Although reciprocal chromosomal translocations are not typical for B-cell chronic lymphocytic leukemia (B-CLL), we identified the novel t(1;6)(p35.3;p25.2) in eight patients with this disorder. Interestingly, all cases showed lack of somatically mutated IgVH. Clinical, morphological, immunologic, and genetic features of these patients are described. Briefly, the age ranged from 33 to 81 years (median: 62.5 years) and the sex ratio was 6M:2F. Most of the patients (6/8) presented with advanced clinical stage. Therapy was required in seven cases. After a median follow-up of 28 months, five patients are alive and three died from disease evolution. Three cases developed transformation into diffuse large B-cell lymphoma. Translocation t(1;6) was found as the primary karyotypic abnormality in three patients. Additional chromosomal aberrations included changes frequently found in unmutated B-CLL, that is, del(11)(q), trisomy 12 and 17p aberrations. Fluorescence in situ hybridization analysis performed in seven cases allowed us to map the t(1;6) breakpoints to the 1p35.3 and 6p25.2 chromosomal bands, respectively. The latter breakpoint was located in the genomic region coding for MUM1/IRF4, one of the key regulators of lymphocyte development and proliferation, suggesting involvement of this gene in the t(1;6). Molecular characterization of the t(1;6)(p35.3;p25.2), exclusively found in unmutated subtype of B-CLL, is in progress.
B-cell chronic lymphocytic leukemia (B-CLL) is the most frequently occurring leukemia in adults in Western countries. This disease arises mainly from accumulation of mature-looking malignant monoclonal B cells in peripheral blood, bone marrow, and lymph nodes. B-CLL has a heterogeneous clinical course. Although the median survival is around 10 years, the prognosis is extremely variable in individual patients, ranging from a very short to a normal lifespan. The clinical staging systems, independently developed by Rai et al1 and Binet et al2 in the early 1980s, are based on easily obtainable biological and clinical variables and represent useful prognostic parameters.1, 2, 3, 4 However, these systems are not accurate enough to identify subgroups of patients with progressive disease, leading to continuous efforts to identify new prognostic factors. Different groups showed that the mutational status of the variable region of the immunoglobulin genes (IgVH) identifies distinct disease subsets and has a prognostic value3, 4, 5, 6, 7, 8 which is independent of clinical stage.3, 4 Patients with unmutated IgVH genes suffer from a disease possibly arising from a naïve B cell and have a poor outcome, whereas those displaying mutated IgVH genes have a more indolent disease possibly arising from a memory B cell.9
Clonal aberrations have been found by conventional cytogenetic techniques in about 50% and by interphase fluorescence in situ hybridization (FISH) in about 80% of the patients with B-CLL. The most recurrent are deletions at 6q21, 11q22–q23, 13q14, 17p13, and trisomy 12.10, 11, 12 Most importantly, these aberrations are prognostically significant, that is deletions involving chromosome 11q and 17p are associated with adverse outcome, whereas del(13q) and trisomy 12 are associated with favorable and intermediate outcome, respectively.3, 8, 10, 13 Balanced chromosomal translocations are rare in CLL and, when present, are usually mediated by immunoglobulin gene loci, that is, t(14;19)(q32;q13), t(2;14)(p13;q32), t(12;22)(p13;q11), or t(14;18)(q32;q21).14 Recent studies showed that chromosomal and/or genomic abnormalities in CLL are related to the IgVH mutation status,15 but were of independent prognostic information in multivariate analysis.3, 8
Given the importance of recognizing novel clinicobiological and cytogenetic associations in B-CLL, we identified a previously unreported recurrent chromosomal aberration, the t(1;6)(p35.3;p25.2), and characterized clinical, hematological, and genetic features associated with this translocation.
Materials and methods
Leukemic patients with the t(1;6)(p36∼p33;p25∼p23) were collected from databases of two Belgian Cytogenetic Institutes.
Cytogenetic and FISH analyses
Cytogenetic analyses were performed on peripheral blood and/or bone marrow cells cultured for 72 h after stimulation with 12-O-tetradecanoyl phorbol-13-acetate (TPA). In some patients, lymph node was analyzed after overnight culture without mitogen. In each case at least six R- and/or G-banded metaphases were karyotyped. Results are reported according to guidelines of International System for Cytogenetic Nomenclature.16 Cytogenetic specimens stored at −20°C were used for further FISH analysis.
FISH followed previously reported protocols.17, 18 The selected BAC and PAC clones obtained from the Roswell Park Cancer Institute libraries (http://www.chori.org/BACPAC) are listed in Table 3. DNA probes were labeled either indirectly with biotin-11-dUTP and digoxigenin-16-dUTP, or directly with SpectrumOrange-dUTP and SpectrumGreen-dUTP (Vysis, Downers Grove, IL, USA). Between 2 and 6 abnormal metaphases were analyzed in each experiment. When possible, these studies were supplemented by interphase FISH analysis. FISH data were collected on a Leica DMRB (Wetzlar, Germany) fluorescence microscope equipped with a triple band-pass filter and a cooled black and white charged couple device camera (Photometrics, Tuscon, AZ, USA) run by Quips SmartCaptureTM FISH Imaging Software (Vysis, Downers Grove, IL, USA).
VH mutation analysis
Rearranged IgVH genes were amplified using FR1IgH polymerase chain reaction method.19 A mixture of the FR1 consensus primer and the antisense primers JH1245, JH3, and JH6 were used. Monoclonality was detected with heteroduplex analysis.20 Direct sequencing was performed on both DNA strands using the same primers as in the amplification. The sequencing procedure was performed using an ABI PRISM 3100 variant (Applied Biosystems), following the manufacturer's procedure. Mutations were identified by comparison of the sequence (adjusted with SeqMan II) with the germline IgH sequence of IMGT/V-Quest (http://imgt.cines.fr). The cutoff of 98% homology to the germline sequence was chosen to discriminate between mutated (less than 98% homology) and unmutated (98% or more homology) cases.
Translocation t(1;6) was identified in eight patients who suffered from B-CLL and in one patient suffering from a mature B-cell proliferation which could not be characterized further due to lack of archival material. These cases represent 0.5% of cases with B-CLL, and 0.75% of cytogenetically aberrant cases with B-CLL referred for cytogenetic evaluation during the same period (1993–2003).
Salient clinical characteristics at the time of diagnosis of the eight patients with B-CLL are summarized in Table 1. There were six males and two females ranging in age from 33 to 81 years (median: 62.5 years). White blood cell counts were usually not high, ranging from 10.1 to 103 × 109/l (median 12.22 × 109/l). In seven of eight patients with available data, LDH values were normal. Two patients presented with Binet stage A, four with stage B, and two with stage C.1, 2 None of the patients initially presented with massive adenopathy. Disease progression to stage B was observed in patient no. 1 at 62 months after diagnosis. Seven patients required therapy for disease evolution shortly (median 0.5, range 0.5–62 months) after diagnosis. Even when a response to first-line therapy was achieved, two or more lines of chemotherapy had to be administered because of refractoriness to first-line therapy or relapse. Of note, two patients underwent autologous stem cell transplantation in complete remission. One (case 1) relapsed 6 months after transplantation and died from infection. The second (case 4) remains in remission and even achieved molecular remission 5 months after transplantation. Follow-up from the time of diagnosis ranged from 14 to 98 months (median: 28 months). Three patients (nos. 1, 2, and 7) died from disease or disease-related complications, 76, 29, and 95 months after diagnosis, respectively. Three patients (nos. 2, 3, and 7) developed histologically well-characterized diffuse large B-cell lymphoma (DLBCL) (Richter's syndrome), 28, 96, and 95 months after diagnosis.
Morphological review showed infiltration by small mature lymphocytes, compatible with CLL,21 in all patients. Cases were further classified as morphologically typical (nos. 1, 2, 6, and 8) or atypical (nos. 3, 4, 5, and 7).22 On immunophenotyping, all cases had a Matutes/Moreau score between 4/5 and 5/5,23 indicative for CLL. Surface immunoglobulin staining for light chains showed a kappa positivity in three and a lambda positivity in four cases.
Cytogenetic analysis of bone marrow and/or peripheral blood, and/or lymph node samples was performed at the time of diagnosis (seven cases) and/or during disease progression (five cases). Results are detailed in Table 2. The t(1;6), shown in Figure 1, was detected at the time of diagnosis in seven patients and during disease evolution in one patient (case 7 for which there is no karyotype available at diagnosis). Abnormal metaphases represented 30 to 100% of analyzed cells. Complexity of karyotype varied and ranged from a simple karyotype with the t(1;6) as the sole karyotypic aberration to more complex karyotype containing up to seven additional chromosomal aberrations. Altogether, the t(1;6) was found as the sole karyotypic change in three cases (cases 2, 3, and 5), including one with additional related subclones (case 2).
Chromosomal abnormalities found in addition to t(1;6) include trisomy 12 (three cases: nos. 1, 6, and 7), del(11q) (two cases: nos. 1 and 4), and aberrations of chromosome 17p (two cases: nos. 2 and 8), 9q (three cases: nos. 2, 7, and 8) and 17q (two cases: nos. 2 and 4). Interestingly, lymph nodes could be analyzed in two patients (nos. 3 and 7) at the time of evolution to DLBCL, and showed the presence of the t(1;6), indicating a clonal relationship between both diseases.
Original cytogenetic description of the t(1;6) differed from case to case and breakpoints were roughly assigned to the 1p34p36 and 6p25p23 regions. In order to characterize both translocation breakpoints on a genomic level and to check whether the t(1;6) found in each case reflects the same molecular aberration, we undertook metaphase FISH analysis with sets of 1p and 6p clones. Initial FISH mapping was performed on patients 1 and 4 with the highest range of abnormal metaphase spreads. As shown in Table 3, six distally mapped 1p clones (RP1-159A19 to RP11-290H1) hybridized to the der(6), while hybridization signals from the remaining proximal clones (RP11-442N24 to RP4-657M3) were found on the der(1). These results indicate that the 1p breakpoint occurred at the p35.3 region flanked by RP11-290H1 and RP11-442N24. The reciprocal 6p breakpoint was mapped using nine BAC/PAC clones covering the first 4.7 Mb region of 6p, including GS-36I2, the first-generation subtelomeric probe (NIH consortium), and GS-62I11, the second-generation subtelomeric probe.24 Six of these clones (including GS-36I2) hybridized to the der(6), two (including GS-62I11) hybridized to the der(1) and one clone, RP11-233K4, showed a split hybridization signal on both derivative chromosomes heralding the 6p25.2 breakpoint. Critical probes flanking/spanning the affected 1p and 6p regions (RP11-290H1 and RP11-442N24, and RP11-233K4, respectively) were further applied to the remaining six cases with available cytogenetic material. The same hybridization pattern was found in all analyzed cases establishing that the translocation breakpoints were identical at the FISH level of resolution. Examples of FISH analyses are illustrated in Figure 2 (supplemental material). Interphase FISH using the same probe set identified the presence of the t(1;6) in the lymph node of patient 3 at the time of transformation into DLBCL.
IgVH mutational analysis was performed in all cases. The monoclonal IgVH rearrangements were amplified and sequenced. They were potentially functional. In all analyzed cases, an unmutated status of IgVH was found. The most frequently used VH family was VH3 (three cases), besides two VH4, two VH1, and one VH5 case (Table 1).
We report here clinical, cytological, immunophenotypic, and genetic features of eight B-CLL cases characterized by the novel t(1;6)(p35.3;p25.2). Particularly interesting is that these cases showed lack of somatically mutated IgVH genes, a feature that has been associated with adverse prognosis and which underlies a subdivision of B-CLL into two, mutated and unmutated, subtypes.3, 4, 5, 7, 8, 10 Indeed, clinicobiological features of the presently reported patients with t(1;6) are in line with those described for unmutated B-CLL. Briefly, the 6M:2F sex ratio found in our series is similar to the 3:1 ratio observed in unmutated B-CLL and contrasts with the 1:1 ratio of mutated B-CLL.3 Moreover, our patients presented with quite advanced clinical stage at diagnosis and a therapy requiring disease. Massive organomegaly or elevated LDH values were not noted. The follow-up period in the present series is too short to determine the prognostic value of the t(1;6). Clinical features at presentation, and death of three patients from their disease 76, 29, and 95 months after diagnosis, however, indicate rather a progressive than benign clinical course. In addition, evolution to DLBCL further demonstrates the disease aggressivity. Interestingly, a recent study suggests that clonal transformation of B-CLL to DLBCL occurs only in unmutated CLL.25
It has been shown that B-CLL is associated with several nonrandom cytogenetic aberrations of which del(11)(q), trisomy 12, and 17p aberrations are the most frequently found in unmutated subtype of CLL.3, 26 Interestingly, these abnormalities were also present in the karyotypes of, respectively, 2, 3, and 2 cases with a t(1;6) (Table 2). Since the t(1;6) was identified in all abnormal metaphases and occurred as a sole chromosomal aberration in the stemline in three cases, this translocation likely represents a primary molecular aberration. It is worth noting that trisomy 12, when found, appeared in all cells with a t(1;6), while del(11)(q) and add/der(17)(p) were found in subclones, or appeared at time of relapse (patient no. 1).
In the first step towards identification of pathognomonic genes affected by this translocation, we undertook metaphase FISH studies of both t(1;6) breakpoints. Using sets of BAC/PAC clones selected for the terminal 1p and 6p regions, we mapped both breakpoints in the respective 1p35.3 and 6p25.2 chromosomal bands. The 1p breakpoint was narrowed down to the approximately 500 kb region that maps between the BACs RP11-290H1 and RP11-442N24 (www.ensembl.org). Unfortunately, the incomplete molecular characterization of this genomic area currently prevents further FISH investigation of this breakpoint region. The 6p breakpoint was successfully localized in the region inserted into the BAC clone RP11-233K4 (AL589962) that hybridized to both derivatives of t(1;6). This clone contains the following gene sequences: tel, DUSP22, MUM/IRF4, and part of SEC5, cen. Some FISH data suggest that the breakpoint occurs within or just upstream of MUM1/IFR4, making this gene a key candidate gene. MUM1/IFR4, the multiple myeloma-1/interferon regulatory factor-4 gene, is a member of a gene family known to be active in the control of B-cell proliferation and differentiation. Experimental studies indicate that IRF4 is essential for mature T- and B-lymphocyte function.27 Iida et al28 identified MUM1/IFR4 as the partner of the multiple myeloma-associated t(6;14)(p25;q32), which results in IgH-mediated activation of this gene and demonstrated oncogenic activity of MUM1/IRF4 in vitro. Further molecular studies are required to determine whether MUM1/IRF4 is indeed targeted by the t(1;6) in B-CLL and to identify the 1p35.3 partner gene.
In conclusion, a novel recurrent cytogenetic aberration, the t(1;6)(p35.3;p25.2), associated with B-CLL has been identified. This novel aberration, exclusively found in unmutated CLL and often observed with high-risk chromosomal aberrations (i.e. deletions of 11q and 17p), seems associated with an aggressive clinical course.
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This work presents research results of the Belgian program of Interuniversity Poles of attraction initiated by the Belgian State, Prime Minister's Office, Science Policy Programming. The scientific responsibility is assumed by the authors. The study was partially supported by the Fund for Scientific Research of Flanders (FWO - Vlaanderen), Grant no. G.0338.01.
We thank U. Pluys for excellent technical assistance, P. Vannuffel for performing IgVH analyses, V.Deneys for performing immunophenotypic analyses, and R. Logist for secretarial assistance.
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)
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Michaux, L., Wlodarska, I., Rack, K. et al. Translocation t(1;6)(p35.3;p25.2): a new recurrent aberration in ‘unmutated’ B-CLL. Leukemia 19, 77–82 (2005). https://doi.org/10.1038/sj.leu.2403543
- B-cell chronic lymphocytic leukemia (B-CLL)
- chromosomal translocation, t(1;6)
- unmutated subtype
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