1. RECURRENT CHROMOSOMAL COPY NUMBER ALTERATIONS IN CHORDOMA G. Petur Nielsen; John Iafrate; Zhenfeng Duan; Ramnik Xavier; Joseph Schwab; Andrew Rosenberg; Julie Miller; Edwin Choy; David Harmon; Thomas DeLaney; Vajiya Ramesh; Vikram Deshpande; Francis Hornicek MGH Chordoma Research Group DF/Harvard Cancer Center

2. D efi nition Malignant tumor with a phenotype that recapitulates the notochord and usually arises within the bones of the axial skeleton.

3. Incidence – 0.5-1 per million –300 cases/yr in US Gender – M:F 1:1 in skull base and 2:1 in spine and sacrum Age – mean 5 th -6 th decade

4. Symptoms – depend on location Skull – diplopia, headaches, cranial nerve palsies Spine – pain, nerve compression Sacrum – pain, constipation, incontinence, bladder dysfunction

6. Treatment Surgical resection - primary Radiation therapy - adjuvant Local recurrence - problem

7. Prognosis Median survival – 6.29 years 5 yr survival-68% 10 yr survival-40% –Alive with local recurrence Survival

8. What is known? Little to no data concerning the presence of somatic mutations in chordoma Handful of studies have looked at karyotype or Comparative Genomic Hybridization (CGH) No correlations with anatomic site or outcome

9. Clinical Characteristics of 21 Patients with Chordoma FeatureDetailed featurePatients GenderMale, n (%)15 (71) Female, n (%)6 (29) AgeMedian age, yr (range)63 (46-83) Location of the tumorSkull base, n (%)2 (10) Sacrum, n (%)12 (57) Mobile spine, n (%)7 (33)

10. Results Average time to death: 8.2 years Local Recurrence: 14 patients Average patient follow-up: 6.1 years

11. Red= loss in patient. Yellow shading Yellow shading= normal. Green= gain in patient Reference DNAChordoma DNA Array-CGH detection of chromosomal abnormalities  Agilent 244,000 spot oligonucleotide DNA array  Frozen tissue from the 21 chordomas: H&E slides reviewed  Genomic DNA extracted using Gentra Puregene isolation kit  1 microgram of tumor DNA labeled with CY5  1 microgram of normal DNA labeled with CY3  2 day array competitive hybridization  Chromosome plots are log2 ratios of normalized CY5:CY3 signals

12. 13

13. Frequent Chromosomal Aberrations by Array CGH Chromosomal aberration Overall (n=21) Skull base (n=2) Mobile spine (n=7) Sacrum (n=12) Loss of 1p212712 Loss of 3p202611 Loss of 411137 Loss of 5q4013 Gain of 76123 Loss of 9p182511 Loss of 10q192611 Loss of 117043 Loss of 13q13247 Loss of 14q152310 Loss of 18q10235 Gain of 197124 Loss of 22q10145

14. Chromosomal Aberrations of Chordomas in Comparison to Chondrosarcoma Chromosomal aberration % Chordomas (n=21) % Chondrosarcoma (Rozeman et al., 2006) p-values (Fisher’s exact test; 2-tailed) Loss of 1p21/215/18<0.001 Loss of 3p20/212/18<0.001 Loss of 411/214/180.055 Loss of 5q4/214/181.000 Gain of 76/215/181.000 Loss of 9p18/217/180.0027 Loss of 10q19/215/180.0003 Loss of 117/212/180.135 Loss of 13q13/215/180.031 Loss of 14q15/215/180.010 Loss of 18q10/212/180.016 Gain of 197/216/181.000 Loss of 22q10/212/180.016

15. VHL p16 PTEN RB SMAD4 NF2

16. 123456 7 8 9 101112 Cen 131415161718 19 20 21 22 TP73 RUNX3 CDKN2C 1

17. Runx3 Actin Osteoblast GB60 Ch8 UCH1 Chordoma tissue

19. 3 Loss 3p (only one orange signal Ch8

20. 9

21. Hallor et al. 2008, British Journal of Cancer CDKN2A deletion commonly observed in Chordoma

22. p16 immunostaining

23. 12356 7 8 9 10 Cen A B 10

25. PTEN 10

26. PTEN

27. mTOR Signaling Pathway mTOR PROTEIN SYNTHESIS & CELL GROWTH S6 P S6K P 4EBP1 P Hamartin Tuberin Low Energy Ras MEK1/2 ERK1/2 RSK1 LKB1 AMPK Rheb PI3K Akt Growth Factor Stimulation P P PTEN rapamycin NF1 P P P

28. EGFR (55.0) Oncogenes on Chromosome 7 (7/22) BRAF (140.0) HGF (81.0) SMO (128.0) SHH (155.0) MET(116.0) Chordomas do not possess common cancer gene mutations

29. HDAC4 Actin Osteoblast GB60 CH8 UCH1 Met

30. miRNA-1 miRNA-1 targets: FoxP1 MET HDAC4 Decreased expression of miRNA-1 in chordoma

31. N1 N2 T2 T1 miRNA-1 miRNA- RNU48 N1 N2 T1 T2 miRNA-1 Relative Expression 28S 18S miRNA-1 N1N2 T1 T2 TC1 TC2 AB C D Real-time RT-PCR and Northern blot detection of mature miRNAs in chordoma

32. Absorbance (cell growth) Concentration of pre-miRNA-1 (nmol) miRNA-1 miRNA-control Hours after pre-miRNA transfection AB CD Met Actin miRNA-1 miRNA- control UCH1 Transfection of miRNA-1 into chordoma cells UCH1suppresses Met expression and inhibits cell growth

33. Expression of hepatocyte growth factor (HGF) and c-MET In skull based chordoma Naka, Kuester, Boltze, Scheil-Bertram, Samii,Herold, Ostertag, Krueger, Roessner Cancer 2008; 112: 104-110.

34. Summary Chordomas have recurrent and remarkably stereotypic chromosomal profile 1p loss is observed in 100% of tumors 9p (CDKN2A; p16) or chromosome 9 loss was seen tumors (77%) Common losses also include 3p, 4, 10, 13, 14, 18, 22 Gains include 7, 19 These alterations point to genes for follow-up studies – therapies?

35. Thank you