Molecular Characterization of sessile serrated adenoma/polyps from a large African American cohort

INTRODUCTION

Colorectal cancer is the second leading cause of cancer-related deaths in the United States and rates are highest among African Americans (AAs) (1). Sessile serrated adenoma/polyps (SSA/Ps) may be precursors of up to 30% of colorectal cancers (2). Flat and mucinous features make SSA/Ps difficult to detect and similarities to histological features of benign hyperplastic polyps (HPs) make them difficult to be accurately diagnosed. As such, there is a need for specific sensitive molecular biomarkers for accurate and reliable diagnosis. Our aim was to assess the diagnostic value of molecular biomarkers that may distinguish SSA/Ps from HPs among AA patients.

MATERIALS AND METHODS

Patients and Specimens.

We conducted a retrospective study of colonoscopies performed at Howard University Hospital (2010–2015, N=12,085). Of these, 83% (N=10,027) were from AA patients. Pathology reports revealed 4,070 AA cases with polyps inclusive of 252 SSA/P patients. For this study, we analyzed SSA/P patients without dysplasia (N=22) with 29 total polyps compared to HP patients (N=18) with 26 total polyps to establish discriminatory biomarkers between these lesions. Four uninvolved normal colon tissues were also included for comparison.

DNA and RNA isolation.

An AllPrep Qiagen DNA/RNA kit was used for DNA and RNA extraction from microdissected tissue blocks according to manufacturer’s instructions.

Gene expression, MSI and BRAF V600E analysis.

Gene expression and mutation frequency were analyzed in 44 patients (59 specimens: 29 SSA/Ps, 26 HP, and 4 uninvolved colons). From a panel of 51 candidate transcripts, we assessed expression levels of the top 4 transcripts (MUC6, FSCN1, SEMG1, and TRNP1) using qRT-PCR with commercially-available TaqMan assays. DNA was also genotyped for the BRAF V600E mutation and MSI status.

CpG Island Methylator Phenotype (CIMP).

Primers and probes for CACNA1G, IGF2, NEUROG1, RUNX, and SOCS were used as previously described (3).

IHC:

MLH1 immunohistochemistry was performed on a tissue microarray using an MLH1 antibody. Scoring was performed as previously described (4).

Statistical analysis:

Demographic and clinical variables were described using median (IQR) or frequency table. Continuous and categorical variables’ distribution was compared by student’s t-test, and Chi-squared exact test, respectively.

RESULTS

Demographic Profile.

Mean age was similar between SSA/P and HP patients (56.7 vs. 55.3 years), with 63% of SSA/Ps patients and 50% HP patients female. Eleven of 22 (50%) SSA/P patients and 13/18 (72%) HP patients had a BMI >30. Twenty of 29 (69%) SSA/Ps and 24/26 (92%) HPs were in the left colon.

Gene Expression Profile in SSA/Ps and HPs.

MUC6, SEMG1, TRNP1, and FSCN1 expression were statistically higher in SSA/Ps compared to HPs (Figure 1A). MUC6 showed the highest fold-change in SSA/Ps (37.2X), followed by SEMG1 (10.7X), TRNP1 (5.8X) and FSCN1 (2.5X). Fold changes in SSA/Ps versus uninvolved normal colon were similar to those noted in comparison to HPs, except for MUC6 which showed a lower fold difference of 12.9.

Figure 1:

(A) Gene Expression of MUC6, FSCN1, SEMG1, and TRNP1 in SSA/Ps vs HPs vs Uninvolved Colons. (B) Frequency of BRAF V600E, CIMP-positive, loss of MLH1 IHC expression and presence of MSI in SSA/Ps in comparison with HPs. Bars show the frequency (%) in SSA/Ps (blue) and HPs (orange).

BRAF V600E, CIMP and MSI analyses.

BRAF V600E was identified in 15 of 27 (55.6%) SSA/Ps compared to 3 of 25 (12.0%) HPs (P=0.001). Although CIMP-positive (6/22, 27.3% vs. 3/23, 13.0%) and loss of MLH1 expression (3/22, 13.6% vs. 1/23, 4.3%) were more prevalent in SSA/Ps, MSI frequency was higher in HPs (5/29, 17.2% vs. 10/25, 40%), the differences did not reach statistical significance (Figure 1B).

MLH1 IHC expression.

SSA/P lesions demonstrated higher MLH1 staining intensity (19/27, 70.3% showing >2+ staining) in comparison to HPs (3/7, 42.9%).

DISCUSSION

The prevalence of SSA/Ps in our study was 2.5% (252/10,027 colonoscopies) which is higher than that published by other groups. Moreover, these lesions were primarily distal which could associate with a different molecular profile. Indeed, neither CIMP nor MSI appear to be potential markers for these distal SSA/Ps. In addition to location, age might also be a determining factor for CIMP and MSI status. Indeed, Lui et al. reported that SSA methylation increased dramatically with age and associated with dysplasia (5). Our SSA/Ps patients were generally young (mean 56 years) and displayed no dysplasia. This is possibly one of the reasons why CIMP was not a distinguishing criterion in our cohort. MSI, a by-product of CIMP when it affects MLH1, was also not a differentiating feature between SSA/Ps and HPs in our AA population.

Nearly 60% of our SSA/P cases displayed BRAF V600E mutation which has been previously associated with hypermethylation. This is not the case in our study, perhaps because the CIMP marker panel we used was not specific for SSA/Ps, particularly distally located ones. In a genome-wide DNA methylation and gene expression study in SSA/Ps and conventional adenomas, Parker et al. established a test panel of six hypermethylated regions (LOX, LEF1-AS1, SFRP4, ZNF793, SYT9, and LINC00693) that distinguished pre-cancers from normal mucosa and SSA/Ps from conventional adenomas with high accuracy (6). The test panel we used in our study differed from the ones established in their study.

MUC6 demonstrated the highest fold-difference suggesting that its high expression might correlate with SSA/P pathogenesis. Three other genes (SEMG1, TRNP1 and FSCN1) also showed significantly higher expression in AA SSA/Ps. Several studies have shown that among four mucin genes clustered on chromosome 11 (MUC2, MUC5AC, MUC5B and MUC6), MUC2 and MUC5B are highly subject to DNA methylation and histone modification, whereas MUC5AC is rarely influenced by epigenetic regulation and MUC6 does not undergo significant epigenetic regulation (7). Indeed, Parker et al. also demonstrated that SSA/Ps have high expression of MUC6 and SEMG1, among others (6). It is worth noting that MUC6 fold difference in SSA/Ps vs. HPs (37.2X) was much higher than that noted for SSA/Ps compared to uninvolved normal colon (12.9X). Therefore, these difficult-to-diagnose lesions could be evaluated for MUC6 and SEMG1 expression in addition to BRAF V600E to differentiate SSA/Ps from HPs. Among AA SSA/Ps, it remains to be seen if TRNP1 and FSCN1 elevated expression solely segregates with race. However, these factors individually, or collectively, may lead to better SSA/P detection and early diagnosis, particularly through non-invasive colorectal cancer screening approaches (8).

Abbreviations used:

AA

African American

SSA

sessile serrated adenoma/polyps

HP

hyperplastic polyps

TA

tubular adenomas

MSI

microsatellite instability

CRC

colorectal cancer

MMR

DNA mismatch repair

CIMP

CpG island methylator phenotype

TSAs

traditional serrated adenomas