Vaginal foreign body in the pediatric patient: A systematic review.

BACKGROUND: Vaginal foreign bodies represent a clinical and diagnostical challenge in pediatric gynecology. Several case reports, case series and retrospective studies have been published, highlighting rare or complex cases. A comprehensive systematic review is lacking. METHODS: Published English-language articles on vaginal foreign objects in patients aged 16 years and younger, with full-text availability were included. Articles on adult patients and patients with an object migrating from the abdominal cavity into the vagina were excluded. RESULTS: Out of the 215 screened articles 75 were included, comprising a total of 522 patients. The age ranged from 6 months to 16 years, with an average of 6 years and 3 months. The presenting symptoms were documented in 340 patients, with the two most common being vaginal bleeding (n = 172) and vaginal discharge (n = 134). Toilet paper or tissue was the most common object, in 155 out of 447 patients. Ultrasonography was the most utilized diagnostic method, with a sensitivity of 79.9 %. Radiography showed more false-negative than true-positive results, with a sensitivity of 33.3 %. Complications were reported in 35 patients. Evidence of sexual abuse was found in a small group of 16 patients. Vaginoscopy under sedation was the most frequently used therapeutic approach. CONCLUSION: A swift and accurate diagnosis is crucial, with clinical examination and ultrasonography playing pivotal roles. Vaginoscopy is the gold standard for definitive diagnosis and therapy. Attention should be given to a potential context of sexual abuse.

Constitutively activating mutation in WASP causes X-linked severe congenital neutropenia.

The Wiskott-Aldrich syndrome protein (WASP; encoded by the gene WAS) and its homologs are important regulators of the actin cytoskeleton, mediating communication between Rho-family GTPases and the actin nucleation/crosslinking factor, the Arp2/3 complex. Many WAS mutations impair cytoskeletal control in hematopoietic tissues, resulting in functional and developmental defects that define the X-linked Wiskott-Aldrich syndrome (WAS) and the related X-linked thrombocytopenia (XLT). These diseases seem to result from reduced WASP signaling, often through decreased transcription or translation of the gene. Here we describe a new disease, X-linked severe congenital neutropenia (XLN), caused by a novel L270P mutation in the region of WAS encoding the conserved GTPase binding domain (GBD). In vitro, the mutant protein is constitutively activated through disruption of an autoinhibitory domain in the wild-type protein, indicating that loss of WASP autoinhibition is a key event in XLN. Our findings highlight the importance of precise regulation of WASP in hematopoietic development and function, as impairment versus enhancement of its activity give rise to distinct spectra of cellular defects and clinical phenotypes.

A new disease categorization of low-grade myelodysplastic syndromes based on the expression of cytopenia and dysplasia in one versus more than one lineage improves on the WHO classification.

Multilineage dysplasia was advanced by the World Health Organization to increase prognostic accuracy in myelodysplastic syndromes (MDS) classification. We performed a structured cytomorphological examination of bone marrow (BM) in 221 low-grade MDS patients, this in conjunction with strict guidelines for cytopenias. A dysplasia scoring system was developed utilizing dysplastic changes, which were associated with worse outcome on univariate and multivariate analysis corrected for the International Prognostic Scoring System (IPSS). Dysplasia >or=10% in one BM lineage and one cytopenia constituted the low-risk category UCUD or Unilineage Cytopenia and Unilineage Dysplasia. The high-risk category comprised patients with cytopenia in >or=2 lineages and dysplasia in >or=2 BM lineages, namely MCMD or Multilineage Cytopenia and Multilineage Dysplasia. Intermediate-risk patients had one cytopenia and multilineage dysplasia, or cytopenia in >or=2 lineages and unilineage BM dysplasia, designated UCMD/MCUD or Unilineage Cytopenia and Multilineage Dysplasia/Multilineage Cytopenia and Unilineage Dysplasia. This system utilizing cytopenia-dysplasia scoring at diagnosis enabled comprehensive categorization of low-grade MDS cases that predicted for overall as well as leukemia-free survival. Cytopenia-dysplasia categorization added additional prognostic values to the lower risk IPSS categories. This suggests that a standardized dysplasia scoring system, used in conjunction with cytopenia, could improve diagnostic and prognostic sub-categorization of MDS patients.

A retrospective comparison of autologous and unrelated donor hematopoietic cell transplantation in myelodysplastic syndrome and secondary acute myeloid leukemia: a report on behalf of the Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT).

Hematopoietic cell transplantation (HCT) is an effective treatment for myelodysplasia (MDS) and secondary acute myeloid leukemia (sAML). In this study, outcome of 593 patients with MDS/sAML after autologous and allogeneic HCT from a matched unrelated donor (MUD) were compared. A total of 167 (28%) patients received HCT from MUD without prior chemotherapy (MUD-U). The rest received HCT in first complete remission (CR1) (Autologous (Auto-CR1), n=290 (49%), HCT from MUD (MUD-CR1), n=136 (23%)). Survival at 3 years was best in MUD-CR1 (50%) compared to Auto-CR1 (41%) and MUD-U (40%) (P=0.01). Similarly, disease-free survival was 44% for MUD-CR1 compared to Auto-CR1 (28%) and MUD-U (34%) (P=0.03). Treatment-related mortality was 17% in Auto-CR1 compared to MUD-CR1 (38%) and MUD-U (49%) (P<0.001). Relapse for Auto-CR1 was 62% compared to 24 and 30% for MUD-CR1 and MUD-U, respectively (P<0.001). Outcome was best for patients with low tumor burden transplanted 6-12 months after diagnosis. Factors influencing outcome at 3 years were mainly significant in the first 6 months. Only, relapse after autologous HCT remained constant over time. Outcomes after allogeneic HCT in patients of 20-40 and >40 years were similar. Autologous and Allogeneic HCT from MUD offer the possibility of long-term survival to patients with MDS/sAML.

Adult umbilical cord blood transplantation: a comprehensive review.

Recent registry studies have established umbilical cord blood (UCB) transplantation as a safe and feasible alternative to bone marrow transplantation in adults when no sibling donor is available. There is, however, no gold standard to guide optimal treatment choices. We review here factors leading to the choice of the 'best available donor' and 'best available unit' in the case of UCB. For instance, it is clear that higher cell dose may partially overcome the negative impact of certain histocompatibility leukocyte antigen (HLA) disparities in UCB transplantation, leading us to choose the more closely HLA-matched unit with a cell dose >2.5 x 10(7)/kg. New approaches in adult UCB transplantation are systematically covered, with a quantitative appreciation of the evidence available to date. Reduced intensity conditioning, for example, broadens the range of potential recipients by reducing transplant-related mortality, but suffers from unproven risks and benefits long term. Potential advantages of multiple units over single unit transplants are discussed, with a particular emphasis on confounding factors that impact interpretation. The limited clinical results of ex vivo UCB expansion, the possible benefits of co-infusion of haploidentical cells and controversial issues (e.g. killer immunoglobulin-like receptor matching and alternative graft sources) are also addressed with a debate on the future of UCB transplantation.

Epoetin beta (NeoRecormon) therapy in patients with solid tumours receiving platinum and non-platinum chemotherapy: a meta-analysis.

BACKGROUND: Anaemia is a common complication of chemotherapy (CT), including both non-platinum (Pt)-based as well as Pt-based CT. PATIENTS AND METHODS: Patients from three controlled trials with solid tumours receiving either Pt- or non-Pt-based CT, who had been randomised to epoetin beta treatment or standard care, were included in this meta-analysis (n=255, n=199, respectively), to see if epoetin beta was equally effective in both CT types. The primary endpoint was haemoglobin (Hb) change. Secondary end-points included transfusion requirement, adverse events (AEs), survival, time to tumour progression and thromboembolic events (TEEs). RESULTS: All patients responded rapidly to epoetin beta treatment, showing a median Hb increase of > or = 1 g/dl from baseline at week 4. A median Hb of 12.2, 12.5 and 11.8 g/dl was achieved in all patients, those receiving Pt-based CT and those receiving non-Pt-based CT, respectively, after 16 weeks of treatment. Transfusion risk reductions associated with epoetin beta treatment of 53% (p<0.0001), 61% (p<0.0001) and 26% (non significant) were observed for all patients, Pt- and non-Pt-based CT patients, respectively. Overall, for all three populations, there were no risks identified for tumour progression or overall survival. There was a statistically non-significant incidence of TEEs (5.9% versus 4.5%) and no marked differences were observed between groups for frequency or type of AEs reported. CONCLUSION: The type of CT has no impact on the ability of epoetin beta to rapidly increase Hb in patients with solid tumours and CT-induced anaemia.

CD34+ marrow progenitors from MDS patients with high levels of intramedullary apoptosis have reduced expression of alpha4beta1 and alpha5beta1 integrins.

Excessive intramedullary apoptosis is central in the pathogenesis of myelodysplastic syndromes (MDS). Growth-inhibiting cytokines, the Fas/FasLigand pathway, and autoreactive cytotoxic T-lymphocytes have been identified to be important proapoptotic factors in MDS. In normal hematopoiesis, alpha4beta1 and alpha5beta1 integrin-mediated interactions between progenitors and fibronectin are critical for progenitor cell survival. In this study, we have used flow cytometry to quantify the expression levels of members of the beta1 integrin family on CD34(+) marrow progenitors in 27 untreated patients with MDS, three with s-AML, and 25 control subjects. In MDS, we observed that nonapoptotic progenitors significantly downregulate cell surface expression levels of alpha4 and beta1 integrin chains compared with healthy controls. Downregulation of alpha4, beta1, and also alpha5 was present in MDS patients with > or =25% apoptotic progenitors, irrespective of their French, American, British subcategory. Reduced cell surface expression levels of alpha4, alpha5, and beta1 did also correlate with decreased in vitro adhesiveness to fibronectin fragments. Therefore, our observations suggest that downregulation of alpha4beta1 and alpha5beta1 integrins on CD34(+) progenitors could be a newly identified proapoptotic mechanism in MDS.

A new recurrent inversion, inv(7)(p15q34), leads to transcriptional activation of HOXA10 and HOXA11 in a subset of T-cell acute lymphoblastic leukemias.

Chromosomal translocations with breakpoints in T-cell receptor (TCR) genes are recurrent in T-cell malignancies. These translocations involve the TCRalphadelta gene (14q11), the TCRbeta gene (7q34) and to a lesser extent the TCRgamma gene at chromosomal band 7p14 and juxtapose T-cell oncogenes next to TCR regulatory sequences leading to deregulated expression of those oncogenes. Here, we describe a new recurrent chromosomal inversion of chromosome 7, inv(7)(p15q34), in a subset of patients with T-cell acute lymphoblastic leukemia characterized by CD2 negative and CD4 positive, CD8 negative blasts. This rearrangement juxtaposes the distal part of the HOXA gene cluster on 7p15 to the TCRbeta locus on 7q34. Real time quantitative PCR analysis for all HOXA genes revealed high levels of HOXA10 and HOXA11 expression in all inv(7) positive cases. This is the first report of a recurrent chromosome rearrangement targeting the HOXA gene cluster in T-cell malignancies resulting in deregulated HOXA gene expression (particularly HOXA10 and HOXA11) and is in keeping with a previous report suggesting HOXA deregulation in MLL-rearranged T- and B cell lymphoblastic leukemia as the key factor in leukaemic transformation. Finally, our observation also supports the previous suggested role of HOXA10 and HOXA11 in normal thymocyte development.

Additional prognostic value of bone marrow histology in patients subclassified according to the International Prognostic Scoring System for myelodysplastic syndromes.

PURPOSE: The most recent and powerful prognostic instrument established for myelodysplastic syndromes (MDS) is the International Prognostic Scoring System (IPSS), which is primarily based on medullary blast cell count, number of cytopenias, and cytogenetics. Although this prognostic system has substantial predictive power in MDS, further refinement is necessary, especially as far as lower-risk patients are concerned. Histologic parameters, which have long proved to be associated with outcome, are promising candidates to improve the prognostic accuracy of the IPSS. Therefore, we assessed the additional predictive power of the presence of abnormally localized immature precursors (ALIPs) and CD34 immunoreactivity in bone marrow (BM) biopsies of MDS patients. PATIENTS AND METHODS: Cytogenetic, morphologic, and clinical data of 184 MDS patients, all from a single institution, were collected, with special emphasis on the determinants of the IPSS score. BM biopsies of 173 patients were analyzed for the presence of ALIP, and CD34 immunoreactivity was assessable in 119 patients. Forty-nine patients received intensive therapy. RESULTS: The presence of ALIP and CD34 immunoreactivity significantly improved the prognostic value of the IPSS, with respect to overall as well as leukemia-free survival, in particular within the lower-risk categories. In contrast to the IPSS, both histologic parameters also were predictive of outcome within the group of intensively treated MDS patients. CONCLUSION: Our data confirm the importance of histopathologic evaluation in MDS and indicate that determining the presence of ALIP and an increase in CD34 immunostaining in addition to the IPSS score could lead to an improved prognostic subcategorization of MDS patients.

Activity of oral fludarabine phosphate in previously treated chronic lymphocytic leukemia.

PURPOSE: A prospective, multicenter, open-label phase II clinical trial was conducted to assess the efficacy and safety of oral fludarabine phosphate. Reference to an historical group of patients treated with the intravenous (IV) formulation allowed the investigators to compare the two formulations. PATIENTS AND METHODS: Efficacy was assessed using the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) and National Cancer Institute (NCI) criteria for complete remission (CR), partial remission (PR), stable disease, or disease progression. Safety monitoring included World Health Organization (WHO) toxicity grading for all adverse events. RESULTS: Seventy-eight (96.3%) of 81 recruited patients with previously treated B-cell chronic lymphocytic leukemia (CLL) received 10-mg tablets of fludarabine phosphate to a dose of 40 mg/m(2)/d for 5 days, repeated every 4 weeks, for a total of six to eight cycles. According to IWCLL criteria, the overall remission rate was 46.2% (CR, 20.5%; PR, 25.6%). The comparative figures using NCI criteria were 51.3% (CR, 17.9%; PR, 33.3%). Overall, 30 incidents of severe adverse events were reported for 22 patients. WHO grade 3 or grade 4 hematologic toxicities included granulocytopenia (53.8%), leukocytopenia (28.2%), thrombocytopenia (25.6%), and anemia (24.4%). Gastrointestinal adverse events were more common with the oral formulation than previously reported with IV fludarabine phosphate. However, these events were generally mild to moderate. CONCLUSION: This study demonstrates that oral fludarabine phosphate has similar clinical efficacy to the IV formulation and a safety profile that is both predictable and essentially similar to that of the IV formulation.

Economic analysis of a randomized clinical trial to compare filgrastim-mobilized peripheral-blood progenitor-cell transplantation and autologous bone marrow transplantation in patients with Hodgkin's and non-Hodgkin's lymphoma.

PURPOSE: High-dose chemotherapy (HDC) with peripheral-blood progenitor cell (PBPC) and autologous bone marrow (ABM) transplant (T) has documented survival benefits for relapsed Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL). Treatment costs associated with HDC and its supportive care have restricted its use both on and off clinical trial. In a prospective randomized clinical trial, filgrastim-mobilized PBPCT resulted in faster recovery of bone marrow function, with less hospitalization and supportive care than ABMT. This study was undertaken to analyze the costs of the two strategies using prospectively collected data from a randomized clinical trial that compared filgrastim-mobilized PBPCT versus ABMT. PATIENTS AND METHODS: Clinical results and resource utilization from a randomized clinical trial that compared filgrastim-mobilized PBPCT versus ABMT following carmustine, etoposide, cytarabine, and melphalan (BEAM) HDC for HD and NHL are presented. The trial was performed in six centers in Germany, the United Kingdom, and Belgium. Resource utilization data were used to project costs and Massay Cancer Center (MCC) in the United States incurred the cost of treating the cohort. Costs were projected to the United States, because the economic implications to United States centers are significant, costs of care vary markedly among countries but resource utilization on this trial did not, and a randomized trial is unlikely to be performed in the United States. RESULTS: Fifty-eight patients with relapsed HD or NHL underwent HDC with BEAM. The PBPCT and ABMT groups had similar short-term survival after BEAM. PBPCT patients had a shorter hospitalization (median, 17 v 23 days; P = .002), neutrophil recovery (11 v 14 days; P = .005), platelet recovery to > or = 20 x 10(9)/L (16 v 23 days; P = .02), and days of platelet transfusions (6 v 10; P < .001). Estimated costs were $8,531 for ABM harvest and $5,760 for PBPC collection, including filgrastim mobilization. The total estimated average cost was $59,314 for each ABMT patient versus $45,792 for each PBPCT patient. Cost savings of $13,521 (23%) were due to shorter hospitalizations with less supportive care. CONCLUSION: PBPCT is as safe and more effective than ABMT for HD and NHL in the short term. PBPCT represents a significant cost savings due to lower autograft collection costs, shorter hospital stays, and less supportive care. The savings exceed the costs for filgrastim mobilization and PBPC collection. Actual savings will vary depending on local practice patterns, charges, and costs.

Value of different modalities of granulocyte-macrophage colony-stimulating factor applied during or after induction therapy of acute myeloid leukemia.

PURPOSE: The hematopoietic growth factors (HGFs) introduced into induction chemotherapy (CT) of acute myeloid leukemia (AML) might be of benefit to treatment outcome by at least two mechanisms. HGFs given on days simultaneously with CT might sensitize the leukemic cells and enhance their susceptibility to CT. HGFs applied after CT might hasten hematopoietic recovery and reduce morbidity or mortality. MATERIALS AND METHODS: We set out to evaluate the use of granulocyte-macrophage colony-stimulating factor (GM-CSF; 5 microg/kg) in a prospective randomized study of factorial design (yes or no GM-CSF during CT, and yes or no GM-CSF after CT) in patients aged 15 to 60 years (mean, 42) with newly diagnosed AML. GM-CSF was applied as follows: during CT only (+/-, n = 64 assessable patients), GM-CSF during and following CT (+/+, n = 66), no GM-CSF (-/-, n = 63), or GM-CSF after CT only (-/+, n = 60). RESULTS: The complete response (CR) rate was 77%. At a median follow-up time of 42 months, probabilities of overall survival (OS) and disease-free survival (DFS) at 3 years were 38% and 37% in all patients. CR rates, OS, and DFS did not differ between the treatment groups (intention-to-treat analysis). Neutrophil recovery (1.0 x 10(9)/L) and monocyte recovery were significantly faster in patients who received GM-CSF after CT (26 days v 30 days; neutrophils, P < .001; monocytes, P < .005). Platelet regeneration, transfusion requirements, use of antibiotics, frequency of infections, and duration of hospitalization did not vary as a function of any of the therapeutic GM-CSF modalities. More frequent side effects (eg, fever and fluid retention) were noted in GM-CSF-treated patients predominantly related to the use of GM-CSF during CT. CONCLUSION: Priming of AML cells to the cytotoxic effects of CT by the use of GM-CSF during CT or accelerating myeloid recovery by the use of GM-CSF after CT does not significantly improve treatment outcome of young and middle-aged adults with newly diagnosed AML.

Cytogenetic effects on cells derived from patients with myelodysplastic syndromes during treatment with hemopoietic growth factors.

Hemopoietic growth factors are used with increasing frequency in the treatment of patients with myelodysplastic syndromes (MDS). While a response occurs regularly, it has not been unequivocally resolved whether this effect is due to the stimulation of normal hemopoiesis or to induced maturation of the abnormal clone. To determine whether selective responses to colony-stimulating factors of normal versus abnormal clones occurred, cytogenetic analysis was performed on bone marrow cells of MDS patients before and during in vivo treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF) or recombinant human erythropoietin (rhEPO). A proliferation of additional clones could be demonstrated by karyotypic analysis in one patient during GM-CSF therapy and in two patients during rhEPO treatment. Two patients, initially with completely normal cytogenetics, developed a mixture of normal and abnormal metaphases during treatment. Two patients, initially with all abnormal metaphases, developed normal metaphases during treatment with GM-CSF. A mosaic of normal and abnormal metaphases was present in six patients. The percentage of abnormal metaphases increased in three patients during GM-CSF treatment, and in one patient during rhEPO therapy. The cytogenetic anomalies in one patient persisted after clinical response to treatment, suggesting that GM-CSF enhanced maturation of the abnormal clone. These data indicate that cytokine therapy in MDS may have diverse effects on hematopoiesis.

Detection of P-glycoprotein with a rapid flow cytometric functional assay using Fluo-3: evaluation of sensitivity, specificity and feasibility in multiparametric analysis.

The specificity and sensitivity of a flow cytometric assay simultaneously measuring expression and transport function of the multidrug resistance associated P-glycoprotein (Pgp) was evaluated. The monoclonal antibody (mAb), MRK16 was used to detect phenotypic Pgp expression while Fluo-3-AM was used as a fluorescent substrate in a Pgp functional transport assay. The specificity of the functional assay was examined in two vinblastine selected human leukemic cell lines (K562/VLB2.5 and CCRF-CEM/VLB50) with acquired Pgp overexpression. Downmodulation of Pgp function in these cell lines could be demonstrated with different substances (verapamil, vinblastine, trifluoperazine, cyclosporin A, progesterone and quinidine) and was proven to be consistently higher in the vinblastine selected cells than in their non-selected drug sensitive counterparts. Unexpectedly, modulator activity was also observed in drug sensitive K562 and CCRF-CEM cell lines despite the inability to detect Pgp in those cells by MRK16 flow cytometrically. Low level expression of the MDR1 gene encoding Pgp in sensitive K562 cells was however demonstrated with a sensitive RT-PCR procedure. The small effect of Pgp modulators in non-drug selected cells could therefore be attributed to low level basal expression of Pgp and illustrates the sensitivity of the functional assay. Also, the effect of various Pgp modulators on Pgp function was more pronounced in a subpopulation of Pgp expressing lymphocytes than in lymphocytes which did not express Pgp. Finally, a correlation was found between discrete variations in Pgp expression and Pgp function of CD4+ lymphocytes, underscoring the feasibility of the functional assay in a triple parametric procedure. The triple parametric assay holds promise to detect Pgp expression and function in clinical samples containing mixtures of malignant and non-malignant cells.

Measurement of serum cytokine levels in patients with myelodysplastic syndromes.

Sera of 25 healthy controls and 75 patients suffering from myelodysplastic syndromes (MDS) were investigated for serum concentration of interleukin-1 alpha (IL-1 alpha), IL-3, IL-6, granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), erythropoietin (Epo), and tumor necrosis factor-alpha (TNF-alpha). According to French-American-British (FAB) classification, 21 refractory anemia (RA), seven refractory anemia with ring sideroblasts (RARS), 15 chronic myelomonocytic leukemia (CMML), 12 refractory anemia with excess of blasts (RAEB), and 20 RAEB in transformation (RAEBt) were examined. TNF-alpha levels were inversely correlated with lower levels of hemoglobin concentration (r = -0.31, p = 0.005), irrespective of the requirements for transfusion in anemic MDS patients. Significant differences in TNF-alpha levels between CMML (26.2 +/- 5.9 pg/ml) and the FAB subgroups (16.1 +/- 1.6 pg/ml) were detected. There was an overall inverse relationship between the level of erythropoietin and the degree of anemia, but a wide range of Epo response between patients with similar hemoglobin concentrations. Serum levels of IL-1 alpha and GM-CSF were undetected in most of the patients. In 57% of the samples there were detectable levels of G-CSF, without a correlation of the serum levels with blood cell counts, nor with any of the FAB subcategories. Overall, 29% and 25% of the patient sera exhibited elevated IL-3 and IL-6 levels, respectively. There was no correlation of the serum levels with any of the blood counts, other cytokines, nor FAB subcategories. In conclusion, simple negative feedback mechanism between a specific cytokine and the production of blood cells seems not to be the case in MDS, except for red cell production and erythropoietin concentration. Our data may suggest the involvement of TNF-alpha in the pathogenesis of anemia in MDS.

High CD33-antigen loads in peripheral blood limit the efficacy of gemtuzumab ozogamicin (Mylotarg) treatment in acute myeloid leukemia patients.

Gemtuzumab ozogamicin (Mylotarg) induces remission in approximately 30% of relapsed AML patients. We previously demonstrated that gemtuzumab infusion results in near-complete CD33 saturation in peripheral blood, and that saturating gemtuzumab levels result in continuous binding and internalization of gemtuzumab due to renewed CD33 expression. We now demonstrate that a high CD33-antigen load in peripheral blood is an independent adverse prognostic factor, likely due to peripheral consumption of gemtuzumab. Indeed, CD33 saturation in bone marrow is significantly reduced (40-90% saturation) as compared with CD33 saturation in corresponding peripheral blood samples (>90%). In vitro, such reduced CD33 saturation levels were strongly related with reduced cell kill. Apparently, high CD33-antigen loads in blood consume gemtuzumab and thereby limit its penetration into bone marrow. Consequently, CD33 saturation in bone marrow is reduced, which hampers efficient cell kill. Therefore, gemtuzumab should be administered at higher or repeated doses, or, preferably, after reduction of the leukemic cell burden by classical chemotherapy.

A single course of remission reinduction chemotherapy for acute myelogenous leukemia relapsing after allogeneic bone marrow transplantation is complicated by graft-versus-host disease and followed by sustained complete remission.

Graft-versus-host disease (GVHD) remains a major immunological complication after allogeneic bone marrow transplantation (allo-BMT), but also favors development of the beneficial graft-versus-leukemia (GVL) effect. A patient with AML-M4 (inv (16)) is described, who was given non-myeloablative remission reinduction therapy for leukemic relapse (inv (16), trisomy 8) diagnosed on day 184 after HLA-compatible sibling BMT. On day 236, ie about 6 weeks after completion of this course, a clinical syndrome suggestive of acute GVHD grade 3 had developed. Skin biopsy confirmed the clinical diagnosis of GVHD, with a compatible liver biopsy. Transfusion-associated GVHD was ruled out by analysis of short tandem repeat (STR) alleles in the skin biopsy, revealing alleles from donor and recipient but not from third party origin. Cyclosporin A (CsA) therapy, which had been tapered between days 150 and 175, was resumed, resulting in a favorable response and gradual transition to limited chronic GVHD. The patient has since remained in complete remission with an excellent performance status for more than 40 months, without further chemotherapy. Thus this biopsy proven case of GVHD was induced by marrow donor lymphocytes more than 200 days after transplantation and apparently triggered by remission reinduction chemotherapy. The case indicates that intensive non-myeloablative chemotherapy can cure AML relapsing after allo-BMT. The therapeutic effect in this case probably involved a direct pharmacological suppression of the leukemic clone followed by a GVL effect initiated by donor-derived alloreactive T lymphocytes.

CD40 triggering of chronic lymphocytic leukemia B cells results in efficient alloantigen presentation and cytotoxic T lymphocyte induction by up-regulation of CD80 and CD86 costimulatory molecules.

Freshly collected chronic lymphocytic leukemia B cells (B-CLL cells) are known to be inefficient at stimulating allogeneic T cells, and to lack significant expression of B7 (CD80 and CD86) costimulatory molecules. We investigated the potential of CD40 triggering to up-regulate the expression of adhesion and costimulatory molecules on B-CLL cells, and to enhance their immunogenicity towards allogeneic T cells. B-CLL cells cocultured with human CD40 ligand-expressing mouse fibroblasts rapidly up-regulated CD54 and CD58 adhesion molecules and B7-1 (CD80) and B7-2 (CD86) costimulatory molecules, and acquired a strong stimulatory capacity towards CD4+ as well as isolated CD8+ allogeneic T cells. Costimulation by both CD80 and CD86 proved critical for allogeneic T cell proliferation and CD25 and HLA-DR expression, since these were strongly inhibited by anti-CD80 or anti-CD86 monoclonal antibodies, and completely abrogated by CTLA4-Ig fusion protein, which blocks both CD80 and CD86. B7 costimulation also proved critical for restimulation of primed B-CLL-reactive T cells. Most importantly, priming of purified CD8+ T cells with CD40-triggered allogeneic B-CLL cells resulted in cytotoxic activity against the unstimulated B-CLL cells. These findings raise the possibility that CD40 triggering of B-CLL cells might be exploited in immunotherapeutic protocols.

CD57+/CD28- T cells in untreated hemato-oncological patients are expanded and display a Th1-type cytokine secretion profile, ex vivo cytolytic activity and enhanced tendency to apoptosis.

Three-color flow cytometry immunophenotyping revealed significant increases of CD57+ and CD28- cells among both circulating CD4+ and CD8+ T lymphocytes of untreated hemato-oncological patients (n = 54) as compared to healthy donors (n = 55), with CD57 and CD28 expression on the patients' T cells being largely reciprocal. Marked expansion of CD57+ cells among circulating CD4+ T lymphocytes was frequently detected in patients with chronic leukemia of B cell origin (B-CLL, hairy cell leukemia) but not in patients with chronic myeloid leukemia, suggesting a causal relation with the tumor's major histocompatibility complex class II expression. Using immunomagnetic separation techniques, we further demonstrate that the patients' CD57+/CD28- T cells display a typical Th1-type cytokine secretion profile upon anti-CD3 stimulation, with a markedly higher secretion of the Th1-type cytokines IL-2, IFN-gamma, and TNF-alpha than their CD57-/CD28+ counterparts. Cytotoxic activity of circulating CD8+ T lymphocytes, measured ex vivo in an anti-CD3-redirected assay, was almost exclusively exerted by the CD57+/CD28- subset. Moreover, a marked cytotoxic activity was detected within CD4+CD57+ T cells from some B-CLL patients. Finally, the patients' CD57+/CD28- T cells displayed an increased tendency to apoptosis in culture. Collectively, our results indicate that the expanded CD57+/CD28- T cells in hemato-oncological patients represent differentiated effector cells, similar to their (quantitatively minor) counterpart in healthy donors. The reason for their expansion and their pathophysiologic significance, however, remains unclear.

Circulating myeloid and lymphoid precursor dendritic cells are clonally involved in myelodysplastic syndromes.

Circulating myeloid and lymphoid precursor dendritic cell (pDC) counts were determined in peripheral blood from 22 patients with myelodysplastic syndromes (MDS) by a single-platform flow cytometric protocol. The absolute count of myeloid and lymphoid pDC, as well as their relative number (as proportion of mononuclear cells or total leukocytes) was significantly lower in MDS (n=22) than in healthy controls (n=41). In 11 patients with chromosomal aberrations, purified pDC were examined by interphase fluorescence in situ hybridization. This revealed clonal involvement of myeloid as well as lymphoid pDC in all of them. These data therefore strongly suggest that myeloid and lymphoid pDC share a common precursor. Whether reduced peripheral blood counts of pDC contribute to the immunological abnormalities observed in MDS remains to be investigated.