Lesson 8, Volume 15—Fluorescence and Ultrasound Bronchoscopy

By Stephen Lam, MD, FCCP; and Heinrich D. Becker, MD, FCCP

Objectives

1. Outline the rationale for developing fluorescence and ultrasound bronchoscopy.
2. Characterize the differences in tissue autofluorescence in normal, preinvasive, and invasive lung cancer.
3. Understand how tissue autofluorescence can be used to detect early lung cancer.
4. Describe the clinical trial results of fluorescence bronchoscopy.
5. Understand endobronchial ultrasound technology.
6. Describe the role of endobronchial ultrasound in lung cancer staging.
7. Describe the clinical results of endobronchial ultrasound.

Key words

endobronchial ultrasonography (EBUS); fluorescence bronchoscopy; lung cancer

Abbreviations

NAD = nicotinamide-adenine dinucleotide; NADH = the reduced form of NAD

Lung cancer is the most common cause of cancer death in the United States and many parts of the world. There are more patients dying from lung cancer than breast, colorectal, and prostate cancers combined. Former smokers retain an increased risk for lung cancers years after they stop smoking. With a large reservoir of current and former smokers and the increasing incidence of lung cancer among women, lung cancer will remain a major health issue for several decades. Less than 15% of all patients diagnosed with invasive lung cancer survive 5 years or more after treatment. Primary prevention measures aiming at curbing tobacco smoking, especially among young people, should remain a priority of government policy. However, in order to significantly reduce lung cancer mortality over the next several decades, evaluation, implementation and further development of novel strategies for the detection of early, preinvasive, or microinvasive disease is required. Curative treatments such as photodynamic therapy, electrocautery, or cryotherapy are now available in addition to surgery for early, preinvasive, or microinvasive lung cancer.¹

Rationale for Fluorescence and Ultrasound Bronchoscopy

At the present time, the only noninvasive method that can detect preinvasive (stage 0) lung cancer is sputum cytology examination. By standardizing the collection procedure, processing method, and interpretation criteria, Kennedy and coworkers² detected carcinoma in situ in 1.2% of current and former smokers with COPD. Moderate and severe atypia were found in another 24%. Over 10% of the participants with high-grade atypia have developed lung cancer during follow-up (T. Kennedy, MD, FCCP; personal communication; 2000). Newer methods such as computer-assisted image analysis of exfoliated sputum cells,³ immunostaining of transformed epithelial cells,⁴ and polymerase chain reaction–based assays to detect mutations in nuclear or mitochondrial DNA^5,6 hold promise to detect early lung cancer with even higher sensitivity. However, more sensitive methods would mean that the size of the lesions discovered by these tests would likely be smaller than that found by conventional sputum cytology examination.

Preinvasive bronchial cancers are usually very small. A study by Woolner⁷ showed that the median surface diameter of carcinoma in situ was 8 mm. Since these lesions are small and only a few cell layers thick, they may not produce any changes on gross examination or may only appear as thickening of the bifurcation, loss of the normal mucosal sheen, slight granularity of the epithelial surface, or mild stenosis. As a result, only 30% of these lesions were visible to experienced bronchoscopists on conventional white-light bronchoscopy prior to surgery in the study by Woolner.⁷ Obvious lesions, such as polypoid or nodular lesions, unless < 5 mm, are usually invasive. Loss of the circular striations, widening of the longitudinal folds, or increase in vascularity indicates tumor invasion to the subepithelial layer or extension to the peribronchial space. If there is no visible abnormality, brushing of individual segmental bronchi or repeated bronchoscopies are required for localization. The size of dysplastic lesions has not been studied in detail. A recent study on the size of clonal patches suggested that many dysplastic lesions might be only 200 cells in cross-sectional diameter.⁸ These lesions usually have no visible abnormality on white-light bronchoscopy or show nonspecific thickening that cannot be distinguished from inflammation or squamous metaplasia without dysplasia. The recent development of fluorescence bronchoscopy allows bronchoscopists to visualize and directly biopsy lesions suspicious of carcinoma in situ or high-grade dysplasia for pathologic confirmation.⁹

When a bronchial biopsy shows carcinoma in situ, the decision regarding endobronchial treatment versus surgical resection is dependent on whether part of the tumor has already invaded deep into the subepithelial layers or even extrabronchially. If the tumor has already invaded beyond the cartilage layer, it is usually not curable by endobronchial treatment such as photodynamic therapy.¹⁰ The depth of tumor infiltration can now be determined by endoscopic ultrasound.¹¹

Principles of Tissue Autofluorescence

When the bronchial surface is illuminated by light, the light can be back-scattered, absorbed, or transmitted, or it can induce autofluorescence. Conventional white-light bronchoscopy (reflectance imaging) makes use of the absorption and back-scattering properties of bronchial tissues to broad-band visible light. It provides information on the structure of the bronchial tree such as the architecture or luminal diameter of the airways and morphologic features such as mucosal thickness, sheen, smoothness, or vascularity.

Biochemical or functional changes in tissue may not be evident from examining the morphologic changes alone. Tissue autofluorescence reflects the electronic structure of absorption chromophores. The major chromophores in bronchial tissues are elastin, collagen, flavins, nicotinamide-adenine dinucleotide (NAD) and the reduced form of NAD (NADH), and porphyrins.¹² When these chromophores are excited by light of specific wavelengths to higher electronic states, fluorescence is emitted when the electrons return to ground level. Most of the tissue fluorescence is from the subepithelial layers; the epithelium itself contributes < 5% of the overall fluorescence detectable on the bronchial surface.^13,14

Upon illumination by violet or blue light (400 to 450 nm), normal tissues fluoresce strongly in the green (500 to 520 nm).¹⁵ As the bronchial epithelium changes from normal to dysplasia, carcinoma in situ, and then invasive cancer, there is a progressive decrease in the fluorescence intensity, especially in the green region, with comparatively less reduction in the red.15 This reduction in fluorescence intensity is due to a decrease in the concentration of short-lifetime chromophores such as reduced or protein-bound flavins, increase in the epithelial thickness that impedes the emission of the fluorescent light to the bronchial surface, and an increase in angiogenesis in premalignant and malignant tissues.^12-16

Fluorescence Bronchoscopy Devices

The differences between normal and abnormal tissues are very subtle. They are not detectable by the unaided human eye unless the lesion is very large. Image-intensified cameras are usually required for fluorescence detection of small preinvasive and microinvasive bronchial cancers. Direct observation of tissue autofluorescence had been tried in 1933,¹⁷ but this approach was abandoned in the 1950s because of the poor sensitivity in detecting small early cancers.

In the current fluorescence imaging device (LIFE-Lung; Xillix Technologies Corp; Richmond, BC, Canada) approved by the Food and Drug Administration, two image-intensified cameras are used to amplify the red and green fluorescence intensity differences between normal and abnormal tissues.^18,19 Because the green autofluorescence is much stronger than the red, normal tissue appears green. In dysplasia or cancer, there is a progressive decrease in the green autofluorescence while the red autofluorescence remains unchanged (or becomes higher, as in the case of necrotic tumor, due to accumulation of endogenous porphyrins). Thus, the lesion appears brown, purplish, or red (Fig 1, Fig 2). Other devices, such as the D-Light/AF system (Karl Storz; Tuttlingen, Germany)²⁰ and the SAFE-1000 (Pentax; Tokyo, Japan)²¹ are currently undergoing clinical trials. In the D-Light/AF system, a nonimage-intensified color charge-coupled device camera is used. However, to record the weak fluorescence, the exposure time has to be increased to 1/8 to 1/15 s, instead of the conventional video rate of 1/32 s, in order to collect enough light for visualization. In addition, a small amount of reflected blue light is used to increase the brightness of the image.²⁰ Because of the time delay, smooth, slower insertion of the bronchoscope is required to avoid movement artifacts. The SAFE-1000 system consists of a single image-intensified camera.²¹ The design is similar to an earlier version of the LIFE-Lung system²² except that a nonlaser light source is used. Areas with abnormal fluorescence appear as a different intensity of the same color on the monitor compared to normal.

Figure 1. Left upper lobe, LIFE image. The carcinoma in situ lesion appears brownish red in appearance while the adjacent normal mucosa appears green.

Figure 2. The same area as in Figure 1 under white-light examination. Only minimal increase in redness in the same area was seen.

Results of LIFE-Lung Clinical Trials

Published data on the use of the LIFE-Lung device in more than 1,400 patients worldwide showed that white-light bronchoscopy alone localized 40% of the high-grade dysplasia and carcinoma in situ, with a range of 27 to 51% in different countries. The addition of fluorescence examination increased the detection rate to an average of 79%, a two-fold improvement.²³ Variation in the reported detection rates of white-light and fluorescence bronchoscopy is probably related to differences in the patient population, the number of patients in the study, the skill of the bronchoscopists, and differences in pathology interpretation.^24,25 In referral centers that specialize in endobronchial therapy for early lung cancer, the detection rate of white-light bronchoscopy is usually higher, since many of the carcinoma in situ lesions or early invasive cancers would have already been diagnosed by white-light bronchoscopy prior to enrollment into the study. Inclusion of these patients would lead to an improved sensitivity with white-light bronchoscopy and a lower ratio of relative sensitivity of fluorescence vs white-light bronchoscopy.^26,27 Significant variations also exist among pathologists in their interpretation of dysplasia and carcinoma in situ.²⁸ This may be one of the reasons for the low detection rate in some of the published reports.²⁹ The recently published World Health Organization classification of preinvasive lung cancer³⁰ will help improve the accuracy of the histopathology diagnosis. Development of objective classification methods such as quantitative image cytometry will further improve the diagnostic accuracy and minimize interobserver variation.²⁸ False-positive abnormal fluorescence can occur in patients with suction trauma, bronchial asthma, severe mucous gland hyperplasia, or acute purulent bronchitis. However, patients with COPD do not show an increased false-positive rate.

Local Staging of Early Lung Cancer

Once a small endobronchial tumor has been detected, the decision regarding the most appropriate treatment depends on the extent of invasion into the bronchial wall and the surrounding structures. If the tumor has already infiltrated beyond the cartilage layer, local endoscopic treatment is unlikely to be curative. Surgical resection would be the treatment of choice. Therefore, it is essential to have precise assessment of the depth of tumor invasion.

In an extensive prospective study evaluating the current staging procedures, the accuracy of radiologic procedures was examined.³¹ The study showed that CT scan is not reliable for evaluating the local extent of tumors and lymph node metastasis.³¹ Only 50% of lymph nodes involved by tumor were correctly classified. Assessment of local extent of tumor invasion into the bronchial wall was impossible with current CT due to the poor resolution between the pathologic and anatomic structures. Endoscopic ultrasound was found to be superior.^11,32

Endobronchial Ultrasound Technology

Regular ultrasonic devices cannot be used inside the airways because of their large diameter. Miniaturized probes were developed that can be inserted through the biopsy channels of regular fiberoptic bronchoscopes. At the tip of these probes is a small piezoelectric crystal that is rotated by a mechanical driving unit. By turning on an alternating electric current, the crystal is set into mechanical vibration that initiates the emission of sound waves.

The frequency for medical imaging devices usually ranges from 3.5 to 20 MHz. The sonic waves are transmitted to the tissues and reflected according to the impedance (resistance) to sound waves of different tissue structures. In between the generation of sound waves, the crystal also serves as a receiver. The acoustic signals initiate mechanical vibrations that are transformed into electrical signals. These in turn are transformed into gray-scale images on the monitor. The intensity of the echo is presented by its brightness. The time elapsed from sending and receiving the signal is shown as the distance from the probe. The lower the frequency of the sound waves, the deeper is the penetration into the tissues and the lower the spatial resolution and vice versa.

The small ultrasonic probes have very limited contact with the bronchial wall and the surrounding air reflects most of the sound waves. Therefore, the probes are constructed with balloons at the tip. By filling the balloon with water, close contact to the bronchial wall is established and filling the balloon with water can transmit the sound waves at 360 degrees into the surrounding structures. As water enhances the transmission of sound waves, a resolution well below 1 mm and a penetration depth of up to 4 cm can be achieved with a 20-MHz probe. This is sufficient to examine all the structures necessary for local staging.³²

Results of Endoscopic Ultrasound Clinical Trials

The EBUS system (Olympus; ______) has been under clinical investigation for a few years and has been on the market since October 1999.³² Several prospective studies by international groups have demonstrated its potential usefulness in the following applications.

Staging of Small Endobronchial Tumors. The sonographic anatomy of the bronchial wall has been established as a delicate structure of seven layers: epithelium and subepithelial layers, cartilage with internal and external surface echo, and the double layer structure of surrounding tissue.¹¹ Each of these layers has a thickness of < 1 mm and can be visualized in normal tissue.

In resected specimens of small cancers, Kurimoto et al³³ showed an almost 100% correlation between the EBUS image and the histologic findings. In the experience of one of us (H.B.), the accuracy of diagnosis of preinvasive lung cancer using autofluorescence bronchoscopy can be improved from 60% to more than 90% using EBUS to define the intactness of the epithelial and subepithelial layers (Fig 3, Fig 4, Fig 5).

Figure 3. Right upper lobe, white-light bronchoscopy image. Secretion and slight irregularity of the mucosa.

Figure 4. Corresponding LIFE image in right upper lobe. The tumor area appeared brownish red in color.

Figure 5. Endoscopic ultrasound image of the right upper lobe. The black arrow points to the normal bronchial wall. The white arrow points to the tumor area. The tumor has already invaded beyond the cartilage layer and hence is a microinvasive cancer and not carcinoma in situ.

The usefulness of EBUS to guide therapy was illustrated by a case study of a patient with adenoid cystic carcinoma reported by Miyazu et al.³⁴ In a recent unpublished paper, the same investigators showed how EBUS now is guiding their decisions to use local or surgical treatment for small endobronchial tumors. In addition to determining the depth of invasion, EBUS can also be used to localize adjacent lymph nodes that are as small as 3 mm¹¹ and, in our experience, to improve the accuracy of transbronchial lymph node needle aspiration biopsy to > 80%.³²

Other Applications of Endobronchial Ultrasound. Preliminary study suggests EBUS may be useful in differentiating between benign and malignant peripheral lung lesions by analyzing their internal echo structure. Invasion of adjacent mediastinal structures by central airway tumors may be more reliably diagnosed by ultrasound than by radiologic imaging. The extent of the tumor, involvement of neighboring structures such as the pulmonary arteries, patency of the airway distal to the tumor, presence of functioning lung tissue, and the safe distance from large blood vessels can now be determined during bronchoscopy without other diagnostic procedures. The information obtained by EBUS is useful in guiding endobronchial therapy. Endobronchial ultrasound will play an important role in the future as a diagnostic and navigation tool in the context of other procedures such as virtual bronchoscopy by ultrafast three-dimensional CT, optical coherence tomography, endobronchial MRI, and others.

References

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