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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 1315 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. 1316 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. 1. Jemal, A. et al. Cancer statistics, 2008. CA Cancer J. Clin. 58, 71–96 (2008). 2. Gondos, A., Bray, F., Hakulinen, T. & Brenner, H. Trends in cancer survival in 11 European populations from 1990 to 2009: a model-based analysis. Ann. Oncol. 20, 564–573 (2009). 3. Aletti, G.D., Gallenberg, M.M., Cliby, W.A., Jatoi, A. & Hartmann, L.C. Current management strategies for ovarian cancer. Mayo Clin. Proc. 82, 751–770 (2007). 4. Winter, W.E. III et al. Tumor residual after surgical cytoreduction in prediction of clinical outcome in stage IV epithelial ovarian cancer: a Gynecologic Oncology Group Study. J. Clin. Oncol. 26, 83–89 (2008). 5. Ibeanu, O.A. & Bristow, R.E. Predicting the outcome of cytoreductive surgery for advanced ovarian cancer: a review. Int. J. Gynecol. Cancer 20 (suppl. 1), S1–S11 (2010). 6. Troyan, S.L. et al. The FLARE intraoperative near-infrared fluorescence imaging system: a first-in-human clinical trial in breast cancer sentinel lymph node mapping. Ann. Surg. Oncol. 16, 2943–2952 (2009). 7. Luker, G.D. & Luker, K.E. Optical imaging: current applications and future directions. J. Nucl. 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Timing of debulking surgery in advanced ovarian cancer. Int. J. Gynecol. Cancer 18 (suppl. 1), 11–19 (2008). 24.Aletti, G.D. et al. Aggressive surgical effort and improved survival in advanced-stage ovarian cancer. Obstet. Gynecol. 107, 77–85 (2006). 25.Koppe, M.J., Boerman, O.C., Oyen, W.J. & Bleichrodt, R.P. Peritoneal carcinomatosis of colorectal origin: incidence and current treatment strategies. Ann. Surg. 243, 212–222 (2006). 26.Low, P.S. & Antony, A.C. Folate receptor-targeted drugs for cancer and inflammatory diseases. Adv. Drug Deliv. Rev. 56, 1055–1058 (2004). 27.Parker, N. et al. Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Anal. Biochem. 338, 284–293 (2005). 28.Troy, T., Jekic-McMullen, D., Sambucetti, L. & Rice, B. Quantitative comparison of the sensitivity of detection of fluorescent and bioluminescent reporters in animal models. Mol. Imaging 3, 9–23 (2004). © 2011 Nature America, Inc. All rights reserved. 8. Tromberg, B.J. et al. Assessing the future of diffuse optical imaging technologies for breast cancer management. Med. Phys. 35, 2443–2451 (2008). 9. Kirsch, D.G. et al. A spatially and temporally restricted mouse model of soft tissue sarcoma. Nat. Med. 13, 992–997 (2007). 10.von Burstin, J. et al. Highly sensitive detection of early-stage pancreatic cancer by multimodal near-infrared molecular imaging in living mice. Int. J. Cancer 123, 2138–2147 (2008). 11.Sevick-Muraca, E.M. et al. Imaging of lymph flow in breast cancer patients after microdose administration of a near-infrared fluorophore: feasibility study. Radiology 246, 734–741 (2008). 12.Tagaya, N. et al. Intraoperative identification of sentinel lymph nodes by nearinfrared fluorescence imaging in patients with breast cancer. Am. J. Surg. 195, 850–853 (2008). 13.Stummer, W. et al. Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial. Lancet Oncol. 7, 392–401 (2006). 14.Kalli, K.R. et al. Folate receptor alpha as a tumor target in epithelial ovarian cancer. Gynecol. Oncol. 108, 619–626 (2008). 15.Markert, S. et al. Alpha-folate receptor expression in epithelial ovarian carcinoma and non-neoplastic ovarian tissue. Anticancer Res. 28, 3567–3572 (2008). 16.Mathias, C.J. et al. Indium-111-DTPA-folate as a potential folate-receptor-targeted radiopharmaceutical. J. Nucl. Med. 39, 1579–1585 (1998). 17.Armstrong, D.K. et al. Exploratory phase II efficacy study of MORAb-003, a monoclonal antibody against folate receptor alpha, in platinum-sensitive ovarian cancer in first relapse. J. Clin. Oncol. 26, 5500 (2008). nature medicine VOLUME 17 | NUMBER 10 | OCTOBER 2011 1319 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). 32.Themelis, G., Yoo, J.S. & Ntziachristos, V. Multispectral imaging using multiplebandpass filters. Opt. Lett. 33, 1023–1025 (2008). doi:10.1038/nm.2472