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|Title: ||Gene expression profiling of non-small cell lung cancer using cDNA microarrays|
|Other Titles: ||Li yong cDNA wei zhen lie jing pian ji shu yan jiu fei xiao xi bao fei ai de ji yin biao da pu|
利用 cDNA 微陣列晶片技術研究非小細胞肺癌的基因表達譜
|Authors: ||Au, Siu Kie (區兆基)|
|Department: ||Department of Biology and Chemistry|
|Degree: ||Doctor of Philosophy|
|Issue Date: ||2009|
|Publisher: ||City University of Hong Kong|
|Subjects: ||Gene expression.|
Lungs -- Cancer -- Chemotherapy.
|Notes: ||CityU Call Number: QH450 .A823 2009|
xviii, 147 leaves : ill. (some col.) 30 cm.
Thesis (Ph.D.)--City University of Hong Kong, 2009.
Includes bibliographical references (leaves 133-147)
|Abstract: ||In the Chinese communities, there is a high incidence of nonsmoking-
related lung cancer, almost invariably of adenocarcinoma (ADC)
histology. Recent evidence suggests that it is a distinct biological entity
different from lung cancer of other aetiologies. On the other hand,
squamous cell carcinoma (SCC) of lung is almost always associated with
In the current project, the mRNA expression profiles of ADC and
SCC samples taken from 84 patients undergoing surgery were studied in
relation to the histology, demographics, stage (lymph node metastases)
and EGFR mutation status. The 10K human clone set from Incyte
Genomics, Inc. (Palo Alto, California, USA) was used as targets in the
spotted cDNA microarray.
In phase I of the study, RNA were pooled from patients stratified
according to (1) smoking history (ever-smoker versus never-smoker) (2)
lymph node metastases (present or absent) (3) histology (ADC versus
SCC). Transcript profiling was done by cDNA microarray using the full
set of 10K targets. There were only 6 pools of RNA because SCC occurred
in smokers exclusively. The experiments were repeated in triplicate for
each pool of RNA. In Phase II of the study, based on the intensities of
gene expression and the differential expression between the strata, a
subset of 1385 genes were selected for the fabrication of a lower density
cDNA microarray for profiling of all 84 lung cancer individual samples
using normal lung parenchymal tissue from the same individual as
control. The data were filtered by pre-set quality criteria and normalized
by localized weighted regression (LOWESS).
There were 40 ADC (all never-smokers) and 44 SCC (22 current
and 22 ex-smokers) cases. EGFR mutations occurred in 24 ADC (60%)
but none in SCC. The gene expression profiles were significantly different
with respect to parameters including histology, EGFR mutation, gender,
lymph node metastases and age. According to pre-set criteria, 53 genes
and 17 genes were found to be differentially expressed between tumour
and normal lung parenchyma in SCC and ADC respectively. Sixteen
genes were differentially expressed between SCC and ADC. Unsupervised
clustering clearly segregated ADC from SCC and mutated EGFR from
wild-type EGFR tumours. By support vector machine (SVM) or K-nearest
neighbor (KNN) algorithms based on the 16 top significant genes with
respect to each corresponding parameter, the accuracies of correct class
prediction of histology, EGFR mutation status and gender were 82 - 87%,
54.5 - 70.1% and 67.8 - 75% respectively. The KEGG “cell
communication” pathway was the most significant pathway overlapping
with those differentially expressed genes in SCC. No significantly
overlapping pathway was identified for ADC, probably because of the
smaller number of genes with differential expression identified.
For validation of the microarray results, 11 genes (A2M, ADH1B,
CAV1, CCT5, CD74, CEACAM6, HIST1H2AE, HIST1H2BD, SFTPC,
SPTBN1 and TACSTD1) were selected (based on literature review of their
biological relevance to the pathogenesis of malignancies) for real-time
reverse-transcription PCR (qRT-PCR). The results between microarray
and qRT-PCR were consistent although statistical significance of
correlation was reached only for 6 out of the 11 genes.
DNA sequence analysis of exons 18-21 of the EGFR gene showed
that the most common mutations were deletion in exon 19 (16.7%) and
substitution L858R in exon 21 (66.7%). Most of the cases had a single
mutation (91.7%) and the incidence of double mutations was 8.3%.
According to pre-set criteria, 7 genes were found to be differentially
expressed between mutated and wild-type EGFR ADC cases.
Unsupervised clustering segregated distinct transcript expression profiles
between the 2 groups.
Because of its great potential of being a therapeutic target, CD74
and its ligand MIF were further studied by qRT-PCR and
immunohistochemisty (IHC) in 41 primary ADC, 46 primary SCC and 11
metastatic CA of the lung patients. By qRT-PCR, the transcript
expression level of CD74 was near normal in primary ADC but markedly
suppressed in both primary SCC and metastatic CA. On the other hand,
mRNA expression level of MIF in all 3 groups were markedly elevated. By
IHC, CD74 protein expression was strong in primary ADC but absent in
the other 2 groups. MIF protein expression was strong for all 3 groups.
Studies of blockade by siRNA in cell lines are in progress to elucidate the
functions of these two genes on tumour cell survival.
We have confirmed that there are distinct mRNA expression
profiles between ADC and SCC of lung, and between those ADC of
wild-type or mutated EGFR gene. CD74 and MIF are potentially
useful biomarkers for ADC of lung at both transcript and protein
levels. The differential expression of biomarkers in different
subgroups of lung cancer in cDNA microarray has been confirmed
by the qRT-PCR results. Further functional or larger clinicopathological
studies on the biomarkers discovered are warranted.|
|Online Catalog Link: ||http://lib.cityu.edu.hk/record=b2374949|
|Appears in Collections:||BCH - Doctor of Philosophy |
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