The purpose of this study was to document the frequency and distribution of karyotypic changes present at diagnosis in 103 non-MALT marginal zone cell lymphoma (MZL) patients. This cytogenetic analysis of a large cohort extends previous observations and allows the identification of new cytogenetic features. Abnormalities identified in more than 15% of patients included +3/+3q (37%), 7q deletions (31%), +18/+18q (28%), 6q deletions (19%), +12/+12q (15%) and 8p deletions (15%). Trisomy 3/3q, 7q deletions, +18 and +12 were seen in different combinations in more than 30% of patients in comparison to 2% in lymphocytic lymphomas/chronic lymphocytic leukemias, 1% in mantle cell lymphomas and 7% in follicular lymphomas. The marked propensity of these abnormalities to be recurrently associated with the same tumoral clone of individual karyotypes allowed the delineation of a cytogenetic profile that may help to distinguish non-MALT MZL among other mature B-cell neoplasms. If +3/3q, +12/+12q, and 6q, 7q and 8p deletions were significantly associated with clinical prognostic factors previously reported to influence survival and time to progression, patients displaying these abnormalities did not experience a significantly shorter time to progression.
Among the new small B-cell lymphomas (SBCL) listed in the recent World Health Organization (WHO) classification, marginal zone lymphomas (MZL) have been separated into three distinct entities including primary nodal, primary splenic, and extranodal lymphoma of the mucosa-associated lymphoid tissue (MALT) type.1 Few series, including a limited number of patients, have described karyotypic changes associated with non-MALT MZL, due in part to difficulty in producing analyzable metaphases.2, 3, 4 In addition to trisomies of chromosomes 3, 12 and 18 which are the most frequent aberrations, translocations such as t(2;7)(p12;q21-22) involving the cyclin-dependent kinase 6 (CDK6) gene, t(6;14)(p21;q32) leading to deregulated cyclin D3 expression, t(3;14)(q27;q32) involving BCL6 locus and t(11;14)(q13;q32) involving the cyclin D1 gene have also been nonrandomly reported but with lower incidences.5 The present study is focused on the occurrence of karyotypic changes in a comprehensive series of 103 non-MALT MZL cases, from a single Institution, that have been diagnosed using stringent histological, morphologic and immunologic criteria.
Materials and methods
Patient selection and tissue samples
Cases were selected from 285 patients with MZL referred in the Hematology Department between March 1993 and March 2003. Selection criteria consisted in (1) a diagnosis of non-MALT MZL confirmed on biopsy material, (2) absence of previous treatment for the disease and (3) identification of abnormal metaphases. A total of 158 patients responded to the first step of selection, 133 to the second and 103 fulfilled all criteria. Patients with primary extra-nodal localizations were excluded. All cases were classified according to the WHO classification. Immunologic characterization was performed on fresh frozen sections, paraffin sections, imprints and/or cell suspensions. The monoclonal antibodies (Ab) used were directed against CD3, CD5, CD19, CD20, CD23 (Dako, Carpinteria, CA, USA) and CD10 (Becton Dickinson, San Jose, CA, USA) or consisted in anti-IgG, anti-IgA, anti-IgM, anti-IgD, anti-κ and anti-λ (Dako, Carpinteria, CA, USA).
Staging procedure and clinical data
Initial staging procedures included age, stage and sites of involvement, computed tomography of the thorax, abdomen and pelvis, gastrointestinal tract endoscopic examination in cases with symptoms, bone marrow (BM) aspiration and biopsy, serum lactic dehydrogenase (LDH) and β2-microglobulin, peripheral blood (PB) count with morphologic examination. Blood involvement was defined by the presence of abnormal lymphocytes or a lymphocyte count greater than 4 × 109/l. Performance status (PS) was evaluated according to the Eastern Cooperative Oncology Group (ECOG) scale. Patients were staged according to the Ann Arbor system. All patient's files were, retrospectively, analyzed to collect complete information about disease history. Patients were treated according to physician standard practice.
Cytogenetic studies were performed on fresh tissue samples used for morphologic and immunologic investigations in 66 cases. In the remaining 37 patients with BM biopsy-proven diagnosis, BM and/or –PB were karyotyped. Methods for cytogenetic analysis have been previously reported.6 Chromosomal abnormalities were described according to the International System for Human Cytogenetic Nomenclature.7 An average of 15–30 metaphases were examined to document the features of the abnormal clones. Additional fluorescent in situ hybridization (FISH) experiments were performed according to standard methods, as previously reported.8 Patients with unidentifiable marker chromosomes were studied for the detection of masked trisomies 3, 12 and 18. An additional 52 patients were investigated using painting probes (Cambio, Cambridge, UK), centromeric and specific probes (Qbiogene, IllKrich, France) to confirm some abnormalities.
In the same time period, 722 cases diagnosed as small lymphocytic lymphoma or chronic lymphocytic leukemia (SLL/CLL, 462 pt), mantle cell lymphoma (MCL, 94 pt) and follicular lymphoma (FL, 166 pt) with clonal karyotypic abnormalities were studied. This group of SBCL was used to evaluate the incidence of some particular chromosomal changes.
A chi-square (χ2) test was used to determine significant differences in the distribution of the chromosomal aberrations with the clinical and biological parameters used for the staging procedure. Overall survival (OS) was defined as the interval of time between diagnosis (first biopsy) and death or last follow-up. Freedom-from-progression (FFP) survival was defined as the duration from the onset of therapy to the date of first relapse or disease progression or last follow-up. Survival was analyzed according to the Kaplan–Meier method and differences between groups were evaluated with the log-rank test (P<0.05).
As shown in Table 1, the group consisted of 49 male and 54 female subjects with a median age of 64 years (extremes 29 and 89 years). With regard to the localization of the involved sites at diagnosis, 77 patients presented a spleen enlargement consistently associated to BM or PB infiltrations but without nodal involvement, whereas eight patients had isolated enlarged peripheral lymph nodes. According to the WHO description, these patients were classified as primary splenic and primary nodal non-MALT MZL, respectively. A disseminated disease including splenic, nodal and/or extra nodal localizations was observed in 18 patients. These cases probably represented late stage of the disease for either the splenic or nodal non-MALT MZL subtypes.
Immunohistochemical and flow cytometry analysis showed expressions of CD19 and/or CD20 in all cases. MZL is usually a CD5−, CD10− and CD23− proliferation, but in spite of the classical morphological aspect, some cases were found with the presence of one of these antigens. No case displayed a positive reaction for CD10 antigen. A double expression for CD5 and CD23 was observed in nine (9%) patients (Table 2). Of note, CD5 positivity, when scored positive by flow cytometry, was low or absent on histological analysis in all but three cases.
Most karyotypes (99%) were pseudo diploid or pseudo near diploid. A single abnormal clone was detected in 48 (47%) cases, whereas 55 (53%) cases displayed evidence of clonal evolution with related sideline clones. If all chromosomes were involved in numerical or structural changes, only abnormalities that occurred at the 15% or greater level were analyzed (Table 3). Owing to their low incidences in the whole series, the lymphoma-related translocations were separately evaluated and no statistical analysis could be performed.
Numerical and structural changes
Trisomy 3 was identified in 38 patients and in all but two, it was found in the stemline clone. In five cases, it occurred as an isolated abnormality but clonal evolution was evident in sideline(s). A total of 16 cases (42%) presented with an additional chromosome 3, whereas in 22 cases (58%) additional material of the long arm of chromosome 3 (+3q) was found to be involved in different unbalanced translocations leading to the presence of a derivative chromosome. These cases, including six patients previously reported,6 were further studied by FISH using probes specific for the centromeric and telomeric (3q29–qter) sequences of chromosome 3. In all cases, the over-representation of the 3q region was demonstrated. Only two cases, who presented a +del/add(3)(p13–14), had retained both the centromeric and telomeric sequences of chromosome 3. Conventional analysis supplemented by FISH experiments allowed the delineation of a 3q24–3qter over-represented region. Additional FISH experiments identified multiple partners in the derivative chromosomes (5q, 6p, 7q, 8p, 8q, 10q, 12q, 13p, 14p, 18p, 19q). Chromosome 7q deletions were the second most frequent change occurring in 32 patients, always in the stemline clone. In nine patients, it was an isolated abnormality. A total of 24 cases resulted from interstitial or terminal deletions, whereas in eight cases chromosomal material of unknown origin was attached and replaced the normal 7q region. The consensus deleted region was delineated on karyotype between bands 7q33 and 7q35. Trisomy 18 was identified in 29 patients. It was the sole change in one patient (case 6, Table 2). Six cases demonstrated trisomy for the long arm of chromosome 18, resulting from an isochromosome 18q in three patients and from unbalanced translocations involving chromosomes 1p36, 3q21 and 6p12. An 18q12–18qter over-represented region was delineated. Deletions of the long arm of chromosome 6 were observed in 20 cases. Overall, there was no single region of minimal loss. However, when deletions were stratified according to the interstitial (eight cases) vs terminal (12 cases) criteria, two deleted regions could be defined involving bands q22–q23.1 and bands q26–qter, respectively. Trisomy 12 was observed in 15 patients, occurring as a whole trisomy in 11 cases. In all but three cases, it was seen in the stemline clone. In three cases, +12 was the sole clonal change. These cases demonstrated a negative immunophenotypic expression for the CD5. A 12q13–12q21 over-represented region was delineated. Abnormalities involving chromosome 8 and leading to an 8p deletion were observed in 15 patients. An isochromosome 8q was identified in three cases.
Lymphoma-associated translocations occurred with a low incidence in the whole series (13%). The typical MCL translocation t(11;14) and the t(3;14)(q27;q32) or variants were observed in four cases, each. One of the t(11;14) cases has been previously reported.9 Three patients presented a t(9;14)(p13;q32). This translocation occurred in addition to a +3 and a 7q deletion in the first case, to a 7q deletion and a +18 in the second case, whereas a +3, a 7q deletion and a +18 were observed in the last case. Two patients presented a t(14;19)(q32;q13.1). The abnormality occurred either with a +12 or a +3 and +18. Neither PAX-5 nor BCL3 status were investigated in these patients. A t(2;7)(p12;q21–22) translocation was identified in one patient. No other lymphoma-related translocation was observed. An unbalanced der(7)t(3;7) translocation was identified in four patients, leading to a trisomy 3q and to a 7q deletion. All rearrangements were confirmed using FISH experiments. In two of them, similar breakpoints were localized on the 3q13.1 and 7q31 regions (Figure 1).
Association between abnormalities and comparison with SLL/CLL, MCL and FL
When examining the distribution of +3, 7q deletions, +18 and +12 in the stemline clone of individual karyotypes, it turned out that 75 patients presented at least one of these four abnormalities. A total of 43 patients presented exclusively one change, whereas 26 and five cases presented at least two and three abnormalities, respectively. In addition, one patient exhibited all the four aberrations. As shown in Table 4, +3 and +18 were the more frequently associated changes (19 cases), followed by +3/7q deletions (10 cases), +3/+12 (six cases), whereas 7q deletions/+12, 7q deletions/+18 and +12/+18 occurred in four cases, each. With regard to the relative frequency of such associations in other SBCL, it appeared that among the 722 abnormal karyotypes, the associated abnormalities mentioned above were identified in eight (2%) SLL/CLL, one (1%) MCL and 12 (7%) FL.
Correlation with clinical presentation, OS and FFP
Correlations with distinct clinical features were observed for all abnormalities (Table 3). Four distinct clinical features were associated with only one chromosomal change: circulating lymphoma cells and high β2-microglobulin values with +3; spleen involvement with 7q deletions; and abdominal/thoracic LN with +12. Clinical outcome was fully available for 84 patients and after a median follow-up of 51 months, 46 patients experienced disease progression and 20 died. In this series, no classical clinical or biological criteria were found to significantly influence the time to progression, while age and PS were associated with a worst OS (respectively, P=0.013 and P=0.04). The cytogenetic alterations that occurred in more than 15% of the patients were not found to be associated with a particular outcome. Of note, the two patients with an identical t(3;7)(q13.1;q31) translocation experienced a rapid disease progression (respectively, 13 and 28 months) as compared to 44 months for the whole group.
Most of the recurrent abnormalities that characterized SBCL are translocations that usually involved immunoglobulin (Ig) loci, IgH (14q32), Igκ (2p12) and Igλ (22q11). The main rearrangements included the t(11;14) which is observed in more than 90% of MCL and the t(14;18) which represent the hallmark of FL. Unlike the previous subtypes, non-MALT MZL, which also represent mature small B-cell neoplasms, have been rather characterized by +3, +12, +18 and chromosome 7q deletions than by recurrent translocations involving Ig gene loci.2, 3, 4, 5 If at least one of these four abnormalities was identified as a primary aberration in more than 70% of cases, the new cytogenetic finding of the present data is the marked propensity of those changes to occur in different combinations within individual karyotypes. Associations of +3/3q, 7q deletions, +12 and +18 were observed in virtually 30% of patients, +3/+18 being the most frequently associated changes. Despite their higher frequencies in non-MALT MZL, those abnormalities are also observed in a large variety of other SBCL. Trisomy 3 is encountered in 50% of MCL,10 +12 is recurrently associated with SLL/CLL,11 +18 is identified in 20% of FL,12 and 7q deletions have been reported in either SLL/CLL, MCL and lymphoplasmacytoid lymphomas.13 However, the consistent association of these four chromosomal changes in nearly 1/3 of individual karyotypes has to be underlined in non-MALT MZL as compared in SLL/CLL, MCL and FL in which the same features are observed in only 2%, 1 and 7% of cases, respectively. Thus, as compared to t(11;14) and t(14;18) which represent the hallmark of MCL and FL, such a characteristic cytogenetic profile in non-MALT MZL constitutes a distinctive relevant feature that may contribute to the diagnosis. Nine patients with a typical morphologic presentation were observed with an atypical immunophenotype consisting of the co-expression of the CD5 and CD23 antigen. In mature SBCL, SLL/CLL and MCL are the two entities thought to correspond to CD5/CD23-positive B cells, even if the CD23 expression is usually weakly positive in MCL. Among the CD5/CD23-positive patients, no cases revealed neither partial or complete +12 nor t(11;14). Conversely, eight patients (89%) presented one of the most frequent abnormalities of non-MALT MZL, and associations such as +3/+18 and +3/7q deletion were observed in four of them (cases 1, 3, 5, 8). These findings strongly support that cytogenetic investigations remain a powerful tool for differential diagnosis in atypical cases of non-MALT MZL. Four patients (4%), including one case previously reported, presented a t(11;14). Eventhough this abnormality is considered to be characteristic of MCL, it has already been described in about 15% of patients with splenic MZL.14 We recently demonstrated that some MZL cases harboring the t(11;14) had a transcriptional profile similar to that of other MZL and distinct from the one observed in MCL patients.9 Furthermore, this abnormality can be missing in rare cases of MCL.15 Therefore, the existence of a t(11;14) in rare cases of lymphoma with marginal zone morphologic characteristics should not lead to refuse the diagnosis of non-MALT MZL.
To encompass the technical problems due to the difficulty in producing abnormal metaphases, most of the reported data have turned to interphase FISH studies, loss of heterozygosity analysis (LOH) and comparative genomic hybridization (CGH) experiments.2, 3, 4, 5, 16, 17, 18 However, FISH and LOH only provide answers to specific predetermined questions and CGH analysis, which is limited to the detection of unbalanced rearrangements, requires more than 50% abnormal cells. In addition, these technologies underestimate the actual distribution of changes within a tumoral clone and preclude the analysis of the clonal evolution of tumors. By the means of interphase FISH studies the incidence of +3 reached from 18 to 60% of cases.5 In most reports, this abnormality is investigated using a chromosome 3 centromeric probe. In the present data, nearly 60% of cases with trisomy 3 resulted from a partial +3q. In these cases, FISH experiments demonstrated that the centromeric sequences were rarely involved, as opposed to the telomeric sequences which were identified in three copies in 20 out of the 22 (91%) patients. Therefore, the use of telomeric rather than centromeric probes seems to be more appropriate in detecting +3/+3q by FISH analysis and could explain the variability in the percentages previously reported. Using CGH experiments, two over-represented regions have been narrowed to 3q23–25 and 3q25–29.5, 16 The localization of the proximal and distal breakpoints of the over-represented region in the present series was similar to those reported by others. However, in contrast to karyotype, CGH analysis did not identify the partner chromosomes in cases where +3q resulted from translocations. It is of particular importance because chromosome 7q was identified here as a partner in four cases. In two of them, similar breakpoints were observed, allowing the identification of a new recurrent unbalanced der(7)t(3;7)(q13;q31) translocation, which might be associated with an adverse outcome. In addition, even if +3 and 7q deletion have been previously reported to be mutually exclusive,4 the present data shows that both abnormalities were identified in 10 patients. Significant differences in the frequency of 7q deletions in patients with or without spleen enlargement (P=0.017) were observed. These findings strongly support previous data indicating that patients with 7q deletions frequently present with splenic MZL.16, 17
If clinical and biological prognostic factors such as BM involvement, elevated levels of β2-microglobulin, presence of an M component and high lymphocyte counts have been previously described to influence the OS and the FFP in non-MALT MZL, the prognostic significance of chromosomal changes has not yet been clearly evaluated. Up to now, such studies have been hindered by the small size of series and by the methodologies used for cytogenetic analysis. In only one report, correlations between 7q deletions and tumor progression have demonstrated a borderline statistical significance.18 Although non-MALT MZL remains mostly indolent, histological transformations into aggressive lymphoma can occur in some cases.19 In the present data, +3, 6q deletions, 7q deletions, 8p deletions and +12 were significantly associated with one of the adverse prognostic factors mentioned above. We were, however, unable to show that patients with these abnormalities experienced a significantly shorter time to progression, but the numbers of patients with those features were small. It is also possible that other genetic changes may influence patient's outcome in non-MALT MZL.20
In conclusion, further cytogenetic studies in non-MALT MZL patients are required and will necessitate a dual strategy, involving complete karyotype evaluation as well as prospective analysis of karyotypic evolution in individual cases at sequential time points. This comprehensive approach will facilitate molecular identification of the critical genetic events associated with disease development and progression and may allow a more accurate stratification of patients into prognostic categories. Conversely, the combined analysis of chromosomal abnormalities with the gene expression changes characterized in this disease may help in identifying critical pathways that are deregulated by gene deletions or amplifications. In addition, this may set the stage for the development of new clinical interventions in the form of targeted gene therapies.
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Boonstra R, Bosga-Bouwer A, Van Imhoff GW, Krause V, Palmer M, Coupland RW et al. Splenic marginal zone lymphomas presenting with splenomegaly and typical immunophenotype are characterized by allelic loss in 7q31–32. Mod Pathol 2003; 16: 1210–1217.
Mateo M, Mollejo M, Villuendas R, Algara P, Sanchez-Beato M, Martinez P et al. 7q31-32 allelic loss is a frequent finding in splenic marginal zone lymphoma. Am J Pathol 1999; 154: 1583–1589.
Berger F, Felman P, Thieblemont C, Pradier T, Baseggio L, Bryon PA et al. Non-MALT marginal zone B-cell lymphomas: a description of clinical presentation and outcome in 124 patients. Blood 2000; 95: 1950–1956.
Algara P, Mateo MS, Sanchez-Beato M, Mollejo M, Navas IC, Romero L et al. Analysis of the IgV(H) somatic mutations in splenic marginal zone lymphoma defines a group of unmutated cases with frequent 7q deletion and adverse clinical course. Blood 2002; 99: 1299–1304.
This work was supported by grants from la Ligue Nationale contre le Cancer (Comité du Rhône).
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