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Published ahead of print on February 11, 2005, doi:10.1164/rccm.200501-046OC
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American Journal of Respiratory and Critical Care Medicine Vol 171. pp. 1178-1184, (2005)
© 2005 American Thoracic Society
doi: 10.1164/rccm.200501-046OC

Original Article

Measurement of Hypoxia-related Parameters in Bronchial Mucosa by Use of Optical Spectroscopy

Martin P. L. Bard, Arjen Amelink, Vincent Noordhoek Hegt, Wilfried J. Graveland, Henricus J. C. M. Sterenborg, Henk C. Hoogsteden and Joachim G. J. V. Aerts

Department of Respiratory Diseases, Center for Optical Diagnostics and Therapy; Departments of Radiation Oncology and Statistics, Erasmus Medical Centre Rotterdam; and Departments of Pathology and Respiratory Diseases, Sint Franciscus Hospital, Rotterdam, the Netherlands

Correspondence and requests for reprints should be addressed to Joachim Aerts, M.D., Ph.D., Department of Respiratory Diseases, Sint Franciscus Hospital, Kleiweg 500, Rotterdam 3045 PM, the Netherlands. E-mail: j.aerts{at}sfg.nl


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Tumor hypoxia has both prognostic and therapeutic consequences for solid tumors. We developed a novel noninvasive technique, differential path-length spectroscopy (DPS), which allows the measurement of hypoxia-related parameters in the superficial microvasculature of tissue. Objectives: The aim of this study was to measure the microvascular oxygenation of histologically normal endobronchial mucosa and of neoplastic lesions during bronchoscopy using DPS. Methods: Sixty-four patients with known or suspected malignancies of the lung were studied. One hundred and five endobronchial lesions (38 histologically normal, 37 metaplastic/mild dysplastic lesions, and 30 invasive carcinomas) were detected by white and/or autofluorescence bronchoscopy and measured using DPS. Results: We observed that bronchial tumors are characterized by a lower blood oxygen saturation and a higher blood content than normal mucosa. No differences were observed between normal and metaplastic/mild dysplastic mucosa. Conclusion: DPS is a new optical technique allowing the noninvasive study of endobronchial tumor hypoxia.

Key Words: cancer • hypoxia • lung • optical spectroscopy • premalignant

Tumor hypoxia has been associated with a lower survival rate, a higher degree of invasiveness, and a higher risk of regional and distant metastases in uterine cervical carcinoma (1–4), head and neck carcinoma (5, 6), and soft tissue sarcoma (7). In addition, hypoxia limits the response of tumor cells to ionizing radiation and to some chemotherapies (8, 9). Therefore, tumor hypoxia has both prognostic and therapeutic consequences, indicating the importance of its measurement (8).

In vivo measurement of tissue oxygenation is challenging in the lung. Invasive techniques, such as polarographic oxygen microelectrodes, cannot be used in the lung because of the inaccessibility of the bronchial tree. Histologic techniques, such as the study of exogeneous (pimonidazole) and endogeneous (hypoxia inducible factor, carbonic anhydrase IX, vascular endothelial growth factors) hypoxic factors, or the measurement of tissue microvessel density (MVD), are indirect methods that can only be used ex vivo on biopsy specimens (9). Functional imaging techniques, such as magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography, are noninvasive techniques that have the advantage to investigate nonaccessible organs such as the lungs and to give information about the metabolic activity, the blood flow, and the microcirculation of tumors (10–13). However, these techniques are expensive, are limited by their spatial resolution hampering their use in case of tumors of size less than 1 cm, and are not sensitive to the early changes associated with cancerous transformations occurring in the epithelium of mucosa.

Noninvasive measurement of relevant parameters in the superficial layer of bronchial mucosa would be particularly interesting because the majority of lung cancers arise in the epithelium and are preceded by precancerous changes that affect only the surface epithelium. Previous authors have reported that both the expression of hypoxic factors and an increased tumor MVD are related to a poor prognosis in lung cancer (14, 15). Interestingly, it was also reported that these hypoxia-related changes could be observed in cases of early-stage (intraepithelial) bronchial carcinomas (16, 17) and in cases of premalignant lesions, such as dysplasia. These results suggest that the modifications of the bronchial mucosal oxygenation occur early during oncogenesis. An in vivo confirmation of these findings requires the development of an original technique sensitive to the oxygenation changes occurring in the bronchial epithelium.

White-light reflectance spectroscopy is a noninvasive technique that allows the analysis of tissue optical properties. Because blood is the dominant light absorber in the visible wavelength range, both the local blood content and the blood oxygen saturation can be extracted from reflected light signal. Visible and near-infrared diffuse reflectance spectroscopy has been used in animal models and in humans to analyze the oxygenation of tumoral tissues (18–22). However, the reflectance spectroscopic techniques used by these authors were characterized by large source-detector separations and a relatively long path length. As a consequence, the detected photons have traveled a long distance through the tissue, and the extracted optical properties represent average values over a relatively large tissue volume. This results in a decreased sensitivity of these optical techniques to changes in the epithelial tissue layer. We recently developed a novel spectroscopic technique, differential path-length spectroscopy (DPS), that allows the in vivo measurement of blood oxygenation, blood volume, and vessel diameter in the most superficial layer of tissue (23). This fiber-based technique can be used during an endoscopic procedure, and preliminary data in the bronchial tree have shown that cancerous bronchial mucosa has a lower capillary oxygenation, a higher blood volume fraction, and a larger average capillary diameter than normal bronchial mucosa (24).

The aim of this study was to measure the oxygenation of histologically normal bronchial mucosa and of endobronchial neoplastic lesions during bronchoscopy in a large population of patients to confirm our preliminary results for a larger variety of histologic tissue types.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Population
Patients with known or suspected malignancies of the lung and with a medical indication for a bronchoscopy were invited to participate. All patients were older than 18 years and provided signed informed consent. The study was approved by the Medical Ethics Review Board of the Erasmus Medical Center Rotterdam, the Netherlands.

Reflectance Probe
Spectra were measured using a custom-made instrument using a fiber-optic probe small enough to be led through the 2.8-mm working channel of the bronchoscope (Figure 1). The fiber probe consisted of two 400-µm-diameter optical fibers fitted into a small metal tube. The two fibers touch one another to minimize the distance between them. One fiber (delivery and collection dc-fiber) is used for both delivery and detection of light. The second fiber (collection c-fiber) is used for only detection of reflected light from the tissue. A tungsten-halogen lamp (Model HL-2000-FHSA; Ocean Optics, Duiven, the Netherlands) is used to light up the bronchial mucosa through the dc-fiber, and the remitted light collected in the dc- and c-fiber is analyzed in a dual-channel spectrometer (SD2000; Ocean Optics). The data presented in this article are part of a larger study using a combination of reflectance and autofluorescence spectroscopy during bronchoscopy for the detection of bronchial (preneoplastic) lesions (25). The autofluorescence of the bronchial lesions is induced by a blue-violet light source, and the autofluorescence spectra are collected through the c-fiber. To attenuate the short-wavelength background caused by the blue-violet light source, the autofluorescence spectra are filtered through a long-pass glass filter (GG435; Schott, Tiel, the Netherlands). As a consequence, the white-light reflectance spectra measured by the c-fiber are very noisy below 435 nm.



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  		Figure 1. Setup used for the differential path-length spectroscopy (DPS) measurement in the bronchial tree. The bifurcated optical fiber probe is small enough to be led through the working channel of the bronchoscope. A tungsten-halogen lamp is used to light up the bronchial mucosa through the dc-fiber, and the remitted light collected in the dc- and c-fiber is analyzed in a dual-channel spectrometer.

 
The difference of the dc- and c-fiber collection signals is the differential reflectance signal R({lambda}). In the range of parameters relevant for biological tissue, we have previously reported that the differential reflectance signal R({lambda}) can be simply modeled by the following equation: R({lambda}) = C1µs'({lambda})exp[–{tau}µa({lambda})], where µs'({lambda}) is the reduced scattering coefficient, µa({lambda}) is the absorption coefficient, {tau} is the apparent differential path length, and C1 is a proportionality constant (23). We have previously demonstrated that the apparent differential path length ({tau}) is independent of the optical properties of the tissue and depends on the fiber diameter only. For fibers with a diameter of 400 µm, the apparent differential path length is only 320 µm, allowing the analysis of photons reflected in the most superficial layer of tissue (within ~ 160 µm of the surface).

Examination Procedure
The endoscopic examination of the bronchial tree was performed with a commercially available flexible fluorescence bronchoscope (11004BI; Karl Storz, Tuttlingen, Germany). All lesions that appeared abnormal at blue- and/or white-light imaging were measured. The probe was led through the working channel of the bronchoscope and placed in gentle contact with the bronchial mucosa (Figure 2). The duration of reflectance spectral acquisition was less than 1 second during which the light source of the bronchoscope was switched off. An average of three measurements was done on each location to take into account the tissue heterogeneity. Finally, bronchial biopsies of the lesions were obtained. Biopsy specimens were transported in formaldehyde and fixed in paraffin. Hematoxylin-eosin–stained slides were evaluated without knowledge of the bronchoscopic and spectroscopic findings. The pathologic diagnoses were coded referring to the World Health Organization lung cancer classification (26).



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  		Figure 2. Three-stage examination procedure for DPS measurements in the bronchial tree. (A) Lesions of the bronchial mucosa are detected with a flexible fluorescence bronchoscope (white light image). (B) The probe is led through the working channel of the bronchoscope and placed in gentle contact with the bronchial mucosa. (C) Bronchial biopsies of the bronchial lesion are obtained.

 
Analysis of Spectra
In accordance with Lorentz-Mie scattering theory, we assumed that µs'({lambda}) = {alpha}{lambda}–b, where the Mie parameter (b) is a constant related to the size of the scattering particles (27). Furthermore, the main absorber in the measured wavelength range (350–1,000 nm) is blood. The complete model to which the differential path-length spectra are fitted is given by the following equation (24):

(1)
where {rho} is the blood volume fraction, StO2 is the microvascular blood oxygenation, µaHbO2({lambda}) is the absorption coefficient of fully oxygenated whole blood, µaHb({lambda}) is the absorption coefficient of fully deoxygenated whole blood, C1' is a proportionality constant, and Ccor is the correction factor that accounts for the inhomogeneous distribution of blood in tissue (28). For whole blood contained in infinitely long cylindric vessels, the correction factor is given by Ccor = {1 – exp[µabl({lambda})Dvessel]}/[µabl({lambda})Dvessel], where µabl({lambda}) is the absorption coefficient of whole blood and Dvessel is the vessel diameter. Blood volume fraction ({rho}) and microvessel diameter (Dvessel) are extracted from Equation 1 assuming cylindric vessels and a blood hemoglobin content of 150 g/L, which is typical for whole blood. However, the size and shape of capillaries vary in the microvasculature, and the hematocrit of blood in the capillaries is lower than the hematocrit of whole blood and may vary between normal and cancerous tissue. As a consequence, the fitted values for these parameters may actually deviate from their absolute values. An additional difficulty for proper analysis of the vessel diameter arises from the large noise observed in the reflectance spectra below 435 nm caused by the presence of the long-pass glass filter. The measured reflectance signal is affected by the size of the blood vessels most notably around 420 nm; the large noise in that wavelength region reduces the accuracy in the fit of the vessel diameter considerably.

Fitting the data to our equation yields values for the local blood oxygenation (StO2), local blood volume fraction ({rho}), apparent average vessel diameter (Dvessel), and the Mie parameter (b). An additional parameter was chosen to appreciate the amplitude of the scattering signal. This scattering amplitude parameter (a) corresponds to the amplitude of the scattering signal measured at 800-nm wavelength, where blood absorption is minimal.

Statistical Analysis
The difference of optical parameters between the normal, metaplastic/mild dysplastic, and cancerous lesions were evaluated with a Kruskal-Wallis test (29). We have chosen this test because some optical parameters have a skewed distribution, which makes the Student t test inappropriate. p values less than 0.05 were regarded as significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Endobronchial Lesions
Sixty-four patients (39 men and 25 women with a median age of 65 ± 12 years) were studied during a 1-year period. One hundred and ten endobronchial lesions were detected by white and/or autofluorescence bronchoscopy and measured using DPS. An adequate amount of lesions was obtained in three histologic types: normal mucosa (38 lesions), metaplastic/mild dysplastic mucosa (37 lesions), and invasive carcinoma (30 lesions). The histologic repartition of the invasive carcinomas were as follows: 10 squamous cell, 9 adenocarcinomas, 5 large cell carcinomas, and 6 non–small cell carcinomas. In addition, two severe dysplastic/carcinoma in situ (CIS) lesions and three necrotic lesions were measured, but because of their low numbers, no statistical analysis was performed on these types of lesions.

Reflectance Spectra
Figure 3 illustrates four examples of DPS spectra measured in normal bronchial mucosa with increasing local blood volume fractions ({rho}). The blood volume fraction ({rho}) is proportional to the depth of the signal dips observed below 600 nm corresponding to the absorption of light by hemoglobin (Figure 3). Figure 4 illustrates examples of DPS spectra, fits, and residues measured in normal (Figure 4, upper panel) and cancerous (Figure 4, lower panel) mucosa, respectively. Note the difference in the shape of the blood absorption signals for wavelengths below 600 nm caused by a difference in microvascular saturations (StO2). A high StO2 (100%) is measured for the normal mucosa (Figure 4, upper panel), and a low StO2 (45%) is measured for the cancerous lesion (Figure 4, lower panel).



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  		Figure 3. Illustration of four DPS spectra (A–D) with increasing local blood volume fraction ({rho}) measured in normal bronchial mucosa. The signal dips observed below 600 nm are caused by the absorption of blood.

 


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  		Figure 4. Illustration of DPS spectra, fits, and residues (= spectrum – fit) measured on normal (upper panel) mucosa and on a cancerous (lower panel) lesion. A difference in oxygen blood saturation (StO2) is observable through a difference in the shape of the blood absorption dips below 600 nm.

 
Because both the StO2 and the apparent average vessel diameter (Dvessel) are related to the shape of the blood absorption dips below 600 nm, the accuracy of the fits for these two parameters is limited in case of small blood volumes (see, e.g., Figure 3A). Therefore, we decided to restrain the calculation of these two parameters to spectra for which the blood volume fraction ({rho}) was higher than 1% (24). As a consequence, these parameters were calculated for 25, 27, and 28 normal, metaplastic/mild dysplastic, and invasive carcinomas, respectively. In addition, Figure 5 illustrates an example of a DPS spectrum measured in necrotic cancerous tissue. Compared with spectra measured in normal mucosa or nonnecrotic cancerous mucosa, an additional absorption dip is visible in the 600- to 650-nm wavelength range. This additional absorption dip is related to an (as yet unidentified) additional absorber present in the necrotic tissue that is not accounted for in Equation 1 but should be incorporated for adequate fitting.



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  		Figure 5. Illustration of a DPS spectrum measured in necrotic cancerous tissue showing an additional absorption dip in the 600–650-nm wavelength range.

 
Two parameters extracted from the spectra contain information about the scattering properties of the tissue: the Mie parameter (b), which corresponds to the slope of the DPS spectra, and the scattering amplitude parameter (a), which corresponds to the amplitude of the scattering signal at 800 nm. Both the slope (b) and amplitude (a) are not affected by the local blood volume fraction ({rho}) because they are measured in a wavelength range where the absorption of blood is negligible.

Optical Parameters
Multiple measurements were systematically done on each bronchial lesion to take into account the intralesion heterogeneity. The optical parameters extracted from all the spectra measured on the same lesion were averaged, and these averaged values were used to optically characterize each bronchial lesion. The bronchial lesions were clustered into three histologic groups: normal mucosa, metaplastic/mild dysplastic mucosa, and invasive carcinoma. Table 1 summarizes the average values and standard deviations of the optical parameters in each of these three histologic groups. Invasive carcinomas were characterized by a lower oxygen saturation, a higher blood volume, and a larger average vessel diameter compared with normal or metaplastic/mild dysplastic mucosa (statistically significant for blood saturation and blood volume, p < 0.001 using a Kruskal-Wallis test; not significant for the vessel diameter). The scattering amplitude parameter (a) was significantly lower in cancer (p < 0.001 using a Kruskal-Wallis test), but no significant difference in the scattering slope (b) was observed between invasive carcinoma and both normal and metaplastic/mild dysplastic mucosa. Furthermore, no significant difference in any of the parameters was observed between normal and metaplastic/mild dysplastic lesions.


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  		TABLE 1. Average values and sds of blood oxygenation, vessel diameter, blood volume, mie parameter, and scattering amplitude parameter measured in normal mucosa, metaplastic/mild dysplastic mucosa, and invasive carcinoma of the bronchial tree

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We used DPS to study the oxygenation and the microvasculature of bronchial lesions in vivo. This is to our knowledge the first report of an optical analysis of human bronchial mucosa during bronchoscopy. DPS allows a real-time and noninvasive measurement of tissue hypoxia–related parameters, such as the blood oxygenation, the blood content, and the microvessel size. Moreover, DPS informs about the scattering properties of the bronchial mucosa. We observed that endobronchial tumors are characterized by a lower blood oxygenation, a higher blood content, and a smaller scattering amplitude than normal or metaplastic/mild dysplastic bronchial mucosa.

Bronchial epithelium is the location of premalignant lesions and in situ carcinomas, which are precursors of the majority of lung cancers. The development of an instrument allowing the in vivo analysis of this superficial layer would help to better understand the pathophysiology of these early lesions and possibly improve their detection. Because DPS uses a fiber-optic probe that is guided through the working channel of a bronchoscope, the technique is limited to lesions of the proximal bronchial tree. We have demonstrated in tissue phantoms that DPS using 400-µm fibers is sensitive to the light scattered in approximately the first 150 µm of the tissue surface (30). Such depth corresponds to the epithelium thickness encountered in cases of metaplastic/dysplastic lesions or in situ carcinomas. In cases of invasive carcinomas, the tumor thickness can attain several centimeters, and DPS informs only about the most superficial layer of these larger tumors. At this point, we must emphasize that DPS was developed with the intention to study the microvasculature and the scattering properties of the most superficial layer of bronchial tissue only. Little is known about the relation between the vasculature at the bronchial epithelial surface and at the center of larger tumors. The use of an animal lung cancer model would allow the study of the correlation between the superficial and deep bronchial tumor oxygenation, but this is beyond the scope of this article.

Tumor hypoxia is a heterogeneous process that depends on several factors, including an increased consumption of oxygen related to the high metabolism and high proliferation rate of the cancerous cells, a lower quality of the tumoral microcirculation related to architectural and functional abnormalities of the tumor microvessels, a decreased capacity of oxygen to diffuse to the cells because of the increased distance between the microvessels and the cancerous cells, and a sluggish blood flow in the tumor microcirculation as a consequence of the increased resistance in the microvessels and the increased viscosity of the blood (8, 9). A correct description of the oxygenation of solid tumors requires the concomitant study of these various parameters (31). It must be noted that DPS measures only a part of these hypoxia-related parameters—that is, blood oxygenation, blood volume, and microvessel size. DPS does not measure blood flow, nor does it measure the MVD. An increased MVD would result in an increased blood volume fraction ({rho}), but an increased blood volume fraction may also be caused by an increase in the microvessel size. As a consequence, we cannot simply relate our parameters to commonly used parameters such as MVD. However, alternative techniques for the in vivo assessment of hypoxia-related parameters, such as tissue oxygenation, MVD or blood flow in bronchial mucosa, do not exist to our knowledge. Moreover, exogeneous hypoxia markers, such as pimonidazole, are hard to measure in the small-size bronchial biopsies obtained during bronchoscopy, and large tumor samples are generally not available in the majority of patients suffering from lung cancer.

Tumor hypoxia has previously been reported in various kinds of solid cancers (2, 4, 5, 32). Oxygenation of tumors is facilitated by the creation of new blood vessels by the tumor itself (neoangiogenesis). This process is regulated depending on a balance of angiogenic and angiostatic factors (33). In response to hypoxemia, tumors secrete angiogenic cytokines, such as vascular endothelial growth factor, inducing the formation of microvessels from the surrounding host vasculature. However, the delivery of oxygen to the neoplastic and stromal cells is frequently altered by structural abnormalities of the microcirculation. We observed that hypoxia is a common event in the mucosal capillary blood of bronchial tumors. In addition to the measurement of capillary blood oxygenation, DPS allows us to study the microvascular geometry of the superficial layer of bronchial mucosa. An increased blood content and enlarged microvessel diameters were observed in bronchial tumors in comparison with normal mucosa, although the latter was not significant. The fact that no significant differences were observed in microvessel size may at least in part be explained by the low signal-to-noise ratio below 435 nm, as explained in METHODS. Lung tumors are known to be characterized by an increase in microvessel count (33). An increase in the size of microvessels is also characteristic of the tumoral microvasculature. Tumor blood vessels are commonly tortuous and irregular, have venous shunts and blind ends, and lack smooth muscles and innervation (34). This increase in vessel size, associated with a decrease of local blood pressure and an increase of blood viscosity, induces tumor blood flow alterations leading to hypoxia (35).

The development of hypoxia and microvasculature alterations during oncogenesis is poorly known. Neoangiogenesis has been reported to be increased in smokers and in bronchial preneoplastic lesions with the expression of angiogenic and proliferation markers and an increase of the MVD (36–38). Moreover, the increased microvascular density observed in solid cancer is directly linked to the angiogenesis process, and previous authors have reported a correlation between increased vessel density and tumor stage (39, 40). A direct visualization of the microvasculature in bronchial mucosa has recently been reported using high-magnification bronchovideoscopy (41, 42). These authors have shown that larger blood vessels were present in dysplastic lesions of the bronchial mucosa. We observed no significant change in oxygenation, blood content, and microvessel size in the metaplastic/mild dysplastic mucosa in comparison with normal mucosa. However, these alterations of the bronchial mucosa are frequent (particularly in smokers), tiny, and mostly spontaneously reversible, and are not considered to be premalignant. Our data show that the onset of macroscopic changes in the microvasculature of bronchial mucosa must occur at a later stage than metaplasia/mild dysplasia. Unfortunately, concerning the alterations occurring in more advanced mucosal lesions, such as severe dysplasia and carcinoma in situ, no conclusion can be drawn from the very small number of these types of lesions measured in our study. The use of DPS during high-magnification bronchoscopy would be particularly interesting, allowing a comparison of these two different techniques for the study of the bronchial microvasculature.

When light enters tissue it is scattered whenever it encounters refractive index variations. The fact that membranes, nuclei, mitochondria, and other organelles all have a different refractive index from the surrounding cytoplasm makes tissue a highly scattering medium. Although the detailed dependence of the scattering signal on the tissue composition is not completely understood, most experimental evidence suggests that the mitochondria contribute most to the light scattering in the backward directions (43–46). In our study, the Mie parameter (b) was not modified in cancerous tissue compared with normal or metaplastic tissue, suggesting that the size of the light scatterers in the cancerous epithelium are not significantly modified. The decreased amplitude of the scattering signal observed in bronchial tumors may be related to a decrease in the mitochondrial content of tumors (44) or to changes in the refractive index of the cytoplasm because of an increased protein and enzyme content, but this is highly speculative and requires further investigation into the microscopic origins of light scattering in tissue.

Recent improvements in the endoscopic technology, such as fluorescence endoscopy and high-magnification videoscopy, have allowed the development of highly sensitive bronchoscopes for the detection of bronchial mucosal lesions. However, these bronchoscopic techniques are characterized by a low specificity (i.e., a lot of false-positives), inducing unnecessary biopsies at greater costs and a longer duration of the endoscopic examination. The concomitant use of DPS during bronchoscopy may be helpful to increase the specificity of bronchoscopy. However, it must be emphasized that the objective of the present study was to measure the oxygenation and the microvasculature in the most superficial layer of tissue noninvasively during routine bronchoscopy for various histologic tissue types. The fact that statistically significant differences are found between the microvascular saturations of normal and cancerous tissue does not imply that it is possible to use the measured saturation for classification purposes. The classification of lesions based on saturation measurements alone requires not only a statistically significant difference in the mean values of the groups but a relatively small standard deviation within each group as well. The latter condition is clearly not fulfilled for our data, and any attempt to classify the lesions using the extracted saturation parameter (StO2) alone will be futile. The question of whether DPS can be used to classify lesions using more advanced statistical techniques (e.g., using combined classifiers and linear discriminant analysis) will be answered in a separate study.

In conclusion, DPS allows a real-time, noninvasive analysis of the oxygenation, the microvasculature, and the scattering properties in the most superficial layer of tissue. This technique is particularly interesting for the analysis of cancer developing in superficial tissues, such as epithelium. Its use during an endoscopic procedure is easy and well tolerated, and opens up a wide field of investigation in various organs. Potentially, DPS can be used for a better understanding of the oxygenation alterations that occur during the bronchial oncogenesis, an improvement of our capacity to detect the preneoplastic and intraepithelial cancers, the analysis of the prognostic significance of endobronchial tumor oxygenation, the survey of antihypoxia or antiangiogenesis therapies, and the real-time monitoring of oxygen-dependent therapies, such as radiotherapy or photodynamic therapy.


   FOOTNOTES
 
Supported by the Dutch Technology Foundation (STW).

Conflict of Interest Statement: M.P.L.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; V.N.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; W.J.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; H.J.C.M.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; H.C.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.G.J.V.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form January 11, 2005; accepted in final form February 7, 2005


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 DISCUSSION
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