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Chitinase 3-like 1 is associated with the response to interferon-beta treatment in multiple sclerosis
JNI-476480; No of Pages 4 Journal of Neuroimmunology xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short communication
Chitinase 3-like 1 is associated with the response to interferon-beta treatment in multiple sclerosis Clara Matute-Blanch a,⁎, Jordi Río a, Luisa M Villar b, Luciana Midaglia a, Sunny Malhotra a, José C Álvarez-Cermeño b, Angela Vidal-Jordana a, Xavier Montalban a, Manuel Comabella a,⁎ a Servei de Neurologia-Neuroimmunologia, Centre d'Esclerosi Múltiple de Catalunya (Cemcat), Institut de Recerca Vall d'Hebron (VHIR), Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain b Departments of Neurology and Immunology, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigacion Sanitaria, Madrid, Spain.
a r t i c l e
i n f o
Article history: Received 3 October 2016 Received in revised form 2 December 2016 Accepted 12 December 2016 Available online xxxx Keywords: Chitinase 3-like 1 Interferon-beta Glatiramer acetate Biomarkers Multiple sclerosis
a b s t r a c t Chitinase 3-like 1 (CHI3L1) plays a prognostic role in patients with multiple sclerosis (MS). Here, we investigated a potential association between CHI3L1 and the response to interferon-beta (IFNβ) and glatiramer acetate (GA). Serum CHI3L1 levels were measured by ELISA in 117 relapsing-remitting MS (RRMS) patients, 76 IFNβ-treated and 41 GA-treated patients. CHI3L1 levels were increased by GA (p = 0.014) but unchanged by IFNβ (p = 0.830). CHI3L1 was associated with IFNβ response and levels were higher in non-responder group (p = 0.020), while GA showed no responder effect (p = 0.943). These results suggest a role for CHI3L1 as response biomarker to IFNβ in RRMS patients. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Chitinase 3-like 1 (CHI3L1) is a member of the glycoside hydrolase 18 chitinase family that targets chitin but lacks enzymatic activity (Renkema et al., 1998). It is produced by a wide range of cells such as macrophages, chondrocytes, synovial cells, osteoblasts, neutrophils, and vascular smooth muscle cells (Hakala et al., 1993; Johansen, 2006; Kawada et al., 2007). Although its physiological function remains elusive, recent studies point to a clear prognostic role for CHI3L1 in patients with multiple sclerosis (MS), particularly at the time of the first neurological event or clinically isolated syndrome (CIS). In this regard, high cerebrospinal fluid CHI3L1 levels in CIS patients have been associated with the conversion to MS (Canto et al., 2015; Hinsinger et al., 2015; Modvig et al., 2015) and with the development of neurological disability (Canto et al., 2015). Studies of CHI3L1 in peripheral blood of MS patients are scarce. In a previous study conducted by our group in plasma samples from MS patients, CHI3L1 levels were found elevated in patients with primary progressive and secondary progressive MS, suggesting a Abbreviations: CHI3L1, chitinase 3-like 1; MS, multiple sclerosis; RRMS, relapsingremitting multiple sclerosis; CIS, clinically isolated syndrome; DMT, disease modifying therapies; IFNβ, interferon-beta; GA, glatiramer acetate. ⁎ Corresponding authors at: Centre d'Esclerosi Múltiple de Catalunya (Cemcat), Institut de Recerca Vall d'Hebron (VHIR), Hospital Universitari Vall d'Hebron, Pg. Vall d'Hebron 119-129, 08035 Barcelona, Spain. E-mail addresses: [email protected] (C. Matute-Blanch), [email protected] (M. Comabella).
role for CHI3L1 in the progressive forms of the disease (Canto et al., 2012). In this same study (Canto et al., 2012), plasma levels of CHI3L1 were also measured in a cohort of relapsing-remitting MS (RRMS) patients before and after treatment with interferon-beta (IFNβ). The finding of a trend towards decreased CHI3L1 in treated patients suggested that peripheral blood CHI3L1 levels could also be modulated by IFNβ treatment. However, the response to IFNβ treatment was not specifically addressed in this particular study (Canto et al., 2012). Building on these previous observations, in the present study we aimed to investigate a potential association between CHI3L1 and the response to firstline disease modifying therapies (DMT) such as IFNβ and glatiramer acetate (GA). 2. Methods 2.1. Ethics statement The study was approved by the Hospital ethics committee, and all patients gave their informed consent. 2.2. Patients This is a prospective study of naive RRMS patients treated with IFNβ or GA at the outpatient clinic of the Centre d'Esclerosi Multiple de Catalunya (Cemcat) and the Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS) and included into a follow-up protocol collecting
http://dx.doi.org/10.1016/j.jneuroim.2016.12.006 0165-5728/© 2016 Elsevier B.V. All rights reserved.
Please cite this article as: Matute-Blanch, C., et al., Chitinase 3-like 1 is associated with the response to interferon-beta treatment in multiple sclerosis, J. Neuroimmunol. (2016), http://dx.doi.org/10.1016/j.jneuroim.2016.12.006
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C. Matute-Blanch et al. / Journal of Neuroimmunology xxx (2016) xxx–xxx
Table 1 Demographic and baseline clinical characteristics of the whole cohort of MS patients treated with GA. Characteristics
R
NR
P valuesc
N Age (years) Female/male (% women) Duration of disease (years) EDSSa Number of relapsesb Number of Gd-enhancing lesions
28 38.6 (10.4) 17/11 (60.7) 6.5 (6.0) 1.8 (1.5–2.0) 1.7 (0.9) 8.1 (10.7)
13 32.1 (9.8) 7/6 (53.8) 4.7 (3.8) 1.7 (1.0–2.5) 1.8 (1.1) 12.1 (9.4)
– 0.064 0.678 0.276 0.561 0.674 0.286
Data are expressed as mean (standard deviation) unless otherwise stated. a Data are expressed as mean (interquartile range). b Refers to the number of relapses in the two previous years. EDSS: Expanded Disability Status Scale. Gd: gadolinium. R: responders to GA. NR: non-responders to GA. c P values: Refers to p values obtained following comparisons between responders and non-responders by means of Student-t-test or Mann-Whitney's test depending on the applicability conditions (age, duration, EDSS, and number of relapses) and chi-square test (gender).
and the intra-assay and inter-assay coefficients of variation were 7.7% and 11.7% respectively. 2.4. Statistical analysis Statistical analysis was performed by using the IBM SPSS Statistics for Windows version 20.0 (IBM Corp, Armonk, NY). An analysis of variance (ANOVA) for repeated measures was used to analyze: (i) time effect, which addresses the question whether CHI3L1 protein levels are modified by treatment during the follow-up period; (ii) responder effect, which addresses the question whether responders and non-responders to DMT differ in the mean CHI3L1 protein levels; and (iii) responder by time interactions, which addresses the question whether responders and non-responders differ in the CHI3L1 protein levels during the follow-up period. Comparisons of mean serum CHI3L1 levels before and after treatment with DMT in responders and non-responders were assessed using a paired Student-t-test. 3. Results
basal and longitudinal clinical data. Clinical and radiological response to first-line DMT was assessed after 1 year of treatment (Rio et al., 2009; Rio et al., 2014). Non-responders to DMT were patients fulfilling 2 or 3 of the following criteria: (i) presence of 1 or more relapses; (ii) confirmed increase at 6 months of 1 or more points in the EDSS score; (iii) presence of 3 or more active lesions (gadolinium enhancing lesions or new or enlarging T2 lesions) on the 1-year brain MRI. The remaining patients were considered responders to DMT. A total of 117 RRMS patients were included in the study, 76 treated with IFNβ and 41 with GA. Of these, 14 patients were classified as non-responders to IFNβ and 13 to GA. Tables 1 and 2 and Supplementary Tables 1 and 2 summarize demographic and main clinical characteristics of patients stratified by treatment and inclusion center.
2.3. Determination of CHI3L1 levels in serum samples by ELISA Peripheral blood was collected by standard venipuncture and allowed to clot spontaneously for 30 min. Serum was obtained by centrifugation and stored frozen at −80 °C until used. CHI3L1 levels were determined in serum samples at baseline and after 12 months of treatment with DMT by enzyme-linked immunosorbent assay (ELISA; MicroVue YKL-40 EIA Kit, Quidel) according to the manufacturers' recommendations. Undiluted serum samples were measured in duplicate
Table 2 Demographic and baseline clinical characteristics of the whole cohort of MS patients treated with IFNβ. Characteristics
R
NR
P valuesc
N Age (years) Female/male (% women) Duration of disease (years) EDSSa Number of relapsesb Number of Gd-enhancing lesions Type of IFNβ [n (%)] IFNβ 1a IM IFNβ 1b SC IFNβ 1a SC
62 35.0 (8.7) 45/17 (72.6) 4.2 (4.6) 1.8 (1.0–2.0) 1.6 (1.1) 9.0 (12.2)
14 36.7 (8.7) 12/2 (85.7) 6.3 (5.4) 2.6 (1.4–3.6) 1.4 (1.1) 11.3 (14.3)
– 0.496 0.305 0.138 0.062 0.632 0.536
30 (48.4) 10 (16.1) 22 (35.5)
6 (42.9) 5 (35.7) 3 (21.4)
0.271
Data are expressed as mean (standard deviation) unless otherwise stated. a Data are expressed as mean (interquartile range). b Refers to the number of relapses in the two previous years. EDSS: Expanded Disability Status Scale. Gd: gadolinium. IM: intramuscular. SC: subcutaneous. R: responders to IFNβ. NR: non-responders to IFNβ. c P values: Refers to p values obtained following comparisons between responders and non-responders by means of Student-t-test or Mann-Whitney's test depending on the applicability conditions (age, duration, EDSS, and number of relapses) and chi-square test (gender and type of IFNβ).
3.1. Serum levels of CHI3L1 are increased by GA We first investigated whether serum CHI3L1 protein levels were modulated by first-line DMT in RRMS patients. As shown in Fig. 1, a statistically significant time effect was observed for GA, and CHI3L1 levels were significantly increased by the effect of GA (F = 6.604, p = 0.014). In contrast, time effect was not statistically significant for IFNβ and mean serum CHI3L1 levels were similar between the baseline and the 12 months time point in the whole group of IFNβ-treated patients (F = 0.046, p = 0.830; Fig. 1). 3.2. Serum CHI3L1 levels are associated with the response to IFNβ treatment We next assessed whether mean serum CHI3L1 levels differed between responders and non-responders during the first year of treatment with DMT. As shown in Fig. 2, a statistically significant responder effect was observed for IFNβ (F = 5.652, p = 0.020). Mean CHI3L1 levels were significantly higher in non-responders compared to responders after 12 months of IFNβ treatment (p = 0.013), whereas a trend towards increased serum CHI3L1 levels was already observed at baseline in the non-responder group (p = 0.055). When we investigated the responder effect of CHI3L1 in GA-treated patients, mean CHI3L1 levels in serum did not significantly differ over time between responders and non-responders (F = 0.005, p = 0.943; Fig. 2). Finally, when the group by time interactions were analyzed no statistically significant differences were observed between responders and non-responders to IFNβ (F = 0.392, p = 0.533) or GA (F = 0.289, p = 0.594). 4. Discussion CHI3L1 measured in the CSF represents a promising prognostic biomarker in patients with CIS (Canto et al., 2015). Here, we aimed to investigate the role of CHI3L1 measured in peripheral blood with the response to IFNβ and GA, two established first-line DMT known to be partially effective and associated with a significant proportion of MS patients who do not respond to treatment (Rio et al., 2002; Rio et al., 2014). CHI3L1 levels were measured in serum samples at baseline and after 12 months of treatment, the latter corresponding to the time point at which the response to IFNβ and GA was evaluated by applying both clinical and radiological criteria (Rio et al., 2009; Rio et al., 2014). Regarding GA, CHI3L1 levels were modulated by the effect of treatment, and serum protein levels were significantly increased in the treated time point compared with the baseline. However, despite the
Please cite this article as: Matute-Blanch, C., et al., Chitinase 3-like 1 is associated with the response to interferon-beta treatment in multiple sclerosis, J. Neuroimmunol. (2016), http://dx.doi.org/10.1016/j.jneuroim.2016.12.006
C. Matute-Blanch et al. / Journal of Neuroimmunology xxx (2016) xxx–xxx
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Fig. 1. Time effect of CHI3L1 in GA- and IFNβ-treated RRMS patients. Mean serum levels of CHI3L1 in the whole group of patients treated with GA (N = 41) and IFNβ (N = 76) before treatment (T0) and after 12 months of treatment (T12). Significant p-values are shown in bold. Error bars represent standard error of the mean. CHI3L1: chitinase 3-like 1.
increase in serum CHI3L1 levels induced by GA, overall mean protein levels did not significantly differ between responders and non-responders, thus explaining the non-significant responder effect observed for this treatment. These results suggest that the changes in serum CHI3L1 levels induced by GA maybe related with the mechanism of action of GA but are not associated with the clinical response to this treatment. Contrary to GA, IFNβ did not seem to modify serum CHI3L1 levels and, actually, protein levels fluctuated very little by the effect of treatment. However, it is important to remark that CHI3L1 was associated with a significant responder effect to IFNβ, and mean CHI3L1 levels were overall increased in treatment non-responders compared to responders. Interestingly, the difference in serum CHI3L1 levels was already present at baseline, finding that may suggest higher disease activity in IFNβ non-responders. It should be mentioned that the lack of a placebo group does not certainly allow discriminating between
lack of response to IFNβ and natural evolution of the disease. Nevertheless, the fact that clinical and radiological variables were comparable between responders and non-responders at baseline, particularly the number of relapses in the two previous years and the number of contrast-enhancing lesions in the MRI, and the absence of similar differences in serum CHI3L1 levels at baseline in the GA-treated cohort, both factors suggest that the significant responder effect observed for IFNβ in our study is due to a true lack of response to treatment rather than to differences in disease activity between IFNβ responders and non-responders. In summary, the aggregate results of the study further expand the prognostic involvement of CHI3L1 in the disease, particularly in patients with CIS, and may suggest a role for CHI3L1 as an IFNβ treatment response biomarker in RRMS patients. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jneuroim.2016.12.006.
Fig. 2. Responder effect of CHI3L1 in GA- and IFNβ-treated RRMS patients. Mean serum levels of CHI3L1 in patients classified into responders (R) and non-responders (NR) according to clinical and radiological criteria after one year of treatment, as described in the Methods section. T0: CHI3L1 levels at baseline. T12: CHI3L1 levels at the 12 month time point. Significant pvalues are shown in bold. Error bars represent standard error of the mean. CHI3L1: chitinase 3-like 1.
Please cite this article as: Matute-Blanch, C., et al., Chitinase 3-like 1 is associated with the response to interferon-beta treatment in multiple sclerosis, J. Neuroimmunol. (2016), http://dx.doi.org/10.1016/j.jneuroim.2016.12.006
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C. Matute-Blanch et al. / Journal of Neuroimmunology xxx (2016) xxx–xxx
Author contribution CM, MC designed the study. CM, SM performed the experiments. JR, LMV, LM, JCAV, AVJ, XM provided the samples and collected clinical data. CM, MC analyzed the data. CM, MC wrote the article. CM, SM, JR, LMV, LM, JCAV, AVJ, XM, MC revised the article and approved the final version of the manuscript. Competing interests The authors declare that they have no competing interests. Acknowledgements The authors thank the “Red Española de Esclerosis Múltiple (REEM)” sponsored by the “Fondo de Investigación Sanitaria” (FIS), Ministry of Science and Innovation, Spain, and the “Ajuts per donar Suport als Grups de Recerca de Catalunya”, sponsored by the “Agència de Gestió d'Ajuts Universitaris i de Recerca” (AGAUR), Generalitat de Catalunya, Spain. References Canto, E., Reverter, F., Morcillo-Suarez, C., Matesanz, F., Fernandez, O., Izquierdo, G., et al., 2012. Chitinase 3-like 1 plasma levels are increased in patients with progressive forms of multiple sclerosis. Mult. Scler. 18, 983–990.
Canto, E., Tintore, M., Villar, L.M., Costa, C., Nurtdinov, R., Alvarez-Cermeno, J.C., et al., 2015. Chitinase 3-like 1: prognostic biomarker in clinically isolated syndromes. Brain 138, 918–931. Hakala, B.E., White, C., Recklies, A.D., 1993. Human cartilage gp-39, a major secretory product of articular chondrocytes and synovial cells, is a mammalian member of a chitinase protein family. J. Biol. Chem. 268, 25803-10. Hinsinger, G., Galeotti, N., Nabholz, N., Urbach, S., Rigau, V., Demattei, C., et al., 2015. Chitinase 3-like proteins as diagnostic and prognostic biomarkers of multiple sclerosis. Mult. Scler. 21, 1251–1261. Johansen, J.S., 2006. Studies on serum YKL-40 as a biomarker in diseases with inflammation, tissue remodelling, fibroses and cancer. Dan. Med. Bull. 53, 172–209. Kawada, M., Hachiya, Y., Arihiro, A., Mizoguchi, E., 2007. Role of mammalian chitinases in inflammatory conditions. Keio J. Med. 56, 21–27. Modvig, S., Degn, M., Roed, H., Sorensen, T.L., Larsson, H.B., Langkilde, A.R., et al., 2015. Cerebrospinal fluid levels of chitinase 3-like 1 and neurofilament light chain predict multiple sclerosis development and disability after optic neuritis. Mult. Scler. 21, 1761–1770. Renkema, G.H., Boot, R.G., FL, A., Donker-Koopman, W.E., Strijland, A., Muijsers, A.O., et al., 1998. Chitotriosidase, a chitinase, and the 39-kDa human cartilage glycoprotein, a chitin-binding lectin, are homologues of family 18 glycosyl hydrolases secreted by human macrophages. Eur. J. Biochem./FEBS. 251, 504–509. Rio, J., Castillo, J., Rovira, A., Tintore, M., Sastre-Garriga, J., Horga, A., et al., 2009. Measures in the first year of therapy predict the response to interferon beta in MS. Mult. Scler. 15, 848–853. Rio, J., Nos, C., Tintore, M., Borras, C., Galan, I., Comabella, M., et al., 2002. Assessment of different treatment failure criteria in a cohort of relapsing-remitting multiple sclerosis patients treated with interferon beta: implications for clinical trials. Ann. Neurol. 52, 400–406. Rio, J., Rovira, A., Tintore, M., Sastre-Garriga, J., Castillo, J., Auger, C., et al., 2014. Evaluating the response to glatiramer acetate in relapsing-remitting multiple sclerosis (RRMS) patients. Mult. Scler. 20, 1602–1608.
Please cite this article as: Matute-Blanch, C., et al., Chitinase 3-like 1 is associated with the response to interferon-beta treatment in multiple sclerosis, J. Neuroimmunol. (2016), http://dx.doi.org/10.1016/j.jneuroim.2016.12.006
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short communication
Chitinase 3-like 1 is associated with the response to interferon-beta treatment in multiple sclerosis Clara Matute-Blanch a,⁎, Jordi Río a, Luisa M Villar b, Luciana Midaglia a, Sunny Malhotra a, José C Álvarez-Cermeño b, Angela Vidal-Jordana a, Xavier Montalban a, Manuel Comabella a,⁎ a Servei de Neurologia-Neuroimmunologia, Centre d'Esclerosi Múltiple de Catalunya (Cemcat), Institut de Recerca Vall d'Hebron (VHIR), Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain b Departments of Neurology and Immunology, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigacion Sanitaria, Madrid, Spain.
a r t i c l e
i n f o
Article history: Received 3 October 2016 Received in revised form 2 December 2016 Accepted 12 December 2016 Available online xxxx Keywords: Chitinase 3-like 1 Interferon-beta Glatiramer acetate Biomarkers Multiple sclerosis
a b s t r a c t Chitinase 3-like 1 (CHI3L1) plays a prognostic role in patients with multiple sclerosis (MS). Here, we investigated a potential association between CHI3L1 and the response to interferon-beta (IFNβ) and glatiramer acetate (GA). Serum CHI3L1 levels were measured by ELISA in 117 relapsing-remitting MS (RRMS) patients, 76 IFNβ-treated and 41 GA-treated patients. CHI3L1 levels were increased by GA (p = 0.014) but unchanged by IFNβ (p = 0.830). CHI3L1 was associated with IFNβ response and levels were higher in non-responder group (p = 0.020), while GA showed no responder effect (p = 0.943). These results suggest a role for CHI3L1 as response biomarker to IFNβ in RRMS patients. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Chitinase 3-like 1 (CHI3L1) is a member of the glycoside hydrolase 18 chitinase family that targets chitin but lacks enzymatic activity (Renkema et al., 1998). It is produced by a wide range of cells such as macrophages, chondrocytes, synovial cells, osteoblasts, neutrophils, and vascular smooth muscle cells (Hakala et al., 1993; Johansen, 2006; Kawada et al., 2007). Although its physiological function remains elusive, recent studies point to a clear prognostic role for CHI3L1 in patients with multiple sclerosis (MS), particularly at the time of the first neurological event or clinically isolated syndrome (CIS). In this regard, high cerebrospinal fluid CHI3L1 levels in CIS patients have been associated with the conversion to MS (Canto et al., 2015; Hinsinger et al., 2015; Modvig et al., 2015) and with the development of neurological disability (Canto et al., 2015). Studies of CHI3L1 in peripheral blood of MS patients are scarce. In a previous study conducted by our group in plasma samples from MS patients, CHI3L1 levels were found elevated in patients with primary progressive and secondary progressive MS, suggesting a Abbreviations: CHI3L1, chitinase 3-like 1; MS, multiple sclerosis; RRMS, relapsingremitting multiple sclerosis; CIS, clinically isolated syndrome; DMT, disease modifying therapies; IFNβ, interferon-beta; GA, glatiramer acetate. ⁎ Corresponding authors at: Centre d'Esclerosi Múltiple de Catalunya (Cemcat), Institut de Recerca Vall d'Hebron (VHIR), Hospital Universitari Vall d'Hebron, Pg. Vall d'Hebron 119-129, 08035 Barcelona, Spain. E-mail addresses: [email protected] (C. Matute-Blanch), [email protected] (M. Comabella).
role for CHI3L1 in the progressive forms of the disease (Canto et al., 2012). In this same study (Canto et al., 2012), plasma levels of CHI3L1 were also measured in a cohort of relapsing-remitting MS (RRMS) patients before and after treatment with interferon-beta (IFNβ). The finding of a trend towards decreased CHI3L1 in treated patients suggested that peripheral blood CHI3L1 levels could also be modulated by IFNβ treatment. However, the response to IFNβ treatment was not specifically addressed in this particular study (Canto et al., 2012). Building on these previous observations, in the present study we aimed to investigate a potential association between CHI3L1 and the response to firstline disease modifying therapies (DMT) such as IFNβ and glatiramer acetate (GA). 2. Methods 2.1. Ethics statement The study was approved by the Hospital ethics committee, and all patients gave their informed consent. 2.2. Patients This is a prospective study of naive RRMS patients treated with IFNβ or GA at the outpatient clinic of the Centre d'Esclerosi Multiple de Catalunya (Cemcat) and the Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS) and included into a follow-up protocol collecting
http://dx.doi.org/10.1016/j.jneuroim.2016.12.006 0165-5728/© 2016 Elsevier B.V. All rights reserved.
Please cite this article as: Matute-Blanch, C., et al., Chitinase 3-like 1 is associated with the response to interferon-beta treatment in multiple sclerosis, J. Neuroimmunol. (2016), http://dx.doi.org/10.1016/j.jneuroim.2016.12.006
2
C. Matute-Blanch et al. / Journal of Neuroimmunology xxx (2016) xxx–xxx
Table 1 Demographic and baseline clinical characteristics of the whole cohort of MS patients treated with GA. Characteristics
R
NR
P valuesc
N Age (years) Female/male (% women) Duration of disease (years) EDSSa Number of relapsesb Number of Gd-enhancing lesions
28 38.6 (10.4) 17/11 (60.7) 6.5 (6.0) 1.8 (1.5–2.0) 1.7 (0.9) 8.1 (10.7)
13 32.1 (9.8) 7/6 (53.8) 4.7 (3.8) 1.7 (1.0–2.5) 1.8 (1.1) 12.1 (9.4)
– 0.064 0.678 0.276 0.561 0.674 0.286
Data are expressed as mean (standard deviation) unless otherwise stated. a Data are expressed as mean (interquartile range). b Refers to the number of relapses in the two previous years. EDSS: Expanded Disability Status Scale. Gd: gadolinium. R: responders to GA. NR: non-responders to GA. c P values: Refers to p values obtained following comparisons between responders and non-responders by means of Student-t-test or Mann-Whitney's test depending on the applicability conditions (age, duration, EDSS, and number of relapses) and chi-square test (gender).
and the intra-assay and inter-assay coefficients of variation were 7.7% and 11.7% respectively. 2.4. Statistical analysis Statistical analysis was performed by using the IBM SPSS Statistics for Windows version 20.0 (IBM Corp, Armonk, NY). An analysis of variance (ANOVA) for repeated measures was used to analyze: (i) time effect, which addresses the question whether CHI3L1 protein levels are modified by treatment during the follow-up period; (ii) responder effect, which addresses the question whether responders and non-responders to DMT differ in the mean CHI3L1 protein levels; and (iii) responder by time interactions, which addresses the question whether responders and non-responders differ in the CHI3L1 protein levels during the follow-up period. Comparisons of mean serum CHI3L1 levels before and after treatment with DMT in responders and non-responders were assessed using a paired Student-t-test. 3. Results
basal and longitudinal clinical data. Clinical and radiological response to first-line DMT was assessed after 1 year of treatment (Rio et al., 2009; Rio et al., 2014). Non-responders to DMT were patients fulfilling 2 or 3 of the following criteria: (i) presence of 1 or more relapses; (ii) confirmed increase at 6 months of 1 or more points in the EDSS score; (iii) presence of 3 or more active lesions (gadolinium enhancing lesions or new or enlarging T2 lesions) on the 1-year brain MRI. The remaining patients were considered responders to DMT. A total of 117 RRMS patients were included in the study, 76 treated with IFNβ and 41 with GA. Of these, 14 patients were classified as non-responders to IFNβ and 13 to GA. Tables 1 and 2 and Supplementary Tables 1 and 2 summarize demographic and main clinical characteristics of patients stratified by treatment and inclusion center.
2.3. Determination of CHI3L1 levels in serum samples by ELISA Peripheral blood was collected by standard venipuncture and allowed to clot spontaneously for 30 min. Serum was obtained by centrifugation and stored frozen at −80 °C until used. CHI3L1 levels were determined in serum samples at baseline and after 12 months of treatment with DMT by enzyme-linked immunosorbent assay (ELISA; MicroVue YKL-40 EIA Kit, Quidel) according to the manufacturers' recommendations. Undiluted serum samples were measured in duplicate
Table 2 Demographic and baseline clinical characteristics of the whole cohort of MS patients treated with IFNβ. Characteristics
R
NR
P valuesc
N Age (years) Female/male (% women) Duration of disease (years) EDSSa Number of relapsesb Number of Gd-enhancing lesions Type of IFNβ [n (%)] IFNβ 1a IM IFNβ 1b SC IFNβ 1a SC
62 35.0 (8.7) 45/17 (72.6) 4.2 (4.6) 1.8 (1.0–2.0) 1.6 (1.1) 9.0 (12.2)
14 36.7 (8.7) 12/2 (85.7) 6.3 (5.4) 2.6 (1.4–3.6) 1.4 (1.1) 11.3 (14.3)
– 0.496 0.305 0.138 0.062 0.632 0.536
30 (48.4) 10 (16.1) 22 (35.5)
6 (42.9) 5 (35.7) 3 (21.4)
0.271
Data are expressed as mean (standard deviation) unless otherwise stated. a Data are expressed as mean (interquartile range). b Refers to the number of relapses in the two previous years. EDSS: Expanded Disability Status Scale. Gd: gadolinium. IM: intramuscular. SC: subcutaneous. R: responders to IFNβ. NR: non-responders to IFNβ. c P values: Refers to p values obtained following comparisons between responders and non-responders by means of Student-t-test or Mann-Whitney's test depending on the applicability conditions (age, duration, EDSS, and number of relapses) and chi-square test (gender and type of IFNβ).
3.1. Serum levels of CHI3L1 are increased by GA We first investigated whether serum CHI3L1 protein levels were modulated by first-line DMT in RRMS patients. As shown in Fig. 1, a statistically significant time effect was observed for GA, and CHI3L1 levels were significantly increased by the effect of GA (F = 6.604, p = 0.014). In contrast, time effect was not statistically significant for IFNβ and mean serum CHI3L1 levels were similar between the baseline and the 12 months time point in the whole group of IFNβ-treated patients (F = 0.046, p = 0.830; Fig. 1). 3.2. Serum CHI3L1 levels are associated with the response to IFNβ treatment We next assessed whether mean serum CHI3L1 levels differed between responders and non-responders during the first year of treatment with DMT. As shown in Fig. 2, a statistically significant responder effect was observed for IFNβ (F = 5.652, p = 0.020). Mean CHI3L1 levels were significantly higher in non-responders compared to responders after 12 months of IFNβ treatment (p = 0.013), whereas a trend towards increased serum CHI3L1 levels was already observed at baseline in the non-responder group (p = 0.055). When we investigated the responder effect of CHI3L1 in GA-treated patients, mean CHI3L1 levels in serum did not significantly differ over time between responders and non-responders (F = 0.005, p = 0.943; Fig. 2). Finally, when the group by time interactions were analyzed no statistically significant differences were observed between responders and non-responders to IFNβ (F = 0.392, p = 0.533) or GA (F = 0.289, p = 0.594). 4. Discussion CHI3L1 measured in the CSF represents a promising prognostic biomarker in patients with CIS (Canto et al., 2015). Here, we aimed to investigate the role of CHI3L1 measured in peripheral blood with the response to IFNβ and GA, two established first-line DMT known to be partially effective and associated with a significant proportion of MS patients who do not respond to treatment (Rio et al., 2002; Rio et al., 2014). CHI3L1 levels were measured in serum samples at baseline and after 12 months of treatment, the latter corresponding to the time point at which the response to IFNβ and GA was evaluated by applying both clinical and radiological criteria (Rio et al., 2009; Rio et al., 2014). Regarding GA, CHI3L1 levels were modulated by the effect of treatment, and serum protein levels were significantly increased in the treated time point compared with the baseline. However, despite the
Please cite this article as: Matute-Blanch, C., et al., Chitinase 3-like 1 is associated with the response to interferon-beta treatment in multiple sclerosis, J. Neuroimmunol. (2016), http://dx.doi.org/10.1016/j.jneuroim.2016.12.006
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Fig. 1. Time effect of CHI3L1 in GA- and IFNβ-treated RRMS patients. Mean serum levels of CHI3L1 in the whole group of patients treated with GA (N = 41) and IFNβ (N = 76) before treatment (T0) and after 12 months of treatment (T12). Significant p-values are shown in bold. Error bars represent standard error of the mean. CHI3L1: chitinase 3-like 1.
increase in serum CHI3L1 levels induced by GA, overall mean protein levels did not significantly differ between responders and non-responders, thus explaining the non-significant responder effect observed for this treatment. These results suggest that the changes in serum CHI3L1 levels induced by GA maybe related with the mechanism of action of GA but are not associated with the clinical response to this treatment. Contrary to GA, IFNβ did not seem to modify serum CHI3L1 levels and, actually, protein levels fluctuated very little by the effect of treatment. However, it is important to remark that CHI3L1 was associated with a significant responder effect to IFNβ, and mean CHI3L1 levels were overall increased in treatment non-responders compared to responders. Interestingly, the difference in serum CHI3L1 levels was already present at baseline, finding that may suggest higher disease activity in IFNβ non-responders. It should be mentioned that the lack of a placebo group does not certainly allow discriminating between
lack of response to IFNβ and natural evolution of the disease. Nevertheless, the fact that clinical and radiological variables were comparable between responders and non-responders at baseline, particularly the number of relapses in the two previous years and the number of contrast-enhancing lesions in the MRI, and the absence of similar differences in serum CHI3L1 levels at baseline in the GA-treated cohort, both factors suggest that the significant responder effect observed for IFNβ in our study is due to a true lack of response to treatment rather than to differences in disease activity between IFNβ responders and non-responders. In summary, the aggregate results of the study further expand the prognostic involvement of CHI3L1 in the disease, particularly in patients with CIS, and may suggest a role for CHI3L1 as an IFNβ treatment response biomarker in RRMS patients. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jneuroim.2016.12.006.
Fig. 2. Responder effect of CHI3L1 in GA- and IFNβ-treated RRMS patients. Mean serum levels of CHI3L1 in patients classified into responders (R) and non-responders (NR) according to clinical and radiological criteria after one year of treatment, as described in the Methods section. T0: CHI3L1 levels at baseline. T12: CHI3L1 levels at the 12 month time point. Significant pvalues are shown in bold. Error bars represent standard error of the mean. CHI3L1: chitinase 3-like 1.
Please cite this article as: Matute-Blanch, C., et al., Chitinase 3-like 1 is associated with the response to interferon-beta treatment in multiple sclerosis, J. Neuroimmunol. (2016), http://dx.doi.org/10.1016/j.jneuroim.2016.12.006
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C. Matute-Blanch et al. / Journal of Neuroimmunology xxx (2016) xxx–xxx
Author contribution CM, MC designed the study. CM, SM performed the experiments. JR, LMV, LM, JCAV, AVJ, XM provided the samples and collected clinical data. CM, MC analyzed the data. CM, MC wrote the article. CM, SM, JR, LMV, LM, JCAV, AVJ, XM, MC revised the article and approved the final version of the manuscript. Competing interests The authors declare that they have no competing interests. Acknowledgements The authors thank the “Red Española de Esclerosis Múltiple (REEM)” sponsored by the “Fondo de Investigación Sanitaria” (FIS), Ministry of Science and Innovation, Spain, and the “Ajuts per donar Suport als Grups de Recerca de Catalunya”, sponsored by the “Agència de Gestió d'Ajuts Universitaris i de Recerca” (AGAUR), Generalitat de Catalunya, Spain. References Canto, E., Reverter, F., Morcillo-Suarez, C., Matesanz, F., Fernandez, O., Izquierdo, G., et al., 2012. Chitinase 3-like 1 plasma levels are increased in patients with progressive forms of multiple sclerosis. Mult. Scler. 18, 983–990.
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Please cite this article as: Matute-Blanch, C., et al., Chitinase 3-like 1 is associated with the response to interferon-beta treatment in multiple sclerosis, J. Neuroimmunol. (2016), http://dx.doi.org/10.1016/j.jneuroim.2016.12.006