1. Theradiagnostics for cancer
Patrick Willems
GENDIA
Antwerp, Belgium

2. Treatment of cancer Personalised treatment Surgery Radiation
Chemotherapy
Personalised treatment

4. Personalized cancer treatment
Immunotherapy
to modulate immune response :
Interferon (IFN) alfa-2b, IL2 (interleukin 2)
CTLA-4 inhibitors
PD-1 inhibitors
PD-L1 inhibitors
Targeted therapy with designer drugs
to target the genetic cause of the tumor
EGFR inhibitors
BRAF inhibitor
MEK inhibitor

5. Bottlenecks in personalized cancer treatment
Immunotherapy
Extremely expensive ( Euro/year)
Few biomarkers (companion diagnostics)
Targeted therapy with designer drugs Very expensive ( Euro/year)
Biomarkers (companion diagnostics)

6. Bottlenecks in personalized cancer treatment
The very high cost of personalised treatment makes companion diagnostics (cancer biomarkers) necessary These are referred to as Theradiagnostics

8. Theradiagnostics Tumor DNA (FFPE - biopsy)
Circulating tumor DNA (ctDNA in liquid biopsy)

9. Market for theradiagnostics
TARGETS
DRUGS
SEQUENCING
Theradiagnostics market : 40 Billion USD per year

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. The changing face of cancer diagnosis
Haematologist
Main interest pathogen. AML
Developing interest in molecular diagnostics
... and I will talk to you about the potential impact of advances in DNA sequencing technologies on the field of cancer Δ , albeit with a slight overrepresentation of examples drawn from haematological cancers

13. Cancer Morbidity and Mortality

14. New cancers per year in Belgium 11 million inhabitants
Lung :
Colon :
Prostate :
Breast :
TOTAAL :

15. Treatment of cancer Surgery Radiation Chemotherapy
Personalised treatment :
Immunotherapy
Targeted therapy with designer drugs

16. Immunotherapy for cancer
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 , MEDI4736, MPDL3280A and MSB C
Other checkpoints : TIM3, LAG3, VISTA, KIR, OX40, CD40, CD137

17. Inhibition immune checkpoints

18. Biomarkers for immunotherapy for Colorectal cancer
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)

19. MSI as biomarker for immunotherapy
MMR deficiency Genomic instability Large mutation load in CRC Many mutant proteins – neo antigens Immune response with immunotherapy

20. MSI as biomarker for immunotherapy in CRC
MMR deficiency Genomic instability Large mutation load in CRC Many mutant proteins – neo antigens Immune response with immunotherapy

21. Treatment of cancer Surgery Radiation Chemotherapy
Personalised treatment :
Immunotherapy
Targeted therapy with designer drugs

22. Targeted treatment for cancer
Personalised targeted treatment inhibits
specific mutations that cause cancer
These mutations are patient-specific
Mutations can be detected by molecular studies of :
. tumor material (biopsy) : FFPE, fresh or frozen
. blood (liquid biopsy)
Therapy is dependent upon the specific mutation
Personalised medicine

23. Which genetic anomalies cause cancer ?

24. Genetics 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 : 3-5%
Prostate cancer : low
Lung cancer : very low

25. Cancer gene mutations Germline Somatic Genes Wellknown genes
Limited number of genes
Cancer-specific genes
Still largely unknown
Large number of genes
Cancer-specific (APC) and aspecific (BRAF, EGFR, KRAS) genes
Mutations
1 or 2 mutations per patient
Large number of different mutations
Unknown (novel-private) mutations
Inactivating sequence mutations
Many mutations in each patient
Limited number
Recurrent mutations
Few inactivating sequence mutations
Inactivating hypermethylation (MLH1)
Activating sequence variants
Amplification of genes (HER2)
Deletions of genes (PTEN)

26. Two step cancer theory (Knudson)
Retinoblastoma (RB1 gene) Mesothelioma Uveal melanoma (BAP1 gene)

27. Two step cancer theory (Knudson)
Inherited cancer :
Germline mutation in all cells
Somatic mutation in cancer cell
Sporadic cancer :
No germline mutation
Somatic mutations in the 2 gene copies in cancer cell

28. Multistep cancer theory (Vogelstein)
Vogelstein et al, Science Aug 22, 2013

29. Cancer genes and mutations
140 driver genes
60 % Tumor suppressor genes
40 % Oncogenes
> 1000 driver gene mutations
(Most tumors 2-10 driver gene mutations)
Millions of passenger gene mutations
(Most tumors passenger gene mutations)

30. 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

31. 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

32. Inactivating mutation Activating point mutations
Somatic mutations
GENE
MECHANISM
TARGETED THERAPY
APC
Inactivating mutation
TP53
EGFR
Activating point mutations
Gene Amplification
Overexpression ligands
Overexpression nuclear EGFR
Cetuximab, panitumumab
erlotinib, gefitinib, afatinib
KRAS
Activating point mutations
Tipifarnib, lonafarnib
BRAF
Dabrafenib, sorafenib,vemurafenib,
NRAS
MEK162
PIK3CA P

33. Inactivating somatic mutations in cancer
Breast
Lung
Colon
Prostate
Cancer-specific gene
BRCA
---
APC
MLH1
TP53 23 34 48 16 P

34. Activating somatic mutations in cancer
Melanoma
Breast
Lung
CRC
Prostate
KRAS 17 35 5
NRAS
13-25
3-5
BRAF
10-50
1-4
8-15
PIK3CA 26 4 22 2
EGFR 34 ??
CTNNB1
2-3 48 P

35. Cell growth and survival pathway

36. Cell growth pathway Ligands Receptors : EGFR
Secondary messengers : 2 pathways :
1. MAPK / RAS pathway : RAS, BRAF, MEK, ERK, Cyclins, CDK4/6
2. mTOR / AKT pathway : PIK3CA, PTEN, AKT, mTOR

37. Classical treatment in colon cancer
Surgery
Chemotherapy
In case of EGFR mutation or overexpression
Start anti EGFR therapy :
mAB : cetuximab, panitumumab
TKI : erlotinib, gefitinib, afatinib

38. EGFR mutations Lung Ca : activating mutations in TK domain
Glioblastoma : activating mutations in Extracellular domain
Colorectal ca : unclear :
Overexpression membrane EGFR (mEGFR)
Overexpression nuclear EGFR (nEGFR)
Gene Amplification
Overexpression ligands
Activating point mutations

39. EGFR status

40. Anti-EGFR therapy mAB : cetuximab, panitumumab
TKI : erlotinib, gefitinib, afatinib

41. EGFR Resistance : T790M mutation
Inhibitors of EGFR with the T790M mutation :
AZD9291
CO-1831

42. EGFR resistance : KRAS and BRAF mutations
TREATMENT
RELAPSE
EGFR
KRAS
WILD

43. EGFR resistance in CRC: KRAS and BRAF mutations
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

44. EGFR resistance in CRC : PIK3CA mutation
Resistance against EGFR therapy
PIK3CA mutation : %
PTEN loss
Addition of mTOR inhibitor

45. PIK3CA PIK3CA encodes p110 subunit of Phosphatidylinositol 3-kinase
PIK3 phosphorylates PI
PI is central in AKT/mTOR pathway
PIK3CA driver mutations in :
Breast cancer (26 %)
Endometrium (23 %)
Colon (22 %)
Non-tumor : somatic overgrowth syndromes
(Cowden and Clove syndrome)
Therapy : PIK3, AKT, mTOR inhibitors

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

47. Genetic studies in cancer
Blood DNA
If CRC occurs in different family members :
Genetic studies on DNA from blood to identify a germline mutation (BRCA)
Tumor
MSI : in order to determine sensitivity for immunotherapy
Mutations in EGFR, KRAS, BRAF, NRAS, PIK3CA
to determine sensitivity for targeted therapy
Liquid biopsy
Initial theradiagnostics if tumor material is unavailable
Follow up during cancer treatment
Screening of high risk patients (HNPCC carriers, BRCA carriers)

48. Genetic studies of somatic mutations
DNA studies on tumor material
Analysis of DNA from tumor (FFPE, fresh, frozen)
Circulating tumor DNA (ctDNA) in Liquid biopsy
Analysis of circulating tumor DNA (ctDNA) in blood

49. Circulating tumor DNA (ctDNA)

50. ctDNA ctDNA from tumor tissue is released
through secretion, necrosis and apoptosis,
but mainly through apoptosis.
ctDNA

51. 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

52. 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

53. Tissue biopsy
TISSUE BIOPSY
EGFR TREATMENT
RELAPSE
EGFR
KRAS
WILD

54. Liquid biopsy
LIQUID BIOPSY
TREATMENT
EGFR
KRAS
BRAF
WILD

55. Companies focusing on Theradiagnostics
Cynvenio
BGI
Agena Bioscience
Boreal Genomics
Chronix Biomedical
Genomic Health
Guardant Health
Inivata
Molecular MD
Pangaea Biotech
Myriad Genetics
Pathway Genomics
Natera
Personal Genome Diagnostics
Sysmex Inostics
Trovagene
ETC
Theradiagnostics market : 40 Billion USD per year

56. ct DNA testing on liquid biopsy for CRC
1. DESCRIPTION : ct DNA testing on liquid biopsies :
90 mutations in 9 cancer genes :
EGFR
TP53
KRAS
BRAF
PIK3CA
2. SAMPLE : blood in specific test kits with Streck tubes (GENDIA)
3. TURNAROUND TIME : 3 weeks 4. PRICE : 650 Euro
NRAS
CTNNB1
GNAS
FOXL2

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