Peripheral Lung Cancer Detection by Vascular Tumor ... - Springer Link

Peripheral Lung Cancer Detection by Vascular Tumor Labeling Using In-Vivo Microendoscopy under Real Time 3D CT Image Guided Intervention Miguel Valdivia y Alvarado, Tian Cheng He, Zhong Xue, Stephen Wong, and Kelvin Wong* The Center for Bioengineering and Informatics, Department of Radiology, The Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, TX [email protected]

Abstract. We designed and evaluated a real time 3D CT Image Guided Intervention system that integrates in-vivo microendoscopic imaging for on-thespot visualization of ICG contrast uptake by tumor vessel in peripheral lung tumors. The performance of the system was evaluated in seven rabbits where VX2 cells were implanted in the chest to create peripheral lung tumors. Two weeks later the animal underwent a chest CT scan which was used for creating a real time 3D vision and navigation tracking. ICG was injected fifteen minutes prior to the needle puncture to allow adequate contrast leakage inside the tumor and plasma clearance. After the needle puncture, the microendoscope was introduced inside the tumor for imaging. Visualization of tumor leaky vasculature was possible in all the tumors. The experiment demonstrated that real-time microendoscopy of deep solid organs under a 3D CT image-guided system is possible while providing enough accuracy in reaching tumors without complications. Keywords: Microendoscopy, Real Time 3D CT Image Guided Intervention, Indocyanine Green (ICG), Tumor Leaky Vasculature, Lung Cancer Detection.

1 Introduction Currently lung cancer has one of the highest mortality and morbidity rates, despite the efforts and investments made in new detection and treatment methods the long-term survival rate of lung cancer does not improve. The key to improve the long-term survival rate relies in early diagnosis, accurate localization and novel targeted therapies [1] for which new imaging techniques are expected to play a significant role. Among the news imaging techniques microendoscopy seems very promising as it allows direct observation of pathologic changes at the microscopic level, moreover fibered microscoendoscopy provides a clear, in-focus image of a thin section within a biological sample, [2] and it is capable of imaging fluorescent labeled biological *

Corresponding author.

H. Liao et al. (Eds.): MIAR 2010, LNCS 6326, pp. 494–502, 2010. © Springer-Verlag Berlin Heidelberg 2010

Peripheral Lung Cancer Detection by Vascular Tumor Labeling

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structures in vivo at exceptionally high spatial resolutions. These technologies can be translated to the thoracic oncology field by assisting in early detection of precancerous and cancer conditions through improved biopsy selection. Considering that microendoscopic probes are able to image tissue with unprecedented spatial (less than 1 micron lateral resolution) and high temporal resolution (usually video rate) using low-cost, portable devices, this technology has the potential to decrease mortality and morbidity by optimizing lung tumor detection. A major limitation of microendoscopy is the capacity to reach deep seated targets such as the lung [3]. To that end our group has developed an image guided intervention based on real time 3D CT images [4] and coupled with electromagnetic tracking system to enable microendoscopic probes to reach deep seated-targets. The integration of microscopic imaging with macroscopic image navigation will allow a closer look to small targets [3, 4], and may improve the accuracy of biopsies. Here we report the results of a study where we coupled microendoscopic imaging with the image guided intervention system we created for peripheral lung cancer detection. We evaluated its utility in small peripheral lung tumor diagnosis by validating the insertion accuracy of the 3D CT image guided intervention system in thoracic percutaneous punctures by using in vivo fibered microendoscopy for tumor detection using vascular labeling with Indocyanine Green contrast.

2 Materials and Methods 2.1 Microendoscopy Probes A CellVizio Lung microendoscopic imaging system was used in the study. The CellVizio Lung system is a commercial system approved by the FDA for human lung application. This system offers a depth of observation between 0-50 µm, a lateral resolution of 3.5 µm, and excitation wavelength between 488 to 600 µm. The field of view ranges from 500 µm to 600 µm, covering a relatively large area of tissue and capturing optical image sequence of the fine tumor vasculature [5]. The video images were obtained at a rate of 12 frames per second. The manufacturer of the CellVizio system, Mauna Kea Technologies, provides us with a 1 mm diameter probe that allows using a smaller needle size for a percutaneous thoracic puncture, this probe will pass through the needle cover after the puncture is successfully achieved and the needle is inside the tumor. 2.2 Contrast Agents Among the various contrast agents available Indocyanine Green (ICG) was selected because it is the only contrast agent approved by FDA for vasculature imaging; therefore a positive result can be translatable to the patient bedside faster. ICG is a sterile solution of a nontoxic tricarbocyanine dye with a peak spectral absorption of 790 nm, [6]. Experiments using near infra-red (NIR) reflected light have showed that it is possible to image sub-cellular features in the epithelial tissue with depth exceeding 400 µm. [7] NIR provides quantitative functional information that cannot be obtained by conventional radiological methods and also can provide in vivo measurements of the oxygenation and vascularization states. ICG is also a blood

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pooling agent and has different delivery behavior between normal and cancer vasculature. In normal tissue, ICG acts as a blood flow indicator in tight capillaries of normal vessels. In tumors, ICG acts as a diffusible (extravascular) flow in leaky capillary of vessels. 2.3 3D CT Real-Time-Image Guided Intervention Our image-guided intervention system consists of the following components (see Figure 1): An electromagnetic (EM) tracking device for real-time tracking of the needle introducer (Aurora Electromagnetic Measurement System); coherent software for real-time localization and visualization of multiple devices being tracked on the intra-procedural CT images and one fine needle (Traxtal Percunav, Biopsy Introducer 18G) for percutaneous lung puncture. The workflow of the system was implemented by using a modular multimodality image guidance platform (MIMIG) [8]. Diagnosis of the tumor mass inside the chest was accomplished based on morphologic and molecular imaging information obtained from different imaging modalities. Novel image computing tools including segmentation, registration, and microendoscopy image sequence processing were developed for MIMIG. The pre-procedural images were segmented for better visualization during surgical planning, and since fast segmentation is needed for intra-procedural images, the segmentation results were transformed onto the intra-procedural images for visualization during the intervention. Using these tools fast and accurate image segmentation and registration algorithms were developed for better visualization during surgical planning, intervention, and alignment of pre-procedural and intra-procedural images. 2.4 Animal Model The animal protocol was revised and approved by the Comparative Medicine Program and IACUC committee at our institution. Solitary lung tumors in seven White New

Fig. 1. Pictures showing various components of the real time 3D CT Image Guided Navigation System developed by our group: the upper left picture displays the connection trackers for EM tracking, the upper right picture shows the field generator that tracks the needle (white arrow). The bottom left picture shows the 3D image display available for the physician to design a preoperative path planning in order to guide the needle to the tumor, and the bottom right picture shows the needle insertion inside the animal chest.


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Zealand rabbits (2.0-2.2 kg) using VX2 carcinoma cells were created for the study. The first cell line was obtained from our cancer research center partner and injected directly on the limbs of one rabbit. The tumors first grew in the hind legs of a carrier rabbit for two weeks. This animal was sacrificed and the VX2 tumors were harvested from the legs to create a cell suspension at a density of 8x106 cells/mL. From two VX2 tumors three needles with 0, 5 ml cell suspension were created, two needles were injected into the limbs of another rabbit for tumor line preservation and the third one was administered in one animal tumor recipient. The cell suspension was administered in the right lower lung of the recipient rabbits using a 22-gauge Chiba needle percutaneously inserted using fluoroscopy. [9, 10] After the fifth day from the lung inoculation a CT scanning was performed weekly to follow the growth of the VX2 tumor. Tumors of ≤15 mm in diameter were confirmed at days 12-14 after inoculation in all the animals’ models. 2.5 Experiments Once the animal model has the desired tumor size it was anesthetized and transported to the CT room for the image-guided procedure. First, a pre-procedural CT scan of the thorax was obtained using a SPECT/CT (Symbia TruePoint from Siemens) with the following parameters: 1.25mm helical acquisition, pitch of 1.55 and a reconstruction of 1.2mm. The chest scan information was transferred from the CT scanner to the Image Guided Intervention system where the physician chooses a path planning for the percutaneous needle puncture according to the tumor location. The real time 3D vision and tool tracking was possible superimposing the electromagnetic tracking data (information about the instruments location) to the pre-procedural scan in real time, thus the display appears modulated by the real-time tracking data. The percutaneous accuracy puncture accuracy was later validated by coregistering pre- and postpunctures CT images. Indocyanine Green (ICG-125 mg) contrast was injected IV 15 minutes before the percutaneous thoracic puncture; this period was set as the time necessary for adequate contrast leakage inside the tumor and contrast systemic clearance from the plasma. Before the puncture the mechanical ventilation was stopped to decrease the respiratory movement to the minimum. Once the tumor target was reached the needle was fixated and a post-procedural scan was performed to verify the needle location inside the tumor. After the image-guided needle puncture, the needle was retracted, leaving the needle introducer in place. Then, the 1mm O.D. Cellvizio microendoscope was inserted through the needle introducer and video recording from the VX2 tumors vessels started (Fig.2). At the end of the video recording a tumor biopsy was taken using a Biopsy Needle (Quick-Core, Cook Medical) that pass through the same needle introducer used for tumor imaging. After completing the biopsy the animals were euthanized, the chest was opened and the tumors were recovered. Samples from the needle biopsy and tumor were sent to histological analysis by a research vet tech blinded to the tumor source. For detecting the ICG expression in the VX2 tumor vessels a pre-defined threshold of two-fold specific labeling over background were used. With these thresholds, the in situ ICG vessel expression inside the tumor can be visualized in real time. The percentage of tumor vessel expressing high uptake of ICG per frame was calculated

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Fig. 2. In the left picture the animal model is undergoing the pre-procedural scan, the picture in the middle shows the introduction of the Cellvizio microendoscope probe inside the VX2 tumor and finally the right picture shows the imaging of tumor vessels labeled with Indocyanine Green.

after doing a post-processing with the pre-defined threshold. To increase reliability motion correction and scene change detection were applied along the video sequences. The ICG fluorescence microendoscopy was validated by semi-quantitative data analysis of fluorescence video intensity in normal lung and tumor tissues at different regions of the lung. The tumor was sampled 5 times while the normal tissue was sampled 2 times due to the low standard deviation of mean fluorescence video intensity in normal lung tissue and larger variation in tumor tissues.

3 Results The tumors grew to a size equal or less than 15 mm measured by CT scan (average 13 mm) in the seven animal models used in the study. Complications related to thoracic puncture such as pneumothorax were not seen in the second scan. In the confirmatory scan the needle tip reached the VX2 tumors in the seven experiments, therefore it was not necessary to adjust the needle once inside the thorax. The needle tip was seen in the center of the tumor in the post-procedural CT scan in five animals. In the remaining two animals the needle tip was located in a peripheral zone of the tumor. Four to six videos were recorded in each animal and each video had a duration period between 35 to 55 seconds. A higher contrast was expected because the micron-sized resolution and shadow reception of the microendoscope removed the depth dependence and partial volume effect. Figure 3 shows the fluorescent microendoscopic images recorded during the experiments, according to an intensity color map. In the left side of figure 3 we can appreciate frames from a video recorded with the needle tip in the peripheral zone of the VX2 tumor while in the right side frames from a centrally located needle tip case are showed. The first frame shows the original fluorescent image, and the next one corresponds to the segmented fluorescent image. The tumors vessels have a heterogeneous and high expression/uptake of ICG compared to areas with less vasculature, which had an intensity value between 40-60. This was particularly evident in the experiments where the needle tip was in a peripheral location, which is a zone less vascular compared to the videos recorded from the tumor center where the uptake of ICG was higher, due to the high angiogenesis rate. Also some dark areas were seen in the videos corresponding to necrotic areas due to hypoxia.


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Fig. 3. Images captured from a VX2 rabbit model experiment cases where the needle tip had a peripheral location (left) and a case where the needle tip had a central location (right) inside the VX2 tumor. Note the stronger vascular labeling and expression obtained from the tumor with a central needle tip location (zones labeled with red and yellow colors).

A more dramatic example about the angiogenesis phenomena can be appreciated in figure 4 where the video sequences recorded showed regions with high uptake of contrast at the center of the tumor; the contrast intensity decreased gradually when the needle and microendoscope were pulled-back to a peripheral zone of the tumor and later disappeared when the microendoscope was outside the tumor (needle position confirmed with CT scan).

Fig. 4. In vivo microendoscopy images showing ICG-labeled vascular architecture inside the VX2 tumor first and later structure of the normal parenchyma when the needle was pulled back from the tumor

The biopsies taken at the end of the procedure had a positive result for VX2 carcinoma, which was compared to samples of the tumor excised from the lung at the end of the procedure (Figure 5).

Fig. 5. The first slide was made from a sample of the recovered tumor after euthanasia, while the second slide comes from the sample obtained through the biopsy (both are from the same tumor). In both samples cells corresponding to VX2 carcinoma were reported.

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4 Discussion In chest medicine, microendoscopy technology has been applied in bronchoscopic procedures previously [11], where it was possible to obtain high-quality images through a fiber-optic probe of 1-mm diameter introduced in the working channel of a bronchoscope. Even more, image registration for displaying the position of flexible bronchoscope on CT scan during transbronchial bronchoscopy needle aspiration has been applied successfully [12, 13]. However, despite its effectiveness, the bronchoscopic approach is useless as a diagnosis or treatment tool for tumors outside the airways, therefore not an option for peripheral lung tumors. For this kind of tumors the work-up plan usually includes a diagnosis made through a percutaneous biopsy and the treatment is either performed with surgery or ablation. Using the real time 3D CT Image Intervention system microendoscopic imaging for tumor diagnosis can be performed through a percutanoues biopsy needle as we proved in our study. The time necessary to do the thoracic puncture with the help of this system, ICG injection and clearance, and microendoscopic imaging does not add a significant amount of time to a regular puncture biopsy, the path planning can be performed in a couple of minutes, while ICG is been cleared (15 minutes) from the plasma. Besides, the radiation from repetitive CT scans is drastically reduced because only two scans are needed. The use of this system may help to overcome the current limitations microendoscopy imaging must face in order to become clinically translatable: shallow penetration and emission signal in deep-seated tissues or organs. This is achieved by bringing the imaging tools closer to the target. Accuracy to reach the tumor also may increase when using the system, in our series the imaging of the tumor vessel were recorded mainly from the tumor center where a later biopsy will have more reliable result, this was validated by the presence of VX2 carcinoma cells in all the biopsies. The use of ICG takes advantages of the high angiogenesis rate seen in tumors, making the leaky vasculature [14] easy to visualize by microendoscopy imaging. Our results showed a difference in contrast between lung parenchyma and the region recognized as the VX2 tumor in CT scan where the contrast was higher whenever the fibered microendoscope was closer to the tumor core. This differentiation based on tumor angiogenesis also gives us different contrast level inside the tumor as we showed when comparing cases where the needle tip and fibered microendoscope had a peripheral location, a zone usually more fibrotic and therefore less vascular. This contrast differentiation could give an extra tool to the physician to determine a region for biopsy, first to distinguish normal tissue and then a zone more vascular from one less vascular. Pharmacokinetics of ICG enables the potential to provide new tools for tumor detection, through the assessment of the leakiness of large ICG molecules from the microvasculature; this permeability is a characteristic of the poorly developed vasculature observed in tumor angiogenesis. The increase in local microvasculature density is also expected to induce a high perturbation in the optical signal from intercapillary vessel inside the tumor. Therefore, considering all the characteristics offered by the use of ICG in conjunction with microendoscopic imaging, this approach may add functional information to morphology, all in real time. The clinical impact of this technology could be extremely relevant considering that biopsy is a sine qua non condition for tumor diagnosis. Due to tumors heterogeneity,


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it can be difficult to establish the optimal site for tissue biopsy and sampling error may lead to incorrect diagnoses. The use of microendoscopy probes for tumor detection using ICG assisted by real time 3D CT image navigation is a technique that could improve the sensitivity of tumor detection, several factors contribute to this end: 1) Continuous tracking of the needle after it has been introduced to the chest without taking more CT scans, 2) noticeable visualization of tumor regions based on their vascularity, and 3) easy recognition of necrotic regions, capsule, and lung parenchyma from tumor (Fig. 4) as they represent non-vascular areas and therefore ICG cannot exert its diffusible (extravascular) flow in leaky capillaries. In our experiments, all the biopsies taken from the tumors were positive; these biopsies were taken from the regions were a higher ICG uptake was detected by microendoscopic imaging. Among the limitations we faced in our study we have a relative small series of cases, use of VX2 line rather than an adenocarcinoma line, and lack of sensors attached to the microendoscope to extend the electromagnetic tracking capabilities to the microendoscope. The VX2 line was chosen based on their easiness of handling, survival rate, and time to growth. To address the last limitation our group has been working in adding EM-sensors to track the fiberoptic image probe to ease fusion of microendoscopy video with 3D lung CT volume, therefore a better correlation and localization among needle, microendoscope, and tumor vessel imaging can be obtained, all together and in real time. A minimally invasive technique that provides in vivo optical expression in realtime would radically improve clinical diagnosis by offering a better assessment of changes in tissues. Microendoscopic imaging can meet this important need by delivering real-time, in vivo images with sub-cellular resolution, and the addition of optical-specific information to anatomic images may benefit the survival rate of patients with peripheral lung tumors by providing an earlier and more specific detection of the disease.

5 Conclusions Our research findings indicates that using real-time 3D CT image guided intervention to guide a microendoscopic probe inside a tumor is fast and effective. This new approach could be extremely relevant in lung cancer detection where repeated rounds of CT scans are necessary both for screening, diagnosis and post-treatment follow-up. This system allows an accurate position of the needle and the microendoscope inside the tumor for in situ microendoscopic imaging. The addition of in situ microendoscopic imaging is especially useful during biopsy since it may allow a better characterization and guidance. Moreover, the addition of tumor vessels information to the tumor images provides by ICG has the potential to provide earlier and more specific tumor detection. These same features also can contribute to a more selective tumor biopsy or ablation. To prove this concept our group is designing a clinical trial in collaboration with interventional radiologists to assess the real time 3D CT Image Navigation System and microendoscopic imaging with ICG for peripheral lung tumor detection and treatment in selected patients.

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