The sentinel lymph node biopsy (SLNB) procedure has dramatically revolutionized breast surgery during the last decades (1). In fact, SLNB with staging intent has progressively replaced complete axillary lymph node dissection (CALND), previously intended with a curative purpose. Probably in the future, even this procedure might be abandoned in favor of a non-surgical lymph node evaluation to predict patients’ prognosis and better tailor subsequent therapies (1, 2).

For what concerns the technique, we assisted in a first evolution to reduce the number of interventions with the introduction of intraoperative frozen section evaluation of the SLNB. Performing in the same surgical session, the primary breast surgery, the SLNB, and eventually the CALND according to the intraoperative lymph node assessment reduces the patients’ surgical burden and the healthcare system costs (3–5). Secondly, an intraoperative molecular-based lymph node staging has been adopted in place of the traditional morphological examination to minimize the operative time and enhance accuracy (6). In particular, the one-step nucleic acid amplification (OSNA) system consists of the amplification of cytokeratin (CK) 19 mRNA directly from the lysate to distinguish positive from negative samples (7–9). This second advance, besides ensuring a reduction in surgical sessions per patient and the costs for the healthcare system, allows reducing operating times and the pathologist workload (4, 6).

Although OSNA is considered the most accurate intraoperative lymph node staging technique (10), the literature lacks cohort studies, with everyday routine data, comparing survival analysis between OSNA and other lymph node staging methods (intraoperative frozen section or definitive histology).

Our study evaluates the OSNA method for the intraoperative analysis of sentinel lymph node biopsy compared with frozen section and definitive histological examination among patients affected by breast cancers classified as TNM stage I and II considering disease-free survival and overall survival.

We included in this study 2412 patients with invasive breast cancer classified as TNM stage I and II and operated on during the considered period. A definitive histological examination of SLNB was performed in 727 cases (group A), intra-operative FS in 697 patients (group B), and OSNA in 988 cases (group C).

Mean patient age resulted in 60.24 years ( ± 12.1), mean BMI was 25.23 kg/m2 ( ± 4.77), and 79.35% of women were in their post-menopausal period. The prevalence of familial cancer history and previous use of estrogens were respectively 30.29% and 35.28%. In most cases, definitive breast surgery was conservative in most cases (62.94%), while mastectomy was definitively performed in 37.06% of cases. Adjuvant hormonal therapy was administered in 84.82% of women, adjuvant radiotherapy in 62.16%, and adjuvant chemotherapy in 31.84% (759/2384).

ITCs were found in 2.32% of cases, micrometastasis in 9.12%, and macrometastases in 13.64%. The extracapsular lymph node invasion was found in 0.5% of cases, and non-axillary loco-regional lymph node metastases were found in 1.37% of cases. Definitive CALND was performed in 22.18% of patients. Among 535 CALND, 312 were performed after detecting macrometastases, 173 micrometastases, 9 ITC, and 41 cases because of sentinel node detection failure. In addition, CALND was not performed in 47 patients with ITCs and 47 with micrometastases.

The most frequent histotype was invasive carcinoma non-special type (previously named invasive ductal carcinoma, 79.1%), followed by invasive lobular carcinoma (12.73%), other special types of invasive carcinoma (3.98%), and the combined ductal and lobular invasive carcinoma (4.19%). The most common molecular subtype was luminal A (50.08%), followed by luminal B (27.57%), basal-like (7.59%), luminal Her (5.85%), and Her-enriched (3.23%). In 5.68%, the molecular subtype was not specified. Tumor grading G2 accounted for 59.37% of cases. The majority of tumors were classified as T1 (85.66%), N0 (77.24%), and TNM stage I (75.17%).

All patients with definitive histological examination had two surgical interventions, while FS and OSNA were performed in the same surgical session as the primary breast tumor. In addition, the surgical operation was significantly longer in cases assessed intra-operatively by FS than by OSNA (70.1 minutes ±10.5 vs. 42.1 minutes ±5.1; p <0.05).

In Table 1 , we report the different characteristics of the three studied groups. The definitive histological examination group had a significantly higher prevalence of BCS than FS or OSNA ones. In addition, FS was associated with a lower prevalence of CALND than the definitive histological examination or OSNA ( Table 1 ). The prevalence of Mib-1>20% was significantly higher in OSNA than FS and definitive histological examination. Table 2 shows the differences in terms of tumor characteristics between the definitive histological examination and OSNA or FS groups. The prevalence of luminal A subtype was significantly higher in FS than in definitive histological examination and OSNA ( Table 2 ). In addition, the prevalence of positive nodes was significantly higher in the OSNA group than in the other two groups ( Table 2 ). Concurrently, the OSNA group had a significantly higher prevalence of N1 tumors ( Table 3 ) and a higher prevalence of both macro- and micro-metastases ( Table 2 ) than the other two groups.

Figure 1 shows the Kaplan-Meier analysis, and no significant differences have been observed in OS, DSF, cumulative local or distant metastases ( Figures 1A–D ). At 5 years follow-up the OS in definitive histological examination group, FS, and OSNA was respectively 99.16% (95% C.I. 98.49-99.83%), 99.12% (95% C.I. 98.43-99.82%), and 99.20% (95% C.I. 98.61-99.80%) while the DFS was respectively 96.38% (95% C.I. 95.02-97.75%), 96.37% (95% C.I. 94.98-97.78%), and 96.51% (95% C.I. 95.32-97.72%). The mortality rates in the definitive histological examination group, FS, and OSNA were respectively 1.681 deaths/1000 patients/year, 1.757 deaths/1000 patients/year, and 1.600 deaths/1000 patients/year. The local recurrence incidence rates in the definitive histology group, FS, and OSNA were respectively 3.383 cases/1000 patients/year, 4.740 cases/1000 patients/year, and 4.853 cases/1000 patients/year. The distant metastases recurrence incidence rates in the definitive histological examination group, FS, and OSNA were respectively 4.236 cases/1000 patients/year, 3.237 cases/1000 patients/year, and 2.753 cases/1000 patients/year. Table 4 also shows univariate and multivariate cox analysis, and no significant differences have been found in OS and DFS among the three studied groups. The analysis in Table 4 was stratified for N0, and no significant differences were found. DFS was assessed separately for macrometastases. In the univariate analysis and multivariate analysis, no significant differences were observed. The multivariate adjustment for DFS in the sub-group of macrometastases resulted for intraoperative FS of HR 2.44 (95% C.I. 0.44 - 13.38) (p=0.305) in reference to definitive histology and for Intraoperative OSNA of HR 1.05 (95% C.I. 0.18 - 6.1) (p=0.957). The multivariate adjustments were performed according to the most predictive factors and the possible confounders found in the univariate analysis. No other stratifications were performed due to the limited number of events.

Surgical procedure length was significantly shortened by the intraoperative OSNA technique. In addition, despite the long follow-up considered, no significant differences have been observed among the three groups (intraoperative OSNA or FS and definitive histology) regarding OS, DSF, cumulative local or distant metastases, apart from a non-significant increased risk of local recurrences related to FS and OSNA method.

Previous studies demonstrated a peak of local recurrences after SLNB between the third and the sixth year of follow-up, which are mostly included in our study (11). Only a limited number of studies performed a survival analysis considering OSNA, and none compared in the same population in all three groups we considered based on the SLNB evaluation technique (10, 27, 28). Recently, Shimazu and coworkers found a significantly improved DFS in N0 status detected by OSNA, if compared with traditional histology, suggesting the better staging efficiency of the OSNA method (28). Our data did not confirm this advantage. Instead, we showed a non-significant increased recurrence risk in the case of intraoperative OSNA, in comparison with definitive histology, which may be explained by the higher incidence of unfavorable prognostic factors found in the group of patients who underwent OSNA evaluation of their SLNB. Indeed, the OSNA method resulted significantly associated with tumor features, which are usually expressions of a more aggressive biological behavior of the disease, such as tumor multifocality/multicentricity, extensive intraductal component, peritumoral vascular invasion, comedo-like necrosis, and Mib-1>20% (29). And in our opinion, this fact simply reflects the progressive extension of SLNB indications.

In the literature, the OSNA system allowed a more efficient detection of micrometastasis, consequently decreasing the number of false-negative histological examinations resulting from the small size of micrometastases, which may not be included in any microscopical section (28, 30, 31). Also, in our experience, a significant increase in the prevalence of micrometastasis was found compared to FS and definitive histology. In addition, the prevalence of macrometastases was similar to the definitive histology, while FS had a significantly lower prevalence of macrometastases than definitive histology. This last finding could be due to a significantly higher detection rate of definitive histology than FS or simply to a better selection of subjects undergoing FS than definitive histology. Along with the increased number of detected micrometastases compared to FS and definitive histology, the OSNA technique also increased the number of diagnosed macrometastases than FS, correlating with a higher prevalence of node-positive disease and consequently a higher prevalence of secondary CALNDs. However, Hintzen and coworkers recently demonstrated that the increased rate of CALND after OSNA could be limited by broader adoption of the criteria that emerged from the Z0011 and AMAROS trials for axilla treatment (32–34). In particular, they found that the use of the OSNA method, in association with these emerging criteria for axilla treatment, does not lead to more CALNDs, axilla radiotherapy, or adjuvant systemic therapies (34).

As expected, the OSNA technique resulted in an evident improvement in breast surgery in our center. In particular, in accordance with the literature (4), it succeeded in significantly reducing the surgical time from a mean operation length of 70.1 ( ± 10.5) minutes in the case of FS to a mean operation length of 42.1 ( ± 5.1) minutes using OSNA. However, this result required accurate compliance with some technical premises, such as the strong limitation of the number of excised nodes. Consequently, both OSNA and FS correlated with a smaller number of excised sentinel nodes than definitive histological examination, resulting in nearly one single node. Furthermore, recently Saruta and coworkers found that in Japan, the adoption of the OSNA technique, in addition to reducing the burden on the patient (limiting the number of surgeries and the duration of surgical procedures), also reduced the breast cancer healthcare costs per patient (35).

The main limitations of this study are the retrospective and non-randomized nature of the chart review and the unavailability of detailed data about cost-effectiveness and side effects. Among the strengths of this study, we can emphasize the broad cohort and the remarkable follow-up data. In addition, another essential strength is the uniform management due to regular multidisciplinary meetings in a single-center experience.

Our findings add further proof to the body of evidence supporting the wider adoption of this innovative technology that enables a safe reduction in patient surgical burden and healthcare costs. The reduction in costs also comprises a lower workload for the pathologist than intraoperative FS and definitive histology.

In conclusion, no difference was found in OS and DFS when comparing OSNA, FS, and definitive histology. At the same time, the OSNA system was advantageous in reducing single-session surgical operating time and the pathologist workload.

The datasets presented in this article are not readily available because the data that support the findings of this study are available, but restrictions apply to the availability of these data, which was used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of the Internal Review Board. Requests to access the datasets should be directed to serena.bertozzi@asufc.sanita.fvg.it.

The studies involving human participants were reviewed and approved by Internal Review Board of the Department of Medical Area (University of Udine). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

Substantial contributions to conception and design or acquisition of data or to analysis and interpretation of data: SB, AL, MB, LS, DA, AP, MA, MO, LM, and CC. Drafting the article or revising it critically for important intellectual content: SB, AL, MB, LS, DA, AP, MA, MO, LM, and CC. All authors have read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.