A new diagnostic method for rapid detection of lymph node metastases using a one-step nucleic acid amplification (OSNA) assay in endometrial cancer.

BACKGROUND: To improve lymph node (LN) metastasis identification for patients with endometrial cancer (EC), this study assessed the usefulness of molecular biologic techniques using a one-step nucleic acid amplification (OSNA) assay. METHODS: Using quantitative reverse transcription polymerase chain reaction (qRT-PCR), an optimal mRNA marker was selected, and its expression was compared between histopathologically positive and negative LNs using an OSNA assay. The authors determined copy number cutoff values and evaluated the diagnostic performance of this OSNA assay using sentinel lymph nodes (SLNs). They also investigated whether an OSNA assay could detect LN metastases with sensitivity and specificity equivalent to the 2-mm-interval histopathology method. RESULTS: For analysis of EC samples, cytokeratin 19 (CK19) was selected as a useful mRNA marker for the OSNA assay. When the cutoff value was set at 250 copies (using 215 LNs from 70 patients), an OSNA assay using CK19 mRNA had a sensitivity of 93.3%, a specificity of 99.5%, and a concordance rate of 99.1%. For performance evaluations using SLNs (120 histopathologically negative LNs and 17 histopathologically positive LNs from 35 patients), a OSNA assay using CK19 mRNA had a sensitivity of 82.4%, a specificity of 99.2%, a positive predictive value of 93.3%, and a concordance rate of 97.1%. Thus, an OSNA assay using CK19 mRNA provided results equivalent to those with the 2-mm-interval histopathology method. CONCLUSIONS: The study data demonstrated that an OSNA assay using CK19 mRNA was applicable for detecting LN metastases in EC. Combined analysis using an OSNA assay and SLNs may improve individualized treatments according to LN metastatic status.

Detection of sentinel lymph node metastases in cervical cancer: assessment of KRT19 mRNA in the one-step nucleic acid amplification (OSNA) method.

OBJECTIVE: The purpose of this study was to examine the utility of the one-step nucleic acid amplification (OSNA) assay using cytokeratin (CK) 19 (KRT19) messenger RNA (mRNA) for the detection of sentinel lymph node (SLN) metastases in cervical cancer patients. METHODS: To determine a cutoff value, KRT19 mRNA was assessed by OSNA assay using 239 lymph nodes (LNs) (217 histopathologically negative LNs and 22 positive LNs). A cutoff value was determined by statistical analysis of the copy numbers obtained by OSNA assay. Subsequently, performance evaluation of the OSNA assay (applying the cutoff value above) on 130 SLNs (32 patients) was used to investigate (through concordance) whether the OSNA assay exhibited diagnostic performance equivalent to the two-mm interval histopathological examination. RESULTS: Two hundred fifty copies/µL of KRT19 mRNA in the OSNA assay appeared to be an optimal cutoff value. In performance evaluation of the OSNA assay, we identified five positive SLNs and 125 negative SLNs by OSNA assay using KRT19 mRNA, exhibiting 96.2% agreement with two-mm interval histopathological examination. CONCLUSIONS: Our results indicated that the KRT19 mRNA OSNA assay can detect LN metastases as accurately as two-mm interval histopathological examination and thus may be an effective additional or alternative method for a rapid intra-operative examination of SLNs in cervical cancer.

An optimal mRNA marker for OSNA (One-step nucleic acid amplification) based lymph node metastasis detection in colorectal cancer patients.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

New chemiluminescent enzyme immunoassay for quantitative measurement of Mac-2 binding protein glycosylation isomer in chronic liver disease.

BACKGROUND: This study aimed to evaluate the quantitative measurement of Mac-2 binding protein glycosylation isomer (M2BPGi) levels using the new chemiluminescent enzyme immunoassay. METHODS: The data of a total of 347 patients with hepatitis C virus (HCV) infection and 150 health volunteers from 13 locations in Japan were evaluated. The quantitative system for measuring M2BPGi-Qt levels was based on a new chemiluminescent enzyme immunoassay. We evaluated the reproducibility and quantitation range in quantitative M2BPGi-Qt measurement. We also investigated the confidence ratio of M2BPGi-Qt levels measured by the new quantitative system to M2BPGi levels measured by the current semi-quantitative system for validating the clinical utility of the new method. RESULTS: The reproducibility of M2BPGi-Qt in HCV samples with negative, positive 1+, and positive 2+ was 0.77 ± 0.02 AU/mL, 2.25 ± 0.03 AU/mL, and 6.55 ± 0.21 AU/mL, respectively, and the corresponding coefficient of variation (CV)s were 2.1%, 1.3%, and 3.2%, respectively. The range of quantification assessment resulted that all CVs showed less than 5% in investigated range. Sample stability testing found that the mean percentage difference between the pre- and post-storage values of 6 samples ranged between 96.2 and 103.9%. The correlation coefficient between M2BPGi and M2BPGi-Qt in patients with HCV and the healthy volunteers was 0.986 and 0.991, respectively. M2BPGi-Qt could be quantitatively assessed in a patient with over 20 C.O.I. CONCLUSION: Compared with qualitative methods, the M2BPGi quantitative measurement system could provide a numerical value unaffected by interpretation bias, and measurements are more precise at high M2BPGi levels.

One-step nucleic acid amplification (OSNA) for the application of sentinel node concept in gastric cancer.

PURPOSE: This study was designed to apply safely the sentinel node navigation surgery (SNNS) to the malignancies, an accurate and prompt intraoperative diagnosis of SN is essential, and micrometastasis has been frequently missed by conventional frozen sections. Recently, a novel molecular-based rapid diagnosis for the lymph node (LN) metastases has been developed using (OSNA) in breast cancer, which takes approximately 30 min to obtain a final result. We evaluated the efficacy of OSNA in terms of the intraoperative diagnosis of LN metastasis in patients with gastric cancer. METHODS: A total of 162 LNs dissected from 32 patients with gastric cancer was included in this study; 45 LNs were pathologically diagnosed as metastatic LNs and 117 LNs were negative. The LNs were bisected; halves were examined with H&E stain, and the opposite halves were subjected to OSNA analyses of CK19 mRNA. The CK19 mRNA expression was examined in the positive or negative metastatic LNs, and the correlation between the tumor volume and CK19 mRNA expression in the metastatic LNs was examined. RESULTS: The CK19 mRNA expressions in the positive metastatic LNs were significantly higher than those of negative LNs. When 250 copies/µl was set as a cutoff value, the concordance rate was 94.4%, the sensitivity was 88.9%, and the specificity was 96.6%. The OSNA expression was significantly correlated with the estimated tumor volumes in the metastatic LNs. CONCLUSIONS: The OSNA method is feasible and acceptable for detecting LN metastases in patients with gastric cancer. This should be applied for the intraoperative diagnosis in the SN-navigation surgery in gastric cancer.

One-step nucleic acid amplification for intraoperative detection of lymph node metastasis in breast cancer patients.

PURPOSE: Detection of sentinel lymph node (SLN) metastasis in breast cancer patients has conventionally been determined by intraoperative histopathologic examination of frozen sections followed by definitive postoperative examination of permanent sections. The purpose of this study is to develop a more efficient method for intraoperative detection of lymph node metastasis. EXPERIMENTAL DESIGN: Cutoff values to distinguish macrometastasis, micrometastasis, and nonmetastasis were determined by measuring cytokeratin 19 (CK19) mRNA in histopathologically positive and negative lymph nodes using one-step nucleic acid amplification (OSNA). In an intraoperative clinical study involving six facilities, 325 lymph nodes (101 patients), including 81 SLNs, were divided into four blocks. Alternate blocks were used for the OSNA assay with CK19 mRNA, and the remaining blocks were used for H&E and CK19 immunohistochemistry-based three-level histopathologic examination. The results from the two methods were then compared. RESULTS: We established CK19 mRNA cutoff values of 2.5 x 10(2) and 5 x 10(3) copies/muL. In the clinical study, an overall concordance rate between the OSNA assay and the three-level histopathology was 98.2%. Similar results were obtained with 81 SLNs. The OSNA assay discriminated macrometastasis from micrometastasis. No false positive was observed in the OSNA assay of 144 histopathologically negative lymph nodes from pN0 patients, indicating an extremely low false positive for the OSNA assay. CONCLUSION: The OSNA assay of half of a lymph node provided results similar to those of three-level histopathology. Clinical results indicate that the OSNA assay provides a useful intraoperative detection method of lymph node metastasis in breast cancer patients.

Rapid detection of CEA mRNA in peritoneal washes using One-Step Nucleic acid Amplification (OSNA) for gastric cancer patients.

BACKGROUND: Carcinoembryonic antigen (CEA) mRNA expression in peritoneal washes from gastric cancer patients has been reported as an indicator for survival or peritoneal recurrence. The whole process of CEA mRNA detection is time- and labor-intensive. We report the potential of One-Step Nucleic acid Amplification (OSNA) as a rapid and simple system for CEA mRNA detection in peritoneal washes. METHODS: A total of 128 peritoneal washes were analyzed by cytological examination including immunocytochemistry. After the cytological examination, the CEA mRNA concentration in the residual cells was measured using the OSNA system. The CEA mRNA concentration in peritoneal washes was compared with the results of the cytological examination. RESULTS: CEA mRNA at concentrations from 10 to 10(7)copies/µL was detected by the OSNA system within 10 min, and an excellent correlation was observed between the logarithmic CEA mRNA concentration and the detection time (r=0.998). The CEA mRNA cutoff value for distinguishing positive and negative cases through cytological examination was identified as 25 copies/µL. At this cutoff value, the concordance rate with the cytological examination was 93.8%. The overall survival in CEA mRNA-positive versus -negative cases identified using the OSNA system was statistically significant. CONCLUSION: This CEA mRNA detection system shows potential for cancer cell detection and for routine use in the clinical laboratory because of its simplicity and rapidity.

An accurate and rapid detection of lymph node metastasis in non-small cell lung cancer patients based on one-step nucleic acid amplification assay.

A sublobar resection is currently recognized as an option for early small-sized non-small cell lung cancer (NSCLC), and intraoperative rapid and accurate lymph node assessment is required for a complete resection. To solve this issue, we investigated the clinical utility of one-step nucleic acid amplification (OSNA) assay, an automated rapid molecular diagnostic method and its optimal mRNA marker for detection of lymph node metastasis in lung cancer. We extracted 16 target candidate mRNA markers with high expression in lung cancer from a genetic database, and then quantified their expression levels by quantitative RT-PCR using surgically dissected lymph nodes with or without metastasis. Cytokeratin 19 (CK19), cytokeratin 7 (CK7), stratifin (SFN), and anterior gradient homolog 2 (AGR2) showed significant differences for mRNA expression between metastasis-negative and -positive lymph nodes in quantitative-RT-PCR screening. CK19 and CK7 were finally selected as potential target markers and were quantified using OSNA assay findings of 165 dissected lymph nodes obtained from 49 lung cancer patients. The OSNA assay with CK19 and CK7 were completed within 40 min and their positive predictive value, negative predictive value, and accuracy comparing to pathological diagnosis with hematoxylin-eosin staining and immunohistochemistry were shown to be 95.0%, 99.3%, and 98.8%, and 85.0%, 97.9%, and 96.4%, respectively, using a cut-off value of 250 copies/µL. Among the 165 lymph nodes tested, 1 false negative result was due to massive necrosis of cancer cells and 1 false positive was caused by the allocation bias of cancer cells in the sampling in patient with pleural dissemination. The best performance was observed when CK19 was used as a marker, while the addition of CK7 mRNA as a marker did not increase sensitivity or specificity. In conclusion, an OSNA assay using CK19 could be effective for molecular diagnosis of lymph node metastasis in lung cancer. This is the first report suggesting the potential clinical utility of OSNA assay for intraoperative rapid diagnosis of nodal status in lung cancer.

Rapid diagnosis of lymph node metastasis in lung cancer with loop-mediated isothermal amplification assay using carcinoembryonic antigen-mRNA.

We investigated the clinical utility of our novel loop-mediated isothermal amplification (LAMP) assay developed as a rapid molecular diagnostic method, using carcinoembryonic antigen (CEA)-mRNA as a marker for detecting tumor cells in patients with non-small cell lung cancer (NSCLC). We evaluated the sensitivity of our LAMP technique using a known quantity of synthesized standard CEA-mRNA. On the basis of those results, we performed LAMP analysis of clinical specimens of 22 primary tumors and 144 lymph nodes obtained from 22 NSCLC patients, and compared the results with those of conventional reverse transcription-polymerase chain reaction (RT-PCR). Standard curves were obtained from the amplification products within 25 min using the LAMP method, which indicated that the limitation to detect extracted CEA-mRNA copies was 100 copies. Further, CEA-mRNA was detected in all 22 primary tumors. Of the 12 lymph nodes shown to be metastasis-positive by hematoxylin-eosin (H-E) staining, 10 showed significant amplification of products in the LAMP assay, while 2 micrometastatic nodes with less than 100 copies of CEA-mRNA were not detectable. Of the 132 histologically non-metastatic lymph nodes, 23 (17%) were judged to be metastasis-positive by the LAMP assay. As compared to the results obtained from conventional RT-PCR used as a control, the LAMP assay was 81% sensitive and 100% specific, while the negative and positive predictive values were 91% and 100%, respectively. These results suggest that our LAMP method using CEA-mRNA as a target molecule has potential to rapidly diagnose nodal metastasis in patients with NSCLC.