Indocyanine Green Fluorescence Plus Blue Dye for Sentinel Lymph Node Biopsy in Patients Undergoing Neoadjuvant Chemotherapy for Breast Cancer: A Multicenter, Prospective Cohort Study
Introduction Neoadjuvant chemotherapy (NAC) is increasingly used for patients with operable breast cancer to tailor the surgery in the breast and axilla [ , , ]. The increasing use of NAC has brought about inquiries regarding the optimal approach to staging the axilla, including whether it can be used to avoid axillary lymph node dissection (ALND) in patients with initially node‐positive (cN+)disease [ , , , , , ]. Sentinel lymph node biopsy (SLNB) is the gold standard for assessing axillary lymph node status in patients with clinically node‐negative (cN0) breast cancer . and major guidelines recommend omission of ALND in patients with negative SLNB . In patients with initially cN+ disease, some studies on SLNB after NAC have been done to guide ALND [ , , , , , , , , , , ]. The false negative rate (FNR) at < 10% has been achieved in patients who have completed NAC for cN+ breast cancer by: (1) using dual radioisotope and blue dye tracers and limiting the patient population to 3 or more retrieved SLNs; (2) removal and inspection of the clipped nodes; and (3) immunohistochemistry for SLNs [ , , , ]. Marking axillary lymph nodes with radioactive iodine seeds (MARIs) yielded 7% FNR . Targeted axillary dissection (TAD) that involves a combination biopsy of SLNs and targeted lymph nodes resulted in a further reduction to 2%–6% FNR [ , , , , , ]. Despite these improvements, significant barriers remain in practice settings at the global scale due to regulatory/logistic barriers, requirement for multiple procedures, and patient fear of isotope use . Displaced and missing marker clips have also been reported . Indocyanine green (ICG) fluorescence imaging has been used for SLNB [ , , , , , , , ] and recommended as an alternative tracer in patients with cN0 breast cancer by professional societies in Europe and China . Promising results with ICG in combination with radioisotope, i.e., higher detection rate versus radioisotope alone, have also been reported in a trial in patients undergoing NAC for initially cN+ breast cancer . We conducted a multicenter, prospective cohort study to examine diagnostic performance of SLNB using ICG plus methylene blue in patients who underwent NAC for initially cN1 breast cancer. 

Methods 2.1 Study Design and Patients This was a multicenter, prospective cohort study (participating centers listed in Appendix ). The study protocol was approved by the Ethics Committee of all participating centers and registered at ClinicalTrials.gov . All patients provided written informed consent. Adult women (20–80 years of age) scheduled to undergo NAC with pathologically confirmed cT0‐3N1M0 primary invasive breast cancer through core needle tissue biopsy were consecutively enrolled from March 9, 2018 to August 31, 2022. Patients who were pregnant, had inflammatory disease, had any contraindication to surgery, or had previous axillary surgery/radiotherapy were excluded. Informed consent was procured prior to NAC. Pre‐NAC and post‐NAC of primary breast tumors and lymph nodes were evaluated by physical and imaging examinations including ultrasonography, mammogram, and magnetic resonance imaging (MRI). cN1 was defined as suspicious palpation on movable ipsilateral level I, II axillary lymph nodes based on the eighth edition of the American Joint Committee on Cancer Staging Manual (AJCC 8th) , and fine‐needle aspiration (FNA) cytology was recommended but not mandatory in all patients. ycN0 was defined as no suspicious lymph nodes on palpation and ultrasound findings, while ycN+ was defined as suspicious lymph nodes on palpation and ultrasound findings based on AJCC 8th. No uniformly accepted standard for sonographic lymph node assessment is available. We classified nodes as suspicious in case of the focal or diffuse cortical thickening (≥ 3 mm), disparity in sizes, rounded appearance , and/or effacement of the fatty hilum of the node . 2.2 Neoadjuvant Chemotherapy All the patients were pathologically confirmed with breast cancer by using core needle tissue biopsy and immunohistochemistry examination. NAC regimens were based on the latest National Comprehensive Cancer Network (NCCN) guidelines at the time and were based on 6–8 cycles of anthracycline/taxane‐based and carboplatin/taxane‐based regimens (e.g., TAC/AC‐T/AC‐TP) in HER2 negative patients and six cycles of TCbH(P) regimen in HER2 positive patients. 2.3 Surgical Procedure All the enrolled patients were scheduled to be performed SLNB and sequent ALND. SLNB was performed within 3–4 weeks after completion of NAC. For SLNB, methylene blue (Jizhou Pharmaceutical, Suzhou, China) and ICG (Dandong Pharmaceutical, Jilin, China) were diluted to a final concentration of 1% and 2.5 mg/mL, respectively. After administration of general anesthesia and sterilization, 1 mL of 1% methylene blue was subdermally injected into the periareolar region immediately before surgery. Five minutes later, 1 mL of ICG solution was intradermally injected into one to two sites in the same periareolar area region. The movement of the tracer in the periareolar region was facilitated by massage. ICG fluorescence was detected using a hand‐held fluorescence detector (Vascular Imaging System MDM‐I, MingDe Biomedical Technology Company Limited, Lang Fang, China) at 780 nm excitation and 830 nm acquisition. Percutaneous ICG projection of lymph vessels and ICG‐stained lymph nodes was navigated in real time (Figure ). Lymph nodes that were fluorescent, blue, or suspicious on palpation were considered SLNs, resected, and submitted for histopathological evaluation. ALND included level I (low‐axilla) and II (mid‐axilla) dissection. Both SLNB and ALND procedures were done in the same surgical setting. Patients were carefully monitored during and after surgery to exclude possible allergic reactions. Breast surgery was either lumpectomy or mastectomy. Post‐operation follow‐up was regularly performed every 3 months for the first 2 years and every 6 months for the 2–5 years. 2.4 Histopathological Evaluation Resected lymph nodes were formalin‐fixed and paraffin‐embedded for histopathological examination. Serial slices perpendicular to the long axis were prepared at 2 mm interval and stained with hematoxylin and eosin for histopathological evaluation. Metastases were categorized as macrometastases (> 2 mm), micrometastases (> 0.2 mm but and ≤ 2.0 mm), and isolated tumor cells (≤ 0.2 mm). The axillary pathological complete response (pCR) was defined as the absence of any residual disease (including micrometastases and isolated tumor cells). The pathological response of the breast was evaluated using the Miller and Payne grading score . Estrogen receptor (ER) and progesterone receptor (PR) were considered positive by expression in at least 1% of cancer cells in immunohistochemistry (IHC). Positive HER2 expression was defined by 3+ in IHC or 2+ protein staining plus gene amplification by fluorescence in situ hybridization based on the ASCO/CAP guideline. 2.5 Statistical Analysis The primary outcome was DR (number of patients with at least one SLN divided by the total number of patients). Secondary outcomes included FNR (number of patients with negative SLNs divided by the number of patients with pathologically confirmed residual axillary disease) and adverse events associated with the tracers. The negative predictive value (NPV) was defined as the number of true negative results divided by the total number of patients with true negative and false negative test findings. The subgroup analysis was stratified by the number of SLNs retrieved and the ycN status, which was not predefined. We planned to enroll at least 150 patients to ensure a meaningful DR and FNR, which was addressed in the registration in ClinicalTrials.gov . Data analyses are descriptive. Trial reporting complied with the Standards for the Reporting of Diagnostic accuracy studies (STARD) criteria  (Appendix ). 

Study Design and Patients This was a multicenter, prospective cohort study (participating centers listed in Appendix ). The study protocol was approved by the Ethics Committee of all participating centers and registered at ClinicalTrials.gov . All patients provided written informed consent. Adult women (20–80 years of age) scheduled to undergo NAC with pathologically confirmed cT0‐3N1M0 primary invasive breast cancer through core needle tissue biopsy were consecutively enrolled from March 9, 2018 to August 31, 2022. Patients who were pregnant, had inflammatory disease, had any contraindication to surgery, or had previous axillary surgery/radiotherapy were excluded. Informed consent was procured prior to NAC. Pre‐NAC and post‐NAC of primary breast tumors and lymph nodes were evaluated by physical and imaging examinations including ultrasonography, mammogram, and magnetic resonance imaging (MRI). cN1 was defined as suspicious palpation on movable ipsilateral level I, II axillary lymph nodes based on the eighth edition of the American Joint Committee on Cancer Staging Manual (AJCC 8th) , and fine‐needle aspiration (FNA) cytology was recommended but not mandatory in all patients. ycN0 was defined as no suspicious lymph nodes on palpation and ultrasound findings, while ycN+ was defined as suspicious lymph nodes on palpation and ultrasound findings based on AJCC 8th. No uniformly accepted standard for sonographic lymph node assessment is available. We classified nodes as suspicious in case of the focal or diffuse cortical thickening (≥ 3 mm), disparity in sizes, rounded appearance , and/or effacement of the fatty hilum of the node . 

Neoadjuvant Chemotherapy All the patients were pathologically confirmed with breast cancer by using core needle tissue biopsy and immunohistochemistry examination. NAC regimens were based on the latest National Comprehensive Cancer Network (NCCN) guidelines at the time and were based on 6–8 cycles of anthracycline/taxane‐based and carboplatin/taxane‐based regimens (e.g., TAC/AC‐T/AC‐TP) in HER2 negative patients and six cycles of TCbH(P) regimen in HER2 positive patients. 

Surgical Procedure All the enrolled patients were scheduled to be performed SLNB and sequent ALND. SLNB was performed within 3–4 weeks after completion of NAC. For SLNB, methylene blue (Jizhou Pharmaceutical, Suzhou, China) and ICG (Dandong Pharmaceutical, Jilin, China) were diluted to a final concentration of 1% and 2.5 mg/mL, respectively. After administration of general anesthesia and sterilization, 1 mL of 1% methylene blue was subdermally injected into the periareolar region immediately before surgery. Five minutes later, 1 mL of ICG solution was intradermally injected into one to two sites in the same periareolar area region. The movement of the tracer in the periareolar region was facilitated by massage. ICG fluorescence was detected using a hand‐held fluorescence detector (Vascular Imaging System MDM‐I, MingDe Biomedical Technology Company Limited, Lang Fang, China) at 780 nm excitation and 830 nm acquisition. Percutaneous ICG projection of lymph vessels and ICG‐stained lymph nodes was navigated in real time (Figure ). Lymph nodes that were fluorescent, blue, or suspicious on palpation were considered SLNs, resected, and submitted for histopathological evaluation. ALND included level I (low‐axilla) and II (mid‐axilla) dissection. Both SLNB and ALND procedures were done in the same surgical setting. Patients were carefully monitored during and after surgery to exclude possible allergic reactions. Breast surgery was either lumpectomy or mastectomy. Post‐operation follow‐up was regularly performed every 3 months for the first 2 years and every 6 months for the 2–5 years. 

Histopathological Evaluation Resected lymph nodes were formalin‐fixed and paraffin‐embedded for histopathological examination. Serial slices perpendicular to the long axis were prepared at 2 mm interval and stained with hematoxylin and eosin for histopathological evaluation. Metastases were categorized as macrometastases (> 2 mm), micrometastases (> 0.2 mm but and ≤ 2.0 mm), and isolated tumor cells (≤ 0.2 mm). The axillary pathological complete response (pCR) was defined as the absence of any residual disease (including micrometastases and isolated tumor cells). The pathological response of the breast was evaluated using the Miller and Payne grading score . Estrogen receptor (ER) and progesterone receptor (PR) were considered positive by expression in at least 1% of cancer cells in immunohistochemistry (IHC). Positive HER2 expression was defined by 3+ in IHC or 2+ protein staining plus gene amplification by fluorescence in situ hybridization based on the ASCO/CAP guideline. 

Statistical Analysis The primary outcome was DR (number of patients with at least one SLN divided by the total number of patients). Secondary outcomes included FNR (number of patients with negative SLNs divided by the number of patients with pathologically confirmed residual axillary disease) and adverse events associated with the tracers. The negative predictive value (NPV) was defined as the number of true negative results divided by the total number of patients with true negative and false negative test findings. The subgroup analysis was stratified by the number of SLNs retrieved and the ycN status, which was not predefined. We planned to enroll at least 150 patients to ensure a meaningful DR and FNR, which was addressed in the registration in ClinicalTrials.gov . Data analyses are descriptive. Trial reporting complied with the Standards for the Reporting of Diagnostic accuracy studies (STARD) criteria  (Appendix ). 

Results 3.1 Cohort Characteristics A total of 350 patients were screened during a period from March 9, 2018 to August 31, 2022; 156 patients were enrolled (Figure ). The median age was 51 years (range: 24–74). Disease stages were II in 133 (85.3%) patients and III in the remaining 23 (14.7%) patients. Molecular subtypes were HR+/HER2‐ in 49 (31.4%) patients, HR‐/HER2‐ in 28 (18.0%) patients, HR+/HER2+ in 42 (26.9%) patients, and HR‐/HER+ in the remaining 37 (23.7%) patients. FNA was conducted in 143 patients and demonstrated lymph node involvement in 134 of these patients. One hundred and forty‐three and 13 patients underwent mastectomy and lumpectomy, respectively (Table ). 3.2 Surgical Treatment and Pathology Evaluation All 156 patients underwent SLNB after NAC. Four patients (all ycN0) decided not to undergo ALND after SLNB as originally planned, due to the higher requirements for postoperative quality of life and the fear of the risk of lymphedema, paresthesia, and pain syndromes arising from ALND. Postoperative pathological results revealed no lymph node involvement in these four patients. None of the four patients developed local recurrence and distant metastasis within the median 21 months follow‐up (range: 18 to 26 months). ALND was actually performed in the 152 patients. All these patients had a total at least 10 lymph nodes dissection (range 10–46) for pathological evaluation. Breast pCR rate was 45.5% (71/156) and axillary pCR rate was 59.2% (90/152) (Table ). The axillary pCR rate was 75.9% (82/108) versus 18.2% (8/44) in patients with ycN0 versus ycN+ status. Among the 62 patients not achieving axillary pCR after NAC, 61 had macrometastases and 1 had micrometastases. There was no patients of local recurrence in the breast or axilla within the median 28 months follow up (range 8–61 months). 3.3 Detection Rate The DR of SLNB with dual ICG and methylene blue tracing was 97.4% (152/156; 95% CI, 93.6%–99.0%). The number of SLNs retrieved during SLNB was 1 in 20 (12.8%) patients, 2 in 38 (24.4%) patients, 3 in 28 (17.9%) patients, 4 in 22 (14.1%) patients, and ≥ 5 in 44 (28.2%) patients. The median number of SLNs was 3 (range: 0–11; interquartile range [IQR]: 2–5) (Table ). According to the stratification on the ICG tracing alone and methylene blue alone, the DR was 96.8% (151/156, 95% CI, 92.7%–98.6%) and 87.8% (137/156, 95% CI, 81.8%–92.1%), respectively. The median number of SLNs was 3 (range: 0–11; [IQR]: 2–5) in the ICG tracing group and 2 (range: 0–10; [IQR]: 1–3) in the methylene blue tracing group (Table ). 3.4 False Negative Rate and Negative Predictive Value Among the 156 patients, four cases were performed SLNB without the sequent ALND, and four cases failed to detect SLNs and subsequently underwent ALND. The FNR and NPV were calculated based on the 148 patients with successful SLNB followed by ALND. Among the 148 patients, 92 cases had negative SLNs and 56 had positive SLNs detected by SLNB. SLNB correctly identified lymph node status as positive in 56 patients and mistakenly reported lymph node status as negative in 4 of 92 patients. The FNRs were 15.2% (7/46; 95% CI, 7.6%–28.2%) in the methylene blue tracing group and 6.8% (4/59; 95% CI, 2.7%–16.2%) in ICG fluorescence tracing group. The NPVs were 92.6% (87/94; 95% CI, 85.4%–96.3%) in methylene blue and 95.7% (88/92; 95% CI, 89.4%–98.3%) in the ICG fluorescence tracing group. When ICG fluorescence was combined with methylene blue tracing, the FNR was 6.7% (4/60; 95% CI, 2.6%–15.9%) and the NPV was 95.7% (88/92; 95% CI, 89.4%–98.3%) (Table ). The detailed clinicopathologic status of four patients with false‐negative results is shown in Table . In the subgroup analysis stratified by the number of SLNs retrieved, the FNR varied with 0% (0/7; 95% CI, 0%–35%) in the 1 SLN subgroup ( n = 20), 0% (0/12; 95% CI, 0%–24%) in the 2 SLN group ( n = 38) and 9.8% (4/41; 95% CI, 3.9%–22.6%) in the ≥ 3 SLN group ( n = 90). And according to the ycN status, the FNR was 4.0% (1/25; 95% CI, 0.7%–19.5%) in the ycN0 subgroup ( n = 105) versus 8.6% (3/35; 95CI, 3.0%–22.4%) in the ycN+ subgroup ( n = 43) (Table ). 3.5 Adverse Events No allergic reaction was reported. Temporary urine discoloration occurred in 110 (70.5%) patients. Skin staining (both methylene blue and ICG) was reported in 4 out of the 13 patients who underwent lumpectomy. At the six‐month follow‐up, staining with methylene blue remained in 2 patients; ICG was no longer visible in any patient. No palpable subcutaneous nodules were noticed. 

Cohort Characteristics A total of 350 patients were screened during a period from March 9, 2018 to August 31, 2022; 156 patients were enrolled (Figure ). The median age was 51 years (range: 24–74). Disease stages were II in 133 (85.3%) patients and III in the remaining 23 (14.7%) patients. Molecular subtypes were HR+/HER2‐ in 49 (31.4%) patients, HR‐/HER2‐ in 28 (18.0%) patients, HR+/HER2+ in 42 (26.9%) patients, and HR‐/HER+ in the remaining 37 (23.7%) patients. FNA was conducted in 143 patients and demonstrated lymph node involvement in 134 of these patients. One hundred and forty‐three and 13 patients underwent mastectomy and lumpectomy, respectively (Table ). 

Surgical Treatment and Pathology Evaluation All 156 patients underwent SLNB after NAC. Four patients (all ycN0) decided not to undergo ALND after SLNB as originally planned, due to the higher requirements for postoperative quality of life and the fear of the risk of lymphedema, paresthesia, and pain syndromes arising from ALND. Postoperative pathological results revealed no lymph node involvement in these four patients. None of the four patients developed local recurrence and distant metastasis within the median 21 months follow‐up (range: 18 to 26 months). ALND was actually performed in the 152 patients. All these patients had a total at least 10 lymph nodes dissection (range 10–46) for pathological evaluation. Breast pCR rate was 45.5% (71/156) and axillary pCR rate was 59.2% (90/152) (Table ). The axillary pCR rate was 75.9% (82/108) versus 18.2% (8/44) in patients with ycN0 versus ycN+ status. Among the 62 patients not achieving axillary pCR after NAC, 61 had macrometastases and 1 had micrometastases. There was no patients of local recurrence in the breast or axilla within the median 28 months follow up (range 8–61 months). 

Detection Rate The DR of SLNB with dual ICG and methylene blue tracing was 97.4% (152/156; 95% CI, 93.6%–99.0%). The number of SLNs retrieved during SLNB was 1 in 20 (12.8%) patients, 2 in 38 (24.4%) patients, 3 in 28 (17.9%) patients, 4 in 22 (14.1%) patients, and ≥ 5 in 44 (28.2%) patients. The median number of SLNs was 3 (range: 0–11; interquartile range [IQR]: 2–5) (Table ). According to the stratification on the ICG tracing alone and methylene blue alone, the DR was 96.8% (151/156, 95% CI, 92.7%–98.6%) and 87.8% (137/156, 95% CI, 81.8%–92.1%), respectively. The median number of SLNs was 3 (range: 0–11; [IQR]: 2–5) in the ICG tracing group and 2 (range: 0–10; [IQR]: 1–3) in the methylene blue tracing group (Table ). 

False Negative Rate and Negative Predictive Value Among the 156 patients, four cases were performed SLNB without the sequent ALND, and four cases failed to detect SLNs and subsequently underwent ALND. The FNR and NPV were calculated based on the 148 patients with successful SLNB followed by ALND. Among the 148 patients, 92 cases had negative SLNs and 56 had positive SLNs detected by SLNB. SLNB correctly identified lymph node status as positive in 56 patients and mistakenly reported lymph node status as negative in 4 of 92 patients. The FNRs were 15.2% (7/46; 95% CI, 7.6%–28.2%) in the methylene blue tracing group and 6.8% (4/59; 95% CI, 2.7%–16.2%) in ICG fluorescence tracing group. The NPVs were 92.6% (87/94; 95% CI, 85.4%–96.3%) in methylene blue and 95.7% (88/92; 95% CI, 89.4%–98.3%) in the ICG fluorescence tracing group. When ICG fluorescence was combined with methylene blue tracing, the FNR was 6.7% (4/60; 95% CI, 2.6%–15.9%) and the NPV was 95.7% (88/92; 95% CI, 89.4%–98.3%) (Table ). The detailed clinicopathologic status of four patients with false‐negative results is shown in Table . In the subgroup analysis stratified by the number of SLNs retrieved, the FNR varied with 0% (0/7; 95% CI, 0%–35%) in the 1 SLN subgroup ( n = 20), 0% (0/12; 95% CI, 0%–24%) in the 2 SLN group ( n = 38) and 9.8% (4/41; 95% CI, 3.9%–22.6%) in the ≥ 3 SLN group ( n = 90). And according to the ycN status, the FNR was 4.0% (1/25; 95% CI, 0.7%–19.5%) in the ycN0 subgroup ( n = 105) versus 8.6% (3/35; 95CI, 3.0%–22.4%) in the ycN+ subgroup ( n = 43) (Table ). 

Adverse Events No allergic reaction was reported. Temporary urine discoloration occurred in 110 (70.5%) patients. Skin staining (both methylene blue and ICG) was reported in 4 out of the 13 patients who underwent lumpectomy. At the six‐month follow‐up, staining with methylene blue remained in 2 patients; ICG was no longer visible in any patient. No palpable subcutaneous nodules were noticed. 

Discussion The DR in the current study was 97.4%. The FNR was 6.7% (95% CI, 2.6%–15.9%) and the NPV was 95.7% (95% CI, 89.4%–98.3%). No ICG‐related adverse events, except temporary urine discoloration and skin staining in a few cases, were reported. The DR in the current study (97.4%) is higher than that reported for conventional SLNB (radioisotope plus blue dye or radioisotope alone) in the SENTINA and SN FNAC trials (87.8% and 87.6%)  as well as in the subgroup of patients with cN1 disease in the Z1071 trial (92.9%)  and is comparable to that reported for SLNB using triple tracers (i.e., carbon nanoparticle, radioisotope and blue dye) (95.0%)  and radioisotope mapping plus ICG (98.3%) . In the overall analysis that included all patients regardless of number of retrieved SLNs during SLNB, the current study achieved the FNR of 6.7%, whereas the FNR was 12.6% in the Z1071 trial, 14.2% in the SENTINA trial and 13.3% without immunohistochemical examination of the retrieved lymph nodes in the SN FNAC trial [ , , ]. Previous studies revealed that FNRs varied with the number of SLNs retrieved and < 10% FNR was achieved only in the subgroup analysis of patients with ≥ 3 SLNs in previous trials using radioisotope plus blue dye for SLNB . In the Z1071 and SENTINA trial, the FNRs of ≥ 3 SLNs subgroup were 9.1% and 4.9%, while the FNRs of < 3 SLNs group were 21.1% and 21.8%, respectively . In the current study of subgroup analysis, the FNRs were 9.8% for patients with ≥ 3 SLNs and 0% for patients with < 3 SLNs. In the study, resecting more SLNs did not decrease the FNR as the reported in the previous studies. The reasons may include: the effect of chemotherapy (fibrosis, obstruction of lymphatic channels, etc.) which may lead to aberrant lymphatic drainage, and the heterogeneity between the primary tumors and the metastatic lymph nodes which may lead to the different response of metastatic lymph nodes, especially in patients with high tumor burden . According to our knowledge, this study is the first one aimed to SLNB for cN1 patients receiving NAC. However, there may be bias with a few pathological N2 even N3 patients. All four false‐negative cases in this study were patients with high tumor burden, including two cases of cT2 (close to cT3) and two cases of cT3 prior to chemotherapy; three cases of ycN+ and one case of ycN0 but cT3. Among the four patients, each one had > 3 retrieved SLNs. All the 4 patients had a total of 15 to 21 lymph nodes resected. Though the 4 patients had negative SLNs and positive non‐SLNs (all macrometastases), reactive hyperplasia was seen in 1 or 2 SLNs of the 4 patients which indicated the chemotherapy effect to the positive nodes. So this result showed ICG and methylene blue tracing SLNB for the patients with high tumor burden still need more study. In addition, the small sample size of the study may be another possible reason of the result. According to the available data, NCCN indicates that highly selected patients with initial cN+ disease, who then converted to ycN0 after NAC, may undergo SLNB by using dual tracer and removing ≥ 3 sentinel nodes and removal of targeted nodes for the improved < 10% FNR . Unlike in the SENTINA trial , conversion from cN+ to ycN0 was not required for enrollment in the current study. In the current study, the FNR in the patients of cN+ to ycN+ subgroup (8.6%) was higher than that in ycN0 subgroup, but nevertheless under the commonly acceptable standard of 10%. It has been reported that 20%–30% patients showed inconsistency between clinical assessment of axillary lymph nodes and pathological response [ , , ]. Similarly, our study showed 18.2% of the patients with ycN+ disease after NAC achieved pCR. It has never been studied on the feasibility of SLNB in the group of ycN+ patients, our preliminary analysis showed that ALND is not always necessary in ycN+ patients to obtain the actual nodal status., SLNB in ycN+ patients is a reasonable choice. Two recent studies reported that the post‐NAC FNA of a clipped metastatic lymph node was associated with a high FNR of 57.6%–60% in assessing nodal response to NAC , indicating the unreliability of FNA in predicting nodal response, and highlighting the need for surgical staging of the axilla after NAC. However, taking into account the assessment of suspicious lymph nodes (ycN+) by FNA after NAC, in Liu's study , post‐NAC FNA might help triage patients and tailor the axillary surgery. In view of that ultrasound‐guided FNA is a low‐risk procedure, post‐NAC FNA could allow patients, who are deemed as ycN+ and positive cytology, to avoid unnecessary SLNB. While for the patients who are deemed as ycN+ and negative cytology, a de‐escalation of axillary surgery might be feasible. So post‐NAC FNA for the ycN+ patients may be a convenient and alternative procedure to tailor SLNB or ALND. Combination of ICG and methylene blue improved the DR and FNR of SLNB than methylene blue alone for the patients with NAC in this study. And ICG tracing alone was comparable with dual ICG and methylene blue tracing in this study. ICG has been used for SLNB tracing in early breast cancer patients for nearly twenty years but mostly combined with other mapping methods such as blue dye and radioisotope [ , , ]. In some smaller studies, ICG has shown an equal sensitivity as a single‐tracer as 99mTc‐nanocolloid with SLN detection rates varying from 93.1% to 100% [ , , , , ] The INFLUENCE trail also supported the use of ICG fluorescence as a single‐tracer during SLNB as an alternative to 99mTc‐nanocolloid in clinically node‐negative patients with early‐stage breast cancer . While if ICG alone could be used for patients after NAC, it still needs more rigorously designed trials with large samples. Targeted axillary dissection (TAD), including biopsy of clipped lymph node and sentinel lymph nodes, had been shown to reduce the FNR in patients after NAC [ , , , , , , ]. While, whether the reduction in FNR observed with TAD translates into a significant reduction in the rate of axillary recurrence is unknown. The recent trials had shown that compared to SLNB, TAD did not obtain a significantly lower axillary recurrence in the cN+ patients who downstaged to ycN0 and had omission of ALND . Furthermore, the recent retrospective study from Memorial Sloan Kettering Cancer Center (MSKCC) found that in patients with cN1 disease downstaged to ycN0 after NAC, with 3 or more negative SLNs with SLNB alone, axillary recurrence rate was as low as 0.4% without routine lymph node clipping , which is consistent with the rate of 0%–1.6% observed in the existing literature [ , , ]. These findings suggest that the TAD‐free procedure using SLNB alone without nodal clipping may be feasible in selected patients. However, there is no consensus on which axillary staging procedure should be used in the post‐NAC setting with initially cN+ disease. Our study suggested that SLNB with ICG plus methylene blue might be an optional choice, especially in the areas which is inaccessible to radioisotope and clips. The NPV in the current study (95.7%) was higher than that reported for conventional SLNB in a meta‐analysis of 17 studies on the diagnostic accuracy of SLNB in patients with cN+ breast cancer after NAC (57%–86%) , as well as for the MARI procedure (83.3%–86.3%) in previous trials , and is comparable to that reported for SLNB using triple tracers (93.3%)  and RISAS procedure (92.8%) . The higher NPV in the current study is primarily driven, in our opinion, by the higher rate of axillary pCR in the study (59.2%) than the reported in previous studies , which in turn could be attributed to the higher proportion of HER‐2 positive patients (50.6%) than the previous data (29.7% in Z1071 trials , 30.7% in RISAS trials ), the enrolled patients with a slightly lower tumor burden (cT0‐3N1M0), as well as the advance in NAC regimens. Another contributing factor to the high NPV in the current study is the low rate of FNR in this study. Since NPV is more important than FNR in guiding decision‐making for individual patients in clinical practice, the criteria for acceptable FNR should be re‐evaluated with the now much higher pCR rate in patients undergoing NAC. Since the inaccessibility of radioisotope and required equipment in the majority hospitals in China, it is regrettable we did not compare dual ICG and methylene blue tracing with the standard methods of radioisotope and blue dye. So we performed this cohort study first, hoping to accumulate the evidence for conducting a large sample trial in the future. The SLNB using ICG plus methylene blue was well tolerated by all patients in the current study. In keeping with the previous studies, the dual ICG plus methylene blue tracing method can be applied without toxic side effects. The safety profile of SLNB using ICG plus methylene blue in the current study was generally consistent with previous studies [ , , ]. The current study has several limitations. First, it was conducted in cN1 patients exclusively. Whether dual ICG and methylene blue SLNB could achieve high DR and low FNR in initially cN2 and cN3 patients remains unknown. Second, it is a single‐arm observational cohort study; no comparison group was included due to the unavailability of radioisotopes in most hospitals in China. Third, the sample size was relatively small and the subgroups were not predefined. 

Conclusion SLNB using dual ICG and methylene blue tracers had high DR and low FNR after NAC in primary invasive breast cancer patients. The FNR was 6.7% for the overall cohort, 4.0% in the ycN0 subgroup, and 8.6% in the ycN+ subgroup. Despite the lack of a comparison group in the study, the findings encourage further large samples and comparative investigation of dual ICG and methylene blue as an alternative method for SLNB in patients undergoing NAC for initially node‐positive breast cancer, even for initially cN2 and cN3 breast cancers. 

Conceptualization: Shu Wang, Miao Liu, Bin Hua, Rui Feng, Mengyuan Wang, and Jun Jiang. Data curation: Shu Wang, Miao Liu, Yang Yang, Bin Hua, Rui Feng, Mengyuan Wang, Danhua Shen, Lei Chen, Xiaowei Qi, and Jun Jiang. Formal analysis: Miao Liu and Yang Yang. Funding acquisition: Shu Wang. Investigation: All authors. Methodology: Miao Liu, Yang Yang, Bin Hua, Rui Feng, Mengyuan Wang, and Xiaowei Qi. Project administration: Miao Liu, Yang Yang, Bin Hua, Rui Feng, Mengyuan Wang, and Xiaowei Qi. Resources: Shu Wang, Miao Liu, Bin Hua, Rui Feng, Mengyuan Wang, Danhua Shen, Lei Chen, and Jun Jiang. Supervision: Shu Wang, Miao Liu, Yang Yang, Bin Hua, Rui Feng, Mengyuan Wang, Jun Jiang, and Danhua Shen. Validation: Shu Wang, Miao Liu, Bin Hua, Rui Feng, Mengyuan Wang, and Danhua Shen. Visualization: Miao Liu and Yang Yang. Writing – original draft: Yang Yang and Miao Liu. Writing – review and editing: Shu Wang, Bin Hua, Rui Feng, Mengyuan Wang, Xiaowei Qi, and Jun Jiang. 

The study protocol was approved by the Ethics Committee of all participating centers. 

Informed consent was obtained from all individual participants included in the study. 

The authors declare no conflicts of interest. 

