Chromosome aberrations in HAEMATOLOGIC MALIGNANCIES

1. Chromosome aberrations in HAEMATOLOGIC MALIGNANCIES

2. Increased growth Blockage of differentiation Less apoptosis Haematological malignancies

3. HAEMATOLOGIC MALIGNANCIES MAY BE CLASSIFIED BY: 1.CLINICAL COURSE: acute leukaemias or chronic leukaemias 2. LINEAGE: lymphoid lineage myeloid lineage 3.PRIMARY SITE leukaemia: originates in the bone marrow - flows in the peripheral blood lymphoma: originates in the lymph nodes - invades bone marrow and blood

4. AML-M2t(8;21)(q22;q22) Starring Ali MacGraw, Ryan O'Neal, John Marley Erich Segal, 1970

5. History • 1956 Correct number of human chromosome • 1960 Ph chromosome • 1970 Banding technique 1973 t(8;21) 1977 t(15;17), t(4;11) 1979 High-resolution banding technique • 1980 1982 t(9;11) in AML-M5a, inv(3) in AML 1983 del(16q), inv(16) in AML-M4E 1984 t(1;3) in AML with dysmegakaryopoiesis t(1;19) in ALL ISCN (1985,1991,1995) 5

6. Most Patients with Acute Leukemia have Characteristic gene rearrangement • 1980 1983 MYC-IGH --- t(8;14) 1984 ABL-BCR --- t(9;22) IGH-BCL2 --- t(14;18) 1988 ATRA in APL Tx • 1990 1991 MLL --- t(4;11) PBX1-E2A --- t(1;19) PML-RARA --- t(15;17) AML1-ETO --- t(8;21) DEK-CAN --- t(6;9) 1993 CBFB-MYH11 --- inv(16) MLL-AF9 --- t(9;11) 6

7. 40 000 35 000 30 000 25 000 20 000 15 000 10 000 5 000 1975 1980 1985 1990 1995 2000 Database of Chromosome Aberrations in Cancer No. of cases year

8. Hematological disorders 62% Lymphomas 9% Database of Chromosome Aberrations in Cancer Solid tumors 29%

9. Recurrent Balanced Chromosome Rearrangements in Neoplasia Tumor type No of No of cloned No of genes aberrations breakpoints involved Hematologic disorders 362 213 84 Mesenchymal tumors 63 26 20 Epithelial tumors 46 8 8 Total 471 247 108 Mitelman et al: Recurrent Chromosome Aberrations in Cancer (2002) http://cgap.nci.nih.gov/Chromosomes/RecurrentAberrations

10. HEMATOLOGY and CLINICAL GENETICS Connection? Importance?

11. 1. 4. 3. 2. 1. BM morphology 2. Immunological features 3. Chromosome analysis 4. Condition of the patient   

12. What´s the use of chromosomal changes? 1. Diagnosis 2. Prognosis 3. Choice of treatment 4. Evaluation of treatment (e.g. interphase-FISH)

13. Culture media and additives Standard media RPMI 1640 Eagle’s MEM Ham’s F12 McCoy’s 5A medium

14. Cell Culture procedure 2183-98 medium + cells Glass flask Peripheral blood Falcon flask

15. HARVESTING PROCEDURES Glass flask Colcemide 30 min. Nismyth et al 1996 Falcon flask Hypotonic treatment (0.075 M KCl) metaphase Fixation (3:1) methanol: acetic acid Spreading

17. interphase Fluorescence in situ hybridization (FISH) ds DNA metaphase ss DNA ss PROBES

18. ACUTE MYELOID LEUKAEMIA (AML) • malignancy affecting myeloid progenitor cells. • the cell involved is an immature blast cell • Most patients are aged over 50 years • the most studied human malignancy as regards acquired genetic changes • >8500 cases with clonal cytogenetic abnormalities • the disease is classified (FAB) according to the morphological and immunological features of this cell.

19. 50% of cases of all AML have a clone with chromosomal aberrations in the bone marrow • 80% of these cases show non-random changes. • t(8;21) 6.9% • t(15;17) 6% • 11q23 2.5% • inv(16) 1.7 Frequency of specific chromosome abnormalities in AML The 4 most common

20. t(8;21)(q22;q22) • Large blast cells, basilophilic cytoplasm, BM eosinophilia, Auer rods, aberrant CD19 (normally B-cells) and a tendency to extramedullary disease i.e., granulocytsarcoma • Fusion of CBFA2 (AML1) at 21q22 and ETO at 8q22. • In cases with a “ typical” t(8;21)-morphology where t(8;21) is not identified by banding can have a CBFA2/ETO fusion nevertheless. • Additional changes e.g. loss of sex chromosomes, del(7q), and del(9q) are common - no prognostic significance

21. t(15;17)(q22;q12-21) • Pathognomonicacute promyelocytic leukaemia (APL) or AML M3 • Any age, but frequent in the young adults • Difficult to identify when poor chromosome morphology • Fusion PML (15q22) and RARA (17q12-21) • Additional changes e.g.+8 is of unknown importance

22. highly granulated blast cells: • NUCLEUS • size and shape irregular, variable (kidney, bilobed) • CYTOPLASM • densely packed large granules • stain pink/red/purple by MGG • large Auer rods (Faggot cells) hypergranular promyelocytic leukemia

23. t(15;17)(q22;q12-21) • Treatment is an emergency in APL! Intra vascular coagulation - 10-40% early mortality (intra cerebral haemorrhage) • oral trans-retinoic acid (ATRA - overcomes the maturation arrest) with combined chemotherapy effective when PML/RARA - Results: 80-90% remission rate with ATRA treatment • APL-like conditions with t(5;17) or t(11;17) involving RARA but not PML respond bad/worse to ATRA • Identification of t(15;17) is crucial!

24. Cases: 25% areinfants (<1 yr) children and adults each represent 50% of cases • Clinics: organomegaly; CNS involvement (5%); • both AML and ALL • MLL - a promiscuous gene • in 70% of infant leukaemias 6q27, 9p21-22 10p11-15 19p13 11q23 (MLL) • The most common translocationpartners are: • Extremely hard to identify t(6;11), t(9;11), and t(11;19). 11q23-aberrations PROGNOSIS VERY POOR in general

25. inv(16)(p13q22) N inv(16) • Eosinophilia, aberrant CD2 (normally in T-cells), increased risk for involvement in the central nervous system • Difficult to identify (especially with R-banding) • Fusion of the genes CBFB and MYH11. • Additional changes: del(7q),+8,+21, and +22 are of unknown prognostic importance • Leukemic infiltrates in lymphoid tissue (head and neck) may be a negative prognostic sign

26. inv(16)(p13q22) • Morphology: • excess of monocytes • variable number of eosinophilic granules larger than normal, purple-violet in colour

27. ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) • Cytogenetic analysis of ALL has been hampered by the difficulty of obtaining good quality chromosomes: poor spreading fuzzy chromosomes indistinct bands • A chromosome abnormality is identified in 66% • 5000 aberrant ALL cases are published • A large number of more or less specific aberrations of clinical significance have been identified

28. Random 30% TEL/AML1 20% Hyperdiploidy (above 50) 25% E2A/PBX1 5% BCR/ABL 4% MLL rearrangements 6% Miscellaneous 10% Random 25% Infant MLL rearr 75% Childhood Random 40% BCR/ABL1 25% Miscellaneous 17% MLL rearrangments 7% Hyperdiploidy 6% E2A/PBX1 3% TEL/AML1 2% Prevalence of genetic changes in ALL with respect to different age groups Adult

29. t(4;11)(q21;q23) • the most common in ALL (B-ALL) • young age • hyperleucocytosis, enlarged liver and spleen, involvement of the central nervous system, 90% blasts in the blood • bad prognosis, complete remission obtained but is promptly followed by relapse • treatment: BM transplantation highly indicated • MLL gene

30. Uckun et al, Blood 1998: ”MLL/AF4 fusion transcripts detectable by RT-PCR assay are frequently generated in patients whose cells lack cytogenetically detectable t(4;11) and that expression of MLL/AF4 fusion transcripts is not a significant prognostic factor for these patients.” Cytogenetics is very important!

31. this translocation is typical for B-ALL (but also described in Burkitt´s lymphoma) • Fusion of the genes IGH and MYC. t(8;14)(q24;q32) t(12;21)(p13;q22) • can NOT be detected by ordinary cytogenetic methods. FISH or PCR is necessary • the most common translocation in ALL among children (very rare in adults) • good prognosis (?). • Fusion ETV6 and CBFA2 wcp- whole chromosome painting probes

32. t(9;22)(q34;q11) in ALL Philadelphia chromosome • Cytogenetically and sometimes molecular genetic identical with t(9;22) in CML • Frequent CNS involvement, even at diagnosis • Treatment: BMT is indicated • Prognosis: very poor • Fusion of BCR and ABL • Additional anomalies: +der(22),-7,del(7q) or +8

33. t(1;19)(q23;p13.3) • t(1;19)(q23;p13.3) leading to formation of the chimeric fusion gene occurs in 25% of the pre-B ALL. • E2A-PBX1 childhood ALL • Prognosis is improved with intensified therapy and is presently not considerd a high risk category. • Identical t(1;19) breakpoints occur rarely in early B-progenitor ALL but not involving E2A or PBX1, and the prognosis is excellent without the need for intensified therapy! Cytogenetics is not enough...

34. der(19)t(1;19)(q23;p13)

35. the presence of non-random translocations such as t(9;22), t(4;11), and t(12;21) indicate that the translocation is most likely the primary change and that the hyperdiploidy is probably a secondary event -different prognostic impact! • Additional anomalies translocations and other structural chromosome abnormalities are present in approximately half of high hyperdiploid cases e.g. dup(1q) and del(6q) - no known prognostic significance wcp-whole chromosome painting probes Hyperdiploid ALL • The number of chromosomes are usually 52-57 • common extra copies of chromosomes; X, 4, 6, 10, 14, 17, 18, and 21 • A good prognosis

36. Prognostic Significance of Cytogenetic Changes in Childhood ALL 100% t(12;21) t(1;19) 50% t(4;11) t(9;22) 1 2 3 4 5 Years

37. Rowley, 1973

38. CHRONIC MYELOID LEUKAEMIAS (CML) • CML is not a specific entity associated with a single anomaly i.e. The Ph chromosome. • Predominantly a disease of adults (median 45-55 years) • splenomelgaly, hepatomegaly, anaemia, sweating, weight loss, bleeding, abdominal fullness, thrombocytosis, fatigue

39. t(9;22)(q34;q11) • 1960 Nowell and Hungerford - the Philadelphia chromosome • Janet Rowley 1973 - t(9;22)(q34;q11) • The first: -specific chromosomal abnormality identified in neoplasia associated with a characteristic cytogenetic evolution pattern that correlated with the clinical behaviour of a disease -to be cloned and characterised at the molecular level - the treatment that specifically targets the cells harbouring a genetic change • t(9;22)(q34;q11) is characteristic for CML. However, not specific as it also can be detected in ALL, and more seldom in AML.

40. t(9;22)(q34;q11) • t(9;22) is at the diagnosis of CML most often the sole anomaly • At blast crisis (transformation to acute leukaemia) additional changes occur in 80% of the cases e.g. +8, i(17)(q10) and an extra Philadelphia chromosome • Gleevec Imatinib mesylate (STI571)

41. normal t(9;22)(q34;q11) BCR/ABL fusion • The t(9;22) yields the gene fusion BCR/ABL1 • In some occasional % of typical CML is one not able to identify the BCR/ABL1 fusion. fusion interphase FISH metaphase FISH

42. A normal karyotype does not exclude a BCR/ABL fusion! t(8;22)(p11;q11) ABLinserted in BCR CML cases with variants of the t(9;22) - 10% of the cases

43. Chronic lymphoproliferative disorders • The blood film - increase in maturelymphocytes. • elderly patients • with marked lymphocytic infiltration of the bone marrow, leading to an immunocompromised state and progressive marrow failure. • spleen may be massively enlarged.

44. Chronic lymphocytic leukemia • Very difficult to retrieve metaphases from the neoplastic cells • normal karyotype or isolated 13q14 deletions - good prognosis • patients with deletions of 17p13 or 11q23 do very badly indeed. Trisomy 12 lower surivival.

45. B-CLL or B-PLL (B-cell prolymphocytic leukaemia) • t(14;18)(q32;q21)-little or no prognostic significance, may be overlooked with R-banding, • 6q- (variable breakpoints) lower survival • patients often present with advanced stage disease : • this disease is always progressive; poor response to therapy • inv(14)(q11q32), t(14;14)(q11;q32) involving  T-cell receptor (TCRA) and IGH • at advanced age, progresses rapidly • generally more aggressive than B-PLL; prognosis: poor response to chemotherapy T-CLL or T-PLL

46. Myelodysplastic syndromes (MDS) • A closely related group of acquired BM disorders - the haemopoiesis is generally ineffective with increased cell death in the BM leading to various cytopenias • may be primary or may evolve in the course of other BM diseases of be secondary to previous exposure to cytotoxic chemotherapy, irradiation or other environmental toxins • occurs predominantly in the elderly • In contrast to the ones in acute leukaemias are the chromosomal changes in MDS most often not specific, but can be very characteristic. • approximately 3000 cases of MDS with chromosomal aberrations have been published

47. Monosomy 5 • loss of the entire chromosome 5 is less common than 5q- in MDS and is almost always accompanied with other aberrations such as -7/7q-, and -12/der(12p), and is often seen in therapy-related MDS. del(5q) • the most common chromosomal aberration in MDS • most often involved in complex karyotypes (bad prognosis) • can appear as the sole anomaly i.e. the 5q- syndrome (elderly women with macrocytic anaemia, good prognosis) • The breakpoints for the deletion on 5q varies and it is yet unknown which gene/genes of pathogenetic importance.

48. del(7q) • Deletion of parts of the long arm of chromosome 7 • Often seen together with aberrations in complex karyotypes and is then said to be a bad prognostic sign The combination of 5q- and -7/7q- is seen in MDS treated with alkylating agents • The second most common chromosomal aberration in MDS • Can be detected as sole anomaly, but most common together with other changes. • Loss of chromosome 7 is associated with a bad prognosis Monosomy 7

49. Trisomy 8 • 3rd most common aberrations in MDS (20%) and is seen in the same frequencies of all the MDS subtypes. del(20q) • deletions of the long arm of chromosome 20 can be identified in 5-10% of MDS (rarely in CMML) • as sole anomaly - god prognosis Loss of the Y chromosome • 5-10% of MDS • -Y as a sole anomaly is also seen in elderly males without any haematological malignancy - should not be taken as evidence of malignant change when seen alone.

50. Burkitt lymphoma • lymphomas tend to contain karyotypic changes more often than in most leukaemis and are more complex • Manolov and Manolova described in 1972 the t(8;14)(q24;q32) in 75-85% of the cases • MYC/IGH • t(8;14) present in the endemic (Africa) and in the nonendemic (America, Japan, Europe) • variant translocations: t(8;22)(q24;q11) and t(2;8)(p12;q24)MYC, IGK, IGL