Identification of a biomarker panel using a multiplex proximity ligation assay improves accuracy of pancreatic cancer diagnosis

Abstract

Background: Pancreatic cancer continues to prove difficult to clinically diagnose. Multiple simultaneous measurements of plasma biomarkers can increase sensitivity and selectivity of diagnosis. Proximity ligation assay (PLA) is a highly sensitive technique for multiplex detection of biomarkers in plasma with little or no interfering background signal.

Methods: We examined the plasma levels of 21 biomarkers in a clinically defined cohort of 52 locally advanced (Stage II/III) pancreatic ductal adenocarcinoma cases and 43 age-matched controls using a multiplex proximity ligation assay. The optimal biomarker panel for diagnosis was computed using a combination of the PAM algorithm and logistic regression modeling. Biomarkers that were significantly prognostic for survival in combination were determined using univariate and multivariate Cox survival models.

Results: Three markers, CA19-9, OPN and CHI3L1, measured in multiplex were found to have superior sensitivity for pancreatic cancer vs. CA19-9 alone (93% vs. 80%). In addition, we identified two markers, CEA and CA125, that when measured simultaneously have prognostic significance for survival for this clinical stage of pancreatic cancer (p < 0.003).

Conclusions: A multiplex panel assaying CA19-9, OPN and CHI3L1 in plasma improves accuracy of pancreatic cancer diagnosis. A panel assaying CEA and CA125 in plasma can predict survival for this clinical cohort of pancreatic cancer patients.

Figure 1

Plasma levels of 21 tumor markers in pancreatic cancer patients and healthy controls measured by proximity ligation assay. Each boxplot corresponds to a single tumor marker measured in 95 samples by proximity ligation assay. Pancreatic cancer cases (52) are depicted at left, healthy controls (43) at right. Y-axis corresponds to log₂ PLA units. Central bars show the median for each cohort, boxes represent the interquartile 50^th percentile (IQ50). Whiskers represent 1.5 times the IQ50.

Figure 2

A tumor marker panel consisting of CA19-9, OPN, and CHI3L1 predicts the presence of pancreatic cancer more accurately than CA19-9 alone. (A) Each row corresponds to 1 of 20 randomly assigned pancreatic cancer cases or healthy controls in the test set. Each column represents a tumor marker. Cells depict normalized log₂ PLA units. (B) Rows are as A. Columns represent either a three-marker panel consisting of CA19-9, OPN, and CHI3L1, or CA19-9 alone. Cells depict the model-outputted probability that a given sample is either pancreatic cancer or healthy control, with a cutoff of p > 0.5 to be considered pancreatic cancer.

Figure 3

CEA and CA125 are significantly prognostic for advanced, unresectable pancreatic cancer. (A) Kaplan-Meier plot depicting survival of 52 cases of advanced, unresectable pancreatic cancer. Cohort divided into tertiles by CEA plasma levels measured by proximity ligation assay. Red line denotes highest 33% by CEA plasma level, green line medial 33%, and blue line lowest 33%. Tick marks represent right censored data. (B) Cohort divided into tertiles by CA125 plasma levels measured by proximity ligation assay. Otherwise as A. (C) Cohort divided into tertiles by combined, rank-ordered levels of CEA and CA125 as measured in plasma by PLA. Otherwise as A.