1. F.I.S.H. in PATHOLOGY; Applications,Answers..and More Allan Kennedy Lead Biomedical Scientist (Molecular Pathology) Glasgow Royal Infirmary

2. Samples  Tissue Imprints  Blood  Formalin-fixed, paraffin embedded sections-usually 1µm thick!

4. TECHNIQUE  ACCESS THE TARGET  DENATURE  HYBRIDISE  COUNTERSTAIN  VIEW

5. F.I.S.H. WORK STATION

6. Probe use  Enumeration; Loss or Amplification  Breakapart; specific gene targeted  Dual Fusion; specific translocations detected

7. ENUMERATION PROBES e.g. Her2, CLL

9. Ideogram Her2 probe set

10. Normal Her2

11. AMPLIFIED HER2

12. Her2 ; Polysomy

13. Monosomy 17 with Her2 Amplification

14. Chronic Lymphocytic Leukemia (C.L.L.)

15. CLL PROBE SET

16. CLL P53 (17p13.1) Red ATM (11q22.3) Green;

17. CLL P53 (17p13.1) Red ATM (11q22.3) Green; Loss of ATM associated with progressive disease

18. CLL:Loss of p53 associated with progressive disease and Fludarabine resistance P53 (17p13.1)Red ATM (11q22.3) Green

19. Oligodendroglial Tumours Loss of Heterozygosity 1p/19q ?

20. 1p36 Red 1q25 Green Loss of 1p36

21. 19p13 green 19q13 red Loss of 19q

22. DUAL COLOUR DUAL FUSION PROBES e.g.(t11:14, t14:18)

23. Molecular Pathogenetic Mechanisms in Rhabdomyosarcoma and Ewing’s Sarcoma

27. t14:18 Negative Chr14 Green (IgH) Chr18 Red (bcl2) CEP18 Aqua

28. T14:18 Positive Chr14 Green (IgH) Chr18 Red (bcl2) CEP18 Aqua Translocations Yellow

29. BREAKAPART PROBES e.g. cMyc

32. cMyc Negative

33. C-Myc (8q24) Positive

34. ANSWERS

35. ? Burkitt’s Lymphoma ?DLBCL LCA,CD10,CD20,bcl6 ;Positive TdT,CD5,MUM-1; Negative  FISH REQUEST FOR c-Myct8:14t14:18

36. C-Myc (8q24) Positive

37. t8:14 Positive

38. t14:18 Negative - Burkitt’s Lymphoma

39. ? Burkitt ?Follicular Lymphoma  CD20,CD10,; Positive  CD5,Cyclin D1,CD23,bcl2; Negative

40. Myc negative

41. t14:18 (IgH / bcl 2) positive

42. Bcl6 (3q27) translocation negative; Follicular Lymphoma

43. Sarcomas

44. Molecular Diagnosis of Sarcomas TumorTranslocation Fusion Gene Ewing/PNETt(11;22)(q24;q12)EWS/FLI1 t(21;22)(q22;q12)EWS/ERG Alveolar Rhabdomyosarcoma t(2;13)(q35;q14)PAX3/FKHR t(1;13)(p36;q14)PAX7/FKHR Desmoplastic small round cell tumor t(11;22)(p13;q12)EWS/WT1 Synovial Sarcoma t(X;18)(p11.2;q11.2)SYT/SSX1+2 Congenital Fibrosarcoma t(12;15)(p13;q25)ETV6/NTRK3 Clear Cell Sarcoma t(12;22)(q13;q12)EWS/ATF1

45. Sarcoma  ? Ewings Sarcoma  ? Synovial sarcoma

46. Syt-Negative

47. EWS Positive-Ewings Sarcoma

48. ? ALVEOLAR RHABDOMYOSARCOMA TEST FOR; t1:13t2:13 FKHR breakapart

49. t1:13 (Pax7 green/FKHR red ) Negative

50. t2:13 (Pax 3 green /FKHR red) Positive

51. FKHR (13q14) Positive

52. ANSWERS…and MORE

53. CLL P53 (17p13.1) Red ATM (11q22.3) Green;

54. t11:14 IgH 14q32 Green Bcl2 18q21 Red CEP11 aqua

55. Amplified Her2 Ratio 2.3

56. Amplified Her2 Ratio 10

57. Her2 ; Fusions?

58. Chromosome 17

59. Her2 gene

60. Her2 ; Triple Bandpass filter

61. Her2 gene

62. Chromosome 17

63. Her2 ; Homogeneously Stained Regions (HSR’s)

64. Her2;Double Minutes

65.  Need to record all observations-whether expected or not!  Need to collaborate across centres  Need close co-operation with Pathologists  Boundaries between Pathology, Genetics and Haematology rapidly blurring

66. Thank You