Cytokeratin 19 mRNA Expression Research Articles

Advancing canine mammary tumor diagnostics: Unraveling the diagnostic potential of Cytokeratin 19 through droplet digital PCR analysis

Cytokeratin 19 (CK19) is a complex intracytoplasmic cytoskeletal protein primarily localized in the ducts of the mammary gland and skin epithelial cells. In humans, the expression of CK19 gene within circulating tumor cells (CTCs) extracted from blood samples of breast cancer patients reflects tumor cell activity, offering valuable insights for predicting early metastatic relapse or monitoring treatment effectiveness. However, knowledge of serum tumor markers is limited in veterinary oncology. Recently, droplet digital PCR (ddPCR), has been employed to explore rare target genes due to its heightened sensitivity and accuracy as a novel molecular diagnostic tool. The objectives of this study were to investigate the expression of the CK19 mRNA in CTCs, non-neoplastic mammary tissues, and both benign and malignant canine mammary tumors (CMTs) through ddPCR analysis. In Study I, we optimized the discard volume for blood samples to reduce CK19 contamination from skin epithelial cells post-venipuncture. The results revealed that discarding the initial 3 mL of blood was adequate and effective in eliminating CK19 mRNA contamination. In Study II, after the removal of the initial 3 mL of blood, we investigated CK19 mRNA-positive CTCs in the peripheral blood of normal healthy dogs, including those with benign and malignant CMTs. Intriguingly, CK19 mRNA was undetectable in all blood samples. The expression of CK19 mRNA in mammary tissues was investigated in Study III. The copy number (CN) ratios of the CK19 gene in non-neoplastic mammary tissues (14.77 ± 14.65) were significantly higher (P < 0.05) than those in benign (4.23 ± 3.35) and malignant groups (6.56 ± 5.64). Notably, no difference was observed between the benign and malignant groups. In conclusion, CK19 mRNA appeared unlikely to be a suitable candidate as a biomarker in the peripheral blood of CMTs, while the CN ratio in mammary tissues could serve as a potential discriminator between non-neoplastic and CMT groups, complementing the gold standard of histopathological examination.

Longitudinal Analysis of Circulating Tumor Cells in Colorectal Cancer Patients by a Cytological and Molecular Approach: Feasibility and Clinical Application.

IntroductionLiquid biopsies allowing for individualized risk stratification of cancer patients have become of high significance in individualized cancer diagnostics and treatment. The detection of circulating tumor cells (CTC) has proven to be highly relevant in risk prediction, e.g., in colorectal cancer (CRC) patients. In this study, we investigate the clinical relevance of longitudinal CTC detection over a course of follow-up after surgical resection of the tumor and correlate these findings with clinico-pathological characteristics.MethodsIn total, 49 patients with histologically proven colorectal carcinoma were recruited for this prospective study. Blood samples were analyzed for CTC presence by two methods: first by marker-dependent immunofluorescence staining combined with automated microscopy with the NYONE® cell imager and additionally, indirectly, by semi-quantitative Cytokeratin-20 (CK20) RT-qPCR. CTC quantification data were compared and correlated with the clinico-pathological parameters.ResultsDetection of CTC over a post-operative time course was feasible with both applied methods. In patients who were pre-operatively negative for CTCs with the NYONE® method or below the cut-off for relative CK20 mRNA expression after analysis by PCR, a statistically significant rise in the immediate post-operative CTC detection could be demonstrated. Further, in the cohort analyzed by PCR, we detected a lower CTC load in patients who were adjuvantly treated with chemotherapy compared to patients in the follow-up subgroup. This finding was contrary to the same patient subset analyzed with the NYONE® for CTC detection.ConclusionOur study investigates the occurrence of CTC in CRC patients after surgical resection of the primary tumor and during postoperative follow-up. The resection of the tumor has an impact on the CTC quantity and the longitudinal CTC analysis supports the significance of CTC as a prognostic biomarker. Future investigations with an even more extended follow-up period and larger patient cohorts will have to validate our results and may help to define an optimal longitudinal sampling scheme for liquid biopsies in the post-operative monitoring of cancer patients to enable tailored therapy concepts for precision medicine.

Cytokeratins (CK7 and CK20) Genes Expression Association with Clinicopathological Indices in Oral Squamous Cell Carcinoma and Dysplastic Oral Epithelium.

High prevalence of oral squamous cell carcinoma (OSCC) demands the additional novel biological markers. Due to the established roles of cytokeratin in the prognosis of metastasis evaluation the relation of expression of both CK7 and CK20 in OSCC compared to the dysplastic oral epithelium biopsies with clinicopathological factors were investigated. We examined the coordinate mRNA expression of cytokeratin 7 (CK7) and cytokeratin 20 (CK20) using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 110 biopsies of oral squamous epithelium samples including 72 tumoral and 38 dysplastic biopsies. We also collected demographic and pathological data including tumor stage and grade from our patients. There was a significant difference in CK7 and CK20 gene expression between OSCC and dysplastic samples (p< 0.001). Further, their mean expression in OSCC samples was significantly higher compared to dysplastic samples. Relative mRNA levels of CK7 and CK20 showed that their mean expression in OSCC grade I was significantly lower than other grades (p< 0.01). The relationship between CK7 and CK20 mRNA expression and age or gender was not significant (p> 0.05). Samples in the advanced stage of disease had significantly higher CK7 and CK20 expression compared to early-stage samples of OSCC specimens (p= 0.001). We found an increase in CK7 and CK20 mRNA levels in grade III OSCC samples compared to other grades. This finding suggests a potential role for CK7 and CK20 in oral mucosal carcinogenesis and OSCC prognosis.

Relationship between circulating lung-specific X protein messenger ribonucleic acid expression and micrometastasis and prognosis in patients with early-stage nonsmall cell lung cancer.

To investigate the diagnostic value of lung-specific X protein (LUNX) messenger ribonucleic acid (mRNA) expression in peripheral blood of patients with nonsmall cell lung cancer (NSCLC) in micrometastasis. Peripheral blood samples of 112 patients with NSCLC were collected, and the expression of LUNX, cytokeratin 19 (CK19), and carcinoembryonic antigen (CEA) mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR). The expression of LUNX, CK19, and CEA mRNA was increased in peripheral blood of patients with NSCLC compared with that of patients with benign lung disease (P < 0.05), and the sensitivity of LUNX mRNA was higher than that of CK19 and CEA mRNA (P < 0.05). LUNX-positive expression was also associated with lymph node metastasis, tumor-node-metastasis (TNM) staging, and reduced 5-year survival rate of patients in our cohort (P < 0.05). Further, the 5-year survival improved for those LUNX-positive patients who became LUNX negative following adjuvant chemotherapy compared to those who remain LUNX positive (P < 0.05). Multivariate analysis showed that lymph node metastasis, TNM stage, and LUNX mRNA expression in peripheral blood were independent prognostic factors. The detection of LUNX expression in peripheral blood of patients with NSCLC by RT-PCR is a highly specific and sensitive detection method for tumor micrometastasis that may be used for molecular diagnosis of tumor micrometastasis. LUNX mRNA expression in peripheral blood is an independent factor affecting prognosis of NSCLC and thus may reliably predict NSCLC prognosis and guide appropriate adjuvant chemotherapy treatment.

Circulating tumor cells (CTCs) and cytokeratin 19 (CK19) mRNA as prognostic factors in heavily pretreated patients with metastatic breast cancer

IntroductionCirculating tumor cell (CTC) count and cytokeratin 19 (CK19) mRNA expression have a prognostic value for patients with metastatic breast cancer (MBC), but their clinical utility remains controversial. We studied CTC count and CK19 mRNA expression in the peripheral blood samples from heavily pretreated patients with MBC and their correlations with prognosis and response to the subsequent line of therapy. MethodsThis prospective observational study included 67 consecutive patients with MBC who were on progression to systemic therapy, and criteria for a new line of systemic treatment were proposed outside a clinical trial. CTC counts and CK19 mRNA expression were measured by the CellSearch® and RT-PCR methods, respectively, before and after the first cycle of treatment. Progression-free survival (PFS) was defined as the time elapsed between the initiation of the treatment and either the date of clinical or radiological tumor progression or death from any cause or the last follow-up. Cox proportional hazards regression model was used to assess the univariate prognostic value of CTC and CK19 mRNA expression on PFS and Kaplan–Meier estimates. A multivariate Cox model was also used to additionally account for phenotype and visceral disease. ResultsThe mean age was 60 (range 35–86) years, and the average number of previous treatments was 3 (range 1–10); 42 patients (62.6%) were ER+ and 38 patients (56.7%) had visceral disease. The median PFS rate was 8 months (95% CI: 3.7–8.2). Univariate analyses showed a significant effect of the initial value of CK19 mRNA expression (HR = 2.00; 95% CI: 1.05–3.8; p = 0.03) and for the second value of CTC (HR = 2.18; 95% CI: 1.22–3.9; p = 0.009) but did not reach statistical significance for the initial value of CTC and the second value of CK19 mRNA expression. The estimated PFS rates at 6 and 12 months were 75% and 31% for patients with a low initial value of CK19 mRNA expression and 36% and 10% for those with a high initial value of CK19 mRNA expression, respectively (p: 0.022). Further, the estimated PFS rates at 6 and 12 months were 86% and 65% for patients with a low second value of CTC and 76% and 47% for those with a high second value of CTC, respectively (p: 0.004). In the multivariate analysis adjusted for phenotype, visceral disease, and the last treatment performed, only the effect of the second value of CTC remained significant (HR = 2.7, p = 0.004). ConclusionsCK19 mRNA expression and CTC count appeared clinically meaningful in pretreated patients with MBC, even when adjusted for phenotype and visceral disease involvement. These results support the use of CK19 and CTC as relevant biomarkers for predicting clinical response in MBC.

Expression and Clinical Significance of Cytokeratin-19 and Thymidine Kinase-1 in Advanced Gastrointestinal Cancer

Background:As the clinical value of cytokeratin-19 (CK19) and thymidine kinase-1 (TK1) in advanced gastrointestinal cancer remains controversial, we investigated their expression and clinical significance in this disease.Methods:A total of 171 advanced gastrointestinal cancer patients were prospectively enrolled in this study. The mRNA level of CK19 was detected using quantitative real-time reverse transcription-polymerase chain reaction (PCR) in all patients, along with a control group of fifty healthy individuals. Furthermore, detection of TK1 protein was carried out in 96 patients using a chemiluminescence dot blot assay. The primary endpoint was overall survival (OS) time.Results:Positive CK19 mRNA expression was detected in 74 (43.3%) of the 171 patients and positive TK1 expression was detected in 66 (68.8%) of the 96 patients. Furthermore, of the 96 patients, 36 (37.5%) were positive for both TK1 protein and CK19 mRNA, 30 (31.3%) were negative for TK1 protein, and 15 (15.6%) were negative for TK1 protein and positive for CK19 mRNA. The results indicated that patients who were positive for CK19 mRNA expression had significantly shorter OS times than those who were negative for it (median OS 7.7 vs. 9.7 months, respectively; P = 0.02). Moreover, patients who were positive for CK19 mRNA and TK1 protein expression had shorter OS times (median OS 6.1 months) than those who were positive for CK19 mRNA and negative for TK1 protein expression (median OS 9.1 months; P = 0.028). Positive CK19 mRNA expression was significantly associated with shorter OS in the univariate analysis (P = 0.027). Based on a multivariate Cox regression analysis, CK19 mRNA together with TK1 protein expression (P = 0.024) was an independent predictor for OS in gastrointestinal cancer patients.Conclusions:Our results suggest that positive expression of CK19 mRNA and TK1 protein is closely correlated with poor prognosis in advanced gastrointestinal cancer. Furthermore, both CK19 and TK1 are possible gastrointestinal cancer biomarkers.

Laser-capture microdissection for analysis of cell type-specific gene expression of muscarinic receptor subtypes in the rat bladder with cyclophosphamide-induced cystitis.

This study examined whether the laser-capture microdissection (LCM) method can achieve separation of urothelial cells from detrusor cells or superficial urothelial cells from intermediate/basal urothelial cells, using α-smooth muscle actin (SMA) and cytokeratin 20 (CK20). In addition, we investigated the changes in expression of muscarinic receptors in laser-captured urothelial and detrusor cells in rats with chronic cystitis. Female SD rats were injected with cyclophosphamide (75 mg/kg) intraperitoneally at day 1, 4, 7 and 10 to induce chronic cystitis. Saline was injected in the same protocol for controls. Bladder specimens were cut at 8 μm thickness, fixed in 70% ethanol and lightly stained by hematoxylin and eosin, and then superficial urothelium, intermediate/basal urothelium and detrusor muscles were laser-captured separately. Real-time PCR was performed to examine expressions of α-SMA, CK20, muscarinic 2 receptors (M2R) and muscarinic 3 receptors (M3R). The expression of α-SMA mRNA in detrusor muscle cells was 200 times higher than that in urothelial cells in controls. CK20 mRNA expression in apical urothelial cells was 55 times more than that in detrusor muscle and four times more than that in intermediate/basal urothelial cells. Expressions of M2R and M3R mRNA were increased in urothelial cells and decreased in detrusor muscles following chronic cystitis. The LCM could be useful for tissue collection of detrusor muscle and different layers of urothelial cells with minimal contamination of other cell types, and cell type-specific changes in molecular expression could accurately be analyzed. Increased expression of urothelial MR might enhance urothelial-afferent interactions to induce bladder overactivity/pain conditions associated with bladder inflammation.

P0060 Cytokeratin 20: Could it be a promising molecular marker of urinary bladder cancer?

<h3>Background</h3> The objective of this study was to investigate the clinical use of cytokeratin 20 (CK20) in urothelial cancer and in the evaluation of efficiency of therapy. <h3>Methods</h3> This study was conducted in 45 patients with bladder cancer and 20 age-matched and sex-matched controls. The patient group included nine patients with superficial urothelial cancer (group I); 16 patients with locally invasive cancer (group II); and 20 patients with metastasising urothelial cancer (group III), including eight patients with locoregional lymph node metastasis and 12 patients with distant metastasis. Of the metastatic group, ten patients were reassessed after chemotherapy and reclassified according to the pathological type of the tumour. Patients with superficial and localised type tumours were reclassified into group I, II, or III according to tumour grade. All individuals in this study were subjected to detection of CK20 mRNA. <h3>Findings</h3> CK20 mRNA positive cells were detected in patients with urothelial cancers of varying stages. There was a significant decline in CK20 mRNA expression after cancer chemotherapy (Chi-square –12.4, <i>p</i> < 0.001).The detection rate of CK20 positive cells was significantly associated with the pathological type of the tumour. <h3>Interpretation</h3> CK20 positive cells were detected in the urinary sediment of patients with urothelial cancer. The decline of CK20 mRNA expression with a good response to systematic chemotherapy lends support to its use as a tumour marker to monitor efficiency of therapy.

MRNA expression of CK19, EGFR and LUNX in patients with lung cancer micrometastasis

To evaluate the clinical significance of mRNA expression of cytokeratin 19 (CK19), epidermal growth factor receptor (EGFR) and lung-specific X protein (LUNX), a total of 42 patients who were diagnosed with non-small cell lung cancer (NSCLC) by pathology were studied retrospectively. The messenger RNA (mRNA) expression levels of CK19, EGFR and LUNX in the peripheral blood were analyzed using reverse transcription-polymerase chain reaction (RT-PCR). The expression of CK19 mRNA did not differ significantly according to the location, size, clinical stage or differentiation of the primary tumor (all P>0.05). However, there was a significant difference in the level of CK19 mRNA expression between squamous carcinoma and adenocarcinoma. The positive rates of EGFR mRNA in the patient and the healthy control groups were 69.0 and 12.5%, respectively, and were significantly different (P<0.05). The positive rates of LUNX mRNA in the two groups were 40.5 and 0%, respectively, and were significantly different (P<0.05). The results indicate that the mRNA expression of CK19, EGFR and LUNX in the peripheral blood is of significant clinical value for the diagnosis of micrometastasis and the prognosis of lung cancer.

Abstract 720: Identification of novel molecular markers for detecting lymph node metastasis of oral squamous cell carcinoma

Abstract Cervical lymph node metastasis is a potent prognostic factor in oral squamous cell carcinoma (OSCC). Management of occult metastasis is an important issue for the surgeons. Sentinel lymph node biopsy (SLNB) is highlighted for its role in deciding making of neck dissection. We have performed the SLNB for cN0 cases routinely and reported the clinical usefulness. Accurate diagnosis of the metastasis in sentinel lymph nodes is important but depends on a heavy workload of the pathologist. We carried out a clinical study to evaluate a novel automated assay system for cytokeratin 19 (CK19) mRNA, the one-step nucleic acid amplification (OSNA) method, to detect the lymph node metastasis of OSCC. The accuracy of OSNA assay was nearly 95%, but it could not detect occult metastasis in case of no or low expression of CK19 mRNA in the primary tumor. In this study, we attempted to identify novel molecular markers to find the harboring cancer cells in the lymph node. First, we determined the gene expression profiles in 7 metastatic lymph nodes from patients with OSCC, 1 benign lymph node, and 5 salivary gland tissues from non-cancerous patients by microarray analysis. We found the overexpression of 36 genes in all metastatic lymph nodes but not in benign lymph node and salivary glands. Subsequently, we examined the expression of these genes in newly 15 metastatic lymph nodes and 9 benign lymph nodes by real-time quantitative RT-PCR method. Among the 36 genes, the expression of annexin A8-like 2 (ANXA8L2) and desmoglein 3 (DSG3) was commonly detected in metastatic lymph nodes at much higher level but not in benign lymph nodes at all. Furthermore, we retrospectively evaluated the usefulness of these markers with the use of 330 lymph nodes that were histopathologically metastasis-positive in 62 and metastasis-negative in 268. The accuracy of each marker CK19, ANXA8L2 and DSG3 is all near 90%. However, the combination of these markers improved the sensitivity up to 96-100%. Expression of ANXA8L2 and DSG3 was also detected in approximately 3% of the histopathologically metastasis-negative lymph nodes. These results suggested that ANXA8L2 and DSG3 could be useful molecular markers and improve detection rate of occult metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 720. doi:1538-7445.AM2012-720

Prognostic Significance of Real-Time RT-PCR Detection of Disseminated Tumour Cells in Bone Marrow and Circulating Tumour Cells in Patients with Breast Cancer.

Abstract Background: Despite optimal staging and treatment, up to 40% of stage I and II breast cancer (BC) patients will develop recurrent disease over time. These patients are believed to have disseminated tumour cells (DTC) at the time of diagnosis. Detection of minimal disease in bone marrow (BM) has been suggested to be a more direct approach to select metastasis-prone patients among this 'good prognosis group'. Peripheral blood (PB) sampling however is more convenient. The aim of this study was to evaluate whether the detection of DTC in either PB or BM predicts overall survival (OS). The initial analysis was published with a mean follow up time of 786 days (Benoy et al., BrJC 2006). We now report on these data after a mean follow up of nearly 5 years.Material and methods: PB and BM samples were collected from 148 patients with primary (M0, n=116) and metastatic (M+, n=32) BC before the initiation of any local or systemic treatment. PB of healthy volunteers and BM of patients with a nonmalignant breast lesion or a haematological malignancy served as control group. DTC were detected by measuring relative gene expression (RGE) for cytokeratin 19 (CK19) and mammaglobin (MAM), using a quantitative RT-PCR detection method. The 95 percentile of the RGE for CK19 and MAM of the control group was used as cutoff to determine elevation in BC patients. Kaplan-Meier analysis was used to predict OS.Results: Mean follow up time was 1518 days (+/- 719). Elevated CK19 expression was detected in 42 (28%) BM samples and in 22 (15%) PB samples. MAM expression was elevated in 20% (both PB and BM) of the patients with BC. There was a 68% (CK19) and 75% (MAM) concordance between PB and BM samples when classifying the results as either positive or negative. Patients with an elevated CK19 or MAM expression in BM had a worse OS than patients without elevated expression levels (p=0.002 (CK19) and p=0.001 (MAM)). For PB, no statistical significant difference in OS was observed between patients with or without elevated CK19 (p=0.227), but a strong trend for predicting OS was observed according to MAM status (p=0.054). Separate analyses of M0 and M+ patients revealed only a marked difference in OS according to BM CK19 in the M+ patient group. In M0 patients disease free survival (DFS) was not significantly predicted by CK19 and/or MAM status in BM alone (p=0.173 (CK19), p=0.219 (MAM)), but the presence of the double positive phenotype showed a trend for predicting DFS (p=0.059).Conclusions: DTC measured as elevated CK19 or MAM mRNA expression, could be detected in both PB and BM of patients with BC. Only the presence of DTC in BM was highly predictive for OS. However, with longer follow up differences in OS between patients with or without elevated CK19 and particularly MAM expression in PB also tend to become more significant. Furthermore, on the long term, double positivity for CK19/MAM in BM seems to predict DFS in patients with localized BC. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3019.

Expression of cytokeratin19 mRNA and integrin alpha v beta 3 in breast cancer patients with negative axillary lymph nodes

Expression of cytokeratin19(CK19)mRNA was detected by reverse transcription-PCR in bone marrow and integrin α_vβ_3 was detected by immunohistochemistry(IHC streptavidin-biotinperoxidase SP)in breast tissue from 62 breast cancer patients with negative axillary lymph nodes.Out of 62 patients 15cases(24%)presented positive CK19,in which all had positive integrin α_vβ_3;36 cases(58%)presented positive integrinα_vβ_3.Two markers were positively correlated with each other(kappa=0.6839,P＜0.05),and correlated with the size of tumor(P＜0.01).The results indicate that combined detection of CK19 in bone marrow and integrin α_vβ_3 in cancer tissues may be helpful for guiding therapy and estimating prognosis in breast cancer patients with negative axillary lymph nodes. Key words: Keratins; Breast neoplasms

Detection of disseminated tumour cells in mediastinoscopic lymph node biopsies and endobronchial ultrasonography-guided transbronchial needle aspiration in patients with suspected lung cancer

Purpose Ultrasound-guided transbronchial needle aspiration of mediastinal lymph nodes (EBUS-TBNA) is apparently more accurate for cancer diagnosis than standard transbronchial needle aspiration (TBNA), but it is less sensitive than mediastinoscopy. The detection of disseminated tumour cells in transbronchial needle aspiration and mediastinoscopic biopsies could improve staging and might be helpful concerning indications for neoadjuvant regimen. The goal of this study was to develop a quantitative method for the detection of disseminated tumour cells (DTCs) in lymph node samples from patients with suspected lung cancer. Patients and methods We compared in a prospective trail EBUS-TBNA ( n = 58 patients, 86 samples) and mediastinoscopy ( n = 22 patients, 37 samples) in two largely independent cohorts of lung cancer patients. Eleven patients, 14 samples were analysed using both methods. Patients without evidence of malignant disease were available as controls for EBUS-TBNA ( n = 20 patients, 28 samples) and mediastinoscopy ( n = 6 patients, 8 samples). Real-time quantitative mRNA analysis was performed for the cytokeratin 19 (CK19) and MAGE-A genes (MAGE-A 1–6, MAGE-A12) as markers, using a LightCycler 480 instrument. Results CK19 mRNA expression in EBUS-TBNA samples was detected in 84/86 (98%) and in 28/28 control samples (100%). After mediastinoscopy 16/37 (43%) samples of lung cancer patients were CK19 mRNA positive while controls showed no CK19 mRNA expression (0/8). MAGE-A expression was detectable in 42/86 (49%) EBUS-TBNA samples and in 13/37 (35%) mediastinoscopy samples. MAGE-A expression was detected in EBUS-TBNA controls in 3/28 (11%) and 1/8 (12%) mediastinoscopy controls. High MAGE-A expression correlated with increased tumour stage. Conclusion Since CK19 expression was detected in all EBUS-TBNA samples from the control patients, but not in mediastinoscopy samples, we conclude that CK19 is not suitable as a marker for disseminated tumour cells in samples attained by EBUS-TBNA. One possible explanation is a contamination with epithelial cells from the bronchial tubes. MAGE-A genes are promising markers for disseminated tumour cells in lymph nodes in patients with suspected lung cancer which merit further investigation.

Detection of Cytokeratin 19 mRNA and CYFRA 21–1 (Cytokeratin 19 Fragments) in Blood of Tunisian Women with Breast Cancer

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, tumor differentiation and CK19 mRNA transcripts was investigated. We found that CK19 mRNA expression in blood (as a direct index of the presence of circulating tumor cells) was not correlated with CYFRA 21-1.

Isolated Tumor Cells and Circulating CK20 mRNA in pN0 Colorectal Cancer Patients

Micrometastases in lymph nodes and blood may provide important prognostic information. In this study, cytokeratin 20 (CK20) positive cells in lymph nodes and circulating CK20 mRNA were studied using 57 paraffin-embedded lymph node specimens and blood from 24 patients with pN0 colorectal cancer (CRC), respectively. Results showed that 29 out of 56 (52%) lymph node specimens had CK20-positive cells (range: 1-35). Follow-up of the patients for 12 months indicated that 4 patients (7%) had CRC metastases to liver, lung, and bone. In addition, 8 out of 24 (33%) samples had at least 2-fold circulating CK20 mRNA expression higher than the pooled normal sample. This study provides evidence that CK20-positive cells were found in the lymph nodes and differentially expressed circulating CK20 mRNA was also detected in the blood from patients with pN0 CRC. Long-term follow-up is necessary to study their prognostic use in patients with non-metastatic CRC.

Intra‐operative rapid diagnostic method based on CK19 mRNA expression for the detection of lymph node metastases in breast cancer

Staging by sentinel node (SN) biopsy is the standard procedure for clinically node-negative breast cancer patients. Intra-operative analysis of the SN allows immediate axillary lymph node (ALN) dissection in SN positive patients, but a quick, reliable and reproducible method is lacking. We tested the suitability of a quantitative cytokeratin 19 (CK19) mRNA one step nucleic acid amplification (OSNA#) technique (OSNA-CK19) for intra-operative SN analysis. OSNA-CK19 involves a short manual sample preparation step and subsequent fully automated amplification of CK19 mRNA based on reverse transcription loop-mediated isothermal amplification, with results available within 30–40 min. OSNA-CK19 was compared to histological staining (Hematoxylin&Eosin and CAM5.2 and CK19 immunostaining) of 346 frozen ALNs from 32 breast cancer patients, using half of the lymph node for each method. 267 samples were negative and 61 positive by both methods. Three samples were histology positive and OSNA-CK19 negative. Fifteen samples were histology negative and OSNA-CK19 positive, 11 of which had copy numbers close to the cut-off level of OSNA-CK19. Seven of these 15 samples were RT-PCR positive for epithelial markers and/or showed CK19 protein expression by Western blot suggesting the presence of tumor deposits in the lymph node part investigated by OSNA-CK19. Concordance with histology was 94.8%, and 96.8% after exclusion of the latter 7 discordant cases. Sensitivity was 95.3% and specificity was 94.7% before and 97.1% after discordant case investigation. Our results indicate that OSNA-CK19 can potentially be useful in an intra-operative clinical setting to detect SN tumor involvement in breast cancer patients.

Detection of cytokeratin 19 mRNA and CYFRA 21-1 (cytokeratin 19 fragments) in blood of Tunisian women with breast cancer

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, tumor differentiation and CK19 mRNA transcripts was investigated. We found that CK19 mRNA expression in blood (as a direct index of the presence of circulating tumor cells) was not correlated with CYFRA 21-1.

Detection of disseminated pancreatic cells by amplification of cytokeratin-19 with quantitative RT-PCR in blood, bone marrow and peritoneal lavage of pancreatic carcinoma patients

To evaluate the diagnostic potential of cytokeratin-19 (CK-19) mRNA for the detection of disseminated tumor cells in blood, bone marrow and peritoneal lavage in patients with ductal adenocarcinoma of the pancreas. Sixty-eight patients with pancreatic cancer (n = 37), chronic pancreatitis (n = 16), and non-pancreatic benign surgical diseases (n = 15, control group) were included in the study. Venous blood was taken preoperatively, intraoperatively and at postoperative d 1 and 10. Preoperative bone marrow aspirates and peritoneal lavage taken before mobilization of the tumor were analyzed. All samples were evaluated for disseminated tumor cells by CK-19-specific nested-PCR and quantitative fluorogenic RT-PCR. CK-19 mRNA expression was increased in 24 (64%) blood samples and 11 (30%) of the peritoneal lavage samples in the patients with pancreatic cancer. In 15 (40%) of the patients with pancreatic cancer, disseminated tumor cells were detected in venous blood and bone marrow and/or peritoneal lavage. In the peritoneal lavage, the detection rates were correlated with the tumor size and the tumor differentiation. CK-19 levels were increased in pT3/T4 and moderately/poorly differentiated tumors (G2/G3). Pancreatic cancer patients with at least one CK-19 mRNA-positive sample showed a trend towards shorter survival. Pancreatic cancer patients showed significantly increased detection rates of disseminated tumor cells in blood and peritoneal lavage compared to the controls and the patients with chronic pancreatitis. Disseminated tumor cells can be detected in patients with pancreatic ductal adenocarcinoma by CK-19 fluorogenic RT-PCR. In peritoneal lavage, detection rate is correlated with tumor stage and differentiation. In the clinical use, CK-19 is suitable for the distinction between malignant and benign pancreatic disease in combination with other tumor-specific markers.

Quantitative real-time RT-PCR detection for CEA, CK20 and CK19 mRNA in peripheral blood of colorectal cancer patients

This study is aimed at establishing a sensitive approach to detect disseminated tumor cells in peripheral blood and evaluate its clinical significance. A total of 198 blood samples including 168 from colorectal carcinoma (CRC) patients and 30 from healthy volunteers were examined by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to evaluate the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and cytokeratin 19 (CK19) mRNA. CEA mRNA was detected in 35.8% of patients and 3.3% of controls, CK20 mRNA in 28.3% of patients and 6.7% of controls, and CK19 mRNA in 41.9% of patients and 3.3% of controls. CEA and CK20 mRNA positive ratio increased with the advancing Dukes stages, but there was no significant difference in positive ratio between any two stages (P>0.05). Also, relatively high positive ratio of CEA, CK20 and CK19 mRNA expression was observed in some CRC patients with earlier Dukes stages. A higher positive ratio was obtained when two or three detection markers were combined compared to a single marker. Our study indicates that quantitative real-time RT-PCR detection for CEA, CK20 and CK19 mRNA in peripheral blood is a valuable tool for monitoring early stage dissemination of CRC cells in blood circulation.

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.