Clinical trial NCT01379573
Conditions: Congenital Heart Block Neonatal Lupus Autoantibody-Associated Heart Block
Keywords: hydroxychloroquine Plaquenil congenital heart block neonatal lupus systemic lupus erythematosus Sjogren's syndrome autoantibodies SSA/Ro SSB/La autoimmunity pregnancy immunoglobulin
Sponsor: NYU Langone Health
Start date: 2011-01-01; Completion date: 2020-07-16
Enrollment: 74 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Women with antibodies to proteins called SSA/Ro and or SSB/La face a 2% chance of having a child with a life threatening heart condition regardless of whether they have very active lupus, are in remission, or have only vague symptoms. This heart problem is referred to as congenital heart block (the most serious being third degree complete block) and represents damage thought to be caused by these autoantibodies. The heart beats abnormally slowly and almost all children require permanent pacemakers before the age of 20. Importantly, women who have had one child with heart block have a ten-fold higher risk of having another child with the same heart condition. Unfortunately, even close monitoring by special techniques during pregnancy does not reverse complete heart block once it is observed. Thus, treatments aimed at prevention are critical. This study will evaluate for the first time whether hydroxychloroquine, a drug used by many patients with SLE, prevents the development of this heart condition. Data from laboratory experiments suggests that this drug, which crosses the placenta, may decrease the inflammation initiated by the passage of anti-Ro antibodies to the fetus. The study uses a Simon's 2-Stage design, and plans to enroll 19 patients in Stage 1 and 35 patients in Stage 2 if Stage 1 is successful. Patients can already be on hydroxychloroquine or will be started as soon as pregnancy is confirmed. The hope is that fewer than 3 cases of heart block will occur in Stage 1, and fewer than 6 cases will occur out of all 54 patients if Stage 2 is reached. The results of this study are expected to become an integral part of the counseling of women with anti-Ro/La antibodies who are considering pregnancy.

Detailed description:
One of the strongest clinical associations with autoantibodies directed to components of the Ro/La ribonucleoprotein complex is the development of congenital heart block (CHB) in an offspring, an alarming prospect facing 2% of primigravid mothers with these reactivities. The risk is 10-fold higher in women who have had a previously affected child. Despite the attempt of large multicenter studies to forestall disease by serial in utero monitoring, irreversible block and extensive myocardial injury have been documented within 7 days of a normal rhythm and PR interval. CHB is associated with a substantial mortality and morbidity. Two recent prospective studies (20 mothers from U.S. and 15 from Europe) utilizing an identical protocol of IVIG at replacement doses demonstrated 1) this intervention does not prevent the recurrence of CHB 2) the recurrence rate of 17-18% is robust 3) recruitment of patients is feasible. During the time period of the IVIG trials, basic science exploring the pathogenesis of disease supported the notion that Toll Like Receptor (TLR) signaling following ligation of ssRNA (hY3) complexed to the Ro protein contributes to fibrosis. This observation led to in vitro studies addressing inhibition of endosomal acidification by chloroquine and subsequent translation to patients by evaluating the use of hydroxychloroquine (HCQ) in an extensive retrospective chart review. The combined data suggest efficacy of HCQ. Accordingly, the goal of this study is to: To determine whether hydroxychloroquine use during pregnancy prevents CHB in a high risk population. The trial is open-label and employs the Simon's 2-stage optimal design to allow for early stopping due to absence of treatment efficacy. The first stage requires 19 subjects. Despite the rarity of disease and the requirement of a previous CHB child, based on the US Research Registry for Neonatal Lupus, this proposal is feasible. If 3 or more mothers have a child with 2nd or 3rd degree CHB, the study is terminated after the first stage. If this does not occur, funds will be sought to enroll an additional 35 mothers in the second stage for a total of 54 subjects. Treatment will be considered efficacious if fewer than 6 mothers of 54 have a child with advanced CHB. With this design, the study has 90% power to conclude that hydroxychloroquine is preventive if the true recurrence rate with the treatment is 5%. In addition, the probability of rejecting the treatment for further study is 95% if the true recurrence rate is 18%. Serial echocardiograms (monitor PR interval) and blood drawing (IFNƒÑƒnsignatures, antibody titers) will be included in the protocol. The results of this study are expected to become an integral part of the counseling of women with anti-SSA/Ro-SSB/La antibodies who are considering pregnancy.

Arms: arm 1: 400 mg/day Hydroxychloroquine
Interventions: intervention 1: Hydroxychloroquine
Intervention descriptions: intervention 1: Nineteen women meeting eligibility criteria will receive 400mg per day of HCQ beginning as soon as pregnancy is established and informed consent obtained. Mothers already on HCQ will remain on 400mg, or escalate to 400mg if on 200mg. Hydroxychloroquine is taken in 200mg pill form - 400mg = 2 200mg pills.

Protocol documents:
Prot_SAP_000.pdf: 
1
-407HUMAN SUBJECTS PROTOCOL
Protocol No.  11-00369
Title:
Preventive Approach to Congenital Heart Block with Hydroxychloroquine (PATCH)
Sponsor:
NIH 
Principal Investigator: Jill P. Buyon, MD.
New York University School of Medicine
Hospital for Joint Disease
Department of Medicine, Division of Rheumatology
301 East 17th Street, Suite 1410, 14th Floor
New York, NY
10003
Tel: 212-598-6110
Email: jill.buyon@nyumc.org
Version date: August 6, 2019
 
P.I. Signature:
__________________________________
08/6/2019
Jill P. Buyon, M.D., Professor of Medicine
Date
Purpose of the Study and Background
Purpose of the Study 
One of the strongest clinical associations with autoantibodies (Ab) directed to components of the SSA/Ro-
SSB/La ribonucleoprotein complex is the development of congenital heart block (CHB) in an offspring, 
an alarming prospect facing 2% of primigravid mothers with these reactivities [reviewed in 1].  The risk is 
10-fold higher in women who have had a previously affected child. Once 3rd degree block is identified, 
permanent reversal has never been achieved. Despite the attempts of large multicenter studies to 
forestall disease by careful monitoring, irreversible block and extensive myocardial injury have been 
documented within 7 days of a normal rhythm and PR interval.  CHB is associated with a significant 
mortality and morbidity. Current prophylactic and treatment strategies include maternal steroids, 
plasmapheresis, sympathomimetics, and in utero cardiac pacing but none have significantly altered 
mortality, justifying the need for continued research. Two recent studies [2,3] utilizing an identical protocol 
of IVIG demonstrated that 1) this intervention does not  prevent the recurrence of CHB 2) the recurrence 
rate of 17-18% is robust and reproducible 3) recruitment of patients is feasible.  During the time period of 
the IVIG trials, basic science exploring the pathogenesis of disease supported the notion that 
macrophage Toll Like Receptor (TLR) signaling following ligation of the ssRNA complexed to the Ro 
protein contributes to cardiac fibrosis [4]. TLR signaling and fibrotic endpoints could be abrogated by 
chloroquine (CQ), which inhibits endosomal acidification. This in vitro observation was “translated” to 
patients by evaluating the use of hydroxychloroquine (HCQ) in an extensive retrospective chart review 
[5]. The combined data suggested a possible efficacy of HCQ, raising the consideration of this drug as a 
highly promising candidate for the prevention of CHB.   
Specific Aim: To determine whether hydroxychloroquine use during pregnancy prevents the 
recurrence of CHB.
This will be approached in an open label Phase II trial entitled: Preventative Approach to Therapy 
for Congenital Heart Block with Hydroxychloroquine (PATCH), in pregnant women who have had a 
previous child with CHB.  This is designed as an open-label trial employing Simon’s 2-stage optimal 
NCT01379573


Human Subjects Protocol # 11-00369
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design to allow for early stopping due to absence of treatment efficacy. The first stage requires 19 
subjects, which are expected to be enrolled in the year of this award.  Despite the rarity of disease and 
the requirement of a previous child with CHB, based on the US Research Registry for Neonatal Lupus 
(RRNL) and data from the UK, this proposal is feasible.  If 3 mothers have a child with 2nd or 3rd degree 
CHB, the study is terminated after the first stage.  Serial echocardiograms (monitor PR interval) and 
evaluation of biomarkers (HCQ levels, IFNa signatures, and Ab titers) will be part of the protocol.  The 
results of this study should become an integral component of the counseling of women with anti-Ro/La 
Ab who are considering pregnancy. 
Background 
CHB associated with maternal anti-Ro Ab has a high mortality (20-30%, primarily fetal/neonatal) and 
morbidity (67% require permanent pacing before adulthood) [6,7].  In addition to conduction disease, 
10-15% of affected offspring will have a life-threatening cardiomyopathy [8,9]. Histopathologic studies 
constitute a major basis for formulating hypotheses regarding the pathogenesis of CHB, which in turn 
guide the rationale for therapeutic approaches.  Studies of several fetal hearts identified in utero with 
CHB or isolated myocarditis revealed that fibrosis is extensive and replaces the AV node, even in 20 
and 22 week hearts [10], supporting the clinical observation that 3rd degree CHB is irreversible.  The 
identification of a macrophage infiltration in fetuses dying most proximate to the time of diagnosis 
suggests that inflammation may be integral to the development of fibrosis [10].
Prenatal Considerations
From an immunologic perspective, reduction of a generalized inflammatory response has traditionally 
been considered a logical approach to the prevention or treatment of CHB. The use of maternal oral 
dexamethasone has been popularized by several groups but its scientific merit is underwhelming, and 
the risks may be substantial [11-13].  The PRIDE (PR Interval Dexamethasone Evaluation) in CHB 
study revealed no reversibility of 3rd degree CHB with dexamethasone, a higher mortality with 
dexamethasone (albeit these may have been sicker fetuses), and inconsistent effects on reversal of 
incomplete blocks [14]. 
Intravenous Immunoglobulin G (IVIG): A prospective study in which feasibility of recruitment and 
reproducibility of the recurrence rate was demonstrated but prevention of CHB was not achieved: 
The consideration of IVIG in the prevention of CHB was based on our working hypothesis of the 
pathogenesis of disease. Tissue injury in the fetus is presumed to depend on the FcRn-mediated 
transplacental passage of maternal IgG Ab [15]. Anti-Ro/La Ab, by binding to translocated antigens on 
the surface of apoptotic cardiocytes generated during remodeling of the conduction system and 
surrounding tissue, may inhibit the normal physiologic removal of these cells [16]. Uncleared opsonized 
apoptotic cardiocytes may be subsequently efferocytosed by infiltrating macrophages with release of 
pro-inflammatory and profibrosing cytokines which transdifferentiate cardiac fibroblasts to a scarring 
phenotype [17]. This scenario supported the consideration of prophylactic therapy with IVIG based on 
2 presumed mechanisms of efficacy.  The first relates to the saturation of FcRn by IVIG, which would 
be expected to decrease fetal exposure to anti-Ro/La Ab by accelerating IgG catabolism in the maternal 
circulation and by decreasing placental transport [18]. The second relates to the potential attenuation 
of anti-inflammatory responses by increasing the macrophage expression of FcRIIB [19]. This would 
represent a downstream effect in the targeted organ.
Accordingly, a multicenter, prospective, open-label study in the U.S. was initiated [2] (Preventative IVIG 
Therapy for CHB, PITCH). Enrollment criteria included the presence of anti-Ro Ab in the mother, birth 
of a previous child with CHB/neonatal lupus rash, current treatment with 20 mg/day of prednisone, and 
<12 weeks pregnant. IVIG (400 mg/kg) was given every 3 weeks from gestational week 12 - 24. The 
primary outcome was the development of 2nd or 3rd degree CHB.  Twenty mothers completed the IVIG 
protocol before the predetermined stopping rule of 3 cases of advanced CHB was reached. CHB was 
detected at 19, 20, and 25 weeks; none occurred following a prolonged PR interval. One of these 3 
mothers had 2 previous children with CHB. One child without CHB developed a transient rash consistent 


Human Subjects Protocol # 11-00369
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Version date: 08/06/2019
with neonatal lupus. Sixteen children had no manifestations of neonatal lupus at birth. No significant 
changes in maternal titers of Ab to Ro60, Ro52, or La48 were detected over the course of therapy or at 
delivery. There were no safety issues. 
In parallel with enrollment of the PITCH study, a European study was initiated in December, 
2004.  We and the P.I., Dr. Munther Khamashta, had agreed on the protocol.  The study was terminated 
after 3 recurrent cases of CHB were identified following enrollment of 15 mothers [3].  Combining data 
generated from the U.S. and the European PITCH studies, there were 6 (18%) recurrences in 33 women 
who had previous pregnancies complicated by CHB. Each study was originally designed to conclude 
inefficacy of IVIG if 6 cases of 54 were identified. However, since the chances of one further recurrence 
in the next 21 (if combined US and UK) was greater than 98% assuming the 18% recurrence rate 
observed, the trial was deemed futile and terminated.  
Hydroxychloroquine: A potential prophylactic approach
Antimalarials are among the most frequently prescribed medications in SLE patients. The 
prevention of flares and reduction of mortality are associated with the use of these medications as 
supported by a recent systematic review of high quality published evidence [20]. Serious adverse events 
such as cardiotoxicity are rare, even after prolonged use. With regard to retinal toxicity, CQ appears to 
be more toxic than HCQ [20].
          The use of antimalarials to prevent SLE flares during pregnancy has been addressed by several 
studies.  In a small, randomized double-blind trial of 10 SLE pregnant patients receiving HCQ 
(surprisingly, dose not stated) compared to 10 on placebo, the drug was administered between 8 – 18 
weeks of gestation [21].  In the active drug group, there were no flares of disease activity compared to 
3/10 in the placebo group. Importantly, neither congenital abnormalities at birth nor ophthalmologic or 
auditory abnormalities were detected up to a minimum follow up of 1.5 years.  A second study, non-
randomized, compared three groups of pregnant patients: those continuously exposed to HCQ before 
and throughout pregnancy (N = 56), those with no exposure (N = 163), and those in whom HCQ was 
discontinued prior to or during the first trimester (N = 38) [22].  The patients who discontinued HCQ 
were reported to have a higher degree of SLE activity and an increased frequency of flares.  
Ophthalmologic and auditory tests were limited to routine infant care although the authors did not report 
any clinically significant fetal abnormalities attributable to HCQ exposure. A third study showed that 
discontinuation of HCQ at the onset of pregnancy was associated with increased lupus activity [23].  
Our own data from the Prospective Predictors of pRegnancy Outcome: bioMarkers In Antiphospholipid 
Antibody Syndrome and Systemic Lupus Erythematosus (PROMISSE) study also suggested the benefit 
of HCQ.  Specifically, 105 patients with SLE were taking HCQ throughout pregnancy [24].   Overall, 
mild/moderate and severe flares were infrequent (<10%) and maintaining HCQ during pregnancy 
appeared to be of benefit in preventing flares. One child was reported to have an abnormal audiology 
screen at birth, which was considered inconsequential upon subsequent testing. 
         Despite accumulating evidence supporting the prevention of SLE flares during pregnancy, some 
physicians may remain reluctant to prescribe HCQ given isolated case reports of auditory toxicity [25] 
and retinal toxicity [26] in fetuses exposed to HCQ, in addition to data from an animal model regarding 
retinal deposition [27]. Currently, the FDA lists both CQ and HCQ as pregnancy risk category C (safety 
in human pregnancy has not been determined).  However, recent reviews of the literature suggest that, 
in particular, HCQ is safe to use during pregnancy [20,28,29]. None of the studies (inclusive of over 250 
children) found an increased frequency of congenital malformation.  In 58 children tested, maternal use 
of HCQ was not associated with either hearing or visual abnormalities [28]. Available ECGs revealed 
no differences with regard to duration of the PR or QTC intervals between unexposed or exposed 
children [28].              
The rationale for HCQ rests on translation of the pathophysiology of CHB which likely involves 
a complex cascade linking antibody to ultimate scar. Dual receptor signaling via uptake of the anti-
Ro/La immune complexes by FcγR on macrophages, and delivery of associated ssRNA to the 
endosomal compartment for ligation with Toll Like Receptors (TLR7/8) may be critical steps toward 


Human Subjects Protocol # 11-00369
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conduction damage. Since endosomal TLR binds ligand at low pH, pharmacologic approaches to 
attenuate TLR-dependent readouts have utilized CQ and bafilomyocin which interfere with acidification 
[30,31]. Our own laboratory has recently demonstrated that both macrophage transfection with non-
coding ssRNA that bind Ro60 and an immune complex generated by incubation of Ro60-ssRNA with 
an IgG fraction from a CHB mother or affinity purified anti-Ro60 significantly increased TNF secretion, 
an effect not observed using control RNAs or normal IgG. Dependence on TLR was supported by the 
significant inhibition of TNF release by IRS661 (inhibits TLR7) and CQ. Fibrosis markers were 
noticeably increased in fetal cardiac fibroblasts after incubation with supernatants generated from 
macrophages transfected with ssRNA or incubated with the immune complex, an effect abrogated by 
IRS661 or CQ [4]. These in vitro findings generate the hypothesis that HCQ, which reduces the 
acidification required for optimal ligation of TLR and has been shown to be safe in pregnancies, as 
detailed above, may prevent cardiac tissue injury associated with antibodies to these ssRNA-complexed 
proteins.
Another potential mechanism favoring efficacy in preventing CHB, is that HCQ by virtue of 
reducing endosomal acidification might also decrease transplacental transport of maternal antibodies.  
This possibility is supported by the fact that maternal IgG is pinocytosed at the apical surface of the 
syncytiotrophoblast into an acid endosomal compartment containing FcRn, and migrates via transport 
vesicles to the basolateral surface of the cell, where at physiologic pH it uncouples from the receptor 
(15).   
Study Design 
Nineteen women in Stage 1 and an additional 51 women in Stage 2 (if reached, for a total of 
70 subjects) meeting eligibility criteria will receive 400mg per day of HCQ beginning as soon as 
pregnancy is established and informed consent obtained.  Mothers already on HCQ will remain on 
400mg, or escalate to 400mg if on 200mg. The primary investigator, Jill Buyon, M.D., holds a U.S. 
Federal Drug Administration-issued IND (Investigational New Drug) number (112357) for HCQ.
A. Screening visit: Prior to the qualifying visit, the consent form will be reviewed and signed. 
Laboratory tests will include: CBC, liver-function tests (routine chemistries; albumin will be important 
to standardize the effect of physiologic hemodilution of pregnancy in gauging changes in autoantibody 
titers), urinalysis, and a 24 h collection for measurement of creatinine clearance and protein excretion 
as deemed appropriate. Serologic profiles will include: measurement of ANA, anti-DNA, C3 and C4.  
Blood will be drawn for baseline anti-Ro/La titer HCQ level, and IFNαactivity. Only the former will be 
available prior to qualification for enrollment. In total, 35 ml of blood will be drawn at screening.
B. Qualifying Visit: This is done as soon as Ab status is established, which should take several days 
(ship overnight and assay in Dr. Buyon’s laboratory is one day turnaround time; for Khamashta, it is 
equivalent). A detailed history and physical examination will be performed. The patient will either be 
given a prescription for HCQ 200mg, 2 tabs hs, and advised to see an ophthalmologist, or told to 
maintain dose if 400mg. For women who have SLE, data will be collected to calculate the Systemic 
Lupus Erythematosus Pregnancy Disease Activity Index (SLEPDAI). Rheumatologists will complete 
assessment forms, which will be reviewed by Drs. Buyon and Izmirly to address any effect of 
treatment on underlying SLE activity per se. Of note, Dr. Buyon has provided these forms for use in 
the ongoing NIH-supported PROMISSE trial.
C. Follow-up Assessments: Fetal echocardiograms will be performed after enrollment (16-18 weeks) 
and weekly thereafter until 26 weeks, then biweekly until 34 weeks. Fetal echocardiographic scans 
will be performed according to standard methods [14,33,39], recorded on VHS videotape or 
DVD/Optical Disc by the pediatric cardiologist or obstetrician, and sent to the core echocardiographic 
lab and quantified on data abstraction forms by Dr. Friedman. After the final fetal echo at 34 weeks, 
patients will continue with routine obstetric and rheumatologic care as appropriate.  Neonates will 
have an EKG and echocardiogram at birth and at 1-year follow-up (it is acknowledged that this will be 


Human Subjects Protocol # 11-00369
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out of the window of LFM award but not affect outcome since to date CHB has not occurred de novo 
after birth).  Each woman will undergo routine obstetrical ultrasounds as per the treating obstetrician.  
Targeted Level 2 ultrasounds will be required between 21-24 weeks, and between 30-32 weeks to 
assess fetal well-being. Mothers enrolled in Europe will have their echocardiograms initially read by 
their primary cardiologists with the tapes then forwarded to Dr. Friedman. 
Between 20-25 weeks and 30-35 weeks, and at delivery for all women, 20 ml of blood will be 
drawn (10 for serum, 10 for plasma and isolation of DNA). Cord blood will also be obtained.  Both 
HCQ levels and IFN-α activity will be evaluated in each sample to serve both as a proxy for 
compliance and as a potential biomarker of efficacy, respectively.  Samples will be sent overnight to 
Dr. Buyon’s lab at each visit (screening, 2nd trimester, 3rd trimester, and delivery), rather than having 
each site process the bloods, which may be unduly cumbersome for busy obstetrical practices.  For 
patients enrolled in Europe, samples will be stored in Dr. Khamashta’s lab and shipped in batch. 
Serum samples will be divided into aliquots of 500 l: 2 will have sodium azide added (final 
concentration 0.1%) and be stored at 4oC; remaining samples will be stored without azide at -70oC to 
form the serum specimen bank available to other investigators with approved research protocols (as 
in the RRNL).  Plasma will be similarly stored.  DNA will be stored for future investigations as 
spawned by the clinical results. 
Enrolled patients will continue usual care dictated by their underlying rheumatic disease. 
Outcome Measures: The primary outcome is recurrence of advanced heart block: i.e., 
echocardiogram reveals 2nd or 3rd degree AV block. First-degree block [PR >150 msec, i.e., 
>(normal mean +3 SD)] [33,39] does not constitute a primary outcome unless it progresses to more 
advanced block. In these situations the mother will be fully counseled regarding morbidity and 
mortality of 2nd and 3rd degree block, and discussion regarding data on dexamethasone will be 
provided. Follow-up will be continued by the patient’s treating physician. Secondary outcomes include: 
1) Prolonged mechanical PR interval (>150 msec) is detected but does not progress to more 
advanced AV block.  EKG at birth must confirm 1st degree AV block.  It is also possible that a fetus 
developing 1st degree block on study medication might have developed more advanced block in the 
absence of study medication. The detection of 1st degree block will prompt discussion with the 
steering committee, treating physician and patient which may lead to treatment with dexamethasone. 
The mother will be informed of the PRIDE results in which 2 women elected to take dexamethasone 
and the block reverted to normal sinus rhythm [33].  However, the patient will be informed that we do 
not know the natural progression or regression of 1st degree block, and that 1st degree block may 
spontaneously reverse.  The mother will continue in the study. 2) Any sign of myocardial injury, 
without change in cardiac rate or rhythm: a) shortening fraction <28% = 2 SD below normal mean or 
qualitatively reduced systolic function; b) cardio-thoracic ratio >0.33; c) hydropic changes; d) 
moderate/severe tricuspid regurgitation. 3) Echocardiographic densities consistent with EFE 
confirmed postnatally. 4) Fetal death not related to cardiac dysfunction. An autopsy with full evaluation 
of the heart will be encouraged but cannot be mandated.  If AV block or evidence of a cardiomyopathy 
can be “proven,” then these will provide the basis for final categorization.  If not possible, the death 
will not be considered a recurrence rate but will be reported. 5) NL rash. 6) Prematurity (gestational 
age <37 weeks at birth.  7) Birth weight <10% in the context of gestational age.  8) Abnormal fluid 
collection. 
Evaluation of Ab Levels:  This will be done exactly as described for the IVIG trial, using recombinant 
proteins Ro60, Ro52, and La48 in an ELISA [2]. 
 HCQ levels, This assay will be done in Dr. Costedoat-Chalumeau’s laboratory in France.  At each 
patient encounter, the interval between the last ingestion of HCQ and blood sampling is recorded. 
HCQ is assayed by HPLC with fluorometric detection as described [35] with minor modifications. The 
detection limit is 10ng/ml, and the between-day and within-day coefficients of variation are <8%.
IFNα activity: This assay will be done in Dr. Peggy Crow’s laboratory and will utilize the WISH cell 
assay, as we have previously reported in anti-Ro positive mothers of children with neonatal lupus [40]. 


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The reporter WISH cells are incubated with 50% patient plasma or serum for 6 hours.  Total mRNA is 
purified from WISH cell lysates and cDNA synthesized from total mRNAs. Forward and reverse 
primers are used for the genes MX1, PKR, and IFIT1, which are highly and specifically induced by 
type I IFN. The relative expression of each of the 3 tested IFN-induced genes is calculated as a fold 
increase compared to its expression in WISH cells cultured with media alone.  The ability of patient 
serum to cause IFN-induced gene expression in the reporter cells is then compared to the mean and 
SD induced by healthy donor serum. The number of SD above the mean of healthy donors for each 
gene is calculated.  Data are expressed quantitatively as the sum of the number of SD of each of the 
three genes above the mean of healthy donors.  The 3 transcripts chosen for measurement represent 
coordinate activation of the IFN-α pathway as would be expected after ligation of the type I IFN 
receptor, instead of measurement of only one IFN-α-induced transcript which may not accurately 
represent pathway activation.  Patients are considered to have high type I IFN activity if either of the 
two following criteria are met:  1) 2/3 tested IFN-induced genes are expressed >1 SD above the mean 
of healthy donors, and at least 1 is greater than or equal to 2 SD above the mean of healthy donors, 
or 2) 1 IFN-induced gene is expressed greater than 4 SD above the mean of healthy donor sera.  If 
neither criterion is met, the sample is considered to have low type I IFN activity.  Two-sided Fisher’s 
exact test (sum of small p’s method for observed ≥ expected) is used to analyze the categorical data, 
and Mann-Whitney non-parametric t-test is used to compare quantitative data. All evaluations will be 
done in batch (with the exception of screening anti-Ro/La) when the patient has completed the study.
Characteristics of the Research Population 
Number of Subjects and Subject Recruitment
Stage 1, 19 subjects will be given HCQ.  If fewer than 3 recurrences are observed, an additional 
51subjects will be treated in Stage 2 (Years 2-3, seeking a renewal and/or NIH funding). However, if 3 
or more recurrences are observed after Stage 1, it will be concluded that HCQ is not effective in 
preventing CHB.  Ultimately, HCQ will be considered efficacious for CHB if fewer than 6 CHB cases 
occur among 70 subjects evaluated on no potentially confounding medications. 
While some subjects will be prescribed the study drug (HCQ) by the PI, Dr. Buyon, or a designated 
study team member (covered by Consent Form Version 1), many patients will be referred to the study 
from other sites.  In these cases, patients’ own doctors will take responsibility for prescribing the study 
drug, and patients will consent to have their bloods and medical records sent to the investigators at 
NYU for data and sample analysis (as delineated in Consent Form Version 2).  Dr. Buyon or another 
study team member will discuss the study with each potential subject and complete the informed 
consent process prior to enrollment.  All subjects (74 in total) will be considered enrolled at NYU and 
consented using NYU IRB-approved consent forms.   
  Gender of Subjects
Because the aim of this study is to determine whether hydroxychloroquine use during pregnancy 
prevents congenital heart block, only female subjects will be recruited.  
Age of Subjects
18-45
Racial and Ethnic Origin
No subject will be excluded from participation in this study on the basis of race or ethnic origin.  
Eligibility (Inclusion) Criteria
1) Mothers must have anti-Ro and/or anti-La Ab documented in the NYU immunology laboratory 
(CLIA-approved), which utilizes an ELISA (Immunovision) as well as reactivity on ELISA to at least 
one of three recombinant antigens (48La, 52Ro, 60Ro, JB laboratory).  Dr. Khamashta will likewise 


Human Subjects Protocol # 11-00369
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verify Ab in his laboratory. 2) Mothers must have a previous child with cardiac NL, defined herein as: 
the presence of heart block (1st, 2nd, or 3rd degree) documented by electrocardiogram (EKG), 
echocardiogram, pacemaker, or statement in the medical record, and/or; presence of cardiac injury, 
which specifically includes autopsy evidence of a mononuclear infiltrate in the endocardium, 
myocardium, and pericardium and/or EFE on echocardiogram always associated with cardiac 
dysfunction.  In PITCH, we included women with a prior child with rash; however, recent data 
generated from the RRNL suggest that recurrence of CHB following rash is 11%, not 18% [34]. Thus, 
inclusion of previous rash could lead to a falsely lowered recurrence rate, and will therefore be 
excluded. 3) Intrauterine pregnancy 12 weeks. 4) Mother may be taking ≤20 mg prednisone 
because, in our experience, CHB has developed in the presence of this dose. 5) Mother may be 
asymptomatic, or have a rheumatic disease such as SLE or SS.  Maternal health status has not been 
considered an influence on the development of CHB [1].  6) Mother may or may not already be taking 
HCQ.  This latter point was discussed with Dr. Nathalie Costedoat-Chalumeau, who has published 
extensively on measurement of HCQ [35,36].  While it might be optimal for the mothers anticipating 
enrollment in the study to all have been on HCQ prior to conception, this is impractical.  Some may 
never achieve pregnancy and not want to take HCQ unless they conceive (especially those 
asymptomatic).  On the other hand, women with SLE are likely to already be on HCQ and it would 
limit enrollment to exclude these patients if all must initiate HCQ only at enrollment in the first 
trimester. Although the accepted dogma is that HCQ requires several months for maximal efficacy in 
treating rheumatic disease, it is unknown whether this would apply to transplacental passage or fetal 
levels (which are impossible to measure).   Dr. Costedoat-Chalumeau suggests that HCQ is probably 
a three compartment model which includes the circulation, tissues and cells.  In the circulation, the 
half life is approximately 7 days and in the tissues, it is 40 days [37,38]. In Dr. Costedoat-Chalumeau’s 
experience, steady state blood levels of HCQ are achieved in 4-6 weeks. Thus, dosing the mother no 
later than 10 weeks gestation should provide sufficient fetal exposure before the vulnerable period of 
CHB which is generally accepted to span 18-24 wks.  Furthermore, the placenta has to be formed for 
HCQ to gain access to the fetus and it may be effective quickly for the biology we are considering. 
Exclusion Criteria
1) Mother does not have Ab to Ro or La. 2) Identification of any of the following structural lesions 
considered causal for CHB, i.e., those that could account for block because of fibrous disruption 
between the atrium and AV node or due to absence of the penetrating bundles of the AV node: a) 
atrioventricular septal defects; b) single ventricle c) developmental tricuspid valve disease; d) L-
transposition of the great arteries; e) heterotaxia. 3) Mother is taking >20 mg prednisone/day. 
Although unlikely to be preventative, higher doses will constitute an exclusion.  The final point of 
intense discussion centered around whether another exclusion should be the use of HCQ in the first 
pregnancy in which CHB occurred.  While one could argue that in these mothers HCQ was not 
effective and perhaps will not be again, this assumption remains speculative and thus prior absence 
of efficacy of HCQ will not constitute an exclusion criteria. 
Vulnerable Subjects
This study will examine two groups of vulnerable subjects – pregnant women and their fetuses.  
Congenital heart block associated with maternal anti-Ro antibodies, the rare disease this study seeks 
to understand and ultimately prevent, affects the fetuses of mother who carry these anti-Ro 
antibodies.  It is therefore essential to include these vulnerable populations in this study, as our  
ultimate goal is to protect these mothers and their children by understanding and ultimately preventing 
this devastating disease.  
Methods and Procedures
Methods and Procedures


Human Subjects Protocol # 11-00369
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With over 400 families now enrolled in the RRNL and our success at rapid enrollment in PITCH, we 
are well positioned to enroll in the current study. Most of the women enrolled in the RRNL are of 
childbearing age, motivation is quite high, and based on review of overall data, it is likely that at least 
20% will again become pregnant and available for this study. Importantly, PRIDE and PITCH solidified 
a referral base of obstetricians and pediatric cardiologists. Furthermore, Dr. Khamashta has agreed to 
participate given that all 19 patients must be recruited in one year. The DSMB for the U.S. based 
PITCH study (Dr. Ware Branch, maternal-fetal medicine, Dr. Bonnie Bermas, rheumatologist with 
expertise in pregnancy and women’s health issues, and Dr. Joan Merrill, rheumatologist with expertise 
in design and monitoring of clinical trials in SLE) in conjunction with Drs. Jill Buyon, Peter Izmirly, 
Deborah Friedman (pediatric cardiologist), and Mimi Kim (statistician) have had extensive discussions 
regarding the results of PITCH and the design of this current study. With regard to a randomized trial 
comparing any drug with placebo, Dr. Kim prepared the following analysis:
Assumptions 1. Recurrence rate of CHB (whether previous child had CHB or rash) is approx 19%. 
2. Clinically meaningful outcome would be at least 50% reduction in recurrence rate.
Power Calculations
19% to 10% recurrence rate: 261 per group (N = 522) 
19% to 5% recurrence rate: 97 per group  (N = 194)
Data Analysis And Data Monitoring 
Statistical considerations for overall study:  
The proposed design has the following operating characteristics. Let p1 = recurrence 
rate definitely not worthy of further investigation; p2 = recurrence definitely worthy of further 
investigation. In this study, we define p1 = 18% [41] and p2 = 5%. According to Simon’s optimal 2-
stage approach [32], the expected sample size is 29.8 subjects with a probability of early termination 
of 0.69 when the recurrence rate is equal to p1. The probability of accepting the treatment for further 
study is at most 5%  if the recurrence rate is unacceptably high (>18%) with that treatment. In 
contrast, there is a 90% probability of accepting the treatment for further study if the true recurrence 
rate is 5%. .It is important to emphasize that the purpose is to obtain critical data on the efficacy of 
HCQ in the prevention of recurrent CHB and determine whether this preventive approach should be 
studied further. At study completion, the proportion of mothers having a child with advanced CHB will 
also be computed, along with corresponding 95% confidence intervals. Secondary endpoints that are 
binary (i.e., occurrence of 1st degree block, abnormal fluid collection) will be analyzed by similar 
approaches. Continuous variables (e.g., shortening fraction, gestational age) will be summarized by 
estimating means and standard deviations.  The effect of therapy on HCQ levels, IFN-α signatures, 
and Ab titers will be evaluated by fitting mixed effects linear models to repeated assessments of titers 
obtained during follow-up. In addition, change in any of these analytes at specific time points from 
baseline will be analyzed using the paired t-test or Wilcoxon signed rank test, depending on 
distribution of the data. 
Safety committee. An independent DSMB will convene by teleconference three times during the year.  
The members include Drs. Joan T. Merrill, Ware Branch, and Bonnie Bermas (all of whom served in 
PITCH). 
Data Storage and Confidentiality 
Data management. Study information will be recorded at the patients’ obstetrical and rheumatology 
sites. Standardized, computer-adapted case report forms (CRFs) have been developed.  Within 2 
weeks of each visit, completed CRFs and tapes of the echocardiograms will be sent to Dr. Friedman. 
Following review, CRFs will be faxed to the coordinator at NYU for final error checks and entered into 
the computer database. The coordinator will maintain a calendar for each patient, monitor completion 


Human Subjects Protocol # 11-00369
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of data collection forms, and mail delinquency lists to investigators and Drs. Friedman and Buyon 
monthly.  The coordinator will generate monthly reports summarizing accrual by institution and 
country; demographic and clinical characteristics; adherence to treatment and follow-up; and 
unexpected or severe complications of treatment.  Raw data will be transferred to Dr. Kim for analysis.  
Separate CRFs for demographics, SLEPDAI (as appropriate), medications, physical exam, laboratory 
data, and adverse events will be sent within 2 weeks to the coordinator who will review each form with 
Drs. Buyon and Izmirly.  Serious adverse events will be reported to the local IRB and the DSMB within 
24 h. A database structure and computer data entry interface has been developed using database 
management software. Data entry procedures will include quality control features such as duplicate 
record entry and comparison for verification, logic checks and range checks, computer logging of 
record entry and editing, and automated back-up. The computer is protected with a password at the 
BIOS level.  Data on patients enrolled in Europe will be forwarded to the coordinating center at NYU.  
Blood collected as part of this study will be stored for future research
SPECIMEN BANKING
1. The samples will be labeled with anonymous and unique codes generated by the FreezerWorks 
database.  
2. In the FreezerWorks database, only authorized users with password will be able to access PHI.  
Paper charts will be kept in a locked room in MSB room 611.   
3. Samples will be stored for future use for an indefinite time period.  
4. The samples will be stored in MSB room 606 and Dr. Buyon will have access to them. 
5. The type of research that will be done on stored samples at this time is not known at this time.  
6. Genetic testing will not be done on these samples.  
7. If a subject requests withdrawal of sample(s) the samples will be appropriately discarded and 
confirmation of this will be provided to the subject.  
Risk/Benefit Assessment
Risk
The potential physical risks and discomforts to you which may relate to the treatment with 
hydroxychloroquine are discussed below.   
Hydroxychloroquine is part of a group of medications initially used to treat malaria, but usage has 
expanded to rheumatologic conditions such as systemic lupus and rheumatoid arthritis.  Even though 
the U.S. Food and Drug Administration (FDA) lists hydroxychloroquine as a medication that has not 
been determined to be safe in pregnancy, extensive review of the medical literature suggests that this 
drug is safe during pregnancy.  Results from animal studies showed that hydroxychloroquine could 
have visual side effects on the baby and there are also isolated case reports of hearing and visual 
problems in babies whose mothers received this drug during pregnancy. However, none of the clinical 
studies (inclusive of over 300 children) using hydroxychloroquine during pregnancy have found a 
higher frequency of hearing problems, visual problems or any other malformation in the babies 
exposed to the medication during pregnancy than in pregnancies in which the baby’s mother did not 
take hydroxychloroquine. The potential side effects for the mothers are rare and consist of temporary 
gastrointestinal discomfort, discoloration of the skin and visual problems.  A patient taking this drug 
should be seen by an eye doctor once per year and therefore if you have never been on this drug and 
are taking it only for this study, you should see an eye doctor. Two pills each day is the dose of 
hydroxychloroquine used for this study and both pills can be taken together.   


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With regard to having blood drawn, minor bruising at the site of blood drawing is common.  Less 
common are larger bruises, inflammation of the vein where the blood was drawn, infection, and blood 
clots.  Rarely, people faint from having blood drawn.  We will draw your blood while you are sitting or 
lying on your back and ask you to remain in that position for several minutes to further minimize the 
risk of fainting.  The risk of removing the amount of blood required for the study is minor.  The 
additional amount of blood drawn only for this study is not large (1 tablespoon at each visit) and does 
not pose an additional risk to you or your unborn child.
No independently confirmed adverse effects on the fetus resulting from prenatal diagnostic ultrasound 
exposure have been reported.  Acoustic output (the amount of sound applied to the mother's 
abdomen to produce the ultrasound image of the fetal heart) will be kept below acceptable limits. 
Protection Against Risks
Safety committee. An independent DSMB will convene by teleconference three times during the year.  
The members include Drs. Joan T. Merrill, Ware Branch, and Bonnie Bermas (all of whom served in 
PITCH). 
Individual stopping rules: Fetal: 1) Development of advanced block (2nd or 3rd degree); 1st degree 
block will not constitute a formal stopping point. 2) Decrease of amniotic fluid index <5 cm. 3) 
Intrauterine growth restriction (IUGR) - no fetal growth over 2 weeks. 4) Hydrops or myocardial 
dysfunction or EFE. 5) Death. Maternal: 1) Serious reaction to study drug, e.g. severe rash or 
intractable G.I. intolerance. 2) Withdrawal of consent. If isolated 1st degree heart block occurs, 
dexamethasone may be offered, and the patient may continue in the study. If a patient achieves an 
individual stopping point due to CHB and hydropic changes, she will be offered dexamethasone and 
the outcome of the pregnancy will be prospectively followed. 
Study stopping rules: Enrollment is stopped if 3 fetuses develop 2nd or 3rd degree block.  
Potential Benefits to the Subjects
We hope that this study shows that treatment with hydroxychloroquine prevents the development of 
heart block in your baby. 
The potential benefits are substantial in relation to the small risks posed by participation. 
This study is important because there is information from basic science research suggesting that 
hydroxychloroquine may be effective in preventing the inflammation and fibrosis which may result in 
heart block.  This is only testing done with test tubes, not animals or humans.  There is also a large 
study that reviewed the charts of many women with anti-Ro antibodies and this study found that 
hydroxychloroquine may be beneficial. However, we also are not sure whether for those mothers who 
received hydroxychloroquine, they may have had normal babies even without taking the drug.  The 
results of this study will be used not only for a potential benefit to your own baby but also as a guide to 
help other pregnancies in which the baby is at risk for heart disease.  
Subject Identification, Recruitment and Consent/Assent 
Method of Subject Identification and Recruitment
With over 400 families now enrolled in the RRNL and our success at rapid enrollment in PITCH, we 
are well positioned to enroll in the current study. Most of the women enrolled in the RRNL are of 
childbearing age, motivation is quite high, and based on review of overall data, it is likely that at least 
20% will again become pregnant and available for this study. Importantly, PRIDE and PITCH solidified 
a referral base of obstetricians and pediatric cardiologists. Furthermore, Dr. Khamashta has agreed to 
participate given that all 19 patients must be recruited in one year.


Human Subjects Protocol # 11-00369
PI: BUYON, Jill P., MD
Version date: 08/06/2019
Innovative Plans for recruitment: 1.) Recruiter for patients: Sterling Larnerd is a mother enrolled in the 
RRNL and has agreed to participate in this project. She has two children with CHB and moderates 
Heart Block Kids (http://health.groups.yahoo.com/group/heartblockkids/), a support group for parents 
of children affected by neonatal lupus. The group will organize forums and educational panels to 
address CHB mothers’ inquiries regarding planning pregnancy and participating in preventive trials 
and existing therapies. This approach allows us to create a link with an established group of mothers 
actively seeking counsel who are motivated to participate. 2.) Conference Call for Patients: This will 
take place every 3 months between Drs. Buyon, Izmirly, and Friedman, and patients who would like 
information about participation in the study. 
Process of Consent 
Each subject meeting the above-described eligibility requirements (or the authorized representative of 
the subject) will have the opportunity to review a consent form specific to this study and approved by 
New York University School of Medicine’s Institutional Review Board.  Either the P.I. of this study, Jill 
Buyon, M.D., or another individual authorized to obtain consent will review the consent form with each 
subject and will answer any questions the subject may have.  Subjects will give their consent in writing 
by signing their name in the appropriate sections of the consent form, and this will only be done once 
the P.I. and/or other study personnel believes that the subject truly understands the significance of 
what they are signing.  
Subject Capacity 
All subjects will have the capacity to give informed consent to participate in this study.  
Subject/Representative Comprehension 
The initials and/or signatures at the appropriate sections of the consent form will provide the indication 
that the subject or subject’s authorized representative has understood the information presented.  The 
opportunity that will be provided each subject to review the consent form with authorized study 
personnel and to address any questions or concerns she may have will provide further assurance that 
the signatures and initials on the consent form indicate true “consent.”  
Debriefing Procedures 
Not applicable.  
Consent Forms 
A consent form has been submitted.  The P.I. is responsible for ensuring that valid consent is 
obtained and documented for all subjects.  
Costs to the Subject
It will not cost subjects anything to participate in this study.  Subjects (or their insurance company) will 
be charged or held responsible for the costs of their routine care (the care they would have received if 
they were not in this study).
Any study-related costs associated with subjects being in this study that are not part of their routine 
care will be paid by study funds.   
This study is being sponsored by a grant.  Portions of Dr. Buyon’s and her research team’s salaries 
are being paid by this grant. 


Human Subjects Protocol # 11-00369
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Payment for Participation 
You will not receive any reimbursement for participating in this study.