Characteristic pattern of chromosomal gains and losses in ... - Nature

Leukemia (1997) 11, 747–758  1997 Stockton Press All rights reserved 0887-6924/97 $12.00

Characteristic pattern of chromosomal gains and losses in marginal zone B cell lymphoma detected by comparative genomic hybridization J Dierlamm1, C Rosenberg2,3, M Stul1, S Pittaluga4, I Wlodarska1, L Michaux1,5, M Dehaen1, G Verhoef6, J Thomas7, W de Kelver1, T Bakker-Schut2,3, JJ Cassiman1, AK Raap2, C De Wolf-Peeters4, H Van den Berghe1 and A Hagemeijer1 Center for Human Genetics and Flanders Institute of Biotechnology, Departments of 4Pathology, 6Hematology and 7Oncology, University of Leuven; 5 Department of Hematology, UCL St-Luc, Brussels, Belgium; 2Department of Cytochemistry and Cytometry, University of Leiden; and 3Laboratory of Experimental Patho-Oncology, Dr Daniel den Hoed Cancer Center, Academic Hospital Rotterdam, Rotterdam, The Netherlands

1

Marginal zone B cell lymphoma (MZBCL) represents a distinct subtype of B cell non-Hodgkin’s lymphoma, which has been recently recognized and defined as a disease entity. We investigated 25 cases (18 at primary diagnosis and seven during the course of disease) of MZBCL by comparative genomic hybridization (CGH) and compared these results with cytogenetic, fluorescence in situ hybridization (FISH), and Southern blot data. Twenty of the 25 cases (80%) showed gains (total 49) or losses (total 15) of genetic material. In extranodal, nodal, and splenic MZBCL, material of chromosomes 3 (52% of cases), 18 (32%), X (24%), and 1q (16%) was most frequently gained, whereas losses predominantly involved chromosomes 17 (16%) and 9 (12%). High-level amplifications involving the regions 18q21-23 and 18q21-22, respectively, were detected in two cases. Gains of chromosomes 1q and 8q and losses of chromosome 17 or 17p occurred more frequently in relapsed or progressive lymphomas. For all of the frequently affected chromosomes, CGH allowed narrowing of the relevant subregions including 3q2123, 3q25-29 and 18q21-23. By Southern blot analysis, the BCL2, BCL6, and CMYC proto-oncogenes were found to be a part of the over-represented regions in two cases, one case, and two cases, respectively, with gains involving 18q, 3q or 8q. In 13 cases, CGH revealed chromosomal imbalances which were not detected by cytogenetic analysis but could be confirmed by FISH or Southern blot analysis in all cases investigated. On the other hand, CGH failed to detect trisomy 3, trisomy 18, and deletion 7q in three cases with a low proportion of tumor cells bearing these abnormalities, as shown by interphase FISH. The characteristic pattern of chromosomal gains and losses detected in this study confirms the distinct nature of MZBCL and may point to chromosomal regions involved in the pathogenesis of these neoplasms. Keywords: marginal zone B cell lymphoma; CGH; molecular cytogenetics

Introduction Marginal zone B cell lymphoma (MZBCL) represents a distinct subtype of B cell non-Hodgkin’s lymphoma (NHL), which includes lymphoma of the mucosa-associated lymphoid tissue (MALT lymphoma),1 monocytoid B cell lymphoma,2 and splenic MZBCL.3 So far, little is known about pathogenesis, biology, and natural history of MZBCL, in part due to the fact that these lymphomas are relatively rare and have only recently been recognized and defined as a disease entity.4 The morphologic appearance is rather heterogeneous and characterized by a mixture of centrocyte-like cells, small lymphocytes, monocytoid B cells, plasma cells, and larger blastCorrespondence: A Hagemeijer, Center for Human Genetics, Herestraat 49, B-3000 Leuven, Belgium This text presents research results of the Belgian programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming. The scientific responsibility is assumed by its authors. Received 20 January 1997; accepted 12 February 1997

like cells showing a parafollicular growth pattern with expansion of the marginal zone of the B follicle, not seldomly with secondary colonization of the follicle center. The different cell components belong to the same neoplastic population; they exhibit monotypic light chain restriction, express IgM and B cell antigens, and are typically negative for CD5, CD10, CD23 and IgD.4,5 MALT lymphomas tend to present with localized extranodal disease and, in this condition, display a favorable prognosis even with local treatment. Nodal MZBCL share a similar indolent clinical course with frequent localized disease at diagnosis.6,7 However, extranodal and nodal MZBCL at advanced clinical stages behave less favorably.8,9 Splenic MZBCL typically reveal bone marrow and peripheral blood involvement in the absence of peripheral lymphadenopathy at diagnosis10,11 and have recently been proposed to represent the histologic counterpart of splenic lymphoma with villous lymphocytes (SLVL).12 SLVL respond well to splenectomy and often show an indolent clinical course.13,14 Chromosomal abnormalities have been described in 27 extranodal,15–23 15 nodal22–25 and eight splenic MZBCL.22,23 Whole or partial trisomy 3 has been reported most consistently,15–17,19,20,22,23 occurring in larger series in 56–78% of cytogenetically abnormal cases16,19,22 and in up to 60% of cases when interphase FISH had been applied.23,26 Trisomy 18,15,19,20,22,24,25 trisomy 7,19,20,22 and trisomy 1215,18,19,20,22 have been observed non-randomly but less frequently than trisomy 3. A t(11;18)(q21;q21) was found in three extranodal lymphomas17,27 and structural aberrations of chromosome 1 recurrently involved the chromosomal regions 1p22,15,19 1p3422,25 and 1q21.22 The distinct nature of MZBCL is further underlined by the fact that known lymphoma-associated chromosomal translocations or rearrangements of the BCL1, BCL2, BCL3 and BCL6 oncogenes have not been detected in these lymphomas.3,16,17,22,28,29 Rearrangements of CMYC and biallelic inactivation of P53 have been associated with high-grade transformation of gastric MALT lymphomas.30,31 In the present study, 25 histologically and immunophenotypically well characterized cases of extranodal, nodal, and splenic MZBCL were investigated with the recently developed technique of CGH.32 CGH is a double color hybridization procedure, which provides in a single experiment an overview of genomic imbalances, such as partial or complete trisomies, monosomies, or amplifications within the tumor genome. In contrast to cytogenetic analysis, CGH does not require metaphase preparations from tumor cells and selection of subclones due to cell culturing is avoided. CGH results were compared with karyotypic, FISH, and Southern blot data.

Chromosomal gains and losses in MZBCL by CGH J Dierlamm et al

748

Materials and methods

Patient samples All cases histologically diagnosed as MZBCL at the Department of Pathology of the University of Leuven, of which stored DNA was available, were included in the present study. One part of the fresh tumor sample was cultured and further processed for cytogenetic and FISH analysis, the other part was used for DNA extraction and subsequent gene rearrangement studies; remaining DNA was stored and used in the present study for CGH analysis and gene amplification studies. The diagnoses of MZBCL were established according to the proposed ‘revised European–American classification of lymphoid neoplasms’.4 Routine hematoxylin and eosin staining and immunohistochemistry on frozen sections using an avidin–biotin complex (ABC) technique and the monoclonal antibodies CD3, CD5, CD10, CD20, CD23, CD35, anti-IgG, anti-IgA, anti-IgM, anti-IgD, anti-k, and anti-l were performed. Additionally, 21 cases (all except cases 7, 11, 13 and 21) were tested for BCL2 and BCL6 protein expression. All biopsies available from each patient as well as clinical data were reviewed. Cytogenetic, histologic, clinical, and gene rearrangement data of 17 patients (cases 1–3, 5–9, 11, 12, 15, 16, 19, 21, 22, 24 and 25)8,22 and interphase FISH data on trisomy 3 of nine cases (cases 2, 5, 6, 8, 12, 15–17 and 25)23 have been published previously.

Figure 1 Comparative genomic hybridization in marginal zone B cell lymphoma. (a) CGH experiment showing a normal metaphase spread hybridized with DNA from case 2 detected with FITC (green) and reference DNA (46,XY) labeled with lissamine (red). Chromosomes are counterstained with DAPI. Chromosomal regions over-represented in the tumor genome appear with enhanced green staining (chromosomes 3 and 18), whereas under-represented regions show more intense red fluorescence. Strong green over-representation of the whole X chromosome and red over-representation of the Y chromosome is due to the sexes of the tumor DNA (female) and the reference DNA (male) (the combination of XX and XY DNAs was chosen for demonstration purposes; note the difference in green fluorescence intensity of chromosomes 3 and 18 compared to the X chromosome). On the right side, the complete green-to-red fluorescence average profile of the same case is shown. The central line indicates a ratio value of 1.0 and the right (green) and left (red) lines represent the diagnostic thresholds for over- (1.2) and under-representation (0.8), respectively. The thick bar next to the chromosomal ideogram shows regions of over-representation (green bar on the right of the ideogram) and under-representation (red bar on the left of the ideogram). The centromeric/heterochromatic regions are excluded from evaluation (ie under-represented centromeric regions of chromosomes 13, 14, 15, 21, 22), because they contain highly repetitive DNA sequences, which are suppressed by Cot1-DNA. The finding of over-representation of the whole chromosomes 3 and 18 is in accordance with the presence of trisomy 3 and 18 as shown by cytogenetic analysis. (b) In the lower part of the figure, representative partial karyotypes revealing whole or partial over-representation of chromosomes 3 and 18 and the green-to-red fluorescence average profiles of the respective chromosomes are shown. In case 17, over-representation of the whole chromosomes 3 and 18 is evident, whereas in the remaining cases only part of the chromosomes is gained: 3q in cases 3 and 15, 3q2529 in case 18, 18q12-23 in case 3 and 18p and 18q21-22 in case 1.

Comparative genomic hybridization DNA was extracted from fresh, uncultured tumor tissue and from blood lymphocytes of normal individuals by phenol– chloroform extraction after proteinase K digestion according to standard methods. CGH was performed as described by Kallioniemi and coworkers33 with minor modifications. Briefly, tumor DNA (test DNA) and normal DNA (reference DNA) were labeled by nick-translation with Biotin-16-dUTP (Boehringer, Mannheim, Germany) and Lissamine-5-dUTP (DuPont, Boston, MA, USA), respectively. In all cases, reference DNA and tumor DNA were derived from individuals with the same gender; in some cases an additional experiment using DNAs of different gender was performed as an internal control for hybridization efficacy (Figure 1). The size of the nick-translated fragments ranged from 400 to 2000 bp. Equal amounts (200 ng) of labeled tumor DNA and normal DNA, and 10 mg of unlabeled human Cot-1 DNA (GIBCO/BRL, Gaithersburg, MD, USA) were combined in 10 ml hybridization mixture (50% deionized formamide, 2 × SSC, 10% dextran sulfate), denatured, preannealed for 60 min, and applied to a denatured slide with normal metaphase spreads. After hybridization for 72 h at 37°C, post-hybridization washes were performed to a stringency of 0.1 × SSC at 60°C. Biotinlabeled tumor DNA was detected with one layer of avidinfluorescein isothiocyanate (FITC) (Vector Laboratories, Burlingame, CA, USA). The slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI) and mounted with antifade solution. Gray levels images of each of the three fluorochromes were acquired using an epifluorescence microscope (Leica DMRB; Leitz, Wetzlar, Germany) equipped with a high-resolution cooled CCD camera (Photometrics, Tuscon, AZ, USA). A three-color image was built up by overlay of the three images in pseudocolors matching the original colors of the fluoroch-

romes and analyzed using the QUIPS software package for CGH analysis (XL System, Vysis, Stuttgart, Germany) according to methods described elsewhere.34 The ratio of FITC/Lissamine fluorescence intensities was calculated along each individual chromosome. Ratio values obtained from five to 10 metaphase spreads were averaged and the resulting profile was plotted next to the chromosomal ideograms (Figure 1). Ratios above 1.20 and below 0.80 were considered to represent chromosomal gain and loss, respectively. These cutoff values are based on our results from a series of control experiments using two normal DNAs. Over-representations were considered as high-level amplifications when the fluorescence ratio values exceeded 2.0 or when a distinct band-like hybridization signal of the tumor DNA beyond the diagnostic threshold for over-representation was seen. The centromeric regions, heterochromatic blocks of chromosomes 1, 9 and 16, the satellite regions of the acrocentric chromosomes, and the Y chromosome were excluded from evaluation because of the abundance of highly repetitive DNA sequences. CGH results were described, according to the International System for Human Cytogenetic Nomenclature (ISCN).35

Cytogenetic analysis Cytogenetic analysis of extranodal, lymph node, or splenic tissue was performed after unstimulated short term culture (24 h) in RPMI 1640 medium supplemented with 15% fetal calf serum, glutamine, and pencillin. Peripheral blood cells were cultured for 3 days in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA). After exposure to colcemid, harvesting was carried out according to standard methods using hypotonic KCl solution and 3:1 methanol:acetic acid as a fixative. Metaphases were G-banded with Wright’s stain.


749


750

Karyotypes were described according to the ISCN.35 Remaining methanol/acetic acid fixed cells were stored at −20°C until used.

Fluorescence in situ hybridization FISH was performed in all cases with available fixed cells where CGH and cytogenetic data were discrepant. In addition, cases with a normal karyotype or with insufficient mitotic yield were screened with alpha-satellite probes specific for chromosomes 3 and 18, respectively (these chromosomes were found to be most frequently gained by CGH). The following DNA probes were used: alpha-satellite probes specific for chromosome 2 (D2Z), 3 (D3Z1), 9 (D9Z1), 11 (D11Z1), 18 (D18Z1) and X (DXZ1), respectively (all from Oncor, Gaithersburg, MD, USA); a library probe for chromosome 18 (WCP 18, Vysis); yeast artificial chromosome (YAC) clones 886A4 (mapped to 6p21), 309G6 (1q31), 766F8 (3q2728), 928C1 (7q31); and cosmid clones B5-2 (3q27) mapped to the BCL6 gene (kindly provided by Dr T Miki, Tokyo Medical and Dental University, Tokyo, Japan) and c53.2 (17p13) mapped to the P53 gene (isolated from the human genomic library CMLW-25383, kindly provided by Dr E Schoenmakers, University of Leuven, Belgium). YAC inserts were amplified by Alu-polymerase chain reaction (PCR) according to a protocol published by Lengauer and coworkers.36 FISH was performed on stored, previously cultured, methanol/acetic acid fixed cells according to standard methods as previously described.37 Slides were viewed and pictures were captured with the same system as described for CGH analysis. For interphase studies, 500 cells were analyzed for each probe in each case. For the probes used for interphase FISH, a proportion of 1–5% of cells showing three hybridization signals and 4–15% of cells with one signal was found in normal controls. Therefore, more than 5% of interphase cells bearing three hybridization signals were considered to indicate the presence of a trisomy and more than 15% of cells with only one hybridization signal were considered to indicate a monosomy of the respective chromosome in the tumor cell population.

(a 3.5-kb EcoRI fragment, kindly provided by TH Rabbitts, MRC, Cambridge, UK) mapped to the T cell receptor d locus on 14q11. The Cd probe served as an internal control and was selected because neither cytogenetics nor CGH analysis revealed changes involving the region 14q11 in any of the patients. The hybridization signals were quantitated by densitometric analysis using a Phosphor Imager and Image Quant 3.0 software (Molecular Dynamics, Sunnyvale, CA, USA). The amplification factors were calculated as a ratio of a BCL2, BCL6 or CMYC hybridization signal, respectively, to a Cd signal normalized to the respective ratio observed in placenta, which was referred to an amplification factor of 1. Additionally, gene rearrangement studies for the immunoglobulin heavy and light chain genes, T cell receptor genes, BCL1, BCL2, BCL3, BCL6 and CMYC genes were performed in all cases as previously described.22

Results

Clinical data Clinical data are summarized in Table 1. The 14 female and 11 male patients ranged in age between 41 and 84 years (mean 60 years). Eighteen cases were studied at primary diagnosis and seven during the course of disease after treatment with chemotherapy (cases 5, 12, 13, 14 and 23–25) and radiotherapy (cases 5 and 13). Five patients (cases 1–5) showed extranodal disease at primary diagnosis and nine patients (cases 6–14) presented with nodal involvement; in the remaining cases, the massively enlarged spleen, leading to hypersplenism or abdominal discomfort, clearly represented the main tumor burden. At diagnosis, most of the latter patients additionally showed bone marrow involvement with or without peripheral blood spread in the absence of lymphadenopathy (all cases except cases 18 and 20). The overall survival ranged from 4 to 228+ months. Seven of the 25 patients were deceased 4–68 months after diagnosis and all but one of the latter presented with stage IV disease.

Southern blot analysis Southern blot analysis was applied to investigate possible amplifications of the BCL2, BCL6 and CMYC genes in cases which showed amplification/over-representation of the chromosomal regions 18q, 3q, and 8q, respectively, by CGH. Eight micrograms of tumor DNA and control placental DNA were digested with the restriction endonucleases EcoRI (for BCL2 analysis), BglII (for BCL6) or PstI (for CMYC) and size fractionated in different dilutions (1:1, 1:2 and 1:4) on a 0.7% agarose gel. After alkali blotting onto Hybond N+ membranes (Amersham, Buckinghamshire, UK) hybridization was carried out using DNA probes labeled with 32P-dCTP using the random priming method. The BCL2 gene was analyzed using probe BCL2B (a 2.8-kb EcoRI–HindIII fragment, kindly provided by Dr Y Tsujimoto, Wistar Institute, Philadelphia, PA, USA), the BCL6 gene was investigated with probe BCL6 (a 4kb SacI fragment, kindly provided by Dr R Dalla-Favera, University Medical Center, New York, NY, USA), and the CMYC gene was studied with probe pP20 (a 1.6-kb PstI–XbaI fragment, kindly provided by Dr D Mathieu-Mahul, Hoˆpital SaintLouis, Paris, France). The blots were hybridized simultaneously with one of these probes and the Cd R21EE probe38

Histopathology The detailed description of all cases but eight (cases 4, 10, 13, 14, 17, 18, 20, 23) has been given previously.22 These additional cases (one extranodal (orbit), three nodal, and four splenic MZBCL) as well as those previously described were composed of a mixture of cells encompassing the full spectrum of marginal zone B cells. In one case (case 10), a pronounced plasmacytic component expressing the same light chain as the remaining tumor cell population was found. Residual reactive follicle centers with preserved lymphocytic coronae were seen in three cases (cases 4, 14 and 17) regardless of the anatomical location of these lymphomas. In all cases but one (case 13), k (cases 4, 10, 14, 18, 20, 23) or l (case 17) light chain restriction was demonstrated. None of the cases expressed CD5 and CD23, while a weak IgD expression was noted in four cases (cases 14, 17, 18 and 23). BCL2 expression was observed in 16 (all cases but cases 8–10, 20 and 24) of the 21 cases analyzed and only three cases showed a weak BCL6 expression in a limited number of cells (cases 4, 6 and 20).

F/41 P

F/74 P

M/65 P

F/71 P

M/65 R/193

M/59 P

F/55 P

M/84 P

F/78 P

F/41 P

F/59 P

2

3

4

5

6

7

8

9

10

11

Soft tissue, LN

IIIEA

IIA

IIA

IVB

IIA

IIA

LN

LN

LN, Pleura, Spleen

LN

LN

LN

Orbit

IEA

IIA

Ileum, Pleura, LN

Breast

IEA

IVB

Stomach

Organ involvement

IEA

Status/ Clinical Time stage b after primary diagnosis (months)a

Orbit

Ileum

Breast

LFU

LN

12+/15+ LN LFU

43+/48+ LN

CT 6+/11+ (CHOP)/ CR CT (CHOP)/ 21/72+ CR

LN

LN

Karyotype

No mitosis

83-85,XXY,+X,−Y,−1,−1,−2,+der(3;6) (q10;p10)x2,+der(3)x2,−4,−4, −6,−6,−7,−8,−9,−12,−15,−18, +1−4mar[cp6]/46,XY[2]

50,XX,der(1)inv(1)(p34q21)t(1;14) (q21;q32),+add(3)(q26),add(7)(p22), add(9)(p11),der(14)t(1;14)(q21;q32), −17,add(18)(p11),+4mar[7]/ 50,idem,der(11)t(11;12)(q13;q24)[2]/ 51,idem,+X[2]/51,idem,+X,del(5) (q31q33)[3] 47,XX,+3[4]/47,idem,+7,+18,−22,−22[2] 49,idem,+2mar[6]/46,XX[2]

rev ish enh(18)

rev ish enh(3,XC)

rev ish enh(9p24-q32), dim(17p)

rev ish enh(3q,7p13-21, 9q22-34,11p13-15,11q14-23, 12p11-12,12q21-22,13q21-31, 18q12-23,Xp21-22),dim(1p3236,1p13-q24,1q32-44,6q21-23, 7q32-36,9p13-q21,17p13-q24, 18p11-q11,19),amp(18q21-23) No copy number changes

rev ish enh(3,18)

rev ish enh (3p14-26,3q21-22, 18p,18q21-22),dim(9p13-24), amp(18q21-22)

CGH

46,XX[13]

49,XX,ins(1;?)(q21;?),add(3)(q28), +der(3)ins(3;12)(p21;?)ins(3;12)(q23;?), +i(5)(p10),del(11)(q14q21),+der (12)t(3;12)(?;q22)[7]/ 46,XX[2] 46,X,t(X;6)(q13;q27),t(1;14)(p34;q32), add(15)(q15)[9]/46,XX[2]

No copy number changes


rev ish enh(3p21-26,5p)

49,XY,+X,+3,+11,del(14)(q24)[9]/46,XY[2] rev ish enh(3,11),amp(X)

47,XX,+18[9]

48,XXYc,+3[12]

132/228+ Soft tissue 46,XY[5]

5+/8+

0/4

0/20

77+/77+ Stomach

CT 0/9+ LFU LN (chloramb.)/ SD

CT (CHVmPBV), RT/CR CT (CHVmPBV), RT/CR LFU

RT/CR

CT (Chloramb.; PDN)/SD Tumor resection, CT (ProMACEMOPP/ CytaBOM; VIM) PD RT/CR

Gastrectomy/ CR

Treatment/ Survivalc Sampled Response DFS/OS (months)

Clinical, cytogenetic, CGH and FISH data from 25 patients with marginal zone B cell lymphoma

1

Case Sex/ Age

Table 1

DXZ1: 2 sig. (88%), 3 sig. (12%)

D3Z1: 3 sig. (2%); D18Z1: 3 sig. (1%) D9Z1: 3 sig. (37%), 4 sig. (10%); D3Z1: 3 sig. (15%); D18Z1: 3 sig. (2%); c53.2 (17p13): 1 sig. (57%)

FISH


751

M/57 PD/64

M/55 R/15

F/49 PD/156

F/60 P

F/44 P

M/69 P

M/42 P

F/57 P

M/70 P

13

14

15

16

17

18

19

20

(Continued)

12

Table 1

IVB

IVB

IV

IVB

IVA

IVB

Spleen, LN, BM, PB

Spleen/BM

Spleen, LN, BM, PB

Spleen, BM

Spleen, BM, PB

Spleen, BM, PB

LN, BM

LN, Stomach, Pleura

LN, Liver, BM (after splenectomy)

0/156+ LFU

5/17

0/68

Splenectomy, CT (CHOP)/ CR Splenectomy, CT (Chloramb.)/SD 0/2+

22/31+

Splen0/42+ ectomy/ SD Splen24+/28+ ectomy, CT (CHOP; BEAM), PBSCT/CR Splen1/12 ectomy, CT(CHOP)/ NR SplenLFU ectomy

LFU

CT (ProMACEMOPP/ CytaBOM)/ PR CT (CEP)/PD

Spleen

Spleen

Spleen

Spleen

Spleen

Spleen

LN

LN

LN

rev ish enh(1q24-31,3,8q21-24, 18,Xq21-24,dim(17p13-q21, 17q24-25,19)

rev ish enh(3,18),dim(21)

rev ish enh(3q13-29)

rev ish enh(2p25-q33,3q,6p,X)

rev ish enh(4,Xp21-22)

rev ish enh(X)

48,XY,der(1)add(1)(p34)add(1)(q21), rev ish enh(1q25-31,5q15-23) add(5)(p11),der(7)t(1;7)(q21;q31), del(12)(p12),dic(12;17)(p11;p11),t(12;14) (p11;q32),add(17)(p13),−19,−20,−22,+2−4mar [11]/96,idem[2]/ 46,XY[5]

No mitosis

46,XY,del(7)(q22q35),−14,−15,+2mar[3]/ rev ish enh(3q25-29) 46,idem,add(22)(p11)[3]/ 46,XY[10]

48,XY,ins(1;?)(q21;?),+3,t(3;9)(q24;p12), +18,add(19)(q13)[10]

47,XX,+3,+18,−21[14]

47,XX,+del(3)(p13)[17]

49-51,XY,t(1;9)(q21;q34),der(4)(q), add(7)(p22),add(14)(q32),+15,+21, +1-3mar[6] 45-53,X,-X[2],+del(3)(p13)[5], +4[4],+add(9)(p21)[4],add(14)(q32)[6], +1-6mar[4],+2dmin[2][cp8]

70-73,XXY,add(1)(p34),trp(1)(q21q31)x2, rev ish enh(1q22-32,3,11q23-25, +add(3)(q29),+del(6)(q15),−9,+12,−14, 12,18q22-23) −15[cp11]

cB5-2 (3q27): 3 sig. (21%), 4 sig. (30%), 5 sig. (18%), 6 sig. (5%); Y766F8 (3q2728): 3 sig. (23%), 4 sig. (36%), 5 sig. (15%), 6 sig. (2%); Y928C1 (7q31): 1 sig. (27%) D3Z1: 3 sig. (5%); D18Z1: 3 sig. (2%); D11Z1: 3 sig. (2%) D3Z1: 3 sig. (1%); D18Z1: 3 sig. (3%); DXZ1: 3 sig. (33%)

ins(1;?)(q21;?).ish dup(1)(Y309G6+ +); c53.2 (17p13): 1 sig. (55%)

D2Z: 3 sig. (55%) 4 sig. (6%); Y886A4 (6p21): 3 sig.(55%), 4 sig. (31%); DXZ1: 3 sig. (56%), 4 sig. (9%)

752

IV

IVB

IVB


F/63 P

M/66 SD/7

M/53 PD/18

F/55 PD/95

22

23

24

25

IVB

IVB

IVA

IV

IVB

Splenectomy, CT, (ProMACEMOPP)/ PD Spleen, BM, PB Splenectomy Spleen, Liver, CT BM, PB (CVP)/SD, Splenectomy/SD Spleen, LN, BM, CT PB (Pro MACEMOPP/ CytaBOM; CEP; VIM)/SD; Splenectomy, CT (DHAP)/PR Spleen, LN, BM, SplenPB ectomy, CT, VIM; DHAP)/PR

Spleen, BM

0/99+

0/29

0/9+

?/84+

0/13

Spleen

Spleen

Spleen

PB

Spleen


rev ish enh(7p,8q22-24), dim(7q31-36)

48,XX,+X,+3,add(17)(p11),+18,−20[7]/ 46,XX[18]


46,XY,t(2;7)(p26;q22),−17,der(18)t(17;18) rev ish dim(17p) (q11;q11),+mar[9]/46,idem,add(12)(p11) [2]/ 46,idem,add(12)(p13)[3] 48,XY,ins(1;1)(q21;q32q21),t(2;6)(p13; rev ish enh(1q23-31,3,5q14-22, p23),t(3;18)(q29;q21),+der(3)t(3;18) 18),dim(9q34) (q29;q21),+18[2]/48,XY,der(1)add(1)(p13) ins(1;1)(q21;q32q21),t(3;18),+der(3)t(3;18), +18[2]/48,Y,t(X;21)(p11;p11),ins(1;1),t(3;18), +der(3)t(3;18),der(14)t(8;14)(q12;q32), +18[15]/ 46,XY[10]

46,XX[12]

47,XX,t(1;14)(p34;q32),+7,del(19)(q13) [14]

D3Z1: 3 sig. (30%); D18Z1: 3 sig. (27%)

+mar.ish der(18) (D18Z1+,WCP18+) D18Z1: 3 sig. (55%)

Cytogenetic, FISH, and CGH results are described according to the ISCN nomenclature; 35 enh (enhanced) and dim (diminished) indicate relative increase or decrease, respectively of the copy number with regard to a basic euploid stage and amp refers to amplification; bold letters indicate CGH data, which provide additional information compared with karyotypic findings. a Status at the time the sample was taken. b Clinical stage according to the Ann Arbor classification. c Survival from diagnosis. d Sample, which was processed for cytogenetic, CGH, FISH and Southern blot analysis. P, primary diagnosis; R, relapse; PD, progressive disease; SD, stable disease; NR, no response; LN, lymph node; BM, bone marrow; PB, peripheral blood; DFS, disease-free survival; OS, overall survival; +, indicates that the patient is alive; CT, chemotherapy; PBSCT, peripheral blood stem cell transplantation; RT, radiation therapy; CR, complete remission; PR, partial remission; LFU, lost to follow-up; sig., signals in interphase nuclei; Chloramb., chlorambucil; PDN, prednisone; proMACE-MOPP, prednisone, doxorubicin, cyclophosphamide, etoposide, nitrogen mustard, vincristine, procarbazine, methotrexate; ProMACE-CytaBOM, prednisone, doxorubicin, cyclophosphamide, etoposide, cytosine-arabinoside, bleomycin, vincristine, methotrexate; VIM, etoposide; ifosfamide, mitoxantrone; CHVmP-BV, cyclophosphamide, doxorubicin, teniposide, prednisone, bleomycin, vincristine; CHOP, cyclophosphamide, doxorubicin, vincristine, prednisone; CEP, CCNU, etoposide, prednimustine; BEAM, BCNU, etoposide, cytosine-arabinoside, melphalan; CVP, cyclophosphamide, vincristine, prednisone; DHAP, dexamethasone, cytosine-arabinoside, cisplatin.

F/74 P

Continued

21

Table 1


753


754

Comparative genomic hybridization Complete data from all patients are shown in Table 1 and summarized in Figure 2. Twenty of the 25 patients (80%) showed gains (total 49) or losses (total 15) of genetic material. Irrespective of the localization of the lymphoma, material of chromosomes 3 (52% of cases), 18 (32%), X (24%) and 1q (16%) was most frequently gained, whereas losses mainly involved chromosomes 17 (16%) and 9 (12%). Gain of the whole X chromosome was observed in a patient with known Klinefelter syndrome and constitutional XXY abnormality (case 6); this chromosomal gain was excluded from evaluation because of its constitutional nature. Most of the cases showed more than one chromosomal imbalance (mean 2.8) with overrepresentation of chromosomes 3 and 18 being most frequently associated (seven cases). In six cases, a single imbalance was identified: gain of material of chromosomes 3 (cases 6, 15 and 18), 18 (case 7), and X (case 19) and loss of chromosome 17p (case 23). Gains of chromosome 1q and 8q and losses of chromosome 17 or 17p were mainly seen in relapsed previously treated lymphomas (cases 5, 12, 23 and 24) or progressive lymphomas defined by stage IVB disease, progressive disease despite intensive chemotherapeutic treatment, and short survival (cases 3, 17 and 21). Strong over-representations (>1.5) were detected in seven instances and five patients and involved the following chromosomal regions: 3q21-22 (case 3), 3q26-27 (case 18), 18q12-23 (case 3), 18q21-22 (case 1), 6p11-21 (case 14), 9q31-33 (case 3) and the X chromosome (case 8) (see thick bars in Figure 2). High-level amplifications (.2) were detected in two cases and involved the regions 18q21-23 (case 3) and 18q21-22 (case 1). Examples of the CGH experiments are given in Figure 1. On each of the frequently affected chromosomes, CGH analysis allowed us to delineate a minimal common region of over- or under-representation (Figures 1 and 2): both arms of chromosome 3 were over-represented in eight cases, whereas in four cases only the long arm was involved (cases 3, 14, 15 and 18). Two cases showed a partial gain of 3q affecting the regions 3q21-23 (case 1) and 3q25-29 (case 18), respectively. On chromosome 18, the commonly over-represented region was mapped to bands 18q21-23, defined by three cases (cases 1, 3 and 12), two of which showed a high-level amplification in this region (cases 1 and 3). For chromosomes X and 1, a consensus region of overlap in Xp22 and 1q25-31, respectively, could be defined. Two cases (cases 17 and 21) showed over-representation of 8q with a common region in 8q22-24. The regions commonly involved by loss of genetic material could be delineated to the short arm of chromosome 17 (cases 3, 5, 17 and 23), the regions 9p13 (cases 1 and 3) and 7q3236 (cases 3 and 21), and chromosome 19 (cases 3 and 17).

Southern blot analysis Gene amplification studies confirmed CGH results in all instances. The following amplification factors were found: 2.1 (case 1) and 2.0 (case 3) for BCL2; 2.7 (case 18) for BCL6; and 1.9 (case 17) and 2.1 (case 21) for CMYC (data not shown). All cases showed rearrangement of the immunoglobulin heavy chain locus. Ck (cases 4, 6, 9–11, 13, 15, 17–19, 21– 24) or Cl (cases 1–3, 5, 7, 8, 12, 14, 16, 25) light chain gene rearrangements were found in 14 and 10 cases, respectively.

All cases revealed germline configuration for the T cell receptor genes and the BCL1, BCL2, BCL3, BCL6 and CMYC genes.

Comparison of CGH results with cytogenetic, FISH and Southern blot data Detailed data are given in Table 1. For most of the non-balanced abnormalities, cytogenetic analysis and CGH showed similar results. Additionally, CGH revealed chromosomal imbalances not detected by cytogenetic analysis in 13 cases (11 of 20 cases with abnormal karyotype, one of three cases with normal karyotype, and one of two cases with insufficient mitotic yield). Most of these cases revealed complex karyotypes including marker chromosomes and/or cytogenetically unidentifiable chromosomal material (cases 1, 3, 9, 12–14, 17, 18, 20 and 24). In eight of the 13 cases, apparent discrepancies were further investigated with FISH (cases 5, 14, 17, 18 and 19) or Southern blot analysis (cases 1, 3, 17, 18 and 21), and in all instances CGH data were confirmed. Especially, over-representations of 18q (cases 1 and 3), 3q (case 18) and 8q (cases 17 and 21) could be confirmed by Southern blot using probes for the BCL2, BCL6 and CMYC genes, respectively. The chromosomal gain involving the region 3q26-27 (case 18) was also analyzed by interphase FISH using a BCL6/3q27-specific cosmid probe (Figure 3) and a YAC probe hybridizing to the region 3q27-28 distal to BCL6. With both probes, between three and five hybridization signals were observed in the majority of interphase cells. However, CGH did not detect trisomy 3, trisomy 18 and deletion 7q in three cases (cases 5, 25 and 18) with a low proportion of tumor cells bearing these abnormalities (15–30%), as shown by interphase FISH. Discussion The rapidly growing number of studies applying the recently introduced CGH technique impressively demonstrate the potential of this approach to detect chromosomal gains and losses in tumor genomes.32,39–44 Using CGH, we identified novel genetic abnormalities including high-level amplifications involving the chromosomal region 18q and regions commonly affected by gain or loss of genetic material in 25 cases with MZBCL. In extranodal, nodal, as well as splenic MZBCL, material of chromosomes 3 (52% of cases), 18 (32%), X (24%), and 1q (16%) was most frequently gained, whereas losses predominantly involved chromosomes 17 (16%) and 9 (12%). The frequent involvement of chromosome 3 is in accordance with previous cytogenetic and FISH studies.15–17,19,20,22,23,26 Interestingly, in four of our 13 cases with over-representation of chromosome 3, only material of the long arm was found to be gained with two cases showing partial gain of 3q affecting the regions 3q21-23 and 3q25-29, respectively. These data indicate that the latter regions are of particular importance and might point to genes involved in the pathogenesis of MZBCL. Several candidate genes are located on these chromosomal regions, eg a gene coding for the surface antigen B7, which can enhance CD28-mediated T cell interactions and thereby increase the production of various lymphokines, particularly interleukin 2;45 the PBX2 homeobox gene located on 3q22-23;46 a gene coding for a subunit of interleukin 12;47 and the BCL6 proto-oncogene mapped to 3q27, which is frequently rearranged in diffuse


755

Figure 2 Summary of chromosomal gains and losses in 25 cases of marginal zone B cell lymphoma. Gains are shown on the right side of the chromosomal ideogram and losses on the left side. The number on top of each line refers to the case number as shown in Table 1. Chromosomal imbalances found at primary diagnosis are indicated with solid lines and those detected during the course of disease with dotted lines. Chromosomal gains .1.5 are shown with thick bars.


756

Figure 3 Fluorescence in situ hybridization in case 18 using a biotin-labeled cosmid probe mapped to the BCL6 gene on 3q27 (green signals) and a digoxigenin-labeled chromosome 3-specific alpha-satellite probe (red signals). In interphase nuclei, three to six green signals and two red signals can be seen in the majority of cells (see also Table 1). These results are in accordance with the finding of over-representation of the chromosomal region 3q25-29 as shown by CGH.

large cell lymphomas arising at extranodal sites.48 Rearrangements of the BCL6 gene have not been detected in MZBCL (Ref. 22 and present study)2 and amplification of this gene has, to the best of our knowledge, not been described so far in any disease entity. Using Southern blot analysis with a probe for the BCL6 gene, we found a 2.7-fold over-representation in a case with a chromosomal gain involving the region 3q26-27. Further investigations with FISH using probes for BCL6 and a region in 3q27-28 distal to BCL6 showed with both probes between three and five hybridization signals in the majority of interphase nuclei, underlining that the BCL6 gene is only a part of the over-represented region. In this respect, it is interesting to note that by immunohistochemistry only three of our cases showed a weak BCL6 protein expression and that there was no apparent correlation between over-representation of the chromosomal region 3q and expression of the BCL6 protein. Gain of material of chromosome 18 was observed in eight of the 25 cases. The commonly over-represented region could be delineated to bands 18q21-23. Two cases showed highlevel amplifications in this region, which were confirmed by Southern blot analysis using a probe for the BCL2 gene on 18q21.3. Interestingly, the regions 18q21-2344 and 18q212242 were also found to be commonly gained in two recent CGH studies investigating diffuse large cell lymphoma and follicle center cell lymphoma, respectively. Monni and coworkers44 demonstrated amplification but no rearrangement of BCL2 by Southern blot in all six cases with gain of 18q. The pathogenetic significance of BCL2 amplification is not known. Rearrangement of this gene by juxtaposition to regulatory sequences of the immunoglobulin genes is known to result in BCL2 overexpression leading to inhibition of apoptosis and thereby conferring a survival advantage to the affected cells.49 BCL2 gene rearrangement or the corresponding t(14;18)(q32;q21) are found in the majority of follicle center cell lymphoma, in contrast to MZBCL where this rearrangement has not been detected so far (Refs 3, 16, 22, 28, 29 and

present study). In the present study, BCL2 protein expression was observed in 16 of the 21 cases investigated including six of seven analyzed cases with over-representation of chromosome 18 and 10 of 14 cases without copy number changes of chromosome 18. Another proto-oncogene on 18q21 is the FVT1 gene (follicular lymphoma variant translocation 1 gene), which maps 10 kb 59 to BCL2.50 It is also worth mentioning, that an as yet unidentified gene on 18q21 is involved in a translocation (11;18)(q21;q21), which has been described in one case of gastric MALT lymphoma17 and two other extranodal lymphomas of possible marginal zone origin.27 Further studies are necessary to identify the target gene(s) of the 18q21-23 over-representation. In the present study, gains of chromosomes 1q and 8q and losses of chromosome 17 or 17p were predominantly observed in relapsed lymphomas or progressive, clinically advanced lymphomas with an unfavorable prognosis, indicating that these chromosomal changes are likely of secondary nature. Duplication of the long arm of chromosome 1 is one of the most common chromosomal abnormalities in human neoplasias and is known to occur frequently as a secondary change during disease progression.51–54 In the present study, all four patients with gain of 1q showed stage IVB disease and an aggressive clinical course; two of them were analyzed during disease progression and three deceased 4–12 months after detection of the abnormality. CGH allowed us to narrow the consensus region of overlap to 1q25-31. Two cases showed over-representation of 8q with a common region in 8q22-24 containing CMYC. Both patients presented with stage IVB disease and died 12 and 13 months, respectively, after diagnosis due to rapid disease progression. Amplification of CMYC has mainly been reported in solid tumors,55 but also in some cases of chronic lymphocytic leukemia40,56 and follicle center cell lymphoma,42 demonstrating that its activation via amplification represents a mechanism of disease progression in a wide spectrum of malignant disorders. Loss of material of chromosome 17, especially of 17p, was the most frequent chromosomal loss observed in the present study. In two cases investigated by FISH, only one hybridization signal for a P53/17p13.1-specific probe was found in the majority of interphase cells. All patients had relapsed with rapidly progressive lymphoma. In accordance with our findings, alterations involving 17p or P53 have been associated with disease progression and resistance to treatment in a variety of lymphoid malignancies.57–61 Alterations of P53 have also been described in a considerable proportion of gastric MALT lymphomas31,62 and splenic lymphomas of possible marginal zone origin28 and have been associated with highgrade transformation of gastric MALT lymphomas.31 Comparing the CGH results with data obtained by karyotypic analysis, overlapping results were found for most of the non-balanced abnormalities. Additionally, in 13 of the 25 cases, CGH revealed chromosomal imbalances, which were not detected by conventional cytogenetic analysis, but could be confirmed by FISH or Southern blot analysis in all cases subsequently investigated. Most of these cases revealed complex karyotypes including marker chromosomes and/or cytogenetically unidentifiable chromosomal material. On the other hand, CGH failed to detect trisomy 3, trisomy 18, and deletion 7q in three cases with a low number of tumor cells bearing these abnormalities (15–30%), as shown by interphase FISH. Similar discrepancies have been described pre-


viously40,41,44,63 and are thought to result from the facts that: (1) CGH detects chromosomal imbalances in the tumor as a composite, whereas conventional cytogenetic analysis shows abnormalities of specific subclones that proliferate in culture; (2) cytogenetically not identifiable chromosomal material such as marker chromosomes may appear as chromosomal gain with CGH; and (3) if less than 50% of tumor cells bear the abnormality, the respective profiles are likely not to reach the thresholds for over- or under-representation. The data presented here, underline the properties of CGH in detecting chromosomal imbalances and amplifications in non-Hodgkin’s lymphoma and demonstrate that CGH, cytogenetic analysis, and FISH complement each other well. The characteristic pattern of chromosomal gains and losses observed in MZBCL is in agreement with its classification as a distinct disease entity and may point to chromosomal regions involved in the pathogenesis of these neoplasms. Acknowledgements We are grateful to Reinhilde Thoelen, Lutgarde Polleunis, Betty Emanuel and the technicians of the leukemia laboratory and the molecular genetic laboratory for their expert technical assistance. We also thank Dr E Schoenmakers for stimulating discussions and providing the YAC probes and Rita Logist for her help in preparation of the manuscript. Judith Dierlamm was supported by a grant from the Deutsche Krebshilfe, Dr Mildred Scheel Stiftung fu¨r Krebsforschung. Carla Rosenberg and Tom Bakker-Schut were supported by the Post-Graduate School for Pathophysiology of Growth and Differentiation (OPGD). References 1 Isaacson PG, Wright DH. Malignant lymphoma of mucosa-associated lymphoid tissue. Cancer 1983; 52: 1410–1416. 2 Sheibani K, Sohn CC, Burke JS, Winberg CD, Wu AM, Rappaport H. Monocytoid B-cell lymphoma. A novel B-cell neoplasm. Am J Pathol 1986; 124: 310–318. 3 Schmid C, Kirkham N, Diss T, Isaacson PG. Splenic marginal zone cell lymphoma. Am J Surg Pathol 1992; 16: 455–466. 4 Harris NL, Jaffe ES, Stein H, Banks PM, Chan JKC, Cleary ML, Delsol G, De Wolf-Peeters C, Falini B, Gatter KC, Grogan TM, Isaacson PG, Knowles DM, Mason DY, Mueller-Hermeling H-K, Pileri SA, Piris MA, Ralfkiaer E, Warnke RA. A revised European– American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood 1994; 84: 1361–1392. 5 Isaacson PG, Norton AJ. Extranodal Lymphomas. Churchill Livingstone: Edinburgh, 1994. 6 Sheibani K, Burke JS, Swartz WG, Nademanee A, Winberg CD. Monocytoid B-cell lymphoma: clinicopathologic study of 21 cases of a unique type of low grade lymphoma. Cancer 1988; 62: 1531–1538. 7 Ngan B-Y, Warnke RA, Wilson M, Takagi K, Cleary ML, Dorfman RF. Monocytoid B-cell lymphoma: a study of 36 cases. Hum Pathol 1991; 22: 409–421. 8 Pittaluga S, Verhoef G, Criel A, Wlodarska I, Dierlamm J, Mecucci C, Van den Berghe H, De Wolf-Peeters C. ‘Small’ B-cell nonHodgkin’s lymphomas with splenomegaly at presentation are either mantle cell lymphoma or marginal zone cell lymphoma. Am J Surg Pathol 1996; 20: 211–223. 9 Fisher RI, Dahlberg S, Nathwani BN, Banks PM, Miller TP, Grogan TM. A clinical analysis of two indolent lymphoma entities: mantle cell lymphoma and marginal zone lymphoma (including the mucosa-associated lymphoid tissue and monocytoid B-cell subcategories): a Southwest Oncology Group study. Blood 1995; 85: 1075–1082.

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