1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

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%; = 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%; = 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.[3] 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.[6]

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.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References


From February to December 2010, 552 consecutive breast cancer patients scheduled for SLNB were enroled in this study from five centers. The study was approved by the ethics committee of each center and each patient provided informed consent. The patients who had undergone previous ipsilateral axillary surgery were excluded from this study.

Sampling method

Sentinel lymph nodes were defatted after SLNB. If the node weighed less than 100 mg, the node was only assessed by histology postoperatively. If the node weighed more than 100 mg, the node was sliced to equal blocks according to the length of the short axis. If the length was <4 mm, the node was sliced into two blocks along the long axis (a, b). Intraoperatively, blocks a and b were tested by TIC, and block a was prepared for OSNA. Postoperatively, block b was assessed by histology. If the length was more than 4 mm, the node was sliced into four blocks (ad). Intraoperatively, all blocks (ad) were tested by TIC, and blocks a and c were prepared for OSNA. Postoperatively, blocks b and d were subjected to histology. In addition, 211 patients from two centers (center A and center D) also received FS evaluation and the block subjected to histology was evaluated by FS intraoperatively. For the patients who accepted SLNB as a substitute for ALND, ALND was only carried out if any of the intraoperative tests were positive.

One-step nucleic acid amplification assay

All the assay operators attended a 3-day training course before the study. All OSNA assays were carried out according to the manufacturer's instructions (Sysmex). Three different calibrators with defined CK-19 mRNA copy concentrations were used to establish a standard curve on an RD-100i instrument (Sysmex). Node tissues were then homogenized in 4 mL homogenizing buffer LYNORHAG (Sysmex). The homogenate was briefly centrifuged and directly used as a template for amplification. Amplification of CK-19 mRNA was automatically carried out in the RD-100i instrument with a ready-to-use reagent LYNOAMP (Sysmex) kit, which consisted of a primer–nucleotide mix, enzymes, and CK19 mRNA calibrators, as well as homogenate, diluted homogenate, positive and negative controls. All the results were presented on the RD-100i instrument in qualitative categories (++, +, I, −) and further specified by CK-19 mRNA copy number/lL: (−), <250 copies in homogenate; (I), <250 copies in homogenate and >250 copies in dilution homogenate; (+), 250–5000 copies in homogenate; and (++), >5000 copies in homogenate. The results (++, +, I) were considered as positive results. The result (+) was comparable to the presence of a micrometastasis, and (++) to macrometastasis.

Histological evaluation

All node blocks used for histological evaluation were fixed in 10% buffered formalin and paraffin embedded. Four 4- to 6-μm-thick slides, 200 μm apart, were taken from each block. Immunohistochemistry was also done on the OSNA assay positive/histology negative or the OSNA assay negative/histology positive cases. Metastases larger than 0.2 mm were considered positive in this study. Metastases were classified according to the 6th criterion of American Joint Cancer Committee.[7] Macrometastases (≥2 mm) and micrometastases (0.2–2 mm, pT1mic) were considered node positive. Isolated tumor cells (≤0.2 mm, pT0[i+]) were considered node negative. All the slides were reviewed by a senior pathologist from another center. When there was a disagreement, a third senior pathologist was attended to make the final diagnosis. All the pathologists were blinded to the OSNA results.

Statistical methods

The primary goal was the sensitivity, specificity, PPV, and NPV of the OSNA assay. Secondary goals included the time taken to carry out the assay and the PPV value of the assay result (++) on the node macrometastases and nSLN metastases. The McNemar test was used to compare the rate between groups. The t-test was used to compare the difference of means between groups. P < 0.05 was considered to be statistical significant.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

A total of 1188 SLNs were removed (median 2, mean 2.6) from 552 patients. Of the 552 patients, 107 had macrometastases, and 31 had micrometastases. Of the 1188 SLNs, 141 had macrometastases, and 48 had micrometastases. The clinicopathological characteristics of the 552 enroled patients is shown in Table 1.

Table 1. Clinicopathological characteristics of breast cancer patients enroled in this study
  1. ALND, axillary lymph node dissection; ER, estrogen receptor; HER-2, human epidermal growth factor receptor; SLN, sentinel lymph node; SLNB, sentinel lymph node biopsy; Tis, ductal carcinoma in situ.

Mean age, years (range)50 (24–83)
Tumor size
Not known45
Blue dye213
Combining use of blue dye and isotope227
Histopathologic diagnosis
Invasive ductal carcinoma383
Invasive lobular carcinoma23
Ductal carcinoma in situ70
Other types76
ER status
Not known32
HER-2 status
Not known38
Breast surgery
Breast conserving surgery236
Axillary surgery
ALND (irrespective of SLN status)289
ALND (following positive SLN)121

Patient-based analysis

As shown in Table 2, the accuracy of the OSNA assay was 89.1% (95% CI, 86.3–91.5%; 492/552). The performance was: sensitivity 87.7% (95% CI, 81.0–92.7%; 121/138); specificity 89.6% (95% CI, 86.3–92.4%; 371/414); PPV 73.8% (121/164); and NPV 95.6% (371/388). For TIC, the results were 79.7% (110/138), 97.3% (403/414), 83.5% (101/121), and 93.5% (403/431), respectively. The sensitivity of the OSNA assay was higher than that of TIC (not significant, P = 0.052).

Table 2. Performance of one-step nucleic acid amplification (OSNA) assay, touch imprint cytology (TIC), and frozen section (FS) in assessing breast sentinel lymph node
 Sensitivity (%)Specificity (%)PPV (%)NPV (%)
  1. NPV, negative predictive value; PPV, positive predictive value.

Patient-based analysis
Node-based analysis

The sensitivity, specificity, PPV, and NPV of FS for 211 patients were 81% (47/58), 98.7% (151/153), 95.9% (47/49), 93.2% (151/162), respectively. The sensitivity (52/58, 89.7%) of the assay was higher than that of FS (not significant, P = 0.227).

Node-based analysis

The accuracy of the OSNA assay compared to histological evaluation was 91.4% (1086/1188; Table 2). The OSNA assay performance was: sensitivity 83.7% (159/190); specificity 92.9% (927/998); PPV 69.1% (159/230); and NPV 96.8% (927/958). The TIC performance in 1087 nodes was: sensitivity 76.2% (144/189); specificity 97.3% (971/998); PPV 84.2% (144/171); and NPV 95.6% (971/1016). The sensitivity of the OSNA assay (83.6%) was significantly higher than that of TIC (P = 0.044).

Results for FS in 459 nodes were: sensitivity 69.7% (53/76); specificity 99.5% (381/383); PPV 96.4% (53/55); and NPV 94.3% (381/404). The sensitivity of the OSNA assay (59/76, 77.6%) was higher than that of FS (not significant, P = 0.286).

When assessing nodes with macrometastases, the sensitivity of the OSNA assay was similar to that of FS (51/59, 86.4%; 49/59, 83.1%; P = 0.791) and TIC (128/141, 90.8%; 127/141, 90.1%; P = 1.00). However, when assessing nodes with micrometastases, the sensitivity of the OSNA assay was higher than that of FS (8/17, 47.1%; 4/17, 23.5%; not significant, P = 0.289) and was significantly higher than that of TIC (30/48, 62.5%; 17/48, 35.4%; P = 0.007).

Discordant case investigation

There were 43 cases that were OSNA assay positive/histology negative. Of these patients, five had positive CK-19 IHC results, three had positive TIC results, three had SLN ITCs, and one was found to have nSLN metastases. Given potential sampling error, it is likely that these 12 patients are not true false-positive cases. Performance of the assay, after adjustment, becomes: accuracy 91.3% (504/552); sensitivity 88.7% (133/150); specificity 92.3% (371/402); PPV 81.1% (133/164); and NPV 95.6% (371/388).

There were 138 SLN positive cases in total in this study. After adjusting the TIC positive/histology negative cases, the FS positive/histology negative cases, the CK-19 IHC positive/histology negative cases, the nSLN positive/histology negative cases, and the SLN ITCs cases, the positive cases increased to 175. The performance of the assay became: accuracy 86.1% (475/552); sensitivity 74.9% (131/175); and specificity 91.3% (344/377). Adjustments also changed the sensitivity of TIC to 69.1% (121/175). The sensitivity of the assay was higher than TIC (P = 0.144). For FS, adjustments changed the sensitivity to 60.5% (49/81). The sensitivity of the assay (75.3%, 61/81) was significantly higher than FS (P = 0.01).

After adjusting the TIC positive/histology negative nodes, the FS positive/histology negative ones, the CK-19 IHC positive/histology negative ones, and the SLN ITCs ones, the positive nodes increased from 190 to 235. In the node-based analysis, the sensitivity of the assay was higher than TIC (77.0%, 181/235 vs 72.7%, 171/235; P = 0.261), and was significantly higher than FS (74.3%, 81/109 vs 54.1%, 59/109; < 0.001).

Turnaround time

The mean turnaround time of the assay (the time from receiving the nodes to acquiring the results) was 37.3 min. There was no significant difference in the turnaround times at each center (P = 0.074). There seemed to be a learning curve for the time taken to complete the assay, as shown in Table 3. In the initial 20 cases, the OSNA assay was carried out in a mean time of 40.2 min. In the median 40 cases, the assay was carried out in a mean time of 36.9 min. For the latter cases, OSNA was done in a mean time of 36.1 min.

Table 3. Turnaround time to complete one-step nucleic acid amplification at each study center
 Center ACenter BCenter CCenter DCenter EMeanP-value
Turnaround time (min)37.237.435.739.637.137.30.074
Initial cases (cases 1–20)41.841.840.640.536.340.2
Median cases (cases 21–60)37.137.635.
Latter cases (cases 60 and above)35.334.834.043.837.336.1

Predictive value of assay results

The PPV value of the assay result (++) on node macrometastases was 83.2% (89/107), and the result (+) on micrometastases was 26.2% (11/42; Table 4). The PPV value of the assay results (++) and (+) on nSLN metastases was 42.2% (43/102) and 12.2% (4/33), respectively. Accordingly, the quantitative molecular assessment of the OSNA assay allowed the distinction of the size of the metastasis, and OSNA (++) could predict the likehood of nSLN metastases more accurately than (+).

Table 4. Predictive value of assay results on the type of metastases and non-sentinel axillary lymph node (nSLN) metastases
OSNA assay resultsSLNTotal
Macromet.Micromet.ITCsNo ITCs
OSNA assay resultsnSLNTotal
  1. Assay results are expressed as the CK-19 mRNA copy number/μL: (I), <250 copies in homogenate and >250 copies in dilution homogenate; (+), 250–5000 copies in homogenate; and (++), >5000 copies in homogenate. Macromet., macrometastases; Micromet., micrometastases; ITCs, isolated tumor cells; OSNA, one-step nucleic acid amplification; SLN, sentinel lymph node.



  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

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.[8] 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.[9] 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.[14] 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.[14] 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.[15] 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.[16] 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.[9] The American Pathological Association advised three-level step sections each with 2-mm SLN blocks.[17] 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.[26] 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.[27] Meta-analysis has shown that TIC has a sensitivity of 63%, and the sensitivity for macrometastases and micrometastases was 81% and 22%, respectively.[5] 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.


axillary lymph node dissection


confidence interval


frozen section




isolated tumor cells


negative predictive value


non-sentinel axillary lymph node


one-step nucleic acid amplification


positive predictive value


sentinel lymph node


sentinel lymph node biopsy


touch imprint cytology


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
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