Detection of early pancreatic ductal adenocarcinoma with thrombospondin-2 and CA19-9 blood markers

Abstract

Markers are needed to facilitate early detection of pancreatic ductal adenocarcinoma (PDAC), which is often diagnosed too late for effective therapy. Starting with a PDAC cell reprogramming model that recapitulated the progression of human PDAC, we identified secreted proteins and tested a subset as potential markers of PDAC. We optimized an enzyme-linked immunosorbent assay (ELISA) using plasma samples from patients with various stages of PDAC, from individuals with benign pancreatic disease, and from healthy controls. A phase 1 discovery study (n = 20), a phase 2a validation study (n = 189), and a second phase 2b validation study (n = 537) revealed that concentrations of plasma thrombospondin-2 (THBS2) discriminated among all stages of PDAC consistently. The receiver operating characteristic (ROC) c-statistic was 0.76 in the phase 1 study, 0.84 in the phase 2a study, and 0.87 in the phase 2b study. The plasma concentration of THBS2 was able to discriminate resectable stage I cancer as readily as stage III/IV PDAC tumors. THBS2 plasma concentrations combined with those for CA19-9, a previously identified PDAC marker, yielded a c-statistic of 0.96 in the phase 2a study and 0.97 in the phase 2b study. THBS2 data improved the ability of CA19-9 to distinguish PDAC from pancreatitis. With a specificity of 98%, the combination of THBS2 and CA19-9 yielded a sensitivity of 87% for PDAC in the phase 2b study. A THBS2 and CA19-9 blood marker panel measured with a conventional ELISA may improve the detection of patients at high risk for PDAC.

Figure 1. Phase 1 validation studies and THBS2 expression in PDAC and other human tumors

(A) AUC analysis of blinded ELISA data for the proteinsMMP2, MMP10, and THBS2 in plasma samples from 10 patients with PDAC at various stages of disease compared to 10 healthy controls. (B) Boxplots of THBS2 mRNA expression measured in various human tumors (sample sizes in parentheses) assessed by RNA-seq. Tumors are sorted in order of decreasing median expression of THBS2 mRNA. Of the pancreatic cancer samples from the TGCA database (n=179), we analyzed only PDAC (n=134). All expression values are log2(RSEM values =1) transformed.

Figure 2. THBS2 and CA19-9 concentrations in plasma samples from patients with PDAC versus healthy controls

(A, CD) Scatter plots of THBS2 concentrations in plasma samples from patients at all stages of PDAC versus controls for the phase 2a (A) and phase 2b (D) validation studies. (B, D, E) ROC curves of THBS2, CA19-9, and THBS2+CA19-9 concentrations in plasma samples from patients with all stages of PDAC versus healthy controls for phase 2a (PDAC n=81, controls n=80) (B) and phase2b (PDAC n=197, controls n=140) (D) studies. P values are shown. (E, F) Scatter plots showing THBS2 and CA19-9 concentrations in plasma samples in patients with all stages of PDAC cases versus healthy controls for Phase 2a (F) and Phase 2b (G) studies.

Figure 3. THBS2 and CA19-9 concentrations in plasma samples from all PDAC cases versus versus benign pancreatic disease cases

(A–D)Shown are ROC curves for THBS2, CA19-9, and THBS2+CA19-9 concentrations in plasma samples from patients with PDAC in the Phase 2b study (n=197) versus pancreatitis (n=55, A), PDAC vs. intraepithelial pancreatic mucinous neoplasm (n=115, B), PDAC vs. PNET (n=30, C), and PNET (n=30) vs. healthy controls (n=140, D).

Figure 4. Expression of THBS2 in human PanIN tissue and PDAC tumor tissue

(A) Representative THBS2 immunohistochemistry analysis of incidental PanIN stage I–II tissue derived from the head and neck of a pancreas from a patient with pancreatic periampullary cancer using two different antibodies. The arrows indicate PanIN2 tissue staining positively for THBS2; dotted arrows indicate weak or negative staining of PanIN1 tissue. THBS2 expression, designated by arrows, was also confirmed in stage II PDAC (C–E) and stage III (F–K) pancreatic cancer tissue arrays. Competitive assays were performed for antibody #2 by pre-incubating the antibody with a 10-fold excess of antigen peptide (E, H, K), to confirm target specificity. Brown color indicates THBS2 staining and blue color indicates hematoxylin nuclear staining. THBS2 was detected in the epithelial cells of non-invasive lesions (PanINs and intraepithelial pancreatic neoplasms) and poorly differentiated PDAC tissue as well as in fibroblasts in invasive PDAC tissue (see Table S8 and Figure S6).