Comparison of serum metabolomics in women with breast Cancer Prior to Chemotherapy and at 1 year: cardiometabolic implications
Authors: Lyon Debra E. , Yao Yingwei , Garrett Timothy , Kelly Debra Lynch , Cousin Lakeshia , Archer Kellie J. 
Published: May 2023

Abstract
Objective
Early-stage breast cancer (BC) is the second most common malignancy in women, worldwide. Early-detection and treatment advances have led to 5-year survival rates of 90% for early-stage breast cancer. However, the long-term morbidity of breast cancer remains high, with a majority of survivors facing increased risk of cardiometabolic conditions as well as secondary cancers. In particular, African American women with breast cancer experience higher morbidity and mortality than other women. Metabolomics is the comprehensive study of metabolites in biological samples to elucidate the role of monosaccharides, amino acids, and their respective metabolic pathways. Although some studies have found differential metabolites in women with breast cancer compared to normal controls, there has been little study of women with breast cancer across time and the active treatment trajectory. This study examines and compares the serum metabolomic profile of women with BC, prior to initial chemotherapy and at 1 year after inception of chemotherapy.

Methods
This study examined serum metabolites through a secondary analysis of a longitudinal parent study (EPIGEN) of women diagnosed with early-stage BC. Participants were evaluated across 5 time points: prior to their receipt of chemotherapy (T1), at the time of their 4th chemotherapy treatment (T2), 6 months after the initiation of chemotherapy (T3), one year after the initiation of chemotherapy (T4) and two years after the initiation of chemotherapy (T5). This analysis focused on the metabolomic data from 70 participants from T1 to T4. Using ultra high-pressure liquid chromatography high resolution mass spectrometry (UHPLC-HRMS), we performed Friedman Rank Sum Test followed by Nemenyi post-hoc pairwise tests to identify which metabolite levels differed between time points, focusing on metabolites with a Benjamini-Hochberg false discovery rate (FDR) from the overall Friedman test < 0.05 and then specifically examined the p-values from the T1 vs. T4 pairwise comparison.

Results
The untargeted serum metabolomics yielded a total of 2,395 metabolites identified on the basis of the accurate mass and MS/MS fragmentation, 1,264 of which were significant after Friedman’s test (FDR < 0.05). The analysis then focused on the levels of 124 metabolites from the T1 vs. T4 post-hoc comparison that had a combined FDR < 0.05 and fold change (FC) > 2.0. Metabolite set enrichment analysis (MSEA) as part of Metaboanalyst 3.0 was performed to identify pathways that were significantly altered. The known metabolites identified from the functional analysis were used to evaluate the up and down regulated pathways. The 40metabolites from the Functional Analysis were mainly attributed to amino acids (specifically lysine regulation), fatty acids (particularly unsaturated) and steroid hormone synthesis (lysophosphatidic acid).

Conclusion
There were multiple significant changes in the serum metabolomic profile of women with breast cancer at one-year post inception of chemotherapy compared to pre-chemotherapy, most notably associated with lysine degradation, branched-chain amino acid synthesis, linoleic acid metabolism, tyrosine metabolism and biosynthesis of unsaturated fatty acids as the top 5 metabolic pathways. Some of these changes could be associated with metabolic perturbations that are consistent with heightened risk of cardiometabolic morbidity. Our results provide new insights into the mechanisms underlying potential heightened cardiovascular health risks in this population.