420

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FEBRUARY

1977

Successful bone marrow transplant for Fanconi's anaemia A J BARRETT, W D BRIGDEN, J R HOBBS, K HUGH-JONES, J G HUMBLE, S RETSAS, T R F ROGERS, S SELWYN, P SNEATH, J G WATSON

British Medical Journal, 1977, 1, 420-422

Summary A 15-year-old boy with Fanconi's anaemia, who required four units of blood each month, received a bone marrow graft from his 9-year-old brother, who was HLA identical and compatible on mixed lymphocyte reaction. Considerable immunosuppression was used and bacterial infection was prevented by vigorous decontamination in a Vickers-Trexler isolator. After the graft the patient's blood counts remained satisfactory for nine months, but it took six months before qualitative immune function was normal. Introduction There have been two reported cases of bone marrow transplantation in Fanconi's anaemia, one of which was successful.' 2 Confronted with the need for increasing transfusions in a boy with severe congenital hypoplastic anaemia, we attempted an elective bone marrow transplant. Methods The donor was initially selected after we had carried out a family study using a two-stage microcytotoxicity method.3 Selection was based on identity of HLA A and B loci. The C loci were not tested. Mixed lymphocyte reactions (MLR) were carried out in both directions in one-way culture using mitomycin-treated stimulating cells.3 A transformation index of less than 1-6 was regarded as acceptable. Lymphocyte response was assessed using phytohaemagglutinin4 and a pool of allogeneic cells from five donors.3 T and B cells were enumerated by sheep red blood cell rosette technique5 and direct immunofluorescence using a polyvalent anti-immunoglobulin.6 Immunosuppression and transplantation-Procarbazine, antithymocyte globulin (ATG), and cyclophosphamide7 were used for immunosuppression as shown in the figure. Bone marrow transplantation was carried out using a procedure previously described.8 Methotrexate was given intravenously as post-graft immunosuppression, 15 mg/M2 on the first day after the graft and 10 mg/M2 on the 3rd, 6th, 11th, 18th, 27th, and 33rd days.

Case report Fanconi's anaemia was diagnosed when the patient presented at the age of 9 years with pancytopenia, skeletal abnormalities, and skin Westminster Children's Hospital, London SWlP 2NS A J BARRETT, MRCPATH, research lecturer in haematology (now Leukaemia Research Fund fellow, H6pital St Louis, Paris 75010) W D BRIGDEN, MRCP, MRC clinical research fellow J R HOBBS, MD, FRCPATH, professor in chemical pathology K HUGH-JONES, MD, FRCP, consultant paediatrician J G HUMBLE, FRCP, FRCPATH, professor in haematology D C 0 JAMES, MD, FRCPATH, consultant in blood transfusion S RETSAS, MRCP, lecturer in oncology T R F ROGERS, LRCPI & LM, LRCSI & LM lecturer in bacteriology S SELWYN, MD, MRCPATH, reader in medical microbiology P SNEATH, MRCP, DCH, resident medical officer (now lecturer in paediatrics, Queen Elizabeth Hospital, Hackney Road, London EC2) J G WATSON, MRCP, DCH, lecturer in paediatrics

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pigmentation.9 Chromosomal breaks, gaps, and endoreduplications were detected on lymphocyte culture. A brother was found to be similarly affected. Another brother and one sister had normal blood findings and chromosomes. At the age of 11 the patient was treated with oxymethalone, but despite the addition of prednisolone his blood counts showed a slowly progressive decline in all elements. From the age of 14, when his haemoglobin fell to 6 5 g/dl, blood transfusions using red cells preserved in liquid nitrogen were given because of a continued fall in haemoglobin of 1-5 g/dl each week. Investigation showed a slightly decreased red cell half-life with increased uptake of chromium-labelled red cells by liver and spleen. The bone marrow was hypocellular with a particular reduction in the number of megakaryocytes. The direct Coombs test was negative, and neither mycoplasma complement-fixing antibodies nor cold agglutinins, which were detected at initial presentation, were found. During this time his platelet count fell to 35 x 109/1 (35 000/mm3) and he had spontaneous bruising. His polymorph count fell to about 0-2 x 109/l (200/mm3), and he developed two episodes of cellulitis from infected acne. Bone marrow transplantation was considered and his haematologically normal brother, who was aged 9, was found to be a compatible donor. He was prepared for an elective marrow transplant. Dental cavities were filled maxillary sinuses washed out and his acne treated with clindamycin and comedo extraction and Quinoderm (potassium hydroxyquinoline and benzoylperoxide). A week of intensive surface decontamination was performed to suppress the body microflora. This includes application of Naseptin nasal cream; aqueous chlorhexidine 0 020, sprays to the throat, ears, and foreskin; chlorhexidine dental gel and mouthwashes. The patient then entered a sterile Vickers-Trexler isolator.'0 Bowel decontamination was started. The antibiotics used were framycetin (500 mg four times a day), colistin sulphomethate (500 000 IU four times a day), and nystatin (1 million units four times a day). These drugs were continued throughout the isolation period of 72 days. After 17 days of isolation and decontamination only scanty Staphylococcus epidermidis and Streptococcus faecalis were cultured. Immunosuppression was started 10 days before transplantation. Only a single dose of ATG was used because of a severe local and systemic reaction. A bone marrow transplant of 6 x 108 cells/kg was given on the 28th day of isolation. For a week after the graft he had a pyrexia of unexplained origin. He was given ampicillin, gentamicin, flucytosine, and buffy coat transfusions from normal donors and from a patient with chronic granulocytic leukaemia. He also required whole blood and platelets. Blood products were irradiated to 1500 or 10 000 rads. Diarrhoea, vomiting, and severe dysphagia posed further problems. Fourteen days after the graft haemopoietic recovery started with a reticulocytosis followed by a rising leucocyte count. By the 17th day after the graft the neutrophil count was 2 x 109/l (2000/mm3). He was removed from the isolator tent 44 days after the graft, when the neutrophil count was 32 x 109/1 (3200/mm2). Platelets and blood transfusions were required until 28 days after the graft, but subsequently he maintained an almost normal blood count. A bone marrow examination 78 days after the graft showed hypercellularity of all

elements. The most serious problem after the transplant was continuing severe dysphagia, which produced considerable weight loss, but a barium swallow examination performed during isolation showed no oesophageal abnormality. A barium meal examination 50 days after the graft showed antral spasm and lesser curve ulceration. With a bland diet he became symptom-free and gained weight. Moderate haemorrhagic cystitis developed 40 days after the graft. This was managed with analgesics and fluids but recurred briefly 80 days after the giaft. He was kept in hospital until his lymphocyte function started to improve, and he was discharged home 89 days after transplantation. Three weeks later he was readmitted with severe trigeminal herpes zoster. The eye was not affected. Convalescent chickenpox plasma and anti-varicella human immunoglobulin were given, and each new lesion was treated for three days with 400' idoxuridine. Over the next 10

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Immunological regeneration

days he developed systemic chickenpox. Despite superinfection with Staphylococcus aureus, all these lesions healed, leaving some minimal scarring. During this time (five months after the graft) he developed orbital cellultis in association with a staphylococcal sinusitis. This responded to antral drainage and antibiotic treatment. At the time of writing his blood count had remained satisfactory for 11 months.

IgG Normal range:

Before graft After graft: 2 Weeks 6 Weeks 12 Weeks 18 Weeks 24 Weeks 28 Weeks

Results Haematological changes during transplantation are shown in fig 1. Haemoglobin F levels initially fell from pregraft values of 9O' but rose three months after transplantation to 35°,,. This level progressively fell over the next six months to 13",. This normal level was maintained. Immunological regeneration-The course of regeneration is shown in the table. The numbers of both T and B lymphocytes rose to our lower limit of normal about three months after the graft. But not until 17 weeks after the graft did the immunoglobulins reach normal levels, and the T-cell function, as assessed by MLR and the phytohaemagglutinin test, reached normal levels only 28 weeks after the graft (see table).

(mg/mi2)

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16 Redcell packs

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11 00

30 5-8 1*5 7-0 11-8 160

0-10 0-77 1-45 3-83

0-50 1 10 4 25 2-80 1 40 2-71

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1-42

! 11 900 1290 7400 1150 5240 29 539

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be considered. Only after frank discussion with the parents and the patient was the transplant planned. The successful haemopoietic restoration after bone marrow transplantation indicates that congenital hypoplastic anaemia of this type is probably a stem-cell defect susceptible to correction by transplantation of normal stem cells. There was, however, no direct evidence of engraftment. Both the patient and his brother, the donor, were identical at the HLA A and B loci. Red cell genotyping of 20 different antigens showed only one discrepancy: that the donor was P - and the recipient P-. We have not yet detected P- cells in the recipient. Chromosome studies failed to distinguish two populations of peripheral

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*IgA and IgM levels closely followed the pattern seen in IgG. tMLR is quoted as cpm of stimulated culture minus cpm of background. Phytohaemagglutinin response followed a similar pattern.

Discussion The decision to perform a bone marrow transplantation was not straightfoward. Although the prognosis of Fanconi's anaemia is poor, the condition in this patient was not immediately lifethreatening, and the risk of death due to the procedure had to

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lymphocytes. The restoration of fetal haemoglobin levels to normal strongly suggests, however, that considerable clones of normal erythrocyte precursors exist. MLR performed between donor and recipient 18 weeks after the graft showed no significant transformation, but the recipient's lymphocyte function was still considerably depressed at that time (see table). Although engraftment has probably occurred, there have been reports of haemopoietic reconstitution in aplastic anaemia after immunosuppression without engraftment.1' Immunosuppression was not started until the patient was effectively decontaminated. The Vickers-Trexler isolator and ready provision of blood products from leukaphoresis and the blood transfusion service were essential prerequisites to the procedure. We hoped that the use of frozen stored red cells would diminish the risk of HLA sensitisation from multiple transfusions. As a precaution against graft rejection, the extended procarbazine, ATG, and cyclophosphamide suppressive regimen was used because it may permit marrow engraftment in sensitised individuals where cyclophosphamide alone fails.12 The gastrointestinal and bladder complications due to this regimen were considerable, however, and contributed to the severe cachexia that occurred after the graft. Impaired resistance to Candida 21 weeks after the graft was a cellular defect. This may have been a consequence of the varicella infection. Staphylococcal killing, assessed one week later, was normal, but this difference might have reflected inadequate programming of the granulocytes by relatively incompetent T lymphocytes. The nitroblue tetrazolium test gave a normal result 23 weeks after the graft. Bone marrow grafting should certainly be considered in Fanconi's anaemia when patients show deterioration and are resistant to medical treatment. We should like to thank Sister J Meyers and her nurses, Westminster Children's Hospital; Dr I M Anderson for help and en-

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couragement; Lt-Col J G Winwick and W 0 I Bushrod of the Army Blood Supply Depot, Aldershot, for supplies of frozen red cells; Dr J Kersey for supplying ATG; Miss M O'Riordan, MRC cytogenics Unit, Edinburgh, for performing the chromosome studies; and Mr P C Trexler and his assistants at the Royal Veterinary College for help in running the isolator. We are grateful to the Andrew Bostic Fund, which paid most of the isolator costs and to the Anthony Nolan Fund, which completely supports the tissue typing laboratory. Without the joint assistance of these funds, this work would have been impossible. Miss Susan English typed the manuscript.

ADDENDUM-Since this report was written another two-way mixed lymphocyte reaction between the patient and the donor has been performed (11 months after the graft). There was no evidence that the patient had become sensitised to the donor. The T-lymphocyte function of the recipient has remained normal, as have his haematological values.

References

1Storb, R, et al, Blood,

1974, 44, 1. Dooren, L, Paper presented at IlIrd International Meeting of Experimental Haematologists, Houston, USA, 1974. (Abstracts p 50.) 3 Yamamura, M, Nikbin, B, and Hobbs, J R, 3ournal of Immunological Methods, 1976, 10, 367. 4Yamamura, M, Clinical and Experimental Immunology, 1973, 14, 457. 5 Dondal, M, Holm, G, and Wigzel, H, Jouirnal of Experimental Medicine, 1972, 136, 207. 6 McLaughlin, H, et al, British.journal of Haematology, 1973, 25, 7. 7 Storb, R, et al, Blood, 1974, 43, 157. 8 Humble, J G, and Barrett, A J, Proceedings of the Royal Society of Medicine, 1975, 68, 580. Beard, M E J, et al, Quarterly Journal of Medicine, 1973, 42, 403. ''Trexler, P C, Spiers, A S D, and Gaya, H, British Medical Journal, 1975, 4, 549. Speck, B, et al, Experimental Haematology, 1976, 4, 131. 12 Kersey, J, et al, Transplantation, 1975, 15, 92. 2

(Accepted 13 December 1976)

Why hypertensive patients vary in their response to oral debrisoquine J H SILAS, M S LENNARD, G T TUCKER, A J SMITH, S L MALCOLM, T R MARTEN British Medical Journal, 1977, 1, 422-425

Summary The relation between dose, systemic availability, and response to oral debrisoquine was studied in 13 hypertensive patients receiving no other treatment. In 11 who received the same daily dose (40 mg) the fall in mean standing systolic blood pressure varied between 0 3 and 44-4 mm Hg. There was a ninefold difference in the daily

Section of Therapeutics, Academic Division of Medicine, University of Sheffield, and Sheffield Hypertension Clinic, Royal Infirmary, Sheffield S6 3DA J H SILAS, MB, MRCP, research assistant M S LENNARD, MSC, research assistant G T TUCKER, BPHARM, PHD, lecturer A J SMITH, DM, FRCP, consultant physician and associate in medicine Department of Drug Metabolism, Roche Products Limited, Welwyn Garden City, Hertfordshire AL7 3AY S L MALCOLM, BSC, research biochemist T R MARTEN, BSC, DPHIL, senior research biochemist

urinary excretion and pre-dose plasma concentration of unchanged drug but an inverse correlation between daily urinary excretion of debrisoquine and its 4hydroxy metabolite (r= -0 86), suggesting that a low recovery of debrisoquine occurs because of extensive metabolism. There was a significant correlation between the fall in standing systolic blood pressure and the mean daily urinary excretion (r= +0-82) and pre-dose plasma concentration (r= +0 82) of unchanged debrisoquine. In contrast, there was a significant inverse correlation between the urinary recovery of the metabolite and the fall in blood pressure (r = -082). The availability of debrisoquine is the major determinant of response to this drug. In the absence of side effects a poor response may be an indication to increase the daily dose rather than add another hypotensive agent. Introduction Variation in response to antihypertensive agents may be due to differences in their availability and concentration at the site of action, in their uptake into this site, in receptor "sensitivity," or

Successful bone marrow transplant for Fanconi's anaemia.

420 BRITISH MEDICAL JOURNAL 12 FEBRUARY 1977 Successful bone marrow transplant for Fanconi's anaemia A J BARRETT, W D BRIGDEN, J R HOBBS, K HUGH...
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