Quantitative CK19 biomarker detection in breast cancer cell lines (original) (raw)
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The International journal of biological markers
Cytokeratin 19 (CK19) is an acidic protein of 40 kDa that is part of the cytoskeleton of epithelial cells. It is highly expressed by all epithelial cells and represents a useful indicator of epithelial differentiation. The soluble fragment of CK19 (CYFRA 21-1) can be a useful circulating tumor marker and can be detected in the serum of cancer patients. The development of metastasis in patients with cancer of epithelial origin is due to the migration of tumor cells from the original tumor to distant organs. In order to detect micrometastasis in patients with breast cancer, we evaluated and compared CK19 gene expression using RT-PCR in blood samples collected from 80 healthy women and 80 patients with localized or metastatic breast cancer. The concentration of the soluble CK19 fragment CYFRA 21-1 was measured in serum of all study subjects by radioimmunoassay employing specific monoclonal antibodies. The relationship between the expression of this molecular marker and clinical stage, ...
Assessment of Cytokeratin Expression in Carcinoma Breast
Journal of Evolution of Medical and Dental Sciences, 2020
BACKGROUND Breast cancer is the most common malignancy in females and an important cause of cancer deaths worldwide. With advances in oncopathology, breast cancers are now diagnosed and treated at a molecular level. To distinguish and subdivide the type of cancer with the suitable markers and to foresee its prognosis and therapeutic approach, cytokeratin can be used which is prognostic tumour marker and has a number of different advantages as it is an important IHC marker, cytokeratin (Ck) is epithelial intermediate filament, which is expressed in a tissue specific manner. Expression of the intermediate filaments, predominantly cytokeratins (Ck) reflects the epithelial cell type. In breast, the luminal epithelial cells express CK 8 / 18, CK 7 and CK 19, while basal cell expresses CK 5 / 6, CK 14 and CK 17. Bloom Richardson grading (BR) system has a powerful prognostic value. It is also incorporated in the algorithm for the determination in the use of adjuvant chemotherapy. Moreover, as a tumour marker cytokeratin can accurately predict disease status as it is easy, inexpensive and a reliable tool for efficient management. CK and BR grading can be used as cost effective diagnostic tools in hormone positive breast carcinoma for diagnosis as well as treatment. Very early detection of breast cancer reduces the mortality and morbidity.
CK19 Expression in breast tumors and lymph node metastasis after neoadjuvant therapy
Histopathology, 2015
Aims: Neoadjuvant therapy is used in many patients with breast cancer before surgery, with the aim of reducing the tumour size, allowing conservative resections. Sentinel node biopsy is a conservative procedure for handling the axilla in breast cancer; however, the use of this technique after neoadjuvant treatment is under discussion. For sentinel node assay, methods based on the detection of cytokeratin 19 (CK19) mRNA, such as one-step nucleic acid amplification (OSNA), are available. However, if systemic therapy could alter protein expression, then CK19 would not be a good target for analysing these nodes. The aim of this study was to evaluate the immunohistochemical expression of CK19 within different cancer types, and to compare its expression in breast tumours and axillary nodes before and after treatment. Methods and results: CK19 immunostaining was studied in 162 tumour and node samples before and after treatment. Statistical studies using the McNemar test and chi-square test were performed. CK19 expression was found in 155 cases. We compared CK19 expression in tumour and node biopsies before and after treatment, and we found a lack of significant CK19 expression changes. Conclusions: Our study has confirmed the preservation of CK19 protein expression in breast cancer cells after neoadjuvant therapy. On the basis of these results, quantification-based methods such as the OSNA CK19 assay, could be an accurate tool with which to analyse the sentinel nodes, regardless of whether they had been obtained before or after treatment.
Clinical Biochemistry, 2001
Objectives: To develop a highly sensitive quantitative RT-PCR hybridization assay for the determination of CK-19 mRNA in peripheral blood of patients with breast cancer. Patients and methods: Quantification of CK-19 mRNA was based on the coamplification of CK-19 mRNA with a recombinant CK-19 RNA internal standard (CK-19 RNA-IS) through RT-PCR. The biotinylated amplification products were immobilized on steptavidin coated wells, hybridized with digoxigenin labeled probes and determined through an antidigoxigenin antibody conjugated to alkaline phosphatase by luminometric detection. The developed luminometric hybridization assay was validated with samples containing total RNA of known amounts from CK-19 expressing cells (MCF-7) in the presence of 1 g total RNA isolated from peripheral blood mononuclear cells (PBMC) of healthy controls and a constant amount of CK-19 RNA-IS. The method was applied for the quantitative determination of CK-19 mRNA in the peripheral blood of 26 healthy volunteers, 14 patients with stage IV breast cancer and 37 patients with stage I/II breast cancer before chemotherapy. Results: Luminescence ratios for CK-19 mRNA and CK-19 RNA-IS were linearly related to the number of MCF-7 cells within the range of 1 to 2000 cells. The overall reproducibility of the assay (between-run) varied between 8.9% and 13.4%. The method can clearly detect CK-19 mRNA from 1 MCF-7 cell in the presence of 10 6 normal PBMC and is highly specific as none of the 26 healthy controls tested had detectable CK-19 mRNA levels, while 10 out of 14 (71.4%) and 9 out of 37 (24.3%) patients with stage IV and stage I/II breast cancer, respectively, were tested positive. Conclusion: The developed quantitative RT-PCR hybridization assay for CK-19 is reproducible, highly sensitive and specific, and can be used for a large-scale prospective evaluation of clinical samples.
Clinical Cancer Research, 2008
To investigate the prognostic value of the molecular detection of circulating tumor cells (CTCs) using three markers [cytokeratin 19 (CK19), mammaglobin A (MGB1), and HER2] in early breast cancer. Experimental Design: CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected using real-time (CK19) and nested (MGB1 and HER2) reverse transcription-PCR in the peripheral blood of 175 women with stage I to III breast cancer before the initiation of adjuvant chemotherapy. The detection of CTCs was correlated with clinical outcome. In 10 patients, immunofluorescence staining experiments were done to investigate the coexpression of cytokeratin, MGB1, and HER2 in CTCs. Results: CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected in 41.1%, 8%, and 28.6% of the 175 patients, respectively. Patients had one of the following molecular profiles: CK19mRNA+/MGB1mRNA+/HER2 mRNA+ (n = 8), CK19mRNA+/MGB1mRNA+/ HER2mRNA-(n = 1), CK19mRNA+/MGB1mRNA-/HER2mRNA+ (n = 42), CK19mRNA+/ MGB1mRNA-/HER2mRNA-(n = 21), CK19mRNA-/MGB1mRNA+/HER2mRNA-(n = 5), and CK19mRNA-/MGB1mRNA-/HER2mRNA-(n = 98). Double-immunofluorescence experiments confirmed the following CTC phenotypes: CK+/MGB1+, CK+/MGB1-, CK-/MGB1+, CK+/ HER2+, CK+/HER2-, MGB1+/HER2-, and MGB1+/HER2+. In univariate analysis, the detection of CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells was associated with shorter diseasefree survival (DFS; P < 0.001, P = 0.001, and P < 0.001, respectively), whereas the detection of CK19mRNA+ and MGB1mRNA+ cells was associated with worse overall survival (P = 0.044 and 0.034, respectively). In multivariate analysis, estrogen receptor^negative tumors and the detection of CK19mRNA+ and MGB1mRNA+ cells were independently associated with worse DFS. Conclusion: The detection of peripheral blood CK19mRNA+ and MGB1mRNA+ cells before adjuvant chemotherapy predicts poor DFS in women with early breast cancer.
2012
Purpose Breast cancer is a leading cause of cancer-related deaths in women Worldwide. The clinical course of this disease is highly variable and clinicians continuously search for prognostic parameters that can accurately predict prognosis. peripheral blood cytokeratin-19 (CK-19) mRNA-positive cells and its correlation with well established prognostic factors including pathologic parameters, hormonal status and biologic marker; HER 2/ neu in breast female Egyptian cancer patients was studied . Patients and Methods A total of 60 peripheral blood specimens were collected for study. Patients were forty newly diagnosed breast cancer and 10 patients with benign breast lesions .The10 apparently healthy donors and patients with benign lesions were used as the control group. They were analyzed for the presence of CK-19 mRNA-positive cells using nested reverse transcription polymerase chain reaction assay (RT-PCR). Immunohistochemical staining for HER 2/neu, estrogen and progesterone recepto...
Laboratory Investigation, 2004
To determine the correlation of the levels of circulating tumor cells (CTC) in breast cancer patients with clinical parameters like disease-free survival and response to therapy, we employed a previously reported quantitative real-time reverse-transcription polymerase chain reaction (QRT-PCR) assay for cytokeratin-19 (Ck19; Ann Oncol 2001; 12: 39-46). Our preliminary results based on this assay (Protocol A) prompted us to optimize the experimental conditions. Here we report an improved assay (Protocol B), which more sensitively and specifically detects CTC in breast cancer patients.
Clinical Cancer Research, 2011
To investigate the associations between baseline and posttreatment circulating tumor cell (CTC) gene expression and outcome of patients enrolled in four North Central Cancer Treatment Group metastatic breast cancer (MBC) trials in which specimens were shipped (at 4°C) from community-based sites to a reference laboratory (Mayo Clinic, Rochester, MN). Blood was collected at treating sites from MBC patients before (baseline), during, and at the end of treatment with erlotinib + gemcitabine (N0234), sorafenib (N0336), irinotecan + cetuximab (N0436), or paclitaxel-poliglumex + capecitabine (N0437). CTCs from 10 mL of EDTA blood were enriched with CD45 depletion, 24 to 30 hours postblood collection. Reverse transcription/quantitative PCR was used to determine cytokeratin-19 (CK19) and mammaglobin (MGB1) mRNA levels in CTCs from up to 13 (N0234), 16 (N0336), 18 (N0436), and 39 (N0437) patients. The gene expressions were normalized to β(2)-microglobulin and calibrated to healthy blood using the 2(-ΔΔCq) algorithm; positivity was defined as 2 or more. CK19+mRNA cells were detected in 56% to 75% and MGB1+mRNA cells in 23% to 38% of 86 patients at baseline. CK19+mRNA cells were detected in 30% to 67% and MGB1+mRNA cells in 14% to 64% of 110 postbaseline serial samples. The presence of baseline CK19+mRNA cells (P = 0.01) but not MGB1+mRNA cells (P = 0.14) was significantly associated with shorter overall survival. A decrease in MGB1+mRNA levels (baseline-week 8) seemed to be associated with clinical response (P = 0.05). CTC gene expression analysis conducted by a reference laboratory is feasible when blood is collected from treating sites and processed 24 to 30 hours postcollection. The presence of baseline CK19+mRNA CTCs was associated with poor prognosis; a decrease in MGB1+mRNA CTCs may help predict response to therapy of MBC patients.
Romanian Journal of Morphology and Embryology, 2008
Purpose: The aim of our study was to characterize and describe the different immunohistochemical expression patterns of cytokeratin 8/18 (CK8/18) in breast tumors and to make a correlation between histopathology, immunohistochemistry for CK8/18 and its possible diagnostic value of this pair of keratins for molecular classification of breast cancers. Material and methods: Forty cases of breast tumors immunostained with monoclonal antibodies against CK8/18 using a polymer based detection system and diaminobenzidine as chromogen were microscopically evaluated in normal and tumor breast tissue concerning the intensity, distribution and density of positive cells. Association with histopathology and nuclear grade were also studied. Results: Three different models of positive reaction were found: (1) normal cytoplasmic with intense and diffuse pattern, (2) aberrant membrane pattern and (3) aberrant cytoplasmic granular pattern associated with membranous positive reaction. Normal expression of CK8/18 was found in 23 cases of breast cancer, aberrant membranous in nine cases and aberrant with granular pattern in four cases. Further studies will be needed to elucidate these differences and possible correlation with other molecular markers.