1. Advancing Precision Medicine:
MicroRNA Prognostic Signatures and
Prediction Models for Breast Cancer
By Natalie Ng
Ingenuity Systems and Stanford University, School of Medicine

2. Motivation
● In the United States alone, there are over 500 new cases of breast cancer
and more than 100 breast cancer related deaths each day.
● One in every eight women will develop breast cancer in their lifetime.
● Today, 85-90% of breast cancer patients are given chemotherapy, while
only 30% need it and benefit from it. The other 70% experience the
unnecessary, severe side-effects of chemotherapy.
Goals
1. Develop a tool that advances precision medicine by enabling doctors
to predict which patients will develop cancer recurrence or
metastasis, evaluate potential benefits, and screen candidates for
chemotherapy.
2. Design the tool to have maximum accessibility in the clinic, by using
as few biomarkers as possible without sacrificing predictability.

3. Research Outline
1. Phase 1: Create prognostic models predictive of distant
metastasis-free survival (time to metastasis) for breast cancer
patients
a. Use microRNAs (miRNA) instead of traditional protein coding
genes.
b. Integrate both miRNA and mRNA data, to increase
predictability by drawing from two sets of genomic information
c. Computationally validate the signatures.
2. Phase 2: Experimentally validate the prognostic signature in vitro
a. Measure miRNA expressions and metastatic characteristics
(migration, invasion, proliferation) in breast cancer cell lines.
b. Determine if there is a correlation between metastatic
characteristics and miRNA expressions.

4. Why miRNAs?
● What are miRNAs?
○ Small, conserved RNA, 19-25 nucleotides long
○ Bind to both DNA and RNA to inhibit both transcription and
translation
● I hypothesize that miRNAs can provide better prediction with
fewer biomarkers.
○ miRNAs act as regulators of networks in cells
○ One miRNA can bind to as many as 200 targets. The
dysregulation of even one miRNA can change the phenotype
of cells in drastic ways.
● 50% of miRNAs originate from unstable regions of the genome
that are involved in cancer tumorigenesis.

5. Dataset Selection
Phase 1: In Silico Discovery
● GSE22220: Superseries dataset that comprises of linked mRNA
and miRNA expression profiles of 207 breast cancer patients.
○ Has both ER+ and ER- patients
● Survival Curves generated using Kaplan
Meier analysis
○ Represent percentage of patients who
are metastasis-free after a given
number of years
● ER- has a significantly poorer prognosis
than ER+
Create two signatures,
one for ER+ and
another for ER-

6. In Silico Discovery Workflow
● Goal: to select miRNAs that are not only differentially expressed
but also able to regulate downstream mRNAs
First demonstration of integrating mRNA and miRNA data through a knowledge based tool.
Phase 1: In Silico Discovery

7. ER+ Output
Phase 1: In Silico Discovery
● Differentially expressed mRNAs used to predict miRNAs.
● Confidence Settings: Experimental and Highly Predicted

8. ER- Output
Phase 1: In Silico Discovery
● Differentially expressed mRNAs used to predict miRNAs.
● Confidence Settings: Experimental and Highly Predicted

9. Phase 1: In Silico Discovery
Cox Regression & Model Selection
● Two Strategies of Cox Regression
○ Forward Step Wise Selection: Employs a combination of
univariate and multivariate analysis.
○ Penalized Cox Regression (L1, L2, L1+L2): Used to create
more parsimonious models and tune parameters.
● Model Selection based on two criteria
○ Maximize Area Under Curve (AUC) of a Receiver Operating
Characteristic (ROC)
○ Most parsimonious model
What does an ROC Curve Represent?
● Every test is a balance of sensitivity and
specificity.
● Only in an ideal test is the area under an
ROC curve 1
● Goal of model fitting is to maximize the
area under the ROC curve.

10. Phase 1: In Silico Discovery
Cox Regression & Model Selection
● Two Strategies of Cox Regression
○ Forward Step Wise Selection: Employs a combination of
univariate and multivariate analysis.
○ Penalized Cox Regression (L1, L2, L1+L2): Used to create
more parsimonious models and tune parameters.
● Model Selection based on two criteria
○ Maximize Area Under Curve (AUC) of a Receiver Operating
Characteristic (ROC)
○ Most parsimonious model
What does an ROC Curve Represent?
● Every test is a balance of sensitivity and
specificity.
● Only in an ideal test is the area under an
ROC curve 1
● Goal of model fitting is to maximize the
area under the ROC curve.
Formulations of Area Under Curve (AUC)
● L1 Penalized Cox Regression was selected because the models
maximized the area under the ROC curve (shown by AUC-WGE and
AUC-RS) and were the most parsimonious (fewest covariates).

11. ER+ Model
Clear separation
between a high
risk and low risk
group for
metastasis
Coefficient for Cox Regression: The larger the Coefficient,
the more that particular miRNA drives the model.
Phase 1: In Silico Discovery

12. ER- Model
Also, a clear
separation
between a high
risk and low risk
group for
metastasis
Coefficient for Cox Regression: The larger the Coefficient,
the more that particular miRNA drives the model.
Phase 1: In Silico Discovery

13. Computational Validation
Phase 1: In Silico Discovery
● Models were validated with independent patient samples by
computing the Area Under Curve (AUC) of a ROC curve
Models still showed predictive value when extended to new data,
especially the prognostic model for ER- breast cancer.

14. Network Maps
Phase 1: In Silico Discovery
Many of the downstream targets are related to metastasis.
This confirms the validity of the proposed workflow. Not only are the miRNAs
differentially expressed, but they are also able to create regulated expression
in downstream mRNAs related to metasasis.

15. Network Maps
Phase 1: In Silico Discovery
Many of the downstream targets are related to metastasis.
This confirms the validity of the proposed workflow. Not only are the miRNAs
differentially expressed, but they are also able to create regulated expression
in downstream mRNAs related to metasasis.

16. Experimental Validation In Vitro
Phase 2: In Vitro Validation
● Determine the correlation between miRNA expressions and in vitro
metastatic characteristics
○ For now, I focused on the ER- model because it is the more invasive
form of breast cancer.
● Cell Lines that represented a range of metastatic potential based on
prior publications were selected. MCF10, non-malignant cells, were used
as a control.

17. Experimental Design
Phase 2: In Vitro Validation
1. Measure metastatic characteristics (migration, invasion, proliferation) in vitro.
2. Measure miRNA expression using qPCR.
3. Determine if miRNA expressions correlate to in vitro metastatic characteristics.

18. Migration and Invasion
Phase 2: In Vitro Validation
MDA-231 had the greatest ability to
migrate and invade, with a response
specific to the chemoattractant.
All experiments repeated twice.
Transwell Assay Results
● Over time, starved cells migrate through the
transwell towards the chemoattractant.
● Greater the number of cells below the transwell,
the greater the migration/invasion.
● In the invasion assay, transwell is coated with
matrigel, an analog of the extra-cellular matrix.

19. Proliferation
Phase 2: In Vitro Validation
All experiments repeated twice.
● Yellow MTT is reduced to Purple Formazan in
dividing and viable cells by mitochondrial
reductace.
● The greater the absorbance at 540 nm, which
corresponds to the color of purple formazan, the
greater the proliferation.
MTT Assay Results
Among the cancer cell lines, SKBR-3, followed by MDA-231 and MDA-
436 had abilities to proliferate.

20. miRNA Expression Profiling
Phase 2: In Vitro Validation
● Of the 12 miRNAs, 5 were detected in significant amounts.
● The reason that not more were detected is that cell lines are not comprehensive
representations of actual tumors, which have a much wider variety of cell types.
All experiments repeated twice.

21. Correlation
Phase 2: In Vitro Validation
In the highly metastatic cell line MDA-231, 4 of the 5
detectable miRNAs match model prediction.
Indicates that model prediction holds up experimentally.

22. Major Accomplishments
I developed a novel in silico discovery
workflow to identify a unique
combination of miRNAs that can be
used to predict metastasis.
I validated the prognostic signatures in
vitro. Correlation was observed
between miRNA expressions in the
ER- signature and in vitro metastatic
characteristics, indicating that model
prediction holds up experimentally.
1
2

23. Further Research
1. Could miR-210 be an independent indicator of metastasis? miR-210 is
the only difference between MDA-231 and MDA-453. I will investigate the
effects of miR-210 upregulation and knockdown through lentiviral infection.
2. Perform validation using in vivo models.
3. Invesitage pathways regulated by the miRNAs to identify their mechanism
of action.

24. Acknowledgements
● Chen Lab, Stanford University (supported experimental
studies by providing laboratory access and training)
○ Professor Chang-Zheng Chen
○ Dr. Rita Fragoso
● Ingenuity Systems (provided summer internship and
computational resources)
○ Dr. Stuart Tugendreich
○ Dr. Debra Toburen
I conducted this independent research from June 2012 to April 2013. Part of
this research has been presented at the 2012 Personal Genome and Medical
Genomics Meeting, Cold Spring Harbor Laboratory.