The goal of the present study was to establish a library of OCT images with the corresponding pathology finding to determine if OCT can discriminate dysplasia and CIS from normal, hyperplasia, or metaplasia. Our data show that invasive cancer can be distinguished from CIS and that dysplasia can be distinguished from metaplasia, hyperplasia, or normal. Using quantitative measurement, a progressive increase in the epithelial thickness was found to parallel the severity of the histopathology grade. The nuclei of the cells also became more discernible as darker less light scattering objects in lesions that are moderate dysplasia or worse. The basement membrane became disrupted or disappeared with invasive carcinoma.
There is considerable uncertainty about the natural history of bronchial IEN lesions. Sequential biopsies of the same sites in volunteer smokers with bronchial dysplasia showed a high regression rate at the end of 6 months in those who were in the placebo arm of the chemoprevention trial (13
). Twenty percent of the current smokers and 50% of the former smokers had complete resolution of their dysplasia to hyperplasia or normal (13
). Other studies also attempted to clarify the natural history of preneoplastic lesions and CIS using serial bronchoscopy and biopsy (10
). These studies were relatively small (~50 patients or less). Similar to our shorter-term studies (13
), >50% of the dysplastic lesions regressed spontaneously on follow-up (8
). The extent to which mechanical removal of the dysplastic lesion contributes to the apparently high regression rate of dysplasia is unknown. The high regression rate of bronchial dysplasia complicates the evaluation of chemopreventive agents. A nonbiopsy method would help to clarify the natural history of these lesions and the effect of chemopreventive intervention.
Currently, there are two imaging modalities that have sufficient spatial resolution and tissue depth penetration to study the bronchial epithelial and subepithelial changes associated with lung cancer development. Confocal microendoscopy is an attractive tool as it offers spatial resolution down to the submicron range. However, cells do not emit strong autofluorescence. Although the basement membrane and upper submucosa can be imaged with superb quality, the epithelial cells are not visible (23
). In addition, because contact with the bronchial surface is required, the fragile epithelium can be scrapped off during the imaging procedure. Motion artifacts due to cardiac pulsation and respiratory movements can also lead to suboptimal imaging of cellular details. OCT is a noncontact method that delivers near-IR light to the tissue and allows imaging of cellular and extracellular structures from analysis of the back scattered light with a spatial resolution of 4 to 15 µm and a depth penetration of ~2 mm to provide near-histologic images in the bronchial wall (17
). The fiberoptic probes can be miniaturized to enable imaging of airways down to the terminal bronchiole beyond the range of a standard bronchoscope. The procedure is simple and adds <5 min to a standard bronchoscopic procedure under local anesthesia and conscious sedation. The in vivo
imaging findings in invasive carcinoma and CIS in the present study are similar to the preliminary study by one of us (N.I.; ref. 20
) and the ex vivo
study by Whiteman et al. (25
). We have extended these earlier studies to show that dysplasia (especially high grade) and CIS can be distinguished from lower-grade lesions in vivo
Our study has several important strengths. To our knowledge, this is the first study that combines the large area imaging capability of autofluorescence endoscopy and the microscopic imaging resolution of OCT. Autofluorescence bronchoscopy makes use of fluorescence and absorption properties to provide information about the biochemical composition and metabolic state of bronchial tissues. The fluorescence properties of bronchial tissue are determined by the concentration of the cellular and extracellular fluorophores, their metabolic state, the tissue architecture, and the wavelength-dependent light attenuation due to the concentration as well as distribution of nonfluorescent chromophores such as hemoglobin (3
). Collagen and elastin are the most important structural fluorophores. Examples of fluorophores involved in cellular metabolism include NAD+
and flavins. The autofluorescence yield in the subepithelial tissue is ~10 times higher than the epithelium. As the bronchial epithelium changes from normal to dysplasia, and then to CIS and invasive cancer, there is a progressive decrease in green autofluorescence but proportionately less decrease in red fluorescence intensity. This change is due to a combination of several factors, such as a decrease in the extracellular collagen and elastin, an increase in the number of cell layers associated with dysplasia or cancer, decrease in the fluorescence measured in the bronchial surface due to reabsorption of fluorescent light by a thickened epithelium, increase in absorption of the blue excitation light, and reduced fluorescence due to an increase in the microvascular density/blood volume as well as a reduction in the amount of flavins and NAD+
in premalignant and malignant cells (3
). Because the microvasculature and blood volume is increased in inflammatory lesions and the epithelial thickness is increased with marked goblet cell hyperplasia or metaplasia, false-positive fluorescence can occur in a benign epithelium. Thus, although autofluorescence provides useful information on the biochemical and functional changes in the bronchial epithelium and autofluorescence bronchoscopy serves as a rapid scanning tool to localize preneoplastic and neoplastic lesions, autofluorescence alone cannot be used to study the natural history of these lesions without biopsy confirmation. We systematically examined the changes in the bronchial epithelium associated with the development of squamous cell carcinoma using OCT as a nonbiopsy optical imaging method to provide architectural information in the bronchial epithelium from a large cohort of heavy smokers at risk of developing lung cancer as well as patients with invasive carcinoma. The multilayer epithelium associated with bronchial dysplasia can be clearly seen. The ability to distinguish dysplasia from lower-grade lesions or inflammation opens the possibility that the effect of chemopreventive agents can be more accurately studied in short-term phase II trials without taking a biopsy before treatment. The same sites can be revisited to document the changes at the end of the treatment period (typically 3–6 months) first by OCT imaging and then by biopsy for histologic confirmation. The spontaneous regression rate of IEN lesions can also be studied in subjects who are treated with placebo. Thus, OCT can complement the rapid scanning ability of autofluorescence bronchoscopy by providing morphologic information to characterize potentially abnormal sites without a biopsy.
Certain limitations to the current study deserve consideration. Different grades of dysplasia could not be distinguished from one another and from CIS using quantitative measurement of the epithelial thickness alone. However, image analysis techniques can be implemented to further investigate the ability of OCT to statistically distinguish different grades of dysplasia from CIS. These techniques include quantifying the SD in OCT signal within a region of interest (27
) or texture analysis (28
). Architectural measurement of the epithelial changes similar to what has been achieved in morphometric measurements in biopsy specimens (29
) may provide an objective grading that is better than the visual grading of the nuclear changes in the present study. Morphometric measurements in OCT images require better spatial resolution than our current OCT device. Systems with higher resolution and Doppler capability that can measure cellular structures in greater detail and quantify vascular density are becoming available for clinical investigation (30
). Measurement of second harmonic signal and two-photon excitation coupled with Doppler OCT would further improve the imaging down to the molecular level (32
In summary, we have shown that autofluorescence endoscopy-guided OCT imaging of bronchial lesions is technically feasible. OCT may be a promising nonbiopsy tool for in vivo imaging of preneoplastic bronchial lesions to study their natural history and the effect of chemopreventive agents.