J. Quincy Brown

Quantitative Optical Spectroscopy: A Robust Tool for Direct Measurement of Breast Cancer Vascular Oxygenation and Total Hemoglobin Content In vivo

We propose the use of a robust, biopsy needle-based, fiber-optic tool for routine clinical quantification of tumor oxygenation at the time of diagnostic biopsy for breast cancer. The purpose of this study was to show diffuse reflectance spectroscopy as a quantitative tool to measure oxygenation levels in the vascular compartment of breast cancers in vivo via an optical biopsy technique. Thirty-five patients undergoing surgical treatment for breast cancer were recruited for the study at Duke University Medical Center. Diffuse reflectance spectroscopy was performed on the tumors in situ before surgical resection, followed by needle-core biopsy of the optically measured tissue. Hemoglobin saturation and total hemoglobin content were quantified from 76 optical spectra-tissue biopsy pairs, consisting of 20 malignant, 23 benign, and 33 adipose tissues. Hemoglobin saturation in malignant tissues was significantly lower than nonmalignant tissues (P<0.002) and was negatively correlated with tumor size and pathologic tumor category (P<0.05). Hemoglobin saturation was positively correlated with total hemoglobin content in malignant tissues (P<0.02). HER2/neu-amplified tumors exhibited significantly higher total hemoglobin content (P<0.05) and significantly higher hemoglobin saturation (P<0.02), which is consistent with a model of increased angiogenesis and tumor perfusion promoted by HER2/neu amplification. Diffuse reflectance spectroscopy could aid in prognosis and prediction in breast cancer via quantitative assessment of tumor physiology at the time of diagnostic biopsy.

Optical Redox Ratio Differentiates Breast Cancer Cell Lines Based on Estrogen Receptor Status

Autofluorescence spectroscopy is a powerful imaging technique that exploits endogenous fluorophores. The endogenous fluorophores NADH and flavin adenine dinucleotide (FAD) are two of the principal electron donors and acceptors in cellular metabolism, respectively. The optical oxidation-reduction (redox) ratio is a measure of cellular metabolism and can be determined by the ratio of NADH/FAD. We hypothesized that there would be a significant difference in the optical redox ratio of normal mammary epithelial cells compared with breast tumor cell lines and that estrogen receptor (ER)-positive cells would have a higher redox ratio than ER-negative cells. To test our hypothesis, the optical redox ratio was determined by collecting the fluorescence emission for NADH and FAD via confocal microscopy. We observed a statistically significant increase in the optical redox ratio of cancer compared with normal cell lines (P < 0.05). Additionally, we observed a statistically significant increase in the optical redox ratio of ER(+) breast cancer cell lines. The level of ESR1 expression, determined by real-time PCR, directly correlated with the optical redox ratio (Pearsons correlation coefficient = 0.8122, P = 0.0024). Furthermore, treatment with tamoxifen and ICI 182,870 statistically decreased the optical redox ratio of only ER(+) breast cancer cell lines. The results of this study raise the important possibility that fluorescence spectroscopy can be used to identify subtypes of breast cancer based on receptor status, monitor response to therapy, or potentially predict response to therapy. This source of optical contrast could be a potentially useful tool for drug screening in preclinical models.

Advances in Quantitative UV-Visible Spectroscopy for Clinical and Pre-clinical Application in Cancer

Methods of optical spectroscopy that provide quantitative, physically or physiologically meaningful measures of tissue properties are an attractive tool for the study, diagnosis, prognosis, and treatment of various cancers. Recent development of methodologies to convert measured reflectance and fluorescence spectra from tissue to cancer-relevant parameters such as vascular volume, oxygenation, extracellular matrix extent, metabolic redox states, and cellular proliferation have significantly advanced the field of tissue optical spectroscopy. The number of publications reporting quantitative tissue spectroscopy results in the UV-visible wavelength range has increased sharply in the past three years, and includes new and emerging studies that correlate optically measured parameters with independent measures such as immunohistochemistry, which should aid in increased clinical acceptance of these technologies.

Rapid noninvasive optical imaging of tissue composition in breast tumor margins.

BACKGROUND In women undergoing breast conserving surgery (BCS), up to 60% can require re-excision. Our objective is to develop an optically based technology which can differentiate benign from malignant breast tissues intraoperatively through differences in tissue composition factors. METHODS A prospective study of optical imaging of BCS margins is being performed. Optical images are transformed into tissue composition maps with parameters of total hemoglobin concentration, b-carotene concentration and scattering. The predicted outcome is then compared to the margin-level pathology. RESULTS Fifty-five margins from 48 patients have undergone assessment. Within 34 specimens with pathologically confirmed positive margins, the ratio map of b-carotene/scattering showed the most significant difference reflecting a decrease in adipose and an increase in cell density within malignant margins (p=.002). These differences were notable in both in-situ and invasive disease. CONCLUSIONS We present a novel optical spectral imaging device that provides a rapid, non-destructive assay of the tissue composition of breast tumor margins.

Performance metrics of an optical spectral imaging system for intra-operative assessment of breast tumor margins

As many as 20-70% of patients undergoing breast conserving surgery require repeat surgeries due to a close or positive surgical margin diagnosed post-operatively [1]. Currently there are no widely accepted tools for intra-operative margin assessment which is a significant unmet clinical need. Our group has developed a first-generation optical visible spectral imaging platform to image the molecular composition of breast tumor margins and has tested it clinically in 48 patients in a previously published study [2]. The goal of this paper is to report on the performance metrics of the system and compare it to clinical criteria for intra-operative tumor margin assessment. The system was found to have an average signal to noise ratio (SNR) >100 and <15% error in the extraction of optical properties indicating that there is sufficient SNR to leverage the differences in optical properties between negative and close/positive margins. The probe had a sensing depth of 0.5-2.2 mm over the wavelength range of 450-600 nm which is consistent with the pathologic criterion for clear margins of 0-2 mm. There was <1% cross-talk between adjacent channels of the multi-channel probe which shows that multiple sites can be measured simultaneously with negligible cross-talk between adjacent sites. Lastly, the system and measurement procedure were found to be reproducible when evaluated with repeated measures, with a low coefficient of variation (<0.11). The only aspect of the system not optimized for intra-operative use was the imaging time. The manuscript includes a discussion of how the speed of the system can be improved to work within the time constraints of an intra-operative setting.

Optical Assesssment of Tumor Resection Margins in the Breast

Breast conserving surgery, in which the breast tumor and the surrounding normal tissue are removed, is the primary mode of treatment for invasive and in situ carcinomas of the breast, conditions that affect nearly 200 000 women annually. Of these nearly 200 000 patients who undergo this surgical procedure, between 20%-70% of them may undergo additional surgeries to remove tumor that was left behind in the first surgery, due to the lack of intraoperative tools that can detect whether the boundaries of the excised specimens are free from residual cancer. Optical techniques have many attractive attributes that may make them useful tools for intraoperative assessment of breast tumor resection margins. In this paper, we discuss clinical design criteria for intraoperative breast tumor margin assessment and review optical techniques applied to this problem. In addition, we report on the development and clinical testing of quantitative diffuse reflectance imaging (Q-DRI) as a potential solution to this clinical need. Q-DRI is a spectral imaging tool, which has been applied to 55 resection margins in 48 patients at Duke University Medical Center. Clear sources of contrast between cancerous and cancer-free resection margins were identified with the device, and resulted in an overall accuracy of 75% in detecting positive margins.

Optical spectral surveillance of breast tissue landscapes for detection of residual disease in breast tumor margins.

We demonstrate a strategy to “sense” the micro-morphology of a breast tumor margin over a wide field of view by creating quantitative hyperspectral maps of the tissue optical properties (absorption and scattering), where each voxel can be deconstructed to provide information on the underlying histology. Information about the underlying tissue histology is encoded in the quantitative spectral information (in the visible wavelength range), and residual carcinoma is detected as a shift in the histological landscape to one with less fat and higher glandular content. To demonstrate this strategy, fully intact, fresh lumpectomy specimens (n = 88) from 70 patients were imaged intra-operatively. The ability of spectral imaging to sense changes in histology over large imaging areas was determined using inter-patient mammographic breast density (MBD) variation in cancer-free tissues as a model system. We discovered that increased MBD was associated with higher baseline β-carotene concentrations (p = 0.066) and higher scattering coefficients (p = 0.007) as measured by spectral imaging, and a trend toward decreased adipocyte size and increased adipocyte density as measured by histological examination in BMI-matched patients. The ability of spectral imaging to detect cancer intra-operatively was demonstrated when MBD-specific breast characteristics were considered. Specifically, the ratio of β-carotene concentration to the light scattering coefficient can report on the relative amount of fat to glandular density at the tissue surface to determine positive margin status, when baseline differences in these parameters between patients with low and high MBD are taken into account by the appropriate selection of threshold values. When MBD was included as a variable a priori, the device was estimated to have a sensitivity of 74% and a specificity of 86% in detecting close or positive margins, regardless of tumor type. Superior performance was demonstrated in high MBD tissue, a population that typically has a higher percentage of involved margins.

Preferential accumulation of 5-aminolevulinic acid-induced protoporphyrin IX in breast cancer: a comprehensive study on six breast cell lines with varying phenotypes

We describe the potential of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence as a source of contrast for margin detection in commonly diagnosed breast cancer subtypes. Fluorescence intensity of PpIX in untreated and ALA-treated normal mammary epithelial and breast cancer cell lines of varying estrogen receptor expression were quantitatively imaged with confocal microscopy. Percentage change in fluorescence intensity integrated over 610-700 nm (attributed to PpIX) of posttreated compared to pretreated cells showed statistically significant differences between four breast cancer and two normal mammary epithelial cell lines. However, a direct comparison of post-treatment PpIX fluorescence intensities showed no differences between breast cancer and normal mammary epithelial cell lines due to confounding effects by endogenous fluorescence from flavin adenine dinucleotide (FAD). Clinically, it is impractical to obtain pre- and post-treatment images. Thus, spectral imaging was demonstrated as a means to remove the effects of endogenous FAD fluorescence allowing for discrimination between post-treatment PpIX fluorescence of four breast cancer and two normal mammary epithelial cell lines. Fluorescence spectral imaging of ALA-treated breast cancer cells showed preferential PpIX accumulation regardless of malignant phenotype and suggests a useful contrast mechanism for discrimination of residual cancer at the surface of breast tumor margins.

Advancing Optical Imaging for Breast Margin Assessment: An Analysis of Excisional Time, Cautery, and Patent Blue Dye on Underlying Sources of Contrast

Breast conserving surgery (BCS) is a recommended treatment for breast cancer patients where the goal is to remove the tumor and a surrounding rim of normal tissue. Unfortunately, a high percentage of patients return for additional surgeries to remove all of the cancer. Post-operative pathology is the gold standard for evaluating BCS margins but is limited due to the amount of tissue that can be sampled. Frozen section analysis and touch-preparation cytology have been proposed to address the surgical needs but also have sampling limitations. These issues represent an unmet clinical need for guidance in resecting malignant tissue intra-operatively and for pathological sampling. We have developed a quantitative spectral imaging device to examine margins intra-operatively. The context in which this technology is applied (intra-operative or post-operative setting) is influenced by time after excision and surgical factors including cautery and the presence of patent blue dye (specifically Lymphazurin™, used for sentinel lymph node mapping). Optical endpoints of hemoglobin ([THb]), fat ([β-carotene]), and fibroglandular content via light scattering () measurements were quantified from diffuse reflectance spectra of lumpectomy and mastectomy specimens using a Monte Carlo model. A linear longitudinal mixed-effects model was used to fit the optical endpoints for the cautery and kinetics studies. Monte Carlo simulations and tissue mimicking phantoms were used for the patent blue dye experiments. [THb], [β-carotene], and were affected by <3.3% error with <80 µM of patent blue dye. The percent change in [β-carotene], , and [β-carotene]/ was <14% in 30 minutes, while percent change in [THb] was >40%. [β-carotene] and [β-carotene]/ were the only parameters not affected by cautery. This work demonstrates the importance of understanding the post-excision kinetics of ex-vivo tissue and the presence of cautery and patent blue dye for breast tumor margin assessment, to accurately interpret data and exploit underling sources of contrast.

Optimization of a widefield structured illumination microscope for non-destructive assessment and quantification of nuclear features in tumor margins of a primary mouse model of sarcoma.

Cancer is associated with specific cellular morphological changes, such as increased nuclear size and crowding from rapidly proliferating cells. In situ tissue imaging using fluorescent stains may be useful for intraoperative detection of residual cancer in surgical tumor margins. We developed a widefield fluorescence structured illumination microscope (SIM) system with a single-shot FOV of 2.1×1.6 mm (3.4 mm2) and sub-cellular resolution (4.4 µm). The objectives of this work were to measure the relationship between illumination pattern frequency and optical sectioning strength and signal-to-noise ratio in turbid (i.e. thick) samples for selection of the optimum frequency, and to determine feasibility for detecting residual cancer on tumor resection margins, using a genetically engineered primary mouse model of sarcoma. The SIM system was tested in tissue mimicking solid phantoms with various scattering levels to determine impact of both turbidity and illumination frequency on two SIM metrics, optical section thickness and modulation depth. To demonstrate preclinical feasibility, ex vivo 50 µm frozen sections and fresh intact thick tissue samples excised from a primary mouse model of sarcoma were stained with acridine orange, which stains cell nuclei, skeletal muscle, and collagenous stroma. The cell nuclei were segmented using a high-pass filter algorithm, which allowed quantification of nuclear density. The results showed that the optimal illumination frequency was 31.7 µm−1 used in conjunction with a 4×0.1 NA objective ( = 0.165). This yielded an optical section thickness of 128 µm and an 8.9×contrast enhancement over uniform illumination. We successfully demonstrated the ability to resolve cell nuclei in situ achieved via SIM, which allowed segmentation of nuclei from heterogeneous tissues in the presence of considerable background fluorescence. Specifically, we demonstrate that optical sectioning of fresh intact thick tissues performed equivalently in regards to nuclear density quantification, to physical frozen sectioning and standard microscopy.