Targeted sequencing of refractory myeloma reveals a high incidence of mutations in CRBN and Ras pathway genes

Abstract

In this study, targeted sequencing to screen 50 multidrug refractory multiple myeloma (rMM) patients was performed by using the Multiple Myeloma Mutation Panel. Patients were pretreated with both immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs), and 88%, 78%, and 68% were refractory to an IMiD, a PI, or both, respectively. The majority of patients had progressive (82%) or refractory (78%) disease immediately before sampling, with 43% being IMiD refractory and 46% being PI refractory in the most recent line of therapy. Compared with newly diagnosed MM, an increased prevalence of mutations in the Ras pathway genes KRAS, NRAS, and/or BRAF (72%), as well as TP53 (26%), CRBN (12%), and CRBN pathway genes (10%) was observed. Longitudinal analyses performed in 3 patients with CRBN mutations at time of IMiD resistance confirmed that these mutations were undetectable at earlier, IMiD-sensitive time points. Furthermore, the functional introduction of these mutations in MM cells conferred lenalidomide resistance in vitro. These data indicate a differential genetic landscape in rMM associated with drug response.

Figure 1

Genomic mutations, adverse cytogenetic aberrations, and treatment characteristics of 50 relapsed/refractory MM patients. All patients are identified by individual numbers. Refractory status to IMiDs or PIs at any time before sampling is shown as well as PD and refractory (IMiD, PI, other drug classes) status immediately before sampling. Information on the adverse cytogenetic aberrations gain 1q21 (>2 copies), deletion (17p), and translocations t(4;14) and t(14;16) are depicted. Genes mutated in at least 1 patient are displayed. Genes (from top to bottom) and patients (from left to right) are sorted according to gene mutation frequencies. Treatment characteristics and cytogenetic aberrations are color-coded: red, yes; green, no; gray, unknown/not available. Refractory drugs immediately prior to sampling are also color-coded: purple, IMiD refractory; blue, PI refractory; orange, other drug classes (including cytotoxic agents and antibodies); green, not refractory to treatment immediately prior to sampling.

Figure 2

Localization of mutations within the CRBN gene. CRBN mutations were found in 6 patients. All mutations found in our cohort occurred at critical sites, and they had the potential to impact CRBN-IMiD interactions by truncating, affecting splicing, or being located within the IMiD-binding site of CRBN. Each mutation is described at the corresponding site of the CRBN protein. CRBN, cereblon; LON, long undivided filaments upon ultraviolet irradiation (homo-oligomeric ATP-dependent protease domain).

Figure 3

Clinical course and treatment history of 3 MM patients with acquired IMiD resistance. CD138-purified tumor samples were obtained after progression to an IMiD-based treatment and from earlier IMiD-responsive disease stages in each patient. A mutation in CRBN was identified at IMiD-resistant disease stage (black circles). This mutation, however, was undetectable at earlier IMiD-sensitive disease stages in all 3 patients (white circles), suggesting a correlation with the acquired IMiD resistance in these patients. Patient #12 (CRBN mutation Q327TER) was a 72-year-old female diagnosed with MM immunoglobulin G kappa (IgG κ). She was initially treated with bortezomib (BTZ), rapidly relapsed, and died as a result of PD during lenalidomide (LEN) treatment (detection of CRBN mutation). Patient #32 (CRBN mutation P411H) was a 44-year-old female diagnosed with MM IgG κ. She was treated with several chemotherapy agents, high-dose melphalan (HDM), and novel agents, including BTZ, carfilzomib (CFZ), LEN, and pomalidomide (POM), and she died during PD from POM-refractory disease (detection of CRBN mutation). Patient #24 (CRBN mutation F381C) was a 64-year-old male diagnosed with MM IgA λ. Initial treatments included tandem (tand.) HDM and interferon (IFN) maintenance. After a long disease course with several short retreatments including BTZ, LEN, and HDM, the disease became lenalidomide-refractory (detection of CRBN mutation) and conventional chemotherapy was begun. Black arrows indicate refractoriness to the indicated treatment. CTX, chemotherapy; mt., maintenance; RIC-allo, reduced-intensity conditioning allogeneic stem cell transplantation; Thal, thalidomide.

Figure 4

CRBN mutations identified in our cohort confer lenalidomide resistance in OCI-MY5 cells. OCI-MY5 cells are resistant to IMiD treatment because of low CRBN expression levels. IMiD sensitivity can be restored by CRBN wt overexpression. (A) All 3 CRBN mutants failed to resensitize the cell line; thus, the mutations conferred IMiD resistance. (B) Protein expression by western blot.