Transcriptomic analysis and mutational status of IDH1 in paired primary-recurrent intrahepatic cholangiocarcinoma.

BACKGROUND: Effective target therapies for intrahepatic cholangiocarcinoma (ICC) have not been identified so far. One of the reasons may be the genetic evolution from primary (PR) to recurrent (REC) tumors. We aim to identify peculiar characteristics and to select potential targets specific for recurrent tumors. Eighteen ICC paired PR and REC tumors were collected from 5 Italian Centers. Eleven pairs were analyzed for gene expression profiling and 16 for mutational status of IDH1. For one pair, deep mutational analysis by Next Generation Sequencing was also carried out. An independent cohort of patients was used for validation. RESULTS: Two class-paired comparison yielded 315 differentially expressed genes between REC and PR tumors. Up-regulated genes in RECs are involved in RNA/DNA processing, cell cycle, epithelial to mesenchymal transition (EMT), resistance to apoptosis, and cytoskeleton remodeling. Down-regulated genes participate to epithelial cell differentiation, proteolysis, apoptotic, immune response, and inflammatory processes. A 24 gene signature is able to discriminate RECs from PRs in an independent cohort; FANCG is statistically associated with survival in the chol-TCGA dataset. IDH1 was mutated in the RECs of five patients; 4 of them displayed the mutation only in RECs. Deep sequencing performed in one patient confirmed the IDH1 mutation in REC. CONCLUSIONS: RECs are enriched for genes involved in EMT, resistance to apoptosis, and cytoskeleton remodeling. Key players of these pathways might be considered druggable targets in RECs. IDH1 is mutated in 30% of RECs, becoming both a marker of progression and a target for therapy.

Detection of breast cancer cell contamination in leukapheresis product by real-time quantitative polymerase chain reaction.

Identification of sensitive techniques for breast cancer cell detection might be relevant for high-dose chemotherapy programs with autologous stem cell transplantation. We investigated the feasibility of Maspin, Mammaglobin and c-ErbB-2 amplification by real-time quantitative polymerase chain reaction (RQ-PCR) for the detection of breast cancer cells in leukaphereses. Expression of the three markers was determined in primary breast cancers and cell lines. Peripheral blood (PB), bone marrow (BM), and leukapheresis samples from patients with malignancies other than breast cancer were used as controls. Sensitivity was evaluated by dilution of primary tumors and cell lines with mononuclear blood cells. We found expression of the three markers in all primary tumors and most cell lines. No blood specimen from control patients had the Maspin transcript, while only one was positive for Mammaglobin. Weak c-ErbB-2 expression was detectable in most PB, all BM and all leukapheresis samples from controls. We observed a low sensitivity of Maspin RQ-PCR and a sensitivity of Mammaglobin RQ-PCR up to one tumor cell in 10(6) mononuclear cells. One out of 18 leukaphereses from breast cancer patients screened for the presence of Mammaglobin mRNA was positive. We conclude that Mammaglobin RQ-PCR might be a useful tool for detection of leukapheresis contamination.

The involvement of human-nuc gene in polyploidization of K562 cell line.

During megakaryocyte differentiation, the immature megakaryocyte increases its ploidy to a 2(x) DNA content by a process called endomitosis. This leads to the formation of a giant cell, the mature megakaryocyte, which gives rise to platelets. We investigated the role of human-nuc (h-nuc), a gene involved in septum formation in karyokynesis in yeast, during megakaryocytic polyploidization. Nocodazole and 12-O-tetradecanoylphorbol-13-acetate (TPA) were used to induce megakaryocytic differentiation in K562 cell line. The ploidy distribution and CD41 expression of treated K562 cells were evaluated by flow cytometry. Using quantitative reverse transcriptase polymerase chain reaction (RT-PCR), we analyzed the h-nuc mRNA expression on treated K562 cells. Mature megakaryocyte-like polyploid cells were detected at day 5-7 of treatment with nocodazole. TPA also had a similar effect on K562 cells, but it was much weaker than that of nocodazole. The analysis of ploidy of nocodazole-treated K562 cells showed that nocodazole preferentially induced polyploidization of K562 cell line with a pronounced increase of the cells 8N at day 7 of culture. Expression of CD41, a differentiation-related phenotype, was significantly induced by TPA after 7 days of treatment, showing that functional maturation was mainly induced by TPA. In contrast, there was no significant increase in CD41 expression in nocodazole-treated K562 cells, suggesting that polyploidization and functional maturation are separately regulated during megakaryocytopoiesis. RT-PCR analysis indicated that h-nuc mRNA increased after 72 hours in the presence of nocodazole, preceding the induction of polyploidization. Our data indicate that h-nuc might play a role in polyploidization during megakaryocytic differentiation via inhibition of septum formation.

Negative influence of IL3 on the expansion of human cord blood in vivo long-term repopulating stem cells.

Identification of culture conditions that support expansion or even long-term maintenance of in vivo repopulating human hematopoietic stem cells is still a major challenge. Using a combination of FLT3 ligand (FL), Stem Cell Factor (SCF), Thrombopoietin (TPO) and Interleukin 6 (IL6), we cultured cord blood (CB) CD34+ cells for up to 12 weeks and transplanted their progeny into sublethally irradiated NOD/SCID mice. Bone marrow engraftment was considered successful when recipients contained measurable numbers of human CD45+, CD71+ and Glycophorin A+(GpA) cells 8 weeks after transplantation. Twelve-week expanded cells with FL+SCF+TPO+IL6 successfully engrafted all of the recipients and human CD45(+)+CD71(+)+GpA(+) cells represented 4.3 to 22.4% of bone marrow. Substitution of IL6 with IL3 led to an even better expansion of cells and a similar clonogenic progenitor output in the first 8 weeks of culture; however, LTC-IC output increased up to week 6 and then decreased and disappeared. By contrast, with FL+SCF+TPO+IL6, LTC-IC kept increasing up to week 12. Four-week cultured cells with FL+SCF+TPO+IL3 less efficiently engrafted NOD/SCID mice, both as measured by frequency of positive recipients (4 out of 10) and percentage of engrafted human cells (< or =2%). Six-week expanded cells failed to engraft. This study provides evidence that many, but not all, of the so-called "early acting" cytokines, can sustain long-term maintenance and even expansion of human primitive in vivo repopulating stem cells. In particular, in the culture conditions used in this study, the presence of IL3 greatly reduces the repopulating potential of expanded CD34+ CB cells.

Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34(+) cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells.

Understanding the repopulating characteristics of human hematopoietic stem/progenitor cells is crucial for predicting their performance after transplant into patients receiving high-dose radiochemotherapy. We have previously reported that CD34(+) cord blood (CB) cells can be expanded in vitro for several months in serum containing culture conditions. The use of combinations of recombinant early acting growth factors and the absence of stroma was essential in determining this phenomenon. However, the effect of these manipulations on in vivo repopulating hematopoietic cells is not known. Recently, a new approach has been developed to establish an in vivo model for human primitive hematopoietic precursors by transplanting human hematopoietic cells into sublethally irradiated nonobese diabetic severe combined immunodeficient (NOD/SCID) mice. We have examined here the expansion of cells, CD34(+) and CD34(+)38(-) subpopulations, colony-forming cells (CFC), long-term culture initiating cells (LTC-IC) and the maintenance or the expansion of SCID-repopulating cells (SRC) during stroma-free suspension cultures of human CD34(+) CB cells for up to 12 weeks. Groups of sublethally irradiated NOD/SCID mice were injected with either 35,000, 20,000, and 10,000 unmanipulated CD34(+) CB cells, which were cryopreserved at the start of cultures, or the cryopreserved cells expanded from 35,000, 20,000, or 10,000 CD34(+) cells for 4, 8, and 12 weeks in the presence of a combination of early acting recombinant growth factors (flt 3/flk2 ligand [FL] + megakaryocyte growth and development factor [MGDF] +/- stem cell factor [SCF] +/- interleukin-6 [IL-6]). Mice that had been injected with >/=20,000 fresh or cryopreserved uncultured CD34(+) cells did not show any sign or showed little engraftment in a limited number of animals. Conversely, cells that had been generated by the same number of initial CD34(+) CB cells in 4 to 10 weeks of expansion cultures engrafted the vast majority of NOD/SCID mice. The level of engraftment, well above that usually observed when the same numbers of uncultured cells were injected in the same recipients (even in the presence of irradiated CD34(-) cells) suggested that primitive hematopoietic cells were maintained for up to 10 weeks of cultures. In addition, dilution experiments suggest that SRC are expanded more than 70-fold after 9 to 10 weeks of expansion. These results support and extend our previous findings that CD34(+) CB stem cells (identified as LTC-IC) could indeed be grown and expanded in vitro for an extremely long period of time. Such information may be essential to design efficient stem cell expansion procedures for clinical use.

GAS6 inhibits granulocyte adhesion to endothelial cells.

GAS6 is a ligand for the tyrosine kinase receptors Rse, Axl, and Mer, but its function is poorly understood. Previous studies reported that both GAS6 and Axl are expressed by vascular endothelial cells (EC), which play a key role in leukocyte extravasation into tissues during inflammation through adhesive interactions with these cells. The aim of this work was to evaluate the GAS6 effect on the adhesive function of EC. Treatment of EC with GAS6 significantly inhibited adhesion of polymorphonuclear cells (PMN) induced by phorbol 12-myristate 13-acetate (PMA), platelet-activating factor (PAF), thrombin, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), but not that induced by FMLP and IL-8. GAS6 did not affect adhesion to resting EC. Titration experiments showed that high concentrations of GAS6 were needed to inhibit PMN adhesion and that inhibition was dose-dependent at the concentration range of 0.1 to 1 microg/mL. One possibility was that high concentrations were needed to overwhelm the effect of endogenous GAS6 produced by EC. In line with this possibility, treatment of resting EC with soluble Axl significantly potentiated PMN adhesion. Analysis of localization of GAS6 by confocal microscopy and cytofluorimetric analysis showed that it is concentrated along the plasma membrane in resting EC and treatment with PAF induces depletion and/or redistribution of the molecule. These data suggest that GAS6 functions as a physiologic antiinflammatory agent produced by resting EC and depleted when proinflammatory stimuli turn on the proadhesive machinery of EC.

GAS6, the ligand of Axl and Rse receptors, is expressed in hematopoietic tissue but lacks mitogenic activity.

GAS6, a gene previously identified as growth arrest specific, has been demonstrated to be the ligand of Axl, a novel tyrosine kinase receptor widely expressed in both normal and neoplastic hematopoietic tissue. We have observed previously that GAS6 mRNA was present in whole bone marrow. This preliminary finding prompted us to investigate the presence of GAS6 in hematopoietic tissue and the possible role of this molecule in controlling the proliferation of hematopoietic precursors. We report here that the protein GAS6 is diffusely present in hematopoietic tissue, both in stromal and in hematopoietic cells, and that, among these cells, positivity is observed in megakaryocytes and myelomonocytic precursors. Furthermore, our data suggest that GAS6 is not a growth factor for hematopoietic progenitors or stromal fibroblasts. Despite the fact that both the Axl receptor and its ligand, GAS6, are expressed in hematopoietic tissue, the biological role of their interactions remains to be determined.

Protein kinase C-dependent release of a functional whole extracellular domain of the mast cell growth factor (MGF) receptor by MGF-dependent human myeloid cells.

The mast cell growth factor (MGF) affects migration, proliferation and differentiation of erythroid and myeloid progenitor cells by binding to a transmembrane receptor tyrosine kinase encoded by the c-Kit proto-oncogene. By using MGF-dependent human myeloid cell lines (M-07e and TF-1), here we show that a Kit-related 100 kDa protein is associated with the cell but it undergoes release into the medium upon treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C. Immunological analysis with a series of antibodies to Kit indicated that the released protein (p100Kit) contains the whole glycosylated extracellular portion of the transmembrane Kit protein (p145Kit). The secreted protein retained the ability to specifically bind MGF. Moreover, p100Kit was able to block the mitogenic effect of MGF on cultured M-07e cells, suggesting that the soluble protein may function as a physiological antagonist of MGF.