Molecular Testing of lung cancer in routine practice

Philippe Taniere

Histopathology/Molecular Pathology,

Queen Elizabeth Hospital, Birmingham

Current situation

• EGFR mutation and ALK translocation testing within 3/5 working days at the time of diagnosis

• Challenges– Small specimens, including cytology specimens more

and more– Uncertainty on how to assess ALK (IHC/FISH)– ISO/Accreditation requirements; quality control

schemes

No commissioning system in place

Challenges for the future

• EGFR, BRAF KRAS, PIK3CA, HER2 mutations• ALK, ROS, RET translocations• C-MET, HER2, FGFR1 amplifications• Secondary mutations within EGFR or ALK in patients

under targeted therapy• Monitoring level of mutations in blood for patients under

treatment• PDL1 expression• Tumour infiltrating lymphocytes• ERCC1 level of expression

Molecular profiling of solid tumours in routine practice

Mutation testing in

tissue

Single target by real time PCR or pyrosequencing

Multiplex testing on NGS

Mutation testing in blood

Surrogate to tissue for diagnosis

Monitoring patients under

TKI

Microscope based tests

IHC

Molecular: ALK, MET, PDL1

Multiplex (TILs)

FISH

Molecular Pathology NHS Diagnostic Laboratory

• Multiplex testing–More than one–Less than too many

Clinical relevance

• Diagnostic • Prognostic • Predictive

–Targeted drugs–Non targeted drugs

Clinical relevance

• How do we decide what tests will be part of our panel?

Evidence based• Licensing• Trials• Research papers

Multiplex testingA single panel on next generation sequencing

• Advantages:– Extensive tumour profiling upfront to guide treatment– Choice of therapy if several actionable mutations

• Pitfalls– No consensus on number of genes– DNA “hungry” technology: not suitable for at least

30% of real life specimens– Over two weeks turn around time– Not accredited technology– Complex reports

Next Generation Sequencing

NGS in practice

• Development of clinically relevant panels– SMP2 panel which includes ALK, ROS, RET: lung

specific; trials mainly (Illumina) (50 ng DNA)– Qiagen commercial panels– Illumina commercial panels– OGT– Roche– Etc,..

QIAGEN NGS panel

• Tumor Actionable Mutations panel V2– BRAF, EGFR, IDH1, IDH2, KIT, KRAS, NRAS,

PDGFRA (20 ng DNA)• Clinically relevant tumor panel V2

– AKT1, IDH2, ALK, KIT, AR, KRAS, BRAF, MAP2K1, CTNNB1, MET, DDR2, NRAS, EGFR, PDGFRA, ERBB2, PIK3CA, FGFR3, PTEN, GNA11, RET, GNAQ, STK11, IDH1, TP53 (40 ng)

BIOINFORMATICS

Tumor Actionable Mutations panel V2

Tumor Actionable Mutations panel V2

Mutation testing in blood

Liquid biopsy

• Free DNA: plasma– Diagnostic– Prognostic– Predictive

• Response to targeted therapy• Early relapse

Liquid biopsyin practice

• Working from plasma has become realistic thanks to tubes delaying cell lysis (PAXGENE)

• Sensitivity: around 70%; BEAMing Digital PCR technology: over 90%?

• Targeted: single genes• Commercial kits available

– therascreen EGFR Plasma RGQ PCR Kit exon 19 deletions and exons 20 and 21 substitutions (T790M and L858R respectively); IVD

Liquid biopsyin practice

• Surrogate to tissue testing• Detecting resistance mutation (T790M) in patients under

anti EGFR therapy• Monitoring response to treatment (assuming mutation

was detected in blood at diagnosis) and diagnosing early relapses: ?to switch to second generation of tki

Microscope based tests

• IHC• FISH• Digital Pathology

“Molecular IHC”

• Antibody set up for accurate assessment of level of expression to be clinically relevant– ALK – MET– BRAF– PDL1

FDA inspired; locked protocols

Validated against clinical significance

Fluorescent In Situ HybridizationFISH

• Very accurate• Still extensively used in trials (ALK, ROS,Her2)

• BUT– Labour intensive and expensive – Dark room…

• To be “modernised”– Scanning for digitalisation of the images– Applying algorithms for interpretation

Digital pathology

FISH/IHC interpretation from digitalised slides

• Fluorescent sections scanned• Areas of interest selected on screen• Software calculates rate of positivity with detailed report

on number of cells examined, ratio normal/abnormal etc,..

Anti PD1 therapy

Anti PD1 therapy

PDL1 IHC

Perspective Digital pathology

Tumour infiltrating lymphocytes

• CD3/CD45RO• CD3/CD8 • CD8/CD45RO• More

Immunoscore

Immunoscore

Immunoscore

Predictive markers to response to non targeted chemotherapy

Platinum based drugs and ERCC1

Molecular Diagnostic ServiceDNA analysis in FFPE specimensSingle mutation testingNGS panelsMethylation assays

DNA analysis in blood

ImmunohistochemistrySingle test, IVD protocolsQuantification on digitalised slides

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Optimal molecular diagnostic service