Fred Hutchinson Cancer Research Center and the
University of Washington School of Medicine, Seattle, Washington,
Marrow grafting from genotypically HLA-identical siblings for the
treatment of chronic myelocytic leukemia (CML) began at the Fred
Hutchinson Cancer Research Center in the early 1970s (1,2). A review
of results in the first 629 patients transplanted shows event-free
survivals at 10 years which are at slightly above 60% for patients
transplanted in chronic phase, 38% for those in second chronic phase,
30% for those in accelerated phase, and 12% for those in blast crisis.
Major problems encountered were acute graft-versus-host disease
(GVHD), cytomegalovirus-associated interstitial pneumonia, and leukemic
relapse. Relapse was seen in approximately 25% of patients transplanted
in chronic phase, 45% of those in accelerated phase, and 75% of
those transplanted in blast crisis. During subsequent years, studies
were carried out addressing each of the three major problems. The
introduction of a combination of methotrexate and cyclosporine in
lieu of either methotrexate or cyclosporine alone led to a significant
reduction in the incidence of acute GVHD and transplant-related
mortality (3,4). The incidence of leukemic relapse remained at 20%.
Event-free long-term survival for patients transplanted in chronic
phase of the disease rose to 70-75% at 7 years. In regards to the
prevention of cytomegalovirusassociated pneumonia, a randomized
placebo controlled study showed an acyclovir derivative, ganciclovir,
to be effective in preventing serious disease (5). Ganciclovir prophylaxis
should have an impact on survival of future patients. Other studies
asked whether the risk of leukemic relapse could be altered by alterations
in the conditioning programs. In the first study, 116 patients were
randomized to receive either cyclophosphamide and 12 ay of fractionated
TBI or cyclophosphamide and 15.75 ay of fractionated TBI (6). None
of the patients given 15.75 Gy TBI relapsed compared to 25% of those
given 12 ay of TBI. With the increase in TBI dose, however , increases
in transplanted-related toxicity and mortality were seen, offsetting
the gain made by the reduction in leukemic relapse. Accordingly,
long-term event-free survival for both groups of patients was the
same, approximately 70% . The next study randomized 115 patients
to receive either cyclophosphamide and 12 Gy of TBI or cyclophosphamide
and 14 mg of busulfan per kg (7). All patients were given methotrexate/cyclosporine
for avHD prevention. Preliminary results show 80% survival at 2
years for both groups of patients and a relapse rate of so far 10%.
Thus, busulfan may not improve the problem of leukemic relapse.
In regards to patients with accelerated phase, small improvements
have been made. Patients were either given a combination ofbusulfan/cyclophosphamide
along with 12 ay of TBI or cyclophosphamide and 15.75 ay of TBI
(R. Clift et al., unpublished). The intensification of the conditioning
program decreased the relapse rate in both groups of patients to
less than 20% .Survival of patients receiving the busulfan-containing
regimen was 55% at 3 years compared to a 48% survival among the
patients given cyclophosphamide and 15.75 ay of TBI. Only 35% of
patients with CML have an HLA-identical sibling donor. In less than
10% of patients, another suitably matched family member can be identified,
either a phenotypically HLA-matched donor or a I-HLA-Iocus mismatched
haploidentical donor (8,9). Patients in chronic phase of CML have
been conditioned with cyclophosphamide and 12 Gy TBI. The risk of
graft failure increased from less than 2% to 7% , and that of avHD
from 30% to 70%. Despite the increase in transplant-related problems,
survival of patients with haploidentical donors was comparable to
that of patients with genotypically HLA-identical siblings, because
of a significant decrease in the risk of leukemic relapse. For 30%
of patients with CML, either fully HLAmatched or 1-HLA-locus mismatched
unrelated donors can be identified, although this figure may increase
as the number of volunteer donors in the international registries
increases (10,11). Patients with CML in chronic phase have been
conditioned with cyclophosphamide and 12 ay TBI while those in more
advanced phase of their disease have been administered cyclophosphamide
and either 13.2 ay (> 18 years of age) or 14.4 Gy «18 years of age)
ofhyperfractionated TBI. Graft failure was seen in 5% , and GVHD
in 75 % of cases, but relapse was rare, presumably the result of
a graft-versus-Ieukemia effect. Largely because of increased transplant-related
mortality, event-free survival at 3 years for recipients of marrow
from unrelated donors, either fully phenotypically matched or 1-HLA-Iocus
mismatched has been on the order of 50% compared to the 75% seen
with genotypically HLAidentical sibling donors. Review of these
results and those obtained in other centers worldwide shows the
frequent inability of the conditioning programs to eradicate the
last leukemic cell to be a persistent major problem. At our Center
, preclinical animal experimental work has focused on different
ways of delivering TBI and on the use of radiolabeled monoclonal
antibodies directed against hematopoietic cells. Most of the clinically
used TBI programs deliver radiation at low dose rates, between 5
and 8 cay per minute. We explored whether higher dose rates might
offer advantages. Specifically, we compared marrow toxicity , gastrointestinal
toxicity , immunosuppressive properties, and late organ toxicities
in dogs given single-dose versus fractionated-dose TBI administered
either at low dose rates of 5 to 10 cay per minute or at high dose
rates of 70 to 80 cay per minute. At 5 to 10 cay per minute, single-dose
and fractionated TBI had comparable marrow toxicity (12). Also,
the early gastrointestinal toxicity of fractionated TBI was similar
to that seen with single-dose TBI (13). However, late organ toxicity
was significantly reduced, and long-term survival improved with
fractionation. While the sparing of nonhematopoietic tissues by
fractionation of radiation is desirable, we were concerned whether
fractionated TBI was as immunosuppressive as single-dose TBI. A
study in recipients of marrow from DLA-identical littermates showed
fractionated TBI delivered at a dose rate of 7 cay per minute to
be significantly less immunosuppressive than single-dose TBI as
measured by the criterion of graft rejection (14,15). We hypothesized
that the greatest benefit of fractionation compared to single-dose
TBI may be obtained at higher dose rates. Results (unpublished)
showed that single-dose TBI delivered at 80 cay per minute was considerably
more toxic to the gastrointestinal tract than TBI given at 5 to
10 cay per minute. The LDo dose of single-dose TBI fell from 14
ay at 5 cay per minute to 7 ay at 80 cay per minute. By comparison,
the LDo dose with fractionated TBI fell from 14 ay at 5 cay per
minute to only 10 ay at 80 cay per minute. This suggests a relative
sparing of gut epithelial cells with fractionation when TBI is given
at high dose rates. What was true for gastrointestinal toxicity
was also found to be true for the other two parameters studied.
Dogs given 300 cay of TBI and no subsequent marrow infusion served
to study the question of marrow toxicity . Seven of 21 animals given
single-dose TBI delivered at 10 cay per minute survived compared
to six of ten given fractionated-dose TBI, a difference which was
not statistically significant. None of five dogs given single-dose
TBI delivered at 75 cay per minute survived, compared to seven of
ten given fractionated TBI, a result which was statistically significantly
different. Thus, single-dose TBI at 75 cay per minute is significantly
more toxic to the marrow than at 10 cay per minute while fractionated
TBI at 75 cay and 10 cay per minute has comparable toxicity .Results
suggest that fractionation at 75 cay per minute spares the myeloid
marrow compartment. Results on immunosuppression were similar to
those on marrow toxicity .To study the immunosuppressive effect
of TBI, dogs were given 450 cay of TBI and marrow grafts from DLA-identicallittermates.
None of 15 dogs given either single-dose or fractionated-dose TBI
delivered at 7 cay per minute showed sustained allogeneic engraftment.
Six of seven dogs given single-dose TBI at 70 cay per minute engrafted,
a result which was significantly better than that seen at 7 cay
per minute (none of 10 dogs engrafted). Only two of ten dogs given
fractionated TBI at 70 cay per minute showed sustained allogeneic
engraftment, a result which was significantly worse than that seen
with single-dose TBI at 70 cay per minute and barely better than
that seen with fractionated TBI delivered at 7 cay per minute (none
of 5 dogs engrafted). We concluded from these studies that fractionated
TBI delivered at dose rates of 70 to 80 cay per minute spared in
an equal manner the gastrointestinal epithelial cells, the myeloid
compartment of the marrow, and the lymphoid system when compared
to single-dose TBI given at 70 to 80 cay per minute. At low dose
rates of 5 to 10 cay per minute, gastrointestinal and myeloid toxicities
of single-dose and fractionated TBI were indistinguishable, and
significant sparing effects with fractionation were seen only with
regard to the immune system and late nonhematopoietic organ toxicities.
Findings imply that fractionated TBI delivered at high dose rates
of 70 to 80 cay per minute is not likely to improve the therapeutic
ratio of TBI. One way to deliver radiation to hematopoietic tissues
while avoiding dose-limiting toxicity to other organs might be to
target radiotherapy specifically using monoclonal antibody coupled
to radionuclides. In pursuit of this objective, we have carried
out studies in dogs using 131I-labeled anti-la and anti-CD44 antibodies.
We chose 1311 as the radionuclide because it is readily available,
inexpensive, attaches easily to the antibody and does not harm it,
and because it has gamma and beta components which enable use of
the same radionuclide for imaging and therapy. Antibodies to la
and CD44 were selected because the antigens they recognize are expressed
in high numbers on most lymphohematopoietic cells in dogs (16-18).
We found that, when trace labeled, the antibodies localized to spleen,
marrow, and lymph node more than to any other organ, and that when
labeled at high activity , the antibodies ablated marrow function,
an effect that could be reversed by infusing autologous marrow.
Another antibody, DM5, directed against a wide spectrum of myeloid
precursor cells and mature granulocytes, appeared even more effective
than the other two antibodies, particularly when used with cold
antibody pretreatment before the infusion of the radiolabeled antibody
(19). Based on studies in preclinical models, a clinical study has
been initiated in patients with advanced leukemia, either in relapse
or in second or third remission of acute leukemia (20). A 131I-labeled
antibody to CD45 is used. CD45 is expressed on all hematopoietic
cells. Doses to the marrow have ranged from 403 to 892 cay, doses
to the spleen from 769-2216 cay, and those to the liver have been
approximately 350 cay. Treatment with radiolabeled antibody was
followed 7-10 days later by the standard conditioning program for
an allogeneic transplant, which included 60 mg of cyclophosphamide
per kg on each of two successi ve days and 6 x 200 cay of TBI. Preliminary
results suggest that 131I-labeled anti-CD45 antibody can target
marrow effectively, and at the doses used so far, can be combined
with a conventional conditioning program without untoward toxicity
.Optimal dose and dose schedule are as yet unknown. It is likely
that combinations of antibody radionuclide conjugates and conventional
conditioning therapy will effectively reduce the risk of leukemic
relapse after marrow transplantation with tolerable toxicity.
This work was supported by grants HL36444, CA18221, CA18029, CA31787,
and CA15704 from the National Institutes of Health, DHHS.
The author thanks Bonnie Larson and Harriet Hefton for typing the
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