Pb1874: btk gene mutations in russian patients treated with ibrutinib: as-pcr versus ngs for allelic load assessment

Background: Ibrutinib is highly effective for CLL treatment. It inactivates Bruton’s tyrosine kinase (BTK) by forming a covalent bond in the enzyme catalytic domain at position C481. Ibrutinib resistance in 50-80% of cases is associated with the C481S mutation in exon 15 of the BTK gene (Ahn et al, 2017; Woyach et al, 2017), which corresponds to the c.1441T>A and c.1442G>C substitutions, still, other variants of gene mutations at this position are also reported. Monitoring allelic load of the BTK gene mutations conferring resistance to ibrutinib is extremely important for studying the mechanism of CLL progression during treatment with BTK inhibitors. Aims: Development and validation of a real-time AS-PCR approach for assessing the relative allelic load of BTK gene mutations in Russian CLL patients treated with ibrutinib. Methods: The study included DNA samples from 101 patients (pts) with CLL: 39 pts with disease progression on ibrutinib therapy (67% male, median age 64.5 years; 33% female, median age 64 years); 24 pts with progression after FCR or FCR-lite therapy (62% men, median age 62 years; 38% women, median age 67 years); 38 primary pts (76% men, median age 64 years; 24% women, median age 60 years). 89 pts with non-neoplastic hematological diseases were selected as control samples. All DNA samples were tested using a real-time AS-PCR with the following primers and probes: c.1441T (5’-CTGAGTACATGGCCAATGGCT-3’); c.1441T>A (5’-CTGAGTACATGGCCAATGGCA-3’); c.1442G (5’-CTGAGTACATGGCCAATGGCTG-3’); c.1442G>C (5’-CTGAGTACATGGCCAATGGCTC-3’); c.1442G>A (5’-TGAGTACATGGCCAATGGCAA-3’); c.1442G>T (5’-TGAGTACATGGCCAATGGGTT-3’); REV (5’-CTGCTACTTCCACCCCATCA-3’); Probe (5’-R&G-TGCTAGAGATGTGCAAGGATGTCTGTGAAG-RTQ1-3’). All DNA samples from pts treated with ibrutinib were analyzed by NGS on a MiSeq genetic analyzer (Illumina, USA). Results: Initially, AS-PCR was performed only for the most common mutations c.1441T>A and c.1442G>C, however, after NGS of the BTK gene in pts with progression during ibrutinib therapy, other options were added to the test system. Using AS-PCR, out of 39 Russian pts with CLL progressing on the background of ibrutinib therapy, a single c.1442G>C mutation was detected in 19 (48.7%) pts (including 3 samples with a low mutant load); 2 (5.1%) had a single mutation c.1442G>T (C481F). In 1 (2.6%) patient, two mutations were detected simultaneously: c.1441T>A and c.1442G>C, and in another 1 (2.6%) patient, 3 mutations were detected simultaneously: c.1442G>C, c. 1442G>T (C481F) and c.1442G>A (C481Y). No mutations in the BTK gene were found in 16 (41%) Russian pts. Table 1 shows a good agreement between the results of NGS with AS-PCR when sequencing the BTK gene in this group of pts, both in identifying the type of mutation and in determining the relative mutational load. To determine the presence or absence of mutations in the studied samples, the amplification curves and Ct values of the normal and mutant alleles were compared. Thus, mutations in the BTK gene leading to an amino acid substitution at position C481 were found in 59% of Russian pts with CLL treated with ibrutinib. In groups of pts with CLL without prior treatment, after FCR/FCR-lite regimens, and in the control group of pts with non-neoplastic diseases, no mutations were found. Image:Summary/Conclusion: AS-PCR protocol effective for the analysis of the BTK gene mutations leading to an amino acid substitution at position C481 has been developed and validated. Prospective studies of low burden BTK mutations in the pathogenesis of relapse in CLL patients treated with BTK inhibitors have been started using this approach.