Fraction Of Positive Cells Research Articles

KIT D816V mutation burden does not correlate to clinical manifestations of indolent systemic mastocytosis

Clinical manifestations of indolent systemic mastocytosis (ISM) comprise mediator-related symptoms, anaphylaxis, and osteoporosis. A new sensitive method for KIT D816V mutation detection allows quantification of the level of mutation-positive cells. To investigate whether the fraction of KIT D816V positive cells in peripheral blood (PB) or bone marrow (BM) aspirate in adult patients with ISM correlates with clinical manifestations of the disease. We included 48 adult patients with ISM (28 females/20 males) from our center in whom the KIT D816V mutation level in both BM aspirate and PB was analyzed. For each patient, the severity of mediator-related symptoms (skin, gastrointestinal, musculoskeletal, and neuropsychiatric) and episodes of anaphylaxis were evaluated by interview and medical record files. Bone mineral density was determined by using dual-energy x-ray absorptiometry. Median fraction (range) of KIT D816V positive cells was 0.6 (0.01%-90%) in BM and 0.3 (0.003%-49%) in PB. Mutation level did not differ between patients with none/mild symptoms and patients with moderate/severe symptoms, patients with and without anaphylaxis, or patients with osteoporosis/osteopenia and normal bone mineral density. No significant differences in clinical profile were detected in patients with different levels of mutation except for an indication of longer disease duration and age in patients with highest mutation levels. To our knowledge, this is the first report on the clinical impact of the fraction of KIT D816V mutation positive cells in ISM, which in the present study does not seem to correlate with clinical manifestations of the disease.

Lineage depletion of stromal vascular fractions isolated from human adipose tissue: a novel approach towards cell enrichment technology

The therapeutic rationale for tissue repair and regeneration using stem cells is at its infancy and needs advancement in understanding the role of individual component's innate capability. As stem cells of adipose tissue reside in a more heterogeneous population of stromal vascular fractions, cell separation or sorting becomes an eminent step towards revealing their unique properties. This study elucidates the comparative efficacy of lineage depleted adipose derived stromal vascular fraction (SVF) and their innate ability using magnetic activated cell sorter (MACS). To this end, isolated SVF from human adipose tissue was lineage depleted according to the manufacturer's instructions using specific antibody cocktail through MACS. The enriched lineage negative (lin-) and lineage positive (lin+) cell fractions were cultured, phenotypically characterized for the panel of cell surface markers using flowcytometry and subjected to osteoblastic and adipogenic differentiation. The expression profile obtained for lin- cells was CD34-/CD45-/HLADR-/CD49d-/CD140b-/CD31-/CD90+/CD105+/CD73+/CD54+/CD166+/CD117- when compared to Lin+ cells expressing CD34+/CD45+/HLADR-/CD49d-/CD140b+/CD31-/CD90+/CD105+/CD73+/CD54+/CD166+/CD117+ (CD-cluster of differentiation). These results, thus, advances our understanding on the inherent property of the individual cell population. Furthermore, both the fractions exhibited mesodermal lineage differentiation capacity. To conclude, this research pursuit rationalized the regenerative therapeutic applicability of both lin- and lin+ cultures of human adipose tissue for disorders of mesodermal, haematological and vascular origin.

Aldehyde dehydrogenase 1/epidermal growth factor receptor coexpression is characteristic of a highly aggressive, poor-prognosis subgroup of high-grade serous ovarian carcinoma

In models of triple-negative breast cancer (TNBC), it has recently been shown that the epidermal growth factor receptor (EGFR) pathway is up-regulated in the aldehyde dehydrogenase 1 (ALDH1)-positive cancer stem cell fraction. Because high-grade serous ovarian carcinoma (HGSC) reveals strong molecular similarities to TNBC, we aimed to investigate the potential link between ALDH1 and EGFR in this entity. Expression of ALDH1 was investigated in 131 primary HGSCs using immunohistochemistry. Expression data were correlated with EGFR expression as well as with clinicopathologic parameters and survival. Forty-two carcinomas (32.1%) were positive for ALDH1 protein expression. Data on EGFR expression were available for 112 tumors. In these cases ALDH1 was significantly linked to EGFR expression (P < .0001). ALDH1 positivity was a significant negative prognostic factor for overall survival both in univariate (P = .010) and in multivariate survival analyses (P = .041). Tumors that were positive for both ALDH1 and EGFR had an exceptionally bad prognosis as compared with carcinomas with 1 or both markers negative in univariate analysis (P < .0001) and in the multivariate setting (P = .004). Our study suggests that similar to TNBC, there is a link between ALDH1 and EGFR expression in HGSC. Double positivity for both markers identifies a subgroup of highly aggressive, poor-prognosis cancers for which alternative treatment options-potentially EGFR-targeting drugs-should be evaluated.

The Yersinia Virulence Effector YopM Binds Caspase-1 to Arrest Inflammasome Assembly and Processing

Inflammasome assembly activates caspase-1 and initiates the inflammatory cell death program pyroptosis, which is protective against numerous pathogens. Consequently, several pathogens, including the plague causing bacterium Yersinia pestis, avoid activating this pathway to enhance their virulence. However, bacterial molecules that directly modulate the inflammasome have yet to be identified. Examining the contribution of Yersinia type III secretion effectors to caspase-1 activation, we identified the leucine-rich repeat effector YopM as a potent antagonist of both caspase-1 activity and activation. YopM directly binds caspase-1, which both inhibits caspase-1 activity and sequesters it to block formation of the mature inflammasome. Caspase-1 activation antagonizes Yersinia survival invivo, and consequently YopM inhibition of caspase-1 is required for Yersinia pathogenesis. Thus, a bacterium obstructs pyroptosis utilizing a direct mechanism of caspase-1 inhibition that is distinct from known viral or host inhibitors.

Cytoplasmic NANOG-Positive Stromal Cells Promote Human Cervical Cancer Progression

Tumor development has long been known to resemble abnormal embryogenesis. The embryonic stem cell gene NANOG, a divergent homeodomain transcription factor that is independent of leukemia inhibitory factor, has been reported to be expressed in germ cells and in several tumor types. However, the short-term expression and role of NANOG in cervical cancer remain unclear. In the present study, we demonstrate that NANOG exhibits cellular shuttling behavior and increasing stromal distribution during the progression of cervical cancer. Our molecular data using RT-PCR and restriction enzyme digestion show that NANOG is mainly transcribed from the NANOG gene in cervical cancer. In addition, IHC using confocal microscopy suggests that mesenchymal stem cells (MSCs) are one type of cytoplasmic NANOG-positive cells in cervical cancer stroma. Co-culture of cervical cancer-derived MSCs with SiHa cells showed increased proliferation characteristics in vitro and enhanced tumor growth in vivo. Our results show, for the first time to our knowledge, that MSCs are a source of cytoplasmic NANOG expression in the cervical cancer stroma and that they participate in the progression of cervical cancer both in vitro and in vivo. Our study provides evidence that NANOG is a cervical cancer progression marker and also serves as a starting point for a more extensive exploration of the cellular translocation of NANOG and the multifunctionality of the stromal microenvironment.

Abstract 3248: Amphiregulin interacts with Wnt signaling to regulate cancer cell growth and survival

Abstract Epidermal growth factor receptor (EGFR) initiates signaling via several ligands that include but are not limited to EGF, amphiregulin (Areg) and epiregulin. These ligands are believed to differentially affect various physiological and cellular processes such as cell survival, proliferation and differentiation. This study is designed to delineate how amphiregulin alters the biology of both breast and ovarian cancer cells that overexpress amphiregulin and activate EGFR in an autocrine manner. AREG knock down drastically reduces the rate of proliferation of both a breast cancer cell line, SUM149 and an ovarian cancer cell line, OV1 that overexpress amphiregulin. Stable, clonal populations of SUM149 cells with AREG knockdown grow poorly in serum free media, but can be rescued by addition of exogenous AREG or other EGFR ligands to the culture medium. Clones with the most effective AREG knock down are completely unable to survive or proliferate in serum free media. Our Micro array analyses show that inhibiting amphiregulin in SUM149 cells dysregulates the expression of several important genes, including up-regulating DKK1, an antagonist of Wnt signaling. DKK1 up regulation upon AREG knockdown was confirmed by ELISA, with one of the clones, C2 showing a 16 fold increase in DKK1 secretion following AREG knock-down. Flow cytometetric analysis of SUM-149 cells and AREG knock-down derivatives demonstrated that 48% of control cells expressing a non-silencing shRNA construct were CD44+/CD24- but only a small population (7%) expressed CD24 alone. AREG knockdown induced significant reduction in the fraction of CD44 positive cells, down to ∼4% while, simultaneously increasing the non-stem cell fraction, CD44-/CD24+ to 60% of the total population. Since CD44hi/CD24lo is a characteristic of undifferentiated cells that have the potential to initiate tumor formation, we are currently examining the in vivo growth potential of SUM-149 cells and AREG knock-down derivatives. To determine the tumorigenicity of SUM149 cells three different cell innocula of unsorted parental SUM149 cells and SUM149 cells expressing a non-silencing shRNA construct (SUM149-NS) were transplanted into the mammary fat pad of NOD-SCID mice. As expected, the tumorigenic activity of the cells is proportional to the cell titer. In the parental SUM149 cells, the injection of 5x105 cells resulted in measurable tumors beginning at day 28 while SUM149-NS displays tumors at day 24, while transplantation of 2.5x105 cells per fat pad yielded tumor formation in 31 and 28 days respectively. Experiments to measure the in vivo growth potential of AREG knock-down SUM-149 cells is in progress. Thus, amphiregulin has a unique biological function to enhance cell survival and differentiation in vitro and in vivo. Future studies will focus on the role of AREG mediated WNT signaling in the malignant potential of SUM-149 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3248. doi:1538-7445.AM2012-3248

Hydrogen sulfide increases hepatic differentiation in tooth-pulp stem cells

The toxicity of hydrogen sulfide (H2S), an oral malodorous compound, is well reported. We have recently established an experimental model of hepatic differentiation from human tooth-pulp stem cells (HTPC) using serum-free medium. The objective of the present study is to determine the effect of H2S on hepatic differentiation. The CD117 positive cell fraction was obtained from deciduous HTPC using magnetic cell sorting. After 3–4 passages, cells were grown in Dulbecco's modified Eagle's medium supplemented with insulin-transferrin-selenium-x (ITS-x), embryotrophic factor (ETF) and hepatocyte growth factor (HGF) for hepatic commitment (five days). For hepatic differentiation the cells were cultured in Iscove's modified Dulbecco's medium supplemented with ITS-x, ETF, oncostatin, HGF and dexamethasone for 15 days in air containing 5% CO2, with or without H2S at 0.05 ng ml−1. Cells were assayed for the expression of hepatic markers α-fetoprotein, albumin or carbamoyl phosphate synthetase, and urea concentrations and glycogen synthesis were also determined. The panel of hepatic markers was expressed more in the test groups exposed to H2S than in the control groups. Urea and glycogen production were also increased, especially glycogen which was approximately five times greater compared to the control (p < 0.01). We concluded that H2S at physiological concentrations increased the ability of HTPC to undergo hepatogenic differentiation.

Association of the expression of CD133 in locally advanced rectal cancer with tumor regression and survival after neoadjuvant radiochemotherapy.

479 Background: CD133 has been described as a stem cell marker in colorectal cancer and is associated with higher tumorigenic potential and resistance to radiochemotherapy (RCT). In this study the expression of CD133 was evaluated in pre-treatment tumor biopsies and corresponding post-treatment surgical specimens of locally advanced rectal cancer patients undergoing multimodal therapy and was correlated to histopathological features and clinical follow-up. Methods: 97 patients with UICC II/III rectal cancer treated with preoperative 5-FU based RCT within the German Rectal Cancer Trials were investigated. Pre- and post-treatment CD133 expression levels were determined using immunhistochemistry and correlated with histopathologic parameters, tumor regression, tumor recurrence and disease-free survival (DFS). Results: As compared to pre-treatment biopsies we observed a significantly higher CD133 expression in post-treatment tumor specimens (p=0.01). There was, however, no correlation for both biopsies and tumor specimens between CD133 expression levels, pathological characteristics and survival. In matched analyses of corresponding biopsy/tumor pairs, patients with a decreased number of CD133 positive cells after preoperative RCT showed significantly lower post-treatment tumor stages (p=0.03) and higher histopathological tumor regression (p&lt;0.01). Moreover, these patients had a significantly improved DFS in uni- (p&lt;0.001) and multivariate analyses (p=0.001). Conclusions: CD133 expression displays a marker with prognostic and monitoring validity in rectal cancer patients. A decrease of the CD133 positive cell fraction in post-treatment tumor tissue is associated with a more favorable outcome after preoperative RCT.

Systemic mastocytosis is uncommon in KIT D816V mutation positive core-binding factor acute myeloid leukemia

The KIT D816V mutation is detected in the vast majority of adult cases of systemic mastocytosis (SM). The mutation is also frequently detected in core-binding factor acute myeloid leukemia (CBF AML) defined by the presence of t(8;21)(q22;q22); RUNX1-RUNX1T1 or inv(16)(p13.1;q22)/t(16;16)(p13.1;q22); CBFB-MYH11 chromosomal rearrangements, but whether the mutation is indicative of associated SM is unclear. In the present study, patients with CBF AML were therefore analyzed for the KIT D816V mutation and mutation positive cases subsequently analyzed for the presence of SM. The KIT D816V mutation was detected in eight of 20 cases of CBF AML, with the frequency in t(8;21)(q22;q22) and inv(16)(p13.1;q22) positive cases being 31% and 57%, respectively. The fraction of KIT D816V mutation positive cells was highly variable among the eight mutation positive patients, with levels ranging from 0.04 to 98% in a pretreatment blood sample. Five of the eight cases carried the mutation in a cell fraction below one-tenth of the blast cell fraction, thus suggesting that KIT mutation is often a late event in leukemogenesis. None of the eight KIT D816V mutation positive cases fulfilled the World Health Organization diagnostic criteria of SM. The presence of the KIT D816V mutation in the CBF AML subgroup can therefore not be considered indicative of associated SM.

MITF Expression in Cutaneous Malignant Melanoma

The MITF protein has a central role in the differentiation and survival of melanocytes. The aim of the study was to investigate whether MITF can be employed as a prognostic marker in patients operated on for cutaneous malignant melanoma. A cohort study design, based on information collected from population-based registries, was employed. For included patients (n=264) tissue microarrays were stained with immunohistochemistry to study the protein expression of MITF in primary malignant melanoma tumors by estimating the fraction of positive tumor cells and the staining intensity. Most of the tumors (84%) expressed MITF in >25% of the tumor cells and for 87% of the tumors the staining intensity was strong. Tumors with cell fraction >75% had a better prognosis compared with those with <75% (HR 0.44, 95% CI: 0.20-1.0, P=0.05). Tumors with a strong staining intensity tended to have a better prognosis compared with the weaker staining ones (HR 0.59, 95% CI: 0.26 -1.36, P=0.22). When cell fraction and intensity were combined, a high-risk group dying of malignant melanoma was identified as those patients with 25- 75% of tumor cells staining with weak intensity (HR 2.9, 95% CI: 0.94-8.7, P=0.06) and those with <25% of tumor cells staining with strong intensity (HR 2.5, 95% CI: 1.1- 6.1, P=0.04). However, the majority of deaths occurred in the lower risk groups. In conclusion, a high-risk group for death in malignant melanoma was identified but MITF is not suitable as a prognostic marker due to the distribution of that particular expression in the population.

Proliferation of acid-secretory cells in the kidney during adaptive remodelling of the collecting duct.

The renal collecting duct adapts to changes in acid-base metabolism by remodelling and altering the relative number of acid or alkali secreting cells, a phenomenon termed plasticity. Acid secretory A intercalated cells (A-IC) express apical H+-ATPases and basolateral bicarbonate exchanger AE1 whereas bicarbonate secretory B intercalated cells (B-IC) express basolateral (and apical) H+-ATPases and the apical bicarbonate exchanger pendrin. Intercalated cells were thought to be terminally differentiated and unable to proliferate. However, a recent report in mouse kidney suggested that intercalated cells may proliferate and that this process is in part dependent on GDF-15. Here we extend these observations to rat kidney and provide a detailed analysis of regional differences and demonstrate that differentiated A-IC proliferate massively during adaptation to systemic acidosis. We used markers of proliferation (PCNA, Ki67, BrdU incorporation) and cell-specific markers for A-IC (AE1) and B-IC (pendrin). Induction of remodelling in rats with metabolic acidosis (with NH4Cl for 12 hrs, 4 and 7 days) or treatment with acetazolamide for 10 days resulted in a larger fraction of AE1 positive cells in the cortical collecting duct. A large number of AE1 expressing A-IC was labelled with proliferative markers in the cortical and outer medullary collecting duct whereas no labeling was found in B-IC. In addition, chronic acidosis also increased the rate of proliferation of principal collecting duct cells. The fact that both NH4Cl as well as acetazolamide stimulated proliferation suggests that systemic but not urinary pH triggers this response. Thus, during chronic acidosis proliferation of AE1 containing acid-secretory cells occurs and may contribute to the remodelling of the collecting duct or replace A-IC due to a shortened life span under these conditions.

Inhibition of telomerase activity preferentially targets aldehyde dehydrogenase-positive cancer stem-like cells in lung cancer

BackgroundMortality rates for advanced lung cancer have not declined for decades, even with the implementation of novel chemotherapeutic regimens or the use of tyrosine kinase inhibitors. Cancer Stem Cells (CSCs) are thought to be responsible for resistance to chemo/radiotherapy. Therefore, targeting CSCs with novel compounds may be an effective approach to reduce lung tumor growth and metastasis. We have isolated and characterized CSCs from non-small cell lung cancer (NSCLC) cell lines and measured their telomerase activity, telomere length, and sensitivity to the novel telomerase inhibitor MST312.ResultsThe aldehyde dehydrogenase (ALDH) positive lung cancer cell fraction is enriched in markers of stemness and endowed with stem cell properties. ALDH+ CSCs display longer telomeres than the non-CSC population. Interestingly, MST312 has a strong antiproliferative effect on lung CSCs and induces p21, p27 and apoptosis in the whole tumor population. MST312 acts through activation of the ATM/pH2AX DNA damage pathway (short-term effect) and through decrease in telomere length (long-term effect). Administration of this telomerase inhibitor (40 mg/kg) in the H460 xenograft model results in significant tumor shrinkage (70% reduction, compared to controls). Combination therapy consisting of irradiation (10Gy) plus administration of MST312 did not improve the therapeutic efficacy of the telomerase inhibitor alone. Treatment with MST312 reduces significantly the number of ALDH+ CSCs and their telomeric length in vivo.ConclusionsWe conclude that antitelomeric therapy using MST312 mainly targets lung CSCs and may represent a novel approach for effective treatment of lung cancer.

Analysis of EpCAM positive cells isolated from sentinel lymph nodes of breast cancer patients identifies subpopulations of cells with distinct transcription profiles

IntroductionThe presence of tumor cells in the axillary lymph nodes is the most important prognostic factor in early stage breast cancer. However, the optimal method for sentinel lymph node (SLN) examination is still sought and currently many different protocols are employed. To examine two approaches for tumor cell detection we performed, in sequence, immunomagnetic enrichment and RT-PCR analysis on SLN samples from early stage breast cancer patients. This allowed us to compare findings based on the expression of cell surface proteins with those based on detection of intracellular transcripts.MethodsEnrichment of EpCAM and Mucin 1 expressing cells from fresh SLN samples was achieved using magnetic beads coated with the appropriate antibodies. All resulting cell fractions were analyzed by RT-PCR using four chosen breast epithelial markers (hMAM, AGR2, SBEM, TFF1). Gene expression was further analyzed using RT-PCR arrays and markers for epithelial to mesenchymal transition (EMT).ResultsBoth EpCAM and Mucin 1 enriched for the epithelial-marker expressing cells. However, EpCAM-IMS identified epithelial cells in 71 SLNs, whereas only 35 samples were positive with RT-PCR targeting breast epithelial transcripts. Further analysis of EpCAM positive but RT-PCR negative cell fractions showed that they had increased expression of MMPs, repressors of E-cadherin, SPARC and vimentin, all transcripts associated with the process of epithelial to mesenchymal transition.ConclusionsThe EpCAM IMS-assay detected tumor cells with epithelial and mesenchymal-like characteristics, thus proving to be a more robust marker than pure epithelial derived biomarkers. This finding has clinical implications, as most methods for SLN analysis today rely on the detection of epithelial transcripts or proteins.

Abstract 4250: A novel thrombospondin-1 mimetic peptide, ABT-898, decreases angiogenesis in a mouse model of glioblastoma multiforme

Abstract Prognostic outlook for patients with glioblastoma multiforme (GBM) still appears bleak in spite of medical advances in treatment of other malignant tumors in the past decades. With optimal therapy, of surgical resection, radiation therapy, and chemotherapy, a median survival merely approximates to 12 months. However, a recent dogma that the microvascular niche of such tumors plays a critical role in their maintenance has led to development of “anti-angiogenenic” therapies that target it. To evaluate the apoptotic effect of ABT-898, a TSP-mimetic, anti-angiogenic peptide on endothelial cells, we used human umbilical vein endothelial cells (HUVEC) to model human brain microvasculature. Furthermore, to then assess its clinical applicability, an orthotopic xenograft mouse model was established using human-derived GBM cells lines, and its therapeutic effect evaluated in vivo. We found that ABT-898 induced apoptosis on HUVEC as evidenced by increased fraction of annexin-V positive cells on flow cytometry. Systemic treatment of BTSC-derived immunodeficient mouse tumors with ABT-898, either alone or in combination with temozolomide, significantly reduced tumor size at day 14 post-injection of GBM cells, compared with that of a vehicle, which was used as a control. In addition, treatment with ABT-898 allowed increase in median survival in the xenograft models compared to the control mice. These observations suggest that ABT-898 is a plausible candidate for use in future treatment of GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4250. doi:10.1158/1538-7445.AM2011-4250

A mouse model analyzing the influence of dietary fat intake on liver apoptosis

Dietary fat intake is associated with hepatobiliary cancers which carry a poor prognosis causing over 20,000 deaths per year in the US alone. We hypothesized that excess lipid accumulation in the liver promotes hepatic cancer through inflammation and oxidative DNA damage. In order for eukaryotic cells to protect genomic integrity, the protein kinase ataxia telangiectasia mutated (ATM) responds to oxidative DNA damage and DNA strand breaks. Failure to activate ATM following damage leads to defective cell cycle control and impaired DNA repair. In this study, ATM knockout mice were used as a sensitized background to assess the effects of oxidative stress and DNA damage associated with hepatosteatosis. ATM-deficient and control mice were fed a high fat diet for eight weeks, and then liver tissue was analyzed for apoptosis. It was expected that more apoptotic cells would be found in ATM-deficient mice fed the high fat diet than in control mice due to DNA break repair deficiencies. Liver tissues were sectioned and stained by TUNEL assay, a method for detecting DNA fragmentation that occurs during apoptosis. The TUNEL immunohistochemistry protocol was first optimized for hepatocytes. Positive cells were counted in multiple 40X fields from each tissue section. Statistical differences between groups were determined by comparison of the fraction of positive cells. There were more apoptotic cells in livers from mice fed a high fat diet relative to those fed the normal diet. Interestingly, among mice fed the high fat diet there was a slight decrease in apoptosis in ATM deficient mice relative to controls. Although this difference did not reach statistical significance, this may indicate that ATM is required for inducing hepatic apoptosis in response to stresses associated with increased dietary fat consumption. Additional staining for cell proliferation as well as DNA damage response activation will be performed in the future. This study will begin to elucidate the interaction between lipid metabolism, oxidative stress, DNA damage and hepatobiliary oncogenesis and will establish a new mouse model that will provide a powerful tool for future mechanistic and translation studies of hepatobiliary disease.

Clinical Relevance of Vascular Endothelial Growth Factor In Myelodysplastic Syndromes

AbstractAbstract 4030 INTRODUCTION:An increased bone marrow angiogenesis might be involved in the pathophysiology of a number of haematological malignancies, including myelodysplasia (MDS). Many angiogenic factors have been investigated in MDS, and vascular endothelial growth factor (VEGF) and its significance has been evaluated the most. VEGF mediates its biological effects by binding to two transmembrane tyrosine kinase receptors, VEGFR-1 and VEGFR-2. The VEGF/receptor signaling system is involved in the regulation of two fundamental processes: the formation of blood vessels (angiogenesis) and of blood cells (hematopoiesis). VEGF regulates the two processes by different mechanisms: hematopoietic stem cell survival and repopulation mainly via an autocrin loop, and angiogenesis via a paracrine loop. PATIENTS:The study population included 79 patients (pts) (48 men and 31 women; median age, 70 years). The patients were given a diagnosis of 10 RA, 6 RARS, 17 RCMD, 7 5q-syndrome, 25 RAEB-1 e 14 RAEB-2 according to the WHO classification (2008). In agreement with the International Prognostic Scoring System (IPSS), 28 pts were graded as Low, 28 as Int-1, 17 as Int-2 and 6 as High. According to the WHO classification based prognostic scoring system (WPSS), 14 pts were graded as Very Low, 17 as Low, 11 as Int, 25 as High and 12 Very High. The leukemic evolution occurred in 23 pts. The median leukemia free survival (LFS) was 825 days. Exitus occurred in 49 pts. The median overall survival (OS) was 902 days. We included in the study 20 controls (NC). METHODS:The MicroVessel Density (MVD) was evaluated through CD34 immuno-histochemical reactivity. Each BMB was first totally scanned at low magnification (10×) in order to identify 5 “hot spot” zones and then examined at high magnification (40×). MVD was estimated by hot-spot “method” (MVD-HS) by counting all of the positive-endothelial cells. Lumens were required to consider a structure as microvessel. Sinusoid-like structures, with the exception of arterioles, were also included in the count. To avoid any bias related to BMB cellularity, we calculated a VEGF expression index (VEGFi), defined as the cellularity of the BMBs multiplied by the fraction of VEGF positive cells and expressed as a number between 0 and 1 [(% of BMB cellularity × %VEGF-positive cells)/10⋀4]. Two VEGFi expression classes, respectively Low and High (L-VEGFi and H-VEGFi), were determined considering as cut-off level the mean VEGF(i) expression value + 2 standard deviations. RESULTS:MVD-HS analysis showed increased levels in MDS in comparison to NCs (p=.009). MVD did not differ among IPSS and WPSS categories. MVD did not predicted leukemia free and overall survival (LFS and OS). VEGF expression in bone marrow cells was found as a weak cytoplasmic granular protein expression in proeritroblasts; normoblasts were not immunoreactive. In the myeloid lineage VEGF expression was more intense in immature cells. The macrophages and megakaryocytes showed a granular weak cytoplasmic positivity. The plasmacells showed an intense granular cytoplasmic positivity. VEGFi expression was evenly distributed among the different IPSS and WPSS categories and was higher in MDS in comparison to NCs (p=.006). L-VEGFi MDS pts presented a significant lower transfusional need in the first years from diagnosis (p=.02). We observed that L-VEGFi MDS pts had a longer LFS compared with H-VEGFi ones (p=.006). Moreover, L-VEGFi MDS pts had a significant better OS compared to H-VEGFi pts (p=.03). At multivariate analysis IPSS and WPSS, as expected, predicted OS (p<0.0001). Interestingly, VEGFi at diagnosis maintained its significant prognostic role on LFS also in multivariate analysis(p=.02), together with IPSS and WPSS (p=.002 and .001). CONCLUSIONS:We show that in MDS pts at diagnosis bone marrow VEGFi evaluation predicts OS and LFS. The influence of VEGFi on LFS is retained also in multivariate analysis in a model including IPSS and WPSS. This finding is even more interesting considering that IPSS and WPSS are multiparametric score systems, and that WPSS necessitates the quantification of the transfusional need in the first months from diagnosis, while VEGFi is a single immunohistochemical parameter evaluated on BMB specimens at diagnosis. Further studies are warranted to confirm the prognostic role of VEGFi in this disease setting and its clinical implications. Disclosures:No relevant conflicts of interest to declare.

Association of TBX21 promoter polymorphisms with type 1 autoimmune hepatitis in a Chinese population

The T-box transcription factor T-bet is a key regulator for the lineage commitment in CD4 Th1 cells and CD8 T cells by activating the hallmark production of interferon-γ, and its expression level is linked to autoimmune diseases. T-1993C and T-1514C polymorphisms in the TBX21 gene (encoding T-bet) promoter can affect transcription activity. We investigated the distributions of these functional polymorphisms in 84 adult patients with type 1 autoimmune hepatitis (AIH-1) and 318 healthy controls. Intracellular T-bet staining of polarized CD4 Th1 cells from healthy controls corresponding to T-1993C genotypes were analyzed by flow cytometry. The −1993C allele frequency was 3.0% in AIH-1 and 11.8% in controls ( p = 0.000 25). Individuals carrying the −1993C allele had a decreased risk to AIH-1 compared with those without the −1993C allele ( p = 0.0016, odds ratio [OR] = 0.22, 95% confidence interval = 0.09–0.56). No association was found between the T-1514C polymorphism and AIH-1. The onset age of AIH-1 was also accelerated among −1993TT homozygotic individuals ( p = 0.013). The fractions of T-bet positive Th1 cells in the -1993TT homozygotes were 2.2-fold higher than those in -1993CC homozygotes ( p = 0.002). Our results suggest that the T-1993C polymorphism in the TBX21 promoter influences susceptibility to AIH-1 in a Chinese population.

Spatial and temporal distribution of Ki-67 proliferation marker, Bcl-2 and Bax proteins in the developing human tooth

Objective To investigate the spatial and temporal expression of proliferation Ki-67 marker, pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins during early development of the human tooth. Materials and methods Histological sections of eight human conceptuses, 5–10 postovulatory weeks old, were used for immunolocalization for Ki-67, Bax and Bcl-2 markers. Quantification was performed by calculating the fraction of Ki-67 positive cells, expressed as a mean ± SD, and analysed by Mann–Whitney test, Kruskal–Wallis and Dunn's post hoc test. Results In 6th–7th developmental weeks, the tooth germ and dental crest contained 37% of proliferating cells, which increased to 40% in the 8th week, and then decreased to 15% in the 10th week, whilst the proliferation in the ectomesenchyme subsequently dropped from 37% to 23%. Epithelial parts of the enamel organ displayed similar proliferation activity (31–36%), dental crest 10%, whilst enamel knot showed no proliferating activity. The tooth ectomesenchyme contained more proliferating cells (50%) than the jaw ectomesenchyme (35%), and both dropped to 28% in the 10th week. Ectomesenchyme between the tooth germs contained 23%, whilst the jaw ectomesenchyme contained 15% of proliferating cells. Bcl-2 expression had following pattern: strong in proliferating cells, moderate in tooth germs and dental crest, and weak in the ectomesenchyme. Bax co-expressed with Bcl-2 in the tooth germ and dental crest. In the reticulum and inner enamel epithelium Bcl-2 had prevalent expression, whilst Bax prevailed in the outer enamel epithelium and tooth ectomesenchyme. Conclusions Proliferating cells most likely influence growth of the tooth germ, Bcl-2 affects proliferation and differentiation of specific cell lineages, whilst Bax influences process of cell death.

Cyclin-dependent Kinase 2 (CDK-2) Expression in Nonmelanocytic Human Cutaneous Lesions

Lesions originating from different types of skin cells differ significantly with respect to their pathologic importance. The aim of this work was to examine as to what extent the differences in the origin are reflected in expression levels of CDK-2 and to investigate whether CDK-2 expression might be considered as potential marker useful for diagnostics and assessment of invasiveness of human nonmelanocytic lesions. We conducted comparative immunohistochemical studies of expression of cyclin-dependent kinase 2 (CDK-2) in 16 benign epithelial skin lesions, 11 precancerous lesions, 19 cases of basal cell carcinoma (first such study), 14 squamous cell carcinomas (SCCs), and 7 fibromas. Development of benign epithelial skin lesions was not associated with considerable increase of the CDK-2 expression. Increase of the CDK-2 level was observed in precancerous lesions, and the expression was strongest in SCCs. The level of CDK-2 may be related to invasiveness of skin cancers, as squamous cell carcinomas expressed the enzyme significantly stronger than basal cell carcinomas. Higher percentage fraction of CDK-2 positive cells observed in SCC compared with precancerous lesions may be useful for histopathologic diagnostics of this cancer. Moreover, strong immunohistochemical CDK-2 staining of the cancer cells present deep in dermis may facilitate their detection in histopathologic examinations.

HIF-1α and HIF-2α Are Differentially Regulated In vivo in Neuroblastoma: High HIF-1α Correlates Negatively to Advanced Clinical Stage and Tumor Vascularization

Hypoxia is considered to be a major driving force behind tumor angiogenesis. The stabilization and activation at hypoxia of the hypoxia-inducible factors HIF-1alpha and HIF-2alpha and the concomitant induction of expression of vascular endothelial growth factor (VEGF) and other proangiogenic factors provide a molecular frame for hypoxia-driven tumor angiogenesis. This study has investigated how HIF and VEGF protein levels relate to each other with regard to vascularization, tumor stage, and overall survival in neuroblastoma. Tissue cores taken from tumor specimens representing 93 children with neuroblastoma were arranged on a microarray and stained for HIF-1alpha, HIF-2alpha, VEGF, and CD31 proteins. Both fraction of positive cells and staining intensity were evaluated and protein levels were correlated with each other and with clinical variables. Although high levels of both HIF-1alpha (P < 0.001) and HIF-2alpha (P < 0.001) correlated positively to VEGF expression, they did not fully correlate with each other. Moreover, HIF-1alpha (P = 0.002) and VEGF (P < 0.001), but not HIF-2alpha, correlated negatively to vascularization as determined by CD31 staining abundance. VEGF expression or degree of vascularization did not correlate with tumor stage or overall survival. High HIF-1alpha levels correlated with low tumor stage (P < 0.001) and were associated with a favorable patient prognosis (P = 0.08). The discordant results on expression of HIF-1alpha and HIF-2alpha suggest that these two proteins are differentially regulated in vivo, thus reflecting distinctive protein expression/stabilization mechanisms. The association between HIF-1alpha and favorable outcome stresses the importance of discriminating HIF-2alpha from HIF-1alpha expression and has implications for using HIFs as treatment targets.