J. M. Vossen

Allogeneic bone marrow transplantation for chronic myelomonocytic leukemia in childhood: a report from the European Working Group on Myelodysplastic Syndrome in Childhood.

PURPOSEnTo evaluate the role of allogeneic bone marrow transplantation (BMT) in children with chronic myelomonocytic leukemia (CMML).nnnPATIENTS AND METHODSnForty-three children with CMML given BMT and reported to the European Working Group on Myelodysplastic Syndrome in Childhood (EWOG-MDS) data base were evaluated. In 25 cases, the donor was a human leukocyte antigen (HLA)-identical or a one-antigen-disparate relative, in four cases a mismatched family donor, and in 14 a matched unrelated donor (MUD). Conditioning regimens consisted of total-body irradiation (TBI) and chemotherapy in 22 patients, whereas busulfan (Bu) with other cytotoxic drugs was used in the remaining patients.nnnRESULTSnSix of 43 patients (14%), five of whom received transplants from alternative donors, failed to engraft. There was a significant difference in the incidences of chronic graft-versus-host disease (GVHD) between children transplanted from compatible/one-antigen-mismatched relatives and from alternative donors (23% and 87%, respectively; P < .005). Probabilities of transplant-related mortality for children given BMT from HLA-identical/one-antigen-disparate relatives or from MUD/ mismatched relatives were 9% and 46%, respectively. The probability of relapse for the entire group was 58%, whereas the 5-year event-free survival (EFS) rate was 31%. The EFS rate for children given BMT from an HLA-identical sibling or one-antigen-disparate relative was 38%. In this latter group, patients who received Bu had a better EFS compared with those given TBI (62% v 11%, P < .01).nnnCONCLUSIONnChildren with CMML and an HLA-compatible relative should be transplanted as early as possible. Improvement of donor selection, GVHD prophylaxis, and supportive care are needed to ameliorate results of BMT from alternative donors.

Final height and hormonal function after bone marrow transplantation in children.

AIMnTo analyze final height and hormonal function in long-term survivors of bone marrow transplantation (BMT).nnnPATIENTSnGroup 1 consisted of 16 patients (10 boys) with a hematologic malignancy, mostly leukemia, conditioned for BMT with total body irradiation (TBI), 7.5 to 12 Gy, and cyclophosphamide. Group 2 consisted of 14 patients (9 boys) with severe aplastic anemia, conditioned with chemotherapy only.nnnRESULTSnIn group 1, patients achieved a reduced final height after BMT. The difference between the height standard deviation score (SDS) at BMT and the height SDS at final height was -1.96 (0.82) SDS in boys and -0.92 (0.71) SDS in girls (p = 0.0001, and p = 0.02 respectively). Final height was also lower than target height (boys, p = 0.01; girls, p = 0.03). Prepubertal growth in the first 3 years after BMT was normal but pubertal height gain was decreased. The patients in group 2 achieved normal height. Thyroid function and adrenal function were normal in all patients, and no growth hormone deficiency was detected. Serum follicle-stimulating hormone values after BMT were increased in all group 1 patients, with return to normal in two patients. Serum luteinizing hormone values were increased in all group 1 girls, with recovery in one girl. Normal serum luteinizing hormone values and spontaneous puberty were found in all group 1 boys. In group 2, disturbances in gonadotropins were seen only in three boys and two girls.nnnCONCLUSIONnIn patients treated in childhood with BMT after chemotherapy and TBI with 7.5 Gy or more, final height is compromised because of blunted growth in puberty. Patients who had not received TBI suffered no height loss. In the majority of patients, the combination of chemotherapy and TBI also resulted in irreversible disturbances of gonadal function.

Pubertal development and growth after total-body irradiation and bone marrow transplantation for haematological malignancies

Abstract Pubertal development after total-body irradiation (TBI) was investigated in 40 children (21 boys) treated with allogeneic bone marrow transplantation (BMT) for haematological malignancies at a mean age of 11.3 years. The mean age at the last visit was 19.0 years. Twenty-five patients (15 boys) were prepubertal at BMT. Data on secondary sexual characteristics, the pituitary-gonadal axis and longitudinal growth were retrospectively collected from the medical records. In boys not receiving additional testicular irradiation (nu2009=u200919), penile growth and pubic hair development was normal and all had serum testosterone levels within the adult range. The majority of them, however, had incidental elevations of LH, suggesting minor Leydig cell damage. Testicular volume at last measurement was small (mean: 10.5u2009ml) and serum FSH levels were elevated in all boys, with normalisation in only one, suggesting severe impairment of reproductive gonadal function. Of the ten girls who received BMT before puberty, six had a spontaneous onset of puberty and menarche; the four other girls needed hormonal substitution therapy. Recovery of gonadal function after cessation of substitution was seen in one girl, who became pregnant but had a spontaneous abortion. Decrease in height SDS was seen in the majority of patients and was positively correlated with male gender and lower age at the time of BMT.nConclusion Careful monitoring of both gonadal function and growth after bone marrow transplantation and total body irradiation is warranted in order to detect disturbances early and ensure normal pubertal development in children treated for haematological malignancies.

Disturbances of growth and endocrine function after busulphan-based conditioning for haematopoietic stem cell transplantation during infancy and childhood

Summary:It is generally assumed that busulphan/cyclophoshamide (Bu/Cy)-based conditioning regimens for haematopoietic stem cell transplantation (SCT) do not affect growth. We evaluated growth and endocrine function after Bu/Cy-based conditioning in 64 children without a history of irradiation. Mean height standard deviation scores remained stable, but unexplained disturbances of growth after SCT were found in 17/48 (35%) of the children without growth-limiting disorders (10/23 in patients treated for haematological malignancies). In 10 patients, growth hormone (GH) secretion status was evaluated, and insufficient GH secretion was diagnosed in four patients. Thyroid function was evaluable in 52 patients. Two developed antibody-mediated thyroid disorders and 10 (19%) compensated primary hypothyroidism. Gonadal function was evaluable in 21 patients and was normal in all seven patients treated with low-dose Bu (8u2009mg/kg), whereas seven of the 14 children receiving high-dose Bu (16–20u2009mg/kg) developed gonadal failure; the majority of these patients had not been exposed to gonadotoxic therapy prior to Bu/Cy. Of the 49 evaluable patients, 16 developed subclinical hyperparathyroidism. We conclude that, besides gonadal and thyroid dysfunction, impaired growth and hyperparathyroidism often occur after Bu/Cy conditioning for SCT and that growth impairment may be the result of insufficient GH secretion.

Detection of mixed chimaerism in flow-sorted cell subpopulations by PCR-amplified VNTR markers after allogeneic bone marrow transplantation.

Summary The amplification of Variable Number of Tandem Repeats (VNTR) by the polymerase chain reaction (PCR) was used to determine the extent of chimaerism in flow sorted lymphoid and myeloid cell populations following allogeneic bone marrow transplantation (BMT). Pre‐BMT screening with a set of five VNTR revealed that at least one marker was maximally informative in 95% of donor‐recipient pairs. Mixing reconstruction experiments indicated that detection of 1–5% of the minor cell population in a sample of 5 ± 103 nucleated cells is feasible. Flow sorted post‐transplant peripheral blood B‐ and T‐lymphocyte, natural killer and monocyte cell populations were subjected to PCR‐VNTR marker analysis. It was shown that this procedure can be used for the early detection of engraftment and the identification of mixed chimaerism in various haematopoietic cell lineages in patients with leukaemia or severe combined immune deficiency, treated with allogeneic BMT.

Reconstitution of lymphocyte subpopulations after paediatric bone marrow transplantation.

We prospectively studied the reconstitution of lymphocyte subpopulations in a group of 22 children, who survived disease-free at least 6 months after allogeneic BMT for a haematological malignancy. Absolute counts of total lymphocytes, B lymphocytes, T lymphocytes, and CD4+ helper T lymphocytes reached the 5th percentile (p5) of age-matched reference values within 6 months after BMT in 15, 17, 7 and 2 patients, respectively. In particular, CD4+ helper T lymphocyte reconstitution was very slow. Unexpectedly, CMV reactivation had a profound positive influence upon the number of CD4+ helper T lymphocytes in the children. In five patients, absolute B lymphocyte counts above the 95th percentile were reached from 6 months after BMT onwards, mimicking normal ontogeny. Unlike normal ontogeny, the percentages of helper T lymphocytes expressing the ‘naive’ CD45RA isoform were low and those expressing the ‘memory’ CD45RO isoform were high in the first 3 months after BMT, as described before. Thereafter, the CD45RA:CD45RO ratio slowly normalised. Also, CD7 expression was absent on up to 90% of T lymphocytes in the first months after BMT, and on a steadily decreasing percentage thereafter, as recently described in adults. However, the absolute counts of CD45RO+/CD4+ and CD7−/CD4+ helper T lymphocytes did not change significantly. So, we found no evidence of peripheral expansion of previously primed donor-derived ‘memory’ T lymphocytes during the follow-up period which spanned 1–18 months after BMT. The absolute counts of ‘naive’ CD45RA+ helper T lymphocytes did not show a faster increase after BMT than in adults, despite the presumed presence of a non-involuted thymus in children. Bone Marrow Transplantation (2000) 25, 267–275.

Preemptive alloimmune intervention in high-risk pediatric acute lymphoblastic leukemia patients guided by minimal residual disease level before stem cell transplantation

Relapse of pediatric acute lymphoblastic leukemia (ALL) remains the main cause of treatment failure after allogeneic stem cell transplantation (alloSCT). A high level of minimal residual disease (MRD) before alloSCT has been shown to predict these relapses. Patients at risk might benefit from a preemptive alloimmune intervention. In this first prospective, MRD-guided intervention study, 48 patients were stratified according to pre-SCT MRD level. Eighteen children with MRD level ⩾1 × 10−4 were eligible for intervention, consisting of early cyclosporine A tapering followed by consecutive, incremental donor lymphocyte infusions (n=1–4). The intervention was associated with graft versus host disease ⩾grade II in only 23% of patients. Event-free survival in the intervention group was 19%. However, in contrast with the usual early recurrence of leukemia, relapses were delayed up to 3 years after SCT. In addition, several relapses presented at unusual extramedullary sites suggesting that the immune intervention may have altered the pattern of leukemia recurrence. In 8 out of 11 evaluable patients, relapse was preceded by MRD recurrence (median 9 weeks, range 0–30). We conclude that in children with high-risk ALL, immunotherapy-based regimens after SCT are feasible and may need to be further intensified to achieve total eradication of residual leukemic cells.

Simultaneous detection of X and Y chromosomes by two-colour fluorescence in situ hybridization in combination with immunophenotyping of single cells to document chimaerism after sex-mismatched bone marrow transplantation

A powerful approach to documenting engraftment after allogeneic BMT is the quantification of the degree of chimaerism in distinct haematopoietic cell lineages. This cannot be achieved by the recently developed, quantitative, modifications of PCR amplification of highly polymorphic DNA markers, unless this technique is applied to separated cell populations. Here, we report the development of a new method, in which cells are simultaneously characterized by enzymatic immuno- phenotyping and identified for their origin by two- colour fluorescence in situ hybridization with X and Y chromosome-specific DNA probes (XY-FISH/immunostaining). The method enables the rapid, reliable and quantitative analysis of chimaerism within distinct cell lineages after sex-mismatched BMT, without the requirement for cell separation techniques. This is illustrated by investigation of the pattern of chimaerism in patients receiving a sex-mismatched BMT for the treatment of primary immunodeficiencies. The results obtained with the quantitative XY-FISH/immuno staining method show a good correlation with the data generated by the semi-quantitative analysis of PCR amplified minisatellites in FACS-sorted cell fractions. In addition, XY-FISH/immunostaining was successfully applied to detect materno-fetal engraftment of T cells in a SCID patient.

Early reconstitution of haematopoiesis after allogeneic bone marrow transplantation: a prospective histopathological study of bone marrow biopsy specimens.

To study early haematopoietic reconstitution after bone marrow transplantation bone marrow biopsy specimens taken in the third week after transplantation were evaluated. Cellularity was highly variable; localisation of the various cell lineages and the ratios of myeloid cells to erythroid cells were abnormal. Clustering of cells of the same lineage in the same stage of maturation was prominent. The bone marrow stroma showed many anomalies, including increased fibre content, periodic acid Schiff positivity of fat cells, oedema, sinus ectasia and granulomas. A comparison of biopsy findings with clinical and laboratory data showed a correlation between the amount of erythroid cells and the day of appearance of reticulocytes, as well as the number of reticulocytes. Absence of clustering of haematopoietic cells in four of five patients was associated with either failure of engraftment or early leukaemic relapse. Variables such as infections and administration of possibly myelosuppressive drugs did not influence bone marrow biopsy findings.

Dot-immunobinding assay as an accurate and versatile technique for the quantification of human IgG subclasses

A dot-immunobinding assay on nitrocellulose membranes has been developed for the quantification of human IgG subclasses using subclass-specific monoclonal antibodies. The advantages of this technique can be summarized as follows: (1) possibility of rapid semi-quantitative evaluation and/or precise quantitation from the same dot-pattern; (2) simple procedure with very good reproducibility; (3) sensitivity for nanogram concentrations of individual subclasses, therefore applicable not only to serum but also to other body fluids with a low content of IgG; (4) very small amounts of test material needed; (5) very good correlation of results with other techniques (ELISA, radial immunodiffusion) but without some of the inherent problems of the latter methods.