Diagnostic value of IgG4 Indices in IgG4-Related ...

JNI-475812; No of Pages 5 Journal of Neuroimmunology xxx (2013) xxx–xxx

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Diagnostic value of IgG4 Indices in IgG4-Related Hypertrophic Pachymeningitis☆,☆☆,★ Emanuel Della-Torre a,b,⁎, Laura Galli c, Diego Franciotta d, Enrica Paola Bozzolo b, Chiara Briani e, Roberto Furlan f, Luisa Roveri g, Maria Sessa g, Gabriella Passerini h,1, Maria Grazia Sabbadini a,b,1 a

Università Vita-Salute San Raffaele, School of Medicine, San Raffaele Scientific Institute, via Olgettina 58, 20132, Milan, Italy Unit of Medicine and Clinical Immunology, San Raffaele Scientific Institute, via Olgettina 60, 20132, Milan, Italy Infectious Diseases Department, San Raffaele Scientific Institute, Via Stamira d'Ancona 20, 20100, Milan, Italy d Laboratory of Neuroimmunology, National Neurological Institute C. Mondino, Pavia, Italy e Department of Neurosciences, University of Padova, Via Giustiniani 5, 35128, Padova, Italy f Clinical Neuroimmunology Unit, Institute of Experimental Neurology, Division of Neuroscience, San Raffaele Scientific Institute, via Olgettina 58, 20132, Milan, Italy g Department of Neurology, San Raffaele Scientific Institute, via Olgettina 60, 20132, Milan, Italy h Diagnostica e ricerca, San Raffaele Scientific Institute, via Olgettina 60, 20132, Milan, Italy b c

a r t i c l e

i n f o

Article history: Received 27 September 2013 Accepted 16 October 2013 Available online xxxx Keywords: IgG4 IgG4-related disease IgG4-related pachymeningitis Hypertrophic pachymeningitis Pachymeningitis Cerebrospinal fluid

a b s t r a c t Diagnosis of IgG4-Related Hypertrophic Pachymeningitis (IgG4-HP) relies on meningeal biopsies, because cerebrospinal fluid (CSF) diagnostic biomarkers are lacking. Here, we determined whether IgG4 intrathecal production could distinguish IgG4-HP from other disorders presenting with HP (OHP). In patients with IgG4HP, the median CSF IgG4 concentration, IgG4 Index and IgG4Loc were significantly higher than in both controls and OHP. CSF IgG4 levels higher than 2.27 mg/dL identified 100% of IgG4-HP and 5% of OHP. An IgG4Loc cut-off of 0.47 identified 100% of IgG4-HP and no cases of OHP. Our results support CSF IgG4 quantification and IgG4 Indices as alternatives to meningeal biopsy for the diagnosis of IgG4-HP when this procedure is contraindicated or uninformative. © 2013 Elsevier B.V. All rights reserved.

1. Introduction IgG4-related disease (IgG4-RD) is a fibrotic disorder characterized by often elevated serum IgG4 and responsiveness to glucocorticoid treatment (Cheuk and Chan, 2010). The disease was first defined in relation to the pancreas but many other sites of involvement have been progressively recognized on the basis of striking similar

☆ DATA ACCESS, RESPONSIBILITY and ANALYSIS: EDT had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. ☆☆ SOURCE of SUPPORT: Study design, data collection, data analysis, data interpretation, and writing of the report had no outside sponsorship or financial support. ★ APPROVAL of ETHIC COMMITTEE: All subjects provided written informed consent for invasive tests such as meningeal biopsy and lumbar puncture that were performed for diagnostic purposes. Therefore, this retrospective analysis was not reviewed by an institutional ethics committee, but all tests were performed in accordance with institutional guidelines as well as with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. ⁎ Corresponding author at: Unit of Medicine and Clinical Immunology, San Raffaele Scientific Institute, via Olgettina 60, 20132, Milan, Italy. Tel.: + 39 02 26434078; fax: + 39 02 26433786. E-mail address: [email protected] (E. Della-Torre). 1 The last two authors equally contributed.

histopathological features (Hamano et al., 2001; Zen and Nakanuma, 2011): namely, diffuse lymphoplasmacytic infiltration by IgG4-positive plasma cells, occasional eosinophils, storiform fibrosis and obliterative phlebitis (Deshpande, 2012). Central nervous system (CNS) involvement has been initially demonstrated in the form of hypophysitis and subsequently described in a subset of patients with idiopathic hypertrophic pachymeningitis (IHP) (Wallace et al., 2013). We recently described three patients affected by IgG4-related hypertrophic pachymeningitis (IgG4-HP) and demonstrated an intrathecal IgG synthesis with oligoclonal pattern. We also evidenced cerebrospinal fluid (CSF) IgG4 production and suggested that an IgG4 Index would be a relatively non-invasive test as an adjunct to the diagnosis of IgG4-HP (Della-Torre et al., 2012, 2013). In effect, due both to the rarity of this condition and the extensive differential diagnosis, the evaluation of IHP might be extremely challenging and often requires an invasive bioptic procedure. For these reasons, a comprehensive definition of CSF findings in IgG4HP could be crucial for diagnostic, prognostic, and therapeutic purposes, mainly in those patients with exclusive pachymeningeal involvement and/or normal serum IgG4 levels. In the present work we sought to determine whether IgG4 intrathecal production, as assessed by the measurement of IgG4 concentrations within the CSF, and IgG4 Indices could distinguish IgG4-HP from other

0165-5728/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jneuroim.2013.10.008

Please cite this article as: Della-Torre, E., et al., Diagnostic value of IgG4 Indices in IgG4-Related Hypertrophic Pachymeningitis, J. Neuroimmunol. (2013), http://dx.doi.org/10.1016/j.jneuroim.2013.10.008

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E. Della-Torre et al. / Journal of Neuroimmunology xxx (2013) xxx–xxx

inflammatory, infectious, and neoplastic disorders that occasionally present with HP, namely, other HP (OHP). 2. Material and methods 2.1. Patients The retrospective case–control study is based on 30 patients (22 men, 8 women) who were referred to our tertiary care centres (San Raffaele Hospital, Milan, Italy and Department of Neuroscience, Padua, Italy) between November 2007 and November 2012 with evidence of HP on magnetic resonance imaging (MRI). Three patients, reported in detail elsewhere (Della-Torre et al., 2012, 2013) (median age, 62years; interquartile range (IQR) 52–67) were diagnosed with definite IgG4-HP based on comprehensive diagnostic criteria: compatible organ involvement, serum IgG4 levelsN135mg/dL, pathological findings on meningeal biopsies showing lymphoplasmacytic infiltration of IgG4 bearing plasma cells with an absolute number N 50/HPF and a percentage of IgG4+/IgG+ cells N 40%, surrounded by storiform fibrosis (Deshpande, 2012). Their demographic characteristics and pathology features are shown in Table 1. Cerebrospinal fluid IgG subclasses concentration and related intrathecal production Indices from patients with IgG4-HP were compared to those of nine control subjects and those of 21 patients with OHP, matched in a 1:3 and 1:7 ratio respectively, by age (±3 year) and sex. The group of control subjects was used to determine normal ranges for all the measures considered and included patients undergoing elective trans-sphenoidal surgery for pituitary non-secreting adenomas

without elevation of serum inflammatory markers or IgE levels, peripheral blood eosinophilia, allergies to foods or air pollens, blood– CSF barrier damage or oligoclonal bands on CSF analysis, nor evidence of meningitis on MRI. The group of subjects with OHP included patients with granulomatosis with polyangiitis (GPA, formerly Wegener's) (n = 3); sarcoidosis (n = 3) (Della Torre et al., 2013); Behcet's disease (n = 1); diffuse large B-cell lymphoma (DLBCL)(n = 2); POEMS syndrome (n = 2); meningeal carcinomatosis related to metastatic lung adenocarcinoma, melanoma, and breast cancer (n = 1 each); tuberculosis, cysticercosis, nocardiosis, and syphilis (n = 1 each); and IHP (n = 3). Behcet's disease and GPA were diagnosed according to internationally recognized classification criteria (Watts et al., 2007; Davatchi, 2012); the diagnoses of sarcoidosis, DLBCL, and metastatic cancer were confirmed by histological evaluation of meningeal biopsies. Tuberculosis, cysticercosis, nocardiosis, and syphilis were diagnosed according to CSF microbiological and immunological results. POEMS syndrome was diagnosed according to the current criteria (Dispenzieri, 2012). The diagnosis of IHP was made when a thorough clinical, microbiological, serological, radiological, and histological work out was unrevealing. All subjects provided written informed consent for invasive tests such as meningeal biopsy and lumbar puncture that were performed for diagnostic purposes. 2.2. Laboratory tests Paired CSF and serum samples were collected before the administration of any specific therapy, centrifuged within 30 min and stored at − 20 °C. Albumin and total IgG concentrations were

Table 1 Cerebrospinal fluid, serological and microbiological findings in the three patients with IgG4-HP, and comparison with Control subjects and patients with Other forms of Hypertrophic Pachymeningits (OHP). (*) Normal values for serum IgG subclasses correspond to reference ranges used at San Raffaele Institute. (§) Rheumatoid factor, anti-nuclear, anti-cyclic citrullinated peptide, and anti-neutrophil cytoplasmic antibodies. (°) PCR for Herpes simplex virus types 1 and 2, Varicella-Zoster virus, Cytomegalovirus, Epstein-Barr virus, Enterovirus, Mycobacterium tuberculosis; Gram, acid-fast stain and microbiological cultures; search for Aspergillus fumigatus and Cryptococcus neoformans antigens; serology for Lyme disease, brucellosis, syphilis. (^) Albumin Quotient measures the blood–CSF barrier permeability. (#) Calculated vs 5 patients with OHP who exhibited positive IgGLoc. (a) IgG4-HP vs Controls subjects vs OHP patients by use of the Kruskal–Wallis test. (b) IgG4-HP vs Controls subjects by use of the Wilcoxon rank sum test. (c) IgG4-HP vs OHP patients by use of the Wilcoxon rank sum test. Normal values*

Age, years Sex, males, n (%) Organ involvement

IgG4 Related Hypertrophic Pachymeningitis (n = 3)

Other forms of Hypertrophic Pachymeningits (n = 21)

P-valuea P-valueb P-valuec

Patient 1

Patient 2

Patient 3

Median (Q1–Q3)

Median (Q1–Q3)

Median (Q1–Q3)

52 Female Dura mater, Periorbital tissue

62 Male Dura mater

67 Male Dura mater, Retroperitoneal fibrosis

62 (52–67) 2 (67%)

58 (55–63) 6 (67%)

60 (50–70) 14 (67%)

0•918 0•999

0•999 0•999

0•861 0•999

3350 (2370––3430) 907 (652–930)

3540 (3450–3890) 1130 (1081–1295)

3610 0•187 (3015–3770) 876 (712–1173) 0•195

0•065

0•221

0•096

0•663

17 (12–32) 0 (0–1) 2•5 (2–4•5) 0•5 (0•4–0•5) 0

41 (27–83) 1 (1–3) 9•0 (3•7–26•5) 0•6 (0•5–1) 5•7 (0•4–20)

0•096 0•065 0•016 0•016 N/A

0•793 0•998 0•257 0•015 0•686#

Serum analysis Albumin (mg/dL)

(3500–5000)

3350

2370

3430

IgG (mg/dL)

(840–1600)

651

607

930

Autoimmunity ACE (U/l)

Negative (37–137)

Negative 29

Negative 41

Negative 68

CSF analysis Appearance Total protein (mg/dL) Glucose (mg/dL) Cells (μL) Cytology

Colorless, clear (12–60) (40–80) (0–1)

Microbiology° Oligoclonal bands Albumin (mg/dL) Albumin Quotient^ IgG (mg/dL) IgG Index IgGLoc

Negative Absent (10–30) (b0•7) (0•8–3•8) (b0•70) (0)

Colorless, clear 44 59 2 (lymphocytes) No atypical cells Negative Present 25 0•8 8•7 1•8 5•5



§

Control subjects (n = 9)

76 (44–82) 42 (59–73) 2 (1–32)

38 (25–85) 1•1 (0•8–3•6) 18•8 (8•7–17•4) 1•8 (0.8–1•8) 5•5 (1•0–12•4)

0•011 0•007 0•003 0•008 N/A



determined by nephelometry (BN II System, Siemens, Germany), and IgG1-4 subclasses by ELISA (Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer's instructions (dilutions: CSF, 1:25; serum, 1:2500 as a starting point). The albumin quotient (CSF albumin/serum albumin × 100) was used to assess the degree of blood–CSF barrier permeability, which, in turn, is an index of intrathecal inflammation (Tibbling et al., 1977). IgG Index and IgGLoc were used to calculate the intrathecal production of total IgG and IgG subclasses (Tibbling et al., 1977; Reiber and Peter, 2001). CSF and serum concentrations for immunoglobulins and albumin were analysed within the same analytical series. As internal control, for both CSF and serum analysis, the sum of IgG subclasses measured by ELISA corresponded to total IgG values measured by nephelometry with a variability that did not exceed 10%. Readers of the laboratory tests and pathologists evaluating meningeal biopsies were blind to the results of the other test. 2.3. Statistical analysis Assuming a CSF IgG4 mean value of 0.8 mg/dL and a standard deviation of 2.0 for subjects with OHP, we evaluated that a sample size of three subjects with IgG4-HP and 12 subjects with OHP would allow 80% power to detect, at 0.05 alpha level, differences between the two groups of at least 4.4 mg/dL or more. Given the limited number of available subjects with IgG4-HP, the sample size was estimated according to the method proposed by Whitley, which allows an unbalanced distribution of the expected total sample size according to a K ratio between the two groups (Hsieh, 1987; Whitley and Ball, 2002). Such a sample size also allows, at 0.05 alpha level, 90% statistical power to detect differences in mean values of CSF IgG4 between subjects with IgG4-HP and control subjects (assuming for controls subjects a CSF IgG4 mean value of 0.1 mg/dL and a standard deviation of 0.1). As for IgG4Loc, assuming a proportion of 10% of subjects with OHP presenting an IgG4Loc N 0 as compared to 100% of subjects with IgG4HP, we calculated that a sample size of three subjects with IgG4-HP and nine with OHP achieved 84% power to detect a difference of 90% between the two groups using a two-sided chi-square test with continuity correction and an alpha level of 0.05. According to the estimated sample size and based on the number of available subjects in each group, IgG4-HP patients were matched by sex and age (±3year), in a ratio of 1:3 or 1:7 (depending) with controls and OHP subjects, respectively. Results were reported as median (quartile 1 – quartile 3) or as frequency (%), as appropriate. The following four IgG4 subclass measures were evaluated for their performance in differentiating IgG4-HP and OHP from control subjects and patients with IgG4-HP from OHP: serum and CSF absolute concentrations, IgG4Index, and IgG4Loc. Of note, since IgG4Loc is pathologic when N 0, we included only positive values of this Index in the analysis and thus considered the three patients with IgG4-HP and five patients with OHP; none of the control subjects exhibited positive IgG4Loc values. Chi square or Fisher exact tests were used to compare qualitative variables between the three groups. The Wilcoxon rank-sum and Kruskal–Wallis tests were applied to compare values distributions of continuous variables among the three groups, as appropriate. The 5th and the 95th percentiles of each specific IgG4 measure determined among control subjects were used as thresholds for defining normal ranges; indeed, the 95th percentile of each specific IgG4 measure determined among patients with OHP was evaluated for its accuracy as a cut-off for the diagnosis of IgG4-HP. Diagnostic performance of each IgG4 measure in identifying IgG4-HP was measured by sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV); exact binomial method was used in order to calculate 95% confidence intervals (CI) for sensitivity, specificity, positive, and negative predictive value. All statistical tests were two-sided at the 5% level and were calculated using SAS Software (version 9.2; SAS Institute).

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3. Results Table 1 summarizes the results of routine serum and CSF analysis in the three patients with IgG4-HP and compares them to controls subjects and OHP. Six patients were excluded from the analysis since they did not undergo histological examination of the meninges. Two patients with IgG4-HP presented with multiorgan involvement and one with localized meningeal disease. CSF analysis exhibited intrathecal inflammation in all of them, as evidenced by a moderated-to-severe damage of the blood–CSF barrier; microbiological, immunological and cytological studies were unrevealing. Control subjects exhibited a preserved blood–CSF function (Albumin Quotient b0.7) without intrathecal synthesis of IgG antibodies, while patients with IgG4-HP and OHP showed a similar degree of blood–CSF barrier damage and CSF IgG production (p = 0.998 and 0.257, respectively); only five subjects with OHP demonstrated pathological values of IgG Index and IgGLoc. Table 2 compares serum and CSF IgG subclasses levels, IgG4 Index, and IgG4Loc between patients with IgG4-HP, control subjects and patients with OHP. Serum IgG4 concentrations in the IgG4-HP group were significantly higher than in controls (p=0.04), but not significantly different from those in patients with OHP (p = 0.08); interestingly, the two IgG4-HP patients with multiorgan involvement had increased serum IgG4 levels as opposite to the patient with localized meningeal disease who showed normal IgG4 concentration. Conversely, CSF IgG4 levels in IgG4-HP patients were significantly higher than in both control subjects and OHP (p=0.02 in both groups), with a median concentration of 5.2 mg/dL (IQR 4.9–5.4). Remarkably, among patients with OHP, elevated CSF IgG4 concentrations were found in the case of meningeal tuberculosis (2.3 mg/dL) and in one case of POEMS syndrome (9.0 mg/dL) with elevated serum IgG4 levels. Intrathecal production of IgG4 antibodies was quantified by means of IgG4 Index and IgG4Loc. Both the IgG4 Indices were normal in control subjects and significantly higher in IgG4-HP compared to OHP (p ≤ 0.03). Of note, among subjects with OHP, IgG4 Index and IgG4Loc were increased in five cases: Behcet's disease (1.31 and 0.11 respectively), GPA (2.26 and 0.47), cysticercosis (5.02 and 0.18), nocardiosis (3.12 and 0.27), and POEMS syndrome (0.81 and 0.46). Indeed, among patients with IgG4-HP, pathological values of IgG Index and IgGLoc were found for each IgG subclass, demonstrating that this condition implies a global intrathecal production of IgG that is not restricted to IgG4 antibodies (data not shown). Cut-off values for differentiating IgG4-HP from OHP are presented in Fig. 1. Serum IgG4 level was confirmed to be an unreliable marker of disease since a cut-off of 156 mg/dL allowed the identification of 1/3 cases of IgG4-HP and 1/21 cases of OHP (p = 0.24). Similarly, values of IgG4 Index higher than a cut-off of 3.12 identified 2/3 cases of IgG4HP and 2/21 cases of OHP (p = 0.06); however, by excluding patients with infectious pachymeningitis, for whom the final diagnosis was suggested by first level CSF analysis as well as by microbiological studies, a newly obtained IgG4 Index cut-off of 2.27 identified 100% of IgG4-HP and no cases of OHP (p b 0.0001). Cut-offs obtained from CSF IgG4 level and IgG4Loc demonstrated the highest diagnostic performance in correctly identifying IgG4-HP patients. In particular, CSF IgG4 levels higher than 2.27mg/dL diagnosed 100% of IgG4-HP and 5% of OHP (p = 0.002) while an IgG4Loc cut-off of 0.47 identified 100% of IgG4-HP and no cases of OHP (p b 0.0001). Interestingly, the combined evaluation of the cut-offs calculated for these two measures led to 100% sensitivity, specificity, PPV, and NPV in differentiating IgG4-HP from OHP (p b 0.0001). 4. Discussion IgG4-related disease has been described in virtually every organ system, and organ-specific diagnostic guidelines have been proposed by a number of investigators (Deshpande, 2012). However, CNS


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Table 2 Comparison of serum and CSF IgG subclasses levels, IgG4 Index, and IgG4Loc between patients with IgG4-HP, Control subjects and patients with Other forms of Hypertrophic Pachymeningits (OHP). (*) Normal values for serum IgG subclasses correspond to reference ranges used at San Raffaele Institute. Normal values for CSF IgG subclasses and IgG4 Index were defined using the 5th and 95th percentiles of Control subjects. (^) Calculated vs 5 patients with OHP who exhibited positive IgG4Loc. (a) IgG4-HP vs Controls subjects vs OHP patients by use of the Kruskal–Wallis test. (b) IgG4-HP vs Controls subjects by use of the Wilcoxon rank sum test. (c) IgG4-HP vs OHP patients by use of the Wilcoxon rank sum test. Normal values*

IgG1 IgG2 IgG3 IgG4

Serum IgG1 (mg/dL) CSF IgG1 (mg/dL) Serum IgG2 (mg/dL) CSF IgG2 (mg/dL) Serum IgG3 (mg/dL) CSF IgG3 (mg/dL) Serum IgG4 (mg/dL) CSF IgG4 (mg/dL) IgG4 Index IgG4Loc

(180–1280) (1•2–3•3) (34–640) (0•4–1•8) (13–160) (0•07–0•46) (3–230) (0•01–0•33) (0•25–2•11) 0

IgG4 Related Hypertrophic Pachymeningitis (n = 3)

Control subjects (n = 9)

Other forms of Hypertrophic Pachymeningits (n = 21)

Patient 1

Patient 2

Patient 3

Median (Q1–Q3)

Median (Q1–Q3)

Median (Q1–Q3)

295 2•77 108 0•78 39 0•25 210 4•94 3•16 4•09

617 10•42 170 0•5 74 1•1 47 5•36 3•21 3•91

524 10•98 217 1•97 37 0•72 153 5•17 3•07 4•02

524 (295–617) 10•42 (2•77–10•98) 170 (108–217) 0•78 (0•50–1•97) 39 (37–74) 0•72 (0•25–1•10) 153 (47–210) 5•17 (4•94–5•36) 3•16 (3•07–3•21) 4•02 (3•91–4•09)

785 (719–970) 1•30 (1•25–2•85) 197 (183–240) 0•68 (0•45–1•11) 73 (43–148) 0•15 (0•08–0•29) 20 (15–41) 0•05 (0•05–0•09) 0•62 (0•33–0•72) 0

604 (439–731) 6•41 (1•87–13•64) 196 (169–334) 1•87 (0•92–3•27) 48 (42–65) 0•26 (0•17–1•00) 25 (12–63) 0•24 (0•11–0•45) 0•53 (0•48–0•70) 0•22 (0•11–0•46)

involvement appears to be rather rare and unexplored, as IgG4-related hypophysitis and pachymeningitis have been reported only sporadically (Wallace et al., 2013). For these reasons, IgG4-RD CNS involvement remains poorly defined and the nature of immunological perturbations occurring across the blood–CSF barrier in these patients remains to be fully characterized. At present, HP (i.e. thickening of the dura mater) represents the most frequently described neurological complication of IgG4-RD with a dozen of reported cases (Della-Torre et al., 2012, 2013; Wallace et al., 2013). Meningeal disease can occur either in isolation or in association with other systemic manifestations (e.g. autoimmune

P-valuea

P-valueb

P-valuec

0•087 0•010 0•456 0•022 0•226 0•024 0•122 0•001 0•037 N/A

0•096 0•065 0•267 0•096 0•459 0•042 0•042 0•016 0•016 N/A

0•386 0•681 0•257 0•257 0•663 0•600 0•081 0•015 0•018 0•028^

pancreatitis, retroperitoneal fibrosis, orbital pseudotumor) and neurological symptoms typically arise when CNS structures are compressed by the growing fibrotic mass. A timely diagnosis is therefore warranted since prompt therapy may avoid prolonged nerve compression and consequent neurologic sequelae. However, the differential diagnosis of IHP is challenging because of its breadth and the need for multiple investigations for precise diagnosis. Moreover, patients with IgG4-HP have non-specific findings on routine CSF analysis and might lack typical systemic involvement as well as serum IgG4 elevation. Consequently, the final diagnosis often requires meningeal biopsy, an invasive surgical procedure,

Fig. 1. Scatterplot for serum and CSF IgG4, IgG4 Index and IgG4LOC in the three different groups (n). IgG4-HP(3), Control subjects(9) and patients with Other form of Hypertrophic Pacymeningitis (OHP)(21). Cut-off values were calculated as (a) the 95th percentile of subjects with OHP (n = 21) and (b) the 95th percentile of subjects with OHP without including infectious form of HP (n = 17).



with possible uninformative sampling due to technical difficulties. Thus, since IgG4 production is a key pathological feature of IgG4-RD and CSF analysis mirrors CNS inflammatory processes, we sought to determine whether CSF IgG4 concentration and IgG4 Indices could reliably distinguish IgG4-HP from other inflammatory, infectious and neoplastic disorders presenting with HP. Here we demonstrated that patients with IgG4-HP exhibit a moderate-to-severe blood–CSF barrier damage (which likely depends on the meningeal inflammatory infiltrates), oligoclonal IgG bands and high CSF IgG4 concentrations (which are the direct productive expression of such infiltrates). Of note, we previously reported that CSF oligoclonal bands in patients with IgG4-HP are preferentially of the IgG4 subclass, thus implying the presence of oligoclonally restricted IgG4 positive plasma cells in meningeal inflammatory niches (Della-Torre et al., 2013); in analogy with what observed in autoimmune cholangitis, this phenomenon could further support a pathogenic hypothesis of an immune reaction against a still unknown antigen(s) (de Buy Wenniger, 2013). As a result, we found pathologic IgG4 Index and IgG4Loc values in patients with IgG4-HP; such values were significantly higher than in subjects with OHP. IgG4Loc, which calculates and integrates serum and CSF IgG4 concentrations in relation to the blood–CSF barrier, demonstrated the best diagnostic performance, given that, when the latter is damaged, false positive IgG Index results may occur (Reiber and Peter, 2001). In particular, IgG4Loc values higher than 0.47 achieved 100% of both sensitivity and specificity in differentiating IgG4-HP from OHP. Interestingly, infectious forms of HP exhibited the highest IgG4 intrathecal synthesis among the group of OHP. This data is of particular relevance since the differential diagnosis with chronic meningeal infections has crucial therapeutic implications. However, infectious HPs are typically diagnosed on the basis of systemic inflammatory symptoms, suggestive serology, routine CSF analysis and microbiological exams. Therefore, CSF analysis for intrathecal IgG4 production should not substitute a thorough clinical, serological and microbiological evaluation, but rather integrate a comprehensive diagnostic process. Given the rarity of both IgG4-RD and IHP, our case series of IgG4-HP, although limited in number, is actually the most detailed in medical literature and has enough power to show consistent results and provide strong evidence. Moreover, the present work provides clinicians with a new diagnostic tool for approaching cases of IHP as well as a commercially available and easy replicable technique for confirming or ruling out the diagnosis of IgG4-HP. In particular, even-though histological evaluation remains the mainstay for diagnostic purposes, our results support CSF IgG4 and IgG4 Indices measurement as valid alternatives to meningeal biopsy when this procedure is contraindicated, technically risky, or uninformative. Moreover, the analysis of CSF for IgG4 production and IgG4 Indices in patients with IgG4-HP may be useful for monitoring therapeutic outcomes since efficacious immunosuppressive treatment results in normalization of IgG4 CSF levels (Della-Torre et al., 2013). In conclusion, given the lack of established defining criteria for extrapancreatic IgG4-RD involvement and the risks related to meningeal biopsies, we propose the analysis of CSF for intrathecal IgG4 production as a useful, non-invasive, and cost-affordable tool for diagnosing IgG4HP.

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Conflicts of interests All authors have not received any financial support or other benefits from commercial sources for the work reported on in the manuscript; authors have no financial interests, activities, relationships, and affiliations in large studies or other groups which could create a potential conflict of interest or the appearance of a conflict of interest with regard to the work. Acknowledgments The authors thank Prof. John H. Stone (Massachusetts General Hospital, Rheumatology Unit) for helpful review of the manuscript. The authors thank Dr. Marco Losa (Neurosurgery Department, San Raffaele Scientific Institute) and Dr. Dacia Dalla Libera (Neurology Department, San Raffaele Scientific Institute) for helpful discussion of clinical cases. References Cheuk, W., Chan, J.K.C., 2010. IgG4-related sclerosing disease. A critical appraisal of an evolving clinicopathologic Entity. Adv. Anat. Pathol. 17 (5), 303–332. Davatchi, F., 2012. Diagnosis/classification criteria for Behcet's disease. Pathol. Res. Int. 2012, 607921. de Buy Wenniger, L.J., Doorenspleet, M.E., Klarenbeek, P.L., Verheij, J., Baas, F., Elferink, R.P., Tak, P.P., de Vries, N., Beuers, U., 2013. IgG4+ clones identified by nextgeneration sequencing dominate the b-cell receptor repertoire in IgG4-associated cholangitis. Hepatology 57 (6), 2390–2398. Della-Torre, E., Bozzolo, E.P., Passerini, G., Doglioni, C., Sabbadini, M.G., 2012. IgG4-related pachymeningitis: evidence of intrathecal IgG4 on cerebrospinal fluid analysis. Ann. Intern. Med. 156 (5), 401–403. Della-Torre, E., Passerini, G., Furlan, R., Roveri, L., Chieffo, R., Anzalone, N., Claudio, Doglioni, Elisabetta, Zardini, Maria Grazia, Sabbadini, Diego, Franciotta, 2013. Cerebrospinal fluid analysis in IgG4-related hypertrophic pachymeningitis. J. Rheumatol. 40 (11), 1927–1929. Deshpande, V., Zen, Y., Chan, J.K., Yi, E.E., Sato, Y., Yoshino, T., Klöppel, G., Heathcote, J.G., Khosroshahi, A., Ferry, J.A., Aalberse, R.C., Bloch, D.B., Brugge, W.R., Bateman, A.C., Carruthers, M.N., Chari, S.T., Cheuk, W., Cornell, L.D., Fernandez-Del Castillo, C., Forcione, D.G., Hamilos, D.L., Kamisawa, T., Kasashima, S., Kawa, S., Kawano, M., Lauwers, G.Y., Masaki, Y., Nakanuma, Y., Notohara, K., Okazaki, K., Ryu, J.K., Saeki, T., Sahani, D.V., Smyrk, T.C., Stone, J.R., Takahira, M., Webster, G.J., Yamamoto, M., Zamboni, G., Umehara, H., Stone, J.H., 2012. Consensus statement on the pathology of IgG4-related disease. Mod. Pathol. 25 (9), 1181–1192. Dispenzieri, A., 2012. POEMS syndrome: update on diagnosis, risk-stratification, and management. Am. J. Hematol. 87 (8), 804–814. Hamano, H., Kawa, S., Horiuchi, A., Unno, H., Furuya, N., Akamatsu, T., Fukushima, M., Nikaido, T., Nakayama, K., Usuda, N., Kiyosawa, K., 2001. High serum IgG4 concentrations in patients with sclerosing pancreatitis. N. Engl. J. Med. 344 (10), 732–738. Hsieh, F.Y., 1987. A simple method of sample size calculation for unequal-sample-size designs that use the logrank or t-test. Stat. Med. 6 (5), 577–581. Reiber, H., Peter, J.B., 2001. Cerebrospinal fluid analysis: disease-related data patterns and evaluation programs. J. Neurol. Sci. 184 (2), 101–122. Tibbling, G., Link, H., Ohman, S., 1977. Principles of albumin and IgG analyses in neurological disorders. I. Establishment of reference values. Scand. J. Clin. Lab. Invest. 37 (5), 385–390. Wallace, Z.S., Carruthers, M.N., Khosroshahi, A., Carruthers, R., Shinagare, S., Stemmer-Rachamimov, A., Deshpande, V., Stone, J.H., 2013. IgG4-related disease and hypertrophic pachymeningitis. Med. (Baltimore) 92 (4), 206–216. Watts, R., Lane, S., Hanslik, T., Hauser, T., Hellmich, B., Koldingsnes, W., Mahr, A., Segelmark, M., Cohen-Tervaert, J.W., Scott, D., 2007. Development and validation of a consensus methodology for the classification of the ANCA-associated vasculitides and polyarteritis nodosa for epidemiological studies. Ann. Rheum. Dis. 66 (2), 222–227. Whitley, E., Ball, J., 2002. Statistics review 4: sample size calculations. Crit. Care 6 (4), 335–341. Zen, Y., Nakanuma, Y., 2011. Pathogenesis of IgG4-related disease. Curr. Opin. Rheumatol. 23 (1), 114–118.
