Sentinel Node Biopsy using Magnetic Nanoparticles: A prospective multicentre phase II non-randomised clinical trial to compare sentinel node biopsy using magnetic nanoparticles vs.
standard technique

The SentiMag Multicentre Trial is a continuation of two earlier conducted
studies (see section 1.3 and 1.4 in protocol). The initial clinical pilot study
was conducted by the Chief Investigator, Mr Michael Douek, at University
College Hospital. Ten patients with newly diagnosed breast cancer scheduled for
sentinel node biopsy were recruited prior to surgery. These patients received a
radioisotope injection and underwent lymphoscintigraphy a day prior to surgery.
On the morning of surgery, they received a subcutaneous injection of a SPIO
tracer (Endorem, Guerbet, Paris) in the MRI Department. Dynamic axillary MRI
before and after injection of the SPIO identified lymphatic tracts and sentinel
nodes in 5/6 patients (1 scan failed for technical reasons). Under general
anaesthetic, patients routinely received an intradermal injection of patent
blue dye (Guerbet, Paris). The sentinel node(s) were localised using both a
gamma probe and magnetometer prototype I. Skin localisation with magnetometer
prototype I and the gamma probe, were identical. A total of 19 sentinel nodes
were resected from 9 patients. Intra-operative localisation using the combined
technique was successful in detecting 19/19 (100%) nodes and using magnetometer
prototype I alone in 19/19 (100%). Once found by the surgeon, most sentinel
nodes were easily identified as black from SPIO deposition.

Following the initial pilot study, several challenges were identified,
including interference from large metal objects, the shape of the probe and the
stability of the magnetic field. These were rectified and a second prototype
developed (prototype II). The Chief Investigator relocated to Guy*s Hospital
and a further 43 patients were recruited into an extended phase I/II trial. The
overall ex-vivo SentiMAG Multicentre SLN detection rate was 86% (37/43
patients) and was higher in patients who received SPIO more than 1 hour prior
to surgery (93%, 14/15). Data on the laboratory performance of the CE-marked
SentiMag and prototype II, were comparable and was included in the application
for CE approval. The first
sentinel node biopsy using the CE-marked SentiMag was performed successfully on
the 6th December 2010. Ethics Committee approval to commence a phase II
clinical trial at Guy*s Hospital to recruit 130 patients, has already been
granted.

The principal objective of the study is to determine if the performance of the
new technique (magnetic tracer and magnetometer) is equivalent to the
performance of the standard technique (patent blue dye and radioisotope; or
radioisotope alone).

The primary objective of the axillary MRI is to localize the SLN, the secundary
objective of the axillary MRI and the ex-vivo MRI is to determine if MRI can be
used as a non-invasive method for identification of breast cancer metastases in
lymph nodes.

The SentiMAG Multicentre Trial is a phase II paired equivalence trial. It will
initially involve 6 centres and will be co-ordinated from King*s College London
(Guy*s Hospital) by the Chief Investigator. The trial aimed to recruit 176
patients (assuming up to 10% patient drop-out). Centres will be invited to
recruit 30 patients and the trial will be closed once the target number of 160
complete patient datasets has been reached.

Patients will receive a radioisotope injection and a subcutaneous, periareolar
injection of Sienna+. This may be given between up to thirty minutes before
surgery. In centre's that also participate in the MRI subprotocol, patients
will undergo a pre-operative axillary MRI scan before and a after injection of
Sienna+. At the Medisch Spectrum Twente pre-operative axillary MRI scans will
be performed depending on the availibility of the MRI scanning slots.

Intra-operatively, patients will receive an intradermal injection of patent
blue (Guerbet, Paris). All sentinel nodes detected intra-operatively using
either the gamma probe or SentiMag; or demonstrating blue or black staining
will be excised. In the lead centre (and any other sites participating in the
MRI subprotocol), ex-vivo MRI scans of the excised nodes will be undertaken
using a high-resolution MRI scanner. In Enschede the ex-vivo MRI scans will be
undertaken at the University of Twente using the 14.1T MRI scanner.
Furthermore, at the University of Twente in Enschede the amount of iron in each
excised node will be measured using a quantitative magnetometer.

All lymph nodes will be assessed histologically and the nodal status will be
related back to the SLNB detection rate with each technique.

Patients will be followed up post-operatively (7-14 days after surgery) to
assess if staining occurs or for any other adverse event. If staining is
present, photographs will be taken. Further follow-up is at 3 months and at 1
year. Patients will be followed up for a total of 5 years, in accordance with
current local policies.

Following completion, a selection of centres that achieved at least a 92%
sentinel node detection (per patient), will be invited to proceed with the
phase III randomised controlled trial.

The Medisch Spectrum Twente will participate in the MRI subprotocol. As part of
the MRI-subprotocol, patients will undergo an MRI scan of the axilla prior to
the scheduled operation. The scan will take roughly 60 minutes and involves
lying down in a tubular scanner. Once positioned in the scanner, an initial
scan will be undertaken. Following the initial scan, an subcutaneous
periareolar injection of 2ml Sienna+ is given into the breast and a second scan
is performed. The scan may need to be repeated at 2 hours or at 24 hours after
the initial scan but further injections will not be required.

The operation will be performed as already planned by the surgeon. In addition
to the normal blue dye and radioactive injection, an additionial injection of
Sienna+ is administered into the breast close to the tumour or nipple (an
additional Sienna+ injection is only required if the MRI was undertaken over
24hours prior to surgery). During surgery, the sentinel nodes will be detected
with the normal detector (gamma probe), the magnetometer (SentiMag) and
visually (blue and black-brown colour). The detected nodes will be excised and
are taken to the University of Twente for ex-vivo MRI scanning and quantitative
magnetometer measurements. Ex-vivo means that the nodes are already outside
your body at the moment of scanning, so the patient is not involved in this
procedure. After the ex-vivo MRI, the lymph nodes are send to the
Histopathology Laboratory for analysis. The analysis in the laboratory is
performed routinely and does not form part of this study.

As already mentioned in section E9, there is the risk of tattooing of the skin
following injection of Sienna+. However, this is a prevalent problem with the
use of blue dye and not with the use of a magnetic tracer. In the previous
SentiMag studies (section 1.3 and section 1.4 in protocol), we used a magnetic
dye that is similar to Sienna+, called Endorem. Injection of Endorem resulted
in minimal skin discolouration in only 6 out of 51 patients.

A potential risk might be the chance of developing adverse reactions to
Sienna+. However, the available evidence on the use of similar materials to
Sienna+ ( for example Resovist, when only less than 1% of patients developed
adverse reactions to intravenous injection of the dye in much higher
concentrations ) and the CE marking of Sienna+ show that there are no
additional risks to participants other than the discolouration / staining noted
previously with Endorem.

Sienna + has been reviewed and tested as specified in EN 10993-1:2009 based on
the specified site of injection and showed no serious reaction after injection.