Mismatch Repair Deficiency Testing Kenneth J. Bloom, MD, FCAP Chief Medical Officer, Clarient, a GE...

Mismatch Repair Deficiency TestingKenneth J. Bloom, MD, FCAPChief Medical Officer, Clarient, a GE Healthcare Company

2 Mismatch Repair Deficiency Testing

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Colorectal Cancer

• 3rd leading cancer and 2nd leading cause of cancer death in males and females

• 150,000 new cases and 55,000 deaths in USA in 2006

• Most CRCs are preceded by adenomas

• Screening (Currently - FOBT, Endoscopy) (Future - Virtual Colonoscopy, Stool DNA)

• Overall 60% 5 yr. survival

• 93% if detected early (Stage I)

• 8% after distant metastasis (Stage IV)


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Normal

Micro-adenoma

APC 5q21

Adenoma

KRAS 12p

Adenoma HGD

DCC/DPC4 18q21

Carcinoma

p53 17p13

Metastasis

Other

Kinzler-Vogelstein-Fearon

Sequential steps in APC pathway

•At least 7 mutations•After APC the order is variable


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Pathways to CRC

3 Possible Pathways AND not linear

Chromosomal Instability 50-70%MSS sporadic CRCFAP

Microsatellite Instability 15%MSI sporadic CRCHNPCC

APC

p53

MMR

MLH1MSH2

TGFβRII, IGF2R, BAX

CpG Island MethylatorPhenotype (CIMP) 20-35%

Tend to have BRAF mutation EXCEPT in HNPCC

Tend to have KRAS mutation


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APC

Regulation of cell cycle and apoptosis

Mediation of intercellular adhesion

Stabilization of cytoskeleton

Signal transductionCell proliferation

Chromosomalstability

Cell migration

APC, 5q21, tumor suppressor gene and a “gatekeeper”

Chromosomal Instability Pathway


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E-Cadherin

B-Catenin

B-Catenin

B-Catenincomplex

p p

APCSerine/Threoninekinase

Degradation

Binds to TCF (T-cell factor) whichtriggers transcriptionof genes that promotecell division

Nucleus

APC - B-Catenin Interaction


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Sporadic MSS CRC

• The majority of CRCs (50-70% of CRC)• MSS, often aneuploid (chromosomal instability)• Left>right sided, M>F• Common phenotype (dirty necrosis, tumor budding, no TILs) • Initial APC somatic mutation• Often KRAS mutation (unresponsive to Anti-EGFR therapy)• Often p53 mutation (usual stage dependant prognosis)


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HNPCC

• 2-4% of CRCs• Phenotype mimics sporadic CRC, precursor adenoma• MSI-H, often diploid• Germline mutation in MMR (MSH2, MLH1)• No BRAF mutation• Rare or no p53 mutation• Better prognosis, but poor response to 5-FU• R>L• Microscopic features include

• Tumor infiltrating lymphocytes (TIL)• Crohn’s-like lymphoid reaction• Poor differentiation• Mucinous change


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MSI-H Sporadic CRC

• 12-15% of CRCs• Precursor is sessile serrated adenoma• MSI-H, often diploid• Methylation of MLH-1 promoter gene• Often BRAF mutation• Rare or no p53 mutation• R>L, F>M• Better prognosis, but poor response to

5-FU• Microscopic phenotype is the same as

HNPCC TIL, mucinous, poor differentiation

• No need for prophylactic colectomy or hysterectomy

• No response to Anti-EGFR therapy


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CpG Island Methylator Pathway

(CIMP) Pathway • 20-35% cases of CRC• MSS (or MSI-L)• Methylation of CpG islands (includes some MHL-1, TP16

and others)• Generalized increase in de novo methylation • Often diploid• Rare or no p53 mutation• Precursor serrated adenoma• Often BRAF mutation, some also have KRAS mutation• Frequent family history (but no germline mutation)• R>L, F>M• Generally no TILs (unlike MSI-H) tumors

• Poor prognosis compared to MSI CRCs• But responsive to 5-FU


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Microsatellite Instability Pathway• Errors in DNA occur constantly during replication

• Mis-Match-Repair system identifies, excises and corrects the errors “Caretaker” genes that do not initiate tumor but cause rapid progression

• MMR defect is identified by studying DNA microsatellites (tri and tetranucleotide repeat sequences of unknown function distributed throughout the human genome)

• Similar short sequences also occur within some tumor suppressor genes (TGFβRII, IGF2R, BAX), which then may become mutated

1 base pair mismatch

3 base pairs mismatch

MSH2 protein is the “scout” identifying an error has occurred.

Heterodimeric complex is formed with•MSH6 if a single base pair mismatch

•MSH3 if there is a 2-8 nucleotide insertion or deletion.



MSH2/MSH6 complex repairs 1 base pair mismatchMSH2/MSH3 complex repairs 3 base pair mismatch



MLH1/PMS1 complex is recruited to excise the mismatched nucleotides

The MMR System

Vilar E et al. Nat Rev Clin Onc (2010) 7: 153

Genes Affected in dMMR

Hewish M et al. Nat Rev Clin Onc (2010) 7: 197-208


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Microsatellite Instability (MSI)

BAT25 Mononucleotide

BAT26 Mononucleotide

D5S346 Dinucleotide

D2S123 Dinucleotide

D17S250 Dinucleotide MSS (stable) = 0/5

MSI-L (low grade) = 1/5MSI-H (high grade) = 2 or >/5


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Unstable

Stable

Unstable

Unstable


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MSH-2 Wild-type and Mutated (likely HNPCC)

MLH-1 Wild-type and Mutated (can be sporadic or HNPCC)

Microsatellite Instability by IHC

IHC Findings in dMMR


Protein Interpretation

Inactivated Gene

Microsatellite Instability

MLH1 MSH2 MSH6 PMS2

+ + + + Intact MMR MSS

- + + - Deficient MMR MLH1 MSI

+ - - + Deficient MMR MSH2 MSI

+ + - + Deficient MMR MSH6 MSI or MSS

+ + + - Deficient MMR PMS2 MSI


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Problems with IHC as a Surrogate

Mutation involves other than MLH-1, MSH-2

Mutations which encode a nonfunctional protein

•Missense

•Frameshift

•Splice


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Rarely Nonsense Mutations Can Occur

Frameshift mutation Splice mutation

Wahlberg et al. Cancer Research (2002)

Known MLH1 germline mutation


Webinar 04/21/23Gatalica Z and Torlakovic E. Familial Cancer 2008;7:15-26

Testing for MSI

Genes Affected in dMMR


Affected Gene

% of Lynch

Syndrome

Age at Presentat

ion

IHC Finding

Sensitivity of IHC

Sensitivity of PCR

MLH1 32 45 Loss of MLH1 and

PMS2

92 92

MSH2 39 45 Loss of MSH2 and

MSH6

93 93

MSH6 14 56 Loss of MSH6

100 25

PMS2 15 59 Loss of PMS2

100 67

All 100 45-60 83-94 83-85


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Reasons for assessing dMMR

• Better classification of colon tumors

• Prognostic implications

• Predictive implications

• Rule out germline mutation

• Select patients for further testing


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Survival Differences dMMR

Ward et al. Gut (2001)


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Impact of dMMR on CRC Management


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Novel Strategy for dMMR



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Effect of 5-FU in dMMR



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Effect of 5FU in dMMR



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5-FU induced death in intact MMR


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Evidence of 5-FU Resistance

Cell line with methylation of MLH1 - MSI Resistant to

5FU

Demethylated cell line- MSS Sensitive to

5FU

MSI closely related to CIMP group, ?MGMT methylation


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Effect of Irinotecan in dMMR



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“Oh, if only it were so simple.”


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Thank You!

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Mismatch Repair Deficiency Testing Kenneth J. Bloom, MD, FCAP Chief Medical Officer, Clarient, a GE...

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