Table 6. Clinical characteristics of 112 patients of IDH-mutant, 1p19q-codeleted gliomas.

Features

Number of cases(n=112)Frequency (%) PFS (p value)

OS (p value)Age<55 years old≥55 years old

93
1983.0
17.0

0.477

0.032

MaleFemale

71
41

63.4
36.6

0.544

0.985

Hemisphere
Non-hemispheric
Not available

1036392.0
5.4
2.70.728

0.009

Gross total resection
Non-total resection
Not available

6642458.9
37.5
3.6

0.030

0.090

Yes
No
Not available

47596

42.0
52.7
5.4

0.946

0.020

Yes
No
Not available

6839
560.7
34.8
4.5

0.732

0.013

Yes
No

4072

35.7
64.3

0.009

0.728

Yes
No

12100

10.7
89.3

0.552

0.168

Deletion
No deletion

1399

11.6
88.4

0.829

0.304

Amplification
No amplification

1111

0.9
99.1

0.098

0.113

Amplification
No amplification

7105

6.3
93.8

0.207

0.858

Amplification
No amplification

12100

10.7
89.3

0.419

0.912

Sex

Location

OperationTemozolomide

Radiotherapy

ALT

TERT rearrangement

CDKN2A/B

EGFR

MYC

PDGFRASupplementary Online Materials and Methods
FISH analyses
FISH studies were performed for ALT, EGFR, MYC, PDGFRA and TERT-rearrangement aspreviously used by us and others. ALT phenotype was examined with the Telomere PNA FISH
kit (K532511, Dako) [1, 2]. TERT-rearrangement was assessed with FISH break-apart probes wereported in previous publications and the probes were directly labeled [1, 2]. Other FISH methods
were also used in our previous studies [1-5]. Vysis LSI CDKN2A SpectrumOrange/CEP 9
SpectrumGreen Probes (Vysis) was used to investigate CDKN2A deletion. EGFR amplification
was detected with the BAC clone (CTD-2199A14) which consists of the genomic sequences of
7p11.2 and a centromere probe (CEP7, Vysis). MYC amplification was detected with the BACclone CTD-3056O22 which spans the genomic region 8q24.21 and a centromere probe (CEP8,Vysis). For PDGFRA amplification, PDGFRA probes (CTD-2054G11 and RP11-231C18) andcentromere probe (CEP 4, Vysis) were used. In brief, tumors areas on 4-μm thickness FFPE
sections were identified and marked for evaluation. Tissue sections were then deparaffinized,
treated with sodium thiocyanate, digested with pepsin, rinsed and dehydrated. The labeled probes
were denatured and hybridized to the sections overnight. Sections were then washed, stained with
Vectashield mounting medium and visualized under a Zeiss Axioplan fluorescence microscope.
At least 100 non-overlapping signals were counted and analysed in each case. Tumors wereconsidered ALT-positive when (1) they displayed ultrabright nuclear foci (telomere FISH signalof 10-fold greater than the signal of individual non-neoplastic cells); and (2) ≥ 5% of tumour cells
exhibited large, very bright intranuclear foci of telomere FISH signals [6-8]. Areas of necrosis
were excluded from analysis. TERT-rearrangement was considered when break-apart signal insamples was found in >5% of evaluated nuclei [1-2, 8]. EGFR, MYC and PDGFRA amplifications
were recorded when >5% examined cells displayed many tight clusters or a ratio of target toreference signal >2 [3, 9]. CDKN2A homozygous deletion was recorded when >20% of tumor
cells showed loss of two signals [10]. For positive controls for the FISH biomarkers, we used
FFPE sections from cases known to be aberrated for the individual FISH markers from previous
publications from our group [1, 2, 11]. For negative controls, we used sections from normal brain.

Targeted sequencing
For targeted sequencing, a panel of customized genes commonly altered in CNS tumors was used
[1, 2, 4]. DNA was extracted with GeneRead DNA FFPE kit (Qiagen). DNA were then evaluatedwith QIAseq DNA QuantiMIZE Assay (Qiagen) to check for quality and quantity. Library
preparation was completed with a custom QIAseq Targeted DNA Panel, that examined codingexons or hotspots of CNS relevant genes (Supplementary Table 3). The DNA libraries were thenqualified prior to DNA sequencing with MiSeq v3 (Illumina).
Paired-end reads were aligned to the hg19 (GRCh37) build of the human reference genome withBWA-MEM algorithm on GeneGlobe platform (Qiagen). smCounter2 and wANNOVAR wereused in variant calling and annotation respectively. Variants that met the following criteria wereexcluded for further analysis: 1. not passing quality filters; 2. variant allele fractions ≤10%; 3.
variant allele counts ≤5, or 4. minor allele frequencies >1% in overall human population or EastAsians or documented in public databases (1000 Genomes, ExAc, gnomAD exome and genomedatabases).

Online references
1. Liu EM, Shi ZF, Li KK, Malta TM, Chung NY, Chen H, Chan JY, Poon MF, Kwan JS, ChanDT, Noushmehr H, Mao Y, Ng HK. Molecular landscape of IDH-wild type, pTERT-wild typeadult glioblastomas. Brain Pathol. 2022; 32(6): e13107.
2. Ng HK, Li KK, Chung NY, Chan JY, Poon MF, Wong QH, Kwan JS, Poon WS, Chen H, ChanDT, Shi ZF, Mao Y. Molecular landscapes of longitudinal NF2/22q and non-NF2/22q
meningiomas show different life histories. Brain Pathol. 2023; 33(3): e13120.
3. Yang RR, Li KK, Liu APY, Chen H, Chung NY, Chan AKY, Li F, Chan DT, Mao Y, Shi ZF,Ng HK. Low-grade BRAF V600E mutant oligodendroglioma-like tumors of children may show
EGFR and MET amplification. Brain Pathol. 2021; 31(1): 211-214.
4. Wong QH, Li KK, Wang WW, Malta TM, Noushmehr H, Grabovska Y, Jones C, Chan AK,Kwan JS, Huang QJ, Wong GC, Li WC, Liu XZ, Chen H, Chan DT, Mao Y, Zhang ZY, Shi ZF,Ng HK. Molecular landscape of IDH-mutant primary astrocytoma Grade IV/glioblastomas. ModPathol. 2021; 34(7): 1245-1260.
5. Chan AK, Shi ZF, Li KK, Wang WW, Chen H, Chung NY, Chan DT, Poon WS, Loong HH,Liu XZ, Zhang ZY, Mao Y, Ng HK. Combinations of Single-Gene Biomarkers Can PreciselyStratify 1,028 Adult Gliomas for Prognostication. Front Oncol. 2022; 12: 839302.
6. Heaphy CM, Subhawong AP, Hong SM, Goggins MG, Montgomery EA, Gabrielson E, NettoGJ, Epstein JI, Lotan TL, Westra WH, Shih IeM, Iacobuzio-Donahue CA, Maitra A, Li QK,Eberhart CG, Taube JM, Rakheja D, Kurman RJ, Wu TC, Roden RB, Argani P, De Marzo AM,Terracciano L, Torbenson M, Meeker AK. Prevalence of the alternative lengthening of telomeres
telomere maintenance mechanism in human cancer subtypes. Am J Pathol. 2011; 179(4): 160815.
7. Minasi S, Baldi C, Gianno F, Antonelli M, Buccoliero AM, Pietsch T, Massimino M, Buttarelli
FR. Alternative lengthening of telomeres in molecular subgroups of paediatric high-grade glioma.
Childs Nerv Syst. 2021; 37(3): 809-818.
8. Diplas BH, He X, Brosnan-Cashman JA, Liu H, Chen LH, Wang Z, et al. The genomic
landscape of TERT promoter wildtype-IDH wildtype glioblastoma. Nat Commun. 2018; 9(1):2087.
9. Horbinski C, Miller R, Perry A. Gone FISHing: Clinical Lessons Learned in Brain Tumor
Molecular Diagnostics Over the Last Decade. 2011; 21(1): 57-73.
10. Horbinski C, Nikiforova MN, Hagenkord JM, Hamilton RL, Pollack IF. Interplay among
BRAF, p16, p53, and MIB1 in pediatric low-grade gliomas. Neuro Oncol. 2012; 14(6): 777-789.
11. Mu Q, Chai R, Pang B, Yang Y, Liu H, Zhao Z, Bao Z, Song D, Zhu Z, Yan M, Jiang B, MoZ, Loi SC, Tam SS, Chan AK, Wu AR, Liu Z, Poon WS, Ng HK, Chan DTM, Iavarone A, NamDH, Jiang T, Wang J. Identifying predictors of glioma evolution from longitudinal sequencing.
Science Translational Medicine, accepted.