Mean Percentage Of Positive Cells Research Articles

Expression of Cathepsin B and Cystatin A in Oral Lichen Planus.

ABSTRACTObjective:Cathepsin B (Cat-B), a cysteine protease, and cystatin A (Cys-A), a protease inhibitor, are involved in the immune response. This study determined Cat-B and Cys-A expression in oral lichen planus (OLP) by immunohistochemistry.Materials and Methods:Thirty specimens each of OLP and healthy gingiva (HG) were included. The expression pattern, the number of positive cells, the staining intensity, and the immunoreactive score (IRS) of Cat-B and Cys-A were investigated. The data were analyzed by using unpaired t-test, Chi-square, and Spearman’s rank correlation.Results:The Cat-B expression in OLP was observed as cytoplasmic staining in the epithelial cells, whereas Cys-A expression was exhibited in the nucleus and cytoplasm of the epithelium. An increase in Cat-B staining intensity was also observed in the basal cells. Conversely, the high staining intensity of Cys-A was observed in the stratum spinosum, but not the stratum basale. In HG, Cat-B expression demonstrated a relatively consistent intensity in the epithelial layer. The Cys-A expression in HG was similar to OLP with a lower staining intensity. The mean percentage of positive cells and the IRS score of Cat-B and Cys-A in OLP were significantly higher than HG (P < 0.05). There was no correlation between Cat-B and Cys-A levels in OLP. Interestingly, Cat-B expression in erosive OLP was greater than in non-erosive OLP (P < 0.05).Conclusion:The Cat-B and Cys-A expression in OLP was more outstanding than in HG, suggesting possible roles for the process of OLP pathogenesis. In addition, Cat-B expression may be an indicator of the disease severity.

E-cadherin-mediated impairment increases anti-apoptotic mechanism through upregulation of Bcl-2: An immunohistochemical study in various patterns of invasion of oral squamous cell carcinoma.

Bcl-2 and E-cadherin proteins are known to be involved in the control of apoptotic cell death and invasive potential, respectively, which is an important hallmark of tumor regulation that influences their biologic behavior. This study investigates the relationship of Bcl-2 and E-cadherin immunoexpression in various Bryne's patterns of invasion. Immunohistochemical analyses for Bcl-2 and E-cadherin were performed on paraffin-embedded tissue sections on 40 cases (32 cases of Oral squamous cell carcinoma and eight cases of controls) and were scored using qualitative and quantitative (percentage positive) analysis. The resulting data were analyzed using SPSS software version 19. Correlation between patterns of invasion and qualitative scores of Bcl-2 and E-cadherin was calculated using Spearman rho correlation. Difference of mean percentage of positive cells of Bcl-2 and E-cadherin in different patterns of invasion was tested by ANOVA followed by Tukey HSD test. Bcl-2 and E-cadherin immunoreactivity was positively correlated with Bryne's pattern of invasion (P value<.05). An inverse relation was found between Bcl-2 and E-cadherin expression with Bryne's patterns 1-5 of invasion. The results pointed to the antagonistic role of E-cadherin and Bcl-2 and thus provide the opportunity for cell survival along with increased invasive potential.

P1.02-043 Multiplexed FISH (ALK/ROS1, RET, NTRK1) in Lung Adenocarcinomas: Novel Dual ALK/ROS1 Probe and Automated Scanning System

Although the use of NGS (next-generation sequencing) is indeed feasible for the study of druggable rearrangements, the sensitivity and specificity of targeted NGS has been challenged on several grounds: use of fixed tissue, poor quality/insufficient sample, RNA-contamination risk or long turn-around time. Our relatively high rate of failures (7.6%) with the RNA workflow of targeted NGS (data not shown), prompted us to investigate possible alternatives. The objective of this study is to demonstrate an efficient solution based on multiplexed fluorescence in situ hybridization (FISH) for the comprehensive characterization of fusions (ALK, ROS1, RET and NTRK1) in lung adenocarcinomas (ACs). We have implemented for the first time in the literature a novel four-color ALK/ROS1 probe and used an automated scanning system for scoring these four translocations. A total of 64 patients with early stage lung ACs who underwent surgery at HM Sanchinarro University Hospital were considered. All slides were carefully reviewed to identify the different histological patterns. Afterwards we performed FISH to study ALK, ROS1, RET and NTRK1 in each pattern. We used both commercially (RET and NTRK1) and non-commercially (ALK/ROS1 dual) available Vysis break-apart probes (Abbott Molecular, USA). Slides were captured and scored with the BioView (Rehovot, Israel) automated imaging and analysis system, using dedicated applications for each probe. Positive cases were all confirmed with targeted NGS (Oncomine Focus AssayTM, Life Technologies, USA). Seven out of 420 slides did not hybridize (1.6%). We found two ALK (2.8%) and two ROS1 (2.8%) positive tumors, while no translocations were identified in RET or NTRK1. The rearrangements were present in all the different histological patterns within a given tumor, with a similar proportion of positive cells. The two major patterns of positivity were represented. The mean percentage of positive cells was 65% (range 46 to 84%). The NGS results confirmed the FISH findings. Simultaneous FISH testing for ALK and ROS1 is feasible on a single slide. The strategy reported herein provided reduced overall scoring time and ensured sensitive counting. This model might be easier to reconcile (lower cost, shorter turn-around time, and less failure rate) with subsequent comprehensive genotyping. Therefore, it could be used prospectively in advanced lung ACs to align the use of several technologies.

Ex Vivo Evaluation of VLA-4 Expression in Primary Human Multiple Myeloma Bone Marrow Samples and In Vivo Mobilization of Murine Multiple Myeloma Cells with Small Molecule VLA-4 Inhibitors

Background: The interaction of multiple myeloma (MM) cells with their microenvironment in the bone marrow (BM) affects disease progression and provides resistance to therapeutic agents. Very-late-antigen 4 (VLA-4, α4β1 integrin, CD49d/CD29) is a noncovalent, heterodimeric transmembrane receptor that is strongly implicated in the pathogenesis of MM via altering cell trafficking, proliferation and drug resistance. LLP2A is a high-affinity peptidomimetic ligand for activated VLA-4. We recently reported (Soodgupta et al. J. Nucl. Med 2016) the sensitive and specific molecular imaging of activated VLA-4 in mouse MM tumors using 64Cu-LLP2A and LLP2A-Cy5. Here we extended these studies by further characterizing VLA-4 expression in primary human MM samples and malignant plasma cells in mouse models of MM.Methods: We evaluated VLA-4 expression in 5 human MM cell lines (U266, OPM2, H929, RPMI-8226 and MM1.S), one mouse MM cell line (5TGM1) and seventeen primary human MM bone marrow samples by flow cytometry using LLP2A-Cy5, soluble VCAM-1/Fc recombinant protein and CD49d (α4) and CD29 (β1) antibodies. The relative mean fluorescence intensity (RMFI) of LLP2A-Cy5 binding was calculated by dividing the MFI of LLP2A-Cy5 binding in the absence of BIO5192 (small molecule VLA-4 inhibitor) by the MFI of LLP2A-Cy5 binding in the presence of excess BIO5192. The 5TGM1/KaLwRij immunocompetent mouse model of MM was used for in vivo study.Results: The expression of activated VLA-4 on MM cell lines as measured by LLP2A-Cy5+ mean fluorescent intensity (MFI) varied 10-fold as follows (LLP2A-Cy5 MFI in parentheses): 5TGM1 (23.7) > U266 (16.1) > OPM2 (4.6) > H929 (3.4) > RPMI-8226 (3.2) > MM1.S (2.1). We observed similar variable expression of LLP2A-Cy5 binding to primary human CD138+CD38+ MM plasma cells (PCs), with 76.47% (13/17) of MM patients exhibiting greater than 20% LLP2A-Cy5+ PCs. expressing VLA-4 on CD138+CD38+ cells. Overall, the mean percentage of positive cells and LLP2A-Cy5 relative MFI (RMFI) on malignant CD138+ PCs from these 13 patients were 78.2% (43.8-98.3%) and 4.3 (1.7-10.8), respectively. Other hematopoietic cells within the BM samples expressed less VLA-4 in descending order as follows; monocytes (58.2%, RMFI 3.0), T-lymphocytes (34.4%, RMFI 2.1) and B-lymphocytes (21.6%, RMFI 1.6). These levels of VLA-4 expression on normal cell subsets within MM patients were comparable to normal blood donors. In general, there was good correlation between LLP2A-Cy5 binding and expression of CD49d and CD29 on CD138+ PCs in MM patients. To our surprise, the four MM patients with <20% LLP2A-Cy5 binding demonstrated high expression of CD49d (92.1%) but very low percentages of CD29 positive cells (17.3%). Using BIO5192 (VLA-4 inhibitor), we found that the LLP2A-Cy5 reagent allowed more accurate detection of activated VLA-4 than the soluble VCAM-1 binding assay as determined by the magnitude of inhibition of binding in the presence of inhibitor. We next evaluated targeting VLA-4 molecule in murine MM model. Preliminary mouse mobilization studies demonstrated that VLA-4 inhibitors effectively and rapidly mobilized murine 5TGM1 MM cells from the bone marrow to the blood (2.49-fold increase in circulating GFP+CD138+ cells) within 1 hour of injection.Summary:This study is the first demonstration that activated VLA4 can be detected on primary human MM cells using LLP2A. These data support the continued development of LLP2A as a molecular diagnostic imaging reagent for MM and as a potential therapeutic target of VLA-4 in MM. Ongoing studies are testing whether small molecule VLA-4 inhibitors can sensitize MM cells to cytotoxic therapy in vivo. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 1849: CXXC5 is overexpressed in human prostate epithelial cancer cell lines in culture and human prostate cancer tissue

Abstract Background: CXXC5 encodes a retinoid-inducible nuclear factor that plays an essential role in human hematopoiesis (1, 2). We have previously identified the differential expression of this gene in rat cancer cell lines with different cancer phenotypes (3). However, knowledge on the expression of this gene in human prostate cell lines or tissue is lacking, both at the transcript and protein level. Methods: Prospective study conducted between January-2012 through June-2015 at the University of Cartagena, Colombia. Expression of CXXC5 was evaluated by quantitative PCR, both in prostate cancer cell lines and tissue biopsies from patients undergoing transrectal ultrasound-guided prostate needle biopsy for suspected prostate cancer (PCa). A total of 65 prostate tissue samples, consisting of 24 benign prostatic tissue biopsies and 41 prostate cancer tissue biopsies were included in the study. Transcript levels in individual biopsies from each group were normalized to transcript levels of beta-actin of the corresponding sample and compared. The protein expression level of CXXC5 was analyzed by immunohistochemistry in benign and malignant prostate tissue. A final score was determined by multiplying the highest intensity of staining by the mean percentage of positive cells stained to yield a value between 0 and 300 for each case. For statistical analysis we compared parameters between group of cases with PCa and without PCa. This study was approved by the Ethical Review Board of the academic institution. Results: CXXC5 transcript expression levels were significantly lower in immortalized non-tumoral prostate cell line PWR-1E compared to PCa cell lines: LNCaP (Fold change: 10.5, p = 0.001), and PC3 (Fold change: 2.8, p = 0.014). In prostate tissue we observed a significant higher expression in low grade prostate cancer compared to benign prostate tissue (p = 0.034). IHC evaluation showed that CXXC5 staining was higher in tissue samples from patients with prostate cancer compared to patients with benign disease (p = &amp;lt;0.001). In the prostate cancer group, immunostaining was also stronger in malignant acini than in adjacent, benign acini, within the same patient sample. Conclusions: CXXC5 exhibits differential expression in prostate cell lines with different cancer phenotypes and between cancer and benign prostate tissue. Additional studies are required to determine the biological and clinical significance of CXXC5 in prostate cancer.

Aldehyde dehyderogenase (ALDH1A1) delineating the normal and cancer stem cells in spectral lung lesions: An immunohistochemical appraisal

Background and aimALDH1A1 is one of the promising cancer stem cell markers, it has been found in different cancers, including lung cancer. We aimed to assess its immunohistochemical expression in spectral lung lesions (neoplastic versus non neoplastic) and to correlate its expression with certain pathological parameters (e.g. histopathological type and tumor grade). Materials and methodsThe study included 105 specimens of spectral lung lesions (20 adjacent normal tissues, 64 non small cell carcinoma (NSCLCs), 16 small cell carcinoma (SCLCs) and six inflammatory pseudotumors, in addition to nine metastatic tumors to lymph nodes. Both H&E and immunohistochemical stained sections were assessed and evaluated according to quick immunoreactivity score. ResultsALDH1A1 expression showed a broad dynamic range of immunoreactivity score (0–8) in different types of lung cancers with strong reactivity of normal stem cells. Significant statistical associations were found between ALDH1A1 expression and squamous cell carcinoma & SCLC (p=0.02 and 0.003, respectively), with very high mean percentage of positive cells in SCLC (58.50±10.62), followed by adenocarcinoma and squamous cell carcinoma (38.37±35.12 and 22.77±13.89, respectively). Adenocarcinomas were classified according to the IASLC/ATS/ERS classification and grouped by architectural grade into low, intermediate and high. Heterogeneity of ALDH1A1 immunoreactivity scores within adenocarcinoma can reflect histological patterns heterogeneity with a strong association to the grade. Strong diffuse expression was seen in inflammatory pseudotumors and impressive membranous expression was noticed in metastatic tumors. Important pitfalls of ALDH1A1 immunostaining were applied for accurate interpretations. ConclusionWe ascertained the presence of strong association between ALDH1A1 expression and both histological subtype & tumor grade of NSCLCs. We report a fascinating finding; ALDH1A1 heterogeneity within adenocarcinoma can be mapped to its histological patterns. Furthermore, we elucidated its presence in SCLCs and inflammatory pseudotumors. Also a comparative image was drawn between its expressions in the primary lung tissue and in the metastatic lymph nodes.

Immunohistochemical evaluation of myofibroblast density in odontogenic cysts and tumors.

Background. The aim of this study was to investigate myofibroblast (MF) density in a broad spectrum of odontogenic cysts and tumors and the relation between the density of MFs and the clinical behavior of these lesions.Methods. A total of 105 cases of odontogenic lesions, including unicystic ameloblastoma (UAM), solid ameloblastoma (SA), odontogenic keratocyst (OKC), dentigerous cyst (DC), radicular cyst (RC) (15 for each category), and odontogenic myxoma (OM), adenomatoid odontogenic tumor (AOT), calcifying odontogenic cyst (COC) (10 for each category), were immunohistochemically stained with anti-α-smooth muscle actin antibody. The mean percentage of positive cells in 10 high-power fields was considered as MF density for each case.Results. A statistically significant difference was observed in the mean scores between the study groups (P < 0.001). The intensity of MFs was significantly higher in odontogenic tumors compared to odontogenic cysts (P < 0.001). There was no statistically significant difference between odontogenic tumors, except between UAM and OM (P = 0.041). The difference between OKC and odontogenic tumors was not statistically significant (P > 0.05). The number of MFs was significantly higher in OKC and lower in COC compared to other odontogenic cysts (P = 0.007 and P = 0.045, respectively).Conclusion. The results of the present study suggest a role for MFs in the aggressive behavior of odontogenic lesions. MFs may represent an important target of therapy, especially for aggressive odontogenic lesions. Our findings support the classification of OKC in the category of odontogenic tumors.

Investigating the association of cytoplasmic and nuclear HIF-2 expression with cancer specific survival (CSS) in clear cell renal cell carcinoma.

387 Background: Studies support the prognostic importance of HIF-2α for ccRCC. Interestingly, a recent study has implicated HIF-2α as part of a protein translational initiation complex, a cytoplasmic function that goes far beyond its role as a nuclear transcription factor. We hypothesized that both the absolute expression as well as the subcellular localization of HIF-2α would predict clinicopathological features and CSS in ccRCC. Methods: A tissue microarray (TMA) study was conducted on 308 ccRCC patients. Survival differences were investigated with the log rank test and associations with CSS with uni- and multivariate Cox regression analyses. Recursive partition tree analysis was used to identify relevant cutoff values. Results: The median follow-up was 2.29 years (IQR 11.82). The mean percentage of positive cells was 22.45±18.00 and 0.12 ± 0.29 for HIF-2α nuclear (N) and cytoplasmic (C), respectively. High HIF-2α N (cutoff&gt;32%) expression was associated with smaller tumor sizes (p = 0.002) and less advanced Fuhrman grades (p = 0.044). To the contrary, tumors with high HIF-2α C (&gt;0%) more often had lymph node (p = 0.004), distant metastases (p = 0.021), and higher Fuhrman grades (p&lt;0.0001). Univariate analyses showed an association of high HIF-2α N (p = 0.035) with better CSS. This was not found when HIF-2α N was used as a continuous variable (p = 0.067). In contrast, HIF-2α C demonstrated an association with CSS when examined as both a continuous (p &lt; 0.0001) and as a dichotomized variable (p &lt; 0.0001). After adjustment for TNM stage, ECOG PS, and Fuhrman grade, both continuous (p &lt; 0.0001) and dichotomized (p &lt; 0.0001) HIF-2α C variables remained significant predictors of CSS, while neither HIF-2α N variable was retained. The ratio of HIF-2α C/N was the strongest predictor of CSS in multivariate analysis (p = 0.001; HR 4.83 [95% CI: 1.99 – 11.76]). Conclusions: Our investigation suggests an important role for HIF-2α as a cytoplasmic protein translational initiation complex in ccRCC. This novel observation warrants further molecular and genetic studies examining biological pathways that are involved in the tumor promotive properties of HIF-2α in the cytoplasm.

IMP3, a Proposed Novel Basal Phenotype Marker, is Commonly Overexpressed in Adenoid Cystic Carcinomas but not in Apocrine Carcinomas of the Breast

Insulin-like growth factor-II mRNA-binding protein 3 (IMP3) is a member of the insulin-like growth factor-II signaling pathway, and has recently been described as a biomarker of basal-like breast carcinomas. This study explored IMP3 expression in adenoid cystic carcinomas of the breast, a special type of basal-like, triple-negative (estrogen receptor/progesterone receptor/human epidermal growth factor receptor 2/neu protein negative) carcinoma and compared it with a group of apocrine carcinomas, which are an example of estrogen receptor/progesterone receptor negative, special type of breast carcinoma. Eighteen breast adenoid cystic carcinomas (16 primary and 2 corresponding metastases) and 18 apocrine carcinomas (16 invasive and 2 in situ) were evaluated for the expression of IMP3 protein using immunohistochemical method. A cut-off value for IMP3 positivity was set at 10%. Thirteen of 16 (81.3%) primary adenoid cystic carcinomas overexpressed IMP3 protein, predominantly in membranous distribution. The mean percentage of positive cells among primary adenoid cystic carcinomas was 50%. Both metastatic adenoid cystic carcinomas also strongly overexpressed IMP3 protein (70% and 80% of the tumor cells, respectively). In contrast, only 4 of 16 invasive apocrine carcinomas (25%) exhibited IMP3 positivity with significantly lower percentage of positive cells (27%, P<0.001). Two in-situ apocrine carcinomas were negative. Our results indicate that IMP3 may be an additional basal-type marker in breast carcinoma whose expression can be occasionally seen in other types of breast carcinomas such as apocrine type.

Reciprocal Altered Expression of E-Cadherin and P-Cadherin in Mucous Membrane Pemphigoid

E- and P- cadherins are involved in the selective adhesion of epidermal cells. To gain insight into the role of cadherins on the acantholysis of keratinocytes and further investigate the pathogenesis of Mucous Membrane Pemphigoid, we examined the expression of P-cadherin and E-cadherin, in normal human oral mucosa, lesional and peri-lesional mucosa in MMP. Twenty-nine samples from paraffin-embedded specimens of MMP were used for the study. Five specimens of healthy oral mucosa were evaluated as control group. To evaluate the E- and P-Cadherin expression, a mean percentage of positive cells was determined from the percentage of positive cells derived from the analysis of 100 cells in ten random areas at x400 magnification. It was observed that E-cadherin was weakly and discontinuously expressed on the epithelial layers of pemphigoid mucosa, while it was intensively expressed on all keratinocytes in normal human skin. In contrast, P-cadherin was strongly expressed throughout the entire epidermal layer in MMP samples, although its expression is restricted to the basal cell layer in normal human skin. Statistical analyses showed that the percentage of E-cadherin positive cells in the epithelium of pemphigoid cases was significantly decreased compared with that in normal human mucosa. There was a significant increase in the percentage of P-cadherin positive cells in the epithelial layers of MMP compared with normal human mucosa. The present study showed that there is downregulation of E-cadherin expression and upregulation of P-cadherin expression in MMP mucosa, which may be involved in the pathogenesis of MMP.

CD133+ Acute Myeloid Leukemia (AML) Cells Exhibit Higher Clonogenic Capacity and Higher Levels of pAKT and Bcl-2 Proteins Than Their Negative Counterpart

AbstractAbstract 1721 Introduction:CD133 is a transmembrane protein expressed by hematopoietic stem cells and other cell types, whose physiological functions remain unknown. To determine its potential prognostic value in AML, we analyzed its expression on blasts from 76 patients at diagnosis. Moreover, since leukemic stem cells play a critical role in the resistance to chemotherapeutic drugs and may be responsible for relapse, we compared the growth potential and activated cellular pathways of CD133+ and CD133- cells from 20 AML patients. Methods:First, bone marrow samples were collected at diagnosis from 76 AML patients (median age: 65, range: 1–84). CD133 expression was evaluated on blasts cells and CD34+ cells using 6-color flow cytometry (FC) and the monoclonal antibody AC133. The mean fluorescence intensity ratio (MFIR) was calculated by dividing the MFI of CD133 by the MFI of its isotypic control. Second, purified CD34+, CD34+CD133+ and CD34+CD133- cells were isolated from 20 AML bone marrows using the magnetic separation system Minimacs (Miltenyi Biotec). These fractions were submitted to an in vitro colony forming assay during 14 days. Finally, the level of expression of survival proteins involved in the PI3K/AKT, Mitogen-Activated Protein Kinase (MAPK) and Bcl-2 pathways, as well as CXCR4 and Focal Adhesion Kinase (FAK) migration proteins, was analyzed by FC in these fractions. Results:The level of CD133 expression on blasts cells was significantly different between AML patients with favorable (n=12), intermediate (n=38) and unfavorable karyotypes (n=23) (p=0.007). In particular, it was significantly higher in patients with unfavorable karyotype than in patients with intermediate karyotype (p=0.032) or favorable karyotype (p=0.005). Moreover, when considering NPM1 and FLT3 mutation status in patients with intermediate karyotype, the level of CD133 expression was significantly higher in patients with an intermediate molecular profile than in patients with a favorable molecular profile (p=0.004). Most interestingly, we showed that the level of CD133 expression has an impact on overall survival in multivariate analysis, independently of leukocytosis, karyotype and achievement of complete remission after induction therapy (p=0.04). Second, in 20 patients, we found that AML progenitor cells (AML CFU) were present in all sorted fractions, but the CD34+CD133+ fraction gave rise to larger and more numerous colonies (74.9±39.6) than the CD34+CD133- fraction (11,25±6). Moreover, the CD34+CD133+ fraction was the only one able to form secondary colonies (52.1±31.8). Finally, we found that the expression of PI3K (mean percentage of positive cells: 78±20.1), pAKT (80±19.8), pERK (80.1±19), CXCR4 (71.6±22.7), pFAK (81.2±19.8), Bcl-2 (72.9±26.1), Bcl-xL (45.7±24.8), and pBad (63.9±36.1) was significantly upregulated in CD34+CD133+ cells compared to CD34+CD133- cells, whereas Bax was downregulated. Discussion:CD133 is known to be expressed in hematopoietic progenitors, but its role in the resistance to chemotherapy is not well studied. In our work, the level of expression of CD133 on AML blasts appeared as an independent immunophenotypic prognostic factor, encouraging its further evaluation in a larger number of cases. We also showed that CD133+ cells have greater replicative properties than CD133- cells, and exhibit activation of various signaling pathways. Since CD133+ cells might escape from apoptosis through activation of PI3K/AKT and ERK, these proteins could represent potential therapeutic targets. In conclusion, our results revealed that CD133+ cells exhibit more aggressive behavior than CD133- ones, and that CD133+ AML progenitors might contribute to resistance to chemotherapy through preferential activation of PI3K/AKT, MAPK and Bcl-2 survival response. Disclosures:No relevant conflicts of interest to declare.

Expression of alpha-dystroglycan correlates with tumour grade and predicts survival in oral squamous cell carcinoma

Dystroglycan (DG) is a non-integrin adhesion molecule connecting the extracellular matrix to the actin cytoskeleton. Decreased expression of DG has been reported in several human cancers and related to tumour aggressiveness. Expression of the alpha-DG subunit was evaluated by immunostaining in a series of oral squamous cell carcinoma (OSCC) and its relation with traditional prognostic indicators and with the clinical outcome of the patients was evaluated. Alpha-DG expression was easily detected in normal epithelium with a mean percentage of positive cells >80% but was undetectable in a significant fraction (59%) of OSCC. Loss of alpha-DG staining correlated with higher tumour grade (p = 0.04) and stage (p = 0.01), with nodal involvement (p = 0.001) and with an increased risk of recurrence (p = 0.002) and death (p = 0.004) in a univariate analysis, but it was not confirmed as an independent predictor of clinical outcome in a multivariate analysis. Loss of alpha-DG expression, which corresponds to loss of a functional DG complex, is a frequent event in human OSCC. Further studies are warranted on the role of this molecule in the entire multistep process of oral squamous tumorigenesis.

Genetic modification of primary chronic lymphocytic leukemia cells with a lentivirus expressing CD38

Studies of the role of individual genes in chronic lymphocytic leukemia (CLL) have been hampered by the inability to consistently transfect primary tumor cells. Here, we describe a highly efficient method of genetically modifying primary CLL cells using a VSVG pseudotyped lentiviral vector. We transduced CD38 negative CLL cells with a lentiviral vector encoding CD38 which caused increased surface CD38 expression in all the samples tested (n=17) with no evidence of plasmacytoid differentiation. The mean percentage of positive cells expressing CD38 was 87%+/-8.5% and the mean cell viability 74%+/-17%. This high level of transduction of all the CLL cell samples tested demonstrates the utility of this technique which should prove applicable for the introduction and analysis of other genes in these non-dividing cells.

Accelerated cellular senescence in myelodysplastic syndrome

To investigate the contribution of cellular senescence to the progression and prognosis of myelodysplastic syndrome (MDS). We have analyzed the expression of p16INK4a in bone marrow mononuclear cells or CD34(+) cells from 53 patients with MDS, 12 acute myeloid leukemia (AML), and 11 healthy controls. Additionally, We have assessed quantitatively senescence-associated beta-galactosidase (SA-beta-gal) staining on bone marrow mononuclear cells from MDS and AML patients, HL60 and SHI-1 leukemia cell lines, and healthy control cells. An upregulated expression of senescence-associated molecular marker p16INK4a was found in MDS compared with healthy controls, while a lower expression of p16INK4a was observed in AML compared with healthy controls. International Prognostic Scoring System score was negatively correlated with the percentage of p16INK4a-positive cells. The SA-beta-gal activity measured by mean percentage of positive cells was significantly higher in MDS cases when compared with controls. Meanwhile, percentage of SA-beta-gal-positive cells was also remarkably higher in dysplastic cells of MDS when compared to nondysplastic cells from MDS. These results of our present study suggested an accelerated cellular senescence occurred in MDS, and the cellular senescence may be involved in the progression and prognosis of MDS.

Over‐expression of metallothionein predicts chemoresistance in breast cancer

Metallothionein (MT) plays a role in fundamental cellular processes such as proliferation, apoptosis and differentiation. We examined MT expression in women with invasive breast ductal carcinoma who underwent mastectomy/lumpectomy without neo-adjuvant treatment. We showed that MT was over-expressed in 87.9% of breast cancer tissues examined, with the mean percentage of positive cells at 30%. There were two patterns of MT expression: predominantly cytoplasmic in 75.9% and nuclear in 24.1% of MT-positive cases. Higher MT scores were associated with poorer histological grade (p = 0.009) but were independent of age, tumour size and oestrogen receptor status. For patients who were treated with adjuvant chemotherapy (cyclophosphamide/methotrexate/5 fluorouracil- or doxorubicin-based regimes), those with high MT expression had a significantly lower recurrence-free survival (p = 0.048), suggesting a role of MT in predicting disease recurrence. Down-regulation of MT in MCF-7 cells by silencing the MT-2A gene (the most abundantly expressed of the 10 known functional MT isoforms) increased chemosensitivity of the cells to doxorubicin. To examine the mechanisms underlying these clinical data, we used siRNAs to decrease MT-2A mRNA expression and protein expression. In MT down-regulated cells challenged with the IC(50) concentration of doxorubicin, we observed a significant reduction in cell viability. Cell cycle analysis also revealed a corresponding increase in apoptosis in the MT down-regulated cells following doxorubicin exposure, showing that down-regulation of MT increased susceptibility to doxorubicin cytotoxicity. The data suggest that MT could be a potential marker of chemoresistance and a molecular therapeutic target.

Expression of Cellular Senesence Associated B-Galactosidase (SABG) Is up-Regulated in Melodysplastic Syndrome

Age and mutagen related stem cell depletion may contribute to emergence of neoplastic clones. While telomere shortening or dysfunction may initiate replicative senescence and apoptosis, oncogene-induced senescence arising from induction of inflammatory cytokines may precede telomeric changes in neoplastic cells (Cell 2008; 133: 1019). Activation of cellular senescence is associated with up-regulation of b-galactosidase activity at pH6 known as ‘senescence-associated’ b-galactosidase (SABG). To investigate the role of senescence in myelodysplastic syndromes (MDS), we evaluated SABG activity in bone marrow cells of newly diagnosed MDS patients (n=18) and age-matched controls (n=9).METHODS: MDS cases that received no prior treatment other than growth factors were included. All MDS diagnoses were histologically confirmed by bone marrow examination, classified by WHO and categorized into low risk {RA, RCMD, RCMD-RS and del (5q)} (n=10) and high risk groups { RAEB-1, RAEB-2} (n=8). Controls bone marrows were obtained from staging marrows for lymphoid malignancy after confirmed absence of medullary tumor involvement. Cytospins were prepared from bone marrow aspirates and stained using a SABG staining Kit. SABG expression was characterized by average staining intensity and quantitated by the percentage of positive cells according to cell lineage (i.e., megakaryocyte, myeloid, erythroid and lymphoid cells). Staining differences were compared using Vander Waerden Two-Sample Test. Correlations between myeloid SABG expression and, blast percentage or, absolute neutrophil count (ANC); megakaryocyte positivity and platelet count; average positivity and IPSS score among the two WHO groups was analyzed using Spearman coefficient.RESULTS: SABG expression as measured by mean percentage of positive cells was significantly higher in low risk MDS cases (mean (SD) = 9% (0.090476)) when compared with controls (mean (SD) =1% (0.030923)) (p 0.02). Percentage of SABG-positive myeloid cells was significantly also higher in low risk MDS cases (mean (SD) = 8% (0.077201)) when compared to controls (mean (SD) =1%) (p 0.02). High risk MDS cases also had increased SABG expression as measured by mean percentage of positive cells (mean (SD) = 14% (0.164099)) and percentage of SABG-positive myeloid cells (mean (SD) = 24 % (0.290369)). Similarly, the percentage of SABG positive megakaryocytes was higher in low risk MDS cases (mean (SD) =20 %( 0.312681)) and high risk MDS cases (mean (SD) = 14% (0.2258)) compared to controls (mean (SD) =0% (0.007559)) (p 0.13 and 0.21 respectively). In high risk MDS we found a positive correlation between the percentage of SABG staining myeloid cells and marrow blast percentage (mean (SD) = 9.3750(4.5019)) (0.07702). Moreover, in low risk MDS there was a negative correlation between percentage myeloid cell positivity and absolute neutrophil count (mean (SD) = 2.2 (1.27)) (−0.40122). There was no significant difference in average percentage of SABG staining among IPSS risk categories. SABG positive cells lacked cytologic features of apoptosis or mitosis.CONCLUSION: MDS is associated with activation of cellular senescence programs in affected hematopoietic precursors. The linkage between myeloid SABG expression and neutropenia and blast percentage suggests that cellular senescence may contribute to maturation impairment and ineffective myelopoiesis.

Dystroglycan expression correlates with tumor grade and with outcome in renal cell carcinoma patients

15554 Background: The dystroglycan (DG) complex is a transmembrane glycoprotein that forms a continuous link from the extracellular matrix to the actin cytoskeleton. Deregulated expression of DG has been reported in a variety of human malignancies and related to tumor differentiation and aggressiveness. Methods: In this study, the expression of the a-subunit of DG was evaluated by immunostaining in a series of 125 renal cell carcinomas (RCCs) and its relation with disease progression and cancer-specific survival was evaluated. Results: to date, median follow-up is 19 months (range 1–96). We found that a-DG expression was lost in a significant fraction of tumors (66%) with 18% being the mean percentage of positive cells (median = 0; range = 0–80%). Loss of DG staining correlated with higher tumor grade (p=0.039) but not with tumor stage or size. Recurrence (p=0.014) and death (p=0.041) from RCCs were significantly more frequent in patients whose tumors displayed reduced staining for DG compared with patients whose tumors were positive for a-DG expression. Kaplan-Meier analysis showed a significant separation between high vs low a-DG expression for both disease-free (p=0.0094) and overall (p=0.0023) survival. In a multivariate analysis, loss of a-DG expression was the only independent risk predictor for recurrence (HR=6.509, p=0.0012) and death (HR=4.701, p=0.012) from RCCs when tumor size as well as tumor grade and stage were included in the model. Conclusions: These findings demonstrate that loss of a-DG expression, which correspond to loss a functional DG complex, is a frequent event in human renal tumorigenesis and is associated with an aggressive phenotype of the disease. They also suggest that evaluation of DG expression has the potential to offer important prognostic information and warrant further studies to better understand the role(s) of this molecule in term of renal cancer development and as a new prognostic marker for RCC patients. No significant financial relationships to disclose.

Biodegradable chitosan scaffolds containing microspheres as carriers for controlled transforming growth factor-β1 delivery for cartilage tissue engineering

Natural articular cartilage has a limited capacity for spontaneous regeneration. Controlled release of transforming growth factor-beta1 (TGF-beta1) to cartilage defects can enhance chondrogenesis. In this study, we assessed the feasibility of using biodegradable chitosan microspheres as carriers for controlled TGF-beta1 delivery and the effect of released TGF-beta1 on the chondrogenic potential of chondrocytes. Chitosan scaffolds and chitosan microspheres loaded with TGF-beta1 were prepared by the freeze-drying and the emulsion-crosslinking method respectively. In vitro drug release kinetics, as measured by enzyme-linked immunosorbent assay, was monitored for 7 days. Lysozyme degradation was performed for 4 weeks to detect in vitro degradability of the scaffolds and the microspheres. Rabbit chondrocytes were seeded on the scaffolds containing TGF-beta1 microspheres and incubated in vitro for 3 weeks. Histological examination and type II collagen immunohistochemical staining was performed to evaluate the effects of released TGF-beta1 on cell adhesivity, proliferation and synthesis of the extracellular matrix. TGF-beta1 was encapsulated into chitosan microspheres and the encapsulation efficiency of TGF-beta1 was high (90.1%). During 4 weeks of incubation in lysozyme solution for in vitro degradation, the mass of both the scaffolds and the microspheres decreased continuously and significant morphological changes was noticed. From the release experiments, it was found that TGF-beta1 could be released from the microspheres in a multiphasic fashion including an initial burst phase, a slow linear release phase and a plateau phase. The release amount of TGF-beta1 was 37.4%, 50.7%, 61.3%, and 63.5% for 1, 3, 5, and 7 days respectively. At 21 days after cultivation, type II collagen immunohistochemical staining was performed. The mean percentage of positive cells for collagen type II in control group (32.7% +/- 10.4%) was significantly lower than that in the controlled TGF-beta1 release group (92.4% +/- 4.8%, P < 0.05). Both the proliferation rate and production of collagen type II in the transforming growth factor-beta1 microsphere incorporated scaffolds were significantly higher than those in the scaffolds without microspheres, indicating that the activity of TGF-beta1 was retained during microsphere fabrication and after growth factor release. Chitosan microspheres can serve as delivery vehicles for controlled release of TGF-beta1, and the released growth factor can augment chondrocytes proliferation and synthesis of extracellular matrix. Chitosan scaffolds incorporated with chitosan microspheres loaded with TGF-beta1 possess a promising potential to be applied for controlled cytokine delivery and cartilage tissue engineering.

α-methylacyl-CoA racemase (P504S)/34 βE12/p63 triple cocktail stain in prostatic adenocarcinoma after hormonal therapy

Alpha-methylacyl-CoA racemase (AMACR) has recently been shown to be a highly sensitive marker for the diagnosis of prostate cancer. However, there is limited information concerning its utility as a marker for prostate carcinoma after hormonal therapy. Our current investigation was conducted to evaluate the expression of AMACR in patients with prostate carcinoma after hormonal therapy and assess its diagnostic utility in combination with p63 and high molecular weight cytokeratin (34betaE12) staining. Prostate tissues from 49 patients who had been treated with hormonal therapy were immunohistochemically analyzed for AMACR, 34betaE12, and p63 expression by a triple antibody cocktail stain. The staining intensities and the percentages of positively staining tumor cells were recorded. The correlations between AMACR expression and metastatic status, associated hormonal therapy regimens, and the extent of hormone therapy effect were analyzed. All malignant acini were completely negative for both basal cell markers (34betaE12 and p63). Tumor cells failed to demonstrate expression of AMACR in 14 (29%) of 49 cases. In the remaining 35 cases (71%), positive immunostaining for AMACR was noted, but with variable intensities and percentages of cells stained. Positive staining for AMACR in benign glands was not seen in any case. In all cases, basal cells were strongly stained by p63 in benign acini with a mean positive percentage of 96%. Similarly, basal cells in benign acini displayed moderate staining intensities for 34betaE12 in 3 (7%) of 41 cases and strong immunostaining for this marker in the remaining 38 cases (93%); the mean percentage of positive cells was 92%. alpha-methylacyl-CoA racemase expression may be substantially diminished or entirely lost in prostate carcinoma after hormonal therapy. This variation in AMACR expression does not correlate with the metastatic status, the modality of hormonal therapy, or the extent of therapy-related effect. It is important that pathologists be aware that some hormonally treated prostate carcinomas do not express AMACR, and that immunostaining in such cases must be interpreted with caution. A triple cocktail stain using AMACR, 34betaE12, and p63 can be helpful in evaluating prostate specimens for the presence of residual or recurrent carcinoma after hormonal therapy for cancer.

Aberrant expression of α-dystroglycan in cervical and vulvar cancer

Objectives. Cervical and vulvar cancers develop through well-defined precursor lesions but their exact pathogenesis is still unknown. The dystroglycan complex is a transmembrane glycoprotein that forms a continuous link from the extracellular matrix to the actin cytoskeleton. Deregulated expression of dystroglycan has been reported in human malignancies and related to tumor differentiation and aggressiveness. In this study, expression of dystroglycan was evaluated in the multistep cervical and vulvar tumorigenesis. Methods. Expression of the dystroglycan complex was evaluated by immunostaining in lesions representing different stages of vulvar and cervical tumorigenesis using a monoclonal antibody which recognizes carbohydratic epitopes on the α-dystroglycan subunit. Results. α-dystroglycan was constantly detected in normal cervical epithelium with a mean percentage of positive cells higher than 80%. A progressive significant reduction in the mean percentage of positive cells was observed in low (67%) and high grade SIL (14%) and in invasive carcinomas (2.6%) of the cervix. In cancers, no differences were observed in terms of percentage of positive cells when cases were stratified according with either tumor grade or stage. A progressive significant reduction in the mean percentage of positive cells was also observed from normal vulvar epithelium (90%) to VIN1 (66%), VIN2 (28%) and invasive vulvar carcinomas (22%). No significant decrease in the α-dystroglycan staining was observed in squamous cell hyperplasia lesions (85%) while lichen sclerosus displayed a percentage of positive cells (47%) significantly lower than normal epithelium. Conclusions. Detection of α-dystroglycan is frequently lost in human cervical and vulvar tumorigenesis and further studies are warranted to verify whether evaluation of this molecule might serve as marker of risk progression of preneoplastic lesions and to better understand its significance in terms of cancer development.