# Successful hematopoietic stem cell transplantation for Fanconi anemia from an unaffected HLA-genotype–identical sibling selected using preimplantation genetic diagnosis

Satkiran S. Grewal, Jeffrey P. Kahn, Margaret L. MacMillan, Norma K. C. Ramsay and John E. Wagner

## Abstract

The only proven cure for Fanconi anemia (FA)-associated bone marrow failure is successful allogeneic hematopoietic stem cell transplantation (HSCT). However, HSCT with donors other than HLA-identical siblings is associated with high morbidity and poor survival. Therefore, we used preimplantation genetic diagnosis (PGD) to select an embryo produced by in vitro fertilization (IVF) that was unaffected by FA and was HLA-identical to the proband. The patient was a 6-year-old girl with FA and myelodysplasia previously treated with oxymetholone and prednisone. After her parents underwent 5 cycles of IVF with intrauterine transfer of 7 embryos over a span of 4 years, successful pregnancy ensued. Twenty-eight days after delivery, the patient underwent transplantation with her newborn sibling donor's HLA-identical umbilical cord blood hematopoietic stem cells (HSCs). Neutrophil recovery occurred on day 17 without subsequent acute or chronic graft-versus-host disease. Currently, 2.5 years after transplantation, the patient is well and hematopoiesis is normal. In summary, we have described the first successful transplantation, using IVF and PGD, of HSCs from a donor selected on the basis of specific, desirable disease and HLA characteristics. The medical, legal, and ethical issues involved with this approach are discussed. (Blood. 2004;103:1147-1151)

## Introduction

Fanconi anemia (FA), an autosomal recessive disease, is characterized by varied congenital physical anomalies, progressive bone marrow failure, and increased predisposition for acute leukemia and other cancers.1-4 The only proven long-term cure of the bone marrow manifestations is successful allogeneic hematopoietic stem cell transplantation (HSCT). HSCT in FA is associated with a particularly high risk for transplantation-related events, including graft failure, graft-versus-host disease (GVHD) and opportunistic infections.1,5-8 Best results have been achieved with HLA-genotype-identical sibling donors.1,7,8 However, most FA patients do not have unaffected HLA-identical sibling donors. HSCT using non-genotype-identical donors is associated with increased cost, transplantation-related events, and poor survival.1,5-7,9,10

Preimplantation genetic diagnosis (PGD) was developed to help couples at high risk for transmitting genetic disease to accurately identify unaffected embryos before implantation and thereby eliminate the potential need for termination.11,12 However, the technology also allows for positive selection of other genetic traits, such as specific HLA haplotypes.

We present the case of a 6-year-old girl with FA-associated bone marrow failure and myelodysplasia who did not have an HLA-identical related donor. Using PGD and in vitro fertilization (IVF) techniques, embryos HLA-identical to the patient and unaffected by FA were selected for intrauterine transfer. In the fifth clinical cycle, a single preselected embryo was transferred, and pregnancy established. The umbilical cord blood (UCB) of the healthy infant was harvested at birth and used as the source of HLA-identical hematopoietic stem cells (HSCs) to reconstitute normal hematopoiesis in his affected sister.

## Patient, materials, and methods

### History

A 3-year-old girl was brought to the University of Minnesota for evaluation of bone marrow failure associated with FA. At birth it was noted that she had multiple congenital malformations, including bilateral radial ray anomalies with absent thumbs, bilateral congenital hip dislocation, and deafness in the left ear. The diagnosis of FA was established based on excessive chromosomal breakage in lymphocytes cultured with diepoxybutane (DEB).13 When she was 2, pancytopenia was first observed (Figure 1); her absolute neutrophil count (ANC) was 0.81 × 109/L, and her platelet count was 104 × 109/L. Therapies included trials of oxymetholone, oral steroids, and a short course of erythropoietin. Subsequently, though the hemoglobin values remained stable (10-12 g/dL), the platelet count persisted typically below 50 × 109/L, and the ANC persisted below 0.5 × 109/L. Bone marrow examination when she was 4 showed features of myelodysplasia (MDS) without an excess of blasts.

Figure 1.

Progression of hematologic disease in the patient (bottom) and time course of 5 PGD/IVF attempts by her parents (top).

### Selection of the donor

Both parents are heterozygotes, and the patient is homozygous for the IVS 4 + 4 A>T mutation. Standard IVF techniques (Colorado Center for Reproductive Medicine, Denver, CO) were combined with PGD (Reproductive Genetics Institute, Chicago, IL).14 Embryo genotyping was performed by obtaining single blastomere biopsy samples of day 3 cleaving embryos using micromanipulation techniques as detailed elsewhere.14 In brief, single-cell polymerase chain reaction (PCR)-amplified DNA was analyzed for IVS 4 + 4 A>T using polyacrylamide gel analysis of ScaI restriction-enzyme-digested product, distinguishing the mutant allele (131-base pair [bp] band) from the wild-type allele (108-bp band) and for HLA type using previously described nested PCR techniques15,16 for allele-level identification of the HLA-A and -B subtype of each embryo.14

Embryos HLA identical to the patient and unaffected by FA (homozygous wild-type allele or heterozygous mutation) were selected for transfer. In the fifth clinical cycle, a single preselected embryo was transferred, and full-term pregnancy resulted (Table 1). Confirmatory genetic testing was performed using chorionic villus sampling (CVS) in the first trimester for FA mutation and HLA type. The UCB was harvested from the placenta after the birth of the child in Denver, Colorado and was delivered to the University of Minnesota, where it was cryopreserved.17 An aliquot was obtained for testing before the UCB unit was frozen (Table 2).

Table 1.

Summary of the 5 PGD/IVF cycles

Table 2.

Characteristics of the sibling umbilical cord blood

### Evaluation for HSCT

The diagnosis of FA was reconfirmed at our institution using DEB and mitomycin C testing of peripheral blood lymphocytes.13 Routine laboratory tests revealed serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) values of 228 U/L and 60 U/L, respectively (normal ranges, 0-35 U/L and 0-50 U/L, respectively). Serum bilirubin and alkaline phosphatase values were in the normal ranges. Cardiac function assessment using 2-dimensional echocardiography with M-mode and glomerular filtration rate was normal. Bone marrow examination revealed 60% cellularity with dysgranulopoiesis and dyserythropoiesis. No excess of blasts was seen. Cytogenetic evaluation revealed 46,XX,der(1)t(1;3) (p36.1;q21),der(17)t(1;17)(q23;p11.2), which was detected in all 20 metaphase cells analyzed.

### Conditioning regimen and HSCT

The University of Minnesota Institutional Review Board reviewed and approved the protocol and the informed consent documents. The patient was admitted to a high-efficiency particulate air (HEPA)-filtered room in the Pediatric Blood and Marrow Transplantation unit. Because she also had MDS, conditioning therapy consisted of cyclophosphamide and total body irradiation. Cyclophosphamide 10 mg/kg per day was given intravenously over 1 to 2 hours on days -6 to -3 (total dose, 40 mg/kg). Mesna 10 mg/kg was administered intravenously on the days of cyclophosphamide infusion. Intravenous hyperhydration was maintained during the administration and for 24 hours after the administration of the last dose of cyclophosphamide. Total body irradiation of 450 cGy in a single fraction was administered using a linear accelerator on day -1, at a dose of 26 cGy/min. It was prescribed to the midplane of the patient, at the level of the umbilicus, and was delivered with right and left lateral fields.

Cyclosporine A was given as GVHD prophylaxis. Peripheral blood hemoglobin and platelet values were maintained above 8 gm/dL and 10 × 109/L, respectively. Leukocytes were filtered from all blood products, and the blood products were then irradiated with 2500 cGy before infusion. Oral itraconazole (antifungal prophylaxis) 3 mg/kg daily was prescribed for the month preceding transplantation, and intravenous cefazolin was given for streptococcal prophylaxis until the neutrophil count exceeded 0.5 × 109/L. Intravenous and oral acyclovir and oral cotrimoxazole were given to prevent herpes simplex virus and Pneumocystis carinii infection, respectively. Parenteral nutrition was provided for the duration of anorexia.

On the day of transplantation, the sibling donor cord blood was infused through a central venous catheter after standard processing.17 In brief, the cryopreserved UCB unit was identified, retrieved, placed in a sterile zip-locked bag, and sealed. The bag was inserted in a 37°C bath; thawing was accelerated by moving the product in the bath and by gentle kneading. The liquefied product was washed (with 10% dextran 40 and 5% albumin) and resuspended in 10% dextran with 5% albumin before infusion.

### Engraftment

Myeloid engraftment was defined as an ANC of 0.5 × 109/L or higher on the first of 3 consecutive days. Platelet engraftment was defined as an untransfused platelet count of 50 × 109/L or higher on the first of 7 consecutive days. Hematopoietic chimerism was estimated on bone-marrow-derived DNA by quantitative PCR analysis of an informative variable number of tandem-repeat regions.18

## Results

### Engraftment and immune reconstitution

Neutrophil and platelet engraftment occurred at days 17 and 30, respectively (Figure 2), with more than 90% donor chimerism at all time points and 100% donor chimerism at 1- and 2-year studies (Figure 3). The last blood product transfusion occurred 26 days after HSC infusion. Assessment at 24 months after HSCT showed an ANC of 3.1 × 109/L, hemoglobin of 14.8 gm/dL, and platelet count of 304 × 109/L; bone marrow evaluation showed 70% cellularity with normal trilineage hematopoiesis and no evidence of myelodysplasia. Serum immunoglobulin levels were in the normal range 6 months after transplantation.

Figure 2.

Recovery of neutrophil and platelet counts after HSCT.

Figure 3.

Assessment of donor chimerism. Electrophoretic profiles of informative variable number of tandem repeat PCR products18 of the recipient and donor (before transplant) and serial posttransplant samples from the recipient bone marrow at indicated time points after HSCT. The donor and recipient are both heterozygous with one shared allele. Thus, one informative donor allele (644 bp) and one informative recipient allele (578 bp) are observed.

### Transplantation-related events

Acute or chronic GVHD did not develop in the patient. Adenovirus-associated gastroenteritis, which developed 1 month after HSC infusion, was clinically mild and resolved by 3 months after HSCT. No opportunistic infections were documented after recovery from adenovirus infection. Asymptomatic hypertransaminasemia, first detected 3 months before transplantation, persisted after HSCT, with serum ALT levels typically between 100 and 200 U/L (range, 20-385 U/L). The etiology has been unclear. No infectious agent was identified. Liver biopsy 1 year after HSCT showed a mild inflammatory pattern consistent with drug-related toxicity. At last follow-up (24 months after HSCT), all medications had been discontinued, the serum ALT level was 141 U/L, the total serum bilirubin level was 0.3 mg/dL, and the serum alkaline phosphatase level was normal.

### Parental motivation

Finally, the motives of parents who engage in the creation of an HLA-compatible donor raise important ethical issues. Will parents conceive a child with the intention of raising him as a loved and cherished member of the family, or will they be motivated merely by the prospect of creating a life-saving hematopoietic stem cell donor for their sick child? Although it is unethical to use a person as a means to an end, either for parents or for a sick sibling, policy protections are insufficient to prevent parents from placing the donor child up for adoption after birth and after collection of the UCB.

Even when parents love and cherish the donor child, there are concerns regarding the level of risk potentially placed on the donor. For example, if the UCB transplant fails, parents may be faced with a decision about a bone marrow harvest from the infant in the first months of life, exposing the child to procedure-associated risks. At what point would the risk to the donor child be ethically unacceptable, and who should decide? Parents are conflicted in that they must consider the interests of the donor child and the recipient child. A final concern is that some couples may use PGD to select a disease-free or an HLA-compatible embryo with the intent to harvest tissue only and not to bring another child into the world. This scenario would, for example, entail an induced abortion at some point during gestation and the collection of HSCs from the fetal liver. Although such directed donation of tissue from an induced abortion would violate federal law,24 some couples have already inquired about this possibility. Clinics performing PGD and physicians working with patients attempting to create a stem cell donor must be aware of this possibility.

Based on discussions of the issues outlined, the first attempts to use PGD to produce an unaffected HLA-identical donor were limited to 3 highly motivated couples after considerable individualized counseling. Each of these couples had an affected child who could benefit from HSCT, and they had a heritable disorder that could be diagnosed using PGD. In addition, all 3 couples independently desired additional healthy children. After 5 unsuccessful attempts, the first couple (β-thalassemia) continues to pursue IVF with PGD. After 9 unsuccessful attempts, the second couple (FA) withdrew and proceeded to HSCT with an unrelated donor graft. The third couple represents the index case.

With the success and publicity surrounding the index case, there have been a large number of requests regarding this procedure. Over time a number of other issues have surfaced, including the potential use of surrogate (family) gamete donors, collection of fetal liver as a source of hematopoietic stem cells in the event of a spontaneous abortion, and requests for gender selection in addition to HLA. Requests have also been received for selection and storage of HLA-identical embryos for potential future use as a source of embryonic stem cells. Some parents of children with newly diagnosed leukemia have asked to use this technology to have an HLA-identical sibling donor available in the possible event of disease relapse, when the time may be too short to initiate this process.

Clearly, these are not the only issues to consider. For example, what is the fate of the unused embryos? As demonstrated in this case (Table 1), the use of IVF plus PGD to create a stem cell donor leads to the creation of many excess embryos. When IVF is used for infertility, small numbers of embryos are often left and frozen for later disposition. However, to have sufficient probability of success in finding an embryo that is disease free and HLA matched, many more embryos must be made and tested, adding to the estimated 400 000+ embryos frozen in laboratories in the United States alone.25

These are among the ethical issues posing challenges for the wider application of the methodology described. Although the true range of applications of this technology is yet to be fully realized, it is clear that (1) there is significant interest in using this technology by couples who might consider HSCT for a child in the family, (2) physicians must be aware that such technology exists and that families who might have benefited from the technology have been involved in lawsuits against their physicians and medical institutions for not counseling them about it,26,27 (3) HSCT physicians must be intimately involved from the outset because the patient's condition may require HSCT before the birth of an HLA-identical sibling, and (4) significant ethical, legal, and policy issues must be addressed, from the clinic and institution levels to the government and society levels.

## Footnotes

• Reprints:
John E. Wagner, MMC 366, University of Minnesota, 420 Delaware St SE, Minneapolis, MN 55455; e-mail: wagne002{at}umn.edu.
• Prepublished online as Blood First Edition Paper, September 22, 2003; DOI 10.1182/blood-2003-02-0587.

• Supported by the Fanconi Anemia Research Fund and the Children's Cancer Research Fund.