Investigation of potential breath biomarkers for the early diagnosis of breast cancer using gas chromatography–mass spectrometry

Highlights

• We identified and assessed exhaled straight aldehydes as potential BC biomarkers.

• Exhaled hexanal, heptanal, octanal, and nonanal levels were higher in BC.

• Breath biomarkers for BC early diagnosis were successfully verified.

• These aldehydes might be BC biomarkers and their joint use had superior result.

Abstract

Background

Breast cancer (BC) remains the most commonly diagnosed malignancy in women. We investigated 4 straight aldehydes in the exhaled breath as potential early BC diagnostic biomarkers.

Methods

End-tailed breath were collected by Bio-VOC® sampler and assayed by gas chromatography–mass spectrometry. Kruskal–Wallis one-way analysis of variance test and binary logistic regression were used for data analysis. The diagnostic accuracies were evaluated by receiver operating characteristic curves. A predictive model/equation was generated using the 4 biomarkers and validated by leave-one-out cross-validation.

Results

All four potential biomarkers demonstrated significant differences in concentrations between BC and healthy controls (HC) (p < 0.05). The areas under the curves (AUCs) in HC vs BC_I–II model using hexanal, heptanal, octanal, and nonanal were 0.816, 0.809, 0.731, and 0.830, respectively. The AUC for their combined use was 0.934 (sensitivity 91.7%, specificity 95.8%) in the early diagnosis of BC. The predictive model/equation exhibited good sensitivity (72.7%) and specificity (91.7%) in distinguishing between HC and BC (cross-validation: sensitivity 68.2% and specificity 91.7%).

Conclusions

The diagnostic values of 4 exhaled straight aldehydes as early diagnostic biomarkers for BC were successfully verified and the diagnostic accuracy improved in their combined use.

Introduction

Breast cancer (BC) remains the most commonly diagnosed malignancy and the second leading cause of cancer-related deaths in women [1]. Early diagnosis is very important for reducing the BC mortality rate. However, the early diagnosis of BC is limited because the disease usually develops asymptomatically, while the conventional diagnostic techniques still have some shortcomings [2]. Therefore, there is still an urgent clinical need to improve or alternate these methods. The combination of molecular screening methods with these conventional methods may help improve this situation [2]. Cancer cells release molecular biomarkers into the blood during their growth, which can provide prognostic symbols that could be useful in early cancer diagnosis through breath, blood, and urine analyses [3], [4], [5], [6]. Breath analysis is noninvasive, easy to perform, painless, and present no risks, thus it may allow the development of convenient and safe method that could complement blood and urine tests [7].

Human exhaled breath (EB) mainly comprises N_2, CO_2, O_2, water vapor, inert gases, volatile organic compounds (VOCs) and nonvolatile substances [8], [9]. These VOCs are either produced endogenously via metabolism or absorbed exogenously from the environment. Some additional compounds are produced and/or their concentrations are changed in pathological states of the body [10]. Endogenous biomarkers are commonly used for diagnostic purposes.

There is a considerable experimental evidence that the volatile components in EB can be used to detect neoplasms [9], [11], [12]. The theoretical basis for diagnosing BC based on breath analysis is speculative as follows. BC is accompanied by increased oxidative stress (OS), which can lead to DNA, proteins and lipid damages. The induction of cytochrome P450 enzymes leads to lipid peroxidation of polyunsaturated fatty acids in the cell membranes and resulting in overexpression of volatile alkanes and alkane-derivatives in the breath, and eventually affecting the abundance of VOCs in the EB [13], [14], [15]. Besides, free radical-induced oxidation of amino acids and proteins has also been demonstrated to result in the generation of some hydrocarbons along with malonaldehyde [16], [17], [18].

Only a limited number of studies have been performed to investigate the feasibility of BC diagnosis based on breath analysis. The published data show that the patterns of VOCs in the EB can distinguish patients with and without BC [19], [20], [21]. Unfortunately, the reported potential breath biomarkers for BC differ among studies [22]. Therefore, there is still a need to continue searching breath biomarkers for BC.

The growing interest in the discovery of breath biomarkers of the OS associated with lung cancer, especially straight aldehydes [23], [24], [25], led us to consider whether straight aldehydes are potential effective breath biomarkers for BC. Besides, there have been articles reported that formaldehyde [26] and heptanal [20] may be potential BC breath biomarkers. Aldehydes are secondary breakdown products of lipids oxidation [27], [28]. In particular, C5–C10 monofunctional aldehydes (including hexanal, heptanal, octanal, and nonanal) have been identified to be derived from lipid peroxidation [28]. The well-understood biochemical pathways of these aldehydes simplify the evaluation of their diagnostic values. In addition, volatile aldehydes have low solubility in the blood, so they are excreted into the breath within minutes after their formation in tissues [9], [29]. Furthermore, hexanal, heptanal, octanal and nonanal were relatively stable than low molecular weight aldehydes and they all could be detected by our method. Moreover, we found that their levels seem to be different between HC and BC in our preliminary experiment results. Therefore, among a variety of straight aldehydes, we choose to study hexanal, heptanal, octanal and nonanal.

Elevated levels of aldehydes imply enhanced OS and they have been proposed as a measure for cancer status diagnosis [30], [31], [32]. Moreover, the pattern of aldehydes appeared to be unique to each form of cancer [31]. Hexanal, heptanal, some alkanes, alkane derivatives, and benzene derivatives [33], pentanal, hexanal, octanal, and nonanal [24] were reported to be potential breath biomarkers for lung cancer. Furthermore, formaldehyde [26] and heptanal [20] was also reported as potential breath biomarkers for BC. Therefore, we and other researchers have proposed that exhaled aldehydes are potential diagnostic biomarkers for various human cancers, including BC [20], [22], [24], [26], [33], [34].

Although Fuchs et al. [24] found out that exhaled aldehydes could be used as lung cancer biomarkers, they only focus on lung cancer. Peng et al. [35] achieved the discrimination between ‘healthy’ and ‘cancerous’ breath in a variety of human cancers (including lung, breast, colorectal, and prostate cancers) by a nanosensor array. To the best of our knowledge, no previous studies have evaluated in detail the clinical value of identifying BC using specific volatile straight aldehydes in EB by simple direct gas chromatography–mass spectrometry (GC–MS) analysis without any preconcentration.

Therefore, we designed a systematic study to investigate and identify novel potential breath aldehyde biomarkers for the early diagnosis of BC. Based on literature survey [22], [23], [24], [31], [32] and preliminary experiment results, we choose hexanal, heptanal, octanal and nonanal (which were relatively stable than low molecular weight aldehydes, and were often reported as potential tumor biomarkers, as well as their biochemical pathways were well understood) as our target compounds.