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-M2 t(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
Banding technique
t(8;21)
t(15;17), t(4;11)
High-resolution banding technique
1980
t(9;11) in AML-M5a, inv(3) in AML
del(16q), inv(16) in AML-M4E
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
MYC-IGH t(8;14)
ABL-BCR t(9;22)
IGH-BCL2 --- t(14;18)
ATRA in APL Tx
1990
MLL t(4;11)
PBX1-E2A --- t(1;19)
PML-RARA t(15;17)
AML1-ETO t(8;21)
DEK-CAN t(6;9)
CBFB-MYH inv(16)
MLL-AF t(9;11) 6

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

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

9. Recurrent Balanced Chromosome Rearrangements in Neoplasia
Tumor type No of No of cloned No of genes
aberrations breakpoints involved
Hematologic disorders
Mesenchymal tumors
Epithelial tumors
Total
Mitelman et al: Recurrent Chromosome Aberrations in Cancer (2002)

10. HEMATOLOGY and CLINICAL GENETICS
Connection?
Importance?

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

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
Peripheral blood
Glass
flask
Falcon flask
medium +
cells

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

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

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. Frequency of specific chromosome abnormalities in AML
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%
11q %
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)
Pathognomonic acute 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. hypergranular promyelocytic
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 % 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% are infants (<1 yr)
Cases: 25% are infants (<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
11q23-aberrations
The most common translocationpartners are:
Extremely hard to identify t(6;11), t(9;11), and t(11;19).
6q27, 9p21-22
10p11-15
19p13
11q23 (MLL)
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. Frequent CNS involvement, even at diagnosis
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 number of chromosomes are usually 52-57
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
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
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!

36. Prognostic Significance of Cytogenetic Changes in Childhood ALL
50%
100%
Years 4 2 3 1 5
Prognostic Significance of
Cytogenetic Changes in Childhood ALL

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 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 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. 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.
normal
fusion
interphase FISH
metaphase FISH

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

43. Chronic lymphoproliferative disorders
The blood film - increase in mature lymphocytes.
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. del(5q) Monosomy 5 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.
loss of the entire chromosome 5 is less common than 5q- in MDS and is almost always accompanied with other aberrations such as /7q-, and -12/der(12p), and is often seen in therapy-related MDS.
Monosomy 5

48. 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
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
del(7q)
The combination of 5q- and -7/7q- is seen in MDS treated with alkylating agents

49. Loss of the Y chromosome
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

51. MYELOPROLIFERATIVE DISORDERS (MPD)
the haemopoiesis is generally effective
MPDs: Polycythemia vera, essential trombocytopenia, and idiopathic myelofibrosis, CML
Chromosomal aberration patters in MPD are not often specific.

52. Idiopathic myelofibrosis
most cases have an apparently normal karyotype at the time of diagnosis,
but the number of PV with chromosomal changes increase during the progression of the disease e.g. 20q-, +8, +9, 13q- and gain of 1q
Polycythemia vera
the number of aberrant cases varies between different series, 20-70%
the most common changes are -7,+8,+9,5q-,13q-, and 20q-
monosomy 7 syndrome - disorder of childhood, mostly boys, repeated infection episodes, hepatosplenomegaly, progression to AML poor prognosis
Idiopathic myelofibrosis

53. Hairy cell leukaemia
mainly a disease of middle-aged men, only 20% of the patients are female
finger-like cytoplasmic projections visible on the cell surface - hairs
low spontaneous mitotic activity and are difficult to stimulate into mitosis
add(14)(q32), 6q-, del(14q)

54. Hodgkin´s disease Follicular lymphoma MALT lymphoma (NHL)
t(11;14)(q13;q32)
CCND1/IGH
various other changes; including der(1q), t(3;22)(q27;q11), der(6q), t(14;18)(q32;q21)
Hodgkin´s disease
Follicular lymphoma
t(14;18)(q32;q21)
IGH/BCL2
MALT lymphoma (NHL)
t(11;18)(q21;q21)- most often seen as a sole anomaly
API2/MALT1
confer a growth advantage to MALT lymphoma cells

55. Gleevec® Indications and Usage
Gleevec® (imatinib mesylate) is indicated for the treatment of: • Newly diagnosed adult and pediatric patients with Philadelphia chromosome     positive chronic myeloid leukemia (Ph+ CML)in chronic phase. Follow-up is limited. • Patients with Philadelphia chromosome positive chronic myeloid leukemia (Ph+ CML) in     blast crisis, accelerated phase,or in chronic phase after failure of interferon-alpha therapy.     Gleevec is also indicated for the treatment of pediatric patients with Ph+ chronic phase CML     whose disease has recurred after stem cell transplant or who are resistant to interferon-alpha     therapy. There are no controlled trials in pediatric patients demonstrating a clinical benefit, such as     improvement in disease-related symptoms or increased survival. • Adult patients with relapsed or refractory Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL). • Adult patients with myelodysplastic/myeloproliferative diseases (MDS/MPD) associated with PDGFR (platelet-derived     growth factor receptor) gene rearrangements. • Adult patients with aggressive systemic mastocytosis (ASM) without the D816V c-Kit mutation or with c-Kit mutational     status unknown. • Adult patients with hypereosinophilic syndrome (HES) and/or chronic eosinophilic leukemia (CEL) who have the     FIP1L1-PDGFRa fusion kinase (mutational analysis or FISH demonstration of CHIC2 allele deletion) and for patients with     HES and/or CEL who are FIP1L1-PDGFRa fusion kinase negative or unknown. • Adult patients with unresectable, recurrent and/or metastatic dermatofibrosarcoma protuberans (DFSP). • Patients with Kit (CD117) positive unresectable and/or metastatic malignant gastrointestinal stromal tumors (GIST).     (See CLINICAL STUDIES, Gastrointestinal Stromal Tumors.) The effectiveness of Gleevec in GIST is based on objective     response rate (see CLINICAL STUDIES). There are no controlled trials demonstrating a clinical benefit, such as improvement     in disease-related symptoms or increased survival.

56. Cancer free
Thank You