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Cancer Genet Cytogenet 118:48–51 (2000)

Elsevier Science Inc., 2000. All rights reserved.655 Avenue of the Americas, New York, NY 10010

FISH Analysis of an AML-M5a with Segmental Rearrangements Involving 11q23-

MLL

Region

K. S. Reddy, L. Parsons, S. Wang, L. Mak, P. Dighe, and T. L. Yu

ABSTRACT:

A 66-year-old man was diagnosed 10 years ago with thrombocytosis and treated withhydroxyurea. Recently, his condition deteriorated and he was found to have 68% blasts in the bloodand AML-M5a. Conventional cytogenetic analysis and fluorescence in situ hybridization studies using11-painting and MLL probes showed that chromosome 11 had duplications or triplications; the markerswere also derived from the chromosome 11-MLL region. Therefore, we have demonstrated segmentalrearrangement of chromosome 11 involving the

MLL

region resulting in multiple copies of the

MLL

gene. © Elsevier Science Inc., 2000. All rights reserved.

INTRODUCTION

Segmental rearrangements involving chromosome 11, the

MLL

gene region, has been previously reported [1–3].Tanaka et al. [2] identified jumping translocations of thechromosomal segments containing the

ABL

or

MLL

onco-genes as a new form of gene amplification in treatment-related leukemias with complex karyotypes. The leuke-mias were of French–American–British (FAB) M1, M2, orM4 morphologic subtypes and followed various classes ofDNA-damaging chemotherapy and radiation used for themanagement of adult solid tumors. The karyotypes alsocontained monosomy 5 or 7, findings typical of alkylatingagent-induced leukemias. Multiple copies of the

ABL

or

MLL

oncogenes dispersed throughout the genome and ex-trachromosomally characterized the segmental jumpingtranslocations. Felix et al. [1] studied a treatment-relatedacute myeloid leukemia (t-AML) case with a complex kary-otype. Fluorescence in situ hybridization (FISH) analysisshowed multiple copies of the

MLL

gene consistent withsegmental jumping translocation. A genomic region in-cluding CD3

e

,

MLL

, and a segment of band 11q24 was notrearranged, but MLL was amplified by Southern blot analy-sis. Their results indicate that loss of wild-type p53 may beassociated with genomic instability after DNA-damagingchemotherapy and radiation, manifesting as a complex

karyotype and gene amplification in some cases of t-AML.Cuthbert et al. [3] described a jumping translocation of11q23 with

MLL

gene rearrangement and interstitial telom-eric sequences. In this paper, we present yet another casewith segmental rearrangements involving the

MLL

region.

CASE REPORT

A 66-year-old Caucasian male was diagnosed with a my-eloproliferative disorder (MPD) 10 years ago. The thromb-ocytosis was treated with hydroxyurea for 1.5 years. In1994 and 1997, the patient was again given hydroxyureaand frequent blood transfusions. Reverse transcriptase-polymerase chain reaction (RT-PCR) was negative for the

BCR/ABL

fusion transcript. Recently, he developed a per-sistent cough. A chest x-ray showed bilateral interstitialpulmonary infiltrates. Progressive abdominal distention,hepatosplenomegaly, weakness, anorexia, and weight losswere noted. Ecchymosis of both arms and legs was ob-served. He also had profuse sweating without fever.

His hemogram had 294.8

3

10

9

/L white blood cells, 8.2g/dL of hemoglobin, and 134

3

10

9

/L platelets. The differ-ential was: segments 21%, neutrophilic bands 8%, lym-phocytes 1%, metamyelocytes 2%, blasts 68%, and mye-locytes 4%. Marked dyserythropoiesis was present.Among the nucleated marrow cells, 75% belonged to thegranulocyte series of immature mononuclear cells. In ad-dition, there were a few dysplastic neutrophils, and giantmetamyelocytes with nuclear cytoplasmic asynchrony.Megakaryocytes were decreased and were frequently hy-persegmented. Many ringed sideroblasts were observed.The smear was significant for marked leukocytosis, nor-mocytic hypochromic anemia, and thrombocytopenia.Frequent basophilic and polychromic erythroblasts andred blood cells with basophilic stippling were noted.

From the Quest Diagnostics Inc. (K. S. R., L. P., S. W., L. M.),San Juan Capistrano, California, USA; Stockton HematologyOncology Medical Group (P. D.), Stockton, California, USA; andthe Department of Pathology and Laboratory Medicine, St. Joseph’sHospital Medical Center (T. L. Y.), Stockton, California, USA.

Address reprint requests to: Dr. Kavita S. Reddy, CytogeneticsDepartment, Quest Diagnostics Inc., San Juan Capistrano, CA92690.

Received June 15, 1999; accepted August 25, 1999.

Segmental Rearrangements Involving 11q23-

MLL

49

White blood cells showed 68% abnormal mononuclearmonocytes negative for periodic acid-Schiff (PAS) andchloracetate (CAE) stains and weakly positive for nonspe-cific esterase (NSE). A few dysplastic neutrophils and my-eloid precursors were also noted. Some giant platelets aswell as agranular/hypogranular platelets were seen. Theacute myeloid leukemia favored the subclass M5a, andflow cytometry studies supported this diagnosis. The pres-ence of trilineage dysplasia raised the possibility of treat-ment-related acute myeloid leukemia.

MATERIALS AND METHODS

Unstimulated bone marrow cultures were harvested after24 and 48 hours. The slides were GTG-banded.

Fluorescence in situ hybridization was performed us-ing coatasome-11, MLL, TP53 and BCR/ABL probes (On-cor Inc.). The GTG-analyzed slide was used for MLL,coatasome 11 and TP53 hybridization. The slides were de-

natured at 70

8

C for 2 minutes. After overnight hybridiza-tion at 37

8

C in a humid chamber, the slides were washed in2

3

SSC (MLL and p53), 1

3

SSC (coatasome 11), or 0.5

3

SSC (BCR/ABL) at 73

8

C for 5 minutes.

RESULTS

Twenty cells were analyzed from the GTG-banded slides.One normal and four related abnormal clones were ob-served. The karyotype was 45

z

47,X,

2

Y[9],add(1)(p35)[9],add(11)(q23)[15],del(17)(p13)[9],add(17)(p11)[4],

2

10[11],

2

20[4],mar1[11],mar2[9][cp15]/89,XXYY,add(11)(q23)

3

2,

2

10,

2

12,

2

16[2]/46,XY[3]. Fluorescence in situ hybridiza-tion was performed using MLL and TP53 to characterizethe add(11) and del(17)(p13). The bcr/abl probe was usedto rule out a cryptic Philadelphia rearrangement or

ABL

amplification. Three copies of the

MLL

gene were foundon the add(11)(q23) in the near tetraploid cells and coata-some-11 painted add(11) end to end (Fig. 1b and 1c). In

Figure 1 (a) G-banded chromosomes from the near-tetraploidclone showing add(11) (arrow). (b) The chromosome 11 paintingprobe hybridized to both add(11) end to end (arrow). (c) The MLLprobe was found to be present in three copies on add(11) (arrow).

Figure 2 (a) A DAPI-converted G-banded chromosome from main line with add(11)(q23) (arrowhead) and twomarkers (arrow). (b) The 11-paint hybridized to the add(11) (arrow) and the markers (arrowhead) end to end. (c)Two copies of MLL probe was found on the add(11) (arrow) and the markers also had MLL signals (arrowhead).

Figure 3 DAPI-converted G-banded chromosome with a dele-tion on 17p13 (arrow). (b) The deletion on 17p13 was missingTP53 (arrow).

50

K. S. Reddy et al.

the main line, add(11)(q23) had two copies of

MLL

, andthe two markers also had

MLL

signals (Fig. 2c); coatasome11 painted the add(11) and the markers end to end (Fig.2b). The FISH studies identified segmental rearrangementsof the 11q23 region. The

TP53

was deleted from del(17)(p13) in the main cell line (Fig. 3b). A Philadelphia rear-rangement was not detected and the

ABL

gene did not ap-pear amplified in interphases (Fig. 4).

DISCUSSION

In this study, acute myeloid leukemia-M5a was found in apatient with multiple chromosome abnormalities. This pa-tient was diagnosed 10 years ago with MPD and was

treated intermittently over a long period with hydrox-yurea. The changes included duplication and triplicationof the 11q23 segment on chromosome 11 and markers de-rived from the same region. The present case was com-pared to other three reported cases with 11q23-MLL seg-mental rearrangements (Table 1) [1–3]. Three were maleand one was female. Their ages ranged from 2 to 74 years.Although one was a primary acute myeloid leukemia case,the majority were secondary acute myeloid leukemias fol-lowing treatment with 5-fluorouracil for breast cancer,vincristine, actinomycin D, ifosfamide, cyclophospha-mide, etopside, doxorubicin, and carboplatinum for em-bryonal rhabdomyosarcoma, and hydroxyurea for myelo-proliferative disorder. The treatment-related acute myeloidleukemia were AML-M1, AML-M5, and AML-5a. The seg-mental rearrangement of the chromosome 11-MLL regionappeared to be predominantly associated with AML-5.The duration between primary malignancy and secondaryleukemia ranged from 2 to 10 years. Survival after the sec-ondary leukemia ranged from 2 days to 2.3 months. Apartfrom monosomy 5 and 7, which are associated with treat-ment-related acute leukemias, duplication of the

MLL

re-gion on 11q23 is yet another therapy-related abnormalityto consider in the future.

The common karyotype change in three of the four cases(cases 2, 3, and 4) was segmental duplication or triplicationof the

MLL

region. In our experience, the segmental duplica-tions were difficult to detect under the microscope. There-fore, in cases with t-AML M5, the 11q region should be care-fully scrutinized for subtle changes, and a FISH study todetect

MLL

gene amplification should be considered.

Figure 4 In interphase, a Philadelphia rearrangement BCR/ABL fusion signal was not detected, and the ABL gene (arrow) didnot appear amplified.

Table 1

Summary of reported cases with segmental rearrangements of the 11q23 region

Reference No.Sex/Age

Primarymalignancy Treatment

Duration(years)

Secondarymalignancy

Survivaltime(months) Karyotype

Tanakaet al. [2]

1 F/63 Breast 5-FU 7.3 AML-M1 2.3 46,XX,dup(1)(q25q44),del(4)(q25q28), del(5)(q13q31),i(11)(q10),der(12)t(11;12)(q23;p12),del(13)(q12q14),

2

14,

2

17,add(19)(q13),der(19)t(?;?11;19)(?;q13;q13.4)[27]. nuc ish9q34(ABLx2-5), ish i(11)(CD3

2

,MLL

11

),der(12) (CD3

2

,MLL

1

)Felix

et al. [1]2 M/2 Embryonal

rhabdomyo-sarcoma

VinAct-D

2 AML-M5 Deceased 45,XY,der(5)t(5;11)(5pter

→

5q12::11q23

→

11qter),der(11)(pter

→

q12::q24

→

q12::q13

→

qter),

2

17Cuthbert

et al. [3]3 M/74 AML-M5a — — 2 days 46,XY,add(11)(q23). ish trp(11)

(q23) (wcp11

1

,MLL

111

)Present

Case4 M/66 Myeloprolif-

erativedisorder

HU 10 AML-M5a 1 week 46

z

47,X,

2

Y,add(1)(p35),

2

10, add(11)(q23),del(17)(p13),

1

mar x 2 [cp9].ish dup(11)(q23qter)(MLL

11

,wcp11

1

), del(17)(p13)(p53

2

), mar

3

2(MLL

1

,wcp11

1

)/46,XY,add(11)(q23), add(17)(p11),

2

20[4]. ish dup(11)(q23qter) (MLL

11

,wcp11

1

)/45

z

46,XY,

2

10,add(11)(q23),

1

mar[2]. ish dup (11)(q23qter)(MLL

11

,wcp11

1

), mar(MLL

1

,wcp11

1

)/89,XXYY,add(11)(q23)

3

2,

2

10,

2

12,

2

16[2]. ish trp(11)(q23qter) (MLL

111

,wcp11

1

)/46,XY[3].

Abbreviations

: FU

5

fluorouracil; Vin

5

vincristine; Act-D

5

actinomycin D; HU

5

hydroxyurea; AML

5

acute myeloid leukemia with the subclass M1, M5, M5a.

Segmental Rearrangements Involving 11q23-

MLL

51

REFERENCES

1. Felix CA, Megonigal MD, Chervinsky DS, Leonard DGB,Tsuchida N, Kakati S, Block AMW, Fisher J, Grossi M, Salh-any KI, Jani-Sait SN, Aplan PD (1998): Association of germlinep53 mutation with MLL segmental jumping translocation intreatment-related leukemia. Blood 91:4451–4456.

2. Tanaka K, Arif M, Eguchi M, Kyo T, Dohy H, Kamada N (1997):

Frequent jumping translocations of chromosomal segmentsinvolving ABL oncogene alone or in combination with CD3-MLL genes in secondary leukemias. Blood 89:596–600.

3. Cuthbert G, McCullough S, Finney R, Breese G, Brown N(1999): Jumping translocation at 11q23 with MLL gene rear-rangement and interstitial telomeric sequences. Genes Chro-mosom Cancer 24:295–298.