Real-time polymerase chain reaction in multiple myeloma: quantitative analysis of tumor contamination of stem cell harvests.
OBJECTIVE
Autologous transplantation of bone marrow (BM) and peripheral blood progenitor cells (PBPC) is commonly used for treatment of multiple myeloma (MM). Although both stem cell sources harbor residual clonal cells, a quantitative evaluation of their level of tumor contamination (LTC) still needs to be performed through highly accurate and reproducible approaches. In this study, we used a validated real-time polymerase chain reaction (PCR) strategy to evaluate LTC of BM and PBPC samples obtained from MM patients.


METHODS
The patients underwent two different mobilization courses (defined as early or late course) following two cycles of cyclophosphamide 5 g/m(2). LTC was evaluated by measuring the number of clonal immunoglobulin heavy-chain rearrangements followed by normalization of samples using the GAPDH gene.


RESULTS
Overall, 26 PBPC and 12 BM samples were analyzed. Main results are as follows. 1) PBPC harvests are less contaminated than BM samples taken immediately after each mobilization course (median difference 2.68 logs; range 1.7 to 4.6) (p < 0.0001). 2) LTC of PBPC harvests has only minimal variation among different leukaphereses performed during the same mobilization course (median difference 0.45 logs; range 0.22 to 1.2). 3) No difference was observed among PBPC and BM samples obtained after the late mobilization course as compared to the early mobilization course (median reduction 0.21 logs; range -0.39 to 1.3) (p = 0.84). 4) In PBPC but not in BM samples, there is a clear overestimation of the percentage of plasma cells when flow cytometric evaluation of CD38(bright) cells is compared to real-time PCR results. This suggests that in PBPC, most CD38(bright) cells do not belong to the neoplastic clone.


CONCLUSIONS
Real-time PCR using the IgH rearrangement proved an effective tool for monitoring LTC in stem cell harvests from MM patients. The smaller LTC of PBPC harvests supports the role of PBPC as stem cell rescue for MM patients compared to BM cells.
Ladetto
Marco
M
Divisione Universitaria di Ematologia, Azienda Ospedaliera S. Giovanni Battista, Torino, Italy. marco.ladetto@unito.it
Omedè
Paola
P
Sametti
Selina
S
Donovan
John W
JW
Astolfi
Monica
M
Drandi
Daniela
D
Volpato
Federica
F
Giaccone
Luisa
L
Giaretta
Fulvia
F
Palumbo
Antonio
A
Bruno
Benedetto
B
Pileri
Alessandro
A
Gribben
John G
JG
Boccadoro
Mario
M
D000328:Adult; D001854:Bone Marrow Cells / Q000473:pathology; D003520:Cyclophosphamide / Q000627:therapeutic use; D005434:Flow Cytometry; D015321:Gene Rearrangement; D019650:Hematopoietic Stem Cell Mobilization / Q000379:methods*; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000473:pathology*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007166:Immunosuppressive Agents / Q000627:therapeutic use; D008875:Middle Aged; D009101:Multiple Myeloma / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
D007143:Immunoglobulin Heavy Chains; D007166:Immunosuppressive Agents; D003520:Cyclophosphamide