• Originally described 6 hallmarks of cancer include (Cancer Discov 2022;12:31, Cell 2011;144:646)
  • Sustaining proliferative signaling
  • Evading growth suppressors
  • Activating invasion and metastasis
  • Enabling replicative immortality
  • Inducing angiogenesis
  • Resisting cell death
• In cancer cells, accumulation of DNA damage with rapid proliferation and signaling imbalances can act as intracellular apoptosis triggers
• Several of the apoptosis regulating proteins interact through shared BH3 (BCL2 homology domain 3) domains / motifs; BH3 mimetics, such as venetoclax, exert anticancer effect by liberating the proapoptotic proteins (such as Bax and Bak) from their binding sites with prosurvival proteins (such as BCL2), thus allowing apoptosis to proceed (Cancers 2020;12:3353)
• BCL2 dysregulation occurs in various hematologic malignancies through different mechanisms (see table below)
• BCL2 dysregulation has also been described in myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and multiple myeloma with t(11;14) (Leukemia 2016;30:761)
• Venetoclax is used as a therapeutic drug in some of these entities (such as myelodysplastic syndrome and acute myeloid leukemia) and is being investigated for use in others (Am J Hematol 2023;98:1307, Hematology 2024;29:2343604, Curr Oncol 2022;29:4117)
• Several other drugs with a similar mechanism of action are also being investigated for potential clinical use (Blood 2021;138:1120)
  
  

  Disease	Molecular mechanism(s) of BCL2 dysregulation	Proportion of patients with the aberration
  Follicular lymphoma	t(14;18)(q32;q21)	90%
  	BCL2 mutations
  Diffuse large B cell lymphoma, germinal center B cell type	t(14;18)(q32;q21)	30%
  	BCL2 mutations	10%
  	Hypermutation of BCL2 promoter
  Diffuse large B cell lymphoma, activated B cell type	18q21 amplifications	20%
  	t(14;18)(q32;q21)	5%
  Mantle cell lymphoma	18q21 gain	20%
  Chronic lymphocytic leukemia	13q14 deletion with loss of negative regulatory miRNA-15a / 16-1 of BCL2	50%

  Adapted from Int J Mol Sci 2021;22:10157

• Diagnosing follicular lymphoma: coexpression of BCL2 with germinal center markers such as BCL6, CD10, MEF2B, HGAL, GCET1 or LMO2 supports the diagnosis of follicular lymphoma over follicular hyperplasia
• Diagnosing diffuse large B cell lymphomas (DLBCL) / high grade B cell lymphomas (HGBCL) with MYC and BCL2 rearrangements (WHO 5th edition)
  • Alternatively named high grade B cell lymphoma, double hit (HGBCL DH BCL2) (ICC 2022): immunohistochemical coexpression of BCL2 (≥ 50%) and MYC (≥ 40%) proteins (double expressors) in diffuse large B cell lymphomas is suggestive of concurrent MYC and BCL2 gene rearrangements (Mod Pathol 2018;31:1470)
• Diagnosing high grade B cell lymphomas, triple hit (HGBCL TH), which are aggressive large B cell lymphomas with coexisting MYC, BCL2 and BCL6 gene rearrangements
• Diagnosing T cell lymphomas: loss of BCL2 expression may be seen in T cell lymphomas (Appl Immunohistochem Mol Morphol 2014;22:99)
• Detecting immature enteric ganglion cells in pediatric intestinal pseudo-obstruction (Am J Surg Pathol 2005;29:1017)
• Ruling out Burkitt lymphoma: strong BCL2 expression is not compatible with a diagnosis of Burkitt lymphoma (Cytometry B Clin Cytom 2020;98:412)
• Using an alternate BCL2 antibody clone may be useful in some cases of suspected follicular lymphomas that do not demonstrate BCL2 expression on initial immunohistochemical studies, which is usually due to mutations in BCL2 gene (Hum Pathol 2023;135:84)
• In particular, multiple antibody clones are available for immunohistochemical assessment of BCL2; these include (Hum Pathol 2013;44:1817)
  • Clone 124 (mouse monoclonal antibody): one of the first to be used clinically and raised against amino acids 41 to 54 of the BCL2 protein; may yield false negative results due to epitope masking as a result of the translocation or mutations in the BCL2 gene impacting the protein structure (Pathology 2010;42:212)
  • E17 (raised to an adjacent region of BCL2, corresponding to amino acids 61 - 76), EP36 and SP66 (rabbit monoclonal antibodies) may detect a subset of apparently BCL2- cases by the 124 clone and in DLBCL (Hum Pathol 2021;107:1, Pathology 2010;42:212, Hum Pathol 2014;45:2144, Hum Pathol 2023;135:84)

Contributed by Shikha Malhotra, M.B.B.S., M.D., Genevieve M. Crane, M.D., Ph.D. and AFIP

Images hosted on other servers:

Contributed by Mahsa Khanlari, M.D. and AFIP

• Lymph node, retroperitoneal, biopsy:
  • Involvement by aggressive B cell lymphoma, germinal center B cell type (Hans criteria), pending ancillary testing (see comment)
  • Comment: The findings are consistent with involvement by a CD10 positive aggressive B cell lymphoma. Additional testing is underway to exclude a possible double hit lymphoma and the diagnosis will be clarified in an addendum. Given the focal nodularity in the process as well as interspersed sclerosis, the possibility that this may have arisen from an underlying follicular lymphoma cannot be excluded. Please also correlate with concurrent flow cytometry.
  • Biomarker results
    • Cell of origin (Hans classifier): germinal center
    • MYC expression (% tumor cells by IHC): < 40%
    • BCL2 expression (% tumor cells by IHC): > 50%
    • Ki67 expression (% tumor cells by IHC): 70 - 80%

Which lymphoma is generally characterized by a rearrangement involving chromosomes 14 and 18, often resulting in aberrant positive staining for BCL2?

1. Burkitt lymphoma
2. Follicular lymphoma
3. Mantle cell lymphoma
4. Pediatric type follicular lymphoma
5. Small lymphocytic lymphoma

B. Follicular lymphoma (FL). > 90% of follicular lymphoma is associated with an IGH::BCL2 rearrangement or t(14;18). This is termed classic follicular lymphoma in the WHO 5th edition. Follicular lymphoma is thought to arise from a germinal center B cell, which is normally negative for BCL2 expression. Aberrant expression by this rearrangement promotes survival. Answers A and D are both incorrect because these entities are not associated with the IGH::BCL2 rearrangement and are characteristically negative for BCL2 by immunohistochemistry, although weak or partial expression may be seen in some cases. Answer C is incorrect because the cells from which mantle cell lymphoma (MCL) arises are nongerminal center cells and normally express BCL2. In addition, mantle cell lymphoma is associated with the IGH::CCND1 rearrangement. Answer E is incorrect because small lymphocytic lymphoma may show a range of genetic alterations but is generally not associated with IGH::BCL2 rearrangement. These small lymphoid cells typically express BCL2 but not as an aberrant marker.

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Reference: BCL2

Why can it be useful to test multiple BCL2 antibody clones in the diagnosis of follicular lymphoma?

1. Cross reaction with BCL6 may occur
2. Different BCL2 isoforms may be expressed
3. Mutations in the BCL2 gene or masking of BCL2 epitopes are common
4. Notorious lot variability by manufacturers

C. Mutations in the BCL2 gene or masking of BCL2 epitopes are common. Various studies report different frequencies of BCL2 gene mutations in follicular lymphoma, ranging from 12% to ~50% of total cases (Blood 2015;125:658, Blood Cancer J 2023;13:81). A subset of these mutations can result in alteration of the antibody clone binding site. Hence, follicular lymphoma may be positive for one BCL2 antibody clone but undetectable by another, as shown in the above images. Answers A, B and D are incorrect because while other issues with detection may occur, this phenomenon is not typically related to isoforms, variability in manufacture of the antibodies or cross reaction with BCL6. In particular, BCL6 is nuclear while BCL2 shows a cytoplasmic pattern.

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Reference: BCL2