« Molecular Pathology for Dummies - BGDO · 2019. 5. 29. · BGDO DIGESTIVE ONCOLOGY COURSE 2019 Molecular pathology for dummies Nicky D Haene Guinney et al. Nature med 2015 ; TCGA

BGDO DIGESTIVE ONCOLOGY COURSE 2019 Molecular pathology for dummies Nicky D Haene

« Molecular Pathology for Dummies »

D’Haene Nicky, MD PhD

Department of Pathology

Erasme Hospital – Université Libre de Bruxelles (ULB)

PLAN

Introduction

Technology of Next Generation Sequencing Report generation and clinical interpretation

Applications in Oncology

Our experience

Conclusions

23 mai 2019 2


Guinney et al. Nature med 2015 ; TCGA Nature 2012

23 mai 2019 44

The Sequencing Explosion

Sequencing : determining the order of nucleotide bases

1990 – 2003: 1st sequencing of human genome (13 years )

Sequence the 3 billion base of the human genome

Discover the 20.000 – 25.000 human genes

Cost 3 billion $ (1$/base)

NGS


5

23 mai 2019 6

Brennan et al. Cell 2013

Discovery of new driver cancer genes


23 mai 2019 7

Lung Adenocarcinoma

Melanoma Thyroid Carcinoma

Molecular characterization

23 mai 2019 8


23 mai 2019 9

The right drug, the right dose for the right patient at the right time

Stricker – Semin Oncol 2011

23 mai 2019


23 mai 2019

June 2015

2019: + Olaparib – Ovary cancer ‐ BRCA+ Osimertinib – NSCLC ‐ EGFR T790M+ Crizotininb‐ NSCLC – ROS1+ Pembrozilumab – PDL‐1….

23 mai 2019 12

Tumor Biomarker

Tests should be performed on tumor cells

tests on the histological specimens


Pathological diagnosis: methodology

Integration

Macroscopy Organ TNM/ Staging

Microscopy Tissue/cell Classification/grading

Immunohistochemistry Protein DiagnosticPrognosticTheranostic

Molecular pathology DNA, mRNA DiagnosticPrognosticTheranostic

Pathological diagnosis: methodology

Macroscopy Organ TNM/ Staging

Microscopy Tissue/cell Classification/grading

Immunohistochemistry Protein DiagnosticPrognosticTheranostic

Molecular pathology DNA, mRNA DiagnosticPrognosticTheranostic

BiomarkersMolecular Pathology


“CENTRAL DOGMA”

GTGCATCTGACTCCTGAGGAGAAGCACGTAGACTGAGGACTCCTCTTC

GUGCAUCUGACUCCUGAGGAGAAG

--V-----H-----L----T------P-----E-----E-----K--

DNA (transcription)

RNA (translation)

Protein

GENETIC ALTERATIONS IN CANCER

Development and progression of cancer is associated with alterations in the cancer DNA including:

• Structural aberrations (translocations,inversions) ex : NTRK fusion• Copy number alterations (duplication, amplifications) ex : HER2 amplification• Deletions, insertions • Base substitutions (point mutations) ex : RAS mutations


TOOLBOX OF THE MOLECULAR PATHOLOGIST

23 mai 2019 17

IHC FISH PCR NGS

Target Proteinexpression

1 Generearrangement, amplification,

deletion

1GeneDNA: mutation

RNA : rearrangement

GenesDNA: mutation,

indels, amplification

RNA : rearrangement

23 mai 2019 18



23 mai 2019 19

IHC FISH PCR NGS

Target Protein Gene Gene Gene

Precision + ++ + +++

Hands on time ‐ +++ + ++

Cost +‐++ ++ +‐++ +++‐++++

TAT 48h 10 days 10 days 10 days


23 mai 2019 20

IHC FISH PCR NGS

Target Protein Gene Gene Gene

Example MSI HER2 amplification

BRAF V600 Gene panels

MLH1 MSH2

PMS2 MSH6


SANGER SEQUENCINGFIRST GENERATION SEQUENCING

23 mai 2019 21Moorcraft et al. Crit rev oncol 2015

23 mai 2019 22

Up to 1000 bases1 region at a time


Definition :Technologies that share the ability to massively sequence millions of

DNA templates in parallel

DNA Library Clonal amplification sequencing

NEXT GENERATION SEQUENCING

23 mai 2019 24


23 mai 2019 25

23 mai 2019 26

1 bead = 1 read

11 millions wells


DATA ANALYSIS

23 mai 2019 27

23 mai 2019 28

Li et al. J Mol Diagn 2017


23 mai 2019 29

http://cancer.sanger.ac.uk/cosmic

23 mai 2019 30

www.mycancergenome.org


https://pct.mdanderson.org

31

23 mai 2019 32

Clinical Interpretation

« Sequencing tumor board » / « molecular rounds » Up to 15 faculty members

Cancer genomics, bioinformatics, pathology, clinical genetics, bioethics, clinical oncology, experimental therapeutics

Not many mutations have been validated with a high enough level of evidence to predict for response to targeted treatment

New challenges: Is this an activating or inactivating mutation? Does this mutation engender sensitivity to specific targeted therapy? How to select therapy in case of multiple genomic alterations? Is this a germline mutation?

Tumor context ! Quid preclinical studies?

DIENSTMANN ET AL. MOL ONCOL 2014


23 mai 2019 3333

Sanger Sequencing Low throughput (100kb) High cost Slow Low sensitivity (20-30% of mutant

DNA) -> low coverage depth

Next generation sequencing High throughput (1-100 Gb) Low cost Fast High sensitivity

-> high coverage depth

SANGER SEQUENCING / NGS

34

Coverage

Sensitivity = 5%

Coverage 20x ‐> 1 mutated read ???

Coverage 1000x ‐> 50 mutated reads

Definition : number of times a nucleotide (or a region) is read during the sequencing process.


35

Coverage

coverage : coverage depth (x) = numbers of times a nucleotide is sequenced

Mean coverage = average number of times a base is sequenced

36

Next Generation Sequencing: Applications in oncology


37

Whole Genome Sequencing (WGS)

Structural variation (SV)

Single nucleotide variation (SNV), also in non coding regions

Copy number variation (CNV)

Comparison of the tumor genome with a paired normal genome

Meyerson et al., Nat Rev Genet 2010

38

Exome Sequencing (WES)

Exome : coding part of the genome -> 1% of the genome (30Mb)

Genome = 3 Gb

‐> 90 Gb for 30x coverage

Exome

‐> 3Gb for 75x coverage

Lower frequency variant

Less data -> Cost, analysis !


39

Applications in Oncology Clinical practice ???

Adapted from Roychowdhury et al. Sci Transl Med; 2011

NGS : APPLICATION

40

Inherent characteristics of tumor samples Aneuploidy

Tumor heterogeneity

Contamination with normal tissue

Low frequency variant detection : High coverage necessity

Samples characteristics Quantity : small biopsy, cytology

Quality : FFPE -> DNA integrity

Purity : tumor infiltration

Cancer Specific challenges


41

Coverage

% tumor cells : 10‐90%

5 – 45% of mutated DNA

2 – 40% of mutated DNA

Sensitivity = 5%

Coverage 20x ‐> 1 mutated read ???

Coverage 1000x ‐> 50 mutated reads

Germinal mutation (heterozygote) ‐> 50 % of mutated DNA ‐> ½ mutated read

Somatic mutation

42



43

Targeted DNA Sequencing

Only the target of interest -> gene panels

Applications -> Daily practice

Tumor molecular diagnostic -> Known Hotspots of several genes (tumor tissue

testing)

Commercial panels

Custom panel

Design flexibility

Coverage

Number of amplicons (targeted region)

Number of patients -> barcodes

‐> 100 amplicons, 10 patients, 1000x coverage

‐> 1000 amplicons, 1 patient, 1000x coverage

‐> 1000 amplicons, 10 patients, 100x coverage

23 mai 2019 4444

Ion AmpliseqTM Colon & lung Panel = 22 genes

PIK3CA

NRAS

NOTCH1

MET

MAP2K1

KRAS

FGFR3EGFR

FGFR2DDR2

TP53FGFR1CTNNB1

STK11FBXW7BRAF

SMAD4ERBB4ALK

PTENERBB2AKT1

PIK3CA

NRAS

NOTCH1

MET

MAP2K1

KRAS

FGFR3EGFR

FGFR2DDR2

TP53FGFR1CTNNB1

STK11FBXW7BRAF

SMAD4ERBB4ALK

PTENERBB2AKT1



4545

Ion AmpliseqTM Comprehensive Cancer Panel = 409 genes


23 mai 2019 46


Validation of targeted NGS for solid tumors using the different platforms. FFPE

Cytology

96 to 100% concordance between NGS and conventional testing

NGS outperforms Sanger sequencing Sensitivity

Time

Cost

23 mai 2019 47

D'Haene, PlosOne 2015; Le Mercier Histopathology 2015; Cottrell. J Mol Diagn.2014; Hadd J Mol Diagn. 2013; Singh J Mol Diagn. 2013;de Biase PLoS One ;TopsBMC cancer. 2015; Endris J Mol Diagn. 2013; Deeb Arch pathol lab med. 2015

48



23 mai 2019 49

RNA seq for gene fusion detection

DNA

RNA

23 mai 2019 50

NGS: Erasme experience

Platform : Ion Torrent – PGM

AmpliSeqTM Colon and Lung Panel

Evaluation of the use of targeted NGS for CRC routine samples


51Siena, JNCI 2009

23 mai 2019 52

Material and methods

Prospective study

CRC samples from 14 different institutions (Nov 2013-Dec 2015)

formalin-fixed paraffin-embedded cell blocks

Ion Torrent AmpliSeq colon/lung cancer panel

DNA from FFPE block

PCR multiplex amplification with colon/lung cancer panel (22 genes)

Library purification and barcoding

Clonal amplification on ISP(Ion One Touch 2)

Sequencing on PGM (318 chip)

Data Analysis (Variant caller, IGV)

10 ng

PIK3CA

NRAS

NOTCH1

MET

MAP2K1

KRAS

FGFR3EGFR

FGFR2DDR2

TP53FGFR1CTNNB1

STK11FBXW7BRAF

SMAD4ERBB4ALK

PTENERBB2AKT1

PIK3CA

NRAS

NOTCH1

MET

MAP2K1

KRAS

FGFR3EGFR

FGFR2DDR2

TP53FGFR1CTNNB1

STK11FBXW7BRAF

SMAD4ERBB4ALK

PTENERBB2AKT1


REPORTING Mutations > 4% with a minimum of 30 variant reads (& min 250x

total reads per amplicon)

Amplicons < 250x are reported Non-informative

Variant < 4% for key mutations (KRAS, NRAS….)

comment in the conclusion

ask for new sample

Only mutations from the COSMIC database

Interpretation :

3 categories

Known clinical impact

Potential clinical impact

Unknown clinical impact

23 mai 2019 54

Clinical series (n= 741)

N= % Primary tumor/metastasis (n=696) 584/112 83.9/16.1 % Sample Types (n=708)

Cell block Biopsy Surgical resection

7 311 390

1 % 44% 55%

% of Tumor Cells (n=735) <10% 55 7.5 % 10-50% 559 76 % >50% 121 16.5 %

Sequencing performance (n=741) Non-informative 14 1.9% Informative 727 98.1%

Results


Results

Number of mutations per tumor ranged from 0 to 5 (mean: 1.6)The most frequent mutations were found in TP53 (61.8%) and KRAS (46.1%)

0 1 2 3 4 5

number of mutations

0

50

100

150

200

250

300

350

Nu

mb

er

of

ob

ser

va

tio

ns

650 samples (89.4%) showed at least one mutation

23 mai 2019 56

Genes Present study cBioPortal database

KRAS 46.1% 42-55%

NRAS 4.4% 2.8-9%

BRAF 10.7% 4.3-9.9%

PIK3CA 13.8% 14.8-30.6%

ERBB2 mutation 0.4%amplification 0.3%

mutation 2.8-4%amplification 3.1%

AKT1 0.1% 0.9-1.4%www.cbioportal.org

CRC mutational profiles


Turn Around Time (TAT)

23 mai 2019 57

The median TAT between reception of the sample in the laboratory and report release was 8 calendar days. For 75.7% of the cases, the report was released within 10 calendar days after receiving the samples. Only 17 cases (2.3%) had a TAT above 2 weeks.

Workflow 1 to 2 run per week

Collection of sample (Thursday)

DNA extraction (Friday)

Library prep (Monday)

Clonal amplification + sequencing (Tuesday)

Analysis + reporting : Wednesday

Agreement with some centers to receive samples Wednesday or Thursday

one week workflow


59

TAT

wet-bench (from sample reception to sequencing : 3-4 days)

dry-bench (4 – 24 hours)

Analysis

Answer within 10 working days

COST

INAMI/RIZIV reimbursement

Limitations

Conclusions

Requirements for clinical testing include the test must be performed on routine samples with low DNA

content

the test results must be delivered rapidly

the test results must be accurate and facilitate clinical decision making.

targeted next generation sequencing

Attractive new technology Low DNA amount (10 ng) – FFPE Blocks

Gene panel testing

Mutational profile

Personalised medicine23 mai 2019 60


Conclusions

www.CAP.org

23 mai 2019 62

ACKNOWLEDGMENTS

Department of Pathology – Erasme Hospital

Isabelle Salmon

Molecular Pathology Lab

Claude Van Campenhout

Sarah De Clercq

Nancy De Nève

Oriane Blanchard

[email protected]

02/555 33 35


23 mai 2019 63

Documents

« Molecular Pathology for Dummies - BGDO · 2019. 5. 29. · BGDO DIGESTIVE ONCOLOGY COURSE 2019 Molecular pathology for dummies Nicky D Haene Guinney et al. Nature med 2015 ; TCGA