Early detection of bone marrow engraftment by amplification of hypervariable DNA regions.
OBJECTIVE
After allogeneic bone marrow transplantation (BMT) it is important to be able to distinguish between the host and donor origin of cells in order to monitor the engraftment process. However, identifying whether the hematopoietic stem cells are of donor or recipient origin may be a difficult task. DNA studies using Southern blotting techniques or the amplification by PCR of regions in the human genome with high polymorphic neutral sequence variation showing Mendelian inheritance as variable number of tandem repeats (VNTR) can detect the origin of host bone marrow after BMT. We have tried to apply these sensitive systems of detection to the early stages of BMT when small numbers of regenerated cells are available for analysis.


METHODS
We used in vitro polymerase chain reaction (PCR) amplification of three single-locus simple repetitive DNA sequences, all of which vary extensively in their repeat number among different individuals (VNTR D1S80, ApoB, and D17S5), to evaluate post transplant engraftment in six patients who showed no signs of peripheral blood engraftment at 2-3 weeks after transplant. We tested 2 patients with chronic myelogenous leukemia (CML), 2 with B-acute lymphoblastic leukemia (B-ALL), 1 with T-acute lymphoblastic leukemia (T-ALL), and 1 with aplastic anemia (SAA), all in prolonged aplasia following allogeneic bone marrow transplantation (BMT).


RESULTS
In a sequential analysis protocol with the different loci, the donor was distinguishable from the recipient in all pairs with at least one of the three markers used. After 16 days (median 16.2; range 15 to 20 days) we found that complete chimerism was present in 5 patients: 4 of donor origin (= engraftment) and one of host origin (= rejection), this last case being one of mixed chimerism. In the 2 cases in which the presence (one complete and one partial) of host DNA was detected, rejection of the donor bone marrow followed, and in 1 patient a second BMT was necessary. The other 4 patients with complete chimerism of donor origin achieved hematological reconstitution and we documented complete engraftment of donor bone marrow a few months later.


CONCLUSIONS
Utilizing PCR to document early post-transplant engraftment and chimerism in the first month after BMT has the advantage over Southern blotting of being more sensitive and requiring small amounts of sample. It may also be useful for guiding subsequent therapeutic decisions.
Martinelli
G
G
Institute of Hematology and Medical Oncology Seragnoli, University of Bologna, Italy.
Trabetti
E
E
Farabegoli
P
P
Testoni
N
N
Bandini
G
G
Motta
M R
MR
Vittone
A
A
Terragna
C
C
Pignatti
P F
PF
Tura
S
S
D015415:Biomarkers; D015139:Blotting, Southern; D016026:Bone Marrow Transplantation; D004247:DNA / Q000032:analysis* / Q000235:genetics; D005260:Female; D006085:Graft Survival / Q000235:genetics*; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D014184:Transplantation, Homologous
D015415:Biomarkers; D004247:DNA