Detection Of CK19 mRNA Research Articles

Abstract P3-03-14: Clinical utility of one-step nucleic acid amplification (OSNA) in axillary surgery after neoadjuvant chemotherapy (NAC)

Abstract Introduction NAC has been used for downsizing of the tumour in breast and axilla to allow more conservative surgery. In the NAC setting, intraoperative assessment of sentinel lymph node(s) (SLN) is still considered necessary1. Current awareness of the prognostic value for axillary nodal down-staging has renewed interest in analysis of SLN post-NAC. In this study we want to examine the clinical utility of OSNA (based on CK19 mRNA detection) as a method of intra-operative analysis of SLN to assist real-time decision-making for axillary surgery post-NAC in early breast cancer (EBC). Methods Retrospective analysis of prospective data on 399 consecutive patients with EBC who received NAC followed by breast surgery with SLN biopsy (408 axillae) and assessment by OSNA, from September 2011 to January 2018 at the Royal Marsden Hospital (UK). OSNA readouts from the Sysmex RD-100i were collected separate to and blinded from clinico-pathological data. A negative or benign pre-treatment axillary ultrasound scan or indeterminate ultrasound with negative or benign axillary cytology/histology prior to NAC was considered cN0. Univariate analysis (significance at p<0.05) was used to identify risk of recurrence. Patients had a median (mean) follow up of 32.5 (36) months. Results The median age at diagnosis was 49 years, median BMI 26, 41 EBC (10%) were screen-detected, 292 (72%) were grade 3 and the most frequent phenotype was receptor triple negative (n=132, 32%). Of 408 axillae, 248 (60%) were initially cN0, of which 113 (46%) had a pathological complete response (pCR) in the breast. SLN in 54 (22%) cN0 patients were positive on OSNA, of which only 6 (9%) had further involved axillary nodes all 6 of which were ER+ Her2-. The remaining 160 (40%) axillae were cN1 of which 87 (54%) had conversion to ypN0 including 55 (34%) with both ypT0ypN0. Axillary lymphadenectomy (AL) was performed in 79 (19%) patients overall, of which n=22 (28%) were cN0 and 57 (72%) were cN1. Of these, 30 (53%) of the cN1 and 6 of 22 (45%) of cN0 had at least 1 additional positive AL node. Overall 59 (14.4%) patients relapsed. A significantly worse rate of relapse was observed in cN1 compared to cN0 patients (37/159 (23.3%) versus 22/244 (9%), p<0.001). Combined pCR of both breast and axilla (in cN1, n=54) was associated with a significantly reduced risk of relapse and death (p<0.001) compared to those without pCR of either breast or axilla (n=62). Of the latter 18 (29%) relapsed (including 10 deaths). The mean of both the single highest node tumour load (and total nodal tumour load), as measured by CK19mRNA copies/ul on OSNA, were significantly higher at 90,000 (98,300) for those who relapsed versus 23,100 (25,100) for those without relapse (p=0.027). Conclusions The OSNA assay is an accurate tool for axillary SLN analysis in patients after NAC and was helpful in intra-operative axillary management. OSNA reduces the need for a second surgery for AL in 20% of breast cancer patients with a positive-SLN after NAC and might offer additional prognostic value. Reference 1. NCCN. National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology Breast Cancer.2016.Version 2.2016. Citation Format: Muscara F, Christaki G, Richardson C, O'Connell R, Padmanabhan P, Warwick J, Lee Y, Smith I, Nerurkar A, Osin P, Krupa K, Rusby J, Roche N, Gui G, MacNeil F, Barry P. Clinical utility of one-step nucleic acid amplification (OSNA) in axillary surgery after neoadjuvant chemotherapy (NAC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-03-14.

Detection of CK19 mRNA Using One-step Nucleic Acid Amplification (OSNA) in Prostate Cancer: Preliminary Results.

Background: Accurate histopathological evaluation of lymph nodes (LNs) is essential for reliable staging in prostate cancer. In routine practice, conventional techniques only examine parts of the LN. Molecular nodal staging methods are limited by their high costs and extensive time requirement. One-step nucleic acid amplification (OSNA) determines the metastatic status of the complete LN and allows for rapid intraoperative detection of LN metastases. OSNA has been proposed for diagnosis of LN metastases from breast cancer by quantifying the CK19 mRNA copy number. To provide basic data for OSNA development for prostate cancer, we conducted an investigation of CK19 and OSNA in prostate cancer specimens.Methods: OSNA is based on a short homogenization step and subsequent automated amplification of CK19 mRNA directly from the sample lysate, with results available in 30-40 min. A total of 20 prostate cancer specimens from consecutive patients with intermediate or high-risk prostate cancer (Gleason-Score ≥7) were investigated by both OSNA and conventional histopathology (H&E staining, CK19 immunohistochemistry). OSNA was performed on frozen samples using a ready-to-use amplification kit in an automated real-time detection system. Samples were defined as 'negative' or 'positive' according to mRNA copy number: >5000 copies/µl (++), 250-5000 copies/µl (+), and <250 copies/µl (-).Results: Histopathological analysis confirmed prostate cancer in all samples: Gleason score 7 (n=11), Gleason score 8 (n=2), and Gleason score 9 (n=6). Gleason score could not be given for one patient who previously underwent hormonal treatment. OSNA analysis detected CK19 expression in 100% of the specimens and high numbers of CK19 mRNA copies in all cases (9 samples ++; 11 samples +). Immunohistochemistry confirmed CK19 expression in 19 of 20 cases. In the immunohistochemistry CK19-negative patient, a Gleason score 9 prostate cancer was diagnosed.Conclusions: This is the first study using OSNA to detect CK19 expression in prostate cancer. Initial data indicate that this rapid method for molecular LN staging reliably identifies CK19 mRNA in prostate cancer. These results suggest that the OSNA assay may be suitable to improve (intraoperative) LN staging in prostate cancer. For further verification, OSNA analysis of LN specimens from prostate cancer patients is required.

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.

A Comparison of Three Methods for the Detection of Circulating Tumor Cells in Patients with Early and Metastatic Breast Cancer

Background: We directly compared CTC detection rates and prognostic significance, using three different methods in patients with breast cancer (BC). Methods: Early (n=200) and metastatic (n=164) patients were evaluated before initiating adjuvant or first-line chemotherapy, using the CellSearch^TM System, an RT-qPCR for CK-19 mRNA detection and by double immunofluorescence (IF) microscopy using A45-B/B3 and CD45 antibodies. Results: Using the CellSearch^TM System, 37% and 16.5% of early BC patients were CTC-positive (at ≥1 and ≥2 CTCs/23 ml of blood), 18.0% by RT-qPCR and 16.9% by IF; no agreement was observed between methods. By the CellSearch^TM 34.8% and 53.7% (at≥ 5 and ≥ 2 CTCs/7.5 ml) of metastatic patients were CTC-positive, 37.8% by RT-qPCR and 28.5% by IF. A significant agreement existed only between the CellSearch^TM and RT-qPCR. In 60.8% of cases, differential EpCAM and CK-19 expression on CTCs by IF could explain the discrepancies between the CellSearch^TM and RT-qPCR. CTC-positivity by either method was associated with decreased overall survival in metastatic patients. Conclusion: A significant concordance was observed between the CellSearch^TM and RT-qPCR in metastatic but not in early BC. Discordant results could be explained in part by CTC heterogeneity. CTC detection by all methods evaluated had prognostic relevance in metastatic patients.

Label-Free Isolation and mRNA Detection of Circulating Tumor Cells from Patients with Metastatic Lung Cancer for Disease Diagnosis and Monitoring Therapeutic Efficacy.

We develop an inertial-based microfluidic cell sorter combined with an integrated membrane filter, allowing for size-based, label-free, and high-efficiency separation and enrichment of circulating tumor cells (CTCs) in whole blood. The cell sorter is composed of a double spiral microchannel that hydrodynamically focuses and separates large CTCs from small blood cells. The focused CTCs with the equilibrium position around the midline of microchannel are further captured and enriched by a membrane filter (pore size of 8 μm) attached at the middle outlet. This integrated microfluidic device can process 1 mL of whole blood containing spiked tumor cells (A549, human lung adenocarcinoma epithelial cell line) within 15 min, with the capture efficiency of 74.4% at the concentration as low as tens of A549 cells per mL of whole blood. This microfluidic cell sorter is further adopted for isolation of CTCs from peripheral blood samples of patients with metastatic lung cancer. The immunostaining and CK-19 mRNA detection are applied for identification of captured CTCs, showing that our method can detect 90% of metastatic lung cancer patients before therapy, whereas the commercially used system can only detect 40% of the same patients. We also use the expression of CK-19 mRNA from captured CTCs as an indicator for monitoring the therapeutic efficiency, which correlates well with X-ray computed tomography (CT) assessment of the disease.

Assessment of suitability of the one step nucleic acid amplification (OSNA) assay as an intraoperative procedure for detection of metastasis in sentinel lymph nodes of breast cancer

AimWe aimed to assess the one step nucleic acid amplification (OSNA) assay as an intraoperative method in comparison with frozen sections (FS) for detection of metastasis in sentinel lymph nodes...

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.

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.

Assessment of the effect of surgery on the kinetics of circulating tumour cells in patients with operable breast cancer based on cytokeratin-19 mRNA detection

Aims The aim of this study was to evaluate the effect of surgery on the kinetics of CTCs in breast cancer patients. Methods The detection of CK-19 mRNA-positive CTCs in the blood by RT-PCR was analysed in 104 stage 0-IIIA patients at 4 time-points: prior to surgery, upon completion, 24 h after surgery and 15 days after surgery. Furthermore, a late sample was assessed prior to initiation of adjuvant chemotherapy in a subgroup of 53 patients. As negative controls, peripheral blood was obtained from 50 female patients undergoing excision of benign breast lesions and from 11 female patients receiving surgery for early-stage colorectal cancer. Results A significant percentage of blood samples from breast cancer patients (14.4%) were negative for CK-19 preoperatively but turned transiently positive early postoperatively. However, no significant difference in CK-19 mRNA detection was noted among the first 4 examined time-points. There was no significant correlation between CK-19 mRNA-positive cells and classic prognostic factors. A significant increase in CK-19 mRNA-positivity (32.1%) was observed in a late sample of the subgroup of 53 patients before adjuvant chemotherapy after a median of 54 days, postoperatively. Conclusions Surgery may result in CTC detection in a small proportion of early breast cancer patients. There is no clear correlation to indicate which patients are expected to have detectable CTCs. Although CTCs are detected in a small proportion of patients during the perioperative period, the detection rate may increase over time and with longer follow-up.

634 Patient-derived breast cancer xenografts: Molecular characteristics and growth properties

The aim of this study was to evaluate the effect of surgery on the kinetics of CTCs in breast cancer patients.The detection of CK-19 mRNA-positive CTCs in the blood by RT-PCR was analysed in 104 stage 0-IIIA patients at 4 time-points: prior to surgery, upon completion, 24 h after surgery and 15 days after surgery. Furthermore, a late sample was assessed prior to initiation of adjuvant chemotherapy in a subgroup of 53 patients. As negative controls, peripheral blood was obtained from 50 female patients undergoing excision of benign breast lesions and from 11 female patients receiving surgery for early-stage colorectal cancer.A significant percentage of blood samples from breast cancer patients (14.4%) were negative for CK-19 preoperatively but turned transiently positive early postoperatively. However, no significant difference in CK-19 mRNA detection was noted among the first 4 examined time-points. There was no significant correlation between CK-19 mRNA-positive cells and classic prognostic factors. A significant increase in CK-19 mRNA-positivity (32.1%) was observed in a late sample of the subgroup of 53 patients before adjuvant chemotherapy after a median of 54 days, postoperatively.Surgery may result in CTC detection in a small proportion of early breast cancer patients. There is no clear correlation to indicate which patients are expected to have detectable CTCs. Although CTCs are detected in a small proportion of patients during the perioperative period, the detection rate may increase over time and with longer follow-up.

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.

Molecular Detection and Prognostic Value of Circulating Cytokeratin-19 Messenger RNA–Positive and HER2 Messenger RNA–Positive Cells in the Peripheral Blood of Women with Early-Stage Breast Cancer

Purpose The aim of this study was to evaluate the clinical relevance of the simultaneous detection of cytokeratin (CK)–19 messenger RNA (mRNA)– and HER2 mRNA–positive cells in peripheral blood of women with early-stage breast cancer. Patients and Methods CK-19 mRNA– and HER2 mRNA–positive cells were detected using a real-time and a nested reverse-transcriptase polymerase chain reaction assay, respectively, in a cohort of 185 women with early-stage breast cancer before the initiation of any adjuvant systemic treatment. Detection of CK-19 mRNA– and HER2 mRNA–positive cells in the peripheral blood was correlated with clinical outcome. Results Overall, 63 of the 185 patients (34%) had detectable CK-19 mRNA–positive cells, and 33 (52.3%) also had detectable HER2 mRNA–positive cells. Patients with CK-19/HER2 mRNA–negative cells showed a trend toward longer disease-free survival (DFS) compared with patients with CK-19 mRNA–positive/HER2 mRNA–negative cells ( P = .054) and had longer DFS than patients with CK-19/HER2 mRNA–positive cells ( P < .001). Similarly, overall survival (OS) was higher in patients with CK-19/HER2 mRNA–negative cells compared with patients with CK-19 mRNA–positive/HER2 mRNA–negative cells ( P = .039) or CK-19/HER2 mRNA–positive cells ( P < .001). Patients with CK-19/HER2 mRNA–positive cells had shorter DFS but not OS compared with patients with CK-19 mRNA–positive/HER2 mRNA–negative cells. In multivariate analysis, the simultaneous detection of CK-19 mRNA– and HER2 mRNA–positive cells was independently associated with early relapse. Conclusion The simultaneous detection of CK-19 mRNA– and HER2 mRNA–positive cells in peripheral blood predicts poor clinical outcome for women with early-stage breast cancer.

Clinical relevance of circulating CK-19 mRNA-positive cells detected during the adjuvant tamoxifen treatment in patients with early breast cancer

The purpose of this study was to evaluate the effect of adjuvant treatment with tamoxifen on the CK-19 mRNA+ cells in patients with early-stage breast cancer. CK-19 mRNA+ cells were prospectively and longitudinally detected using a specific real-time PCR assay for CK-19 mRNA in 119 patients with estrogen and/or progesterone receptor-positive tumors during the period of tamoxifen administration. Twenty-two (18.5%) patients had detectable CK-19 mRNA+ cells after the completion of adjuvant chemotherapy and in 15 (68.2%) of them adjuvant tamoxifen could not eliminate these cells (persistently positive). In 68 (57.1%) patients, no CK-19 mRNA+ cells could be detected throughout the follow-up period (persistently negative). Seven (46.7%) of the 15 persistently positive and six (8.8%) of the 68 persistently negative patients developed disease recurrence (P = 0.00026). Persistency of CK-19 mRNA+ cells was associated with a significantly lower median disease-free interval (P = 0.0001) and overall survival (P = 0.0005). Multivariate analysis revealed that the detection of CK-19 mRNA+ cells during the administration of tamoxifen was associated with an increased risk of relapse [hazard ratio (HR) = 22.318, P = 0.00006] and death (HR = 13.954, P < 0.00001). The detection of CK-19 mRNA+ cells throughout the period of adjuvant tamoxifen treatment is an independent poor prognostic factor in patients with early breast cancer.

Clinical relevance of circulating CK-19 mRNA-positive cells detected during the period of adjuvant tamoxifen treatment in patients with early breast cancer

10534 Background: To evaluate the effect of adjuvant treatment with tamoxifen on the CK-19 mRNA+ circulating tumor cells (CTCs) in patients with early-stage breast cancer. Methods: CTCs were prospectively and longitudinally detected using a specific real-time PCR for CK-19 mRNA in 119 patients with estrogen and/or progesterone receptor-positive tumors during the period of tamoxifen administration. Results: Twenty-two (18.5%) patients had detectable CK-19 mRNA+ CTCs after the completion of adjuvant chemotherapy and in 15 (68.2%) of them adjuvant tamoxifen could not eliminate these cells (persistently positive). In 68 (57.1%) patients no CK-19 mRNA+ CTCs could be detected throughout the follow up period (persistently negative). Seven (46.7%) of the 15 persistently positive and six (8.8%) of the 68 persistently negative patients developed disease recurrence (p=0.00026). Persistence of CK-19 mRNA+ CTCs was associated with a significantly shorter median disease-free interval (p=0.0001) and overall survival (0.0005). Multivariate analysis revealed that the detection of CK-19 mRNA+ CTCs during the administration of tamoxifen was associated with an increased risk of relapse (HR=22.318, p=0.00006) and death (HR=13.954, p&lt; 0.00001). Conclusions: The detection of CK-19 mRNA+ CTCs throughout the period of adjuvant tamoxifen treatment is an independent poor prognostic factor in patients with early breast cancer. No significant financial relationships to disclose.

One Step Nucleic Acid Amplification (OSNA) for intra-operative detection of lymph node metastases in breast cancer patients

21053 Background: Sentinel lymph node metastasis in breast cancer patients has conventionally been determined by intra- operative histopathological examination of frozen sections followed by definitive post-operative examination of permanent sections. The purpose of this study is to evaluate a more efficient method for intra-operative detection of lymph node metastasis in a clinical setting. Therefore, a new rapid method of detection of CK19 mRNA by One Step Nucleic Acid Amplification (OSNA) and Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) was tested against standard histological techniques. Methods: The clinical study was conducted at two facilities using 188 lymph nodes (46 patients). Lymph nodes from axillary dissected patients with breast cancer were divided into four slices. Alternate slices were used for the new method of CK19 mRNA detection (OSNA) and histopathological examination by hematoxylin &amp; eosin and cytokeratin staining. For the analysis of concordance between both methods, the slices for histopathology were cut in 4 μm sections. For the OSNA method the other 2 slices were homogenised in a short sample preparation step. Afterwards CK19 mRNA was directly amplified from the tissue lysate within 16 minutes. Results: In the clinical study, an overall concordance rate between the OSNA assay and histopathology was 92%. Several discordant results are explainable with the expected uneven distribution of metastases within the lymph nodes. Conclusions: The study indicates that OSNA is a reliable and fast diagnostic tool for the detection of lymph node metastases in breast cancer patients. This method could be beneficial to avoid diagnostic delay for the patient or second surgery based on a post-operatively diagnosed positive lymph node. No significant financial relationships to disclose.

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.

A New Nested Primer Pair Improves the Specificity of Ck-19 Mrna Detection by Rt-Pcr in Occult Breast Cancer Cells

Reverse transcription polymerase chain reaction (RT-PCR) of cytokeratin-19 (CK-19) has been widely used to detect small numbers of circulating malignant epithelial cells in the bone marrow or the peripheral blood of patients with breast cancer. However, a high percentage of false positive results has been recorded and conflicting reports question the clinical relevance of this technical approach. We demonstrate that the use of a new nested primer pair for CK-19 RT-PCR avoids false positive results without affecting the sensitivity of the assay. Our experiments were carried out using MCF-7 cells alone or mixed with peripheral blood mononuclear cells (PBMNC) of healthy donors. The results were also validated in a large series of healthy donors and in a preliminary study on a limited number of patients with breast cancer, thus suggesting that our assay is feasible for application in the clinical evaluation of occult malignant epithelial cells.

Association of CK19 mRNA detection of occult cancer cells in mediastinal lymph nodes in non-small cell lung carcinoma and high risk of early recurrence

This study was designed to screen occult cancer cells by CK19 mRNA detection using reverse transcriptase-polymerase chain reaction (RT-PCR) in mediastinal lymph nodes stations (MLNS) in non-small cell lung carcinoma (NSCLC). In 49 NSCLC patients free of mediastinal adenopathy on computed tomograph, 254 MLNS were evaluated by histopathology, immunohistochemistry (IHC) and RT-PCR. Of 225 non-tumoral MLNS on histopathology, 32 (14.2%) were positive by RT-PCR. IHC did not provide significant additional results. Seventeen patients were without mediastinal tumoral extension on histopathology and RT-PCR (Group 1), 16 were upgraded by RT-PCR (Group 2) and 16 pN2 on histopathology (Group 3). The two-year cancer-related death survival in Groups 1 (100%) and 2 (64.5%) was significantly different ( P = 0.04). The relative risk of recurrence in Group 2 compared with Group 1, evaluated by the Cox model multivariate analysis, was 5.61 ( P = 0.02). In conclusion, CK19 mRNA detected by RT-PCR in MLNS was significantly associated with an increased risk of rapid recurrence.

Detection of CK19 and CEA mRNA expression for the diagnosis of peripheral blood micrometastases in patients with non-small cell lung cancer

To explore the sensitivity and clinical significance of detection of CK19 mRNA and CEA mRNA expression for the diagnosis of micrometastases in the peripheral blood of patients with non small cell lung cancer (NSCLC). Nested reverse transcription-polymerase chain reaction (RT-PCR) was used to detect CK19 mRNA and CEA mRNA in the peripheral blood of 35 patients with NSCLC. Twenty patients with pulmonary benign lesions and 20 normal healthy volunteers were served as controls. The positive rates of CK19 mRNA and CEA mRNA for NSCLC patients were 40% (14/35) and 57% (20/35) respectively, 14 cases (40%) were positive for both mRNA, and 15 cases (43%) were both negative for both mRNA. No blood samples from patients with pulmonary benign lesions was positive for CEA mRNA, but 2 samples (10%) were positive for CK19 mRNA. Of these 20 samples from normal volunteers, none was positive for both CK19 mRNA and CEA mRNA. The distant metastasis rate of patients with positive CK-19 mRNA , positive CEA mRNA and both negative in the peripheral blood were 50%, 56% and 27% respectively. Both CK-19 mRNA and CEA mRNA are good markers to detect micrometastasis in the peripheral blood of patients with NSCLC. Detection of CK19 and CEA mRNA expression might be helpful to predict the prognosis of patients with lung cancer.

Clinical significance of molecular diagnosis of micrometastasis in lymph nodes, peripheral blood and bone marrow in the surgical treatment of patients with non-small cell lung cancer

To explore the clinical significance of detecting micrometastasis in lymph node, peripheral blood and bone marrow from patients with lung cancer for selection of surgical indication and the correlation among them. The CK19 mRNA expression was detected in lymph nodes, peripheral blood and bone marrow from 29 lung cancer patients and 11 benign pulmonary lesion patients by nested RY-PCR. The sensitivity of RT-PCR technique was 10(-6) in this study. The positive rate of CK19 mRNA were 54. 5% ( 55/ 101) , 34. 5% ( 10/ 29) and 20. 7% ( 6/ 29) in lymph node, peripheral blood and bone marrow from lung cancer patients respectively, and a highly positive correlation existed among the three groups ( P < 0. 05) . The micrometastasis in peripheral blood and bone marrow were closely related to the pathological classification, cell differentiation of the cancer and P-TNM stage of the disease ( P < 0. 05 or P < 0. 01) . RT-PCR technique is a specific and sensitive method to detect micrometastasis in patients with lung cancer. The detection of CK19 mRNA might have important clinical significance in molecular diagnosis and surgical indication in patients with lung cancer.