Optical biopsy of lymph node morphology using optical coherence tomography

Optical diagnostic imaging techniques are increasingly being used in the clinical environment, allowing for improved screening and diagnosis while minimizing the number of invasive procedures. Diffuse optical tomography, for example, is capable of whole-breast imaging and is being developed as an alternative to traditional X-ray mammography. While this may eventually be a very effective screening method, other optical techniques are better suited for imaging on the cellular and molecular scale. Optical Coherence Tomography (OCT), for instance, is capable of high-resolution cross-sectional imaging of tissue morphology. In a manner analogous to ultrasound imaging except using optics, pulses of near-infrared light are sent into the tissue while coherence-gated reflections are measured interferometrically to form a cross-sectional image of tissue. In this paper we apply OCT techniques for the highresolution three-dimensional visualization of lymph node morphology. We present the first reported OCT images showing detailed morphological structure and corresponding histological features of lymph nodes from a carcinogen-induced rat mammary tumor model, as well as from a human lymph node containing late stage metastatic disease. The results illustrate the potential for OCT to visualize detailed lymph node structures on the scale of micrometastases and the potential for the detection of metastatic nodal disease intraoperatively. Key words: Lymph node; Optical coherence tomography; Optical imaging; High-resolution imaging; Metastasis; and Breast cancer. Introduction Breast Cancer Breast cancer continues to be one of the most frequently diagnosed cancers among women and the second leading source of cancer deaths in women. According to the most recent American Cancer Society statistics, approximately 211,240 new cases (or 32% of all new cancer cases amongst women) of invasive breast cancer and 58,490 new cases of in situ breast cancer are expected to be reported in the U.S. in 2005 (1). The number of U.S. deaths attributed to breast cancer this year is estimated at 40,870, second only to lung cancer (1). Over the years, the decrease in the number of breast cancer deaths has largely been attributed to increased awareness, earlier detection, and improved treatment. There is a continued emphasis on the early diagnosis of breast cancer in order to provide better managed care. The ability to detect and remove a tumor prior to metastasis is essential to lowering the breast cancer mortality rate. rate for U.S. breast cancer patients when the disease is detected at a localized stage is currently 97.5% (2). However, according to the American Cancer Society statistics, approximately 37% of the newly reported breast cancer cases in the U.S. are diagnosed at later stages when the tumor is no longer localized (2). One major method currently used to detect metastatic breast disease is axillary lymph node dissection (ALND). It is well known that as cancer cells metastasize from the primary tumor site they often travel through the lymphatic system as part of the natural immunological mechanism for processing and filtering out abnormal cells and foreign bodies. Cancerous cells tend to aggregate and cause the reactive enlargement of lymph nodes located near the primary tumor site and downstream in the lymphatic drainage pattern. In breast cancer patients, the two most common patterns drain into the axillary nodes (88-100%) and the internal mammary chain region (10-52%) and are highly dependent on the location of the primary breast cancer (3). In the past, it was common for a patient to undergo a complete axillary lymph node resection in order to histologically analyze all of the nodes suspected to contain metastases. Standard axillary lymph node dissection led to the removal of 10-30 lymph nodes and often adjacent non-diagnostic tissue. This extensive surgical procedure frequently resulted in a number of post-operative long-term complications such as numbness, sensitivity, and lymphedema in the dissection area and extremity. A more recent and less complicating surgical procedure is the sentinel lymph node biopsy (SLNB) (4). This technique has revolutionized the management of melanoma and solid tumors that metastasize through the lymphatic system (5, 6). The sentinel lymph node is the first node in the lymphatic drainage pattern leading away from the tumor site. This diagnostic procedure drastically reduces the number of lymph nodes removed in the axillary area to one to three nodes and eliminates the need for a complete ALND for patients with a negative SLNB. In addition to providing an equally valid diagnosis (7), reducing the patient's discomfort, and decreasing the associated costs, the SLNB has been shown in a number of clinical studies to be an effective method for the staging of breast cancer especially in assessing whether the cancer has metastasized to the axillary lymph nodes (7). An accurate staging of the metastatic state is the single most predictive factor of 10-and 20-year patient survival (8). The SLNB is performed using techniques to aid in the mapping of the breast lymphatic system and the identification of the sentinel node. One method is the use of a radioactive tracer, technetium-99, and a dye, methylene blue, to locate the sentinel nodes (8). A small dose of the technetium-99 is injected into the breast along with the blue dye and allowed to circulate through the lymphatic system for a period of 30 minutes to 8 hours (8). The dye aids in the visualization of node location and the level of radioactivity is used as a diagnostic marker to guide the removal of the entire lymph node. Although these can be used individually, a lower rate of false negatives is achieved when combined. Other traditional lymphatic mapping methods include standard X-ray and computed tomography (CT). Recent developments in contrast agents such as near-infrared (NIR) quantum dots (9) and indocyanine green (ICG) dye (10) allow for a more accurate visualization of sentinel lymph node location. Magnetic resonance imaging (MRI) contrast agents such as G6 Once a lymph node has been identified and resected, a pathological assessment is made to determine its status, thereby assessing disease progression, a process referred to as staging. OCT may provide beneficial alternatives to current methods for lymph node assessment, and subsequently affect the staging procedure. OCT is a low-cost and minimally invasive technique that is capable of imaging morphological structures at cellular resolutions as well as identifying the location of lymph nodes in tissue without the use of radioactive tracers or dyes. In comparison to other optical biomedical imaging techniques, OCT is also capable of imaging 2-3 mm in depth in opaque samples, allowing for the real-time in vivo evaluation of the tissue prior to its resection. Optical Coherence Tomography Optical coherence tomography is a nondestructive optical imaging modality that uses NIR light to form cross-sectional images of tissue morphology (12). Light reflected from structures within the tissue is coherence-gated to form a depth-resolved image of the specimen, in much the same way that ultrasound images are formed using acoustic pulses. OCT, however, has achieved resolutions approaching that of conventional histopathology and is able to resolve cellular features due to the very short wavelengths in the optical regime and the precision afforded by interferometric detection Since its introduction in 1991, OCT has undergone rapid growth, extending across a wide range of applications in medical and laboratory research. Commercially available equipment is now in widespread clinical use by ophthalmologists for the imaging of retinal pathologies and other ocular diseases (15). OCT imaging of more dense scattering tissues (16) has yielded a wide array of potential applications. In order to achieve imaging depths of 2-3 mm in non-transpar- OCT is also a highly robust and adaptable imaging modality. Advances in broadband optical sources have yielded OCT axial resolutions of less than 2 µm (30-32). Real-time imaging has also been reported, using either swept-source optical frequency domain imaging (OFDI) or broadband spectral domain (SD) systems with axial scan-line rates of up to 29,000 per second (33-35). Capabilities such as Doppler analysis (36, 37) and polarization sensitivity (38) have been incorporated into OCT systems, as well, enabling the study of blood flow and skin burns, respectively. Contrast agents have also been developed, adding molecular sensitivity to OCT images and thereby enabling the potential detection of chemical disease markers prior to evident morphological changes (39, 40). OCT imaging systems have been integrated into a variety of modern clinical instruments. In the surgical suite, for exam- Optical Biopsy of Lymph Nodes using OCT 541 Technology ple, the depth scanning capabilities of OCT have been used to augment the capabilities of standard surgical microscopes (24). Endoscopic, catheter, and needle probes have also been developed, allowing for high resolution analysis of the GI tract and vasculature, or in conjunction with soft tissue biopsy procedures Materials and Methods Animal Model A clinically relevant animal model, already being employed in parallel studies, was adopted for this study (44, 45). The induction of mammary tumors of virgin female rats by injection with the direct-acting carcinogen N-methyl-N-nitrosourea (MNU) is the most widely used animal model for studying breast cancer development (46-48). Mammary carcinogenesis in this model mimics numerous characteristics associated with human breast carcinogenesis, including hormone dependency and histopathological features (46). Invasion of the regional lymph node chain has been observed under this rat model, and distant metastases to the lung have also been reported (46). Similar patterns of invasion and metastasis are observed in the human disease (49). The induced tumors emulate the carcinogenesis of human ductal carcinoma, first as ductal carcinoma in situ, then as locally invasive disease, and finally as metastatic disease to the liver, lung, and spleen (43, 44, 48, 50, 51). Using this animal model, we were able to validate the use of OCT for the visualization of microscopic tumor morphology and the identification of tumor margins (43). In this study, we demonstrate the application of OCT as an "optical biopsy" technique for the evaluation of lymph node morphology in this rat tumor model. All animal procedures were performed according to a protocol approved by the Institutional Animal Care and Use Committee of the University of Illinois at UrbanaChampaign. Female Sprague-Dawley rats were purchased from Harlan (Indianapolis, IN) at three to four weeks of age, individually housed in rooms with controlled temperature and lighting, and fed rodent laboratory chow (Harlan Teklad, Madison, WI). At four weeks and six weeks of age, six rats were administered one dose of MNU (Sigma Chemical, St. Louis, MO; i.p. 55 mg/kg in saline) as part of parallel ongoing studies of carcinogenesis in this tumor model. Two control rats were injected with an equal volume of a saline vehicle. Beginning two weeks post-MNU injection, animals were palpated weekly. Rats that developed mammary masses around 3 cm in size, and corresponding control animals, were euthanized by carbon dioxide asphyxiation prior to OCT imaging. A midline and two lateral incisions were made in the abdominal skin to reflect back the abdominal wall, exposing the peritoneal cavity and the mesenteric lymph nodes. The animals were placed on an OCT microscope stage for initial imaging. Subsequently, the mesenteric lymph nodes were dissected, placed in Petri dishes, and imaged using OCT. All eight animals were imaged for this study with an average of three lymph nodes per animal and 256 OCT images per lymph node. Human Tissue Specimens Specimens of surgically-resected human tissue, including two lymph nodes, were provided fresh and stored in 0.9% normal saline after a pathological diagnosis was made. Specimens were imaged using OCT within 24 hours, followed by standard histological processing. Both lymph nodes were imaged in three-dimensions, acquiring a total of 400 cross-sectional OCT images. Tissue procurement and imaging was performed under protocols approved by the Institutional Review Boards of Carle Foundation Hospital, Urbana, Illinois, and the University of Illinois at UrbanaChampaign. Tissue and Image Analysis After imaging, tissue was fixed in 10% neutral buffered formalin solution for standard histological processing. Samples were embedded in paraffin and sectioned 5 µm thick with a rotary microtome (RM2255, Leica Microsystems, Inc.). Sections were stained with hematoxylin and eosin (H&E) and prepared on glass slides according to standard protocol for light microscopy observation. Light microscopy was performed (BH-2, Olympus, Japan) and digital images were captured (Spot RT Slider, Diagnostic Instruments) for comparison with OCT images. Image analysis yielded the identification and correlation of microstructural features. The OCT and histological images presented here represent the best match based on the observed architectural morphology. Three-dimensional data sets were constructed and analyzed using commercially available software (Slicer Dicer® 4.0, PIXOTEC) on a personal computer. Three-dimensional projections were generated and rotated along the X-Y-Z coordinates for the visualization of node features. Optical Coherence Tomography Instrument The OCT instrument in this study Luo Technology in Cancer Research & Treatment, Volume 4, Number 5, October 2005 The broad bandwidth yielded an axial imaging resolution of approximately 2 µm in tissue. A 20 mm achromatic lens was used to focus approximately 10 mW of light down to a 15 µm spot size (transverse resolution) onto the sample from a single-mode 50/50 fiber optic splitter (Gould Fiber Optics, Inc.) that also coupled light reflected from a galvanometerbased reference delay operating at a rate of 30 scan lines per second. Spatial scanning in the X-Y plane was accomplished via a pair of galvanometer-mounted mirrors (Cambridge Technology, Inc.). Time-domain detection was achieved via a dual-balanced detection scheme (52) using a 125 kHz auto-balanced photoreceiver (New Focus Inc., Model #2007) and data acquisition was performed by a dedicated computer cards (National Instruments, Model #PCI-6110, PCI-6711) with a 10MHz sampling rate, a 12-bit quantizer, and a ±5V input range. Data acquisition in the spectral domain was achieved using a diffraction grating with 830 grooves/mm and blazed for 828nm (Richardson Grating Laboratory, Rochester, New York) to disperse the light, and a lens to focus onto a line scan camera (Model #L104K, Basler Vision Technologies). The time domain and spectral domain systems have measured signal-to-noise ratios (SNR) of approximately 100dB and 90dB, at acquisition rates of 10 lines/sec and 29,000 lines/sec, respectively. The human breast cancer tissue specimen and the rat lymph nodes were both imaged using time-domain OCT (TD-OCT). Recent technological advances have led to the addition of Fourier-domain optical detection in OCT, enabling significantly faster acquisition speeds with minimal corresponding loss of sensitivity. Employing a method referred to as spectral-domain OCT (SD-OCT) (33-35), the same broadband light source was employed and the signal was measured using a spectrometer (see Results Mesenteric rat lymph nodes were imaged in vivo and in vitro using OCT. During three-dimensional OCT imaging, 256 2-D images of each specimen were acquired in 10 µm spatial increments. The images of two representative in vitro rat lymph nodes are presented in In this rat lymph node morphology imaging study, no significant differences were detected between the OCT images of lymph nodes from tumor-bearing animals and those from control animals. The H&E stained sections from these animals were examined by a board-certified pathologist in Veterinary Pathobiology to confirm that no micrometastases or isolated tumor cells were found in the rat lymph nodes. For comparison with the previous normal-appearing lymph nodes, images were acquired from a late-stage metastatic human lymph node Discussion The results reported here represent the first demonstration of OCT imaging of lymph node morphology. The resulting images show the internal structures of the lymph nodes at a high resolution (2 µm axial, 15 µm transverse) and as deep as 1 mm in the highly scattering lymphatic tissue. The observed internal lymph node architecture is clearly identifiable when imaged from the external surface, and strongly correlated with corresponding histological observations, showing that lymph nodes are a promising target for clinical OCT application. Despite the depth penetration limits of Pixel dimensions in the 3-D set are 2 µm, 7 µm, and 3 µm in the x, y, and z directions, respectively. Scale bars = 100 µm. Pixel dimensions in the 3-D set are 2 µm, 8 µm, and 3 µm in the x, y, and z directions, respectively. Scale bars = 100 µm