MOLECULAIRE ONCOLOGIE
INHOUDSOPGAVE
1.1 Inleiding...................................................................................................................................................... 6
1.1.1 Hallmarks of cancer ............................................................................................................................... 6
1.1.2 Kankerbehandeling ................................................................................................................................ 6
1.2 Doelgerichte kankerbehandeling ............................................................................................................... 7
1.2.1 Algemeen: definitie ............................................................................................................................... 7
1.2.2 Voorbeelden van doelgerichte therapie ................................................................................................ 8
1.2.2.1 Hormonale therapie ...................................................................................................................... 8
1.2.2.2 Behandelingen gericht op intracellulaire signaaltransductie ....................................................... 9
1.2.2.3 Monoklonale antilichamen (mAb) .............................................................................................. 10
1.2.2.4 Tyrosinekinase inhibitoren (TKI’s) ............................................................................................... 11
1.2.2.5 Antiangiogene therapie .............................................................................................................. 11
1.2.3 Uitdagingen van doelgerichte therapie ............................................................................................... 12
1.3 Voorbeeld: de EGFR-pathway als aangrijpingspunt ................................................................................. 12
1.3.1 Algemeen: de EGFR pathway............................................................................................................... 12
1.3.2 Anti-EGFR therapie .............................................................................................................................. 13
1.3.2.1 Tyrosine kinase inhibitoren (TKI) ................................................................................................ 13
1.3.2.2 Monoklonale antilichamen ......................................................................................................... 14
1.4 Biomerkers ............................................................................................................................................... 14
1.4.1 Types biomerkers ................................................................................................................................ 14
1.4.1.1 Biomerker voor aanleg ................................................................................................................ 15
1.4.1.2 Screeningsbiomerker .................................................................................................................. 15
1.4.1.3 Diagnostische biomerker ............................................................................................................ 15
1.4.1.4 Prognostische biomerker ............................................................................................................ 16
1.4.1.5 Predictieve biomerker ................................................................................................................ 16
1.4.1.6 Farmacodynamische biomerker ................................................................................................. 16
1.4.1.7 Herval biomerker ........................................................................................................................ 17
1.4.1.8 Uitdagingen ................................................................................................................................. 17
1.4.2 EGFR en KRAS als biomerker ............................................................................................................... 17
1.4.2.1 EGFR ............................................................................................................................................ 18
1.4.2.2 KRAS ............................................................................................................................................ 18
2.1 HIF activeert angiogene factoren ............................................................................................................. 20
2.1.1 Hypoxie-induceerbare factor 1 (HIF 1) ................................................................................................ 20
2.1.2 HIF 1 activeert angiogene factoren ..................................................................................................... 21
1
,2.2 De vorming van nieuwe bloedvaten d.m.v. sprouting ............................................................................. 22
2.2.1 Het proces van ‘sprouting’................................................................................................................... 22
2.2.2 De ‘tip’cel ............................................................................................................................................. 22
2.2.2.1 De ‘tip’cel .................................................................................................................................... 22
2.2.2.2 Selectie van de ‘tip’cel ................................................................................................................ 22
2.2.2.3 Laterale inhibitie door Dll4/Notchsignalisatie ............................................................................ 23
2.2.2.4 Invasie van de ‘tip’cel .................................................................................................................. 23
2.2.3 De ‘stalk’cel.......................................................................................................................................... 23
2.2.3.1 De ‘stalk’cel ................................................................................................................................. 23
2.2.3.2 Elongatie van de ‘stalk’cel ........................................................................................................... 23
2.2.3.3 Rol van VEGFR-1 (Flt 1) in de ‘stalk’cellen ................................................................................... 24
2.2.3.4 ‘Stalk’cellen en de vorming van een lumen ................................................................................ 24
2.2.4 De ‘falanx’cel ....................................................................................................................................... 24
2.2.5 Maturatie van het gevormde bloedvat ............................................................................................... 24
2.3 Hypoxie veroorzaakt een abnormale tumorvasculatuur ......................................................................... 25
2.4 Antiangiogene middelen in de behandeling van kanker .......................................................................... 25
2.4.1 Inhibitie van angiogenese via VEGF ..................................................................................................... 25
2.4.2 Successen van anti-VEGF therapie ...................................................................................................... 26
2.4.3 Uitdagingen van anti-VEGF therapie ................................................................................................... 26
2.5 Normalisatie van de tumorvasculatuur ................................................................................................... 26
2.6 Bijkomende mechanismen die leiden tot verhoogde bloedtoevoer ....................................................... 28
2.6.1 ‘Intussusceptive’ microvasculaire groei ............................................................................................... 28
2.6.2 Vasculaire co-optie .............................................................................................................................. 28
2.6.3 Vasculogenese: rol van EPC’s in tumor neovascularisatie ................................................................... 29
2.6.4 Vasculogene ‘mimicry’ ......................................................................................................................... 29
2.6.5 Rol van kankerstamcellen in tumor neovascularisatie ........................................................................ 29
3.1 Inleiding.................................................................................................................................................... 30
3.2 DNA methylatie ........................................................................................................................................ 31
3.3 Histon modificaties .................................................................................................................................. 32
3.3.1 Histon modificatie mechanismen ........................................................................................................ 32
3.3.2 Interactie epigenetica .......................................................................................................................... 33
3.3.3 Tumor initiatie ..................................................................................................................................... 33
3.3.4 Epigenetische landschap in kanker ...................................................................................................... 34
3.3.5 Interactie genetica – epigenetica ........................................................................................................ 35
3.4 MicroRNA’s .............................................................................................................................................. 36
3.5 Technieken ............................................................................................................................................... 37
3.5.1 Bisulfiet behandeling ........................................................................................................................... 37
2
, 3.5.2 PCR ....................................................................................................................................................... 37
3.5.3 Pyrosequencing ................................................................................................................................... 38
3.5.4 array..................................................................................................................................................... 38
3.5.5 NGS ...................................................................................................................................................... 39
3.6 Therapie ................................................................................................................................................... 40
4.1 Inleiding.................................................................................................................................................... 41
4.2 Technieken ............................................................................................................................................... 42
4.2.1 Next generation sequencing ................................................................................................................ 42
4.2.2 Microarray ........................................................................................................................................... 42
4.2.2.1 Microarray – SNP ........................................................................................................................ 42
4.2.2.2 Microarray – methylatie ............................................................................................................. 43
4.2.2.3 Microarray – genexpressie.......................................................................................................... 43
4.3 Databases ................................................................................................................................................. 44
4.3.1 The cancer genome atlas ..................................................................................................................... 44
4.3.2 Gene Expression Omnibus ................................................................................................................... 44
4.3.3 Cosmic ................................................................................................................................................. 44
4.3.4 ICGC ..................................................................................................................................................... 44
4.4 Wat doen we met deze data? .................................................................................................................. 45
4.4.1 Classificatie van CRC ............................................................................................................................ 45
4.4.2 Voorbeeld uit het lab: biomerker onderzoek ...................................................................................... 46
5.1 Companion diagnostics (CDx) for precision medicine: tissue and body fluid analysis ............................. 47
5.1.1 General concepts ................................................................................................................................. 47
5.1.1.1 Precision medicine ...................................................................................................................... 47
5.1.1.2 Companion diagnostics ............................................................................................................... 47
5.1.1.3 Types biomerkers ........................................................................................................................ 48
5.1.2 Technieken .......................................................................................................................................... 49
5.1.2.1 Histologische technieken ............................................................................................................ 49
5.1.2.2 Moleculaire technieken .............................................................................................................. 51
5.1.3 Considerations ..................................................................................................................................... 52
5.1.4 Liquid biopsy ........................................................................................................................................ 54
5.2 Moleculaire pathologie ............................................................................................................................ 55
6.1 Drug development proces........................................................................................................................ 57
6.1.1 Drug discovery and development timeline ......................................................................................... 57
6.1.2 Het proces............................................................................................................................................ 57
6.1.3 GCP ...................................................................................................................................................... 57
6.2 Ontwikkeling van geneesmiddelen: Een onderneming met een hoog risico ........................................... 58
6.3 Clinical trials ............................................................................................................................................. 58
3
, 6.3.1 Verloop ................................................................................................................................................ 58
6.3.2 Fase 1 proefopzetten ........................................................................................................................... 59
6.3.2.1 Dosislimiterende toxiciteit (DLT)................................................................................................. 60
6.3.2.2 Andere termen ............................................................................................................................ 60
6.3.2.3 Startdoses voor fase 1 trials ........................................................................................................ 61
6.3.2.4 Target population van fase 1 non-oncology studies ................................................................... 62
6.3.3 Response Evaluation ............................................................................................................................ 62
7.1 Introductie ............................................................................................................................................... 64
7.2 Concept van immuunontwijking .............................................................................................................. 64
7.2.1 Concept................................................................................................................................................ 64
7.2.2 Bewijzen van interactie ....................................................................................................................... 64
7.2.3 Belangrijke immuuncellen betrokken bij de antitumor immuunrespons............................................ 65
7.3 Kanker immunotherapie .......................................................................................................................... 65
7.3.1 T-cellen ................................................................................................................................................ 65
7.3.1.1 Signaal 1: specificiteit.................................................................................................................. 66
7.3.1.2 Signaal 2: activatie of remming................................................................................................... 67
7.3.1.3 Signaal 3: differentiatie ............................................................................................................... 67
7.4 Immuunontwijkingsmechanismen van tumorcellen ................................................................................ 68
7.5 Immuun checkpoint blockers ................................................................................................................... 68
7.6 Hoe weerstand overwinnen? ................................................................................................................... 69
8.1 Introductie ............................................................................................................................................... 71
8.1.1 Oorsprong en afstamming van NK-cellen ............................................................................................ 71
8.1.2 Een overvloed aan receptoren ............................................................................................................ 72
8.2 CD16 en antilichaam-afhankelijke cellulaire cytotoxiciteit (ADCC) .......................................................... 72
8.3 Cytokine stimulatie .................................................................................................................................. 73
8.3.1 De IL-2 ‘common γ chain’ cytokine familie .......................................................................................... 73
8.3.2 IL-15 modificaties ................................................................................................................................ 73
8.4 Resistentie tegen immunosuppressie ...................................................................................................... 74
8.4.1 Gene editing ........................................................................................................................................ 74
8.4.2 TGFβRII KO ........................................................................................................................................... 75
8.5 Manieren om NK-celtherapie te verbeteren ........................................................................................... 75
8.5.1 Checkpoint blokkade ........................................................................................................................... 75
8.5.2 Non-self (celtherapie) .......................................................................................................................... 75
8.5.3 chimeric antigen receptors .................................................................................................................. 76
8.5.4 BiKE, BiTE and TriKE ............................................................................................................................. 76
9.1 De basiskenmerken van kanker ............................................................................................................... 77
9.1.1 De levensloop van kankercellen .......................................................................................................... 77
4
INHOUDSOPGAVE
1.1 Inleiding...................................................................................................................................................... 6
1.1.1 Hallmarks of cancer ............................................................................................................................... 6
1.1.2 Kankerbehandeling ................................................................................................................................ 6
1.2 Doelgerichte kankerbehandeling ............................................................................................................... 7
1.2.1 Algemeen: definitie ............................................................................................................................... 7
1.2.2 Voorbeelden van doelgerichte therapie ................................................................................................ 8
1.2.2.1 Hormonale therapie ...................................................................................................................... 8
1.2.2.2 Behandelingen gericht op intracellulaire signaaltransductie ....................................................... 9
1.2.2.3 Monoklonale antilichamen (mAb) .............................................................................................. 10
1.2.2.4 Tyrosinekinase inhibitoren (TKI’s) ............................................................................................... 11
1.2.2.5 Antiangiogene therapie .............................................................................................................. 11
1.2.3 Uitdagingen van doelgerichte therapie ............................................................................................... 12
1.3 Voorbeeld: de EGFR-pathway als aangrijpingspunt ................................................................................. 12
1.3.1 Algemeen: de EGFR pathway............................................................................................................... 12
1.3.2 Anti-EGFR therapie .............................................................................................................................. 13
1.3.2.1 Tyrosine kinase inhibitoren (TKI) ................................................................................................ 13
1.3.2.2 Monoklonale antilichamen ......................................................................................................... 14
1.4 Biomerkers ............................................................................................................................................... 14
1.4.1 Types biomerkers ................................................................................................................................ 14
1.4.1.1 Biomerker voor aanleg ................................................................................................................ 15
1.4.1.2 Screeningsbiomerker .................................................................................................................. 15
1.4.1.3 Diagnostische biomerker ............................................................................................................ 15
1.4.1.4 Prognostische biomerker ............................................................................................................ 16
1.4.1.5 Predictieve biomerker ................................................................................................................ 16
1.4.1.6 Farmacodynamische biomerker ................................................................................................. 16
1.4.1.7 Herval biomerker ........................................................................................................................ 17
1.4.1.8 Uitdagingen ................................................................................................................................. 17
1.4.2 EGFR en KRAS als biomerker ............................................................................................................... 17
1.4.2.1 EGFR ............................................................................................................................................ 18
1.4.2.2 KRAS ............................................................................................................................................ 18
2.1 HIF activeert angiogene factoren ............................................................................................................. 20
2.1.1 Hypoxie-induceerbare factor 1 (HIF 1) ................................................................................................ 20
2.1.2 HIF 1 activeert angiogene factoren ..................................................................................................... 21
1
,2.2 De vorming van nieuwe bloedvaten d.m.v. sprouting ............................................................................. 22
2.2.1 Het proces van ‘sprouting’................................................................................................................... 22
2.2.2 De ‘tip’cel ............................................................................................................................................. 22
2.2.2.1 De ‘tip’cel .................................................................................................................................... 22
2.2.2.2 Selectie van de ‘tip’cel ................................................................................................................ 22
2.2.2.3 Laterale inhibitie door Dll4/Notchsignalisatie ............................................................................ 23
2.2.2.4 Invasie van de ‘tip’cel .................................................................................................................. 23
2.2.3 De ‘stalk’cel.......................................................................................................................................... 23
2.2.3.1 De ‘stalk’cel ................................................................................................................................. 23
2.2.3.2 Elongatie van de ‘stalk’cel ........................................................................................................... 23
2.2.3.3 Rol van VEGFR-1 (Flt 1) in de ‘stalk’cellen ................................................................................... 24
2.2.3.4 ‘Stalk’cellen en de vorming van een lumen ................................................................................ 24
2.2.4 De ‘falanx’cel ....................................................................................................................................... 24
2.2.5 Maturatie van het gevormde bloedvat ............................................................................................... 24
2.3 Hypoxie veroorzaakt een abnormale tumorvasculatuur ......................................................................... 25
2.4 Antiangiogene middelen in de behandeling van kanker .......................................................................... 25
2.4.1 Inhibitie van angiogenese via VEGF ..................................................................................................... 25
2.4.2 Successen van anti-VEGF therapie ...................................................................................................... 26
2.4.3 Uitdagingen van anti-VEGF therapie ................................................................................................... 26
2.5 Normalisatie van de tumorvasculatuur ................................................................................................... 26
2.6 Bijkomende mechanismen die leiden tot verhoogde bloedtoevoer ....................................................... 28
2.6.1 ‘Intussusceptive’ microvasculaire groei ............................................................................................... 28
2.6.2 Vasculaire co-optie .............................................................................................................................. 28
2.6.3 Vasculogenese: rol van EPC’s in tumor neovascularisatie ................................................................... 29
2.6.4 Vasculogene ‘mimicry’ ......................................................................................................................... 29
2.6.5 Rol van kankerstamcellen in tumor neovascularisatie ........................................................................ 29
3.1 Inleiding.................................................................................................................................................... 30
3.2 DNA methylatie ........................................................................................................................................ 31
3.3 Histon modificaties .................................................................................................................................. 32
3.3.1 Histon modificatie mechanismen ........................................................................................................ 32
3.3.2 Interactie epigenetica .......................................................................................................................... 33
3.3.3 Tumor initiatie ..................................................................................................................................... 33
3.3.4 Epigenetische landschap in kanker ...................................................................................................... 34
3.3.5 Interactie genetica – epigenetica ........................................................................................................ 35
3.4 MicroRNA’s .............................................................................................................................................. 36
3.5 Technieken ............................................................................................................................................... 37
3.5.1 Bisulfiet behandeling ........................................................................................................................... 37
2
, 3.5.2 PCR ....................................................................................................................................................... 37
3.5.3 Pyrosequencing ................................................................................................................................... 38
3.5.4 array..................................................................................................................................................... 38
3.5.5 NGS ...................................................................................................................................................... 39
3.6 Therapie ................................................................................................................................................... 40
4.1 Inleiding.................................................................................................................................................... 41
4.2 Technieken ............................................................................................................................................... 42
4.2.1 Next generation sequencing ................................................................................................................ 42
4.2.2 Microarray ........................................................................................................................................... 42
4.2.2.1 Microarray – SNP ........................................................................................................................ 42
4.2.2.2 Microarray – methylatie ............................................................................................................. 43
4.2.2.3 Microarray – genexpressie.......................................................................................................... 43
4.3 Databases ................................................................................................................................................. 44
4.3.1 The cancer genome atlas ..................................................................................................................... 44
4.3.2 Gene Expression Omnibus ................................................................................................................... 44
4.3.3 Cosmic ................................................................................................................................................. 44
4.3.4 ICGC ..................................................................................................................................................... 44
4.4 Wat doen we met deze data? .................................................................................................................. 45
4.4.1 Classificatie van CRC ............................................................................................................................ 45
4.4.2 Voorbeeld uit het lab: biomerker onderzoek ...................................................................................... 46
5.1 Companion diagnostics (CDx) for precision medicine: tissue and body fluid analysis ............................. 47
5.1.1 General concepts ................................................................................................................................. 47
5.1.1.1 Precision medicine ...................................................................................................................... 47
5.1.1.2 Companion diagnostics ............................................................................................................... 47
5.1.1.3 Types biomerkers ........................................................................................................................ 48
5.1.2 Technieken .......................................................................................................................................... 49
5.1.2.1 Histologische technieken ............................................................................................................ 49
5.1.2.2 Moleculaire technieken .............................................................................................................. 51
5.1.3 Considerations ..................................................................................................................................... 52
5.1.4 Liquid biopsy ........................................................................................................................................ 54
5.2 Moleculaire pathologie ............................................................................................................................ 55
6.1 Drug development proces........................................................................................................................ 57
6.1.1 Drug discovery and development timeline ......................................................................................... 57
6.1.2 Het proces............................................................................................................................................ 57
6.1.3 GCP ...................................................................................................................................................... 57
6.2 Ontwikkeling van geneesmiddelen: Een onderneming met een hoog risico ........................................... 58
6.3 Clinical trials ............................................................................................................................................. 58
3
, 6.3.1 Verloop ................................................................................................................................................ 58
6.3.2 Fase 1 proefopzetten ........................................................................................................................... 59
6.3.2.1 Dosislimiterende toxiciteit (DLT)................................................................................................. 60
6.3.2.2 Andere termen ............................................................................................................................ 60
6.3.2.3 Startdoses voor fase 1 trials ........................................................................................................ 61
6.3.2.4 Target population van fase 1 non-oncology studies ................................................................... 62
6.3.3 Response Evaluation ............................................................................................................................ 62
7.1 Introductie ............................................................................................................................................... 64
7.2 Concept van immuunontwijking .............................................................................................................. 64
7.2.1 Concept................................................................................................................................................ 64
7.2.2 Bewijzen van interactie ....................................................................................................................... 64
7.2.3 Belangrijke immuuncellen betrokken bij de antitumor immuunrespons............................................ 65
7.3 Kanker immunotherapie .......................................................................................................................... 65
7.3.1 T-cellen ................................................................................................................................................ 65
7.3.1.1 Signaal 1: specificiteit.................................................................................................................. 66
7.3.1.2 Signaal 2: activatie of remming................................................................................................... 67
7.3.1.3 Signaal 3: differentiatie ............................................................................................................... 67
7.4 Immuunontwijkingsmechanismen van tumorcellen ................................................................................ 68
7.5 Immuun checkpoint blockers ................................................................................................................... 68
7.6 Hoe weerstand overwinnen? ................................................................................................................... 69
8.1 Introductie ............................................................................................................................................... 71
8.1.1 Oorsprong en afstamming van NK-cellen ............................................................................................ 71
8.1.2 Een overvloed aan receptoren ............................................................................................................ 72
8.2 CD16 en antilichaam-afhankelijke cellulaire cytotoxiciteit (ADCC) .......................................................... 72
8.3 Cytokine stimulatie .................................................................................................................................. 73
8.3.1 De IL-2 ‘common γ chain’ cytokine familie .......................................................................................... 73
8.3.2 IL-15 modificaties ................................................................................................................................ 73
8.4 Resistentie tegen immunosuppressie ...................................................................................................... 74
8.4.1 Gene editing ........................................................................................................................................ 74
8.4.2 TGFβRII KO ........................................................................................................................................... 75
8.5 Manieren om NK-celtherapie te verbeteren ........................................................................................... 75
8.5.1 Checkpoint blokkade ........................................................................................................................... 75
8.5.2 Non-self (celtherapie) .......................................................................................................................... 75
8.5.3 chimeric antigen receptors .................................................................................................................. 76
8.5.4 BiKE, BiTE and TriKE ............................................................................................................................. 76
9.1 De basiskenmerken van kanker ............................................................................................................... 77
9.1.1 De levensloop van kankercellen .......................................................................................................... 77
4
