DOI: https://doi.org/10.21203/rs.3.rs-2452342/v1
the purpose of this study is proving the contrast-enhanced ultrasound (CEUS) identified sentinel lymph node (SLN) could accurately represent the axillary nodes status in early-stage breast cancer.
A total of 109 consecutive consenting patients with clinical node negative and T1-2 breast cancer were included in this study. All patients received CEUS to identify SLN before surgery and deploying a guidewire to locate the SLN in those who were CEUS explored successfully. All patients underwent sentinel lymph node biopsy (SLNB), and blue dye was also used to help in tracing sentinel lymph node during the surgery. Whether to perform axillary lymph node dissection (ALND) depends on the intraoperative pathological result of the SLN identified by CEUS (CE-SLN). Comparing the pathological results of the CE-SLN with the remaining dyed SLN and axillary dissection nodes.
4 patients was failed to identify the CE-SLN, the detection rate of the CEUS is 96.3%. Among the 105 successful patients, 18 were CE-SLN positive by intraoperative frozen section, another one with CE-SLN micrometastasis was diagnosed by paraffin section. No additional lymph node metastases were found for all CE-SLN negative patients, the false negative rate of CEUS-guided SLNB is 0%.
For patients with clinical node negative and small tumor burden breast cancer, SLN located by CEUS can accurately represent the status of axillary lymph nodes.
Sentinel lymph node biopsy (SLNB) is recognized as the preferred operation for axillary node staging in clinical node-negative breast cancer patients. Common sentinel lymph node tracers are ⁹⁹mTechnetium-sulfur colloid and blue dye. Prior studies had shown that the dual-tracer method which combined isotope and dye was the best approach with the highest success rate, [1, 2] and a meta-analysis showed that the combination method increased the success rate of sentinel lymph node biopsy to 96–99%.[3] However, due to the high cost and poor popularity of isotope, most institutions just use single dye. But the success rate of the single dye is relatively low. False negative rate (FNR) is an important index to evaluate the accuracy of SLNB. B32 study showed that in order to reduce the false negative rate, two or more SLNs should be obtained in the operation, and the more SLNs obtained, the lower the false negative rate.[1] ALMANAC study also reached a similar conclusion.[2] This makes surgeons doubt the accuracy of a situation that only one SLN is marked and obtained in clinical practice. Most of those patients need to undergo more extensive lymph node excision or axillary lymph node dissection to obtain perhaps more accurate axillary staging, but it can also lead to excessive surgery. In addition, sometimes the tracer may recognize a lot of SLNs. For instance, the maximum number of SLNs removed reported in the ALMANAC study was 9.[4] The reason for that may be caused by the following factor, due to the access of tracer to secondary lymphatic vessels, which leads to excessive axillary exploration and over resection of lymph nodes, that may indeed reduce the risk of false negative somewhat, but increasing the risk of postoperative complications, which is also contrary to the primary purpose of the SLNB.
Contrast enhanced ultrasound (CEUS) is a novel lymph node tracing method, and its application in SLNB surgery for breast cancer has been reported in several studies. [5–11] The tracer used in this method is a microbubble contrast agent that can enhances ultrasonography of lymphatic vessels and lymph nodes. Due to the higher molecular weight of the microbubble relative to blue dye and similar feature of rapid entry into the lymphatic vessels like the blue dye, it can detect the SLN within minutes after drug injection and also not flow into the secondary lymphatic vessels immediately. Because of these features, the SLN identified by CEUS is significantly less than isotope or dye. [12–14] Due to less number of SLNs identified, CEUS was often used in combination with isotope or blue dye. Some studies have shown that, in case with positive SLN, the SLN identified by CEUS must be or be included in positive nodes. [9, 14] A meta-analysis that we published earlier suggested that the positive rate of sentinel lymph nodes identified by CEUS(CE-SLN) are significantly higher than those by conventional methods.[15] The reason was that other secondary lymph nodes that are not identified by CEUS are eliminated. Those lymph nodes are not real SLN, but are over-identified by conventional tracer methods, and most of them are negative. In addition, another advantage of CEUS is to clarify the location and drainage route of sentinel lymph nodes before operation, and reduce the difficulty of sentinel lymph node biopsy through the method of skin lineation and guide wire positioning, so as to make surgical exploration more accurate, shorten the operation time and reduce the surgical trauma. Especially for those node negative patients, it may be sufficient to remove the SLN identified by CEUS. Therefore, we conducted a prospective cohort study to investigate whether the pathological status of axillary lymph nodes could be accurately determined by just removing the SLN identified by CEUS.
Study population
Patients’ inclusion criteria: 1. pathologically confirmed breast invasive carcinoma; 2. the tumor is unifocal and less than 5cm in maximum diameter; 3. the clinical assessment is axillary lymph node negative. Exclusion criteria: 1. distant metastasis was diagnosed before surgery; 2. there was a history of surgery around areola region or in the outer upper quadrant of the breast and axillary, including tumor excision biopsy of this time; 3. Prior radiotherapy to the ipsilateral chest wall and axilla. The axillary node status was estimated with ultrasonography performed by two professional sonographers, and those who had suspicious lymph node were eliminated. The study was approved by the Institutional Ethics Committee, and all patients signed an informed consent before surgery.
CEUS for locating SLN
Contrast enhanced ultrasound is performed after the patient was generally anaesthetized and before surgery. The contrast agent we use is a microbubble(SonoVue™ BRACCO Imaging, S.p.A, Milan, Italy), that was dissolved with 5 ml of saline (NaCl 0.9%). After repeated shocks, 0.5ml liquid was respectively taken out and subcutaneous injected immediately at the areola edges 12, 3, 6, and 9 o 'clock and massaged for 1–3 minutes. Lymphatic flow from the breast to the axilla was identified by contrast enhanced ultrasound using Logiq E9 with XDclear (GE Healthcare, Tokyo, Japan). The specific operation method can refer to the previous literature.[16] If the enhanced lymph node is not explored within 5 minutes, repeat the above operation again, and if the enhanced lymph node is still not found, the operation is not repeated. After successfully exploring the enhanced sentinel lymph node, the operation needs to be repeated once time to confirm the number and location of the nodes. Depicting the direction of the lymphatic tube and the position of the sentinel lymph nodes, respectively, on the skin. Finally, under the guidance of ultrasound, the positioning hook wire (20G, LW0077, BARD) pierce into the lymph node. Before the study beginning, a training set of 30 patients was operated and achieving a success rate of more than 90%, and these patients were not included. The entire SLN identification and positioning process as shown in Fig. 1.
Surgery
The SLNB surgery was initiated immediately after SLN localization was completed. Before skin incision, 1ml methylene blue was subcutaneously injected in the same position as the microbubble injected and kneaded gently for several minutes, then the surgeon made an incision of about 3cm in length in axilla according to the marked SLN. Exploring and removing the SLN along the direction of the guide wire. Pathological status of CE-SLN was intraoperatively diagnosed by frozen section. While waiting for intraoperative pathological result, the surgeon will continue to explore and remove remaining dyed lymph nodes. If the CE-SLN was pathological positive, axillary lymph node dissection was performed, and if the CE-SLN was pathological negative, except for the remaining dyed lymph nodes, peripheral non-dyed lymph nodes were also removed until the number of SLNs reached 6. These supplementary SLNs and axillary dissection specimens were directly diagnosed by paraffin section, without intraoperative frozen section. The operation time is recorded as the CE-SLN operation time and the BD-SLN (blue dye identified SLN) operation time, respectively. CE-SLN operation time is recorded from skin incision to excision of CE-SLN, the time of each dyed lymph node excision is also recorded, and the time from skin incision to the last dyed lymph node was removed is defined as BD-SLN operation time.
Data were subjected to χ2 test or Fisher’s exact test using SPSS statistical software version 18.0 (SPSS Inc. Chicago, IL, USA). The level of significance was set at P < 0.05.
From May 1, 2020 to May 31, 2022, a total of 204 female patients were eligible and 109 patients were agreed the protocol and formed the initial study population, 105 of which were successfully located the SLN by CEUS, and to be the main study population. Another 4 patients were accepted dye method only. So, the detection rate of CEUS was 96.3%. The clinical characteristics of patients were listed in Table 1. The whole process of this study is as shown in Fig. 2.
| Characteristics | Number of patients (%) |
|---|---|
| Age | |
| < 50y | 34(31.2%) |
| ≥ 50y | 75(68.8%) |
| T stage | |
| T1 | 91(83.5%) |
| T2 | 18(16.5%) |
| Grade | |
| I | 2(1.8%) |
| II | 68(62.4%) |
| III | 39(35.8%) |
| ER/PR status | |
| ER positive and/or PR positive | 85(78.0%) |
| ER and PR negative | 24(22.0%) |
| Her2 status | |
| Positive | 20(18.3%) |
| Negative | 89(81.7%) |
| Pathological N status | |
| negative | 87(79.8%) |
| positive | 22(20.2%) |
| ≥ 2 positive nodes | 5(4.6%) |
| Surgical management | |
| mastectomy | 86(78.9%) |
| breast-conserving surgery | 23(21.1%) |
| Method of diagnosis | |
| core needle biopsy | 74(67.9%) |
| excision biopsya | 35(32.1%) |
| a: excision biopsy was permitted when the tumor is located outside the outer upper quadrant and areola region | |
Among the 4 unsuccessful patients, three of them were failed to find the sentinel lymph node, even by the dye method, and the final pathological results showed that 3 patients were lymph node metastasis. Among the 105 successful patients, 72 patients were located one CE-SLN, 33 patients were located two CE-SLNs, and the mean number of CE-SLNs was 1.3, while the mean number of dyed SLNs was 2.5, contained all of the CE-SLNs. The pathological node metastasis status of the main study population showed 86 were axillary node negative, and 19 were positive. In the group of node positive patients, at least one CE-SLN was metastatic for every patient, and 17 of them were CE-SLN positive alone. It is worth noting that two of the patients had CE-SLN micrometastases, and one of them was found in paraffin pathology, neither of these two patients received ALND. All 19 node positive patients are listed in Table 2.
| CASE No. | Age (y) | Grade | Surgical management | Maximal diameter (mm) | Number of CE-SLN | CEUS operation times | Number of dyed SLN | Intraoperative pathology of CE-SLN (positive number/total number) | size of CE-SLN metastasis (macro/micro) | pathological status of other ALNs or SLNs |
|---|---|---|---|---|---|---|---|---|---|---|
| Case4* | 85 | II | Mastectomy | 20 | 1 | 1 | 3 | 0/1 | Micro | 0/5 |
| Case10 | 64 | II | Mastectomy | 15 | 2 | 2 | 2 | 1/2 | Macro | 0/16 |
| Case19 | 42 | III | Mastectomy | 20 | 2 | 2 | 2 | 1/2 | Macro | 0/16 |
| Case29 | 48 | III | BCS | 25 | 1 | 2 | 2 | 1/1 | Macro | 1/18 |
| Case36 | 51 | II | BCS | 15 | 1 | 1 | 3 | 1/1 | Macro | 0/7 |
| Case39 | 64 | II | Mastectomy | 20 | 1 | 1 | 1 | 1/1 | Macro | 0/17 |
| Case42 | 59 | III | Mastectomy | 20 | 2 | 1 | 2 | 1/2 | Macro | 0/13 |
| Case45 | 48 | II | BCS | 30 | 1 | 1 | 1 | 1/1 | Macro | 0/12 |
| Case59 | 73 | II | Mastectomy | 19 | 1 | 1 | 3 | 1/1 | Micro | 0/5 |
| Case64 | 63 | II | Mastectomy | 17 | 2 | 2 | 3 | 1/2 | Macro | 0/19 |
| Case72 | 46 | II | Mastectomy | 15 | 1 | 2 | 2 | 1/1 | Macro | 0/15 |
| Case73 | 42 | III | Mastectomy | 20 | 1 | 2 | 2 | 1/1 | Macro | 0/13 |
| Case76** | 27 | III | BCS | 20 | 1 | 2 | 3 | 1/1 | Macro | 2/17 |
| Case80 | 50 | II | BCS | 15 | 1 | 1 | 2 | 1/1 | Macro | 0/13 |
| Case88 | 48 | II | BCS | 22 | 1 | 1 | 1 | 1/1 | Macro | 0/15 |
| Case92 | 63 | II | Mastectomy | 20 | 1 | 1 | 2 | 1/1 | Macro | 0/20 |
| Case97 | 57 | III | Mastectomy | 23 | 2 | 1 | 2 | 1/2 | Macro | 0/12 |
| Case104 | 60 | II | Mastectomy | 25 | 1 | 2 | 1 | 1/1 | Macro | 0/18 |
| Case105 | 45 | III | Mastectomy | 18 | 1 | 2 | 2 | 1/1 | Macro | 0/19 |
| * The intraoperative frozen section was negative, and CE-SLN micrometastasis was found in the paraffin pathology ** In this patient, the two positive non-CE-SLNs were dyed, and one of them was micrometastasis BCS, breast-conserving surgery | ||||||||||
CEUS operation records showed that 84 patients were successfully displayed and positioned the SLN through once injection of contrast agent, and only 11.9% (10/84) patients were node positive. On the contrary, 21 patients were positioned the SLN after the second injection, and 42.8% (9/21) were node positive. The latter had a significantly higher risk of lymph node metastasis than the former. Among node-positive patients, 42.1% (8/19) had only CE-SLN dyeing, compared with 20.9% (18/86) of node-negative patients. Above analysis results are listed in Table 3. The median number of dyed lymph nodes was 2 in node-positive patients and 3 in node-negative patients. The mean operation time of CE-SLN in this study was 415 ± 134 seconds, while it is 536 ± 201 seconds for BD-SLN.
| Node positive | Node negative | OR (95%CI) | P value | |
|---|---|---|---|---|
| CEUS operation times | ||||
| 1 | 10 | 74 | 0.278 (0.130–0.596) | .003 |
| 2 | 9 | 12 | ||
| Dyed node except CE-SLN | ||||
| Yes | 11 | 68 | 0.453 (0.204–1.003) | 0.101 |
| No | 8 | 18 |
Since the 1990s, when sentinel lymph node biopsy was applied to the surgical staging of lymph node of breast cancer, the widely accepted tracer method has been dyeing and isotopic method. [17–19] Although isotopic method is acknowledged the most accurate, it is limited to a certain extent to be extensively applied, because of its high cost and potential radiological hazards. Even though the dyeing method is simple and low-cost, there are also many problems such as tattoo effect and allergic reaction. [20–21] For patient who accepted nipple-sparing mastectomy and immediate implant-based reconstruction, the periareolar coloration caused by the dye may increase the risk of nipple necrosis. [22] CEUS-guided sentinel lymph node biopsy has been reported for more than a decade, especially in the last five years, correlative researches have increased year by year. By using ultrasonic enhanced agent, simple operation similar to dyeing method, with the assistance of ultrasound professional doctor, clinical surgeon can accurately know the location of the sentinel lymph node before the surgery, and when combination with wire positioning, the operation of sentinel node biopsy will be easier, the damage caused by the exploration of SLN may further reduced.
Based on the results of several prior prospective studies, the detection rate of CEUS-localized SLN was more than 90%. Li et al.'s study, which included a total of 453 patients, with a detection rate of 98.23%. [10] Zhong et al.'s study, that included 126 patients, with a detection rate of 100%. [23] In our result, the detection rate of SLN is 96.3%, that is consistent with previous researches and not inferior to the isotope and dye. In addition, consistent with previous reports, [12–14] the average number of SLNs identified by CEUS in our study was 1.3, which was significantly smaller than that obtained by dyeing. This is mainly attributed to differences in molecular weight between the tracers. [6, 7] The smaller the tracer molecular weight and the longer the tracer duration, the more likely it is to enter the secondary lymphatic vessels, which may lead to the removal of more non-sentinel lymph nodes, potentially increasing the risk of postoperative complications. Previous studies, like B32 and ALMANAC, also suggested that although SLNB has significantly reduced the risk of upper limb complication compared with ALND, about 8%~41% of patients still have upper limb paresthesia and other complications. [24] Retrospective studies and meta-analyses have demonstrated that there is still a certain morbidity of upper limb complications after SLNB surgery, the most common is axillary pain, which torments more than 10 percent of patients. [25–27] The reason may be largely due to excessive exploration of SLN. Previous study has suggested that, in a small minority of patients, the SLN for the breast and upper limb may be at the same station. [28] The CEUS method may be able to find the real SLN more quickly and accurately, reduce the exploration of other secondary lymph nodes, and may further reduce the occurrence of upper limb complications. Nowadays, a number of RCT studies about omitting sentinel lymph node biopsy are currently under way,[29] but the persuasive results and conclusions still need a long time of follow-up to be obtained, which naturally cannot be an optional strategy in current clinical practice. Therefore, reducing the surgical trauma of sentinel lymph node biopsy while ensuring the accuracy of its diagnosis may be a more practical approach at this stage, like the CEUS method.
In this study, all preoperatively located CE-SLNs were dyed. However, previous reports have shown that a few SLNs identified by CEUS may not be dyed, [9, 30] which may be related to the lymphatic drainage pattern of patients. It has been reported that the lymphatic drainage pattern from mammary gland to axilla can be divided into four modes: a single primary lymphatic vessel corresponds to a single SLN, a single primary lymphatic vessel after branching corresponds to two or more SLNs, multiple primary lymphatic vessels correspond to multiple SLNs, and multiple primary lymphatic vessels when aggregated correspond to a single SLN. [13] The first two modes account for more than 80% of the total, and the third is the rarest. So, if CE-SLN is found undyed, the first lymph node corresponding to the dyed primary lymphatic vessel must be removed to minimize the false-negative rate.
In our study, Three-quarters of the patients who failed to identified CE-SLN were node metastatic. Further analysis of CEUS operating records found that, in all lymph node positive patients, 47.4% belongs to the secondary operation group, and patients of this group had a sentinel lymph node positive rate of 42.8%, which may be related to poor flow of lymphatic tubes in patients with lymph node metastasis. [31] That suggested, in the clinical practice, the success rate of SLNB can be well predicting by the CEUS method, and in patients with CEUS failure, double tracing may be necessary to increase the success rate. On the contrary, in the patients with successful SLN localization after once CEUS operation, most of them are node negative, or only a mild tumor burden in the SLN. By this method, the axillary state or lymph node load can be accurately predicted before surgery.
Compared with the single dye method, the operation time of CEUS-guided SLNB is significantly reduced. Furthermore, the operation time of the dye method in this study might be shortened because of the guidance of guide wire, and the operation time of the single dye method may be longer. This technology may be able to shorten the learning curve of surgeons and improve the accuracy of SLN exploration. [32] The results indicated the false negative rate of CE-SLN was 0%. Although these patients did not undergo further ALND, but at least six lymph nodes including all dyed nodes were removed, and previous studies have shown that the risk of false negative rate is very small when more than 5 lymph nodes are removed. [1, 2]
Our study also has some limitations. Firstly, 83.5% of patients was in early breast cancer with stage T1, and the application value of this method to patients with larger tumor diameters still needs to be further studied. This study only investigated the methodological feasibility, and the effect on long-term recurrence are still lacking data. Meanwhile, the impact of this method on upper limb function needs to be further evaluated, proving whether its security is indeed superior to the traditional methods. The intraoperative tracer in this study only used blue dye, which is not the best single tracer, nor can it avoid the tattoo effect and other defects. CEUS combined with isotope may better avoid the defects of dye, and can also be used to verify the results of this study. More clinical studies still needed to confirm these results.
For clinical node negative breast cancer patients, SLNB under CEUS guidance is a high-efficiency new method, avoiding probable excessive exploration of SLNs. Of course, more clinical studies still needed to confirm these results.
Ethical Approval
The study was approved by the institutional ethics committee of Changshu Hospital Affiliated to Nanjing University of Chinese Medicine (IRB approval number is 2020028). Informed consent was obtained from all enrolled patients. The study meets the requirements of the Declaration of Helsinki.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CQX and DL wrote the main manuscript text. XJL and LJL completed data collating and prepared figures 1-2 and table 1-3. XJL, CQX, DL and LJL participated in and completed the operation and data collection records of SLNB surgery. SY, THJ and ZXF participated in and completed the operation and data collection records of CEUS and CE-SLN positioning. XJL provided guidance for the study. All authors reviewed and approved the final manuscript.
Funding
This work was supported by the maternal and child health research project of Jiangsu province (F201951 to Jialei Xue)
Availability of data and materials
All data analysed during this study are included in this published article.