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Published byJanel Roberts Modified over 9 years ago
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Molecular Testing of lung cancer in routine practice
Philippe Taniere Histopathology/Molecular Pathology, Queen Elizabeth Hospital, Birmingham
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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
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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
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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
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Molecular Pathology NHS Diagnostic Laboratory
Multiplex testing More than one Less than too many
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Clinical relevance Diagnostic Prognostic Predictive Targeted drugs
Non targeted drugs
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Clinical relevance How do we decide what tests will be part of our panel? Evidence based Licensing Trials Research papers
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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
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Next Generation Sequencing
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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,..
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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
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Tumor Actionable Mutations panel V2
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Tumor Actionable Mutations panel V2
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Mutation testing in blood Liquid biopsy
Free DNA: plasma Diagnostic Prognostic Predictive Response to targeted therapy Early relapse
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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
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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
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Microscope based tests
IHC FISH Digital Pathology
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“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
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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
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Digital pathology
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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,..
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Anti PD1 therapy
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Anti PD1 therapy
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PDL1 IHC
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Perspective Digital pathology
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Tumour infiltrating lymphocytes
CD3/CD45RO CD3/CD8 CD8/CD45RO More
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Immunoscore
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Immunoscore
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Immunoscore
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Predictive markers to response to non targeted chemotherapy
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Platinum based drugs and ERCC1
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Molecular Diagnostic Service
Optimal molecular diagnostic service DNA analysis in FFPE specimens Single mutation testing Methylation assays NGS panels DNA analysis in blood Quantification on digitalised slides Single test, IVD protocols Immunohistochemistry Partial automation of interpretation on digitalised slides FISH
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