• Test Code:
  • Department:
    Hematological Malignancies
  • Test Synonyms:
    PLCG2Ibrutinib resistance
  • CPT Code(s):

Mutations in BTK and PLC-gamma-2 (PLCG2) are associated with acquired ibrutinib resistance in B cell neoplasms, particularly chronic lymphocytic leukemia.  The use of BTK inhibitors against a wider range of cancers, including T-cell and myeloid malignancies, raises the possibility that BTK resistance mutations will present in those cases as well. In a germline setting, pathogenic variants in BTK are associated with X-linked agammaglobulinemia (XLA). Ibrutinib, a BTK inhibitor, has been used to treat CLL and other B-cell neoplasms but 2nd and 3rd generation BTK inhibitors with fewer adverse effects are now available with fewer adverse effects and improved tolerability.

Clinical Utility:
BTK and PLCG2 hotspot mutations lead to resistance to ibrutinib and other BTK inhibitors in CLL and other B-cell neoplasms.


This test is performed by PCR-based Next Generation Sequencing of DNA extracted from formalin fixed paraffin embedded (FFPE) tissue or fresh tissue including peripheral blood and bone marrow.  Selected exons in BTK and PLCG2are 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).

Clinical Sensitivity:
BTK and/or PLCG2 mutations have been detected in approximately 80% of patients with acquired Ibrutinib resistance.

Clinical Specificity:
BTK and PLCG2 mutations may be identified across a spectrum of lymphomas. Interpretation of this test requires clinical, morphologic, and immunophenotypic correlation.

Specimen Requirements:

  • 5-10 mL of blood or bone marrow — yellow (ACD) or purple (EDTA) tube
  • Formalin-fixed paraffin-embedded (FFPE) tissue blocks
  • 10 unstained slides (4-5 microns)
  • Fresh Tissue:
    • Stabilize in Allprotect Tissue Reagent (Qiagen) and ship at room temperature – OR
    • Suspend in sterile culture media (RPMI or DMEM) or non-bacteriostatic normal saline in a sterile container and shipped at room temperature – OR
    • Snap Frozen and shipped on dry ice
  • If sending DNA: please send 200ng at a minimum of 10ng/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.

Test Performed (Days):

Once per week

Turn Around Time:

14-18 days

Shipment Sensitivity Requirements:

  • Package and ship specimen to remain cold, but not frozen. 
  • Ship via overnight express, using the FedEx priority overnight label provided. 
  • Contact Client Services for shipping kits and instructions at (855) 535-1522.


  1. Xu et al. Acquired mutations associated with ibrutinib resistance in Waldenstrom macroglobulinemia. Blood. 2017. 129(18):2519-2525.
  2. Woyach et al. Resistance mechanisms for the Bruton’s tyrosine kinase inhibitor ibrutinib. N Engl J Med. 2014 370(24):2286-2294.
  3. Hendriks et al. Targeting Bruton’s tyrosine kinase in B cell malignancies. Nat Rev Cancer. 2014. 14(4):2190-232.
  4. Shirley. Bruton Tyrosine Kinase Inhibitors in B-Cell Malignancies: Their Use and Differential Features. 2022. 17(1):69-84.
  5. Tabaro et al. The Role of BTK Inhibition in the Treatment of Chronic Lymphocytic Leukemia: A Clinical View. J Exp Pharmacol. 2021. 13:923-935.
  6. Zhu et al. Clinical Trials of the BTK Inhibitors Ibrutinib and Acalabrutinib in Human Diseases Beyond B Cell Malignancies. 2021. Front Oncol. 11:737943

Additional Info: