2.1.

Patient Population and Measurement Procedure

An ex vivo study to evaluate partial mastectomy specimens with diffuse optical spectroscopy in patients undergoing surgery for breast malignancies was approved by the Institutional Review Board at Duke University as detailed in previous publications.^3–5 The following patient characteristics were collected (if available): mammographic breast density (MBD), menopausal status, neo-adjuvant treatment status (chemotherapy or endocrine therapy), age, BMI, and surgical re-excision status. Based on preoperative mammograms, each patient was assigned an MBD based on a four-point scale indicative of increasing amounts of fibroglanduar tissue: 1 (fatty), 2 (scattered fibroglandular), 3 (heterogeneously dense), and 4 (extremely dense). Women were designated as postmenopausal if they had one of the following: either a bilateral salpingo-oopherectomy or lack of a menstrual cycle for more than 1 year.

The surgical procedures, optical measurements, and the histological processing of the tissue are described elsewhere.⁴ A total of 38 malignant sites and 595 benign sites from the previous study⁴ were examined for this analysis. The corresponding hematoxylin & eosin (H&E) slides for each of these sites were requested from the Duke surgical pathology archives. Each H&E slide was imaged, quantified and classified to determine the proportion of glands, collagen, and adipose tissues as well as the size and density of adipocytes in predominantly fatty tissues. This process is shown in Fig. 1.

Fig. 1

Flowchart demonstrating the study design. Diffuse reflectance spectra were measured and processed using the Monte Carlo model of reflectance to extract tissue optical properties. The corresponding site was inked and then submitted for pathology. The corresponding $5\mu \mathrm{m}$ H&E sections from the optically interrogated sites were imaged and detailed morphological composition was quantified (A, adipose; C, collagen; G, glands).

The 595 benign sites were categorized by the study pathologist (JG) as follows: A, Mx, V, FA, FG, F, FCC, and FN. The 38 malignant sites were similarly categorized as follows: IDC, DCIS, ILC, and LCIS. In order to focus on the underlying sources of scattering from adipocytes, collagen, and glands, we excluded sites that had significant additional components [i.e., V ($n=64$)] as well as sites that lacked a large enough sample size to draw conclusions regarding trends [i.e., FCC ($n=4$), FN ($n=1$), ILC ($n=1$), and LCIS ($n=3$)]. When specifically examining the adipose sites, the only features quantified from these sites were adipocyte size and density as shown on the left-hand side of Fig. 1. The black circles within the adipose H&E image are illustrative of the adipocyte features. The remaining benign sites were comprised of two to three of the following components: adipose, collagen, and glands and the fraction of each of these components were quantified as shown on the right side of Fig. 1. Although some of the sites contained vessels or fluid filled cysts; these were accounted for but not included in this analysis. In regions with inflammatory cells, the box was classified according to the predominant component. Inflammatory cells have a range of diameters: 10 to $12\mu \mathrm{m}$ in neutrophils, eosinophils, and basophils; 6 to $15\mu \mathrm{m}$ in lymphocytes; and $\sim 18\mu \mathrm{m}$ in monocytes.¹² These inflammatory components mimic densely packed epithelial cells; thus, when inflammatory cells comprised the majority of the $200\mu \mathrm{m}\times 200\mu \mathrm{m}$ area, the tissue section was designated as glandular or nucleated, but this was a rare event. When most of the section contained collagen with a few inflammatory cells, the tissue section was designated as collagen. A total of 526 benign sites (A, Mx, FA, FG, and F) and 34 malignant sites (IDC and DCIS) were included in the imaging protocol. Figure 2 shows the included and excluded sites for this study; the exclusion and inclusion criteria for the imaging analysis are further detailed in Sec. 2.2.1 for adipose sites and Sec. 2.2.2 for nonadipose sites.

Fig. 2

REMARK diagram illustrating the excluded and retained sites for study investigation.

2.2.

Imaging and Quantification of Detailed Morphological Composition of Breast Tissue Histological Sections

The criteria for inclusion of the sites in this analysis were as follows: (1) the measurement ink could be clearly identified, (2) the imaged area corresponded with the pathologist’s diagnosis, and (3) the tissue was intact throughout the area of interest.

3.1.

Patient Characteristics

The patient characteristics in terms of age, BMI, BD, menopausal status, and chemotherapy status are shown in Table 1. The average age and BMI of the population were 58.5 years and 30.2, respectively. The investigated population represented all four breast density categories with a higher prevalence of MBD-2 and MBD-3. The population studied was skewed toward postmenopausal and chemo-naïve patients.

Table 1

Patient characteristics from the investigated population.

3.2.

Representative Data and Summary Variables

Representative wavelength-dependent ${\mu }_{\mathrm{a}}$ and ${\mu }_{\mathrm{s}}^{\prime }$ spectra and histological images are shown in Fig. 3. This figure demonstrates that adipose tissue contributed to increased absorption [Fig. 3(a)] and fibroglandular tissue contributed to increased scattering [Fig. 3(b)]. Additionally, both collagen and glands played a role in determining tissue scattering, while adipose sites exhibited a baseline scattering signature. The corresponding H&E stained histology images for the fibroglandular (FG), fibroadipose (FA), and adipose (A) sites are shown in Figs. 3(c)–3(e).

Fig. 3

(a) The corresponding mean absorption coefficient ($\langle {\mu }_{\mathrm{a}}\rangle$) and (b) the mean reduced scattering coefficient ($\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$) for fibroglandular (FG), fibroadipose (FA), and adipose (A). (C–E) The corresponding histology images for the fibroglandular (FG), fibroadipose (FA), and adipose (A). This figure demonstrates that adipose tissue contributes to increased absorption and fibrogalndular tissue contributes to increased scattering. The scale bars represent $500\mu \mathrm{m}$. The adipose H&E was imaged at $100\times$ while the FG and FA images were acquired at $25\times$.

Table 2 shows the mean and standard deviation for the optical and histological variables as a function of tissue type. A Kruskal–Wallis test (one-way ANOVA on ranks) demonstrated there is a statistically significant difference in the distribution of $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ among the five tissue types shown in Table 2 ($\mathrm{chi}\text{-}\text{squared}=44.612$, $\mathrm{df}=4$, $p\text{-}\text{value}=4.787\times {10}^{-9}$).

Table 2

Sample sizes, mean values, and standard deviations for the optical and histological variables by tissue type. The mean and range of values demonstrate that FA has the highest % adipose, FG has the highest quantified collagen and μs′, and malignant (IDC and DCIS) sites have the highest quantified glandular parameters.

The inter-rater agreement was $\kappa =0.69$ indicating a high degree of agreement across raters. The 394 sites imaged were taken from 93 patients. Of the 394 total sites, 370 were benign and 24 were malignant. The latter included 15 foci of IDC and 9 foci of DCIS. The benign sites did not include specific lesions such as FCC, fibroadenomas or papillomas. The highest scattering was seen in the FG sites followed by Mal, Mix, FA, and A sites. [$\beta$-carotene] was highest in Mal sites followed by A, FA, Mix, and FG sites. Although [$\beta$-carotene] was the highest in Mal, the values were not statistically significantly different than those observed in the adipose sites ($p=0.26$). The percent adipose tissue decreased in the following order: A, FA, Mix, Mal, and FG, respectively. The percent collagen decreased as follows: FG, Mal (lower than FG due to inclusion of DCIS and IDC), Mix, FA, and A. The percent glandular tissue was highest in Mal followed by FG and Mix; FA sites had minimal glandular tissue if any.

3.3.

Patient Characteristics in Sites with Low and High Percentages of Collagen

Table 3 shows the breakdown according to sites with either a low or high percentage of collagen using the median value (44%) of all sites as the cutoff. There was an even distribution of number of sites and patients with high and low percentage collagen. Sites were evenly distributed between MBD-2 and MBD-3. Sites from MBD-4 exhibited a higher fraction of collagen while sites from MBD-1 exhibited lower fractions of collagen, as expected. The sites with low percentage of collagen were from older patients ($p<0.00005$). Sites with a low percentage of collagen were also weighted toward postmenopausal patients consistent with increased age. Sites with a higher fraction of collagen were evenly split between pre- and postmenopausal patients; FG sites only fell into the higher percent collagen category but FA, Mix, and Mal all exhibited high and low collagen morphologies. The cancer stage and invasive grade populated both high and low collagen categories and was not included (data not shown).

Table 3

Summary table for the nonadipose H&E images and corresponding spectra. The data are separated to show the number of data points above and below the median % collagen. The summary shows the number of sites, MBD, age, menopausal status, and tissue type. Sites with a high percentage of collagen were mostly from MBD of 2 or higher. FG sites had at least 56% or higher collagen.

3.4.

Scattering versus Collagen in Benign Sites

The relationship between $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ and percent collagen was examined; a positive correlation in benign sites is shown in Fig. 4. $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ exhibited greater variability in sites with a higher percentage of collagen as shown in Fig. 4(a). Using the adipose sites, scattering was shown to have no significant correlation to adipose tissue morphology as shown in Figs. 4(b) and 4(c).

Fig. 4

The relationship between scattering and the % collagen in benign sites. $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ is expected to be related to collagen and glands (nuclei and subnuclear components). When considering benign sites (FA, FG, and Mix), (a) $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ shows a positive correlation to the % collagen. The sites with collagen greater than 44% exhibit greater variability in $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ that cannot be explained by the % adipose tissue. $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ shows no correlation to (b) the log of the average adipocyte cell density or (c) the average adipocyte cell area.

3.5.

Effect of Patient Characteristics and Percentage Collagen on Scattering

In order to investigate the relationship between the patient characteristics and morphology with respect to $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$, the importance of each variable was analyzed after adjusting for percent collagen. The 213 adipose images were not included in this analysis. The correlations were examined by high and low percent collagen to determine whether the relationships changed with collagen level for benign sites (FA, FG, and Mix) and all sites (FA, FG, Mix, and Mal). The results of the correlations, linear fits, and interactions are shown in Table 4. $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ was negatively correlated to age in benign ($r=-0.32$, $p=4.7\times {10}^{-5}$) and all ($r=-0.32$, $p=1.4\times {10}^{-5}$) sites and this correlation varied significantly by the collagen level ($r=-0.40$ in high % collagen versus $r=-0.13$ in low % collagen sites, $p=0.01$). $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ was negatively correlated to BMI in benign ($r=-0.32$, $p=4\times {10}^{-5}$) and all sites ($r=-0.31$, $p=2.8\times {10}^{-5}$) but this relationship did not vary with collagen level. Age was found to vary with percentage collagen, resulting in a stronger negative correlation to $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ in sites with a high percentage of collagen. Age was positively correlated to BMI ($r=0.36$) but BMI remained significant in describing $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ after correcting for age. BMI did not have a dependence upon percentage collagen yet remained significant after adjusting for age by percent collagen.

Table 4

Correlations of Age, BMI, and MBD to high and low % collagen. Each characteristic was fit as an independent variable with 〈μs′〉 as the dependent variable using Pearson’s correlations and linear fits to establish relationships. Age and BMI were found to be independently associated with 〈μs′〉. 〈μs′〉 decreased with both age and BMI but exhibited a stronger negative relationship to age in sites with a high percentage of collagen. MBD was positively correlated with 〈μs′〉 but not independent of age and BMI.

3.6.

Effects of Patient Characteristics and Adipocyte Morphology on Scattering

To confirm that adipocytes did not contribute to the positive correlation between collagen and $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$, the relationship between $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ with adipocyte size and log (adipocyte density) as well as age and BMI, stratified by MBD was examined and is shown in Table 5 for the 213 adipose samples. This variability was not dependent upon the size and density of the adipocytes, nor age and BMI ($p>0.05$). Adipose tissues, however, did contribute to a baseline scattering value. After adjusting the morphology components by breast density, a stronger relationship between average cell area (ACA) and $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ was seen in sites from high breast density patients ($r=-0.29$, $p=0.026$), but the relationship to cell density remained nonsignificant. Analyzing the relationships between scattering and characteristics associated with MBD shows that $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ is not correlated to patient characteristics or adipocyte morphology in adipose sites ($n=213$, $p>0.05$).

Table 5

Analyzing the relationships between scattering and characteristics associated with MBD shows 〈μs′〉 is not correlated to patient characteristics or adipocyte morphology in adipose sites; n=213, all p>0.05. ACA is significantly larger in sites from high breast density patients (r=−0.29) versus low breast density patients (r=0.02). Ø indicates no correlation; NS, not significant.

3.7.

Scattering, Collagen Density and Menopausal Status

To determine how these scattering differences might affect optical contrast in patients of different breast compositions, scattering was compared across sites with low and high percentages of collagen as well as pre- and postmenopausal status as a binary surrogate for age. Menopausal status was used because it was highly positively correlated with age ($r=0.75$). Scattering was found to be significantly lower in benign sites with low percentage of collagen in postmenopausal women compared to benign sites with a high percentage of collagen in premenopausal women (Fig. 5, unadj. $p=5.3\times {10}^{-6}$) as would be expected. However, unexpectedly, benign sites with a high percentage of collagen in postmenopausal women were not statistically different from those with low percentage collagen in either pre- or postmenopausal women. These sites from postmenopausal women with high percentage of collagen were statistically lower than that of sites from premenopausal women with a high percentage of collagen (Fig. 5, unadj. $p=1.4\times {10}^{-4}$).

Fig. 5

Box plots demonstrating menopausal status analysis with respect to high and low % collagen. Sites from postmenopausal patients with low and high % collagens exhibit the lowest scattering values. High % collagen sites from premenopausal patients exhibit the highest scattering values. The difference in $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ values for malignant and benign sites is greater in postmenopausal patients.

3.8.

Relative Contributions of Collagen versus Glands to Scattering

Lastly, the relationship between percent collagen and percent glands in benign, malignant, and all sites was analyzed to determine the relative contributions of stroma versus epithelium to $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$. The relationships among $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$, the percent collagen, and percent glands are shown in Fig. 6 in order to determine the contribution of collagen and glands to $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$, respectively.

Fig. 6

Statistical modeling results show that both % collagen and % glands independently relate to scattering in a positive direction with no relationship to one another. The relationship between scattering and % collagen (a) or % glands (b) in malignant sites demonstrates a slightly stronger dependence upon glands.

The linear models (Table 6) identified that both percent collagen and percent glands were independently associated with $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ in all sites and sites with glands (FG, Mix, and Mal). The analysis of collagen versus glands was narrowed to investigate sites with glands between 0% and 35% (the dynamic range of the data). The linear model reflected an equivalent relationship to scattering from glands and the collagen in benign sites ($r=0.18$ versus $r=0.17$). However, as shown in Table 6, the malignant sites had a slightly stronger positive relationship of percent glands ($r=0.64$, $p=0.005$) to $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ compared to the collagen content and $\langle {\mu }_{\mathrm{s}}^{\prime }\rangle$ ($r=0.52$, $p=0.03$).

Table 6

Examining the dynamic range of the data includes restricting sites to % glands <40%. Here, the % glands and % collagen were found to independently contribute to 〈μs′〉 in both benign and malignant sites. 〈μs′〉 increased with both collagen and glands; 〈μs′〉 exhibited a stronger positive relationship to glands in malignant sites.