Manuscript.doc Click here to view linked References

Manuscript.doc Click here to view linked References

Leukemia Research, Original Article

The Origin of Deletion 22q11 in Chronic Lymphocytic Leukemia Is Related to the Rearrangement of Immunoglobulin Lambda Light Chain Locus

1,2,*

Marek Mraz

, Katerina Stano Kozubik

2

1,2,*

, Karla Plevova

3

1,2,*

1,2

1,2

1,2

, Katerina Musilova , Boris Tichy , 2

1,2

Marek Borsky , Petr Kuglík , Michael Doubek , Yvona Brychtova , Jiri Mayer , Sarka Pospisilova 1 2

1,2

CEITEC, Center of Molecular Medicine, Masaryk University, Brno, Czech Republic Dept. of Hematology and Oncology, University Hospital Brno and Faculty of Medicine Masaryk

University, Brno, Czech Republic 3

Dept. of Genetics and Molecular Biology, Institute of Experimental Biology, Faculty of Science,

Masaryk University, Brno, Czech Republic *

These authors contributed equally to the study

Correspondence: Sarka Pospisilova, Ph.D. Dept. of Hematology and Oncology, University Hospital Brno Cernopolni 9, 625 00, Brno, Czech Republic Tel.: +420-532234622 Fax: +420-532234623 [email protected]

Keywords: CLL, 22q11, immunoglobulin lambda light chain locus, IGL, IGLV, PRAME Word count: 2300 words

1

Abstract

The technology of array comparative genomic hybridization (array-CGH) enabled the identification of novel genomic aberrations in chronic lymphocytic leukemia (CLL) including the monoallelic and biallelic deletions affecting 22q11 locus. In contrast to previous publications, we hypothesized that the described 22q11 deletions are a consequence of the rearrangement of immunoglobulin lambda light chain locus (IGL) segments surrounding several protein-coding genes located in this region. Indeed, using array-CGH and PCR analysis we show that all deletions (n=7) affecting the 22q11 locus in our cohort (n=40) are based on the physiological mechanism of IGL rearrangement. This demonstrates that this loss of genetic material is likely not pathogenic and in fact is merely a marker of IGL rearrangement.

2

Introduction

Chromosomal aberrations examined by classical karyotype analysis or fluorescent in situ hybridization (FISH) can be detected in ≥80% of chronic lymphocytic leukemia (CLL) patients. Deletions of 13q14, 17p13, 11q23, and trisomy 12 are frequently used to define prognosis of CLL patients

1-3

. The precise

molecular analysis of these regions allowed for the elucidation of the affected genes (like miR-15a-161, TP53, ATM) and understanding of their functions in CLL pathogenesis and/or progression

2,4

.

Recently, array comparative genomic hybridization (array-CGH) enabled the identification of other 5-8

genomic aberrations in CLL cells with potential pathogenetic relevance

.

Significantly, several groups have performed array-CGH analysis in large cohorts of CLL patients and described the monoallelic and biallelic deletions of chromosome 22q11 in CLL largest study by Gunn and colleagues

9

7,9-11

. The

identified these deletions in 15% of CLL cases (28/187) and

analyzed their size in detail. In that study, 22q11 deletion was the second most frequent abnormality after 13q deletion in 187 CLL cases screened by BAC array-CGH. The subsequent breakpoint mapping based on Agilent Human Genome 44K CGH Arrays led to the description of minimally deleted region (0.34Mb) containing protein-coding genes PRAME (preferentially expressed antigen in melanoma), GGTLC2 (gamma-glutamyltransferase light chain 2), ZNF280A (zinc finger protein 280A), 9

and ZNF280B (zinc finger protein 280B) . Authors suggested that the PRAME gene is the candidate 9

tumor-suppressor localized in 22q11 , because it was previously associated with the biology and aggressiveness of both solid tumors and myeloid hematological malignancies

12-15

.

The repeated description of cases with aberrations in 22q11 locus prompted us to characterize in detail such deletions and their possible consequences on gene expression. This genomic region contains the segments (subgenes) for immunoglobulin lambda light chain (IGL) together with above mentioned protein coding genes and one microRNA gene (miR-650)

16

. This is a unique feature of IGL

locus, because immunoglobulin kappa light chain locus (IGK) or heavy chain locus (IGH) does not contain any protein-coding genes or microRNAs. Recently, we reported that the microRNA gene (miR650) located in this locus can be activated by specific rearrangement of IGL

16

. However, in contrast to

previous authors, we hypothesized that the described deletions at 22q11 are a consequence of the rearrangement of IGL segments surrounding these protein-coding genes. Indeed, we show that all deletions (n=7) affecting the 22q11 locus in our cohort (n=40) are based on the physiological mechanism of IGL rearrangement.

Methods CLL patients and sample characterization The study was performed on peripheral blood samples from 40 CLL patients monitored at the Department of Internal Medicine – Hematology and Oncology, University Hospital Brno. CLL diagnosis was based on standard immunophenotypic criteria

17

, and all samples were processed with written

informed consent in accordance with the Declaration of Helsinki under protocols of the University Hospital Brno. Peripheral blood mononuclear cells (PBMC) were isolated using Histopaque-1077 (Sigma-Aldrich). In samples with lower proportion of CLL cells, enrichment for B-lymphocytes was

3

performed by RosetteSep B Cell Enrichment Cocktail (StemCell Technologies, Vancouver, Canada). Leukemic cell proportion (CD5+CD19+) was determined by flow cytometry and exceeded 90% in all samples. The expression of immunoglobulin light chain on B cells was determined by flow cytometry (mouse Anti-IGL/IGK; Invitrogen, Carlsbad, CA, USA). I-FISH for del(13)(q14), del(11)(q22), del(17)(p13) and trisomy 12 was performed using locusspecific probes from Abbott Vysis, Inc. (Illinois, USA) according to manufacturer instructions. At least 200 interphase nuclei per slide were evaluated using a LUCIA KARYO/FISH/CGH imaging system (Laboratory Imaging, Prague, Czech Rep.). The 5% cut-off level for deletion detection was determined by analysis of 15 samples obtained from healthy bone marrow donors.

Immunoglobulin Locus Rearrangement Immunoglobulin gene PCR amplification and direct sequencing were used to analyze the immunoglobulin heavy chain IGH rearrangements as described previously

18,19

. The utilized

immunoglobulin light chain (kappa vs. lambda) and the exact IGL variable (V) segment were determined as previously described rearrangements

were

analyzed

18,20

using

ImMunoGeneTics database, www.imgt.org)

. The obtained sequences of immunoglobulin gene

IMGT

V-QUEST

database

and

tools

(International

19,21-23

.

Array-CGH Total DNA isolated (Dneasy Blood & Tissue Kit, Qiagen) from CLL samples was fragmented, labeled and hybridized to array-CGH (CGH Microarray 4x44 K, Agilent) according to manufacturer’s instructions and compared to human reference DNA (Promega, Madison, WI, USA) as described 8

previously . The arrays were scanned by DNA Microarray Scanner (Agilent Technologies Santa Clara, CA, USA) and data generated and checked for quality by Feature Extraction software, v 9.5 (Agilent Technologies). Data were subsequently analyzed by CGH Analytics software v. 3.4 (Agilent Technologies). Criteria for quality were set as DLR (derivative log ratio) standard deviation lower than 0.3 log unit and signal to background ration greater than 30 for each color. We utilized the Aberration Detection Method 1 (ADM-1, cut-off 7.0) to identify losses and gains of genetic material. All identified regions were subsequently checked manually and loss/gain of at least 3 probes was necessary to consider a region as aberrant.

Gene expression analysis Total RNA was isolated by TRI-Reagent (MRC Inc, Cincinnati, OH, USA)

24

, and gene expression

(PRAME, ZNF280A, ZNF280B, GSTT1) was analyzed by commercially available TaqMan Assays (Applied Biosystems, Foster City, CA, USA). Geometric mean of GAPDH cycle threshold (Ct) values served as a normalization gene. Sequence Detection Software (version 1.3.1; Applied Biosystems) was used to analyze the fluorescence emission data after the Real-Time PCR, and results were subjected to 2-∆∆Ct analysis. The differences in gene expression were tested by a non-parametric Mann-Whitney U-test (GraphPad Prism v. 5.00, GraphPad Software, San Diego, CA).

4

Results Detection of 22q11 deletion by array-CGH

We analyzed genomes of 40 CLL patients using array-CGH and I-FISH (del(13)(q14), del(11)(q22), del(17)(p13), +12), and in particular focused on detailed characterization of 22q11 deletion. Using array-CGH (n=40), additional aberrations to routinely performed FISH were found in 88% of samples. The results of the FISH and array-CGH analysis are summarized in Table 1. Deletion of 22q11 locus was the most frequent additional aberration observed by array-CGH present in 17.5% (7/40) of patients (CLL 1-7 in Table 1). Deletion size was ranging from 0.34 Mb up to 0.77 Mb. However, the precise size of the deletions could not be exactly determined due to a relatively low density of the probes on the CGH Microarray 4x44K in this region. Immunoglobulin light chain locus rearrangement leads to “deletions” in 22q11

We next analyzed the usage of immunoglobulin light chain by PCR detection (see methods) and Sanger sequencing in patients with del(22)(q11) detected by array-CGH (n=7). We found that all patients with deletion detected by array-CGH at 22q11 had a rearrangement of the immunoglobulin lambda light chain locus. The identification of kappa vs. lambda light chain by PCR was concordant with flow cytometry for surface immunoglobulin light chain expression in all cases. This strongly suggests that the deletion is a consequence of IGL rearrangement. Indeed, the breakpoint region of these aberrations corresponded to the consequence of a productive VJ rearrangement that created an in-frame Complementarity Determining Region (CDR3) responsible for antigen recognition in 6 out of 7 cases. The characterization of the IGL rearrangements and their alignment according to the IGMT database is depicted in Table 2 and Fig. 1. Figure 2 illustrates that 5 out of the 7 samples had a rearrangement leading to the deletion of protein-coding genes ZNF280A, ZNF280B, and PRAME (CLL 2-6), whereas 2 patients had a rearrangement utilizing IGL segments that are more proximal to the JC cluster (CLL 1 and 7). We then determined the usage of IGL variable (V) segments in all other patients (n=33) that had an intact 22q11 region according to array-CGH results. We observed that certain patients also had an IGL rearrangement (n=8, CLL8-16 in Table 1). In these cases, the loss of genetic material was not reliably detected by array-CGH, because these CLL B cells utilized some of the V segments that are proximal to the J-C cluster and/or the deletion of the probe(s) did not fulfill the criteria for an aberration as defined by our algorithm. Altogether in our cohort, rearrangements of 22q11 locus involving protein-coding genes (i.e. PRAME/ZNF280A/ZNF280B) constituted 12.5% (n=5), and 25% of patients (n=10) harbored smaller rearrangement of IGL not involving protein-coding genes. Loss of genetic material at 22q11 is frequently detected in CLL patients, but our data in particular demonstrates that it is caused by the physiological process of immunoglobulin lambda light chain locus rearrangement. As a result, we believe that it is not suitable to define any kind of minimally deleted region (MDR).

5

The expression of protein coding genes localized in 22q11 locus

Gunn et al. demonstrated that the deletion of protein-coding genes (PRAME, ZNF280A and ZNF280B) at 22q11 is associated with significant down-regulation of their expression compared to samples without 22q11 deletion. We tested this observation by performing Real-Time PCR for PRAME, ZNF280A, and ZNF280B genes. In our cohort (n=23) we compared gene expression in samples with rearrangement of lambda light chain at 22q11 locus (n=13) to samples that express kappa light chain (IGK) and do not have the rearrangement of IGL (n=10). We included 23 samples from the initial cohort from which material was available for RNA isolation. The group with lambda light chain rearrangement included 5 samples (CLL 2-6 in Table 1) that had a deletion involving protein coding genes and 8 samples with the IGL rearrangement not affecting these genes (CLL1,7,8-13 in Table 1). Figure 3 indicates that there is no tendency for differential expression of PRAME, ZNF280A, and ZNF280B in samples with their deletion (for all genes p>0.15 for comparison: deletion of PRAME/ZNF280A/ZNF280B vs. no deletion PRAME/ZNF280A/ZNF280B). Moreover, all genes had very high Ct values (median Ct value: 38.0, 38.0, 30.4 for PRAME, ZNF280A, and ZNF280B respectively) reflecting their very weak expression in CLL B cells.

Discussion This study identifies the origin of 22q11 deletion that was repeatedly described in CLL

7,9-11

. We

demonstrate that deletion of 22q11 is frequently detected in CLL patients, but it is caused by the physiological process of immunoglobulin lambda light chain locus rearrangement rather than driven by the selection pressure against protein-coding genes in this region. This implies that they are likely not pathogenic and are in fact only a marker of IGL rearrangement. In the most recent study, Kolquist et al. identified 17% of patients harboring deletion in 22q11.23 region

11

. All these deletions, identified by 135K-feature whole genome microarray

(Signature OncoChip), were more distant from telomere, and authors claimed that it seemed not to overlap with the deletion described by Gunn et al. All cases identified in this study were deletions of 49kb to 56kb that included GSTT1 gene, which is a member of the glutathione S-transferase family. However, due to a relatively low coverage in this region, we believe that authors cannot exclude the possibility that these events are also a consequence of IGL rearrangement. The status of the immunoglobulin lambda light chain locus was not analyzed or commented in that study. We did not observe any such deletion outside the IGL cluster in our cohort. Moreover, the expression of GSTT1 did not differ between cases with large rearrangement versus small rearrangement of IGL in our cohort (Fig. 4). Presented data recognizes that it is unlikely that described 22q11 deletions are directly linked to the development of leukemia, because identical rearrangements are occurring in normal B cells during the IGLV-IGLJ-IGLC recombination. However, we have recently demonstrated that a microRNA gene located in 22q11 region (miR-650) is regulated by the promoter of several IGL V segments and influences the biology of CLL B-cells. This microRNA can be deleted or activated based on IGL

6

rearrangement, and signaling properties of IGL/IGK in general can be important for the biology of chronic lymphocytic leukemia

16,25,26

. The discovery of loss of genetic material at 22q11 can be

alarming in non B cells but an expected phenomenon in 1/3 of all B cells, because approximately this proportion of B cells has a monoallelic or biallelic rearrangement of the lambda light chain for immunoglobulin

27-29

. A similar rule also applies for losses in the IGK and IGH regions that can be 30

observed in CLL samples by array-CGH as noted previously . In summary, “chasing” the potential gene coding a tumor-suppressor in 22q11 locus would likely not bring results relevant for CLL pathogenesis. Moreover, protein-coding genes that might be deleted during physiological rearrangement of IGL are not differentially expressed in cases with such loss of DNA material.

7

References

1. Dohner H, Stilgenbauer S, Benner A, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 2000;343:1910-1916. 2. Zenz T, Mertens D, Kuppers R, Dohner H, Stilgenbauer S. From pathogenesis to treatment of chronic lymphocytic leukaemia. Nat Rev Cancer. 2010;10:37-50. 3. Trbusek M, Smardova J, Malcikova J, et al. Missense mutations located in structural p53 DNA-binding motifs are associated with extremely poor survival in chronic lymphocytic leukemia. J Clin Oncol. 2011;29:2703-2708. 4. Mraz M, Pospisilova S, Malinova K, Slapak I, Mayer J. MicroRNAs in chronic lymphocytic leukemia pathogenesis and disease subtypes. Leuk Lymphoma. 2009;50:506-509. 5. Ouillette P, Collins R, Shakhan S, et al. Acquired genomic copy number aberrations and survival in chronic lymphocytic leukemia. Blood. 2011;118:30513061. 6. Ouillette P, Erba H, Kujawski L, Kaminski M, Shedden K, Malek SN. Integrated genomic profiling of chronic lymphocytic leukemia identifies subtypes of deletion 13q14. Cancer Res. 2008;68:1012-1021. 7. Pfeifer D, Pantic M, Skatulla I, et al. Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays. Blood. 2007;109:1202-1210. 8. Stano-Kozubik K, Malcikova J, Tichy B, et al. Inactivation of p53 and amplification of MYCN gene in a terminal lymphoblastic relapse in a chronic lymphocytic leukemia patient. Cancer Genet Cytogenet. 2009;189:53-58. 9. Gunn SR, Bolla AR, Barron LL, et al. Array CGH analysis of chronic lymphocytic leukemia reveals frequent cryptic monoallelic and biallelic deletions of chromosome 22q11 that include the PRAME gene. Leuk Res. 2009;33:1276-1281. 10. Tyybakinoja A, Vilpo J, Knuutila S. High-resolution oligonucleotide array-CGH pinpoints genes involved in cryptic losses in chronic lymphocytic leukemia. Cytogenet Genome Res. 2007;118:8-12. 11. Kolquist KA, Schultz RA, Slovak ML, et al. Evaluation of chronic lymphocytic leukemia by oligonucleotide-based microarray analysis uncovers novel aberrations not detected by FISH or cytogenetic analysis. Mol Cytogenet. 2011;4:25. 12. Oehler VG, Guthrie KA, Cummings CL, et al. The preferentially expressed antigen in melanoma (PRAME) inhibits myeloid differentiation in normal hematopoietic and leukemic progenitor cells. Blood. 2009;114:3299-3308. 13. Qin Y, Zhu H, Jiang B, et al. Expression patterns of WT1 and PRAME in acute myeloid leukemia patients and their usefulness for monitoring minimal residual disease. Leuk Res. 2009;33:384-390. 14. Santamaría C, Chillón MC, García-Sanz R, et al. The relevance of preferentially expressed antigen of melanoma (PRAME) as a marker of disease activity and prognosis in acute promyelocytic leukemia. Haematologica. 2008;93:1797-1805. 15. Steinbach D, Hermann J, Viehmann S, Zintl F, Gruhn B. Clinical implications of PRAME gene expression in childhood acute myeloid leukemia. Cancer Genet Cytogenet. 2002;133:118-123. 16. Mraz M, Dolezalova D, Plevova K, et al. MicroRNA-650 expression is influenced by immunoglobulin gene rearrangement and affects the biology of chronic lymphocytic leukemia. Blood. 2012;119:2110-2113. 17. Hallek M, Cheson BD, Catovsky D, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop 8

on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood. 2008;111:5446-5456. 18. van Dongen JJ, Langerak AW, Brüggemann M, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia. 2003;17:2257-2317. 19. Agathangelidis A, Darzentas N, Hadzidimitriou A, et al. Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies. Blood. 2012;119:4467-4475. 20. Langerak AW, Davi F, Ghia P, et al. Immunoglobulin sequence analysis and prognostication in CLL: guidelines from the ERIC review board for reliable interpretation of problematic cases. Leukemia. 2011;25:979-984. 21. Lefranc MP. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. 2003;31:307-310. 22. Yousfi Monod M, Giudicelli V, Chaume D, Lefranc MP. IMGT/JunctionAnalysis: the first tool for the analysis of the immunoglobulin and T cell receptor complex V-J and V-D-J JUNCTIONs. Bioinformatics. 2004;20 Suppl 1:i379385. 23. Giudicelli V, Brochet X, Lefranc MP. IMGT/V-QUEST: IMGT standardized analysis of the immunoglobulin (IG) and T cell receptor (TR) nucleotide sequences. Cold Spring Harb Protoc. 2011;2011:695-715. 24. Mraz M, Malinova K, Mayer J, Pospisilova S. MicroRNA isolation and stability in stored RNA samples. Biochemical and Biophysical Research Communications. 2009;390:1-4. 25. Hadzidimitriou A, Darzentas N, Murray F, et al. Evidence for the significant role of immunoglobulin light chains in antigen recognition and selection in chronic lymphocytic leukemia. Blood. 2009;113:403-411. 26. Kostareli E, Sutton LA, Hadzidimitriou A, et al. Intraclonal diversification of immunoglobulin light chains in a subset of chronic lymphocytic leukemia alludes to antigen-driven clonal evolution. Leukemia. 2010;24:1317-1324. 27. Hieter PA, Korsmeyer SJ, Waldmann TA, Leder P. Human immunoglobulin kappa light-chain genes are deleted or rearranged in lambda-producing B cells. Nature. 1981;290:368-372. 28. van der Burg M, Barendregt BH, Szczepański T, van Wering ER, Langerak AW, van Dongen JJ. Immunoglobulin light chain gene rearrangements display hierarchy in absence of selection for functionality in precursor-B-ALL. Leukemia. 2002;16:1448-1453. 29. van der Burg M, Tümkaya T, Boerma M, de Bruin-Versteeg S, Langerak AW, van Dongen JJ. Ordered recombination of immunoglobulin light chain genes occurs at the IGK locus but seems less strict at the IGL locus. Blood. 2001;97:1001-1008. 30. Bernheim A, Dessen P, Lazar V, et al. Cryptic del(13q14.2) and physiological deletions of immunoglobulin genes detected by high-resolution array comparative genomic hybridization in a patient with indolent chronic lymphocytic leukemia. Cancer Genet Cytogenet. 2007;176:89-91.

9

Table 1.doc

Table 1. Results of I-FISH and array-CGH analysis in 40 CLL patients (ND stands for “Not determined”) chromosome 17

chromosome 11

Age at IGHV Sample diagnosis/ germ-line arrayCGHarrayCGHID FISH sex homology FISH locus locus del(17)(p13) del(11)(q23) 17p13 11q23

CLL01

69/M

99%

Neg

Neg

98%

del 11q14.1q23.2

CLL02

71/F

100%

Neg

Neg

92%

CLL03

69/F

100%

43%

CLL04

44/F

100%

Neg

del 17p11.1p13.3 Neg

CLL05

64/F

100%

98%

CLL06

69/F

ND

CLL07

57/F

CLL08

chromosome 13

chromosome 12

Immunoglobulin Locus Rearrangement

arrayCGH-chromosome 22

arrayCGHarrayCGH- FISH arrayCGH- Start of FISH arrayCGHlocus trisomy deletion at the del. del(13)(q14) trisomy 12 13q14 12 22q11 (1st del.probe)

arrayCGHarrayCGHEnd of the Size of the del.(last del. (bp) del.probe)

Immunoglobulin Lambda Light Chain Locus Rearrangement

detection of utilized IGLV segments

del 13q14.2-3

Neg

Neg

Yes

21004565

21514827

510262

Yes

IGLV3-21

del11q22.1Neg q23.2

Neg

70%

amp 12p + amp Yes 12q11-q15

20747615

21514827

767212

Yes

IGLV6-57

Neg

Neg

82%

del 13q14.3

Neg

Neg

Yes

21174548

21514827

340279

Yes

IGLV5-39

Neg

Neg

18%

Neg

Neg

Neg

Yes

21174548

21514827

340279

Yes

IGLV1-44

del 17p

Neg

Neg

Neg

Neg

Neg

Yes

21174548

21514827

340279

Yes

IGLV1-44

Neg

Neg

Neg

Neg

70%

Neg

Neg

Yes

21004565

21514827

510262

Yes

IGLV1-51

100

Neg

Neg

Neg

Neg

80%

Neg del 13q14.2-3 del 13q14.2

Neg

Neg

Yes

20894376

21514827

620451

Yes

IGLV3-19

75/F

96,50%

73%

Neg

Neg

62%

del 13q14.3

Neg

Neg

No

—

—

—

Yes

IGLV2-23

CLL09

71/M

100%

85%

del 17p11.1p13.3 del 17p

Neg

Neg

Neg

Neg

Neg

No

—

—

—

Yes

IGLV2-5

CLL10

50/M

100

Neg

Neg

85%

Neg del 11q22.123.2

30%

del 13q14.2

Neg

Neg

No

—

—

—

Yes

IGLV2-23

CLL11

83/M

100%

9%

Neg

Neg

Neg

Neg

Neg

Neg

No

—

—

—

Yes

IGLV3-21

CLL12

61/F

93,50%

Neg

Neg

Neg

Neg

86%

74%

12

No

—

—

—

Yes

IGLV3-27

CLL13

56/F

100

Neg

Neg

Neg

Neg

Neg

Neg

Neg

No

—

—

—

Yes

IGLV2-8

CLL14

66/M

ND

62%

del 17p

Neg

Neg

62%

Neg

Neg

No

—

—

—

Yes

IGLV2-14

CLL15

68/M

100%

35%

del 17p11.1p13.3

Neg

neg

25%

del 13q14

Neg

Neg

No

—

—

—

Yes

IGLV3-21

average age: 63

>98% n=21

del n=8

del n=8

del n=6

del n=6

del n=13

del n=13

F/M: 6/19