Hematological Malignancies MYD88 Mutation Analysis

The L265P activating mutation in the MYD88 gene is a common recurrent mutation in several B-cell lymphoproliferative disorders. This mutation is present in nearly all (67-100%) patients with Waldenstroms Macroglobulinemia/lymphoplasmacytic lymphoma (WM/LPL) and approximately 50% of IgM MGUS patients. This mutation is also quite common in diffuse large B-cell lymphoma, especially DLBCL’s at immune privileged sites such as the skin, testis, and CNS (including ocular). It can also be detected in rare cases of low-grade B-cell lymphoma/leukemia such as marginal zone lymphoma (4%), and chronic lymphocytic leukemia/small lymphocytic lymphoma (2%). The MYD88 gene encodes a cytoplasmic adaptor protein that plays a central role in innate and adaptive immunity and functions as a key linker protein in the Toll Like Receptor (TLRs) and IL-1 receptor signaling pathway. The L265P mutation favors cell survival through IRAK1/IRAK4 and NF-kB signaling. Novel agents such as the BTK inhibitor ibrutinib are a proven effective therapy for WM and are showing promise for the treatment of DLBCL.

Reasons for Referral:
The assay is designed to allowing an accurate diagnosis of Waldenstroms Macroglobulinemia/lymphoplasmacytic lymphoma (WM/LPL) and exclusion of other lymphoproliferative disorders with overlapping features, providing prognosis and predicting responses to targeted therapy.

This test is performed by PCR-based Next Generation Sequencing of DNA extracted from formalin fixed paraffin embedded (FFPE) tissue, body fluids (including vitreous and CSF), or fresh tissue including peripheral blood and bone marrow.  The entire coding region of MYD88 is sequenced using massively parallel sequencing (next-generation sequencing) with a combination of multiplexed PCR (customized QIAseq Targeted DNA panel) and sequencing on an Illumina platform.  An in-house bioinformatics analysis pipeline has been used that employs multiple established variant calling tools (FreeBayes, MuTect2 and Scalpel) and variant annotation tools.  The genomic variants have been interpreted in accordance with the 2017 guideline recommendations by AMP/ASCO/CAP (PMID: 27993330).  The assay is validated in accordance with the AMP guidelines (PMID: 28341590).

For samples sent as formalin-fixed paraffin-embedded tissue (FFPE), the tissue was subjected to pathologist review and, if needed, enrichment (macrodissection) of involved areas of tissue – prior to extraction.

Please send one of the following:

• Blood: 5mL EDTA (lavender top tube) or ACD (yellow top)
  
  
• Bone Marrow Aspirate: 2-5mL EDTA (lavender top tube) or ACD (yellow top)
  
  
• Paraffin block
  • 10 unstained sections of a bone marrow biopsy or a lymph node (4-5 microns)
• CSF: 0.5-2.0 ml in a sealed small-volume microtube without RPMI or other additives – (ship cold, not frozen)
  
  
• Vitreous fluid:
  • RPMI must be added to raw vitreous fluid within 1-2 hours to stabilize it for shipment.
  • Add 2-3 ml of RPMI media to each 0.5mL (or smaller) vitreous sample.
  • Ship the RPMI-diluted sample cold (but not frozen) to KDL lab, to arrive within 24 hours of collection. KDL can provide steril single-use aliquots of RPMI media (contact us).
  • Vitreous fluid cytology report MUST accompany sample for interpretation of results.
  • Vitreous fluid samples arriving in KDL without RPMI will be discarded as inadequate.

• DNA: 200ng at a minimum of 50-100ng/µL (DNA must be extracted in a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or CMS).

Pathology report MUST accompany sample for interpretation of results

A REQUISITION FORM MUST ACCOMPANY ALL SAMPLES.  Please include detailed clinical information.

Weekly

7-10 days

• Package and ship specimen at room temperature (blood or bone marrow); on cold-pack for vitreous or CSF.
• Contact Client Services at (855) 535-1522 for shipping kits and instructions. 
• Ship the specimen within 24 hours of collection overnight express, using the FedEx priority overnight label provided. 

1. Varettoni et al.Prevalence and clinical significance of the MYD88 (L265P) somatic mutation in Waldenstrom's macroglobulinemia and related lymphoid neoplasms.Blood. 2013. 121(13):2522-2528.
2. Landgren et al. MYD88 and beyond: novel opportunities for diagnosis, prognosis and treatment in Waldenström's Macroglobulinemia.Leukemia. 2014. 28(9):1799-803.