Mean Percentage Of Cells Research Articles

Invadopodia formation assay: a promising experiment for targeting breast cancer invasion and metastasis

Abstract Introduction/Objective Invadopodia has been found to invade the extracellular matrix (ECM) in multiple cancers, including breast, lung, brain, and prostate, which is highly associated with cancer metastasis. Invadopodia have been shown to form in various cancer cells, such as the MDA-MB-231 breast cancer cell line. Objectives In-vitro testing to validate the anti-cancer invasion drugs using an invadopodia formation assay. Methods/Case Report MDA-MB-231 triple-negative breast cancer cell line was used in this study due to its highly invasive characteristics obtained from American Type Culture Collection. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM), a high-glucose medium containing FBS 10% and 1% penicillin/streptomycin. Cells were maintained in a humidified atmosphere of 95% O2 and 5% CO2 incubator at 37 °C for normal conditions. Cells with low passage numbers were used in order to get accurate results. Then, the gelatin-coated coverslips were prepared for invadopodium assay, washed three times with ddH2O, and transferred to slides containing a drop of antifade, a mounting solution. For invadopodia quantification, invadopodia images on the F-actin, gelatin, and nucleus were taken using the same fluorescence microscope settings (Confocal Laser Leica fluorescent microscope, German). Cells were scored for the presence of underlying invadopodia. The mean percentage of cells with invadopodia formation was calculated for drug-validated tests. Results (if a Case Study enter NA) The study revealed how MDA-MB-231 cells penetrate the gelatin that mimics the ECM-like basement membrane in the human body; this is thought to be a key point during intravasation and extravasation steps in metastasis. The study showed actin-rich filament as red dots in the center of the MDA-MB-231 cells, representing invadopodia in a 3D image under normal conditions. In contrast, in a 2D image, invadopodia are represented as black dots on gelatin in a merged image. Conclusion Breast cancer is one of the leading causes of cancer-related death in the advanced stage. The study helps to promote using invadopodia assay to validate the effectiveness of the potential anti-cancer drugs and predict cancer metastasis in clinical studies. However, invadopodia formation assay can be especially useful for patients with early-stage cancers; it can help to predict which patients are more likely to benefit from aggressive treatment or further monitoring rather than immediate surgical intervention.

Detection of genetic instability and proliferative activity in urinary bladder intraepithelial flat lesions using UroVysion fluorescence in-situ hybridization and flow cytometry

Background Chromosomal abnormalities play a key role in neoplastic potential for flat urothelial lesions, irrespective of whether cytologic atypia is present or not. Having information about lesions with malignant potential in the urinary bladder can help clinicians in taking logical actions concerning management after urinary bladder-conserving resections.Aim The aim of the study was to differentiate various flat urothelial lesions by detecting its genetic abnormalities and proliferative activity using UroVysion fluorescence in-situ hybridization and flow cytometry.Patients and methods Ninety-eight paraffin-embedded tissue sections from 35 patients with infiltrative bladder cancer and flat urothelial lesions [reactive atypia, hyperplasia, dysplasia, carcinoma in situ (CIS), squamous metaplasia, glandular metaplasia, histologically normal urothelium in patients with tumor and control] were assessed by flow cytometry and fluorescence in-situ hybridization using UroVysion targeted against the band 9p21 locus and pericentromeric areas of chromosomes 3, 7, and 17.Results Gain of at least one of the chromosomes was observed in 60% of samples being more than 90% in CIS, dysplasia, and infiltrating bladder tumors. The mean percentage of cells with polysomy in reactive atypia, flat urothelial hyperplasia, glandular metaplasia, keratinizing squamous metaplasia, and histologically normal urothelium in patients with tumors ranged from 12 to 20%, but in CIS and dysplasia it was 67 and 44%, respectively. Deletion of the P16 locus was detected in 49% of lesions. Flow cytometry displayed aneuploidy pattern in 60% of lesions with higher percentage in CIS, dysplasia, and infiltrating bladder tumors. High S-phase fraction (SPF) was observed in 44% of samples. High S phase showed significant association with polysomy and DNA ploidy (P=0.02 and 0.01, respectively). In addition, there was association between polysomy expression with del 9p21 (P=0.03) and between del 9p21 with DNA ploidy and SPF (P=0.003 and 0.02).Conclusions Our analysis has shown that combined evaluation of flat urothelial lesions using UroVysion, DNA ploidy, and SPF help in the recognition of these lesions, especially those with higher genetic abnormalities and having higher S phase because follow up of these lesions showed a higher tendency for malignant transformation. Furthermore, estimation of continuing chromosomal instability such as polysomy, DNA aneuploidy, and high SPF might deliver further interpretation of urinary bladder cancer risk assessment away from the position and histopathology of precancerous lesions.

CD25+CD127+Foxp3- Cells Represent a Major Subpopulation of CD8+ T Cells in the Eye Chambers of Normal Mice.

The aim of this study has been to determine whether eye chambers constitute part of the normal migratory pathway of naive CD4+ and CD8+ T cells in mouse and if natural CD4+CD25+Foxp3+ and CD8+CD25+Foxp3+ regulatory T cells are present within these eye compartments. To this aim, the cells obtained from aqueous humor (AH) of normal mice were phenotyped in terms of the expression CD4, CD8, CD25, CD127 and transcription factor Foxp3. The mean percentage of CD8+ T cells in the total AH lymphocyte population was as high as 28.69%; the mean percentage of CD8high and CD8low cells in this population was 34.09% and 65.91%, respectively. The presence of cells with the regulatory phenotype, i.e. CD25+Foxp3+ cells, constituted only 0.32% of CD8+ T cell subset. Regarding the expression of CD25, AH CD8+ T cells were an exceptional population in that nearly 85% of these cells expressed this molecule without concomitant Foxp3 expression. Despite having this phenotype, they should not be viewed as activated cells because most of them co-expressed CD127, which indicates that they are naive lymphocytes. With regard to the markers applied in the present research, CD8+CD25+CD127+Foxp3- T cells represent the most numerous subset of AH CD8+ cells. The results suggest that eye chambers in mice are an element in the normal migratory pathway of naive CD8+ T cells. The study presented herein demonstrated only trace presence of CD4+ cells in the eye chambers, as the mean percentage of these cells was just 0.56. Such selective and specific homing of CD8+ and CD4+ cells to the eye chambers is most clearly engaged in the induction and maintenance of ocular immune privilege.

Characterization of CD44+ALDH1+Ki-67- Cells in Non-malignant and Neoplastic Lesions of the Breast.

Cancer stem cells are tumor cells that present self-renewal, clonal tumor initiation capacity and clonal long-term repopulation potential. We have previously demonstrated that the co-expression of the breast cancer stem cell (BCSC) markers hyaluronan receptor (CD44) and aldehyde dehydrogenase-1 (ALDH1) in ductal carcinomas in situ could be determinant for disease progression. Combining these established BCSC markers with Ki-67 to evaluate quiescence we sought to identify, evaluate the distribution and estimate the mean percentages of CD44(+)ALDH1(+)Ki-67(-) breast cells. Triple-immunohistochemistry for CD44, ALDH1 and Ki-67 was applied in a series of 16 normal, 54 non-malignant and 155 malignant breast tissues. Clinical relevance was inferred by associations with markers of breast cancer behavior, progression and survival. The mean percentages of cells with this phenotype increased significantly from non-malignant lesions to high-grade ductal carcinomas in situ, decreasing in invasive ductal carcinomas, as also evidenced by an inverse correlation with histological grade and tumor size. The mean percentage of CD44(+)ALDH1(+)Ki-67(-) cells was also significantly higher in women who developed distant metastasis and died due to breast cancer, and a significant association with human epidermal growth factor type 2 (HER2) negativity was observed. Our novel findings indicate that CD44(+)ALDH1(+)Ki-67(-) tumor cells may favor distant metastasis and can predict overall survival in patients with ductal carcinomas of the breast. More importantly, quiescence may have a crucial role for tumor progression, treatment resistance and metastatic ability of BCSCs.

Abstract 264: The cell cycle checkpoint kinase 1/2 inhibitor, LY2606368 with PARP inhibition results in synergistic cytotoxicity in high-grade serous ovarian cancer (HGSOC) at lower than physiologically administered concentrations

Abstract Background: Chk1/2 are major cell cycle regulators in p53-deficient tumors, such as HGSOC. Chk1 plays a critical role in DNA repair, facilitating the BRCA2-RAD51 interaction by phosphorylating the BRCA2 C-terminal domain and Thr309-RAD51. Clinical activity with the Chk1/2 inhibitor, LY2606368 mesylate monohydrate (LY), has been observed in solid tumors. We hypothesize that Chk1 inhibition would sensitize HGSOC to PARP inhibition (PARPi) by preventing nuclear RAD51 foci formation, thus impairing DNA repair. We investigated potential synergy of the combination of LY and the PARPi, olaparib (O), in HGSOC cell lines, testing lower concentrations than clinically attained. Materials and Methods: We examined cytotoxicity, DNA damage, and nuclear RAD51 foci formation by LY and/or O using XTT assay, comet assay and immunofluorescence (IF), in 3 HGSOC cell lines: two BRCA1/2 wild type (CAOV3, OV90), and one with BRCA2 mutation (PEO1). We examined a dose range based on clinically achievable concentrations of LY (0.53μM–1.34μM) and O (7.8μM–11.0μM) and calculated IC50 concentrations for cytotoxicity. The combination index (CI) was calculated to evaluate synergism. DNA damage and nuclear RAD51 foci formation were examined. DMSO was used as vehicle control. All experiments were performed in at least three replicates in all cell lines. Results are presented as mean ± SEM. Results: LY alone yielded cytotoxicity in CAOV3, OV90, and PEO1 with IC50 6.34nM, 35.2nM, 12.6nM, respectively. O 5μM monotherapy resulted in 47%, 13%, and 69% cytotoxicity in CAOV3, OV90, and PEO1. LY/O combination showed 51%, 58% and 82% cell injury in CAOV3, OV90 and PEO1. CI values indicated cytotoxicity synergism with the combination. LY/O (5nM/5μM) treatment showed greater DNA damage than O alone in OV90 and PEO1 (p<0.05), and a similar trend was observed in CAOV3 (20.2% ±3.10 vs. 6.71% ±1.08, p = 0.05). The mean percentage of cells with ≥ 5 nuclear RAD51 foci decreased with LY/O (5nM/5μM) combination therapy compared to O alone in CAOV3 (9.03% ±1.85 vs. 36.3% ±7.45, p<0.01) and OV90 (3.66% ±1.84 vs. 22.9% ±3.08, p<0.01), but not in PEO1 (1.26% ± 1.26 vs. 3.73% ±3.73). Ongoing work includes cell cycle analysis and immunoblotting to examine biochemical changes in DNA repair and cell cycle checkpoint pathways. Conclusions: Our preliminary results suggest synergistic activity of the combination of LY and PARPi in HGSOC cell lines using concentrations that are lower than clinically attainable in patients. Citation Format: Yeong-ran Ahn, Takuhei Yokoyama, Minshu Yu, Nicolas Gordon, Elise C. Kohn, Jung-min Lee. The cell cycle checkpoint kinase 1/2 inhibitor, LY2606368 with PARP inhibition results in synergistic cytotoxicity in high-grade serous ovarian cancer (HGSOC) at lower than physiologically administered concentrations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 264.

Expression of VEGF-A in Intraepithelial and Invasive Cervical Neoplasia

Angiogenesis seems to be an early event that is associated with disease progression in cervical intraepithelial neoplasia (CIN). The aim of this study was to describe and analyze VEGF-A expression in CIN 3 and invasive cervical carcinoma, in tumor cells and stromal cells. This study comprised three groups: Group 1: 53 cases with CIN 3; Group 2: 24 cases with both CIN 3 and invasive carcinoma components; Group 3: 36 cases with invasive carcinoma. Protein expression was investigated in tumor and stromal cells by immunohistochemistry. Statistical analysis was carried out, considering the mean percentage of immunopositive cells. VEGF-A expression was higher in stromal cells in cases of invasive carcinoma (Group 3) than in CIN 3 cases (Group 1) and this difference was statistically significant. VEGF-A expression showed a borderline association between stromal cells in invasive carcinoma (Group 3) and invasive carcinoma associated with CIN 3 (Group 2). The mean percentage of cells expressing VEGF-A was significantly higher in tumor cells in CIN 3 than in stromal cells in CIN 3 (Group 1). The progression of CIN 3 to invasive carcinoma seems be determined by complex interactions between tumor and stromal cells. These findings reinforce the evidence of the role of stromal cells in tumor invasiveness.

'Soya milk Tris-based phytoextender reduces apoptosis in cryopreserved buffalo (Bubalus bubalis) spermatozoa'.

The objective of this study was to investigate the effect of newly developed soya milk Tris (SMT)-based phytoextender as an alternative to egg yolk Tris (EYT) extender used for cryopreservation of buffalo (Bubalus bubalis) spermatozoa on apoptosis. Fresh buffalo semen (control without dilution) was cryopreserved in conventional EYT (20% egg yolk v/v in Tris) and SMT (25% soya milk v/v in Tris) extender and used for the assessment of expression of apoptotic proteins. Proteins extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random were separated using SDS-PAGE followed by immunoblotting against caspase-8, caspase-9, caspase-3, poly(ADP-ribose)polymerase (PARP), cytochrome c and apoptosis inducing factor (AIF). In addition, fluorescence microscopy was used for the detection of mitochondrial membrane potential (JC-1 assay) and apoptotic cells (annexin V-FITC/PI assay). The results obtained clearly indicate the significant (p<0.05) reduction in the expression of caspase-3 (27kDa), caspase-8 (53kDa), caspase-9 (50kDa) precursor and cytochrome c (17kDa) in semen cryopreserved in SMT extender in comparison with EYT extender. A non-significant (p>0.05) reduction in expression of PARP-DNA-binding subunit (24kDa) was observed in SMT extender. No expression of AIF was found in cryopreserved semen samples. A significant (p<0.05) increase in the mean percentage of cells having high mitochondrial membrane potential and a non-significant (p>0.05) decrease in late apoptotic cells (AN+/PI+) was observed in SMT extender when compared to EYT extender. The results demonstrated that cryopreservation of buffalo semen in SMT-based phytoextender can replace the traditional egg yolk extenders as it reduces the expression of apoptotic proteins maintaining high mitochondrial membrane potential and gives better protection to sperms in terms of its non-animal origin.

Prognostic value of cancer stem cells, epithelial-mesenchymal transition and circulating tumor cells in lung cancer

The epithelial-mesenchymal transition (EMT) is a program involved in embryonic development that is often activated during cancer invasion and metastasis. CD133 is the main marker identifying cancer stem cells (CSCs) in lung cancer. Circulating tumor cells (CTCs) are demonstrated to be useful as a biomarker for the diagnosis and treatment of cancer. The aim of this study was to correlate EMT, CSCs and CTCs with patient prognosis to verify whether they can contribute to better stratification of lung cancer patients at risk for recurrent and metastatic disease. Pulmonary venous blood was drawn after major pulmonary surgery in 45 patients with resectable non-small cell lung cancer (NSCLC) in order to identify CTCs. For the same patients, we also constructed prognostic lung tissue microarrays (TMA) for CD133 and c-kit and evaluated CSC and EMT markers using flow cytometry. Cytokeratin-positive cells were detectable in 11 (23.9%) cases. c-kit expression was heterogeneous in prognostic TMAs while CD133 expression was low or absent which was also confirmed by flow cytometry and RT-PCR. Flow cytometric analysis showed that the mean percentage of cells with CD133 expression was 1.6%. CD90 and CD326 markers were co-expressed with a mean percentage of 10.41%. When CD133 and CD90/CD326 expression was correlated with follow-up, CD133 showed a higher correlation with deceased patients when compared with CD90/CD326 co-expression (32.5 vs. 9.5%). CD133 expression demonstrated a strong significant association with patients exhibiting progressive disease when compared to CD90/CD326 expression (15 vs. 7.1%). CD133 may be significantly associated with invasion and metastatic spread of NSCLC. The co-expression of CD90, CD326 and CD133 has definite prognostic value in patients with NSCLC.

Nanoparticles – a Novel Approach to Chronic Lymphocytic Leukemia Treatment?

AbstractAbstract 4601Treatment of chronic lymphocytic leukemia (CLL), including conventional therapy based on alkylating agents, purine nucleoside analogues (cladribine, fludarabine (FA)) or monoclonal antibodies (rituximab (Rit), alemtuzumab), significantly improved overall and complete responses. Although there are many treatment possibilities, this disease still remains incurable. Therefore, searching for new therapeutical strategies in CLL is vital.Nanotechnology, a new and promising field of scientific research, may be of use in medicine and the pharmaceutical industry. Dendrimers, nanoparticles (polymers) of dendritic architecture, used as carriers of drugs, nucleic acid and photosensitizers for targeted delivery, contrast agents in magnetic resonance imaging (MRI) have already been reported. It seems likely that dendrimers themselves might be damaging for neoplastic cells.The aim of our study was to preliminarly assess the clinical value of treating CLL patients with poly(propylene imine) (PPI) dendrimers. The assessment was based on the in vitro induction of cytotoxicity and apoptosis by fourth generation PPI dendrimers, with the surface amino groups substituted with maltotriose [Mal-III] residues. PPI-G4-OS-Mal-III dendrimers have, in contrast to the parental cationic PPI-G4, a virtually neutral surface charge. Therefore, they are preferentially involved in H-bond driven interactions.The study was conducted in 15 untreated CLL patients (7 men, 8 women) who were diagnosed in accordance with IWCLL criteria and followed at the Hematology Department, Medical University, Lodz, Poland. The Ethics Committee of the Medical University of Lodz, Poland approved the study (RNN/75/10/KE). Informed consent was obtained from all patients involved in the study. The mean age of CLL patients was 63.80 yrs.Peripheral blood mononuclear cells (MNCs) were separated from EDTA fresh blood. MNCs were incubated with dendrimers at concentrations of 4, 6 and 8 mg/ml.The cultures without dendrimers served as controls.Dendrimers in which approximately 35% of peripheral amino groups were coated with Mal-III (Leibniz Institute of Polymer Research, Dresden, Germany) have been defined as PPI-G4-OS-Mal-III. The abbreviation OS describes the open shell structure of carbohydrate-modified dendrimers (Fig. 1). The molar mass of this PPI dendrimer was 31000 g/mol.Apoptosis was measured by the Annexin-V/Propidium Iodide test. A decrease in mitochondrial membrane potential (one of the earliest events in the apoptotic pathway) was evaluated by the CMXRos technique, using flow cytometry. The percentage of cells with lower mitochondrial membrane potential (MMP) (DYmlow/Gly-A−) was determined.The percentage of apoptotic MNCs induced by PPI-G4-OS-Mal-III after 24 h and 48 h incubations was significantly higher than the percentage of spontaneous apoptotic leukemic cells. PPI glycodendrimers induced MNC apoptosis to a greater degree after 48 h than 24 h. The largest differences were observed for Ann-V+JP+ cells at a concentration of 8 mg/ml (p = 0.007). The IC50 value after 48-h-incubation was calculated as 8.24 mg/ml.Moreover, the CMXRos technique revealed apoptosis induction by PPI-G4-OS-Mal-III at each examined concentration in comparison with control cultures (Table 1).Table 124hControlPPI-G4-OS-Mal-III 4 mg/mlPPI-G4-OS-Mal-III 6 mg/mlPPI-G4-OS-Mal-III 8 mg/mlFA 1.6 μMRit 10 μg/mlX23.751.060.164.462.928.048hX28.367.871.979.389.029.4X – mean percentage of cells with lower mitochondrial potential (DYmlow/Gly-A− [%])Finally, PPI-G4-OS-Mal-III was not observed to have any harmful effects on erythrocytes (RBCs) or platelets (PLTs). The studied dendrimer induced PLT aggregation at a concentration of 50 mg/ml. Hemolysis induced by the PPI-G4-OS-Mal-III dendrimer is not important from the biological point of view.To summarise, the PPI-G4-OS-Mal-III dendrimer demonstrated higher cytotoxicity towards CLL cells than healthy donor cells. Its potency to trigger apoptosis is similar to many PNAs and monoclonal antibodies widely used in CLL therapy. Thus, dendrimers are a potential tool for CLL treatment.The study was partially supported by Grant No. DEC-2011/01/B/NZ5/01371 from the National Science Centre, Poland. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Whole Transcriptome Sequencing Reveals Recurrent NOTCH1 Mutations in Mantle Cell Lymphoma

Abstract 436 Background:Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma that is characterized by the hallmark t(11;14)(q13;q32) translocation, as well as a high number of secondary chromosomal alterations. Further, a small number of genes such as TP53, ATM and CCND1 have been reported to be recurrently mutated in MCL, but do not fully explain the biology and do not adequately account for the wide spectrum of clinical manifestations, response to treatment and prognosis. The aim of this study was to discover new somatic mutations that could contribute to our understanding of the pathogenesis of MCL. Methods:In our discovery cohort, we sequenced the transcriptomes of 18 clinical samples (11 diagnostic and 7 progression biopsies) and 2 mantle cell lymphoma-derived cell lines (Mino and Jeko-1). For this purpose, whole transcriptome shotgun sequencing was performed on RNA extracted from fresh frozen tissue. We assembled an extension cohort of 103 diagnostic patient samples and 4 additional cell lines (Rec-1, Z-138, Maver-1, JVM-2), and performed Sanger sequencing of NOTCH1 exons 26, 27 and 34 on genomic DNA. We further exposed the 6 cell lines to 1 μM of the γ-secretase inhibitor XXI (compound E) for 7 days and measured cellular proliferation with an EdU incorporation assay. Survival analysis was carried out in the 113 patients with diagnostic biopsies and available outcome data. Results:NOTCH1 mutations were found in 14 out of 121 patient samples (11.6%) and in 2 out of 6 cell lines, Mino and Rec-1 (33.3%). The majority of these mutations (12 out of 14) lie in exon 34 that encodes the PEST domain of NOTCH1 and consist of either small frameshift-causing indels (10 cases) or nonsense mutations (2 cases). These mutations are predicted to cause truncations of the C-terminal PEST domain. To gain further insight into functional relevance, we treated 6 cell lines with compound E, an inhibitor of the γ-secretase complex that plays a critical role in the release of the intracellular domain of NOTCH1 after ligand-induced activation. In Rec-1, that harbours a NOTCH1 mutation, we observed a significant decrease in proliferation (mean percentage of cells in culture incorporating EdU decreasing from 47.5% to 1.4%, p<.001). No effect of compound E was observed in Mino, the other cell line with a NOTCH1 mutation, nor in the 4 cell lines that are wild type for NOTCH1. Outcome correlation analysis showed that NOTCH1 mutations are associated with poor overall survival (1.56 versus 3.86 years respectively, p=.001), but not with significantly shortened progression-free survival (0.88 versus 1.73 years respectively, p=.07). Discussion:We have identified recurrent mutations in NOTCH1 in a subset of patients with MCL (11.6%). The frequency and the pattern of mutations are strikingly similar to what has recently been reported in chronic lymphocytic leukemia, the other major CD5 positive B-cell malignancy (Nature, 2011 Jun 5, 475:101–105 and J Exp Med, 2011 Jul 4, 208:1389–1401). NOTCH1 mutations are associated with adverse prognosis as evidenced by shortened overall survival. This latter finding, however, should ideally be validated in a larger and uniformly treated cohort. Finally, the sensitivity of the Rec-1 cell line to compound E suggests that NOTCH1 mutations could serve as the target for tailored therapy in mantle cell lymphoma. Disclosures:Sehn:Roche/Genentech: Consultancy, Honoraria, Research Funding. Connors:Roche: Research Funding.

Gamma-tubulin and 53BP1 as candidate biomarkers of human papillomavirus-associated anal dysplasia

Background HIV+ men who have sex with men (MSM) are at increased risk of developing anal squamous cell carcinoma. High-grade anal intraepithelial neoplasia (HG-AIN or AIN2/3) is the consequence of persistent infection with high-risk human papillomaviruses and is a precursor to cancer. No biomarkers exist to identify patients with HGAIN at risk of malignant progression. We investigated two candidate biomarkers of genomic instability, gamma-tubulin and 53BP1, in a series of anal biopsies from HIV+ MSM by immunofluorescence microscopy. Gamma-tubulin staining is used to assess centrosome abnormalities as a potential source of cell division errors and aneuploidy, while staining for 53BP1 nuclear foci reflects the presence of DNA double strand breaks. Materials and methods Anal biopsies were obtained from HIV+ MSM undergoing high-resolution anoscopy and reported using the Bethesda 2001 classification system as AIN1 through 3 or carcinoma. Normal colonic tissue served as controls. The percentage of cells with abnormal centrosome number (more than two per cell) and DNA damageassociated 53BP1 foci was assessed and correlated with the histopathological diagnosis. Eighty-eight biopsies were stained for gamma-tubulin, and ninety biopsies were stained for 53BP1. Results The mean percentage of cells with abnormal centrosomes was 1.5% in AIN1, 1.3% in AIN2, and 0.9% in AIN3. No controls showed centrosome aberrations. A single biopsy diagnosed as carcinoma contained 16.1% cells with abnormal centrosomes. The mean percentage of cells with abnormal 53BP1 foci was 2.5% in controls, 17% in AIN1, 18.5% in AIN2, and 5.2% in AIN3. Higher levels of abnormal 53BP1 foci were seen in AIN1 and AIN2 compared to controls based on pair-wise comparisons between control and AIN1 (p=0.0008) and control and AIN2 (p=0.0030) using the Mann Whitney test. Lower levels of 53BP1 foci were seen in AIN3 compared to AIN1 and 2 based on pair-wise comparisons between AIN1 versus AIN3 (p=0.0034) and between AIN2 versus AIN3 (p=0.0056). Conclusion

Abstract LB-329: Defining rgulatory T-cells as CD4+CD25hiCD127- or as CD4+CD25hiFoxP3+ in immune monitoring of cancer vaccine clinical trials

Abstract Background: Regulatory T cells (Treg) are widely accepted to play a key role in the immune tolerance and escape of cancer cells. Originally Tregs were identified as being CD4+ T cells expressing high levels of CD25 (IL2 receptor). The identification of the transcription factor FoxP3 resulted in defining Tregs as CD4CD25hiFoxP3+ T cells by staining permeablized cells. Recently Tregs have been identified as CD4CD25hi T cells with low levels of CD127 (IL7 receptor) expressing a CD4+CD25hiCD127- cell surface phenotype. In an ongoing clinical trial with a MHC class II HER2/neu pep tide vaccine we have evaluated these various definitions of Treg phenotypes for immunological monitoring. Methods: Tregs were quantified in disease-free, high risk BCa patients enrolled in adjuvant peptide-based breast cancer vaccine trials. Peripheral blood mononuclear cells (PBMCs) were both permeabilized and stained with CD4, CD25, and FoxP3 monoclonal antibodies and surface stained with CD4, CD25, and CD127. Samples were then analyzed by flow cytometry. The means are compared with t-test and correlation is determined with Pearson's correlation coefficient. Results: 196 PBMC samples from 72 patients were available for Treg quantification. There was a greater mean percentage of cells identified as Treg in permeabilized than nonpermeabilized cells by staining with CD4+CD25hi (5.9±0.1% vs 4.8 ±0.1%, p&amp;lt;0.01) and excellent correlation between the tests (r=0.78, p&amp;lt;0.01). There was a greater mean percentage of cells identified as Tregs by CD4+CD25hi than CD4+CD25hiFoxP3+ (5.9±0.1% vs 3.6±0.1%, p&amp;lt;0.01) with good correlation (r=0.68, p&amp;lt;0.01). There was a greater mean percentage of Tregs by CD4+CD25+ than CD4+CD25+CD127- (4.8±0.1 vs 4.0±0.1, p&amp;lt;0.01) with excellent correlation (r=0.89, p&amp;lt;0.01). There was a greater mean percentage of Tregs by CD4+CD25hiCD127- than Tregs by CD4+ CD25hiFoxP3+ although levels were similar (4.0±0.1 vs 3.6±0.1, p&amp;lt;0.01) and the two tests had good correlation (r=0.58, p&amp;lt;0.01). Conclusion: There was good correlation between various methods of defining Tregs with and without cell membrane permeabilization. Defining Treg cells as either CD4+CD25hiFoxP3+ or CD4+CD25+CD127- appear to be more specific than defining Tregs as CD4+CD25hi alone. Furthermore, defining Treg populations by surface staining as CD4+CD25hiCD127- yields similar levels with good correlation as CD4+CD25hiFoxP3+ and may be the preferred method for immune monitoring. 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 LB-329.

Detection of chromosomal anomalies in endometrial atypical hyperplasia and carcinoma by using fluorescence in situ hybridization

Endometrial cancer is the most common pelvic gynecological malignancy. The diagnosis of well-differentiated endometrial adenocarcinoma, atypical hyperplasia, and hyperplasia is often challenging. The authors sought to investigate the utility of chromosomal anomalies for the detection of endometrial hyperplasia and carcinoma using multitarget fluorescence in situ hybridization (FISH). Samples were collected by endometrial Tao brush and processed by liquid-based cytological preparation protocol from consecutive cases to include 50 benign, 50 hyperplasia without atypia, 47 atypical hyperplasia, and 53 endometrial cancers. Each was hybridized using fluorescence-labeled DNA probes to chromosomes 1, 8, and 10. The FISH signals were enumerated in 100 cells per case, and the chromosomal anomalies were correlated with pathologic findings, including histologic diagnoses on matched endometrial tissue samples. Numeric chromosomal anomalies were found in 0% (0 of 50) of benign, 20% (10 of 50) of hyperplasia, 74% (35 of 47) of atypical hyperplasia, and 87% (46 of 53) of carcinoma specimens. The mean percentage of cells with chromosomal changes was 55% in cancer specimens, which was significantly higher than that in hyperplasia without atypia (13%, P < .0001) and atypical hyperplasia (32%, P = .003). The most frequent chromosomal anomaly was gain of chromosome 1. FISH anomalies had an overall sensitivity of 81% and specificity of 90% for the detection of atypical hyperplasia and/or endometrial carcinoma. There was no association with grade of endometrial carcinoma. Multitarget FISH appears to be useful for the differential diagnosis of hyperplasia, atypical hyperplasia, and endometrial adenocarcinoma, with a high level of sensitivity and specificity. It is also a potential tool for the early detection of neoplastic cells in endometrial cytology specimens. Endometrial hyperplasia with FISH-detected chromosomal anomalies may represent a clinically significant subset of cases that warrant close clinical follow-up.

Sputum cellularity in pulmonary tuberculosis: A comparative study between HIV-positive and -negative individuals

To compare sputum cellularity between HIV-positive and -negative individuals with pulmonary tuberculosis. A cross-sectional study was conducted in patients with pulmonary tuberculosis. Sputum samples were collected and processed within two hours after collection. The absolute number of squamous cells of a total of 400 cells was counted, as well as the absolute number (× 106 cells/ml) and percentage of eosinophils, lymphocytes, macrophages and neutrophils and total cellularity and viability were determined. Comparisons of the means of each cell type were held in a significance level of 95% (p < 0.05). Pearson’s correlation coefficient between the identified cell types was calculated. Results: Assessment was performed in a cohort of 40 subjects, mean age 40 years, 77.5% male, 70% Caucasian, 40% HIV-positive (mean age 35.9 years). Mean percentage viability in the samples was 56.1%. The average value of squamous cells was 58.8. Mean percentages of cells were: 33.7% neutrophils, 1.7% eosinophils, 50.7%, macrophages and 12.3% lymphocytes. The average total cell count was 1.9 x 106cells/ml. The average CD4+ T-cell count in HIV-positive was 95.4 cells/mm3. Association of radiological patterns was present in 72.5% of cases. Pearson’s correlation coefficient was 0.08 (p < 0.01) between absolute counts of eosinophils and lymphocytes, eosinophils and macrophages and macrophages and neutrophils. Inverse relationship was observed between the percentage of macrophages and neutrophils. There was no statistically significant difference between cell count of HIV-positive and -negative individuals. Key words: Sputum, tuberculosis, HIV.

Evaluation of cell proliferation rate in non-dysplastic leukoplakias

Analyze whether the most frequent cases of non-dysplastic leukoplakias, hyperkeratosis (H), acanthosis (A), and hyperkeratosis with acanthosis (HA) have similar cell proliferation rates and to compare them with epithelial dysplastic (ED) leukoplakias and normal oral epithelium (NOE). The sample comprised 10 cases of normal oral epithelium, 10 cases of hyperkeratosis, 10 cases of acanthosis, 10 cases of hyperkeratosis with acanthosis and 10 cases of epithelial dysplasia. The mean number of AgNORs per nucleus (mAgNOR) and the mean percentage of cells with 1, 2, 3 and 4 or more AgNORs per nucleus (pAgNOR) were recorded. The results of mAgNOR showed differences between disorders in the evaluation of the basal layer, of the parabasal layer, and in the overall evaluation. mAgNOR and pAgNOR=2 increased progressively from normal oral epithelium to hyperkeratosis with acanthosis, hyperkeratosis, acanthosis and epithelial dysplasia (p<0.05). Cell proliferation rate was different between different subtypes of non-dysplastic leukoplakias and this group presented a higher proliferative behavior when compared to normal oral epithelium. It may be suggested that non-dysplastic leukoplakias had different characteristics regarding cell proliferation rates and sometimes showed a proliferative behavior similar to that found in epithelial dysplasia. More studies should be conduced to increase knowledge about the biological profile of non-dysplastic leukoplakias, especially as it pertains to acanthosis.

The Nuclear Receptor FXR Links Metabolism with Breast Cancer.

Abstract Background:Abnormal metabolism, as measured by obesity, has been associated with an increase risk in both the development of breast cancer as well as breast cancer recurrence and death. To date, identification of a molecular link between obesity and breast cancer remains elusive. Farnesoid X receptor (FXR) is a ligand dependent transcriptional factor and plays a critical role in bile acid, cholesterol and carbohydrate metabolism. Little is known of the function of FXR in cancer although several recent studies have shown that FXR may contribute to breast, colorectal and hepatocellular cancer. Given the role of FXR in metabolism and emerging data suggesting a role for FXR in cancer, we hypothesize that expression of FXR in breast tissue may be related to metabolism. In addition, changes in FXR expression may also be associated with breast cancer development and progression.Methods:Clinical data on estrogen/progesterone receptor positive, Stage I and II breast cancer patients were extracted from an institutionally approved prospective database established in 2006. Paired formaldehyde fixed, paraffin embedded normal, in situ, and invasive tissues from these patients were identified from the COH tissue repository. Immunohistochemical staining of FXR using a validated polyclonal antibody was completed with appropriate positive and negative controls. The slides were graded independently by two investigators using an agreed upon scale to detect the percentage of positively stained cells to the nearest 10th percentile. Clinical data were correlated with expression analysis. Statistical analyses were performed with ANOVA tests followed by Fisher's t-tests for pair-wise comparisons. In determination of factors accounting for FXR expression, a multivariate analysis was completed. A p-value of 0.05 was considered significant in all analyses.Results:Paired normal tissue and invasive cancer was identified in all 43 patient specimens. In 40% (17/43) of these specimens, in situ disease was also identified. FXR expression in normal breast tissue was significantly less when compared to both invasive and noninvasive cancer (p≤ 0.001). The mean percentage of cells staining positive for FXR in normal breast tissue was 60%, non-invasive 77% and invasive 83% (p &amp;lt; 0.0001). FXR expression did not correlate with grade, histology, nodal status, Her 2 neu status, HTN, DM or hyperlipidemia in normal, in situ and invasive tissue on multivariate analysis. BMI strongly correlated with FXR expression in normal tissues (p = 0.02) and may correlate in in situ tissues (p = 0.07). However, there was no correlation of BMI with FXR expression in invasive cancer (p = 0.55).Conclusions:FXR expression is upregulated in invasive cancer and increases progressively along the continuum from normal breast tissue to malignancy. Expression of FXR correlates with BMI in normal breast tissue, suggesting that FXR may be a molecular link between obesity and the development and progression of breast cancer. Our results warrant further investigation into the relationship of FXR, obesity and breast cancer. These findings may provide support for the development of targeted FXR agents in prevention and treatment of obesity related cancers. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5153.

Clinical significance of hTERC gene amplification detection by FISH in the screening of cervical lesions

This study evaluated the clinical significance of hTERC gene amplification detection by fluorescence in situ hybridization (FISH) in the screening of cervical lesions. Cervical specimens of 50 high risk patients were detected by thin liquid-based cytology. The patients whose cytological results were classified as ASCUS or above were subjected to the subsequent colposcopic biopsies. Slides prepared from these 50 cervical specimens were analyzed for hTERC gene amplification using interphase FISH with the two-color hTERC probe. The results of the cytological analysis and those of subsequent biopsies, when available, were compared with the FISH-detected hTERC abnormalities. It was found that the positive rates of hTERC gene amplification in NILM, ASCUS, LSIL, HSIL, and SCC groups were 0.00, 28.57%, 57.14%, 100%, and 100%, respectively. The positive rates of hTERC gene amplification in HSIL and SCC groups were significantly higher than those in NILM, ASCUS and LSIL groups (all P<0.05). The mean percentages of cells with hTERC gene amplification in NILM, ASCUS, LSIL, HSIL, and SCC groups were 0.00, 10.50%, 36.00%, 79.00%, and 96.50%, respectively. Patients with HSIL or SCC cytological diagnoses had significantly higher mean percentages of cells with hTERC gene amplification than did patients with NILM, ASCUS or LSIL cytological diagnoses (all P<0.05). It was concluded that two-color interphase FISH could detect hTERC gene amplification to accurately distinguish HSIL and ISIL of cervical cells. It may be an adjunct to cytology screening, especially high-risk patients.

Detection of chromosomal anomalies in uterine endometrial carcinoma using fluorescence in situ hybridization (UteroFISH)

5533 Background: Endometrial cancer is the most common pelvic gynecological malignancy. The diagnosis of well-differentiated endometrial adenocarcinoma, atypical hyperplasia, and marked hyperplasia is often challenging. We sought to investigate the utility of chromosomal anomalies for the detection of uterine endometrial carcinoma using multitarget fluorescence in situ hybridization (FISH). Methods: Samples were collected by endometrial brush and processed by liquid-based thin-layer cytological preparation protocol. For study, we collected cytology slides from consecutive cases to include 50 benign, 50 hyperplasia without atypia, 50 atypical hyperplasia, and 50 endometrial cancers. Each was hybridized using fluorescence labeled DNA probes to chromosomes 1, 8, and 10 (UteroFISH). The FISH signals were enumerated in 100 cells per case, and the chromosomal anomalies were correlated with pathologic findings, including histologic diagnoses on endometrial tissue samples. Results: Numeric chromosomal anomalies were found in 0% (0/50) of benign, 20% (10/50) of hyperplasia, 76% of atypical hyperplasia (38/50), and 86% (43/50) of carcinoma specimens. The mean percentage of cells with chromosomal changes was 54% in cancer specimens, significantly higher than that in hyperplasia without atypia (13%, p&lt; 0.0001) and atypical hyperplasia (34%, p&lt; 0.0001). The most frequent chromosomal anomaly was gain of chromosome 1. FISH anomalies had an overall sensitivity of 81% and specificity of 90% for the detection of atypical hyperplasia and/or endometrial carcinoma. There was no association with grade of endometrial carcinoma. Conclusions: Multi-target UteroFISH appeared to be useful for the differential diagnosis of reactive hyperplasia, atypical hyperplasia, and endometrial adenocarcinoma, with a high level of sensitivity and specificity. Endometrial hyperplasia with FISH-detected chromosomal anomalies may require close clinical follow-up. No significant financial relationships to disclose.

A pilot study to compare FXR expression in normal and malignant tissue in receptor-positive early-stage breast cancer

e14645 Background: Farnesoid X receptor (FXR), a nuclear receptor, is a ligand dependent transcriptional factor regulating cholesterol and carbohydrate metabolism. Recently, FXR was shown to have a contributing role in colorectal cancer. We hypothesize that FXR expression changes from normal to premalignant to malignant tissue in patients with breast cancer. Methods: We identified 16 paired formaldehyde fixed, paraffin embedded tissue (normal, premalignant, and malignant) from patients with receptor positive, early stage breast cancer. Clinical information was extracted from a prospective database initiated in 2006 under institutional approval. Immunohistochemical staining of FXR using a validated polyclonal antibody was completed with appropriate positive and negative controls. The slides were graded independently by two investigators using an agreed upon scale to detect the percentage of positively stained cells to the nearest 10th percentile. Statistical analysis was performed by ANOVA and Student's t-test. A p-value of 0.05 was considered significant in all analyses. Results: Normal tissue and invasive cancer was identified in all 16 patient specimens. Of the 16 invasive cancers, 12 were ductal and 4 were lobular. 8/16 (50%) of the specimens also contained non-invasive cancer. 5/16 patients (31%) had N1 disease. FXR expression did not correlate with grade, histology, stage, or lymph node status. However, FXR expression increases with malignant transformation of the breast cancer cell. The mean percentage of cells staining positive for FXR in normal breast tissue was 58%, non-invasive 72% and invasive 79%. FXR staining in normal breast tissue was significantly less when compared to both invasive and noninvasive cancer (p&lt; 0.007). Conclusions: FXR expression is upregulated in breast cancer when compared with expression in normal tissue and appears to progressively increase along the continuum of malignancy. Our pilot study results warrant further evaluation into FXR as a predictive biomarker for breast cancer, given the ability to target FXR via development of non-toxic oral ligands. No significant financial relationships to disclose.

Analysis of the cellular centrosome in fine-needle aspirations of the breast

BackgroundThe purpose of the present investigation is to determine whether centrosome amplifications are present in breast tumor cells, whether there are differences of centrosome amplification between benign breast lesions and breast carcinomas, and whether centrosomal analysis can be of value in the diagnosis and prognosis of breast carcinoma.MethodsUsing immunofluorescence analysis with an antibody against γ-tubulin, we analyzed centrosome abnormalities in fine-needle aspirations of 100 breast lesions (25 cases with benign lesions and 75 cases with carcinomas).ResultsWe found that centrosome amplifications, including numerical centrosome amplification and structural centrosome amplification, were present in most breast tumors. Cells with numerical centrosome amplification were found in 23 of 25 benign lesions, and in all 75 cases of breast carcinomas. Cells with structural centrosome amplification were found in three of 25 benign lesions, and in 69 of 75 breast carcinomas. The breast carcinomas showed a mean percentage of cells with numerical centrosome amplification of 4.86% and a mean percentage of cells with structural centrosome amplification of 3.98%. These percentages were significantly higher than those in benign lesions, with a numerical centrosome amplification of 2.77% and a structural centrosome amplification of 0.10%. Furthermore, the mean percentage of cells with structural centrosome amplification was significantly associated with HER2/neu overexpression (P < 0.05) and with negative estrogen receptor status (P < 0.05), and had a borderline association with negative progesterone receptor status (P = 0.056) in breast carcinomas.ConclusionStructural centrosome amplification may bear a close relationship with breast carcinoma and may be a potential biomarker for diagnosis and prognosis of breast carcinoma.