In the United States, the overall 5-year survival rate for patients with squamous carcinoma of the oral cavity is only 54%, 1 of the lowest rates of all major cancers; in developing countries, survival rates drop below 30%.1,2
Patients with early lesions have better chances for cure and less treatment-associated morbidity, yet despite the easy accessibility of the mouth, most patients present with advanced tumors, when treatment is more difficult, more expensive, and less successful compared to earlier interventions.3
Improved screening and early diagnosis of oral cancer could benefit the global population substantially.
Optical imaging technologies have the potential to improve early detection of oral neoplasia by noninvasively assessing the morphologic and biochemical changes associated with precancer and cancer in real time at the point-of-care.4–6
Driven by advances in consumer electronics, high quality optical images can now be obtained with low-cost devices; tandem advances in digital signal processing provide the ability to automate image analysis. These advances have the potential to improve screening in settings ranging from tertiary care centers to regions with limited personnel and infrastructure.
Enhanced imaging of broad areas of oral mucosa should enable clinicians to determine optimal sites for pinpoint probe interrogation and/or biopsy, reducing the chance of sampling error. Normally, clinicians observe reflected white light because this is the dominant light–tissue interaction and the intensity of reflected light far exceeds the intensity of induced fluorescence. However, it is also possible to directly observe tissue autofluorescence. Several groups have demonstrated that imaging systems that record the spatial distribution of fluorescence intensity at specific excitation/emission wavelength combinations can be used to survey large areas of oral cavity mucosa for neoplastic changes. A commercially available device, the VELscope (LED Dental, Inc., Burnaby, BC, Canada), uses a metal–halide lamp with emission peaks at 405 and 436 nm to excite autofluorescence; images indicate a characteristic loss of fluorescence associated with neoplasia.7,8
Results from 50 biopsies taken from areas with loss of fluorescence in 44 patients showed a sensitivity of 98% and specificity of 100% for discriminating normal tissue from severe dysplasia, carcinoma in situ, or invasive carcinoma, using histology as the gold standard. An important finding was the ability of fluorescence visualization to aid clinicians to see early neoplastic lesions that were initially missed during traditional white light examination.9
Additional results indicated that this device enhanced the ability of surgeons to visualize the peripheral extension of histologic and molecular abnormalities around neoplastic lesions to facilitate more accurate determination of resection margins.10
Although wide-field autofluorescence visualization can aid in the detection of early neoplastic changes, there is increasing concern that benign changes, such as inflammation, may also exhibit loss of fluorescence and may reduce specificity, especially in low-prevalence populations.11
High-resolution optical imaging can be used to directly visualize changes in epithelial morphology in suspicious regions of tissue, and can be used to complement such wide-field systems by distinguishing neoplastic from benign processes in regions with abnormal autofluorescence.12,13
A number of studies have explored the use of high-resolution optical imaging for improved detection of oral neoplasia.14,15
Reflectance confocal microscopy has been used to image changes in cell and nuclear morphology, nuclear-to-cytoplasmic ratio, and epithelial architecture associated with early oral neoplasia.16
A fiber-optic reflectance confocal microscope, comprising a single optical fiber and a resonating tuning fork at the distal tip, has been integrated into an endomicroscope platform to enable high-resolution fluorescence imaging of suspicious lesions in the oral cavity.17
Fiber-optic systems have also been developed to perform in vivo confocal fluorescence microscopy imaging. One such device, the Cellvizio (Mauna Kea Technologies, Paris, France) uses a galvanometric scanner to raster scan laser light across the proximal tip of a coherent fiber bundle to enable confocal fluorescence imaging of tissue at the distal tip of the device. This technique has been used successfully to image gastrointestinal and respiratory epithelium and other tissues in vivo.18,19
Although these fiber-optic confocal microscopes can obtain high-resolution images of tissue in real time, they are technically complex and expensive.
An alternative to confocal microscopy is high-resolution microendoscopy.20,21
The high-resolution microendoscope (HRME) uses a coherent fiber bundle to obtain high-resolution fluorescence images of the tissue in contact with the distal tip of the device without the need for complex mechanical scanning systems and associated control electronics.22,23
The system uses a low-cost light-emitting diode to provide illumination and a consumer-grade charge coupled device camera to capture high-resolution digital images on a laptop computer.
A topically applied fluorescent dye, proflavine (Sigma–Aldrich, St. Louis, MO), is used to preferentially stain cell nuclei. Proflavine is an acridine-derived dye which binds to DNA in a reversible and non-covalent manner.24,25
Proflavine has been safely used for years as 1 of the main components of triple dye, applied to the umbilicus of newborns to prevent infection.26
In addition, a number of in vivo imaging studies have been performed using topically delivered proflavine as a contrast agent.27,28
Proflavine is the principal component of acriflavine and has been used for fluorescence imaging in the European, Asian, and Australian gastrointestinal literature without any adverse effects noted.27
Moreover, proflavine has been used clinically as an antibacterial agent for decades. In neonatal care, triple dye, a combination of brilliant green, proflavine hemisulfate, and gentian violet is routinely used as a topical antibacterial agent on the umbilical stump of newborn babies,26
with a recent review of the practice categorizing toxicity as rare.29
The concentrations of proflavine solution required for successful imaging (0.01% to 0.05%)27
are substantially lower than that of the proflavine component in commercial triple dye, 0.11% (w/v; Kerr Triple Dye, VistaPharm). The quantity of solution required for diagnostic imaging is approximately the same as that used in neonatal care (0.65 mL per single-use swab). The additional exposure to light, which will occur during imaging, can also be compared to that received by newborn babies undergoing phototherapy for jaundice. The high-resolution fiber-optic microendoscope proposed for use here delivers 0.5 mW of 455 nm light to the tissue through a 0.8 mm diameter fiber-optic bundle, corresponding to an irradiance level of 100 mW/cm2
. The American Academy of Pediatrics defines intensive phototherapy as a spectral irradiance of at least 30 μW/cm2
per nanometer over the 430 to 490 nm spectral band, equivalent to a total irradiance of 1.8 mW/cm2
Although the irradiance level is over 50 times higher with the fiber microendoscope system, a typical imaging session of 30 minutes (including imaging for routine care) is approximately 50 times shorter than a typical 24-hour (1440 minutes) phototherapy incubation, leading to an equivalent light dose in each scenario. Proflavine has also been safely used in previous clinical studies evaluating its effect as a photosensitizing agent for the treatment of genital herpes simplex virus.31
The ability of proflavine to stain epithelial cell nuclei is useful for precancer and cancer imaging applications because it allows for visualization of features such as nuclear-to-cytoplasmic ratio. Because digital images are acquired, quantitative analysis of this and other image metrics can be easily performed to aid in image analysis.32
Such high-resolution imaging techniques can complement wide-field autofluorescence imaging systems discussed earlier. Wide-field imaging devices, such as the VELScope, have the ability to survey large mucosal surface areas to detect regions with loss of autofluorescence, which are suspicious for dysplasia. The HRME device can complement these types of wide-field imaging systems by providing high-resolution image data at specific lesions first identified by loss of fluorescence. Other studies show that the type of data acquired by the HRME—information about the spatial distribution of nuclei in the epithelium—is less affected by removal from the body than is autofluorescence.33
The purpose of this study was to evaluate the ability of high-resolution microendoscopy to identify oral neoplasia. HRME images from each site were analyzed qualitatively by visual inspection and quantitatively using digital image analysis algorithms to determine whether the imaged site contained neoplastic tissue. Results of image analysis were compared to the gold standard of histopathology to assess the ability of HRME to correctly identify the presence of neoplasia.