Journal of Cancer Therapy, 2012, 3, 343-351
http://dx.doi.org/10.4236/jct.2012.324045 Published Online September 2012 (http://www.SciRP.org/journal/jct)
343
Detection of Gene Dosage in Circulating Free Plasma DNA
as Biomarker for Lung Cancer
Alba Mayerly Alvarez1, Sandra Janneth Perdomo Lara1,2, Diana M. Palacios3,4,
Edward Fabián Carrillo1,5, Luis Gerardo García Herreros3, Fidel Camacho Durán3,
Paulina Ojeda León6, Fabio A. Aristizábal1
1Departamento de Farmacia, Facultad de Ciencias, Universidad Nacional de Colombia, Bogotá, Colombia; 2Facultad de Odontología;
Unidad de Investigación Básica Oral (UIBO), Universidad el Bosque, Bogotá, Colombia; 3Departamento de Patología, Fundación
Santa Fe de Bogotá (FSFB), Bogotá, Colombia; 4Departamento de Patología, Facultad de Medicina, Universidad Nacional de Co-
lombia, Bogotá, Colombia; 5Instituto de Investigaciones Biomédicas, Universidad Libre, Cali, Colombia; 6Departamento de Pa-
tología, Hospital Santa Clara ESE, Bogotá, Colombia.
Email: faaristizabalg@unal.edu.co
Received July 10th, 2012; revised August 14th, 2012; accepted August 28th, 2012
ABSTRACT
The increase in the number of gene copies at specific loci is a genetic alteration frequently associated with over expres-
sion of the related protein in cancer cells. Genes whose dose is consistently augmented in cancer include those in-
volved in cell cycle control, proliferation, apoptosis, and angiogenesis among others. In this study, gene dose of onco-
genes MYCL1, MYCN, MYC, EGFR, ERBB2 and AKT2 in DNA obtained from lung tissue and blood plasma, of pa-
tients with primary lung cancer was evaluated with respect to normal lung tissue and plasma DNA of healthy individu-
als, to determine the capacity of these genes to discriminate normal and neoplastic phenotypes. The number of copies of
each gene was determined using real-time (2-∆∆CT). The AKT2 oncogene was found to be amplified frequently in
plasma DNA from patients (74% of cases). This marker showed a noticeable ability to discriminate normal and neo-
plastic phenotypes, with a 76% to 89% probability of correctly recognize a plasma sample provided by a lung cancer
patient or a healthy individual. For this reason, this detection could be a very useful tool to supplement the existing di-
agnostic methods in pulmonary cancer.
Keywords: Lung Cancer; Gene Amplification; Plasma; EGFR Family; MYC Family
1. Introduction
Lung cancer is the cancer with the highest incidence and
mortality worldwide. According to statistics from Glo-
bocan in 2008, 1.38 million people died from lung cancer,
corresponding to 18.2% of all cancer deaths [1]. This
mortality is due in large part because they are diagnosed
in advanced stages of the disease, where treatment strat-
egies are limited and the 5-year survival does not exceed
15% [2]. Therefore, one of the current goals of cancer
research is to generate diagnostic methods that allow
early detection of disease. The first changes experienced
by cells towards a neoplastic phenotype are at the mo-
lecular level [3], among them gene amplification is asso-
ciated with an over expression of the proteins involved.
Identifying these changes and molecular markers of can-
cer can allow for an early diagnosis and personalized
treatments that lead to a better prognosis for survival.
After identifying a molecular marker of cancer, it is im-
portant for the clinical practice to evaluate biological
samples obtained non-invasively. Blood plasma, serum,
and sputum have been proposed as suitable biological
fluids to assess molecular markers [4], since an increase
in the amount of free nucleic acids in these fluids have
been detected, and it has been shown that the origin of
this DNA is mainly from apoptotic and/or necrotic cells
derived from tissue that is undergoing carcinogenic trans-
formation [5]. In this study, the amplification status of
oncogenes MYC, MYCN, MYCL1, EGFR, ERBB2, and
AKT2 was evaluated with respect to the reference gene
β-actin, in DNA obtained from plasma and lung tissue of
lung cancer patients and healthy volunteers. Then the
amplification state of these genes was assessed in order
to verify if these genes could differentiate between a nor-
mal and cancerous phenotype, and if plasma was a suit-
able biological sample to detect molecular tumor charac-
teristics.
The MYC family genes (MYC, MYCN and MYCL1)
encode a number of transcription factors involved in
multiple cellular functions, including activating DNA
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Detection of Gene Dosage in Circulating Free Plasma DNA as Biomarker for Lung Cancer
344
synthesis and cell cycle progression [6]. The genes
EGFR, ERBB2, and AKT2 are involved in important
signaling pathways related to cellular functions such as
proliferation, apoptosis, and angiogenesis [7].
2. Materials and Methods
2.1. Samples
A total of 55 samples of blood plasma from patients di-
agnosed with primary lung cancer (PC) treated in the
Hospital Santa Clara and Fundación Santa Fe de Bogotá
between 2004-2009 were included. Of these 55 samples
of plasma, there were 27 paired samples that included
both the plasma sample and paraffin-embedded tumor
tissue (T) (Table 1).
A case-control study was conducted with blood sam-
ples, using 55 plasmas of healthy volunteers (PH), non-
smokers of the same-sex and age of the cancer patients
enrolled in the study. Additionally, 35 samples of normal
lung tissue (N), paraffin embedded, supplied by the De-
partment of Pathology, Hospital Santa Clara were in-
cluded.
2.2. Collection and Processing of Samples
Plasma: With the informed consent of the patients, 4 mL
of peripheral blood was collected in EDTA tubes. The
sample was centrifuged at 358 g for 10 minutes, and the
plasma was separated into tubes of 1.5 mL to be further
centrifuged at 2700 g for 10 minutes to eliminate conta-
mination by lymphocytes. The plasma obtained was
stored at –70˚C before DNA extraction.
Lung tissue: The cases obtained were reviewed by a
pathologist to confirm the diagnosis. In each paraffin
block, 3 consecutive histological cuts were performed
and then stained with H&E: a histological cut 3 μm thick
with delineated areas of interest in both neoplastic and
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