47-mG 2a : A Mouse IgG 2a -Type of PcMab-47 Useful for Detecting Podocalyxin in Esophageal Cancers by Immunohistochemistry

Introduction

Esophageal cancer is one of the highly malignant tumors with respect to prognosis and mortality rate. It is the eighth most common cancer and most common cause of cancer-related death. More than 450,000 new cases and 400,000 deaths have been reported worldwide.^(1,2) Esophageal cancer comprises two common histological types: squamous cell carcinoma (SCC) and adenocarcinoma (AC). SCC accounts for about 90% of esophageal cancers.^(3,4) Despite improvement in treatment strategies, prognosis remains poor with overall 5-year survival rates ranging from 15% to 25%.⁽⁵⁾ This cancer is treated by surgery that can be complemented by radiotherapy with or without chemotherapy. Preoperative neoadjuvant chemotherapy with cisplatin accompanied by 5-fluorouracil can be effective and is regarded as standard treatment.^(6,7) In contrast, adjuvant therapy is ineffective in many cases.^(8,9) Several clinical trials testing the treatment potential of monoclonal antibodies (mAbs) against esophageal cancer are ongoing.^(10–12) However, few molecular targeting drugs against this disease have been approved.

Podocalyxin (PODXL) is a type-I transmembrane protein highly glycosylated with N-glycan or O-glycan⁽¹²⁾ belonging to the CD34 family of sialomucins. It is expressed in normal tissues, such as kidney, heart, breast, and pancreas. PODXL upregulation has been correlated with tumor progression, invasion, and metastasis.⁽¹³⁾ It is used as a diagnostic marker in numerous cancers, including brain,⁽¹⁴⁾ colorectal,⁽¹⁵⁾ oral,⁽¹⁶⁾ lung,^(17,18) bladder,⁽¹⁹⁾ prostate,⁽²⁰⁾ and ovarian cancers.⁽²¹⁾ Indeed, Borg et al. reported that PODXL expression in esophageal AC is an independent prognostic biomarker for poor overall survival.⁽²²⁾ Therefore, specific and sensitive anti-PODXL mAbs are required.

We previously established anti-PODXL mAbs PcMab-47 (mouse IgG₁, kappa)⁽²³⁾ and mouse IgG_2a-type (47-mG_2a).⁽²⁴⁾ In this study, we investigated their utility for immunohistochemical analysis of esophageal squamous cell carcinoma (ESCC).

Materials and Methods

Results and Discussion

Previously, we immunized mice with recombinant PODXL and produced PcMab-47 (mouse IgG₁, kappa).⁽²³⁾ This antibody was then engineered into a mouse IgG_2a-type mAb (47-mG_2a).⁽²⁴⁾ 47-mG_2a exhibited a much higher PODXL-binding affinity compared with PcMab-47. Immunohistochemical analysis of oral cancer tissues revealed that those mAbs sensitively stain cells in a cytoplasmic pattern. In this study, we performed immunohistochemical analysis using PcMab-47 and 47-mG_2a against ESCC.

Representative immunohistochemistry results of PcMab-47 and 47-mG_2a against ESCC are shown in Figure 1. Both antibodies stained cancer cells in a cytoplasmic pattern (Fig. 1). As shown in Table 1 and Supplementary Table S1, PcMab-47 stained 123/130 (94.6%) and 47-mG_2a 127/130 (97.7%) of ESCCs, indicating that both antibodies can be used to detect PODXL in ESCC using this method. Among more than 3+ cases, 47-mG_2a was more sensitive than PcMab-47 with 56/127 (44.1%) and 41/123 (33.3%) occurrences, respectively (Table 1). In conclusion, 47-mG_2a can be used to detect PODXL in ESCC by immunohistochemical analysis.

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FIG. 1.

Immunohistochemical analysis by anti-PODXL antibodies against esophageal squamous cell carcinoma (Case-26). Sections were incubated with 5 μg/mL of PcMab-47 (A, D) and 0.5 μg/mL 47-mG_2a (B, E) for 1 hour at room temperature followed by treatment with Envision+ kit for 30 minutes. Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB) for 2 minutes, and sections were then counterstained with hematoxylin. (C, F) HE staining; scale bar = 100 μm. HE, hematoxylin and eosin; PODXL, podocalyxin.