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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
Low-dose total body irradiation followed by allogeneic lymphocyte infusion may
induce remission in patients with refractory hematologic malignancy
Karen K. Ballen, Pamela S. Becker, Robert V. B. Emmons, Thomas J. Fitzgerald, Chung C. Hsieh, Qin Liu, Christine Heyes,
Yeteive Clark, William Levy, Jean Francois Lambert, Frank Chiafari, Irma Szymanski, Sarah Rososhansky,
Mark A. Popovsky, F. Marc Stewart, and Peter J. Quesenberry
Allogeneic stem cell transplantation is
curative for certain cancers, but the high
doses of chemotherapy/radiotherapy lead
to toxicity. Here, we treat patients with
refractory cancer with 100 cGy total body
irradiation (TBI) followed by infusion of
nonmobilized pheresed allogeneic peripheral blood cells. Twenty-five patients, with
a median age of 47 years, with refractory
cancers were enrolled. Eighteen patients
received sibling and 7 received unrelated
cord blood cells. Donor chimerism was
assessed at weeks 1, 2, 3, 4, and 8 after
transplantation. Seven patients with solid
tumors received a sibling transplant and
6 received a cord blood transplant; none
achieved donor chimerism, but 1 treated
at the higher dose level of 1 ⴛ 108 CD3ⴙ
cells/kg had a transient nodal response.
Twelve patients with hematologic malignancies were treated; 1 received a cord
blood transplant and 11 received sibling
donor cells. Nine of these 11 patients
achieved donor chimerism, ranging from
5% to 100%. Four patients had sustained
complete remission of their cancers, including one patient with transient 5%
donor chimerism. The development of
chimerism correlated with hematologic
malignancy (P < .001), total previous my-
elotoxic chemotherapy (P < .001), T-cell
dose (P ⴝ .03), and graft-versus-host disease (P ⴝ .01). Tumor response correlated with donor chimerism (P ⴝ .01). Engraftment was achieved in patients with
hematologic malignancies who had been
heavily pretreated, suggesting the degree
of immunosuppression may be a determinant of engraftment. Low-dose TBI and
allogeneic lymphocyte infusion may induce remission in patients with refractory
hematologic malignancy. (Blood. 2002;
100:442-450)
© 2002 by The American Society of Hematology
Introduction
Myeloablative chemotherapy and radiotherapy followed by allogeneic stem cell transplantation has been shown to be curative for
certain hematologic malignancies, aplastic anemia, and genetic
diseases.1-3 The morbidity of the procedure has limited allogeneic
transplantation to younger patients in good physical condition.4,5
Many centers have set age eligibility criteria at younger than 50 or
60 years.6,7 However, the majority of patients with leukemia and
lymphoma are over the age of 50 years.8 Therefore, novel strategies
are needed to care for these patients.
Work from our laboratory, in a syngeneic murine model, has
shown that in a nonablative transplant model, engraftment can be
achieved using a high stem cell dose.9 Using a BALB/c male-tofemale transplant model, we have shown that 40% donor chimerism can be achieved by injection of 200 million cells into the
nonirradiated mice. Using the same murine model, we have shown
that a dose of 100 cGy radiation (minimal myeloablation) permits
60% to 80% donor chimerism, with injection of only 40 million
marrow cells.10 These studies indicate that in this syngeneic murine
model, the dose of 100 cGy is toxic to stem cells, but not
myelotoxic, and suggest that the final host and donor ratio may be
determined by competition between host and donor stem cells.
Recently, Slavin et al, Khouri et al, and others have used
minimally ablative doses of chemotherapy (“mini-transplants”) to
treat older patients with hematologic malignancies.11-13 These
studies were based on previous data that showed that donor
lymphocyte infusions can induce remissions in patients who have a
relapse after allogeneic transplantation, illustrating a graft-versusleukemia effect.14-16 In the experience at the M. D. Anderson Center
(Houston, TX), 15 patients with advanced leukemia or myelodysplastic syndromes, who were not candidates for conventional
transplantation because of age or comorbid medical conditions,
received an allogeneic stem cell transplant after conditioning with a
purine analog-based nonmyeloablative chemotherapy regimen.
Thirteen patients had engraftment, with one treatment-related
death; 3 patients are alive without disease 6 months after treatment.17 Porter et al treated 18 patients with interferon and donor
lymphocyte infusions.18 Prior autologous transplantation was associated
with late chimerism, and one patient developed pancytopenia.
“Mini-transplants” have been studied in patients with diseases
traditionally treated with stem cell transplantation, such as leukemia and lymphoma that is responsive to chemotherapy, and in
patients with matched or one antigen-mismatched sibling donors.
In this phase 1 study, we treated patients with refractory cancer,
including solid tumors not traditionally treated with transplantation. Because the majority of patients do not have a family matched
donor, we also extended this therapy to patients without family
donors through the use of umbilical cord blood.19,20 Umbilical cord
blood has been a useful alternative stem cell source for patients
From the Cancer Center and Departments of Medicine, Radiation Oncology,
and Pathology, University of Massachusetts Memorial Health Care, Worcester;
Baltimore Rh Typing Laboratory, Baltimore, MD; and the American Red Cross
Cord Blood Program, Dedham, MA.
Reprints: Karen K. Ballen, Massachusetts General Hospital, 100 Blossom
Street, Boston, MA 02114; e-mail: [email protected].
Submitted March 26, 2001; accepted March 12, 2002.
Supported in part by a grant from the Our Danny Cancer Fund, University of
Massachusetts Medical School.
442
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2002 by The American Society of Hematology
BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
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BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
without family donors, but it has not been studied after a
low-dose radiation-conditioning regimen.21-24 We selected the
dose of 100 cGy based on our preclinical studies, which used a
syngeneic model.
Patients and methods
Umbilical cord blood collections and processing
Cord blood units were obtained from the American Red Cross Cord Blood
Program. All cord blood donors signed an informed consent approved by
the Institutional Review Board of the University of Massachusetts Medical
School, Memorial Health Care, or St Vincent’s Hospital, (Worcester, MA)
depending on the hospital of delivery. Cord blood was collected only from
single birth, term, low-risk deliveries, and the blood was collected with the
placenta in utero after cleansing the cord with alcohol and Betadine. The
blood was collected into a standard blood donor bag containing 35 mL
citrate phosphate dextrose (CPD; Baxter Healthcare, Deerfield, IL). Cord
blood was processed and cryopreserved according to the methods of
Rubinstein et al.20 Cord blood was stored in the liquid phase of liquid
nitrogen until ready for use. Cord blood was thawed just prior to
transplantation according to methods of Kurtzberg et al.22
Patient eligibility
Patients between the ages of 18 and 75 years with histologically proven
malignancy refractory to conventional therapy were eligible for this study.
Patients required an HLA-identical or one-antigen mismatched family
donor or a 4 of 6, 5 of 6, or 6 of 6 antigen-matched umbilical cord blood
unit. Patients were required to have pulmonary function tests with carbon
monoxide diffusion in the lung 50% or greater predicted and a creatinine
clearance more than 50 mL/min. Patients and donors signed written
informed consents approved by the Institutional Review Board of University of Massachusetts Medical School.
LOW-DOSE IRRADIATION
443
chimerism was performed at 16 weeks, and then every 6 months in the
long-term survivors. Chimerism was performed by the Baltimore Rh
Typing Laboratory (BRT) and was performed on nucleated cells isolated
from whole blood. Short tandem repeat testing in multiplex battery was
performed by BRT.26,27 The samples were processed using a modified
guanidinium-based isolation method and isolated DNA quantitated in a test
gel.28 DNA (1 ng) was subjected to Taqstart antibody-regulated polymerase
chain reaction (PCR) amplification with Promega Power Plex 1.1 and 2.1
primer sets (Madison, WI).29 The amplified DNA was run in a 43-cm
denaturing polyacrylamide gel and scanned using an FMBIOII fluorescent
imager. Relative genomic copy number of donor and recipient cells in each
posttransplantation sample was estimated by analyzing the area under the
signal spectrum peaks corresponding to relevant alleles. The reproducible
level of sensitivity in our assay, as monitored by control mixture lanes, was
5% to 7%. Percent donor engraftment or chimerism was defined as the
percent of total cells derived from estimates of donor DNA.
Statistical analysis
Disease (hematologic malignancy versus solid tumor), graft type (cord
blood versus sibling), T-cell dose (1 ⫻ 108 CD3 cells/kg versus other),
development of GVHD, number of prior chemotherapy drugs, days of
neutropenia, and number of platelet and red blood cell (RBC) transfusions
were studied as predictive factors for 2 end points of donor chimerism and
tumor response. In univariate analysis, the Fisher exact test was used to test
the difference of proportional distribution of donor chimerism and tumor
response among different categories of all these 7 factors. The association
between donor chimerism and tumor response was tested using the
McNemar test. A multivariate, exact logistic regression analysis (Cytel
Statistical Software, Cambridge, MA) was fitted to examine disease type,
T-cell dose, graft type, and number of prior chemotherapies as predictors for
chimerism. All statistical analyses were conducted with SAS 8.0.30
Results
Donor lymphocyte collection
Patient characteristics
Donors received no filgrastim or chemotherapy priming. Leukapheresis was
performed using conventional techniques for mononuclear cell collection.
The target dose was initially set at 1 ⫻ 107 CD3⫹ cells/kg (2 patients) and
was then increased to 1 ⫻ 108 CD3⫹ cells/kg. Donors were collected a
second consecutive day if the target dose was not reached on the first day
of collection.
From June 1998 to June 2000, 25 patients with refractory cancer
enrolled in this phase 1 study. Patient and disease characteristics are
summarized in Table 1. In brief, the median age was 47 years
(range, 24-68 years). Thirteen patients had solid tumors: 3 patients
with lung cancer, 2 patients each with melanoma and esophageal
Conditioning regimen
Patients were treated with 100 cGy total body irradiation (TBI) given by
lateral opposing fields at a dose rate of 10 cGy/min. Individual patient
specific compensators were used to provide x-ray dose uniformity throughout the patient. There was no normal tissue (pulmonary or renal) shielding.
Treatment was given in the morning, and the pheresis product was given 4
to 6 hours after the TBI. Treatment was given to recipients as outpatients
and no graft-versus-host (GVHD) disease prophylaxis was administered. A
second pheresis product infusion (no TBI) was offered at 8 weeks after the
first infusion for patients with less than 50% donor chimerism.
Table 1. Patient characteristics
Diagnosis and graft
n
Solid tumors, N ⫽ 13
Lung cancer
3
Melanoma
2
Esophageal cancer
2
Hepatocellular cancer
1
Neuroendocrine cancer
1
Adrenocortical cancer
1
Follow-up after transplantation
Pancreatic cancer
1
Renal cell cancer
1
Patients were followed in the outpatient clinic weekly for 4 weeks, then at 8
weeks, 16 weeks, and then at 6-month intervals. The assessment included
blood counts, GVHD assessment, and chimerism analysis. No routine
prophylactic antibiotics, growth factors, or immunosuppression were given.
GVHD was graded according to the Glucksberg criteria.25 At 8 weeks after
infusion, a bone marrow aspirate and biopsy and a formal tumor staging
(computed tomography [CT] scans as appropriate) were performed.
Sarcoma
1
Chimerism analysis
The primary end point of the study was donor-recipient chimerism
measured at 1, 2, 3, 4, and 8 weeks after infusion. After the first 8 weeks,
Hematologic malignancies, N ⫽ 12
Lymphoma
7
Myeloma
2
Leukemia
3
Graft source, N ⫽ 25
6/6 matched sibling
17
5/6 matched sibling
1
6/6 matched cord
1
5/6 matched cord
3
4/6 matched cord
3
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444
BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
BALLEN et al
Table 2. Prior chemotherapy
Table 2. Prior chemotherapy (continued)
Unique
patient
ID
Unique
patient
ID
Diagnosis
No. of
prior
regimens
14
Lymphoma
5
Diagnosis
No. of
prior
regimens
Chemotherapy
Radiation
Solid tumor patients
large cell
3
Esophageal
1
4
cancer
Hepatocellular
0
5
Neuroendocrine
2
1. 5-FU/cisplatin
4. Rituxan
No
1. VP-16/cisplatin
1. Mitotane
5. Etoposide/prednisone/vincristine/
No
Cytoxan
20
6
1. CHOP
10
Sarcoma
0
Yes
3. Cytoxan
13
Melanoma
0
Yes
4. Auto BMT
15
Lung
3
1. Cisplatin/etoposide
Yes
Lymphoma
1
large cell
No
(Cytoxan/BCNU/etoposide)
5. Taxol/topotecan
3. Taxol/Carbo
16
17
Pancreatic
Renal cell
1
0
18
Esophageal
3
1. Taxol
1. Cisplatin/5-FU
6. Rituxan
Yes
Yes
23
CLL
5
Yes
4. Chlorambucil
5. Rituxan
Yes
24
2. Navelbine
21
22
Lung
Melanoma
1
3
3.
4.
5.
1.
1.
2.
3.
Gemcitabine/Herceptin
Camptosar
Taxol
Taxol/Carbo
Interferon
Vaccine/IL-2
Dacarbazine
Lymphoma
4
Large cell
1. Cytoxan/vincristine/Adriamycin/
25
Yes
Yes
Yes
2. Etoposide/Solu-Medrol/ARA-C/
cisplatin (ESHAP)
3. Cytoxan
4. Autologous stem cell
transplantation (BMT)
2
Lymphoma
3
large cell
6
Myeloma
(Cytoxan/etoposide/BCNU)
1. CHOP
Yes
2. ESHAP
6
3. Rituxan
1. Melphalan/prednisone
2
AML
1. CHOP
Yes
2. ESHAP
3
1. Idarubicin/ARA-C
No
2. Mitoxantrone/etoposide
prednisone (CHOP)
Follicular mixed
Lymphoma
large cell
Hematologic malignancy patients
1
Yes
3. Adriamycin-etoposide-vincristine
3. Camptosar
1. Taxol/carbo
1. Fludarabine-Cytoxan
2. Solu-Medrol
2. Taxol/5FU
5
No
2. ICE
2. Topotecan
Lung
Yes
3. ICE
Adrenocortical
19
Radiation
2. ESHAP
Yes
2. Carbo/Taxol/VP-16
7
Chemotherapy
1. CHOP
Yes
2. Vincristine/Cytoxan/Adriamycin/
prednisone
3. Cytoxan
3. Daunorubicin/ARA-C
5-FU indicates 5-fluorouracil; carbo, carboplatin; and IL-2, interleukin 2.
cancer, and 1 patient each with hepatocellular carcinoma, neuroendocrine carcinoma, adrenocortical cancer, pancreatic cancer, sarcoma, and renal cell carcinoma. Twelve patients had hematologic
malignancies: 7 patients with lymphoma, 2 patients with myeloma,
and 3 with leukemia. The median time from diagnosis to transplantation was 13 months (range, 4-105 months).
Prior treatment before transplantation is outlined in Table 2. For
patients with solid tumors, the median number of prior chemotherapy regimens was 1 (range, 0-5). For patients with hematologic
malignancies, the median number of prior chemotherapy regimens
was 4 (range, 2-6). This group included 5 patients with prior
autologous transplants and 1 patient who had received a prior
allogeneic transplant from another donor. Ten of the 12 patients
with hematologic malignancy, but none with solid tumors, had
received cyclophosphamide.
4. Auto BMT
Donor characteristics
(busulfan/Cytoxan/thiotepa)
5. Interferon
6. Decadron/Cytoxan/etoposide/
8
Myeloma
3
prednisone
1. VAD (vincristine/Adriamycin
Yes
Decadron)
2. Cytoxan
9
Chronic
2
myelogenous
11
leukemia
Lymphoma
3. Auto BMT (melphalan)
1. Idarubicin/vincristine/prednisone
Yes
2. Allo BMT (Cytoxan/TBI)
4
large cell
1. CHOP
No
2. Ifosfamide/carboplatin/etoposide
(ICE)
3. ESHAP
12
Lymphoma
lymphoblastic
3
4. CHOP
1. CHOP
2. Cytoxan
3. Auto BMT (Cytoxan/BCNU/
etoposide)
No
Family donors. Eighteen donors were family donors. The median
donor age was 47 years (range, 22-71 years). Donors were either
fully HLA compatible siblings (n ⫽ 17) or 1 antigen-mismatched
sibling (n ⫽ 1). Eight donors were men and 10 donors were
women; 4 donor-recipient pairs were sex mismatched. Thirteen
donors underwent 1 pheresis procedure and 5 underwent 2 pheresis
procedures. The median number of CD34⫹ cells/kg infused was
5.2 ⫻ 104 (range, 1.5 ⫻ 104 to 2.8 ⫻ 105 cells/kg). Two patients,
early in the study, received 1 ⫻ 107 CD3⫹ cells/kg; the other 16
patients received 1 ⫻ 108 CD3⫹ cells/kg.
Cord blood donors. Cord blood donors were selected from the
American Red Cross Cord Blood Program and selection was based
on HLA match and the number of nucleated cells/kg of the
recipient. One patient received a cord unit matched at 6 of 6 HLA
loci. Three patients received a cord unit matched at 5 of 6 HLA loci,
and 3 patients received a unit matched at 4 of 6 HLA loci. The
median number of CD34⫹ cells/kg infused was 3.1 ⫻ 104 (range,
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BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
LOW-DOSE IRRADIATION
1.1 to 10.7 ⫻ 104 cells/kg). The median number of CD3⫹ cells/kg
infused was 1.7 ⫻ 106 (range, 0.5 to 3.7 ⫻ 106 cells/kg).
Engraftment
Patients’ blood counts dropped 3 to 4 weeks after therapy. The
median days of neutropenia (absolute neutrophil count [ANC]) in
the first 8 weeks after transplantation was 6 days (range, 0-48 days)
for the sibling transplants and 0 days for the cord blood transplants
(range, 0-5 days), and occurred a median of 21 days after
transplantation. For the sibling transplants, 2 patients with a solid
tumor and 6 patients with hematologic malignancies had a neutrophil count lower than 500. The median days of neutropenia for the
9 chimeric patients was 14 days (range, 0-48 days). Twenty patients
required blood transfusions; the median number of RBC transfusions was 2 (range, 0-42). The median number of RBC transfusions
for the chimeric patients was 6 (range, 0-42). The median number
of platelet transfusions for all patients was 1 (range, 0-62). The
median number of platelet transfusions for the chimeric patients
was 6 (range, 0-62).
Response and chimerism
Cord blood recipients. Seven patients received a cord blood graft.
Six of these patients had solid tumors and one patient had
lymphoma. Of these 7 patients, none achieved a tumor response or
evidence of donor chimerism.
Patients with solid tumors. Thirteen patients were treated for
refractory solid tumors. Six received a cord blood infusion. Seven
patients received a transplant from a sibling; of these, 2 patients
were treated at the lower cell dose of 1 ⫻ 107 CD3⫹ cells/kg. These
2 patients did not show engraftment nor did they have a tumor
response. Five patients were treated at the higher cell dose of
1 ⫻ 108 CD3⫹ cells/kg. None of these 5 patients developed any
evidence of donor chimerism. One patient, a woman with adrenocortical cancer, achieved a nodal response between 4 and 8 weeks
after infusion. She was the only patient with a solid tumor to
receive a second lymphocyte infusion of 1 ⫻ 108 CD3⫹ cells/kg as
planned 8 weeks after infusion. She did not develop any evidence
of donor chimerism after the second infusion either; her disease
later progressed and she died of recurrent cancer.
445
Patients with hematologic malignancy. Twelve patients with
hematologic malignancies were treated. One patient received a
cord blood transplant. The response and chimerism analyses of the
11 patients treated with a sibling transplant are displayed in Table 3.
Eleven patients received a sibling infusion at a dose of 1 ⫻ 108
CD3⫹ cells/kg. Nine of 11 patients achieved some degree of donor
chimerism. Three patients achieved 100% donor chimerism in the 4
weeks after infusion, and 2 patients achieved 67% chimerism. Two
patients achieved 10% donor chimerism, and 2 patients achieved
5% donor chimerism. At 8 weeks after infusion, 3 of the 7 surviving
patients showed 100% donor cells, and 1 patient showed 5% donor
cells. In 3 of the 9 patients, evidence of donor DNA was transient,
and there was conversion to recipient DNA.
Four patients achieved a complete remission (CR) of their
cancer. One patient achieved CR with transient 5% donor chimerism, and the other 3 patients who achieved CR had 100% donor
chimerism. The first patient achieved a CR although she had only
transient 5% donor chimerism. This patient, a 51-year-old woman
with large cell lymphoma, had a relapse after autologous transplantation. Histology at the time of relapse was a follicular mixed
lymphoma. She had been treated with radiotherapy and salvage
chemotherapy without response. At the time of treatment, she had
extensive pretracheal, subcarinal, and hilar adenopathy. Her course
is outlined in Table 4. She developed 5% donor chimerism at 1
week after transplantation. Subsequent chimerism studies performed weekly through 4 weeks, at 8 weeks, and every 6 months
have shown only recipient cells. There was depression of blood
counts, which had increased by the week 8 evaluation. Figure 1
demonstrates serial CT images before treatment and at 8 weeks
after treatment. The CT scans show a dramatic resolution of the
chest adenopathy. The patient is alive and well, 42 months after
transplantation. Restaging CT scans at 36 months also showed no
evidence of disease and the patient’s blood showed all recipient cells.
Three other patients achieved CR from their cancers; these 3
patients had 100% donor chimerism. One patient, a 56-year-old
woman with a T-cell lymphoblastic lymphoma, had a recurrence
after autologous transplantation. She had 100% donor chimerism at
4 weeks after infusion. She was in CR on her 8-week postinfusion
restaging CT scans and remained persistently 100% chimeric at 8
weeks after infusion. She developed mild skin GVHD, requiring
Table 3. Chimerism and tumor responses of the 11 patients with hematologic malignancies treated with a sibling graft
Donor chimerism
Patient
no.
Wk
1
Disease
1
Lymphoma
5%
0
2
Lymphoma
10%
10%
Wk 2
Wk 4
Wk 8
Disease
state at 8 wk
Survival
days
0
0
0
CR
1285*
Not obtained
Patient died of progressive
Wk 3
28
disease
6
Myeloma
0
8
Myeloma
10%
9
CML
11
Lymphoma
0
5%
0
Not obtained
100%
100%
CR
10%
0
0
0
Stable
116
229
0
67%
5%
5%
No response
129
Not obtained
Not obtained
Patient died of progressive
29
disease
12
Lymphoma
10%
20
Lymphoma
0
40%
90%
100%
100%
0
0
67%
Patient died of progressive
CR
222
37
disease
23
CLL
0
0
0
0
0
24
Lymphoma
0
0
0
0
Patient died of progressive
PR
179
40
disease
25
AML
0
100%
CML indicates chronic myelogenous leukemia.
*Patient alive at last follow-up of December 31, 2001.
100%
100%
100%
CR
630*
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446
BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
BALLEN et al
Table 4. Time course of patient 1
Time
0
White cell count, 109/L
Hematocrit, %
Platelets, 109/L
Wk 2
Wk 3
Wk 4
Wk 8
3.8
3.2
3.1
2.3
5.0
6.6
36.6
34.9
34.6
32.2
29.1
24.5
39.6
282
83
7
123
0
0
0
0
0
No nodes ⬎ 1 cm
No nodes ⬎ 1 cm
adenopathy and retroperitoneal
on chest and
on chest CT
adenopathy
pelvic CT scan
scan
0
260
3y
122
Chimerism
Disease state
Wk 1
5.4
5%
Peritracheal hilar/subcarinal
treatment with steroids and cyclosporine. She had a relapse 5
months after infusion and died of recurrent disease.
The third patient who achieved CR was a 55-year-old woman
with multiple myeloma, who had recurrence after autologous stem
cell transplantation, and multiple other chemotherapies, resulting in
294
pancytopenia at the time of treatment. She developed complete
donor chimerism by 4 weeks after infusion and was in CR by bone
marrow aspirate and biopsy. Her course was complicated by skin
GVHD, pancytopenia, and Pseudomonas sepsis. Therefore, at 8
weeks after infusion, she received a second infusion of granulocyte
Figure 1. CT scans from patient 1. (A) Chest CT scan
images before treatment. (B) Chest CT scan images at 8
weeks after treatment shows resolution of adenopathy.
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BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
colony-stimulating factor (G-CSF)–primed stem cells (0.5 ⫻ 106
CD34⫹ cells/kg) from the same donor. She remained completely
100% donor chimeric. She developed GVHD of the liver and died
of liver failure 3.5 months after infusion. Autopsy revealed liver
GVHD and no evidence of myeloma.
The fourth patient who achieved CR was a 24-year-old woman
with acute myelogenous leukemia (AML), who had failed 3
different induction regimens, and was treated in frank relapse. She
developed CR, documented by bone marrow histology and flow
cytometry, and demonstrated persistent 100% donor chimerism
after transplantation. Her course was complicated by pancytopenia
and she received an infusion of ␣␤ T-cell–depleted donor bone
marrow (courtesy of Chimeric Therapies, Sharon Hill, PA), which
was tolerated well. She is now in remission, documented by bone
marrow histology and flow cytometry, and 100% donor cells, 20
months after her initial infusion.
In addition, one patient with chronic lymphocytic leukemia
(CLL) who did not show any evidence of donor chimerism had a
transient 75% reduction in lymph node size.
GVHD
Acute GVHD occurred in 6 patients. Five of the 9 patients (55%)
who developed donor chimerism developed acute GVHD. Two
patients, who did not have tumor response and were 10% donor
chimerism, had grade II skin GVHD, which responded to steroids.
One of these patients had histologic documentation. Another
patient with CLL, with transient partial remission (PR), also
developed histologically documented skin GVHD, but did not have
any evidence of donor chimerism, as assayed by our methods, and
studied at 1, 2, 3, 4, and 8 weeks after infusion. The 3 patients with
100% donor chimerism also developed GVHD; 2 patients had
histologic documentation of grade II skin GVHD; the other patient
(as discussed above) had liver GVHD. One patient (who was 100%
donor chimeric and has had a CR—the fourth patient outlined
above) developed chronic GVHD, but only 6 patients had donor
engraftment and were alive at 100 days to be evaluable for
chronic GVHD.
Toxicity
There was one treatment-related death as described above. This
patient also had Pseudomonas sepsis, thought secondary to prolonged pancytopenia, which predated the primary infusion. Five
additional patients (including 3 of the 4 patients with a CR)
required admission for fever and neutropenia, with positive blood
cultures for Klebsiella and Pseudomonas in one patient and
positive blood cultures for Enterobacter in another patient. Four of
the 6 patients who were admitted for fever and neutropenia were
chimeric patients and 2 were nonchimeric. Two of the 4 responding
patients did require subsequent stem cell infusions. One of these
patients was pancytopenic prior to initiating treatment, as discussed
above. There were no other significant infections in this cohort of
heavily pretreated patients. The low-dose TBI was well tolerated
without evidence of acute morbidity.
Survival
One patient died of a treatment-related death (4%). Twenty patients
have died of progressive disease between 1 and 12 months after
transplantation. Two patients survive in CR, at 42 and 20 months
after therapy. Both these patients had hematologic malignancies
LOW-DOSE IRRADIATION
447
and had some evidence of donor chimerism. One patient with
sarcoma who did not develop chimerism survives with stable
disease, 24 months after therapy. The development of a partial
tumor response without donor chimerism was seen in 2 patients,
suggesting a possible effect of the 100-cGy dose alone. However,
these responses were seen between 4 and 8 weeks after transplantation, in between the time points that chimerism assays were
performed (a limitation of the study), also suggesting the possibility of a missed transient chimerism.
Associations with donor chimerism
We examined the following variables as univariate associations for
the development of donor chimerism: disease (hematologic malignancy versus solid tumor), graft type (cord blood versus sibling),
T-cell dose (1 ⫻ 108 CD3⫹ cells/kg versus other), development of
GVHD, number of prior chemotherapy drugs, days of neutropenia,
and number of platelet and RBC transfusions. The number of prior
chemotherapy regimens was evaluated on a myelotoxicity index,
with chemotherapy regimens with one drug given a score of 1, 2
drugs a score of 2, and so on. A prior bone marrow transplant
(BMT) was given a score of 5. For example, a patient who had
received CHOP chemotherapy plus a BMT would receive a score
of 9. Prior chemotherapy was highly statistically correlated with
chimerism (P ⬍ .001). Other variables shown to have a significant
association with donor chimerism were a diagnosis of hematologic
malignancy (P ⬍ .001), higher T-cell dose (P ⫽ .03), sibling graft
(P ⫽ .03), more than 5 platelet transfusions (P ⫽ .01), and the
presence of GVHD (P ⫽ .01). These factors are outlined in Table 5.
It is possible that transfusion use and GVHD are the result of
chimerism rather than a predictor of donor chimerism.
A multivariate, exact logistic regression analysis was fitted to
examine disease type, T-cell dose, graft type, and number of prior
chemotherapies as predictors of chimerism. Due to the high
correlation between disease type and T-cell dose, both variables
could not be simultaneously fit in the same model. In a model with
T-cell dose, graft type, and number of prior chemotherapies as
predictors, only number of prior chemotherapies remained to be a
significant predictor of chimerism (P ⫽ .006, odds ratio of chimerism associated with each one additional score was 1.58, 95% CI,
1.10-3.08).
Associations with tumor response
We studied also univariate correlations with tumor response. Donor
chimerism was shown to be associated with tumor response
(P ⫽ .01).
Discussion
Traditional transplantation is associated with high doses of myeloablative chemotherapy and radiotherapy.1-7 In this study, we treated
patients with low-dose TBI and have demonstrated that 100 cGy is
sufficient to achieve engraftment in some patients with
refractory cancer.
Because most patients do not have a matched family donor,
umbilical cord blood has been used as an alternative donor
source.21-24,31 Unrelated cord blood banks have been developed in
New York, St Louis, Worcester, Milan, Dusseldorf, Barcelona, and
other sites.20,32-36 Conditioning regimens that have been used
include cyclophosphamide/TBI (1375 cGy)/antithymocyte globulin,
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448
BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
BALLEN et al
Table 5. Associations with donor chimerism
Donor
chimerism
Characteristic
Yes (%)
No
P from Fisher exact test
.0001*
Diagnosis
Hematologic tumor
9 (75.0)
3
Solid tumor
0 (0.0)
13
Dose of T cell
1 ⫻ 108
8 (57.1)
6
Other
1 (9.1)
10
Allo cord
0 (0.0)
7
Allo PB
9 (50.0)
9
Less than 5
0 (0.0)
3
5 or more
4 (36.4)
7
Less than 5
3 (20.0)
12
5 or more
6 (60.0)
4
Less than 5
3 (17.7)
14
5 or more
6 (75.0)
2
.03*
Graft type
.03*
Days to ANC more than 500
.51
Number of RBC transfusion
.09
Number of platelet transfusion
.01*
Acute GVHD
Yes
5 (83.3)
1
No
4 (21.1)
15
0 (0.0)
4
.01*
Prior myelotoxic therapy score†
0
.001*
(P values from MantelHaenszel ␹2 test for trend)
1
0 (0.0)
2
2
0 (0.0)
2
3
0 (0.0)
1
4
1 (33.3)
2
5
0 (0.0)
1
6
0 (0.0)
1
8
1 (50.0)
1
9
3 (75.0)
1
10
1 (100.0)
0
11
1 (100.0)
0
12
0 (0.0)
1
13
2 (100.0)
0
Allo cord indicates allogeneic cord blood; allo PB, allogeneic peripheral blood.
*Achieve significance level of .05.
†“Score” indicates grading for previous myelotoxic therapy. Score ⫽ (no. of prior
chemotherapy drugs) ⫹ 5 ⫻ (prior bone marrow transplantation), where “prior bone
marrow transplantation” ⫽ 1 if patient had prior bone marrow transplantation,
otherwise “prior bone marrow transplantation” ⫽ 0.
etoposide/cytosine arabinoside/cyclophosphamide/TBI (1200 cGy),
melphalan/TBI (1350 cGy), antithymocyte globulin, and others.22,37
In our study, using 100 cGy TBI, engraftment with cord blood
was not demonstrated. The lack of engraftment may be related to
the conditioning regimen or cord blood factors. Because engraftment was demonstrated in similar patients receiving sibling
transplants using 100 cGy TBI, we postulate that the lack of
engraftment was due to the lower T-cell dose in the cord blood
units. However, 6 of the 7 cord blood transplants were performed in
patients with solid tumors, who showed no engraftment even with
sibling donors. The median CD3⫹ dose in the cord blood units was
1.7 ⫻ 106/kg as compared to 1 ⫻ 108/kg in the sibling recipients, a
difference of 2 logs. Therefore, it is unlikely that pooling of 2 to 3
cord blood units would produce a T-cell dose comparable to the
sibling grafts.
The majority of our patients had end-stage solid tumors. In
these patients, there was no evidence of donor engraftment and
only one transient nodal response. Eligibility criteria for this
phase 1 study included life expectancy at greater than 1 month;
therefore, many terminal patients enrolled. The protocol will now
be amended to include a 3-month life expectancy. The patients with
solid tumors were less heavily pretreated than the patients with
hematologic malignancies were, and the types of chemotherapy
received were less immunosuppressive than those used in the
patients with hematologic cancers. Ten of 12 patients with hematologic malignancy had received cyclophosphamide as noted above.
We postulate that previous chemotherapy, particularly transplant
doses of chemotherapy and high doses of alkylating agents, may
facilitate engraftment and chimerism, on the basis of immune
competition issues, such as the lack of ability of the heavily
pretreated patients to reject the donor cells. Alternatively, the prior
chemotherapy may deplete host stem cells severely and facilitate
engraftment on the basis of stem cell competition.10 It is possible
that more pretransplantation myelosuppressive therapy might improve engraftment in these patients. However, additional immunosuppression could increase the risk of aplasia and might be
performed more safely using G-CSF–mobilized stem cells.
The most promising results were seen in the patients with
hematologic malignancies treated with sibling transplants. Eleven
patients were treated with this approach, and 9 showed some
evidence of donor chimerism. Because the chimerism assay studied
only the nucleated cell population, we are unable to comment on
specific lineage engraftment, except that the patients with 100%
engraftment represented multilineage engraftment. A limitation of
the study is the absence of data on specific T-cell chimerism; this
may explain the lack of identifiable chimerism in one patient who
developed GVHD and those patients who had disease regression
but no chimerism. Our subsequent studies will assess T-cell–
specific chimerism. Childs et al recently reported regression in
patients with refractory renal cell cancer treated with cyclophosphamide and fludarabine followed by allogeneic peripheral blood stem
cell transplantation.38 In our study, 4 of 11 patients with refractory
hematologic cancer achieved CR. Although we cannot prove
definitively that the responses were not related to the effects of 100
cGy alone, we believe that is unlikely because all 3 patients who
developed 100% donor chimerism were complete responders. The
disease in patient 1 had not responded to higher doses of
radiotherapy administered before this treatment, and it would be
unlikely to have such a dramatic response to 100 cGy alone.
Patients who did not develop chimerism, as assayed in this study,
did not have a sustained response. The transplant-related mortality
was low at less than 5% (1 patient with GVHD) for all patients and
11% for chimeric patients. The patient who died of GVHD was 1 of
the 3 patients with 100% chimerism. Ferrara and others have
postulated that GVHD is related to a cytokine storm, and that
higher TBI doses lead to an increase in GVHD severity by causing
higher levels of inflammatory cytokines.39 We speculate that the
extensive pretreatment many of these patients received contributed
to their ability to engraft donor cells with a low-dose TBI therapy.
Our patients received nonmobilized peripheral blood cells,
which contained a target T-cell dose of 1 ⫻ 108 CD3⫹ cells/kg.
Initially, a nonmobilized product was chosen because of the
different lymphocyte properties compared to a G-CSF–stimulated
product and because of the literature on donor lymphocyte
infusions.15,16 No target dose of CD34⫹ cells was required;
however, the CD34⫹ cells/kg in the sibling donor patients ranged
from 1.5 ⫻ 104 to 2.8 ⫻ 105 with a median dose of 5.2 ⫻ 104
CD34⫹ cells/kg in the product, about 2 logs lower than in
traditional transplantation. Stem cell dose was not the object of the
study but an interesting finding was that chimerism did occur with a
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BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
LOW-DOSE IRRADIATION
low CD34⫹ cell/kg dose. Nonmobilized blood has CD34⫹ cells and
can give at least short-term 100% chimerism. However, a higher
rate of chimerism might have been seen with a higher CD34 dose.
In addition, 2 patients required subsequent infusions of stem cells,
which is a drawback of this approach; one of these patients
(myeloma) was pancytopenic at the time of the first infusion. The
use of a G-CSF–mobilized product may reduce the risk of
pancytopenia, and will be used in future studies.
The patient with the most durable response had only transient
chimerism to a level of 5%. She also had transient pancytopenia
that corresponded with the tumor response. There may be several
mechanisms for response. The goal is to cure patients with partial
or transient chimerism, thereby reducing the toxicity of a full
myeloablative regimen. Ability to achieve chimerism was associated with the presence of a hematologic malignancy and the
development of GVHD. Both permanent and transient chimerisms
were associated with tumor responses.
The 100-cGy TBI as a conditioning regimen is novel. It is
unlikely that the therapeutic benefit in this trial is related to the 100
cGy alone, because donor chimerism was associated with tumor
response. Alternatively, it is possible that engraftment could have
occurred without any irradiation, given the immunosuppressive
state of the patients. The marrow effects of the 100 cGy can be
illustrated in the 11 nonchimeric patients who also experienced
decreased blood counts. In a study examining 100 cGy as salvage
therapy in canine lymphoma, thrombocytopenia has been a major
toxicity (personal communication, Angela Frimberger). Storb et al
449
have used several approaches in the canine model, using 100 to 200
cGy, with and without CTLA4Ig, which blocks T-cell costimulator
activity.40,41 McSweeney et al have reported the use of the
nonmyeloablative approach in 44 patients with hematologic malignancies who were not end stage; 80% showed engraftment beyond
2 months. The transplant-related mortality was 6.8%, and 16 of
the 42 patients had a major disease response.42,43 Porter et al
treated 18 patients with interferon followed by donor lymphocyte infusion; a fatal case of marrow aplasia occurred, which
may have been related to interferon, a known marrow suppressant.18 Other strategies have included a 550-cGy regimen and
low-dose fludarabine/Cytoxan.44,45
The optimal low dose–conditioning regimen has yet to be
defined. The lower the dose of radiation that can be given, the lower
the potential toxicity to the patient. In this preliminary study, we
have shown that engraftment can occur in humans with a radiation
dose as low as 100 cGy. Tumor response can be seen with either
complete or partial chimerism, and immune suppression may be a
determinant of engraftment.
Acknowledgments
The authors acknowledge Shelly Anderson and Christina Giza for their
excellent technical assistance in preparation of the manuscript. The
authors thank Chimeric Therapies for a graft manipulation.
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2002 100: 442-450
doi:10.1182/blood.V100.2.442
Low-dose total body irradiation followed by allogeneic lymphocyte
infusion may induce remission in patients with refractory hematologic
malignancy
Karen K. Ballen, Pamela S. Becker, Robert V. B. Emmons, Thomas J. Fitzgerald, Chung C. Hsieh,
Qin Liu, Christine Heyes, Yeteive Clark, William Levy, Jean Francois Lambert, Frank Chiafari, Irma
Szymanski, Sarah Rososhansky, Mark A. Popovsky, F. Marc Stewart and Peter J. Quesenberry
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