Macroscopic optical physiological parameters correlate with microscopic proliferation and vessel area breast cancer signatures

Abstract

Introduction: Non-invasive diffuse optical tomography (DOT) and diffuse correlation spectroscopy (DCS) can detect and characterize breast cancer and predict tumor responses to neoadjuvant chemotherapy, even in patients with radiographically dense breasts. However, the relationship between measured optical parameters and pathological biomarker information needs to be further studied to connect information from optics to traditional clinical cancer biology. Thus we investigate how optically measured physiological parameters in malignant tumors such as oxy-, deoxy-hemoglobin concentration, tissue blood oxygenation, and metabolic rate of oxygen correlate with microscopic histopathological biomarkers from the same malignant tumors, e.g., Ki67 proliferation markers, CD34 stained vasculature markers and nuclear morphology.

Methods: In this pilot study, we investigate correlations of macroscopic physiological parameters of malignant tumors measured by diffuse optical technologies with microscopic histopathological biomarkers of the same tumors, i.e., the Ki67 proliferation marker, the CD34 stained vascular properties marker, and nuclear morphology.

Results: The tumor-to-normal relative ratio of Ki67-positive nuclei is positively correlated with DOT-measured relative tissue blood oxygen saturation (R = 0.89, p-value: 0.001), and lower tumor-to-normal deoxy-hemoglobin concentration is associated with higher expression level of Ki67 nuclei (p-value: 0.01). In a subset of the Ki67-negative group (defined by the 15 % threshold), an inverse correlation between Ki67 expression level and mammary metabolic rate of oxygen was observed (R = -0.95, p-value: 0.014). Further, CD34 stained mean-vessel-area in tumor is positively correlated with tumor-to-normal total-hemoglobin and oxy-hemoglobin concentration. Finally, we find that cell nuclei tend to have more elongated shapes in less oxygenated DOT-measured environments.

Conclusions: Collectively, the pilot data are consistent with the notion that increased blood is supplied to breast cancers, and it also suggests that less conversion of oxy- to deoxy-hemoglobin occurs in more proliferative cancers. Overall, the observations corroborate expectations that macroscopic measurements of breast cancer physiology using DOT and DCS can reveal microscopic pathological properties of breast cancer and hold potential to complement pathological biomarker information.

Fig. 1

Instrumental setup for the diffuse optical tomography (DOT) system, and DOT images acquired from a 53-year-old woman with a 2.2-cm (longest dimension) invasive ductal carcinoma. Bottom left depicts a three-dimensional tumor region (red). The images of rHb, rHbO ₂ and rStO ₂ are for relative (i.e., tumor-to-normal ratio) deoxyhemoglobin and oxyhemoglobin concentration and tissue oxygenation, respectively. Black solid line in the images indicates the region identified as tumor. FD frequency domain, CCD charge-coupled-device camera

Fig. 2

a Ki67-expresseing nuclei were stained by MIB-1 and appear as brown spots. Abnormal extreme dark spots caused errors in automated analyses and were excluded manually (see blue loops). b Dark blue regions designate cells in which the nuclei did not express Ki67, and yellow and orange regions designate cells with Ki67-expressing nuclei (note, orange signifies more intense expression). c CD34-stained vessels appear brown. d Detected vessels were highlighted in neon green using the Aperio algorithm. e The raw image was segmented into cancer (pink) versus non-cancerous (green) areas as shown in f. g Individual cells were detected as neon green loops. Based on nuclear shape, the compactness of the nuclei was calculated using inForm 1.0.2 (Perkin Elmer)

Fig. 3

Correlation between a relative tissue oxygen saturation (rStO₂) and rKi67 and b relative oxyhemoglobin concentration (rHbO₂) and rKi67 (n = 9). Dotted lines indicate 95 % confidence interval for the mean of the linear fit. These pilot results suggest that more oxygen is present in the more proliferative cancer tissues

Fig. 4

a Relative deoxyhemoglobin concentration (rHb) versus Ki67 expression in breast cancer tissues. Lower rHb is observed in Ki67-positive cancer compared to Ki67-negative cancer (p-value 0.01, n = 3 for Ki67-positive and n = 15 for Ki67-negative cancer). Squares and circles in the box plots show the values for each individual subject. On each box, the central bar is the median, the edges of the box mark the 25th and 75th percentiles and the whiskers extend to the most extreme data points not considered outliers. b Correlation between mammary metabolic rate of oxygen (rMMR₂) and Ki67 in cancer for a subset of the Ki67-negative group (n = 5), for whom diffuse correlation spectroscopy flow measurements were available. Dotted lines indicate the 95 % confidence interval of the mean of the linear fit

Fig. 5

Correlation between a relative total hemoglobin concentration (rTHC) and b relative oxyhemoglobin concentration (rHbO₂) versus mean vessel area (MVA, μm²) (n = 19). Dotted lines indicate the 95 % confidence interval of the mean of the linear fit. These pilot results suggest that diffuse optical tomography is measuring an increased blood supply in the larger-diameter blood vessels of these cancer tissues