translating cancer genomes and transcriptomes for precision oncology
TRANSCRIPT
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Translating Cancer Genomes and
Transcriptomes for
Precision OncologySameek Roychowdhury, MD, PhD1,2,3; Arul M. Chinnaiyan, MD, PhD4,5,6,7,8,9
KALLEE VibhaMOWLABACCUS WafaaRAMAH UrshilaSWEETMAN SairahTOOLSEE Karishma
MD Year 2March 2016
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Diagnosis, prognosis & treatment of cancer
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• Defining genome and t ranscr iptome• DNA sequenc ing us ing ngs technolog ies• Exome sequenc ing and chromosomal
karyotyp ing and banding to revea l mutat ions in the genome in cancer
• Rna sequenc ing and microarray for t ranscr iptome profi l ing
• Biomarkers found • Att i tudes of phys ic ians and pat ients to
th is new pract ice• Areas of improvement and cha l lenges .
Discuss current and future impact of genome and transcriptome sequencing
for patient care in clinical oncology.
How we can integrate and use this information for precision oncology?
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Genome + transcriptome
CANCER
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Defining the omics…
Previously,
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• Defining genome and transcriptome• DNA sequencing using NGS technologies• Exome sequencing and chromosomal karyotyping
and banding to reveal mutations in the genome in cancer
• RNA sequencing and microarray for transcriptome profiling
• Biomarkers found • Attitudes of physicians and patients to this new
practice• Areas of improvement and challenges.
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NGS sequencing
• Advantages of NGS approaches:
Detecting multiple genomic alterations types when the etiology is not apparent
Early screening for relatives carrying the same mutation as the patient.
It makes it possible to find uncommon genetic changes in patients who test negative for the common mutation.
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Is it feasible to sequence the whole genome?
•
• 1% of the genome was sequenced.
Why?• Only 1% of the genome contains the approximately 20 000 known
genes• Reduces cost by 100 fold,
Exome sequencing!
Whole genome sequencing is expensive!!
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• Defining genome and transcriptome• DNA sequencing using NGS technologies• Exome sequencing and chromosomal karyotyping and
banding to reveal mutations in the genome in cancer• RNA sequencing and microarray for transcriptome
profiling• Biomarkers found • Attitudes of physicians and patients to this new practice• Areas of improvement and challenges.
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How does exome sequencing work?
Exome sequencing uses baits to hybridize and capture corresponding regions of the genome, focusing on the coding regions of the genome.
Exome sequencing can include the whole exome (about 20,000 genes), comprising just over 1% of the genome;
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What has exome sequencing revealed? Definition Example
Point mutation Single base substitution that can change the function of the gene product
Present in melanoma
Copy number variation Refers to extra or missing copies of a gene
Extra e.g(HER2) seen in breast cancer
Loss of copy e.g(PTEN) - seen in prostate and other cancers
Translocation Gene arrangement which causes gene fusion
Usually involve kinases in cancer
Can cause dysregulation of transcription factors
Chronic myeloid leukemiaFusion of philadephia chromosome and BCR-ABL1 gene arrangement
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Gene fusion
• Enable the detection of chromosomal rearrangement
• this helped the identification of novel oncogenes and tumor suppressors in cancer can help to know more about there function in cancer biology.
Chomosomal karyotyping and
banding
• Can help to find novel gene fusion in solid tumors
Cancer genome and
transcriptome sequencing
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• Defining genome and transcriptome• DNA sequencing using NGS technologies• Exome sequencing and chromosomal karyotyping and
banding to reveal mutations in the genome in cancer• RNA sequencing and microarray for transcriptome
profiling• Biomarkers found • Attitudes of physicians and patients to this new practice• Areas of improvement and challenges.
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Transcriptome profiling
Transcriptome profiling
Microarray
Classify disease into clusters
E.g B cell lymphoma was
divided into activated b-cell
lymphoma(better prognosis) and germinal centre
lymphoma
RNA sequencing ( whole Transcriptome shotgun
sequencing )
Advantages over micro arrayPrecise detail about base pairs
Ability to detect novel RNA that cannot be detected on microarray
Detect multiple gene arrangement as well as novel ones
Uses Classify cancer subtypes using gene expressionCan detect multiple fusion and rearrangement
of genes unlike FISH and PCR which can detect only one gene at a time
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RNA sequencing -extraction of RNA from cell
-Isolation of specific RNA species
-Convertion of RNA to cDNA using reverse transcriptase
-Construction of a sequencing library
-PCR amplication and sequencing
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What is a gene signature?
Gene signatures
A gene signature is a group of genes in a cell whose combined expression pattern is uniquely characteristic of a biological phenotype or medical condition.
What have we learnt from it?
classify cancer types into molecular subsets that have clinical relevance
aid in classifying using point mutations
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Cancer genome and transcriptome. What have we
learnt ?
Mutational pattern can help in classification trough an underlying mechanism such as defects in DNA repair , radiation exposure and tobacco exposure
Over 10 000 cases of cancer have undergone DNA sequencing • Revealed heterogeneity within and across cancer types classified by tissue
of origin (e.g lung, breast)
Genome and transcriptome profiling has measurably affected clinical outcomes by moving from a histology to genomics-based classification .• Lung cancer
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Acute myloid leukemia have few point mutation but rather more copy number variation and gene fusion
Abundant point mutation due to carcinogen exposure of tobacco smoke and UV
Mutational heterogeniety with and across cancers
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• Defining genome and transcriptome• DNA sequencing using NGS technologies• Exome sequencing and chromosomal
karyotyping and banding to reveal mutations in the genome in cancer
• RNA sequencing and microarray for transcriptome profiling
• Biomarkers found • Attitudes of physicians and patients to this new
practice• Areas of improvement and challenges.
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Types of Molecular Biomarkers
What is a biomarker?The National Cancer Institute (NCI) definition of
biomarker is: "A biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or disease.
Also called molecular marker and signature molecule.
A biomarker may be used to see how well the body responds to a treatment for a disease or condition.
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Factors of an ideal biomarker
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Molecular Biomarkers
Research discoveries derived through cancer genome and transcriptome studies have the potential for clinical impact as biomarkers.
There are three key types of biomarkers used for clinical decision making:
diagnosticprognosticpredictive biomarkers.
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Types of Biomarkers
• facilitate the identification of a cancer type or subtype
DIAGNOSTIC
• aid clinicians in determining the risk of relapse or disease progression after therapy
PROGNOSTIC
• to select one therapy over others, based on associations between biomarker results and the likelihood of response to certain therapies
PREDICTIVE
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Each type of biomarker could be assayed to detect changes in :
1. a tumor’s genome (DNA)
2. transcriptome (RNA)3. proteome (protein)4. phenotypic
characteristics (such as histopathologic classification)
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Methods to detect biomarkers
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Methods to detect biomarkers
DNA-based predictive biomarker
guide therapy selection
RNA-based biomarkers
FISH methods are used for standard
diagnostic sub-typing
IMMUNOHISTOCHEMSTRY
Used to detect estrogen receptor protein in breast
cancer
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However, before any biomarker can be translated into the clinic for use in standard practice ….
The clinical utility of the biomarker must be
tested through clinical trials to establish its impact and association with clinical outcomes.
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However, it is costly to develop FISH and Sanger sequencing tests for single genes and their relevant mutations…..
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NGS has been translated to the clinic to cost-effectively broaden the number of genes and types of mutations tested
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Integrating Whole-Exome (DNA) and Transcriptome (RNA) Sequencing
There is an increasing need for multidisciplinary team effort
Oncologists
Genomic Scientists
Bio-informatician
sPathologists
Genetic counselors
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Established by National Human Genome
Research Institute
• Aim: To study and provide guidelines for bringing
genomics to clinical practice.
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• Defining genome and transcriptome• DNA sequencing using NGS technologies• Exome sequencing and chromosomal karyotyping
and banding to reveal mutations in the genome in cancer
• RNA sequencing and microarray for transcriptome profiling
• Biomarkers found • Attitudes of physicians and patients to this new
practice• Areas of improvement and challenges.
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SURVEY:PHYSICIAN AND PATIENT ATTITUDES…
• 22%: “low confidence in genomic knowledge”
• Therefore, they require guidelines and education.
Physicians
• Interested to improve their cancer care
• Some: Concerned about discrimination, incidental findings etc.
Patients
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CLINICAL INTERPRETATION OF TUMOR SEQUENCING
RESULTS…Availability of many software tools to predict potential impacts of mutation.
However-Need for expert clinical annotation of specific mutations.
Why?
Numerous genetic mutations exist with multiple pathways!
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TYPES OF MUTATIONS
Driver mutations
Aid survival, growth, spread of
cancer
Passenger mutations
-Variants of unknown
significance-Present in
greater number
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ASSAYS…
•Must undergo analytic validation, based on its sensitivity and specificity in detecting mutations.
•Results are tested by another assay. E.g. Sanger sequencing, PCR or FISH. •All tests are performed in laboratory inspected by a certifying body.
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• Defining genome and transcriptome• DNA sequencing using NGS technologies• Exome sequencing and chromosomal
karyotyping and banding to reveal mutations in the genome in cancer
• RNA sequencing and microarray for transcriptome profiling
• Biomarkers found • Attitudes of physicians and patients to this new
practice• Areas of improvement and challenges.
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UNMET NEEDS - CANCERS OF UNKNOWN PRIMARY…
.
Cancer of unknown primary
Inability to identify the origin of the tumor using traditional radiologic imaging and immunohistochemical assessment.
Difficult to select appropriate treatment.
Several assays have been developed
To classify tissue of origin
Based in mRNA and miRNA signatures
Can aid in choosing chemotherapy
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GENOMICS-BASED CLASSIFICATION OF CANCER HAS CHANGED THE WAY CLINICAL
TRIALS ARE DESIGNED!
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Based on cancer type Can be characterised and split into
molecular subsets
Therefore, clinical designs can be deeper
1. Treatment based on molecular eligibility
2. It is possible to focus solely on those with a certain mutation and test treatment efficiency.
Classification of cancer
NowPreviously
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Patients with any solid tumorPhase 1 trial:
Patients with BRAF V 600E-activating mutations more
likely to respond
Observation:
Patients with BRAF mutations enrolled
Second phase:
80% respondedObservation:
EXAMPLE-DETERMINING DOSING AND ASSESSING TOXICITY OF BRAF INHIBITERS IN
MELANOMA
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LIMITATIONS
•Number of eligible patients with molecular eligibility is dramatically reduced.
-For instance, a clinical trial for a mutation with prevalence of 1% in a common cancer type will be difficult to accrue and complete.
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MUTATION-BASED AND PATHWAY-BASED ELIGIBILITY: ANOTHER EMERGING APPROACH!
Patients receive an oral pan-FGFR inhibitor (ponatinib)
To identify clinical responders in disease or mutation
subsets
Patients with any solid tumor that has alterations in FGFRs
To guide future trials and drug development
Selection Criteria:
Therapy:
Endpoints:
Aim:
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LEARNING FROM EXCEPTIONAL RESPONDERS IN TRIALS…
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SOME RARE PATIENTS EXPERIENCE EXCEPTIONAL COMPLETE RESPONSE TO A
THERAPY!
Efforts are made to learn from them.
Their phenotypic changes are observed.
Retrospective genomic and transcriptome sequencing of patient’s tumor is
performed.
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Patients with metastatic bladder
mTOR inhibitor
EXAMPLE…
One patient had a complete response
Majority did not respond
Point mutation in TSC1
Therefore, other patients with TSC1 mutations are more likely to respond to mTOR inhibition.
Selection Criteria:
Therapy:
Observation:
Whole-Genome Sequencing:
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EXCEPTIONAL RESPONDERS INITIATIVE…
Established by NCI
Mission
To identify and confirm patients who have had remarkable
responses to systemic therapy
To use genomic technologies to characterise their tumors to study the molecular
mechanisms underlying why these patients benefit from systemic therapy
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Challenges in precision oncology
1) LIMITED SAMPLING Researchers have only detected a partial genomic landscape of cancer despite profiling thousands of people.
2) Implementing precision cancer medicine will require multidisciplinary collaborations and novel molecular diagnostics for cancer genomic testing.
3) The majority of cancer genome data available are based on primary tumors
rather than advanced metastatic tumors, that behave more aggressively.
4) Clinical trials will require an integrated network to coordinate tumour samples to offer access to novel therapies
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Summary points:
Cancer Molecular aspect
Whole and transcriptome sequencing
Other complexities to consider :
1) How methylation regulates gene expression. For a better view of individual
cancers.
2) New ‘omics’ approaches for proteome and metabolome. To integrate data analysis in clinical
trial
3) Additional aspects of cancer biology
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Additional aspects of
cancer biology
Tumour heterogen
ity
Drug Resistan
ce
Tumour micro
environment
Stem cell propertie
s
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Therapies considered
Cancer Therapy
Genomics guided
Combination
TherapiesImmunotherapy
Oncolytic Viruses
Stem Cell / Metabolism targeting inhibitor
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Precision Oncology - The Future…
Molecular classification of cancer based on genomics and transcriptome alterations may reveal novel biomarkers for diagnosis, prognosis and predicting response to therapies.These will open new doors to enable precision cancer medicine.
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Any Questions…