Molecular Analysis of RAS-RAF Tyrosine-Kinase Signaling Pathway Alterations in Multiple Myeloma

Abstract 2876

Multiple myeloma (MM) is a malignancy of abnormal plasma cells and a correlation with poor outcome has been described for immunoglobulin heavy-chain (IgH) translocations, deletions of 13q or 17p. Thus far, a convincing relationship between specific mutations, disease onset and progression has not been fully established. Aberrant impairment of important signaling pathways can drive oncogenesis and contribute to MM development. We therefore chose to study NRAS, KRAS and BRAF, three members of the RAS-RAF signaling pathway, as well as TP53 and CCND1, two fundamental genes in cell cycle control. We here investigated 41 MM cases to further elucidate molecular mechanisms underlying this disease. Bone marrow (n=35) or, in case of plasma cell leukemia, peripheral blood (n=6) specimens were collected between 12/2006 and 6/2011 and molecular analyses using a deep-sequencing assay (454, Branford, CT) in combination with the 48.48 Access Array technology (Fluidigm, South San Francisco, CA) were performed on mononuclear cells after Ficoll enrichment or magnetic activated plasma cell sorting using anti-CD138 beads (RoboSep, STEMCELL Technologies SARL, France). The cohort included 16 female and 25 male patients at first diagnosis, with a median age of 63 years (range: 33–84 years). Based on fluorescence in situ hybridization (FISH), the cohort was characterized as follows: IgH rearrangements were detected in 54.3% of patients (19/35: n=6 with t(4;14), n=9 with t(11;14), n=3 with t(14;16), n=1 other; data not available: n=5). A deletion 13q14 was present in 64.9% of patients (24/37; data not available: n=4). Trisomy 3 was detected in 48.0% of patients (12/25; data not available: n=16), trisomy 9 was detected in 50.0% of patients (12/24; data not available: n=17), trisomy 11 was detected in 46.4% of patients (13/28; data not available: n=13), and trisomy 15 was detected in 56.2% of patients (9/16; data not available: n=25), respectively. Interestingly, in all cases where FISH data was available (n=36), at least 1 aberration was detectable. Further, we studied the occurrence of somatic mutations in NRAS, KRAS, BRAF, TP53 and CCND1. In our cohort, we detected an overall mutation rate within the RAS pathway of 41.4% (17/41), in line with a recent report (Chapman et al., Nature, 2011). KRAS was the most frequently mutated gene with 21.9% of cases with mutations (9/41 patients), followed by NRAS (19.5%; 8/41 patients). Recently, BRAF V600E mutations have gained clinical interest since they became manageable by targeted treatment in melanoma. Interestingly, Chapman et al. discovered a mutational rate of 4% by sequencing of 161 MM patients (Nature, 2011). Even if BRAF is not a frequently mutated gene in MM, it justifies upfront diagnostic screening since these patients may benefit from new treatments. In our cohort, 2/41 patients harbored BRAF V600E mutations. Moreover, because of their involvement in the same signaling pathway, we also noticed that mutations affecting NRAS, KRAS or BRAF were predominantly mutually exclusive, except for one patient who concomitantly harbored a BRAF and a NRAS mutation. Additionally, we observed an overall molecular TP53 mutation rate of 12.2% (5/41 patients). In these 5 patients, in total 7 mutations (5 missense substitutions; 2 frame-shift mutations) were detected. 1/4 cases concomitantly harbored a deletion of the TP53 gene, as detected by FISH. Finally, we were interested in the analysis of CCND1, which is located on 11q13, a region frequently involved in chromosomal translocations (9/20 IgH translocated cases in our cohort). Here, we were able to detect 2/41 (4.8%) CCND1 mutated cases. Concerning the correlation between IgH rearrangements and molecular aberrations we observed that 21.9% (9/35; n=5 IgH status not available) of patients that were IgH rearranged, concomitantly carried a TP53 or RAS-RAF mutation. In more detail, 2/5 TP53 mutated patients and 50.0% (8/16) RAS-RAF mutated cases concomitantly harbored an IgH rearrangement. Taken together, MM patients are currently stratified in part based on cytogenetic/FISH classification. We demonstrated that deep-sequencing analyses support an additional molecular characterization. In our cohort, all patients carried mutations detected by FISH and 23/41 (56.1%) carried a molecular mutation. Future clinical studies need to confirm the frequencies of these mutations as well as their association with response to therapy and outcome.