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