OncoGene ® MAGAZINE Home of Biomarkers in Oncology

Expert Opinion

Quick Facts

Double- and Triple-Hit Lymphomas

B-cell lymphomas with 2 or 3 recurrent chromosomal breakpoint aberrations (rare) are referred to as double- or triple-hit lymphomas. These usually involve the MYC oncogene in association with the BCL2 and/or BCL6 gene rearrangements. These are classified by WHO as a “high-grade B-cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements” (Swerdlow, WHO, 2016).

It is important to identify these high-grade lymphomas as they tend to manifest aggressive behavior and respond poorly to traditional chemotherapy.

  • Follicular lymphoma
  • High-grade lymphomas (rare)


NCCN Clinical Practice Guidlines in Oncology.B Cell Lymphomas

Version1. 2020

The 2016 updated WHO classification of lymphoid neoplasias

2016 revision of the WHO classification, non‐Hodgkin lymphomas

Which Breakpoints Are Used to Identify Double- or Triple-Hit Lymphomas?


Which Tests are recommended as diagnostic workup?

  • Individual probes
    • Chromosome FISH, Interphase 
  • Panels
  • Aggressive B-Cell Lymphoma Reflex Panel by FISH, Tissue – if MYC (8q24) gene rearrangement by FISH is positive, then IGH-BCL2 fusion t(14;18) by FISH is added; if IGH-BCL2 fusion, t(14;18) by FISH is negative, then BCL6 (3q27) gene rearrangement by FISH will be added Lymphoma (Aggressive) Panel by FISH – probes include IGH/BCL2, BCL6, and MYC
  • For histologically aggressive B-cell lymphomas with either Burkitt-like or diffuse large cell morphologies in adults demonstrating a CD20+, CD10+ phenotype with high proliferative index OR CD20+ neoplasms with morphologic features indeterminate between DLBCL and Burkitt lymphoma, it is reasonable to test forMYC, BCL2, and BCL6 rearrangements using FISH
    • IGH-BCL2 Fusion, t(14;18) by FISH
    • IGH-MYC t(8;14) by FISH
    • MYC (8q24) Gene  Rearrangement by FISH
    • BCL6 (3q27) Gene Rearrangement by FISH

Which Genetic mutations are related to prognosis ?

  • Follicular lymphoma
    • Adverse prognosis associated with del 17p, trisomy 12 and abnormalities of 6p
  • Diffuse large B-cell lymphoma (DLBCL)
    • Better prognosis associated with t(14;18)
    • Adverse prognosis with MYC oncogene; BCL2 gene, p53(+)
  • Follicular and DLBCL
    • Dismal outcomes for 8q24/MYC in association with 18q21/BCL2 or 3q27/BCL6
  • Double-hit and triple-hit lymphomas
    • Refer to Key Points for double- and triple-hit lymphomas
  • CLL
    • CD38 – mutation status correlates inversely with prognosis

Determination of lymphocyte clonality

Whereas a normal immune response typically generates a polyclonal population of lymphocytes with a multitude of different antigen receptors and antibodies, malignant lymphoproliferations derived from B- and T-cells usually show monoclonal rearrangements of the immunoglobulin and T-cell receptor genes, respectively, demonstrating their origin from a single clonal precursor cell. This fact is exploited for molecular diagnosis of lymphoma, because the presence of a clonal population of B- or T-cells is a strong indication of a malignant lymphoproliferation in the right context.


Indications for clonality determination: case studies

Benign versus malignant lymphoproliferations and determination of clonal relationship


  • Pearls and pitfalls of clonality testing

1- Use multiple targets and primer combinations, such as IG heavy chain FR1-3 and Kappa VJ and Kde for B-cells for high rates of clonality detection
2- Make sure your sample contains enough lymphoid cells and shows sufficient DNA quality to avoid pseudoclonal results
3-Have a clear plan how the outcome of clonality studies will influence your diagnostic decision this avoids difficult explanations of unexpected results
4- Don’t perform clonality on clearly reactive samples
5- Don’t perform clonality on clear-cut lymphomas, except for specific purposes, such as showing clonal relationship
6- Follicular lymphoma and diffuse large B-cell lymphoma show extensive somatic hypermutation of IGH genes, leading to lack of primer binding and false negative results in 10e15% of cases, if only IGH primers are used
7-Premalignant lymphoproliferations such as monoclonal gammopathy of uncertain significance (MGUS) and monoclonal B-cell lymphocytosis (MBL), as well reactive clonal expansions due to infection of autoimmunity can result in overcalling of malignancy

  • Virus testing
    • EBV – posttransplant lymphomas, endemic nasopharyngeal lymphomas
    • HHV8 – AIDS-related lymphomas
  • Bacteria testing
    • Helicobacter – mucosa-associated lymphoid tissue (MALT) lymphoma
  • Kappa and lambda light-chain clonality in situ hybridization
  • Testing prior to treatment with immunosuppressive therapies
    • HBV – surface antigen and core antibodies for all patients considering rituximab
      • Also include HBV e antigen testing for patients with relevant risk factors
    • HCV – for high-risk patients considering rituximab
    • CMV – PCR quantitative