Abstract Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research
Background: Despite the fact that activating mutations in the RAS-ERK cascade genes quite often detected in MM, the literature data on their prognostic value are contradictory. To assess the heterogeneity of MM, not only the tumor substrate in the bone marrow and plasmacytoma, but also in plasma circulating tumor DNA (ctDNA) could be effectively analyzed.
Aims: To study the mutational status of KRAS, NRAS, BRAF genes in the tumor substrate from different loci in MM.
Methods: The single-center study from October 2021 to January 2023 included 70 patients with symptomatic MM (29 men, 41 women) aged 35 to 84 years (median - 58). The diagnosis was established in accordance with the criteria of IMWG-2014. In 66% of patients with MM, according to CT data, plasmacytomas were detected (in 40 patients - bone and in 6 - extramedullary). A FISH study of CD138+ cells was performed using DNA probes to detect translocations of 14q32/IgH, 8q24/MYC; deletions of 17p13/TP53, 13q14, 1p32; amplification of 1q21; and multiple trisomies (MetaSystems, Altlussheim, Germany). Upon detection of t(4;14), t(14;16), del17p13, amplification of 1q21, the patient was assigned to a high cytogenetic risk group. DNA was isolated from samples of various localization: CD138+ bone marrow cells (n=60), ctDNA (n=19), bone plasmacytoma (n=9), extramedullary plasmacytoma (n=6). The mutational status of KRAS, NRAS, and BRAF genes was studied in the tumor substrate from different loci. KRAS and NRAS genes mutations were identified by Sanger sequencing on the Nanophor 05 genetic analyzer (Institute for Analytical Instrumentation Russian Academy of Science, Russia) and by the NGS on the MiSeq genetic analyzer (Illumina, USA). The BRAF V600E mutation was determined by real-time allele-specific PCR with the device CFX96 Touch (Bio-Rad, USA).
Results: KRAS gene mutations were detected in 16% of patients (11 out of 70), of which less than a third of patients (27%) had high-risk cytogenetic abnormalities. NRAS gene mutations were detected in another 16% of patients, while more than half of the patients (55%) were assigned to a high cytogenetic risk group. BRAF gene mutations was found in 9% of patients (6 out of 70), one third of whom had high-risk aberrations (Figure 1). Paired tumor samples (plasma ctDNA and CD138+ bone marrow cells) were analyzed in 15 patients with MM. In 11 patients, mutations in any of the three genes were found in the bone marrow, while in five patients (45%), similar mutations were also detected in a paired sample of tumor ctDNA isolated from plasma. No cases with KRAS, NRAS, or BRAF gene mutation detected in the plasma and the absence of the corresponding mutation in the bone marrow were found. The mutational status of the three genes was analyzed in 15 plasmacytoma samples (9 - bone, 6 - extramedullary). It turned out that only KRAS gene mutations (7% of cases) were detected in the samples of bone plasmacytomas, and only NRAS gene mutations (50% of cases) were detected in the samples of extramedullary plasmacytomas.
Summary/Conclusion: There was a trend towards higher frequency of high-risk cytogenetic aberrations in patients with NRAS gene mutations compared to patients with KRAS gene mutations (55% vs. 27%). It was also determined that the NRAS gene was mutated in 50% of extramedullary plasmacytomas samples. In 45% of the cases with KRAS, NRAS, or BRAF gene mutation detected in the bone marrow substrate, similar mutations were also detected in the tumor ctDNA isolated from plasma.
Keywords: Multiple myeloma, Mutation status, Ras