Spectroscopy enhances the information content of optical mammography

Near-infrared (NIR) diffuse optical spectroscopy and imaging may enhance existing technologies for breast cancer screening, diagnosis, and treatment. NIR techniques are based on quantitative measurements of functional contrast between healthy and diseased tissue. In this study we measured the spectral dependence of tissue absorption (mu(a)) and reduced scattering (mu'(s)) in the breasts of 30 healthy women and one woman with a fibroadenoma using a seven-wavelength frequency-domain photon migration probe. Subjects included pre- and postmenopausal women between the ages of 18 and 64. Multi-spectral measurements were used along with a four-component fit to determine the concentrations of de-oxy and oxy-hemoglobin, water and lipids in breast. The scattering spectral shape was also quantified. Our measurements demonstrate that the measured concentrations of NIR analytes correlate well with known breast physiology. Although the tissue scattering at a single wavelength was found to have little value as a functional parameter, the dependence of the scattering on wavelength provided key insights into breast composition and physiology. Lipids and scattering spectra in the breast were found to increase and decrease, respectively, with increasing body mass index. Simple calculations are also provided to demonstrate potential penalties from ignoring the contributions of water and lipids in breast measurements. Finally, water is shown to be a possible indicator for detecting a fibroadenoma, whereas the hemoglobin saturation was found to be a poor indicator. Multi-spectral measurements, compared to measurements restricted to one or two wavelengths, provide additional information that may be useful in managing breast disease.     

Quantification of optical properties of a breast tumor using random walk theory

For the first time we use a random walk methodology based on time-dependent contrast functions to quantify the optical properties of breast tumors (invasive ductal carcinoma) of two patients. Previously this theoretical approach was successfully applied for analysis of embedded objects in several phantoms. Data analysis was performed on distributions of times of flight for photons transmitted through the breast which were recorded in vivo using a time-domain scanning mammograph at 670 and 785 nm. The size of the tumors, their optical properties, and those of the surrounding tissue were reconstructed at both wavelengths. The tumors showed increased absorption and scattering. From the absorption coefficients at both wavelengths blood oxygen saturation was estimated for the tumors and the surrounding tissue.     

Assessing the future of diffuse optical imaging technologies for breast cancer management

Diffuse optical imaging (DOI) is a noninvasive optical technique that employs near-infrared (NIR) light to quantitatively characterize the optical properties of thick tissues. Although NIR methods were first applied to breast transillumination (also called diaphanography) nearly 80 years ago, quantitative DOI methods employing time- or frequency-domain photon migration technologies have only recently been used for breast imaging (i.e., since the mid-1990s). In this review, the state of the art in DOI for breast cancer is outlined and a multi-institutional Network for Translational Research in Optical Imaging (NTROI) is described, which has been formed by the National Cancer Institute to advance diffuse optical spectroscopy and imaging (DOSI) for the purpose of improving breast cancer detection and clinical management. DOSI employs broadband technology both in near-infrared spectral and temporal signal domains in order to separate absorption from scattering and quantify uptake of multiple molecular probes based on absorption or fluorescence contrast. Additional dimensionality in the data is provided by integrating and co-registering the functional information of DOSI with x-ray mammography and magnetic resonance imaging (MRI), which provide structural information or vascular flow information, respectively. Factors affecting DOSI performance, such as intrinsic and extrinsic contrast mechanisms, quantitation of biochemical components, image formation/visualization, and multimodality co-registration are under investigation in the ongoing research NTROI sites. One of the goals is to develop standardized DOSI platforms that can be used as stand-alone devices or in conjunction with MRI, mammography, or ultrasound. This broad-based, multidisciplinary effort is expected to provide new insight regarding the origins of breast disease and practical approaches for addressing several key challenges in breast cancer, including: Detecting disease in mammographically dense tissue, distinguishing between malignant and benign lesions, and understanding the impact of neoadjuvant chemotherapies.     

Locating inhomogeneities in tissue by using the most probable diffuse path of light

Characterization of human tissue using near-IR (NIR) light is becoming increasingly popular. The light signal transmitted from the tissue contains information concerning inhomogeneities in tissue, such as size, position, and pathological states (benign or malignant). We discuss the most probable diffuse path (MPDP) related to frequency-domain diffuse photon density waves (DPDWs) propagating inside turbid media. We find that for a medium of finite size, the existence of boundaries between tissue and nonscattering media would have considerable impact on the path shape. It is also demonstrated that such paths can be used to obtain higher accuracy in localizing absorbers embedded in a homogeneous background. Based on the proposed MPDP, a new method for 3-D localization of heterogeneities in turbid media is proposed, which is validated by experiments using Intralipid and pork fat. The experiments are performed with an NIR breast cancer detection system designed and assembled in our lab, using 780-nm NIR light. In Intralipid, when the size of a single absorber is less than 1 cm, the localization error is about 2 mm. The results from pork fat are also acceptable.     

Spatial variations in optical and physiological properties of healthy breast tissue

Near-infrared (NIR) diffuse optical spectroscopy (DOS) and diffuse optical imaging (DOI) show promise as noninvasive clinical techniques for breast cancer screening and diagnosis. Since NIR methods are based on optical contrast between healthy and diseased tissue, it is essential to characterize the sources of endogenous contrast in normal subjects. We report intra- and inter-subject variation and bilateral asymmetry of the optical and physiological parameters of 31 women using a seven-wavelength NIR frequency-domain photon migration (FDPM) instrument. Wavelength-dependent absorption and reduced scattering parameters (micro(a) and micro(s'), respectively) were measured in four major quadrants and the areolar regions of left and right breasts. These values were used to determine tissue concentrations of oxy-(HbO(2)) and deoxy-(Hb-R) hemoglobin, lipid content, water concentration, and tissue "scatter power." Mean total hemoglobin for premenopausal (PRE) women (20 to 30 microM) is approximately two-fold higher than for postmenopausal (POST) subjects at all positions. POST women have approximately 50% higher lipid content (50 to 60%) than PRE at all positions. Water concentration on average is 1.8-fold higher for PRE subjects (30 to 40%) than POST. These differences are most pronounced when comparing the areolar complex to the other regions of the breast. In premenopausal women, the areolar regions have 40 to 45% increased total hemoglobin concentration (THC), 20 to 25% lower lipid content, and 30 to 60% higher scatter power versus the quadrants. Small-scale (3 cm) changes in optical properties are negligible compared to large-scale variations over all quadrants, where the intrinsic spatial heterogeneity of healthy breast tissue is 20 to 40% for micro(a) and 5 to 12% for micro(s'). Although no consistent right-left differences are observed in the study population, relative differences between symmetric positions ranged from 18 to 30% for THC, 10 to 40% for adipose, 10 to 25% for water, and 4 to 9% for scattering (674 nm) within an individual.     

Multilayered, core/shell nanoprobes based on magnetic ferric oxide particles and quantum dots for multimodality imaging of breast cancer tumors

Multilayered, core/shell nanoprobes (MQQ-probe) based on magnetic nanoparticles (MNPs) and quantum dots (QDs) have been successfully developed for multimodality tumor imaging. This MQQ-probe contains Fe(3)O(4) MNPs, visible-fluorescent QDs (600?nm emission) and near infrared-fluorescent QDs (780?nm emission) in multiple silica layers. The fabrication of the MQQ-probe involves the synthesis of a primer Fe(3)O(4) MNPs/SiO(2) core by a reverse microemulsion method. The MQQ-probe can be used both as a fluorescent probe and a contrast reagent of magnetic resonance imaging. For breast cancer tumor imaging, anti-HER2 (human epidermal growth factor receptor 2) antibody was conjugated to the surface of the MQQ-probe. The specific binding of the antibody conjugated MQQ-probe to the surface of human breast cancer cells (KPL-4) was confirmed by fluorescence microscopy and fluorescence-activated cell sorting analysis in?vitro. Due to the high tissue permeability of near-infrared (NIR) light, NIR fluorescence imaging of the tumor mice (KPL-4 cells transplanted) was conducted by using the anti-HER2 antibody conjugated MQQ-probe. In?vivo multimodality images of breast tumors were successfully taken by NIR fluorescence and T(2)-weighted magnetic resonance. Antibody conjugated MQQ-probes have great potential to use for multimodality imaging of cancer tumors in?vitro and in?vivo.     

High-sensitivity detection of breast tumors in vivo by use of a pH-sensitive near-infrared fluorescence probe

We investigated the potential of the pH-sensitive dye, CypHer5E, conjugated to Herceptin (pH-Her) for the sensitive detection of breast tumors in mice using noninvasive time-domain near-infrared fluorescence imaging and different methods of data analysis. First, the fluorescence properties of pH-Her were analyzed as function of pH and/or dye-to-protein ratio, and binding specificity was confirmed in cell-based assays. Subsequently, the performance of pH-Her in nude mice bearing orthotopic HER2-positive (KPL-4) and HER2-negative (MDA-MB-231) breast carcinoma xenografts was compared to that of an always-on fluorescent conjugate Alexa Fluor 647-Herceptin (Alexa-Her). Subtraction of autofluorescence and lifetime (LT)-gated image analyses were performed for background fluorescence suppression. In mice bearing HER2-positive tumors, autofluorescence subtraction together with the selective fluorescence enhancement of pH-Her solely in the tumor's acidic environment provided high contrast-to-noise ratios (CNRs). This led to an improved sensitivity of tumor detection compared to Alexa-Her. In contrast, LT-gated imaging using LTs determined in model systems did not improve tumor-detection sensitivity in vivo for either probe. In conclusion, pH-Her is suitable for sensitive in vivo monitoring of HER2-expressing breast tumors with imaging in the intensity domain and represents a promising tool for detection of weak fluorescent signals deriving from small tumors or metastases.     

Image reconstruction of effective Mie scattering parameters of breast tissue in vivo with near-infrared tomography

A method for image reconstruction of the effective size and number density of scattering particles is discussed within the context of interpreting near-infrared (NIR) tomography images of breast tissue. An approach to use Mie theory to estimate the effective scattering parameters is examined and applied, given some assumptions about the index of refraction change expected in lipid membrane-bound scatterers. When using a limited number of NIR wavelengths in the reduced scattering spectra, the parameter extraction technique is limited to representing a continuous distribution of scatterer sizes, which is modeled as a simple exponentially decreasing distribution function. In this paper, image formation of effective scatterer size and number density is presented based on the estimation method. The method was evaluated with Intralipid phantom studies to demonstrate particle size estimation to within 9% of the expected value. Then the method was used in NIR patient images, and it indicates that for a cancer tumor, the effective scatterer size is smaller than the background breast values and the effective number density is higher. In contrast, for benign tumor patients, there is not a significant difference in effective scatterer size or number density between tumor and normal tissues. The method was used to interpret magnetic resonance imaging-coupled NIR images of adipose and fibroglandular tissues, and it indicated that the fibroglandular tissue has smaller effective scatterer size and larger effective number density than the adipose tissue does.     

In vivo absorption, scattering, and physiologic properties of 58 malignant breast tumors determined by broadband diffuse optical spectroscopy

Diffuse optical imaging (DOI) may be a beneficial diagnostic method for women with mammographically dense breast tissue. In order to evaluate the utility of DOI, we are developing broadband diffuse optical spectroscopy (DOS) to characterize the functional origins of optical signals in breast cancer patients. Broadband DOS combines multifrequency intensity-modulated and continuous-wave near-infrared light to quantify tissue absorption and scattering spectra from 650 to 1000 nm. Values of intrinsic physiological properties (oxy- and deoxy-hemoglobin, water, lipid, and scatter power) derived from absorption and scattering spectra provide detailed information on breast physiology. We present the results of clinical studies of 58 stage II/III malignant breast tumors using a noninvasive, handheld, broadband DOS probe. On average, eight positions were scanned over tumor and contralateral normal breast for each subject. Intrinsic physiological properties were statistically significantly different for malignant vs. normal tissues for all subjects, without patient age or tumor size/type stratification. Breast tissues containing malignant tumors displayed reduced lipid content ( approximately 20%) and increased water, deoxy-, and oxy-hemoglobin (>50% each) compared to normal breast tissues. Functional perturbations by the tumor were significantly larger than functional variations in normal tissues. A tissue optical index (TOI) derived from intrinsic physiological properties yielded an average two-fold contrast difference between malignant tumors and intrinsic tissue properties. Our results demonstrate that intrinsic optical signals can be influenced by functional perturbations characteristic of malignant transformation; cellular metabolism, extracellular matrix composition, and angiogenesis. Our findings further underscore the importance of broadband measurements and patient age stratification in breast cancer DOI.     

Near-infrared fluorescent labeled CGRRAGGSC peptides for optical imaging of IL-11Rα in athymic mice bearing tumor xenografts

The interleukin-11 (IL-11) and the IL-11 receptor α-subunit (IL-11Rα) have been demonstrated to regulate the invasion and proliferation of tumor cells. Our study intends to evaluate a noninvasive imaging of IL-11Rα expression in breast tumors using near-infrared (NIR) fluorescent dye Cy7-labeled IL-11 mimic peptide CGRRAGGSC. This work evaluated the IL-11Rα expression of breast tumor cells and the binding status of this peptide to IL-11Rα in vitro and in vivo by using Western blotting, immunofluorescence staining and near-infrared fluorescence imaging. Our biochemical study showed that IL-11Rα was overexpressed in breast tumor cells (MCF-7). The cell-binding assay demonstrated specific binding of peptide CGRRAGGSC to MCF-7 cells in vitro. In vivo imaging results showed that NIR fluorescent signals of Cy7-CGRRAGGSC were selectively accumulated in tumor and metabolic organs. While in the blocking experiment, free CGRRAGGSC obviously blocked the concentration of the Cy7-CGRRAGGSC in the tumors. These results suggested that IL-11Rα may be used as a potential target for noninvasive imaging in IL-11Rα overexpressed tumors. Furthermore, the imaging agent of near-infrared fluorescent dye Cy7-labeled CGRRAGGSC is suitable for IL-11Rα expression imaging study in vivo.     

In vivo water state measurements in breast cancer using broadband diffuse optical spectroscopy

Structural changes in water molecules are related to physiological, anatomical and pathological properties of tissues. Near infrared (NIR) optical absorption methods are sensitive to water; however, detailed characterization of water in thick tissues is difficult to achieve because subtle spectral shifts can be obscured by multiple light scattering. In the NIR, a water absorption peak is observed around 975 nm. The precise NIR peak's shape and position are highly sensitive to water molecular disposition. We introduce a bound water index (BWI) that quantifies shifts observed in tissue water absorption spectra measured by broadband diffuse optical spectroscopy (DOS). DOS quantitatively measures light absorption and scattering spectra and therefore reveals bound water spectral shifts. BWI as a water state index was validated by comparing broadband DOS to magnetic resonance spectroscopy, diffusion-weighted MRI and conductivity in bound water tissue phantoms. Non-invasive DOS measurements of malignant and normal breast tissues performed in 18 subjects showed a significantly higher fraction of free water in malignant tissues (p < 0.0001) compared to normal tissues. BWI of breast cancer tissues inversely correlated with Nottingham-Bloom-Richardson histopathology scores. These results highlight broadband DOS sensitivity to molecular disposition of water and demonstrate the potential of BWI as a non-invasive in vivo index that correlates with tissue pathology.     

Elastic registration of x-ray mammograms and three-dimensional magnetic resonance imaging data

Major problems in treating breast cancer are the early detection of tumors and accurate biopsy of small volumes of breast (mamma) tissue. This report presents an elastic registration algorithm of two x-ray mammograms and a corresponding magnetic resonance imaging (MRI) volume. To cope with the soft tissue deformation of the breast during mammography, a two-dimensional model of breast deformation behavior is used as an elastic transformation. Normalized mutual information is employed as a measure of similarity. Regions of interest in the uncompressed x-ray mammograms are projected into the MRI volume to determine their three-dimensional origin.     

Characterization of hemoglobin, water, and NIR scattering in breast tissue: analysis of intersubject variability and menstrual cycle changes

Near-infrared imaging was used to quantify typical values of hemoglobin concentration, oxygen saturation, water fraction, scattering power, and scattering amplitude within the breast tissue of volunteer subjects. A systematic study of the menstrual variations in these parameters was carried out by measuring a group of seven premenopausal normal women (aged 41 to 47 years) in the follicular (days 7 to 14 of the cycle) and secretory phases (days 21 to 28) of the cycle, for two complete menstrual cycles. An average increase in hemoglobin concentration of 2.6 microM or 13% of the background breast values was observed in the secretory phase relative to the follicular phase (p<0.0001), but no other average near-infrared parameter changes were significant. While repeatable and systematic changes were observed in all parameters for individual subjects, large intersubject variations were present in all parameters. In a survey of thirty-nine normal subjects, the total hemoglobin varied from 9 to 45 microM, with a systematic correlation observed between total hemoglobin concentration and breast radiographic density. Scattering power and scattering amplitude were also correlated with radiographic density, but oxygen saturation and water fraction were not. Images of breast lesions indicate that total hemoglobin-based contrast can be up to 200% relative to the background in the same breast. Yet, since the background hemoglobin values vary considerably among breasts, the maximum hemoglobin concentrations observed in cancer tumors may vary considerably as well. In light of these observations, it may be important to use hemoglobin contrast values relative to the background for a given breast, rather than absolute hemoglobin contrast when trying to compare the features of breast lesions among subjects.     

X-ray scattering for classifying tissue types associated with breast disease

Collagen types I and III can be characterized at the molecular level (at the tens to hundreds of nanometers scale) using small angle x-ray scattering (SAXS). Although collagen fibril structural parameters at this length scale have shown differences between diseased and nondiseased breast tissues, a comprehensive analysis involving a multitude of features with a large (>50) patient cohort has not previously been investigated. Breast tissue samples were excised from 80 patients presenting with either a breast lump or reduction mammoplasty. From these, invasive carcinoma, benign tissue, and normal parenchyma were analyzed. Parameters related to collagen structure, including longitudinal (axial) and lateral (equatorial) features, polar angle features, total scattering intensity, and tissue heterogeneity effects, were extracted from the SAXS patterns and examined. The amplitude of the third-order axial peak and the total scattering intensity (amorphous scatter) showed the most separation between tissue groups and a classification model using these two parameters demonstrated an accuracy of over 95% between invasive carcinoma and mammoplasty patients. Normal tissue taken from disease-free patients (mammoplasty) and normal tissue taken from patients with presence of disease showed significant differences, suggesting that SAXS may provide different diagnostic information from that of conventional histopathology.     

Intrinsic tumor biomarkers revealed by novel double-differential spectroscopic analysis of near-infrared spectra

We develop a double-differential spectroscopic analysis method for broadband near-infrared (NIR, 650 to 1000 nm) absorption spectra. Application of this method to spectra of tumor-containing breast tissue reveals specific cancer biomarkers. In this method, patient-specific variations in molecular composition are removed by using the normal tissue as an internal control. The effects of concentration differences of the four major tissue absorbers (oxyhemoglobin, deoxyhemoglobin, water, and bulk lipid) between the tumor and normal tissue are accounted for to reveal small spectral components unique to cancer. From a pilot study of 15 cancer patients, we find these spectral components to be characterized by specific NIR absorption bands. Based on the spectral regions of absorption at about 760, 930, and 980 nm, we identify these biomarkers with changes in state or addition of lipid and/or water. To quantify spectral variation in the absorption bands, we construct the specific tumor component (STC) index. The STC index identifies regions of the breast with tumors.     

Wavelet-based feature extraction applied to small-angle x-ray scattering patterns from breast tissue: a tool for differentiating between tissue types

This paper reports on the application of wavelet decomposition to small-angle x-ray scattering (SAXS) patterns from human breast tissue produced by a synchrotron source. The pixel intensities of SAXS patterns of normal, benign and malignant tissue types were transformed into wavelet coefficients. Statistical analysis found significant differences between the wavelet coefficients describing the patterns produced by different tissue types. These differences were then correlated with position in the image and have been linked to the supra-molecular structural changes that occur in breast tissue in the presence of disease. Specifically, results indicate that there are significant differences between healthy and diseased tissues in the wavelet coefficients that describe the peaks produced by the axial d-spacing of collagen. These differences suggest that a useful classification tool could be based upon the spectral information within the axial peaks.     

Intrinsic Near-Infrared Spectroscopic Markers of Breast Tumors

We have discovered quantitative optical biomarkers unique to cancer by developing a double-differential spectroscopic analysis method for near-infrared (NIR, 650–1000 nm) spectra acquired non-invasively from breast tumors. These biomarkers are characterized by specific NIR absorption bands. The double-differential method removes patient specific variations in molecular composition which are not related to cancer, and reveals these specific cancer biomarkers. Based on the spectral regions of absorption, we identify these biomarkers with lipids that are present in tumors either in different abundance than in the normal breast or new lipid components that are generated by tumor metabolism. Furthermore, the O-H overtone regions (980–1000 nm) show distinct variations in the tumor as compared to the normal breast. To quantify spectral variation in the absorption bands, we constructed the Specific Tumor Component (STC) index. In a pilot study of 12 cancer patients we found 100% sensitivity and 100% specificity for lesion identification. The STC index, combined with other previously described tissue optical indices, further improves the diagnostic power of NIR for breast cancer detection.     

A large-scale study of the ultrawideband microwave dielectric properties of normal, benign and malignant breast tissues obtained from cancer surgeries

The development of microwave breast cancer detection and treatment techniques has been driven by reports of substantial contrast in the dielectric properties of malignant and normal breast tissues. However, definitive knowledge of the dielectric properties of normal and diseased breast tissues at microwave frequencies has been limited by gaps and discrepancies across previously published studies. To address these issues, we conducted a large-scale study to experimentally determine the ultrawideband microwave dielectric properties of a variety of normal, malignant and benign breast tissues, measured from 0.5 to 20 GHz using a precision open-ended coaxial probe. Previously, we reported the dielectric properties of normal breast tissue samples obtained from reduction surgeries. Here, we report the dielectric properties of normal (adipose, glandular and fibroconnective), malignant (invasive and non-invasive ductal and lobular carcinomas) and benign (fibroadenomas and cysts) breast tissue samples obtained from cancer surgeries. We fit a one-pole Cole-Cole model to the complex permittivity data set of each characterized sample. Our analyses show that the contrast in the microwave-frequency dielectric properties between malignant and normal adipose-dominated tissues in the breast is considerable, as large as 10:1, while the contrast in the microwave-frequency dielectric properties between malignant and normal glandular/fibroconnective tissues in the breast is no more than about 10%.     

Time-domain scanning optical mammography: II. Optical properties and tissue parameters of 87 carcinomas

Within a clinical trial on scanning time-domain optical mammography reported on in a companion publication (part I), craniocaudal and mediolateral projection optical mammograms were recorded from 154 patients, suspected of having breast cancer. Here we report on in vivo optical properties of the subset of 87 histologically validated carcinomas which were visible in optical mammograms recorded at two or three near-infrared wavelengths. Tumour absorption and reduced scattering coefficients were derived from distributions of times of flight of photons recorded at the tumour site employing the model of diffraction of photon density waves by a spherical inhomogeneity, located in an otherwise homogeneous tissue slab. Effective tumour radii, taken from pathology, and tumour location along the compression direction, deduced from off-axis optical scans of the tumour region, were included in the analysis as prior knowledge, if available. On average, tumour absorption coefficients exceeded those of surrounding healthy breast tissue by a factor of about 2.5 (670 nm), whereas tumour reduced scattering coefficients were larger by about 20% (670 nm). From absorption coefficients at 670 nm and 785 nm total haemoglobin concentration and blood oxygen saturation were deduced for tumours and surrounding healthy breast tissue. Apart from a few outliers total haemoglobin concentration was observed to be systematically larger in tumours compared to healthy breast tissue. In contrast, blood oxygen saturation was found to be a poor discriminator for tumours and healthy breast tissue; both median values of blood oxygen saturation are the same within their statistical uncertainties. However, the ratio of total haemoglobin concentration over blood oxygen saturation further improves discrimination between tumours and healthy breast tissue. For 29 tumours detected in optical mammograms recorded at three wavelengths (670 nm, 785 nm, 843 nm or 884 nm), scatter power was derived from transport scattering coefficients. Scatter power of tumours tends to be larger than that of surrounding healthy breast tissue, yet the 95% confidence intervals of both medians overlap.