Session: 651. Multiple Myeloma and Plasma Cell Dyscrasias: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research, Plasma Cell Disorders, Diseases, Lymphoid Malignancies, Technology and Procedures, omics technologies
Methods: Bone marrow aspirates from patients with multiple myeloma or plasma cell leukemia from the Indiana Myeloma Registry underwent CD138+ MACSorting and cells were viably frozen. All patients had been previously treated with 2-7 lines of therapy. Thawed CD138+ cells were injected into mice using the SCID-Rab model. Briefly, rabbit kit femurs were cut in half and inserted under the flank of NOD-SCID-gamma (NSG) mice, followed by injection of cells into the rabbit bone 6-8 weeks later. Mice (n=17) were injected with seven unique samples with each sample being injected into 2-4 mice each. At the endpoint, mice were euthanized, the rabbit bone removed, and PDX cells underwent CD138+ MACSorting. An average of 59 million CD138+ cells were isolated per mouse. The PDX cells and the original patient sample underwent whole genome sequencing and RNA-sequencing. Mutations, copy number abnormalities, and structural variants were identified by Strelka, AscatNGS, and Manta, respectively. Also, CD138+ patient samples underwent single-cell multiome sequencing (10X Genomics) and copy number variation was determined using inferCNV.
Results: Of the seven patient samples injected into mice three were t(11;14), one was t(4;14), and three were hyperdiploid. All samples bore high-risk markers: six of the seven had TP53 abnormalities (of which five were biallelic), four had a deletion of 1p32 (CDKN2C), and five had gain/amp 1q. In addition, all seven had at least one Ras mutation (KRAS/NRAS or BRAF).
Notable copy number abnormalities that changed in the PDX clones included the status of CDKN2C, TP53, and 1q. In one patient sample injected into two mice, both PDX clones had a similar copy number profile, but one clone had acquired gain 1q. By examining single cell data we were not able to identify these high-risk cells with the same profile in the patient sample indicating that the PDX cells derived from a low abundance clone in the patient or had subsequently evolved in the mouse.
Some other mouse-specific variations were identified. From one patient with biallelic deletion of CDKN2C, a clone in one mouse retained the deletion of both alleles but a clone in another mouse had a combination of deletion and mutation of alleles. In another patient sample with mutation of both alleles of TP53, a clone in one mouse retained the two TP53 mutations, but a clone in a second mouse had a combination of deletion and mutation of alleles. In both instances the result is biallelic inactivation of these key tumor suppressor genes, indicating ongoing convergent evolution of high-risk markers in independent subclones within patient samples.
Another patient sample contained subclonal NRAS and KRAS mutations. In three mice the subclone with the KRAS mutation rose to dominance, and in one mouse the NRAS and KRAS subclones remained at the original frequencies seen in the patient sample. In the three mice with dominant KRAS mutations there was evidence of on-going evolution with the accumulation of private mutations not seen in other mice or the original patient sample. Across all PDX samples, there was an accumulation of private mutations but the rate of accumulation differed across patients and between mice, mean=169 (range 17-579). The majority of the private mutations were not in known driver genes of myeloma and are likely to be passenger mutations. However, a faster mutation rate will increase the likelihood of acquiring a driver event, which we estimate to occur in 3% of non-silent mutations. There was no obvious association between the rate of increase and genomic markers, such as TP53 status, or lines of treatment.
In conclusion, we have examined PDX cells derived from treated myeloma patients and see evidence of convergent evolution, on-going evolution through acquisition of mutations, and the acquisition of high-risk markers in subclones that were not detectable in the original patient sample. These data indicate the potential for sub-clonal diversity that can lead to treatment resistance and can be studied using these in vivo models.
Disclosures: Johnson: Genentech: Research Funding. Abu Zaid: Pharmacyclics: Research Funding; Pieris: Current equity holder in publicly-traded company; Ossium Health: Consultancy; Cormedix: Current equity holder in publicly-traded company; Genentech: Research Funding; BMS: Research Funding; Janssen: Research Funding. Abonour: GSK: Honoraria, Research Funding; Janssen: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria; Takeda: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Prothena: Honoraria. Walker: Genentech: Research Funding; Bristol Myers Squibb: Research Funding.
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