The assessment compared tumour testing strategies involving microsatellite instability (MSI) testing, immunohistochemistry (IHC) testing (both with and without further testing to exclude sporadic colorectal cancers), and comprehensive mismatch repair (MMR) gene mutation testing with a single comparator. Comprehensive MMR gene mutation testing was also used as the reference standard for assessing the accuracy of the tumour testing strategies.
3.1 Microsatellites are repetitive sequences of DNA that are at increased risk of copying errors during replication. In tumours of people without an effective DNA MMR system, errors in copying microsatellite sequences cause them to vary in length. This is known as MSI.
3.2 MSI testing can, therefore, be used to assess whether the DNA MMR system is working effectively by detecting the size of microsatellite regions in tumour samples from people diagnosed with colorectal cancer. Deficiencies in DNA MMR show that a person's cancer may have developed because they have Lynch syndrome.
3.3 MSI testing is a polymerase chain reaction (PCR) based method that amplifies DNA at several microsatellite sites from a person's tumour tissue sample and also a healthy tissue sample. MSI tests can differ in the panel of microsatellite marker sites they assess, both in terms of their number and genetic location.
3.4 IHC uses antibodies to detect decreased or abnormal expression of MMR proteins in colorectal tumour tissue samples. Absent or reduced nuclear staining of 1 or more MMR proteins suggests that there may be a pathogenic mutation in a gene encoding these proteins.
3.5 MMR proteins detected by IHC are MLH1, MSH2, MSH6 and PMS2. Laboratories may differ in the source of the antibodies used to carry out these tests.
3.6 Although deficient DNA MMR systems (identified with MSI testing or IHC) indicate that a person may have Lynch syndrome, they can also be seen in sporadic colorectal cancers (that is, cancers not caused by Lynch syndrome). Sporadic colorectal cancers can show loss of MLH1 protein expression caused by changes in the MLH1 gene promoter. MLH1 promoter hypermethylation testing can be used to directly test for these changes, or BRAF V600E mutation testing can be used, because this mutation is associated with MLH1 promoter hypermethylation. Using these tests can identify sporadic colorectal tumours that are MSI positive or have abnormal MLH1 protein expression in people who are not at risk for Lynch syndrome, and therefore prevent unnecessary further genetic testing.
3.7 Comprehensive screening for constitutional mutations in the MMR genes, and also possibly the EPCAM gene, is the gold standard for diagnosing Lynch syndrome. This involves gene sequencing to detect point mutations and small insertions or deletions in these genes, and also multiplex ligation-dependent probe amplification to detect larger structural changes to genes, such as deletions, duplications or rearrangements.
3.8 Comprehensive screening for constitutional mutations in MMR genes can identify novel sequence variations in these genes that are of unknown significance, that is, it is unknown whether they are pathological or non-pathological. It can therefore be uncertain as to whether people with such sequence variants should be diagnosed as having Lynch syndrome or not.