CK19 mRNA Research Articles

Prognostic significance of quantitative carcinoembryonic antigen and cytokeratin 20 mRNA detection in peritoneal washes of gastric cancer patients.

Peritoneal carcinomatosis is the most common recurrence type in gastric cancer. Disseminated tumor cells derived from serosal invasion may be indicators of early peritoneal seeding and subsequent peritoneal dissemination. Reverse-transcriptase polymerase chain reaction (RT-PCR) techniques have been introduced to aid in detection of disseminated tumor cells in peritoneal washes, however, use of a single molecular marker lacks adequate sensitivity. We sought to improve both sensitivity and specificity in detecting disseminated tumor cells in peritoneal washes by using two markers; carcinoembryonic antigen (CEA) and cytokeratin 20 mRNA (CK20 mRNA). Between July 2007 and June 2010, peritoneal washing samples were collected from 131 patients who underwent surgery for histologically proven gastric cancer. CEA and CK20 mRNA levels were quantified using a Light Cycler. Analysis using of the two markers had higher sensitivity (93.9%) and specificity (87.7%) than single marker detection (p<0.01, p<0.001 respectively). These analyses also correlated with various clinicopathological factors, and aided in predicting survival and peritoneal recurrence. Two-marker analysis has a significant correlation of survival or peritoneal recurrence in gastric cancer, and this analysis may be more useful as a prognostic predictor of peritoneal recurrence compared with RT-PCR mediated detection of CEA or CK20 alone.

Multiplex PCR-Based Detection of Circulating Tumor Cells in Lung Cancer Patients Using CK19, PTHrP, and LUNX Specific Primers

IntroductionThe aim of this study was to develop a multiplex polymerase chain reaction (PCR)-based method for detection of circulating tumor cells in peripheral blood of lung cancer (LC) patients. Patients and MethodsPeripheral blood was collected from 71 healthy donors and 125 LC patients at different pathological stages. Samples were analyzed using multiplex PCR, and specific primers for CK19, PTHrP, and LUNX mRNA. The sensitivity of our method was set at 10 LC cells (A549 cells) in 3 mL of peripheral blood of healthy donors using spiking experiments. ResultsThe detection rates in LC patients for CK19, PTHrP, and LUNX were 45.6%, 64.8%, and 28%, and in healthy individuals were 7%, 7%, and 5.6%, respectively. Overall, our method produced 77.8% positive detections for at least 1 molecular marker. Twenty-eight (22.2%) were negative for expression of all markers, 39 (31.2%) were positive for expression of 1 marker, 42 (33.6%) were positive for expression of 2 markers, and 17 (13.6%) were positive for expression of all 3 markers. Detection of CK19 mRNA expression positively correlated with LC stage and distant metastases. PTHrP mRNA detection correlated positively with LC stage, presence of bone metastasis, and squamous cell carcinoma, and LUNX mRNA detection correlated with lymph node involvement. Combined detection of 2 or 3 markers was significantly correlated with metastatic disease, and negative detection of all 3 molecular markers was correlated with early stage nonmetastatic disease. ConclusionMultiple PCR-based detection of CK19, PTHrP, and LUNX mRNA expression provides useful information for disease stage and dissemination in LC patients.

Metasin—An Intra-Operative RT-qPCR Assay to Detect Metastatic Breast Cancer in Sentinel Lymph Nodes

Nodal status is one of the most important prognostic factors in breast cancer. Established tests such as touch imprint cytology and frozen sections currently used in the intra-operative setting show variations in sensitivity and specificity. This limitation has led to the development of molecular alternatives, such as GeneSearch, a commercial intra-operative real-time quantitative Polymerase Chain Reaction (RT-qPCR) assay that allows the surgeon to carry out axillary clearance as a one-step process. Since GeneSearch has been discontinued, we have developed the replacement Metasin assay, which targets the breast epithelial cell markers CK19 and mammaglobin mRNA and identifies metastatic disease in sentinel lymph nodes. The optimised assay can be completed within 32 min (6 min for RNA preparation and 26 min instrument run time), making its use feasible in the intraoperative setting. An analysis by Metasin of 154 archived lymph node homogenates previously analysed by both parallel histology and GeneSearch showed concordance for 148 cases. The sensitivity and specificity of Metasin compared with GeneSearch were 95% (CI 83%–99%) and 97% (CI 91%–99%) respectively; compared with histology they were 95% (CI 83%–99%) and 97% (CI 91%–99%), respectively. The sensitivity and specificity of GeneSearch compared with histology were 90% (CI 77%–96%) and 97% (CI 93%–99%) respectively. The positive predictive value of Metasin was 90% and negative predictive value was 98% for both histology and GeneSearch. The positive predictive value of GeneSearch was 92% and the negative predictive value was 97% compared to histology. The discordance rates of Metasin with both GeneSearch and histology were 3.89%. In comparison, the discordance rate of GeneSearch with histology was 4.5%. Metasin’s robustness was independently evaluated on 193 samples previously analysed by GeneSearch from the Jules Bordet Institute, where Metasin yielded comparable results.

Association of peripheral CK19 mRNA expression with micrometastasis in patients with breast carcinoma

Objective To detect the expression of CKI9 mRNA in peripheral blood in patients with breast carcinoma and to explore whether this expression can be used as a molecular biological marker for predicting micrometastasis and prognosis. Methods Peripheral serum samples were collected from 75 patients with breast carcinoma, 21 patients with benign breast lesions, and 20 healthy subjects. Expression of CK19 mRNA was detected by RT-PCR. Results The positive rate of CK19 mRNA was 36.0% in patients with breast carcinoma and 0% in those with benign breast lesions and in healthy subjects; and there were significant differences in the expression of CK19 mRNA among patients with breast carcinoma, those with benign breast lesions, and healthy subjects （P〈0.001）. The positive rate of CK19 mRNA positively correlated with the clinical stages （P〈0.05）. Conclusions CK19 mRNA can be detected in peripheral serum of breast carcinoma patients. CK19 mRNA expression closely correlates with the clinical stages in patients with breast carcinoma, suggesting this index can be used to predict micrometastasis and prognosis. Key words: Breast neoplasms; Metastasis; Keratin 19; RNA; Messenger

Surgery-induced peritoneal metastasis and its intraoperative treatment.

4092 Background: A large number of advanced gastric cancer patients undergoing curative gastrectomy with D2 lymph node dissection (D2 gastrectomy) show peritoneal metastasis. The source of these metastatic cells and their treatment remain unclear. We examined the mechanism of surgery-induced peritoneal metastasis and determined the appropriate intraoperative treatment. Methods: (1) Curative gastrectomy was performed for 102 gastric cancer patients. Peritoneal lavage fluid was collected before and after gastrectomy. Cytology, RT-PCR, and cell culture were used to determine the presence of cancer cells. Proliferative potential of tumor cells was evaluated using Ki-67 staining. Tumorigenic capacity was assessed by cell injection into the peritoneal cavity of NOD/ShiJic-scid mice. (2) Fifty clinical T3(SE) or T4(SI) advanced gastric cancer patients undergoing curative D2 gastrectomy prospectively received intraoperative hyperthermic intraperitoneal chemotherapy (HIPEC) in a phase II trial. HIPEC comprised 50 mg CDDP, 10 mg MMC, and 1000 mg 5-FU in 5 L saline maintained at 42–43C° for 30 min. Results: (1) Of 102 peritoneal lavage fluid samples obtained before gastrectomy, 57 from both early and advanced cancer patients did not contain CEA or CK20 mRNA amplification products or cancer cells. Of these 57 samples, CEA or CK20 mRNA was detected in 35 and viable cancer cells were identified in 24 after gastrectomy. Viable cancer cells in all 24 cases showed Ki-67 positivity, indicating proliferative activity. Cultured viable cancer cells developed into peritoneal tumor nodules after spill over into the peritoneal cavity in NOD/ShiJic-scid mice. (2) Fifty patients were eligible for the phase II clinical trial. The overall 5-year survival rate for all patients was 92.4%. This rate in patients with pT2(ss) (n = 12), pT3(se) (n = 35), and pT4(si) (n = 3) disease was 90.0%, 92.3%, and 100%, respectively. Only 2 patients (4%) showed peritoneal relapse. Conclusions: Viable tumorigenic cancer cells spilled over the peritoneal cavity during curative gastrectomy. Intraoperative HIPEC following curative D2 gastrectomy effectively prevented peritoneal metastasis, thereby potentially improving the prognosis of patients with advanced gastric cancer.

Prediction of non-sentinel lymph node metastasis in early breast cancer by assessing total tumoral load in the sentinel lymph node by molecular assay

The one-step nucleic acid amplification (OSNA) is a molecular procedure that yields a semiquantitative result for detection of nodal metastasis. Size of metastasis in the sentinel lymph node (SLN) by conventional histology has been described as a predictive factor for additional axillary metastasis. The objective of this study is to quantify intraoperatively the total tumoral load (TTL) in the positive SLNs assessed by OSNA and to determine whether this TTL predicts non-SLN metastasis in patients with clinically node negative early stage breast cancer. 306 patients with cT1-3N0 invasive breast cancer who had undergone intraoperative SLN evaluation by OSNA were included. TTL was defined as the addition of CK19 mRNA copies of each positive SLN (copies/μL). TTL was a predictive factor of additional non-SLN metastasis in the complete axillary lymph node dissection (cALND) (OR, 1.67; 95% CI, 1.18-2.35). In the multivariate analysis, the TTL was a predictor of non-SLN metastasis in HR positive patients (OR, 1.69; 95% CI, 1.19-2.41). In our cohort of patients, with a TTL ≤1.2 × 10(5) copies/μL, there was a specificity of 85.3% and negative predictive value (NPV) of 80%. If we consider only the HR positive patients, with a TTL ≤5 × 10(5) copies/μL there was a specificity of 86.7% and NPV of 83.7%. TTL assessed by OSNA assay predicts for additional non-SLN metastasis and this intraoperative tool can help guiding decisions on performing a cALND in breast cancer patients.

Intraoperative molecular analysis of total tumor load in sentinel lymph node: a new predictor of axillary status in early breast cancer patients

To assess the intraoperative positive sentinel lymph node (SLN) total tumor load (TTL, defined as the amount of CK19 mRNA copies [copies/μL] in all positive SLNs) obtained by one-step nucleic acid amplification (OSNA) and to determine whether it is predictive of non-SLNs involvement. SUMMARY/BACKGROUND/DATA: The OSNA assay (Sysmex Corporation, Kobe, Japan) is a new diagnostic technique that uses molecular biological techniques to analyze SLN that has been validated as an accurate method for detection of positive SLN. Although the American College of Surgeons Oncology Group Z0011 trial has defined a select cohort of patients in whom a completion axillary lymph node dissection (cALND) may be safely omitted, there are a still a number of patients where prediction of non-SLN metastasis may be helpful for cALND decision making. Multiple studies suggest that specific pathologic characteristics of the primary tumor and the SLN metastases are associated with an increased likelihood of additional positive non-SLN. This is a retrospective multicentric cohort study of 697 patients with cT1-3N0 breast cancer, who had had intraoperative SLN evaluation by OSNA assay with a cALND. TTL is defined as the amount of CK19 mRNA copies number in all positives SLN (copies/μL). Univariate logistic regression showed that, in addition to TTL (p < 0.001), the number of affected SLNs (p < 0.001), tumor size (p < 0.001), HER2 status (p = 0.007), and lymphovascular invasion (LVI, p < 0.001) were predictive of ALND status. The multivariate logistic regression analysis showed that TTL is an independent predictor of metastatic non-SLNs, after adjusting for the tumor size, HER2 status, LVI and, in particular, the number of affected SLNs. TTL by OSNA is a newly standardized and automated tool that predicts axillary node status better and independently of the number of affected SLNs and the type of surgery. This value can then help clinicians to personalize surgical treatment. Prospective studies will be carried out to determine the clinical impact of this variable in the management of patients.

A novel molecular tool for lymph node upstaging in colon cancer patients: Results of a prospective European, multicenter study.

464^ Background: A new diagnostic semi-automated system, One Step Nucleic Acid Amplification (OSNA), was designed for the detection of cytokeratin 19 (CK 19) mRNA in lymph node (LN) metastases of colon cancer (CC) patients (Güller U et al. CANCER 2012: DOI 10.1002/cncr.27667). Recently it has been shown that occult metastases detected in pN0 patients have an adverse effect on survival based on stage migration. In this study we investigated whether pN0 patients diagnosed by standard histopathology can be upstaged by OSNA analysis to stage III. Methods: In this international, multicenter (4 institutions: CH, G [2], E) study 103 patients-originally diagnosed as pN0 by the standard use of 1 single section haematoxylin &amp; eosin (H&amp;E)-were subjected to OSNA analysis. LN were isolated from fresh tissue, homogenised, and directly subjected to OSNA without prior isolation of mRNA. OSNA results were provided in qualitative categories (- / + / ++), along with the exact CK19 mRNA copy number (+: 250-5000 copies, ++: &gt;5000 copies). Results: In total, 1,594 LN from 103 CC patients (median 14 LN / patient, range 1-46) were analyzed with OSNA. From the 103 histology pN0 patients 26 were positive with OSNA, resulting in an upstaging rate of 25.2%. Sixteen patients had 1 OSNA positive LN, three had 2, three had 3, and one had 4 positive LNs, resulting in a total of 35 positive LN. Patients with pT1, pT2, and pT4 tumors only had 1 positive LN, while all patients with multiple positive LN were from the pT3 group which also contained 9/10 patients with a (++) OSNA result. Concerning stage categories, 6/37 (16.2%) CC patients with stage I were upstaged and 20/66 (30.3%) patients with stage II. Conclusions: OSNA is a novel molecular tool for LN upstaging in CC patients with similar performance as very intensive histological investigation (Güller U et al. CANCER: 2012 DOI 10.1002/cncr.27667). The present study confirms that OSNA improves staging in CC patients and provides a standardized, observer-independent technique for a more accurate staging than the standard histopathology.

An Optimal mRNA Marker for OSNA (One-step Nucleic Acid Amplification) Based Lymph Node Metastasis Detection in Colorectal Cancer Patients

We previously reported that the one-step nucleic acid amplification assay is effective for lymph node metastasis detection in breast cancer patients. This paper describes the identification of CK19 mRNA as an optimal marker and its cut-off value for use in the detection of one-step nucleic acid amplification-based lymph node metastasis in colorectal cancer patients. Candidate mRNA markers selected from the genome-wide expressed sequence tag database were evaluated by quantitative RT-PCR using a mixture of metastasis-positive and another mixture of metastasis-negative lymph nodes (n = 5 each), followed by quantitative RT-PCR using metastasis-positive and -negative lymph nodes (n = 10 each) from 20 patients. The one-step nucleic acid amplification assay for mRNA markers selected above was examined using 28 positive lymph nodes from 19 patients and 38 negative lymph nodes from the 11 pN0 patients. Quantitative RT-PCR analyses of the 98 mRNAs selected from the genome-wide expressed sequence tag database and the subsequent quantitative RT-PCR analyses of the nine mRNAs selected above indicated that CK19 and CEA mRNAs have the highest capability for distinguishing between positive and negative lymph nodes. CK19, CEA and CK20 mRNAs were evaluated by the one-step nucleic acid amplification assay. An area under a receiver-operating-characteristic curve for CK19 mRNA (0.999) was slightly larger than that for CEA mRNA (0.946; P = 0.062) and significantly larger that than for CK20 mRNA (0.875; P = 0.006). We found that CK19 mRNA has the best diagnostic performance and its cut-off value for discriminating positive from negative lymph nodes can be set in the range of 75-500 copies/µl with 96.4% sensitivity and 100% specificity.

Detection of micrometastasis in lymph nodes of oral squamous cell carcinoma: A comparative study

Background:The annual mortality rate from head and neck squamous cell carcinoma (HNSCC) is over 11,000 worldwide. Squamous cell carcinoma of the head and neck (SCCHN) frequently metastasizes to the regional lymph nodes which are the first site of arrest of tumor cells that have invaded the peritumoral lymphatics, hence the strongest predictor of disease prognosis and outcome.Aim:The present study aims to compare the efficacy of frozen sections (cryosection), step-serial sectioning conventional H and E staining, immunohistochemistry (IHC) and RT-PCR analysis in detection of lymph node micrometastasis.Materials and Methods:A prospective series of 30 patients who were diagnosed with primary squamous cell carcinoma of the oral cavity and underwent surgical treatment including unilateral or bilateral selective neck dissection were considered for the study.Result:Metastatic carcinomatous cells were observed in H and E staining of frozen section in 18 lymph nodes (54%) and in 19 lymph nodes (57%) in step-serial sectioned H and E-stained sections of the 78 lymph nodes from 30 patients. Carcinomatous cells were immunolabeled with pancytokeratin in 18 lymphnodes (54%). CK19 mRNA was detected in 33 lymph nodes of 16 patients. RT-PCR gave positive signals for 24% and 23% of lymph nodes positive by histopathology and immunohistochemistry.Conclusion:Our study demonstrated that RT-PCR is far more sensitive in detection of micrometastasis than any other technique used in routine procedures and immunohistochemistry. Fifty-three percent patients with micrometastasis detected by RT-PCR had large T3/T4 tumors. Prognosis was poor for patients who were positive for micrometastasis detected only by RT-PCR, among which two patients died within a period of 6 months.

Abstract P1-01-11: Is OSNA mRNA copy number in sentinel lymph node biopsy predictive of further disease in the axilla?

Abstract Introduction: Intra-operative assessment of sentinel nodes (SLNs) allows immediate completion axillary dissection (cALND) in breast cancer patients. Molecular assessment such as one-step nucleic acid amplication (OSNA) promises greater sensitivity and provides a more accurate quantitative assessment than traditional methods. Our unit policy is to proceed to cALND in patients with macrometastases but not for micrometastases. However, evidence of upstaging has led us to seek to raise the threshold for proceeding to cALND. The CK19 mRNA copy number is an expression of the metastatic burden in the SLN and may be related to the presence of additional disease in the cALND. Since the original copy number threshold between micro (250–5000 copies/microliter) and macrometastasis (&amp;gt;5000 copies/microliter) was based on few patients and serial pathological sections, we investigated the mRNA copy number in patients with and without additional disease in the cALND. Methods: All patients in our unit undergo pre-operative axillary ultrasound with fine needle aspiration cytology of any suspicious nodes. Those with malignant cytology proceed directly to ALND. Radiologically and cytologically node negative patients undergo sentinel lymph node biopsy (SLNB) and OSNA. Electronic records of consecutive patients with invasive breast cancer undergoing SLNB with OSNA from August 2011 to March 2012 were retrospectively reviewed. Two parameters of mRNA copy number were examined: Copy number of the highest copy number SLN and the summed copy numbers of all positive SLNs. Their relationship to the presence of further disease in the axilla was examined using Student's t test. Results: Of 201 SLNBs, 45 (22%) had macrometastasis-positive OSNA and therefore underwent cALND (1 patient declined). Twenty patients (45%) had no further positive nodes (a negative cALND) with a total axillary metastatic burden of 1–2 in 11–27 nodes. Twenty four (55%) showed further disease (a positive cALND) with a burden of 2–20 in 9–30 nodes, including the SLNs. There was no significant difference in tumour size or grade between patients with additional positive nodes in the cALND compared with those with no further disease. There was no significant difference in the copy number of the highest copy number positive SLN (p = 0.44) or in the summed copy number of all positive SLNs (p = 0.36) between the cALND positive and negative groups. Conclusion: OSNA CK19 mRNA copy number does not correlate with the cALND metastatic burden. Therefore, raising the copy number threshold may be too simplistic as a method to better select patients with high probability of a positive cALND. A predictive model will be derived based on multivariate analysis of the larger patient population (&amp;gt;400 patients) that will have undergone SLNB with OSNA by the time of SABCS. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-01-11.

Abstract P1-01-29: Intraoperative molecular analysis of sentinel lymph node as a new predictor of axillary status in early breast cancer patients.

Abstract The one-step nucleic acid amplification (OSNA) assay (Sysmex Corporation, Kobe, Japan) is a new diagnostic device that uses molecular biological techniques to analyze sentinel lymph node (SLN). Intraoperative SLN assessed by OSNA has been validated as an accurate method for detection of SLN metastasis compared to conventional histological examination. Although recent reports have shown that breast cancer patients with &amp;lt;2 positive SLNs can be spared of a complete axillary lymph node dissection (cALND), there are still a number of patients for whom prediction of non SLN metastasis may be helpful for cALND decision making. The aim of the present study is to assess the intraoperative positive SLN total tumor load (TTL, defined as the amount of CK19 mRNA copies [copies/μL] in all positive SLNs) obtained by OSNA and to determine whether it is predictive of non-SLNs metastasis independently of the number of affected SLN and the type of surgery. Data were collected during the month of June 2012 from medical records and include age, tumor size and grade, estrogen and progesterone receptor status, HER2 status, Ki67, presence of lymphovascular invasion (LVI), total number of SLN and non-SLN, number of positive and negative non-SLN, size of SLN and non-SLN metastasis, and TTL in each SLN. A total number of 701 patients were recruited, of which 697 (99,4%) met the study selection criteria. Univariate logistic regression showed that, in addition to TTL (p &amp;lt; 0,001), the number of affected SLNs (p &amp;lt; 0,001), tumor size (p &amp;lt; 0,001), HER2 status (p = 0,007), and LVI (p &amp;lt; 0,001) were predictive of ALND status. The multivariate logistic regression analysis showed that TTL is an independent predictor of metastatic non-SLNs, after adjusting for the tumor size, HER2 status, LVI and, in particular, the number of affected SLNs. Moreover, the ROC curve analysis showed that, as compared to the number of affected SLN, TTL has a better ROC curve, as measured by the AUC: LogTTL 0.709 (95% CI, 0.667–0.760); number of affected SLN 0.610 (95% CI, 0.570–0.652), p &amp;lt; 0.001. Furthermore, in patients possessing a TTL&amp;lt;15000, the frequency of non-SLN metastasis was 14,7% (NPV = 85,3%, PPV = 41,1%, Sensitivity = 76,7%, Specificity = 55,2%). Taking this value as a cutpoint, 85 patients with mastectomy may have spared a cALND considering the predictive results of the TTL. In seven patients with &amp;gt; 3 positive SLN the TTL was &amp;lt; 15000 so this group, even with 3 positive SLNs, have 14.7% of having additional non SLN metastasis. In conclusion, TTL by OSNA is a newly standardized, automated, and reproducible tool that predicts axillary node status better and independently of the number of affected SLNs and the type of surgery. This value can then help clinicians to personalize surgical treatment. Prospective studies will be carried out to determine the clinical impact of this variable in the management of patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-01-29.

Differentiation of adipose-derived adult stem cells into epithelial-like stem cells

Adipose-derived adult stem (ADAS) cells can be easily obtained in large quantities. Previous studies have suggested that all-trans-retinoic acid (ATRA) plays an important role in the differentiation of mesenchymal stem cells toward an epithelial lineage. In order to verify that ADAS-cells can differentiate into an epithelial lineage retaining most of the characteristics of stem cells, ADAS-cells were isolated and cultured. They were induced to differentiate toward an epithelial lineage in vitro. Differentiated epithelial cells were assayed as to whether they retain characteristics of stem cells by RT-PCR and cell cycle stage analysis, and were further induced to differentiate toward an osteogenic lineage. RT-PCR analysis revealed that no CK5, CK10 or CK19 mRNA was detected in ADAS-cells, CK19 but not CK5 or CK10 mRNA was detected in differentiated cells at passage 1, CK10 and CK19 expression but not CK5 mRNA was detected in differentiated cells at passage 10. After induction, the expression of CK19 was observed by immunofluorescent staining. Positive staining with alkaline phosphatase (ALP) and Von Kossa staining verified that differentiated epithelial cells still had potential to further differentiate toward an osteogenic lineage. These experiments provide proof that ADAS-cells can differentiate into an epithelial lineage retaining most of the characteristics of stem cells.

CK19 expression should be tested prior to OSNA analysis of sentinel lymph nodes in breast cancer

Dear Editor, The observations of Remoundos et al. [1] are quite interesting. Effectively, there is not a direct relationship between presence of mRNA and expression of protein. In the first case of breast cancer in which we found lack of CK19 expression, absence of CK19 mRNA was confirmed by Sysmex S.L. by means of a RT-PCR, which confirmed that truly CK19-negative tumours can be found among those with a luminal phenotype and not only among those with a triple-negative phenotype. After terminating the study, we found five more cases lacking CK19 expression. For various reasons, we did not confirm the mRNA status of these tumours, but the one-step nucleic acid amplification (OSNA) assay was not performed and sentinel lymph nodes (SLN) were studied by means of standard procedures. Even though the proportion of breast cancers lacking CK19 mRNA is most likely lower than that of immunohistochemically CK19-negative tumours, we think it may be hazardous to perform the OSNA assay in cases lacking expression of CK19. In our paper [2], we reported a case of a luminal-A breast carcinoma expressing CK19 in only very few cells, both in the primary tumour and in the matched SLN macrometastasis. This metastasis probably would have been detected by the OSNA analysis, not as macrometastasis but with a diagnosis of micrometastasis or even as isolated cells. Dr. Remoundos affirms that they found on OSNA analysis a micrometastasis in a SLN from a patient with a breast cancer lacking CK19 expression. How do they know that that metastasis was a micrometastasis and not a macrometastasis?

Detection of circulating tumor cells in peripheral blood of colorectal cancer patients without distant organ metastases

Recently, the detection of circulating tumor cells (CTCs) in peripheral blood has become an important tool for the non-invasive assessment of micrometastases and to predict clinical outcome. The objective of this study was to investigate if the presence of CTCs in peripheral blood influences the prognosis in colorectal cancer (CRC) patients without distant organ metastases. The GCC mRNA and CK20 mRNA levels in peripheral blood and the serum levels of CEA of 92 CRC patients without distant organ metastasis were analyzed by quantitative RT-PCR and ELISA, respectively. Its associations with overall survival (OS) and disease-free survival (DFS) rates were analyzed. Univariate analyses showed that lower OS and DFS rates were significantly associated with GCC and CK20 mRNA levels, the presence of lymph node metastases, the presence of mesenteric root lymph node metastases, and the presence of tumor emboli in vessels (p < 0.05), but not with CEA levels. Multivariate analyses showed a significant association between 1) OS and GCC mRNA levels and differentiation types and 2) DFS and the presence of tumor emboli in the vessels. Kaplan-Meier curves showed that DFS was significantly associated with the presence of poorly differentiated cells, the presence of mesenteric root lymph node metastases having received prior chemotherapy, and the presence of tumor emboli in vessels. The detection of CTCs in peripheral blood may be useful for the prediction of clinical outcome in CRC patients without distant organ metastases.

CK19 can be used to predict the early recurrence and prognosis of HBV-related hepatocellular carcinoma patients with low AFP serum concentration after R0 radical hepatectomy

To investigate the expression of CK19 in HBV-related hepatocellular carcinoma (HCC) tissues in patients with low serum AFP concentration and the relationship between them and the recurrence and prognosis of HCC after R0 radical hepatectomy. The expressions of CK19 and Ki67 in HCC tissues of 235 cases were examined using tissue microarray and two-step methods of PV-6000 immunohistochemistry. The expression of CK19 mRNA in 20 frozen HCC specimens was examined by RT-PCR. The correlation between gene expressions and tumor recurrence and prognosis was analyzed. Among the 235 HBV-related HCC patients after R0 radical hepatectomy, the median disease-free survival (DFS) was 31.2 months in the patients with serum AFP < 400 µg/L and 13.8 months in the patients with serum AFP ≥ 400 µg/L (P = 0.041), the overall survival (OS) was 84.0 and 58.6 months in the two subgroups (P = 0.125), and the tumor recurrence within one year was in 43 cases (27%) and 37 cases (49.3%), respectively, (P = 0.001). The DFS was 11.6 months in the CK19-positive cases and 27.0 months in the CK19-negative cases (P > 0.05). The OS was significantly lower in the CK19-positive cases than that in the CK19-negative cases (P = 0.023). Both DFS and OS in the CK19-positive cases with AFP < 400 µg/L were significantly lower than those in the CK19-negative cases with AFP < 400 µg/L (both P < 0.05). The CK19 expression was significantly correlated with histological differentiation (P = 0.023), number of tumor foci (P = 0.044), vascular tumor embolism (P = 0.005), regional lymph node metastasis (P = 0.023), and 1-year recurrence (P = 0.006). Among the patients with AFP < 400 µg/L, the 1-year recurrence was 53% in the CK19-positive cases and 23% in the CK19-negative cases (P < 0.001), the median DFS was 11.3 months in CK19-positive cases and 34.0 months in CK19-negative cases (P = 0.010), and the median OS was 19.5 months in the CK19-positive cases, significantly lower than 84.0 months in the CK19-negative cases (P = 0.001). Cox regression analysis showed that CK19-positive expression was an independent factor affecting early HCC recurrence and prognosis. In HBV-related HCC patients after radical hepatectomy with AFP < 400 µg/L, positive expression of CK19 indicates a higher proliferation and invasiveness of HCC, and is an important factor of early recurrence and poor prognosis.

163. A new development in sentinel lymph node biopsy in breast cancer using a combination of molecular and histological methods

Background: Sentinel lymph node biopsy(SLNB)has become a standard surgical method for clinically node-negative breast cancer. In addition to histological diagnosis using frozen sections, new intraoperative diagnostic performance of OSNA assay (one-step nucleic acid amplification test that amplifies CK19 mRNA) is expected to detect lymph node metastases more precisely, even in molecular level. However, significance and validity of OSNA assay with or without histological method has not been studied enough, and the significance of positive sentinel lymph node only by OSNA method (pN0mol+) need to be evaluated.

524. Usefulness of OSNA for Intraoperative Analysis of Breast Cancer SNS

Background: SN (sentinel node) biopsy is a common procedure in surgical treatment of clinically node-negative breast cancer patients. OSNA (One-step nucleic acid amplification) is a semi-automated examination using molecular biological technique and allows straightforward diagnosis of SN metastasis without a pathologist by quantitative evaluation of CK19 m-RNA as a copy number of OSNA. In this study, we compared OSNA analysis to histological investigation by pathologists for determining the suitability of OSNA, and we also examined that the copy number of OSNA is related with primary tumor characteristics or not.

Diagnostic value of cytokeratin 19 fragment in nasopharyngeal carcinoma

To explore the clinical significance of cytokeratin 19 fragments test in the diagnosis of nasopharyngeal carcinoma. The study included 102 cases of nasopharyngeal carcinoma, 90 cases of nasal polyp/nasopharyngitis, and 150 healthy individuals. RT-PCR was used to detect CK19 mRNA expression and Western blot to detect CK19 fragment protein expression in tissues of nasopharyngeal carcinoma. Expression of CK19-2G2 was examined by immunohistochemistry. Chemiluminescence analysis was used to detect the serum levels of CK19-2G2, and ELISA to detect that of EB-VCA IgA. Among 102 cases of nasophryngeal carcinoma, 64 showed CK19 mRNA expression by RT-PCR, 60 showed CK19 protein fragments in tumor tissues by Western blot, and 66 showed expression of CK19-2G2 by immunohistochemistry in nasopharyngeal carcinoma, including strong positivity in 20 cases, moderate in 34 cases and weak in 12 cases. The sensitivity and specificity of CK19-2G2 in the diagnosis of nasopharyngeal carcinoma were 49.0% and 89.2%, and those of EB-VCA IgA were 52.9% and 85.4%, respectively. The combined detection of CK19-2G2 and EB-VCA IgA increased the sensitivity to 73.5% while the specificity remained at 80.0%. High levels of CK19-2G2 fragment expressed in tissue and serum are present in patients with nasopharyngeal carcinoma. The serum level of CK19-2G2 is helpful in the diagnosis of nasopharyngeal carcinoma. Furthermore, the combination of serum CK19-2G2 and EB-VCA IgA improves the detection sensitivity.

Intraoperative detection of lymph node metastasis using one-step nucleic acid amplification (OSNA) in breast cancer patients: Effect on second surgery rate and delay for adjuvant therapy.

10517 Background: Sentinel lymph node (SLN) analysis is conventionally analysed using HES and CK19 immunohistochemistry. In case of SLN involvement, a second surgery is required for axillary lymph node (ALN) resection thus delaying the initiation of adjuvant therapies. Methods: 381 pts with invasive breast cancer were considered in this retrospective study. SLN were detected using combined radio-isotope and color detection. SLN involvement was analysed using OSNA for CK19 mRNA, in 100 pts (group 1) and compared to conventional histopathology in 281 pts (reference population, group 2). In all cases, control cytology was performed. Results: No significant difference was found between group 1 and 2 regarding patients characteristics, tumor localization, size, grade, steroid receptors and HER2 expression and the mean number of SLN analysed per pt, 2.4 (range 1-7) and 2.5 (range 1-8), respectively. Considering positive SLN as “++” (CK19 mRNA copy number&gt;5000), “+” (250 &lt; CK19 mRNA copy number &lt; 5000) and positive by inhibition in group 1 and macro-, micrometastases and isolated tumor cells in group 2, no difference in lymph node involvement rate was found between the two groups with 29.0 and 29.9% of positive SLN, respectively. Only one discordance case was observed with negative OSNA analysis and positive cytology with isolated tumor cells. Using OSNA intraoperatively, the mean time to process SLN was 42 min (range 10-104) allowing immediate ALN resection. Significant (P&lt;.01) reduction of re-intervention rate was observed when OSNA was used (9 vs 39%), including margins insufficiency (5 vs 13%), ALN resection (3 vs 15%) or both (1 vs 11%). Delay before adjuvant therapy was also significantly (P &lt;.01) reduced when OSNA was used with 43 (range 20-82) vs 59 (range 14-212) days. Conclusions: Results achieved with OSNA are fully consistent with those achieved using conventional immunohistochemistry analysis. SLN analysis using OSNA avoids a second operation in most patients for ALN resection and shortens the delay for adjuvant therapy.