Circulating nucleosomes as epigenetic biomarkers in pancreatic cancer

Abstract

Background: To improve the prognosis of patients with pancreatic cancer, new biomarkers are required for earlier, pre-symptomatic diagnosis. Epigenetic mutations take place at the earliest stages of tumorigenesis and therefore offer new approaches for detecting and diagnosing disease. Nucleosomes are the repeating subunits of DNA and histone proteins that constitute human chromatin. Because of their release into the circulation, intact nucleosome levels in serum or plasma can serve as diagnostic disease biomarkers, and elevated levels have been reported in various cancers. However, quantifying nucleosomes in the circulation for cancer detection has been challenging due to nonspecific elevation in sera of patients with benign diseases. Here, we report for the first time differential, disease-associated epigenetic profiles of intact cell-free nucleosomes (cfnucleosomes) containing specific DNA and histone modifications as well as histone variants circulating in the blood. The study comprised serum samples from 59 individuals, including 25 patients with resectable pancreatic cancer, 10 patients with benign pancreatic disease, and 24 healthy individuals using Nucleosomics(®), a novel ELISA method.

Results: Multivariate analysis defined a panel of five serum cfnucleosome biomarkers that gave an area under the curve (AUC) of 0.95 for the discrimination of pancreatic cancer from healthy controls, which was superior to the diagnostic performance of the common pancreatic tumor biomarker, carbohydrate antigen 19-9 (CA 19-9) with an AUC of 0.87. Combining CA 19-9 with a panel of four cfnucleosome biomarkers gave an AUC of 0.98 with an overall sensitivity of 92 % at 90 % specificity.

Conclusions: The present study suggests that global epigenetic profiling of cfnucleosomes in serum using a simple NuQ(®) immunoassay-based approach can provide novel diagnostic biomarkers in pancreatic cancer.

Keywords: DNA; Diagnosis; Epigenetics; NuQ® assays; Nucleosomes; Pancreatic cancer; Screening; Serum.

Fig. 1

Discrimination of five NuQ^® assay panel for pancreatic cancer, benign disease, and healthy controls. Significant separation (p < 0.001) between the pancreatic cancer (n = 25), the benign samples (n = 10), and healthy controls (n = 24) was achieved with pre-processed ELISA data from five nucleosomal biomarkers. A linear model (Fisher’s linear discriminant) was used to generate a weighted sum of values assigned as arbitrary units (AU) = −0.825 (5MC) − 2.909 (H2AZ) + 2.641 (H2A1.1) − 1.050 (H3K4Me₂) − 0.551 (H2AK119Ub). P value was determined by the Mann-Whitney U test. Box plots indicate the median and 25th and 75th percentiles. Whiskers indicate the 5th and 95th percentiles

Fig. 2

ROC curve for discrimination of cancer vs. healthy and benign. The area under the curve for an optimal panel of five nucleosomal biomarkers (0.92) selected from a panel of nine was significantly higher than that of CA 19-9 (0.85), the current gold standard for pancreatic cancer. The AUC was further improved by replacing the lowest weighted nucleosomal biomarker in model 1 with CA 19-9 in a panel with the four nucleosomal biomarkers (0.94) to give a second, mixed biomarker, model

Fig. 3

Discrimination of four NuQ^® assay panel combined with CA 19-9 for pancreatic cancer, benign disease, and healthy controls. Improved separation between the pancreatic cancer (n = 25), the benign samples (n = 10), and healthy controls (n = 24) was achieved with pre-processed ELISA data from four nucleosomal biomarkers combined with CA 19-9. A linear model (Fisher’s linear discriminant) was used to generate a weighted sum of values assigned as arbitrary units (AU) = −0.788 (5MC) − 2.338 (H2AZ) + 1.959 (H2A1.1) + 0.672 (H3K4Me₂) + 0.782 (CA 19-9). P value was determined by the Mann-Whitney U test. Box plots indicate the median and 25th and 75th percentiles. Whiskers indicate the 5th and 95th percentiles

Fig. 4

ROC curve for discrimination of cancer vs. healthy. The area under the curve for an optimal panel of five nucleosomal biomarkers (0.95) selected from a panel of nine was significantly higher than that of CA 19-9 (0.87), the current gold standard for pancreatic cancer. As for discrimination of cancer vs. healthy and benign, the AUC was further improved by replacing the lowest weighted nucleosomal biomarker in model 1 with CA 19-9 in a panel with the four nucleosomal biomarkers (0.98) to give a second, mixed biomarker, model

Fig. 5

Discrimination of CA 19-9 for pancreatic cancer, benign disease, and healthy controls. No significant separation between the pancreatic cancer (n = 25), the benign samples (n = 10), and healthy controls (n = 24) was achieved. The performance of CA 19-9 alone is relatively poor with a large degree of overlap between cancer and healthy, cancer as well as benign and healthy emphasizing the poor suitability for CA 19-9 as a biomarker in a screening setting. P value was determined by Mann-Whitney U test. Box plots indicate the median and 25th and 75th percentiles. Whiskers indicate the 5th and 95th percentiles