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- Materials and Methods
- Disclosure Statement
Conventional procedures for the intraoperative assessment of breast cancer sentinel lymph nodes (SLNs) are frozen section (FS) and touch imprint cytology (TIC). The one-step nucleic acid amplification (OSNA) assay is a novel molecular technique. The aim of this study was to evaluate the optimal approach by comparing OSNA assay, FS, and TIC. Five hundred and fifty-two consecutive patients were enroled from five study centers in China. The SLNs were cut into alternating 2 mm blocks. The odd blocks were tested by the OSNA assay intraoperatively, and the even ones were assessed by postoperative histology (four 4- to 6-μm-thick sections were taken every 200 μm per block). In addition, intraoperative histological assessments were carried out on the even blocks of 211 patients by FS and all blocks of 552 patients by TIC. Overall performance of the assay compared to postoperative histology was: accuracy 91.4%; sensitivity 83.7%; and specificity 92.9%. The sensitivity of the assay was higher than FS (211 patients, 77.6% vs 69.7%; not significant, P = 0.286) and was significantly higher than TIC (552 patients, 83.6% vs 76.2%; P = 0.044). When assessing nodes with micrometastases, the sensitivity of the assay was higher than FS (17 nodes, 47.1% vs 23.5%; not significant, P = 0.289) and was significantly higher than TIC (48 nodes, 62.5% vs 35.4%; P = 0.007). The study indicated that the OSNA assay is an accurate and rapid intraoperative assay for assessing breast SLNs and it can replace FS and TIC for application in general medical practice. The trial was registered as: OSNA assay China Registration Study. Clinical trial registration number: China Breast Cancer Clinical Study Group 001c.
Sentinel lymph node biopsy has become the standard staging technique for clinically node-negative breast cancer patients and reduced the morbidity associated with ALND.[1, 2] The accurate and rapid intraoperative assessment of the SLN has the advantage of enabling ALND to be carried out at the time of primary breast surgery if the SLNs are identified as positive for metastases, thus avoiding the morbidity, inconvenience, and cost of a second, separate operation. However, adequate diagnostic standardization has not yet been established and protocols for the evaluation are highly variable among different centers. Conventional procedures for intraoperative assessments of SLNs are FS and TIC. They both require experienced pathologists and are not standardized, and they also show low sensitivity in SLN micrometastases.[4, 5]
Reverse transcription–PCR was the first molecular approach used for the detection of SLN metastases, which analyzed tumor-specific mRNAs. The GeneSearch breast lymph node assay (Veridex, LLC, Raritan, NJ, USA) was approved by the US FDA in 2007. The OSNA assay (Sysmex, Kobe, Japan) is a molecular technique that combines node tissue homogenization and subsequent reverse-transcription loop-mediated isothermal amplification of CK-19 mRNA in a single quick step. The assay could accurately detect nodal metastases of >0.2 mm, comparable to conventional pathological examination, and yields quantitative results.
The China Breast Cancer Clinical Study Group-001c is a prospective multicenter clinical study group that was established to evaluate the intraoperative OSNA assay in China. We compared the performance of the OSNA assay with the present standard histological evaluation, and also made a comparative analysis of OSNA assay with FS and TIC.
- Top of page
- Materials and Methods
- Disclosure Statement
At the 11th St Gallen conference, most experts agreed that SLNB is the standard treatment for patients with clinical node-negative invasive carcinoma, except for those patients with T4d stage disease. As the indications for SLNB have expanded, there is a need for more sensitive and accurate assessments of SLNs.
The panel of the American Society of Clinical Oncology recommended ALND for patients with macrometastatic and micrometastatic SLNs, and follow-up for those with ITCs. However, there was still controversy regarding the treatment of patients with SLN micrometastases and ITCs. The MIRROR clinical trial indicated that micrometastases or ITCs were associated with a reduced disease-free survival among women with favorable early-stage breast cancer who did not receive adjuvant therapy. The trial also affirmed that ALND should be carried out for patients with SLN micrometastases, and axillary treatment should not be omitted for patients with ITCs unless primary tumor characteristics are favorable.[10, 11] In contrast, the American College of Surgeons Oncology Group Z0011 trial indicated that, among patients with limited SLN metastases treated with breast conservation therapy and adjuvant therapy, the uses of SLNB compared with ALND did not result in inferior survival.[12, 13] This trial also indicated that the use of ALND seemed unsuitable for SLN micrometastases and ITCs. At the 12th St Gallen conference, most experts agreed that micrometastases and ITCs in a single SLN were not considered to constitute an indication for ALND regardless of the type of breast surgery. In China, we thought it was vitally important to identify SLN macrometastases. As the clinical significance of micrometastases was ambiguous, it was at least necessary to identify node micrometastases. The SLN ITCs could be ignored as evidence on their effect on prognosis was limited.
The routine use of IHC for low-volume metastases in SLNs is not indicated. Step section or serial section H&E slides is recommended as the gold standard of SLN by many guidelines. In Europe, serial section or step section was used in 90% laboratories. However, absolute consensus regarding the optimal interval has not yet been reached. The European Working Group for Breast Screening Pathology recommended serial sectioning at an interval of 200 μm. The American Society of Clinical Oncology guidelines advised top level plus one or two step sections cut at 200–500 μm intervals of each 2-mm-thick block. The American Pathological Association advised three-level step sections each with 2-mm SLN blocks. However, it should be pointed out that such narrow interval sections recommended in almost all guidelines are time-consuming and labor intensive.
The OSNA assay is a reverse-transcription loop-mediated isothermal amplification-based assay that accurately detects the presence of metastases in breast lymph nodes by quantitatively measuring the expression of CK-19. The assay could significantly minimize the sampling limitation compared to histological evaluations that evaluate <5% of the SLN tissues. The OSNA is largely an automated assay and standardized with objective criteria, thus largely avoiding subjective errors. Based on the patients, the performance of the assay compared with histology was: accuracy 89.1%; sensitivity 87.7%; specificity 89.6%; PPV 73.8%; and NPV 95.6%. Based on the nodes, the performance was 91.4%, 83.7%, 92.9%, 69.1%, and 96.8%, respectively. Our results were similar to the published research.[7, 18-25]
The assay is easy to operate and it can test four SLNs from different patients at the same time. After training and 10 learning cases, a laboratory technician could accomplish the test within 40 min, which assists in reducing the workload of experienced pathologists. The time for completing a mastectomy or tumor resection is also approximately 40 min. As the OSNA assay results can inform surgeons just after the breast operation is finished, the whole process seems to fit together compactly with little if any downtime.
As mentioned above, intraoperative tests have their advantages and traditional approaches have their limitations. As the tissue used for FS is limited, it provides relatively accurate information regarding identifying SLN macrometastases, but it is not suitable for the identification of SLN micrometastases. It was reported by van de Vrande et al. that the sensitivity of FS was 71.6%, and it was less sensitive for ascertaining micrometastases compared to macrometastases (sensitivity 61.1% vs 84.0%). Touch imprint cytology is more subjective. The false-negative cases of TIC are usually due to the existence of micrometastases and the primary lobular carcinoma, and the false-positive cases are often correlated with active endothelial cells or epithelioid cells. Meta-analysis has shown that TIC has a sensitivity of 63%, and the sensitivity for macrometastases and micrometastases was 81% and 22%, respectively. In our study, FS and TIC had a sensitivity of 76.2% and 69.7%, respectively, based on the nodes; they both had poor sensitivities of 23.5% and 35.4% when testing nodes with micrometastases.
Another molecular assay for the diagnosis of SLNs is the GeneSearch breast lymph node assay. We have participated in the validation studies of both the breast lymph node assay and the OSNA assay and are aware of their relative merits.[28-30] The breast lymph node assay offers a qualified result as it lacks an external control and it could not differentiate SLN macrometastases and micrometastases. In contrast, the OSNA assay gives an absolute quantification of the number of CK-19 mRNA copies and can offer a prediction of macrometastases and micrometastases. We showed that the PPV value of the OSNA assay result (++) on node macrometastases was 83.2% and the assay could distinguish macrometastases and micrometastases. This is important, as mentioned above. The OSNA result (++) can also predict the chance of nSLN metastases. As approximately 50% of cases are SLN positive/nSLN negative, some mathematic models that could predict nSLN status with SLN involvement in breast cancer patients has been produced, such as the Memorial Sloan-Kettering Cancer Center nomogram, the Mayo nomogram, the Cambridge nomogram, the Stanford nomogram, and the Tenon axilla scoring system. It will be necessary to carry out another study to compare the performance of the OSNA predictive model with other models. From a technical aspect, the breast lymph node assay is more complex than OSNA, in that RNA extraction requires some experience in molecular laboratory techniques. This is not the case with the OSNA assay, as it has a low technical complexity and high degree of automation.
It is important to point out that it is exceedingly difficult to evaluate the real sensitivity of the OSNA assay because there is no SLN tissue left for histological evaluation after the molecular procedure. The small metastatic deposit may remain confined exclusively to the sample undergoing one test, escaping detection by the other. Thus, we introduced alternative slicing to minimize this sampling discrepancy, reducing the number of false-negative and false-positive results to a great extent. The accuracy of the OSNA assay in this study was excellent and the sensitivity is more reliable than current methods in the current study. The OSNA assay is an accurate and rapid intraoperative assay for assessing breast SLNs and it can replace FS and TIC in general medical practice.