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Single Circulating Multiple Myeloma Cells Harbor Mutations Seen in Bulk Samples

NEW YORK (GenomeWeb) – A Broad Institute-led team of researchers has developed a new approach to pluck single circulating tumor cells from peripheral blood samples for analysis.

The Broad's Todd Golub and his colleagues further found that, for multiple myeloma patients, analysis of single circulating tumor cells reflects what has been found through analysis of bulk bone marrow samples and, occasionally, exhibited greater sensitivity in uncovering mutations. As they reported in Science Translational Medicine today, this suggests that circulating tumor cells could be used to monitor multiple myeloma patients and guide therapy, a process that would be less painful than repeat bone marrow biopsies.

"The ability to track the genetic evolution of [multiple myeloma] could reveal emerging drug resistance mechanisms that might point to early intervention with mechanism-driven therapies," Golub and his colleagues wrote in their paper.

The researchers developed a way to isolate single circulating multiple myeloma tumor cells using CD138+CD45 cell enrichment, along with serial dilution and single-cell micromanipulation using live fluorescence microscopy. They reported that this approach enabled them to isolate at least 12 single circulating multiple myeloma tumor cells from 24 patients, while a flow cytometry-based method was only able to do so for 13 of the 24 patients.

They then performed single-cell whole-genome amplification followed by PCR amplification of 35 loci, chosen to include most genes mutated in multiple myeloma. For comparison, the researchers also isolated and analyzed single multiple myeloma cells obtained from bone marrow samples as well as CD19+ B lymphocytes and CD45+CD138 white blood cells. They sequenced a total of 568 cells from these groups.

To assess the approach's sensitivity, Golub and his colleagues also analyzed 80 single cells from four multiple myeloma cell lines with known mutations. Seventy-two of those cells passed quality control checks and, in those, the researchers reported finding all known mutations.

The researchers further reported that their analyses of single circulating tumor cells largely recapitulated findings from bulk analyses of bone marrow biopsy samples.

For one patient, the researchers uncovered a KRAS gene mutation in the bulk bone marrow sample — a mutation the patient was known to harbor based on clinical testing — and they further found this mutation in 12 of the 19 single bone marrow multiple myeloma cells they analyzed, a finding they said provided confidence in their approach.

When they expanded their mutational analysis to blood and bone marrow samples from a cohort of nine patients, the researchers uncovered 12 mutations. Two of these patients had previously undergone bulk analysis, which had indicated that they harbored KRAS and NRAS mutations, and here, the KRAS mutation was found in seven of the 11 circulating tumor cells obtained from one patient and the NRAS mutation was identified in 11 of the 14 circulating tumor cells from the other.

In addition, the investigators compared the mutational profiles of single circulating tumor cells to single cells isolated from bone marrow to find that they harbored the same mutations. That is, Golub and his colleagues said, all of the mutations found through bulk clinical-grade analysis of bone marrow could be detected through single-cell analysis.

He and his colleagues also reported that their single-cell analysis could sometimes detect mutations with greater frequency than bone marrow analyses. For instance, for three patients, the proportion of circulating tumor cells with TP53, BRAF, and NRAS mutations was higher among circulating tumor cells than among single cells isolated from bone marrow of the same patient.

These and additional findings suggested to Golub and his colleagues that genomic characterization of circulating tumor cells should be investigated further. "We propose that it is now time to incorporate deep molecular analysis of the peripheral blood into the characterization of [multiple myeloma] patients undergoing clinical investigation," they wrote.

Single-cell analyses could, they added, help guide treatment. For instance, through gene-expression analysis of single cells isolated from plasma, the researchers were able to uncover differences in the expression of therapeutically relevant transcripts, like CD38, SLAMF7, and BCMA. This suggested that gene expression profiling of circulating tumor cells could also help diagnose or classify multiple myeloma subtypes.