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Chronic Lymphocytic Leukemia With t(14;19)(q32;q13) Is Characterized by Atypical Morphologic and Immunophenotypic Features and Distinctive Genetic Features

Yang O. Huh MD, Carmen D. Schweighofer MD, Rhett P. Ketterling MD, Ryan A. Knudson, Francisco Vega MD, PhD, Ji E. Kim MD, Rajyalakshmi Luthra PhD, Michael J. Keating MB, BS, L. Jeffrey Medeiros MD, Lynne V. Abruzzo MD, PhD
DOI: http://dx.doi.org/10.1309/AJCPOEFP3SLX6HXJ 686-696 First published online: 1 May 2011

Abstract

The t(14;19)(q32;q13) involving the IGH@ and BCL3 loci is an infrequent cytogenetic abnormality detected in B-cell malignancies. We describe the clinicopathologic, cytogenetic, and molecular genetic characteristics of 14 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) with t(14;19)(q32;q13). All patients (10 men and 4 women) had lymphocytosis; 10 had lymphadenopathy. Blood and bone marrow lymphocytes were predominantly small, but cytologically and immunophenotypically atypical. In all cases, t(14;19) was found in the neoplastic stem line; it was the sole abnormality in 4. Ten cases showed additional cytogenetic abnormalities, including trisomy 12 in 9 and complex karyotypes in 7. Fluorescence in situ hybridization demonstrated IGH@/BCL3 fusion gene in all cases. In all cases, the IGHV genes were unmutated, but only 7 expressed ZAP70. Seven cases preferentially used IGHV4-39. Our results indicate that t(14;19)(q32;q13) identifies a subset of CLL/SLL with distinctive clinicopathologic and genetic features. Furthermore, t(14;19) may represent an early, possibly primary, genetic event.

Key Words:
  • Chronic lymphocytic leukemia
  • t(14;19)(q32;q13)
  • BCL3
  • Atypical morphology
  • IGHV somatic mutation

Reciprocal chromosomal translocations have a major role in the pathogenesis of many lymphoid neoplasms and often define disease entities. For example, translocations involving MYC are considered a primary genetic event in Burkitt lymphoma. Similarly, the t(11;14)(q13;q32) and t(14;18)(q32;q21) are found in almost all cases of mantle cell and follicular lymphoma, respectively. Unlike these well-defined translocations in B-cell malignancies, the clinical significance of the t(14;19) (q32;q13) is unclear, in part because it is uncommon, reported to occur in fewer than 0.1% of all B-cell neoplasms.1 In most cases, this translocation juxtaposes the BCL3 gene at chromosome 19q13 with the immunoglobulin heavy chain (IGH@) locus at chromosome 14q32. Since it was first reported by Bloomfield and coworkers2 in 1983, many B-cell neoplasms associated with the t(14;19)(q32;q13) have been classified as chronic lymphocytic leukemia (CLL).1,3,4 However, these cases often have features that are unusual for CLL, including younger patient age, an aggressive clinical course, atypical morphologic and immunophenotypic features, and an association with trisomy 12.1,3

To more clearly define the clinicopathologic features of cases classified as CLL that were associated with t(14;19)(q32;q13), we performed clinicopathologic, cytogenetic, and molecular genetic studies on 14 cases identified at The University of Texas M. D. Anderson Cancer Center, Houston (MDACC). These studies included immunohistochemical staining for the BCL3 protein, fluorescence in situ hybridization (FISH) for IGH@/BCL3 rearrangement, and DNA sequence analysis of the immunoglobulin heavy chain variable region (IGHV) genes. We confirmed that these cases are characterized by atypical morphologic and immunophenotypic features and are frequently associated with trisomy 12. We demonstrated by FISH analysis that the t(14;19) results from fusion of the IGH@ and BCL3 genes and that BCL3 protein is overexpressed. Sequence analysis of the IGHV genes demonstrated that all cases lacked somatic mutations, and half used the same heavy chain variable region family, IGHV4-39. Our results indicate that CLL cases with the t(14;19)(q32;q13) have distinctive clinicopathologic and genetic features. Furthermore, the t(14;19) may represent an early and, possibly, primary genetic event.

Materials and Methods

Case Selection

We searched the database of the Clinical Cytogenetics Laboratory, Department of Hematopathology, MDACC, for cases with t(14;19)(q32;q13) or the variant translocations t(2;19)(p13;q13) and t(19;22)(q13;q11) for the period between January 1993 and August 2009. We identified 13 cases with t(14;19)(q32;q13) and 1 case with t(10;14;19)(q22;q32;q13.2), all classified as CLL/small lymphocytic lymphoma based on morphologic and immunophenotypic criteria, 4 of which have been reported previously.5 We identified no cases with t(2;19) or t(19;22) variant translocations. We obtained clinical data by review of the medical records.

Morphologic Examination

In each case, we reviewed the peripheral blood and bone marrow aspirate smears, touch imprints, and aspirate clot and core biopsy specimens. Peripheral blood smears were stained with May-Grünwald-Giemsa stain, and a manual 100-cell differential WBC count was performed. Differential 500-cell counts were performed on bone marrow aspirate smears or touch imprints stained with Wright-Giemsa stain. Particular attention was given to lymphocyte cytologic features with respect to atypical features, including indented or clefted nuclei, plasmacytoid features, and the presence of prolymphocytes, large cells, or immature-appearing cells. Bone marrow aspirate clot and decalcified core biopsy specimens were routinely processed and stained with H&E. Bone marrow cellularity was assessed, and the pattern of lymphocytic infiltration was classified as nodular, interstitial, diffuse, or a combination of these patterns. Excisional lymph node biopsy specimens and core needle biopsy specimens of extramedullary tissue infiltrates, when available, were routinely processed, and H&E-stained sections were examined.

Immunophenotypic Analysis

Immunophenotypic analysis by flow cytometry was performed on bone marrow aspirate specimens assessed by 3- or 4-color flow cytometry, as described previously.6 Briefly, the lymphocyte population was gated using right-angle light scatter and CD45 expression. The panel of monoclonal antibodies used included reagents specific for CD5, CD10, CD11c, CD19, CD20, CD22, CD23, CD38, CD45, CD79b, FMC7, and immunoglobulin light chains. In total, 10,000 events were acquired by a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA), and the data were analyzed using CellQuest software (Becton Dickinson).

For each case, we calculated the CLL score according to the system of Matutes and coworkers7 as subsequently modified by Moreau and coworkers.8 Scores were based on 5 variables: expression of dim surface immunoglobulin, 1 point; CD5, 1 point; CD23, 1 point; dim or absent CD22/CD79b, 1 point; and absent FMC-7, 1 point. Cases with a score of 4 to 5 were considered to have typical immunophenotypes; cases that deviated from this pattern of antigen expression were considered to have atypical immunophenotypes.

Immunohistochemical stains were performed using routinely fixed, paraffin-embedded tissue sections of lymph node and other extramedullary tissue core biopsy specimens, as described previously.9 The panel included antibodies specific for CD3 (dilution 1:100; DAKO, Carpinteria, CA), CD5 (4C7, dilution 1:20; Labvision/NeoMarkers, Fremont, CA), CD10 (56C6, dilution 1:70; Leica Microsystems, Bannockburn, IL), CD20 (L26, dilution 1:1,400; DAKO), BCL2 (100, dilution 1:200; BioGenex, San Ramon, CA), Ki-67 (MIB-1, dilution 1:100; DAKO), and cyclin D1 (SP4, dilution 1:40; Labvision/Neomarkers). Staining with monoclonal antibodies specific for ZAP70 (dilution 1:100, Upstate Cell Signaling Systems, Lake Placid, NY) and BCL3 (clone 1E8, dilution 1:50; Novocastra, Newcastle upon Tyne, England) was performed, as described previously.10,11

Somatic Mutation Analysis of the IGHV Genes

Sequence analysis of the IGHV genes was performed using total RNA extracted from bone marrow aspirate material or DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue sections obtained from bone marrow aspirate clot sections, as described previously.12 To determine the degree of somatic mutation, patient’s IGHV sequences were aligned to germline sequences using the international ImMunoGeneTics (IMGT) information system and database tools (IMGT/V-Quest, http://imgt.org).13,14 The IGHV somatic 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.15

Conventional Cytogenetic and FISH Analyses

Conventional cytogenetic analysis was performed on metaphase cells prepared from bone marrow aspirate specimens from all patients that were cultured for 24 hours without mitogens or 72 hours with lipopolysaccharide, using standard techniques. We analyzed 20 Giemsa-banded metaphases, and the results were reported using the International System for Human Cytogenetic Nomenclature.

FISH analysis for common abnormalities associated with CLL was performed on interphase nuclei obtained from cultured bone marrow cells using a probe panel designed to detect deletions of 11q22.3 (ATM), 13q14.3 (D13S319), 17p13.1 (TP53), and trisomy 12 (12p11.1-q11) according to the manufacturer’s instructions (Abbott Molecular, Abbott Park, IL). FISH analysis for the t(11;14)(q13;q32) was performed using the LSI IGH/CCND1 dual-color, dual fusion translocation probe (Abbott Molecular) on interphase nuclei obtained from cultured bone marrow cells or on FFPE bone marrow biopsy clot sections.

FISH analysis to detect the IGH@/BCL3 fusion gene was performed in all cases using a dual-color, dual fusion probe on interphase nuclei obtained from cultured bone marrow cells or on FFPE tissue sections obtained from bone marrow aspirate clot specimens. FISH analysis was also performed on FFPE tissue sections obtained from lymph node excisional or core biopsy specimens. Direct-labeled FISH probes were designed from bacterial artificial chromosomes and validated according to standard methods, as described previously.16 FISH analysis using these probes was performed on FFPE bone marrow clot specimens using standard methods.17

Results

Clinical Findings

Clinical and laboratory data are summarized in Table 1. There were 10 men and 4 women, with median ages of 51.5 years at the time of diagnosis of CLL (range, 29–79 years) and 52.5 years (range, 29–84 years) at admission to the MDACC. Seven patients were previously untreated (cases 2, 4–6, 9, 11, and 12) and 6 had received therapy before coming to MDACC (cases 1, 3, 7, 8, 10, and 14). Treatment information was unavailable for 1 case (case 13). Of the 7 previously untreated patients, 5 were admitted to MDACC within 1 year of diagnosis and 2 at 5 and 6 years after diagnosis. The 6 previously treated patients were admitted to MDACC at a median of 5.5 years after diagnosis (range, 1–11 years).

Physical examination at MDACC revealed lymphadenopathy in 9 patients (cases 1–5, 7–9, and 12) and splenomegaly in 1 (case 8). All patients had absolute lymphocytosis (lymphocyte count range, 5,000–162,000/μL [5–162 × 109/L]; reference range, 1,000–4,800/μL [1–4.8 × 109/L]), with a median lymphocyte count of 55,500/μL (55.5 × 109/L). In 9 patients, anemia was found (cases 2, 5, 6, 8–12, and 14), with a median hemoglobin level of 11.6 g/dL (116 g/L; range, 9.1–16.5 g/dL [91–165 g/L]; reference range, men, 14.0–18.0 g/dL [140–180 g/L]; women, 12.0–16.0 g/dL [120–160 g/L]). The platelet count was normal in 13 patients, and 1 patient (case 11) had slight thrombocytopenia (platelet count, 120 × 103μ/L [120 × 109/L]); the a median platelet count was 191 × 103μ/L (191 × 109/L; range, 120–364 × 103μ/L [120–364 × 109/L]; reference range, 140–440 × 103/μL [140–440 × 109/L]). Nine patients reported systemic symptoms (cases 1–5, 7, 10, 11, and 14), most frequently night sweats and fatigue. The serum lactate dehydrogenase level was increased in 11 patients (cases 1–3, 5–10, 12, and 14), with a median level of 895 U/L (14.9 μkat/L; range, 574–1,895 U/L [9.6–31.6 μkat/L]; reference range, 313–618 U/L [5.2–10.3 μkat/L]). The serum β2-microglobulin level was elevated in all patients, with a median of 3.5 mg/L (range, 2.2–7.8 mg/L; reference range, 0.7–1.8 mg/L).

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Table 2

At MDACC, 10 patients received treatment with multi-agent chemotherapy (cases 1–10), and 2 patients are currently being observed (cases 11 and 12). Follow-up data were unavailable for 2 patients (cases 13 and 14). At a median follow-up of 36.5 months for 12 patients, 7 were alive, 1 without evidence of disease (case 5), and 6 with residual disease: 1 in lymph nodes (case 4), 2 in bone marrow (cases 2 and 6), and 3 in bone marrow and lymph nodes (cases 8, 11, and 12). An inguinal lymph node in 1 patient (case 11) demonstrated herpes simplex virus lymphadenitis while the patient was being observed without treatment. Five patients have died, 3 of CLL (cases 3, 7, and 9), 1 of diffuse large B-cell lymphoma (DLBCL; Richter syndrome) 67 months after the initial diagnosis of CLL (case 10), and 1 of metastatic carcinoma of unknown primary origin while the t(14;19)+ small B-cell leukemia was in remission, 66 months after diagnosis (case 1).

Morphologic Findings

The morphologic features are summarized in Table 2. In all cases, lymphocytes in peripheral blood and bone marrow aspirate smears were cytologically atypical, and the lymphoid infiltrates in bone marrow were cytologically heterogeneous Image 1A, Image 1B, Image 1C, and Image 1D. In all cases, most cells were small, often with nuclear indentations, and with occasional admixed medium and large cells. In 1 case (case 2), the cells ranged from small to medium-sized, with barely discernible cytoplasm, resulting in a high nuclear/cytoplasmic ratio (Image 1A). Prolymphocytes were increased in only 1 case (case 7), in which they constituted 10% to 12% of the lymphoid cells. The cellularity of the bone marrow core biopsy specimens ranged from 25% to 90% (median, 50%). Lymphoid cells infiltrated the bone marrow in an interstitial or interstitial and partially diffuse pattern. The infiltrates were composed of predominantly small cells with occasional admixed larger cells (Images 1C and 1D). Proliferation centers were identified in the bone marrow in 1 case (case 4).

Lymph node biopsy specimens were obtained from 6 patients during the course of the disease. Excisional lymph node biopsy specimens from 3 patients (cases 4, 5, and 13) showed complete architectural effacement in a diffuse pattern by small lymphocytic lymphoma with proliferation centers Image 1E. In 2 of these cases (cases 5 and 13), large cells were numerically increased, but there was no morphologic evidence of DLBCL. In 1 patient (case 8), core needle biopsy specimens obtained from breast and pelvic masses showed diffuse infiltrates composed predominantly of small to medium-sized lymphocytes with admixed large cells, but with no morphologic evidence of DLBCL. However, in 1 case (case 10) a core needle biopsy of a right inguinal lymph node showed sheets of large B cells, indicating histologic progression to DLBCL, ie, Richter syndrome. In another patient (case 11), an excisional biopsy of an inguinal lymph node revealed herpes simplex virus lymphadenitis 1 year after admission to MDACC, which resolved following treatment with antiviral therapy (valacyclovir hydrochloride).

Immunophenotypic Findings

The results of immunophenotypic analysis by flow cytometry are summarized in Table 3. The CLL score, using the modified Matutes system, was 2 or 3 in all cases except for case 13, for which we lacked the data to determine the precise score. As expected for CLL, all cases were positive for B-cell antigens and CD5. All cases tested expressed CD79b, and 9 were positive for CD22 (cases 1–4, 6, 9, 11, 13, and 14). Three cases coexpressed surface IgM and IgD (cases 3, 6, and 10), and 3 expressed surface IgM without detectable IgD (cases 4, 7, and 9). Ten cases (cases 1, 4–9, and 12–14) expressed CD38. However, 8 cases were negative or dimly positive for CD23 (cases 1, 2, 4–7, 12, and 14), and 6 were positive for FMC7 (cases 2, 4, 5, 9, 11, and 12). Of the cases, 7 were negative for surface IgM and IgD (cases 1, 2, 5, 8, 11, 12, and 14); the 1 case that was assessed for expression of surface IgG (case 12) was brightly positive. Thus, all cases for which complete immunophenotypic data were available were atypical.

Image 1

A (Case 2), A peripheral blood smear shows predominantly small lymphoid cells with prominent indented nuclei (May-Grünwald-Giemsa, ×1,000). B and C (Case 14), Lymphoid cells in the peripheral blood smear vary in size and show irregular nuclear features (B, May-Grünwald-Giemsa, ×1,000). The corresponding bone marrow aspirate smear shows variably-sized lymphoid cells with frequent nuclear indentations (C, Wright-Giemsa, ×1,000). D and E (Case 4), The bone marrow contains a heterogeneous mixture of small and medium-sized lymphoid cells with indented nuclei (D, May-Grünwald-Giemsa, ×1,000). The lymph node architecture is effaced by a diffuse proliferation of predominantly small lymphoid cells with pale proliferation centers (E, H&E, ×400). F (Case 5), Lymphoid cells in a bone marrow core biopsy specimen show strong nuclear staining for BCL3 (×400).

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Table 3

Immunohistochemical stains performed on bone marrow biopsy specimens showed that the neoplastic cells in all cases were moderately to intensely positive for BCL3 in a nuclear pattern Image 1F. Seven cases were positive for ZAP70 (Table 2). Staining for Ki-67 revealed a low fraction of proliferating cells (<10%). The neoplastic cells were negative for cyclin D1 in all cases (data not shown). Immunohistochemical stains performed on lymph node biopsy specimens showed that the lymphoid cells were positive for CD5, CD20, and BCL2 and were negative for CD3, CD10, and cyclin D1.

Somatic Mutation Status of the IGHV Genes

Sequence analysis of the IGHV genes was performed using total RNA extracted from bone marrow aspirate material (cases 4–12 and 14) or DNA extracted from FFPE tissue sections obtained from bone marrow aspirate clot sections (cases 1–3 and 13; Table 2). These studies demonstrated that all cases were unmutated and 9 cases used IGHV4 family genes, including IGHV4-39 in 7 patients (cases 1, 2, 5, 8, and 12–14).

Cytogenetic Findings

The results of conventional karyotypic and FISH analyses are summarized in Table 4. In all cases, the t(14;19) was identified on samples obtained at the time of admission to MDACC; the results of cytogenetic studies before admission were unavailable. In all cases, the t(14;19) was identified in the stem line, the most basic clone of the neoplastic population. It was the sole abnormality in the stem line in 4 cases (cases 4 and 11–13). Seven cases showed a complex karyotype, in the stem line in 5 cases (cases 2, 5–7, and 9) and in a sideline (an additional deviating subclone) in 2 cases (cases 1 and 4). Trisomy 12 was identified in 9 cases (cases 1, 2, 3, 6, 8–10, 12, and 14). In 1 case (case 12) trisomy 12 was found in a sideline, but not in the stem line, suggesting that it was acquired as a secondary abnormality. Trisomy 12 was found in the stem line in 8 cases (cases 1, 2, 3, 6, 8–10, and 14), 4 of which demonstrated a complex karyotype (cases 1, 2, 6, and 9). A representative karyogram (case 6) is shown in Image 2.

FISH analysis was performed on cultured bone marrow cells in 9 cases with material available for analysis (cases 2, 5, and 7–13) to assess for common abnormalities in CLL, ie, deletions of 11q22.3, 13q14.3, 13q34, and 17p13.1 and trisomy 12. In all cases, the results of conventional cytogenetic and FISH analyses were concordant. By FISH analysis, 2 cases showed no abnormalities (cases 11 and 13), 5 cases showed only trisomy 12 (cases 2, 8–10, and 12), and 1 case showed only del(17)(p13.1) (case 5). In 1 case (case 7), FISH analysis showed absence of the signals for both probes on chromosome 13 (D13S319 and LAMP1) and absence of the signal for the TP53 probe on chromosome 17, consistent with the monosomies of chromosomes 13 and 17 identified on conventional cytogenetic analysis.

In all cases, FISH analysis for IGH@/BCL3 fusion gene, performed on cultured bone marrow cells (cases 7–12 and 14) or on FFPE bone marrow aspirate clot sections (cases 1–6), was positive for the rearrangement Image 3. FISH analysis performed on lymph node specimens in 3 cases (cases 4, 10, and 13) also showed IGH@/BCL3 fusion gene (data not shown). FISH analysis for the IGH@/CCND1 rearrangement, performed on cultured bone marrow cells (cases 2, 4, 5, and 7) or on FFPE bone marrow aspirate clot sections (cases 1 and 3), was negative for this rearrangement (data not shown).

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Table 4

Discussion

The contribution of the t(14;19)(q32;q13) to the pathophysiology of a subset of small B-cell neoplasms generally classified as CLL is unclear. We identified the t(14;19) as the sole abnormality in the most basic neoplastic clone in 4 patients, 3 of whom were previously untreated and underwent cytogenetic analysis within 1 year of diagnosis; treatment information was not available for the fourth patient. Consistent with other studies, we found an association between t(14;19)(q32;q13) and trisomy 12 in 9 of 14 cases. In 1 of these cases, the t(14;19) was the sole abnormality in the neoplastic stem line, and trisomy 12 was identified in a subclone. Similarly, Martin-Subero and coworkers18 identified the t(14;19) as the sole abnormality in 2 cases of CLL and in the stem line in 1 case that also demonstrated trisomy 12 in a subclone. Taken together, these findings suggest that, in at least a subset of cases, the t(14;19) is an early, possibly primary, event and that acquisition of the extra chromosome 12 occurs after the t(14;19).

It is interesting that only 1 neoplasm (of 9 evaluable using the CLL FISH panel) was found to have del(13)(q14), the most common cytogenetic abnormality in CLL, identified in at least 50% of cases by FISH analysis.19 This region contains the microRNA genes MIR15a and MIR16-1, whose products are important regulators of BCL2 expression and are believed to have an important role in the pathogenesis of the CLL cases that have this deletion.20 Our finding raises the possibility that cases with the t(14;19) have a pathogenesis distinct from that of common cytogenetic subtypes of CLL.

The t(14;19) has been reported in a wide variety of B-cell neoplasms other than CLL.4 In a recent European multicenter study, the largest to date, Martin-Subero and coworkers18 described 56 cases of B-cell neoplasms with the t(14;19) and IGH@/BCL3 fusion gene, which included 29 B-cell non-Hodgkin lymphomas (B-NHLs) in addition to 27 cases of CLL.18 The B-NHLs included a variety of histologic types: marginal zone lymphoma, splenic marginal zone lymphoma, follicular lymphoma, and DLBCL. These cases were characterized by a relatively large number of chromosomal changes and predominantly mutated IGHV genes. Cases classified as CLL demonstrated relatively fewer additional chromosomal changes, frequent trisomy 12, and unmutated IGHV genes.

Differences between our studies likely reflect differences in the patient populations. First, all of our cases had been classified as CLL with atypical morphologic and immunophenotypic features. Martin-Subero and coworkers,18 in contrast, were unable to assess the morphologic features of most of their CLL cases because the diagnostic material, in most cases peripheral blood smears, was unavailable for central review. Second, almost half of their cases were B-NHL, whereas our study included only atypical CLL cases. Because MDACC is a tertiary care center, most patients admitted with a diagnosis of lymphoma have had tissue biopsy specimens obtained elsewhere, and cytogenetic studies were not available for review. Thus, they would not have been included in the MDACC Clinical Cytogenetics Laboratory database. In contrast, patients who are referred with a diagnosis of CLL routinely undergo bone marrow sampling and a complete laboratory evaluation that includes cytogenetic studies.

Image 2

(Case 6), The karyogram demonstrates the t(14;19) (q32;q13) in a complex karyotype: 47,XY,–2,+12,add(14) (q32),t(14;19)(q32;q13.2),+mar. Both chromosomes 14 are abnormal. The abnormal chromosome 14 that is involved in the t(14;19) translocation is on the left. The abnormal chromosome 14 on the right has unidentified chromosomal material added at band q32.

Image 3

(Case 6) Fluorescence in situ hybridization analysis demonstrates IGH@/BCL3 fusion gene. The IGH@ locus is labeled with SpectrumGreen, and the BCL3 locus is labeled with SpectrumOrange. The IGH@/BCL3 fusion gene is indicated by the yellow fusion signal.

The t(14;19) may be more frequent than has been reported previously. In our experience, the abnormality on the long arm of the derivative chromosome 19 can be subtle and easily missed on conventional cytogenetic analysis. The karyogram may be misinterpreted as having only a small amount of material added to the long arm of chromosome 14, ie, add(14)(q32), rather than having a reciprocal translocation between chromosomes 14 and 19. Thus, in CLL cases that demonstrate add(14q32) on conventional karyotypic analysis, we would recommend performing FISH analysis for IGH@/BCL3 rearrangement. With commercially available probes, mapping back to a metaphase using a break-apart probe to the IGH@ locus would demonstrate a split signal, located on the long arms of chromosomes 14 and 19. Additional FISH analysis using a dual-color break-apart probe to BCL3 would establish if the t(14;19) involved BCL3. 21

Molecular genetic analysis of the IGHV somatic mutation status in patients with CLL has identified 2 prognostic subtypes. Patients whose CLL cells lack somatic mutations have a poorer prognosis than patients whose CLL cells contain somatic mutations, with median survival of 8 and 24 years, respectively.22,23 Similar to other reports on small B-cell leukemias with BCL3 translocation,18,21 all cases were classified as unmutated. Expression of ZAP70 protein, assessed by flow cytometry or immunohistochemical stains, has been used as a surrogate marker for the somatic mutation status of the immunoglobulin genes.11,24 Since the original report by Crespo and coworkers,24 the concordance rate between ZAP70 expression and IGHV mutation status has been shown to be about 80%.25 Recent studies suggest that ZAP70 expression may be a better marker of the need for treatment than the IGHV somatic mutation status.26,27 In our series, we found that half of our cases showed discordance between ZAP70 expression and somatic mutation status, ie, 7 of 14 unmutated cases were negative for ZAP70 by immunohistochemical stain. Unfortunately, the sample size and the length of follow-up are inadequate for determining whether mutation status or ZAP70 expression is a better marker of prognosis in patients with the t(14;19). The concordance between ZAP70 expression and mutation status in cases with the t(14;19) has not been addressed in other studies.

It has been demonstrated clearly that the repertoire of IGHV use in CLL cells differs from that of healthy aging adults. This skewing, or bias, in IGHV use is a feature of CLL.28 Bias in immunoglobulin gene use in CLL is associated with somatic mutation status; some IGHV genes are used preferentially in unmutated rearrangements, eg, IGHV1-69*01, whereas others are used preferentially in mutated rearrangements, eg, IGHV4-34. Bias also varies with geographic location, which may reflect genetic differences in patient populations or variability in the environment.28 The results of gene expression profiling and immunophenotypic studies have demonstrated that mutated and unmutated cases express markers associated with antigen exposure, indicating that both are likely derived from antigen-experienced B cells.2931 Taken together, these findings strongly support the concept that antigen selection and stimulation have important roles in development and evolution of CLL.32

The biased use of IGHV4-39 in almost half of the cases in our study is remarkable. Bias in the use of IGHV4-39 has been described previously in CLL.15,18,32 In the study by Martin-Subero and coworkers,18 5 of 14 cases classified as CLL, but none of 10 B-NHLs, used IGHV4-39. Another group described 7 CLL cases that used IGHV4-39; all showed minimal deviation from germline sequences, and 5 had undergone isotype switch to express IgG.15,32 However, in these studies, the morphologic findings, immunophenotype other than isotype, and cytogenetic findings were not reported. We found that 7 of our cases were positive for surface immunoglobulin but were negative for surface IgM and IgD; the 1 case tested for surface IgG was strongly positive. It is interesting that all 7 cases used IGHV4-39, which suggests that they had undergone isotype switch, consistent with the findings of previous studies. We subsequently performed a complete sequence analysis of the IGHV and immunoglobulin light chain variable region genes in 11 of the 14 cases with material available for analysis.33 We found that the cases that showed biased use of IGHV4-39 also demonstrated homology in their heavy and light chain complementarity determining region 3 motifs, using rearranged IGHV4-39–D6-13/19–J5 and IGKV1-39–J1/J2 genes, respectively. This finding suggests that at least some cases of CLL with t(14;19) are antigen-driven and that antigen drive through distinct antigen receptors may induce or select for specific chromosomal translocations that confer unique clinical and biologic characteristics.

In summary, CLL cases with t(14;19)(q32;q13) have distinctive features, including morphologically atypical lymphocytes and an atypical immunophenotype, a frequent association with trisomy 12 and complex cytogenetics, an infrequent association with del(13q14), unmutated IGHV genes, and stereotypy of the B-cell antigen receptor in the cases that use the IGHV4-39 family.

CME/SAM

Upon completion of this activity you will be able to:

  • describe the morphologic and immunophenotypic characteristics of lymphocytes in peripheral blood and bone marrow aspirate of chronic lymphocytic leukemia (CLL) with t(14;19)(q32;q13).

  • discuss the most common additional cytogenetic abnormality in CLL with t(14;19).

  • list the distinctive genetic features in CLL with t(14;19).

The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this educational activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module.

The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

Questions appear on p 796. Exam is located at www.ascp.org/ajcpcme.

Footnotes

  • Supported in part by grants from the European Hematology Association-American Society of Hematology, The Hague, the Netherlands (Dr Schweighofer), the CLL Global Research Foundation, Houston, TX (Drs Schweighofer and Abruzzo), and R01CA123252-3 from the National Cancer Institute, Bethesda, MD (Dr Abruzzo).

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View Abstract