Microsatellite instability and other molecularmarkers: how useful are they ?

Pr Frédéric Bibeau, MD, PhD

Head, Pathology department

CHU de Caen,

Normandy University,

France

ESMO preceptorship, Barcelona, 20.10.17

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 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 expressed

Immunohistochemistry

Loss of hML1 hMSH2 +

hMSH6 + Parallel loss of PMS2

Negative tumour Positive tumour

perso

nn

al caselF. B

ibeau

*MisMatch Repair

Instable tumour(MSI): extinction of MMR proteins

Immunohistochemistry

CIN pathway MSI pathwayCIMP pathway≈20 % 15- 20 %80-85 %

Lynch syndromeSerratedtumours

Conventionnal carcinomaCancer 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

Autosomic dominant transmission

MLH1MHS2MSH6PMS2

Germline mutation Constant MSI

Early onset CRC Multiple locations

Familial contextOther cancers

Lynch syndrome screening

Lynch syndrome spectrum HNPCC: Hereditary Non Polyposis Colorectal Cancer

Other cancers

Cancer risk : 75% CCR, 50% endometrium, 15% others

Lynch syndrome screening

Colorectal

Small bowelUrinary tract

Endometrium

MLH1MSH2MSH6PMS2

Germile mutation Constant MSI

Mutation of thecorresponding gene

DNA

RNA

PROTEINS

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 ansPersonal 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

Cetuximab

PanitumumabAnti-EGFR antibodies

Angiogenesis

Growth

MotilityMetastases

ChemotherapyRadiotherapy

Cell cycle activation

RAS

Résistance: mutations KRAS

Normal différenciation, proliferationand growth

Adapté de Van Krieken et al. Virchows 2008;453:417-431

abnormal différenciation, proliferationand growth

Recommendations

Primary CRC Metastasis

RAS testing mandatory before anti-EGFR therapy on

Or

Molecular techniques

Selection Macrodissection Mutation ?

Quality of the pre-analytique step

Quality of the pre-analytique step

Use the pretreatment biopsy

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 pembrolizumab)

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

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 PuigLecture)

CRC molecular profile

- Predictive impact ?

- Bevacizumab: CMS 1? 2-3?

- Anti-EGFR: CMS 2-4 ? Guinney Nature Med 2015

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 syndromeSerratedtumours

Conventional carcinomaCancer 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

MLH1MSH2MSH6PMS2

Germile mutation Constant MSI

Time consumingHighly specialized laboratories

Lynch syndrome screening

Microsatellite instability

Diploidy

No /or few loss of heterozygocity

(ou soustraction)

Normal DNA

MSI tumour

Loss or

gain of

nucleotides

Molecular profileMicrosatellite Instability

(ou soustraction)

Normal DNA

MSI tumour

NucleotidesLoss or gain

MisMatch Repair system (MMR)

4 proteinsfor DNA reparation

*Marisa et al. Plos One 2016, * * Guinney Nature Med 2015

Molecular profile

Signatures

Poor pronosis

Addition of immune signature ?

Adapté de Pagès et al., New Engl J Med 2005

CMS**ConsensusMolecularsignature

*Marisa et al. Plos One 2016, * * Guinney Nature Med 2015

Molecular profile

Signatures

Adapté de Pagès et al., New Engl J Med 2005

CMS**ConsensusMolecularsignature

Reliable on fixed tissue ? Reliable by using immunohistochimistry ?

Prediction of response to FOLFIRI, Cetuximab ?

Quality control

Quality control

Quality control