research article a novel cryptic three-way translocation t...

Research ArticleA Novel Cryptic Three-Way Translocationt(2918)(p232p213q2133) with Deletion of TumorSuppressor Genes in 9p213 and 13q14 in a T-Cell AcuteLymphoblastic Leukemia

Moneeb A K Othman1 Martina Rincic12 Joana B Melo34 Isabel M Carreira34

Eyad Alhourani1 Friederike Hunstig5 Anita Glaser1 and Thomas Liehr1

1 Jena University Hospital Friedrich Schiller University Institute of Human Genetics Kollegiengasse 10 07743 Jena Germany2 Croatian Institute of Brain Research Salata 12 10000 Zagreb Croatia3 Laboratory of Cytogenetics and Genomics Faculty of Medicine University of Coimbra Azinhaga Santa CombaPolo Ciencias da Saude 3000-548 Coimbra Portugal

4 Centro de Investigacao em Meio Ambiente Genetica e Oncobiologia (CIMAGO) Rua Larga 3004-504 Coimbra Portugal5 Jena University Hospital Friedrich Schiller University Department of Internal Medicine II (Oncology and Hematology)07749 Jena Germany

Correspondence should be addressed toThomas Liehr thomasliehrmeduni-jenade

Received 25 July 2014 Revised 18 September 2014 Accepted 20 September 2014 Published 8 October 2014

Academic Editor Daniela Cilloni

Copyright copy 2014 Moneeb A K Othman et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Acute leukemia often presents with pure chromosomal resolution thus aberrations may not be detected by banding cytogeneticsHere a case of 26-year-old male diagnosed with T-cell acute lymphoblastic leukemia (T-ALL) and a normal karyotype afterstandard GTG-banding was studied retrospectively in detail by molecular cytogenetic and molecular approaches Besidesfluorescence in situ hybridization (FISH) multiplex ligation-dependent probe amplification (MLPA) and high resolution array-comparative genomic hybridization (aCGH) were applied Thus cryptic chromosomal aberrations not observed before weredetected three chromosomes were involved in a cytogenetically balanced occurring translocation t(2918)(p232p213q2133)Besides a translocation t(1014)(q24q11) was identified an aberration known to be common in T-ALL Due to the three-waytranslocation deletion of tumor suppressor genes CDKN2AINK4Ap16 CDKN2BINK4Bp15 andMTAPARFp14 in 9p213 tookplace Additionally RB1 in 13q14 was deleted This patient considered to have a normal karyotype after low resolution bandingcytogenetics was treated according to general protocol of anticancer therapy (ALL-BFM 95)

1 Introduction

T-cell acute lymphoblastic leukemia (T-ALL) is a quite rareand heterogeneous disease more common in males than infemales It accounts for 15 of childhood and 25 of adultALL cases [1] Underlying genetic causes of T-ALL are poorlyunderstood and this is highlighted by the fact that T-ALL isassociated with a normal karyotype in 30ndash50 of the cases [23] In abnormal karyotypes recurrent chromosomal aberra-tions are reported [4] Regularly promoter and enhancer ele-ments of genes involved in T-cell development are juxtaposed

with translocations in close proximity of oncogenes [5 6]Themost common structural chromosomal abnormalities in T-ALL are TCR (T-cell receptor) loci rearrangements Break-points in 14q11 (TCRAD) and 7q34 (TCR120573) are observedfrequently Besides deletions in the long arm of chromosome6 may be found the common deleted region involves mainlysubband 6q16 however candidate gene(s) have not beenformally identified yet [7 8] Also tumor suppressor geneshave been seen to be involved in T-ALL [9]

Cryptic structural chromosomal abnormalities are stilla challenge in cytogenetic standard diagnostics of acute

Hindawi Publishing CorporationLeukemia Research and TreatmentVolume 2014 Article ID 357123 7 pageshttpdxdoiorg1011552014357123

2 Leukemia Research and Treatment

leukemia However many cryptic aberrations have beenidentified by molecular cytogenetics already Examples inT-ALL are cryptic deletions in 9p21 involving the genesCDKN2AINK4Ap16 CDKN2BINK4Bp15 and MTAPARFp14 leading to loss of G1 checkpoint control of the cellcycle or the RB1 locus in 13q14 which also plays a role astumor suppressor gene in cell cycle regulation [9]

Here a case of a young adult T-ALL patient with a novelcryptic three-way translocation a reciprocal translocationand submicroscopic deletions is reported

2 Material and Methods

21 Clinical Description A 26-year-old male presented in1998 initially with a total white blood cell count of 202 times109L hemoglobin of 92mmolL and platelets of 126 times109L Bone marrow examination was consistent with T-ALLhaving 91 blast cells According to flow cytometry theimmunophenotype of bone marrow lymphocytes was asfollows the cells were positive for CD2 (96) CD8 (96)CD4 (92) CD7 (92) CD1A (89) CD10 (87) CyCD3(86) and TdT (85) and negative for 120572F1 120573F1 CD3 CD13CD19 CD20 CD24 CD33 CD34 HLA-DR MPO-7 slgTZR-120572120573 and TZR120574120575 The patient was treated accordingto ALL-BFM 95 protocol and died eight months after initialdiagnosis from serious infections and severe complicationswhile being in complete hematological remission

22 Test Done at Diagnosis GTG-banding was done accord-ing to standard procedures A total of 7 metaphases wereavailable for cytogenetic evolution derived from unstimu-lated bone marrow of the patient and were analyzed ona banding level of 180ndash250 bands per haploid karyotype[11] and determined as 46XY [7 12] RT-PCR performedfor TELAML1 and BCRABL fusion genes was reported tobe negative and fluorescence in situ hybridization (FISH)analysis carried out according to manufacturerrsquos instructionsfor the same loci was negative (probes used LSI BCRABLand LSI TELAML1 Abbott MolecularVysis MannheimGermany)

23 Test Done in Retrospective

231 Molecular Cytogenetics FISH was done according tostandard procedures and manufacturerrsquos instructions for thefollowing commercially available probes LSI 13 in 13q142(RB1 Abbott MolecularVysis Mannheim Germany) LSIIGHBCL2 (IGH in 14q32 BCL2 in 18q21 Abbott Molec-ularVysis Mannheim Germany) SPEC ALK2q11 (ALKin 2p23 Zytovision GmbH Bremerhaven Germany) SPECp16CEN9 (p16 in 9p213 Zytovision GmbH Bremer-haven Germany) SPEC BIRC3MALT1 (BIRC3 in 11q222MALT1 in 18q2132 Zytovision Bremerhaven Germany) andPOSEIDONMLLMLLT3 (MLL in 11q233MLLT3 in 9p213Kreatech Diagnostics Amsterdam Netherland)

Whole chromosome painting (WCP) probe for chromo-somes 2 9 10 14 and 18 and bacterial artificial chromo-some probes (BACs) for chromosomes 2 and 9 (Table 1)

were homemade [13] The homemade multitude multicolor-banding (mMCB) and chromosome specific high resolutionarray-proven multicolor-banding (aMCB) probe sets werealso applied as previously reported [10 14 15]

A total of 10ndash15 metaphase spreads were analyzedusing a fluorescence microscope (AxioImagerZ1 mot Zeiss)equipped with appropriate filter sets to discriminate betweenamaximumof five fluorochromes and the counterstain DAPI(Diaminophenylindol) Image capturing and processing werecarried out using an ISIS imaging system (MetaSystemsAltluszligheim Germany)

232 DNA Isolation GenomicDNAwas extracted from cellsfixed in acetic acid methanol (1 3) by Puregene DNA Purifi-cation Kit (Gentra Systems Minneapolis MN USA) DNAconcentration was determined by a Nanodrop spectropho-tometer The quality of DNA was checked using agarose gelelectrophoresis DNA samples extracted from fixed cells of 2healthymales and 2 healthy females by the samemethodwereused as reference samples

233 Multiplex Ligation-Dependent Probe Amplification(MLPA) The P377-A1 hematologic malignancies probemixand SALSA reagents were used for this study (MRC-Hol-land Amsterdam The Netherlands) Amplified probes andGenescan 500ROX standardwere separated by capillary elec-trophoresis using a 4-capillary ABI-PRISM 3130XL GeneticAnalyzer (Applied Biosystems Foster City USA) Sizing ofpeaks and quantification of peak areas and heights wereperformed using GeneMarker v19 software (Applied Biosys-tems) A minimum of 4 healthy control samples were includ-ed in each run

234 High Resolution Array-Comparative Genomic Hybridi-zation (aCGH) aCGH was performed using Agilent Sure-Print G3 Human Genome microarray 180K (Agilent Tech-nologies Santa Clara CA USA) an oligonucleotidemicroar-ray containing approximately 180000 probes 60-mer with a17 kb average probe spacing Genomic DNA of patient wascohybridized with a male control DNA (Agilent Technolo-gies Santa Clara CA USA) Labeling was performed usingAgilent Genomic DNA enzymatic labeling kit (Agilent)according to the manufacturersrsquo instructions After hybridi-zation the aCGH slide was scanned on an Agilent scannerand processed with Feature Extraction software (v107) andresults were analyzed using Cytogenomics (v2913) usingADM2 as aberration algorithm

3 Results of Retrospective Analysis

As an initial test of retrospective analysis a genome wideFISH-banding applying mMCB was performed Thereby apreviously unrecognized reciprocal and apparently balancedtranslocation between the three chromosomes 2 9 and18 was identified Besides a known recurrent translocationof chromosomes 10 and 14 was recognized and the kary-otype was suggested as 46XYt(2918)(p232p213q2133)t(1014)(q24q11) (Figure 1) aMCB and WCP probes as

Leukemia Research and Treatment 3

Table 1 (a) Probes used for characterization of the three-way translocation their location and obtained results (b) Probes used forcharacterization of the in aCGH detected deletions their location and obtained results

(a)

Cytoband Location [hg19] Probe Result for derivative chromosomes

2p243 chr2 RP11-119F22 Signal on der(9) no split signal16014784ndash16140647

2p233 chr2 RP11-106G13 Signal on der(9) no split signal26967697ndash27136688

2p232 chr2 SPEC ALK Signal on der(9) no split signal29415640ndash29447593

9p221 chr9 RP11-503K16 Signal on der(18) no split signal18717972ndash18718524

9p221 chr9 RP11-513M16 Signal on der(18) no split signal19371384ndash19371943

9p213 chr9 RP11-15P13 Signal on der(18) no split signal20182493ndash20361132

9p213 chr9 MLLT3 MLLT3-gene signal on der(18) no split signal20344968ndash20621872

9p213 chr9 SPEC p16 Deletion on der(9) andor der(18)21967751ndash21975132

9p213 chr9 RP11-946B6 Deletion on der(9) andor der(18) ish 9p213(RP11-946B6x0)[8]23608612ndash23790449

9p212 chr9 RP11-438B23 Signal on der(9) no split signal27937615ndash27944495

18q2132 chr18 MALT1 MALT1-gene signal on der(18) no split signal56338618ndash56417370

18q21 chr18 BCL2 BCL2-gene signal on der(2) no split signal60985282ndash60985899

(b)


9p213 chr921967751ndash21975132 SPEC p16

ish 9p213(p16x1)[4]nuc ish 9p21(p16x0)[64]9p21(p16x1)[83]

9p21(p16x2)[53]

13q142 chr1348920000ndash49140000 LSI 13 = 1198771198611

nuc ish 13q142(1198771198611x0)[36]13q142(1198771198611x1)[43]13q142(1198771198611x2)[121]

shown in Figure 2 confirmed these suggestions Locusspecific probes narrowed down the breakpoints as shownin Table 1(a) Unfortunately there was no sufficient cellpellet available to characterize the breakpoints in moredetail than listed in Table 1(a) Even though closely locatedto the observed chromosomal breakpoints direct involve-ment of the following oncogenes was excluded usinglocus specific FISH-probes for ALK in 2p232 MLLT3 in9p213 and MALT1 and BCL2 in 18q2133 However MLPA(result not shown) and aCGH (Figure 3) revealed that thet(2918) is not really balanced a deletion in 9p213 includ-ing CDKN2AINK4Ap16 CDKN2BINK4Bp15 andMTAPARFp14 could be found as chr9 21252517ndash21798676x1and 21817082ndash23515821x0 (hg19) (Figure 3 Table 1(b))Moreover a deletion in 13q142 was detected as chr13

48982000ndash49062000x1 (hg19 Figure 3) FISH showed amosaic condition of mixed heterozygous and homozygousdeletion of 9p213 and 13q142 (Table 1(b))

4 Discussion

Chromosomal translocations are considered to be the pri-mary cause of leukemia for both acute and chronic phase Inthis study we retrospectively identified previously undetectedbalanced and unbalanced chromosomal and subchromoso-mal changes by application of molecular cytogenetics includ-ing FISH-banding locus-specific FISH-probes and aCGHplus MLPA FISH-banding especially mMCB allows theidentification of balanced and unbalanced inter- and intra-chromosomal rearrangements of the whole human karyotype


1 2 3 4 5

6 7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 X Y

Figure 1 Application of mMCB showed no normal karyotypebut derivative chromosomes 2 9 10 14 and 18 (arrows) mMCBresults are shown as overlay of three of the six used color channelsEvaluation was done as previously reported [10] using all 6 colorchannels and pseudocoloring Breakpoints were determined as2p232 9p213 10q24 14q11 and 18q2133

in one single experiment [10] It might be indicated to applymMCBor comparable FISH-banding approaches routinely inT-ALL cases exhibiting poor quality of the metaphase thatis not well spreading ones with chromosomes appearing asfuzzy with indistinct margins [16 17]

In this study one well-known and one yet unreportedbalanced translocation event were identified for a T-ALLas t(1014)(q24q11) and t(2918)(p232p213q2133) respec-tively While a direct involvement of the cancer-relatedoncogenes ALK in 2p232 MLLT3 in 9p213 and BCL2 in18q2133 could be excluded loss of two tumor suppresser geneloci in 9p21 and in 13q14 was found

Data from the literature confirmed that the oncogenestested and located nearby the chromosomal breakpoints ofthe three-way translocation were not yet found to be involvedin T-ALL ALK located in 2p232 was previously detected ina variety of B- and T-cell lymphomas and nonhematopoieticsolid tumors [18ndash23] the BCL2 gene is overexpressed inlymphomas [24 25] and theMLLT3 genewas one of themosthighly upregulated transcripts and the most common fusionpartner of MLL in de novo acute myeloid leukemia (AML)subtype M5 and therapy-related AML [26ndash28] howeverMeyer et al [29] found that MLLT3 also plays a role inpediatric rather than adult ALL

In the present case an additional chromosomal translo-cation t(1014)(q24q11) known as sole abnormality in 10of T-ALL patients was identified Also it is present in 5 ofpediatric and 30 of adult T-ALL [20 30 31] The TLX1 geneat 10q24 is a transcription factor becoming overexpressed asoncogene due to its juxtaposition to a strong promoter andenhancer elements of the TCR loci at 14q11 [5 32ndash34] Afavorable outcome was reported in pediatric and adult T-ALL

MCB 2

MCB 9

MCB 18

Normal der(2) der(9) der(18)

(a)

wcp10wcp14

der(14)der(10)10 14

(b)

Figure 2 (a) Results of aMCB probe sets for chromosomes 2 9 and18 are shown in pseudocolor depiction which confirmed the char-acterization of these three chromosomes involving rearrangementas t(2918)(p232p213q2133) (b)Whole chromosomepaints (wcp)for chromosomes 10 and 14 confirmed that the t(1014)(q24q11) wasindependent of the t(2918)

to be associatedwith the t(1014) or TLX1 gene overexpression[5 20 35]

Even though balanced rearrangements are known to betypical for hematopoietic malignancies to date only a limitednumber of studies have used whole genome directed FISHapproaches to identify cryptic chromosomal abnormalitiesin ALL patients [36ndash38] Still in ALL it is uncommon tosee three-way translocations However due to lowmetaphaseresolution in ALL the real incidence of three-way transloca-tions is currently unknown

The present report highlights that after identificationof apparently balanced chromosomal aberrations it is stillnecessary to screen for further unbalanced submicroscopicabnormalities by molecular approaches such as MLPA andaCGH However also a confirmation of the results by molec-ular cytogenetics is necessary as aCGH was partially mis-classified a mix of homo- and heterozygote deletions as purehomozygote ones

9p213 deletions which lead to the loss of CDKN2AINK4Ap16 CDKN2BINK4Bp15 and MTAPARFp14tumor suppressor genes expression are the most predomi-nant aberrations seen in precursor B-cell ALL (sim20 of thecases) and T-ALL (gt60 of the case) [39ndash42] Besides alsoa deletion of RB1 gene resulting in inactivation of anothertumor suppressor gene expression was identified RB1 israrely reported to be deleted in T-ALL In contrast deletionof RB1 has been detected in 30 of B-ALL and nearly to 60in B-CLL cases [43 44] Thus RB1 pathway was identified aspotential targets for therapy of ALL [45 46]


p12p112

q1212q122q131q1411q1412q143q211q212

q2132q221q311

q312q321q323q332q34

p241p223p213p212p133p131p112

q13

q32

q2112q212

q2133q2231q2233q312

q332q3411q342

Chromosome 9 Chromosome 13

Figure 3 aCGH confirmed deletions in 9p213 and 13q142 (arrows) detected initially byMLPA (result not shown) FISH confirmed presenceof these deletions in metaphase andor interphase Examples for heterozygote deletions of 9p213 and 13q142 are depicted probes specific forthe corresponding tumor suppressor genes were labeled in red centromeric probe for chromosome 9 (D9Z3) was labeled in green

5 Conclusion

In conclusion we report a case of T-ALL with complex chro-mosomal aberrations Even if at time of diagnosis the deletionon 9p213 would have been detected and accordingly treatedit remains unclear what influence the other tumor suppres-sors and oncogenes (possibly) activated by the complex rear-rangements would have had for the clinical outcome Overallthe present case stresses the necessity to study hematologicalmalignancies by different means to get a comprehensive pic-ture of the genetic changes in connection with the acquireddisease as aCGH or MLPA alone would only have identifiedthe imbalanced rearrangements while molecular cytogenet-ics predominantly gave hints on the presence of balancedrearrangements

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research paper is supported in part by the DAAD andKAAD

References

[1] C-H Pui M V Relling and J R Downing ldquoMechanismsof disease acute lymphoblastic leukemiardquo The New EnglandJournal of Medicine vol 350 no 15 pp 1535ndash1548 2004

[2] C J Harrison ldquoKey pathways as therapeutic targetsrdquo Blood vol118 no 11 pp 2935ndash2936 2011

[3] A Vitale A Guarini C Ariola et al ldquoAdult T-cell acute lym-phoblastic leukemia Biologic profile at presentation and corre-lation with response to induction treatment in patients enrolledin the GIMEMA LAL 0496 protocolrdquo Blood vol 107 no 72 pp473ndash479 2006

[4] C Graux J Cools L Michaux P Vandenberghe and AHagemeijer ldquoCytogenetics and molecular genetics of T-cellacute lymphoblastic leukemia from thymocyte to lymphoblastrdquoLeukemia vol 20 no 9 pp 1496ndash1510 2006


[5] A A Ferrando D S Neuberg R K Dodge et al ldquoPrognosticimportance of TLX1 (HOX11) oncogene expression in adultswith T-cell acute lymphoblastic leukaemiardquoTheLancet vol 363no 9408 pp 535ndash536 2004

[6] S H Oram J Thoms J I Sive et al ldquoBivalent promotermarks and a latent enhancermayprime the leukaemia oncogeneLMO1 for ectopic expression in T-cell leukaemiardquo Leukemiavol 27 no 6 pp 1348ndash1357 2013

[7] P B Sinclair A Sorour M Martineau et al ldquoA fluorescencein situ hybridization map of 6q deletions in acute lymphocyticleukemia Identification and analysis of a candidate tumorsuppressor generdquo Cancer Research vol 64 no 12 pp 4089ndash4098 2004

[8] B Cauwelier N Dastugue J Cools et al ldquoMolecular cytoge-netic study of 126 unselected T-ALL cases reveals high inci-dence of TCR120573 locus rearrangements and putative new T-celloncogenesrdquo Leukemia vol 20 no 7 pp 1238ndash1244 2006

[9] P van Vlierberghe and A Ferrando ldquoThe molecular basis ofT cell acute lymphoblastic leukemiardquo Journal of Clinical Inves-tigation vol 122 no 10 pp 3398ndash3406 2012

[10] A Weise A Heller H Starke et al ldquoMultitude multicolorchromosome banding (mMCB)mdasha comprehensive one-stepmulticolor FISH banding methodrdquo Cytogenetic and GenomeResearch vol 103 no 1-2 pp 34ndash39 2003

[11] U Claussen S Michel P Muhlig et al ldquoDemystifying chro-mosome preparation and the implications for the concept ofchromosome condensation during mitosisrdquo Cytogenetic andGenome Research vol 98 no 2-3 pp 136ndash146 2002

[12] FMitelman Ed ISCN 1995 An International System forHumanCytogenetic Nomenclature (1995) S Karger Basel Switzerland1995

[13] AWeise H Starke AHeller et al ldquoChromosome 2 aberrationsin clinical cases characterised by high resolution multicolourbanding and region specific FISH probesrdquo Journal of MedicalGenetics vol 39 no 6 pp 434ndash439 2002

[14] A Weise K Mrasek I Fickelscher et al ldquoMolecular definitionof high-resolution multicolor banding probes first within thehuman DNA sequence anchored FISH banding probe setrdquoJournal of Histochemistry and Cytochemistry vol 56 no 5 pp487ndash493 2008

[15] T Liehr A Heller H Starke et al ldquoMicrodissection based highresolution multicolor banding for all 24 human chromosomesrdquoInternational Journal of Molecular Medicine vol 9 no 4 pp335ndash339 2002

[16] H Mkrtchyan M Glaser M Gross et al ldquoMulticolor-FISHapplied to resolve complex chromosomal changes in a case ofT-ALL (FAB L2)rdquo Cytogenetic and Genome Research vol 114no 3-4 pp 270ndash273 2006

[17] W Al Achkar A Wafa H Mkrtchyan F Moassass and TLiehr ldquoA unique complex translocation involving six differentchromosomes in a case of childhood acute lymphoblasticleukemiawith the Philadelphia chromosome and adverse prog-nosisrdquo Oncology Letters vol 1 no 5 pp 801ndash804 2010

[18] A Barreca E Lasorsa L Riera et al ldquoAnaplastic lymphomakinase in human cancerrdquo Journal of Molecular Endocrinologyvol 47 no 1 pp R11ndashR23 2011

[19] R Chiarle C Voena C Ambrogio R Piva and G InghiramildquoThe anaplastic lymphoma kinase in the pathogenesis of can-cerrdquo Nature Reviews Cancer vol 8 no 1 pp 11ndash23 2008

[20] S Chiaretti and R Foa ldquoT-cell acute lymphoblastic leukemiardquoHaematologica vol 94 no 2 pp 160ndash162 2009

[21] R R Einerson P J Kurtin G A Dayharsh T K Kimlingerand E D Remstein ldquoFISH is superior to PCR in detect-ing t(1418)(q32q21)-IgHbcl-2 in follicular lymphoma usingparaffin-embedded tissue samplesrdquo The American Journal ofClinical Pathology vol 124 no 3 pp 421ndash429 2005

[22] M Soda Y L Choi M Enomoto et al ldquoIdentification of thetransforming EML4-ALK fusion gene in non-small-cell lungcancerrdquo Nature vol 448 no 7153 pp 561ndash566 2007

[23] W Wan M S Albom L Lu et al ldquoAnaplastic lymphomakinase activity is essential for the proliferation and survival ofanaplastic large-cell lymphoma cellsrdquo Blood vol 107 no 4 pp1617ndash1623 2006

[24] J Iqbal V T Neppalli G Wright et al ldquoBCL2 expression is aprognostic marker for the activated B-cell-like type of diffuselarge B-cell lymphomardquo Journal of Clinical Oncology vol 24 no6 pp 961ndash968 2006

[25] L Impera F Albano C Lo Cunsolo et al ldquoA novel fusion51015840AFF331015840BCL2 originated from a t(218)(q112q2133) translo-cation in follicular lymphomardquo Oncogene vol 27 no 47 pp6187ndash6190 2008

[26] L Tsao H Y Draoua I Osunkwo et al ldquoMature B-cellacute lymphoblastic leukemia with t(9111) translocation adistinct subset of B-cell acute lymphloblastic leukemiardquoModernPathology vol 17 no 7 pp 832ndash839 2004

[27] N Blin F Mechinaud P Talmant et al ldquoMature B-cell lym-phoblastic leukemia with MLL rearrangement an uncommonand distinct subset of childhood acute leukemiardquo Leukemia vol22 no 5 pp 1056ndash1059 2008

[28] M de Braekeleer FMorelM-J le Bris AHerry andNDouet-Guilbert ldquoTheMLL gene and translocations involving chromo-somal band 11q23 in acute leukemiardquo Anticancer Research vol25 no 3 pp 1931ndash1944 2005

[29] C Meyer B Schneider S Jakob et al ldquoTheMLL recombinomeof acute leukemiasrdquo Leukemia vol 20 no 5 pp 777ndash784 2006

[30] I Riz T S Hawley T V Luu N H Lee and R G HawleyldquoTLX1 and NOTCH coregulate transcription in T cell acutelymphoblastic leukemia cellsrdquo Molecular Cancer vol 9 article181 2010

[31] M D Kraszewska M Dawidowska T Szczepanski and MWitt ldquoT-cell acute lymphoblastic leukaemia recent molecularbiology findingsrdquo British Journal of Haematology vol 156 no 3pp 303ndash315 2012

[32] I D Dube S C Raimondi D Pi and D K Kalousek ldquoA newtranslocation t(1014)(q24q11) in T cell neoplasiardquo Blood vol67 no 4 pp 1181ndash1184 1986

[33] K de Keersmaecker and A A Ferrando ldquoTLX1-induced T-cellacute lymphoblastic leukemiardquo Clinical Cancer Research vol 17no 20 pp 6381ndash6386 2011

[34] S Padhi R Sarangi P Mohanty et al ldquoCytogenetic profile ofpediatric acute lymphoblastic leukemia (ALL) analysis of 31cases with review of literaturerdquo Caryologia vol 64 no 1 pp33ndash41 2011

[35] S C Raimondi ldquoT-lineage acute lymphoblastic leukemia (T-ALL)rdquo Atlas of Genetics and Cytogenetics in Oncology andHaematology no 4 2007

[36] J D Rowley S Reshmi K Carlson and D Roulston ldquoSpectralkaryotype analysis of T-cell acute leukemiardquo Blood vol 93 no6 pp 2038ndash2042 1999

[37] B Poppe B Cauwelier H van Limbergen et al ldquoNovel crypticchromosomal rearrangements in childhood acute lymphoblas-tic leukemia detected by multiple color fluorescent in situhybridizationrdquo Haematologica vol 90 no 9 pp 1179ndash11852005


[38] C Karst M Gross D Haase et al ldquoNovel cryptic chromoso-mal rearrangements detected in acute lymphoblastic leukemiadetected by application of new multicolor fluorescent in situhybridization approachesrdquo International Journal of Oncologyvol 28 no 4 pp 891ndash897 2006

[39] D Mirebeau C Acquaviva S Suciu et al ldquoThe prognosticsignificance of CDKN2A CDKN2B and MTAP inactivation inB-lineage acute lymphoblastic leukemia of childhood Resultsof the EORTC studies 58881 and 58951rdquo Haematologica vol 91no 7 pp 881ndash885 2006

[40] C G Mullighan S Goorha I Radtke et al ldquoGenome-wideanalysis of genetic alterations in acute lymphoblastic leu-kaemiardquo Nature vol 446 no 7137 pp 758ndash764 2007

[41] M Kim S-H Yim N-S Cho et al ldquoHomozygous deletionof CDKN2A (p16 p14) and CDKN2B (p15) genes is a poorprognostic factor in adult but not in childhood B-lineage acutelymphoblastic leukemia a comparative deletion and hyper-methylation studyrdquo Cancer Genetics and Cytogenetics vol 195no 1 pp 59ndash65 2009

[42] T Szczepanski C J Harrison and J J van Dongen ldquoGeneticaberrations in paediatric acute leukaemias and implications formanagement of patientsrdquoThe Lancet Oncology vol 11 no 9 pp880ndash889 2010

[43] H Cave H Avet-Loiseau I Devaux et al ldquoDeletion ofchromosomal region 13q143 in childhood acute lymphoblasticleukemiardquo Leukemia vol 15 no 3 pp 371ndash376 2001

[44] C J Schwab L Chilton H Morrison et al ldquoGenes commonlydeleted in childhood B-cell precursor acute lymphoblasticleukemia association with cytogenetics and clinical featuresrdquoHaematologica vol 98 no 7 pp 1081ndash1088 2013

[45] C J Harrison and L Foroni ldquoCytogenetics and moleculargenetics of acute lymphoblastic leukemiardquo Reviews in Clinicaland Experimental Hematology vol 6 no 2 pp 91ndash113 2002

[46] K de Keersmaecker PMarynen and J Cools ldquoGenetic insightsin the pathogenesis of T-cell acute lymphoblastic leukemiardquoHaematologica vol 90 no 8 pp 1116ndash1127 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


leukemia However many cryptic aberrations have beenidentified by molecular cytogenetics already Examples inT-ALL are cryptic deletions in 9p21 involving the genesCDKN2AINK4Ap16 CDKN2BINK4Bp15 and MTAPARFp14 leading to loss of G1 checkpoint control of the cellcycle or the RB1 locus in 13q14 which also plays a role astumor suppressor gene in cell cycle regulation [9]

Here a case of a young adult T-ALL patient with a novelcryptic three-way translocation a reciprocal translocationand submicroscopic deletions is reported

2 Material and Methods

21 Clinical Description A 26-year-old male presented in1998 initially with a total white blood cell count of 202 times109L hemoglobin of 92mmolL and platelets of 126 times109L Bone marrow examination was consistent with T-ALLhaving 91 blast cells According to flow cytometry theimmunophenotype of bone marrow lymphocytes was asfollows the cells were positive for CD2 (96) CD8 (96)CD4 (92) CD7 (92) CD1A (89) CD10 (87) CyCD3(86) and TdT (85) and negative for 120572F1 120573F1 CD3 CD13CD19 CD20 CD24 CD33 CD34 HLA-DR MPO-7 slgTZR-120572120573 and TZR120574120575 The patient was treated accordingto ALL-BFM 95 protocol and died eight months after initialdiagnosis from serious infections and severe complicationswhile being in complete hematological remission

22 Test Done at Diagnosis GTG-banding was done accord-ing to standard procedures A total of 7 metaphases wereavailable for cytogenetic evolution derived from unstimu-lated bone marrow of the patient and were analyzed ona banding level of 180ndash250 bands per haploid karyotype[11] and determined as 46XY [7 12] RT-PCR performedfor TELAML1 and BCRABL fusion genes was reported tobe negative and fluorescence in situ hybridization (FISH)analysis carried out according to manufacturerrsquos instructionsfor the same loci was negative (probes used LSI BCRABLand LSI TELAML1 Abbott MolecularVysis MannheimGermany)

23 Test Done in Retrospective

231 Molecular Cytogenetics FISH was done according tostandard procedures and manufacturerrsquos instructions for thefollowing commercially available probes LSI 13 in 13q142(RB1 Abbott MolecularVysis Mannheim Germany) LSIIGHBCL2 (IGH in 14q32 BCL2 in 18q21 Abbott Molec-ularVysis Mannheim Germany) SPEC ALK2q11 (ALKin 2p23 Zytovision GmbH Bremerhaven Germany) SPECp16CEN9 (p16 in 9p213 Zytovision GmbH Bremer-haven Germany) SPEC BIRC3MALT1 (BIRC3 in 11q222MALT1 in 18q2132 Zytovision Bremerhaven Germany) andPOSEIDONMLLMLLT3 (MLL in 11q233MLLT3 in 9p213Kreatech Diagnostics Amsterdam Netherland)

Whole chromosome painting (WCP) probe for chromo-somes 2 9 10 14 and 18 and bacterial artificial chromo-some probes (BACs) for chromosomes 2 and 9 (Table 1)

were homemade [13] The homemade multitude multicolor-banding (mMCB) and chromosome specific high resolutionarray-proven multicolor-banding (aMCB) probe sets werealso applied as previously reported [10 14 15]

A total of 10ndash15 metaphase spreads were analyzedusing a fluorescence microscope (AxioImagerZ1 mot Zeiss)equipped with appropriate filter sets to discriminate betweenamaximumof five fluorochromes and the counterstain DAPI(Diaminophenylindol) Image capturing and processing werecarried out using an ISIS imaging system (MetaSystemsAltluszligheim Germany)

232 DNA Isolation GenomicDNAwas extracted from cellsfixed in acetic acid methanol (1 3) by Puregene DNA Purifi-cation Kit (Gentra Systems Minneapolis MN USA) DNAconcentration was determined by a Nanodrop spectropho-tometer The quality of DNA was checked using agarose gelelectrophoresis DNA samples extracted from fixed cells of 2healthymales and 2 healthy females by the samemethodwereused as reference samples

233 Multiplex Ligation-Dependent Probe Amplification(MLPA) The P377-A1 hematologic malignancies probemixand SALSA reagents were used for this study (MRC-Hol-land Amsterdam The Netherlands) Amplified probes andGenescan 500ROX standardwere separated by capillary elec-trophoresis using a 4-capillary ABI-PRISM 3130XL GeneticAnalyzer (Applied Biosystems Foster City USA) Sizing ofpeaks and quantification of peak areas and heights wereperformed using GeneMarker v19 software (Applied Biosys-tems) A minimum of 4 healthy control samples were includ-ed in each run

234 High Resolution Array-Comparative Genomic Hybridi-zation (aCGH) aCGH was performed using Agilent Sure-Print G3 Human Genome microarray 180K (Agilent Tech-nologies Santa Clara CA USA) an oligonucleotidemicroar-ray containing approximately 180000 probes 60-mer with a17 kb average probe spacing Genomic DNA of patient wascohybridized with a male control DNA (Agilent Technolo-gies Santa Clara CA USA) Labeling was performed usingAgilent Genomic DNA enzymatic labeling kit (Agilent)according to the manufacturersrsquo instructions After hybridi-zation the aCGH slide was scanned on an Agilent scannerand processed with Feature Extraction software (v107) andresults were analyzed using Cytogenomics (v2913) usingADM2 as aberration algorithm

3 Results of Retrospective Analysis

As an initial test of retrospective analysis a genome wideFISH-banding applying mMCB was performed Thereby apreviously unrecognized reciprocal and apparently balancedtranslocation between the three chromosomes 2 9 and18 was identified Besides a known recurrent translocationof chromosomes 10 and 14 was recognized and the kary-otype was suggested as 46XYt(2918)(p232p213q2133)t(1014)(q24q11) (Figure 1) aMCB and WCP probes as



(a)














(b)


9p213 chr921967751ndash21975132 SPEC p16


9p21(p16x2)[53]

13q142 chr1348920000ndash49140000 LSI 13 = 1198771198611

nuc ish 13q142(1198771198611x0)[36]13q142(1198771198611x1)[43]13q142(1198771198611x2)[121]



4 Discussion



1 2 3 4 5

6 7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 X Y






MCB 2

MCB 9

MCB 18


(a)

wcp10wcp14

der(14)der(10)10 14

(b)







p12p112

q1212q122q131q1411q1412q143q211q212

q2132q221q311

q312q321q323q332q34

p241p223p213p212p133p131p112

q13

q32

q2112q212

q2133q2231q2233q312

q332q3411q342



5 Conclusion




Acknowledgments


References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)














(b)


9p213 chr921967751ndash21975132 SPEC p16


9p21(p16x2)[53]

13q142 chr1348920000ndash49140000 LSI 13 = 1198771198611

nuc ish 13q142(1198771198611x0)[36]13q142(1198771198611x1)[43]13q142(1198771198611x2)[121]



4 Discussion



1 2 3 4 5

6 7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 X Y






MCB 2

MCB 9

MCB 18


(a)

wcp10wcp14

der(14)der(10)10 14

(b)







p12p112

q1212q122q131q1411q1412q143q211q212

q2132q221q311

q312q321q323q332q34

p241p223p213p212p133p131p112

q13

q32

q2112q212

q2133q2231q2233q312

q332q3411q342



5 Conclusion




Acknowledgments


References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1 2 3 4 5

6 7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 X Y






MCB 2

MCB 9

MCB 18


(a)

wcp10wcp14

der(14)der(10)10 14

(b)







p12p112

q1212q122q131q1411q1412q143q211q212

q2132q221q311

q312q321q323q332q34

p241p223p213p212p133p131p112

q13

q32

q2112q212

q2133q2231q2233q312

q332q3411q342



5 Conclusion




Acknowledgments


References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















p12p112

q1212q122q131q1411q1412q143q211q212

q2132q221q311

q312q321q323q332q34

p241p223p213p212p133p131p112

q13

q32

q2112q212

q2133q2231q2233q312

q332q3411q342



5 Conclusion




Acknowledgments


References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article a novel cryptic three-way translocation t...

Documents