1,6)Fucosyltransferase expression is an independent prognostic factor for disease-free survival in colorectal carcinoma

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Human Pathology (2011) 42, 1740–1750


Original contribution

α(1,6)Fucosyltransferase expression is an independent prognostic factor for disease-free survival in colorectal carcinoma☆ L. Muinelo-Romay PhD a , S. Villar-Portela PhD a , E. Cuevas Alvarez MD, PhD b , E. Gil-Martín PhD a,1 , Almudena Fernández-Briera PhD a,⁎,1 a

Department of Biochemistry, Genetics and Immunology, Faculty of Biology, University of Vigo, Spain Pathology Service, University Complex Hospital of Ourense, Spain


Received 22 November 2010; revised 23 January 2011; accepted 28 January 2011

Keywords: Colorectal cancer; Glycosylation; Core-fucosylation; α(1,6)Fucosyltransferase; Tumor biomarker; Prognostic factor

Abstract We previously reported that α(1,6)fucosyltransferase (Enzyme class activity and expression are increased in colorectal cancer, suggesting a role for this enzyme in tumor development and progression. However, the possible impact of α(1,6)fucosyltransferase activity or expression on clinical outcomes in colorectal cancer patients has never been studied. Thus, the present study was conducted to determine the value of α(1,6)fucosyltransferase as a prognostic factor for colorectal cancer. α(1,6)Fucosyltransferase expression was analyzed using immunohistochemistry in 141 colorectal tumors, and α(1,6)fucosyltransferase activity was determined in 39 tumors. A complete standardized follow-up of patients was documented until the end of the observation period of 5 years or patient death. Univariate analysis demonstrated the absence of a correlation between enzyme activity and disease evolution. However, in patients with moderate or strong α(1,6)fucosyltransferase expression, a significant decrease in the overall (P = .04) and disease-free (P = .03) survival rates was observed. In addition, when local and distant disease recurrence were considered separately, enzyme expression was found to correlate with local tumor recurrences (P = .01). Furthermore, multivariate analysis showed that α(1,6)fucosyltransferase expression has independent value for predicting tumor recurrences and, specifically, local recurrences. These findings suggest that α(1,6) fucosyltransferase expression may be a good indicator of poor prognosis in colorectal cancer and, therefore, a helpful tool to choose the most effective treatment. © 2011 Elsevier Inc. All rights reserved.

1. Introduction ☆

This project was partially supported by the “Xunta de Galicia” Grant INCITE08PXIB310249PR. ⁎ Corresponding author. Almudena Fernández-Briera, Department of Biochemistry, Genetics and Immunology, Faculty of Biology, University of Vigo, Campus As Lagoas-Marcosende, 36310, Vigo, Spain. E-mail address: [email protected] (A. Fernández-Briera). 1 These authors contributed equally to this work. 0046-8177/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2011.01.021

Colorectal carcinoma (CRC) is one of the most frequent tumors in the Western world. Despite the fact that survival rates have improved over the last decades, nearly one half of patients with CRC develop local recurrences or distant metastasis after surgery [1,2]. Therefore, the identification of new biomolecules implicated in tumor progression could

α(1,6)FT expression is a prognostic factor for CRC be helpful for recognizing patients at high risk for relapse and to select adequate postoperative therapy. We have previously reported that α(1,6)fucosyltransferase [α(1,6)FT (Enzyme class] activity and expression is increased in CRC, suggesting a role for this enzyme in tumor development and progression [3]. Similar results have been described for other human malignant processes, such as hepatocellular carcinoma [4,5], thyroid papillary carcinoma [6], and ovarian adenocarcinoma [7]. The α(1,6)FT enzyme catalyzes the transfer of fucose from guanosine diphosphate (GDP)–fucose to the innermost GlcNAc of hybrid and complex N-linked oligosaccharides via an α(1,6)linkage, resulting in core-fucosylated glycoproteins [8]. This catalytic activity constitutes a form of posttranslational modification, because it modulates the steric conformational properties of the core-fucosylated glucide antenna and, consequently, the action of other FTs on carbohydrate side chains and the biological function of resulting glycoproteins [9]. In support of this, several studies have demonstrated an important role for α(1,6) fucosylation in regulating the activity of proteins strongly implicated in tumor growth (eg, epidermal growth factor receptor [EGFR], transforming growth factor receptor β1 [TGFR-β1], and vascular endothelial growth factor receptor 2 [VEGFR-2]) [10-12] and tumor dissemination (E-cadherin [13,14], α3β1, and α5β1 integrins [5,15]). Interestingly, inhibition of these proteins is currently a therapeutic strategy against several different neoplasias [16,17]. Consequently, the response of tumor cells to these new treatments could be partially regulated by the action of the α(1,6)FT enzyme [18]. In addition, increased expression levels of α(1,6)fucosylated glycoproteins, coupled with an enhancement of α(1,6) FT activity, have been described in a wide variety of human tumors, suggesting their potential usefulness as biomarkers. The most representative case is the α(1,6)fucosylated α-fetoprotein, which is used as a specific tumor marker for hepatocellular carcinoma, whereas other unusually corefucosylated serum glycoproteins, such as haptoglobin or Gp73, have recently been proposed as potential diagnostic markers for this malignancy [19,20]. Although many studies have reported an association between altered α(1,6)FT status [3,6,21] or its catalytic products [13,22] and the clinicopathologic parameters of advanced and aggressive tumors, to our knowledge, the influence of α(1,6)FT on tumor progression remains unknown. In fact, only one recent report has analyzed the prognostic value of α(1,6)FT activity for predicting the response of metastatic breast cancer patients treated with the anti-EGFR antibody, trastuzumab, with negative results [18]. In this context, the present study was undertaken to assess the possible impact of α(1,6)FT activity and expression on clinical outcomes (local recurrence, distant metastasis, disease-free, and overall survival rates) of curatively CRC resected patients, in an attempt to clarify the real value of α(1,6)FT as a prognostic factor for this malignant tumor and, therefore, its utility in helping to select the most effective treatment strategy.


2. Materials and methods 2.1. Patients and specimens A total of 141 tumor colorectal tissue samples were obtained from CRC patients who underwent curative surgery (1995-2004) at the University Complex Hospital of Ourense (Ourense, Spain), after receiving approval from the appropriate local institutional review board (Comité Ético de Investigación Clínica de Galicia, Spain). Sixty-one women and 80 men were included in the present study, with a mean age at diagnosis of 71.65 ± 0.77 years. Patients with metastasis at the time of surgery or who died within 30 days postoperation were excluded from this study. For all 141 patients, α(1,6)FT expression was determined by immunohistochemistry, whereas α(1,6)FT activity was measured for 39 cases. Specimens used for immunohistochemical analysis were fixed in formalin (10%) and embedded in paraffin (60°C). Specimens used for α(1,6) FT assays were washed with ice-cold saline buffer and stored frozen at −85°C until use. Tumors were macroscopically and microscopically examined to determine their location, size, and growth, as well as the histologic type and tumor staging based on Dukes [23] and TNM [24] classifications, respectively. For all analyzed patients, a complete standardized follow-up was documented until the end of the observation period (60 months) or patient death. Follow-up information included local and distant metastasis (determined by x-ray, computed tomography scan, or endoscopy biopsy), as well as patient death.

2.2. Immunohistochemistry Immunohistochemistry was performed as previously described [3,6]. Tissue sections (2-3 μm) were deparaffinized in xylene and rehydrated in a graded ethanol series (100°, 96°, and 70°). To quench endogenous peroxidase activity, sections were incubated with 0.5% (vol/vol) hydrogen peroxide in methanol. After rinsing with phosphate-buffered saline (PBS), bovine serum was applied to block nonspecific binding of the antibody. Sections were then incubated with primary antibody (dilution 1:40, antihuman α(1,6)FT, 15C6; Fujirebio Corp, Tokyo, Japan) at 4°C overnight. After rinsing with PBS, sections were incubated with secondary antibody bound to horseradish peroxidase; horseradish peroxidase activity was visualized by incubating with 3,3-diaminobenzidine. Finally, after a wash in water, sections were counterstained with hematoxylin, dehydrated in a graded ethanol series (70°, 96°, and 100°), washed in xylene, and mounted on a glass slide. Negative controls were performed using PBS instead of primary antibody. Semiquantitative staining analysis was performed by a pathologist from the University Complex Hospital of Ourense (Ourense, Spain). The immunostaining


L. Muinelo-Romay et al. 2.3.2. Enzyme activity assays Standard α(1,6)FT activity assays were performed as previously described [3], using 100 μmol/L donor substrate [0.5 μmol/L GDP-L-[14C]-fucose (Amersham Bioscience Europe GMBH, Uppsala, Sweden; 270-283 mCi/mmol), 99.5 μmol/L GDP-L-fucose] and 0.32 mg acceptor substrate (asialoagalactofetuin). The radioactivity incorporated in the proteins was measured in a Wallac 1409-12 scintillator system (Pegasus Scientific Inc., Rockville, MD, USA), using Ecoscint H as a scintillation counting mixture (National Diagnostics, Atlanta, GA, USA). Enzyme activity was expressed as microunits per milligram of protein = picomoles of fucose incorporated in asialoagalactofetuin per minute per milligram of protein.

2.4. Statistical analyses

Fig. 1 Immunohistochemical analysis of α(1,6)FT expression in healthy (A) and cancerous (B) colorectal mucosa. The signal was clearly located in the cytoplasm of the epithelial cells.

Statistical analyses were performed using SPSS, version 15.00, for Windows XP (SPSS, Chicago, IL, USA). χ2 and Fisher exact probability tests were used to evaluate the differences between categorical data. For continuous data, we used the Mann-Whitney U test and the Kruskal-Wallis test. Survival time was calculated as the number of months postsurgery to the final event (local recurrence, distant metastasis, or death). Data from patients who were alive without recurrence at the end of the 60 months of evaluation were censored. Survival curves were calculated by the Kaplan-Meier method, and the significance of differences was estimated by the log-rank test. Finally, multivariate analysis was performed using Cox regression model. Results were considered statistically significant when P b .05.

3. Results expression pattern was classified as follows: 0, no staining; 1, weak, less than 10% staining; 2, moderate, 10% to 50%, and 3, strong, more than 50% of the tissue was stained.

2.3. Determination of α(1,6)FT activity 2.3.1. Preparation of samples for enzyme activity assays Colorectal tissues were homogenized in 6 volumes of 0.01 mol/L Tris-HCl buffer (pH 7.4), containing 0.25 mol/L sucrose. The homogenate was centrifuged for 10 minutes at 500×g, at 4°C. The resulting supernatant was centrifuged at 33 000×g for 60 minutes, at 4°C. Subsequently, the pellet was resuspended in 1.5 mL of 0.01 mol/L Tris -HCl buffer (pH 7.4) and centrifuged at 145 000×g for 45 minutes, at 4°C. The final pellet, containing the total cell membrane fraction, was resuspended in 300 μL of 0.01 mol/L Tris-HCl buffer (pH 7.4) and stored at −20°C until use in enzymatic assays. Total protein content in the final membrane preparation was determined using the Bicinchoninic acid assay, with bovine serum albumin as a standard.

3.1. Association of α(1,6)FT activity and immunohistochemical expression with clinicopathologic parameters Expression of α(1,6)FT, as evidenced by immunohistochemical brown staining clearly located in the cytoplasm of epithelial cells (Fig. 1), was observed in 84 (59.57%) of the 141 CRC patients analyzed. Of these, the intensity of staining, and therefore α(1,6)FT expression, was weak in 38 (26.9%) cases, moderate in 31 (22%) cases, and strong in 15 (10.67%) cases. After correlation analysis (Table 1), no statistically significant differences between α(1,6)FT expression and clinicopathologic features such as age, tumor size, localization, pattern growth, differentiation, or lymph node infiltration were found. Nevertheless, the percentage of cases with moderate (2) or strong (3) α(1,6)FT expression was higher in women than in men (P = .05). In addition, α(1,6)FT expression was more frequent in patients with advanced Dukes stage (P = .05) tumors or higher infiltration rates into

α(1,6)FT expression is a prognostic factor for CRC

1743 T3 stage tumors (TNM classification), with a mean activity of 54.1 ± 10.9 μU/mg, in comparison with earlier stage tumors (Tis, T1, and T2) with 33.1 ± 5.8 μU/mg. Finally, we evaluated the possible correlation between α(1,6)FT immunohistochemical expression and activity in 39 patients. For this purpose, 2 groups of enzyme activity were established (b28.6 μU/mg, low activity; N28.6 μU/mg, high activity) and compared with low (0, 1) and high (2, 3) immunohistochemical expression groups. No correlation was found between them (data not shown).

the intestinal wall (P = .07; Table 1), although for the latter, this difference was not statistically significant. In contrast, correlation analysis between α(1,6)FT activity and standard clinicopathologic features considered in our study revealed a significant decrease in enzyme activity levels in tumors with nonpolypoid growth. Specifically, the mean of α(1,6)FT activity was 24.6 ± 4.1 μU/mg in nonpolypoid tumors and 57.9 ± 10.0 μU/mg in polypoid tumors (P = .001, by Mann-Whitney U test). In advanced Dukes stage B and C tumors, the mean of α(1,6)FT activity was 33.1 ± 5.7 and 36.0 ± 10.0 μU/mg, respectively. Interestingly, stage A tumors showed higher α(1,6)FT activity levels (52.9 ± 11.4 μU/mg) than the other 2 stages (P = .03, by Kruskal-Wallis test). In addition, we found a nearly statistically significant decrease (P = .08, by MannWhitney U test) in α(1,6)FT activity in advanced, T4, and Table 1

3.2. Univariate analysis of patient survival rates 3.2.1. Overall survival At the end of the 60-month follow-up period, 112 (79.4%) of the 141 CRC patients were alive, with a mean survival time

Relationship between α(1,6)FT expression and clinicopathologic features


Sex Male Female Age (y) b67 67-77 N77 Tumor location Proximal colon Distal colon Rectum Size (cm) b4 4-5 N5 Tumor differentiation High Moderate Poor Growth type Polypoid Nonpolypoid Dukes stage A B C TNM classification T Tis/T1/T2 T3/T4 N N0 N1 N2

No. of patients (%); expression Negative


37 (26.24) 20 (14.18)

43 (30.50) 41 (29.08)

17 (12.06) 25 (17.73) 15 (10.64)


No. of patients (%); expression


(0, 1)

(2, 3)


60 (42.55) 36 (25.53)

20 (14.18) 25 (17.73)

.048 ⁎

30 (21.28) 25 (17.73) 29 (20.57)


32 (22.70) 37 (26.24) 27 (19.15)

15 (10.64) 13 (9.22) 17 (12.06)


10 (7.19) 18 (12.95) 29 (20.86)

22 (15.83) 18 (12.95) 42 (30.22)


19 (13.67) 27 (19.42) 50 (35.97)

13 (9.35) 9 (6.47) 21 (15.11)


28 (20.29) 11 (7.97) 17 (12.32)

32 (23.19) 29 (21.01) 21 (15.22)


41 (29.71) 25 (18.12) 29 (21.01)

19 (13.77) 15 (10.87) 9 (6.52)


4 (2.88) 50 (35.97) 3 (2.16)

6 (4.32) 71 (51.08) 5 (3.60)


6 (4.32) 85 (61.15) 5 (3.60)

4 (2.88) 36 (25.90) 3 (2.16)


26 (18.71) 30 (21.58)

39 (28.06) 44 (31.65)


42 (30.22) 53 (38.13)

23 (16.55) 21 (15.11)


16 (11.35) 23 (16.31) 18 (12.77)

10 (7.09) 40 (28.37) 34 (24.11)

.050 ⁎

21 (14.89) 38 (26.95) 37 (26.24)

5 (3.55) 25 (17.73) 15 (10.64)


18 (12.77) 39 (27.66)

15 (10.64) 69 (48.94)


23 (16.31) 73 (51.77)

10 (7.09) 35 (24.82)


39 (27.66) 13 (9.22) 5 (3.55)

50 (35.46) 22 (15.60) 12 (8.51)


59 (41.84) 24 (17.02) 13 (9.22)

30 (21.28) 11 (7.80) 4 (2.84)


NOTE. P, calculated by χ2 or Fisher exact probability test. Abbreviations: T, primary tumor extent; N, lymph node metastasis; Tis, in situ carcinoma. ⁎ P ≤ .05.

1744 Table 2 Feature

L. Muinelo-Romay et al. Univariate analysis with respect to overall and disease-free survival of CRC patients n

Sex Male 80 Female 61 Age (y) b67 47 67-77 50 N77 44 Tumor location Proximal colon 32 Distal colon 36 Rectum 71 Size (cm) b4 60 4-5 40 N5 38 Tumor differentiation High 10 Moderate 1211 Poor 8 Growth type Polypoid 65 Nonpolypoid 74 Dukes stage A 26 B 63 C 52 TNM classification T Tis/T1/T2 33 T3/T4 108 N N0 89 N1 35 N2 17 α(1.6)FT activity (μU/mg) b24.4 15 24.3- 44.1 13 N44.1 11 α(1.6)FT expression Negative 57 Positive 84 (0, 1) 96 (2. 3) 45

Mean survival time (mo)

Survival rate (%)


Overall survival

Disease-free survival

Overall survival

Disease-free survival

Overall survival

Disease-free survival

52.5 54.2

47.8 48.6

76.8 77.7

62.7 70.9



55.2 54.5 49.6

45.7 50.8 48.1

84.3 72.8 66.6

61.4 74.6 58.8



49.1 53.8 54.6

42.3 50.1 50.0

73.5 81.5 77.8

48.7 72.3 71.9


.052 ⁎

52.4 53.0 54.4

50.2 45.3 49.0

78.9 74.2 78.4

73.1 61.2 67.6



47.9 53.7 57.4

44.6 48.4 54.7

70.0 77.7 85.7

56.3 67.0 87.5



54.0 52.4

49.0 47.1

76.5 76.9

70.1 62.4



56.3 53.9 50.9

52.6 50.8 43.0

85.6 80.7 68.5

79.8 71.9 53.8


.034 ⁎

52.7 51.1

51.1 47.4

74.8 77.7

76.7 63.5



54.6 50.1 52.7

51.3 46.4 36.6

82.3 66.1 74.8

74.0 63.8 35.3


.002 ⁎

56.2 53.7 55.3

47.6 46.5 56.3

86.7 66.6 87.5

65.2 60.6 90.0



55.3 51.8 54.6 50.2

51.6 45.9 51.0 42.1

84.4 72.4 82.1 65.7

74.9 60.8 71.4 55.0



.047 ⁎

.029 ⁎

NOTE. P, calculated by log-rank test. Abbreviations: T, primary tumor extent; N, lymph node metastasis; Tis, in situ carcinoma. ⁎ P ≤ .05.

of 51.4 months. With the exception of α(1,6)FT expression, univariate analysis showed total independence between all variables evaluated in the present study and overall patient survival rate. The presence of moderate or strong α(1,6)FT expression significantly correlated with a fatal outcome in CRC patients (P = .047; Table 2 and Fig. 2A).

3.2.2. Disease-free survival During follow-up evaluation period, 44 (31.2%) patients had disease recurrence, and the overall mean disease-free survival time was 48.2 months. Interestingly, a clear correlation between lymph node infiltration and disease recurrence was found (P b .01; Table 2). Specifically, the

α(1,6)FT expression is a prognostic factor for CRC disease-free survival rate was 74.0% in patients without lymph node invasion (N0), 63.8% in patients with 1 to 3 lymph nodes affected (N1), and 35.3% in patients with 4 or more nodes infiltrated (N2) (Table 2). Detection of the tumor in an advanced Dukes stage or location of the tumor in the proximal colon was also associated with a poor disease-free survival rate (P = .03 and P = .05, respectively). In addition, patients with moderate or strong α(1,6)FT expression had a lower disease-free survival rate (55.0%) than did patients with weak or no α(1,6)FT expression (71.4%; P = .029; Table 2 and Fig. 2B). 3.2.3. Local recurrence-free survival Local tumor recurrence after surgery developed in 18 (22.8%) of the 141 patients included in this study; the mean survival time without local recurrence was 54.7 months. The results of univariate analysis for local recurrence are

1745 summarized in Table 3. Patient age was significantly correlated with local recurrence (P = .02), with patients younger than 67 years displaying better local recurrence-free survival rates (75.4%). Furthermore, although α(1,6)FT activity levels were not associated with the development of local recurrence, the difference in local recurrence-free survival rates in patients with moderate or strong α(1,6)FT expression (76.3%) and weak or no α(1,6)FT expression (90.5%) was statistically significant (P = .01; Fig. 2C). 3.2.4. Distant metastasis-free survival Distant metastasis developed in 35 patients (24.8%), 9 of whom had both a local recurrence and distant metastatic spread. The mean survival time without distant metastasis was 50.3 months but was significantly lower in patients with lymph node invasion (P = .03; Table 3). The location of the tumors in the proximal colon was associated with a poor

Fig. 2 Kaplan-Meier curves of overall survival (A), disease-free survival (B), local recurrence-free survival (C), and distant metastasis-free survival (D), according to α(1,6)FT expression in patients with CRC. (0, 1), absent or weak α(1,6)FT expression; (2, 3), moderate or strong α(1,6)FT expression. P, calculated by log rank test.

1746 Table 3 Feature

L. Muinelo-Romay et al. Univariate analysis with respect to local recurrence and distant metastasis-free survival of CRC patients n

Sex Male 80 Female 61 Age (y) b67 47 67-77 50 N77 44 Tumor location Proximal colon 32 Distal colon 36 Rectum 71 Size (cm) b4 60 4-5 40 N5 38 Tumor differentiation High 10 Moderate 121 Poor 8 Growth type Polypoid 65 Nonpolypoid 74 Dukes stage A 26 B 63 C 52 TNM classification T Tis/T1/T2 33 T3/T4 108 N N0 89 N1 35 N2 17 α(1.6)FT activity (μU/mg) b24.4 15 24.3- 44.1 13 N44.1 11 α(1.6)FT expression Negative 57 Positive 84 (0, 1) 96 (2. 3) 45

Mean survival time (mo)

Survival rate (%)

Local recurrence

Distant metastasis

Local recurrence

55.9 52.9

49.2 51.7

51.3 58.3 54.4

P Distant metastasis

Local recurrence

Distant metastasis

88.5 82.7

69.6 76.6



51.9 44.9 43.1

75.4 95.6 88.1

75.0 74.8 65.7

.027 ⁎


50.6 55.6 55.7

44.6 53.2 51.6

80.1 86.4 87.9

53.3 81.4 77.5


.030 ⁎

56.3 54.0 52.7

51.9 47.8 51.5

90.8 83.9 83.7

76.0 68.4 80.4



54.6 56.7 –

47.2 50.7 54.7

64.3 87.4 100

67.5 73.5 87.5



53.7 55.3

52.2 48.4

83.0 88.3

79.7 66.3



57.0 54.4 53.7

53.6 52.2 46.5

91.2 86.4 82.7

84.0 76.5 62.7



54.7 54.6

54.5 49.1

86.2 86.0

86.2 69.0



55.2 56.7 48.1

52.6 49.2 40.9

87.8 89.6 69.5

78.6 69.0 49.9


.031 ⁎

52.7 55.4 –

47.9 46.5 56.3

86.7 92.3 100

66.6 67.7 90.0



57.1 52.9 57.0 49.5

53.2 48.3 52.3 45.8

89.1 83.7 90.5 76.3

78.3 68.9 77.1 62.8



.012 ⁎


NOTE. P, calculated by log-rank test. Abbreviations: T, primary tumor extent; N, lymph node metastasis; Tis, in situ carcinoma. ⁎ P ≤ .05.

disease-free survival rate and a worse distant metastasis-free survival prognosis (P = .03). In addition, more metastasis events were observed in patients with moderate or strong α(1,6)FT expression than those in patients with weak or no α(1,6)FT expression, although this difference was not significant (P = .072; Fig. 2D).

3.3. Multivariate analysis With the goal of determining their independent prognostic value for predicting CRC patient survival, both α(1,6)FT activity and expression levels were included in a Cox regression model, together with clinicopathologic parameters.

α(1,6)FT expression is a prognostic factor for CRC Table 4


Multivariate analysis of CRC patient survival


Disease recurrence RR (95% CI)

N N0 1 N1 1.8 (0.86-3.94) N2 3.9 (1.85-8.51) α(1,6)FT expression (0, 1) 1 (2, 3) 1.9 (1.02-3.78)

Local recurrence P

RR (95% CI)

.002 ⁎ .041 ⁎

1 3.6 (1.35-9.78)

Distant metastasis P

RR (95% CI)



1 1.8 (0.79-4.35) 3.5 (1.48-8.63)

.027 ⁎

.010 ⁎


Abbreviations: RR, relative risk; CI, confidence interval; NS, not statistically significant; N, lymph node metastasis. ⁎ P ≤ .05, calculated by Cox regression analysis.

No independent prognostic factors were identified for the prediction of overall survival. However, both lymph node status and α(1,6)FT expression levels displayed independent prognostic value for predicting tumor recurrence, taking into consideration both local recurrence and distant metastasis (Table 4). In particular, the presence of tumor infiltration in 4 or more lymph nodes increased the relative risk of disease recurrence 3.9-fold [95% confidence interval (CI), 1.85-8.51; P = .002), whereas moderate or strong α(1,6)FT expression increased the relative risk of disease recurrence 1.9-fold (95% CI, 1.02-3.78; P = .04). For local recurrence, only α(1,6)FT expression levels showed true prognostic utility, with increased α(1,6)FT expression levels associated with a 3.6-fold increased risk for local recurrence (95% CI, 1.35-9.78; P = .01). Finally, in the present study, lymph node infiltration was the only independent prognostic factor able to predict metastasis development in CRC patients and was associated with a 3.5-fold increased risk for distant metastasis (95% CI, 1.48-8.63; P = .02; Table 4).

4. Discussion During neoplastic transformation, numerous changes in the composition and structure of cell-surface glycoproteins are frequently observed [25]. Accumulating evidence suggests that many of these alterations are influenced by the production of extensive fucosylated sugar chains [22]. In fact, altered levels of α(1,6)FT activity and expression have been described in a variety of human tumors and are normally associated with an advanced disease stage [3-5]. In a previous study, we demonstrated that increased α(1,6)FT activity and expression are associated with altered α(1,6)fucosylation in human CRC as a result of malignant transformation [3]. In support of this, elevated α(1,6)FT messenger RNA levels were also reported in human colorectal tumor tissues and colon cell lines [6,26]. With respect to the role of α(1,6)FT in tumor malignancy, we observed a close association between α(1,6)FT activity or expression levels and colorectal tumor aggressiveness [3]. However, the impact of α(1,6)FT on CRC patient outcome

has never been analyzed, and therefore, its prognostic value for CRC or other tumor types remains unknown. In this context, we conducted the present study to determine possible correlations between α(1,6)FT activity and expression, and CRC progression. In agreement with our previous results, α(1,6)FT activity levels in tumor tissues analyzed in the present study decreased progressively with the degree of tumor infiltration into the intestinal wall. Consistent with these results, increased α(1,6)FT activity was observed in polypoid tumors, which are more localized and less invasive than nonpolypoid tumors. In contrast, the percentage of cases with positive α(1,6)FT immunohistochemical staining was higher in advanced stage tumors than early stage tumors. In fact, this absence of a correlation between α(1,6)FT expression and activity has been previously described [27,28] and has been proposed to be due to a posttranslational modification or alternative enzyme activation mechanism that modulates the levels of active catalytic protein [28]. Interestingly, α(1,6)FT expression was found to be less in men than in women. Consistent with this finding, the decrease in biantennary core-fucosylated structures reported for patients older than 40 to 50 years occurs earlier in women than in men [29]. Most patients included in the present study were older than 40 to 50 years; and no differences in α(1,6) FT status were observed among the 3 age groups considered. In our opinion, sex-associated α(1,6)FT expression differences could be influenced by sex hormone balance. Several studies have described modulation of estrogen and progesterone levels in colorectal epithelium homeostasis [30,31]. Consequently, the glycosylation dynamics of colorectal cells could be also influenced by these hormone modulators. In any case, despite this hypothetical correlation between sex and α(1,6)FT status, the possible clinical utility of α(1,6)FT is not invalidated, assuming the determination of appropriate normality reference intervals for each sex. With respect to the prognostic value of the standard clinicopathologic features included in the present study, as expected, the presence of lymph node invasion correlated with more aggressive tumor progression at the 5-year followup in terms of disease-free survival and, specifically, with the

1748 appearance of distant metastasis. The predictive power of lymph node metastasis for cancer evolution is well established in the literature, and the lymphatic system is considered the main pathway by which cancer cells disseminate [32]. In agreement with this, tumors in an advanced Dukes stage were associated with poor disease-free survival rates. However, remarkably, overall survival rates were observed to be independent of the clinicopathologic variables analyzed. Considering that metastasis is the major cause of death in cancer patients with solid tumors, a correlation between lymph node infiltration and overall survival rates for CRC patients was expected. Nevertheless, in older populations such as the one included in the present study, it is frequently difficult to identify with precision cancer-related deaths, because most patients present with synergistic pathologies. Thus, we consider disease-free survival rates more informative about CRC progression than overall survival rates. The anatomical location of the primary tumor was found to correlate with CRC disease progression. Specifically, tumors localized to the proximal colon (right and transverse colon) presented poor rates of disease-free survival. Consequently, in terms of metastasis development, tumors localized to the distal colon and rectum are associated with better outcomes. Traditionally, rectum lesions correlate with unfavorable prognoses after surgery, because of the difficulty of removing these tumors per se. However, right colon tumors are usually detected in an advanced stage, because symptoms typically present later than in left colon tumors, and thus, they are frequently more aggressive [33]. In addition, the group of patients older than 67 years analyzed in our study had a higher rate of local disease recurrence. Although some studies of colon and rectum tumors have described a rapid downhill course, characteristic of young cancer patients, others reported the contrary [34,35]. Interestingly, the present study demonstrates the useful of α(1,6)FT expression levels for predicting CRC recurrence after the surgical resection. Specifically, patients with moderate or strong α(1,6)FT expression had lower diseasefree survival rates than did patients with weak or no α(1,6)FT expression. The negative influence of α(1,6)FT expression was also observed for local recurrence after curative surgery, with less aggressive tumor progression in patients with low immunohistochemical α(1,6)FT levels. The same trend was observed between distant metastasis and α(1,6)FT expression, although a nonstatistically significant association was found between both variables. In addition, α(1,6)FT expression was the only factor in this study that correlated with overall CRC survival rates. Therefore, for the first time, α(1,6)FT tissue expression has been correlated with the survival of cancer patients. To the best of our knowledge, only one previous study has analyzed the utility of plasma α(1,6)FT activity levels for predicting clinical outcomes. This study of breast cancer patients [18] indicated a lack of correlation between plasma α(1,6)FT activity and the response of metastatic breast tumors to treatment with

L. Muinelo-Romay et al. trastuzumab, a potent anticancer agent used against tyrosine kinase-type cell receptor HER2 overexpressing tumors [36]. α(1,6)Fucosylation of EGFR regulates its intracellular signaling pathway and sensitivity to EGFR tyrosine kinase inhibitors. However, in the aforementioned study, plasma α(1,6)FT catalytic levels seemed to be irrelevant for predicting patient response to treatment [18]. The goal of the present study was to define the clinical usefulness of α(1,6)FT status as a prognostic factor in A, B, and C stage tumors, independently of the adjuvant therapy used. The results of multivariate analysis revealed that α(1,6) FT immunohistochemical expression in colorectal tumors is an independent predictor of tumor recurrence, even more so than the most conventional clinicopathologic factors. Hence, the relative risk of disease reappearance was doubled in patients with moderate or strong α(1,6)FT expression, whereas the relative risk of local recurrence in this group of patients was increased 3.6-fold. Moreover, based on univariate analysis, the only clinicopathologic factor useful for predicting disease recurrence in this study (specifically, distant metastasis development) was lymph node infiltration, as expected. Based on our results, the evaluation of α(1,6)FT immunohistochemical expression in colorectal tumors adds important prognostic information about cancer recurrence and could help specialists select the appropriate treatment strategy to prevent CRC progression. A molecular explanation for the correlation between α(1,6)FT expression and CRC disease outcome should be defined in future studies. In fact, it is well known that molecular determinants of proximal and distant tumor recurrences are quite different, with local recurrences essentially occurring as a result of the presence of treatment-resistant tumor cells, which maintain their unrestrained proliferative dynamics. Several studies have demonstrated that core-fucosylation regulates the biological function of numerous molecules implicated in cell proliferation, differentiation, and apoptosis. In addition to the α(1,6) FT-mediated modulation of EGFR function mentioned above, other receptors, such as TGFβ-R or VEGFR, also must be core-fucosylated to activate their respective intracellular signaling pathways. Moreover, some recent reports have shown that α(1,6)fucosylation of E-cadherin modulates the processing of its oligosaccharide antennae, as well as the turnover of this adhesion molecule and, consequently, cell-cell interactions in tumor cells [13,14]. These E-cadherin–mediated adhesions could also modulate tyrosine kinase or Wnt signaling receptors, both of which upregulate the signaling pathways in different types of tumors [37,38]. In particular, β-catenin/Wnt plays an important role in the control of colon epithelium homeostasis [39]. In this molecular context, we propose that high α(1,6)FT levels in colorectal cancer cells could be decisive in maintaining its proliferative status and in modulating its response to different chemotherapy agents. However, for distant metastases development, cancer cells must dissociate from the original tumor and migrate via

α(1,6)FT expression is a prognostic factor for CRC blood vessels or the lymph system to colonize specific target organs. Distant metastasis is a multistep process in which adhesive interactions play a critical role. In turn, α(1,6) fucosylation of different adhesion molecules, such as adhesins or integrins, modifies their functional activity and may be a relevant factor for determining the acquisition of a migratory phenotype by epithelial tumor cells. For example, core-fucosylation is a prerequisite for neural cell adhesion molecule (NCAM) polysialylation [40]. Furthermore, expression of the polysialylated form of NCAM has been demonstrated in some malignant tumors, allowing prostate specific antigen (PSA) positive cancer cells to detach from the primary tumor by attenuating the adhesive properties of NCAM and, consequently, by increasing the metastatic potential of tumor cells [41-43]. Interestingly, a study of lung cancer cells demonstrated that E-cadherin is core-fucosylated in highly metastatic cells, whereas this type of glycosylation is absent in low metastatic cells. These authors proposed that core-fucosylation on E-cadherin inhibits cellcell aggregation and promotes matrix metallopeptidases 9 and 2 (MMP 9 and MMP 2) activity, contributing to lung cell migration and invasiveness [13]. Likewise, other molecules implicated in cell-extracellular matrix interactions and cancer cell invasiveness, such as α3β1 or α5β1 integrins, are α(1,6) fucosylated in their active form [5,15,44]. Probably, the high α(1,6)FT expression observed in colon cancer cells promotes cell interactions mediated by these integrins and, therefore, facilitates tumor progression. In summary, the present study has demonstrated, for the first time, that α(1,6)FT expression may be a good prognostic factor for predicting colorectal cancer progression after surgery. The role of α(1,6)FT in tumor cell aggressiveness may be explained by its effects on the activity of several molecules strongly implicated in critical malignancy processes, including cell proliferation and dissemination. Although further, multicentric studies will be required to validate the use of α(1,6)FT immunohistochemical expression for the therapeutic management of CRC, our findings suggest that α(1,6)FT and its corefucosylated products could be promising tumor markers and therapeutic targets for this pathology.

Acknowledgments The members of the Pathology Service of the University Complex Hospital of Ourense (Ourense, Spain) have played a very important role in the immunohistochemical evaluation used in this study, and this kind collaboration is acknowledged.

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