1. MICROARRAYS AND
DATA ANALYSIS
FINAL PROJECT
Kiranmayee Bakshy
08/19/2014

2. Introduction
• Expression data from 46 cultured human ovarian
carcinoma cell lines with and without Cisplatin
treatment
• Array: A-AFFY-141 - Affymetrix GeneChip Human
Gene 1.0 ST Array [HuGene-1_0-st-v1] (GPL6244)
• Technology type: in situ oligonucleotide
• Experiment type: transcription profiling by array
• Samples: 171
• NCBI GEO accession no. GSE47856
(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GS
E47856)
Epithelial-mesenchymal status renders differential responses to cisplatin in ovarian cancer. Miow QH,
Tan TZ, Ye J, Lau JA, Yokomizo T, Thiery JP, Mori S. , Europe PMC 24858042

3. Background
• Chemo-resistance to platinum in anti-cancer drugs such
as cisplatin is critical in the treatment of cancer.
• Epithelial-mesenchymal transition (EMT) is linked with
the drug resistance as a contributing mechanism.
• The current study is designed to explore the
connection between cellular responses to cisplatin with
EMT in ovarian cancer.
• Expression microarrays were utilized to estimate the
EMT status as a binary phenotype
• Various bioassays such as cell number, proliferation
rate and apoptosis were conducted to quantify
phenotypic responses to Cisplatin treatment.

4. Data Analysis pipeline
Load raw CEL
files into R and
normalize using RMA
Outlier analysis (CV vs mean plot, Hierarchical
clustering dendrogram and Average correlation
plot)
Run statistical tests and fold change to select
differentially expressed genes
Dimensionality
reduction/clustering (PCA)
Classification (QDA)
Report top 5 up-
regulated and down-
regulated gene names
and their functions
Dataset:
Total no. of samples: 171
Total no. of probesets: 33297
Annotation classes:
Epithelial-like: 86 samples
Mesenchymal-like: 85 samples

5. Outlier analysis
Outlier-GSM1160845

6. Outlier-GSM1160845
Outlier analysis

7. Outlier-GSM1160845
Outlier analysis
 The outlier - GSM1160845 was removed from the data matrix
 13707 genes that have low expression values (mean < 5) were also
deleted

8. Statistical analysis
Student’s t-test and fold change
No. of probes with p-value:
< 0.05 9507
< 0.01 7074
< 0.05/no. of probes 2133
Linear fold change:
Min. -8.651798
Max. 33.05966
Threshold for selecting
differentially
expressed genes:
p-value < 0.05/no. of probes
and
fold change > log2(2)

9. Visualization of differentially
expressed genes
656 differentially expressed genes were selected from the analysis based
on the threshold

10. Dimensionality reduction of
differentially expressed genes
Around 50% of variability in data can be explained by the first two eigenfunctions
of PCA
Principle component analysis

11. Spectral k-means clustering of 50 random
differentially expressed genes
Spectral k-means clustering is useful in this case as the variability can be
best summarized in a few eigenfunctions.

12. Classification – Quadratic Discriminant Analysis
Epithelial
-like
Mesenchymal-
like
Epithelial-like 36 0
Mesenchymal-
like
0 34
Confusion matrix from QDA
predicted membership
Actualmembership
 Training set: 50 epithelial-like
50 mesenchymal-like
 Test set: 36 epithelial-like
34 mesenchymal-like
 QDA was performed on the first three
components of principle component
analysis of training set.
QDA predicted all the samples of the test set correctly

13. AFFYMETRIX_EX
ON_GENE_ID
GENE NAME
GENE
SYMBOL
FUNCTION
8102792 protocadherin 18
PCDH18
Potential calcium-dependent cell-
adhesion protein
7899167
lin-28 homolog(C.
elegans)
LIN28A
Acts as a 'translational enhancer',
driving specific mRNAs to polysomes
and thus increasing the efficiency
of protein synthesis
7906878
discoidin domain
receptor tyrosine
kinase-2
DDR2
This tyrosine kinase receptor for
fibrillar collagen mediates
fibroblast migration and
proliferation
7906900*
discoidin domain
receptor tyrosine
kinase 2
DDR2
This tyrosine kinase receptor for
fibrillar collagen mediates
fibroblast migration and
proliferation
7926368 vimentin
VIM
class-III intermediate filaments
found in mesenchymal cells
DAVID functional annotations of top 5 discriminant genes
(Negative)
* Unmapped in DAVID; information obtained from NetAffx
No pathways or GO information was suggested by DAVID.

14. AFFYMETRIX_EX
ON_GENE_ID
GENE NAME
GENE
SYMBOL
FUNCTION
8026490
urothelial cancer
associated-1
UCA1 role in bladder cancer progression and
embryonic development
8041853
epithelial cell
adhesion molecule
EpCAM
carcinoma-associated antigen EpCAM up
regulates c-myc and induces cell
proliferation
8098439
epithelial cell
adhesion molecule
EpCAM
carcinoma-associated antigen EpCAM up
regulates c-myc and induces cell
proliferation
8147351
mal, T-cell
differentiation
protein-2
MAL-2 Member of the machinery of polarized
transport
8148040
epithelial splicing
regulatory protein-
1
Esrp-1
mRNA splicing factor that regulates the
formation of epithelial cell-specific
isoforms
DAVID functional annotations of top 5 discriminant genes
(Positive)
No pathways or GO information was suggested by DAVID.

15. Conclusions:
• The outlier observed in this dataset was GSM1160845 which is
a mesenchymal-like ovarian cancer cell line treated with
Cisplatin.
• 656 out of 19590 genes were selected as differentially
expressed genes based on the threshold.
• The QDA classification model trained using 100 samples
predicted the classes of test set with 70 samples successfully.
• All the top 5 positively and negatively regulated genes obtained
in this analysis are involved in cellular processes such as cell
adhesion, migration, proliferation and protein synthesis.
• The authors have reported an epithelial gene set consisting of
known epithelial cell markers such as DDR1, KRT8, KRT18,
CDH1, CDH3, CLDN3, CLDN4 and EPCAM, and a mesenchymal
gene set consisting of known mesenchymal cell markers ZEB1,
CDH2, VIM and TWIST1.