case report complex variant of philadelphia translocation...

Case ReportComplex Variant of Philadelphia TranslocationInvolving Chromosomes 9 12 and 22 in a Case withChronic Myeloid Leukaemia

F Malvestiti1 C Agrati1 S Chinetti1 A Di Meco1 S Cirrincione2

M Oggionni2 B Grimi1 F Maggi1 G Simoni1 and F R Grati1

1 Research and Development Cytogenetics and Molecular Biology TOMA Advanced Biomedical Assays SpA2527 Francesco Ferrer Street 21052 Busto Arsizio Varese Italy

2 Treviglio Caravaggio Hospital 1 Pittori Cavenaghi Square 24043 Caravaggio Bergamo Italy

Correspondence should be addressed to F Malvestiti fmalvestititomalabcom

Received 20 March 2014 Accepted 27 May 2014 Published 18 June 2014

Academic Editor Gopalrao Velagaleti

Copyright copy 2014 F Malvestiti et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder included in the broader diagnostic category ofmyeloproliferative neoplasms associated with fusion by BCR gene at chromosome 22q11 to ABL1 gene at chromosome 9q34 withthe formation of the Philadelphia (Ph) chromosome In 2ndash10 of CML cases the fusion gene arises in connection with a varianttranslocation involving chromosomes 9 22 and one or more different chromosomes consequently the Ph chromosome couldbe masked within a complex chromosome rearrangement In cases with variant Ph translocation a deletion on der(9) may bemore frequently observed than in cases with the classical one Herein we describe a novel case of CML with complex variant Phtranslocation involving chromosomes 9 12 and 22 We present the hematologic response and cytogenetic response after Imatinibtreatment We also speculated the mechanism which had originated the chromosome rearrangement

1 Introduction

Chronic myeloid leukemia (CML) is a hematopoietic stemcell disease included in the broader diagnostic category ofmyeloproliferative neoplasms [1] that is characterized by neo-plastic overproduction of mainly granulocytes CML is con-sistently associatedwith fusion by chromosome translocationof the breakpoint cluster region gene (BCR) at chromosome22q11 to the Abelson gene (ABL1) at chromosome 9q34 Thisfusion gene BCRABL1 encodes for an oncoprotein (P210more rarely P190 or P230) with a strong constitutive activatedtyrosine kinase activity inducing several downstream signalscausing the transformation of hemopoietic stem cells [2]Thetranslocation t(922) may be detected by routine karyotypeas Philadelphia (Ph) chromosome although in 2ndash10 ofthe cases the fusion gene arises from a variant transloca-tion [3] Two variant subgroups have been recognized thesimple variant group with the 22q segment translocated on

chromosome other than 9 and the complex variant translo-cation involving chromosomes 9 22 and one or moreadditional chromosomes Consequently the Ph chromo-some could be masked within a complex chromosome rear-rangement Although all chromosomes could be involved inthese variant translocations there is a marked clustering tospecific chromosomal bands suggesting that specific regionsare particularly prone to breakage In addition in variantcases a deletion on der(9) may be more frequent thanin cases with the classical Ph translocation (40 versus14) [4] Prognostic evaluation of different complex vari-ants was attempted in a limited number of CML casesgiving controversial and inconclusive results [5] Hereinwe describe a novel CML case with complex variant Phtranslocation involving chromosomes 9 12 and 22 Weevaluated the response to the Imatinib treatment and spec-ulated the molecular events underlying this chromosomerearrangement

Hindawi Publishing CorporationCase Reports in GeneticsVolume 2014 Article ID 691630 4 pageshttpdxdoiorg1011552014691630

2 Case Reports in Genetics

2 Case Report

The patient a 72-year-old woman had a clinical historyof immune-mediated thrombocytopenia During routinelaboratory analysis an unexpected increase of white bloodcount (WBC) was found and a CML was suspected Thelaboratory data showed aWBC count of 392 times 103mcL with60 of neutrophils 21 of lymphocytes 10 of monocytes2 of eosinophils 2 of basophils 4 of myelocytes and 1of metamyelocytes Hemoglobin concentration of 135 gdLwas within the normal range while the platelet count was low(101 times 103mcL) Cytogenetic analysis on bone marrow andRT-PCR on peripheral blood were carried out Conventionalcytogenetic analysis was performed on unstimulated 24-and 48-hour bone marrow cultures Cells were culturedand processed by standard methods [6] and chromosomeswere stained by QFQ-banding The analysis was performedaccording to the Italian and European Acquired Cytogeneticsand the ESMO (European Society of Medical Oncology)clinical practice guidelines [7ndash9] FISH analysis usingBCRABL1 t(922) Triple-Color and Dual-Fusion probeand Sub-Telomere 9qter probe (Kreatech DiagnosticsVlierweg 20 1032 LG Amsterdam The Netherlands) wasdone following the manufacturer procedures Karyotyperesult was described according to the ISCN 2013 [10]Reverse-transcription quantitative polymerase chainreaction (RT-PCR) for chimeric BCR-ABL1 transcript onperipheral blood was performed with Philadelphia p210Q-PCR Alert kit (Nanogen Inc San Diego CA USA)based on TaqMan technology RNA extraction and RT-PCR were performed following the insert kit instructions(Nanogen Inc San Diego CA USA) The measurement ofthe cDNAof P210 was normalized to the cDNAof ABL1 geneConventional cytogenetic analysis on bone marrow showedon 22 metaphases a reciprocal translocation involving thelong arm of chromosomes 12 and 22 t(1222) without theinvolvement of chromosome 9 (Figure 1(a)) The presenceof a cryptic BCRABL1 fusion transcript was detected byRT-PCR and subsequently by interphase FISH analyses onbone marrow Quantitative RT-PCR analysis for BCRABL1on peripheral blood revealed the major chimeric transcriptwith a BCR-ABL1(P210)ABL1 ratio of 1495 (InternationalScale) FISH analysis with BCRABL1 t(922) Triple-Colorand Dual-Fusion probe was performed to characterize thet(1222) translocation and to detect the localization of thefusion gene The probe set is a mixture of ASS-ABL1 probelabeled in red and of BCR probe with the proximal BCRregion labeled in blue and the distal one in green FISH on200 metaphases and nuclei showed the following (i) onepurple (bluered) fusion signal representing the fusion gene(BCRABL1) on der(22) (ii) one green signal of 31015840 BCRsequences on chromosome 12 involved in translocationt(1222) (iii) a greenblue signal on normal chromosome22 and (iv) a red signal on normal chromosome 9 (Figures1(b) and 1(c)) The reciprocal fusion ABL1BCR signal wasnot detected FISH analysis on 200 nuclei and metaphasesusing the subtelomeric 9qter probe was performed tofurther investigate the involvement of chromosome 9 in thecomplex rearrangement it showed a normal signal pattern

In summary FISH disclosed the deletion of the 51015840 ABL1sequences including the ASS gene on der(9) and allowedto map the breakpoint of t(1222) within the sequencesdistal to BCR gene The BCR probe gave a splitted signal onder(22) and on der(12) respectivelyThe ISCN karyotype was46XXder(9)del(9)(q34q34)ins(229)(q112q34q34)der(12)t(1222)(q13q112)der(22)ins(229)t(1222)[22] All theseresults were consistent with the CML diagnosis and thepatient started the treatment with Imatinib mesylate(Glivec) After three months of therapy the WBC countwas 51 times 103mcL with 497 of neutrophils 378 oflymphocytes 76 of monocytes 43 of eosinophils 06of basophils the hemoglobin concentration was 124 gdLand platelets count was 211 times 103mcL The molecularcytogenetic followup by interphase FISH with BCRABL1probe on 200 nuclei after 4 and 6 months of therapy showeda normal signal pattern while the chromosome analysis atsix months revealed a new abnormal clone detected in the5 (2 out of 5 metaphases and 10 out of 200 interphase nucleianalyzed by FISH with chromosomes 8 and 9 centromericprobes) of the sample with trisomies 8 and 9 (48XX+8+9)

3 Discussion

Wedescribe a patient with CML associated with a novel cryp-tic complex variant t(922) involving chromosome 12 besideschromosomes 9 and 22 which was unmasked and character-ized by RT-PCR and FISH analyses In agreement with ESMOclinical practice guidelines this case report proves the role ofthese molecular approaches in detecting cryptic fusion genein some types of variant translocations with masked Ph andder(9) chromosomes As previously reported the breakpointslocation of complex variant t(922) is nonrandom with amarked clustering to specific chromosome bands suggestingthat some regions are more prone to breakage This findingcould be explained by the presence of a specific genomicstructure mediating the recombination Indeed a significantclustering was described for high CG content regions Alurepeats LINE genes and miRNA explaining the presenceof recombination hotspots [11 12] The 12q13 chromosomeregion involved in our case was described by Costa et al[13] in association with complex Philadelphia translocationand in some cases of three-way translocation t(922) [11] Inaddition this region is involved both in other chromosomaltranslocations originating chimeric genes related to differentsubtypes of leukemia as reported in Mitelman et al [14] andin Atlas of chromosome in cancer databases [15] and inthe fragile site FRA12A which is caused by an expandedCGG repeat in the 5-prime untranslated region of the DIP2Bgene (OMIM 611379) [16] Combining all these data we canspeculate that the presence of specific genomicmotif in 12q13such as CGG repeats could have caused the variant t(922)observed in our patient To the best of our knowledge this isthe first case with this type of variant translocation in a CMLpatient

We can also hypothesize that this chromosomal rear-rangement was arisen by one-step mechanism with atleast four simultaneous breaks and joints because (i) at

Case Reports in Genetics 3

1 2 3 4 5

6 7 8 9 10 11 12

13

19

14

20 21 22

15 16 17 18

X Y

(a)

der(22)

chr 22

der(12)chr 9

der(9)

(b)

Chr 9 chr 12 chr 22

der(9)der(12)

der(22)

ASS-ABL1(9q34)

BCR (22q11) 5998400BCRABL1

3998400BCR

9q34

12q13 22q11

(c)

Figure 1 (a)QFQkaryotype derived frombonemarrow cellsThe arrows indicate the derivative chromosomes involved in the rearrangement(b) BCRABL1 FISH signal pattern on metaphase The arrows indicate the rearranged chromosomes and the normal chromosomes 9 and 22(c) Ideogram of the rearrangement identified in our CML case with the schematic representation of the FISH probe signals

diagnosis we did not detect additional clonal abnormalitiesand (ii) on der(22) only one breakpoint occurred which islocated within the BCR gene and that originated both thefusion gene and the t(1222) Conversely other cases showedthe coexistence of standard and complex translocation inthe same patient suggesting that two or more consecutivetranslocations caused the formation of the complex varianttranslocation [4]

Prognostic data on response to Imatinib in cases withcomplex Philadelphia translocation are contradictory andthe poor prognostic outcome in some patient of this groupwas explained by an increased frequency of the concomi-tant deletion on der(9) rather than to the type of chro-mosome rearrangement [5] Our patient has been treatedwith Imatinib and at 3 months of therapy she achievedthe hematological and cytogenetics responses despite thepresence of the deletion on der(9) while at six months oftherapy she developed a clone with trisomies 8 and 9 Thesetrisomies have apparently no prognostic significance in CMLIn more detail trisomy 8 may arise after interferon andorImatinib treatment with unknown significance and trisomy 9is assumed to represent a gain-of-function mechanism withrespect to the JAK2 gene on 9p24 coding for the JAK2 kinase

with no prognostic impact according to follow-up studies oflimited sample sizes [17]

Up to now our patient showed a good response toImatinib treatment but further studies are needed to confirmthis finding

Conflict of Interests

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

References

[1] J W Vardiman J Thiele D A Arber et al ldquoThe 2008 revisionof the World Health Organization (WHO) classification ofmyeloid neoplasms and acute leukemia Rationale and impor-tant changesrdquo Blood vol 114 no 5 pp 937ndash951 2009

[2] A Quintas-Cardama H Kantarjian and J Cortes ldquoTyrosinekinase inhibitors for chronic myelogenous leukemiardquo The NewEngland Journal ofMedicine vol 357 no 15 pp 1557ndash1558 2007

[3] B Johansson T Fioretos and F Mitelman ldquoCytogenetic andmolecular genetic evolution of chronic myeloid leukemiardquoActaHaematologica vol 107 no 2 pp 76ndash94 2002


[4] A G Reid B J P Huntly C Grace A R Green and EP Nacheva ldquoSurvival implications of molecular heterogeneityin variant Philadelphia-positive chronic myeloid leukaemiardquoBritish Journal of Haematology vol 121 no 3 pp 419ndash427 2003

[5] M M T El-Zimaity H Kantarjian M Talpaz et al ldquoResults ofimatinib mesylate therapy in chronic myelogenous leukaemiawith variant Philadelphia chromosomerdquo British Journal ofHaematology vol 125 no 2 pp 187ndash195 2004

[6] A Babu and R S Verma Human Chromosomes Principles andTechniques McGraw Hill Texas Tex USA 2nd edition 1995

[7] ldquoCitogenetica delle neoplasie oncoematoloficherdquo httpwwwsigunet

[8] R Hastings R Howell D Betts et al Guidelines and QualityAssurance for Acquired Cytogenetics ECA European Cyto-geneticists Association Newsletter No 31 2013 httpe-c-aeufilesdownloadsNL31 Acquired Guidelinespdf

[9] M Baccarani S Pileri J-L Steegmann M Muller S Soveriniand M Dreyling ldquoChronic myeloid leukemia ESMO clinicalpractice guidelines for diagnosis treatment and follow-uprdquoAnnals of Oncology vol 23 no 7 Article ID mds228 pp vii72ndashvii77 2012

[10] L G Shaffer JMcGowan-Jordan andM Schmid InternationalSystem for Human Cytogenetic Nomenclature Karger BaselSwitzerland 2013

[11] F Albano L Anelli A Zagaria et al ldquoNon random distributionof genomic features in breakpoint regions involved in chronicmyeloid leukemia cases with variant t(922) or additionalchromosomal rearrangementsrdquoMolecular Cancer vol 9 article120 2010

[12] A M Fisher P Strike C Scott and A V MoormanldquoBreakpoints of variant 922 translocations in chronic myeloidleukemia locate preferentially in the CG-richest regions of thegenomerdquo Genes Chromosomes and Cancer vol 43 no 4 pp383ndash389 2005

[13] D Costa A Carrio I Madrigal et al ldquoStudies of complex Phtranslocations in cases with chronic myelogenous leukemia andone with acute lymphoblastic leukemiardquo Cancer Genetics andCytogenetics vol 166 no 1 pp 89ndash93 2006

[14] F Mitelman B Johansson and F Mertens The MitelmanDatabase of Chromosome Aberrations in Cancer httpwwwcgapncinihgovChromosomesMitelman

[15] Atlas of Chromosome in Cancer httpatlasgeneticsoncol-ogyorgindexhtml

[16] OMIM httpwwwncbinlmnihgovomimterm=OMIM[17] U Bacher T Haferlach W Hiddemann S Schnittger W

Kern and C Schoch ldquoAdditional clonal abnormalities inPhiladelphia-positive ALL and CML demonstrate a differentcytogenetic pattern at diagnosis and follow different pathwaysat progressionrdquo Cancer Genetics and Cytogenetics vol 157 no 1pp 53ndash61 2005

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2 Case Report

The patient a 72-year-old woman had a clinical historyof immune-mediated thrombocytopenia During routinelaboratory analysis an unexpected increase of white bloodcount (WBC) was found and a CML was suspected Thelaboratory data showed aWBC count of 392 times 103mcL with60 of neutrophils 21 of lymphocytes 10 of monocytes2 of eosinophils 2 of basophils 4 of myelocytes and 1of metamyelocytes Hemoglobin concentration of 135 gdLwas within the normal range while the platelet count was low(101 times 103mcL) Cytogenetic analysis on bone marrow andRT-PCR on peripheral blood were carried out Conventionalcytogenetic analysis was performed on unstimulated 24-and 48-hour bone marrow cultures Cells were culturedand processed by standard methods [6] and chromosomeswere stained by QFQ-banding The analysis was performedaccording to the Italian and European Acquired Cytogeneticsand the ESMO (European Society of Medical Oncology)clinical practice guidelines [7ndash9] FISH analysis usingBCRABL1 t(922) Triple-Color and Dual-Fusion probeand Sub-Telomere 9qter probe (Kreatech DiagnosticsVlierweg 20 1032 LG Amsterdam The Netherlands) wasdone following the manufacturer procedures Karyotyperesult was described according to the ISCN 2013 [10]Reverse-transcription quantitative polymerase chainreaction (RT-PCR) for chimeric BCR-ABL1 transcript onperipheral blood was performed with Philadelphia p210Q-PCR Alert kit (Nanogen Inc San Diego CA USA)based on TaqMan technology RNA extraction and RT-PCR were performed following the insert kit instructions(Nanogen Inc San Diego CA USA) The measurement ofthe cDNAof P210 was normalized to the cDNAof ABL1 geneConventional cytogenetic analysis on bone marrow showedon 22 metaphases a reciprocal translocation involving thelong arm of chromosomes 12 and 22 t(1222) without theinvolvement of chromosome 9 (Figure 1(a)) The presenceof a cryptic BCRABL1 fusion transcript was detected byRT-PCR and subsequently by interphase FISH analyses onbone marrow Quantitative RT-PCR analysis for BCRABL1on peripheral blood revealed the major chimeric transcriptwith a BCR-ABL1(P210)ABL1 ratio of 1495 (InternationalScale) FISH analysis with BCRABL1 t(922) Triple-Colorand Dual-Fusion probe was performed to characterize thet(1222) translocation and to detect the localization of thefusion gene The probe set is a mixture of ASS-ABL1 probelabeled in red and of BCR probe with the proximal BCRregion labeled in blue and the distal one in green FISH on200 metaphases and nuclei showed the following (i) onepurple (bluered) fusion signal representing the fusion gene(BCRABL1) on der(22) (ii) one green signal of 31015840 BCRsequences on chromosome 12 involved in translocationt(1222) (iii) a greenblue signal on normal chromosome22 and (iv) a red signal on normal chromosome 9 (Figures1(b) and 1(c)) The reciprocal fusion ABL1BCR signal wasnot detected FISH analysis on 200 nuclei and metaphasesusing the subtelomeric 9qter probe was performed tofurther investigate the involvement of chromosome 9 in thecomplex rearrangement it showed a normal signal pattern

In summary FISH disclosed the deletion of the 51015840 ABL1sequences including the ASS gene on der(9) and allowedto map the breakpoint of t(1222) within the sequencesdistal to BCR gene The BCR probe gave a splitted signal onder(22) and on der(12) respectivelyThe ISCN karyotype was46XXder(9)del(9)(q34q34)ins(229)(q112q34q34)der(12)t(1222)(q13q112)der(22)ins(229)t(1222)[22] All theseresults were consistent with the CML diagnosis and thepatient started the treatment with Imatinib mesylate(Glivec) After three months of therapy the WBC countwas 51 times 103mcL with 497 of neutrophils 378 oflymphocytes 76 of monocytes 43 of eosinophils 06of basophils the hemoglobin concentration was 124 gdLand platelets count was 211 times 103mcL The molecularcytogenetic followup by interphase FISH with BCRABL1probe on 200 nuclei after 4 and 6 months of therapy showeda normal signal pattern while the chromosome analysis atsix months revealed a new abnormal clone detected in the5 (2 out of 5 metaphases and 10 out of 200 interphase nucleianalyzed by FISH with chromosomes 8 and 9 centromericprobes) of the sample with trisomies 8 and 9 (48XX+8+9)

3 Discussion

Wedescribe a patient with CML associated with a novel cryp-tic complex variant t(922) involving chromosome 12 besideschromosomes 9 and 22 which was unmasked and character-ized by RT-PCR and FISH analyses In agreement with ESMOclinical practice guidelines this case report proves the role ofthese molecular approaches in detecting cryptic fusion genein some types of variant translocations with masked Ph andder(9) chromosomes As previously reported the breakpointslocation of complex variant t(922) is nonrandom with amarked clustering to specific chromosome bands suggestingthat some regions are more prone to breakage This findingcould be explained by the presence of a specific genomicstructure mediating the recombination Indeed a significantclustering was described for high CG content regions Alurepeats LINE genes and miRNA explaining the presenceof recombination hotspots [11 12] The 12q13 chromosomeregion involved in our case was described by Costa et al[13] in association with complex Philadelphia translocationand in some cases of three-way translocation t(922) [11] Inaddition this region is involved both in other chromosomaltranslocations originating chimeric genes related to differentsubtypes of leukemia as reported in Mitelman et al [14] andin Atlas of chromosome in cancer databases [15] and inthe fragile site FRA12A which is caused by an expandedCGG repeat in the 5-prime untranslated region of the DIP2Bgene (OMIM 611379) [16] Combining all these data we canspeculate that the presence of specific genomicmotif in 12q13such as CGG repeats could have caused the variant t(922)observed in our patient To the best of our knowledge this isthe first case with this type of variant translocation in a CMLpatient

We can also hypothesize that this chromosomal rear-rangement was arisen by one-step mechanism with atleast four simultaneous breaks and joints because (i) at


1 2 3 4 5

6 7 8 9 10 11 12

13

19

14

20 21 22

15 16 17 18

X Y

(a)

der(22)

chr 22

der(12)chr 9

der(9)

(b)

Chr 9 chr 12 chr 22

der(9)der(12)

der(22)

ASS-ABL1(9q34)

BCR (22q11) 5998400BCRABL1

3998400BCR

9q34

12q13 22q11

(c)








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

19

14

20 21 22

15 16 17 18

X Y

(a)

der(22)

chr 22

der(12)chr 9

der(9)

(b)

Chr 9 chr 12 chr 22

der(9)der(12)

der(22)

ASS-ABL1(9q34)

BCR (22q11) 5998400BCRABL1

3998400BCR

9q34

12q13 22q11

(c)








References
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















case report complex variant of philadelphia translocation...

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