This document discusses microsatellite instability (MSI), which refers to changes in repeated sequences of DNA called microsatellites during DNA replication due to defects in the DNA mismatch repair system. The key mechanisms, detection methods, clinical significance and applications of MSI in various cancers are described. MSI is an important factor in tumor development and progression. Detection of MSI using techniques such as PCR and immunohistochemistry can aid in cancer diagnosis and predict response to immunotherapy. Cancers with high MSI (MSI-H) often respond well to immune checkpoint inhibitors.

1. Micro
satellite
instability
D E P A R T M E N T O F S U R G I C A L
O N C O L O G Y
C E N T R E F O R O N C O L O G Y
G R H , R O Y A P E T T A H

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Journal
Bio- Medical Central journal – UK based non profit open access publisher
Impact Factor is 7.86
Kai Li, Haiqing Luo, Lianfang Huang , Hui Luo2 and Xiao Zhu
Guangdong Medical University, Zhanjiang 524023, China

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Introduction
• Microsatellite (MS), also called Short Tandem Repeats (STRs) or Simple Sequence Repeat
(SSRs), consists of repeated sequences of 1–6 nucleotides.
-widely distributed and mostly is located near the coding region.
- Can be located others region like intron or non-coding region
-- Each MS specific site  two parts: the central core and the peripheral flanks,
 the specificity of MS is mainly due to the change in the number of
core repeating units

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Definition : Change in cells where number of inherited repeated bases of micro
satellite are different
Mechanism: i) to be DNA slippage in the process of replication
ii) Defective DNA match in replication
Mismatch repair (MMR) can correct in the process of DNA replication.
MSI is an important factor in the occurrence and development of tumours

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High microsatellite instability (MSI-H), low microsatellite instability (MSI-L) and microsatellite
stability (MSS).
Presently MSI (L) and MSS are classified as single entity
Colorectal cancer – Microsatellite instability mechanisms sporadic and Lynch syndrome
MSI H tumours respond well to immunotherapy

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Fluorescent PCR and Capillary
electrophoresis.
- Polymerase Chain Reaction (PCR) is to compare the microsatellite loci detected in tumor
tissues
with normal DNA,
- Two single nucleotide repeat loci BAT25 and BAT-26 and three multi-nucleotide repeat loci
D2S123, D5S346 and D17S250 as microsatellite markers to determine the status of MSI.
-. Fluorescence multiplex PCR and CE is used to detect genes after amplification after
fluorescence labeled PCR amplification.
- The gold standard for MSI detection.

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Immunohistochemistry:
- Detection of MMR gene deletion can indirectly reflect the status of MSI.
- Detects the expression of MMR protein which consists of hMLH1, hPMS2, hMSH2 and
hMSH6.
- MMR deficient and MMR proficient.
- molecular analysis to IHC and MSI analysis can reduce the incompatibility of results .

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- Single molecule inversion probes:
- MANTIS
MSI score after calculating the allele distribution difference value of each microsatellite site
by comparing tumor and normal samples.
quantitative analysis

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Mechanism of MMR:
Slipped Strand Mispairing : in the process of DNA replication and synthesis, the allele
region of MS repeat sequence between new chain and template chain may be mismatched.
This results in the instantaneous separation of new chain and template chain or the
formation of stable single chain structure by several repeat units.
When slip mutation occurs, MS with small number of repeat units expands more frequently,
MMR deficient:

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- the germline mutations of MMR gene, POLE (polymerase E)/POLD1 (human DNA
polymerase δ) are more common.
- The loss of miRNA (microRNA) mediated regulation.
- A class of noncoding RNA molecules can trigger human immune response and accelerate
the development of cancer.
- MSI-H cases occur some tumor specific loci.
- The occurrence of frameshift MSI in TGFBR2 is more common in Colon adenocarcinoma
and Stomach adenocarcinoma.
- MSI occurs mostly in ion-binding genes in gastric adenocarcinoma

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Treatment options in MSI tumours:
- MSI-H treatment, such as PD-L1 (programed cell death ligand 1) immunosuppressant, can
produce heteroantigens that are easy to be recognized by T cells.
- RecQDNA helicase WRN (Werner syndrome, RecQ helicase-like) is an essential factor for
the MSI model  Silencing WRN can induce DNA double-strand breakage

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Clinical Applications:
- dMMR is common in patients with Lynch syndrome (LS), so patients with MSI-H or dMMR
tumors can predict Lynch syndrome through MSI related tests.
- NCCN panel includes MMR genes (hMLH1, hMSH2, hMSH6, hPMS2) and EpCAM genes.
- the hMLH1 promoter needs to be tested to determine whether there is methylation.
- when EpCAM immunostaining is negative  indicates hMSH2 mutation.
- combination of IHC and genetic evaluation  ideal

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Colorectal Cancers
- Tumours are infltrated with dense cytotoxic T cells.
- NR-21, BAT-26 and BAT-25 markers play an important role in judging MSI status
- Prognosis of MSI-H colorectal cancer was good while the prognosis of MSS and MSI-L
CRC was poor.
- Early stage cancers vs late stage cancers.

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Gastric Cancers:
Choi et al. suggested that microsatellite instability is also found in gastric cancer.
Using hMLH1 and hMSH2 in IHC and MSI analysis system, patients with MSI related gastric
cancer can be detected.
MSI-H patients with gastric cancer is common in women and most of them occur in non-
cardia area.
Good prognostic factor
PD L1 , CD8 expression
Siewerts type 2 and 3 tumours .

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Breast
- BRCA1 expression function can affect the silencing mechanism.
- The mutation of BRCA1, which can make the repair function of DNA loss, will cause
microsatellite instability and abnormal cell
-- Poor prognosis
PROSTATE:
-pathogenic embryonic mutants carrying Lynch syndrome-related genes
- targetable mutation  good prognosis

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CHOLANGIOCARCINOMA:
Non Trematode related cholangiocarcinoma
MSI-H were young patients with atypical tissue morphology
MSI-H/dMMR patients with cholangiocarcinoma have good prognosis for the anti-PD-1/PD-L1
treatment.
CHRONIC MYELOID LEUKEMIA:
Analysis of MSI related loci D17S261 and D3S643 is helpful to identify MSI related chronic
leukemia
PDL1 responsiveness

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BLADDER CANCER:
IHC detection of hMLH1, hMSH2 and hMSH6 and MSI analysis can detect MSI associated
bladder cancer.
a low risk of bladder cancer when they get MSI-H Lynch syndrome.
MSI related loci D16S476, D9S171 of MSI-H patients with bladder cancer
MSI of urine sediment can be considered as a clinical assistant diagnosis.

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OVARIAN CANCER:
CD8+, PD-1+, and TILS in MSI patients.
The patients with Clearcell ovarian carcinoma (CCOCs) are likely to beneft from
immunotherapy. MSI analysis.
ENDOMETRIAL CARCINOMA:
MSI analysis and IHC MMR related protein detection can be used to identify MSI endometrial
carcinoma.
Stage of disease and prognosis

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PANCREATIC CANCER:
Poor prognosis
THYROID CANCERS:
Prolonged survival and seen in Follicular Carcinoma Thyroid.
ADRENAL CORTICAL CARCINOMA:
Prognosis is not yet determined

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Preventive Measures
MSI or MMR testing should be considered for all types of colorectal cancer.
Aspirin/Sulindac may play a preventive role in reducing the risk of Lynch syndrome-related
cancer
HLA-A0201-restricted cytotoxic T cell epitope (FSP11) is expected to become an integral part
of MSI-H tumor vaccine in the future,

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- 5-FU is not used as adjuvant chemotherapy for patients with MSI-H and dMMR
characteristics for the reason that patients with MSI-H/dMMR have poor results.
- Early vs late stage cancer.
- Benson et al - MMR status of patients with stage III to IV CRC does not affect the outcome
of 5-FU treatment.
MSI-H can be used as one of the predictors of the efficacy of immunotherapy at present.
FDA approved Nivolumab with advanced metastatic colon cancer

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The FDA approved the combination therapy of Nivolumab and Ipilimumab as the frst
immunosuppressive combination therapy for metastatic CRC with worsened MSI-H/dMMR
after first line treatment.
Check mate 142 trial.
Bevaczimumab in MSI H tumours
PDL1 inhibtors role in gastric cancer – doubtful
Immune check point inhibitors combined with radiotherapy is recommended as a treatment
plan for patients with advanced biliary tumors

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- Tumors with high mutation rates may respond well to checkpoint inhibitors (CPI and scope
for additional testing.

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Conclusions and Perspectives:
The development of PCR and IHC promote the development of MSI detection.
The need of large scale studies to expand uses
Early diagnosis of MSI is of great significance to the prognosis and treatment of MSI.
Early use of aspirin in patients with hMSH2 and hMLH1 gene changes is of great signifcance
to reduce the risk of cancer related to Lynch syndrome
Anti tumour vaccines

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More clinical studies on the relationship between MSI related sites and tumor drug resistance
are needed to improve the therapeutic effect of chemotherapy.
MSI will open up a new field for the diagnosis, prevention and treatment of diseases