Cytogenetic follow-up by karyotyping and fluorescence in situ hybridization: implications for monitoring patients with myelodysplastic syndrome and deletion 5q treated with lenalidomide

by Gudrun Göhring, Aristoteles Giagounidis, Guntram Büsche, Winfried Hofmann,Hans Heinrich Kreipe, Pierre Fenaux, Eva Hellström-Lindberg,and Brigitte Schlegelberger

Haematologica 2010 [Epub ahead of print]

Citation: by Göhring G, Giagounidis A, Büsche G, Hofmann W, Kreipe HH, Fenaux P,Hellström-Lindberg E, and Schlegelberger B. Cytogenetic follow-up by karyotyping and fluorescence in situ hybridization: implications for monitoring patients with myelodysplastic syndrome and deletion 5q treated with lenalidomide. Haematologica. 2010; 95:xxx doi:10.3324/haematol.2010.026658

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Copyright 2010 Ferrata Storti Foundation.Published Ahead of Print on November 25, 2010, as doi:10.3324/haematol.2010.026658.

Cytogenetic follow-up by karyotyping and fluorescence in situ hybridization:

implications for monitoring patients with myelodysplastic syndrome and

deletion 5q treated with lenalidomide

Gudrun Göhring,1 Aristoteles Giagounidis,2 Guntram Büsche,3 Winfried Hofmann,1

Hans Heinrich Kreipe,3 Pierre Fenaux,4 Eva Hellström-Lindberg,5

and Brigitte Schlegelberger1

1Institute of Cell and Molecular Pathology, Hannover Medical School, Germany; 2Department of Haematology, Oncology and Clinical Immunology, Johannes

Hospital, Duisburg, Germany; 3Institute of Pathology, Hannover Medical School,

Germany; 4Service d'Hématologie Clinique, Hôpital Avicenne, Assistance Publique

Hôpitaux de Paris, Paris, Bobigny, France; and 5Division of Haematology and Centre

of Experimental Haematology, Department of Medicine, Karolinska Institutet,

Karolinska University Hospital, Stockholm, Sweden

Correspondence Gudrun Göhring, Institute of Cell and Molecular Pathology, Hannover Medical

School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.

Phone: international +49.511.5324517. Fax: international +49.511.5324521.

E-Mail: goehring.gudrun@mh-hannover.de

Acknowledgments The authors would like to thank Gillian Teicke for her help in preparing the manuscript, Michael Morgan for critically reading the manuscript and Aspasia Stamatoullas, Odile Beyne-Rauzy, Ghulam Mufti, Petra Muus, Lars Nilsson, Guillermo F. Sanz and Dominik Selleslag for the recruitment of patients. Finally, we thank Dr. Robert Knight and Dr. Jay Backström, Celgene, for allowing us to use data obtained within the Celgene MDS-003 and MDS-004 clinical trials. Funding Supported by COST Action BM0801 (EuGESMA) and the Excellence cluster Rebirth.

DOI: 10.3324/haematol.2010.026658

Abstract

In patients with low and intermediate risk myelodysplastic syndrome and deletion 5q

(del(5q)) treated with lenalidomide, monitoring of cytogenetic response is mandatory,

since patients without cytogenetic response have a significantly increased risk of

progression. Therefore, we have reviewed cytogenetic data of 302 patients.

Patients were analyzed by karyotyping and fluorescence in situ hybridization.

In 85 patients, del(5q) was only detected by karyotyping. In 8 patients undergoing

karyotypic evolution, the del(5q) and additional chromosomal aberrations were only

detected by karyotyping. In 3 patients, del(5q) was only detected by fluorescence in

situ hybridization, but not by karyotyping due to a low number of metaphases.

Karyotyping was significantly more sensitive than fluorescence in situ hybridization in

detecting the del(5q) clone. In conclusion, to optimize therapy control of

myelodysplastic syndrome patients with del(5q) treated with lenalidomide and to

identify cytogenetic non-response or progression as early as possible, fluorescence

in situ hybridization alone is inadequate for evaluation. Karyotyping must be

performed to optimally evaluate response.

(clinicaltrials.gov identifier: NCT01099267 and NCT00179621)

DOI: 10.3324/haematol.2010.026658

Introduction

Myelodysplastic syndrome (MDS) associated with isolated del(5q) is a distinct entity

with a risk of evolution into acute myeloid leukemia (AML) of approximately 10% (1-

2). It is characterized by anemia with or without other cytopenias and/or

thrombocytosis. Myeloblasts comprise <5% of bone marrow and <1% of peripheral

blood (1). Lenalidomide is particularly active in MDS patients with del(5q), with over

two-thirds of low and intermediate risk MDS patients with 5q deletions achieving

transfusion independence (3). In this cohort with an a priori increased risk, about one

third of the patients underwent clonal evolution and AML progression (4-5). Patients

without hematologic or cytogenetic response had a significantly increased risk of

progression (5). Therefore, careful monitoring of the cytogenetic response may add

important prognostic information in MDS patients treated with lenalidomide.

Gold standard for the detection of aberrations is karyotyping (6-8). Aberrations are

detected in about half of MDS patients (9). Additional fluorescence in situ

hybridization (FISH) analyses are recommended to detect deletions 5q, 7q, 17p or

20q, monosomy 7 and trisomy 8 (6). Recently, it was suggested that FISH should be

performed in cases of a suspected ‘5q- syndrome’ and/or if karyotyping shows no

metaphases or an aberrant karyotype involving chromosome 5 (10).

In order to optimize therapeutic control of MDS patients treated with lenalidomide, we

have evaluated the cytogenetic data of 302 del(5q) patients treated with lenalidomide

and compared the results obtained by karyotyping and FISH.

Design and Methods

Patients

Our cohort included 302 patients with transfusion-dependent anemia due to low- or

intermediate-1-risk myelodysplastic syndrome (according to the International

DOI: 10.3324/haematol.2010.026658

Prognostic Scoring System (IPSS)) associated with a 5q deletion with or without

additional cytogenetic abnormalities. This cohort includes all European patients

enrolled in the CC-5013-MDS-003 (ClinicalTrials.gov Identifier: NCT01099267) and

all patients enrolled in the CC-5013-MDS-004 study (ClinicalTrials.gov Identifier:

NCT00179621) for MDS patients with del(5q) treated with lenalidomide. Cytological

and morphological diagnoses were made centrally at St. Johannes Hospital Duisburg

and Hannover Medical School. Patients were enrolled in the studies MDS-003 (n=42)

or MDS-004 (n=260) and treated with lenalidomide (starting dose, 10mg daily)

according to the study protocols (3). Written informed consent was provided

according to the Declaration of Helsinki and approval from the Ethics Committee of

Hannover Medical School (MHH) was obtained.

Lenalidomide therapy is still ongoing in responding patients. Dose adjustments were

performed in the majority of patients due to side effects such as neutropenia.

Regular follow-up investigations were performed every 6 months of treatment or

more frequently, depending upon clinical features. As many as 11 cytogenetic

analyses were performed for each patient. In total, we reviewed 1075 cytogenetic

analyses.

Standardized cytogenetic investigations: Karyotyping and FISH

Bone marrow was received from European study centers for central cytogenetic

review. Karyotyping was performed according to standard procedures (11).

Whenever possible, 25 metaphases were analyzed. Description of the karyotype, the

chromosomal aberrations, followed the recommendations of the International System

for Cytogenetic Nomenclature (12). In all patients, an interstitial del(5q) containing the

commonly deleted region 5q31 was present (Figure 1A-C). Fluorescence in situ

hybridization (FISH) for del(5q) was included in each investigation (probes supplied

by Abbott, Wiesbaden, Germany). FISH was performed on fixed cells from the

DOI: 10.3324/haematol.2010.026658

cytogenetic culture according to standard procedures (Figure 1D-F) (11). For each

FISH analysis, at least two hundred interphase nuclei were analyzed. In agreement

with the literature, mean cut-off levels were determined to be 8% (+/- 3SD) by

analysis of at least 1000 cells from 10 healthy donors (10).

Cytogenetic response

Cytogenetic response was applied to karyotyping only. Complete cytogenetic

response (CCyR) was defined as disappearance of the 5q deletion or any other

chromosomal aberration. Partial cytogenetic response (PCyR) was defined as a

reduction of aberrant metaphases of more than 50% compared to the previous

karyotyping (3). Cytogenetic relapse was defined as reappearance of a metaphase

with a deletion in 5q after reaching a complete cytogenetic response.

Statistical analyses

Analysis of del(5q) detection rates in metaphases and interphases was done by

Wilcoxon test (13). Values of p<0.05 were considered statistically significant.

Results and Discussion

We compare the sensitivity of detecting the del(5q) clone by karyotyping and FISH,

respectively, in a large cohort of MDS patients treated with lenalidomide (n=302). A

complete cytogenetic investigation based on at least 25 metaphases and FISH based

on the analyses of at least 200 interphase nuclei was performed in 835 (78%) of the

investigations. Here, we focus on discrepant results of karyotyping and FISH that

would have led to a different judgement of whether the patient is eligible for treatment

with lenalidomide, or to a different diagnosis of cytogenetic remission, relapse or

progression during follow-up.

DOI: 10.3324/haematol.2010.026658

Detection of del(5q) at study entry before treatment with lenalidomide

Twelve of 302 patients (4%) (patients 1-10, 23 and 86) showed discrepant

karyotyping and FISH results already in the first cytogenetic investigation at study

entry (Table 1). In patients 1-10, del(5q) was not detected by FISH but only by

karyotyping. In 5 of these 10 patients less than 10% of the metaphases showed a

deletion in 5q at study entry. In contrast, a deletion in 5q was detected in 72% and

100% of the metaphases in two of those patients (patients 6 and 10) (Supplement 1).

In patients 23 and 86, del(5q) was detected by FISH only. The del(5q) was detected

in 21% and 32% of the interphase nuclei, respectively. However, in both

investigations, less than 10 metaphases could be analyzed. 17 patients showed one

additional aberration and 12 patients an independent clone, e.g. +8 or t(2;11).

Follow-up after 6 months of treatment

Six months after treatment with lenalidomide, in 27 of 232 patients (12%) del(5q) was

detected only by karyotyping and not by FISH. In two of these patients, a clonal

evolution occurred. Neither the clonal evolution nor the del(5q) clone would have

been detected if only FISH had been used. At this time point, in no case was del(5q)

detected by FISH only (Table 1, Supplement 2).

Follow-up after 12 months of treatment

After 12 months of treatment, in 39 of 169 patients (23%) del(5q) was detected only

by karyotyping. In one patient, del(5q) was detected only by FISH. In three patients, a

clonal evolution occurred. In 6 patients, a cytogenetic relapse occurred at this time

point. This relapse would not have been detected if only FISH had been used (Table

1).

Third and later follow-up investigations after 18 months and later of treatment

In 85 of 267 patients (32%), a discrepancy was detected after at least 18 months of

treatment. In only one patient was del(5q) detected by FISH only. In the other cases,

DOI: 10.3324/haematol.2010.026658

del(5q) was detected by karyotyping only and not by FISH. In 31 of these patients, a

cytogenetic relapse occurred and would not have been detected if only FISH had

been used. In five of them, a progress with a clonal evolution was identified by

karyotyping. Neither the clonal evolution nor the del(5q) clone would have been

detected if only FISH had been used (Table 2). There was no difference regarding

the detection of the del(5q) comparing patients with a discrepancy at the first time

point of analysis and others.

Detection of a complete cytogenetic response

24 of 163 patients (15%) with a discrepancy between karyotyping and FISH reached

a CCyR. Eleven patients reached CCyR after 6 months of treatment with

lenalidomide. Three patients reached CCyR after 12 months, 9 patients after 18 and

one patient after 24 months (median of 15 months). Nine of these 24 patients (37%)

reached a partial response and 15 patients (63%) did not reach a partial response

beforehand. The partial response occurred once after 6 months, 7 times after 12

months and once after 18 months of treatment. Patient 35, for example, showed a

CCyR after 12 months without reaching a partial remission beforehand (Supplement

2). On the other hand, patient 15 reached a partial remission after 6 and a CCyR

after 12 months of treatment (Supplement 1).

In 19 of 302 patients (6%), independent clones were detected by karyotyping. The

independent clones contained trisomy 8 in 12 patients, loss of the Y chromosome in

4 patients, monosomy 7 in 3 patients, del(20q) in 2 patients, and del(11q), trisomy 14

and der(1;7) in one patient each. In four of these patients, two or three independent

clones were found. In 8 of 16 patients undergoing a karyotypic evolution, at 17 time

points, neither the del(5q) nor the additional chromosomal aberrations were detected

by FISH – in contrast to karyotyping. In patients 34, 35 and 36, karyotypic evolution

occurred after they had reached a complete cytogenetic response.

DOI: 10.3324/haematol.2010.026658

Clinical consequences

This study has three major implications for monitoring MDS patients with del(5q)

treated by lenalidomide. Firstly, the del(5q) clone was detected significantly more

sensitively by karyotyping than by FISH, thus making the diagnosis of non-remission

and cytogenetic relapse possible only by karyotyping in a significant number of

patients. Secondly, without prior knowledge of the additional chromosome

aberrations, clonal evolution can be identified only by karyotyping. At the time point of

clonal evolution, even del(5q) was detected only by karyotyping in a significant

number of patients. Thirdly, detection of the del(5q) clone was sometimes only

possible by FISH, making the diagnosis of non-remission and cytogenetic relapse in

these patients only possible by FISH analysis. Thus, all cytogenetic follow-up

investigations must include karyotyping. An additional FISH analysis should at least

be performed, if karyotyping does not give sufficient results, e.g. due to poor

chromosome morphology or due to a low number of metaphases (< 25 metaphases).

Karyotyping was significantly more sensitive than FISH for detection of del(5q)

(p<2.2e-16). This was true for the time point of study entry as well as during follow-

up. In contrast, a deletion in 5q was only detected by FISH in three patients. In these

cases, less than 10 metaphases were available for analysis. As recommended by the

European LeukemiaNet, at least 25 metaphases are necessary for a reliable

karyotyping (6). In the literature, several studies described discrepant results of

karyotype and FISH analyses (10,14-17). In most instances, the cases with a

discrepant result (positive by FISH only) had an insufficient number of metaphases or

the chromosome morphology was poor. Pitchford et al. recommended FISH in MDS

patients only if an adequate karyotyping with at least 20 metaphases was not

possible, since they were unable to demonstrate a benefit of FISH in the setting of an

optimal banding analysis (7). FISH is a valuable method to quantify the clone size

DOI: 10.3324/haematol.2010.026658

with cytogenetic aberrations known from diagnosis. It may be discussed whether it is

sufficient only to perform FISH during follow-up analyses. FISH may be used to

monitor for the presence of a specific chromosomal abnormality that was present

before treatment (18). Obviously, this approach has positive aspects, such as being

cost- and time-effective as no culturing is needed since only interphase nuclei are

analyzed. However, our findings question the proposal that FISH analysis alone is

sufficient for follow-up analysis. Using this approach in our study, cytogenetic non-

response or relapse and even clonal evolution would not have been detected in a

significant number of patients.

One reason for the higher sensitivity of karyotyping in detecting the del(5q) clone

might be that a small clone with proliferative advantage over normal cells may be

more easily identified by karyotyping than by FISH. FISH is a quantitative method

counting interphase nuclei with a normal signal constellation and with a signal

constellation indicative of a del(5q). A small clone with a del(5q) might be missed if it

is below the cut-off level for the detection of a deletion, which is typically between 3

and 10 % and was 8% in our study (7,9,19). In contrast, karyotyping is based on

metaphases, i.e. cells undergoing spontaneous cell division in vitro. Therefore, the

amount of metaphases with a del(5q) identified by karyotyping does not necessarily

represent the proportion of these cells within the bone marrow. To monitor

cytogenetic response in chronic myeloid leukemia (CML), Fugazza et al. did not find

an advantage of karyotyping or FISH. As in our study, one or the other method was

superior in some patients (20). In contrast, the GIMENA group recently showed that

FISH is more sensitive than banding analysis in CML and should be used for

monitoring (21). However, due to the FISH probe designed to detect the Philadelphia

translocation, the cut-off level was below 1%.

DOI: 10.3324/haematol.2010.026658

A very important advantage of karyotyping is the possibility to identify additional

aberrations. Only previously selected loci can be analyzed by FISH. Moreover, an

additional multicolor FISH can be applied to detect cryptic aberrations not seen by

karyotyping. In our study, clonal evolution was identified in 8 patients. In most

patients, clonal evolution was associated with leukemic progression as described

before (5,22,23). In these patients, chromosomal instability leading to clonal evolution

seems to be the driving force behind the expansion of tumor cell populations (24).

In conclusion, to optimize monitoring of MDS patients with del(5q) treated with

lenalidomide and to detect cytogenetic non-response, cytogenetic relapse or clonal

evolution as early as possible, FISH alone is inadequate for evaluation. Karyotyping

must be performed to optimally evaluate response.

Authorship and Disclosures

GG and BS were the principal investigators and take primary responsibility for the

paper. AG, PF and EHL recruited the patients. GG, GB, and HHK performed the

laboratory work for this study. WH participated in the statistical analysis. GG and BS

wrote the paper. The authors reported no potential conflicts of interest.

DOI: 10.3324/haematol.2010.026658

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Table 1. Discrepancy in detecting del(5q) by karyotyping and FISH in patients with MDS and del(5q) at study entry before treatment with lenalidomide (time point 0) and after 6 and 12 months of treatment (time point 1 and 2).

Time point Patient No FAB ISCN-Karyotyp ISCN FISH % MP 5q- % IP 5q- Response*0 1 RARS 46,XY,del(5)(q?14q34)[1]/46,XY[24] 5q31(EGR1x2) 4 1 n.e.0 2 RA 46,XX,del(5)(q22q34)[4]/46,XX[21] 5q31(EGR1x2) 16 3 n.e.0 3 RA 46,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 4 n.e.0 4 RA 46,XX,del(5)(q14q34)[3]/46,XX[22] 5q31(EGR1x2) 12 6 n.e.0 5 punctio sicca 47,XX,+8[4]/45,XX,del(5)(q1?q34),-7[3]/46,XX[48] 5q31(EGR1x2) 5 4 n.e.0 6 punctio sicca 46,XX,del(5)(q13q34)[1]/46,idem,add(20)(q13)[2] 5q31(EGR1x2) 100 2 n.e.0 7 RA 46,XX,del(5)(q23q34)[4]/46,XX[7] 5q31(EGR1x2) 36 6 n.e.0 8 RARS 46,XY,del(5)(q?21q?34)[2]/46,XY[23] 5q31(EGR1x2) 8 5 n.e.0 9 RA 46,XY,del(5)(q21q34)[1]/46,XY[24] 5q31(EGR1x2) 4 6 n.e.0 10 RA 46,XY,del(5)(q14q34),inv(11)(p13 p14q22 q23)[18]/46,XY[7] 5q31(EGR1x2) 72 0 n.e.0 23 RAEB 46,XX[2] 5q31(EGR1x1) 0 21 n.e.0 86 punctio sicca 46,XX[3] 5q31(EGR1x1) 0 32 n.e.1 8 RARS 46,XY,del(5)(q21q34)[1]/46,XY[19] 5q31(EGR1x2) 5 4 no CyR1 11 RA 46,XY,del(5)(q21q34)[3]/46,XY[22] 5q31(EGR1x2) 12 4 no CyR1 12 RA 46,XX,t(X;16)(p11;q23),del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 4 no CyR1 14 RA 46,XX,del(5)(q21q34)[2]/46,XX[23] 5q31(EGR1x2) 8 5 no CyR1 15 RARS 46,XX,del(5)(q14q34)[1]/46,XX[19] 5q31(EGR1x2) 5 0 no CyR

1 30 RA47,XX,+8[7]/46,XX,del(20)(q12q13)[4]/46,XX,del(5)(q14q34)[2]/ 46,XX,del(5)(q14q34),del(20)(q12q13)[1]/46,XX[11] 5q31(EGR1x2) 12 2

progress

1 31 RA 47,XX,+8[2]/46,XX,del(5)(q14q34)[1]/46,XX[22] 5q31(EGR1x2) 12 2 progress1 32 RARS 46,XX,del(5)(q13q34)[2]/46,idem,dup(1)(q21q32)[4]/47,XX,+8[5]/46,XX[14] 5q31(EGR1x2) 24 7 progress

1 37 RA46,XX,t(2;11)(p16;q23)[11]/46,idem,-11,+der(11)t(2;11) (p16;q23)[13]/46,idem,del(5)(q14q34)[1] 5q31(EGR1x2) 4 7

no CyR

1 39 RA 46,XX,del(5)(q14q34)[1]/46,XX,del(20)(q12q13)[2]/ 46,XX[22] 5q31(EGR1x2) 4 0 no CyR1 41 RARS 46,XX,del(5)(q14q34)[4]/46,XX[21] 5q31(EGR1x2) 16 5 no CyR1 46 RA 46,XY,del(5)(q22q34)[1]/46,XY[10] 5q31(EGR1x2) 9 0 no CyR1 51 RA 46,XX,del(5)(q14q34)[4]/46,XX[12] 5q31(EGR1x2) 25 4 no CyR1 60 RA 46,XX,del(5)(q14q34)[8]/46,XX[17] 5q31(EGR1x2) 32 5 no CyR1 61 RA 46,XX,del(5)(q14q34)[8]/46,XX[5] 5q31(EGR1x2) 62 6 no CyR1 66 RA 46,XX,del(5)(q21q34)[2]/46,XX[23] 5q31(EGR1x2) 8 2 no CyR1 68 RA 46,XX,del(5)(q21q34)[1]/46,XX[24] 5q31(EGR1x2) 4 3 no CyR1 69 RA 46,XX,del(5)(q14q34)[6]/47,XX,+8[14]/46,XX[5] 5q31(EGR1x2) 24 7 no CyR1 70 RA 47,XX,+8[25]/45,XX,-7[2]/46,XX,del(5)(q14q34)[1] 5q31(EGR1x2) 4 6 no CyR1 73 RA 46,XX,del(5)(q21q34),del(11)(q21)[3]/47,XX+8[7]/46,XX[17] 5q31(EGR1x2) 11 2 no CyR1 75 RARS 46,XX,del(5)(q21q34),t(6;13)(p?23;q?13)[1]/47,XX,+8[21]/46,XX[9] 5q31(EGR1x2) 3 1 no CyR1 76 RA 46,XX,del(5)(q?14q?34)[1]/46,XX[11] 5q31(EGR1x2) 8 7 no CyR1 77 RA 46,XX,del(5)(q13q34)[5]/47,XX,+8[2]/46,XX[18] 5q31(EGR1x2) 20 6 no CyR1 80 RA 46,XX,del(5)(q14q34)[6]/46,XX[19] 5q31(EGR1x2) 24 2 no CyR1 83 RARS 46,XY,del(5)(q14q34)[1]/46,XY[19] 5q31(EGR1x2) 5 0 no CyR1 84 RA 46,XY,del(5)(q14q34)[2]/46,XY[23] 5q31(EGR1x2) 8 6 no CyR1 85 RARS 46,XY,del(5)(q14q34)[1]/46,XY[19] 5q31(EGR1x2) 5 0 no CyR2 9 RA 46,XY,del(5)(q14q34)[1]/46,XY[24] 5q31(EGR1x2) 4 2 no CyR2 10 RA 46,XY,del(5)(q14q34),inv(11)(p13 p14q22 q23)[2]/ 46,XY[23] 5q31(EGR1x2) 8 4 relapse2 13 RA 46,XX,del(5)(q14q34)[1]/46,XX[25] 5q31(EGR1x2) 4 4 no CyR2 23 RA 46,XX,del(5)(q14q34)[2]/46,XX[23] 5q31(EGR1x2) 8 1 relapse2 24 RA 46,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 0 relapse2 26 RA 46,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 3 relapse2 27 RARS 46,XX,del(5)(q21q34)[1]/46,XX[24] 5q31(EGR1x2) 4 1 relapse

2 29RA

46,XX,del(5)(q14q34)[1]/48-50,idem,+21,+21,+idic(21)(q21~q22), +mar[cp4]/46,XX[20] 5q31(EGR1x2) 20 6

progress

2 30 RA 46,XX,del(20)(q12q13)[6]/46,XX,del(5)(q14q34)[1]/47,XX,+8[1]/46,XX[14] 5q31(EGR1x2) 5 4 progress2 31 RA 46,XX,del(5)(q14q34)[1]/46,idem,del(11)(q21)[2]/46,XX[22] 5q31(EGR1x2) 12 3 progress2 33 RA 46,XX,del(5)(q14q34)[3]/46,XX[3] 5q31(EGR1x2) 50 4 no CyR2 36 RA 45,X,-Y[13]/46,XY,del(5)(q14q34)[5]/ 46,idem,del(13)(q14q31)[4]/ 46,XY[3] 5q31(EGR1x2) 36 2 relapse, progress2 38 RA 46,XY,del(5)(q14q34)[3]/46,XY[22] 5q31(EGR1x2) 12 5 no CyR2 39 RA 46,XX,del(20)(q12q13)[13]/46,XX,del(5)(q14q34)[1]/46,XX[11] 5q31(EGR1x2) 4 3 no CyR2 43 RA 45,XX,-7[3]/46,XX,del(5)(q14q34)[2]/46,XX[20] 5q31(EGR1x2) 8 3 no CyR2 45 RA 46,XY,del(5)(q14q34)[1]/46,XY[24] 5q31(EGR1x2) 4 0 no CyR2 46 RA 46,XY,del(5)(q22q34)[1]/46,XY[11] 5q31(EGR1x2) 9 0 no CyR2 47 RA 46,XY,del(5)(q21q34)[4]/46,XY[21] 5q31(EGR1x2) 16 3 no CyR2 49 RA 47,XX,+8[6]/46,XX,del(5)(q14q34)[4]/46,XX[15] 5q31(EGR1x2) 16 6 no CyR2 50 RA 46,XX,del(5)(q14q34)[1]/46,XX[6] 5q31(EGR1x2) 14 2 no CyR2 53 RA 46,XY,t(2;11)(q16;q24)[23]/46,idem,del(5)(q14q34)[2] 5q31(EGR1x2) 8 4 no CyR2 56 RA 46,XY,del(5)(q14q34)[4]/46,XY[21] 5q31(EGR1x2) 16 6 no CyR2 57 RA 46,XX,del(11)(q14)[3]/46,XX,del(5)(q13q34)[2]/46,XX[20] 5q31(EGR1x2) 8 0 no CyR2 59 RA 46,XX,add(1)(q31),del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 96 3 no CyR2 60 RA 46,XX,del(5)(q14q34)[2]/46,XX[24] 5q31(EGR1x2) 8 6 no CyR2 63 punctio sicca 46,XX,del(5)(q14q34)[4]/46,XX[13] 5q31(EGR1x2) 24 3 no CyR2 64 RA 46,XX,del(5)(q21q34)[3]/46,XX[22] 5q31(EGR1x2) 12 4 no CyR2 66 RA 46,XX,del(5)(q14q34)[3]/46,XX[23] 5q31(EGR1x2) 12 5 no CyR2 67 RA 46,XX,add(5)(q14)[1]/46,XX[13] 5q31(EGR1x2) 7 3 no CyR2 68 punctio sicca 46,XX,del(5)(q21q34)[1]/46,XX[24] 5q31(EGR1x2) 4 1 no CyR2 69 RA 46,XX,del(5)(q14q34)[3]/46,XX[22] 5q31(EGR1x2) 12 6 no CyR2 71 RA 46,XX,del(5)(q22q34)[4]/46,XX[21] 5q31(EGR1x2) 16 4 no CyR2 72 RARS 46,XX,del(5)(q21q34)[2]/46,XX[23] 5q31(EGR1x2) 8 7 no CyR2 74 RA 46,XX,del(5)(q14q34),del(13)(q12q14[1]/46,XX[24] 5q31(EGR1x2) 4 1 no CyR2 79 RA 46,XY,del(5)(q13q34)[4]/46,XY[14] 5q31(EGR1x2) 22 4 no CyR2 80 RA 46,XX,del(5)(q14q34)[2]/46,XX[18] 5q31(EGR1x2) 10 5 no CyR2 81 RA 46,XX,del(5)(q14q34)[14]/46,XX[11] 5q31(EGR1x2) 56 3 no CyR2 82 RA 46,XX,del(5)(q14q34)[8]/46,XX[7] 5q31(EGR1x2) 53 0 no CyR2 85 RARS 46,XY,del(5)(q14q34)[1]/46,XY[25] 5q31(EGR1x2) 4 0 no CyR2 87 RA 46,XX[3] 5q31(EGR1x1) 0 17 no CyR

MP: metaphases. IP: interphases. ISCN: International System for Human Cytogenetic Nomenclature. FISH: fluorescence in situ hybridization. RA: refractory anemia. RARS: RA with ring sideroblasts. n.e.:not evaluable. no CyR: no cytogenetic response. Bold: all discrepancies between karyotyping and FISH, including discrepancies in the detection of independent clones and clonal evolution. *Cytogenetic response was applied to karyotyping only. Complete cytogenetic response (CCyR) was defined as disappearance of the 5q deletion or any other chromosomal aberration. Partial cytogenetic response (PCyR) was defined as a reduction of aberrant metaphases of more than 50% compared to the previous karyotyping. Cytogenetic relapse was defined as reappearance of a metaphase with a deletion in 5q after reaching a complete cytogenetic response. At study entry, in 10 patients, del(5q) was detected only by karyotyping and not by FISH. Therefore, patients 1-10 would not have been eligible for treatment with lenalidomide, if only FISH had been used. In 2 patients, del(5q) was detected only by FISH and not by karyotyping. After 6 months of treatment, in 27 patients del(5q) was detected only by karyotyping and not by FISH. In three of

DOI: 10.3324/haematol.2010.026658

these patients, a clonal evolution occurred. Neither the clonal evolution nor the del(5q) clone would have been detected, if only FISH had been used. After 12 months of treatment, in 39 patients del(5q) was detected only by karyotyping. In one patient, del(5q) was detected only by FISH. In four patients, a clonal evolution occurred. In 6 patients, a cytogenetic relapse occurred at this time point. This relapse would not have been detected, if only FISH had been used.

DOI: 10.3324/haematol.2010.026658

Table 2. Discrepancy in detecting del(5q) by karyotyping and FISH in patients with MDS and del(5q) under at least 18 months (time point 3) of treatment with lenalidomide.

Time point Patient No FAB ISCN-Karyotyp ISCN FISH % MP 5q- % IP 5q- Response*3 14 RA 46,XX,del(5)(q21q34)[3]/46,XX[23] 5q31(EGR1x2) 12 7 relapse3 15 RARS 46,XX,del(5)(q14q34)[3]/46,XX[22] 5q31(EGR1x2) 12 0 relapse3 17 RA 46,XX,del(5)(q14q34)[1]/46,XX,[24] 5q31(EGR1x2) 4 4 relapse3 19 RA 46,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 3 relapse3 21 RA 46,XX,del(5)(q21q34)[10]/46,idem,del(13)(q12q14)[16] 5q31(EGR1x2) 100 1 relapse3 23 RA 46,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 0 relapse3 24 RA 46,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 3 relapse3 25 RA 46,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 3 relapse3 26 RA 46,XX,del(5)(q14q34)[3]/46,XX[29] 5q31(EGR1x2) 9 6 relapse3 30 RA 46,XX,del(20)(q12q13)[3]/46,XX,del(5)(q14q34)[2]/ 46,XX[20] 5q31(EGR1x2) 8 2 progress

3 31RA

46,XX,del(5)(q14q34),del(11)(q21)[8]/46,idem,?t(X;1)(q11;q36)[2]/47,idem, ?t(X;1)(q11;q36),+8[2]/46,XX[13] 5q31(EGR1x2) 48 4

progress

3 33 RA 46,XX,del(5)(q14q34)[2]/46,idem,del(13)(q13q21)[2]/ 46,XX[21] 5q31(EGR1x2) 8 6 progress3 34 RA 46,XY,del(5)(q14q34)[4]/46,XY,del(11)(q14)[2]/46,XY[19] 5q31(EGR1x2) 16 4 relapse, progress

3 37 RA46,XX,t(2;11)(p16;q23)[21]/46,idem,del(5)(q14q34)[2]/46,idem,der(18)t(9;18) (p10;q10)[2] 5q31(EGR1x2) 8 4

no CyR

3 38 RA 46,XY,del(5)(q14q34)[1]/46,XY[15] 5q31(EGR1x2) 6 8 no CyR3 40 RA 46,XX,del(5)(q14q34)[9]/46,XX[16] 5q31(EGR1x2) 36 4 no CyR3 44 RA 46,XX,inv(9)(p11q12)c[24]/46,idem,del(5)(q14q34)[1] 5q31(EGR1x2) 4 5 no CyR3 46 RA 46,XY,del(5)(q14q34)[3]/46,XY[22] 5q31(EGR1x2) 12 4 no CyR3 47 RA 46,XY,del(5)(q21q34)[10]/46,XY[15] 5q31(EGR1x2) 40 1 no CyR3 48 RA 45,X,-Y[4]/46,XY,del(5)(q21q34)[2]/46,XY[19] 5q31(EGR1x2) 8 5 no CyR3 50 RA 46,XX,del(5)(q14q34)[1]/46,XX[4] 5q31(EGR1x2) 20 3 no CyR3 52 RA 46,XX,del(5)(q14q34)[1]/46,XX[6] 5q31(EGR1x2) 14 3 no CyR3 53 RA 46,XY,t(2;11)(p16;q24)[24]/46,idem,del(5)(q14q34)[2] 5q31(EGR1x2) 8 6 no CyR3 54 RA 47,XX,del(5)(q13q34),+21[9]/46,XX[1] 5q31(EGR1x2) 90 2 no CyR3 55 RA 46,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 3 no CyR3 60 RA 46,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 0 no CyR3 63 punctio sicca 46,XX,del(5)(q14q34)[3]/46,XX[22] 5q31(EGR1x2) 12 4 no CyR3 66 RA 46,XX,del(5)(q14q34)[2]/46,XX[23] 5q31(EGR1x2) 8 1 no CyR3 67 RA 46,XX,del(5)(q14q34)[2]/46,XX[12] 5q31(EGR1x2) 14 2 no CyR3 68 punctio sicca 46,XX,del(5)(q21q34)[2]/46,XX[23] 5q31(EGR1x2) 8 3 no CyR3 71 RA 46,XX,del(5)(q22q34)[2]/46,XX[5] 5q31(EGR1x2) 29 3 no CyR3 72 RARS 46,XX,del(5)(q21q34)[4]/46,XX[21] 5q31(EGR1x2) 16 6 no CyR3 74 RA 46,XX,del(5)(q14q34),del(13)(q12q14)[3]/46,XX[22] 5q31(EGR1x2) 12 2 no CyR3 79 RA 46,XY,del(5)(q13q34)[1]/46,XY[25] 5q31(EGR1x2) 4 2 no CyR4 11 RA 46,XY,del(5)(q21q34)[2]/46,XY[23] 5q31(EGR1x2) 8 4 relapse4 12 RA 46,X,t(X;16)(p11;q23),del(5)(q21q34)[3]/46,XX[23] 5q31(EGR1x2) 12 4 relapse4 13 RA 46,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 5 relapse4 18 RA 46,XX,del(5)(q14q34)[3]/46,XX[22] 5q31(EGR1x2) 12 5 relapse4 20 RA 46,XX,del(5)(q15q34)[5]/46,XX[20] 5q31(EGR1x2) 20 7 relapse4 22 RARS 45,X,-Y[5]/46,XY,del(5)(q14q34)[2]/46,XY[20] 5q31(EGR1x2) 7 1 relapse4 23 RA 46,XX,del(5)(q14q34)[1]/46,idem,del(1)(p32p35)[1]/ 46,XX[23] 5q31(EGR1x2) 8 1 relapse4 24 RA 46,XX,del(5)(q14q34)[2]/46,XX[23] 5q31(EGR1x2) 8 4 relapse4 25 RA 46,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 1 relapse4 28 RARS 46,XX,del(5)(q14q34)[2]/46,XX[23] 5q31(EGR1x2) 8 2 relapse

4 30 RA46,XX,del(1)(p32p36),del(5)(q14q34)[11]/46,XX,del(20)(q12q13)[4]/46,XX[10] 5q31(EGR1x2) 44 3

progress

4 34 RA 46,XY,del(11)(q14)[5]/46,XY,del(5)(q14q34)[3]/46,XY[17] 5q31(EGR1x2) 12 6 relapse, progress4 38 RA 46,XY,del(5)(q21q34)[2]/46,XY[3] 5q31(EGR1x2) 40 6 no CyR4 42 RA 46,XX,del(5)(q21q34)[10]/46,XX[19] 5q31(EGR1x2) 34 2 no CyR4 47 RA 46,XY,del(5)(q14q34)[1]/46,XY[5] 5q31(EGR1x2) 17 5 no CyR4 48 RA 46,XY,del(5)(q21q34)[11]/45,X,-Y[2]/46,XY[12] 5q31(EGR1x2) 44 7 no CyR4 50 RA 46,XX,del(5)(q14q34)[3]/46,XX[22] 5q31(EGR1x2) 12 2 no CyR4 52 RA 46,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 2 no CyR4 60 RA 46,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 2 no CyR4 65 RA 46,XX,del(5)(q14q34)[10]/47,idem,+21[1]/46,XX[10] 5q31(EGR1x2) 52 6 no CyR4 66 RA 46,XX,del(5)(q14q34)[1]/46,XX[28] 5q31(EGR1x2) 3 4 no CyR4 67 RA 46,XX,del(5)(q14q34)[3]/46,XX[14] 5q31(EGR1x2) 18 0 no CyR4 68 punctio sicca 46,XX,del(5)(q21q34)[1]/46,XX[24] 5q31(EGR1x2) 4 4 no CyR4 78 RA 47,XX,+X[24]/47,idem,del(5)(q14q34)[1] 5q31(EGR1x2) 4 2 no CyR4 79 RA 92,XXYY,del(5)(q13q34)x2[1]/46,XY[4] 5q31(EGR1x2) 20 4 no CyR5 11 RA 46,XY,del(5)(q21q34)[2]/46,XY[23] 5q31(EGR1x2) 8 2 relapse5 12 RA 46,XX,t(X;16)(p11;q23),del(5)(q21q34)[5]/46,XX[20] 5q31(EGR1x2) 20 4 relapse5 13 RA 46,XX,del(5)(q14q34) [3]/46,XX[22] 5q31(EGR1x2) 12 4 relapse5 16 RA 46,XX,del(5)(q23q34)[20]/46,XX[5] 5q31(EGR1x2) 80 0 relapse5 24 RA 46,XX,del(5)(q14q34)[2]/46,XX[23] 5q31(EGR1x2) 8 5 relapse5 25 RA 46,XX,del(5)(q14q34)[2]/46,XX[23] 5q31(EGR1x2) 8 1 relapse

5 30 RA46,XX,del(1)(p32p36),del(5)(q14q34)[7]/46,idem,del (20)(q12q13)[1]/ 46,XX,del(20)(q12q13)[8]/46,XX[9] 5q31(EGR1x2) 32 1

progress

5 35 RA 46,XX,del(5)(q14q34),-7,-17,+mar[2]/ 46,XX[24] 5q31(EGR1x2) 8 0 relapse, progress

5 37 RA46,XX,t(2;11)(p16;q23)[3]/46,idem,-11,+der(11)t(2;11) (p16;q23)[20]/ 46,idem,del(5)(q14q34)[2] 5q31(EGR1x2) 8 6

no CyR

5 40 RA 46,XX,del(5)(q14q34)[2]/46,XX[23] 5q31(EGR1x2) 8 5 no CyR5 47 RA 46,XY,del(5)(q21q34)[10]/46,XY[12] 5q31(EGR1x2) 45 0 no CyR5 53 RA 46,XY,t(2;11)(p16;q24)[24]/46,idem,del(5)(q14q34)[1] 5q31(EGR1x2) 4 1 no CyR5 66 RA 46,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 1 no CyR5 67 RA 46,XX,del(5)(q14q34)[6]/46,XX[19] 5q31(EGR1x2) 24 5 no CyR5 68 punctio sicca 46,XX,del(5)(q21q34)[1]/46,XX[26] 5q31(EGR1x2) 4 1 no CyR5 87 RA 46,XX[3] 5q31(EGR1x1) 0 56 no response6 11 RA 46,XY,del(5)(q21q34)[2]/46,XY[24] 5q31(EGR1x2) 8 2 relapse6 16 RA 46,XX,del(5)(q23q34)[1]/46,XX[24] 5q31(EGR1x2) 4 2 relapse6 33 RA 46,XX,del(5)(q14q34)[2]/46,idem,del(13)(q13q21)[6]/ 46,XX[17] 5q31(EGR1x2) 8 3 progress

6 37 RA46,XX,t(2;11)(p16;q23)[3]/46,idem,-11,+der(11)t(2;11 (p16;q23)[20]/46,idem,del(5)(q14q34)[3] 5q31(EGR1x2) 12 2

no CyR

6 58 punctio sicca 46,XX,del(5)(q14q34)[5]/46,XX[20] 5q31(EGR1x2) 20 4 no CyR6 60 RA 46,XX,del(5)(q14q34)[2]/46,XX[23] 5q31(EGR1x2) 8 2 no CyR7 11 RA 46,XY,del(5)(q21q34)[4]/46,XY[21] 5q31(EGR1x2) 16 5 relapse

7 37 RA46,XX,t(2;11)(p16;q23),der(11)t(2;11)(p16;q23)[23]/ 46,idem,del(5)(q14q34), -der(11)t(2;11)(p16;q23)[2] 5q31(EGR1x2) 8 2

no CyR

8 62 RA 68 - 178,3 - 8n ,del(5)(q14q34),+21[cp2]/46,XX[9] 5q31(EGR1x2) 18 1 no CyR9 62 RA 47,XX,del(5)(q21q34),+21[1]/46,XX[24] 5q31(EGR1x2) 4 2 no CyR

MP: metaphases. IP: interphases. ISCN: International System for Human Cytogenetic Nomenclature. FISH: fluorescence in situ hybridization. RA: refractory anemia. RARS: RA with ring sideroblasts. n.e.: not evaluable. No CyR: no cytogenetic response. Bold: all discrepancies between karyotyping and FISH, including discrepancies in the detection of independent clones and clonal evolution.*Cytogenetic response was applied to karyotyping only. Complete cytogenetic response (CCyR) was defined as disappearance of the 5q deletion or any other chromosomal aberration. Partial cytogenetic response (PCyR) was defined as a reduction of aberrant metaphases of more than 50% compared to the previous karyotyping. Cytogenetic relapse was defined as reappearance of a metaphase with a deletion in 5q after reaching a complete cytogenetic response. In 85 cases, a discrepancy was detected. In only one patient was del(5q) detected by FISH only. In the other cases, del(5q) was detected by karyotyping only and not by FISH. In 31 cases, a cytogenetic relapse occurred and would not have been detected, if only FISH had been used. In 9 cases, a progress with a clonal evolution could be detected by karyotyping. Neither the clonal evolution nor the del(5q) clone would have been detected, if only FISH had been used.

DOI: 10.3324/haematol.2010.026658

Figure 1 (A) Karyogram of a patient with myelodysplastic syndrome and a deletion in

the long arm of chromosome 5 (fluorescence R-banding). (B) More detailed analysis

identified the deletion as an interstitial deletion. (C) Comparison with the ideogram of

chromosome 5 shows that the breakpoints are located in 5q14 and 5q34.

(D) Scheme of fluorescence in situ hybridization to detect a deletion in 5q. The probe

localized to the short arm of chromosome 5 (5p21) generates a green control signal.

The probe binding to the commonly deleted region 5q31 gives an orange signal.

(E) Interphase nucleus with a normal signal constellation, i.e. two green and two

orange signals. (F) Interphase nuclei with a signal constellation indicative of a

deletion in 5q, i.e. two green signals and one orange signal.

DOI: 10.3324/haematol.2010.026658

Figure 1

A

C c

D

F

B

E

A

C c

D

F

B

E

DOI: 10.3324/haematol.2010.026658

Supplementary Table 1

Patient No ISCN Karyotype ISCN FISH % MP 5q- % IP 5q-

1 46,XY,del(5)(q?14q34)[1]/46,XY[24] 5q31(EGR1x2) 4 146,XY[25] 5q31(EGR1x2) 0 346,XY[28] 5q31(EGR1x2) 0 2

2 46,XX,del(5)(q22q34)[4]/46,XX[21] 5q31(EGR1x2) 16 33 46,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 44 46,XX,del(5)(q14q34)[3]/46,XX[22] 5q31(EGR1x2) 12 6

46,XX,del(5)(q14q34)[24]/46,XX[1] 5q31(EGR1x1) 96 3446,XX,del(5)(q14q34)[11]/46,XX[14] 5q31(EGR1x2) 44 546,XX,del(5)(q14q34)[15]/46,XX[10] 5q31(EGR1x1) 60 1746,XX,del(5)(q14q34)[15]/46,XX[10] 5q31(EGR1x1) 60 2446,XX,del(5)(q14q34)[16]/46,XX[9] 5q31(EGR1x1) 64 3746,XX,del(5)(q14q34)[15]/46,idem,der(1)t(1;10)(p11;?),der(9)t(1;9)(?;?),der(10)t(9;10)(?;q21) [2]/46,XX[8] 5q31(EGR1x1) 68 3746,XX,del(5)(q14q34)[12]/46,idem,der(1)t(1;10)(p11;?),der(9)t(1;9)(?;?),der(10)t(9;10)(?;q21)/47,idem,der(1)t(1;10)(p11;?),+9,der(9)t(1;9)(?;?)x2,der(10)t(9;10)(?;q21)[2]/45,idem,der(1) t(1;10)(p11;?),-7,der(9),t(1;9)(?;?),der(10)t(9;10)(q;q21)[3]/46,idem,der(1)t(1;10)(p11;?),-7, der(9)t(1;9)(?;?),der(10)t(9;10)(?;q21),+mar[2]/46,XX[5] 5q31(EGR1x1) 80 4546,XX,del(5)(q14q34)[2]/46,idem,der(1)t(1;10)(p11;?),der(2)t(2;4)(p25;q26),inv(6)(p12q15), der(9)t(1;9)(?,?),der(10)t(9;10)(?;q21)[12]/44,idem,-7,dic(12;15)(p11;p11),-17,+mar[5]/ 46,XX[1] 5q31(EGR1x1) 95 35

5 47,XX,+8[4]/45,XX,del(5)(q1?4q34),-7[3]/46,XX[48] 5q31(EGR1x2) 5 46 46,XX,del(5)(q13q34)[1]/46,idem,add(20)(q13)[2] 5q31(EGR1x2) 100 2

46,XX,del(5)(q14q34)[21]/46,XX[4] 5q31(EGR1x1) 84 297 46,XX,del(5)(q23q34)[4]/46,XX[7] 5q31(EGR1x2) 36 6

46,XX,del(5)(q23q34)[20]/46,XX[5] 5q31(EGR1x1) 80 5846,XX,del(5)(q23q34)[1]/46,XX[2] 5q31(EGR1x1) 33 26no metaphases 5q31(EGR1x1) - 35

8 46,XY,del(5)(q?21q?34)[2]/46,XY[23] 5q31(EGR1x2) 8 546,XY,del(5)(q21q34)[1]/46,XY[19] 5q31(EGR1x2) 5 4

9 46,XY,del(5)(q21q34)[1]/46,XY[24] 5q31(EGR1x2) 4 646,XY,del(5)(q14q34)[15]/46,XY[9] 5q31(EGR1x1) 63 2646,XY,del(5)(q14q34)[1]/46,XY[24] 5q31(EGR1x2) 4 246,XY[25] 5q31(EGR1x2) 0 446,XY[14] 5q31(EGR1x2) 0 546,XY,del(5)(q21q34)[9]/46,idem,inv(1)(p12p35),t (1;4)(p36;q32)[3]/ 46,XY[15] 5q31(EGR1x1) 44 1846,XY,del(5)(q21q34)[3]/46,idem,inv(1)(p12p35),t (1;4)(p36;q32)[7]/ 46,XY[5] 5q31(EGR1x1) 67 1646,XY,del(5)(q21q34)[10]/46,idem,inv(1)(p12p35),t(1;4)(p36;q32)[12]/46,XY[3] 5q31(EGR1x1) 88 46

10 46,XY,del(5)(q14q34),inv(11)(p13~p14q22 q23)[18]/46,XY[7] 5q31(EGR1x2) 72 045,X,-Y[11]/46,XY[14] 5q31(EGR1x2) 0 046,XY,del(5)(q14q34),inv(11)(p13~p14q22 q23)[2]/ 46,XY[23] 5q31(EGR1x2) 8 445,X,-Y[11]/46,XY[14] 5q31(EGR1x2) 0 346,XY[25] 5q31(EGR1x2) 0 046,XY[25] 5q31(EGR1x2) 0 0

11 47,XY,del(5)(q21q34),+8[3]/46,XY[2] 5q31(EGR1x1) 60 7346,XY,del(5)(q21q34)[3]/46,XY[22] 5q31(EGR1x2) 12 446,XY,del(5)(q21q34)[3]/46,XY[22] 5q31(EGR1x1) 12 346,XY[20] 5q31(EGR1x2) 0 446,XY,del(5)(q21q34)[2]/46,XY[23] 5q31(EGR1x2) 8 446,XY,del(5)(q21q34)[2]/46,XY[23] 5q31(EGR1x2) 8 2

DOI: 10.3324/haematol.2010.026658

Supplementary Table 1

46,XY,del(5)(q21q34)[2]/46,XY[24] 5q31(EGR1x2) 8 246,XY,del(5)(q21q34)[4]/46,XY[21] 5q31(EGR1x2) 16 5

12 46,XX,t(X;16)(p11;q23),del(5)(q14q34)[23]/46,XX[2] 5q31(EGR1x1) 92 7346,XX,t(X;16)(p11;q23),del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 446,XX[25] 5q31(EGR1x2) 0 246,XX[25] 5q31(EGR1x2) 0 546,XX,t(X;16)(p11;q23),del(5)(q21q34)[3]/46,XX[23] 5q31(EGR1x2) 12 446,XX,t(X;16)(p11;q23),del(5)(q21q34)[5]/46,XX[20] 5q31(EGR1x2) 20 446,XX,t(X;16)(p11;q23),del(5)(q21q34)[10]/46,XX[15] 5q31(EGR1x1) 40 9

13 46,XX,del(5)(q14q34)[19]/46,XX[6] 5q31(EGR1x1) 76 71

46,XX,del(5)(q14q34)[1]/46.XX[14]no interphase nuclei 7 -

46,XX,del(5)(q14q34)[1]/46,XX[25] 5q31(EGR1x2) 4 446,XX[24] 5q31(EGR1x2) 0 346,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 546,XX,del(5)(q14q34) [3]/46,XX[22] 5q31(EGR1x2) 12 446,XX,del(5)(q14q34)[12]/46,XX[13] 5q31(EGR1x1) 48 18

14 46,XX,del(5)(q21q34)[11]/46,XX[5] 5q31(EGR1x1) 69 1246,XX,del(5)(q21q34)[2]/46,XX[23] 5q31(EGR1x2) 8 546,XX[26] 5q31(EGR1x2) 0 246,XX,del(5)(q21q34)[3]/46,XX[23] 5q31(EGR1x2) 12 746,XX,del(5)(q21q34)[9]/46,XX[16] 5q31(EGR1x1) 36 3846,XX,del(5)(q21q34)[19]/47,idem,+21[1]/46,XX[5] 5q31(EGR1x1) 80 36

15 46,XX,del(5)(q14q34)[15]/46,XX[6] 5q31(EGR1x1). 71 5946,XX,del(5)(q14q34)[1]/46,XX[19] 5q31(EGR1x2) 5 046,XX[20] 5q31(EGR1x2) 0 046,XX,del(5)(q14q34)[3]/46,XX[22] 5q31(EGR1x2) 12 0

16 46,XX,del(5)(q14q34)[20]/46,XX[5] 5q31(EGR1x1) 80 5947,XX,+14[2]/46,XX[23] 5q31(EGR1x2) 0 346,XX,del(5)(q23q34)[22]/46,XX[3] 5q31(EGR1x1) 8 7447,XX,+8[5]/46,XX,del(5)(q14q34)[2]/46,XX[18] 5q31(EGR1x1) 8 1146,XX[25] 5q31(EGR1x2) 0 146,XX,del(5)(q23q34)[20]/46,XX[5] 5q31(EGR1x2) 80 046,XX,del(5)(q23q34)[1]/46,XX[24] 5q31(EGR1x2) 4 2

17 46,XX,del(5)(q14q34)[20] 5q31(EGR1x1) 100 3745,XX,-7[16]/47,XX,+8[4]/46,XX,del(5)(q14q34)[3]/46,XX[2] 5q31(EGR1x1) 12 347,XX,+8[1]/46,XX[24] 5q31(EGR1x2) 0 046,XX,del(5)(q14q34)[1]/46,XX,[24] 5q31(EGR1x2) 4 446,XX,+1,der(1;7)(q10;p10)[5]/46,XX[20] 5q31(EGR1x2) 0 246,XX,+1,der(1;7)(q10;p10)[9]/46,XX[16] 5q31(EGR1x2) 0 346,XX,+1,der(1;7)(q10;p10)[12]/46,XX[13] 5q31(EGR1x2) 0 146,XX,+1,der(1;7)(q10;p10)[9]/46,XX[16] 5q31(EGR1x2) 0 2

18 no metaphasesno interphase nuclei - -

no metaphases 5q31(EGR1x2) - 546,XX[25] 5q31(EGR1x2) 0 346,XX[28] 5q31(EGR1x1) 0 446,XX,del(5)(q14q34)[3]/46,XX[22] 5q31(EGR1x2) 12 546,XX,del(5)(q14q34)[1]/46,XX[2] 5q31(EGR1x1) 33 25

19 46,XX,del(5)(q14q34)[14]/46,idem,del(12)(p12p13)[5]/ 46,XX[6] 5q31(EGR1x1) 76 62

DOI: 10.3324/haematol.2010.026658

Supplementary Table 1

46,XX,del(5)(q14q34)[6]/46,XX[19] 5q31(EGR1x1) 24 846,XX[25] 5q31(EGR1x2) 0 546,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 346,XX,del(5)(q14q34)[14]/46,idem,del(12)(p12p13)[5]/ 46,XX[6] 5q31(EGR1x1) 76 8

20 46,XX,del(5)(q15q34)[25] 5q31(EGR1x1) 100 8146,XX,del(5)(q15q34)[18]/46,XX[7] 5q31(EGR1x1) 72 4446,XX,del(5)(q15q34)[3]/46,XX[17] 5q31(EGR1x1) 15 1046,XX[25] 5q31(EGR1x2) 0 246,XX,del(5)(q15q34)[5]/46,XX[20] 5q31(EGR1x2) 20 746,XX,del(5)(q15q34)[13]/46,idem,del(17)(p12)[6]/ 46,XX[6] 5q31(EGR1x1) 76 2746,XX,del(5)(q15q34)[24]/46,idem,del(17)(p12)[1] 5q31(EGR1x1) 100 5846,XX,del(5)(q15q34)[25]/46,XX[1] 5q31(EGR1x1) 96 5246,XX,del(5)(q15q34)[23]/46,idem,del(17)(p12)[2]/ 46,XX[1] 5q31(EGR1x1) 96 4146,XX,del(5)(q15q34)[23]/46,XX[2] 5q31(EGR1x1) 92 64

21 46,XX,del(5)(q21q34)[10]/46,idem,del(13)(q12q14)[16] 5q31(EGR1x1) 100 8146,XX[7] 5q31(EGR1x2) 0 446,XX[23] 5q31(EGR1x2) 0 246,XX,del(5)(q21q34)[10]/46,idem,del(13)(q12q14)[16] 5q31(EGR1x2) 100 146,XX[7] 5q31(EGR1x2) 0 146,XX[25] 5q31(EGR1x2) 0 346,XX[25] 5q31(EGR1x2) 0 246,XX,del(5)(q21q34),del(13)(q12q14)[2]/46,XX[25] 5q31(EGR1x1) 7 8

22 46,XX,del(5)(q14q34)[8]/46,XX[17] 5q31(EGR1x1) 32 2245,X,-Y[5]/46,XY[23] 5q31(EGR1x2) 0 245,X,-Y[10]/46,XY[16] 5q31(EGR1x2) 0 145,X,-Y[5]/46,XY[21] 5q31(EGR1x2) 0 145,X,-Y[5]/46,XY,del(5)(q14q34)[2]/46,XY[20] 5q31(EGR1x2) 7 1

23 46,XX[2] 5q31(EGR1x1) 0 2146,XX[25] 5q31(EGR1x2) 0 146,XX,del(5)(q14q34)[2]/46,XX[23] 5q31(EGR1x2) 8 146,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 046,XX,del(5)(q14q34)[1]/46,idem,del(1)(p32p35)[1]/ 46,XX[23] 5q31(EGR1x2) 8 146,XX[26] 5q31(EGR1x2) 0 246,XX[25] 5q31(EGR1x2) 0 2

24 46,XX,del(5)(q14q34)[20]/46,XX[6] 5q31(EGR1x1) 77 6346,XX[25] 5q31(EGR1x2) 0 446,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 046,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 346,XX,del(5)(q14q34)[2]/46,XX[23] 5q31(EGR1x2) 8 446,XX,del(5)(q14q34)[2]/46,XX[23] 5q31(EGR1x2) 8 5

25 46,XX,del(5)(q14q34)[23]/46,XX[2] 5q31(EGR1x1) 92 5146,XX[3] 5q31(EGR1x2) 0 646,XX[25] 5q31(EGR1x2) 0 346,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 346,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 146,XX,del(5)(q14q34)[2]/46,XX[23] 5q31(EGR1x2) 8 146,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x1) 4 8

26 46,XX,del(5)(q14q34)[25] 5q31(EGR1x1) 100 6346,XX[25] 5q31(EGR1x2) 0 446,XX,del(5)(q14q34)[1]/46,XX[24] 5q31(EGR1x2) 4 3

DOI: 10.3324/haematol.2010.026658

Supplementary Table 1

46,XX,del(5)(q14q34)[3]/46,XX[29] 5q31(EGR1x2) 9 627 46,XX,del(5)(q21q34)[7] 5q31(EGR1x1) 100 64

46,XX[6] 5q31(EGR1x2) 0 246,XX,del(5)(q21q34)[1]/46,XX[24] 5q31(EGR1x2) 4 1

no metaphasesno interphase nuclei - -

46,XX[25] 5q31(EGR1x2) 0 246,XX[27] 5q31(EGR1x2) 0 546,XX[14] 5q31(EGR1x2) 0 1

28 46,XX,del(5)(q14q34)[3]/46,XX[23] 5q31(EGR1x1) 12 846,XX[25] 5q31(EGR1x2) 0 046,XX[9] 5q31(EGR1x2) 0 246,XX[24] 5q31(EGR1x2) 0 446,XX,del(5)(q14q34)[2]/46,XX[23] 5q31(EGR1x2) 8 2

DOI: 10.3324/haematol.2010.026658

Supplementary Table 2

Patient No ISCN Karyotype ISCN FISH % MP 5q- % IP 5q-29 46,XX,del(5)(q14q34)[10]/47,idem,+21[4] 5q31(EGR1x1) 100 42

46,XX,del(5)(q14q34)[11]/50,idem,+idic(21)(q22)x5[1]/ 46,XX[13] 5q31(EGR1x1) 48 12

46,XX,del(5)(q14q34)[1]/48-50,idem,+21,+21,+idic(21)(q21q22), +mar[cp4]/46,XX[20] 5q31(EGR1x2) 20 6

30 46,XX,del(5)(q14q34)[24]/46,XX[1] 5q31(EGR1x1) 96 7247,XX,+8[7]/46,XX,del(20)(q12q13)[4]/46,XX,del(5)(q14q34)[2]/ 46,XX,idem,del(20)(q12q13)[1]/46,XX[11] 5q31(EGR1x2) 12 246,XX,del(20)(q12q13)[6]/46,XX,del(5)(q14q34)[1]/ 47,XX,+8[1]/ 46,XX[14] 5q31(EGR1x2) 5 446,XX,del(20)(q12q13)[3]/46,XX,del(5)(q14q34)[2]/ 46,XX[20] 5q31(EGR1x2) 8 246,XX,del(1)(p32p36),del(5)(q14q34)[11]/46,XX,del(20)(q12q13) [4]/ 46,XX[10] 5q31(EGR1x2) 44 346,XX,del(1)(p32p36),del(5)(q14q34)[7]/46,idem,del (20)(q12q13)[1]/ 46,XX,del(20)(q12q13)[8]/46,XX[9] 5q31(EGR1x2) 32 1

31 46,XX,del(5)(q14q34)[21]/46,XX[4] 5q31(EGR1x1) 84 4547,XX,+8[2]/46,XX,del(5)(q14q34)[1]/46,XX[22] 5q31(EGR1x2) 12 2

46,XX,del(5)(q14q34)[1]/46,idem,del(11)(q21)[2]/46,XX[22] 5q31(EGR1x2) 12 346,XX,del(5)(q14q34),del(11)(q21)[8]/46,idem,?t(X;1)(q11;q36)[2]/47,idem,?t(X;1)(q11;q36),+8[2]/46,XX[13] 5q31(EGR1x2) 48 446,XX,del(5)(q14q34),del(11)(q21)[6]/47,idem,t(X;1)(q11;q36), +8[16]/46,idem,del(12)(p12p13)[1]/46,XX[2] 5q31(EGR1x1) 92 82

32 46,XX,del(5)(q1?3q34)[22] 5q31(EGR1x1) 100 6046,XX,del(5)(q13q34)[2]/46,idem,dup(1)(q21q32)[4]/ 47,XX,+8[5]/ 46,XX[14] 5q31(EGR1x2) 24 7

33 no metaphasesno interphase nuclei - -

46,XX,del(5)(q?14q34)[1] 5q31(EGR1x1) 100 2746,XX,del(5)(q14q34)[3]/46,XX[3] 5q31(EGR1x2) 50 446,XX,del(5)(q14q34)[2]/46,idem,del(13)(q13q21)[2]/ 46,XX[21] 5q31(EGR1x2) 8 646,XX[27] 5q31(EGR1x2) 0 346,XX,del(5)(q14q34)[6]/46,idem,del(13)(q13q21)[4]/ 46,XX[15] 5q31(EGR1x1) 40 1246,XX,del(5)(q14q34)[2]/46,idem,del(13)(q13q21)[6]/ 46,XX[17] 5q31(EGR1x2) 8 3

34 46,XY,del(5)(q14q34)[23]/46,XY[2] 5q31(EGR1x1) 92 8146,XY,del(5)(q14q34)[25] 5q31(EGR1x1) 100 8946,XY[25] 5q31(EGR1x2) 0 546,XY,del(5)(q14q34)[4]/46,XY,del(11)(q14)[2]/46,XY[19] 5q31(EGR1x2) 16 446,XY,del(11)(q14)[5]/46,XY,del(5)(q14q34)[3]/46,XY[17] 5q31(EGR1x2) 12 6

35 46,XX,del(5)(q21q34)[6] 5q31(EGR1x1) 100 6846,XX,del(5)(q21q34)[21] 5q31(EGR1x1) 100 5946,XX[25] 5q31(EGR1x2) 0 146,XX[25] 5q31(EGR1x2) 0 046,XX[25] 5q31(EGR1x2) 0 245,XX,del(5)(q),-7,-17,+mar[2]/ 46,XX[24] 5q31(EGR1x2) 8 044,XX,del(5)(q21q34),-7,idic(8;?17)(p11;p11),del(12)(p12p13), 18[24]/46,XX[1] 5q31(EGR1x1) 96 31

36 46,XY,del(5)(q14q34)[8]/46,XY[5] 5q31(EGR1x1) 62 1345,X,-Y[19]/ 46,XY[7] 5q31(EGR1x2) 0 545,X,-Y[13]/46,XY,del(5)(q14q34)[5]/ 46,idem,del(13)(q14q31)[4]/ 46,XY[3] 5q31(EGR1x2)

36 2

DOI: 10.3324/haematol.2010.026658