Polmonare terapia dell'ipertensione arteriosa può essere sicuro ed efficace nei pazienti con sclerosi sistemica e borderline pugnale pressione arteriosa polmonare borderline ipertensione arteriosa polmonare (PAH), caratterizzata da un aumento indotta da esercizio marcato della pressione arteriosa polmonare (PAP), con valori a riposo normali, possono precedere PAH palese nella sclerosi sistemica (sclerodermia). Abbiamo intrapreso questo studio per indagare se il trattamento PAH è sicuro in questi pazienti e potrebbe attenuare la progressione emodinamica. pazienti affetti da SSc con borderline PAH sono stati sottoposti a destra cateterizzazione cardiaca al basale, dopo un periodo di osservazione di 12 mesi, e, successivamente, dopo 6 mesi di terapia bosentan. Le variazioni di PAP media a 50W durante il periodo di osservazione contro durante la terapia sono stati confrontati. Dieci pazienti hanno completato lo studio. Media PAP a riposo, a 50W, e durante l'esercizio massimale aumentato in modo significativo durante il periodo di osservazione (media plusmn SD aumenta di 2,5 plusmn 3,0 millimetri Hg P 0,03, 4,0 plusmn 2,9 millimetri Hg P 0,002 e 6,8 plusmn 4,1 millimetri Hg P 0,0005, rispettivamente, ) e tendeva a diminuire durante il periodo di trattamento (diminuzioni di 2,5 plusmn 3,9 millimetri Hg P 0.07, 1.5 plusmn 4,5 millimetri Hg P 0,32 e 1,8 plusmn 7,0 millimetri Hg P 0.43, rispettivamente). Le variazioni intervenute nel periodo di osservazione rispetto al periodo di terapia erano significativamente differenti (P 0,03 a riposo, P 0.01 a 50W end point primario, e P 0,02 durante l'esercizio massimale). Le variazioni di riposo resistenza vascolare polmonare sono stati anche significativamente diversi durante il periodo di osservazione (incremento di 8 plusmn 25 dine secondi middot middot cm minus5) rispetto durante il periodo della terapia (diminuzione di 45 plusmn 22 dine secondi middot middot cm minus5) (P lt 0,0005 ). Le variazioni di pressione arteriosa a riposo di cuneo polmonare non erano significativamente differenti tra il periodo di osservazione e il periodo di trattamento, nonostante l'aumento significativo durante il periodo di osservazione (2,6 plusmn 2,5 millimetri Hg P 0,01). Non sono stati segnalati effetti avversi rilevanti. Conclusione In pazienti affetti da SSc con emodinamica borderline anomalo polmonare, di riposo e l'esercizio fisico PAP può aumentare in modo significativo entro 1 anno di osservazione. Bosentan potrebbe essere sicuro ed efficace per attenuare questi cambiamenti. studi randomizzati controllati sono garantiti per confermare i risultati esplorativi di questo studio pilota ipotesi che generano. Arteriosa polmonare (PAH) è una malattia progressiva delle piccole arterie polmonari che porta ad una diminuzione della capacità di esercizio, insufficienza ventricolare destra, e alla fine, la morte. Riposo pressione media dell'arteria polmonare (PAP) di 8ndash20 mm Hg è considerata normale (1), e PAH viene diagnosticata quando significa PAP è GE25 mm Hg e polmonare pressione arteriosa cuneo (PAWP) è LE15 mm Hg (2). Anche se i dati di esercizio non sono ancora standardizzati, i risultati di recenti studi hanno suggerito che un marcato aumento indotta da esercizio fisico in PAP può indicare vasculopatia polmonare precoce e rappresentano una fase intermedia tra il PAP normale e IPA (3. 4). In uno studio britannico, 19 dei pazienti con sclerosi sistemica (sclerodermia SSc) con un incremento PAP media indotta da esercizio di GT30 mm Hg progredito a manifestarsi PAH entro 3 anni (5). In accordo con questo, il nostro gruppo ha dimostrato che borderline elevazione del PAP e un aumento indotta da esercizio marcato della PAP nei pazienti SSc è stato associato a riposo ridotta capacità di esercizio (6). A parte alcuni casi clinici promettenti (7), non è chiaro se i pazienti con sclerodermia senza PAH palese ma con emodinamica polmonare anormale borderline, caratterizzata da un aumento indotto da esercizio fisico marcata nel PAP, potrebbero beneficiare di una terapia mirata PAH. Gli studi che coinvolgono follow-invasiva di PAH borderline nei pazienti con sclerodermia non sono stati segnalati fino ad oggi. L'obiettivo di questo studio pilota esplorativo è stato quindi di monitorare invasivo il corso naturale di questa entità malattia e per studiare gli effetti del bosentan sull'emodinamica e la capacità di esercizio in questi pazienti. Abbiamo ipotizzato che l'emodinamica polmonare sarebbero deteriorarsi nel tempo e che questo potrebbe essere attenuata dalla somministrazione di una terapia mirata PAH come bosentan. PAZIENTI E METODI Pazienti in cui SSc era l'unico fattore di rischio per la PAH sono stati arruolati in un programma di screening per la PAH come descritto in precedenza (6. 8). Tutti i pazienti soddisfatti l'American College of Rheumatology criteri per SSC (9). SSc è stato classificato in base ai criteri proposti da LeRoy et al (10). I pazienti sono stati esclusi se avessero saputo PAH manifesto, ostruttiva sintomatica o malattia polmonare restrittiva (pre-broncodilatatore capacità vitale forzata in 1 secondo lt65 del predetto), sistolica o insufficienza ventricolare sinistra diastolica (frazione di eiezione LT50, insufficienza diastolica di più di un grado lieve) ( 11), emodinamicamente significativa malattia valvolare, ipertensione arteriosa sistemica, o clinicamente rilevante artrite o miosite che potrebbe avere sostanzialmente influenzato i test da sforzo. Il disegno dello studio e il protocollo sono stati in conformità con la Dichiarazione di Helsinki e sono stati approvati dal comitato etico locale. Tutti i pazienti hanno fornito il consenso informato. I pazienti con una media di PAP a riposo e GT30 mm Hg LT25 mm Hg (e PAWP di LE15 mm Hg) durante l'esercizio fisico (concomitante test da sforzo cardiopolmonare) a cateterizzazione cardiaca destra (RHC) erano eleggibili per questo studio pilota. Test di sei minuti a piedi, test di funzionalità polmonare, e test di laboratorio compresi i test per N-terminale prondashbrain peptide natriuretico (NT-proBNP) sono state eseguite entro 24 ore dalla RHC. Dopo questa linea di base RHC ci fu un periodo di osservazione di 12 mesi, durante il quale sono stati ammessi cambiamenti nella farmaco emodinamico. Dopo questo periodo, un secondo RHC è stata eseguita nelle stesse condizioni della valutazione di base, e le prove descritte sono stati ripetuti. Questa è stata seguita da un periodo di trattamento di 6 mesi, in cui i pazienti hanno ricevuto bosentan (62,5 mg due volte al giorno per 1 mese, poi 125 mg due volte al giorno per 5 mesi). Dopo questo, RHC e tutti gli altri esami descritte sono state ripetute. Gli end point dello studio e dei test statistici sono stati definiti a priori e approvati dal comitato etico locale. I punti finali sono basate sui risultati di un precedente studio esplorativo che suggerisce che significa che i valori PAP a 50W ed a riposo, così come i valori resistenza vascolare polmonare (PVR) a riposo, potrebbe essere di rilevanza clinica nei pazienti con sclerodermia con PAP borderline (6 ). L'endpoint primario in questo studio era la variazione PAP media al 50W nel corso del periodo di trattamento rispetto al periodo di osservazione. endpoint secondari includevano il cambiamento di riposo ed esercizio PVR e la variazione di picco V O 2 durante il periodo di trattamento rispetto al periodo di osservazione. Le variazioni di questi parametri sono stati confrontati con 2 lati t-test accoppiato. Per gli endpoint secondari, così come per il riposo PAP media, PAWP, e la distanza percorsa in 6 minuti, la stessa analisi è stato utilizzato in maniera esplorativa. I risultati sono stati espressi come media plusmn SD. Dieci pazienti con sclerosi sistemica (6 con limitata SSc cutanea, 1 con diffusa cutanea SSc, 3 con sovrapposizione media plusmn SD età 55.4 plusmn 8,9 anni, la durata media plusmn SD malattia 8.3 plusmn 9,8 anni) sono stati arruolati. Durante i periodi di osservazione e di trattamento, non sono stati osservati eventi avversi o effetti collaterali rilevanti di bosentan. Nessun paziente ha interrotto la partecipazione allo studio prematuramente. Non ci sono state violazioni del protocollo. Le variazioni di emodinamica e capacità di esercizio durante i periodi di osservazione e trattamento sono riportati nella Tabella 1. Media PAP a 50W è aumentato in modo significativo durante il periodo di osservazione (media plusmn SD 4.0 plusmn 2,9 millimetri Hg P 0,002) ed era immutata durante il periodo di trattamento (minus1. 5 plusmn 4,5 millimetri Hg P 0,32). Le variazioni intervenute nel periodo di trattamento rispetto al periodo di osservazione (end point primario) erano statisticamente differenti (P 0,01) (Tabella 1 e Figura 1 a). Tabella 1. Confronto di parametri emodinamici e di funzionalità respiratoria e la capacità di esercizio nei 10 pazienti con sclerosi sistemica al basale, dopo il periodo di osservazione, e dopo il periodo di trattamento variazioni individuali della pressione arteriosa polmonare media (PAP) a 50W (a), polmonare a riposo resistenza vascolare (PVR) (b), e durante il periodo di osservazione e il periodo di trattamento medio PAP (c) di riposo. RHC-0, -1, -2 e cateterizzazione lato destro del cuore al basale, alla fine del periodo di osservazione di 12 mesi, e alla fine del periodo di trattamento bosentan 6 mesi, rispettivamente. lowast P 0.03 lowastlowast P le 0.002. Tra gli endpoint secondari, riposo PVR tendeva ad aumentare durante il periodo di osservazione (media plusmn SD 8 plusmn 25 dyne secondi middot middot cm minus5 P 0,32) ed è diminuito in modo significativo durante il periodo di trattamento (minus45 plusmn 22 dyne secondi middot middot cm minus5 P lt 0,0005) (Tabella 1 e Figura 1 b). Le variazioni tra i 2 periodi erano altamente significativamente differenti (P lt 0,0005), e PVR alla fine del periodo di trattamento era ancora significativamente inferiore rispetto al basale (P 0,01). Le variazioni di PVR durante l'esercizio massimale hanno seguito lo stesso schema (10 plusmn 35 dine secondi middot middot cm minus5 contro minus35 plusmn 26 dyne secondi middot middot cm minus5 p = 0,02). PVR a 50W è aumentata anche durante il periodo di osservazione e diminuito durante il periodo di trattamento (4 plusmn 24 dine secondi middot middot cm minus5 contro minus25 plusmn 38 dyne secondi middot middot cm minus5), tuttavia, la differenza nei cambiamenti tra i 2 periodi non ha raggiunto significatività statistica (P 0,13). C'è stata una tendenza verso un miglioramento picco di VO 2 durante il periodo di trattamento (0,6 plusmn 1.2 mlminutekg) rispetto al periodo di osservazione (minus1.1 plusmn 2,0 mlminutekg), ma questi cambiamenti non erano significativamente differenti (P 0,08) (Tabella 1 ). Tra altri parametri di interesse, che riposano media PAP è aumentata durante il periodo di osservazione (media plusmn SD 2,5 plusmn 3,0 millimetri Hg P 0,03), e questo è stato invertito durante il periodo di trattamento (minus2.5 plusmn 3,9 millimetri Hg P 0,07). I cambiamenti erano significativamente differenti tra i 2 periodi (P 0,03) (Tabella 1 e Figura 1 c). Allo stesso modo, significare PAP a livello sforzo massimale aumentato durante il periodo di osservazione (6,8 plusmn 4,1 millimetri Hg P 0,0005) ed era invariata durante il periodo di trattamento (minus1.8 plusmn 7,0 millimetri Hg P 0,43), e le variazioni durante i periodi di 2 erano significativamente diverso (p = 0,02). Tutti i pazienti avevano normale PAWP a riposo (definito come LE15 mm Hg) a tutti e 3 gli esami RHC. Durante il periodo di osservazione, PAWP aumentato in modo significativo, sia a riposo che durante l'esercizio fisico, e di riposo PAWP leggermente diminuita durante il periodo di trattamento (Tabella 1). Sei minuti di distanza a piedi ha mostrato un lieve peggioramento nel corso del periodo di osservazione (media plusmn SD minus12 plusmn 33m) e un leggero aumento durante il periodo di trattamento (7 plusmn 31m), tuttavia, la differenza di questi cambiamenti non è stato significativo (P 0,31). I cambiamenti nei risultati dei test di funzionalità polmonare, capacità di monossido di carbonio, o livelli di NT-proBNP diffondenti non erano significativamente differenti tra i periodi di trattamento e di osservazione. Il Borg Dispnea Index al termine del test del cammino di 6 minuti è rimasta stabile nel corso dello studio. Durante il periodo di osservazione di 12 mesi, come media PAP aumentata, gittata cardiaca ridotta (da sim0.5 litersminute). Dopo ldquocorrectionrdquo per la diminuzione della gittata cardiaca, valori medi PAP sarebbe aumentato del mm Hg 3ndash5 invece di 2,5 mm Hg. Dopo il periodo di trattamento di 6 mesi, la pendenza di uscita media PAPcardiac quasi completamente restituito al basale (Figura 2). Diagramma Pressureflow di pressione media arteriosa polmonare (PAP) rispetto gittata cardiaca. I valori sono la media al basale (blu), al termine del periodo di osservazione (rosso), e alla fine del periodo di trattamento (verde). DISCUSSIONE In questo studio pilota esplorativo, abbiamo ipotizzato che il trattamento PAH mirato potrebbe attenuare la progressione emodinamica nei pazienti SSc senza PAH manifesto ma con emodinamica polmonare anormale borderline. Come un marcatore abbiamo utilizzato la definizione precedentemente accettato di PAH indotta dall'esercizio, indicato da un aumento della PAP GT30 mm Hg. Sulla base di rapporti pubblicati, questi cambiamenti emodinamici possono essere relativamente comune tra i pazienti SSc (4). A nostra conoscenza, questo è il primo studio per valutare i cambiamenti emodinamici e per studiare la sicurezza e gli effetti potenziali della terapia mirata PAH in questo gruppo di pazienti, utilizzando il monitoraggio emodinamico invasivo. I risultati suggeriscono che in questa prima fase, la progressione di riposo e di esercizio emodinamica verso PAH è stato attenuato e parzialmente invertito dal trattamento bosentan. Il significato clinico delle piccole variazioni assolute osservati nei parametri emodinamici deve essere convalidato da followup a lungo termine e con un approccio prospettico randomizzato in uno studio sufficientemente alimentato. I risultati del periodo di osservazione nel nostro studio ha dimostrato che un aumento significativo di PAP media a riposo e durante l'esercizio sono stati osservati nel giro di 1 anno. Quattro dei 10 pazienti avevano valori medi di PAP 20ndash25 mm Hg alla fine del periodo di osservazione, mentre il valore era in questo intervallo in solo 1 dei 10 al basale. I nostri dati suggeriscono che, ipotizzando una variazione lineare nel tempo, alcuni pazienti possono sperimentare progressione relativamente rapida da normali valori medio di PAP e sviluppare PAH entro un breve periodo di tempo. Questi risultati sono supportati da dati del Registro Sclerodermia francese, dimostrando che sim55 di pazienti con PAH sclerodermia associata avevano sviluppato IPA entro 5 anni dopo la diagnosi di SSc (dopo il primo nonndashRaynauds fenomeno sintomo) (12). I dati di un registro sclerodermia britannico suggeriscono che i pazienti con un PAP di 20ndash25 mm Hg e quelli con un marcato aumento della PAP indotta da esercizio fisico possono essere particolarmente inclini a rapido sviluppo di palese, e in ultima analisi fatale, PAH (5). I pazienti con significativa disfunzione diastolica sono stati esclusi dalla iscrizione nel nostro studio, e di conseguenza tutti i pazienti avevano valori normali di riposo PAWP (LE15 mm Hg) durante tutte le indagini RHC. Durante il periodo di osservazione, PAWP leggermente, ma significativamente sia aumentata a riposo e durante l'esercizio fisico, che può aver contribuito all'aumento osservata in PAP media. Questo aumento di PAWP spiega perché l'aumento PVR calcolato era minore e non ha raggiunto la significatività statistica, anche se la gittata cardiaca è diminuita in modo significativo. Si suggerisce inoltre che sia lasciato funzione diastolica ventricolare e vasculopatia polmonare possono aver contribuito all'aumento della PAP. Questo concetto è supportato da studi precedenti che dimostrano che entrambi i meccanismi sono comuni in SSc (13). Tuttavia, le limitazioni tecniche di misura PAWP durante l'esercizio e l'assenza di un consenso internazionale su normali intervalli ben definiti di PAWP a determinati livelli di esercizio rendono l'analisi e l'interpretazione di questi dati difficile. Pertanto, data la piccola coorte di pazienti nel nostro studio, interpretazione del ruolo di PAWP rimane speculativo. indagini futuro della potenziale rilevanza dei cambiamenti longitudinali PAWP durante il riposo e l'esercizio fisico nei pazienti con sclerodermia potrebbe fornire indicazioni importanti. Durante il periodo di trattamento, l'esercizio fisico e riposo valori medi PAP sono stati stabilizzati e PVR è diminuito. Il numero di pazienti con riposo significa PAP tra 20 e 25 mm Hg diminuita: dopo 6 mesi di terapia, significa PAP rimasto in questo intervallo in solo 1 dei 10 pazienti. Il miglioramento emodinamico può rappresentare un'inversione di parete dei vasi polmonari proliferazione Andor costrizione. Entrambi i fattori (proliferazione e costrizione) contribuiscono a vasculopatia polmonare. l'osservazione a lungo termine è garantito per determinare se bosentan inverte o semplicemente attenua e vasculopatia polmonare ritardi all'inizio del scleroderma. The regime di trattamento con bosentan nei pazienti dello studio sembrava essere al sicuro, senza effetti negativi significativi. Durante tutto il periodo di trattamento, i livelli di transaminasi non erano significativamente elevati (volte GT3 il limite superiore della norma) in nessuno dei pazienti. Otto dei 10 pazienti hanno continuato il trattamento dopo la fine dello studio (per un SD plusmn medio di 22 plusmn 5 mesi), senza effetti collaterali rilevanti. Tre pazienti avevano edema periferico di transizione durante le prime settimane di terapia. Un paziente ha sviluppato la conta dei leucociti bassi durante il periodo di trattamento dello studio, ma questo era probabilmente dovuto alla terapia azatioprina in corso, ed è migliorato dopo la riduzione del dosaggio azatioprina. Non c'erano segni di eventuali interazioni farmacologiche o cambiamenti significativi nel livello di emoglobina nel corso dello studio. La capacità di esercizio, come valutato dal picco V O 2 e la distanza percorsa in 6 minuti, non è cambiata significativamente durante il periodo di osservazione o il periodo di trattamento. La ragione potrebbe essere che lo studio non era sufficientemente alimentato per questo scopo. Inoltre, l'assenza di cambiamento di capacità di esercizio può riflettere il deterioramento basale minima dei pazienti. Alcuni potenziali limitazioni di questo studio devono essere considerati. E 'importante sottolineare che lo studio è stato condotto in pazienti con sclerosi sistemica, e il risultato potrebbe non essere applicabile ad altri soggetti con PAH indotta da esercizio fisico. Il piccolo numero di pazienti dello studio impedisce anche conclusioni generali. Abbiamo eseguito un confronto sequenziale piuttosto che un confronto a gruppi paralleli e utilizzato diverse lunghezze di tempo per l'osservazione ed i periodi di trattamento, che può aver introdotto bias. Questo disegno è stato impiegato a causa del numero limitato di pazienti e le restrizioni di tempo in questo studio pilota. Il vantaggio era i dati completi impostati senza necessità di imputazione dati. Farmaco è stato dato su una base in aperto, che potrebbe anche essere prevenuto alcuni risultati. Tuttavia, mentre questo può essersi verificato per le misure di capacità di esercizio, è improbabile che possa aver influenzato l'emodinamica invasiva misurati. E 'possibile che le proprietà vasodilatatrici del bosentan, piuttosto che le proprietà antiproliferativa, hanno contribuito agli effetti emodinamici. Tuttavia, questo è un problema generale con farmaci che hanno proprietà vasodilatatrici. misure PAWP durante l'esercizio fisico possono essere imprecisi per motivi tecnici, che è una limitazione generale di studi di valutazione emodinamica esercizio. In conclusione, l'esercizio fisico PAP a riposo ed ha aumentato in modo significativo entro 1 anno di follow-up nei pazienti con sclerodermia senza PAH palese ma con emodinamica polmonare anormale borderline. Tale incremento è stato significativamente attenuata dalla terapia bosentan. studi randomizzati e controllati, compresa la valutazione di eventuali cambiamenti nella funzione ventricolare sinistra, sono garantiti per confermare i risultati di questo studio ipotesi generatrice. Autore contributi Tutti gli autori sono stati coinvolti nella stesura l'articolo o sua revisione critica per importante contenuto intellettuale, e tutti gli autori hanno approvato la versione finale da pubblicare. Dr. Kovacs aveva pieno accesso a tutti i dati nello studio ed è responsabile per l'integrità dei dati e l'accuratezza dell'analisi dei dati. Studio ideazione e progettazione. Kovacs, Aberer, Brodmann, Graninger, Hesse, Rubin, Olschewski. Acquisizione dei dati. Kovacs, Maier, Aberer, Brodmann, Graninger, Kqiku, Scheidl, Troumlster, Assia, Olschewski. Analisi e interpretazione dei dati. Kovacs, Maier, Kqiku, Rubin, Olschewski. Ruolo dello sponsor STUDIO Actelion Austria ha avuto alcun coinvolgimento nella progettazione e realizzazione di questo studio e l'interpretazione dei risultati. La pubblicazione di questo articolo non è stato contigent previa approvazione Actelion Austria. Ringraziamenti Vorremmo ringraziare il Prof. Andrea Berghold (Direttore dell'Istituto di Informatica, Statistica e Documentazione, Università Medica di Graz) per la consultazione statistiche e la signora Eugenia Lamont (Divisione di Chirurgia Sperimentale e Divisione di Gastroenterologia ed Epatologia, Università Medica di Graz ) per un'attenta revisione linguistica. Articolo Informazioni Storia di pubblicazione Edizione on-line: 2 Mar 2012 Versione di registrazione on-line: 27 marzo 2012 accettate manoscritto on-line: 29 novembre 2011 Manoscritto accettate: 1 Novembre 2011 Manoscritto ricevute: 19 aprile 2011 RIFERIMENTI 1 Kovacs G. Berghold A. Scheidl S. Olschewski H . la pressione arteriosa polmonare durante il riposo e l'esercizio fisico in soggetti sani: una revisione sistematica. Eur Respir J 2009 34. 888 ndash 94. CrossRef PubMed CAS Web di Sciencereg tempi citati: 91 2 Galie N. Hoeper MM. Humbert M. Torbicki A. Vachiery JL. Barbera JA. et al, la Task Force per la diagnosi e il trattamento di Ipertensione polmonare della Società Europea di Cardiologia (ESC) e della European Respiratory Society (ERS). approvato dalla International Society of Heart Lung Transplantation (ISHLT). Linee guida per la diagnosi e il trattamento di ipertensione polmonare pubblicato erratum appare in Eur Heart Journal 201132: 926. 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Borderline pressione arteriosa polmonare è associata ad una diminuzione della capacità di esercizio nella sclerodermia. Am J Respir Crit Care Med 2009 180. 881 ndash 6. CrossRef PubMed Web di Sciencereg tempi citati: 31 7 Yagi S. Akaike M. Iwase T. Kusunose K. Niki T. Yamaguchi K. et al. Bosentan migliorato indotta da esercizio fisico ipertensione arteriosa polmonare complicata con sclerosi sistemica. Intern Med 2010 49. 2309 ndash 12. CrossRef PubMed Web di Sciencereg tempi citati: 1 8 Kovacs G. Maier R. Aberer E. Brodmann M. Scheidl S. Hesse C. et al. La valutazione della pressione arteriosa polmonare durante l'esercizio fisico in malattie vascolari del collageno: ecocardiografia contro il cateterismo cardiaco destro. Petto 2010 138. 270 ndash 8. CrossRef PubMed Web di Sciencereg tempi citati: 10 9 Sottocommissione per la sclerodermia criteri dell'American Rheumatism Association Diagnostico e Comitato criteri terapeutici. criteri preliminari per la classificazione della sclerosi sistemica (sclerodermia). Arthritis Rheum 1980 23. 581 ndash 90. Wiley online Biblioteca PubMed 10 LeRoy CE. Nero C. Fleischmajer R. Jablonska S. Krieg T. Medsger TA Jr. et al. Sclerodermia (sclerosi sistemica): classificazione, sottoinsiemi e patogenesi. J Rheumatol 1988 15. 202 ndash 5. PubMed CAS Web di Sciencereg tempi citati: 1852 11 Redfield MM. Jacobsen SJ. Burnett JC Jr. Mahoney DW. Bailey KR. Rodeheffer RJ. Onere della disfunzione ventricolare sistolica e diastolica nella comunità: apprezzare la portata dell'epidemia insufficienza cardiaca. JAMA 2003 289. 194 ndash 202. CrossRef PubMed Web di Sciencereg tempi citati: 904 12 Hachulla E. Launay D. Mouthon L. Sitbon O. Berezne A. Guillevin L. et al, per i francesi PAH-SSC rete. È l'ipertensione arteriosa polmonare in realtà una complicanza tardiva della sclerosi sistemica petto 2009 136. 1211 ndash 9. CrossRef PubMed Web di Sciencereg tempi citati: 27 13 Tyndall AJ. Bannert B. Vonk M. Airo P. Cozzi F. Carreira PE. et al. Cause e fattori di rischio per la morte nella sclerosi sistemica: uno studio dalle prove EULAR la sclerodermia e la banca dati di ricerca (Eustar). Ann Rheum Dis 2010 69. 1809 ndash 15. CrossRef PubMed Web di Sciencereg tempi citati: 41 contenuti correlati Articoli correlati a quello che si sta visualizzando Abilitare Javascript per visualizzare il relativo contenuto di questo articolo. Citando Literature Numero di volte citato. 23 1 Deepika Misra. Ante Kendes. Roxana Sulica. Carabello Blase. Indotta da esercizio fisico ipertensione polmonare da stress ecocardiografia: prevalenza e correlazione con emodinamica del cuore destro, International Journal of Cardiology. 2017. 228. 518 CrossRef 2 Edmund M. Lau. Denis Chemla. Kenneth Whyte. Gabor Kovacs. Horst Olschewski. Philippe Herve. Si esercita ipertensione polmonare esistere. Current Opinion in Pulmonary Medicine. 2016. 22. 5, 400 CrossRef 3 Mike O. Becker. Gabriela Riemekasten. Fattori di rischio per la gravità e manifestazioni nella sclerosi sistemica e la previsione di decorso della malattia, Expert Review di Immunologia Clinica. 2016. 12. 2, 115 CrossRef 4 J. Sawalla Guseh. Il paesaggio in evoluzione dei indotta da esercizio fisico Ipertensione polmonare, Current opzioni di trattamento in medicina cardiovascolare. 2016. 18. 6 CrossRef 5 Gabor Kovacs. Horst Olschewski. pressione polmonare borderline in sclerodermia - una condizione arteriosa hypertensionrsquo lsquopre-polmonare. Artrite Terapia amp Research. 2015. 17. 1 CrossRef 6 C. Gabriels. P. Lancellotti. A. Van De Bruaene. D. Voilliot. P. De Meester. R. Buys. M. Delcroix. W. Budts. significato clinico di dinamica resistenza vascolare polmonare in due popolazioni a rischio di ipertensione arteriosa polmonare, European Heart Journal - imaging cardiovascolare. 2015. 16. 5, 564 CrossRef 7 A. V. Volkov. I. A. Kurmukov. N. N. Yudkina. S. I. Glukhova. E. V. Nikolaeva. Impatto della terapia bosentan sulla ipertensione polmonare indotta da stress nei pazienti con sclerodermia Systematica, Terapevticheskii arkhiv. 2015. 87. 1, 49 CrossRef 8 Kosei Tsuchida. Hidehiro Yamada. Yoshioki Yamasaki. Kengo Suzuki. Ken Kongoji. Yoshihiro Akashi. Shoichi Ozaki. anomalie cardiache lasciato in connettivo pazienti affetti da malattie del tessuto con ipertensione polmonare pre-capillare così come borderline pressione arteriosa polmonare, moderna reumatologia. 2015. 25. 5, 744 CrossRef 9 Christian Nagel. Philipp Henn. Nicola Ehlken. Antonello DrsquoAndrea. Norbert vuoto. Eduardo Bossone. Anke Boumlttger. Christoph Fiehn. Christine Fischer. Hanns-Martin Lorenz. Frank Stoumlckl. Ekkehard Gruumlnig. Benjamin Egenlauf. Lo stress ecocardiografia Doppler per la diagnosi precoce di sistemico ipertensione arteriosa polmonare sclerosi associata, Arthritis Research amp Therapy. 2015. 17. 1 CrossRef 10 Courtney J. McCray. Maureen D. Mayes. Aggiornamento sulla sclerosi sistemica, Allergia e Asma Current Report. 2015. 15. 5 CrossRef 11 Gabor Kovacs. Alexander aviaria. Maria Tscherner. Vasile Foris. Gerhard Bachmaier. Andrea Olschewski. Horst Olschewski. Caratterizzazione dei pazienti con Borderline arteriosa polmonare pressione, Chest. 2014. 146. 6, 1486 CrossRef 12 Gyoumlngyveacuter Koumlltx151. Reacuteka Faludi. Daacuteniel Aradi. Barbara Bartos. Gaacutebor Kumaacutenovics. Tuumlnde Minier. Laacuteszloacute Czirjaacutek. Andraacutes Komoacutecsi. Impatto del coinvolgimento cardiaco sul rischio di mortalità tra i pazienti con sclerosi sistemica: a 5 anni di follow-up di un unico centro di coorte, Reumatologia clinica. 2014. 33. 2, 197 CrossRef 13 L Miller. S Chartrand. M Koenig. J-R Goulet. Eacute Rich. AS Chin. C Chartrand-Lefebvre. M Abrahamowicz. J-L Seneacutecal. T Grodzicky. malattie del cuore sinistro: una frequente causa di ipertensione polmonare precoce nella sclerosi sistemica, non correlato ai livelli di NT-proBNP elevati o la fibrosi cardiaca manifesta, ma associata ad un aumento dei livelli di MR-proANP e MR-proADM: analisi retrospettiva di una coorte canadese francese, Scandinavian Journal di Reumatologia. 2014. 43. 4, 314 CrossRef 14 Nicolas Dumoitier. Seacutebastien Lofek. Luc Mouthon. Fisiopatologia della sclerosi sistemica: Stato dell'arte nel 2014, La Presse Meacutedicale. 2014. 43. 10, e267 CrossRef 15 Saman Ahmed. Harold I. Palevsky. Ipertensione arteriosa polmonare correlati a Malattia del tessuto connettivo, malattia reumatica Clinics of North America. 2014. 40. 1, 103 CrossRef 16 Eric D. Austin. Steven M. Kawut. Mark T. Gladwin. Steven H. Abman. Ipertensione polmonare: NHLBI Workshop sulla prevenzione primaria delle malattie croniche polmonari, Annals of American Thoracic Society. 2014. 11. Supplemento 3, S178 CrossRef 17 Martin Johnson. Stephen Thomson. Il ruolo di test da sforzo nella moderna gestione delle ipertensione arteriosa polmonare, malattie. 2014. 2. 2, 120 CrossRef 18 Christopher J. Valerio. Benjamin E. Schreiber. Clive E. Handler. Christopher P. Denton. John G. Coghlan. Borderline media polmonare Arteria di pressione nei pazienti con sclerosi sistemica: transpolmonare gradiente predice rischio di sviluppare ipertensione polmonare, l'artrite amp Rheumatism. 2013. 65. 4, 1074 Wiley online Biblioteca 19 Luna Gargani. Rosa Sicari. Nuovi aspetti di ecocardiografica Valutazione della Ipertensione polmonare, attuali cardiovascolari Imaging Report. 2013. 6. 6, 507 CrossRef 20 Veronica Codullo. Roberto Caporali. Giovanna Cuomo. Stefano Ghio. Michele Dalto. Chiara Fusetti. Elena Borgogno. Carlomaurizio Montecucco. Gabriele Valentini. Lo stress ecocardiografia Doppler in sclerosi sistemica: La prova per un ruolo nella previsione di ipertensione polmonare, l'artrite amp Rheumatism. 2013. 65. 9, 2403 trattamento Wiley linea LibraryFresolimumab diminuisce biomarcatori e migliora i sintomi clinici nella sclerosi sistemica pazienti 1 Boston University School of Medicine, Dipartimento di Medicina Interna, Sezione di Reumatologia, Boston, Massachusetts, Stati Uniti d'America. 2 Hospital for Special Surgery di New York, New York, Stati Uniti d'America. 3 Geisel School of Medicine presso Dartmouth University Medical School, Hanover, New Hampshire, Stati Uniti d'America. 4 Boston University School of Public Health, Boston, Massachusetts, Stati Uniti d'America. Indirizzo corrispondenza: Robert Lafyatis, Boston University School of Medicine, 72 E. Concord St Boston, Massachusetts 02118, Stati Uniti d'America. Telefono: 617.638.5388 E-mail: lafyatisbu. edu. 1 Boston University School of Medicine, Dipartimento di Medicina Interna, Sezione di Reumatologia, Boston, Massachusetts, Stati Uniti d'America. 2 Hospital for Special Surgery di New York, New York, Stati Uniti d'America. 3 Geisel School of Medicine presso Dartmouth University Medical School, Hanover, New Hampshire, Stati Uniti d'America. 4 Boston University School of Public Health, Boston, Massachusetts, Stati Uniti d'America. Indirizzo corrispondenza: Robert Lafyatis, Boston University School of Medicine, 72 E. Concord St Boston, Massachusetts 02118, Stati Uniti d'America. Telefono: 617.638.5388 E-mail: lafyatisbu. edu. 1 Boston University School of Medicine, Dipartimento di Medicina Interna, Sezione di Reumatologia, Boston, Massachusetts, Stati Uniti d'America. 2 Hospital for Special Surgery di New York, New York, Stati Uniti d'America. 3 Geisel School of Medicine presso Dartmouth University Medical School, Hanover, New Hampshire, Stati Uniti d'America. 4 Boston University School of Public Health, Boston, Massachusetts, Stati Uniti d'America. Indirizzo corrispondenza: Robert Lafyatis, Boston University School of Medicine, 72 E. Concord St Boston, Massachusetts 02118, Stati Uniti d'America. Telefono: 617.638.5388 E-mail: lafyatisbu. edu. 1 Boston University School of Medicine, Dipartimento di Medicina Interna, Sezione di Reumatologia, Boston, Massachusetts, Stati Uniti d'America. 2 Hospital for Special Surgery di New York, New York, Stati Uniti d'America. 3 Geisel School of Medicine presso Dartmouth University Medical School, Hanover, New Hampshire, Stati Uniti d'America. 4 Boston University School of Public Health, Boston, Massachusetts, USA. Address correspondence to: Robert Lafyatis, Boston University School of Medicine, 72 E. Concord St. Boston, Massachusetts 02118, USA. Phone: 617.638.5388 E-mail: lafyatisbu. edu . 1 Boston University School of Medicine, Department of Internal Medicine, Rheumatology Section, Boston, Massachusetts, USA. 2 Hospital for Special Surgery, New York, New York, USA. 3 Geisel School of Medicine at Dartmouth University Medical School, Hanover, New Hampshire, USA. 4 Boston University School of Public Health, Boston, Massachusetts, USA. Address correspondence to: Robert Lafyatis, Boston University School of Medicine, 72 E. Concord St. Boston, Massachusetts 02118, USA. Phone: 617.638.5388 E-mail: lafyatisbu. edu . 1 Boston University School of Medicine, Department of Internal Medicine, Rheumatology Section, Boston, Massachusetts, USA. 2 Hospital for Special Surgery, New York, New York, USA. 3 Geisel School of Medicine at Dartmouth University Medical School, Hanover, New Hampshire, USA. 4 Boston University School of Public Health, Boston, Massachusetts, USA. Address correspondence to: Robert Lafyatis, Boston University School of Medicine, 72 E. Concord St. Boston, Massachusetts 02118, USA. Phone: 617.638.5388 E-mail: lafyatisbu. edu . 1 Boston University School of Medicine, Department of Internal Medicine, Rheumatology Section, Boston, Massachusetts, USA. 2 Hospital for Special Surgery, New York, New York, USA. 3 Geisel School of Medicine at Dartmouth University Medical School, Hanover, New Hampshire, USA. 4 Boston University School of Public Health, Boston, Massachusetts, USA. Address correspondence to: Robert Lafyatis, Boston University School of Medicine, 72 E. Concord St. Boston, Massachusetts 02118, USA. Phone: 617.638.5388 E-mail: lafyatisbu. edu . 1 Boston University School of Medicine, Department of Internal Medicine, Rheumatology Section, Boston, Massachusetts, USA. 2 Hospital for Special Surgery, New York, New York, USA. 3 Geisel School of Medicine at Dartmouth University Medical School, Hanover, New Hampshire, USA. 4 Boston University School of Public Health, Boston, Massachusetts, USA. Address correspondence to: Robert Lafyatis, Boston University School of Medicine, 72 E. Concord St. Boston, Massachusetts 02118, USA. Phone: 617.638.5388 E-mail: lafyatisbu. edu . 1 Boston University School of Medicine, Department of Internal Medicine, Rheumatology Section, Boston, Massachusetts, USA. 2 Hospital for Special Surgery, New York, New York, USA. 3 Geisel School of Medicine at Dartmouth University Medical School, Hanover, New Hampshire, USA. 4 Boston University School of Public Health, Boston, Massachusetts, USA. Address correspondence to: Robert Lafyatis, Boston University School of Medicine, 72 E. Concord St. Boston, Massachusetts 02118, USA. Phone: 617.638.5388 E-mail: lafyatisbu. edu . 1 Boston University School of Medicine, Department of Internal Medicine, Rheumatology Section, Boston, Massachusetts, USA. 2 Hospital for Special Surgery, New York, New York, USA. 3 Geisel School of Medicine at Dartmouth University Medical School, Hanover, New Hampshire, USA. 4 Boston University School of Public Health, Boston, Massachusetts, USA. Address correspondence to: Robert Lafyatis, Boston University School of Medicine, 72 E. Concord St. Boston, Massachusetts 02118, USA. Phone: 617.638.5388 E-mail: lafyatisbu. edu . 1 Boston University School of Medicine, Department of Internal Medicine, Rheumatology Section, Boston, Massachusetts, USA. 2 Hospital for Special Surgery, New York, New York, USA. 3 Geisel School of Medicine at Dartmouth University Medical School, Hanover, New Hampshire, USA. 4 Boston University School of Public Health, Boston, Massachusetts, USA. Address correspondence to: Robert Lafyatis, Boston University School of Medicine, 72 E. Concord St. Boston, Massachusetts 02118, USA. Phone: 617.638.5388 E-mail: lafyatisbu. edu . 1 Boston University School of Medicine, Department of Internal Medicine, Rheumatology Section, Boston, Massachusetts, USA. 2 Hospital for Special Surgery, New York, New York, USA. 3 Geisel School of Medicine at Dartmouth University Medical School, Hanover, New Hampshire, USA. 4 Boston University School of Public Health, Boston, Massachusetts, USA. Address correspondence to: Robert Lafyatis, Boston University School of Medicine, 72 E. Concord St. Boston, Massachusetts 02118, USA. Phone: 617.638.5388 E-mail: lafyatisbu. edu . 1 Boston University School of Medicine, Department of Internal Medicine, Rheumatology Section, Boston, Massachusetts, USA. 2 Hospital for Special Surgery, New York, New York, USA. 3 Geisel School of Medicine at Dartmouth University Medical School, Hanover, New Hampshire, USA. 4 Boston University School of Public Health, Boston, Massachusetts, USA. Address correspondence to: Robert Lafyatis, Boston University School of Medicine, 72 E. Concord St. Boston, Massachusetts 02118, USA. Phone: 617.638.5388 E-mail: lafyatisbu. edu . 1 Boston University School of Medicine, Department of Internal Medicine, Rheumatology Section, Boston, Massachusetts, USA. 2 Hospital for Special Surgery, New York, New York, USA. 3 Geisel School of Medicine at Dartmouth University Medical School, Hanover, New Hampshire, USA. 4 Boston University School of Public Health, Boston, Massachusetts, USA. Address correspondence to: Robert Lafyatis, Boston University School of Medicine, 72 E. Concord St. Boston, Massachusetts 02118, USA. Phone: 617.638.5388 E-mail: lafyatisbu. edu . 1 Boston University School of Medicine, Department of Internal Medicine, Rheumatology Section, Boston, Massachusetts, USA. 2 Hospital for Special Surgery, New York, New York, USA. 3 Geisel School of Medicine at Dartmouth University Medical School, Hanover, New Hampshire, USA. 4 Boston University School of Public Health, Boston, Massachusetts, USA. Address correspondence to: Robert Lafyatis, Boston University School of Medicine, 72 E. Concord St. Boston, Massachusetts 02118, USA. Phone: 617.638.5388 E-mail: lafyatisbu. edu . 1 Boston University School of Medicine, Department of Internal Medicine, Rheumatology Section, Boston, Massachusetts, USA. 2 Hospital for Special Surgery, New York, New York, USA. 3 Geisel School of Medicine at Dartmouth University Medical School, Hanover, New Hampshire, USA. 4 Boston University School of Public Health, Boston, Massachusetts, USA. Address correspondence to: Robert Lafyatis, Boston University School of Medicine, 72 E. Concord St. Boston, Massachusetts 02118, USA. Phone: 617.638.5388 E-mail: lafyatisbu. edu . First published June 22, 2015 - More info Published in Volume 125, Issue 7 (July 1, 2015) J Clin Invest. 2015125(7):27952807. doi:10.1172JCI77958. xA Copyright 2015, American Society for Clinical Investigation First published June 22, 2015 Received: September 16, 2014 Accepted: May 14, 2015 BACKGROUND . TGF - has potent profibrotic activity in vitro and has long been implicated in systemic sclerosis (SSc), as expression of TGF-regulated genes is increased in the skin and lungs of patients with SSc. Therefore, inhibition of TGF - may benefit these patients. METHODS . Patients with early, diffuse cutaneous SSc were enrolled in an open-label trial of fresolimumab, a high-affinity neutralizing antibody that targets all 3 TGF - isoforms. Seven patients received two 1 mgkg doses of fresolimumab, and eight patients received one 5 mgkg dose of fresolimumab. Serial mid-forearm skin biopsies, performed before and after treatment, were analyzed for expression of the TGF-regulated biomarker genes thrombospondin-1 ( THBS1 ) and cartilage oligomeric protein ( COMP ) and stained for myofibroblasts. Clinical skin disease was assessed using the modified Rodnan skin score (MRSS). RESULTS . In patient skin, THBS1 expression rapidly declined after fresolimumab treatment in both groups ( P 0.0313 at 7 weeks and P 0.0156 at 3 weeks), and skin expression of COMP exhibited a strong downward trend in both groups. Clinical skin disease dramatically and rapidly decreased ( P lt 0.001 at all time points). Expression levels of other TGF-regulated genes, including SERPINE1 and CTGF . declined ( P 0.049 and P 0.012, respectively), and a 2-gene, longitudinal pharmacodynamic biomarker of SSc skin disease decreased after fresolimumab treatment ( P 0.0067). Dermal myofibroblast infiltration also declined in patient skin after fresolimumab ( P lt 0.05). Baseline levels of THBS1 were predictive of reduced THBS1 expression and improved MRSS after fresolimumab treatment. CONCLUSION . The rapid inhibition of TGF-regulated gene expression in response to fresolimumab strongly implicates TGF - in the pathogenesis of fibrosis in SSc. Parallel improvement in the MRSS indicates that fresolimumab rapidly reverses markers of skin fibrosis. TRIAL REGISTRATION . Clinicaltrials. gov NCT01284322. FUNDING . This study was supported by the Boston University Clinical and Translational Science Institute, the NIHs National Center for Advancing Translational Sciences Clinical and Translational Sciences, the National Institute of Arthritis Musculoskeletal and Skin Disease: Scleroderma Core Centers and Scleroderma Center of Research Translation, the Boston University Medical Campus Microarray Core, the Kellen Foundation at Hospital for Special Surgery, the Scleroderma Research Foundation, the Dr. Ralph and Marian Falk Medical Research Trust, and Novartis. Soon after its discovery as a transforming growth factor, TGF - was recognized to have potent profibrotic activity ( 1. 2 ). TGF - promotes collagen synthesis, secretion, processing, and cross-linking ( 3 ) as well as secretion of other matrix molecules, such as fibronectin and thrombospondin ( 4 ). All 3 TGF - isotypes (TGF-1, TGF-2, and TGF-3) interact with the same receptor complex and have wide-ranging, context-specific effects on cell proliferation and differentiation, embryological processes, and the immune system ( 5 ). Fibrosis in patients with systemic sclerosis (SSc) affects the skin in addition to internal organs: lungs, heart, and gastrointestinal tract ( 6 ). It represents a major unmet therapeutic challenge, as treatment for SSc fibrotic disease is currently limited to cyclophosphamide, which has modest effects ( 7 ) immunoablation and stem cell transplant offer a more dramatic benefit, which is associated with significant treatment-associated morbidity and mortality ( 8 ). Further, fibrosis is an end-stage process in many diseases, including many common diseases, such as liver cirrhosis complicating viral or drug-induced hepatitis, renal fibrosis as the pathological feature leading to many forms of end-stage renal disease, cardiac fibrosis after myocardial infarctions and associated with hypertension, and pulmonary fibrosis, either idiopathic or associated with a variety of other diseases or environmental exposures ( 9 ). Although skin disease in SSc does not cause death, extensive skin disease is associated with considerable morbidity and severity of internal organ disease as well as disease-associated mortality ( 10 ). In addition, skin fibrosis shares pathogenic features with fibrotic disease at other sites in patients with SSc, suggesting that agents successfully treating skin might show efficacy for other involved organs ( 11 ). As skin can be easily biopsied, it provides a window into understanding pathogenesis and also a methodology for assessing response of a target organ. Several factors have slowed development of therapeutics targeting fibrosis in SSc. The variable trajectory of disease progression in different organs in patients with SSc has led to particular challenges in identifying efficacy in early-phase clinical trials this has included slow rates of progression of interstitial lung disease ( 12 ) and spontaneous improvement of skin disease ( 13 ). In addition, the relatively slow rate of change in skin disease, as assessed by the main clinical outcome, the modified Rodnan skin score (MRSS), and the relative insensitivity of the MRSS to change has led to the notion that changes in the MRSS require long periods of observation to detect. The clinical heterogeneity of the disease and years-long progression have made it difficult to demonstrate efficacy in large, placebo-controlled, 12- to 24-month clinical trials in Ssc, even in the early phase of clinical development ( 14 ), presenting a significant obstacle for discovering effective therapies for SSc. Biomarkers that correlate with the MRSS might act as surrogate outcome measures for the degree of skin disease in patients with SSc and thus provide an approach for more quickly detecting potential drug efficacy in early-phase SSc clinical trials. Biomarkers can be measured objectively and might be expected to change more quickly than clinical outcome measures such as the MRSS. We previously described a 4-gene, pharmacodynamic biomarker of SSc skin disease, based on gene expression in a mid-forearm skin biopsy ( 15 ). Two of the four genes making up the biomarker, thrombospondin-1 ( THBS1 ) and cartilage oligomeric protein ( COMP ), are highly regulated by TGF-. Thus, to test the importance of TGF - in SSc skin fibrosis as well as to test a biomarker-based approach to early-phase clinical trial design, we treated patients with early, diffuse SSc with fresolimumab, a first-in-class human IgG4 monoclonal antibody capable of neutralizing all mammalian isoforms of TGF-. The predefined primary efficacy outcome for this trial was change in COMP and THBS1 mRNA expression in skin after treatment compared with that at baseline. Patients. Patients were mostly female, with a median age of 51 years and median MRSS of 24 (Table 1 ). Only one patient showed signs of interstitial lung disease, with a median forced vital capacity of 90 predicted. Four patients (27) had anti-Scl70 autoantibodies, and eight patients (53) had antiRNA polymerase (anti-Pol3) III autoantibodies. Anti-Pol3 autoantibodies are typically seen in only about 11 of all patients with SSc ( 16 ) but are much more common in patients with diffuse cutaneous disease ( 17 ). Since anti-Pol3 autoantibodies are associated with more severe skin disease and less severe interstitial lung disease ( 17 ), our entry criteria, which included only patients with diffuse cutaneous disease and excluded patients with significant lung disease, likely enriched for patients with this autoantibody specificity. Two groups of patients were treated with fresolimumab: the first group (group 1, n 7) was treated with 2 doses of 1 mgkg fresolimumab 4 weeks apart, and the second group (group 2, n 8) was treated with 1 dose of 5 mgkg fresolimumab (Figure 1 and Figure 2A ). Skin biopsies were performed at baseline (the day of first infusion) and at 3 weeks (group 1) or 4 weeks (group 2), 7 weeks, and 24 weeks after the baseline biopsy (Figure 2A ). CONSORT flow diagram. Schematic of patient screening, enrollment, and completion of the trial in the two study groups. Study design and pharmacokinetics of fresolimumab in two dosing groups. ( A ) Trial protocol for each group shows dosing levels and frequency and timing of skin biopsies. Red arrows indicate skin biopsies. ( B ) Fresolimumab levels measured in sera at 3, 7, 11, 17, and 24 weeks in group 1, which received 2 doses of 1 mgkg at weeks 0 and 4 (blue diamonds), and group 2, which received a single dose of 5 mgkg fresolimumab at week 0 (red squares). Error bars indicate SD. Baseline demographic and clinical characteristics of study patients ( n 15) Skin biomarker gene expression of THBS1 shows a rapid decline after fresolimumab treatment. The 2 groups of patients showed dramatically different serum levels of fresolimumab, with group 2 reaching approximately 5 times higher serum levels 3 weeks after treatment than group 1 (Figure 2B ). Even after the first group of patients received a second 1 mgkg dose of study medication, the relative levels of circulating fresolimumab were nearly twice as high in the second group (receiving a single 5 mgkg dose) at 7 weeks and subsequent time points due to the circulating half-life of approximately 22 days. Both treatment groups separately showed rapid declines in THBS1 and COMP gene expression, the primary efficacy outcome measure (Figure 3, AD. and Supplemental Table 1A supplemental material available online with this article doi:10.1172JCI77958DS1 ). Patients in the first dosing group showed a significant decline in median THBS1 expression (67.2 of baseline) at 7 weeks compared with that at baseline ( P 0.0313, Figure 3A and Supplemental Figure 1A ), and a trend toward decreased COMP expression (77.1 of baseline) ( P 0.218, Figure 3C and Supplemental Figure 1C ). Patients in the second dosing group showed a more rapid decrease in THBS1 (70.0 of baseline P 0.0156, Figure 3B and Supplemental Figure 1B ) and COMP (66.7 of baseline P 0.0781, Figure 3D and Supplemental Figure 1D ), which reached a nadir at week 3. Decreases in gene expression seen at weeks 3 or 4 and week 7 were lost at week 24 (Figure 3, AD ), a time when circulating fresolimumab was mostly metabolized (Figure 2B ). However, in the first dosing group, the loss of significant change at week 24 might reflect the fewer biopsies available for analysis. Changes in biomarker gene expression and MRSS by study group. ( A and B ) THBS1 and ( C and D ) COMP gene expression and ( E and F ) MRSS from study patients in ( A . C . and E ) group 1 ( n 7), which received 2 doses of 1 mgkg fresolimumab, and ( B . D . and F ) group 2 ( n 8), which received 1 dose of 5 mgkg fresolimumab. Dot plots show median (horizontal bars) and statistical significance of THBS1 and COMP gene expression (Wilcoxon signed-rank test, using available data). Box plots show median (horizontal line), interquartile (box), and 95 confidence limits (whiskers) of MRSS. Line graphs showing changes in individual patients over time are shown in Supplemental Figure 1 . Changes in gene expression in study patients generally correlated with changes in MRSS (compare Supplemental Figure 1, A and C with Supplemental Figure 1E and Supplemental Figure 1, B and D with Supplemental Figure 1F ). Examining the correlation between the MRSS and biomarkers in all biopsies revealed a high correlation for THBS1 ( r 0.63) and COMP ( r 0.62) (data not shown), similar to correlations we reported previously between the MRSS and gene expression ( 15 ). Baseline THBS1 and COMP gene expression was strikingly higher in both SSc patient cohorts than in healthy control skin, as we have reported previously (Figure 3, AD. and ref. 15 ). Median baseline values were higher in the first cohort ( THBS1 11.63, COMP 36.84) compared with those in the second cohort ( THBS1 5.97, COMP 16.20 Figure 3, AD ), as were median baseline MRSS values (25 and 22.5, respectively). Summary statistics using all available data for patients entered into both treatment groups showed that THBS1 and COMP expression declined rapidly after fresolimumab treatment (Table 2 ). Gene expression results from skin biopsies following treatment in the first group at 3 weeks and in the second group at 4 weeks were combined for these analyses. Median THBS1 levels at week 3 or 4 were 77 of baseline, reaching a nadir at week 7 of 61 of baseline THBS1 expression. Median COMP levels at week 3 or 4 and week 7 were 60 and 75 of baseline COMP expression. rispettivamente. Examining gene expression of evaluable, paired biopsies from both treatment groups, median THBS1 expression decreased by 1.5 ( P 0.0135) and 1 ( P 0.004) at week 3 or 4 and week 7, respectively (from baseline median of 8), and median COMP expression decreased by 7.9 ( P 0.058) and 4.3 ( P 0.153) at week 3 or 4 and week 7, respectively (from baseline median of 22.6). Summary statistics on all available data (dosing groups combined) To verify the results using an analytic method robust in accounting for missing-at-random data, we used a repeated-measures model to take advantage of the multiple measurements per subject. Controlling for dose group, the predicted mean changes from baseline ( coefficients) closely paralleled the results obtained using all available data (Table 3 ). Changes in THBS1 and COMP expression were largest at week 7, with predicted decreases of 3.4 ( P 0.018) and 3.6 ( P 0.135), respectively. The dose by time interaction terms were significant for both THBS1 ( P 0.0012) and COMP ( P 0.0492), indicating that the patterns of change over time differed by dose group. Repeated-measures model results To better understand the relationship between the skin score at the biopsy site and gene expression, we compared gene expression of THBS1 and COMP to the local forearm skin score, the 03 score performed on the same day as the biopsy. The local skin score correlated highly with the MRSS and also with expression of both THBS1 and COMP (Supplemental Figure 2 ). We also examined autoantibody levels after treatment (week 11) compared with those at baseline (Supplemental Figure 3 ). Anti-Scl70 autoantibody levels fell modestly in all patients (4 of 4), but our patient groups size was not sufficient to indicate a statistically meaningful result. Anti-Pol3 levels showed no consistent change, rising in some patients and falling in others. We also examined the effects of autoantibody status on the clinical response. The change in MRSS in anti-Pol3negative or anti-Scl70negative patients (median change 12) was higher than that in anti-Scl70positive (median change 5) or anti-Pol3positive patients (median change 2), but these groups were too small to make a meaningful statistical comparison. The change in MRSS was different when comparing anti-Pol3positive patients (median change in MRSS 2) to anti-Pol3negative patients (grouping anti-Scl70positive patients with patients negative for both anti-Scl70 and anti-Pol3 mean change in MRSS 6, P 0.014) at week 7 but not at week 3 or 4 ( P 0.33). Some studies have shown that longer disease duration is associated with spontaneous improvement in skin disease in patients with SSc ( 18 ). However, in patients in this early disease cohort, we found no relationship between the baseline disease duration and the subsequent change in MRSS (Supplemental Figure 4 ). Skin disease shows rapid improvement after fresolimumab treatment. The primary clinical outcome in this trial, the MRSS, declined rapidly in most patients, generally within several weeks of the infusion (Table 2 ). Examining the change in MRSS in each study group separately showed similar seeming response kinetics, with both groups showing rapid decreases in mean MRSS as soon as the first follow-up study visit, and then increasing skin scores by the week 24 study visit, apparently representing recurrent disease (Figure 3, E and F. and Supplemental Table 1B ). The median change in MRSS, using all available data across both dose groups, was most striking at weeks 11 and 17, 6 ( P 0.0005) and 9.5 ( P 0.0024), respectively (Table 2 ). Many patients then showed recurrent disease, i. e. an increase in the MRSS at a later point in the trial. The repeated-measures model reinforced the analysis of all available data (Table 3 ), with a nadir in the predicted change of 8 at week 11 ( P 0.0002). There was no statistically significant dose by time interaction in the repeated-measures model, indicating that the patterns of change over time in MRSS were somewhat similar for the 2 dose groups ( P 0.4168). Decreased expression of a coregulated cluster of TGF-regulated genes and macrophage-associated genes after fresolimumab treatment. To further understand the genes regulated by fresolimumab in the study patients, biopsies were analyzed by microarray. Clustering of these genes revealed a readily recognizable cluster of TGF-regulated genes that included both of the genes predefined as the primary outcome measures: THBS1 and COMP (Figure 4 ). All of the genes in this cluster were found to correlate highly with the MRSS (Figure 4 ). Genes clustering with the primary outcome measures included several genes regulated by TGF-: connective tissue growth factor ( CTGF . also known as CCN2 ) ( 19 ), cadherin 11 ( CAD11 ) ( 20 ), NADPH oxidase 4 ( NOX4 ) ( 21 ), collagen X 1 ( COL10A1 ) ( 22 ), and collagen XI, 1 ( 23 ). We quantified expression of several TGF - and collagen genes by RT-PCR. CTGF . SERPINE1 . and COL10A1 expression levels all declined after fresolimumab treatment compared with those at baseline (Figure 5, AC ). Hierarchical clustering of skin gene expression in study patients. The gene cluster, including both primary outcome measures, THBS1 and COMP . is shown. Samples from each study patient ( n 15) are shown as columns of gene expression (red, high gene expression green, low gene expression black, intermediate gene expression), labeled by study visit, starting with the baseline biopsy, followed sequentially by subsequent biopsies (top of figure), with a blank column between each patient. Gene names and the correlation between each gene and the MRSS are shown to the right and far right, respectively. GalNAc-T1, UDP-N-acetyl--D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 1. Changes in gene expression before and after fresolimumab treatment. Skin biopsy RNAs were analyzed for expression of TGF-regulated genes, ( A ) CTGF . ( B ) SERPINE1 . ( C ) and COL10A1 . and a macrophage marker, ( D ) CD163 . at baseline and after fresolimumab treatment. Levels from 5 healthy controls skin samples are also shown. CTGF . SERPINE1 . and COL10A1 mRNA expression was assayed by RT-PCR CD163 expression was assayed by NanoString. Statistical significance was assessed by Wilcoxon signed-rank. Error bars indicate SEM. We also examined the microarray expression of inflammatory genes after fresolimumab treatment. CD14 . CD163 . and MS4A4A . all genes expressed mainly in monocytesmacrophages, clustered together (data not shown). Expression of CD163 . tested by NanoString, trended lower after fresolimumab treatment (Figure 5D ). Decreased expression of a SSc skin pharmacodynamic biomarker after fresolimumab treatment. We tested the effect of treatment with fresolimumab on a recently developed, multianalyte, longitudinal pharmacodynamic biomarker (2GSSc skin biomarker ref. 24 ). This biomarker is composed of weighted values for THBS1 and MS4A4A mRNA expression assessed by NanoString. The 2GSSc skin biomarker showed reduced expression at both week 3 or 4 and week 7 after fresolimumab treatment compared with baseline expression levels (Figure 6A ). We compared the 2GSSc biomarker in samples from a clinical trial of nilotinib in SSc. This trial included very similar patients with early, diffuse SSc (Supplemental Table 2 ), but skin biopsies were analyzed at baseline, 6 months, and 12 months. This longer time interval between biopsies would be expected to show more dramatic changes in the 2GSSc skin biomarker than seen in samples from fresolimumab-treated patients, due to the natural history of the disease. In contrast to samples from fresolimumab-treated patients, nilotinib-treated patients showed no significant change in 2GSSc skin biomarker at 6 and 12 months compared with that at baseline (Figure 6B ). Decreased 2GSSc biomarker level in skin after fresolimumab treatment. Skin RNAs from patients entered into ( A ) the fresolimumab trial or ( B ) a clinical trial of nilotinib were assayed by NanoString for 2GSSc skin biomarker levels in baseline, week 3 or 4, and week 7 (fresolimumab) or at baseline and 6 and 12 months (nilotinib). Statistical significance was assessed by Wilcoxon signed-rank. Error bars indicate SEM. Inhibition of myofibroblasts and plasminogen activator protein-1 in fresolimumab-treated patients. TGF - stimulates fibroblast differentiation into myofibroblasts, the cell type consistently implicated in fibrosis in many diseases ( 25 ). Myofibroblasts are sensitive markers of SSc skin disease, and the intensity of myofibroblast infiltration correlates highly with the severity of skin disease, as assessed by the MRSS ( 26 ). In several patients, fresolimumab treatment was associated with a particularly rapid, dramatic decline in myofibroblast infiltration of the deep dermis (Figure 7, AC ). All study patients showed a decrease in myofibroblast infiltration at week 3 or 4 after treatment with fresolimumab (Figure 7D. P 0.022). The degree of myofibroblast infiltration changed more consistently in patients receiving the higher dose of fresolimumab (second treatment group data not shown). Myofibroblast infiltration in fresolimumab-treated patients. Sequential biopsies from patient GC17 are shown, comparing ( B ) week 4 and ( C ) week 7 to ( A ) baseline myofibroblast infiltration (original magnification, 10). Arrows indicate myofibroblasts. Insets show myofibroblast staining in the deep dermis (original magnification, 40). ( D ) Skin biopsies stained for smooth muscle actin (SMA) were scored by an observer blinded to biopsy sample for intensity of myofibroblast infiltration. Median values for each time point are shown by the red line with rectangular symbols ( P lt 0.01, comparing week 4 to baseline, Wilcoxon ranked-sum). Sections were also stained for the SERPINE1 gene product, plasminogen activator protein-1 (PAI-1). PAI-1 has long been recognized as a target of TGF - ( 27 ). We show above that SERPINE1 mRNA is highly upregulated in SSc skin and its levels are reduced after fresolimumab treatment. PAI-1 protein staining was also reduced in SSc skin (Supplemental Figure 5A ). Additionally, we examined dermal thickness in sections from patients after staining with trichrome. However, skin thickness was not found to change after fresolimumab treatment. (Supplemental Figure 5B ). Baseline expression of TGF-regulated genes predicts response to fresolimumab. In order to understand whether expression of some genes might predict patient response to fresolimumab, we examined the relationship between microarray gene expression at baseline to the change in expression of THBS1 at week 3 or 4 and week 7 compared with baseline. A negative correlation would indicate genes whose level of expression at baseline predicts a decrease in THBS1 expression after fresolimumab treatment. Clustering only genes showing correlations r lt 0.6 revealed two known TGF-regulated genes, ADAM metallopeptidase domain 12 ( ADAM12 ) and fibronectin ( FN ), clustering adjacent to each other ( r 0.7 for both, Figure 8A ). THBS1 also clustered with these genes, correlating almost as highly as ADAM12 and FN ( r 0.67). Patients stratified dichotomously on the basis of having higher than median baseline ADAM12 expression were found to significantly and more frequently respond to fresolimumab (decreased THBS1 expression compared with baseline Figure 8B. P lt 0.05). THBS1 and ADAM12 expression predicts response to fresolimumab. ( A ) 58 genes showing correlations with the change in THBS1 expression at week 3 or 4 and week 7 compared with baseline of less than 0.6 were clustered. Baseline ADAM12 . THBS1 . and FN cluster together, showing strongly negative correlations with the change in THBS1 expression after fresolimumab treatment at week 3 or 4 and week 7 (red, higher expression green, lower expression). Pearson correlations are shown to the right. ( B ) Patients were stratified into high ( n 14) and low ( n 14) baseline ADAM12 expression groups, based on whether they exhibited greater or less than the median level of ADAM12 expression on microarray. Patients were then graphed according to week 3 or 4 and week 7 THBS1 expression, compared with baseline (change in THBS1 ). Patient samples showing high baseline ADAM12 expression showed greater decreases in THBS1 after fresolimumab than patient samples with low baseline ADAM12 expression ( P lt 0.05, Fishers exact test). ( C ) Patient samples were stratified into high ( n 14) and low ( n 14) baseline THBS1 expression groups, based on whether they exhibited greater or less than median level of THBS1 expression on microarray. Samples were then graphed according to week 3 or 4 and week 7 THBS1 expression, compared with baseline (change in THBS1 ). Samples showing high baseline THBS1 expression showed greater decreases in MRSS after fresolimumab than samples with low baseline THBS1 expression ( P lt 0.05, Fishers exact test). We also examined whether expression of genes might predict the observed change in the MRSS. Baseline THBS1 expression robustly correlated with the change in MRSS at week 3 or 4 and week 7 compared with that at baseline ( r 0.38). Patients stratified dichotomously on the basis of having a higher median baseline THBS1 expression were found to respond more frequently to fresolimumab, both biochemically (decreased THBS1 expression compared with baseline, P lt 0.05, data not shown) and clinically (decreased MRSS compared with baseline, P lt 0.05, Figure 8C ). Safety outcomes. The most significant adverse events were several bleeding episodes and the common occurrence of anemia (Table 4 ). Two patients developed clinically significant gastrointestinal bleeding from gastric antral vascular ectasia (GAVE) one patient (GC08) with a history of GAVE required hospitalization, and both required transfusion. Both lesions were treated effectively with argon laser photocoagulation. Gingival bleeding andor epistaxis were described by 3 patients, and 2 patients showed subconjunctival hemorrhages. Hemoglobin levels decreased by more than 10 at some point in the trial in 10 of 15 (66.7) patients (Table 4 and Supplemental Figure 6 ), in most cases resolving without further treatment. Most anemic patients had iron studies consistent with anemia of chronic disease, although several also appeared iron deficient. Possibleprobable drug-related adverse events in study patients One patient (GC13) with severe skin disease, who experienced rapid improvement in skin sclerosis (MRSS 37 at baseline, improving to 30 at 3 weeks) after receiving one dose of fresolimumab (5 mgkg), subsequently developed recurrent skin disease (MRSS 35 at week 11) and developed shortness of breath 12 weeks after fresolimumab dosing. This patient was subsequently diagnosed with severe congestive heart failure and died of heart failure 10 days later. A premorbid cardiac biopsy of this patient showed severe cardiac fibrosis, a known complication of SSc ( 28 ). In another patient, one premalignant lesion was identified, a low-grade squamous intraepithelial cervical lesion found on routine PAP smear 17 weeks after fresolimumab dosing subsequent colposcopy showed mild dysplasia. This patient had a history of abnormal PAP smears, a low-grade squamous intraepithelial lesion, and a positive test for HPV DNA. Subsequent to the abnormal study PAP smear, the patient has had 2 normal PAP smears. Based on extensive in vitro and animal data, TGF - has long been suspected as an important mediator of fibrosis in SSc as well as a variety of other fibrotic diseases, including renal, pulmonary, cardiac, and liver fibrosis ( 29 31 ). However, to our knowledge there has never previously been clinical data directly supporting its role in humans. Our data show that fresolimumab leads to a rapid decrease of TGF-regulated biomarker genes. For the predetermined primary efficacy outcome measures for the trial, THBS1 expression in SSc skin declined significantly in both fresolimumab-treated groups, while COMP trended strongly toward decreased expression in both groups. In addition microarray analyses showed that downregulation of these genes was associated with downregulated expression of a larger cluster of known TGF-regulated genes, including NOX4 and CTGF . both implicated as secondary mediators of TGF - signaling ( 21. 32 ). Several collagen genes were also included in this cluster, consistent with a role for fresolimumab on genes directly mediating fibrosis. Expression of all of the genes in this cluster, most with r values greater than 0.4, was found to correlate with the MRSS, suggesting that these genes are part of a cluster of TGF-regulated genes that appear to play a driving role in TGF-mediated skin fibrosis in SSc. Further supporting the pivotal role of TGF - in SSc pathogenesis, patients treated with fresolimumab showed a rapid, significant decrease in MRSS, which correlated closely with the degree and speed of decline in the skin biomarkers. Thus, the biomarker data strongly reinforce the clinical data, while robustly supplementing it as an objective measure. The rate of decline of the skin score in these patients is unprecedented in clinical trials of even much longer duration ( 18 ). The natural history of patients with early, diffuse SSc, extracting from 7 large, placebo-controlled, negative large trials ( n 429), showed median MRSS improvements (decreases in MRSS) of 2.9 and 3.4 at 6 and 12 months ( 13 ). Thus, much larger and more rapid decreases in MRSS were seen in fresolimumab-treated patients (decreases of 5.1, 6.3, and 8 at week 3 or 4, 7, and 11, respectively). TGF - is also known to induce myofibroblasts ( 25 ), and the intensity of myofibroblast staining correlates highly with the degree of skin disease ( 26 ). Thus, decreased staining of myofibroblasts after fresolimumab treatment further supports the role of TGF - in SSc pathogenesis as well as the observed clinical effect on skin disease. Expression of genes known to be highly regulated by TGF-, CTGF and SERPINE (which encodes PAI-1), was also lower in patient skin samples 3 or 4 weeks after fresolimumab treatment, strongly supporting the role of TGF - in pathogenesis and expression of these genes. On the other hand, skin thickness did not change significantly after fresolimumab treatment. The MRSS is primarily designed to detect changes in skin thickness however, the effect of fresolimumab on the MRSS is likely due to changes in skin tightness and tethering that result from blocking TGF-. These clinical manifestations of skin tightness and tethering might be due more to the presence of myofibroblasts in SSc skin and to loss of subcutaneous fat, respectively, than to increased skin thickness. Our data show that at least one of these features, myofibroblast number, changes more rapidly than skin thickness after TGF - inhibition. Notably, the statistically significant improvements in both clinical score and biomarker expression observed early after fresolimumab treatment were lost by the last study visit (week 24), 7 or 8 drug half-lives after the last dose given in the first and second patient groups, respectively. Thus, these data further support the role of fresolimumab in the observed declines in MRSS and biomarker gene expression and indicate a need for longer treatment in future studies. Collectively, these observations strongly support a key role for TGF - in SSc pathogenesis. Patients treated with fresolimumab showed a trend of decreased levels of the macrophagemonocyte-associated genes, CD163 and MS4A4A . The latter of these genes is the second of two genes in the 2GSSc skin biomarker ( 24 ). The change in macrophage marker genes was less dramatic (and not statistically significant in this small trial) but might suggest that TGF - also contributes to the perivascular inflammation seen in this disease. Indeed, TGF - is known to induce monocyte chemotaxis, in addition to its antiinflammatory properties ( 33 ), and induces Th2, Th17, and plasmacytoid dendritic cell migration into the skin of mice with mutations found in patients with stiff skin syndrome that mimic dermal fibrosis of SSc ( 34 ). We found that baseline expression of THBS1 and ADAM12 was a predictive biomarker for improved THBS1 expression after fresolimumab treatment and baseline THBS1 expression was predictive of improvement in the MRSS after fresolimumab treatment. These observations suggest that patients might be stratified for their likely response to TGF - inhibition on the basis of their pretreatment gene expression. Both THBS1 and ADAM12 are known to be induced by TGF - ( 35. 36 ). THBS1 can also activate TGF-, while ADAM12 has also been shown to contribute to TGF - signaling through an interaction with the type II TGF - receptor ( 37 ). Notably, in recent studies, ADAM12 pericytes were shown to be progenitors of myofibroblasts induced after skin or muscle injury, and deletion of ADAM12 cells blocked fibrosis ( 38 ). Thus, it is intriguing to consider a possible role for TGF - in stimulating ADAM12 pericytes in the generation of myofibroblasts and fibrosis in SSc skin. An earlier study of the TGF - mAb CAT-192 in SSc failed to show any difference in change in MRSS between treatment groups ( 39 ). Importantly, CAT-192 targets only TGF-1 ( 40 ), with a dissociation constant of 150 nM, whereas fresolimumab is a much higher-affinity antibody for TGF-1 but also targets TGF-2 and TGF-3, with dissociation constants of 2.3 nM, 2.8 nM, and 1.4 nM, respectively (Genzyme, unpublished Biacore analyses). Thus, the neutralizing activity against TGF-2 and TGF-3 or much higher target affinity may be critical in the activity of fresolimumab in SSc. The antiTGF - antibody fresolimumab (also known as GC1008) has been tried previously in patients for several diseases. In patients with focal segmental glomerulosclerosis (FSGS), fresolimumab treatment was associated with a decline in proteinuria in some patients, suggesting possible efficacy in this disease, for which TGF - is also strongly implicated in pathogenesis ( 41. 42 ). Like in SSc, THBS1 is overexpressed in FSGS renal glomeruli ( 43. 44 ), suggesting that it may provide a strong biomarker in other TGF-mediated diseases. Patients with malignant mesothelioma ( n 13) were treated with 3 mgkg fresolimumab for up to 6 doses, showing little clinical effect but also few adverse effects. One patient developed erythematous, verrucous papules on biopsy, showing atypical keratinocyte proliferation ( 45 ). In patients with malignant melanoma ( n 28) or renal cell carcinoma ( n 1), patients received up to 4 doses of 15 mgkg fresolimumab over a period of 56 days, with 5 patients also receiving an additional 4 doses ranging from 0.1 to 1 mgkg ( 46 ). Minor bleeding (epistaxis and gingival bleeding) was seen frequently in these patients. In addition, several patients developed epithelial lesions suggestive of squamous cell carcinoma, rather than keratoacanthoma, which is more commonly seen in patients receiving higher doses of medication. All but one of these patients was ultimately diagnosed pathologically as having keratoacanthomas, and the lesions resolved spontaneously after fresolimumab treatment was complete, a clinical course more consistent with keratoacanthoma. We did not see keratoacanthomas in our study however, several patients had epistaxis, gum bleeding, or subconjunctival eye hemorrhage, as reported in other studies of fresolimumab ( 46. 47 ). Although the incidence of GAVE in this study group was similar to that expected in such a patient cohort, the timing and severity of these and other bleeding incidences in our study patients dictates careful observation of this complication in future trials. Anemia in the study patients has not been reported in past trials and may be related to bleeding, SSc, andor its treatment. Iron studies did not clearly distinguish iron deficiency from anemia of chronic disease (or both) in most patients. No patients showed evidence of hemolysis, and most showed an inappropriate response to the anemia (low or normal reticulocyte counts). Most patients were also on proton pump inhibitors, known to block iron absorption, and severe anemia associated with GAVE, though not correcting with oral iron, responded promptly to intravenous iron, consistent with underlying iron deficiency and preexisting gastrointestinal blood loss. Thus, future studies of fresolimumab in patients with SSc should include careful baseline evaluation for indolent gastrointestinal bleeding and iron deficiency. Despite recent studies clarifying the natural history of diffuse cutaneous SSc as a disease that frequently remits spontaneously, the disease often progresses relentlessly and is associated with considerable mortality through a variety of complications ( 48 ). Although patients do not typically die directly of skin disease, the underlying pathogenesis for fibrotic complications, including fatal complications of the lung and bowel, is likely similar. Thus, these complications may also be driven by TGF - and potentially amenable to inhibition by fresolimumab. Fresolimumab has been well tolerated in several trials (described above) at higher doses than those used in this study and multiple doses. The concern for side effects from TGF - inhibition needs to be carefully weighed against the prognosis of SSc and currently available therapeutics. Cyclophosphamide and immunoablation with stem cell transplant are both associated with neoplastic complications, and stem cell transplant is associated with considerable transplant-associated mortality ( 8 ). Fresolimumab is potentially a safer alternative. These complications, as well as the wide array of other diseases associated with organ fibrosis, may also be driven by TGF - and respond to fresolimumab. Thus, fresolimumab holds promise as a potent antifibrotic therapeutic, which needs further study for the safety of longer-term use and for treatment of skin, lung, and gastrointestinal tract fibrosis. Patients. This single-center study was approved by the Boston University Medical Campus Institutional Review Board and registered with clinicaltrials. gov identifier NCT01284322 prior to patient recruitment. The study was conducted after FDA review under Investigational New Drug 110704. A Data and Safety Monitoring Board, composed of two physicians external to the study investigators, reviewed all adverse events. Patients 18 years of age were included who had SSc ( 49 ) with diffuse cutaneous disease ( 50 ), a MRSS 15, and early disease, as defined by occurrence of the first non-Raynauds disease symptom within 2 years study entry. Patients with substantial pulmonary compromise (forced vital capacity less than 80 predicted, diffusion capacity less than 70 predicted, or gt20 fibrosis on computerized chest tomography) recent (within 6 months of study entry) scleroderma renal crisis or elevated creatinine gt2.0 significant arrhythmia, unstable angina, or clinical heart failure elevated liver function tests gt2.5 times normal bowel obstruction requiring hospitalization within 3 months or malabsorption requiring parenteral nutrition recent bleeding, anemia (Hgb lt8.5), or thrombocytopenia (platelet count lt100,000mm 3 ) active infection or any history of malignancy or history of premalignancy within 5 years were excluded. Patients were required to be on a stable dose of 10 mg per day or less of prednisone and no other immunosuppressive medication. None of the patients received any immunosuppressive medications before the primary outcome measures, the biomarker skin biopsies, were performed at week 3 or 4 and week 7. Five patients were started on immunosuppressive therapy during the safety follow-up. GC02 was started on methotrexate at week 11, which was discontinued, and the patient started on cyclophosphamide at week 17. GC03 was started on mycophenolate on week 15 after study withdrawal at the week 4 visit. GC11 and GC12 were started on methotrexate at week 11. GC13 was started on methotrexate on week 9. Study design. Patients meeting inclusion criteria at the screening visit returned to the Boston University Medical Campus General Clinical Research Unit within 4 weeks. After an interim history and physical examination, two 3-mm skin biopsies were performed on the dorsal surface of the mid-forearm: one biopsy was immediately placed into 10 buffered formalin, and the other was placed into RNAlater (Qiagen). Study medication was then administered by intravenous infusion. The first cohort, receiving 1 mgkg fresolimumab, returned after 3 weeks for safety laboratory tests and repeat skin biopsies at sites approximately 5 mm from but immediately adjacent to the initial biopsies, followed by a repeat 1 mgkg dose of fresolimumab at week 4. Fresolimumab was supplied by Genzyme under Investigational New Drug 110704, granted to the Principal Study Investigator, Robert Lafyatis. The second cohort, receiving a single dose of 5 mgkg fresolimumab, returned at week 4 for safety laboratory tests and skin biopsies. Both cohorts returned for follow-up safety monitoring at weeks 7, 11, 17, and 24, with skin biopsies performed again at week 7 and, optionally, if the patient was willing, at week 24 at sites approximately 5 mm from but immediately adjacent to the most recent biopsy. The MRSS was assessed at each study visit by the same study physician. The scleroderma-modified health assessment questionnaire was administered at weeks 0, 7, and 24. Safety assessments. Safety was assessed at each study visit using Common Terminology Criteria for Adverse Events v4.0. Based on FDA protocol recommendations, all adverse events were considered related to the study drug unless there was a clear and identifiable reason or explanation to reject casual relation. Due to concerns regarding possible promotion of tumor progression by blocking TGF-, in addition to routine physical exam, patients were monitored by complete skin, oropharyngeal, and nasal exams for the second cohort, patients were monitored by urinalysis for hematuria at baseline and all subsequent study visits. A complete blood count with differential and an extended metabolic panel, including electrolytes and liver and renal function tests, were performed at each study visit. Skin biomarker measurements. RNA was collected from skin samples and placed directly at the bedside into RNAlater. The skin was minced and homogenized with a Polytron homogenizer, RNA was purified using the RNeasy Mini Kit (Qiagen), and the concentration of total RNA was measured (Nanodrop 1000 ThermoScientific). 200 ng of RNA was used to make cDNA, according to the SuperScript II RT (Invitrogen) protocol using random primers, and tested for THBS1 . COMP . CTGF . SERPINE1 . and COL10A1 levels by RT-PCR, normalizing values to 18S RNA and then normalizing the fold change to the average expression of RNAs from 5 healthy control skin samples. Primers were purchased from Applied Biosystems: THBS1 (Hs00962908m1) COMP (Hs00164359M1) CTGF (Hs01026927g1) SERPINE1 (Hs01126606m1) and COL10A1 (Hs0016657m1). RNA was also analyzed by microarray on Affymetrix U133A2.0 microarray chips, and data were normalized using the MAS 5.0 algorithm. Gene expression values were clustered using Cluster 2.0 ( 51 ). After filtering for genes showing differences of greater than 200 across all samples, genes were mean centered, normalized, clustered by complete linkage, and visualized using Java Treeview. Further exploratory analyses for inflammatory genes were carried out by clustering only genes selected on the basis of an expression correlating with the MRSS gt0.4. RNA from skin biopsies was prepared and analyzed by microarray on Affymetrix U133A2.0 microarray chips. After filtering for genes showing differences of greater than 200 across all samples, genes were mean centered, normalized, clustered by complete linkage using Cluster 2.0 ( 51 ), and visualized using Java Treeview. All microarray data have been deposited in the GEO database (accession GSE55036). Immunohistochemical staining was carried out on formalin-fixed, paraffin-embedded sections. For antigen retrieval, slides were incubated in citrate buffer pH 6.1 (Dako North America) for 20 minutes at 90C. Sections were then incubated with a rabbit antiPAI-1 (NBP2-13298, Novus Biologicals 1:25 dilution) or mouse anti-SMA (clone 1A4, M0851, Dako 1:400 dilution). After washing, the sections were incubated with polymerHRP solution (K5361, Dako) and developed using DAB. Staining intensity was assessed by an observer blinded to the patient and visit, as described previously ( 26 ). Collagen thickness on trichrome-stained slides was measured using a Nikon Eclipse E400 microscope and SPOT Software Advanced version 4.5. Measurements were taken on each biopsy from the dermal epidermal junction to the point at which collagen staining disappeared. Statistiche. Summary statistics, including the mean, median, standard deviation, and 95 confidence limits, are reported overall and by dosing group for THBS1 . COMP . and MRSSs at each time point and for change from baseline to each follow-up time point. The Wilcoxon signed-rank test was used to assess statistically significant changes in each outcome for all available data for both dosing groups, combined and separately, and graphed using Prism software (GraphPad). Spearmans correlations were calculated to examine the relationships between gene expression and MRSS. For each outcome, a general linear model for correlated data was used to incorporate all of the data into a single model and account for potential correlation between repeated measurements on an individual. This approach uses all available data and is robust to missing-at-random data mechanisms. An unstructured covariance was used for all analyses. Each model contained terms for dosing group, categorical time, and their interaction. The interaction was removed to assess the main effect of time. Coefficients and corresponding standard errors are reported. These analyses were performed using SAS v9.3. The difference in immunohistochemical staining intensities and autoantibody titers, comparing baseline and after treatment in each patient, were analyzed using Wilcoxon signed-rank test. Statistical differences in the changes in skin score in patient groups showing different autoantibody status were compared using the Mann-Whitney test. For predictive biomarker identification, Pearson correlations between expression of each gene analyzed by microarray and the difference in THBS1 expression at week 3 or 4 and week 7 compared with baseline were calculated. Changes in expression from baseline to week 3 or 4 and week 7 were treated as independent values for these analyses. Genes showing correlations with fresolimumab-associated change in THBS1 expression of r lt 0.4 or r lt 0.6 were selected for clustering. Using Cluster 2.0, genes were mean centered, normalized, clustered by complete linkage, and visualized using Java Treeview. Fishers exact test was used to calculate statistical significance on a contingency table, based on patients having higher or lower than median baseline ADAM12 expression and having increased or decreased THBS1 gene expression from baseline. Study approval. The study was reviewed and approved by the Boston University Medical Campus Institutional Review Board, Boston, Massachusetts, USA. All subjects provided informed consent prior to their participation in the study. The authors would like to thank Richard Polisson, Patrick Finn, Eileen Budri, Michael Halpin, and Marta Frisinger for help with regulatory and safety review during the study. The authors thank Kate Brennan for help with manuscript preparation and Marc Lenburg and Yuriy Alekseyev for help with microarray analyses. This study was supported by the NIHs National Center for Advancing Translational Sciences Clinical and Translational Science Award National Institute of Arthritis Musculoskeletal and Skin Disease grants: Scleroderma Core Centers (5P30AR061271), Scleroderma Center of Research Translation (1P50AR060780), and 2R01AR051089 a Clinician Scientist Development Award through the Kellen Foundation at Hospital for Special Surgery to J. Gordon and Scleroderma Research Foundation and Dr. Ralph and Marian Falk Medical Research Trust grants to M. L. Whitfield. The nilotinib clinical trial was also supported by an investigator-initiated grant from Novartis and by the Rudolph Rupert Scleroderma Program at Hospital for Special Surgery. Conflict of interest: Robert F. Spiera has received grants from the Rudolph Rupert Scleroderma Program at Hospital for Special Surgery, Novartis, GenentechRoche, Human Genome SciencesGlaxoSmithKline, Actelion, United Therapeutics, ChemoCentryx, and Bristol-Myers Squibb and consulting fees from Boehringer Ingelheim and Alexion Pharmaceuticals. Jessica K. Gordon has received grants from the Kellen Foundation at Hospital for Special Surgery, the Rudolph Rupert Scleroderma Program at Hospital for Special Surgery, and Novartis. Michael L. Whitfield is a scientific founder of Celdara Medical LLC and has filed patents and received patent royalties and fees on gene expression biomarkers for SSc but received no consulting fees or patent royalties for the work conducted in this manuscript. Robert W. Simms received grants from Actelion, Celgene, Reata Pharmaceuticals, Bayer, GenentechRoche, and InterMune and both grants and consulting fees from Cytori Therapeutics Inc. and Gilead Sciences. Robert Lafyatis has received both grants and consulting fees from Genzyme (now a fully owned subsidiary of Sanofi), Shire, Regeneron, Biogen, Bristol-Myers Squibb, Inception Sciences, Precision Dermatology, PRISM, UCB, Pfizer, and GenentechRoche. He has received consulting fees from Lycera, Novartis, Celgene, Amira, Celdara, Celltex, Dart Therapeutics, Idera, InterMune, MedImmune, Promedior, Zwitter Technology, Actelion, EMD Serono, Akros, Extera Partners, Reneo, Scholar Rock, and Human Genome Sciences. Role of funding source: The NIH reviewed and funded the grant supporting the study but otherwise played no direct role in the study conduct. Genzyme supplied the study drug and assisted with regulatory aspects, supplying the Investigational New Drug cross-reference letter and reviewing adverse events, but provided no other financial support for the study. Reference information: J Clin Invest . 2015125(7):27952807. doi:10.1172JCI77958. 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