Tracking the Progression of Triple Negative Mammary Tumors over Time by Chemometric Analysis of Urinary Volatile Organic Compounds

Abstract

Previous studies have shown that volatile organic compounds (VOCs) are potential biomarkers of breast cancer. An unanswered question is how urinary VOCs change over time as tumors progress. To explore this, BALB/c mice were injected with 4T1.2 triple negative murine tumor cells in the tibia. This typically causes tumor progression and osteolysis in 1–2 weeks. Samples were collected prior to tumor injection and from days 2–19. Samples were analyzed by headspace solid phase microextraction coupled to gas chromatography–mass spectrometry. Univariate analysis identified VOCs that were biomarkers for breast cancer; some of these varied significantly over time and others did not. Principal component analysis was used to distinguish Cancer (all Weeks) from Control and Cancer Week 1 from Cancer Week 3 with over 90% accuracy. Forward feature selection and linear discriminant analysis identified a unique panel that could identify tumor presence with 94% accuracy and distinguish progression (Cancer Week 1 from Cancer Week 3) with 97% accuracy. Principal component regression analysis also demonstrated that a VOC panel could predict number of days since tumor injection (R2 = 0.71 and adjusted R2 = 0.63). VOC biomarkers identified by these analyses were associated with metabolic pathways relevant to breast cancer.

Chemometric Analysis of Urinary Volatile Organic Compounds to Monitor the Efficacy of Pitavastatin Treatments on Mammary Tumor Progression over Time

“…Interestingly, many of the VOCs have been previously reported as biomarkers for breast cancer. For example, 2-HEX and many other ketone bodies have been reported by Silva et al [30] and in our previous studies [17,23] to be potential biomarkers of breast cancer, as they are biological products of lipid peroxidation. Ketones have also been implicated as potential biomarkers for other diseases, including hypoglycemia and different cancer types [4,[31][32][33].…”

“…Results of this study identified a unique panel of VOCs that discriminated between healthy and tumor-bearing mice with 94% accuracy and differentiated the end points (cancer week 1 and cancer week 3) with 97% accuracy. In the same study, we also reported a principal component regression model that predicts the number of days after tumor injection with R 2 equal to 0.71, indicating the capability of VOCs to noninvasively monitor breast cancer progression in real time as early as one week after induced tumor injection [23]. Another study based on the same cohort of mice investigated the use of pitavastatin as a therapeutic agent.…”

“…VOCs in the pita high class are expressed similarly to the healthy controls, whereas the VOCs in the pita low samples are more similar to cancer week 1-3 samples. In a previous paper, we identified VOCs of breast cancer, some of which did change over time (modeling cancer progression) and some of which did not [23]. In this case, statistical methods were not trained to identify differences due to tumor progression, and therefore, were more in favor of those VOCs remaining consistently dysregulated.…”

“…However, urine samples were not analyzed in mice administered pitavastatin at low doses, and the previous studies did not profile VOCs over the course of time. Herein, we extend these previous studies using the same cohorts of mice [23,29] to analyze the ability of urinary VOCs to determine the efficacy of pitavastatin (pita) at high and low doses over the course of three weeks using two approaches (approach 1 and 2).…”

Application of Mass Spectrometry in Pancreatic Cancer Translational Research

“…Currently, the GC-MS is the benchmark for comprehensive VOC analysis. Many studies have reported the benefits of GC-MS in the search for tumor patient VOC biomarkers ( 10 , 11 ). In addition to VOCs, GC-MS is also widely used to detect metabolites in cancer patients’ plasma and urine samples.…”