The Contribution of Induced Glycolysis to the Exhaled Volatile Organic Compounds' (VOCs) Signature in Lung Cancer
The Contribution of induced Glycolysis to the Exhaled Volatile Organic Compounds' (VOCs)
Signature in Lung Cancer
Lung cancer is infamous for its cruel mortality rates as it is the most lethal cancer in
both men and women. Unfortunately, most lung cancers are detected and diagnosed at a very
advanced stage and this complicates treatment options. Therefore, there is dire, unmet need
to develop an effective, highly sensitive, highly specific and non-invasive screening
method. The solution to this problem may be found in volatile organic compounds (VOCs) which
are organic molecules identified in exhaled breath. These VOCs differ between those
individuals who suffer from lung cancer and healthy individuals. Seeing as increased
glycolysis is one of the characteristic patterns of malignancies, the purpose of this study
is to determine the contribution of induced glycolysis to these exhaled VOCs. Therefore,
this study will compare exhaled breath samples in individuals who suffer from lung cancer
before and after a glucose challenge test. This study aims to better understand the
mechanisms involved in order to identify the distinguishing VOCs. This may aid in developing
the so necessary diagnostic means for lung cancer screening.
Lung cancer is the moth lethal cancer in both men and women.  There are two main
classes of lung cancer: the first, Small Cell Lung Cancer (SCLC) which is often
referred to as Oat Cell Cancer, and the second being Non-Small Cell Lung Cancer
(NSCLC), which constitutes the majority of cases of lung cancer.  Unfortunately,
most lung cancer detection and diagnosis is performed when the cancer is in an
advanced, metastatic stage in which surgery is not a viable treatment option.  Thus,
detection and diagnosis its early stages may significantly improve the five year
survival rate as well as provide the possibility of a cure.
An effective, non-invasive lung cancer screening method for distinguishing biological
markers found in high-risk groups has yet to be developed. Lately, researchers have
begun considering Volatile Organic Compounds' (VOCs) templates as a possible means for
early detection. These VOCs can be retrieved and isolated from exhaled breath samples.
The VOCs reflect the unique metabolic and biochemical activity in malignant cells.
This association has been proven in several researchers as well as in our own group 
who has shown that not only can VOCs distinguish between healthy and affected
individuals, but they can even discern between SCLC and NSCLC. [5, 6] The current
research will focus on the detection, identification and the characterization of these
unique biological markers found in the exhaled breath of those who suffer from lung
cancer. Amongst those VOCs which are detected, this research will specifically focus on
those VOCs which hold the potential to be incorporated into an effective, non-invasive
screening method for lung cancer.
Carcinogenesis is a process that is bound to somatic evolution. One of the most
popular evolutionary advantages found in malignant, metastatic cells is the increased
uptake of glucose through the process of glycolysis. This glycolysis process is unique
to malignant cell because it is most pronounced in anaerobic conditions, whereas
healthy cells most often perform glycolysis in aerobic conditions. Anaerobic
glycolysis- the process of the conversion of glucose to lactic acid in the presence of
oxygen- is also known as the "Warburg Effect" after Otto Warburg who discovered this
Thus, some of the distinguishing processes found in malignant cells are the exceeded
rate of substance exchange and increased glucose degradation (Warburg Effect). These
processes release organic compounds, some of which are released into exhaled breath.
Therefore, this research will examine the relationship between a glucose challenge test
and its effect on the pattern on VOCs in exhaled breath.
2. Research Goals:
The template for organic particles in exhaled breath discriminates between those
individuals who suffer from active lung cancer from those individuals who are
considered healthy. The purpose of this study is to examine the contribution of the
process of glycolysis to this unique template. Being that the process of glycolysis is
accelerated in malignant cells, we hypothesize that the glucose challenge test will
express VOC in those individuals who suffer from the disease. These findings can
contribute to the development of diagnostic tools for lung cancer.
We hypothesize that the Warburg Effect is an essential component in the creation of
VOCs that are unique to lung cancer, and therefore, that the glucose challenge test
will increase the production of these VOCs. These markers hold the potential to create
a more effective, non-invasive screening method with improved sensitivity and
4.1 Research Design: This research is an observational case-control study assesses the
comparison between the VOCs samples obtained from exhaled breath samples and blood tests
taken before and after a glucose challenge test. The glucose challenge test involves
drinking a solution which contains 75 grams of glucose and a waiting period of 90 minutes
between the collection of exhaled breath and blood samples. There is a need to fast for six
hours before this test. In addition, a clinical assessment will be performed based on the
medical anamnesis and on existing, current medical information.
4.2 Study Population: The goal of this research is to determine if the process of glycolysis
contributes to the unique VOC pattern in the exhaled breath of those who are affected with
lung cancer. The participants will be chosen from individuals that are currently being
treated in pulmonology clinics for various reasons. The control group will include
participants that have been identified as high-risk individuals for lung cancer, whereas the
study group will include participants who suffer from active lung cancer but have not yet
begun any form of medical treatment.
The recruitment process will be performed in two consecutive stages. In the first stage,
twenty patients from the study group will be compared to the twenty participants from the
control group. At this point, a preliminary assessment of the data will be performed to find
differences in the VOCs between these two groups. Only in the event that these differences
are found will the research go on to the second stage, in which 30 more participants will be
recruited and tested for the study group.
The control group as previously mentioned will include 20 participants that do not have lung
cancer, but are similar to the research group in terms of age, gender, medical history, and
history of smoking. This classification will be performed based off the self-report surveys
completed by the participants.
Exclusion criterions for the study population are as follows:
1. Patients that are uninterested to participant and/or unable to sign a consent form
2. Lung Cancer patients who have begun treatment prior to this research
3. Patients who are unable to complete this research and/or the follow up visits
4. Individuals that suffer from diabetes
4.3 Research Variables:
The main variables which will be used to test our hypothesis are as follows:
- Epidemiological variables of the participants
- Quantitative data defining the malignant cells ( TMN, the size of the tumor, number of
metastases and their location)
- The glucose levels( via a dipstick) before and after the glucose challenge test
(requires a fast of 6 hours prior to the test)
- The VOCs pattern obtained from the exhaled breath samples and the characterization of
these molecules via the GCMS method.
4.4 Research Methods:
A collection of the details of the patient's medical history will be obtained. Information
such as gender, age, medical history, consumption of tobacco and family history will be
noted. In addition, information which quantifies and qualifies the malignant cells (if
present) will be collected and recorded. This includes details such as the type of tumor as
well as its place, volume, histological characteristics and imaging results. Some of the
medical information will be obtained from pre-existing updated medical records.
Exhaled Breath Test:
Each participant will undergo an exhaled breath test before and after the glucose challenge
test with a lay period of 90 minutes in between the tests.
The detailed explanation regarding the process of collection of the exhaled breath samples:
The patient will exhale 3 breaths (with 750 ml in each exhaled breath) into appropriate
Mylar bags in a sterile area with technology that ensures that the exhaled breath is
isolated and unaffected from the external environment. The contents of the Mylar bags will
be stored in TENAX samples which preserves the samples for a period of six months. The
samples will undergo additional analysis at the Israel Institute of Technology (Technion)
under the auspices of Professor Haim Hussam. Each sample will be analyzed for the purposes
of identifying VOCs via the GC-MS method. The sample will undergo additional processing such
as SPME (pre-concentration into solid-phase micro extraction). The SPME will be transferred
to the GC-MS. The GC-MS method allows for the identification of the compounds in a short
period of time via the use of chromatographic and spectroscopic instruments. The
spectroscopic instrument will undergo additional analysis via the AOS instrument. The AOS
includes detectors which are conjugated to nano-particles of gold. Each detector identifies
a wide variety of VOCs in accordance to their location and concentration which cause varying
levels of resistance. This response is recorded and will undergo statistical analysis via
PCA to focus on the specific VOC patterns found in the sample.
The detailed explanation of the process of the glucose challenge test is as follows:
The participant is requested to drink a sugary solution (273 ml) constituted from 75 grams
of glucose and water. The test requires that the participant fast for a period of 6 hours
before the test. After drinking the solution, the participant must wait a lay period of 90
minutes before the retest of his or her exhaled breath. In addition, the level of glucose
will be tested by a dipstick before drinking the solution, and after the 90 minute lay
4.5 Statistical Analysis: The sample size is 70 persons ( 40 persons in the first stage, and
an additional 30 participants in the second stage as explained above).
The expectation is that there will be a significant difference between the VOC template
before and after the glucose challenge test. The estimated difference of effect is around
30%, with the assumption that the sampling error stands at around 10% and with a confidence
interval of 95%. The number of necessary participants needed in order to achieve results
with statistical significance and size effect ranges from approximately 20-25 persons.
The information from the AOS which recorded the changes in resistance will be analyzed via
PCA to focus on the specific VOC pattern changes obtained in each sample.
The remaining variables which were measured quantitatively will be presented in averages
with their corresponding standard deviations. Those variables that were obtained
quantitatively will be presented as percentages.
Statistical analysis for significance will be performed by the following statistical tests:
Student's T Test, Chi-Square, Analysis of Variance (ANOVA), and the Kruskal-Wallis test. All
statistical tests will be performed on the SPSS computer program.
Observational Model: Case Control, Time Perspective: Prospective
Diagnostic VOC signature for lung cancer
Each breath sample will be analyzed to identify VOCs via the GC-MS and Artificaial olfactory System AOS. We expect a significant difference between the VOC template before and after the glucose challenge test. The estimated difference of effect ~ 30%, with a sampling error ~ 10% and CI ~ 95% and sample size of 20-25 persons. The information from the AOS which recorded the changes in resistance will be analyzed via PCA to focus on the specific VOC pattern changes obtained in each sample. The remaining variables which were measured quantitatively will be presented in averages with their corresponding standard deviations. Those variables that were obtained quantitatively will be presented as percentages. Statistical analysis for significance will be performed by the following statistical tests: Student's T Test, Chi-Square, Analysis of Variance (ANOVA), and the Kruskal-Wallis test.
Nir Peled, MD PhD FCCP
Sheba Medical Center
Israel: Ministry of Health