acute myeloid leukemias: conventional cytogenetics, FISH, and multiplex FISH

Cancer Genetics and Cytogenetics 168 (2006) 133–145

Cytogenetic studies of a series of 43 consecutive secondary myelodysplastic syndromes/acute myeloid leukemias: conventional cytogenetics, FISH, and multiplex FISH Wei Shalia,1, Catherine He´liasa, Ce´cile Fohrerb, Ste´phanie Struskia, Carine Gervaisa, Annie Falkenrodta, Vincent Leymarieb, Bruno Lioureb, Pascale Rabyc, Raoul Herbrechtb, Michel Lessarda,* a

Laboratoire d’He´matologie, bDe´partement d’ Onco-He´matologie, Hoˆpitaux Universitaires de Strasbourg, Avenue Molie´re, 67098 Strasbourg, France c De´partement d’ Onco-He´matologie, Hoˆpital Pasteur, 39 Avenue de la Liberte´, 68021 Colmar, France Received 9 September 2005; received in revised form 31 January 2006; accepted 7 February 2006

Abstract

We report a series of 43 consecutive therapy-related myelodysplastic syndromes (t-MDS) or acute myeloid leukemias (t-AML) observed for 6 years. This series consisted of 26 women and 17 men, ages ranging from 9 to 85 years. These cases were classified into three groups according to the primary diagnosis. Conventional cytogenetic and flurosecence in situ hybridization (FISH)/ multiplex FISH (M-FISH) methods were used to analyze cytogenetic characteristics of secondary MDS/ AML. The features of chromosomal abnormalities were linked to the nature of the therapy and protocols used. A considerable proportion of recurrent balanced translocations characterized t-AML secondary to therapy. FISH techniques showed that conventional cytogenetics often underestimated associated translocations; some deletions were in fact derivative chromosomes associated with deletions. After treatment for lymphomas and chronic myeloproliferative diseases, there were more complex unbalanced abnormalities than the control group. Compared to other series, recurrent translocations appeared to be more numerous (25%), probably reflecting an evolution of therapeutic modalities. Ó 2006 Elsevier Inc. All rights reserved.

1. Introduction Therapy-related acute myeloid leukemias (t-AML) and myelodysplastic syndromes (t-MDS) arise as a result of cytotoxic chemotherapy and/or radiation therapy. These neoplasms were thought to be the direct result of mutational events induced by cytotoxic therapy. Several distinct cytogenetic and clinical subtypes of t-AML/t-MDS are recognized to be closely associated with the nature of preceding treatment or environmental exposure [1,2]. This close relationship existing between the origin of MDS/ AML and some cytotoxic agents have been known since

1 Present address: Department of Reproductive Physiology, University of Medical Sciences, Yixueyaun Road, YuDistrict, Chongqing 400016, China. * Corresponding author. Tel.: 1 33-3-88-12-75-53; Fax: 133-3-88-1275-55. E-mail address: [email protected] (M. Lessard).

0165-4608/06/$ – see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2006.02.006

the 1970s; somatic acquired chromosomal abnormalities had been observed in cells of hematopoietic tissues [3–6]. There recently has been considerable technical progress that has enabled more precise analysis of chromosomal abnormalities, first with FISH and then with M-FISH. FISH can specifically demonstrate or confirm a known chromosomal abnormality, and M-FISH is a global approach that analyzes and/or reveals simultaneously all the unknown rearrangements in a metaphase, regardless of the number of aberrations (within the limits of its sensitivity) [7]. Secondary hemopathies often present complex chromosomal abnormalities (three or more by metaphase), so FISH and M-FISH may be particularly useful in these cases. We report here on the results of a conventional cytogenetic study (RHG banding) completed by FISH and/or M-FISH in a series of 43 consecutive secondary MDS/AML observed in our laboratory for 6 years. We aimed to estimate the frequency of recurrent translocations versus other well-known abnormalities such as del(5q), del(7q), del(12p), and

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del(17q), and to use FISH techniques to distinguish ‘‘simple’’ (pure) deletions from translocation-associated deletions.

2. Materials and methods 2.1. Patients characteristics General data, sex, age, previous history, clinical data, and therapy are summarized in Table 1. Patients were retrospectively selected on the basis of their clinical and biologic data, previous history of therapy for a malignant pathology, or administration of drugs known for their mutagenic or genotoxic capacity. Criteria for diagnosis were made according to French–American–British classification [8] and were reviewed for application of World Health Organization (WHO) classification [9]. When necessary, cytochemistry and immunophenotyping were used to classify hemopathies. The 43 patients were 26 women and 17 men; the mean age was 60 for women (range 5 9–85 years) and 53 for men (range 5 25–69 years). Only 10 patients were still alive, 32 were deceased, and 1 experienced loss of sight. At the time of the first cytogenetic study, there were 33 overt AML (WHO definition, 20% blasts or more) versus 10 MDS cases. For the overt leukemia cases, 2 were promyelocytic (AML-M3) and 13 had monocytic differentiation. The median latency period between the diagnosis of the primary disease and the diagnosis of t-MDS/AML was 93 months (range 5 4–324). This latency period may depend on the cumulative dose and dose intensity of the preceding cytotoxic therapy, as well as exposure to specific agents. As shown in Table 1, only 10/43 patients are known to be alive. The median time from diagnosis of t-MDS/ AML to death was 8.8 months (range 5 0.5–51). The median survival for patients with t-MDS was 9.8 months compared to 8.4 months for patients with t-AML. At the time of t-MDS/AML diagnosis, the global percent of chromosomal abnormalities was high, 95%, with only 2/ 43 cases being normal (cases 9 and 20). In our series, we distinguished three different groups according to primary pathology: solid tumors (Table 2), various hematologic primary pathologies (Table 3), and chronic myeloproliferative disease (CMPD; Table 4). CMPD was distinguished from other hematologic primary pathologies because of the difficulty in determining whether AML is the natural evolution of CMPD or therapy related. 2.2. Cytogenetics Blood and bone marrow cells were cultured for 24 and 48 hours with 5-fluoro-2-deoxyuridine synchronization using the technique of Weber and Garson [10] to improve the quality of the banding. Metaphase chromosome spreads were obtained using classic procedures, and standard karyotypic analysis were performed after RHG banding and were classified according to the International System for human Cytogenetic Nomenclature (ISCN 1995) [11].

2.3. FISH techniques After conventional cytogenetics, additional FISH with whole chromosome painting probes, centromeric probes, region- or gene-specific probes, and unique sequence probes (BAC and PAC) was performed to confirm or characterize chromosomal rearrangements. Total Chromosome Paint Probes (Q-Biogene, MP Biomedicals, Illkirch, France) were used for all human chromosomes. The following region- and gene-specific probes were used: LSI EGR1 (5q31), LSI CSF1R (5q33), LSI MYC (8q24), LSI TEL/ AML1 (TEL/ETV6: 12p13; AML1/RUNX1: 21q22), LSI PML/RARA (PML: 15q22; RARA: 17q21.1), LSI IGH/ BCL2 (IGH: 14q32; BCL2: 18q21) (Vysis-Abbott, Voisins Le Bretonneux, France), chromosome 1, 5, 19 a-satellite probe, chromosome 8 a-satellite probe (D8Z2), chromosome 17 a-satellite probe ((D17Z1) (Q-Biogene, MP Biomedicals, Illkirch, France), and 13q14.3 deletion probe (D13S19, D13S25) (AmpliTech, Compie`gne, France). All probes were hybridized according to the manufacturers’ recommendations. The following BAC and PAC probes were used: BAC RP11-33A1 (EVI1: 3q26.2), BAC RP11211G3 (BCL6: 3q27), BAC RP11-589C21 and RP11313J8 (MOZ: 8p11), BAC RP11-336O12 and RP11-73E6 (AF9: 9p22), PAC dJ852F10 (ETV6, exon 2: 12p13) and PAC dJ846M9 (ETV6, exons 6–8: 12p13), BAC RP111072J2 and RP11-619A23 (CBP: 16p13.3) (BACPAC Resources Center, Oakland, CA), BAC RP11-120E20 (NUP98: 11p15, kindly provided by Dr. M. Rocchi, Bari, Italy), PAC dJ217A21 and dJ167K13 (MLL: 11q23, kindly provided by Dr. E Schuuring, Leiden, The Nederlands). 2.4. M-FISH technique The ‘‘24Xcyte’’ probe kit was purchased from MetaSystems (Altlussheim, Germany). This probe kit consists of 24 different chromosome painting probes obtained from degenerated oligonucleotide polymerase chain reaction. A panel of five fluorochromes (plus 40 ,6-diamidino-2-phenylindole counterstain) are used in combinatorially labeled chromosome painting probes. In a single hybridization experiment, normal or marker chromosomes, complex chromosomal rearrangements, and all numeric aberrations can be visualized simultaneously with two limiting factors: the combination of fluorochromes, some of them being more legible than others, and the size of the translocated chromosomal segments, depending on the degree of chromosomal stretching and the quality of the slide preparation [12]. The procedure was carried out according to the manufacturer’s instructions. 2.5. Cytogenetic analysis Conventional cytogenetics and FISH/M-FISH methods were used to analyze the cytogenetic characteristics of secondary MDS/AML. Analysis of clonal cytogenetic

W. Shali et al. / Cancer Genetics and Cytogenetics 168 (2006) 133–145

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Table 1 Clinical data of the 43 patients First disease therapy Patient Age First no. (yr) /sex disease

Anthr Antimetabolite or Inh or Alk Topo II Hydrea RT

ASCT or BMT

1 2 3 4 5 6 7 8 9 10

65/F 68/M 43/F 69/F 59/F 71/F 59/F 64/M 47/F 47/F

No Yes Yes Yes No Yes Yes Yes Yes Yes

Yes No Yes Yes Yes Yes Yes No Yes Yes

Yes No No Yes No No No No Yes Yes

No No Yes No No Yes Yes No Yes Yes

No No No No No No Yes No No No

11 12

85/F 57/M

No Yes

No No

No No

Yes No Yes No

WHO MPS-MDS MDS / CMML AML-M4 AML-M4 AML-M4 AML-M5 AML-M3 CMML AML AML-M4 /M5b 2005-04 AML-M5a MDS / AML

13 14 15 16

32/M 47/M 66/F 52/F

Yes Yes Yes Yes

Yes Yes Yes Yes

No No No No

Yes Yes No No

Yes Yes Yes Yes

AML AML-M4 AML-M5 AML-M5b

17 18 19 20 21 22

37/F 58/M 56/F 72/M 69/M 25/M

Yes Yes Yes Yes Yes Yes

Yes Yes Yes Yes No Yes

No No No Yes No Yes

Yes No Yes No No Yes

23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

68/F 59/F 56/M 64/M 64/F 9/F 40/M 30/M 56/M 56/F 46/M 76/F 67/F 77/F 61/F

Yes Yes Yes Yes Yes Yes Yes Yes No No No No No Yes Yes

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No Yes

No No Yes No No No Yes Yes Yes No Yes No Yes No No

No No No No Yes Yes Yes No No No No Yes No No Yes

Yes Yes Yes No No ASCT 1 BMT No Yes No No Yes Yes Yes Yes Yes No Yes No No No No

38 39 40 41 42 43

76/F 64/M 54/M 65/F 84/F 69/F

No No No Yes Yes Yes

No No No No No No

Yes Yes Yes Yes No Yes

No No No No No No

No No No No No No

Ovarian C Bladder C Breast C Breast C Breast C Breast C Breast C Lung C Breast C Kidney C 1999 Breast C 2002 Breast C MGUS 1 cutaneous mucinosis Hodgkin Hodgkin Hodgkin Hodgkin scleronodular stage IV Bb Hodgkin NHL Hodgkin DLBC-NHL Waldenstro¨m FL stage IV 1993 Testis C 1997 NHL DLBC-NHL B-CLL MM MM ALL Pre B-ALL ALL MDS AML-M4 MDS / RAEB-t treated with Thorotrast* ET PV Breast C 1990 ET 1994 ET ET PV PV PV PV

Second hematologic malignancy

Latency Survival period after AML/ (mo) Status MDS (mo) 52 12 24 12 10 14 24 41 22 25

Dead Dead Dead Dead Dead Dead Alive Alive Alive Alive

5 19 11 7 5 13 60 1

360 130

Alive Dead

1 2

76 38 36 72

Dead Dead Dead Dead

5 5 2 10

MDS/ AML MDS / AML-M5 MDS / AML-M6 RAEB / AML MDS / AML MDS

84 46 168 95 72 96

Dead Dead Dead Dead Dead Alive

5 7 12 1.5 24

RAEB /AML AML-M4 MDS / AML-M4/M5 RAEB RAEB MDS / AML MDS / AML-M5 AML AML MDS trilineage AML AML-M3 AML 1 TL dysplasia AML-M2 TL dysplasia - MDS / RAEB / AML RAEB AML MDS / AML MDS / AML MDS / AML-M1 RAEB

132 52 11 48 144 26 10 108 23 44 5 180 204 324 168

Dead Dead Dead Dead Alive Dead Dead Alive Dead Alive Dead Alive Dead Dead Dead

4 8 0.2 9 5 13 3 8 51 38 51 5 6 5 7

132 156 240 180 228 ?

Dead Dead Dead Dead Dead LS

1.5 7 6 0.5 15 ?

4

Latency period refers to the time elapsed from the therapy of the first disease until the diagnosis of the t-MDS/AML. Survival refers to the time elapsed between the beginning of the therapy of the t-MDS/AML and death. Abbreviations: Alk, alkylating agent (cyclophosphamide, melphalan, ifosfamide, busulfan, nitrosureas, metal salts); Anthr or Inh Topo, anthracyclines and topoisomerase inhibitors (doxorubicin, daunorubicin, epirubicin, mitoxantrone, idarubicin); Antimetabolite or hydrea, folic acid antagonist, pyrimidine antagonist, purine antagonist, or hydroxyurea; C, cancer; RT, radiotherapy; CT, chemotherapy; ASCT, autologous stem cell transplantation; BMT, bone marrow transplantation; WHO, World Health Organization; Hodgkin, Hodgkin’s disease; NHL, Non-Hodgkin lymphoma; DLBC-NHL, diffuse large B-cell NonHodgkin lymphoma; FL, follicular lymphoma; B-CLL, B chronic lymphocytic leukemia; MM, multiple myeloma; MGUS, monoclonal gammopathy of unknown significance; ET, essential thrombocythemia; PV, polycythemia vera; CMML, chronic myelo-monocytic leukemia; MDS, myelodysplastic syndromes; RAEB, refractory anemia with excess blasts; TL, tri lineage; LS, loss of sight. * Human carcinogen used in the past as intravascular contrast agent.

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Table 2 Solid tumors Secondary disease

1 Ovarian C

MPS/MDS

2 Bladder C 3 Breast C

CMML AML- M4

4 Breast C

Karyotypes

FISH

M-FISH

ish der(5)t(3;5)(q24;q31) (wcp51,EGR12,CSF1R-,wcp31,BCL61), i(8)(q11)(wcp81,MYC11),11q23 .nuc ish(MLLx2)

46,XX,der(5)t(3;5)(q24;q31),18,113,215,220[7]/ 45,X,-X,der(5)t(3;5)(q24;q31),der(7)t(X;7) (?;q31),i(8)(q11),217[3]

ish del(7)(q21q35)(wcp71) ish t(8;16)(p11;p13)(wcp81, MOZ1,wcp161, CBP1; wcp161, CBP1,wcp81,MOZ1)

ND ND

AML-M4

46,XX,add(5)(q31),add(8)(p11)[2]/45,idem,2X[9]/ 47,XX,add(8)(p11),1add(8)(p11)[1]/48,XX,add (5)(q31),18,113[7]/49,XX,18,113,1mar[1]/46, XX[1] 46,XY,del(7)(q21q35)[3]/47,idem,18[18]/46,XY[18] (10/2001): 46,XX,t(8;16)(p11;p13)[16] (06/2002): 46,XX,t(8;16)(p11;p13)[12]/46,idem, del(18)(q21)[6]/46,idem,add(13)(p11)[3]/46,XX[3] 46,XX,t(9;11)(p22;q23)[20]/46,XX[5]

ND

5 Breast C

AML- M4

46,XX,add(12)(p13)[12]/46,XX[6]

6 Breast C

AML-M5

46,XX,t(9;11)(p22;q23)[7]/46,idem,t(2;8)(p21;q24) [4]/46,XX[12]

7 Breast C

AML-M3

46,XX,t(15;17)(q22;q21)[3]/46,XX[17]

8 Lung C 9 Breast C 10 Breast C 1 kidney C 11 Breast C

CMML AML AML-M4/M5b

45,XY,27[27]/46,XY[1] 46,XX[30] 46,XX,t(9;11)(p22;q23) [25]

AML-M5

46,X,2X,16,i(8)(q11),del(10)(q11),der(17)(?)[9]/47, idem,1i(8)(q10)[3]/45,idem,2del(10)(q11)[cp7]

ish t(9;11)(p22;q23)(30 MLL1,AF91; 50 MLL1,AF91) ish t(3;12)(q26;p13)(wcp31, EVI11,wcp121, 50 ETV61; wcp121,30 ETV61,wcp31, EVI11),11q23(MLLx2) ish t(9;11)(p22;q23)(wcp91,AF91,wcp111,30 MLL1;wcp111,50 MLL1,wcp91,AF91),t(2;8) (p21;q24)(30 MYC2;50 MYC1) ish t(15;17)(q22;q21)(PML1,RARA1; RARA-,PML1) ish 12p12(ETV6x2) ish 12p12(ETV6x2),11q23(MLLx2) ish t(9;11)(p22;q23)(wcp91,AF91,wcp111, 30 MLL1;wcp111,50 MLL1,wcp91,AF91) ND

Abbreviation: ND, not done (because often lack of metaphase cells).

46,XX,t(3;12)(q26;p13)[8]/46,XX[4]

ND

ND ND ND ND 46,X,2X,16,i(8)(q10),del(11)(q11),der (17)(11qter/11q11::17p1?2/17q2?3::8p? /8pter) [7]/45,idem,2del(11)(q11)[2]/47,idem,1i(8)(q10)[1]

W. Shali et al. / Cancer Genetics and Cytogenetics 168 (2006) 133–145

Identification and history

Table 3 Hodgkin’s disease, non-Hodgkin’s lymphomas, acute lymphoblastic, and myeloid leukemias, and exposure to genotoxins (23 cases) Secondary disease

Karyotypes

FISH

M-FISH

12 Dermatosis

MDS

45,XY,del(5)(q?),del(7)(q11),28[6]/46,XY[2]

ish del(5)(q31)(EGR12) ish del(5)(q33)(CSF1R2)

13 Hodgkin

AML

ND

14 Hodgkin

AML-M4

15 Hodgkin

AML-M5

47,XY,del(7)(q21q34),del(12)(p11p12), del(20)(q11qter),121[4]/46,idem,27[2]/47, idem,221,1mar[6] 47,XY,?add(3)(pter),del(8)(p12), del(9)(p21),add(11)(q22),del(12)(p?) or add(12)(p?),121[16] 46,XX,add(1)(q?),del(7)(q?), del(9)(q?),add(11)(q?)[19]/49,idem,117,118,120[1]

44,XY,der(5)t(5;9)(q11;?),der(6)t(Y;6) (q?;q25),del(7)(q11),29,der(12) t(9;12)(?;p11),der(14)t(6;14)(q26;q31), 216,der (19)t(16;19)(?;?)[4]/46,XY[2] 47,XY,der(7)t(3;7)(p2?3;q2?1), del(12)(p11p12),del(20)(q11),121[5]

16 Hodgkin

AML-M5b

17 Hodgkin

MDS/AML

18 NHL

Cytopenia / 46,XY,del(11)(q23)[7]/46,XY[7] RAEB / AML-M5

19 Hodgkin

AML-M6

20 DLBC-NHL AML 21 Waldenstro¨m MDS 22 FL 1 testis C MDS

23 FL

RAEB

47,XX,t(1;13)(q11;q34),1der(13)t(1;13)(q11;q34) [cp12]/46,XY[7]/46,XX[1] 45~47,XX,add(1)(p?),del(3)(q?),add(13)(p?), ?i(17)(q10),2221mar,1dmin[cp22]

44,XX,t(3;12)(p?14;p?13),del(5)(q?), 27,216,218,120[19]/45,idem,18[3]/46,XX[1]

ND

47,XY,del(8)(p12),del(9)(p21),121[12]/46, XY,19,215[2]

ish der(19)t(11;19)(q23;p13)(MLL1)

46,XX,t(1;7)(q4?1;q2?1),ins(11;9) (11pter/11q11::9?::11q11/qter),der(19) (19qter/19p13::11q23/qter)[19] ND

ish t(1;13)(q11;q34) (wcp11,wcp131),der (13)t(1;13)(q11;q34)(wcp11,wcp131) ND

ish t(11;16)(q23; p13)(wcp111,50 MLL1, wcp161,CBP1 ;wcp161,CBP1,wcp111, 30 MLL1) ish del(5)(q31)(EGR12)ish del(5)(q33) (CSF1R2)ish t(3;12)(p?14;p?13) (ETV6-;ETV62),.nuc ish11q23(MLLx2) ND ND ND

46,XY,-7,1mar[2]/46,XY[19] 47,XY,18[24] 46,XY,del(8)(q?)[1]/46,XY,inv(7)(?),del(10)(q?)[1]/ 46,XY,del(7)(q?)[1]/46,XY,del(7)(q?),del(5)(q?)[1]/46, XY[18] 43,XX,der(3)t(3;22)(q11;?),der(5) ish der(5)(q31)(EGR12) t(5;17)(?q11;?q11),del(7)(q?21),der(8)dic(2;8) kish der(5)(q33)(CSF1R2) (?p16;?q24),der(13)t(3;2;13)(?;?;p11),214, 214,del(16)(q?),217,219,der(21)t(20;21) (?p11;?p11),1mar[cp23]

24a DLBC-NHL AML-M4

46,XX,t(9;11)(q34;p15)[21]/46,XX[15]

25 B-CCL

AML-M4/M5

26 MM IgGl

Pancytopenia / RAEB

46,XY,add(6)(q?),t(8;16)(p11;p13) [cp2]/40~43,idem,del(9)(q?)[cp3] / 40~46,idem,1mar[cp4]/46,XY[17] 46~53,XY,del(20)(q?)[cp13]/46,XY[20]

W. Shali et al. / Cancer Genetics and Cytogenetics 168 (2006) 133–145

Identification and history

46,XX,t(1;3)(p35;q23),i(13)(q11),t(17;20) (p12?;?),i(22)(q11),der(22)t(20;22)(q11;?) [cp15] ND

44,XX,t(3;12)(p?14;p?13),der(5)t(5;7) (q1?1;?),27,del(16)(q2?1),-18[5] 46,XY[15] ND 46,XY,t(8;10)(q2?1;q2?3)[2]/46,XY[9]

43,XX,der(3)(3pter/3q11::22q11/ 22qter),der(5)(5pter/5q11::17q12/ 17qter),del(7)(q?21),der(8)(8pter/8qter:: 8qter/8q12::14q?2/14qter),der(13) (13qter/13p11::14?::3?),214,214,del(16) (q?),217,219,der(21)(21qter/21?p11:: 20?p11/20qter),13~8mar[10] 46,XX,t(9;11)(q34;p15)[10]

ish t(9;11)(ABL2, NUP981,MLL2;ABL1, NUP981,MLL1),12p13 (ETV6x2) ish t(8;16)(p11;p13)(wcp81, MOZ1,wcp161, ND CBP1; wcp161, CBP1, wcp81,MOZ1), .nuc ish11q23 (MLLx2), 12p13 (ETV6x2) ish del(20)(q?)(wcp201) 46,XY,del(20)(q?)[6]/51,X,2Y,13,15, 17,19,-13,der(14)t(1;14)(?;p?11),115, 115,119[2]/46,XY[3]

137

(Continued)

138

Table 3 Continued Identification and history 27 MM

Secondary disease

Karyotypes

43~45,X,del(X)(q?),del(1)(q2?),add(6)(pter), add(7)(q?),add(9)(q?),1?add(14)(q32), 1? add(15)(q?),217,218,220[cp11] / 43~44,idem,?del(5) (q?),add(12)(p?)[cp4]/46,XX[8] Dysplasia/RAEB 45,XX,der(5)t(5;17)(p10;p10),217[5]/46,XX[27] 28b BII-ALL /AML (01/26/2004): 45,XX,der(5)t(5;17)(p10;p10),-17[1]/ 44,idem,der(3)(?),1?der(5),add(6)(q?),218, add(21)(q?)[cp15]/46,XX[4] 29c B-ALL AML-M5 (02/2002): 46,XY,del(7)(p?)[3]/46,XY,del(9)(q?)[2]/ (04/2002): 46,XY,del(5)(p15)[3]/ 46,XY,del(7)(p14p15)[3]/46,XY,inv(1)(p22q32 ?)[2]/ 46,XY,r(6)[2]/46,XY,del(9)(q2?2)[2] 46,XY[2] 30d Bipheno ALL AML 92,XXYY,add(5)(q13), t(11;18)(q23;q21)[cp10]/91, idem, 2der(18)t(11;18)(q23;q21) [4]/91,idem, ?del(7)(p?),214[1]/91,idem,27[1]/46,XY[6] 31 MDS AML 46,XY,del(13)(q?)[1]/45,idem,22,?del(4)(q?), der(6)t(2;6)(q?;?) [15]/46,XX[4]

M-FISH

ND

44,XX,t(1;20)(q?11;?),t(5;11)(q?11;q1?1), der(5)t(5;7)(p13;?),t(6;9)(p11;q11),27,218[3]/ 44,idem,del(10)(q25),der(12)(12qter/12p?::10?::5?::3q? / 3qter)[3]/46,XX[2] (01/26/2004): 44,XX,der(3)del(3)(p2?1) del(3)(q2?1),dic(5;17)(q10;p10),der(6)t(6;18) (p22;q?),dup(6)(q?),-17,-18,i(21)(q11)[6]/46, XX[7] ND

ish der(5)t(5;17)(D5Z11,D17Z11)

ND

ish add(5)(q31)(EGR1-) ish add(5)(q33) (CSF1R2) ish t(11;18)(q23;q21)(5 MLL1, BCL21;3MLL1, BCL22) ND

32 AML-M4 33 MDS

MDS AML

46,XX,t(3;18)(p14;p11)[3]/46,XX[19]/46,XX[9]

nuc ish 8cen(D8Z2x2) ND

34 Thorotrast

AML-M3

46,XX,del(15)(q1?3q?25)[3]/46,XX[21]

ish t(15;17)(q22;q21)(PML1, RARA1;PML1,RARA2)

Abbreviations: ND, not done (because often lack of metaphase cells); TCLL, T chronic lymphocytic leukemia. a This case is reported in reference 19. b,d Normal karyotype at diagnosis. c 46,XY,t(14;19)(q32;q13)[12]/46,idem,idic(8)(p11)[6]/46,XY[7] at diagnosis.

92,XXYY,der(5)t(5;6)(q13;q26),t(11;18)(q23; q21)[6]/91,idem,2der(18)t(11;18) (q23;q11) [3]/46,XY[2] 45,XY,der(2)t(2;4)(p1?4;p2?1),24,der(6) (2?qter/2?q11::6p24/6q24::4q11/4qter), t(13;18)(q21;q12)[cp8]/46,XY[5]/46,XX[4] ND 85,XXYY,der(3)t(3;22)(p3?;q1?1)x2,del(4)(q2?5) x2,del(7)(q3?1)x2,19,der(11)t(3;11)(?;q1?1)x2,der (12)t(12;22)(p11;q12)x2,115,der(16)t(5;16) (?;?),der(16)t(15;16)(?;?)x2,der(17)t(5;17) (q1?1;q1?1)x2,der(18)t(18;20)(p11;?q)x3, 218,220,220,der(22)(18?::22p11/ 22q11:: 11?)x2,9~16dim[3] ND

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MDS: pancytopenia

FISH

Table 4 Nine patients with myeloproliferative syndromes Secondary disease 1 latency period

35 ET

TL AML 204 months

36 PV

MDS /AML 324 months

37 ET

TL MDS / RAEB / AML 168 months

42~44,X,der(X)t(X;14)(?q11;?q11),der(5)t(5;12)(p?;?), ?del(7)(q?),del(9)(q13q32),der(12)t(5;12;?20)(?;p?;?), del(14)(q?)x2,der(17)t(14;17)(?;p?),220[cp15]/46, XX,del(12)(p?)[3] / 46,XX[5]

ND

38 ET

RAEB 132 months

45,XX,217[3]/46,XX,add(17)(p?)[cp6]/45,XX,del(17) (q?),221[cp14]/44~45,idem,214,add(16)(q?)[cp2]/46,idem, del(5)(q?),add(8)(q?)[1]/45~46,idem,add(15)(p?)[cp3]

39 ET

MDS / AML 156 months

47,XY,24,?der(8),del(9)(q13), del(18)(?),12mar[cp12]/47, XY,del(13)(q13q21),del(18)(?),1r(?) [cp7]/46,XY[4]

ish t(16;17)(wcp161,wcp171) / ish der (16)t(16;21)(wcp161,wcp211), der(17) del(17)(q?)add(17)(q?) (wcp171,wcp172), add(17)(p?)(wcp171,wcp172) ish 13 (D13S252D13S319x1, 13qterx2)

40 PV

AML 240 months

45,XY,del(5)(q?),add(7)(q?),hsr(9)(p13),217[16]/ 45,XY,del(5)(q?),27,hsr(9)(p13),220,1mar[3]/46,XY[3]

ish der(5)t(3;5)(q25;q12)(wcp51,EGR12, CSF1R2,wcp31,BCL61),der(7)t(7;17) (q31;?)ins(7;17)(q11;?)(wcp71,wcp 171, p532), hsr(9)(p13)(wcp91)

41 PV

acute myelofibrosis 180 months

59~82,XXX,24,25,add(6)(q?),18,18, del(9)(q?)x2,add (10)(q?),del(11)(p14),211,del(13)(q?)x2,214,214, 215,217,217,220,220,?i(21)(q11)x2,221,221, 222,222,222,14mar[cp23]

ish der(9)t(9;21)(q12;q21)(AML11)

42 PV

AML-M1 228 months RAEB ?

46,XX,del(20)(q?)[3]/46, idem,der(7)t(1;7)(q12;q11)[13]/ 46,idem,del(9)(q13q34)[7] 51,XX,del(5)(q14q33),15mar[15]/46,XX[7]

ish der(7)t(1;7)(wcp71,wcp72), del(20) (q?)(wcp201),del(9)(q13q34)(wcp91) ish del(5)(q31)(EGR12) ish del(5)(q33) (CSF1R2) ish 9p22 (AF9x7), 11q23 (MLLx7)

43 PV

Karyotypes

FISH

M-FISH

46,XX,del(5)(q12q35),del(7)(q?)[1]/48~50,XX,del(5) (q12q35),del(9)(q?),1del(9)(q?),1del(11)(q?)x3, add(19)(q?)[cp6]/ 48~50,idem,add(1),12mar [cp4]/46,XX[15] 46,XX,t(3;17)(p11;q12),del(5)(q?),del(7)(q?),18, 212,add(15)(q?),217,119[27]/46,XX[1]

ish del(5)(q31)(EGR12) ish del(5)(q33) (CSF1R2) ish 12p13 (ETV6x2), 11q23 (MLLx2), 11p15 (NUP98x2, D11S1363x2)

50,XX,del(5)(q12q35),i(9)(p11),der(19)(19?::11?:: 19?::11?::9?),19~11dmin[10]

ish del(5)(q31)(EGR12) ish del(5)(q33) (CSF1R2) ish 12p13 (ETV6x1), 11q23 (MLLx2), 11p15 (NUP98x2, D11S1363x2)

46~47,XX,der(3)t(3;17)(p11;q12),der(5)t(5;17) (q1?;?),der(7)t(7;12)(q31;?),18,212,ins(15;12) (15pter/15q?::12?::15?::12?::15q? /15qter), 217,119[cp7]/45~47,idem,218[cp6] 42~44,X,t(X;13)(q21;q13),der(5)(12pter/12p 11::5p13/5q31::20p11/20qter),t(5;12)(p?;?),27, del(9)(q13q32),der(12)(20qter/20p11::12q11/ 12q24::5q31/5qter),t(14;17)(q11;p11),t(14;17) (q24;p11), 220[cp10]/46,XX[2] ND

46,XY,24,del(9)(q13),218,1del(21)(q21)x2[cp4]/ 47,idem,1der(9)t(9;9)(?;?)[7]/47,idem,del(7) (q21qter),1der(9)t(9;9)(?;?)[2]/47,XY,del(13) (q13q21),del(18)(?),1r(11)[2] 45,XY,der(5)t(3;5)(q25;q12),der(7)(7pter/ 7q1?1::17?::7q1?1/7q31::17q? /17qter),hsr(9) (p13),217[6]/45,XY,der(5)t(3;5)(q25;q12),der(7) (7pter/q1?1::17?::7q1?1/7q31::17q? /17qter), der(9)t(8;9)(q1?2;p?)hsr(9)(p?),217[1]/47,XY,der(5) t(3;5)(q25;q12),1del(8)(?), hsr(9)(p13),der(19) (19pter/19q11::21?::19?::21?),221,1dmin(4)[3] 59~82,XXX,24,25,der(6)t(6;14)(q2?4;q3?1),18,18, der(9)t(9;21)(q12;q21)x2,der(10)t(6;10)(?;q26),del (11)(p14),der(12)t(12;22)(p1?2;?)x2,der(13)t(11;13) (?;p11),214,214,215,del(16)(?p12),216,217,217, der(20)t(20;22)(?;?)x2,1der(20)t(20;22)(?;?)x2,221, 221,der(22)t(1;22)(q11;p11),222,222[cp10] ND

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Identification and history

51,XX,der(5)(5pter/5q11::18q11/18qter), 1der(9)t(9;11)(p?;q23q25)x5[15]

Abbreviation: ND, not done (because often lack of metaphase cells). 139

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abnormalities, based on the primary diagnosis, are shown in Tables 2–4.

3. Results and discussion 3.1. Eleven cases secondary to therapy for solid tumors In this group of 11 patients (ages 47–85, mean 5 61.5), there were one MDS, two chronic myelo-monocytic leukemia, and eight AML cases. Eight cases were characterized by a monocytic differentiation. A total of 25 chromosomal abnormalities were listed (2.3 abnormalities per case). Among seven balanced translocations, there were six ‘‘classic’’ recurrent translocations: one t(8;16)(p11;p13) with MOZ/CBP rearrangements, three t(9;11)(p22;q23) with MLL/AF9 rearrangements, and one t(15;17)(q21;q22), which was promyelocytic, with PML/RARa rearrangements. A case of AML-M4 was associated with a recurrent t(3;12)(q26;p13) that implicated EVI1/ETV6. A balanced t(2;8)(p21;q24) was present as a second translocation in association with a t(9;11)(p22;q23). Chromosome 8 was the most implicated, with two balanced translocations, two trisomies, and two i(8q). There were only three deletions (7q, 11q, and 18q) and four unbalanced translocations of chromosomes 5, 7, 13, and 17. There were no del(5q) in this group, but FISH revealed a der(5)t(3;5)(q24;q31) associated with a deletion of EGR1 and CSF1R genes.

3.2. Twenty-three cases with various primary hematologic pathologies This group was the most heterogeneous according to age and previous history. The subjects’ ages ranged between 9 and 76 years, and the mean value was 50.6 years. This group was composed of Hodgkin’s disease (six cases), non-Hodgkin’s lymphomas (seven cases), multiple myeloma (two cases), monoclonal gammapathy of undertermined significance with dermatosis (one case), B-ALL (three cases), MDS/AML followed by different MDS/ AML (three cases), and one promyelocytic leukemia (AML-M3) after the use of Thorotrast (a radioactive colloidal suspension of thorium dioxide which had been used in the past as an intravascular contrast agent). One patient, who is currently alive (case 22), had a previous history of two consecutive malignant diseases (follicular lymphoma followed 4 years later by a testis carcinoma). This group included 6 t-MDS and 17 t-AML. Cytologically, monocytic M4-M5 forms were the most frequent (seven cases). We observed 81 chromosomal abnormalities (3.5 aberrations per patient). Chromosome 5 was modified 11 times, chromosome 7 was modified 10 times, and chromosome 18 was modified 9 times. Six monosomies, 4 trisomies, 2 isochromosomes, 1 dicentric, 1 insertion, 1 inversion, 1 ring, 1 duplication, 1 amplification, 19 deletions, and 45 translocations were present (example in Fig. 1). With regard to translocations, 15 were balanced and 30 were unbalanced, including one whole-arm translocation resulting

Fig. 1. M-FISH image of a complex karyotype in patient 31 (Table 3). Identification of a der(2)t(2;4)(p1?4;p2?1), a der(6)(2?qter/2?q11::6p24/ 6q24::4q11/4qter) and a t(13;18)(q21;q12).

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in a dic(5;7)(q10;q10). Besides ‘‘classic’’ balanced recurrent translocations such as t(8;16)(p11;p13) and t(11;16) (q23;p13), which implicate MOZ and CBP, and MLL and CBP, respectively, we observed a der(15)t(15;17)(q22;q21) (with PML/RARA fusion) secondary to Thorotrast use, as well as t(11;19)(q23;p13), a translocation with MLL rearrangement. Within this cluster of patients, there was also a case with a double amplification for material arising from chromosomes 9 and 16. 3.3. Nine cases of CMPD This group was composed of five polycythemia vera (PV) and four essential thrombocythemia (ET) in ‘‘acute transformation’’ after a latency period ranging from 132 to 324 months (mean 5 204 months). In this group, patients were older than in the two other groups, with ages ranging from 52 to 84 years (mean age 5 66.9 years). One of these patients had a history of breast cancer in 1990 followed by ET 4 years later. There were a lot of abnormalities (57, mean of 6.3 per patient), including 12 deletions. Only 4 balanced versus 22 unbalanced translocations, 7 monosomies, and 2 trisomies were observed. Amplifications such as homogeneously staining regions (hsr) or double minutes (dmin) were observed in three patients. The most altered chromosomes were chromosomes 9 (seven times), 17 (six times), 5 (six times), and 7 (five times). It should be noted that there were two cases of amplification on chromosome 9 in two out of five PV cases: case 40, t(8;9)(q1?2;p?) and hsr(9)(p13), and case 43, der(9)t(9;11)(p?;q23q25)x5 with unrearranged MLL amplification. 3.4. Numeric abnormalities In t-MDS/AML, numeric aberrations are multiple, and hypodiploid forms are generally the most frequent. Among the 43 patients in our series, there were 15 hypodiploidies that were single or associated with hyper- or pseudodiploid clones. These cases were unequally distributed, depending on the group. In Table 2 (t-MDS/t-AML in post-solid tumors), diploid cases were majority (6/11 patients, and in 5 of them, the diploidy was accompanied by a balanced translocation). The five other cases were associations of hyperploidy [2], with hypo- and/or pseudodiploid clones. In these cases, the clones were always related (clonal evolution). In Table 3, there were eight hypo-, seven pseudo-, four hyper-, and four diploidies with balanced translocation. In case 26, the two clones were unrelated; it was in fact multiple myeloma with secondary MDS [del(20q)] and another hyperploid clone corresponding to abnormal plasmacytic cells [13]. For the nine patients in Table 4 (post-CMPD), there were two hyperploidies (linked to amplifications) and three pseudodiploidies, one of them with a hypodiploid clone. In one out of the three cases of hypodiploidy, a hyperploid clone

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was associated with an amplification. The last case could be considered as hypotetraploid. 3.5. Structural abnormalities The M-FISH technique, which was completed by FISH studies, allowed the identification of unbalanced translocations and cryptic translocations in 89% and 30% of the cases studied, respectively. Deletions Every structural abnormality suggesting a possible deletion was studied by FISH techniques (painting and unique sequence probes). There were 36 deletions in the 43 patients. In our series, deletions confirmed by FISH and MFISH were del(5q): 11 times (26%); del(7q): 11 times (26%); del(9q): 8 times (19%); followed by del(18q), del(20q), and del(21q), two times each (5%). All cases showing chromosome abnormalities were studied by FISH with the EGR1 (5q31) probe. Among the 11 del(5q) confirmed by FISH, only one was a ‘‘pure,’’ simple deletion (without associated chromosome 5 translocation). FISH techniques showed that conventional cytogenetics often underestimated associated translocations, with some deletions being in fact derivative chromosomes associated with deletion [14]. Seven patients (16%) had both del(5q) and del(7q). Balanced translocations In this series of 43 patients, we observed 26 balanced translocations that were recurrent (n 5 9) or not recurrent (n 5 17). Six of these nine recurrent balanced translocations (67%) were from the group in Table 2 (6/11 patients). It is obvious that the nature of the therapeutics used was important in the appearance of these abnormalities. According to the WHO classification of hematopoietic tumors [9], two major types of therapy-related malignancies are now recognized on the basis of the causative agents: the alkylating agents/radiation-related MDS/AML and the topoisomerase II inhibitor–related AML. The first one has a latency period varying from a few months to several years, while the second may have a very short (few weeks) or even no latency period, beginning immediately after the post-therapy aplastic phase. 11q23/MLL translocations are mainly linked to the use of topoisomerase II inhibitors [15,16]. 11q23 rearrangements in treatment-related leukemias were thought to be found mainly after treatment with anti–topoisomerase II (epipodophyllotoxins) or with an intercalating topoisomerase II inhibitor (anthracyclins), as in the case of some 21q22 rearrangements. They may also be found after treatment with alkylating agents and/or radiotherapy, with varying previous types of cancer: breast cancer, non-Hodgkin’s lymphoma, Hodgkin’s disease, leukemia, lung carcinoma, and other malignancies. Various 11q23 rearrangements may be found [16]. In our series, there were three cases of t(9;11)(p22;q23) in Table 2

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(cases 4, 6, and 10), as well as one t(11;16)(q23;p13) (case 18, Table 3) and one t(11;18)(q23;q21) (case 30, Table 3) implicating the MLL gene. The published cases of t(11;16)(q23;p13) were always therapy-related, and no de novo cases have been described [17]. In this series, there were five rearrangements implicating MLL, and all expressed an AML phenotype. For the two t(8;16)(p11;p12) implicating MOZ and CBP (case 3, Table 2; case 25, Table 3), a correlation with topoisomerase II inhibitor use has also been reported [18]. For the t(9;11)(q34;p15) (case 24, Table 3), FISH demonstrated a NUP98 rearrangement and molecular biology identified PRRX2 as the partner gene [19]. The t(11;18)(q23;q11) (case 30, Table 3), which showed MLL rearrangement, will also be a recurrent one. For the recurrent translocation t(3;12)(q26;p13) with ETV6/EVI1 rearrangement, we were unable to confirm the presence of a transcript, due to the lack of material. To date, the other 14 balanced translocations do not appear to be recurrent. Further characterization of the genes involved in these aberrations are needed to determine whether the translocated segment leads to functional fusion proteins or if other mechanisms are involved [20]. Unbalanced translocations These usually appear as ‘‘del’’ or ‘‘add’’ if studied by conventional cytogenetics. Only FISH or M-FISH can determine the exact nature of the translocation and can distinguish the real deletions. In this series, there were 56 unbalanced translocations. Derivative chromosome 5 was implicated most often (11 times). Such der(5) were often interpreted as del(5q) by conventional cytogenetics. The other derivatives were der(7) and der(12), five times each; der(6), der(9), der(17), and der(19), four times each; and der(3), three times. It can be noted that all unbalanced translocations of chromosome 9 affected cases secondary to CMPD (Table 4). These abnormalities (structural and numeric) of chromosome 9 are a well-known characteristic of CMPD [21–24]. An infrequent form of unbalanced translocation is ‘‘whole-arm translocation’’ of chromosomes 5, 7, and 17, which is sometimes observed after exposure to alkylating agents and hydroxyurea, leading to dicentric chromosomes [25]. Some of these dicentrics are described in treated MDS/MPS, and a particular form, which is correlated with cell morphology (Pelger cells with vacuoles and lack of cytoplasmic granulations), is the monosomy 17p [26,27]. Patient 28 (Table 3), a 9-year-old girl, is an example of monosomy 17p with monosomy 5q. She presented with acute lymphoblastic leukemia with normal karyotype. After induction treatment, she was in clinical and cytologic remission. A dysplasia (refractory anemia with excess blasts) appeared in a few weeks. At the same time, the cytogenetic follow-up after the first course of treatment revealed a dic(5;17)(q10;p10) without cytologic abnormality. Thirteen successive analyses were performed. Additional abnormalities appeared while the blasts augmented, she became

resistant to the treatment, and finally developed overt AML. This case emphasizes the very unfavorable prognosis of dicentric chromosomes involving chromosomes 5 and 17, which has already been mentioned by others [28]. Amplification An interesting application of M-FISH is the study of amplifications. In this series, six different amplifications were present in four patients, and we used M-FISH to identify the origin of the amplified material in these four cases. For patient 33 (Table 3, AML after bone marrow transplant, Fig. 2A), M-FISH allowed the identification of two kinds of dmin in the same cell; some are identified as material of chromosome 9 and others as material of chromosome 16. For patient 43 (Table 4, AML post-PV; Fig. 2B), the bicolor MLL probe hybridized on derivative chromosome der(9)t(9;11)(?;?) without splitting. This absence of MLL rearrangement when amplified has been already described [29,30]. MLL was amplified not as an hsr or dmin, but as an MLL low-copy gain because of the retention of MLL copies on derivative chromosomes der(9)t(9;11)(?;?). Finding extra copies of the MLL gene is a recurrent change found in approximately 20% of MDS and AML. It is often associated with dysplastic bone marrow changes or complex karyotypes suggestive of genotoxic exposure and associated with extremely poor survival [30]. For patient 35 (Table 4, AML post-ET; Fig. 2C), the karyotype revealed four minutes: three of them were composed of chromosome 11, and the fourth one of chromosome 9. A der (19)(19?::11?::19?::11?::9?) was also present. These rearranged chromosomes were not hybridized by the MLL probe. The karyotype of patient 40 (Table 4, AML post-PV; Fig. 2D) presents a der(9)hsr(9p), which was confirmed by M-FISH homogeneous hybridization. Moreover, we observed one minute from chromosome 4 in some metaphase cells. It can be emphasized that amplification of chromosome 9 material in our series was restricted to the post– myeloproliferative (MPS) syndrome cases. For MPS, it is difficult to say whether the evolution in AML is a spontaneous mechanism (natural history of the disease) or if this AML is secondary to the treatment [31]. Considering the cytogenetic abnormalities (numerous chromosome 9 and 17 abnormalities), the two mechanisms probably coexisted [32]. In conclusion, the majority of published reports highlight the complexity of cytogenetic abnormalities in secondary MDS/AML. The first series published in the 1980s, when the effects of alkylating agents were demonstrated, emphasized the rearrangements of chromosomes 5, 7, 12, and 17 [33]. In our series of t-MDS/AML, the complexity of abnormalities and implicated chromosomes did not seem to be different from de novo MDS/AML rearrangements. In 20% of the patients studied, we found the same recurrent translocations [t(8;16)(p11;p13) with

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MOZ/CBP rearrangements, t(9;11)(p22;q23) with MLL/ AF9 rearrangements, t(15;17)(q21;q22) with PML/RARa rearrangements, t(3;12)(q26;p13), with EVI1/ETV6 rearrangements, and t(11;16)(q23;p13), implicating MLL and CBP] as in de novo MDS/AML. This observation agrees

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with the established notion that de novo MDS/AML in older patients is close to secondary forms. In the literature, a selection criterion of cases is often the karyotype complexity. If we analyze some recent series that group together secondary and de novo cases [30,34], it appears

Fig. 2. Representative M-FISH images and R-banded chromosomes illustrating the various types of amplifications in four patients. (A) Two types of dmin coexisting in patient 33 (Table 3). It was identified as chromosomes 9 and 16. (B) Identification of markers (der(9)t(9;11)(?;q23)) in patient 43 (Table 4). FISH image with MLL dual-color probe showing seven copies of the MLL gene. (C) Two types of dmin in patient 35 (Table 4), dmin from chromosomes 9 and 11. (D) Two types of non-coexisting amplification in patient 40 (Table 4) [hsr(9) and dmin from chromosome 4].

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that the patient age reduces the differences between the two categories. There is not much available data on unselected consecutive secondary MDS/AML. It is possible that the frequency of secondary recurrent translocations (which represent 25% of the abnormalities in our series) has been underestimated by lack of available information.

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