Autoimmune Lymphoproliferative Syndrome (ALPS) Disease and ALPS Phenotype: Are They Two Distinct Entities?

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited disorder of lymphocyte homeostasis classically due to mutation of FAS, FASL, and CASP10 genes (ALPS-FAS/CASP10). Despite recent progress, about one-third of ALPS patients does not carry classical mutations and still remains gene orphan (ALPS-U, undetermined genetic defects). The aims of the present study were to compare the clinical and immunological features of ALPS-FAS/CASP10 versus those of ALPS-U affected subjects and to deepen the genetic characteristics of this latter group. Demographical, anamnestic, biochemical data were retrieved from medical record of 46 ALPS subjects. An enlarged panel of genes (next-generation sequencing) was applied to the ALPS-U group. ALPS-U subjects showed a more complex phenotype if compared to ALPS-FAS/CASP10 group, characterized by multiorgan involvement (P = 0.001) and positivity of autoimmune markers (P = 0.02). Multilineage cytopenia was present in both groups without differences with the exception of lymphocytopenia and autoimmune neutropenia that were more frequent in ALPS-U than in the ALPS-FAS/CASP10 group (P = 0.01 and P = 0.04). First- and second-line treatments were able to control the symptoms in 100% of the ALPS-FAS/CASP10 patients, while 63% of ALPS-U needed >2 lines of treatment and remission in some cases was obtained only after target therapy. In the ALPS-U group, we found in 14 of 28 (50%) patients 19 variants; of these, 4 of 19 (21%) were known as pathogenic and 8 of 19 (42%) as likely pathogenic. A characteristic flow cytometry panel including CD3CD4-CD8-+TCRαß+, CD3+CD25+/CD3HLADR+, TCR αß+ B220+, and CD19+CD27+ identified the ALPS-FAS/CASP10 group. ALPS-U seems to represent a distinct entity from ALPS-FAS/CASP10; this is relevant for management and tailored treatments whenever available.

Multicenter analysis of neutrophil extracellular trap dysregulation in adult and pediatric COVID-19.

Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of this disease, including multisystem inflammatory syndrome in children (MIS-C) and chilblain-like lesions (CLLs), otherwise known as "COVID toes," remains unclear. Studying multinational cohorts, we found that, in CLLs, NETs were significantly increased in serum and skin. There was geographic variability in the prevalence of increased NETs in MIS-C, in association with disease severity. MIS-C and CLL serum samples displayed decreased NET degradation ability, in association with C1q and G-actin or anti-NET antibodies, respectively, but not with genetic variants of DNases. In adult COVID-19, persistent elevations in NETs after disease diagnosis were detected but did not occur in asymptomatic infection. COVID-19-affected adults displayed significant prevalence of impaired NET degradation, in association with anti-DNase1L3, G-actin, and specific disease manifestations, but not with genetic variants of DNases. NETs were detected in many organs of adult patients who died from COVID-19 complications. Infection with the Omicron variant was associated with decreased NET levels when compared with other SARS-CoV-2 strains. These data support a role for NETs in the pathogenesis and severity of COVID-19 in pediatric and adult patients.

Multicenter analysis of neutrophil extracellular trap dysregulation in adult and pediatric COVID-19.

Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of this disease including MIS-C and chilblain-like lesions (CLL), otherwise known as "COVID toes", remains unclear. Studying multinational cohorts, we found that, in CLL, NETs were significantly increased in serum and skin. There was geographic variability in the prevalence of increased NETs in MIS-C, in association with disease severity. MIS-C and CLL serum samples displayed decreased NET degradation ability, in association with C1q and G-actin or anti-NET antibodies, respectively, but not with genetic variants of DNases. In adult COVID-19, persistent elevations in NETs post-disease diagnosis were detected but did not occur in asymptomatic infection. COVID-19-affected adults displayed significant prevalence of impaired NET degradation, in association with anti-DNase1L3, G-actin, and specific disease manifestations, but not with genetic variants of DNases. NETs were detected in many organs of adult patients who died from COVID-19 complications. Infection with the Omicron variant was associated with decreased levels of NETs when compared to other SARS-CoV-2 strains. These data support a role for NETs in the pathogenesis and severity of COVID-19 in pediatric and adult patients. Summary: NET formation and degradation are dysregulated in pediatric and symptomatic adult patients with various complications of COVID-19, in association with disease severity. NET degradation impairments are multifactorial and associated with natural inhibitors of DNase 1, G-actin and anti-DNase1L3 and anti-NET antibodies. Infection with the Omicron variant is associated with decreased levels of NETs when compared to other SARS-CoV-2 strains.

Reduction of CFU-GM and circulating hematopoietic progenitors in a subgroup of children with chronic neutropenia associated with severe infections and delayed recovery.

Myelopoiesis was evaluated in 66 pediatric patients with chronic neutropenia who were positive for anti-neutrophil antibodies (median age at diagnosis: 11 months, median neutrophil count at diagnosis: 419/µl). Other causes of neutropenia were excluded. Bone marrow morphology, clonogenic tests and/or the peripheral blood CD 34+ cell count, and apoptotic rate were evaluated in 61 patients with neutropenia lasting > 12 months or severe infections. The peripheral blood CD 34+ cell count and apoptotic rate were evaluated in five patients with shorter neutropenia. The median follow-up time was 29 months (range 7-180 months). Forty-seven patients (71.2%) had a spontaneous recovery after 7-180 months (median 29 months). The group of patients younger than 24 months at diagnosis (n = 50) had a higher probability of recovery (40/50 vs. 7/16 χ2 p<0.01) with a shorter period of neutropenia (median 26 versus 47 months, Kaplan-Meier analysis p = 0.001). The colony-forming units-granulocyte-macrophage (CFU-GM) were significantly decreased in 26/35 patients (74%) evaluated for clonogenic tests. All patients with normal CFU-GM recovered (9/9 patients); whereas, neutropenia persisted in 12/26 patients with reduced CFU-GM (46%, Pearson χ2 p = 0.02). In 36/55 (65%) patients evaluated by flow cytometry we observed reduced circulating CD34+ cells compared with controls of the same age. An increase in the circulating CD34+ cell apoptotic rate was observed in 28/55 patients (51%). Infections requiring hospitalization were observed in 9/18 (50%; Pearson χ2, p = 0.03) patients with both decreased circulating CD34+ cells and increased CD34+ apoptotic rates. In the group aged < 24 months, we observed a significant correlation between the persistence of neutropenia and decreased circulating CD34+ cells (Pearson χ2 p = 0.008). In conclusion, reduced CFU-GM and circulating hematopoietic progenitors were observed in a subgroup of children with chronic neutropenia who were positive for anti-neutrophil antibodies and had a higher incidence of severe infections and delayed spontaneous remission.

Mycophenolate mofetil for the treatment of children with immune thrombocytopenia and Evans syndrome. A retrospective data review from the Italian association of paediatric haematology/oncology.

Mycophenolate mofetil (MMF) has been shown to be effective in children with immune thrombocytopenia (ITP) and Evans syndrome (ES), but data from larger series and details on the timing of the response are lacking. We evaluated 56 children treated with MMF for ITP (n = 40) or ES (n = 16), which was primary or secondary to autoimmune lymphoproliferative syndrome -related syndrome (ARS). Thirty-five of the 54 evaluable patients (65%) achieved a partial (18%) or complete (46%) response after a median (range) of 20 (7-137) and 37 (7-192) d, respectively. ITP and ES patients responded in 58% and 81% of cases (P = not significant, ns), with complete response in 32% and 81% (P = 0·01), respectively. 60% and 73% of children with primary disease and ARS responded (P = ns) with complete response in 34% and 68% of cases (P = 0·01), respectively. Six of 35 (17%) children relapsed after a median of 283 d (range 189-1036). Limited toxicity was observed in four patients. The median durations of treatment and follow-up were seven and 12·7 months, respectively. This is the largest reported cohort of patients treated with MMF for ITP/ES. The results show that MMF is effective and safe and provides a relatively quick response, suggesting that it has a potential role as an alternative to more aggressive and expensive second/further-line treatments.

Paroxysmal nocturnal hemoglobinuria clones in children with acquired aplastic anemia: a multicentre study.

A multicentre study evaluating the presence of glycosil phosphatidyl-inositol (GPI)-negative populations was performed in 85 children with acquired aplastic anemia (AA). A GPI-negative population was observed in 41% of patients at diagnosis, 48% during immune-suppressive therapy (IST), and 45% in patients off-therapy. No association was found between the presence of a GPI-negative population at diagnosis and the response to IST. In addition, the response rate to IST did not differ between the patients who were GPI-positive at diagnosis and later developed GPI-negative populations and the 11 patients who remained GPI-positive. Two patients with a GPI-negative population >10%, and laboratory signs of hemolysis without hemoglobinuria were considered affected by paroxysmal nocturnal hemoglobinuria (PNH) secondary to AA; no thrombotic event was reported. Excluding the 2 patients with a GPI-negative population greater than 10%, we did not observe a significant correlation between LDH levels and GPI-negative population size. In this study monitoring for laboratory signs of hemolysis was sufficient to diagnose PNH in AA patients. The presence of minor GPI-negative populations at diagnosis in our series did not influence the therapeutic response. As occasionally the appearance of a GPI-negative population was observed at cyclosporine (CSA) tapering or AA relapse, a possible role of GPI-negative population monitoring during IST modulation may need further investigation.

Functional evaluation of circulating hematopoietic progenitors in Noonan syndrome.

Noonan syndrome (NS) is an autosomal dominant disorder, characterized by short stature, multiple dysmorphisms and congenital heart defects. A myeloproliferative disorder (NS/MPD), resembling juvenile myelomonocytic leukemia (JMML), is occasionally diagnosed in infants with NS. In the present study, we performed a functional evaluation of the circulating hematopoietic progenitors in a series of NS, NS/MPD and JMML patients. The different functional patterns were compared with the aim to identify a possible NS subgroup worthy of stringent hematological follow-up for an increased risk of MPD development. We studied 27 NS and 5 JMML patients fulfilling EWOG-MDS criteria. The more frequent molecular defects observed in NS were mutations in the PTPN11 and SOS genes. The absolute count of monocytes, circulating CD34+ hematopoietic progenitors, their apoptotic rate and the number of circulating CFU-GMs cultured in the presence of decreasing concentrations or in the absence of granulocyte-macrophage colony-stimulating factor (GM-CSF) were evaluated. All JMML patients showed monocytosis>1,000/µl. Ten out of the 27 NS patients showed monocytosis>1,000/µl, which included the 3 NS/MPD patients. In JMML patients, circulating CD34+ cells were significantly increased (median, 109.8/µl; range, 44-232) with a low rate of apoptosis (median, 2.1%; range, 0.4-12.1%), and circulating CFU-GMs were hyper-responsive to GM-CSF. NS/MPD patients showed the same flow cytometric pattern as the JMML patients (median, CD34+ cells/µl, 205.7; range, 58-1374; median apoptotic rate, 1.4%; range, 0.2-2.4%) and their circulating CFU-GMs were hyper-responsive to GM-CSF. These functional alterations appeared 10 months before the typical clinical manifestations in 1 NS/MPD patient. In NS, the CD34+ absolute cell count and circulating CFU-GMs showed a normal pattern (median CD34+ cells/µl, 4.9; range, 1.3-17.5), whereas the CD34+ cell apoptotic rate was significantly decreased in comparison with the controls (median, 8.6%; range, 0-27.7% vs. median, 17.6%; range, 2.8-49.6%), suggesting an increased CD34+ cell survival. The functional evaluation of circulating hematopoietic progenitors showed specific patterns in NS and NS/MPD. These tests are a reliable integrative tool that, together with clinical data and other hematological parameters, could help detect NS patients with a high risk for a myeloproliferative evolution.

Transcriptional hallmarks of Noonan syndrome and Noonan-like syndrome with loose anagen hair.

Noonan syndrome (NS) is among the most common nonchromosomal disorders affecting development and growth. NS is genetically heterogeneous, being caused by germline mutations affecting various genes implicated in the RAS signaling network. This network transduces extracellular signals into intracellular biochemical and transcriptional responses controlling cell proliferation, differentiation, metabolism, and senescence. To explore the transcriptional consequences of NS-causing mutations, we performed global mRNA expression profiling on peripheral blood mononuclear cells obtained from 23 NS patients carrying heterozygous mutations in PTPN11 or SOS1. Gene expression profiling was also resolved in five subjects with Noonan-like syndrome with loose anagen hair (NS/LAH), a condition clinically related to NS and caused by an invariant mutation in SHOC2. Robust transcriptional signatures were found to specifically discriminate each of the three mutation groups from 21 age- and sex-matched controls. Despite the only partial overlap in terms of gene composition, the three signatures showed a notable concordance in terms of biological processes and regulatory circuits affected. These data establish expression profiling of peripheral blood mononuclear cells as a powerful tool to appreciate differential perturbations driven by germline mutations of transducers involved in RAS signaling and to dissect molecular mechanisms underlying NS and other RASopathies.

In vitro anti-neuroblastoma activity of saquinavir and its association with imatinib.

Neuroblastoma (NB) has a poor prognosis when in advanced stages, highlighting the need for new therapeutic options. The human immunodeficiency virus (HIV) protease inhibitor saquinavir is active in vitro against chronic myeloid leukaemia cells, in synergy with the tyrosine kinase inhibitor imatinib. Here, we evaluated the effects of saquinavir, alone or in association with imatinib, on cell proliferation (count of viable cells after trypan blue exclusion), apoptosis (Annexin V binding) and invasion (through a transwell membrane coated with Matrigel) in SJ-N-KP, IMR5, AF-8, SK-N-SH and SK-N-BE NB lines, all expressing c-kit and PDGF-R (determined by flow cytometry). Saquinavir showed a dose-dependent anti-proliferative and anti-invasive activity on NB lines, increased by the association with imatinib when the two drugs were utilized at clinically attainable concentrations. The same low saquinavir concentrations inhibited in NB cells the nuclear activation of NF-κB (Western immuno-blotting for nuclear NF-κB p50 and p65). Saquinavir at high concentrations also exerted a pro-apoptotic activity on NB lines, significantly increased by the association with imatinib. In conclusion, saquinavir and imatinib are both drugs utilized for long-term therapies, with good oral bioavailability and a well-known toxicity profile. The anti-NB activity of saquinavir and of its association with imatinib suggests a potential usefulness in the treatment of NB, particularly for remission maintenance.

Prospective study of hemostatic alterations in children with acute lymphoblastic leukemia.

In a group of newly diagnosed acute lymphocytic leukemia (ALL) children we evaluated a number of hemostatic and inflammatory markers at diagnosis and at different time points during chemotherapy for the remission induction to identify alterations in the plasma levels of prothrombotic markers before and during the course of chemotherapy. The following plasma markers were evaluated: thrombin-antithrombin complex (TAT), D-Dimer, plasminogen activator inhibitor 1 (PAI-1), antithrombin, fibrinogen, von Willebrand factor (VWF) antigen and high molecular weight VWF (HMW-VWF) multimers, P-selectin, tumor necrosis factor alpha (TNF-alpha), and interleukin 6 (IL-6). Plasma samples were collected at the following time points: at T0 (baseline) and T1 (+24 days of therapy), T2 (+36 days therapy), and T3 (+64 days therapy). The results show that, at diagnosis, ALL children presented with laboratory signs of increased thrombin generation and fibrin formation (i.e. high TAT and D-dimer levels), fibrinolysis inhibition (i.e. high PAI-1 level), endothelial activation (i.e., high HMW-VWF and soluble P-selectin levels) and inflammation (i.e. high TNF-alpha and IL-6 levels). After starting induction therapy, the thrombin generation markers and inflammatory cytokines significantly decreased. To the opposite, PAI-1 and P-selectin significantly increased, suggesting an insult by chemotherapy on the vascular endothelium. These effects were more evident during steroid administration. Symptomatic venous thromboembolism (VTE) episodes developed in two cases during induction therapy, which did not allow the evaluation of the predictive value for VTE of laboratory markers.

Fibroblasts from patients with Diamond-Blackfan anaemia show abnormal expression of genes involved in protein synthesis, amino acid metabolism and cancer.

BACKGROUND: Diamond-Blackfan anaemia (DBA) is a rare inherited red cell hypoplasia characterised by a defect in the maturation of erythroid progenitors and in some cases associated with malformations. Patients have an increased risk of solid tumors. Mutations have been found in several ribosomal protein (RP) genes, i.e RPS19, RPS24, RPS17, RPL5, RPL11, RPL35A. Studies in haematopoietic progenitors from patients show that haplo-insufficiency of an RP impairs rRNA processing and ribosome biogenesis. DBA lymphocytes show reduced protein synthesis and fibroblasts display abnormal rRNA processing and impaired proliferation. RESULTS: To evaluate the involvement of non-haematopoietic tissues in DBA, we have analysed global gene expression in fibroblasts from DBA patients compared to healthy controls. Microarray expression profiling using Affymetrix GeneChip Human Genome U133A 2.0 Arrays revealed that 421 genes are differentially expressed in DBA patient fibroblasts. These genes include a large cluster of ribosomal proteins and factors involved in protein synthesis and amino acid metabolism, as well as genes associated to cell death, cancer and tissue development. CONCLUSION: This analysis reports for the first time an abnormal gene expression profile in a non-haematopoietic cell type in DBA. These data support the hypothesis that DBA may be due to a defect in general or specific protein synthesis.

In vitro antiproliferative and antimigratory activity of dasatinib in neuroblastoma and Ewing sarcoma cell lines.

Neuroblastoma (NB) and Ewing sarcoma (ES) are neuroectodermal tumors typical of pediatric age that, despite aggressive treatment, still present a poor prognosis when in advanced stages. Studies indicate that c-KIT and platelet-derived growth factor receptor (PDGFR) play a substantial role in the proliferation and survival of NB and ES cells. Dasatinib, an oral multi-targeted inhibitor of several kinases including BCR-ABL and SRC-family kinases, is also active against c-KIT and PDGFR. Here, we evaluated the effect of dasatinib on the NB cell lines SJ-N-KP, SK-N-BE, AF8 and IMR5, and on the ES lines PDE02, TC106 and 6647. Proliferation and viability assays showed that dasatinib exerts an antiproliferative activity with a peak effect occurring at 24 h. After a 24-h exposure to dasatinib at 100 nM, proliferation was inhibited by 29.4+/-5.7% in SJ-N-KP, 41.3+/-11.7% in IMR5, 35.3+/-7.6% in PDE02 and 14+/-10.6% in 6647. Dasatinib did not induce apoptosis in NB and ES cell lines. A possible antimigratory activity of dasatinib was evaluated by scratch test. Dasatinib at 100 nM inhibited the migration of NB and ES cell lines by a mean of 30.2 and 25.3%, respectively. This activity suggests a possible role of dasatinib in inhibiting metastasis and appears of particular interest, given the association between metastatic disease and poor prognosis in these tumors. In conclusion, the cytostatic and antimigratory activity of dasatinib in NB and ES cell lines and the lack of pro-apoptotic activity suggests a possible use for this compound in the treatment of these tumors as a combination with other cytotoxic therapy.

Co-inherited mutations of Fas and caspase-10 in development of the autoimmune lymphoproliferative syndrome.

BACKGROUND: Autoimmune lymphoproliferative syndrome (ALPS) is a rare inherited disorder characterized by defective function of Fas, autoimmune manifestations that predominantly involve blood cells, polyclonal accumulation of lymphocytes in the spleen and lymph nodes with lymphoadenomegaly and/or splenomegaly, and expansion of TCRalphabeta+ CD4/CD8 double-negative (DN) T cells in the peripheral blood. Most frequently, it is due to Fas gene mutations, causing ALPS type Ia (ALPS-Ia). However, other mutations, namely of the FasL gene (ALPS-Ib) and the caspase-10 gene (ALPS-II) are occasionally detected, whereas some patients do not present any known mutations (ALPS-III). Recently, mutations of the NRAS gene have been suggested to cause ALPS-IV. RESULTS: This work reports two patients that are combined heterozygous for single nucleotide substitutions in the Fas and caspase-10 genes. The first patient carried a splice site defect suppressing allele expression in the Fas gene and the P501L substitution in caspase-10. The second had a mutation causing a premature stop codon (Q47X) in the Fas gene and the Y446C substitution in caspase-10. Fas expression was reduced and caspase-10 activity was decreased in both patients. In both patients, the mutations were inherited from distinct healthy parents. CONCLUSION: These data strongly suggest that co-transmission of these mutation was responsible for ALPS.

Inhibition of heat shock proteins (HSP) expression by quercetin and differential doxorubicin sensitization in neuroblastoma and Ewing's sarcoma cell lines.

Neuroblastoma (NB) and Ewing's sarcoma (ES) represent the most common extracranial solid tumors of childhood. Heat shock proteins (HSP) are elevated in cancer cells and their over-expression was correlated to drug-resistance. In this work we identified the HSP by a sensitive proteomic analysis of NB and ES cell lines, then, we studied the HSP response to doxorubicin. Some identified HSP were constitutively more expressed in NB than in ES cells. Doxorubicin-stimulated HSP response only in NB cells. Quercetin was found to inhibit HSP expression depleting heat shock factor 1 (HSF1) cellular stores. Quercetin caused a higher anti-proliferative effect in NB (IC(50): 6.9 +/- 5.8 mumol/L) than in ES cells (IC(50): 85.5 +/- 53.1 mumol/L). Moreover, quercetin caused a very pronounced doxorubicin sensitizing effect in NB cells (241 fold IC(50) decrease) and a moderate effect in ES cells. HSP involvement in NB cells sensitization was confirmed by the silencing of HSF1. Quercetin treatment and HSF1 silencing increased the pro-apoptotic effect of doxorubicin. In conclusion, the higher HSP levels, observed in NB cells, did not confer increased resistance to doxorubicin; on the contrary, HSP inhibition by quercetin or gene silencing caused higher sensitization to doxorubicin. These results may have a potential application in the treatment of NB.

The effects of saquinavir on imatinib-resistant chronic myelogenous leukemia cell lines.

We evaluated the effect of the human immunodeficiency virus (HIV) protease inhibitor saquinavir on the imatinib-sensitive and imatinib-resistant chronic myelogenous leukemia cell lines. Saquinavir, which is also a proteasome blocker, showed dose- and time-related anti-proliferative activity, particularly on the imatinib-resistant lines and a pro-apoptotic effect. Association with imatinib caused a significant increase of activity.

Multilineage engraftment of refrozen cord blood hematopoietic progenitors in NOD/SCID mice.

Seven cord blood (CB) units were tested for their capacity to repopulate irradiated NOD/SCID mice after one or two successive cryopreservation procedures. In primary transplants with frozen or refrozen CB cells we observed equivalent human colonies and percentages of human CD45+ cells, with multilineage engraftment. In secondary transplants flow cytometry and polymerase chain reaction for the a satellite region of chromosome 17 showed equivalent levels of human engraftment. Since CB units have, to date, mainly been stored in individual bags, our results suggest new options for optimizing the timing of infusions of expanded and non-expanded progenitors in transplants.

Flow cytometric evaluation of circulating CD34+ cell counts and apoptotic rate in children with acquired aplastic anemia and myelodysplasia.

OBJECTIVE: Identification of a rapid and noninvasive test for the follow-up of aplastic anemia (AA) patients during immunosuppressive therapy (IST) to evaluate its functional effect on hematopoietic progenitors (HPC) and for early detection of progression to myelodysplasia or relapse. MATERIALS AND METHODS: Absolute count and apoptotic rate (AR) of peripheral blood (PB) CD34+ cells were evaluated by three-color flow cytometry for CD45, CD34, and annexin V in cord blood (CB), normal children, and adults, as well as in pediatric patients with AA at diagnosis and during IST, Fanconi anemia (FA), chronic immune cytopenia, and refractory anemia with excess blasts (RAEB). RESULTS: In normal subjects, the AR of PB CD34+ cells showed a progressive increase (p < 0.05), while their counts decreased (p < 0.05) from birth to adulthood. In very severe AA (vSAA) and severe AA (SAA) at diagnosis, the AR was 91.6% +/- 2.8%, higher than controls (p < 0.05), and PB CD34+ cell count was 2.6 +/- 2.4/microL. In FA patients, the PB CD34+ AR was again significantly increased (54.2% +/- 13.7%) with an absolute count of 3.7 +/- 1.2/microL. Conversely, in RAEB the AR was 11.7% +/- 3.5% and the absolute count 85.1 +/- 48.2/microL (p < 0.05). Chronic immune cytopenias did not significantly differ from controls. CONCLUSIONS: Flow cytometry evaluation of PB CD34+ AR and counts is a noninvasive and feasible first-step method for the differentiation of AA and myelodysplasia (MDS), and it might be useful for monitoring AA during IST to secure the early detection of relapse or transformation to MDS.

Imatinib mesylate (STI571) interference with growth of neuroectodermal tumour cell lines does not critically involve c-Kit inhibition.

A therapeutic role of STI571 (imatinib mesylate) has been anticipated in patients with c-Kit positive neuroectodermal tumors. We examined the efficacy of STI571 to inhibit expansion of c-Kit positive neuroectodermal tumor cell lines in vitro and in a mouse model inoculated with ES (Ewing sarcoma) derived tumor cells, and investigated the molecular mechanism of STI571 action. Eleven tumor lines of ES, PNET (primitive neuroectodermal tumors) and NB (neuroblastoma) were assayed in the presence of 1, 5, 10, 15, 20 or 30 micro M STI571 for 24, 48, 72 h and 7 days. The mechanism of STI571 action was investigated using a microphysiometer cytosensor that determines cellular metabolic rates in the presence of STI571. c-Kit and global protein phosphorylation was assayed by immunoprecipitation and a direct enzyme-linked immunoadsorbent assay after 72 h of 10 micro M STI571. Apoptosis was investigated by propidium iodide (PI), Annexin V staining and by enzymatic activity of caspase-3. Moreover, apoptotic gene expression was investigated using microarray technology. In nude mice, tumor volume and histology were analyzed in STI571 treated and untreated mice, and apoptotic gene expression analysis was performed on tumor masses. A decrease in cell proliferation and increase of cell apoptosis was caused by STI571 in a concentration- and time-dependent manner. Cytosensor microphysiometer and immunoprecipitation experiments demonstrated a time- and concentration-dependent decrease of cellular metabolic activity and global protein dephosphorylation after STI571 exposure. The inhibition by STI571 appeared at least to some extent independent of c-Kit inhibition since cells remained sensitive to SCF stimulation. Tumor volume was significantly reduced in STI571-treated mice compared to tumors from control inoculated non-treated mice. The apoptosis pathway in response to STI571 appeared not to be dependent on caspase activation, while gene expression profiles suggested accumulation of reactive oxygen species (ROS) resulting in cell death after exposure to STI571. The results point to the potential relevance of STI157 for neuroectodermal tumor therapy in view of its inhibitory effect on tumor cell growth, in spite of the observation that the inhibition of the c-Kit signaling pathway is not critically involved.