For clinical investigation
Patients
After screening to rule out systemic lymphoma by positron emission tomography (PET) or whole-body computed tomography (CT), the patients with preoperative diagnosis of PCNSL exclusively located within the CNS underwent biopsy at our department. After confirming the histological diagnosis of PCNSL based on criteria published by the World Health Organization [20], HD-MTX therapy was conducted between January 2001 and December 2014 at our institute and affiliated hospitals according to our PCNSL protocol (Fig. 1). Paraffin-embedded tissues were obtained from 82 of the 113 patients who were newly evaluated with consecutive immunocompetent PCNSL. Under the approval for genetic and molecular analysis using patient specimens by the Research Ethics Committee of the Institutional Review Board of Kumamoto University Hospital [30, 32], this study was conducted after obtaining written informed consent from all participating patients or their family members.
PCNSL protocol
HD-MTX-based chemotherapy with LV rescue was performed according to our previously reported protocol [21]. Induction therapy consisted of a cycle of high-dose MTX (3.5 g/m2) delivered intravenously over 3 h on days 1, 22, and 43. LV rescue was initiated 24 h after the start of MTX infusion, and 15 mg of LV was intravenously administered nine times every 3 h, followed by five times every 6 h. The repeated intravenous administration of LV was continued until MTX clearance (< 0.1 μM). Procarbazine (60 mg/m2) was administered orally on days 1 through 7, 22 through 28, and 43 through 49. The initial betamethasone treatment dose was tapered from 16 mg to 2 mg every 4 days. The first-line therapy did not include any rituximab therapy. One course was administered every 3 weeks, and three such courses were performed (Fig. 1). Patients with PCNSL and aged < 60 years were supposed to undergo radiotherapy (RT) from 4 weeks after the completion of three courses of HD-MTX therapy. By contrast, patients aged ≥60 years were monitored for complete response (CR) to the therapy during follow-up evaluation after completion of the three courses. Alternatively, they underwent RT or other chemotherapies. If the progressive disease (PD) occurred before the three courses of HD-MTX were completed, patients aged < 60 years underwent RT, whereas those aged ≥60 years underwent RT or other chemotherapies. The second-line therapy was not uniform in cases of recurrence after completing HD-MTX therapies with or without RT.
Evaluation of therapeutic response to HD-MTX
The therapeutic response to HD-MTX was evaluated using CT or magnetic resonance imaging (MRI), with contrast-enhancement according to the response criteria published by the International PCNSL Collaborative Group [1]. Evaluation of the therapeutic response to HD-MTX was performed just before the initiation of each course of HD-MTX, 3 weeks after the completion of the three courses, and during the follow-up period. We evaluated all patients every 3 months for the first 2 years and every 6 months thereafter. CR was assumed in cases of both CR and unconfirmed CR (CRu). For patients unable to complete the three courses of HD-MTX due to adverse events associated with MTX, MRI data obtained after the final HD-MTX treatment was used for the evaluation.
Immunohistochemistry (IHC)
IHC was performed on formalin-fixed paraffin-embedded (FFPE) tumor specimens with validation of positive and negative controls according to our previously reported protocol [29, 31]. The antibodies for polyglutamylation (mouse monoclonal; GT335; AdipoGen AG, Liestal, Switzerland) were used at 1:2000 dilution. A glioblastoma (GBM) specimen was used as a positive control for polyglutamylation, and specimens of meningioma and pituitary adenoma were used as negative controls, as was a GBM specimen without primary antibody treatment (Fig. 2a). Polyglutamylation positivity was quantified by manual counting. The antibodies for CD20 (rabbit monoclonal; SP32; Spring Bioscience, Pleasanton, CA, USA), a common B cell lymphoma marker, were used at 1:100 dilution. To classify the cell of origin of diffuse large B cell lymphoma into the germinal center B-cell-like (GCB) or non-GCB group using the algorithm established by Hans et al. [15], antibodies against the following proteins were used: CD10 (mouse monoclonal; 56C6; Leica Biosystems Newcastle Ltd., UK) at 1:25 dilution, Bcl-6 (mouse monoclonal; LN22; Leica Biosystems Newcastle Ltd., UK) at 1:100 dilution, and MUM1 (mouse monoclonal; MUM1p; Dako Cytomation, Glostrup Denmark) at 1:50 dilution.
Immunofluorescence of tissue sections
Multicolored immunofluorescence using different primary antibodies for polyglutamylation (AdipoGen) and CD20 (Spring Bioscience) was performed as reported previously [29]. To subtract the autofluorescence in FFPE tumor specimens, a Mantra system was used (PerkinElmer, Waltham, MA, USA) [25].
Area under the concentration-time curve of MTX (AUCMTX)
According to population pharmacokinetic analysis using the nonlinear mixed-effects modeling program (NONMEM, version 7.3.0) [17], we examined AUCMTX in 67 patients with PCNSL whose plasma MTX concentrations were available. We also assessed the correlation between clinical response and AUCMTX according to polyglutamylation status.
Survival analysis
As previously reported [21], overall survival (OS) was measured as the time from initial diagnosis to death from any cause, and progression-free survival (PFS) as the time from diagnosis to the first PD. Cases in which extracranial lesions were found by PET or contrast-enhanced CT after treatment were also considered PD. Patients whose day of death was uncertain and patients who were alive on the day of analysis were censored, with the time from the first diagnosis to the last physical interaction or clinic visit used as the censoring time. Patients whose day of PD was uncertain and patients without PD on the day of analysis were censored with the time from diagnosis to that of the last MRI/CT showing a response. We investigated whether polyglutamylation could be a predictor of OS and PFS using Kaplan-Meier survival curves and multivariate analysis. For survival analyses, we used the log-rank test to compare the Kaplan-Meier curves for OS or PFS in patients who did and did not manifest polyglutamylation. To estimate the treatment response to MTX, we applied the Cox proportional-hazards model. Univariate analysis was used to estimate the prognostic relevance of polyglutamylation status, a predictive marker for HD-MTX treatment, and of patient age, sex, preoperative Karnofsky performance status (KPS), MSKCC prognostic scoring [2], cell of origin (GCB vs non-GCB) [15], lactate dehydrogenase (LDH) levels, and tumor location, in which the tumors were divided into deep (corpus callosum, basal ganglia, brain stem, and cerebellum) or non-deep location included in the International Extranodal Lymphoma Study Group score [9]. The variables were included in the Cox model and subjected to multivariate analysis.
For experimental investigation
Cell lines
Human lymphoma cell lines, namely, HKBML, a human PCNSL-derived cell line; RAJI, a Burkkit lymphoma cell line; and TL-1, a lymph node B-lymphoma cell line, were purchased from RIKEN BioResource Center (Tsukuba, Japan). These floating cells were maintained as previously reported [26].
Knockdown of folpolyglutamate synthetase (FPGS) in lymphoma cells
FPGS induces the accumulation of high levels of MTX polyglutamates in childhood leukemia [28]. To decrease this accumulation in lymphoma cell lines, we established stable, genetically modified cell lines in which FPGS was knocked down by a lentivirus system. Lentiviruses were prepared as reported previously [29]. PMD2.G (envelope vector) and psPAX2 (packaging vector) were purchased from Addgene (Cambridge, MA). FPGS-specific short-hairpin (sh)RNA constructs in pLKO.1 vectors were also obtained from Dharmacon (Lafayette, CO, USA), and an empty vector was used as a scrambled-sequence control. Cells were selected and maintained with puromycin (0.5–1 μg/mL).
Cell viability assay
We used the Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) to evaluate cell viability after individual treatment, as previously reported [3, 4]. Cells were treated with 100 nM MTX (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 24 h, followed by the addition of LV (Pfizer Japan, Inc., Tokyo, Japan) at a final concentration of 3 μg/mL and culturing for an additional 24 h. Cell viability assay was performed 48 h later. Histone-deacetylase inhibitors (HDACIs) enhance the antitumor effect of MTX by upregulating FPGS expression, thereby causing intracellular accumulation of long-chain MTX polyglutamates in childhood acute lymphoblastic leukemia (ALL) [19]. Sodium butyrate (NaBu; Sigma-Aldrich, St Louis, MO, USA), a pan-HDACI, was used in this study. Lymphoma cell lines were treated with 100 nM MTX with or without 1 mM NaBu for 24 h prior to the addition of LV. Cell viability was assessed 48 h later.
Western blot
Western blot was performed as previously described [29]. The primary antibodies used were anti-FPGS (1:1000; rabbit polyclonal; Spring Bioscience), anti-DHFR (1:10,000; rabbit monoclonal; EPR5285; Abcam, Cambridge, MA, USA), and anti-α-tubulin (1:5000; mouse monoclonal; Sigma-Aldrich).
Immunofluorescence of lymphoma cells
Lymphoma cells were collected, attached to glass slides using the cytospin method, and processed for immunofluorescence as previously reported [4, 29]. To evaluate polyglutamylation levels in cells, anti-polyglutamylation antibodies (AdipoGen) were used at 1:2000 dilution. 4′,6-Diamidino-2-phenylindole (DAPI; FluoroPure grade; Thermo Fisher Scientific, Waltham, MA, USA) was used for counterstaining.
Statistical analyses
Statistical differences were assessed by Mann-Whitney U test, chi-squared test, log-rank test, and Student’s t test. Differences were determined to be statistically significant if p < 0.05. The data were represented as the mean ± standard deviation (SD) of at least three replicates for each experiment. The Statistical Package for the Social Sciences (SPSS version 19; IBM corp., Armonk, NY, USA) was used for all statistical analyses.