1. Molecular Testing of lung cancer in routine practice
Philippe Taniere
Histopathology/Molecular Pathology,
Queen Elizabeth Hospital, Birmingham

2. 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

3. 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

4. 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

5. Molecular Pathology NHS Diagnostic Laboratory
Multiplex testing
More than one
Less than too many

6. Clinical relevance Diagnostic Prognostic Predictive Targeted drugs
Non targeted drugs

7. Clinical relevance
How do we decide what tests will be part of our panel?
Evidence based
Licensing
Trials
Research papers

8. 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

9. Next Generation Sequencing

10. 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,..

11. 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

12. Tumor Actionable Mutations panel V2

13. Tumor Actionable Mutations panel V2

14. Mutation testing in blood Liquid biopsy
Free DNA: plasma
Diagnostic
Prognostic
Predictive
Response to targeted therapy
Early relapse

15. 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

16. 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

17. Microscope based tests
IHC
FISH
Digital Pathology

18. “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

19. 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

20. Digital pathology

21. 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,..

22. Anti PD1 therapy

23. Anti PD1 therapy

24. PDL1 IHC

25. Perspective
Digital pathology

26. Tumour infiltrating lymphocytes
CD3/CD45RO
CD3/CD8
CD8/CD45RO
More

27. Immunoscore

28. Immunoscore

29. Immunoscore

30. Predictive markers to response to non targeted chemotherapy

31. Platinum based drugs and ERCC1

32. 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