Effect of trehalose on cryopreservation of pure peripheral blood stem cells.

Stem cells are an important tool for the study of hematopoiesis. Despite developments in cryopreservation, post-thaw cell death remains a considerable problem. Cryopreservation protocol should limit cell damage due to freezing and ensure the recovery of the functional cell characteristics after thawing. Thus, the use of cryoprotectants is essential. In particular, the efficacy of trehalose has been reported for clinical purposes in blood stem cells. The aim of the current study was to establish an efficient method for biological research based on the use of trehalose, to cryopreserve pure peripheral blood stem cells. The efficacy of trehalose was assessed in vitro and the cell viability was evaluated. The data indicate that trehalose improves cell survival after thawing compared with the standard freezing procedure. These findings could suggest the potential for future trehalose application for research purposes in cell cryopreservation.

MicroRNA-based molecular classification of papillary thyroid carcinoma.

MicroRNA (miRNA) expression is dysregulated in many human malignancies, and a growing number of studies are focused on their potential use as tumor biomarkers. To identify a miRNA signature for papillary thyroid carcinomas (PTC), we investigated miRNA expression profiles in two independent cohorts of PTCs, which included major histological subtypes [classical-type (PTC­CT), follicular-variant (PTC­FV), and tall-cell variant (PTC­TCV)] and cases with low or intermediate risk of recurrence. Using TaqMan® Array Human MicroRNA A+B Cards v3.0, we first performed microRNA profiling of normal and neoplastic thyroid tissues from 29 PTC patients. Promising candidates were then investigated in a second, independent cohort of 76 PTCs using Custom TaqMan® Array MicroRNA Cards. We identified a molecular signature of 11 miRNAs that were significantly upregulated (miR­146b-5p, miR­146b-3p, miR­221-3p, miR­222­5p, miR­222­3p) or downregulated (miR­1179, miR­486­5p, miR­204-5p, miR­7-2-3p, miR­144-5p, miR­140-3p) in PTC tissues vs. normal thyroid tissue. Upregulation of miR­146b-5p and miR­222­3p was also significantly associated with an increased risk of recurrence. Higher than normal expression of miR­146b-5p and miR­146b-3p characterized PTC­CT and PTC­TCV but not PTC­FV, whereas miR­21-5p was significantly upregulated only in PTC­TCV. When PTC­FV were subclassified as encapsulated (PTC­EFV) or infiltrative (PTC­IFV), miR­204-5p was downregulated in all histological subtypes except PTC­EFV, which displayed expression levels similar to those of normal thyroid tissues. These findings provide new insights into the molecular classification of PTC, showing that different miRNA expression profiles are associated with different histological types of PTC and different risks of recurrence.

KRAS and BRAF mutational status in colon cancer from Albanian patients.

BACKGROUND: Numerous clinical studies have shown that anti-EGFR therapies are effective only in a subset of patients with colorectal cancer. Mutations in the KRAS and BRAF genes have been confirmed as negative predictors of the response to EGFR-targeted therapies.In this study we evaluated KRAS and BRAF status in 159 colorectal cancer samples obtained from the University of Tirana. METHODS: We evaluated KRAS mutations in codons 12, 13, 61, 146 and in codon 600 of BRAF by direct sequencing. 90 patients were male (57%) and 69 female (43%); the patients' ages ranged from 17 to 85 (median 61.7). 24 patient were stage I, 36 stage II, 84 stage III and 15 stage IV. RESULTS: Out of the 159 cases, 28 (17,6%) showed KRAS mutation (13 G12D, 4 G12C, 4 G12V, 3 G12A, 2 G13 D, 1 G12S and 1 A146T), and 10 (6,3%) showed BRAF mutation (all V600E). No significant correlations between KRAS and BRAF mutations and various clinicopathological parameters was found.This is the first report of KRAS and BRAF status in Albanian patients with colorectal carcinoma (CRC) and though the relatively small sample size might not provide enough statistics power. CONCLUSIONS: The results of KRAS and BRAF mutation analysis could be used in the selection of patients for anti-EGFR therapy. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_187.

Assessment of MAGE-A expression in resected non-small cell lung cancer in relation to clinicopathologic features and mutational status of EGFR and KRAS.

Non-small cell lung cancer (NSCLC) is a major public health problem, accounting for more cancer-related deaths than any other cancer. Both immunotherapy, based on the expression of tumor-specific antigens, and targeted therapy, based on the presence of oncogenic mutations, are under development for NSCLC. In this study, we analyzed the expression of MAGE-A, a cancer-testis antigen, in tumors from a cohort of patients with resected NSCLC with respect to their clinicopathologic characteristics and their mutational status for the EGFR and KRAS genes. We found MAGE-A expression by IHC in 43% of the tumors. MAGE-A expression was significantly more frequent in squamous tumors than in adenocarcinomas, did not correlate with disease stage, but was correlated significantly with high tumor grade and worse survival. EGFR and KRAS mutations were present in adenocarcinomas, but not in squamous tumors. Whereas the presence of EGFR mutations did not seem to affect survival, the presence of KRAS mutations was associated with early-stage disease and better survival. MAGE-A expression was absent from adenocarcinomas with KRAS mutations, but not significantly different in tumors with or without EGFR mutations. Together, the reported results provide guidance for the design of combination therapies in patients with NSCLC.

Mek inhibition results in marked antitumor activity against metastatic melanoma patient-derived melanospheres and in melanosphere-generated xenografts.

One of the key oncogenic pathways involved in melanoma aggressiveness, development and progression is the RAS/BRAF/MEK pathway, whose alterations are found in most patients. These molecular anomalies are promising targets for more effective anti-cancer therapies. Some Mek inhibitors showed promising antitumor activity, although schedules and doses associated with low systemic toxicity need to be defined. In addition, it is now accepted that cancers can arise from and be maintained by the cancer stem cells (CSC) or tumor-initiating cells (TIC), commonly expanded in vitro as tumorspheres from several solid tumors, including melanoma (melanospheres). Here, we investigated the potential targeting of MEK pathway by exploiting highly reliable in vitro and in vivo pre-clinical models of melanomas based on melanospheres, as melanoma initiating cells (MIC) surrogates. MEK inhibition, through PD0325901, provided a successful strategy to affect survival of mutated-BRAF melanospheres and growth of wild type-BRAF melanospheres. A marked citotoxicity was observed in differentated melanoma cells regardless BRAF mutational status. PD0325901 treatment, dramatically inhibited growth of melanosphere-generated xenografts and determined impaired tumor vascularization of both mutated- and wild type-BRAF tumors, in the absence of mice toxicity. These results suggest that MEK inhibition might represent a valid treatment option for patients with both mutated- or wild type-BRAF melanomas, affecting tumor growth through multiple targets.

Large cell neuroendocrine carcinoma (LCNEC) of the urinary bladder: a case report.

Neuroendocrine carcinoma of the urinary bladder is a rare entity, accounting less then 1% of urinary bladder malignancies. The vast majority of the neuroendocrine carcinoma of the urinary bladder is represented by small cell neuroendocrine carcinoma while just few cases of large cell neuroendocrine carcinoma (LCNEC) have been reported. In this cases report we describe a rare case of primary bladder LCNEC. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2474700528951562.

Disulfiram, an old drug with new potential therapeutic uses for human hematological malignancies.

Disulfiram (DSF) is an aldehyde dehydrogenase inhibitor currently used for the treatment of alcoholism. Here, we show that multiple myeloma (MM) cell lines and primary cells from newly diagnosed and relapsed/resistant patients affected by MM, acute myeloid and lymphoblastic leukemia are significantly sensitive to DSF alone and in combination with copper. These effects are present at doses lower than those achievable in vivo after DSF standard administration. The cytotoxic effect achieved by this treatment is comparable to that obtained by conventional chemotherapy and is absent in normal hematopoietic cells. In addition, we found that DSF plus copper induces loss of mitochondrial membrane potential, triggers reactive oxygen species (ROS) production and activates executioner caspases. DSF-copper-induced apoptosis and caspases activation are strongly reversed by antioxidant N-acetylcysteine, thus indicating a critical role of ROS. These results might suggest the use of the old drug DSF, alone or in combination with copper, in the treatment of hematological malignancies.

Prevention of chemotherapy-induced anemia and thrombocytopenia by constant administration of stem cell factor.

PURPOSE: Chemotherapy-induced apoptosis of immature hematopoietic cells is a major cause of anemia and thrombocytopenia in cancer patients. Although hematopoietic growth factors such as erythropoietin and colony-stimulating factors cannot prevent the occurrence of drug-induced myelosuppression, stem cell factor (SCF) has been previously shown to protect immature erythroid and megakaryocytic cells in vitro from drug-induced apoptosis. However, the effect of SCF in vivo as a single myeloprotective agent has never been elucidated. EXPERIMENTAL DESIGN: The ability of SCF to prevent the occurrence of chemotherapy-induced anemia and thrombocytopenia was tested in a mouse model of cisplatin-induced myelosuppression. To highlight the importance of maintaining a continuous antiapoptotic signal in immature hematopoietic cells, we compared two treatment schedules: in the first schedule, SCF administration was interrupted during chemotherapy treatment and resumed thereafter, whereas in the second schedule, SCF was administered without interruption for 7 days, including the day of chemotherapy treatment. RESULTS: The administration of SCF to cisplatin-treated mice could preserve bone marrow integrity, inhibit apoptosis of erythroid and megakaryocytic precursors, prevent chemotherapy-induced anemia, and rapidly restore normal platelet production. Treatment with SCF increased the frequency of Bcl-2/Bcl-XL-positive bone marrow erythroid cells and sustained Akt activation in megakaryocytes. Myeloprotection was observed only when SCF was administered concomitantly with cisplatin and kept constantly present during the days following chemotherapy treatment. CONCLUSIONS: SCF treatment can prevent the occurrence of chemotherapy-induced anemia and thrombocytopenia in mice, indicating a potential use of this cytokine in the supportive therapy of cancer patients.

NF-κB localization in multiple myeloma plasma cells and mesenchymal cells.

Several reports demonstrated that the activation of Nuclear Factor-kappa B NF-κB is essential for the pathogenesis of multiple myeloma (MM). We analyzed the nuclear localization of NF-κB in MM-cells derived from 60 different patients with MM at presentation and in relapse, as well as in three myeloma cell lines. Nuclear localization (the active form) of NF-κB was detected in only one MM-sample from a refractory patient and in two samples from relapsed patients, while all the other samples, including the MM-cell lines, almost exclusively express the cytoplasmic (inactive) form of NF-κB. In mesenchymal cells from MM-patients NF-κB was clearly present in the nucleus. In addition, the proteasome inhibitor Bortezomib, which is described to antagonize NF-κB activity, had a consistent antitumor activity against both chemoresistant and chemosensitive MM-cells, regardless the NF-κB localization, thus suggesting the existence of other molecular targets of proteasome inhibitors in MM.

Chemotherapy-induced thrombocytopenia derives from the selective death of megakaryocyte progenitors and can be rescued by stem cell factor.

Thrombocytopenia is a common side effect of chemotherapy, responsible for increased risk of bleeding and delay of treatment schedules in cancer patients. It is currently unknown how chemotherapeutic agents affect platelet production and whether the platelet precursors megakaryocytes represent a direct target of cytotoxic drugs. We investigated the effects of chemotherapeutic agents on primary megakaryocytes by using a culture system that recapitulates in vitro human megakaryopoiesis and found that cytotoxic drugs predominantly destroyed megakaryocytic progenitors at early stages of differentiation. Immature megakaryocytes could be protected from chemotherapeutic agents by the cytokine stem cell factor (SCF), which binds the c-kit receptor expressed on hematopoietic stem and progenitor cells. In chemotherapy-treated megakaryocytes, SCF activated Akt, neutralized the mitochondrial apoptotic machinery, and inhibited caspase activity. Interfering with Akt activation abrogated the antiapoptotic effects of SCF, whereas exogenous expression of constitutively active Akt inhibited drug-induced apoptosis of primary megakaryocytes, indicating the Akt pathway as primarily responsible for SCF-mediated protection of megakaryocyte progenitors. These results indicate apoptosis of megakaryocyte progenitors as a major cause of chemotherapy-induced thrombocytopenia and suggest that SCF may be used to prevent platelet loss in cancer patients with c-kit-negative tumors.