• Microsatellite instability (MSI) results from abnormal function of one or more mismatch repair genes (MLH1, PMS2, MSH2, MSH6)
• 10 - 15% of colorectal adenocarcinomas (CRC) are MSI / MMR deficient (dMMR)
• dMMR colorectal cancer arises due to sporadic hypermethylation of the MLH1 promoter or due to germline mutations in MMR genes (Lynch syndrome)
• In pathology, colorectal cancer is initially screened for MSI by assessing the presence of MMR proteins using immunohistochemistry
• Loss of one or more MMR proteins is followed by either MLH1 hypermethylation testing or BRAF mutation testing; the absence of MLH1 hypermethylation or a BRAF mutation prompts germline mutation to assess for Lynch syndrome
• Identifying dMMR colorectal cancer is significant as these tumors have a better stage adjusted survival compared to microsatellite stable (MSS) tumors

• Lynch syndrome was formerly known as hereditary nonpolyposis colorectal cancer (HNPCC) syndrome
• Biallelic germline mutation in an MMR related gene is termed constitutional mismatch repair deficiency

• MMR pathway (see diagram 1) (J Gastroenterol 2020;55:15)
  • Corrects DNA base substitution mismatch, insertions, deletions or slippage made during DNA replication
  • Errors are corrected through the action of two protein complexes:
    • MutSα: heterodimer of mutS homologue 2 (MSH2) and mutS homologue 6 (MSH6) proteins
    • MutSβ: heterodimer of MSH2 and mutS homologue 3 (MSH3) proteins
  • MutSα or MutSβ bind to the area with the defect and recruit MutLα (heterodimer of mutL homologue 1 [MLH1] and postmeiotic segregation increased 2 [PMS2] proteins)
  • PMS2 endonuclease makes a nick at 5’ to the mismatch (upstream)
  • Exonuclease 1 is recruited and activated by MSH2 or MLH1
    • Catalyzes excision of the nascent DNA strand up to and slightly beyond the mismatch
  • DNA excision gap is resynthesized by polymerase δ and nick is sealed by DNA ligase I
• MLH1 is required for the function of PMS2 (when MLH1 is lost, PMS2 must also be lost)
• MSH2 is required for the function of MSH6 (when MSH2 is lost, MSH6 must also be lost)

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• Lynch syndrome is associated with increased risk of carcinoma at other sites
  • Gastrointestinal: colon, small intestine, stomach, hepatobiliary, pancreas
  • Genitourinary: kidney, bladder, prostate
  • Gynecological: endometrium, ovary

Contributed by Phoenix D. Bell, M.D.

Contributed by Phoenix D. Bell, M.D.

• MSS / pMMR colorectal cancer: all MMR proteins intact (MLH1, PMS2, MSH2, MSH6)

• MSI / dMMR colorectal cancer: 1 or more MMR proteins absent (MLH1, PMS2, MSH2, MSH6)
• Note: Several pitfalls exist when interpreting MMR (Mod Pathol 2019;32:1)

• BRAF somatic mutation testing
  • DNA change C.1799T>A
  • Amino acid change: p.V600E (Val600Glu)
• BRAF mutation absent: send for somatic tests to evaluate tumor for Lynch syndrome
• Germline mutation testing
  • PCR amplification of seven markers including five mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27) and two pentanucleotide repeat markers (Penta C and Penta D) followed by separation of the PCR products by capillary electrophoresis
    • ≥ 2 unstable loci = MSI-H, < 1 unstable loci = MSI-L, 0 unstable loci = MSS
• Reference: Cancer Res 1998;58:5248

Contributed by Phoenix D. Bell, M.D.

• Interpretations for MSI immunohistochemistry, BRAF mutation or hypermethylation status should be included in a colorectal adenocarcinoma pathology report. Examples are as follows:
  • MSI (by IHC) interpretation:
    • pMMR colorectal cancer:
      • Immunohistochemical stains for the mismatch repair proteins MLH1, MSH2, MSH6 and PMS2 were performed. The tumor cells demonstrate retained nuclear staining for all four proteins, indicating the tumor is MMR proficient (pMMR) and likely microsatellite stable.
    • dMMR colorectal cancer:
      • Immunohistochemical stains demonstrate the tumor is mismatch repair protein deficient (dMMR) with loss of expression of MLH1 and PMS2, while nuclear expression of MSH2 and MSH6 is retained. BRAF mutational analysis (or methylation status) is being performed to determine if this represents a sporadic type carcinoma.
  • BRAF mutation interpretation:
    • Absence of mutation:
      • BRAF mutational analysis was performed and the V600E mutation was not identified. These are somatic tests being performed to evaluate the tumor phenotype. dMMR tumors may be sporadic or associated with Lynch syndrome / hereditary nonpolyposis colorectal cancer. Referral to a hereditary cancer screening program is recommended.
    • Presence of mutation:
      • Molecular testing demonstrated the presence of BRAF V600E mutation, which correlates with sporadic hypermethylation of the MLH1 promoter. However, if there is a strong family history, caution should be exercised in excluding patients from germline screening on the basis of BRAF V600E mutations.

A 64 year old woman presents for routine colonoscopy and a mass is identified in the ascending colon. She undergoes a partial hemicolectomy. Representative histologic sections of the mass are shown. Which of the following features is associated with MSI-H colorectal carcinoma?

1. Extensive intraluminal (dirty) necrosis
2. Lack of a Crohn-like reaction
3. Micropapillary subtype
4. Mucinous features

D. Mucinous features. Histologically, traditional MSS colorectal adenocarcinomas are seen as invasive irregular glands surrounded by a desmoplastic stroma. The cells are composed of elongated hyerchromatic nuclei, surrounding luminal (dirty) necrosis. In contrast, MSI / dMMR colorectal carcinomas are associated with mucinous / poorly differentiated / medullary / signet ring cell subtypes, a Crohn-like reaction (dense lymphoid aggregates), increased intratumor / peritumoral lymphocytes, relative lack of intraluminal necrosis and tumoral heterogeneity. Although many institutions regularly test colorectal carcinoma cases for MSI, these features in particular should signal one to consider an MSI / dMMR tumor.

Reference: Microsatellite instability pathway

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Sporadic MMR deficient colorectal carcinoma is due to promoter hypermethylation of which of the following genes?

1. EPCAM
2. MLH1
3. MSH2
4. MSH6
5. PMS2

B. Hypermethylation of the MLH1 (heterodimer of mutL homologue 1) promoter is the most common cause of sporadic microsatellite unstable (MSI) CRC. MLH1 is part of a heterodimer, which also includes post-meiotic segregation increased 2 (PMS). MLH1/PMS2 are members of the DNA mismatch repair (MMR) pathway, which fixes mutations in microsatellite regions made during DNA replication. Defects in any of the MMR genes (including MLH1) prevent these mutations from being repaired, resulting in MSI/MMR deficient (dMMR) CRC. In contrast to promoter hypermethylation that leads to sporadic MMR deficient colorectal carcinoma, Lynch syndrome is due to germline mutations in any of the MMR genes.

Reference: Microsatellite instability pathway

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