Cytokeratin labeling of breast cancer cells extracted from paraffin‐embedded tissue for bivariate flow cytometric analysis

Glogovac, Jeri; Porter, Peggy L.; Banker, Deborah E.; Rabinovitch, Peter S.

doi:10.1002/(sici)1097-0320(19960701)24:3<260::aid-cyto9>3.3.co;2-g

Cited by 13 publications

(15 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To restrict the inclusion of non-tumor cells from analysis, formalin-fixed, paraffin-embedded tumor samples were sorted by flow cytometry with a cytokeratin label as described previously (25). Briefly, thick sections of tumor samples were dissected to exclude normal breast epithelium, digested to prepare a cell suspension, and sorted by bivariate analysis based on cytokeratin expression and DNA content with 4Ј,6-diamidino-2-phenylindole fluorescence.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Array Comparative Genomic Hybridization Analysis of Genomic Alterations in Breast Cancer Subtypes

Loo¹,

Grove²,

Williams³

et al. 2004

Cancer Research

Self Cite

185

157

View full text Add to dashboard Cite

In this study, we performed high-resolution array comparative genomic hybridization with an array of 4153 bacterial artificial chromosome clones to assess copy number changes in 44 archival breast cancers. The tumors were flow sorted to exclude non-tumor DNA and increase our ability to detect gene copy number changes. In these tumors, losses were more frequent than gains, and gains in 1q and loss in 16q were the most frequent alterations. We compared gene copy number changes in the tumors based on histologic subtype and estrogen receptor (ER) status, i.e., ER-negative infiltrating ductal carcinoma, ER-positive infiltrating ductal carcinoma, and ER-positive infiltrating lobular carcinoma. We observed a consistent association between loss in regions of 5q and ER-negative infiltrating ductal carcinoma, as well as more frequent loss in 4p16, 8p23, 8p21, 10q25, and 17p11.2 in ER-negative infiltrating ductal carcinoma compared with ER-positive infiltrating ductal carcinoma (adjusted P values < 0.05). We also observed high-level amplifications in ER-negative infiltrating ductal carcinoma in regions of 8q24 and 17q12 encompassing the c-myc and c-erbB-2 genes and apparent homozygous deletions in 3p21, 5q33, 8p23, 8p21, 9q34, 16q24, and 19q13. ER-positive infiltrating ductal carcinoma showed a higher frequency of gain in 16p13 and loss in 16q21 than ER-negative infiltrating ductal carcinoma. Correlation analysis highlighted regions of change commonly seen together in ER-negative infiltrating ductal carcinoma. ER-positive infiltrating lobular carcinoma differed from ER-positive infiltrating ductal carcinoma in the frequency of gain in 1q and loss in 11q and showed high-level amplifications in 1q32, 8p23, 11q13, and 11q14. These results indicate that array comparative genomic hybridization can identify significant differences in the genomic alterations between subtypes of breast cancer.

show abstract

Section: Methodsmentioning

confidence: 99%

“…Briefly, thick sections of tumor samples were dissected to exclude normal breast epithelium, digested to prepare a cell suspension, and sorted by bivariate analysis based on cytokeratin expression and DNA content with 4Ј,6-diamidino-2-phenylindole fluorescence. This resulted in Ͼ90% purity of tumor cells in the sorted sample (25).…”

Section: Methodsmentioning

confidence: 99%

Array Comparative Genomic Hybridization Analysis of Genomic Alterations in Breast Cancer Subtypes

Loo¹,

Grove²,

Williams³

et al. 2004

Cancer Research

Self Cite

185

157

View full text Add to dashboard Cite

show abstract

“…We also pursued cytological studies to characterize the makeup of the cells in whole saliva using immunostaining for cytokeratin expression, a well described marker for epithelial cells (32,48). Because many researchers are interested in …”

Section: Figmentioning

confidence: 99%

Proteomics Analysis of Cells in Whole Saliva from Oral Cancer Patients via Value-added Three-dimensional Peptide Fractionation and Tandem Mass Spectrometry

Xie

Onsongo

Popko

et al. 2008

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

Whole human saliva possesses tremendous potential in clinical diagnostics, particularly for conditions within the oral cavity such as oral cancer. Although many have studied the soluble fraction of whole saliva, few have taken advantage of the diagnostic potential of the cells present in saliva, and none have taken advantage of proteomics capabilities for their study. We report on a novel proteomics method with which we characterized for the first time cells contained in whole saliva from patients diagnosed with oral squamous cell carcinoma. Our method uses three dimensions of peptide fractionation, combining the following steps: preparative IEF using free flow electrophoresis, strong cation exchange step gradient chromatography, and microcapillary reverse-phase liquid chromatography. We determined that the whole saliva samples contained enough cells, mostly exfoliated epithelial cells, providing adequate amounts of total protein for proteomics analysis. From a mixture of four oral cancer patient samples, the analysis resulted in a catalogue of over 1000 human proteins, each identified from at least two peptides, including numerous proteins with a role in oral squamous cell carcinoma signaling and tumorigenesis pathways. Additionally proteins from over 30 different bacteria were identified, some of which putatively contribute to cancer development. The combination of preparative IEF followed by strong cation exchange chromatography effectively fractionated the complex peptide mixtures despite the closely related physiochemical peptide properties of these separations (pI and solution phase charge, respectively). Furthermore compared with our two-step method combining preparative IEF and reversephase liquid chromatography, our three-step method identified significantly more cellular proteins while retaining higher confidence protein identification enabled by peptide pI information gained through IEF. Thus, for detecting salivary markers of oral cancer and possibly other conditions of the oral cavity, the results confirm both the potential of analyzing the cells in whole saliva and doing so with our proteomics method. Molecular & Cellular Proteomics 7:486 -498, 2008.Whole human saliva is easily collected in the clinic in a non-invasive, on-demand manner and in relatively large, easily stored quantities, making it an optimal bodily fluid for clinical diagnostics (1, 2). Diagnosis of oral cancer could benefit greatly from the development of whole saliva-based clinical tests given the physical proximity of the site of cancer development with the diagnostic fluid. In fact, oral cancer, usually diagnosed in the form of oral squamous cell carcinoma (OSCC), 1 has not seen a drop in its 50% mortality rate over the last 30 years (3), motivating the development of new and reliable, saliva-based clinical diagnostic tests for the early detection of OSCC. Such tests would lead to more informed treatment of patients and a reduction in the suffering and death caused by this cancer.To develop these tests, molecular markers that ar...

show abstract

“…19 The ploidy level of cytokeratin-positive tumor cells was compared with benign, cytokeratin-negative diploid cells to create a DNA index. To allow for instrument error of 5%, the diploid tumors have DNA index of 0.95-1.05.…”

Section: Flow Cytometry Analysis Of Msi and Immunohistochemistrymentioning

confidence: 99%

Progressive methylation during the serrated neoplasia pathway of the colorectum

et al. 2005

View full text Add to dashboard Cite

Serrated adenoma is a recently described entity characterized by having combined architectural features of hyperplastic polyps and classical adenoma. To understand the role of gene regulation in the progression of the serrated neoplasia pathway, we examined the methylation profiles of the promoter regions of 19 genes, DNA ploidy, and mutator phenotype status. In all, 40 sporadic, classical serrated adenomas were pathologically reviewed and divided into four pathologic groups according to their histologic grades. Methylation-specific PCR was performed using primers for p16, hMLH1, RASSF1A, APC, HIC-1, DAPK, MGMT, SLC5A8, RB1, HCadherin, E-Cadherin, TIMP3, PTEN, THBS1, LKB1, p14, p15, FHIT, and VHL. Dual flow-cytometric analyses using cytokeratin and DAPI and MSI studies using BAT26 were also performed. Methylation was observed in 2.5-82.5% (mean 33.9%) of the CpG islands in the promoter regions of 16 genes. The tumors with higher histologic grades, including carcinomas, showed more extensive methylation compared to those with lower grades, and serrated adenomas in the right colon showed more frequent methylation than those in the left (Po0.05). Tumor-specific promoter methylation of SLC5A8 was observed in 33 (82.5%) of the serrated adenomas. Aneuploidization with near-diploid DNA indices was detected in four out of 28 cases examined (14.3%); two were low-grade serrated adenomas and two were carcinomas in the left colon. The high mutator phenotype was not observed in any of the cases examined. Our results indicate that: (1) aberrant, widespread methylation of CpG islands increases with the histological progression of serrated adenomas; (2) methylation of SLC5A8 is an early event; and (3) additional methylation of the p16, p14, MGMT, TIMP3, and FHIT genes are important tumorigenic steps in the serrated neoplasia pathway.

show abstract

Cytokeratin labeling of breast cancer cells extracted from paraffin‐embedded tissue for bivariate flow cytometric analysis

Cited by 13 publications

References 0 publications

Array Comparative Genomic Hybridization Analysis of Genomic Alterations in Breast Cancer Subtypes

Array Comparative Genomic Hybridization Analysis of Genomic Alterations in Breast Cancer Subtypes

Proteomics Analysis of Cells in Whole Saliva from Oral Cancer Patients via Value-added Three-dimensional Peptide Fractionation and Tandem Mass Spectrometry

Progressive methylation during the serrated neoplasia pathway of the colorectum

Contact Info

Product

Resources

About