Brown, J.M.

Cha, S.

Formankova, D.

Mocarski, E.S.

Potena, L.

Sharma, A.

Vana, M.L.

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75
INCREASED SENSITIVITY OF TWO-STEP CYTOMEGALOVIRUS PCR
ANALYSIS ON PERIPHERAL BLOOD LEUKOCYTES PERMITS DETAILED
ANALYSIS OF REACTIVATION AND IMMUNE RECONSTITUTION
Vana, M.L., Formankova, D., Cha, S., Sharma, A., Potena, L.,
Brown, J.M., Mocarski, E.S. Stanford University School of Medicine,
Stanford, CA.
Although current methods to detect human cytomegalovirus
(CMV) infection following hematopoietic cell transplantation
(HCT) have adequate sensitivity to guide preemptive treatment,
we chose to monitor CMV with a more sensitive PCR approach to
try to better understand the degree and timing of reactivation. The
purpose of this study was to compare the occurrence of CMV
reactivation during the ﬁrst 100 days post HCT in patients receiv-
ing a non-myeloablative (NMA) or myeloablative (MA) regimen
prior to allogeneic HCT. We analyzed reactivation in 98 consec-
utive patients undergoing HCT (MA  48 and NMA  50) with
the COBAS AMPLICOR™ quantitative PCR on plasma (n  98)
and a more sensitive two-step PCR (qualitative nested PCR and
quantitative real-time PCR) on peripheral blood leukocytes (PBL,
n  77). The estimated cumulative incidence of reactivation was
similar in both patient groups using either AMPLICOR™ (63%
NMA, 68% MA) or nested PCR (80% NMA, 100% MA). How-
ever, reactivation was detected earlier in the NMA group com-
pared to the MA group using either method (AMPLICOR™ P 
.012, nested PCR P  .002). Of the 77 patients followed by both
AMPLICOR™ and nested PCR, 9 patients in each of the NMA
(21%) and MA (26%) groups had levels of CMV DNA detectable
with nested PCR but not with AMPLICOR™. When levels of
viral DNA were quantiﬁed using real-time PCR, we found that the
CMV copy number was lower in the NMA group compared to the
MA group at the ﬁrst positive PCR result (P  .038) and at the
peak viral load (P  .02), suggesting NMA patients are better able
to control CMV early post-transplant. However, more NMA pa-
tients had late CMV disease. We also compared the timing of
CMV reactivation in relation to acute graft vs. host disease
(aGvHD) in the NMA and MA groups, as differences between
these patient groups are unclear. When using nested PCR we
found that all of the NMA patients (n  5 of 5, 100%) reactivated
before aGvHD, in contrast to the MA group where few patients
(n  2 of 10, 20%) reactivated before aGvHD (P  .037). There
was a similar trend with AMPLICOR™, as almost half of the
NMA patients (n 3 of 7, 43%) and none of the MA patients (n
0 of 10) reactivated before aGvHD. These data suggest that PCR
on PBL may be an effective way to further understand the timing
of CMV reactivation in relation to aGvHD in the NMA and MA
groups. In addition, detection of CMV with PCR on PBL may be
a sensitive method with which to monitor immune reconstitution
after HCT.
76
UNRELATED UMBILICAL CORD BLOOD TRANSPLANTATION USING TBI/
FLAG CONDITIONING REGIMEN FOR ADVANCED HEMATOLOGICAL MA-
LIGNANCIES
Okada, M.1, Misawa, M.1, Kai, S.2, Nakajima, T.1, Nomura, K.1,
Okikawa, Y.1, Satake, A.1, Itoi, H.1, Takatsuka, H.1, Itsukuma, T.3,
Nishioka, K.3, Fujimori, Y.3, Ogawa, H.1 1Division of Hematology,
Department of Internal Medicine; 2Division of Blood Transfusion; 3Lab-
oratory of Cell Transplantation, Institute for Advanced Medical Sciences,
Nishinomiya, Hyogo, Japan.
A combined chemotherapy using ﬂudarabine (Flu), cytosine ar-
abinoside and granulocyte colony-stimulating factor (FLAG) has
been reported to be effective in the treatment of relapsed or
refractory leukemias. Rationale for FLAG therapy is (i) Flu infu-
sion before Ara-C increases the accumulation of the active metab-
olite ara-C triphosphate (ara-CTP) (ii) G-CSF may sensitize leu-
kemic blasts to S-phase speciﬁc Ara-C by recruiting quiescent cells
into cell cycle and increasing Ara-C phosphorylation. Flu is also
highly immunosuppressive agent and frequently used in prepara-
tive regimen for stem cell transplantation. We here report thirty-
two patients with advanced hematological malignancies who un-
derwent cord blood transplantation (CBT) with a conditioning
regimen composed of 1200 cGy total body irradiation (TBI) and
FLAG therapy (TBI/ FLAG). Graft-versus-host disease (GVHD)
prophylaxis was FK506 or cyclosporin A and/or methotrexate. The
cumulative incidence of sustained donor engraftment was 91%.
The median number of days to an absolute neutrophil count of
500/ml was 22 (range, 19-31 days). The median time to an un-
transfused platelet count of 50000/ml was 46 (range, 21-208 days).
The incidence of grades II-IV acute GVHD was 67%. Eleven
patients were alive at a median follow-up of 22 months (range, 5-58
months). Three year overall survival and disease-free survival rates
were 34 and 26%, respectively. The result suggests that this novel
regimen is well tolerable and offers rapid donor cell engraftment.
Thus, this study demonstrated the feasibility of FLAG/TBI as a
conditioning regimen in CBT in advanced hematological malig-
nancies.
77
DEPLETION OF RECIPIENT NK CELLS SIGNIFICANTLY INCREASES EN-
GRAFTMENT OF HAPLO-MATCHED HIGHLY PURIFIED HEMATOPOIETIC
STEM CELLS
Smith-Berdan, S.K., Gille, D., Fong, T., Christensen, J. Cellerant
Therapeutics, San Carlos, CA.
Hematopoietic stem cell transplantation has been shown to be a
potential curative treatment for malignant and nonmalignant he-
matopoietic disorders. However, many patients lack an HLA iden-
tical matched donor, and haplo-matched transplanted patients are
at risk of developing acute and chronic graft versus host disease
(GvHD). Preparations of puriﬁed hematopoietic stem cells (HSC)
depleted of T cells greatly decrease the risk of GvHD. However,
transplantation of allogeneic haplo or completely mismatched pu-
riﬁed HSC results in greater resistance to engraftment compared
to whole bone marrow. An earlier study demonstrated that barriers
to engraftment of allogeneic HSC may be decreased by increasing
the stem cell dose (Uchida et al., 1998); however, the mechanism of
inhibition of engraftment of puriﬁed HSC was not shown. Our
study evaluated hematopoietic recovery kinetics (WBC, RBC, and
platelets) and long-term functional immune restoration of highly
puriﬁed mouse KTLS HSC in syngeneic, MHC-matched unre-
lated (MUD) and haplo-identical transplantation models. Mice
transplanted with haplo-identical HSC exhibited slower recovery
kinetics, decreased levels of donor chimerism and survival at all
HSC doses tested as compared to syngeneic or MUD HSC. Pre-
treatment of recipients with antibody to clear residual NK cells
improved recovery kinetics, donor chimerism and survival of
haplo-identical HSC transplants equal to levels observed with
syngeneic HSC transplants. Analysis of transplanted mice revealed
no quantitative differences of donor B and T lymphoid, myeloid,
or NK cells in the bone marrow, spleen, thymus, lymph nodes and
peripheral blood. Progenitor compartments and B and T lymphoid
maturation pathways appeared normal. Immunization of haplo-
identical HSC transplanted mice with ova, elicited a speciﬁc T-
mediated B cell response in all three transplant models. These data
illustrate that depletion of residual host NK cell activity in prep-
aration for puriﬁed haplo-identical HSC transplantation results in
rapid early immune recovery and a quantitative and qualitative
restoration of the adaptive immune response equivalent to fully
matched HSC. Our studies suggest the potential clinical use of
puriﬁed haplo-identical HSC could be safely broadened by deple-
tion of host NK cells in transplant patients without a matched
related donor.
78
GENOTYPING KILLER IMMUNOGLOBULIN-LIKE RECEPTORS USING
MALDI-TOF MASS SPECTROMETRY FOR STEM CELL TRANSPLANT DO-
NOR SELECTION
Trachtenberg, E.A.1, Nichols, R.J.1, Boal, H.E.1, Maiers, M.2,
Houtchens, K.A.1 1C.H.O.R.I., Oakland, CA; 2N.M.D.P., Minneapolis,
MN.
The highly polymorphic receptors known as killer immunoglob-
ulin-like receptors (KIR) are one of several families of receptors
involved in regulating the activity of natural killer (NK) cells, a
Poster Session I
30


Increased sensitivity of two-step cytomegalovirus PCR analysis on peripheral blood leukocytes permits detailed analysis of reactivation and immune reconstitution 

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Increased sensitivity of two-step cytomegalovirus PCR analysis on peripheral blood leukocytes permits detailed analysis of reactivation and immune reconstitution

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