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Analysis of complex chromosomal rearrangements in adult patients with MDS and AML by multicolor FISH
Leukemia Research 31 (2007) 39–47
Analysis of complex chromosomal rearrangements in adult patients with MDS and AML by multicolor FISH Libuse Babicka a,∗ , Sarka Ransdorfova b , Jana Brezinova b , Zuzana Zemanova a , Lenka Sindelarova a , Magda Siskova c , Jacqueline Maaloufova b , Jaroslav Cermak b , Kyra Michalova a,b a
c
Center of Oncocytogenetics, Institute of Clinical Biochemistry and Laboratory Diagnostics, General Faculty Hospital and 1st Faculty of Medicine of Charles University, Prague, Czech Republic b Institute of Hematology and Blood Transfusion, Prague, Czech Republic 1st Medical Department, General Faculty Hospital and 1st Medical Faculty, Charles University, Prague, Czech Republic Received 28 February 2006; received in revised form 28 February 2006; accepted 4 March 2006 Available online 9 May 2006
Abstract We analyzed complex chromosomal aberrations in 37 adult patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) using classical cytogenetic method, FISH with locus-specific probes, multicolor FISH (mFISH) and multicolor banding (mBAND). Unbalanced structural aberrations, leading to a gain or loss of chromosomal material, were frequently observed in bone marrow cells. In 30 patients (81.1%) loss or rearrangement of chromosome 5, 7 and/or 11 was found. The most frequent numerical change was trisomy 8 as expected (detected in six patients—16.2%) and the most frequent breakpoints 5q13, 5q33, 7q31, 10p12, 11q23, 12p13, 17p11 and 21q22 were determined. © 2006 Elsevier Ltd. All rights reserved. Keywords: MDS; AML; Complex chromosomal aberrations; mFISH; mBAND
1. Introduction Cytogenetic findings in bone marrow cells of patients with myelodysplastic syndrome—refractory anemia with excess of blasts in transformation (MDS RAEB-T) and acute myeloid leukemia (AML) are essential for precise diagnosis and classification of the disease. Karyotype of leukemic cells is one of the important prognostic factors [1–6] and it was proved by multivariate analyses that it is an independent predictor of therapy response, remission duration and survival [7]. Acquired cytogenetic aberrations are detected in 55–75% of newly diagnosed patients with AML [8]. Most of karyotypic abnormalities are associated with specific disease subtypes, characteristic morphologic and immunologic profiles ∗
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0145-2126/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2006.03.010
and distinct therapeutic and prognostic implications [2,8,9]. However, approximately 10–15% of AML with cytogenetic aberrations have no specific rearrangements, but do have complex chromosomal aberrations [8,10]. Similarly, patients with myelodysplastic syndrome (MDS) have at diagnosis pathological cytogenetic findings in 50–60% of cases. Specific and recurrent clonal chromosomal abnormalities observed in leukemic cells of patients with MDS and AML can be used to classify them into groups with consistent clinical features and similar prognosis. Three major translocations found in AML, all indicators of good prognosis, are associated with particular FAB subtypes and/or morphological features: t(8;21)(q22;q22) with M2 and granulocytic maturation, t(15;17)(q22;q21) with M3, inv(16)(p13q22) with M4 and eosinophilia [2,11]. According to Grimwade et al. [2] the group of patients with 11q23 abnormalities, +8, +21, +22, del(9q), del(7q) or other structural or numerical defects have an intermediate prognosis, patients with complex karyotype
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L. Babicka et al. / Leukemia Research 31 (2007) 39–47
and −5, del(5q), −7, or abnormalities of 3q have very poor prognosis [2,5]. Complex chromosomal aberrations (CCAs) involving more than two chromosomes and/or more than three breakpoints are associated with rather poor prognosis and respond poorly to antileukemic treatment and it was suggested that some of these rearrangements contribute to drug resistence and disease progression [2,8,12–17]. Reported complete remission rates in AML vary from 21 to 46%, with a median overall survival between 1 and 5 months [2,18]. We performed mFISH study of complex karyotypes in bone marrow cells of 60 patients with MDS and/or with primary or secondary AML, 37 of them were finally included into cohort and evaluated in detail. We detected balanced and/or unbalanced chromosomal aberrations, determined exact breakpoints, investigated whether unbalanced rearrangements lead to random or non-random chromosomal gains or losses. The most frequent breakpoints were determined on chromosomes 5, 7, 10, 11, 12, 17 and 21. 2. Patients From January 1998 to December 2004 we examined 311 adult patients with MDS or AML by classical cytogenetic method. Selection criteria for mFISH study were: diagnosis of AML, secondary AML (sAML) or MDS RAEB-T, according to the FAB and WHO classifications, presence of chromosomal aberrations involving more than two chromosomes and/or more than three breakpoints. Sixty patients with complex rearrangements were examined by mFISH, mBAND, locus-specific probes (LSI) and/or whole chromosome painting (WCP) probes. There were 30 males, 30 females, with an average age of 53.3 years (range 20–81 years). From this group of patients we excluded those who had specific translocations (eight patients, three with t(8;21), two with t(15;17) and one with inv(16), one patient with constitutive chromosomal translocation t(14;22)). Acquired chromosomal changes, probably as posttreatment karyotype evolution were found in 10 patients and they were excluded from this study as well. Clinical data and description of complex karyotypes confirmed by all methods in bone marrow cells of 37 patients are presented in Table 1 .
3.2. FISH analyses FISH analyses were performed according to the result of G-banding using commercially available probes following the manufacturer’s recommendations. Locus-specific DNA probes (LSI Abbott VYSISTM and Q BIOgeneTM ) were used to detect the (1) fusion genes: PML/RARA [t(15;17) LSI Kit], AML1/ETO (LSI AML/ETO Kit ES) and CBF/MYH11 (LSI CBFB Dual Color Probe [inv (16)]), (2) rearrangements and/or deletions: 5p15/5q31 (LSI EGR1/D5S721, D5S23), 7q22 and 7q35 (7q22/7q35 Dual-Color Specific Probe), 7q31 (LSI D7S486 7q31/CEP 7), 11q13 (LSI Cyclin D1/CEP 11 Kit), 11q22.3 (LSI ATM [11q22.3]) and 11q23 (LSI MLL Dual Color Rearrangement Probe). Whole chromosome painting probes (CAMBIOTM ) were used to define more precisely structural rearrangements. The number of mitoses examined by classical cytogenetics and FISH analyses depended on their availability on the chromosomal preparations; usually 20 mitoses were evaluated. At least 200 interphase nuclei were analyzed. To rule out the presence of the false positive results, cut-off level was established at 2.5% based on analyses of bone marrow samples from 10 healthy individuals. The signals were detected using an AXIOPLAN 2 Imaging microscope (Zeiss) or AX 70 PROVIS microscope (Olympus) equipped with computer analysis system ISIS (MetaSystemsTM ). 3.3. mFISH, mBAND mFISH was carried out using 24 XCyte MetaSystems color kit with combinatorially labeled painting probes specific for all autosomes and sex chromosomes. Hybridization and post-hybridization washes were described in detail by manufacturer. Analyses were done on AX 70 PROVIS fluorescence microscope (Olympus) or AXIOPLAN 2 Imaging microscope (Zeiss) with filters for FITC, Spectrum OrangeTM , TexasRed® , DEAC, Cy5TM and DAPI using ISIS software (MetaSystemsTM ). In some cases multicolor banding mBAND was carried out using XCyte color kits (MetaSystemTM ) for chromosomes 3, 5 and 11.
3. Methods
4. Results
3.1. Conventional cytogenetics
Complex chromosomal aberrations were identified by conventional cytogenetic methods in bone marrow cells of 60 patients out of 311 (19.3%), 37 (11.9%) were included into this study and the karyotypes of bone marrow cells examined at diagnosis are presented in Table 1. The most frequently observed aberrations were unbalanced chromosomal translocations, leading to the gain or loss of chromosomal material. In these translocations the most often were involved chromosomes 5, 7 and 11 (Fig. 2).
Bone marrow cells were cultivated for 24 h in RPMI 1640 medium with 10% of fetal calf serum without any stimulation and were harvested using conventional techniques. Cytogenetic analyses were done on G-banded chromosomes using computer analysis for karyotyping (MetaSystemsTM ). Karyotypes were described according to the International System of Human Cytogenetic Nomenclature (ISCN, 1995, 2005) [19,20].
L. Babicka et al. / Leukemia Research 31 (2007) 39–47
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Table 1 Clinical data of examined patients, karyotypes according to G-banding and mFISH analyses No.
Sex/age
Diagnosis
Surviv.
Karyotype
1
F/48
AML M1–M2
6
2 3
F/75 F/78
AML M5 AML M4
7 1
4 5
M/38 F/63
AML M0 AML M1
9 14
6
F/41
AML
3
7
F/66
AML M1
0.6
8
F/64
AML
0.2
9 10
F/76 F/68
MDS → AML AML M2
NA NA
11
M/62
RAEB-T
3
12
M/25
AML M5
6
13
F/65
AML M2
4
14
F/26
AML M2
NA
15 16 17
F/72 M/55 M/71
AML M4 AML M0 AML M6
0.2 4 6
18
F/64
MDS → AML
15
19 20
M/66 F/58
AML M4 RAEB-T
9 NA
21
M/80
RAEB-T
9+
22
F/67
AML
26
23 24 25
M/47 F/78 M/45
AML M5a AML M0 AML M0
5 1 6
26 27
M/70 M/73
MDS → AML AML M4
NA 1
28
M/62
AML M2
2
29
M/67
RAEB-T
2
46, XX, der(6)t(6;11)(q27;q23), del(11)(q23) [2]/48, XX, der(6)t(6;11)(q27;q23), t(10;17)(q21;q12), +10, del(11)(q23), +17 [4] 46, XX, der(10)t(10;11)(p12;q23), der(11)t(10;11)(p?;q12)t(1;10)(q?;p?) [11] 46, XX [2]/46, XX, der(11)t(1;11)(?;q23) [19]/46, XX, idem, i(8)(q10) [5]/46, XX, der(22)t(1;22)(?;p11.2) [4]/ 46, XY [2]/47, XY, +X, del(5)(q22q33), t(10;11)(p12;q21) [6] 47, XY, +X, idem, −22 [3] 44, XX, der(2)t(2;17)(p16;?), −3, der(4)t(4;5)(q21;?), −5, del(6)(p24), del(8)(q24), del(11)(q24), −12, −13, der(15)t(4;12;15)(?;?;q15), der(15)t(12;15;16)(?;q15;?), der(16)del(16)(p11.2)del(16)(q13), +der(16)t(15;16)(q15;q13), der(17)t(8;17)(q24;q12), der(20)ider(20)(q10)ins(20;12)(p12;?), +der(22)t(11;12;22)(q24;?;q11) [5]/43, idem, −17 [5]/44, idem, −17, +del(8)(q?) [2] 45, XX, der(2)t(2;8)(q24;?), der(3)t(3;5)(q13.3;p13), del(3)(p12);t(4;10)(p15.3;p13), del(5)(p13), del(6)(q25), −10, −11, del(13)(q13), del(14)(q24), del(15)(q25), +18, der(21)t(11;21)(?;q22), dup(22)(q?) [10] 46, XX [2]/46, XX, del(5)(q13q33), del(7)(q21q33), dup(11)(q13q23), −17, der(20)t(17;20)(q12;q11) [7]/46, XX, del(5)(q13q33), del(7)(q21q33), +8, dup(11)(q23q23), −14, der(17)t(14;17)(?;q12), der(20)t(17;20)(q12;q11) [7]/54, XX, +4, del(5)(q13q33), +5, del(7)(q21q31), +8, +8, +16, −17, +18, +18, +19, der(20)t(17;20)(q12;q11), −20 [1] 45, XX, −14, der(16)t(16;21)(p12;?), der(17)t(17;19;21)(p11.2;p13.1;?), der(19) t(14;19)(?;p13), dic(21;21)(q22.1q22.1) [2]/46, idem, +der(16)del(16)(p11.2)del(16)(q12) [4] 42, XX, −5, der(6)t(6;17;18)(p12;?;?), der(7)t(7;16)(p13;?), der(11)t(6;11;13;18)(?;p15;q13;?) [6] 45, XX, del(5)(q14q33), der(5)t(1;5)(?;q35), der(7)t(7;12)(q21;?), +8, −12, der(16)ins(16;17)(q?;?), −17, der(20)t(17;20)(?;q11.2) [9] 45, XY, del(5)(q13q33), del(7)(q22q35), dup(11)(q?), der(17)t(17;21)(p11.2;?), der(20)t(20;21)(q12;?), −21 [2]/44, XY, del(4)(q32), del(5)(q13q33), der(7)t(3;7)(q?;q21), −15, der(16)t(15;16)(?;q22), der(17)t(17;21)(p11.2;?), der(20)t(20;21)(q12;?), −21 [4] 47, XY, del(8)(q11.2), +del(8)(q11.2), der(13)t(8;13)(q11.2;?) [3]/48, idem, +10 [3]/49, idem, +del(8)(q11.2), +10 [2] 46, XX [4]/42, XX, der(4)t(4;5;19)(q13;?;?), der(5)t(5;8;12)(p14q14;?;?), −7, der (8)t(8;12;17)(p12;?;?), +11, −12, der(13)t(13;19)(p11;?), −17, −18, −19 [14] 47, XX, i(8)(q10), +der(8)t(8;10)(q23;?), der(10)t(10;22)(p14;?), +19 [27]/48, idem, t(4;6)(q26;q22) [2] 44, XX, del(5)(q13q33), −7, t(8;12)(q24.2;p13), −16, del(18)(q21) [20] 46, XY, del(7)(p12) [18]/46, XY, idem, t(2;2)(q31;q35) [23] 48, XY, del(5)(q13q33), der(7)t(7;19)(p14;?), del(13)(q33), +der(22)t(20;22)(?;p12), +der(22)t(13;20;22)(?;?;p12q?) [6]/49, idem, +del(5)(q13q33) [2] 46, XX [14]/45, XX, der(1)ins(1;11)(p11;?), der(2)t(2;5)(q33;?), del(5)(q12q33), −11 [8]/46, idem, +der(1)ins(1;11)(p11;?), der(5)t(2;5)(?;q12), der(7)t(7;12;15)(p21q31;p13;q26), der(12)t(7;12)(?;p13), del(15)(q26) [1] 50, XY, del(8)(q11.2), +12, der(13)t(8;13)(q11.2;p11.2), +21, +21 [16]/49, idem, −8 [10] 46, XX, del(5)(q14q33), der(7)del(7)(q31)t(7;12)(q33?;q24), der(12)del(12)(p13)t(7;12)(q33?;q24), der(16)t(1;16)(?;q22) [22] 42, XY, der(5)t(5;7)(p12;?), −7, der(12)t(12;16)(p11;?), −16, der(19)t(19;21)(p13;q22), −19, der(20) t(20;22)(q12;q13), del(21)(q22), −22 [8]/44, XY, del(1)(p?), der(5)t(5;7)(p12;?), −7, der(12)t(12;14)(p11;q12), del(14)(q12), der(19)t(19;21)(p13;q22), −19, der(20)t(20;22)(q12;q13), del(21)(q22), +der(21)t(1;21)(?;q21), −22 [2] 46, XX [2]/51, XX, der(2)t(2;5)(p11.2;?), +der(2)t(2;5)(p11.2;?), der(3)t(2;3)(p11.2;p11), +der(3)t(2;3)(p11.2;p11), −5, +6, +8, +13, +21 [8] 50, XY, +5, +6, +18, +der(19)t(X;19)(?;p13.2) [8] 46, X, t(X;7)(p22.1;p15) [13]/47, idem, +22 [12] 46, XY [3]/69, XY, +X, +1, +2, +4, +4, +5, +6, +7, +8, +10, t(10;11)(p12;q23), +12, +13, +13, +13, +14, +17, +18, +19, +19, +19, +20, +21, +21, +21, −22 [19] 43, XY, der(5)t(5;11;16)(q12;?;?), der(6)t(6;11)(q27;q23), −11, −16, −18 [24] 52, XY+6, +8, +9, der(11)t(11;22)(p15;?)dup(11)(q?), +der(11)t(11;22)(p15;?)dup(11)(q?), +13, der(17)t(7;17)(?;p11), +der(17)t(7;17)(?;p11), +19, der(21)t(11;21)(?;p11)dup(11)(q?), −22 [3]/53, idem, +der(7)del(7)(p)del(7)(q) [5]/44, XY, −7, r(11)(p14q24), der(13)t(4;13;21)(?;p11;?), der(17)t(7;17)(?;p11), −21 [2] 43, XY, der(2)t(2;17)(q36;q?), −5, der(12)t(5;12)(?;q13), der(17)t(17;18;20)(p11q12;?;?), −18, −20 [9] 72, XY, +Y, +del(1)(p?), +2, +2, +3, −4, del(5)(q13.1q33.3), +6, +6, +8, +8, +der(9)t(4;9)(?;q11), +der(9)t(4;9)(?;q11), der(10)t(10;18)(p12;?), +der(10)t(10;18)(p12;?), +11, +13, +13, +del(14)(q21), +15, +15, +17, +19, +der(19)t(1;19)(p?;q13), +20, +21, +21, +der(22)t(14;22)(q21;q13) [4]/46, XY, del(5)(q13.1q33.3) [5]
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L. Babicka et al. / Leukemia Research 31 (2007) 39–47
Table 1 (Continued ) No.
Sex/age
Diagnosis
Surviv.
Karyotype
30
F/41
RAEB-T
?
31 32
M/20 M/57
AML M0 AML M5a
3 0.1
33
M/61
AML M1–2
4+
34
F/81
MDS → AML
7
35
M/78
AML
10
36
F/80
AML M2
0.3
37
F/58
AML M6
NA
46, XX [8]/46, XX, der(1)t(1;13;17)(q21;?;q22), del(2)(p22), der(3)t(2;3)(p22p24;q26q27), del(5)(q13q33), del(8)(q24.1), der(9)ins(9;8)(q22.3;q24.1q24.3), der(12)t(9;12)(?;p13), del(13)(q32), der(17)t(1;17;19)(q21;q22;?) [12] 48, XY, +4, t(4;7)(q31;q31), del(13)(q33), der(21)ins(21;7)(q21;q?), +der(21)ins(21;7)(q21;q?) [30] 44, XY, der(2)t(2;5)(q13;p13.2), der(5)del(5)(p13.2)del(5)(q12), der(7)ins(7;11)(q21;?)t(7;11)(q31;?), −11, −12, der(17)t(2;17)(q13;p11.2), der(17)t(12;17)(q11;p11.2) [1]/45, XY, idem, der(20)t(12;20)(p11;q12) [3]/43, XY, der(2)t(2;5)(q13;p13.2), t(4;6)(q21;q22), −5, −5, der(7)ins(7;11)(q21;?)t(7;11)(q31;?), del(9)(q22), −11, der(17)t(2;17)(q13;p11.2), der(20)t(9;20)(q22;q12) [2] 45, XY, del(5)(q13q33), t(6;16)(q12;q22), −7, der(10)t(10;14)(p14;q?), del(12)(p12), der(14)t(6;14) (q?;q23), del(18)(p11.3), der(21)ider(21)(q10)t(1;21)(?;p11.1) [3]/45, XY, idem, der(Y)ins(Y;12)(q11.2;p12p13) [1] 45, XY, idem, der(Y)ins(Y;12)(q11.2;p12p13), der(22)t(22;22)(q11.1;q11.1) [1] 45, XY, idem, der(4)t(4;11)(q24;?) [1] 46, XX [1]/44, X, der(X)t(X;7)(q26;?), t(3;11)(q26;p13), t(5;13)(q23.3;q12), der(5)t(X;5)(?;q11.2), der(7)t(5;7)(q11.2;?), der(12)t(12;22)(p13;q11), der(14)t(14;15)(q10;q10), −15, 22p+ [5]/43, idem, −7 [3] 46, XY [1]/44, XY, −4, −5, der(7)t(5;7;10)(q13;p11;?), der(10)t(4;5;10)(?;p15.5q23;p?q?), der(11)t(4;11)(?;p14) [21] 44, XX, der(1)t(1;3)(q32;?), −3, der(5)del(5)(q12q33)t(5;18)(p13;p11), −7, der(18)t(1;3;18)(?;?;p11) [7] 46, XX [1]/49, XX, del(5)(q14q33), t(17;20)(p11.2;q12), +19, +21, +22 [12]
Surviv.—survival in months, survival + patients live in complete remission (November 2004), NA—not available, MDS → AML—myelodysplastic syndrome transformed in acute myeloid leukemia, BMT—bone marrow transplantation.
4.1. Changes of chromosome 5, 7 and 11 Numerical and structural aberrations of chromosome 5 were ascertained in 22 patients (59.5%). Monosomy was found in five cases (Nos. 5, 9, 22, 28 and 35). Rearrangements of long arms of chromosome 5 were proved in four patients (Nos. 10, 13, 26 and 34) and deletion of long arm was detected in 14 patients, in all of them deletion of the critical region 5q31 was confirmed by FISH. The most frequent deletion was del(5)(q13q33) found in bone marrow cells of seven patients, in three of them deletion del(5)(q14q33) was ascertained and in two deletion del(5)(q12q33) was determined. Changes of chromosome 7 were presented in 12 patients (32.4%). Monosomy of chromosome 7 was found in five cases (Nos. 13, 15, 21, 33 and 36). We found aberrations of the long arm of chromosome 7 in four patients, in all of them (Nos. 10, 20, 31 and 32) with rearrangements or losses of 7q31 region. Deletion of long arms of chromosome 7 was found in three cases and critical region 7q31 was lost in all three as was proved by FISH with locus-specific probes. Aberrations of chromosome 11 were detected in 13 patients (35.1%), monosomy was found in three cases (Nos. 6, 18 and 32) and FISH with LSI MLL Dual Color Rearrangement Probe was performed in all of them. In three patients (Nos. 1, 2 and 25) FISH proved rearrangements of the MLL gene. In one patient (No. 18) deletion of the MLL gene and in two cases (Nos. 9 and 11) amplification of the MLL gene was determined. In four others (Nos. 3–6) the MLL gene was not altered. In total, aberrations of chromosomes 5 and/or 7 and/or 11 were detected in 30 (81.1%) out of 37 patients. Rearrangements of chromosome 5 associated with changes of
chromosome 7 were found in six patients, rearrangements of chromosome 5 together with chromosome 11 were detected in four patients and rearrangements of chromosomes 7 and 11 were determined in one patient. In two patients complex karyotypes were associated with aberrations of all these three chromosomes (see Fig. 1). 4.2. Other aberrations The majority of structural abnormalities were unbalanced. We found translocation der(13)t(8;13)(q11.2;p11.2), del(8)(q11.2) in two patients, i.e. Nos. 12 and 19. In patient No. 12 translocation was associated with duplication of del(8)(q11.2) and trisomy of chromosome 10. In patient No. 19 translocation was associated with trisomy of chromosome 21, tetrasomy of chromosomes 21 and monosomy of chromosome 8. We did not detect any other repeatedly occurring translocations in our cohort. The other aberrations were insertions, isochromosomes, dicentric and ring chromosomes. Insertions were detected in six patients (Nos. 5, 10, 18, 30, 31 and 32), isochromosome i(8)(q10) was proved in two cases (Nos. 3 and 14). Isochromosomes der(20)ider(20)(q10)ins(20;12) and der(21)ider(21)(q10)t(1;21)(?;p11.2) were ascertained only once (cases Nos. 5 and 33, respectively). One ring chromosome r(11)(p24q24) was determined (case No. 27). Dicentric chromosome involving chromosome 21 was found in patient No. 8: dic(21;21)(q22.1;q22.1). 4.3. The breakpoints associated with complex aberrations Chromosomal breakpoints that could be unequivocally assigned by comparison of results obtained with different
L. Babicka et al. / Leukemia Research 31 (2007) 39–47
43
Fig. 1. Incidence of chromosomes 5, 7 and 11 aberrations as determined by FISH with locus-specific probes, mFISH and mBAND in 37 patients with complex aberrant karyotype.
types of classical and FISH analyses are depicted in Fig. 2. Losses and rearrangements attributed to structural abnormalities numerously involved were chromosome arms 5q, 7q, 11q and 10p, 12p and 17p. The most frequent breakpoints were found to be: 5q33 (12x), 5q13 (8x), 5q12 (5x), 7q31 (4x), 11q23 (6x), 10p12 (4x), 12p13 (7x) and 17p11.2 (7x). Interestingly, both sex chromosomes X and Y were sporadically involved in complex aberrations (Fig. 2).
5. Discussion Precise identifications of clonal chromosomal aberrations and delineation of breakpoints in bone marrow cells of patients at diagnosis could lead to a better understanding of the genetic events during leukemogenesis and disease progression, as well as guiding further molecular studies of genes involved in leukemogenesis, and could be providing clinically relevant information that can assist in the development of risk-adapted therapeutic strategies. Moreover, cytogenetic data until now are particularly useful for determining prognosis of the disease and/or prediction of response to certain therapeutic treatment. Several studies have shown that specific chromosomal aberrations t(8;21)(q22;q22), inv(16)(p13q22) or t(15;17)(q22;q21) are associated with a good prognosis [13,15,17,21–24] on contrary to aberrations of chromosomes 5, 7, inv(3)/t(3;3) and complex aberrant karyotype, which are all indicators of a very poor prognosis [2,13,15,22,23,25,26]. The aberrations seen in AML cases with complex chromosomal rearrangements have often been referred as random, because the same chromosomal rearrangement is rarely identified in more than one patient. However, in the past few years, new cytogenetic methods, such as mFISH, SKY and CGH [27–29], with significantly higher resolution than Gbanding have been introduced and allowed a more detailed characterization of CCAs. In our study we have analyzed complex rearrangements in 37 patients with MDS RAEB-T
or AML by mFISH method to identify exactly all numerical and structural changes and to confirm origin of every marker chromosome, supernumerary chromosomes and chromosomal parts, which are involved in CCAs and tried to establish the frequency of non-random changes. In already published reviews one-third of deletions found by G-banding were shown to represent cryptic translocations (both balanced and unbalanced) or insertions using mFISH or SKY techniques. In agreement with the literature we found complex chromosomal rearrangements in 11.9% of patients with de novo AML or MDS (37 patients out of 321) at diagnosis, with majority of structural unbalanced rearrangements with variable breakpoints within a small region of single chromosomes. Complex unbalanced rearrangements lead to loss rather than amplification of chromosome material, suggesting possible involvement of tumor suppressor genes. In our study chromosomes most often involved in CCR were found to be 5, 7, 8, 10, 11, 12, 17 and 21 with aberrations of chromosomes 11, 5 and 7 as the most frequent ones. According to Heim and Mitelman [11] and Bredeson et al. [30] chromosomal abnormalities involving long arm of chromosome 11, especially of 11q23 region, have been described in 5–10% of published cases of adult patients with de novo AML and in 80–90% therapy-related AML and they were connected with topoisomerase II inhibitors. In our study of complex rearrangements chromosomal aberrations of 11q were detected in 13 patients (35.1%). Using locus-specific probes the rearrangement of MLL gene was observed in three patients, deletion of one MLL allele in one patient and duplication of the MLL gene in two cases. Extra copies of MLL gene are a recurrent genetic change found in approximately 20% of MDS and AML [31,32]. It is often associated with dysplastic bone marrow changes or complex karyotypes suggestive of genotoxic exposure and with poor prognosis for the patient [33]. Our results correlate well with reported data. In our group with 11q aberrations all patients died and median overall survival of this group was found to be 4.5 months.
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Fig. 2. Incidence of chromosomal breakpoints assigned by FISH with locus-specific probes, mFISH, mBAND and G-banding.
As expected, the majority of cases (22 patients; 59.2%) showed aberrations associated with chromosome 5—loss of 5q material, monosomy or unbalanced rearrangements of 5q. Deletion of 5q can be detected in 10% of de novo AML [34]. The size of deletion varies among patients, del(5)(q13q33) is
the most frequent one, with common deleted segment 5q31 [35]. Our findings correspond with the data quoted in the literature. In our cohort of patients deletion of the critical region 5q31 was found in 14 patients and the most frequent deletion del(5)(q13q33) was determined in eight patients.
L. Babicka et al. / Leukemia Research 31 (2007) 39–47
45
Fig. 3. Karyotype of patient No. 4 with complex chromosomal rearrangements, revealed by multicolor FISH: 46, XY, +X, del(5)(q22q33), t(10;11)(p12;q21), −22.
Monosomy or the loss of part of chromosome 7 is a consistent finding in malignant myeloid disorders, occurring in all subtypes of MDS and AML. The minimal deleted segment or critical region, in which the putative tumor suppressor genes could be located has been variously reported as 7q22, 7q31 and 7q33-q34 [36,37] suggesting that 7q may contain more than one myeloid leukemia-related gene. Aberrations associated with chromosome 7 were detected in our cohort in 12 patients (32.4%). Monosomy was found in five patients. In all cases with del 7q (three patients) region 7q31 was deleted and in four patients with aberrations involving 7q rearrangements of 7q31 region was proved. Schoch et al. [38], analyzed complex aberrant karyotypes associated with the most frequently rearranged chromosomes—aberrations of chromosomes 5, 7 and/or 11. In our study it was 30 patients (81.1%) out of 37 who had loss or rearrangement of chromosomes 5, 7 and/or 11. We also proved an excess of chromosomes 5, 7 and 11 abnormalities in the group of patients with complex karyotype compared to patients with simple karyotype. Our findings correspond well with the results of studies of Limbergen et al. [32] and Schoch et al. [38]. The current data elucidate the role of loss of chromosomal material, especially of 5q and 7q, in the pathogenesis of AML with a
complex aberrant karyotype. Lindvall et al. (2004) have combined transcriptional profiles with cytogenetic data from 15 AML patients with normal and abnormal complex karyotypes [8]. Their results show that AML with complex karyotypes exhibit specific gene expression profiles. The differentially expressed genes included several which are located on 5q and 7q, as well as genes involved in control of cell division. Further analyses of expression of genes located on 5q and 7q will help to specify which genes show a reduced expression and may be active during progression of the disease (Figs. 3 and 4). In agreement with previously published data [38,39] trisomy of chromosome 8 was observed as the most frequent numerical change and was confirmed by mFISH in six patients. We did not determine any new recurrent change in our cohort, but we identified chromosomal parts and breakpoints occurring repeatedly in complex translocations. The most frequently involved in CCAs were found to be 5q, 7q, 11q, 21q, 10p, 12p and 17p and breakpoints: 5q13 (8x), 5q33 (12x), 7q31 (4x), 11q23 (6x), 21q22 (4x), 12p13 (7x), 17p11 (7x) and 10p12 (4x). Our results correlate with data already published by Limbergen et al. [32] who analyzed 36 patients with AML and MDS and complex karyotype by mFISH and
Fig. 4. Rearrangements of chromosome 5 in bone marrow cells of patient No. 35 detected by mBAND.
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L. Babicka et al. / Leukemia Research 31 (2007) 39–47
found as the most frequent breakpoints 3q26, 5q13, 5q33, 7q22, 11q23, 12p13, 17p11 and 22q11.2. Barouk-Simonet et al. [40] demonstrated in 28 patients with AML and MDS the most frequent breakpoints in region between 5q11 and 5q35, 7q11 to 7q34, and 20q11 to 20qter. MLL gene was involved in rearrangements in 14% of patients in Barouk-Simonet et al. study [40], in our cohort it was 16%. Defects of chromosome arm 5q have been shown previously closely associated with mutations of p53 [10,38,40–43]. In our cohort we did not check the loss of 17p13, i.e. p53 gene by FISH with LSI probes in every patient, however, we found aberration of 5q together with aberration of 17p or −17 in six patients. The simultaneous use of G-banding, FISH, mFISH and mBAND allowed precise delineation of complex rearrangements of chromosomes and proved the existence of cryptic chromosomal changes which are under resolution of classical methods, by mFISH technique high genomic instability of malignant cells on chromosomal level was ascertained as well. In a few cases mFISH brought complete reinterpretation of the marker chromosomes and helped to identify unbalanced and cryptic translocations and small insertions. This technique also unequivocally determined autosomes or sex chromosomes involved in numerical changes. Improvement in precise karyotyping might be helpful in defining subgroups of patients with complex chromosomal rearrangements. Precise identification of deleted regions by mBAND could point to the genes involved in progression of the disease and knowing them could help us to identify the patients who have poor prognosis and therefore they could be eligible for more intensive therapy. Acknowledgments This work was supported by grants GACR 301-04-0407, MSM LC535 and IGA MZCR 7995-3. References [1] Bloomfield CD, Lawrence D, Byrd JC, Carroll A, Pettenati MJ, Tantravahi R, et al. Frequency of prolonged remission duration after high-dose cytarabine intensification in acute myeloid leukemia varies by cytogenetic subtype. Cancer Res 1998;58:4173–9. [2] Grimwade D, Walker H, Oliver F, Wheatley K, Harrison Ch, Harrison G, et al. The importence of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered Ito the MRD AML 10 trial. The Medical Research Council Adult and Children’s Leukemia Working Parties. Blood 1998;92:2322–33. [3] Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, Vardiman J, et al. World Health Organization classification of neoplastic diseases of the hematopoetic and lymphoid tissues: report of the Clinical Advisory Committee Meeting-Airlie House, Virginia, November 1997. J Clin Oncol 1999;17:3835–49. [4] L¨owenberg B, Downing JR, Burnett A. Acute myeloid leukemia. N Engl J Med 1999;341:1051–62. [5] Rowley JD, Reshmi S, Carlson K, Roulston D. Spectral karyotype analysis of the T-cell acute leukemia. Blood 1999;93:2038–42. [6] Mr´ozek K, Heinonen K, Bloomfield CD. Clinical importance of cytogenetics in acute myeloid leukaemia. Best Pract Res Clin Haematol 2001;14:19–47.
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L. Babicka et al. / Leukemia Research 31 (2007) 39–47 [26] Fonatsch C, Gudat H, Lengfelder E, Wandt H, Silling-Engelhardt G, Ludwig WD, et al. Correlation of cytogenetic findings with clinical features in 18 patients with inv(3)(q21q26) or t(3;3)(q21;q26). Leukemia 1994;8:1318–26. [27] Schr¨ock E, Dumanoir S, Veldman T, et al. Multicolor spectral karyotypingof human chromosomes. Science 1996;273:494–7. [28] Speicher M, Ballard SG, Ward DS. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet 1996;12:368–75. [29] Tanke HJ, Wiegant J, van Gijlswijk RP, Bezrookove V, Pattenier H, Heetebrij RJ, et al. New strategy for multi-color fluorescence in situ hybridization: COBRA: combined binary ratio labelling. Eur J Hum Genet 1999;7:2–11. [30] Bredeson CN, Barnett MJ, Horsman DE, Dalal BI, Ragaz J, Philips GL. Therapy-related acute myelogenous leukemia associated with 11q23 chromosomal abnormalities and topoisomerase II inhibitors: report of four additional cases and brief commentary. Leuk Lymphoma 1993;11:141–5. [31] Andersen MK, Christiansen DH, Kirchhoff M, Pedersen-Bjergaard J. Duplication or amplification of chromosome band 11q23, including the unrearranged MLL gene, is recurrent abnormality in therapy-related MDS and AML, and is closely related to mutation of the TP53 gene and the previous therapy with alkylating agents. Genes Chromosomes Cancer 2001;31:33–41. [32] Limbergen HV, Poppe B, Michaux L, Herens Ch, Brown J, Noens L, et al. Identification of cytogenetic subclasse and recurring chromosomal aberrations in AML and MDS with complex karyotypes using mFISH. Genes Chromosomes Cancer 2002;33:60–72. [33] Michaux L, Wlodarska I, Stul M, Dierlamm J, Mugneret F, Herens C, et al. MLL amplification in myeloid leukemias: a study 14 cases with multiple copies of 11q23. Genes Chromosomes Cancer 2000;29:40–7. [34] Westbrook CA, Kein¨an MJ. Myeloid malignancies and chromosome 5 deletions. Baillierie’s Clin Haematol 1992;5:931–42. [35] Pedersen B, Jensen IM. Clinical and prognostic implications of chromosome 5 deletions: 96 high resolution studied patients. Leukemia 1991;5:566–73.
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[36] Tosi S, Scherer SW, Giudici G, Czepulkowski B, Biondi A, Kearney L. Delineation of multiple deleted regions in 7q in myeloid disorders. Genes Chromosomes Cancer 1999;25:384–92. [37] Koike M, Tasaka T, Spira S, Tsuruoka A, Koeffler HP. Allelotyping of acute myelogenous leukemia: loss of heterozygosity at 7q31.1 (D7S486) and q33-34 (D7S498, D7S505). Leuk Res 1999;23: 307–10. [38] Schoch C, Haferlach T, Bursch S, Gerstner D, Schnittger S, Dugas M, et al. Loss of genetic material is more common then gain in acute myeloid leukemia with complex aberrant karyotype: a detailed analysis of 125 cases using conventional chromosome analysis and Fluoroscence In Situ Hybridization including 24-Color FISH. Genes Chromosomes Cancer 2002;35:20–9. [39] Fr¨ohling S, Skelin S, Liebisch C, Scholl C, Schlenk RF, D¨ohner H, et al., Acute Myeloid Leukemia Study Group (Ulm). Comparison of cytogenetic and molecular cytogenetic detection of chromosome abnormalities in 240 consecutive adult patients with acute myeloid leukemia. J Clin Oncol 2002;10:2480–5. [40] Barouk-Simonet E, Soenen-Cornu V, Roumier Ch, Cosson A, Lai JL, Fenaux P, et al. Role of multiplex FISH in identifying chromosome involvement in myelodysplastic syndromes and acute myeloid leukemias with complex karyotypes: a report on 28 cases. Cancer Genet Cytogenet 2005;157:118–26. [41] Christiansen DH, Andersen MK, Pedersen-Bjergaard J. Mutations with loss of heterozygosity of p53 are common in therapy-related myelodisplasia and acute myeloid leukemia after exposure to alkyllating agents and significantly associated with deletion or los sof 5q a komplex karyotype and poor prognosis. J Clin Oncol 2001;19:1405–13. [42] Castro PD, Liang JC, Nagarajan L. Deletions of chromosome 5q13.3 and 17p loci cooperate in myeloid neoplasms. Blood 2000;95:2138– 43. [43] Martinez-Ramirez A, Urioste M, Alvarez S, Vizmanos JL, Cigudosa JC, Benitez J. Cytogenetic profile of myelodisplastic syndromes with complex karyotypes: an analysis using spectral karyotyping. Cancer Genet Cytogenet 2004;153:39–47.
Analysis of complex chromosomal rearrangements in adult patients with MDS and AML by multicolor FISH Libuse Babicka a,∗ , Sarka Ransdorfova b , Jana Brezinova b , Zuzana Zemanova a , Lenka Sindelarova a , Magda Siskova c , Jacqueline Maaloufova b , Jaroslav Cermak b , Kyra Michalova a,b a
c
Center of Oncocytogenetics, Institute of Clinical Biochemistry and Laboratory Diagnostics, General Faculty Hospital and 1st Faculty of Medicine of Charles University, Prague, Czech Republic b Institute of Hematology and Blood Transfusion, Prague, Czech Republic 1st Medical Department, General Faculty Hospital and 1st Medical Faculty, Charles University, Prague, Czech Republic Received 28 February 2006; received in revised form 28 February 2006; accepted 4 March 2006 Available online 9 May 2006
Abstract We analyzed complex chromosomal aberrations in 37 adult patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) using classical cytogenetic method, FISH with locus-specific probes, multicolor FISH (mFISH) and multicolor banding (mBAND). Unbalanced structural aberrations, leading to a gain or loss of chromosomal material, were frequently observed in bone marrow cells. In 30 patients (81.1%) loss or rearrangement of chromosome 5, 7 and/or 11 was found. The most frequent numerical change was trisomy 8 as expected (detected in six patients—16.2%) and the most frequent breakpoints 5q13, 5q33, 7q31, 10p12, 11q23, 12p13, 17p11 and 21q22 were determined. © 2006 Elsevier Ltd. All rights reserved. Keywords: MDS; AML; Complex chromosomal aberrations; mFISH; mBAND
1. Introduction Cytogenetic findings in bone marrow cells of patients with myelodysplastic syndrome—refractory anemia with excess of blasts in transformation (MDS RAEB-T) and acute myeloid leukemia (AML) are essential for precise diagnosis and classification of the disease. Karyotype of leukemic cells is one of the important prognostic factors [1–6] and it was proved by multivariate analyses that it is an independent predictor of therapy response, remission duration and survival [7]. Acquired cytogenetic aberrations are detected in 55–75% of newly diagnosed patients with AML [8]. Most of karyotypic abnormalities are associated with specific disease subtypes, characteristic morphologic and immunologic profiles ∗
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and distinct therapeutic and prognostic implications [2,8,9]. However, approximately 10–15% of AML with cytogenetic aberrations have no specific rearrangements, but do have complex chromosomal aberrations [8,10]. Similarly, patients with myelodysplastic syndrome (MDS) have at diagnosis pathological cytogenetic findings in 50–60% of cases. Specific and recurrent clonal chromosomal abnormalities observed in leukemic cells of patients with MDS and AML can be used to classify them into groups with consistent clinical features and similar prognosis. Three major translocations found in AML, all indicators of good prognosis, are associated with particular FAB subtypes and/or morphological features: t(8;21)(q22;q22) with M2 and granulocytic maturation, t(15;17)(q22;q21) with M3, inv(16)(p13q22) with M4 and eosinophilia [2,11]. According to Grimwade et al. [2] the group of patients with 11q23 abnormalities, +8, +21, +22, del(9q), del(7q) or other structural or numerical defects have an intermediate prognosis, patients with complex karyotype
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L. Babicka et al. / Leukemia Research 31 (2007) 39–47
and −5, del(5q), −7, or abnormalities of 3q have very poor prognosis [2,5]. Complex chromosomal aberrations (CCAs) involving more than two chromosomes and/or more than three breakpoints are associated with rather poor prognosis and respond poorly to antileukemic treatment and it was suggested that some of these rearrangements contribute to drug resistence and disease progression [2,8,12–17]. Reported complete remission rates in AML vary from 21 to 46%, with a median overall survival between 1 and 5 months [2,18]. We performed mFISH study of complex karyotypes in bone marrow cells of 60 patients with MDS and/or with primary or secondary AML, 37 of them were finally included into cohort and evaluated in detail. We detected balanced and/or unbalanced chromosomal aberrations, determined exact breakpoints, investigated whether unbalanced rearrangements lead to random or non-random chromosomal gains or losses. The most frequent breakpoints were determined on chromosomes 5, 7, 10, 11, 12, 17 and 21. 2. Patients From January 1998 to December 2004 we examined 311 adult patients with MDS or AML by classical cytogenetic method. Selection criteria for mFISH study were: diagnosis of AML, secondary AML (sAML) or MDS RAEB-T, according to the FAB and WHO classifications, presence of chromosomal aberrations involving more than two chromosomes and/or more than three breakpoints. Sixty patients with complex rearrangements were examined by mFISH, mBAND, locus-specific probes (LSI) and/or whole chromosome painting (WCP) probes. There were 30 males, 30 females, with an average age of 53.3 years (range 20–81 years). From this group of patients we excluded those who had specific translocations (eight patients, three with t(8;21), two with t(15;17) and one with inv(16), one patient with constitutive chromosomal translocation t(14;22)). Acquired chromosomal changes, probably as posttreatment karyotype evolution were found in 10 patients and they were excluded from this study as well. Clinical data and description of complex karyotypes confirmed by all methods in bone marrow cells of 37 patients are presented in Table 1 .
3.2. FISH analyses FISH analyses were performed according to the result of G-banding using commercially available probes following the manufacturer’s recommendations. Locus-specific DNA probes (LSI Abbott VYSISTM and Q BIOgeneTM ) were used to detect the (1) fusion genes: PML/RARA [t(15;17) LSI Kit], AML1/ETO (LSI AML/ETO Kit ES) and CBF/MYH11 (LSI CBFB Dual Color Probe [inv (16)]), (2) rearrangements and/or deletions: 5p15/5q31 (LSI EGR1/D5S721, D5S23), 7q22 and 7q35 (7q22/7q35 Dual-Color Specific Probe), 7q31 (LSI D7S486 7q31/CEP 7), 11q13 (LSI Cyclin D1/CEP 11 Kit), 11q22.3 (LSI ATM [11q22.3]) and 11q23 (LSI MLL Dual Color Rearrangement Probe). Whole chromosome painting probes (CAMBIOTM ) were used to define more precisely structural rearrangements. The number of mitoses examined by classical cytogenetics and FISH analyses depended on their availability on the chromosomal preparations; usually 20 mitoses were evaluated. At least 200 interphase nuclei were analyzed. To rule out the presence of the false positive results, cut-off level was established at 2.5% based on analyses of bone marrow samples from 10 healthy individuals. The signals were detected using an AXIOPLAN 2 Imaging microscope (Zeiss) or AX 70 PROVIS microscope (Olympus) equipped with computer analysis system ISIS (MetaSystemsTM ). 3.3. mFISH, mBAND mFISH was carried out using 24 XCyte MetaSystems color kit with combinatorially labeled painting probes specific for all autosomes and sex chromosomes. Hybridization and post-hybridization washes were described in detail by manufacturer. Analyses were done on AX 70 PROVIS fluorescence microscope (Olympus) or AXIOPLAN 2 Imaging microscope (Zeiss) with filters for FITC, Spectrum OrangeTM , TexasRed® , DEAC, Cy5TM and DAPI using ISIS software (MetaSystemsTM ). In some cases multicolor banding mBAND was carried out using XCyte color kits (MetaSystemTM ) for chromosomes 3, 5 and 11.
3. Methods
4. Results
3.1. Conventional cytogenetics
Complex chromosomal aberrations were identified by conventional cytogenetic methods in bone marrow cells of 60 patients out of 311 (19.3%), 37 (11.9%) were included into this study and the karyotypes of bone marrow cells examined at diagnosis are presented in Table 1. The most frequently observed aberrations were unbalanced chromosomal translocations, leading to the gain or loss of chromosomal material. In these translocations the most often were involved chromosomes 5, 7 and 11 (Fig. 2).
Bone marrow cells were cultivated for 24 h in RPMI 1640 medium with 10% of fetal calf serum without any stimulation and were harvested using conventional techniques. Cytogenetic analyses were done on G-banded chromosomes using computer analysis for karyotyping (MetaSystemsTM ). Karyotypes were described according to the International System of Human Cytogenetic Nomenclature (ISCN, 1995, 2005) [19,20].
L. Babicka et al. / Leukemia Research 31 (2007) 39–47
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Table 1 Clinical data of examined patients, karyotypes according to G-banding and mFISH analyses No.
Sex/age
Diagnosis
Surviv.
Karyotype
1
F/48
AML M1–M2
6
2 3
F/75 F/78
AML M5 AML M4
7 1
4 5
M/38 F/63
AML M0 AML M1
9 14
6
F/41
AML
3
7
F/66
AML M1
0.6
8
F/64
AML
0.2
9 10
F/76 F/68
MDS → AML AML M2
NA NA
11
M/62
RAEB-T
3
12
M/25
AML M5
6
13
F/65
AML M2
4
14
F/26
AML M2
NA
15 16 17
F/72 M/55 M/71
AML M4 AML M0 AML M6
0.2 4 6
18
F/64
MDS → AML
15
19 20
M/66 F/58
AML M4 RAEB-T
9 NA
21
M/80
RAEB-T
9+
22
F/67
AML
26
23 24 25
M/47 F/78 M/45
AML M5a AML M0 AML M0
5 1 6
26 27
M/70 M/73
MDS → AML AML M4
NA 1
28
M/62
AML M2
2
29
M/67
RAEB-T
2
46, XX, der(6)t(6;11)(q27;q23), del(11)(q23) [2]/48, XX, der(6)t(6;11)(q27;q23), t(10;17)(q21;q12), +10, del(11)(q23), +17 [4] 46, XX, der(10)t(10;11)(p12;q23), der(11)t(10;11)(p?;q12)t(1;10)(q?;p?) [11] 46, XX [2]/46, XX, der(11)t(1;11)(?;q23) [19]/46, XX, idem, i(8)(q10) [5]/46, XX, der(22)t(1;22)(?;p11.2) [4]/ 46, XY [2]/47, XY, +X, del(5)(q22q33), t(10;11)(p12;q21) [6] 47, XY, +X, idem, −22 [3] 44, XX, der(2)t(2;17)(p16;?), −3, der(4)t(4;5)(q21;?), −5, del(6)(p24), del(8)(q24), del(11)(q24), −12, −13, der(15)t(4;12;15)(?;?;q15), der(15)t(12;15;16)(?;q15;?), der(16)del(16)(p11.2)del(16)(q13), +der(16)t(15;16)(q15;q13), der(17)t(8;17)(q24;q12), der(20)ider(20)(q10)ins(20;12)(p12;?), +der(22)t(11;12;22)(q24;?;q11) [5]/43, idem, −17 [5]/44, idem, −17, +del(8)(q?) [2] 45, XX, der(2)t(2;8)(q24;?), der(3)t(3;5)(q13.3;p13), del(3)(p12);t(4;10)(p15.3;p13), del(5)(p13), del(6)(q25), −10, −11, del(13)(q13), del(14)(q24), del(15)(q25), +18, der(21)t(11;21)(?;q22), dup(22)(q?) [10] 46, XX [2]/46, XX, del(5)(q13q33), del(7)(q21q33), dup(11)(q13q23), −17, der(20)t(17;20)(q12;q11) [7]/46, XX, del(5)(q13q33), del(7)(q21q33), +8, dup(11)(q23q23), −14, der(17)t(14;17)(?;q12), der(20)t(17;20)(q12;q11) [7]/54, XX, +4, del(5)(q13q33), +5, del(7)(q21q31), +8, +8, +16, −17, +18, +18, +19, der(20)t(17;20)(q12;q11), −20 [1] 45, XX, −14, der(16)t(16;21)(p12;?), der(17)t(17;19;21)(p11.2;p13.1;?), der(19) t(14;19)(?;p13), dic(21;21)(q22.1q22.1) [2]/46, idem, +der(16)del(16)(p11.2)del(16)(q12) [4] 42, XX, −5, der(6)t(6;17;18)(p12;?;?), der(7)t(7;16)(p13;?), der(11)t(6;11;13;18)(?;p15;q13;?) [6] 45, XX, del(5)(q14q33), der(5)t(1;5)(?;q35), der(7)t(7;12)(q21;?), +8, −12, der(16)ins(16;17)(q?;?), −17, der(20)t(17;20)(?;q11.2) [9] 45, XY, del(5)(q13q33), del(7)(q22q35), dup(11)(q?), der(17)t(17;21)(p11.2;?), der(20)t(20;21)(q12;?), −21 [2]/44, XY, del(4)(q32), del(5)(q13q33), der(7)t(3;7)(q?;q21), −15, der(16)t(15;16)(?;q22), der(17)t(17;21)(p11.2;?), der(20)t(20;21)(q12;?), −21 [4] 47, XY, del(8)(q11.2), +del(8)(q11.2), der(13)t(8;13)(q11.2;?) [3]/48, idem, +10 [3]/49, idem, +del(8)(q11.2), +10 [2] 46, XX [4]/42, XX, der(4)t(4;5;19)(q13;?;?), der(5)t(5;8;12)(p14q14;?;?), −7, der (8)t(8;12;17)(p12;?;?), +11, −12, der(13)t(13;19)(p11;?), −17, −18, −19 [14] 47, XX, i(8)(q10), +der(8)t(8;10)(q23;?), der(10)t(10;22)(p14;?), +19 [27]/48, idem, t(4;6)(q26;q22) [2] 44, XX, del(5)(q13q33), −7, t(8;12)(q24.2;p13), −16, del(18)(q21) [20] 46, XY, del(7)(p12) [18]/46, XY, idem, t(2;2)(q31;q35) [23] 48, XY, del(5)(q13q33), der(7)t(7;19)(p14;?), del(13)(q33), +der(22)t(20;22)(?;p12), +der(22)t(13;20;22)(?;?;p12q?) [6]/49, idem, +del(5)(q13q33) [2] 46, XX [14]/45, XX, der(1)ins(1;11)(p11;?), der(2)t(2;5)(q33;?), del(5)(q12q33), −11 [8]/46, idem, +der(1)ins(1;11)(p11;?), der(5)t(2;5)(?;q12), der(7)t(7;12;15)(p21q31;p13;q26), der(12)t(7;12)(?;p13), del(15)(q26) [1] 50, XY, del(8)(q11.2), +12, der(13)t(8;13)(q11.2;p11.2), +21, +21 [16]/49, idem, −8 [10] 46, XX, del(5)(q14q33), der(7)del(7)(q31)t(7;12)(q33?;q24), der(12)del(12)(p13)t(7;12)(q33?;q24), der(16)t(1;16)(?;q22) [22] 42, XY, der(5)t(5;7)(p12;?), −7, der(12)t(12;16)(p11;?), −16, der(19)t(19;21)(p13;q22), −19, der(20) t(20;22)(q12;q13), del(21)(q22), −22 [8]/44, XY, del(1)(p?), der(5)t(5;7)(p12;?), −7, der(12)t(12;14)(p11;q12), del(14)(q12), der(19)t(19;21)(p13;q22), −19, der(20)t(20;22)(q12;q13), del(21)(q22), +der(21)t(1;21)(?;q21), −22 [2] 46, XX [2]/51, XX, der(2)t(2;5)(p11.2;?), +der(2)t(2;5)(p11.2;?), der(3)t(2;3)(p11.2;p11), +der(3)t(2;3)(p11.2;p11), −5, +6, +8, +13, +21 [8] 50, XY, +5, +6, +18, +der(19)t(X;19)(?;p13.2) [8] 46, X, t(X;7)(p22.1;p15) [13]/47, idem, +22 [12] 46, XY [3]/69, XY, +X, +1, +2, +4, +4, +5, +6, +7, +8, +10, t(10;11)(p12;q23), +12, +13, +13, +13, +14, +17, +18, +19, +19, +19, +20, +21, +21, +21, −22 [19] 43, XY, der(5)t(5;11;16)(q12;?;?), der(6)t(6;11)(q27;q23), −11, −16, −18 [24] 52, XY+6, +8, +9, der(11)t(11;22)(p15;?)dup(11)(q?), +der(11)t(11;22)(p15;?)dup(11)(q?), +13, der(17)t(7;17)(?;p11), +der(17)t(7;17)(?;p11), +19, der(21)t(11;21)(?;p11)dup(11)(q?), −22 [3]/53, idem, +der(7)del(7)(p)del(7)(q) [5]/44, XY, −7, r(11)(p14q24), der(13)t(4;13;21)(?;p11;?), der(17)t(7;17)(?;p11), −21 [2] 43, XY, der(2)t(2;17)(q36;q?), −5, der(12)t(5;12)(?;q13), der(17)t(17;18;20)(p11q12;?;?), −18, −20 [9] 72, XY, +Y, +del(1)(p?), +2, +2, +3, −4, del(5)(q13.1q33.3), +6, +6, +8, +8, +der(9)t(4;9)(?;q11), +der(9)t(4;9)(?;q11), der(10)t(10;18)(p12;?), +der(10)t(10;18)(p12;?), +11, +13, +13, +del(14)(q21), +15, +15, +17, +19, +der(19)t(1;19)(p?;q13), +20, +21, +21, +der(22)t(14;22)(q21;q13) [4]/46, XY, del(5)(q13.1q33.3) [5]
42
L. Babicka et al. / Leukemia Research 31 (2007) 39–47
Table 1 (Continued ) No.
Sex/age
Diagnosis
Surviv.
Karyotype
30
F/41
RAEB-T
?
31 32
M/20 M/57
AML M0 AML M5a
3 0.1
33
M/61
AML M1–2
4+
34
F/81
MDS → AML
7
35
M/78
AML
10
36
F/80
AML M2
0.3
37
F/58
AML M6
NA
46, XX [8]/46, XX, der(1)t(1;13;17)(q21;?;q22), del(2)(p22), der(3)t(2;3)(p22p24;q26q27), del(5)(q13q33), del(8)(q24.1), der(9)ins(9;8)(q22.3;q24.1q24.3), der(12)t(9;12)(?;p13), del(13)(q32), der(17)t(1;17;19)(q21;q22;?) [12] 48, XY, +4, t(4;7)(q31;q31), del(13)(q33), der(21)ins(21;7)(q21;q?), +der(21)ins(21;7)(q21;q?) [30] 44, XY, der(2)t(2;5)(q13;p13.2), der(5)del(5)(p13.2)del(5)(q12), der(7)ins(7;11)(q21;?)t(7;11)(q31;?), −11, −12, der(17)t(2;17)(q13;p11.2), der(17)t(12;17)(q11;p11.2) [1]/45, XY, idem, der(20)t(12;20)(p11;q12) [3]/43, XY, der(2)t(2;5)(q13;p13.2), t(4;6)(q21;q22), −5, −5, der(7)ins(7;11)(q21;?)t(7;11)(q31;?), del(9)(q22), −11, der(17)t(2;17)(q13;p11.2), der(20)t(9;20)(q22;q12) [2] 45, XY, del(5)(q13q33), t(6;16)(q12;q22), −7, der(10)t(10;14)(p14;q?), del(12)(p12), der(14)t(6;14) (q?;q23), del(18)(p11.3), der(21)ider(21)(q10)t(1;21)(?;p11.1) [3]/45, XY, idem, der(Y)ins(Y;12)(q11.2;p12p13) [1] 45, XY, idem, der(Y)ins(Y;12)(q11.2;p12p13), der(22)t(22;22)(q11.1;q11.1) [1] 45, XY, idem, der(4)t(4;11)(q24;?) [1] 46, XX [1]/44, X, der(X)t(X;7)(q26;?), t(3;11)(q26;p13), t(5;13)(q23.3;q12), der(5)t(X;5)(?;q11.2), der(7)t(5;7)(q11.2;?), der(12)t(12;22)(p13;q11), der(14)t(14;15)(q10;q10), −15, 22p+ [5]/43, idem, −7 [3] 46, XY [1]/44, XY, −4, −5, der(7)t(5;7;10)(q13;p11;?), der(10)t(4;5;10)(?;p15.5q23;p?q?), der(11)t(4;11)(?;p14) [21] 44, XX, der(1)t(1;3)(q32;?), −3, der(5)del(5)(q12q33)t(5;18)(p13;p11), −7, der(18)t(1;3;18)(?;?;p11) [7] 46, XX [1]/49, XX, del(5)(q14q33), t(17;20)(p11.2;q12), +19, +21, +22 [12]
Surviv.—survival in months, survival + patients live in complete remission (November 2004), NA—not available, MDS → AML—myelodysplastic syndrome transformed in acute myeloid leukemia, BMT—bone marrow transplantation.
4.1. Changes of chromosome 5, 7 and 11 Numerical and structural aberrations of chromosome 5 were ascertained in 22 patients (59.5%). Monosomy was found in five cases (Nos. 5, 9, 22, 28 and 35). Rearrangements of long arms of chromosome 5 were proved in four patients (Nos. 10, 13, 26 and 34) and deletion of long arm was detected in 14 patients, in all of them deletion of the critical region 5q31 was confirmed by FISH. The most frequent deletion was del(5)(q13q33) found in bone marrow cells of seven patients, in three of them deletion del(5)(q14q33) was ascertained and in two deletion del(5)(q12q33) was determined. Changes of chromosome 7 were presented in 12 patients (32.4%). Monosomy of chromosome 7 was found in five cases (Nos. 13, 15, 21, 33 and 36). We found aberrations of the long arm of chromosome 7 in four patients, in all of them (Nos. 10, 20, 31 and 32) with rearrangements or losses of 7q31 region. Deletion of long arms of chromosome 7 was found in three cases and critical region 7q31 was lost in all three as was proved by FISH with locus-specific probes. Aberrations of chromosome 11 were detected in 13 patients (35.1%), monosomy was found in three cases (Nos. 6, 18 and 32) and FISH with LSI MLL Dual Color Rearrangement Probe was performed in all of them. In three patients (Nos. 1, 2 and 25) FISH proved rearrangements of the MLL gene. In one patient (No. 18) deletion of the MLL gene and in two cases (Nos. 9 and 11) amplification of the MLL gene was determined. In four others (Nos. 3–6) the MLL gene was not altered. In total, aberrations of chromosomes 5 and/or 7 and/or 11 were detected in 30 (81.1%) out of 37 patients. Rearrangements of chromosome 5 associated with changes of
chromosome 7 were found in six patients, rearrangements of chromosome 5 together with chromosome 11 were detected in four patients and rearrangements of chromosomes 7 and 11 were determined in one patient. In two patients complex karyotypes were associated with aberrations of all these three chromosomes (see Fig. 1). 4.2. Other aberrations The majority of structural abnormalities were unbalanced. We found translocation der(13)t(8;13)(q11.2;p11.2), del(8)(q11.2) in two patients, i.e. Nos. 12 and 19. In patient No. 12 translocation was associated with duplication of del(8)(q11.2) and trisomy of chromosome 10. In patient No. 19 translocation was associated with trisomy of chromosome 21, tetrasomy of chromosomes 21 and monosomy of chromosome 8. We did not detect any other repeatedly occurring translocations in our cohort. The other aberrations were insertions, isochromosomes, dicentric and ring chromosomes. Insertions were detected in six patients (Nos. 5, 10, 18, 30, 31 and 32), isochromosome i(8)(q10) was proved in two cases (Nos. 3 and 14). Isochromosomes der(20)ider(20)(q10)ins(20;12) and der(21)ider(21)(q10)t(1;21)(?;p11.2) were ascertained only once (cases Nos. 5 and 33, respectively). One ring chromosome r(11)(p24q24) was determined (case No. 27). Dicentric chromosome involving chromosome 21 was found in patient No. 8: dic(21;21)(q22.1;q22.1). 4.3. The breakpoints associated with complex aberrations Chromosomal breakpoints that could be unequivocally assigned by comparison of results obtained with different
L. Babicka et al. / Leukemia Research 31 (2007) 39–47
43
Fig. 1. Incidence of chromosomes 5, 7 and 11 aberrations as determined by FISH with locus-specific probes, mFISH and mBAND in 37 patients with complex aberrant karyotype.
types of classical and FISH analyses are depicted in Fig. 2. Losses and rearrangements attributed to structural abnormalities numerously involved were chromosome arms 5q, 7q, 11q and 10p, 12p and 17p. The most frequent breakpoints were found to be: 5q33 (12x), 5q13 (8x), 5q12 (5x), 7q31 (4x), 11q23 (6x), 10p12 (4x), 12p13 (7x) and 17p11.2 (7x). Interestingly, both sex chromosomes X and Y were sporadically involved in complex aberrations (Fig. 2).
5. Discussion Precise identifications of clonal chromosomal aberrations and delineation of breakpoints in bone marrow cells of patients at diagnosis could lead to a better understanding of the genetic events during leukemogenesis and disease progression, as well as guiding further molecular studies of genes involved in leukemogenesis, and could be providing clinically relevant information that can assist in the development of risk-adapted therapeutic strategies. Moreover, cytogenetic data until now are particularly useful for determining prognosis of the disease and/or prediction of response to certain therapeutic treatment. Several studies have shown that specific chromosomal aberrations t(8;21)(q22;q22), inv(16)(p13q22) or t(15;17)(q22;q21) are associated with a good prognosis [13,15,17,21–24] on contrary to aberrations of chromosomes 5, 7, inv(3)/t(3;3) and complex aberrant karyotype, which are all indicators of a very poor prognosis [2,13,15,22,23,25,26]. The aberrations seen in AML cases with complex chromosomal rearrangements have often been referred as random, because the same chromosomal rearrangement is rarely identified in more than one patient. However, in the past few years, new cytogenetic methods, such as mFISH, SKY and CGH [27–29], with significantly higher resolution than Gbanding have been introduced and allowed a more detailed characterization of CCAs. In our study we have analyzed complex rearrangements in 37 patients with MDS RAEB-T
or AML by mFISH method to identify exactly all numerical and structural changes and to confirm origin of every marker chromosome, supernumerary chromosomes and chromosomal parts, which are involved in CCAs and tried to establish the frequency of non-random changes. In already published reviews one-third of deletions found by G-banding were shown to represent cryptic translocations (both balanced and unbalanced) or insertions using mFISH or SKY techniques. In agreement with the literature we found complex chromosomal rearrangements in 11.9% of patients with de novo AML or MDS (37 patients out of 321) at diagnosis, with majority of structural unbalanced rearrangements with variable breakpoints within a small region of single chromosomes. Complex unbalanced rearrangements lead to loss rather than amplification of chromosome material, suggesting possible involvement of tumor suppressor genes. In our study chromosomes most often involved in CCR were found to be 5, 7, 8, 10, 11, 12, 17 and 21 with aberrations of chromosomes 11, 5 and 7 as the most frequent ones. According to Heim and Mitelman [11] and Bredeson et al. [30] chromosomal abnormalities involving long arm of chromosome 11, especially of 11q23 region, have been described in 5–10% of published cases of adult patients with de novo AML and in 80–90% therapy-related AML and they were connected with topoisomerase II inhibitors. In our study of complex rearrangements chromosomal aberrations of 11q were detected in 13 patients (35.1%). Using locus-specific probes the rearrangement of MLL gene was observed in three patients, deletion of one MLL allele in one patient and duplication of the MLL gene in two cases. Extra copies of MLL gene are a recurrent genetic change found in approximately 20% of MDS and AML [31,32]. It is often associated with dysplastic bone marrow changes or complex karyotypes suggestive of genotoxic exposure and with poor prognosis for the patient [33]. Our results correlate well with reported data. In our group with 11q aberrations all patients died and median overall survival of this group was found to be 4.5 months.
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L. Babicka et al. / Leukemia Research 31 (2007) 39–47
Fig. 2. Incidence of chromosomal breakpoints assigned by FISH with locus-specific probes, mFISH, mBAND and G-banding.
As expected, the majority of cases (22 patients; 59.2%) showed aberrations associated with chromosome 5—loss of 5q material, monosomy or unbalanced rearrangements of 5q. Deletion of 5q can be detected in 10% of de novo AML [34]. The size of deletion varies among patients, del(5)(q13q33) is
the most frequent one, with common deleted segment 5q31 [35]. Our findings correspond with the data quoted in the literature. In our cohort of patients deletion of the critical region 5q31 was found in 14 patients and the most frequent deletion del(5)(q13q33) was determined in eight patients.
L. Babicka et al. / Leukemia Research 31 (2007) 39–47
45
Fig. 3. Karyotype of patient No. 4 with complex chromosomal rearrangements, revealed by multicolor FISH: 46, XY, +X, del(5)(q22q33), t(10;11)(p12;q21), −22.
Monosomy or the loss of part of chromosome 7 is a consistent finding in malignant myeloid disorders, occurring in all subtypes of MDS and AML. The minimal deleted segment or critical region, in which the putative tumor suppressor genes could be located has been variously reported as 7q22, 7q31 and 7q33-q34 [36,37] suggesting that 7q may contain more than one myeloid leukemia-related gene. Aberrations associated with chromosome 7 were detected in our cohort in 12 patients (32.4%). Monosomy was found in five patients. In all cases with del 7q (three patients) region 7q31 was deleted and in four patients with aberrations involving 7q rearrangements of 7q31 region was proved. Schoch et al. [38], analyzed complex aberrant karyotypes associated with the most frequently rearranged chromosomes—aberrations of chromosomes 5, 7 and/or 11. In our study it was 30 patients (81.1%) out of 37 who had loss or rearrangement of chromosomes 5, 7 and/or 11. We also proved an excess of chromosomes 5, 7 and 11 abnormalities in the group of patients with complex karyotype compared to patients with simple karyotype. Our findings correspond well with the results of studies of Limbergen et al. [32] and Schoch et al. [38]. The current data elucidate the role of loss of chromosomal material, especially of 5q and 7q, in the pathogenesis of AML with a
complex aberrant karyotype. Lindvall et al. (2004) have combined transcriptional profiles with cytogenetic data from 15 AML patients with normal and abnormal complex karyotypes [8]. Their results show that AML with complex karyotypes exhibit specific gene expression profiles. The differentially expressed genes included several which are located on 5q and 7q, as well as genes involved in control of cell division. Further analyses of expression of genes located on 5q and 7q will help to specify which genes show a reduced expression and may be active during progression of the disease (Figs. 3 and 4). In agreement with previously published data [38,39] trisomy of chromosome 8 was observed as the most frequent numerical change and was confirmed by mFISH in six patients. We did not determine any new recurrent change in our cohort, but we identified chromosomal parts and breakpoints occurring repeatedly in complex translocations. The most frequently involved in CCAs were found to be 5q, 7q, 11q, 21q, 10p, 12p and 17p and breakpoints: 5q13 (8x), 5q33 (12x), 7q31 (4x), 11q23 (6x), 21q22 (4x), 12p13 (7x), 17p11 (7x) and 10p12 (4x). Our results correlate with data already published by Limbergen et al. [32] who analyzed 36 patients with AML and MDS and complex karyotype by mFISH and
Fig. 4. Rearrangements of chromosome 5 in bone marrow cells of patient No. 35 detected by mBAND.
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found as the most frequent breakpoints 3q26, 5q13, 5q33, 7q22, 11q23, 12p13, 17p11 and 22q11.2. Barouk-Simonet et al. [40] demonstrated in 28 patients with AML and MDS the most frequent breakpoints in region between 5q11 and 5q35, 7q11 to 7q34, and 20q11 to 20qter. MLL gene was involved in rearrangements in 14% of patients in Barouk-Simonet et al. study [40], in our cohort it was 16%. Defects of chromosome arm 5q have been shown previously closely associated with mutations of p53 [10,38,40–43]. In our cohort we did not check the loss of 17p13, i.e. p53 gene by FISH with LSI probes in every patient, however, we found aberration of 5q together with aberration of 17p or −17 in six patients. The simultaneous use of G-banding, FISH, mFISH and mBAND allowed precise delineation of complex rearrangements of chromosomes and proved the existence of cryptic chromosomal changes which are under resolution of classical methods, by mFISH technique high genomic instability of malignant cells on chromosomal level was ascertained as well. In a few cases mFISH brought complete reinterpretation of the marker chromosomes and helped to identify unbalanced and cryptic translocations and small insertions. This technique also unequivocally determined autosomes or sex chromosomes involved in numerical changes. Improvement in precise karyotyping might be helpful in defining subgroups of patients with complex chromosomal rearrangements. Precise identification of deleted regions by mBAND could point to the genes involved in progression of the disease and knowing them could help us to identify the patients who have poor prognosis and therefore they could be eligible for more intensive therapy. Acknowledgments This work was supported by grants GACR 301-04-0407, MSM LC535 and IGA MZCR 7995-3. References [1] Bloomfield CD, Lawrence D, Byrd JC, Carroll A, Pettenati MJ, Tantravahi R, et al. Frequency of prolonged remission duration after high-dose cytarabine intensification in acute myeloid leukemia varies by cytogenetic subtype. Cancer Res 1998;58:4173–9. [2] Grimwade D, Walker H, Oliver F, Wheatley K, Harrison Ch, Harrison G, et al. The importence of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered Ito the MRD AML 10 trial. The Medical Research Council Adult and Children’s Leukemia Working Parties. Blood 1998;92:2322–33. [3] Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, Vardiman J, et al. World Health Organization classification of neoplastic diseases of the hematopoetic and lymphoid tissues: report of the Clinical Advisory Committee Meeting-Airlie House, Virginia, November 1997. J Clin Oncol 1999;17:3835–49. [4] L¨owenberg B, Downing JR, Burnett A. Acute myeloid leukemia. N Engl J Med 1999;341:1051–62. [5] Rowley JD, Reshmi S, Carlson K, Roulston D. Spectral karyotype analysis of the T-cell acute leukemia. Blood 1999;93:2038–42. [6] Mr´ozek K, Heinonen K, Bloomfield CD. Clinical importance of cytogenetics in acute myeloid leukaemia. Best Pract Res Clin Haematol 2001;14:19–47.
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