12-11-2019
1
MOLECULAR TESTING
Prof. Fernando SchmittDepartment of Pathology and Oncology, Medical Faculty of Porto University
Head of Molecular Pathology Unit, IPATIMUPPresident of the International Society of Breast Pathology
General-Secretary of the International Academy of Cytology
No financial disclosures
UPS AND DOWNS OF CYTOLOGY
1930 60-80 1990
1904 1950 20092000
M
S
K
C
C
P
A
P
K
S
C
N
B
L
U
N
G
Molecular
Lack of expertise
Cancer Biomarker Drug % eligible
Breast HER2 gene amplification Trastuzumab 30%
Lung EGFR mutation Gefitinib/Erlotinib 12%
Lung EML4-ALK translocation Crizotinib 5%
Colon KRAS mutation Cetuximab/Panitumumab 55%
CML BCR-ABL translocation Imatinib 95%
GIST KIT/PDGFRA mutation Imatinib 90%
Gastric HER2 gene amplification Trastuzumab 20%
Melanoma BRAF mutation Vemurafenib 42%
Biomarkers for therapy selection
PATHOLOGY CONSIDERATIONS FOR
GOOD PRACTICE
• Small biopsy and cytology samples should be
managed not only for diagnosis but also to maximize
the amount of tissue available for molecular studies.
DIAGN CYTOPATHOL 19: 395-397, 1998 DIAGN CYTOPATHOL 27: 210-213, 2002
• Ten years ago, the use of molecular techniques to detect EGFR in lung cancer,
KRAS in colon cancer and cKIT in GISTs was just starting.
• Today, this is routine and cytology frequently is the only available material to be
tested, especially in lung cancer.
• Curiously, a technique that changed cancer genomics, next-generation sequencing
was not mentioned at that time. This technology is rapidly replacing the classical
use of PCR and Sanger sequencing, allowing to study not only DNA but also RNA
alterations.
12-11-2019
2
LUNGCARCINOMA
Small cell morphology
YesP63
Positive
No
No
SqCC-small cell type
Chromogranin/
Synaptophysin/CD56-
Positive
Yes SCLC
P40/CK5
TTF1/Napsin AADC
+++-
SqCC- Or -/+
+++
EGFR
Positive
Yes
Tyrosine kinase
inhibitors
Yes
NoALK
Positive
CrizotinibCancer Cytopathology 2011
NoROS1
Positive
Crizotinib
NoRETPositive
Cabozantinib
No
Molecular testing in NSCLC
EGFR Mutation (10-20% of Tumours)
EML4-ALK Translocation
(3-5%)
Rarer Mutations? (BRAF, MEK1, AKT1, PIK3CA)
No Further Testing
+
–
–
+
Major problems• Tissue limitations• Slow turnaround time• Cost effectiveness
Schmitt FC & Machado JC, 2013
Diagnostic algorithm for ALK testing
NSCLC
ALK IHC
Negative Positive
ALK FISH
NegativePositive
No ALKrearrangement
ALK rearrangement
HER 2
RET
MET
BRAF
KRAS
ROS 1
• Lung cancer is a molecularly heterogeneous disease andunderstanding its biology is crucial for the developmentof effective therapy.
• There are more than 300 non-synonymous mutationsper lung cancer but only a minority can promotetumorigenesis.
• Establish targets: EGFR,ALK,ROS1,BRAF and PDL1
• ESMO recommendation for genetic test: non-squamousand squamous in minimal or non smokers
Genomic Analysis in Lung CancerLUNG CANCER AND PERSONALIZED THERAPY
12-11-2019
3
• Ten years ago, the idea that all of the genes
altered in cancer could be identified at base-
pair resolution would have seemed like
science fiction.
• Today, such genome-wide analysis, through
sequencing of the exome or of the whole
genome, is routine….
Vogelstein et al. Science 2013
The Human Genome Project
The Cancer Genome Project
The Molecular Tools
Why Now?
Screening lung cancer clinically relevant alterations
1. Pao & Hutchinson, Nature Medicine 2012; 2. Socinski MA, et al. The oncologist. 2016.
Diagnosis Progression
NSCLC: 40-50% diagnosed by cytology Precision OncologyMore biology from smaller samples
Smaller tumours/targets Smaller samples
Cytology or blood sample
More biology
12-11-2019
4
Comprehensive testing with less tumor tissue
TOMORROW: multiplex assays
through NGS panels
H&E2 slides8-10 slidesBiopsy
3 slides3 slides
1
Comprehensive
tumor Analysis
2DNA
Panels
Ex: EGFR, KRAS,HER2, MET
RNA Panels
Ex: ALK, ROS, RET
8-10 slidesBiopsy
H&E + squamous /non-sq IHC
3-5 slides
EGFR, KRAS
3 slides
ALK
3 slides
Met4 slides
Based onGuidelines
1
2
3
?
TODAY: serial single assays
“tissue is the issue”
Tissue often not sufficient for
all assays
PAST PRESENT
Cytology specimens are suitable for NGS
Any kind of cytological material can be used for NGS
Lung Cancer Molecular Testing Guidelines - CAP, IASLC and AMP
• Cytopathology has established itself as independent diagnostic modality to guide clinical
management in many different settings.
• While earlier studies have demonstrated that single biomarker testing is feasible on cytology,
currently this information is insufficient to guide patient care.
• More recently, multigene mutational assays, such as NGS have gained popularity because
provide genomic information on multiple genes.
• Cytopathologist plays a key role in ensuring success of NGS in cytology by influencing pre-
analytical steps and selecting adequate material.
NGS in cytology
Conclusions
Good Molecular only with Good Material
• The variability in material and fixatives is still a major factor preventing standardization of some
procedures using cytology.
• Not only the different types of cytological preparations affect the management of cytological
material for further studies, the vials and devices used, quantity and quality of the material
obtained, the type of pathology studied and the molecular technique that will be applied can
influence the success of the test.
• There are three key factors that affect the validity of a molecular assay : the proportion of tumor
cells in the sample, nucleic acid quality and nucleic acid quantity.
Schmitt F. Cytopathology 2019
12-11-2019
5
Tumour cell content:
Which molecular test?
ONCOMINE FOCUS ASSAY
20%
50%
• Cases with < 20% tumor cells are reported to have >20% cells in 38% of the cases possibly
causing false negative results.
• In conclusion, estimates of tumor cell percentages on stained slides are not accurate, which
could result in misinterpretation of test results.
• Reliability could possibly be improved by using a training set with feedback.
Polyploidy – a challenging aspect in
tumor cell quantification for molecular
analysis
•Polyploidy should have been take in account in tumor cell quantification because can be a cause of
discrepancy with AF detected by NGS.
•Morphological correlation with molecular results is essential for a correct interpretation of molecular
tests.
Quadros C et al. 2019
Cancer Cytopathology 2017
•Genomic reference standards representative of routinecytology clinical practice showed highly reproducibleresults across all laboratories in detection of mutations
down to 5% of AF despite the difference in smearsstaining and sequencing practices.
Cancer Cytopathology 2019
Molecular Cytopathology Group
12-11-2019
6
NGS in oncology
TÍTULO DA PALESTRA
SUBTÍTULO
Título
• Lorem ipsum dolor sit amet, consectetur adipiscing elit. • Nunc et cursus nibh. • Ut nec lorem congue, rhoncus nunc euismod, imperdiet purus. • Nam ultrices velit ut mi pulvinar sodales. • Vestibulum imperdiet neque et dolor egestas• Sit amet gravidapurus rhoncus.
• Science still early.
• More data should be in public domain.
• Many variations not clinically relevant.
• Costs still needs to go down.
• Ethical issues in testing individual genotype.
• Still Unclear how to deliver information to the practitioner.
There is still lots to figure out…
PERSONALIZED HEALTHCARE IN
THE NEXT 10 YEARS
12-11-2019
7
Biomarkers for Immunotherapy
Cancer Immunosurveillance
Adapted from Schreiber RD et al. Science 331:1565-1579, 2011
Checkpoint inhibitors
• Ipilimumab (CTLA4)
• Tremelimumab (CTLA4)
Checkpoint inhibitors
• Nivolumab (PD-1)
• Pembrolizumab (PD-1)
• Atezolizumab (PD-L1)
• Durvalumab (PD-L1)
As preanalytical processing varies significantly
from histology specimens, especially for conventional
cytology specimens, cytology specific PD-L1
protocols need to be established and validated
12-11-2019
8
Biomarker
candidates
Cellular
markers
Cancer stem cells1
Immune cells5
DNA-
based
markers
Chromosomal
aberrations9
Specific gene
mutations7
Microsatellite
instability6
Tumour mutational
burden4
SNPs8
Immune checkpoints12
Protein
markers
Neoantigens4
Cell-surface receptors2
Tumour antigens1
Carbohydrate
determinants3
RNA-
based
markers
Transcripts10
Regulatory RNAs11
Potential biomarkers for Immunotherapy Tumor Mutation Load (TML)
• TML is a measure of the number of mutations within a tumour
genome, defined as the total number of mutations per coding area of
a tumour genome.
• There is large variability of in ML within tumour types, ranging from
few to thousands of mutations.
• TML can be determined by whole-exome sequencing but can be
inferred from sequencing a smaller panel of genes (ex. 324).
Immunogenic vs. non-Immunogenic
non-Immunogenic Immunogenic
Carcinogen-induced
CancersHematologic &
Childhood
CancersOvarian,
Breast,
Prostate
Cancers
Clinical Trials defining a TMB threshold for ICB benefit
12-11-2019
9
Liquid Biopsy
Beca F & Schmitt F. 2017
Three sample types can be used for EGFR and T790M mutation testing in advanced NSCLC:
2. Tumour biopsy
samples
Gold standard sample
type for all EGFR
mutation testing in
advanced NSCLC1–3
3. Cytology samples
Suitable alternative if a
tumour biopsy sample is
not available4–9
1. Plasma1
Rapid turnaround time
to facilitate treatment
decisions
Preferred sample for
testing at progression
EGFR T790M mutation testing on disease progression
12-11-2019
10
ADC SqCC
ADC SCC
ADC LNEC
CYTOLOGY IS ALWAYS PIONNER!CYTOPATHOLOGIST NEED TO BE IN THE
FRONTLINE
THANK YOU
Molecular Pathology Unit
@fcshmitt