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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.

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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

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• 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

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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

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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

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NGS in oncology

TÍTULO DA PALESTRA

SUBTÍTULO

Título

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• 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

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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

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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

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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

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ADC SqCC

ADC SCC

ADC LNEC

CYTOLOGY IS ALWAYS PIONNER!CYTOPATHOLOGIST NEED TO BE IN THE

FRONTLINE

THANK YOU

Molecular Pathology Unit

fschmitt@ipatimup.pt

@fcshmitt