Molecular profiling 2012

Next Generation Molecular Profiling

Lab for Bioinformatics and
computational genomics
10 “genome hackers”
mostly engineers (statistics)

42 scientists
technicians, geneticists, clinicians

>100 people
hardware engineers,
mathematicians, molecular biologists

Overview

Personalized Medicine,
Biomarkers …
… Molecular Profiling

First Generation Molecular Profiling
Next Generation Molecular Profiling
Next Generation Epigenetic Profiling

Concluding Remarks

Personalized Medicine
• The use of diagnostic tests (aka biomarkers) to identify in advance
which patients are likely to respond well to a therapy
• The benefits of this approach are to
– avoid adverse drug reactions
– improve efficacy
– adjust the dose to suit the patient
– differentiate a product in a competitive market
– meet future legal or regulatory requirements
• Potential uses of biomarkers
– Risk assessment
– Initial/early detection
– Prognosis
– Prediction/therapy selection
– Response assessment
– Monitoring for recurrence

Biomarker

First used in 1971 … An objective and
« predictive » measure … at the molecular
level … of normal and pathogenic processes
and responses to therapeutic interventions
Characteristic that is objectively measured and
evaluated as an indicator of normal biologic
or pathogenic processes or pharmacologic
response to a drug
A biomarker is valid if:
– It can be measured in a test system with well
established performance characteristics
– Evidence for its clinical significance has been
established

Rationale 1:
Why now ? Regulatory path becoming more clear

There is more at stake than
efficient drug
development. FDA
« critical path initiative »
Pharmacogenomics
guideline

Biomarkers are the
foundation of « evidence
based medicine » - who
should be treated, how
and with what.

Without Biomarkers
advances in targeted
therapy will be limited and
treatment remain largely
emperical. It is imperative
that Biomarker
development be
accelarated along with
therapeutics

Why now ?

First and maturing second generation molecular
profiling methodologies allow to stratify clinical
trial participants to include those most likely to
benefit from the drug candidate—and exclude
those who likely will not—pharmacogenomics-
based
Clinical trials should attain more specific results
with smaller numbers of patients. Smaller
numbers mean fewer costs (factor 2-10)
An additional benefit for trial participants and
internal review boards (IRBs) is that
stratification, given the correct biomarker, may
reduce or eliminate adverse events.

Molecular Profiling

The study of specific patterns (fingerprints) of proteins,
DNA, and/or mRNA and how these patterns correlate
with an individual's physical characteristics or
symptoms of disease.

Generic Health advice

• Exercise (Hypertrophic Cardiomyopathy)
• Drink your milk (MCM6 Lactose intolarance)
• Eat your green beans (glucose-6-phosphate
dehydrogenase Deficiency)
• & your grains (HLA-DQ2 – Celiac disease)
• & your iron (HFE - Hemochromatosis)
• Get more rest (HLA-DR2 - Narcolepsy)

Generic Health advice (UNLESS)

• Drink your milk (MCM6 Lactose intolarance)

Generic Health advice (UNLESS)

• Drink your milk (MCM6 Lactose intolerance)

Before molecular profiling …


• Flow cytometry correlates surface markers,
cell size and other parameters
• Circulating tumor cell assays (CTC’s)
quantitate the number of tumor cells in the
peripheral blood.
• Exosomes are 30-90 nm vesicles secreted by
a wide range of mammalian cell types.
• Immunohistochemistry (IHC) measures
protein expression, usually on the cell
surface.


• Gene sequencing for mutation detection

• Microarray for m-RNA message detection
• RT-PCR for gene expression

• FISH analysis for gene copy number
• Comparative Genome Hybridization (CGH) for
gene copy number

Basics of the ―old‖ technology

• Clone the DNA.
• Generate a ladder of labeled (colored)
molecules that are different by 1 nucleotide.
• Separate mixture on some matrix.
• Detect fluorochrome by laser.
• Interpret peaks as string of DNA.
• Strings are 500 to 1,000 letters long
• 1 machine generates 57,000 nucleotides/run
• Assemble all strings into a genome.

Genetic Variation
Among People
Single nucleotide polymorphisms
(SNPs)
GATTTAGATCGCGATAGAG
GATTTAGATCTCGATAGAG

0.1% difference among
people

The genome fits as an e-mail attachment

Basics of the ―new‖ technology

• Get DNA.
• Attach it to something.
• Extend and amplify signal with some color
scheme.
• Detect fluorochrome by microscopy.
• Interpret series of spots as short strings of
DNA.
• Strings are 30-300 letters long
• Multiple images are interpreted as 0.4 to 1.2
GB/run (1,200,000,000 letters/day).
• Map or align strings to one or many genome.

Next Generation Technologies

• Roche (454)
–Emulsion PCR
–Polymerase
–Natural Nucleotides
• 100-500 Mb for 5-15k
–1% error rate
–Homopolymers

Read Length is Not As Important For Resequencing

100%
% of Paired K-mers with Uniquely
90%
80%
Assignable Location

70%
60%
E.COLI
50%
HUMAN
40%
30%
20%
10%
0%
8 10 12 14 16 18 20
Length of K-mer Reads (bp)
Jay Shendure

Short Read Techologies

• Illumina GA (HiSeq, MySeq)

• ABI SOLID

Other second generation technology: (ABI) SOLID

Second generation DNA/RNA profiling

Second Generation DNA profiling

• Enrichment Sequencing
• ChIP-Seq (Chromosome
Immunoprecipitation)
• A substitute for ChIP-chip
• Eg. to find the binding sequence of
proteins (TFBS)

Paired End Reads are Important!

Known Distance

Repetitive DNA
Read 1Unique DNA 2
Read

Single read maps to
multiple positions


• Exome Sequencing (aka known as
targeted exome capture) is an
efficient strategy to selectively
sequence the coding regions of the
genome to identify novel genes
associated with rare and common
disorders.
• 160K exons

Second Generation RNA profiling

Besides the 6000 protein coding-genes …

140 ribosomal RNA genes
275 transfer RNA gnes
40 small nuclear RNA genes
>100 small nucleolar genes

Function of RNA genes

pRNA in 29 rotary packaging motor (Simpson
et el. Nature 408:745-750,2000)
Cartilage-hair hypoplasmia mapped to an RNA
Contents-Schedule

(Ridanpoa et al. Cell 104:195-203,2001)
The human Prader-Willi ciritical region (Cavaille
et al. PNAS 97:14035-7, 2000)


RNA genes can be hard to detects

UGAGGUAGUAGGUUGUAUAGU

C.elegans let-27; 21 nt
(Pasquinelli et al. Nature 408:86-89,2000)

Often small
Sometimes multicopy and redundant
Often not polyadenylated
(not represented in ESTs)
Immune to frameshift and nonsense
mutations
No open reading frame, no codon bias
Often evolving rapidly in primary sequence


Although details of the methods vary, the concept
behind RNA-seq is simple:
• isolate all mRNA
• convert to cDNA using reverse transcriptase
• sequence the cDNA
• map sequences to the genome
The more times a given sequence is detected, the
more abundantly transcribed it is. If enough
sequences are generated, a comprehensive and
quantitative view of the entire transcriptome of an
organism or tissue can be obtained.


• Comparing to microarray
– Microarray
• Closed technology: Prior knowledge required
• Affected by pseudo-genes (homologous of real genes)
• Low sensitivity
– RNA-Seq
• Open technology: No prior knowledge required
• Not affected by pseudo-genes because exact
sequence is measured
• Other information could be yielded (SNP, Alternative
splicing)

ncRNAs in human genome

tRNA 600 SRP RNA 1
18S rRNA 200 RNase P RNA 1
5.8S rRNA 200
Telomerase RNA 1
28S rRNA 200
RNase MRP 1
5S rRNA 200
Y RNA 5
snoRNA 300
miRNA 250 Vault 4
U1 40 7SK RNA 1
U2 30 Xist 1
U4 30 H19 1
U5 30 BIC 1
U6 20
U4atac 5
Antisense RNAs 1000s?
U6atac 5
Cis reg regions 100s?
U11 5
U12 5 Others ?

Mapping Structural Variation in Humans
>1 kb segments
- Thought to be Common
12% of the genome
(Redon et al. 2006)
- Likely involved in phenotype
variation and disease
CNVs
- Until recently most methods for
detection were low resolution
(>50 kb)

Size Distribution of CNV in a Human Genome

Next next generation sequencing
Third generation sequencing
Now sequencing

Ultra-low-cost SINGLE molecule sequencing

Pacific Biosciences: A Third Generation Sequencing Technology

Eid et al 2008

Second Generation Protein profiling

• Proteomics MS-MS-based
exclusively in discovery mode
• Automate diagnostics assay
generation (next generation
proteomics)
• Aptamers as alternative to antibodies
• ImmunoPCR

MS/MS identification
pipeline overview
pipeline
Goal
filter dataset Goal
prior to multi-tiered
database
Goal
search
database
Bonanza define PTMs search
profile
prior to
database
search

Bonanza + IggyPep

Defining Epigenetics
Genome

DNA  Reversible changes in gene
expression/function
 Without changes in DNA
Chromatin sequence
Epigenome
 Can be inherited from
precursor cells
Gene Expression  Allows to integrate intrinsic
with environmental signals
Phenotype
(including diet)

CONFIDENTIAL

Methylation I Epigenetics | Oncology | Biomarker
I NEXT-GEN | PharmacoDX | CRC

CONFIDENTIAL


Epigenetic Regulation:
Post Translational Modifications to Histones and Base Changes in DNA

 Epigenetic modifications of histones and DNA include:
– Histone acetylation and methylation, and DNA methylation

Histone
Methylation
Me Me
Histone
Me
Acetylation
Ac

DNA Methylation

CONFIDENTIAL


MGMT Biology
O6 Methyl-Guanine
Methyl Transferase
Essential DNA Repair Enzyme

Removes alkyl groups from damaged guanine
bases

Healthy individual:
- MGMT is an essential DNA repair enzyme
Loss of MGMT activity makes individuals susceptible
to DNA damage and prone to tumor development

Glioblastoma patient on alkylator chemotherapy:
- Patients with MGMT promoter methylation show
have longer PFS and OS with the use of alkylating
agents as chemotherapy

CONFIDENTIAL


MGMT Promoter
Methylation Predicts
Benefit form DNA-Alkylating Chemotherapy
Post-hoc subgroup analysis of Temozolomide Clinical trial with primary glioblastoma
patients show benefit for patients with MGMT promoter methylation

Median Overall Survival
25
21.7 months
20 plus
temozolomide
15
12.7 months

10 radiotherapy

radiotherapy
5
Adapted from Hegi et al.
NEJM 2005
0 352(10):1036-8.
Non-Methylated Methylated Study with 207 patients
MGMT Gene MGMT Gene
CONFIDENTIAL


Genome-wide methylation
by methylation sensitive restriction enzymes

CONFIDENTIAL


by probes

CONFIDENTIAL


…. by next generation sequencing

# markers

Discovery

Verification

Validation

# samples CONFIDENTIAL


MBD_Seq

Condensed Chromatin DNA Sheared

Immobilized
Methyl Binding Domain
DNA Sheared

CONFIDENTIAL


MBD_Seq

Immobilized
Methyl binding domain

MgCl2

Next Gen Sequencing
GA Illumina: 100 million reads

CONFIDENTIAL


MBD_Seq
MGMT = dual core

CONFIDENTIAL


# markers

1-2 million
MBD_Seq
methylation
cores
Discovery



Data integration
Correlation tracks

expression expression

Corr =-1 Corr = 1

methylation methylation

CONFIDENTIAL

99

Correlation track
in GBM @ MGMT

+1

-1
CONFIDENTIAL

100
I NEXT-GEN | PharmacoDX |


# markers

MBD_Seq

Discovery

454_BT_Seq
Verification MSP
Validation


I NEXT-GEN | PharmacoDX |

Deep Sequencing

unmethylated alleles

methylated alleles less methylation

more methylation

CONFIDENTIAL
GCATCGTGACTTACGACTGATCGATGGATGCTA

Deep MGMT
Heterogenic complexity

CONFIDENTIAL


CONFIDENTIAL

105

Bioinformatics, a life science discipline …

Math

(Molecular)
Informatics
Biology


Math

Computer Science Theoretical Biology

(Molecular)
Informatics
Biology
Computational Biology


Math


Bioinformatics

(Molecular)
Informatics
Biology

Bioinformatics, a life science discipline … management of expectations

Math

NP AI, Image Analysis
Datamining structure prediction (HTX)
Bioinformatics

Interface Design Expert Annotation
Sequence Analysis (Molecular)
Informatics
Biology

Bioinformatics, a life science discipline … management of expectations

Math

NP AI, Image Analysis
Datamining structure prediction (HTX)
Bioinformatics
Discovery Informatics – Computational Genomics
Interface Design Expert Annotation
Sequence Analysis (Molecular)
Informatics
Biology

Translational Medicine: An inconvenient truth

• 1% of genome codes for proteins, however
more than 90% is transcribed
• Less than 10% of protein experimentally
measured can be ―explained‖ from the
genome
• 1 genome ? Structural variation
• > 200 Epigenomes ??

• Space/time continuum …

Translational Medicine: An inconvenient truth

• 1% of genome codes for proteins, however
more than 90% is transcribed
• Less than 10% of protein experimentally
measured can be ―explained‖ from the
genome
• 1 genome ? Structural variation
• > 200 Epigenomes …

• ―space/time‖ continuum

Cellular programming

Epigenetic (meta)information = stem cells

Cellular reprogramming

Tumor

Tumor
Development
and
Growth

Epigenetically
altered, self-
renewing cancer
stem cells

Cellular reprogramming

Gene-specific
Epigenetic
reprogramming

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Molecular profiling 2012

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