1. DNA diagnosis of lung cancer
Patrick Willems
GENDIA
Antwerp, Belgium

2. Treatment of Lung Cancer
Small Cell Lung Cancer (SCLC)
chemotherapy
radiation
Non-Small Cell Lung Cancer (NSCLC)
surgery
targeted treatment
immunotherapy

4. 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

5. Problems 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. 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. Mortality
UK,

13. Cancer Morbidity and Mortality
Canada, 2007

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

15. Lung cancer 14 % of all cancer
80 % is non–small cell lung cancer (NSCLC)
Belgium : 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.
Lung cancer

16. Treatment of Lung Cancer
Small Cell Lung Cancer (SCLC)
chemotherapy
Radiation
Non-Small Cell Lung Cancer (NSCLC)
surgery
radiation
immunotherapy
personalised targeted treatment

17. 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 , MPDL3280A

18. Inhibition immune checkpoints

19. 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)

20. MSI as Biomarker for immunotherapy
MMR deficiency Genomic instability Large mutation load in tumor (driver and passenger) Many mutant proteins - neoantgens Immune response

21. Immunotherapy for NSCLC
Extremely expensive
( Euro/year)
No biomarkers to select patients

22. 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

23. Receptor antibodies inhibit receptor kinases
by interfering with ligand-receptor binding
Preventing intracellular signaling
Receptor antibodies

24. 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 %)
Herceptin

25. Tyrosine kinase inhibitors (TKI)
TKI inhibits a Tyrosine kinase
by binding to its kinase domain
Preventing phosphorylation (activation) of target

26. 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)

27. Targeted treatment Non-Small Cell Lung Cancer (NSCLC) surgery
radiation
chemotherapy
personalised targeted treatment
immunotherapy
Small Cell Lung Cancer (SCLC)

28. 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)

29. Progress in lung ca treatment

30. 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

31. 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

32. Inheritance of lung cancer
NO germline mutations
MANY somatic mutations

33. Driver and passenger gene mutations
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
Young age
Vogelstein et al, Science Aug 22, 2013
NEJM May 30, 2015

34. Somatic mutations in cancerP
Breast
NSCLC
Colon
Prostate
TP53 23 34 48 16
KRAS
< 10
15-25 35 5
PIK3CA 26 4 22 2
EGFR
10-30
MLL3 7 10 12
CTNNB1

35. Somatic mutations in adeno ca NSCLC
TP53 : 34 %
EGFR : %
KRAS : %
MLL3 : 10 %
STK11 : 9 %
CDKN2A : 8 %
ALK fusions : 5 %
HER 2 : 2%
BRAF : 1-2 %
Ros fusions : 2 %
PTEN : 25 % (loss) P

36. Somatic mutations in adeno ca NSCLC

37. Cell growth and survival pathway

38. 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

39. EGFR Mutations in lung cancer
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

40. EGFR mutations

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. KRAS Mutations in lung cancer
KRAS mutations : % in NSCLC
smokers
90% of KRAS mutations : codon 12 (90 %)
codon 13 (5-10 %)
KRAS Mutations are contraindications for EGFR TKI

44. BRAF Mutations in lung cancer
BRAF mutations : 1-4 % in NSCLC
55 % of KRAS mutations : V600E
BRAF Mutations are contraindications for EGFR TKI

45. 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)

46. ROS1 Mutations in lung cancer
ROS1 activation is caused by ROS1 fusion to different partners
ROS1 mutations are sensitive to Crizotinib (Xalkori) P

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

48. Prognosis according to EGFR mutations

49. Prognosis according to BRAF mutations

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

51. Monitoring recurrence - metastasis
Clinical : imaging
Tumor markers : CEA
Circulating tumor cells (CTC)
Circulating tumor DNA (ctDNA)

52. Diagnostic tests on tumor DNA
Solid tumor biopsy
FFPE
Frozen
Fresh
Liquid biopsy : Circulating tumor DNA (ctDNA)
Blood plasma/serum

53. 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

54. 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

55. Circulating tumor DNA (ctDNA)

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

57. 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

58. 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

59. 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

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

61. Liquid biopsy
LIQUID BIOPSY
TREATMENT
EGFR
KRAS
BRAF
WILD

62. 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

63. 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”

64. Sensitivity to detect mutant sequence
Sanger sequencing : 10 %
Next gen sequencing (NGS) : 1 %
NGS + specific technology : 0.1 %

65. 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

66. Mutant Allele - specific PCR
PCR amplification of wild type allele is blocked
by PNA probe, while mutant is allele is PCR-amplified

67. 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

68. How offer ctDNA testing to your patients ?
Refer to our consultation :
to ask for an appointment
Send blood :
to ask for tubes