MYC, BCL2, and BCL6 Rearrangements by FISH: Defining High-Grade B-Cell Lymphoma

What the test measures

Aggressive B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL) and high-grade B-cell lymphoma (HGBL), are biologically heterogeneous. A subset carries chromosomal rearrangements affecting three genes central to B-cell biology: MYC, BCL2, and BCL6.

MYC (chromosome 8q24) encodes a master transcription factor that drives cell proliferation and metabolism. BCL2 (18q21) encodes an anti-apoptotic protein that blocks programmed cell death. BCL6 (3q27) encodes a transcriptional repressor important in germinal-center B-cell function. When one or more of these genes is placed under the control of a strong regulatory element—most commonly an immunoglobulin (IG) enhancer through a translocation—the result can be deregulated overexpression. The combination of a MYC rearrangement with a BCL2 rearrangement is biologically potent: unchecked proliferation is paired with resistance to apoptosis. This co-occurrence defines a distinct, aggressive lymphoma category recognized in current classifications [1][2][3].

How it is tested

The standard assay is fluorescence in situ hybridization (FISH) performed on formalin-fixed, paraffin-embedded (FFPE) tissue. Two probe designs are used:

• Break-apart probes flank the gene of interest with differently colored fluorophores. In the normal (intact) state the colors overlap; when the locus is rearranged, the signals separate ("split"), indicating a translocation regardless of the partner gene.
• Fusion probes are designed to detect a specific partnering, most classically the IG-MYC or IG-BCL2 fusion, where signals from two loci come together.

A practical limitation of standard break-apart probes is that they detect a rearrangement but not the partner. Because the prognostic and classification impact of a MYC rearrangement depends in part on whether the partner is an IG locus versus a non-IG gene, some laboratories supplement break-apart testing with fusion or partner-specific probes [3].

Scoring is reported simply as presence or absence of a rearrangement at each locus, based on the proportion of nuclei showing an abnormal signal pattern above a validated laboratory cutoff. Preanalytic quality matters: adequate tumor cellularity, appropriate fixation, and section thickness all affect signal quality and interpretability in FFPE material.

What each result state means

Each locus is reported independently, and the combination determines the diagnostic category:

• MYC-R present/absent — A MYC rearrangement alone does not define the double-hit category. Its significance depends on context and partner.
• BCL2-R present/absent — A BCL2 rearrangement in isolation is common in follicular lymphoma and some DLBCL and does not by itself define high-grade disease.
• BCL6-R present/absent — A BCL6 rearrangement is reported but, importantly, no longer contributes to the double-hit definition (see below).
• Double-hit MYC+BCL2 — The concurrent presence of MYC and BCL2 rearrangements defines the entity variably termed DLBCL/HGBL with MYC and BCL2 rearrangements in current classifications. This is the category with established adverse prognostic implications [1][2][3].
• Triple-hit — Rearrangements of MYC, BCL2, and BCL6 together. These are grouped with the MYC/BCL2 double-hit category by virtue of the shared MYC+BCL2 combination [1][2].

A crucial classification change deserves emphasis: a MYC plus BCL6 rearrangement without a BCL2 rearrangement no longer qualifies for the double-hit high-grade category. In earlier schemes MYC/BCL6 "double-hit" cases were included, but the biology and outcomes of these cases proved heterogeneous, and current WHO (5th edition) and ICC 2022 frameworks restrict the molecularly defined high-grade entity to MYC+BCL2 (with or without BCL6) [1][2][3].

What it determines for treatment eligibility

The primary clinical value of this panel is diagnostic classification, which in turn carries prognostic weight. Identification of a MYC+BCL2 double-hit (or triple-hit) lymphoma establishes an entity associated with an aggressive clinical course and inferior outcomes compared with DLBCL not otherwise specified [3][4]. This classification informs whether a patient's disease falls into a category for which more intensive chemoimmunotherapy regimens—rather than standard-intensity regimens—are considered by treating clinicians [3].

Framed appropriately for education: a double-hit result identifies eligibility to be considered for intensified chemoimmunotherapy approaches as a disease class, and does not by itself dictate any specific individual therapy. Treatment selection integrates stage, performance status, and other clinical factors and is the province of the treating team. This article does not offer treatment recommendations.

Caveats and what is evolving

Several areas remain nuanced or contested:

• Partner identity. The prognostic meaning of a MYC rearrangement is influenced by whether the partner is an IG locus or a non-IG gene. Standard break-apart FISH does not resolve this, and testing strategies vary between laboratories [3].
• Cytogenetics vs. protein expression. FISH-defined double-hit lymphoma is distinct from "double-expressor" lymphoma, in which MYC and BCL2 proteins are overexpressed by immunohistochemistry without underlying rearrangements. Double-expressor status carries its own prognostic implications but is not the same entity and is not diagnosed by FISH [3].
• Reclassification of MYC/BCL6. As noted, the removal of MYC/BCL6-only cases from the high-grade double-hit category reflects evolving understanding and means that older literature and newer criteria are not directly comparable [1][2].
• Concordance between classifications. WHO-HAEM5 and ICC 2022 largely align on the centrality of MYC+BCL2, but terminology and boundary definitions differ in detail, and the field continues to refine morphologic and molecular criteria [1][2][3].

The evidence suggests that FISH for MYC, BCL2, and BCL6 remains an essential tool for identifying the most aggressive molecularly defined large B-cell lymphomas, while acknowledging that testing algorithms and category boundaries continue to evolve.

References

1. Alaggio R, Amador C, Anagnostopoulos I, et al. The 5th edition of the WHO Classification of Haematolymphoid Tumours: Lymphoid Neoplasms. Leukemia. 2022. doi:10.1038/s41375-022-01620-2

2. Campo E, Jaffe ES, Cook JR, et al. The International Consensus Classification of Mature Lymphoid Neoplasms. Blood. 2022. doi:10.1182/blood.2022015851

3. Davies AJ. The high-grade B-cell lymphomas: double hit and more. Blood. 2024. doi:10.1182/blood.2023020780

4. Schmitz R, et al. High grade B-cell lymphoma with rearrangements of MYC and BCL2 and/or BCL6. Blood (PMC5572137). 2017.