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Leukemia Research 35 (2011) 1114– 1116

Contents lists available at ScienceDirect

Leukemia Research

jou rna l h omepa g e: www.elsev ier .com/ locate / leukres

rief communication

volutionary sequence of cytogenetic aberrations during the oncogenesis oflasma cell disorders. Direct evidence at single cell level

sófia Nagya, Béla Kajtára, Pál Jáksóa, Mariann Dávidb, Szabolcs Kosztolányib, Judit Hermesza,ászló Kereskaia, László Pajora, Donát Alpára,∗

Department of Pathology, University of Pécs, Pécs, Hungary1st Department of Internal Medicine, University of Pécs, Pécs, Hungary

r t i c l e i n f o

rticle history:eceived 19 January 2011eceived in revised form 22 January 2011ccepted 8 February 2011

a b s t r a c t

Bone marrow specimens from 185 patients with plasma cell disorders (PCD) were investigated by fluores-cence in situ hybridization (FISH) in order to determine the temporal sequence of cytogenetic aberrations.In 25 cases combined FISH analysis has also been performed at single cell level. Clonal evolution wasobserved in 16% of cases. The �13 was preceded by t(4;14)(p16;q32) and t(14;16)(q32;q23) translo-

vailable online 5 March 2011

eywords:lasma cell disorderISHotorized microscopy

cations. Deletion of p53 gene was a secondary aberration compared to �13 and t(11;14)(q13;q32)translocation. In 22% of all cases with recurrent IGH translocation, this aberration was presented onlyin a subset of purified plasma cells questioning its initiating role.

© 2011 Elsevier Ltd. All rights reserved.

ytogenetic evolutionecurrent IGH translocation

. Introduction

Genetic subtypes of plasma cell disorders (PCD) are associatedith unique clinicopathological features and divergent outcome

1]. Cytogenetic markers most investigated in the recent pastre monosomy or deletion of chromosome 13 (�13), deletionf p53 gene and the most frequent recurrent immunoglob-lin heavy-chain gene (IGH) translocations: t(4;14)(p16;q32),(11;14)(q13;q32) and t(14;16)(q32;q23). Although there is nooubt about the biological significance of these cytogenetic changes1], the exact time of their development during the pathogenesisf PCD is poorly known. The recurrent IGH translocations are con-idered as early events but their initiating impact is uncertain [2,3].ontroversial data are available about the time of �13 [1,2,4,5],hile deletion of p53 is thought to be correlated with disease pro-

ression [1]. Current models of oncogenesis are mainly supportedy indirect evidence such as the comparison of the incidence ofberrations in monoclonal gammopathy of undetermined signif-

cance (MGUS) and in plasma cell myeloma (PCM) or plasma celleukemia (PCL) [2,7]. Some workgroups analyzed the ratio of certainytogenetic anomalies within the purified plasma cell populationut the presence of various aberrations was not examined in the

∗ Corresponding author at: Department of Pathology, University of Pécs, 12 Szigetitr., 7624 Pécs, Hungary. Tel.: +36 72 536 282; fax: +36 72 536 281.

E-mail address: donat.alpar@kk.pte.hu (D. Alpár).

145-2126/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.leukres.2011.02.010

same nuclei [4–7]. Investigating the relationship of different cloneswithin one sample may provide information regarding the tempo-ral sequence of the detected abnormalities. For example, if thereare two clones in the sample, one with only abnormality A, andanother one, with abnormalities A and B, we can conclude that Aoccurred prior to B, excluding the unlikely occurrence of acquiring,and than losing an abnormality.

In this study, we have analyzed the correlation of cytogeneticchanges to each other and the incidence of abnormalities within thepurified plasma cell population. Combined interphase fluorescencein situ hybridization (FISH) analysis allowed correlated parametercollection at single cell level providing direct evidence regardingcytogenetic evolution of PCD.

2. Materials and methods

Diagnostic bone marrow samples from a total of 181 patients with PCM and4 patients with PCL were investigated. Aspirated plasma cells were purified withimmunomagnetic cell separation (BD IMagTM, BD Biosciences, CA) according to themanufacturer’s instructions using CD56 and CD138 antibodies. The results of theprocess were validated by flow cytometry (CyFlow® space, Partec GmbH, Germany).Ethical Committee approval and written informed consent from the patients havebeen obtained for the study.

FISH was performed on cells fixed in Carnoy-solution and dropped on slides.

Commercial probe sets from Vysis (Downers Grove, IL) were used for the detection of�13 (LSI D13S319/13q34), deletion of p53 gene (LSI TP53/CEP17), disruption of theIGH locus (LSI IGH BA) and the specific identification of the three IGH translocationsmost frequent in PCD (LSI IGH/FGFR3 DF, LSI IGH/CCND1 XT DF and LSI IGH/MAFDF). FISH procedures were performed according to the manufacturer’s instructionsand the signal patterns of 200 nuclei were analyzed. During evaluation an increased

esearch 35 (2011) 1114– 1116 1115

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Table 1Number of patients with more than one cytogenetic abnormality detected by inter-phase FISH.

Aberrations Number of cases

�13 + IgH 25�13 + t(4;14) 17�13 + t(11;14) 7�13 + t(14;16) 5�13 + del(17)(p13) 1del(17)(p13) + IgH 1del(17)(p13) + t(4;14) 1

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Z. Nagy et al. / Leukemia R

ttention was paid to avoid potential pitfalls emphasized earlier by the Europeanyeloma Network [8].

Patients’ samples with more than one specific aberration were chosen forhe combined FISH analysis. After FISH labeling for the first aberration, slidesere scanned by a motorized microscope (Zeiss Axioplan2ie MOT; Metasystems,ltlussheim, Germany). At least 500 nuclei were evaluated per sample with inter-ctive confirmation but collection of nuclei was extended until at least 200 cellsrom both positive and negative populations have been acquired. To investigate fur-her cytogenetic aberrations in the same nuclei the slides were ‘stripped’ using 50%ormamide/2×SSC (2 × 30′ , 65 ◦C) and re-hybridized with the second or third probeets. Individual cells were identified by their previously recorded coordinates. Allamples were analyzed by two independent investigators.

Following the recommendation of the European Myeloma Network a conserva-ive cut-off level of 10% was considered for positive results using dual-fusion andreak-apart probes, a cut-off value of 20% was used for numerical abnormalities oringle-fusion results with dual-fusion probes [8].

. Results

In the initial screening, the �13, p53 deletion and IGHisruption were found in 47.2, 7.5 and 58.9% of cases, respec-ively. The incidence of t(4;14)(p16;q32), t(11;14)(q13;q32) and(14;16)(q32;q23) within the group with IGH rearrangement were2.6, 21.7 and 6.6%, respectively. 64 samples (34.6%) harboringore than one genetic anomaly have been found; aberrations were

dentifiable specifically in 35 out of 64 cases (Table 1).Sufficient amount of bone marrow aspirates from 25 patients

24 PCM and 1 PCL cases) were available for further combined FISHnalyses. In 21 cases only one cell clone was identified harboringll abnormalities preventing the determination of the sequence ofytogenetic evolution. In 4 out of 25 cases (16%) more than oneell clone were observed (Fig. 1). Cell clones identified by the com-

ig. 1. Interphase FISH pattern of cell clones detected in bone marrow aspirate of patients

3. The colocalized signal pairs (fusion signals) refer to the juxtaposition of IGH (immueceptor 3 gene, 4p16, red) genes. Probes specific for the D13S319 and 13q34 regions arerformed twice in consecutive steps. An automated microscope scanning system was uhe 2F1R1G1A1Go (2 fusion, 2 red, 1 green, 1 aqua and 1 gold) signal pattern indicates th3. (C) IGH/c-MAF+ nucleus with normal chromosome 13 constitution. The IGH and c-MAFreen and red colors, respectively. (D) IGH/c-MAF+ nucleus with monosomy of chromosocyclin D1 gene, 11q13) genes are signed with green and red colors, respectively. Probes

ndicated by lilac and orange pseudo-colors. (F) IGH/CCND1+ nucleus with monoallelic dnd normal chromosome 17 constitution. (H) Nucleus harboring monoallelic deletion of

perture oil objective in a Zeiss Axioplan2ie MOT fluorescence microscope (Zeiss) equipJAI Corporation) and the Isis FISH Imaging System Version 5.0 (Metasystems). The figureoftwares.

del(17)(p13) + t(11;14) 1�13 + del(17)(p13) + IgH 3�13 + del(17)(p13) + t(4;14) 3

bined FISH analysis and their proportion within the whole purifiedplasma cell population are summarized in Table 2. The �13 waspreceded by the t(4;14)(p16;q32) and t(14;16)(q32;q23) translo-cations. Deletion of p53 gene was a secondary aberration comparedto the �13 and the t(11;14)(q13;q32) translocation. In 6 cases,dual-fusion probe sets resulted various abnormal FISH patternsindicating further abnormalities of the genes involved in the IGHtranslocation attributable to cytogenetic evolution. In these casesconsidering only the expected abnormal FISH pattern of two fusionswould have resulted in significant underestimation of the num-

ber of cells harboring the IGH translocation, possibly leading to thefalse conclusion that IGH translocation was preceded by �13. Datapresented here show that the recurrent IGH translocations are theearliest aberrations investigated in this study followed by �13 andp53 deletion.

with clonal evolution processes. (A) IGH/FGFR3+ nucleus with normal chromosomesnoglobulin heavy-chain gene, 14q32, green) and FGFR3 (fibroblast growth factore indicated by aqua and gold pseudo-colors, because red-green FISH labeling wassed for relocation and signal pattern evaluation. (B) IGH/FGFR3+ nucleus with �13.e balance nature of the translocation and the total loss of one of the chromosomes

(musculoaponeurotic fibrosarcoma gene, 16q23) genes are visualized by probes ofme 13. (E) IGH/CCND1+ nucleus with normal chromosomes 17. The IGH and CCND1specific for the p53 gene (17p13) and the �-satellite region of chromosome 17 areeletion of p53 gene. (G) Nucleus with monoallelic deletion of the D13S319 locus

the D13S319 and the p53 loci. FISH images were acquired using 63×/1.25 numericped with the appropriate filter sets (AHF) and documented using a CV-M1 camera

was composed using GraphicConverter X 6.7 and Adobe Photoshop Elements 4.0

1116 Z. Nagy et al. / Leukemia Resear

Table 2Chromosomal abnormalities and plasma cell clones detected by combined FISHanalysis in patients tested for clonal evolution.

Patient Abnormalities % of sorted PCsa Clonal evolution

A+ AB+ B+

#26 A: t(4;14) B: �13 – 61.4 – No#41 A: t(4;14) B: �13 69.2 29.8 – Yes#43 A: t(4;14) B: �13 – 94.8 – No#53 A: t(4;14) B: �13 – 80.7 – No#62 A: t(4;14) B: �13 – 36.3 – No#66 A: t(4;14) B: �13 – 91.1 – No#79 A: t(4;14) B: �13 – 92.8 – No

#119 A: t(4;14) B: �13 – 94.6 – No#132 A: t(4;14)b B: �13 – 93.7 – No#148 A: t(4;14)b B: �13 – 29.5 – No#149 A: t(4;14) B: �13 – 85.1 – No#151 A: t(4;14) B: �13 – 88.9 – No#181 A: t(4;14) B: �13 – 90.9 – No

#61 A: t(11;14)b B: �13 – 66.9 – No#90 A: t(11;14)b B: �13 – 82.7 – No

#131 A: t(11;14) B: �13 – 97.8 – No#143 A: t(11;14) B: �13 – 77.3 – No#182 A: t(11;14)b B: �13 – 89.1 – No

#40 A: t(14;16) B: �13 – 61.9 – No#45 A: t(14;16)b B: �13 17.9 78.9 – Yes

#138 A: t(14;16) B: �13 – 55.8 – No#124 A: t(11;14) B: del(17p) 10.2 88.6 – Yes#159 A: �13 B: del(17p) 56.7 38.2 – Yes

A+ B+ C+ AB+ BC+ AC+ ABC+

#3 A: t(4;14) B: �13 C: del(17p) – – – – – – 61.5 No#111 A: t(4;14) B: �13 C: del(17p) – – – – – – 87.3 No

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a The plasma cells were purified by CD56 and CD138 magnetic bead separationsnd the result of the process was validated by flow cytometry.b Various abnormal (positive) signal patterns were observed.

In 6 (25%) out of the 24 IGH positive cases investigated byombined FISH analysis the recurrent IGH translocations were pre-ented only in a subset (range: 29.5–61.9%) of purified plasmaells (Table 2). Subsequently we extended our observation to all85 patients screened in this study. In 21.8% of cases harboringpecifically detected recurrent IGH translocation (IGH/FGFR3 20.0%,GH/CCND1 16.7% and IGH/c-MAF 28.6%) the aberration was pre-ented in less than two-thirds of purified plasma cell population.he difference of incidence across the three groups was not statisti-ally significant (Kruskall–Wallis test: IGH/FGFR3 vs. IGH/CCND1 vs.GH/c-MAF, p = 0.786). The presence of two different IGH transloca-ions in the same sample was excludable in these particular cases.

. Discussion

Recently, Chiecchio et al. reported a comprehensive

tudy in which they have found that �13 occurs later than(14;20)(q32;q11), t(11;14)(q13;q32) and t(6;14)(p21;q32)hile they could not determine the temporal order of �13 and

(4;14)(p16;q32) or t(14;16)(q32;q23) with non-combined FISHnalyses [5]. Combined FISH analysis addressing the issue of clonal

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ch 35 (2011) 1114– 1116

evolution of structural cytogenetic aberrations in patients withPCD has not been published yet; therefore, our results not onlysupport the current model of the oncogenesis of PCD [2,3], but alsoprovide the first direct evidence at single cell level.

Other workgroups investigating patients with MGUS and smol-dering myeloma have also found that sometimes recurrent IgHtranslocations are present in a subset of plasma cells [6,7]. In theseearly lesions, the presence of normal plasma cells may confoundresults, since the count of abnormal plasma cells is relatively low.However, it is widely recognized that this diluting effect of normalplasma cells is negligible at the time of diagnosis of PCM or PCL[8]. Consequently, our results suggest that recurrent IGH translo-cations investigated in this study are not primary events at least inapproximately one-fifth of cases.

Conflict of interest

All authors have no conflict of interest to report.

Acknowledgements

The authors would like to thank Csilla Radvánszky for her excel-lent technical assistance. This work was supported in part by theMolecular Pathology Foundation.

Contributions. B.K. and D.A. designed the experiments; M.D., S.K.and L.K. provided tumor samples; Z.N., B.K., P.J., J.H., L.P. and D.A.generated and/or analyzed experimental data; Z.N. and D.A. wrotethe paper; L.P. and D.A. supervised the study. All authors criticallyreviewed and approved the final draft of the manuscript.

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