OVERVIEW
High-grade B-cell lymphomas/Diffuse large B-cell lymphomas are tumors of B-cells with aggressive appearing histology that encompass a variety of disease entities using current WHO criteria, including diffuse large B-cell lymphoma, Burkitt lymphoma, high-grade B-cell lymphoma, NOS, etc.
INDICATION
Testing is indicated for all patients with diffuse B-cell lymphoma or high grade B-cell lymphoma to aid in the diagnosis and treatment decisions. LExA will replace FISH as the first-tier test for all diffuse large B-cell lymphomas and high grade B-cell lymphomas. FISH for MYC and BCL2 will be performed as a reflex test on any case that is dark zone positive or indeterminant by LExA or any cases that fails to provide an interpretable result by LExA. BCL6 will not be part of the reflex FISH performed but remains a test option for follicular lymphoma, Burkitt lymphoma and others as indicated.
TEST REQUIREMENTS
The following is required to complete LExA and reflex FISH as necessary. Despite FISH being a reflex test, all cases should continue to have FISH slides submitted to allow quick reflex FISH to ensure patient results. Any specimen received without the required components listed below will be returned without testing.
For LExA testing request, the following are required or the case will be returned without testing:
- Tumour content estimate for the whole section written in the space provided in the Specimen section
- Tissue block – which will be returned when the test is completed
- Completed CGL Lymphoid Testing requisition form
A minimum tumour content of 40% is required for LExA in the whole section to proceed with testing.
For FFPE FISH requests (reflex testing), the following are required or the case will be returned without testing:
- One H&E stained slide with the area for FISH scoring circled on the H&E slide
- Tumour content estimate (see below) of the circled FISH region written in the space provided in the Specimen section
- Three 4-5um unstained tissue sections on positively changed slides
- Tissue block – which will be returned when the test is completed
- Completed CGL Lymphoid Testing requisition form
There must be a minimum of 200 tumour nuclei within each section or area to perform FISH analysis.
Tumour content refers to the proportion (%) of tumour cell nuclei relative to non-tumour cell nuclei in a given area.
NOTE: Decalcified specimens are not amenable to FISH or LExA testing.
TURN-AROUND TIME
If reflex MYC and BCL2 FISH is performed, a separate report will be issued when results are available.
RESULTS REPORTING
LExA Assay
Results will be reported qualitatively for each of the three gene signatures detectable by the assay:
• Cell-of-origin signature – Germinal Centre B-cell-like (GCB) Activated B-cell-like (ABC), Unclassifiable
• Dark Zone signature – Positive, Negative, Unclassifiable
• Signature associated with primary mediastinal large B-cell lymphoma (PMBL) – PMBCL, DLBCL, Unclassifiable
Reflex FISH Testing
MYC and BCL2 FISH will be performed on any case that is dark zone positive or indeterminant by LExA or any cases that fails to provide an interpretable result by LExA. The MYC and BCL2 status will be reported together in a single report.
METHODS
LExA Assay
RNA is extracted from the FFPE tissue scrolls and run on the Nanostring platform. The LExA assay is performed to identify tumour cell-of-origin and the dark zone signature. The LExA assay can also identify a signature that can discriminates primary mediastinal large B-cell lymphoma from diffuse large B-cell lymphoma, NOS in in the clinical correct setting. See here for further details.
Reflex FISH Testing
MYC and BCL2 FISH is performed using a BCL2 FISH break-apart probe (Vysis). NOTE: BCL6 is no longer performed as a reflex probe for high grade B-cell lymphomas but remains available for follicular lymphoma, Burkitt lymphoma and other indications as necessary.
REFERENCES
- Scott et al. Determining cell-of-origin subtypes of diffuse large B-cell lymphoma using gene expression in formalin-fixed paraffin-embedded tissue Blood (2014) PMID:24398326
- Mottok et al. Molecular classification of primary mediastinal large B-cell lymphoma using routinely available tissue specimens Blood (2018) PMID:30257882
- Ennishi et al. Double-Hit Gene Expression Signature Defines a Distinct Subgroup of Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma J. Clin. Oncol. (2019) PMID:30523716
- Duns et al. Characterization of DLBCL with a PMBL gene expression signature Blood (2021) PMID:33684939
- Alduaij et al. Molecular determinants of clinical outcomes in a real-world diffuse large B-cell lymphoma population Blood (2023) PMID:36302166