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Background. A systematic review of purine analogs revealed heterogeneity between trials in treatment effects on response and progression free survival, but not survival, perhaps partly due to variations in analysis methods. In addition, combination treatments required evaluation. Design and Methods. Individual patient data were sought for all randomized trials in untreated chronic lymphocytic leukemia which involved a purine analog, but not including antibody therapies. Results. Sixteen trials were found, addressing seven comparisons. Eight trials, with 2753 patients, showed that single agent purine analog improved progression free survival (Odds ratio = 0.71; 95% confidence interval =0.63-0.79). Heterogeneity remained substantial. Three trials, with 1403 patients, showed that progression free survival was further improved by the addition of cyclophosphamide (Odds ratio = 0.54; 0.47-0.62). Fewer data were available on the addition of other drugs to purine analog, and none showed clear benefit. Two trials, with 544 patients, suggested cladribine improved progression free survival compared to fludarabine (Odds ratio = 0.77; 0.63-0.95). No differences were seen in overall survival for any comparisons. Conclusions. Purine analogs, particularly combined with cyclophosphamide, significantly improve progression free survival but not survival. Some groups, such as the elderly, may not see the same benefits and maximising doses may be important for all treatments, including chlorambucil. Longer follow-up, consistent definitions and detailed reporting of trials should be encouraged.

Complementary and alternative medicine (CAM) is common in patients with cancer and its use is steadily increasing over time. We performed a multicenter survey in which the use of CAM in 442 Italian patients with chronic lymphocytic leukemia (CLL), the commonest form of leukemia in Western countries, was assessed. Data were collected by means of a face-to-face standardized questionnaire with several items. Mean age was 69 years; 258 patients (58%) were male and 184 (42%) female. Seventy-three patients (16.5%) were found to be CAM users. The most common CAM therapies were green tea, aloe formulations and high dose vitamins. Predictors of CAM use were female gender, younger age, higher education level, internet availability and newspaper reading. The reasons for CAM popularity among these patients are complex. Given the number of patients combining therapy with CAM and its possible drug interactions, doctor interest as well as patient education about CAM should be improved.

After consuming comparable amounts of ethanol, women have higher blood ethanol concentrations than men, even with allowance for differences in size, and are more susceptible to alcoholic liver disease. Recently, we documented significant "first-pass metabolism" of ethanol due to its oxidation by gastric tissue. We report a study of the possible contribution of this metabolism to the sex-related difference in blood alcohol concentrations in 20 men and 23 women. Six in each group were alcoholics. The first-pass metabolism was determined on the basis of the difference in areas under the curves of blood alcohol concentrations after intravenous and oral administration of ethanol (0.3 g per kilogram of body weight). Alcohol dehydrogenase activity was also measured in endoscopic gastric biopsies. In nonalcoholic subjects, the first-pass metabolism and gastric alcohol dehydrogenase activity of the women were 23 and 59 percent, respectively, of those in the men, and there was a significant correlation (rs = 0.659) between first-pass metabolism and gastric mucosal alcohol dehydrogenase activity. In the alcoholic men, the first-pass metabolism and gastric alcohol dehydrogenase activity were about half those in the nonalcoholic men; in the alcoholic women, the gastric mucosal alcohol dehydrogenase activity was even lower than in the alcoholic men, and first-pass metabolism was virtually abolished. We conclude that the increased bioavailability of ethanol resulting from decreased gastric oxidation of ethanol may contribute to the enhanced vulnerability of women to acute and chronic complications of alcoholism.

Given the lack of effective treatments for Hepatocellular carcinoma (HCC), the development of novel therapeutic approaches is very urgent. Here, siRNAs were delivered to HCC cells by a synthetic polymer containing α,β-poly-(N-2-hydroxyethyl)-D,L-aspartamide-(PHEA) derivatized with diethylene triamine (DETA) and bearing in the side chain galactose (GAL) linked via a polyethylene glycol (PEG) to obtain (PHEA-DETA-PEG-GAL, PDPG). The GAL residue allows the targeting to the asialo-glycoprotein receptor (ASGPR), overexpressed in HCC cells compared to normal hepatocytes. Uptake studies performed using a model siRNA or a siRNA targeted against the enhanced green fluorescence protein, demonstrated the PDPG specific delivery of siRNA to HuH7 cells, a human cellular model of HCC. GAL-free copolymer (PHEA-DETA-PEG-NH2, PDP) or the chemical block of ASGPR, impaired PDPG targeting effectiveness in vitro. The specificity of PDPG delivery was confirmed in vivo in a mouse dorsal skinfold window chamber assay. Functional studies using siRNAs targeting the mRNAs of HCC-related genes (eEF1A1, eEF1A2 and E2F1) delivered by PDPG, significantly decreased HuH7 vitality/number and down regulated the expression of the target genes. Only minor effectiveness was in contrast observed for PDP. In IHH, a human model of normal hepatocytes with reduced ASGPR expression, PDPG barely reduced cell vitality. In a subcutaneous xenograft mouse model of HCC, PDPG-siRNAs reduced HCC tumor growth compared to controls without significant toxic effects. In conclusion, our study demonstrates the valuable potentials of PDPG for the specific delivery of siRNAs targeting HCC-related genes.

Despite the advances in the treatment of rheumatoid arthritis (RA) achieved in the last few years, several patients are diagnosed late, do not respond to or have to stop therapy because of inefficacy and/or toxicity, leaving still a huge unmet need. Tissue-specific strategies have the potential to address some of these issues. The aim of the study is the development of a safe nanotechnology approach for tissue-specific delivery of drugs and diagnostic probes. CD34 + endothelial precursors were addressed in inflamed synovium using targeted biodegradable nanoparticles (tBNPs). These nanostructures were made of poly-lactic acid, poly-caprolactone, and PEG and then coated with a synovial homing peptide. Immunofluorescence analysis clearly demonstrated their capacity to selectively address CD34 + endothelial cells in synovial tissue obtained from human, mouse, and rat. Biodistribution studies in two different animal models of rheumatoid arthritis (antigen-induced arthritis/AIA and collagen-induced arthritis/CIA) confirmed the selective accumulation in inflamed joints but also evidenced the capacity of tBNP to detect early phases of the disease and the preferential liver elimination. The therapeutic effect of methotrexate (MTX)-loaded tBNPs were studied in comparison with conventional MTX doses. MTX-loaded tBNPs prevented and treated CIA and AIA at a lower dose and reduced administration frequency than MTX. Moreover, MTX-loaded tBNP showed a novel mechanism of action, in which the particles target and kill CD34 + endothelial progenitors, preventing neo-angiogenesis and, consequently, synovial inflammation. tBNPs represent a stable and safe platform to develop highly-sensitive imaging and therapeutic approaches in RA targeting specifically synovial neo-angiogenesis to reduce local inflammation.

Purpose: In chronic lymphocytic leukemia (CLL), TP53 mutations are associated with reduced survival and resistance to standard chemoimmunotherapy (CIT). Nevertheless, the clinical impact of subclonal TP53 mutations below 10% to 15% variant allele frequency (VAF) remains unclear. Experimental Design: Using a training/validation approach, we retrospectively analyzed the clinical and biological features of TP53 mutations above (high-VAF) or below (low-VAF) the previously reported 10.0% VAF threshold, as determined by deep next-generation sequencing. Clinical impact of low-VAF TP53 mutations was also confirmed in a cohort (n = 251) of CLL treated with fludarabine-cyclophosphamide-rituximab (FCR) or FCR-like regimens from two UK trials. Results: In the training cohort, 97 of 684 patients bore 152 TP53 mutations, while in the validation cohort, 71 of 536 patients had 109 TP53 mutations. In both cohorts, patients with the TP53 mutation experienced significantly shorter overall survival (OS) than TP53 wild-type patients, regardless of the TP53 mutation VAF. By combining TP53 mutation and 17p13.1 deletion (del17p) data in the total cohort (n = 1,220), 113 cases were TP53 mutated only (73/113 with low-VAF mutations), 55 del17p/TP53 mutated (3/55 with low-VAF mutations), 20 del17p only, and 1,032 (84.6%) TP53 wild-type. A model including low-VAF cases outperformed the canonical model, which considered only high-VAF cases (c-indices 0.643 vs. 0.603, P < 0.0001), and improved the prognostic risk stratification of CLL International Prognostic Index. Clinical results were confirmed in CIT-treated cases (n = 552) from the retrospective cohort, and the UK trials cohort. Conclusions: TP53 mutations affected OS regardless of VAF. This finding can be used to update the definition of TP53 mutated CLL for clinical purposes.

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Hepatocellular carcinoma (HCC) is the sixth most common type of tumor and the second leading cause of tumor-related death worldwide. Liver cirrhosis is the most important predisposing factor for HCC. Available therapeutic approaches are not very effective, especially for advanced HCC, which is the most common form of the disease at diagnosis. New therapeutic strategies are therefore urgently needed. The use of animal models represents a relevant tool for preclinical screening of new molecules/strategies against HCC. However, several issues, including animal husbandry, limit the use of current models (rodent/pig). One animal model that has attracted the attention of the scientific community in the last 15 years is the zebrafish. This freshwater fish has several attractive features, such as short reproductive time, limited space and cost requirements for husbandry, body transparency and the fact that embryos do not show immune response to transplanted cells. To date, two different types of zebrafish models for HCC have been developed: the transgenic zebrafish and the zebrafish xenograft models. Since transgenic zebrafish models for HCC have been described elsewhere, in this review, we focus on the description of zebrafish xenograft models that have been used in the last five years to test new molecules/strategies against HCC.

The hepatitis C virus-positive (HCV+) mixed cryoglobulinaemia (MC) is associated with haematological alterations such as monoclonal B-cell lymphocytosis or non-Hodgkin lymphomas (NHLs). Antiviral therapy for MC, based on interferon and ribavirin, has been shown to be able to eliminate the viral replication as well as the B-cell monoclonal alterations. Many studies have reported the efficacy of direct-acting antivirals (DAAs) in the treatment of HCV+ MC. However, some authors noticed the persistence of haematological diseases despite HCV eradication. To verify the effects of DAAs on B-cell proliferation, we evaluated 67 patients with HCV+ MC. Six patients had an overt NHL and 30% had monoclonal B-lymphocytosis. In 20% of the patients, the mutation L265P of the myeloid differentiation factor 88 (MYD88) gene was detected in peripheral blood. All patients had negative HCV viraemia at week 12; one had a breakthrough, while two cases relapsed. A complete clinical response of vasculitis was seen in 60% of the patients. Among the six patients with NHL, one showed a complete response, whereas in the others there were no changes in the number and size of the nodes. Among the patients carrying a clonal population in peripheral blood, only 22% became negative. These data indicate that DAAs are not able to eliminate the clonal alterations induced by HCV in a large proportion of cases.

We present a laboratory-based prognostic calculator (designated CRO score) to risk stratify treatment-free survival in early stage (Rai 0) chronic lymphocytic leukemia developed using a training-validation model in a series of 1,879 cases from Italy, the United Kingdom and the United States. By means of regression analysis, we identified five prognostic variables with weighting as follows: deletion of the short arm of chromosome 17 and unmutated immunoglobulin heavy chain gene status, 2 points; deletion of the long arm of chromosome 11, trisomy of chromosome 12, and white blood cell count>32.0x103/microliter, 1 point. Low, intermediate and high-risk categories were established by recursive partitioning in a training cohort of 478 cases, and then validated in four independent cohorts of 144/395/540/322 cases, as well as in the composite validation cohort. Concordance indices were 0.75 in the training cohort and ranged from 0.63 to 0.74 in the four validation cohorts (0.69 in the composite validation cohort). These findings advocate potential application of our novel prognostic calculator to better stratify early-stage chronic lymphocytic leukemia, and aid case selection in risk-adapted treatment for early disease. Furthermore, they support immunocytogenetic analysis in Rai 0 chronic lymphocytic leukemia being performed at the time of diagnosis to aid prognosis and treatment, particularly in today's chemo-free era.

Background The effectiveness of therapies for chronic lymphocytic leukemia (CLL), the most common leukemia in Western countries adults, can be improved via a deeper understanding of its molecular abnormalities. Whereas the isoforms of the eukaryotic elongation factor 1A (eEF1A1 and eEF1A2) are implicated in different tumors, no information are available in CLL. Methods eEF1A1/eEF1A2 amounts were quantitated in the lymphocytes of 46 CLL patients vs normal control (real time PCR, western blotting). eEF1A1 role in CLL was investigated in a cellular (MEC-1) and animal model of CLL via its targeting by an aptamer (GT75) or a siRNA (siA1) delivered by electroporation (in vitro) or lipofection (in vivo). Results eEF1A1/eEF1A2 were elevated in CLL lymphocytes vs control. eEF1A1 but not eEF1A2 levels were higher in patients which died during the study compared to those surviving. eEF1A1 targeting (GT75/siA1) resulted in MEC-1 viability reduction/autophagy stimulation and in vivo tumor growth down-regulation. Conclusions The increase of eEF1A1 in dead vs surviving patients may confer to eEF1A1 the role of a prognostic marker for CLL and possibly of a therapeutic target, given its involvement in MEC-1 survival. Specific aptamer/siRNA released by optimized delivery systems may allow the development of novel therapeutic options.

In chronic lymphocytic leukemia (CLL), stabilizing mutations of NOTCH1, affecting up to 10–15% of cases, have been associated to poor prognosis, disease progression and refractoriness to chemotherapy. NOTCH1 mutations are significantly overrepresented in trisomy 12 CLL, a disease subset frequently expressing CD49d, the α4 chain of the very-late-activation-4 integrin, a well-known key regulator of microenviromental interactions, and negative prognosticator in CLL. In the present study, by analysing a wide cohort of 1180 CLL, we observed a very strong association between the presence of NOTCH1 mutations and the expression of CD49d (Po0.0001), occurring also outside the trisomy 12 CLL subset. Using both the MEC-1 CLL-like cells stably transfected with the NOTCH1 intracellular domain and primary CLL cells bearing a mutated or wild-type NOTCH1 gene configuration, we provide evidence that triggering of the NOTCH1 pathway resulted in a positive CD49d expression regulation, which was driven by a NOTCH1-dependent activation of nuclear factot-κB (NF-κB). Consistently, pharmacological inhibition of the NOTCH1 and/or of the NF-κB pathways resulted in impaired NF-κB nuclear translocation with consequent down-modulation of CD49d expression. Altogether, our data link for the first time NOTCH1 mutations to CD49d expression regulation through the involvement of the NF-κB pathway in CLL.

Current approaches for the treatment of chronic lymphocytic leukemia (CLL) have greatly improved the prognosis for survival, but some patients remain refractive to these therapeutic regimens. Hence, in addition to reducing the long-term sideeffects of therapeutics for all leukemia patients, there is an urgent need for novel therapeutic strategies for difficult-to-treat leukemia cases. Due to the cytotoxicity of drugs, the major challenge currently is to deliver the therapeutic agents to neoplastic cells while preserving the viability of non-malignant cells. In this study, we propose a therapeutic approach in which high doses of hydroxychloroquine and chlorambucil were loaded into biodegradable polymeric nanoparticles coated with an anti-CD20 antibody.We first demonstrated the ability of the nanoparticles to target and internalize in tumor B-cells. Moreover, these nanoparticles could kill not only p53-mutated/deleted leukemia cells expressing a low amount of CD20, but also circulating primary cells isolated from chronic lymphocytic leukemia patients. The safety of these nanoparticles was also demonstrated in healthy mice, and their therapeutic effects were shown in a new model of aggressive leukemia. These results showed that anti-CD20 nanoparticles containing hydroxychloroquine and chlorambucil can be effective in controlling aggressive leukemia and provided a rationale for adopting this approach for the treatment of other B-cell disorders. [Figure not available: see fulltext.]

Leukemia cutis (LC) is described as cutaneous infiltration by neoplastic leukocytes into the epidermidis, dermis, or subcutis, resulting in clinically various skin lesions. When the infiltrate is characterized by neoplastic granulocytic precursors, LC is defined as granulocytic sarcoma. Multiple, erythematous, and infiltrated papules and nodules localized on the legs, arms, and trunk are the most common clinical presentation. Here we report a case of granulocytic sarcoma in a patient with a previous diagnosis of acute myeloid leukemia currently in hematological remission.

The limited efficacy of available treatments for hepatocellular carcinoma (HCC) requires the development of novel therapeutic approaches. We synthesized a novel cationic polymer based on α,β-poly-(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) for drug delivery to HCC cells. The copolymer was synthesized by subsequent derivatization of PHEA with diethylene triamine (DETA) and with a polyethylene glycol (PEG) derivative bearing galactose (GAL) molecules, obtaining the cationic derivative PHEA-DETA-PEG-GAL. PHEA-DETA-PEG-GAL has suitable chemical-physical characteristics for a potential systemic use and can effectively deliver a siRNA (siE2F1) targeted against the transcription factor E2F1, a gene product involved in HCC. The presence of GAL residues in the polyplexes allows the targeting of HCC cells that express the asialo-glycoprotein receptor (ASGP-R). In these cells, but not in ASGP-R non-expressing cells, PHEA-DETA-PEG-GAL/siE2F1 polyplexes induce the reduction of the mRNA and protein levels of E2F1 and of E2F1-regulated genes, all involved in the promotion of the G1/S phase transition. This results in a decrease of cell proliferation with a G1/G0 phase cells accumulation. Notably, removal of GAL residue almost completely abrogates the targeting capacity of the developed polyplexes. In conclusion, the generated polyplexes demonstrate the potential to effectively contributing to the development of novel anti-HCC therapeutic approaches via a siRNA-targeted delivery.

First-line treatment for young/fit patients with chronic lymphocytic leukemia (CLL) is the combination of fludarabine, cyclophosphamide and rituximab (FCR), which has improved these patients’ progression-free survival and overall survival,1 but is poorly tolerated by elderly patients or patients with comorbidities.2 Such patients have been historically treated with chlorambucil, which is well tolerated but does not improve survival.3 To improve outcomes, chlorambucil has been combined with anti-CD20 monoclonal antibodies. Three prospective studies4–6 and one retrospective7 one investigated the combination of chlorambucil with rituximab (Chl-R) as front-line treatment for elderly CLL patients or for younger patients unsuitable for fludarabine-based therapies. Overall response rates ranging from 66% to 84% have been reported, with complete response rates of 8–26% and progression-free survival from 16.3 to 34.7 months.

Sara Capolla,1 Chiara Garrovo,2 Sonia Zorzet,1 Andrea Lorenzon,3 Enrico Rampazzo,4 Ruben Spretz,5 Gabriele Pozzato,6 Luis Núñez,7 Claudio Tripodo,8 Paolo Macor,1,9 Stefania Biffi2 1Department of Life Sciences, University of Trieste, 2Institute for Maternal and Child Health – IRCCS “Burlo Garofolo”, Trieste, 3Animal Care Unit, Cluster in Biomedicine (CBM scrl), Trieste, Italy; 4Department of Chemistry “G. Ciamician”, University of Bologna, Bologna, Italy; 5LNK Chemsolutions LLC, Lincoln, NE, USA; 6Department of Medical, Surgery and Health Sciences, University of Trieste, Trieste, Italy; 7Bio-Target, Inc., University of Chicago, Chicago, IL, USA; 8Department of Human Pathology, University of Palermo, Palermo, Italy; 9Callerio Foundation Onlus, Institutes of Biological Researches, Trieste, Italy Abstract: The expectations of nanoparticle (NP)-based targeted drug delivery systems in cancer, when compared with convectional therapeutic methods, are greater efficacy and reduced drug side effects due to specific cellular-level interactions. However, there are conflicting literature reports on enhanced tumor accumulation of targeted NPs, which is essential for translating their applications as improved drug-delivery systems and contrast agents in cancer imaging. In this study, we characterized biodegradable NPs conjugated with an anti-CD20 antibody for in vivo imaging and drug delivery onto tumor cells. NPs’ binding specificity mediated by anti-CD20 antibody was evaluated on MEC1 cells and chronic lymphocytic leukemia patients’ cells. The whole-body distribution of untargeted NPs and anti-CD20 NPs were compared by time-domain optical imaging in a localized human/mouse model of B-cell malignancy. These studies provided evidence that NPs’ functionalization by an anti-CD20 antibody improves tumor pharmacokinetic profiles in vivo after systemic administration and increases in vivo imaging of tumor mass compared to non-targeted NPs. Together, drug delivery and imaging probe represents a promising theranostics tool for targeting B-cell malignancies. Keywords: active targeting, optical imaging, tumor accumulation

OBJECTIVES: Antiviral treatment in chronic hepatitis C (CHC) involves ribavirin, a hemolytic agent. We planned a prospective study to evaluate whether drug-induced iron perturbation is clinically relevant as it relates to therapeutic outcome. METHODS: Iron variables were sequentially assessed in 206 CHC patients undergoing antiviral therapy and were correlated with pretreatment iron status and histology, hemolysis, and therapeutic outcome. RESULTS: At week 1 of therapy, serum iron (SI), transferrin saturation (TS), and serum ferritin (SF) increased markedly in all patients. All iron parameters correlated with hemolysis up to week 4; this correlation was lost for SF at later time points. SF rise during treatment was inversely related to baseline SF and iron deposits in hepatic mesenchymal/Kupffer cells. Both baseline SF and mesenchymal iron significantly correlated with fibrosis at multivariate analysis (P=0.015 and 0.008, respectively). Interestingly, baseline SF, despite good specificity (89%), had low sensitivity in predicting siderosis (25%). During therapy, SI, TS, and hemolysis parameters did not correlate with sustained virological response (SVR), whereas SF rise became an independent predictor of therapeutic response: a 2.5-fold increase of SF at week 12 associated with higher likelihood of SVR (odds ratio 1.91, P=0.032). Accordingly, lack of mesenchymal iron deposits at the baseline biopsy correlated with SVR (odds ratio 3.02, P=0.043). CONCLUSIONS: In CHC, SF is a useful marker for assessing disease duration and progression before starting treatment and for predicting therapeutic response while on therapy. SF rise during antiviral therapy is largely independent of hemolysis and likely indicates activation of macrophages in response to antivirals.

Current B-cell disorder treatments take advantage of dose-intensive chemotherapy regimens and immunotherapy via use of monoclonal antibodies. Unfortunately, they may lead to insufficient tumor distribution of therapeutic agents, and often cause adverse effects on patients. In this contribution, we propose a novel therapeutic approach in which relatively high doses of Hydroxychloroquine and Chlorambucil were loaded into biodegradable nanoparticles coated with an anti-CD20 antibody. We demonstrate their ability to effectively target and internalize in tumor B-cells. Moreover, these nanoparticles were able to kill not only p53 mutated/deleted lymphoma cell lines expressing a low amount of CD20, but also circulating primary cells purified from chronic lymphocitic leukemia patients. Their safety was demonstrated in healthy mice, and their therapeutic effects in a new model of Burkitt's lymphoma. The latter serves as a prototype of an aggressive lympho-proliferative disease. In vitro and in vivo data showed the ability of anti-CD20 nanoparticles loaded with Hydroxychloroquine and Chlorambucil to increase tumor cell killing in comparison to free cytotoxic agents or Rituximab. These results shed light on the potential of anti-CD20 nanoparticles carrying Hydroxychloroquine and Chlorambucil for controlling a disseminated model of aggressive lymphoma, and lend credence to the idea of adopting this therapeutic approach for the treatment of B-cell disorders.