Intraoperative palpation increases the eciency of sentinel lymph node biopsy but is not necessary for every operation

Although intraoperative palpation (IOP) is recommended during the sentinel lymph node biopsy (SLNB), its actual effect on SLNB remains unclear. Meanwhile, controversy exists regarding whether IOP is necessary for all SLNB. The study examined the impacts of IOP on SLNB and determined whether IOP is necessary for every operation. Methods 126 patients with early stage breast cancer underwent SLNB using Indocyanine green (n = 64) and Methylene blue (n = 62). Traced lymph nodes (TLNs) were resected, followed by the palpated lymph nodes (PLNs) surrounding the original TLNs. and we analyzed the results (with and without IOP) in terms of detection rate, number of removed SLNs and detection rate of metastatic SLNs.


Introduction
Sentinel lymph node biopsy (SLNB) is proved to be alternative to axillary lymph node dissection (ALND) by the ACOSOG Z0011 trial, and has been the standard operation for axillary assessment in patients with early-stage breast [1]. Even intraoperative palpation (IOP) of axillary tissue is recommended during the procedure of SLNB [1,2], the nodes indenti ed by tracers (TLNs) were exclusively regarded as sentinel lymph node(SLNs) in most previous studies, but nodes missed by tracer but could be detected by IOP were not assessed. Without releazing the impacts of IOP in clinical practice, many surgons may be prone to neglect intraoperative palpation when TLNs are detected already.
There is limited research investigating the impacts of IOP on SLNB, but in one study investigating SLNB using standard tracing method (the combination of blue dye and radioisotope tracers), the author observed that 2.4% patients had SLNs detected solely by IOP, and seven patients had clinically suspicious nodes identi ed solely by palpation, in addition to sentinel lymph nodes detected by tracer. In ve of the seven patients, the nodes harbored metastasis [3]. The impacts of IOP on SLNB using common tracing methods such as indocyanine green and methylene blue remains unknown. Moreover, compared with standard tracing method, these tracing methods are inferior in detection rate, mean number of SLN and false negative rate but more available and adopted widely in the world [4,5]. Meanwhile, controversy exists regarding whether IOP is necessary for every SLNB operation.
We conducted this study to examined the impacts of IOP on SLNB and determined whether IOP is necessary for every patient.

Patients
This study included 126 breast cancer patients who underwent SLNB with ICG and MB from October 2017 to December 2019 in our department. The study was in accordance with Helsinki Declaration and approved by the Institutional Review Board of Zhengzhou People's Hospital. The IRB numbers was 2017CTN0118, and all patients signed informed consent.
The patient inclusion criteria consisted of pathological diagnosis of breast cancer; maximum tumor diameter of 5 cm; no identi cation of clinically metastatic axillary lymph nodes through ultrasound, chest computed tomography or breast magnetic resonance imaging. And the exclusion criteria included palpable axillary lymph nodes; previous axillary surgery; neoadjuvant chemotherapy or endocrine therapy.

Surgical procedure
SLNs identi cation using ICG: One minute before surgery, 1 ml of ICG solution (Yichuang Pharmaceutical LLC, Dandong, China.) injected subcutaneously and intradermally at the areola. Following gentle breast massage, ICG uorescence was stimulated and detected by a hand-held photodynamic eye camera (MingDe Biomedical Technology Corporation, Hebei, China). Later, an incision was made to start the SLNB where the uorescence disappeared. After excising the uorescent nodes, the area of a 3 cm diameter around the rst TLNs was gently palpated, and any enlarged or hard palpable suspicious LNs were also excised.
PLNs were more offwhite in color and more pliable and tough in texture than fat or glandular tissue. When suspicious nodes were identi ed, they were excised and handled as if they were lymph nodes.
SLNs identi cation using MB: Ten minutes before surgery, 1 ml of 1% MB (Jichuan Pharmaceutical, Taizhou, China) was subdermally injected into the periareolar region of 4 quadrants of the breast (clockwise). Following gentle massage, a small transverse incision was made one centimeter below the axillary line to detect blue-stained nodes.
After that, palpable suspicious nodes around the blue-stained nodes were also excised.
After the complication of SLNB procedure, we divided all removed lymph nodes into two parts: traced lymph nodes (TLNs) and palpated lymph nodes (PLNs). TLNs were de ned as any uorescent lymph nodes located by photodynamic eye camera or any node stained by MB. PLNs were any lymph nodes that were clinically suspicious on systematic palpation of the area of a 3 cm diameter around the rst TLNs. All these SLNs were examined by intraoperative histopathology examination of sliced 2-mm sections of frozen tissues to detect SLN metastasis. In patients displaying malignant cells in the nodesof frozen sections, ALND was performed as a standard axillary treatment.

Statistical analysis
Chi-squared test were used to compare detection rate of SLNs, number of SLNs and detection rate of metastatic nodes between the results with palpation and those without palpation. All statistical analyses were performed using IBM SPSS statistics version 23 (SPSS Inc., Chicago, IL, USA) software, and p-value of < 0.05 represented statistical signi cance.

Patients and Tumor Characteristics
The patients ranged in age from 26 to 75 years (mean±SD, 47.7±11.4 years) and other characteristics of patients and tumors were presented in Table 1.
Detection rate of SLNB Detection rate of metastatic nodes 12(18.8%) and 10(16.1%) patients were detected with13 and 12 traced metastatic SLNs in the two groups. However, 2 and 2 more patients were detected with only palpated metastatic node even there were additional TLNs. As a consequence, the detection rate of metastatic nodes increased to 14 (21.9%) and 12(20.9%), respectively. The metastatic SLNs in the two groups increased to 17 and 14, respectively . The above detailed results were shown in Table2 and Table 3.
The bene cial impacts of intraoperative palpation may lie on that tracer and intraoperative palpation can complement each other. Tracers such as ICG helped doctors to quickly locate lymph nodes [14,15]. But leakage caused by cuts in the lymphatic ducts after the excision of the rst node made it di cult to detect other uorescent nodes in the surgical eld. MB can stain lymph nodes blue, facilitating their identi cation during opreation. But the visual interference caused by intraoperative bleeding will obstruct SLNs identi cation, particularly in obese patients [10,16]. Surgeons can continue to detect lymph nodes by systematically palpating neighboring axillary contents. In our study, 183 (28.9%) nodes were detected merely by IOP, indicating that these nodes may be undetected if merely traced lymph nodes were reoved and considered as the SLNs. Besides that, we observed that in the 26 patients detected with metastatic nodes, 4 (15.4%) were detected with only palpated metastatic nodes,even there were additional TLNs were detected before. It suggested that if only TLNs were removed and examinated, some metastatic lymph nodes would be undetected, a false-negative does occur inevitably. So PLNs should be counted as part of SLNs to reduce the false-negative rate(FNR).
IOP could raise the number of SLNs removed and many researchers have con rmed the association between the number of SLNB and the FNR. But the optimal number of removed SLNs has always been controversial. Kenny S concluded that two SLNs were enough to predicte the status of axillary lymph nodes [17]. While Melanie A observed all positive SLNs were identi ed within the rst three SLNs detected; removal of more than three SLNs did not increase the accuracy of nding a positive node [18]. Min Yi found that removing up to 5 SLNs was su cient to identify metastatic carcinoma in > 99% of patients [19]. But Martin D thought that there is no absolute upper threshold for the number of SLNs that should be removed [20]. In our study, The rst positive SLN was the rst node removed in 20 patients (76.9%), following the second node in 22 patients (84.6%) and was removed in all patients by the third SLN. The correlation between the number of lymph nodes removed and cumulative percentage with a positive SLN was shown in Fig. 1 [18,[21][22][23]. Accoding to previous studies [24][25][26][27], the likelihood of a false-negative SLN nding was signi cantly decreased, when SLNs detection number reaches 3, the FNR can reached an acceptable level of less than 5%, the relationship between SLNs removed and FNR was shown in Fig. 2.
Whereas, the number of SLNs removed is associated with the morbidity of SLNB. Removing too many additional lymph nodes may expose patients to increased morbidity of lymphedema, pain, stiffness and shoulder weakness and brachial plexus injuries [28]. Data from the ACOSOG Z0010 trial found an increase in rates of axillary seroma formation and wound infections in patients who had more than four SLN removed [28]. Cost-effectiveness and cost-utilization studies have found that excision of increasing numbers of SLN is associated with increased OR time, increased pathology costs, and increased procedure costs [21].
Thus, We need to nd a balance piont between the bene ts and risks of IOP to ensure accuracy and minimize morbidity and procedure costs. We suggest that when the number of TLNs reaches 3 or more, intraoperative palpation of axillary tissue was not necessary to remove redundant lymph nodes.

Conclusion
Intraoperative palpation increases detection rate and number of SLNs and reduced the potential falsenegative rate. But intraoperative palpation is not necessary for all SLNB when the number of TLNs reaches three or more. Declarations Acknowledgements Not applicable.

Authors' contributions
Xingsong Q performed the experiments and the data analysis. Xingsong Q and Zhiwei L conceived and designed the study. Zhiwei L and Wei L contributed to the acquisition of the data. Xinsong Q, Xinglei Q and Xinyu Z contributed to the analysis and interpretation of data. Xingsong Q wrote the manuscript. Zhiwei L, Wei L and Xinyu Z reviewed and edited the manuscript. All authors read and approved the nal manuscript.

Funding
No sources for funding are to be declared.
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The study was in accordance with Helsinki Declaration and approved by the Institutional Review Board of Zhengzhou People's Hospital. The IRB numbers was 2017CTN0118, and all patients signed informed consent.

Consent for publication
Not applicable.    Figure 1 The Number of Lymph Nodes Removed and Cumulative percentage with a positive SLN When two SLNs was removed, around 90% of the rst positive SLN was detected; when the number of SLNs reaches 3, more than 95% of the rst positive SLN was included.