Lynch syndrome is an autosomal-dominant inherited condition defined by the identification of a germline mutation in a DNA mismatch repair (MMR) gene (MLH1, MSH2, PMS2 or MSH6), or in the EPCAM gene, leading to constitutional epigenetic silencing of the downstream MSH2 gene.1 Individuals who carry a MMR gene mutation are at an increased risk of developing cancers at multiple sites, most notably colorectal and endometrial carcinomas, but also carcinomas from the upper urinary tract, pancreas, hepatobiliary tract, stomach, small intestine and ovaries.2

The current diagnostic approach for the identification of individuals with an MMR gene mutation is a multistep process in which pathologists play an instrumental role. Tumours arising in individuals with an MMR gene mutation demonstrate high levels of microsatellite instability (MSI) secondary to altered DNA MMR mechanisms in tumour cells. Immunohistochemistry for DNA MMR proteins is widely used to identify MMR deficiency in colorectal carcinomas (CRCs) as a screen for MMR gene carriers.3 Of all abnormal patterns of immunohistochemical results, loss of expression of MLH1 and PMS2 is the most common. MLH1 and PMS2 function as a stable heterodimer that, along with MSH2, MSH6 and EXO1, corrects small errors involving mispaired nucleotides which are introduced by DNA polymerase during DNA replication. A functional defect in MLH1 results in the degradation of both MLH1 and PMS2, whereas a defect in PMS2 results only in the degradation of PMS2. Consequently, loss of expression of MLH1 and PMS2 in CRC generally indicates an alteration in MLH1, either by somatic methylation of the MLH1 promoter region (sporadic cases) or by a MLH1 germline mutation (Lynch syndrome), and solitary loss of PMS2 expression generally indicates an underlying germline defect in PMS2.

Inconsistent immunohistochemical results have been reported, in particular the retained expression of MLH1 in tumours from individuals with a germline MLH1 mutation.4–8 This phenomenon can be misleading if PMS2 immunostaining is not performed. We sought to confirm that germline mutations in MLH1 may underlie a substantial proportion of CRC with solitary loss of PMS2 expression. To address this question, we performed mutation analysis of the MLH1 and PMS2 genes in individuals from the Colon Cancer Family Registry Cohort whose tumours showed solitary loss of PMS2.

The study included 90 CRCs from 88 individuals demonstrating loss of PMS2 expression and normal retained MLH1 expression by immunohistochemistry. They had a mean age at CRC diagnosis of 51.7±SD 12.4 years and included 57% males. MSI status was available for 46/90 CRCs (51%), with high levels of MSI observed in 42/46 (91%) cases. MLH1 methylation and/or a BRAF^V600E mutation were present in 4 of the 90 CRCs that were excluded from the study. Six CRCs (7%) also showed loss of MSH6 protein expression. Four individuals were not tested for PMS2 and MLH1 mutations due to the unavailability of blood-derived DNA, and complete gene testing was not possible for a further 14 individuals (figure 1). The final study group consisted of 66 individuals with complete screening for germline mutations in the PMS2 and MLH1 genes. A pathogenic PMS2 germline mutation was identified in 49 individuals (74%; see online supplementary table S2), some of which were reported previously.18 Variants in the MLH1 gene were identified in 11 individuals (17%). In eight individuals, the variants were classified as pathogenic mutations (class 5); in the other three individuals, variants were unclassified but predicted to be damaging by SIFT and PolyPhen-2 algorithms (table 1 and figure 2).

Immunostained slides were reviewed in 5 of these 11 cases, confirming the retained expression of MLH1 and the loss of PMS2 expression in carcinoma cells. No mutation within PMS2 or MLH1 could be found in the remaining six individuals (9%). The clinicopathological characteristics of the PMS2 mutation carriers, the MLH1 pathogenic mutation and UV carriers and those individuals tested but found not to have a mutation in PMS2 or MLH1 are shown in table 2.

The mean age at CRC diagnosis of the individuals with a MLH1 mutation or UV was significantly younger than those individuals with a PMS2 mutation (p=0.046). Amsterdam criteria I or II were less frequently found in PMS2 mutation carriers compared with MLH1 variant carriers (p=0.001).

Missense variants were the most common MLH1 alteration identified, in eight individuals (83%). The MLH1 c.113A>G p.Asn38Ser variant was found in two related individuals (cases 2 and 3). One individual who carried the intronic MLH1 germline mutation c.677+3A>G p.Gln197Argfs*8, which leads to the skipping of exon 8, developed two CRCs both of which retained MLH1 expression (cases 5 and 6). One individual carried a splice site mutation leading to an in-frame deletion of two exons (case 4) and one individual carried a small insertion resulting in a frameshift mutation (case 7; table 1).

To assess the possible role of MLH1 mutations in CRCs showing solitary loss of PMS2 expression by immunohistochemistry, we studied a series of 90 CRCs from 88 individuals from the Colon Cancer Family Registry Cohort with this immunophenotype. Among the 66 individuals with complete germline mutation analysis, we identified a pathogenic PMS2 mutation in 49 cases (74%) and a pathogenic MLH1 mutation in 8 cases (12%). A further three cases (4%) had a variant of uncertain clinical significance in MLH1 predicted to be damaging, and six cases (9%) had no identifiable variant likely to have clinical significance in either gene. Moreover, a high proportion of the MLH1 variants identified resulted in missense changes, suggesting that a non-functional MLH1 protein that retains its MLH1 antigenicity is a conceivable explanation.

Immunohistochemistry for the DNA MMR proteins MLH1, PMS2, MSH2 and MSH6 in CRC is a highly sensitive test to screen for Lynch syndrome, with 93–100% concordance with MSI testing.3 4 However, false-negative results for MLH1 immunohistochemistry have been reported in small series. In a study evaluating the benefit of adding PMS2 to MLH1 staining, de Jong et al4 found eight MLH1 mutations (42%) compared with only three PMS2 mutations (16%) out of 19 CRCs demonstrating solitary loss of PMS2 expression. When considering all the MLH1 mutations identified in their study, a high proportion (8/35; 23%) showed loss only of PMS2 expression while retaining expression of MLH1. A large deletion of exons 14–19 of MLH1 was also reported in 2 of 8 (25%) CRC with solitary PMS2 loss of expression in a separate study.5 A recent study of 16 CRCs and 16 endometrial carcinomas from 31 individuals, all with solitary loss of PMS2 expression, explored the frequency of MLH1 mutations in this group.19 Of the 17 individuals who subsequently had germline mutation testing of the MLH1 and PMS2 genes, six had pathogenic mutations in PMS2 (35%), two had variants of uncertain clinical significance in PMS2 (12%), four had MLH1 pathogenic mutations (24%) whereas five had no mutation identified in either gene (29%). When restricted to patients with a CRC, a deleterious germline mutation in MLH1 was reported in two of nine tested patients (22%). Compared with these studies, our rate of PMS2 mutation in 66 tested individuals was higher at 74% and the rate of MLH1 deleterious mutation slightly lower at 12%. Two cousins (tumours 2 and 3) who carried the same MLH1 mutation both had CRC with solitary PMS2 loss. Similarly, one individual, who carried the MLH1 c.677+3A>G p.Gln197Argfs*8 mutation, developed two CRCs with solitary PMS2 loss. Both these examples suggest that it is the nature of the mutation rather than a technical anomaly associated with tissue fixation or staining quality that is the cause of this differential staining pattern. In support of this, Zighelboim et al20 described two sisters who carried the same MLH1 mutation: one developed endometrial cancer at 48 years and the other CRC at 45 years and endometrial cancer at 53 years; all tumours showed solitary loss of PMS2 expression and the presence of MLH1 expression.

A trend towards universal CRC tumour immunohistochemistry will increase the detection of abnormal staining patterns that require interpretation. This allows the most probable cause to be decided and thus the most appropriate management instituted. A solitary loss of PMS2 expression is suggestive of Lynch syndrome with a primary defect in the PMS2 gene. Interestingly, we identified MLH1 methylation or the somatic BRAF^V600E mutation in four cases, indicating that isolated PMS2 loss of expression can occur outside Lynch syndrome. It may therefore be useful to test PMS2-deficient CRC for BRAF^V600E mutation or MLH1 methylation to exclude sporadic tumour. Screening for PMS2 mutations has been problematic due a large number of homologous sequences within pseudogenes that closely flank the functional gene and most likely accounts for the lower proportion of PMS2 mutations reported in previous studies. The recent development of new methods incorporating long-range PCR and MLPA has eliminated most of the previous problems, such that the identification of large-scale deletions of exons 3 and/or 4 are now the only difficulty. The results from this study, representing the largest number of CRC with solitary loss of PMS2, support germline mutation screening of MLH1 when no mutation in PMS2 has been found. However, a substantial proportion of MMR-deficient CRCs with no evidence of MLH1 methylation or BRAF^V600E mutation remain unexplained and are referred to as Lynch-like or suspected Lynch syndrome. A number of potential causes for the underlying loss of PMS2 protein expression in these cases, including biallelic somatic mutations and cryptic mutations, have been described in a recent review.21 In a large population-based study of the Colon Cancer Family Registry Cohort, 5.6% (271/4 853) of all CRCs were classified as Lynch-like syndrome, representing 56% of all MMR-deficient CRCs not secondary to MLH1 methylation. In our study, six CRCs showed concurrent loss of MSH6 and PMS2. The most likely explanation for the loss of MSH6 expression in these six cases is the somatic frameshift mutation in the (C)8 microsatellite in exon 5 of the MSH6 gene secondary to the loss of MMR function resulting from the PMS2 defect.22 The use of panel testing rather than a single-gene approach would be useful; this is of particular interest clinically, where the PMS2 gene has lower penetrance than other MMR genes23 and family history is a suboptimal way of finding potentially high-risk families, where risk assessment and risk management has improved outcomes. However, PMS2 testing remains challenging even by next generation sequencing due to its complex structure.

Our study included the largest reported sample of CRCs with solitary loss of PMS2 to date. Testing for germline PMS2 mutations used in this study employed the most up-to-date and comprehensive approaches described,18 24 as demonstrated by the high rate of identified PMS2 mutations. One limitation of this study is the multicentre setting which may affect the consistency in the formalin fixation conditions of tissue blocks and lead to immunostaining artefacts. Other limitations include the absence of other Lynch syndrome-associated tumours, and the lack of mutation screening data for 20 (24%) cases. Moreover, our results may not reflect the actual rate of PMS2-deficient CRC in the general population and the mutation rates in PMS2 and MLH1, as these cases were selected in young individuals with strong family history of CRC.

In conclusion, the findings from this study suggest that CRCs in MLH1 mutation carriers can demonstrate a normal pattern of MLH1 expression and justify the testing for MLH1 germline mutation in individuals with a CRC showing solitary loss of PMS2 expression when a PMS2 mutation is not identified.