Histologic features distinguish microsatellite-high from microsatellite-low and microsatellite-stable colorectal carcinomas, but do not differentiate germline mutations from methylation of the MLH1 promoter

Summary

The detection of microsatellite-unstable (microsatellite instability [MSI]) colorectal carcinomas (CRCs) has prognostic value and can help screen for Lynch syndrome. We determined which histologic features are associated with MSI status and presence of germline mutation and/or methylation of MLH1 promoter. Patients diagnosed with CRC were offered participation in the Columbus-area hereditary nonpolyposis colorectal cancer syndrome study regardless of age or family history. Tumors were evaluated for MSI using a modified Bethesda panel of microsatellite markers. Methylation status of the MLH1 promoter was evaluated by methylation-specific polymerase chain reaction and bisulfite PCR followed by restriction digestion of tumor DNA. All patients with microsatellite-unstable tumors underwent mutation analysis of the MLH1, MSH2, and MSH6 genes by full sequencing of genomic DNA and by multiplex ligation probe assay of MLH1 and MSH2. Histologic end points were tumor type, grade, percentage of mucin, border, and lymphoid host response. Of the 482 CRCs, 87 were MSI with 69 MSI high (MSI-H), 18 MSI low (MSI-L), and 395 microsatellite stable (MSS). Of 87 MSI tumors, 12 had germline mutations and 34 had methylation of the MLH1 promoter. Younger age, but not histologic features, was significantly associated with a germline mutation. Percentage of mucin, histologic type, grade, and lymphoid host response differed significantly between MSI-H when compared with MSI-L or MSS. No difference was found between MSI-L versus MSS. Histologic features are associated with MSI-H CRC and are helpful to differentiate MSI-H from MSI-L and MSS. These features are not useful to distinguish MSI-L from MSS carcinomas, and those with a deleterious germline hereditary nonpolyposis colorectal cancer syndrome mutation from those with methylation of the MLH1 promoter region.

Introduction

Microsatellite instability (MSI) occurs when tissues acquire numerical nucleotide variability in repetitive DNA sequences because of abnormalities in the mismatch repair proteins. Microsatellite instability is detected in 50% to 80% of tumors from patients with hereditary nonpolyposis colorectal cancer syndrome (HNPCC) [1], [2], [3] and is present in 15% to 20% of unselected colorectal cancer cases [4], [5], [6], some of which may be due to Lynch syndrome.

Lynch syndrome is defined as carcinoma having MSI due to a germline mutation in one of the DNA mismatch repair genes, MLH1, MSH2, MSH6, or PMS2. In sporadic colorectal cancer, MSI is related to epigenetic silencing of the MLH1 gene by hypermethylation of the promoter region [7]. The inability to repair somatic gene mutations raises the possibility that mutations will accumulate within a cell. These accumulated mutations are the underlying basis for cancer development.

Because most HNPCC-associated colorectal tumors also represent Lynch syndrome and have an analyzable gene defect, testing for MSI is an important screening/diagnostic tool. Microsatellite instability testing identifies patients and families who need a detailed study of germline DNA to confirm germline mutation to make the diagnosis of Lynch syndrome. The detection of MSI in sporadic colorectal carcinomas (CRCs) has prognostic value in that microsatellite-unstable cancers have an improved overall survival and may have a different response to adjuvant therapy [8], [9]. In addition, recent studies have suggested an association between low level of MSI and poor cancer-specific survival [10], [11].

Certain histologic features of microsatellite-unstable tumors have been described, including 2 patterns of adenocarcinoma. One pattern is a mucinous adenocarcinoma that is seen predominantly in the ascending colon. The tumors are well circumscribed, well to moderately differentiated with a prominent lymphocytic infiltrate and tumor-infiltrating lymphocytes (TIL) that may be evident in nonmucinous areas. The second pattern is a poorly differentiated adenocarcinoma with epithelium disposed in small clusters and irregular trabeculae or large aggregates. Tumors are well circumscribed and lack abundant desmoplastic stroma. Some tumors are peppered with TIL. A Crohn's-like lymphocytic reaction may also be present. This subtype has been described as medullary or undifferentiated, although most contains subclones in which glandular differentiation is evident [5], [12], [13], [14], [15], [16], [17]. Among the histologic features, mucinous histology and intraepithelial lymphocytosis correlate the most with MSI [5], [18], [19].

The aim of this study was to confirm the histologic features that are associated with MSI status. Furthermore, we wanted to evaluate if histologic features may help discriminate the between MSI-H and MSI-L tumors and between those with germline mutations or methylation of the MLH1 promoter.