Microsatellite instability read more

Microsatellite instability – MSI

Microsatellite instability (MSI) is the mutational signature found in colorectal cancers (CRCs) that evolve as a result of inactivation of the DNA mismatch repair (MMR) system. Genes in the MMR pathway are responsible for identifying and repairing single nucleotide mismatches and insertion or deletion loops that occur as cells grow and divide [1]. Defects in the genes involved in mismatch repair lead to an accumulation of somatic mutations in a cell, which may result in the cell becoming malignant.

Microsatellites are repeating sequences of nucleotide bases (e.g., AAAAA or CGCGCGCG) of unknown function within the genome. The length of  microsatellites is highly polymorphic in human populations, but appear stable during the life span of an individual. Microsatellites do not cause a malignancy to develop, but fluctuations in the length of microsatellites (termed instability) can mean that mismatch repair genes are not functioning correctly. MSI can be found in approximately 15% of all CRCs. Approximately 3% of all CRCs are a consequence of Lynch Syndrome, and nearly all Lynch Syndrome CRCs have MSI [2].

Microsatellite instability testing can be performed to determine if a tumor exhibits microsatellite instability (MSI) by comparing the microsatellites in the tumor specimen to a normal tissue of the same individual. If the tumor specimen exhibits alterations within the microsatellite regions, it is indicative of a probable defect in the mismatch repair genes. MSI testing demonstrating instability in the tumor specimen is suggestive of HNPCC, although not diagnostic since 10-15% of sporadic colon cancers will also exhibit MSI.

For the determination of MSI, we are using fragment analysis based test of six markers: five microsatellite markers of the Bethesda consensus panel (BAT25, BAT26, D5S346, D2S123, and D17S250) [3] one additional mononucleotide marker (BAT40) [4]. The panel of six markers is used to assess microsatellite instability in tumor tissue and normal tissue.

Patterns of normal and tumor genotypes are compared for each marker and scored as:

>> MSI-high if more than 30% of the markers show instability
>> MSI-low if fewer than 30% of the markers show instability
>> MSI-stable if 0% of the markers show instability

Patient with a microsatellite instability-high (MSI-H) tumor may be further tested for germline mutation in a mismatch repair gene and, if found, the patient and blood relatives may be counseled about strategies for early cancer detection and risk reduction. On the other hand, the majority of MSI-H tumors will be identified as sporadic by further testing, which greatly reduces the likelihood of developing new malignancies.

References:
[1]    Boland CR: Evolution of the nomenclature for the hereditary colorectal cancer syndromes. Fam Cancer 4:211-218, 2005.
[2]    Dietmaier W, Wallinger S, Bocker T et al. Diagnostic microsatellite instability: Definition and correlation with mismatch repair protein expression. Cancer Res 1997; 57: 4749–56.
[3]     Boland C.R., Thibodeau S.N., Hamilton S.R., Sidransky D., Eshleman J.R, Burt R.W., Meltzer S.J., Rodriguez-Bigas M.A., Fodde R., Ranzani G.N., Srivastava S. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res. 1998; 58: 5248–57.
[4]    Samowitz W.S., Slattery M.L., Potter J.D., Leppert M.F. BAT-26 and BAT-40  Instability in Colorectal Adenomas and Carcinomas and Germline Polymorphisms. American Journal of Pathology. 1999;154:1637-1641.

Lynch Syndrome/Hereditary Non-Polyposis Colon Cancer – HNPCC

Lynch Syndrome
NGS panel

Genes: MLH1, MSH2, MSH6

Price / TAT: 1030 EUR / 6-9 weeks

Specimen requirements: 2-4 ml of blood with anticoagulant EDTA

1 µg DNA in TE, AE or pure sterile water at 100-250 ng/µl
The A260/A280 ratio should be 1.8-2.0. DNA sample should be run on an agarose gel as a single band, showing no degradation, alongside with a quantitative DNA marker.


Ordering information: Go to online ordering or download sample submission form

Single Gene Sequencing

Genes: MLH1, MSH2, MSH6

Lab method: Sanger sequencing

Price / TAT: MLH1 gene – 515 EUR / 2-4 weeks
MSH2 gene – 515 EUR / 2-4 weeks
MSH6 gene – 515 EUR / 2-4 weeks

Specimen requirements: 2-4 ml of blood with anticoagulant EDTA

2 µg DNA in TE, AE or pure sterile water at 100-250 ng/µl
The A260/A280 ratio should be 1.8-2.0. DNA sample should be run on an agarose gel as a single band, showing no degradation, alongside with a quantitative DNA marker.


Ordering information: Go to online ordering or download sample submission form

Deletion/duplication analysis

Genes: MLH1, MSH2, MSH6

Lab method: MLPA

Price / TAT: MLH1, MSH2 genes – 310 EUR / 4-6 weeks
MSH6 gene – 310 EUR / 4-6 weeks

Specimen requirements: 2-4 ml of blood with anticoagulant EDTA

2,5 µg DNA in TE, AE or pure sterile water at 100-250 ng/µl
The A260/A280 ratio should be 1.8-2.0. DNA sample should be run on an agarose gel as a single band, showing no degradation, alongside with a quantitative DNA marker.


Ordering information: Go to online ordering or download sample submission form

Referral for molecular testing of Lynch syndrome:

  1. Tumour tissue analysis to evaluate the MMR proteins expression by immunohistochemical analysis (IHC) and DNA microsatellite instability (MSI) testing is suggested for the individuals meeting Amsterdam II/Bethesda criteria.
  2. If absence of the MLH1/PMS2 proteins expression is observed by IHC, methylation analysis of the MLH1 gene promoter and/or testing of the somatic BRAF V600E mutation is recommended in order to exclude sporadic colorectal cancer cases.
  3. If tumour with MMR deficiency and MSI high is detected, further mutation analysis from peripheral blood/normal tissue of the MMR genes is indicated.

Indications for mutation analysis:

    1. Testing of individuals meeting Amsterdam II/Bethesda criteria
    2. Testing of individuals with family history of colorectal cancer or other Lynch syndrome-related cancers
    3. Testing of at-risk family members for known mutations
    4. Genetic counseling

Lynch syndrome, also called hereditary non-polyposis colon cancer (HNPCC), is characterized by an increased risk of colon cancer and other cancers (e.g., of the endometrium, ovary, stomach, small intestine, hepatobiliary tract, upper urinary tract, brain, and skin). Lynch syndrome is inherited in an autosomal dominant manner and it is associated with germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. Mutation carriers have a lifetime risk of up to 80% for colorectal cancer, 20-60% risk of endometrial cancer, as well as other tumors. Lynch syndrome is associated with early onset of cancer, the average age of diagnosis is 45 years.