GLUT1 and ASCT2 Protein Expression in Papillary Thyroid Carcinoma Patients and Relation to Hepatitis C Virus: A Propensity-Score Matched Analysis

Abstract

Purpose: Recently, glucose and amino acid transporters have gradually become a hot topic in thyroid gland biology and cancer research. We aimed to investigate the expressions of glucose transporter 1 (GLUT1) and glutamine transporter 2 (ASCT2) in papillary thyroid carcinoma (PTC) and their clinical significance and relation to HCV-related hepatitis.

Patients and methods: Screening 202 TC tissue samples against the selection criteria using a propensity-score matched analysis to adjust for age, sex, side of tumor, histopathological variants, TNM staging system, and the positivity for HCV yielded 51 matched (17 HCV positive and 34 HCV negative) PTC samples. The expressions of GLUT1 and ASCT2 expressions were detected by immunohistochemical staining. Kaplan-Meier survival curves were generated for disease-free and overall survival, and multivariate Cox regression analysis was applied to identify predictors for mortality.

Results: Of 51 thyroid cancer tissues, 85% showed positive GLUT1 cytoplasmic staining, and 26% had a high expression score. All thyroid cancer specimens demonstrated ASCT2 cytoplasmic staining with membranous accentuation. Of these, 78% showed a high expression score, and 22% showed weak staining. On stratifying the study cohort based on the HCV status, HCV negative cohort showed a significantly higher immunoreactivity score for GLUT1 (p = 0.004) but not ASCT2 (p = 0.94) than HCV positive group. The expressions of the studied transporters showed no significant associations with the prognostic features of PTC nor the disease-free/overall survival.

Conclusion: GLUT1 and ASCT2 immunohistochemical staining showed positive expression with variable intensity in nearly 85% and 100% of PTC tissue samples compared to normal ones, respectively. Furthermore, GLUT1 protein expression, not ASCT2, showed a higher immunoreactivity score in PTC patients who are negative for HCV than cancer patients with positive HCV. Meanwhile, the expression of both protein markers was not associated with the clinicopathological characteristics of the studied PTC patients. Further large-scale multicenter studies are recommended to validate the present findings.

Keywords: HCV; glucose transporter 1; glutamine transporter 2; immunohistochemistry; papillary thyroid cancer.

Figure 1

Workflow for the selection process of the study population.

Figure 2

Immunohistochemistry staining of GLUT1 protein. (A) Non-neoplastic thyroid tissue with negative staining (x400), (B) papillary thyroid carcinoma (Classic variant) with diffuse cytoplasmic solid staining with membranous accentuation (x200), (C) papillary thyroid carcinoma with diffuse cytoplasmic solid staining with membranous accentuation (x400), (D) papillary thyroid carcinoma (Tall cell variant) with weak staining and positive inflammatory cells in the papillary core (x400), (E) papillary thyroid carcinoma (follicular variant) showing moderate diffuse cytoplasmic staining (x200), (F) papillary thyroid carcinoma (follicular variant) showing weak cytoplasmic staining (x400).

Figure 3

Immunohistochemistry staining of ASCT2 protein in well-differentiated thyroid cancer specimens. (A) Non-neoplastic thyroid tissue with negative staining (x400), (B) papillary thyroid carcinoma with diffuse cytoplasmic solid staining with membranous accentuation (x200), (C) papillary thyroid carcinoma with diffuse cytoplasmic solid staining with membranous accentuation (x400), (D) papillary thyroid carcinoma with negative staining (x400), (E) papillary thyroid carcinoma (follicular variant) showing partial cytoplasmic staining (x200), (F) papillary thyroid carcinoma (follicular variant) showing partial cytoplasmic staining (x400).

Figure 4

Differential expression of GLUT1 and ASCT2 proteins in thyroid cancer patients according to their hepatitis C virus (HCV) infection status. The immune histochemical staining score (IHS) was determined by combining an estimate of the percentage of immunoreactive cells (quantity score) with the staining intensity (staining intensity score). For the percentage of staining, 0 indicated no staining; 1: 1–10% of cells stained; 2: 11–50%; 3: 51–80%; 4: 81–100%. Staining intensity scores were as follows: 0: negative; 1: weak; 2: moderate; and 3: strong. The raw data were converted to the IHS by multiplying the quantity and staining intensity scores. The IHS ranged from 0 to 12, with 0 indicating negative, 1 to 4 indicating weak, 5 to 8 indicating moderate, and 9 to 12 indicating strong immunoreactivity. (A) Overall GLUT1 protein expression is categorized according to protein staining severity scores. (B) GLUT1 protein immunohistochemistry quantitative score (from 0 to 12) according to the presence of HCV antibodies. Median and interquartile range was 4 (2–6) in HCV negative group, compared to 2 (0.5–4) in HCV positive group. Mann–Whitney U-test was used. (C) GLUT1 protein immunoreactivity categories are classified according to the presence of HCV. A two-sided Chi-square test was used. (D) Overall ASCT2 protein expression is categorized according to protein staining severity scores. (E) ASCT2 protein immunohistochemistry quantitative score (from 0 to 12) according to the presence of HCV antibodies. The median and interquartile range was 6 (6–9.75) in HCV negative group, compared to 6 (4.5–9) in HCV positive group. Mann–Whitney U-test was used. (F) ASCT2 protein immunoreactivity categories are classified according to the presence of HCV. A two-sided Chi-square test was used. Statistical significance was set at p-value < 0.05.

Figure 5

Disease-free survival (DFS) analysis. Cohorts with moderate and intense staining scores were considered high expression (scores 5–12) compared to negative/weak ones who considered low expression (scores 0–4). (A) Kaplan–Meier curve for survival analysis based on GLUT1 protein expression. (B) Kaplan–Meier curve for survival analysis based on ASCT2 protein expression. Log Rank test was applied for significance test. (C) Independent risk factors for disease progression. Cox proportional hazard regression analysis for disease-free survival (DFS) was employed to predict putative independent risk factors. Hazard ratio (HR) and 95% confidence interval (CI) are shown.