Screening of salivary volatiles for putative breast cancer discrimination: an exploratory study involving geographically distant populations

Abstract

Saliva is possibly the easiest biofluid to analyse and, despite its simple composition, contains relevant metabolic information. In this work, we explored the potential of the volatile composition of saliva samples as biosignatures for breast cancer (BC) non-invasive diagnosis. To achieve this, 106 saliva samples of BC patients and controls in two distinct geographic regions in Portugal and India were extracted and analysed using optimised headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME/GC-MS, 2 mL acidified saliva containing 10% NaCl, stirred (800 rpm) for 45 min at 38 °C and using the CAR/PDMS SPME fibre) followed by multivariate statistical analysis (MVSA). Over 120 volatiles from distinct chemical classes, with significant variations among the groups, were identified. MVSA retrieved a limited number of volatiles, viz. 3-methyl-pentanoic acid, 4-methyl-pentanoic acid, phenol and p-tert-butyl-phenol (Portuguese samples) and acetic, propanoic, benzoic acids, 1,2-decanediol, 2-decanone, and decanal (Indian samples), statistically relevant for the discrimination of BC patients in the populations analysed. This work defines an experimental layout, HS-SPME/GC-MS followed by MVSA, suitable to characterise volatile fingerprints for saliva as putative biosignatures for BC non-invasive diagnosis. Here, it was applied to BC samples from geographically distant populations and good disease separation was obtained. Further studies using larger cohorts are therefore very pertinent to challenge and strengthen this proof-of-concept study. Graphical abstract ᅟ.

Volatomic pattern of breast cancer and cancer-free tissues as a powerful strategy to identify potential biomarkers

“…The main VOMs identified for these families were phenol, toluene and acetic acid, respectively. These metabolites were already identified in several reports in the literature in biological matrices such as urine, 10,12 cancer cell lines, 11,18 saliva, 14 and exhaled breath. 18,19 Several studies reported the potential of VOMs from different biological matrices on differentiation between cancer patients and healthy subjects.…”

“…It can be observed that the pathway with the highest impact was the pyruvate and sulfur metabolism due to acetic acid. This metabolite according to the human metabolome database is normally found in most tissues (liver, kidney, among others) and in several biofluids, namely saliva 14 and urine. 10 Moreover, limonene was also included as a significant metabolite belonging to the class of monoterpenoids involved in monooxygenase activity through cytochrome P450 and the mevalonate pathway.…”

“…The most common procedure used in the extraction of volatile compounds in biological samples is solid-phase microextraction (SPME), normally in the headspace mode (HS-SPME) being used in several biological matrices. [10][11][12][13] GC-qMS was used to screen salivary volatiles for putative BC as an exploratory study involving geographically distant populations, 14 also to establish the metabolomic signature of human BC cell lines 11 and to discriminate different types of cancer based on urinary volatomic biosignatures. 13 In the first study, up to 120 VOMs from distinct chemical families, with significant variations among the groups, were identified, 14 whereas Silva et al 11 and Porto-Figueira et al 13 identified 60 and 130 VOMs in BC cell lines and urine, respectively.…”

“…[10][11][12][13] GC-qMS was used to screen salivary volatiles for putative BC as an exploratory study involving geographically distant populations, 14 also to establish the metabolomic signature of human BC cell lines 11 and to discriminate different types of cancer based on urinary volatomic biosignatures. 13 In the first study, up to 120 VOMs from distinct chemical families, with significant variations among the groups, were identified, 14 whereas Silva et al 11 and Porto-Figueira et al 13 identified 60 and 130 VOMs in BC cell lines and urine, respectively. On the other hand, Budczies et al 15,16 used a gas chromatography time of flight mass spectrometry (GC-TOFMS) framework to evaluate the glutamate enrichment as a new diagnostic opportunity in BC and to accomplish comparative metabolomics of estrogen receptor positive (ER + ) and estrogen receptor negative (ER − ) samples.…”

Unravelling the Potential of Salivary Volatile Metabolites in Oral Diseases. A Review

“…Cancer development and progression causes metabolic shifts that can be detected in different biofluids, including in saliva [52]. In this regard, volatomic fingerprints able to discriminate BC from healthy individuals have been already proposed [95,96]. Taware et al applied the same principle to oral cancer and analysed the salivary volatile composition of a small cohort of 32 oral cancer patients and 27 controls.…”