Prognostic significance of flow-cytometry evaluation of minimal residual disease in children with acute myeloid leukaemia treated according to the AIEOP-AML 2002/01 study protocol.

In children with acute myeloid leukaemia (AML), assessment of initial treatment response is an essential prognostic factor; methods more sensitive than morphology are still under evaluation. We report on the measurement of minimal residual disease (MRD), by multicolour flow-cytometry in one centralized laboratory, in 142 children with newly diagnosed AML enrolled in the Associazione Italiana di EmatoOncologia Pediatrica-AML 2002/01 trial. At the end of the first induction course, MRD was <0·1% in 69, 0·1-1% in 16 and >1% in 51 patients. The 8-year disease-free survival (DFS) of 125 children in morphological complete remission and with MRD <0·1%, 0·1-1% and ≥1% was 73·1 ± 5·6%, 37·8 ± 12·1% and 34·1 ± 8·8%, respectively (P < 0·01). MRD was also available after the second induction course in 92/142 patients. MRD was ≥0·1% at the end of the first induction course in 36 patients; 13 reached an MRD <0·1% after the second one and their DFS was 45·4 ± 16·7% vs. 22·8 ± 8·9% in patients with persisting MRD ≥0·1% (P = 0·037). Multivariate analysis demonstrated that MRD ≥0·1% after first induction course was, together with a monosomal karyotype, an independent adverse prognostic factor for DFS. Our results show that MRD detected by flow-cytometry after induction therapy predicts outcome in patients with childhood AML and can help stratifying post-remission treatment.

Detection and role of minimal disseminated disease in children with lymphoblastic lymphoma: The AIEOP experience.

BACKGROUND: The use of intensive chemotherapy regimens in children with lymphoblastic lymphoma (LBL) has significantly improved outcome, but the salvage rate for these patients is still poor. The aim of this study was to evaluate the prognostic value of minimal disseminated disease (MDD), studied by multiparametric flow cytometry (MFC), in pediatric patients with T- and B-lineage LBL. PROCEDURE: We examined bone marrow (BM) and peripheral blood (PB) samples from a series of 65 children affected by T- (52) and B-lineage (13) LBL using an MFC method; 10 of them were also analyzed for clonality of T-cell receptor gene rearrangements. RESULTS: MDD was detected in 49% (32/65) of BM samples, whereas only 21% (14/65) were positive at standard morphological evaluation. Findings from MFC analyses of paired BM and PB samples were highly concordant. We analyzed the prognostic significance of MDD results detected at diagnosis in morphologically negative patients, as almost all relapsed cases (10/11) did not have any morphological involvement of BM at diagnosis. Using an MDD cut-off level of 3% by FCM (75th percentile), 5-year event-free survival (EFS) was 60% (SE ± 22) for patients with MDD >3% LBL cells versus 83% (SE ± 6) for the remaining patients (P = 0.04). No statistically significant difference in EFS was observed between LBL patients considering all the other clinical characteristics. CONCLUSIONS: Our data demonstrated that MDD studied at diagnosis by MFC could represent a useful prognostic tool in childhood LBL and further application for better stratification is warranted.

Methylation of the IL-12Rbeta2 gene as novel tumor escape mechanism for pediatric B-acute lymphoblastic leukemia cells.

Previous studies have shown that the interleukin-12 receptor beta2 (IL-12Rbeta2) gene is expressed in normal naive, germinal center and memory B cells but not in their malignant counterparts. The aim of this study was to investigate (i) whether the IL-12Rbeta2 gene is silenced in B-cell acute lymphoblastic leukemia (B-ALL) cells, and (ii) what the functional implications of such silencing for tumor growth are. Here, we show that although mature B cells expressed both chains of the IL-12R, normal pro-B and pre-B cells failed to express the IL-12Rbeta2 chain. Similarly, primary tumor cells from pediatric pro-B, early pre-B, and pre-B ALL (30 cases) did not express the IL-12Rbeta2 chain. IL-12Rbeta2 gene silencing in B-ALL was found to depend on methylation of a CpG island in exon 1. Such methylation was not detected in normal early B cells that when differentiated into mature B cells expressed the IL-12Rbeta2 gene. Detection of IL-12Rbeta2 mRNA and protein in the tumorigenic 697 pre-B-ALL cell line allowed to perform functional experiments in severe combined immunodeficient/nonobese diabetic mice receiving 697 cells with or without human recombinant IL-12 (hrIL-12). hrIL-12 administration reduced tumor growth and metastasis through antiproliferative and proapoptotic rather than antiangiogenic, activities. In conclusion, epigenetic silencing of the IL-12Rbeta2 gene represents a novel mechanism of tumor escape for B-ALL cells.

Induction of apoptosis by photoexcited tetracyclic compounds derivatives of benzo[b]thiophenes and pyridines.

The antiproliferative activity, upon UVA irradiation, of two tetracyclic derivatives of benzo[b]thiophenes and pyridines, a benzo[b]thienopyridopyrimidone (1) and a thienocarboline (2), has been investigated in a panel of human tumor cell lines. The two compounds present a remarkable cytotoxicity after UVA irradiation (365 nm), reaching an IC50 of 0.1 microM in the leukaemia cell lines and 0.3-0.5 microM in the solid tumour cell lines. Their effect on the cell cycle was measured by flow cytometry in Jurkat cells. The compounds induce cell cycle perturbations and trigger a massive apoptosis as revealed by the externalisation of Annexin V-targeted residues at the outer plasmatic membrane. Furthermore the drugs induce, upon UVA irradiation significant variations of the mitochondrial potential (Deltapsi(mt)) measured by flow cytometry using the fluorochrome JC-1. In addition we characterized the mitochondrial production of reactive oxygen species (ROS) using the probe dihydroethidine (HE) and the oxidations of the mitochondrial phospholipid cardiolipin using the interacting probe nonyl acridine orange (NAO). Both compounds stimulate the production of ROS, and remarkably induce oxidation of cardiolipin. We have investigated the DNA-binding properties of these two compounds by means of UV-Vis spectroscopy and fluorescence. The two compounds exhibit a low affinity toward the macromolecule. The mode of binding was also investigated by means of flow linear dichroism (LD) which has revealed that the two compounds do not efficiently intercalate into DNA. Finally, the DNA-photocleavaging properties of the test compounds were studied on pBR322 plasmid DNA as a model. Only compound 1 is able to induce a significant production of single strand breaks only after digestion with the base excision repair enzyme Endo III. Altogether these data suggest that DNA is not a preferential target of these molecules and other subcellular structures may be responsible for their high phototoxic activity.

Synthesis and photochemotherapeutic activity of thiopyrano[2,3-e]indol-2-ones.

A series of derivatives of the new ring system thiopyrano[2,3-e]indol-2-one was prepared with the aim of obtaining new photochemotherapeutic drugs. Biological screenings were performed on this new class of photoactivable drugs and a strong antiproliferative effect was observed upon irradiation with UVA light. The compound bearing a methyl substituent at the pyrrole nitrogen resulted as the most interesting showing IC50 in the nanomolar range.

Differential response of linear and angular psoralens in PUVA-induced apoptosis in HL-60 cells.

Treatment of cells with UVA radiation in combination with linear psoralens induces a cell-cycle block and subsequent apoptosis, whereas angular derivatives produce apoptosis without affecting the cellular cycle.

Synthesis, cytotoxicity, and apoptosis induction in human tumor cells by geiparvarin analogues.

A series of geiparvarin analogues modified on the unsaturated alkenyloxy bridge, where a H-atom replaced the 3'-Me group, were synthesized and evaluated against a panel of human tumor cell lines in vitro. These compounds demonstrated a stronger increase in growth inhibitory activity when compared to the parent compound geiparvarin (8). In particular, the activity increased even further in the series of demethylated compounds when a Me substituent in the coumarin moiety is introduced. On the contrary, the same modifications exerted on the parent compound led to an activity reduction. Interestingly, the new derivatives proved to be fully inhibitory to drug-resistant cell lines, thus suggesting that they are not subject to the pump-mediating efflux of antitumor drugs. On the basis of their cytotoxic profiles, the most-active compounds were selected for further biological evaluation. The extracellular acidification rate by the new geiparvarin analogues was measured with the Cytosensor microphysiometer. The new derivatives significantly increased the acidification rate during the 24-48 h of incubation in a concentration-dependent manner. Cell-cycle analysis, evaluated by flow cytometry, revealed a strong apoptotic induction by these compounds confirmed by DNA laddering and observation by electron microscopy. Interestingly, the apoptotic pathway did not appear to be mediated by the activation of caspase-3.