Mitogen activated protein kinase kinase kinase 3 (MAP3K3/MEKK3) overexpression is an early event in esophageal tumorigenesis and is a predictor of poor disease prognosis

The Institutional Human Ethics Committee of the All India Institute of Medical Sciences (AIIMS), New Delhi, India, approved this study prior to its commencement. Tissue specimens were obtained by diagnostic or therapeutic procedures from patients with clinically defined esophageal dysplasia (n = 61) attending the Outpatient Clinic of the Departments of Surgical Disciplines and Gastrointestinal Surgery, AIIMS. Tissue specimens were also collected from 93 ESCC patients undergoing curative cancer surgery during the period 2005–2010, after obtaining the patients’ written consent. Wherever possible, non-malignant tissues were taken, each from a site distant from the surgically resected ESCC. Non-malignant esophageal tissues were also collected from the patients attending the Endoscopy clinic in the Outpatient Department of Gastroenterology, after obtaining the patients’ written consent. Taken together, these 47 non-malignant esophageal tissues with histological evidence of normal epithelia constituted the normal group. After excision, tissues were immediately snap-frozen in liquid nitrogen and stored at −80°C in the Research Tissue Bank till further use; one part of the tissue was collected in 10% formalin and embedded in paraffin for histopathological and immunohistochemical analyses. Histologically confirmed esophageal normal epithelia, dysplasia, and ESCC as revealed by hematoxylin and eosin (H&E) staining were used for immunohistochemistry [21, 22]. Patient demographic, clinical, and pathological data were recorded in a pre-designed Performa as described previously to establish a clinical database [21, 22]. The information documented included clinical TNM staging (tumor, node, and metastasis based on the Union International Center le Cancer TNM classification of malignant tumors 2002), site of the lesion, histopathological differentiation, age and gender. All the ESCC tissues analyzed in this study had more than 80% tumor cells in H&E sections.

Eighty two of 93 ESCC patients who underwent treatment from 2005–2010 could be investigated and evaluated in the esophageal cancer follow-up clinic at regular time intervals, while 11 patients did not report in the follow up clinic. Survival status of the ESCC patients was verified and updated from the records of the Tumor Registry, Department of Gastrointestinal Surgery, AIIMS, as of June 2013. ESCC patients were monitored for a maximum period of 7.5 years. Disease-free survival time is defined as the time from completion of primary treatment till the patient showed any clinical and radiological evidence of local or regional disease, or distant metastasis at the time of the last follow-up of patients monitored in this study. Thirty one patients who did not show recurrence were alive until the end of the follow-up period. Only disease-free survival (expressed as the number of months from the date of surgery to loco-regional relapse/death) was evaluated in the present study, as the number of deaths due to disease progression did not allow a reliable statistical analysis.

Paraffin-embedded sections (5 μm) of human esophageal histological normal (n =47), dysplasia (n = 61) and ESCC (n = 93) were collected on gelatin-coated slides. In brief, the sections were deparaffinized in xylene, hydrated in gradient alcohol, and pre-treated in a microwave oven for 10 min at 800 W and 5 min at 480 W in Citrate buffer (0.01 M, pH = 6.0) for antigen retrieval. The sections were incubated with hydrogen peroxide (3% v/v) in methanol for 30 min to quench the endogenous peroxidise activity, followed by blocking with 1% bovine serum albumin (BSA) to preclude non-specific binding. Thereafter, the slides were incubated with rabbit polyclonal anti-MEKK3 antibody (0.5 mg/ml, sc-28769, Santa Cruz Biotechnology, San Diego, CA) for 16 h at 4°C. The primary antibody was detected using the streptavidin-biotin complex with the Dako LSAB plus kit (Dako Cytomation, Glostrup, Denmark) and diaminobenzidine as the chromogen as described previously [23]. In the negative control tissue sections, the primary antibody was replaced by isotype specific non-immune mouse IgG. A section from breast cancer tissue was used as a positive control in each batch of immunohistochemistry.

Each tissue section was evaluated for MEKK3 immunostaining using a semi-quantitative scoring system for both staining intensity and the percentage of positive epithelial cells [22]. For MEKK3 protein expression, sections were scored as positive if epithelial cells showed immunopositivity in the nucleus/cytoplasm when observed independently by three of us (MRH, RS, SDG), who were blinded to the clinical outcome (the slides were coded and the scorers did not have prior knowledge of the local tumor burden, lymphonodular spread, and grading of the tissue samples). The tissue sections were scored based on the% of immunostained cells as: ≤10% = 0; 11–30% = 1; 31–50% = 2; 51–70% = 3 and > 70% = 4. Sections were also scored semi-quantitatively on the basis of staining intensity as negative = 0; mild = 1; moderate = 2; intense =3. Finally, a total score was obtained by adding the scores of percentage positivity and intensity. The scoring by the three observers was discrepant in about 5% cases and a consensus on the final result was reached by re-evaluation of these slides and discussion. Based on sensitivity and specificity values for MEKK3, a total score cut-off value of 3 was defined as MEKK3 immunopositivity.

The immunohistochemical data were subjected to statistical analyses using the SPSS 13.0 software (Chicago, IL). Sensitivity and specificity were calculated and quantified using receiver operating characteristic (ROC) analyses. The positive predictive value (PPV) describes the proportion of the correctly classified cases. A total score cut-off value of 3 was defined as MEKK3 immunopositivity for statistical analyses. The relationships between MEKK3 protein expression and clinicopathological parameters were tested using Chi-Square and Fischer’s exact test. Two-sided p values were calculated and p < 0.05 was considered to be significant. Similarly, PPV was calculated for esophageal dysplasia and ESCC with respect to normal tissues. The correlation of MEKK3 staining with patient survival was evaluated using life tables constructed from survival data with Kaplan-Meier plots. Multivariate analysis was carried out using Cox regression model [24].

To determine the clinical significance of MEKK3 protein in ESCC, its expression was analyzed in clinical specimens from, histologically normal esophageal tissues, dysplasia, and ESCC using a specific anti-MEKK3 antibody by immunohistochemistry. Of the 47 normal tissues analyzed, 37 (79%) cases did not show detectable MEKK3 immunostaining in nucleus/cytoplasm of epithelial cells [Figure 1 (i)]; moderate staining was observed in differentiated epithelial cells in 10/47 (21%) normal tissues. Chi square trend analysis showed significant increase in MEKK3 expression (cytoplasmic/nuclear) in tissues obtained from different stages of esophageal tumorigenesis (normal, dysplasia and ESCC; Table 1, p_trend < 0.001). Notably, significant increase in cytoplasmic/nuclear localization of MEKK3 was observed in 34 of 61 (55.7%) dysplasia cases (p < 0.001, odd’s ratio (OR) = 4.6, 95% CI = 1.9-11.0) compared to normal esophageal tissues [Table 1 and Figure 1 (ii)]. Similar localization pattern of MEKK3 immunostaining was observed in ESCC as well [Figure 1 (iii, iv)]. Sixty three of 93 (67.7%) ESCCs showed cytoplasmic/nuclear localization of MEKK3 in tumor cells as compared to the normal tissues (p < 0.001, OR = 7.77, 95%, CI = 3.41-17.7). However, no significant correlations were observed between clinicopathological parameters of ESCC and MEKK3 expression (Table 1). No immunostaining was observed in ESCC tissue sections used as negative controls where the primary antibody was replaced by isotype specific IgG (Figure 1v). Receiver Operating Characteristic (ROC) analysis was used to determine the area-under-the-curve (AUC) - 0.68 and 0.80, with sensitivity of 55.56% and 52.46% for dysplasia and ESCC respectively, and specificity of 78.72% for both (Figure 2A and B; Table 2).

Kaplan–Meier survival analysis showed significantly reduced disease-free survival (median disease free survival = 10 months) in ESCC patients harbouring increased MEKK3 expression compared with the patients showing no nuclear/cytoplasmic MEKK3 immunostaining (p = 0.04, median DFS = 19 months), (Figure 3a). Notably, ESCC patients showing nodal positivity and MEKK3 overexpression had significantly reduced median DFS = 9 months in comparison node negative patients with low MEKK3 expression (median DFS = 21 months; range 1–90 months; p = 0.01) (Figure 3b).

Cox regression analysis was carried out to determine the prognostic potential of MEKK3 overexpression for ESCC in comparison with the other clinical parameters - nodal status (Table 3). MEKK3 overexpression in combination with nodal metastasis emerged as the most significant prognostic marker for ESCC (p = 0.015 HR = 2.082, 95% CI = 1.154- 3.756) (Table 3).