Download Leukemia SUDAN HEM MAL

Chromosome aberrations in
HAEMATOLOGIC
MALIGNANCIES
Haematological malignancies
Increased growth
Blockage of differentiation
Less apoptosis
HAEMATOLOGIC MALIGNANCIES MAY BE CLASSIFIED BY:
1. CLINICAL COURSE:
acute leukaemias or chronic leukaemia
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
AML-M2
t(8;21)(q22;q22)
Starring Ali MacGraw,
Ryan O'Neal,
John Marley
Erich Segal, 1970
History




1956
1960
1970
1973
1977
1979
1980
1982
1983
1984
Correct number of human chromosome
Ph chromosome
Banding technique
t(8;21)
t(15;17), t(4;11)
High-resolution banding technique
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
Most Patients with Acute Leukemia
have Characteristic gene
rearrangement

1980
1983
1984
1988

1990
1991
1993
MYC-IGH
ABL-BCR
IGH-BCL2
--- t(8;14)
--- t(9;22)
--- t(14;18)
ATRA in APL Tx
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-MYH11
--- inv(16)
MLL-AF9
--- t(9;11)
6
Database of Chromosome
Aberrations in Cancer
No. of
cases
40 000
35 000
30 000
25 000
20 000
15 000
10 000
5 000
1975
1980
1985
year
1990
1995
2000
Solid tumors
29%
Hematological
disorders
62%
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
HEMATOLOGY and
CLINICAL GENETICS
Connection?
Importance?
1. BM morphology
2. Immunological features
3. Chromosome analysis
1.
4. Condition of the patient

4.
2.
3.
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 additives
Standard media
RPMI 1640
Eagle’s MEM
Ham’s F12
McCoy’s 5A medium
Cell Culture procedure
medium
Glass
flask
+
2183-98
cells
Peripheral blood
Falcon flask
HARVESTING PROCEDURES
Glass
flask
Falcon flask
Colcemide 30 min.
Hypotonic treatment
(0.075 M KCl)
Fixation (3:1) methanol:
acetic acid
Spreading
Nismyth et al 1996
metaphase
Fluorescence in situ hybridization (FISH)
ds DNA
metaphase
ss DNA
interphase
ss PROBES
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.
Frequency of specific chromosome abnormalities in AML
The 4 most common
t(8;21)
6.9%
t(15;17)
6%
11q23
2.5%
inv(16)
1.7
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
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
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
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!
11q23-aberrations
•Cases: 25% are infants (<1 yr)
children and adults each represent 50%
of cases
•Clinics: organomegaly;
CNS involvement (5%);
•both AML and ALL
6q27,
9p21-22
•MLL - a promiscuous gene
11q23 (MLL)
•in 70% of infant leukaemias
10p11-15
19p13
•The most common
translocationpartners are:
•Extremely hard to identify
t(6;11), t(9;11), and t(11;19).
PROGNOSIS VERY POOR in general
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
inv(16)(p13q22)
Morphology:
•excess of monocytes
•variable number of eosinophilic granules larger than normal,
purple-violet in colour
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
Infant
Random
25%
Random 30%
Sektor 1
Sektor 2
Sektor 3
Sektor 4
TEL/AML1 20%
Hyperdiploidy (above 50) 25%
MLL rearr
75%
E2A/PBX1 5%
BCR/ABL 4%
Childhood
Random 40%
MLL rearrangements 6%
BCR/ABL1 25%
Miscellaneous 10%
Miscellaneous 17%
Prevalence of genetic
changes in ALL with
respect to different age
groups
MLL rearrangments 7%
Hyperdiploidy 6%
E2A/PBX1 3%
TEL/AML1 2%
Adult
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
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!
t(8;14)(q24;q32)
•this translocation is typical for B-ALL (but also
described in Burkitt´s lymphoma)
•Fusion of the genes IGH and MYC.
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
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
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 Bprogenitor ALL but not involving E2A or PBX1, and the
prognosis is excellent without the need for intensified
therapy! Cytogenetics is not enough...
der(19)t(1;19)(q23;p13)
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
•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 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
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
Rowley, 1973
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
•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
normal
interphase FISH
metaphase FISH
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
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.
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.
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
T-CLL or T-PLL
•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
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
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.
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.
Monosomy 7
•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
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
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.
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
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.
Polycythemia vera
•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
Idiopathic myelofibrosis
•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
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)
Hodgkin´s disease
•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)
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
Gleevec®
Gleevec® Indications and Usage
(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