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Aug 17, 2013 - Magnetic sentinel lymph node biopsy and localization properties of a magnetic tracer in an in vivo porcine model. Bauke Anninga • Muneer ...
Breast Cancer Res Treat (2013) 141:33–42 DOI 10.1007/s10549-013-2657-0

PRECLINICAL STUDY

Magnetic sentinel lymph node biopsy and localization properties of a magnetic tracer in an in vivo porcine model Bauke Anninga • Muneer Ahmed • Mieke Van Hemelrijck • Joost Pouw • David Westbroek • Sarah Pinder • Bennie ten Haken Quentin Pankhurst • Michael Douek



Received: 4 July 2013 / Accepted: 28 July 2013 / Published online: 17 August 2013 Ó Springer Science+Business Media New York 2013

Abstract The standard for the treatment of early non-palpable breast cancers is wide local excision directed by wireguided localization and sentinel lymph node biopsy (SLNB). This has drawbacks technically and due to reliance upon radioisotopes. We evaluated the use of a magnetic tracer for its localization capabilities and concurrent performance of SLNB using a handheld magnetometer in a porcine model as a novel alternative to the current standard. Ethical approval by the IRCAD Ethics Review Board, Strasbourg (France) was received. A magnetic tracer was injected in varying volumes (0.1–5 mL) subcutaneously into the areolar of the left and right 3rd inguinal mammary glands in 16 mini-pigs. After 4 h magnetometer counts were taken at the injection sites and in the groins. The magnetometer was used to localize any in vivo Bauke Anninga and Muneer Ahmed are joint first authors and contributed equally to this study. B. Anninga  M. Ahmed  D. Westbroek  S. Pinder  M. Douek (&) Breast Surgery, Department of Research Oncology, King’s College London, 3rd Floor Bermondsey Wing, Guy’s Hospital Campus, Great Maze Pond, London SE1 9RT, UK e-mail: [email protected] B. Anninga  J. Pouw  B. ten Haken MIRA Institute for Biomedical Technology and Technical Medicine, Universiteit Twente, Enschede 7500 AE, The Netherlands M. Van Hemelrijck Cancer Epidemiology Group, School of Medicine, Guy’s Hospital, King’s College London, Great Maze Pond, London SE1 9RT, UK Q. Pankhurst Institute of Biomedical Engineering, University College London, Gower Street, London WC1E 6BT, UK

signal from the draining inguinal lymph nodes. Magnetic SLNB followed by excision of the injection site was performed. The iron content of sentinel lymph nodes (SLNs) were graded and quantified. All excised specimens were weighed and volumes were calculated. Univariate analyses were performed to evaluate correlation. Magnetic SLNB was successful in all mini-pigs. There was a significant correlation (r = 0.86; p \ 0.01) between magnetometer counts and iron content of SLNs. Grading of SLNs on both H&E and Perl’s staining correlated significantly with the iron content (p = 0.001; p = 0.003) and magnetometer counts (p \ 0.001; p = 0.004). The peak counts corresponded to the original magnetic tracer injection sites 4 h after injection in all cases. The mean volume and weight of excised injection site specimens was 2.9 cm3 (SD 0.81) and 3.1 g (SD 0.85), respectively. Injection of C0.5 mL magnetic tracer was associated with significantly greater volume (p = 0.05) and weight of excision specimens (p = 0.01). SLNB and localization can be performed in vivo using a magnetic tracer. This could provide a viable alternative for lesion localization and concurrent SLNB in the treatment of non-palpable breast cancer. Keywords Superparamagnetic iron oxide nanoparticles (SPION)  Sentinel lymph node biopsy (SLNB)  Sentinel node and occult lesion localization (SNOLL)  Non-palpable breast cancer  Magnetic tracer

Introduction Approximately one-third of all breast cancers are non-palpable at diagnosis [1]. The current standard for the treatment of these cancers in the presence of a clinically normal axilla pre-operatively is by surgical excision directed by wire-

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guided localization (WGL) [2] and staging of the axilla using sentinel lymph node biopsy (SLNB) [3–8]. This approach has its drawbacks in terms of technical difficulties and high re-excision rates for the localization component. The reliance of SLNB upon radioisotopes limits its uptake making it only available to 60 % of patients in the developed world [9] and almost negligible uptake in the rest of the world [10]. As a consequence of these drawbacks alternative localization techniques in the form of radio-guided localization (RGL) have been developed and have been proven beneficial over WGL in meta-analyses [11–14]. However, uptake of RGL has been low due to reliance upon radioactive materials, which have cost and waste management disadvantages. Despite advancements in imaging modalities [15–17] no alternative to surgical axillary staging using SLNB has a diagnostic accuracy of greater than 90 % for determining the axillary nodal status in breast cancer and SLNB remains justified as the standard of care [3–8]. Magnetic nanoparticles (MNPs) possess unique properties, which make them attractive to medical applications. These properties include their small size (10–160 9 10-9 m), high surface to volume ratio and their ability to carry other compounds [18]. We developed a non-invasive method for identifying the sentinel lymph node (SLN) using super-paramagnetic iron oxide (SPIO) contrast agent (Endorem, Guerbet, France), injected subcutaneously into the breast rather than intravenously. We demonstrated proof of principle to identify SLNs using a handheld prototype magnetometer [19]. When injected intravenously, SPIOs have been used as contrast agents for MRI and their characteristics are well recognized. When injected subcutaneously, SPIO moves into SLNs within minutes and iron deposition is seen predominantly within sinuses and in macrophages. In the event of metastatic involvement of the node, SPIOs are seen to deposit within uninvolved areas of the node only [20]. The nodes can be visualized on MRI [21] and at operation, are often coloured brown or black. This research work led on to the development of two devices: an injectable magnetic tracer (Sienna?, Endomagnetics Ltd., UK) and a handheld magnetometer (SentiMag, Endomagnetics, UK). The magnetic tracer is a blackish-brown sterile aqueous suspension of super-paramagnetic carboxydextran-coated iron oxide particles (SPIONs) that is intended for use with the handheld magnetometer device, and is CE marked as an injectable device. The carboxydextran coating prevents agglomeration while maintaining biocompatibility. The z-averaged particle diameter, including organic coating, is 60 nm (\0.25 polydispersity), ideally suited for SLNB and equivalent in particle size to radiolabeled nanocolloid. This diameter enables the SLNs to selectively filter out particles. This technology is the subject of a NIHR-adopted UK multicentre trials [22] in breast cancer (SentiMAG

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Multicentre Trial) and melanoma (MELAMAG Multicentre Trial) [23]. We decided to evaluate the use of the same magnetic tracer for its localization capabilities and concurrent performance of SLNB using the handheld magnetometer in a porcine model. This is a new clinical indication for this technology and is potentially more important than that of SLNB alone.

Materials and methods This study was conducted at the IRCAD institute, Strasbourg (France); King’s College London (UK) and the Universiteit Twente, Enschede (The Netherlands). Ethical permission was granted for animal experimentation, by the IRCAD Ethics Review Board, Strasbourg, France (Reference number: 38.2012.01.047). Mini-pigs used for the IRCAD laparoscopic general surgical skills course were surgically prepped and anaesthetized for the purpose of the course. Prior to commencement of the laparoscopic skills course a magnetic tracer (Sienna?, Endomagnetics, UK) and Patent V Blue dye (Guerbet, France) acting as a control, were injected subcutaneously into the areolar of the left and right 3rd inguinal mammary glands in 16 minipigs. The performance of SLNB in porcine model Magnetic tracer was injected in 0.5, 1 and 2 mL neat and these volumes diluted to 5 mL with normal saline. The blue dye was injected neat at 2 mL, diluted to 5 mL with normal saline and combined with magnetic tracer (2 mL blue dye; 2 mL magnetic tracer; 6 mL normal saline) to act as a control. Bilateral SLNB of groin nodes was undertaken 4 h after injection. A handheld magnetometer (SentiMag, Endomagnetics Ltd, UK) was then used to localize any in vivo signal from draining inguinal lymph nodes. SLNB was undertaken at the site of magnetic ‘hot spots’. Those lymph nodes with a magnetometer signal higher than 10 % of the hottest node and/or black staining, were considered to be SLNs, and excised with ex vivo counts taken (Fig. 1a). The harvested nodes were fixed in formalin and sent to Universiteit Twente, Enschede (The Netherlands), where the quantification of iron in each excised node was performed using Vibrating Sample Magnetometry (VSM). The measurement was performed using a magnetic field of ±4.0 T, which is suitable to bring the MNPs into saturation. The amount of magnetic tracer in the lymph nodes was determined by comparing the obtained amplitude of the magnetization to known calibration samples. These nodes were then sent to King’s College London (UK) and underwent fixation, thin slicing, processing and paraffin wax embedding and haematoxylin and eosin (H&E)

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neat and each of these volumes was diluted to a total volume of 0.5 and 1 mL using normal saline and injected subcutaneously into the areolar of the left and right 3rd inguinal mammary glands. Bilateral SLNB of groin nodes was undertaken 4 h after injection and all harvested nodes underwent VSM and histopathological assessment (as mentioned above). Magnetic localization For the lower volumes of injected magnetic tracer between 0.1 and 0.5 mL, the handheld magnetometer was used to perform counts at the site of injection to confirm that the peak magnetometer signal counts corresponded with the original injection site 4 h after injection. This was followed by the performance of a magnetic-guided excision of the mammary gland injection site (central segmentectomy) as shown in Fig. 1b. All tissue positive for a magnetometer signal was excised until no residual activity remained in the vicinity of the operating field. The excised specimens were then weighed and their volumes were calculated using water displacement before being placed in formalin and sent to King’s College London where they were processed in the same way as the excised nodes. Statistical analysis

Fig. 1 The operative procedures performed: a In vivo SLNB with the injection site indicated with the white arrow and the brown discoloured node in vivo indicated with the red arrow; b In vivo magnetic-guided excision of the mammary gland injection site (white arrow indicates brown staining from injection of magnetic tracer)

staining. Each node was examined for the presence of SPIO using standard microscopy and confirmation of iron deposition was performed using Perl’s Prussian blue (Sigma-Aldrich, Poole, UK) staining. The grading of iron deposition within each node was recorded, using a 5-point grading scale (0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked) for the extent of iron deposition using H&E and Perl’s stained SLN specimens jointly by an experienced pathologist and a second observer (SP; BA or MA) (Fig. 2) as previously described [20]. Magnetic tracer dose escalation for SLNB SLNB was undertaken with lower volumes of magnetic tracer injected. The volume of magnetic tracer was reduced to allow a dual localization and SLNB procedure to be performed. The magnetic tracer was injected in 0.1 mL incremental volumes between the range of 0.1 and 0.5 mL

All associations were evaluated with univariate analyses. The correlation between different continuous variables (handheld magnetometer readings and iron content measurements of the excised SLNs) was calculated using Pearson’s correlation coefficient (r). Associations between categorical and continuous covariates (i.e. volume of injected tracer, total injected volume, magnetometer readings, iron content, Perl’s and H&E grading, weight and volumes of excised specimens) were assessed with analyses of variance (ANOVA). All analyses were performed with Statistical Analysis Systems (SAS) release 9.2 (SAS Institute, Cary, NC).

Results SLNB and dose escalation of magnetic tracer A total of 32 SLNB procedures were performed on 16 mini-pigs. All 32 were successful and at least 1 ‘hot’ node was identified in each procedure. In vivo magnetic ‘hot spots’ from the draining inguinal lymph nodes were identified transcutaneously prior to surgical incision using the handheld magnetometer in all cases of administered magnetic tracer. In total 65 nodes were harvested with a mean

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Fig. 2 Examples of sentinel lymph nodes that were scored 1–4 after H&E (a, c, e and g) and 1–4 after Perl’s (b, d, f and h) staining clearly showing the brown discoloration predominantly in the cortex of the nodes

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of 2.25 nodes (SD 1.48). All nodes harvested by the magnetic technique were macroscopically brown or black and identified by the magnetometer. Although SLNs were harvested in all procedures with all volumes of magnetic tracer injected (0.1–5 mL), increasing the volume of magnetic tracer injected during the procedure did not show any significant correlation with the handheld magnetometer counts (p = 0.36) as demonstrated by the boxplots in Fig. 3a. In two control cases where blue dye was administered, SLNs were visualized with blue staining, with a mean value of two blue nodes excised (SD 1.4). The remaining case, which consisted of injection with a combination of blue dye and magnetic tracer (2 mL blue dye; 2 mL magnetic tracer; 6 mL normal saline) did not permit blue visualization of SLNs due to the masking of any blue staining by the black staining from the magnetic tracer.

ANOVA p = 0.360

Magneto meter Counts

The histopathological grading of SLNs Iron was identified in all SLNs on histopathological assessment, with no SLNs receiving a Grade 0. A significant correlation between magnetometer counts and grading of SLNs on H&E (p \ 0.001) and Perl’s staining (p = 0.004) was identified, with the boxplots in Fig. 5a and b demonstrating the greater mean and median magnetometer counts as the recorded grade increased. A significant correlation was also recorded for the iron content of SLNs and their grading on H&E (p = 0.001) and Perl’s (p = 0.003). The mean and median iron content of the SLNs was demonstrated to increase with an increase in grade as shown in Fig. 5c and d. Magnetic localization

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The peak magnetometer counts corresponded to the original magnetic tracer injection sites 4 h after injection in all 15 procedures using volumes of magnetic tracer between 0.1 and 0.5 mL. The boxplots demonstrate a trend towards an increasing volume of excised specimens as the volume of magnetic tracer injected was increased as is shown in Fig. 6a. The boxplots of the weight of excised specimens versus the volume of magnetic tracer demonstrate the same trend of Pearson’s correlation r: 0.859 p < 0.001

Iron Content (µg)

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Increasing the volume of magnetic tracer injected demonstrated a significant correlation with the iron content of the SLNs excised (p = 0.02). The boxplot in Fig. 3b demonstrates that for volumes between 0.1 and 0.5 mL the mean and median iron contents closely resemble each other but become elevated at greater volumes. A significant linear relationship was demonstrated between the handheld magnetometer counts and the iron content of SLNs recorded on VSM (r = 0.86; p \ 0.01) as shown in Fig. 4.

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Fig. 3 The relationships for variations in the volume of magnetic tracer: a Boxplot of the volume of magnetic tracer versus magnetometer counts from excised sentinel lymph nodes; b Boxplot of the volume of magnetic tracer versus iron content of sentinel lymph nodes

Iron Content (µg)

Fig. 4 Graph demonstrating the relationship between the iron content and magnetometer counts of excised sentinel lymph nodes

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ANOVA p < 0.001

ANOVA p = 0.004

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Magneto meter Counts

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ANOVA p = 0.003

ANOVA p = 0.001

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Fig. 5 The relationships for the histopathological grading of excised sentinel lymph nodes: a Histopathological grading (H&E) versus magnetometer counts of excised sentinel lymph nodes; b Histopathological grading (Perl’s) versus magnetometer counts of excised

sentinel lymph nodes; c Histopathological grading (Perl’s) versus iron content of excised sentinel lymph nodes; d Histopathological grading (H&E) versus iron content of excised sentinel lymph nodes

greater weights of excised specimens as the volume of injected tracer is increased (Fig. 6b). However, in both these cases there was no significant correlation between the volume of injected magnetic tracer and either the volume of the excised specimens (p = 0.096) or their weights (p = 0.157). Due to small numbers we combined the volumes of injected magnetic tracer between 0.1 and 0.4 mL into a single group and compared to 0.5 and 1 mL. There was a statistically significant association between the injected volumes of magnetic tracer and the volumes (p = 0.05) and weights of excised specimens (p = 0.01). The boxplot in Fig. 6c demonstrates that the mean volume of excised specimens is doubled from 2 to 4 cm3 between injected volumes of 0.1–0.4 mL when compared to 1 mL. A similar near doubling in the weights of excised specimens from a mean of 2 to 3.7 g was seen for the same corresponding injected volumes of magnetic tracer as shown in Fig. 6d.

radiologically negative axilla [3–8]. The current combined technique (radioisotope and blue dye) has proven to be successful for detecting the SLN in 96 per cent of cases with a mean false negative rate of 7.3 % [4]. Although widely used, this technique has drawbacks regarding the use of radioisotopes [9, 10] and adverse reactions related to blue dye [24]. The mini-pig provides a suitable model for SLNB as shown by the feasibility of its performance using blue dye alone. Our study has demonstrated that the use of a magnetic tracer combined with a handheld magnetometer can be successfully used for the performance of SLNB in a porcine model. It has been demonstrated in this study that injected volumes of magnetic tracer as low as 0.1 mL can be successfully identified by the handheld magnetometer in the performance of SLNB. In the clinical context of using the technique in the operating room no significant correlation was identified between the volume of injected magnetic tracer and magnetometer counts (p = 0.36) during the SLNB procedures. This is most likely explained by heterogenous distribution of the magnetic tracer within the SLNs allowing high magnetometer counts to be acquired at focal points of magnetic tracer accumulation, whilst not

Discussion SLNB is the standard of care for axillary staging of patients with early breast cancer who possess both a clinically and

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b Results Kruskal-Wallis Test p = 0.096

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Results ANOVA p= 0.005

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Results Kruskal-Wallis Test p = 0.157

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Fig. 6 The relationships of the volume of magnetic tracer injected and excised injection site specimens: a volume of magnetic tracer injected versus the volume of excised specimens, b volume of magnetic tracer injected versus the weight of excised specimens,

c volume of magnetic tracer injected versus the volume of excised specimens with 0.1–0.4 mL considered as a single group for analysis, d volume of magnetic tracer injected and weight of excised specimen with 0.1–0.4 mL considered as a single group for analysis

representative of overall iron content. Therefore, whilst magnetic tracer was easily identified using the handheld magnetometer, it was not possible to distinguish between the volumes of magnetic tracer injected on the basis of intra-operative magnetometer counts. This lack of intraoperative quantification of magnetic tracer volume is of minimal clinical relevance, since a standard dose is usually administered in the clinical setting. The operating surgeon simply requires the knowledge of the presence or absence of magnetic tracer uptake by lymph nodes in order to successfully identify SLNs and for this purpose the handheld magnetometer is perfectly functional. Based on the average of 2.25 nodes excised per SLNB procedure and no residual background counts the magnetic tracer is retained by the first echelon nodes and the technique is comparable to the number of SLNs excised using the current combined technique [25]. The VSM technique has been used to quantify the iron content of SLNs after injection with SPIO to an accuracy of ±0.5 lg [26]. A significant correlation was found between increasing volumes of the magnetic tracer and the iron

content of the SLNs (p = 0.02) as recorded on VSM. Increasing the volume of magnetic tracer injected significantly increases the iron content of the SLNs excised. The sharp rise in the mean iron content of SLNs when 1 mL or greater of magnetic tracer was administered suggests that SLNs have a capacity for the uptake of iron which is underutilized at lower volumes of injected magnetic tracer. In order to assess if there was truly a saturation point for iron uptake, greater volumes of magnetic tracer beyond 2 mL would have to be injected and iron contents have to be calculated. However, administration of 0.1 mL of magnetic tracer deposits sufficient iron within the SLNs to allow recordable magnetometer counts in ex vivo SLNs. Therefore, overloading of the SLNs by administering increasing volumes of magnetic tracer is not clinically beneficial. When the handheld magnetometer readings were compared to the VSM calculations of iron content of SLNs a strong correlation was observed (r = 0.86; p \ 0.01). Therefore, magnetometer readings can be used for quantification against the calculated VSM iron content scores for the SLNs harvested. Quantification of the iron content

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within SLNs could be applied to the development and assessment of newer SPIOs, which have a higher magnetic yield within SLNs and clinical magnetometers to replace VSM [26]. The magnetic tracer was deposited within the sinuses and sub-capsular space of the SLNs as has been documented in previous studies but a higher proportion of SLNs in the porcine model reached the highest grading level compared to when assessed in humans [20]. The grading of the SLNs on both H&E and Perl’s showed a significant association with magnetometer counts and iron content of the SLNs. This demonstrates that the grading of SLNs used in this study accurately quantifies the iron content of excised SLNs as corroborated by VSM measurements and handheld magnetometer counts. In each procedure at least 1 node was graded 3 or 4, which would assist successful localization. The presence of iron did not interfere with the histological assessment of lymph nodes.

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This study has demonstrated that a magnetic tracer can successfully localize at the site of injection. In all cases we found that the peak magnetometer count persisted at the site of injection 4 h later in the porcine model above the maximal detection count. This clearly demonstrated that widespread dispersal of the magnetic tracer was not an issue in vivo as already has been demonstrated in vitro [27]. The magnetic tracer was taken up by macrophages at the site of injection but no other intracellular uptake was identified (Fig. 7a). The majority of magnetic tracer was found surrounding the lactiferous ducts at the site of injection within the stroma (Fig. 7b). A significant correlation was identified for greater volumes and weights of excised specimens at the sites of injection when volumes of magnetic tracer of 0.5 mL or greater were used. However, even with the greatest volume of magnetic tracer used (1 mL) the mean volume and weight of excised specimens were only 3.5 cm3 and 4 g, respectively. During the ROLL and WGL procedures for the treatment of non-palpable breast cancers, volumes of excised specimens in the range of 9–71cm3 and 23–64 cm3, respectively, are documented in the published literature of randomized controlled trials of ROLL versus WGL [28–33]. The weights of excised specimens are in the range of 14–68.1 g and 28–67.3 g for ROLL and WGL, respectively [28–33]. This suggests that our results are very favourable compared to ROLL and WGL. The magnetic tracer has limited dispersal and unnecessarily large volume excisions are unlikely.

Conclusion

Fig. 7 Histopathological assessment of the magnetic tracer injection sites: a The presence of intermittently dispersed brown-stained macrophages (white arrow) at the site of injection (magnification 9 40). b Perl’s staining of the excised injection site demonstrating the blue discolouration of the stained iron deep to the skin surface (white arrow) surrounding the lactiferous ducts (low power)

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Magnetic SLNB is feasible in an in vivo porcine model using volumes of magnetic tracer as low as 0.1 mL for injection. The magnetic tracer demonstrates localization at the site of injection in vivo. The ability to perform magnetic SLNB and the localizing properties of the magnetic tracer when combined could allow for the performance of a localization procedure for non-palpable breast cancers and concurrent SLNB using a single radiologically guided intra-tumoural injection. Such a technique would avoid the limitations of WGL in terms of wire insertion and eliminate the drawbacks of RGL and the combined technique of SLNB, in terms of radioisotope dependence. Such a technique could also provide access to SLNB and to non-palpable lesion localization, in centres and countries without readily available access to nuclear medicine facilities. Further evaluation of this technique should be undertaken within a prospective clinical trial. Acknowledgments We would like to thank Professor Marescaux and his team at the IRCAD Institute, Strasbourg (France) for their kind assistance in allowing us to perform this study.

Breast Cancer Res Treat (2013) 141:33–42 Conflict of interest The authors have no disclosures to make concerning financial and personal relationships with other people or organizations that could inappropriately influence their work.

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