Breast Cancer DOI 10.1007/s12282-014-0551-1
ORIGINAL ARTICLE
Triple assessment of sentinel lymph node metastasis in early breast cancer using preoperative CTLG, intraoperative fluorescence navigation and OSNA Mohamed Mokhtar • Yukiko Tadokoro • Misako Nakagawa Masami Morimoto • Hirokazu Takechi • Kazuya Kondo • Akira Tangoku
•
Received: 26 December 2013 / Accepted: 30 June 2014 Ó The Japanese Breast Cancer Society 2014
Abstract Background Sentinel lymph node biopsy (SLNB) became a standard surgical procedure for patients with early breast cancer; however, the optimal method of sentinel lymph node (SLN) identification remains controversial. The current study presents the protocol of our institution for preoperative and intraoperative SLN detection. Methods Fifty female patients with early breast cancer and clinically node-negative axilla were enrolled in this study. All patients underwent preoperative CT lymphography (CTLG), intraoperative SLNB using fluorescence navigation, intraoperative one-step nucleic acid amplification (OSNA) and postoperative hematoxylin and eosin histopathological analysis. Prediction of metastasis by CTLG and detection of metastasis by OSNA were compared to results of histopathology as standard reference.
Results SLN were identified by preoperative CTLG and intraoperative SLNB with fluorescence navigation in all patients, the identification rate was 100 %. SLN metastases were detected as positive by OSNA in 9 patients (18 %), 4 were (??), 4 were (?) and 1 was (?I). SLN metastases were detected as positive by histopathology in 10 patients (20 %). The concordance rate between OSNA and permanent sections was 90 %. The negative predictive value of CTLG was 80 %. Conclusion Use of CTLG and fluorescence navigation made performing SLNB with high accuracy possible in institutions that cannot use the radioisotope method. OSNA provided accurate intraoperative method, allowing for completion of axillary node dissection during surgery and avoidance of second surgical procedure in patients with positive SLNs, thereby reducing patient distress and, finally, saving hospital costs. Keywords Sentinel lymph node biopsy Computed tomographic lymphography One-step nucleic acid amplification Breast cancer
M. Mokhtar K. Kondo Department of Oncological Medical Services, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8505, Japan M. Mokhtar Department of Oncological Surgery, Minia Oncology Institute, Minya 61111, Egypt Y. Tadokoro (&) M. Nakagawa M. Morimoto H. Takechi A. Tangoku Department of Thoracic, Endocrine Surgery and Oncology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8505, Japan e-mail: [email protected]
Abbreviations ALND Axillary lymph node dissection BCS Breast conserving surgery BMI Body mass index CTLG Computed tomography lymphography H&E Hematoxylin and eosin NPV Negative predictive value OSNA One-step nucleic acid amplification PPV Positive predictive value QRT-PCR Quantitative reverse transcription-PCR RI Radioisotope SLN Sentinel lymph node SLNB Sentinel lymph node biopsy
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Introduction
Patients and methods
SLNB became a standard surgical procedure for patients with early breast cancer. SLNB benefits patients without metastatic nodes by avoiding unnecessary axillary lymph node dissection (ALND) with its concomitant morbidities [1, 2]. False negative of SLNB is a critical problem, as it may lead to inadequate adjuvant therapy, which can negatively affect patient’s prognosis [3]. The optimal method of SLN identification remains controversial. The most commonly used methods to identify the SLN are dye staining and radioisotope (RI) incorporation. The combination of the RI and dye-staining methods is the most accurate way to identify SLN [4–6]. However, in Japan, most hospitals cannot use RI because of institutional limitations. The identification rate with the dye method alone is inferior to the RI combination method. The correct SLNs with afferent and efferent lymphatic routes surrounding detailed anatomy were imaged preoperatively, and detected by blue dye imaging based on CTLG navigation. With CTLG, preoperative diagnosis of SLN metastasis is feasible as mentioned before [7, 8]. SLN metastasis is generally detected by conventional means including the intraoperative H&E-based histopathologic examination of frozen section or cytological observation of touch imprints, followed by definitive postoperative histopathologic examination of permanent sections [10, 11]. However, the sensitivity of these intraoperative methods is not high; many investigators have reported that the intraoperative H&E-based histopathologic examination has a false-negative rate of 5–52 % [12]. Furthermore, these methods provide subjective rather than objective results, which may differ from one pathologist to another. On the other hand, the definitive postoperative histopathologic examination generally requires 5–10 days for assessment [13, 14]. To overcome the shortcomings of histopathological methods, molecular assays have been developed for detection of lymph node metastasis in breast cancer patients [15, 16]. OSNA that amplifies CK19 mRNA (OSNA, Sysmex, Kobe, Japan) was recently developed to accurately detect metastasis measuring 0.2 mm or more. OSNA method is characterized by the quantitative measurement of a target mRNA in a metastatic lymph node, a brief reaction time for the OSNA process, a high specificity for the target mRNA, and an absence of genomic DNA amplification [17, 18]. Our aim was to overcome the downsides of conventional methods for detection of SLN in early breast cancer through a new protocol including preoperative CTLG, intraoperative fluorescence navigation and OSNA in detection of SLN metastasis using final histopathology as a reference standard.
Patients
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From September 2012 to September 2013, 50 female patients with early breast cancer and clinically node-negative axilla were enrolled in this study. All patients underwent preoperative CTLG, intraoperative SLNB using fluorescence imaging with indocyanine green (ICG) navigated by CTLG as reported before [19, 20], intraoperative OSNA and postoperative H&E histopathological examination in Tokushima University Hospital. The results were analyzed in relation to the SLN identification and various clinicopathological parameters, including age, body mass index (BMI), tumor size, clinical stage, nuclear grade and histological type. Patients’ characteristics and tumor status are shown in Table 1. 3D CTLG CTLG was performed using multidetector row helical CT scanner (Siemens Volume Zoom; Siemens-Asahi Medical
Table 1 Patients’ characteristics of and tumor status
Age (mean ± SD)
60 ± 11.5
BMI (mean ± SD)
23 ± 3.4
Tumor size (%) Tis
11/50 (22)
T1
27/50 (54)
T2
12/50 (24)
Clinical stage (%) 0
11/50 (22)
I
27/50 (54)
II
12/50 (24)
Nuclear grade (%) Grade 1 30/50 (60) Grade 2
15/50 (30)
Grade 3
5/50 (10)
Histological type (%) DCIS
12/50 (24)
IDCA
29/50 (58)
ILCA
2/50 (4)
Others
7/50 (14)
Operation type (%) Total Partial DCIS ductal carcinoma in situ, IDCA invasive ductal carcinoma, ILCA invasive lobular carcinoma
19/50 (38) 31/50 (62)
SLN positive
10/50 (20 %)
SLN negative
40/50 (80 %)
Breast Cancer
Technologies Ltd., Tokyo, Japan). Each patient was placed in supine position with arms positioned in a cranial direction. After local anesthesia with subcutaneous injection of a total of 0.5 ml of 1 % lidocaine hydrochloride, a total 4-ml dose of non-ionic water-soluble iodinated contrast medium (Iopamidol; Iopamiron 370, Nihon Shering, Osaka, Japan) was injected into the peritumor (the parenchyma of the breast), and the subareolar skin (2 ml each) using 26-gauge, 5/8-inch hypodermic needle attached to 2.5-ml syringe. Iopamidol is a commercially available contrast agent with a molecular weight of 777.09 Dalton, iodine concentration of 370 mg/ ml, osmolality of 780 mOsm/kg. Injected parts were gently massaged for about 60 s to facilitate the migration of the contrast agent into the draining lymphatics. CT was performed (Once Contiguous 1-mm thick and once Enhanced). Contiguous 2-mm-thick CT images from the upper thorax to axillary regions were obtained once prior to administration of the contrast agent and successively at 3 min after massage of the injection sites. CT scanning with a detector of 0.5 mm 9 2 row was operated at 120 kV, 330 mA, a 45-cm field of view, 512 9 512 matrix, section spacing of 5 mm, and table speed of 1.53 mm/0.5 s. The number of sections (36–40) was adapted to each individual to ensure coverage of the breast and axillary regions, and acquisition time ranged from 22 to 25 s. During CT image acquisition, each patient was placed in as nearly the same position as possible, and breath-hold was performed at a tidal inspiration level. The transaxial CT images were reconstructed with a 0.5-mm pitch and slice thickness of 0.3 mm. 3D CT images were then reconstructed from the post-contrast CT images at each time point with maximum intensity projection or surface-rendering techniques [7–9]. CTLG imaging patterns SLN detection with CTLG CTLG allows easy visualization of direct connection between primary SLN and its afferent lymphatic channels, and by providing detailed anatomy [7] (Fig. 1). Metastatic SLN Tumor occupation by the tumor resulted in dilation (Fig. 2a), stagnated non-staining of lymph vessels (Fig. 2a), and shadow defect of lymph nodes or partially stained LN as shown in Fig. 2a, b. Incomplete staining of SLN (mottled pattern: left), unstained (right upper) and partially stained lymph nodes in Fig. 2c. Lymph vessel was stagnant and detoured to another route as shown in Fig. 2d. The lymphatic route was rerouted by tumor occupied SLN
SSD image
MIP image
Not stained SLN Non SLN (Level II)
Non SLN (Level II)
SLN
SLN
Areola
Areola Tumor
Tumor
Fig. 1 SLN detection with CTLG allows easy visualization of direct connection between primary SLN and its afferent lymphatic channels, and by providing detailed anatomy [7] (Fig. 1)
and neighboring LN was often misdiagnosed as SLN (Fig. 3). SLNB and backup dissection Either breast conserving surgery (BCS) or mastectomy was performed. During the operation, 1–2 ml of dye (50 % mixture of indigo carmine and ICG) (Daiichi, Osaka, Japan) was injected into the periareolar skin, followed by massage for 30 s. Using the fluorescence imaging, subcutaneous lymphatic streams could be detected over the skin usually within 1 or 2 min (Fig. 4a). In BCS, a 3-cm incision was made on the lateral aspect of the mammary gland within the area covered by a bra, and green lymphatic channels were carefully dissected and traced until the first drained lymph node was then dissected. When lymphatic channels or lymph nodes were not clearly visible as green, fluorescence imaging was used for guidance. The lymphatic vessels and nodes receiving indigo carmine and ICG appeared as shining streams and spots in fluorescence image (Fig. 4b). In mastectomy, SLNB was performed through the skin incision made on the anterior chest wall for the mastectomy. SLNs are green lymph nodes and shining lymph nodes in fluorescence image. Not green but shining lymph nodes were biopsied as SLN referring CTLG. OSNA assay A fresh lymph node with a short axis of 4–12 mm was divided into four blocks (a, b, c, d) at 1 or 2 mm intervals using our original cutting device. Blocks a and c were used for the OSNA assay. Blocks b and d were used for histopathologic examination with H&E (Fig. 5). The OSNA assay for lymph nodes was described in detail in a previous
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a
b Non stained SLN Non stained LNs Dilated lymph vessel
Stagnated lymph vessel
Areola Tumor Tumor Areola
c
d SLN
LN metastasis SLN
LN metastasis
Rerouting lymph vessel
Tumor
LN metastasis
Areola Areola
Tumor
Fig. 2 Diagnostic criteria of SLN on CTLG. Tumor occupation by the tumor resulted in dilation (a), stagnant of lymph vessels (a), and shadow defect or partially stained LN as shown in a, b. In c incomplete staining of SLN (mottled pattern: left), unstained (right upper) and partially stained lymph nodes (right lower). d Lymph
vessel was stagnant and detoured to another route; the lymph node adjacent to true SLN were stained like SLN as shown in d, because the lymph vessels was occupied by tumor and rerouted to another route
report [17]. Briefly, after removing the extranodal tissue and lipid, a whole lymph node was homogenized with 4 ml of a lysis buffer solution (Lynorhag; Sysmex) and centrifuged at 100009g at room temperature. A 20-ll sample of the supernatant was analyzed with the RD-100i system (Sysmex), an automated gene amplification detection system using a reverse transcription loop-mediated isothermal amplification method and with the Lynoamp BC (Sysmex). The resulting change in turbidity was in turn correlated to CK19 mRNA copy number/ll of the original lysate via a standard curve that was established beforehand with 3 calibrators containing different CK19 mRNA concentrations. A standard positive control sample containing 5000 copies/ll of CK19mRNA and a negative control sample not containing any CK19 mRNA were used for validation in every assay. Lymph nodes that exceeded the specified maximum weight of 600 mg were cut into 2 or
more pieces and processed as separate nodes. Four lymph nodes can be analyzed in one run. The number of CK19 mRNA copies per microliter was calculated, and based on this number, the result (positive/negative) was assessed in accordance with the cut-off level determined by Tsujimoto et al. [17]. The OSNA assay can classify a positive lymph node into 3 categories: (??), (?), and (?I) (positive with reaction inhibited). In the present study, OSNA (??) considered to be equivalent to AJCC macrometastases, and OSNA (?) to AJCC micrometastases, criteria are shown in Table 2 as determined by Tsujimoto et al. [17]. Turnaround time was defined as the period from arrival of sentinel lymph nodes in our pathology department to getting the full results. The following situations were excluded from calculation of turnaround time: (1) simultaneous submission with another patient’s lymph nodes and (2) submission of lymph nodes twice.
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Histopathology of SLN Blocks for histopathology were stored in 10 % buffered formalin, processed overnight, and sectioned in serial 2.0mm-thick slices. All slices were embedded in paraffin and examined by H&E staining. In the histopathologic examination, macrometastases and micrometastases were defined according to the TNM classification of the Unio Internationale Contra Cancrum sixth and American Joint Committee on Cancer sixth editions. Conflicting results were settled consensually. The performance of the OSNA assay was compared with the histopathology. The OSNA assay analyzed different blocks from those used in the histopathologic examination. Therefore, in this protocol, the sensitivity and specificity of the OSNA assay could not be
Metastatic LN
calculated based on the histopathologic results. For this reason, we evaluated the performance of the OSNA assay as a concordance rate with the histopathologic examination. All procedures were approved by the Institutional Review Board of the Tokushima University School of Medicine. Informed consent was obtained from all patients before the procedures. Statistical analysis Quantitative values were presented as a mean and standard deviation. To evaluate diagnostic value of CTLG and OSNA, we computed positive predictive value (PPV), negative predictive value (NPV) according to concordance rate with histopathology as the reference method. We used Fisher’s exact test and Chi-square test to evaluate the relationship between SLN identification and clinicopathological features, P value of \0.05 was considered to indicate statistical significance.
Results Detection of SLN in CTLG
Stagnated lymph vessel
Often misdiagnosed as SLN Tumor Areola
Fig. 3 The lymphatic route was rerouted by tumor occupied SLN, and neighboring LN was often misdiagnosed as SLN
SLN was identified by preoperative CTLG in all patients, the identification rate was 100 %. Lymph vessels were detected in all patients. Oval-shaped lymph nodes with diameters greater than those of lymph vessels were easily identified (Fig. 1). We referred to the direction of lymphatic vessels draining into SLN, the depth of SLN from the skin surface, SLN size and the detailed anatomical relation between the visualized lymphatic routs and chest wall muscles, ribs and primary tumors using the highly spatial 3D CT. Number of cases was 50. Number of SLN detected was 67 (1.3 ± 0.46 in each case). Forty-one SLN showed negative metastatic criteria. Nine of 50 cases (18 %)
Fig. 4 SLN detection with ICG. a Subcutaneous lymphatic channels were detected by fluorescence, two streams from the areola joined together before they drained into the axilla, the point to be incised was marked. b SLN which received fluorescent dye appeared as shining spots in the fluorescent image
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Table 3 Characteristics of OSNA positive patients
Copy number of CK19 mRNA (N)
OSNA assessment
Size of metastasis (M)
Patient criteria
N \ 250
-
None, M \ 0.2 mm
Patient age
250 B N \ 5000
?
0.2 B M \ 2 mm
5000 B N
??
2 mm B M
a b c d Fig. 5 How to examine the SLN metastasis. A fresh lymph node with a short axis of 4–12 mm was divided into four blocks (a, b, c, d) at 1or 2-mm intervals using our original cutting device. Blocks a and c were used for the OSNA assay. Blocks b and d were used for histopathologic examination with H&E
suspected to have metastatic nodes preoperatively with CTLG. Only one case had micrometastasis with pathology in SLN non-metastasis was diagnosed with CTLG. The NPV of CTLG was 9/10 (90 %).
Number of OSNA positive patients (%)
P value
\60 years
5/22 (22)
0.4
[60 years
4/28 (14)
Tumor size T1
5/27 (18)
T2
4/12 (33)
Clinical stage I II
5/27 (18)
0.4
0.4
4/12 (33)
Nuclear grade 1
3/30 (10)
2
3/15 (20)
0.3
3
3/5 (60)
0.03
IDCA
8/29 (28)
0.5
ILCA
1/2 (50)
Histological type
clinical stage were also associated with positive OSNA (although insignificant) as shown in Table 3. All OSNA assays were completed during surgery within 39 min for each case.
Adverse events Detection of metastases by H&E histopathology There were no adverse events, such as anaphylaxis or skin reactions, because of the contrast medium or dye in this study. Detection of SLN in SLNB SLN was identified by intraoperative SLNB in all patients, the identification rate was 100 %. Even with small skin incision the detection of SLN was easy. The number of SLN detected was 100 (2 ± 1.03 in each case). Almost all SLNs were located in the second rib to third intercostal space, 2- to 4-cm beneath the deep thoracic fascia. Fluorescent nodes were sampled referring CTLG images, therefore the number of biopsied nodes was larger compared with diagnosed SLNs by CTLG. However in 22/50 cases, the number of SLN detected in both methods was identical.
SLN metastases were detected as positive in 10 cases (20 %). One SLN involved isolated tumor cells (ITC) and this node was considered as negative. There were 10 cases with metastases. Six cases have micrometastases and 4 cases have macrometastases in SLN. NonSLN metastases were detected in 2 cases after backup dissection. Metastasis of non-SLN was recognized in OSNA (??) cases more than that in OSNA (?) cases (2/4 vs. 0/4). OSNA (??) was recognized in two cases. False-negative case of CTLG False negative with CTLG was only one case, which showed normal CTLG pattern. There was a micrometastasis in permanent pathology (Fig. 6).
Detection of metastases by OSNA
False-negative cases of OSNA
SLN metastases were detected as positive in 9 patients (18 %), 4 were (??), 4 were (?) and 1 was (?I). There were no false-positive cases and only one false-negative case. Positive OSNA was significantly associated with higher nuclear grade. Tumor size [20 mm and higher
Only one case was false negative in OSNA, but positive in permanent section. This case was DCIS and showed micrometastases in permanent section. This case did not need additional dissection because level I dissection was already done in same setting.
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Sampled SLNs
This node had micro metastasis Tumor Areola Fig. 6 False-negative CTLG case. False negative with CTLG was only one case, which showed normal CTLG pattern. There was a micrometastasis in permanent pathology
Concordance rate between OSNA and H&E histopathology The concordance rate between OSNA and histopathology was 9/10 (90 %). The PPV of OSNA was 100 % and NPV of OSNA was 97.5 %. Recurrence of axillary lymph nodes No recurrence has been detected in any case for 14 months (8–20 months) of median follow-up time.
Discussion In early breast cancer, the presence of metastasis in axillary lymph nodes is an important factor in prognosis and further treatment. SLNB can detect such metastases and provide information that may obviate the need for ALND, because the sentinel SLN is the first lymph node drained of lymph from the tumor [1, 2]. False negative of SLNB is a critical problem, as it may lead to inadequate adjuvant therapy, which can negatively affect a patient’s prognosis [3]. The optimal method of SLN identification remains controversial. Here we presented, in current study, our institution’s protocol of preoperative and intraoperative SLN detection. Our study gave additional proof that the combination of CTLG and fluorescence navigation made performing SLNB with high accuracy possible in institutions that
cannot use the RI method. CTLG and fluorescence navigation potentially enhance the surgeon’s ability to perform SLNB and backup dissection, by localizing the targeted lesion and the critical lesion that should be avoided, the surgical navigation helps to achieve effective and safe surgery while minimizing the invasiveness of the surgery. Our study showed that SLNs were easily identified even with small skin incision. The procedure requires only about 1 min after injection of contrast medium into the peritumor parenchyma and around the nipple to represent SLN precisely. Because CTLG has detailed spatial resolution activity, we believe that CTLG allows easy visualization of direct connection between primary SLN and its afferent lymphatic channels, and by providing detailed anatomy of the breast lymphatics. Fluorescence imaging provided us with real-time observation of the lymphatic streams on the skin, and superior recognition of lymphatic vessels and LNs in comparison with dye staining alone. The identification rate was improved to 100 % by using CTLG and fluorescence navigation. The number of detected SLN was larger in combined CTLG and fluorescence navigation (2 ± 1.03 in each case) than in CTLG alone (1.3 ± 0.46 in each case). The non-ionic contrast media used in CTLG have a larger particle size than dyes [21]. But both iopamidol and ICG figured out not only SLN, but second and third nodes. SLNs were diagnosed from the connection of SLNs and afferent lymph routes from the tumor on CTLG. SLNB was performed using ICG fluorescent methods referring CTLG image, therefore fluorescent lymph nodes beside the true SLN were also sampled. Many studies reported that the identification rate improved more by combining CTLG with ICG fluorescence navigation [1, 22]. For identification of SLN by CTLG, the patient age, tumor location, surgical biopsy, the tumor size and the presence or absence of lymph node metastasis have been reported to influence identification of the SLN [7, 23, 24]. In our patients’ series, there was no significant related factor. No false-negative case was noted with SLNB using fluorescent method navigated CTLG. Our study revealed that the use of OSNA increases the accuracy of intraoperative detection of SLN metastases to 90 % with PPV of 100 % and NPV of 97.5 % that was much higher than conventional frozen section. Positive OSNA was significantly associated with higher nuclear grade. Tumor size [20 mm and higher clinical stage were also associated with positive OSNA but were not significant. SLNs were cut into four equal slices and alternate slices were prepared for OSNA and histology, which was considered as the standard method. As different parts of the node were used for each examination, because each technique required different tissue preparation, it was reported that discrepancies between OSNA and histological results were expected and tolerated [25]. In this study, the
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concordance rate between OSNA and permanent histological sections was 90 %, the false-negative case had a micrometastasis in permanent section. This might be due to different part was examined. OSNA test using whole LN might be obtain better results [26]. All OSNA assays were carried out during surgery and were completed within 39 min for each case, which is equal to time needed by frozen section, but with higher accuracy. In our study, OSNA allowed precise differentiation of micro- from macrometastases, and the CK19 copy number predicts the probability of tumor load in other axillary lymph nodes and might help to find adequate adjuvant treatment options. This objective method may reduce the pathologic workload and the risk of secondary operative interventions with all associated costs and stress for the patients. The use of the OSNA assay can be expected to decrease the number of women who require a second surgical procedure for ALND, because its sensitivity is higher than intraoperative frozen section evaluation. All comparative studies to date are supportive of OSNA as a method of metastasis detection in SLN of breast cancer patients [27]. SLN is the first lymph node in which tumor cells reach and make lymph node metastasis theoretically. The theory is true only in node-negative cases. But preoperative diagnosis of N0 is very difficult even with CT scan or/and PETCT [28]. Ten cases (20 %) were examined using OSNA. Of the 10 metastatic SLNs, 9 were suspected to have metastasis by CTLG preoperatively. Only one case missed its micrometastasis by CTLG. Percentage of pathologically positive SLNs among evaluated T1N0 cases for SLNB was lower compared with other reports [26], because metastatic LN was ruled out using a criteria for metastasis on CTLG; shadow defect of SLN by occupying tumor and/or obstruction, dilation and rerouting of lymphatic vessels [7, 9]. In our patients’ series, no recurrence has been detected in any case for 14 months of median follow-up time. Proudly, we can add this result to the accuracy scale of our protocol in detection of SLN metastases. Finally, our protocol seemed to be accurate, reliable and practical, and serve to increase patients’ quality of life by avoidance of unnecessary ALND, or second time surgery. This scenario has an influence on both the psychological and physical condition of the patient as a second operation is associated with extra-emotional stress. In addition to clinical and procedural benefits that our protocol offered to breast cancer patients, there are marked financial advantages from avoiding second time surgery and extensive postoperative analysis, which is more demanding on an operative level for the surgeon and might delay a necessary adjuvant treatment. Besides this, extra bed and hospital time is a disadvantage from an economic point of view.
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In conclusion, using CTLG and fluorescence navigation made performing SLNB with high accuracy possible in institutions that cannot use the radioisotope method. Application of CTLG and fluorescence navigation obtains safe and early identification of SLN. OSNA provided accurate and rapid intraoperative method, allowing for the completion of axillary node dissection during surgery and avoidance of second surgical procedure in patients with positive SLNs, thereby reducing patient distress, increase patient quality of life, and finally, saving hospital costs. Conflict of interest of interest.
The authors declare that they have no conflict
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ORIGINAL ARTICLE
Triple assessment of sentinel lymph node metastasis in early breast cancer using preoperative CTLG, intraoperative fluorescence navigation and OSNA Mohamed Mokhtar • Yukiko Tadokoro • Misako Nakagawa Masami Morimoto • Hirokazu Takechi • Kazuya Kondo • Akira Tangoku
•
Received: 26 December 2013 / Accepted: 30 June 2014 Ó The Japanese Breast Cancer Society 2014
Abstract Background Sentinel lymph node biopsy (SLNB) became a standard surgical procedure for patients with early breast cancer; however, the optimal method of sentinel lymph node (SLN) identification remains controversial. The current study presents the protocol of our institution for preoperative and intraoperative SLN detection. Methods Fifty female patients with early breast cancer and clinically node-negative axilla were enrolled in this study. All patients underwent preoperative CT lymphography (CTLG), intraoperative SLNB using fluorescence navigation, intraoperative one-step nucleic acid amplification (OSNA) and postoperative hematoxylin and eosin histopathological analysis. Prediction of metastasis by CTLG and detection of metastasis by OSNA were compared to results of histopathology as standard reference.
Results SLN were identified by preoperative CTLG and intraoperative SLNB with fluorescence navigation in all patients, the identification rate was 100 %. SLN metastases were detected as positive by OSNA in 9 patients (18 %), 4 were (??), 4 were (?) and 1 was (?I). SLN metastases were detected as positive by histopathology in 10 patients (20 %). The concordance rate between OSNA and permanent sections was 90 %. The negative predictive value of CTLG was 80 %. Conclusion Use of CTLG and fluorescence navigation made performing SLNB with high accuracy possible in institutions that cannot use the radioisotope method. OSNA provided accurate intraoperative method, allowing for completion of axillary node dissection during surgery and avoidance of second surgical procedure in patients with positive SLNs, thereby reducing patient distress and, finally, saving hospital costs. Keywords Sentinel lymph node biopsy Computed tomographic lymphography One-step nucleic acid amplification Breast cancer
M. Mokhtar K. Kondo Department of Oncological Medical Services, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8505, Japan M. Mokhtar Department of Oncological Surgery, Minia Oncology Institute, Minya 61111, Egypt Y. Tadokoro (&) M. Nakagawa M. Morimoto H. Takechi A. Tangoku Department of Thoracic, Endocrine Surgery and Oncology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8505, Japan e-mail: [email protected]
Abbreviations ALND Axillary lymph node dissection BCS Breast conserving surgery BMI Body mass index CTLG Computed tomography lymphography H&E Hematoxylin and eosin NPV Negative predictive value OSNA One-step nucleic acid amplification PPV Positive predictive value QRT-PCR Quantitative reverse transcription-PCR RI Radioisotope SLN Sentinel lymph node SLNB Sentinel lymph node biopsy
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Introduction
Patients and methods
SLNB became a standard surgical procedure for patients with early breast cancer. SLNB benefits patients without metastatic nodes by avoiding unnecessary axillary lymph node dissection (ALND) with its concomitant morbidities [1, 2]. False negative of SLNB is a critical problem, as it may lead to inadequate adjuvant therapy, which can negatively affect patient’s prognosis [3]. The optimal method of SLN identification remains controversial. The most commonly used methods to identify the SLN are dye staining and radioisotope (RI) incorporation. The combination of the RI and dye-staining methods is the most accurate way to identify SLN [4–6]. However, in Japan, most hospitals cannot use RI because of institutional limitations. The identification rate with the dye method alone is inferior to the RI combination method. The correct SLNs with afferent and efferent lymphatic routes surrounding detailed anatomy were imaged preoperatively, and detected by blue dye imaging based on CTLG navigation. With CTLG, preoperative diagnosis of SLN metastasis is feasible as mentioned before [7, 8]. SLN metastasis is generally detected by conventional means including the intraoperative H&E-based histopathologic examination of frozen section or cytological observation of touch imprints, followed by definitive postoperative histopathologic examination of permanent sections [10, 11]. However, the sensitivity of these intraoperative methods is not high; many investigators have reported that the intraoperative H&E-based histopathologic examination has a false-negative rate of 5–52 % [12]. Furthermore, these methods provide subjective rather than objective results, which may differ from one pathologist to another. On the other hand, the definitive postoperative histopathologic examination generally requires 5–10 days for assessment [13, 14]. To overcome the shortcomings of histopathological methods, molecular assays have been developed for detection of lymph node metastasis in breast cancer patients [15, 16]. OSNA that amplifies CK19 mRNA (OSNA, Sysmex, Kobe, Japan) was recently developed to accurately detect metastasis measuring 0.2 mm or more. OSNA method is characterized by the quantitative measurement of a target mRNA in a metastatic lymph node, a brief reaction time for the OSNA process, a high specificity for the target mRNA, and an absence of genomic DNA amplification [17, 18]. Our aim was to overcome the downsides of conventional methods for detection of SLN in early breast cancer through a new protocol including preoperative CTLG, intraoperative fluorescence navigation and OSNA in detection of SLN metastasis using final histopathology as a reference standard.
Patients
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From September 2012 to September 2013, 50 female patients with early breast cancer and clinically node-negative axilla were enrolled in this study. All patients underwent preoperative CTLG, intraoperative SLNB using fluorescence imaging with indocyanine green (ICG) navigated by CTLG as reported before [19, 20], intraoperative OSNA and postoperative H&E histopathological examination in Tokushima University Hospital. The results were analyzed in relation to the SLN identification and various clinicopathological parameters, including age, body mass index (BMI), tumor size, clinical stage, nuclear grade and histological type. Patients’ characteristics and tumor status are shown in Table 1. 3D CTLG CTLG was performed using multidetector row helical CT scanner (Siemens Volume Zoom; Siemens-Asahi Medical
Table 1 Patients’ characteristics of and tumor status
Age (mean ± SD)
60 ± 11.5
BMI (mean ± SD)
23 ± 3.4
Tumor size (%) Tis
11/50 (22)
T1
27/50 (54)
T2
12/50 (24)
Clinical stage (%) 0
11/50 (22)
I
27/50 (54)
II
12/50 (24)
Nuclear grade (%) Grade 1 30/50 (60) Grade 2
15/50 (30)
Grade 3
5/50 (10)
Histological type (%) DCIS
12/50 (24)
IDCA
29/50 (58)
ILCA
2/50 (4)
Others
7/50 (14)
Operation type (%) Total Partial DCIS ductal carcinoma in situ, IDCA invasive ductal carcinoma, ILCA invasive lobular carcinoma
19/50 (38) 31/50 (62)
SLN positive
10/50 (20 %)
SLN negative
40/50 (80 %)
Breast Cancer
Technologies Ltd., Tokyo, Japan). Each patient was placed in supine position with arms positioned in a cranial direction. After local anesthesia with subcutaneous injection of a total of 0.5 ml of 1 % lidocaine hydrochloride, a total 4-ml dose of non-ionic water-soluble iodinated contrast medium (Iopamidol; Iopamiron 370, Nihon Shering, Osaka, Japan) was injected into the peritumor (the parenchyma of the breast), and the subareolar skin (2 ml each) using 26-gauge, 5/8-inch hypodermic needle attached to 2.5-ml syringe. Iopamidol is a commercially available contrast agent with a molecular weight of 777.09 Dalton, iodine concentration of 370 mg/ ml, osmolality of 780 mOsm/kg. Injected parts were gently massaged for about 60 s to facilitate the migration of the contrast agent into the draining lymphatics. CT was performed (Once Contiguous 1-mm thick and once Enhanced). Contiguous 2-mm-thick CT images from the upper thorax to axillary regions were obtained once prior to administration of the contrast agent and successively at 3 min after massage of the injection sites. CT scanning with a detector of 0.5 mm 9 2 row was operated at 120 kV, 330 mA, a 45-cm field of view, 512 9 512 matrix, section spacing of 5 mm, and table speed of 1.53 mm/0.5 s. The number of sections (36–40) was adapted to each individual to ensure coverage of the breast and axillary regions, and acquisition time ranged from 22 to 25 s. During CT image acquisition, each patient was placed in as nearly the same position as possible, and breath-hold was performed at a tidal inspiration level. The transaxial CT images were reconstructed with a 0.5-mm pitch and slice thickness of 0.3 mm. 3D CT images were then reconstructed from the post-contrast CT images at each time point with maximum intensity projection or surface-rendering techniques [7–9]. CTLG imaging patterns SLN detection with CTLG CTLG allows easy visualization of direct connection between primary SLN and its afferent lymphatic channels, and by providing detailed anatomy [7] (Fig. 1). Metastatic SLN Tumor occupation by the tumor resulted in dilation (Fig. 2a), stagnated non-staining of lymph vessels (Fig. 2a), and shadow defect of lymph nodes or partially stained LN as shown in Fig. 2a, b. Incomplete staining of SLN (mottled pattern: left), unstained (right upper) and partially stained lymph nodes in Fig. 2c. Lymph vessel was stagnant and detoured to another route as shown in Fig. 2d. The lymphatic route was rerouted by tumor occupied SLN
SSD image
MIP image
Not stained SLN Non SLN (Level II)
Non SLN (Level II)
SLN
SLN
Areola
Areola Tumor
Tumor
Fig. 1 SLN detection with CTLG allows easy visualization of direct connection between primary SLN and its afferent lymphatic channels, and by providing detailed anatomy [7] (Fig. 1)
and neighboring LN was often misdiagnosed as SLN (Fig. 3). SLNB and backup dissection Either breast conserving surgery (BCS) or mastectomy was performed. During the operation, 1–2 ml of dye (50 % mixture of indigo carmine and ICG) (Daiichi, Osaka, Japan) was injected into the periareolar skin, followed by massage for 30 s. Using the fluorescence imaging, subcutaneous lymphatic streams could be detected over the skin usually within 1 or 2 min (Fig. 4a). In BCS, a 3-cm incision was made on the lateral aspect of the mammary gland within the area covered by a bra, and green lymphatic channels were carefully dissected and traced until the first drained lymph node was then dissected. When lymphatic channels or lymph nodes were not clearly visible as green, fluorescence imaging was used for guidance. The lymphatic vessels and nodes receiving indigo carmine and ICG appeared as shining streams and spots in fluorescence image (Fig. 4b). In mastectomy, SLNB was performed through the skin incision made on the anterior chest wall for the mastectomy. SLNs are green lymph nodes and shining lymph nodes in fluorescence image. Not green but shining lymph nodes were biopsied as SLN referring CTLG. OSNA assay A fresh lymph node with a short axis of 4–12 mm was divided into four blocks (a, b, c, d) at 1 or 2 mm intervals using our original cutting device. Blocks a and c were used for the OSNA assay. Blocks b and d were used for histopathologic examination with H&E (Fig. 5). The OSNA assay for lymph nodes was described in detail in a previous
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a
b Non stained SLN Non stained LNs Dilated lymph vessel
Stagnated lymph vessel
Areola Tumor Tumor Areola
c
d SLN
LN metastasis SLN
LN metastasis
Rerouting lymph vessel
Tumor
LN metastasis
Areola Areola
Tumor
Fig. 2 Diagnostic criteria of SLN on CTLG. Tumor occupation by the tumor resulted in dilation (a), stagnant of lymph vessels (a), and shadow defect or partially stained LN as shown in a, b. In c incomplete staining of SLN (mottled pattern: left), unstained (right upper) and partially stained lymph nodes (right lower). d Lymph
vessel was stagnant and detoured to another route; the lymph node adjacent to true SLN were stained like SLN as shown in d, because the lymph vessels was occupied by tumor and rerouted to another route
report [17]. Briefly, after removing the extranodal tissue and lipid, a whole lymph node was homogenized with 4 ml of a lysis buffer solution (Lynorhag; Sysmex) and centrifuged at 100009g at room temperature. A 20-ll sample of the supernatant was analyzed with the RD-100i system (Sysmex), an automated gene amplification detection system using a reverse transcription loop-mediated isothermal amplification method and with the Lynoamp BC (Sysmex). The resulting change in turbidity was in turn correlated to CK19 mRNA copy number/ll of the original lysate via a standard curve that was established beforehand with 3 calibrators containing different CK19 mRNA concentrations. A standard positive control sample containing 5000 copies/ll of CK19mRNA and a negative control sample not containing any CK19 mRNA were used for validation in every assay. Lymph nodes that exceeded the specified maximum weight of 600 mg were cut into 2 or
more pieces and processed as separate nodes. Four lymph nodes can be analyzed in one run. The number of CK19 mRNA copies per microliter was calculated, and based on this number, the result (positive/negative) was assessed in accordance with the cut-off level determined by Tsujimoto et al. [17]. The OSNA assay can classify a positive lymph node into 3 categories: (??), (?), and (?I) (positive with reaction inhibited). In the present study, OSNA (??) considered to be equivalent to AJCC macrometastases, and OSNA (?) to AJCC micrometastases, criteria are shown in Table 2 as determined by Tsujimoto et al. [17]. Turnaround time was defined as the period from arrival of sentinel lymph nodes in our pathology department to getting the full results. The following situations were excluded from calculation of turnaround time: (1) simultaneous submission with another patient’s lymph nodes and (2) submission of lymph nodes twice.
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Histopathology of SLN Blocks for histopathology were stored in 10 % buffered formalin, processed overnight, and sectioned in serial 2.0mm-thick slices. All slices were embedded in paraffin and examined by H&E staining. In the histopathologic examination, macrometastases and micrometastases were defined according to the TNM classification of the Unio Internationale Contra Cancrum sixth and American Joint Committee on Cancer sixth editions. Conflicting results were settled consensually. The performance of the OSNA assay was compared with the histopathology. The OSNA assay analyzed different blocks from those used in the histopathologic examination. Therefore, in this protocol, the sensitivity and specificity of the OSNA assay could not be
Metastatic LN
calculated based on the histopathologic results. For this reason, we evaluated the performance of the OSNA assay as a concordance rate with the histopathologic examination. All procedures were approved by the Institutional Review Board of the Tokushima University School of Medicine. Informed consent was obtained from all patients before the procedures. Statistical analysis Quantitative values were presented as a mean and standard deviation. To evaluate diagnostic value of CTLG and OSNA, we computed positive predictive value (PPV), negative predictive value (NPV) according to concordance rate with histopathology as the reference method. We used Fisher’s exact test and Chi-square test to evaluate the relationship between SLN identification and clinicopathological features, P value of \0.05 was considered to indicate statistical significance.
Results Detection of SLN in CTLG
Stagnated lymph vessel
Often misdiagnosed as SLN Tumor Areola
Fig. 3 The lymphatic route was rerouted by tumor occupied SLN, and neighboring LN was often misdiagnosed as SLN
SLN was identified by preoperative CTLG in all patients, the identification rate was 100 %. Lymph vessels were detected in all patients. Oval-shaped lymph nodes with diameters greater than those of lymph vessels were easily identified (Fig. 1). We referred to the direction of lymphatic vessels draining into SLN, the depth of SLN from the skin surface, SLN size and the detailed anatomical relation between the visualized lymphatic routs and chest wall muscles, ribs and primary tumors using the highly spatial 3D CT. Number of cases was 50. Number of SLN detected was 67 (1.3 ± 0.46 in each case). Forty-one SLN showed negative metastatic criteria. Nine of 50 cases (18 %)
Fig. 4 SLN detection with ICG. a Subcutaneous lymphatic channels were detected by fluorescence, two streams from the areola joined together before they drained into the axilla, the point to be incised was marked. b SLN which received fluorescent dye appeared as shining spots in the fluorescent image
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Table 3 Characteristics of OSNA positive patients
Copy number of CK19 mRNA (N)
OSNA assessment
Size of metastasis (M)
Patient criteria
N \ 250
-
None, M \ 0.2 mm
Patient age
250 B N \ 5000
?
0.2 B M \ 2 mm
5000 B N
??
2 mm B M
a b c d Fig. 5 How to examine the SLN metastasis. A fresh lymph node with a short axis of 4–12 mm was divided into four blocks (a, b, c, d) at 1or 2-mm intervals using our original cutting device. Blocks a and c were used for the OSNA assay. Blocks b and d were used for histopathologic examination with H&E
suspected to have metastatic nodes preoperatively with CTLG. Only one case had micrometastasis with pathology in SLN non-metastasis was diagnosed with CTLG. The NPV of CTLG was 9/10 (90 %).
Number of OSNA positive patients (%)
P value
\60 years
5/22 (22)
0.4
[60 years
4/28 (14)
Tumor size T1
5/27 (18)
T2
4/12 (33)
Clinical stage I II
5/27 (18)
0.4
0.4
4/12 (33)
Nuclear grade 1
3/30 (10)
2
3/15 (20)
0.3
3
3/5 (60)
0.03
IDCA
8/29 (28)
0.5
ILCA
1/2 (50)
Histological type
clinical stage were also associated with positive OSNA (although insignificant) as shown in Table 3. All OSNA assays were completed during surgery within 39 min for each case.
Adverse events Detection of metastases by H&E histopathology There were no adverse events, such as anaphylaxis or skin reactions, because of the contrast medium or dye in this study. Detection of SLN in SLNB SLN was identified by intraoperative SLNB in all patients, the identification rate was 100 %. Even with small skin incision the detection of SLN was easy. The number of SLN detected was 100 (2 ± 1.03 in each case). Almost all SLNs were located in the second rib to third intercostal space, 2- to 4-cm beneath the deep thoracic fascia. Fluorescent nodes were sampled referring CTLG images, therefore the number of biopsied nodes was larger compared with diagnosed SLNs by CTLG. However in 22/50 cases, the number of SLN detected in both methods was identical.
SLN metastases were detected as positive in 10 cases (20 %). One SLN involved isolated tumor cells (ITC) and this node was considered as negative. There were 10 cases with metastases. Six cases have micrometastases and 4 cases have macrometastases in SLN. NonSLN metastases were detected in 2 cases after backup dissection. Metastasis of non-SLN was recognized in OSNA (??) cases more than that in OSNA (?) cases (2/4 vs. 0/4). OSNA (??) was recognized in two cases. False-negative case of CTLG False negative with CTLG was only one case, which showed normal CTLG pattern. There was a micrometastasis in permanent pathology (Fig. 6).
Detection of metastases by OSNA
False-negative cases of OSNA
SLN metastases were detected as positive in 9 patients (18 %), 4 were (??), 4 were (?) and 1 was (?I). There were no false-positive cases and only one false-negative case. Positive OSNA was significantly associated with higher nuclear grade. Tumor size [20 mm and higher
Only one case was false negative in OSNA, but positive in permanent section. This case was DCIS and showed micrometastases in permanent section. This case did not need additional dissection because level I dissection was already done in same setting.
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Sampled SLNs
This node had micro metastasis Tumor Areola Fig. 6 False-negative CTLG case. False negative with CTLG was only one case, which showed normal CTLG pattern. There was a micrometastasis in permanent pathology
Concordance rate between OSNA and H&E histopathology The concordance rate between OSNA and histopathology was 9/10 (90 %). The PPV of OSNA was 100 % and NPV of OSNA was 97.5 %. Recurrence of axillary lymph nodes No recurrence has been detected in any case for 14 months (8–20 months) of median follow-up time.
Discussion In early breast cancer, the presence of metastasis in axillary lymph nodes is an important factor in prognosis and further treatment. SLNB can detect such metastases and provide information that may obviate the need for ALND, because the sentinel SLN is the first lymph node drained of lymph from the tumor [1, 2]. False negative of SLNB is a critical problem, as it may lead to inadequate adjuvant therapy, which can negatively affect a patient’s prognosis [3]. The optimal method of SLN identification remains controversial. Here we presented, in current study, our institution’s protocol of preoperative and intraoperative SLN detection. Our study gave additional proof that the combination of CTLG and fluorescence navigation made performing SLNB with high accuracy possible in institutions that
cannot use the RI method. CTLG and fluorescence navigation potentially enhance the surgeon’s ability to perform SLNB and backup dissection, by localizing the targeted lesion and the critical lesion that should be avoided, the surgical navigation helps to achieve effective and safe surgery while minimizing the invasiveness of the surgery. Our study showed that SLNs were easily identified even with small skin incision. The procedure requires only about 1 min after injection of contrast medium into the peritumor parenchyma and around the nipple to represent SLN precisely. Because CTLG has detailed spatial resolution activity, we believe that CTLG allows easy visualization of direct connection between primary SLN and its afferent lymphatic channels, and by providing detailed anatomy of the breast lymphatics. Fluorescence imaging provided us with real-time observation of the lymphatic streams on the skin, and superior recognition of lymphatic vessels and LNs in comparison with dye staining alone. The identification rate was improved to 100 % by using CTLG and fluorescence navigation. The number of detected SLN was larger in combined CTLG and fluorescence navigation (2 ± 1.03 in each case) than in CTLG alone (1.3 ± 0.46 in each case). The non-ionic contrast media used in CTLG have a larger particle size than dyes [21]. But both iopamidol and ICG figured out not only SLN, but second and third nodes. SLNs were diagnosed from the connection of SLNs and afferent lymph routes from the tumor on CTLG. SLNB was performed using ICG fluorescent methods referring CTLG image, therefore fluorescent lymph nodes beside the true SLN were also sampled. Many studies reported that the identification rate improved more by combining CTLG with ICG fluorescence navigation [1, 22]. For identification of SLN by CTLG, the patient age, tumor location, surgical biopsy, the tumor size and the presence or absence of lymph node metastasis have been reported to influence identification of the SLN [7, 23, 24]. In our patients’ series, there was no significant related factor. No false-negative case was noted with SLNB using fluorescent method navigated CTLG. Our study revealed that the use of OSNA increases the accuracy of intraoperative detection of SLN metastases to 90 % with PPV of 100 % and NPV of 97.5 % that was much higher than conventional frozen section. Positive OSNA was significantly associated with higher nuclear grade. Tumor size [20 mm and higher clinical stage were also associated with positive OSNA but were not significant. SLNs were cut into four equal slices and alternate slices were prepared for OSNA and histology, which was considered as the standard method. As different parts of the node were used for each examination, because each technique required different tissue preparation, it was reported that discrepancies between OSNA and histological results were expected and tolerated [25]. In this study, the
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concordance rate between OSNA and permanent histological sections was 90 %, the false-negative case had a micrometastasis in permanent section. This might be due to different part was examined. OSNA test using whole LN might be obtain better results [26]. All OSNA assays were carried out during surgery and were completed within 39 min for each case, which is equal to time needed by frozen section, but with higher accuracy. In our study, OSNA allowed precise differentiation of micro- from macrometastases, and the CK19 copy number predicts the probability of tumor load in other axillary lymph nodes and might help to find adequate adjuvant treatment options. This objective method may reduce the pathologic workload and the risk of secondary operative interventions with all associated costs and stress for the patients. The use of the OSNA assay can be expected to decrease the number of women who require a second surgical procedure for ALND, because its sensitivity is higher than intraoperative frozen section evaluation. All comparative studies to date are supportive of OSNA as a method of metastasis detection in SLN of breast cancer patients [27]. SLN is the first lymph node in which tumor cells reach and make lymph node metastasis theoretically. The theory is true only in node-negative cases. But preoperative diagnosis of N0 is very difficult even with CT scan or/and PETCT [28]. Ten cases (20 %) were examined using OSNA. Of the 10 metastatic SLNs, 9 were suspected to have metastasis by CTLG preoperatively. Only one case missed its micrometastasis by CTLG. Percentage of pathologically positive SLNs among evaluated T1N0 cases for SLNB was lower compared with other reports [26], because metastatic LN was ruled out using a criteria for metastasis on CTLG; shadow defect of SLN by occupying tumor and/or obstruction, dilation and rerouting of lymphatic vessels [7, 9]. In our patients’ series, no recurrence has been detected in any case for 14 months of median follow-up time. Proudly, we can add this result to the accuracy scale of our protocol in detection of SLN metastases. Finally, our protocol seemed to be accurate, reliable and practical, and serve to increase patients’ quality of life by avoidance of unnecessary ALND, or second time surgery. This scenario has an influence on both the psychological and physical condition of the patient as a second operation is associated with extra-emotional stress. In addition to clinical and procedural benefits that our protocol offered to breast cancer patients, there are marked financial advantages from avoiding second time surgery and extensive postoperative analysis, which is more demanding on an operative level for the surgeon and might delay a necessary adjuvant treatment. Besides this, extra bed and hospital time is a disadvantage from an economic point of view.
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In conclusion, using CTLG and fluorescence navigation made performing SLNB with high accuracy possible in institutions that cannot use the radioisotope method. Application of CTLG and fluorescence navigation obtains safe and early identification of SLN. OSNA provided accurate and rapid intraoperative method, allowing for the completion of axillary node dissection during surgery and avoidance of second surgical procedure in patients with positive SLNs, thereby reducing patient distress, increase patient quality of life, and finally, saving hospital costs. Conflict of interest of interest.
The authors declare that they have no conflict
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