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1.
Clin Genet ; 93(3): 665-670, 2018 03.
Article in English | MEDLINE | ID: mdl-28776642

ABSTRACT

Next-generation sequencing (NGS) has opened up novel diagnostic opportunities for children with unidentified, but suspected inherited diseases. We describe our single-center experience with NGS diagnostics in standard clinical scenarios in pediatric hepatology. We investigated 135 children with suspected inherited hepatopathies, where initially no causative pathogenic variant had been identified, with an amplicon-based NGS panel of 21 genes associated with acute and chronic hepatopathies. In 23 of these patients, we detected pathogenic or likely pathogenic variants in 10 different genes. We present 6 novel variants. A total of 14 of these patients presented with the characteristic phenotype of the related hepatopathy. Nine patients showed only few or atypical clinical symptoms or presented with additional signs. In another 13 out of 135 cases, we detected variants of unknown significance (VUS) in 9 different genes. Only 2 of these patients showed characteristic phenotypes conclusive with the detected variants, whereas 11 patients showed unspecific or atypical phenotypes. Our multi-gene panel is a fast and comprehensive tool to diagnose inherited pediatric hepatopathies. We also illustrate the challenge of dealing with genetic variants and highlight arising clinical questions, especially in patients with atypical phenotypes.


Subject(s)
Genetic Association Studies , Genetic Predisposition to Disease , High-Throughput Nucleotide Sequencing , Liver Diseases/diagnosis , Liver Diseases/genetics , Child , Female , Genetic Association Studies/methods , Genetic Testing , High-Throughput Nucleotide Sequencing/methods , Humans , Male , Phenotype
2.
Leukemia ; 31(12): 2807-2814, 2017 12.
Article in English | MEDLINE | ID: mdl-28443606

ABSTRACT

We conducted a cytogenetic analysis of 642 children with de novo acute myeloid leukemia (AML) treated on the AML-Berlin-Frankfurt-Münster (BFM) 04 protocol to determine the prognostic value of specific chromosomal aberrations including monosomal (MK+), complex (CK+) and hypodiploid (HK+) karyotypes, individually and in combination. Multivariate regression analysis identified in particular MK+ (n=22) as a new independent risk factor for poor event-free survival (EFS 23±9% vs 53±2% for all other patients, P=0.0003), even after exclusion of four patients with monosomy 7 (EFS 28±11%, P=0.0081). CK+ patients without MK had a better prognosis (n=47, EFS 47±8%, P=0.46) than those with MK+ (n=12, EFS 25±13%, P=0.024). HK+ (n=37, EFS 44±8% for total cohort, P=0.3) influenced outcome only when t(8;21) patients were excluded (remaining n=16, EFS 9±8%, P<0.0001). An extremely poor outcome was observed for MK+/HK+ patients (n=10, EFS 10±10%, P<0.0001). Finally, isolated trisomy 8 was also associated with low EFS (n=16, EFS 25±11%, P=0.0091). In conclusion, monosomal karyotype is a strong and independent predictor for high-risk pediatric AML. In addition, isolated trisomy 8 and hypodiploidy without t(8;21) coincide with dismal outcome. These results have important implications for risk stratification and should be further validated in independent pediatric cohorts.


Subject(s)
Genetic Variation , Genotype , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/mortality , Chromosome Aberrations , Chromosomes, Human, Pair 7 , Clinical Trials as Topic , Female , Humans , Karyotype , Leukemia, Myeloid, Acute/diagnosis , Male , Monosomy , Mutation , Prognosis , Survival Analysis
3.
Klin Padiatr ; 227(3): 123-30, 2015 May.
Article in English | MEDLINE | ID: mdl-25985447

ABSTRACT

BACKGROUND: The response to initial glucocorticoid (gc) treatment is a reliable stratification factor in childhood acute lymphoblastic leukemia (ALL) and may predict the response to multi-agent chemotherapy. In a former study we detected that the valosin-containing protein (VCP, cdc48), a member of the ubiquitin proteasome degradation system (UPS), is altered in gc-resistant leukemic cells suggesting that the associated pathways might be involved in chemotherapy resistance in childhood ALL. METHODS: Human B-cell precursor leukemia cell lines, gc-resistant MHH-cALL-2 and gc-sensitive MHH-cALL-3, were treated with prednisolone and various concentrations of bortezomib. Viability and apoptosis rates were determined. RESULTS: Both cell lines showed a dose-dependent increase in caspase activity after bortezomib single treatment. The gc-sensitive cells showed an additive effect after combined treatment with prednisolone and bortezomib. In contrast, both cell lines showed a reduced viability and enhanced propidium iodide positivity after combined treatment as determined by flow cytometry. Western blot analyses of poly-(ADP-ribose) polymerase 1 (PARP-1) suggested that combined treatment promote necrotic cleavage of PARP-1 in gc-resistant cells. Furthermore, after prednisolone treatment the UPS associated proteins VCP and NFκB-inhibitor IκBα were differentially modulated in gc-resistant cells. CONCLUSIONS: The proteasome inhibitor bortezomib seems to sensitize gc-resistant childhood ALL cells for prednisolone-induced cell death.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Bortezomib/pharmacology , Cell Survival/drug effects , Drug Resistance, Neoplasm , Glucocorticoids/pharmacology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Adenosine Triphosphatases/genetics , Apoptosis/genetics , Cell Cycle Proteins/genetics , Cell Death/drug effects , Cell Line, Tumor/drug effects , Cell Proliferation , Child , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/genetics , Flow Cytometry , Gene Expression Regulation, Neoplastic/drug effects , Humans , I-kappa B Proteins/genetics , NF-KappaB Inhibitor alpha , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Prednisolone/pharmacology , Valosin Containing Protein
4.
Mol Psychiatry ; 16(1): 97-107, 2011 Jan.
Article in English | MEDLINE | ID: mdl-19786962

ABSTRACT

Dyslexia is one of the most common learning disorders affecting about 5% of all school-aged children. It has been shown that event-related potential measurements reveal differences between dyslexic children and age-matched controls. This holds particularly true for mismatch negativity (MMN), which reflects automatic speech deviance processing and is altered in dyslexic children. We performed a whole-genome association analysis in 200 dyslexic children, focusing on MMN measurements. We identified rs4234898, a marker located on chromosome 4q32.1, to be significantly associated with the late MMN component. This association could be replicated in an independent second sample of 186 dyslexic children, reaching genome-wide significance in the combined sample (P = 5.14e-08). We also found an association between the late MMN component and a two-marker haplotype of rs4234898 and rs11100040, one of its neighboring single nucleotide polymorphisms (SNPs). In the combined sample, this marker combination withstands correction for multiple testing (P = 6.71e-08). Both SNPs lie in a region devoid of any protein-coding genes; however, they both show significant association with mRNA-expression levels of SLC2A3 on chromosome 12, the predominant facilitative glucose transporter in neurons. Our results suggest a possible trans-regulation effect on SLC2A3, which might lead to glucose deficits in dyslexic children and could explain their attenuated MMN in passive listening tasks.


Subject(s)
Chromosomes, Human, Pair 4 , Dyslexia/genetics , Evoked Potentials, Auditory/genetics , Glucose Transporter Type 3/genetics , Speech Perception/genetics , Adolescent , Case-Control Studies , Child , Contingent Negative Variation/genetics , Discrimination, Psychological/physiology , Female , Genome-Wide Association Study , Humans , Male , Reference Values , Young Adult
5.
Leukemia ; 21(12): 2420-7, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17805334

ABSTRACT

In chronic myeloid leukemia (CML), imatinib may reverse bone marrow fibrosis (MF). Whether the unfavorable prognosis of MF is also reversed and whether imatinib guarantees against evolution of MF are unclear as yet. Fifty-nine patients with Ph+ CML treated with > or = 400 mg imatinib/day were examined for MF in 6- to 12-month intervals. Imatinib effectively reversed initial MF (P<0.0005). However, during a follow-up period of up to 4.8 years, small foci with abnormal fiber increase (FFI) emerged in 8 of 30 pretreated and 6 of 29 non-pretreated patients. Patients with FFI showed a significantly lower probability of achieving a complete cytogenetic or major molecular response (36 versus 81%; P<0.007). During the further follow up, 57% of patients with FFI but none of the other patients suffered from full-blown MF (P=0.00005). None of the patients with FFI or MF showed a Janus kinase-2 mutation (V617F). Evolutions of FFI and MF were independent significant predictors of imatinib failure (P=0.0031), accelerated phase and death of patients (P=0.0001; multivariate analyses). Imatinib effectively reverses initial MF in CML, but neither eliminates its unfavorable prognosis nor guarantees completely against new evolution of MF.


Subject(s)
Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Piperazines/therapeutic use , Primary Myelofibrosis/drug therapy , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Benzamides , Biomarkers, Tumor/analysis , Bone Marrow/drug effects , Bone Marrow/pathology , Disease Progression , Follow-Up Studies , Fusion Proteins, bcr-abl/analysis , Humans , Imatinib Mesylate , Interferon-alpha/therapeutic use , Janus Kinase 2/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/complications , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Male , Middle Aged , Pilot Projects , Piperazines/pharmacology , Primary Myelofibrosis/etiology , Primary Myelofibrosis/pathology , Prognosis , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Recurrence , Remission Induction , Salvage Therapy
6.
Leukemia ; 20(5): 820-6, 2006 May.
Article in English | MEDLINE | ID: mdl-16541142

ABSTRACT

The response to initial glucocorticoid therapy in childhood acute lymphoblastic leukaemia (ALL) reliably predicts the response to multiagent chemotherapy. Patients resistant to glucocorticoids (prednisone poor responders (PPR)) have a poorer event-free survival compared to glucocorticoid-sensitive patients (prednisone good responders (PGR)). A case-control study was performed to investigate differential protein expression in leukaemic blasts from PGR and PPR childhood ALL patients. Two-dimensional gel electrophoresis (2-DE) was used for an unsupervised screening and surface enhanced laser desorption/ionisation-time of flight mass spectrometry (SELDI-TOF MS) for the characterisation of protein spots. In difference maps of average gels for the proteomes of each responder group, differentially expressed proteins were identified after tryptic digestion and spotting onto H4-SELDI-TOF-MS chips. Proteins overexpressed in PPR were Catalase, RING finger protein 22 alpha, Valosin-containing protein (VCP) and a G-protein-coupled receptor. Proteins overexpressed in PGR were protein kinase C and malate dehydrogenase. Valosin-containing protein was chosen for validation and quantification by Western blot analysis in a second case-control group of ALL patients. In this second independent cohort, median VCP expression (P25-P75) was 0.15 (0.11-0.28) in PGR and 0.34 (0.14-0.99) in PPR patients (P = 0.04). We conclude that high VCP expression is associated with poor prednisone response in childhood ALL patients.


Subject(s)
Biomarkers, Tumor/analysis , Cell Cycle Proteins/analysis , Drug Resistance, Neoplasm , Glucocorticoids/pharmacology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Prednisone/therapeutic use , Proteome/analysis , Adenosine Triphosphatases , Blotting, Western , Case-Control Studies , Catalase/biosynthesis , Cell Cycle Proteins/biosynthesis , Cell Cycle Proteins/drug effects , Child , Child, Preschool , Electrophoresis, Gel, Two-Dimensional , Female , Glucocorticoids/therapeutic use , Humans , Infant , Infant, Newborn , Malate Dehydrogenase/biosynthesis , Male , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Prednisone/pharmacology , Protein Kinase C/biosynthesis , Receptors, G-Protein-Coupled/biosynthesis , Sensitivity and Specificity , Valosin Containing Protein
7.
Cancer Genet Cytogenet ; 153(2): 144-50, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15350304

ABSTRACT

Combining fluorescence R-banding, fluorescence in situ hybridization and spectral karyotyping allowed us to precisely define chromosomal breakpoints, gains, losses and a newly detected amplification in the human mantle cell lymphoma (MCL) cell line GRANTA-519. GRANTA-519 is characterized by the t(11;14)(q13;q32) resulting in overexpression of cyclin D1, a key player in cell cycle control. Hitherto unresolved complex rearrangements involve 1p, 1q, 3cen, 9p, 11q, 12p, 12q, 16p, 17p, and 18cen. Moreover, a 4- to 6-fold gain of sequences on 18q leads to a low-level amplification of the BCL2 gene and to an overexpression of the BCL2 protein. These results provide the basis for the identification of not only candidate oncogenes responsible for MCL in gained regions, but also for the identification of putative tumor suppressor genes in commonly deleted regions like 1p22, which would eventually enable functional studies of these genes.


Subject(s)
Lymphoma, Mantle-Cell/genetics , Translocation, Genetic/genetics , Cell Culture Techniques/methods , Cell Line, Tumor , Chromosome Banding , Chromosome Mapping , Cytogenetics , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Lymphoma, Mantle-Cell/pathology , Metaphase
8.
J Dent Res ; 82(10): 814-8, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14514762

ABSTRACT

Previous in vivo studies have revealed that resins may generate a persistent inflammation of oral tissues and cell death as well. Apoptosis is an important regulated process that results in rapid cell death. This study tested the hypothesis that the comonomer triethyleneglycol-dimethacrylate (TEGDMA) causes apoptosis. The effects of TEGDMA on proliferation and apoptosis in primary oral fibroblasts were analyzed by light microscopy and flow cytometry (FACS; Annexin V-assay). TEGDMA at 5 and 7.5 mM inhibited proliferation after 24 hrs. No increased frequency of apoptosis or necrosis was observed with 1 mM or 2.5 mM TEGDMA after 24 hrs. Apoptosis and Annexin V-positive cells were observed with 5 mM and 7.5 mM TEGDMA by light microscopy after 24 hrs. A dramatic increase in apoptotic cells was detected by FACS after 24 hrs with 7.5 mM TEGDMA. Thus, TEGDMA was cytotoxic and "apoptotic" in a dose- and time-dependent manner.


Subject(s)
Apoptosis/drug effects , Composite Resins/toxicity , Dental Materials/toxicity , Fibroblasts/drug effects , Gingiva/drug effects , Polyethylene Glycols/toxicity , Polymethacrylic Acids/toxicity , Analysis of Variance , Annexin A5 , Cell Culture Techniques , Cell Division/drug effects , Enzyme Inhibitors , Flow Cytometry , Gingiva/cytology , Humans , Necrosis , Time Factors
9.
Infect Immun ; 71(5): 2970-5, 2003 May.
Article in English | MEDLINE | ID: mdl-12704181

ABSTRACT

In this study we report that extracellular Burkholderia pseudomallei rhamnolipid induced cytopathic changes characterized by retraction, rounding up, and, finally, detachment in phagocytic and nonphagocytic cell lines. These changes were due to a progressive reorganization of the F-actin network resulting in impaired cell cycle progression and a reduced phagocytic function of macrophages.


Subject(s)
Burkholderia pseudomallei/pathogenicity , Glycolipids/toxicity , Actins/metabolism , Animals , Cell Cycle/drug effects , Cell Line , Cytoskeleton/drug effects , Flow Cytometry , GTP Phosphohydrolases/physiology , Mice , Microscopy, Fluorescence , Phagocytosis
10.
Exp Hematol ; 29(12): 1410-6, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11750099

ABSTRACT

OBJECTIVE: Anti-CD20 chimeric monoclonal antibody rituximab (Mabthera; IDEC-C2B8) is currently tested in several clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). In the present study, we investigated whether rituximab therapy may select for CD20(-) subclones. MATERIALS AND METHODS: Leukemic B-CLL cells were isolated from patients with B-CLL and sensitivity to rituximab-induced cell death was examined. Levels of CD20 protein and mRNA were determined using flow cytometry and real-time PCR, respectively. Clonality analyses of leukemic cells throughout rituximab therapy were performed by GeneScan analysis of patient clone specific rearrangements of the complementarity determining region III of the heavy chain immunoglobulin. RESULTS: Cytotoxicity of rituximab in vitro did not depend on the protein levels of CD20. During therapy with rituximab CD20(+) B-CLL cells were depleted and CD20(-) leukemic cells emerged. After treatment, the initial CD20(+) B-CLL cell clone reexpanded. CD20(-) B-CLL cells retained their capacity to synthesize the CD20 molecule. CONCLUSIONS: These data support the concept that in B-CLL rituximab treatment may not lead to the emergence of CD20(-) leukemic variants. Our findings support clinical studies investigating the benefit of prolonged period of rituximab therapy in B-CLL disease.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antigens, CD20/genetics , Antineoplastic Agents/therapeutic use , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Antibodies, Monoclonal, Murine-Derived , Base Sequence , DNA Primers , Gene Expression Regulation, Neoplastic , Genetic Variation , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Polymerase Chain Reaction , RNA, Messenger/genetics , Rituximab , Transcription, Genetic
11.
Exp Hematol ; 28(10): 1187-96, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11027838

ABSTRACT

OBJECTIVE: Autologous peripheral blood stem cell (PBSC) transplantation is increasingly being used in patients with chronic lymphocytic leukemia (CLL). As the autografts are frequently contaminated with large numbers of tumor cells, we have prospectively investigated the feasibility and efficacy of ex vivo double purging of PBSC grafts in an open, nonrandomized, single-center phase I/II clinical study. MATERIALS AND METHODS: Twenty consecutive patients with poor-risk CLL underwent uniform stem cell mobilization with chemotherapy and granulocyte colony-stimulating factor (G-CSF). Double B-cell depletion of the harvested PBSC products was performed using immunomagnetic CD34(+) cell selection (Isolex300i Nexell, Irvine, CA) followed by a negative step with anti-CD19/20/23/37-labeled immunomagnetic beads. The purified PBSC were reinfused after myeloablative treatment with TBI/CY. RESULTS: A total of 25 separation runs was accomplished using collection products containing 3.4% (1.1-8.1) CD34(+) cells and 1.2% (0.1-42) CD19(+)CD5(+) CLL cells. After double selection, 33% (15-67) CD34(+) cells were recovered with a purity of 98.8% (89.1-99. 8). CLL cells were undetectable by high-resolution flow cytometry in 15 of 25 final products; median purging efficacy was 5 (4.1-6) log. The CD34(+) content of the 20 final grafts was 4.6 (2.2-6.5) x 10(6)/kg. Rapid and durable engraftment developed in all cases. With a median follow-up of 20 (6-29) months, 17 patients live in complete clinical remission, two have recurrent disease, and one patient died due to pulmonary embolism five months after transplant. Persistence of the leukemic clone on the molecular level was demonstrated by dot blotting with clone-specific CDR3 probes in an additional five patients. Serious or unexpected infectious complications did not occur. CONCLUSIONS: Positive/negative purging with the Isolex system allows preparation of highly purified CD34(+) fractions and up to six log of tumor cell depletion in patients with B-CLL and can be safety reinfused after myeloablative therapy without affecting hematopoietic engraftment.


Subject(s)
B-Lymphocytes , Blood Component Removal , Hematopoietic Stem Cell Transplantation , Leukemia, Lymphocytic, Chronic, B-Cell/surgery , Adult , Antigens, CD34/analysis , Feasibility Studies , Female , Flow Cytometry , Hematopoietic Stem Cell Transplantation/adverse effects , Humans , Immunomagnetic Separation , Infections/etiology , Leukapheresis , Male , Middle Aged , Prognosis , Prospective Studies , Treatment Outcome
12.
Leukemia ; 14(8): 1419-25, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10942238

ABSTRACT

PCR of clonally rearranged immunoglobulin heavy chain (IgH) gene sequences is increasingly used for detection of minimal residual disease (MRD) in lymphoid malignancies. Inherent quantitating problems are the main drawbacks of traditional PCR technologies. These limitations have been overcome by the recently developed real-time quantitative PCR (RQ PCR) technology. However, clinical application of the few published RQ PCR assays targeting immune gene rearrangements is hampered by the expensive and time-consuming need for individual hybridization probes for each patient. We have developed a new RQ PCR strategy targeting clonally rearranged IgH sequences that solves this problem. The method uses only two different JH hybridization probes and four downstream JH primers homologous to consensus germline JH gene segments. In combination with an allele-specific upstream (ASO) primer the consensus JH probes and primers allow quantitation of about 90% of possible IgH rearrangements. In a series of 22 B-lineage ALL the new assay allowed the detection of one to 10 blasts in a background of 10(5) normal cells. To prove the clinical utility we quantified MRD in 23 follow-up samples of six ALL patients with the new assay in comparison with a published RQ PCR technique that used individually designed primer/probe sets. We showed that the sensitivity of the new RQ PCR assay was slightly higher for four of the six cases and about 100-fold higher for one case, enabling detection of an increasing MRD level as an indicator of subsequent relapse 44 weeks earlier compared to the ASO probe assay in this particular patient. The results suggest, that the novel RQ PCR assay is a rapid, technically simple, reliable, and sensitive alternative to traditional quantification assays and simplifies current approaches of monitoring MRD in clinical trials.


Subject(s)
Burkitt Lymphoma/pathology , Base Sequence , DNA Primers , Fluorescent Dyes , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Neoplasm, Residual , Polymerase Chain Reaction , Sensitivity and Specificity
14.
Bone Marrow Transplant ; 24(2): 153-61, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10455343

ABSTRACT

In a prospective study, we have investigated CD34+ selection of peripheral blood progenitor cells (PBPC) for autotransplantation in patients with lymphoma. Twenty-six consecutive patients (10 follicular lymphomas, seven mantle cell lymphomas, seven B-CLL, two immunocytomas) were mobilized using chemotherapy plus G-CSF. Sufficient numbers of PBPC could be collected from 24 patients and were immunoselected with the semiautomated Isolex 300 (n = 17) or the fully integrated Isolex 300i (n = 7) devices. The selection products were assayed by PCR amplification of clonal CDRIII or t(14;18) rearrangements for residual tumor cell content. Residual disease and long-term hematopoietic and immune recovery were studied by assessing the following parameters at 3, 6, and 12 months post-transplant: CDRIII or t(14;18) PCR, platelet count, lymphocyte subsets, serum IgG, serum IgA, and measles titer. With the Isolex 300 device 26% (10-65) of input CD34+ cells were recovered with a median purity of 89.2% (49.4-98.9) after CD34+ selection. The Isolex 300i device allowed significantly better recoveries (46% (22-86)) and purities of CD34+ cells (98.8% (92.2-99.2)). The overall purging efficacy was 3.2 (0.6-5.1) log. Twenty patients have been reinfused with CD34+ selected grafts after myeloablative preparation. Rapid engraftment occurred in all patients. With a median follow-up of 28 (19-42) months, 14 patients are alive without clinical or molecular evidence of disease recurrence, whereas five have relapsed and one additional patient shows persistent presence of the disease-specific molecular marker without clinical progression. Cellular and serological parameters of hematopoietic and immune functions were largely normal at 12 months post-transplant including the measles titer which was present in all patients. Kinetics of immunohematopoietic recovery were similar to those of 12 control patients who had received unmanipulated PBPC during the same time period except for the recovery of CD4+ CD45RA+ T cells which was significantly delayed in the CD34+ group. During the first year post-transplant, transient monoclonal or oligoclonal gammopathies were observed in seven of 16 study patients. We conclude that CD34+ selection with the Isolex system allows preparation of highly purified CD34+ fractions and effective tumor cell depletion. The CD34+ products can be reinfused safely after myeloablative treatment and result in sustained hematopoietic and immune recovery. The fact that all patients retained their specific measles immunity suggests that myeloablative treatment with reinfusion of highly purified CD34+ PBPC is not immunoablative.


Subject(s)
Graft Survival/immunology , Hematopoietic Stem Cell Mobilization , Hematopoietic Stem Cell Transplantation , Lymphoma/therapy , Adult , Female , Hematopoiesis , Humans , Lymphoma/immunology , Lymphoma/pathology , Male , Middle Aged , Prospective Studies , Transplantation, Autologous , Treatment Outcome
15.
Br J Haematol ; 106(1): 115-8, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10444173

ABSTRACT

Waldenström's macroglobulinaemia (WM) is an incurable lymphoproliferative disorder. The purpose of this study was to investigate the role of autologous peripheral blood stem cell transplantation (ASCT) for the treatment of WM. Seven patients (untreated or after first-line therapy) with symptomatic WM underwent two or three cycles of Dexa-BEAM chemotherapy + G-CSF with stem cell harvesting and proceeded to total body irradiation and high-dose cyclophosphamide followed by reinfusion of ex-vivo B-cell-depleted stem cells. Engraftment was prompt, and procedure-related deaths did not occur. A strong reduction or normalization of BM infiltration and serum IgM levels occurred in all evaluable patients, but immunofixation electrophoresis revealed persistent paraproteinaemia in five of them. With 3-30 months of follow-up, all patients are alive without clinical or serological signs of disease progression. This pilot trial shows for the first time that high-dose radiochemotherapy with purged stem cells is effective and may improve the course of patients with WM. In the majority of cases, however, complete eradication of the disease does not appear to be possible with ASCT alone.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Hematopoietic Stem Cell Transplantation/methods , Waldenstrom Macroglobulinemia/therapy , Whole-Body Irradiation/methods , Adolescent , Adult , Aged , Female , Hematopoietic Stem Cell Mobilization/methods , Humans , Male , Middle Aged
16.
Bone Marrow Transplant ; 22(2): 161-6, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9707024

ABSTRACT

Two different molecular genetic methods were compared for their suitability for monitoring minimal residual disease in patients with follicular lymphoma (FL) treated with high-dose therapy and autologous stem cell transplantation. Fifteen patients were selected because of a specific PCR-amplifiable t(14;18) mbr translocation. PCR amplification of rearrangements of the complementary region III (CDRIII) of the immunoglobulin heavy chain gene was also carried out. After autologous stem cell transplantation, patients were prospectively monitored with both molecular genetic methods. Seven of the 15 patients with detectable t(14;18) prior to transplantation were persistently negative during follow-up to 32 months post transplant. None of these patients relapsed, whereas four of eight patients with positive PCR signals post transplant relapsed. Comparing t(14;18) and PAGE results, we observed six patients showing clonal signals in CDRIII PAGE in spite of persistent negativity of t(14;18) PCR. We concluded that in patients with FL, t(14;18) PCR is superior to CDRIII PCR in terms of sensitivity and specificity. A positive t(14;18) PCR during the first year post transplant is highly predictive for disease recurrence. CDRIII PCR may be used for monitoring in t(14;18) negative lymphomas. However, due to the poor specificity of conventional gel electrophoresis PCR, the use of clone-specific probes is highly desirable.


Subject(s)
Chromosomes, Human, Pair 14 , Chromosomes, Human, Pair 18 , Hematopoietic Stem Cell Transplantation , Lymphoma, Follicular/therapy , Neoplasm, Residual/genetics , Translocation, Genetic , Adult , Female , Genetic Markers , Humans , Lymphoma, Follicular/genetics , Male , Middle Aged , Neoplasm, Residual/diagnosis , Transplantation, Autologous
17.
Br J Cancer ; 77(12): 2291-7, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9649148

ABSTRACT

B-cell chronic lymphocytic leukaemia (CLL) cannot be cured by conventional therapy. To improve the prognosis of patients with CLL, we have designed a sequential treatment strategy that comprises intensive chemotherapy for mobilization of peripheral blood progenitor cells (PBPCs) and induction of minimal disease, followed by high-dose radiochemotherapy with stem cell reinfusion and post-transplant molecular monitoring by polymerase chain reaction (PCR) amplification of the complementary determining region III (CDRIII) gene. In a prospective study, we have evaluated this protocol in 18 patients with CLL, also including early stages of the disease. The median age was 49 (29-61) years; Binet stages were A, six; B, nine; and C, three. Adverse prognostic factors [high lymphocyte count and/or diffuse bone marrow (BM) infiltration] were present in 16 out of 18 patients. All patients showed a clone-specific molecular marker as demonstrated by PCR amplification of CDRIII rearrangements. For stem cell mobilization and reduction of tumour load, one to two cycles of Dexa-BEAM chemotherapy were administered, resulting in minimal disease (circulating lymphoma cells <1 x 10(9) l(-1); BM infiltration <20%; lymphomas <2 cm) in 16 out of 18 patients, including four patients who already had minimal disease before Dexa-BEAM. Stem cell harvesting was successful in 14 patients. All grafts [three BM, 11 peripheral blood (PB)] were purged from leukaemic cells using immunomagnetic methods. Thirteen patients having achieved minimal disease were reinfused with purged autologous stem cells (ASC) after preparation with total body irradiation and cyclophosphamide. Engraftment was delayed in patients receiving BM (n = 3) but prompt [neutrophils >0.5 x 10(9) l(-1) after 10 (9-13) days, platelets >20 x 10(9) l(-1) after 11 (9-214) days] in patients restored with PBPCs (n = 10). Procedure-related deaths did not occur. Although the results of CDRIII PCR suggest persistence or recurrence of the leukaemic clone in at least three cases, to date only one patient has relapsed, whereas all others survive without clinical evidence of disease with a maximum follow-up of 48 months. We conclude that sequential high-dose therapy using Dexa-BEAM and autologous stem cell transplantation is a safe and highly effective treatment for patients with CLL. However, a longer follow-up is needed to assess whether definite cures can be achieved using this strategy.


Subject(s)
Hematopoietic Stem Cell Transplantation , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carmustine/administration & dosage , Chlorambucil/administration & dosage , Cisplatin/administration & dosage , Combined Modality Therapy , Cyclophosphamide/administration & dosage , Cytarabine/administration & dosage , Dexamethasone/administration & dosage , Dose-Response Relationship, Drug , Doxorubicin/administration & dosage , Etoposide , Female , Fluorouracil/administration & dosage , Follow-Up Studies , Hematopoietic Stem Cell Mobilization , Humans , Hydrocortisone/administration & dosage , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Male , Melphalan/administration & dosage , Methotrexate/administration & dosage , Methylprednisolone/administration & dosage , Middle Aged , Mitomycins/administration & dosage , Prednisone/administration & dosage , Prospective Studies , Treatment Outcome , Vincristine/administration & dosage
18.
Bone Marrow Transplant ; 20(5): 415-20, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9339759

ABSTRACT

Autologous PBPC transplantation is a potentially curative treatment for patients with chronic lymphocytic leukemia (CLL). As the autografts are frequently contaminated with large numbers of tumor cells, we have studied double purging of PBPC using immunomagnetic CD34+ cell selection (Isolex 300i) followed by negative depletion with anti-CD19/20/23/37-labeled immunomagnetic beads. In four small-scale experiments using PBPC from patients with CLL or leukemic low-grade lymphoma, double purging resulted in CD34+ enrichment from 0.9-4.4% to 95.8-99.4%. Lymphoma cells were always undetectable by FACS and PCR (CDR3 or t(14;18)) after negative depletion. Next, seven heavily contaminated full grafts from five patients with CLL or lymphoplasmocytoid immunocytoma were subjected to the double purging procedure, resulting in a CD34+ enrichment from 1.6% (0.7-5.6) to 98.5% (96-99.8). The CD34+ yield after double purging was 1.3-6.3 x 10(6)/kg, according to a median recovery of 32%. The overall reduction of lymphoma cells was 5.6 (>4.6-6) log. Although CLL cells were completely absent after purging in five cases as assessed by FACS, all grafts remained PCR positive. The first two patients have been reinfused with double selected products after myeloablative radiochemotherapy and showed prompt and uneventful hematopoietic engraftment. We conclude that without significant loss of CD34+ cells, negative depletion adds 2 log of CLL cell depletion to CD34+ selection, resulting in an overall purging efficacy of more than 5 log. This combination of positive and negative selection can be successfully applied even to heavily contaminated PBPC grafts.


Subject(s)
Bone Marrow Purging , Hematopoietic Stem Cell Transplantation , Immunomagnetic Separation/methods , Adult , Antigens, CD34/blood , Cell Separation , Flow Cytometry , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Lymphocyte Depletion/methods , Middle Aged , Polymerase Chain Reaction , Transplantation, Autologous
20.
Ann Oncol ; 8(4): 401-3, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9209672

ABSTRACT

BACKGROUND: Mantle cell lymphoma (MC) is not curable with conventional chemotherapy. To improve the prognosis of patients with this disease, we prospectively studied an intensive sequential therapy consisting of the Dexa-BEAM regimen (dexamethasone, BCNU, etoposide, ara-C, melphalan) followed by myeloablative therapy with autologous stem cell reinfusion. PATIENTS AND METHODS: Nine consecutive patients with stage III/IV MC were included. Two had untreated disease, four were in first remission, whereas three had more advanced disease. All patients underwent one to two cycles of Dexa-BEAM chemotherapy to reduce the tumor load and to mobilize peripheral blood progenitor cells (PBPC). Subsequently, patients were treated with high-dose radiochemotherapy followed by PBPC reinfusion and were prospectively analyzed for residual disease by clinical methods as well as by PCR amplification clonal CDRIII rearrangements. RESULTS: With an overall response rate of 100%, the initial Dexa-BEAM cycles effectively reduced the tumor load. All patients proceeded to high-dose therapy and subsequent stem cell rescue. Engraftment was prompt, and procedure-related deaths did not occur. With a median follow-up of 12 (3-33) months post transplant, all patients are alive in continuing clinical and molecular remission. CONCLUSIONS: Sequential intensive therapy consisting of Dexa-BEAM and high-dose radiochemotherapy appears to be a highly effective treatment for patients with MC. However, the data are still preliminary, and larger patient numbers and a longer follow-up are required.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Hematopoietic Stem Cell Transplantation , Lymphoma, Non-Hodgkin/therapy , Adolescent , Adult , Aged , Carmustine/administration & dosage , Combined Modality Therapy , Cytarabine/administration & dosage , Dexamethasone/administration & dosage , Dose-Response Relationship, Drug , Etoposide , Female , Humans , Immunophenotyping , Lymphoma, Non-Hodgkin/pathology , Male , Melphalan/administration & dosage , Middle Aged , Neoplasm Staging , Prospective Studies , Transplantation, Autologous
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