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Research strategy
(a) SIGNIFICANCE
Cancer is a leading cause of death worldwide. Estimated by the International Agency for
Research on Cancer (IARC), there were 12.7 million new cancer cases in 2008 worldwide, 44%
of which occurred in economically developed countries and 56% in economically developing
countries [1]. The World Health Organization (WHO) reported that cancer is causing around 7.6
million deaths (second most common cause of all deaths, comprised of approximately 13% of
death) worldwide each year [2]. Of these deaths, around 70% are now occurring in low- and
middle-income countries, making the shift of heavy burden in cancer control from the developed
countries to disadvantaged populations. It is predicted to increase morbidity, mortality, and
economic cost in the next 20 years in developing countries. By 2030, the global burden is
expected to grow to 21.4 million new cancer cases and 13.2 million cancer deaths [3].
Breast cancer is the most frequently diagnosed cancer and leading cause of cancer death in
women worldwide with an estimated 1.4 million new cases and estimated 458,400 deaths in
2008 [3]. About half of these cases occurred in economically developing countries. Despite the
great progress achieved in the treatment of breast cancer, breast cancer is still causing around
40,000 deaths in the U.S. each year, according to the American Cancer Society. Significant
improvements in breast cancer survival over the past 25 years have shown success, credited to
early detection and improved treatment [4]. Currently available diagnostic techniques for breast
cancer detection include mammogram and the molecular analysis of tumor biomarkers in nipple
aspirate fluid or in ductal lavage [5]. However, these methods are somewhat expensive, time-
consuming, and discomfort for patients, and require skilled medical staff. Furthermore, these
options for early detection in developing countries are often lacking, not accessible or not
affordable. The need to develop point-of-care, portable and cost effective screening and
diagnostic technologies is urgent.
A long quest has been undertaken in searching for tumor markers for early diagnosis of
cancer. The search for tumor specific antigens has been focused on proteins, carbohydrates, or
lipids produced by tumor cells; unfortunately these attempts have only identified molecules that
are over-expressed or ectopically expressed in tumors as compared with their normal cell
progenitors, thus generally limiting the specificity of these cancer markers and the early
detection of cancer [6]. To search for other biomarkers, research has shown promising
evidences using trained animals to detect scents in urine from bladder, prostate, and lung
cancer patients [7,8,9] indicating that volatile organic compounds (VOCs) may be differentially
produced by normal and cancer cells both in human and animal models. These findings were
further supported by detection of VOCs in the lung, breast, colorectal, and prostate cancer
patients breath and blood samples by gas chromatographymass spectrometry (GC/MS)
[10,11,12,13,14,15,16,17,18]. However, detection of VOCs in exhaled breath or blood is
technically challenging and invasive; therefore its applicability is limited. On the other hand,
urine samples can be easily collected and stored for long periods; and generally VOCs are
present at much higher concentrations in urine than in other body fluids [19]. Urine sample
collection is more patient friendly, and the samples are easier to be stored and analyzed as
compared to the procedures involved with breath samples. In addition, some disease-
associated VOCs released from urinary biochemical materials contain rich information about
individual physiological conditions and have been explored in cancer diagnosis [20,21,22].
In recent years, efforts were made to develop analytical methods for VOCs in biological
samples, such as blood and breath. The most successful technique in this field is Solid-phase
microextraction (SPME) coupled with GC/MS [5,23,24]. SPME was first introduced by Arthur
and Pawliszyn in 1990 [25] and has been successfully applied for extracting organic compounds
from various sample matrices [26,27,28,29], including human breath [30] and blood [31]. In this
project, we propose to use a similar technique, yet with higher extraction capacity and therefore
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better sensitivity [32], called Stir Bar Sorptive Extraction (SBSE) which was later developed in
1999 [33,34]. This methodology has been applied to analyze VOCs in environmental, food, and
biological analysis [35,36]. Using SBSE coupled with GC/MS, this project will develop an
innovative technique for early diagnosis of breast cancer using VOCs in urine. Optimized
analytical method for urinary VOCs will allow a potential implementation of point-of care cancer
screening. This study will identify the VOCs needed to be used for breast cancer screening. In
addition, we will study the pathways and the mechanisms for the differential expressions of
VOCs in breast cancer by cell and animal model for future development of breast cancer
treatment.
(b) INNOVATION
Studies indicate analysis of VOCs in breath can be successfully used for cancer diagnosis
[14-16] but very limited research had performed to use urinary VOCs as biomarkers for breast
cancer diagnosis. This project is to combine a solvent-less analytical method to extract,
characterize and measure the presence of VOCs in urine samples from nave-treatment breast
cancer patients of early and advanced stages, and healthy subjects. The SBSE technique
coupled with Thermal Desorption (TD)-GC/MS, is a fast, easy, and sensitive analytical method
which offers many advantages including the elimination of interference from the biological matrix
and polar compounds in the sample, no solvent use, and the use of small quantities of sample
(20 mL) for analysis [37]. This project could provide a quick and non-invasive tool for the
subjects involved in future cancer monitoring. It is also a relatively cost-effective screening
method for breast cancer detection. The knowledge obtained from this research has the
potential to expand the technique for other types of cancer, such as prostate and colon cancer.
Using VOCs as biological screeners for different forms of cancer could be a promising
technique but its application for breast cancer is understudied. Even less studies on the VOC
origins, biochemical pathways, and the mechanisms for their differential expressions are
available. Research questions remain open whether specific VOCs in urine appear at very early
stages of breast cancer or only at advanced stages of the disease during tumor decomposition.
This project will examine the application of urinary VOCs in breast cancer and enhance our
understanding of the mechanism of the VOC biomarker production.
(c) APPROACH
Specific Aim 1. Determine the urinary VOC profiles of
breast cancer patients. (Directed by the PI, Wen-Yee Lee)
The project will identify and measure the presence of
VOCs in urine samples from nave-treatment breast cancer
patients (female) and their age-matched healthy controls. A
brief experimental design is illustrated in Figure 1.
Subject recruitment: Urine samples from three groups will be
collected (1) healthy individuals (control), (2) early stages
breast cancer patients, and (3) advanced stages breast
cancer patients. A published study showed 6 metabolites
were found to be different between breast cancer and
control groups. The percent change for different metabolites
ranges from 29.1 to 934.2 [5]. Postulating similar change
from control to early stage and further early stage to
advanced stage and assuming maximum variation provided
a sample size of 17 each group (51). Thus, we propose to
include 51 subjects (17 per group) which will have more than
80% power to detect significant differences at the .01 level
of significance using one way analysis of variance. We
adjusted level of significance due to multiple comparisons.
Figure 1 A flow chart of the
experimental design for specific aim 1.
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The female subjects who are positively diagnosed of breast cancer with no history of prior
treatment will be recruited. Two cohorts of the same subject size and categories as
aforementioned will be recruited in year 1 and year 2 of the study respectively. Recruiting will
be held at the Sadie and Annabelle Garbar Breast Care Center (GBCC) supervised by our
collaborator, Dr. Zeina Nahleh, the chief of hematology-oncology at Texas Tech University
Health Sciences Center Paul L. Foster School of Medicine. At GBCC, more than 10 new breast
cancer patients are admitted on a monthly basis. The patient pool is much larger than our
proposed subject recruitment size. A questionnaire (included in the supplementary material) will
be developed to acquire basic information of the patients (such as age, family medical history,
weight and height, and medication.)
Urine Sample Collection: Following recruitment of the participants the urine samples (50 mL) will
be collected and stored at 4
o
C. Within 48 hours upon collection, the urine samples will be
analyzed for VOCs by SBSE and GC/MS.
Chemical Analysis: A 20mL urine sample will be adjusted to a pH of 3. An internal standard
(mirex) will be added to the sample to achieve a final internal standard concentration of 10 ng/L
in urine. A stir bar (Twisters
TM
, 10 mm 1 mm) will be added to the sample and stirred at 1000
rpm for two hours to extract VOCs from the urine. Upon completion, the stir bar will be removed
and thermally desorbed in a Thermal Desorption unit (TDU) under splitless mode. VOC
analyses will be performed on a 6890 GC system and a 5973 N Mass Selective Detector
(Agilent Technologies, Wilmington DE). A fused-silica capillary HP-5MS column (30 m, 0.25mm,
i.d. 0.25mm film thickness; Agilent) will be used for separation with helium as the carrier gas.
Identification of VOCs: Specific VOCs in the urine samples will be identified as potential
biomarker for early diagnosis of breast cancer. ChemStation software (Agilent Technologies,
U.S.A.) with NIST (National Institute of Standards and Technology) Mass Spectral Library will
be used to process the chromatograms and to identify VOCs which are unique in cancer
patients for disease discrimination. The identification of biomarkers will be categorized in three
groups: (1) Present and absent of any specific urinary VOCs in breast cancer patients as
compared with the healthy subjects; (2) atypical levels of any specific VOCs in urine of breast
cancer patients as compared with healthy controls; and (3) trend in the atypical levels of any
specific VOCs in early vs. late stage breast cancer patients.
Statistics: Data analysis will be performed using SAS 9.3 and Stata 12.1 by our collaborator Dr.
Alok Dwivedi, Assistant Professor, Division of Biostatistics and Epidemiology, TTUHSC, El Paso.
First, principal component analysis (PCA) will be performed to reduce the dimension of VOCs.
Significant differences among the groups will be assessed using a one-way analysis of variance
(ANOVA) followed by post hoc analysis. Bonferroni correction will be employed in post hoc
analysis. Data will be subjected to partial least-squares and discriminate analysis (PLS-DA) and
orthogonal partial least-squares and discriminate analysis (OPLS-DA), where a model is built
and used to identify the putative VOC markers with higher discriminatory power. The identified
VOCs combined and individual performance will be examined using receiver operating
characteristic (ROC) analysis.
Study limitations: There are three types of polymers coated on the stir bar: PDMS, polyacrylate,
and silicone, with PDMS being the most commonly used coating material. PDMS has the
capability to absorb hydrophobic compounds from aqueous solution. Therefore, this study may
be limited to analyze only hydrophobic urinary VOCs but not those of water soluble (hydrophilic)
components in the samples. We can use derivatization to convert hydrophilic organics to
hydrophobic compounds or use SPME (which has more coating fiber options) to compensate
the extraction of hydrophilic compounds.
Many VOCs from exogenous sources are detected in urine; these may be derived from
foods, personal care products, and medicines [19]. Some VOCs can be derived from both
endogenous and exogenous sources, while some VOCs in urine could be produced from
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mammalian metabolism of exogenous compounds. For example, dimethylamine is produced
endogenously and is also a metabolite of ingested foods [38]; 4-heptanone is an endogenous
metabolite of di-(2-ethylhexyl)-phthalate [39]. The occurrence of these diet-derived VOCs
varies between individuals. Therefore they can potentially complicate the data analysis.
Questions regarding food intake should be acquired during the subject interview. When
suspicious diet-derived VOCs are detected, a second sample from the individual will be
obtained within 7 days of the first sampling so that the diet-derived VOCs could be ruled out.
Specific Aim 2: Identify the pathway and mechanism of production of VOC biomarkers.
(Directed by the collaborator, Dr. Adarsh Kumar)
Though VOCs in breath, urine,
tissue and blood have been identified as
cancer biomarkers, little is understood
about the origins of many endogenous
VOCs. Study showed that protein
changes in malignant cells cause lipid
peroxidation in the cell membrane
during tumour growth [8]. Under
oxidative stress, polyunsaturated fatty
acids can undergo peroxidation
producing VOCs such as alkanes,
methylalkanes, aldehydes and ketones,
as shown in Figure 2 [40]. For instance,
hexanal, like other aldehydes, is a
biological VOC produced by lipid
peroxidation but it is also directly
cytotoxic to cells. Its imbalance between
cell injury and repair could lead to development of cancer [41]. 1-Octen-3-ol is an antioxidant
which can reduce aldehyde levels in blood so it is often linked to cell injury and oncogenesis.
Increased oxidative stress and induction of polymorphic cytochrome P-450 mixed oxidase
enzymes are often observed in breast cancer [14]. These two processes have been found to
affect the abundance of VOCs in the breath. We will conduct experiment to study the pathways
of production of atypical and abnormal levels of VOCs in urine and mammary tissues of the rats.
This will indicate the mechanism by which cancer specific VOCs develop and evolve in animals.
Animal model: A batch of 24 female SpragueDawley rats (weighed 190210 g) will be used for
the animal study at the Department of Toxicology, All India Institute of Medical Sciences, New
Delhi, India. The rats will be divided in 2 groups: control and experimental. DMBA (7,12-
dimethylbenz(a)anthracene), as the most favorable inducing agent in rats aged 50-65 days, will
be used for the induction of mammary cancer [42]. DMBA will be dissolved in arachis oil and
administered in 1 mL doses by gastric tubes fortnightly to the rats in the experimental group.
Control animals will be given 1 mL of arachis oil. The mammary tumor should begin to develop
in four weeks following the first feeding of DMBA and the feeding will continue 3-4 times per
week until autopsy. Throughout the experimental period, urine samples of the animals will be
collected on Day 1, 15, 30 (or after early cancer stage is developed), 45, 60 and 75 days (or
until the tumor is developed to a size of 20-40 mm, i.e. advanced stage). On Day 30 and 75,
animals from both groups will be sacrificed to get the liver cell and mammary tissue for the VOC
study which is described as follows. An experimental design is summarized in Figure 3.

Figure 2: Products generated by oxidation of
polyunsaturated fatty acids (40).
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Figure 3 An illustration of the experimental design for Specific Aim 2.

Collection of urine: The collection of urine will be done twice prior to the development of early
stage cancer (i.e. Day 1 and 15), and twice between the development of early and advanced
stage of cancer (Day 45 and 60). Two additional urine collections will take place when the early
and advance stage cancers are observed and that will be around 30 and 75 days respectively.
Urine samples will be collected from SpragueDawley rats overnight (12 h) in metabolism cages
in polypropylene tubes. After centrifugation at 4000 RPM for 10 min to remove residues, urine
samples were mixed and stored in aliquots at 10
o
C until use. Urine samples will be analyzed
for VOCs by GC/MS. The same statistical analysis mentioned in Specific Aim 1 will be applied
to study the VOC profiles in animal model.
Autopsy of the rats: The procedure [43] will be performed after 30 days to isolate the tumor cells
in mammary and liver tissues. Liver sections will be cut and minced in three portions of 2-3
grams. Hepatocellular carcinoma cells will be incubated in specially designed head-space glass
bottles sealed for 24 hours prior to measurements. Mammary tissues will be isolated and
incubated in glass bottles for 24 hours. Identification and quantification of VOCs released and
consumed by cells under study will be performed by GC/MS coupled with head-space needle
trap device extraction using an established method [44]. The VOCs will be used to study the
possible pathways and the mechanism of VOC production.
Study Limitation: The small sample size (6 in control and 10 in experimental group) of the
animal study could render only preliminary data. The results, nonetheless could enhance our
knowledge and understanding in the origins and biochemical pathways for the differential
expressions of VOCs in breast cancer.
Specific Aim 3: Optimize analytical methods for improving the biomarker detection using
solventless sample preparation techniques coupled with thermal desorption/gas
chromatography/mass spectrometry (GC/MS). (Directed the PI, Wen-Yee Lee)
Optimization of the analytical method will include but not limited to temperature, pH and
preservatives on the stability of VOCs in urine. Three temperature settings will be selected: 4
o
C,
25
o
C and 40
o
C to represent cold, room, and hot weather temperatures. The urine sample would
be stored at different temperatures accordingly and analyzed at different time periods to
establish the rate of degradation of VOCs in urine. This analysis will be performed at regular
intervals (i.e. every week) at each selected temperatures for a period of 2 months. Blanks (in
pure water), spiked controls (VOC spiked in pure water), and controls (urine of healthy subject)
will also be analyzed for VOCs along with the breast cancer subject samples.
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To study the effect of preservatives, acid, base, salt contents will be tested. The pH in urine
will be controlled at 4, 7, and 10 respectively; salt (NaCl) contents will be tested at 200 mg, 400
mg, 600 mg, and 1000 mg in 20-mL urine sample. The chemical analysis will be performed on
a GC/MS as aforementioned in Specific Aim 1.
Study Limitation: There are more than 75 VOCs existing in urine. They usually are
hydrocarbons, aldehydes, amines, and fatty acids. These compounds have different
hydrophobicity, and may respond to PDMS differently under the optimized condition. Multiple
optimized conditions may have to be developed for different chemical groups.
(d) Preliminary studies
Method Development for VOC analysis: The PIs group has tested the application of
SBSE/GC/MS for the urinary VOC analysis. A selection of 35 VOCs was used in method
optimization. The VOCs chosen were based on their hydrophobicity determined by their
octanol-water partition coefficient (Kow). The compounds included alkanes, alkenes, aromatics,
alcohols, aldehydes and ketones with a wide range of boiling points (80 - 242C) and Kow
values (0.35 8.54). The preliminary result showed that the SBSE-GC/MS could analyze a
broad range of VOCs qualitatively and quantitatively. The effect of acidifying urine showed that
the response and number of peaks increased significantly when the urine samples were in
acidic condition. This is because many of the VOCs are in their dissociated forms in urine so
when samples are acidified, they are protonated to their non-ionic form and they can be better
absorbed by the PDMS phase on the stir bar.
VOCs in Urine Samples. Twenty healthy female subjects have donated their urine samples for
testing. More than 60 VOCs were identified (Figure 4) and about 27 VOCs were commonly
found in urine of healthy females (Table 1). The VOCs found in the urine samples of the healthy
subjects were related to breast cancer (such as nonadecane, cyclooctane, 1-nonadecanol). The
results provided us a breath of VOCs that could be targeted in this proposed study.




Table 1 Common urinary VOCs detected in healthy female subjects.
N-benzyl-N-ethyl-p-
isopropylbenzamide
1-(1,5-dimethyl-4-hexenyl)-4-
methylbenzene
2-methyl-cycloheptanone
Bis(N-methoxyl-N-
methylamino)methane
1,6-dimethyl-4-(1-methylethyl)-
Naphthalene
2-methyl-5-(1-methylethenyl)-2-
cyclohexen-1-one
1,4-bis(1-methylethenyl)benzene 4-Heptanone 1-Nonadecanol
2-n-butyladamantane 2-Heptanone & 4-Nonanone Nonadecane
Cyclohexadecane Hexamethyl benzene Nonanal
Cyclooctane Menthol nonyl- cyclopropane
Cyclotetradecane 2-Mercapto-4-phenylthiazole (E)-5-Octadecene
2-Cyclohexen-1-one, 2-methyl-5-
(1-methylethenyl)-
1-(2-methoxyethoxy)butane
5-methyl-2-(1-
methylthyl)cyclohexanone
Decanal 1-Methyl-2-(1-methylethyl)benzene 1-Pentenyl benzene
Figure 4 A typical chromatogram of VOCs in urine.