IMPORTANCE OF CYTOGENETICS
IN
HEMATOLOGIC MALIGNANCIES
Marie Jarošová
and Sabina Sevcikova
IHOK FN Brno
and BMG UPF LF MU
What is cytogenetics?
 Study of number and structure of chromosomes, their
properties, behavior during cell division, influence on
phenotype
 Any changes in number or structure may lead to disease
 Changes lead to disrupted genes leading to proteins that
do not function correctly
 Depending on size, location, and timing, structural changes
in chromosomes can lead to birth defects, syndromes or
even cancer
Cytogenetics
 All cells are effected
 Changes are stable
 Tumor cytogenetics (acquired alterations)
 Only a portion of cells is effected
 Clonal evolution or regression
 Role of sensitivity of any method
 Clinical cytogenetics (germline alterations)
 A tumor is a genetic disease that arises as a consequence of
accumulation of various genetic changes
 Every 3rd person will get a tumor disease
 In the Czech Republic, more than 70 thousand people are
diagnosed with a tumor every year
 Tumor cells are characterized by chromosomal changes:
numerical or structural changes of chromosomes
Tumor genetics
 Studies of acquired chromosomal changes of tumor cells
 Analyses of numerical and structural changes
of chromosomes
 Basic method – G- banding
 One analysis checks the entire genome
Tumor cytogenetics
History of cytogenetics
Early cytogenetics
• 1842 – Carl Nageli- cell division (anomaly), botanist
• 1879 – Walther Flemming – chromosomal movement
during mitosis
• 1882 – published his work
• 1888 – Waldeyer - chromosome
1940s – the sex revolution
Dr. Barr and Dr. Bertram discovered sex-chromatin, now known
as the Barr body, while working at The University of Western
Ontario, in 1949.
GLCC 50th anniversary
GLCC 50th anniversary
1950s – the hypotonic revolution
• T. C. Hsu discovered the
utility of hypotonic solution
in 1952
• Tijo and Levan reported the
correct chromosome
number in humans in vitro.
• This was confirmed within a
year, in vivo by Ford and
Hamerton (1956) They could count the chromosomes but
classification was rough and approximate.
1970s – the banding revolution
GLCC 50th anniversary
High Resolution G-banding,
Yunis, 1975
Klasická cytogenetika
 80s – imaging – automated imaging
 90s – colors – unique locus probes
 00s – genomics – SNP arrays, next gen sequencing
80s, 90s, 00s
GLCC 50th anniversary
Cytogenetics FISH
arrayCGH/SNP array
Cytogenetic methods
Profase Prometafase Metaphase
Anafase Telofase
1-2ml
cultivation
2/24/72 hrs
+
colcemide
COLCEMIDE BLOCKS MITOSIS IN METAPHASE
bone marrow
peripheral blood
lymph node
Molecular cytogenetics
 Methods based on fluorescence in
situ hybridization (FISH) – based on
molecular as well as classical
cytogenetics
 Methods use the basic property of
single stranded DNA to bind
together based on complementarity
Denaturate
Hybridize
centromeric
Whole chromosome
gene
Types of probes
Multicolor fluorescence in situ hybridization
(M-FISH)
M-FISH is based on hybridization of 24 fluorescently
labeled whole chromosome probes that allow the
staining of all chromosomal pairs by different colors
• Combines paint probes
specific for a region of a
chromosome
• Banding covers the whole
chromosome
Mband FISH
Převzato I.Chudoba
aCGH in MM
J. Smetana
Analysis
J. Smetana
 Diagnosis
 Prognosis
 Treatment decisions
Cytogenetics in hematology
Genetic changes in
hematological malignancies
 90-95% of CML
 60-80% of AML
 60% of MDS
 50-80% of CLL
 60-90% of NHL
 70-90% of MM
 70-90% of ALL
Philadelphia chromosome (Ph)
First specific chromosomal aberration linked to a tumor
CYTOGENETIKA CML
90-95% Ph1 result of translocation
t(9;22)(q34;q21)
Cytogenetics of CML
Additional chromosomal changes
Diagnosis: ~12%
Accelerated phase: ~30%
Blast crisis : ~70%
Diagnosis
Prognosis
Philadelphia chromosome
Philadelphia chromosome (Ph)
 1960 – Peter Nowell and
David Hungerford described
abnormal chromosome in
CML
 First genetic signature of
cancer – growth advantage
for abnormal cells?
 Cause or consequence?
 Janet Rowley in the 1972 –
t(9,22)
Philadelphia chromosome (Ph)
 1983 abl (Heisterkamp)
 1984 bcr (Groffen)
 1990 bcr-abl cause of CML (Daley)
 1990 - bcr-abl- abnormal tyrosine kinase activity (Lugo)
 Chronic phase, accelerated phase, blast crisis
 Bad prognosis
Gleevec - Imatinib mesylate (1993)
 Activity against CML colonies (Druker 1996)
 2 years later – clinical study, 31 patients, 98% response
 Clinical trial phase III – 16 countries, 177 centers, 1100
patients – closed early
 All patients moved to Gleevec arm
 Survival 95%, 65% in blast crisis
 Molecular positivity still a problem
 Dasatinib, nilotinib….
https://www.google.cz/search?q=gleevec+cml&dcr=0&source=lnms&tbm=isch&sa=X&ved=0ahUK
EwjokaX99NnWAhXMDMAKHUfAAisQ_AUICigB&biw=1366&bih=604#imgrc=TNlp3Ot1Yx6sbM:
Type of Response Definition
CHR
Complete Hematologic
Response
Normal differential, WBC &
platelets ≤ ULN
MCyR
Major cytogenetic
Response
0–35% Ph+marrow metaphases
CCyR
Complete Cytogenetic
Response
0% Ph+marrow metaphases
MMR Major Molecular Response
BCR-ABL/ABL ≤ 0.1%
(International Scale)
MR4.0 BCR-ABL/ABL ≤ 0.001% (IS) “4-log
reduction”
MR4.5 BCR-ABL/ABL ≤ 0.003% (IS) “4.5log
reduction”
CMR
Complete Molecular
Response
Undetectable BCR-ABL (test of
sensitivity ≥4.5 logs)
MRD in CML
WHO Classification 2008
WHO Classification
 Since 2008, cytogenetics is part of diagnosis and
classification for many hematological malignancies
 Cytogenetics is a part of WHO classification of AML
 Together with cytomophology stratifies MDS patients
 Classification of lymphomas – histology, cytogenetics and
FISH confirm classification
 Is part of prognostic stratification of MM
WHO classification of AML
WHO prognostic stratification of AML
Grimwade D et al. Blood 2010;116:354-365
Stratification based on cytogenetics
APL t(15;17)(q22;q12) / PML-RARA
CBFB-Rearrangement
t(15;17)(q22;q12)
PML-RARA
Janet D Rowley et al. Lancet 1977
Targeted treatment of APL
 heterogeneous disease with monoclonal proliferation and
expansion of lymphoid cells in BM, PB and other organs
 Cytogenetics- prognostic significance
 Imunophenotyping – diagnostic significance
ALL
Distribution of cytogenetic abnormalities from data collected from UK childhood ALL treatment trials.
Christine J. Harrison Hematology 2013;2013:118-125
©2013 by American Society of Hematology
Pediatric ALL cca 30% of all pediatric tumors
Frequency of cytogenetic subtypes of pediatric ALL
Charles G. Mullighan Hematology 2012;2012:389-396
©2012 by American Society of Hematology
46,XY,dic(2;12)(?;p?12)t(2;16)(?;q?),der(10)t(2;10)(q?12;q?12),t(12;21)(p13;q22),+21
Boy b 1998, dg. BCP-ALL 2003
 de novo MDS 40-60%
 t-MDS or secondary MDS 90%
 SNPs+arrayCGH 70%
Chromosomal changes in MDS
Clinical heterogeneity is mirrored in heterogeneity of acquired genetic changes
MDS
Refractory cytopenia with unilineage dysplasia (RCUD)
Refractory anemia with ringed sideroblasts (RARS)
Refractory cytopenia with multilineage dysplasia (RCMD)
Refractory anemia with excess blasts-1 (RAEB-1)
Refractory anemia with excess blasts-2 (RAEB-2)
Myelodysplastic syndrome, unclassified (MDS-U)
Myelodysplastic syndrome associated with isolated del(5q)
WHO classification
MDS
Prognostic stratification of MDS
46,XX,del(5)(q31)
5q- SYNDROME
 10% of patients
 good prognosis
 5-16 % progress into AML
Döhner H, Stilgenbauer S, Benner A, Leupolt E, Krober A,
Bullinger L, Dohner K, Bentz M, Lichter P: Genomic
aberrations and survival in chronic lymphocytic leukemia.
N Engl J Med 2000; 343:1910-1916.
Prognostic significance of
cytogenetics in CLL
CLL and genetic abnormalities:
Probability of survival
Döhner et al. N Engl J Med 2000
Months
Patientssurviving
(%)
100
80
60
40
20
0
0 24 48 72 96 120 144 168
13q deletion
17p (p53)
deletion
11q deletion
12q trisomy
Normal
N=26
(4.0%)
NOTCH1 M
SF3B1 M
BIRC3 M
del13q14
+12
TP53 M
del11q22-q23
MYD88 M
BIRC3 del
del17p13 *
* **
Rossi et al. Blood 2013
0 2 4 6 8 10 12 14
0.00.20.40.60.81.0
Years from diagnosis
CumulativeprobabilityofOS(%)
del13q14
Normal/+12
NOTCH1 M/SF3B1 M/
del11q22-q23
TP53 DIS/BIRC3 DIS
Matched general popul.
Mutational and cytogenetic model
of CLL
Puiggros et al, BMRI 2014
CLL – prognostic and treatment stratification
NON HODGKIN LYMPHOMA
 Heterogenous group of tumors of the lymphatic tissues
 Arise from genetic changes in originally normal cells
 Classification – histopathology WHO 2008
 Cytogenetics and FISH used for classification
Folicular lymphoma (FL)
indolent B cell lymphoma
~20% of all lymphomas
Clinically heterogenous, OS up to 20 years
90% of patients t(14;18)(q32;q21)
Fúze IGH/BCL2
MANTLE CELL LYMPHOMA
 Aggressive disease (OS 3-5 years)
 ∼ 6 % všech NHL
 Diagnostics:
 Morphology
 Imunohistochemistry
 Imunophenotyping
 Genetics:
 cytogenetics
 FISH
 Molecular genetics - PCR
• FISH
dual color dual fusion probes
IgH/CCND1 DC
DF Kreatech
• Conventional
cytogenetics
t(11;14)
MANTLE CELL LYMPHOMA
NON-HODGKIN LYMPHOMAS IN CHILDREN
Převzato: Hochberg et al, BJH 2008
 4-7% of tumors in children and young adults
 Incidence increases with changes
 WHO classification 2008
 frequency of histological subtypes different from adults
48,X,-Y,del(1)(p13pter),+der(1)del(1)(q?24q?ter)t(1;4)(q23;?q?),
+ider(1)(q11)del(1)(q?24q?ter)t(1;4)(q23;?q?),del(6)(q?15),+7,t(8;14)(q24;q32)(1.klon-56%)
11/2011
BURKITT LYMPHOMA
MULTIPLE MYELOMA
Walker B, Wardell CP, Melchor L et al., Submitted
SUMMARY - CYTOGENETICS
 Diagnostics and prognostic stratifications of hematologic
malignancies
 Can analyze the entire genome in one run
 Allows for clarification of diagnosis by specific
chromosomal aberration
 Recurrent nonrandom changes determine prognosis of
disease
 Aberration classification allows monitoring of treatment
efficacy
Thanks for your attention