Mark Bettington

The serrated pathway to colorectal carcinoma: current concepts and challenges

Approximately 30% of colorectal carcinomas develop via a serrated neoplasia pathway, named for the pattern of crypts in the precursor polyps. Molecular abnormalities consistently involve methylation of CpG islands [CpG island methylator phenotype (CIMP)] of low degree (CIMP‐L) or high degree (CIMP‐H), and activating mutations of the mitogen‐activated protein kinase pathway components BRAF or KRAS. Microsatellite instability (MSI) of a high level (MSI‐H) is often present, allowing for a molecular classification of serrated pathway carcinoma as: (i) BRAF mutant/CIMP‐H with either a) MSI‐H or b) microsatellite stable (MSS); and (ii) KRAS mutant/CIMP‐L/MSS. Precursor polyps include sessile serrated adenoma (SSA), characterized by proximal location, crypt architectural disturbance, and BRAF mutation. Microvesicular hyperplasic polyp (MVHP) probably precedes the development of SSA, and borderline lesions between MVHP and SSA occur. Cytological dysplasia in SSA portends advanced genetic abnormality and a high risk of progression to carcinoma. The traditional serrated adenoma has a predilection for the left colon, tubulovillous architecture, eosinophilic cytoplasm, and frequent KRAS mutation. Serrated morphology carcinoma is a new World Health Organization subtype with well‐differentiated, mucinous or trabecular patterns. It has frequent KRAS or BRAF mutations and a poor prognosis. This review provides an insight into the histology and molecular mechanisms driving these serrated pathway lesions.

A clinicopathological and molecular analysis of 200 traditional serrated adenomas

The traditional serrated adenoma is the least common colorectal serrated polyp. The clinicopathological features and molecular drivers of these polyps require further investigation. We have prospectively collected a cohort of 200 ordinary and advanced traditional serrated adenomas and performed BRAF and KRAS mutational profiling, CpG island methylator phenotype analysis, and immunohistochemistry for a panel of 7 antibodies (MLH1, β-catenin, p53, p16, Ki67, CK7, and CK20) on all cases. The mean age of the patients was 64 years and 50% were female. Of the polyps, 71% were distal. Advanced histology (overt dysplasia or carcinoma) was present in 19% of cases. BRAF mutation was present in 67% and KRAS mutation in 22%. BRAF mutant traditional serrated adenomas were more frequently proximal (39% versus 2%; P≤0.0001), were exclusively associated with a precursor polyp (57% versus 0%; P≤0.0001), and were more frequently CpG island methylator phenotype high (60% versus 16%; P≤0.0001) than KRAS mutant traditional serrated adenomas. Advanced traditional serrated adenomas retained MLH1 expression in 97%, showed strong p53 staining in 55%, and nuclear β-catenin staining in 40%. P16 staining was lost in the advanced areas of 55% of BRAF mutant traditional serrated adenomas compared with 10% of the advanced areas of KRAS mutant or BRAF/KRAS wild-type traditional serrated adenomas. BRAF and KRAS mutant traditional serrated adenomas are morphologically related but biologically disparate polyps with distinctive clinicopathological and molecular features. The overwhelming majority of traditional serrated adenomas retain mismatch repair enzyme function indicating a microsatellite-stable phenotype. Malignant progression occurs via TP53 mutation and Wnt pathway activation regardless of mutation status. However, CDKN2A (encoding the p16 protein) is silenced nearly exclusively in the advanced areas of the BRAF mutant traditional serrated adenomas. Thus, the BRAF mutant traditional serrated adenoma represents an important precursor of the aggressive BRAF mutant, microsatellite-stable subtype of colorectal carcinoma.

Clinicopathological and molecular features of sessile serrated adenomas with dysplasia or carcinoma

Objective Sessile serrated adenomas (SSAs) are the precursors of at least 15% of colorectal carcinomas, but their biology is incompletely understood. We performed a clinicopathological and molecular analysis of a large number of the rarely observed SSAs with dysplasia/carcinoma to better define their features and the pathways by which they progress to carcinoma. Design A cross-sectional analysis of 137 SSAs containing regions of dysplasia/carcinoma prospectively collected at a community GI pathology practice was conducted. Samples were examined for BRAF and KRAS mutations, the CpG island methylator phenotype (CIMP) and immunostained for MLH1, p53, p16, β-catenin and 0-6-methylguanine DNA methyltransferase (MGMT). Results The median polyp size was 9 mm and 86.5% were proximal. Most were BRAF mutated (92.7%) and 94.0% showed CIMP. Mismatch repair deficiency, evidenced by loss of MLH1 (74.5%) is associated with older age (76.7 versus 71.0; p<0.0029), female gender (70% versus 36%; p<0.0008), proximal location (91% versus 72%; p<0.02), CIMP (98% versus 80%; p<0.02) and lack of aberrant p53 (7% versus 34%; p<0.001) when compared with the mismatch repair-proficient cases. Loss of p16 (43.1%) and gain of nuclear β-catenin (55.5%) were common in areas of dysplasia/cancer, irrespective of mismatch repair status. Conclusions SSAs containing dysplasia/carcinoma are predominantly small (<10 mm) and proximal. The mismatch repair status separates these lesions into distinct clinicopathological subgroups, although WNT activation and p16 silencing are common to both. Cases with dysplasia occur at a similar age to cases with carcinoma. This, together with the rarity of these ‘caught in the act’ lesions, suggests a rapid transition to malignancy following a long dwell time as an SSA without dysplasia.

Critical appraisal of the diagnosis of the sessile serrated adenoma.

The sessile serrated adenoma (SSA) is a relatively recently described polyp that can present diagnostic difficulties for the practicing pathologist. The frequency of SSA diagnoses varies dramatically in the reported literature. In addition, the histologic interface between the microvesicular hyperplastic polyp (MVHP) and the SSA continues to be a diagnostic problem. The trend in recent years has been toward a lower threshold for SSA diagnosis. Herein, we have performed a cross-sectional study of 6340 colorectal polyps received at a high-volume community-based pathology practice over a 3-month period. After central review, with strict application of the diagnostic criteria outlined in the 2010 edition of the World Health Organization Classification of Tumours of the Digestive Tract, we found that SSAs represented 12.1% of all polyps. In addition, we developed novel diagnostic subcategories in an attempt to determine the most appropriate cutoff for the interface between the MVHP and the SSA. We found that serrated polyps (MVHPs or SSAs) with any SSA-like crypts had clinical features more in common with the SSA than the MVHP and that this diagnostic cutoff showed good reproducibility between pathologists. This supports the position of a recent consensus publication proposing that polyps with as few as 1 SSA-type crypt should be diagnosed as an SSA. Applying these criteria to our cohort yields an overall SSA rate of 14.7%. In summary, we believe that SSAs continue to be underdiagnosed in pathologic practice and that this may result in inadequate surveillance and thus contribute to interval colorectal carcinomas.

Traditional serrated adenoma: an update

Although recognized 25 years ago, the traditional serrated adenoma (TSA) remains an ongoing source of diagnostic and biologic debate. Recent research has greatly improved our understanding of the morphological and molecular aspects of these polyps. In particular, the recognition of ectopic crypt foci (ECFs) in combination with typical cytology and slitlike serrations improves diagnostic reproducibility. Awareness that many TSAs, particularly BRAF-mutated TSAs, arise in precursor microvesicular hyperplastic polyps and sessile serrated adenomas can aid in making this diagnosis and should not be confused with a sessile serrated adenoma with dysplasia. At a molecular level, TSAs can be divided into 2 groups based on their BRAF or KRAS mutation status. The development of overt cytologic dysplasia is accompanied by TP53 mutation, Wnt pathway activation, and, in some cases, silencing of CDKN2A. Importantly, however, mismatch repair enzyme function is retained. Thus, the TSA is an important precursor of aggressive molecular subtypes of colorectal carcinoma.

Genome-wide DNA methylation analysis of formalin-fixed paraffin embedded colorectal cancer tissue

Formalin fixation and embedding of clinical tissue samples in paraffin is a common method for archiving biological material. These samples are often well annotated and provide an invaluable resource for research. However, this process of fixation and storage of tissue leads to DNA damage and fragmentation. The use of DNA from formalin fixed, paraffin‐embedded (FFPE) tissue to interrogate methylation levels on a genome‐wide scale can pose challenges. We compared fresh and matched FFPE tissue DNA samples using the Illumina Infinium HD Human Methylation 450K BeadChip platform with a companion application for repair and “restoration” of DNA from FFPE tissue. Our results showed good correlation between fresh and FFPE sample data. FFPE DNA captured 99% of the CpG sites on the array on average. Significant cancer subgroups based on the CpG island methylator phenotype (CIMP) were clearly distinguished for both fresh and FFPE sample sets with cluster and scaling analysis. The DNA methylation status for the five standard CIMP panel genes which was evaluated for all samples by the MethyLight assay was correctly assigned in both fresh and FFPE samples by the array data. We conclude that the “restoration” method followed by assay on the Infinium HD Human Methylation 450K microarray can produce good quality data for DNA from FFPE samples.

RNF43 and ZNRF3 are commonly altered in serrated pathway colorectal tumorigenesis

Serrated pathway colorectal cancers (CRCs) are characterised by a BRAF mutation and half display microsatellite instability (MSI). The Wnt pathway is commonly upregulated in conventional CRC through APC mutation. By contrast, serrated cancers do not mutate APC. We investigated mutation of the ubiquitin ligases RNF43 and ZNRF3 as alternate mechanism of altering the Wnt signal in serrated colorectal neoplasia. RNF43 was mutated in 47/54(87%) BRAF mutant/MSI and 8/33(24%) BRAF mutant/microsatellite stable cancers compared to only 3/79(4%) BRAF wildtype cancers (p<0.0001). ZNRF3 was mutated in 16/54(30%) BRAF mutant/MSI and 5/33(15%) BRAF mutant/microsatellite stable compared to 0/27 BRAF wild type cancers (p=0.004). An RNF43 frameshift mutation (X659fs) occurred in 80% BRAF mutant/MSI cancers. This high rate was verified in a second series of 25/35(71%) BRAF mutant/MSI cancers. RNF43 and ZNRF3 had lower transcript expression in BRAF mutant compared to BRAF wildtype cancers and less cytoplasmic protein expression in BRAF mutant/MSI compared to other subtypes. Treatment with a porcupine inhibitor reduced RNF43/ZNRF3 mutant colony growth by 50% and synergised with a MEK inhibitor to dramatically reduce growth. This study suggests inactivation of RNF43 and ZNRF3 is important in serrated tumorigenesis and has identified a potential therapeutic strategy for this cancer subtype.

Tropical sprue: revisiting an underrecognized disease.

Tropical sprue is an acquired chronic diarrheal disorder of unclear etiology affecting residents of and visitors to tropical regions. Patients usually present with profuse diarrhea, weight loss, and malabsorption, notably of vitamin B12 and folate. The histologic changes typically resemble that of gluten-sensitive enteropathy. Reports of tropical sprue have become infrequent in the literature, and the diagnosis is often not considered either clinically or pathologically. This disease may, however, cause significant morbidity, although it is eminently treatable with broad-spectrum antibiotics. In this study, we report the clinical presentation of 12 tropical sprue patients along with the histologic changes of the intestinal mucosa and compare it with those of a series of 150 cases of gluten-sensitive enteropathy, the condition with which it is most frequently misdiagnosed. The cohort comprised 6 men and 6 women with a median age of 59 years (range, 38 to 78 y) with a history of residence or visitation in South Asia or Papua New Guinea. Partial villous blunting in the duodenal mucosa was present in 75% of cases, and a marked intraepithelial lymphocytosis was observed in all cases (mean per 100 epithelial cells 77.3; range, 42 to 124). A villous tip accentuation of intraepithelial lymphocytosis was not appreciable in most cases. No case of complete villous blunting (Marsh stage 3c) was identified in tropical sprue, contrasting with 25% in gluten-sensitive enteropathy cases. A duodenal mucosa eosinophil infiltrate was present in all cases with significantly higher counts compared with untreated gluten-sensitive enteropathy patients (26.6/HPF vs. 14.6/HPF; P=0.009). The ileal mucosa displayed more severe villous blunting with higher Marsh stages than in the corresponding duodenum from 5 patients. There was a mild intraepithelial lymphocytosis and eosinophil infiltrate in the colonic mucosa of half of the cases. Follow-up biopsies in 6 patients demonstrated a histologic response after oral folates and doxycycline treatment. In summary, tropical sprue is a pan-enteric inflammatory process often mistaken for gluten-sensitive enteropathy. Histologic findings suggesting tropical sprue in the appropriate clinical context include incomplete duodenal villous blunting without development of flat mucosa, frequent involvement of the terminal ileum with more marked inflammation and villous blunting than in the duodenum, and a conspicuous eosinophil infiltrate in the lamina propria. With the expansion of tourism and increasing employment opportunities in tropical regions, pathologists in the West are increasingly likely to encounter cases of tropical sprue and should be aware of this diagnosis.

The challenging diagnosis of Cronkhite-Canada syndrome in the upper gastrointestinal tract: a series of 7 cases with clinical follow-up.

Cronkhite-Canada syndrome is a rare protein-losing enteropathy, classically characterized by ectodermal changes and gastrointestinal polyposis. The etiology remains obscure but immune dysregulation may be important. The diagnosis of Cronkhite-Canada syndrome in the upper gastrointestinal tract is challenging, frequently resulting in delayed patient management. In this study, we described the initial clinical presentations, upper gastrointestinal endoscopic appearances, clinical follow-up, and histologic diagnoses in 7 patients who were subsequently diagnosed with Cronkhite-Canada syndrome. Histology slides were reviewed, and IgG4 immunohistochemical analysis was performed. The most common initial endoscopic impressions were antral malignancy and gastric infection, but gastroduodenal polyposis was not described. On histologic review, the main findings in the gastric mucosa were a prominent mucosal edema, a mixed inflammatory infiltrate rich in eosinophils, and architectural changes with gland dilatation and withering. In the duodenal mucosa, total or subtotal duodenal villous atrophy, inflammation, crypt distortion, and increased apoptotic bodies were the most common features. Three patients died of the disease, and 4 patients were asymptomatic at a mean follow-up of 3.5 years. No intestinal malignancy had been diagnosed. In 2 patients foci of dysplasia in colonic polyps were identified. In only 1 patient, a significant increase in IgG4-positive plasma cells was observed in a colonic polyp. In summary, we found that patients with Cronkhite-Canada syndrome have histologic features commonly found in other immune disorders of the gastrointestinal tract that may help in establishing the diagnosis and further supports the hypothesis that Cronkhite-Canada syndrome may represent an immune dysregulation syndrome, different from IgG4-related disease.

Autoimmune gastritis: novel clues to histological diagnosis

Aims: To compare the histological features of a consecutive biopsy series of autoimmune gastritis (AIG) with other forms of chronic gastritis to identify morphological clues to the diagnosis. Methods: Morphological features in gastric biopsies were examined in 42 prospectively identified patients with AIG without concomitant neoplasia (n = 184 biopsies), 50 normal controls (NGB; n = 118 biopsies), 52 Helicobacter associated gastritis (HPG; n = 123 biopsies), 16 multifocal atrophic gastritis (MAG; n = 62 biopsies) and 50 chronic pangastritis without defined cause (CG; n = 117 biopsies). Results: In AIG, inflammation was characterised by lymphocyte infiltration into the epithelium (98%), basal lymphoid aggregates (82.7%), basal predominance (59.6%), eosinophil infiltration [mean 34.5/high power field (HPF), >30 eosinophils/HPF in 46.1%] and neutrophil infiltration (44.2%). Architectural abnormality was characterised by muscularis mucosae thickening (92.9%) and gland irregularity (86.5%). Pancreatic metaplasia was identified in 21.2% of cases. Compared to NGB, HPG, MAG and CG, AIG displayed more eosinophil infiltration (p < 0.001) and more frequent lymphocyte infiltration of the epithelium, basal predominant inflammation and pancreatic acinar metaplasia (all p < 0.05). Conclusions: Basal predominance of inflammation, lymphocyte infiltration into gland epithelium, prominent eosinophils, architectural irregularity and muscularis mucosae thickening are common in AIG and are helpful when biopsy site and/or clinical history is uncertain.