Humoral CSF parameters in the differential diagnosis of hematologic CNS neoplasia Weller M, Stevens A, Sommer N, Schabet M, Wietholter H. Humoral C SF parameters in the differential diagnosis of hematologic CNS neoplasia. Acta Neurol Scand 1992: 86: 129-133. Cerebrospinal fluid (CSF) and serum samples of 20 patients with central nervous system manifestations of hematological malignancies including primary cerebral lymphoma (n = 5) and disseminated non-Hodgkin lymphoma (n = 7) were examined for albumin, IgG, IgM, fibronectin, P,-microglobulin, interleukin-6, soluble interleukin-2 receptor, tumor necrosis factor alpha, and oligoclonal immunoglobulin bands. Although a broad range of abnormalities were detected, no reliable CSF parameter for the diagnosis of leptomeninged spread from hematological neoplasias could be identified. An analysis of 61 repeat lumbar punctures added little to the findings of the first CSF examinations. Currently, immunochemical studies of CSF cell surface markers and early biopsy have probably more clinical value than the determination of the humoral CSF parameters included in this study. However, analysis of cytokine synthesis by single CSF cells using molecular biology techniques may improve the differential diagnosis of hematological neoplasia of the brain and spinal cord in the future.
The diagnostic evaluation of CSF in suspected primary or secondary hematologic malignancy of the CNS has attracted new interest because of recent developments in neurology and general medicine. Improved systemic management of leukemia has caused a shift to neurological complications due to CNS relapse (1). The introduction of intrathecal chemotherapy for neoplastic meningitis is associated with a need for monitoring parameters in the CSF (2). Owing to an increasing number of patients suffering from the acquired immunodeficiency syndrome and of irradiated and otherwise immunosuppressed patients, the incidence of primary cerebral lymphoma (PCL) is rising. PCL, however, is difficult to diagnose by CSF analysis, mostly because of equivocal cytological findings. Therefore, we studied a set of several CSF parameters of potentially diagnostic value in 20 patients with leptomeningeal neoplasia from different hematologic malignancies and with PCL. Patients
Paired lumbar CSF and serum samples were obtained from patients with PCL (n = 5), leptomeningeal seeding from disseminated non-Hodgkin lymphoma (D-NHL) (n=7), and from patients with other hematological malignancies with neurological complications such as leptomeningeal metastasis and spinal cord compression, including acute lym-
M. Weller, A. Stevens, N. Sommer, M. Schabet, H. Wietholter Department of Neurology, University of Tubingen, Germany
Key words: leptomeningeal metastasis; primary cerebral lymphoma; tumor markers; CSF; ELISA. Michael Weller, National Institute of Health, Clinical Neuroscience Branch, Building 10, Room 3N256, Bethesda, MD, 20892, USA.
phocytic leukemia (n = l), Hodgkin’s disease (n = 2), acute myelogenous leukemia (n = 3), and multiple myeloma (n = 2). Diagnosis was based on biopsy, positive CSF cytology, and neuroradiological findings. The diagnosis of primary CNS disease (PCL) was supported by the absence of laboratory abnormalities in peripheral blood suggestive of systemic malignancy. All PCL patients had negative chest radiographic studies and no enlarged lymph nodes on abdominal ultrasound. A control group consisted of 20 patients suffering from tension headaches or from patients with suspected but eventually disproven neurological disease. Material and methods
Albumin, IgG, IgM, 0,-microglobulin (02M), interleukin-6 (IL-6), soluble interleukin-2 receptor (sIL-~R),and tumor necrosis factor alpha (TNFa) were determined in CSF and serum. Additional CSF studies included lactate, glucose, cell counts, cytology, fibronectin, and analysis of oligoclonal immunoglobulin bands on isoelectric focusing of CSF proteins. Albumin and IgG were measured by routine nephelometry. Previously described noncompetitive Elisa procedures were used to quantify IgM, fibronectin, 02M, IL-6, s I L - ~ R ,and TNFa (Table 1) (3-7). Statistical analysis included student’s t-test, linear correlation analysis, and ANOVA. 129
Weller et al.
Of five patients with biopsy-proven PCL, only one had unequivocally positive routine CSF cytology (without specific immunohistological analysis). These first lumbar punctures had all been performed prior to biopsy. In two of seven patients with DNHL, the diagnosis of CNS seeding could not be established by CSF cytology but only by biopsy. In one patient, CNS seeding was the presenting clinical manifestation of systemic NHL, the other with known NHL was referred for biopsy of a brain stem lesion before initiation of radiotherapy. Of a total of 20 patients, 10 patients had a positive cytology on their first spinal tap and 2 more on a repeat lumbar puncture (PCL, l/5; D-NHL, 5/7; Hodgkin’s disease, 1/2; multiple myeloma, 2/2; acute myelogeneous leukemia, 2/3; acute lymphocytic leukemia 1/ 1). The laboratory findings in patients with PCL, DNHL, and controls are summarized in Tables 2, 3, 4. The patient with acute lymphoblastic leukemia had a cell count of 377/p1 and mild blood brain
barrier dysfunction but no CSF cytokines detected. Of the two patients with Hodgkin’s disease, one had elevated CSF IL-6 (44 ng/l), CSF sIL-2R (76 Ujml) as well as elevated IgG and IgM indices and CSF fibronectin. The other had spinal cord compression and no abnormalities of CSF immune parameters. Two of three patients with acute myelogenous leukemia had pleocytosis as the only major CSF abnormality. The third patient who failed to exhibit evidence for extracerebral acute myelogeneous leukemia, had very high IL-6 (427ng/l) and sIL2R (527 Ujml) in the CSF. Of two patients with multiple myeloma, cord compression, and positive cytology, one had increased IL-6 (1 18 ng/l) and sIL-2R (223 U/ml) in the CSF. Oligoclonal immunoglobulin bands in CS F, but not serum, were detected in one of 5 PCL patients in 2 of 6 D-NHL patients, and in the abovementioned atypical patient with primary cerebral acute myelogenous leukemia. The oligoclonal CSF bands were associated with a pathological IgG index only in the PCL patient and in the patient with acute myelogenous leukemia, but not in the two D-NHL patients. Corresponding oligoclonal bands in CSF and serum were observed in one patient with PCL and in one patient with Hodgkin’s disease. Isoelectric focusing for oligoclonal bands was not performed in four patients (acute lymphocytic leukemia, acute myelogeneous leukemia, D-NHL, multiple myeloma) because of limited CSF volume available. Table 3 indicates the frequency of pathological results for single parameters in different diagnostic subgroups. The diagnostic yield of a total of 61 repeat punctures is provided in the same table. Cytokine data are summarized in Table 4. No further cytokine elevations on subsequent lumbar punctures were detected. As compared to controls, CSF cell count, CSF albumin, CSF IgG, CSF IgM, IgG and IgM indices,
Table 2. Mean values and standard error of the me an (SEM) for CSF cell counts h - ~ r n ~ ) , CSF albumin, CSF IgG, CSF IgM (all mg/l), IgG index, IgM index, CSF and serum PZM (mg/I), CSF fibronectin (mg/l), and lactate (pnolll), in patients with PCL, D-NHL, and in controls (n=20)on the first spinal fluid examination. indicates significant elevation compared to controls.
Table 3. Incidenceof pathological findings for parameters of Table 2 in PCL, D-NHL, and all 20 patients with hematological CNS malignancies pooled, on first CSF analysis. The additional diagnostic yield of 61 repeated lumbar punctures is given in brackets. The cumulative number of repeat punctures was 24 in PCL, 24 in secondary CNS NHL, and 13 in the other patients.
Table 1. Reagents for Elisa protocols. Cytokine assays (slt-2R. IL-6, TNFr) were performed by means of commercial Elisa kits. Fibronectin
IgM
P2M
It-6 ~lt-2R TNFr
Monoclonal anti-human fibronectin (341646) Polyclonal anti-human fibronectin (341640) Calbiochem, La Jolla, USA Fibronectin (F-2006)Sigma, St. Louis, USA Monoclonal anti-human IgM (1-6385) Polyclonal anti-human IgM (1-2386) IgM (1-86201, all Sigma Polyclonal anti-human PZM (A072) Polyclonal anti-human PZM peroxidase (P174)Dakopatts, Glostrup, Denmark P2M (M4890)Sigma Biotechnology, Oxford, UK Eurogenetics, Tessenderloo, Belgium Biotechnology, Oxford, UK
Results
’
Cell Counts CSF Albumin CSF IgG CSF IgM IgG Index IgM Index CSF P2M Serum PZM CSF Fibronectin CSF Lactate
130
PCL
D-NHL
Controls
7+3‘ 467 f 65’ 72f22’ 2.lfl.l’ 0.65f0.06’ 0.12f0.03’ 2.5k0.5’ 2.0f0.7 4.2f0.1’ 2.5f0.4
llf7’ 767f421‘ 116f63‘ 2.7f1.0’ 0.61f0.06’ 0.33f0.13‘ 1.9k0.4’ 1.3f0.1 6.9f1.4’ 2.8f0.5
1f1 177f10 21 f 2 0.3f0.04 0.47f0.0 1 0.08+0.03 1.3fO.l 1.6fO.l 2.3i0.2
The diagnostic evaluation of CSF in suspected primary or secondary hematologic malignancy of the CNS has attracted new interest because of recent developments in neurology and general medicine. Improved systemic management of leukemia has caused a shift to neurological complications due to CNS relapse (1). The introduction of intrathecal chemotherapy for neoplastic meningitis is associated with a need for monitoring parameters in the CSF (2). Owing to an increasing number of patients suffering from the acquired immunodeficiency syndrome and of irradiated and otherwise immunosuppressed patients, the incidence of primary cerebral lymphoma (PCL) is rising. PCL, however, is difficult to diagnose by CSF analysis, mostly because of equivocal cytological findings. Therefore, we studied a set of several CSF parameters of potentially diagnostic value in 20 patients with leptomeningeal neoplasia from different hematologic malignancies and with PCL. Patients
Paired lumbar CSF and serum samples were obtained from patients with PCL (n = 5), leptomeningeal seeding from disseminated non-Hodgkin lymphoma (D-NHL) (n=7), and from patients with other hematological malignancies with neurological complications such as leptomeningeal metastasis and spinal cord compression, including acute lym-
M. Weller, A. Stevens, N. Sommer, M. Schabet, H. Wietholter Department of Neurology, University of Tubingen, Germany
Key words: leptomeningeal metastasis; primary cerebral lymphoma; tumor markers; CSF; ELISA. Michael Weller, National Institute of Health, Clinical Neuroscience Branch, Building 10, Room 3N256, Bethesda, MD, 20892, USA.
phocytic leukemia (n = l), Hodgkin’s disease (n = 2), acute myelogenous leukemia (n = 3), and multiple myeloma (n = 2). Diagnosis was based on biopsy, positive CSF cytology, and neuroradiological findings. The diagnosis of primary CNS disease (PCL) was supported by the absence of laboratory abnormalities in peripheral blood suggestive of systemic malignancy. All PCL patients had negative chest radiographic studies and no enlarged lymph nodes on abdominal ultrasound. A control group consisted of 20 patients suffering from tension headaches or from patients with suspected but eventually disproven neurological disease. Material and methods
Albumin, IgG, IgM, 0,-microglobulin (02M), interleukin-6 (IL-6), soluble interleukin-2 receptor (sIL-~R),and tumor necrosis factor alpha (TNFa) were determined in CSF and serum. Additional CSF studies included lactate, glucose, cell counts, cytology, fibronectin, and analysis of oligoclonal immunoglobulin bands on isoelectric focusing of CSF proteins. Albumin and IgG were measured by routine nephelometry. Previously described noncompetitive Elisa procedures were used to quantify IgM, fibronectin, 02M, IL-6, s I L - ~ R ,and TNFa (Table 1) (3-7). Statistical analysis included student’s t-test, linear correlation analysis, and ANOVA. 129
Weller et al.
Of five patients with biopsy-proven PCL, only one had unequivocally positive routine CSF cytology (without specific immunohistological analysis). These first lumbar punctures had all been performed prior to biopsy. In two of seven patients with DNHL, the diagnosis of CNS seeding could not be established by CSF cytology but only by biopsy. In one patient, CNS seeding was the presenting clinical manifestation of systemic NHL, the other with known NHL was referred for biopsy of a brain stem lesion before initiation of radiotherapy. Of a total of 20 patients, 10 patients had a positive cytology on their first spinal tap and 2 more on a repeat lumbar puncture (PCL, l/5; D-NHL, 5/7; Hodgkin’s disease, 1/2; multiple myeloma, 2/2; acute myelogeneous leukemia, 2/3; acute lymphocytic leukemia 1/ 1). The laboratory findings in patients with PCL, DNHL, and controls are summarized in Tables 2, 3, 4. The patient with acute lymphoblastic leukemia had a cell count of 377/p1 and mild blood brain
barrier dysfunction but no CSF cytokines detected. Of the two patients with Hodgkin’s disease, one had elevated CSF IL-6 (44 ng/l), CSF sIL-2R (76 Ujml) as well as elevated IgG and IgM indices and CSF fibronectin. The other had spinal cord compression and no abnormalities of CSF immune parameters. Two of three patients with acute myelogenous leukemia had pleocytosis as the only major CSF abnormality. The third patient who failed to exhibit evidence for extracerebral acute myelogeneous leukemia, had very high IL-6 (427ng/l) and sIL2R (527 Ujml) in the CSF. Of two patients with multiple myeloma, cord compression, and positive cytology, one had increased IL-6 (1 18 ng/l) and sIL-2R (223 U/ml) in the CSF. Oligoclonal immunoglobulin bands in CS F, but not serum, were detected in one of 5 PCL patients in 2 of 6 D-NHL patients, and in the abovementioned atypical patient with primary cerebral acute myelogenous leukemia. The oligoclonal CSF bands were associated with a pathological IgG index only in the PCL patient and in the patient with acute myelogenous leukemia, but not in the two D-NHL patients. Corresponding oligoclonal bands in CSF and serum were observed in one patient with PCL and in one patient with Hodgkin’s disease. Isoelectric focusing for oligoclonal bands was not performed in four patients (acute lymphocytic leukemia, acute myelogeneous leukemia, D-NHL, multiple myeloma) because of limited CSF volume available. Table 3 indicates the frequency of pathological results for single parameters in different diagnostic subgroups. The diagnostic yield of a total of 61 repeat punctures is provided in the same table. Cytokine data are summarized in Table 4. No further cytokine elevations on subsequent lumbar punctures were detected. As compared to controls, CSF cell count, CSF albumin, CSF IgG, CSF IgM, IgG and IgM indices,
Table 2. Mean values and standard error of the me an (SEM) for CSF cell counts h - ~ r n ~ ) , CSF albumin, CSF IgG, CSF IgM (all mg/l), IgG index, IgM index, CSF and serum PZM (mg/I), CSF fibronectin (mg/l), and lactate (pnolll), in patients with PCL, D-NHL, and in controls (n=20)on the first spinal fluid examination. indicates significant elevation compared to controls.
Table 3. Incidenceof pathological findings for parameters of Table 2 in PCL, D-NHL, and all 20 patients with hematological CNS malignancies pooled, on first CSF analysis. The additional diagnostic yield of 61 repeated lumbar punctures is given in brackets. The cumulative number of repeat punctures was 24 in PCL, 24 in secondary CNS NHL, and 13 in the other patients.
Table 1. Reagents for Elisa protocols. Cytokine assays (slt-2R. IL-6, TNFr) were performed by means of commercial Elisa kits. Fibronectin
IgM
P2M
It-6 ~lt-2R TNFr
Monoclonal anti-human fibronectin (341646) Polyclonal anti-human fibronectin (341640) Calbiochem, La Jolla, USA Fibronectin (F-2006)Sigma, St. Louis, USA Monoclonal anti-human IgM (1-6385) Polyclonal anti-human IgM (1-2386) IgM (1-86201, all Sigma Polyclonal anti-human PZM (A072) Polyclonal anti-human PZM peroxidase (P174)Dakopatts, Glostrup, Denmark P2M (M4890)Sigma Biotechnology, Oxford, UK Eurogenetics, Tessenderloo, Belgium Biotechnology, Oxford, UK
Results
’
Cell Counts CSF Albumin CSF IgG CSF IgM IgG Index IgM Index CSF P2M Serum PZM CSF Fibronectin CSF Lactate
130
PCL
D-NHL
Controls
7+3‘ 467 f 65’ 72f22’ 2.lfl.l’ 0.65f0.06’ 0.12f0.03’ 2.5k0.5’ 2.0f0.7 4.2f0.1’ 2.5f0.4
llf7’ 767f421‘ 116f63‘ 2.7f1.0’ 0.61f0.06’ 0.33f0.13‘ 1.9k0.4’ 1.3f0.1 6.9f1.4’ 2.8f0.5
1f1 177f10 21 f 2 0.3f0.04 0.47f0.0 1 0.08+0.03 1.3fO.l 1.6fO.l 2.3i0.2
