Hematological Malignancies BTK, Exon 14, Sequencing

The mutation of p.C481S has been characterized as a known contributor to the resistance of the drug ibrutinib in patients being treated for CLL.

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 hotspot exons in BTK 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).

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.

Preferred specimens:

• Blood or Bone Marrow: 5-10 mL purple (EDTA) or yellow (ADC) tube (unspun)

Acceptable specimens:

• Paraffin block or 10 unstained sections of tumor or bone marrow
• DNA: 150ng 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)

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

Weekly

7-10 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 at (855) 535-1522 for shipping kits and instructions.

1. Woyach J et al. Resistance mechanisms for the Bruton’s tyrosine kinase inhibitor Ibrutinib. N Engl J Med 2014;370:2286-94.

2. Chiron et al. Cell cycle reprogamming for PI3K inhibition overrides a relapse-specific C481S BTK mutation revealed by longitudinal functional genomics in mantle cell lymphoma. Cancer Discov; 4(9); 00-00.2014 AACR.