1. DNA diagnosis in malignant melanoma
Patrick Willems
GENDIA
Antwerp, Belgium

3. 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

4. Treatment of Malignant melanoma
surgery
radiation
Chemotherapy
Targeted treatment
BRAF inhibitor (Vemurafenib)
MEK inhibitor
Immunotherapy
Interferon (IFN) alfa-2b, IL2 (interleukin 2)
CTLA-4 inhibitors (Ipilimumab)
PD-1 inhibitors (Pembrolizumab and nivolumab)

5. Problems in personalized cancer treatment
Immunotherapy
Very Expensive ( Euro/year
Few biomarkers (companion diagnostics)
Designer drugs
Expensive ( Euro/year)
Biomarkers (companion diagnostics)

6. Problems in personalized cancer treatment
The very high cost of personalised treatment makes companion diagnostics (cancer biomarkers) necessary

8. Cancer biomarkers
tumor material (biopsy)
blood (liquid biopsy)

9. Market for tumor biomarkers in Liquid biopsies
TARGETS
DRUGS
SEQUENCING
Liquid biopsy market for tumor biomarkers: 40 Billion USD per year (Illumina estimate)

10. Current paradigm PATIENT PHYSICIAN PATHOLOGIST general treatment visit
Result
Pathological studies
sample
PATHOLOGIST
Lab

11. Future paradigm PATIENT PHYSICIAN PHARMA LAB Personalised treatment
visit
PHYSICIAN
PHARMA
Result
Molecular testing
sample
LAB
Pathologist

12. Cancer Morbidity and Mortality
Melanoma : 1-8 %

13. New cancers per year in Belgium
Lung :
Colon :
Prostate :
Breast :
MM :
TOTAAL :

14. Incidence MM Higher in sunny countries Higher in light skin people
Increasing everywhere

15. Skin cancer Basal cell carcinoma :75 % Spinocellular epithelioma: 5%
Melanoma : 10 %
Other : 10 %

16. Malignant melanoma Melanoma is a malignant tumor of melanocytes.
Fifth most common cancer in men and the seventh in women
new cases in 2014 in the US
9.710 deaths in 2014 in the US
Five-year survival rates for patients with metastatic disease < 10%

17. Personalised targeted treatment of MM
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) blood (liquid biopsy)

18. Why liquid biopsies for MM ?
Common cancer
High mortality
High load of driver oncogenic mutations
Druggable targets

19. Inheritance of cancer
Majority of cancers are caused by genetic anomalies in the tumor (somatic mutations)
Minority of cancers is inherited (germline mutations) :
Breast Cancer : 10 %
Colon cancer : %
Prostate cancer : low
Lung cancer : very low
Melanoma : 10 %

20. Germline mutations in MM
Gene/Locus
Protein
Function
CDKN2A
(cyclin-dependent kinase inhibitor 2) AD %
p16 (INK4)
p14 (ARF)
p16 : CDK inhibitor
p14 : binds MDM2- p53
CDK4
(cyclin-dependent kinase 4)
<10 fam
control of cell proliferation
MC1R
melanocortin-1 receptor
XRCC3
Risk factor
X-ray repair cross-complementing protein 3
DNA repair protein
MITF
microphthalmia-associated transcription factor
transcription factor
TERT
telomerase reverse transcriptase
Telomerase integrity
POT1
ACD
POT1-interacting protein 1
TERF2IP
TERF2-interacting protein
BAP1
Breast cancer associated prtotein P

21. Inheritance of MM
10 % germline mutations
MANY somatic mutations

22. 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 passenger gene mutations)

23. Driver and passenger gene mutations
TUMOR
MUTATIONS
EXPLANATION
HNPCC
1782
Genomic instability
Lung
150
Mutagen (smoke)
Melanoma 80
Mutagen (sun)
Tumors with high mutation load
due to Mutagens or genomic instability
form many neoantigens
and are candidates for immunotherapy

24. Somatic mutations in cancer
Melanoma
Breast
Lung
Colon
Prostate
TP53 10 23 34 48 16
KRAS
Few
< 10 19 35 5
NRAS
13-25
BRAF
10-50
1-4
8-15
PIK3CA 26 4 22 2
EGFR
MLL3 7 12
CTNNB1
2-3 P

25. Somatic mutations in MM
Gene
% Mutations
Targeted therapy
BRAF
Activating point mutations
10-50
Dabrafenib, vemurafenib
NRAS
13-25
MEK162
KIT
2-6
Dasatinib, imatinib
MEK1 6
Trametinib, MEK162
CTNNB1
2-3
Cyclin D1 inhibitor
CDKN2A
Deletions 50
CDK4 10 LY
GNA11 2
PTEN
20-40
p53
GNAQ 1
PIC3CA 5
Overall
60-70 P

26. Somatic mutations in MM
Gene
% Mutations
Skin
Normal
Sun
Much sun
Mucosa
Acra
Eye
BRAF +
50-60 10
5-10
15-25
< 1
NRAS 20
10-15
5-15
<1
KIT 2 15
CDK4
CCND1
Low
High
CDKN2A _
CNV
MANY
Other
BAP1
GNAQ
GNA11 P

27. Somatic mutations in uvual MM
Gene
% Mutations
in MM
in uveal MM
BRAF %
< 1 %
NRAS
13-25 %
MEK1 %
KIT %
CTNNB1 %
GNA11 %
32 %
GNAQ %
50 %
BAP1
< % P

29. Cell growth and survival pathway

30. Cell growth pathway Ligands Receptors : KIT (EGFR, HER2, MET)
Secondary messengers : 2 pathways :
MAPK pathway : RAS, BRAF, MEK, ERK, Cyclins, CDK4/6
PI3K / AKT pathway : PI3K, PTEN, AKT, mTOR

31. Designer molecules

32. DNA testing to orient personalised treatment
Gene
% Mutations
Targeted therapy
BRAF
10-50
Dabrafenib, vemurafenib
NRAS
13-25
MEK162
MEK1 6
Trametinib, MEK162
KIT
2-6
Dasatinib, imatinib
CTNNB1
2-3
Cyclin D1 inhibitor
CDK4 10 LY P

33. DNA testing to follow treatment and detect metastasis and resistance
Gene
% Mutations
Targeted therapy
Respons
Resistance
Relaps
BRAF
10-50
Dabrafenib, vemurafenib
50 %
Most
NRAS
13-25
MEK162 P

34. Resistance to BRAF inhibitors with reactivation opf MAPK pathway
Gene
Mechanism
BRAF
Amplification
Splice variants
NRAS
Activating point mutation
MEK1
MEK2
PTEN loss
Activating PI3K/AKT pathway
PI3CA P

35. Cell growth and survival pathway

36. Combination therapy BRAF en MEK inhibitors
Dabrafenib
Trametinib
Vemurafenib
Cobimetinib P

37. Resistance to BRAF-MEK inhibitors combi with reactivation of MAPK pathway or PI
Gene
Mutation
Mechanism
BRAF
Amplification
Splice variants
Activation MAPK pathway
NRAS
Activating point mutation
MEK1
MEK2
PTEN
loss
Activating PI3K/AKT pathway
PI3CA P

38. Cell growth and survival pathway

39. Resistance to BRAF-MEK inhibitors combi with reactivation of MAPK or PI3K pathway
Mechanism
Therapy
Re-Activation
MAPK pathway
Inhibition
distal MAPK pathway
ERK inhibitors
Activating
PI3K/AKT pathway
PI3K inhibitors
AKT inhibitors
mTOR inhibitors P

40. Why perform genetic studies on tumor DNA ?
Initial diagnosis and prognosis
Monitoring recurrence – metastasis

41. On which tissue should genetic studies be performed ?
If melanoma occurs in different family members :
Genetic studies on DNA from blood to identify a germline mutation :
CDKN2A - CDK4 (melanoma)
BAP1 (uveal melanoma, mesothelioma)
If melanoma is sporadic :
Genetic studies on Tumor DNA or liquid biopsy to identify a somatic mutation
BRAF
NRAS
KIT .

42. Genetic studies to identify somatic mutations
FFPE material of the tumor
Analysis of DNA
from Formaldehyde Fixed-Paraffin Embedded
Melanoma tissue
Liquid biopsy
Analysis of DNA from circulating tumor cells in blood (ctDNA)

43. 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

44. 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) : 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

45. 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

46. for detection of cancer
ctDNA
circulating tumor DNA
testing in blood
for detection of cancer

47. 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

48. 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

49. ct DNA testing on liquid biopsy for malignant melanoma
1. DESCRIPTION : ct DNA testing on liquid biopsies :
BRAF: %
V600E : 80–90%
V600K : 5-12%
V600R or V600D : 5%
NRAS : %
positions 12, 13, or 61
2. SAMPLE : blood in specific test kits with Streck tubes provided by GENDIA
3. TURNAROUND TIME : 3 weeks 4. PRICE : < 1000 Euro

50. How offer ctDNA testing to your patients ?
Refer to our consultation :
to ask for an appointment
Take blood yourself :
to ask for kits