Karyotypic complexity and multiclonality (either cytogenetically unrelated clones or cytogenetically related clones) represent two parameters of prognostic significance in management of patients with Myelodysplastic syndromes. Presented by Georgia Bardi, PhD at MDS workshop in Perugia 2008. Cytogenetic data produced by Eugenia Gourgouveli and Despina Iakovaki.

1. KARYOTYPIC COMPLEXITY AND
MULTICLONALITY:
TWO CYTOGENETIC PARAMETERS TO BE
CONSIDERED IN MDS PROGNOSIS
Georgia Bardi
BioAnalytica-GenoType SA
Molecular Cytogenetic Research and Applications,
Athens, Greece
No. 350ISO 15189

2. Why are we still doing
conventional cytogenetics
in MDS?
MDS:
 umbrella diagnosis
 No single genomic target per subgroup
 unknown genomic changes associated
with early stages
 unknown changes that trigger the
progression to advanced stages and to
AML
Karyotypic analysis:
 a genome-wide picture and
 information on inter- and intra-
neoplasmic heterogeneity

3. Karyotype: a variable in IPSS of MDS
Low risk: no clonal aberration
-Y as the sole anomaly
5q- as the sole anomaly
20q- as the sole anomaly
High risk: 7q-/-7
≥3 aberrations
? ? ?
Intermediate:
other
aberrations
(all the rest)
4-5000 cytogenetically abnormal MDS in the literature
 Correlation analysis: cytogenetic and clinicopathologic features (IPSS)
more cytogenetic data needed: improvement in IPSS cytogenetic classification

4.  122 bone marrow
specimens
 patients with de-novo
MDS at the time of
diagnosis
 2½-year period
Sex: 68% ♂- 32% ♀
average age: 73 years old
WHO classification:
RA/RARS: 34%
RCMD: 27%
RAEB-I και –ΙΙ: 17%
5q-: 3%
Unclassified: 12%
CMML:7%
Follow-up time: 1-30 months
Progression to AML: 15%
Cytogenetic analysis of MDS

5. Abnormal
49% Normal
51%
62 had a normal karyotype
60 showed clonal chromosome aberrations

6. 1p
1q
2p
2q
3p
3q
4p
4q
5p
5q
6p
6q
7p
7q
8p
8q
9p
9q
10p
10q
11p
11q
12p
12q
13p
13q
14p
14q
15p
15q
16p
16q
17p
17q
18p
18q
19p
19q
20p
20q
21p
21q
22p
22q
X
Y
Gain
1p
3p
5p
5q
6q
7p
7q
9q
12p
12q
13q14p
17p17q 19p
20q
21p
21q
Y
8p
8q
0
5
10
15
20
25
30
%involvementinabnormalMDS
Chromosome arm
Chromosomal imbalances in MDS
Gain
Loss
Genomic loss is more frequent than gain in MDS

7. Distribution of no of aberrations per sample (abnormal MDS)
1 ab
61%
2 ab
16%
3 ab
8%
4 ab
2%
5 ab
2%
6 ab
2%
7 ab
2%
8 ab
3%
12 ab
2%
13 ab
2%
Distribution of abnormal samples according to the no. of aberrations
The average number of chromosome aberrations per sample was 2 (range, 1-13)

8. 46,ΧY,t(3;3)(q21;q26)[3]/46,idem,+der(
2)t(2;?;20)(q14;?;p13),-7, +der(20)
t(2;?;20)(q14;?;p13)[17]/44-45,idem,
+der(2)t(2;?;20)(q14;?;p13),-7,-
19,+der(20) t(2;?;20)(q14;?;p13)[3]/43-
45,idem,+der(2)t(2;?;20)(q14;?;p13),-7,
+der(20)t(2;?;20)(q14;?;p13),-21[3]
46,ΧX,del(20)(q12)[19]/46,XX[6]

9. GOOD
70%
POOR
15%
INTERMEDIATE
15%
Low risk: no clonal aberration, -Y, 5q-, 20q- as the sole clonal change
High risk: 7q-/-7 &≥3 aberrations
Intermediate risk: other aberrations
Distribution of abnormal samples according to IPSS criteria

10. 11%
related
clones
6%
unrelated
clones
6%
single clone
38%
normal
50%
More than one cytogenetically abnormal clones
12%

11. Karyotype
No of
clones
No of
aberrations
45,Χ,-Υ[15]/45,idem,+del(12)(p13)[3]/46,XY[5] 2 2
46,ΧY,t(3;3)(q21;q26)[3]/46,idem,+der(2)t(2;?;20)(q14;?;p13),-7, +der(20)
t(2;?;20)(q14;?;p13)[17]/44-45,idem, +der(2)t(2;?;20)(q14;?;p13),-7,-19,+der(20)
t(2;?;20)(q14;?;p13)[3]/43-45,idem,+der(2)t(2;?;20)(q14;?;p13),-7,
+der(20)t(2;?;20)(q14;?;p13),-21[3]
4 6
45,ΧΥ,-7[10]/46,ΧΥ,-7,+13[16]/46,ΧΥ[2] 2 2
47,ΧΥ,-1,+add(1)(p13)x2[6]/47,idem,del(20)(q13)[16]/46,XY[2] 2 3
45,ΧΥ,-3,+del(3)(p13p25)x2,del(5)(q21q33),del(12)(q14q15),-18,-20[21]/46,XY,-
3,+del(3)(p13p25)x2,-5,del(12)(q14q15),-
20,+mar[2]/44,XY,del(3)(p13p25),del(5)(q21q33),del(12)(q14q15),-18,-20[2]
3 8
46,ΧΥ,der(2)t(2;9)(p23;p21),del(9)(p21)[11]/46,XY,idem,+del(8)(q22q24)[9]/92,XX
YY,idemx2,+del(8)(q22q24)x2[4]/46,XY[2]
3 3
46,ΧΥ,der(1)t(1;?;5)(p36 ;?;q13),-5,+8,der(12)t(12;17)(q13 ;p11),-
14,der(17)t(12;17)(q13;p11),+mar[21]/ 45,idem,-mar[2]/46,XY[2
2 7
Cytogenetically Related abnormal clones
 More than one abnormal, cytogenetically related clones (2-4) were
identified in 6 % of the cases:

12. Karyotype
abnormal
clones x no of
aberrations Total aberrations
45,Χ,-Υ[7]/46,XY,del(20)(q12)[3]/46,XY[19] 2x1 2
46,ΧΥ,add(11)(q23)[3]/46,ΧΥ,del(12)(p12)[6] /(46,XΥ[12] 2 2
45,ΧΥ,-14[3]/45,ΧΥ,-21[3]/46,ΧΥ,del(5)(q22q23)[2]/
46,ΧΥ,add(7)(q32)[3]/46,XY[11] 4x1 4
45,Χ,-Υ[15]/ 46,ΧΥ,del(5)(q15q33)[6]/46,ΧΥ[5] 2x1 2
82-84,ΧΧΥΥ,-4,-5,-5,-5,-11,-12,-14,-15,-15,del(17)(q11)x2,
der(19)t(8;19) (q12;p12),add(19)(p13)[4]/46,XY,del(5)(q32q34)
[2]/46,XY[15] 1x9+1x1 10
45-47,ΧΥ,add(6)(q23)[5],add(11)(q23)[5][cp9] /45,XY,-13[3]/
46,XY[12] 1x2+1x1
3
46,ΧΥ,del(6)(q16)[3]/46,XY,del(9)(q31q32)[4]/46,XY[22] 2 2
Cytogenetically Unrelated abnormal clones
 More than one abnormal, cytogenetically unrelated clones (2-4) were
identified in 6 % of the cases

13. Correlation analysis
1. Age
2. Sex
3. WHO subgroup
4. Progression to
AML
1. Abnormal karyotype
2. Cytogenetic complexity (no
of chromosome aberrations
per case)
3. Cytogenetic heterogeneity
(more than one abnormal
clone: related/unrelated)
ClinicopathologyCytogenetics

14.  The percentage of abnormal karyotypes was higher in men than in women (p=0,04)
64
36
44
56
51
49
0
10
20
30
40
50
60
70
%
Female Male Total
Sex-Karyotype
Normal
Abnormal
Karyotype and Sex

15.  The mean no of chromosome aberrations in men was higher than that in
women (F:0,41 / M:1,43)
0,00
0,50
1,00
1,50
2,00
2,50
3,00
3,50
4,00
4,50
F M
Sex
Noab ± Standard deviation
p=0,0005
Karyotype and Sex

16.  The percentage of abnormal karyotypes increased from 39% in RA/RARS
to 55% in RCMDS/RCMD and 70% in RAEB-1/RAEB-2
39
61
55
45
70
30
0
10
20
30
40
50
60
70
Frequency(%)
RA/RARS RCMD RAEB-I&II
WHO subgroup
ABNORMAL
NORMAL
Karyotype and WHO subgroups

17.  The mean number of aberrations per case in WHO subgroups provide strong evidence of
increasing karyotypic complexity from RA/RARS to RCMDS/RCMD and RAEB-1/RAEB-
2 (0,50 in RA/RARS, 1,31 in RCMD and 2 in RAEB-1/RAEB-2)
0,00
0,50
1,00
1,50
2,00
2,50
3,00
3,50
4,00
4,50
5,00
5q- RA/RARS RCMD RAEB-I/II MDS-U
WHO
Noab
± Standard deviation
Karyotype and WHO subgroups

18. 0
10
20
30
40
50
60
70
80
%Frequency
5q- RA/RARS RCMD Unclassified RAEB
WHO subgroups
Frequncy of multiple abnormal clones in WHO subgroups
Related
Unrelated
 The frequency of unrelated abnormal clones was higher in RCMD, suggesting clonal heterogeneity
whereas that of related clones was higher in RAEB-1/RAEB-2, documenting clonal evolution at the
cytogenetic level.
p=3,30887E-33
Frequency of multiple abnormal clones in WHO subgroups

19. 9
91
21
79
0
10
20
30
40
50
60
70
80
90
100
NORMAL ABNORMAL
PROGRESSION TO AML NO PROGRESSION
p=0,07
 9% of patients with normal karyotypes developed AML compared to 21% of those
with abnormal karyotypes.
Karyotype and progression to AML

20.  The MDS that at the end of the follow-up had progressed to AML, displayed a higher
number of aberrations per case at diagnosis (2,56) compared to those who had not
(0,88).
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
Y N
AMLProgr
Noab
± Standard deviation
p=0,008
Karyotype and progression to AML

21. Karyotypic features correlate with clinicopathologic characteristics of patients with de novo
MDS (Sex, WHO subgroups, risk to AML progression).
Karyotypic complexity increases from RA towards RAEB
Cytogenetically unrelated clones are more common in RCMD than any other subgroup: they
may represent cell populations with different genetic alterations present in the bone marrow of
patients at the early stages in the development of multilineage dysplasia. One of these cell
populations with growth advantages might become dominant and evolve to AML.
Cytogenetically related clones are more common in RAEB than any other subgroup: they
probably represent cytogenetic evidence of clonal evolution of the neoplastic cell population at
later stages of MDS towards AML development.
Normal independent clones one dominant clone
subclones
AML
Early
events
Late
events
SUMMARY

22. IPSS refinement: karyotypic complexity and multiclonality to be
considered in MDS prognostic evaluation
Independent abnormal clones with 1 anomaly:
to classify the case according to the one with the worse prognosis
Related abnormal clones:
Classify the case as high risk independently of the no of
aberrations present in each of the clones
Prognostic assessment of karyotypic complexity and multiclonality in
multivariate analysis, using classical clinicopathological parameters
as covariates, including survival of the patients
Cytogenetic Multiclonality and WHO classification (European Database)
What is next in MDS cytogenetics?

23. Δ Ιακωβάκη, Ιωάννης Τσίρκας, Γεωργία Μπάρδη
1. Μαρία Δήμου, Παναγιώτης Παναγιωτίδης: Λαϊκό Νοσοκομείο
2. Μάρκος Φισφής, Αικατερίνη Στεφανουδάκη: Νοσοκομείο Αμαλία Φλέμιγκ
3. Ειρήνη Παναγιώτη, Ευφημία Βρακίδου-Λιούρη, Δημήτρης Γρενζελιάς:
Θεραπευτήριο Υγεία
4. Ελένη Χανδρινού, Ελισάβετ Βερβεσού, Αλίκη Μανιάτη: ΓΝ Ερρίκος Ντυνάν
5. Δέσποινα Μπαρμπαρούση, Χάρις Ματσούκα: Νοσοκομείο Αλεξάνδρας
6. Χρήστος Ποζιόπουλος: 7401 ΓΣΝΕ
7. Ιωάννης Αποστολίδης, Νικόλαος Χαρχαλάκης: Νοσοκομείο Ευαγγελισμός