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Webinar SeriesWebinar SeriesScienceScienceThe Personal Side of Sequencing:The Personal Side of Sequencing:

Exploring the Role of Cancer Gene Panels and NGS in Clinical ResearchExploring the Role of Cancer Gene Panels and NGS in Clinical Research 23 October, 201323 October, 2013

Sponsored by:

Participating Experts:

Brought to you by the Science/AAAS Custom Publishing Office

Harriet Feilotter, Ph.D.Queen’s UniversityKingston, Ontario, Canada

José Luis Costa, Ph.D.IPATIMUPPorto, Portugal



Sandra Balladares, Ph.D.Life TechnologiesMexico City, Mexico

The Personal Side of Sequencing:Exploring the Role of Cancer Gene Panels and

NGS in Clinical Research

José Luis Costa

AAAS – Technology webinar2013‐10‐23

The Personal Side of Sequencing:Exploring the Role of Cancer Gene Panels and NGS in

Clinical Research

Harriet Feilotter, Ph.D.José Luis Costa, Ph.D.

Sandra Balladares, Ph.D.

NGS in Cancer Research

Bentchtop sequencers Full (Lab) sequencers

NGS in Cancer Research

Increasing complexity

Gene panels

Exome sequencing

Genome sequencing

Bentchtop sequencers Full (Lab) sequencers

NGS in Cancer Basic Research

Exome sequencing

Genome sequencing

Full (Lab) sequencers

NGS in Cancer Clinical Research

Gene panels

Bentchtop sequencers


Gene panels


• Therapy selection• Disease monitoring• Drug resistance

Sporadic(Biomarkers)

Sporadic Cancer Gene Panels Clinical Utility

Cancer Biomarker DrugCML BCR‐ABL Imatinib

Melanoma BRAF Vemurafenib

Ovarian BRCA1/2 Olaparib, Veliparib

Breast BRCA1/2 Olaparib, Veliparib

GIST c‐Kit Imatinib

Lung EGFR Erlotinib, Gefitinib

Lung EML4‐ALK fusion Crizotinib

Breast HER2 Trastuzumab

Colon KRAS/NRAS Cetuximab, Panitumumab

Gastric HER2 Trastuzumab

Sporadic Cancer Gene Panels Clinical Utility

Biomarker DrugBCR‐ABL Imatinib

BRAF Vemurafenib

BRCA1/2 Olaparib, Veliparib

c‐Kit Imatinib

EGFR Erlotinib, Gefitinib

EML4‐ALK fusion Crizotinib

HER2 Trastuzumab

KRAS/NRAS Cetuximab, Panitumumab

HER2 Trastuzumab

Gene panels

Comprehensive and unbiased screening of biomarkers


Gene panels


• Therapy selection• Disease monitoring• Drug resistance

Sporadic(Biomarkers)

• Diagnosis• Genetic counseling• Treatment options

Hereditary(Genetic cause)

Hereditary Cancer Gene Panels Clinical Utility

Cancer GenesBreast and Ovarian (HBOC) BRCA1 and BRCA2Colon (FAP) APC

Colon (HNPCC) MSH2, MLH1, MSH6and PMS2

Childhood Rb and TP53 …Endocrine MEN and RET …

Comprehensive and faster screening

Breast cancer affects 1 in 8 women

When a BRCA gene is mutated the risk of breast cancer rises from 12% to 60%

Women with a BRCA1 gene mutation have 60‐90% lifetime risk and who have BRCA2 gene mutations have a 45‐85% lifetime risk

12%

60%

BRCA1

BRCA2

Lifetime risk

Screening BRCA1/2 Background

Adapted from NHS

Structural aspects

• Size—15.8 Kb

• Many homopolymer regions

BRCA1 (on chromosome 17)

5.6 kb, 1863 aa

BRCA2 (on chromosome 13)

10.3 kb, 3418 aa


BRCA1/2 18 homopolymer 6 mer7 homopolymer 7 mer3 homopolymer 8 mer

Structural aspects

• Size—15.8 Kb

• Many homopolymer regions

• Genes highly polymorphic with mutations scattered throughout the

genes (no hot spots) ‐ nonsense, frameshifts, indels


BRCA1

BRCA2

Exon

Mutation

Exon

Mutation Adapted from NCI – Cancer.gov

BRCA 1 and BRCA2 Global Consortia

Prof. Jeffrey N. Weitzel Division of Clinical Cancer GeneticsCity of Hope Cancer Center. Los Angeles, United States

Prof. Harriet Feilotter Department of Pathology at Queen's University. Ontario Canada

Dr. Alfredo Hidalgo Miranda, National Institute of Genomic Medicine. Mexico City, Mexico

Dr. Nicola Williams Southern General HospitalGlasgow, United Kingdom

Dr. José Carlos MachadoDr. José Luis CostaIPATIMUP Medical Faculty of Porto, Portugal

Dr. Marjolijn J.L. Ligtenberg, Dr. Arjen R. MensenkampRadboud University NijmegenMedical Centre, The Netherlands

Dr. Arif B. EkiciInstitute of Human GeneticsFriedrich‐Alexander‐University of Erlangen‐Nürnberg, Germany

Dr. Rosella PetraroliDr. Chrysanthi Ainali

BRCA1 and BRCA2 Global Consortium

Goal: Develop a BRCA1 and BRCA2 NGS panel with Ion AmpliSeq™ technology and Ion PGM™ Sequencer

1. Coverage of targets: 100% coverage of all coding exons and exon‐intron boundaries (‐20 to +20)Amplicons covering exons are overlapping

2. European Molecular Genetics Quality Network GuidelinesPrimers do not overlapNo validated SNPs in the last five nucleotides of primerMax 3 validated SNPs per primer

3. Adoptable by other research labs ‐ accurate, affordable & easySingle day workflowMultiplex at least 6 samples per chip (316)Reliable and easy data analysis – e.g. Ion Reporter ™ Software

Ion AmpliSeq™ BRCA1 & BRCA2 PanelProject Design

• Design following collaborators’ requirements• First analytical verification on 20 archived samples

• 9‐mer homopolymer variants

Phase 1:Design and test

• 30 archived samples with 65 known different variants tested and exchanged across 2 labs:

•Homopolymer stretches ✓

•Deletions/insertions ✓

•Point mutations ✓

•Exon deletions ✓

• Multiplex 8 samples per Ion 316™ Chip

Phase 2:Analytical verification and reproducibility

Panel launch

• Global verification will be performed in 8 labs on additional 200 archived samples with known variant status

Phase 3:Global consortium

verification

For research use only

Ultra‐highMultiplex PCR

Partially digest primers

Adapter ligation Amplification

Construct library

3.5 h

Prepare template

4 h

Run sequence

3 h

Analyze data

3 h

Ion AmpliSeq™ BRCA1 & BRCA2 Panel• Resulting design meets requirements

• 100% coverage of target regions• 167 amplicons• 200 bp design (single 349 bp)• No SNP in the 3’ end of the primer• EMQN Best Practices Guidelines

Ion AmpliSeq™ BRCA1 & BRCA2 Panel

Trim adapters

Remove poor signal reads

Split reads per barcodeAssembly

AlignmentCoverage analysis

Variant calling

Read generation

Read mapping

Variant filtering

Variantconfirmation

Identify known SNPs

Identify synonymous

Verify variants found

Ion Reporter™ Software

Ion Reporter pre-configured workflow

Report

FilterAnalyze

Detection Rate‐ Analysis Results from six labs

• Data: Nijmegen – Porto – Erlangen – Canada – Glasgow ‐ COH• Analysis: Ion Reporter pre‐configured BRCA Workflow

• Workflow contains modified parameters for calling homopolymers• Not including in the sensitivity the samples with large exon deletions, the LOH

variants

Ion Reporter™ Software

Type of Mutation Unique Mutations Samples Sensitivity

In long homopolymer 11 12/12 100%Indel 79 79/79 100%

point mutations 63 67/67 100%155 165/165 100%

Positive Predictive Value (PPV) was found to be > 94% and specificity > 84%

IPATIMUP 2012

• Ion PGM – Multiplex PCR of 77 amplicons + MLPA• 10‐15 samples per month, reduction of 75% TAT (3‐5 weeks)

• Ion PGM – Ampliseq panel or in house muliplex PCR (+ MLPA) • 20‐35 samples per month, TAT 3‐5 weeks

IPATIMUP 2013

Our approach for BRCA1/2 Screening

Genomic DNA

Target genes

Ion PGM

Interpretation

CE Seq

AmpliSeq

Ion library prep

Bioinformatics Bioinformatics

Target genes

Thermocycler

MLPA

Bioinformatics

Small alterations Large alterations

Gene Panels in Clinical Cancer Research

Genomic DNA

Target genes

Ion PGM

Interpretation

CE Seq

AmpliSeq

Ion library prep

Bioinformatics Bioinformatics

Target genes

Thermocycler

MLPA

Bioinformatics

Small / Large alterations Large alterations

Gene Panels in Clinical Cancer Research

Sponsored by:








The evolution of clinical research: BRCA as a model

Dr Harriet Feilotter, PhD, FCCMGDept of Path and Molecular Medicine

Queen’s UniversityKingston, Ontario

Canada

Challenges of variant detection

• Increasing numbers of genes• Multiplex requirements• Analytical sensitivity• Clinical sensitivity• Cost• Throughput• Data interpretation

Evolution of BRCA clinical research

• Clinical research to identify BRCA1/2 mutations has changed

• Evolution has been driven by available technology

PTT DHPLC Sanger NGS

1990s 2000 2006 2013

More than a decade ago• Early mutation methods focused identification of truncating mutations

• Protein truncation testingX

PTT in reality

• Requires multiple steps• Analytical interpretation

difficult

• Multiple steps• Requires follow up assay

• Interpretation difficult

• Sensitivity low• Clinical interpretation easy

DHPLC

• DHPLC methodology• Advantage of scanning entire coding region

DHPLC and scanning methods

• Many individual assays• Requires follow up assay• Sensitive to assay conditions• Sensitivity high• Cost moderate

Sanger sequencing

Removes requirement for follow up assaysMany individual reactions‐ F and RMany opportunities for errorsHigh sensitivity, high cost

Promise of next generation sequencing

• Equipment• More equipment?• Library preps• Analysis• Data storage

Solutions for Clinical Research?• Consortium approach appealing• 4‐6 month validation window• Shared validation results• Shared experience

4 month TAT~$1500/sample

1.0FTE

2 week TAT~$300/sample

0.2 FTE

Challenges remain?

• Yes‐ new challenges emerge• New work flow for data analysis• Data handling and storage• Follow up with Sanger?

Ion Community ™ Panels

Develop applications that satisfy customer needs Content and workflow defined by International Consortia

Analytical verification part of the development process Panel tested on clinical research samples at collaborator’s lab

Complete workflow including software solution Include collaborators need to use the panel in their settings

Share experiences of it with other users Be part of a community

OncoNetwork Global ConsortiumQueen's University, Ontario

Canada

IPATIMUP Medical Faculty of Porto. Portugal

Radboud University NijmegenMedical Centre, Netherlands

Centro Ricerche OncologicheMercogliano, Italy

St James's Hospital Dublin, Ireland

Warwick Medical School United Kingdom

Université Paris Descartes, Paris, France

Institut Gustave Roussy Paris, France

ARC-NET University of Verona Italy

VIOLLIER AG Basel , Switzerland

Faculty of Medicine, Kinki University Osaka, Japan

ARUP- Institute for Clinical and Experimental Pathology

Utah, USA

Sponsored by:








The Personal Side of Sequencing:In pursuit of a world without cancer

Sandra Balladares

AAAS – Technology webinar2013‐10‐23

• Molecular Biomedicine PhD• 10 year working experience on DNA

technology leading company providers• Healthy habits, diet, exercise• No family history of Breast Cancer• Two children at the age of 33 and 35 • Breastfeed• May 10th, 2010 • Self exploration• Breast Cancer Patient at the age of 36

About me…

• PET‐CT Solid tumor 3x2.6cm SUV 10.08, 4 more nodules of 0.6‐0.9 cm SUV 4.6

• Radical Mastectomy• Clean surgical borders, negative

sentinel lymph nodes• Immunochemistry: HER2+, p53

+ (85%), Ki‐67 + (60%)• Multicentric IDC, Stage IIA• Adjuvant therapy

recommended

Identifying the enemy and preparing the battle

Surgery and Chemo, pain + pain

• 4 x Epirubicin/Cyclophosphamide 4 x Taxotere and 12 x Herceptin

• Serious or life‐threatening side effects: Neutropenia, allergy, lethal hepatic toxicity, carcinogenesis, mutagenesis, second malignancies, leukemia

• Personalized Medicine: the right drug at the right dose for the right patient

• July 1st to Dec 12th 2010

Pharmacogenetics: slow metabolizer?

• Cancer Genomic Signature• Prospective clinical validation

to identify how aggressive a breast tumor is

• Gene expression of cancer progression related genes (21‐70)

• 50% of women receiving chemo at early stage do not get the benefits, but do get the side effects

• Estimate the risk of recurrence in 10 years

• Breast cancer at age 50 or younger• Ovarian cancer at any age• Male breast cancer at any age• Two breast cancers in the same person or on the

same side of the family• Triple negative breast cancer at any age• Pancreatic cancer and an HBOC‐associated* cancer

in the same person or on the same side of the family

• There are three family members with breast cancer in the same side of the family

• A previously identified BRCA1 or BRCA2 mutation in your family

Genetic Risk Assessment….

PDQ® Cancer Information Summary. National Cancer Institute; Bethesda, MD. Genetics of Breast and Ovarian Cancer (PDQ®) ‐ Health Professional. Date last modified 04/24/2009. Available at: http://www.cancer.gov/cancertopics/pdq/genetics/breast‐and‐ovarian/healthprofessional. Accessed 05/15/2009.National Cancer Institute. SEER Cancer Statistics Review, 1975–2005. Retrieved April 20, 2009, from: http://seer.cancer.gov/csr/1975_2005/index.html.

The Crusaders: Dr Jeffrey Weitzel and Dr Kathy Blazer

• BRCA genes mutation expert• Develop Genetic Awareness in

Mexico and Latin America• Build a BRCA analysis network and

working group• Awareness workshops in México,

Brazil, Colombia, Peru, Panama, Ecuador

• Wet lab training for Mutation Analysis of BRCA

• Genetic counseling training for data interpretation

A journey of 3 years and three crusaders…

In pursuit of a world without cancer• Cancer is a multifactorial disease

that causes multiple pains, physical and psychological

• Genetic and Genomic tools are critical in order to determine the right diagnostic, treatment and follow up for cancer patients

• It is necessary to join efforts to make any contribution that increases awareness around the Genetic and Genomic tools.

• Joint decision making

In pursuit of a world without cancer

Genetic and Genomic Testing save lives and help to improve the condition of cancer patientsHope never dies…..

Sponsored by:



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Science Webinar Series Slides_Breast Can… · Science Webinar Series ... Breast cancer affects 1 in 8 women When a BRCA gene is mutated the risk of ... • 30 archived samples with

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