Cytokeratin-19 mRNA-positive Cells Research Articles

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.

CK-19 mRNA-positive cells in peripheral blood predict treatment efficacy and survival in small-cell lung cancer patients

Small-cell lung caner (SCLC) is the most aggressive form of lung cancer. The aim of this study was to investigate whether the presence of cytokeratin-19 (CK-19) mRNA-positive circulating tumor cells (CTCs) predicts treatment response, progression-free survival (PFS), and overall survival (OS) in SCLC patients who received standard therapy. Fifty-five SCLC patients were enrolled in this single-center prospective study. CK-19 mRNA-positive CTCs in blood samples were detected using real-time quantitative-PCR assay before the initiation of chemotherapy (B0) and after one chemotherapy cycle (B1) and three chemotherapy cycles (B3). The association with known prognostic factors and the effect of CK-19 mRNA-positive CTCs on patients' prognosis were analyzed. Patients with positivity for CK-19 mRNA-positive CTCs at B0, B1, and B3 time points had shorter PFS and OS compared with patients without (P=0.014 and P=0.01, respectively, at B0; P=0.008 and P=0.002, respectively, at B1; P=0.003 and P=0.001, respectively, at B3). Conversion of initial positivity for CK-19 mRNA-positive CTCs to negativity at B1 and B3 time points was associated with longer PFS and OS compared with patients with persistent positivity at three time points (P=0.008 and P=0.010, respectively). Multivariate analysis demonstrated that the presence of CK-19 mRNA-positive CTCs at B0, B1, and B3 time points remained strong predictors of PFS and OS after adjustment for clinically significant factors. In conclusion, detection of CK-19 mRNA-positive CTCs before and during chemotherapy is an accurate indication of subsequent disease progression and mortality for SCLC patients.

1092 Detection of cytokeratin-19 mRNA-positive cells in peripheral blood and bone marrow of patients with operable breast cancer

1092 Detection of cytokeratin-19 mRNA-positive cells in peripheral blood and bone marrow of patients with operable breast cancer

Detection of cytokeratin-19 mRNA-positive cells in the peripheral blood and bone marrow of patients with operable breast cancer

Background:To compare detection rates and evaluate the clinical relevance of cytokeratin-19 (CK-19) mRNA-positive cells in the peripheral blood (circulating tumour cells, CTCs) and bone marrow (disseminated tumour cells; DTCs) of patients with early breast cancer.Methods:Paired samples of peripheral blood and bone marrow were obtained from 165 patients with stage I–II breast cancer before the initiation of adjuvant chemotherapy. In 84 patients, paired blood and bone marrow samples were also available after chemotherapy. The detection of CK-19 mRNA-positive CTCs and DTCs was assessed by real-time PCR.Results:CK-19 mRNA-positive CTCs and DTCs were detected in 55.2 and 57.6% of patients before chemotherapy, respectively. After chemotherapy, CTCs and DTCs were identified in 44 (52.4%) and 43 (51.2%) of the 84 patients, respectively. There was a 93.9% (McNemar; P=0.344) and 72.6% (McNemar; P=0.999) concordance between blood and bone marrow samples before and after chemotherapy, respectively. The detection of CK-19 mRNA-positive CTCs or DTCs before chemotherapy was associated with decreased overall survival (P=0.024 and P=0.015, respectively). In addition, their simultaneous detection was also associated with an increased incidence of disease-related death and decreased overall survival (P=0.016).Conclusions:The detection of CK-19 mRNA-positive CTCs using reverse transcription-PCR (RT–PCR) both before and after chemotherapy is correlated with the detection of CK-19 mRNA-positive DTCs in patients with early-stage breast cancer. The determination of the CTC status by RT–PCR conveys clinically relevant information that is not inferior to DTC status and, owing to the ease of sampling, warrants further evaluation as a tool for monitoring minimal residual disease.

Predictive and Prognostic Value of Peripheral Blood Cytokeratin-19 mRNA-Positive Cells Detected by Real-Time Polymerase Chain Reaction in Node-Negative Breast Cancer Patients

To evaluate the predictive and prognostic value of peripheral blood cytokeratin-19 (CK-19) mRNA-positive cells in axillary lymph node-negative breast cancer patients. Peripheral blood was obtained from 167 node-negative breast cancer patients before the initiation of any systemic adjuvant therapy, and was analyzed for the presence of CK-19 mRNA-positive cells using a real time polymerase chain reaction assay. The association with known prognostic factors and the effect of CK-19 mRNA-positive cells on patients' prognosis was investigated. CK-19 mRNA-positive cells were detected in the blood of 36 (21.6%) of the 167 patients. There was no correlation between the detection of CK-19 mRNA-positive cells in the peripheral blood and the various known pathologic and clinical prognostic factors; only overexpression of HER2 receptor (score 2+/3+) on the primary tumor was associated with a higher incidence of CK-19 mRNA-positive cell detection (P = .033). Multivariate analysis revealed that detection of peripheral blood CK-19 mRNA-positive cells was associated with early clinical relapse (P < .00001) and disease-related death (P = .008). Detection of peripheral-blood CK-19 mRNA-positive cells is an independent predictive and prognostic factor for reduced disease-free interval and overall survival, respectively, in node-negative breast cancer patients.

Isolation by size of epithelial tumor cells in peripheral blood of patients with breast cancer: correlation with real-time reverse transcriptase–polymerase chain reaction results and feasibility of molecular analysis by laser microdissection

The aim of this study is the counting and the immunomorphological and molecular characterization of circulating tumor cells (CTCs) by the isolation by size of epithelial tumor cells (ISET) method in the peripheral blood of patients with breast cancer. An evaluation of the method's ability to reveal the presence of occult carcinoma cells in blood of a patient with breast cancer was performed and the results compared with those obtained by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for the evaluation of cytokeratin-19 (CK-19) mRNA expression. The feasibility of molecular analysis of CTCs after laser microdissection of filters used in ISET was illustrated, referring to HER-2 amplification. Blood samples drawn from 44 patients with breast cancer were preoperatively analyzed by ISET. From the same samples, total RNA was extracted and submitted to quantitative real-time RT-PCR for the detection of CK-19 mRNA-positive cells using TaqMan technology. HER-2 amplification was measured by real-time RT-PCR on DNA extracted from cells recovered by laser microdissection from 7 selected ISET-positive filters. Of 44 samples, 12 (27%) showed the presence of epithelial cells on the filter (mean +/- SE: 8.5 +/- 2.4 cells per milliliter of blood). A statistically significant agreement (P = .001) was observed between real-time RT-PCR results and those obtained by ISET. With regard to HER-2 amplification, a good correspondence was found between the results obtained from microdissected CTCs and those obtained using DNA extracted from the primary tumor (R = 0.918; P < .01), as well as the immunohistochemistry results. The ISET method allows for the collection of breast carcinoma cells by filtration despite their smaller dimension relative to other carcinoma cell types. The sensitivity and specificity of the method is comparable with those obtained using the quantitative real-time RT-PCR assay for the evaluation of CK-19 mRNA expression. Moreover, the laser microdissection technique allows for the recovery of nucleic acids for further molecular analysis and CTC characterization.

Detection of CK-19 mRNA-positive cells in the peripheral blood of breast cancer patients with histologically and immunohistochemically negative axillary lymph nodes

BackgroundTo investigate the incidence of direct hematogenous spread of cancer cells in patients with early-stage breast cancer by studying the presence of occult tumor cytokeratin-19 (CK-19) mRNA+ cells in the peripheral blood in relation to the status of sentinel (SLNs) and (ALNs) axillary lymph nodes. Patients and methodsSLNs and ALNs from 111 patients with operable stage I–II breast adenocarcinoma were evaluated for the presence of tumor cells by hematoxylin–eosin (H&E) staining and, if negative, by immunohistochemistry (IHC) using an anti-CK-19 antibody. Peripheral blood was also analyzed for the presence of CK-19 mRNA+ cells by nested RT–PCR, before the initiation of adjuvant treatment and in CK-19 mRNA+ patients following the completion of adjuvant chemotherapy and hormonal treatment. ResultsAfter both H&E staining and IHC analysis, 29 (26%) patients were ALN negative (N0). In 78 (70%) patients H&E staining and in four (3.6%) IHC analysis revealed tumors cells, and these patients were considered as ALN positive (N+). Peripheral blood CK-19 mRNA+ cells were detected in nine (31%) out of 29 N0 and in 31 (38%) out of 82 N+patients (P=0.5) before any adjuvant treatment. Adjuvant chemotherapy and hormone treatment resulted in the disappearance of the CK-19 mRNA+ cells in all N0 patients and in 15 out of 31 N+patients. After a median follow-up of 40 months, all the N0 CK-19 mRNA+ patients were relapse-free whereas four (13%) N+CK-19 mRNA+ patients had relapsed. ConclusionsDirect hematogenous dissemination of occult tumor cells may occur in a substantial proportion of patients with early-stage breast cancer. The prognostic implication of the detection of these cells requires long follow-up periods and further studies.

Trastuzumab Administration Can Effectively Target Chemotherapy-Resistant Cytokeratin-19 Messenger RNA–Positive Tumor Cells in the Peripheral Blood and Bone Marrow of Patients With Breast Cancer

The detection of disseminated occult breast cancer cells in peripheral blood and bone marrow is associated with poor prognosis. Since a high proportion of these cells express the HER-2 receptor, we evaluated the effectiveness of the anti-HER-2 antibody trastuzumab (Herceptin) administration to eliminate them. Thirty patients with prior chemotherapy exposure were recruited to the study on the basis of having detectable cytokeratin-19 (CK-19) mRNA transcripts by nested reverse transcription (RT)-PCR in the peripheral blood and/or bone marrow. There were 13 patients with stage I, II, or III breast cancer and 17 with stage IV disease. They were treated in two cohorts with either 4 to 8 weekly infusions of trastuzumab at 2 mg/kg (4 mg/kg loading dose; 20 patients) or 2 to 3 infusions every 3 weeks at 6 mg/kg (8 mg/kg loading dose; 10 patients). All of the patients' samples were also analyzed for HER-2 by nested RT-PCR, but detectable HER-2 messenger RNA (mRNA) was not required for inclusion in the study. After trastuzumab infusions, patients were closely monitored by nested RT-PCR and real-time RT-PCR for the detection of CK-19 mRNA-positive cells. Before trastuzumab infusions, CK-19 mRNA-positive cells were detected in the peripheral blood (n = 10), bone marrow (n = 14), or both (n = 6). In 25 of 30 patients (83%), HER-2 mRNA expression was detected by nested RT-PCR in the pretrastuzumab CK-19-positive sample. After trastuzumab infusions, overall, 28 of 30 (93%) patients became CK-19 mRNA negative by nested RT-PCR and 20 of 30 (67%) by real-time RT-PCR. After a median follow-up of 6 months (range 2 to 22+), the median duration of CK-19 mRNA negativity by nested RT-PCR was 9, 12, and 6 months for stage I/II, III, and IV disease, respectively. Therapy-resistant CK-19 mRNA-positive cells in the peripheral blood and bone marrow can be effectively targeted by trastuzumab administration. Further studies are needed to evaluate the prognostic significance of the disappearance of these cells.

Molecular detection of cytokeratin-19-positive cells in the peripheral blood of patients with operable breast cancer: evaluation of their prognostic significance.

To evaluate the prognostic significance of molecular detection of cytokeratin 19 (CK-19) mRNA-positive cells by nested reverse transcriptase polymerase chain reaction (RT-PCR) in the peripheral blood of women with stages I and II breast cancer before adjuvant chemotherapy. The sensitivity and specificity of CK-19 mRNA detection by nested RT-PCR were investigated using MCF-7 and ARH-77 cells and blood from healthy women and patients with hematologic malignancies, metastatic colorectal cancer, and early and metastatic breast cancer. Peripheral blood from 148 patients with operable breast cancer, obtained before initiation of any adjuvant therapy, was tested for the presence of CK-19 mRNA-positive cells. The nested RT-PCR assay for CK-19 mRNA detected one MCF-7 tumor cell in 10(6) normal peripheral blood mononuclear cells in four of five experiments; no signal was detected with the CK-19-negative ARH-77 cells. CK-19 mRNA was detected in the peripheral blood of 3.7% of healthy blood donors, 14.3% of patients with hematologic malignancies, and 3.2% of patients with metastatic colorectal cancer. Detection rates for CK-19 mRNA-positive cells in the bone marrow/blood of patients with early or metastatic breast cancer were 63%/30% and 74%/52%, respectively. For stages I and II breast cancer, detection of CK-19-positive cells in the peripheral blood before adjuvant therapy was associated with reduced disease-free interval (P =.0007) and overall survival (P =.01). In multivariate analysis, detection of peripheral-blood CK-19-positive cells was an independent prognostic factor for disease relapse and death. Molecular detection of CK-19 mRNA-positive cells by RT-PCR in the peripheral blood of patients with stages I and II breast cancer before initiation of adjuvant therapy has independent prognostic value as a marker of poor clinical outcome.