Calreticulin is a B cell molecular target in some gastrointestinal malignancies

Abstract

Calreticulin, upon translocation to the cell surface, plays a critical role in the recognition of tumour cells and in experimentally induced cellular anti-tumour immunity. However, less is known about anti-calreticulin antibodies and their role in malignancies. Using enzyme-linked immunosorbent assay (ELISA), we found immunoglobulin (Ig)A and/or IgG anti-calreticulin antibodies in sera of approximately 63% of patients with hepatocellular carcinoma (HCC), 57% of patients with colorectal adenocarcinoma (CRA) and 47% of patients with pancreatic adenocarcinoma (PACA), while healthy controls, patients with viral hepatitis C and with chronic pancreatitis reached only 2%, 20% and 31% seropositivity, respectively. We found significantly elevated mean levels of IgA anti-calreticulin antibodies (P < 0.001) in patients with HCC (78.7 +/- 52.3 AU, mean +/- standard deviation), PACA (66.5 +/- 30.9 AU) and CRA (61.8 +/- 25.8 AU) when compared to healthy controls (41.4 +/- 19.2 AU). Significantly elevated mean levels of IgG anti-calreticulin antibodies (P < 0.001) were detected in patients with HCC (121.9 +/- 94.2 AU), gall bladder adenocarcinoma (118.4 +/- 80.0 AU) and PACA (88.7 +/- 55.6 AU) when compared to healthy controls (56.7 +/- 22.9 AU). Pepscan analysis revealed a large number of antigenic epitopes of calreticulin recognized by both IgA and IgG antibodies of patients with HCC and PACA, indicating robust systemic immune response. Moreover, significantly elevated levels of antibodies against peptide KGEWKPRQIDNP (P < 0.001) in these patients, tested by ELISA, confirmed the distinct character of antibody reactivity against calreticulin. The high occurrence and specificity of serum anti-calreticulin autoantibodies in the majority of patients with some gastrointestinal malignancies provide the evidence for their possible clinical relevance.

Fig. 1

Shown is a scatter graph of individual values of serum immunoglobulin (Ig)A (a) and IgG (b) anti-calreticulin antibodies tested by enzyme-linked immunosorbent assay (ELISA) in patients with hepatocellular carcinoma (HCC, n = 43), viral hepatitis C (VHC, n = 20), pancreatic adenocarcinoma (PACA, n = 55), chronic pancreatitis (CP, n = 16), colorectal adenocarcinoma (CRA, n = 30), gall bladder adenocarcinoma (GBA, n = 29) and healthy controls (C, n = 56). The anti-calreticulin antibody serum levels are expressed in arbitrary units (AU). Solid lines indicate the mean. Cut-off values are 80 AU for IgA and 102 AU for IgG antibodies. ***P < 0·001; **P < 0·01; n.s., not significant.

Fig. 2

Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, serum immunoglobulin (Ig)A and IgG antibody reactivity with human recombinant calreticulin (CRT, ∼60 kDa) in patients with hepatocellular carcinoma (HCC) and pancreatic adenocarcinoma (PACA) was analysed. Lane 1 represents the marker of molecular weights, and lane 2 is SDS-PAGE-subjected calreticulin stained by Coomassie brilliant blue. Nitrocellulose-blotted recombinant calreticulin was incubated with sera of HCC (lanes 3–5) and PACA (lanes 6–8) patients, and for control purposes, with specific rabbit anti-calreticulin antibodies (lane 12) and sera of healthy individuals (lanes 14, 15). The binding of anti-calreticulin antibodies was detected by peroxidase conjugated anti-human IgA (lanes 3, 4, 6, 7, 14), anti-human IgG (lanes 5, 8, 15) or anti-rabbit secondary antibodies (lane 12). Lane 9 shows the reactivity of secretory IgA antibodies of patient with HCC. As a control, secondary antibodies were used (lanes 10, 11, 13).

Fig. 4

Recognition profiles (—) of serum immunoglobulin (Ig)A and IgG antibodies against calreticulin decapeptides (each overlapping by eight amino acids) in patients with hepatocellular carcinoma (a, n = 10), pancreatic adenocarcinoma (b, n = 10) and healthy controls (----, n = 12). Peaks of antibody reactivity [expressed as index of reactivity (RI)] which exceeded the horizontally dotted line (*) represent calreticulin peptides that are recognized frequently by antibodies of patients in the tested group. The synthetic peptides 1–88, 89–143 and 144–201 correspond to the N-, P- and C-domains (distinguished by vertically dotted lines) of the calreticulin molecule, respectively.

Fig. 3

This figure shows the reactivity of serum immunoglobulin (Ig)A (a,b) and IgG (c,d) antibodies from patients with hepatocellular carcinoma (a,c), pancreatic adenocarcinoma (b,d), and healthy controls [e (IgA), f (IgG)] with calreticulin decapeptides using Pepscan (no. = number of calreticulin peptides).

Fig. 5

The serum levels of immunoglobulin (Ig)A antibodies against peptides KGEWKPRQIDNP (a), VQFTVKHEQNID (b) and GVTKAAEKQMKD (c) were tested by enzyme-linked immunosorbent assay (ELISA) in patients with hepatocellular carcinoma (HCC, n = 16), pancreatic adenocarcinoma (PACA, n = 16) and healthy controls (C, n = 16). Data are expressed as optical density (OD). Solid lines represent the mean values. ***P < 0·001; n.s., not significant.