1. CBTTC project 0011: Targeting Alternative Lengthening of Telomeres (ALT) in Pediatric CNS malignancies
Kristina A. Cole, MD/PhD
The Children’s Hospital of Philadelphia
University of Pennsylvania School of Medicine

2. ALT in pediatric brain tumors (Alternative Maintenance of Telomeres)
ALT is a telomere maintenance mechanism
required for continued cellular proliferation (vs. telomerase)
20-40% of pediatric high grade gliomas use ALT, including:
All H3F3A G34 mutant tumors (Korshunev et al, Gessi et al)
23% H3F3A K27M diffuse midline gliomas
often associated with ATRX, DAXX and TP53 mutations
12% of sPNET, 23% of choroid plexus carcinomas use ALT
Studies are limited by a lack of pediatric brain tumor models with ALT
Tumors that use ALT may have therapeutic vulnerabilities

3. CBTTC 0011: Downstream studies
CBTTC 0011 proposes to generate patient derived (PD) cell lines of known ALT status
They will be used for:
testing of published proposed inhibitors of ALT tumors (ie ATRi, CHK1i)
validation of candidates from drug and CRISPR screens (i.e. isogenic ATRX lines)
functional analysis of candidate TS and OG from large genomic studies in primary tumors
TS = tumor suppressor
OG =oncogene

4. CBTTC 0011 Proposal and Timeline (proposal approved in the summer 2016) 38 HGG/PNET samples with freezing media
Aim 1: Analysis of corresponding primary tumor DNA for prioritization and downstream validation
ALT (CCA) assay of primary tumor (3 months) - completed
Sequencing of H3F3A for K27M, G34R/V (3 months) – completed
Identification of mutations by WGS (n= 17 overlapping sets) - ongoing
Aim 2: Generation of Cell lines
Adherent and suspension lines (6 m - 1 yr) – 30/38 in various stages of production - ongoing
Aim 3: Cell line validation and characterization
H3F3A targeted sequencing (9/9 tested to date match primary tumor)
Targeted sequencing of known BRAF, IDH1, ATRX, TP53 etc mutations
growth kinetics, tumorgenecity, ICC/IHC, ALT assays, Telomerase assays, WB for ATRX etc
Sequencing
CCA = c-circle assay. This is a rolling circle amplification assay (based on tel sequence) that is read out on a dot blot. It only requires < 50 ng of DNA.
WGS = whole genome sequencing
ICC = immunocytochemistry and/or IHC immunohistochemistry for typical glioma/PNET markers
WB = western blot

5. Potential Characteristics of Cell lines (based on primary tumor DNA analysis)
Of 38 samples
2 are diagnostic / relapse pairs
17 tumor are midline, and 13 are hemispheric gliomas
8 samples pathology is “PNET”
ALT status
9/35 primary tumor DNA samples are ALT (CCA) positive
Targeted sequencing of H3F3A showed that 3/3 (100%) of G34R mutant, 2/8 (25%) of K27M mutant and 4/24 (17%) of H3F3A wild-type tumors are ALT positive.
17 of 38 had WGS. 4 of these were from ALT positive tumors.
The ALT positive tumors have known pathogenic mutations in TP53 (4/4), ATRX (2/4), H3F3A (2/4) and IDH1 (1/4).
The reason only 35 out of 38 have CCA (ALT) testing is because the other 3 were added to the project later.

6. Thank-you !
CBTTC: Martha Williams, Mateusz Koptyra, Jen Mason, Elizabeth Appert, Adam Resnick and Angela Waanders
Cole lab: Heba Ijaz
U Penn: Robert Dilley and Roger Greenberg
Bioinformatics: Pichai Raman, Yuankun Zhu, Bo Zhang
All CBTTC investigator sites !
Questions/comments/recommendations, please
Kristina at: