Molecular purging of multiple myeloma cells by ex-vivo culture and retroviral transduction of mobilized-blood CD34+ cells

Abstract

Background: Tumor cell contamination of the apheresis in multiple myeloma is likely to affect disease-free and overall survival after autografting.

Objective: To purge myeloma aphereses from tumor contaminants with a novel culture-based purging method.

Methods: We cultured myeloma-positive CD34+ PB samples in conditions that retained multipotency of hematopoietic stem cells, but were unfavourable to survival of plasma cells. Moreover, we exploited the resistance of myeloma plasma cells to retroviral transduction by targeting the hematopoietic CD34+ cell population with a retroviral vector carrying a selectable marker (the truncated form of the human receptor for nerve growth factor, DeltaNGFR). We performed therefore a further myeloma purging step by selecting the transduced cells at the end of the culture.

Results: Overall recovery of CD34+ cells after culture was 128.5%; DeltaNGFR transduction rate was 28.8% for CD34+ cells and 0% for CD138-selected primary myeloma cells, respectively. Recovery of CD34+ cells after DeltaNGFR selection was 22.3%. By patient-specific Ig-gene rearrangements, we assessed a decrease of 0.7-1.4 logs in tumor load after the CD34+ cell selection, and up to 2.3 logs after culture and DeltaNGFR selection.

Conclusion: We conclude that ex-vivo culture and retroviral-mediated transduction of myeloma leukaphereses provide an efficient tumor cell purging.

Figure 1

MPB CD34⁺ cells of myeloma patients before and after culture, and transduction. (A) Phenotypic analyses of CD34+ cells before and after cytokine culture, ΔNGFR transduction, and selection of transduced cells. (B) Relative fold expansion in culture, and recovery after transduction and selection of total cells (open bars) and CD34+ cells (black bars), were measured, in comparison with the initial cell population (fresh cells).

Figure 2

Transduction pattern of myeloma plasma cells. CD138+ selected myeloma bone marrow plasma cells were transduced with the ΔNGFR vector in a bulk, and in a clonal culture (B and C). Part of the cells was mixed before transduction with CD34+ cells of the same patient (A). Culture conditions were the same as in the CD34+ cell transductions.