www.nature.com/bmt. Prenatal HLA-matching to determine suitability for allogeneic bone marrow transplantation. H van den Berg1, M Verjaal2, K Boer3 and NM ...
Bone Marrow Transplantation (2000) 25, 579–581 2000 Macmillan Publishers Ltd All rights reserved 0268–3369/00 $15.00 www.nature.com/bmt
Prenatal HLA-matching to determine suitability for allogeneic bone marrow transplantation H van den Berg1, M Verjaal2, K Boer3 and NM Lardy4 1 Emma Children Hospital, Department of Paediatric Oncology, Academic Medical Center, University of Amsterdam; 2Department of Clinical Genetics, Academic Medical Center, University of Amsterdam; 3Department of Obstetrics and Gynaecology, Academic Medical Center, University of Amsterdam; and 4HLA Laboratory, Central Laboratory of the Blood Transfusion Service of the Dutch Red Cross, Amsterdam, The Netherlands
Summary: For several haematological malignancies, allogeneic stem cell transplantation is the treatment of choice. In most cases an HLA-identical sibling is required. If the mother of a patient is pregnant, cord blood from a related donor, which can be used for stem cell transplantation, might be obtainable in the near future. For the patient, knowledge of the foetal HLA-type can be important since it might influence choice of treatment and timing of transplantation. If the foetus is HLA compatible, as would be the situation in 25% of cases, the delivery has to be arranged in such a way that cord stem cells can be collected. As a result, in the other 75% of cases (spontaneous) delivery can take place in the home/local setting. Here we report four cases in which amniocentesis was performed and HLA-typing influenced treatment of the patient and delivery of the sibling. Bone Marrow Transplantation (2000) 25, 579–581. Keywords: stem cell transplantation; HLA-typing; amniocentesis; delivery; foetus
For some years stem cell transplantations have been performed using cells collected from cord blood.1–4 Recently, we have had four cases in which prenatal determination of HLA-type was an important factor in the decision making process of both treatment of a child with leukaemia and scheduling time and place of delivery of a sibling of the patient. Materials and methods Amniocyte cultures These were done according to standard procedures. Briefly, four amniocyte cultures were started from amniotic fluid samples, using different Chang D (Irvine Scientific, Santa Ana, CA, USA), Chang A + supplement (Irvine Scientific), Amniomax C100 + supplement (Life Technologies, Pais-
Correspondence: Dr H van den Berg, Emma Children Hospital AMC, PO Box 22700, 1100 DE Amsterdam, The Netherlands Received 16 August 1999; accepted 13 November 1999
ley, UK) and Amnioma Chrome II + supplement (Biowhittaker, Walkersville, MD, USA). After trypsinization new cultures were started, until four 75-cm2 monolayers were obtained. Cells were harvested and transported in a physiologic saline solution for HLA typing. HLA class I and class II PCR-SSP analysis HLA PCR-SSP genotyping was carried out by means of the commercially available Dynal PCR-SSP typing kits (Dynal AS, Oslo, Norway), according to the manufacturer’s instructions. Briefly, 100 ng genomic DNA was prepared using the Qiagen DNA isolation kit (Qiagen, Hilden, Germany) and amplified in a 10 l PCR mixture containing 0.25 m allele- and group-specific primers, 500 mm KCl, 15 mm MgCl2, 100 mm Tris-HCl pH 8.3, 0.01% w/v gelatin, 10 mm of each of the four deoxyribonucleotide phosphates and 0.375 units of Taq polymerase (Promega, Madison, WI, USA). DNA amplification consisted of one cycle for 2 min at 94°C followed by 10 cycles each for 10 s at 94°C, 60 s at 65°C and 20 cycles each for 10 s at 94°C, 50 s at 61°C and 30 s at 72°C. Following PCR amplification and addition of loading dye, 10 l of the contents of each well were electrophoresed and visualised on a 2% agarose gel. The resultant gel was photographed for documentation. Lanes identified with specific primer amplification were analysed and the HLA class I and/or class II allele specificities were determined.5,6 Patients Case 1: A diagnosis of a myelodysplastic syndrome was made in a 2-year-old child with trisomy 21 (FAB-type refractory anaemia with excess of blasts was based on 19% blasts in the bone marrow and 48,XX,+21c,+8 [2/28]). The child had no HLA-identical siblings and a matched unrelated bone marrow transplant was considered. The child was treated with Ara-C, daunorubicin and etoposide. Bone marrow morphology and cytogenetics (karyotyping and FISH) showed complete remission afterwards. During the following intensification course with Ara-C and etoposide cerebritis, typhlitis and endophthalmitis occurred. Total recovery ensued, although blindness persisted for half a year. During treatment the mother became pregnant. Amniocentesis was performed at 15 weeks. Primary indication for this amniocentesis was, however, exclusion of
Prenatal HLA-typing prior to cord blood harvesting H van den Berg et al
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Down’s syndrome. The foetal karyotype was 46,XX and the HLA-type, diagnosed from cultured amniocytes, was incompatible. Spontaneous delivery at term in the home setting (100 miles from our centre) took place. Cord blood cells were not collected. No congenital anomalies were found. In view of the severe complications after standard chemotherapy we had decided that stem cell transplantation was only appropriate for this patient if the myelodysplastic syndrome evolved into a leukaemia. If the sibling had been HLA-identical, stem cells collected from the cord blood at delivery would have replaced a bone marrow harvest in the event of a leukaemia developing. Case 2: A diagnosis of acute megakaryoblastic leukaemia (FAB-M7) was made in a previously healthy 1-year-old girl. Treatment with a protocol for acute non-lymphoblastic leukaemia was given. Five months after cessation of treatment relapse occurred. Two courses of ADE (MRCAML12) were given. Since the mother was pregnant when the relapse was diagnosed, amniocentesis followed by culturing of amniocytes and subsequent DNA analysis was performed. The foetus appeared not to be HLA-compatible. In view of the transplant-related complication and mortality rate in matched unrelated transplants and the very high rate of second relapses in FAB-M7 leukaemia which relapses early, the parents decided not to proceed to a matched unrelated or HLA-haplo-identical transplant. Mild palliative chemotherapy was given. Six months later a healthy girl was born at home. Stem cells were not collected. The patient died 3 months afterwards. Case 3: Acute lymphoblastic leukaemia (ALL) with a rearranged mll-gene was diagnosed in a 2-year-old boy. Treatment according to a high risk ALL protocol was started. If a suitable HLA-identical donor had been available stem cell transplantation would have been planned. Unfortunately the child had no siblings. During treatment the mother became pregnant. Amniocentesis followed by culturing of fibroblasts and DNA analysis revealed that the foetus was not HLA-compatible. Treatment continued according to the initial chemotherapy regimen for high risk ALL. Delivery at home is planned. Case 4: Philadelphia-chromosome positive acute lymphoblastic leukaemia (Ph+ALL) was diagnosed in a 1-yearold girl. Treatment with a high risk ALL protocol was started. A suitable matched unrelated donor was available. During the initial treatment the mother became pregnant. Amniocentesis followed by culturing amniotic fluid cells and subsequent DNA analysis revealed that the foetus was not HLA-compatible. After the intensification courses maintenance treatment was not given and the patient was transplanted with bone marrow from the matched unrelated donor. If the foetus had been HLA-compatible a stem cell transplant with cells harvested from cord blood would have been performed after delivery. Delivery is now planned in a local hospital.
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Discussion Our cases indicate that prenatal determination of HLAtypes may be of importance in determining whether stem cells should be collected from the cord blood or not. Since 25% of foetuses are HLA-compatible with their siblings, delivery has to be planned in a medical centre where stem cells can be processed in only 25% of cases. The time of delivery has to be scheduled at a time convenient for the stem cell laboratory. In unexpected, very rapid deliveries, especially after previous pregnancies, there is a serious risk of missing the graft. In all our cases there were arguments for performing amniocentesis, but even more important is the fact that in three out of four cases the results of the HLA-typing had major importance for the patient in relation to the choice of treatment. Another argument is that when the sibling is compatible, an earlier time of grafting may be possible and this might be beneficial with respect to outcome. The scheduling of transplantation at an earlier time is due to the fact that cord blood cells are available at least 6 months before conventional bone marrow harvesting can be performed. All pregnancies in our series were started to fulfil the parents wish for another child; however all the parents were disappointed to know that the new-born was not HLAidentical. In this report, the benefits of prenatal HLA-typing varied from case to case. In case 1 the direct benefit was the possibility of a normal, spontaneous delivery in the home setting. Since the primary indication for amniocentesis was exclusion of Down’s syndrome, knowledge of the HLAtyping was merely coincidental and did not influence treatment. In case 2, the parents wanted to weigh the risks of the various transplantation types. Early after initiating relapse treatment, knowing the incompatibility, the high risk of relapse related to this type and stage of the leukaemia and the relatively high complication rate using bone marrow or cord blood originating from a matched unrelated donor, the parents themselves decided to adopt a palliative form of treatment. In case 3 the situation is more complex. The prognosis of infants under the age of 6 months suffering from ALL with a rearranged mll-gene is poor. However for cases above the age of 1 year no clear data exist, but the prognosis might be relatively good.7 This was the reason we decided, in the event of HLA-incompatibility, not to proceed to a graft from a matched unrelated donor. However, if the foetus had been HLA-compatible, a transplant would have been considered. In case 4 the benefit is most obvious, since a transplant for this high risk ALL was performed prior to delivery of the new-born. Although the risk related to amniocentesis is very low (up to 2%), amniocentesis can interfere with well being of the foetus.8–10 This ethical problem should be discussed with both parents. Thus, we consider that amniocentesis and subsequent HLAtyping should only be performed when results influence treatment and a better ultimate outcome for the recipient of the stem cells is anticipated.
Prenatal HLA-typing prior to cord blood harvesting H van den Berg et al
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