Microsatellite instability testing is an important part in diagnostics in Metastatic cancer settings after the FDA has given approval for tissue agnostic indications in almost all solid cancers. MSI by PCR and MMR status by IHC is also helpful for evaluation of genetic risk in Colon and Endometrial cancers

1. MICROSATELLITE
INSTABILITY –
WHAT IS IT? HOW
TO TEST IT?
DR Venkata Pradeep Babu K
Medical Oncology

2. MICROSATELLITE INSTABILITY- ROLE IN
ONCOLOGY
• Initially evolved in colon cancer
• Colon cancers - 9% of all cancers
sporadic
Familial
Hereditary

3. The types of colon cancer
Sporadic disease- 70 percent of
all CRCs - It is most common
over the age of 50 - Dietary and
environmental factors have
been etiologically implicated.
The least well understood
pattern is known as "familial"
CRC - 25 percent of cases.
Affected patients have a family
history of CRC, but the pattern
is not consistent with one of the
inherited syndromes.
The risk is high but not as high
as inherited syndromes.

4. Inherited colon cancers
• Less than 10 percent of patients have a true inherited predisposition to
CRC.
• Inherited cancers are divided according to whether Colonic Polyps are
present or not as a disease manifestation.

5. Inherited colon cancer
With polyposis
familial adenomatous polyposis (FAP)
MUTYH-associated polyposis (MAP),
The hamartomatous polyposis syndromes
(eg, Peutz-Jeghers, juvenile polyposis)
The phosphatase and tensin homolog
[PTEN] hamartoma tumor [Cowden]
syndrome)
Without polyposis
HNPCC aka Lynch
syndrome (MSI
related)

6. Let’s start
with some
Basics….
• Cancer is a genetic disease
• Any cancer occurs due to the abnormalities in
one of the four important classes of genes.
• Tumor suppressor
• Proto-oncogenes
• DNA repair genes
• Apoptosis related genes

7. For the simplest of organism- Gene is a basic unit

8. For human

9. DNA basic
structure
• A pairs with T
• G pairs with C

10. How much defect in DNA can cause cancer?
The information in A,T,G,C s
if printed on A4 sheets, will
occupy ten 8*10*10 rooms
filled with those papers.
And to cause cancer the
mistake needed is just one
base in some cancers!!

11. DNA mistakes
– how these
happen and
how these are
corrected?
DNA repair
systems

12. DNA REPAIR
SINGLE STRAND REPAIR DOUBLE STRAND REPAIR

13. Double Strand Repair
• Homologous Recombination Mechanism
• Non-Homologous recombination Mechanism

14. Single strand repair
3 types
Nucleotide excision repair
Base excision repair
Mismatch repair

15. MISMATCH REPAIR
• The mismatch can occur any where in DNA, during replication (ATGC)
• The mismatch is more prone in repetitive sequences of DNA.
• These repetitive sequences many a times escape surveillance by DNA polymerase
system, prone to mutations.
• These repetitions are DNA sequences of Base”Pairs”.
• The group of base pairs can be repeated many times and named accordingly.
• Example: AGAGAG doublet repeated 3 times in tandem.

16. What is the importance of Repetitive DNA?
• We, all humans share 99% of DNA
• 1% of remaining have variations resulting in diversity, except in
identical twins.
• Majority of this diversity is due to repetitive DNA, which is unique for
everyone.

23. Our area of interest is
Microsatellite
• These repetitive areas are
more prone to mutations,
most importantly
“MISMATCH”

24. This mismatch
is repaired by
MMR proteins
• MMR proteins recognizes and binds to
mismatch area
• Excises the mismatch area using Nucleases
• DNA polymerase reforms the DNA strand
in a proper fashion

27. • The important proteins are MSH2,MLH1,MLH3,MSH3, MSH6 and PMS2
• The mismatch repair proteins are coded by MMR genes.
• These genes are mutated in some individuals and can result in cancer – MMR
deficiency related cancers. These mutations can be somatic or Germline.
• The MMR protein deficiency leads to uncorrected and uncontrolled mutations and
mismatches in Microsatellites, leading to Microsatellite Instability. (MSI-H cancers).

28. • These microsatellites occur in non-coding regions.
• In MMR deficiency, they continue to replicate with
errors and no mechanism to correct it through various
cell division cycles.
• As those cells continue to divide, increasing errors
accumulate in a random selection of genes.
• Occasionally one of those errors will occur in an
oncogene or tumor suppressor gene, potentially
giving that cell a growth advantage and transforming
the tissue into a malignant tumor.

29. MSI
• This propensity for uncorrected errors
(ie, mutations) is called the "mutator
phenotype“ – Microsatellite instability.
• It can serve as a molecular marker for
HNPCC at the individual tumor level.
• But how to detect it?

30. TESTS to detect MSI
• First, we should understand what we are testing and what test can be
used.
• MMR gene mutations (Germline or somatic) is the cause.
• When the genes are mutated, Proteins are not produced – Expression
is lost.
• Because of absence of corrections by MMR proteins, MSI with
hypermutability is the effect.

32. What are the options?
• To test MMR gene mutations – NGS. If these genes are mutated,
probably we are dealing with Lynch syndrome
• MMR proteins loss can be detected by IHC. How ever protein loss can
be seen in genetic mutations as well as epigenetic changes like
methylation of genes (Sporadic event).
• The effect (hypermutability) of Microsatellites – can be detected by
PCR.

33. IHC and PCR

34. • PCR with 5 markers (BAT52 etc) detects the effect (MSI)
• IHC of MMR proteins – loss of protein expression indirectly infers
mutation in corresponding genes. (The cause).
• Both the above tests are not diagnostic of Lynch syndrome.
• Because MSI can be high in sporadic tumors also – detected by PCR
• IHC loss of protein can be seen due to epigenetic inactivation of MMR
genes.
• We call it as a lynch only when directly gene is affected by mutation.
(Not epigenetic changes)

35. MSI testing
• BAT25, BAT26, D2S123, D5S346, and D17S25 are markers used in PCR.
• Positive cases are stratified into MSI-High (MSI-H), in which at least 30% of the markers
show instability (or at least 2 of the 5 markers if the basic NCI panel is used)
• MSI-Low (MSI-L), in which less than 30% of the markers (or only one of the markers in the
NCI panel) show instability.
• Negative cases, in which none of the markers shows instability, are designated MSI-Stable
[MSS].

36. MSI-H
• Previously, MSI-H results are considered clinically
important regarding potential genetic etiology and
prognosis. (Now an indication and eligibility for
Immunotherapy).
• Regarding etiology, the finding of MSI suggests that the
tumor may be part of the HNPCC syndrome.
• MSI-H status can alert the patient that other blood
relatives should undergo surveillance (colonoscopy).
• Also for the patient himself or herself should be
monitored for the appearance of other malignancies
associated with the syndrome, such as those originating
in the stomach, bladder, and especially the endometrium.

37. Testing in Non-cancer relative of
Lynch patient
• Keep in mind that screening of healthy relatives must be either clinical or by direct DNA
sequencing of the mismatch repair genes (MSH2, MLH1, etc.) themselves.
• In the absence of a colon tumor, no MSI testing can be done on germline (ie, non-tumor)
DNA alone.

38. IS MSI-H by PCR always Lynch syndrome?
• 15% of sporadic tumors show MSI on PCR.
• Only further studies can distinguish the sporadic from HNPCC tumors.
One is complete sequencing of the mismatch repair genes, which is
offered in only a few select laboratories and is quite expensive.
• A faster and less expensive alternative is to perform
immunohistochemistry (IHC) for expression of these genes in the
tumor using specific antibodies against their protein products.

40. Clinical significance
of MSI-dMMR

41. MSI testing in daily practice
• MSI testing assesses the functionality of the MMR system and has different
clinical significance for sporadic and hereditary cancers.
• It has an established role in the identification of hereditary cancer syndromes and
is of prognostic significance in surgically resected gastrointestinal cancers.
• It also has an emerging potential predictive value of response to immunotherapy.
• These findings have recently increased the clinical request for MSI molecular
testing as a predictive biomarker for immunotherapy - avoid excessive and
fruitless costs for the health system.

42. Hereditary cancer syndromes associated with MSI cancers
• Lynch syndrome
• These patients are characterized by early onset of tumours (average age <45 years),
mainly colorectal and endometrial but also tumours in other organs, and usually present
germline mutations in MLH1 or MSH2
• Lynch syndrome due to TACSTD1 germline mutations
• Biallelic mismatch repair deficiency syndrome: GI, Brain, Hemat and CALM
• Muir-Torre Syndrome: Sebaceous gland tumors with internal malignancies
• Turcot’s syndrome: Early Brain and colorectal cancers (APC, MLH1, PMS2)

43. MSI in different sporadic cancer types
• Colorectal cancer:
• 15% of sporadic CRCs harbor MSI - Related to MLH1 promoter
hypermethylation
• MSI sporadic CRCs are characterized by specific clinicopathological
features: mainly female gender, older age, right colon location, high
grade, mucinous differentiation, signet ring or medullary histology,
peritumoural lymphocytic infiltrate and Crohn-like inflammatory
reaction, diploid status, lower stage and better prognosis

44. • MSI is considered a favourable prognostic factor in early stage CRCs,
with longer disease free and overall survival (DFS and OS).
• Some authors hypothesize that their better prognosis may be partly
explained by the increased immune response found in dMMR
neoplasm
• Pt3no MSS MSI - treatment

45. • dMMR (MSI) negatively affects the response of CRCs to chemo drugs
such as cisplatin, TMZ, Procarbazine.
• 5-FU may not benefit MSI-H patients, but may be beneficial in MSS.
• A retrospective analysis has shown a statistically significant survival
benefit for patients with dMMR tumours by the addition
of bevacizumab to adjuvant FOLFOX therapy compared with patients with
proficient MMR tumours

46. • The utility of MSI status as a promising predictive marker for response
to anti-PD-1 therapy in stage IV CRCs has been recently reported10,11.
• MSI can be acquired during chemotherapy by selective mutations in
MMR genes.

47. • Gastric cancer: MSI in gastric cancer may be considered a favourable
prognostic indicator for both earlyand advanced stages.
• Endometrial cancer (EC). EC is associated with defective MMR in up
to 33% of cases.
• The universal screening of ECs for MSI has been suggested to identify
Lynch syndrome patients
• Ovarian cancer – 10% ; Cervical Cancer- 5%, Breast : 0-1%

48. Sporadic hepatic, pancreatic and biliary tract cancers
• MSI is almost non-existent in sporadic pancreatic ductal
adenocarcinoma (PDAC) occurring in less than 1% of cases based on
molecular MSI testing.
• MSI is also found in the peculiar and rare medullary subtype of
pancreatic carcinoma – a rare type associated with Lynch syndrome.

49. Sporadic skin tumours and melanoma
• Sebaceous gland skin tumours (sebaceous hyperplasias, sebaceous
adenomas, and sebaceous carcinomas) are ‘sentinel’ pathologies of
Muir-Torre syndrome.
• About 25% of sporadic sebaceous skin tumours show MSI 104,105.
• Due to such high prevalence MSI testing is recommended in
all sebaceous neoplasms regardless of patient's age or other clinical
characteristics

50. • Melanoma:
• MSI status increases from benign nevi (0%) through primary
melanoma (11%) to metastatic melanoma (21%-77%)

51. • Lung cancer:
• MSI is absent in SCLC (0%) and exceedingly rare in NSCLC (0-1%), and
thus no prognostic or predictive value of dMMR status exist in lung
cancer
• Glioma. MSI is extremely rare (0.16%) in gliomas of adults.
• MSI was reported in a significant proportion (between 18% and 33%)
of high grade, paediatric and young adult gliomas, also in the setting
of Turcot’s syndrome

52. • Prostate cancer: 1% in primary to 12% in metastatic cancers
• Head and neck squamous cell cancer. In only around 1%
• Renal cell carcinoma. MSI is practically absent in renal cell carcinoma
(0%-0.7%).

53. MSI and immunotherapy
• dMMR - High TILs - upregulation of immune checkpoint proteins –
Increased TMB- Immune response
• somatic hypermutation creating putative neoepitopes is generated
not only by MSI/dMMR but also by a high mutational load of
nonsynonymous mutations due to mutations in DNA
polymerases POLE or POLD1
• Such hypermutation may reveal higher predictive power than MSI
status as immunotherapy response biomarkers.