A review on the molecular diagnostics of Lynch syndrome: a central role for the pathology laboratory

Abstract

Lynch syndrome (LS) is caused by mutations in mismatch repair genes and is characterized by a high cumulative risk for the development of mainly colorectal carcinoma and endometrial carcinoma. Early detection of LS is important since surveillance can reduce morbidity and mortality. However, the diagnosis of LS is complicated by the absence of a pre-morbid phenotype and germline mutation analysis is expensive and time consuming. Therefore it is standard practice to precede germline mutation analysis by a molecular diagnostic work-up of tumours, guided by clinical and pathological criteria, to select patients for germline mutation analysis. In this review we address these molecular analyses, the central role for the pathologist in the selection of patients for germline diagnostics of LS, as well as the molecular basis of LS.

Fig 1

The MMR system. During DNA replication, insertions or deletions of one or more nucleotides and single nucleotide mismatches may occur. For example: (A) A single nucleotide mismatch occurs (G>T in red). (B) MSH2 and MSH6 form a heterodimer and recognize the mismatch. (C) MLH1 and PMS2 dimerize and bind to the MSH2-MSH6 complex. (D) The complex of four proteins activates an exonuclease to perform the DNA repair.

Fig 2

MSI. A schematic microsatellite is indicated (poly A track). When the tumour cells have an intact MMR system the size of the microsatellite will be the same in DNA isolated from normal (N) and from tumour (T) cells: microsatellite stable (MSS) tumour. In case of a defect in MMR the size of the microsatellite (number of repeat units) can change (in most cases becomes shorter) when comparing N with T DNA: microsatellite unstable (MSI) tumour. Asterisks indicate the microsatellite unstable tumour DNA fragment.

Fig 3

Five CRC cases; haematoxylin and eosin staining and MLH1, PMS2, MSH2 and MSH6 immunohistochemistry (IHC) results. Case 1: normal IHC in the tumour cells. Case 2: absence of MLH1 and PMS2 in the tumour cells. Case 3: absence of PMS2 in the tumour cells. Case 4: absence of MSH2 and MSH6 in the tumour cells. Case 5: absence of MSH6 in the tumour cells. Arrows point to IHC⁺ tumour cells, filled arrow heads point to IHC^– tumour cells and open arrow heads point to IHC⁺ stromal cells.

Fig 4

Flowchart for molecular diagnostics of LS in The Netherlands.

Fig 5

(A, B, C) Manual macrodissection of a normal tissue fragment (arrow) and a region of the tumour composed of a high percentage of tumour cells (arrow head), before, during and after macrodissection, respectively. (D, E, F) Laser capture microdissection of a small tumour tissue fragment surrounded by abundant stromal cells, before, during and after laser capture microdissection, respectively. Insert in (F) shows the microdissected fragment.

Fig 6

MSI analysis of four cases of paired normal (N) and tumour (T) DNA. (A) Case 1: N1 and T1, CRC no MSI: MSS. Case 2: N2 and T2, CRC demonstrates clear MSI. (B) Case 3: N3 and T3, endometrial carcinoma with MSI (subtle microsatellite shifts). Case 4: N4 and T4, CRC without MSI but with heterozygous NR-21 microsatellite alleles (present in normal and tumour DNA). Arrow heads indicate the MSI shifts, arrows indicate the variant NR-21 allele.

Fig 6

MSI analysis of four cases of paired normal (N) and tumour (T) DNA. (A) Case 1: N1 and T1, CRC no MSI: MSS. Case 2: N2 and T2, CRC demonstrates clear MSI. (B) Case 3: N3 and T3, endometrial carcinoma with MSI (subtle microsatellite shifts). Case 4: N4 and T4, CRC without MSI but with heterozygous NR-21 microsatellite alleles (present in normal and tumour DNA). Arrow heads indicate the MSI shifts, arrows indicate the variant NR-21 allele.

Fig 7

MLH1 promoter hypermethylation assay; MS-MLPA analysis of a MSI CRC with absence of MLH1 and PMS2 expression. Results are shown of paired undigested normal (Nu) and undigested tumour (Tu) DNA and of the methylation-sensitive endonuclease HhaI digested normal (Nd) and tumour (Td) DNA. Arrows indicate six MLH1 promoter probes, nr 1 representative for a fragment without and nrs 2–6 representative for fragments with a HhaI restriction site. In the HhaI digested DNA, probes 2–6 are clearly present in the tumour DNA (Td) and not in the paired normal DNA (Nd) indicating tumour-specific methylation of the MLH1 promoter fragments 2–6.

Fig 8

Mutation analysis of codon 600 of the BRAF gene. (A) Tumour DNA without BRAF codon 600 mutation. (B) Tumour DNA with a heterozygous oncogenic mutation GTG-GAG, leading to a V600E amino acid substitution.