DNA diagnosis for colorectal carcinoma

Patrick Willems

GENDIA

Antwerp, Belgium

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Treatment of Colorectal carcinoma

• surgery

• radiation

• Chemotherapy

• Targeted treatment

• Immunotherapy

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

Monoclonal antibodies (mAB): Herceptin

Tyrosine kinase inhibitors (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

The changing face of cancer diagnosis

Cancer Morbidity and Mortality

New cancers per year in Belgium

• Lung : 7.100

• Colon : 6.500

• Prostate : 8.800

• Breast : 9.700

• Melanoma : 1.500

TOTAAL : 65.000

Colorecal carcinoma (CRC)

• second leading cause of cancer related mortality (12.2 %)

• 132.700 new cases anticipated in 2015 in the US

• 49.700 deaths in 2015 in the US

• Five-year survival rates for patients with metastatic disease still low

Treatment of CRC

• surgery• radiation• Chemotherapy• Targeted treatment

– BRAF inhibitor– MEK inhibitor

• Immunotherapy – CTLA-4 inhibitors– PD-1 inhibitors– PD-L1 inhibitors

Immunotherapy for CRC

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

ipilimumab, tremelimumab

 

• PD-1 (programmed death-1) :

nivolumab, pembrolizumab, Lambrolizumab, pidilizumab

 

• PD-L1 (programmed death-1 ligand) :

BMS-935559, MEDI4736, MPDL3280A and MSB0010718C

 

• Other checkpoints : TIM3, LAG3, VISTA, KIR, OX40, CD40, CD137

Inhibition immune checkpoints

Biomarkers for immunotherapy for CRC

Few biomarkers for immunotherapy

First real biomarker : MicroSatellite Instability (MSI)

Response to pembrolizumab (PD-1 inhibitor) in CRC

MMR-proficient : 0 %

MMR-deficient : 40 %

NEJM : May 30, 2015 (Vogelstein group)

MSI as Biomarker for immunotherapy in CRC

MMR deficiency

Genomic instability

Large mutation load in CRC (driver and passenger)

Many mutant proteins - neoantgens

Immune response

Microsatellite instability (MSI)

Targeted treatment for CRC

Personalised targeted treatment

inhibits specific somatic mutations

that cause MM

These mutations are patient-specific

These mutations can be detected

by molecular studies of :

tumor material (biopsy) : FFPE, fresh or frozen

blood (liquid biopsy)

Why liquid biopsies for CRC ?

• Common cancer

• High mortality

• High load of driver oncogenic mutations

• Druggable targets

Inheritance of cancer

• Breast Cancer : 10 %

• Colon cancer : 3-5%

• 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 CRC

3-5 % germline mutations

MANY somatic mutations

Germline mutations in Colon cancer

Polyposis coli: APC gene (Autosomal dominant)

MUTYH (Autosomal recessive)

Hereditary Non Poliposis Coli (HNPCC) :

Autosomal dominant mutations in :

MLH1, MSH2, MSH6, PMS1

HNPCC

Autosomal dominant germline mutation :

1. MLH1, MSH2, MSH6, PMS2 : majority

2. Constitutional (germline) epimutation in MLH1

3. Germline deletion EPCAM gene leading to epigenetic change (methylation-downsilencing of MSH2)

Two step cancer theory (Knudson)

Retinoblastoma (RB1 gene)

Mesothelioma

Uveal melanoma (BAP1 gene)

Multistep cancer theory (Vogelstein)

Vogelstein et al, Science Aug 22, 2013

Colon cancer

Cancer genes and mutations

• 140 driver genes • 60 % TSG• 40 % oncogenes

• > 1000 driver gene mutations(Most tumors 2-10 driver gene mutations)

• Millions (?) passenger gene mutations(Most tumors 10-100 passenger gene mutations)

Mutations in cancer

• Gate keeper mutations : transforms normal cell into tumor cell

Rb in retinoblastoma

APC in colon cancer

• Driver mutations : confers growth advantage to tumor cell

HER2 in breast cancer

KRAS in colon cancer

• Passenger mutations : accidental mutation not conferring

growth advantage to tumor cell

Any gene

Also driver gene

Mutations in cancer

• Inactivation of tumor suppressor genes

TP53 in breast cancer

APC in colon cancer

• Activation of oncogenes HER2 in breast cancer

KRAS in colon cancer

• Inactivation of DNA repair genesBRCA1/2 in breast cancer

MLH1, MSH2, MSH6 in colon cancer

Mutations in cancer

• Inactivation of tumor suppressor gene

or DNA repair gene :– Intragenic inactivating mutation– Promotor Methylation– Gene Loss

• Activation of oncogenes : _ Intragenic activating mutation– Gene amplification

Driver and passenger gene mutations

Tumors with high mutation load

due to Mutagens or genomic instability

form many neoantigens

and are candidates for immunotherapy

TUMOR MUTATIONS EXPLANATION

HNPCC 1782 Genomic instability

Lung 150 Mutagen (smoke)

Melanoma 80 Mutagen (sun)

Somatic mutations in cancer

P

Breast Lung Colon Prostate

EGFR < 10 34 20-80 4

KRAS < 10 19 36-40 5

NRAS 1-6

BRAF Few 1-4 8-15 Few

PIK3CA 26 4 10-30 2

TP53 23 34 48 16

MLL3 7 10 12 5

CTNNB1 < 10 < 10 < 10 4

Somatic mutations in CRC

P

Gene Mechanism % Mutations Targeted therapy

EGFR Activating point mutations Gene Amplification Overexpression ligands Overexpression nuclear EGFR

20-80 %

KRAS Activating point mutations 36-40 Tipifarnib, lonafarnib

BRAF Activating point mutations 8-15 Dabrafenib, vemurafenib,

sorafenib

NRAS Activating point mutations 1-6 MEK162

PIC3CA Activating point mutations 10-30

mTOR

Cell growth and survival pathway

Cell growth pathway

• Ligands

• Receptors : EGFR

• Secondary messengers : 2 pathways :

1. MAPK pathway : RAS, BRAF, MEK, ERK, Cyclins, CDK4/6

2. PI3K / AKT pathway : PI3K, PTEN, AKT, mTOR

1. MAPK pathway : KRAS, BRAF, NRAS

2. PI3K / AKT pathway : PIK3CA

Driver mutations in CRC

Classical treatment in colon cancer

• Surgery

• Chemotherapy

• If pathology shows EGFR overexpression

Start anti EGFR therapy : – mAB : Cetuximab, panitumumab– TKI : erlotinib, gefitinib, afatinib

EGFR overexpression in CRC

• In Lung Ca : activating mutations TK domain of EGFR

• In Glioblastoma : activating mutations Extracellular domain of EGFR

• In CRC : unclear :

Overexpression membrane EGFR (mEGFR)

Overexpression nuclear EGFR (nEGFR)

Gene Amplification

Overexpression ligands

Activating point mutations

EGFR overexpression

Overexpression membrane EGFR (mEGFR)

Overexpression nuclear EGFR (nEGFR)

Gene Amplification

Overexpression ligands

Activating point mutations

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EGFR status

Anti-EGFR therapy

mAB : cetuximab, panitumumab

TKI : erlotinib, gefitinib, afatinib

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

EGFR resistance in CRC

Resistance against EGFR therapy

– KRAS mutation : 40 %

– BRAF mutation : 8-15 %

– NRAS mutation : 1-6 %

• Mostly pre-existent – selection due to anti-EGFR treatment

• Also new due to ongoing mutagenesis ?

Addition of BRAF or MEK inhibitor

BRAF en MEK inhibitors

P

BRAF MEK

Dabrafenib Trametinib

Vemurafenib Cobimetinib

EGFR resistance treatment in CRC

Resistance against EGFR therapy

PIC3CA mutation : 10-30 %

PTEN loss

Addition of mTOR inhibitor

PIK3CA Driver gene

• PIK3CA encodes p110 subunit of Phosphatidylinositol 3-kinase

PIK3 phosphorylates PI

PI is central in AKT/mTOR pathway

• PIK3CA driver mutations in :– Breast cancer (25 -40 %)– Endometrium (23 %)– Ovarium– Colon– Non-tumor : somatic overgrowth syndromes

(Cowden and Clove syndrome)

• Therapy : PIK3, AKT, mTOR inhibitors

Resistance to BRAF-MEK inhibitors combi with reactivation of MAPK pathway or PI

P

Gene Mutation Mechanism

BRAF Amplification Splice variants

Activation MAPK pathway

KRAS Activating point mutation Activation MAPK pathway

MEK1 Activating point mutation

Activation MAPK pathway

PTEN loss Activating PI3K/AKT pathway

PI3CA Activating PI3K/AKT pathway Activating PI3K/AKT pathway

Why perform genetic studies on tumor DNA ?

• Initial diagnosis and prognosis

• Monitoring recurrence – metastasis

On which tissue should genetic studies be performed ?

• If CRC occurs in different family members :

Genetic studies on DNA from blood to identify a germline mutation :

Polyposis coli : APC, MUTYH

HNPCC : MLH1, MESH2, MSH6, PMS1

• If CRC is sporadic :

Genetic studies on Tumor or liquid biopsy to identify a somatic mutation :

EGFR

KRAS

BRAF

.

Genetic studies to identify somatic mutations

• FFPE material of the tumor

Analysis of DNA from Formaldehyde Fixed-Paraffin Embedded

(FFPE) CRC tissue

• Liquid biopsy

Analysis of DNA from circulating tumor cells in blood (ctDNA)

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

circulating tumor (ctDNA) is only a small fraction of cfDNA in blood

cell-free DNA (cfDNA) testing

• 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) : 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 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

Tissue biopsy

EGFR

KRAS

WILD

EGFR TREATMENT RELAPSE

TISSUE BIOPSY

Liquid biopsy

EGFR

KRAS

BRAF

WILD

TREATMENT

LIQUID BIOPSY

Technology to detect mutations in ctDNA

Next gen sequencing (NGS) + specific technology

• Digital PCR (dilution over many wells)

• Epcam selection for epithelial tumors

• Selection of mutant sequence

Mutant Allele - specific PCR

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

ct DNA testing on liquid biopsy for CRC

1. DESCRIPTION : ct DNA testing on liquid biopsies :

• EGFR• KRAS• BRAF• PIK3CA

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. Take blood yourself :

Email ctDNA@GENDIA.net to ask for kits

www.circulatingtumorDNA.net

www.circulatingtumorDNA.net