Detection of Volatile Organic Compounds (VOCs) in Urine via Gas Chromatography-Mass Spectrometry QTOF to Differentiate Between Localized and Metastatic Models of Breast Cancer

Abstract

Breast cancer is the most common cancer detected in women and current screening methods for the disease are not sensitive. Volatile organic compounds (VOCs) include endogenous metabolites that provide information about health and disease which might be useful to develop a better screening method for breast cancer. The goal of this study was to classify mice with and without tumors and compare tumors localized to the mammary pad and tumor cells injected into the iliac artery by differences in VOCs in urine. After 4T1.2 tumor cells were injected into BALB/c mice either in the mammary pad or into the iliac artery, urine was collected, VOCs from urine headspace were concentrated by solid phase microextraction and results were analyzed by gas chromatography-mass spectrometry quadrupole time-of-flight. Multivariate and univariate statistical analyses were employed to find potential biomarkers for breast cancer and metastatic breast cancer in mice models. A set of six VOCs classified mice with and without tumors with an area under the receiver operator characteristic (ROC AUC) of 0.98 (95% confidence interval [0.85, 1.00]) via five-fold cross validation. Classification of mice with tumors in the mammary pad and iliac artery was executed utilizing a different set of six VOCs, with a ROC AUC of 0.96 (95% confidence interval [0.75, 1.00]).

Figure 1

(a) Volcano plot where statistical significance via the Student’s T-test is plotted against log 2-Fold Change between classes for metabolites present in at least half of one class, distinguishing between mouse urine with and without cancer (5-OCT = 5-Octen-1-ol, BERG = Bergamotene, BNAT = Benzeneacetaldehyde, BNZA = Benzaldehyde, THIO = Thiophene, 2-pentyl, FRNS = Farnesene), (b) Volcano plot in a similar fashion produced to distinguish between mouse urine with localized and metastasized breast cancer (MENA = Menadione, TRIM = 2,6,6-Trimethyl-2-cyclohexene-1,4-dione, 6-DMH = 6,6-Dimethylhepta-2,4-diene, BNZE = Benzene, 4-ethenyl-1,2-dimethyl-, DLIM = D-Limonene).

Figure 2

Hierarchical heatmap of the 43 VOCs (p-value < 0.1) different between mouse urine samples with and without breast cancer. Full compound names which are associated with the illustrated abbreviation can be observed in Table 1.

Figure 3

Hierarchical heatmap of the 30 VOCs that are (p-value < 0.1) different between mouse urine samples with localized breast cancer and metastasized breast cancer. Full compound names which are associated with the illustrated abbreviation can be observed in Table 2.

Figure 4

PCA utilizing (a) 43 VOCs to discriminate between mouse urine with and without breast cancer, (b) 30 VOCs to discriminate between mouse urine that was collected from mice that had cancer injected in the mammary pad (localized) and in the iliac artery (metastasized), (c) 20 VOCs to discriminate between mouse urine that was collected from all three classes (localized, metastasized and no cancer).

Figure 5

(a) LDA utilizing six VOCs to discriminate between mouse urine with and without breast cancer with 100% sensitivity and specificity, (b) LDA utilizing six different VOCs to discriminate between mouse urine that was collected from mice that had cancer injected in the mammary pad (localized) and mice that had cancer cells injected in the iliac artery (metastasized) with 100% sensitivity and specificity and (c) LDA using nine VOCs to perfectly discriminate between mouse urine that was collected from all three classes (localized, metastasized and no cancer).