Before Bone Marrow Transplantation Research Articles

Determination of Relationship between Infection and Serum Levels of Prohepcidin in Pediatric Patients before and after Bone Marrow Transplantation

Since the immune system is suppressed before bone marrow transplantation (BMT), severe infections might arise. This clinical article considers the role of prohepcidin, in addition to interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in infections occurring after BMT. Serum prohepcidin, IL-6, TNF-α, and C-reactive protein (CRP) levels were measured in 10 pediatric BMT patients by enzyme-linked immunosorbent assay before and after BMT. The results were evaluated statistically. As a result of the analysis, it was observed that there was a significant correlation between serum prohepcidin and IL-6, TNF-α, and CRP levels in the pretransplantation, posttransplantation, and discharge periods of the patients. The relationship between CRP levels and prohepcidin levels suggests that the serum prohepcidin level might be an important parameter for transplantation patients in consideration of infection. Verification of these results from future studies may confirm the clinical importance of hepcidin.

Restoration of ovarian function after allografting of ovarian cortex between genetically non-identical sisters

Aggressive chemotherapy generally results in the loss of both endocrine and reproductive functions. For some women, however, oocyte, embryo or ovarian tissue cryopreservation were not proposed at the time. For three such women, orthotopic allotransplantation of fresh ovarian tissue from their genetically non-identical sister was performed. Three women, aged 20, 15 and 12 years, respectively, underwent chemotherapy and total body irradiation before bone marrow transplantation (BMT), the donor in each case being their HLA-compatible sister. Years later, HLA group analysis revealed complete chimerism, and ovarian allografting was performed, with the ovarian tissue donor being the sister who had already donated bone marrow. No immunosuppressive therapy was administered. No sign of rejection was observed. Restoration of ovarian function occurred in all three cases, respectively, 6, 3.5 and 3.5 months after transplantation. The timing of the first estradiol peaks and the persistence of ovarian function were probably related to the primordial follicle density of donor ovarian tissue. Even in the absence of immunosuppressive therapy, ovarian allografting between genetically non-identical sisters allowed restoration of ovarian function in cases where previous BMT from the HLA-compatible sister resulted in full chimerism, avoiding the threat of rejection.

Short-term inhibition of p53 combined with keratinocyte growth factor improves thymic epithelial cell recovery and enhances T-cell reconstitution after murine bone marrow transplantation

AbstractMyeloablative conditioning before bone marrow transplantation (BMT) results in thymic epithelial cell (TEC) injury, T-cell immune deficiency, and susceptibility to opportunistic infections. Conditioning regimen–induced TEC damage directly contributes to slow thymopoietic recovery after BMT. Keratinocyte growth factor (KGF) is a TEC mitogen that stimulates proliferation and, when given before conditioning, reduces TEC injury. Some TEC subsets are refractory to KGF and functional T-cell responses are not fully restored in KGF-treated BM transplant recipients. Therefore, we investigated whether the addition of a pharmacologic inhibitor, PFT-β, to transiently inhibit p53 during radiotherapy could spare TECs from radiation-induced damage in congenic and allogeneic BMTs. Combined before BMT KGF + PFT-β administration additively restored numbers of cortical and medullary TECs and improved thymic function after BMT, resulting in higher numbers of donor-derived, naive peripheral CD4+ and CD8+ T cells. Radiation conditioning caused a loss of T-cell zone fibroblastic reticular cells (FRCs) and CCL21 expression in lymphoid stroma. KGF + PFT-β treatment restored both FRC and CCL21 expression, findings that correlated with improved T-cell reconstitution and an enhanced immune response against Listeria monocytogenes infection. Thus, transient p53 inhibition combined with KGF represents a novel and potentially translatable approach to promote rapid and durable thymic and peripheral T-cell recovery after BMT.

The CXCR4 antagonist 4F-benzoyl-TN14003 stimulates the recovery of the bone marrow after transplantation

Cytopenia represents a significant complication after chemotherapy, irradiation before bone marrow (BM) transplantation or as a therapy for cancer. The mechanisms that determine the pace of BM recovery are not fully understood. During the recovery phase after chemotherapy or irradiation, the signals for retention of white blood cells within the BM increase significantly. This leads to a delay in the release of WBC, which can be overcome by targeting the CXCR4 axis with the antagonist 4F-benzoyl-TN14003 (T140). The delay in the release of WBC is also accompanied by suppression in the production of progenitor cells and mature cells by the BM stroma. Administration of T140 to mice transplanted with BM cells stimulates the production of all types of progenitors and mature cells, and increases the exit of mature cells to the periphery. Moreover, addition of T140, but not AMD3100, to BM stromal cultures stimulates the production of mature cells and progenitors from all lineages. The unique ability of the CXCR4 antagonist, T140 to stimulate the production and exit of WBC cells may be used as a novel therapeutic approach to overcome cytopenia associated with treatments for cancer and BM transplantation.

Acquisition of Vernal and Atopic Keratoconjunctivitis After Bone Marrow Transplantation

Vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC) result from genetic and environmental factors. We present patients who had no history of atopic disorders before bone marrow transplantation (BMT) and who seem to have acquired VKC or AKC from their donors, who had atopic disorders. Observational case series. The patients in this study were part of a cohort of patients who had undergone allogeneic hemapoietic stem cell transplantation (HSCT) from January 1997 through December 2007. Of 621 HSCT recipients, four recipients who were free of allergic disorders acquired VKC or AKC from their afflicted donors after HSCT. Each patient underwent complete ophthalmologic examination, determination of the total serum immunoglobulin (Ig) E, and conjunctival scrapings. Four (0.64%) of 621 patients who had undergone HSCT acquired VKC or AKC after BMT. The donors had VKC or atopic dermatitis. In addition, in two of these four patients, asthma developed. One patient had elevated total serum IgE. Conjunctival scrapings of all four patients revealed the presence of eosinophils. One patient had concurrent graft-versus-host disease. VKC and AKC are systemic allergic disorders characterized by local ocular manifestations. This report suggests the possibility of the acquisition of VKC or AKC after BMT by adoptive transfer.

Allograft of ovarian cortex between two genetically non-identical sisters: Case Report

Aggressive chemotherapy and radiotherapy generally result in the loss of both endocrine and reproductive functions. In 1990, a woman aged 20 years, presenting with beta-thalassemia major, underwent chemotherapy (busulfan and cyclophosphamide) and total body irradiation (TBI) before bone marrow transplantation (BMT), the donor being her 17-year-old HLA-compatible sister. The treatment resulted in premature ovarian failure. In 2006, after excision of ovarian cortical fragments from the HLA-compatible sister, these fragments were immediately sutured to the ovarian medulla of the patient. Both procedures were performed by laparoscopy. Six months after reimplantation, vaginal ultrasonography and hormone concentrations indicated recovery of ovarian secretion and function. From 6 to 11 months, the patient experienced menstrual bleeding and the development of a follicle concomitant with high estradiol levels. Eleven months after reimplantation, two follicles were detected and punctured under vaginal ultrasonographic control. Two mature oocytes were retrieved and inseminated by ICSI. Two embryos (2- and 3-cell) were obtained. Allotransplantation of fresh ovarian tissue was laparoscopically performed between two genetically non-identical sisters. Restoration of ovarian function was achieved after six months. Oocyte retrieval and embryo development were demonstrated.

Oral cryotherapy reduces mucositis and opioid use after myeloablative therapy—a randomized controlled trial

Mucositis is a major complication in myeloablative therapy, which often necessitates advanced pharmacological pain treatment, including i.v. opioids. Attempts to prevent oral mucositis have included oral cryotherapy, which has been shown to reduce mucositis, but there is a lack of knowledge concerning the effect of oral cryotherapy on opioid use by reducing the mucositis for patients treated with myeloablative therapy before bone marrow transplantation (BMT). The aim of the present study was to evaluate if oral cryotherapy could delay or alleviate the development of mucositis and thereby reduce the number of days with i.v. opioids among patients who receive myeloablative therapy before BMT. Eighty patients 18 years and older, scheduled for BMT, were included consecutively and randomised to oral cryotherapy or standard oral care. A stratified randomisation was used with regard to type of transplantation. Intensity of pain, severity of mucositis and use of opioids were recorded using pain visual analogue scale (VAS) scores, mucositis index scores and medical and nursing charts. This study showed that patients receiving oral cryotherapy had less pronounced mucositis and significantly fewer days with i.v. opioids than the control group. In the autologous setting, cryotherapy patients also needed significantly lower total dose of opioids. Oral cryotherapy is an effective and well-tolerated therapy to alleviate mucositis and consequently reduce the number of days with i.v. opioids among patients treated with myeloablative therapy before BMT.

Prognosis value of atrial natriuretic peptide and brain natriuretic peptide for heart failure in patients undergoing allogeneic bone marrow transplantation

It is essential to evaluate the organ function of the recipient before bone marrow transplantation (BMT). This study investigated the usefulness of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) levels as indicators of cardiac function.Seventy-five consecutive patients undergoing allogeneic BMT were enrolled. All of them had an ejection fraction of 55% or more on echo cardiography. Six of the 75 patients died of heart failure after transplantation and these 6 patients were compared with the other 69 patients to assess the prognostic value of the two natriuretic peptides. Both peptides remained normal from before conditioning until recovery from leukopenia in all 69 surviving patients. Among the 6 patients who died of heart failure, however, BNP was increased in all 6 patients and ANP was increased in five of them at an average of 43.6 ± 16.7 days before the onset of heart failure.Monitoring of these peptides may not only be useful for assessment of cardiac function but also for predicting the occurrence of heart failure.

Keratinocyte growth factor (KGF) is required for postnatal thymic regeneration

AbstractKeratinocyte growth factor (KGF) is a member of the fibroblast growth factor family that mediates epithelial cell proliferation and differentiation in a variety of tissues, including the thymus. We studied the role of KGF in T-cell development with KGF-/- mice and demonstrated that thymic cellularity and the distribution of thymocyte subsets among KGF-/-, wildtype (WT), and KGF+/- mice were similar. However, KGF-/- mice are more vulnerable to sublethal irradiation (450 cGy), and a significant decrease was found in thymic cellularity after irradiation. Defective thymopoiesis and peripheral T-cell reconstitution were found in KGF-/- recipients of syngeneic or allogeneic bone marrow transplant, but using KGF-/- mice as a donor did not affect T-cell development after transplantation. Despite causing an early developmental block in the thymus, administration of KGF to young and old mice enhanced thymopoiesis. Exogenous KGF also accelerated thymic recovery after irradiation, cyclophosphamide, and dexamethasone treatment. Finally, we found that administering KGF before bone marrow transplantation (BMT) resulted in enhanced thymopoiesis and peripheral T-cell numbers in middle-aged recipients of an allogeneic BM transplant. We conclude that KGF plays a critical role in postnatal thymic regeneration and may be useful in treating immune deficiency conditions. (Blood. 2006;107:2453-2460)

A simple approximation for busulfan dose adjustment in adult patients undergoing bone marrow transplantation.

Busulfan is an alkylating agent used in preparative regimens before bone marrow transplantation (BMT). Busulfan concentrations in plasma, expressed as the area under the concentration-time curve (AUC), were reported to correlate with treatment outcome. Because busulfan is administered in 16 doses of 1 mg/kg every 6 hours for 4 days, the opportunities to "correct" the dose as a consequence of the measured AUC are limited to the 16-dosage protocol. In the present research busulfan pharmacokinetics were prospectively evaluated in 27 adult patients treated according to the above protocol by measuring the first, second, and fifth dose AUC. The pharmacokinetic analysis was based on a noncompartment model for extravascular absorption, but calculations according to a 1-compartment model gave similar results. A simple mathematical approximation allowed prediction of the AUC of the second dose from that of the first and the busulfan concentration at trough. Fifteen patients had the dose adjusted at the fourth dose to obtain an AUC within the "therapeutic window" of 950-1500 microM-min. This procedure was then validated by the measurement of the fifth dose AUC. It appears that this simple pocket calculator method allows a rapid evaluation of the need and the extent of dose adjustment and proved to be a valuable tool to improve busulfan administration in pre BMT treatment.

Degeneration of stroma reduces retention of homed cells in bone marrow of lethally irradiated mice.

Cytotoxic drugs or irradiation are generally administered before bone marrow (BM) transplantation because of the idea that host bone marrow 'niches' become available to the donor cells for engraftment. How BM stromal cells respond to the radiation, which ultimately modulates grafting of donor cells, is poorly understood. In this study, we examined homing and marrow retention of PKH26+ donor cells in BM of age-matched C57BL/6J mice conditioned at different doses of irradiation. When we injected donor cells into mice that received 900 cGy, the percent homing was highest (15.8 +/- 1.5%) as compared to the lower doses of radiation. Despite the highest levels of homing of donor cells in these mice, about 70% (p < 0.005) homed cells were detached from the marrow within 72 h of transplantation. In contrast, a 2- to 2.5-fold (p < 0.03) multiplication of homed PKH-26+ Sca-1+ cells was observed in sublethally irradiated mice. While determining that CD45- CD106+ cells in BM of the mice received 900 cGy, we found that more than 80% of cells were depleted. It was also revealed from this investigation that grafted cells conferred partial protection to the endogenous myeloid colony-forming cells from radiation injury. Collectively, the present study implicates radiation-induced degeneration of stroma as a cause of poor retention of donor cells in BM of lethally irradiated mice. These results may have important clinical implications in designing conditioning regimens for BM transplantation.

Long-term acceptance of composite tissue allografts through mixed chimerism and CD28 blockade.

Clinical composite-tissue (hand) transplantation between genetically disparate individuals currently requires potent, nonspecific immunosuppressive agents that are neither completely successful in preventing acute episodes of rejection nor free from complications. The reliance on long-term immunosuppression has prompted this study to achieve donor-specific transplantation tolerance in adult recipients using a nontoxic, nonmyeloablative protocol. Fully mismatched, 4- to 6-week-old ACI (RT1Aa) and Wistar Furth (WF) rats were used as donors and recipients, respectively. Recipients were administered CTLA4-Ig at 2 mg/kg per day (alternate days) in combination with tacrolimus at 1 mg/kg per day (daily) from day 0 through day +10, antilymphocyte serum at 10 mg at day +10 (single dose), and total-body irradiation t 300 cGy (day 0) before bone-marrow transplantation (BMT) (day 0) with 100 x 10(6) T-cell-depleted bone marrow cells. Hindlimb transplants were performed 4 weeks postBMT. Multilineage donor hematopoiesis was determined pre- and posttransplant using flow cytometry. In vitro T-cell responses were evaluated by mixed lymphocyte reactivity assays. CD28 blockade in a transplant model of mixed chimerism effectively aborts T-cell clonal expansion in vitro and in vivo, inhibits the development of acute and chronic rejection of vascularized hindlimb allografts in rats (ACI limbs to ACI-->WF chimeras, n=5; WF limbs to ACI-->WF chimeras, n=4), and subsequently leads to long-term survival of allogeneic skin grafts (n=9). Third-party (F344, n=4) transplants were uniformly rejected within 14 days posttransplant. Multilineage donor hematopoiesis was demonstrated pre- and posttransplant. Donor chimerism, present postBMT, increased throughout the study (pretransplant range 2-28%, mean 17%; posttransplant range 5-49%, mean 34%). Transplant recipients maintained full reactivity to respond to third-party antigens without harmful manifestations of graft-versus-host disease. Although efforts have been made to induce tolerance to composite tissue allografts in adult recipients, thus far, none have succeeded without toxic, myeloablative host preconditioning. Our demonstration that tolerance can be achieved with minimal preconditioning provides a rationale for application to large animals and humans and suggests that although composite tissue allografts may have a significant skin component (and are therefore felt to be highly antigenic), protocols used to induce tolerance to organ transplants may be equally applicable to composite-tissue allotransplantation.

Eye shielding during total body irradiation for bone marrow transplantation in children transplanted for a hematological disorder: risks and benefits.

This is a retrospective analysis of 188 children who underwent total body irradiation (TBI) in one or two fractions before bone marrow transplantation (BMT) for a hematological disorder. While 139 children had eye shielding during TBI to decrease cataract formation, 49 did not. The blocks used for shielding caused cylindrical areas of decreased dose intensity in the brain. The aim of the study was to determine if there was an increased risk of relapse in the eyes or in the CNS after shielding of the eyes. The probability and severity of cataract formation with and without shielding were also evaluated. None of the 49 children without shielding had a relapse in their eyes or in the CNS after BMT. Of the children with shielding, none had a relapse in the eyes but two of the 139 (1.4%) had a CNS relapse. The incidence of cataracts without shielding was 90% (19 of 21 evaluable patients), while with shielding it was 31% (20 of 64). Severe cataracts were present in eight of 21 (38%) patients without and two of 64 (3%) patients with shielding. The probability of staying cataract free for at least five years was 0.77 with and 0.33 without shielding, at 8 years it was 0.53 and 0.24 respectively. The relative risk of developing a cataract without shielding vs shielding was three (95% CI=1.5; 5.9). It appears that the incidence of relapse in the eyes and CNS is not increased when the eyes are shielded during TBI. Shielding increased the latency time of cataract formation and decreased the severity of cataracts.

Renal toxicity after total body irradiation

Purpose: To evaluate the incidence of renal dysfunction after total body irradiation (TBI). Methods and Materials: Between 1990 and 1997, 64 patients (median age 50 years) received TBI as part of the conditioning regimen before bone marrow transplantation (BMT). Five patients with abnormal renal function at the beginning of treatment or with incomplete data were excluded. All patients received a total of 12 Gy (6 fractions twice daily for 3 consecutive days) prescribed to the peak lung dose (corrected for lung transmission) at a dose rate of 7.5 cGy/min. Renal shielding was not used. Renal dysfunction was assessed on the basis of the serum creatinine levels measured at the start and end of TBI and at 6, 12, 18, and 24 months after completion of BMT. Cox proportional hazard analysis was used to evaluate the various factors known to affect renal function. Results: Only 4 patients had elevated serum creatinine levels at 12 months and subsequently only 2 of the 33 surviving patients had persistent elevated renal serum creatinine levels 24 months after BMT. A fifth patient developed proteinuria and mildly elevated serum creatinine levels at 2.5 years. In 2 patients, the elevation coincided with disease relapse and normalized once remission was achieved. In the third patient, the elevation in serum creatinine levels coincided with relapse of multiple myeloma and the presence of Bence-Jones proteinuria. The fourth patient was the only patient who developed chronic renal failure secondary to radiation nephritis at 2 years. The etiology of the fifth patient’s rise in creatinine was unknown, but may have been secondary to radiation nephritis. On univariate analysis, but not on multivariate analysis, a significant correlation was found between TBI-related renal dysfunction and hypertension before and after BMT. Conclusion: A dose of 12 Gy at 2 Gy/fraction resulted in only 1 case of radiation nephritis in the 59 patients studied 24 months after the completion of TBI and BMT.

Value of panoramic radiographs in paediatric pre-bone marrow transplantation oral evaluation

Abstract.Panoramic radiographs (orthopantomogram [OPT]) are, beside clinical examination, helpful in detecting possible dental foci of infections before bone marrow transplantation (BMT). The value of an OPT in paediatric BMT-recipients was assessed by the results of dental evaluation, consisting of an OPT and a consultation by an oral surgeon, in 161 children between 1987 and 1999. Eleven out of 161 children had at least one oral focus of infection that required treatment before myeloablative therapy. In seven children the foci were detectable both clinically and radiographically and in two children the foci were visible only clinically. Only two children had a focus that was only detectable by means of radiographs. In both patients it concerned erupting third molars. In conclusion, we recommend an OPT only when the child has clinically obvious abnormalities. In addition, in older children an OPT will be indicated to evaluate the third molars.

Mixed allogeneic chimerism as a reliable model for composite tissue allograft tolerance induction across major and minor histocompatibility barriers.

Although prolonged composite tissue allograft (CTA) survival is achievable in animals using immunosuppressive drugs, long-term immunosuppression of CTAs in the clinical setting may be unacceptable for most patients. The purpose of this study was to develop a model for reliable CTA tolerance induction in the adult rat across a major MHC mismatch without the need for long-term immunosuppression. Mixed allogeneic chimeras were prepared by using rat strains with strong MHC incompatibility [WF (RT1Au), ACI (RT1Aa)] WF + ACI-->WF, n=23. The bone marrow (BM) of recipient animals was pretreated with low-dose irradiation (500-700 cGy), followed by reconstitution with a mixture of T cell-depleted syngeneic (WF) and allogeneic (ACI) cells. Additionally, the recipient animals received a single dose of anti-lymphocyte serum (10 mg) 5 days before bone marrow transplantation (BMT) and tacrolimus (1 mg/kg/day) from the day before BMT to 10 days post-BMT. Hindlimb transplants were performed 12 months after BMT. Five animals received a limb allograft irradiated (1000 cGy) just before transplantation. Rat chimeras were characterized (percentage of donor cells present within the bloodstream) by flow cytometry at 3 and 12 months after BM reconstitution and after hindlimb transplantation. Peripheral blood lymphocyte chimerism (WF/ACI) remained stable >12 months after BM reconstitution in 18/23 animals. Multi-lineage chimerism of both lymphoid and myeloid lineages was present, suggesting that engraftment of the pluripotent rat stem cell had occurred. In animals with donor chimerism >60% (n=18) no sign of limb rejection was present for the duration of the study. All animals with chimerism <20% (n=5) developed moderate signs of rejection clinically and histologically. Gross motor and sensory reinnervation (weight bearing, toe spread) developed at >60 days in 14/21 rats. Postoperative flow cytometry studies demonstrated stable chimerism in all animals studied (n=10). Five out of five animals with irradiated limb transplants showed no sign of GVHD at >100 days. Stable mixed allogeneic chimerism can be achieved in a rat hindlimb model of composite tissue allotransplantation. Hindlimb allografts to mixed allogeneic chimeras exhibit prolonged, rejection-free survival. Partial functional return should be expected. The BM transplanted as part of the hindlimb allograft plays a role in the etiology of GVHD. Manipulating that BM before transplantation may influence the incidence of GVHD. This represents the first reliable rat hindlimb model demonstrating rejection-free CTA survival in an adult animal across a major MHC mismatch without the long-term need for immunosuppressive agents.

Long-term complications of total body irradiation in adults

Purpose: To report long-term pulmonary, thyroid, and ocular complications in patients who had conditioning regimens including total body irradiation (TBI) before bone marrow transplantation (BMT). Methods and Materials: Between June 1986 and December 1995, 478 patients received TBI in our institution. The present study includes 186 adult patients who had complete remission lasting one year or more after BMT. There were 108 males and 78 females. Median age was 36.5 years (range 15–60). Initial diagnoses were lymphomas (50%), acute lymphoid leukemias (16%), acute myeloid leukemias (16%), chronic myeloid leukemia (13%), aplastic anemia (3%), and myelodysplasia (2%). At the time of BMT, 43.5% of patients were in complete response and 56.5% in partial response. Treatment consisted of a single dose TBI at 10 Gy in 9% and fractionated TBI delivering 12 to 13.5 Gy in 6 fractions in 91%. From 1986 to October 1991, TBI was performed in lateral position with 9 MV energy (57% of patients) and thereafter in alternate prone and supine positions with 15 MV energy (43%). Chemical conditioning regimen was cyclophosphamide (60 mg/kg at D-4 and D-3) in 69% and CBV (cyclophosphamide 1500 mg/m 2 from D-6 to D-3, BCNU 300 mg/m 2 at D-6, VP-16 200 mg/m 2 from D-6 to D-4) in 25%. Fifty eight percent of patients received autologous and 42% allogeneic BMT. All patients had clinical, biologic, and functional examinations at one-year intervals. Results: Median follow-up from BMT was 49 months (range 12–136). Late pulmonary effects were observed only in functional explorations, without clinical effect, including restrictive syndrome in 8% and alteration in the diffusing capacity of carbon monoxide in 12%. No patient showed clinical thyroid symptoms, and 10% developed biologic dysfunction: hypothyroidism (6.5%), thyroiditis (3%), and Basedow disease (0.5%). Ocular complications occurred in 29.5%, including cataract (15%), dry syndrome (13%), and keratitis (1.5%). In univariate and multivariate analysis, pulmonary complications were statistically increased by chronicle graft vs. host disease (GVHD) vs. no ( p = 0.02), prone and supine vs. lateral TBI position ( p = 0.02), and with 15 MV vs. 9 MV beam energy ( p = 0.02). Cataract occurred less frequently with fractionated than with single-dose TBI ( p = 0.000002). No differences were observed regarding age, sex, initial diagnosis, status at the time of BMT, conditioning chemotherapy regimen, and total dose of TBI. Conclusion: From this retrospective study it was shown that long-term complications of TBI were not symptomatic in most patients. The role of parameters of irradiation and especially position of treatment and beam energy should be emphasized and assessed with a longer follow-up.

471 poster Total Body Irradiation (TBI) before Bone Marrow Transplantation (BMT): technical and clinical experience

471 poster Total Body Irradiation (TBI) before Bone Marrow Transplantation (BMT): technical and clinical experience

Validation de la dosimétrie biologique chez des patients conditionnés par irradiation corporelle totale : étude cytogénétique conventionnelle et hybridation in situ (FISH)

Purpose. – Validation of biological dosimetry versus physical dosimetry in malignant haemopathy patients conditioned by total body irradiation (TBI) before bone marrow transplantation (BMT). Patients and methods. – The scoring of chromosomal aberrations in peripheral lymphocytes irradiated in vivo was used to perform the biological dosimetry. The data were compared to those obtained with healthy volunteers' total blood exposed to in vitro irradiation with linear accelerator doses (0.2, 0.5, 0.75, 1, 2, 3, 4 and 5 Gy) for dose-response curves. In experimental animal models, can in vivo and in vitro responses be considered as being the same? All the published human data are based on retrospective dose evaluation with very large uncertainties on the dose precisely delivered to the subject. TBI before BMT was taken as a model where the dose calculation results from the physical method, with homogeneous beam and dose delivered precisely along the entire organism. In vivo response allows us to validate biological dosimetry in 15 adult patients (female + male), before (D = 0 Gy) and after the first fraction of 1.8 Gy, delivered by a linear accelerator (18 MV, dose-rate of 15.8 cGy/min –1). Two methods, conventional cytogenetics (CCG) and fluorescent in situ hybridization (FISH painting) of chromosome 4 were respectively used to analyze the unstable chromosome aberrations and stable chromosome aberrations. Results. – Healthy volunteer lymphocytes, before irradiation, yielded 0.1% dicentrics and 0.3% translocations of chromosome 4, with 2.5% for the whole genome. Patients before irradiation had 2% dicentrics and 11.48% chromosome 4 translocations for the whole genome. In the 15 patients, for a physical dose of 1.8 Gy, the evaluated biological dose was 1.93 Gy (95% CI: 1.85–2.05 Gy) with conventional cytogenetics and 2.06 Gy (95% CI: 1.75–2.15 Gy) with FISH. Conclusion. – These results, in which the biologically estimated dose is in complete agreement with the dose calculated by physical dosimetry in the homogeneous irradiation model, suggest the validation of biological dosimetry in TBI conditioning.

Cytotoxic T-lymphocyte precursor frequency (CTLp-f) as a tool for distinguishing permissible from non-permissible class I mismatches in T-cell-depleted allogeneic bone marrow transplantation

Matching for HLA has been the gold standard in bone marrow donor selection. But, with the ever increasing number of identified HLA alleles, it is becoming more difficult to find a fully HLA-identical donor other than a sibling. Retrospective analysis revealed that HLA mismatches do not necessarily give rise to acute graft-versus-host-disease (GVHD). However, we have no means of defining these ‘permissible’ mismatches before bone marrow transplantation (BMT). Thus, we set out to establish whether functional matching by means of helper and cytotoxic T-lymphocyte precursor frequency analysis (HTLp-f and CTLp-f respectively) can be applied to this end. Fifty-five recipient–donor pairs other than HLA-identical siblings, the recipient of which received a T-cell-depleted graft, were analysed by high-resolution HLA typing and/or HTLp-f/CTLp-f analysis. The predictive value of the CTLp-f assay for development of acute GVHD was confirmed. More importantly, our data indicate that the CTLp-f assay was able to discriminate permissible from non-permissible HLA-A, -B or -Cw mismatches, but not for DRB/DQB mismatches. The absolute number of alloreactive CTLs present in the graft correlated with the risk of acute GVHD. Although HTLp-f and CTLp-f together had a high negative predictive value, HTLp-f outcome by itself was not correlated with acute GVHD. As we have no evidence yet that HTLp-f or CTLp-f can identify permissible DRB/DQB mismatches, high-resolution matching for these antigens remains the best option. The combination of high-resolution DRB/DQB typing and the CTLp-f assay would enable the accurate prediction of the risk of acute GVHD while extending the pool of potential donors. Furthermore, it would enable adjustment of the number of T- cells in the graft accordingly to improve clinical outcome.