Microsatellite Instability and its

Detection by IHC
By Dr. Ankush Nayyar
MBBS, MD, Fellowship in Hematopathology
Indus International Hospital
Derabassi
 Content
- What are microsatellites?

- What is microsatellite instability?

- What happens if it is Increased?

- Which cancers are caused by increased MSI?

- How is it Detected?

- Why should it be detected?

- Conclusion
What are Microsatellites?
• Microsatellites are stretches of DNA with a repetitive sequence of
DNA Motifs, each motif composed of 1 to 6 nucleotides, (e.g., AAAAA
or CGCGCGCG), Repeated typically around 5-50 times.
• They are also called SSTR (Single sequence Tandom repeats)
• DNA has highly repetitive sequences present in large number of
copies.
• These repeat sequences can be arranged at irregular intervals
(Dispersed repeated DNA) or Clustered together (Tandomly repeated
DNA)
• These stretches are particularly susceptible to acquiring errors during
DNA replication, due to “DNA Polymerase slippage” or “Replication
Slippage”
• When DNA breaks apart to replicate and the reforms, it separates. It
comes back together in a specific order because the base pairs can
only match up in that specific order.
• Adenine (A) and thymine (T) can only pair with each other. The same
is true for cytosine (C) and guanine (G). However, in a microsatellite, it
becomes much more likely for one of them to get skipped when the
DNA comes together.
What is MMR and MSI?
Specific genes monitor the DNA for mistakes and then repair them. This
built-in ability to error-correct is called mismatch repair genes.
These genes and their products make microsatellites usually stable
when the DNA replicates.
• Loss of function of these genes results in Mismatch repair defects

• Microsatellite instability (MSI) is the condition of genetic

predisposition to mutation that results from impaired DNA mismatch
repair (MMR)- the cell has lost its ability to fix its own errors.
How do we detect MSI?

MMR detection by IHC detects presence or absence of Proteins involved in mismatch repair

MSI By PCR determines the functional activity of these proteins

MSI Detection by IHC
• MMR by IHC uses Labelled protein specific Antibodies to dtermine
each specific protein. One section for detection of each protein.
• IHC loss of protein- MMR deficient or D-MMR
• Mutations in MLH1, MSH2, MSH6 and PMS2, which are one of the
main causes of deficient mismatch repair and subsequent MSI are
detected by IHC
• In some cases, proteins may be present, but are in fact dysfunctional.
So IHC detects them, but actually, MSI is high.
• IHC detects expression or loss of proteins
• The proteins form heterodiamers - MLH1 with PMS2 and MSH2 with
MLH6.
• Concurrent Loss of MLH1 and PMS2 on one hand , And MSH2 and
MSH6 on the other.

• However, sporadic loss of PMS2 and MSH6 can be there

• 4 antibiotic screening is done.
 Immunohistochemistry
Stable tumour (MSS): 4 MMR proteins expressed
 Immunohistochemistry
Microsatellite Instable tumour(MSI): Loss of MMR proteins
Loss of MLH1 MSH2 +

Negative tumour Positive tumour

personna l casel F. Bibeau

MSH6 + Parallel loss of PMS2

*MisMatch Repair
CAP Protocol for interpretation of MSI
• Immunohistochemistry Testing for Mismatch Repair (MMR) Proteins-
• MLH1 , MSH2 , MSH6 , PMS2 All are nuclear markers, Intact nuclear expression or
Loss of nuclear expression is determined. If it Cannot be determined, It is recorded
as indeterminate.
• Background non-neoplastic tissue/internal control with intact nuclear expression.
• Patchy expression is normal

Interpretation –
• MSI – stable (MSS) -All 4 proteins show nuclear positivity- No loss of nuclear
expression of MMR proteins: low probability of microsatellite instability
• MSI Low- Loss of only 1 marker
• MSI High- Loss of 2 or more nuclear markers
• MSI Indeterminate- disagreement or difficulties in interpreting IHC or in the case of
MSI by PCR
• MSI by PCR Detects changes in DNA microsatellites, caused by Loss of
MMR protein activity

• To do this mononucleotide sequences are amplified from both tumor

and matched normal DNA sample. The amplified samples are resolved
by size and changes in size indicate MSI is present 9MSI High or MSI H.
USES Of MSI detection
• Diagnostic

• Prognostic

• Predictive
Diagnostic Utility
• Screening for HNPCC/Lynch syndrome
• Lynch syndrome- Every newly diagnosed case of CRC is recommended
to be screened for MSI. About 15% of patients having MSI tumors
have Lynch Syndrome, while the remaining have a sporadic form of
CRC; 95% of patients with Lynch Syndrome have MSI.
• CRC associated with lynch syndrome can have specific Clinical features
like early onset – less than 50 years age, multiple cancers etc or
specific morphological features like Right sided location, poor
differentiation, mucin production and Crohn like features,
Cap protocol for MSI and Lynch syndrome
Diagnosis
• Loss of nuclear expression of MLH1 and PMS2 both: testing for
methylation of the MLH1 promoter and/or mutation of BRAF is
indicated, absence of both MLH1 methylation and of BRAF mutation
suggests the possibility of Lynch syndrome
• Loss of nuclear expression of MSH2 and MSH6: high probability of
Lynch syndrome
• Loss of nuclear expression of MSH6 only: high probability of Lynch
syndrome
• Loss of nuclear expression of PMS2 only: high probability of Lynch
syndrome
MSI High, is it a bad news?

• A. It’s a bad news

• B. It’s a good news
MSI High, is it a bad news?

A. It’s a bad news

B. It’s a good news

• Answer- It Depends
Prognostic Significance of MSI
• MSI-related CRC has a distinct clinico-pathological profile, favorable
prognosis, and adjuvant therapy does not improve survival, especially
in stage II cancers.
• Recurrent or metastatic setting the MSI tumors tends to have negative
prognostic role with reduced Overall Survival as compared to the MSS
CRC
• There is a survival advantage for MSI-H colorectal carcinoma stage for
stage compared with MSI-low and MSI stable cancer
•MSI-H predicts poor response to fluorouracil-based
chemotherapy regimens
Predictive role of MSI
• MSI status as a promising biomarker which positively correlates with
survival outcomes
• Enables predicting the efficacy of immune checkpoint blockade
therapy in solid tumors.
• MSI High tumors have higher incidence of DNA defects in tumor cells.
• This results in their easier detection by Immune cells and hence
better host response
• This also makes them better target for immunotherapy
Difference in the response to immune checkpoint therapy between microsatellite-stable (MSS) tumors and
microsatellite instability-high or mismatch repair deficiency (MSI-H/dMMR) tumors.
High mutation burden in MSI-H/dMMR tumor leads to the synthesis of mutation-associated neoantigens (small
circles) presented by major histocompatibility complex (MHC) class I
CAP Recommendations for Microsatellite
Instability Testing for Immune Checkpoint
Inhibitor Therapy/Immunotherapy
Strong Recommendation for
• CRC being considered for immune checkpoint inhibitor therapy
• Gastroesophageal and small bowel cancer being considered for
immune checkpoint inhibitor therapy
• Endometrial cancer being considered for immune checkpoint
inhibitor therapy
• cancer patients being considered for immune checkpoint inhibitor
therapy, if an MMR deficiency consistent with Lynch Syndrome is
identified in the tumor
Conclusion
• MSI forms an important pathway for development of Cancers
• CRC Pathogenesis, one of the pathways is MSI
• MSI detection has a diagnostic role in Lynch syndrome
• MSI High tumors may predict Familial predisposition to cancers
• MSI high tumors predict poor response to Chemotherapy
• MSI High tumors predict good prognosis to Immunotherapy
• Overall predictive value of MSI depends on the stage of tumor
References
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70
• Kern SE, Fearon ER, Tersmette KW, et al. Clinical and pathological
associations with allelic loss in colorectal carcinoma [corrected] JAMA.
• Vogelstein B, Fearon ER, Kern SE, et al. Allelotype of colorectal
carcinomas. Science.
• Ionov Y, Peinado MA, Malkhosyan S, et al. Ubiquitous somatic
mutations in simple repeated sequences reveal a new mechanism for
colonic carcinogenesis.
THANK YOU