Rectal Optical Markers for In Vivo Risk Stratification of Premalignant Colorectal Lesions.

PURPOSE: Colorectal cancer remains the second leading cause of cancer deaths in the United States despite being eminently preventable by colonoscopy via removal of premalignant adenomas. In order to more effectively reduce colorectal cancer mortality, improved screening paradigms are needed. Our group pioneered the use of low-coherence enhanced backscattering (LEBS) spectroscopy to detect the presence of adenomas throughout the colon via optical interrogation of the rectal mucosa. In a previous ex vivo biopsy study of 219 patients, LEBS demonstrated excellent diagnostic potential with 89.5% accuracy for advanced adenomas. The objective of the current cross-sectional study is to assess the viability of rectal LEBS in vivo. EXPERIMENTAL DESIGN: Measurements from 619 patients were taken using a minimally invasive 3.4-mm diameter LEBS probe introduced into the rectum via anoscope or direct insertion, requiring approximately 1 minute from probe insertion to withdrawal. The diagnostic LEBS marker was formed as a logistic regression of the optical reduced scattering coefficient [Formula: see text] and mass density distribution factor D. RESULTS: The rectal LEBS marker was significantly altered in patients harboring advanced adenomas and multiple non-advanced adenomas throughout the colon. Blinded and cross-validated test performance characteristics showed 88% sensitivity to advanced adenomas, 71% sensitivity to multiple non-advanced adenomas, and 72% specificity in the validation set. CONCLUSIONS: We demonstrate the viability of in vivo LEBS measurement of histologically normal rectal mucosa to predict the presence of clinically relevant adenomas throughout the colon. The current work represents the next step in the development of rectal LEBS as a tool for colorectal cancer risk stratification.

In vivo risk analysis of pancreatic cancer through optical characterization of duodenal mucosa.

OBJECTIVES: To reduce pancreatic cancer mortality, a paradigm shift in cancer screening is needed. Our group pioneered the use of low-coherence enhanced backscattering (LEBS) spectroscopy to predict the presence of pancreatic cancer by interrogating the duodenal mucosa. A previous ex vivo study (n = 203) demonstrated excellent diagnostic potential: sensitivity, 95%; specificity, 71%; and accuracy, 85%. The objective of the current case-control study was to evaluate this approach in vivo. METHODS: We developed a novel endoscope-compatible fiber-optic probe to measure LEBS in the periampullary duodenum of 41 patients undergoing upper endoscopy. This approach enables minimally invasive detection of the ultrastructural consequences of pancreatic field carcinogenesis. RESULTS: The LEBS parameters and optical properties were significantly altered in patients harboring adenocarcinomas (including early-stage) throughout the pancreas relative to healthy controls. Test performance characteristics were excellent with sensitivity = 78%, specificity = 85%, and accuracy = 81%. Moreover, the LEBS prediction rule was not confounded by patients' demographics. CONCLUSION: We demonstrate the feasibility of in vivo measurement of histologically normal duodenal mucosa to predict the presence of adenocarcinoma throughout the pancreas. This represents the next step in establishing duodenal LEBS analysis as a prescreening technique that identifies clinically asymptomatic patients who are at elevated risk of PC.

Buccal spectral markers for lung cancer risk stratification.

Lung cancer remains the leading cause of cancer deaths in the US with >150,000 deaths per year. In order to more effectively reduce lung cancer mortality, more sophisticated screening paradigms are needed. Previously, our group demonstrated the use of low-coherence enhanced backscattering (LEBS) spectroscopy to detect and quantify the micro/nano-architectural correlates of colorectal and pancreatic field carcinogenesis. In the lung, the buccal (cheek) mucosa has been suggested as an excellent surrogate site in the "field of injury". We, therefore, wanted to assess whether LEBS could similarly sense the presence of lung. To this end, we applied a fiber-optic LEBS probe to a dataset of 27 smokers without diagnosed lung cancer (controls) and 46 with lung cancer (cases), which was divided into a training and a blinded validation set (32 and 41 subjects, respectively). LEBS readings of the buccal mucosa were taken from the oral cavity applying gentle contact. The diagnostic LEBS marker was notably altered in patients harboring lung cancer compared to smoking controls. The prediction rule developed on training set data provided excellent diagnostics with 94% sensitivity, 80% specificity, and 95% accuracy. Applying the same threshold to the blinded validation set yielded 79% sensitivity and 83% specificity. These results were not confounded by patient demographics or impacted by cancer type or location. Moreover, the prediction rule was robust across all stages of cancer including stage I. We envision the use of LEBS as the first part of a two-step paradigm shift in lung cancer screening in which patients with high LEBS risk markers are funnelled into more invasive screening for confirmation.

Spatially resolved optical and ultrastructural properties of colorectal and pancreatic field carcinogenesis observed by inverse spectroscopic optical coherence tomography.

Field carcinogenesis is the initial stage of cancer progression. Understanding field carcinogenesis is valuable for both cancer biology and clinical medicine. Here, we used inverse spectroscopic optical coherence tomography to study colorectal cancer (CRC) and pancreatic cancer (PC) field carcinogenesis. Depth-resolved optical and ultrastructural properties of the mucosa were quantified from histologically normal rectal biopsies from patients with and without colon adenomas (n=85) as well as from histologically normal peri-ampullary duodenal biopsies from patients with and without PC (n=22). Changes in the epithelium and stroma in CRC field carcinogenesis were separately quantified. In both compartments, optical and ultra-structural alterations were consistent. Optical alterations included lower backscattering (µb) and reduced scattering (µs') coefficients and higher anisotropy factor g. Ultrastructurally pronounced alterations were observed at length scales up to ∼450 nm, with the shape of the mass density correlation function having a higher shape factor D, thus implying a shift to larger length scales. Similar alterations were found in the PC field carcinogenesis despite the difference in genetic pathways and etiologies. We further verified that the chromatin clumping in epithelial cells and collagen cross-linking caused D to increase in vitro and could be among the mechanisms responsible for the observed changes in epithelium and stroma, respectively.

End-binding protein 1 (EB1) up-regulation is an early event in colorectal carcinogenesis.

End-binding protein (EB1) is a microtubule protein that binds to the tumor suppressor adenomatous polyposis coli (APC). While EB1 is implicated as a potential oncogene, its role in cancer progression is unknown. Therefore, we analyzed EB1/APC expression at the earliest stages of colorectal carcinogenesis and in the uninvolved mucosa ("field effect") of human and animal tissue. We also performed siRNA-knockdown in colon cancer cell lines. EB1 is up-regulated in early and field carcinogenesis in the colon, and the cellular/nano-architectural effect of EB1 knockdown depended on the genetic context. Thus, dysregulation of EB1 is an important early event in colon carcinogenesis.

Ultrastructural alterations in field carcinogenesis measured by enhanced backscattering spectroscopy.

Optical characterization of biological tissue in field carcinogenesis offers a method with which to study the mechanisms behind early cancer development and the potential to perform clinical diagnosis. Previously, low-coherence enhanced backscattering spectroscopy (LEBS) has demonstrated the ability to discriminate between normal and diseased organs based on measurements of histologically normal-appearing tissue in the field of colorectal (CRC) and pancreatic (PC) cancers. Here, we implement the more comprehensive enhanced backscattering (EBS) spectroscopy to better understand the structural and optical changes which lead to the previous findings. EBS provides high-resolution measurement of the spatial reflectance profile P(rs) between 30 microns and 2.7 mm, where information about nanoscale mass density fluctuations in the mucosa can be quantified. A demonstration of the length-scales at which P(rs) is optimally altered in CRC and PC field carcinogenesis is given and subsequently these changes are related to the tissue's structural composition. Three main conclusions are made. First, the most significant changes in P(rs) occur at short length-scales corresponding to the superficial mucosal layer. Second, these changes are predominantly attributable to a reduction in the presence of subdiffractional structures. Third, similar trends are seen for both cancer types, suggesting a common progression of structural alterations in each.

Biological mechanisms underlying structural changes induced by colorectal field carcinogenesis measured with low-coherence enhanced backscattering (LEBS) spectroscopy.

We previously reported the utility of Low-Coherence Enhanced Backscattering (LEBS) Spectroscopy in detecting optical changes in uninvolved rectal mucosa, changes that are indicative of the presence of advanced colorectal adenomas elsewhere in the colon (field carcinogenesis). We hypothesized that the alterations in optical signatures are due to structural changes in colonocytes. To elucidate those colonocyte changes, we used LEBS and an early time point in an animal model of colorectal field carcinogenesis--rats treated with azoxymethane (AOM). Changes in LEBS markers in intact mucosa from AOM-treated rats could be at least partially attributed to changes in colonocytes. To investigate the molecular mechanisms underlying the colonocyte abnormalities in premalignant colon, we took a candidate approach. We compared expression profiles of genes implicated directly or indirectly in cytoskeletal dysregulation in colorectal tissues from saline-treated versus AOM-treated rats. Our data suggest that a number of genes known to affect colon tumorigenesis are up-regulated in colonocytes, and genes previously reported to be tumor suppressors in metastatic cancer are down-regulated in colonocytes, despite the colonocytes being histologically normal. To further understand the role of the cytoskeleton in generating changes in optical markers of cells, we used pharmacological disruption (using colchicine) of the cytoskeleton. We found that differences in optical markers (between AOM- and control-treated rats) were negated by the disruption, suggesting cytoskeletal involvement in the optical changes. These studies provide significant insights into the micro-architectural alterations in early colon carcinogenesis, and may enable optimization of both bio-photonic and molecular risk stratification techniques to personalize colorectal cancer screening.

Open source software for electric field Monte Carlo simulation of coherent backscattering in biological media containing birefringence.

ABSTRACT. We present an open source electric field tracking Monte Carlo program to model backscattering in biological media containing birefringence, with computation of the coherent backscattering phenomenon as an example. These simulations enable the modeling of tissue scattering as a statistically homogeneous continuous random media under the Whittle-Matérn model, which includes the Henyey-Greenstein phase function as a special case, or as a composition of discrete spherical scatterers under Mie theory. The calculation of the amplitude scattering matrix for the above two cases as well as the implementation of birefringence using the Jones N-matrix formalism is presented. For ease of operator use and data processing, our simulation incorporates a graphical user interface written in MATLAB to interact with the underlying C code. Additionally, an increase in computational speed is achieved through implementation of message passing interface and the semi-analytical approach. Finally, we provide demonstrations of the results of our simulation for purely scattering media and scattering media containing linear birefringence.

A fiber optic probe design to measure depth-limited optical properties in-vivo with low-coherence enhanced backscattering (LEBS) spectroscopy.

Low-coherence enhanced backscattering (LEBS) spectroscopy is an angular resolved backscattering technique that is sensitive to sub-diffusion light transport length scales in which information about scattering phase function is preserved. Our group has shown the ability to measure the spatial backscattering impulse response function along with depth-selective optical properties in tissue ex-vivo using LEBS. Here we report the design and implementation of a lens-free fiber optic LEBS probe capable of providing depth-limited measurements of the reduced scattering coefficient in-vivo. Experimental measurements combined with Monte Carlo simulation of scattering phantoms consisting of polystyrene microspheres in water are used to validate the performance of the probe. Additionally, depth-limited capabilities are demonstrated using Monte Carlo modeling and experimental measurements from a two-layered phantom.

Polarized Enhanced Backscattering Spectroscopy for Characterization of Biological Tissues at Subdiffusion Length-scales.

Since the early 1980's, the enhanced backscattering (EBS) phenomenon has been well-studied in a large variety of non-biological materials. Yet, until recently the use of conventional EBS for the characterization of biological tissue has been fairly limited. In this work we detail the unique ability of EBS to provide spectroscopic, polarimetric, and depth-resolved characterization of biological tissue using a simple backscattering instrument. We first explain the experimental and numerical procedures used to accurately measure and model the full azimuthal EBS peak shape in biological tissue. Next we explore the peak shape and height dependencies for different polarization channels and spatial coherence of illumination. We then illustrate the extraordinary sensitivity of EBS to the shape of the scattering phase function using suspensions of latex microspheres. Finally, we apply EBS to biological tissue samples in order to measure optical properties and observe the spatial length-scales at which backscattering is altered in early colon carcinogenesis.

Measurement of the spatial backscattering impulse-response at short length scales with polarized enhanced backscattering.

In this Letter, we describe an easy to implement technique to measure the spatial backscattering impulse-response at length scales shorter than a transport mean free path with resolution of better than 10 µm using the enhanced backscattering phenomenon. This technique enables spectroscopic measurements throughout the visible range and sensitivity to all polarization channels. Through a combination of Monte Carlo simulations and experimental measurements of latex microspheres, we explore the various sensitivities of our technique to both intrinsic sample properties and extrinsic instrumental properties. We conclude by demonstrating the extraordinary sensitivity of our technique to the shape of the scattering phase function, including higher order shape parameters than the anisotropy factor (or first moment).

Multiple scattering model for the penetration depth of low-coherence enhanced backscattering.

Low-coherence enhanced backscattering (LEBS) is a depth-selective self-interference phenomenon that originates from light traveling time-reversed paths in a scattering medium. The depth selectivity of LEBS and its sensitivity to optical properties of the scattering medium has made it a promising technique for probing the structure of biological tissue with applications to disease diagnosis and, in particular, precancerous conditions. The ability to accurately predict the penetration depth of the LEBS signal is important in targeting an optimal tissue depth for detecting precancerous cells. This prediction is further complicated by the variation in optical properties of different tissue types. In this paper, the effects of the reduced scattering coefficient (µ(s)'), the phase function and the instrument spatial coherence length (L(sc)) on the LEBS penetration depth are quantified. It is determined that the LEBS penetration depth is primarily dependent on L(sc), µ(s)', and the anisotropy factor (g), but has minimal dependence on higher moments of the phase function. An empirical expression, having a similar form as the double scattering approximation for LEBS, is found to accurately predict the average penetration depth in the multiple scattering regime. The expression is shown to be accurate for a broad range of experimentally relevant optical properties and spatial coherence lengths.

Measurement of optical scattering properties with low-coherence enhanced backscattering spectroscopy.

Low-coherence enhanced backscattering (LEBS) is a depth selective technique that allows noninvasive characterization of turbid media such as biological tissue. LEBS provides a spectral measurement of the tissue reflectance distribution as a function of distance between incident and reflected ray pairs through the use of partial spatial coherence broadband illumination. We present LEBS as a new depth-selective technique to measure optical properties of tissue in situ. Because LEBS enables measurements of reflectance due to initial scattering events, LEBS is sensitive to the shape of the phase function in addition to the reduced scattering coefficient (µ(s) (*)). We introduce a simulation of LEBS that implements a two parameter phase function based on the Whittle-Matérn refractive index correlation function model. We show that the LEBS enhancement factor (E) primarily depends on µ(s) (*), the normalized spectral dependence of E (S(n)) depends on one of the two parameters of the phase function that also defines the functional type of the refractive index correlation function (m), and the LEBS peak width depends on both the anisotropy factor (g) and m. Three inverse models for calculating these optical properties are described and the calculations are validated with an experimental measurement from a tissue phantom.

Alternate formulation of enhanced backscattering as phase conjugation and diffraction: derivation and experimental observation.

Enhanced backscattering (EBS), also known as weak localization of light, is derived using the Huygens-Fresnel principle and backscattering is generally shown to be the sum of an incoherent baseline and a phase conjugated portion of the incident wave that forms EBS. The phase conjugated portion is truncated by an effective aperture described by the probability function P(s) of coherent path-pair separations. P(s) is determined by the scattering properties of the medium and so characterization of EBS can be used for metrology of scattering materials. A three dimensional intensity peak is predicted in free space at a point conjugate to the source and is experimentally observed.

Characterization of light transport in scattering media at sub-diffusion length scales with Low-coherence Enhanced Backscattering.

Low-coherence enhanced backscattering (LEBS) is a technique that has recently shown promise for tissue characterization and the detection of early pre-cancer. Although several Monte Carlo models of LEBS have been described, these models have not been accurate enough to predict all of the experimentally observed LEBS features. We present an appropriate Monte Carlo model to simulate LEBS peak properties from polystyrene microsphere suspensions in water. Results show that the choice of the phase function greatly impacts the accuracy of the simulation when the transport mean free path (ls*) is much greater than the spatial coherence length (L(SC)). When ls* < L(SC), a diffusion approximation based model of LEBS is sufficiently accurate. We also use the Monte Carlo model to validate that LEBS can be used to measure the radial scattering probability distribution (radial point spread function), p(r), at small length scales and demonstrate LEBS measurements of p(r) from biological tissue. In particular, we show that pre-cancerous and benign mucosal tissues have different small length scale light transport properties.

Depth-resolved measurement of mucosal microvascular blood content using low-coherence enhanced backscattering spectroscopy.

Low-coherence enhanced backscattering (LEBS) spectroscopy is a light scattering technique which uses partial spatial coherence broadband illumination to interrogate the optical properties at sub-diffusion length scales. In this work, we present a post-processing technique which isolates the hemoglobin concentration at different depths within a sample using a single spectroscopic LEBS measurement with a fixed spatial coherence of illumination. We verify the method with scattering (spectralon reflectance standard and polystyrene microspheres) and absorbing (hemoglobin) phantoms. We then demonstrate the relevance of this method for quantifying hemoglobin content as a function of depth within biological tissue using the azoxymethane treated animal model of colorectal cancer.