DNA diagnosis of lung cancer

Patrick Willems

GENDIA

•Antwerp, Belgium

Treatment of Lung Cancer

– Small Cell Lung Cancer (SCLC)• chemotherapy• radiation

– Non-Small Cell Lung Cancer (NSCLC)• surgery• radiation• chemotherapy• targeted treatment• immunotherapy

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Personalized cancer treatment

• Immunotherapy to stimulate immune response to cancer

PD-1 inhibitors

PD-L1 inhibitors

CTLA-4 inhibitors

• Targeted therapy with designer drugs

that target the genetic cause of the tumor

mAB: Herceptin

TKI: Gleevec

Problems in personalized cancer treatment

• Immunotherapy

Extremely expensive (100-300.000 Euro/year)

Few biomarkers (companion diagnostics)

• Targeted therapy with designer drugs

Very expensive (50-100.000 Euro/year)

Biomarkers (companion diagnostics)

Problems in personalized cancer treatment

The very high cost of personalised treatment makes

companion diagnostics (cancer biomarkers) necessary

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Cancer biomarkers

tumor material (biopsy)

blood (liquid biopsy)

Market for tumor biomarkers in Liquid biopsies

TARGETS DRUGS SEQUENCING

Liquid biopsy market

for tumor biomarkers:

40 Billion USD per year

(Illumina estimate)

PHYSICIAN

Current paradigm

sampleResult

Pathological studies

PATIENT

PATHOLOGIST

general

treatmentvisit

Lab

PHYSICIAN

Future paradigm

sampleResult

Molecular testing

PHARMA

PATIENT

LAB

Personalised

treatmentvisit

Pathologist

Mortality

UK, 2009-2011

Cancer Morbidity and Mortality

Canada, 2007

New cancers per year in Belgium

• Lung : 7.100

• Colon : 6.500

• Prostate : 8.800

• Breast : 9.700

TOTAAL : 65.000

Lung cancer

• 14 % of all cancer

• 80 % is non–small cell lung cancer (NSCLC)

• Belgium : 7.100 new cases per year

• Worldwide : 10 million new cases per year

• Worldwide : 8 million fatalities per year

•   The main cause (20-30%) of cancer-related death

in both men and women :

• More women die of lung ca than breast, cervical and uterine ca combined.

• More men die of lung ca than prostate and colorectal ca combined.

Treatment of Lung Cancer

– Small Cell Lung Cancer (SCLC)• chemotherapy• Radiation

– Non-Small Cell Lung Cancer (NSCLC)• surgery• radiation• chemotherapy• immunotherapy• personalised targeted treatment

Immunotherapy for NSCLC

• CTLA-4 (cytotoxic T-lymphocyte–associated antigen 4) :

ipilimumab

 

• PD-1 (programmed death-1) :

nivolumab, pembrolizumab

 

• PD-L1 (programmed death-1 ligand)

BMS-935559, MPDL3280A

 

Inhibition immune checkpoints

Biomarkers for immunotherapy for Lung Ca

Few biomarkers for immunotherapy

First real biomarker : Tumor load

(amount of mutations-driver and passenger)

Response to pembrolizumab (PD-1 inhibitor)

better if high mutation load

Science, April 3, 2015 (Rizvi et al)

MSI as Biomarker for immunotherapy

MMR deficiency

Genomic instability

Large mutation load in tumor (driver and passenger)

Many mutant proteins - neoantgens

Immune response

Immunotherapy for NSCLC

– Extremely expensive

(100-300.000 Euro/year)

– No biomarkers to select patients

Targeted therapy with designer drugs

• Receptor antibodies (---- ab)

• HER2 : Trastuzumab (Herceptin)

• EGFR : Cetuximab, Pertuzumab

• MET : AMG102

• VEGF : Befacizumab

• Tyrosine Kinase Inhibitors : TKI (---- ib)

• BRC-ABL : Imatinib (Gleevec)

• KRAS : Tipifarnib

• BRAF : Sorafenib

• MEK

• ERK

• mTOR : Everolimus

Receptor antibodiesinhibit receptor kinases

by interfering with ligand-receptor binding

Preventing intracellular signaling

Herceptin Herceptin (Trastuzumab) Inhibits HER2 dimerisation / activation

and the downstream signaling pathways MAPK and AKT/mTOR

Active when there is HER2 overexpression

• Breast ca (25 %)

• Gastric ca (20 %)

Tyrosine kinase inhibitors (TKI)TKI inhibits a Tyrosine kinase

by binding to its kinase domain

Preventing phosphorylation (activation) of target

Gleevec

Gleevec (Imatinib) inhibits Tyrosine kinases

by binding to its kinase domain

Thereby preventing phosphorylation (activation) of targets :

• BCR-ABL (CML)

• cKIT (GIST, Mastocytosis)

• PDGFR (GIST)

Targeted treatment

– Non-Small Cell Lung Cancer (NSCLC)• surgery• radiation• chemotherapy• personalised targeted treatment• immunotherapy

– Small Cell Lung Cancer (SCLC)• chemotherapy• radiation

Targeted treatment of NSCLC

Expensive, but many biomarkers to select patient

Personalised targeted treatment

targets specific somatic mutations

that cause NSCLC

These mutations are patient-specific

These mutations can be detected

by molecular studies of :

tumor (biopsy)

blood (liquid biopsy)

Progress in lung ca treatment

Problems in targeted cancer treatment

The very high cost of personalised treatment makes

companion diagnostics (cancer biomarkers) necessary

The mutations leading to lung ca

are the biomarkers to guide targeted therapy

Inheritance of cancer

• Breast Cancer : 10 %

• Colon cancer : 5-10 %

• Prostate cancer : low

• Lung cancer : very low

• Majority of cancers are caused by genetic anomalies in the tumor

(somatic mutations)

• Minority of cancers is inherited (germline mutations) :

Inheritance of lung cancer

• NO germline mutations

• MANY somatic mutations

Driver and passenger gene mutations

Vogelstein et al, Science Aug 22, 2013

NEJM May 30, 2015

TUMOR MUTATIONS EXPLANATION

HNPCC 1782 Genomic instability

Lung 150 Mutagen (smoke)

Melanoma 80 Mutagen (sun)

Colon with MSS 73

Breast 60

Prostate 40

Leukemia 10 Fast (acute) tumor

Pediatric tumors 10 Young age

Somatic mutations in cancer

P

Breast NSCLC Colon Prostate

TP53 23 34 48 16

KRAS < 10 15-25 35 5

PIK3CA 26 4 22 2

EGFR < 10 10-30 < 10 4

MLL3 7 10 12 5

CTNNB1 < 10 < 10 < 10 4

Somatic mutations in adeno ca NSCLC

TP53 : 34 %

EGFR : 10-30 %

KRAS : 15-25 %

MLL3 : 10 %

STK11 : 9 %

CDKN2A : 8 %

ALK fusions : 5 %

HER 2 : 2%

BRAF : 1-2 %

Ros fusions : 2 %

PTEN : 25 % (loss)

P

Somatic mutations in adeno ca NSCLC

Cell growth and survival pathway

Genetic testing for lung cancer

– EGFR: deletions in exon 19

L858R mutation in exon 21

T790M mutation in exon 20

– KRAS: mutations of codons 12 and 13

– BRAF: V600E, G469A and D594G mutations

– ALK-EML4 fusion

EGFR Mutations in lung cancer

EGFR mutations :

10 % (Europe)

30 % (Asia)

women, non-smokers, adenocarcinoma (NSCLC)

90% of EGFR mutations :

L858R in exon 21 (Sensitivity to TKIs)

Small deletions in exon 19 (Sensitivity to TKIs)

T790M in exon 20 (Resistance to TKIs)

First-generation EGFR tyrosine-kinase inhibitors :

Erlotinib (Tarceva)

Gefitinib (Iressa)

Second-generation EGFR tyrosine-kinase inhibitors :

Dacomitinib

Afatinib (Gilotrif)

P

EGFR mutations

EGFR Resistance : T790M mutation

Inhibitors of EGFR with the T790M mutation :

AZD9291

CO-1831

EGFR resistance : KRAS and BRAF mutations

EGFR

KRAS

WILD

TREATMENT RELAPSE

KRAS Mutations in lung cancer

KRAS mutations : 15-25 % in NSCLC

smokers

90% of KRAS mutations : codon 12 (90 %)

codon 13 (5-10 %)

KRAS Mutations are contraindications for EGFR TKI

BRAF Mutations in lung cancer

BRAF mutations : 1-4 % in NSCLC

55 % of KRAS mutations : V600E

BRAF Mutations are contraindications for EGFR TKI

ALK Mutations in lung cancer

• ALK mutations : 5 % in NSCLC

• ALK activation is caused by EML4-ALK fusion generated by

inv(2)(p21p23)

• ALK mutations are sensitive to ALK inhibitors :

Crizotinib (Xalkori)

Ceritinib (Zykadia)

ROS1 Mutations in lung cancer

ROS1 mutations : 2 %

ROS1 activation is caused by ROS1 fusion to different partners

ROS1 mutations are sensitive to Crizotinib (Xalkori)

P

Why perform genetic studies on tumor DNA ?

• Initial diagnosis and prognosis

Initial therapy

• Monitoring recurrence – metastasis

Secundary therapy

Prognosis according to EGFR mutations

Prognosis according to BRAF mutations

Why perform genetic studies on tumor DNA ?

• Initial diagnosis and prognosis

Initial therapy

• Monitoring recurrence – metastasis

Secundary therapy

Monitoring recurrence - metastasis

• Clinical : imaging

• Tumor markers : CEA

• Circulating tumor cells (CTC)

• Circulating tumor DNA (ctDNA)

Diagnostic tests on tumor DNA

• Solid tumor biopsy

FFPE

Frozen

Fresh

• Liquid biopsy : Circulating tumor DNA (ctDNA)

Blood plasma/serum

Advantages liquid biopsies

• No tissue biopsy needed

• No FFPE fixation

• Profiling the overall genotype of cancer

• primary cancer

• circulating cells

• metastases

• Better evaluation of :

• reaction to therapy

• development of resistance

Why liquid biopsies for Lung cancer ?

• The main cause (20-30%) of cancer-related death

• High percentage of driver oncogenic mutations

• Druggable targets

• Lung biopsy difficult – liquid biopsy easy

• Relatively cheap test

• Association with expert group of Rafael Rosell

Circulating tumor DNA (ctDNA)

ctDNA

•

ctDNA from tumor tissue is released

through secretion, necrosis and apoptosis,

but mainly through apoptosis

Ct DNA

cell-free DNA (cfDNA) is released from healthy, inflamed or cancerous tissue undergoing apoptosis or necrosis

a small fraction of cfDNA is circulating tumor (ctDNA)

very sensitive technology is needed to detect mutations in ct DNA

cell-free DNA (cfDNA)

• Cell-free DNA (cfDNA) in plasma of healthy individuals : Mandel and Métais (1948)

• A proportion of cfDNA in pregnant women is fetus-derived (cffDNA) : Lo et al. (1997)

• Non-Invasive Prenatal testing (NIPT) for Down syndrome:

2012 : start

2015 : > 1 million tests

  Market : 4 billion USD

• Increased concentrations of cfDNA in the circulation of cancer patients : Leon et al. (1977)

• A proportion of cfDNA is tumor-derived : Stroun et al. (1987)

• Circulating tumor DNA (ctDNA) testing (liquid biopsy) :

2015 : start

  Market : 40 billion USD

Advantages of liquid biopsies vs FFPE

• No biopsy needed

• Better representation of :

• Total mutation load• Mutations in metastatic cells• Reaction to therapy• Development of resistance

Tissue biopsy

EGFR

KRAS

WILD

• EGFR TREATMENT • RELAPSE

• TISSUE BIOPSY

Liquid biopsy

EGFR

KRAS

BRAF

WILD

• TREATMENT

• LIQUID BIOPSY

Companies focusing on ctDNA

• Pangaea Biotech• Cynvenio• BGI• Agena Bioscience • Boreal Genomics • Chronix Biomedical • Genomic Health • Guardant Health• Inivata• Molecular MD • Myriad Genetics• Natera • Personal Genome Diagnostics• Sysmex Inostics• Trovagene

Liquid biopsy market

for tumor biomarkers:

40 Billion USD per year

Companies focusing on ctDNA

Most companies have an expensive test (5000 USD)

based upon NGS (Next Generation sequencing)

detecting many mutations in many cancer genes

of which the majority are currently “nondruggable”

Sensitivity to detect mutant sequence

• Sanger sequencing : 10 %

• Next gen sequencing (NGS) : 1 %

• NGS + specific technology : 0.1 %

Pangaea biotech

• Simple, relatively cheap test for lung cancer

• Spin off company from IOR (Instituto oncologico Rosell)

• CEO : Rafael Rosell

• Focusing on Lung cancer

• Technology : Mutant Allele - specific PCR using PNAs

Mutant Allele - specific PCR

PCR amplification of wild type allele is blocked

by PNA probe, while mutant is allele is PCR-amplified

ctDNA testing for lung ca

1. DESCRIPTION : ct DNA testing on liquid biopsies :

• EGFR: deletions in exon 19, L858R mutation in exon 2, T790M mutation in

exon 20

 • KRAS: codon 12 and 13 mutations

 • BRAF: V600E, G469A and D594G mutations

2. SAMPLE : blood in specific test kits with Streck tubes provided by GENDIA

3. TURNAROUND TIME : 3 weeks

4. PRICE : < 1000 Euro

How offer ctDNA testing to your patients ?

1. Refer to our consultation :

Email ctDNA@GENDIA.net to ask for an appointment

2. Send blood :

Email ctDNA@GENDIA.net to ask for tubes

www.circulatingtumorDNA.net