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.
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 MarkerViewTM 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.
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 M, sensitivity and specificity were 79.2% and 78.4%, respectively.
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.
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