An Autoantibody-Mediated Immune Response to Calreticulin Isoforms in Pancreatic Cancer

Abstract

The identification of circulating tumor antigens or their related autoantibodies provides a means for early cancer diagnosis as well as leads for therapy. We have used a proteomic approach to identify proteins that commonly induce a humoral response in pancreatic cancer. Aliquots of solubilized proteins from a pancreatic cancer cell line (Panc-1) were subjected to two-dimensional PAGE, followed by Western blot analysis in which sera of individual patients were tested for primary antibodies. Sera from 36 newly diagnosed patients with pancreatic cancer, 18 patients with chronic pancreatitis, 33 patients with other cancers, and 15 healthy subjects were analyzed. Autoantibodies were detected against either one or two calreticulin isoforms identified by mass spectrometry in sera from 21 of 36 patients with pancreatic cancer. One of 18 chronic pancreatitis patients and 1 of 15 healthy controls demonstrated autoantibodies to calreticulin isoform 1; none demonstrated autoantibodies to isoform 2. None of the sera from patients with colon cancer exhibited reactivity against either of these two proteins. One of 14 sera from lung adenocarcinoma patients demonstrated autoantibodies to calreticulin isoform 1; 2 of 14 demonstrated autoantibodies to isoform 2. Immunohistochemical analysis of calreticulin in pancreatic/ampullary tumor tissue arrays using an isoform nonspecific antibody revealed diffuse and consistent cytoplasmic staining in the neoplastic epithelial cells of the pancreatic and ampullary adenocarcinomas. The detection of autoantibodies to calreticulin isoforms may have utility for the early diagnosis of pancreatic cancer.

Tumor-associated Antigen Arrays for the Serological Diagnosis of Cancer

“…Interestingly, although glucose-6-phosphate dehydrogenase is widely expressed in normal cells, the breast cancer sera reacted preferentially with some isoforms of the protein detected by MS, suggesting that autoantibodies in these sera might be driven by posttranslational modifications (8), which is consistent with previous observations that intracellular proteins targeted by autoantibodies in systemic autoimmune diseases often undergo these modifications (41). The SERPA approach has also been used to identify calreticulin and DEAD-box protein 48 (DDX48) as target autoantigens in pancreatic cancer (33,38) and the Rho GDP dissociation inhibitor 2 as a major candidate TAA in leukemia (26). Although a powerful approach, SERPA also presents some drawbacks, including the difficulty in generating reproducible gels, which leads to variations in the migration of spots of interest from gel to gel; the difficulty in resolving certain classes of proteins; the requirement of highly expensive and sophisticated MS and imaging equipment and appropriately trained personnel; and the dependence on Western blotting for the serological screening, which has an inherent bias toward the detection of denatured epitopes (8,135,136).…”

“…These autoantibodies have been detected in several human cancers, and significant advances have been made in the identification of their target antigens, particularly in lung cancer (28,30), colorectal cancer (36), breast cancer (29), prostate cancer (27,37), leukemia (26), non-Hodgkin lymphoma (24), hepatocellular carcinoma (25,32,34), ovarian cancer (31), pancreatic cancer (33,38), and paraneoplastic neurological syndromes (35). Although the mechanisms leading to autoantibody production in cancer patients are not clearly understood, emerging evidence indicates that most TAAs are cellular proteins whose aberrant regulation or function could be linked to malignancy (3).…”

Seromic profiling of ovarian and pancreatic cancer

“…Arrays can be used with high throughput to determine patterns of antigens recognized by autoantibodies during the course of diseases, such as autoimmunity or cancer (1)(2)(3)(4)(5)(6)(7)(8)(9), as well as a way to characterize the repertoire of serological responses in healthy individuals (10). Defined protein collections assembled from phage expression systems or purified from recombinant sources were used to detect antibody responses in the serum of ovarian (11,12), breast (13), colorectal (2), pancreatic (14), or lung (15,16) cancer patients, and some of these responses appear to have diagnostic significance. In addition, protein array tools are also useful to measure changes in antibody responses to vaccination or immunotherapies, although it has yet to be applied to the monitoring of cancer treatments (17,18).…”

Étude de la réaction immunitaire humorale aux cancers par des approches protéomiques

“…Cette approche, qui a été nommée par la suite SERPA (serological proteome analysis) [18], Proteomex [19,20] ou SPEAR (serological and proteomic evaluation of antibody response) [21], a été utilisée avec succès pour identifier des AAT dans plusieurs autres types de cancers. Sans vouloir être exhaustif, on peut citer, par exemple, la protéine peroxirédoxine 6 dans le carcinome squameux de l'oesophage [22,23], les protéines triosephosphate isomérase (Tim) et superoxyde dismutase (MnSOD) dans le carcinome squameux du poumon [22,23], la calréticuline dans les cancers hépatique [24] et du pancréas [25] et RS/DJ1 dans le cancer du sein [26].…”