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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