Identification of hypoxanthine as a urine marker for non-Hodgkin lymphoma by low-mass-ion profiling

Abstract

Background: Non-Hodgkin lymphoma (NHL) is a hematologic malignancy for which good diagnostic markers are lacking. Despite continued improvement in our understanding of NHL, efforts to identify diagnostic markers have yielded dismal results. Here, we translated low-mass-ion information in urine samples from patients with NHL into a diagnostic marker.

Methods: To minimize experimental error, we tested variable parameters before MALDI-TOF analysis of low-mass ions in urine. Urine from 30 controls and 30 NHL patients was analyzed as a training set for NHL prediction. All individual peak areas were normalized to total area up to 1000 m/z. The training set analysis was repeated four times. Low-mass peaks that were not affected by changes in experimental conditions were collected using MarkerView software. Human Metabolome Database (HMDB) searches and ESI LC-MS/MS analyses were used to identify low-mass ions that exhibited differential patterns in control and NHL urines. Identified low-mass ions were validated in a blinded fashion in 95 controls and 66 NHL urines to determine their ability to discriminate NHL patients from controls.

Results: The 30 highest-ranking low-mass-ion peaks were selected from the 60-urine training set, and three low-mass-ion peaks with high intensity were selected for identification. Of these, a 137.08-m/z ion showed lower mass-peak intensity in urines of NHL patients, a result that was validated in a 161-urine blind validation set (95 controls and 66 NHL urines). The 130.08-m/z ion was identified from HMDB searches and ESI LC-MS/MS analyses as hypoxanthine (HX). The HX concentration in urines of NHL patients was significantly decreased (P < 0.001) and was correlated with the mass-peak area of the 137.08-m/z ion. At an HX concentration cutoff of 17.4 microM, sensitivity and specificity were 79.2% and 78.4%, respectively.

Conclusions: The present study represents a good example of low-mass-ion profiling in the setting of disease screening using urine. This technique can be a powerful non-invasive diagnostic tool with high sensitivity and specificity for NHL screening. Furthermore, HX identified in the study may be a useful single urine marker for NHL screening.

Figure 1

Selection of two low-mass ions with differential peak areas in urines from controls and NHL patients after establishing MALDI-MS conditions for low-mass-ion profiling. A. The resolution of mass peaks acquired at variable focus mass. Linear resolution of low-mass peaks was obtained at a focus mass of 500 m/z. B. The effect of laser intensity on overall mass spectrum acquisition. Laser intensity was positively correlated with the area of low-mass peaks (< 1000 m/z). Because low-mass peaks with the highest intensity become saturated at a laser intensity of 5500, the laser intensity for data acquisition was fixed at 5250. C. Typical pattern of mass spectra from urines from controls and NHL patients. D. Box plot of total area of low-mass peaks (< 1000 m/z). The total area of low-mass peaks was obtained from four replicate experiments (cases A to D) using urines from 12 controls (green) and 11 NHL patients (pink).

Figure 2

Discriminating NHL patients from controls based on low-mass ions present in urine. A. Principal components analysis (PCA). The first principle component (PC1) explains the greatest amount of variance, and PC2 represents the next largest amount. Left panel: Unsupervised PCA showing that profiling of low-mass ions in urine may discriminate NHL patients. Right panel: Supervised PCA describing low-mass ions with differential mass-peak intensities in urines from controls and NHL patients. B. Thirty low-mass ions selected by t-test analysis. C. Profile plot focused on 137.08 m/z peaks. The profile plot demonstrates a higher peak intensity of the 137.08-m/z ion in urines from NHL patients compared to that in controls in both training and validation groups. Lower panel describes overlapped mass spectra of four urine samples from NHL patients and controls from blue box in profile plot.

Figure 3

Identical ESI-MS/MS pattern obtained from 137.08 m/z ion in urine and hypoxanthine. A. Mass shift of the 137.08-m/z ion in urine in LTQ-XL analysis. In a direct urine analysis without LC separation, the urine candidate 137.08-m/z ion and hypoxanthine (HX) were monitored as 137.70 m/z. B. The ESI-MS/MS pattern of the 137.08-m/z ion in urine was identical to that of HX. C. Mass-peak area of the 137.08-m/z ion and HX concentration were positively correlated; however, this relationship did not reach statistical significance.

Figure 4

Significant decrease of hypoxanthine and xanthine in NHL urine. The levels of hypoxanthine (HX) (A) and its oxidative product, xanthine (X) (B) were significantly lower in NHL urines (both P < 0.001). Area under the curve, sensitivity and specificity obtained from blind validation of 221 urines samples (125 control, and 96 NHL) are shown.