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
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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
Mutation
testing in blood
Microscope
based tests
Single target by
real time PCR or
pyrosequencing
Surrogate to
tissue for
diagnosis
IHC
Molecular: ALK,
MET, PDL1
Multiplex (TILs)
Multiplex testing
on NGS
Monitoring
patients under
TKI
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 testing
A 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 biopsy
in 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 biopsy
in 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 Hybridization
FISH
• 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
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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 Service
DNA analysis in FFPE specimens
Single mutation testing
NGS panels
Methylation assays
DNA analysis in blood
Optimal molecular
diagnostic service
Immunohistochemistry
Single test, IVD protocols
Quantification on digitalised slides
FISH
Partial automation of interpretation
on digitalised slides