Quantitative optical spectroscopy: A robust tool for direct measurement of breast cancer vascular oxygenation and total hemoglobin content in vivo

doi:10.1158/0008-5472.CAN-08-3370

Cancer Res. Author manuscript; available in PMC 2010 April 1.

Published in final edited form as:

Cancer Res. 2009 April 1; 69(7): 2919–2926.

Published online 2009 March 17. doi: 10.1158/0008-5472.CAN-08-3370

PMCID: PMC2677720

NIHMSID: NIHMS94037

Quantitative optical spectroscopy: A robust tool for direct measurement of breast cancer vascular oxygenation and total hemoglobin content in vivo

J. Quincy Brown,¹ Lee G. Wilke,² Joseph Geradts,³ Stephanie A. Kennedy,¹ Gregory M. Palmer,⁴ and Nirmala Ramanujam¹

¹ Department of Biomedical Engineering, Duke University, Durham, NC, USA

² Department of Surgery, Duke University Medical Center, Durham, NC, USA

³ Department of Pathology, Duke University Medical Center, Durham, NC, USA

⁴ Department of Radiation Oncology, Duke University Medical Center, Durham, NC USA

Corresponding Author: J. Quincy Brown, Dept. of Biomedical Engineering, 136 Hudson Hall, Box 90281, Durham, NC, USA 27708. Email: quincy.brown/at/duke.edu

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Abstract

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 demonstrate 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 non-malignant tissues (p < 0.002), and was negatively correlated with tumor size and pathologic tumor category (pT) (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.

Keywords: breast cancer, optical spectroscopy, tumor hypoxia, HER2/neu, needle biopsy

I. Introduction

Tumor growth is facilitated by hypoxia, or low oxygen tension in tissue (1). Direct measurement of tumor hypoxia has been linked to decreased overall survival and increased risk of local recurrence in several organ sites, including the uterine cervix (2), head and neck cancers (3), and soft tissue sarcomas (4). Over-expression of biomarkers related to hypoxia (HIF1-α and CA IX) in breast cancer has also been linked to a poor prognosis (5). Hypoxia has been implicated in the resistance of tumors to radiation and some types of chemotherapy (6, 7) and has been shown to be a predictor of response (8, 9). The aggressiveness of hypoxic tumors is likely related to the selective adaptation of hypoxia-resistant tumor phenotypes, which can thrive in low oxygen environments. Numerous studies have investigated the link between clinical outcomes and hypoxia using a variety of different methods to date (8). All of these studies have demonstrated that hypoxia is clearly related to clinical outcome, which motivates the importance of measuring hypoxia in the clinical setting.

Based on the number of studies which relate various measures of hypoxia with clinical outcomes in the breast, it is clear that routine measurement of pre-treatment tumor oxygenation in the breast would be attractive to physicians due to its potential prognostic and predictive value. However, the combined disadvantages of currently available methods have translated to the lack of routine clinical measurements of breast tumor hypoxia. We propose the use of a simple to use, robust, fiber-optic probe which is compatible with biopsy needles commonly used in routine diagnostic biopsy of the breast, to provide an immediate and non-destructive measurement of tumor vascular oxygenation in vivo using a technique called diffuse reflectance spectroscopy. This optical method is label-free, does not require an oxygen standard for calibration, has a signal-to-noise ratio that is independent of oxygenation, does not consume oxygen, and is not susceptible to electromagnetic interference. Diffuse reflectance spectroscopy provides a measurement of the absorption, and scattering, of the tissue from cubic-millimeter (UV-visible wavelengths) to cubic-centimeter volumes (near infrared (NIR) wavelengths). The absorption and scattering, or optical properties, of the tissues are reflective of their physiological and structural composition. The primary absorbers in breast tissue include oxygenated (HbO₂) and deoxygenated (Hb) hemoglobin (absorption in the UV, visible and NIR wavelengths), β-carotene (visible wavelengths), as well as lipids and water (NIR wavelengths).

Our group has developed a novel optical toolbox to perform quantitative physiology in vivo, which has been extensively tested in a laboratory setting (10, 11) and in pre-clinical models (12, 13). This toolbox consists of an optical spectrometer coupled to custom-designed fiber-optic probes for delivery and collection of light from tissue, which are compatible with commonly used biopsy or endoscopy devices. In addition, the toolbox includes a series of stochastic Monte Carlo models, which extract tissue absorption and scattering from diffuse reflectance measurements (11) as well as the contribution of intrinsic tissue fluorophores (14) from the tissue fluorescence spectra. The objective of the current study was to demonstrate the capability and utility of this toolbox to directly measure breast tumor oxygenation in vivo. Ultrasound guidance was used to interface the fiber-optic probe with normal and cancerous tissues in patients undergoing surgical treatment for stage I–IIIA breast cancer prior to surgical resection, ensuring that measurements came from tissues in their vascularized and nascent state. The measurements were made on patients undergoing surgery rather than on those undergoing core needle biopsy to increase the tumor measurement yield. The focus of this paper is to present our quantitative measurements of tumor physiology via diffuse reflectance spectroscopy, and to demonstrate its potential utility as a prognostic/predictive tool.

II. Materials and Methods

Instrumentation

Custom-designed fiber-optic probes were manufactured by Polymicro Technologies, Inc. (Pheonix, AZ) and RoMack, Inc. (Williamsburg, VA) with surgical stainless steel jacketing. Two probe tip designs were used: Probe A had an outer diameter of 3.47mm while Probe B had an outer diameter of 2.1mm. The lengths and diameters of Probe A and Probe B were designed to fit into the lumens of 10G and 14G biopsy cannulas, respectively. Each probe shared a common illumination and collection fiber arrangement, namely, a bundle of 19, 200μm illumination fibers arranged to have an effective illumination area with diameter of 1mm, surrounded by a concentric ring (Probe A: 4, Probe B: 18) of 200μm collection fibers. Probes A and B have been described in our previous publications (15) and (10), respectively. Although Probe A has 16 fewer collection fibers than Probe B, in both probes the collection fibers are close-packed and evenly spaced around the 19-fiber illumination core. Thus, the average fiber-to-fiber spacing between the two probes is not significantly different, ensuring that the sensing depth of each probe was approximately the same (simulated to be 0.6–2mm, dependent on wavelength-dependent optical properties). However, because Probe A had fewer collection fibers, the collected signal was lower than Probe B requiring longer CCD integration times, in general, during data collection. The probes were designed such that, when fully inserted, they extended past the end of the biopsy cannula only far enough to fill the cavity left by the needle used to introduce the cannula. This was to minimize, as much as possible, any artifact due to variations in pressure, although there currently is no mechanism to provide feedback to the user as to how much pressure is being applied. The fiber optic probes were coupled to an optical spectrometer. The optical spectrometer used in this study consisted of a 450W xenon arc lamp, double excitation monochromator (Gemini 180), imaging spectrograph (TRIAX 320) and Peltier-cooled CCD (Symphony), from Jobin-Yvon HORIBA (Edison, NJ). The fiber probes were sterilized with low-temperature ethylene-oxide gas prior to use in the patient studies.

Clinical Study Design

This study was approved by the Institutional Review Board at Duke University in accordance with assurances filed with and approved by the Department of Health and Human Services. Informed consent for study participation was obtained from 35 eligible participants. Patients undergoing either a modified radical mastectomy or partial mastectomy for invasive and non-invasive breast malignancies, with lesions of at least 1 cm diameter were recruited for study participation. Demographic and pathologic data were collected for each patient who offered informed consent.

Optical spectroscopy of the breast in vivo was performed in the operating room prior to resection of the tumor or removal of the breast. Patients in this study underwent local anesthesia via thoracic paravertebral block, which is the standard of care at our institution. The sterilized optical probes were handled by the surgeon (LGW) under the sterile field, while the other end that couples to the instrument was handed to the spectrometer operators for connection to the light source and detector. The operating room lights were turned off during optical spectroscopy, and the theater lights were directed away from the operating field during each measurement to prevent contamination from ambient light. The surgeon located the lesion under ultrasound guidance; then, either a 10G or 14G biopsy needle coaxial cannula (Bard “Vacora,” Tempe, AZ or Cardinal Health “Achieve,” Dublin, OH) was guided through a small incision in the skin into the region of interest. After the cannula was in the region of interest, the needle was removed (leaving the cannula in place), and the cannula was flushed with sterile saline to remove any residual blood in the field. Then, the appropriate fiber-optic probe (10G or 14G diameter) was inserted through the cannula and interfaced with the tissue at a distance of 2 mm past the cannula. A diffuse reflectance measurement (350–600 nm) was collected, the optical probe was retracted, and a biopsy needle (Bard Vacora 10G, Bard Vacora 14G, or Cardinal Achieve 14G) was inserted through the cannula and a biopsy sample removed. This resulted in the removal of a typically 20 mm long cylinder of tissue, the proximal end of which corresponded to the volume optically measured by the probe. Co-registration of the biopsied tissue with the optically-measured area was independently verified in a separate experiment on reduction mammoplasty tissue. Both biopsy devices maintained the orientation of the tissue upon removal of the tissue from the biopsy device. The end of the biopsy core distal to the optical probe was marked with India ink to preserve orientation, and immediately placed in 10% buffered formalin. The above procedure was typically repeated at 1–2 more sites for each patient (2–3 sites total). The entire process took on average 10 minutes of operating time. Immediately after collection of tissue spectra, reflectance calibration measurements were performed by inserting the tip of the probe into an integrating sphere (Labsphere, North Sutton, NH). Tissue reflectance spectra were calibrated for the wavelength-dependent throughput of the system by dividing the tissue spectra point-by-point by the integrating sphere diffuse reflectance spectrum for each patient study.

Tissue processing, pathologic review and tissue classification

Biopsy samples were processed via standard histological procedures. After paraffin embedding, the cores were sectioned along the long axis of the biopsy cores. Tissue sections were obtained longitudinally at three levels (denoted top, middle, and bottom of the core) and stained with hematoxylin and eosin (H&E). The resulting tissue sections were scored by a board-certified pathologist (JG), with special attention being paid to the proximal 3 mm of the core corresponding to the optical measurement, opposite the end marked with India ink. Both the proximal ends and whole cores were given an overall classification as normal, benign, or malignant. The proximal end of the core only was also scored for tissue composition - percent epithelium, stroma, fat, and hemorrhage for normal/benign tissues; and percent benign epithelium, stroma, fat, in situ carcinoma, invasive carcinoma, and hemorrhage for malignant tissues. Table 1 contains a detailed histological breakdown of the tissue samples used in the study.

Table 1

Histological breakdown of tissue samples used for data analysis.

Pathologically-confirmed final tumor size, tumor category, nodal status, and Nottingham combined tumor grade was determined from the surgical pathology report. Menopausal status and chemotherapy status (neo-adjuvant chemotherapy versus no prior chemotherapy) were obtained from the patients’ medical records. Estrogen receptor (ER), progesterone receptor (PR), and epidermal growth factor receptor (EGFR) status were determined via IHC. HER2/neu receptor status of tumor tissues was determined via a combination of IHC and fluorescence in-situ hybridization (FISH). To dichotomize the results as positive and negative for HER2, IHC membrane-expression scores (Dako Herceptest) of 0+ and 1+ were classified as HER2 negative, and scores of 3+ were classified as HER2 positive. For tissues with an IHC score of 2+, the results of HER2 gene amplification by FISH were used to classify these tissues as HER2 negative (HER2 gene not amplified) or HER2 positive (HER2 gene amplified).

Extraction of tissue optical properties

An inverse Monte-Carlo model previously developed by our group was used to extract scattering and absorption properties of the measured tissues. Details about this model are available elsewhere (11, 16). Diffuse reflectance spectra measured from tissue were fit over the wavelength range of 400–600 nm. The free parameters related to absorption were the concentrations of the intrinsic absorbers assumed to be present in this wavelength range, namely, Hb, HbO₂, and β-carotene. Lymphazurin^™ (Tyco Healthcare), a blue dye used to locate the sentinel node during surgery, was also included as an extrinsic absorber since it was present in some of the samples. The fixed absorption parameters were the extinction coefficients of oxygenated and deoxygenated hemoglobin and β-carotene, which were obtained from an online database, whereas the absorption spectrum of Lymphazurin^™ was measured separately in our laboratory. A Gaussian-shaped absorber with a center wavelength of 515nm and a full width half maximum (FWHM) of 10 nm was used as well, to account for deviations in the model fits thought to be due to differences in the absorption of β-carotene in vivo (15). For scattering, the fixed parameters were the refractive indices of the scatterers and the surrounding medium, and the anisotropy factor, whereas the free parameters were the size and density of the spherical scatterers. All fits and subsequent data analysis were performed using MATLAB 7.1 (The Mathworks, Natick, MA).

Statistical analysis

For data analysis, the percent tissue types for each tissue sample were averaged over the three section levels to give the average percent tissue type for each core, assumed to be representative of the entire proximal 3 mm of the biopsy core. For comparison purposes, the tissue samples were grouped into several classes. Tissue samples were re-classified from the proximal tissue type percentages as adipose (>50% fat content, without malignant pathology), benign (normal or benign pathology with <50% fat), or malignant (any in situ or invasive carcinoma present). Tissue samples were further classified as simply non-malignant or malignant based on this reclassification. Non-malignant samples were grouped by menopausal status, while malignant samples were grouped by receptor status, menopausal status, and chemotherapy status for more detailed statistical analysis. Unpaired student t-tests and Wilcoxon rank-sum tests were used (on the means and medians, respectively) to determine whether statistically significant differences between tissue types, subgroups, and diagnoses existed in parameters extracted from the diffuse reflectance spectra. Correlations were evaluated by computing Spearman’s rho (rank correlation). All statistical analyses were performed using the Statistics Toolbox in MATLAB.

III. Results

Representative diffuse reflectance spectra (calibrated for system throughput) are shown in Figure 1 for benign and malignant tissues. As expected, the benign tissue spectrum (Figure 1A) is dominated by oxy-hemoglobin absorption as evidenced by the prominent α and β absorption bands between 550–580nm. Figures 1B and 1C, however, demonstrate the variability in oxygenation status for malignant tissues, with the malignant spectrum in Figure 1C being dominated by deoxy-hemoglobin (as evidenced by the lack of distinct α and β absorption bands and the slight red-shift of the Soret band at around 420nm) in contrast to the more oxygenated malignant tissue of Figure 1B. Extracted sO₂ values, quantified using the inverse model (11, 16), for each of these tissues are given in the figures as well. The malignant sample of Figure 1B was positive for the HER2/neu receptor, whereas the malignant tissue of Figure 1C was negative for HER2. Table 2 gives THb and sO₂ of breast tissue vasculature in vivo, stratified by tissue type. The mean and median values of sO₂ for malignant tissues were significantly lower than benign, adipose, and all non-malignant tissue types. Both the mean and median sO₂ values were approximately 50% of the values for the non-malignant categories. The sO₂ of benign and adipose tissues were not statistically different. Neither the mean nor the median value of THb for malignant tissues was statistically different from those of benign, adipose, or all non-malignant tissues. However, the mean value of THb for malignant tissues was ~4μM higher than those of benign, adipose, and all non-malignant tissues, which only varied by 0.2μM between the three categories. Whereas there appears to be a (non-significant) trend in a higher mean THb in malignant tissues, that same trend was not observed in the median THb values.

Figure 1

Representative calibrated diffuse reflectance spectra and model fits for benign (A), HER2-positive malignant (B), and HER2-negative malignant (C) tissues. Corresponding extracted hemoglobin saturation (sO₂) values are indicated above the figure legends. (more ...)

Table 2

Results of hemoglobin saturation (sO2) and total hemoglobin content (THb) measurements for all tissues, stratified by tissue type. The p-values for the mean (unpaired Students’ t-test) and median (Wilcoxon rank-sum) are given to indicate which (more ...)

Figure 2 contains sO₂ histograms given in relative frequency for (A) malignant, (B) all nonmalignant, (C) benign, and (D) adipose tissues. These histograms are useful in graphically representing the distribution of sO₂ for tissues in both non-malignant and malignant categories. Thirty percent (30%) of malignant tissues exhibit sO₂ values between 0–2.5%, and the majority (60%) of malignant tissues exhibit sO₂ values lower than 75%. For malignant samples with sO₂ values higher than 75%, most (75%) were from Stage I and Stage IIA lesions, while the remaining 25% were equally split between Stage IIB and Stage IIIA lesions. None of the samples from Stage I lesions had sO₂ values lower than 75%. Sixty percent (60%) of Stage II+ cancers had sO₂ values lower than 75%. Although the trend was for decreased sO₂ with increased cancer stage, no significant correlation between sO₂ and cancer stage could be established.

Figure 2

Tissue hemoglobin saturation histograms for: A) malignant, B) non-malignant, C) benign, and D) adipose tissues.

Non-malignant tissues (Figure 2B–D) demonstrate a probability distribution weighted towards higher sO₂ values. Thirty-four percent of non-malignant tissues exhibited saturation values between 95–100% sO₂. The majority (73%) of non-malignant tissue samples exhibited hemoglobin saturations greater than 75%. Benign tissues (Figure 2C) appeared to exhibit less variability in sO₂ than adipose tissues (Figure 2D); although the overall range of sO₂ values was similar between the two categories, unlike benign tissues, adipose tissues were distinct in that a small percentage (6%) exhibit sO₂ values below 40%.

Whereas there was no significant correlation between sO₂ and cancer stage (which incorporates tumor size, axillary lymph node status, and distant metastases), there was a correlation between sO₂ and tumor size alone. Figure 3A shows a plot of tissue hemoglobin saturation versus tumor size for all malignant tissues. sO₂ of tumor tissue was negatively correlated with overall invasive tumor size (rho = −0.50, p < 0.05), as well as pathologic tumor category, pT (rho = −0.49, p < 0.05). The observed relationship between sO₂ and tumor size is consistent with a study in an ectopic rodent model which noted the same relationship (17). sO₂ was not correlated with percent malignancy in the biopsied tissue (rho = 0.12, p = 0.62), nor was it correlated with lymph node status or Nottingham histological grade. Figure 3B presents sO₂ as a function of THb content in malignant tissues only. sO₂ was positively correlated with THb (p < 0.02) in malignant tissues, but not in non-malignant tissues (p = 0.60, not shown).

Figure 3

Tissue hemoglobin saturation as a function of A) tumor size and B) total hemoglobin content, in malignant tissues only. Spearman’s rho and corresponding p value are given in the inset.

Malignant and non-malignant tissues were further divided into subgroups based on menopausal status, chemotherapy status, and tumor receptor status (malignant tissues only). No significant differences were observed in non-malignant tissues based on menopausal status or chemotherapy status. Table 3 contains the results of comparisons of sO₂ and THb between each subgroup in malignant tissues only. In the case of ER and EGFR, statistical testing could not be performed due to the presence of only one ER− and EGFR+ sample, respectively. No statistical differences were observed in either the mean or the median sO₂ and THb in PR+ versus PR− tumor tissues. However, HER2+ tumor tissues exhibited a statistically significant higher mean and median sO₂, and higher mean and median THb, than tissues not over-expressing the HER2/neu receptor.

Table 3

Mean and median hemoglobin saturation and total hemoglobin content for subgroups of malignant tissues only. P-values are given to indicate significant differences in each subgroup (n.s. = not significant, -- = not calculated).

No significant differences between malignant tissues taken from pre-menopausal and post-menopausal women could be determined in either sO₂ or THb, although the trend was for pre-menopausal tissues to have lower sO₂ and higher THb than post-menopausal tissues. Two (2) of the sampled tumor tissues were from a single patient who had neo-adjuvant chemotherapy; although there were not enough neo-adjuvant samples to determine significant differences in sO₂ and THb from tissues not exposed to chemotherapy, the neo-adjuvant samples exhibited markedly lower sO₂ and THb values than the mean and median for tissues from patients not undergoing neo-adjuvant chemotherapy. Longitudinal studies, with pre- and post-chemotherapy measurements, will be needed to determine whether chemotherapy has the general effect of reducing sO₂ and THb, or whether the current results can be attributed to inter-patient variability.

Discussion

Significant differences in tissue hemoglobin saturation exist between malignant and non-malignant tissues in the breast, as has been seen in previous studies of the breast (18), oral cavity (19), and rectum/anus (20). It is well accepted that solid tumors are associated with decreased oxygen tension, due to the increased oxygen demand of metabolically active cancer cells in combination with deficiencies in perfusion caused by a disordered and inefficient microvasculature created during the angiogenic process (21–26). In the important study by Vaupel et al. (26), polarographic oxygen electrodes were used to measure oxygen tension from approximately 50 sites in each of 16 normal tissues, 15 cancer tissues, and 5 fibrocystic tissues, in the breast. In that report, the result of pooling all measurements for each tissue type together demonstrated that breast cancer tissues exhibited a bimodal distribution of oxygen tension, with an overall lower median than normal tissues, whereas normal tissues exhibited a nearly Gaussian distribution of oxygen tension centered at higher values. Although our measurements arise from the vascular compartment, our results are consistent with those in that report, in that there is clearly tumor-to-tumor variability in oxygenation, giving rise to a non-uniform, multimodal tissue saturation distribution for malignant tissues. We observed a weighted distribution of sO₂ values for non-malignant tissues, with the greatest number of tissues exhibiting 95–100% sO₂.

The presence of a significant number of malignant tissues with hemoglobin saturation values greater than 80% prompted a deeper investigation into the characteristics of these tumors, particularly with regard to menopausal and hormone receptor status. Although the malignant samples were evenly balanced between pre- and post-menopausal, no relationship was found between hemoglobin saturation or content, and menopausal status in these samples. No relationship between hemoglobin saturation and content, and ER or EGFR status could be calculated, due to 19 of 20 malignant samples being ER negative and EGFR positive, respectively. Furthermore, no significant differences in hemoglobin saturation or content were observed between PR− and PR+ samples. However, we found that both hemoglobin saturation and total hemoglobin content were positively correlated with the extent of HER2/neu over-expression, and that four of the five hypoxic tumors sampled in this study were negative for the HER2/neu receptor. HER2/neu over-expression is associated with a poor prognosis, and has been implicated in tumors which are particularly aggressive and resistant to systemic therapies and local radiotherapy (27, 28). Breast cancers which overexpress HER2/neu are also associated with an increase in angiogenesis (29), primarily through up-regulation of HIF-1α and resultant VEGF expression, via activation of the PI3K/Akt pathway (28, 30). Our results show that hemoglobin saturation in tumors is positively correlated with the total hemoglobin concentration. This indicates that tumors which contain higher blood volume (and thus likely more vasculature due to angiogenesis), are better perfused and oxygenated than those with less dense vasculature. These findings are consistent with a model of increased angiogenesis and decreased tumor hypoxia promoted by HER2/neu over-expression. Thus, our results seem to indicate that a) HER2/neu over-expression promotes angiogenesis in breast tumors, and b) this results in more highly-perfused and well-oxygenated tumors.

Our results are supported by previous studies which noted the relationship between HER2/neu status, tumor angiogenesis, and tumor hypoxia (29, 31). In one study by Blackwell et al. (29), HER2/neu amplification was correlated with decreased tumor hypoxia and increased angiogenesis, as determined by fluorescence in situ hybridization (FISH) analysis of HER2/neu amplification, and IHC analysis of micro-vessel density (von Willebrand factor), the hypoxia marker CA IX, and vascular endothelial growth factor (VEGF), on previously biopsied tissues. In another study, Hohenberger et al. (31) analyzed pO₂ electrode measurements of breast tissues in vivo with respect to molecular growth determinants, including HER2/neu over-expression. The authors observed that tumors with higher HER2/neu over-expression (as determined by IHC) had higher oxygenation, which is in agreement with our results.

The merits of the pO₂ electrode vs. UV-visible optical spectroscopy are discussed below in the context of the type of tumor micro-environments that these technologies can probe and their methods of implementation A number of studies have investigated breast tumor oxygenation using the pO₂ electrode (26, 31–36). The advantages and disadvantages of pO₂ histography are well outlined in the recent review by Vaupel et al. (37). The primary attributes of the pO₂ electrode for measurement of tissue oxygenation are, it can quantify actual tissue pO₂, and it can measure both perfusion-mediated (acute) as well as chronic hypoxia (at tissues far from the vasculature), since it does not rely on a measurement of vascular oxygenation alone. While the pO₂ electrode can in principle isolate chronic from acute hypoxia, in practice the electrode may be placed in the vasculature, interstitial space, or intracellularly, for any given measurement (37). This uncertainty in electrode placement is usually overcome by taking tens to hundreds of measurements in any given tumor, with the resulting histograms likely reflective of both chronic and acute hypoxia. In contrast, the measurement reported with UV-visible optical spectroscopy is a volume-averaged measurement of the vascular oxygenation (including afferent and efferent vessels), and like other methods which measure vascular oxygenation, it is related to perfusion-mediated, or acute hypoxia in tissues adjacent to the vasculature (38). Theoretical oxygen transport studies have indicated that although vascular O₂ supply is deterministic for tissue pO₂, vascular oxygenation is not always reflective of tissue oxygenation in all tumors due to inter-tumor variations in vascular geometry and arrangement (39–41). However, simultaneous measurement of total hemoglobin content (a surrogate for vascular extent) could aid in interpretation of vascular oxygenation measurements, as it could identify tumors with sparse vasculature (where the difference between vascular and tissue pO₂ is expected to be greatest) from more well-perfused tumors (where vascular and tissue pO₂ are expected to be linearly related) (40). In any case, it is not likely that a chronically-hypoxic tumor would be characterized by well-oxygenated vasculature, meaning that measurement of vascular oxygenation can still identify a poorly-oxygenated tumor, although there may not be a quantitative relationship between average vascular pO₂ and average tissue pO₂. Since vascular oxygenation cannot always be directly translated to clinical definitions of tumor hypoxia (which pertain to tissue pO₂ specifically), this suggests the need for further study to establish empirical relationships between tumor vascular oxygenation and radio- and chemo-resistance, with the aim of identifying “sensitivity thresholds” for vascular oxygenation (as has been done for tissue oxygenation (1)).

With respect to implementation, UV-visible optical spectroscopy offers several benefits over the pO₂ electrode in that it can continuously monitor changes in vascular oxygenation over short periods of time (because the probe does not consume oxygen) and it requires only a few measurements and thus less time to get an overall representation of acute hypoxia in a solid tumor because of its inherently larger sampling volume per placement (several mm³) compared to that of the pO₂ electrode (limited primarily to the surface of the electrode). Although this manuscript specifically discussed vascular oxygenation measurements with light, UV-visible optical spectroscopy is sensitive to additional parameters including tissue blood volume (via measurements of total hemoglobin absorbance), cell density and/or proliferative status (via measurements of scattering (42)), and can also be extended to measure cellular metabolism (via measurements of the fluorescence of cellular NADH and FAD), and can be extended into the near-infrared regime for measurement of blood volume and saturation of superficial tumors from the tissue surface, as demonstrated by Chance (43, 44), Tromberg (45, 46), and others (47, 48).

In conclusion, direct measurement of tumor vascular oxygenation via optical methods could be a viable method for clinical application. Due to its compatibility with commonly used biopsy needles, optical spectroscopy could be used at the time of core-needle biopsy, providing an immediate measurement related to the extent of acute hypoxia in the tumor. This information could be used in conjunction with immunohistological markers for chronic hypoxia, to provide guidance to the oncologist about potential therapy options. In addition, measurement of acute hypoxia at diagnostic biopsy could aid in the interpretation of standard prognostic factors, for example estrogen receptor status, which is a useful biomarker for endocrine therapy, but which has also been shown to be modulated by tumor hypoxia (49, 50). A previous study employed quantitative UV-visible optical spectroscopy in the breast on a smaller number of patients (18). Although the sampling volume of that technique is roughly two orders of magnitude smaller than the one reported here, our results are consistent with those of that report, and expand the available data on vascular oxygenation of the normal and diseased breast. Limitations of this study include 1) the ability to definitively measure the extent of chronic hypoxia in tissues distant from the vasculature, and 2) the lack of available data on the clinical outcomes for the patients enrolled in this study, precluding any interpretation of the prognostic or treatment-predictive value of these measurements. In future studies, we will perform longitudinal measurements in patients undergoing neo-adjuvant systemic therapy, which could lead to the identification of relationships between vascular oxygenation, and response or resistance to therapy.

Acknowledgments

Financial support is acknowledged from the National Institutes of Health RO1 CA100559 and the Duke Comprehensive Cancer Center. JQ Brown acknowledges fellowship support from the NIH/NCI F32 CA1240582.

Financial support for this work was provided by NIH-National Cancer Institute RO1 CA100559, and generous support from the Duke Comprehensive Cancer Center. J.Q. Brown acknowledges fellowship support from the NIH-NCI (F32 CA1240582). Dr. Mark Dewhirst and Dr. Karthik Vishwanath are acknowledged for helpful discussions. The authors would like to thank the staff of the Duke University Ambulatory Surgery Center for their invaluable assistance.

Footnotes

There are no potential conflicts of interest to disclose.

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