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Chronic Lymphocytic Leukemia With t(2;14)(p16;q32) Involves the BCL11A and IgH Genes and Is Associated With Atypical Morphologic Features and Unmutated IgVH Genes

C. Cameron Yin MD, PhD, Katherine I-Chun Lin MD, Rhett P. Ketterling MD, Ryan A. Knudson, L. Jeffrey Medeiros MD, Lynn L. Barron, Yang O. Huh MD, Rajyalakshmi Luthra PhD, Michael J. Keating MD, Lynne V. Abruzzo MD, PhD
DOI: http://dx.doi.org/10.1309/AJCPXLY46UPFLISC 663-670 First published online: 1 May 2009


The t(2;14)(p16;q32) has been reported previously in only 12 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). The clinicopathologic features have been incompletely described. We describe 6 new cases of CLL/SLL with t(2;14)(p16;q32). All had marrow involvement, 4 had absolute lymphocytosis, 4 had lymphadenopathy, and 3 had hepatosplenomegaly. All showed atypical lymphocyte morphologic features with plasmacytoid differentiation and irregular nuclei; 3 had increased prolymphocytes. Flow cytometry demonstrated typical immunophenotypes in 5 and an atypical immunophenotype in 1. All expressed ZAP70; 5 assessed showed unmutated IgVH genes. Karyotyping identified t(2;14)(p16;q32) as the sole abnormality in 1, primary abnormality in 2, and part of a complex karyotype in 3. Fluorescence in situ hybridization analysis revealed BCL11A/IgH rearrangement in all. After chemotherapy, 3 patients died of disease and 3 were alive with disease (median follow-up, 80 months). We conclude that CLL/SLL with t(2;14) (p16;q32) and BCL11A/IgH rearrangement is characterized by atypical morphologic features and unmutated IgVH genes.

Key Words:
  • Chronic lymphocytic leukemia/small lymphocytic lymphoma
  • t(2;14)(p16;q32)
  • IgVH mutation
  • BCL11A
  • Atypical morphologic features

Many B-cell lymphomas and leukemias are characterized by chromosomal translocations that involve the immunoglobulin genes.1 The best known of these include the t(8;14) (q24;q32), t(2;8)(p12;q24), and t(8;22)(q24;q11) in Burkitt lymphoma/leukemia; the t(14;18)(q32;q21) in follicular lymphoma; the t(3;14)(q27;q32) in diffuse large B-cell lymphoma; and the t(11;14)(q13;q32) in mantle cell lymphoma. The translocations most commonly involve the immunoglobulin heavy chain (IgH) locus at chromosome 14q32. Juxtaposition of IgH enhancer elements with the translocation partner gene drives the inappropriate overexpression of the partner gene product. Unlike many other mature B-cell neoplasms, chromosome translocations are relatively infrequent in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL).

The t(2;14)(p16;q32) is a rare, recurrent chromosomal aberration that has been identified predominantly in B-cell malignancies, including CLL/SLL,214 acute lymphoblastic leukemia,1517 and non-Hodgkin lymphoma.18,19 To date, only 12 cases of CLL/SLL with the t(2;14)(p13~16;q32) have been reported.214 Of the 4 previously reported cases with complete clinical histories, at diagnosis, the patients had marked lymphocytosis, generalized lymphadenopathy, and splenomegaly,2,4,6 and 2 were children.2,4 Molecular genetic studies, performed in 4 cases, demonstrated that this translocation juxtaposes the BCL11A gene at 2p16 with the IgH gene at 14q32.3,5,7,20,21 These results suggest that t(2;14)-positive cases of CLL/SLL represent a distinct clinicopathologic entity.

We report the clinical, morphologic, immunophenotypic, cytogenetic, and molecular genetic features of 6 adults with CLL/SLL with the t(2;14), the largest series to date. Furthermore, we verify that the t(2;14)(p16;q32) in CLL/SLL involves the BCL11A and IgH genes in all cases.

Materials and Methods

Case Selection

We searched the files of the Clinical Cytogenetics Laboratory, Department of Hematopathology, the University of Texas M. D. Anderson Cancer Center, Houston, for cases with the t(2;14). We identified 6 cases (0.14%) of mature B-cell neoplasms, all classified as CLL/SLL, out of 4,422 untreated and previously treated patients with CLL/SLL seen at our institution between November 1, 1993, and January 31, 2008. The diagnosis of CLL/SLL was based on morphologic and immunophenotypic criteria as specified in the World Health Organization classification.22 The clinical data were obtained by review of medical records.

Morphologic Examination

We reviewed bone marrow aspirate smears, touch imprints, and clot and core biopsy specimens in all cases. Differential 500-cell counts were performed on bone marrow touch imprints or bone marrow aspirate smears stained with Wright-Giemsa. Particular attention was given to the cytologic features of the lymphocytes with respect to atypical morphologic features, including indented or clefted nuclei, plasmacytoid features, and the presence of prolymphocytes. The percentages of plasmacytoid lymphocytes, defined as cells with eccentrically placed nuclei, moderately abundant cytoplasm, and/or cartwheel-like chromatin, and lymphocytes with indented nuclei were recorded. Bone marrow aspirate clot and decalcified core biopsy specimens were routinely processed and stained with H&E. Bone marrow cellularity and the pattern of lymphocytic infiltration were assessed in the bone marrow core biopsy specimens. The pattern was classified as nodular, interstitial, diffuse, or a combination of these patterns. Excisional lymph node biopsy specimens available in 3 cases were routinely processed, and H&E-stained sections were examined.

Immunophenotypic Analysis

Immunophenotypic analysis by flow cytometry was performed on a bone marrow aspirate specimen from each patient using a FACScan or FACSCalibur instrument (Becton Dickinson Biosciences, San Jose, CA) as described previously.23 The lymphocyte population was gated using right-angle side scatter and CD45 expression. The panel of monoclonal antibodies included reagents specific for CD3, CD5, CD10, CD11c, CD19, CD20, CD22, CD23, CD38, CD79b, FMC-7, immunoglobulin light chains, and surface IgM and IgD.

We calculated the CLL score in each case according to the system proposed by Matutes and colleagues24 and subsequently modified by Moreau and colleagues.25 Scores were based on 5 variables: expression of dim surface immunoglobulin (1 point), CD5 (1 point) and CD23 (1 point); dim or absent CD22/CD79b (1 point); and absent FMC-7 (1 point). The immunophenotype of cases with a score of 4 or 5 was considered to be typical. Cases that deviated from this pattern of antigen expression were considered to have an atypical immunophenotype.

Immunohistochemical stains were performed on formalin-fixed, paraffin-embedded bone marrow core biopsy or clot sections. Staining with a monoclonal antibody specific for ZAP70 (dilution 1:500, Upstate Cell Signaling Systems, Lake Placid, NY) was performed as described previously.26 The cases with nuclear staining in more than 20% of the neoplastic cells were considered positive. Nuclear staining of admixed nonneoplastic T cells served as an internal control. We also performed immunohistochemical staining for cyclin D1 (SP4, Lab Vision/NeoMarkers, Fremont, CA).

Conventional Cytogenetic and Fluorescence In Situ Hybridization Analyses

In all cases, conventional cytogenetic analysis was performed on metaphase cells prepared from bone marrow aspirate or peripheral blood specimens cultured for 24 hours without mitogens and for 72 hours with lipopolysaccharide (LPS), using standard techniques. Giemsa-banded meta-phases were analyzed, and the results were reported using the International System for Human Cytogenetic Nomenclature, 2005. In 1 case (case 4), the conventional cytogenetic study was performed at an outside laboratory, and we reviewed the report and karyograms.

Fluorescence in situ hybridization (FISH) analysis for common cytogenetic abnormalities associated with CLL was performed in 3 cases on interphase nuclei obtained from cultures of bone marrow or peripheral blood using a panel of probes designed to detect trisomy 12 and deletions of 13q14.3, 13q34, TP53 at 17p13, and ATM at 11q22.3, according to the manufacturer’s instructions (Vysis/Abbott Laboratories, Des Plaines, IL).

FISH analysis to assess the BCL11A and IgH genes was performed in all cases using a dual-color, dual-fusion probe on tissue sections prepared from formalin-fixed, paraffin-embedded bone marrow clot sections or on inter-phase nuclei prepared from short-term cultures of bone marrow or whole peripheral blood. Direct-labeled FISH probes were designed from bacterial artificial chromosomes (BAC) and validated according to standard methods.27 For the BCL11A gene (2p16.1), 3 BAC clones (RP11-158I21, RP11-440P5, and RP11-416L21) were labeled with Spectrum Orange-dUTP (Vysis/Abbott Laboratories) to make a 467-kb probe. For the IgH gene (14q32), 11 BAC clones (RP11-151B17, CTD-3148C6, CTD-3074B5, RP11-47P23, CTD-2011A5, RP11-44N21, RP11-1087P8, RP11-521B24, RP11-731F5, RP11-815D20, and RP11-417P24) were labeled with Spectrum Green-dUTP (Vysis/Abbott Laboratories) to make an approximately 1.4-Mb probe. Under the fluorescence microscope, the BCL11A signal is red, the IgH signal is green, and the fusion product appears yellow.

Tissue sections were incubated at 90°C for 15 minutes and then deparaffinized with xylene at room temperature (RT) (2 times, 15 minutes each), dehydrated in 100% ethanol at RT for 5 minutes, and incubated at 80°C in 10 mmol/L of citric acid (pH 6.0) for 45 minutes. Following this, the slides were immersed in 2x standard saline citrate (SSC) at 37°C for 5 minutes followed by digestion in 0.2% pepsin working solution (1.2 g of pepsin/600 mL 0.9% NaCl, pH 2.5) at 37°C for 48 minutes. Immediately after digestion, the tissue sections were dehydrated using 70%, 85%, and then 100% ethanol at RT for 2 minutes each. A working solution of each probe was prepared by mixing 3 μL of concentrated probe with 7 μL of LSI/WCP hybridization buffer (Vysis/Abbott Laboratories). The working solutions were then mixed (2:1 green/red ratio) and applied to the target areas. The slides were coverslipped, sealed with rubber cement, denatured in a HYBrite system (Vysis/Abbott Laboratories) at 80°C for 5 minutes, and incubated overnight at 37°C in a humidified oven. Following hybridization, the slides were soaked in 2x SSC/0.1% NP-40 at RT to remove the coverslips, placed in 2x SSC/0.1% NP-40 at 74°C for 2 minutes, and then placed in 2x SSC/0.1% NP-40 at RT for 2 minutes. The tissue sections were stained with 4′-6,-diamidino-2-phenylindole (DAPI; Vysis/Abbott Laboratories) and coverslipped. Interphase nuclei prepared from short-term cultures of peripheral blood were subjected to standard FISH methods, as described previously.28 Both sample types were analyzed using standard fluorescence microscopy techniques.28

Somatic Mutation Status of the IgVH Genes

Sequence analysis of the immunoglobulin heavy chain variable region (IgVH) genes was performed in 5 cases using total RNA extracted from bone marrow aspirate material or peripheral blood, or DNA extracted from formalin-fixed, paraffin-embedded bone marrow clot sections, as described previously.29 To determine the level of somatic mutation, patients’ sequences were aligned to the germline sequences in the V-BASE 2 database.30 The IgVH mutation status was designated as unmutated if there were fewer than 2% mutations (≥98% homology to germline sequences) or as mutated if there were 2% or more mutations (< 98% homology to germline sequences) compared with the germline sequences.31


Clinical Findings

We identified 6 cases of CLL and t(2;14). The clinical and laboratory data are summarized in Table 1. There were 5 men and 1 woman, with a median age of 44 years (range, 39–65 years). Two patients (cases 1 and 2) were previously untreated. At the time of initial diagnosis at outside institutions, 2 patients (cases 5 and 6) had lymphadenopathy, 3 (cases 1, 3, and 4) had lymphocytosis detected on routine examination, and 1 (case 2) was found to have lymphocytosis during an evaluation for chest pain.

On evaluation at our institution, 4 patients (cases 1, 2, 4, and 6) reported systemic symptoms that included fever, fatigue, night sweats, and weight loss. Physical examination revealed generalized lymphadenopathy in 4 patients (cases 2, 4, 5, and 6) and splenomegaly in 3 patients (cases 2, 3, and 5). Four patients (cases 1–4) had an absolute peripheral blood lymphocytosis (lymphocyte count, range, 1,600–610,100/μL [1.6–610.1 x 109/L]; median, 36,900/μL [36.9 x 109/L]; reference range, 1,000–4,800/μL [1.0–4.8 x 109/L]). Four patients (cases 2–5) were anemic (hemoglobin level, range, 8.8–14.6 g/dL [88–146 g/L]; median, 13.1 g/dL [131 g/L]; reference range, 14.0–18.0 g/dL [140–180 g/L] for men and 12.0–16.0 g/ dL [120–160 g/L] for women), and 3 (cases 2–4) were thrombocytopenic (platelet count, range, 58–309 x 103/μL [58–309 x 109/L]; median, 156 x 103/μL [156 x 109/L]; reference range, 140–440 x 103/μL [140–440 x 109/L]). Serum lactate dehydrogenase levels were elevated in 4 patients (cases 2, 3, 4, and 6; range, 412-1,841 U/L; median, 671 U/L; reference range, 313-618 U/L), as were β2-microglobulin levels in all 6 patients (2.1–9.7 mg/L; median, 3.3 mg/L; reference range, 0.7–1.8 mg/L).

View this table:
Table 1

All patients received treatment with multiagent chemotherapy. In a median follow-up of 80 months from the time of initial diagnosis (range, 42–145 months), 3 patients were alive with disease and 3 died of disease (Table 1).

Morphologic Findings

The morphologic features of the bone marrow specimens are summarized in Table 2. Bone marrow aspirate smears showed a mixture of small and larger lymphocytes, with a subset of plasmacytoid lymphocytes, representing 12% to 22% (median, 20%) of total lymphocytes. Prolymphocytes ranged from 3% to 47% (median, 8%) and 3 patients (cases 2, 3, and 5) had increased (≥10%) prolymphocytes Image 1. In addition, all cases contained a subset of cytologically atypical lymphoid cells with indented and irregular nuclear contours, representing 6% to 18% (median, 15%) of total lymphocytes, and these lymphoid cells were conspicuous in 2 cases (cases 2 and 5). The bone marrow core biopsy specimens were hyper-cellular for age (range, 55%–95%; median, 75%). Lymphoid cells infiltrated the bone marrow in a diffuse (cases 2 and 4), interstitial (cases 1 and 5), nodular (case 6), or mixed interstitial and diffuse (case 3) pattern.

Three patients (cases 1, 5, and 6) underwent excisional lymph node biopsy. In 2 patients (cases 1 and 5), the lymph node biopsies were performed at the same time that the t(2;14) was identified in the bone marrow. In both cases, the lymph nodes demonstrated CLL/SLL with increased paraimmunoblasts/prolymphocytes. In 1 case (case 6), the lymph node specimen, obtained 37 months before the t(2;14) was identified in the bone marrow, showed typical CLL/SLL.

Immunophenotypic Findings

Immunophenotypic analysis performed by flow cytometry on bone marrow aspirate samples demonstrated that the neoplastic cells in all cases were positive for CD5, CD19, CD20 (5 moderate, 1 bright), CD23, surface monotypic immunoglobulin light chain (5 κ and 1 λ), and surface IgM/ IgD (5 dim, 1 moderate). In addition, the neoplastic cells were positive for CD38 in 5 (100%) of 5, CD79b (dim) in 2 (50%) of 4, CD11c in 3 (50%) of 6, and FMC-7 (dim) in 2 (33%) of 6 cases. In all cases, the cells were negative for CD3, CD10, and CD22. In 5 cases (cases 1, 2, 4, 5, and 6) the immunophenotype was typical, with a CLL score of 4 or 5. One case (case 3) had an atypical immunophenotype with a CLL score of 3, with moderate expression of CD20 and surface IgM/IgD, as well as FMC-7.

View this table:
Table 2
Image 1

(Case 3) Morphologic findings. A, The bone marrow core biopsy specimen contains an interstitial infiltrate of generally small lymphoid cells (H&E, x200). B, The aspirate smear demonstrates that many of the cells are cytologically atypical, with irregular nuclear contours and increased prolymphocytes (Wright-Giemsa, x1,000).

Immunohistochemical stains performed on formalin-fixed, paraffin-embedded tissue sections of bone marrow core biopsy or clot specimens demonstrated that all neoplasms were positive for ZAP70, with more than 90% of cells positive (Table 2). All were negative for cyclin D1.

Cytogenetic Findings

The results of conventional karyotypic analysis are summarized in Table 3. The t(2;14)(p16;q32) was identified in all cases. In bone marrow specimens (cases 1–5), the t(2;14) was identified in unstimulated and LPS-stimulated cultures; in the peripheral blood specimen (case 6), it was identified in the LPS-stimulated culture. The t(2;14) was the sole abnormality in 1 case (case 5). In 2 cases, the t(2;14) was the primary abnormality, with subclones that contained additional clonal abnormalities (cases 2 and 4). The t(2;14) was part of a complex karyotype in 3 cases, 2 of which also showed trisomy 12 (cases 1, 3, and 6). The t(2;14) was identified at the time of initial diagnosis of CLL in 1 case (case 2) Image 2A. The results of conventional cytogenetic analysis performed at the time of diagnosis were not available for the other cases.

In 3 patients (cases 2, 4, and 6), FISH analysis was performed to assess for abnormalities of 11q22.3, 13q14.3, 13q34, and 17p13.1 and trisomy 12. The studies were performed on interphase nuclei from bone marrow (case 2) or peripheral blood (cases 4 and 6) specimens obtained within 1 month of the conventional cytogenetic study that had demonstrated the t(2;14). Additional abnormalities that were not apparent in the conventional karyotype were identified in 1 case (case 4), which showed del(13)(q14.3) and del(17)(p13).

View this table:
Table 3
Image 2

Cytogenetic findings. A (Case 2), Conventional cytogenetic analysis demonstrating the t(2;14)(p16;q32). B (Case 1), Fluorescence in situ hybridization analysis was performed on interphase cells using a dual-color, dual-fusion probe to BCL11A at 2p16 and IgH at 14q32. Reciprocal translocation involving the BCL11A and IgH genes generates yellow fusion signals on both derivative chromosomes. The normal chromosome 14 shows a green signal, and the normal chromosome 2 shows a red signal. A cell that contains the translocation is on the left; a cell without the translocation is on the right.

FISH analysis to assess the BCL11A and IgH genes was performed in all cases using a dual-color, dual-fusion probe on tissue sections prepared from formalin-fixed, paraffin-embedded bone marrow clot sections (cases 2, 3, and 5) or interphase nuclei prepared from short-term cultures of bone marrow (case 1) or whole peripheral blood (cases 4 and 6). All cases demonstrated yellow fusion signals, indicating the presence of BCL11A/IgH translocation Image 2B.

Somatic Mutation Status of the IgVH Genes

Sequence analysis of the IgVH genes was performed using total RNA extracted from bone marrow aspirate material and peripheral blood (case 2) or peripheral blood only (cases 4 and 6) or DNA extracted from formalin-fixed, paraffin-embedded bone marrow clot sections (cases 1 and 5). These studies demonstrated that the IgVH genes lacked somatic mutations in all 5 cases assessed (Table 2).


The BCL11A (B-cell lymphoma/leukemia 11A) gene was discovered as a site of retroviral integration in the mouse and was originally named EVI9.32,33 Activation of this gene by retroviral integration into the mouse genome induced myeloid leukemias.32,33 The human homolog, BCL11A, was discovered in CLL cases with the t(2;14).6 The gene was originally assigned to region 1, band 3 of the short arm of chromosome 2, ie, 2p13. It has subsequently been reassigned to 2p16.1. In normal tissues, with few exceptions, expression of BCL11A protein is restricted to hematopoietic cells. It is expressed at low levels, primarily in the nuclei of B cells in the germinal centers and mantle zones, a subset of cells in the interfollicular areas of tonsil and lymph nodes, and in the splenic marginal zones.34 Only rare BCL11A+ cells are observed in the bone marrow and fetal thymus.34 BCL11A encodes a novel zinc finger protein that interacts directly with BCL6, a known human B-cell proto-oncogene product that serves a crucial role in lymphocyte development.35 These findings suggest that BCL11A is a proto-oncogene that may be activated through chromosomal translocation or amplification, leading to myeloid leukemias in mice and lymphoid malignancies in humans. Our finding that BCL11A is the translocation partner of the IgH gene in CLL cases with the t(2;14) supports this hypothesis.

In this series, the t(2;14)(p16;q32) was identified in 6 cases and was the sole abnormality in 1 case. In 2 cases, the t(2;14) was the primary abnormality, with subclones that contained additional clonal abnormalities, and it was part of a complex karyotype in 3 cases. In 5 of 6 cases, the translocation was identified in metaphases obtained from unstimulated cultures and from cultures that had been stimulated with mitogen. In 2 cases, the t(2;14) was identified before the patients had received treatment; the results of cytogenetic studies obtained before treatment were not available for the other 4 patients. Similarly, Kuppers and colleagues7 identified the t(2;14) as the sole abnormality in 3 of 4 cases. In the 12 previously reported cases with t(2;14)(p13;q32), the t(2;14) was found at initial diagnosis in 2 patients, both children with CLL,24 and after treatment in 2 patients.9,10 The timing of the identification of the t(2;14) with respect to diagnosis or treatment was not reported for the other 8 patients.6,8,1114 In aggregate, these findings suggest that the t(2;14) is an early event in CLL cases that harbor this translocation.

All cases in our series showed atypical cytologic features. In the bone marrow, all showed a heterogeneous mixture of small and larger cells, all showed plasmacytoid differentiation, 3 showed increased prolymphocytes (cases 1, 2, and 5), and 2 showed conspicuous nuclear indentations (cases 2 and 5). By using a system proposed by Matutes and coworkers36 that defines more than 15% of cells having cleaved nuclei and/or lymphoplasmacytic features as atypical morphologic features, atypical morphologic features have been correlated with trisomy 12, which was identified in 2 cases in our study. Atypical morphologic features also have been correlated with an unfavorable prognosis.37,38

Despite the presence of atypical cytologic features, the immunophenotype of these neoplasms was typical in 5 of 6 cases. CLL has a characteristic immunophenotype: expression of CD5, CD19, CD23, dim (weak) intensity of surface immunoglobulin and CD20, weak or negative CD79b/CD22, and absence of FMC-7.22 A scoring system based on the immunophenotype has been developed to distinguish CLL from other B-cell neoplasms.24,25 This system is helpful in most cases. However, a small but significant percentage of patients with CLL have an atypical immunophenotype.39,40 Five cases had a score of 4 or 5 indicating a typical immunophenotype. One case had an atypical profile, with moderate expression of CD20 and surface IgM/IgD and expression of FMC-7. All cases were negative for cyclin D1.

Molecular genetic studies of the IgVH somatic mutation status in CLL have identified 2 prognostic subtypes. Patients whose CLL cells lack somatic mutations have an unfavorable prognosis (median survival, 8 years) compared with patients whose CLL cells have somatic mutations (median survival, 25 years).4143 Gene expression profiling studies subsequently identified ZAP70 as being expressed preferentially in unmutated CLL cases.44,45 ZAP70 is a member of the syk tyrosine kinase family that is expressed predominantly by T cells and natural killer cells.44,45 It is essential for T-cell activation, but also for B-cell maturation from the pro-B to pre-B cell stage.44,45 ZAP70 expression, assessed by flow cytometry or immunohistochemical analysis, has been used as a surrogate marker for the mutation status of the immunoglobulin genes.26,41,43 Immunohistochemical staining for ZAP70 demonstrated that all 6 cases with the t(2;14) were positive. Sequence analysis of the IgVH genes demonstrated that all 5 cases assessed lacked somatic mutations, similar to the results of Kuppers and colleagues.7 The association of the t(2;14) with unmutated IgVH somatic mutation status raises the possibility that these patients may have a more aggressive clinical course. However, our number of patients is too small to conclude this with certainty.

We have described 6 patients with CLL/SLL associated with the t(2;14)(p16;q32). In addition, we have confirmed that the t(2;14) involves the BCL11A and IgH genes. These neoplasms may represent a distinct clinicopathologic entity characterized by atypical morphologic features, ZAP70 expression, and unmutated IgVH genes.


We thank Gary Lu, MD, for critically reviewing the manuscript and Navnit Mitter, MD, for providing additional cytogenetic information.


  • Supported in part by a grant from the CLL Global Research Foundation, Houston, TX.


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