MLH1, BRAF and p53 - searching for significant markers to predict evolution towards adenocarcinoma in colonic sessile serrated lesions

Abstract

Background and aim: Colonic serrated lesions are premalignant lesions, using an alternative malignization pathway, including multiple genetic and epigenetic alterations, as: mismatch repair deficiency due to MutL homolog 1 (MLH1) promoter methylation, tumor protein p53 (TP53) mutations, activating mutations of v-Raf murine sarcoma viral oncogene homolog B (BRAF) and Kirsten rat sarcoma viral oncogene homolog (KRAS). Our study aims to evaluate MLH1, BRAF and p53 immunohistochemical (IHC) status in sessile serrated lesions (SSLs), with and without dysplasia.

Materials and methods: This is a retrospective case-control study including 20 SSLs with dysplasia and 20 SSLs without dysplasia (matching sex and age). IHC expression of MLH1, BRAF and p53 was evaluated as the percent of nuclear loss of MLH1, cytoplasmic positivity of BRAF and nuclear positivity of p53. Data concerning age, sex, localization of the lesion, dysplasia and IHC results were statistically processed using Microsoft Excel.

Results: We had very polymorphous patterns of IHC expression for BRAF, MLH1 and p53, especially in the dysplastic group. Thus, two patients were BRAF+∕MLH1-∕p53+, three were BRAF+∕MLH1-∕p53-, one was BRAF+∕MLH1+∕p53- and six were BRAF+∕MLH1+∕p53+. Dysplastic lesions without BRAF mutation exhibited the following phenotype: one case BRAF-∕MLH1-∕p53+, four BRAF-∕MLH1-∕p53- and three BRAF-∕MLH1+∕p53+. In the control group (SSLs without dysplasia), there was a more homogenous distribution of cases: eight cases BRAF+∕MLH1+∕p53-, seven BRAF-∕MLH1+∕p53-, one BRAF-∕MLH1-∕p53+, two BRAF-∕MLH1-∕p53- and two BRAF-∕MLH1+∕p53+.

Conclusions: There are more routes on the serrated pathway, with different mutations and time of acquisition of each genetic or epigenetic lesion with the same morphological result. These lesions should be stratified according to their risk to poor outcome and their need to further surveillance.

Figure 1

SSLs with LGD. Note the branching crypts with dilated base (a) and the hyperchromatic nuclei with minimal atypia and pseudostratification (b). HE staining: (a) ×100; (b) ×200. HE: Hematoxylin–Eosin; LGD: Low-grade dysplasia; SSLs: Sessile serrated lesions

Figure 2

SSL with HGD. Although architectural distortion is not severe, there are significant cellular atypia: large irregular nuclei with visible nucleoli and abnormal nuclear/cytoplasmic ratio. HE staining, ×200. HGD: High-grade dysplasia

Figure 3

Invasive serrated carcinoma developed in a SSL with dysplasia (LGD and HGD) (left inferior corner). HE staining, ×100

Figure 4

Invasive serrated adenocarcinoma arising from a SSL with dysplasia. Anti-CD34 antibody immunostaining highlighting capillaries in the immediate vicinity of an invasive saw-toothed gland, ×200. CD34: Cluster of differentiation 34

Figure 5

Distribution of SSLs in case-group (dysplastic lesions) and in control group (non-dysplastic lesions). N/A/: Not available

Figure 6

Age distribution of patients with dysplastic lesions. IC: Invasive carcinoma

Figure 7

Mutation of BRAF is becoming increasingly frequent as the degree of dysplasia is rising. BRAF: v-Raf murine sarcoma viral oncogene homolog B

Figure 8

Anti-BRAF antibody immunostaining revealing intense positivity in epithelial cells in a LGD SSL, ×100

Figure 9

MLH1 loss revealed in all SSLs, with a strong tendency to dysplastic lesions. MLH1: MutL homolog 1

Figure 10

MLH1 loss in a non-dysplastic SSL (a) and in a HGD SSL (b). There is a higher percent of negative cells in the dysplastic lesion. Anti-MLH1 antibody immunostaining: (a) ×100; (b) ×200

Figure 11

Mutation of p53 significantly more frequent in HGD SSLs

Figure 12

p53 aberrant immunoexpression in a SSL with HGD. Anti-p53 antibody immunostaining, ×40

Figure 13

High polymorphism of immunophenotype in dysplastic SSLs. MSI: Microsatellite instability; MSS: Microsatellite stable

Figure 14

Mild polymorphism of immunophenotype in non-dysplastic SSLs