Sentinel Lymph Node Analysis Research Articles

Axillary dissection compared to sentinel node biopsy for the treatment of pathologically node-negative breast cancer: a meta-analysis of four randomized trials with long-term follow up.

Sentinel lymph node biopsy is now accepted as the initial approach for women with early stage breast cancer with clinically node-negative disease. We performed a pooled analysis of trials comparing axillary lymph node dissection to sentinel lymph node biopsy in patients with early stage breast cancer and pathologically negative sentinel lymph node analysis. A systematic MEDLINE review identified four randomized trials of axillary dissection versus sentinel lymph node biopsy in lymph node-negative early stage breast cancer patients. A meta-analysis was performed for survival and relapse. The combined analyses of these four trials found no significant difference in overall survival (relative risk [RR] 1.15; P=0.16; 95% CI: 0.95–1.39), breast cancer-specific (RR 1.03; P=0.85; 95% CI: 0.751.43) and disease-free survival (RR 1.07; P=0.3; 95% CI: 0.94–1.21), distant metastases (RR 1; P=0.98; 95% CI: 0.76–1.32), and ipsilateral breast recurrence (RR 1.64; P=0.34; 95% CI: 0.60–4.47) associated with sentinel lymph node biopsy. In particular, a similar rate of nodal recurrences was seen after sentinel lymph node biopsy (RR 1.74; P=0.13; 95% CI: 0.86–3.53). Axillary dissection does not confer a survival benefit nor prevent further nodal relapses in the setting of early stage, pathologically lymph node-negative breast cancer.

Axillary dissection compared to sentinel node biopsy for the treatment of pathologically node-negative breast cancer: a meta-analysis of four randomized trials with long-term follow up

Sentinel lymph node biopsy is now accepted as the initial approach for women with early stage breast cancer with clinically node-negative disease. We performed a pooled analysis of trials comparing axillary lymph node dissection to sentinel lymph node biopsy in patients with early stage breast cancer and pathologically negative sentinel lymph node analysis. A systematic MEDLINE review identified four randomized trials of axillary dissection versus sentinel lymph node biopsy in lymph node-negative early stage breast cancer patients. A meta-analysis was performed for survival and relapse. The combined analyses of these four trials found no significant difference in overall survival (relative risk [RR] 1.15; P=0.16; 95% CI: 0.95-1.39), breast cancer-specific (RR 1.03; P=0.85; 95% CI: 0.75- 1.43) and disease-free survival (RR 1.07; P=0.3; 95% CI: 0.94-1.21), distant metastases (RR 1; P=0.98; 95% CI: 0.76-1.32), and ipsilateral breast recurrence (RR 1.64; P=0.34; 95% CI: 0.60-4.47) associated with sentinel lymph node biopsy. In particular, a similar rate of nodal recurrences was seen after sentinel lymph node biopsy (RR 1.74; P=0.13; 95% CI: 0.86- 3.53). Axillary dissection does not confer a survival benefit nor prevent further nodal relapses in the setting of early stage, pathologically lymph node-negative breast cancer.

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.

Intraoperative pathologische Sofortuntersuchung in der Mammachirurgie

In breast surgery intraoperative frozen sections for the diagnosis of malignancy has lost impact and has largely been replaced by preoperative core needle biopsies. Nevertheless, there is still need for immediate pathological investigation of native breast tissue during surgery due to three reasons: (1) macroscopic and microscopic evaluation of resection margins, (2) the histological analysis of sentinel lymph nodes in order to circumvent secondary axillary surgery and (3) the preparation of native tissue for tumor banking or measurement of biomarkers. Because histology provides only a facultative component of immediate pathological examination of breast specimens during surgery, the term frozen section does not seem to be appropriate anymore. Intraoperative evaluation of resection specimens by pathologists provides surgically relevant information immediately, guarantees standardized preparation and fixation of specimens and enables tissue banking of native tissue for assessment of biological markers.

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 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>5000), “+” (250 < CK19 mRNA copy number < 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<.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 <.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.

Scoring system for prediction of having three or more involved axillary nodes for breast cancer.

1133 Background: Completion axillary lymph node dissection (ALND) is currently the standard of care in the event of a positive sentinel lymph node biopsy (SLNB). However, result from Z0011 indicate that women with a one or two involved axillary nodes and clinical T1-T2 tumors undergoing lumpectomy with radiation therapy followed by systemic therapy do not benefit from completion of ALND in terms of survival. The purpose of this study was to define possible predictors of having three or more involved axillary node to provide information for surgeons making decision about sparing intraoperative frozen section analysis of sentinel lymph node and completion ALND. Methods: We reviewed the records of 1215 patients with clinical T1-T2 invasive breast cancer. None of these patients were in situ cancer on initial gun biopsy nor received neoadjuvant chemotherapy. Factors associated with having three or more involved axillary nodes were evaluated by univariate and multivariate logistic regression analysis. Results: Among 1215 patients, 321 patients had three or more positive nodes. On a multivariate analysis, having three or more positive nodes was associated with primary tumor size by breast US, axillary LN grade according to cortical thickness by US, presence of axillary LN enlargement on chest CT and age. A scoring system to predict the probability of having three or more nodes based on patients’ data and preoperative image findings was developed from the multivariate logistic regression model. The area under the ROC curve was 0.827 (95% CI: 0.793-0.860), and negative predictive value was 90.2% for a score ≤2.7. Similar findings were observed for a validation dataset of 505 patients. Conclusions: Patients with a low probability of having three or more positive nodes can be identified from preoperative image finding. The scoring system developed will be helpful to surgeons making decision about sparing intraoperative frozen section analysis of sentinel lymph node and completion ALND.

Impact of immunohistochemistry in sentinel lymph node biopsy in head and neck cancer

To determine the sensitivity, specificity, negative predictive value (NPV) and accuracy of hematoxylin-eosin (HE) staining compared to immunohistochemistry (IHC) in sentinel lymph node (SLN) histological analyses of head and neck squamous cell carcinoma. The Clinical prospective study was carried out at Tertiary referral university hospital. Patients with oral, lip and oropharyngeal squamous cell carcinoma undergoing elective neck dissection with clinically and radiologically negative necks were included. All patients were submitted to computer tomography scan for the evaluation of lymphatic metastases. The surgical procedure consisted of tumor resection, SLN sampling and elective neck dissection. Negative SLNs via HE were then submitted for IHC analysis of cytokeratin AE1/AE3 and step serial section (SSS). The main outcome measures were the negative predictive value of conventional HE staining techniques in the diagnosis of lymphatic metastases with the SSS/IHC analysis. Of 46 patients undergoing 63 neck procedures, 53 were SLN negative and 10 were positive on HE analysis. Using SSS/IHC analysis of these 53 negative SLNs on HE, two (3.8 %) were found to be positive. For HE, the sensitivity, specificity, NPV and accuracy were 77, 100, 94, and 95 %, respectively. With subsequent analysis with SSS/IHC, these values increased to 92, 100, 98 and 98 %, respectively. SSS/IHC is important in SLN analysis as the false negative rate decreased significantly while increasing the inherent sensitivity of the analyses.

Importance of assessing CK19 immunostaining in core biopsies in patients subjected to sentinel node study by OSNA

Analysis of sentinel lymph node (SLN) by means of One-Step Nucleic Acid Amplification (OSNA) is being used increasingly as a very sensitive and quick method for intraoperative axillary staging in patients with breast cancer. This molecular diagnostic assay detects the expression level of cytokeratin 19 (CK19), a luminal epithelial cell marker broadly expressed in most breast carcinomas and not normally found in lymph nodes. Almost all breast cancers express this cytoskeleton protein, but some breast tumors have been found to lose the expression of CK19. CK19 immunostaining in core biopsies has been recommended in selecting patients eligible for OSNA analysis because SLNs with metastatic involvement by CK19-negative breast cancers may result in a false negative result by OSNA. However, the real frequency of CK19-negative breast cancer has to be elucidated. In this study, we have assessed the frequency and molecular profile of CK19-negative breast carcinomas in three series of cases. The first is a prospective series of 197 breast carcinomas, 111 of which were subjected to SLN evaluation by OSNA. The second is a retrospective series of 41 triple-negative (TN) breast carcinomas, and the third is a retrospective series of 68 breast cancer patients (matched core biopsies and metastatic lymph nodes) that had been evaluated by conventional procedures before the OSNA methodology was adopted in our institution. Our results not only demonstrate that lack of expression of CK19 is infrequent in breast cancers but also that performing CK19 immunohistochemical staining is important on diagnostic core biopsies in taking the decision of using OSNA methodology in the evaluation of sentinel nodes in breast cancer patients.

Abstract 4548: Prognostic value of occult metastases detected by cytokeratin 20 and mucin 2 mRNA quantification in sentinel lymph nodes from colon cancer patients

Abstract BACKGROUND: Lymph node (LN) metastasis status is among the strongest prognostic factors in colon cancer. Despite this, 20-30% of LN negative patients experience disease recurrence. These patients may have occult LN metastases that are not detected by routine microscopy. The aim of the present study was to investigate the prognostic value of occult metastases detected by cytokeratin 20 (CK20) and mucin 2 (MUC2) mRNA quantification in sentinel lymph nodes (SLNs) from patients curatively resected for colon cancer. METHODS: Resection specimens from consecutive patients undergoing surgery for localized colon cancer were subjected to ex vivo SLN mapping. The isolated SLNs were examined for the presence of metastases by routine HES staining and quantitative CK20 and MUC2 mRNA measurements. Primary tumor microsatelite instability- (MSI), KRAS and BRAF mutation status were determined for the included patients. Recurrence-free and disease-specific survivals were analyzed by Kaplan-Meier survival estimates and log rank tests. RESULTS: SLNs were identified in 206 (97%) of the 213 included patients. Routine histological analysis of SLNs and other regional lymph nodes revealed LN metastases in 63 patients (pN+), of which 42 (67%) had metastases detected in one or more SLNs (sensitivity 67%, false negative rate 33%). When analyzing the SLNs by CK20 and MUC2 mRNA quantification, occult metastases were suggested in 86 (60%) and 52 (36%) of the 143 otherwise LN negative (pN0) patients, respectively. Survival analysis with a median 3.6 year follow-up revealed a significant prognostic value of MUC2 mRNA quantification in SLNs from all the patients, but not for the pN0 patients. For CK20 mRNA levels in SLNs we observed no statistically significant survival differences. Patient stratification according to primary tumor MSI, KRAS mutation and BRAF mutation status did not reveal any significant survival differences related to the suggested occult SLN metastases, neither. CONCLUSIONS: Occult SLN metastases detected by quantitative CK20 and MUC2 mRNA measurements did not demonstrate any prognostic value in this study. 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 4548. doi:1538-7445.AM2012-4548

The intraoperative molecular analysis of sentinel lymph node metastases and micro-metastases in breast cancer patients using One Step Nucleic Acid Amplification on whole nodes

Introduction: Intraoperative sentinel lymph node (SLN) analysis in breast cancer should be automated, concordant with histopathology and practical. One step nucleic acid amplification (OSNA), a highly sensitive validated intraoperative assay of cytokeratin 19 mRNA, is used for the detection of SLN macro- and micro-metastases in breast cancer. We present our 35 month data since introduction in December 2008.

Repeat surgery following Breast Conservation and Intra-operative Sentinel Lymph Node Analysis for Breast Cancer

Abstract Introduction Intra-operative sentinel node analysis (IOA) for breast cancer reduces the need for a second operation by revealing metastasis intra-operatively, allowing immediate axillary clearance. Critics argue that the number of patients deriving benefit is limited, as further surgery is often required for reasons other than nodal status. Aim To identify the proportion of women avoiding further surgery by using IOA excluding those who require further surgery for reasons other than axillary node metastasis. Patients and methods All patients undergoing sentinel node biopsy with IOA over one year were reviewed. Patient demographics, margin positivity, sentinel node metastasis, requirement for further surgery, and cavity shave involvement were analysed. Results 322 patients were analysed: 253 undergoing breast-conserving surgery [BCS] and 69 undergoing mastectomy). IOA revealed metastasis in 81 (25.2.%) patients [25 undergoing mastectomy and 56 undergoing BCS], who underwent immediate axillary clearance. 43 BCS patients (17%) did not require further surgery other than for sentinel node involvement. 39 patients required further oncological surgery: 16 excision of margins; 13 completion mastectomy; 6 excision of margins followed by mastectomy; 3 completion axillary clearance; and 1 excision of recurrence. 20.6% had involvement of any circumferential histological margin. Cavity shaves were performed in 28.5% patients at initial surgery, the majority of which were clear of malignancy. 20 mastectomy patients had concordant definitive histology, avoiding a second operation. In total, 19.6% of this cohort avoided a second operation through the use of IOA. Discussion Approximately 15% of patients undergoing breast conservation surgery for breast cancer require further surgery. However, a further 17% were saved subsequent surgery by utilising IOA, since they had immediate axillary clearance. When also considering patients undergoing mastectomy, this proportion is even higher.

Do we need intraoperative sentinel lymph node analysis when preoperative ultrasound staging is so accurate?

Introduction: Preoperative axillary ultrasound is a mandatory staging investigation for patients with breast cancer. We analysed data to evaluate the accuracy of ultrasound in our unit and correlated this with postoperative histopathology. In particular we aimed to determine if those patients with lymph node metastasis, in whom a preoperative diagnosis was not made, have low volume metastatic disease. Following publication of the results of ACOSOG Z0011, axillary lymph node dissection may not be required for all patients with a positive sentinel lymph node, especially those with low volume disease.

How Much is Enough? Pathologic Evaluation of Sentinel Lymph Nodes

It is well accepted that detailed analysis of sentinel lymph nodes (SLNs) may upstage breast cancer, but generally involves the identification of low-volume metastases (including isolated tumor cells and clusters). Although several guidelines recommend therapeutic interventions for micrometastases, there is also evidence supporting therapeutic interventions only for macrometastases. There is also evidence in support of means of regional disease control other than axillary lymph node dissection (ALND). The pathologic evaluation of SLNs should consider the clinical setting. Intraoperative assessment is indicated only if ALND is an immediate interventional option for SLN positivity. Generally, hematoxylin and eosin-stained sections taken at 2-mm step intervals are sufficient to disclose macrometastases, but if micrometastases are also considered in further treatment planning, smaller intervals and immunohistochemistry as well as quantitative molecular methods may also be considered.

Is there a requirement for axillary lymph node dissection following identification of micro-metastasis or isolated tumour cells at sentinel node biopsy for breast cancer?

IntroductionRecent decades have seen a significant shift towards conservative management of the axilla. Increasingly, immunohistochemical analysis of sentinel nodes leads to the detection of small tumour deposits, the significance of which remains uncertain. The aims of this study are to examine patients whose sentinel lymph nodes are positive for macro-metastasis, micro-metastasis or isolated tumour cells (ITCs) and to determine the rate of further nodal disease after axillary lymph node dissection (ALND). MethodsA retrospective analysis of all patients undergoing a sentinel lymph node biopsy (SLNB) between January 2007 and December 2010 in a tertiary referral breast unit was performed. Patients who underwent an axillary lymph node dissection for macro-metastasis, micro-metastasis or ITCs were identified. Demographics, histological data and the rate of further axillary disease were examined. ResultsIn total, 664 breast cancer patients attended the symptomatic breast unit during the study period, 360 of whom underwent a SLNB. Seventy patients had a SLNB positive for macro-metastasis. All of these patients underwent ALND. A positive SLNB with either micro-metastasis or ITCs was identified in 58 patients. Only 41 of the 58 patients went on to have an ALND, due primarily to variations in surgeons' preferences. Nineteen patients with micro-metastasis underwent an ALND. Four patients had further axillary disease (21%). Twenty-two patients had ITCs identified, of whom only one had further disease (4.5%). No statistically significant difference was found between the two groups in terms of tumour size, grade, lymphovascular invasion or oestrogen receptor status. ConclusionALND should be considered in patients with micro-metastasis at SLNB. It should rarely be employed in the setting of SLNB positive for ITCs.

The prognostic value of a reverse transcriptase-PCR assay of sentinel lymph node biopsy for patients with cutaneous melanoma

Sentinel lymph node biopsy (SLNB) is the standard of care for staging melanoma. However, limited research has been carried out on the prognostic value of SLNB in patients with primary cutaneous melanoma in Asian countries. The objective is to evaluate the efficacy of SLNB in Japanese patients with primary cutaneous melanoma and to elucidate whether reverse transcriptase (RT)-PCR analysis of sentinel lymph nodes (SLNs) is valuable for predicting patient outcome. A total of 101 patients with primary cutaneous melanoma underwent SLNB at the Department of Dermatology, Kyushu University (Fukuoka, Japan), between May 2001 and December 2009. The removed nodes were stained with hematoxylin-eosin and with immunohistochemical stains for HMB-45, tyrosinase, MART-1, and MITF and multiple-mRNA marker (MART-1, tyrosinase, and GP-100) RT-PCR assays were conducted. The following clinicopathological variables were evaluated: age, sex, histological type, tumor site, Breslow thickness, disease-free survival (DFS), and melanoma-specific survival (MSS). Several parameters were analyzed for DFS and MSS using the Kaplan-Meier method and Cox proportional hazards model. The success rate of identifying SLNs was 98% (99 of 101 cases). Tumor-positive SLNs were significantly correlated with higher Breslow thickness, stage, tumor subtype, and tumor site. Patients with tumor-positive SLNs had a significantly shorter MSS and DFS than those with tumor-negative SLNs (P=0.0153 and 0.0004, respectively). Patients with at least two positive markers in the RT-PCR assay had a significantly shorter DFS than those with less than one marker (P=0.013). SLNB and multimarker RT-PCR analysis are useful for predicting the prognosis of patients with melanoma.

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.

P3-07-23: Intraoperative Molecular Analysis of Sentinel Lymph Nodes in Breast Cancer Using One Step Nucleic Acid Amplification (OSNA).

Abstract Introduction: The OSNA method for the intra-operative analysis of sentinel lymph nodes (SLNs) in breast cancer has been introduced in 3 UK centres since 2007. The methodology uses a polymerase chain reaction to quantitate CK19, a cytokeratin specific to breast duct epithelial cells. OSNA provides “real-time” results on SLNs analysed as negative (−) or positive with either micro (+) or macrometastases (++). Method: This is a single-centre prospective pilot study of all patients undergoing breast cancer surgery including sentinel node biopsy from February 2010 to May 2011. SLN identification was performed using a dual localization technique with peri-areolar Patente V blue dye and Tc99 radio-active isotope. SLNs were cut into 4 slices labeled as A, B, C and D, respectively after the removal of all perinodal fat. In all SLNs, slices A and C were processed in OSNA and slices B and D underwent histological assessment by H&E staining. Slices A and C were prepared for OSNA analysis with the required reagent (Lynorhag), followed by centrifugation and homogenization. Micro-pipetted samples were processed against control specimens, to produce normalized and quantitative curve results. The primary outcome measure was to correlate the results of OSNA with histopathology for each SLN. A negative SLN resulted in no further axillary surgery compared to a level I axillary sampling following the presence of micrometastases and a level II dissection following the detection of macrometastases. The total duration of OSNA for all SLNs was recorded in relation to delays in the completion of the intended breast surgery in each patient. Results: 251 SLNs were analysed in 112 patients (mean age of 55 years). Comparisons between OSNA and histopathology were made in 116 nodes (34 SLNs analysed by OSNA only). SLN positivity was evident in 30 nodes (26%) comprising either macrometastases (n=13) or micrometastases (n=17). The OSNA sensitivity and specificity of was 93% and 89%, respectively. Accounting for a tissue allocation bias in the presence of micrometastases only, the specificity rose to 94% if these cases were excluded in the analysis. There was no correlation between SLN positivity and tumour grade, size or receptor status. The time to OSNA results were analysed in 75 patients undergoing 45 wide local excisions (WLE), of which 18 were wire localized; 23 mastectomies and 22 SLNB alone. The mean time for OSNA was 40.5, 51.8, 54 and 61.5 minutes for 1,2,3, and 4 sentinel lymph nodes respectively. Operation time was prolonged by a median of 20 minutes (range −48 to +65 minutes) WLEs were delayed by the greatest time Exclusions: Nodes that were not available for histological comparison i) nodes weighing <0.05g (n=34) were processed whole. ii) Departmental agreement from mid March 2011 to process nodes whole via OSNA (n=51); 6 had micro or macrometastases. Conclusion: OSNA prevented staged axillary surgery in 24 (21%) of patients. A median time of 20 minutes for the OSNA procedure is comparable with acceptable operating times. Current experience supports the use of OSNA for each individual whole SLN analysis. The pilot data has resulted in a prescribed change in policy to analyse the whole SLN using this technique. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-07-23.

P3-07-28: One Step Nucleic Acid Amplification (OSNA) for Intraoperative Molecular Detection of Lymph Node Metastases and Micro-Metastases in Breast Cancer.

Abstract Introduction: Ideally, intraoperative sentinel lymph node analysis in breast cancer should be automated, concordant with histopathology and practically applicable. One step nucleic acid amplification (OSNA), a highly sensitive intraoperative assay of cytokeratin 19 mRNA, is used for the detection of sentinel lymph node (SLN) macro- and micro-metastases in breast cancer. Guildford adopted the intraoperative OSNA “live” in December 2008 after undertaking a multicentre evaluation of its accuracy and high concordance with histopathology and here we present our two year data since its introduction. Methods: Data was collected prospectively from 2008–10. All patients eligible for sentinel node biopsy were offered OSNA and operations were performed by five consultant breast surgeons. On detection of micro-metastasis (+) and positive but inhibited metastases a level 1 axillary nodal clearance (ANC) and for a macro-metastasis (++), a level 3 ANC was performed. Results: 471 patients had 999 SLN analysed, median age being 61. All except one were females. 72% (n=340) had wide local excision, 26% (n=120) underwent mastectomy and 2% (8) SNB alone. Mean tumour size was 18.3 mms. 80% (n=371) of the cases were IDC and 55% (n=256) had grade II tumour. 34% (n=161/471) had positive SLN who had further ANC. Of these, 48% (n=78/161) had macro-metastases, 37% (n=59/161) had micro-metastases and 15% (n=24/161) had positive but inhibited results. 17% (10/59) of the patients with micrometastases had positive non-SLN (NSLN), four (4/59, 6.8%) had four positive nodes (SLN+NSLN) thus receiving adjuvant radiotherapy. 8% (2/24) of those with positive but inhibited results and 39% (30/78) of those with macro-metastases had positive NSLN. Conclusion: Over a third of patients had OSNA positive SLN and underwent axillary surgery at the same operation. This technique eliminates the need for a second operation in sentinel lymph node positive patients and avoids the anxious wait for results in all, streamlining the patient's cancer journey. OSNA upstages patients with micro-metastases and long term studies are needed to determine the clinical relevance of molecular micro-metastatic disease. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-07-28.

Frozen section analysis of sentinel lymph nodes in patients with breast cancer does not impair the probability to detect lymph node metastases

Intra-operative frozen section analysis (FS analysis) of sentinel lymph nodes (SLNs) in patients with breast cancer can prevent a second operation for axillary lymph node dissection. In contrast, loss of tissue during FS analysis may impair the probability to detect lymph node metastases. To determine the effect of tissue loss on the probability of detection of metastases, dimensions and tissue loss resulting from intra-operative frozen section analysis were measured for 21 SLNs. In a mathematical model, the influence of tissue loss on the probability to detect metastases was calculated in relation to SLN size for various pathology protocols: an American, a widely used European, the extensive ‘Milan’ and the Dutch protocol. For median-sized SLN 11 × 8 × 5 mm (length × width × height), FS analysis led to a median loss of 680 μm (13.6%) of the height of the SLN. Irrespective of SLN size or used pathology protocol, the probability of detecting 2 mm metastases remained unchanged or even increased (0–12.8%). Moreover, the probability to detect 0.2 mm metastases increased for the majority of tested combinations of SLN size, tissue loss and used protocol. Only when combining maximum tissue loss and smallest SLN size in the Dutch protocol, or when applying the extensive Milan protocol on a median-sized SLN, the probability to detect 0.2 mm metastases decreased by 2.7% and 14.3%, respectively. Contrary to ‘common knowledge’, doing FS analysis of SLNs does not impair the probability to detect lymph node metastases.