Percentage Of Ki-67 Positive Cells Research Articles

Survivin expression in canine lymphomas in relation with proliferative markers.

Survivin is a member of apoptosis inhibiting proteins family. Apart from its antiapoptotic activity it plays a critical role in regulating the cell cycle and mitosis. It is overexpressed in most human malignancies. While the prognostic significance of survivin expression is widely investigated in human non-Hodgkin's lymphomas, little is known about its expression in canine lymphomas. The aim of the study was to evaluate the expression of survivin in canine lymphomas in relation to proliferation markers (mitotic index and percentage of Ki67-positive cells). Survivin was found in all examined lymphomas belonging to 6 different morphological subtypes with nuclear immunoreactivity. In most of lymphomas (18/25) survivin expression ranged 10%-25% of positive cells. Only single cases had lower (0-10% positive cells, 1/25) or higher (25-50% and > 50% positive cells, 5/25 and 1/25, respectively) index of survivin. Neither mitotic index nor proliferative index correlated with survivin expression when the values quantified randomly in whole specimens were compared. However, when survivin expression were quantified in selected tumor areas of low and high proliferation activity the high correlations between survivin expression and proliferation index were found. The results indicated that survivin is commonly expressed in canine lymphomas. Nuclear labelling together with the relation of its expression and proliferative activity in highly proliferative areas of neoplastic tissue suggest a potential role of survivin in cell cycle activation in canine lymphoma cells. However, further studies of the relation between expression of survivin and other proteins involved in cell cycle regulation are needed. Moreover, the results suggest that survivin may pose the therapeutic target in canine lymphomas.

Microrna Mir-21 Is Upregulated after Different Microenvironmental Stimuli and Controls Proliferation, Chemotaxis and Chemoresistance in Chronic Lymphocytic Leukemia

Aberrant expression of diverse microRNAs (miRNAs) has been related to pathogenesis and clinical outcome in patients with CLL. miR-21 is overexpressed in a wide variety of neoplasms where it participates in oncogenic events such as proliferation, resistance to treatment and metastasis. In CLL, miR-21 expression has been associated to refractoriness to fludarabine and to shorter overall survival. In addition, high expression of ZAP-70 protein in CLL is related to shorter overall survival and higher probability of progression. Several biological mechanisms have been described explaining the adverse prognosis associated with high ZAP-70 expression. In this sense, ZAP-70 protein increases the capability of CLL cells to respond to different survival and migration signals provided by the microenvironment. In a previous work, we found that transfected B-cell lines with a ZAP-70 expressing vector (Calpe at al, Blood 2011) had an increased expression of several molecules, including miR-21. Against this background, we studied the relationship between ZAP-70 protein and miR-21. For this, we firstly analyzed the correlation of ZAP-70 with miR-21 in primary CLL cells from 32 patients. In this series we observed that miR-21 expression analyzed by QRT-PCR was significantly higher in patients with high expression of ZAP-70 compared to patients with low expression of ZAP-70 (mean miR-21 in high ZAP-70 (N=17) = 5.781 ± 1.517; mean miR-21 in low ZAP-70 (N=15) = 0.6783 ± 0.2730; p=0.0082). In order to further analyze the molecular mechanisms regulating the induction of miR-21 and the potential role of ZAP-70 protein in this process, we co-cultured primary CLL cells (N=16) in conditions mimicking the microenvironment in the proliferation centers (bone marrow stromal cells with concomitant stimulation of CD40 and TLR9). In these conditions, besides ZAP-70 activation, we observed a 3.6 ± 0.78 mean fold induction in miR-21 expression after 48 hours compared to cells in suspension. Interestingly, this increase was only significant in patients with high expression of ZAP-70 (N=8; p= 0.0379). To define the role of ZAP-70 in miR-21 regulation, we stably transfected Ramos B-cells with ZAP-70 protein and found that both MAPK and STAT3 participate in the induction of miR-21 expression after ZAP-70 activation upon BCR crosslinking. Moreover, using this system we observed downregulation of the tumor suppressors PTEN, PDCD4 and PIAS3, all of which have been found to be targeted by miR-21 in malignant cells. Next, we aimed to study the functional role of miR-21 in primary CLL cells co-cultured in conditions mimicking the microenvironment from the proliferation centers. For this, we analyzed survival, proliferation and response to fludarabine after inhibition of miR-21 expression by transfecting primary CLL cells with antisense miR-21 inhibitor. The co-culture of primary CLL cells significantly increased their survival after 48 hours regardless the inhibition of miR-21. However, the induction of proliferation (measured as percentage of Ki-67 positive cells) was significantly inhibited by the suppression of miR-21 (N=13; p=0.022). Next, the analysis of a small pilot cohort showed a consistent but not yet significant overcoming of the chemoresistance induced by the co-culture after the inhibition of miR-21 Interestingly, the sensitivity to fludarabine of primary CLL cells cultured in suspension was also increased by the inhibition of miR-21. Finally, we examined the ability of primary CLL cells to migrate towards CXCL12 and observed that inhibition of miR-21 resulted in a 2.62-fold reduction of the migration index (ratio of cells migrating towards culture media to cells migrating towards media with 200ng/mL CXCL12). In conclusion, we have showed the correlation and participation of ZAP-70 protein in the regulation of miR-21. We have also observed that miR-21 contributes to CLL proliferation, resistance to fludarabine, and chemotaxis towards CXCL12. Although further experiments are warranted in order to fully elucidate the regulation and functional role of miR-21 in CLL, these results help to enlighten the biology behind the adverse clinical outcome of patients with CLL and high expression of ZAP-70, and could potentially be exploited for the development of new treatments in a near future. DisclosuresBosch:Roche: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Abstract 4913: Development of Fe3O4@TiO2 core-shell nanocomposites as radiosensitizers

Abstract Purpose: To test in vivo, in a rabbit VX2 liver cancer model, a nanocomposite made with Fe3O4 core and TiO2 shell as a possible radiosensitization agent. Methods: This study was approved by the institutional ACUC. Rabbits were implanted in liver with pairs of VX2 tumors distantly enough to allow irradiation of only one of the two tumors. The tumors were allowed to grow until clearly detectable by magnetic resonance imaging, followed by Computed Tomography (CT) simulation to identify and contour the tumors. Each rabbit was anesthetized, and immobilized in supine position. Isocenter was placed during the CT simulation and the rabbit was tattooed. After the CT, each rabbit was injected with nanoparticles and put under observation until recovery. The core-shell nanoparticles were surface covered with glucose (glut-NPs) and delivered intravenously to 6 rabbits. A radiation oncologist contoured the target volumes. The gross target volume included a hepatic tumor in one lobe; 1 - 3 mm expansion was done to create the clinical target volume and another 1-2 mm expansion to create the planning target volume (PTV). The control hepatic tumor was contoured and the treatment plan objective was to deliver more than 95% of the prescribed dose to the PTV while sparing the control tumor. A certified radiation physicist created the treatment plan. Dose Volume Histograms were created and the radiation oncologist approved the plan. Irradiation usually utilized 2 - 3 beams, delivering 2 Gy as a single fraction using Linear Accelerator. All 6 rabbits treated with nanoparticles the day before (14-18h previously) and 3 radiation-only (not glut-NPs treated) animals were irradiated so that only one of the tumors received radiation. The animals were terminated 3 days after irradiation and the tumors were harvested. Several view-fields with at 3-10 x 103 cells were automatically scored using the NanoZoomer and percentage of Ki67 positive cells, as a marker of proliferation was determined in each tumor. TUNEL assay was done as well. Results: In each rabbit the larger tumor of the pair was irradiated. In no-NP treated animals that led to the fact that irradiated tumors at 3 days after irradiation generally still had greater number of proliferating cells than the non-irradiated tumor sections. Conversely, in glut-NPs treated rabbits, irradiation led to a decrease of Ki-67 positive cells compared to the paired non-irradiated glut-NPs treated tumors. Three-day time-point was better suited for evaluation of tumor proliferation changes. TUNEL analysis showed higher apoptotic cell numbers in glut-NPs treated tumors (p=0.049). Combination of radiation with glut-NP treatment showed only a mild trend for increased apoptosis compared to non-irradiated glut-NPs treated tumors, this trend was not statistically significant (p=0.53). Conclusion: These data suggest that the glut-NPs show a considerable promise for radiosensitization. Note: This abstract was not presented at the meeting. Citation Format: Tamer Refaat, Derek West, kathleen R. Harris, Vamsi Parini, William Liu, Beau Wanzer, Lydia Finney, Andrew C. Larson, Jonathan Bautista, Vythialinga Sathiaseelan, Bharat Mittal, Tatjana Paunesku, Gayle Woloschak. Development of Fe3O4@TiO2 core-shell nanocomposites as radiosensitizers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4913. doi:10.1158/1538-7445.AM2014-4913

Nuclear location of tumor suppressor protein maspin inhibits proliferation of breast cancer cells without affecting proliferation of normal epithelial cells

BackgroundMaspin, which is classified as a tumor suppressor protein, is downregulated in many types of cancer. Several studies have suggested potential anti-proliferative activity of maspin as well as sensitizing activity of maspin for therapeutic cytotoxic agents in breast cancer tissue culture and animal models. All of the experimental data gathered so far have been based on studies with maspin localized cytoplasmically, while maspin in breast cancer tumor cells may be located in the cytoplasm, nucleus or both. In this study, the effect of maspin cytoplasmic and nuclear location and expression level on breast cancer proliferation and patient survival was studied.MethodsTissue sections from 166 patients with invasive ductal breast cancer were stained by immunohistochemistry for maspin and Ki-67 protein. The localization and expression level of maspin were correlated with estimated patient overall survival and percent of Ki-67-positive cells. In further studies, we created constructs for transient transfection of maspin into breast cancer cells with targeted cytoplasmic and nuclear location. We analyzed the effect of maspin location in normal epithelial cell line MCF10A and three breast cancer cell lines - MCF-7, MDA-MB-231 and SKBR-3 - by immunofluorescence and proliferation assay.ResultsWe observed a strong positive correlation between moderate and high nuclear maspin level and survival of patients. Moreover, a statistically significant negative relationship was observed between nuclear maspin and Ki-67 expression in patients with invasive ductal breast cancer. Spearman’s correlation analysis showed a negative correlation between level of maspin localized in nucleus and percentage of Ki-67 positive cells. No such differences were observed in cells with cytoplasmic maspin. We found a strong correlation between nuclear maspin and loss of Ki-67 protein in breast cancer cell lines, while there was no effect in normal epithelial cells from breast. The anti-proliferative effect of nuclear maspin on breast cancer cells was statistically significant in comparison to cytoplasmic maspin.ConclusionsOur results suggest that nuclear maspin localization may be a prognostic factor in breast cancer and may have a strong therapeutic potential in gene therapy. Moreover, these data provide a new insight into the role of cytoplasmic and nuclear fractions of maspin in breast cancer.

Prostate Epithelium-Specific Deletion of the Selenocysteine tRNA Gene Trsp Leads to Early Onset Intraepithelial Neoplasia

Although various lines of evidence suggest that oxidative stress plays a role in human prostate cancer initiation and progression, there is a paucity of direct evidence for its role in tumor initiation. To begin to address this issue, we developed a novel tumorigenesis model by reducing the expression of multiple selenoproteins (SPs) in mouse prostatic epithelium. This was accomplished via the prostate-specific deletion of Trsp, a gene that encodes a transfer RNA (Sec tRNA) required for the insertion of selenocysteine residues into SPs during their translation. By 6 weeks of age, Trsp-deficient mice exhibited widespread prostatic intraepithelial neoplasia lesions in all prostatic lobes, which then progressed to high-grade dysplasia and microinvasive carcinoma by 24 weeks. In contrast to other murine prostate cancer models, Trsp-deficient mice required neither the deletion of a tumor suppressor nor the transgenic introduction of an oncogene for prostatic intraepithelial neoplasia lesion development. In keeping with the antioxidant functions of several SPs, we found increases in lipid peroxidation markers in Trsp-deficient epithelial cells. This novel model of prostate neoplasia provides evidence for the existence of a selenoprotein or selenoproteins capable of acting as a tumor suppressor in the murine prostate.

Selected immunohistochemical features of conventional renal cell carcinomas coexpressing P53 and MDM2.

Renal clear cell carcinoma (CCRCC) is an aggressive tumor for which new prognostic factors are needed. It has been suggested that CCRCCs co-expressing P53 and MDM2 could represent a special subgroup; therefore the aim of this study was to explore their immunohistochemical features. The material studied consisted of 470 cases of CCRCC. Immunohistochemistry for MDM2, P53, Ki-67, VEGF-A, VEGF-C, VEGF-D, GLUT1, CA9, and CK 7 was performed on tissue microarrays and assessed semi-quantitatively. On average, 6.6% or 5.3% of cases were P53+/MDM2+, depending on the P53 antibody used. The mean percentage of Ki-67 positive cells was 0.6% and p53-positive MDM2-positive cases showed significantly higher expression of Ki-67. The other immunohistochemical parameters studied did not differ between p53-positive MDM2-positive cases and the rest of the subtypes studied. Expression of almost all immunohistochemical markers differed with respect to pT stage; only for CA9 was the difference not significant. Furthermore, almost all immunohistochemical markers studied differed with respect to differences in grade; only for GLUT1 was the difference not significant. Our results suggest that with the exception of Ki-67, there are no significant associations between analyzed markers and the double P53+/MDM2+ phenotype.

Parthenolide reduces cell proliferation and prostaglandin estradiol synthesis in human endometriotic stromal cells and inhibits development of endometriosis in the murine model

To evaluate the effects of parthenolide on human endometriotic cells and murine endometriotic lesions. Experimental study. University hospital and laboratory of animal science. Twenty women with ovarian endometrioma and 30 mice. Ectopic endometrial tissue from the endometrioma was collected. Human endometriotic stromal cells (ESCs) were pretreated with parthenolide and exposed to tumor necrosis factor (TNF)-α. Interleukin 8 (IL-8) and COX-2 gene expressions were evaluated by real-time reverse transcription-polymerase chain reaction. Interleukin-8 protein, prostaglandin E₂ (PGE₂) level, and intranuclear p65 protein concentration were determined by ELISA. Cell proliferation was assessed by 5-bromo-2'-deoxyuridine-ELISA. Phosphorylation of signaling pathways in ESCs was evaluated by Western blotting. Gene expression and proliferative activity in murine endometriosis-like lesions were assessed by real-time reverse transcription-polymerase chain reaction and Ki67 staining, respectively. With parthenolide pretreatment, TNF-α-induced IL-8 gene and protein expression in ESCs were diminished. Tumor necrosis factor α-induced COX-2 expression and PGE2 synthesis were also inhibited. Adding parthenolide repressed TNF-α-induced 5-bromo-2'-deoxyuridine incorporation and IκB phosphorylation in ESCs. As in vivo experiments, administering parthenolide reduced the number, surface area, and weight, the level of Vegf, Il-6, Mcp-1, and Lif gene expression, and the percentage of Ki67-positive cells in murine endometriosis-like lesions. Parthenolide repressed the development of endometriosis by suppressing the inflammatory peritoneal environment through the nuclear factor κB pathway.

Characterization of Different Osteosarcoma Phenotypes by PET Imaging in Preclinical Animal Models

The aim of this study was to characterize the different phenotypes of osteosarcoma by PET, comparing the uptake of 3 tracers ((18)F-FDG, (18)F-fluoromisonidazole [(18)F-FMISO], and (18)F-fluoride) in preclinical mouse models that reflect the heterogeneity of the human disease. Mouse LM8 osteosarcoma, human 143B, and Caprin-1 stably overexpressing SaOS-2 cells were injected intratibially in C3H and severe-combined immunodeficient mice. PET imaging with (18)F-FDG, (18)F-FMISO, and (18)F-fluoride was performed in these mouse models, and a ratio between the standardized uptake value of the primary tumor and a control area of bone was calculated and compared among the models. Histology and immunohistochemistry were performed to confirm the PET findings. The pattern of tracer uptake differed among the primary tumors of the 3 models in accordance with the histology and immunohistochemistry on primary tumor sections. The osteolytic tumors in the 143B model showed the highest uptake of (18)F-FDG, an indicator of glucose metabolism, which was significantly higher (P < 0.05) than in the SaOS-2/Caprin-1 model and correlated with the percentage of Ki67-positive cells in the primary tumors. Hypoxia, indicated by (18)F-FMISO accumulation, was higher in the SaOS-2/Caprin-1 and 143B cell line-derived tumors (P < 0.01). Finally (18)F-fluoride, a marker of bone remodeling, correlated with the osteoblastic phenotype. The SaOS-2/Caprin-1 cell-derived tumors showed a significantly higher uptake than the moderately osteoblastic LM8 (P < 0.05) and the osteolytic 143B (P < 0.01) cell line-derived tumors. Differential PET imaging with tracers indicating metabolic activity, hypoxia, or bone remodeling will be helpful for the characterization of different osteosarcoma phenotypes and subsequent evaluation of more specific treatment modalities targeting the processes that are predominant in each specific tumor type or subtype.

Effect of ultraviolet radiation on the exp ression of p53, matrix metalloproteinase-2 and-9 in actinic keratosis lesions and normal human skin

Objective To estimate the effect of different doses of ultraviolet (UV) radiation on the proliferation of and apoptosis in kertatinocytes,as well as on the expression of p53,matrix metalloproteinase-2 (MMP2) and-9 (MMP9) in actinic keratosis (AK) lesions and normal human skin.Methods Tissue specimens were obtained from the lesions of 20 patients with AK and sun-exposed normal skin of 20 healthy human subjects,and subjected to an air-exposed culture.Each of the specimens was divided into 4 areas to remain untreated (control area) or be irradiated with UV of 5,10 and 20 J/cm2 (irradiated areas) for 4 consecutive days.After another 24-hour culture,the tissue cultures were collected followed by the evaluation of apoptosis in and proliferation of keratinocytes by using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and Ki-67 staining,and determination of mRNA and protein expressions of p53,MMP2 and MMP9 by using real time PCR and immunohistochemistry respectively.Results A statistical increase was observed in the percentage of apoptotic cells in the normal skin irradiated with UV of 10 and 20 J/cm2 (46.8％ ± 2.1％ and 56.7％± 2.4％,both P ＜ 0.05) and in the AK lesions irradiated with UV of 20 J/cm2 (43.5％ ± 1.5％,P ＜ 0.05)compared with the corresponding unirradiated tissues.The normal skin showed a higher percentage of apoptotic cells than the lesional skin after irradiation with UV of 10 and 20 J/cm2 (both P ＜ 0.05).The percentage of Ki67-positive cells was significantly decreased in the normal skin after irradiation with UV of 20 J/cm2 (3.34％ ±0.76％,P ＜ 0.05),but experienced no statistical changes in the lesional skin after different doses of UV irradiation (all P ＞ 0.05).There was a statistical elevation in the expression of p53 mRNA (5 J/cm2:1.106 ± 0.025,10 J/cm2: 1.259 ± 0.045,20 J/cm2:1.425 ± 0.053,all P ＜ 0.05) and protein(10 J/cm2:0.1169 ± 0.0032,20 J/cm2:0.1454 ± 0.0047,both P＜ 0.05) in the normal skin,but a statistical reduction in the expression of p53 mRNA(10 J/cm2.0.611 ± 0.050,20 J/cm2:0.578 ± 0.070,both P ＜ 0.05) and protein (20 J/cm2:0.0404 ± 0.0027,P＜ 0.05) in the lesional skin after irradiation compared with the corresponding unirradiated skin tissues.Further more,a statistical increment was observed in MMP2 mRNA and protein expression in normal skin irradiated with UV of 10 J/cm2 (1.086 ± 0.013,0.0843 ± 0.0024,respectively,both P ＜ 0.05) and 20 J/cm2 (1.417 ± 0.036,0.1236 ±0.0042,respectively,both P ＜ 0.05) and in lesional skin irradiated with UV of 20 J/cm2 (1.296 ± 0.028,0.0744± 0.0032,respectively,both P ＜ 0.05),as well as in MMP9 mRNA and protein expression in normal skin irradiated with UV of 20 J/cm2 (1.395 ± 0.026,0.3065 ± 0.0162,respectively,both P ＜ 0.05) and in lesional skin irradiated with UV of 10 J/cm2 (1.298 ± 0.035,0.0992 ± 0.0053,respectively,both P ＜ 0.05) and 20 J/cm2(1.286 ± 0.032,0.1010 ± 0.0063,respectively,both P ＜ 0.05) compared with the corresponding unirradiated tissues.Conclusion Ultraviolet may accelerate the progression of AK by down-regulating p53 expression but up-regulating MMP2 and MMP9 expression. Key words: Actinic keratosis; Ultraviolet rays; Genes,p53; Matrix metalloproteinase 2; Matrix metalloproteinase 9

Cell Proliferation (Ki-67) As Prognostic Marker in Mantle Cell Lymphoma.

AbstractAbstract 2677 Introduction:The percentage of proliferating cells evaluated on diagnostic tumor samples has been shown to be of high prognostic relevance in Mantle Cell Lymphoma (MCL) patients. As MCL is relatively rare, evaluation of proliferation has so far mostly been based on smaller patient cohorts that were retrospectively collected and inhomogenously treated. In 2004, the European MCL Network initiated two large European randomized trials for younger (“MCL Younger” trial) and older (“MCL Elderly” trial) MCL patients, primary results of which have recently been reported (Kluin-Nelemans et al., NEJM 2012, Hermine et al., ASH 2010). We aimed to clarify the prognostic relevance of the proliferation marker Ki-67 using pooled data from these two trials. Patients and Methods:Patients with histologically confirmed and previously untreated MCL of stages II-IV up to 65 years of age were randomly assigned in “MCL Younger” to either 6 cycles R-CHOP followed by myeloablative radio-chemotherapy and autologous stem cell transplantation (ASCT), or 6 cycles alternating R-CHOP/R-DHAP followed by high-dose-Ara-C containing conditioning and ASCT. Patients aged 60 years or older and not eligible for high-dose therapy were randomly assigned in “MCL Elderly” to either 8 cycles of R-CHOP or 6 cycles of R-FC; responding patients were subsequently randomized to either interferon-alpha or rituximab maintenance until progression. Histological diagnosis was confirmed by central review within the European MCL Pathology Panel. The percentage of Ki-67 positive cells was counted on diagnostic lymphoma samples among 2 times 100 cells by the central pathology labs according to published consensus guidelines (Klapper et al., J Hematopathology 2009). The outcome measures were time to treatment failure (TTF) from treatment initiation to stable disease, progression, or death from any cause, and overall survival (OS) from trial registration to death from any cause. We investigated the prognostic value of proliferation as a quantitative marker with regards to TTF and OS in univariable Cox regression and evaluated the previously established cut-off values of 10% and 30% (Determann et al., Blood 2008) using Kaplan-Meier estimates and log rank tests. We also adjusted for clinical prognostic factors (MIPI, Hoster et al., Blood 2008). Results:Counted Ki-67 values were available in 51% (543) of 1057 randomized patients (material not available, 30%; Ki-67 evaluation not possible due to technical reasons, 16%). The origin of tumor tissue was lymph node in 81%, gastrointestinal tract in 12%, bone marrow in 4% and other in 3%. The median proliferation rate was 20% (range, 0–97%; interquartile range, 12–34%) and did not significantly differ between tissue origins. In univariable analysis, a 10% higher proliferation rate was associated with hazard ratios of 1.18 (95% confidence interval, 1.12 to 1.25, p<0.0001) for TTF and 1.23 (95% CI, 1.15 to 1.31, p<0.0001) for OS. Patients with Ki-67 ≥ 30% had median TTF and OS of 19 and 45 months compared to 64 months and not reached with Ki-67 < 30% (p<0.0001 each). Patients with Ki-67 < 30% and either ≥ 10% or < 10% had similar TTF and OS. The separation of a high risk group as defined by Ki-67 ≥ 30% was consistently seen within “MCL Younger” and “MCL Elderly” as well as within the 4 different induction treatment arms. The prognostic impact of proliferation was independent of the MIPI prognostic score (adjusted hazard ratio for TTF, 1.11, 95% CI, 1.05 – 1.17, p=0.0005; for OS, 1.14, 1.07–1.23, p=0.0001), which was also independently highly prognostic (p<0.0001). Almost identical results were seen when the analyses were restricted to lymph node samples. Conclusions:Cell proliferation was confirmed as important biological prognostic marker independent of clinical prognostic factors on a large cohort of MCL patients uniformly treated within clinical trials. Since the evaluation of Ki-67 has been standardized, guidelines (e.g. Dreyling et al., Ann Onc, in press) recommend applying this parameter in clinical routine. Further analyses will focus on the joint correlation of Ki-67, MIPI and minimal residual disease with outcome to potentially allow a more individualized therapeutic approach in MCL patients.On behalf of the European Mantle Cell Lymphoma Network. Disclosures:No relevant conflicts of interest to declare.

Abstract 3271: Targeting fibroblast growth factor receptor 4 decreases prostate cancer growth and metastasis

Abstract FGFR4 up-regulation occurs in advanced prostate cancers. Multiple FGFs have been found upregulated in prostate cancers, including FGF1, FGF2, FGF6, FGF8 and FGF17, all of which can activate FGFR4. Furthermore, a SNP changing the sense codon 388 from glycine to arginine in the transmembrane domain of the human FGFR4 gene has been linked to prostate cancer initiation, progression and poor prognosis. In this study, we determined whether downregulation of FGFR4 expression using small hairpin RNA (shRNA) against FGFR4 inhibits prostate cancer progression in vitro and in vivo. We used two well-characterized prostate adenocarcinoma cell lines, PC3 and LNCaP to derive prostate cancer cells stably expressing a shRNA targeting FGFR4 (PC3-sh-FGFR4 and LNCaP-sh-FGFR4) by using lentivirus vector infection. Real-time quantitative RT-PCR shows that FGFR4 mRNA expression was decreased by 87% (PC3-sh-FGFR4) and 78% (LNCaP-sh-FGFR4) compared to scramble control shRNA cells PC3-shV and LNCaP-shV, respectively. In vitro analysis showed that the targeted knockdown of FGFR4 gene in PC3 and LNCaP cells with sh-FGFR4 resulted in significantly inhibited cell proliferation and invasion. The reduction of Akt phosphorylation on both Ser473 and Thr-308 sites detected by western blot suggests that FGFR4 signaling is implicated in Akt activation. The upregulated expression of Caspase 8 and activated Caspase 3 in FGFR4 knockdown cells measured with immunoblot indicates that FGFR4 signaling inhibits prostate cancer cell apoptosis. The prostate cancer orthotopic xenograft models established with PC3-sh-FGFR4 and scramble shRNA control PC3 cells reveal that FGFR-4 knockdown significantly inhibited tumorigenicity in this model (P=0.01). In mice with pathologically confirmed primary tumors, primary tumor weight was decreased 63% in PC3-shFRGR-4 tumors (p&amp;lt;0.05) and the proportion of mice with lymph node metastasis was decreased from 93.3% to 50% (p&amp;lt;0.01). The Ki67 immunohistochemistry showed a significant decrease of Ki67 positive cell percentage in PC3-sh-FGFR4 xenograft tumors (19.85%) compared to control group (35.93%, p&amp;lt;0.001) indicating that the reduced primary tumor size was associated with the decreased cell proliferation. The orthotopic xenograft tumors produced with LNCaP-shFGFR4 showed 67.5% decreased weight compared to the tumors of control LNCaP-shV cells (p=0.0157). Our data indicated that FGFR4 can be a promising target in prostate cancer therapy. 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 3271. doi:1538-7445.AM2012-3271

Generation and characterization of orthotopic murine models for endometrial cancer

We describe the generation of two orthotopic murine models for endometrial cancer (EC).The first model is generated from endometrial Hec-1A cancer cells transfected with luciferase and injected directly into the uterus of female mice. This model allows a follow-up with bioluminescence imaging (BLI) along the experiment and generates abdominal dissemination and lymphatic and hematogenous metastases in high percentages, also detectables with BLI. The dissemination pattern of this model imitates the advanced stages of EC in patients, and its molecular profile corresponds to aggressive type 2 EC (p53 positive, hormone receptors negative, high percentage of Ki67 positive cells). The second model is derived from endometrioid human tissue collected from surgical pieces. By injecting this tissue inside the uterine cavity of a mouse we obtain orthotopic growth with pelvic dissemination and lymph node metastasis. The molecular pattern observed in human type 1 endometrioid EC (p53 negative, low Ki67 index, presence of hormone receptors) is conserved after the murine growth in orthotopic tumor and metastases. This model supposes a singular pre-clinical tool to study therapeutic agents, though it mimics clinical and molecular behavior of endometrioid EC, which is the most common histology in the patient.

NASA-Approved Rotary Bioreactor Enhances Proliferation of Human Epidermal Stem Cells and Supports Formation of 3D Epidermis-Like Structure

The skin is susceptible to different injuries and diseases. One major obstacle in skin tissue engineering is how to develop functional three-dimensional (3D) substitute for damaged skin. Previous studies have proved a 3D dynamic simulated microgravity (SMG) culture system as a “stimulatory” environment for the proliferation and differentiation of stem cells. Here, we employed the NASA-approved rotary bioreactor to investigate the proliferation and differentiation of human epidermal stem cells (hEpSCs). hEpSCs were isolated from children foreskins and enriched by collecting epidermal stem cell colonies. Cytodex-3 micro-carriers and hEpSCs were co-cultured in the rotary bioreactor and 6-well dish for 15 days. The result showed that hEpSCs cultured in rotary bioreactor exhibited enhanced proliferation and viability surpassing those cultured in static conditions. Additionally, immunostaining analysis confirmed higher percentage of ki67 positive cells in rotary bioreactor compared with the static culture. In contrast, comparing with static culture, cells in the rotary bioreactor displayed a low expression of involucrin at day 10. Histological analysis revealed that cells cultured in rotary bioreactor aggregated on the micro-carriers and formed multilayer 3D epidermis structures. In conclusion, our research suggests that NASA-approved rotary bioreactor can support the proliferation of hEpSCs and provide a strategy to form multilayer epidermis structure.

Compromised Tissue Renewal in the Ageing Human Colonic Epithelium

BACKGROUND: The intestinal epithelium is the most rapidly renewing tissue in the body. It is widely believed that this attribute minimises the accumulation of age-related molecular damage. Increasing evidence suggests that this protective mechanism is undermined by agerelated molecular changes that accumulate in long-lived stem/progenitor cells. Age-related molecular damage in the intestinal epithelium of flies and rodents manifests as a hyperproliferative state that exhibits a greater degree of apoptosis, clonogen/stem cell number and reduced regenerative potential following damage. Furthermore, the human colonic epithelium is subject to age-related accumulation of mutations in mitochondrial DNA. Given that ageing is a major risk factor for cancer, it is surprising that the status of tissue renewal in the ageing human colonic epithelium has received little attention. AIM: To investigate age-related changes in the renewal of the human colonic epithelium.METHODS: Tissue biopsies obtained at sigmoidoscopy (Ethical approval) from young ( 70 years, N= 12) individuals with no apparent pathology were immediately fixed or processed for crypt isolation. Isolated crypts were observed in 3D culture by digital time-lapse microscopy. Native crypts obtained by microdissection of fixed biopsy tissue were subjected to morphometric analysis and immunofluorescence for detection of Ki67 (proliferation marker), beta catenin, c-Myc and axin2 (all markers for Wnt signals), and OLFM4 (an intestinal stem cell marker). RESULTS: Crypts from young subjects (n=116 crypts) were significantly longer (p< 0.05) than those derived from the older cohort (117 crypts), 375± 10 um versus 330 ± 10 um, respectively. The percentage of Ki67 positive cells in all regions along the cryptaxis was significantly greater (p<0.05) in tissue from older subjects : e.g. crypt base 35% (young) versus 52% (old); mid region 42%% (young) versus 62% (old) and upper region 10% (young) versus 17% (old). The number of cell divisions observed under timelapse microscopy was reduced for crypts derived from older subjects suggesting that the increased Ki67 labelling index in the older group reflected a slower cell cycle time. Although the nature of the proliferating cell type is not known at this stage, an increase in OLFM4+ (stem) cell number was observed along the axis of crypts derived from older subjects. The above traits were associated with a trend towards an extended profile of immunolabelling intensity for nuclear beta catenin and Wnt target gene expression along the crypt-axis of tissue derived from older subjects. CONCLUSIONS: Age-related changes in crypt length, cell proliferation and markers for intestinal stem cells and Wnt signalling components along the crypt-axis suggest that tissue renewal is compromised in the ageing human colonic epithelium.

Abstract 1106: Fibroblast growth factor FGF19 signaling promotes prostate cancer progression

Abstract Aberrant fibroblast growth factor (FGF) signaling can promote tumor development by directly driving cancer cell proliferation and survival, and by supporting tumor angiogenesis. Multiple FGFs have been found upregulated in prostate cancers, including FGF1, FGF2, FGF6, FGF8 and FGF17, all of which can activate FGF receptor 4 (FGFR4). FGFR4 is overexpressed in prostate cancer (PCa) and positively associated with aggressive PCa. FGF19 is a distinct member of FGF family in that it predominantly binds to FGFR4 with high affinity. In this present study we aimed to study the role of FGF19 in human PCa progression, and to determine whether the targeted suppression of FGF19/FGFR4 signaling has potential therapeutic benefits in PCa. Our results demonstrated that FGF19 is upregulated in human PCa compared to normal prostate tissues. FGF19 is expressed in an autocrine manner by all tested PCa cell lines. Exogenous FGF19 stimulates PCa cell proliferation, anchorage-independent growth, adhesion and invasion in vitro. The mRNAs of FGF19 co-receptors αKlotho and ßKlotho are expressed in 97.5% and 27.5% of PCa clinical samples, respectively; but αKlotho is expressed in only 57% normal prostate tissues, ßKlotho is barely detected in normal prostate tissues. Suppression of FGF19 by short hairpin RNA (shRNA) targeting FGF19 gene inhibits PCa cell proliferation, adhesion and invasiveness in vitro. Immunoprecipitation and western blot assays showed that FGF19 stimulates phosphorylation of FGFR4, FRS2α, Erk1/2 and p-38 MAPK, as well as MEK1/2 in both PC3 and DU145. Our data also revealed that FGF19 induced the serine/threonine protein kinase Akt phosphorylation in PCa cells. Lentiviral shRNA delivery was used for stable FGFR4 gene silencing in PC3 and LNCaP cells. The targeted knockdown of FGFR4 in PC3 and LNCaP cells resulted in significantly inhibited cell proliferation, invasion and remarkably reduced Akt phosphorylation. The upregulated expression of activated Caspase 8 and activated Caspase 3 in FGFR4 knockdown cells indicates that FGFR4 signaling inhibits PCa cell apoptosis. To determine if FGFR4 suppression impacts prostate tumor growth and metastasis in vivo we generated a PCa orthotopic xenograft model in which PC3-sh-FGFR4 or PC3-shV cells are injected directly into the prostates of nude mice in each group. The results revealed that the FGFR4 suppression in PCa cells significantly inhibited tumorigenicity in this model (P=0.01). The primary tumor weight was decreased by 63% in sh-FRGR4 tumors (p&amp;lt;0.05) and the proportion of mice with lymph node metastasis was decreased from 93.3% to 50% (p&amp;lt;0.01). The Ki67 immunohistochemistry showed a significant decrease of Ki67 positive cell percentage in PC3-sh-FGFR4 xenograft tumors (19.85%) compared to control group (35.93%, p&amp;lt;0.001). Our data indicated that FGF19/FGFR4 signaling may be a promising target in PCa therapy. 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 1106. doi:10.1158/1538-7445.AM2011-1106

Proliferation and apoptosis in normal bitch mammary tissues in relation to progesterone level

The objective of this study was to investigate the proliferating activity and apoptosis in canine normal mammary tissues in different cycle stages. Mammary tissues were collected from five beagle bitches at six different estrous stages: anestrus, proestrus, estrus, early diestrus, mid diestrus, and late diestrus. The expression of Ki-67 protein and apoptotic bodies was evaluated by avidin–biotin–peroxidase complex (ABC) method and TUNEL assay, respectively. Percentage of Ki-67 positive cells and apoptotic bodies was calculated. The significant highest percentage of Ki-67 positive cells and apoptotic bodies was observed at mid diestrus. Positive correlation was found mainly in the alveolar epithelium between progesterone levels and percentage of Ki-67 positive cells, as well as between progesterone levels and apoptosis. In conclusion, the results from the present study suggested that progesterone may influence on proliferation and apoptosis in canine mammary tissue.

Expression of fragile histidine triad (FHIT) protein and Ki-67 in transformed epithelial cells induced by Yunnan tin mine dust

To study the expression and significance of fragile histidine triad (FHIT) and Ki-67 in transformed epithelial cells induced by Yunnan tin mine dust. Every second generation of immortalized human bronchial epithelial cells (BEAS-2B) and human embryo lung fibroblasts (WI-38) were exposed to 100 µg/ml Yunnan tin mine dust for 72 h, until the ninth generation. The cells were subsequently co-cultured from the 11th generation. Experimental setup: B group, B (W) group, B (W 100) group, B100 group, B100 (W) group, B100 (W100) group. The expressions of FHIT and Ki-67 in epithelial cells were determined by the method of immunocytochemistry at the 16th, 26th and 36th generation. The percentage of Ki-67 positive cells was calculated as proliferation index. The expression of FHIT was observed in BEAS-2B cells. The expression levels of FHIT among B group, B (W) group and B (W 100) group had not instinctive difference. At the 16th generation, the expression of FHIT in the B100 group was decreased compared with that in the B group and the expression of FHIT between B100 (W) group and B100 (W100) group was lower than that in the B100 group. At the 26th generation, the expression of FHIT was decreased compared with that at the 16th generation in the B100, B100 (W) and B100 (W100) groups. However, At the 36th generation, positive expression were observed again in the B100, B100 (W) and B100 (W100) groups and the expression levels were in incremental order. At the 16th, 26th and 36th generation, the proliferation indexes of B group, B (W) group and B (W 100) group were all < 3%. The proliferation indexes of B100, B100 (W) and B100 (W100) were increased step by step with the generation elongation. FHIT could be a target at which Yunnan tin mine dust induces transformation of BEAS-2B cells. The proliferation activation of BEAS-2B cells can be improved by Yunnan tin mine dust.

Abstract 5367: Medroxyprogesterone acetate impedes 5α-dihydrotesterone induced androgen receptor signaling in normal and malignant human breast epithelial cells

Abstract Combined hormone replacement therapy (cHRT) comprised of equine estrogens and the synthetic progestin medroxyprogesterone acetate (MPA) increases breast cancer risk in post-menopausal women [1], via as yet undetermined mechanisms. In addition to the progesterone receptor (PR), MPA binds with high affinity to the androgen receptor (AR), and therefore has the potential to elicit actions distinct to those of natural progesterone, which does not bind AR. We have proposed that MPA increases breast cancer risk by binding to the AR and interfering with the ability of endogenous AR ligands to inhibit proliferation of normal breast epithelial cells [2, 3]. To further investigate this proposal, we examined the effect of MPA on 5α-dihydrotestosterone (DHT)-induced AR signaling in normal human breast tissue and in a breast cancer cell line (ZR75-1) that expresses estrogen receptor alpha (ERα), PR, and AR. Normal human breast tissues was obtained from 12 post-menopausal women following surgery for benign or malignant disease. Normal histology was confirmed by a pathologist. Breast tissues (3mm3) were cultured in triplicate for 48 hours on gelatine sponges soaked in media containing 10% steroid deplete fetal calf serum (FCS) and DHT (1nM), MPA (1nM), or the AR antagonist, bicalutamide (Bic; 1uM), alone and in combination. Following culture, expression of AR, Ki67 (a marker of cell proliferation) and bcl-2 (a pro-survival factor) was assessed by immunohistochemistry. ZR75-1 cells were cultured for 24 and 48 hours in 10% steroid deplete FCS supplemented with DHT, MPA or the combination, followed by immunoblot analysis to detect AR and an androgen-regulated protein, FKBP5. Statistical analysis was preformed by Wilcoxon signed-rank test unless otherwise stated. Normal human breast tissues possessed significantly higher levels of immunoreactive AR than ERα or PR (p&amp;lt;0.0001; t-test for unequal variances). Treatment with DHT significantly increased AR expression (p=0.02) and decreased the percentage of Ki67 positive cells (p=0.03), but had no effect on expression of bcl-2. The effects of DHT on AR and Ki67 were reversed by co-incubation with Bic (p&amp;lt;0.05) or MPA (p&amp;lt;0.05). In ZR75 cells, DHT increased AR protein levels at 24 and 48 hours and increased FKBP5 protein levels at 48 hours. Co-incubation with MPA reversed the effects of DHT on AR and FKBP5 proteins. Collectively, these data support our hypothesis that MPA interferes with the action of endogenous AR ligands in normal and malignant human breast epithelial cells. This interference of androgen action corresponds to an increased proliferation index in normal human breast epithelial cells, which feasibly contributes to an increased risk of developing breast cancer. 1. Chlebowski., R.T., et al., Jama, 2003. 289: 3243-53 2. Birrell, S.N., et al., Faseb J, 2007. 21; 2285-93 3. Peters, A.A., et al., Cancer Res, 2009. 69: 6131-40 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5367.

Abstract 2373: Heterogeneity of permeability in a preclinical model of brain metastases of breast cancer does not correlate with vascular density, HIF-1α expression, or tumor survival

Abstract Introduction: High vascular density of brain metastases of breast cancer has been correlated with both poor patient prognosis and with potential chemotherapeutic success. Inversely a low vascular density implies there would be decreased distribution of anti-cancer chemotherapeutics, and over time reduced efficacy of tumor kill. Given that there is heterogeneity in response to chemotherapeutics in human brain metastases and metastases are different than primary tumors; we used a novel brain metastases of breast cancer model to characterize vascular density, and corresponding changes in tumor permeability, hypoxia and survival. Methods: Brain seeking metastatic tumor lines MDA-MB-231-Br and 4T1-Br5 were injected via the left cardiac ventricle in separate experiments and brain metastases developed for ∼28 days and ∼15 days respectively. After development, both 14C-AIB and indocyanine green (ICG) were injected and allowed to circulate for 10 and 1 min respectively. Near infrared imaging was used to identify ICG filled vessels to quantify vascular density of lesions compared to normal brain. To determine permeability, concentrations of 14C-AIB in blood and brain tissue were calculated using autoradiography. Immunofluorescent analysis was performed using anti-HIF-1α and anti Ki67 antibodies and co-stained with DAPI. Results: A significant amount of metastatic lesions were observed to have developed in each model. Lesions varied in size from 0.028 to 2.3mm2. Vascular density was significantly decreased (MDA 176 ± 96 and 4T1 201 ± 76) compared to control brain (429 ± 25) and to an RG2 implanted glioma used as a positive control (736 ± 168). Based on accumulation of 14C-AIB, metastases (range: 22 nCi/g to 1682 nCi/g) were considered to be either permeable (3 fold or greater uptake compared to adjacent control tissue) or non-permeable. There was no strong correlation between lesion size and permeability (r2=0.23) or vascular density (r2 = 0.06). HIF-1α was expressed in both permeable and non-permeable lesions. Lastly, no significant difference was seen in the percentage of Ki67 positive cells in the MDA (permeable 41 ± 2.9, n=7; and non-permeable 40.8 ± 2.7, n=14) and 4T1 model (permeable 52.0 ± 4.6, n=6; and non-permeable 45.0 ± 2.8, n=6). Discussion: This data provides a correlation of permeability to vascular density, HIF-1α and Ki67 in a brain metastases model. While, both brain model systems exhibited a high degree of heterogeneous permeability, in neither model did permeability strongly correlate with vascular density, lesion size, HIF-1α expression and positivity for Ki67. Based upon the data, this experimental model may reflect the heterogeneity seen in human brain metastases of breast cancer and suggests there are other biological factors besides vascular density which correlate with drug distribution and or tumor/survival growth. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2373.

The TEAM Trial Pathology Study Identifies Potential Prognostic and Predictive Biomarker Models for Postmenopausal Patients Treated with Endocrine Therapy.

Abstract Background: The Tamoxifen and Exemestane Adjuvant Multinational (TEAM) trial included prospectively planned biomarker studies to identify prognostic and predictive biomarkers for patients receiving endocrine therapy. Quantitative IHC data for ER/PgR (Can Res 69:83S, SABCS2008), HER2, HER3 and Ki67 was available for the current analysis relative to outcome of estrogen receptor–positive (ER+) early postmenopausal breast cancer (BC) patients treated with exemestane versus tamoxifen.Patients &amp; Methods: Pathology blocks from 4598 TEAM patients were collected and tissue microarrays constructed. Quantitative analysis of hormone receptors (HER2/3) by conventional IHC, and image analysis derived continuous scores for Ki67/ER/PgR were analyzed relative to disease-free survival and treatment on an intent to treat basis using survival data for the first 2.75 years of the TEAM trial. Data on HER2FISH and EGF Receptor IHC will be presented.Results: Of 4595 eligible cases samples received, 16 were excluded, 271 had incomplete biomarker data, leaving 4308 patients for the final biomarker analysis. 1275 (30%) cases were HER2/3 positive.A significant treatment by marker effect was observed for exemestane versus tamoxifen with HER2/3 negative cases deriving benefit from aromatase inhibitor treatment (HER2/3-ve HR=0.69 95%CI, 0.53-0.88; HER2/3 pos HR, 1.13; 95%CI, 0.82–1.55; p=0.016 for interaction in multivariate analysis). By conventional and STEPP analysis no predictive effect of Ki67 was observed. In multivariate regression analysis increased HER2 expression (P=0.0001) decreased PgR expression (P&amp;lt;0.0001) and increased percentage of Ki67 positive cells (P=0.004) as continuous IHC variables were independently prognostic as were size (P=0.0001), nodal status (P&amp;lt;0.0001), grade (P=0.03) and age (P&amp;lt;0.0001).Conclusion: Multiple biological parameters (HER2/PgR/Ki67) are independently prognostic in ER+ve early postmenopausal BC. Modelling will be explored to derive prognostic and potentially predictive biomarker signatures for application in BC. Preferential exemestane versus tamoxifen treatment benefit was seen in HER2/3 negative cases, whilst HER2/3 positive cases had a poor prognosis in this population receiving hormonal therapy (suggesting resistance to endocrine therapy), and no evidence of benefit from AIs versus tamoxifen. Type I receptor tyrosine kinases may identify breast cancers with relative resistance to all forms of endocrine therapy. Whilst Ki67 alone was not predictive of benefit from Ais, Ki67, HER2 and PgR were independent prognostic variables and modelling of predictive/prognostic effects may further inform treatment selection in early postmenopausal breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 75.