One-step Nucleic Acid Amplification Assay Research Articles

Abstract P1-01-12: The Performance of the One Step Nucleic acid Amplification (OSNA) Assay in Breast Cancer Patients with Receiving Preoperative Systemic Therapy

Abstract Background: The OSNA (One Step Nucleic acid Amplification) assay is a semi-automated lymph node examination method using molecular biological technique. The OSNA assay has been validated for breast cancer patients without receiving preoperative systemic therapy (PST) by several clinical studies and has currently become more popular as sentinel lymph node (SLN) examination method with the following two main advantages; 1) to allow examination of the whole portion of a node, 2) to allow intraoperative judgment of metastasis positive or negative. However, the feasibility of the OSNA assay in breast cancer patients treated by PST has never been confirmed. In this multi-central clinical study, we compared the judgments of the OSNA assay and of pathological examination on lymph nodes dissected after receiving PST to evaluate the performance of the OSNA assay. Material & Methods: Three hundred two nodes dissected from the 80 breast cancer patients who received PST were examined. Each lymph node was divided at 2mm intervals and the slices were alternately applied to the OSNA assay and pathological examination with H&E staining and CK19 immunohistochemical staining of permanent-section. In pathological examination, judgments of metastasis positive or negative were determined by one central-review pathologist according to the criteria of AJCC 7th edition (“positive” if >0.2mm metastases were detected). Result: The overall concordance rate between the OSNA assay and pathological examination was 91.1% (275/302) with sensitivity of 88.3% (53/60) and specificity of 91.7% (222/242) (Table). These results are very similar to those of the Japanese clinical validation study in breast cancer patients without receiving PST which was conducted by the almost same protocol (Tamaki Y, et al. Clin Cancer Res, 2009, 15: 2879–2884). Conclusion & Discussion: These results indicate the OSNA assay can be applicable for breast cancer patients after receiving PST as well as breast cancer patients without receiving PST. The OSNA assay will enable to examine the whole portion of nodes, leading to more detection of metastases (especially micrometastases) and more exact nodal staging for breast cancer patients treated by PST. Also, for the patients who receive sentinel lymph node biopsy after PST, the OSNA assay will be useful as intraoperative examination method of SLNs because it is expected to provide more correct judgments than current intraoperative methods such as frozen-section or touch-print cytology. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-01-12.

Abstract P1-01-10: Comparison of sentinel lymph node positivity rates pre and post introduction of OSNA molecular analysis

Abstract Introduction: Intra-operative assessment (IOA) of the axillary sentinel lymph node (SLN) allows immediate completion axillary lymph node dissection (cALND) for a positive SLN. One-step nucleic acid amplification assay (OSNA, (Sysmex)), a molecular technique, is very sensitive and may upstage the axilla compared with other forms of intra-operative assessment and with formal, paraffin-embedded haematoxylin and eosin (H&E) histopathology. This would result in an increased cALND rate at a time when efforts are being made to reduce unnecessary axillary surgery in line with the Z0011 trial (Giuliano et al. 2011). This study compares node positive rates before and after two changes in practice: the introduction of OSNA and the unit policy to perform cALND for macrometastatic SLN disease only. 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 SLNB. Consecutive electronic records were examined for all SLNB procedures in two groups: Pre-OSNA from June 2006 to December 2010 and OSNA from August 2011 to March 2012. Pre-OSNA, patients were offered cALND for micro-or macroscopic SLN disease identified by H&E histology. Following Z0011, patients in the OSNA group proceeded to cALND for SLN macrometastases but not for micrometastases. The chi-squared test was used to compare results between the two groups. Results: The groups were similar in terms of age, tumour size, grade and receptor status. Sentinel node and cALND results are shown in table 1. In summary, testing by OSNA resulted in a higher node positive rate and a greater proportion reported with micrometastases (40% compared with 23% pre-OSNA). The OSNA macrometastasis rate was equal to the pre-OSNA overall node positive rate (22%). Among patients proceeding to cALND (all node-positive pre-OSNA patients and OSNA patients with macrometastases) there was no significant difference in the proportion with additional positive nodes in the cALND or the proportion with a total of four or more positive nodes. Conclusion: The introduction of OSNA has increased the proportion of patients identified as node positive, and has increased the proportion reported to have macrometastasis. Simultaneously, the decision not to perform cALND for patients with micrometastasis was intended to limit the additional surgery performed in the SLNB group as a whole. However, since this decision was based on data from studies using H&E assessment, we should look for ways to raise the threshold for cALND such that it includes only the patients at the highest risk of axillary recurrence. Reference Giuliano AE et al. JAMA 2011; 305(6): 569–75. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-01-10.

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 <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 < 0,001), the number of affected SLNs (p < 0,001), tumor size (p < 0,001), HER2 status (p = 0,007), and 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. 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 < 0.001. Furthermore, in patients possessing a TTL<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 > 3 positive SLN the TTL was < 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.

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?

Comparative study of one‐step nucleic acid amplification assay, frozen section, and touch imprint cytology for intraoperative assessment of breast sentinel lymph node in Chinese patients

Conventional procedures for the intraoperative assessment of breast cancer sentinel lymph nodes (SLNs) are frozen section (FS) and touch imprint cytology (TIC). The one-step nucleic acid amplification (OSNA) assay is a novel molecular technique. The aim of this study was to evaluate the optimal approach by comparing OSNA assay, FS, and TIC. Five hundred and fifty-two consecutive patients were enroled from five study centers in China. The SLNs were cut into alternating 2mm blocks. The odd blocks were tested by the OSNA assay intraoperatively, and the even ones were assessed by postoperative histology (four 4- to 6-μm-thick sections were taken every 200μm per block). In addition, intraoperative histological assessments were carried out on the even blocks of 211 patients by FS and all blocks of 552 patients by TIC. Overall performance of the assay compared to postoperative histology was: accuracy 91.4%; sensitivity 83.7%; and specificity 92.9%. The sensitivity of the assay was higher than FS (211 patients, 77.6% vs 69.7%; not significant, P=0.286) and was significantly higher than TIC (552 patients, 83.6% vs 76.2%; P=0.044). When assessing nodes with micrometastases, the sensitivity of the assay was higher than FS (17 nodes, 47.1% vs 23.5%; not significant, P=0.289) and was significantly higher than TIC (48 nodes, 62.5% vs 35.4%; P=0.007). The study indicated that the OSNA assay is an accurate and rapid intraoperative assay for assessing breast SLNs and it can replace FS and TIC for application in general medical practice. The trial was registered as: OSNA assay China Registration Study. China Breast Cancer Clinical Study Group 001c.

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 40min 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.

9. Studies of sentinel lymph node biopsy after preoperative chemotherapy in breast cancer patients

S 735 of the study will promote a more structured breast cancer surgical care and better patient information service. To our best knowledge, this is the largest related preoperative investigation in the English language literature and the first one in the Central and Eastern European region. 9. Studies of sentinel lymph node biopsy after preoperative chemotherapy in breast cancer patients T. Kinoshita, T. Hojo, J. Suzuki, K. Jimbo, K. Taruno, S. Asaga, M. Yoshida, H. Tsuda 1 National Cancer Centre Hospital, Surgical Oncology Division, Tokyo, Japan National Cancer Centre Hospital, Pathology Division, Tokyo, Japan Background: Despite the increasing use of both sentinel lymph node biopsy (SLNB) and preoperative chemotherapy (PST) in patients with operable breast cancer, there is still limited information on the feasibility and accuracy of SLNB following PST. In this study, the feasibility and accuracy of SLNB for breast cancer patients with clinically negative lymph nodes after PSTwere investigated. In addition, we evaluated a new automated assay system for cytokeratin 19 mRNA, the one-step nucleic acid amplification (OSNA) assay (Sysmex), to detect lymph node metastasis of breast cancer after PST. Materials and methods: Between 2003 and 2008, 200 patients with Stage II and III breast cancer previously treated with PST were enrolled in this study. The eligible criteria for PST were (a) primary tumor > 3cm or (b) positive axillary lymph node status on initial examination. FNA biopsy was performed for clinically or ultrasonographically suspicious axillary lymph nodes. The patients then underwent SLNB, which involved a combination of intradermal injection over the tumor of radiocolloid and subareolar injection of blue dye. This was followed by Level I/ II axillary lymph node dissection (ALND). Surgically obtained 84 lymph nodes from 21 breast cancer patients received PST were examined with both OSNA assay and HE five hundred & seventy patients of stage I to III breast carcinoma have autologous breast reconstruction with modified extended LDF with added vascularised chest wall fat; 47% had SSM and the remaining had NSM. Age ranges from 23 to 53 years (median 1⁄4 40.5). Results: Subjective patient satisfaction was excellent in 71%, good in 20%, fair in 7% & poor in 2% of cases. Bilateral size & shape symmetry are excellent in 56%, good in 26%, fair in 12% & poor in 6% patient. The overall RT-related complications are 9 %, the most common complications are skin burns (5%) & fat necrosis (4%). Patients are followed for mean follow up of 75.5 months (2-96). Conclusion: Modified extended latissimus dorsi myocutaneous flap with added vascularised chest wall fat is a single stage totally autologous breast reconstruction allows reconstruction without the additional cost of an implant, many complications of synthetic implants, micro vascular procedure second stage surgery or surgical manipulation in the other breast. In addition the overall survival & local recurrence rates were similar to MRM.

Whole sentinel lymph node analysis by a molecular assay predicts axillary node status in breast cancer

Background:The one-step nucleic acid amplification (OSNA) assay is a rapid procedure for the detection of lymph node (LN) metastases using molecular biological techniques. The aim of this study was to assess the reliability of the whole sentinel lymph node (SLN) analysis by the OSNA assay as a predictor of non-SLN metastases.Methods:Consecutive 742 patients with breast cancer were enroled in the study. The association of non-SLN or ⩾4 LN metastases with clinicopathological variables was investigated using multivariate logistic analysis.Results:In total, 130 patients with a positive SLN who underwent complete axillary LN dissection were investigated. The frequency of non-SLN metastases in patients who were OSNA+ and ++ was 19.3% and 53.4%, respectively, and that in patients with ⩾4 LN metastases who were OSNA+ and ++ was 7.0% and 27.4%, respectively. The cytokeratin 19 (CK19) mRNA copy number (⩾5.0 × 103; OSNA++) in the SLN was the most significant predictors of non-SLN metastases (P=0.003). The CK19 mRNA copy number (⩾1.0 × 105) in the SLN was the only independent predictor of ⩾4 LN metastases (P=0.014).Conclusion:Whole SLN analysis using the OSNA assay could become a valuable method for predicting non-SLN and ⩾4 LN metastases.

One-step nucleic acid amplification assay (OSNA) for sentinel lymph node biopsy

The one-step nucleic acid amplification assay (OSNA) has been developed for the intraoperative rapid detection of sentinel lymph node metastasis. Pooled analysis of recent studies comparing OSNA with pathology indicated that OSNA is as accurate as pathology (96.3% concordance rate) and is useful for making the decision to omit axillary dissection for OSNA-negative patients (97.4% negative predictive value). The advantage of OSNA over pathology is that the former allows the semiquantitative evaluation of total tumor volume in the node when a whole node is examined. OSNA is expected to be a powerful tool for the estimation of risk of non-sentinel lymph node metastasis and also patient prognosis, though further studies about this issue with larger numbers of patients is needed.

Encuesta nacional sobre la actitud ante el hallazgo de metástasis en el ganglio centinela por cáncer de mama en distintas unidades hospitalarias

AimThe indication for axillary lymphadenectomy has changed as a main aspect in the management of breast cancer in the last few years, overall since the introduction of sentinel node biopsy. The objective of this study was to find out the current attitude as regards the involvement of the sentinel node. Patients and methodsA descriptive study was performed using the data obtained from a survey aimed specifically at Spanish Breast Units as regards the surgical and adjuvant treatment decisions made when sentinel node metastases were found. ResultsSixty-six (60%) of the 110 questionnaires sent out were completed. Sentinel node analysis is mainly performed during surgery (84.8%) and by using the one-step nucleic acid amplification assay (OSNA) (69.7%). The diagnosis of isolated tumour cells does not lead to a lymphadenectomy, although there was a wide variation when macrometastases or micrometastases were found (axillary dissection was performed in 86.3% and 33.3%, respectively). There was notable lack of uniformity in the criteria to indicate whether or not to perform a lymphadenectomy, as well as in the adjuvant therapy combination to use. ConclusionsThere is currently a wide variation in the attitudes of different Breast Units toward the finding of disease in the sentinel node, particularly when there is a low tumour load (micrometastases). This variability was seen in the indication of axillary lymphadenectomy as well as in the indication of adjuvant therapies in these cases.

Intraoperative Molecular Assessment for Lymph Node Metastasis in Head and Neck Squamous Cell Carcinoma Using One-Step Nucleic Acid Amplification (OSNA) Assay

Conventional intraoperative pathological examination for Sentinel node navigation surgery (SNNS) has been controversial. We evaluated the efficacy of one-step nucleic acid amplification (OSNA) assay for intraoperative diagnosis of cervical lymph node (CLN) metastasis compared with histopathological examination in patients with head and neck squamous cell carcinoma (HNSCC). A total of 175 CLNs dissected from 56 patients with HNSCC who underwent surgery at Aichi Cancer Center, Kyorin University, Gunma University or Fukushima Medical University, between April 2008 and December 2011 were enrolled. CLN samples were sectioned into four equal pieces, with two of each used for OSNA assay and other histopathological examinations. The diagnostic value of OSNA assay in HNSCC patients in predicting the results of histopathological diagnosis was evaluated using the area under the receiver operating characteristic (AUROC) curve. OSNA assay showed acceptable efficacy in the detection of pathological CLN metastasis (AUROC 0.918, 95% confidence interval [CI] 0.852-0.984). Regarding the CK19mRNA cutoff value, the optimum cutoff point in HNSCC patients was 131copies/μl (sensitivity: 82.4, 95% CI 65.5-93.2; specificity: 99.3, 95% CI 96.1-100.0; positive likelihood ratio 116.1; negative likelihood ratio 0.2]. We demonstrated that OSNA assay is useful in intraoperative diagnosis for CLN metastasis in patients with HNSCC. OSNA assay could be applied for SNNS in HNSCC patients.

Evaluation of a multiparametric system able to predict nonsentinel lymph node status in breast cancer patients with a micrometastatic sentinel node assessed by the one step nucleic acidamplification (OSNA) assay.

1050 Background: Axillary lymph node dissection (ALND) may not be necessary in women with breast cancer (BC) who have micrometastasis in a sentinel lymph node (SLN), owing to the low risk of non-SLN (NSLN) involvement. In our Institute we validated and adopted the molecular diagnostic tool OSNA based on the quantitative measurement of Cytokeratin 19 (CK19) mRNA.The aims of our work in a subgroup of women with micrometastatic SLN, were: 1) to correlate the copy numbers of CK19 mRNA with the risk of additional positive NSLNs; 2) to assess the relationships between the molecular subtype classification and the probability of a positive ALND; 3) to verify whether a combination of the new above mentioned parameters is able to identify a subgroup of patients with a micrometastatic SLN and a negligible risk of positive NSLNs in whom ALND may be avoided. Methods: The SLN lysates from 709 patients were analyzed by OSNA assay. We considered only patients with a micrometastatic SLN (copy numbers between 250 and 5000/μL) and the probability of having a positive ALND was calculated by the logistic regression model. This series of BC patients were divided into four main subtypes taking in account the BC classification as defined by a combination of estrogen, progesteron receptors and HER2 status. Results: OSNA positivity for micrometastasis was reported in 91/709 cases (12,8%).The number of patients with positive ALND was 20 (22%). The statistical analyses showed that the metastatic involvement of NSLNs is associated with SLNs with a high copy numbers (>2000) of CK19 mRNA together with HER2 subtype. Otherwise none of the luminal A patients with a positive SLN but presenting a copy number <1000, had a positive NSLNs. Conclusions: We showed that biologically-driven analyses may be able to build new models with higher performance in terms of breast cancer axillary status prediction after positive SLN biopsy for micrometastasis. The copy numbers of CK19 mRNA and the molecular subtypes are more advantageous than traditional parameters because they are not pathologist-dependent and therefore they are more reliable and reproducible.

Incidence and possible pathogenesis of sentinel node micrometastases in ductal carcinoma in situ of the breast detected using molecular whole lymph node assay

Background:The pathogenesis of lymph node metastases in preinvasive breast cancer – ductal carcinoma in situ (DCIS) – remains controversial. The one-step nucleic acid amplification (OSNA) assay is a novel molecular method that can assess a whole node and detect clinically relevant metastases. In this retrospective cohort study, we determined the performance of the OSNA assay in DCIS and the pathogenesis of node-positive DCIS.Methods:The subjects consisted of 623 patients with DCIS who underwent sentinel lymph node (SN) biopsy. Of these, 2-mm-sectioned nodes were examined using frozen-section (FS) histology in 338 patients between 2007 and 2009, while 285 underwent OSNA whole node assays between 2009 and 2011. The SN-positivity rate was compared between cohorts, and the characteristics of OSNA-positive DCIS were investigated.Results:The OSNA detected more cases of SN metastases than FS histology (12 out of 285, 4.2% vs 1 out of 338, 0.3%). Most of the metastases were micrometastases. The characteristics of high-risk DCIS (i.e., mass formation, size, grade, and comedo) and preoperative breast biopsy (i.e., methods or time to surgery) were not valid for OSNA assay–positive DCIS.Conclusion:The OSNA detects more SN metastases in DCIS than FS histology. Further examination of the primary tumours and follow-up of node-positive DCIS are needed to elucidate the pathogenesis.

One-step nucleic acid amplification for detecting lymph node metastasis of head and neck squamous cell carcinoma

Lymph node stage is an important prognostic factor in head and neck squamous cell carcinoma (HNSCC). We previously reported the clinical usefulness of sentinel lymph node biopsy diagnosed by genetic analysis using quantitative RT-PCR. However, this method takes about 3h. In this study, we attempted to develop a more efficient method for the intraoperative genetic detection of lymph node metastasis in HNSCC. A total of 312 lymph nodes (65 patients) were diagnosed by the one-step nucleic acid amplification (OSNA) method using GD-100. OSNA consists of a short homogenization step followed by amplification of cytokeratin 19 (CK19) mRNA directly from the lysate. Each lymph node was divided into two to diagnose metastasis. One half was used for the OSNA assay, and the other was subjected to semi-serial sectioning, sliced at 200-μm intervals and examined by H&E and cytokeratin AE1/AE3 immunohistochemical staining. The accuracy of OSNA assay was evaluated based on histopathological diagnosis. Sixty-one of 312 lymph nodes were pathologically metastasis-positive. The overall concordance rate between the OSNA assay using breast cancer criteria and histopathology was 94.2%. The optimal cut-off for the copy number of CK19 mRNA in assessing lymph node metastasis of HNSCC was 300 copies/μl, which had the highest diagnostic accuracy (95.2%). The OSNA assay can be completed within 30 min. The OSNA assay, which shows high sensitivity and specificity, suggests the possibility to be used as a novel tool for the genetic detection of lymph node metastasis in HNSCC patients.

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

Reliability of Whole Sentinel Lymph Node Analysis by One-Step Nucleic Acid Amplification for Intraoperative Diagnosis of Breast Cancer Metastases

To assess the reliability of using the One-Step Nucleic Acid Amplification (OSNA) assay as a single test on whole sentinel lymph nodes (SLN) as a method of intraoperative diagnosis and staging of SLNs in breast cancer. Combining histological and molecular assessment of metastasis on the same SLN may not fully reproduce the actual load of cancer cells present in the SLN and create problems in decisions regarding axillary dissection. Selection criteria for the whole SLN OSNA test required that the primary tumor expressed CK19 in more than 80% of tumor cells. Imprint cytology analysis of SLNs was performed together with the OSNA. Of the 279 patients enrolled for SLN evaluation, 123 gave consent to the OSNA protocol and 156 to the standard histology. Thirteen patients were excluded from OSNA evaluation because of low CK19 gene expression in the primary tumor; only 2.3% were truly negative. The kappa of concordance between the imprint cytology and OSNA results was 0.52. The rate of macrometastases determined by OSNA was 11% versus 20% determined by histology, whereas the rate of OSNA-micrometastases (18%) was significantly higher than that determined by histology (8%). The rate of SLN-negative cases was similar between the 2 protocols. Macrometastases correlated with the presence of vascular invasion in both protocols. The rate of axillary lymph node metastases was consistent with SLN tumor load. Intraoperative OSNA assay performed on the whole SLN gave objective and reproducible results that were useful for directing decisions regarding axillary dissection and for accurately defining the SLN stage.

Routine clinical use of the one‐step nucleic acid amplification assay for detection of sentinel lymph node metastases in breast cancer patients

The objective of this study was to confirm, by means of a multicenter study conducted in Japan, the reliability and usefulness of the one-step nucleic acid amplification (OSNA) assay in routine clinical use for sentinel lymph node biopsy (SLNB) of breast cancer patients. Patients with Tis-T2N0M0 breast cancer who underwent SLNB before systemic chemotherapy comprised the study cohort. A whole sentinel lymph node (SLN) was examined intraoperatively with the OSNA assay except for a 1-mm-thick, central slice of the lymph node, which underwent pathologic examination after the operation. For patients who underwent axillary dissection, non-SLNs were examined with routine pathologic examination. In total, 417 SLNBs from 413 patients were analyzed. SLN metastases were detected with greater sensitivity by the OSNA assay than by pathologic examination (22.5% vs 15.8%; P < .001), as expected from the difference in size of the specimens examined. Patients who had SLN metastases assessed with the OSNA assay proved to harbor non-SLN metastases with an overall risk ratio of 33.7%. The risk of non-SLN metastasis was significantly lower for patients who had positive SLNs assessed as OSNA+ than for those who had SLNs assessed as OSNA++ (17.6% vs 44%; P = .012). The OSNA assay can be used for routine clinical SLNB, and its assessment for volume of metastasis may be a powerful predictive factor for non-SLN metastasis. Further studies with more patients are needed to confirm the usefulness of this assay for selection in the clinical setting of patients who do not need axillary dissection.

Clinical application of the one-step nucleic acid amplification method to detect sentinel lymph node metastasis in breast cancer

The one-step nucleic acid amplification (OSNA) method can assess the expression level of cytokeratin 19 mRNA in sentinel lymph nodes in breast cancer. We compared the time required for diagnosis and concordance of results between the OSNA method and conventional intraoperative pathological examination. We then examined the relationship between the frequency of non-sentinel lymph node metastasis and (1) the expression level of CK19 mRNA in the sentinel lymph nodes and (2) clinico-pathological features of the primary tumor. In the comparison study, pairs of sentinel lymph node sections from 53 consecutive patients were examined: one section by hematoxylin-eosin staining and the other by OSNA assay. The latter involved reverse-transcription loop-mediated isothermal amplification of cytokeratin 19 mRNA, assessed quantitatively. In the second phase, 306 sentinel lymph nodes were removed from 248 consecutive patients, and whole sentinel lymph nodes were examined by OSNA assay alone. OSNA assay was a little more time-consuming than conventional pathological diagnosis (34-45 vs. 22-25 min, p < 0.0001). Concordance between the two methods was 93%. The frequency of non-sentinel lymph node metastasis (p < 0.0001) and the total number of lymph node metastases (p < 0.0001) increased with the amount of cytokeratin 19 mRNA on OSNA assay. We found no significant relationship between the amount of cytokeratin 19 mRNA in sentinel lymph nodes and breast cancer immunohistochemical subtype. The OSNA method is suitable to detect sentinel lymph node metastasis and to predict the possibility of non-sentinel metastasis. This semi-automated quantitative analysis system reduces the burden on pathologists.

P3-07-11: Multicenter Comparative Study between One-Step Nucleic Acid Amplification (OSNA) Whole Node Assay and Standard Frozen Section Histology: Intraoperative Molecular Assay for Sentinel Lymph Node Metastases in Early Breast Cancer Can Avoid a Second Surgery.

Abstract Studies on intraoperative assessment of sentinel nodes by frozen section in breast cancer have reported low sensitivity rates. Recently, a molecular-based procedure to detect lymph node metastases, one-step nucleic acid amplification (OSNA) assay, has been developed. OSNA assay can assess a whole lymph node and yields definitive semi quantitative results. The aim of this study was to evaluate the ability of intraoperative assessment comparing sentinel lymph node (SLN) metastases with OSNA assay versus routine H&amp;E frozen section (FS) histology. METHODS: A cohort of 478 consecutive patients diagnosed in 2010 with early breast cancer in two centers was analyzed. Frozen section was performed intraoperatively in 228 patients and OSNA assay in 250 patients. Patient characteristics were evaluated in both groups and rates of metastasis detected by both methods were compared. RESULTS: Patients and tumor characteristic are summarized in the table. A median of 1,95 SLNs were assessed by FS and a median of 2,06 SLNs by OSNA (p=0.15). Intraoperative histopathological assessment detected 67 SLNs metastasis by FS and 107 SLNs metastasis by OSNA assay (p=0.07). There were no differences in rates of macrometastasis (54 by FS and 74 by OSNA) and micrometastasis (13 by FS and 33 by OSNA) (p=0.09). When compared the OSNA assay with permanent section, 92 SLNs had metastasis by permanent section (63 macrometastasis and 29 micrometastasis) with no statistically significant differences (p=0.5). SLNs metastasis were found in 71 patients (31,1 %) by permanent section and in 83 (33,2 %) by OSNA assay (p=0.69). Axillary lymph node dissection (ALND) for metastatic SLN was performed in 148 patients, 79 (31,6 %) in OSNA group and 69 (30,2 %) in the permanent section group (p=0.68). All patients diagnosed by OSNA had a complete ALND during the initial surgical procedure. On the other hand, ALND was performed in 51 patients (73,9 %) in the permanent section in the initial surgery, and ALND was performed in a second surgical procedure in18 patients (26 %), due to false negative results of the FS (p&amp;lt;0.001). CONCLUSIONS: The OSNA assay can detect SLN metastasis as accurately as conventional pathology, with no increased detection of positive SLNs. Given the definitive pathology of the SLN intraoperatively, the use of OSNA can reduce the need for a second surgery in 26 % of patients with breast cancer and a positive SLN. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-07-11.

P3-07-03: One-Step Nucleic Acid Amplification (OSNA) for the Diagnosis of Sentinel Lymph Nodes of Breast Cancer – Results of the China Multicenter Study CBCSG-001c.

Abstract Background With the adoption of sentinel lymph node (SLN) biopsy as the standard of care, there is an increasing need for the rapid and accurate intra-operative diagnosis of SLNs. CBCSG-001c was a prospective multicenter trial to validate the One-step nucleic acid amplification (OSNA) assay in China. The primary endpoint was the concordance rates of intraoperative OSNA assay with the in-depth permanent histological analyses based both on cases and SLNs. Methods: From Feb. to Dec. 2010, 1188 SLNs from 552 breast cancer patients were enrolled in the CBCSG-001c study at 5 centers. SLNs were cut into alternating ∼2mm sections. One half of the sections were sampled for H&amp;E, with 4 sections at different intervals. The other half was fully tested with the OSNA assay. Predetermined cutoffs were calibrated so only metastases &amp;gt;0.2 mm were detected. Results: The concordance rate was 89.1% (95% CI, 86.3−91.5%), sensitivity 87.7% (95% CI, 81.0−92.7%), and specificity 89.6% (95% CI, 86.3−92.4%) based on 552 cases, and the concordance rate was 91.4% (95% CI, 89.7−92.9%), sensitivity 83.7% (95% CI, 77.7−88.6%), and specificity 92.9% (95% CI, 91.1−94.4%) based on the 1188 SLNs. This quantitative molecular assessment allows the distinction of the size of the metastasis, and the PPV of OSNA [++] for macrometastases was 83.2% (95% CI, 75.0- 89.1%). Discordant results were thought to be partly due to the fact that different tissue sections were used for OSNA assay and histology, and SLNs with ITCs were not considered as histological positive nodes. After discordant case investigation, the senstivity of OSNA assay was significantly higher than that of intraoprative frozen section and touch imprint cyctology. Discussion: As the largest OSNA study to date, our results, together with that of Japan, Germany, and France study, proved the OSNA assay based on CK19 mRNA expression to be a reliable and standardized tool for the intraoperative detection of SLN metastases of breast cancer patients as compared to in-depth permanent histology. The high sensitivity of OSNA assay means reducing the risk of second operation for ALND, medical care costs and patients anxiety. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-07-03.