Leukemia Research 38 (2014) 170–175
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Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Clonal evolution in chronic lymphocytic leukemia detected by fluorescence in situ hybridization and conventional cytogenetics after stimulation with CpG oligonucleotides and interleukin-2: A prospective analysis Martin Brejcha a , Martina Stoklasová b , Yvona Brychtová c , Anna Panovská c , c ˇ epanovská c , Gabriela Vanková ˇ Kristina Stˇ , Karla Plevová c,d , Alexandra Oltová c , b ˇ c,d Kateˇrina Horká , Sárka Pospíˇsilová , Jiˇrí Mayer c,d , Michael Doubek c,d,∗ a
Department of Hematology, Hospital Novy Jicin, Czech Republic Laboratory of Medical Genetics – Department of Cytogenetics, AGEL Research and Training Institute – Novy Jicin Branch, AGEL Laboratories, Czech Republic Department of Internal Medicine – Hematology and Oncology, University Hospital, Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic d Central European Institute of Technology, Masaryk University, Brno, Czech Republic b c
a r t i c l e
i n f o
Article history: Received 31 August 2013 Received in revised form 20 October 2013 Accepted 22 October 2013 Available online 29 October 2013 Keywords: Chronic lymphocytic leukemia Clonal evolution Cytogenetics CpG oligonucleotides Fluorescence in situ hybridization Interleukin-2
a b s t r a c t Chronic lymphocytic leukemia (CLL) patients may acquire new chromosome abnormalities during the course of their disease. Clonal evolution (CE) has been detected by conventional chromosome banding (CBA), several groups also confirmed CE with fluorescence in situ hybridization (FISH). At present, there are minimal prospective data on CE frequency determined using a combination of both methods. Therefore, the aim of our study was to prospectively assess CE frequency using a combination of FISH and CBA after stimulation with CpG oligonucleotides and interleukin-2. Between 2008 and 2012, we enrolled 140 patients with previously untreated CLL in a prospective trial evaluating CE using FISH and CBA after stimulation. Patients provided baseline and regular follow-up peripheral blood samples for testing. There was a median of 3 cytogenetic examinations (using both methods) per patient. CE was detected in 15.7% (22/140) of patients using FISH, in 28.6% (40/140) using CBA, and in 34.3% (48/140) of patients by combining both methods. Poor-prognosis CE (new deletion 17p, new deletion 11q or new complex karyotype) was detected in 15% (21/140) of patients and was significantly associated with previous CLL treatment (p = 0.013). CBA provides more complex information about cytogenetic abnormalities in CLL patients than FISH and confirms that many patients can acquire new abnormalities during the course of their disease in a relatively short time period. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction The clinical course of chronic lymphocytic leukemia (CLL) is remarkably heterogeneous. Approximately half of CLL patients never require treatment; however, there is a group of patients with a very poor prognosis and a median overall survival (OS) of 1–2 years regardless of repeated therapies [1–4]. A significant prognostic factor correlating with the clinical course of CLL and OS is the presence of some chromosomal aberrations [5,6]. At present, fluorescent in situ hybridization (FISH) is the standard method used
∗ Corresponding author at: University Hospital and Masaryk University, Jihlavská 20, 62500 Brno, Czech Republic. Tel.: +420 532 233 642; fax: +420 532 233 603. E-mail address: [email protected] (M. Doubek). 0145-2126/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2013.10.019
to detect genomic aberrations in CLL [6]; however, this method is not able to detect all chromosomal changes which can also play an important role in CLL pathogenesis. Conventional chromosome banding analysis (CBA) has not been used in CLL as malignant Blymphocytes did not proliferate in vitro [5]. Nevertheless, after introducing the new CLL cell stimulation methodology with CpG oligonucleotides and interleukin-2 (IL-2) [7], detecting chromosomal aberrations using CBA has become possible in up to 80% of CLL cases [8,9]. It is of importance that additional aberrations modifying the CLL patients’ prognostic stratification (prognosis defined according to FISH) can be detected using CBA [10]. Chromosomal translocations are found in up to 34% of CLL cases [10]. Although the original concept considered CLL as a genetically stable disease [11], clonal evolution (CE) with the acquisition of new cytogenetic aberrations occurs in 17–26% of patients during the course of the disease
M. Brejcha et al. / Leukemia Research 38 (2014) 170–175
171
Table 1 Baseline patients’ characteristics and clinical data at the start of the study. All patients (n = 140) Median age (years) 64 (37–75) 86/54 Male/female ratio Rai stage 36 (25.7%) 0 50 (35.7%) I 8 (5.7%) II 19 (13.6%) III IV 27 (19.3%) IGHV mutational status (n = 135) 60 (42.9%) Mutated Unmutated 75 (53.6%) CD38 107 (76.4%) Negative Positive 33 (23.6%) ZAP-70 (n = 129) 60 (42.9%) Negative 69 (49.3%) Positive Therapy before CE 54 No 86 Yes
Patients with poor prognosis CE (n = 21)
Patients without poor prognosis CE (n = 119)
p-Value (poor prognosis CE vs. no poor prognosis CE)
64 (38–75) 15/6
63 (37–73) 71/48
NS NS
5 6 1 4 5
31 44 7 15 22
NS
8 13
52 62
NS
15 6
92 27
NS
6 13
54 56
0.157
3 18
51 68
0.013
CE, clonal evolution; poor prognosis CE, clonal evolution with new poor prognosis abnormalities detected during the follow-up (new 17p deletion, new 11q deletion, or new complex karyotype); NS, not significant.
[12–18]. CE is associated with shorter OS and a new finding of deletion 17p is important to determine a treatment strategy [19–21]. In the majority of published analyses to date, CE has been monitored using FISH [13–18]. Therefore, the aim of the present study was to prospectively assess CE frequency using the combination of both FISH and CBA after stimulating with CpG oligonucleotides and IL-2 in CLL patients. We compared these methods and evaluated how frequently new adverse cytogenetic findings occurred when correlated with treatment and other prognostic factors (therapy, variable part of the immunoglobulin heavy chain [IGHV] gene mutation status, CD38 and ZAP-70 expression). 2. Patients and methods 2.1. Patients Between 2008 and 2012, we enrolled 140 patients with previously untreated CLL in a prospective trial evaluating CE by FISH and CBA. In cases enrolled more than 1 year after CLL diagnosis, FISH data from previous tests were available. The patients provided baseline and follow-up (every 12 months in untreated patients with the stable disease or earlier, before each therapy, in treated patients) peripheral blood samples for testing. All blood samples were processed with written informed consent in accordance with the Declaration of Helsinki under protocols approved by the local ethical committee.
2.2. FISH FISH analyses were performed on the interphase nuclei of cultured peripheral blood cells using a panel of DNA probes to detect prognostically significant aberrations: deletion 13q and trisomy 12 (LSI D13S319/LSI 13q34/CEP 12, Vysis, Downers Grove, IL, USA; or XL DLEU/LAMP/12cen and XL DLEU/LAMP/12cen MetaSystems, Altlussheim, Germany), deletion 11q and deletion 17p (LSI p53/LSI ATM, Vysis, Downers Grove, IL, USA; or XL ATM/p53 probe, MetaSystems, Altlussheim, Germany), rearrangement 14q32 (LSI IgH DC Break Apart Probe; Vysis, Downers Grove, IL, USA). Two hundred interphase nuclei were evaluated. The cut-off level for each individual probe was determined based on a negative sample analysis and calculated as the mean +3SD. Chromosomal aberrations were categorized according to Döhner’s hierarchical model [6].
Cytogenetic Nomenclature (ISCN 2009). Complex aberrations were assessed using M-FISH and M-BAND methods (MetaSystems, Altlussheim, Germany). 2.4. Mutation status of IGHV The IGHV gene’s mutation status was examined using polymerase chain reaction (PCR) followed by PCR amplicon sequencing. The sequences obtained were analyzed using an IMGT/V-QUEST tool and database. The unmutated IGHV gene was defined using sequence identity to germ-line sequence ≥98%; the mutated IGHV gene was defined by identity 12?::6q22->6q24::3p?->3p?::1q42->1qter),der(6)(8qter->8q2?::6p12>6q1?::12q?->12q?::6q1?->6q22:),der(8)t(8;12)(q24;p?),−12[5]/46,XY[17] 44,XY,−4,−6,der(19)t(6;19)(q13;q13),der(19)t(4;19)(q21;q13)[16]/44,XY,−6,der(10)t(10;15)(p14;q12),−15,der(19)t(6;19) (q13;q13),der(19)t(Y;19)(?;q13)[3]/46,XY[10]
M/70
4/2009 7/2011
II I
Chl
Norm. Norm.
46,XY[23] 46,XY,del(12)(q23),der(14)t(12;14)(q23;q22)[7]/46,XY[54]
F/53
12/2008 2/2010
IV IV
FC
13q− 11q−,13q−
46,XX[31] 46,XX,del(11)(q22q24)[13]/46,XX[7]
M/64
6/2009 6/2011
0 I
No
Norm. 13q−
47,XY,+21[3]/46,XY[29] 47,XY,+21[2]/46,XY,del(13)(q14)[1]/46,XY,der(1)t(1;13)(q12;q13),del(13)(q14)[6]/46,XY,der(6)t(6;20)(q?;q12),del(6)(q12), t(7;8;19)(p15.?;p21.?;q12),der(14)ins(6;14)(q?;q21q24)[6]/46,XY[3]
M/50
1/2009 3/2010
I I
FCR
Norm. 13q−
46,XY[31] 46,XY,der(8)t(8;13)(p11.2;q21),der(13)del(13)(q14)t(8;13)(p11.2;q21)[12]/46,XY,t(1;8)(p34;p11.2),t(7;22)(q11.2;q11.2) [2]/45,X,−Y,t(1;8)(p34;p11.2),t(7;22)(q11.2;q11.2),der(10)t(Y;10)(?;p11.2)[5]/45,XY,t(1;8)(p34;p11.2)t(7;22)(q11.2;q11.2), der(10)t(10;18)(p11.2;q11.2),−18[3]/46,XY[7]
F/60
2/2008 1/2012
0 III
RCOP
13q− 13q−
46,X,inv(X)(p11.4q21)c,del(13)(q14.3q31)[20]/46,X,inv(X)(p11.4q21)c[5] 46,X,inv(X)(p11.4q21)c,t(1;5)(p3?3q35)[2]/46,X,inv(X)(p11.4q21)c,del(13)(q14.3q31)[7]/46,X,inv(X)(p11.4q21)c[12]
F/75
2/2009 3/2010
I III
FC
Norm. Norm.
45,XX,der(3)t(3;4)(q28;q12)[4]/46,XX[30] 45,XX,der(3)t(3;4)(q28;q12),der(4)del(4)(p12)del(q12)[4]/46,XX[23]
M/60
2/2008 6/2012
I III
Norm. 17p−
46,XY[22] 45,XY,dic(8;17)(p11.1;p11.1)[5]/45,X,−Y[3]/46,XY[33]
M/66
1/2010 1/2011
0 III
+12 +12,13q−
47,XY,+12[8]/46,XY[4] 47,XY,+12,t(18;22)(q21;q11)[10]/47,XY,der(3)t(3;13)(p?21;q14),del(13)(q14),+12,t(18;22)(q21;q11)[10]/46,XY,t(13;14) (q21;q31),t(18;22)(q21;q11)[4]/46,XY[6]
F/60
5/2008 3/2011
0 0
FCR
Norm. 11q−
46,XX,i(4)(q10)[25]/46,XX[2] 46,XX,i(4)(q10)[14]/46,XX[7]
M/61
12/2008 4/2010
IV IV
FC
Norm. 17p,11q
46,XY[30] 44,XY,−4,der(17;18)t(17;18)(p13;p11.3)t(4;17)(q27;q25)[12]/44,XY,−4,der(6)t(6;11)(q23;q23),der(11)del(11)(q22.3)t(6;11) (q23;q22.3),der(17;18)t(17;18)t(4;17)[3]/46,XY[12]
M/59
2/2008 8/2010
III II
FCR
Norm. 11q,13q
46,XY,t(7;8)(q34;p12)[11]/46,XY[21] 46,XY,t(7;8)(q34;p12)[15]/46,XY,del(11)(q14q23)[11]/3.klon:46,XY[6]
M/69
1/2009 4/2010
I I
RCHOP
Norm. Norm.
46,XY[30] 47,XY,t(1;5)(q21;p14),+der(3)t(3;18)(p12;q21)[16]/46,XY[15]
M/66
12/2008 1/2011
IV 0
FCR
13q− 17p−,13q−
46,XY[30] 45,XY,der(6)t(6;13)(?q23;q31),del(13)(q14),dic(17;18)(p11.2;q11.1)[10]/45,XY,dic(17;18)(p11.2;q11.1)[27]/46,XY[11]
M/38
FCR
17p−
FCR and Rdexa FCR
M. Brejcha et al. / Leukemia Research 38 (2014) 170–175
FISH result
Therapy before poor-prognosis CE
173
0 8/2010
Chl, chlorambucil; FC, fludarabine and cyclophosphamide regimen; FCR, rituximab, fludarabine, cyclophosphamide regimen; R-COP, rituximab, cyclophosphamide, vincristin, prednisone regimen; R-CHOP, rituximab, doxorubicin, vincristin, prednisone regimen; R-dexa, rituximab and dexamethasone regimen.
47,XX,+12,t(14;19)(q32;q13)[19]/47,XX,idem,der(13)t(11;13)(q?;q12)[6]/47,XX,idem,der(6)t(5;6)(q?;q16)[3]/48,XX,idem, del(6)(q16),der(13)t(11;13)(q?;q12),+21[2] 46,XX,+12,dic(12;17)(p12;p11),t(14;19)(q32;q13),der(17)t(1;17)(q21;q25)[26]/46,XX,der(5)t(5;12)(q11;?q)del(12)(?q), t(14;19)(q32;q13),der(17)t(1;17)(q21;q25),der(17)t(5;17)(q11;p11)[5] +12,13q− F/52
8/2008
III
FCR
17p−,+12
46,XY,del(11)(q22q23)[3]/46,XY[27] 46,XY,t(4;7;13)(p14;q22;q13)[11]/46,XY,der(11)t(11;17)(q21;?q24)[3]/46,XY[39] 11q−,13q− 11q−,13q− FCR 1/2009 1/2011 M/69
III 0/I
47,XY,+21[4]/46,XY[26] 47,XY,+21[1]/46,XY[25] 13q− 17p−,13q− Rdexa 6/2009 5/2011 M/69
IV IV
46,XY,der(18)t(2;18)(p12;p1?)[12]/46,XY[10] 46,XY,der(18)t(2;18)(p12;p11.3)[14]/46,XY,del(13)(q14q21),der(18)t(2;18)(p12;p11.3)[6]/46,XY[2] Norm. 13q− No 6/2008 5/2011 M/69
0 I
Date (first analysis date/date of poor-risk CE) Patient (gender/age)
Table 4 (Continued)
Rai stage
Therapy before poor-prognosis CE
CBA result
M. Brejcha et al. / Leukemia Research 38 (2014) 170–175
FISH result
174
ZAP-70 positivity or IGHV mutation status, whereas Berková et al. [15] reported a link between poor-prognosis CE and positivity of all these factors. In our analysis, no statistically significant correlation between poor-prognosis CE was found in relation to the prognostic markers monitored (IGHV mutational status, ZAP-70, CD38). In our study, poor-prognosis CE was significantly associated only with previous CLL treatment. According to valid guidelines for CLL management [20], patients should be treated only in cases where there is clear evidence of disease progression. In addition to other factors, therapy could select resistant CLL clones [2,4,17,22]. Our results strongly confirm these findings. Nevertheless, some authors did not prove a significant negative influence of treatment on CE [15,18]. Absence of therapy, however, does not exclude poorprognosis CE [14,16]. It is possible that a higher poor-prognosis CE frequency in pre-treated patients in our study may not only be related to chemotherapy, but may also be related to the fact that patients who underwent treatment had a more advanced disease. Shanafelt et al. [13] reported that the risk of CE increases with the duration of follow-up with the median time of new cytogenetic abnormality acquirement 7.8 (range 1.9–11) years from the first FISH examination. In other published studies, the median time of CE occurrence was 40–64 months [14,17,18]. In all these studies, only FISH was used to detect cytogenetic abnormalities. In our study, the median time to CE occurrence was shorter: 16 (range 6–52) months. This shorter time to detect CE could be explained by the regularity of cytogenetic examinations and by using a combination of two methods for these analyses: FISH and CBA. In our analysis, the median time for a new aberration occurring was shorter using CBA than FISH. Moreover, our results show that CBA after stimulating with CpG oligonucleotides and IL-2 significantly assisted in increasing the number of chromosomal abnormalities detected in CLL patients at diagnosis and also during follow-up. When comparing both methods, FISH can reveal cryptic deletions in cases with a loss of submicroscopic amounts of genetic material escaping detection using CBA. On the other hand CBA allows the detection of additional changes which are not routinely examined by FISH, and makes it possible to detect cases with complex karyotype. 5. Conclusions This clinical trial confirms prospectively that the spectrum of cytogenetic abnormalities detected by CBA after stimulation with CpG oligonucleotides and IL-2 is broader than with FISH analysis, suggesting that many patients (more than in studies published to date) acquire new abnormalities during the course of their disease. Treated patients are at a higher risk of experiencing CE. These findings have important implications for the clinical management of CLL patients. Conflict of interest The authors have had costs of participating in certain scientific meetings reimbursed by the pharmaceutical industry. Acknowledgements Supported in part by Research Grants MSMT CR CZ.1.05/1.1.00/02.0068 (CEITEC), MSMT CR CZ.1.07/2.3.00/20.0045 (SuPReMMe), MSM0021622430; by the European Commission under the Health Theme of the 7th Framework Programme for Research and Technological Development GA 306242; by the grant from the Internal Grant Agency, Ministry of Health, Czech Republic, NT13493-4/2012; by the grant MUNI/A/0723/2012, and by the Czech Leukemia Study Group – for Life (CELL). The authors
M. Brejcha et al. / Leukemia Research 38 (2014) 170–175
thank Dr. Matthew Smith and David P. Figgitt PhD, Content Ed Net, for providing editorial assistance. Contributions. MB was involved in the design, acquisition of data, data interpretation, and manuscript writing. MS, KS, GV, AO and KH were involved in cytogenetic analyses. YB and AP were involved in data acquisition. SP was involved in data analysis and manuscript revision. JM was involved in conception, design, data interpretation and manuscript revision. MD was involved in conception, design, data acquisition, data interpretation, and manuscript writing. All authors read and approved the final manuscript.
[10]
[11] [12] [13]
[14]
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Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Clonal evolution in chronic lymphocytic leukemia detected by fluorescence in situ hybridization and conventional cytogenetics after stimulation with CpG oligonucleotides and interleukin-2: A prospective analysis Martin Brejcha a , Martina Stoklasová b , Yvona Brychtová c , Anna Panovská c , c ˇ epanovská c , Gabriela Vanková ˇ Kristina Stˇ , Karla Plevová c,d , Alexandra Oltová c , b ˇ c,d Kateˇrina Horká , Sárka Pospíˇsilová , Jiˇrí Mayer c,d , Michael Doubek c,d,∗ a
Department of Hematology, Hospital Novy Jicin, Czech Republic Laboratory of Medical Genetics – Department of Cytogenetics, AGEL Research and Training Institute – Novy Jicin Branch, AGEL Laboratories, Czech Republic Department of Internal Medicine – Hematology and Oncology, University Hospital, Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic d Central European Institute of Technology, Masaryk University, Brno, Czech Republic b c
a r t i c l e
i n f o
Article history: Received 31 August 2013 Received in revised form 20 October 2013 Accepted 22 October 2013 Available online 29 October 2013 Keywords: Chronic lymphocytic leukemia Clonal evolution Cytogenetics CpG oligonucleotides Fluorescence in situ hybridization Interleukin-2
a b s t r a c t Chronic lymphocytic leukemia (CLL) patients may acquire new chromosome abnormalities during the course of their disease. Clonal evolution (CE) has been detected by conventional chromosome banding (CBA), several groups also confirmed CE with fluorescence in situ hybridization (FISH). At present, there are minimal prospective data on CE frequency determined using a combination of both methods. Therefore, the aim of our study was to prospectively assess CE frequency using a combination of FISH and CBA after stimulation with CpG oligonucleotides and interleukin-2. Between 2008 and 2012, we enrolled 140 patients with previously untreated CLL in a prospective trial evaluating CE using FISH and CBA after stimulation. Patients provided baseline and regular follow-up peripheral blood samples for testing. There was a median of 3 cytogenetic examinations (using both methods) per patient. CE was detected in 15.7% (22/140) of patients using FISH, in 28.6% (40/140) using CBA, and in 34.3% (48/140) of patients by combining both methods. Poor-prognosis CE (new deletion 17p, new deletion 11q or new complex karyotype) was detected in 15% (21/140) of patients and was significantly associated with previous CLL treatment (p = 0.013). CBA provides more complex information about cytogenetic abnormalities in CLL patients than FISH and confirms that many patients can acquire new abnormalities during the course of their disease in a relatively short time period. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction The clinical course of chronic lymphocytic leukemia (CLL) is remarkably heterogeneous. Approximately half of CLL patients never require treatment; however, there is a group of patients with a very poor prognosis and a median overall survival (OS) of 1–2 years regardless of repeated therapies [1–4]. A significant prognostic factor correlating with the clinical course of CLL and OS is the presence of some chromosomal aberrations [5,6]. At present, fluorescent in situ hybridization (FISH) is the standard method used
∗ Corresponding author at: University Hospital and Masaryk University, Jihlavská 20, 62500 Brno, Czech Republic. Tel.: +420 532 233 642; fax: +420 532 233 603. E-mail address: [email protected] (M. Doubek). 0145-2126/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2013.10.019
to detect genomic aberrations in CLL [6]; however, this method is not able to detect all chromosomal changes which can also play an important role in CLL pathogenesis. Conventional chromosome banding analysis (CBA) has not been used in CLL as malignant Blymphocytes did not proliferate in vitro [5]. Nevertheless, after introducing the new CLL cell stimulation methodology with CpG oligonucleotides and interleukin-2 (IL-2) [7], detecting chromosomal aberrations using CBA has become possible in up to 80% of CLL cases [8,9]. It is of importance that additional aberrations modifying the CLL patients’ prognostic stratification (prognosis defined according to FISH) can be detected using CBA [10]. Chromosomal translocations are found in up to 34% of CLL cases [10]. Although the original concept considered CLL as a genetically stable disease [11], clonal evolution (CE) with the acquisition of new cytogenetic aberrations occurs in 17–26% of patients during the course of the disease
M. Brejcha et al. / Leukemia Research 38 (2014) 170–175
171
Table 1 Baseline patients’ characteristics and clinical data at the start of the study. All patients (n = 140) Median age (years) 64 (37–75) 86/54 Male/female ratio Rai stage 36 (25.7%) 0 50 (35.7%) I 8 (5.7%) II 19 (13.6%) III IV 27 (19.3%) IGHV mutational status (n = 135) 60 (42.9%) Mutated Unmutated 75 (53.6%) CD38 107 (76.4%) Negative Positive 33 (23.6%) ZAP-70 (n = 129) 60 (42.9%) Negative 69 (49.3%) Positive Therapy before CE 54 No 86 Yes
Patients with poor prognosis CE (n = 21)
Patients without poor prognosis CE (n = 119)
p-Value (poor prognosis CE vs. no poor prognosis CE)
64 (38–75) 15/6
63 (37–73) 71/48
NS NS
5 6 1 4 5
31 44 7 15 22
NS
8 13
52 62
NS
15 6
92 27
NS
6 13
54 56
0.157
3 18
51 68
0.013
CE, clonal evolution; poor prognosis CE, clonal evolution with new poor prognosis abnormalities detected during the follow-up (new 17p deletion, new 11q deletion, or new complex karyotype); NS, not significant.
[12–18]. CE is associated with shorter OS and a new finding of deletion 17p is important to determine a treatment strategy [19–21]. In the majority of published analyses to date, CE has been monitored using FISH [13–18]. Therefore, the aim of the present study was to prospectively assess CE frequency using the combination of both FISH and CBA after stimulating with CpG oligonucleotides and IL-2 in CLL patients. We compared these methods and evaluated how frequently new adverse cytogenetic findings occurred when correlated with treatment and other prognostic factors (therapy, variable part of the immunoglobulin heavy chain [IGHV] gene mutation status, CD38 and ZAP-70 expression). 2. Patients and methods 2.1. Patients Between 2008 and 2012, we enrolled 140 patients with previously untreated CLL in a prospective trial evaluating CE by FISH and CBA. In cases enrolled more than 1 year after CLL diagnosis, FISH data from previous tests were available. The patients provided baseline and follow-up (every 12 months in untreated patients with the stable disease or earlier, before each therapy, in treated patients) peripheral blood samples for testing. All blood samples were processed with written informed consent in accordance with the Declaration of Helsinki under protocols approved by the local ethical committee.
2.2. FISH FISH analyses were performed on the interphase nuclei of cultured peripheral blood cells using a panel of DNA probes to detect prognostically significant aberrations: deletion 13q and trisomy 12 (LSI D13S319/LSI 13q34/CEP 12, Vysis, Downers Grove, IL, USA; or XL DLEU/LAMP/12cen and XL DLEU/LAMP/12cen MetaSystems, Altlussheim, Germany), deletion 11q and deletion 17p (LSI p53/LSI ATM, Vysis, Downers Grove, IL, USA; or XL ATM/p53 probe, MetaSystems, Altlussheim, Germany), rearrangement 14q32 (LSI IgH DC Break Apart Probe; Vysis, Downers Grove, IL, USA). Two hundred interphase nuclei were evaluated. The cut-off level for each individual probe was determined based on a negative sample analysis and calculated as the mean +3SD. Chromosomal aberrations were categorized according to Döhner’s hierarchical model [6].
Cytogenetic Nomenclature (ISCN 2009). Complex aberrations were assessed using M-FISH and M-BAND methods (MetaSystems, Altlussheim, Germany). 2.4. Mutation status of IGHV The IGHV gene’s mutation status was examined using polymerase chain reaction (PCR) followed by PCR amplicon sequencing. The sequences obtained were analyzed using an IMGT/V-QUEST tool and database. The unmutated IGHV gene was defined using sequence identity to germ-line sequence ≥98%; the mutated IGHV gene was defined by identity 12?::6q22->6q24::3p?->3p?::1q42->1qter),der(6)(8qter->8q2?::6p12>6q1?::12q?->12q?::6q1?->6q22:),der(8)t(8;12)(q24;p?),−12[5]/46,XY[17] 44,XY,−4,−6,der(19)t(6;19)(q13;q13),der(19)t(4;19)(q21;q13)[16]/44,XY,−6,der(10)t(10;15)(p14;q12),−15,der(19)t(6;19) (q13;q13),der(19)t(Y;19)(?;q13)[3]/46,XY[10]
M/70
4/2009 7/2011
II I
Chl
Norm. Norm.
46,XY[23] 46,XY,del(12)(q23),der(14)t(12;14)(q23;q22)[7]/46,XY[54]
F/53
12/2008 2/2010
IV IV
FC
13q− 11q−,13q−
46,XX[31] 46,XX,del(11)(q22q24)[13]/46,XX[7]
M/64
6/2009 6/2011
0 I
No
Norm. 13q−
47,XY,+21[3]/46,XY[29] 47,XY,+21[2]/46,XY,del(13)(q14)[1]/46,XY,der(1)t(1;13)(q12;q13),del(13)(q14)[6]/46,XY,der(6)t(6;20)(q?;q12),del(6)(q12), t(7;8;19)(p15.?;p21.?;q12),der(14)ins(6;14)(q?;q21q24)[6]/46,XY[3]
M/50
1/2009 3/2010
I I
FCR
Norm. 13q−
46,XY[31] 46,XY,der(8)t(8;13)(p11.2;q21),der(13)del(13)(q14)t(8;13)(p11.2;q21)[12]/46,XY,t(1;8)(p34;p11.2),t(7;22)(q11.2;q11.2) [2]/45,X,−Y,t(1;8)(p34;p11.2),t(7;22)(q11.2;q11.2),der(10)t(Y;10)(?;p11.2)[5]/45,XY,t(1;8)(p34;p11.2)t(7;22)(q11.2;q11.2), der(10)t(10;18)(p11.2;q11.2),−18[3]/46,XY[7]
F/60
2/2008 1/2012
0 III
RCOP
13q− 13q−
46,X,inv(X)(p11.4q21)c,del(13)(q14.3q31)[20]/46,X,inv(X)(p11.4q21)c[5] 46,X,inv(X)(p11.4q21)c,t(1;5)(p3?3q35)[2]/46,X,inv(X)(p11.4q21)c,del(13)(q14.3q31)[7]/46,X,inv(X)(p11.4q21)c[12]
F/75
2/2009 3/2010
I III
FC
Norm. Norm.
45,XX,der(3)t(3;4)(q28;q12)[4]/46,XX[30] 45,XX,der(3)t(3;4)(q28;q12),der(4)del(4)(p12)del(q12)[4]/46,XX[23]
M/60
2/2008 6/2012
I III
Norm. 17p−
46,XY[22] 45,XY,dic(8;17)(p11.1;p11.1)[5]/45,X,−Y[3]/46,XY[33]
M/66
1/2010 1/2011
0 III
+12 +12,13q−
47,XY,+12[8]/46,XY[4] 47,XY,+12,t(18;22)(q21;q11)[10]/47,XY,der(3)t(3;13)(p?21;q14),del(13)(q14),+12,t(18;22)(q21;q11)[10]/46,XY,t(13;14) (q21;q31),t(18;22)(q21;q11)[4]/46,XY[6]
F/60
5/2008 3/2011
0 0
FCR
Norm. 11q−
46,XX,i(4)(q10)[25]/46,XX[2] 46,XX,i(4)(q10)[14]/46,XX[7]
M/61
12/2008 4/2010
IV IV
FC
Norm. 17p,11q
46,XY[30] 44,XY,−4,der(17;18)t(17;18)(p13;p11.3)t(4;17)(q27;q25)[12]/44,XY,−4,der(6)t(6;11)(q23;q23),der(11)del(11)(q22.3)t(6;11) (q23;q22.3),der(17;18)t(17;18)t(4;17)[3]/46,XY[12]
M/59
2/2008 8/2010
III II
FCR
Norm. 11q,13q
46,XY,t(7;8)(q34;p12)[11]/46,XY[21] 46,XY,t(7;8)(q34;p12)[15]/46,XY,del(11)(q14q23)[11]/3.klon:46,XY[6]
M/69
1/2009 4/2010
I I
RCHOP
Norm. Norm.
46,XY[30] 47,XY,t(1;5)(q21;p14),+der(3)t(3;18)(p12;q21)[16]/46,XY[15]
M/66
12/2008 1/2011
IV 0
FCR
13q− 17p−,13q−
46,XY[30] 45,XY,der(6)t(6;13)(?q23;q31),del(13)(q14),dic(17;18)(p11.2;q11.1)[10]/45,XY,dic(17;18)(p11.2;q11.1)[27]/46,XY[11]
M/38
FCR
17p−
FCR and Rdexa FCR
M. Brejcha et al. / Leukemia Research 38 (2014) 170–175
FISH result
Therapy before poor-prognosis CE
173
0 8/2010
Chl, chlorambucil; FC, fludarabine and cyclophosphamide regimen; FCR, rituximab, fludarabine, cyclophosphamide regimen; R-COP, rituximab, cyclophosphamide, vincristin, prednisone regimen; R-CHOP, rituximab, doxorubicin, vincristin, prednisone regimen; R-dexa, rituximab and dexamethasone regimen.
47,XX,+12,t(14;19)(q32;q13)[19]/47,XX,idem,der(13)t(11;13)(q?;q12)[6]/47,XX,idem,der(6)t(5;6)(q?;q16)[3]/48,XX,idem, del(6)(q16),der(13)t(11;13)(q?;q12),+21[2] 46,XX,+12,dic(12;17)(p12;p11),t(14;19)(q32;q13),der(17)t(1;17)(q21;q25)[26]/46,XX,der(5)t(5;12)(q11;?q)del(12)(?q), t(14;19)(q32;q13),der(17)t(1;17)(q21;q25),der(17)t(5;17)(q11;p11)[5] +12,13q− F/52
8/2008
III
FCR
17p−,+12
46,XY,del(11)(q22q23)[3]/46,XY[27] 46,XY,t(4;7;13)(p14;q22;q13)[11]/46,XY,der(11)t(11;17)(q21;?q24)[3]/46,XY[39] 11q−,13q− 11q−,13q− FCR 1/2009 1/2011 M/69
III 0/I
47,XY,+21[4]/46,XY[26] 47,XY,+21[1]/46,XY[25] 13q− 17p−,13q− Rdexa 6/2009 5/2011 M/69
IV IV
46,XY,der(18)t(2;18)(p12;p1?)[12]/46,XY[10] 46,XY,der(18)t(2;18)(p12;p11.3)[14]/46,XY,del(13)(q14q21),der(18)t(2;18)(p12;p11.3)[6]/46,XY[2] Norm. 13q− No 6/2008 5/2011 M/69
0 I
Date (first analysis date/date of poor-risk CE) Patient (gender/age)
Table 4 (Continued)
Rai stage
Therapy before poor-prognosis CE
CBA result
M. Brejcha et al. / Leukemia Research 38 (2014) 170–175
FISH result
174
ZAP-70 positivity or IGHV mutation status, whereas Berková et al. [15] reported a link between poor-prognosis CE and positivity of all these factors. In our analysis, no statistically significant correlation between poor-prognosis CE was found in relation to the prognostic markers monitored (IGHV mutational status, ZAP-70, CD38). In our study, poor-prognosis CE was significantly associated only with previous CLL treatment. According to valid guidelines for CLL management [20], patients should be treated only in cases where there is clear evidence of disease progression. In addition to other factors, therapy could select resistant CLL clones [2,4,17,22]. Our results strongly confirm these findings. Nevertheless, some authors did not prove a significant negative influence of treatment on CE [15,18]. Absence of therapy, however, does not exclude poorprognosis CE [14,16]. It is possible that a higher poor-prognosis CE frequency in pre-treated patients in our study may not only be related to chemotherapy, but may also be related to the fact that patients who underwent treatment had a more advanced disease. Shanafelt et al. [13] reported that the risk of CE increases with the duration of follow-up with the median time of new cytogenetic abnormality acquirement 7.8 (range 1.9–11) years from the first FISH examination. In other published studies, the median time of CE occurrence was 40–64 months [14,17,18]. In all these studies, only FISH was used to detect cytogenetic abnormalities. In our study, the median time to CE occurrence was shorter: 16 (range 6–52) months. This shorter time to detect CE could be explained by the regularity of cytogenetic examinations and by using a combination of two methods for these analyses: FISH and CBA. In our analysis, the median time for a new aberration occurring was shorter using CBA than FISH. Moreover, our results show that CBA after stimulating with CpG oligonucleotides and IL-2 significantly assisted in increasing the number of chromosomal abnormalities detected in CLL patients at diagnosis and also during follow-up. When comparing both methods, FISH can reveal cryptic deletions in cases with a loss of submicroscopic amounts of genetic material escaping detection using CBA. On the other hand CBA allows the detection of additional changes which are not routinely examined by FISH, and makes it possible to detect cases with complex karyotype. 5. Conclusions This clinical trial confirms prospectively that the spectrum of cytogenetic abnormalities detected by CBA after stimulation with CpG oligonucleotides and IL-2 is broader than with FISH analysis, suggesting that many patients (more than in studies published to date) acquire new abnormalities during the course of their disease. Treated patients are at a higher risk of experiencing CE. These findings have important implications for the clinical management of CLL patients. Conflict of interest The authors have had costs of participating in certain scientific meetings reimbursed by the pharmaceutical industry. Acknowledgements Supported in part by Research Grants MSMT CR CZ.1.05/1.1.00/02.0068 (CEITEC), MSMT CR CZ.1.07/2.3.00/20.0045 (SuPReMMe), MSM0021622430; by the European Commission under the Health Theme of the 7th Framework Programme for Research and Technological Development GA 306242; by the grant from the Internal Grant Agency, Ministry of Health, Czech Republic, NT13493-4/2012; by the grant MUNI/A/0723/2012, and by the Czech Leukemia Study Group – for Life (CELL). The authors
M. Brejcha et al. / Leukemia Research 38 (2014) 170–175
thank Dr. Matthew Smith and David P. Figgitt PhD, Content Ed Net, for providing editorial assistance. Contributions. MB was involved in the design, acquisition of data, data interpretation, and manuscript writing. MS, KS, GV, AO and KH were involved in cytogenetic analyses. YB and AP were involved in data acquisition. SP was involved in data analysis and manuscript revision. JM was involved in conception, design, data interpretation and manuscript revision. MD was involved in conception, design, data acquisition, data interpretation, and manuscript writing. All authors read and approved the final manuscript.
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