Immunogenetics features and genomic lesions in splenicmarginal zone lymphoma

Andrea Rinaldi,1 Francesco Forconi,2

Luca Arcaini,3 Michael Mian,1 Elena

Sozzi,2 Silvia Zibellini,3 Luca Baldini,4

Silvia Franceschetti,5 Gianluca Gaidano,5

Roberto Marasca,6 Manuela Mollejo,7

Miguel A. Piris,7 Alessandra Tucci,8

Fabio Facchetti,8 Govind Bhagat,9

Riccardo D. Favera,9 Paola M. V.

Rancoita,1,10 Emanuele Zucca,1 Ivo

Kwee1,10 and Francesco Bertoni1

1Laboratory of Experimental Oncology and

Lymphoma Unit, Oncology Institute of Southern

Switzerland (IOSI), Bellinzona, Switzerland,2Division of Haematology and Transplant,

Department of Clinical Medicine and

Immunological Sciences, University of Siena,

Siena, 3Division of Haematology, Fondazione

IRCCS Policlinico San Matteo, University of

Pavia, Pavia, 4UO Ematologia 1/CTMO,

Universita degli Studi di Milano, Dipartimento di

Scienze Mediche, Ospedale Maggiore Policlinico,

IRCCS, Milano, 5Division of Haematology,

Department of Clinical and Experimental

Medicine & BRMA, Amedeo Avogadro University

of Eastern Piedmont, Novara, 6Division of

Haematology, Department of Oncology and

Hematology–University of Modena and Reggio

Emilia, Modena, Italy, 7Programa de Patologia

Molecular, Centro Nacional de Investigaciones

Oncologicas (CNIO), Madrid, Spain,8Department of Pathology, University of Brescia, I

Servizio di Anatomia Patologica, and Division of

Haematology, Spedali Civili di Brescia, Brescia,

Italy, 9Institute for Cancer Genetics, Departments

of Pathology and Genetics & Development, and

the Herbert Irving Comprehensive Cancer Center,

Columbia University, New York, NY, USA, and10Istituto Dalle Molle di Studi sull’Intelligenza

Artificiale (IDSIA), Manno, Switzerland

Received 13 April 2010; accepted for publication

27 May 2010

Correspondence: Dr Francesco Bertoni,

Laboratory of Experimental Oncology,

Oncology Institute of Southern Switzerland

(IOSI), via Vincenzo Vela 6, 6500 Bellinzona,

Switzerland.

E-mail: frbertoni@mac.com

Summary

Splenic marginal zone lymphomas (MZL) express mutated (M)) or

unmutated (U)) immunoglobulin heavy chain (IGHV) genes. To

investigate the IGHV mutational status impact on genetic lesions, this

study combined single nucleotide polymorphism-arrays and IGHV

sequencing in 83 cases. Clinical features and outcome were similar between

U- and M-IGHV cases. Recurrent lesions frequency was higher in U-IGHV

cases, including poor prognosticators. Frequencies differed among cases

bearing individual IGHV genes or lambda light chains. In conclusion, SMZL

comprises subgroups based on genetic abnormalities and immunogenetic

status. Genomic lesion frequency differed and was higher in U-IGHV cases,

possibly affecting the outcome.

Keywords: immunogenetics, immunoglobulin genes, lymphomas, spleen,

CGH, Microarray.

short report

First published online 29 September 2010ª 2010 Blackwell Publishing Ltd, British Journal of Haematology, 151, 435–439 doi:10.1111/j.1365-2141.2010.08347.x

Sequencing analysis of the rearranged immunoglobulin heavy

(IGHV) chain genes expressed by B cell lymphomas has

contributed to our knowledge of the normal counterparts of

the neoplastic B cells. Importantly, IGH@ mutation status has

emerged as an important prognosticator in chronic lymphocytic

leukaemia (CLL): patients bearing somatically mutated IGHV

(M-IGHV) have longer survival than patients carrying unmu-

tated IGHV (U-IGHV). Splenic marginal zone lymphoma

(MZL) is a small B-cell, indolent lymphoma (Mollejo et al,

2005; Matutes et al, 2008; Swerdlow et al, 2008). Similar to CLL,

splenic MZL also comprises cases with M- or U-IGHV, but the

impact of IGHV mutations on the clinical outcome of splenic

MZL is unclear (Algara et al, 2002; Ruiz-Ballesteros et al, 2005;

Novara et al, 2009; Salido et al, 2010). M-IGHV cases seem to be

associated with a better clinical outcome than U-IGHV cases,

but the difference is not as strong as observed among CLL

patients. In CLL, U-IGHV cases are associated with the presence

of high-risk, genetic lesions, such as del(17p13) (TP53) and

del(11q23) (ATM), that are uncommon in the general CLL

population. In contrast, splenic MZL cases with U-IGHV have

been associated with a higher prevalence of del(7q31), which is

the most common genetic lesion in this disorder (Algara et al,

2002; Novara et al, 2009; Salido et al, 2010). With the aim of

investigating the impact of the IGHV mutational status on

genetic lesions in splenic MZL, we analysed a series of over 80

cases with a genome-wide high-density single nucleotide poly-

morphism (SNP)-array and IGHV sequencing.

Methods

Eighty-three cases of splenic MZL were analysed for IGHV

status and for the presence of genomic lesions. Table SI

presents the clinical characteristics at diagnosis of the cohort.

Diagnosis of splenic MZL was made on spleen histology (39/

83), peripheral blood (9/83), bone marrow (32/83) or lymph

node (1/83), incorporating immunophenotype and clinical

data, based on the criteria proposed by the World Health

Organization (WHO) classification (Swerdlow et al, 2008) and

by Matutes et al (2008). Cases were selected based upon the

availability of frozen material with a fraction of neoplastic cells

in the specimen representing >70% of the overall cellularity as

determined by morphological and/or immunophenotypic

studies. All clinical specimens were derived from involved

sites and obtained in the course of routine diagnostic

procedures before therapy initiation. The study was approved

by the Bellinzona ethical committee.

Samples for DNA extraction were from spleen in 29/83 cases

(35%), lymph node in five (6%), bone marrow in 32 (39%)

and peripheral blood in 17 (15%). IGHV sequences were

obtained as previously described (Hawkins et al, 1994; Novara

et al, 2009). IGHV sequences were considered mutated or

unmutated using the canonical cut-off of 2% mismatch from

germline IGHV sequences. Genomic DNA profiles were

obtained using the GeneChip Human Mapping 250K NspI

(Affymetrix, Santa Clara, CA, USA), and data mining was

performed as previously described (Scandurra et al, 2010). For

minimal common regions (MCR) occurring in at least 15% of

cases, differences in frequencies between subgroups were

evaluated using Fisher’s exact test followed by multiple test

correction (False Discovery Rate, q-value). In order to evaluate

the impact of the immunoglobulin status and of genetic

aberrations on overall survival (OS), univariate analysis was

performed with the Log-Rank test. OS was calculated from

diagnosis to the last follow up or death from any cause. The

actuarial durations of OS were plotted as curves according to

the Kaplan-Meier method. Statistical analyses were performed

with the Statistical Package for the Social Sciences (spss),

version 17.0.2 (SPSS, Chicago, IL, USA).

Results and discussion

The IGHV gene was mutated in 51/83 (61%) and unmutated

in 32/83 (39%) cases. Among the 96 MCRs detected, the most

frequent aberrations were del(7q31-q32) and gains at 3q. There

were no statistical differences in terms of clinical features and

outcome between cases with U-IGHV and cases bearing

M-IGHV (Fig S1). However, U-IGHV cases showed a higher

frequency of recurrent lesions (Fig 1). In particular, the

presence of U-IGHV was associated with a significantly higher

frequency of gains at 1q, 3q (NFKBIZ, BCL6), del(7q31-32)

(POT1, MIR29A, MIR29B-1) and del(8p) (TNFRSF10A,

TNFRSF10B) (Table I). Thus, despite the lack of statistical

differences in terms of OS or clinical features, U-IGHV status

was associated with an increased occurrence of genomic

lesions, some reported as poor prognosticators in other B-cell

tumours. Gains of 1q confer poor outcome in multiple

myeloma (Avet-Loiseau et al, 2009), gain of 3q in diffuse large

B-cell lymphoma (DLBCL) (Lenz et al, 2008) and mantle cell

lymphoma (MCL) (Salaverria et al, 2007), and del(8p) in CLL,

DLBCL and MCL (Salaverria et al, 2007; Forconi et al, 2008;

Scandurra et al, 2010). Very recently, the presence of a high

rate of IGHV somatic mutations (>5%) has been associated

with an indolent course in MCL (Fernandez et al, 2010): the

application of a similar cut-off in our series gave results

overlapping with the standard 2% (data not shown).

As previously reported (Algara et al, 2002; Salido et al, 2010;

Zibellini et al, 2010), splenic MZL presented a biased IGHV

usage, with IGHV1-2 (21/83, 25%) and IGHV3-23 (13/83, 16%)

being the two most commonly observed genes. When compared

with the remaining cases, IGHV1-2 positive cases presented

more commonly with del(7q31-q32) (POT1, MIR29A, MIR29B)

and del(9p21Æ3) (CDKN2A) (Fig S2). CDKN2A is an important

cell cycle regulator, whose inactivation, mainly by genomic

losses or promoter methylation, is associated with a poor

outcome in B-cell lymphomas (Lenz et al, 2008). In the

present study, almost all cases of splenic MZL with loss of

CDKN2A carried IGHV1-2 (5/7), and splenic MZL cases with

IGHV1-2 and del(9p21Æ3) had a poorer outcome compared to

the rest of the cases (P = 0Æ004) (Fig S3). Splenic MZL bearing

IGHV3-23 displayed a low frequency of aberrations, but no

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statistically significant differences were observed between these

and other cases, possibly due to the small number of cases

(Fig S2). These data, together with the recent demonstration of

subsets of splenic MZL cases with stereotyped B cell receptors

(Zibellini et al, 2010) strongly suggest the existence of a

relationship between pattern of IGHV rearrangement and

underlying genetic events.

The immunoglobulin light chain was kappa in 57/80 cases

(71%) and lambda in 23/80 (29%), with no impact on OS.

Splenic MZL cases bearing immunoglobulin lambda light

chains had a higher frequency of del(13q14Æ2-14Æ3) (MIR15A/

MIR16-1) and del(17p13) (TP53) when compared with kappa-

positive cases (Fig S4). The del(13q14Æ3) is common in CLL,

but can also be observed in other lymphomas. The deletion of

the short arm of chromosome 17, including TP53, is a

relatively common lesion in splenic MZL, possibly associated

with poorer outcome (Salido et al, 2010). Four of the five

(80%) lambda-positive cases with del(13q14Æ3) had U-IGHV

(two IGHV1-2, one IGHV3-23, one IGHV3-21), but additional

cases have to be analysed to assess whether these features

determine a specific subset of splenic MZL.

In conclusion, splenic MZL appears to be composed of

different subsets based on the genetic abnormalities and

immunogenetic status. The frequency of occurrence of geno-

mic lesions differed and was higher in cases bearing U-IGHV,

possibly affecting the outcome. Only the analysis of a very large

number of cases will clearly identify the individual small

prognostic subgroups of splenic MZL.

Acknowledgements

Work supported by Oncosuisse grant OCS-02034-02-2007;

Swiss National Science Foundation grant 205321-112430;

Fondazione per la Ricerca e la Cura sui Linfomi (Lugano,

Fig 1. Frequency of genomic lesions in splenic MZL according to IGHV mutational status: Frequency of DNA gains (up, red) and losses (down, blue)

in 32 cases with unmutated (upper panel) and 51 cases with mutated IGHV (lower panel). X-axis, chromosome localisation and physical mapping;

Y-axis, percentage of cases showing the aberrations.

Table I. Association between immunogenetics and specific genomic lesions in splenic MZL as evaluated by applying Fisher’s exact test (P-value)

followed by multiple test correction (q-value).

Associated region U-IGHV % M-IGHV % P value q value

1q gain 5/32 16 0/51 0 0Æ0069 0Æ093

del(8p) 10/32 31 4/51 8 0Æ013 0Æ093

del(7q31-32) 13/32 41 8/51 16 0Æ018 0Æ093

3q gain (NFKBIZ, BCL6) 10/32 31 5/51 10 0Æ019 0Æ093

IGHV1-2 % other IGHV % P value q value

del(7q31-q32) 10/21 48 11/62 18 0Æ0099 0Æ09

del(9p21.3) (CDKN2A) 5/21 24 2/62 3 0Æ01 0Æ09

IgL lambda % IgL kappa % P value q value

del(13q14.2-14.3) (MIR15A/MIR16-1) 5/23 22 1/57 2 0Æ007 0Æ19

del(17p13) (TP53) 9/23 39 7/57 12 0Æ012 0Æ19

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ª 2010 Blackwell Publishing Ltd, British Journal of Haematology, 151, 435–439 437

Switzerland); Cantone Ticino ‘‘Ticino in rete’’. M.M.

is recipient of fellowship from Alto Adige Bolzano-AIL Onlus.

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Supporting information

Additional Supporting Information may be found in the

online version of this article:

Fig S1. Kaplan Meier according to IGHV mutational status:

45 patients carrying M-IGHV (green) and 30 with U-IGHV

(blue) IGHV genes status (upper panel) (P = 0Æ58).

Fig S2. Frequency of DNA gains and losses in splenic MZL

with different VH genes. S2A: 21 cases of splenic MZL with

IGVH1-02 vs 66 VH1-02 negative. S2B: 14 cases of splenic

MZL with VH3-23 vs 72 IGVH3-23 negative. For each panel,

frequency of DNA gains (up, red) and losses (down, blue).

X-axis, chromosome localisation and physical mapping;

Y-axis, percentage of cases showing the aberrations.

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438 ª 2010 Blackwell Publishing Ltd, British Journal of Haematology, 151, 435–439

Fig S3. Kaplan Meier for OS according to the concomitant

presence of IGVH1-02 and del(9p21) in splenic MZL: four

patients with both features (green) and 71 without

(P = 0Æ004).

Fig S4. Frequency of DNA gains (up, red) and losses (down,

blue) in 57 splenic MZL cases with kappa IgL chains (upper

part) and in 23 splenic MZL with lambda IgL chains. For each

panel, frequency of DNA gains (up, red) and losses (down,

blue). X-axis, chromosome localisation and physical mapping;

Y-axis, percentage of cases showing the aberrations.

Table SI. Clinical characteristics of 83 SMZL cases according

to the immunogenetic status.

Please note: Wiley-Blackwell are not responsible for the

content or functionality of any supporting materials supplied

by the authors. Any queries (other than missing material)

should be directed to the corresponding author for the article.

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