Hematological Malignancies PLCG2 Mutation Testing

PLCG2 is a transmembrane protein that plays a role in immune-receptor signaling.  Activating, hotspot mutations in PLCG2 occur in CLL under treatment, typically along with co-occurring BTK mutations, and are known to cause resistance to BTK inhibitors in CLL.  The mutations often show up several months before relapse and typically display VAFs of under 10%, although VAFs of up to 80% have been reported.  The mutations lead to hypersensitivity of PLCG2 to activation by Rac2.  Combination therapies of ibrutinib plus venetoclax or umbralisib plus ublituximab are being tested, but the most promising treatment of BTK-inhibitor resistant CLL will likely be targeted antibodies against surface proteins such as CD3 and CD19.

In germline settings variants in PLCG2 can lead to immune disorders such as PLCG2-associated antibody deficiency and immune dysregulation (PLAID) and autoinflammatory PLAID (APLAID).

Clinical Utility:
PLCG2 mutations in CLL lead to resistance to BTK inhibitors and are associated with relapse, often showing up months before clinical presentation.

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 PLCG2 are 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).

• 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.

Once per week

14-18 days

• 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. Quinquenel et al. Prevalence of BTK and PLCG2 mutations in a real-life CLL cohort still on ibrutinib after 3 years: a FILO group study. Blood. 2019. 134(7):641-644.
2. Sedlarikova et al. Resistance-Associated Mutations in Chronic Lymphocytic Leukemia Patients Treated With Novel Agents. Front Oncol. 2020. 10:894.
3. Walliser et al. The Phospholipase Cγ2 Mutants R665W and L845F Identified in Ibrutinib-resistant Chronic Lymphocytic Leukemia Patients are Hypersensitive to the Rho GTPase Rac2 Protein. J Biol Chem. 2016. 291(42)22136-22148.
4. Koss et al. Dysfunction of phospholipase Cγ in immune disorders and cancer. Trends Biochem Sci. 2014. 39(12):603-611.
5. Jones et al. PLCG2 C2 domain mutations co-occur with BTK and PLCG2 resistance mutations in chronic lymphocytic leukemia undergoing ibrutinib treatment. Leukemia. 2017. 31(7):1645-1647.
6.  Ahn et al. Targeting Bruton’s Tyrosine Kinase in CLL. Front Immunol. 2021. 12:687458.