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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
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
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
NGS in Cancer Clinical Research
Gene panels
Bentchtop sequencers
• 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
NGS in Cancer Clinical Research
Gene panels
Bentchtop sequencers
• 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
Screening BRCA1/2 Background
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
Screening BRCA1/2 Background
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:
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
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
Sandra Balladares, Ph.D.Life TechnologiesMexico City, Mexico
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:
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
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
Sandra Balladares, Ph.D.Life TechnologiesMexico City, Mexico
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:
Participating Experts:
Brought to you by the Science/AAAS Custom Publishing Office
To submit your questions, type them into the text box
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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
Harriet Feilotter, Ph.D.Queen’s UniversityKingston, Ontario, Canada
José Luis Costa, Ph.D.IPATIMUPPorto, Portugal
Sandra Balladares, Ph.D.Life TechnologiesMexico City, Mexico
Look out for more webinars in the series at:webinar.sciencemag.org
For information related to this webinar, go to:www.lifetechnologies.com/ampliseqbrca
To provide feedback on this webinar, please e‐mailyour comments to [email protected]
Sponsored by:
Brought to you by the Science/AAAS Custom Publishing Office
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