1. GENETIC STUDIES OF C-MYC ONCONGENE AND ROR1 GENE OVEREXPRESSION IN EPENDYMOMAS
YUEMIN DING
Neuro-oncology Group
Department of Molecular Neuroscience
Institute of Neurology
Queen Square, London
Supervisor: Dr Tracy Warr

3. BACKGROUND
Ependymomas are glial tumors that arise from the ependymal lining of the ventricular system of the CNS.
They represent the third most frequent brain tumor in children. (Hamilton RL et al. 1997)
The genetic events that contribute to the pathogenesis of paediatric ependymoma are poorly defined. (Ward S et al. 2001)

4. BACKGROUND Previous finding of our group
The expression of >12,000 genes in a set of 11 ependymoma samples were determined using oligonucleotide micrarrays analysis. Oncogene c-myc and gene ROR1 were identified to be highly expressed in the tumour samples.
Previous experiments failed to show extra copies of c- myc gene in ependymomas by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH).

5. BACKGROUND c-myc oncogene CHR # 8 c-Myc Cellular processes apoptosis
Cell growth
proliferation
differentiation

6. BACKGROUND
c-Myc c-Myc c-Myc
c-Myc c-Myc !
c-Myc c-Myc
c-Myc c-Myc c-Myc
— c-myc has emerged as a central oncogenic switch in many human cancers. (Stella Pelengaris 2002)

7. Mechanisms lead to gene overexpression Chromosome duplication
BACKGROUND
Mechanisms lead to gene overexpression
Chromosome duplication
Gene amplification
Point mutation
Regulators dysfunction

8. overexpression of normal product
gene
amplification
point mutation
chromosome
duplication
A - T
C - G
G - C
T - A

9. overexpression of normal product
Loss of function mutation

10. BACKGROUND Previous finding of our group
The expression of >12,000 genes in a set of 11 ependymoma samples were determined using oligonucleotide micrarrays analysis. Oncogene c-myc and gene ROR1 were identified to be highly expressed in the tumour samples.
Previous experiments failed to show extra copies of c- myc gene in ependymomas by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH).

11. HYPOTHESIS
The regulator upstream in the c-Myc pathway was abnormal or dysfunctional, eg. loss of function mutation in MM-1.

12. BACKGROUND Receptor tyrosine kinase-like orphan receptor 1 (ROR1)
FRZ: frizzled module
Wnt pathway
Oncogene WNT5A

13. HYPOTHESIS
ROR1 plays a role in the pathogenesis of ependymomas partially mediated through Wnt-ROR pathway.

14. AIMS
To investigate the mutation status of the MM-1 gene, which may play a role in the development of ependymomas by deregulating c-myc expression.
To identify the association between the genetic changes of the ROR1 gene and the pathogenesis of ependymomas via Wnt pathway.

15. MATERIALS AND METHODS Tumour specimens PCR/RT-PCR SSCP analysis
Ependymoma case information (sourse: cell culture)
Key to table:
IN1: Institute of Neurology assigned number
Age2: Age in years at diagnosis
Sex3: M = male; F = female
Grade4: SE = subependymoma; E = ependymoma;
AE = Anaplastic ependymoma
P/R5: P = primary sample; R = recurrent sample
Survival6: survival in months from date of diagnosis
Gains7 and losses7: detected by CGH
MATERIALS AND METHODS
Tumour specimens
PCR/RT-PCR
SSCP analysis
DNA sequencing

16. MATERIALS AND METHODS
Tumour specimens
PCR/RT-PCR
SSCP analysis
DNA sequencing

17. MATERIALS AND METHODS
Tumour specimens
PCR/RT-PCR
SSCP analysis
DNA sequencing

18. MATERIALS AND METHODS Tumour specimens PCR/RT-PCR SSCP analysis1 2 3 4
MATERIALS AND METHODS
Tumour specimens
PCR/RT-PCR
SSCP analysis
DNA sequencing

19. Amplify target gene by PCR technique Detect mutations by SSCP analysis
Protocol
Search the Genomic Database and design the primers
Extract DNA from frozen cell cultures of ependymoma samples
Amplify target gene by PCR technique
Detect mutations by SSCP analysis
Direct DNA sequencing

20. Expression level of c-Myc SSCP analysis of MM-1 DNA sequencing of MM-1
RESULTS
Fig 1 RT-PCR studies of c-myc. cDNAs were synthesized from RNA of the tumour samples and the normal brain (CC). β-actin was used as the internal control. DNA marker used is the 100bp ladder. c-myc was overexpressed in tumour samples compared with the CC. A B
Fig. 2 Representative results of SSCP analysis of MM-1. Lanes 1–10, samples of ependymomas. A, MM-1 exon 4-5; B, MM-1 exon 6. Arrowhead highlights abnormal migrating SSCP bands.
MM-1
Expression level of c-Myc
SSCP analysis of MM-1
DNA sequencing of MM-1

21. RESULTS
ROR1
Expression level of ROR1
SSCP analysis of ROR1
DNA sequencing of ROR1

22. RESULTS
Fig. 3 RT-PCR analysis of ROR1. cDNAs were synthesized from RNA of the tumour samples and the normal brain (CC). β-actin was used as the internal control. DNA marker used is the 100bp ladder. Note ROR1 was overexpressed in tumour samples compared with CC.

23. A B
Fig. 4 Representative results of SSCP analysis of ROR1. Lanes 1–11, samples of ependymomas. A, ROR1 exon 7; B, ROR1 exon 8. Arrowhead highlights abnormal migrating SSCP bands.

24. 3008 For
3008 Rev
2941 For
2941 Rev A B
Fig. 5 Sequence confirmation of mutations identified by SSCP analysis (ROR1 gene). Black arrows indicate the heterozygous C → T mutation in exon 7 of the ependymoma samples, which were confirmed in both sequencing reactions. (A, sample IN3008; B, sample IN2941)

25. A B C
1258 For
1258 Rev
2443 Rev
2443 For
2682 For
2682 Rev
Fig. 6 Sequence confirmation of mutations identified by SSCP analysis (ROR1 gene). Black arrows indicate the heterozygous (A and B) or homozygous (C) A → G mutation in exon 8 of the ependymoma samples, which were confirmed in both sequencing reactions. (A, sample IN1258; B, sample IN2443; C, IN2682)

26. RESULTS
Table 1 Sequence changes in the ROR1 gene

27. NM_ [1p32_p31] 937 aa
348:
T→T
451:
V→V
Immunoglobulin domain (72 – 133) FRZ domain (160 – 297)
Kringle domain (313 – 391)
Phabdovirus spike glycoprotein (411 – 457)
Protein kinase domain (473 – 746)
Fig. 4.2 The structures of ROR1 and SNP location. The silent mutation on codon 348 was located at Kringle domain and the other mutation on codon 451 was at Phabdovirus spike glycoprotein.

28. CONCLUSION
We failed to detect mutations in MM-1 in ependymomas by DNA sequencing. But it didn’t preclude the possibility that mutations in MM-1 abolished its suppressor function, which lead to c-Myc overexpression, because loss of function mutations may have been missed in the present sequencing analysis.
Two silent mutations were found in ROR1 but they present little relevance in the Wnt pathway in ependymomas. Association of these mutations in ROR1 to cancer risk require further studies.