CNS Lymphoma Patients Research Articles

Dynamic Changes in Macrophage Polarization Correlate with Progression and Response in CNS Lymphoma: Insights Into the Transcriptome of Lymphoma-Associated M2 Macrophages,

Abstract 3219 Background:Macrophages are an important component of the tumor microenvironment and the immune response to malignancy. Classically activated (M1-polarized) macrophages exhibit anti-tumor effect, while alternatively activated (M2-polarized) macrophages promote tissue repair, angiogenesis, immunosuppression and tumor progression. To date, the vast majority of studies on macrophage phenotype and polarization have been based upon in vitro studies or murine model systems. We tested the hypothesis that distinct macrophage subsets can be identified within the microenvironment of lymphoid tumors and that the relative proportion of M1:M2 macrophages correlates with therapeutic response and/or resistance. In addition, we postulated that transcriptional analysis of distinct macrophage subpopulations would provide insights into mechanisms by which M2 macrophages promote tumor progression and reveal novel mechanisms which direct the interconversion of activated macrophages between classical and alternative activation states. Our focus has been on the evaluation of the tumor microenvironment in CNS lymphomas, the cerebrospinal fluid (CSF), which is enriched in inflammatory cells including activated CD14+ macrophages that we hypothesized would exhibit phenotypic features consistent with M1 and M2 polarization. Methods:We developed a novel 9-parameter flow-cytometric method to isolate distinct subpopulations of activated macrophages from CSF and peripheral blood. The method identifies macrophages with M1 features based upon expression of nitric oxide synthase (iNOS) and M2 macrophages on the basis of low-expression of iNOS and high expression of scavenger receptors CD206, CD209 and CD36. Subpopulations of macrophages were quantified and analyzed by FACS and transcriptional profiling (Affymetrix Gene Chip 1.0 ST). Serial analysis of CSF was performed in CNS lymphoma patients to correlate macrophage polarization states with response and/or progression. Parallel analyses were performed in blood in patients with non-Hodgkin lymphoma (NHL) to control conditions including infections and/or sarcoid. Results:At least four distinct subpopulations of activated macrophages were identified in CSF in association with CNS lymphoma (N=30) and controls (N=28). A greater than six-fold increase in the proportion of M2 macrophages was detected in CSF of CNS lymphoma patients compared to control subjects (p<0.001). By contrast, the proportion of CSF macrophages with M1 features was similar between NHL and controls. Moreover, peripheral blood macrophages from CNS lymphoma patients did not demonstrate significant M2-polarization. Serial analysis demonstrated that M2 polarization of CSF macrophages correlated with tumor progression and/or response. Transcriptional profiling analysis was conducted on macrophage subpopulations isolated from CSF of CNS lymphoma patients and controls. While few significant transcriptional differences were identified between M1 and unpolarized (Mnaive) macrophages, the transcriptional comparison of M2 vs. M1 as well as M2 vs. Mnaive macrophage subpopulations revealed several hundred differentially expressed genes with significant adjusted p-values. Pathway Analysis (Ingenuity) suggested mechanisms strongly associated with the M2 phenotype including activation of Notch signaling. Transcripts associated with short survival were most strongly expressed by M2 macrophages and included Ephrin A4 and lymphoid chemokines such as CXCL-13. The prognostic significance of high CXCL-13 and Ephrin A4 expression were confirmed by ELISA in an independent validation set of cases. Conclusions:We believe this to be the first application of flow-cytometry to define the phenotypes and dynamic interconversion of intratumoral macrophages within the lymphoma microenvironment and the first correlation of M2 macrophages with the evolution of resistance to therapy. In addition, this dataset provides the first in-depth transcriptional profiling analysis of in vivo human macrophage subpopulations and suggests novel mechanisms by which tumor-associated macrophages may facilitate lymphoma progression via the regulation of the metabolic microenvironment, angiogenesis, tumor invasion and immunosuppression. Based upon our additional preliminary data, we hypothesize that these results may be relevant to a variety of lymphoid tumors. Disclosures:Off Label Use: We will discuss the use of rituximab within the leptomeningeal compartment to treat recurrent/refractory CNS lymphomas.

Metabolic Profiling of CNS Lymphoma and Its Microenvironment

Abstract 588 Background:Establishing the pathologic basis of focal brain lesions caused by disease processes such as CNS lymphoma is often difficult. Results of standard MRI-based neuro-imaging are non-specific and cytological testing of cerebrospinal fluid (CSF) is highly insensitive. We tested the hypothesis that the metabolic constituents of the CSF reflect pathologic processes within the brain and thus might lead to the identification of novel diagnostic and prognostic biomarkers and provide insight into mechanisms of CNS lymphoma pathogenesis and its impact on cognitive function. We have pursued two novel approaches to define and model the metabolic microenvironment in CNS lymphoma: (1) Differential metabolite profiling of CSF; (2) Metabolic imaging using hyperpolarized 13C probes using preclinical models involving intracranial xenografts of CNS lymphoma. Methods:We are using GC/MS, in collaboration with Metabolon, to profile a broad spectrum of metabolites (> 140) in CSF from CNS lymphoma patients at diagnosis, relapse as well as from control subjects without brain tumors. In parallel, we are using high resolution MRI (14T) to perform anatomic and contrast enhanced imaging plus hyperpolarized metabolic imaging using 13C-metabolic probes such as [1-13C] pyruvate to monitor the production of intratumoral metabolites such as lactate within highly infiltrative xenograft models of CNS lymphoma. Results:Metabolite concentrations in CSF from 15 subjects with active CNS lymphoma were compared to 15 controls without brain tumors. Seventeen metabolites were significantly upregulated in CSF (> two-fold) compared to controls (p<0.05). Notably, despite similar tumor burdens, the CSF concentrations of lactate, malate and succinate were highest in patients with refractory lymphoma compared to chemotherapy-sensitive tumors. Another TCA intermediate, citrate, was detected at similar concentrations in CSF from controls and lymphoma patients. CSF concentrations of N-acetyl-aspartyl-glutamate, a neurotransmitter, were significantly lower in subjects with CNS lymphoma compared to controls. Metabolic imaging of murine CNS lymphoma xenograft (Raji lymphoma) using hyperpolarized [1-13C] pyruvate demonstrated intratumoral Warburg metabolism with a striking elevation of [1-13C] lactate that was apparent beyond the anatomical margins of the contrast enhancing lymphoma, as demonstrated by T1-weighted post-gadolinium images. Conclusions:These preliminary results suggest that 13C imaging may be able to identify regions of metabolic abnormality in CNS lymphoma outside those identified by 1H methods, and thus potentially provide insight into the metabolic features of the lymphoma as well as its stromal microenvironment. Our hypothesis is that MRI-based, non-invasive metabolic imaging will provide insights into the molecular differences between newly-diagnosed and relapsed cases as well as provide novel, non-invasive diagnostic and prognostic biomarkers. Supported by a grant from CFAR and the Sandler Program for Breakthrough Biomedical Research. Disclosures:No relevant conflicts of interest to declare.

Secondary Central Nervous System Lymphoma: A Single Center Experience

Abstract 5181 Background:Secondary CNS non-Hodgkin lymphoma (SCNSL) is estimated to occur in ∼5% of patients with non-Hodgkin lymphoma, and it carries a poor prognosis. Early CNS relapse (i.e. during treatment or within the first 6 months following chemotherapy) may represent subclinical CNS lymphoma present at the time of diagnosis. DLBCL is the most common histology seen in SCNSL and primary CNS (PCNSL) lymphoma. PCNSL DLBCL is usually characterized by an activated B-cell like (ABC) phenotype; it is unknown whether SCNSL follows the same pattern or if it includes germinal center B-cell (GCB) and non-ABC/non-GCB phenotypes. Methods:We queried our IRB approved clinicopathologic database of hematologic malignancies for patients with lymphoma diagnosed between 1999 and July 2011 who had brain or spine MRI, head CT, lumbar puncture or intrathecal chemotherapy. Clinical data and patient characteristics were extracted. We also obtained pathologic features of tumors including immunohistochemistry and cytogenetics, when available. Descriptive statistics were used to characterize patients at baseline. Overall survival (OS) is defined as the time from time from systemic lymphoma diagnosis (i.e. disease outside of the CNS) to death (event), or censored at last known date of survival. Overall survival curves were obtained using the Kaplan-Meier method with 95% confidence intervals calculated using Greenwood's formula. We did an analysis of CNS lymphoma diagnosis within the first 6 months from systemic lymphoma diagnosis (Early CNS involvement) versus greater than 6 months from systemic lymphoma diagnosis (Late CNS relapse), based on Multivariable logistic regression. The associations between potential risk factors and OS after CNS relapse have been explored using the stepwise Cox regression models. Results:One-hundred and twelve patients met the criteria of systemic lymphoma with CNS relapse/involvement with 68% DLBCL and the remainder including Mantle Cell lymphoma (5%), BL (5%), CLL/SLL (4%) and others. Of the DLBCL patients, 25/76 (33%) were GCB type, 20/76 (26%) were non-GCB, and the remaining unclassifiable with the data available. The median OS for all patients was 23 mo (95% CI=11–41), and 6.7 mo (95% CI=4.6–9.9) after CNS lymphoma diagnosis. Sixty-one of 112 patients (54%) were found to have CNS lymphoma within 6 mo of systemic lymphoma diagnosis including 40 patients (36%) where CNS involvement was present at the time of, or within the first month of, systemic diagnosis, suggesting that these are concurrent presentations. Median OS for patients with early (i.e. concurrent systemic and CNS lymphoma and early CNS relapse patients) v. late CNS relapse was 8.5 mo (95% CI=5.5–10.4) compared to 57.8 mo (95% CI=33.2–94.7), (p < 0.003). The OS after CNS diagnosis was similar between the two groups at 5.5 mo (95% CI=2.85–11) and 8.2 mo (95% CI=3.9–9.7), respectively (p=0.5). Regression analysis of clinical and pathological features (e.g. age, sex, stage, IPI score, LDH, histology, cytogenetics, immunohistochemistry, initial treatment and others) was performed to determine if there were factors associated with early versus late CNS lymphoma involvement and none were significant or clinically relevant. Hazard ratios (HR) for OS after CNS relapse favored patients who obtained a CNS complete response (CR1, HR=0.18, p=<0.01, 95% CI=0.08–0.40) or partial response (PR1, HR=0.31, p=0.004, 95% CI=0.14–0.68). Fifteen patients underwent autologous stem cell transplantation (ASCT) for CNS lymphoma therapy; the median OS after CNS relapse was over 1000 days (HR=0.19, p=0.002, 95% CI=0.07–0.56), suggesting a benefit for intensive therapy in selected patients. HR’s for OS after CNS relapse were worse for DLBCL patients with non-GCB phenotype (HR=5.61, 95% CI=2.85–11) and those with an elevated LDH and >1 site of EN disease (HR=2.50, p=0.004, 95% CI=1.34–4.68) at the time of systemic diagnosis, perhaps reflecting more aggressive disease at initial presentation. Conclusion:The prognosis of secondary CNS lymphoma remains poor, though selected patients who undergo high-dose chemotherapy with ASCT may enjoy prolonged remissions. In contrast to PCNSL, both GCB and non-GCB DLBCL relapse in the CNS, with the non-GCB subtype associated with an inferior prognosis. For patients at high-risk of CNS involvement, efforts must be directed at prophylactic strategies and novel therapeutic approaches. Disclosures:No relevant conflicts of interest to declare.

Abstract 2728: Multivariate analysis of cerebrospinal fluid protein biomarkers in central nervous system lymphoma patients and controls

Abstract Establishing the diagnosis of primary central nervous system lymphoma (PCNSL) is often difficult as the clinical presentation and radiographic features are nonspecific. The gold standards for diagnosis are cytologic analysis of the CSF, which has poor sensitivity, or brain biopsy, which may be associated with significant morbidity and a high rate of diagnostic failure. We are testing the hypothesis that the cerebrospinal fluid (CSF) proteome contains information which may facilitate early and noninvasive diagnosis of CNS lymphoma as well as provide prognostic information. We evaluated the CSF concentrations of three candidate CNS lymphoma protein biomarkers: CXCL-13, a B-cell homing chemokine, Interleukin-10 (IL-10), an autocrine B-cell growth factor, and antithrombin-III (ATIII), a candidate tumor biomarker which we previously identified by mass spectrometric analysis of the CSF. Determinations were performed by ELISA of CSF diagnostic specimens from 228 patients: 75 specimens were from patients with a confirmed diagnosis of non-Hodgkin's lymphoma of the brain and 153 controls including 15 specimens from patients with astrocytic neoplasms, 16 specimens from secondary brain tumors including metastases from breast and lung cancer, and 122 other controls including patients with established systemic lymphoma who staged negative for CNS involvement. Mean concentrations of CXCL-13, IL-10 and ATIII were each significantly elevated in the CSF of CNS lymphoma patients compared to the controls (p&amp;lt;0.0003). By contrast, mean CSF albumin concentration, measured to control for disruption of the blood-brain barrier in the two groups, was similar in CNS lymphoma versus controls (p=0.17). The potential utility of each of these candidate biomarkers for CNS lymphoma was evaluated by means of receiver operating characteristic (ROC) curve analysis, both individually as well as in combination. Bivariate determination of CXCL-13 and IL-10 concentrations yield the best potential discrimination of CNS lymphoma versus all other controls. Detection of IL-10 in combination with CXCL-13 in CSF identified CNS lymphoma with the highest accuracy with area under the ROC curve equal to 0.858 (79% sensitivity and 92.2% specificity), (p&amp;lt;0.0001). In addition, we noted a potential relationship between high CSF concentration of CXCL-13 and short survival at first relapse. These preliminary data support a role for CXCL-13 and IL-10 in the pathogenesis of B-cell non-Hodgkin's lymphoma involving the brain and support our hypothesis that molecular signals within the CSF may be rapidly evaluated in combination to facilitate early and relatively non-invasive diagnosis. This approach may also be applied with other diagnostic modalities such as neuroimaging and flow-cytometry. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2728.

Use of PET in the diagnosis of primary CNS lymphoma in patients with atypical MR findings

The diagnosis of primary central nervous system lymphoma (PCNSL) in immunocompetent patients with atypical magnetic resonance (MR) findings such as disseminated lesions or no (non-enhancing) lesion is sometimes difficult because of mimicking other tumorous and non-tumorous diseases. Positron emission tomography (PET) with (18)F-fluorodeoxyglucose (FDG) and (11)C-methionine (MET) can measure the glucose and amino acid metabolism in the lesions and may provide useful information for diagnosing PCNSL in patients with such subtle MR findings. We performed PET studies with FDG and MET in 17 histologically proven PCNSL and compared the uptake of FDG and MET qualitatively and quantitatively in the tumors between 12 typical and 5 atypical MR findings. All typical PCNSL showed strong uptake of FDG and MET; however, visual analysis of FDG and MET uptake in atypical PCNSL was not very useful for finding lesions in the brain. Semiquantitative FDG and MET uptake values (SUVmax) and quantitative FDG influx rate constant (K ( i )) in the tumors are significantly lower in atypical PCNSL compared with those in typical PCNSL. These values obtained in the lesions with atypical MR findings were also not useful for differentiating PCNSL from other tumorous and non-tumorous diseases. The k (3) values evaluated by FDG kinetic analysis in atypical PCNSL were similar to those obtained in typical PCNSL. Visual analysis of FDG and MET uptake in atypical PCNSL was not useful for finding the lesions in the brain. Semiquantitative and quantitative values obtained in the lesions with atypical MR findings were also not useful for differentiating PCNSL from other tumorous and non-tumorous diseases. The k (3) values evaluated by FDG kinetic analysis in atypical PCNSL may provide valuable information in the diagnosis of PCNSL.

Macrophage Polarization and Acquired Resistance to Rituximab in CNS Lymphoma.

Abstract 3749Poster Board III-685Macrophages are a critical component of anti-tumor immunity but may be subverted from the classically-activated, or M1 phenotype, which mediates tumor elimination, to an alternatively-activated, M2 phenotype, which promotes tumor progression. Interleukin-4 (IL-4) signaling is a pivotal regulator of macrophage polarization to the M2 phenotype.The efficacy of rituximab appears to be mediated predominantly by antibody-dependent cellular cytotoxicity (ADCC) with tumor-associated macrophages being the dominant effector cell. We hypothesize that the phenotype of tumor-associated macrophages (M1 vs. M2) may be a potential determinant of rituximab efficacy or resistance. Relevant to this hypothesis is our demonstration of the intratumoral expression of IL-4 by CNS lymphoma (Blood, 2006; Clin Cancer Res., 2009) as well as our observation that the M2 polarization of macrophages by IL-4 treatment in vitro (18 h) results in an 8-fold decrease in rituximab-mediated ADCC of Raji lymphoma cells compared to ADCC-mediated by naïve or M1 polarized macrophages (p<0.001).There is an extreme paucity of molecular information regarding the role and phenotype of tumor macrophages in non-Hodgkin lymphoma in general, including CNS lymphoma. While macrophages from the cerebrospinal fluid (CSF) are routinely quantified in cytospin analyses, to date there is no established information regarding the phenotype and state of differentiation of macrophages within the leptomeningeal compartment. We have developed a novel flow-cytometry-based protocol for the isolation and phenotypic characterization of macrophages from the CSF of patients with CNS lymphoma. We have used candidate markers of M2 differentiation based upon our gene expression studies of CD14+ activated CSF macrophages, including CD206 and Factor XIII (each of which have previously been shown to be induced in macrophages upon IL-4 stimulation). We have identified M1 differentiation of CSF CD14+ macrophages by high fluorescence when incubated with DAF-FM diacetate, a cell-permeable marker of nitric oxide synthase (iNOS). Using flow-cytometry to evaluate the relative expression of DAF-FM and CD206, we demonstrate the presence of at least four subpopulations of activated macrophages in the CSF of CNS lymphoma patients. M1 macrophages, which highly express iNOS are denoted as DAF-FM(+)/CD206(-). M2 macrophages, which weakly express iNOS but which highly express CD206 are denoted as DAF-FM (-)/CD206(+). In addition, we have detected a mixed phenotype with features of both M1 and M2 macrophages, DAF-FM(+)/CD206(+), which we have termed dual-activated. A fourth subpopulation of activated macrophages within the CSF do not express iNOS or CD206.Thus far, CSF macrophage subpopulations have been characterized and sorted from thirty-five subjects: 18 patients with CNS lymphoma and 17 control subjects with non-neoplastic conditions. We demonstrate for the first time the association of M2 macrophages within the CSF with the pathogenesis of CNS lymphoma: there was a greater than six-fold increase in the proportion of macrophages with M2 features in immunocompetent subjects with CNS lymphoma compared to controls (p<0.001). By contrast, the proportion of macrophages with M1 features was similar between lymphoma and controls.In addition, we reproducibly detected an increase in the ratio of M1:M2 macrophages which correlated with therapeutic response to intrathecal methotrexate or cytarabine (within 94 h) in CNS lymphoma patients. By contrast, the intraventricular administration of rituximab was reproducibly associated with a greater than three-fold increase in the relative ratio of M2:M1 macrophages which was sustained compared to macrophages analyzed pre-intrathecal rituximab (p<0.001). In each of four cases, increases in M2 macrophage polarization anticipated the onset of intrathecal rituximab resistance and tumor progression.We believe this to be the first application of flow-cytometry to define the polarization states of intratumoral macrophages in non-Hodgkin lymphoma as well as the first description of dynamic changes in macrophage phenotypes during the evolution of resistance to rituximab therapy. The elucidation of distinct macrophage subpopulations based upon the expression of candidate markers of M1 vs. M2 phenotype may provide insight into tumor pathogenesis and prognosis. Disclosures:Off Label Use: While we describe the use of intrathecal administration of rituximab in patients with recurrent CNS and intraocular lymphomaa, the focus of our study is on the relationship between M2 macrophage polarization and the pathogenesis of CNS lymphoma as well as the potential relationship of macrophage polarization to acquired resistance to rituximab in CNS lymphoma patients.

Immuno-Chemotherapy with Rituximab, Methotrexate, Lomustine, and Procarbazine for the Treatment of Primary CNS Lymphoma in Patients >65 Years.

Abstract 3712Poster Board III-648 IntroductionPrimary central nervous system lymphoma (PCNSL) is an aggressive extranodal non-Hodgkin lymphoma (NHL) confined to the CNS and/or eyes at presentation. PCNSL have a poor prognosis dispite good initial response to steroids and whole brain irradiation (WBRT). Addition of high-dose methotrexate (MTX) to therapy has improved the prognosis of patients with PCNSL, resulting in median survival rates of up to 60 months (mo). However, most patients eventually relapse. Surviving patients, particularly elderly treated with combined radio-chemotherapy are at substantial risk of developing leukoencephalopathy. We developed a protocol with high-dose MTX combined with the lipophilic alkylating agents procarbacine and lomustine and the anti-CD20 monoclonal antibody rituximab (R-MPL protocol), especially adapted for elderly patients with PCNSL. Here we report the results of 28 patients treated with combined immuno-chemotherapy with R-MPL protocol within a monocentric study. MethodsPatients ≥65 yrs with PCNSL were treated with up to 3 cycles of R-MPL protocol: rituximab (375mg/m2 d-6, 1, 15, 29), MTX (3g/m2 d2, 16, 30), procarbazine (60 mg/m2 p.o., d1-10) and lomustine (110 mg/m2 p.o., d1). Cycles were repeated d42. Inclusion criterias were age ≥65 yrs and biopsy proven PCNSL. There was no lower limit of Karnofsky Performance Score. Results28 patients (median age 75 yrs., range 65-83 yrs.) received R-MPL protocol. 2 patients died, one due to pulmonary embolism 2 weeks after initiation of treatment, the other had a perforation of the sigmoid and both were not evaluable for response. Objective response was seen in 25 of 26 patients (96,2%) with 19 CR and 6 PR. In 5 patients, MTX was not tolerated after 1 (n=4) and 2 (n=1) applications due to cholestatic hepatitis (n=1) and renal impairment (n=4). 2 patients with refractory disease could successfully be salvaged with AraC/thiotepa (n=1) and HDT and ASCT (n=1), respectively. 5 patients experienced relapse and could not be salvaged. After a median follow-up of 26 mo (range 2-35) 10 patients (35,7 %) are alive and disease-free. By intend-to-treat-analysis the 12-mo and 24-mo is 69% and 52% respectively. Severe leukoencephalopathy has not been observed. Most recent follow-up data will be presented in detail. ConclusionThe immuno-chemotherapy protocol presented here is safe and shows high efficacy in treating elderly patients with PCNSL. A prospective phase-II trial will be initiated. Disclosures:No relevant conflicts of interest to declare.

Relapse of Central Nervous System Lymphoma after systemic Lymphoma (SCNSL): Clinical Features and Outcome as compared to Primary CNS Lymphoma (PCNSL) Patients

Background: Data on relapsed central nervous system lymphoma (CNSL) is limited. Therefore, we have evaluated the clinical characteristics and outcome of relapsed CNSL patients at our centers. A central nervous system relapse is a serious complication of aggressive lymphomas. The prognosis is generally regarded as poor and standard therapies of relapsed CNSL have not yet been established.Patients and Methods: All pts with primary and secondary CNSL who were treated at Bonn University Hospital and Cologne University Hospital from 09/1995 – 12/2007 were included into the study. 125 pts could be identified; 102 with newly diagnosed primary CNSL (PCNSL) and 23 pts with secondary CNSL (SCNSL). First-line- treatment for pts with PCNSL consisted of high-dose methotrexate (MTX) in combination with vincristine, ifosfamide, prednisolone and cytarabine (Bonn protocol). Patients with SCNSL received a CHOP-like combination chemotherapy first-line. At cerebral relapse, for 17/23 pts SCNSL-treatment was Bonn polychemotherapy, 4/23 pts received an acute leukaemia regimen (GMALL-B-ALL protocol) at relapse and 2/23 received radiotherapy only. 81/125 pts (64%) were male with a median age of 62 yrs (range 27–77) and 72/125 pts (57%) were older > 60 years.Results: Overall response rate (CR+PR) was 63% (49CR +19PR) for PCNSL and 65% (12CR+3PR) for SCNSL. Median overall survival was 15.8 months for all 125 pts and 23 vs. 7.1 months for PCNSL and SCNSL. There was a difference in OS of 15 months between pts >60 yrs (median 13 months) vs. pts <60 yrs (median 28 months). After a median follow-up of 12 months (range 1–119) 80/125 (64%) pts with PCNSL and SCNSL relapsed (37) or progressed (43). All 80 patients relapsed/progressed again within the CNS. 65/80 pts initially suffered from PCNSL, 15/80 pts from SCNSL. Prognosis of relapsed/progressed PCNSL and SCNSL was dismal. 56/80 patients died. Median OS was short with only 8.5 months (range 1–124) survival time after relapse. Treatment at relapse was radiotherapy alone (16), radiotherapy in combination with temozolomide (2), radiotherapy in combination with MTX, dexamethasone and cytarabine (1), PVC-Chemotherapy (1), BEAM-Polychemothapy (1), Bonn polychemotherapy (7), cytarabine and etoposide (CYVE) with autologous stem cell transplantation (5) and “wait and see” (4). Response to relapse treatment was in 12 cases CR, in 9 cases PR and in 16 cases PD. Treatment at progress was radiotherapy alone (16), radiotherapy in combination with temozolomide (3), radiotherapy in combination with MTX, dexamethasone and cytarabine (1), BEAM-polychemotherapy (1), Bonn polychemotherapy (1), cytarabine and etoposide (CYVE) with autologous stem cell transplantation (2) and “wait and see”/no therapy (16). Response to progress treatment was in 3 cases CR, in 3 cases PR and in 37 cases PD.Conclusions: HD-MTX chemotherapy according to the Bonn Protocol is equally effective in PCNSL and SCNSL pts although the prognosis of pts with SCNSL seems to be worse. Prognosis of relapsed and refractory CNSL pts is dismal. Median OS was only 8.5 months. Therefore, new treatment strategies are urgently needed.

Prospective cognitive follow-up in primary CNS lymphoma patients treated with chemotherapy and reduced-dose radiotherapy.

High-dose chemotherapy and whole brain radiotherapy (WBRT) can prolong survival in primary CNS lymphoma (PCNSL) patients, but is often associated with clinically significant cognitive decline. In this study we assessed neuropsychological functioning prospectively in newly diagnosed PCNSL patients treated with induction chemotherapy followed by reduced-dose WBRT. Twelve patients underwent neuropsychological evaluations at diagnosis, after induction chemotherapy, and 6 and 12 months after WBRT. Nine patients completed additional cognitive evaluations 18 and 24 months post-treatment. At diagnosis, patients had impairments in Executive Functions, Verbal Memory, and Motor Speed. There was a significant improvement in Executive Functions (P < 0.01) and Verbal Memory (P < 0.05) following induction chemotherapy, and scores remained relatively stable up to 12 months post-treatment. Among the nine patients who completed a 2-year follow-up, there was a significant improvement in the Executive domain (P < 0.05) and a trend toward a decline in the Verbal Memory domain. Executive and Verbal Memory functions improved following induction chemotherapy, likely due to decreased tumor burden and discontinuation of corticosteroid and anticonvulsant medications. There was no significant cognitive decline up to 24 months post-chemotherapy and reduced-dose WBRT in this group of PCNSL patients, however, difficulties in Verbal Memory and Motor speed persisted over the follow-up period.

CXCL12 and CXCL13 Levels in Cerebrospinal Fluid (CSF) of Patients with CNS Lymphoma.

Background: The mechanisms leading to development of CNS lymphoma (CNSL) are still poorly understood. Homing of malignant lymphocytes to the CNS may play an important role in the pathogenesis of CNSL. Expression of chemokine receptors CXCR4 and CXCR5 has been shown on tumor cells of primary CNSL. In this study we examined the level of chemokines CXCL12 and CXCL13 which are ligands of CXCR4 and CXCR5 respectively in the CSF of CNSL patients and compared it to non-CNSL controls.Methods: Lumbar CSF and peripheral blood from 16 patients with primary and one with secondary CNSL, as well as control CSF from 8 patients with hydrocephalus, glioblastoma, peripheral lymphoma without CNS involvement or unspecific neurological symptoms were collected, and after centrifugation supernatant and serum were cryopreserved at −80°C until analysis. Chemokine concentrations were measured using commercially available ELISA kits (CXCL13/BCA-1 and CXCL12/SDF-1α Quantikine Immunoassay, R&D Systems, Minneapolis) according to the manufacturer's instructions. Results were confirmed by duplicate measurements.Results: 22 CSF samples and 11 serum samples from CNSL patients and nine CSF samples from controls were evaluated (see table). There was no correlation between serum and CSF chemokine levels in CNSL patients. Median concentrations of both CXCL12 and CXCR13 were higher in patients with CNS lymphoma compared to controls, CXCL13 being significantly raised (p=0.0019). In one CNSL patient three serial CSF samples could be analysed and a ten-fold decrease of CXCL12 during succesfull chemotherapy was found whilst concentration of CXCL13 remained stable.Conclusion: CXCL12 and CXCL13 can be measured in the CSF of CNS lymphoma patients at higher concentration as compared to non-CNSL patients. Analysis of a larger population is warranted in order to define the role of these chemokines in CNSL and to establish their value for diagnosis and follow up.SerumCSFCNSL patientsCNSL patientscontrolsprangemedian (range)median (range)CXCL121788–2933 pg/ml481 (55–2079) pg/ml115 (0–2442) pg/ml0.067CXCL1336–657 pg/ml457 (18–657) pg/ml16.9 (0.6–187) pg/ml0.00019

Salvage whole brain radiotherapy for recurrent or refractory primary CNS lymphoma

High-dose methotrexate (MTX) and whole brain radiation therapy (WBRT) prolong survival in primary CNS lymphoma (PCNSL) patients but have been associated with delayed neurotoxicity. Consequently, patients are often treated with chemotherapy alone, and WBRT is deferred until relapse. We performed a retrospective study to evaluate the safety and efficacy of salvage WBRT. Radiographic response, survival, and late neurotoxicity were assessed as the main endpoints. Forty-eight patients received salvage WBRT for PCNSL progression or recurrence. After WBRT, 58% achieved a complete radiographic response, 21% achieved a partial response, 6% had stable disease, and 15% progressed. The median survival from initiation of WBRT was 16 months, and 54% were alive 1 year after WBRT. The median time to PCNSL progression was 10 months; 15 patients (31%) had no subsequent disease recurrence after WBRT. Age younger than 60 years and complete response to WBRT were associated with better outcome. Treatment-related neurotoxicity was observed in 22% of patients. Patients older than 60 years and those treated less than 6 months from MTX therapy were at increased risk for development of neurotoxicity. Salvage whole brain radiation therapy (WBRT) is effective for recurrent and refractory primary CNS lymphoma. Reserving WBRT until tumor recurrence is a reasonable strategy to minimize or delay the risk of treatment-related neurotoxicity.

Identification of CSF Biomarkers for the Diagnosis of CNS Lymphoma.

CNS lymphoma is an aggressive form of non-Hodgkin's lymphoma. The current means for establishing its diagnosis are:brain biopsy, associated with risk of brain hemorrhage;cytological analysis of the cerebrospinal fluid (CSF), which is insensitive in over 50% of cases.The identification of novel, sensitive and specific biomarkers are required to facilitate early, non-invasive diagnosis of lymphomatous involvement of the CNS. Moreover, elevation of CSF protein is an established adverse prognostic variable in patients with primary CNS lymphoma; however specific peptide markers which relate to prognosis have not previously been identified within the CSF. We are testing the hypothesis that peptide biomarkers for CNS lymphoma are present in the CSF and that these may facilitate noninvasive, early diagnosis, provide prognostic information and enhance clinical monitoring in the setting of minimal residual disease. We describe a two-dimensional liquid chromatographymass spectrometry-based sensitive method for differential quantification and identification of several hundred CSF proteins. Proprietary spectral interpretation and intensity-normalization software was used to quantify differential expression of proteins between controls and lymphoma patients in CSF. An application of this approach to biomarker discovery in CSF is presented. We performed two sets of analyses using independent CSF specimens. In the experimental set we compared the pattern of expression in 9 control vs 9 cases of CNS lymphoma. 130 peptides were differentially expressed between the two groups (p<0.001). In a second, validation set of cases consisting of 7 controls and 7 cases of CNS lymphoma, we observed 131 peptides which exhibited significant differential expression between the two groups (p<0.001). At least 28 candidate biomarker proteins were found to exhibit differential expression in both experimental and validation sets of cases. Unsupervised clustering of the relative expression of these candidate biomarkers identifies peptides which correlate with adverse prognosis (p<0.003). In addition, we have validated the differential expression of four candidate biomarkers by immunoblot analysis of CSF. Quantitative measurement of two such novel CNS lymphoma peptide biomarkers has also been determined by ELISA methods. One of these biomarkers, a serine protease inhibitor, termed L1, was shown to be expressed within CNS lymphoma tumors by microarray as well as by quantitative RT-PCR. Immunohistochemistry localizes L1 expression to tumor cells and tumor vasculature. In addition, both gene expression analyses and ELISA reveal that high L1 concentrations in CSF correlate with significantly worse prognosis. Receiver operator characteristic curve analysis of L1 suggests that this marker may distinguish CNS lymphoma from a variety of non-malignant neurologic conditions with promising sensitivity and specificity. A second novel biomarker which we have identified and validated by immunoblot and by ELISA is a lymphoid chemokine, also found to be expressed in CNS lymphomas by gene expression analysis. Elevated concentrations of this chemokine in the CSF were also shown to be associated with adverse outcome in CNS lymphoma patients.

Necessity of Intraventricular Chemotherapy with the Modified Bonn Protocol in Primary CNS Lymphoma (PCNSL) Patients.

Background: Treatment of primary CNS lymphoma (PCNSL) with a combined systemic and intraventricular chemotherapy (Bonn protocol) has achieved an overall response rate (ORR) of 84% and long term complete remissions in a substantial fraction of patients younger than 60 years.Purpose: Due to a high infection rate of the Ommaya reservoir the question was addressed if intraventricular treatment is dispensable in this polychemotherapy protocol.Patients and Methods: Fifty patients with histologically confirmed PCNSL were enrolled onto a phase II-study evaluating chemotherapy without radiotherapy and without intraventricular treatment. A high-dose methotrexate (MTX) (cycles 1,2,4,5) and cytarabine (ara-C) (cycles 3,6) based systemic therapy (including dexamethasone, vinca-alkaloids, ifosfamide and cyclophosphamide) was administered.Results: In an ongoing trial thirty-five of 50 patients (18 pat. < 60 years, 17 pat. over 60 years) are yet assessable for response after a median follow up of nine months (range: 1 to 26 months). In 18 patients < 60 years, the ORR was 78%. However, median time to treatment failure (TTF) was eight months, and median progression free survival (PFS) only 7 months according to frequent early relapses.Conclusions: Early relapses are frequent in younger patients treated with the modified Bonn protocol without intraventricular treatment despite a high ORR. These preliminary results support the assumption that intraventricular treatment is essential to achieve sustained remissions after successful treatment of PCNSL.

FDG-PET versus Thallium-201 SPECT in the evaluation of putative CNS lymphoma in AIDS patients

1537 Background: The evaluation of CNS lesions in AIDS patients is challenging as both CNS lymphoma and non-neoplastic lesions can have similar clinical presentations and imaging findings. Both Tl-201 SPECT and FDG PET imaging have been used to differentiate malignancy from infection. This study investigates the accuracy of FDG PET compared to Tl-201 SPECT in the diagnosis of CNS lymphoma in patients with AIDS-associated CNS lesions. Methods: Nine patients with AIDS who were found to have rim enhancing brain lesions on MRI underwent both Tl-201 SPECT and FDG PET imaging prior to diagnosis. Diagnosis was made by stereotactic brain biopsy in 6 pts and clinical follow-up in the remaining 3. FDG PET of the brain was performed using a dedicated PET/CT instrument (GE Discovery) 1 hour after 444 MBq FDG. Brain SPECT was performed on a dual head gamma camera (GE Hawkeye) with 185 MBq Tl-201. Images were reviewed along with MRI for anatomic correlation by 2 experienced nuclear medicine physicians blinded to the diagnosis. Results: 43 MRI lesions in 9 patients were evaluated. Clinically, 5 pts (18 lesions) were diagnosed with lymphoma (4 biopsy proven, 1 with known history of lymphoma), and 4 pts (25 lesions) were diagnosed with toxoplasmosis (2 with negative biopsies for malignancy, 2 with clinical course consistent with toxoplasmosis). FDG PET had true positive results for lymphoma in 5 of 5 pts, while Tl-201 SPECT was true positive in only 2 of 5. FDG PET had true negative results in 4 of 4 pts with toxoplasmosis, while Tl-201 had false positive results in 2 of 4. FDG PET had positive and negative predictive values of 100%, while Tl-201 SPECT had PPV 50% and NPV 40%. On a lesion basis, PET showed increased uptake of FDG in 8 of 18 brain lesions in patients with lymphoma, while SPECT showed increased uptake of Tl-201 in 4 of 18 lesions. Tl-201 was positive in 2 lesions in patients diagnosed with toxoplasmosis, while FDG had no false positive lesions. Conclusion: In this limited study, FDG PET proved to be superior to Tl-201 SPECT in the differentiation of CNS lymphoma from toxoplasmosis (overall accuracy 100% vs 44%, respectively). With the increasing availability of FDG PET, this test should probably supplant Tl-201 SPECT as the study of choice following conventional imaging (CT, MRI) of CNS lesions in AIDS patients. No significant financial relationships to disclose.

Secondary Analysis of Radiation Therapy Oncology Group study (RTOG) 9310: An Intergroup Phase II Combined Modality Treatment of Primary Central Nervous System Lymphoma

To determine whether a lower dose of hyperfractionated whole brain radiation reduces central nervous system morbidity without compromising survival for primary CNS lymphoma (PCNSL) patients receiving combined modality treatment. One hundred and two patients received a course of pre-radiation chemotherapy, followed by whole brain radiation, followed by cytosine-arabinoside. Initial radiation dose was 45 Gy/25 fractions (RT) then the study was amended to reduce this dose for complete responders to induction chemotherapy to 36 Gy/30 fractions/3 weeks (HFX). Eighty-two patients received radiotherapy and were evaluable for toxicity analysis (66 RT patients and 16 HFX patients). MMSE scores and survival for the 40 patients who received radiotherapy after complete response to chemotherapy (27 RT and 13 HFX) were compared. There were no notable differences in pre-treatment patient characteristics between the RT and HFX groups. Neurotoxicity: By 4 years, there were 8/82 (10%) grade 5 neurotoxicities which included 2/16 (13%) grade 5 encephalopathies and 0/27 in the RT group of complete responders to chemotherapy. Survival: There was no statistically significant difference in overall or progression-free survival (PFS) between the chemotherapy-complete responders who received RT and HFX. Cognitive function testing: MMSE scores improved at 8 months across both treatment groups. Analysis of the area under the MMSE curve at 8 months showed no statistically significant difference between RT and HFX groups (P=0.81). Leukoencephalopathy occurred later in the HFX group than in the RT patients. Although the HFX schedule represented a 25% reduction in biologically effective tumor dose in comparison, PFS and overall survival were not significantly affected. The HFX regimen delayed but did not eliminate severe neurotoxicity from chemoradiation in PCNSL patients.

Controversies in primary CNS lymphoma

Primary CNS lymphoma is a non-Hodgkin’s lymphoma confined to the CNS: the brain, eyes and cerebrospinal fluid. Unlike other primary brain tumors, primary CNS lymphoma is uniquely chemo- and radiosensitive. However, the outcome of primary CNS lymphoma patients is significantly inferior to similar non-Hodgkin’s lymphoma patients. Multiple prospective studies using high-dose methotrexate, with or without whole-brain radiotherapy, have reported improved primary CNS lymphoma outcome, but numerous questions remain regarding the optimal care of a given patient.

Positron emission tomography in patients with primary CNS lymphomas.

This article reviews possible clinical applications of positron emission tomography (PET) in patients with CNS lymphomas. PET allows quantitative assessment of brain tumor pathophysiology and biochemistry in vivo. Therefore, it provides different information about tumors when compared to histological or neuroradiological methods. In a diagnostic setting, PET cannot differentiate between primary lymphomas of the CNS, brain secondaries, or malignant gliomas, since various brain tumors share biochemical alterations. In HIV patients with contrast-enhancing brain tumors, however, data from the literature suggest that PET with the tracer F-18 fluoro-deoxyglucose may help to discriminate neoplastic (CNS lymphoma) from inflammatory (e.g. toxoplasmosis) lesions. Assuming that tumor biochemistry is highly abnormal in the most malignant parts of tumors, PET may also assist in defining targets for stereotactic biopsy. With regard to treatment evaluation, the prediction of individual treatment response is among the most challenging clinical applications of PET. On the one hand, this could be achieved on the basis of measures like tumor perfusion, oxygen consumption, or hypoxia. On the other hand, PET tracer methods may allow to quantify the expression of gene products following gene therapy. However, in CNS lymphoma patients these topics have yet not been addressed with PET.

Lack of an epidemiologic link between Kaposi's sarcoma and CNS lymphoma in patients with AIDS

Reply from the Authors: We appreciate the opportunity to reply to the data described suggesting a lack of epidemiologic association between primary CNS lymphoma (1°CNSL) and KS. In our table 1,1 we identified comorbidity in our patients with brain lymphoma. Only 2 of the 20 HIV-1-infected patients with 1°CNSL also had KS. In both cases, the lymphomas were HHV-8-positive. In contrast to these data and the findings of Buskin et al., however, Ahsan and Neugut have found that KS and 1°CNSL are associated with one another in HIV-1-infected individuals. …

CT Appearance of Primary CNS Lymphoma in Patients with Acquired Immunodeficiency Syndrome

Cranial CT studies of 32 patients with biopsy-proven AIDS-related primary CNS lymphoma were reviewed retrospectively. A wide variety of different CT appearances were identified. Mass lesions varied in location, size, and number. Most lesions were either iso- or hyperdense and all enhanced with contrast medium. Several different patterns of enhancement were observed. Mass effect and edema were seen in almost all patients. After radiotherapy, most tumors decreased in diameter, became hypodense, and no longer enhanced with contrast medium. Edema and mass effect decreased or resolved in all but one patient. Postradiotherapy CT scans also revealed interval enlargement of the ventricles and cortical sulci. This study demonstrates the wide diversity of CT appearances of AIDS-related primary CNS lymphoma. The CT findings cannot be used in lieu of biopsy for diagnosis of this disorder. The appearance of postradiotherapy CT scans was consistent with regressing lymphoma.

Central nervous system involvement in non-Hodgkin's lymphoma

Fifty-eight Hong Kong Chinese patients with CNS lymphoma were reviewed (primary seven, secondary 51). The incidence of secondary CNS lymphoma in patients with non-Hodgkin's lymphoma was estimated to be 9.4%. The Working Formulation separated subtypes which had a special propensity to involve the CNS. Significant proportions of our patients with secondary CNS lymphoma had other features which were known to be associated with a high risk of CNS disease including stage IV ( 48 51 , 91.4%), bone marrow ( 26 51 , 50.9%), peripheral blood (7.51, 13.7%), nasal ( 7 51 , 13.7%), orbital ( 3 51 , 5.9%), testicular ( 2 51 , 3.9%) and bulky retroperitoneal ( 6 51 , 11.8%) disease. 82% of patients with secondary CNS lymphoma had concurrent systemic disease and a further 12% had systemic relapse shortly afterward. CNS lymphoma is associated with poor prognosis and only 29% and 14% of the patients with primary and secondary CNS lymphoma respectively survived beyond 1 year. Patients responding to therapy had significantly better survival. 69.9% of the deaths were related to progressive systemic disease.