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Pr Frédéric Bibeau, MD, PhDHead, Pathology department
CHU de Caen,Normandy University,
France
ESMO preceptorship, Valence, 17.05.19
RAS, BRAF, Microsatellite instability and othermolecular markers: how useful are they ? Pitfalls in diagnosis
DISCLOSURE OF INTEREST
Amgen, BMS, MSD, Merck, Sanofi
Content
- Colorectal cancer context
- CRC molecular classification
- Diagnostic value
- Prognostic value
- Therapeutic value
- Perspectives
- Conclusion
Content
- Colorectal cancer context
- CRC molecular classification
- Diagnostic value
- Prognostic value
- Therapeutic value
- Perspectives
- Conclusion
Tis T1 T2 T3 T4
MUCOSA
SUB-MUCOSA
MUSCULARIS
SUB-SEROSA -->SEROSA -->
pT
pN
pM
Muscularis Muscosae -->
N0 : no positive lymph node (LN)N1 : ≤ 3 positive LNN2 : ≥ 4 positive LN
M0 : No distant metastasisM1 : Distant metastasis
Organe infiltrationand / or visceral
peritoneal perforation
TNM UICC 2016 8thClassification
pTNM CRC classification
Early CRC treatment
N+Stage III
Chemotherapy(FOLFOX, 5-FU)
pT3-4 N0 Stage II
No chemotherapyBut rate of relapses: 20%
Need for additionnal prognostic factors
• Chemotherapy: 5FU/oxaliplatin/irinotecan• Targeted therapies:
- Cetuximab (Erbitux®) (IgG1)- Panitumumab (Vectibix®) (IgG2)- Bevacizumab (Avastin®) (IgG1)
Aflibercept (Zaltrap®), Regorafenib(Stivarga®)
Anti-EGFR
Anti-VEGF
Metastatic CRC treatment
Epidermal GrowthFactor Receptor
Vascular EndothelialGrowth Fractor
Need for predictive factors
6 patients/10
Sporadic(majority of cases)
Hereditary(6 % of cases)
CRC context
Screening toolsOptimal management
Content
- Colorectal cancer context
- CRC molecular classification
- Diagnostic value
- Prognostic value
- Therapeutic value
- Perspectives
- Conclusion
small polype advanced polypavancé
cancer metastasesAberrant
crypts
CRC tumour progression
Voies d’oncogenèse CCR
CIN pathway MSI pathwayCIMP pathway
≈20 % 15- 20 %80-85 %
CRC carcinogenesis
Chromosomic Instabilty CpG Island Methylator Phenotype
Epigenetic instability
MicroSatellite Instabilty
KRAS, TP53 mutation
hMLH1, p16, MGMT methylation
BRAF mutation
Molecular profileMicrosatellite Instability
(ou soustraction)
Normal DNA
MSI tumour
NucleotidesLoss or gain
MSI CRC carcinogenesis
4 proteinsfor DNA reparation
Deficient MisMatch Repair (MMR) system
MSI (microsatellite instable)
Terminology
dMMR(deficient mismatch repair)
pMMR(proficient mismatch repair)
MSS (microsatellite stable)
RER+ Phenotype (Replication Error+)
RER- Phenotype(Replication Error-)
Stable tumour (MSS): 4 MMR proteins expressedImmunohistochemistry
Loss of hML1 hMSH2 +
hMSH6 + Parallel loss of PMS2
Negative tumour Positive tumour
personnal caselF. Bibeau
*MisMatch Repair
Instable tumour(MSI): extinction of MMR proteinsImmunohistochemistry
Adapted from Bao et al. Am J Surg Pathol 2010; 34:1798-1804
MSH6 decreased expression after chemoradiation !Immunohistochemistry pitfall
Not MSI !
Biopsie diagnostique pré-thérapeutique
hMSH6 +Adapté de Bao et al. Am J Surg Pathol 2010; 34:1798-1804
MSH6 normal expression on pre-treatment biopsyImmunohistochemistry pitfall
Adapted from Bao et al. Am J Surg Pathol 2010; 34:1798-1804
CIN pathway MSI pathwayCIMP pathway≈20 % 15- 20 %80-85 %
Lynch syndromeSerratedtumoursConventionnal carcinoma
Cancer of the elderly
CRC molecular classification
Lieberkühnian Serrated Médullary/ lymphocytes
Chromosomic instability Epigenetic instability Microsatellite instability
Content
- Colorectal cancer context
- CRC molecular classifications
- Diagnostic value
- Prognostic value
- Therapeutic value
- Perspectives
- Conclusion
Lynch syndrome spectrum HNPCC: Hereditary Non Polyposis Colorectal Cancer
Early onset CRC and other cancers
Cancer risk : 75% CCR, 50% endometrium, 15% others
Lynch syndrome screening
Colorectal Small bowel
Urinary tract Endometrium
MLH1MSH2MSH6PMS2
Germile mutation Constant MSI
Mutation of thecorresponding gene
DNA
RNAPROTEINS
Lynch syndrome screening
Time consumingHighly specialized laboratories
Oncogenetics team consultationGermline mutation determination
Prophylactic surgery …
Clinical, endoscopic, and US(if woman) follow-up
Lynch diagnosis
Familial investigation+
CRC < 60 year-oldPersonal CRC historyCRC familial context
MSI +
Lynch syndrome screening
MSI and hMLH1 loss
Lynch syndrome (2%)Sporadic cancer (15%)
HypermethylationMLH1 promotor
BRAF mutation
Microsatellite instability context
Absent
Absent
Elderly patient Young patient
Content
- Colorectal cancer context
- CRC molecular classifications
- Diagnostic value
- Prognostic value
- Therapeutic value
- Perspectives
- Conclusion
Identification of favorable stage II CRC
No adjuvant chemotherapy (5-FU)Lack of 5-FU efficacy
MSI Normal DNA
MSI tumour
Loss or gain of
nucleotides
• Perforation• Occlusion• pT4• Lymph node < 12• Poorly differenciated tumour• Venous/lymphatic Invasion • Perineural invasion
Adjuvant chemotherapy: to be discussed
(5-FU)
Caracterization of High risk CRC stage II
MSS
Caracterization of agressive stage III CRC
MSSKRAS mut.BRAF mut.
*Taieb et al JAMA Oncol 2016
Intensified chemotherapy: clinical trials Stratification according mutations ?
Identification of agressive stage IV CRC
MSSKRAS mut.BRAF mut.
Metastatic setting
Intensified chemotherapy: FOLFIRINOX+ Bevacizumab (BRAF mut.)Ongoing clinical trials (combined targeted therapies)
Content
- Colorectal cancer context
- CRC molecular classifications
- Diagnostic value
- Prognostic value
- Therapeutic value
- Perspectives
- Conclusion
RAS mutations = marker of resistance
Anti-EGFR targeted therapies
CetuximabPanitumumab
Anti-EGFR antibodies
Angiogenesis
Growth
MotilityMetastases
ChemotherapyRadiotherapy
Cell cycle activation
RAS
Résistance: mutations KRAS
Normal différenciation, proliferationand growth
Adapted from Van Krieken et al. Virchows 2008;453:417-431
abnormal différenciation, proliferationand growth
Recommendations
Primary CRC Metastasis
RAS testing mandatory before anti-EGFR therapy
Or
Molecular techniques
Quick turn around time
Selection Macrodissection Mutation ?
Quality of the pre-analytique step
pitfall !
Quality (age) of the paraffin, type of fixatives…
Quality of the pre-analytique step
Use the pretreatment biopsy
pitfall !
RAS and BRAF WT
Mutation KRAS exon 2
Mut KRAS ex 3, 4
Mut NRAS
50%RAS mutated
HER-2
BRAF
10%
Amplifications: 2,5%Mutations: 1,9%
Anti-HER2Targeted
therapies?
Anti-EGFR resistance ?
SPECTAcolor: Folprecht ESMO 2016, abst 4580
Sartore-Bianchi Lancet Oncol 2016Hurwitz ASCO GI 2016Marsoni AACR 2017
Trastuzumab + lapatinib(HERACLES)
Trastuzumab + pertuzumab
Raghac ASCO 2016
40%
CRCm molecular biomarkers and targets
MSI5%
Immunotherapy
MSI CRC : immunogenic tumour Metastatic MSI CRC
Immuneescape
Crohn like reaction
Lymphocytic infiltrate
ImmunotherapyCheck-points immunity
inhibitors
High response rate(anti-PD1)
Immune enhancement
Le DT et al. N Eng J Med 2015;372:2509-20
MSI CRC : immunogenic tumourT lymphocyte receptor
T lymphocyte receptor
Antigen
Antigen
Tumor cell
Tumor cell
PDL1 inhibitor
PD1inhibitor
ImmunotherapyAnti-PD-1 treatment: overall survival
Selection of patients based on MSI status
CCR MSI
CCR MSS
Mois
Le DT et al. N Eng J Med 2015;372:2509-20
pitfall !
Sample number Local assessment Central review Best response under ICKi IHC PCR IHC PCR
Prospective cohort (N = 36) #47 pMMR MSI pMMR MSS progression disease #115 NE MSI pMMR MSS progression disease #181 dMMR NE pMMR MSS progression disease
Retrospective cohort (N = 92) #29 pMMR MSI pMMR MSS - #41 NE MSI pMMR MSS - #42 NE MSI pMMR MSS - #43 NE MSI pMMR MSS - #46 NE MSI pMMR MSS - #56 NE MSI pMMR MSS - #64 pMMR MSI pMMR MSS - #94 pMMR MSI pMMR MSS - #106 NE MSI pMMR MSS -
MSI Testing MSI : only in expert centres?
False positive vcases: 10% of cases! Mainly PCR interprétation errors !
Performing the 2 méthods : IHC and PCR according the authors…
Cohen R et al. Jama Oncol 2018
pitfall !
Sample number
Local assessment
Central review
Best response under ICKi
IHC
PCR
IHC
PCR
Prospective cohort (N = 36)
#47
pMMR
MSI
pMMR
MSS
progression disease
#115
NE
MSI
pMMR
MSS
progression disease
#181
dMMR
NE
pMMR
MSS
progression disease
Retrospective cohort (N = 92)
#29
pMMR
MSI
pMMR
MSS
-
#41
NE
MSI
pMMR
MSS
-
#42
NE
MSI
pMMR
MSS
-
#43
NE
MSI
pMMR
MSS
-
#46
NE
MSI
pMMR
MSS
-
#56
NE
MSI
pMMR
MSS
-
#64
pMMR
MSI
pMMR
MSS
-
#94
pMMR
MSI
pMMR
MSS
-
#106
NE
MSI
pMMR
MSS
-
Content
- Colorectal cancer context
- CRC molecular classification
- Diagnostic value
- Prognostic value
- Therapeutic value
- Perspectives
- Conclusion
Content
- Colorectal cancer context
- CRC molecular classification
- Diagnostic value
- Prognostic value
- Therapeutic value
- Perspectives
- Conclusion
RAS and BRAF mutationnal status determination
Circulating tumour DNA ?
- Non invasive technique- Monitoring
(cf Pierre Laurent Puiget Clara Montagut 2017
lectures)
Pitfalls
Absence of liver metastases (peritonealcarcinomatosis): main clinical factor associated withinconclusive circulating tumor DNA results
RAS mutation analysis in circulating tumor DNAfrom patients with metastatic colorectal cancer:the AGEO RASANC prospective multicenter study
Accuracy: 94.8%But…
Bachet et al. Annals of Oncology 2018, mdy061, https://doi.org/10.1093/annonc/mdy061
https://doi.org/10.1093/annonc/mdy061
CRC molecular profile
- Predictive impact ? - Bevacizumab: CMS 1? 2-3?- Anti-EGFR: CMS 2-4 ? Guinney Nature Med 2015
In primary CRC !
Quality control
Content
- Colorectal cancer context
- CRC molecular classification
- Diagnostic value
- Prognostic value
- Therapeutic value
- Perspectives
- Conclusion
CIN pathway MSI pathwayCIMP pathway≈20 % 15- 20 %80-85 %
Lynch syndromeSerratedtumoursConventional carcinoma
Cancer of the elderly
Molecular CRC classification- Useful biomarkers
BRAFmutation
RASmutation
Anti-EGFR resistance(predictive factor)
Pronosticfactor
Lynch diagnosis
No 5-FU efficacy
Anti-PD-1 efficacy
PronosticMSI
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