Bcl-6 Immunostaining – Practical Aspects in a Clinical Diagnostic Setting

01. September 2007

The bcl-6 gene is associated with translocations affecting 3q27 in cases of diffuse large B-cell lymphoma (DLBCL). The gene product is a zinc finger protein thought to be involved in control of germinal centre proliferation. Bcl-6 protein can be detected immunohistochemically and positive staining is nuclear in location.

Bcl-6 protein is selectively expressed in cells showing a mature B-cell phenotype but is not present in immature or precursor B-cells. Virtually all B-cells within normal germinal centres are bcl-6 positive and in everyday practice bcl-6 is a useful marker of follicle centre B-cells.

Bcl-6 immunostaining is helpful in a number of diagnostic settings;

  • In the differential diagnosis of small B-cell lymphoma. Follicular lymphoma will show bcl-6 (and CD10) positivity whereas other small B-cell lymphomas are usually negative.
  • In difficult cases of follicular lymphoma bcl-6 can identify an interfollicular component.
  • Bcl-6 is an important prognostic marker in DLBCL, where CD10, bcl-6 and MUM1/IRF4 are used to identify germinal centre and activated B-cell phenotypes.
  • Bcl-6 can be valuable in distinguishing classical Hodgkin lymphoma from nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). The Reed-Sternberg cells of classical Hodgkin lymphoma are bcl-6 negative whereas the large (“L&H”) cells of NLPHL are bcl-6 positive.
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Introduction

The bcl-6 gene was initially detected by its involvement in chromosomal translocations affecting 3q27 in cases of diffuse large B-cell lymphoma (DLBCL).1 Approximately 40% of DLBCL show a rearrangement of the bcl-6 gene, and bcl-6 abnormalities are also found in 5–10% of follicular lymphomas (FL). In these rearrangements the chromosome breakpoints are clustered around a 4 kb region spanning the bcl-6 promoter sequences, and the translocations lead to the fusion of exons 2–10 from bcl-6 with promoter regions on other chromosomes, with consequent over-expression of bcl-6 protein.

Studies of bcl-6 gene product have identified a 95 kD nuclear protein with a Kruppel-type zinc finger protein structure that resembles similar molecules involved in developmental regulation. The protein is a transcription factor thought to be involved in controlling germinal centre proliferation, possibly responding to specific signals provided by antigens, germinal centre cells, follicular dendritic cells or cytokines.2

Bcl-6 protein is selectively expressed in cells showing a mature B-cell phenotype (DLBCL, Burkitt lymphoma (BL)) but not in immature B-cells such as those in precursor B-acute lymphoblastic lymphoma/leukaemia (B-ALL). Virtually all cells within follicle centres express bcl-6, both centroblasts and centrocytes, and this bcl-6 expression is seen in both proliferating and non-proliferating cells.3 The majority of cells in the mantle zones do not express bcl-6 protein, with the exception of a few scattered IgD-positive small lymphocytes. The evidence suggests that most of the bcl-6 positive B-cells in normal germinal centres do not co-express bcl-2. Normal immunoblasts and plasma cells are bcl-6 negative. A percentage of normal T-cells also show bcl-6 expression, and these are seen in germinal centres, mantle zones and the interfollicular region of lymph nodes. The majority of bcl-6 positive T-cells are CD4 positive.4

Immunohistochemical staining of bcl-6 is carried out using antigen retrieval methods. When using Leica Microsystems’ LN22 clone, the best results were obtained with 3 µm sections which were de-waxed and pre-treated on a Bond-maxTM immunostainer (Leica Microsystems UK) with ER2 solution (pH 9.0) for 30 minutes. The sections were then stained with LN22 using the Bond Polymer Refine Detection (peroxidase-DAB). Normal staining is nuclear, and pre-treatment is critical. Over treatment can lead to non-specific staining of all the nuclei on the section.

In everyday practice bcl-6 is a useful marker of follicle centre B-cells. There is some non-specific nuclear staining of spindle-shaped cells in lymph nodes (fibroblastic or myofibroblastic cells). In some settings macrophage cytoplasm can show weak non-specific staining (alveolar macrophages in the lungs, for example). In bone marrow specimens there is some nuclear positivity in immature myeloid cells, which must be taken into account when looking for interstitial infiltrates. Epithelial cell nuclei (both squamous and glandular) can also show weak staining.

Figure 1

A case of reactive lymphoid hyperplasia showing the edge of a lymphoid follicle and the interfollicular zone stained with H&E and bcl-6. The germinal centre cells are positive with bcl-6 but there are only rare positive cells in the interfollicular region.

Bcl-6 as a marker of follicular lymphoma

Both non-neoplastic and neoplastic follicle centre cells show positive nuclear staining with bcl-6. When assessing follicular proliferations in lymph nodes bcl-6 is routinely used together with CD10, another marker of germinal centre B-cells, and with bcl-2, which is over-expressed in the majority of follicular lymphomas (FL). These antibodies can reveal follicular structures in core biopsies of lymph nodes or in distorted nodes, particularly when positive staining is associated with the presence of a follicular dendritic cell (FDC) network, as shown by CD21, CD23 or CD35. The combination of CD20, bcl-6, CD10, bcl-2 and CD21 is particularly helpful in the detection of neoplastic follicles in FL.

Most follicular lymphomas show an interfollicular component, where the tumour cells are present outside the neoplastic follicles. This interfollicular component is not seen in reactive follicular hyperplasia, and can be detected using CD10 and bcl-6.5, 6 The presence of cells with a germinal centre phenotype in the interfollicular region of a node supports the diagnosis of follicular lymphoma. Figures 1 and 2 illustrate bcl-6 staining of the interfollicular region in reactive hyperplasia and follicular lymphoma.

When assessing lymphomatous infiltrates in bone marrow, bcl-6 is useful in a proportion of cases. Nodular deposits of follicular lymphoma within the bone marrow may not contain FDC networks, and whilst both CD10 and bcl-6 can be downregulated in marrow, the presence of nuclear bcl-6 staining in nodules of lymphocytes in a bone marrow biopsy is good evidence of a follicle centre cell origin. It is important to recognise that myeloid precursors also express bcl-6, and that these need to be distinguished from the lymphoma cells.

As a marker of germinal centre cells, bcl-6 can be helpful in the differential diagnosis of small B-cell lymphoma. Lymphomas composed of small B-cells include follicular lymphoma (FL), small lymphocytic lymphoma/chronic lymphocytic leukaemia (CLL), lymphoplasmacytic lymphoma (LPL), mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL). It is often difficult to distinguish these conditions morphologically, and a panel of antibodies is required to identify the individual conditions. CLL and MCL will usually show co-expression of CD20 and CD5. MCL will additionally be cyclin-D1 positive. LPL and MZL may show no specific markers, although IgM is usually positive. Both bcl-6 and CD10 are normally expressed in follicular lymphoma, and these markers will be absent in the other conditions in this group. Similarly bcl-6 in combination with CD10, bcl-2 and CD21 can be helpful in the distinction of cutaneous follicular lymphoma from cutaneous marginal zone lymphoma.7

In practice, although bcl-6 is predominantly a marker of follicle centre cells, it is not completely specific – some mantle cell lymphomas and some marginal zone lymphomas can show positive staining. As with most antibodies, bcl-6 is most informative when used in conjunction with other markers, and should be used as part of a panel of antibodies that includes CD20, CD3, CD10 and CD21.

Figure 2

Follicular lymphoma stained with H&E and bcl-6. The neoplastic follicle is bcl-6 positive and there is a significant number of bcl-6 positive cells in the interfollicular zone.

Bcl-6 as a prognostic marker in diffuse large B-cell lymphoma

Immunohistochemical analysis of both DLBCL and FL indicates that bcl-6 protein is detectable in these tumours independent of the presence of bcl-6 genetic rearrangements. Deregulation of bcl-6 expression may contribute to lymphomagenesis by preventing post-germinal centre differentiation.

Gene expression microarray studies8–10 initially indicated that DLBCL could be sub-classified into germinal centre and post germinal centre categories. The separation into these categories was found to be prognostically significant; those patients whose lymphoma had a germinal centre phenotype responded better to chemotherapy. Subsequently it was discovered that the sub-classification recognised by gene expression can be readily replicated using flow cytometry and immunohistochemistry.10, 11 DLBCL can now be divided into cases with a germinal centre cell phenotype and cases with an activated B-cell phenotype (post germinal centre cell) using bcl-6, CD10 and MUM1/IRF4. The germinal centre subtype shows either co-expression of CD10 and bcl-6 or is CD10 negative, bcl-6 positive and MUM1/IRF4 negative. These tumours can express IgG or IgM and a small number are CD23 positive. In contrast, the activated B-cell/post germinal centre subtype of DLBCL is negative for CD10, is either bcl-6 positive or negative, and is positive with MUM1/IRF4. There are occasional rare cases that are CD10 positive, bcl-6 negative and MUM1/IRF4 negative. A simple algorithm for sub-categorising DLBCL is shown below in Diagram 1, and a bcl-6 positive DLBCL is illustrated in Figure 3.

In practice some cases show focal expression of CD10, bcl-6 and/or MUM1 so that it is not always possible to assign a definite subtype to all cases of DLBCL. A critical assessment of bcl-6 staining in DLCBL reported that whilst positivity is usually defined as >10% of tumour cells staining, a sub-population of cases showed uniform staining of >75% of cells; this uniform pattern was more often associated with CD10 co-expression and was considered more indicative of a germinal centre phenotype.13

More recently there is a preliminary report in the literature indicating that bcl-6 expression in DLBCL may be a biological marker of patient response to the addition of Rituximab to CHOP chemotherapy. In a US Intergroup trial comparing R-CHOP and CHOP in DLBCL, the addition of Rituximab resulted in a significant increase in failure-free survival and overall survival only in those patients where the tumour was bcl-6 negative. Bcl-6 positive cases did not benefit from the addition of Rituximab.14

Bcl-6 expression has also been reported as being associated with longer overall survival in primary central nervous system lymphomas.15 Expression of bcl-6 and MUM1/IRF4 proteins correlates with overall survival in patients with primary cutaneous large B-cell lymphoma.16

Figure 3

A case of diffuse large B-cell lymphoma stained with H&E, CD20, bcl-6 and CD10. MUM1/IRF4 was negative in this case, indicating a germinal centre phenotype.

Diagram 1

DLBCL prognostic stratification (from Berglund et al [12]). GC = germinal centre, ABC = activated B-cell.

Bcl-6 in Hodgkin lymphoma

Bcl-6 staining has a role in distinguishing nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) from classical Hodgkin lymphoma (cHL). This can sometimes be a problem, particularly in cases of the lymphocyte-rich subtype of cHL and where there is CD30 positivity in NLPHL. In the majority of cases of NLPHL the large cells (L&H cells) show strong nuclear bcl-6 expression whereas the Hodgkin/Reed-Sternberg cells of cHL are positive in only a small proportion of cases. In addition the CD3+,CD4+ T-cells seen around L&H cells are also strongly bcl-6 positive, whereas T-cells rosetting classical Hodgkin cells are mainly bcl-6 negative.17, 18 Figures 4 and 5 illustrate bcl-6 staining in NLPHL and cHL.

Figure 4

Nodular lymphocyte predominant Hodgkin lymphoma stained with H&E and bcl-6. The large cells (“L&H” cells) show positive nuclear staining with bcl-6.

Bcl-6 in other lymphomas

A proportion of cases of angioimmunoblastic T-cell lymphoma (AITL) contain neoplastic T-cells that demonstrate co-expression of CD4, CD10 and bcl-6. Identifying these cells can help in the diagnosis of AITL, and may be useful in distinguishing AITL from peripheral T-cell lymphoma, unspecified.5, 19

In human immunodeficiency virus (HIV)-related lymphomas there is some evidence that different clinicopathological groups can be separated by their expression pattern with a panel of antibodies consisting of bcl-6, MUM-1/IRF4 and CD138. In a study of 87 HIV-related lymphomas Burkitt lymphoma and most diffuse large B-cell lymphomas showed a bcl-6+, MUM1/IRF4–, CD138– phenotype. Immunoblastic lymphomas were bcl-6–, MUM1/IRF4+, CD138–, whilst primary central nervous system lymphoma, primary effusion lymphoma, HIV-Hodgkin lymphoma and oral plasmablastic lymphoma were all bcl-6–, MUM1/IRF4+, CD138+.20

Figure 5

Classical Hodgkin lymphoma stained with H&E and bcl-6. The Reed-Sternberg cells are bcl-6 negative.

Bcl-6 in other lymphomas

A proportion of cases of angioimmunoblastic T-cell lymphoma (AITL) contain neoplastic T-cells that demonstrate co-expression of CD4, CD10 and bcl-6. Identifying these cells can help in the diagnosis of AITL, and may be useful in distinguishing AITL from peripheral T-cell lymphoma, unspecified.5, 19

In human immunodeficiency virus (HIV)-related lymphomas there is some evidence that different clinicopathological groups can be separated by their expression pattern with a panel of antibodies consisting of bcl-6, MUM-1/IRF4 and CD138. In a study of 87 HIV-related lymphomas Burkitt lymphoma and most diffuse large B-cell lymphomas showed a bcl-6+, MUM1/IRF4–, CD138– phenotype. Immunoblastic lymphomas were bcl-6–, MUM1/IRF4+, CD138–, whilst primary central nervous system lymphoma, primary effusion lymphoma, HIV-Hodgkin lymphoma and oral plasmablastic lymphoma were all bcl-6–, MUM1/IRF4+, CD138+.20

Summary

Bcl-6 has a variety of roles in the diagnosis of lymphoma, and should be included in antibody panels in a number of settings;

  • Bcl-6 is a valuable marker of follicle centre cells, and when used together with CD20, CD21, bcl-2 and CD10 is helpful in the identification of follicular lymphoma.
  • In cases of diffuse large B-cell lymphoma bcl-6 is important in prognostic stratification and together with CD10 and MUM1/IRF4 can distinguish cases with a germinal centre phenotype from those with an activated B-cell phenotype.
  • Bcl-6 can contribute the distinction of classical Hodgkin lymphoma from nodular lymphocyte predominant Hodgkin lymphoma; in this setting bcl-6 should be part of a panel of antibodies consisting of CD20, CD30, CD15 and CD10.

Although bcl-6 may provide biological information in some cases of lymphoma, long-term studies are needed to determine where and when bcl-6 expression is of clinical significance.

References

  1. Ohno H. Pathogenetic role of BCL6 translocation in B-cell non-Hodgkin’s lymphoma. Histol Histopathol 2004; 19(2): 637–650.
  2. Niu H. The proto-oncogene BCL-6 in normal and malignant B cell development. Hematol Oncol 2002; 20 (4): 155–66.
  3. Cattoretti G, Chang CC, Cechova K, Zhang J, Ye BH, Falini B, et al. BCL-6 protein is expressed in germinal-center B cells. Blood 1995; 86 (1): 45–53.
  4. Falini B, Fizzotti M, Pileri S, Liso A, Pasqualucci L, Flenghi L. Bcl-6 protein expression in normal and neoplastic lymphoid tissues. Ann Oncol 1997; 8 Suppl 2: 101–4.
  5. Ree HJ, Kadin ME, Kikuchi M, Ko YH, Suzumiya J, Go JH. Bcl-6 expression in reactive follicular hyperplasia, follicular lymphoma, and angioimmunoblastic T-cell lymphoma with hyperplastic germinal centers: heterogeneity of intrafollicular T-cells and their altered distribution in the pathogenesis of angioimmunoblastic T-cell lymphoma. Hum Pathol 1999; 30 (4): 403–11.
  6. Dogan A, Bagdi E, Munson P, Issacson PG. CD10 and BCL-6 expression in paraffin sections of normal lymphoid tissue and B-cell lymphomas. Am J Surg Pathol 2000; 24 (6): 846–52.
  7. de Leval L, Harris NL, Longtine J, Ferry JA, Duncan LM. Cutaneous b-cell lymphomas of follicular and marginal zone types: use of Bcl-6, CD10, Bcl-2, and CD21 in differential diagnosis and classification. Am J Surg Pathol 2001; 25 (6): 732–41.
  8. Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000; 403 (6769): 503–11.
  9. Rosenwald A, Staudt LM. Gene expression profiling of diffuse large B-cell lymphoma. Leuk Lymphoma 2003; 44 Suppl 3: S41–7.
  10. Hans CP, Weisenburger DD, Greiner TC, Gascoyne RD, Delabie J, Ott G, et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood 2004; 103 (1): 275–82. Epub 2003 Sep 22.
  11. Barrans SL, Carter I, Owen RG, Davies FE, Patmore RD, Haynes AP, et al. Germinal center phenotype and bcl-2 expression combined with the International Prognostic Index improves patient risk stratification in diffuse large B-cell lymphoma. Blood 2002; 99 (4): 1136–43.
  12. Berglund M, Thunberg U, Amini RM, Book M, Roos G, Erlanson M, et al. Evaluation of immunophenotype in diffuse large B-cell lymphoma and its impact on prognosis. Mod Pathol 2005; 18 (8): 1113–20.
  13. Ree HJ, Ohsima K, Aozasa K, Takeuchi K, Kim CW, Yang WI, et al. Detection of germinal center B-cell lymphoma in archival specimens: critical evaluation of Bcl-6 protein expression in diffuse large B-cell lymphoma of the tonsil. Hum Pathol 2003; 34 (6): 610–6.
  14. Winter JN, Weller EA, Horning SJ, Krajewska M, Variakojis D, Habermann TM, et al. Prognostic significance of Bcl-6 protein expression in DLBCL treated with CHOP or R-CHOP: a prospective correlative study. Blood 2006; 107 (11): 4207–13.
  15. Braaten KM, Betensky RA, de Leval L, Okada Y, Hochberg FH, Louis DN, et al. BCL-6 expression predicts improved survival in patients with primary central nervous system lymphoma. Clin Cancer Res 2003; 9 (3): 1063–9.
  16. Sundram U, Kim Y, Mraz-Gernhard S, Hoppe R, Natkunam Y, Kohler S. Expression of the bcl-6 and MUM1/IRF4 proteins correlate with overall and disease-specific survival in patients with primary cutaneous large B-cell lymphoma: a tissue microarray study. J Cutan Pathol 2005; 32 (3): 227–34.
  17. Falini B, Bigerna B, Pasqualucci L, Fizzotti M, Martelli MF, Pileri S, et al. Distinctive expression pattern of the BCL-6 protein in nodular lymphocyte predominance Hodgkin’s disease. Blood 1996; 87 (2): 465–71.
  18. Kraus MD, Haley J. Lymphocyte predominance Hodgkin’s disease: the use of bcl-6 and CD57 in diagnosis and differential diagnosis. Am J Surg Pathol 2000; 24 (8): 1068–78.
  19. Yuan CM, Vergilio JA, Zhao XF, Smith TK, Harris NL, Bagg A. CD10 and BCL6 expression in the diagnosis of angioimmunoblastic T-cell lymphoma: utility of detecting CD10+ T cells by flow cytometry. Hum Pathol 2005; 36 (7): 784–91.
  20. Carbone A, Gloghini A, Larocca LM, Capello D, Pierconti F, Canzonieri V, et al. Expression profile of MUM1/IRF4, BCL-6, and CD138/syndecan-1 defines novel histogenetic subsets of human immunodeficiency virus-related lymphomas. Blood 2001; 97 (3): 744–51.

Comments

Alan Ramsay, Dr., MD

Alan Ramsay is a Consultant Histopathologist at University College London Hospital NHS Trust, UK. He has been at UCLH since 2005, previously working at Great Ormond Street and Southampton. Since 2006 he has specialized in haematopathology. He is the Haematopathology Lead at UCLH and the Royal Free Hospital, and for the North Central London Cancer Network. He reports over 2000 haematopathology cases each year, approximately 300 of which are personal referral cases.

Alan Ramsay qualified as a doctor and was trained in Pathology at UCLH, and has an MD from Southampton University. He served as Secretary of the British Lymphoma Pathology Group from 1992 to 2000, and as President from 2000 to 2006. As a member of the Haemato-Oncology Task Force of the British Committee for Standards in Haematology he has contributed to a number of national guidelines including the best practice in lymphoma diagnosis and reporting.

a.ramsay@ucl.ac.uk

 

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