Cancer Genetics and Cytogenetics 203 (2010) 297e302

Short communication

The t(6;9)(p22;q34) in myeloid neoplasms: a retrospectivestudy of 16 cases

Monika Guptaa, J. Ashok Kumarb, Usha Sitaramc, S. Neerajb, A. Nancyb,Poonkuzhali Balasubramanianb, Aby Abrahamb, Vikram Mathewsb, Auro Viswabandyab,

Biju Georgeb, Mammen Chandyb, Alok Srivastavab, Vivi M. Srivastavaa,*aCytogenetics Unit, Christian Medical College, Ida Scudder Road, Vellore, 632004, India

bDepartment of Haematology, Christian Medical College, Ida Scudder Road, Vellore, 632004, IndiacDepartment of Transfusion Medicine and Immunohaematology, Christian Medical College, Ida Scudder Road, Vellore, 632004, India

Received 26 April 2010; received in revised form 28 July 2010; accepted 8 August 2010

Abstract Among patients with acute myeloid leukemia

* Corresponding a

E-mail address: c

0165-4608/$ - see fro

doi:10.1016/j.cancerg

(AML), the t(6;9) (p22;q34) is a rare but definedsubset with a poor prognosis. We report 16 patients with the t(6;9), of whom 13 had AML, 2had myelodysplastic syndrome (MDS), and 1 had chronic myeloid leukemia in myeloid blast crisis(CML-BC). All except for one were evaluated at diagnosis. The median age was 34.5 (range: 7e62years), with 12 adults and 12 males. Trilineage dysplasia was present in 13 (81%). Marrow baso-philia was seen in only two patients, one of whom had CML-BC. HLA-DR was positive in all 12patients assessed, CD33 in 11, CD13 in 10, and CD34 in seven. Four patients had one other abnor-mality apart from the t(6;9). These were the t(9;22) in the patient with CML and deletion 9q, addi-tion 13q, and an isochromosome 8q in the other three patients. There were no complex karyotypes.Fms-related tyrosine kinase 3dinternal tandem duplication (FLT3-ITD) mutations were seen inseven of 13 patients. Follow-up details were available for six patients. Three received palliativecare, and follow-up details were not available for the other seven. The response to chemotherapywas poor in the remaining patients. The only patients who survived were three out of the fourwho had allogeneic hematopoietic stem cell transplantation (HSCT). � 2010 Elsevier Inc. Allrights reserved.

1. Introduction

The t(6;9)(p22;q34) is a rare translocation found in about1% of all acutemyeloid leukemia (AML) [1]. It has also beenreported in myelodysplastic syndrome (MDS), chronicmyeloid leukemia (CML), and acute myelofibrosis [2e5].In AML, the t(6;9) is usually seen in younger individuals,more often in de novo than in secondary AML, but otherwisethere are no distinguishing clinical features. Morphologi-cally, the t(6;9) is associated with bone marrow dysplasiaand basophilia and a poor prognosis [6]. Immunophenotypicanalysis is usually positive for CD13, CD33, CD34, CD38,CD45, HLA-DR, and TdT [1,7e9]. The translocationinvolves the DEK gene on chromosome 6 and the NUP214(CAN ) gene on chromosome 9, and results in the formationof an oncogenicDEK/NUP214 fusion gene on the derivativechromosome 6 [10]. A high prevalence (71e88%) of the

uthor. Tel.: þ91-04162282803.

ytogen@cmcvellore.ac.in (V.M. Srivastava).

nt matter � 2010 Elsevier Inc. All rights reserved.

encyto.2010.08.012

FLT3-ITD mutation has been reported in these patients[1,9]. To our knowledge, there is limited literature on thiscondition, and only one previous report from Asia [11]. Thisstudy was undertaken to evaluate the profile of this subset ofpatients among those presenting with myeloid malignancies.

2. Materials and methods

All patients with myeloid neoplasia seen in the Depart-ment of Hematology, Christian Medical College, Vellore,India, between January 2004 andMarch 2009, were includedin the study. Cytogenetic analysis of bone marrow sampleswas performed using standard protocols [12]. For each case,chromosomes from at least two unstimulated overnight (withand without colcemid) cultures of bone marrow were har-vested. G-banded metaphases using trypsin and Leishmanstain (GTL) were analyzed using an automated karyotypingsystem (MetaSystems GmbH, Altlussheim, Germany).Karyotypes were reported in accordance with the Interna-tional System of Human Cytogenetic Nomenclature (ISCN)

298 M. Gupta et al. / Cancer Genetics and Cytogenetics 203 (2010) 297e302

2005 [13]. Cytogenetic findings were correlated with hema-tologic data. AML and MDS were subtyped according toFrencheAmericaneBritish classification. Immunopheno-type was assessed by flow cytometry using four-color anal-ysis on the FACSCalibur instrument (Becton Dickinson,San Jose, CA) using the following markers: CD2, CD3,CD7, CD10, CD13, CD19, CD33, CD34, HLA-DR, andSmIg. FLT3eITD and tyrosine kinase domain (TKD)[D835 and I836] mutation detection was performed bya previously described method using genomic DNA ex-tracted from the available fixed-cell suspension preparedfor conventional cytogenetic analysis [14]. Polymerase chainreaction (PCR) to detect the presence of ITD was carried outusing fluorescence-labeled forward primer and a reverseprimer to amplify exons 14 and 15, alongwith the interveningintron of the FLT3 gene. The size of the wild-type band was330 base pairs (bp), and the size of the ITD varied from 6 to400 bp. The PCR products were further analyzed by capillaryelectrophoresis on the ABI 3130 Genetic analyzer (AppliedBiosystems, Foster City, CA) to identify the tandem duplica-tion using the GeneScan software. The presence of ITD was

Fig. 1. 46,XX,t(6;9)(p22;q34),

further confirmed in representative samples by directsequencing. To detect point mutations in the TKD, exon 17of the FLT3 gene was amplified, using the primers asdescribed previously [15]. The amplified PCR productswere subjected to restriction fragment length polymorphism(RFLP) analysis using 6 base cutter EcoRV enzyme(Fermentas, Ontario, Canada) [16] and size-fractionatedthrough 3% agarose gel. Thewild-type sample has an EcoRVrestriction site, and digestion with the enzyme results in 68-and 46-bp bands, which are absent in the mutated samples.

3. Results

A total of 1,721 patients (1,090 AML, 435 MDS, and196 CML) with myeloid neoplasia were seen during theperiod selected for analysis. The t(6;9) was seen in 16patients (0.9%). Of these, 13 had AML (1.2% of allAML evaluated), 2 had MDS, and 1 had CML in myeloidblast crisis. Twelve of the 16 were males, and there were12 adults. The median age was 34.5 years (range: 7e62).The median white blood cell (WBC) count was

GTL-banded karyotype.

299M. Gupta et al. / Cancer Genetics and Cytogenetics 203 (2010) 297e302

6.9�10 9/L (range: 1.0e148.2), the median hemoglobinlevel was 7.75 g/dL (range: 4.4e11.4), and the medianplatelet count was 46� 109/L (range: 3.0e183).

Cytogenetic analysis was done at diagnosis in all but onepatient who had CML, for whom only a post-treatmentsample was available. Trilineage dysplasia was noted inthe bone marrow of 13/16 patients. Basophilia was foundin only two patients, and one of these had CML. Auer rodswere seen in six patients, and blasts were positive for Sudanblack B in 12/16 patients. Iron stain showed abundant ironstores with siderotic granules in six cases. None of thepatients had ringed sideroblasts in the bone marrow. Immu-nophenotypic analysis was done in 12/16 patients. All 12were positive for HLA-DR, 11 for CD33, 10 for CD13,and seven for CD34. The t(6;9)(p22;q34) was the sole cyto-genetic abnormality in 12 cases (Fig. 1). Additional chro-mosomal abnormalities were found in the remaining four.These included an abnormal chromosome 13 due to addi-tion of material of unknown origin to its long (q) arm,a deletion 9q, and an isochromosome 8q in one patienteach. The fourth was the patient with CML who had thet(9;22). FLT3 mutation analysis was performed in 13/16patients. FLT3-ITD mutations were seen in seven of 13patients. All 13 patients were negative for FLT3-TKDmutations (Fig. 2).

Fig. 2. (A) Agarose gel electrophoresis picture showing detection of FLT3-ITD m

a sample having the ITD mutation. NC, negative control; M, 100-bp DNA ladder.

of the FLT3-wt and FLT3-ITDepositive samples. Peak 330 bp represents wild-ty

gene. (C) Screening for FLT3-TKD mutation by RFLP using the EcoRV restric

ladder.

Follow-up information was available for six patients, fourof whom had AML (Table 1); the rest opted for palliativetreatment. Two of the four patients with AML achievedcomplete remission (CR) after primary induction chemo-therapy and the third after the second induction chemo-therapy. The fourth patient with AML did not achieve CRafter the second induction chemotherapy and was lost tofollow-up. Four of the six patients with follow-up underwentallogeneic HSCT. Three of these patients had AML and thefourth had MDS-refractory cytopenia with multilineagedysplasia (RCMD). One of the four who had HSCT died 2months after the transplant, with relapse of disease. Of theother three, two were alive in remission 18 and 22 monthsafter their first HSCT, while the third, who relapsed 106 daysafter the first HSCT, was alive in remission 45 months afterthe second HSCT. The patient with CML was on treatmentwith Imatinib mesylate for about 24 months with a goodresponse before presenting with blast transformation.

4. Discussion

Fewer than 200 cases of the t(6;9) have been reportedsince it was first identified in 1976 [17]. Most of theseare reports of small numbers of cases [8,18e20]. The

utation. Lanes 1 and 2, negative for ITD mutation in FLT3. Lane 3 shows

(B) Genescan analysis for FLT3-ITD mutation. Capillary electropherogram

pe FLT3, and 412 bp respresents a sample having 112-bp ITD in the FLT3

tion enzyme. Lanes 1 and 2 indicate wild type samples. M, 100-bp DNA

Table 1

Summary of hematologic data, cytogenetic findings, FLT3 mutation analysis, and follow-up details

Case

no. Age Sex

Marrow

diagnosis Karyotype

WBC

count

�109/L

PB

blasts

%

Hb

g/dl

Platelet

count

�109/L

BM

blast %

Marrow

count

�109/L

Tri-

lineage

dysplasia

Sudan

Black

B

FLT3-

ITD

FLT3-

TKD

Treatment

details

Status and

duration of

follow-up

1 30 F MDS-RA 46,XX,t(6;9)(p22;q34)[20] 4.5 0 7.4 14 4.5 36 þ � � � No follow up �2 44 M AML M2 46,XY,t(6;9)(p22;q34)[18]/

46,XY[2]

40.0 37 11.0 49 53 109 þ þ þ � CR achieved

after 2nd

induction chemo

Allogeneic

HSCT

1st relapse after

106 days

2nd transplant

GVHD I-II

Alive, 45

months

after second

HSCT

3 39 F AML M2 46,XX,t(6;9)(p22;q34)[20] 18.9 8 8.8 8 36 26 � þ þ � No follow up �4 41 M AML M2 46,XY,t(6;9)(p22;q34)[20] 35.0 70 7.0 52 70 56 þ þ � � CR achieved

after induction

chemo

Relapsed after

allogeneic

HSCT

8 months; died

2 months

after

HSCT

5 15 M AML M2 46,XY,t(6;9)(p22;q34)[20] 38.4 16 8.2 45 26 234 þ þ þ � No follow-up �6 40 M AML M6 46,XY,t(6;9)(p22;q34)[6]/

46,idem,add(13)(q34)[2]/

46,XY,add(13)(q34)[3]/

46,XY[8]

1.9 0 9.3 119 12 16 þ þ Not

done

Not

done

No follow-up �

7 26 M AML M2 46,XY,t(6;9)(p22;q34)[16]/

92,XXYY[2]

26.2 52 9.3 36 52 245 � þ þ � CR not

achieved

after 2nd

induction

2 months

lost to

follow-up

8 15 M AML M2 46,XY,t(6;9)(p22;q34)[20] 148.2 78 5.6 61 71.5 166 þ þ Not

done

Not

done

No follow-up �

9 7 F AML 46,XX,t(6;9)(p22;q34)[20] 11.3 21 4.4 76 21 26 þ � � � Palliative care �10 62 M AML M2 46,XY,t(6;9)(p22;q34), del

(9)(q21?q23?q33)[12]

1.7 0 7.7 41 65 !1 þ þ Not

done

Not

done

No follow-up �

11 52 M CML BT 46,XY,t(6;9)(p22;q34),

t(9;22)(q34;q11.2)[20]

6.4 0 7.8 19 43 28 þ � � � Dasatinib

therapy

45 days

lost to

follow-up

12 18 M AML M2 46,XY,t(6;9)(p22;q34)[19] 5.2 14 6.3 183 26 13 þ þ � � CR achieved

after induction

chemo

Allogeneic

HSCT

Alive 22 months

post HSCT

13 57 M AML M2 46,XY,t(6;9)(p22;q34)[17]/

92,idem �2[2]

1.0 0 5.1 56 88 35 þ þ þ � No follow-up �

300

M.Gupta

etal./Cancer

Genetics

andCyto

genetics

203(2010)297e

302

14

47

MMDS-

RCMD

46,XY,t(6;9)(p22;q34)[20]

2.7

011.4

22

111

þ�

��

Allogeneic

HSCT

chronic

GVHD

Alive

18months

postHSCT

15

12

FAML

46,XX,t(6;9)(p22;q34)[20]

3.2

50

5.1

394

25

þþ

þ�

Palliative

care

�

16

21

MAMLM1

46,XY,t(6;9)(p22;q34)[3]/

46,idem

,i(8)(q10)[15]/

46,XY{2}

7.5

76

9.1

47

85

56

�þ

þ�

Palliative

care

�

Abbreviations:GVHD,graftversushostdisease;þ

,present;�

,absent.

301M. Gupta et al. / Cancer Genetics and Cytogenetics 203 (2010) 297e302

largest series of t(6;9) comprising 69 patients (38 adults and31 children) noted an incidence of 0.9% in AML. The over-all median age was 23 years, with that of the adults being35 years [1]. Others have also noted an association witha young age [18,19]. The median age of 34.5 years in ourpatients is similar to that reported in the literature. In thisstudy, the translocation was seen in 0.9% of all myeloidneoplasia and 1.2% of AML. While the majority (81%)of our patients had AML, the translocation was also seenin MDS and CML. The t(6;9) is associated with FAB-M2or M4 subtypes in 61e90% of patients [1,2]. Varyingdegrees of dysplasia, usually trilineage, have been re-ported in 67e100% of patients [1,9,18]. Marrow baso-philia has been noted in up to 44% of patients with the t(6;9) in some reports [1,7,9], while others have not notedthis association [20]. Trilineage dysplasia was seen in 80%of our patients d in 12/15 samples obtained at diagnosisand in 10/13 AML. We found basophilia in only twopatients, one of whom had CML.

In most reports, the immunophenotype was positive inall cases for CD13, CD33, CD38, and HLA-DR, withCD34 positivity in 28e92% of patients [1,9,18]. We foundpositivity for HLA-DR in 100%, CD33 in 92%, CD 13 in83%, and CD34 in 58%. CD38 was not assessed. Thet(6;9)(p22;q34) usually occurs as a solitary abnormality atdiagnosis. Additional abnormalities, notably trisomies 8and 13, appear when there is clonal evolution [9,21].We had additional cytogenetic abnormalities in fourcases, three of which were structural abnormalities of chro-mosomes 8, 9, and 13. In the fourth patient, the t(6;9)occurred secondary to the t(9;22) in a patient who hadCML-BC. FLT3-ITD mutations have been reported in20e30% of younger adult patients with AML, and FLT3/TKD (D835) mutations in 7% [22]. These mutations havean adverse impact on prognosis and have been reportedin 71e88 % of patients with the t(6;9) [1,9]. FLT3 muta-tions were present in 54% of our patients, all of whomhad FLT3 eITD.

Follow-up details were available for 6/16 patients. Threeof the remaining 10 received palliative care; the other sevenwere lost to follow-up. The outcome of treatment was poorin the six patients who were on follow-up. Among the fourpatients with AML who received treatment, only oneachieved sustained complete remission with chemotherapy.The only patients who were alive were three of the fourwho underwent allogeneic HSCT at a follow-up rangingfrom 18 to 45 months. These results are similar to the over-all poor outcome of this group reported previously [1].

In conclusion, in this series of patients with the t(6;9),the clinical profile and haematologic features were similarto that reported in the literature. However, we did not finda consistent association with basophilia. The incidence ofFLT3-ITD mutations also appeared lower than what hasbeen reported. There was poor response to chemotherapyin these patients. It is now well recognized that the t(6;9)(p22;q34) defines a subgroup of AML with a poor outcome.

302 M. Gupta et al. / Cancer Genetics and Cytogenetics 203 (2010) 297e302

It has been included as a separate entity in the revised 2008World Health Organization classification of AML [23]. It istherefore important to be aware of this relatively rareabnormality because these patients respond poorly tochemotherapy alone and may require the inclusion of allo-geneic stem cell transplantation early in the treatment plan.

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