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

Diagnosis, prognosis & treatment of cancer

• 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?

Genome + transcriptome

CANCER

Defining the omics…

Previously,

• 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.

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.

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!!

• 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.

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;

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

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

• 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.

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

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

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

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

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

• 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.

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.

Factors of an ideal biomarker

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.

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

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)

Methods to detect biomarkers

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

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.

However, it is costly to develop FISH and Sanger sequencing tests for single genes and their relevant mutations…..

NGS has been translated to the clinic to cost-effectively broaden the number of genes and types of mutations tested

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

Established by National Human Genome

Research Institute

• Aim: To study and provide guidelines for bringing

genomics to clinical practice.

• 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.

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

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!

TYPES OF MUTATIONS

Driver mutations

Aid survival, growth, spread of

cancer

Passenger mutations

-Variants of unknown

significance-Present in

greater number

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.

• 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.

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

DESIGN OF CLINICAL TRIALS

GENOMICS-BASED CLASSIFICATION OF CANCER HAS CHANGED THE WAY CLINICAL

TRIALS ARE DESIGNED!

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

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

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.

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:

LEARNING FROM EXCEPTIONAL RESPONDERS IN TRIALS…

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.

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:

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

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

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

Additional aspects of

cancer biology

Tumour heterogen

ity

Drug Resistan

ce

Tumour micro

environment

Stem cell propertie

s

Therapies considered

Cancer Therapy

Genomics guided

Combination

TherapiesImmunotherapy

Oncolytic Viruses

Stem Cell / Metabolism targeting inhibitor

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.

Any Questions…