70 BIOMEDICAL REPORTS 11: 70-78, 2019 Quantitative proteomic analysis of cerebrospinal fluid from patients with diffuse large B‑cell lymphoma with central nervous system involvement: A novel approach to diagnosis XIAOBEI LIU1*, FEI MO1, HAO ZENG2, SHA ZHU2 and XUELEI MA1* 1Cancer Center, State Key Laboratory of Biotherapy; 2West China School of Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China Received December 7, 2018; Accepted June 10, 2019 DOI: 10.3892/br.2019.1222 Abstract. The outcome of patients with diffuse large B-cell with DLBCL, enabling clinicians to offer prophylactic therapy lymphoma (DLBCL) with central nervous system (CNS) at the time of diagnosis. recurrence is poor. However, there is currently no consensus regarding diagnostic techniques. The aim of the present study Introduction was to investigate the cerebrospinal fluid (CSF) protein profile of DLBCL and identify a potential novel method for the early Diffuse large B-cell lymphoma (DLBCL) is the most common diagnosis of patients with DLBCL at high risk for subsequent type of non-Hodgkin's lymphoma (NHL), accounting for 31% of CNS involvement. The CSF proteomic profiling of patients all cases of NHL in western countries and >40% of NHL cases with DLBCL and a control group were compared using in Asia (1-3). With the currently available options for chemoim- label-free liquid chromatography-tandem mass spectrometry. munotherapy and systemic disease control, the 5-year survival Gene Ontology and pathway analyses were conducted using rate of patients with DLBCL has improved (60-90%) (4,5). the Database for Annotation, Visualization and Integrated Secondary involvement of the central nervous system (CNS) Discovery. The protein interactions were analyzed using the in patients with DLBCL is a relatively uncommon manifes- Search Tool for the Retrieval of Interacting Genes/Proteins tation, encountered in only 5-7% of cases (6-9); however, its database. In the present study, a total of 53 differentially incidence is higher in patients with certain high-risk clinical expressed proteins with >1 log2 fold change (false discovery rate characteristics at the time of diagnosis (10). These risk factors <0.01, P<0.05) were identified and quantified. These proteins include a high International Prognostic Index score (11); appeared to be involved in platelet degranulation, innate involvement of more than two extranodal sites, retroperitoneal immune response and cell adhesion. Two hub gene network lymph node involvement, elevated lactate dehydrogenase level, modules were obtained by protein-protein interaction network or DLBCL originating from high-risk locations, such as the analysis. Of these proteins, secreted protein acidic and rich in bone marrow, paranasal sinuses, testis, breast, adrenal gland cysteine (SPARC) and proenkephalin (PENK) were signifi- and kidney (8,12-16). The outcome following CNS relapse is cantly decreased in the CSF of patients with DLBCL, which poor, with the overall survival shortened to <6 months (17). appeared to be correlated with CNS involvement. The findings Intrathecal (IT) and intravenous high-dose (HD) methotrexate of the present study indicate that decreased expression levels are common methods of CNS prophylaxis (11). Given the of SPARC and PENK in the CSF may serve as early‑phase low CNS relapse rate in DLBCL, and evaluating the benefits biomarkers to evaluate the risk of CNS involvement in patients against the adverse effects, the application of CNS prophylaxis for DLBCL is not widely implemented (12,18). As it is prefer- able that CNS prophylaxis is administered during primary chemotherapy, the identification of patients with DLBCL who are at high risk for subsequent CNS recurrence at the Correspondence to: Dr Xuelei Ma, Cancer Center, State Key time of diagnosis is crucial. There is currently no consensus Laboratory of Biotherapy, West China Hospital, Sichuan University, 37 Guo Xue Alley, Chengdu, Sichuan 610041, P.R. China regarding a diagnostic algorithm for CNS involvement in E-mail: [email protected] DLBCL. Neurological symptoms, CNS imaging, stereotactic biopsy and cerebrospinal fluid (CSF) cytology are the current *Contributed equally methods commonly used for diagnosis and evaluating patients at high risk of, or with suspected, CNS involvement (19). CSF Key words: diffuse large B-cell lymphoma, central nervous system examination includes cytology, flow cytometric analysis and involvement, cerebrospinal fluid, mass spectrometry, proteomic biochemical biomarkers. CSF cytology is a specific diagnostic analysis approach, but it can only detect malignant lymphoid cells in 40% of patients with suspected CNS dissemination (20). Flow cytometric analysis of the CSF has already demonstrated LIU et al: PROTEOMIC ANALYSIS OF DLBCL 71 increased sensitivity (21); however, since the introduction of 20,000 x g at 4˚C for 5 min and quantified using a Bradford rituximab, the majority of CNS relapse events are parenchymal protein assay. Buffer containing 100 mM NH4HCO3 was (65‑70%), and CSF flow cytometry is of limited diagnostic added to equivalent proteins at 100 µg for trypsin diges- value in such cases (22-24). Biochemical biomarker exami- tion. The protein samples were then treated with 5 mM nation of the CSF exhibits higher sensitivity compared with DL-dithioreitol (DTT, Sigma-Aldrich; Merck KGaA) and CSF cytology (58‑85%), but only a moderate improvement in incubated for 60 min at 37˚C. To alkylate the cysteines, iodo- specificity (85%) (25). Therefore, it is necessary to develop a acetamide (IAM; Sigma-Aldrich; Merck KGaA) was added convenient and accurate method for evaluating the risk of CNS to a final concentration of 15 mM, followed by incubation in involvement at diagnosis in order to implement adequate CNS the dark at room temperature for 45 min. A total of 30 mM prophylaxis. L-cysteine (Promega Corporation) was required for blocking Quantitative global proteomics is an advanced approach to redundant IAM. The protein samples were digested with the accurate characterization of proteins in complex biological trypsin (Promega Corporation) overnight at 37˚C at a protein: systems, which is applied to identify unbiased biomarkers or Trypsin ratio of 50:1. The digestion reaction was terminated by key proteins associated with specific physiological and patho- heating the samples to 90˚C to inactivate the enzyme. Finally, logical states (26). The advantages of label‑free quantification C18 ZipTip (Merck KGaA) was used for desalination of the liquid chromatography-mass spectrometry (LC/MS) analysis in-solution digested samples. are as follows: First, the cost, procedure and artificial expenses of labeling samples are eliminated. Second, it has the capacity MS analysis. Prior to being analyzed by LC-MS/MS, coupling to quantify a large number of proteins per LC/MS measure- an Easy nLC1000 nanoflow HPLC system to the Q‑Exactive ment (27). By using proteomics, numerous studies have quadrupole-orbitrap mass spectrometer (Thermo Fisher identified potential CSF biomarkers of neurological diseases, Scientific, Inc.), all samples were lyophilized and resuspended including amyotrophic lateral sclerosis, cerebral malaria and in buffer A [2% acetonitrile (ACN) + 0.1% formic acid tuberculous meningitis (28-30). Quantitative proteomics of (FA)]. A two-column setup was used. Both the trap column CSF samples from serial lumbar punctures during induction in (100 µm x 2 cm) and analytical column (75 µm x 12 cm) patients with acute lymphoblastic leukemia have found poten- were packed in-house with Magic C18 AQ resin (200A, 5 µm; tial predictive markers of CNS thrombosis (31). However, to Michrom Bioresources). The composition (v/v) of the LC buffer the best of our knowledge, global proteomic profiling of CSF was as follows; buffer A: 97.9% water, 2% ACN and 0.1% FA; from patients with DLBCL has not been reported to date. and buffer B: 95% ACN, 4.9% water and 0.1% FA. The mobile Therefore, in the present study, a high-throughput label-free phases were initially 4% B for 3 min, reaching 22% B between quantitative proteomic analysis was performed to identify 3 and 43 min at a flow rate of 400 nl/min. An increase to 30% proteins present in the CSF of patients with DLBCL with CNS B over the next 8 min was at 300 nl/min. An increase to 95% B recurrence compared with those in healthy controls, in order occurred between 52 and 60 min and lasted for the final 5 min. to identify potential CSF biomarkers for patients at high risk The mass spectrometer was set to perform data-dependent of developing CNS recurrence. acquisition in positive ion mode. Full MS spectra were acquired at a resolution of 70,000 over a mass range of 350-1,800 m/z. Materials and methods The automatic gain control (AGC) value was set to 3x106 with maximum fill times of 20 ms. For the MS/MS scans, the 20 Subjects. The subjects included four patients diagnosed most intense parent ions were selected with a 1.6 m/z mass with DLBCL at the West China Medical Center of Sichuan window and fragmented with a normalized collision energy University (Chengdu, China), and six healthy control subjects of 27%. The MS/MS spectra were recorded at a resolution of recruited at the Physical Examination Center of West China 17,500, with the AGC value target set to 1x106 and a maximum Hospital, Sichuan University from January 2016 to January fill time of 64 ms. Parent ions with a single charge or with 2017. The patients with DLBCL were evaluated for CNS unassigned charge states were not selected for fragmentation, recurrence based on the clinical characteristics at the time of and the intensity threshold for selection was set to 3.1x106. diagnosis. The healthy control subjects were defined as indi- Dynamic exclusion with a time window of 30 sec was applied. viduals without active DLBCL or any neurological complaints. Informed consent was obtained from all the participants Data analysis.