One-step Nucleic Acid Amplification Research Articles

P01. Can one-step nucleic acid amplification assay predict 4 or more positive axillary lymph node involvement in breast cancer patients: A single centre retrospective study

P01. Can one-step nucleic acid amplification assay predict 4 or more positive axillary lymph node involvement in breast cancer patients: A single centre retrospective study

Sentinel lymph node processing in gynecological cancer histopathology and molecular biology.

As sentinel lymph nodes (SLNs) are the first nodes receiving drainage from primary tumors, they provide important prognostic information about the nodal status of a tumor. SLN biopsy has modified the lymph node assessment by pathologists. This review highlights the different ways of histopathological and molecular SLN assessment according to the different gynecological cancers. Other than in breast cancer and melanoma, frozen section (FS) analysis of SLN in gynecological malignancies is still considered an important diagnostic tool. Intraoperative evaluation of the SLN allows to determine the need of completing lymph node dissection in case of metastasis. Intraoperative FS has a high negative predictive value (NPV) and is more sensitive than imprint cytology (IC) alone. If on intraoperative examination on FS the SLN is negative, subsequent analysis of the entire lymph node and histological ultrastadification has the potential to detect occult low volume metastases or to ascertain that a SLN is really negative. This reduces the morbidity compared to systematic pelvic and paraaortic lymph node dissection. Inclusion of the entire lymph node tissue in paraffin blocks after cutting it in 2 mm thick slices and histopathological ultrastaging with serial sections provides important prognostic information about the need of adjuvant treatment. Three sections at 200-250 µm seem to identify the majority of micrometastases. This review discusses different histopathological protocols and molecular [qRT-PCR and one-step nucleic acid amplification (OSNA®)] aspects of SLN evaluation in gynecological cancer.

Survey of the clinical practice pattern of using sentinel lymph node biopsy in patients with gynecological cancers in Japan: the Japan Society of Gynecologic Oncology study.

Sentinel lymph node (SN) biopsy is essential for evaluating survival and minimal treatment-related morbidity associated with cervical, endometrial, and vulvar cancer in Japan. As such, our aim in this study was to evaluate the current practice pattern of using SN biopsy for cervical, endometrial, and vulvar cancer in Japan. We deployed a 47-question survey on the use of SN biopsy for gynecological cancers to 216 gynecological oncology training facilities. The survey included information on the use of SN biopsy for uterine (cervical and endometrial) and vulvar cancers; details on the type, timing, and concentration of tracers used; surgical approach used for SN biopsy; method of biopsy and pathological examination; and facilities' experience with clinical research on SN biopsy. The response rate was 84% (181/216), with 40 facilities (22%) having experience in SN biopsy for gynecological cancers, 34 (85%) for uterine cancers, and 15 (37%) for vulvar cancers. Radioisotope, indocyanine green (ICG), and blue dyes were available for the detection of uterine cancers in 21 (52%), 25 (62%), and 19 (48%) facilities and for vulvar cancers in 9 (22%), 3 (7%), and 11 (27%) facilities, respectively. Thirty-four facilities (85%) used intraoperative frozen section procedure for diagnosis when possible, with 24 (71%) of these facilities using 2-mm specimen cuts. Diagnosis included pathological examination (85%), immunostaining (57%), and one-step nucleic acid amplification (5%). Increasing research evidence, providing insurance coverage for radioisotope tracers, and increasing the availability of training are expected to increase the use of SN biopsy in Japan.

Evaluation of Fast Molecular Detection of Lymph Node Metastases in Prostate Cancer Patients Using One-Step Nucleic Acid Amplification (OSNA).

Simple SummaryProstate cancer (PCa) is the second most common cancer and one of the highestcauses of cancer deaths among men. The presence of lymph node metastases (LNM) is the strongest prognostic factor. Histological detection of LNM is the gold standard for LN staging. In clinical practice, only fractions of LNs are examined histopathologically, resulting in missed (micro-)metastases. Biomolecular techniques can enhance the identification of LNM but are not routinely used because of high cost. One-step nucleic acid amplification (OSNA) analyzes the entire LN by detecting cytokeratin 19 mRNA as a surrogate for LNM requiring less effort and allowing intraoperative application. To verify the reliability of OSNA for the first time in PCa, we examined LNs of patients undergoing prostatectomy and sentinel lymphadenectomy. Results were compared with histopathological and immunohistochemistry findings. OSNA identified LNM equivalent to, or in cases of micrometastases, even better than enhanced histopathology, and might be useful in intraoperative decision-making in personalized LN surgery.Background: In clinical routine, only fractions of lymph nodes (LNs) are examined histopathologically, often resulting in missed (micro-)metastases and incorrect staging of prostate cancer (PCa). One-step nucleic acid amplification (OSNA) analyzes the entire LN by detecting cytokeratin 19 (CK19) mRNA as a surrogate for LN metastases requiring less effort than conventional biomolecular techniques. We aimed to evaluate performance of OSNA in detecting sentinel LN (SLN) metastases in PCa. Methods: SLNs (n = 534) of 64 intermediate- or high-risk PCa patients undergoing radical prostatectomy with extended and sentinel-guided lymphadenectomy were cut into slices and alternatingly assigned to OSNA and histopathology (hematoxylin-eosin staining, CK19, and CK AE1/AE3 immunohistochemistry). Sensitivity and specificity of OSNA and concordance and measure of agreement (Cohen’s kappa (κ)) between OSNA and histopathology were assessed. Results: Histopathology revealed metastases in 76 SLNs. Sensitivity and specificity of OSNA were 84.2% and 96.1%, respectively. Discordant results were recorded for 30 of 534 SLNs, revealing high concordance (94.4%). Twenty-four discordant cases were classified as micrometastases, indicating a possible allocation bias. In 18 cases, positive results were conferred only by OSNA resulting in seven LN-positive patients who were missed by histopathology. Overall, the level of agreement was high (κ = 0.78). Conclusions: OSNA provided a diagnosis that was as least as accurate as detailed histological examination and might improve LN staging in PCa.

Lymph Node Tumor Burden Correlates With Tumor Budding and Poorly Differentiated Clusters: A New Prognostic Factor in Colorectal Carcinoma?

INTRODUCTION:Molecular lymph node (LN) staging in early colorectal cancer (CRC) has demonstrated to be more precise than conventional histopathology pN staging. Tumor budding (TB) and poorly differentiated clusters (PDCs) are associated with LN metastases, recurrences, and lower survival in CRC. We evaluated the correlation between the total tumor load (TTL) in LNs from CRC surgical specimens with patient outcome, TB, and PDC.METHODS:In this retrospective multicentre study, 5,931 LNs from 342 stage I–III CRC were analyzed by both hematoxylin and eosin and molecular detection of tumor cytokeratin 19 mRNA by one-step nucleic acid amplification. TB and PDC were evaluated by hematoxylin and eosin and cytokeratin 19 immunohistochemistry.Results:One-step nucleic acid was positive in 38.3% patients (n = 131). Tumor Budding was low in 45% cases, intermediate in 25%, and high in 30%. Poorly Differentiated Clusters were low-grade G1 in 53%, G2 in 32%, and G3 in 15%. TB and PDC correlated with TTL, high-grade, lymphovascular and perineural invasion, pT, pN and stage (P < 0.001). TB, PDC, and TTL ≥ 6,000 copies/µL were associated with worse overall survival (P = 0.002, P = 0.013, and P = 0.046) and disease-free survival (P < 0.001).Discussion:The implementation of more sensitive molecular methods to assess LN status is a promising alternative approach to pN staging, which could be integrated to other factors to help risk stratification and management of patients with early-stage CRC. This study demonstrates the correlation of the amount of LN tumor burden with TB and PDCs. TTL is related to the outcome and could be used as a new prognostic factor in CRC (see Visual , Supplementary Digital Content 2, http://links.lww.com/CTG/A512).

Abstract PS1-04: Utility of rapid- immunohistochemistry using an alternating current electric field for intraoperative diagnosis of sentinel lymph nodes in breast cancer

Abstract Axillary lymph node status and pathological diagnosis of sentinel lymph nodes (SLNs) is an important factor that influences management of postoperative therapy. Recent reports indicate that one-step nucleic acid amplification (OSNA) and hematoxylin and eosin (HE)-stained frozen sections are effective for intraoperative diagnosis of SLNs. In the present study, we report a rapid-immunohistochemical staining (R-IHC) method that enables intraoperative detection of SLN metastases within 16 min using an anti-cytokeratin antibody. With this R-IHC system, we apply a high-voltage, low-frequency AC electric field to lymph node sections while they are incubating with the antibodies. The antibodies are mixed within microdroplets and the opportunity for contact between the antibody and antigen is increased. This greatly reduces the time required for the antigen-antibody reaction. This is the report on SLN diagnosis using R-IHC in patients with breast cancer. We prospectively examined 632 dissected SLNs from 260 breast cancer patients who underwent surgery at our institute between July 2014 and March 2020. The dissected SLNs were sectioned and conventionally stained with HE or immunohistochemically labeled with anti-cytokeratin antibody using R-IHC procedures. Intraoperative R-IHC analyses were completed within 16 min, after which diagnoses were made by two pathologists. The total time required for intraoperative diagnosis was about 20 min. In this study, R-IHC detected four metastatic SLNs that were undetected using conventional HE staining (10/52, 19.2%). Compared with subsequent permanent diagnosis, R-IHC offered 98.0% sensitivity, 100% specificity and 99.8% accuracy while those values for intraoperative HE were 80.4%, 100%, 98.4%, respectively (isolated tumor cells were counted as negative). These findings indicate R-IHC is a clinically applicable technique that enables precise, and quick intraoperative detection of micro- and macrometastases in breast cancer. Moreover, R-IHC is cost-effective method: it’s cost is less than a quarter of OSNA and previous report showed that the concentration of primary antibody could be reduced by more than 90% by using R-IHC because AC mixing activated antigen-antibody reaction. Another benefit is its ability to shorten the time needed for microscopic diagnosis itself. When using R-IHC, pathologists are able to readily find metastatic lesions, even within low-power fields (e.g. 40x). This reduces not only the time needed for intraoperative diagnosis, but also the effort necessary to find unclear lesions like small cancer cells (even though it is macrometastasis), micrometastasis, and artifacts of frozen section. This study was conducted at a single institute. Further investigation in a multi-institutional collaborative prospective study will be needed to confirm the utility of this method. But for now, R-IHC appears to be a cost-effective and clinically applicable method for diagnosis of breast cancer and SLN metastasis. Citation Format: Kaori Terata, Hiroshi Nanjo, Eriko Takahashi, Ryuta Nakamura, Yoichi Akagami, Ayuko Yamaguchi, Misako Yatsuyanagi, Chiaki Kudo, Yoshihiro Minamiya. Utility of rapid- immunohistochemistry using an alternating current electric field for intraoperative diagnosis of sentinel lymph nodes in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS1-04.

Predictive and prognostic value of total tumor load in sentinel lymph nodes in breast cancer patients after neoadjuvant treatment using one-step nucleic acid amplification: the NEOVATTL study

ObjectiveTo evaluate the predictive and prognostic value of total tumor load (TTL) in sentinel lymph nodes (SLNs) in patients with infiltrating breast cancer after neoadjuvant systemic therapy (NST).MethodsThis retrospective multicenter study used data from a Spanish Sentinel Lymph Node database. Patients underwent intraoperative SLN biopsy after NST. TTL was determined from whole nodes using a one-step nucleic acid amplification (OSNA) assay and defined as the total sum of CK19 mRNA copies in all positive SLNs. Cox-regression models identified independent predictive variables, which were incorporated into a nomogram to predict axillary non-SLN metastasis, and identified prognostic variables for incorporation into a disease-free survival (DFS) prognostic score.ResultsA total of 314 patients were included; most had no lymph node involvement prior to NST (cN0; 75.0% of patients). Most received chemotherapy with or without biologic therapy (91.7%), and 81 patients had a pathologic complete response. TTL was predictive of non-SLN involvement (area under the concentration curve = 0.87), and at a cut-off of 15,000 copies/µL had a negative predictive value of 90.5%. Nomogram parameters included log (TTL + 1), maximum tumor diameter and study-defined NST response. TTL was prognostic of disease recurrence and DFS at a cut-off of 25,000 copies/µL. After a 5-year follow-up, DFS was higher in patients with ≤ 25,000 copies/µL than those with > 25,000 (89.9% vs. 70.0%; p = 0.0017).ConclusionsTTL > 15,000 mRNA copies/µL was predictive of non-SLN involvement and TTL > 25,000 mRNA copies/µL was associated with a higher risk of disease recurrence in breast cancer patients who had received NST.

Molecular Lymph Node Staging with One-Step Nucleic Acid Amplification and its Prognostic Value for Patients with Colon Cancer: The First Follow-up Study

BackgroundMolecular lymph node workup with one-step nucleic acid amplification (OSNA) is a validated diagnostic adjunct in breast cancer and also appealing for colon cancer (CC) staging. This study, for the first time, evaluates the prognostic value of OSNA in CC.Patients and methodsThe retrospective study includes patients with stage I-III CC from three centres. Lymph nodes were investigated with haematoxylin and eosin (H&E) and with OSNA, applying a 250 copies/μL threshold of CK19 mRNA. Diagnostic value of H&E and OSNA was assessed by survival analysis, sensitivity, specificity and time-dependent receiver operating characteristic curves.ResultsEighty-seven patients were included [mean follow-up 53.4 months (± 24.9)]. Disease recurrence occurred in 16.1% after 19.8 months (± 12.3). Staging with H&E independently predicted worse cancer-specific survival in multivariate analysis (HR = 10.77, 95% CI 1.07–108.7, p = 0.019) but not OSNA (HR = 3.08, 95% CI 0.26–36.07, p = 0.197). With cancer-specific death or recurrence as gold standard, H&E sensitivity was 46.7% (95% CI 21.3–73.4%) and specificity 84.7% (95% CI 74.3–92.1%). OSNA sensitivity and specificity were 60.0% (95% CI 32.3–83.7%) and 75.0% (95% CI 63.4–84.5%), respectively.ConclusionsIn patients with CC, OSNA does not add relevant prognostic value to conventional H&E contrasting findings in other cancers. Further studies should assess lower thresholds for OSNA (< 250 copies/μL).

Total Tumor Load of mRNA Cytokeratin 19 in the Sentinel Lymph Node as a Predictive Value of Axillary Lymphadenectomy in Patients with Neoadjuvant Breast Cancer.

Although sentinel lymph node biopsy (SLNB) has proved to be able to diagnose axillary lymph node status safely and reliably, there is still not enough evidence to suggest that it can be used in patients who have undergone neoadjuvant chemotherapy (NAC) for lymph node-sparing surgery. The present study used molecular approaches to determine whether SLNB can be reliably used in patients who have been treated with NAC before SLN surgery, and whether the total tumor load of the SLN can be used as a predictive factor in axillary lymphadenectomy (ALD). We used one-step nucleic acid amplification (OSNA) to analyze a total of 111 consecutive patients who presented operable invasive breast carcinomas and who had been treated with NAC. SLN was positive in 55 patients and the identification rate was 100%. In 9 of these 55 patients, ALD showed that other lymph nodes were also involved. In all of the other 46 patients, the only lymph node to be identified as positive was SLN. Metastasis was not found in any of the axillary lymph nodes in the isolated tumor cell group. The total tumor load, defined as the amount of cytokeratin 19 mRNA copy numbers in all positives SLN (copies/µL), showed three risk groups related to the possibility of positive non-sentinel nodes. OSNA is a diagnostic technique that is highly sensitive, specific, and reproducible and it can be used to analyze sentinel lymph nodes after NAC. Total tumor load may be able to help predict additional metastases in axillary lymphadenectomy.

Application of OSNA Nomogram in Patients With Macrometastatic Sentinel Lymph Node: A Retrospective Assessment of Accuracy

Introduction:Almost 50% to 70% of patients who undergo axillary lymph node dissection (ALND) because of a single metastatic sentinel lymph node (SLN) have no further metastatic nodes at the axillary histology. On these grounds, the one-step nucleic acid amplification (OSNA) nomogram was designed and validated. As a mathematical model, calculated through tumor size (expressed in millimeters) and CK19 mRNA copy number, it is thought to predict nonsentinel lymph node (NSLN) status. The aim of the study is to verify the diagnostic accuracy of the OSNA nomogram in a group of patients with macrometastatic SLN, with a retrospective analysis.Methods:The OSNA nomogram was retrospectively applied to a group of 66 patients with macrometastatic SLN who underwent ALND. The result of the final histology of the axillary cavity was compared to the nomogram prediction. We calculated the prevalence of NSLN metastasis in patients who underwent ALND, sensitivity and specificity, negative and positive predictive value of the nomogram.Results:In patients with macrometastasis in SLN, the prevalence of patients with metastatic NSLN was 45%. The sensitivity of the nomogram was excellent (90%). The specificity was low (36%). Positive predictive value amounted to 54%, while negative predictive value was good (81%).Conclusions:These results suggest that the OSNA nomogram is a valid instrument that can help choose the best surgical strategy for the treatment of axillary cavity. The mathematical model is useful to avoid surgery in a selected group of patients because it accurately predicts NSLN status.

Molecular Lymph Node Staging with One-Step Nucleic Acid Amplification in Colon Cancer and its Prognostic Value

Molecular Lymph Node Staging with One-Step Nucleic Acid Amplification in Colon Cancer and its Prognostic Value

Is it possible to predict non sentinel node positivity on the basis of mRNA copy numbers of CK19 receptor in breast cancer?

Background: One step nucleic acid amplification (OSNA) has found wide acceptance as a tool for intraoperative assessment of the sentinel lymph node (SLN). This is a semiquantitative assay based on quantification of mRNA copy numbers of cytokeratin 19 (CK19) receptor. The study aimed to determine if there is any correlation between the number of positive non sentinel lymph nodes (NSLN) and the mRNA copy numbers of CK19 receptor on OSNA in the SLN.

In Vitro System for the Detection of Prostate Cancer Markers via Loop-Mediated Isothermal Amplification

Loop-mediated isothermal amplification (LAMP) of nucleic acids enables the detection of amplification products 10–20 min after the beginning of the reaction. An intraoperative method for the detection of metastases in lymph nodes based on LAMP, one-step nucleic acid amplification (OSNA), allows the rapid detection of the epithelial-specific marker gene KRT19 in lymph nodes. The standard protocol for OSNA was developed more than 10 years ago to detect breast cancer metastases in lymph nodes. Since then, a new version of the key enzyme involved in the reaction (Bst-polymerase) was obtained, but its use in OSNA has remained unexplored. Moreover, the time has come to apply OSNA to the detection of other cancer types, in particular, prostate cancer. The first step is to create an in vitro system that allows LAMP to be carried out on prostate-cancer cell lines. In this work, the LAMP protocol was developed for the new Bst 3.0 polymerase with optimized dNTP concentrations and without reverse transcriptase, and cell lines were selected (DU-145 for prostate cancer and Molt-4 for negative control). As a result, a new, in vitro system for the LAMP-based detection of prostate cancer with the marker gene KRT19 was developed.

Evaluation of accuracy of One Step Nucleic acid Amplification (OSNA) in diagnosis of lymph node metastases of papillary thyroid carcinoma: a comparison between real-time PCR and histopatology

Papillary thyroid carcinoma (PTC) is the most common malignant thyroid neoplasia, representing 80% of thyroid tumors, progressively increased in the last decades by the diffusion of screening ultrasound. The incidence of node metastases is high, ranging from 20% to 90%.

Standard ultrastaging compared to one-step nucleic acid amplification (OSNA) for the detection of sentinel lymph node metastases in early stage cervical cancer

ObjectiveWe compared ultrastaging and one-step nucleic acid amplification (OSNA) examination of sentinel lymph nodes in two homogeneous patient populations diagnosed with early stage cervical cancer. The primary aim of our...

OPTimizing Irradiation through Molecular Assessment of Lymph node (OPTIMAL): a randomized open label trial

BackgroundConservative surgery followed by breast and nodal irradiation is the standard loco-regional early breast cancer (BC) treatment for patients with four or more involved lymph nodes. However, the treatment strategy when fewer nodes are involved remains unclear, especially when lymphadenectomy has not been performed. Sensitive nodal status assessment molecular techniques as the One-Step Nucleic Acid Amplification (OSNA) assay can contribute to the definition and standardization of the treatment strategy. Therefore, the OPTIMAL study aims to demonstrate the feasibility of incidental irradiation of axillary nodes in patients with early-stage BC and limited involvement of the SLN.MethodsBC patients who underwent conservative surgery and whose SLN total tumour load assessed with OSNA ranged between 250–15,000 copies/µL will be eligible. Patients will be randomized to receive irradiation on the breast, tumour bed, axillary and supraclavicular lymph node areas (intentional arm) or only on the breast and tumour bed (incidental arm). All areas, including the internal mammary chain, will be contoured. The mean, median, D5% and D95% doses received in all volumes will be calculated. The primary endpoint is the non-inferiority of the incidental irradiation of axillary nodes compared to the intentional irradiation in terms of 5-year disease free survival. Secondary endpoints comprise the comparison of acute and chronic toxicity and loco-regional and distant disease recurrence rates.DiscussionStandardizing the treatment and diagnosis of BC patients with few nodes affected is crucial due to the lack of consensus. Hence, the quantitative score for the metastatic burden of SLN provided by OSNA can contribute by improving the discrimination of which BC patients with limited nodal involvement can benefit from incidental radiation as an adjuvant treatment strategy.Trial registrationClinicalTrial.gov, NCT02335957; https://clinicaltrials.gov/ct2/show/NCT02335957

Rapid Detection of Free Cancer Cells in Intraoperative Peritoneal Lavage Using One-Step Nucleic Acid Amplification (OSNA) in Gastric Cancer Patients.

Cytokeratin-19 (CK19) has been proven to be commonly expressed by cancer cells in a variety of solid tumors and may serve as a suitable marker of metastases in gastric cancer (GC). Since objective assessment of peritoneal lavage or fluid for free cancer cells (FCC) is essential for clinical decision making in patients with GC, it is important to develop a quantitative and reproducible method for such evaluation. We assessed the possible application of One-Step Nucleic Acid amplification (OSNA) assay as a rapid method for FCC detection in intraoperative peritoneal lavage or fluid of GC patients. Seventy-eight intraoperative peritoneal lavage or fluid samples were eligible for the analysis by conventional cytology and OSNA examination. The concentration of CK19 mRNA in intraoperative peritoneal lavage and fluid was compared with the conventional cytological assessment. CK19 mRNA concentration was detected by OSNA assay. For peritoneal lavage samples, sensitivity and specificity were 83.3% and 87.8%, respectively. In peritoneal fluid, significantly higher CK19 values were observed in patients with serosal infiltration (medians: 100 copies/µL vs. 415.7 copies/µL; p = 0.0335) and lymph node metastases (medians: 2.48 copies/µL vs. 334.8 copies/µL). OSNA assay turns out to be an objective, fast, and reproducible quantitative method of FCC assessment.

Molecular analysis of sentinel lymph nodes in patients with breast cancer using one-step nucleic acid amplification (OSNA): Does not lead to overtreatment in the current era of de-escalating axillary management

PurposeOSNA is a molecular technique for sentinel lymph node (SN) analysis in breast cancer. Compared to histology, OSNA may yield more (micro)metastases and thereby result in more axillary lymph node dissections or radiotherapy. We investigated whether axillary treatments increase when using OSNA, applying current guidelines for de-escalating axillary management. MethodsAll patients treated for cT1-3N0 breast cancer in our hospital between December 2013 and February 2016 were included. In 148 prospectively included patients (January 2015–February 2016), SN's were examined with OSNA. In a retrospective cohort of 123 patients (December 2013–December 2014), SN's were examined with conventional histology. Outcomes were: number of macro and micrometastases, amount of patients receiving axillary dissection or irradiation, number of patients receiving adjuvant systemic therapy. Data were analyzed using Mann-Whitney and Pearson Chi-square test. P < 0.05 was considered statistically significant. Results230 SN's from 123 patients were examined with conventional histology. 229 SN's from 148 patients were evaluated with OSNA. Amount of macrometastases was equal between groups (histology 17.9% versus OSNA 16.2%, p = 0.715). We found significantly more micrometastases when using OSNA (histology 11.4% versus OSNA 25.0%, p = 0.004). Total number of axillary lymph node dissections was comparable in both groups (histology 12.2%, OSNA 12.2%, p = 0.993), as well as number of axillary radiations (histology 8.9%, OSNA 11.5%, p = 0.493). Also, the number of patients receiving adjuvant systemic therapy was similar between conventional histology and OSNA (histology 53.7% versus OSNA 58.1%, p = 0.462). ConclusionOSNA analysis for SN in breast cancer is a highly sensitive technique, detecting more micrometastases than standard histology. When applying current guidelines, OSNA analysis does not lead to overtreatment with more axillary dissections or irradiation.

Axillary staging based on molecular analysis: Results of the B-CLOSER-II study

IntroductionAxillary staging (pN) is a strong predictor of outcome in early stage breast cancer yet following the publication of the Z0011 trial there has been an increasing tendency to spare lymph node dissection. Automated molecular detection of cytokeratin 19mRNA by one-step nucleic acid amplification (OSNA) has been demonstrated to be an accurate method to assess sentinel lymph node (SLN) metastasis. In this study we compare histological and molecular methods following complete axillary lymph node dissection (cALND), determine whether molecular axillary staging affects survival, and evaluate the predictive and prognostic value of total tumor load in ALND (AD-TTL) and in all positive nodes (G-TTL). Material and methodsAxillary lymph nodes were collected from 102 patients with primary breast cancer with histological confirmation of axillary involvement (cN+) or positive SLN. The central 1-mm portion of each non-SLN was processed for hematoxylin-eosin staining and the remaining tissue was analyzed by OSNA. ResultsNon-SLNs were diagnosed as positive in 72 out of 102 patients (70.6 %) on OSNA compared with only 53 (52 %) on histology (p < 0.01). Thirteen patients would have changed staging if the diagnoses provided had been by molecular methods (p < 0.01), but without a change in prognosis. AD-TTL and G-TTL were predictive of recurrence and mortality. ConclusionsCompared to molecular detection, histological examination significantly underestimates the frequency of axillary node metastases. However, the increase in pN did not show a clinical effect on survival in those patients.

180P Development of prognosis prediction model using cytokeratin 19 mRNA copy number of sentinel lymph node metastasis in breast cancer: A multicenter study in Japan

The one-step nucleic acid amplification (OSNATM) assay (Sysmex, Kobe, Japan) is an intraoperative diagnostic procedure performed for the detection of lymph node metastases using molecular techniques to amplify and detect cytokeratin 19 (CK19) mRNA. This multicenter study aimed to construct stratification of prognosis by CK19 mRNA copy number and development of a prognosis prediction model in breast cancer patients.