Breath test for breast cancer using the BREATHLINK*

1.Evidence for breath biomarkers of breast cancer
Clinical studies have shown that volatile biomarkers of breast cancer are
excreted in the breath. In a pilot study of volatile biomarkers of oxidative
stress a breath test for identified breast cancer with 93.8% sensitivity and
84.6% specificity6, 7. In a recent validation study8, volatile organic
compounds (VOCs) in the breath of 54 women with biopsy-proven breast cancer and
204 cancer-free controls were analyzed using automated thermal desorption/gas
chromatography/mass spectroscopy (ATD/GC/MS). Chromatograms were converted into
a series of datapoints by segmenting them into 900 time slices (8 s duration, 4
s overlap) and determiningtheir alveolar gradients (abundance in breath minus
abundance in ambient room air). MonteCarlo simulations identified time slices
with better than random accuracy as biomarkers ofbreast cancer by excluding
random identifiers. Patients were randomly allocated to trainingsets or test
sets in 2:1 data splits. In the training sets, time slices were ranked
according theirC-statistic values (area under curve of receiver operating
characteristic), and the top ten timeslices were combined in multivariate
algorithms that were cross-validated in the test sets. Monte Carlo simulations
identified an excess of correct over random time slices, consistent with
non-random biomarkers of breast cancer in the breath. The outcomes of ten
random data splits (mean (standard deviation)) in the training sets were
sensitivity = 78.5% (6.14), specificity = 88.3% (5.47), C-statistic = 0.89
(0.03) and in the test sets, sensitivity = 75.3% (7.22), specificity = 84.8
(9.97), C-statistic = 0.83 (0.06). These findings demonstrated that volatile
biomarkers in the breath identified breast cancer with accuracy comparable to a
film or digital mammogram.

2.Scientific basis of breath biomarkers in breast cancer
The time slices and VOCs identified as biomarkers were not unique to patients
with breast cancer, but were also observed in the cancer-free controls in
greater or lesser abundance. Previous studies of breath biomarkers of disease
yielded similar findings: in patients with lung cancer, breath biomarkers were
apparently generated by accelerated catabolism of normal metabolic products,
consistent with cancer-associated induction of cytochrome P450 enzymes. A
similar mechanism may account for the volatile biomarkers of breast cancer,
associated with altered metabolism of estrogen (figure 1). Estrogens promote
the proliferation of both normal and neoplastic breast epithelium cells, and
their role as breast carcinogens has been confirmed by epidemiological studies.
The carcinogenic role of estrogens is supported by the finding that aromatase
is expressed at a higher level in human breast cancer tissue than in normal
breast tissue. Aromatase (estrogen synthase) is the cytochrome P450 enzyme
complex that catalyzes estrogen production by converting C19 androgens to C18
estrogens. Other cytochrome P450 enzymes are also activated in breast cancer,
including CYP1A1, CYP1B1 and CYP3A4. Cytochromes P450 are hemoproteins encoded
by a superfamily of genes nearly ubiquitously distributed in different
organisms from all biological kingdoms. The reactions carried out by P450s are
extremely diverse and contribute to bioconversion of xenobiotics, alkanes,
terpenes and aromatic compounds. Since normal human metabolism generates a wide
variety of VOC products including alkane products of oxidative stress 16, the
induced cytochrome P450 activity associated with breast cancer may have
modulated the composition of VOCs excreted in the breath. In addition, a number
of the VOCs identified as candidate biomarkers of breast cancer included
alkanes (e.g. tridecane, dodecane) and methylated alkane derivatives, which are
products of oxidative stress produced by lipid peroxidation of polyunsaturated
fatty acids. Increased oxidative stress has been implicated as a risk factor in
women with breast cancer and increased breath pentane, another alkane, has been
reported in women with breast cancer

3.Clinical Perspective on breath testing
A brief history of breath testing: In the18th century, Lavoisier developed a
breath test for carbon dioxide in the breath. This was the first chemical probe
of metabolism, and it provided the first evidence that foodstuffs are oxidized
in the body. During the 19th century, colorimetric breath tests detected
ethanol in alcohol drinkers, and acetone in diabetics with ketoacidosis.
Radiolabeled drugs in the 20th century enabled new breath tests for digestive
disorders including H. pylori infection and pancreatic insufficiency. In 1971,
Linus Pauling reported a new analytical technique: microanalysis of volatile
organic compounds (VOCs) in the breath, and the remarkable discovery that
normal human breath contains a large number of different VOCs in very low
concentrations. He made this discovery by freezing breath VOCs in a tube
chilled with acetone and dry ice, then analyzing the concentrated sample using
the then-new technology of GC. Subsequent studies have shown that a sample of
human breath contains hundreds of different VOCs, mostly in picomolar (10-12 M)
concentrations.
Breath VOC assays in the laboratory:Laboratory-based assayshave been employed
for proof-of-principle studies, and biomarker discovery. Breath VOC samples are
collected at the clinical study with a breath collection apparatus (BCA):
Breath VOC samples are captured onto sorbent traps that are sealed hermetically
and sent to the laboratory for analysis by ATD/GC/MS (Figure 3). This
methodology has enabled the first-ever large multi-center clinical studies of
breath testing.
Breath biomarkers of oxidative stress: Power plants, biological or man-made,
commonly produce toxic byproducts. Mammalian life is sustained by the energy
produced in mitochondrial power plants which also convert oxygen to toxic and
potentially lethal byproducts. Oxygen is the final acceptor of electrons in
oxidative metabolism, but electron leakage from the mitochondria in the form of
reactive oxygen species (ROS) inflicts oxidative stress, a constant barrage of
oxidative damage to DNA, proteins, lipids and other biologically important
molecules27 (Figure 2). Oxidative stress has been implicated as a pathologic
mechanism in aging and several diseases, but it has proved difficult to measure
its intensity in vivo. Various markers of oxidative stress have been proposed,
including malonaldehyde and conjugated dienes in the blood, and alkanes and in
the breath. Increased breath alkanes, particularly ethane and pentane, have
demonstrated increased oxidative stress in breast cancer, rheumatoid arthritis,
heart transplant rejection, acute myocardial infarction, schizophrenia and
bronchial asthma.

To evaluate the BreathLinkTMin women with breast cancer and in cancer-free
controls, in order to identify their breath VOC profiles and to develop
multivariate predictive algorithms that can identify women with breast cancer.

Study Design
A 12 month multi-center, unblinded study in humans using breath assays to
evaluate the BreathLinkTM to determine breast cancer biomarkers

Procedures:
Patient Screening
a. Healthy subjects scheduled for a screening-mammogram (= group 1) or subjects
with an abnormal mammogram who've had a breast biopsy (= group 2) are screened
to ensure they satisfy the inclusion/exclusion criteria.
b. Informed Consent
c. Medical and medication history
d. Clinical Report Form

Breath Test
1 Breath test using the BreathLinkTM

Safety Variables:
BreathLinkTM: disposable mouthpiece and bacterial filter
List any Adverse Events that occur within 24 hours of breath sample collection.
Compliance with sample collection according to protocol, only by qualified
personel

Study Devices
BreathLinkTM

Duration of Subject Participation
The total time allocated to the study will not exceed 10 minutes.
* Breath tests in Group 1 (screening mammogram) will be performed prior to
screening mammography.
* Breath tests in Group 2 A (post-biopsy, abnormal mammograrm and benigne
biopt) will be performed after breast biopsy.
* Breath tests in Grooup 2B (post-biopsy, abnormal mammogram and breastcancer)
will be performed in the window between biopsy and start treatment.