Clinicopathological and molecular analyses of hyperplastic lesions including microvesicular variant and goblet cell rich variant hyperplastic polyps and hyperplastic nodules—Hyperplastic nodule is an independent histological entity

INTRODUCTION

Serrated lesions are generally classified into hyperplastic polyps (HPs), traditional serrated adenomas, and sessile serrated lesions.1-3 HPs are currently sub-classified into microvesicular variants (MVHPs) and goblet cell-rich variants (GCHPs).1-3 MVHPs and GCHPs differ in terms of histological features and molecular findings.1-3 MVHPs are characterized by admixed goblet and columnar cells with microvesicular mucin, inconspicuous nuclei, and luminal serrations, whereas GCHPs are less serrated and show prominent goblet cells.1, 2 Moreover, while BRAF mutation characterizes MVHPs, KRAS mutation is frequently found in GCHPs.1-3 However, DNA methylation may play a role in the development of both lesions.4-6 These findings suggest that although MVHPs and GCHPs are both neoplastic lesions, they have different natures1, 2 and are independent lesions, both pathologically and clinically.

Apart from these HPs, another hyperplastic lesion characterized by elongated crypts without serration and abundance of goblet cells has been designated as hyperplastic nodule (HN) among Japanese but not Western pathologists.7 Differential diagnosis of HN from GCHP is difficult due to their similar histology (e.g., elongated crypts with little or no serration, abundance of goblet cells).7 The histological and molecular characteristics of GCHPs are well recognized, but those of HN have not been investigated. In the present study, we identified clinicopathological and molecular findings of HNs. In addition, we examined the difference in histological and molecular features between MVHPs, GCHPs, and HNs to define the pathological hallmarks of HN.

MATERIALS AND METHODS Patients

Patients with hyperplastic lesions including 61 GCHPs, 62 MVHPs, and 19 HNs were examined and the clinicopathological findings are shown in Table 1. The histopathological diagnoses of GCHP and MVHP were reached according to the 2019 WHO classification (World Health Organization).2 In brief, MVHPs were histologically characterized by (1) a “sawtooth” appearance of the upper portion of polyp, (2) small regular round nuclei located basally in the luminal half of crypt, (3) elongated, narrow, and hyperchromatic nuclei at the base of the crypt, (4) microvesicular cytoplasm, and (5) a variable but low number of goblet cells.2 GCHPs were histologically characterized by (1) elongated and flat crypts, (2) little serration, (3) goblet cell abundance extending to the surface, and (4) surface tufting.2 Finally, HNs were histologically defined as lesions accompanied by goblet cell abundance and elongated crypts without serration, in accordance with the Japanese criteria.7

Table 1. Clinicopathlogical features of colorectal hyperplastic lesions GCHP (%) HN (%) MVHP (%) p-value Total 61 19 62 Man:Woman 49:12 13:6 48:14 0.5552 Age (years old) Range (mean) 29–84 (61) 35–77 (74) 31–79 (61) N. S. Locus Right 13 (21.3) 5 (21.7) 23 (37.1) 0.1495 Left 48 (78.7) 14 (78.3) 39 (62.9) Size (mm) Range (mean) 2–20 (6) 1–10 (6) 3–13 (7) N. S. Macroscopical type Protruded type 32 (52.5)*,# 17 (89.5)* 45 (72.6)# 0.0044 Flat elevated type 29 (47.5)*,# 2 (10.5)* 17 (27.4)# Abbreviations: GCHP, goblet cell-rich hyperplastic polyp; HN, hyperplastic nodule; MVHP, microvesicular hyperplastic polyp; N. S., not significant. * p < 0.05; #p < 0.05.

To avoid inter-observer variation of histological diagnosis, we discussed differences regarding histological assessment. Agreement could be obtained among four gastrointestinal pathologists (N. U., Y. A., T. A., and T. S.).

Histological features examined in GCHPs, MVHPs, and HNs

To histologically characterize each lesion, we selected nine histological features found among the 142 hyperplastic lesions. As listed in Table S1 and illustrated in Figure S1 these were: (1) goblet cell abundance; (2) limited serrated change, only at the surface of the crypt; (3) serration of the crypt within the upper half of the crypt; (4) branching of the crypt; (5) dilatation of the crypt; (6) lateral spread of the crypt base (e.g., boot-shaped or anchor-shaped crypts); (7) asymmetrical branching; (8) narrowing of the lower crypt; and (9) elongated straightforward crypt without serration.2, 8-12 The first eight factors were defined as a lesion with at least greater than one factors. A finding of “elongated straightforward crypt without serration” was termed as a lesion with more than 50% present within the same tumor. The histological features are illustrated in Figure S1.

To evaluate absorptive epithelial cells within the glands of each lesion, we selected single glands with vertical axes that could be observed from the crypt base to the surface of the crypt. According to this method, we counted the number of absorptive epithelial cells and goblet cells in the single glands selected in each lesion. We measured the ratio of absorptive epithelial cells among the total cell population (absorptive epithelial cells and goblet cells). The ratio of absorptive epithelial cells was significantly higher in MVHPs than in HNs and GCHPs (p < 0.001; Figure S2).

Immunohistochemistry

Immunostaining was carried out on 3-μm-thick paraffin sections. After deparaffinization and rehydration, the sections were heated in Envision FLEX target retrieval solution (pH 6.0 or 9.0; Dako) for 20 min and washed 2 × 5 min in phosphate-buffered saline. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 5 min. Nonspecific binding was blocked with 1.5% normal serum in phosphate-buffered saline for 35 min at room temperature. Immunohistochemical staining was performed as described previously. Immunohistochemical analysis used anti-p53 (DO7; Dako), anti-MUC2 (Ccp58; Novocastra), anti-MUC5AC (CLH2; Novocastra), anti-MUC6 (CLH5; Novocastra), anti-CD10 (56C6; Novocastra), Annexin A10 (polyclonal; Novusbio), and anti-Ki67 (MIB1, monoclonal; DAKO). Detailed data are presented in Table S2.

Assessment of immunohistochemical expression

To avoid being arbitrary, we used the following criteria to analyze staining of mucin markers (MIUC2, MUC5AC, and MUC6), CD10, p53, and annexin A10. Staining intensity scores were divided into no staining, weak or equivocal staining, moderate staining, and strong staining. Both moderate and strong staining were considered positive expression. The scoring of cells with positive expression was: 0 for 0%–10% of cells; 1 for 10% to <30% of cells; 2 for 30% to <60% of cells; 3 for 60% to <100% of cells; and 4 for 100% of cells. In this study, a score >1 was classified as positive expression of the markers, according to previous study.13

To evaluate the Ki67-positive index, two representative adjacent crypts running from the bottom to the surface of the crypt were selected in each specimen. The percentage of Ki67-expressing cells per crypt was separately calculated for the upper, middle, and lower parts of crypt.14 Depending on the result, each case was classified as upper, middle, and lower.

DNA extraction

Tissue for DNA extraction was micro-dissected from the central area of the lesions. DNA was extracted from isolated normal and tumor tissue by sodium dodecyl sulfate lysis and proteinase K digestion, followed by a phenol-chloroform procedure.

Analysis of KRAS and BRAF mutation

Mutations of KRAS and BRAF genes were examined using a pyrosequencer (Pyromark Q24; Qiagen NV) and primers as previously described.15

Pyrosequencing for evaluation of methylation

We used a two-panel method to determine genome-wide methylation status as in a previous report.16, 17 The DNA methylation status of each gene promoter region was established by PCR analysis of bisulfite-modified genomic DNA (EpiTect Bisulfite Kit; Qiagen) using pyrosequencing for quantitative methylation analysis (Pyromark Q24; Qiagen). In brief, six markers (RUNX3, MINT31, LOX, NEUROG1, ELMO1, and THBD) were selected for determination of genome-wide methylation status. After methylation analysis of a panel of three markers (RUNX3, MINT31, and LOX), tumors with high (hypermethylated) methylated epigenomes (HMEs) were defined as those with at least two methylated markers. The remaining tumors were examined using three additional markers (NEUROG1, ELMO1, and THBD). Tumors were designated as having intermediately methylated epigenomes (IMEs) if they had at least two methylated markers. Tumors that were not HMEs or IMEs were designated as having low (hypomethylated) methylated epigenomes (LMEs). The cut-off value for the mutation assay was 15% mutant alleles, while that for the methylation assay was 30% of tumor cells, as previously reported.15

Hierarchical analysis of the expression of histological features

Hierarchical cluster analysis was performed for clustering of the samples according to the histological findings in order to achieve maximal homogeneity for each group, and the greatest difference between the groups, using open-access clustering software (Cluster 3.0 software; bonsai.hgc.jp/~mdehoon/software/cluster/software.htm). The clustering algorithm was set to centroid linkage clustering, which is standard for biological studies.

Statistical analysis

Data obtained for clinicopathological findings, histological features, immunohistochemical markers, mutations (KRAS and BRAF), and methylation status based on each lesion were analyzed using χ2 tests with the aid of Stat Mate-III software (Atom). If statistical differences between the three lesions were found, statistical analysis between two groups was further performed using χ2 tests (Stat Mate-III software). Differences in age distributions between the three lesions were evaluated using Kruskal–Wallis H tests with the aid of Stat Mate-III software (Atom). If statistical differences between the three lesions were observed, statistical differences were further evaluated using the same method. Differences with p values of less than 0.05 were considered significant.

RESULTS

Representative histological and immunohistochemical figures of MVHP, GCHP, and HN lesions are shown in Figure 1.

image

Representative figure of hyperplastic lesions: (a) GCHP, (g) MVHP, and (m) HN. (a–f) Goblet cell-rich variant hyperplastic polyp (GCHP); (a) HE staining, (b) MUC2, (c) MUC5AC, (d) MUC6, (e) CD10, (f) Ki-67; (g–l) microvesicular variant hyperplastic polyp (MVHP); (g) HE staining, (h) MUC2, (i) MUC5AC, (j) MUC6, (k) CD10, (l) Ki-67; (m–r) hyperplastic nodule (HN); (m) HE staining. (n) MUC2, (o) MUC5AC, (p) MUC6, (q) CD10, (r) Ki-67. GCHP, goblet cell-rich hyperplastic polyp; HE, hematoxylin and eosin; HN, hyperplastic nodule; MVHP, microvesicular hyperplastic polyp

Clinicopathological characteristics among GCHP, MVHP, and HN lesions

We compared clinicopathological characteristics with each hyperplastic lesion (Table 1). The frequency of the macroscopic flat elevated type was significantly higher in GCHP than in MVHP and HN cases (p < 0.05; Table 1). There were no significant differences in the frequency of the other clinicopathological variables (sex, age, tumor location, and tumor size) between lesions.

Association of histological features with each lesion

We investigated the association of the nine histological features with GCHP, MVHP, and HN (Table 2). There was a significant difference in the frequency of eight of the features between the three lesions, but not “lateral spread of the crypt base.” In addition, these eight features showed significant differences between each pair of lesions (GCHP vs. MCHP; GCHP vs. HN; MSHC vs. HN). First, we found a significant difference between GCHP and MVHP in the frequency of three features: “goblet cell abundance,” “serration of the crypt within the upper half of the crypt,” and “branching of the crypt.” Second, although the frequency of two features (“limited serrated change, only at the surface of the crypt” and “serration of the crypt with the upper half of the crypt”) were significantly higher in GCHP than in HN, the frequency of “elongated straightforward serrated crypt without serration” was statistically lower in GCHP than in HN. Third, there was a significant difference in the frequency of “goblet cell abundance” and “elongated straightforward crypt without serration” between HN and MVHP, with HN > MCHP, while the statistical difference in the frequency of “serration of the crypt within the upper half of the crypt,” “dilatation of the crypt,” and “narrowing of the lower crypt” between HN and MVHP had HN < MVHP. Finally, although HN consisted of an admixture of goblet cell-rich and absorptive cells, GCHP was predominantly composed of goblet cells. In summary, two features were closely associated with GCHPs: “goblet cell abundance” and “limited serrated change within the superficial area of the crypt.” Four features characterized MVHPs: “serration of the crypt with the upper half of the crypt,” “branching of the crypt,” and “dilatation of the crypt and narrowing of the lower crypt.” Finally, “elongated straightforward serrated crypt without serration” was the distinctive feature of HNs. Detailed findings are summarized in Table 2.

Table 2. Differences of histological findings in colorectal hyperplastic lesions GCHP (%) HN (%) MVHP (%) p-value Total 61 19 62 Goblet cell abundance 61 (100)* 19 (100)# 0*,# 3.78E−42 Limited serrated change at superficial area of the crypt 33 (54.1)*,# 0* 0# 1.91E−16 Serration of the crypt with the upper half of the crypt 25 (41.0)*, 0*,# 62 (100)#, 1.73E−23 Branching of the crypt 18 (29.5)** 5 (26.3) 33 (53.2)** 0.0121 Dilatation of the crypt 16 (26.2)** 2 (10.5)# 31 (50.0)**,# 0.0013 Lateral spread of the crypt base (such as boot- shaped or anchor-shaped crypts) 0 0 0 N. S. Asymmetrical branching 0 0 6 (9.7) 0.0116 Narrowing of lower crypt 0* 0# 36 (58.1)*,# 7.03E−17 Elongated straightforward crypt without serration 5 (8.2)* 19 (100)*,# 0# 1.90E−20 Abbreviations: GCHP, goblet cell-rich hyperplastic polyp; HN, hyperplastic nodule; MVHP, microvesicular hyperplastic polyp; N. S., not significant. * p < 0.01; #p < 0.01; ♭p < 0.01; ** p < 0.05. Differences in immunohistochemical marker expression, gene mutations (KRAS and BRAF), and DNA methylation status among lesions

Although the frequency of MUC5AC was significantly lower in HNs (0/19) than in GCHPs (0/61) and MVHPs (53/62, 85.5%); p < 0.001), the frequency of MUC6 was significantly higher in MVHPs (5/62, 8.1%) than GCHPs (0/61) and HNs (0/19; p = 0.0274; Table 3). To investigate whether morphological changes in hyperplastic lesions were associated with cell proliferation, we performed immunohistochemical examination for Ki67. In HNs, Ki67-positive cells were predominantly found in the lower third of the crypt. In MVHPs, although Ki67-positive cells were primarily observed in the middle third of the crypt, Ki67-positive cells were broadly expanded toward the middle part of the crypt in GCHPs in comparison with HNs and MVHPs. In addition, a low frequency of Annexin A10 expression was common among the lesions. The frequency of KRAS mutations was significantly higher in GCHPs than in MVHPs and HN. By contrast, there was a significant difference in the frequency of BRAF mutations between GCHPs and HNs and between GCHPs and MVHPs (MVHPs > GCHPs and HNs). Detailed mutation data for KRAS and BRAF are summarized in Table S2. No differences in the codon of KRAS mutations were observed among the three lesion types. In addition, the KRAS mutation type did not differ among the lesion types. Finally, no significant differences in methylation status were observed among lesions. The detailed findings are presented in Table 3.

Table 3. Differences of molecular findings in colorectal hyperplastic lesions GCHP (%) HN (%) MVHP (%) p-value Total 61 19 62 MUC2 61 (100) 19 (100) 62 (100) N. S. MUC5AC 0* 0# 53 (85.5)*,# 1.068E−29 MUC6 0 0 5 (8.1) 0.0274 CD10 0 0 0 N. S. Ki-67 distribution Lower type 27 (44.3)*,# 19 (100)*,** 5 (8.1)#,** 2.39E−09 Middle type 34 (55.7)*,# 0*,** 56 (90.3)#,** Upper type 0 0 1 (1.6) p53 0 0 0 N. S. Annexin A10 0 0 6 (9.7) 0.0116 KRAS mutation 30 (49.2)*,# 0* 13 (21.0)# 3.18E−06 BRAF mutation 0* 1 (4.3)# 38 (61.3)*,# 6.99E−17 No mutation 31 (50.8)* 18 (96.7)*,# 11 (17.7)# 6.75E−10 DNA methylation LME 37 (82.2) 12 (75.0) 40 (64.5) 0.3194 IME 7 (15.6) 4 (25.0) 19 (30.6) HME 1 (2.2) 0 3 (4.2) Abbreviations: GCHP, goblet cell-rich hyperplastic polyp; HN, hyperplastic nodule; HME, high-methylation epigenotype; IME, intermediate-methylation epigenotype; MVHP, microvesicular hyperplastic polyp; LME, low-methylation epigenotype; N. S., not significant. * p < 0.01; #p < 0.01; ** p < 0.01. Hierarchical cluster analysis using the nine histological features

We examined the patterns of nine histological features using heat-map hierarchical cluster analysis to identify associations of the histological features with each lesion. Three distinct subgroups were stratified based on the histological features (Figure 2). In clinicop

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