Dose Comparison (1 mL versus 2 mL) of Superparamagnetic Tracer for Sentinel Node Biopsy in Early Breast Cancer: Real-World Analysis from 456 Patients.

Introduction
Sentinel lymph node biopsy (SNB) is a well-established surgical procedure for axillary staging in patients with clinically node-negative early breast cancer (eBC), allowing accurate assessment of lymphatic involvement with a reduced morbidity compared to axillary lymph node dissection [1–4]. One of the first established SNB techniques utilizes a dual-tracer method combining technetium-99m (99mTc) radiocolloid and blue dye [4, 5]. Although 99mTc is an approved and effective tracer, this approach presents logistical challenges due to reliance on nuclear medicine infrastructure, regulatory constraints, limited scheduling flexibility, and exposure to ionizing radiation [6, 7].
Superparamagnetic iron oxide (SPIO) nanoparticles were introduced as radiation-free alternative for SNB [1, 2, 8, 9]. SPIO, administered peritumoral, accumulates in lymphatic tissue and enables intraoperative localization of sentinel lymph nodes (SNs) using a handheld magnetometer [1, 10–12]. Numerous prospective trials and meta-analyses have demonstrated that the detection rates with SPIO are comparable to the standard radiotracer technique [2, 3, 6, 8, 13, 14]. Additionally, SPIO offers practical advantages, including extended injection-to-surgery intervals, elimination of radioactive handling, and applicability in settings without nuclear medicine departments.
Despite the growing evidence supporting SPIO use, the clinical protocols vary, particularly regarding the optimal tracer volume. Clinical trials and real-world analyses have reported the use of different SPIO volumes, with limited data on whether the amount of tracer affects detection rates and whether there are associations with patient-related parameters [12, 15, 16]. This study aimed to evaluate the performance of SPIO (Magtrace®) as a tracer for SNB in patients with eBC in a real-world setting of a single center, with the main focus on comparing the approved amounts of 1 mL versus 2 mL SPIO injection volumes and their impact on SN detection rates and the number of nodes retrieved. By addressing these aspects, our analysis seeks to provide evidence-based recommendation for optimizing SPIO use in diverse clinical settings.

Materials and Methods

Study Population and Treatment
In this retrospective study, all patients with eBC, who received Magtrace®, a SPIO tracer for SNB at the Department of Obstetrics and Gynecology, University Medical Center, Johannes Gutenberg University Mainz, between January 01, 2020 and December 31, 2022 were included. Patients presented with clinically and ultrasonographical node-negative breast cancer. Exclusion criteria included known allergy to iron, iron overload disorders, presence of a pacemaker, or ferromagnetic devices in the chest wall, pregnancy, and lactation.
According to the product information for Magtrace®, either 1 mL (May 2021 – December 2022) or 2 mL of SPIO (January 2020 – April 2021) was injected in the clinic peritumorally according to the protocol. The institutional switch from 2 mL to 1 mL Magtrace® was guided by the available European approval and published clinical data indicating non-inferior SN detection compared with 2 mL [12, 15]. The surgeries were performed by the same four certified breast surgeons at the same center. The SNs were detected intraoperatively using the approved Sentimag® system. Clinicopathological data were obtained from the institutional breast cancer database and pathology reports, including patient and tumor characteristics as well as the surgical procedures including the SPIO administration.

Clinical Outcomes
The primary endpoint of the study was to investigate whether the amount of SPIO injected (1 mL vs. 2 mL) had an impact on the detection rate and the number of SNs removed. Secondary endpoints included (i) number of SNs intraoperatively detected by the surgeon, (ii) number of removed SN reported by pathology, (iii) the impact of the type of breast surgery on the removed SN, and (iv) the impact of tracer timing on the detection rate and number of removed SN. Furthermore, (v) the impact of body mass index (BMI) on detection rate was evaluated. A successful procedure was defined as intraoperative detection of at least one SN and histological confirmation of lymph node tissue in the pathology report. We performed two subgroup analyses. First, we defined two groups of patients according to timing of tracer application: group 1: timeframe ≤ median value and group 2: timeframe > median value. Second, according to the definition of WHO for obesity, two groups of patients were defined (patients of normal weight with a BMI <25 kg/m2 vs. patients with overweight and obesity with a BMI ≥25 kg/m2) [17]. Postoperative complications, defined as postoperative events requiring surgical intervention, were analyzed.

Statistical Analysis
Statistical analyses were conducted using SPSS statistical software program, version 27.0 V5 R (SPSS Inc, Chicago, IL, USA). A two-sided p value <0.05 was considered statistically significant. Patients’ characteristics were analyzed descriptively using median and range for continuous data. For analyses, patients were divided into two groups based on the volume of SPIO administered: 1 mL or 2 mL. Comparisons between both groups according to the study endpoints were conducted using Pearson’s chi square test, Mann-Whitney test, as well as linear regression. No correction for multiple testing was applied; therefore, the reported p values are exploratory and should be interpreted as hypothesis generating. For the purposes of this study, “real-world analysis” refers to the retrospective evaluation of routinely collected clinical data reflecting standard clinical practice, without experimental intervention or predefined research protocols.

Results

Patient Characteristics
This real-world data analysis included 456 patients with eBC who received Magtrace® as a tracer for SNB. Baseline characteristics are summarized in Table 1.
The median age at diagnosis was 61.0 years (range:31–92) in the 1 mL group and 60.0 years (range: 26–86) in the 2 mL group. Most patients (79.4% in the 1 mL group and 80.2% in the 2 mL group) had an invasive ductal carcinoma (no special type); overall, 88.7% and 88.0% of tumors were smaller than 5 cm, respectively. The final postoperative node-negative status was documented in 191 patients (85.7%) in the 1 mL group and 208 patients (89.7%) in the 2 mL group.

Surgical Procedures
Surgical procedures are summarized in Table 2. The majority of patients, 69.5% in the 1 mL group and 66.4% in the 2 mL group, underwent simple wide local excision. The SNB alone was performed in 87.0% and 91.8% of patients (1 mL vs. 2 mL). In 9 (4.0%) patients in the 1 mL group and 7 (3.0%) patients in the 2 mL group, SNB with SPIO failed. Conversion to axillary dissection due to SNB failure or positive SN was documented in 19 (7.5%) patients and 12 (5.2%) patients, respectively, in 1 mL vs. 2 mL groups. When comparing postoperative complications between the 1 mL and 2 mL groups, postoperative hematoma occurred in 10 patients (4.5%) and 6 patients (2.6%), respectively. Surgical site infections requiring surgical intervention were observed in 1 patient (0.4%) in the 1 mL group and 2 patients (0.9%) in the 2 mL group.

Impact of SPIO Volume on Clinical Outcome
The amount of 1 mL and 2 mL SPIO was administered in 223 and 232 patients, respectively. The tracer was injected peritumorally in the affected breast region preoperatively with a median of 4 days (range: day of surgery – 9 days).
The amount of 1 mL and 2 mL SPIO did not show any significant differences in the detection rate and the number of removed SN (Table 3). The SN detection rate was 95.5% (213 patients) in the 1 mL SPIO group and 96.1% (223 patients) in the 2 mL group (p = 0.707). Linear regression also showed no significant association between injection volume and detection rate (B = 0.004, 95% CI: −0.68 to 0.74, p = 0.931). According to the pathology reports, the median number of removed SNs were 2.0 (range: 0–9) in the 1 mL group and 2.0 (range: 0–8) in the 2 mL SPIO group (p = 0.205). The median duration of surgery was 70 min (range: 19–220 min) in the1ml group and 72 min (range: 15–218 min) in the 2 mL group and did not differ significantly between the abovementioned SPIO volumes (p = 0.972). The analysis regarding the type of breast surgery showed no statistically significant difference in the number of SNs removed between the two SPIO volumes (Table 4, p > 0.05).

Impact of the Time of Tracer Application and BMI on Detection Rates and the Number of Removed SNs
The median time of tracer application before surgery was 4 days (range 0–18 days), and this was set as cutoff value for performing a subgroup analysis to investigate the detection rate depending on the time of administration. The detection rate was 97.3% in the group in which SPIO was administered 0 till 4 days prior to surgery and 95.3% in the group with the SPIO application later than 4 days prior to surgery (p = 0.286) (Table 5). The median number of removed SN also did not differ significantly between the two groups (p > 0.05).
The comparison of the detection rates between different tracer volumes (1 mL vs. 2 mL) among patients with BMI <25 kg/m2 and patients with BMI ≥25 kg/m2 showed no significant differences. The results of this subgroup analysis are outlined in Table 6.

Discussion
This real-world data analysis of 456 patients with eBC provides evidence supporting the efficacy and safety of SPIO tracer Magtrace® for SNB. It offers valuable insights into the impact of tracer volume on SN detection across varying clinical and patient-related parameters. To our knowledge, this is one of the largest real-world data analyses on the use of SPIO in patients with eBC. We compared two groups of patients, who received different SPIO volumes (1 mL vs. 2 mL) to evaluate the impact of dosage on SN detection. The analysis focused on the detection rates and the number of lymph nodes removed associated with each volume. This comparison is clinically relevant as removing a higher number of lymph nodes can increase the risk of postoperative morbidity [18–20]. Since SNB is a diagnostic and not a therapeutic procedure, it is essential for the surgeon to consider whether removing more than two nodes is clinically necessary [21, 22]. In such cases, the tumor’s risk profile should guide the decision-making process to balance diagnostic accuracy with the goal of minimizing morbidity. Additionally, we investigated whether the SPIO volume influenced surgery time, which could impact the surgical efficiency and patient outcomes. Our analysis sought to clarify, whether a lower volume of SPIO could achieve comparable detection rates while minimizing potential risks.
Variations in injection dose, site such as peritumoral, intratumoral, periareolar, or subareolar, and timing of injection can potentially influence the lymphatic uptake of magnetic tracers. Previous studies have explored a range of injection volumes – including 0.1 mL [23, 24], 0.5 mL and 1.0 mL [16], 1.5 mL [12, 15, 25], and 2.0 mL [10, 15, 25]. Christenhusz et al. [16] showed in a meta-analysis including three Dutch and five Swedish studies that detection rates remained comparable among different protocols (1 mL: 100% vs. 2 mL: 97.3%). These studies have demonstrated that alternative approaches do not differ significantly from each other in terms of detection rates.
In the present analysis, SN detection rates were comparable between both groups: 95.5% in the 1 mL group and 96.1% in the 2 mL group (p = 0.707), indicating no statistically significant difference. The modestly lower absolute detection rates in our cohort may reflect differences in study design, patient populations, or real-world clinical practice compared with the controlled settings of the studies included in the meta-analysis. Despite minor numerical differences, the equivalence of 1 mL and 2 mL injections in both the meta-analysis and our study supports the conclusion that injection volume does not significantly impact SN detection, and both volumes are suitable for clinical use.
Sundh et al. [23] lately published the results of a multicenter prospective study, investigating the use of ultra-low dose of 0.1 mL intradermal injection of SPIO (Magtrace®) in 216 patients. The Swedish team showed that ultra-low SPIO is non-inferior to 99mTc ± blue dye for SN detection (97.7% vs. 99.5%; p = 0.111). Skin discoloration was limited and further reduced by removal during surgery [23].
When considering the number of removed SN, a few studies investigated the differences among several SPIO doses [15, 16, 26, 27]. In the SUNRISE study with 135 patients, the median number of SNs retrieved by SPIO in different doses (1 mL vs. 1.5 mL vs 2.0 mL) showed no significant differences [15]. Mean number of SNs detected by magnetic tracer was 1.76, 1.64, and 1.89 in each group, respectively. Similarly, in our study the median number of SNs removed, based on pathology reports, was 2.0 in both the 1 mL and 2 mL SPIO group. No significant differences in detection rates were observed between the two groups, even when analyzed across various surgical procedures, including simple wide local excision, oncoplastic breast-conserving surgery, modified radical mastectomy, and skin-sparing mastectomy. However, our study may have been underpowered to demonstrate a difference in this subgroup analysis.
Our findings revealed that reducing the SPIO dose from 2 mL to 1 mL did not negatively influence the detection rate or the number of SN identified. Reducing the number of removed lymph nodes may be clinically beneficial as it can lower the risk of postoperative morbidity without affecting diagnostic accuracy [18]. Additionally, since no significant difference in the detection rate or number of SNs were observed, using a lower SPIO volume may offer advantages in terms of cost-effectiveness [7].
To evaluate whether the timing of SPIO application influences SN detection, a subgroup analysis was conducted using the median interval of 4 days between tracer injection and surgery as a cutoff. No statistically significant differences were observed between both groups in terms of the detection rate (97.3 vs. 95.3, p = 0.286) and SNs removed by the surgeon (median 2.0 in both groups; p = 0.347), or the number of SN confirmed by pathology (median 2.0 in both groups; p = 0.147). These findings indicate that the timeframe between SPIO administration and surgery, whether short or extended beyond 4 days, does not significantly influence the tracer performance. This flexibility in timing may offer logistical advantages in clinical practice without compromising diagnostic efficacy. Our results are consistent with previous published studies [1, 25, 28]. Karakatsanis et al. [1] additionally showed that higher detection rates were found when SPIO was injected 1–28 days ahead of surgery compared to that administered on the day of surgery.
Breast surgeons recognize that an increased BMI is a potential risk factor for failure of the SNB procedure, which may result in higher surgery-associated morbidity, if axillary lymph node dissection is required. Although traditional SN localization methods have shown variable performance in patients with higher BMI, with some studies reporting mapping failure rates correlated with BMI, very few studies have directly investigated the impact of BMI on SPIO-based SN detection in eBC [29, 30]. Hughes et al. [31] investigated in a cohort of 174 patients the impact of obesity on lymphatic mapping and SNB in patients with eBC. The results of their study showed no significant difference (p = 0.379) in the SN detection among normal (96%), overweight (97%), and obese patients (97%) [31]. We observed high detection rates among patients with BMI <25 kg/m2 as well as patients with BMI ≥25 kg/m2, providing direct evidence that SPIO efficacy remains robust in patients with elevated BMI.
The use of SPIO may be associated with potential disadvantages, such as artefacts on magnetic resonance imaging (MRI) and the possibility of skin discoloration at the injection site [32–37]. However, whether skin staining and MRI artefacts are reduced by lower dose was not investigated in this analysis and requires further investigation.
The limitations of this study include its lack of randomization, the retrospective design, the inclusion of all types of breast surgery techniques and missing follow-up data regarding skin discoloration and MRI artefacts. Furthermore, according to current knowledge, SNB can be omitted in postmenopausal patients with clinically unremarkable axillary lymph nodes and small hormone receptor-positive GI or GII tumors who undergo breast-conserving therapy [38–40]. However, the inclusion of well-documented cases and the performance of surgeries and pathological reviews by the same experienced team at a single center enhances the relevance of our results.
In conclusion, our findings demonstrate that reduced SPIO volume of 1 mL is similar to 2 mL in terms of SN detection rates, number of nodes retrieved, and timing between injection and surgery. These results support the clinical feasibility of using lower SPIO doses without compromising diagnostic performance, even in patients with elevated BMI. This real-world analysis reinforces SPIO’s role as an effective and adaptable alternative to conventional tracers in SNB for eBC.

Acknowledgment
A part of the results of this study is included in the doctoral thesis of Mr. Paul Löwe.

Statement of Ethics
The STROBE guidelines were utilized to structure all reporting of reported results. Written informed consent was obtained from all patients, and all clinical investigations were conducted ethically in accordance with ethical and legal standards and in consideration of the Declarations of Helsinki. The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Ethics Committee of Rhineland-Palatinate, Germany (No. 837.139.05).

Conflict of Interest Statement
Ina Shehaj received funding and travel reimbursement from Novartis Pharma GmbH, AstraZeneca, Olympus, and AURIKAMED. Anne-Sophie Heimes received honoraria from Pfizer Pharma GmbH, Roche Pharma AG, Daiichy Sankyo GmbH, Novartis Pharma GmbH, AstraZeneca, Medupdate GmbH, and Streamedup! GmbH. Kathrin Stewen received honoraria from AstraZeneca, Roche Pharma AG, StreamedUp! GmbH, and Pierre Fabre. Annette Hasenburg received honoraria from AstraZeneca, Celgen, GSK, LEO Pharma, MedConcept GmbH, Med update GmbH, Medicultus, Pfizer, Promedicis GmbH, SoftConsult, Roche Pharma AG, Streamedup! GmbH, and Tesaro Bio Germany GmbH. She is a member of the advisory board of AstraZeneca, GSK, LEO Pharma, PharmaMar, Promedicis GmbH, Roche Pharma AG, Tesaro Bio Germany GmbH, MSD Sharp, and Dohme GmbH. Marcus Schmidt reports personal fees from AstraZeneca, BioNTech, Daiichi Sankyo, Eisai, Gilead, Lilly, MSD, Novartis, Pantarhei Bioscience, Pfizer, Roche, and SeaGen outside the submitted work. Institutional research funding from AstraZeneca, BioNTech, Eisai, Genentech, German Breast Group, Novartis, Palleos, Pantarhei Bioscience, Pierre Fabre, and SeaGen. Marcus Schmidt has a patent for EP 2390370 B1 issued and a patent for EP 2951317 B1 issued. Marcus Schmidt was a member of the journal’s Editorial Board at the time of submission. Slavomir Krajnak received speaker honoraria from Roche Pharma AG and Novartis Pharma GmbH Germany, research funding from Novartis Pharma GmbH Germany, and travel reimbursement from PharmaMar and Novartis Pharma GmbH Germany. All other authors declare that they have no conflicts of interest.

Funding Sources
This research received no external funding.

Author Contributions
Conceptualization: S.K.; methodology: S.K. and I.S.; data curation: P.L. and A.L.; and formal analysis and writing – original draft preparation: I.S.; and writing – review and editing: A.L., P.L., A.-S.H., A.L., K.S., A.H., M.S., and S.K. All authors have read and agreed to the published version of the manuscript.