In vitro irradiation system for radiobiological experiments

Background: Although two-dimensional (2-D) monolayer cell cultures provide important information on basic tumor biology and radiobiology, they are not representative of the complexity of three-dimensional (3-D) solid tumors. In particular, new models reproducing clinical conditions as closely as possible are needed for radiobiological studies to provide information that can be translated from bench to bedside. Methods: We developed a novel system for the irradiation, under sterile conditions, of 3-D tumor spheroids, the in vitro model considered as a bridge between the complex architectural organization of in vivo tumors and the very simple one of in vitro monolayer cell cultures. The system exploits the same equipment as that used for patient treatments, without the need for dedicated and highly expensive instruments. To mimic the passage of radiation beams through human tissues before they reach the target tumor mass, 96-multiwell plates containing the multicellular tumor spheroids (MCTS) are inserted into a custom-built phantom made of plexiglass, the material most similar to water, the main component of human tissue. Results: The system was used to irradiate CAEP- and A549-derived MCTS, pre-treated or not with 20 \u3bcM cisplatin, with a dose of 20 Gy delivered in one session. We also tested the same treatment schemes on monolayer CAEP and A549 cells. Our preliminary results indicated a significant increment in radiotoxicity 20 days after the end of irradiation in the CAEP spheroids pre-treated with cisplatin compared to those treated with cisplatin or irradiation alone. Conversely, the effect of the radio- chemotherapy combination in A549-derived MCTS was similar to that induced by cisplatin or irradiation alone. Finally, the 20 Gy dose did not affect cell survival in monolayer CAEP and A549 cells, whereas cisplatin or cisplatin plus radiation caused 100% cell death, regardless of the type of cell line used. Conclusions: We set up a system for the irradiation, under sterile conditions, of tumor cells grown in 3-D which allows for the use of the same dose intensities and schedules utilized in clinical practice. This irradiation system, coupled with 3-D cell cultures, has the potential to generate information that could be used to individually tailor radiotherap

Conditioning regimens in acute myeloid leukemia.

Current intensive consolidation chemotherapy for patients with acute myeloid leukemia (AML) produces median remission duration of 12-18 months, with less than 30% of patients surviving 5 years free of disease. Post-remission therapy is necessary to prevent relapse in most patients with AML; therefore, the aim of post-remission treatment is to eradicate the minimal residual disease. Nevertheless, the optimal form of treatment is still under debate. The choice among the possible approaches (intensive chemotherapy, autologous or allogeneic hematopoietic stem cell transplantation) relies on two main factors: the expected risk of relapse, as determined by biological features, and expected morbidity and mortality associated with a specific option. In this review, we focus on the different preparative regimens before autologous and allogeneic hematopoietic stem cell transplantation in patients with AML, stressing the importance of an adequate conditioning regimen as a mandatory element of a successful AML therapy, in both the allogeneic and the autologous transplant setting

Data sets for studies of flow and dispersion in complex terrain: IV) The 'Rushil' and 'Rusval' wind tunnel experiments (concentration data)

Consiglio Nazionale delle Ricerche - Biblioteca Centrale -. P.le Aldo Moro, 7, Rome / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

The role of the immunosuppressive microenvironment in acute myeloid leukemia development and treatment.

Functional interplay between acute myeloid leukemia (AML) cells and the bone marrow microenvironment is a distinctive characteristic of this hematological cancer. Indeed, a large body of evidence suggests that proliferation, survival and drug resistance of AML are sustained and modulated by the bone marrow immunosuppressive microenvironment, where both innate and adaptive immune responses are profoundly deregulated. Furthermore, the presence of a number of different immunosuppressive mechanisms results in massive immune deregulation, which causes the eventual escape from natural immune control. Modulating the immune system, as documented by 40 years of stem cell transplantation, may improve survival of AML patients, as the immune system is clearly able to recognize and attack leukemic cells. The understanding of the factors responsible for the escape from immune destruction in AML, which becomes more prominent with disease progression, is necessary for the development of innovative immunotherapeutic treatment modalities in AML

Clinical Relevance of ABCB1, ABCG2, and ABCC2 Gene Polymorphisms in Chronic Myeloid Leukemia Patients Treated With Nilotinib

Tyrosine kinase inhibitors (TKIs) have radically changed the outcome of chronic myeloid leukemia (CML) patients in the last 20 years. Moreover, the advent of second generation TKIs, namely nilotinib and dasatinib, have largely increased the number of CML patients achieving deep and sustained molecular responses. However, the possible mechanisms capable of influencing the maintenance of the long-term molecular response are not yet fully known and understood. In this light, polymorphisms in MDR-ABC transporters may influence the efficacy and safety of TKIs. In this study, we examined seven single nucleotide polymorphisms (SNPs) in four ABC transporter genes: ABCC1 rs212090 (5463T>A), ABCC2 rs3740066 (3972C>T), ABCC2 rs4148386 G>A, ABCC2 rs1885301 (1549G>A), ABCG2 rs2231137 (34G>A), ABCG2 rs2231142 G>C, ABCB1 rs1045642 (3435C>T), to determine their effect on the achievement and/or loss of molecular response in 90 CML patients treated with nilotinib. We found that ABCC2 rs3740066 CC and CT as well as the ABCB1 rs1045642 TT genotypes correlated with a higher probability to achieve MR3 in a shorter time (p=0.02, p=0.004, and p=0.01), whereas ABCG2 rs2231137 GG was associated with lower probability of MR3 achievement (p=0.005). Moreover, ABCC2 rs3740066 CC genotype, the ABCB1 rs1045642 CC and TT genotypes were positively correlated with MR4 achievement (p=0.02, p=0.007, and p=0.003). We then generated a predictive model incorporating the information of four genotypes, to evaluate the combined effect of the SNPs. The combination of SNPs present in the model affected the probability and the time to molecular response. This model had a high prognostic significance for both MR3 and MR4 (p=0.005 and p=0.008, respectively). Finally, we found ABCG2 rs2231142 GG genotype to be associated with a decrease risk of MR3 loss. In conclusion, MDR-transporters SNPs may significantly affect the achievement and loss of molecular response in CML patients treated with nilotinib

Environmental nanoparticles are significantly over-expressed in acute myeloid leukemia. PMID: 27669365 Similar articles

The increase in the incidence of acute myeloid leukemia (AML) may suggest a possible environmental etiology. PM2.5 was declared by IARC a Class I carcinogen. No report has focused on particulate environmental pollution together with AML. The study investigated the presence and composition of particulate matter in blood with a Scanning Electron Microscope coupled with an Energy Dispersive Spectroscope, a sensor capable of identifying the composition of foreign bodies. 38 peripheral blood samples, 19 AML cases and 19 healthy controls, were analyzed. A significant overload of particulate matter-derived nanoparticles linked or aggregated to blood components was found in AML patients, while almost absent in matched healthy controls. Two-tailed Student’s t-test, MANOVA and Principal Component Analysis indicated that the total numbers of aggregates and particles were statistically different between cases and controls (MANOVA, P < 0.001 and P = 0.009 respectively). The particles detected showed to contain highly-reactive, non-biocompatible and non-biodegradable metals; in particular, micro- and nano-sized particles grouped in organic/inorganic clusters, with statistically higher frequency of a subgroup of elements in AML samples. The demonstration, for the first time, of an overload of nanoparticles linked to blood components in AML patients could be the basis for a possible, novel pathogenetic mechanism for AML developmen

Effect of small molecules modulating androgen receptor (SARMs) in human prostate cancer models.

"The management of hormone-refractory prostate cancer represents a major challenge in the therapy of this tumor, and identification of novel androgen receptor antagonists is needed to render treatment more effective. We analyzed the activity of two novel androgen receptor antagonists, (S)-11 and (R)-9, in in vitro and in vivo experimental models of hormone-sensitive or castration-resistant prostate cancer (CRPC). In vitro experiments were performed on LNCaP, LNCaP-AR, LNCaP-Rbic and VCaP human prostate cancer cells. Cytotoxic activity was assessed by SRB and BrdU uptake, AR transactivation by luciferase reporter assay and PSA levels by Real Time RT-PCR and ELISA assays. Cell cycle progression-related markers were evaluated by western blot. In vivo experiments were performed on SCID mice xenografted with cells with different sensitivity to hormonal treatment. In hormone-sensitive LNCaP and LNCaP-AR cells, the latter expressing high androgen receptor levels, (R)-9 and (S)-11 exhibited a higher cytotoxic effect compared to that of the reference compound ((R)-bicalutamide), also in the presence of the synthetic androgen R1881. Furthermore, the cytotoxic effect produced by (R)-9 was higher than that of (S)-11 in the two hormone-resistant LNCaP-AR and VCaP cells. A significant reduction in PSA levels was observed after exposure to both molecules. Moreover, (S)-11 and (R)-9 inhibited DNA synthesis by blocking the androgen-induced increase in cyclin D1 protein levels. In vivo studies on the toxicological profile of (R)-9 did not reveal the presence of adverse events. Furthermore, (R)-9 inhibited tumor growth in various in vivo models, especially LNCaP-Rbic xenografts, representative of recurrent disease. Our in vitro results highlight the antitumor activity of the two novel molecules (R)-9 and (S)-11, making them a potentially attractive option for the treatment of CRPC.. .

Prognostic Impact of TS, MTHFR and XRCC1 Genetic Variants in 113 Patients with Myelodysplastic Syndromes

Background: Several studies have suggested that genetic variability related with single nucleotide polymorphisms (SNPs) of the BER system, DNA synthesis and folate-metabolizing pathway genes could modulate DNA repair capacity. Moreover, these genes are supposed to be related to cancer risk. However, the prognostic impact of the association of individual and/or combined genetic variants in patients with myelodysplastic syndromes (MDS) remains undetermined. Methods: We genotyped 113 MDS patients, 54 with IPSS low/int-1 receiving only best supportive care (BSC group) and 59 with IPSS int-2/high treated with azacitidine (AZA-group), for the following polymorphisms: XRCC1 194 and 399, APE1 148, XRCC3 241, TS5'-UTR (2R/3R and G/C) and 3'-UTR (6bp+/6bp-), MTHFR 677 and 1298. Genomic DNA was analyzed by High Resolution Melting assay and restriction digests of PCR products. Overall survival (OS) was calculated using the Kaplan-Meier estimate probabilities, and differences between survival curves were analyzed by the log-rank test. Multivariate analyses were performed using the Cox method. Results: For all the target genes, the distribution of genotypes was consistent with the Hardy-Weinberg equilibrium. Among the baseline characteristics analyzed (age, sex, diagnosis according to WHO, hemoglobin) there was no statistically significant difference in the genotype distribution of studied polymorphisms. In the BSC group, the variants XRCC1 399 GG [Hazard ratio (HR)= 7.07; p=0.02], -6/-6 of TS3’-UTR (HR=4.65; p=0.05), 2R/3G, 3C/3G, 3G/3G of TS5’-UTR (HR=11.44; p=0.02) and TT of MTHFR 677 (HR=67.12; p<0.001), were associated with a statistically significant adverse clinical outcome compared to variant alleles (Table 1). This is consistent with the enzymatic activity reduction attributed to these genetic variants. Multivariable regression model analysis was also performed in the AZA group for the same genetic variants. We found similar results for the association between XRCC1 399 GG(HR=5.71 p=0.002), TS3’-UTR +6/+6(HR=0.097 p=0.004), MTHFR 677 TT (HR=8.58 p<0.001) and survival, but not for SNPs in TS5’-UTR (Table 2). Finally, we performed an exploratory analysis to investigate the combined effect of the unfavorable genotypes on survival. In the BSC group, the 3-year OS was 33% for those patients with ≥2 variant alleles, as compared to 62.5%, and 100%, respectively, for those with 2 or 0/1 variant alleles. The predictive role of the adverse genotypes combination on survival was confirmed also in the AZA group, suggesting that patients with a higher number of genetic variants had a shorter survival. Interestingly, when we compared survival of patients with adverse genotypes between BSC and AZA groups, we did not find any statistically significant difference between the 2 groups (Kaplan-Meyer and Log-rank test). Therefore, we speculated that azacitidine could give a survival advantage to patients with unfavorable genetic variants, independently from IPSS at diagnosis. Conclusion: Our study reveals, for the first time, an associations between genetic variants in TS, MTHFR and XRCC1 genes, BSC, azacitidine and survival in MDS patients. If confirmed, they could represent new prognostic markers able to provide guidance for clinical management of MDS patients. In particular, the presence of adverse genotypes could represent a biomarker to treat patients with low-risk IPSS with azacitidine, if confirmed on larger series. Further studies with larger population are needed to validate these associations, especially in SNPs with low variant allele frequency

TS, MTHFR AND XRCC1 GENETIC VARIANTS INFLUENCE THE OUTCOME OF MDS PATIENTS IRRESPECTIVELY OF IPSS RISK

TS, MTHFR AND XRCC1 GENETIC VARIANTS INFLUENCE THE OUTCOME OF MDS PATIENTS IRRESPECTIVELY OF IPSS RISK G. Visani1, F. Loscocco1, A. Ruzzo2, M. Voso3, E. Fabiani3, C. Finelli4, F. Graziano1, S. Barulli1, A. Volpe5, D. Magro6, P. Piccaluga7, F. Fuligni7, E. Gabucci1, E. Giacomini2, M. Vignetti8, P. Fazi8, A. Piciocchi8, M. Rocchi9, M. Magnani2, A. Isidori1 1AORMN, Hematology and Stem Cell Transplant Center, Pesaro, Italy; 2University of Urbino, Biochemistry and Molecular Biology, Urbino, Italy; 3Tor Vergata University, Hematology, Rome, Italy; 4S.Orsola-Malpighi Hospital, Hematology, Bologna, Italy; 5S.G. Moscati Hospital, Hematology and Transplant Center, Avellino, Italy; 6Pugliese-Ciacco Hospital, Hematology, Catanzaro, Italy; 7S.Orsola-Malpighi Hospital Bologna University, Experimental Diagnostic and Specialty Medicine, Bologna, Italy; 8Fondazione GIMEMA, Gruppo Italiano Malattie Ematologiche dell’Adulto, Rome, Italy; 9University of Urbino, Institute of Biomathematics, Urbino, Italy Background: Loss of genomic integrity is thought to be one of the underlying causes of myelodysplastic syndromes (MDS). We investigated the association between genetic variants in genes encoding main enzymes of BER pathway, DNA synthesis and folate metabolism and survival in MDS patients. Methods: We genotyped 113 MDS patients, 54 with IPSS low/int-1 receiving only best supportive care (BSC) (low risk group) and 59 with IPSS int-2/high treated with azacitidine (high risk group), for the following polymorphisms: XRCC1 194and 399, XRCC3 241, TS5’-UTR (2R/3R and G/C) and 3’-UTR (6bp+/6bp-), MTHFR 677 and1298, APE1 148. OS was calculated using the Kaplan-Meier estimate probabilities, and differences between survival curves were analyzed by the log-rank test. The association between genetic variants and survival was assessed using stepwise logistic regression model. Results: The median period of observation was 27 months for low/ int-1 risk group and 13 months for int-2/high risk group. In the low risk group, XRCC1 399 GG [Hazard ratio (HR)=4.65; p=0.05], TS3’-UTR -6/-6 (HR=7.07; p=0.02), TS5’-UTR 2R/3G, 3C/3G, 3G/3G (HR=11.44; p=0.02) and MTHFR 677 TT (HR=67.12; p=0.0001) genotypes were associated with a shorter survival if compared to the reference group of variant alleles. We then analyzed the high risk group, and we found that 3 out of the 4 genotypes associated with an adverse outcome in the lower group were still significantly associated to lower survival [XRCC1 399 GG (HR=5.71 p=0.002), MTHFR677 TT (HR=8.58 p=0.0001), TS3’-UTR +6/+6 (HR=0.097 p=0.004), this latter with a protective role]. Finally, we performed an exploratory analysis to investigate the effect on survival of the combination of the unfavorable genotypes to each other. In the low risk group, the 3-year OS was 33% for those patients with ≥2 variant alleles, as compared to 62.5%, and 100%, respectively, for those with 2 or 0/1 variant alleles. The predictive role of the adverse genotypes combination on survival was confirmed also in the high risk group. Interestingly, treatment with azacitidine conferred a survival advantage to patients with unfavorable genetic variants, resulting in the absence of a statistical difference in survival between low-risk patients treated with BSC only and high-risk patients treated with azacitidine. Conclusions: These data suggest, for the first time, that genetic variants in TS, MTHFR and XRCC1 genes could independently modulate OS of MDS patients, representing a possible new prognostic biomarkers able to provide guidance for clinical management of MDS patients