Objective Histological evidence is considered the only proof of primary central nervous system vasculitis (PCNSV). However, brain biopsy is often omitted or delayed because of the invasiveness and possible complications of the procedure. Circulating endothelial cells (CEC) were shown to be elevated in patients with active antineutrophil cytoplasmic antibody-associated vasculitis. We hypothesise that CEC are also elevated in patients with active PCNSV and may contribute to the diagnosis.
Methods CEC were assessed in 18 patients, 3 of whom had biopsy-proven PCNSV and 15 clinical, cerebrospinal fluid and imaging data, highly suggestive of PCNSV. In 3 of these 15 patients CEC assessment was performed after initiation of successful immunosuppressive therapy. CEC numbers of all patients were compared to those of 16 healthy volunteers and 123 subjects with cerebrovascular risk factors and/or ischaemic stroke, who had been studied in our group before. CEC were assessed by immunomagnetic isolation from peripheral blood.
Results In patients with proven and suspected active PCNSV, CEC were extremely elevated (>400 cells/ml in most of the patients) and significantly higher than in healthy and disease controls (p≤0.01 for each group). CEC significantly decreased with immunosuppressive treatment.
Conclusions For the first time it is shown that CEC are significantly elevated in patients with active PCNSV in contrast to other pathologies associated with brain infarction and correlate with disease activity. Sensitivity and specificity of the method for diagnosing PCNSV and the use of the method for treatment monitoring should be addressed in future prospective studies with a larger patient group.
- Cerebrovascular Disease
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Primary central nervous system vasculitis (PCNSV) is a poorly understood, rare variant of vasculitis which usually shows no laboratory signs of systemic inflammation. Knowledge about this serious disease is predominantly based on case series.1–4 The clinical symptoms vary from progressive cognitive dysfunction and headache to different focal neurological deficits depending on the localisation of the brain lesions. Diagnostic criteria include a neurological deficit unexplained by other causes, a cerebral angiography with changes characteristic for vasculitis and/or evidence of vasculitis in a brain biopsy.2 ,5 Diagnosis is definite only in case of a positive biopsy result, since angiographic findings are characteristic but not specific for PCNSV. Unfortunately, however, the diagnosis may also be missed with brain biopsy—either because the cortical vessels are not involved, or because biopsy revealed an unaffected part of the vessel, or the tissue obtained is insufficient to prove the diagnosis. Thus diagnostic methods which are less invasive and easier to perform, such as the assessment of blood-borne biomarkers, are eagerly awaited.
In the last decade, circulating endothelial cells (CEC) were established as markers of (inflammatory) endothelial damage.6 Recent studies showed that CEC are elevated in the active course of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, and nearly normalise in case of successful immunosuppressive treatment. These studies revealed CEC as a potential biomarker for the diagnosis of vasculitis.
In this study we analysed the number of CEC in patients with PCNSV. We hypothesised CEC to be increased in an active state of PCNSV followed by a decrease under successful immunosuppressive treatment. We thus aimed to show that CEC are appropriate biomarkers for the diagnosis and monitoring of PCNSV.
Patients and methods
Patient groups, healthy control and disease control groups
Data of 18 patients who had been referred to our clinic between August 2008 and June 2010 (median age: 46 years, range 29–82 years, 11 male) and were suspected for PCNSV were retrospectively analysed. Patients were subdivided into three groups: (a) patients with biopsy confirmed PCNSV (n=3), (b) patients with neurological symptoms highly suspected for PCNSV based on their clinical, angiographic, cerebrospinal fluid (CSF) and brain imaging findings (n=12) (see online supplementary document e1) and (c) patients highly suspected for PCNSV based on the same criteria, who had stabilised after initiation of immunosuppressive therapy (n=3).
Brain biopsy was performed in 10 of the patients. All biopsies were evaluated by two independent neuropathologists.
CEC were isolated and enumerated as described in detail elsewhere (see online supplementary document e1).7
CEC values were not normally distributed, thus they are presented as median and range. The patients’ results were compared to those of healthy controls, subjects carrying cerebrovascular risk factors and disease controls with cardioembolic, lacunar and atherothrombotic stroke who had been studied in our laboratory before (see online supplementary table e1) using the Mann-Whitney U test.8 CEC values of >400 cells/ml were considered as 400 cells/ml in the statistical analysis. Considering the descriptive nature of the analysis and the small number of patients, no correction for multiple comparisons was applied.
The study was approved by the local ethics committee. Written informed consent was obtained from all patients and control subjects prior to blood collection.
The clinical characteristics of all patients including CSF, angiographic, MRI and biopsy results are given in online supplementary tables e2–e5.
Brain biopsy showed evidence of PCNSV in 3 of 10 cases (see online supplementary table e2 and figure e1).
All three patients had been referred to our clinic with suspected stroke and showed acute and chronic bihemispheral ischaemic and haemorrhagic lesions on MRI. CEC levels were above 400 cells/ml in the peripheral blood, and thus were significantly higher than those of healthy controls (p=0.006), patients with cerebrovascular risk factors (hypertension, hyperlipidaemia, smoking, diabetes mellitus, p=0.006; hyperhomocysteinemia, p=0.01) and patients with acute stroke (cardioembolic stroke, p=0.007; atherothrombotic stroke, p=0.006; lacunar stroke, p=0.008) (table 1, figure 1).
Follow-up measurements of CEC showed a significant decrease with clinically successful immunosuppressive therapy in two of these patients (see online supplementary table e5). In the third patient the first CEC measurement was performed after an ischaemic stroke, while the possible diagnostic use of the method had not yet been elaborated. At that time point brain biopsy was not performed in the patient since atherosclerotic microangiopathy was considered far more probable. A few months later the patient suffered another ischaemic stroke, again accompanied by a notably high CEC value. Now, brain biopsy was performed and the diagnosis of PCNSV was confirmed.
The 12 patients (see online supplementary table e3) highly suspected for PCNSV showed a median CEC value of 346 cells/ml (range 152 – >400 cells/ml). CEC numbers in these patients were significantly higher than those of healthy controls, subjects with cerebrovascular risk factors and patients with acute stroke (p<0.0001 for each group). There was no significant difference compared to the biopsy-confirmed PCNSV patients (p>0.05) (table 1, figure 1). Of note, none of these 12 patients received immunosuppressive therapy at time of CEC measurement.
Three patients were examined after immunosuppressive therapy for suspected PCNSV had already been initiated (see online supplementary table e4). All had suffered multiple cerebral infarctions before. After initiation of treatment no further symptoms occurred. Thus all three stood to benefit from immunosuppressive treatment. In this subgroup, the CEC number was significantly lower (median 12 cells/ml, range 0–52 cells/ml) than in the biopsy-confirmed PCNSV group (p=0.037) and highly suspected PCNSV group (p=0.008) and did not differ from those of the healthy or disease control groups (p>0.05) (table 1, figure 1).
Knowledge about PCNSV is scarce because of its infrequent occurrence and difficult diagnosis. The pathogenesis is still unclear and diagnosis and treatment of PCNSV remain challenging.9 ,10 Several retrospective studies in case series provided clinical data that improved understanding and characterisation of the disease.1–4 Two of the most relevant studies are a recent one of 101 patients from the USA2 and a series of 21 cases collected in Germany.1 Due to the poor prognosis of untreated PCNSV on the one hand and the serious side effects of aggressive immunosuppressive treatment on the other, diagnostic criteria and reliable markers are important. To date, brain biopsy is the only means to obtain a definite diagnosis, but its sensitivity is only about 50–75%.5 Thus more and better diagnostic criteria are required.
In this study we found evidence that CEC might serve as a potential diagnostic marker for PCNSV. Interestingly, in all patients with PCNSV-positive biopsy results and also in those with highly suspected PCNSV based on clinical, CSF and imaging data, a significantly elevated number of CEC were detected as compared to all healthy and disease controls. In seven of these highly suspected patients, a brain biopsy had been performed but was not helpful in confirming the diagnosis of PCNSV. In some patients the tissue obtained was insufficient to give a clear diagnosis while others obviously contained disease-unaffected vessels. In four of these seven patients angiography was suggestive for PCNSV. This is in accordance with the findings of Salvarani et al who found angiographic evidence for vasculitis in spite of negative biopsy results in 18 of 32 patients in whom both methods had been applied.2
Of course, in those patients highly suspected for PCNSV we were not able to make a definite diagnosis. With regard to the angiographic findings especially the syndrome of reversible vasoconstriction has to be considered for differential diagnosis as it resembles the features of vasculitis. None of our patients, however, presented with the cardinal symptom of this syndrome—abrupt and severe headache. After re-evaluation of all medical data for each patient, considering all examination results, the recommendations for the diagnosis of PCNSV,5 and the current knowledge about CEC values in different diseases6 ,11 ,12 including stroke of other origin than vasculitis13 we felt justified in maintaining the diagnosis of PCNSV.
Recent studies show that the number of CEC is a reliable surrogate marker of endothelial inflammatory damage.6 In these studies, patients with ANCA-associated small-vessel vasculitis showed highly elevated values of CEC compared to other diseases and controls. Interestingly, cell numbers decreased progressively during successful immunosuppressive therapy.6 Similar findings were also detected in patients with other immunological diseases.14 ,15 This indicates that CEC can be used as markers for diagnosis and therapy monitoring.
Accordingly we showed that the CEC values were significantly higher in patients with an active course of PCNSV while they decreased with successful immunosuppressive treatment. Our observations suggest that CEC might be useful markers of disease activity and treatment effect also in PCNSV.
Of note, stroke and the presence of stroke risk factors are not associated with CEC levels as high as in PCNSV.8 This is important for the differential diagnostic considerations in any individual case of stroke with suspected PCNSV.
Although our study has several limitations such as the small number of patients with proven PCNSV and inconsistencies in the diagnostic work-up of the patients, in summary, our results suggest CEC being a promising, non-invasive tool for diagnosis and monitoring of PCNSV and encourage further studies including larger numbers of patients and standardised diagnostic procedures to assess the sensitivity and specificity of this method for diagnosing PCNSV.
We are grateful to Mrs Kristin Wyss and Mrs Michaela Beese (both: Division of Nephrology, Hannover Medical School, Germany) for their excellent technical assistance. We thank Professor Dr Hartmut Hecker, Institute of Biometry, Hannover Medical School, for statistical advice.
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KW and MH contributed equally.
Contributors MD: Drafting/writing the article and revising the manuscript critically for important intellectual content, including medical writing for content, conception and design, analysis and interpretation of the data, study supervision and coordination, acquisition of data, statistical analysis, final approval of the version published. SG, MH, JL, HW, AG, ABT, SL, WS-S, AB, RD: Revising the article critically for important intellectual content, acquisition of data, final approval of the version published. KW, MH: Conception and design of the study, revising the article critically for important intellectual content, analysis and interpretation of the data, study supervision and coordination, final approval of the version published.
Competing interests HW reports funding from the Hannover Medical School. AG reports funding from the BMBF. ABT reports funding from the BMBF. WS-S received travel expenses and honoraria for lectures/speaking engagement at the EANM Satellite Symposium 2009 by Bayer HealthCare. He serves as an editorial board member of Case Reports in Medicine. He holds a patent on the PET blot method for detection of protein aggregates in formalin-fixed tissues (DE 199 63 198.0–41). He is a consultant for Bayer HealthCare. He serves as a Principle Investigator for a study by Bayer HealthCare, for the study SCHU 1547/2-1 from the DFG, for the study ZN2168 from the Volkswagen Stiftung and the WP-Leader for the Alberta Prion Research (APRI), Canada. RD received consulting fees from Boehringer Ingelheim, Sanofi-Aventis and Pharm-Allergan; honoraria from Boehringer Ingelheim, Ipsen Pharma and Temmler Pharma; travel grants from Boehringer Ingelheim, Ipsen Pharma and Bayer-Schering Pharma. He is editor of Klinische Neurophysiologie and is on the executive board of Clinical Neurophysiology. He receives research support from Boehringer Ingelheim, Merz Pharma, Deutsche Forschungsgemeinschaft and the Government of Lower Saxony, Germany. KW reports funding from the BMBF, serves as a consultant for Boehringer Ingelheim, Merz & Co, Solvay and Abbott.
Ethics approval Local ethics committee.
Provenance and peer review Not commissioned; externally peer reviewed.
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