Context.—Posttransplant B-cell lymphoproliferative disorders (PTLDs) constitute a heterogeneous group that includes hyperplastic and unique polymorphic lesions at one end of the spectrum and monomorphic lymphoid proliferations indistinguishable morphologically from conventional B-cell non-Hodgkin lymphomas (NHLs) at the other end. Almost all the PTLDs are of B-cell origin, with only rare examples of T-cell phenotype described. Despite a plethora of information available on the morphologic spectrum, pathogenetic role of Epstein-Barr virus, and various treatment options, a detailed flow cytometric immunophenotypic evaluation of PTLDs is largely lacking.

Objective.—To evaluate the immunophenotypic profiles of various PTLDs using multiparameter flow cytometric analysis to compare and contrast with conventional de novo B-cell lymphoproliferative disorders and to identify any immunophenotypic patterns useful in diagnosis.

Design.—We retrospectively analyzed data on the immunophenotype of 25 cases of pediatric and adult PTLD (12 cases of monomorphic PTLD [m-PTLD] and 13 cases of polymorphic PTLD [p-PTLD]) using multiparameter flow cytometry in addition to routine morphologic and immunohistochemical evaluation. The flow cytometric immunophenotypic data were also compared and contrasted with 334 cases of various de novo B-cell NHLs during the same period as a control group.

Results.—We observed a much higher incidence of lack of surface immunoglobulin light chains and CD20 expression in B-cell PTLDs using multiparameter flow cytometry in comparison with de novo B-cell NHL as a group (with the exception of small lymphocytic lymphoma). Four (16%) of 25 cases of PTLD (3 m-PTLD and 1 p-PTLD) showed almost complete lack (CD20%/CD19% ratio < 1:9) of CD20 expression in contrast to only 8 (∼2%) of 334 cases of de novo B-cell NHL (P = .007). Several other cases of both m-PTLD and p-PTLD also showed partial and dim expression of CD20. Nine (36%) of 25 cases, including 5 cases of m-PTLD and 4 of p-PTLD, showed either an almost complete lack (light chains%/CD19% ratio < 1:9) or significant loss (>50% loss) of surface immunoglobulin light chains in contrast to less than 5% incidence of light-chain negativity in conventional de novo B-cell NHL. Immunoglobulin light-chain clonality was observed in 9 cases (5 m-PTLD and 4 p-PTLD). Seven cases (5 p-PTLD and 2 m-PTLD) had polyclonal expression of immunoglobulin κ and λ light chains. The m-PTLD showed expression patterns of CD5, CD10, and CD23 similar to their de novo counterparts.

Conclusions.—Both polymorphic and monomorphic PTLDs show a higher incidence of lack of CD20 and surface immunoglobulin light-chain expression. The lack of CD20 expression in these lesions may have therapeutic implications, since anti-CD20 antibody has increasingly become an important modality in the treatment of B-cell lymphoproliferative disorders, including posttransplant disorders.

Posttransplant B-cell lymphoproliferative disorders (PTLDs) constitute a heterogeneous group that includes early hyperplastic lesions, polymorphic B-cell proliferations, monomorphic B-cell lymphomas, and Hodgkin lymphoma or unique Hodgkin lymphoma–like lesions.1 The current World Health Organization (WHO) classification has evolved from earlier classification schemes, and considerable insight has been gained in the pathogenesis and morphologic spectrum of these lesions in the last 2 decades.1–4 Immunophenotyping is an essential component in the lineage and clonality determination of these disorders, and flow cytometry or frozen section immunohistochemistry are the preferred methods over paraffin-based immunohistochemistry.1 Despite their widespread recognition, however, only rare reports have described the immunophenotypic spectrum of these disorders by multiparameter flow cytometry.5,6 The importance of flow cytometric analysis of these disorders usually rests in the evaluation of clonality, which serves as an essential piece of information in the overall treatment strategy. Monoclonal lesions are usually treated with combination chemotherapy, whereas polymorphic polyclonal lesions may regress with reduction in immunosuppressive therapy alone.7 Also, with the availability of anti-CD20 monoclonal antibody, the B-cell PTLDs are increasingly treated with protocols that include the anti-CD20 antibody.8–12 We have evaluated a number of these B-cell PTLDs during the past several years using multiparameter flow cytometry and have noted a higher incidence of lack of CD20 and immunoglobulin light-chain expression, which may bear diagnostic and therapeutic implications. Our results emphasize the need for routine evaluation of these disorders by multiparameter flow cytometry.

All 25 cases of PTLD and 334 cases of de novo B-cell non-Hodgkin lymphoma (NHL; excluding small lymphocytic lymphomas) were retrieved from the files of the Lauren V. Ackerman Laboratory of Surgical Pathology (Washington University School of Medicine, St Louis, Mo) from January 1, 1994 to December 31, 2001. Only cases with a B-cell immunophenotype and a flow cytometric analysis were selected. Cases of small lymphocytic lymphoma were not included in the control group because of the known fact that they show dim and/or partial expression of surface immunoglobulins and CD20. Cases with plasmacytic hyperplasia, plasmacytoma, and Hodgkin-like lesions were also excluded. All cases were reviewed by at least 2 pathologists and conformed strictly to the definition of PTLD as provided by WHO. Cases were classified as monomorphic PTLD (m-PTLD) when they had sufficient architectural and cytologic atypia to be diagnosed as lymphoma on morphologic grounds and had expression of B-cell–associated antigens, whereas polymorphic PTLD (p-PTLD) showed destructive lesions composed of immunoblasts, plasma cells, and intermediate-sized lymphocytes that efface the architecture of lymph nodes or formed destructive extranodal masses.1 

Morphologic Examination

All specimens were obtained and prepared for morphologic examination using standard techniques. The lymph nodes and extranodal soft tissues were fixed in 10% buffered formaldehyde, embedded in paraffin, processed routinely, and the sections were stained with hematoxylin-eosin for light microscopy.

Flow Cytometry

Fresh tissues were immediately transported in RPMI solution to the flow cytometry laboratory, and lymphocytes were disaggregated and released from the solid tissue by RPMI injections and physical crushing of the tissue. Mononuclear cells were stained with various combinations of the following fluorescein isothiocyanate (FITC), phycoerythrin (PE), phycoerythrin-cyanine 5 (PC5), or phycoerythrin-Texas Red (ECD)-labeled monoclonal antibodies against the following antigens: CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20 (clone B9E9; Coulter, Miami, Fla), CD23, HLA-DR, and immunoglobulins κ (Ig-κ) and λ (Ig-λ) light chains (polyclonal; Dako Corporation, Carpinteria, Calif). In several cases, additional markers were used appropriately. The typical combinations included κ-FITC/CD19-ECD, λ-FITC/CD19-ECD, CD19-FITC/CD5-PE, CD19-FITC/CD10-PE, CD23-FITC/CD19-PE, CD7-FITC/CD1-PE, CD4-FITC/CD8-PE, CD3-PC5/CD4-FITC, CD3-PC5/CD8-PE, CD19-FITC/HLA-DR–PE, and CD19-ECD/CD20-PC5. Other combinations of fluorochromes and custom combinations were used whenever indicated. No evaluation was performed for intracytoplasmic Ig-κ and Ig-λ, as it was not the standard procedure at the time of original flow cytometric evaluation.

Two- and 4-color flow cytometric immunophenotyping was performed on FACScan (2-color only; Becton Dickinson, San Jose, Calif) or on the Coulter XL cytometer (4-color; Coulter) by collecting 10 000 ungated list mode events, selecting an appropriate gate on the combination of forward and side scatter, and analyzing cells with the most appropriate gate. In cases with more than 1 lymphocyte gate, the gate with the greatest number of B cells and/or showing aberrant coexpression of antigens and closely corresponding to the cell size of the lymphoma in question was chosen for analysis. An antigen was considered positively expressed when at least 25% of the gated cells expressed that antigen. Cases were classified as monoclonal using Ig-κ and Ig-λ light-chain ratio (κ%/λ% ratio > 4:1 or <0.5:1). These cutoff levels were used based on the results of a study we conducted previously.13 All the gates were set according to the isotype controls, which were run in all cases. All the antibodies were purchased from Becton Dickinson, Coulter, or Dako, and were used according to the manufacturers' guidelines.

Immunohistochemistry

Immunohistochemical analysis was also performed on formalin-fixed, paraffin-embedded nodal and extranodal tissues as deemed necessary. The antigens that were sought included CD20 (L26), CD43 (MT-1), Ki-67, Bcl-2, Epstein-Barr virus latent membrane protein (EBV-LMP), and κ and λ immunoglobulin light chains. Five-micrometer-thick sections were cut and collected on lysine-coated slides and dried in a 60°C paraffin oven for 45 minutes. Sections were deparaffinized in xylene, incubated for 30 minutes in methanolic hydrogen peroxide (0.3% [vol/vol]) to quench for endogenous peroxidase, and rehydrated in graded ethanol solution, followed by rinsing in distilled water and phosphate-buffered saline (pH 7.4). Heat-mediated “antigen retrieval” (epitope retrieval) was carried out for 12 minutes in a microwave oven in the presence of citrate buffer (pH 6.0). The sections were cooled for 20 minutes, followed by rinsing in water and incubation in phosphate-buffered saline. A protein block (Dako) was performed for polyclonal antibodies, with a 5-minute incubation period. Primary antibodies against L26 (1:4000 dilution; Dako), MT-1 (1:100 dilution; Biotest, Denville, NJ), OPD-4 (1:200 dilution; Dako), Bcl-2 (1:200 dilution, Dako), EBV-LMP (1:500 dilution, Dako), and κ (1:150 000 dilution, Dako) and λ (1:100 000 dilution, Dako) light chains were applied, and the sections were incubated for 18 hours at 4°C. Antibody bridge assembly by the avidin-biotin-peroxidase complex method using the Elite ABC kit (Vector Laboratories, Burlingame, Calif) was performed the next day by 2 sequential 1-hour incubations. Chromogenic development was accomplished by immersion of the sections in 3,3′-diaminobenzidine solution (0.25 mg/mL with 0.003% hydrogen peroxide). The slides were immersed in 0.125% osmium tetroxide to enhance chromogenic precipitation, followed by light counterstaining with Harris hematoxylin. The sections were dehydrated in graded ethanol, cleared in xylene, and mounted with Cytoseal medium (Electron Microscopy Sciences, Fort Washington, Pa).

Cases of PTLD included 11 male and 14 female patients, whose ages ranged from 2 to 70 years. The patients underwent the following transplantations: kidney (n = 5), lung (n = 8), heart (n = 8), liver (n = 3), and combined heart and kidney (n = 1) (Table 1). Twelve patients developed m-PTLDs, which were characterized as follows: Burkitt lymphoma (n = 3) and diffuse large B-cell NHL (n = 9, including 5 centroblastic, 3 immunoblastic, and 1 anaplastic). Thirteen patients developed p-PTLD (8 polymorphous B-cell lymphoma, 5 polymorphous B-cell hyperplasia). These PTLDs arose in a variety of organs. Six cases (25%) were negative for EBV-LMP. The distribution of de novo B-cell NHLs in the control group was as follows: follicular lymphoma (n = 122), diffuse large B-cell lymphoma (DLBCL; n = 102), marginal zone B-cell lymphoma (n = 34), mantle cell lymphoma (n = 38), lymphoplasmacytoid lymphoma (n = 16), and Burkitt lymphoma (n = 22).

Table 1.

Clinicopathologic Features of 25 Cases of B-Cell Posttransplant Lymphoproliferative Disorder (PTLD)*

Clinicopathologic Features of 25 Cases of B-Cell Posttransplant Lymphoproliferative Disorder (PTLD)*
Clinicopathologic Features of 25 Cases of B-Cell Posttransplant Lymphoproliferative Disorder (PTLD)*

Four cases (cases 6, 7, 10, and 25) showed almost complete lack (CD20%/CD19% ≤ 1:9) of CD20 expression (CD20 negative) when compared with expression level of pan–B-cell marker CD19 by flow cytometry (Figure 1). Another 8 cases (cases 9, 13–15, 18, 19, 22, and 24) showed partial and/or dim CD20 expression (Table 2). Case 13 also showed lack of CD20 expression by immunohistochemistry. All other cases showed expression of significant CD20 by flow cytometry and also by immunohistochemistry, whenever performed (Table 2). The findings were verified after careful review of the isotype controls for CD20 and review of CD19 expression. Among the de novo B-cell NHLs, only 8 (∼2%) of 334 showed almost complete lack of CD20 expression (P = .007). This group included 6 of 102 DLBCLs, 1 of 122 follicular lymphomas, and 1 of 16 lymphoplasmacytoid lymphomas.

Figure 1.

Flow cytometric evaluation of CD20. The cells showed significant expression of the B-cell marker CD19 (93%) (A) but only 7% of CD19-expressing B cells expressed CD20 (B). ECD indicates phycoerythrin-Texas Red; PC5, phycoerythrin-cyanine 5; and SS, side scatter. Figure 2. Flow cytometric evaluation of surface immunoglobulin light chains. The clonal CD19-positive B cells do not express either Ig-κ (A) or Ig-λ (B) light chains. ECD indicates phycoerythrin-Texas Red; FITC, fluorescein isothiocyanate

Figure 1.

Flow cytometric evaluation of CD20. The cells showed significant expression of the B-cell marker CD19 (93%) (A) but only 7% of CD19-expressing B cells expressed CD20 (B). ECD indicates phycoerythrin-Texas Red; PC5, phycoerythrin-cyanine 5; and SS, side scatter. Figure 2. Flow cytometric evaluation of surface immunoglobulin light chains. The clonal CD19-positive B cells do not express either Ig-κ (A) or Ig-λ (B) light chains. ECD indicates phycoerythrin-Texas Red; FITC, fluorescein isothiocyanate

Close modal
Table 2.

Flow Cytometric Characterization Posttransplant B-Cell Lymphoproliferative Disorders

Flow Cytometric Characterization Posttransplant B-Cell Lymphoproliferative Disorders
Flow Cytometric Characterization Posttransplant B-Cell Lymphoproliferative Disorders

Nine (36%; 5 m-PTLDs and 4 p-PTLDs) of 25 cases showed either almost complete (>90% loss; cases 2, 3, 5, and 10) or significant (>50% loss; cases 1, 14–16, and 21) lack of surface immunoglobulin (sIg) light-chain expression (Figure 2). Five cases of m-PTLD showed unequivocal monoclonal light-chain expression (cases 6, 7, 9, 11, and 12), whereas in 2 of 12 m-PTLDs no apparent light-chain clonal excess could be demonstrated (cases 4 and 8). In case 4, however, only about 75% of CD19-positive B cells (Ig-κ [44%] + Ig-λ [21%]/CD19-positive [86%]) expressed either κ or λ light chains. Furthermore, this case showed clonal expression of IgG heavy chain, thus confirming the monoclonal nature of this PTLD. Likewise in case 8, only about 62% of CD19-positive B cells showed expression of either Ig-κ or Ig-λ light chains, whereas the remaining 38% of CD19-positive B cells did not express light chains. We believe that the apparent lack of light-chain clonality in cases 4 and 8 is most likely due to partial lack of light-chain expression by clonal B cells. Four (31%) of the p-PTLDs showed monoclonal light-chain expression (cases 17, 19, 24, and 25), whereas the remaining 5 cases (cases 13, 18, 20, 22, and 23) expressed Ig-κ and Ig-λ in a polyclonal fashion. Of the 6 cases in which evaluation for immunoglobulin heavy chains was performed, the following distribution of heavy-chain expression was seen: IgG only (cases 4 and 19), IgM only (cases 11 and 17), IgA only (case 6), and IgM and IgD together (case 22). Thus, case 22 did not show clonality by either light-chain analysis or the expression pattern of heavy chains, because naive B lymphocytes also express IgM and IgD on their surface. Only 10 (∼3%) of 334 cases of de novo B-cell NHLs showed a complete lack of sIg light chains, including follicular lymphoma (3/122), DLBCL (5/102), and Burkitt lymphoma (2/22). Three of 16 cases of lymphoplasmacytoid lymphoma showed partial (<50%) but not complete lack of surface light chains. Even after excluding all cases of follicular lymphoma from the comparison group (since none of our PTLD cases resembled follicular lymphoma), the incidence of light-chain negativity (7 [3.3%] of 212) did not change significantly among cases of NHL.

Two (cases 2 and 10) of the 3 cases classified as m-PTLD, Burkitt lymphoma type, showed the typical antigen profile of a de novo Burkitt lymphoma, that is, CD5 negative, CD10 positive, and CD23 negative. The third case, however, did not show expression of CD10. Six of the 9 cases of m-PTLD classified as DLBCL did not express any significant amounts of CD5, CD10, or CD23. Of 3 other cases of DLBCL, 1 case showed expression of both CD5 and CD23 and showed immunoblastic morphology. It is of interest that this immunophenotype may be seen in immunoblastic transformation of B-cell chronic lymphocytic leukemia. Among p-PTLD cases, 1 case each showed expression of CD5 (case 19) and CD23 (case 22). None of the other p-PTLD cases showed any significant expression of CD5, CD10, or CD23, whenever performed.

Six (24%) of 25 cases were negative for EBV-LMP by immunohistochemistry, including both monomorphic and polymorphic PTLD types. Six of the 7 cases showed aberrant expression of T-cell–associated marker CD43. One case of m-PTLD with anaplastic morphology (case 6) also showed CD30 expression. The pattern of CD20 and light-chain expression by immunohistochemistry on several cases concurred with results seen with flow cytometry.

We have noted a higher incidence of lack of CD20 expression in our group of B-cell PTLD cases after solid organ transplantation. This lack of CD20 expression cannot be attributed to the presence of plasma cells in these lesions because of the following factors: (1) most plasma cells are negative when evaluated with flow cytometry on the CD45 gate, in contrast to bright CD45 expression seen with lymphocytes in our group; (2) most plasma cells are also negative for B-cell marker CD19, in contrast to universal expression of CD19 seen in all of our cases; (3) evaluations were done on the lymphocyte gate rather than the plasma cell gate; and (4) many cases showed monoclonal or polyclonal expression of sIg light chains, which is not a feature of plasma cells.14 Also, only 4 CD20-negative/partially positive cases were examples of polymorphic type containing a variable number of plasma cells, whereas the other 5 cases with a CD20-negative/partially positive immunophenotype were of monomorphic variety with no significant number of plasma cells. Partial and/or dim CD20 expression is a frequent feature of B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma.14 The lack of CD20 expression or its dim expression bears important therapeutic implications, since anti-CD20 monoclonal antibody (rituximab) is increasingly being used for the treatment of B-cell lymphoproliferative disorders. This is especially true of B-cell PTLD because of higher mortality associated with combination chemotherapy used in immunosuppressed patients with organ transplants.8–12 Our results emphasize the need for CD20 evaluation in all B-cell PTLD cases by flow cytometry, since interpretation of CD20 by immunohistochemistry may not accurately reflect the true expression density of this antibody on the B cells. Cases that lack CD20 expression may not show the expected response to anti-CD20 antibody and may fare better with other monoclonal antibodies, such as anti-CD52 (CAMPATH).15 

We found no conclusive explanation of this phenomenon. The lack of expression or dim expression of CD20 may be an intrinsic property of B-cell PTLD or may result from varied causes in different neoplasms. The latter explanation appears more plausible, because only 12 of 25 cases displayed this aberrancy. One of 3 cases described by Dunphy et al5 was also negative for CD20 (L26) by immunohistochemistry. CD20 is an integral membrane protein whose expression is limited to normal and neoplastic B cells and which is absent from mature and neoplastic plasma cells and all other leukocytes and tissues.16 Mature B cells differentiating toward plasma cells may loose surface CD20 expression, but this phenomenon was also seen with Burkitt lymphoma, which normally shows bright CD20 expression.14 

We also noted a total or partial lack of sIg light-chain expression in about one third of our cases. This does not reflect the presence of plasma cells (only 4 polymorphic type) for the reasons described above. One of 2 cases of immunoblastic lymphoma and 1 of 4 cases of polymorphic B-cell lymphoma described by Dunphy et al5 were also negative for sIg. The incidence of lack of sIg expression is found disproportionately higher in PTLD than in de novo B-cell NHL (<3.5%), as we described previously.17 The lack of sIg in these lesions may result from varied causes. Defects at any level from gene transcription to translocation of proteins to the cell surface may result in such a phenomenon. Lack of effective rearrangement of immunoglobulin of light-chain genes in clonal B cells may be the earliest abnormality.18 Excess degradation of transcribed messenger RNA may represent another potential faulty step.19 Defects of the translational machinery of the cell may involve ribosomal RNA, signal recognition particle, or abnormalities of physiological processes within the endoplasmic reticulum, such as defective glycosylation, folding, and protein assembly.20 Specific C-terminal sequences in proteins have been shown to cause their retention within the endoplasmic reticulum and a failure to export proteins extracellularly and to the cell surface.21 Furthermore, only fully assembled protein molecules are exported to the cell surface. Proteins that fail these editing steps are not transported to the cell surface and are degraded intracellularly.22 Alternatively, cells may synthesize only those proteins destined to be secreted and that lack the surface membrane–anchoring segment of the molecule.23,24 The normal physiological mechanisms operating in plasma cells (which lack surface immunoglobulins) may also be of interests in these cases.

Clonal expression of sIg light chain and/or heavy chains was seen in the remaining cases of m-PTLD. Five of our 13 cases of p-PTLD showed polyclonal expression of sIg light chains. Our data suggest that the PTLD can exhibit the following patterns of sIg light-chain expression by flow cytometry:

1. Polymorphic PTLD

a. Surface immunoglobulin light chain negative (sIg-κ negative and sIg-λ negative)

b. Monoclonal by immunoglobulin light-chain analysis (κ%/λ% > 4:1 or <0.5:1)

c. Polyclonal by light-chain analysis

2. Monomorphic PTLD

a. Surface immunoglobulin light chain negative (sIg-κ negative and sIg-λ negative)

b. Monoclonal by immunoglobulin light-chain analysis (κ%/λ% > 4:1 or <0.5:1)

The expression of various CD antigens by m-PTLD usually follows the pattern seen with de novo counterparts, such as expression of CD10 and not CD5 or CD23 by Burkitt lymphoma. There appears to be no consistent pattern, however, with the polymorphic type of PTLD. We have seen expression of CD5 only, CD23 only, CD5 with CD23, and CD10 only. Most cases, however, showed no significant expression of CD5, CD10, or CD23. When aberrant expression of 1 or more of these antigens is seen with p-PTLD, it is much more probable that those lesions are monoclonal and lymphomas rather than polymorphic hyperplasia, although this distinction is not justified in the current WHO classification scheme. One notable finding was frequent expression (7 of 8 cases tested) of T-cell–associated antigen CD43 (MT-1) by immunohistochemistry. This frequency is much higher than is seen with de novo DLBCLs.

Our flow cytometric findings reinforce the concept that PTLDs constitute a heterogeneous group of entities that share similarities, yet that are often distinct from de novo B-cell NHL with respect to the immunophenotype. The knowledge of the surface immunophenotype may prove invaluable in distinguishing monoclonal proliferations requiring combination chemotherapy from polyclonal ones that may regress with reduction in immunosuppression alone.25 Although chemotherapy is often used, consensus treatment protocols are largely lacking for various PTLDs; distinction of monoclonal proliferations from polyclonal proliferations may prove very helpful for the clinicians making critical decisions in these circumstances. Also, expression level of CD20 must be determined, preferably by flow cytometry, before instituting anti-CD20 monoclonal antibody.

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Author notes

Reprints: Zahid Kaleem, MD, Hematology and Flow Cytometry Laboratory, Department of Pathology, Creighton University Medical Center, 601 N 30th St, Omaha, NE 68131 ([email protected])