Chromosome aberrations in Chromosome aberrations in HAEMATOLOGIC HAEMATOLOGIC MALIGNANCIESMALIGNANCIES

Increased growth

Blockage of differentiation

Less apoptosis

Haematological malignancies

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

HAEMATOLOGIC MALIGNANCIES MAY BE CLASSIFIED BY:HAEMATOLOGIC MALIGNANCIES MAY BE CLASSIFIED BY:

1. CLINICAL COURSE:

acute leukaemias or chronic leukaemias

2. LINEAGE:

lymphoid lineage

myeloid lineage

Starring Ali MacGraw, Starring Ali MacGraw, Ryan O'Neal, Ryan O'Neal, John Marley John Marley

Erich Segal, 1970Erich Segal, 1970

AML-M2AML-M2t(8;21)(q22;q22)t(8;21)(q22;q22)

History History 1956 1956 Correct number of human Correct number of human

chromosomechromosome 1960 Ph chromosome1960 Ph chromosome 1970 1970 Banding techniqueBanding technique

1973 t(8;21)1973 t(8;21)1977 t(15;17), t(4;11)1977 t(15;17), t(4;11) 1979 1979 High-resolution banding techniqueHigh-resolution banding technique

19801980 1982 t(9;11) in AML-M5a, inv(3) in AML1982 t(9;11) in AML-M5a, inv(3) in AML1983 del(16q), inv(16) in AML-M4E 1983 del(16q), inv(16) in AML-M4E 1984 t(1;3) in AML with 1984 t(1;3) in AML with dysmegakaryopoiesisdysmegakaryopoiesis

t(1;19) in ALLt(1;19) in ALL ISCN (1985,1991,1995)ISCN (1985,1991,1995)

5

19801980 1983 1983 MYC-IGHMYC-IGH --- --- t(8;14)t(8;14) 1984 1984 ABL-BCRABL-BCR --- --- t(9;22)t(9;22)

IGH-BCL2IGH-BCL2 --- --- t(14;18)t(14;18)1988 ATRA in APL Tx1988 ATRA in APL Tx

1990 1990 1991 1991 MLL MLL --- t(4;11)--- t(4;11)

PBX1-E2APBX1-E2A --- --- t(1;19)t(1;19) PML-RARAPML-RARA --- --- t(15;17)t(15;17) AML1-ETOAML1-ETO --- --- t(8;21)t(8;21) DEK-CANDEK-CAN --- t(6;9)--- t(6;9)

1993 1993 CBFB-MYH11CBFB-MYH11 --- --- inv(16)inv(16) MLL-AF9MLL-AF9 --- --- t(9;11)t(9;11)

Most Patients with Acute Most Patients with Acute Leukemia have Characteristic Leukemia have Characteristic gene rearrangementgene rearrangement

6

5 000

10 000

15 000

20 000

25 000

30 000

35 000

40 000

1975 1980 1985 1990 1995 2000

Database of Chromosome Aberrations in Cancer

year

No. of cases

Hematological disorders

62%Lymphomas9%

Solid tumors29%

Recurrent Balanced Chromosome Rearrangements in Neoplasia

Tumor type No of No of cloned No of genesaberrations 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

HEMATOLOGY and CLINICAL GENETICS

Connection?

Importance?

1. BM morphology

2. Immunological features

3. Chromosome analysis

4. Condition of the patient

1.

2. 3.

4.

What´s the use of chromosomal changes?

1. Diagnosis

2. Prognosis

3. Choice of treatment

4. Evaluation of treatment (e.g. interphase-FISH)

Culture media and Culture media and additivesadditives

Standard mediaStandard media

RPMI 1640RPMI 1640

Eagle’s MEMEagle’s MEM

Ham’s F12Ham’s F12

McCoy’s 5A mediumMcCoy’s 5A medium

Cell Culture procedure Cell Culture procedure

Peripheral blood

2183-98

Glass flask

Falcon flask

medium

+

cells

HARVESTING PROCEDURESHARVESTING PROCEDURES

Fixation (3:1) methanol: acetic acid

Hypotonic treatment (0.075 M KCl)

Colcemide 30 min.Colcemide 30 min.

metaphase

Nismyth et al 1996

Spreading

Glass flask

Falcon flask

metaphase

interphase

ds DNA

ss DNA

ss PROBES

Fluorescence in situ hybridization (FISH)

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.

•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) t(8;21) 6.9%6.9%

t(15;17)t(15;17) 6%6%

11q2311q23 2.5%2.5%

inv(16)inv(16) 1.71.7

Frequency of specific chromosome abnormalities in AML

The 4 most common

•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

t(8;21)(q22;q22)t(8;21)(q22;q22)

•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

t(15;17)(q22;q12-21)

hypergranular promyelocytic leukemia

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)

•Treatment is an emergency in APLTreatment is an emergency in APL! Intra vascular coagulation - 10-40% early mortality (intra cerebral haemorrhage)

•oral trans-retinoic acidoral 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 conditionsAPL-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!

t(15;17)(q22;q12-21)

11q23-aberrations11q23-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)

•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

PROGNOSIS VERY POOR in general

•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

inv(16)(p13q22)inv(16)(p13q22)

N inv(16)

Morphology:

•excess of monocytes

•variable number of eosinophilic granules larger than normal, purple-violet in colour

inv(16)(p13q22)inv(16)(p13q22)

•Cytogenetic analysis of ALL has been hampered by the difficulty of obtaining good quality chromosomes:

poor spreadingfuzzy chromosomesindistinct 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

ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)

MLL rearr 75%

Random 25%Infant

Prevalence of genetic changes in ALL with respect to different age groups

Random 30%

TEL/AML1 20%

Hyperdiploidy (above 50) 25%

E2A/PBX1 5%

BCR/ABL 4%

MLL rearrangements 6%

Miscellaneous 10%

Childhood

Random 40%

BCR/ABL1 25%

Miscellaneous 17%

MLL rearrangments 7%

Hyperdiploidy 6%

E2A/PBX1 3%

TEL/AML1 2%Adult

•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

t(4;11)(q21;q23)

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!

•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

•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

t(9;22)(q34;q11) in ALL

Philadelphia chromosome

•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...

t(1;19)(q23;p13.3)

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

•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

Hyperdiploid ALLwcp-whole chromosome painting probes

•Additional anomaliestranslocations 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!

t(12;21)

t(1;19)

t(4;11)

t(9;22)

50%

100%

Years42 31 5

Prognostic Significance ofCytogenetic Changes in Childhood ALL

Rowley, 1973

CHRONIC MYELOID LEUKAEMIAS (CML)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

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.

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)

t(9;22)(q34;q11) BCR/ABL fusion

interphase FISH

normal

metaphase FISH

•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

ABL inserted in BCR

t(8;22)(p11;q11)

CML cases with variants of the t(9;22) - 10% of the cases

A normal karyotype does not exclude a BCR/ABL fusion!

•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.

Chronic lymphoproliferative disordersChronic lymphoproliferative disorders

•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.

Chronic lymphocytic leukemia

• 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-PLLT-CLL or T-PLL

B-CLL or B-PLL (B-cell prolymphocytic leukaemia)B-CLL or B-PLL (B-cell prolymphocytic leukaemia)

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

•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 -7/7q-, and -12/der(12p), and is often seen in therapy-related MDS.

Monosomy 5Monosomy 5

del(5q)del(5q)

•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 7Monosomy 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)del(7q)

The combination of 5q- and -7/7q- is seen in MDS treated with alkylating agents

•3rd most common aberrations in MDS (20%) and is seen in the same frequencies of all the MDS subtypes.

•deletions of the long arm of chromosome 20 can be identified in 5-10% of MDS (rarely in CMML)

•as sole anomaly - god prognosis

•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.

Trisomy 8Trisomy 8

del(20q)del(20q)

Loss of the Y chromosomeLoss of the Y chromosome

•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

Burkitt lymphomaBurkitt lymphoma

• the haemopoiesis is generally effective

•MPDs: Polycythemia vera, essential trombocytopenia, and idiopathic myelofibrosis, CML

•Chromosomal aberration patters in MPD are not often specific.

MYELOPROLIFERATIVE DISORDERS (MPD)

•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 veraPolycythemia 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 myelofibrosisIdiopathic myelofibrosis

•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)

Hairy cell leukaemiaHairy cell leukaemia

•t(14;18)(q32;q21)

•IGH/BCL2

•t(11;18)(q21;q21)- most often seen as a sole anomaly

•API2/MALT1

•confer a growth advantage to MALT lymphoma cells

Follicular lymphomaFollicular lymphoma

MALT lymphoma (NHL)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 diseaseHodgkin´s disease

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.

Cancer free Cancer free