Trial Information

K-RAS Oncogene Mutation in Patients With Advanced Non-Small Cell Lung Cancer Associated With Exposure to Wood Smoke and Tobacco Smoking: Therapeutic Implications

Background:

Lung cancer is the leading cause of cancer-related death among men and women worldwide (1).
In 2007, 213,380 new cases of lung cancer were registered; from these cases, 160,390 deaths
were reported. This number represents 15% of the total cases and 29% of cancer-related
deaths in the world. Only 13% of the patients with lung cancer survive for 5 years
[surveillance epidemiology and end result (seer) statistics http://seer. cancer.gov/],
therefore it still is a serious health problem (2). Clinically speaking, lung cancer is
divided into two main categories that cover small cell lung cancer and non-small cell lung
cancer (NSCLC). About 75% of all lung tumors are NSCLC, including squamous cell carcinoma,
adenocarcinoma, and large cell carcinoma (1). Despite the multimodal treatment using
chemotherapy, radiotherapy and surgery there is a poor prognosis for locally advanced NSCLC.
Survival rates for clinical stage IIIA is 64% at one year and between 23% and 30% after five
years; for clinical stage IIIB 32% and 3% after one and five years, respectively (3). While
advancements in the treatment of NSCLC have been made, few survival rate improvements have
been achieved.

Tobacco smoking is considered the main cause of NSCLC, but there are other risk factors as
well, such as the exposure to asbestos, radon, heavy metals, and wood smoke. The latter has
been described as a human carcinogen and an important risk factor for the development of
NSCLC. The frequency of exposure to wood smoke in patients with NSCLC is 28% (2). Current
data indicates than lung cancer associated with tobacco smoking and the lung cancer
associated with wood smoke exposure present different clinical characteristics, which
suggests that they might also have different genetic alterations, which are a consequence of
tumor etiology. Nevertheless, there are no significant molecular researches that make
possible to determine the differences in the pattern of mutations in oncogenes involved in
lung tumorigenesis, in relation to the risk factors accounted by the patient. Recent
advances in the knowledge of cancer biology have led to the identification of various
potential molecular targets that play a major role in the development and progression of the
disease. One of the most extensively researched signaling pathways involved in the cell
proliferation and survival is the one activated by the epidermal growth factor receptor
(EGFR). When the epidermal growth factor (EGF) binds to the extracellular domain of EGFR the
kinase property of the receptor is activated regulating the activation of different
signaling pathways. One of these pathways leads to the activation of K-RAS, which at the
same time activates the pathway of MAP kinase, involved in the proliferation,
differentiation, migration, and survival of the cell (these are fundamental events in
oncogenesis). Reports indicate that mutations in EGFR can be found in 51% of non-smoking
patients (4) and in 10% of smoking patients, while mutations in K-RAS occur in 30% to 50% of
the lung adenocarcinomas associated with tobacco.

Active mutations in K-RAS not only occur in NSCLC, but they also occur in >50% of the
colorectal adenocarcinomas, 75% of pancreatic tumors, 48% of lung cancer, and 44% of
adrenocortical tumors (5). These mutations seem to be an independent risk factor for the
prognosis of malignant tumors, since more than 90% of them occur at the codon 12 of the
K-RAS gene (6-8), and they are associated with lack of response to erlotinib (9), a
tyrosine-kinase inhibitor. Our research team has recently reported that wood smoke is an
independent factor for the survival and response to a treatment with erlotinib (10). This
information strongly supports the hypothesis that lung cancer tumorigenesis results from
different molecular mechanisms according to the smoker's stage (11) or other carcinogenic
factors associated with the appearance of the malignant neoplasia, such as wood smoke
exposure. Therefore, we believe that, compared to what has been reported in smoking
patients, wood smoke could cause a different frequency and pattern of mutations in the K-RAS
oncogene. It is of clinical relevance to determine whether the patients with NSCLC exposed
to wood smoke present any differences in the frequency and the type of mutations in the
K-RAS gene, with respect to both smoking and non-smoking patients.

Actually, a number of potential biological markers are being investigated with the aim of
selecting patients who will receive more specific therapies and thus achieve better results
regarding the response to treatment and overall survival. This is why the ability to detect
K-RAS mutations associated with risk factors for NSCLC could represent a starting point for
proposing different treatment approaches and benefiting the patients according to the
specific characteristics of each tumor.

General Objective:

To define the frequency and type of mutations at codon 12 of the K-RAS oncogene that occur
in patients with non-small cell lung cancer who have a background of wood smoke exposure or
tobacco smoking.

Materials and Methods:

The presence of mutations at the K-RAS oncogene will be evaluated retrospectively in 50
patients with advanced NSCLC who have a background of wood smoke exposure and 50 patients
with NSCLC who have a background of tobacco smoking. Additionally, 50 patients with NSCLC
with or without a background of exposure to any of these risk factors for lung cancer will
be prospectively evaluated. For the patients that will be prospectively studied, the
inclusion criteria will be as follows: patients diagnosed with advanced NSCLC stage IIIB/IV
who have not received previous chemotherapy, radiotherapy or both and who have tumor tissue
embedded in paraffin blocks or formalin-fixed; these patients must sign an informed consent
letter. The exclusion criteria will be: patients who refuse to participate in the study or
those who decide to withdraw from it, patients without tumor tissue or with a poor quality
sample. The tumor samples, both formalin-fixed or paraffin-embedded, used for the
histological diagnosis of patients will be obtained from the Pathology Departments of the
National Cancer Institute and the National Institute of Respiratory Diseases; these samples
will be used to gather DNA for the analysis of K-RAS mutations. The clinical data of the
patients will be obtained from their medical files.

DNA extraction:

Genomic DNA was extracted by standard procedure from areas of paraffin slides containing
>50% tumor using the QIAamp DNAFFPE Tissue Kit (QIAGEN), according to the manufacturer's
instructions.

Analysis of K-RAS oncogene mutations KRAS gene mutations from the samples were detected by
Therascreen RGQ PCR Kit (QUIAGEN, Scorpions ARMS method), according to the manufacturer's
instructions.

For the statistical analysis of results, continuous variables will be presented as
arithmetic means, medians, and standard deviations, while the categorical variables will be
presented as proportions and 95% confidence intervals. Inferential comparisons will be
performed by Student's t test and Mann-Whitney U test, according to the distribution of data
(normal and abnormal) determined by the Kolmogorov-Smirnov test. Either the chi square test
or the Fisher's exact test will be performed to evaluate significance among categorical
variables. Statistic significance will be determined as a p<0.05 value with a two- tailed
test. Both significant variables and variables with a borderline significance (p<0.1) will
be included in the multivariate logistic regression analysis. Comparisons between groups
will be carried out with the log-rank test.