Photodiagnosis and Photodynamic Therapy (2004) 1, 111—122
REVIEW
Optical spectroscopy and imaging for early lung cancer detection: a review Haishan Zeng, Annette McWilliams, Stephen Lam MD, FRCPC∗ Cancer Imaging Department, British Columbia Cancer Agency, 601 West 10th Avenue, Vancouver, BC, Canada V5Z 1L3 KEYWORDS Fluorescence endoscopy; Lung cancer detection; Fluorescence imaging; Fluorescence spectroscopy; Reflectance spectroscopy; Raman spectroscopy
Summary Over the last 15 years, optical spectroscopy and imaging has been intensively studied to improve the detection and localization of early lung cancer. Autofluorescence bronchoscopy (AFB) is the most successfully developed technique and has significantly improved the detection sensitivity of early lung cancer. In this review, the optical principles behind white-light and autofluorescence bronchoscopy, as well as the role of AFB in the diagnosis of early lung cancer and the overall management of patients with early lung cancer are discussed. Other newest development such as Raman spectroscopy and simultaneous imaging and spectroscopy measurements are also highlighted. © 2004 Elsevier B.V. All rights reserved.
Contents Introduction ..............................................................................................
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Principles of optical spectroscopy and imaging............................................................
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Autofluorescence bronchoscopy devices ..................................................................
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Clinical trials of autofluorescence bronchoscopy ..........................................................
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Clinical applications of autofluorescence bronchoscopy ...................................................
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Research applications of optical spectroscopy and imaging ...............................................
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Summary .................................................................................................
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References................................................................................................
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* Corresponding author. Tel.: +1 604 875 4325; fax: +1 604 877 6077.
E-mail address: [email protected] (S. Lam).
1572-1000/$ — see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/S1572-1000(04)00042-0
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Introduction The tracheobronchial tree is a complex branching system with a surface area that is equivalent to the size of a tennis court. Squamous cell carcinomas and small cell carcinomas are more commonly found in the central airways while adenocarcinomas are usually found in the peripheral airways or lung parenchyma beyond the range of a standard, adultsize flexible bronchoscope. Thoracic CT scan is a sensitive tool in the detection of early peripheral lung cancers especially adenocarcinomas [1—10], but is sub-optimal for the detection of early central squamous cell carcinoma [10]. Squamous cell carcinoma in situ or micro-invasive cancers have high 5-year survival rates of >90% and are therefore clinically important lesions to detect and treat with intention of cure [11,12]. Despite the rising incidence of adenocarcinoma world-wide, squamous cell carcinoma still accounts for a significant proportion of lung cancer cases, ranging from 37% and 20% in men and women, respectively in the United States and Canada, to 47% and 27% in men and women, respectively in Europe [13]. In one recent report that assessed the combined role of CT scan and autofluorescence bronchoscopy in a screening program, approximately 25% of lung cancers detected were CT occult squamous carcinoma in situ or microinvasive cancer diagnosed by autofluorescence bronchoscopy alone [10]. Bronchoscopy complements thoracic CT in the diagnosis of early lung cancer with the added advantage that a cytological or tissue sample can be obtained in the same procedure for definitive diagnosis. In this paper, the optical principles behind whitelight and autofluorescence bronchoscopy (AFB), as well as the role of AFB in the diagnosis of early lung cancer and the overall management of patients with early lung cancer are discussed. Other newest developments such as Raman spectroscopy and simultaneous imaging and spectroscopy measurements are also highlighted.
Principles of optical spectroscopy and imaging When a beam of light reaches the bronchial surface (Fig. 1 and ref. [14]), part of it will be reflected by the surface directly, while the rest will be refracted and transmitted into the bronchial tissue. The direct reflection by the bronchial surface is called specular reflection, and is related only to the refractive index change between air and the epithelium of the bronchial tissue. The light transmitted
Figure 1 Schematic diagram of light pathways in bronchial tissue. A beam of incident light could interact with the bronchial tissue and generate various secondary photons measurable at the tissue surface: specular reflection, diffuse reflection, fluorescence, and Raman scattering. These measurable optical properties can be used for determining the structural features as well as the biochemical composition and functional changes in normal and abnormal bronchial tissues.
into the tissue will be scattered and absorbed by the tissue. After multiple scattering, some of the transmitted light will re-emerge through the bronchial surface into the air. This re-emergence is called diffuse reflection. The amount of diffuse reflection is determined by both scattering and absorption properties of the bronchial tissue. The stronger the absorption, the less the diffuse reflection; the stronger the scattering, the larger the diffuse reflection. Following absorption of a photon by the bronchial tissue, the electrically excited absorbing molecule may rapidly return to a more stable energy state by re-emission of a photon with lower energy, i.e. fluorescence emission. Fluorescence photons are also subjected to tissue re-absorption and scattering before some of these photons reach the tissue surface. Most light scattering in tissue is elastic scattering (or Rayleigh scattering) with no change in photon energy (or frequency). A very small portion of the scattered light, about 1 in 108 , is inelastically scattered (Raman scattering) with a corresponding change in frequency. The difference between the incident and scattered frequencies corresponds to an excitation of the molecular system, most often excitation of vibrational modes (Fig. 2). By measuring the intensity of the scattered photons as a function of the frequency difference, a Raman spectrum is obtained. Raman peaks are typically narrow (a few wavenumbers) and in many cases can be attributed to the vibration of specific
Optical spectroscopy and imaging for early lung cancer detection
Figure 2 Schematic illustration of Raman scattering process. When light with photon energy hν0 interact with a molecule, the molecule is first excited to a virtual state (horizontal dashed line). The molecule immediately returns to a higher energy vibrational state with the emission of an altered photon hν. Part of the energy (E) from the incident photon is transferred to induce molecular vibrations, leaving the scattered photon with reduced energy, hν = hν0 − E.
chemical bonds (or normal mode dominated by the vibration of a single functional group) in a molecule. As such, it is a ‘‘fingerprint’’ for the presence of various molecular species. These tissue optical properties can be used for determining the structural features as well as the biochemical composition and functional changes in normal and abnormal bronchial tissues. Reflectance imaging, of which white-light bronchoscopy is a prime example, makes use of differences in specular reflection and diffuse reflection of broadband visible light to define the structural features and color of epithelial surfaces in order to discriminate normal and abnormal bronchial tissues. Since the development of the flexible fiberoptic bronchoscope by Dr. Shigeto Ikeda in the late 1960s, whitelight bronchoscopy (WLB) has become the standard method for diagnosis of lung cancer, especially early lung cancer, in the central airways. More recently, other optical imaging methods such as confocal microendoscopy [15] and optical coherence tomography [16], are being developed to allow single cell imaging as well as visualization of structures below the bronchial surface. In contrast to reflectance imaging, autofluorescence imaging provides information about the biochemical composition and metabolic state of bronchial tissues. Most endogenous fluorophores are associated with the tissue matrix or are involved in cellular metabolic processes. Collagen and elastin are the most important structural fluorophores and their composition involves cross-
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linking between fluorescing amino acids. Fluorophores involved in cellular metabolism include nicotinamide adenine dinucleotide (NADH) and flavins. Other fluorophores include the aromatic amino acids (e.g. tryptophan, tyrosine, phenylalanine), various porphyrins, and lipopigments (e.g. ceroids, lipofuscin). The fluorescence properties of bronchial tissue is determined by the concentration of these fluorophores, the distinct excitation and emission spectrum of each fluorophore, distribution of various fluorophores in the tissue, the metabolic state of the fluorophores, the tissue architecture and the wavelength-dependent light attenuation due to the concentration as well as distribution of non-fluorescent chromophores such as hemoglobin [17]. When the bronchial surface is illuminated by violet or blue light, normal tissues fluoresce strongly in the green. As the bronchial epithelium changes from normal to dysplasia, and then to carcinoma in situ and invasive cancer, there is a progressive decrease in green autofluorescence but proportionately less decrease in red fluorescence intensity [18]. This change is due to a combination of several factors. The autofluorescence yield in the submucosa is approximately ten times higher than the epithelium [17,19—21]. There is a decrease in extracellular matrix such as collagen and elastin in dysplasia and cancer. Secondly, the increase in the number of cells associated with dysplasia or cancer decreases the fluorescence measured in the bronchial surface due to absorption of the excitation light and re-absorption of the fluorescence light by the thickened epithelium [19,20]. Thirdly, the microvascular density is increased in dysplastic and malignant tissues. The presence of an increased concentration and distribution of hemoglobin results in increased absorption of the blue excitation light and reduced fluorescence. For example, angiogenic squamous dysplasia was found to have decreased autofluorescence [22]. Fourthly, there is a reduction in the amount of flavins and NADH in pre-malignant and malignant cells. Other factors such as pH and oxygenation may also alter the fluorescence quantum yield, spectral peak positions and line widths [23]. The extent to which these metabolic and morphologic changes will alter the fluorescence signal depends on the excitation and emission wavelengths used for illumination and detection in fluorescence imaging devices used clinically in bronchoscopy. The excitation wavelengths producing the highest tumour to normal tissue contrasts are between 400 and 480 nm with a peak at 405 nm [18,24]. The spectral differences between 500 and 700 nm in normal, pre-malignant and malignant tissues serve as the basis for the design of several autofluorescence endoscopic imaging devices
114 for localization of early lung cancer in the bronchial tree. Although white light bronchoscopy is the simplest imaging technique, less than 40% of carcinoma in situ is detectable by standard white-light bronchoscopy [25,26]. This clinical problem, combined with the improvement of endoscopic technology, has driven the development and evaluation of fluorescence bronchoscopy for the localisation of preinvasive lung cancer [17,18,26—28].
Autofluorescence bronchoscopy devices A number of devices have been developed for commercial use including LIFE-Lung device (Xillix Technologies, Richmond, BC, Canada), Storz D-light (Karl Storz GmbH, Tuttlingen, Germany), and SAFE1000 (Pentax, Japan). The first device, the LIFELung system, uses a He-Cd laser (442 nm) for illumination [27,29,30]. A second generation device, the LIFE-Lung II uses a filtered Xe lamp to produce the blue light [26,31]. Two image-intensified CCD sensors are used to capture the emitted fluorescence, one in the green region (480—520 nm) and the other in the red region (≥625 nm). The red and green images are then combined. Because pre-malignant and malignant lesions lose more green autofluorescence than red, these lesions appear reddish-brown against a greenish normal background (Fig. 3a and b). The original LIFE-Lung device has separate light sources for the white light and fluorescence examinations and requires manual change of light source between examinations. In LIFE-Lung II, a filtered arc lamp is used which allows rapid switching between the two examination modes. A unique design in LIFE-Lung II is its real time quantitative display of the red/green autofluorescence ratio to minimise human error of subjective colour interpretation [31]. The Pentax SAFE-1000 system uses a filtered Xe lamp in the 420—480 nm ranges to produce the excitation light, but only detects fluorescence in the green spectrum (490—590 nm) using a single imageintensified CCD sensor [32]. The Wolf system is similar to the Xillix LIFELung system with a filtered 300 W Xe lamp in the ‘‘violet-blue’’ range (390—460 nm) and slightly different band-pass filters for detection: 500—590 nm (green region), and 600—700 nm (red region) [17,33]. The Storz system consists of a RGB CCD camera and a filtered Xe lamp (380—460 nm). It combines a fluorescence image with a blue reflectance image [28]. The lesions appear purple against a bluish-
H. Zeng et al. green background. Frame integration is used to amplify the weak autofluorescence. A third generation device by Xillix (Onco-LIFE, Richmond, Canada) also utilises a combination of reflectance and fluorescence imaging [32]. A red reflectance image is used in combination with the green fluorescence image to enhance the contrast between malignant and normal tissues. Using reflected red light as a reference has the theoretical advantage over reflected blue light in that it is less absorbed by hemoglobin and hence less influenced by changes in vascularity associated with inflammation. As the latest advancement in this field, an integrated endoscopy system for simultaneous imaging and spectroscopy (OmniScopeTM ) [34] has been developed by the Cancer Imaging group at the British Columbia Cancer Agency in collaboration with SpectraVu Medical, Inc. to improve the specificity of autofluorescence bronchoscopy while maintaining its high sensitivity. In this system, a specially designed three-CCD camera in combination with a dedicated light source permits capture of both white-light color images and autofluorescence images without the need to switch between cameras. A mirror with an optical fiber at its center, placed at an interim-imaging plane inside the camera unit, facilitate simultaneous imaging and spectroscopy measurements in either white-light reflectance mode or fluorescence mode. Four types of information: (1) white light color image, (2) autofluorescence image, (3) reflectance spectrum, (4) autofluorescence spectrum, can be obtained without introducing a fiber-optic catheter through the bronchoscope biopsy channel to touch the tissue surface as in conventional endoscopic spectral measurements [18,19,24], This integrated imaging and spectroscopy system presents the potential of using autofluorescence imaging to achieve high diagnostic sensitivity while at the same time using reflectance and fluorescence spectroscopy to achieve high diagnostic specificity. Fig. 4 shows an example of this approach in a patient with a small carcinoma in situ.
Clinical trials of autofluorescence bronchoscopy Most of the published clinical studies on autofluorescence bronchoscopy have been conducted with the Xillix LIFE-Lung devices with smaller studies in the other devices [35—37]. Over the last decade, a number of studies have been conducted in Canada, USA, Europe and Asia, using the
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Figure 3 White-light and autofluorescence images of a carcinoma in situ lesion in the left main bronchus. (a) Whitelight bronchoscopic image showing a subtle nodular lesion. (b) Autofluorescence image of the same area. The tumor area appears brownish red (arrow) against a normal green background (LIFE-LungTM II Device, Xillix Technologies Inc., Richmond, BC, Canada).
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Figure 4 Example WLB images, reflectance spectral curves (a—c) and AFB images and fluorescence spectral curves (d—f) of a CIS lesion and its surrounding normal tissue. (a) WLB image with the optical fiber almost outside of the interim image field; (b) WLB image when the fiber is aligned with the CIS lesion for spectral measurement and shows as a black spot; (c) reflectance spectra obtained from the CIS and its surrounding normal tissue. (d) AFB image with the fiber almost outside of the interim image field; (e) AFB image when the fiber is aligned with the CIS lesion for spectral measurement; (f) fluorescence spectra from the CIS and its surrounding normal tissue (OmniScopeTM Device, SpectraVu Medical Inc., Vancouver, BC, Canada; from ref. [34] with permission).
LIFE-Lung devices involving more than 2000 patients [10,22,26,27,29—31,38—60]. In the majority of these trials, autofluorescence bronchoscopy was performed and evaluated as an adjunct to white light bronchoscopy. The relative sensitivity of autofluorescence bronchoscopy versus white-light examination alone in detecting high-grade dysplasia and carcinoma in situ was found to increase by an average of two-fold (1.5—6.3-fold). Approximately 80% of the lesions (range 43—100%) can be localized by fluorescence examination compared to an average of 40% (range 9—78%) by whitelight examination alone. A randomized study using
a different bronchoscopist to perform either the white-light or the fluorescence examination also showed a significant increase in the relative sensitivity of fluorescence examination versus whitelight examination (68.8% versus 21.9%) [44]. The improved sensitivity was associated with a lower specificity of an average of 60% compared to 81% by white-light examination. The wide range of sensitivity in both examinations most likely reflects the differences in study design and varying experience of endoscopists and pathologists in the interpretation of fluorescence images and bronchial biopsies.
Optical spectroscopy and imaging for early lung cancer detection The results of a multi-center trial of the Storz D-Light autofluorescence device involving 293 patients were recently reported [35]. Autofluorescence bronchoscopy improved the detection rate of pre-invasive lesions and early invasive cancer from 11% to 61%. The specificity of autofluorescence bronchoscopy and white-light bronchoscopy was 75% and 95%, respectively. There has been only one report comparing the performance of the first LIFE-Lung device and the Storz D-light system [38]. Both systems gave comparable results although details of the cross-over design were not given in the report. In the Xillix and Storz trials, most of the falsepositive biopsies were found to be due to inflammation, goblet cell hyperplasia or metaplasia. However, areas with ‘‘benign’’ pathology and abnormal fluorescence have been found to contain more genetic alterations on comparative genomic hybridization than areas with normal fluorescence, suggesting that areas with abnormal fluorescence may represent higher risk lesions [61]. Some areas with abnormal fluorescence and benign pathology have been shown to develop into carcinoma on bronchoscopic follow-up [51,52]. In addition, the presence of three or more sites of abnormal fluorescence was found to predict development of squamous cell carcinoma in high risk patients [50].
Clinical applications of autofluorescence bronchoscopy Up until very recently, autofluorescence bronchoscopy in lung cancer diagnosis has been limited by equipment costs and availability, as well as the need for specialized training. Over the last decade, the principles behind exploiting both the reflectance and autofluorescence properties of bronchial tissues to improve the sensitivity of detecting pre-invasive lesions are becoming familiar to endoscopists. Commercial devices are cheaper and easier to use. Current devices using filtered lamps allow rapid switching between the whitelight and fluorescence modes. The combined examination usually adds five to 10 minutes to a conventional white-light bronchoscopic procedure. The bronchoscopy is well tolerated by patients under local anaesthesia and conscious sedation. Devices with simultaneous display of the white-light and fluorescence images are undergoing development and testing by our group. These new devices will further shorten the procedure time and minimizing re-imbursement issues that may be a barrier in some countries.
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Current evidence supports the use of autofluorescence bronchoscopy in the following clinical situations: 1. Patients with severe atypia or malignant cells in their sputum cytology and a negative chest X-ray or CT scan; 2. Patients with suspected lung cancer where a diagnostic bronchoscopy is indicated; and 3. Patients with potentially curable carcinoma in situ/micro-invasive cancer prior to therapy. Patients who present with malignant cells in sputum cytology examination with a negative chest X-ray or CT scan—–the so-called radiographically occult lung cancer represent a diagnostic challenge. When white-light bronchoscopy fails to localize the source of the malignant cells, repeat bronchoscopies with blind segmental bronchial brushings and multiple bronchial spur biopsies are required for diagnosis [62—64]. Although the procedure can be done under local anaesthesia, it is more commonly done under general anaesthesia. The examination generally takes 1—2 hours. There is also the problem of cross contamination from spilling of cells from one segment or lobe to another with coughing. With the introduction of autofluorescence bronchoscopy, these radiographically occult cancers can now be readily detected with one bronchoscopy as shown by the clinical experience reviewed above. Severe atypia on sputum cytology examination has been reported in several studies to have a risk of developing lung cancer within two years of approximately 45% [65—67]. In the Johns Hopkins Early Lung Cancer Detection Project, moderate atypia was also found to have an increased risk of the subsequent development of lung cancer. Fourteen percent of the participants with moderate atypia developed lung cancer on long-term follow-up, compared to 3% of participants without atypia [66]. In the Colorado SPORE cohort of high-risk smokers and ex-smokers with airflow obstruction, there have been 83 incident lung cancers after more than 4469 years of observation. The relative risks of developing lung cancer, adjusted for age, gender, recruitment year, pack-years and smoking status, for increasing grades of cytologic atypia were 1.0 (normal), 1.10 (mild atypia), 1.68 (moderate atypia), 3.18 (moderate atypia or worse) and 31.4 (worse than moderate atypia) [67]. Sputum cytologic atypia of severe dysplasia or worse clearly carries a risk of lung cancer that is high enough to warrant an aggressive diagnostic approach with combined white-light and fluorescence bronchoscopy. Two groups have reported results of bronchoscopy in subjects with moderate atypia sputum cytology
118 and chest radiographs negative for carcinoma. Fujita reported, in abstract form only, a series of 25 subjects with moderate atypia sputum cytology and negative chest radiographs who underwent bronchoscopy. Two had carcinoma diagnosed [68]. Kennedy and colleagues have reported a series of 79 subjects with moderate atypia sputum cytology and chest radiographs negative for cancer [69]. Of the 79, lung cancer was found at bronchoscopy in 5 (6.3%; 95% CI = 0.7—11%). Two of the cancers were carcinoma in situ lesions and three were invasive. The rates of discovery of cancer at bronchoscopy reported in these studies exceeded the rate of discovery of colon cancer when colonoscopy is performed for a positive fecal occult blood test. While moderate atypia in sputum cytology is not as compelling an indication for bronchoscopy as is severe atypia and the high rate of discovery of unexpected lung cancer in the reported studies may be partly due to chance, publication bias or a selection bias in which only subjects with moderate atypia and additional health problems were examined, consideration of bronchoscopy is certainly reasonable in a concerned patient with moderate atypia. For patients with early lung cancer who are being assessed for curative surgical resection and those with carcinoma in situ who are being evaluated for endobronchial therapies, autofluorescence bronchoscopy is useful in the delineation of tumour margins and to assess the presence of synchronous lesions in the bronchial tree [48,49,70]. Synchronous cancer can be found on autofluorescence bronchoscopy in up to 14% of these patients. Up to 27% of patients may also have other moderate/severe dysplastic lesions that will require bronchoscopic followup [27,29,48,70]. Carcinoma in situ is a potentially curable lesion with endobronchial therapy such as photodynamic therapy, electrocautery or cryotherapy if the lesion is ≤1 cm2 [49,71]. However, white light bronchoscopy is inadequate both for detection and for delineation of the margins of these lesions, whereas autofluorescence bronchoscopy improves staging of these occult cancers and has an impact on their management [49,60]. In addition, during endobronchial therapy with techniques such as electrocautery, autofluorescence can be used to ensure that the lesion is adequately treated. The role of autofluorescence bronchoscopy for surveillance of second primary lung cancer in patients who have curative therapy for non-small cell lung cancer or limited stage small cell lung cancer was examined in several studies. This group of patients has a relatively high rate of development of metachronous tumours; 1—5% per year after non-small cell lung cancer resection and 2—13%
H. Zeng et al. after small cell lung cancer [71—76]. The number of patients in these studies were small. Lesions were identified in 3—6% of these patients who were thought to be disease free [39—41,77]. Patients with prior squamous cell carcinoma appear to be a population that may warrant prospective study of postoperative fluorescence bronchoscopic surveillance [41]. Likewise, the prevalence of preinvasive lesions in patients with cancer in the upper aerodigestive tract (head and neck cancer excluding nasopharyngeal cancer or esophageal cancer) may be sufficiently high due to the field cancerization effect of inhaled tobacco carcinogens to warrant a fluorescence bronchoscopy prior to curative therapy or for surveillance for second primary cancers. In patients with esophageal cancer up to 19% may have synchronous or metachronous cancers in other organs, and lung cancer contributed to approximately 10% of these second tumours [78]. In laryngeal cancer patients, up to 28% have been reported to have synchronous or metachronous tumours, and lung cancer was the most frequent second malignancy, contributing to 41% of second tumours [29,79,80]. Currently, there are no established evidencebased guidelines to aid physicians in the ongoing surveillance of lung cancer patients following curative intent therapy [81]. Although there has been stimulating research into lung cancer screening over the last decade with the emergence of techniques such as low dose thoracic CT scans, autofluorescence bronchoscopy and new sputum biomarkers, we ultimately need the ability to both detect and reduce the development of metachronous pulmonary malignancies to impact lung cancer mortality. Evidence based follow-up protocols need to be developed and their benefits need to be evaluated in larger prospective clinical trials.
Research applications of optical spectroscopy and imaging Autofluorescence bronchoscopy also has a significant role in ongoing lung cancer research. The study of the natural history and genetic alterations in preneoplastic lesions accessible to the fiberoptic bronchoscope will lead to a better understanding of lung cancer pathogenesis and progression. It will in turn lead to development of better biomarkers for early detection and novel molecular targets for intervention. Preliminary study suggests that fluorescence bronchoscopy plays an important role in an early lung cancer detection program in conjunction with spiral CT [10]. The ability to identify pre-neoplastic
Optical spectroscopy and imaging for early lung cancer detection
Figure 5 The mean Raman spectra of normal bronchial tissue (n = 12), adenocarcinoma (n = 6) and squamous cell carcinoma (n =1 0). Each spectrum was normalized to the integrated area under the curve to correct for variations in absolute spectral intensity. Each spectral peak can be assigned to specific biomolecules inside the tissue such as nucleic acids and proteins. The Raman signatures for tumour are significantly different from normal tissue (with permission from ref. [83]).
lesions and perform serial biopsies before and after treatment provides an intermediate endpoint to evaluate novel agents for chemoprevention of lung cancer [82]. Very recently, the promising but technically challenging Raman spectroscopy has also been applied to improve lung cancer detection [83]. Raman signals are very weak but provide fingerprint type signatures of specific molecules in tissue. This study demonstrated that using a specially designed rapidacquisition NIR Raman spectroscopy system, Raman spectra could be obtained from fresh bronchial tissue samples from biopsy or surgical resection in 5 s [83]. Fig. 5 shows the normalized mean Raman spectra of normal and cancerous bronchial tissues. It can be seen that significant Raman spectral differences exist between normal and tumour tissue, while Raman spectra of adenocarcinoma and squamous cell carcinoma are very similar to each other. The ratio of Raman intensities at 1445—1655 cm−1 provided good differentiation between normal and tumour tissues. Tumours showed higher percentage Raman signals for nucleic acid, tryptophan and phenylalanine, but lower percentage signals for phospholipids, proline and valine, compared to normal tissue (Fig. 5). The results of this exploratory study indicate that NIR Raman spectroscopy may have significant potential for improving lung cancer diagnosis based on optical evaluation of biomolecules. The development of an endoscopic Raman probe for in vivo Raman spectral measurements is underway.
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In the future, Raman spectroscopy may be used to reduce the false positive rate from AFB imaging detection. Raman spectroscopy may also be used to identify the structural and compositional differences on proteins and genetic materials between malignant lung cancers, their pre-curses, and normal lung tissues. This knowledge will lead to better understanding, on the biochemical bases, of the evolution process of lung cancers from benign to malignancy. The biochemical information obtained from in vivo Raman measurements may also be helpful for predicting the malignancy potential of pre-invasive and invasive lung cancers.
Summary Among all types of optical imaging and spectroscopy techniques, autofluorescence bronchoscopy is the most successfully developed. AFB is a sensitive technique for detection of intraepithelial neoplasia. In conjunction with white-light bronchoscopy, it has a definite clinical role in localization of preinvasive lung cancer in patients with abnormal sputum cytology and determining the extent of endobronchial spread in patients with early lung cancer prior to curative therapy. It may also play a role in patients with potentially curable oesophageal and head and neck cancer in detection of second primary lung cancer, as well as in the postoperative surveillance of lung cancer patients, but further studies are required. Fluorescence bronchoscopy plays an important part in lung cancer research particularly in the study of the natural history and the molecular biology of pre-neoplastic lesions, in lung cancer screening clinical trials as well as Phase II trials of chemopreventive agents. The decreased specificity associated with AFB could be improved by new technologies currently under development such as Raman spectroscopy and simultaneous AFB imaging with fluorescence spectroscopy and simultaneous WLB imaging with reflectance spectroscopy.
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REVIEW
Optical spectroscopy and imaging for early lung cancer detection: a review Haishan Zeng, Annette McWilliams, Stephen Lam MD, FRCPC∗ Cancer Imaging Department, British Columbia Cancer Agency, 601 West 10th Avenue, Vancouver, BC, Canada V5Z 1L3 KEYWORDS Fluorescence endoscopy; Lung cancer detection; Fluorescence imaging; Fluorescence spectroscopy; Reflectance spectroscopy; Raman spectroscopy
Summary Over the last 15 years, optical spectroscopy and imaging has been intensively studied to improve the detection and localization of early lung cancer. Autofluorescence bronchoscopy (AFB) is the most successfully developed technique and has significantly improved the detection sensitivity of early lung cancer. In this review, the optical principles behind white-light and autofluorescence bronchoscopy, as well as the role of AFB in the diagnosis of early lung cancer and the overall management of patients with early lung cancer are discussed. Other newest development such as Raman spectroscopy and simultaneous imaging and spectroscopy measurements are also highlighted. © 2004 Elsevier B.V. All rights reserved.
Contents Introduction ..............................................................................................
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Principles of optical spectroscopy and imaging............................................................
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Autofluorescence bronchoscopy devices ..................................................................
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Clinical trials of autofluorescence bronchoscopy ..........................................................
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Clinical applications of autofluorescence bronchoscopy ...................................................
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Research applications of optical spectroscopy and imaging ...............................................
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Summary .................................................................................................
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* Corresponding author. Tel.: +1 604 875 4325; fax: +1 604 877 6077.
E-mail address: [email protected] (S. Lam).
1572-1000/$ — see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/S1572-1000(04)00042-0
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Introduction The tracheobronchial tree is a complex branching system with a surface area that is equivalent to the size of a tennis court. Squamous cell carcinomas and small cell carcinomas are more commonly found in the central airways while adenocarcinomas are usually found in the peripheral airways or lung parenchyma beyond the range of a standard, adultsize flexible bronchoscope. Thoracic CT scan is a sensitive tool in the detection of early peripheral lung cancers especially adenocarcinomas [1—10], but is sub-optimal for the detection of early central squamous cell carcinoma [10]. Squamous cell carcinoma in situ or micro-invasive cancers have high 5-year survival rates of >90% and are therefore clinically important lesions to detect and treat with intention of cure [11,12]. Despite the rising incidence of adenocarcinoma world-wide, squamous cell carcinoma still accounts for a significant proportion of lung cancer cases, ranging from 37% and 20% in men and women, respectively in the United States and Canada, to 47% and 27% in men and women, respectively in Europe [13]. In one recent report that assessed the combined role of CT scan and autofluorescence bronchoscopy in a screening program, approximately 25% of lung cancers detected were CT occult squamous carcinoma in situ or microinvasive cancer diagnosed by autofluorescence bronchoscopy alone [10]. Bronchoscopy complements thoracic CT in the diagnosis of early lung cancer with the added advantage that a cytological or tissue sample can be obtained in the same procedure for definitive diagnosis. In this paper, the optical principles behind whitelight and autofluorescence bronchoscopy (AFB), as well as the role of AFB in the diagnosis of early lung cancer and the overall management of patients with early lung cancer are discussed. Other newest developments such as Raman spectroscopy and simultaneous imaging and spectroscopy measurements are also highlighted.
Principles of optical spectroscopy and imaging When a beam of light reaches the bronchial surface (Fig. 1 and ref. [14]), part of it will be reflected by the surface directly, while the rest will be refracted and transmitted into the bronchial tissue. The direct reflection by the bronchial surface is called specular reflection, and is related only to the refractive index change between air and the epithelium of the bronchial tissue. The light transmitted
Figure 1 Schematic diagram of light pathways in bronchial tissue. A beam of incident light could interact with the bronchial tissue and generate various secondary photons measurable at the tissue surface: specular reflection, diffuse reflection, fluorescence, and Raman scattering. These measurable optical properties can be used for determining the structural features as well as the biochemical composition and functional changes in normal and abnormal bronchial tissues.
into the tissue will be scattered and absorbed by the tissue. After multiple scattering, some of the transmitted light will re-emerge through the bronchial surface into the air. This re-emergence is called diffuse reflection. The amount of diffuse reflection is determined by both scattering and absorption properties of the bronchial tissue. The stronger the absorption, the less the diffuse reflection; the stronger the scattering, the larger the diffuse reflection. Following absorption of a photon by the bronchial tissue, the electrically excited absorbing molecule may rapidly return to a more stable energy state by re-emission of a photon with lower energy, i.e. fluorescence emission. Fluorescence photons are also subjected to tissue re-absorption and scattering before some of these photons reach the tissue surface. Most light scattering in tissue is elastic scattering (or Rayleigh scattering) with no change in photon energy (or frequency). A very small portion of the scattered light, about 1 in 108 , is inelastically scattered (Raman scattering) with a corresponding change in frequency. The difference between the incident and scattered frequencies corresponds to an excitation of the molecular system, most often excitation of vibrational modes (Fig. 2). By measuring the intensity of the scattered photons as a function of the frequency difference, a Raman spectrum is obtained. Raman peaks are typically narrow (a few wavenumbers) and in many cases can be attributed to the vibration of specific
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Figure 2 Schematic illustration of Raman scattering process. When light with photon energy hν0 interact with a molecule, the molecule is first excited to a virtual state (horizontal dashed line). The molecule immediately returns to a higher energy vibrational state with the emission of an altered photon hν. Part of the energy (E) from the incident photon is transferred to induce molecular vibrations, leaving the scattered photon with reduced energy, hν = hν0 − E.
chemical bonds (or normal mode dominated by the vibration of a single functional group) in a molecule. As such, it is a ‘‘fingerprint’’ for the presence of various molecular species. These tissue optical properties can be used for determining the structural features as well as the biochemical composition and functional changes in normal and abnormal bronchial tissues. Reflectance imaging, of which white-light bronchoscopy is a prime example, makes use of differences in specular reflection and diffuse reflection of broadband visible light to define the structural features and color of epithelial surfaces in order to discriminate normal and abnormal bronchial tissues. Since the development of the flexible fiberoptic bronchoscope by Dr. Shigeto Ikeda in the late 1960s, whitelight bronchoscopy (WLB) has become the standard method for diagnosis of lung cancer, especially early lung cancer, in the central airways. More recently, other optical imaging methods such as confocal microendoscopy [15] and optical coherence tomography [16], are being developed to allow single cell imaging as well as visualization of structures below the bronchial surface. In contrast to reflectance imaging, autofluorescence imaging provides information about the biochemical composition and metabolic state of bronchial tissues. Most endogenous fluorophores are associated with the tissue matrix or are involved in cellular metabolic processes. Collagen and elastin are the most important structural fluorophores and their composition involves cross-
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linking between fluorescing amino acids. Fluorophores involved in cellular metabolism include nicotinamide adenine dinucleotide (NADH) and flavins. Other fluorophores include the aromatic amino acids (e.g. tryptophan, tyrosine, phenylalanine), various porphyrins, and lipopigments (e.g. ceroids, lipofuscin). The fluorescence properties of bronchial tissue is determined by the concentration of these fluorophores, the distinct excitation and emission spectrum of each fluorophore, distribution of various fluorophores in the tissue, the metabolic state of the fluorophores, the tissue architecture and the wavelength-dependent light attenuation due to the concentration as well as distribution of non-fluorescent chromophores such as hemoglobin [17]. When the bronchial surface is illuminated by violet or blue light, normal tissues fluoresce strongly in the green. As the bronchial epithelium changes from normal to dysplasia, and then to carcinoma in situ and invasive cancer, there is a progressive decrease in green autofluorescence but proportionately less decrease in red fluorescence intensity [18]. This change is due to a combination of several factors. The autofluorescence yield in the submucosa is approximately ten times higher than the epithelium [17,19—21]. There is a decrease in extracellular matrix such as collagen and elastin in dysplasia and cancer. Secondly, the increase in the number of cells associated with dysplasia or cancer decreases the fluorescence measured in the bronchial surface due to absorption of the excitation light and re-absorption of the fluorescence light by the thickened epithelium [19,20]. Thirdly, the microvascular density is increased in dysplastic and malignant tissues. The presence of an increased concentration and distribution of hemoglobin results in increased absorption of the blue excitation light and reduced fluorescence. For example, angiogenic squamous dysplasia was found to have decreased autofluorescence [22]. Fourthly, there is a reduction in the amount of flavins and NADH in pre-malignant and malignant cells. Other factors such as pH and oxygenation may also alter the fluorescence quantum yield, spectral peak positions and line widths [23]. The extent to which these metabolic and morphologic changes will alter the fluorescence signal depends on the excitation and emission wavelengths used for illumination and detection in fluorescence imaging devices used clinically in bronchoscopy. The excitation wavelengths producing the highest tumour to normal tissue contrasts are between 400 and 480 nm with a peak at 405 nm [18,24]. The spectral differences between 500 and 700 nm in normal, pre-malignant and malignant tissues serve as the basis for the design of several autofluorescence endoscopic imaging devices
114 for localization of early lung cancer in the bronchial tree. Although white light bronchoscopy is the simplest imaging technique, less than 40% of carcinoma in situ is detectable by standard white-light bronchoscopy [25,26]. This clinical problem, combined with the improvement of endoscopic technology, has driven the development and evaluation of fluorescence bronchoscopy for the localisation of preinvasive lung cancer [17,18,26—28].
Autofluorescence bronchoscopy devices A number of devices have been developed for commercial use including LIFE-Lung device (Xillix Technologies, Richmond, BC, Canada), Storz D-light (Karl Storz GmbH, Tuttlingen, Germany), and SAFE1000 (Pentax, Japan). The first device, the LIFELung system, uses a He-Cd laser (442 nm) for illumination [27,29,30]. A second generation device, the LIFE-Lung II uses a filtered Xe lamp to produce the blue light [26,31]. Two image-intensified CCD sensors are used to capture the emitted fluorescence, one in the green region (480—520 nm) and the other in the red region (≥625 nm). The red and green images are then combined. Because pre-malignant and malignant lesions lose more green autofluorescence than red, these lesions appear reddish-brown against a greenish normal background (Fig. 3a and b). The original LIFE-Lung device has separate light sources for the white light and fluorescence examinations and requires manual change of light source between examinations. In LIFE-Lung II, a filtered arc lamp is used which allows rapid switching between the two examination modes. A unique design in LIFE-Lung II is its real time quantitative display of the red/green autofluorescence ratio to minimise human error of subjective colour interpretation [31]. The Pentax SAFE-1000 system uses a filtered Xe lamp in the 420—480 nm ranges to produce the excitation light, but only detects fluorescence in the green spectrum (490—590 nm) using a single imageintensified CCD sensor [32]. The Wolf system is similar to the Xillix LIFELung system with a filtered 300 W Xe lamp in the ‘‘violet-blue’’ range (390—460 nm) and slightly different band-pass filters for detection: 500—590 nm (green region), and 600—700 nm (red region) [17,33]. The Storz system consists of a RGB CCD camera and a filtered Xe lamp (380—460 nm). It combines a fluorescence image with a blue reflectance image [28]. The lesions appear purple against a bluish-
H. Zeng et al. green background. Frame integration is used to amplify the weak autofluorescence. A third generation device by Xillix (Onco-LIFE, Richmond, Canada) also utilises a combination of reflectance and fluorescence imaging [32]. A red reflectance image is used in combination with the green fluorescence image to enhance the contrast between malignant and normal tissues. Using reflected red light as a reference has the theoretical advantage over reflected blue light in that it is less absorbed by hemoglobin and hence less influenced by changes in vascularity associated with inflammation. As the latest advancement in this field, an integrated endoscopy system for simultaneous imaging and spectroscopy (OmniScopeTM ) [34] has been developed by the Cancer Imaging group at the British Columbia Cancer Agency in collaboration with SpectraVu Medical, Inc. to improve the specificity of autofluorescence bronchoscopy while maintaining its high sensitivity. In this system, a specially designed three-CCD camera in combination with a dedicated light source permits capture of both white-light color images and autofluorescence images without the need to switch between cameras. A mirror with an optical fiber at its center, placed at an interim-imaging plane inside the camera unit, facilitate simultaneous imaging and spectroscopy measurements in either white-light reflectance mode or fluorescence mode. Four types of information: (1) white light color image, (2) autofluorescence image, (3) reflectance spectrum, (4) autofluorescence spectrum, can be obtained without introducing a fiber-optic catheter through the bronchoscope biopsy channel to touch the tissue surface as in conventional endoscopic spectral measurements [18,19,24], This integrated imaging and spectroscopy system presents the potential of using autofluorescence imaging to achieve high diagnostic sensitivity while at the same time using reflectance and fluorescence spectroscopy to achieve high diagnostic specificity. Fig. 4 shows an example of this approach in a patient with a small carcinoma in situ.
Clinical trials of autofluorescence bronchoscopy Most of the published clinical studies on autofluorescence bronchoscopy have been conducted with the Xillix LIFE-Lung devices with smaller studies in the other devices [35—37]. Over the last decade, a number of studies have been conducted in Canada, USA, Europe and Asia, using the
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Figure 3 White-light and autofluorescence images of a carcinoma in situ lesion in the left main bronchus. (a) Whitelight bronchoscopic image showing a subtle nodular lesion. (b) Autofluorescence image of the same area. The tumor area appears brownish red (arrow) against a normal green background (LIFE-LungTM II Device, Xillix Technologies Inc., Richmond, BC, Canada).
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Figure 4 Example WLB images, reflectance spectral curves (a—c) and AFB images and fluorescence spectral curves (d—f) of a CIS lesion and its surrounding normal tissue. (a) WLB image with the optical fiber almost outside of the interim image field; (b) WLB image when the fiber is aligned with the CIS lesion for spectral measurement and shows as a black spot; (c) reflectance spectra obtained from the CIS and its surrounding normal tissue. (d) AFB image with the fiber almost outside of the interim image field; (e) AFB image when the fiber is aligned with the CIS lesion for spectral measurement; (f) fluorescence spectra from the CIS and its surrounding normal tissue (OmniScopeTM Device, SpectraVu Medical Inc., Vancouver, BC, Canada; from ref. [34] with permission).
LIFE-Lung devices involving more than 2000 patients [10,22,26,27,29—31,38—60]. In the majority of these trials, autofluorescence bronchoscopy was performed and evaluated as an adjunct to white light bronchoscopy. The relative sensitivity of autofluorescence bronchoscopy versus white-light examination alone in detecting high-grade dysplasia and carcinoma in situ was found to increase by an average of two-fold (1.5—6.3-fold). Approximately 80% of the lesions (range 43—100%) can be localized by fluorescence examination compared to an average of 40% (range 9—78%) by whitelight examination alone. A randomized study using
a different bronchoscopist to perform either the white-light or the fluorescence examination also showed a significant increase in the relative sensitivity of fluorescence examination versus whitelight examination (68.8% versus 21.9%) [44]. The improved sensitivity was associated with a lower specificity of an average of 60% compared to 81% by white-light examination. The wide range of sensitivity in both examinations most likely reflects the differences in study design and varying experience of endoscopists and pathologists in the interpretation of fluorescence images and bronchial biopsies.
Optical spectroscopy and imaging for early lung cancer detection The results of a multi-center trial of the Storz D-Light autofluorescence device involving 293 patients were recently reported [35]. Autofluorescence bronchoscopy improved the detection rate of pre-invasive lesions and early invasive cancer from 11% to 61%. The specificity of autofluorescence bronchoscopy and white-light bronchoscopy was 75% and 95%, respectively. There has been only one report comparing the performance of the first LIFE-Lung device and the Storz D-light system [38]. Both systems gave comparable results although details of the cross-over design were not given in the report. In the Xillix and Storz trials, most of the falsepositive biopsies were found to be due to inflammation, goblet cell hyperplasia or metaplasia. However, areas with ‘‘benign’’ pathology and abnormal fluorescence have been found to contain more genetic alterations on comparative genomic hybridization than areas with normal fluorescence, suggesting that areas with abnormal fluorescence may represent higher risk lesions [61]. Some areas with abnormal fluorescence and benign pathology have been shown to develop into carcinoma on bronchoscopic follow-up [51,52]. In addition, the presence of three or more sites of abnormal fluorescence was found to predict development of squamous cell carcinoma in high risk patients [50].
Clinical applications of autofluorescence bronchoscopy Up until very recently, autofluorescence bronchoscopy in lung cancer diagnosis has been limited by equipment costs and availability, as well as the need for specialized training. Over the last decade, the principles behind exploiting both the reflectance and autofluorescence properties of bronchial tissues to improve the sensitivity of detecting pre-invasive lesions are becoming familiar to endoscopists. Commercial devices are cheaper and easier to use. Current devices using filtered lamps allow rapid switching between the whitelight and fluorescence modes. The combined examination usually adds five to 10 minutes to a conventional white-light bronchoscopic procedure. The bronchoscopy is well tolerated by patients under local anaesthesia and conscious sedation. Devices with simultaneous display of the white-light and fluorescence images are undergoing development and testing by our group. These new devices will further shorten the procedure time and minimizing re-imbursement issues that may be a barrier in some countries.
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Current evidence supports the use of autofluorescence bronchoscopy in the following clinical situations: 1. Patients with severe atypia or malignant cells in their sputum cytology and a negative chest X-ray or CT scan; 2. Patients with suspected lung cancer where a diagnostic bronchoscopy is indicated; and 3. Patients with potentially curable carcinoma in situ/micro-invasive cancer prior to therapy. Patients who present with malignant cells in sputum cytology examination with a negative chest X-ray or CT scan—–the so-called radiographically occult lung cancer represent a diagnostic challenge. When white-light bronchoscopy fails to localize the source of the malignant cells, repeat bronchoscopies with blind segmental bronchial brushings and multiple bronchial spur biopsies are required for diagnosis [62—64]. Although the procedure can be done under local anaesthesia, it is more commonly done under general anaesthesia. The examination generally takes 1—2 hours. There is also the problem of cross contamination from spilling of cells from one segment or lobe to another with coughing. With the introduction of autofluorescence bronchoscopy, these radiographically occult cancers can now be readily detected with one bronchoscopy as shown by the clinical experience reviewed above. Severe atypia on sputum cytology examination has been reported in several studies to have a risk of developing lung cancer within two years of approximately 45% [65—67]. In the Johns Hopkins Early Lung Cancer Detection Project, moderate atypia was also found to have an increased risk of the subsequent development of lung cancer. Fourteen percent of the participants with moderate atypia developed lung cancer on long-term follow-up, compared to 3% of participants without atypia [66]. In the Colorado SPORE cohort of high-risk smokers and ex-smokers with airflow obstruction, there have been 83 incident lung cancers after more than 4469 years of observation. The relative risks of developing lung cancer, adjusted for age, gender, recruitment year, pack-years and smoking status, for increasing grades of cytologic atypia were 1.0 (normal), 1.10 (mild atypia), 1.68 (moderate atypia), 3.18 (moderate atypia or worse) and 31.4 (worse than moderate atypia) [67]. Sputum cytologic atypia of severe dysplasia or worse clearly carries a risk of lung cancer that is high enough to warrant an aggressive diagnostic approach with combined white-light and fluorescence bronchoscopy. Two groups have reported results of bronchoscopy in subjects with moderate atypia sputum cytology
118 and chest radiographs negative for carcinoma. Fujita reported, in abstract form only, a series of 25 subjects with moderate atypia sputum cytology and negative chest radiographs who underwent bronchoscopy. Two had carcinoma diagnosed [68]. Kennedy and colleagues have reported a series of 79 subjects with moderate atypia sputum cytology and chest radiographs negative for cancer [69]. Of the 79, lung cancer was found at bronchoscopy in 5 (6.3%; 95% CI = 0.7—11%). Two of the cancers were carcinoma in situ lesions and three were invasive. The rates of discovery of cancer at bronchoscopy reported in these studies exceeded the rate of discovery of colon cancer when colonoscopy is performed for a positive fecal occult blood test. While moderate atypia in sputum cytology is not as compelling an indication for bronchoscopy as is severe atypia and the high rate of discovery of unexpected lung cancer in the reported studies may be partly due to chance, publication bias or a selection bias in which only subjects with moderate atypia and additional health problems were examined, consideration of bronchoscopy is certainly reasonable in a concerned patient with moderate atypia. For patients with early lung cancer who are being assessed for curative surgical resection and those with carcinoma in situ who are being evaluated for endobronchial therapies, autofluorescence bronchoscopy is useful in the delineation of tumour margins and to assess the presence of synchronous lesions in the bronchial tree [48,49,70]. Synchronous cancer can be found on autofluorescence bronchoscopy in up to 14% of these patients. Up to 27% of patients may also have other moderate/severe dysplastic lesions that will require bronchoscopic followup [27,29,48,70]. Carcinoma in situ is a potentially curable lesion with endobronchial therapy such as photodynamic therapy, electrocautery or cryotherapy if the lesion is ≤1 cm2 [49,71]. However, white light bronchoscopy is inadequate both for detection and for delineation of the margins of these lesions, whereas autofluorescence bronchoscopy improves staging of these occult cancers and has an impact on their management [49,60]. In addition, during endobronchial therapy with techniques such as electrocautery, autofluorescence can be used to ensure that the lesion is adequately treated. The role of autofluorescence bronchoscopy for surveillance of second primary lung cancer in patients who have curative therapy for non-small cell lung cancer or limited stage small cell lung cancer was examined in several studies. This group of patients has a relatively high rate of development of metachronous tumours; 1—5% per year after non-small cell lung cancer resection and 2—13%
H. Zeng et al. after small cell lung cancer [71—76]. The number of patients in these studies were small. Lesions were identified in 3—6% of these patients who were thought to be disease free [39—41,77]. Patients with prior squamous cell carcinoma appear to be a population that may warrant prospective study of postoperative fluorescence bronchoscopic surveillance [41]. Likewise, the prevalence of preinvasive lesions in patients with cancer in the upper aerodigestive tract (head and neck cancer excluding nasopharyngeal cancer or esophageal cancer) may be sufficiently high due to the field cancerization effect of inhaled tobacco carcinogens to warrant a fluorescence bronchoscopy prior to curative therapy or for surveillance for second primary cancers. In patients with esophageal cancer up to 19% may have synchronous or metachronous cancers in other organs, and lung cancer contributed to approximately 10% of these second tumours [78]. In laryngeal cancer patients, up to 28% have been reported to have synchronous or metachronous tumours, and lung cancer was the most frequent second malignancy, contributing to 41% of second tumours [29,79,80]. Currently, there are no established evidencebased guidelines to aid physicians in the ongoing surveillance of lung cancer patients following curative intent therapy [81]. Although there has been stimulating research into lung cancer screening over the last decade with the emergence of techniques such as low dose thoracic CT scans, autofluorescence bronchoscopy and new sputum biomarkers, we ultimately need the ability to both detect and reduce the development of metachronous pulmonary malignancies to impact lung cancer mortality. Evidence based follow-up protocols need to be developed and their benefits need to be evaluated in larger prospective clinical trials.
Research applications of optical spectroscopy and imaging Autofluorescence bronchoscopy also has a significant role in ongoing lung cancer research. The study of the natural history and genetic alterations in preneoplastic lesions accessible to the fiberoptic bronchoscope will lead to a better understanding of lung cancer pathogenesis and progression. It will in turn lead to development of better biomarkers for early detection and novel molecular targets for intervention. Preliminary study suggests that fluorescence bronchoscopy plays an important role in an early lung cancer detection program in conjunction with spiral CT [10]. The ability to identify pre-neoplastic
Optical spectroscopy and imaging for early lung cancer detection
Figure 5 The mean Raman spectra of normal bronchial tissue (n = 12), adenocarcinoma (n = 6) and squamous cell carcinoma (n =1 0). Each spectrum was normalized to the integrated area under the curve to correct for variations in absolute spectral intensity. Each spectral peak can be assigned to specific biomolecules inside the tissue such as nucleic acids and proteins. The Raman signatures for tumour are significantly different from normal tissue (with permission from ref. [83]).
lesions and perform serial biopsies before and after treatment provides an intermediate endpoint to evaluate novel agents for chemoprevention of lung cancer [82]. Very recently, the promising but technically challenging Raman spectroscopy has also been applied to improve lung cancer detection [83]. Raman signals are very weak but provide fingerprint type signatures of specific molecules in tissue. This study demonstrated that using a specially designed rapidacquisition NIR Raman spectroscopy system, Raman spectra could be obtained from fresh bronchial tissue samples from biopsy or surgical resection in 5 s [83]. Fig. 5 shows the normalized mean Raman spectra of normal and cancerous bronchial tissues. It can be seen that significant Raman spectral differences exist between normal and tumour tissue, while Raman spectra of adenocarcinoma and squamous cell carcinoma are very similar to each other. The ratio of Raman intensities at 1445—1655 cm−1 provided good differentiation between normal and tumour tissues. Tumours showed higher percentage Raman signals for nucleic acid, tryptophan and phenylalanine, but lower percentage signals for phospholipids, proline and valine, compared to normal tissue (Fig. 5). The results of this exploratory study indicate that NIR Raman spectroscopy may have significant potential for improving lung cancer diagnosis based on optical evaluation of biomolecules. The development of an endoscopic Raman probe for in vivo Raman spectral measurements is underway.
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In the future, Raman spectroscopy may be used to reduce the false positive rate from AFB imaging detection. Raman spectroscopy may also be used to identify the structural and compositional differences on proteins and genetic materials between malignant lung cancers, their pre-curses, and normal lung tissues. This knowledge will lead to better understanding, on the biochemical bases, of the evolution process of lung cancers from benign to malignancy. The biochemical information obtained from in vivo Raman measurements may also be helpful for predicting the malignancy potential of pre-invasive and invasive lung cancers.
Summary Among all types of optical imaging and spectroscopy techniques, autofluorescence bronchoscopy is the most successfully developed. AFB is a sensitive technique for detection of intraepithelial neoplasia. In conjunction with white-light bronchoscopy, it has a definite clinical role in localization of preinvasive lung cancer in patients with abnormal sputum cytology and determining the extent of endobronchial spread in patients with early lung cancer prior to curative therapy. It may also play a role in patients with potentially curable oesophageal and head and neck cancer in detection of second primary lung cancer, as well as in the postoperative surveillance of lung cancer patients, but further studies are required. Fluorescence bronchoscopy plays an important part in lung cancer research particularly in the study of the natural history and the molecular biology of pre-neoplastic lesions, in lung cancer screening clinical trials as well as Phase II trials of chemopreventive agents. The decreased specificity associated with AFB could be improved by new technologies currently under development such as Raman spectroscopy and simultaneous AFB imaging with fluorescence spectroscopy and simultaneous WLB imaging with reflectance spectroscopy.
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