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Keywords:

  • fucose;
  • fucosylated glycoproteins;
  • fucosyltransferase;
  • α-L-fucosidase;
  • fucosylation;
  • oral cancer;
  • oral precancerous conditions

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND.

The objective of the current study was to investigate the clinical usefulness of serum fucose, fucosylated glycoproteins (fucoproteins), fucosyltransferase (FucT), and α-L-fucosidase in oral carcinoma.

METHODS.

Blood samples were collected from 130 patients with untreated oral cancer (OC), from 75 patients with oral precancerous conditions (OPC), and from 100 healthy controls. Cancer patients were followed after the initiation of anticancer treatments, and 75 follow-up samples were also collected. Serum levels of fucose and α-L-fucosidase were measured spectrophotometrically. Fucoproteins were detected by using lectin-affinity chromatography. FucT activity was analyzed by using radioassay.

RESULTS.

Serum levels of fucose and fucoprotein were found to be increased significantly in patients with untreated OC compared with controls, patients with OPC, and complete responders (CR) to treatment; whereas the levels were comparable between untreated patients with OC and nonresponders (NR). A similar trend was observed for serum FucT levels, and changes in enzyme activity correlated well with fucose and fucoprotein alterations. The OPC group had significantly increased fucosylation of serum proteins. Furthermore, serum α-L-fucosidase activity was markedly higher in patients with untreated OC and in patients with OPC compared with controls. Using receiver operating characteristic curves, a cutoff for α-L-fucosidase was determined at >450.6 U/mL, which showed good sensitivity and specificity in OC and OPC compared with controls. The enzyme activity was declined in the CR group but remained higher in the NR group compared with pretreatment levels. Furthermore, various clinicopathologic characteristics were correlated positively with serum fucosylation changes.

CONCLUSIONS.

The findings of the current study suggest that serum fucosylation has clinical usefulness in the detection of early changes and for monitoring treatment response in patients with OC. Among the markers studied, serum α-L-fucosidase was identified as a useful marker for close monitoring of patients during post–treatment follow-up. Cancer 2008. © 2008 American Cancer Society.

Cellular glycosylation changes are associated with diverse types of neoplastic transformation. Many properties of mammalian cells are either expressed at or mediated through the cell surface. Studies suggest that altered glycosylation of cell surface proteins is critically important in cancer progression, especially the terminal epitopes of glycoproteins, which have been proposed to play a significant role in cell-cell interactions, development of cell adhesion, malignant transformation, and metastasis.1, 2 Fucosylation of glycoproteins (the addition of L-fucose at the terminal end of the oligosaccharide chain) is one of the most important features that mediates several specific biologic functions.3 It has been documented that tumor cells modulate their surface by increasing fucosylation levels to escape recognition, which contribute to several abnormal characteristics of tumor cells, such as decreased adhesion and uncontrolled tumor growth.4, 5 Several studies have suggested that monitoring serum/tissue fucose levels could be a promising approach for the early detection, diagnosis, and prognosis of various cancer types6-10

Fucosyltransferases (FucT) (Enzyme Commission [EC] number 2.4.1 [65, 68, 69, 152, 214, 221]) are a group of enzymes that catalyze incorporation of fucose from activated nucleotide donor guanosine diphosphate (GDP)-fucose to the reducing end of complex glycans in a linkage-specific manner. These enzymes are expressed by many tissues and are increased in serum and tumors from cancer patients.8, 11-13 It has been reported that increased fucosylation is associated with elevated FucT activity.11 Cancer cells that are shed or released into circulation from the primary tumor often over express fucosylated glycans on their surface. The expression of fucosylated glycoproteins (ie, fucoproteins) has been detected by means of specific lectins.14 Several lectin-based studies have indicated that fucoproteins are increased in various cancers.15-17 Abnormally fucosylated serum heptoglobin and α-fetoprotein (AFP) are used widely as tumor markers of hepatocellular carcinoma.15, 16, 18 Profound fucosylation of the serum microenvironment may be a factor that interrupts adhesion and influences the formation of metastases. For example, several fucose-containing ‘natural ligands’ reportedly are involved in the migration of tumor cells.19 Increased expression of fucosylated cell surface antigens, such as Lewis x/y (Lex/y) or sialyl Lex/a, and the up-regulation of α1,3/4-FucT have been associated with malignant transformation and increased metastatic potential of tumors, which result in a poor prognosis of patients with cancer.20

α-L-fucosidase (EC number 3.2.1.51) is a lysosomal enzyme that catalyzes the hydrolytic cleavage of terminal fucose residue that is involved in maintaining the homeostasis of fucose metabolism. It has been reported that alterations in serum and/or tissue α-L-fucosidase activity potentially may be useful in the diagnosis and management of cancer patients.5, 8, 21-23 Serum α-L-fucosidase has been proposed as a marker for colorectal and hepatocellular carcinoma.21, 24, 25 Thus, earlier reports have encouraged interest in the use of serum fucosidase for the diagnosis of malignant diseases and as an indicator of tumor burden, metastasis, and response to anticancer treatments.

Oral cancer (OC) is one of the major health hazards in India, and approximately 80,000 new cases are diagnosed annually, mainly attributed to different forms of tobacco consumption.26 OC may serve as an excellent model in which to study the molecular changes that occur in a cancer cell, because OC generally is preceded by early changes termed as oral precancerous conditions (OPC), such as hyperplasia, dysplasia, leukoplakia, and oral submucus fibrosis (OSMF). It has been established that OPC represents an increased risk of developing OC with malignant transformation rates that vary from 0.6% to 36%.27 Although fucose plays an important role, the simultaneous evaluation of fucosylation changes in OC is under studied. Therefore, the objective of the current retrospective study was to determine the clinical significance of altered serum fucose, fucoproteins, FucT, and fucosidase in oral carcinogenesis and in treatment monitoring of patients with cancer. To assess the diagnostic specificity of these markers, patients with OPC were included in the current study who were at risk of cancer and were distinguished from the patients with cancer, because we hoped that the inclusion of patients with OPC would provide an indication of early changes during the development of OC. Healthy individuals (controls) were enrolled in the study to obtain the baseline levels of markers, and serum levels in the OC and OPC groups were compared with levels in the control group.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Study Groups

The study mainly included 3 groups (Table 1): 1) controls (100 healthy individuals who had no major illness in the recent past), 2) pathologic controls (75 patients with OPC that included 50 patients with OSMF and 25 patients with leukoplakia), and 3) cancer patients (130 patients with histopathologically proven, untreated cancer of the oral cavity). Consent was obtained from all individuals who participated in the study. Table 1 shows that the majority of the patients with OPC and OC were tobacco users and that most patients with disease were men. Clinical TNM staging of malignant disease was determined according to International Union Against Cancer norms.28 The distribution of patients with OC with regard to various clinicopathologic characteristics, including stage, tumor differentiation, lymph node status, and tumor grade, is provided in Table 2. All patients with OC underwent surgical resection of their tumor as primary treatment. These patients were followed at various intervals after the initiation of postsurgical anticancer treatment. The study also included post-treatment follow-up serum samples (N = 75) from cancer patients. Patients' responses to anticancer treatment were assessed on the basis of their clinical and radiologic findings during follow-up. The clinical status of patients during and after anticancer treatment was evaluated as suggested by Therasse et al,29 and patients were grouped into complete responders (CR) (N = 52) and nonresponders (NR) (N = 23). Patients who had a partial response (PR) or stable/progressive disease were grouped together with the NR group.

Table 1. Details of the Study Groups
VariableControls (N=100)Patients with OPC (N=75)Cancer patients (N=130)
  1. OPC indicates oral precancerous condition.

Age, y
 Median3328.542
 Range20-7114-8018-77
Sex, %
 Men889294
 Women1286
 Ratio of men to women7.6:111.7:115.7:1
Tobacco habits (frequency), %
 Yes6197.497.7
 No392.62.3
Tobacco habits, %
 Chewing6267.540
 Smoking1913.524
 Chewing and smoking15.51933
 Snuff3.53
Table 2. Clinical Characteristics of the Patients with Oral Cancer
Clinical characteristicPercent of patients (N=130)
Disease site
 Buccal mucosa45.4
 Oral tongue20.8
 Alveolus10.7
 Retromolar trigon7.7
 Lip and others (gum, gingival sulcus, hard palate)15.4
Histopathology
 Squamous cell carcinoma100
Lymph node involvement
 No49.4
 Yes50.6
Stage of disease
 I10.6
 II17
Early stage (I and II)27.6
 III17.9
 IV54.5
Advanced stage (III and IV)72.4
Tumor differentiation
 Well30.8
 Moderate57.7
 Poor11.5
Nuclear grade
 127
 261.5
 311.5

Blood samples were collected by venipuncture from all participants, and sera were separated and stored at −80 °C until analyzed. Each sample was analyzed in duplicate.

Methods

Estimating total serum fucose levels

Fucose was estimated using the method described by Winzler.30 Briefly, serum proteins were precipitated out by 95% ethanol. The pellet obtained from centrifugation was dissolved in 2 mL of 0.2 N NaOH. From this, 0.5 mL of aliquots was used for fucose estimation. Bound fucose was released from complex carbohydrates and was converted into furfural derivatives upon reaction with concentrated H2SO4 followed by boiling for 3 minutes. Free fucose, upon reaction with cystein-hydrochloride, formed yellow chromophores, and absorbance was read at 396 nanometers (nm). A reaction mixture also was read at 430 nm simultaneously to correct the interference caused by chromophores from other sugars. Fucose levels were normalized with serum total proteins (TP) and expressed as mg/g protein.

Isolating fucoproteins using lectin-affinity chromatography

Lectin-affinity chromatography was performed to isolate serum fucoproteins as described by Thompson and Turner.15 The fucose-specific lectin Lotus tetragonolobus (LTA) (Sigma Chemical Company, St. Louis, Mo) was coupled with cyanogen bromide-activated sepharose-4B beads (Amersham Pharmacia) at a final concentration of 2 mg lectin mL−1 packed beads according to the manufacturer's instructions. Before coupling with serum proteins, lectin-sepharose beads were washed with Tris buffer (pH 7.4) that contained KCl, CaCl2, MgCl2, and Nonidet P40. Serum fucoproteins were isolated by mixing an equal concentration of serum proteins with an equal volume of packed lectin-sepharose beads for 30 minutes at room temperature. Then, the beads were washed with Tris buffer to remove unbound serum proteins. Bound fucoproteins were released from lectin beads by solubilizing in 100 μL Tris (pH 6.8) containing sodium dodecyl sulfate (SDS) and ethylenediamine tetracetic acid. Resultant elutes were run on 7.5% SDS-polyacrylamide gel electrophoresis gels under reducing conditions, and the proteins were stained using silver staining method (Bio-Rad Kit).

FucT assay

The assay described by Yazawa et al was used to measure FucT activity.31 Briefly, 0.1 mL assay reaction mixture consisted 50 mM N-(2-hydroxethyl) piperazine-NA-2-ethanesulfonic acid-NaOH buffer (pH 6.8) and 10 mM MnCl2. To this, 4 mg of fucose acceptor ovomucoid and 20 μL enzyme source were added. After equilibration at 37 °C, the reaction was initiated by addition of C14-labeled nucleotide sugar GDP-fucose. A control assay for each sample was run in the absence of acceptor substrate. The reaction was terminated by adding a chilled mixture containing 5% trichloroacetic acid, 2% phosphotungestic acid, and 0.5 N HCl. The resultant pellet was washed twice with the same acid mixture followed by 95% ethanol. Protein precipitates were solubilized in 1 mL NCS solubilizer (Amersham) and placed in 10 mL scintillation cocktail. Enrichment of glycoproteins with fucose was measured by measuring radioactivity using a β counter. Enzyme activity was expressed as continuous passive motion/mg protein/hour.

α-L-fucosidase assay

The fucosidase activity was determined as described by Wiederschain et al32 Briefly, the reaction mixture, which contained 20 μL serum as an enzyme source, 205 μL 0.05 N acetate buffer (pH 5.5) and 25 μL 10 mM p-nitrophenyl (PNP)-α-L-fucopyranoside substrate for enzyme, was incubated at 37 °C for 1 hour. To this, 2.5% ZnSO4 and 0.15 N NaOH were added to terminate the reaction. The samples were centrifuged, and 300 μL supernatant were mixed with 300 μL 0.04 M glycine buffer, pH 10.5. The absorbance of liberated PNP was measured at 410 nm wavelength. One unit of enzyme activity was defined as the amount of enzyme required to hydrolyze 1 nmole of PNP/mL/hour at 37 °C.

Statistical Analysis

Data were analyzed using SPSS statistical software (version 10). The levels of significance were determined by using unpaired and paired Student t tests. Receiver operating characteristic (ROC) curves were constructed to evaluate the diagnostic value of serum α-L-fucosidase in patients by selecting a cutoff level from multiple points on the graph. Multivariate analysis was performed to determine the correlation of serum markers with clinicopathologic parameters. The values were expressed as the mean ± standard error of the mean. P values ≤.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Comparison of Serum Fucose Levels Between Controls and Patients

Figure 1 shows that serum fucose levels were increased significantly in patients who had OC compared with patients who had OPC (P = .003) and compared with controls (P < .001). In addition, patients with OPC had significantly higher levels of serum fucose than controls (P = .002).

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Figure 1. Comparison of serum fucose levels between controls (N = 100), patients with an oral precancerous condition (OPC) (N = 75), and patients with untreated oral cancer (Cancer Pts.) (N = 130). Values (mean ± standard error of the mean) are expressed as mg/g proteins.

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Comparison of Fucoproteins Between Controls and Patients and Serum Fucoproteins as Treatment Response Monitors in Patients With OC

Serum fucoproteins were isolated by using lectin LTA, which detects fucose linked either at the α1,2/α1,6 position in the core or at the α1,3 position as terminal or preterminal residue. The intensity of fucoproteins was quantified using molecular analysis software (Gel Documentation; Bio-Rad), and results expressed as optical density/mm2. Figure 2A shows that the patients with untreated OC had significantly increased levels of serum fucoproteins compared with the control group and compared with the patients with OPC (P = .006 and P <.05, respectively). In addition, increased levels of serum fucoproteins were observed in the patients who had OPC compared with the control group (P = .07). Representative electrophoretic patterns of serum fucoproteins in controls, in patients with OPC, and in 3 patients with untreated OC who had all post-treatment follow-up serum levels available are shown in Figure 2B,C. Increased LTA reactivity was observed especially in ∼43-kilodaltons (kD) and ∼66-kD serum glycoproteins. In Figure 2B, lanes A and B represent fucoprotein expression in a control and in a patient with OPC, respectively. The patterns represent fucoprotein changes in the CR group, the PR group, and the NR group during follow-up. Furthermore, the aggregate presentation of mean fucoprotein levels in the CR, PR, and NR groups were plotted the follow-up of each patient against their pretreatment (PT) levels, as shown in Figure 2D. The fucoprotein levels were higher at the time of diagnosis (PT levels) in all patients with OC (ie, the CR, PR, and NR groups), as depicted in the graph. The serum levels were decreased in the CR group (Fig. 2D, solid line) during follow-up (F1-F4 indicate follow-up at 1 month, 3 months, 7 months, and 10 months, respectively) compared with their PT levels. Conversely, serum fucoproteins remained unchanged or increased in the NR group (Fig. 2D, dotted line) because of postsurgical and/or postradiotherapeutic progression of disease in this group. It is interesting to note that serum fucoproteins in the PR group (Fig. 2D, dashed line) demonstrated a mix response to the anticancer treatment during follow-up. Thus, our data suggest a close correlation between serum fucoprotein levels and response to anticancer treatment and progressive disease in these patients.

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Figure 2. (A) Serum fucosylated glycoproteins (fucoproteins) in controls (N = 100), in patients with an oral precancerous condition (OPC) (N = 75), and in patients with untreated oral cancer (OC) (Cancer Pts.) (N = 130). Serum fucoproteins were isolated with the fucose-specific lectin Lotus tetragonolobu by using lectin-affinity chromatography. The band intensity was quantified and compared between the groups. Values (mean ± standard error of the mean) are expressed as optical density (OD)/mm2. (B) Representative serum fucoprotein electrophoretic pattern and fucose levels in controls, in patients with OPC, and in patients with OC at the time of diagnosis and during follow-up in patients who achieved a complete response (CR) and in patients who achieved a partial response (PR). Lanes A and B indicate serum fucoprotein patterns in a control and in a patient with an OPC, respectively; Lane C, molecular weight (MW) marker; Lanes D-H, serum fucoprotein patterns in a patient who had a CR at pretreatment (PT) (Lane D) and during follow-up (Lanes E-H), serum fucoprotein patterns in a patient who had a PR at pretreatment (Lane I) and during follow-up (Lanes J-L). (C) Representative serum fucoprotein electrophoretic pattern and fucose levels in a nonresponder (NR) at the time of diagnosis and during follow-up. Lane 1, molecular weight (Mol Wt.) markers; Lane 2, serum fucoprotein patterns in an NR before the initiation of anticancer treatment; Lanes 3-6, serum fucoproteins in patients during post–treatment follow-up. (D) Serum fucoprotein levels before and after anticancer treatment in patients who achieved a CR (solid line), in patients who achieved a PR (dashed line), and in NR (dotted line). F1 through F4 indicate follow-up at 1 month, 3 months, 7 months, and 10 months, respectively.

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Furthermore, we observed a significant association of serum fucose levels in these patients before and after anticancer treatment, as indicated at the bottom of the electrophoretic patterns (Fig. 2B and 2C). It is evident from representative patterns and aggregate presentations of the data that serum fucoproteins correlated well with disease persistence, remission, and progression as well as patient outcome (Figs. 2B-D).

Alterations in Serum FucT and α-L-Fucosidase Activity

Increased levels of fucose and fucoprotein led us to examine the activity of FucT. Figure 3 shows that patients who had untreated OC and patients who had OPC had significantly higher levels of FucT activity than controls (P <.05 and P< .05, respectively). A comparison of serum fucosidase activity among the 3 groups is provided in Figure 4A. The mean serum fucosidase levels were found to be elevated significantly in patients who had untreated OC and in patients who had OPC compared with controls (P < .001 and P < .001, respectively), whereas the levels were comparable between patients with OC and patients with OPC.

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Figure 3. Comparison of serum fucosyltransferase activity between the controls (N = 100), patients with an oral precancerous condition (OPC) (N = 75), and patients with untreated oral cancer (Cancer Pts.) (N = 130) analyzed by radioactivity assay. Values (mean ± standard error of the mean) are expressed as counts per minute/mg protein/hour. NS indicates not significant.

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Figure 4. (A) Comparison of serum fucosidase activity between the controls (N = 100), patients with an oral precancerous condition (OPC) (N = 75), and patients with untreated oral cancer (OC) (N = 130) analyzed by p-nitrophenol assay. Values (mean ± standard error of the mean) are expressed as U/mL. (B) Receiver operating characteristic (ROC) curves for preoperative serum α-L-fucosidase. Comparisons were made between 1) patients with untreated OC versus controls; 2) patients with OPC versus controls; and 3) patients with OPC versus patients with untreated OC. ROC curves were constructed for the analysis of sensitivity and specificity of serum fucosidase andits ability to discriminate between the study groups. Fucosidase values (indicated as line graphs) above the reference line on an ROC curve (middle lines in graphs) suggest a good discriminatory efficacy of the marker that is within acceptable limits.

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ROC Curve Analysis for Preoperative Serum α-L-Fucosidase

The ROC curve is a meaningful statistical approach with which to discriminate the groups under analysis that simultaneously considers both the sensitivity and the specificity of the parameters. The ROC curves for serum α-L-fucosidase activity are shown in Figure 4B, and the corresponding sensitivity and specificity values as well as the area under the ROC curves (AUCs) are provided in Table 3. The AUCs for the comparisons of the control group versus the OC group, the control group versus the OPC group, and the OPC group versus the OC group were 0.795, 0.840, and 0.475, respectively. The efficacy of the serum α-L-fucosidase level for discriminating between the control group and the untreated OC group or the OPC group was statistically significant (P < .001 and P < .001, respectively). The ideal cutoff of >450.6 U/mL for α-L-fucosidase was determined from multiple points on the ROC curve that resembled the mean value in the control group. At the chosen cutoff, α-L-fucosidase had 60% sensitivity and a specificity of 89% to discriminate between patients with OC patients and controls. The cutoff also had good discriminatory efficacy for OPC with high sensitivity (60%) and specificity (94%).

Table 3. Comparison of the Discriminatory Efficacy of Serum α-L-Fucosidase Activity in Patients With Oral Cancer and Oral Precancerous Conditions
Groups*Sensitivity, %Specificity, %PAUC
  • OPC indicates oral precancerous condition; AUC, area under the curve; NS, not significant.

  • *

    The cut-off for α-L-fucosidase was determined as >450.6 U/mL, which showed good sensitivity and specificity for oral cancer and OPC.

Controls vs cancer patients6089<.0010.795
Controls vs OPC patients6094<.0010.840
OPC patients vs cancer patients  NS 

Correlation Between Serum Fucosylation Changes and Clinicopathologic Parameters

Multivariate analysis was performed to determine the association of serum fucosylation changes with various clinicopathologic parameters in cancer patients. We observed that increased serum fucose levels were associated significantly with tumor differentiation (P = .019) and stage of the malignant disease (P = .041). Serum fucoproteins also had a positive correlation with lymph node metastasis (P = .065).

Comparison Between PT and Posttreatment Serum Levels of Fucosylation Markers in Patients With OC

Serum fucosylation levels at diagnosis were compared with their follow-up levels. The follow-up samples were categorized into CR and NR. A comparison of fucosylation changes between patients with untreated OC (PT group), the CR group, and the NR group is shown in Figure 5A-D. The CR group had declined serum levels of the markers compared with the PT group and the NR group. Statistical analysis revealed significantly lower levels of serum fucose, fucoproteins, FucT, and fucosidase in the CR group compared with the PT group (P < .001, P = .001, P = .05, and P = .001, respectively); whereas levels in the NR group were markedly higher (P = .001, P < .001, P = .05, and P = .07, respectively) compared with levels in the CR group. Serum fucose, fucoproteins, FucT, and fucosidase revealed comparable levels in the NR group and the PT group. To determine the association of altered serum fucosylation levels with the effectiveness of anticancer treatment, the marker levels at each follow-up were paired with the patient's PT levels and were analyzed. The statistical significance of the alterations during favorable or unfavorable treatment response was confirmed in each patient group using the Student t test for paired data, which revealed a trend comparable to that described above. Serial estimates of the changes in serum fucosylation levels were calculated after the initiation of anticancer treatment to evaluate the efficacy of these markers in treatment monitoring. The patterns of biomarkers at the time of diagnosis and during and/or after anticancer treatment in patients who had different treatment outcomes were studied. Alterations in fucoproteins in 3 representative individuals are shown in Figure 2B and 2C. Levels of fucose, FucT and fucosidase revealed a sharp decrease during follow-up and remained below their PT levels throughout follow-up duration in the CR group. Conversely, the levels rose progressively during follow-up in the NR group. In addition, serum fucosylation levels decreased during the clinical remission phase. Remarkably, marker levels rose before the clinical detection of recurrence. Thus, it is evident from the results that fucosylation changes reflect the response to the anticancer treatment in patients before clinical manifestations of disease.

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Figure 5. Patients with oral cancer (OC) were followed during and/or after anticancer treatment. The post–treatment follow-ups were categorized into complete responders (CR) and nonresponders (NR) as described in the text. The follow-up serum samples were analyzed for fucosylation changes using a Student t test for paired data analysis for each patient's follow-up. Graphs show the comparison of (A) serum L-fucose, (B) fucosylated glycoproteins (fucoproteins), (C) fucosyltransferase (FucT), and (D) α-L-fucosidase between the untreated/pretreatment (PT) patients with OC (N = 130) and patients who achieved a CR (N = 52) or who had an NR (N = 23). Values are shown as the mean ± standard error of the mean. OD indicates optical density; CPM, counts per minute.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The current study demonstrates the association of changes in serum fucosylation with OC development and with treatment response monitoring in patients with OC. We observed that the measurement of serum levels of fucose, fucoproteins, FucT, and fucosidase was useful clinically in patients with OC and OPC. Furthermore, these levels tend to change with the anticancer treatments in patients with OC and may be an important aspect of treatment monitoring and management for these patients.

Currently, OC is the major cause of cancer-related mortality among Indian men because of intense tobacco consumption. In addition, the incidence of OPC continues to increase among the young population. Therefore, the inclusion of OPC in the current study established a link between normal and malignant conditions. The screening and follow-up of patients with minimally invasive serum tests are appealing because of the accessibility to repeated sampling. The observations of this study are noteworthy because, to our knowledge, the simultaneous evaluation of serum fucose, fucoproteins, FucT, and fucosidase in patients with OC has not been reported previously.

Fucose is known as an important constituent of glycoproteins, which may be released into the circulation through increased turnover, secretion, and/or shedding from malignant cells. In the current study, we observed a significant increase in serum fucose levels, fucoprotein levels, and FucT activity in patients with OC and OPC. Increased levels of serum/tissue fucose in patients with cancer have been reported by various investigators and are of considerable interest because of their potential clinical applications.6, 8-10, 33 Rao et al reported higher levels of serum fucose in OC and OPC in a small number of patients.7

Lectins have been used as an important tool for exploring the structure and functions of carbohydrates of glycoproteins, and they contributed tremendously to the advancement of glycobiology.34 Increased fucose levels may lead to altered or unique glycoconjugates. In the current study, it is interesting to note that the quantitative analysis of fucosylation changes in patients with OC and OPC indicated overall increased fucosylation. Among these, the ∼43-kD and ∼66-kD serum glycoproteins produced significant hyperfucosylation in patients who had untreated OC compared with controls and with patients who had OPC. Furthermore, the expression of fucoproteins broadly reflected increased fucose levels, which may have been caused by hyperfucosylation of the ∼43-kD and ∼66-kD serum glycoproteins. Our results are in accordance with previous observations by Thompson and Turner15, 16 and Shirahama et al35 Those authors reported increased fucosylation of serum and tumor glycoproteins as a prognostic marker in patients with cancer that reflected tumor burden and treatment response. Moreover, they reported that the role of linkage-specific fucose in the development of an invasive cancer phenotype reportedly was associated with the expression of fucosylated Lewis antigens.

The turnover of fucose residue in glycoconjugates can be implied by measuring the activity of FucT and fucosidase. Increased FucT leads to hyperfucosylation, which manifests as elevated fucoproteins during neoplastic transformation. We observed a significant rise in FucT activity along with fucose and fucoprotein levels in serum from patients with OC and OPC compared with the activity observed in controls. Increased total and linkage-specific FucT activities in serum or tumor samples have been reported in endometrial, hepatocellular, and ovarian carcinomas.8, 11-13 In a previous study, it was demonstrated shown that α1,6-FucT is responsible for the fucosylation of AFP, which is a useful serum marker for hepatoma.16 Enhanced expression of α-(1-3/4)FucT was associated with increased expression of fucosylated Lewis antigens Lea/b/x/y on the cell surface.20 These Lewis antigens mediate a critical binding of neoplastic cells to activated endothelium through E-selectin, which is one of the key steps in hematogenous metastasis.36 We also observed an association of fucosylated antigen sialyl Lex and FucT activity in malignant and precancerous tissues (data not shown), suggesting that linkage-specific analysis of FucT may yield vital information regarding molecular and phenotypic changes in OC.

Our multivariate analysis indicated a positive association of serum fucose and fucose/total proteins (TP) with tumor differentiation, which suggests that the levels increased as the degree of differentiation decreased from well differentiated to poorly differentiated tumors. Recently, Kossowska et al suggested that the fucosylation status of serum glycoproteins may serve as a promising predictor of survival for patients with lung cancer.17 A positive association has been established between elevated serum fucose levels and a poor prognosis in patients with breast cancer.37 Shirahama et al used qualitative immunohistochemical analysis and observed that the degree of fucosylation of tumor glycoproteins in patients with urinary bladder cancer reflected metastatic potential and poor survival.35

Furthermore, in the current results provide evidence that serum fucosylation changes can be used to monitor treatment in patients with OC. Serum levels of fucose, fucoproteins, and FucT were decreased in patients who had a successful therapeutic response (the CR group) and remained elevated or comparable in the NR group compared with their PT levels. Response to therapy, no response, and persistent disease correlated well with serum fucosylation changes in patients with OC. The results of the current study strongly suggest that serum fucosylation changes can be useful clinically during post–treatment follow-up. The markers may serve as indicators of disease and may help to predict treatment response, lymph node recurrence, or possible therapy failure. Thus, close monitoring of serum fucosylation changes may be promising as a tool for patients with OC. It has been reported that serum FucT activities were reduced in breast and gastric cancers after the removal of tumors, whereas recurrence or metastasis tended to increase FucT activity.38, 39

L-fucosidase, an important enzyme for fucose catabolism, also is responsible for the altered fucosylation status of glycoproteins. We observed that serum fucosidase was elevated significantly in patients with OC and OPC compared with the controls. In the current study, we have demonstrated the efficiency of serum α-L-fucosidase for discriminating the 3 study groups. Considerably higher sensitivity (60%) and specificity (94% for OPC; 89% for OC) of this enzyme were observed at a threshold >450.6 U/mL, which is suggestive of its usefulness for the detection of early changes during cancer development. Similarly, α-L-fucosidase in serum has been reported as a promising marker for the early detection and diagnosis of hepatocellular carcinoma.21, 24, 25, 40 Ayude et al demonstrated the diagnostic and prognostic utility of decreased serum α-L-fucosidase activity in patients with colorectal cancer.21, 24 We observed a positive correlation of serum fucosidase activity with the extent of disease, which may indicate an association of this enzyme activity with tumor progression. In addition, our data indicated that α-L-fucosidase activity was altered in response to anticancer treatment in the CR and NR groups and may be useful for monitoring the treatment response in patients with OC after primary tumor resection. It is noteworthy that fucosidase levels in the CR group were similar to the threshold value of >450.6 U/mL, whereas the NR group had levels that were higher than the threshold. Thus, on the basis of the current data, we propose using the preoperative serum α-L-fucosidase level as a marker for the detection of early changes during OC development and for monitoring postoperative treatment response in patients with OC. The nonsignificant increase in enzyme activity that we observed in patients who had cancer compared with patients who had OPC suggests that fucosidase activity increases during early neoplastic changes to compensate for hyperfucosylation of cell surface glyco-proteins. However, the incorporation of fucose during malignant transformation may be several fold higher than the hydrolytic cleavage of this residue.

In conclusion, the current findings provide clear evidence of increased fucosylation of serum glycoproteins and its association with OC development and treatment response. The evaluation of serum α-L-fucosidase changes in normal, precancerous, and malignant conditions suggest its potential use for the early detection and for monitoring treatment response in patients with OC.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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