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“Double-Hit” Mature B-Cell Lymphomas Show a Common Immunophenotype by Flow Cytometry That Includes Decreased CD20 Expression

David Wu MD, PhD, Brent L. Wood MD, PhD, Russell Dorer MD, PhD, Jonathan R. Fromm MD, PhD
DOI: http://dx.doi.org/10.1309/AJCP7YLDTJPLCE5F 258-265 First published online: 1 August 2010


Lymphomas with 2 translocations involving c-MYC and BCL2 or BCL6 are a subset of biologically aggressive mature B-cell lymphomas now frequently categorized under the entity of B-cell lymphoma, unclassifiable, with features intermediate between Burkitt lymphoma and diffuse large B-cell lymphoma. We identified a cohort of these lymphomas in our databases and retrospectively reviewed corresponding in-house flow cytometric data to determine whether a common immunophenotype might be present. Herein we report on our findings on 10 lymphomas, each with translocations involving c-MYC and BCL2 or BCL6 and show that these cases frequently showed a common immunophenotype that includes decreased expression of CD20. Because these lymphomas often show aggressive biologic behavior and poor clinical outcome, recognition of this relatively common immunophenotype may be useful for identifying cases for confirmatory cytogenetic studies, as often, flow cytometry provides the first assessment of these clinical specimens.

Key Words:
  • Flow cytometry
  • c-MYC
  • Double-hit
  • Dual translocation
  • BCL2
  • BCL6
  • CD20

Mature B-cell lymphomas with dual translocations involving c-MYC and typically BCL2 or alternatively BCL6 are a subset of lymphomas that commonly show aggressive clinical behavior.13 Typically, these tumors appear intermediate or high grade by morphologic review; however, in some cases, tumors with an appearance compatible with a low-grade lymphoma or plasmablastic myeloma have also been described.2 In immunohistochemical studies, these neoplasms may show features of Burkitt lymphoma and diffuse large B-cell lymphoma (DLBCL), but may also not infrequently show features intermediate between the two.4,5 Despite the variable morphologic appearance and immunophenotype of these lymphomas, a commonality of these neoplasms is the identification of dual translocations involving c-MYC and typically BCL2 or alternatively BCL6 in these lesions that together lead to dual oncogenic stimuli resulting in biologically aggressive behavior.5 These translocations are thought to lead to increased proliferation (c-MYC and BCL6) or reduced apoptosis (BCL2), thereby driving aggressive growth characteristics. Because dual-translocation tumors commonly are more aggressive than their morphologic and immunohistochemical counterparts that lack dual translocations, identifying these tumors may be important for patient prognostication and therapy.68

Flow cytometry is a rapid and routine method used in many clinical laboratories to characterize leukocyte antigen expression in complex mixtures, such as those derived from peripheral blood, bone marrow, or tissue samples. Recent data have suggested that a significant proportion of mature B-cell lymphomas with c-MYC translocations may show a common immunophenotype, including increased expression of CD38, as assessed by immunohistochemical analysis9 or flow cytometry.10 As such, we sought to determine whether there might be a common immunophenotype of dual-hit, aggressive B-cell lymphomas that might identify these cases for cytogenetic analysis because this testing might not otherwise be routinely performed. Herein, we report on 10 cases from our cohort and describe their common immunophenotype by multicolor flow cytometry performed at the University of Washington Medical Center (UWMC), Seattle.

Materials and Methods

Case Selection

With institutional review board approval we searched the University of Washington pathology and laboratory medicine databases to identify cases with documented c-MYC and BCL2 or BCL6 translocations, using the terms “c-MYC,” “Burkitt,” “BCL2,” and “BCL6.” Of 202 cases identified with c-MYC translocations from 2001 to 2009, we further identified 10 cases that also had documented translocations involving BCL2 or BCL6 by in-house testing or external report and that further had flow cytometry performed at the UWMC available for rereview.

During our database search, we also identified 1 additional case for which cytogenetic testing was performed at the University of Washington, but flow cytometry was performed at Virginia Mason Medical Center, Seattle. This case is not formally included in this series but showed features similar to our in-house cases, to be described.

Flow Cytometry

Flow cytometry was performed on a modified 4-laser, 10-color Becton Dickinson LSR II flow cytometer (BD Biosciences, San Jose, CA; same instrument used for routine clinical cases at the UWMC) using the following laser-fluorochrome combinations: (1) 405-nm violet laser (1 color) and Pacific blue (PB); (2) 488-nm blue laser (5 colors), fluorescein isothiocyanate (FITC), phycoerythrin (PE), PE–Texas red (ECD/PE-TR), PE-cyanine (Cy)-5, PE-Cy5.5, and PE-Cy7; (3) 594-nm yellow laser (1 color) and Alexa Fluor 594 (A594); and (4) 633-nm red laser (3 colors), allophycocyanin (APC), APC–Alexa Fluor 700 (BD Biosciences), and APC-Cy7. Details of this instrumentation have been previously described.11,12

The specific fluorescently labeled antibodies used in this study were obtained from Beckman Coulter (BC; Fullerton, CA) and Becton Dickinson (BD; San Jose, CA) and were run as either of the following 2 reagent combinations: (1) CD45 PB (BC), CD19 PE-TR (BC), CD20 PE-Cy7 (BC), CD5 APC-Cy7 (BC), CD10 APC (BD), CD38 A594 (BD), κ FITC (BD), and λ PE (BD) or (2) CD45 APC-H7 (BD), CD19 PE-Cy7 (BC), CD20 V450 (BD), CD5 PE-Cy5 (BC), CD10 APC (BD), CD38 A594 (BD), κ FITC (BD), and λ PE (BD). For the study, 100-μL aliquots of whole blood, whole bone marrow, or disaggregated tissue were stained and processed with a standard whole blood lysing procedure using ammonium chloride containing 0.25% ultrapure formaldehyde following 3 washes with phosphate-buffered saline–bovine serum albumin to remove plasma protein, as previously described.10 Data were analyzed using in-house software developed by B.L. Wood, University of Washington (software version 2.7.6). Antigen expression of tumor populations is reported relative to that of its normal counterpart, follicle center B cells. In cases in which normal follicle center B cells were not directly present in the sample, comparison was made indirectly with the expected level of antigen intensity for follicle center B cells, observed in other clinical samples. In our experience, the levels of antigen expression on normal follicle center B-cell populations are consistent and reproducible.13

Morphologic Evaluation

Routine Wright-Giemsa– and/or H&E-stained peripheral blood and bone marrow aspirate smears and cytocentrifuged preparations from disaggregated tissue specimens submitted for flow cytometry were retrieved from the UWMC Laboratory Medicine archives and rereviewed. Select images were digitally captured using a Leica DM1000 microscope with attached digital camera using the manufacturer’s FireCam software (Leica Microsystems, Bannockburn, IL).


Clinical and Histologic Features

Clinical details of the 10 cases identified are shown in Table 1. Similar to prior reports, the morphologic or clinical diagnoses of these lesions were variable and included Burkitt lymphoma, DLBCL, and B-cell lymphoma, unclassifiable, with features intermediate between Burkitt lymphoma and DLBCL. In 3 of 10 patients, a history of a low-grade follicular lymphoma was identified. In the remainder, no history of malignancy was identified in the patient’s medical records.

Flow Cytometry

On rereview of flow cytometric data, 8 (80%) of 10 double-hit lymphomas in our cohort showed an intriguing common immunophenotype, including a marked decrease in expression of CD20 ranging from dim to absent compared with normal follicle center B cells, a feature that, to our knowledge, has not been described in the literature Image 1. This immunophenotype was seen in 7 of 9 tumors with translocations involving c-MYC and BCL2 (Image 1A) and 1 of 1 tumor with translocations involving c-MYC and BCL6 (Image 1B). Other common features of this immunopheno-type include positivity for CD10, variably increased forward and side scatter properties, consistent with a relative increase in cell size, similar to normal follicle center B cells; variably decreased expression of CD45; and variably increased expression of CD38, which has been previously described for some c-MYC+ lymphomas.10 In addition, immunoglobulin light chain restriction with decreased intensity or complete absence was noted in these cases. Details of the flow cytometric immunophenotype for each case are shown in Table 2.

In general, this common immunophenotype for double-hit lymphomas was seen irrespective of the initial site of involvement (data not shown). For example, in 1 patient with multisite disease (case 4), a nearly identical immunophenotype was observed in the various sampled locations, including peripheral blood, pleural fluid, ascites, bone marrow, and lymph node. In a separate patient (case 3), with involvement of peripheral blood, bone marrow, and cerebrospinal fluid, the common immunophenotype was also observed in each specimen.

In 2 (22%) of 9 cases with confirmed dual translocations involving c-MYC and BCL2 (cases 6 and 8), this common marked decrease or absence of CD20 expression was not observed Image 2A (Table 2). Case 6 showed normal to slightly increased expression of CD20, whereas case 8 showed only a slight decrease in CD20 expression compared with the expected level of CD20 for normal follicle center B cells. For case 8, given the minimal decrease observed, this case was not included as part of the other cases that showed a more dramatic decrease or absence of CD20 expression. Both of these cases, however, showed absent (case 6) or decreased (case 8) expression of surface light chains, similar to the other cases in the series. By morphologic features, these cases did not appear dissimilar because high-grade cytologic features, including prominent nucleoli and finely dispersed and immature-appearing chromatin, were present in both Image 2B.

During our database search for double-hit lymphoma cases, we identified 1 additional case of a peripheral blood sample from a 73-year-old woman for which translocations involving c-MYC and BCL2 were confirmed at UWMC. Because flow cytometry was performed at Virginia Mason Medical Center, this case was not formally included in our series. Nevertheless, for this external case, identical immunophenotypic features, namely decreased-to-absent expression of CD20 with absent light chain expression was noted (data not shown).


Mature B-cell lymphomas are routinely diagnosed primarily on the basis of characteristic histologic features, with the use of flow cytometry, immunohistochemical analysis, and cytogenetic analysis as ancillary supporting data to further support or confirm a morphologic impression.4,5 In most cases, the distinction among high-, intermediate-, and low-grade B-cell processes can be readily made with morphologic examination alone. However, in some cases, lymphomas with underlying dual translocations involving c-MYC and typically BCL2 or alternatively BCL6 may have more clinically aggressive behavior compared with morphologically similar-appearing variants lacking such translocations. These double-hit lymphomas arguably should be identified for patient prognostication and for potential treatment with higher-grade (vs intermediate-grade) chemotherapy.4,5

Herein, we report on our single institutional, retrospective review of mature B-cell lymphomas with translocations involving c-MYC and BCL2 or BCL6. We found that many of these double-hit lymphomas showed a common immuno-phenotype that includes, most prominently, variably decreased expression of CD20. These tumors may also show a variable level of expression of CD45, typically increased expression of CD38, and decreased restricted or absent expression of immunoglobulin light chains compared with background lymphocytes. These findings were present in most tumors that had dual translocations involving c-MYC and BCL2 and in our single case that had translocations involving c-MYC and BCL6. Furthermore, these findings were present in 6 of 7 de novo tumors in which the patients lacked a prior diagnosis of lymphoma and in 2 of 3 cases of double-hit lymphomas arising in patients with a history of a low-grade follicular lymphoma.

The findings of a common immunophenotype, including most prominently the variably decreased and, in some cases, absent expression of CD20 in this cohort was unexpected because, to our knowledge, these findings have not previously been described for this subgroup. In general, with the exception of chronic lymphocytic leukemia/small lymphocytic lymphoma, most mature B-cell lymphomas, including Burkitt lymphoma and DLBCL, are thought to show expression of CD20 comparable to nonneoplastic lymphocytes.14 The variably marked decreased to absent expression of CD20 in this double-hit lymphoma cohort seems to be unique and not previously recognized.

Image 1

A, Double-hit B-cell lymphomas with c-MYC and BCL2 translocations. Case 2, top row, lymph node, left supraclavicular; case 4, middle row, ascites; and case 9, bottom row, retroperitoneal mass. Tumor populations are shown in teal. T cells, defined by CD5 positivity and CD19 negativity, are shown in green. Normal B cells are shown in blue (κ light chain) or red (λ light chain). For case 9 only, a second abnormal B-cell population is shown in magenta. B, A double-hit lymphoma case with c-MYC and BCL6 translocation and no evidence of a BCL2 translocation by fluorescence in situ hybridization. A594, Alexa Fluor 594; APC, allophycocyanin; Cy, cyanine; FITC, fluorescein isothiocyanate; FSC-H, forward scatter, height; PB, Pacific blue; PE, phycoerythrin; SSC-H, side scatter, height; TR, Texas red.

Image 2

A, Two double-hit lymphoma cases without marked decreased expression of CD20: case 6, abdominal mass; and case 8, bone marrow. The abnormal B-cell population is shown in fuchsia. T cells, defined by CD5 positivity and CD19 negativity, are shown in green. Normal B cells are shown in blue (κ light chain) or red (λ light chain). In case 8, hematogones are shown in yellow. A594, Alexa Fluor 594; APC, allophycocyanin; Cy, cyanine; FITC, fluorescein isothiocyanate; FSC-H, forward scatter, height; PB, Pacific blue; PE, phycoerythrin; SSC-H, side scatter, height; TR, Texas red. B (Case 6), Wright-Giemsa–stained preparation of a double-hit lymphoma case without decreased CD20 expression showing an immature blast-like appearance (×400). Case 8 had a similar appearance (data not shown).

Although our findings of decreased expression of CD20 in this cytogenetically defined subgroup of lymphomas are based on limited case numbers, our reading of the double-hit lymphoma literature supports the potential validity of these findings. In 2 reports on double-hit lymphomas in which flow cytometric analysis was performed but not reported fully in detail, the thresholds used for determining the positive expression of CD20 by flow cytometry were notably set low, suggesting that variably decreased CD20 expression may have been previously unappreciated.1,6 In the first instance in which 12 double-hit lymphomas were examined (excluding 1 plasmablastic myeloma case), the relative level of expression of CD20 was not reported in detail. However, the criteria used in that study required only that 20% of the cells show CD20 expression to be considered positive.1 In the second instance evaluating 12 DLBCLs with dual translocations, the criteria used for positivity of CD20 expression were similarly low and required only that 30% of the CD19+ B cells show expression.6 This points out the limitation of using thresholds for assessing antigen expression rather than using a reference to the normal level of expression expected for cells of the appropriate lineage and maturational stage. Thus, while the flow cytometric aspects of double-hit lymphomas have been previously studied, it seems likely that our observation of variably decreased expression of CD20 was not previously recognized.

In contrast with the situation of most chronic lymphocytic leukemias/small lymphocytic lymphomas that typically show indolent behavior, it has been recently proposed that decreased CD20 expression may be characteristic of some B-cell lymphomas that are associated with a more aggressive phenotype.15,16 For example, as shown in a recent study of DLBCLs, tumors with decreased expression of CD20 that was “discordant” with respect to the normal level of CD19 expression were found to be associated with a significantly worse clinical outcome.16 These tumors also reportedly showed an increased association with strong bcl-2 protein expression, suggesting possible concomitant dysregulation of this antiapoptotic pathway. In a separate study, patients with B-cell lymphomas that relapsed after rituximab therapy with so-called phenotypic transformation to a CD20-protein–negative state tended also to have a very aggressive clinical course. All 5 of the patients were reported to die within 1 year of change in CD20 expression status.15 As suggested by these authors, decreased CD20 expression may represent a common antigen change seen in transformation toward more aggressive behavior and is consistent with our findings in this series. Unfortunately, we were unable to directly explore this question in our cohort because of the 3 patients who each had a history of low-grade follicular lymphoma, none had flow cytometry of the primary lesion performed at our institution available for reconsideration. As such, additional studies would be needed to further explore this potential correlation.

The mechanisms resulting in decreased expression of CD20 in these dual-translocation lymphomas in particular and in high-grade lymphomas in general are unclear. That diminished expression of CD20 may be due to differences in antibody binding to CD20 antigen cannot be excluded. Theoretically, mutations in CD20 may disrupt the binding of anti-CD20 antibody in flow cytometric studies by directly affecting the extracellular domain or by indirectly affecting the cytoplasmic or transmembrane domains, thereby resulting in conformational changes to the protein. The presence of any such mutations in double-hit lymphomas, however, has not been explored. In the aforementioned cohort of DLBCLs, an attempt to define mutations in the MSA4 gene that encodes CD20 was pursued. However, in 15 cases in which sequencing was successful, no mutations were identified in exon 15, which encodes the extracellular domain recognized by the anti-CD20 antibody.16 This study did not explore the possibility of large deletions in the MSA4 gene resulting in haploinsufficiency, nor did it explore the possibility of hypermethylation of the promoter region. However, either of these changes would not be expected to significantly alter the binding affinities of antibody to the CD20 antigen. Alternatively, diminished expression of CD20 may be the result of pretranscriptional and posttranscriptional mechanisms regulating messenger RNA levels. Indeed, there are some data to suggest that epigenetic changes in other circumstances may alter the stability of or affect the levels of CD20 messenger RNA.15 For double-hit lymphomas specifically, however, this mechanism for altering CD20 expression has also not been explored.17

Clinical Implications

While the optimal treatment for double-hit lymphomas remains to be defined, recognition of this potentially common immunophenotype is likely to be important for patient prognostication and also may have treatment implications.5 In several studies, dual-hit lymphomas were shown to behave in a clinically more aggressive manner6 with early relapse, even if complete response to initial chemotherapy is initially achieved,7 and, therefore, some studies have suggested that patients may benefit from intensified chemotherapeutic regimens.5,18 Tomita et al8 showed that dual-hit lymphomas had a short median survival course of only 4 months from diagnosis and, intriguingly, that 7 cases of so-called triple-hit lymphomas with translocations involving c-MYC, BCL2, and BCL6 had a duration of patient survival that was statistically shorter than that of dual-hit lymphomas. Furthermore, if this immunophenotype is proved to be common for these lymphomas, there may be implications for the use of anti-CD20 antibody therapies. Owing to the decreased expression of CD20 antigen, the use of second-generation monoclonal antibodies targeting CD20 may be fruitful because these engineered antibodies are reportedly more effective than rituximab in inducing complement-dependent cytotoxicity, particularly in tumors with decreased CD20 antigen density.19 Thus, recognition of these neoplasms with unique immunophenotype may be increasingly clinically important.

We report on a cohort of double-hit lymphomas from our institution that seems to show a common immunophenotype that includes variably decreased CD20 expression. If further validated by others, this flow cytometric finding may be helpful in identifying lymphomas that may benefit from confirmatory cytogenetic studies.


We thank H. Greisman for helpful discussions.


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