Poor prognosis for P2RY8-CRLF2 fusion but not for CRLF2 over-expression in children with intermediate risk B-cell precursor acute lymphoblastic leukemia.

Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has achieved an 80% cure rate as a result of a risk-adapted therapy largely based on minimal residual disease (MRD) monitoring. However, relapse is still the most frequent adverse event, occurring mainly in the patients with intermediate MRD levels (intermediate risk, IR), emphasizing the need for new prognostic markers. We analyzed the prognostic impact of cytokine receptor-like factor 2 (CRLF2) over-expression and P2RY8-CRLF2 fusion in 464 BCP-ALL patients (not affected by Down syndrome and BCR-ABL negative) enrolled in the AIEOP-BFM ALL2000 study in Italy. In 22/464 (4.7%) samples, RQ-PCR showed CRLF2 over-expression (≥20 times higher than the overall median). P2RY8-CRLF2 fusion was detected in 22/365 (6%) cases, with 10/22 cases also showing CRLF2 over-expression. P2RY8-CRLF2 fusion was the most relevant prognostic factor independent of CRLF2 over-expression with a threefold increase in risk of relapse. Significantly, the cumulative incidence of relapse of the P2RY8-CRLF2 + patients in the IR group was high (61.1% ± 12.9 vs 17.6% ± 2.6, P<0.0001), similar to high-risk patients in AIEOP-BFM ALL2000 study. These results were confirmed in a cohort of patients treated in Germany. In conclusion, P2RY8-CRLF2 identifies a subset of BCP-ALL patients currently stratified as IR that could be considered for treatment intensification.

Macromolecular composition and drug-loading effect on the delivery of paclitaxel and folic acid from acrylic matrices.

Drug delivery systems based on synthetic polymers are widely employed in the treatment of several pathologies. In particular, the use of implantable devices able to release one or more active principles in a topic site with a controlled delivery kinetic represents an important improvement in this field. However, the release kinetic, that could be affected by different parameters, like polymer composition or chemical nature and initial drug loading, represents one of the problems related to the implantation of delivery systems. In this study, acrylic membranes with different macromolecular composition were prepared and studied analyzing delivery kinetic properties. Drug delivery systems were prepared using as matrix the copolymer poly(methylmethacrylate-co-butylmethacrylate) in three different compositions and folic acid (less hydrophobic) or Paclitaxel (more hydrophobic) as drugs, to evaluate the effect of macromolecular composition and hydrophilicity degree on the release properties. In addition, the effect of the initial drug loading was considered, loading drug delivery systems with four different initial drug percentages. Results showed a direct dependence of kinetics from macromolecular composition, hydrophilicity degree of solutes, and initial drug loading, allowing one to conclude that it is possible to design and to develop drug delivery systems starting from poly(methylmethacrylate-co-butylmethacrylate) matrices with specific properties by varying these three parameters.

Combined drug release from biodegradable bilayer coating for endovascular stents.

In this work, the characterization of a biodegradable bilayer system, used as controlled and combined drug delivery platform, is reported. For this aim, a bilayer system, composed of poly(lactic-co-glycolic acid) and poly(3-hydroxybutyric-co-3-hydroxyvaleric acid), was investigated under physicochemical and functional aspects by evaluating polymer/polymer and polymer/stent material interactions, the kinetic of in vitro degradation, and drug release properties, comparing results with the monolayer reference systems. Obtained results showed that the bilayer system allowed increasing the total amount of eluted Tacrolimus and Paclitaxel drugs with respect to the monolayer systems in the considered testing period and conditions. This evidence was associated to a faster degradation of the tested copolymers in the bilayered configuration, excluding a synergic effect of two drugs on delivery performance. In addition, a macromolecular relaxation process was identified to govern the PLX release from poly(lactic-co-glycolic acid), whereas a pure Fickian diffusion occurred in the delivery of Tacrolimus from poly(3-hydroxybutyric-co-3-hydroxyvaleric acid).

Long-term results of the Italian Association of Pediatric Hematology and Oncology (AIEOP) Studies 82, 87, 88, 91 and 95 for childhood acute lymphoblastic leukemia.

We analyzed the long-term outcome of 4865 patients treated in Studies 82, 87, 88, 91 and 95 for childhood acute lymphoblastic leukemia (ALL) of the Italian Association of Pediatric Hematology and Oncology (AIEOP). Treatment was characterized by progressive intensification of systemic therapy and reduction of cranial radiotherapy. A progressive improvement of results with reduction of isolated central nervous system relapse rate was obtained. Ten-year event-free survival increased from 53% in Study 82 to 72% in Study 95, whereas survival improved from 64 to 82%. Since 1991, all patients were treated according to Berlin-Frankfurt-Muenster (BFM) ALL treatment strategy. In Study 91, reduced treatment intensity (25%) yielded inferior results, but intensification of maintenance with high-dose (HD)-L-asparaginase (randomized) allowed to compensate for this disadvantage; in high-risk patients (HR, 15%), substitution of intensive polychemotherapy blocks for conventional BFM backbone failed to improve results. A marked improvement of results was obtained in HR patients when conventional BFM therapy was intensified with three polychemotherapy blocks and double delayed intensification (Study 95). The introduction of minimal residual disease monitoring and evaluation of common randomized questions by AIEOP and BFM groups in the protocol AIEOP-BFM-ALL 2000 are expected to further ameliorate treatment of children with ALL.

Acrylic copolymers as candidates for drug-eluting coating of vascular stents.

The aim of the present work is the synthesis and characterization of polymer materials showing good adhesion, drug loading, and delivery properties, for potential cardiovascular application. In particular, poly(methylmethacrylate-co-acrylic acid) copolymers are prepared in different compositions by a radical polymerization and investigated as potential materials to coat metallic stents and to carry out a local drug release. Films obtained by dissolving the copolymer in an appropriate organic solvent (also loaded with an anti-restenosis drug, such as tacrolimus) are investigated: physicochemical properties, adhesiveness to metallic stent material, and kinetics of drug release in physiological environment are studied.

Molecularly imprinted poly(ethylene-co-vinyl alcohol) membranes for the specific recognition of phospholipids.

In this paper we concentrated on the possibility of adopting molecular imprinting technology for the preparation of polymeric membranes imprinted with phosphatidylcholine, one of the main phospholipids found in the cell membrane and lipoproteins, via phase inversion, with the intention of applying this method in the ongoing research into the regression of atherosclerosis. The polymer matrix was based on poly(ethylene-co-vinyl alcohol) with an ethylene molar content of 44% and the amount of template molecule was varied so as to obtain three different kinds of membrane. We found that they possessed elevated binding capabilities (78.6% of the initial amount of phosphatidylcholine was found to be adsorbed by the membrane) united with a very high selectivity. Similar phospholipids (phosphatidylinositol and phosphatidylethanolamine) were found to be adsorbed only in very small quantities and mostly due to the porosity of the membrane itself and not to molecular imprinting.

Poly(methyl methacrylate) membranes with controlled porosity for advanced multi-step drug elution.

Most of the systems developed for controlled drug delivery applications depend on membrane technology and their preparation parameters. For some applications, a dense membrane structure used in controlled-release systems can excessively prolong drug release due to the low permeability of the coating to the drug or to the low solubility of the drug in water. In these cases, to increase the drug delivery rate, asymmetric membranes can be prepared by a phase-inversion technique, allowing a different drug delivery approach with respect to dense membranes. In this study, porous poly(methyl methacrylate) membranes with different vacuum degrees were prepared through the phase-inversion process. Ternary homogeneous solutions, obtained by mixing polymer, tetrahydrofuran (THF) and water in the desired amounts, were precipitated by the evaporation of a solvent (THF) and a non-solvent (water) at a controlled temperature and ventilation. Membrane morphology, investigated by scanning electron microscopy, showed it to have a diffuse porosity with a regular arrangement and geometry of pores on the top surface. The porosity degree of the membranes, mainly relying on the starting polymer concentration, was also investigated by the use of the software Image-Pro Plus, indicating the presence of a relationship between porosity and permeability characteristics. Membranes, containing folic acid as a model drug, were tested for their transport characteristics and drug delivery both in diffusive and in convective- diffusive conditions. Transport and release parameters, as well as permeability and effective diffusivity, were found to be dependent on the porosity and vacuum degree, which could be controlled by varying the preparation conditions. Furthermore, these membranes showed high hydraulic permeability and rapid drug release, suggesting their use for applications where an intensive therapy in the first few days is required, followed by a constant and slow release for a longer time (two-step drug delivery).

Composite membranes modified with recognition-able nanobeads as potential adsorbers for purification of biological fluids.

Therapeutic approaches in the clinical field require advanced properties for delivery or recognition of clinical species. The molecular imprinting method allows selective cavities to be inserted into a polymeric material built ""around"" a stamp molecule (template) through polymerization or phase inversion. This study focuses on the application of both methods in the realization of polymeric membranes with selective recognition and adsorption properties. Imprinted polymethacrylic acid (PMAA) particles, exhibiting specific binding sites for cholesterol molecule (template), were realized via precipitation polymerization in the shape of nanobeads and loaded in the bulk or on the surface of methylmethacrylate-acrylic acid P(MMA-co-AA) membranes obtained by the non-solvent induced phase separation (NIPS) technique. In this way, specific cavities were introduced into the membrane network to enhance and specialize uptake performances of the porous membranes taking advantage of the particle characteristics. Rebinding performances towards cholesterol in a physiological environment were tested showing very interesting results: the adsorption of cholesterol molecules from physiological solution was increased by using composite membrane-nanobead systems instead of control membranes (a quantitative increase of 14 mg of cholesterol per g of polymer matrix in respect of blank membrane was detected). The results obtained showed an improved performance of composite membranes, but also an unmodified behavior of loaded nanobeads (with respect to free ones) concerning the recognition capability in aqueous medium, which is the most difficult obstacle to overcome in molecular imprinting. The absolute rebinding capacity and the imprinting efficiency of membranes were in the range (and in some case higher) of other efficient systems, but the real improvement was that molecularly imprinted embranes showed an excellent recognition capacity in physiological medium instead of organic solvents.

Release of proteinic and non-proteinic compounds from novel ionizable hydrogels: Effect of pH on swelling and drug delivery behavior.

Ionizable hydrogels were prepared from new copolymers, poly(vinylalcohol-co-acrylic acid) indicated as P(VA-co-AA), by freezing-thawing processes. These materials are designed as potential controlled delivery devices with specific properties to respond to chemical environment stimuli. The swelling behavior of the P(VA-co-AA) hydrogels was evaluated in response to pH changes in release medium demonstrating a strong dependence with the environmental pH. The release of theophylline (THO) and bovine serum albumin (BSA) incorporated into the hydrogels before freezing-thawing cycles were examined by varying the pH. The release curves of the two different solutes showed a very similar trend depending on the hydrogel porosity and the medium pH. The dependence of THO and BSA release on their size and ionic nature was detected.

Transport properties of EVAl-starch-alpha amylase membranes.

We investigated the influence of various physicochemical parameters on the morphology and time-porosity formation of membranes composed of ethylene-vinyl alcohol, starch, and alpha-amylase. In particular, we determined that (1) it is possible to obtain a membrane with desired porosity by phase inversion in an appropriate water-ethanol mixture and (2) the enzymatic bioerosion is controlled by the amount of alpha-amylase present in the blend. Although no experiments involving drugs were carried out, the delivery properties of the film were determined by measuring the Darcy permeability, the effective diffusivity, and the mean reaction rate of the membranes, relating them to the modality of membrane preparation, the amount of enzyme present within the membrane, and the incubation time of the samples in a buffer solution. Simple theoretical models of the delivery properties of the membranes were developed, leading to predictions that were in good agreement with the experimental results.

Molecularly imprinted bioartificial membranes for the selective recognition of biological molecules. Part 2: release of components and thermal analysis.

Molecularly imprinted membranes imprinted for a large-molecular-weight protein were realised using a blend of natural and synthetic polymers. Bioartificial membranes of synthetic (poly(ethylene-co-vinyl alcohol)-EVAL, Clarene) and biological (Dextran) polymers, molecularly imprinted with alpha-amylase as the template, were prepared and investigated. Dimethyl sulfoxide (DMSO) solutions of the alpha-amylase template, Clarene and Dextran were mixed under stirring in the desired proportions and dipped in DMSO (solvent)/water (non solvent) mixture, to obtain the phase separation. The release of Clarene, Dextran and alpha-amylase in the inversion baths was quantified by spectrophotometric methods and final composition of membranes was established. To study the interactions between the polymer components and between polymeric materials and the template, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were carried out. Results indicated that stable and continuous bioartificial membranes of Clarene and Dextran can be obtained, whereby calorimetric analysis suggested the presence of high interaction between alpha-amylase and the Clarene component.

The relevance of the transfer of molecular information between natural and synthetic materials in the realisation of biomedical devices with enhanced properties.

Past and recent attempts to introduce in synthetic polymers molecular information from natural substances through simple blending, template polymerization and molecular imprinting are reviewed. The most promising approaches that can open the way to the realisation of new materials with improved biocompatibility, antibody- or enzyme-like performances are analysed more deeply. The realisation of bioartificial blends from natural and synthetic polymers, molecularly imprinted nanospheres or membranes that can act as recognition element in (bio)sensing devices, as synthetic enzymes or as key constituents of body fluids purification tools is presented in order to make the reader aware of the fascinating possibilities that these techniques make available to the biomedical science and engineering in the close future. The last part of the paper describes recent attempts to insert recognition elements for large molecules as proteins, DNA segments, viruses or whole cells in synthetic polymer systems, in order to develop new systems in the treatments of diseases and for tissue-engineering applications.

Designing porous bioartificial membranes for clinical use with desired morphological and transport properties by phase inversion control.

Bioartificial membranes of synthetic (poly(ethylene-co-vinyl alcohol) - EVAL, Clarene (R) ) and biological (dextran) polymers with different compositions were prepared through the phase inversion process. Dimethyl sulfoxide (DMSO) solutions of EVAL and dextran were mixed under stirring in the desired proportions and coagulated in water or DMSO (solvent)/water (non-solvent) mixture. Membrane morphologies were shown to be dependent on the synthetic and biological polymer contents and on the coagulation medium composition. Component release during solid membrane formation was evaluated by a UV method and the final composition of the bioartificial membranes established, confirming the successful entrapment of the biological component in the synthetic network. The porosity degree of the bioartificial membranes was also investigated by permeability tests and the effect of morphological characteristics on transport properties was studied. Water flux across the membranes, solute permeability and sieving coefficients were also calculated. The results revealed that the transport properties of these bioartificial membranes, obtained by the phase inversion method, could be controlled by mainly changing the preparation control parameters and the EVAL-dextran ratio.

Acrylic polymeric nanospheres for the release and recognition of molecules of clinical interest.

Cross-linked poly(methylmethacrylate-co-methacrylic acid) nanospheres were imprinted with theophylline through template radical polymerisation in diluted acetonitrile solution. This study will focus on the effect of functional monomer nature used (methylmethacrylate and/or methacrylic acid) in the recognition and in the release of template in order to develop a material with combined properties of drug delivery and rebinding for clinical applications. After template extraction the nanospheres showed satisfactory recognition properties (up to 1mg template/g of polymer). Moreover polymers prepared selectively removed theophylline with a theophylline rebinding of 5.1 times higher than that of caffeine, a compound of similar structure. Drug release properties were also satisfactory (up to 95% of loaded theophylline in 7 days).

Molecularly imprinted bioartificial membranes for the selective recognition of biological molecules.

Membranes of a synthetic (poly(ethylene-co-vinyl alcohol), Clarene) and a biological (dextran) polymer, imprinted with alpha-amylase, of different compositions were prepared by the phase-inversion process. Membrane morphologies were shown to be dependent on the synthetic-biological components composition. The removal of the template from the membranes was performed by extraction with water, while an aqueous solution of alpha-amylase was permeated across the membranes under pressure to obtain the rebinding of the template. The selectivity of alpha-amylase-imprinted membranes was investigated by the same uptake experiment using pepsin, albumin and amyloglucosidase, and the rebinding of these proteins was compared with that of the print molecule. Before and after template extraction and after the rebinding experiment, kinetic measurements of the imprinting molecule were conducted to estimate the activity of the enzyme immobilised in the polymer matrix. Results obtained revealed that the immobilised enzyme maintains a good functionality while in the membrane compared to the free enzyme and the imprinted 'bioartificial' dextran and Clarene membranes, obtained by the phase-inversion method, can establish efficient interaction with alpha-amylase as template molecule, as confirmed by the fair selectivity in rebinding tests.

Transplant-related toxicity and mortality: an AIEOP prospective study in 636 pediatric patients transplanted for acute leukemia.

Hematopoietic stem cell transplantation can cure high-risk acute leukemia (AL), but the occurrence of non-leukemic death is still high. The AIEOP conducted a prospective study in order to assess incidence and relationships of early toxicity and transplant-related mortality (TRM) in a pediatric population. Between 1990 and 1997 toxicities reported in eight organs (central nervous system, heart, lungs, liver, gut, kidneys, bladder, mucosa) were classified into three grades (mild, moderate, severe) and prospectively registered for 636 consecutive children who underwent autologous (216) or allogeneic (420) transplantation, either from an HLA compatible related (294), or alternative (126) donor in 13 AIEOP transplant centers. Overall, 47% of the patients are alive in CR (3-year EFS: 45.2%, s.e.: 2.1), 19% died in CR at a median of 60 days (90-day TRM: 14.3%, s.e.: 1.4), 34% relapsed. Toxicity of any organ, but mucosa and gut, was positively correlated with early death; moderate and severe toxicity to heart, lungs, liver and kidneys significantly increased early TRM, with estimated relative risks of 9.1, 5.5, 2.7 and 2.8, respectively, as compared to absent or mild toxicity. Patients with grade III-IV aGVHD experienced more than double (56% vs. 19%) TRM than patients with grade 0-II aGVHD. A higher cumulative toxicity score, estimating the impact of toxicity on TRM, was significantly associated with transplantation from an alternative donor. Quantitative assessment allowed us to describe the extent to which 'grade' of toxicity and 'type' of involved organs were related to mortality and pre-transplant characteristics and yielded a prognostic score potentially useful to compare different conditioning regimens and predict probability of early death.

Early and late deaths after elective end of therapies for childhood cancer in Italy.

The first cohort of subjects treated for cancer during childhood is now entering adulthood, and it is necessary to determine whether treatment has been sufficient to completely eradicate the neoplastic clone, and whether the cancer itself or treatment-related toxicity may have increased the risk of premature death. For these reasons, long-term survival and causes of death were evaluated in a cohort of subjects treated for childhood cancer who reached the elective end of therapy in continuous remission and were registered until 1992 in the Italian Registry of off-therapy subjects (OTR). The vital status of OTR subjects was ascertained in 1996 by a postal survey through census bureaux; for deceased subjects, the cause of death was defined and compared with the expected rates in the general population. At follow-up, out of 6402 eligible and evaluable subjects, 890 were found to have died; the estimated overall survival at 20 years was 80.7% (95% CI 79.3-82.1). Most of the patients (84.6%) died due to recurrence of the primary cancer, usually within the first 5 years after the OT. The cumulative incidence of death due to recurrence of the primary tumor was greater among subjects treated for solid tumor than among those treated for leukemia/lymphoma (p = 0.0001); in contrast, OT subjects after leukemia and lymphoma were more likely to die due to of medical complications of therapy (p < 0.02). Second cancers were the second most frequent cause of death, with a 12-fold risk compared with the general population; the figures were similar in the 2 cancer groups. Compared with the general population, OT subjects were 32 times more likely than same-age subjects to die. The SMR decreased to 6.1 when only non-cancer deaths were considered. Deaths due to external or avoidable causes occurred among survivors at a rate similar to that of the general population.

Pelvic and paraortic lymph nodal status in advanced ovarian cancer and survival.

BACKGROUND: In order to analyze the prognostic role of node involvement in advanced ovarian cancer, we have analyzed data from a randomized clinical trial on advanced ovarian cancer. METHODS: Cases were 456 women who entered a randomized multicentric clinical trial comparing two cisplatin-based schemes of treatment after cytoreductive surgery for advanced stage III-IV ovarian cancer. They underwent selective pelvic and/or paraortic lymphadenectomy. RESULTS: A total of 161 (35.3%) cases had positive nodes. The frequency of positive nodes was statistically significantly higher in FIGO stage IV than in stage III. Also grade 3 tumors were more likely to have positive nodes than grade 1-2 tumors. No association was observed between nodal status and response to chemotherapy. The 3-year survival was 46.2 (standard error (SE) = 3.4 based on 147 deaths) and 44.6 (SE = 4.4, based on 84 deaths), respectively, in negative and positive node groups. The corresponding values, when the analysis was performed considering only subjects with residual tumor <1 cm or absent, after first-line cytoreductive surgery were 66.2 (SE = 5.7) and 62.4 (SE = 9.6). CONCLUSIONS: We did not find any association between nodal status and survival. Particularly, nodal status was not a prognostic factor for survival in the subgroup of women with residual tumor <1 cm or absent after cytoreductive surgery.