The aim of the study was to evaluate CXCL4 levels in undifferentiated connective tissue disease at risk for SSc (UCTD-SSc-risk) and confirm its increase and investigate its prognostic value. Serum CXCL4 levels were measured in 45 patients and 24 controls. CXCL4 was significantly higher in UCTD-SSc-risk patients than in controls. It resulted higher in patients with a shorter disease duration and in those lacking capillaroscopic alterations. We confirm that CXCL4 levels are increased in UCTD-risk-SSc patients. Further studies are needed to investigate the role of CXCL4 assessment in UCTD-risk-SSc

Bocchino, Marialuisa

Cozzolino, Domenico

Docimo, Ludovico

Irace, Rosaria

Iudici, Michele

Riccardi, Antonella

Sanduzzi Zamparelli, Alessandro

Tolone, Salvatore

Valentini, Gabriele

Vettori, Serena

BOCCHINO, MARIALUISA

SANDUZZI ZAMPARELLI, ALESSANDRO

Archivio della ricerca - Università degli studi di Napoli Federico II

SHORT COMMUNICATIONCXCL4 in undifferentiated connective tissue disease at riskfor systemic sclerosis (SSc) (previously referred to as very earlySSc)Gabriele Valentini1,2,3 • Antonella Riccardi1,2,3 • Serena Vettori1,2,3 •Rosaria Irace1,2,3 • Michele Iudici1,2,3 • Salvatore Tolone1,2,3 • Ludovico Docimo1,2,3 •Marialuisa Bocchino1,2,3 • Alessandro Sanduzzi1,2,3 • Domenico Cozzolino1,2,3Received: 19 July 2016 / Accepted: 8 September 2016! Springer International Publishing Switzerland 2016Abstract The aim of the study was to evaluate CXCL4levels in undifferentiated connective tissue disease at riskfor SSc (UCTD-SSc-risk) and confirm its increase andinvestigate its prognostic value. Serum CXCL4 levelswere measured in 45 patients and 24 controls. CXCL4was significantly higher in UCTD-SSc-risk patients thanin controls. It resulted higher in patients with a shorterdisease duration and in those lacking capillaroscopicalterations. We confirm that CXCL4 levels are increasedin UCTD-risk-SSc patients. Further studies are needed toinvestigate the role of CXCL4 assessment in UCTD-risk-SSc.Keywords Systemic sclerosis ! Undifferentiatedconnective tissue diseaseIntroductionVery early (or early) SSc is a condition characterizedby Raynaud’s phenomenon (RP) plus SSc markerautoantibodies and/or distinct capillaroscopic alterations[1, 2]. It carries a significant, but not absolute risk ofevolution into definite SSc [3, 4]. Because of thisprognostic evidence as well as to avoid any nosographicconfusion with early definite SSc or early diffuse SSc[5–7], it should be properly referred to as undifferen-tiated connective tissue disease (UCTD) at risk for SSc(UCTD-SSc-risk) [8].Van Bon et al. [9] recently reported increased plateletfactor 4 (CXCL4) levels in both the plasma and skin ofpatients with systemic sclerosis (SSc) in whom theyresulted to be associated with distinct clinical features andto predict a poorer prognosis. They also pointed outincreased values in patients with very early SSc, but did notinvestigate any association between them.The aim of this study is to confirm increased CXCL4levels in patients with strictly defined UCTD-SSc-risk andto evaluate whether this finding is associated with anydisease feature and/or is predictive of evolution into defi-nite disease [3, 4].Materials and methodsForty-five patients with UCTD-SSc-risk consecutivelyadmitted from November 1st 2000 to December 31st 2013were enrolled in the study after giving a written informedconsent.ClassificationUCTD-SSc-risk was diagnosed in patients with RP andeither SSc marker autoantibodies or distinct capillaro-scopic alterations or both, who at admission neither sat-isfied the 2013 ACR/EULAR criteria for SSc [10] norpresented any manifestation indicative of SSc sine scle-roderma [11].& Gabriele Valentinigabriele.valentini@unina2.it1 Department of Clinical and Experimental Medicine,Rheumatology Section, Second University of Naples, IIPoliclinico, Building 3, Via Pansini 5, Naples, Italy2 Division of General and Bariatric Surgery, Second Universityof Naples, Naples, Italy3 Division of Respiratory Disease, Department of ClinicalMedicine and Surgery, University Federico II of Naples,Naples, Italy123Clin Exp MedDOI 10.1007/s10238-016-0437-yAssessment at admissionPatients underwent a detailed history, an accurate physicalexamination, routine laboratory investigations, nailfoldcapillaroscopy, autoantibodies detection and investigationof preclinical organ involvement as already described [4].SubsettingPatients were subdivided into 3 subsets [12]: subset I(patients with both SSc marker autoantibodies and capil-laroscopic abnormalities), subset II (patients with onlymarker autoantibodies, i.e., anti-centromere or anti-DNAtopoisomerase I or anti-RNA polymerase III or anti-Fib-rillarin or anti-Th/To or anti-Pm-Scl), and subset III (pa-tients with only capillaroscopic abnormalities, i.e.,megacapillaries and/or avascular areas) (Table 1).Serum marker quantificationTo avoid any influence of platelet-derived chemokines [13],we measured CXCL4 levels in the serum of 45 patients atadmission and in 24 osteoarthritis/fibromyalgia control sub-jects matched for sex and age. Peripheral blood was obtainedat baseline by venipuncture. Serum was separated by cen-trifugation at 1500g for 10 min, aliquoted and stored at-20 "C. CXCL4 levels were measured by a suspension flu-orescence-based immunoassay (Merk Millipore, Billerica,MA, USA) using a Luminex 200 instrument (Luminex Cor-poration, Austin, TX, USA). Serum samples were diluted1:40,000, and final concentrations of CXCL4 were expressedas ng/ml. No patient was under glucocorticoid and/orimmunosuppressive treatment at the time of blood drawing.Follow-upAfter enrollment, patients were re-evaluated at yearlyintervals, up to December 31, 2015, by history taking,clinical examination, EKG, B-mode-echocardiography,esophageal barium X-ray, lung function testing and lunghigh-resolution tomography to assess whether and wheneach of them satisfied the ACR/EULAR classification cri-teria disease score C9) [10] and/or criteria for SSc sinescleroderma [11].Statistical analysisWe used the SPSS for Windows software (version 16.0) forstatistical analysis. Categorical data were analyzed by Chi-square test. Continuous data were compared by the Mann–Whitney U test. A p value\0.05 was considered signifi-cant. Risk was assessed by assessing hazard ratios in uni-variate and step-wise regression analysis.Informed consentInformed consent was obtained from all individual partic-ipants included in the study.The study was approved by the Ethics Committee.ResultsSerum CXCL4 was significantly higher in the 45 UCTD-SSc-risk patients [median 4.84 ng/ml; interquartile range(1.83–10.82)] than in controls [median 1.43 ng/ml;interquartile range (0.21–2.70), p = 0.0001]. In detail,serum CXCL4 resulted to be significantly higher in 28subset I [median 4.74 ng/ml; interquartile range(1.67–10.18), p = 0.0012] and in 13 subset II patients[median 7.0 ng/ml; interquartile range (2.9–16.8),p = 0.0002] than in controls, while no difference wasdetected between 4 subset III patients [median 2.32 ng/ml;interquartile range (1.7–3.3)] and the 24 controls. More-over, serum CXCL4 levels were almost significantly higherin 13 subset II patients (i.e., those lacking microcirculatoryabnormalities as detected by nailfold videocapillaroscopy)than in 32 subset I ? III patients (p = 0.07).No other association was detected between CXCL4levels and either disease duration or autoantibody speci-ficity or presence of any (esophageal and/or cardiac and/orlung) preclinical internal organ involvement.At December 31, 2015, 4 patients had been lost to fol-low-up, 1 patient died (pulmonary embolism). Theremaining 40 had been monitored for 6–101.4 months(median 25.4 months; interquartile range 12.4–47.4; 19.04patient/years follow-up). At the study closure, 26/40(65 %) patients had evolved into definite SSc (at6–101.4 months from enrollment into the study; median24.35 months interquartile range 8–41.5). Of these, 9 onlyhad CXCL4 values at baseline [95th percentile of thevalues recorded in controls (8.30 ng/ml). Nevertheless, 2out of the 3 patients with the highest values (Fig. 1) hadevolved into definite SSc.Figure 2 shows the receiver operator curve devoted toidentify the performance of baseline serum CXCL4 inpredicting the evolution into definite SSc. A baselineCXCL4 value[8.3 ng/ml was found to identify patientsevolved into definite SSc during follow-up with a 30 %sensitivity and 86 % specificity (AUC 0.69).DiscussionCXCL4 is a 7.8-kDa protein that comprises from 2 to 3 %of the protein content of activated platelets and is consid-ered one of the most anti-angiogenic chemokines [13].Clin Exp Med123In their pivotal paper, van Bon et al. [9] reported acorrelation between CXCL4 levels and the extent of skinfibrosis in patients affected by SSc. Moreover, theyfound higher CXCL4 levels were associated with lungfibrosis and pulmonary arterial hypertension and resultedto be predictive of faster decline in DLCO, faster pro-gression of skin fibrosis and higher incidence of lungfibrosis in 79 SSc patients followed up for 18 months.These authors also mentioned increased CXCL4 valuesin ‘‘very early’’ SSc, but did not investigate either thepresence of associations or the prognostic value of thefinding.We undertook the present study in order to address theseaspects. Unlike van Bon et al. [1], who assessed CXCL4levels in plasma, we investigated the chemokine levels inthe serum, because increased levels of chemokine inplasma can depend on its release from platelets [14]. Sinceplatelet activation occurs in SSc, we opted to assessCXCL4 levels in the serum in order to better evaluate theTable 1 Demographic, serological, capillaroscopic features and functional abnormalities indicative of preclinical internal organ involvement in45 patients divided into 3 subsetsFeatures Whole cohort Subset I Subset II Subset IIISex (F/M) 43/2 27/1 12/1 4/0Age (years: median; range) 42 (37–48.5) 41 (34–45) 42 (39.2–50.2) 56 (47–62)Disease duration from RP (years: median; range) 2 (1–5.25) 3 (2–6) 2 (1–4.2) 2.5 (1.5–17)Marker autoantibody positivity 41 28 13 0Anti-centromere 30 (66.6 %) 21/28 (75 %) 9/13 (69.23 %)Anti-DNA topoisomerase I 10/45 (22.2 %) 6/28 (21.4 %) 4/13 (30.7 %)Anti-PM-Scl 1/45 (2.2 %) 1/28 (3.5 %)Capillaroscopic alterations 32 28 0 4Megacapillaries 28/32 (87.5 %) 24/28 (85.7 %) 4/4 (100 %)Avascular areas 4/32 (12.5 %) 4/28 (14.2 %) 0DLCO\80 % of the predicted value 17/45 (37.8 %) 8/28 (28.5 %) 6/13 (46.1 %) 3/4 (75 %)E/A\ 1a 0 0 0 0Low esophageal sphincter pressure\15 mm Hg 8/29 (27.6 %) 5/18 (27.7 %) 2/9 (22.2 %) 1/2 (50 %)RP Raynaud’s phenomenon, DLCO diffusing lung capacity for carbon monoxide, E/A early/atrial ratioa In patients aged\50 years, with an arterial blood pressure\130/85 mmHgFig. 1 CXCL4 levels in healthy controls (HC), in the whole cohort(WC) of UCTD-SSc-risk patientsFig. 2 Receiver operator curve showing the relationship betweenbaseline serum CXCL4 and evolution into definite SScClin Exp Med123amount of the circulating chemokine probably secreted byplasmocytoid dendritic cells, which constitute its mainsource in SSc [9].Despite the different origin of the samples used, weconfirm the original report by van Bon et al. [9] thatCXCL4 levels are higher in UCTD-risk-SSc patients, whohad been referred by them as ‘‘very early SSc.’’We investigated the presence of associations betweenCXCL4 levels and disease duration from the onset of RP,disease subsetting, autoantibody specificity, preclinicalinternal organ involvement as well as the value as aprognostic marker of evolution into definite SSc.We found that CXCL4 levels are almost significantlyhigher in patients lacking microcirculatory abnormalities asdetected by nailfold videocapillaroscopy. These associa-tions should be evaluated further in a larger series ofpatients also assessed for other factors influencing angio-genesis in SSc, for example vascular endothelial growthfactor (VEGF). Notably, megacapillaries, which were theonly microvascular abnormality in most our patients (28/45), may represent a compensatory mechanism, promotedby VEGF that might be inhibited by CXCL4 [15].As far as CXCL4 prognostic value is concerned, ROCanalysis pointed out a significant specificity, suggesting apotential role of its evaluation in the assessment of theUCTD-risk-SSc patient at baseline. Such a role is, how-ever, challenged by its low sensitivity.The prognostic value of CXCL4 in definite SSc pointedout by van Bon et al. [9] has been recently challenged byVolkmann et al. [16] who investigated patients enrolledinto Scleroderma Lung Study 2. This aspect, therefore, alsoawaits to be clarified in definite SSc.In conclusion, we confirmed that increased CXCL4levels are detected in UCTD-SSc-risk patients. Furtherstudies on a larger, possibly multicentric series, are neededto assess the value of CXCL4 and other biomarkers inpredicting the evolution in UCTD-SSc-risk. At present,predicting prognosis in SSc relies on the definition of thesubset [3, 4] and the presence of preclinical organinvolvement at admission [4].Compliance with ethical standardsConflict of interest None.References1. Avouac J, Fransen J, Walker UA, et al. EUSTAR Group. Pre-liminary criteria for the very early diagnosis of systemicsclerosis: results of a Delphi Consensus Study from EULARScleroderma Trials and Research Group. Ann Rheum Dis.2011;70:476–81.2. Matucci-Cerinic M, Bellando-Randone S, Lepri G, Bruni C,Guiducci S. Very early versus early disease: the evolving defi-nition of the ‘many faces’ of systemic sclerosis. Ann Rheum Dis.2013;72:319–21.3. Koenig M, Joyal F, Fritzler MJ, et al. Autoantibodies andmicrovascular damage are independent predictive factors for theprogression of Raynaud’s phenomenon to systemic sclerosis: atwenty-year prospective study of 586 patients with validation ofproposed criteria for early systemic sclerosis. Arthritis Rheum.2008;58:3902–12.4. Valentini G, Marcoccia A, Cuomo G, et al. Early systemicsclerosis: analysis of the disease course in patients with markerautoantibodies or capillaroscopic positivity or both. Arthritis CareRes. 2014;66:1520–7.5. Wuttge DM, Lood C, Tufvesson E, et al. Increased serum type Iinterferon activity in early systemic sclerosis is associated withantibodies against Sjo¨gren’s syndrome antigens and nuclearribonucleoprotein antigens. Scand J Rheumatol. 2013;42:235–40.6. Camargo CZ, Sekiyama JY, Arismendi MI, Kayser C.Microvascular abnormalities in patients with early systemicsclerosis: less severe morphological changes than in patients withdefinite disease. Scand J Rheumatol. 2015;44:48–55.7. Frech TM, Murtaugh M, Gordon JK et al. Longitudinal assess-ment of gastrointestinal symptoms in the prospective registry ofearly systemic sclerosis cohort [abstract]. Arthritis Rheumatol.2015; 67 suppl 10.8. Valentini G. Undifferentiated connective tissue disease at risk forsystemic sclerosis (SSc) (so far referred to as very early/early SScor pre-SSc). Autoimmun Rev. 2015;14:210–3.9. van Bon L, Affandi AJ, Broen J, et al. Proteome-wide analysisand CXCL4 as a biomarker in systemic sclerosis. N Engl J Med.2014;370:433–43.10. Van den Hoogen F, Khanna D, Fransen J, et al. Classificationcriteria for systemic sclerosis: an ACR-EULAR collaborativeinitiative. Arthritis Rheum. 2013;65:2737–47.11. Poormoghin H, Lucas M, Fertig N, Medsger TA Jr. Systemicsclerosis sine scleroderma: demographic, clinical, and serologicfeatures and survival in forty-eight patients. Arthritis Rheum.2000;43:444–51.12. Valentini G, Marcoccia A, Cuomo G, et al. Early systemicsclerosis: marker autoantibodies and videocapillaroscopy patternsare each associated with distinct clinical, functional and cellularactivation markers. Arthritis Res Ther. 2013;15:R63.13. Aidoudi S, Bujakowska K, Kieffer N. Bikfalvi A The CXC-chemokine CXCL4 interacts with integrins implicated in angio-genesis. PLoS ONE. 2008;3:e2657.14. Wiesner T, Bugl S, Mayer F, Hartmann JT, Kopp H-G. Differ-ential changes in platelet VEGF, Tsp, CXCL12, and CXCL4 inpatients with metastatic cancer. Clin Exp Metastasis.2010;27:141–9.15. Distler JHW, Gay S, Distler O. Angiogenesis and vasculogenesisin systemic sclerosis. Rheumatology. 2006;45:iii26–7.16. Volkmann ER, Tashkin DP, Roth M and Scleroderma LungStudy II Group. CXCL4 does not predict extent or progression ofinterstitial lung disease in systemic sclerosis [abstract]. ArthritisRheumatol. 2015; 67 suppl 10.Clin Exp Med123

CXCL4 in undifferentiated connective tissue disease at risk for systemic sclerosis (SSc) (previously referred to as very early SSc)

Gabriele Valentini

Antonella Riccardi

Serena Vettori

Rosaria Irace

Michele Iudici

Salvatore Tolone

Ludovico Docimo

Marialuisa Bocchino

Alessandro Sanduzzi

Domenico Cozzolino

Crossref

Clinical and Experimental Medicine

VALENTINI, Gabriele

VETTORI, Serena

TOLONE, SALVATORE

DOCIMO, Ludovico

Sanduzzi, Alessandro

COZZOLINO, Domenico

Archivio Istituzionale della Ricerca - Università degli Studi della Campania "Luigi Vanvitelli"

CXCL4 in undifferentiated connective tissue disease at risk for systemic sclerosis (SSc) (previously referred to as very early SSc)

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Archivio della ricerca - Università degli studi di Napoli Federico II

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Archivio Istituzionale della Ricerca - Università degli Studi della Campania "Luigi Vanvitelli"