Trial Title:
Gene-expression Profiles in CNS-metastatic Non-small Cell Lung Cancer
NCT ID:
NCT00862173
Condition:
Brain Disease
Metastasis
Lung Cancer
Conditions: Official terms:
Lung Neoplasms
Carcinoma, Non-Small-Cell Lung
Neoplasm Metastasis
Brain Diseases
Conditions: Keywords:
Gene-expression profile
Central nervous system metastasis
Non-small-cell lung cancer
Study type:
Observational
Overall status:
Unknown status
Study design:
Time perspective:
Prospective
Summary:
Non small-cell lung cancer (NSCLC) accounts 85% of all lung cancer.The development of
brain metastasis diminished life expectancy to less than one year with a median survival
of less than three months. In NSCLC cancer, approximately 50% of patients with locally
advanced disease develop brain metastasis at some time during the natural of disease. The
central nervous system constitutes the first site of recurrence in 15 to 40% of these
patients. Microarrays evaluate the diagnosis, treatment and prognosis of lung
cancer.There are no studies that specifically evaluate the relationship between a genetic
profile of NSCLC and metastasis to the CNS, with the purpose of distinguishing a subgroup
of patients that will benefit of prophylactic treatment.What is the association between a
genetic profile on NSCLC and the development of CNS metastasis.Obtaining a genetic
profile from the primary NSCLC tumor cells, by using microarrays, we can predict the
development of CNS metastasis arise a subgroup of patients that could benefit from
prophylactic cranial radiation with which their quality of life and prognosis most
probably will increase.Objective:Determine the association between a genetic profile from
the primary tumor cells and the development of central nervous system metastasis in
patients with non small-cell lung cancer.A genetic profile from the primary tumor cells
are associated with the development of central nervous system metastasis in patients with
NSCLC. A clinical, prospective, analytic, open, non randomized, prognostic and
observational cohort with 66 patients with NSCLC who authorize a biopsy study from
February, 2008 to December, 2012, INMEGEN institute will be in charge of performing the
microarrays and the computer analysis in order to obtain the different genetic profiles
that will be differentially expressed related with CNS metastasis risk profiles. Patients
will be followed-up by means of the external consult of lung neoplasms. The statistical
analysis will be performed using tests like Student's t or Mann-Whitney's U test. A
multivariate analysis of logistic regression will be performed. Global survival time will
be analyzed using Kaplan-Meier's technique and the comparison between groups will be
performed with log-rank test. The adjustment for potential confusors will be performed
using multivariate regression analysis. For result representation, we will use tables and
graphs and pertinent measures will be taken to disclose the study.
Detailed description:
Brain metastases occur in 30-50% of lung adenocarcinoma (LAC) patients and confer a worse
prognosis and quality of life. A better selection of in-risk patients through a more
accurate biomarker could improve the benefit of prophylactic therapies. The aim of this
prospective study was to determine a gene-expression profile of primary tumor associated
with brain metastasis (BM) and to evaluate the overall survival (OS) in patients with
advanced LAC.
Introduction.Lung cancer is the first cause of cancer death in the world. Eighty five
percent of patients are diagnosed yearly with non-small cell lung cancer (NSCLC). Despite
efforts, innovations, and progress in diagnosis and treatment of these patients, overall
survival (OS) at 5 years of diagnosis is only 15%. The central nervous system (CNS) is a
devastating and frequent site of metastasis development in NSCLC. The reported incidence
of CNS metastasis in patients with NSCLC is 54% with an OS of <1 year after diagnosis.
Age, clinical stage, gender, and initial treatment period are some of the reported with
CNS metastasis development-related factors in patients with NSCLC. We recently described
that high carcino-embryonic antigen (CEA) serum levels (>40 ng/dL) at diagnosis and
adenocarcinoma histological type are independently associated with higher risk if brain
metastasis development. However, due to their lack of specificity of all this reported
factors, we are required to detect biomarkers to predict brain metastasis in patients
with NSCLC with the objective to prevent their development.Metastasis process is complex
it involves well-defined molecular-printed steps, as invasion, vascular intravasation,
implantation and growing at the new specific organ. Higher motility, capacity for
extracellular matrix degradation, immune system evasion, and adhesion at the new specific
organ are some of the tumoral cell characteristics required to metastasize. In
particular, molecular events for brain metastasis development from primary NSCLC even if
no at al described, are currently well understood. Gene-expression microarray allow
analyzing the expression changes of thousands of genes simultaneously, distinguishing the
altered expression of neoplastic cell genes from normal tissue. Identification of
biological patterns, pharmacological molecular targets, and biomarkers for prognosis and
evaluation for therapeutic response, and even a more specific neoplasia classification
are some of the results of gene-expression microarray studies in hematological, breast
and NSCLC cancer. Determination of tobacco-smoke transcriptional changes in oncogenes and
antioncogenes had been determined by the use microarray data. A gene-expression profile
of 20 genes differentiates health lung tissue and lung cancer with higher specificity
than histopathologic evaluation. Furthermore, gene-expression microarray studies had been
developed for predicting survival and recurrence in early NSCLC stages for identification
of patients who could have benefit of adjuvant therapy, with promising result.The
objective of this study was to identify a gene-expression profile from primary lung
adenocarcinoma related with brain metastasis, and to evaluate in a prospective manner
their prognostic significance on survival in patients with advanced disease.Experimental
Design These study used clinical, longitudinal, prospective, observational, and
analytical cohort s with the selection of a nonprobabalistic-type sample.In a prospective
manner, from January 2009 to June 2011, patients admitted to our Institute confirmed
histologically confirmed stage IIIb and IV of LAC were eligible for study inclusion.
Analyzed clinical variables comprised smoking history, gender, age, general condition and
brain metastasis development. Primary tumor core-biopsy was performed prior to any
treatment and snap-frozen. A single pathologist evaluated all tumors. Standard
platinum-based chemotherapy was employed for all patients. All patients were submitted to
Magnetic resonance imaging (RM) to evidence the presence or absence of brain metastasis
(BM). During follow-up, we carried out Computed tomography (CT) of the brain stem for
this purpose. Preliminary statistical analysis was performed utilizing the Student t, the
Mann-Whitney U, the χ2, or the Fisher exact test. Once the study ends, we will conduct
logistic-regression multivariate analysis. Global survival time will be analyzed with the
Kaplan-Meier technique, and comparisons among groups will be performed with the log-rank
test. High Risk characteristics as gender, histology, and age related with greater
frequency of development of metastasis to CNS were analyzed.The study was carried out
according to the principles of ClinicalTrials and accepted by the INCan Bioethical and
Scientific Committees (reference numbers INCAN/CC/067/08). A collaboration agreement was
signed with the INMEGEN and was approved within the Health Research Sectorial Fund (FSIS)
CONACyT-México (SALUD-2009-01-115552).
Selection for obtaining tumor biopsies depended on the characteristics of the patient and
of the tumor. The biopsy could be taken by means of CT guided tru-cut needle, open
thoracoscopy, or bronchoscopy with optic fiber. Each tumor sample was divided into two;
one sample was analyzed by the INCan Pathology Service (by a sole observer blinded to the
clinical variables) for their histological clinical diagnosis and quantification of
neoplastic cellularity, and the remaining half was immediately stored at -80°C until
processing for RNA extraction.RNA Extraction:RNA was extracted from frozen tumor
biopsies, weighted and cryofractured in liquid nitrogen. Extraction and purification of
total RNA from tissue (up to 5 mg tissue) procedure was done using RNeasy Micro Kit
(QIAGEN, Germany) (cat. 217084). RNA was analyzed using the Agilent 6000 Chip (Agilent
Technologies, Santa Clara, C.A.). RNA quality consisted on the obtention of RNA Integrity
Number (RIN) > 8. RNA samples were chosen for microarray analysis when quality and
concentration were obtained for this purpose. Microarray Expression:We are using an
Affymetrix platform, which consists of an in situ synthesized oligonucleotide microarray.
The GeneChip® that we are employing is the Human Gene 1.0 ST Array, which allows
analyzing the expression of 28,869 different genes (each transcript is represented by 26
disperse probes along its entire length, present in the human genome, which covers 99% of
the sequences present in the RefSeq data bank of the GenBank. The Two-Cycle Target
Labeling protocol is followed as suggested by the manufacturer and described succinctly
as follows and shown graphically in the figure at the end of this Materials and Methods
section: Total RNA (100 ng/uL) is retro-transcribed using T7-Oligo(dT) Promoter primers
in the synthesis reaction of the first complementary DNA chain (cDNA). After treatment
with RNAase H, the second cDNA chain is synthesized, which will serve as template in the
Transcription reaction in vitro (TVI). The first TVI reaction is carried out with T7
polymerase RNA and unmarked ribonucleotides for the amplification of complementary RNA
(cRNA). These cRNAs are retro-transcribed in the synthesis step of the first cDNA chain
of the second cycle using random primers. Subsequently, T7-Oligo(dT)-Promoter primers are
employed for the synthesis of the second chain of cDNA, generating the double-helix cDNA
template that contains the promoter T7 sequence. This cDNA is amplified and marked in a
second TVI reaction using the T7 polymerase DNA and a mixture of
ribonucleotides/biotinylated nucleotide analog. These target biotinylated cDNAs are
cleaned, fragmented, and hybridized to the GeneChip® expression microarrays. After
hybridization, fluorescent marking was performed with a streptavidin-phycoerythrin and
biotinylated anti-streptavidin antibody amplifier system. Fluorescence is detected with a
high-resolution laser scanner.Reading and Analysis of Expression Microarrays:We will
utilize the Expression Console of Affymetrix software to evaluate the quality of the
microarrays and will correct the background signal with standard methods [49,50]. For
signal intensity-level normalization, we will employ "non-supervised" methods, which use
the data of all of the experiment's microarrays; this will allow us to render these
comparable among themselves, because what a microarray assay evaluates is gene expression
by means of fluorescence intensities; thus, it is important to establish cut-off points
from which we can consider under- or overexpression. Once the data are normalized, the
final stage is to resume the values of all the probes that exist for each gene in the
microarray in a sole value, which is the gene's expression level. For detection of
differentially expressed genes, we will construct a linear model that includes all of the
experiment's microarrays simultaneously; in this manner, we will be able to analyze
contrasts between group A (patients with metastasis to CNS during the follow-up period)
and group B (patients without metastasis to CNS during the same period). As internal
control, we will prove the differential expression of some genes by RT-PCR in real time.
Results will be represented in blocks of heat maps and in tables, in which the most
active genes are listed along with their functional description. For risk-profile
validation, we will perform leave-one-out cross validation method and will postulate a
second cohort for this objective.
Criteria for eligibility:
Study pop:
Patients with confirmed or suggested diagnosis of non-small-cell lung cancer, consulting
at the National Institutes of Cancerology or Respiratory Diseases (INCan, INER; Mexico
City, Mexico)
Sampling method:
Non-Probability Sample
Criteria:
Inclusion Criteria:
- Age > 18 years
- With auto-sufficiency
- Biopsy procedure approved
- Any TNM status
- Consulting at the National Institutes of Cancerology or Respiratory Diseases
- Informed consent signed
Exclusion Criteria:
- Comorbidity with other cancer type, hereditary or autoimmune diseases and treatment
with immunosuppressor drugs
- Previous treatment with adjuvant or neoadjuvant chemo- or radiotherapy
- No specimen availability
- No diagnosis of non-small-cell lung cancer at histopathology sample
Gender:
All
Minimum age:
18 Years
Maximum age:
N/A
Healthy volunteers:
No
Locations:
Facility:
Name:
Instituto Nacional de Cancerología
Address:
City:
Mexico City
Zip:
14080
Country:
Mexico
Status:
Recruiting
Contact:
Last name:
Oscar Arrieta, MD
Phone:
015255 56280400
Phone ext:
832
Email:
ogar@servidor.unam.mx
Investigator:
Last name:
David Saavedra-Pérez, MD
Email:
Principal Investigator
Facility:
Name:
Instituto Nacional de Enfermedades Respiratorias
Address:
City:
Mexico City
Zip:
14080
Country:
Mexico
Status:
Recruiting
Contact:
Last name:
Enrique Guzmán, MD
Phone:
015255 56664539
Phone ext:
5120
Email:
enriqueg@prodigy.net.mx
Investigator:
Last name:
Enrique Guzmán, MD
Email:
Sub-Investigator
Start date:
March 2008
Completion date:
December 2012
Lead sponsor:
Agency:
National Institute of Cancerología
Agency class:
Other
Source:
National Institute of Cancerología
Record processing date:
ClinicalTrials.gov processed this data on November 12, 2024
Source: ClinicalTrials.gov page:
https://clinicaltrials.gov/ct2/show/NCT00862173
