Analytical & Bioanalytical Chemistry

Breast cancer discrimination using saliva: an exploratory

study involving geographic distant populations

Journal: Analytical and Bioanalytical Chemistry

Manuscript ID Draft
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Type of Paper: Research Paper

Date Submitted by the Author: n/a

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Complete List of Authors: Cavaco, Carina; Universidade da Madeira, Centro de Química da Madeira
Pereira, Jorge; Universidade da Madeira, Centro de Química da Madeira
Taunk, Khushman; National Centre For Cell Science, Proteomics Lab
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Taware, Ravindra; National Centre For Cell Science, Proteomics Lab

Rapole, Srikanth; National Centre For Cell Science, Proteomics Lab
Nagarajaram, Hampapathalu; University of Hyderabad School of Life
Sciences, Department of Biotechnology & Bioinformatics; Centre for DNA
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Fingerprinting and Diagnostics, Laboratory of Computational Biology

Câmara, José; Universidade da Madeira, Centro de Química da Madeira;
Universidade da Madeira Faculdade de Ciencias Exatas e da Engenharia
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Saliva, Volatile organic metabolites (VOMs), HS-SPME, GC-MS, breast

Keywords:
cancer diagnosis
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Page 1 of 29 Analytical & Bioanalytical Chemistry

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4 1 Breast cancer discrimination using saliva: an exploratory study involving geographic
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6 2 distant populations
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10 4 Carina Cavaco1, Jorge A. M. Pereira1*, Khushman Taunk2, Ravindra Taware2, Srikanth Rapole2,
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12 5 Hampapathalu Nagarajaram3,4 and José S. Câmara1,5
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16 7 CQM/UMa – Centro de Química da Madeira, Universidade da Madeira, Campus
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18 8 Universitário da Penteada, Funchal 9000-390, Portugal
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20 9 Proteomics Lab, National Centre for Cell Science, Ganeshkhind, Pune 411007, India
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22 10 Laboratory of Computational Biology, Centre for DNA fingerprinting & Diagnostics (CDFD),
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24 11 Nampally, Hyderabad, 500001, India

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26 12 Department of Biotechnology & Bioinformatics, School of Life Sciences, University of
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28 13 Hyderabad, Hyderabad 500 046, India
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30 14 Faculdade de Ciências Exatas e da Engenharia, Universidade da Madeira, Campus
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32 15 Universitário da Penteada, Funchal 9000-390, Portugal
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34 16 *corresponding author
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36 17
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38 18 ORCID IDs
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40 19 JAMP - 0000-0003-0316-5348, JSC - 0000-0003-1965-3151,
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43 20 KT - 0000-0003-2293-2936, SR - 0000-0003-0273-0819,
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45 21 HN - 0000-0001-8568-0620
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 Analytical & Bioanalytical Chemistry Page 2 of 29

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4 22 Abstract
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6 23 Saliva is possibly the easiest biofluids to analyse and, despite its simple composition, contains
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8 24 relevant metabolic information. In this work, we explored the potential of the volatile
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10 25 composition of saliva samples as biosignatures for BC non-invasive diagnosis. To achieve this,
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12 26 106 saliva samples of BC patients and controls in two distinct geographic regions in Portugal
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14 27 and India were extracted and analysed using an optimized headspace solid-phase
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16 28 microextraction gas chromatography-mass spectrometry (HS-SPME/GC-MS, 2 mL acidified
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18 29 saliva containing 10% NaCl, stirred (800 rpm) for 45 min at 38ºC and using the CAR/PDMS
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20 30 SPME fibre) followed by multivariate statistical analysis (MVSA). Over 120 volatiles from
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22 31 distinct chemical classes, with significant variations among the groups, were identified. MVSA
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24 32 retrieved a limited number of volatiles viz. 3-methyl-pentanoic acid, 4-methyl-pentanoic acid,

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26 33 phenol and p-tert-butyl-phenol (Portuguese samples) and acetic, propanoic, benzoic acids, 1,2-
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28 34 decanediol, 2-decanone, and decanal (Indian samples), statistically relevant for the
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30 35 discrimination of BC patients in the populations analysed. This work defines an experimental

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32 36 layout, HS-SPME/GC-MS followed by MVSA, suitable to characterize volatile fingerprints for
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34 37 saliva as putative biosignatures for BC non-invasive diagnosis. Here it was applied to BC
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36 38 samples from geographically distant populations and good disease discrimination was obtained.
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39 Further studies using larger cohorts are therefore very pertinent to challenge and strength this
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40 proof-of-concept study.
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42 Keywords
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43 Saliva, Volatile organic metabolites (VOMs), HS-SPME, GC-MS, breast cancer diagnosis
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50 45 Abbreviations: BC - breast cancer, CAR/PDMS - Carboxen®/ polydimethylsiloxane SPME
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52 46 fibre, CTRL – control healthy subjects, HCA - hierarchical cluster analysis, HCl - hydrogen
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54 47 chloride, HS-SPME/GC-MS - headspace solid-phase microextraction gas chromatography-mass
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56 48 spectrometry, IN – Indian samples (Pune), MCCV - Monte Carlo cross-validation, MVSA -
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Page 3 of 29 Analytical & Bioanalytical Chemistry

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4 49 multivariate statistical analysis, OPLS-DA - Orthogonal Projections to Latent Structures
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6 50 Discriminant Analysis, PCA - principal component analysis, PLS-DA - Partial Least Squares
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8 51 Discriminant Analysis, PT – Portuguese samples (Madeira Island), ROC - receiver operator
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10 52 characteristic, RF - random forest, SPME - solid-phase microextraction, VOMs - Volatile
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12 53 organic metabolites
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 Analytical & Bioanalytical Chemistry Page 4 of 29

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4 55 Introduction
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56 In the last 50 years, and despite the global efforts to decrease the incidence of cancer, this
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57 disease has become the main cause of death [1, 2]. In industrialized countries, breast cancer
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58 (BC) is the most common malignancy and the second most common cause of cancer-related
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59 mortality in women [2]. In the past several years, various therapies have been used to fight
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60 cancer, however the survival rates remain low, particularly in the patients in which the disease is
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16 61 detected at an advanced stage [2]. Unfortunately, this late diagnosis continues to be a hallmark
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18 62 of cancer prevalence, including BC, because the current screening methodologies are mostly
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20 63 expensive, invasive and ineffective in the early stages of cancer development [3, 4]. Volatile
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22 64 organic metabolites (VOMs) are subset of organic compounds that are volatile (or semi-volatile)
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24 65 and are produced endogenously or resulting from exogenous sources (environmental, lifestyle,
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66 biological agents). VOMs can be established as promising cancer biosignatures and several
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28 67 studies have presented evidences that exhaled breath and urine volatile profiles of different
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30 68 cancer patients, including BC, can be discriminative from healthy subject’s profiles [1, 5-10].
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32 69 Human saliva is a highly variable and individualized biological fluid, being a mixture of
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34 70 secretions from the salivary glands, gingival crevicular fluid, bacteria, epithelial cells and food
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71 debris [11-13]. Several factors affect saliva composition, as the circadian rhythm, different
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38 72 physiological and pathological factors, age, menstrual cycle, diet, exercise and psycho-
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40 73 emotional state, drugs of abuse or the individual's hydration status [14]. Therefore, saliva can
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42 74 act as a "mirror of the body´s health" [15], constituting an important source of metabolic
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44 75 information for the detection of pathologic conditions [13, 16]. In fact, saliva is a good indicator
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46 76 of the plasma/serum levels of several substances, such as hormones and drugs. This has been
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48 77 shown in recent years through several reports correlating different drugs, pollutants or hormones
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50 78 present in saliva with bacterial, viral and systemic diseases, as well as cancer [13, 15, 17-19].
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52 79 These correlation have being explored in the evaluation of the severity of some diseases, in the
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54 80 early diagnosis and prognosis and in the post-therapy state monitoring [15, 16, 20]. Saliva,
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56 81 similarly to other biofluids, also contains VOMs in its composition and their potential in disease
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4 82 diagnosis is just recently being unveiled. The non-invasive nature, simplicity in the collection,
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6 83 storage and transport, as well as availability for fast and repeated sampling makes saliva a
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8 84 promising alternative to blood and urine in the determination of different biochemical profiles
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10 85 [15, 16, 21, 22]. This is further supported by the fact that many VOMs detected in other
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12 86 biofluids are also present in saliva [13, 23]. The aim of this study was to establish a standardized
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14 87 protocol to characterize the salivary volatomic profiles of BC patients and healthy individuals
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16 88 and additionally explore the potential of these profiles as biosignatures for BC diagnosis. This
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18 89 study was carried out in two distinct demographic cohorts, namely Portugal (Madeira Island)
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20 90 and India (Pune). Upon the optimization of several parameters, headspace solid-phase
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22 91 microextraction (HS-SPME) using Carboxen®/ polydimethylsiloxane (CAR/PDMS) sorbent
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24 92 was selected to extract the VOMs from 2 mL of acidified saliva sample containing 10% NaCl,
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26 93 stirred (800 rpm) for 45 min at 38 ºC, that were then analysed by GC-MS and processed using
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28 94 MVSA to obtain the respective metabolomic profiles.
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30 95
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32 96 MATERIALS AND METHODS
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34 97 Reagents and materials
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36 98 Sodium chloride (NaCl) was purchased from Panreac (Barcelona, Spain) and hydrogen chloride
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99 (HCl) from Sigma-Aldrich (St Louis, MO, USA). The Cimarec digital stirring hot plate was
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100 provided by Thermo Scientific (Waltham, MA, USA). The SPME holder and glass vials were
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101 acquired from Supelco (Bellefonte, PA, USA). The GC carrier gas used was Helium at a purity
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102 of 99.999 % (Air Liquide, Lisbon, Portugal).
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104 Subjects recruitment and sample collection
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105 The saliva samples used in this study were collected in Funchal (Portugal) and Pune (India). In
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106 Funchal, the samples were donated by healthy volunteers (control group) and patients with BC
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107 (oncological groups) from the Haematology-Oncology Unit of the Hospital Dr. Nélio
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55 108 Mendonça. The saliva samples collected in Pune were obtained from healthy volunteers and BC
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4 109 patients at the Department of Oncology, Armed Force Medical College. The present study was
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6 110 approved by the Ethics Committee of all institutions involved, namely the Ethics Committee of
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8 111 the Hospital Dr. Nélio Mendonça (Funchal), the Institutional Ethics Committee of Armed Force
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10 112 medical College (Pune) and the Institutional Ethics Committee of NCCS (Pune). As this study
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12 113 involved cancer patients, the protocol for saliva collection was the simplest possible and similar
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14 114 to others previously reported [24-27]. Accordingly, all the saliva samples were collected in the
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16 115 morning in an unstimulated fashion after rinsing the mouth with clean water and before
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18 116 breakfast. The number of subjects recruited for the study were 66 for BC (n = 36 from Portugal
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20 117 and n = 30 from India) and 40 for the control group (n = 16 for Portugal and n = 24 for India).
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22 118 Table 1 summarizes the samples used in the study by gender, age and origin of the participants.
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24 119 The samples were collected in 8 mL glass vials and stored at -80°C in aliquots of 2 mL, until
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26 120 further analysis. All the participants enrolled in this study were arbitrarily and voluntarily
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28 121 selected after being properly informed about the study.
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30 122 Table 1 Characterization of the samples

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33 Number of subjects Age range (years)

34 Portugal
35 CTRL 16 18-63
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BC 36 39-73
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India
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39 CTRL 24 23-65
40 BC 30 25-76
41 123 Legend: * female subjects, BC – breast cancer saliva samples, CTRL – saliva samples from
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43 124 healthy subjects
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46 125
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48 126 HS-SPME procedure and optimization
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51 127 To find an optimum HS-SPME procedure, several parameters affecting the extraction of
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53 128 salivary VOMs and particularly this methodology were optimized. These include the adsorptive
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55 129 phase (PDMS 100 µm, PA 85 µm, DVB/CAR/PDMS 50/30 µm, CAR/PDMS 75 µm, and
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4 130 PDMS/DVB 65 µm, all manufactured by Supelco, Bellefonte, USA), sample ionic strength (0,
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6 131 5, 10, 15 and 20 % sodium chloride final concentration), volume (1, 2 and 4 mL) and pH (acid,
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8 132 neutral and basic), agitation and extraction time (30, 45 and 60 min) and temperature (28, 38
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10 133 and 48 °C).
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12 134 The sample used as the matrix for the optimization of the HS-SPME parameters was obtained
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14 135 from normal subjects and kept at -80 °C until used. Prior to the experiments, the frozen saliva
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16 136 samples were thawed at room temperature. The optimum conditions were determined by the
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18 137 totality of the peak areas obtained in each parameter and the number of extracted compounds.
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20 138 The fibres used were daily conditioned in GC injector for 6 min at 250 °C to remove any
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22 139 potential contaminants present in the fibre. In the optimized procedure, 1 mL saliva aliquots
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24 140 placed in a 4 mL-headspace glass vial were adjusted to pH 1-2 (with 0.125 mL of hydrochloric
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26 141 acid 5 M), added 0.1 g of NaCl, stirred (at 800 rpm) and the vial topped with a Teflon (PTFE)
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28 142 septum and an aluminium cap (Chromacol, Hertfordshire, UK). Then, the sample vial was
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30 143 placed in a thermostat bath (37 °C) and the SPME fibre exposed to the headspace for 45 min.
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32 144 After sampling, the SPME fibre was inserted into the injector port (250 °C) of the GC-MS
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34 145 system for 6 min and the metabolites thermally desorbed to the analytical column. Each sample
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36 146 was analysed in duplicate.
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148 GC-MS analysis
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149 The gas chromatograph system was composed by an Agilent Technologies 6890N Gas
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150 Chromatograph (Palo Alto, CA, USA) equipped with a BP-20 fused silica capillary column (60
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151 m×0.25 mm ID×0.25 µm film thickness, SGE, Dortmund, Germany) interfaced with an Agilent
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152 5975 quadrupole inert mass selective detector. The oven temperature was programmed as
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153 follows: 45 °C for 5 min, then ramped 2 °C/min to 150°C, 10 min hold time, another
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154 temperature shift of 15 °C/min up to 220 °C and 15 min hold time. The total GC run time was
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155 87 min. The column flow was maintained constant at 1 mL/min using He (Helium N60, Air
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55 156 Liquide, Portugal) as carrier gas. The injections were performed in splitless mode at 250°C. For
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4 157 the 5975MS system, the operating temperatures of the transfer line, quadrupole and ionization
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6 158 source were 270, 150 and 230°C, respectively. The acquisitions were performed in full scan
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8 159 mode (30–300 m/z) and the electron impact mass spectra were recorded at a 70 eV ionization
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10 160 voltage. Metabolite identification was achieved by manual interpretation of spectra and through
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12 161 the comparison with the NIST11 mass spectral library, using a similarity threshold higher than
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14 162 85%.
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18 164 Statistical Analysis
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20 165 The data obtained was analysed with Metaboanalyst 3.0 [28] and SIMCA 14.1. The analysis
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22 166 with Metaboanalyst 3.0 included a data pre-processing to remove metabolites with missing
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24 167 values (MV), MV imputation of the resulting data (by Bayesian principal component analysis,
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26 168 bPCA method) and normalization (to sample median, data transformation by cubic root and data
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28 169 scaling by autoscaling). The normalised data was further subjected to univariate and
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30 170 multivariate statistical analysis. The univariate analysis, Mann Whitney U test (p <0.05) was
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32 171 performed to evaluate the existence of significant differences between the salivary VOMs of
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34 172 healthy controls and BC patients. In turn, the multivariate statistical analysis (SIMCA), namely
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36 173 partial least squares discriminant analysis (PLS-DA) and orthogonal projections to latent
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174 structures – discriminant analysis (OPLS-DA) was used to determine the distribution of
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175 oncologic and control variables and to detect VOMs that may indicate differences between the
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176 samples sets. Hierarchical cluster analysis (HCA) was performed using the metabolite profiles
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177 of both groups to identify inherent clustering patterns. Ward distance calculation was performed
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178 and the trees were sorted according to size for HCA analysis. The robustness of the data
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179 obtained was assessed using permutation statistic test with 200 iterations.
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52 181 Results and Discussion
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54 182 Optimization of HS-SPME parameters
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4 183 Several parameters that influence the HS-SPME, namely the fibre, the sample ionic strength,
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6 184 volume and pH, agitation and time and temperature of extraction, were optimized. For this
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8 185 purpose, we used a saliva stock solution composed by saliva samples from several individuals
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10 186 without cancer pathology (control group).
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18 189 To select the best fibre to study salivary VOMs, five SPME fibres with different thicknesses and
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stationary phases, PDMS fibre (100 µm), PA (85 µm), the DVB/CAR/PDMS (50/30 µm),
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22 191 CAR/PDMS (75 µm) and PDMS/DVB (65 µm), were assayed using 45 min of extraction of 2
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24 192 mL of saliva at 38 °C, 10% sodium chloride (NaCl), pH 1-2 and constant stirring (800 rpm).
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194 the total area obtained and the reproducibility of the fibre. Fig.1A shows the typical results
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195 obtained. Overall, CAR/PDMS fibre was most effective in the extraction of salivary VOMs,
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196 retrieving higher total area and number of extracted metabolites. This result is certainly due to
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197 the CAR/PDMS fibre coating containing a polar and a nonpolar group that provides a bipolar
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198 character to the fibre, allowing the adsorption of analytes of medium and low polarity [29]. In
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199 turn, the fibres that showed lower extraction efficiency were PDMS and PA.
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42 201 Ionic strength
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45 202 The efficiency of the SPME technique is influenced by the extension of the analyte transference
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47 203 from the sample to the headspace and then to the fibre. This process can be enhanced by
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49 204 increasing the ionic strength of the sample, due to a "salting out" effect that is more effective for
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51 205 analytes with low hydrophobicity. However, this increase in ionic strength can cause a reduction
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53 206 in the movement of analytes to the fibre because the polar molecules can participate in
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207 electrostatic interactions with the ionic salts present in the solution [30]. Therefore, five
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4 209 Fig. 1B, a greater number of metabolites and higher total area was found with NaCl 10 % (w/v)
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213 The volumes of the sample and the headspace available in the sampling tube have an important
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214 role in SPME analysis, affecting the quantity of the extracted analytes and the kinetics of the
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16 215 extraction process [31]. The equilibration time associated to the extraction step, for instance, can
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18 216 be significantly reduced by manipulating the headspace volume or increasing the partition
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20 217 coefficient between the headspace and the sample (by rising the temperature) [32]. In our
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22 218 experiments, we assayed three sample volumes (1, 2 and 4 ml) and verified that a 2 mL-sample
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24 219 volume allowed the most efficient extraction (Fig. 1C). However, the volumes of most saliva
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220 samples we could collect from the recruited subjects, particularly the cancer patients, was lower
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30 222 to allow replicates.

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37 225 The pH of the medium affects the extraction efficiency of acidic and basic metabolites [30, 33]
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39 226 as they can only be extracted by SPME in the protonated form. Furthermore, for metabolites
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41 227 that possess a pH-dependent ionisable group, only the undissociated form is extracted by the
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43 228 SPME fibre coating [30]. Considering these constrains, samples were prepared at acid (pH 1-2
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45 229 adjusted with HCl 5M), neutral (pH 6-7) and basic pH (pH 9-10 adjusted with NaOH 1M)
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47 230 conditions to assess the best pH range for the salivary VOMs extraction and characterization. As
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49 231 Fig.1D shows, the acidic pH is more favourable than the basic or neutral pH, allowing the
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51 232 extraction of a higher total area and number of metabolites.
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55 234 Sample stirring
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4 235 The sample agitation facilitates the mass transference from the sample to the headspace,
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6 236 therefore, reducing the time necessary to achieve the extraction equilibrium. Moreover, agitation
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8 237 is important and necessary for certain biological samples, as saliva, that have a relatively high
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10 238 viscosity and low diffusion coefficients [33]. In this context, we performed extractions with
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12 239 (800 rpm) and without agitation and confirm a significant improvement of the extraction
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14 240 efficiency when agitation was included in the extraction procedure (Fig.1E).
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16 241
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18 242 Extraction time
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20 243 Since the HS-SPME methodology is a process that involves the balance between the analytes
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22 244 present in the sample and in the fibre, it is important to select the optimum time to achieve this
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24 245 equilibrium. Therefore, to evaluate the influence of the extraction time on the extraction
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246 efficiency of the HS-SPME, 1 mL of acidified saliva sample containing 10 % NaCl (w/v) was
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28 247 extracted with the CAR/PDMS fibre for 30, 45 and 60 min, under constant agitation. The results
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30 248 obtained are shown in Fig. 1F and reveal an increase in the total area and in the number of
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32 249 identified metabolites from 30 to 45 min of extraction. To higher extraction times, 60 min, the
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34 250 total area is the same, but the number of detected metabolites decreases from 18 to 13,
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251 suggesting that some metabolite degradation may be occurring. Therefore, 45 min of extraction
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38 252 was selected as the best extraction time for the proposed methodology.
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43 254 Extraction temperature
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45 255 The extraction temperature is another important parameter in the extraction efficiency of HS-
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47 256 SPME. Generally, temperature increments cause an increase of the diffusion of the analytes to
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49 257 the headspace and consequently to the stationary phase, thus allowing a more efficient
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51 258 extraction [33], but at the same time, high temperatures can generate isomerization or
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53 259 degradation of some metabolites [30]. Here, we assayed 28, 38 and 48 °C and verified a
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55 260 continuous increase in total area correlating with temperature (Fig. 1G). However, the number
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4 261 of metabolites extracted and identified is greater at 38 ºC (18 vs 15) and moreover this
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6 262 temperature is closer to normal to body temperature. Therefore, 38 ºC was selected as the most
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8 263 suitable temperature to extract the salivary VOMs.
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10 264
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12 265 <Figure 1>
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14 266
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16 267 Identification of VOMs in BC saliva samples
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18 268 The VOMs present in human saliva samples were extracted via the optimised HS-SPME
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20 269 methodology using a CAR/PDMS SPME fibre and desorbed in the GC-MS for chromatographic
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22 270 separation and mass spectrometric identification. The saliva samples analysed were collected
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24 271 from BC patients recruited in Portugal and India. To establish a saliva volatomic profile, 16 and
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272 24 samples from healthy individuals (CTRL) and 36 and 30 samples from BC patients were
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28 273 processed in Portugal and in India, respectively (Table 1). Fig. 2 shows the typical
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30 274 chromatograms obtained from saliva samples of the two groups analysed (an equivalent result
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32 275 for Indian samples is available in the Supplementary Fig. 1).
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277 <Figure 2>

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38 278
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40 279 Overall, 55 volatile metabolites were identified in the control group and 121 in BC group in the
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42 280 Portuguese subjects and 86 and 90 VOMs in the equivalent groups in India. The metabolites
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44 281 identified were classified according to the major chemical families, as organic acids, alkanes,
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46 282 alkenes, higher alcohols, ketones, nitrogen compounds, sulphur compounds, terpene
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48 283 compounds, benzene derivatives and phenols, and the average areas of VOMs identified are
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50 284 presented in Fig. 3A. The results obtained are quite heterogeneous, but there are a few trends
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52 285 that can be distinguished in the volatile composition of the saliva from both populations, namely
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54 286 the predominance of organic acids above the other chemical families. Notably, however, in the
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56 287 Portuguese BC patients such abundance is partially lost for nitrogen, terpenic and benzenic
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4 288 compounds, and aldehydes and esters, that present levels clearly higher than remain chemical
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6 289 classes and groups analysed. Additionally, higher alcohols are also more abundant in the
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8 290 controls than in cancer patients, regardless of their geographic origin. Considering the other
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10 291 chemical families, alkanes, esters and aldehydes were hardly detected in the Portuguese
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12 292 subjects, while they are abundant in the Indian population. In contrast, terpenic compounds and
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14 293 ketones exhibit the opposite trend. Overall, the variations observed in the volatile composition
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16 294 of saliva for the different groups analysed has the contribution of several factors, particularly the
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18 295 diet and genetic background of the populations analysed. Furthermore, the metabolites
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20 296 identified may have distinct sources, being the endogenous produced during different metabolic
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22 297 processes, while the exogenous metabolites can be assimilated by feeding, air inhalation or
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24 298 absorbed through skin [34]. In this context, it is important to refer that certain volatile organic
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26 299 acids are important intermediates in different biological processes and their presence in a
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28 300 sample is usually associated with bacterial activity. This is the case, for instance, of the
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30 301 degradation of carbohydrates that occurs in the intestine by bacterial anaerobic fermentation
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32 302 [35]. In turn, hydrocarbon production in the body relies in the oxidative stress, being alkanes
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34 303 essentially produced by the peroxidation of polyunsaturated fatty acids, mainly found in cell
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36 304 membranes. The saturated hydrocarbons, such as ethane and pentane, are final products of lipid
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305 peroxidation and both volatiles, as well as the methylated hydrocarbons, have been widely used
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306 as a non-invasive indicators of lipid peroxidation [36]. As for the unsaturated hydrocarbons,
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307 there is a supposition that they are formed along the mevalonic acid pathway of cholesterol
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308 synthesis (e.g. isoprene) [37]. To obtain a more detailed analysis of the volatile composition of
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309 the saliva samples, the major metabolites found in each group were characterized and compared.
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310 As shown in Fig. 4, six of the eight major metabolites present in the saliva samples studied are
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311 organic acids, from which acetic acid, propanoic acid and butanoic acid are the most abundant.
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312 Phenol was also found in all samples, although in much higher levels particularly in the Indian
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313 samples, while 1,2-decanediol was exclusively identified in this population. Overall, the major
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4 314 compounds here identified in saliva have been previously reported in different studies [13, 23,
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6 315 38].
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8 316
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10 317 <Figure 3>
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12 318
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14 319 Statistical analysis applied to saliva samples
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16 320 The data obtained in the collaborating groups in Portugal and India was analysed using the
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18 321 Metaboanalyst 3.0 software package [28]. To obtain a reliable dataset to apply multivariate
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20 322 analysis, the raw data was screened to eliminate spurious results (8 samples were not considered
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22 323 for the analysis due to consistency problems as a very low number of VOMs identified,
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24 324 suggesting problems during sampling procedure, or lack of reproducibility among replicas
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26 325 analysis, among others). Additionally, only VOMs with a frequency of occurrence above 85%
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32 328 Table 2 Altered salivary VOMs with VIP > 1 in Portugal and India datasets
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34 Portuguese dataset Indian dataset
35 VOMs Fold Mann VIP VOMs Fold Mann VIP
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Change Whitney score Change Whitney score

37 U test p- (OPLS- U test p- (OPLS-
38 value DA) value DA)
39 3-methyl- 0.454 5.38E-04 1.01 Acetic acid 4.98 4.25E-07 1.16
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pentanoic acid
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42 4-methyl- 0.06 3.39E-06 1.08 Propanoic acid 8.47 6.3E-10 1.33
43 pentanoic acid
44 Phenol 0.24 3.16E-07 1.68 1,2-Decanediol 0.39 1.56E-05 1.22
45 329 Legend: VIP – variable importance in projection, OPLS-DA - orthogonal partial least squares-
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47 330 discriminant analysis, VOMs – volatile organic metabolites
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49 331
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51 332 Missing values imputation was then applied using Bayesian principal component analysis
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53 333 (PCA), which is the more appropriate methodology for such kind of metabolomics data set [39,
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55 334 40]. Row-wise normalization to sample median, data transformation by cubic root
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4 335 transformation and data scaling by autoscaling were carried out to transform the data to follow a
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6 336 normal distribution (Supplementary Figure 2). Mann Whitney univariate test was used to
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8 337 identify statistically significant metabolites in the datasets. The combination of univariate and
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10 338 multivariate analysis revealed a set of four VOMs in Portuguese while a set of six VOMs were
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12 339 found to be statistically differentially regulated in the Indian dataset. Overall, a total of four and
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14 340 six metabolites in Portugal and India, respectively, with VIP score > 1.0, and p-value < 0.05,
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16 341 emerged as statistically significant VOMs which discriminated BC from healthy controls in the
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18 342 respective dataset (Table 2). Partial least square discrimination analysis (PLS-DA) and
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20 343 orthogonal projections to latent structures – discriminant analysis (OPLS-DA) are regression
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22 344 analysis method for multivariate data which define the latent variables and maximize the
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24 345 covariance between the groups under study [41]. In our study, the OPLS-DA plot demonstrates
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26 346 the importance of the selected VOMs (or features) through the calculated VIP scores (Table 2)
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28 347 towards the classification of the groups. The R2 values represent the goodness of fit for a
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30 348 multivariate model while the Q2 values depict the predictive ability of the model. The R2 value
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32 349 was 0.876 and Q2 value was 0.831 for the OPLS-DA model of Indian dataset. The Portuguese
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34 350 dataset had 0.833 as the R2 and 0.752 as the Q2 value for the OPLS-DA model. As shown in
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36 351 Fig. 4, OPLS-DA clustered the samples according to the disease existence (BC vs healthy
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352 subjects) both in Indian as in Portuguese saliva samples. The same result was obtained using
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353 PLS-DA (Supplementary Fig. 3). HCA for Portuguese and Indian datasets revealed clear class
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354 separation between BC and healthy control groups. The permutation test analysis depicted the
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355 robustness of OPLS-DA model which suggests that this model is not over-fitted. HCA and
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356 permutation test analysis for Portuguese and Indian datasets are shown in Supplementary Figs. 4
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357 and 5.
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359 <Figure 4>
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4 361 Here we developed and optimized a methodology for the characterization of the volatile
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6 362 composition of saliva samples from BC and healthy subjects using HS-SPME/GC-MS. This
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8 363 methodology was then applied to two geographic distant populations, one from Madeira Island,
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10 364 Portugal, and another from Pune, India. The application of MVSA to the volatile fingerprints
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12 365 identified allowed the identification of a few metabolites significantly altered and statically
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14 366 relevant for the discrimination of BC presence in both populations. As far we are aware, this is
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16 367 the first report characterizing the salivary volatile fingerprint of BC patients. Further studies
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18 368 using larger cohorts are therefore very pertinent to challenge and strength this proof-of-concept
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20 369 study on breast cancer.
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22 370
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25 371 Acknowledgments
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372 This research was supported by Fundação para a Ciência e a Tecnologia (FCT) with funds from
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373 the Portuguese Government (PEst-OE/QUI/UI0674/2013), MS Portuguese Networks

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374 (REDE/1508/RNEM/2011), New-INDIGO/0003/2012 project (ERA- NET, FP 7), INNO
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375 Indigo program under NCD-CAPomics-INNO INDIGO EU Project (FCT reference:
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376 INNOINDIGO/0001/2015) and ARDITI - Agência Regional para o Desenvolvimento da
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377 Investigação Tecnologia e Inovação (project M1420-01-0145-FEDER-000005 - Centro de
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378 Química da Madeira - CQM+ (Madeira 14-20), and Project M1420 - 09-5369-FSE-000001 for
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379 the Post-Doctoral fellowships granted to JAMP). SR, KT, and HN gratefully acknowledge the
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380 support of Department of Biotechnology (DBT), Ministry of Science & Technology,
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45 381 Government of India (New Indigo project BT/IN/New Indigo/03/RS/2013). RT acknowledges
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47 382 Council of Scientific and Industrial Research (CSIR), New Delhi, India for research
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49 383 associateship. SR, KT and RT would like to acknowledge Col. Dharmesh Soneji (AFMC, Pune)
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51 384 for sample collection.
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54 385 Conflict of Interest
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386 The authors have any conflict of interest to report.
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4 387 References
5
6
388 1.Silva CL, Passos M, Camara JS. Investigation of urinary volatile organic metabolites as
7
389 potential cancer biomarkers by solid-phase microextraction in combination with gas
8
390 chromatography-mass spectrometry. Br J Cancer. 2011;105(12):1894-904.
9
391 doi:10.1038/bjc.2011.437.
10
392 2.Ullah MF, Aatif M. The footprints of cancer development: Cancer biomarkers. Cancer Treat
11
393 Rev. 2009;35(3):193-200. doi:10.1016/j.ctrv.2008.10.004.
12
394 3.Banin Hirata BK, Oda JM, Losi Guembarovski R, Ariza CB, de Oliveira CE, Watanabe MA.
13
395 Molecular markers for breast cancer: prediction on tumor behavior. Dis Markers.
14
396 2014;2014:513158. doi:10.1155/2014/513158.
15 397 4.Yang Q, Shi X, Wang Y, Wang W, He H, Lu X et al. Urinary metabonomic study of lung
16 398 cancer by a fully automatic hyphenated hydrophilic interaction/RPLC-MS system. J Sep Sci.
17 399 2010;33(10):1495-503. doi:10.1002/jssc.200900798.
18 400 5.Peng G, Hakim M, Broza YY, Billan S, Abdah-Bortnyak R, Kuten A et al. Detection of lung,
19 401 breast, colorectal, and prostate cancers from exhaled breath using a single array of
Fo
20 402 nanosensors. Br J Cancer. 2010;103(4):542-51. doi:10.1038/sj.bjc.6605810.
21 403 6.Buszewski B, Rudnicka J, Ligor T, Walczak M, Jezierski T, Amann A. Analytical and
22 404 unconventional methods of cancer detection using odor. Trac-Trends in Analytical
23
rP

405 Chemistry. 2012;38:1-12. doi:10.1016/j.trac.2012.03.019.

24 406 7.Silva CL, Passos M, Camara JS. Solid phase microextraction, mass spectrometry and
25 407 metabolomic approaches for detection of potential urinary cancer biomarkers--a powerful
26
ee

408 strategy for breast cancer diagnosis. Talanta. 2012;89:360-8.

27 409 doi:10.1016/j.talanta.2011.12.041.
28 410 8.Pereira J, Silva CL, Perestrelo R, Goncalves J, Alves V, Camara JS. Re-exploring the high-
29 411 throughput potential of microextraction techniques, SPME and MEPS, as powerful strategies
rR

30 412 for medical diagnostic purposes. Innovative approaches, recent applications and future
31 413 trends. Anal Bioanal Chem. 2014;406(8):2101-22. doi:10.1007/s00216-013-7527-4.
32 414 9.Sun X, Shao K, Wang T. Detection of volatile organic compounds (VOCs) from exhaled
ev

33 415 breath as noninvasive methods for cancer diagnosis. Anal Bioanal Chem.
34 416 2016;408(11):2759-80. doi:10.1007/s00216-015-9200-6.
35 417 10.Wang C, Ke C, Wang X, Chi C, Guo L, Luo S et al. Noninvasive detection of colorectal
36
iew

418 cancer by analysis of exhaled breath. Anal Bioanal Chem. 2014;406(19):4757-63.

37 419 doi:10.1007/s00216-014-7865-x.
38 420 11.Subrahmanyam MV, Sangeetha M. Gingival crevicular fluid a marker of the periodontal
39 421 disease activity. Indian J Clin Biochem. 2003;18(1):5-7. doi:10.1007/BF02867658.
40 422 12.Dodds MW, Johnson DA, Yeh CK. Health benefits of saliva: a review. J Dent.
41 423 2005;33(3):223-33. doi:10.1016/j.jdent.2004.10.009.
42 424 13.Milanowski M, Pomastowski P, Ligor T, Buszewski B. Saliva - Volatile Biomarkers and
43 425 Profiles. Crit Rev Anal Chem. 2017;47(3):251-66. doi:10.1080/10408347.2016.1266925.
44 426 14.Schipper RG, Silletti E, Vingerhoeds MH. Saliva as research material: biochemical,
45 427 physicochemical and practical aspects. Arch Oral Biol. 2007;52(12):1114-35.
46 428 doi:10.1016/j.archoralbio.2007.06.009.
47 429 15.Shah FD, Begum R, Vajaria BN, Patel KR, Patel JB, Shukla SN et al. A review on salivary
48 430 genomics and proteomics biomarkers in oral cancer. Indian J Clin Biochem. 2011;26(4):326-
49 431 34. doi:10.1007/s12291-011-0149-8.
50 432 16.Liu J, Duan Y. Saliva: a potential media for disease diagnostics and monitoring. Oral Oncol.
51 433 2012;48(7):569-77. doi:10.1016/j.oraloncology.2012.01.021.
52 434 17.Reznick AZ, Hershkovich O, Nagler RM. Saliva--a pivotal player in the pathogenesis of
53 435 oropharyngeal cancer. Br J Cancer. 2004;91(1):111-8. doi:10.1038/sj.bjc.6601869.
54 436 18.Shpitzer T, Hamzany Y, Bahar G, Feinmesser R, Savulescu D, Borovoi I et al. Salivary
55 437 analysis of oral cancer biomarkers. Br J Cancer. 2009;101(7):1194-8.
56 438 doi:10.1038/sj.bjc.6605290.
57
58 17
59
60
 Analytical & Bioanalytical Chemistry Page 18 of 29

1
2
3
4 439 19.Wei J, Xie G, Zhou Z, Shi P, Qiu Y, Zheng X et al. Salivary metabolite signatures of oral
5 440 cancer and leukoplakia. Int J Cancer. 2011;129(9):2207-17. doi:10.1002/ijc.25881.
6 441 20.Zhang L, Farrell JJ, Zhou H, Elashoff D, Akin D, Park NH et al. Salivary transcriptomic
7 442 biomarkers for detection of resectable pancreatic cancer. Gastroenterology. 2010;138(3):949-
8 443 57 e1-7. doi:10.1053/j.gastro.2009.11.010.
9 444 21.Martin HJ, Riazanskaia S, Thomas CL. Sampling and characterisation of volatile organic
10 445 compound profiles in human saliva using a polydimethylsiloxane coupon placed within the
11 446 oral cavity. Analyst. 2012;137(16):3627-34. doi:10.1039/c2an35432b.
12 447 22.Wu JY, Yi C, Chung HR, Wang DJ, Chang WC, Lee SY et al. Potential biomarkers in saliva
13 448 for oral squamous cell carcinoma. Oral Oncol. 2010;46(4):226-31.
14 449 doi:10.1016/j.oraloncology.2010.01.007.
15 450 23.Amann A, Costello Bde L, Miekisch W, Schubert J, Buszewski B, Pleil J et al. The human
16 451 volatilome: volatile organic compounds (VOCs) in exhaled breath, skin emanations, urine,
17 452 feces and saliva. J Breath Res. 2014;8(3):034001. doi:10.1088/1752-7155/8/3/034001.
18 453 24.Al-Kateb H, de Lacy Costello B, Ratcliffe N. An investigation of volatile organic
19 454 compounds from the saliva of healthy individuals using headspace-trap/GC-MS. J Breath
Fo
20 455 Res. 2013;7(3):036004. doi:10.1088/1752-7155/7/3/036004.
21 456 25.Sanchez Mdel N, Garcia EH, Pavon JL, Cordero BM. Fast analytical methodology based on
22 457 mass spectrometry for the determination of volatile biomarkers in saliva. Anal Chem.
23 458 2012;84(1):379-85. doi:10.1021/ac2026892.
rP

24 459 26.Kusano M, Mendez E, Furton KG. Development of headspace SPME method for analysis of
25 460 volatile organic compounds present in human biological specimens. Anal Bioanal Chem.
26 461 2011;400(7):1817-26. doi:10.1007/s00216-011-4950-2.
ee

27 462 27.Perez Anton A, Del Nogal Sanchez M, Crisolino Pozas AP, Perez Pavon JL, Moreno
28 463 Cordero B. Headspace-programmed temperature vaporizer-mass spectrometry and pattern
29 464 recognition techniques for the analysis of volatiles in saliva samples. Talanta. 2016;160:21-7.
rR

30 465 doi:10.1016/j.talanta.2016.06.061.
31 466 28.Xia J, Sinelnikov IV, Han B, Wishart DS. MetaboAnalyst 3.0--making metabolomics more
32 467 meaningful. Nucleic Acids Res. 2015;43(W1):W251-7. doi:10.1093/nar/gkv380.
468 29.Aguirre-Gonzalez M, Taborda-Ocampo G, Dussan-Lubert C, Nerin C, Rosero-Moreano M.
ev

33
34 469 Optimization of the HS-SPME Technique by using Response Surface Methodology for
35 470 Evaluating Chlorine Disinfection by-Products by GC in Drinking Water. Journal of the
36 471 Brazilian Chemical Society. 2011;22(12):2330-6.
iew

37 472 30.Camara JS, Alves MA, Marques JC. Development of headspace solid-phase microextraction-
38
473 gas chromatography-mass spectrometry methodology for analysis of terpenoids in Madeira
474 wines. Anal Chim Acta. 2006;555(2):191-200. doi:10.1016/j.aca.2005.09.001.
39
475 31.Gorecki T, Pawliszyn J. Effect of sample volume on quantitative analysis by solid-phase
40
476 microextraction. Part 1. Theoretical considerations. Analyst. 1997;122(10):1079-86.
41
477 doi:10.1039/A701303E.
42
478 32.Gorecki T, Khaled A, Pawliszyn J. The effect of sample volume on quantitative analysis by
43
479 solid phase microextraction - Part 2. Experimental verification. Analyst. 1998;123(12):2819-
44
480 24. doi:DOI 10.1039/a806788k.
45
481 33.Ulrich S. Solid-phase microextraction in biomedical analysis. J Chromatogr A.
46
482 2000;902(1):167-94. doi:http://dx.doi.org/10.1016/S0021-9673(00)00934-1.
47
483 34.Ulanowska A, Ligor T, Michel M, Buszewski B. Hyphenated and Unconventional Methods
48
484 for Searching Volatile Cancer Biomarkers. Ecological Chemistry and Engineering S-Chemia
49
485 I Inzynieria Ekologiczna S. 2010;17(1):9-23.
50
486 35.Cummings JH. Short chain fatty acids in the human colon. Gut. 1981;22(9):763-79.
51
487 36.Haick H, Broza YY, Mochalski P, Ruzsanyi V, Amann A. Assessment, origin, and
52
488 implementation of breath volatile cancer markers. Chem Soc Rev. 2014;43(5):1423-49.
53
489 doi:10.1039/c3cs60329f.
54
490 37.Miekisch W, Schubert JK, Noeldge-Schomburg GF. Diagnostic potential of breath analysis--
55 491 focus on volatile organic compounds. Clin Chim Acta. 2004;347(1-2):25-39.
56 492 doi:10.1016/j.cccn.2004.04.023.
57
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59
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1
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3
4 493 38.de Lacy Costello B, Amann A, Al-Kateb H, Flynn C, Filipiak W, Khalid T et al. A review of
5 494 the volatiles from the healthy human body. J Breath Res. 2014;8(1):014001.
6 495 doi:10.1088/1752-7155/8/1/014001.
7 496 39.Mandel J, Schmitt P. A Comparison of Six Methods for Missing Data Imputation. Journal of
8 497 Biometrics & Biostatistics. 2015;06(01). doi:10.4172/2155-6180.1000224.
9 498 40.Broadhurst DI, Kell DB. Statistical strategies for avoiding false discoveries in metabolomics
10 499 and related experiments. Metabolomics. 2006;2(4):171-96. doi:10.1007/s11306-006-0037-z.
11 500 41.Westerhuis JA, Hoefsloot HCJ, Smit S, Vis DJ, Smilde AK, van Velzen EJJ et al.
12 501 Assessment of PLSDA cross validation. Metabolomics. 2008;4(1):81-9. doi:10.1007/s11306-
13 502 007-0099-6.
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12 508 (g). The selected condition is highlighted in each parameter. Legend: A – DVD/CAR/PDMS, B
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14 509 – CAR/PDMS, C – PDMS/DVB, D – PDMS, E – PA, Ac – acid, Neu – neutral, Bas – basic
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18 511 Fig. 2 Representative GC-MS chromatograms from healthy subjects (CTRL) and breast cancer
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520 (BC) patients (a) and major VOMs identified in the saliva samples from CTRL and BC subjects
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521 recruited in Madeira and Pune. The relative area from each VOM is indicated inside the
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46 524 Fig. 4 - Application of OPLS-DA to the experimental data obtained for the Portuguese (a) and
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Supplementary Figure 3: Application of PLS-DA to the experimental data obtained for the
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22 control samples, PLS-DA - supervised partial least square discriminant analysis.

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 Analytical & Bioanalytical Chemistry Page 28 of 29

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35 Supplementary Figure 4. Dataset hierarchical clustering (a) and permutation test (b)
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36 for Portuguese samples analyzed by SIMCA 14.1.

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Page 29 of 29 Analytical & Bioanalytical Chemistry

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35 Supplementary Figure 5. Dataset hierarchical clustering (a) and permutation test (b)
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