Molecular Alterations and Association with Clinical Parameters

Lynch syndrome is caused by germline mutations of DNA mismatch repair (MMR) genes, most frequently MLH1 and MSH2. Recently, MMR-deficient crypt foci (MMR- DCF) have been identified as a novel lesion which occurs at high frequency in the intestinal mucosa from Lynch syndrome mutation carriers, but very rarely progress to cancer. To shed light on molecular alterations and clinical associations of MMR-DCF, we systematically searched the intestinal mucosa from Lynch syndrome patients for MMR-DCF by immunohistochemistry. The identified lesions were characterised for alterations in microsatellite-bearing genes with proven or suspected role in malignant transformation. We demonstrate that the prevalence of MMR-DCF (mean 0.84 MMR-DCF per 1 cm2 mucosa in the colorectum of Lynch syndrome patients) was significantly associated with patients’ age, but not with patients’ gender. No MMR-DCF were detectable in the mucosa of patients with sporadic MSI-H colorectal cancer (n = 12). Microsatellite instability of at least one tested marker was detected in 89% of the MMR-DCF examined, indicating an immediate onset of microsatellite instability after MMR gene inactivation. Coding microsatellite mutations were most frequent in the genes HT001 (ASTE1) with 33%, followed by AIM2 (17%) and BAX (10%). Though MMR deficiency alone appears to be insufficient for malignant transformation, it leads to measurable microsatellite instability even in single MMR-deficient crypts. Our data indicate for the first time that the frequency of MMR-DCF increases with patients’ age. Similar patterns of coding microsatellite instability in MMR-DCF and MMR-deficient cancers suggest that certain combinations of coding microsatellite mutations, including mutations of the HT001, AIM2 and BAX gene, may contribute to the progression of MMR-deficient lesions into MMR-deficient cancers

Mismatch Repair-Deficient Crypt Foci in Lynch Syndrome – Molecular Alterations and Association with Clinical Parameters

<div><p>Lynch syndrome is caused by germline mutations of DNA mismatch repair (MMR) genes, most frequently <i>MLH1</i> and <i>MSH2</i>. Recently, MMR-deficient crypt foci (MMR-DCF) have been identified as a novel lesion which occurs at high frequency in the intestinal mucosa from Lynch syndrome mutation carriers, but very rarely progress to cancer. To shed light on molecular alterations and clinical associations of MMR-DCF, we systematically searched the intestinal mucosa from Lynch syndrome patients for MMR-DCF by immunohistochemistry. The identified lesions were characterised for alterations in microsatellite-bearing genes with proven or suspected role in malignant transformation. We demonstrate that the prevalence of MMR-DCF (mean 0.84 MMR-DCF per 1 cm² mucosa in the colorectum of Lynch syndrome patients) was significantly associated with patients’ age, but not with patients’ gender. No MMR-DCF were detectable in the mucosa of patients with sporadic MSI-H colorectal cancer (n = 12). Microsatellite instability of at least one tested marker was detected in 89% of the MMR-DCF examined, indicating an immediate onset of microsatellite instability after MMR gene inactivation. Coding microsatellite mutations were most frequent in the genes <i>HT001</i> (<i>ASTE1</i>) with 33%, followed by <i>AIM2</i> (17%) and <i>BAX</i> (10%). Though MMR deficiency alone appears to be insufficient for malignant transformation, it leads to measurable microsatellite instability even in single MMR-deficient crypts. Our data indicate for the first time that the frequency of MMR-DCF increases with patients’ age. Similar patterns of coding microsatellite instability in MMR-DCF and MMR-deficient cancers suggest that certain combinations of coding microsatellite mutations, including mutations of the <i>HT001</i>, <i>AIM2</i> and <i>BAX</i> gene, may contribute to the progression of MMR-deficient lesions into MMR-deficient cancers.</p></div

Mutation frequency of microsatellite markers.

<p>Black bars represent the observed mutation frequency of the respective microsatellites in MMR-DCF. Mutation frequency reported in the literature for MSI-H colorectal adenoma and carcinoma are displayed by dark grey (adenoma) and white (carcinoma) bars, respectively. Data for adenomas and carcinomas were retrieved from <a href="http://www.seltarbase.org/" target="_blank">www.seltarbase.org</a>. Light grey bars indicate mutation frequency in corresponding carcinomas from the same patients from whom the MMR-DCF were obtained.</p

Histology and molecular phenotype of representative MMR-DCF.

<p>(A) Staining of three representative MMR-DCF. Objective magnification is 20x for all panels. Representative staining results with antibodies specific for the MMR protein corresponding to the germline mutation of the respective patient. Lesions are denoted by patient ID (LYS = Lynch syndrome patient) and lesion ID. (B) Fragment length analysis of triplex PCR with BAT25, BAT26 and CAT25. Compared to two normal intestinal crypts analysed as a reference (lower panels), all three markers show deletion mutations in the examined MMR-DCF (LYS 21, Lesion 1). Deletion mutations are indicated by red arrows in MMR-DCF (upper panel: BAT25, green peaks, -3; BAT26, filled blue peaks, -2; CAT25, open blue peaks, -1). (C) Fragment length analysis of duplex PCR with <i>BAX</i> and <i>HT001</i> of patient LYS 19. Depiction of an <i>HT001</i> (right sided fragments) mononucleotide shift (-1, red arrow) as an example of a coding microsatellite-bearing target gene mutation. The fragment analysis is directly compared to the results obtained from two normal intestinal crypts of the same patient (lower panels).</p

MMR-DCF prevalence and patient age.

<p>Association between age (years) of the patient at operation and prevalence of MMR-DCF (A). The association of MMR-DCF prevalence with patient age is depicted with a black line adjusted for a colon length of 15 cm; dots represent patients. Estimated incident rate ratio of colorectal MMR-DCF was 1.75 (95% CI 1.30–2.35) for a change of age at operation of 10 years. The prevalence of MMR-DCF is not significantly associated with patients’ gender (B) or the MMR gene affected by germline mutation (C).</p