Immunogenicity of SEREX-identified antigens and disease outcome in pancreatic cancer

Abstract

Despite spontaneous or vaccination-induced immune responses, pancreatic cancer remains one of the most deadly immunotherapy-resistant malignancies. We sought to comprehend the spectrum of pancreatic tumor-associated antigens (pTAAs) and to assess the clinical relevance of their immunogenicity. An autologous SEREX-based screening of a cDNA library constructed from a pancreatic T3N0M0/GIII specimen belonging to a long-term survivor (36 months) revealed 18 immunogenic pTAA. RT-PCR analysis displayed broad distribution of the identified antigens among normal human tissues. PNLIPRP2 and MIA demonstrated the most distinct pancreatic cancer-specific patterns. ELISA-based screening of sera for corresponding autoantibodies revealed that although significantly increased, the immunogenicity of these molecules was not a common feature in pancreatic cancer. QRT-PCR and immunohistochemistry characterized PNLIPRP2 as a robust acinar cell-specific marker whose decreased expression mirrored the disappearance of parenchyma in the diseased organ, but was not related to the presence of PNLIPRP2 autoantibodies. Analyses of MIA-known to be preferentially expressed in malignant cells-surprisingly revealed an inverse correlation between intratumoral gene expression and the emergence of autoantibodies. MIA(high) patients were autoantibody-negative and had shorter median survival when compared with autoantibody-positive MIA(low) patients (12 vs. 34 months). The observed pTAA spectrum comprised molecules associated with acinar, stromal and malignant structures, thus presenting novel targets for tumor cell-specific therapies as well as for approaches based on the bystander effects. Applying the concept of cancer immunoediting to interpret relationships between gene expression, antitumor immune responses, and clinical outcome might better discriminate between past and ongoing immune responses, consequently enabling prognostic stratification of patients and individual adjustment of immunotherapy.

Fig. 1

Immunogenicity of SEREX-defined antigens and outcome in patients with pancreatic adenocarcinoma. a The immunogenicity of SEREX-identified MIA, IFITM3 or PNLIPRP2 was determined by ELISA-based detection of circulating autoantibodies serving as a read-out of the humoral immune response. The frequency of antigen-specific and cumulative antibody seropositivity of donors and patients with pancreatic cancer was calculated and related to the survival time. b Kaplan–Meier analysis showed a better outcome trend for the patients carrying autoantibodies against the SEREX-identified antigens

Fig. 2

PNLIPRP2 as an indicator of the acinar loss in pancreatic diseases. a QRT-PCR showed that PNLIPRP2 mRNA expression was absent in the cultured tumor cells, high in normal pancreatic tissues and gradually lost in inflammatory and cancerous pancreatic lesions. b PNLIPRP2 mRNA levels in relation to the proportion of the acinar parenchyma presented in the analyzed specimens. c Western blot analysis showed the loss of PNLIPRP2 protein in pancreatic specimens with various degrees of parenchymal loss. d Immunohistochemistry revealed acinar cells as a single source of PNLIPRP2 in pancreatic tissues

Fig. 3

Association of MIA expression levels with autoantibody response and clinical outcome. The overexpression of MIA mRNA in cancerous tissues (a) did not coincide with anti-MIA antibody response (b), but tended to be associated with shorter survival (p = 0.076; Kaplan–Meier analysis, (c). In contrast, median survival time was significantly (p < 0.05) longer in the low-expressing group carrying autoantibodies to MIA (MIA^low-Ab Pos, (d)

Fig. 4

MIA-based prognostic algorithm and concept of immunoediting. This flow chart shows how viewing MIA mRNA expression levels in the context of immunogenicity (as determined by anti-MIA autoantibodies detected in sera) may enable prognostic stratification of the pancreatic cancer patients. An immunoediting-based interpretation of each antigen–antibody outcome signature is consequently attempted, pointing to the individualized therapeutic options