Non-Hodgkin lymphomas (NHL) are a heterogeneous group of malignancies that arise from lymphoid tissue. They exhibit varied clinical and biological features [1], and their incidence has been increasing over the past several decades [2]. The past decade has seen enormous changes in our understanding of lymphomas, including the identification of better prognostic factors [3]. However, results from efforts to identify good diagnostic factors have been disappointing. Lactate dehydrogenase has been used as an NHL marker [4], but its accuracy in diagnosis has been unsatisfactory. Thus, finding specific tumor markers that are useful for diagnosing and monitoring NHL remains a high priority. In the present study, we introduce a new NHL diagnostic marker obtained by translating the information in low-mass ions (i.e., < 1000 m/z) in urine samples from lymphoma patients.

Previous efforts to find diagnostic factors for NHL have yielded disappointing results compared with studies reporting prognostic factors with clinicopathological correlations [5, 6]. Our previous study showed that the urine level of IL-8 normalized to creatinine could be a possible biomarker with the capacity to discriminate NHL patients from normal controls; thus, we expected that urine might be a valuable biological source for diagnostic markers for NHL [7].

In the present study, we sought to develop a new diagnostic approach for NHL, using MALDI-MS analysis to translate the information of low-mass ions (i.e., < ~1000 m/z) present in urine samples into a tool capable of discriminating NHL patients from normal individuals. There are two motivations for our focus on this low-mass range. The first is the existence of valuable information in the low-mass range, analyzed by mass spectrometry, that has not yet been systematically exploited. Second, ions in the low-mass range provide an enormous amount of information about biological changes that originate from alterations in gene and protein expression. We hypothesized that a new non-invasive cancer-screening protocol could be established if low-mass-ion data were properly collected, statistically translated, and analyzed by MALDI-MS.

Interestingly, a low-mass ion appeared at 137.08 m/z that was significantly different in urine samples from NHL patients and controls (Figure 2B &2C). Searching the Human Metabolome Database (HMDB) yielded a list of candidate metabolites corresponding to the 137.08-m/z ion (Table 2). An ESI-MS/MS analysis of low-mass ions in urine identified 137.08 m/z as hypoxanthine (Figure 3B and 3C). Consistent with this identification, the concentration of hypoxanthine in urine compared favorably with the MALDI-MS profile of the 137.08-m/z ion (Figure 3C).

The Low-mass ion profiling is absolutely depends on the accurate mass measuring technology. Recent mass spectrometers employing either MALDI-, or ESI-based technology provide very accurate mass information. Using this advanced technology, we herein were able to suggest a possible application of low-mass ion profiling for cancer screening. However, a couple of problems are still remained. First, software for low-mass ion profiling is still incomplete. During the normalization process of mass spectra obtained from each individual samples, software sometimes collects the noise on the mass spectrum as a low-intensity peaks with discriminating power. To prevent this nose picking, low-mass ions ranked by software have to be checked again on the raw mass spectra. Furthermore, the low-mass ions with low-intensity, even they have a great discriminating power, may not be proper for identification. This is the reason why the low-mass ion with 137.08 m/z was selected for identification since it showed highest intensity among the low-mass ions ranked within 30^th (Figure 2B).

In patients with gastric cancer or colorectal cancer, purine bases in plasma have been reported to increase in association with a decrease in purine base excretion [8]. Whether hypoxanthine and xanthine levels are altered in urine samples from gastrointestinal cancer patients is still a matter of debate [8]. In plasma from children with acute lymphoblastic leukemia or NHL, hypoxanthine levels were reported to be higher than those in healthy adult controls; these elevated plasma hypoxanthine levels decreased after methotrexate infusion [9]. However, a change in urine levels of hypoxanthine has not yet been reported.

At present, we are unable to explain the underlying mechanism for the change in hypoxanthine, but one possibility may be found in alterations of purine metabolism that occur during tumor development. Intracellular concentrations of hypoxanthine and xanthine are inversely related to adenylate energy changes and, therefore, to the energy currency of cellular ATP [10]. Recently, a classical antifolate has been shown to possess cytotoxic activity against human prostatic cancer cell lines that lacked hypoxanthine, whereas growth was maintained in tumors with hypoxanthine [11]. We reasoned that the level of hypoxanthine in NHL urines might decrease due to consumption by tumor cells. However, a recent study has shown that urinary hypoxanthine is significantly increased when tumor development in mesothelioma-transplanted nude mice was maximized [12]. In addition, changes in the activity or expression of enzymes involved in hypoxanthine or xanthine metabolism, which might affect hypoxanthine and xanthine levels in NHL urines, cannot be ruled out. For example, xanthine oxidoreductase, a key enzyme in the degradation of DNA and RNA, is associated with histological grade of differentiation and extent of disease in colorectal cancer [13], as well as the migratory activity of human breast cancer cells [14, 15].

The present study represents a good example of low-mass-ion profiling in the setting of disease-screening using urine samples. This technique can be a powerful, non-invasive diagnostic tool with high sensitivity and specificity for NHL screening. Furthermore, hypoxanthine identified in this study may be a useful single urine marker for NHL screening. However, the biochemical mechanism responsible for the decreased levels of hypoxanthine and xanthine in urine samples from NHL patients remains to be elucidated.