Download Intraoperative tumor-specific fluorescence imaging in ovarian cancer

Technical Reports
Intraoperative tumor-specific fluorescence imaging
in ovarian cancer by folate receptor-α targeting:
first in-human results
© 2011 Nature America, Inc. All rights reserved.
Gooitzen M van Dam1, George Themelis2, Lucia M A Crane1, Niels J Harlaar1,2, Rick G Pleijhuis1,
Wendy Kelder1, Athanasios Sarantopoulos2, Johannes S de Jong1, Henriette J G Arts3, Ate G J van der Zee3,
Joost Bart4, Philip S Low5 & Vasilis Ntziachristos2
The prognosis in advanced-stage ovarian cancer remains poor.
Tumor-specific intraoperative fluorescence imaging may
improve staging and debulking efforts in cytoreductive surgery
and thereby improve prognosis. The overexpression of folate
receptor-a (FR-a) in 90–95% of epithelial ovarian cancers
prompted the investigation of intraoperative tumor-specific
fluorescence imaging in ovarian cancer surgery using an
FR-a–targeted fluorescent agent. In patients with ovarian
cancer, intraoperative tumor-specific fluorescence imaging with
an FR-a–targeted fluorescent agent showcased the potential
applications in patients with ovarian cancer for improved
intraoperative staging and more radical cytoreductive surgery.
Of all gynecologic malignancies, epithelial ovarian cancer (EOC) is
the most frequent cause of death, both in the United States1 and in
Europe2. The relative absence of a clear, distinctive clinical presentation in early stages, combined with the lack of a screening tool, often
results in the disease being diagnosed only at more advanced stages.
The overall 5-year survival rate is 45%3, and for stages III and IV it is
only 20–25%4,5. Currently, cytoreductive surgery followed by combination chemotherapy is regarded as the most effective treatment. The
degree of cytoreduction, in which minimal residual disease is defined
as tumor deposits <1 cm, is one of the few prognostic factors that can
be actively influenced by the surgeon. Improved staging and possibly
survival could be achieved by better cytoreduction aided by an intraoperative, tumor-specific detection strategy that could assist the surgeon
by providing real-time feedback on residual malignant tissue.
Radiologic approaches such as X-ray, CT, MRI and ultrasound
have been considered for use in assisting surgical procedures, but
these are not tumor specific and generally are not useful for intraoperative applications. In contrast, fluorescence imaging, as an
optical technique, relates naturally to surgical inspection and practice, and it offers superior resolution and sensitivity compared to
preoperative radiological imaging and to visual inspection and palpation during surgery. The combination of accurate real-time imaging
with tumor-specific fluorescent agents can shift the paradigm of surgical inspection by enabling localization of lesions that are difficult
or impossible to detect by visual observation or palpation, possibly
leading to up-staging of patients due to improved detection of tumor
tissue and more radical excision of tumor tissue. In the last decade,
several groups have contributed to progress in the development of
optical imaging systems6 and tumor-specific fluorophores for clinical
applications7,8. Several preclinical9,10 and clinical11–13 studies have
demonstrated the potential use of image-guided surgery.
A promising target in EOC is FR-α. Recently, several studies on
the expression of the FR-α in ovarian cancer have revealed that it is
increased in 90–95% of patients with EOC14,15. Moreover, the absence
of FR-α on healthy cells leads to high tumor-to-normal ratios. As
such, FR-α is potentially a good target for both therapeutic8–10 and
imaging purposes16–18. As a ligand of FR-α, folate has already been
conjugated to DTPA for SPECT/CT imaging16 and to several PET
tracers19. It has also been linked to fluorescein for use in imaging
metastatic disease in murine tumor models18, although this was never
tested in humans. In this study, folate conjugated to fluorescein isothiocyanate (folate-FITC; Fig. 1) for targeting FR-α is used together
with a real-time multispectral intraoperative fluorescence imaging
system20. We report on the results of first-in-human use of intraoperative tumor-specific fluorescence imaging for real-time surgical
visualization of tumor tissue in patients undergoing an exploratory
laparotomy for suspected ovarian cancer.
RESULTS
Patient characteristics
The mean age of all patients was 61.2 ± 11.4 (mean ± s.d.). Four patients
were diagnosed with a malignant epithelial ovarian tumor (two serous
carcinomas, one undifferentiated carcinoma and one mucinous
1Department
of Surgery, Division of Surgical Oncology, BioOptical Imaging Center, University of Groningen, Groningen, The Netherlands. 2Technische Universität
München & Helmholtz Zentrum, München, Germany. 3Department of Gynaecology, Division of Gynaecological Oncology, University of Groningen, Groningen,
The Netherlands. 4Department of Pathology and Molecular Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
5Department of Chemistry, Purdue University, West Lafayette, Indiana, USA. Correspondence should be addressed to G.M.v.D. ([email protected])
or V.N. ([email protected]).
Received 15 October 2010; accepted 11 March 2011; published online 18 September 2011; doi:10.1038/nm.2472
nature medicine VOLUME 17 | NUMBER 10 | OCTOBER 2011
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Technical Reports
a
Folate receptor
b
FITC
Folate
© 2011 Nature America, Inc. All rights reserved.
Tumor cell
Figure 1 The tumor-specific fluorescent agent folate-FITC. (a) Folate
is conjugated through an ethylenediamine spacer to fluorescein
isothiocyanate (FITC), resulting in folate-FITC, with a molecular weight of
917 kDa. (b) A schematic presentation of the targeting of ovarian cancer.
Folate-FITC is targeted toward FR-α and internalized upon binding,
shuttling folate-FITC into the cytoplasm.
carcinoma) and one patient with a serous borderline tumor. Five
patients were diagnosed with a benign ovarian tumor, as confirmed
by histopathology: two fibrothecomas, one cellular fibroma, one cystic
teratoma and one benign multicystic ischemic ovary (Table 1).
positive), widespread tumor-specific fluorescence (white spots) was
present throughout the abdominal cavity (Fig. 2), as confirmed by
ex vivo histopathology. Real-time image-guided excision of fluorescent
tumor deposits of size <1 mm was feasible (Supplementary Fig. 1),
and all fluorescent tissue was confirmed to be malignant by histopathology. In the same patient, the tumor-specific fluorescent signal
originating from disseminated tumor deposits could be detected up
to 8 h after injection during a prolonged procedure.
Detection of tumor deposits by fluorescence ex vivo
Five surgeons independently identified tumor deposits on three separate color images (shown on a representative image in Fig. 3a) and
on their corresponding fluorescence image of precisely the same area
(Fig. 3b). The number of tumor deposits detected by surgeons when
guided by tumor-specific fluorescence (median 34, range 8–81) was
significantly higher than with visual observation alone (median 7,
range 4–22, P < 0.001) (Fig. 3c).
Post-operative histopathological analyses
All excised fluorescent tissue was again analyzed for tumor-specific
fluorescence ex vivo, in which tumor deposits could be visualized with
a resolution of approximately ≤1.0 mm (Supplementary Fig. 1a–f).
Representative examples of postoperative histopathological analyses
are depicted in Figure 4 for three different ovarian tumors (fibrothecoma, serous borderline tumor and high-grade serous carcinoma).
Routine histopathological examination using hematoxylin and eosin
(H&E) staining was performed to determine the nature of the excised
tissue (Fig. 4, top row). All fluorescent tissue samples were confirmed
to contain tumor, whereas nonfluorescent tissue was free of tumor.
Additionally, immunohistochemical (IHC) staining for FR-α revealed
strong expression in the malignant tumors, moderate expression in
the borderline tumor and no expression in the benign lesions (Fig. 4,
middle row). On one of the benign lesions there was considerable
inflammation, with increased number of macrophages. This patient
showed no fluorescence activity either in vivo or ex vivo by fluorescence microscopy (data not shown). Finally, fluorescence microscopy
for folate-FITC showed a strong signal in all malignant tumors with
FR-α expression and no signal in FR-α–negative malignant or benign
lesions (Fig. 4, bottom row). These results show excellent correlation
with the presence and intensity of the intraoperative fluorescence
signal (Table 1).
Intraoperative tumor-specific fluorescence imaging
Use of the intraoperative imaging system did not interfere with the
standard surgical procedure (Supplementary Video 1). The mean
duration for capturing in vivo intraoperative
fluorescence images and video was 10 min
Table 1 Demographics and individual data for patients
(range: 4–36 min).
Age
In vivo
IHC FR-α
Fluorescence was detectable intraopera- Study no.
(years)
Histopathology
FIGO stage fluorescence expression FM FITC
tively in all patients with a malignant tumor Malignant tumor
and FR-α expression but was absent in the 1
72
Serous ovarian carcinoma
III
++
++
++
patient with a malignant tumor but no FR-α 7
76
Serous ovarian carcinoma
III
+
+
+
expression and in those with benign tumors 9
64
Undifferentiated carcinoma
III
–
–
–
(Table 1). Healthy tissue did not show any 10
61
Mucineus ovarian carcinoma
III
+
+
+
fluorescence signal either in vivo, ex vivo Borderline tumor
48
Serous borderline tumor
I
0
+
0/+
or on histopathological validation. In two 5
separate still images of patients with ovar- Benign tumor
59
Fibrothecoma
n.a.
–
–
–
ian cancer, the mean tumor-to-background 2
3
74
Fibrothecoma
n.a.
–
–
–
ratio (as compared to healthy peritoneal sur53
Mature cystic teratoma
n.a.
–
–
–
face) for ten demarcated fluorescent tumor 4
64
Benign multicystic ischemic ovary
n.a.
–
–
–
deposits in each still image was 3.1 (± 0.8 s.d.) 6
8
41
Fibroma
n.a.
–
–
–
(data not shown). In the patient with a highn = 10 patients. ++, strong; +, moderate; 0, weak; −, absent; FIGO, International Federation of Gynecology and
grade serous carcinoma and extensive peri- Obstetrics; IHC FR-α, immunohistochemistry folate-receptor alpha; FM FITC, fluorescence microscopy for folate-FITC;
toneal disseminated disease (stage III, FR-α n.a., not applicable.
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VOLUME 17 | NUMBER 10 | OCTOBER 2011 nature medicine
Technical Reports
Figure 2 Intra-operative multispectral imaging
system. (a) Multispectral fluorescence camera
system. (b) Positioning before draping. (c)
Intraoperative application including draping.
(d–g) Intraoperative screenshots of
simultaneously detected and depicted images in
color (d,f) and corresponding fluorescence during
surgery (e,g) in a patient with high-grade serous
ovarian carcinoma and extensive peritoneal
carcinomatosis (stage III, FR-α positive).
a
CCD 1
Imaging channel 1
Relay lens
Imaging
channel 2 CCD 2
b
Number of tumor spots detected
Median
Minimum
Maximum
80
60
© 2011 Nature America, Inc. All rights reserved.
d
e
f
g
Filter
Dichroic mirrors
Imaging
CCD 3
channel 3
DISCUSSION
Ovarian cancer, known as the ‘silent lady
killer’, is the leading cause of death among
Beam diffuser
gynecologic malignancies in the Western
world. In this proof-of-concept study, we
investigated the potential value of intraoperative tumor-specific fluorescence imaging in the detection
of tumor tissue in ovarian cancer for improvement of optimal
cytoreductive surgery.
In this limited series, we showed that the use of intraoperative
tumor-specific fluorescence imaging of the systemically administered FR-α–targeted agent folate-FITC offers specific and sensitive
real-time identification of tumor tissue during surgery in patients
with ovarian cancer and the presence of FR-α–positive tumors.
Nevertheless, one patient presented with a malignant tumor that did
not express FR-α, and consequently, no fluorescence was detected.
To identify patients suitable for FR-α targeted image-guided surgery,
FR-α may be detected on either tumor cells in ascites or on tumor
tissue obtained during staging laparoscopy or primary surgery.
A folate-targeted technetium scan (EC20, Endocyte Inc.) may be also
used to preoperatively identify FR-α–positive patients21.
In a patient with extensive peritoneal carcinomatosis, a significantly
higher number of fluorescent tumor deposits were detected using the
fluorescence imaging approach than with conventional visual inspection. Although the scoring was not accompanied by tactile information, we feel confident in comparing the two imaging methods
(visual observation versus fluorescence imaging) alone, instead of
adding tactile information, for which a gold standard is lacking.
c
c
Filter
Laser
a
b
P < 0.001
Imaging
optics
White
light
source
Filter
Although tactile information is considered by many surgeons as an
important feature in staging, clear data is lacking in the literature on
the exact sensitivity, specificity and diagnostic accuracy of palpation
in cancer surgery.
In the laparoscopic staging setting, enhanced tumor detection with
tumor-targeted fluorescence may lead to up-staging. Furthermore,
in interval debulking surgeries, vital tumor tissue can be difficult to
distinguish owing to fibrosis and necrosis caused by chemotherapy.
A tumor-targeted agent may help identify vital residual tumor tissue
in the interval debulking setting. As a result of the increasing application of neo-adjuvant chemotherapy in patients with ovarian cancer
and the possible effects of chemotherapy on the expression levels of
FR-α in remaining vital tumor tissue present at interval debulking, it
remains of paramount importance to investigate the presence of the
target. In a tissue microarray study of more than 350 patients at the
University Medical Center Groningen, we found that FR-α expression in remaining vital tumor tissue is not significantly altered after
chemotherapy22 (Crane et al. Cell. Oncol. 2011, in press), which is in
accordance with results reported previously14.
In this pilot study, folate-FITC in an intravenously injected formulation appeared to be safe. Furthermore, it has a pharmacodynamic
profile that facilitates fluorescence imaging over the time course from
2 to 8 h after injection. This systemic administration of a targeted
fluorescent agent in humans provides a versatile platform for intraoperative tumor-specific fluorescence imaging.
The use of targeted fluorescent agents could provide a paradigm
shift in surgical imaging as it allows an engineered approach to
improving tumor staging and the technique of cytoreductive surgery and thereby improving the outcome in ovarian cancer. A major
advantage over current imaging modalities is that an intraoperative
fluorescence imaging system offers a large field of view for inspection
and staging. This, in turn, may permit future patient-tailored ­surgical
interventions and may decrease the number of needless extensive
surgical procedures and the associated morbidity. Besides improved
staging, primarily expected for subjects initially classified in stages I
and II, the second major advantage of intraoperative imaging as
40
20
0
Color
FLI
nature medicine VOLUME 17 | NUMBER 10 | OCTOBER 2011
Figure 3 Quantification of tumor deposits ex vivo. (a,b) Color image
(a) with the corresponding tumor-specific fluorescence image (b) of
a representative area in the abdominal cavity. (c) Scoring was based
on three different color images (median 7, range 4–22) and their
corresponding fluorescence images (FLI) (median 34, range 8–81);
P < 0.001 by five independent surgeons.
1317
Technical Reports
a
b
c
H&E
5x
5x
5x
10x
20x
20x
IHC
This technique did not create unwanted interference with standard
surgical procedures. The combination of optical imaging technologies with tumor-targeting strategies can shift the paradigm of
surgical oncologic imaging, offering the unique opportunity to intraoperatively detect and quantify tumor growth and intra-abdominal
spread. Larger international multicenter studies using standardized,
uniformly calibrated multispectral fluorescence camera systems
combined with folate-FITC are needed to confirm our data and
further elucidate the diagnostic (accuracy, sensitivity and specificity)
and therapeutic value of the reported approach in larger series of
ovarian cancer patients.
Methods
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/naturemedicine/.
FM
10x
20x
20x
© 2011 Nature America, Inc. All rights reserved.
Note: Supplementary information is available on the Nature Medicine website.
Figure 4 Microphotographs of different ovarian tumors. (a–c) Three
different tumor types are shown: fibrothecoma (a), borderline serous
tumor (b) and high-grade serous carcinoma (c). Top row, routine staining
with H&E; middle row, immunohistochemical staining (IHC) for FR-α;
lower row, unstained slides observed with fluorescent microscopy (FM)
to detect the intravenously administered folate-FITC. The fibrothecoma
(a) shows no expression of FR-α nor binding of folate-FITC, which
corresponds with the absence of lesions visualized intraoperatively using
the fluorescence camera system. Both the borderline serous tumor (b) and
the high-grade serous tumor (c) show epithelial expression of FR-α and
binding of folate-FITC, which corresponds with the presence of visible
lesions intraoperatively.
compared to current standard techniques is that it may guide the
surgeon in debulking efforts, thus contributing to more efficient
cytoreduction and ultimately improving the effect of adjuvant chemotherapy in patients with reduced tumor load. Because cytoreduction is
a key aspect in the prognosis of many solid tumors with a peritoneal
dissemination pattern—including ovarian cancer23,24 and colorectal cancer combined, in the so-called hyperthermic intraperitoneal
chemotherapy (HIPEC) procedure25—a more complete debulking
procedure may have a beneficial effect on outcome in different types
of cancer. Improving the detection of cancer deposits to submillimeter
size might ultimately improve survival rates, but whether this is the
case needs to established by additional clinical studies.
In ovarian cancer, the FR-α appears to constitute a good target
because it is overexpressed in 90–95% of malignant tumors, especially
serous carcinomas. Furthermore, the targeting ligand, folate, is attractive as it is nontoxic, inexpensive and relatively easily conjugated to a
fluorescent dye to create a tumor-specific fluorescent contrast agent26.
However, overexpression of FR-α varies strongly between different solid
tumors originating from different organs27, a characteristic that reduces
the general applicability of folate-FITC in cancer. Future developments
in the identification of tumor-specific targets will undoubtedly bridge
this gap and open new possibilities for tumor-specific fluorescence
imaging in cancer surgery for other solid tumors. Subsequently, development of new fluorescent agents in the near-infrared spectrum will
allow for identification of more deeply seated tumors, based on the
stronger penetration properties of near-infrared dyes with an excitation
wavelength >700 nm compared to FITC28.
In summary, our data outline the first in-human proof-of-principle
and the potential benefit of intraoperative tumor-specific ­fluorescence
imaging in staging and debulking surgery for ovarian cancer using
the systemically administered targeted fluorescent agent folate-FITC.
1318
Acknowledgments
The authors wish to thank W. Sluiter for help with statistical analyses, I. Wesselman
for training the operating room personnel in using the camera system and
B. Molmans (Department of Hospital Pharmacy, University Medical Center
Groningen) for pharmaceutical expertise and support. Monoclonal antibody
mAb343 was kindly provided by W. Franklin (University of Colorado Health
Science Center).
Author Contributions
G.M.v.D. supervised the project, designed and performed the study, interpreted
data and wrote the manuscript. G.T. designed the camera system, performed
the study, interpreted data with technical and computer analyses support and
wrote the manuscript. L.M.A.C. designed and performed the study, interpreted
data and wrote the manuscript. N.J.H. performed the study and interpreted
data. R.G.P. performed the study and interpreted data. W.K. performed the
study and interpreted data. A.S. designed the camera system, performed the
study and interpreted data with technical and computer analysis support. J.S.d.J.
performed the study and interpreted data. H.J.G.A. designed and performed
the study, interpreted data and wrote the manuscript. A.G.J.v.d.Z. designed
and performed the study, interpreted data and wrote the manuscript.
J.B. designed and performed the study, interpreted data, performed the
pathology analyses and wrote the manuscript. P.S.L. designed and performed
the study, and interpreted data. V.N. supervised the project, designed and
performed the study, designed and provided the camera system and interpreted
data, and wrote the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturemedicine/.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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nature medicine VOLUME 17 | NUMBER 10 | OCTOBER 2011
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ONLINE METHODS
© 2011 Nature America, Inc. All rights reserved.
Patients. The study was approved by the Investigational Review Board (IRB)
of the University Medical Center Groningen and the Central Committee on
Research Involving Human Subjects (CCMO NL26980.042.09; http://www.
ccmo-online.nl/main.asp) and was registered at the Dutch Trial Register
(NTR1980; http://www.trialregister.nl/trialreg/index.asp) and the European
Clinical Trials Database (EudraCT 2009-010559-29; http://eudract.emea.
europa.eu/) before inclusion. All patients gave their written informed consent
and completed the study.
Between September 2009 and May 2010, patients scheduled to undergo a
staging or debulking laparotomy for suspected ovarian cancer—based on an
ovarian mass and/or the presence of ascites as diagnosed by ultrasound or CT
scan, and/or elevated serum concentration of the CA-125 tumor marker—were
consented for the study. Exclusion criteria were pregnancy, significant renal
failure (creatinine >400 µmol per liter), severe cardiac or pulmonary disease
(ASA III-IV), a history of iodine allergy or anaphylactic reactions to insect
bites or medication, and the presence or history of hyperthyroidism. After
documented informed consent, the operative procedure was planned. Patients
were not limited in their normal behavior or dietary or medication intake
before the study. During surgery, patients were staged according to FIGO
standard guidelines29 via a midline laparotomy.
Tumor-specific fluorescent agent. For targeting of the FR-α in ovarian cancer
in patients, the imaging agent was produced at clinical grade according to GMP
conditions by Endocyte Inc. Folate hapten (vitamin B9) was conjugated with
fluorescein isothiocyanate (FITC), yielding folate-FITC (Fig. 1a), as described
previously by others30,31. Folate-FITC has an excitation wavelength of 495 nm
and emits light at 520 nm. The agent specifically targets FR-α, after which it is
internalized into the cytoplasm (Fig. 1b).
All vials of folate-FITC (produced as EC17) were supplied by Endocyte Inc.
after approval of the Investigational Medicinal Product Dossier documentation according to US Food and Drug Administration (FDA) and European
Medicines Agency (EMEA) regulations by the local IRB and Hospital
Pharmacy at the University Medical Center Groningen. All imaging agents
were stored, recorded and monitored before release by the Hospital Pharmacy
according to FDA/EMEA and Good Clinical Practice (GCP) guidelines. After
intravenous injection of folate-FITC, all patients were monitored during inhospital admission. Folate-FITC was dissolved in 10 ml sterile normal saline
and injected at a dose of 0.3 mg per kg body weight over a period of 10 min.
Four patients experienced mild discomfort in the upper abdominal region after
injection of ~0.1 mg per kg folate-FITC, which disappeared within 10–15 min
but which prompted cessation of the remaining dose according to protocol.
All patients completed the entire study. No serious adverse events (SAEs) were
reported during or after the study.
Multispectral fluorescence camera system. The camera system was developed by the Technical University Munich/Helmholtz Center Munich (by G.T.,
A.S. and V.N.)20,32. In summary (Fig. 2a), the camera system consists of a
nature medicine
charge-coupled digital (EM-CCD) camera (Andor Technology) for sensitive
fluorescence detection and two separate cameras for detection of intrinsic
fluorescence and color (PCO AG). The system is controlled by a synchronized multi-CPU computer system (Dell Computer) for simultaneous processing of raw data and image registration and rendering. During surgery, the
camera was covered with sterile transparent drapes (Carl Zeiss Vision BV,
OPMI Sterile Drape, cat. no. 306071) (Fig. 2b). The system attains a variable
field of view (FOV) of 15 cm × 15 cm to 3 cm × 3 cm, with a corresponding
resolution varying from 150 to 30 µm. Data are acquired in parallel by all
cameras so that color, light attenuation images and fluorescence images are
simultaneously collected. Attention has been given to the development of an
appropriate multispectral strategy that allows color imaging and simultaneous
sensitive fluorescence detection in the visible light spectrum, as appropriate
for FITC imaging. Besides training of the operating room personnel to
cover the camera in sterile draping, no special training is necessary to use the
camera intraoperatively.
Surgical and imaging procedure. In all patients, a standardized imaging
protocol was used before, during and after the surgical procedure. On opening
the abdominal cavity through a midline incision, the surgeon localized the
tumor and inspected and palpated the abdominal and pelvic regions according
to FIGO guidelines28. At this point, fluorescence video inspection at maximum FOV determined the presence of fluorescence activity, and multispectral
images were captured (Fig. 2c,d,f). Next, the surgeon proceeded with the
standard surgical procedure. All tissue suspicious for tumor involvement was
imaged both in vivo, and ex vivo immediately after tissue excision, for the
presence of fluorescence. Thereafter, the surgical field was inspected with the
fluorescence system for remaining fluorescence, and additional multispectral
images were captured. Biopsies were taken of residual fluorescent tissue as
part of the standard operative procedure. For ethical reasons (i.e., the risk of
removing false-positive fluorescent (healthy) tissue situated in the vicinity of
vital organs or structures), it was decided to remove fluorescent tissue only
if it did not impose an additive negative effect on morbidity as judged by the
operating surgeon.
Additional methods. Detailed methodology is described in the Supplementary
Methods.
29.Benedet, J.L., Bender, H., Jones, H. III, Ngan, H.Y. & Pecorelli, S. FIGO staging
classifications and clinical practice guidelines in the management of gynecologic
cancers. FIGO Committee on Gynecologic Oncology. Int. J. Gynaecol. Obstet. 70,
209–262 (2000).
30.Lu, Y. & Low, P.S. Folate targeting of haptens to cancer cell surfaces mediates
immunotherapy of syngeneic murine tumors. Cancer Immunol. Immunother. 51,
153–162 (2002).
31.Lu, Y. et al. Strategy to prevent drug-related hypersensitivity in folate-targeted
hapten immunotherapy of cancer. AAPS J. 11, 628–638 (2009).
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doi:10.1038/nm.2472