BLN Assay Research Articles

Intra-operative GeneSearch(TM) BLN assay for detecting breast cancer metastases in sentinel lymph nodes

To evaluate the clinical value of GeneSearch(TM) BLN Assay as an intra-operative diagnostic method of sentinel lymph node (SLN) for breast cancer patients. A total of 479 consecutive patients from six centers were involved in this prospective study. The SLNs were identified, dissected and then sectioned along the short axis into multiple blocks. The odd blocks were tested intra-operatively by the above-mentioned assay and the even blocks assessed post-operatively by histopathologic examination. The 4 - 6 µm thick stepwise sectioning permanent HE slides were prepared every 150 µm and one block yielded 6 slides. In addition, the even blocks of 136 patients were prepared for frozen section (FS) and all blocks of 156 patients evaluated intra-operatively by touch imprint cytology (TIC). In the node basis analysis, its accuracy, sensitivity, specificity, positive predict value (PPV) and negative predict value (NPV) were 93.0%, 85.6%, 94.6%, 76.6% and 96.9% respectively. Its sensitivity was similar to that of FS (84.9%, P = 0.885) and significantly higher than that of TIC (70.0%, P = 0.007). When assessing nodes with macro-metastases, its sensitivity was similar to that of FS (93.6% vs 95.6%, P = 0.558) and significantly higher than that of TIC (93.6% vs 80.9%, P = 0.011). When assessing nodes with micro-metastases, it had a higher sensitivity than that of FS (57.5% vs 44.4%, P = 0.356) and TIC (57.5% vs 30.8%, P = 0.094). In the patient basis analysis, the accuracy, sensitivity, specificity, PPV and NPV were 91.4%, 87.5%, 92.9%, 81.8% and 95.3% respectively. Its sensitivity was similar to that of FS (84.5%, P = 0.576) and significantly higher than that of TIC (75.0%, P = 0.049). After adjustment, it had the accuracy, sensitivity, specificity, PPV and NPV of 91.7%, 83.5%, 95.2%, 88.3% and 93.0% respectively. Its sensitivity was higher than that of FS (72.1%, P = 0.054) and significantly higher than that of TIC (66.7%, P = 0.011). The two had no significant difference in the sensitivity and specificity. After a learning curve of around 10 cases, it could be performed in a median time of around 35 min. The threshold cycle time values of MG and CK-19 were 36 and 31 respectively. The type of metastases could be estimated approximately according to the cycle time values. The cycle time values of MG under 33 indicated SLN macro-metastases and those of 33-36 denoted micro-metastases. The values of CK-19 under 29 indicated SLN macro-metastases and those of 29-31 denoted micro-metastases. As an accurate and rapid intra-operative assay for breast sentinel lymph nodes, the GeneSearch(TM) BLN Assay may replace FS and TIC in general medical practice.

GeneSearch™ BLN Assay could replace frozen section and touch imprint cytology for intra-operative assessment of breast sentinel lymph nodes

Breast cancer sentinel lymph node (SLN) biopsy has become a common procedure. The GeneSearch™ Breast Lymph Node Assay is a real-time reverse-transcriptase polymerase chain reaction assay for detecting nodal metastases larger than 0.2mm. The trial is a prospective multi-center clinical trial conducted to validate the assay in China. Four hundred and seventy-nine consecutive prospective patients were enrolled from six centers. SLNs were sectioned along the short axis into multiple blocks. Odd blocks were tested by the assay intra-operatively, and even blocks were assessed by post-operative histology. Six 4- to 6-μm-thick sections were taken every 150μm per block. In addition, intra-operative histological assessments were performed on the even blocks of 214 patients by frozen section (FS) and all blocks of 156 patients by touch imprint cytology (TIC). A total of 1046 SLNs were excised. Overall performance of the assay compared to post-operative histology was accuracy of 91.4%, sensitivity of 87.5%, and specificity of 92.9%. There were no significant differences in assay performance of each center. After a learning curve of about 10 cases, the assay could be performed in a median time of about 35min. The sensitivity of the assay was similar to the FS (84.9%, P=0.885) and was significantly higher than the TIC (70.0%, P=0.007) while the specificity of all were comparable. The GeneSearch™ Breast Lymph Node Assay is an accurate and rapid intra-operative assay for breast SLNs and it can replace FS and TIC for application.

RT–PCR technique for the intra-operative assessment of breast sentinel lymph nodes – Is this the way forward?

Background Sentinel lymph node biopsy (SLNB) is the current standard of care for lymphatic staging in early breast cancer patients. The combination of a SLNB with an intra-operative diagnosis of the SLNB enables a single staged procedure, avoiding a delayed axillary lymph node dissection (ALND) in node positive patients. The current conventional pathological techniques are resource intensive, require expert personnel and are not routinely used in many breast units. Methods GeneSearch™ BLN assay is a RT-PCR technique calibrated to detect SLN metastases of >0.2mm. It is designed to detect 2 clinically validated genes Mammaglobin (MG) and Cytokeratin – 19 to provide a rapid diagnosis.The SLN identification was by the combined technique using radiocolloid and patent blue dye. The excised SLNs were sliced in their short axis at intervals of 1.5 mm to 2 mm. The sections were numbered and alternate sections were processed for histopathology and RT-PCR assay.The study was conducted in 2 phases. The first phase was to evaluate the accuracy and feasibility of RT-PCR assay as an intra-operative diagnostic test. The second phase was the real time application of the results to inform decision regarding immediate ALND. Results This prospective study comprised of 166 patients with 164 females and 2 male patients. 86 patients had left breast cancer, 75 patients had right breast cancer and 5 patients had synchronous bilateral breast cancers. A total of 171 SLNB procedures were done and 271 SLNs were excised at an average of 1.58 SLNs per procedure. Only a total of 266 SLNs were included in the analysis as the RT-PCR assay results of 5 SLNs were invalid. All the SLNs underwent RT-PCR assay and the results were compared to the final histology. The results were recorded on per patient and per node basis . The initial validation phase of the study included 53 patients and the RT-PCR assay was performed on 92 SLNs from these patients. The sensitivity and specificity on a per patient basis was 100% and 87.1% respectively. On a per node basis, the sensitivity and specificity was 93.7% and 92.1% respectively. The average processing time for 1 SLN during this phase was 31.6 min and 47.4 min for 2 SLNs. During the 2nd phase of the study, the actioning phase, 113 patients were recruited analysing 174 SLNs from these patients. The sensitivity and specificity of the RT-PCR assay on a per patient basis was 92.3% and 94.3% respectively. On a per node basis, the sensitivity and specificity was 94.1% and 96.4% respectively. The turn around time was significantly better at 21.5 min for 1 SLN and 32.6 min for 2 SLNs. Combining both phases, the sensitivity and specificity on a per patient basis was 95% and 92% respectively. The sensitivity and specificity on a per node basis was 94% and 94.9% respectively. Conclusions RT-PCR assay is a reliable intra-operative diagnostic technique for the detection of clinically relevant breast SLN metastases. It is a good alternative to conventional histopathological tests with better sensitivity but comparably less specificity.

Breast Cancer Sentinel Node Intraoperative Molecular Diagnosis: GeneSearch BLN assay vs. Metasin assay

Introduction: Sentinel lymph node biopsy (SLNB) is the gold standard for clinically and radiologically node-negative breast cancer. Intraoperative sentinel node assessment (IOA) enables immediate axillary clearance when positive. Our unit previously reported the implementation of the ‘GeneSearch BLN assay (Veridex LLC, Warren, New Jersey, USA)’, showing very high acceptability and accuracy. Following its withdrawal, we participated in validating an alternative (Metasin) assay, which also amplifies and detects RNA markers cytokeratin 19 (CK-19) and mammoglobin (MG).

Évaluation de l’analyse peropératoire des ganglions « sentinelle » par biologie moléculaire (technique Gene Search BLN Assay) dans le cancer du sein : à propos de 126 patientes

Intraoperative molecular assay Gene Search BLN Assay (BLN) detects sentinel lymph node (SLN) metastasis in breast cancer. Our objective was to compare BLN to the definitive conventional histologic methods and to experiment the management of BLN in routine. Each SLN was cut into alternate slabs. Half slabs were analysed with the intraoperative BLN molecular method, and the other slabs with the definitive histologic method. Two hundred and thirty four SLN have been analysed (124 patients). Thirty-five SLN had metastasis for 29 patients (23.4%). BLN correctly identified 28 patients. Two cases of discordance between BLN and standard method were found, probably explained by a sample bias. The sensibility of BLN is 96.4%, the sensitivity is 99%, the predictive positive value is 96.4%, the predictive negative value is 99% and the concordance is 98.4%. The surgery time increases and there is a need to adapt the theatre organization accordingly. The Gene Search BLN Assay gives a great interest for the patient, the surgeon and the pathologist because it increases the quality of the intraoperative analysis by comparison with the intraoperative conventional histology.

Value of intraoperative GeneSearch(TM) BLN assay to detect breast cancer metastases in sentinel lymph nodes

To evaluate the value of GeneSearch(TM) BLN assay as an intraoperative diagnostic method of sentinel lymph node metastases in breast cancer patients. Ninety consecutive patients were involved in this study. SLNs were intraoperatively identified and dissected, and then sectioned vertically to the long axis into multiple blocks. The odd blocks were tested by BLN assay and even ones prepared for frozen sectioning (FS), while all blocks were evaluated by touch imprint cytology (TIC). Post-operatively, residual tissues of the even blocks were assessed by histopathologic examination (4 - 6 µm thick serial sectioning permanent H&E slides were performed every 150 µm and one block made 6 slides). BLN assay could be performed within less than 35 min after learning curve of 10 cases. A correlation was found between cycle time values of mammaglobin or cytokeratin-19 and size of metastases, with Spearman correlation coefficients of 0.67 and 0.71, respectively. The accuracy, sensitivity, specificity, positive predict value (PPV) and negative predict value (NPV) of the assay were 95.6%, 93.3%, 96.7%, 93.3% and 96.7%, While FS had the sensitivity, specificity, PPV, NPV of 76.7%, 100%, 100%, 89.6%, and TIC of 73.3%, 100%, 100%, 88.2%, respectively. The sensitivity of the assay was higher than that of FS (P = 0.07), and was significantly higher than that of FS (P = 0.04). When assessing patients with micro-metastases, the assay had a sensitivity of 85.7%, which was significantly higher than that of FS and TIC (P = 0.03). GeneSearch(TM) BLN Assay can replace FS and TIC for the intraoperative assessment of SLN.

Use Pre- and Intra-Operative Data To Predict Probability of Positive Non-Sentinel Lymph Nodes.

Abstract Introduction: Many sentinel lymph node (SLN) positive patients have no further positive nodes, and benefit little from completion axillary node dissection (ALND). Prediction of non-SLN metastases (mets) based on pre- and intra-operative data remains a challenge. We sought to determine if this could be improved using a novel intraoperative RT-PCR based assay.Methods: 728 patients were enrolled in 2 prospective studies of the GeneSearch™ BLN Assay (Veridex LLC) for SLN mets. Of these, 205 had ≥1 positive SLN. Of these, 116 underwent ALND and had complete data for tumor size, BLN assay and frozen section (FS)/touch imprint cytology (TIC). These patients formed the cohort of interest for this study. A BLN Score was calculated using the cycle time (CT) values for mammaglobin (MG) and CK19 based on the BLN assay. These CT values were divided by that of the internal control (IC), and the least of the two was used. The BLN Score=10.2586-2.8053 x min(MG_CT/IC_CT, CK19_CT/IC_CT)+1.7164 x BLN positive rate in SLNs. Stepwise logistic regression models were evaluated for their ability to predict non-SLN status. A final score was created to determine the probability of non-SLN mets based on pre- and intra-operative data alone.Results: The median tumor size in this cohort was 2.20 cm (range: 0.19-22.00). The median number of SLNs removed was 2; the median number of positive SLNs was 1. The median number of non-SLNs removed was 12 (range; 1-38).As tumor size can be estimated from preoperative imaging and/or measured intraoperatively, this formed our initial model. Tumor size was a statistically significant predictor of non-SLN mets (AUC=0.631, p=0.027). In a model with the proportion of positive SLNs by intraoperative analysis (FS/TIC positive rate), both tumor size and FS/TIC positive rate were significant (p=0.022 and 0.006). In the final model that included tumor size, FS/TIC positive rate, and BLN score, BLN score was statistically significant (p=0.012) while tumor size and FS/TIC positive rate became borderline significant (p= 0.071 and 0.084, respectively). The final model brought AUC to 0.750 (p=0.048 compared to the original model). This was somewhat better than the model with BLN score alone (AUC=0.72) and that with BLN score and tumor size (AUC=0.742), although not significantly so.A logistic regression model with dichotomized tumor size data was then fitted. When tumor size was >2 cm, FS/TIC positive rate was ≥50%, and BLN score was >10, the probability of non-SLN mets is >62.5%. When tumor size was ≤2cm, FS/TIC positive rate was <50%, and the BLN score was <7, the probability of non-SLN mets is <5%. Even when tumor size >2 cm or FS/TIC positive rate was ≥50%, the probability of non-SLN mets is < 8% if BLN score<7 (see figure).Conclusions: BLN score is a significant predictor of non-SLN metastases. When combined with tumor size and proportion of SLNs positive by FS/TIC, BLN score may provide accurate estimate of probability of non-SLN mets. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 302.

Génexpresszió mérésén alapuló multigénes prognosztikai és prediktív előrejelzés emlőtumorokban

Patient tailored therapy will serve the fundamentals of future cancer treatment. For this it will be imperative to characterize the tumor and to acquire precise predictive and prognostic information. We can achieve this by using not only monogenic (like ER, PR, HER-2, Ki-67) but also multigene assays, which can provide answers to several diagnostic questions simultaneously. We present a summary of currently available RT-PCR and microarray based multigene tests including MammaPrint, Oncotype DX, BLN Assay, Theros Breast Cancer Index SM, MapQuant DX, ARUP Breast Bioclassifier, Celera Metastatic Score, eXagen BCtm, Invasive Gene Signature, Wound Response Indicator and Mammostrat. Two of these (Oncotype DX and MammaPrint) are already incorporated in several diagnostic protocols. However, multiple unsolved issues deteriorate the value of these tests: generally the validation is poor, the gene sets do not confirm each other, the associated costs are high and the necessary bioinformatics is highly complex.

Evaluation of the histological size of the sentinel lymph node metastases using RT–PCR assay: a rapid tool to estimate the risk of non-sentinel lymph node invasion in patients with breast cancer

A RT-PCR assay (GeneSearch™, Veridex, LLC), FDA approved and CE marked to detect metastases > 0.2 mm in sentinel lymph nodes (SLNs) is used intra-operatively for the management of patients with breast cancer. The assay provides qualitative results by applying cut-off values to cycle times (Ct) for mammaglobin (MG) and cytokeratin-19 (CK19) genes. Aims of this study were to evaluate the performance of the quantitative Ct values to estimate the size of nodal metastases and the risk of additional disease in non-SLNs. SLNs from 367 patients were clinically processed using both BLN assay and post-operative histology. Complementary axillary lymph node dissection (ALND) was performed concurrently in case of BLN assay positivity or tumour size > 2 cm. BLN positivity was reported in 19.6% of the patients for a sensitivity of 89%. BLN specificity (94.5%) and negative predictive value (97.5%) clearly demonstrated its reliability to guide ALND decision. All, except one, residual axillary metastases were found in BLN-positive patients. Considering the 78 patients with SLN positivity or discordant status according to both criteria, the metastases histological size was significantly correlated to the expression level of MG (ρ = 0.62) and CK19 (ρ = 0.64) genes (P < 10E-6). Moreover, ALND status positivity was significantly associated to Ct value of MG (z = 2.4; P = 0.018) and CK19 (z = 3.2; P = 0.001). The high intra-operative quality performance of the BLN assay minimizes the need for second surgeries for ALND. Results from this investigational study suggest that markers Ct value may provide, intra-operatively, valuable metastases size data and a risk prediction of additional disease in non-SLNs.

Use of quantitative RT-PCR assay to predict metastases size of sentinel node from breast cancer patients

621 Background: A RT-PCR Assay (GeneSearch, Veridex, LLC), FDA approved and CE marked to detect metastases &gt; 0.2 mm in sentinel lymph nodes (SLNs) of breast cancer patients, has been in clinical use in our institute for 25 months. The assay gives qualitative (negative/positive) results by applying cutoff values to cycle times (Cts) of mammaglobin (MG) and cytokeratin 19 (CK19) (cutoff values = 30 / 31 Cts). This study evaluates if quantitative Ct values can be used to estimate the probable size of nodal metastases. Methods: To date 384 patients have been clinically tested with the BLN Assay at Institut Jules Bordet. An additional 74 patients were tested in a pilot study. Both sets had SLNs of breast cancer patients divided in ∼2 mm pieces. Alternating pieces were homogenized and intra-operatively processed by the assay. The remaining pieces were used for histological analysis (positive when &gt; 0.2 mm). During clinical use, same-surgery complete axillary dissection was performed when the assay was positive (n=59). Results: Performance of the assay's qualitative results against permanent section H&amp;E was similar for both groups (total N = 458), for a total sensitivity 89% (68/76), specificity 95% (364/382) and overall agreement 94%. The marker Ct values are correlated with metastases size as determined by H&amp;E on adjacent node pieces (r = -0.74 for MG and -0.77 for CK19, p &lt; 0.001). Lower Ct values indicate increased likelihood of macrometastases reported by H&amp;E, as described in the table. Additionally, Assay Ct values in the SLN were predictive of metastases in non-SLN nodes. For example when CK19 and MG &lt; 24 Cts, the non-SLN positivity rate jumps from 29% to 58%. Conclusions: The BLN Assay's high intra- operative qualitative performance minimizes the need for second surgeries for complete axillary dissections. Results from this investigational study examining the marker Ct values suggest that the assay may provide valuable individual tumor volume data intra- operatively or post-operatively. [Table: see text] [Table: see text]

Use of intraoperative breast cancer sentinel lymph node (SLN) assay to predict of ≥4 positive (+) lymph nodes (LN)

530 Background: Predicting which patients will have ≥4 +LN and may need radiation therapy affects immediate breast reconstruction. We hypothesized that model s to predict the likelihood of ≥4+LN may be improved by incorporation of quantitative real time RT-PCR analysis of SLN. Methods: 728 patients were enrolled in 2 prospective studies of the GeneSearch BLN Assay (Veridex LLC) for SLN metastases. 205 (28.2%) were found to have +SLN by hematoxylin-eosin staining. Of these, 115 pts (15.8%) had tumors ≤5 cm in size with &gt;4 total LN removed, forming the cohort of interest for this study. Quantitative cycle times (CT) for mammaglobin (MG) and CK19 were correlated with finding ≥4+LN on final pathology. Results: Median tumor size was 2.0 cm (range; 0.2–5.0 cm). Median number of SLN removed was 3 (range; 1–11). 18 patients (15.7%) had ≥4 +LN on final pathology. Median CT for MG was 29.1 (interquartile range (IQR): 24.7–39.0) in patients with &lt;4 +LN vs. 21.4 (IQR: 18.5–26.8) in those with ≥4 +LN, p&lt;0.001. Median CT for CK19 was 23.7 (IQR: 20.9–28.2) in patients with &lt;4 +LN vs. 19.6 (IQR: 17.8–20.7) in those with ≥4 +LN, p&lt;0.001. Tumor size ≥2cm, proportion of SLN+ &gt;50%, MG CT &lt;25.8 and CK19 &lt;20.8 were correlated with ≥4 +LN on final pathology. On multivariate analysis, tumor size, MG CT and CK19 CT were significant (see table). A simplified CPR was created with 1 point given if tumor size was ≥2cm, 1 point if MG CT &lt;25.8 and 2 points if CK19 CT&lt;20.8. Of the 24 patients (20.9%) with 0 points, only 1 (4.2%) had ≥4 +LN; of the 12 patients (10.4%) with 3 points, 8 (66.7%) had ≥4 +LN on final pathology, p&lt;0.001. Area under the receiver-operator curve was 86.2% (95% CI: 76.1%-96.2%). Conclusions: Intraoperative quantitative RT-PCR of SLN improves prediction of ≥4 +LN; further validation of this model will have clinical utility regarding the use of immediate, delayed, or delayed-immediate reconstruction approaches. [Table: see text] [Table: see text]

Editorial introductions

This article reviews the recent advancements and refinements of the sentinel lymph node (SLN) biopsy technique in breast cancer. Data from four randomized controlled trials conclusively demonstrate that SLN biopsy is associated with less arm morbidity and better quality of life than axillary lymph node dissection. Large observational studies have shown that SLN biopsy is associated with low local recurrence rate and similar survival to axillary lymph node dissection.Preoperative axillary ultrasound and fine-needle aspiration cytology has recently been shown to improve patient selection for sentinel node biopsy. Furthermore, intraoperative assessment of SLN can now be performed rapidly and accurately using the realtime GeneSearch BLN Assay. There is accumulating evidence that SLN biopsy is applicable before or after neoadjuvant chemotherapy and remapping is feasible inpatients who develop breast recurrence following breast conservation and SLN biopsy. SLN biopsy is the new standard of care for nodal staging in early-stage breast cancer patients and contraindications are decreasing with experience. However, the clinical relevance of SLN micrometastasis and isolated tumour cells remains unclear.Definitive data from randomized trials is needed to decide if axillary dissection is needed when the SLN is positive.

Clinical use of an intra-operative RT-PCR assay for the detection of metastases in sentinel lymph nodes of breast cancer patients at the Institut Jules Bordet

11100 Background: A molecular assay (GeneSearch™ BLN Assay, Veridex, LLC), CE marked and approved by the FDA, has been in clinical use at our institution for over 18 months to intra-operatively detect metastases ≥0.2 mm in the sentinel lymph nodes (SLNs) in breast cancer patients. After a small validation study (N=78, sensitivity = 92.3%, specificity = 96.9%) the assay was adopted as the only intra-operative SLN test used at the Institut Jules Bordet. This is the first report of the post-market assay performance. Methods: One SLN per patient is cut into approximately 2 mm sections. Alternating sections are processed fresh in the BLN assay. The assay detects mRNA expression of mammaglobin and CK19. Predetermined cutoffs are set to detect metastases ≥0.2 mm. The BLN Assay results are used to make intra-operative decisions for same surgery axillary lymph node dissections (ALND). The remaining alternating sections of the SLN are examined later by permanent section H&E. Results: Since August 2006, the BLN Assay has been used for intra-operative ALND decision-making on 100 patients. The assay intra-operatively detected all 15 H&E-positive SLNs. The assay was in agreement with 93% of the H&E-negative SLNs (79 of 85) and was positive when H&E was negative for 6 SLNs. One of these 6 SLNs had a 0.3 mm size metastasis detected by IHC alone. The average turn around time for the assay was 39 minutes for all 100 patients, and was 35 minutes for the second 50 patients. Conclusions: The BLN Assay results shows high correlation to permanent section H&E results that take 2–3 days, and its timing permits same-surgery ALND when nodes are found positive for metastases. In clinical use, agreement of assay results and permanent section H&E results (94%, 94/100) is similar to the agreement found in an earlier validation study (96.2%, 75/78). Perfect agreement is not expected since the two tests are evaluating different sections of the node. The BLN Assay is beneficial to our patients by reducing the need for second surgeries while being practical and cost effective for the institution to perform. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Veridex Veridex

Sentinel node staging for breast cancer: intraoperative molecular pathology overcomes conventional histologic sampling errors

Background When sentinel node dissection reveals breast cancer metastasis, completion axillary lymph node dissection is ideally performed during the same operation. Intraoperative histologic techniques have low and variable sensitivity. A new intraoperative molecular assay (GeneSearch BLN Assay; Veridex, LLC, Warren, NJ) was evaluated to determine its efficiency in identifying significant sentinel lymph node metastases (>.2 mm). Methods Positive or negative BLN Assay results generated from fresh 2-mm node slabs were compared with results from conventional histologic evaluation of adjacent fixed tissue slabs. Results In a prospective study of 416 patients at 11 clinical sites, the assay detected 98% of metastases >2 mm and 88% of metastasis greater >.2 mm, results superior to frozen section. Micrometastases were less frequently detected (57%) and assay positive results in nodes found negative by histology were rare (4%). Conclusions The BLN Assay is properly calibrated for use as a stand alone intraoperative molecular test.