CD30 Expression in Follicular Lymphoma.

CD30 is a transmembrane glycoprotein and is a member of the tumor necrosis factor receptor superfamily.[1] CD30 plays a role in regulating the function or proliferation of normal lymphoid cells.[2] The Ki-1 monoclonal antibody was first identified as useful for immunohistochemical staining in identifying Reed-Sternberg cells in Hodgkin lymphoma.[2-4] Subsequently, CD30 proved useful in helping define anaplastic large cell lymphoma (ALCL), which was initially known as Ki-1 lymphoma. However, it is now known that morphology, histology, and molecular identification of the t(2;5)(p23;q35) translocation are most critical in identifying ALCL, since numerous other lymphomas and carcinomas may have CD30 expression as a nonspecific indicator of cellular activation. In addition, the t(2;5) of ALCL can now be readily detected in paraffin tissue by the immunohistochemical evaluation of ALK-1.[5]

[CD30.sup.+] B-cell lymphomas have been placed into other categories, most commonly diffuse large B-cell lymphomas.[2,6] However, occasional to numerous [CD30.sup.+] B cells have been described in sporadic cases of follicular lymphoma (FL),[7] resembling the pattern seen in reactive tonsils and lymph nodes.[8] CD30 may be present on activated B or T cells, but not resting mature or precursor B or T cells.[2,9-11] Benign reactive [CD30.sup.+] cells tend to be large, immunoblastic-appearing cells located at the periphery of germinal centers.[2,11] In infectious mononucleosis or toxoplasmosis, the [CD30.sup.+] cells are also increased in the paracortex.[2,12]

We describe the pattern of CD30 expression in FL and compare it with the pattern described in reactive conditions, as well as with diffuse (non-ALCL) large B-cell non-Hodgkin lymphoma.

INDEX CASE HISTORY

A 69-year-old white woman with cervical lymphadenopathy had a 3.5-cm lymph node surgically excised. The patient had no previous history of malignant lymphoma. The cut surface was homogeneous, tan-white, and of fish-flesh consistency. A portion was submitted in formalin for routine histology, and a portion was submitted in RPMI 1640 medium (Cellgro Mediatech Tissue Culture Media; Fisher Scientific, Pittsburgh, Pa) for flow cytometric analysis.

MATERIALS AND METHODS

Index Case

Cytomorphology/Flow Cytometric Immunophenotyping.--Wright-stained touch preparations of the lymph node were performed. Based on review of these touch preparations, the cell suspension of lymph node prepared in the RPMI medium was analyzed on an Ortho Cytoronabsolute flow cytometer (Ortho Diagnostic Systems, Raritan, NJ) using standard techniques and the following commercially available monoclonal antibodies: CD5, CD10, CD19, and HLA-DR (Ortho); CD2 and CD14 (Coulter Clone, Coulter Immunology, Hialeah, Fla); CD3 and CD20 (Becton Dickinson, San Jose, Calif); CD30, [Kappa] and [Lambda] light chains (Dako Corporation, Carpinteria, Calif); and CD45 (Caltag, Burlingame, Calif). Dual staining was performed as follows: CD19/ CD5, CD20/HLA-DR, CD45/CD10, and [Kappa]/[Lambda].

Histology/Immunohistochemistry.--Sections of the lymph node were stained with hematoxylin-eosin as well as the following immunoperoxidase stains: L26 (CD20), CD23, Bcl-2, CD3, ALK-1, epithelial membrane antigen, [Kappa] light chain, and [Lambda] light chain (Dako), and CD10 and CD30 (Ki-1; Novocastra, Vector Laboratories, Burlingame, Calif). Unmasking antigen retrieval was used with the CD3 and CD30 stains. Specifically, antigen retrieval of the CD30 stain was performed using Decloaker solution (Biocare, Walnut Creek, Calif). A T180 dilution was used for the CD30 stain. Control tissue included tonsil (L26, CD3, CD23, Bcl-2, CD10, [Kappa], and [Lambda]), breast carcinoma (epithelial membrane antigen), and [CD30.SUP.+] ALCL (CD30 and ALK-1).

The immunoperoxidase stains were performed on the Dako autostainer universal staining system using the Dako-labeled streptavidin-biotin-horse radish peroxidase system and Dako 3,3'-diaminobenzidae substrate.

Molecular Analysis.--Polymerase chain reaction (PCR) analysis was performed for Bcl-2 [t(14;18)] on the paraffin block of the lymph node at the University of Utah Health Sciences Center (Salt Lake City, Utah) as outlined below.

Samples were deparaffinized using xylene, rehydrated, and DNA was isolated from three 15-[micro]m slices using a guanidine thiocyanate-based extraction method (IsoQuick Nucleic Acid Extraction Kit, ORCA Research, Bothell, Wash). Amplifiability of the DNA was confirmed using primers specific for the human [Beta]-globin gene.

Major Breakpoint Regiond/[J.sub.H] PCR.--Rapid-cycle PCR analysis was performed[13] using paired otigonucleotide primers specific for the major breakpoint region of the bcl-2 gene and the [J.sub.H] region of the immunoglobulin heavy-chain joining region for the first round of PCR, which consisted of 25 cycles of denaturation (94 [degrees] C for 0 seconds), annealing (60 [degrees] C for 1 second), and extension (72 [degrees] C for 10 seconds) using 50 ng of DNA as the starting template. Amplification was performed using a 1605 Rapid Air Thermocycler (Rapid Cycler, Idaho Technology, Idaho Falls, Idaho).

Heminesting was performed by utilizing 1 [micro]L of the initial amplification product as the template in a second reaction performed under identical conditions using an internal major breakpoint region primer. Polymerase chain reaction products were subjected to electrophoresis at 80 V for 1 hour using ethidium bromide-stained 1.5% agarose gels (FMC Bioproducts, Rockland, Me) and were detected by ultraviolet transillumination.

Minor Cluster Regiond/[J.sub.H].--A heminested rapid-cycle PCR approach was performed using primers specific for the minor cluster region of the bcl-2 gene and the IgH J region. The first round of PCR consisted of 25 cycles of denaturation (94 [degrees] C for 0 seconds), annealing (55 [degrees] C for 0 seconds), and extension (72 [degrees] C for 20 seconds). Heminesting was performed by utilizing 1 [micro]L of the initial amplification product as the template in a second reaction performed under identical conditions using an internal minor cluster region primer. Both amplification reactions were performed using a 1605 Rapid Air Thermocycler (Idaho Technology). Polymerase chain reaction products were subjected to electrophoresis at 80 V for 1 hour using ethidium bromide--stained 1.5% agarose gels (FMC Bioproducts) and were detected by ultraviolet transillumination.

Review/Immunohistochemistry of Additional Cases

The files of the Division of Hematopathology at St Louis University Health Sciences Center (St Louis, Mo) and the Section of Hematopathology, Division of Anatomic Pathology, at the University of Utah Health Sciences Center were reviewed for FLs. Twenty-two cases and the following subtypes were retrieved for immunoperoxidase staining of paraffin sections with CD30: follicular, predominantly small cleaved cells (3 cases); follicular, [+ or -] diffuse, mixed, small cleaved, and large cells (10 cases); follicular, [+ or -] diffuse, large cell (8 cases); and diffuse, large cell (1 case). A representative sample of the spectrum of FL was included with an emphasis on those with a larger cell component since the larger cells were expected to be [CD30.sup.+]. Of the 21 cases with a follicular component, CD10 immunoperoxidase staining was performed in 20 cases. CD10 was positive in the great majority (18/ 20) of these cases. Of the 2 CD10- cases, 1 was positive for Bcl-6 and LN-1 (CDw75). In the other case, stainings for Bcl-6 and LN-1 were not performed. LN-1 staining was performed in only 7 additional cases with a follicular component (all [CD10.sup.+]); 6 were LN-[1.sup.+] and 1 was LN-[1.sup.-]. No additional cases with a follicular component were stained with Bcl-6. The diffuse, large cell lymphoma revealed a monoclonal B-cell immunophenotype by flow cytometric immunophenotyping and was [CD10.sup.+], CDw75 (LN-1)+, and Bcl-[6.sup.+] by immunoperoxidase staining. This immunophenotype supported a follicular origin in this case. Molecular analysis of bcl-2 immunoglobulin heavy-chain gene fusion was performed in only 1 case with a follicular component and was detected. The CD30 immunoperoxidase stain was performed as in the index case.

RESULTS

Index Case

Cytomorphology.--The Wright-stained touch preparations of the lymph node revealed predominantly large lymphoid cells with reniform nuclei, large nucleoli, and a variable amount of cytoplasm, containing Golgi-based vacuoles.

Flow Cytometry.--The flow cytometric results revealed an expanded large cell region (56% of cells). Cells within this region were monoclonal B cells expressing CD45, CD19, CD20, HLA-DR, and CD30, and showed selective bright expression of surface [Lambda] light chain (Figure 1). CD30 was expressed by 79% of cells in this region, and the monoclonal B cells represented 96% of cells in this region; thus, the CD30 must have been coexpressed by the monoclonal B cells. There was no coexpression of CD10, CD5, or other T-cell markers by this monoclonal B-cell population.

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Histology/Immunohistochemistry.--Hematoxylin-eosin-stained tissue sections showed complete effacement of the lymph node architecture by a malignant lymphoma with a nodular growth pattern. The nodules were composed of large pleomorphic cells characterized by vesicular nuclear chromatin, nucleoli, and a small amount of cytoplasm containing small pink globules indenting the nuclear membrane (Figure 2).

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Immunoperoxidase stains performed on the paraffin-embedded tissue block showed intense staining of the large malignant cells by CD20 (membrane), CD10 (membrane), and Bcl-2 (cytoplasmic). CD30 showed membrane and Golgi staining of the large pleomorphic lymphoid cells throughout the nodules and revealed occasional clustering of cells. Reactions for epithelial membrane antigen, ALK-1, CD3, and CD23 were negative in tumor cells. CD3 and CD23 stained small scattered cells, and epithelial membrane antigen stained occasional plasma cells. [Kappa] and [Lambda] light chains revealed staining of occasional plasma cells. The large pleomorphic cells were negative for [Kappa] and [Lambda] light chains.

Molecular Analysis.--Polymerase chain reaction performed at the University of Utah on the paraffin tissue block showed a t(14;18) translocation (Figure 3).

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Summary of Index Case.--The flow cytometric analysis, combined with cytomorphology, histology, immunoperoxidase stains, and molecular analysis, was diagnostic of involvement of the cervical lymph node by a malignant lymphoma, non-Hodgkin, follicular, predominantly large cell type with the presence of [CD30.sup.+] large, pleomorphic, malignant B cells. Although CD10 was not expressed in the index case, lack of CD10 occurs in approximately 9% of FLs, particularly those with a large cell component.[14]

Review/Immunohistochemistry of Additional Cases

Immunoperoxidase staining of the 22 additional cases with CD30 revealed no staining in 11 of 22 cases (Table). The diffuse large cell lymphoma fell in this group of 11 cases. The other half showed varying degrees of CD30 staining. There was no clear correlation between numbers of large cells and the expression of CD30. Sixty-seven percent of grade 1 FLs had [CD30.sup.+] cells, 40% of grade 2 cases had [CD30.sup.+] cells, and 55% of grade 3 cases had [CD30.sup.+] cells. The pattern of CD30 expression was heterogeneous, ranging from intense to faint staining between cases, as well as showing variability within the same case. In 4 cases, there were rare medium-sized cells either in between nodules or within nodules that stained faintly with CD30. These were not obviously malignant cells. However, in the remaining 7 cases, there was definitive staining of large malignant cells, some of which had the characteristic Golgi pattern of staining (Figure 4). In 3 of these 7 cases, the large pleomorphic cells were relatively rare and scattered with faint staining. One of these 3 cases had one pleomorphic cell at the edge of a nodule with intense Golgi staining. In the other 4 cases, the large pleomorphic cells were scattered (1-5 cells per high-power field) but intensely staining (2 cases), numerous (up to 50 cells per highpower field) and faintly staining (1 case), or numerous (up to 50 cells per high-power field) and intensely staining (1 case). The [CD30.sup.+] large pleomorphic cells in these 4 cases were all located in the malignant nodules. In these 4 cases, ALK-1 staining was also analyzed and was negative in all cases. The CD30 staining in the 11 cases did not correlate with evidence of plasmacytoid features.

Patterns of CD30 Staining in 22 Cases of Grades 1-3 Follicle Center
Cell (FCC) Lymphomas

             CD30 Staining Patterns                Grade 1   Grade 2

No staining                                           1         6
Faint staining of rare medium-sized
 cells between nodules                                0         0
Faint staining of rare medium-sized
 cells in and between nodules                         1         0
Faint staining of rare scattered
 large malignant cells in nodules                     0         2
Intense Golgi staining of 1 anaplastic
 cell at edge of nodule; faint staining of
 scattered large malignant cells between nodules      1         0
Intense cytoplasmic Golgi staining of rare
 large malignant cells in nodules                     0         0
Faint staining of numerous large malignant
 cells in nodules                                     0         1(*)
Intense cytoplasmic Golgi staining of numerous
 malignant cells in nodules                           0         1(*)

             CD30 Staining Patterns                Grade 3    Total

No staining                                           4        11
Faint staining of rare medium-sized
 cells between nodules                                2         2
Faint staining of rare medium-sized
 cells in and between nodules                         1         2
Faint staining of rare scattered
 large malignant cells in nodules                     0         2
Intense Golgi staining of 1 anaplastic
 cell at edge of nodule; faint staining of
 scattered large malignant cells between nodules      0         1
Intense cytoplasmic Golgi staining of rare
 large malignant cells in nodules                     2(*)      2
Faint staining of numerous large malignant
 cells in nodules                                     0         1
Intense cytoplasmic Golgi staining of numerous
 malignant cells in nodules                           0         1

(*) These case were additionally ALK-1 negative.

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COMMENT

In our index case, the tumor was clearly of B-cell origin, as determined by both flow cytometry (CD19 and CD20 positivity) and CD20 positivity in paraffin. In addition, the histology was that of a follicular cell lymphoma, large cell type (grade 3). The CD10 positivity by immunoperoxidase staining and molecular detection of t(14;18) supported the follicle center cell origin. CD30 was positive in malignant cells, both by flow cytometry and immunoperoxidase staining.

Review of the literature reveals scant data evaluating the use of CD30 by flow cytometric analysis. We have detected it in a number of cases of ALCL. Testing for CD30 was ordered in our case because on touch preparations the cells were large with reniform nuclei and a perinuclear vacuole. Our differential diagnosis included a [CD30.sup.+] ALCL, other types of non-Hodgkin lymphoma, and Hodgkin lymphoma.

Numerous studies have shown that B-cell lymphomas may be positive for CD30 by immunohistochemistry. These are predominantly diffuse large cell lymphomas, as previously discussed.[2,5,6,15-20] In an article by Noorduyn et al,[21] CD30 expression was investigated using frozen tissue in a series of 146 large B-cell lymphomas. Ten (40%) of 25 cases of large B-cell lymphomas with either anaplastic or immunoblastic features had at least 50% of cells positive for CD30. The pattern of reactivity within these [CD30.sup.+] cells was not described. The remainder (121 cases) were low-grade B-cell lymphomas, including FL; 15 (12%) of these 121 cases had at least 50% of cells staining with CD30, and 27 (22%) of these 121 cases had scattered [CD30.sup.+] cells. A recent article on mediastinal B-cell lymphomas noted the common finding of CD30 positivity in these tumors. Ber-H2 was used in formalin-fixed tissue, and of 51 cases, 35 (69%) stained for CD30, ranging from rare to focal to diffuse membrane and paranuclear staining.[22] We found only 2 references specifically addressing CD30 expression in FL. Piris et al[23] in 1991 described 19 cases of FLs. Of these, 17 of 19 possessed a subpopulation of [CD30.SUP.+] cells that were located in B-cell areas, mainly at the edge of malignant germinal centers but occasionally within them. In addition, these cells were also located in 1 case in the remnant of the mantle zones, and in 3 cases in T-cell areas. These CD30 cells were found only in frozen sections; the authors were not able to identify them in paraffin sections. Morphologically, the cells were described as blastlike with prominent nucleoli. The authors also compared the cases of follicle center cell lymphomas to CD30 staining in reactive tonsils and lymph nodes. They found [CD30.sup.+] cells in 6 of 10 cases of reactive lymphadenitis, with cells predominantly in the paracortical Tcell areas, close to follicles, but also occasionally in the mantle zones and germinal centers. The 3 tonsils studied all contained scattered [CD30.sup.+] cells. The authors concluded that [CD30.sup.+] cells in both FLs and reactive conditions appear similar and are in comparable distribution; thus, they questioned if the [CD30.sup.+] cells in FLs were indeed part of the malignant process. Piris et al did double-labeling with Ki-67 and anti-CD19 and found that the [CD30.sup.+] cells were also positive for Ki-67 and CD19. They concluded that although the [CD30.sup.+] cells were proliferating B cells, mono-clonality could not be determined.

Subsequently, in 1992, Miettinen[24] described [CD30.sup.+] cells in 51% of nodular small cleaved cell lymphomas studied. Most cases contained only a few [CD30.sup.+] cells. The occasional positive cells were large and located either within the periphery of the nodules or between them. In 4 cases, high numbers of [CD30.sup.+] large cells were seen within the nodules admixed with the small cleaved cells, some of which were centroblastic and with complex nuclear outlines. Some of the [CD30.sup.+] cells were of B-cell origin; none were confirmed to be of T-cell origin by immunohistochemistry.

In our index case, the staining pattern was membranous in scattered large malignant cells, including those with vacuoles. In addition, the flow cytometric analysis showed a monoclonal B-cell population that expressed CD30, suggesting that these cells were indeed monoclonal. To further confirm the follicular origin of this lymphoma, the cells had a t(14;18) translocation and ALK-1 immunohistochemical staining was negative.

We reviewed an additional 22 cases of FL (grades 1-3) and performed CD30 immunoperoxidase staining on B-5--or formalin-fixed paraffin sections. Of these, 50% had no staining for CD30. Eighteen percent (4/22) had staining of rare, medium-sized cells in between and within follicles, similar to the pattern previously described by Piris et al[23] in frozen tissues. Thirty-two percent (7/22) had definitive staining of either large or pleomorphic-appearing malignant cells predominantly within the nodules. Of these 7 cases, 4 cases were evaluated for ALK-1 staining and were negative.

Interestingly, Alsabeh et al[25] described transformation of FLs into [CD30.sup.+] large cell lymphomas with anaplastic features. They had 6 cases of malignant lymphoma with both follicular and diffuse/sinusoidal components. The follicular component was [CD30.sup.-], and the diffuse/sinusoidal component was [CD30.sup.+]. In addition, cells were positive for Bcl-2 in 4 of 6 cases. All 6 cases were positive for CD20 and monoclonal immunoglobulin heavy chains. In only 1 of the 6 cases was reactivity for ALK-1 tested; it was negative. None of the cases were positive for t(14;18), as was our index case.

In reviewing the literature for B-cell lymphomas, we found that some articles noted a difference in the percentage of [CD30.sup.+] cases, dependent on whether immunohistochemical stains were performed on frozen or formalin-fixed tissue. There is a difference in CD30 antibodies used for frozen and formalin-fixed tissue. Ki-1 is not reactive in routinely processed histologic material, but may be used in frozen tissue. Ber-H2 is a monoclonal antibody recognizing a formaldehyde-resistant epitope within the same antigen and may be used in frozen or formalin-fixed tissue.[2,26,27] The percentage of CD30 immunoreactivity appears to be higher in frozen tissue.21 We did not have difficulty in using Ber-H2 in formalin-fixed tissue using typical antigen retrieval techniques. No prognostic significance has been found in B-cell lymphomas regarding whether they are positive or negative for CD30.[21]

In summary, we describe a well-defined case of FL with large pleomorphic cells that was [CD30.sup.+] by both flow cytometric and immunohistochemical methods. In addition, we reviewed 22 cases of FL and demonstrated that 32% of cases showed definitive CD30 positivity in malignant cells (some with pleomorphic features) by immunohistochemistry. Thus, the finding of CD30 staining and cellular pleomorphism is not uncommon in the morphologic and immunophenotypic spectrum of FL. The lack of ALK-1 staining in our cases supports the hypothesis that this represents an activation phenotype rather than falling within the spectrum of ALCL. The [CD30.SUP.+] staining was not limited to large cell populations, which are those cells usually described in the literature to express CD30, but involved a spectrum of cell sizes. In addition, CD30 expression was heterogeneous, as previously described, indicating a noninherent quality of the tumor cells. It is uncertain whether the activation of tumor cells imparts different biologic behavior, although studies of [CD30.SUP.+] diffuse large B-cell nonHodgkin lymphoma would suggest no effect on biologic behavior. The effect of CD30 expression on tumor behavior, growth, and response to therapy has not been well investigated in FL. Documentation of CD30 positivity in FL may allow for the use of immunotherapies, such as anti-CD30 alone or in concert with antibodies to CD20, as therapeutic agents.

References

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[3.] Froese P, Lemke H, Gerdes J, et al. Biochemical characterization and biosynthesis of the Ki-1 antigen in Hodgkin-derived and virus transformed human B and T lymphoid cell lines. J Immunol. 1987;139:2081-2087.

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[8.] Segal GH, Kjeldsberg CR, Smith GE Perkins SE. CD30 antigen expression in florid immunoblastic proliferations: a clinicopathologic study of 14 cases. Am J Clin Pathol. 1994;102:292-298.

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[12.] Abbondanzo SL, Sato N, Stauss SC, Jaffe ES. Acute infectious mononucleosis CD30 (Ki-1) antigen expression and histologic correlations. Am J Clin Pathol. 1990;93:698-702.

[13.] Wittwer CT, Ririe KM, Andrew RV, David DA, Gundry RA, Balis UJ. The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. Biotechniques. 1997;22:176-181.

[14.] Dunphy CH. Contribution of flow cytometric immunophenotyping to the evaluation of tissues with suspected lymphoma? Cytometry. 2000;42:296-306.

[15.] Su L, Schnitzer B, Ross CW, et al. The t(2;5) associated p80 NPM/ALK fusion protein in nodal and cutaneous CD30+ lymphoproliferative disorders. J Cutan Pathol. 1997;24:597-603.

[16.] Stansfeld AG, Diebold J, Noel H, et al. Updated Kiel classification for lymphomas. Lancet. 1988;1:292-293.

[17.] Harris NL, Jaffe ES, Stein H, et al. A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood. 1994;84:1361-1392.

[18.] Chan JKC, Ng CS, Hui PK, et al. Anaplastic large cell Ki-1 lymphoma: delineation of two morphological types. Histopathology. 1989;15:11-34.

[19.] Delsol G, Lamant L, Mariame B, et al. A new subtype of large B-cell lymphoma expressing the ALK kinase and lacking the 2;5 translocation. Blood. 1997;89:1483-1490.

[20.] Kuze T, Nakamura N, Hashimoto Y, Abe M. Most of CD30 positive anaplastic large cell lymphoma of B cell type show a somatic mutation in the IgH V region genes. Leukemia. 1998;12:753-757.

[21.] Noorduyn LA, de Bruin PC, van Heerde P, Van de Sandt MM, Ossenkoppele GJ, Meijer CJLM. Relation of CD30 expression to survival and morphology in large cell B cell lymphomas. J Clin Pathol. 1994;47:33-37.

[22.] Higgins JP, Warnke RA. CD30 expression is common in mediastinal large B cell lymphoma. Am J Clin Pathol. 1999;112:241-247.

[23.] Piris M, Garter KC, Mason DY. CD30 expression in follicular lymphoma. Histopathology. 1991;18:25-29.

[24.] Miettinen M. CD30 distribution, lmmunohistochemical study on formaldehyde-fixed, paraffin-embedded Hodgkin's and non-Hodgkin's lymphomas. Arch Pathol Lab Med. 1992; 116:1197-1201.

[25.] Alsabeh R, Medeiros J, Glackin C, Weiss L. Transformation of follicular lymphoma into CD30-positive large cell lymphoma with anaplastic cytologic features. Am J Surg Pathol. 1997;21:528-536.

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Accepted for publication March 9, 2001.

From the Division of Hematopathology, Department of Pathology, St Louis University Health Sciences Center, St Louis, Mo (Drs Gardner, Evans, and Dunphy); the Department of Pathology, University of South Alabama, Mobile (Dr Polski); and the Section of Hematopathology, University of Utah Health Sciences Center, Salt Lake City (Dr Perkins).

Reprints: Cherie H. Dunphy, MD, Department of Pathology and Laboratory Medicine, CB#7525, Brinkhous-Bullitt Bldg, University of North Carolina, Chapel Hill, NC 27599-7525 (e-mail: [e-mail]).