Biomedical subjects
Publications and source records attributed to P Bader.
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We have retrospectively investigated the relationship between the level of minimal residual disease (MRD) detected in bone marrow taken prior to conditioning therapy and outcome following stem cell transplantation for high risk childhood ALL. Forty-one patients, in whom both a molecular marker of MRD and sufficient archival material was available, were included in the study. All were in remission at BMT: eight in CR1, 32 in CR2 and five in greater than CR2. MRD was measured by PCR amplification of antigen receptor gene rearrangements and clone-specific oligoprobing, the median sensitivity of detection being one leukaemic cell in 10000 normals. Results were classified as high-level positive (if a clonal band was evident after electrophoresis), low-level positive (if MRD was detected only after oligoprobing) and negative. MRD was detected at high levels in 17 patients, at low levels in 10 patients and 14 patients were MRD negative at the time of transplant. The 5-year event-free survival for these groups was 23%, 48% and 78%, respectively (P = 0.022). Limited multivariate analysis confirmed the significance of MRD (P = 0.0095) vs CR status, donor type, sex, immunophenotype and acute GvHD. This study confirms the strong relationship between MRD level and outcome following allogeneic transplantation. In contrast to a previous study we observed that a minority of children with high-level pre-BMT MRD can enter long lasting remission. The possible role for acute GVHD coupled with a graft-versus-leukaemia effect in the clearance of high level MRD in patients with ALL is discussed.
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The first step in adopting a practical approach to the management of patients with prostatitis lies in the realization that the etiology of the symptoms often remains unclear and the traditional etiologically based classification system is part of the problem and not the solution. This problem was recognized in 1995 by the National Institutes of Health Consensus Conference on prostatitis. It was suggested that the classification of this disease be changed. The traditional categories "chronic nonbacterial prostatitis" and "prostatodynia" were replaced by the new category "chronic pelvic pain syndrome." The introduction of the term "syndrome" reflects two issues: despite lack of evidence for bacterial involvement based on conventional methods, nonbacterial prostatitis may indeed still be an infectious disease, and the etiology of the symptoms may be caused by a disorder not related to the prostate gland alone.
We performed HLA-mismatched stem cell transplantation with megadoses of purified positively selected mobilized peripheral blood CD34(+) progenitor cells (PBPC) from related adult donors in 39 children lacking an otherwise suitable donor. The patients received a mean number of 20.7 +/- 9.8 x 10(6)/kg purified CD34(+) and a mean number of 15.5 +/- 20.4 x 10(3)/kg CD3(+) T lymphocytes. The first seven patients received short term (<4 weeks) GVHD prophylaxis with cyclosporin A, whereas in all the following 32 patients no GVHD prophylaxis was used. In 38 evaluable patients, five patients experienced primary acute GVHD grade I and one patient grade II. In 32 patients, no signs of primary GVHD were seen and GVHD only occurred after T cell add backs. T cell reconstitution was more rapid if the number of transplanted CD34(+) cells exceeded 20 x 10(6)/kg. Of the 39 patients, 15 are alive and well, 13 died due to relapse and 10 transplant-related deaths occurred. We conclude that the HLA barrier can be overcome by transplantation of megadoses of highly purified mismatched CD34(+) stem cells. GVHD can be prevented without pharmacological immunosuppression by the efficient T cell depletion associated with the CD34(+) positive selection procedure. This approach offers a promising therapeutic option for every child without an otherwise suitable donor.
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Transplantation of haematopoietic stem cells from human leucocyte antigen (HLA)-disparate parental donors presents a promising new approach for the treatment of patients lacking a HLA-matched donor. Success against major obstacles such as graft-versus-host disease (GvHD) and graft rejection has recently been demonstrated, so that immune reconstitution is one of the prime factors that determines the long-term prognosis following transplantation. Twenty children transplanted with megadoses of highly purified CD34(+) haematopoietic stem cells after rigorous T-cell depletion were prospectively monitored for their immune reconstitution during the first post-transplant year. Natural killer (NK) cells showed a marked increase on d +30. T and B cells began to reconstitute on d +72 and +68 respectively. During extended follow-up, their numbers and proliferative capacity upon mitogen stimulation continually increased. Early reconstituting T cells were predominantly of a primed, activated phenotype with severely skewed T-cell receptor (TCR)-repertoire complexity. Naive T cells emerged 6 months post transplantation, paralleled by an increase in TCR-repertoire diversity. All patients self-maintained sufficient immunoglobulin levels after d +200. This study demonstrates that paediatric recipients of highly purified, haploidentical stem cells are able to reconstitute functioning T-, B- and NK-cell compartments within the first post-transplant year. This, together with the absence of significant GvHD, provides a strong indication for this approach to be considered in children who lack a HLA-matched donor.
The rationale for purging of autologous acute myeloid leukemia (AML) grafts is to eradicate contaminating leukemic cells that might contribute to relapse. However, in vitro purging generally delays post-transplant hematopoietic recovery, thus increasing treatment-related complication rates. Theoretically, this prolonged aplasia might be shortened by the additional transplantation of ex vivo-generated progenitor cells. Therefore, we investigated whether nonleukemic progenitors could be expanded ex vivo from AML cell-contaminated CD34(+) peripheral blood progenitor cell (PBPC) preparations. Nonleukemic CD34(+)-selected PBPC and AML cells (Kasumi-1, KG-1, primary AML blasts) were cultured in cytokine-supplemented liquid culture for up to 19 days. Cells were used either unmanipulated or following in vitro purging with mafosfamide (30, 50, 75 microg/ml). Ex vivo-generated cells were assessed by flow cytometry, progenitor cell assays, and polymerase chain reaction. Without prior purging, ex vivo culture markedly amplified AML cells as well as nonleukemic CD34(+) PBPC (day 12: Kasumi-1, 18.5 +/- 0.6-fold; KG-1, 52.2 +/- 2.6-fold; CD34(+), 74.1 +/- 5.6-fold). Co-culture with leukemic cells did not affect CD34(+) cell growth and vice versa. Following in vitro purging, CD34(+) PBPC were expanded even at the highest mafosfamide dose (day 19: 25 +/- 15-fold), whereas leukemic cells were markedly depleted (approx. 1.5 log). Furthermore, normal colony-forming units (CFU) could be effectively recovered (day 19: 10 +/- 3.1% of prepurging input CFU), whereas CFU-L were depleted to undetectable levels in six of seven experiments. Finally, leukemic cells were undetectable following ex vivo co-culture of purged cells (CD34(+) PBPC plus 10% Kasumi-1 cells or primary blasts), but were clearly detectable without purging. Taken together, these data demonstrated that ex vivo expansion of normal progenitors from mafosfamide-purged AML cell-contaminated grafts might be feasible.
The major determinants mediating drug resistance in acute lymphoblastic leukemias (ALL) unresponsive to chemotherapy, are still unclear. For example, it is still unknown whether selection or induction processes are responsible for drug resistance here or whether protein kinase C (PKC) isozymes contribute to the resistant phenotype. Therefore, inducibility of resistance factors or PKC isozymes genes was examined in CCRF-CEM cells treated with diverse anticancer drugs--adriamycin, camptothecin, etoposide or vincristine--at sublethal concentrations for 24 h. MDR1, MRP1, LRP and PKC isozyme alpha, beta(1), beta(2), epsilon, iota, eta, theta, zeta gene expression was determined by cDNA-PCR. We found significant dose-dependent, mostly combined, induction of the MDR1, MRP1 and LRP genes. Significantly enhanced gene expression of the majority of PKC isozyme genes was found after treatment with camptothecin. PKCzeta was upregulated throughout by each anticancer drug applied in this setting. A series of selected CCRF-CEM-derived multidrug resistance (MDR) sublines also showed enhanced expression of the PKC isozymes compared to the parental cell line. MDR1 and PKCeta gene expression levels were correlated highly significantly. Blasts from two patients with ALL during the first week of monotherapy with steroids revealed combined induction of the MDR1, multidrug resistance-associated protein 1 (MRP1), lung cancer resistance-related protein (LRP) and most PKC isozymes, predominantly PKCzeta. Another patient with T-ALL, who failed to respond to four months of intensive chemotherapy, showed an enhanced MRP1 gene expression combined with markedly overexpression of PKCeta and PKCtheta. Furthermore, the camptothecin and etoposide-mediated induction of resistance factors in the CCRF-CEM cell line could be suppressed by staurosporine, a rather unspecific inhibitor of protein kinases. However, selective inhibitors of PKC isozymes (bisindolylmaleimide GO 6850, indolocarbazole GO 6976) produced no significant effects here. Therefore, the PKC isozymes eta, theta and zeta are of interest as potential targets to overcome drug resistance in ALL.
Thirty-eight children with high-risk hematological malignancies underwent transplantation with megadoses of peripheral mobilized CD34+ cells from haploidentical parents (n = 24) or from matched unrelated donors (n = 14). The CD34+ cells were isolated to a purity of > 98% using magnetic-activated cell sorting. This high purity was associated with an almost complete depletion of T lymphocytes. No pharmacological prophylaxis for graft-versus-host disease (GvHD) was used, and significant primary GvHD was not seen. A final engraftment was seen in all patients. Sixteen patients are alive and disease-free with a median follow-up of 24 months. The immunological reconstitution was faster in the patients transplanted with CD34+ stem cells from the haploidentical donors compared to the matched unrelated donors, and the transplantation of large numbers of haploidentical CD34+ stem cells seems to be superior to that of the matched unrelated donors. The phenotypical and functional analysis of the immune reconstitution provided some insights into the biology of transplantation of highly purified CD34+ cells. In this article, we summarize our current results with the transplantation of highly purified stem cells and discuss possible implications for further antileukemic post-transplant therapeutic strategies.
PURPOSE: Continence after radical prostatectomy is thought to depend completely on the striated urethral sphincter. However, some patients complain only of occasional post-void dribbling. Therefore, we determined whether urethral dysfunction may be another cause of incontinence. MATERIALS AND METHODS: The sensory threshold of electric stimulation was measured by double ring electrodes in the membranous urethra and 2.5 cm. distal in 29 men before and in 29 after radical retropubic prostatectomy. In addition, voiding cystourethrography was performed in 66 patients before and in 49 after surgery to determine complete post-void urethral emptying or milking. RESULTS: The mean sensory threshold of the membranous urethra was 15 +/- 3 mA. preoperatively versus 38 +/- 17 postoperatively (p <0.0001). The sensory threshold 2.5 cm. further distal was 12 +/- 5 mA. before and 10 +/- 4 after radical prostatectomy, which was not statistically significant. Postoperatively in completely continent patients and in those with dribbling the mean threshold was 32 +/- 12 and 43 +/- 18 mA. in the membranous urethra (p = 0.09), and 11 +/- 4 and 9 +/- 4 mA. in the bulbar urethra, respectively, which was not statistically significant. Of the 66 patients 36 (55%) showed post-void urethral milking before surgery but only 8 of 49 (16%) showed it postoperatively (chi-square test p <0.0001), including 7 who were completely continent and 1 who complained of occasional post-void dribbling. CONCLUSIONS: After radical prostatectomy sensitivity of the membranous but not of the bulbar urethra is affected, correlating with postoperative continence. In addition, post-void dribbling seems to be associated with the loss of urethral milking. We conclude that preserving urethral function is another important continence factor after radical prostatectomy.
Asymptomatic microhematuria is a common reason for a urological consultation. Uncertainty prevails as to how meticulous the work-up must be, to not miss relevant or even life-threatening underlying diseases. To date, the Urological Associations have not released any guidelines to which extent patients need to be examined for asymptomatic microhematuria, which therefore is managed individually by each urologist. There are various potential examinations that can be applied, ranging from a clinical examination to a kidney biopsy. After reviewing the literature, an algorithm has been developed, which should assure diagnosis of serious disease and at the same time avoid costly, unpleasant and unnecessary examinations.
PCR-based preemptive therapy with ganciclovir has been shown to reduce the incidence of CMV disease after BMT. Failures of this treatment strategy are CMV disease and secondary non-viral infections. Eighty-six consecutive patients at high risk for CMV disease who received PCR-based preemptive therapy with ganciclovir were assessed for treatment failures and possible risk factors. Ganciclovir was initiated in 57 of 86 patients (66%). Only 28 of 86 (32%) patients received 4 or more weeks of ganciclovir. Recurrence of CMV infection after successful treatment was more frequent among recipients of a BMT from an unrelated compared to a sibling donor (P = 0.004). Three (3.5%) patients developed non-fatal early onset CMV disease and seven of 68 (10.3 %) late onset CMV disease (>100 days post transplant). Risk factors for late onset CMV disease were cGVHD (P = 0.0017) and duration of prior antiviral therapy >4 weeks (P = 0. 0073). The incidence of secondary non-viral infections was 28% with the duration of antiviral treatment being a significant risk factor for secondary bacterial (P = 0.0045) and invasive fungal infections (P = 0.006). Thus, PCR-based preemptive treatment with ganciclovir reduces early onset CMV disease, but the duration of antiviral therapy prior to day +100 is a significant risk factor for late onset CMV disease as well as secondary non-viral infections.
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Recently, we have shown that patients with acute leukaemias and myelodysplastic syndromes (MDS), who showed increasing mixed chimaerism (MC) upon serial PCR analysis after transplant, have a significantly increased risk of relapse. To determine whether the increasing MC in these patients is caused by the reappearance of normal recipient haematopoiesis or by the reoccurrence of malignant cells, we purified different leucocyte subpopulations and analysed these subfractions with regard to their donor-recipient ratio by a PCR-based method for the analysis of minisatellite DNA regions. In 14 patients [eight acute lymphoblastic leukaemia (ALL), three acute myelogenous leukaemia (AML) and three MDS] subfractions were analysed when increasing MC was first noted upon serial analysis of the peripheral blood. In seven of these 14 patients (four ALL, two AML and one MDS), subfractions were characterized at the time of frank haematological relapse. In all 14 patients investigated with increasing MC, recipient cells were detected in different mononuclear cell subpopulations. In patients characterized during frank relapse, two distinct distribution patterns were found. Patients who relapsed before day +300 (one ALL, two AML and one MDS) showed recipient-derived (normal) cells in addition to blast populations in different mononuclear subsets as well as granulocytes. In patients with acute leukaemias who relapsed after day +300 (two ALL and one AML), only leukaemic cells were found that were of recipient origin, whereas all other haematopoietic cell lines were donor derived. These data show that persistent MC in the early post-transplant period is caused predominantly by normal recipient haematopoietic cells. This finding further supports the hypothesis that a state of mixed haematopoietic chimaerism may reduce the clinical graft-versus-leukaemia (GVL) effect of alloreactive donor-derived effector cells in patients with acute leukaemias and MDS, and thus facilitate the proliferation of residual malignant cells that may have survived the preparative regimen.
Primary non-engraftment or early rejection after transplantation of haematopoietic stem cells represent life-threatening complications of allogeneic stem cell transplantation. Management of early graft failure has been problematic, as the risk of fatal infectious complications increases with the time of pancytopenia and as a second transplant preceded by a conventional myeloablative conditioning regimen has been associated with high rates of cumulative organ toxicity. For paediatric patients with early graft failure following the transplantation of highly purified major histocompatibility complex (MHC)-disparate haematopoietic stem cells, we have evaluated an immunosuppressive OKT-3/methylprednisolone-based reconditioning regimen with low toxicity in preparation for a secondary transplant of purified haematopoietic stem cells from the same donor. This report presents the results from a 4-year pilot study including six patients with early graft failure. The results demonstrate that this antibody-based regimen can be used effectively to prepare patients for secondary transplantation. Successful engraftment after a second transplant procedure was achieved in five of these six high-risk patients. The median interval between first and second transplant was 27 d (range 22-51 d), and the median time for engraftment was 10 d (range 9-13 d). Chimaerism analysis of microsatellite regions by polymerase chain reaction (PCR) demonstrated complete donor chimaerism in four of these patients within the first month after secondary transplant and revealed mixed chimaerism in one patient who converted to complete chimaerism after T-cell add-back.
Invasive fungal infections are a major cause of mortality in neutropenic cancer patients. To determine whether a polymerase chain reaction (PCR)-based assay enabled the identification of patients at risk for invasive fungal infections, a prospective monitoring once per week was performed during 92 neutropenic episodes in patients receiving chemotherapy for acute leukaemia or high-dose therapy followed by allogeneic or autologous stem cell transplantation, with the investigators blinded to clinical and microbiological data. PCR positivity was documented in 34 out of 92 risk episodes. All patients developing proven invasive fungal infection were found PCR positive, and PCR was found to be the earliest indicator of invasive fungal infection preceding clinical evidence by a mean of 5.75 d (range 0-14 d). In febrile neutropenic patients without a prior history of invasive fungal infection, a sensitivity of 100% and a specificity of 73% of the PCR assay for the development of proven or probable invasive fungal infection was documented. In conclusion, panfungal PCR performed prospectively once a week enabled the identification of patients at high risk for invasive fungal infections.
Peripheral mobilized parental CD34+ progenitors were isolated and used for the hematopoietic reconstitution after a myeloablative therapy in 23 pediatric patients with various diseases. Fourteen donors were human leukocyte antigen (HLA) three-loci mismatches, 6 donors were two-loci and 3 donors were one-locus mismatches. For depletion of T-lymphocytes, a positive selection of the mobilized peripheral CD34+ progenitors using the method of magnetic-activated cell sorting (MACS) was used. The purity of the CD34+ cells after MACS-sorting was 98-99%, the average number of transplanted CD34+ cells was 14.2 x 10(6)/kg (range 5.4-3.9 x 10(6)/kg) and the average number of infused T-lymphocytes was 1.4 x 10(4)/kg. Due to this low T cell number, only a short-term or no prophylaxis of graft-versus-host disease (GVHD) was necessary and no GVHD was seen. A significant GVHD was only seen in patients after add-back of donor T-lymphocytes, which was performed in some patients for prevention of relapse or in patients who showed a transient mixed chimerism. Since the B lymphocyte contamination of the isolated CD34+ cells was low in the range of 0.2%, no Epstein-Barr virus (EBV)-associated lymphoproliferative syndrome was observed. A primary engraftment was seen in 18 patients. Nonengraftment and rejection occurred in three and two patients, respectively. In four of these 5 patients, a second transplant using purified CD34+ cells from the same donor after an immunological reconditioning regimen resulted in a complete and sustained hematopoietic reconstitution. The speed of the immunological recovery was dependent on the number of transplanted CD34+ cells and was more rapid if this number was > 20 x 10(6)/kg. Eleven of the 23 patients are alive and disease free with a median follow-up of 12 months (range 2-30). The main cause of death was relapse (7 patients), and only one fatal infection was seen. Our data suggest that the transplantation of megadoses of haploidentical CD34+ cells is a realistic therapeutic option for patients who otherwise have no suitable donor, and an alternative to the use of unrelated cord blood.