Hematological Malignancies CXCR4 Mutation Testing

CXCR4 is both a cytokine and ubiquitin receptor and regulates signaling pathways in PI3K, RAS, and MAPK. Testing for CXCR4 genetic alterations has been indicated for warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome and LPL/WM. Mutations in CXCR4, typically activating frameshift/nonsense mutations in S338 or S341, are found in around 30% of LPL/WM cases and 20% of IgM MGUS cases.  In WM they are almost always accompanied by MYD88 mutations and are thought to occur after MYD88 within the context of disease progression.  They are associated with a higher disease burden but not reduced OS.  They have also been associated with resistance to some therapies, including inhibitors of BTK, PI3K, and mTOR.  However, there is evidence that CXCR4 mutant cases will respond to anti-CXCR4 antibodies.

In a germline setting, variants in CXCR4 can cause WHIM syndrome and are also associated with an increased risk of CLL.  The mutations found in LPL/WM cases are similar to those found in WHIM and are referred to as WHIM syndrome-like mutations.

Clinical Utility:
CXCR4 mutations can lead to resistance to ibrutinib and other BTK inhibitors in LPL/WM.

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.  The entire coding region of CXCR4 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).

• 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)
• 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. Aghamohammadi et al. Preference of Genetic Diagnosis of CXCR4 Mutation Compared with Clinical Diagnosis WHIM Syndrom. J Clin Immunol. 2017. 37:282-286.
2. Schmidt et al. MYD88 and CXCR4 mutations in lymphoplasmacytic lymphoma identify cases with high disease activity. B Jour Haematol. 2015. 169(6):795-803.
3. Garcia-Reyero et al. Diagnostic value of bone marrow core biopsy patterns in lymphoplasmacytic lymphoma/Waldenström macroglobulinaemia and description of its mutational profiles by targeted NGS. J Clin Pathol. 2020 73(9):571-577.
4. Freusier et al. Shared genomic segment analysis in a large high-risk chronic lymphocytic leukemia pedigree implicates CXCR4 in inherited risk. J Transl Genet Genom. 2021. 5:189-199.
5. Castillo et al. Recommendations for the diagnosis and initial evaluation of patients with Waldenström Macroglobulinaemia: A Task Force from the 8th International Workshop on Waldenström Macroglobulinaemia. Brit J Haematol. 2016. 175(1):77-86.
6. Roccaro et al. C1013G/CXCR4 acts as a driver mutation of tumor progression and modulator of drug resistance in lymphoplasmacytic lymphoma. Blood. 2014. 123(26):4120-4131.