Allogeneic SCT in refractory or relapsed adult ALL is effective without prior reinduction chemotherapy.

We present 60 patients with refractory (n=8) or relapsed (n=52) adult ALL who received allogeneic hematopoietic SCT (HSCT) with (n=41) or without (n=19) prior reinduction chemotherapy. In our center, omission of reinduction is recommended if a suitable donor is promptly available, tumor burden is moderate and disease features suggest a highly aggressive course. Overall survival (OS) of the whole cohort at 1, 2 and 5 years was 42, 33 and 28%, respectively. Leukemia-free survival at 1, 2 and 5 years was 37, 33 and 24%. Deaths were due to relapse (n=25), acute or chronic GVHD (n=7), infections (n=8) or toxicity (n=4). Interestingly, patients who did not receive reinduction before HSCT had better outcomes than patients who received reinduction with OS at 1, 2 and 5 years being 58 vs 34%, 47 vs 25% and 47 vs 18%, respectively (P=0.039). Importantly, even achievement of a second CR after reinduction was not associated with improved survival compared to patients directly proceeding to HSCT. We conclude that patients who undergo HSCT for refractory or relapsed ALL can achieve long-term survival. In selected patients, reinduction chemotherapy can be omitted if immediate HSCT is feasible.

Successful salvage treatment of disseminated cutaneous fusariosis with liposomal amphotericin B and terbinafine after allogeneic stem cell transplantation.

Fusarium infections are associated with high mortality after allogeneic stem cell transplantation. We report on successful treatment of a disseminated cutaneous Fusarium proliferatum infection using liposomal amphotericin B and terbinafine. In vitro susceptibility tests of antifungal drugs suggest that terbinafine is a potent additional antifungal drug for disseminated cutaneous fusariosis.

A pilot study of prophylactic donor lymphocyte infusions to prevent relapse in adult acute lymphoblastic leukemias after allogeneic hematopoietic stem cell transplantation.

The outcome of patients with acute lymphoblastic leukemia (ALL) receiving therapeutic donor lymphocyte infusions (DLIs) in relapse after stem cell transplantation (SCT) is poor. We analyzed the impact of prophylactic DLIs in ALL on chimerism and sustained complete remission (CR). Eighty-five patients with ALL were allografted between January 1998 and September 2004. Twenty-six of them received prophylactic DLIs and were included in this analysis. A total of 12 of 13 patients, who were treated with mixed chimerism (MC) converted to complete donor chimerism (92%) and 10 of 12 patients had persistent donor chimerism and sustained CR during subsequent follow-up. Overall, 18 of 26 patients developed graft-versus-host disease (GVHD) after DLIs (69%), acute GVHD in 46 and chronic GVHD in 62%. After a median follow-up of 42 months (14-72) after SCT, 18 of 26 patients (70%) are alive, 16 in CR. Probability of event-free survival (EFS) for patients treated with DLIs is 62%, and overall survival is 70% at 3 years. Our preliminary data support a graft-versus-leukemia effect of prophylactic DLIs able to induce stable donor chimerism and ongoing CR after SCT. As the accompanying GVHD rate was considerable, careful selection of patients for prophylactic DLIs is mandatory.

Survival after reduced-intensity conditioning is not inferior to standard high-dose conditioning before allogeneic haematopoietic cell transplantation in acute leukaemias.

To assess the role of allogeneic stem cell transplantation (SCT) after reduced-intensity conditioning (RIC) in acute leukaemias, we retrospectively compared 25 patients with acute lymphoblastic leukaemia or acute myelogenous leukaemia after RIC to a historical group of 50 matched controls after high-dose conditioning. Engraftment, acute GvHD and severe infections were comparable in both groups. During the observation period, 1/25 patients (4%) after RIC and 14/50 (28%) after standard SCT died due to transplant-related causes; cumulative nonrelapse mortality (NRM) was 4% after RIC and 24% after standard SCT (P=0.029). In total, 15/25 patients (60%) relapsed after RIC and 20/50 (40%) after standard SCT; probability of disease-free survival (DFS) at 3 years was 43% after RIC and 49% after standard SCT (NS). Overall survival (OS) was 40% after RIC and 37% after standard SCT (NS). Stage of disease, cytogenetic risk profile, acute and chronic GvHD, chimerism status at day 90 and severe infections after transplantation were risk factors with significant impact on DFS and/or OS. In retrospective analysis, patients with acute leukaemias who receive RIC because of contraindications against standard SCT have a comparable outcome to standard SCT, but the higher relapse rate warrants further studies.

Induction of gene expression by 5-Aza-2'-deoxycytidine in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) but not epithelial cells by DNA-methylation-dependent and -independent mechanisms.

The methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR, decitabine) has therapeutic efficacy in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Using microarray analysis, we investigated global changes in gene expression after 5-Aza-CdR treatment in AML. In the AML cell line OCI-AML2, Aza-CdR induced the expression of 81 out of 22 000 genes; 96 genes were downregulated (> or =2-fold change in expression). RT-PCR analysis of 10 randomly selected genes confirmed the changes of expression in AML cells. Similar results were obtained with primary AML and MDS cells after treatment with 5-Aza-CdR ex vivo and in vivo, respectively. In contrast, significantly fewer changes in gene expression and cytotoxicity were detected in normal peripheral blood mononuclear and bone marrow cells or transformed epithelial cells treated with 5-Aza-CdR. Interestingly, only 50.6% of the induced genes contain putative CpG islands in the 5' region. To further investigate the significance of promoter methylation in the induced genes, we analyzed the actual methylation status of randomly selected 5-Aza-CdR-inducible genes. We detected hypermethylation exclusively in the 5' region of the myeloperoxidase (MPO) gene. DNA methylation inversely correlated with MPO expression in newly diagnosed untreated AML patients (P< or =0.004). In contrast, all other analyzed 5-Aza-CdR-inducible genes revealed no CpG methylation in the promoter region, suggesting a methylation-independent effect of 5-Aza-CdR.

Pleural effusion due to Histoplasma capsulatum and idiopathic CD4 lymphocytopenia.

Acute histoplasmosis is usually a benign, self-limited infection in endemic areas. Since protection against Histoplasma capsulatum infection requires specific, cell-mediated immunity, histoplasmosis is well documented in patients with acquired T cell deficiencies e.g. due to HIV infection. We report here for the first time a case of pleural effusion due to H. capsulatum infection in a patient with idiopathic CD4 lymphocytopenia (ICL). A 25-year-old woman presented with chest pain, dyspnea on exertion and a moderate weight loss. Chest X-ray showed a large left pleural effusion, and thoracentesis yielded an exudate. Histologic examination of pleural biopsies identified H. capsulatum. Laboratory tests revealed lymphocytopenia with low CD4+ T cell counts (<100/microl) and a decreased CD4/CD8 ratio. Serology, including HIV, was repeatedly negative. The diagnosis of pleural effusion due to H. capsulatum infection in a patient with idiophatic ICL was established. There was no evidence of any other opportunistic infection. Treatment with itraconazole was initiated and pleural effusion resolved within 2 weeks of treatment. Moreover, the patient was found to have idiopathic thrombocytopenic purpura, as confirmed by the detection of autoantibodies against thrombocytes. In a 1-year follow-up, the patient remained asymptomatic without relapse or any new infection. Treatment with itraconazole was given for 12 months. Because of persistent CD4+ T cell counts below 100/microl, prophylactic antibiotic treatment is continued.

Antisense therapy in oncology: new hope for an old idea?

There is a potential role for antisense oligonucleotides in the treatment of disease. The principle of antisense technology is the sequence-specific binding of an antisense oligonucleotide to target mRNA, resulting in the prevention of gene translation. The specificity of hybridisation makes antisense treatment an attractive strategy to selectively modulate the expression of genes involved in the pathogenesis of diseases. One antisense drug has been approved for local treatment of cytomegalovirus-induced retinitis, and several antisense oligonucleotides are in clinical trials, including oligonucleotides that target the mRNA of BCL2, protein-kinase-C alpha, and RAF kinase. Antisense oligonucleotides are well tolerated and might have therapeutic activity. Here, we summarise treatment ideas in this field, summarise clinical trials that are being done, discuss the potential contribution of CpG motif-mediated effects, and look at promising molecular targets to treat human cancer with antisense oligonucleotides.

Apoptosis: implications of basic research for clinical oncology.

Recent studies have indicated a role for apoptosis in a variety of human diseases. Suppression of apoptosis contributes to carcinogenesis by several mechanisms, including facilitating the accumulation of gene mutations, permitting growth-factor-independent cell survival, promoting resistance to immune-based cytotoxicity, and allowing bypassing of cell-cycle checkpoints, which would normally induce apoptosis. Defects in apoptotic mechanisms also play an important part in resistance to chemotherapy and radiation. The core machinery of the cell death pathway can be reduced to a few critical types of proteins, which are well conserved across animal evolution. This review gives an update on the key players involved in apoptosis as well as an overview of the involvement of apoptosis in disease, and novel diagnostic and therapeutic options derived from the extensive basic research on this topic carried out over the last decade.

Expression and prognostic significance of IAP-family genes in human cancers and myeloid leukemias.

Expression of several inhibitor of apoptosis proteins (IAPs) was investigated in the National Cancer Institute panel of 60 human tumor cell lines, and the expression and prognostic significance of one of these, XIAP, was evaluated in 78 previously untreated patients with acute myelogenous leukemia (AML). XIAP and cIAP1 were expressed in most cancer lines analyzed, with substantial variability in their relative levels. In contrast, NAIP mRNA was not detectable, and cIAP2 was found at the mRNA and protein levels in only 34 (56%) and 5 (8%) of the 60 tumor cell lines analyzed, respectively. Interestingly, XIAP, cIAP1, and cIAP2 mRNA levels did not correlate with protein levels in the tumor lines, indicating posttranscriptional regulation of expression. High levels of XIAP protein in tumor cell lines were unexpectedly correlated with sensitivity to some anticancer drugs, particularly cytarabine and other nucleosides, whereas higher levels of cIAP1 protein levels were associated with resistance to several anticancer drugs. The relevance of XIAP to in vivo responses to cytarabine was explored in AML, making correlations with patient outcome (n = 78). Patients with lower levels of XIAP protein had significantly longer survival (median, 133 versus 52.5 weeks; P = 0.05) and a tendency toward longer remission duration (median, 87 versus 52.5 weeks; P = 0.13) than those with higher levels of XIAP. Altogether, these findings show that IAPs are widely but differentially expressed in human cancers and leukemias and suggest that higher XIAP protein levels may have adverse prognostic significance for patients with AML.

Severe barium sulfate aspirationinto the lung: clinical presentation, prognosis and therapy.

Aspiration of large amounts of barium sulfate is a rare incident during radiographic contrast procedures. Here we describe two patients, who developed acute dyspnea after aspiration of significant amounts of barium into the lung during an upper gastrointestinal radiographic contrast study. The regions of the lung involved depended on the position of the patients during and after aspiration. Arterial blood gas analysis revealed hypoxemia due to alveolar shunt with V/Q distribution disturbances. Bronchoscopy was performed to extract the contrast medium from the tracheobronchial tree. The patients could be discharged a few days later with normal lung function. Long-term prognosis is generally excellent due to the inert character of barium sulfate, even though impressive radiographic findings remain.

IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family. We investigated the antiapoptotic mechanism of Survivin, as well as its expression in 60 human tumor cell lines used for the National Cancer Institute's anticancer drug screening program. In cotransfection experiments, cell death induced by Bax or Fas (CD 95) was partially inhibited (mean +/- SD, 65% +/- 8%) by Survivin, whereas XIAP, another IAP family member, almost completely blocked cell death (93% +/- 4%) under the same conditions. Survivin and XIAP also protected 293 cells from apoptosis induced by overexpression of procaspase-3 and -7 and inhibited the processing of these zymogens into active caspases. In vitro binding experiments indicated that, like other IAP-family proteins, Survivin binds specifically to the terminal effector cell death proteases, caspase-3 and -7, but not to the proximal initiator protease caspase-8. Using a cell-free system in which cytosolic extracts were derived from control- or Survivin-transfected cells and where caspases were activated either by addition of cytochrome c and dATP or by adding recombinant active caspase-8, Survivin was able to substantially reduce caspase activity, as measured by cleavage of a tetrapeptide substrate, AspGluValAsp-aminofluorocoumarin. Similar results were obtained in intact cells when Survivin was overexpressed by gene transfection and caspase activation was induced by the anticancer drug etoposide. Survivin was expressed in all 60 cancer cell lines analyzed, with highest levels in breast and lung cancers and lowest levels in renal cancers. These findings indicate that Survivin, which is commonly expressed in human tumor cell lines, can bind the effector cell death proteases caspase-3 and -7 in vitro and inhibits caspase activity and cell death in cells exposed to diverse apoptotic stimuli. Although quantitative differences may exist, these observations suggest commonality in the mechanisms used by IAP-family proteins to suppress apoptosis.

A single BIR domain of XIAP sufficient for inhibiting caspases.

The inhibitor of apoptosis proteins (IAPs) constitute an evolutionarily conserved family of homologous proteins that suppress apoptosis induced by multiple stimuli. Some IAP family proteins, including XIAP, cIAP-1, and cIAP-2, can bind and directly inhibit selected caspases, a group of intracellular cell death proteases. These caspase-inhibiting IAP family proteins all contain three tandem BIR domains followed by a RING zinc finger domain. To determine the structural basis for caspase inhibition by XIAP, we analyzed the effects of various fragments of this IAP family protein on caspase activity in vitro and on apoptosis suppression in intact cells. The RING domain of XIAP failed to inhibit the activity of recombinant caspases-3 or -7, whereas a fragment of XIAP encompassing the three tandem BIR domains potently inhibited these caspases in vitro and blocked Fas (CD95)-induced apoptosis when expressed in cells. Further dissection of the XIAP protein demonstrated that only the second of the three BIR domains (BIR2) was capable of binding and inhibiting these caspases. The apparent inhibition constants (Ki) for BIR2-mediated inhibition of caspases-3 and -7 were 2-5 nM, indicating that this single BIR domain possesses potent anti-caspase activity. Expression of the BIR2 domain in cells also partially suppressed Fas-induced apoptosis and blocked cytochrome c-induced processing of caspase-9 in cytosolic extracts, whereas BIR1 and BIR3 did not. These findings identify BIR2 as the minimal caspase-inhibitory domain of XIAP and indicate that a single BIR domain can be sufficient for binding and inhibiting caspases.

Dacarbazine and interferon alpha for stage IV malignant melanoma.

Based on encouraging reports of improved response rates with the use of dacarbazine (DTIC) in combination with recombinant interferon alpha-2a (rIFN-alpha-2a) in patients with metastatic malignant melanoma, we conducted a phase II study to determine the efficacy and feasibility of this treatment regimen. 31 patients were treated with an induction dose of rIFN-alpha-2a at 15 MIU/ m2 intravenously (i.v.) daily for 5 days per week for 3 consecutive weeks followed by a continuous maintenance dose of 10 MIU/m2 subcutaneously (s.c.) given 3 days per week; starting on day 22, in conjunction with rIFN-alpha-2a s.c., DTIC was started at a dose of 200 mg/m2 i.v. for 5 continuous days completing a 28-day cycle. Therapy was continued until progression was evidenced. Of the 29 evaluable patients, 7 (24.1%) achieved an objective response (complete plus partial remission) with the highest responses occurring in those patients assessed with pulmonary metastases. The median duration to treatment failure was 2.6 months, while the median survival was 6.9 months. Our data reveal that using rIFN-alpha-2a plus DTIC in combination does not yield better results than those achieved when using DTIC alone. However, 3 of the 7 responders experienced long-term survival ranging up to 42 months. Whether this benefit is achieved by the addition of rIFN-alpha-2a can only be answered by large randomized clinical trials. Conflicting results with some of the current literature are discussed.

Selective interferon-alpha/beta effects on platelet-derived growth factor-stimulated processes in quiescent BALB/c-3T3 fibroblasts.

Interferon-alpha/beta (IFN-alpha/beta) suppresses cell cycle activation by platelet-derived growth factor (PDGF) as well as the induction of the 31-kD (pI) and the 35-kD (pII) proteins in density-arrested BALB/c-3T3 cells. We report that elevation of [Ca2+]i by ionomycin induces the synthesis of the 31-kD protein, but not that of the 35-kD protein. Since IFN blocks the PDGF-induced elevation of [Ca2+]i, these results suggest that IFN treatment may suppress pI induction by impairing this PDGF-activated signal transduction pathway. In contrast, because ionomycin did not induce the 35-kD protein, the suppression by IFN of PDGF-induced pII appears to be mediated via a pathway distinct from that operating in the suppression of pI. In BALB/c-3T3 cells, IFN-alpha/beta did not itself affect the turnover or de novo synthesis of inositol phospholipids and the cellular content of diacylglycerol, nor did IFN block the enhancement of these parameters by PDGF.

E-cadherin distribution in interleukin 6-induced cell-cell separation of ductal breast carcinoma cells.

E-cadherin is expressed in both the ZR-75-1-Tx and the ZR-75-1-Ro sublines of ductal breast carcinoma cells and is concentrated at cell-cell borders as shown by immunocytochemical examination. Free cell borders generally show no or little staining. The localized decrease in E-cadherin expression observed after interleukin 6 (IL-6) treatment of either subline correlates with the increase in free cell borders as IL-6 causes cell-cell separation. As we previously reported, many IL-6-treated ZR-75-1-Tx cells round up and detach from the substratum while ZR-75-1-Ro cells remain adherent and display prominent processes. The results are consistent with the view that E-cadherin expression is not responsible for the marked difference in the IL-6-induced phenotypes in these cell lines, although the localized decrease may play a role in cell-cell separation. ZR-75-1-Tx cells are deficient in desmosomes and show a wider intercellular space than ZR-75-1-Ro cells. Alternative mechanisms involving different aspects of the interlinked cytoskeletal and cell adhesion structures are considered to account for the IL-6-induced antimorphogenetic effect.

Cell-adhesion-disrupting action of interleukin 6 in human ductal breast carcinoma cells.

Recombinant baculovirus-derived interleukin 6 (IL-6) disrupts the attachment of human ductal breast carcinoma subline ZR-75-1-Tx cells to neighbors and the substratum in culture without inhibiting the proliferation of the cells. The nonadherent cells lack pseudopodia and do not translocate directionally. These findings stand in contrast to the earlier observations in the Ro subline of ZR-75-1 cells in which IL-6 induces cell-cell separation without detachment of the cells from the substratum, with the cells displaying pseudopodia, increased motility, and decreased proliferation. The IL-6-induced ZR-75-1-Tx cell detachment and rounding are reversible by incubation of the treated cells in IL-6-free medium for several days. The distinctive changes induced by IL-6 in ZR-75-1-Ro cells are similarly reversible. Either acidic fibroblast growth factor or phorbol 12-myristate 13-acetate can replace serum as a cofactor in IL-6-induced ZR-75-1-Tx cell detachment. Our findings indicate that genetic changes can occur in breast carcinoma cells that through cytokine action markedly affect cell structure, adhesiveness, and motility.