CK19 mRNA In Peripheral Blood Research Articles

CK19 combined with contrast-enhanced ultrasound: A prediction model for non-sentinel lymph node involvement in early breast cancer.

11539 Background: According to Z0011 and AMAROS trials, patients with breast cancer stage cT1~2cN0 and sentinel lymphnode (SLN) 1~2 involvement can avoid axillary lymphnode dissection (ALND). But the risk of non-sentinel lymphnode (nSLN) involvement in those early stage patients is still unclear and it is difficult to predicting the risk before surgery. Our previous study showed CK19 mRNA in peripheral blood had predicative value of nSLN involvement. Also, contrast-enhanced ultrasound (CEUS) is a new effective method exmaming axillary lymph node. We aim to establish a prediction model for nSLN involvement in early breast cancer using CK19 combined with CEUS score. Methods: We identified 119 cases diagnosed early breast cancer (stage cT1~2cN0 and 1~2 SLNs involvement as in Z0011 and AMAROS trials) from Oct 2015 to Nov 2016 in Zhejiang Cancer Hospital. The CK19 mRNA of peripheral blood by RT-PCR and CEUS score of axillary lymph nodes were acquired before surgery. We used logistic regression analysis for filtering out valuable predictive clinical parameters and establishing formulas to calculate the probability of nSLN involvement. Our model was compared with Memorial Sloan Kettering Cancer Center (MSKCC) nomogram, which is one of the most reliable and validated methods for predicting of nSLN. Results: The histological grade, CK19 and CEUS score were screened by logistic regression analysis into the formula to calculate the probability of nSLN involvement. The sensitivity, specificity, total accuracy of this model was 89.13%, 80.82% and 84.03%, respectively. The false negative rate was 10.87%. The model had high quality of consistency (Kappa 0.675, p < 0.01) and goodness of fit (likelihood-ratio test, -2log liklihood = 84.607). The area under curve (AUC) of ROC was significantly higher (P < 0.01) in our model (0.914, 95%CI, 0.863~0.965) than in MSKCC nomogram (0.563, 95%CI, 0.459~0.667). Conclusions: The prediction model based on CK19 and CEUS score has satisfying sensitivity, specificity and accuracy, more effective than MSKCC nomogram. It is a valuable model of evaluating the risk of nSLN involvement in early breast cancer before surgery, picking out the patients who can truly avoid ALND.

Prognostic value of chemotherapy-resistant CK19mRNA-positive circulating tumor cells in patients with advanced/metastatic non-small cell lung cancer.

The detection of circulating tumor cells (CTCs) is of prognostic significance in several tumor types. The present study evaluated the detection and the clinical relevance of CK19mRNA(+) CTCs in patients with advanced/metastatic non-small cell lung cancer before and after front-line chemotherapy. Peripheral blood was obtained from 642 patients with treatment-naïve unresectable stage IIIB and IV non-small cell lung cancer and from 455 patients after the completion of 1st line chemotherapy. RNA was extracted from peripheral blood mononuclear cells and the detection of CK19mRNA-positive cells was performed using a quantitative PCR assay. Based on the detection limit of the assay, 167 (26.0%) patients had detectable CK19mRNA(+) CTCs at baseline. The detection of CK19mRNA(+) CTCs before treatment was not associated with the clinical outcome, but their detection at the end of chemotherapy was associated with significantly decreased PFS and OS [PFS: 2.6 vs 3.8months (p=0.008); OS: 5.7 vs 10.0months (p=0.006) for CK19mRNA(+) vs CK19mRNA(-) patients, respectively]. Multivariate analysis revealed that the detection of CK19mRNA(+) CTCs both before and after chemotherapy emerged as an independent factor associated with reduced PFS (HR: 1.778; p<0.001) and OS (HR: 1.608; p=0.001). The detection of peripheral blood CK19mRNA(+) CTCs before and after the completion of front-line chemotherapy is an adverse prognostic factor associated with poor clinical outcome in patients with stage IIIB/IV non-small cell lung cancer.

CK19 mRNA in blood can predict non-sentinel lymph node metastasis in breast cancer.

Reverse-transcription polymerase chain reaction (RT-PCR) is used to detect CK19 mRNA in sentinel lymph node biopsy (SLNB) tissues from breast cancer patients. We examined whether CK19 mRNA in peripheral blood is predictive of non-sentinel lymph node (nSLN) metastasis. Breast cancer cases diagnosed with clinical stage cT1–3cN0 and registered in our medical biobank were identified retrospectively. This study then included 120 breast cancer cases treated at Zhejiang Cancer Hospital from Aug 2014 to Aug 2015, including 60 SLN-positive and 60 SLN-negative cases. CK19 mRNA levels in peripheral blood samples were assessed using RT-PCR prior to tumor removal. During surgery, if SLNB tissue showed evidence of metastasis, axillary lymph node dissection (ALND) was performed. No ALND was performed if SLNB and nSLN tissues were both negative for metastasis. CK19 expression was higher in nSLN-positive patients than in nSLN-negative patients (p < 0.05). Logistic regression indicated that lymphatic vessel invasion and CK19 levels were predictive of nSLN status (p < 0.05). The area under the ROC curve for CK19 was 0.878 (p < 0.05). We conclude that high CK19 levels in peripheral blood may independently predict nSLN metastasis in breast cancer patients.

Effects of Herceptin on circulating tumor cells in HER2 positive early breast cancer.

The objective of this study was to determine the changes in peripheral blood circulating tumor cells in HER2-positive early breast cancer before and after Herceptin therapy, and to explore the effects of the HER2 gene and Herceptin on circulating tumor cells. CK19 mRNA expression in peripheral blood was evaluated by qRT-PCR as an index of circulating tumor cells in 15 cases of HER-2-positive breast cancer and 18 cases of HER2-negative breast cancer before, and after chemotherapy as well. Ten cases of HER2-positive breast cancer continued on Herceptin therapy for 3 months after chemotherapy, and their peripheral blood was again drawn and assayed for CK-19 mRNA expression. Preoperatively, all cases of HER2-positive cancer were positive for CK19 mRNA in peripheral blood, but 6 cases of HER2-negative breast cancer were positive (33.3%), where there was a substantial difference between the two groups. After 6 cycles of adjuvant chemotherapy, CK19 positive rates in cases of HER2-positive and -negative breast cancer reduced by 93.3 and 11.1%, respectively, with a significant difference still existing. After 3 months of Herceptin therapy, expression of CK19 mRNA declined considerably in 10 cases of HER2 positive breast cancer (113.66 ± 88.65 vs 63.35 ± 49.27, P = 0.025). HER-2 gene expression closely correlated with circulating tumor cells in peripheral blood of early breast cancer patients. Moreover, Herceptin, a monoclonal antibody for HER2, can reduce the number of circulating tumor cells, which can be an early predictive factor for Herceptin therapy effectiveness against breast cancer.

Detection of Circulating Cytokeratin-19 mRNA-Positive Cells in the Blood and the Mitotic Index of the Primary Tumor Have Independent Prognostic Value in Early Breast Cancer

BackgroundPrevious studies showed that molecular detection of CK-19 mRNA in peripheral blood and the mitotic index of primary tumors have prognostic value in early breast cancer. The aim of this study was to assess the association between these variables. Patients and MethodsThe primary tumors of 223 operable breast cancer patients (92 premenopausal and 131 postmenopausal) were evaluated for the MAI classified as either ≤ 5 per 10, 6 to 10 per 10 and > 10 per 10 or < 10 per 10 and ≥ 10 per 10 mitoses per high power field using a standardized protocol previously reported. Peripheral blood was also collected before and after the end of adjuvant chemotherapy for detection of CK-19 mRNA-positive cells using reverse transcription polymerase chain reaction previously described. ResultsAfter a median follow-up of 118 months, 75 patients (33.6%) experienced disease relapse and 56 (25.1%) died of breast cancer. MAI was strongly associated with disease-free survival (DFS) and overall survival (OS) (P < .001 for DFS and OS together). Detecting CK-19 mRNA-positive cells in the peripheral blood before but not after adjuvant chemotherapy was associated with marginally worse DFS (P = .055) and OS (P = .059). Cox regression analysis revealed that MAI and CK-19 mRNA-positive cell detection before adjuvant chemotherapy were independent variables associated with decreased DFS (P < .001 and P = .038, respectively) and OS (P < .001 and P = .029, respectively). There was no significant interaction between MAI and detection of CK-19 mRNA-positive cells. ConclusionMAI of the primary tumor and detection of CK-19 mRNA-positive cells in the blood before adjuvant chemotherapy in early breast cancer patients are 2 independent prognostic factors associated with clinical outcome.

Correlation between expression of CK20 mRNA in peripheral blood and clinicopathological features and prognosis in patients with colorectal cancer

Correlation between expression of CK20 mRNA in peripheral blood and clinicopathological features and prognosis in patients with colorectal cancer

P5-01-16: The Detection of Circulating CK19 mRNA-Positive Cells in the Blood and the Mitotic Index of the Primary Tumor Have Independent Prognostic Value in Early Breast Cancer.

Abstract Background: Previous studies have shown that the molecular detection of CK-19 mRNA in peripheral blood and the mitotic index of primary tumor have prognostic value in early breast cancer. The aim of the present study was to assess the association between these variables. Patients and Methods: The primary tumors of 223 operable breast cancer patients (92 premenopausal and 131 postmenopausal) were evaluated for the mitotic activity index (MAI) classified either as &amp;lt;5/10, 6–10/10 and &amp;gt;10/10 or &amp;lt;10/10 and &amp;gt;10/10 mitoses/hpf using a standardized protocol as previously reported (Baak JP et al, Breast Cancer Res Treat 2009, 115:241–254). Peripheral blood was also collected before the start and after the end of adjuvant chemotherapy for detection of CK-19 mRNA-positive cells by RT-PCR as previously described (Stathopoulou et al, Clin Cancer Res 2003, 9:5145–5151). Results: After a median follow-up of 118 months, 75 (33.6%) patients have experienced disease relapse and 56 (25.1%) have died of breast cancer. MAI was strongly associated with disease-free survival (DFS) and overall survival (OS) (p&amp;lt;0.001 for both DFS and OS). Detection of CK19 mRNA-positive cells in the peripheral blood before but not after adjuvant chemotherapy was marginally associated with worse DFS (p=0.055) and OS (p=0.059). There was no correlation between MAI and CK19 mRNA detection before chemotherapy. Cox regression analysis revealed that both MAI and CK19 mRNA-positive cell detection before adjuvant chemotherapy were independent variables associated with decreased DFS (p&amp;lt;0.001 and p=0.038, respectively) and OS (p&amp;lt;0.001 and p=0.029, respectively). The interaction test showed no significant association between MAI and detection of CK19 mRNA-positive cells. Conclusion: MAI of the primary tumor and detection CK-19 mRNA-positive cells in the blood before the start of adjuvant chemotherapy in women with early breast cancer are two independent prognostic factors associated with clinical outcome. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-16.

Expressions and clinical significances of MUC1-mRNA and CK20-mRNA in esophageal cancer

Objective To investigate the expressions and clinical significances of MUC1-mRNA and CK20-mRNA in peripheral blood of esophageal cancer patients.Methods MUC1-mRNA and CK20-mRNA were detected in 53 patients with esophageal cancer,10 patients with esophageal benign tumor and 20 healthy volunteers by RT-PCR technique.Results The expressions of MUC1-mRNA,CK20-mRNA and combining group were 35.85 % (19/53),49.06 % (26/53) and 62.26 % (36/53) in peripheral blood of 53 esophageal cancer patients.In control group,there was no expression of MUC1-mRNA and CK20-mRNA in peripheral blood of 10 patients with benign esophageal disease and 20 healthy volunteers.The positive rate increased by combining test(x2 =11.0228,P ＜0.05).Conclusion MUC1-mRNA and CK20-mRNA might be specific and sensitive markers to detect circulating tumor cells in peripheral blood and their expressions are closely related to TNM stages of the esophageal cancer patients.The combining test might be of high value of the diagnosis of micrometastasis. Key words: Esophageal neoplasms; Mucins; Keratins; Neoplasm metastasis

Serum vascular endothelial growth factor levels in patients with non-small cell lung cancer and its relations to the micrometastasis in peripheral blood

To examine the relationship between the levels of the serum vascular endothelial growth factor (VEGF) and the micrometastasis of peripheral blood in patients with non-small cell lung cancer (NSCLC), 108 NSCLC patients, including 40 patients with benign lung diseases and 30 healthy controls, were investigated. The serum VEGF levels were detected by ELISA and CK19 mRNA in peripheral blood by reverse transcriptase-polymerase chain reaction (RT-PCR). In NSCLC group, the serum VEGF levels and the positive rate of CK19 mRNA in peripheral blood were 479.8+/-268.5 pg/mL and 66.7%, which were significantly higher than those of the other two groups respectively (P<0.01), and both of them were increased significantly with the progression of clinical stage of the tumors (P<0.01). Serum VEGF levels as well as the positive rate of CK19 mRNA in different pathological types of lung cancer had no significant differences (P>0.05). Serum VEGF levels in the patients positive for CK19 mRNA was 561.7+/-325.6 pg/mL. It is significantly higher than that in the negative patients (P<0.01). There existed a significant correlation between serum VEGF levels and expression of CK19 mRNA in peripheral blood in NSCLC patients (P<0.001). The detection of serum VEGF levels and CK19 mRNA in peripheral blood is helpful in judging the condition and the prognosis of NSCLC patients, and serum VEGF levels and CK19 mRNA are independent of the pathological types of lung cancer. The micrometastasis in peripheral blood of NSCLC patients is significantly associated with serum VEGF levels.

Effects of dendritic cell vaccines on hematogenous micrometastasis of bladder cancer carrying for PBL-SCID mice

To study the effect of dendritic cells loaded with whole tumor antigen on hematogenous micrometastasis of bladder cancer model in hu-PBL-SCID mice. T24-3 cell subset was selected from human bladder transitional cell carcinoma T24 cell line by Boyden chamber system. The SCID mice intraperitoneally injected with 4 × 107 hu-PBL and subcutaneously injected with 3 × 106 T24-3 cells were named hu-PBL-T24-3-SCID model. Human IgG level in the blood plasma of mice was detected by ELISA, and human CD3+, CD4+, CD8+ T cells in blood and spleen cells of mice were detected by FCM analysis for human immune reconstruction study. Human CK20 mRNA expression in mice peripheral blood was detected by RT-PCR to investigate metastasis of tumor cells. The PBMCs were isolated from human peripheral blood, and were induced into DCs by co-culture with rhGM-CSF and rhIL-4 in vitro. The DC vaccines were produced by co-culturing with whole tumor antigen which was purified through freezing and melting T24-3 cell subset. After T24-3 cells injected into SCID mice for 5 weeks, the mice were treated with DC vaccines. All mice were initially treated at 5th week. The expression of CK20 mRNA in peripheral blood of DC vaccines treated mice was the lowest. There was 2 mice showing CK20 mRNA expression and 3 mice with metastasis tumor in PBS group. MMP-7 mRNA expression in tumor tissues of DC vaccines treated mice was statistically lower than that of PBS group (P < 0.01). DC vaccines have a good effect on hu-PBL-SCID mice bladder cancer model by reducing hematogenous micrometastasis.

Expression and clinical significance of CEA-mRNA, CK20-mRNA in peripheral blood of esophageal cancer patients

Objective To establish reverse transcriptase-polymerase chain reaction(RT-PCR) with primers specific for CEA gene and CK20 gene to detect circulating tumor cells in peripheral blood of esophageal cancer pa-tients,and try to find the relationship between the mRNA expression and micrometastasis. Methods The expressions of CEA,CK20 were analyzed by RT-PCR in 53 cases of esophageal tumor tissue and in peripheral blood,compared with 10 patients with benign esophageal disease and 20 healthy volunteers. Results The expressions of CEA-mRNA, CK20-mRNA were 96.23% (51/53), 100% ( 53/53 ) in 53 esophageal tumor tissue and were 52.83% (28/53), 49.06% (26/53) in peripheral blood of 53 esophageal cancer patients. In control group,there was only one expression of CEA-mRNA in peripheral blood of 10 patients with benign esophageal disease,as well as in 20 healthy volunteers. There was no expression of CK20-mRNA in peripheral blood of 10 patients with benign esophageal disease and 20 healthy volunteers. Conclusion CEA-mRNA, CK20-mRNA might be specific and sensitive markers to detect circu-lating tumor cells in peripheral blood and their expression was closely related to TNM stages of the esophageal cancer patients. Key words: Esophageal neoplasms; Neoplasm metastasis; Carcinoembryonic antigen; Keratin-20

Circulating tumor cells (CTCs) monitoring in HER2-negative early breast cancer: Implications for secondary anti-HER2 adjuvant treatment strategies

11502 Background: We have shown that the detection of peripheral blood CK19mRNA+ and HER2mRNA+ cells predicts poor outcome in early breast cancer and that trastuzumab can effectively eliminate these cells. Methods: We used real-time and nested RT-PCR for the detection of peripheral blood CK19mRNA+ and HER2mRNA+ cells, respectively, before and after adjuvant chemotherapy in 260 HER2-negative (HER2 0,1+ by immunohistochemistry) early breast cancer patients. Furthermore, we compared 3 prognosticators that classify a patient into poor vs good prognosis group: (A:CK19mRNA pre-chemotherapy alone; positive vs negative, B:CK19mRNA pre- and post-chemotherapy; at least once positive vs all negative, C:CK19mRNA and HER2mRNA pre- and post-chemotherapy; see Table) Results: Patient groups and their clinical outcome based on CTCs’ monitoring with CK19mRNA and HER2mRNA pre- and post- chemotherapy are depicted in Table. The median follow-up for patients that did not relapse was 52 months (range 10–106 months). The prognos...

Prognostic Value of the Molecular Detection of Circulating Tumor Cells Using a Multimarker Reverse Transcription-PCR Assay for Cytokeratin 19, Mammaglobin A, and HER2 in Early Breast Cancer

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. 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. 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+/HER2mRNA+ (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 disease-free 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. The detection of peripheral blood CK19mRNA+ and MGB1mRNA+ cells before adjuvant chemotherapy predicts poor DFS in women with early breast cancer.

Effect of adjuvant treatment on the circulating CK-19 mRNA-positive tumor cells in patients with early stage breast cancer

20012 Background: To evaluate the effect of adjuvant treatment on the peripheral blood CK-19 mRNA circulating tumor cells (CTCs) in patients with early-stage breast cancer. Methods: CTCs were detected by real-time RT-PCR assay, in 119 patients with estrogen or/and progesterone receptors-positive tumors at the following time points: before the initiation and after the completion of adjuvant chemotherapy and every 3 or 6 months during the period of adjuvant tamoxifen administration. Results: Adjuvant chemotherapy failed to “eliminate” the CTCs in 17 (47%) out of 36 patients with detectable cells in pre-chemotherapy samples; in addition, adjuvant tamoxifen failed to “eliminate” the CTCs in 8 (36.4%) of 22 patients displaying detectable cells in post-chemotherapy samples. The incidence of clinical relapse was significantly (p = 0.017) higher for patients with post-chemotherapy detectable CTCs as well as for patients with persistently detectable CTCs throughout the follow-up period compared with those who did not (65.3% vs 7.3%, respectively; p &lt; 0.001). In multivariate analysis, post-chemotherapy detection of CTCs and their persistence throughout the follow-up period were significantly associated (p = 0.001 and 0.003, respectively) with early relapse. Conclusions: The detection of CK-19 mRNA+ CTCs after adjuvant chemotherapy and during tamoxifen administration is an independent prognostic factor associated with unfavorable outcome. No significant financial relationships to disclose.

Effect of adjuvant tamoxifen and letrozol on the fate of peripheral blood CK-19 mRNA+ cells in patients with early breast cancer

754 Background: To evaluate the effect of adjuvant tamoxifen (TAM) and letrozol (LETR) on the occult peripheral blood (PB) CK-19 mRNA+ cells in correlation with the clinical outcome. Methods: Patients were treated with TAM (group TAM: n= 117 pts), LETR (LETR: n= 34pts) or sequential TAM (2 or 3 years) and LETR (3 or 2 years) (TAM/LETR: n= 46pts) after completion of adjuvant chemotherapy. CK-19 mRNA+ cells were detected by real-time RT-PCR. Results: Post-chemotherapy, CK-19 mRNA+ cells could be detected in 39 (33.3%), 13 (38.2%) and 14 (30.4%) patients treated with TAM, LETR, and TAM/LETR, respectively. Hormone treatment resulted in undetectable levels of PB CK-19 mRNA+ cells in 33 (84.5%) TAM and 11 (85%) LETR patients. In the TAM/LETR group, LETR resulted in undetectable levels of CK-19 mRNA+ cells in 12 (80%) of 15 patients with CK-19 mRNA+ cells after adjuvant TAM. The median duration of CK-19 mRNA-negative peripheral blood was higher in patients receiving LETR (LETR and TAM/LETR groups) than in those ...

Detection of CK20mRNA in peripheral blood of pancreatic cancer and its clinical significance

To detect the expression of CK20mRNA in peripheral blood of pancreatic cancer and evaluate its clinical significance. Expression of CK20mRNA in peripheral blood was detected by fluorogenic qualitative reverse transcription-polymerase chain reaction (RT-PCR) in 40 cases of pancreatic cancer at the night before operation, in 5 cases of benign pancreatic diseases, in 5 cases of healthy individuals. The relationships were investigated between CK20mRNA expression and the clinicopathological variables, and clinical follow-up outcome in those patients with pancreatic cancer having undergone radical resection. Of the 40 patients with pancreatic cancer, 23 (57.5%) cases were positive for CK20mRNA expression. CK20mRNA expression was significantly correlated with lymphatic metastasis (P = 0.008), histopathological grading (P = 0.009), and pathological stage (P = 0.021); there was no significant correlation between CK20mRNA expression and age, gender, tumor diameter, and depth of invasion. The cumulative metastasis rates of patients with CK20mRNA expression were higher than those of patients with no CK20mRNA expression within 6 mo (34.7% vs 5.9%, P = 0.043) or 12 mo (73.9% vs 35.3%, P = 0.02) after operation. CK20mRNA expression in peripheral blood of pancreatic cancer indicated poorer prognosis. The survival rate of patients with CK20mRNA expression was lower than that of patients with negative CK20mRNA expression (Log-Rank = 13.31, P = 0.0003). CK20mRNA is a sensitive and specific molecular marker for the detection of micrometastasis in peripheral blood of patients with pancreatic cancer. The CK20mRNA expression in peripheral blood is correlated with biological characteristic of pancreatic cancer. It can help to predict the prognosis of pancreatic cancer after operation, and to determine which patient will benefit from aggressive adjuvant therapies.

Quantitative RT-PCR luminometric hybridization assay with an RNA internal standard for cytokeratin-19 mRNA in peripheral blood of patients with breast cancer

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 μg 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.

Quantification of CK19 mRNA in peripheral blood (PB) and bone marrow (BM) from primary operable breast cancer (BC) patients pre- and postoperatively to investigate possible shedding of CK 19+ cells during the operation

Quantification of CK19 mRNA in peripheral blood (PB) and bone marrow (BM) from primary operable breast cancer (BC) patients pre- and postoperatively to investigate possible shedding of CK 19+ cells during the operation