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Author, Editor: Tilda Barliya PhD

Surgery is being commonly used to diagnose, treat and even help  prevent cancer. In which the surgeon will cut into the body to remove the cancer along with some surrounding healthy tissue to ensure that all of the cancer is removed. However distinguishing cancer cells from healthy ones during surgery can prove difficult, if not impossible. Sometimes lesions are detected only postoperatively, leading to more surgery down the line. Currently, surgeons rely on vision and touch to detect tumors during surgery but in many cases there is still no good way to determine a tumor’s margins.

In recent years, major progression has been made in imaging-guided surgery and doctors believe that  use of fluorescent dye could boost survival rates by guiding them to tiny clusters of malignant cells.

The first fluorescence-guided surgery in ovarian cancer patients have yielded great results and are summarized herein.

Dr. Phillip Low, a Ralph C. Corely Distinguished Professor of Chemistry from Purdue University has invented a fluorescent imaging agent to a modified form of the vitamin folic acid, which acts as a “homing device” to seek out and attach to ovarian cancer cells (1)

” Of all gynecologic malignancies, epithelial ovarian cancer (EOC) is the most frequent cause of death, both in the United States and in Europe. 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%, and for stages III and IV it is only 20–25%.” Cytoreduction surgery followed by chemotherapy is considereed the most effective treatment.  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. Therefore, a better tumor-specific detection strategy may drastically improve the patient survival.

The overexpression of folate receptor-α (FR-α) in 90–95% of epithelial ovarian cancers prompted the investigation of intraoperative tumor-specific fluorescence imaging in ovarian cancer surgery using an FR-α–targeted fluorescent agent.  Moreover, the absence of FR-α on healthy cells leads to high tumor-to-normal ratios.

Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-α targeting (http://spie.org/newsroom/technical-articles-archive/4003-shifting-the-paradigm-in-surgical-vision-with-fluorescence-molecular-imaging)

As a ligand of FR-α, folate has already been conjugated to DTPA for SPECT/CT imaging and to several PET tracers. It has also been linked to fluorescein for use in imaging metastatic disease in murine tumor models, although this was never tested in humans.

In this article, the authors have conjugated the folate to fluorescein isothiocyanate (FITC) for the use in surgery together with a real-time multispectral intraoperative fluorescence imaging system.

The authors have conducted the first clinical trial using the fluorescence-guided surgery in ovarian cancer patient. Described herein:

Tumor-specific fluorescent agent:

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 (See Fitgure). Folate-FITC has an excitation wavelength of 495 nm and emits light at 520 nm. The conjugate has a very high sensitivity and  clusters of cancer cells as small as one-tenth of a millimeter can be detected, as opposed to the earlier average minimal cluster size of 3 millimeters in diameter based on current methods of visual and tactile detection.

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 and was injected 2 hrs prior to the surgery.

Patients:

10 patients with different stages of the over cancer were recruited, 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 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.

Multispectral fluorescence camera system:

The camera system (developed by the Technical University Munich/Helmholtz Center) consists of a 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. The system allows color imaging and simultaneous sensitive fluorescence detection in the visible light spectrum, as appropriate for FITC imaging.  Surgery and imaging procedure are described in detail in the article (1). Shortly, a live imaging during surgery enabled the surgeon to locate the tumor and remove it, biopsy was taken for further histopathology.

Results:

Fluorescence was detectable intraoperatively in all patients with a malignant tumor and FR-αexpression but was absent in the patient with a malignant tumor but no FR-α expression and in those with benign tumors (Table 1)

Table 1: Demographics an  individual data for patients

Healthy tissue did not show any fluorescence signal either in vivo, ex vivo or on histopathological validation. In two separate still images of patients with ovarian cancer, the mean tumor-to-background ratio (as compared to healthy peritoneal surface) for ten demarcated fluorescent tumor deposits in each still image was 3.1 (± 0.8 s.d.). In the patient with a high-grade serous carcinoma and extensive peritoneal disseminated disease (stage III, FR-α positive), widespread tumor-specific fluorescence (white spots) was present throughout the abdominal cavity, as confirmed by ex vivo histopathology. Real-time image-guided excision of fluorescent tumor deposits of size <1 mm was feasible.

A video of the surgery is presented herein:

http://www.purdue.edu/newsroom/research/2011/110918LowSurgery.html

Detection of Tumor Deposits:

Five surgeons independently identified tumor deposits on three separate color images (shown on a representative image in (Left) and on their corresponding fluorescence image of precisely the same area (Right).

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).

Summary:

In this limited series, the authored 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.

• 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.
• The second major advantage of intraoperative imaging as 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
• 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.

Advantages:

• 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.
  
• 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 agent.

Disadvantages:

• Overexpression of FR-α varies strongly between different solid tumors originating from different organs, a characteristic that reduces the general applicability of folate-FITC in cancer.
  
• Many organs have autofluorescence in the excitation and emission parameters of the FITC dye.

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 FITC.

This is 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. 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.

Note:  Other similar approaches have been explored for  brain tumors (3a, 3b) in human clinical trials using 5-aminolevulinic acid (5-ALA). We will not address this trial in this discussion.

Ref:

1. Gooitzen M van Dam, George Themelis, Lucia M A Crane, Niels J Harlaar, Rick G Pleijhuis, Wendy Kelder, Athanasios Sarantopoulos, Johannes S de Jong, Henriette J G Arts, Ate G J van der Zee, Joost Bart, Philip S Low & Vasilis Ntziachristos. Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-αtargeting: first in-human results. Nature Medicine 17, 1315–1319 (2011). http://www.nature.com.rproxy.tau.ac.il/nm/journal/v17/n10/full/nm.2472.html

http://www.purdue.edu/newsroom/research/2011/110918LowSurgery.html

Video: http://www.youtube.com/watch?v=cPlRP0qrxts

http://www.guardian.co.uk/science/2011/sep/18/ovarian-cancer-fluorescence-detection

2. Lung cancer: http://emoryhealthmagazine.emory.edu/issues/2012/winter/briefs/a-yellow-brick-path/index.html

3a. Stummer W, Pichlmeier U, Meinel T, Wiestler OD, Zanella F, Reulen HJ; ALA-Glioma Study group. Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial. Lancet Oncol  2006 May;7(5):392-401.

3b. Clinical trial set up: http://clinicaltrials.gov/show/NCT01502280