Prospective Molecular Characterization of Multiple Myeloma Patient Samples Identifies High-Risk Patients and Informs Treatment Sequences through Resistance Mechanisms to Immunotherapies

Introduction. Multiple myeloma (MM) genetic diagnostics has traditionally been performed using fluorescence in situ hybridization (FISH), giving prognostic information based on immunoglobulin (Ig) translocations and key copy number abnormalities such as 1p, 1q, and 17p. However, in recent years it has become clear that more mutations, biallelic events, and focal deletions are also prognostically important and can be captured better through molecular techniques such as DNA sequencing. In addition, as targeted and immunotherapies become standard, monitoring the molecular targets will be important in determining the optimal sequence of therapies.

Methods. Bone marrow aspirates from patients with MGUS/amyloid (n=3), smoldering MM (n=3), newly diagnosed MM (n=15), or progression/relapse MM (n=33) were collected. All patients were enrolled in the Indiana Myeloma Registry and consented for use of data for research purposes. CD138+ MACS cell separation was performed in a clinical pathology laboratory and DNA was extracted from CD138+ cells and patient-matched buccal swabs. 100 ng DNA from both the tumor and control sample were processed using the HyperPlus kit (KAPA Biosystems) and hybridized to the Myeloma Genome Project (MGP) sequencing panel in a CLIA-certified laboratory. The panel consisted of 228 genes for mutation and copy number analysis, as well as the immunoglobulin and MYC loci for translocation analysis. Samples were sequenced using paired-end reads to a median depth of 1063x. Data were analyzed using a clinical workflow and genomic abnormalities reported by a pathologist.

Results. Of the 54 patients, Ig translocations were detected in 30 and included the known t(4;14), t(6;14), t(11;14), t(14;16) and t(12;14) as well as rare Ig translocations to partners such as GADD45B, LYN, PPCDC, and RCBTB2, of which over-expression of GADD45B and LYN are potentially targetable. A complex t(1;8;11;14) was also detected involving CCND1, MYC and the IGH loci which would not have been detectable by FISH. High-risk markers, such as gain/amp1q (22.2%) and deletion/mutation TP53 (16.6%) were detected including biallelic TP53 alterations in 5 patients (9.3%), which were mostly deletion plus mutation (4/5) and would not have been detected by traditional techniques.

Of the MM patients who had been previously treated (n=33), 27 had received an IMiD and 30 had received a proteasome inhibitor. One patient showed mutation of CRBN (p.N236D) in conjunction with del3p and another patient with mutation of CUL4B (p.E277Ter) with delX indicating complete inactivation of the genes as a resistance mechanism to IMiDs. In addition, 17 patients had received an immunotherapeutic regimen including anti-CD38 or anti-BCMA. Of those that had received an anti-CD38 treatment (n=17), three had deletion of 4p (the location of CD38) including one patient, who had received three previous anti-CD38 regimens, with focal deletion of CD38. Six patients had also received anti-BCMA therapy, of which one had a mutation in the gene encoding BCMA, TNFRSF17, resulting in premature termination within the extracellular domain (p.S48Ter) in 42% of cells. This sample came from a patient who relapsed after an initial response to a BCMA CAR-T, followed by a progression occurring quickly while on a BCMA bispecific antibody Teclistamab, suggesting the loss of BCMA expression as a mechanism of resistance in both treatments.

Interestingly, two patients harbored somatic mutations in the transmembrane domains of either CD38 (p.S29I) or TNFRSF17 (p.L60S) prior to the start of the respective treatments indicating that not all mutations seen in these genes are a result of treatment resistance.

Conclusions. DNA sequencing approaches are cost-effective alternatives to FISH, the current standard diagnostic technology, to detect genomic abnormalities in multiple myeloma and they can identify clinically meaningful markers related to prognosis and treatment, particularly in the emerging era of immunotherapeutics where antigen evasion is a key mechanism of molecular resistance.