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Lymphoma Cell Burden in Progenitor Cell Grafts Measured by
Competitive Polymerase Chain Reaction: Less Than One Log Difference
Between Bone Marrow and Peripheral Blood Sources
Brigitte M. Léonard,
Francis Hétu,
Lambert Busque,
Martin Gyger,
Robert Bélanger,
Claude Perreault, and
Denis-Claude Roy
From the Division of Hematology-Immunology, Maisonneuve-Rosemont
Hospital; and the Department of Medicine, Université de
Montréal, Québec, Canada.
A controversy persists in autologous transplantation as to which
source of progenitor cells, bone marrow (BM) or peripheral blood (PB),
contains the lowest number of contaminating lymphoma cells, and how
mobilization procedures affect these numbers. To accurately measure the
number of non-Hodgkin's lymphoma (NHL) cells harboring the
bcl-2/immunoglobulin H (IgH) rearrangement in progenitor cell grafts,
we developed a nested quantitative competitive polymerase chain
reaction assay (QC-PCR). DNA from lymph nodes of four patients with NHL
were cloned into the pSK(+) vectors to generate four internal
controls (ICs) (two with major breakpoint region [MBR]
and two with minor cluster region [mcr] rearrangements).
The kinetics of amplification of ICs paralleled those of bcl-2/IgH
rearranged genomic DNA. When used in a QC-PCR assay, these ICs were
accurate at a 0.2-log level and provided reproducible results, as shown
by low intrarun and interrun variability. An excellent correlation
between predicted and observed lymphoma cell content (r = .99) was observed over a range of at least 5 logs of rearranged cells.
This approach was used to measure involvement by NHL cells at the time
of progenitor cell harvest in 37 autologous transplant patients. The
number of bcl-2/IgH rearranged cells in BM, PB, and mobilized PB (mPB)
was found to vary from 1 to 1.1 × 105 per million cells.
The number of lymphoma cells present in BM was significantly higher
than in PB (P = .0001), with a median difference in lymphoma
cell content between BM and PB of 0.48 log of cells (range,  0.7 to 5 logs). In contrast, we found no difference in the concentration of
bcl-2/IgH rearranged cells present in BM versus PB progenitor cells
mobilized with cyclophosphamide and granulocyte colony-stimulating
factor (G-CSF) (mPB) (P = .57). In conclusion, the QC-PCR
assay described in this study could measure accurately and reproducibly
the number of bcl-2/IgH rearranged cells among normal cells.
Differences in levels of contamination by lymphoma cells between BM and
PB were of less than one log (10-fold), and no differences in lymphoma
cell concentrations were observed between BM and mobilized PB. As more
cells are usually infused with mPB than with BM grafts, mPB progenitor
cell grafts may actually be associated with higher levels of
contamination by lymphoma cells. Furthermore, this QC-PCR assay should
provide an important tool to assess the prognostic impact of lymphoma cell burden both in progenitor cell grafts and in vivo.
Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 331-339
© 1998 by The American Society of Hematology.
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