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Predictive Biomarkers for tailoring drug therapy. Outline. What is a predictive biomarker and why are they important? How does genomics facilitates the search for predictive biomarkers? Are there any examples of clinically useful predictive biomarkers?. 1. What is a predictive biomarker?.

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slide1

Predictive Biomarkers

for tailoring drug therapy

outline
Outline
  • What is a predictive biomarker and why are they important?
  • How does genomics facilitates the search for predictive biomarkers?
  • Are there any examples of clinically useful predictive biomarkers?
slide4

Predictive Biomarkers

Biomarker: A gene, protein, or other change that heralds a biomedical phenotype before that phenotype Is clinically apparent.

Biomarkers used for: Diagnosing Disease

Establishing Disease Risk

Establishing Drug Response

Establishing Drug Toxicity

Predicting Drug Response

Predicting Drug Toxicity

slide6

Discuss

LDL

High LDL (low density lipoprotein) levels indicate individuals

At high risk of artherosclerotic plaque formation.

Artherosclerotic plaque formation leads to thrombosis (stroke) and

Embolism (heart attack)

Stroke and heart attach are the major killers in the world

The use of LDL as a biomarker of heart disease has led to the explosion of the statins: multi-billion cholesterol reducing drugs.

slide9

Using the sequence information to probe the transcriptome of the Cell

Transcriptome: the set of all expressed genes (mRNAs) inside a cell.

Different types of technology used to probe the transcriptome:

SAGE (serial analysis of gene expression)

Strategy: isolated mRNA is reversed-transcribed to cDNA, digested into fragments, randomly ligated into clones, and the clones are sequenced. Relative abundance of different mRNAs within a single pool are reflected in the sequenced clones.

DNA microarrays

Strategy: mRNA is reversed transcribed to cDNA, labeled with fluorescent probe,

And hybridized to complementary strand DNA attached to glass slides. By

comparing hybridizationn of two samples, relative abundance of mRNA between samples can be determined

Affymetrix GeneChips. Same as DNA microarrays, except that cDNA is transcribed to cRNA in vitro, which is labeled with fluorescent probes.oligonucleotides are chemically synthesized on chip and the presence/absence of genes is determined based on hybridization between sets of perfectly matched and mismatched probes.

slide10

SAGE: Serial Analysis of Gene Expression

gtac

AAAA

gtac

AAAA

gtac

AAAA

gtac

AAAA

gtac

AAAA

Cut and tag

AAAA

gtac

Total cell mRNA

Concatenate

ATTGCGTACCCCgtacTTGAGAACCCgtacATTGCGTACCCCgtacGCCCAATTCATTTAgtacATTGCGTACCCCgtacAAAACCCCTTTTT

Sequence

slide11

Differential Analysis of Gene Expression with SAGE

Normal Sample

Disease Sample

Gene

A B C D E F

A B C D E F

5 3 12 4 1 12

# SAGETags

4 2 10 5 1 4

Y=x

1SD

2SD

slide12

Probe Preparation and labeling for hybridization experiments

  • Fluorescent probes prepared from two mRNA sources to be compared
  • Cy-dCTP nucleotides added to the cDNA synthesis reaction
    • Cy3 (green)-control sample
    • Cy5 (red)-experimental sample
slide14

Differential Analysis of Gene Expression with GeneChip

Normal Sample (Red)

Disease Sample(Green)

Gene

A B C D E F

A B C D E F

5 3 12 4 1 12

PM-MM

4 2 10 5 1 4

Y=x

1SD

2SD

slide15

Spotted arrays: cDNA microarrays

  • Oligonucleotides are obtained by PCR amplification of cDNA templates and have an amine linking group on 5’ end
  • Glass slides are chemically treated (aldehyde) to be reactive to the group on the 5’ of the oligo
  • A microdroplet of PCR product is placed on slide with use of metal pin. Schiff base reaction links oligo to slide surface via covalent bond.
slide16

Differential Analysis of Gene Expression with Microarrays

Normal Sample (Red)

Disease Sample(Green)

Gene

A B C D E F

A B C D E F

5 3 12 4 1 12

Fluorescence

4 2 10 5 1 4

Y=x

1SD

2SD

slide17

How transcriptome analysis offers insights into potential biomarkers

Differential gene expression in normal tissues

(relating the expression of specific genes to suceptibility

Of certain tissue to specific diseases (e.g. retinal degeneration).

Differential gene expression in disease states.

(comparing normal and disease tissues; class prediction; class

Identification.)

Gene expression in model systems

(comparing drugs to mutations, human to mice, cells to

Living tissues)

Gene expression in pathogens

Gene expression in response to drugs

(drug target identification, drug classification; drug activity,

Drug metabolism)

slide18

Proteomics –Finding Protein Biomarkers

30,000 genes

>200,000 proteins

slide26

BIOMARKERS USED IN CANCER THERAPY

The two-hit hypothesis: Cancer cells need to acquire both gain-of-function (“oncogenic”) and loss-of-function (“tumor suppressor”)

Mutations to become cancerous

Oncogene:gain of function

Tumor suppressor: loss

Of function

slide27

Oncogenic mutation mechanisms

DNA

Chromosomal

rearrangement

Deletion or point mutation

in coding sequence

Gene amplification

DNA

mRNA

protein

slide28

HERCEPTIN

    • Herceptin® (Trastuzumab) is the first targeted, humanized antibody for treatment of women with HER2 (human epidermal growth factor receptor 2) positive metastatic breast cancer. Herceptin is designed to target and block the function of HER2.
    • HER2 positive metastatic breast cancer have a more aggressive disease, greater likelihood of recurrence, poorer prognosis and approximately half the life expectancy of women with HER2 negative breast cancer.
    • The U.S. Food and Drug Administration (FDA) approved Herceptin in September 1998 in combination with paclitaxel for treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein and who have not received chemotherapy for their metastatic disease.
    • Herceptin is one of the few therapies, and the only humanized antibody, that has demonstrated a survival benefit indicated for metastatic breast cancer
slide30

Clinical Trial Results

  • Data showed that patients receiving Herceptin with chemotherapy survived for a median of 25.1 months compared to 20.3 months for patients in the Phase III trial receiving chemotherapy alone, a difference of 24 percent.
    • In addition to survival, when used in combination with chemotherapy, Herceptin was shown to improve overall response rates from 29 percent in women treated with chemotherapy alone to 45 percent with Herceptin added -- a 64 percent increase. The median time to disease progression increased from 4.5 months in the chemotherapy alone group to 7.2 months in the Herceptin plus chemotherapy group -- a 63 percent increase.
    • Additionally, the duration of response of women treated with Herceptin with chemotherapy was 8.3 months, compared to 5.8 months for those women receiving chemotherapy alone.
    • In the trial evaluating Herceptin alone, 14 percent of 222 women who had failed one or two prior chemotherapy regimens had objective tumor responses with tumor shrinkage of 50 percent or greater.1
slide31

Other example: Targeted Inhibitors of BCR-ABL oncogene

  • Chronic Myelogenous Leukemia: a biphasic disease
  • a. chronic phase (associated with t(9;22) translocation.)
  • b. blast crisis (fatal for 85% of Patients)
  • .
  • Allogeneic bone marrow transplantation was the only curative treatment available but is only applicable to a minority of patients (15-20%).
  • A recent advance in the treatment has been the introduction of the Bcr-Abl inhibitor STI571 (Gleevec)
slide32

Diagnosis: t(9;22)

Karyotype

analysis

FISH: FLUORESCENCE IN SITU HYBRIDIZATION

NORMAL

MUTANT

slide33

Gleevec binds Bcr-Abl protein target

Rational design:

STI-571 bound to

ABL (PDB: 1IEP)

slide34

Gleevec clinical results

Initially, 90% Remission with GLEEVEC with recurrence due to resistance (mediated by BCR-ABL mutations)

Anti BCR-ABL cocktails to fight off resistant cells.

Ability to target c-kit kinase has led to additional application: gastric cancer.

Current market value over 1 billion dollars.

slide35

Transcriptome Analysis for Personalized Therapy

Collect tumor sample from patient

Perform transcriptome analysis of tumor sample

relative to control sample (gene expression profiles)

Treat tumor

Establish sets of genes that are up or down-regulated

in responsive vs. non responsive tumors

Use those genes to predict outcome or

Guide treatment decisions