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Setia Pramana 1
The Role of Statisticians in Personalized Medicine: An Overview of Statistical Methods in Bioinformatics
Setia Pramana
STISJakarta, 8 August 2014
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Setia Pramana 2
Outline• Drug Development• Personalized Medicine• Central Dogma• Microarray Data Analysis• Next Generation Sequencing• Summary
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Drug Developments• Takes 10-15 years• Cost millions USD• Who: Pharmaceutical, biotechnology, device companies,
Universities and government research agencies• Regulatory: The US Food and Drug Administration, BP POM• Evaluate:
– Safety – can people take it?– Efficacy – does it do anything in humans?– Effectiveness – is it better or at least as good as what is
currently available?– Do the benefits outweigh the risks?
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Drug Development• The Stages:
- Drug Discovery- Pre-clinical Development- Clinical Development 4 Phases
• Statisticians are involved in all stages• Stages are highly regulated• Result is based on most of patients• But .. Patients are created differently!
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Setia Pramana 5
Patients Heterogeneity
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Patients Heterogeneity• We’re all different in
- Physiological, demographic characteristics- Medical history- Genetic/genomic characteristics
• What works for a patient with one set of characteristics might not work for another!
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Patients Heterogeneity• “One size does not fit all”• Use a patient’s characteristics to determine best
treatment for him/her• Genomic information is a great potential
-- > Personalized medicine:“The right treatment for the right patient at the right
time”
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Personalized Medicine
• The ability to determine an individual's unique molecular characteristics and to use those genetic distinctions to diagnose more finely an individual's disease, select treatments that increase the chances of a successful outcome and reduce possible adverse reactions.
• Personalized medicine also is the ability to predict an individual's susceptibility to diseases and thus to try to shape steps that may help avoid or reduce the extent to which an individual will experience a disease
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Subgroup Identification and Targeted Treatment
• Determine subgroups of patients who share certain characteristics and would get better on a particular treatment
• Discover biomarkers which can identify the subgroup• Focus on finding and treating a subgroup
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Subgroup Identification and Targeted Treatment
Genotype Phenotype Intervention Outcome
Mutations/SNPGene/Protein ExpressionEpigenetics
DiseasesDisabilityEtc.
DrugsTherapiesRegimes
Personalized medicine
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Advanced Biomedical Technologies• High-throughput microarrays and molecular imaging
to monitor SNPs, gene and protein expressions• Next-Generation Sequencing
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First…. Bit Biology
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Central Dogma
http://compbio.pbworks.comSetia Pramana
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Gene• The full DNA sequence of an organism is called its
genome• A gene is a segment that specifies the sequence of
one or more protein.
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Genomics • The study of all the genes of a cell, or tissue, at :– the DNA (genotype), e.g., GWAS SNP, CNV etc…– mRNA (transcriptomics), Gene expression,– or protein levels (proteomics).
• Functional Genomics: study the functionality of specific genes, their relations to diseases, their associated proteins and their participation in biological processes.
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Setia Pramana 16
Microarrays
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Microarray
• DNA microarrays are biotechnologies which allow the monitoring of expression of thousand genes.
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Applications• High efficacy and low/no side effect drug• Genes related disease.• Biological discovery– new and better molecular diagnostics– new molecular targets for therapy– finding and refining biological pathways
• Molecular diagnosis of leukemia, breast cancer, etc.• Appropriate treatment for genetic signature• Potential new drug targets
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Microarray
Overview of the process of generating high throughput gene expression data using microarrays.
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Pipeline• Experiment design Lab work Image processing • Signal summarization (RMA, GCRMA)• Normalization • Data Analysis:
– Differentially Expressed genes– Clustering– Classification– Etc.
• Network / Pathways (GSEA etc..) • Biological interpretations
Setia Pramana
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Microarray Data Structure
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Preprocessed DataGenes C1 C2 C3 T1 T2 T3
G8522 6.78 6.55 6.37 6.89 6.78 6.92G8523 6.52 6.61 6.72 6.51 6.59 6.46G8524 5.67 5.69 5.88 7.43 7.16 7.31G8525 5.64 5.91 5.61 7.41 7.49 7.41G8526 4.63 4.85 5.72 5.71 5.47 5.79G8528 7.81 7.58 7.24 7.79 7.38 8.60G8529 4.26 4.20 4.82 3.11 4.94 3.08G8530 7.36 7.45 7.31 7.46 7.53 7.35G8531 5.30 5.36 5.70 5.41 5.73 5.77G8532 5.84 5.48 5.93 5.84 5.73 5.75
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Challenges• Mega data, difficult to visualize• Too few records (columns/samples), usually < 100 • Too many rows(genes), usually > 10,000• Too many genes likely leading to False positives• For exploration, a large set of all relevant genes is
desired• For diagnostics or identification of therapeutic
targets, the smallest set of genes is needed• Model needs to be explainable to biologists
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Type of Microarray Data Analysis
• Gene Selection–find genes for therapeutic targets
• Classification (Supervised)– identify disease (biomarker study)–predict outcome / select best treatment
• Clustering (Unsupervised)–find new biological classes / refine existing ones–Understanding regulatory relationship/pathway–exploration
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Gene Selection• Modified t-test• Significance Analysis of Microarray (SAM)• Limma (Linear model for microarrays )• Linear Mixed model• Logistics Regression• Lasso (least absolute selection and shrinkage operator)• Elastic-net• Etc,
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Visualization• Dimensionality reduction• PCA (Principal Component Analysis)• Biplot• Heatmap• Multi dimensional scaling• Etc
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Clustering• Cluster the genes• Cluster the
arrays/conditions• Cluster both simultaneously
• K-means• Hierarchical• Biclustering algorithms
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Clustering
• Cluster or Classify genes according to tumors
• Cluster tumors according to genes
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Classification• Linear Discriminant Analysis• K nearest Neighbor• Logistic regression• L1 Penalized Logistic Regression• Neural Network• Support Vector Machines• Random forest• etc
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Aim: To improve understanding of host protein profiles during disease progression especially in
children.
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Classification of Malaria Subtypes
•Identify panel of proteins which could distinguish between different subtypes.•Implement L1-penalized logistic regression
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Penalized Logistic Regression
•Logistic regression is a supervised method for binary or multi-class classification.•In high-dimensional data (e.g., microarray): More variables than the observations Classical logistic regression does not work.•Other problems: Variables are correlated (multicolinierity) and over fitting.•Solution: Introduce a penalty for complexity in the model.
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Penalized Logistic RegressionLogistic model:
Maximize the log-likelihood:
•-Penalization (Lasso):
•
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• Shrinks all regression coefficients () toward zero and set some of them to zero.
• Performs parameter estimation and variable selection at the same time.
• The choice of λ is crucial and chosen via k-fold cross-validation procedure.
• The procedure is implemented in an R package called penalized.
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L1 Penalized Logistic Regression
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Classification of Severe Malaria Anemia vs. Uncomplicated Malaria group
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AUC: 0.86
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Dose-response Microarray Studies
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Dose-response Microarray Studies
Implemented in R package IsoGene and IsoGeneGUI.
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Dose-response Microarray Studies
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Setia Pramana 42
Gene Signature for Prostate Cancer
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Gene Signature for Prostate Cancer
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Gene Signature for Prostate Cancer
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Setia Pramana 45
Next Generation Sequencing
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Next Generation Sequencing
Reading the order of bases of DNA fragments
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NGS used for:• Whole genome re-sequencing• Metagenomics• Cancer genomics• Exome sequencing (targeted)• RNA-sequencing• Chip-seq• Genomic Epidemiology
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Next Generation Sequencing
• Produce Massive Data and fast• Problem is storage and analysis
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RNA-seq Pipeline
• Align to a reference genome using Tophat.
Reference
Pramana, et.al 50NBBC 2013Source: Trapnell et.al, 2010
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RNA-seq Pipeline
• Measure gene expression using Cufflinks: FPKM (Fragments Per Kilobase of transcript per Million mapped reads).
Reference Gene
Transcript 2Transcript 1
Isoform/Transcript FPKM
Gene FPKM
Sample 1
Sample 2
Sample 3
Pramana, et.al 51NBBC 2013 Source: Trapnell et.al, 2013
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Setia Pramana 52
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Subtype-specific Transcripts/Isoforms• Breast invasive carcinoma (BRCA) from the Cancer
Genome Atlas Project (TCGA).• 329 tumor samples.• Platform: illumina• Paired-end reads (length 50 bp).• 20 -100 million reads
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Subtype-specific Transcripts/Isoforms• To discover transcripts/isoforms which are only
significantly (high/low) expressed in a certain cancer subtype.
Pramana, et.al 54NBBC 2013
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Analysis Flow329 samples TCGA
Discovery set179 samples
Validation set- TCGA 150 samples- External samples
Classification to mol-subtypes- Use Swedish microarray data as
training data.- Based on gene level FPKM- Median and variance normalization- K-nearest neighbor- Classifier genes selection
Subtype-specific Transcript- Transcript level FPKM of all
genes- For each transcript: Robust
contrast tests.- Multiple testing adjustment.
Pramana, et.al 55NBBC 2013
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Subtype-specific Transcripts/Isoforms
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Subtype-specific Transcripts/Isoforms
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Subtype-specific Transcripts/Isoforms
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Software?• R now is growing, especially in bioinformatics– Statistics, data analysis, machine learning– Free– High Quality– Open Source– Extendable (you can submit and publish your own package!!)– Can be integrated with other languages (C/C++, Java, Python)– Large active user community– Command-based (-)
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My Current Research• Integration of Somatic Mutation, Expression and Functional
Data Reveals Potential Driver Genes Predictive of Breast Cancer Survival (KI, Ewha Univ, Brescia Univ).
• Molecular Subtyping of Breast Cancers using RNA-Sequence Data (KI, Ewha Univ, Brescia Univ).
• The genomic surveillance of drug-resistant tuberculosis (FKUI, NUS).
• Genomics screening for prostate cancer (KI)• Molecular subtyping of Malaria (KI, Scilab, Eijkman Inst.)• Health Technology Assessment (FKUI, Depkes)
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Summary• Statistics plays important roles in developing
personalized medicine• Multidisciplinary field need collaboration with
different experts. • Bioinformaticians is one of the sexiest job• Big Data in Medicine: Numerous opportunities to be
explored and discovered.
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Thank you for your attention….