Blood, 15 January 2002, Vol. 99, No. 2, pp. 698-701
BRIEF REPORT
Nonmyeloablative conditioning followed by transplantation of
genetically modified HLA-matched peripheral blood progenitor cells for
hematologic malignancies in patients with acquired
immunodeficiency syndrome
Elizabeth M. Kang,
Moniek de Witte,
Harry Malech,
Richard A. Morgan,
Sheila Phang,
Charles Carter,
Susan F. Leitman,
Richard Childs,
A. John Barrett,
Richard Little, and
John F. Tisdale
From the Molecular and Clinical Hematology Branch,
National Institute of Diabetes and Digestive and Kidney Diseases;
National Institute of Allergy and Infectious Diseases; National Human
Genome Research Institute; Department of Nursing and Department of
Transfusion Medicine, Clinical Center; National Heart, Lung, and Blood
Institute; and National Cancer Institute, National Institutes of
Health, Bethesda, MD.
 |
Abstract |
To assess the safety and efficacy of nonmyeloablative
allogeneic transplantation in patients with HIV infection, a clinical protocol was initiated in patients with refractory hematologic malignancies and concomitant HIV infection. The results from the first
2 patients are reported. The indications for transplantation were
treatment-related acute myelogenous leukemia and primary refractory
Hodgkin disease in patients 1 and 2, respectively. Only patient 1 received genetically modified cells. Both patients tolerated the
procedure well with minimal toxicity, and complete remissions were
achieved in both patients, but patient 2 died of relapsed Hodgkin
disease 12 months after transplantation. Patient 1 continues in
complete remission with undetectable HIV levels and rising CD4 counts,
and with both the therapeutic and control gene transfer vectors
remaining detectable at low levels more than 2 years after
transplantation. These results suggest that nonmyeloablative allogeneic
transplantation in the context of highly active antiretroviral therapy
is feasible in patients with treatment-sensitive HIV infection.
(Blood. 2002;99:698-701)
© 2002 by The American Society of Hematology.
| |
Introduction |
Allogeneic bone marrow transplantation (BMT) is a
well-established approach with curative potential for a number of
hematologic malignancies1; however, patients with
concomitant HIV infection have generally been excluded from allogeneic
BMT trials. Indeed, early allogeneic BMT trials as a primary treatment
for HIV infection uniformly failed to control disease
progression.2-4 The demonstration by Kolb et
al5 that donor lymphocytes alone exert a powerful antileukemic effect, however, challenged the notion that high-dose chemoradiotherapy is necessary. This graft-versus-leukemia effect is
most dramatically demonstrated among relapsed allogeneic BMT recipients
receiving transplants for chronic myeloid leukemia; in these patients,
a simple infusion of donor lymphocytes produces a complete and durable
remission in the majority of treated patients6-8 and has
led to intense interest and clinical activity investigating the role of
nonmyeloablative transplant regimens for both malignant and
nonmalignant disorders.9,10 We developed a clinical
research trial to evaluate the use of
cyclophosphamide/fludarabine-based conditioning in HIV- infected
patients with an underlying malignancy, in whom an HIV-negative,
HLA-matched sibling donor was available. Using this strategy, we also
examined the possibility of efficient gene transfer of an HIV
resistance vector carrying a dominant-negative mutant Rev at the level
of the self-renewing stem cell.
| |
Study design |
The protocol was approved by the Institutional Scientific Review
Board and the Institutional Review Board of the National Heart, Lung,
and Blood Institute, the Institutional Biosafety Committee, the Center
for Biologics Evaluation of the Food and Drug Administration, and the
Office of Biotechnology Activities, and patients gave written informed
consent. Eligibility included HIV positivity; an HLA-matched,
HIV-negative sibling donor; and a hematologic malignancy meeting the
standard indications for allogeneic transplantation in HIV-negative
subjects.11 No restrictions were made regarding viral load
or highly active antiretroviral therapy (HAART) sensitivity. Donor
granulocyte colony-stimulating factor-mobilized peripheral
blood was collected by apheresis on days 5 and 6. The day 5 apheresis
product was CD34 enriched by means of a Nexell 300i (Nexell
Therapeutics, Irvine, CA) automated immunomagnetic selection system for
the recipient of genetically modified cells. Target CD34+
cells were split equally for transduction with either (1) GCsapSL3rd3, containing a dominant-mutant or transdominant Rev (TdRev) engineered to
inhibit viral replication through inhibition of wild-type Rev, a key
HIV regulatory protein,12,13 or (2) a control vector encoding human GP91phox14 using previously
established methods.15 Products not subjected to genetic
modification, including the day 6 apheresis product for both patients,
were cryopreserved without manipulation.
Patients were conditioned with a nonmyeloablative regimen16
consisting of 60 mg/kg intravenous cyclophosphamide (IV) per day on
days 
7 and 6, followed by 25 mg/m2 fludarabine IV per
day over 30 minutes on days 5 through 1. Cyclosporin was begun on
day 4. HAART was discontinued 1 week prior to conditioning and was
resumed when the patient was able to tolerate intake by mouth. Patients
received antibiotic prophylaxis including acyclovir, bactrim, and
norfloxacin as well as prophylaxis for mycobacterium avium
complex if indicated. Cytomegalovirus (CMV) reactivation was monitored
by antigenemia and was treated preemptively with ganciclovir until day
100.17
In vitro transduction efficiency was estimated by polymerase chain
reaction (PCR) of DNA isolated from individual clonogenic progenitors
derived from the end of transduction for genetically modified cells as
previously described.18 Lineage-specific assessment of
donor chimerism was determined as previously described.19 Lineage-specific engraftment by genetically modified cells was determined by real-time PCR (Perkin Elmer, Branchburg, NJ) as previously described20 with the use of vector-specific primers.
| |
Results and discussion |
Two patients have been treated to date. Their characteristics as
well as the characteristics of the allograft are given in Table
1. Both patients tolerated the procedure
well and resumed HAART on day 0; they were discharged on day 9 and
11, respectively. In both patients, 100% donor chimerism
was achieved by day 96 in both myeloid and lymphoid lineages. Lymphoid
engraftment preceded myeloid engraftment and was similar to
HIV-negative allogeneic transplant recipients conditioned with the same
regimen.19 Both patients developed CMV antigenemia and
were treated preemptively without evidence of disease. Restaging by
bone marrow examination in patient 1 as well as computerized tomography
and gallium scanning in patient 2 showed no evidence of disease in
either. Grade II "acute" skin graft-versus-host disease (GVHD)
developed in both beyond day 100, necessitating treatment with oral
prednisone and continued cyclosporin. The observation of a clinical
remission in concert with the development of acute GVHD in a patient
with chemo-refractory Hodgkin is supportive of the concept of a
graft-versus-Hodgkin effect.21-23 Control of GVHD in
patient 2 was associated with relapsed Hodgkin disease in the
retroperitoneum on day 180. Biopsy-proven central nervous system
toxoplasmosis also developed and responded to treatment. However,
despite withdrawal of immunosuppression, radiation therapy, interferon,
and eventual administration of 1 × 107 donor lymphocytes
per kilogram, the patient succumbed to progressive disease and died 12 months after transplantation. Patient 1 is currently being treated with
cyclosporine and low-dose prednisone for limited chronic GVHD and
remains in complete remission more than 2 years after
transplantation.
The effect of nonmyeloablative allogeneic transplantation on HIV status
is depicted in Figure 1. HIV was
undetectable in both patients during extended follow-up with the
exception of a brief period in patient 2 during which HAART was
interrupted owing to persistent nausea. An acute febrile illness of 2 weeks' duration, with headache, cerebrospinal fluid pleocytosis, and a
negative evaluation for infectious causes coincided with a rise in the HIV burden to 106 copies per milliliter. This episode may
have represented acute HIV infection of the allograft,24
underscoring the importance of continued antiretroviral therapy
following transplantation. Reinstitution of HAART was associated with
resumed viral control. CD4 counts rose above pretransplantation levels
in both patients after transplantation. In patient 1, the CD4
count has risen as high as 450 cells per microliter, and the pattern of
recovery is similar to that seen in HIV-negative allogeneic transplant recipients who received the same conditioning regimen (unpublished observations, November 1999).
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| Figure 1.
Effect of transplantation on peripheral blood CD4 count
and viral load.
Data for patient 1 are represented by dashed lines and symbols, and for
patient 2 by solid lines and symbols. The CD4 count in cells per
microliter is represented by squares for both patients and is plotted
along the left y-axis. The circles represent the viral load in copies
per milliliter, which is plotted along the right y-axis. Both variables
are plotted against the days after transplantation. The
horizontal lines represent the days of HAART therapy, and the
horizontal arrows represent the beginning of the immunosuppression
taper. The vertical arrow shows the commencement of interferon, and the
arrowhead, the day of donor lymphocyte infusion given to patient 2. Patient 2 died 12 months after transplantation. The CD4 count for both
patients remained at or above what it was prior to transplantation. The
viral load remained undetectable at the latest time point
measured.
|
|
Recent improvements in gene transfer methodologies targeting the
hematopoietic stem cell population have resulted in increased levels of
genetically modified cells circulating after transplantation in
relevant large animals, with levels of 5% to 10% or greater now
achievable.25-27 While an equally high gene-transfer
efficiency to the progenitor population was obtained as estimated by
PCR, with 80% for the TdRev aliquot and 90% for the
GP91phox aliquot, in vivo levels of circulating genetically
modified cells were lower than those obtained in large animals. Overall
marking levels of 0.01% were seen (Figure
2). The differences in marking from the
TdRev and control vector-transduced fractions did not suggest a
survival or proliferative advantage to cells carrying the therapeutic gene; however, the absence of detectable HIV for the duration of the
study suggests the absence of selective pressure as one potential
explanation. Further, a threshold level of engraftment may be necessary
to achieve a therapeutic effect. Finally, an effect by the genetically
modified cells cannot be ruled out, as the recipient of genetically
modified cells continues to show detectable marking from the TdRev
vector with no detectable HIV. The low-level contribution toward
hematopoeisis by genetically modified cells may also reflect
competition by the unmanipulated graft. In murine and nonhuman primate
competitive repopulation experiments, ex vivo cultured cells compete
poorly against fresh cells, and cells cultured for longer periods
compete poorly against cells cultured for shorter
periods.25,28,29
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| Figure 2.
Percentage of transduced cells measured in the
peripheral blood.
The gene-marking levels in patient 1 were analyzed by PCR. The
percentage of cells positive for the GP91phox transgene is
represented by dashed lines, and the percentage positive for
TdRev, by solid lines. Marking in myeloid cells is shown as
circles and in T cells as triangles. Overall marking averaged about
0.01% for both vectors and in both lineages.
|
|
A reluctance to consider allogeneic transplantation in HIV-infected
patients may in part explain our slow accrual. However, these
preliminary results suggest that allogeneic transplantation after
nonmyeloablative conditioning represents a viable option for such
patients. Further improvements in both gene transfer technology and
allogeneic transplantation may allow application to patients with
progressive AIDS without an underlying hematologic malignancy.
| |
Footnotes |
Submitted June 28, 2001; accepted September 7, 2001.
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.
Reprints: John F. Tisdale, Bldg 10, Rm 9N116, 9000 Rockville
Pike, National Institutes of Health, Bethesda, MD 20892; e-mail:
johntis{at}intra.niddk.nih.gov.
| |
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Abstract
Introduction
