Basic Molecular Pathology · 2019. 11. 23. · breast cancer patients within 10 years who is going to be treated with adjuvant endocrine therapy. • Identification of different breast

Chit Chow (PhD)Laboratory Development ManagerDept of Anatomical & Cellular Pathology, The Chinese University of Hong Kong

27th April, 2019

Basic Molecular PathologyPractical Perspective from A Laboratory Scientist

Disclaimer

• No financial interest• I am not a pathologist…• I am not a clinician…• I am a lab scientist of anatomical pathology lab. Thus I will focus my

talk on molecular technologies for anatomical pathology.

Molecular Pathology: A Piece of Jigsaw Puzzle of Pathology

• Study the root cause of diseases at genetics or molecular level

• To provide genetics informationAid diagnosis of anatomical pathologyGuide personalized / precision medicineDisease prognosis

• Like morphology, immunohistochemistry and other assays, molecular pathology is a piece of jigsaw puzzle in the world of pathology.

Cancer: Genetic disorders

• Cancers are genetic disorders• Activation of oncogenes• Inactivation of tumor suppressor genes• Results in uncontrolled cell growth, invasion and

metastasis

• Molecular pathology: Dissecting the root cause of cancer pathogenesis.

• Better diagnosis via detection of hallmark mutations.• Design precision treatment strategies.• Provide prognosis information.• Improve patient management.

Dauglas Hanahan, Robert A. Weinberg, Cell, 2011)

Dauglas Hanahan, Robert A. Weinberg, Cell, 2000

Genetic Abnormalities of CancersEpigenetics

• Promoter Hypermethylation• Promoter hydroxymethylation• Histone modifications

Genomics

• Gene mutations• Gene translocations• Copy number variations

Gene Expression

• Activation of oncogenic pathways

• Down-regulation of tumorsuppressor

• Expression of mutated genes

Sanger / Pyro-Sequencing

Mutation-specific PCR

MALDI-TOF mass

spectrometry

Immunohisto-chemistry

Fluorescence in-situ

hybridization

Array-CGH / SNP Array

RT-PCRMethylation-specific PCR

Methylation array

Gene Expression

Array

RFLP

ChIP-PCR/ ChIP-chip

Dete

ctio

n Pl

atfo

rms

Next-Generation Sequencing

NanoString

Molecular Platforms: General Considerations• Clinical questions• Specimen compatibility

• Fresh specimen vs FFPE vs Cell-free DNA/RNA• Sensitivity requirement

• Homogeneous (Germline variants) vs Heterogeneous (Somatic Variants)• Complexity / Robustness / Cost / Through-put / Turn-around time• Validated platform vs Laboratory-developed test• Laboratory set up

• Space, workflow, ventilation etc• Personnel, training etc…

Considerations on Specimen TypesSpecimen Types Advantage Disadvantage

Fresh / Frozen tissues

Provide the highest DNA / RNA quality Not compatible to routine IHC workflow.Not easy to preserve and store.Not readily available.

FFPE tissues Available as routine histological specimens.Specimen can be archived at room temperature.

DNA and RNA are damaged and cross-linked byformalin- DNA / RNA fragmentation- Introduce artefacts- Low yield

Liquid biopsy Easy for collectionFast TATLow costVirtually unlimited supply of cancer-derived DNA

Variation on ctDNA fraction- Need highly sensitive platforms- Sampling error, false negative resultsDNA / RNA fragmentationGenomic DNA / RNA releases from WBC over time.- Need special blood collection tube

Platforms for Single Gene / Small Panel Mutations

Platforms for Single Nucleotide Variants /Small Insertion-deletions

Platform Mutant coverage Sensitivity (Mutant level)

Specimen compatibility

Fresh / frozen tissue

FFPE tissue

CirculatingcfDNA

Sanger sequencing Whole gene scanning 10-20% + + -

Mutant-specific PCRDigital PCR

Mutant-specific

1-5%0.1%

++

++/-

+/-+

Mass spectrometery(MALDI-TOF) 0.1-5% + + +/-

Restriction fragment length polymorphism 5% + + -

Mutant-specific IHC N.A. +/- + -

Sanger Sequencing: Most Essential Tool for Mutation Detection and Genotyping

• Widely adopted by molecular laboratories.• Determine the DNA sequence of the targeted regions.• Versatile technique:

• Just need a pair of primers for PCR and sequencing.

• TAT: < 24 hours from sample to result.• Robust technology.

DNA extractionPCR andclean up

Terminator-Sequencing Clean up

Capillary electrophoresis

and data analysis

EGFR wild-type

EGFR p.L858RT/G

• Limitation on Analytical Sensitivity: 5-10% mutant DNA on background of wild-type DNA (10-20% tumor cellularity)

• Tedious procedure• Prone to human error• Difficult for high throughput analysis

• Quality of sequencing decreases with length of sequencing product:• Sequence up to 1000bp.• Best sequencing result for up to 600bp.

Sanger Sequencing: Limitation

50% KRASG12D DNA

10% KRASG12D DNA

5% KRASG12D DNA

0% KRASG12D DNA

Mutation-Specific PCR: Real-time PCR

Chemistry ARMS + scorpion probe ARMS + Taqman probe Universal primer + Hyb-probe

Inhibition of wild-type DNA PCR by blocker

Example QIAGEN TheraScreenEGFR RGQ PCR Test

QIAGEN TheraScreenKRAS RGQ PCR Test

Roche cobas EGFR Test Roche KRAS PCR test Diacarta QClamp EGFR TestPanagene PNAClamp EGFR

Test

Sensitivity 1% mutant DNA 5% mutant DNA 5% mutant DNA 0.1% mutant DNA

Mutant DNA

FQ

Melting-curve analysis

Gene-specific primer

X

FQ

X

Mutant DNA

Mutant-specific primerFQ

F Q

X

X

Mutant DNA

Mutant-specific primer F Q

FQ

X

X

Mutant DNAF Q

FQ

WT DNABlocker

No PCR product

X

X

Mutation Detection by Real-time PCR

• Strength:• Simple and straight-forward workflow. Suitable for mid-throughput volume.• High sensitivity (detect 1%-5% mutant DNA; c.f. 10% for Sanger sequencing).• Fast TAT (< 8 hours from sample to result).

• Weakness:• Only cover hotspot mutations, not for whole gene screening.• Difficult to optimize, especially for multiplex detection.• More expensive (for validated test kits).

DNA extraction PCR Data analysis

Running PCR with single copy of DNA Partition PCR reaction in tiny volume

25uL with 100 copies of DNA

0 or 1 moleculein each well

PCR

No. of positive wells

Dilution

Digital PCR: Ultimate Sensitivity and Specificity

Digital PCR for Mutation Detection

Sample with wild-type DNA

WT Droplet

Mutant Droplet

WT Droplet

Mutant Droplet

PCR Analysis

Flow CytometryData Analysis

Droplet with no DNA

Droplet with mutant DNADroplet with WT DNA

Partitioning

WT DNAMutant DNA

Probe for mutant DNAPCR Reagent

Probe for WT DNA

Sample with wild-type and mutant DNA

Digital PCR for Cancer Diagnosis

• High Sensitivity:• High differentiation power of mutant / WT DNA• Most suitable for detection of tumor-derived circulating mutant DNA

• Absolute Quantitation:• Count the no. of mutant / WT DNA copy• Indicator of tumor loading / disease progression

• Applications:• Non-invasive mutation detection for solid cancers using ctDNA (EGFR for lung

cancers, KRAS for CRC etc…)• Detection of drug-resistant mutations of solid cancers• Minimal residue disease detection of leukemia

MALDI-TOF for Mutation Detection• Detection of mutations by MALDI-TOF

after single base-extension.• Strength:

• Suitable for multiplexing and mid-high sample throughput.

• Quantitative results.• Fast and simple workflow (TAT < 8hr).

• Weakness:• High setup and assay costs.

G

A

PCR

G

AG

AG

A

G

A

Primer extension with single base

WT DNA

MuT DNA

C-X

T-X

Mass-spectrometer

Time-of-flight

Sign

alIn

tens

ity CT

Platforms for Copy Number Variants / Gene Translocation

Platforms for Copy Number Variants / Gene Translocations

Platform Single locus / Genome-wide

Resolution Specimen compatibility

Frozen tissue FFPE tissue

Fluorescence in-situ hybridization (FISH)

Single locus 200kb – 1Mb + +

MLPA Single locus / Gene panel Exon level + -

Microarray (SNP / aCGH) Genome-wide >= 40kb + -

Platform Require known breakpoint?

Specimen compatibility

Frozen tissue FFPE tissue

Fluorescence in-situ hybridization (FISH)

No + +

RT-PCR Yes + +/-

Copy

num

ber V

aria

nts

Gene

Tr

anslo

catio

n

• Probe gene-specific locus by fluorescence probes for visualization under microscope.

Fluorescence in-situ Hybridization (FISH) for Gene Translocation and Copy Number Variation Detection

Tissue Section Pretreatment:Digest and permeablize tissue

Hybridization of fluorescence-labelled DNA probe to the

genomic DNA

Wash and visualize signals under fluorescence microscope

Fluorescence in-situ Hybridization (FISH) for Gene Translocation and Copy Number Variation Detection

SS18 3’

SSX1SS18 5’SSX1

ChrXChr18

SS18

Chr17

CEN17

HER2

PDGFBCOL1A1

PDGFB

Chr22Chr17

COL1A1

Break-apart FISH Fusion FISHCopy Number FISH

Fluorescence in-situ Hybridization• Strength:

• Detect genetic abnormality in each individual cells• TAT: 1-2 days (single-day FISH with specific FISH buffer)• Simple laboratory set up: Ovens / hybridizer, water bath and fluorescence

microscope

• Weakness:• Tedious experiment and scoring procedure• Only analyse one or few targets per test• Difficult to detect cryptic rearrangement• Difficult to interpret atypical result

Platforms for Gene Expression Profiling

Molecular Classification of Cancers by Gene Expression Profiling

• Gene expression profile is the Signature of different cancers and their subtypes

• Identification of different subtypes, which cannot be distinguished by histopathology

• Stratification of prognosis of cancer patients

DeRose et al., 2011

Mischel et al., 2004

Gene-Expression Profiling Platforms

Platform qRT-PCR NanoString Microarray

Example Oncotype Dx ProSignaLymph2Cx

Mammaprint

Strength Low cost on equipmentand assay

Very robustAmplification-free GEPCompatible to FFPE RNA

Whole transcriptome GEP or GEP of selected genes

Weakness Limitation on multiplexing

Expansive assay.Limitation on number of targets (

NanoString Technology: Molecular Counting

From NanoString Technologies

Clinical Applications of NanoString Technology• Prognostic test of early stage HR+ breast cancers (ProSigna test)

• Determine risk of distance recurrence of stage I/II post-menopause HR+ breast cancer patients within 10 years who is going to be treated with adjuvant endocrine therapy.

• Identification of different breast cancer molecular subtypes (Luminal A, Luminal B, Basal-like and HER2-enriched)

• Molecular subgroups of medulloblastoma• Differentiation between Wnt, Sonic Hedgehog, Group 3 (MYC amplification),

Group 4 (Iso17q, MYCN amplification, CDK6 amplification)

• Molecular classification of DLBCL (Lym2Cx test)• Differentiation of ABC vs GCB subtypes of DLBCL

• Detection of fusion transcript• Design detection probe to span the junction of fusion transcripts

• Detection of gene amplification on DNA level

Next-generation Sequencing:Powerful Technology for Mutation Detection

Next-generation Sequencing: Versatile Platform for High Throughput Mutation Detection• Detection of Gene Mutation: From Sanger sequencing to

NGS• Sanger Sequencing:

• Gold standard technology for mutation detection.• Routine technology of genetic lab.• Sequence single DNA locus per reaction.• For single gene sequencing test.

• Advance of Next-generation sequencing (NGS)• Massively parallel sequencing.• Sequence multiple DNA pieces simultaneously.• For multiple gene, multiple sample sequencing.

Molecular Classification of Cancers: Era of Targeted Therapies

Non-Small Cell Lung Cancer @ 2019

EGFRMutation

ALKTranslocation

ROS1Translocation

RETTranslocation

METMutation/

Amplification

HER2Mutation

EGFRInhibitor

Gefitinib/Erlotinib/Afatinib

Approved by EMA/FDA

ALK/ROS1InhibitorCrizotinib /

ceritinib/ alectinib/ brigatinib

Approved by EMA/FDA

RETInhibitorVandetanib

ClinicalTrial

METInhibitor

Crizotinib

ClinicalTrial

ClinicalTrial

HER2Inhibitor

NeratinibHerceptin

ALK/ROS1Inhibitor

Crizotinib

Fast-TrackApproved by

FDA

NTRK1/2/3Translocation

FDA-approved / Clinical

Trial

TrkInhibitorLarotrectinibEntrectinib

PD-L1Expression

PD1 / PDL1 Immunotherapy

PembrolizumabNivolumab

Approved by EMA/FDA

BRAFMutation

BRAF+MEKInhibitor

dabrafenib and trametinib

Approved by

FDA

Limitation of Conventional Test Platforms• No multiplexing

• Only one or few targets can be tested in each assay• Time-consuming and labor-intensive for a panel of test

• Requirement of different platforms for different kinds of mutations• EGFR mutation: Allele-specific PCR• BRAF mutation and HER2 mutation: Sanger sequencing• ALK translocation: Immunohistochemistry and FISH• ROS1 RET translocation, NTRK translocations: FISH

• Risk of exhausting tiny materials

Next Generation Sequencing Workflow

Library preparation

Clonal Amplification

Sequencing

Data Analysis Pipelines

Single nucleotide

variance

Small insertion/ deletion

Structural variance

(translocation)

Copy Number Variance

AA T

NGS Chemistries

(http://www.nimr.mrc.ac.uk)

Sequencing-by-synthesis (Illumina Chemistry)

(Life Technologies)

Change of Acidity (H+) (ION Torrent chemistry)

NGS for Cancers: Different StrategiesStrategy Region of Interest Pros Cons

Whole Genome Sequencing

Whole Genome(3 X 109)

Identify mutations in whole genome, including non-coding regions (e.g. promoters)

- Expensive, Low coverage.- Difficult in data handling.

Whole ExomeSequencing

Coding Exome + UTR + non-coding genes(~68Mb)

Identify mutations in coding regions / transcribed regions

- May miss genetic translocations- May miss mutations in introns- Low coverage

Targeted Resequencing

User-define - Achieve high coverage.- Cost-effective for targeted genes.

- Require well-defined region

TranscriptomeSequencing

Whole transcriptome (RNA) - Excellent for translocation.- Excellent for expression level

analysis.

- Not suitable to detect SNV / Indel mutations

Bisulfite sequencing / RRBS

Methylated DNA Genome-wide survey of DNA methylation

- Low sequencing efficiency- Difficult in data analysis

Chromatinimmunoprecipitation -sequencing

Methylated DNA / Hydroxymethylated DNA / DNA on modified histone

Whole epigenomic profiling - Robustness?- Difficult in data analysis.

Target Enrichment: Sequence Capture

M

M

M

Gene-specific probe

Magnetic bead

Probe-targeted DNA

Non-targeted DNA

Target-enriched library

Target Enrichment: Amplicon SequencingRegion of Interest

Primer

PrimerTag2

Tag1

PCR

Indexing PCR

Library

BarcodeBarcode

NGS

• Strength• Cost-effective for panel of genes.• Versatile: detecting 4 types of mutations in a single test.• High multiplexing capacity: different samples, different libraries, different

tests can be sequenced together to share the cost.• Highly sensitive. Detection of mutations in both tissue and ctDNA.

• Weakness• Expansive set up cost and running cost.• Complex data analysis and curations.• TAT from 3 days to 7 days, depends on assays.

No Perfect Platform…

Case Study: Atypical ROS1 FISH

• 60/M patient. NSCLC.• EGFR WT, ALK IHC –ve.• ROS1 IHC: Focally positive• ROS1 break-apart FISH: occasionally

isolated green signals (~15-20%).• Corresponding to the 3’end (kinase domain)

of ROS1.• Positive??

Further Investigations• In-house targeted RNA-seq:

• Negative for ROS1 fusions. However, expression of ROS1 was Detected by RNA-seq.

• Support the finding of ROS1 IHC.• The atypical FISH pattern may not result in

fusion transcript.• POSITIVE for CD47-MET fusion.

• Kinase domain of MET gene fused with 5’end of CD47 gene.

• This fusion was reported in paediatric glioma. (Benders et al., 2016, Nat Med)

Expr

essio

n Le

vel ROS1 expression was detected by RNA-seq

CD47-MET was detected by RNA-seq

Case Study: MET exon 14-skipping Mutation

• 57/F, glioblastoma• Refer to RNA-seq test to determine whether the patient is eligible for

Trk inhibitor clinical trial.• Result: Positive for MET exon 14-skipping mutation, confirmed with

RT-PCR.

ATCTGGGCAGT……. (Exon 14)ATCAGTTTTCC…… (Exon 15)

MET Exon 13

Further Investigation on the DNA Level

• MET exon 14-skipping mutation is caused by mutations at intron 13 – intron 14 of MET gene.

• Sanger sequencing using primers flanking the mutation hotspots of intron 13 – intron 14 of MET gene.

• Primer covering -70bp - +30bp of exon 14, detecting >95% MET exon 14-skipping mutations.

• No mutation found….

Framptom et al., 2015, Cancer Discovery

Further Investigation on the DNA Level by NGS• Capture-sequencing of DNA of Case 3 using probes targeting intron 13 – intron 14

of MET gene.• On structural variant analysis, a complex rearrangement on intron 13 and intron 14 was

discovered.

chr1 pos1 gene1 chr2 pos2 gene2 filter type7 116411705 MET_exon_+ 7 116418251 MET_intron_+ PASS DUP

7 116411932 MET_exon_+ 7 116413958 MET_intron_+ PASS DEL

Duplication

DeletionRearranged

Exon-skipped due to loss of 3’ splicing site

13 14 15

151513 13 1414

Large insertion / duplication missed by PCR sequencing on DNA

Take Home Message

• Many new emerging molecular platforms.• Traditional platforms are robust and cost-effective for single or few

targets.• Next-generation sequencing is powerful and cost-effective for

screening of panel of genes, or even whole genome / whole transcriptome.

• No perfect platform. Understanding the strengths and limitations of the platforms.

• Different platforms are complementary with each others.

Future Direction of Molecular Pathology• New platform development

• CRISPR-based mutation detection platform (SHERLOCK)• Third-generation sequencing (single-molecule

sequencing)• New molecular classifications

• Cancer genome projects• Population genome projects

• Genomic-phenotypic mapping• Understand biological / clinical significance of variants• Development of new therapeutic strategies by

combination of genome sequencing and drug screening on cancer organoids / xenografts model.

Gootenburg, Science 2018

Thank you!

Slide Number 1DisclaimerMolecular Pathology: A Piece of Jigsaw Puzzle of PathologyCancer: Genetic disordersGenetic Abnormalities of CancersMolecular Platforms: General ConsiderationsConsiderations on Specimen TypesPlatforms for Single Gene / Small Panel MutationsPlatforms for Single Nucleotide Variants /Small Insertion-deletionsSanger Sequencing: Most Essential Tool for Mutation Detection and GenotypingSanger Sequencing: LimitationMutation-Specific PCR: Real-time PCRMutation Detection by Real-time PCRSlide Number 14Digital PCR for Mutation DetectionDigital PCR for Cancer DiagnosisMALDI-TOF for Mutation DetectionPlatforms for Copy Number Variants / Gene TranslocationPlatforms for Copy Number Variants / Gene TranslocationsFluorescence in-situ Hybridization (FISH) for Gene Translocation and Copy Number Variation Detection Fluorescence in-situ Hybridization (FISH) for Gene Translocation and Copy Number Variation Detection Fluorescence in-situ HybridizationPlatforms for Gene Expression ProfilingMolecular Classification of Cancers by Gene Expression ProfilingGene-Expression Profiling PlatformsNanoString Technology: �Molecular CountingClinical Applications of NanoString TechnologyNext-generation Sequencing:�Powerful Technology for Mutation DetectionNext-generation Sequencing: Versatile Platform for High Throughput Mutation DetectionSlide Number 30Limitation of Conventional Test PlatformsNext Generation Sequencing WorkflowNGS ChemistriesNGS for Cancers: Different StrategiesTarget Enrichment: Sequence CaptureTarget Enrichment: Amplicon SequencingNGSNo Perfect Platform…Case Study: Atypical ROS1 FISHFurther InvestigationsCase Study: MET exon 14-skipping MutationFurther Investigation on the DNA LevelFurther Investigation on the DNA Level by NGSTake Home MessageFuture Direction of Molecular PathologyThank you!