The real-time intra-operative evaluation of sentinel lymph nodes in breast cancer patients using One Step Nucleic Acid Amplification (OSNA) and implications for clinical decision-making

doi:10.1016/j.ejso.2013.12.007

European Journal of Surgical Oncology (EJSO)

Volume 40, Issue 2, February 2014, Pages 150-157

https://doi.org/10.1016/j.ejso.2013.12.007 Get rights and content

Abstract

Introduction

One Step Nucleic Acid Amplification (OSNA) method for the intraoperative analysis of sentinel lymph nodes (SLNs) in breast cancer, obviates a second operation to the axilla and thereby expedites progression to adjuvant therapy. Recent NICE guidelines have approved OSNA as a method of sentinel node diagnosis to support the above case.¹

Method

This is a single centre prospective cohort analysis of all patients undergoing breast cancer surgery including sentinel node biopsy from February 2010 to June 2012. Patients with negative SLN(s) on OSNA had no further axillary surgery. A validation phase was performed prior to using OSNA routinely. Those with micrometastases underwent a level 1 clearance, and >one SLN with macrometastases, underwent treatment by level 2 axillary dissection. The length of time from sentinel node retrieval to OSNA result was recorded.

Results

Four hundred and forty nodes were analysed in 212 patients with a mean age of 55 years (range 24–98). The sensitivity and specificity of OSNA was 93% and 94% respectively in cases of macrometastases. The process required additional median anaesthesia time of 20 min (range −48 to +65 min). Non-sentinel node positivity was 5% and 48% for micrometastasis and macrometastasis respectively.

Conclusion

OSNA identified 62 of 212 patients with at least one positive sentinel node, thereby sparing 29% from a second procedure to clear the axilla subsequently. The median waiting time of 20 min for node results from completion of breast procedure is acceptable and allows for an efficient operating list. OSNA can be incorporated into routine practice and with improved methods of imaging preoperatively, can be an excellent adjunct to the breast cancer patient pathway of care.

Introduction

The One Step Nucleic Acid Amplification (OSNA) method for the perioperative analysis of sentinel lymph nodes in breast cancer has recently been introduced in approximately 12 UK centres. Current NICE guidelines (2009) suggest that pre-treatment ultrasound evaluation of the axilla should be performed for all patients being investigated for early invasive breast cancer and, if morphologically abnormal lymph nodes are identified, ultrasound-guided needle sampling or core biopsy should be performed.² In patients who have a normal axillary ultrasound or negative fine needle aspiration of a node, it is appropriate to offer sentinel lymph node (SLN) biopsy. It is current practice that a positive result on SLN biopsy should be treated with axillary dissection.³ Furthermore, recent NICE guidelines recommend OSNA for the diagnosis of sentinel node biopsy.¹ Recently, several studies have proposed the intra-operative molecular-based detection of SLN metastasis using standardised quantitative calibration methods in breast cancer.⁵ This obviates a second operation to treat the axilla, thereby expediting progression to adjuvant treatment.⁴ The One Step Nucleic Acid Amplification (OSNA, Sysmex, Kobe, Japan) assay evaluates the number of mRNA copies of CK19,⁵ a duct epithelial cell marker, which is highly expressed in the majority (>95%) of breast cancers. The histopathology protocols to determine the sensitivity and specificity of OSNA have been described (Tsujimoto 2008, Visser 2009, Snook 2010). In this two phase study, we aimed firstly to delineate the concordance of OSNA with routine histopathological diagnosis and secondly to determine how many patients could be saved a second operation, whilst also analysing the impact on theatre operating time.

Section snippets

Patient samples

All breast cancer patients underwent axillary ultrasound ± FNA and only USS or cytologically negative patients underwent SNB. Phase 1 consisted of all patients undergoing SNB from Feb 2010 to Sept 2010 and was the validation phase of the study. During this period, axillary dissections were performed as the unit was completing its training phase in the NEWSTART sentinel node study, a national scheme for verifying competence in the sentinel node procedure. The prospective adoption of SLN analysis

Results

The study comprised 212 patients with normal lymph node morphology on axillary ultrasound (US), or US-guided cytologically normal lymph nodes. Four hundred and forty sentinel lymph nodes (SLNs) were analysed. Following SLN biopsy (SLNB) and OSNA, 62 (29%) patients underwent completion therapeutic axillary dissection, compared to the majority of patients (n = 150; 70.8%) requiring SLNB alone.

Discussion

This prospective cohort study from a single UK centre assesses the pilot data on the overall concordance rate between intra-operative OSNA and post-operative histology as 93–94%. These findings are based on standard methods of subdividing the SLN into four slices for synchronous molecular and histological diagnoses. The reported results confirm similar excellent rates of specificity and sensitivity from other studies,5, 9, 10, 11, 12, 13 as shown in Table 5. It is well described that routine

Conclusion

OSNA processing of SLNs intra-operatively is becoming more widespread in the UK. Pre-operative US diagnosis of SLN positivity is improving but is currently associated with relatively high diagnostic false-negative rates of up to 50%. This may improve with improved methods of imaging and greater use of fine needle cytology of all suspicious and equivocal nodes. We have shown that the analysis of SLN can be undertaken in routine practice with a very high accuracy and only a modest impact on

Conflict of interest

There is no conflict of interest.

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Cited by (36)

Sentinel lymph node study in breast cancer using the OSNA (one-step nucleic acid amplification) method after neoadjuvant treatment at Hospital Universitario de Navarra
2023, Revista de Senologia y Patologia Mamaria
la biopsia selectiva de ganglio centinela (GC) es la técnica estándar para la estadificación axilar en el cáncer de mama. No hay consenso en el empleo del método OSNA (One-Step Nucleic Acid Amplification) para el análisis del GC en las pacientes que recibieron el tratamiento neoadyuvante (TNA). En este trabajo analizamos los resultados obtenidos con OSNA en estas pacientes para justificar su implantación en nuestro centro.
se seleccionaron 42 casos del grupo de 163 pacientes con CM tratadas con TNA en nuestro centro, a las que se realizó OSNA del GC, obteniéndose una media de 2,1 ganglios por paciente. Se analizó además la expresión de citoqueratina 19 (CK19), grado tumoral, fenotipo molecular y el grado de respuesta al TNA de estas pacientes. Se estudiaron los GC mediante técnica OSNA y los no centinelas por el método tradicional.
el grado tumoral fue 2-3 en el 97,6% de los casos, el fenotipo luminal A (17%), luminal B (38%), triple-negativo (26,1%) y HER2 (19%). La respuesta al TNA fue completa en el 59,5% de las pacientes y la expresión de CK19 no se vio modificada. Los ganglios estudiados fueron positivos en 9 pacientes (21,4%) en las que posteriormente se realizó una linfadenectomía y un único caso presentó ganglio no centinela afecto (2,3%).
el método OSNA para el estudio del GC tras el TNA es muy superior al método tradicional, ya que permite la detección intraoperatoria de grupo celular aislado y micrometástasis no detectables con los métodos tradicionales, evitando segundas intervenciones y falsos negativos al analizarse completo el GC, y demuestra que no se altera la expresión de CK19 con el TNA.
Selective sentinel node (SN) biopsy is the standard technique for axillary staging in breast cancer (BC). There is no consensus on the use of OSNA (One-Step Acid Nucleic Amplification) method for SN in patients undergoing neoadjuvant treatment (NAT). We have studied the results obtained in our centre to justify the advantages of its implementation.
42 cases were selected from the group of 163 patients with BC treated with NAT, who underwent OSNA of the SN, obtaining a mean of 2.1 nodes per patient. We also analyzed cytokeratin 19 (CK19) expression, tumour grade, molecular phenotype and the degree of response to NAT in these patients. The SN were studied using the OSNA technique and non-sentinel nodes using the traditional method.
Tumour grade was 2-3 in 97.6% of cases, phenotype luminal A (17%), luminal B (38%), triple-negative (26.1%) and HER2 (19%). The response to NAT was complete in 59.5% of patients and CK19 expression was unchanged. The nodes studied were positive in 9 patients (21.4%) in whom lymphadenectomy was performed and only one case had a non-sentinel node involvement (2.3%).
The OSNA method for the study of SN after NAT is far superior to the traditional method as it: It allows intraoperative detection of isolated cell group and micrometastases not detectable with traditional methods, avoiding second interventions. It avoids false negatives when the whole SN is analyzed. It shows that CK19 expression is not altered by NAT.
Septin 9 methylation analysis of lymph node micrometastases for predicting relapse of colorectal cancer
2022, Annals of Diagnostic Pathology
Citation Excerpt :
The data resulted in an upstaging of 25.2 % of patients with initially histologically negative lymph nodes from UICC-I/II to UICC-III. The same or similar strategy has also been used to detect cancer cells in resected lymph nodes of breast cancer and lung cancer patients for clinical decision-making [7,8]. In addition, immunohistochemistry (IHC) staining of lymph nodes has been described to improve the clinical staging of non-small cell lung cancer and predict prognosis and outcome [9].
Molecular markers for the detection of lymph node micrometastases of malignant tumors have been extensively investigated. However, epigenetic signatures have rarely been reported for identification of metastatic lymph nodes and disease relapse. Septin 9 is the most frequently reported hypermethylated gene in colorectal cancer (CRC). This study aimed to assess the clinical relevance of Septin 9 methylation in regional lymph nodes in recurrence/metastases of CRC.
We analyzed Septin 9 methylation of DNA from resected lymph nodes in 75 CRC patients with or without tumor recurrence using quantitative methylation-sensitive PCR (qMS-PCR).
Of the 30 histologically negative lymph node CRC patients without recurrence (group 1), methylated Septin 9 was detected in 3 (10 %) cases. The positivity rate of methylated Septin 9 in group 2 containing 30 histologically node-negative CRC patients with recurrence was 30 % (9/30). For group 3, lymphatic invasion as well as tumor recurrence, 11 (73 %) out of 15 subjects had Septin 9 methylation-positive lymph nodes. Moreover, patients in group 3 had a higher level of methylated Septin 9 compared to subjects in group 1 and group 2 (p < 0.05). In addition, CRC patients with Septin 9 methylation in lymph nodes had significantly reduced survival (Log-rank P < 0.0001).
Our data support the predictive role of Septin 9 methylation analysis of lymph node micrometastases for tumor relapse after surgery.
Is it Possible to Predict Non Sentinel Node Positivity on the Basis of mRNA Copy Numbers of CK19 Receptor in Breast Cancer?
2021, Clinical Breast Cancer
To determine if there is any correlation between the number of positive non-sentinel lymph nodes (NSLN) and the mRNA copy numbers of cytokeratin 19 receptor on one step nucleic acid amplification (OSNA) in the sentinel lymph node (SLN).
An 8-year retrospective study of consecutive patients who had primary surgery and sentinel node biopsy for breast cancer from January 2011 to December 2018 was carried out. All these patients had intra-operative analysis of sentinel lymph nodes by OSNA. Patients who had neoadjuvant chemotherapy or neoadjuvant endocrine therapy were excluded.
There were 1159 patients with an age range of 24 to 90 years and a mean age of 63 years in this study. A total of 1324 SLNs were analyzed by OSNA. Macrometastasis was found in 120 patients and they underwent axillary lymph node dissection (ALND). A total of 2405 NSLNs were analyzed. Of the patients who had ALND, 51 (43%) patients had negative NSLNs and 69 (57%) had positive NSLNs. The mean mRNA copy numbers respectively for the 2 groups were 853,665 and 609,855. The difference between the means is not statistically significant (P = 0.82). Also the Receiver Operating Characteristic (ROC) Curve of the total CK-19 mRNA copy number for both groups-negative and positive NSLN were almost identical (Figure 3) indicating mRNA copy numbers cannot be used to discriminate between positive and negative non-sentinel lymph nodes.
It is clear from our study that in patients who have ALND due to macromets on OSNA, there is no correlation between the total tumor load as represented by mRNA copy numbers and the likelihood of positive non-sentinel lymph nodes. We therefore cannot rely solely on the mRNA copy numbers to decide on ALND.
The evolving role of one-step nucleic acid amplification (OSNA) for the intra-operative detection of lymph node metastases: A diagnostic accuracy meta-analysis
2021, European Journal of Surgical Oncology
Citation Excerpt :
CK19 is a typical epithelial cell marker expressed in human malignant cells, whilst its expression rate correlates with the size of the metastatic foci [9]. OSNA is an established technique for accurately identifying LN metastases in breast cancer (BC) [10,11]. In light of the high diagnostic performance in BC, its accuracy for detecting LN metastases has also been investigated in the context of other tumour types, including colorectal (ColCa), gastric (GasCa), gynaecological (GynCa), lung (LungCa), head and neck (HNSCC) and thyroid cancer (ThyCa), respectively.
One Step Nucleic Acid Amplification (OSNA) assay has recently emerged as a rapid molecular diagnostic tool for the detection of lymph node (LN) metastases. It is a molecular technique that analyses the entire LN tissue using a reverse-transcriptase loop-mediated isothermal amplification reaction to detect tumour specific cytoceratin 19 mRNA.
To ascertain the diagnostic accuracy of OSNA assay in detecting LN metastases amongst different types of malignancy.
We systematically searched MEDLINE, SCOPUS, ClinicalTrials.gov, and Cochrane Database, from inception up to August 2020. Quality assessment was performed using the Modified Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2). We calculated pooled diagnostic indices using the random-effects model. Meta-regression and sub-group analyses were performed to address heterogeneity.
31 studies were included in this meta-analysis, including four different types of cancer. The risk of bias and the overall quality of included studies was moderate to high. There was no evidence of publication bias. The pooled diagnostic odds ratio (DOR) for detecting LN metastases in gynaecological, head & neck/thyroid, gastrointestinal and lung cancer were 100.38, 76.17, 275.14, and 305.84, respectively.
Our findings suggest that OSNA assay had a high diagnostic accuracy in detecting metastatic LNs in different types of malignancy. This evidence is constrained by the limited studies available for few tumour types and the rather high heterogeneity for few outcomes.
The performance of one-step nucleic acid amplification assay for intraoperative detection of sentinel lymph node macrometastasis in breast cancer: An updated meta-analysis
2018, Breast
One-step nucleic acid amplification (OSNA) assay is a molecular diagnostic method for intraoperative detection of lymph node metastasis by amplifying cytokeratin 19 mRNA. We aim to further evaluate the performance of OSNA assay for the intraoperative diagnosis of sentinel lymph node metastasis in breast cancer.
PubMed, Cochrane Library, Web of Science databases were searched to retrieve related literature published up until December 2017. This meta-analysis was performed using a random-effects model. Risks of bias and quality assessments of included studies were evaluated and subgroup analysis was performed.
Nineteen studies were included in this meta-analysis. For overall metastasis, the pooled sensitivity, specificity and area under the summary receiver-operating characteristic curve (AUC) were 0.90, 0.96 and 0.98, respectively. For macrometastasis, the pooled sensitivity, specificity and AUC were 0.85, 0.98 and 0.94, respectively.
OSNA assay is an accurate molecular diagnosis for intraoperative detection of sentinel lymph node macrometastasis in breast cancer.
Total tumor load assessed by one-step nucleic acid amplification assay as an intraoperative predictor for non-sentinel lymph node metastasis in breast cancer
2017, Breast
Citation Excerpt :
Intraoperative diagnosis of positive SLN can allow ALND in the same surgical procedure when criteria are present, thus avoiding a second surgery to treat the axilla and decreasing the patient's associated discomfort and institutional costs [2,3]. The one-step nucleic acid amplification (OSNA, Sysmex, Kobe, Japan) assay is a molecular method that measures the quantity of cytokeratin (CK)-19 mRNA (a duct epithelial cell marker that is highly expressed in more than 95% of breast cancers) in axillary lymph nodes [4]. Cutoff values were defined to classify macrometastases (more than 5000 copies/μL), micrometastases (250–5000 copies/μL) and negative nodes (fewer than 250 copies/μL) [5].
This study aimed to determine the relationship between CK19 mRNA copy number in sentinel lymph nodes (SLN) assessed by one-step nucleic acid amplification (OSNA) technique, and non-sentinel lymph nodes (NSLN) metastization in invasive breast cancer. A model using total tumor load (TTL) obtained by OSNA technique was also constructed to evaluate its predictability.
We conducted an observational retrospective study including 598 patients with clinically T1-T3 and node negative invasive breast cancer. Of the 88 patients with positive SLN, 58 patients fulfill the inclusion criteria.
In the analyzed group 25.86% had at least one positive NSLN in axillary lymph node dissection. Univariate analysis showed that tumor size, TTL and number of SLN macrometastases were predictive factors for NSLN metastases. In multivariate analysis just the TTL was predictive for positive NSLN (OR 2.67; 95% CI 1.06–6.70; P = 0.036). The ROC curve for the model using TTL alone was obtained and an AUC of 0.805 (95% CI 0.69–0.92) was achieved. For TTL >1.9 × 10⁵ copies/μL we got 73.3% sensitivity, 74.4% specificity and 88.9% negative predictive value to predict NSLN metastases.
When using OSNA technique to evaluate SLN, NSLN metastases can be predicted intraoperatively. This prediction tool could help in decision for axillary lymph node dissection.

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The real-time intra-operative evaluation of sentinel lymph nodes in breast cancer patients using One Step Nucleic Acid Amplification (OSNA) and implications for clinical decision-making

Abstract

Introduction

Method

Results

Conclusion

Introduction

Section snippets

Patient samples

Results

Discussion

Conclusion

Conflict of interest

Lancet Oncol

Eur J Surg Oncol

Eur J Surg Oncol

DG8 Introperative tests (RD-100i OSNA system and Metasin test) for detection of sentinel lymph node metastses in breast cancer: guidance

NICE

Guidelines for the management of symptomatic breast disease

Eur J Surg Oncol

Intraoperative examination of sentinel lumps nodes in breast cancer: is the glass half full or half empty?

Ann Surg Oncol

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

Clin Cancer Res

Current issues in diagnostic breast pathology

J Clin Pathol

American society of clinical oncology. American society of clinical oncology guideline recommendations for sentinel lymph node biopsy in early stage breast cancer

J Clin Oncol