Introduction: The serrated pathway contributes to interval colorectal cancers, highlighting the need for new biomarkers to assess lesion progression risk. The β1,6-GlcNAc branched N-glycans expression in CRC cells was associated with an invasive phenotype and with immune evasion. Therefore, this study aims to identify potential risk factors for progression of serrated lesions (SLs) to malignancy, analyzing the N-glycosylation profile of epithelial/infiltrating immune cells. Methods: A retrospective cohort study was performed with data from 53 colonoscopies (48 patients). Sixty-three serrated pathway lesions (SPLs) were characterized based on N-glycosylation profile (lectin histochemistry/flow cytometry) and MGAT5 expression. Statistical analysis was performed to search for associations between the glycoprofile and clinical variables from each patient. Results: Increased β1,6-GlcNAc branched N-glycans expression in epithelial cells is found associated with age (p = 0.007 in SPL), smoking (p = 0.038 in SL), increased BMI (p = 0.036 in sessile serrated lesions [SSL]), and polyp dimensions ≥10 mm (p = 0.001 in SL), while increased expression of these structures on immune cells is associated with synchronous CA number (CD4+T cells: p = 0.016; CD8+T cells: p = 0.044 in SL) and female gender (p = 0.026 in SL). Moreover, a lower high-mannose N-glycans expression in immune cells is associated with smoking (p = 0.010 in SPL) and synchronous CA presence (p = 0.010 in SPL). Higher expression of these glycans is associated with female (p = 0.016 in SL) and male (p = 0.044 in SL) gender, left colon location (p = 0.028), dysplasia (p = 0.028), and adenocarcinoma (p = 0.010). Conclusions: We identified an association between an abnormal glycoprofile and several clinical risk factors, proposing the N-glycosylation profile as a potential biomarker of tumor progression in the serrated pathway. The N-glycosylation anatomopathological profile analysis could be further used to decide shorter interval follow-up in patients with SPL.

Introdução: A via serreada contribui para os cancros colorretais de intervalo, destacando a necessidade de novos biomarcadores para determinar o risco de progressão destas lesões. A expressão de β1,6-GlcNAc N-glicanos ramificados foi associada a um fenótipo invasivo e a evasão imune. Assim, este estudo tem como objetivo identificar potenciais fatores de risco de progressão das lesões serreadas para malignidade, analisando o perfil de N-glicosilação das células epiteliais/células imunitárias.Métodos: Foi realizado um estudo retrospetivo com dados de 53 colonoscopias (48 doentes). 63 lesões da via serreada foram caracterizadas segundo o perfil de N-glicosilação (histoquímica de lectinas/citometria de fluxo) e expressão de MGAT5. A análise estatística foi realizada para encontrar associações entre o perfil de N-glicosilação e as variáveis clínicas de cada doente.Resultados: O aumento da expressão de β1,6-GlcNAc N-glicanos ramificados nas células epiteliais encontra-se associado com a idade (p = 0.007 nas SPL), tabagismo (p = 0.038 nas SL), aumento do BMI (p = 0.036 nas SSL), e pólipos com dimensões ≥10 mm (p = 0.001 nas SL), enquanto que o aumento destas estruturas nas células imunitárias está associado com o número de CA síncronos (células TCD4+: p = 0.016; células TCD8+: p = 0.044 nas SL) e o género feminino (p = 0.026 nas SL). Além disso, uma diminuição da expressão de N-glicanos ricos em manose está associada ao tabagismo (p = 0.010 para SPL) e a presença de adenomas síncronos (p = 0.010 nas SPL). A expressão aumentada destas estruturas está associado com o género feminino (p = 0.016 nas SSL), género masculino (p = 0.044 nas SSL), localização no cólon esquerdo (p = 0.028), displasia (p = 0–028) e adenocarcinoma (p = 0.010).Discussão/Conclusão: Identificámos uma associação entre um perfil de glicosilação anormal e vários fatores de risco clínicos, propondo o perfil de N-glicosilação como um potencial biomarcador de progressão tumoral na via serreada. A análise anatomopatológica do perfil de N-glicosilação pode vir a ser usada para decidir intervalos de follow-up mais curtos em doentes com SPL.

Colorectal cancer (CRC) is the third most frequent cancer and it is responsible for 10% of cancer mortality worldwide [1]. This cancer type occurs mostly from conventional adenoma (CA)-carcinoma pathway, while serrated pathway is responsible for about 25% of the cases [3]. Serrated lesions (SLs) are known as the precursor lesion in this carcinogenic pathway. SL can be divided into hyperplastic polyps (HPs), sessile serrated lesions (SSLs), sessile serrated lesions with dysplasia (SSLs-D), traditional serrated adenomas, and unclassified serrated adenomas, accordingly to the World Health Organization [4].

The last two decades were marked by advances in the study of the serrated pathway in order to understand the neoplastic mechanisms underlying this disease to prevent the progression to cancer [5]. This pathway is characterized by epigenetic alterations with mismatch repair genes deficiency and by the presence of a CpG island hypermethylation phenotype, with microsatellite instability in the vast majority of the cases [1]. Consequently, the serrated pathway presents a high lymphocytic immune infiltrate and upregulation of immune checkpoints associated with tumor immune evasion [8]. However, there is a gap of knowledge in understanding this pathway, particularly the progression to malignancy and the risk factors involved.

Some association studies defined smoking, alcohol consumption, overweight, red meat consumption, hypertension, and hypertriglyceridemia as risk factors for the SL development. Other researchers identified aging, absence of regular consumption of non-steroidal anti-inflammatory drugs, polyp dimensions ≥10 mm, dysplasia, female gender, and synchronous CA as risk factors for progression to malignancy in the serrated pathway [7, 9–15]. However, these risk factors are not as well established as in the adenoma-carcinoma pathway. Additionally, this CRC subtype is one of the responsibilities for the occurrence of interval cancers. This is presumed to be secondary to the difficult endoscopic identification of these lesions due to their sessile morphology, mucus coverage, and proximal colon location and rapid cancer progression after the development of dysplasia [7]. Thus, the protective effect of CRC screening is expected to decrease in these patients [7]. This represents a challenge to physicians managing these cases due to the lack of biomarkers that could impact therapeutic decisions. Taking this into account, it seems crucial to identify a new biomarker capable of improving risk stratification and further clinical decision.

N-glycosylation has been associated with the malignant transformation process, and it is considered to be a cancer hallmark [11]. This process is a post-translational modification characterized by enzymatic reactions that allow the binding of carbohydrates (glycans) to proteins, lipids, or other saccharides [11]. These glycan structures are found on all cell surfaces, constituting the glycocalyx [12]. The differential glycans profiles are associated with immunologic and epithelial biologic functions [13]. In fact, our group described that the expression of β1,6-GlcNAc branched N-glycans in the conventional colorectal carcinogenesis cascade is considered an important immune checkpoint, demonstrating that these complex N-glycans overexpression in CRC cells was associated with immune escape [14]. Additionally, Demetriou et al. [15] demonstrated that T-cell activity is particularly regulated by β1,6-GlcNAc branched N-glycans on the T-cell receptor that modulates the threshold of T-cell activation and signaling. In line with this and in the context of chronic inflammatory processes such as inflammatory bowel disease, our group showed that these complex N-glycans are capable of regulating T-cell-mediated immune response associated with disease severity [16]. Particularly, we demonstrated that a β1,6-GlcNAc branched N-glycans deficiency, due to a MGAT5 decreased expression, confers an hyperimmune response by decreasing T-cell activation threshold, increasing proinflammatory cytokines production, and increasing T-cell signaling [16]. This highlights the crucial role of the N-glycans pattern on cancer development/progression and immune response regulation. Therefore, in this study, we aimed to identify risk factors for progression to malignancy of serrated pathway lesions (SPLs) based on the N-glycosylation profile of both cancer cells and infiltrating immune cells.

Cohort Characterization

This is retrospective cohort study of patients with lesions of the serrated pathway, followed between September 2014 and 2021. Data were collected from 53 colonoscopies, corresponding to 48 patients.

The N-glycosylation profile was previously obtained from FFPE (formalin-fixed paraffin-embedded) biopsies and fresh biopsies of SPL. The MGAT5 gene (gene that encodes the enzyme N-acetylglucosaminyltransferase-V [GnT-V]), responsible for the expression of β1,6-GlcNAc branched N-glycans, was evaluated by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) in FFPE biopsies. Also, in FFPE biopsies, a lectin histochemistry was performed to evaluate the expression of β1,6-GlcNAc branched N-glycans (complex glycans) in epithelial and stromal cells, obtained by staining with Phaseolus vulgaris leucoagglutinin (L-PHA), as well as the presence of high-mannose N-glycans (simple glycans), identified by labeling Glanthus nivalis agglutinin. The lectin histochemistry evaluation was performed by two independent observers, who gave a score from 0 to 3 according to the degree of staining (0: ≤25%; 1: 26% to 50%, 2: 51% to 75%, and 3: >75%). Flow cytometry of epithelial cells (CD45 cells), CD4+ T cells, CD8+ T cells, and FoxP3+CD25+ T cells (regulatory T cell – Treg) was performed on fresh biopsies, and L-PHA and Glanthus nivalis agglutinin expression was obtained, corresponding to β1,6-GlcNAc branched N-glycans and high-mannose N-glycans expression, respectively, in each of these cell populations.

Statistical Analysis

Descriptive statistics were performed based on the analysis of the mean and standard deviation of the continuous variables under study; percentages were used for the categorical variables. The relationship between the clinical variables and the N-glycosylation profile of the epithelial cells and colonic T cells obtained by RT-qPCR, lectin histochemistry, and flow cytometry was performed using Pearson’s correlation, t test for independent samples, and nonparametric Mann-Whitney U. This relationship was performed for 3 groups: SPL (which includes SL and serrated pathway adenocarcinoma), SL (which includes HP, SSL, and SSL-D), and SSL (with or without dysplasia). The association between continuous variables and non-binary discrete variables was performed using the one-way ANOVA test, using Tukey’s post hoc test. A significance level of 0.05 was considered and the statistical analysis of the variables was performed using the SPSS version 26 program.

Of the 53 colonoscopies performed, 63 samples of SPL were obtained (34 FFPE biopsies and 29 fresh biopsies). This cohort includes 44 (69.8%) SSL without dysplasia, 9 (14.3%) SSL-D, 7 (11.1%) HP, 1 (1.6%) adenocarcinoma of the serrated pathway, and 2 (3.2%) SSL-D with concomitant adenocarcinoma (Table 1). The description of the sample regarding the remaining sociodemographic characteristics, risk factors, and anatomopathological characteristics is depicted in detail in Table 1. The descriptive statistic of N-glycosylation profile is depicted in detail in Table 2.

Table 1.

Cohort characterization

VariableN (%)MeanStandard deviation
Gender 48   
 Male 24 (50.0)   
 Female 24 (50.0)   
Age, years  64.2 11.4 
Smoking 48   
 No 25 (52.1)   
 Yes 23 (47.9)   
 Missing 0 (0)   
BMI, kg/m2 42 27.3 4.9 
Lesion number  3.6 4.5 
SPL number  3.1 4.4 
Synchronous CA number  0.6 1.2 
Synchronous CA 
 No 47 (74.6)   
 Yes 16 (25.4)   
Classification 63   
 SSL without dysplasia 44 (69.8)   
 SSL-D 9 (14.3)   
 HP 7 (11.1)   
 Adenocarcinoma 1 (1.6)   
 Adenocarcinoma + SSL-D 2 (3.2)   
Location 63   
 Right colon 41 (65.1)   
 Transverse colon 15 (23.8)   
 Left colon 7 (11.1)   
 Missing 0 (0)   
Dimension, mm 63 22.8 11.4 
 <10 4 (6.3)   
 ≥10 58 (92.1)   
 Missing 1 (1.6)   
VariableN (%)MeanStandard deviation
Gender 48   
 Male 24 (50.0)   
 Female 24 (50.0)   
Age, years  64.2 11.4 
Smoking 48   
 No 25 (52.1)   
 Yes 23 (47.9)   
 Missing 0 (0)   
BMI, kg/m2 42 27.3 4.9 
Lesion number  3.6 4.5 
SPL number  3.1 4.4 
Synchronous CA number  0.6 1.2 
Synchronous CA 
 No 47 (74.6)   
 Yes 16 (25.4)   
Classification 63   
 SSL without dysplasia 44 (69.8)   
 SSL-D 9 (14.3)   
 HP 7 (11.1)   
 Adenocarcinoma 1 (1.6)   
 Adenocarcinoma + SSL-D 2 (3.2)   
Location 63   
 Right colon 41 (65.1)   
 Transverse colon 15 (23.8)   
 Left colon 7 (11.1)   
 Missing 0 (0)   
Dimension, mm 63 22.8 11.4 
 <10 4 (6.3)   
 ≥10 58 (92.1)   
 Missing 1 (1.6)   

BMI, body mass index; CA, conventional adenoma; HP, hyperplastic polyp; SPL, serrated pathway lesion; SSL, sessile serrated lesion; SSL-D, sessile serrated lesion with dysplasia.

Table 2.

Statistical description of the glycosylation profile of SPLs

Mean±standard deviationMedianMinimumMaximum
MGAT5 gene RT-qPCR (∆ct value) 2.6 × 10−4±4.4 × 10−4 9.30017 × 10−5 1.14743 × 10−5 2.37524 × 10−3 
Histochemistry 
 Epithelial L-PHA 1.2±0.9 1.25 
 Stromal L-PHA 0.9±0.6 
 Epithelial GNA 0.9±0.9 0.5 
 Stromal GNA 1.6±0.9 0.5 
FC 
 Epithelial L-PHA 233.1±156.9 185 20.6 713 
 Epithelial GNA 148.4±129.9 109 25.7 551 
 T CD4+ L-PHA 984.4±805.0 838.5 134 3,497 
 T CD4+ GNA 132.1±146.6 67.5 547 
 T CD8+ L-PHA 1,376.8±1,076.3 1,049 413 4,416 
 T CD8+ GNA 558.2±965.8 73.2 2,843 
 FoxP3 T CD25+ L-PHA 1,906.7±1,960.7 1,742.5 223 8,775 
 FoxP3 T CD25+ GNA 1,172.3±2,138.0 559 50.1 9,862 
Mean±standard deviationMedianMinimumMaximum
MGAT5 gene RT-qPCR (∆ct value) 2.6 × 10−4±4.4 × 10−4 9.30017 × 10−5 1.14743 × 10−5 2.37524 × 10−3 
Histochemistry 
 Epithelial L-PHA 1.2±0.9 1.25 
 Stromal L-PHA 0.9±0.6 
 Epithelial GNA 0.9±0.9 0.5 
 Stromal GNA 1.6±0.9 0.5 
FC 
 Epithelial L-PHA 233.1±156.9 185 20.6 713 
 Epithelial GNA 148.4±129.9 109 25.7 551 
 T CD4+ L-PHA 984.4±805.0 838.5 134 3,497 
 T CD4+ GNA 132.1±146.6 67.5 547 
 T CD8+ L-PHA 1,376.8±1,076.3 1,049 413 4,416 
 T CD8+ GNA 558.2±965.8 73.2 2,843 
 FoxP3 T CD25+ L-PHA 1,906.7±1,960.7 1,742.5 223 8,775 
 FoxP3 T CD25+ GNA 1,172.3±2,138.0 559 50.1 9,862 

FC, flow cytometry; GNA, Glanthus nivalis agglutinin; L-PHA, Phaseolus vulgaris leucoagglutinin; RT-PCR, reverse transcriptase-quantitative polymerase chain reaction.

Altered Premalignant Epithelial N-Glycosylation Profile Is Correlated with Age, Smoking, Increased BMI, Polyp’s Dimensions, and Lesion Location in the Serrated Pathway

The N-glycosylation profile of epithelial cells was correlated with potential risk factors for disease progression in the serrated pathway. Our results indicated a significant correlation between increased age and the β1,6-GlcNAc branched N-glycans expression (L-PHA expression) on epithelial cells in SPL (p = 0.007; r = 0.501) (Table 3). Additionally, tobacco consumption was also associated with an increased MGAT5 gene expression. In fact, smokers presented an evident higher β1,6-GlcNAc branched N-glycans expression in SL comparing to non-smokers (∆ct value: 2.50 × 10−4 ± 2.62 × 10−4 vs. 9.49 × 10−5 ± 9.82 × 10−5; p = 0.038) (Table 4). Furthermore, a statistically significant correlation was also observed between body mass index (BMI) and β1,6-GlcNAc branched N-glycans expression in SPL (p = 0.045; r = 0.432) and in SSL (p = 0.036; r = 0.496), regarding MGAT5 gene expression (Table 4). In addition, our data indicate a higher β1,6-GlcNAc branched N-glycans expression in SL with dimensions ≥10 mm, comparing to SL with dimensions <10 mm, regarding MGAT5 gene expression (∆ct value: 1.70 × 10−4 ± 1.68 × 10−4 vs. 4.40 × 10−5 ± 3.96 × 10−6; p = 0.001) (Table 4). Concerning the SPLs location, there was an increase in the high-mannose N-glycans expression in the epithelial part of the left colon, compared to the right and transverse colon (2.50 ± 0.71 vs. 0.80 ± 0.84 vs. 0.40 ± 0.22; p = 0.009) (Table 4).

Table 3.

N-glycosylation profile results obtained by FC

VariableEpithelial L-PHAEpithelial GNAT CD4+ L-PHAT CD4+ GNAT CD8+ L-PHAT CD8+ GNATreg L-PHATreg GNA
Nmeanp valuenmeanp valuenmeanp valuenmeanp valuenmeanp valuenmeanp valuenmeanp valuenmeanp value
Gender 
 SPL   0.308   0.443   0.396   0.014   0.309   0.044   0.040   0.980 
  Male 13 204.62±90.35  13 139.35±86.77  13 830.54±392.51  14 52.65±38.99  10 1,182.60±432.40  12 778.79±1,084.28  13 1,036.99±990.21  13 1,028.22±2,679.55  
  Female 15 259.87±171.95  14 176.89±152.10  13 1,073.85±935.95  14 178.86±164.04  1,608.78±1,204.08  10 67.98±50.69  11 2,556.36±2,269.88  12 1,049.29±886.34  
 SL   0.587   0.386   0.404   0.016   0.195   0.044   0.026   0.907 
  Male 13 204.62±90.35  13 139.35±86.77  13 830.54±392.51  14 52.65±38.99  10 1,182.60±432.40  12 778.79±1,084.28  13 1,036.99±990.21  13 1,028.22±2,679.55  
  Female 14 227.50±122.14  13 183.19±156.41  12 1,079.83±977.31  13 185.30±168.89  1,739.25±1,217.31  10 67.98±50.69  10 2,736.70±2,308.10  11 1,128.50±883.94  
 SSL   0.822   0.369   0.309   0.017   0.119   0.043   0.029   0.958 
  Male 11 223.76±77.05  11 148.80±87.38  11 820.09±322.43  12 44.083±29.48  1,161.11±452.93  11 844.16±1,112.12  11 916.18±813.00  11 1,138.10±2,916.71  
  Female 11 233.64±121.67  11 200.48±164.97  1,187.78±114.37  11 200.37±179.86  1,892.14±1,229.08  8 65.55±57.13  7 3,005.86±2,759.88  1,083.39±918.62  
Age, years 
 SPL 28 0.501* 0.007 27 −0.107* 0.596 26 0.206* 0.314 28 −0.183* 0.352 19 −0.101* 0.682 22 0.136* 0.547 24 0.167* 0.436 25 −0.067* 0.751 
 SL 27 0.422* 0.028 26 −0.080* 0.697 25 0.212* 0.308 27 −0.182* 0.363 18 −0.028* 0.913 22 0.136* 0.547 23 0.218* 0.317 24 −0.041* 0.848 
 SSL 22 0.224* 0.316 22 −0.227* 0.309 20 0.224* 0.343 23 −0.166* 0.449 16 −0.058* 0.830 19 0.104* 0.672 18 0.242* 0.334 20 0.002* 0.993 
Smoking 
 SPL   0.195   0.773   0.281   0.715   0.763   0.629   0.357   0.179 
  No 14 269.07±143.48  14 151.97±105.63  13 1,106.08±922.12  14 106.28±126.00  11 1,439.00±1,050.68  11 263.58±656.57  11 1,349.36±1,043.60  12 475.91±721.11  
  Yes 14 199.26±133.45  13 166.19±145.55  13 798.31±400.64  14 125.23±144.70  1,309.50±657.32  11 547.81±1,058.95  13 2,057.31±2,294.65  13 1,557.49±2,610.63  
 SL   0.397   0.847   0.287   0.737   0.622   0.629   0.420   0.210 
  No 13 234.92±67.93  13 156.35±108.61  12 1,114.75±962.57  13 107.14±131.10  10 1,526.40±1,064.53  11 363.58±656.57  10 1,049.00±1,080.11  11 502.99±749.88  
  Yes 14 199.36±133.45  13 166.19±145.55  13 798.31±400.64  14 125.23±144.70  1,309.50±657.32  11 547.81±1,058.95  13 2,057.31±2,294.65  13 1,557.49±2,610.63  
 SSL   0.783   0.633   0.251   0.783   0.809   0.549   0.410   0.213 
  No 12 223.17±55.45  12 161.98±111.44  11 1,171.46±988.31  12 110.50±136.34  10 1,526.40±1,064.53  10 391.72±685.06  1,317.67±1,103.91  10 489.49±789.03  
  Yes 10 235.34±138.77  10 189.83±157.19  758.33±358.98  11 127.92±163.41  1,405.17±714.84  654.78±1,153.65  2,140.00±2,697.21  10 1,737.47±2,952.34  
BMI, kg/m2 
 SPL 22 −0.061* 0.787 21 −0.410* 0.065 21 −0.012* 0.958 22 −0.397* 0.067 18 0.142* 0.574 16 −0.242* 0.366 19 −0.230* 0.344 19 −0.487* 0.034 
 SL 21 −0.155* 0.502 20 −0.405* 0.076 20 −0.012* 0.959 21 −0.398* 0.074 17 0.161* 0.537 16 −0.242* 0.366 18 −0.224* 0.371 18 −0.484* 0.042 
 SSL 18 −0.275* 0.270 18 −0.390* 0.110 17 −0.021* 0.937 19 −0.392* 0.097 15 0.131* 0.643 15 −0.216* 0.440 15 −0.292* 0.291 16 −0.489* 0.055 
Dimension, mm 
 SPL                 
  <10         
  ≥10 27 216.48±106.59  26 161.27±125.92  25 950.20±728.67  27 116.52±135.96  18 1,430.00±888.91  22 455.70±864.96  23 1,775.44±1,859.38  24 1,074.18±2,021.98  
 SL                 
  <10         
  ≥10 27 216.48±106.59  26 161.27±125.92  25 950.20±728.67  27 116.52±135.96  18 1,430.00±888.91  22 455.70±864.96  23 1,775.44±1,859.38  24 1,074.18±2,021.98  
 SSL                 
  <10         
  ≥10 22 228.70±99.51  22 174.64±131.51  20 985.55±782.82  23 118.83±146.67  16 1,480.94±924.09  19 516.33±918.90  18 1,728.83±2,043.52  20 1,113.48±2,198.53  
Dysplasia 
 SPL         0.312     0.548     
  No 26 213.19±107.30  25 161.48±128.51  24 948.96±744.32  25 124.14±138.46  17 1,437.35±915.70  20 491.86±900.91  22 1,827.23±1,886.08  23 1,119.37±2,054.99  
  Yes 302.00  156.00  980.00  21.20±29.98  1,305.00  94.05±49.43  636.00  34.70  
 SL         0.312     0.548     
  No 26 213.19±107.30  25 161.48±128.51  24 948.96±744.32  25 124.14±138.46  17 1,437.35±915.70  20 491.86±900.91  22 1,827.23±1,886.08  23 1,119.37±2,054.99  
  Yes 302.00  156.00  980.00  21.20±29.98  1,305.00  94.05±49.43  636.00  34.70  
 SSL         0.336     0.508     
  No 21 225.21±100.58  21 175.53±134.69  19 985.84±804.27  21 128.13±150.24  15 1,492.67±955.29  17 566.01±961.70  17 1,793.12±2,087.57  19 1,170.26±2,243.66  
  Yes 302.00  156.00  980.00  21.20±29.98  1,305.00  94.05±49.43  636.00  34.70  
Synchronous CA 
 SPL   0.699   0.171   0.265   0.096   0.433   0.051   0.398   0.219 
  No 21 228.11±111.51  20 178.38±137.86  19 855.74±514.56  21 131.39±150.07  14 1,227.86±536.31  18 540.11±939.40  17 1,940.65±2,026.58  18 1,347.82±2,277.49  
  Yes 252.51±216.53  102.01±40.83  1,214.00±1,104.78  68.86±41.06  1,832.00±1,508.10  75.85±18.89  1,228.14±1,213.32  242.51±279.52  
 SL   0.298   0.212   0.469   0.297   0.325   0.051   0.486   0.260 
  No 21 228.11±111.51  20 178.38±137.86  19 855.74±514.56  21 131.39±150.07  14 1,227.86±536.31  18 540.11±939.40  17 1,940.65±2,026.58  18 1,347.82±2,277.49  
  Yes 175.77±82.36  104.22±44.57  1,249.33±1,205.89  64.48±43.15  2,137.500±1,540.48  75.85±18.89  1,307.33±1,309.16  253.27±304.61  
 SSL   0.753   0.309   0.496   0.025   0.390   0.053   0.511   0.350 
  No 18 231.97±109.25  18 188.42±142.03  16 874.44±552.45  19 134.44±157.42  13 1,270.85±532.51  16 597.91±984.00  14 1,904.64±2,237.02  16 1,349.86±2,411.30  
  Yes 214.00±36.47  112.65±22.18  1,430.00±1,423.34  4 44.70±13.82  2,391.33±1,781.31  81.23±19.02  1,113.50±1,153.06  167.98±225.08  
Synchronous CA number 
 SPL 25 −0.031* 0.882 24 −0.256* 0.228 23 0.475* 0.022 25 −0.274* 0.185 16 0.602* 0.014 19 −0.116* 0.637 21 −0.134* 0.562 22 −0.282* 0.204 
 SL 24 −0.162* 0.449 23 −0.245* 0.260 22 0.477* 0.025 24 −0.271* 0.201 15 0.655* 0.008 19 −0.116* 0.637 20 −0.121* 0.612 21 −0.273* 0.231 
 SSL 19 −0.120* 0.625 19 −0.264* 0.274 17 0.493* 0.044 20 −0.272* 0.246 13 0.727* 0.005 16 −0.118* 0.664 15 −0.162* 0.564 17 −0.247* 0.339 
Location 
 SPL   0.538   0.739   0.068   0.382   0.022   0.390   0.726   0.928 
  Right colon 19 254.63±165.58  18 155.18±124.04  18 749.67±379.37  19 91.29±120.16  14 1,068.14±430.38  15 632.67±1,007.77  18 1,574.50±2,021.34  18 1,072.38±2,288.92  
  Transverse colon 197.14±46.55  183.21±145.79  1,476.29±1,128.23  164.36±163.98  2,238.75±1,459.49  96.64±44.70  2,078.00±1,150.33  1,131.48±1,086.42  
  Left colon 170.00±16.97  106.10±39.46  929.00  178.05±155.49  2,396.00  26.00±36.77  2,857.00  499.05±609.46  
 SL   0.666   0.755   0.071   0.401   0.033   0.390   0.764   0.922 
  Right colon 18 229.17±126.43  17 158.72±126.92  17 734.82±385.62  18 91.08±123.64  13 1,106.85±421.83  15 632.67±1,007.77  17 1,622.82±2,071.80  17 1,124.99±2,348.12  
  Transverse colon 197.14±46.55  183.21±145.79  1,476.29±1,128.23  164.36±163.98  2,238.75±1,459.49  96.64±44.70  2,078.00±1,150.33  1,131.48±1,086.42  
  Left colon 170.00±16.97  106.10±39.46  929.00  178.05±155.49  2,396.00  26.00±36.77  2,857.00  499.05±609.46  
 SSL   0.537   0.707   0.101   0.479   0.058   0.387   0.825   0.892 
  Right colon 14 245.53±119.87  14 173.91±133.54  13 730.00±350.15  15 90.97±134.94  11 1,122.18±432.27  13 719.09±1,060.26  13 1,571.92±2,312.86  14 1,259.94±2,579.78  
  Transverse colon 209.00±39.14  199.18±152.85  1,548.67±1,217.98  168.75±179.17  2,238.75±1,459.49  102.50±49.35  1,956.75±1,290.87  908.10±1,114.04  
  Left colon 170.00±16.97  106.10±39.46  929.00  178.05±155.29  2,396.00  26.00±36.77  2,857.00  499.05±609.46  
Adenocarcinoma 
 SPL 
  No 27 216.48±106.59  26 161.27±125.92  25 959.20  27 116.52±135.96  18 1,430.00±888.91  22 455.70±864.96  23 1,775.44±1,859.38  24 1,074.18±2,021.98  
  Yes 713.00  95.10  1,002.00  95.10  565.00   753.00  178.00  
VariableEpithelial L-PHAEpithelial GNAT CD4+ L-PHAT CD4+ GNAT CD8+ L-PHAT CD8+ GNATreg L-PHATreg GNA
Nmeanp valuenmeanp valuenmeanp valuenmeanp valuenmeanp valuenmeanp valuenmeanp valuenmeanp value
Gender 
 SPL   0.308   0.443   0.396   0.014   0.309   0.044   0.040   0.980 
  Male 13 204.62±90.35  13 139.35±86.77  13 830.54±392.51  14 52.65±38.99  10 1,182.60±432.40  12 778.79±1,084.28  13 1,036.99±990.21  13 1,028.22±2,679.55  
  Female 15 259.87±171.95  14 176.89±152.10  13 1,073.85±935.95  14 178.86±164.04  1,608.78±1,204.08  10 67.98±50.69  11 2,556.36±2,269.88  12 1,049.29±886.34  
 SL   0.587   0.386   0.404   0.016   0.195   0.044   0.026   0.907 
  Male 13 204.62±90.35  13 139.35±86.77  13 830.54±392.51  14 52.65±38.99  10 1,182.60±432.40  12 778.79±1,084.28  13 1,036.99±990.21  13 1,028.22±2,679.55  
  Female 14 227.50±122.14  13 183.19±156.41  12 1,079.83±977.31  13 185.30±168.89  1,739.25±1,217.31  10 67.98±50.69  10 2,736.70±2,308.10  11 1,128.50±883.94  
 SSL   0.822   0.369   0.309   0.017   0.119   0.043   0.029   0.958 
  Male 11 223.76±77.05  11 148.80±87.38  11 820.09±322.43  12 44.083±29.48  1,161.11±452.93  11 844.16±1,112.12  11 916.18±813.00  11 1,138.10±2,916.71  
  Female 11 233.64±121.67  11 200.48±164.97  1,187.78±114.37  11 200.37±179.86  1,892.14±1,229.08  8 65.55±57.13  7 3,005.86±2,759.88  1,083.39±918.62  
Age, years 
 SPL 28 0.501* 0.007 27 −0.107* 0.596 26 0.206* 0.314 28 −0.183* 0.352 19 −0.101* 0.682 22 0.136* 0.547 24 0.167* 0.436 25 −0.067* 0.751 
 SL 27 0.422* 0.028 26 −0.080* 0.697 25 0.212* 0.308 27 −0.182* 0.363 18 −0.028* 0.913 22 0.136* 0.547 23 0.218* 0.317 24 −0.041* 0.848 
 SSL 22 0.224* 0.316 22 −0.227* 0.309 20 0.224* 0.343 23 −0.166* 0.449 16 −0.058* 0.830 19 0.104* 0.672 18 0.242* 0.334 20 0.002* 0.993 
Smoking 
 SPL   0.195   0.773   0.281   0.715   0.763   0.629   0.357   0.179 
  No 14 269.07±143.48  14 151.97±105.63  13 1,106.08±922.12  14 106.28±126.00  11 1,439.00±1,050.68  11 263.58±656.57  11 1,349.36±1,043.60  12 475.91±721.11  
  Yes 14 199.26±133.45  13 166.19±145.55  13 798.31±400.64  14 125.23±144.70  1,309.50±657.32  11 547.81±1,058.95  13 2,057.31±2,294.65  13 1,557.49±2,610.63  
 SL   0.397   0.847   0.287   0.737   0.622   0.629   0.420   0.210 
  No 13 234.92±67.93  13 156.35±108.61  12 1,114.75±962.57  13 107.14±131.10  10 1,526.40±1,064.53  11 363.58±656.57  10 1,049.00±1,080.11  11 502.99±749.88  
  Yes 14 199.36±133.45  13 166.19±145.55  13 798.31±400.64  14 125.23±144.70  1,309.50±657.32  11 547.81±1,058.95  13 2,057.31±2,294.65  13 1,557.49±2,610.63  
 SSL   0.783   0.633   0.251   0.783   0.809   0.549   0.410   0.213 
  No 12 223.17±55.45  12 161.98±111.44  11 1,171.46±988.31  12 110.50±136.34  10 1,526.40±1,064.53  10 391.72±685.06  1,317.67±1,103.91  10 489.49±789.03  
  Yes 10 235.34±138.77  10 189.83±157.19  758.33±358.98  11 127.92±163.41  1,405.17±714.84  654.78±1,153.65  2,140.00±2,697.21  10 1,737.47±2,952.34  
BMI, kg/m2 
 SPL 22 −0.061* 0.787 21 −0.410* 0.065 21 −0.012* 0.958 22 −0.397* 0.067 18 0.142* 0.574 16 −0.242* 0.366 19 −0.230* 0.344 19 −0.487* 0.034 
 SL 21 −0.155* 0.502 20 −0.405* 0.076 20 −0.012* 0.959 21 −0.398* 0.074 17 0.161* 0.537 16 −0.242* 0.366 18 −0.224* 0.371 18 −0.484* 0.042 
 SSL 18 −0.275* 0.270 18 −0.390* 0.110 17 −0.021* 0.937 19 −0.392* 0.097 15 0.131* 0.643 15 −0.216* 0.440 15 −0.292* 0.291 16 −0.489* 0.055 
Dimension, mm 
 SPL                 
  <10         
  ≥10 27 216.48±106.59  26 161.27±125.92  25 950.20±728.67  27 116.52±135.96  18 1,430.00±888.91  22 455.70±864.96  23 1,775.44±1,859.38  24 1,074.18±2,021.98  
 SL                 
  <10         
  ≥10 27 216.48±106.59  26 161.27±125.92  25 950.20±728.67  27 116.52±135.96  18 1,430.00±888.91  22 455.70±864.96  23 1,775.44±1,859.38  24 1,074.18±2,021.98  
 SSL                 
  <10         
  ≥10 22 228.70±99.51  22 174.64±131.51  20 985.55±782.82  23 118.83±146.67  16 1,480.94±924.09  19 516.33±918.90  18 1,728.83±2,043.52  20 1,113.48±2,198.53  
Dysplasia 
 SPL         0.312     0.548     
  No 26 213.19±107.30  25 161.48±128.51  24 948.96±744.32  25 124.14±138.46  17 1,437.35±915.70  20 491.86±900.91  22 1,827.23±1,886.08  23 1,119.37±2,054.99  
  Yes 302.00  156.00  980.00  21.20±29.98  1,305.00  94.05±49.43  636.00  34.70  
 SL         0.312     0.548     
  No 26 213.19±107.30  25 161.48±128.51  24 948.96±744.32  25 124.14±138.46  17 1,437.35±915.70  20 491.86±900.91  22 1,827.23±1,886.08  23 1,119.37±2,054.99  
  Yes 302.00  156.00  980.00  21.20±29.98  1,305.00  94.05±49.43  636.00  34.70  
 SSL         0.336     0.508     
  No 21 225.21±100.58  21 175.53±134.69  19 985.84±804.27  21 128.13±150.24  15 1,492.67±955.29  17 566.01±961.70  17 1,793.12±2,087.57  19 1,170.26±2,243.66  
  Yes 302.00  156.00  980.00  21.20±29.98  1,305.00  94.05±49.43  636.00  34.70  
Synchronous CA 
 SPL   0.699   0.171   0.265   0.096   0.433   0.051   0.398   0.219 
  No 21 228.11±111.51  20 178.38±137.86  19 855.74±514.56  21 131.39±150.07  14 1,227.86±536.31  18 540.11±939.40  17 1,940.65±2,026.58  18 1,347.82±2,277.49  
  Yes 252.51±216.53  102.01±40.83  1,214.00±1,104.78  68.86±41.06  1,832.00±1,508.10  75.85±18.89  1,228.14±1,213.32  242.51±279.52  
 SL   0.298   0.212   0.469   0.297   0.325   0.051   0.486   0.260 
  No 21 228.11±111.51  20 178.38±137.86  19 855.74±514.56  21 131.39±150.07  14 1,227.86±536.31  18 540.11±939.40  17 1,940.65±2,026.58  18 1,347.82±2,277.49  
  Yes 175.77±82.36  104.22±44.57  1,249.33±1,205.89  64.48±43.15  2,137.500±1,540.48  75.85±18.89  1,307.33±1,309.16  253.27±304.61  
 SSL   0.753   0.309   0.496   0.025   0.390   0.053   0.511   0.350 
  No 18 231.97±109.25  18 188.42±142.03  16 874.44±552.45  19 134.44±157.42  13 1,270.85±532.51  16 597.91±984.00  14 1,904.64±2,237.02  16 1,349.86±2,411.30  
  Yes 214.00±36.47  112.65±22.18  1,430.00±1,423.34  4 44.70±13.82  2,391.33±1,781.31  81.23±19.02  1,113.50±1,153.06  167.98±225.08  
Synchronous CA number 
 SPL 25 −0.031* 0.882 24 −0.256* 0.228 23 0.475* 0.022 25 −0.274* 0.185 16 0.602* 0.014 19 −0.116* 0.637 21 −0.134* 0.562 22 −0.282* 0.204 
 SL 24 −0.162* 0.449 23 −0.245* 0.260 22 0.477* 0.025 24 −0.271* 0.201 15 0.655* 0.008 19 −0.116* 0.637 20 −0.121* 0.612 21 −0.273* 0.231 
 SSL 19 −0.120* 0.625 19 −0.264* 0.274 17 0.493* 0.044 20 −0.272* 0.246 13 0.727* 0.005 16 −0.118* 0.664 15 −0.162* 0.564 17 −0.247* 0.339 
Location 
 SPL   0.538   0.739   0.068   0.382   0.022   0.390   0.726   0.928 
  Right colon 19 254.63±165.58  18 155.18±124.04  18 749.67±379.37  19 91.29±120.16  14 1,068.14±430.38  15 632.67±1,007.77  18 1,574.50±2,021.34  18 1,072.38±2,288.92  
  Transverse colon 197.14±46.55  183.21±145.79  1,476.29±1,128.23  164.36±163.98  2,238.75±1,459.49  96.64±44.70  2,078.00±1,150.33  1,131.48±1,086.42  
  Left colon 170.00±16.97  106.10±39.46  929.00  178.05±155.49  2,396.00  26.00±36.77  2,857.00  499.05±609.46  
 SL   0.666   0.755   0.071   0.401   0.033   0.390   0.764   0.922 
  Right colon 18 229.17±126.43  17 158.72±126.92  17 734.82±385.62  18 91.08±123.64  13 1,106.85±421.83  15 632.67±1,007.77  17 1,622.82±2,071.80  17 1,124.99±2,348.12  
  Transverse colon 197.14±46.55  183.21±145.79  1,476.29±1,128.23  164.36±163.98  2,238.75±1,459.49  96.64±44.70  2,078.00±1,150.33  1,131.48±1,086.42  
  Left colon 170.00±16.97  106.10±39.46  929.00  178.05±155.49  2,396.00  26.00±36.77  2,857.00  499.05±609.46  
 SSL   0.537   0.707   0.101   0.479   0.058   0.387   0.825   0.892 
  Right colon 14 245.53±119.87  14 173.91±133.54  13 730.00±350.15  15 90.97±134.94  11 1,122.18±432.27  13 719.09±1,060.26  13 1,571.92±2,312.86  14 1,259.94±2,579.78  
  Transverse colon 209.00±39.14  199.18±152.85  1,548.67±1,217.98  168.75±179.17  2,238.75±1,459.49  102.50±49.35  1,956.75±1,290.87  908.10±1,114.04  
  Left colon 170.00±16.97  106.10±39.46  929.00  178.05±155.29  2,396.00  26.00±36.77  2,857.00  499.05±609.46  
Adenocarcinoma 
 SPL 
  No 27 216.48±106.59  26 161.27±125.92  25 959.20  27 116.52±135.96  18 1,430.00±888.91  22 455.70±864.96  23 1,775.44±1,859.38  24 1,074.18±2,021.98  
  Yes 713.00  95.10  1,002.00  95.10  565.00   753.00  178.00  

BMI, body mass index; CA, conventional adenoma; GNA, Glanthus nivalis agglutinin; L-PHA, Phaseolus vulgaris leucoagglutinin; SL, serrated lesion; SPL, serrated pathway lesion; SSL, sessile serrated lesion; r, correlation coefficient; Treg, regulatory T cell; FC, flow cytometry.

*Correlation coefficient.

Table 4.

N-glycosylation profile results obtained by MGAT5 gene RT-qPCR and histochemistry

VariableMGAT5 gene RT-qPCREpithelial L-PHAEpithelial GNAStromal L-PHAStromal GNA
Nmean/r*p valuenmean/r*p valuenmean/r*p valuenmean/r*p valuenmean/r*p value
Gender 
 SPL   0.124   0.338   0.387   0.628   0.943 
  Male 16 3.83 × 10−4±5.97 × 10−4  0.92±0.92  1.17±1.13  0.83±0.52  1.54±1.14  
  Female 17 1.35 × 10−4±1.31 × 10−4  1.42±0.80  0.67±0.75  1.00±0.63  1.58±0.80  
 SL   0.240   0.102   0.677   0.601   0.172 
  Male 14 1.37 × 10−4±1.35 × 10−4  0.88±0.85  0.50±0.41  0.75±0.65  0.81±0.24  
  Female 16 2.39 × 10−4±2.88 × 10−4  1.70±0.45  0.70±0.84  1.00±0.71  1.50±0.87  
 SSL   0.273   0.143   0.725   0.553   0.205 
  Male 14 1.43 × 10−4±1.37 × 10−4  0.83±1.04  0.50±0.50  0.67±0.76  0.75±0.25  
  Female 15 2.39 × 10−4±2.88 × 10−4  1.70±0.45  0.70±0.84  1.00±0.71  1.50±0.87  
Age, years 
 SPL 33 0.139* 0.440 12 −0.361* 0.263 12 −0.261* 0.650 12 0.146* 0.412 12 0.040* 0.901 
 SL 30 0.017* 0.930 −0.454* 0.219 −0.329* 0.310 −0.382* 0.387 −0.479* 0.192 
 SSL 29 0.016* 0.933 −0.451* 0.262 −0.335* 0.354 −0.379* 0.418 −0.476* 0.233 
Smoking 
 SPL   0.878   0.265   0.106   0.780   0.010 
  No 15 2.68 × 10−4±5.95 × 10−4  0.67±1.15  1.84±1.26  1.00±0.00  3 2.67±0.58  
  Yes 18 2.45 × 10−4±2.62 × 10−4  1.33±0.75  0.61±0.65  0.89±0.65  9 1.19±0.73  
 SL   0.038        
  No 12 9.49 × 105±9.82 × 105      
  Yes 18 2.50 × 104±2.62 × 104  1.33±0.75  0.61±0.65  0.89±0.65  1.19±0.73  
 SSL   0.053         
  No 11 9.99 × 10−5±1.01 × 10−4      
  Yes 18 2.45 × 10−4±2.62 × 10−4  1.38±0.79  0.63±0.69  0.88±0.69  1.22±0.77  
BMI, kg/m2 
 SPL 22 0.4320.045 0.458* 0.254 −0.115* 0.516 0.271* 0.786 0.371* 0.445 
 SL 19 0.428* 0.068 0.566* 0.320 −0.115* 0.178 −0.711* 0.853 −0.032* 0.959 
 SSL 18 0.4960.036 0.698* 0.302 −0.154* 0.236 −0.764* 0.846 −0.005* 0.995 
Dimension, mm 
 SPL   0.427         
  <10 4.40 × 10−5±3.96 × 10−6       
  ≥10 29 2.52 × 10−4±4.40 × 10−4  12 1.17±0.86  12 0.92±0.95  12 0.92±0.56  12 1.56±0.94  
 SL   0.001         
  <10 3 4.40 × 105±3.96 × 106      
  ≥10 26 1.70 × 104±1.68 × 104  1.33±0.75  0.61±0.65  0.89±0.65  1.19±0.73  
 SSL   0.316         
  <10 4.61 × 10−5±2.26 × 10−6      
  ≥10 26 1.70 × 10−4±1.68 × 10.4  1.38±0.79  0.63±0.69  0.88±0.69  1.22±0.77  
Dysplasia  
 SPL   0.084   0.755   0.241     0.028 
  No 24 1.75 × 10−4±2.38 × 10−4  1.08±0.80  0.58±0.38  0.92±0.80  6 0.96±0.33  
  Yes 4.70 × 10−4±7.28 × 10−4  1.25±0.99  1.25±1.26  0.92±0.20  6 2.17±0.98  
 SL   0.637   0.170   0.913   0.870   0.800 
  No 24 1.75 × 10−4±2.38 × 10−4  1.08±0.80  0.58±0.38  0.92±0.80  0.96±0.33  
  Yes 2.24 × 10−4±1.51 × 10−4  1.83±0.29  0.67±1.15  0.83±0.29  1.67±1.15  
 SSL   0.683   0.229          
  No 23 1.81 × 10−4±2.42 × 10−4  1.10±0.89  0.60±0.42  0.90±0.89  0.95±0.37  
  Yes 2.24 × 10−4±1.51 × 10−4  1.83±0.29  0.68±1.15  0.83±0.29  1.67±1.15  
Synchronous CA 
 SPL   0.646   0.265   0.406   0.780   0.010 
  No 23 2.79 × 10−4±5.15 × 10−4  1.33±0.87  1.06±1.04  0.94±0.53  9 1.83±0.94  
  Yes 10 2.02 × 10−4±1.60 × 10−4  0.67±0.76  0.50±0.50  0.83±0.76  3 0.75±0.25  
 SL   0.778   0.208   0.743   0.870   0.214 
  No 20 1.76 × 10−4±2.51 × 10−4  1.67±0.52  0.67±0.75  0.92±0.66  1.42±0.80  
  Yes 10 2.01 × 10−4±1.60 × 10−4  0.67±0.76  0.50±0.50  0.83±0.76  0.75±0.25  
 SSL   0.845   0.035   0.725   0.907   0.205 
  No 19 1.84 × 10−4±2.56 × 10−4  5 1.80±0.45  0.70±0.84  0.90±0.74  1.50±0.87  
  Yes 10 2.01 × 10−4±1.60 × 10−4  3 0.68±0.76  0.50±0.50  0.83±0.76  0.75±0.25  
Synchronous CA number 
 SPL 27 −0.074* 0.713 10 −0.308* 0.387 10 −0.551* 0.621 10 −0.179* 0.099 10 −0.501* 0.140 
 SL 25 0.037* 0.861 −0.607* 0.110 −0.569* 0.770 −0.124* 0.141 −0.481* 0.227 
 SSL 25 0.037* 0.861 −0.658* 0.108 −0.583* 0.742 −0.154* 0.170 −0.5220.230 
Location 
 SPL 33  0.332 12  0.567 12  0.009 12  0.855 12  0.039 
  Right colon 20 1.90 × 10−4±2.29 × 10−4  1.50±0.87  5 0.80±0.84  1.00±0.35  5 1.35±0.93  
  Transverse colon 2.35 × 10−4±2.42 × 10−4  0.90±0.74  5 0.40±0.22  0.80±0.84  5 1.20±0.57  
  Left colon 4.96 × 10−4±9.31 × 10−4  1.00±1.41  2 2.50±0.71  1.00±0.00  2 3.00±0.00  
 SL   0.362         
  Right colon 20 1.90 × 10−4±2.29 × 10−4  1.50±0.87  0.80±0.84  1.00±0.35  1.35±0.93  
  Transverse colon 2.55 × 10−4±2.59 × 10−4  1.13±0.63  0.38±0.25  0.75±0.96  1.00±0.41  
  Left colon 4.95 × 10−5±4.61 × 10−6      
 SSL   0.362         
  Right colon 19 1.98 × 10−4±2.32 × 10−4  1.50±0.87  0.80±0.84  1.00±0.35  1.35±0.93  
  Transverse colon 2.55 × 10−4±2.59 × 10−4  1.17±0.76  0.33±0.29  0.67±1.15  1.00±0.50  
  Left colon 4.50 × 10−5±4.61 × 10.5      
Adenocarcinoma 
 SPL   0.388   0.265   0.106   0.780   0.010 
  No 30 1.85 × 10−4±2.22 × 10−4  1.33±0.75  0.61±0.65  0.89±0.65  9 1.19±0.73  
  Yes 9.63 × 10−4±1.23 × 10−3  0.67±1.15  1.83±1.26  1.00±0.00  3 2.67±0.58  
VariableMGAT5 gene RT-qPCREpithelial L-PHAEpithelial GNAStromal L-PHAStromal GNA
Nmean/r*p valuenmean/r*p valuenmean/r*p valuenmean/r*p valuenmean/r*p value
Gender 
 SPL   0.124   0.338   0.387   0.628   0.943 
  Male 16 3.83 × 10−4±5.97 × 10−4  0.92±0.92  1.17±1.13  0.83±0.52  1.54±1.14  
  Female 17 1.35 × 10−4±1.31 × 10−4  1.42±0.80  0.67±0.75  1.00±0.63  1.58±0.80  
 SL   0.240   0.102   0.677   0.601   0.172 
  Male 14 1.37 × 10−4±1.35 × 10−4  0.88±0.85  0.50±0.41  0.75±0.65  0.81±0.24  
  Female 16 2.39 × 10−4±2.88 × 10−4  1.70±0.45  0.70±0.84  1.00±0.71  1.50±0.87  
 SSL   0.273   0.143   0.725   0.553   0.205 
  Male 14 1.43 × 10−4±1.37 × 10−4  0.83±1.04  0.50±0.50  0.67±0.76  0.75±0.25  
  Female 15 2.39 × 10−4±2.88 × 10−4  1.70±0.45  0.70±0.84  1.00±0.71  1.50±0.87  
Age, years 
 SPL 33 0.139* 0.440 12 −0.361* 0.263 12 −0.261* 0.650 12 0.146* 0.412 12 0.040* 0.901 
 SL 30 0.017* 0.930 −0.454* 0.219 −0.329* 0.310 −0.382* 0.387 −0.479* 0.192 
 SSL 29 0.016* 0.933 −0.451* 0.262 −0.335* 0.354 −0.379* 0.418 −0.476* 0.233 
Smoking 
 SPL   0.878   0.265   0.106   0.780   0.010 
  No 15 2.68 × 10−4±5.95 × 10−4  0.67±1.15  1.84±1.26  1.00±0.00  3 2.67±0.58  
  Yes 18 2.45 × 10−4±2.62 × 10−4  1.33±0.75  0.61±0.65  0.89±0.65  9 1.19±0.73  
 SL   0.038        
  No 12 9.49 × 105±9.82 × 105      
  Yes 18 2.50 × 104±2.62 × 104  1.33±0.75  0.61±0.65  0.89±0.65  1.19±0.73  
 SSL   0.053         
  No 11 9.99 × 10−5±1.01 × 10−4      
  Yes 18 2.45 × 10−4±2.62 × 10−4  1.38±0.79  0.63±0.69  0.88±0.69  1.22±0.77  
BMI, kg/m2 
 SPL 22 0.4320.045 0.458* 0.254 −0.115* 0.516 0.271* 0.786 0.371* 0.445 
 SL 19 0.428* 0.068 0.566* 0.320 −0.115* 0.178 −0.711* 0.853 −0.032* 0.959 
 SSL 18 0.4960.036 0.698* 0.302 −0.154* 0.236 −0.764* 0.846 −0.005* 0.995 
Dimension, mm 
 SPL   0.427         
  <10 4.40 × 10−5±3.96 × 10−6       
  ≥10 29 2.52 × 10−4±4.40 × 10−4  12 1.17±0.86  12 0.92±0.95  12 0.92±0.56  12 1.56±0.94  
 SL   0.001         
  <10 3 4.40 × 105±3.96 × 106      
  ≥10 26 1.70 × 104±1.68 × 104  1.33±0.75  0.61±0.65  0.89±0.65  1.19±0.73  
 SSL   0.316         
  <10 4.61 × 10−5±2.26 × 10−6      
  ≥10 26 1.70 × 10−4±1.68 × 10.4  1.38±0.79  0.63±0.69  0.88±0.69  1.22±0.77  
Dysplasia  
 SPL   0.084   0.755   0.241     0.028 
  No 24 1.75 × 10−4±2.38 × 10−4  1.08±0.80  0.58±0.38  0.92±0.80  6 0.96±0.33  
  Yes 4.70 × 10−4±7.28 × 10−4  1.25±0.99  1.25±1.26  0.92±0.20  6 2.17±0.98  
 SL   0.637   0.170   0.913   0.870   0.800 
  No 24 1.75 × 10−4±2.38 × 10−4  1.08±0.80  0.58±0.38  0.92±0.80  0.96±0.33  
  Yes 2.24 × 10−4±1.51 × 10−4  1.83±0.29  0.67±1.15  0.83±0.29  1.67±1.15  
 SSL   0.683   0.229          
  No 23 1.81 × 10−4±2.42 × 10−4  1.10±0.89  0.60±0.42  0.90±0.89  0.95±0.37  
  Yes 2.24 × 10−4±1.51 × 10−4  1.83±0.29  0.68±1.15  0.83±0.29  1.67±1.15  
Synchronous CA 
 SPL   0.646   0.265   0.406   0.780   0.010 
  No 23 2.79 × 10−4±5.15 × 10−4  1.33±0.87  1.06±1.04  0.94±0.53  9 1.83±0.94  
  Yes 10 2.02 × 10−4±1.60 × 10−4  0.67±0.76  0.50±0.50  0.83±0.76  3 0.75±0.25  
 SL   0.778   0.208   0.743   0.870   0.214 
  No 20 1.76 × 10−4±2.51 × 10−4  1.67±0.52  0.67±0.75  0.92±0.66  1.42±0.80  
  Yes 10 2.01 × 10−4±1.60 × 10−4  0.67±0.76  0.50±0.50  0.83±0.76  0.75±0.25  
 SSL   0.845   0.035   0.725   0.907   0.205 
  No 19 1.84 × 10−4±2.56 × 10−4  5 1.80±0.45  0.70±0.84  0.90±0.74  1.50±0.87  
  Yes 10 2.01 × 10−4±1.60 × 10−4  3 0.68±0.76  0.50±0.50  0.83±0.76  0.75±0.25  
Synchronous CA number 
 SPL 27 −0.074* 0.713 10 −0.308* 0.387 10 −0.551* 0.621 10 −0.179* 0.099 10 −0.501* 0.140 
 SL 25 0.037* 0.861 −0.607* 0.110 −0.569* 0.770 −0.124* 0.141 −0.481* 0.227 
 SSL 25 0.037* 0.861 −0.658* 0.108 −0.583* 0.742 −0.154* 0.170 −0.5220.230 
Location 
 SPL 33  0.332 12  0.567 12  0.009 12  0.855 12  0.039 
  Right colon 20 1.90 × 10−4±2.29 × 10−4  1.50±0.87  5 0.80±0.84  1.00±0.35  5 1.35±0.93  
  Transverse colon 2.35 × 10−4±2.42 × 10−4  0.90±0.74  5 0.40±0.22  0.80±0.84  5 1.20±0.57  
  Left colon 4.96 × 10−4±9.31 × 10−4  1.00±1.41  2 2.50±0.71  1.00±0.00  2 3.00±0.00  
 SL   0.362         
  Right colon 20 1.90 × 10−4±2.29 × 10−4  1.50±0.87  0.80±0.84  1.00±0.35  1.35±0.93  
  Transverse colon 2.55 × 10−4±2.59 × 10−4  1.13±0.63  0.38±0.25  0.75±0.96  1.00±0.41  
  Left colon 4.95 × 10−5±4.61 × 10−6      
 SSL   0.362         
  Right colon 19 1.98 × 10−4±2.32 × 10−4  1.50±0.87  0.80±0.84  1.00±0.35  1.35±0.93  
  Transverse colon 2.55 × 10−4±2.59 × 10−4  1.17±0.76  0.33±0.29  0.67±1.15  1.00±0.50  
  Left colon 4.50 × 10−5±4.61 × 10.5      
Adenocarcinoma 
 SPL   0.388   0.265   0.106   0.780   0.010 
  No 30 1.85 × 10−4±2.22 × 10−4  1.33±0.75  0.61±0.65  0.89±0.65  9 1.19±0.73  
  Yes 9.63 × 10−4±1.23 × 10−3  0.67±1.15  1.83±1.26  1.00±0.00  3 2.67±0.58  

BMI, body mass index; CA, conventional adenoma; GNA, Glanthus nivalis agglutinin; L-PHA, Phaseolus vulgaris leucoagglutinin; SL, serrated lesion; SPL, serrated pathway lesion; SSL, sessile serrated lesion; r, correlation coefficient; RT-PCR, reverse transcriptase-quantitative polymerase chain reaction.

*Correlation coefficient.

Altered Immune N-Glycosylation Profile Is Correlated with Increased Synchronous CA, Sex, Smoking, Location of the Lesion, and BMI in the Serrated Pathway

The N-glycosylation profile of immune cells was correlated with potential risk factors for disease progression in the serrated pathway. Our results demonstrated a positive correlation between the synchronous CA number in patients with SPL, SL, and SSL and β1,6-GlcNAc branched N-glycans expression in CD4+ T cells (p = 0.022, r = 0.475; p = 0.025, r = 0.477; p = 0.044, r = 0.493) and CD8+ T cells (p = 0.014, r = 0.602; p = 0.008, r = 0.655; p = 0.005, r = 0.727) (Table 3). Furthermore, synchronous CA in patients with SPL was associated with a lower high-mannose N-glycans expression in the stromal cells of SPL (0.75 ± 0.25 vs. 1.83 ± 0.94; p = 0.010) and specifically in CD4+ T cells in patients with SSL (44.70 ± 13.82 vs. 134.44 ± 157.42; p = 0.025) (Tables 3, 4). Furthermore, smokers showed, on average, a lower high-mannose N-glycans expression in the SPL stromal cells, when compared with non-smokers (1.19 ± 0.73 vs. 2.67 ± 0.58; p = 0.010) (Table 4). Regarding the BMI, we observed an inverse relationship between this factor and the expression of high-mannose N-glycans in Tregs in SPL and SL (p = 0.034, r = −0.487; p = 0.042, r = −0.484) (Table 3). Additionally, females presented, on average, a higher high-mannose N-glycans expression in CD4+ T cells in SPL (178.86 ± 164.04 vs. 67.98 ± 50.69; p = 0.020), in SSL (299.87 ± 179.86 vs. 44.08 ± 29.48; p = 0.017), and in SL (178.86 ± 164.04 vs. 52.65 ± 38.99; p = 0.016) comparing to males (Table 3). Moreover, females showed, on average, a higher β1,6-GlcNAc branched N-glycans expression on Tregs in SPL (2,556.36 ± 2,269.88 vs. 1,036.99 ± 990.22; p = 0.040), in SL (2,736.70 ± 2,308.10 vs. 1,036.99 ± 990.21; p = 0.026), and in SSL (3,005.86 ± 2,759.88 vs. 916.18 ± 813.00; p = 0.029), comparing with males (Table 3). On the other hand, males presented, on average, a higher high-mannose N-glycans expression in CD8+ T cells in SPL (778.79 ± 1,084.28 vs. 67.98 ± 50.69; p = 0.044), in SSL (884.16 ± 1,112.12 vs. 65.55 ± 57.13; p = 0.043), and in SL (778.86 ± 1,084.28 vs. 67.78 ± 50.69; p = 0.044) (Table 3). Furthermore, left colon lesions presented a higher high-mannose N-glycans expression in stromal cells compared to the transverse colon (3.00 ± 0.00 vs. 1.20 ± 0.57; p = 0.039) (Table 4). SPL with dysplasia showed a higher high-mannose N-glycans expression in stromal cells (2.17 ± 0.98 vs. 0.96 ± 0.33; p = 0.028), comparing with non-dysplastic SPL (Table 4). Also, in serrated pathway adenocarcinomas and SSL-D with concomitant adenocarcinoma a higher high-mannose N-glycans expression on stroma was observed on average (2.67 ± 0.80 vs. 1.19 ± 0.73; p = 0.010) (Table 4).

SPL follow-up still raises serious concerns due to rapid progression from dysplasia to cancer. Moreover, there are few robust studies of clinical progression risk factors on serrated pathway. The changes in N-glycosylation has been considered as a CRC progression hallmark in epithelial cells [11]. Thus, the main goal of our study was to define potential risk factors for progression to malignancy by analyzing the N-glycosylation profile of the serrated pathway.

We found an increased β1,6-GlcNAc branched N-glycans expression in the SPL epithelial component correlated with increasing age and BMI. In SL, β1,6-GlcNAc branched N-glycans expression in the epithelial component is also correlated with smoking and polyp dimensions ≥10 mm. Previously, our group showed that these types of glycans present an aberrant expression in CRC and are direct immune modulators in the tumor microenvironment, allowing immune evasion [14]. Thus, these clinical variables may intervene as risk factors for the progression to malignancy in the serrated pathway by enabling identification of immunological escape in these lesions. In fact, age is a known risk factor for the CRC development and progression, with immunosenescence potentially playing an important role in the immune escape suggested in our results [1]. Smoking is a studied and validated risk factor for SL development and progression to CRC [17]. This evidence is in line with our results, emphasizing smoking cessation as a method of preventing progression in the serrated pathway. Additionally, BMI has been described in the literature as a possible risk factor for CRC progression [19]. However, it is not fully understood whether it impacts the adenoma-carcinoma pathway or the serrated pathway [19]. Our findings suggest that BMI may contribute to progression to malignancy in the serrated pathway by upregulating β1,6-GlcNAc branched N-glycans in epithelial cells. Thereby, weight loss should be encouraged in individuals with SPL in an attempt to prevent progression to malignancy. Regarding SL dimensions, our results suggest that polyps with dimensions ≥10 mm have greater risk of progression to malignancy, by overexpression of β1,6-GlcNAc branched N-glycans in epithelial cells. This result is in line with the European guideline for post-polypectomy colonoscopy follow-up, which defined a cut-off of 10 mm to perform a shorter interval follow-up [20].

Regarding the immune compartment, it was observed a higher β1,6-GlcNAc branched N-glycans expression with the increasing number of synchronous CA in patients with SPL. Previously, our group had shown that an increasing β1,6-GlcNAc branched N-glycans expression in T cells, by GlcNAc supplementation, controls T-cell immune response in inflammatory bowel disease, by increasing its threshold of activation [16]. Thus, T-cell β1,6-GlcNAc branched N-glycosylation seems to create an immunosuppressive environment disabling T-cell activation and function in SPL, promoting their growth. This immunosuppression promotes the development of more synchronous CA. Therefore, a higher synchronous CA number seems to be a risk factor for progression to malignancy in the serrated pathway. Synchronous CA number has not been described in the literature as a risk factor for development or progression to malignancy in the serrated pathway yet. Thereby, the anatomopathological identification of the N-glycosylation pattern in the presence of a high synchronous CA number can select patients that would benefit from a shorter interval of follow-up, to reduce interval cancers. However, we did not distinguish the dysplasia type of CA (high-grade vs. low-grade dysplasia), which may limit these conclusions.

According to our data, smoking and synchronous CA presence seem to be risk factors for progression to CRC in the serrated pathway, since they are correlated with high-mannose N-glycans downregulation on T cells, suggesting decreased immune function in SPL. Furthermore, it seems that the lower immune system activation by downregulation of high-mannose N-glycans enables the synchronous CA development. In fact, Li et al. [21] verified that the synchronous CA presence in patients with SL confers an increased risk of developing CRC. Regarding this, our results are in accordance with the literature, emphasizing N-glycosylation as a new potential biomarker for malignancy progression in serrated pathway. Contrariwise, we also showed an increased stromal high-mannose N-glycans expression in the presence of dysplasia and adenocarcinoma with concomitant SSL-D and in the left colon lesions. These findings suggest that the immune system is more active both in the presence of dysplasia and adenocarcinoma of the serrated pathway, contradicting what we expected. We predicted a reduced high-mannose N-glycans in immune cells related with T cells inability to recognize neoplastic lesions. Thereby, we need further investigations with a larger sample size to clarify these results. Regarding SPL location, left colon lesions appear to have a more active immunological profile, by having a higher high-mannose N-glycans expression, suggesting that this location is less likely to progress to CRC. In fact, SPL is more frequent in the right colon [22]. These results have a follow-up impact, considering the low potential for progression of a left colon lesion. Therefore, the N-glycosylation profile in colonic immune compartment could be used to guide clinicians on follow-up decision.

As previously mentioned, we found an association between the β1,6-GlcNAc branched N-glycans in epithelial component and a high BMI. An increased BMI was also associated with a reduced high-mannose N-glycans expression on Tregs. These results suggest that Tregs might have an increased immunosuppressive capacity in the presence of elevated BMI, creating an immunosuppressive microenvironment, which contributes to progression to malignancy. Thus, as emphasized earlier, patients with higher BMI may be considered for a closer surveillance of SPL.

Regarding gender, a different N-glycosylation pattern was observed in different immune system cells. In fact, β1,6-GlcNAc N-glycosylation in different genders has never been studied before. Our results showed a higher high-mannose N-glycosylation expression in CD4+ T cells in females and in CD8+ T cells in males in all serrated pathway, highlighting that men and women have higher activation of different T cells. Both CD8+ and CD4+ T cells play a role in tumor eradication by direct action (CD8+ T cells) and cytokine release (CD4+ T cells) [12]. Despite this, CD8+ T cells infiltration in tumor microenvironment is correlated with a better prognosis in CRC, suggesting that men have lower progression to cancer in serrated pathway [23]. By opposition, Tregs in females have a higher β1,6-GlcNAc branched N-glycans expression creating an immunosuppressive environment that allows the progression to malignancy. Regarding this, our results suggest a different N-glycosylation pattern of immune system cells occurring in both genders, which demands further investigation.

To conclude, biomarkers are an essential tool in current medical practice, playing an important role on understanding and identifying several diseases. Our study showed that N-glycosylation could be a potential biomarker of tumor progression in the serrated pathway. This study set the ground for the potential inclusion of β1,6-GlcNAc branched N-glycans and high-mannose N-glycans in the SPL anatomopathological analysis to select those patients who need shorter intervals of follow-up to reduce the interval cancers incidence. Furthermore, according to N-glycosylation profile, we identified smoking, aging, elevated BMI, SL dimensions ≥10 mm, the presence and number of synchronous CA as risk factors to progression to malignancy. These associations allow directed clinical interventions based on risk factors to reduce the progression to malignancy. Taken together, N-glycosylation profile seems to be one key to solve this puzzling pathway with large impact in clinical and molecular research. Despite our results, this study has several limitations, namely, the small sample size, the high missing data value, and the lack of similar articles that prevent us to draw more conclusions.

This study protocol was reviewed and approved by Departamento de Ensino, Formação e Investigação (DEFI), and ethical committee of Centro Hospitalar Universitário de Santo António, number 2021.306 (252-DEFI/260-CE). Informed consent was not required, decided by ethical committee of Centro Hospitalar Universitário de Santo António.

The authors have no conflicts of interest to declare.

This article had the funding support of Liga Portuguesa Contra o Cancro (LPCC) and MultiCare (Grant/Award No. C 3035006C17). This work was also funded by Raquel Seruca Porto Comprehensive Cancer Centre (P.CCC). Catarina M. Azevedo thanks Fundação para a Ciência e Tecnologia (FCT) for a fellowship (2021.07357.BD).

Henrique Fernandes-Mendes: conceptualization; methodology; investigation; formal analysis; and writing – original draft; Catarina M. Azevedo: conceptualization; methodology; investigation; and writing – review and editing. Mónica Garrido: conceptualization and writing – review and editing. Carolina Lemos: methodology and formal analysis. Isabel Pedroto: supervision. Salomé S. Pinho: writing – review and editing and supervision. Ricardo Marcos-Pinto and Ângela Fernandes: conceptualization; methodology; investigation; formal analysis; writing – review and editing; and supervision. All authors approved the final version of the manuscript.

Additional Information

Ricardo Marcos-Pinto and Ângela Fernandes share last authorship.

This article was based on a final master’s thesis of medical training, which will be published on Repositório da Universidade do Porto on June 28, 2023.

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