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J O U R N A L O F T H E A M E R I C A N C O L L E G E O F C A R D I O L O G Y V O L . 7 2 , N O . 1 7 , 2 0 1 8

ª 2 0 1 8 B Y T H E A M E R I C A N C O L L E G E O F C A R D I O L O G Y F O U N D A T I O N

P U B L I S H E D B Y E L S E V I E R

Tenosynovial and Cardiac Amyloidosisin Patients UndergoingCarpal Tunnel Release

Brett W. Sperry, MD,a,b Bryan A. Reyes, MD,c Asad Ikram, MBBS,a Joseph P. Donnelly, MD,a

Dermot Phelan, MD, PHD,a Wael A. Jaber, MD,a David Shapiro, MD,c Peter J. Evans, MD, PHD,c Steven Maschke, MD,c

Scott E. Kilpatrick, MD,d Carmela D. Tan, MD,d E. Rene Rodriguez, MD,d Cecilia Monteiro, MD,e

W.H. Wilson Tang, MD,a Jeffery W. Kelly, PHD,e William H. Seitz, JR, MD,c Mazen Hanna, MDa

ABSTRACT

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BACKGROUND Patients with cardiac amyloidosis often have carpal tunnel syndrome that precedes cardiac manifes-

tations by several years. However, the prevalence of cardiac involvement at the time of carpal tunnel surgery has not

been established.

OBJECTIVES The authors sought to identify the prevalence and type of amyloid deposits in patients undergoing carpal

tunnel surgery and evaluate for cardiac involvement. The authors also sought to determine if patients with soft tissue

transthyretin (TTR) amyloid had abnormal TTR tetramer kinetic stability.

METHODS This was a prospective, cross-sectional, multidisciplinary study of consecutive men age $50 years and

women $60 years undergoing carpal tunnel release surgery. Biopsy specimens of tenosynovial tissue were obtained and

stained with Congo red; those with confirmed amyloid deposits were typed with mass spectrometry and further evalu-

ated for cardiac involvement with biomarkers, electrocardiography, echocardiography with longitudinal strain, and

technetium pyrophosphate scintigraphy. Additionally, serum TTR concentration and tetramer kinetic stability were

examined.

RESULTS Of 98 patients enrolled (median age 68 years, 51% male), 10 (10.2%) had a positive biopsy for amyloid

(7 ATTR, 2 light chain [AL], 1 untyped). Two patients were diagnosed with hereditary ATTR (Leu58His and Ala81Thr),

2 were found to have cardiac involvement (1 AL, 1 ATTR wild-type), and 3 were initiated on therapy. In those patients who

had biopsy-diagnosed ATTR, there was no difference in plasma TTR concentration or tetramer kinetic stability.

CONCLUSIONS In a cohort of patients undergoing carpal tunnel release surgery, Congo red staining of

tenosynovial tissue detected amyloid deposits in 10.2% of patients. Concomitant cardiac evaluation identified

patients with involvement of the myocardium, allowing for implementation of disease-modifying therapy.

(Carpal Tunnel Syndrome and Amyloid Cardiomyopathy; NCT02792790) (J Am Coll Cardiol 2018;72:2040–50)

© 2018 by the American College of Cardiology Foundation.

N 0735-1097/$36.00 https://doi.org/10.1016/j.jacc.2018.07.092

m the aDepartment of Cardiovascular Medicine, Cleveland Clinic Foundation, Cleveland, Ohio; bMid America Heart Institute,

int Luke’s Hospital, Kansas City, Missouri; cDepartment of Orthopaedic Surgery, Cleveland Clinic Foundation, Cleveland, Ohio;

epartment of Pathology, Cleveland Clinic Foundation, Cleveland, Ohio; and the eDepartments of Chemistry and Molecular

dicine, The Scripps Research Institute, La Jolla, California. This study was funded by Dr. Hanna’s Term Chair in Amyloid Heart

ease: Case Western Reserve University/Cleveland Clinic CTSA Grant Number UL1TR000439 from the National Center for

vancing Translational Sciences (NCATS), a component of the National Institutes of Health (NIH). Its contents are solely the

ponsibility of the authors and do not necessarily represent the official view of NCATS or NIH. Dr. Sperry has received consulting

s from GlaxoSmithKline. Dr. Monteiro has been supported by an American Heart Association Predoctoral Grant

PRE31130009). Dr. Tang has been supported by grants from the NIH (R01HL103931) and Collins Family Fund; and has served as

onsultant for the Advisory Board Company. Dr. Kelly has been supported by NIH NIDDK 046335; is a shareholder of FoldRx/

zer equity; and receives tafamidis royalties from Pfizer. Dr. Seitz Jr. has served as a consultant for Stryker, Zimmer/Biomet, and

pp Surgical; and has served as an orthopedic surgery consultant. Dr. Hanna has received funding from the NCATS/NIH

L1TR000439); and has received consulting fees from Pfizer, Ionis, and Eidos pharmaceuticals. All other authors have reported

t they have no relationships relevant to the contents of this paper to disclose.

nuscript received May 10, 2018; revised manuscript received July 23, 2018, accepted July 26, 2018.

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AB BR E V I A T I O N S

AND ACRONYM S

AL = immunoglobulin light

chain amyloidosis

ATTR = transthyretin

amyloidosis

ECG = electrocardiography

NT-proBNP = N-terminal

pro–B-type natriuretic peptide

TcPYP = technetium

A myloidoses are a group of protein misfoldingdisorders in which 1 of 35 distinct proteinspathologically misfolds and aggregates extra-

cellularly as insoluble amyloid fibrils, ultimately lead-ing to organ dysfunction. Each precursor proteindefines the amyloid subtype, which determines theorgans involved. Immunoglobulin light chain (AL)and transthyretin (ATTR) are the 2 primary proteintypes which deposit in the myocardium, ultimatelyleading to symptomatic heart failure and death (1).

SEE PAGE 2051

pyrophosphate

= transthyretin

Transthyretin (TTR), also called prealbumin, ispredominantly a liver-secreted protein that circulatesas a tetramer and is a known carrier for thyroxine andholo-retinol binding protein. In both the wild-typesporadic form (formerly called senile systemicamyloidosis) and in the mutant hereditary form(termed familial amyloid cardiomyopathy), the dis-ease is thought to be linked to tetramer dissociationinto folded monomers. This is considered the ratelimiting step of the ATTR amyloidogenesis cascade.TTR monomers then partially misfold, leading to theformation of non-native oligomers and amyloid fibrils(2). A subunit exchange assay has been developed toassess the kinetic stability of the TTR tetramer inhuman plasma (3).

In addition to deposition in the myocardium, asignificant proportion of patients with ATTR havedeposition in soft tissue structures leading to spinalstenosis, biceps tendon rupture, and carpal tunnelsyndrome. Carpal tunnel syndrome is frequentlybilateral and typically manifests 5 to 10 years prior tocardiac diagnosis, often requiring carpal tunnelrelease surgery. Carpal tunnel syndrome in AL is lesscommon (4), but similarly tends to precede overtcardiac disease. Amyloid deposits have been found inthe flexor tenosynovium and transverse carpal liga-ment of the hand (5–9), raising the potential for earlydiagnosis prior to the development of cardiac symp-toms (Central Illustration). However, the prevalenceof cardiac involvement at the time of carpal tunnelsurgery has not been established.

We sought to determine the prevalence and type ofamyloid deposits in the tenosynovium of patientsundergoing carpal tunnel release surgery and toinvestigate whether simultaneous cardiac involve-ment could be identified using biomarkers andadvanced cardiac imaging. Additionally, we exam-ined measured native TTR plasma concentration andtetramer kinetic stability using a subunit exchangeassay with the hypothesis that those diagnosed withATTR amyloidosis would have less stable tetramers.

METHODS

STUDY DESIGN. This was a prospective,cross-sectional study in which consecutivepatients undergoing carpal tunnel release byparticipating surgeons were screened for in-clusion between May 2016 and May 2017.Four orthopedic hand surgeons participatedin the study operating out of 6 surgical facil-ities in the Cleveland Clinic health system.Men age $50 years and women $60 yearswere included. Patients were excluded if they

had a known diagnosis of amyloidosis or had carpaltunnel syndrome thought to be secondary to traumaor rheumatoid arthritis. The Institutional ReviewBoard of the Cleveland Clinic approved this study.The research protocol was discussed with patients inperson by the surgical team or via telephone by thestudy staff prior to the surgical date. Patients whoagreed to participate were enrolled, and all patientsprovided written informed consent prior to surgery.

SAMPLE COLLECTION, SURGICAL PROCEDURE, AND

CARDIAC EVALUATION. Enrolled patients had ablood sample collected on the day of surgery. Opencarpal tunnel release was performed in all patients.After transection of the transverse carpal ligament,the median nerve and flexor tendons were protected,and a small sample of tenosynovium was excised. Thetenosynovium was then formalin fixed, processedroutinely, and subsequently evaluated by hematox-ylin and eosin and Congo red staining by ClevelandClinic pathologists. Biopsy specimens with confirmedamyloid deposits via Congo red staining were furtheranalyzed using mass spectrometry for subtyping. Ifthere was insufficient tissue for mass spectrometry,immunohistochemistry was attempted for subtyping.Biopsy was not performed in cases with minimalvisible tenosynovium or when disruption of adjacentneural or vascular architecture posed an increasedrisk to the patient.

Patients with evidence of amyloid deposition intenosynovial tissue were further evaluated for cardiacinvolvement with a comprehensive physical exami-nation, electrocardiography (ECG), N-terminal pro–B-type natriuretic peptide (NT-proBNP), troponin T,echocardiography with longitudinal strain imaging,and technetium pyrophosphate (TcPYP) nuclearscintigraphy. ECGs were reviewed for each patientto identify characteristics suggestive of cardiacamyloidosis including different degrees of heartblock, low limb voltage (<5 mm in all limb leads), lowSokolow voltage, and presence of a pseudoinfarctpattern (10).

TTR

CENTRAL ILLUSTRATION Mechanism of Amyloid Deposition

Sperry, B.W. et al. J Am Coll Cardiol. 2018;72(17):2040–50.

Mechanism of amyloid deposition. Two precursor proteins are responsible for >95% of cardiac amyloidosis cases—immunoglobulin light

chain (AL) and transthyretin (ATTR). In AL amyloidosis, aberrant monoclonal plasma cells in the bone marrow secrete light chains that

misfold and aggregate, forming insoluble amyloid fibrils. In ATTR amyloidosis, the liver-derived protein transthyretin, which is normally a

tetramer, inappropriately dissociates into monomers, which misfold and aggregate to form insoluble amyloid fibrils. Both AL and ATTR

amyloid fibrils deposit extracellularly over time (AL more rapidly than ATTR), with bilateral carpal tunnel syndrome often presenting years

before the onset of cardiac symptoms.

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J A C C V O L . 7 2 , N O . 1 7 , 2 0 1 8 Sperry et al.O C T O B E R 2 3 , 2 0 1 8 : 2 0 4 0 – 5 0 Amyloidosis Screening in the Carpal Tunnel

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Echocardiography was performed using Vivid 7 or 9ultrasound systems (GE Medical, Milwaukee, Wis-consin), and parameters including longitudinal strainwere analyzed as previously described using EchoPACsoftware version 113 (Advanced Analysis Technolo-gies, GE Medical Systems) (11,12). 99m-TcPYPscintigraphy was performed after patients received20 mCi � 10% of TcPYP intravenously, and imageswere obtained after a 1-h and 3-h uptake delay aspreviously described (13,14). Single-photon emissioncomputer tomography and planar images were ac-quired with Siemens Symbia T6 cameras (SiemensMedical Solutions, Flanders, New Jersey), and imageswere analyzed using 4DM software (INVIA, AnnArbor, Michigan) by 2 experienced readers (B.W.S.and W.A.J.) blinded to clinical and echocardiographicdata. A semi-quantitative score as well as heart-to-contralateral lung ratio were calculated based uponplanar images (13).

MEASUREMENT OF NATIVE TTR IN PLASMA. NativeTTR tetramer levels in plasma were quantified using aWaters Acquity H-Class Bio-UPLC (Ultra-PerformanceLiquid Chromatography) instrument using a WatersProtein-Pak Hi Res Q ion exchange column (stronganion exchanger, 5-mm particle size, 4.6 � 100-mmcolumn). A standard curve was prepared using re-combinant wild-type TTR (15) at concentrations of 10mmol/l, 5 mmol/l, 2.5 mmol/l, and 1 mmol/l in standardphosphate buffer. The freshly prepared standardcurve samples and patient samples (9 ml) were incu-bated with 1 ml of the fluorogenic small molecule A2(500 mmol/l) (16) for 3 h at room temperature. Afterincubation, the samples were injected into the ionexchange column and separated using a linear 24% to29% buffer B gradient over 10 min (flow 0.5 ml/min,buffer A: 25 mmol/l Tris pH 8; buffer B: same as bufferA, but with 1 mol/l NaCl added). The TTR–(A2)2 fluo-rescent conjugate peak (excitation 328 nm emission430 nm; elution time ¼ 6 min) was integrated, and theconcentration of TTR in patient samples was quanti-fied using the standard curve.

PLASMA-TTR SUBUNIT EXCHANGE ASSAY TO

ASSESS KINETIC STABILITY. Subunit exchange rateswere determined as described previously (3) with thefollowing minor modifications. FT2-WT-TTR (2 ml of a40-mmol/l solution) was added to the plasma samples(38 ml) to afford a final FT2-WT-TTR concentration of2 mmol/l. The samples were incubated at 25�C for 48 hto allow subunit exchange to occur. At 48 h, the re-action was stopped by the addition of the fluorogenicsmall molecule A2 at a final concentration of500 mmol/l. The samples were incubated with A2 forat least 3 h to allow complete covalent labeling of

TTR, before being injected into the ion exchangecolumn and separated using the same gradient asdescribed previously (3,17). The rate of exchange forall subunit exchange experiments was calculated us-ing peak 1, as previously described (3).

STATISTICAL ANALYSIS. Continuous variables areexpressed as median (interquartile range [IQR]) andanalyzed by the Wilcoxon rank-sum test. Categoricalvariables are expressed as n (%) and compared by theFisher exact test. All analyses used double-sidedp values, and <0.05 was considered statistically sig-nificant. Statistical analyses were performed usingStata 13.1 software (StataCorp LP, College Station,Texas) and GraphPad Prism 7 (GraphPad Software, LaJolla, California).

RESULTS

A total of 319 consecutive patients underwent carpaltunnel release between May 2016 and May 2017 byparticipating orthopedic surgeons. An overview ofstudy enrollment is seen in Figure 1. Of 200 eligiblepatients, 98 were enrolled and biopsied. There wereno procedural complications. Demographics, medicalhistory, and results of laboratory testing are seen inTable 1 for the enrolled cohort. The mean age was 68years, and 85% of patients had bilateral symptoms.Ten patients (10.2%; 95% confidence interval: 5% to18%) were found to have amyloid in the tenosynovialbiopsy after Congo red staining. Mass spectrometrywas used to diagnose 7 patients with ATTR and 2patients with AL.

Patients with amyloid-positive biopsies were morelikely to have prior carpal tunnel release surgery. All10 patients with amyloid-positive biopsies had eithersymptoms of bilateral carpal tunnel syndrome or aprevious contralateral carpal tunnel release. A total of6 of 10 patients had a history of intervention fortrigger finger, and 6 had a history of lumbar spinalstenosis (4 ATTR, 2 AL), 3 of whom required surgery.A history of biceps tendon rupture was seen in 2 pa-tients (both ATTR: 1 wild-type and 1 hereditary). Amonoclonal protein was detected on serum immu-nofixation in 4 patients with biopsy-confirmed ATTR.

Tables 2 and 3 detail the cases of amyloid found inthe carpal tunnel and the subsequent cardiac evalu-ation. Of the 10 patients with amyloid, 2 were diag-nosed with cardiac involvement. Of note, no patientsmet criteria for an apical sparing pattern on echo-cardiography (18).

Patient #4 had AL amyloidosis of the lambda sub-type with evidence of cardiac involvement based onechocardiography showing increased septal wall

FIGURE 1 Study Cohort

Assessed for eligibility(n = 319)

Patients eligible (n = 200)

Excluded at screening (n = 119)• Age (n = 101)• Prior amyloidosis (n = 1)• CTS due to trauma or RA (n = 17)

Excluded during consent (n = 90)• Declined to participate (n = 51)• Logistic issues (n = 17)• Unreachable (n = 22)

Excluded during surgery (n = 12)• Anatomy not amenable to biopsy (n = 12)

Consented (n = 110)

Enrolled and biopsied (n = 98)

Overview of the study cohort. CTS ¼ carpal tunnel syndrome; RA ¼ rheumatoid arthritis.

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thickness to 1.3 cm, an elevated NT-proBNP andtroponin T, and a physical examination consistentwith heart failure. She was treated with dexametha-sone, cyclophosphamide, and bortezomib. After5 months of treatment, she had achieved a completehematologic response with normalized lambda lightchains. NT-proBNP decreased from 1,615 to 161 pg/ml,and the patient went from New York Heart Associa-tion functional class III to I symptoms. Patient #3 hadno M protein and only a mildly elevated kappa level,but had kappa light chain deposits in the carpal tun-nel. Patient #5 had ATTR amyloidosis with no muta-tion on TTR genetic testing consistent with thediagnosis of wild-type disease. Although he did nothave signs or symptoms of heart failure and the ECGwas normal, echocardiography did reveal increasedseptal wall thickness (1.3 cm) with borderlineabnormal global longitudinal strain. Evidence ofATTR cardiac amyloidosis was confirmed by TcPYPnuclear scintigraphy showing grade 3 diffusemyocardial uptake (Figure 2). He was placed ondiflunisal, a TTR stabilizer that slows ATTR poly-neuropathy progression based on a randomizedplacebo-controlled trial (19). Patient #9 had TTR am-yloid deposits in the tenosynovium and evidence ofheart failure on examination, but TcPYP scintigraphywas not consistent with cardiac amyloidosis of theATTR type. It was felt that he most likely had diastolic

heart failure from hypertension and obesity and wastreated with a loop diuretic. Patient #8 was not able tobe subtyped due to an insufficient tissue sample tocomplete mass spectrometry or immunohistochem-istry. The patient had no M protein on immunofix-ation of the serum and urine nor on serum proteinelectrophoresis. Despite initially having a mildlyabnormal free light chain ratio, subsequent assaysshowed a normal ratio. A hematology consult wasobtained and it was agreed the overall findings werenot consistent with AL amyloidosis, and she waspresumed to have ATTR.

Two patients were found to harbor known muta-tions in the TTR gene. Patient #1 was diagnosed withhereditary ATTR based on TTR genetic testing, whichrevealed a heterozygous Ala81Thr mutation that isknown to cause late-onset cardiac amyloid. She hadno evidence of heart involvement based on normalbiomarkers, ECG, echocardiogram (septum 0.9 cm),and no cardiac uptake on TcPYP scan. Patient #6 wasdiagnosed with hereditary ATTR due to a Leu58Hismutation, which has been described as familial amy-loid polyneuropathy type 2 of Maryland/German type(20). Upon further questioning, his family originatedin Maryland (of German descent), and several rela-tives had a history of progressive neuropathy.Although he had no cardiac involvement, the patientdid complain of mild neuropathic symptoms in hislower extremities. He was referred to a neurologistwho confirmed the diagnosis of a small fiber axonalsensory neuropathy. Based upon these findings aswell as his known pathogenic TTR mutation, he wasplaced on diflunisal to slow neuropathy progression.Both hereditary ATTR patients and their familieswere offered genetic counseling.

Native TTR plasma concentrations and kineticstability were evaluated, with results depicted inFigure 3. Median TTR concentration was 20.33 mg/dl(IQR: 15.84 to 26.68 mg/dl) in patients with ATTRand 20.46 mg/dl (IQR: 16.26 to 24.52 mg/dl) inpatients without ATTR (p ¼ 0.757), both withinnormal range (Figure 3A). Of the 7 patients with TTR-derived amyloid, 6 had a less stable TTR relative tothe median stability of the whole group, althoughno statistical differences were found between pa-tients with or without ATTR (�4.768 per h [IQR:�4.828 to �4.566 per h] vs. �4.934 per h [IQR: �5.203to �4.711 per h]; p ¼ 0.117) (Figure 3B).

DISCUSSION

In this study, we found that 10.2% of men age $50years and women $60 years undergoing carpal tunnelrelease surgery for idiopathic carpal tunnel syndrome

TABLE 1 Baseline Characteristics

Total Cohort(n ¼ 98)

No Amyloid(n ¼ 88)

Amyloid(n ¼ 10) p Value

Clinical values

Age, yrs 68 (61–74) 68 (60–74) 72.5 (65–80) 0.20

Male 51 (52) 45 (51) 6 (60) 0.74

White 92 (94) 83 (94) 9 (90) 0.49

Hypertension 69 (70) 61 (69) 8 (80) 0.72

Hyperlipidemia 80 (82) 72 (82) 8 (80) 1.00

Coronary artery disease 18 (18) 17 (19) 1 (10) 0.68

Atrial fibrillation 12 (12) 10 (11) 2 (20) 0.35

Diabetes mellitus 27 (28) 23 (26) 4 (40) 0.46

Smoking 57 (58) 51 (58) 6 (60) 1.00

BMI, kg/m2 30.5 (26.0–33.8) 30.7 (26.0–33.9) 28.8 (26.8–30.9) 0.48

Bilateral symptoms 83 (85) 73 (83) 10 (100) 0.35

Prior carpal tunnelrelease surgery

36 (37) 29 (33) 7 (70) 0.035

Systolic bloodpressure, mm Hg

139 (130–155) 140 (129–156) 137 (134–151) 0.80

Diastolic bloodpressure, mm Hg

74 (66–84) 75 (66–85) 74 (67–78) 0.45

Heart rate, beats/min 71 (64–78) 70 (68–78) 72 (60–82) 0.82

Laboratory values

Sodium, mmol/l 140 (139–142) 141 (139–142) 139 (133–141) 0.030

Potassium, mmol/l 4.3 (3.9–4.6) 4.3 (3.9–4.6) 4.25 (4.1–4.5) 0.93

Creatinine, mg/dl 0.94 (0.74–1.06) 0.93 (0.74–1.07) 0.98 (0.73–1.03) 0.67

eGFR, ml/min/1.73 m2 81 (65–90) 81 (65–89) 80 (60–90) 0.96

Total protein, g/dl 7 (6.7–7.4) 7.1 (6.8–7.4) 7 (6.4–7.4) 0.44

Albumin, g/dl 4.3 (4.0–4.4) 4.3 (4.1–4.4) 4.3 (3.9–4.4) 0.66

NT-proBNP, pg/ml 97 (44–212) 94 (39.5–232) 127.5 (63–212) 0.39

Troponin T >0.01 ng/ml 1 (1) 1 (1) 0 (0) 1.00

Values are median (interquartile range) or n (%). In addition, there were no significant differences in white bloodcell count, hemoglobin, platelets, potassium, BUN, total protein, total bilirubin, alkaline phosphatase, and cal-cium between patients with and without amyloid in the carpal tunnel.

BMI ¼ body mass index; BUN ¼ blood urea nitrogen; eGFR ¼ estimated glomerular filtration rate by theModification of Diet in Renal Disease Study equation; NT-proBNP ¼ N-terminal pro-brain natriuretic peptide.

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had amyloid identified on tenosynovial tissue biopsy.Two patients (20% of those with amyloid) hadconcomitant but previously unknown cardiacamyloidosis diagnosed by a comprehensive exami-nation and advanced cardiac imaging, leading tointervention with medical treatment. A third patienthad a mutation in the TTR gene, which leads to aprogressive polyneuropathy and was also treatedwith disease-modifying therapy. Plasma TTR con-centration and TTR kinetic stability were notdifferent between patients with amyloid-positive bi-opsies. Our findings are novel, as we describe theprevalence of amyloid deposition found prospec-tively in the tenosynovial tissue of patients under-going carpal tunnel release in the modern era with thereference standard diagnostic modality (mass spec-trometry). Additionally, concomitant cardiac evalua-tion at the time of soft tissue amyloid diagnosis hasnot previously been described. These findings pointto the importance of recognizing idiopathic carpaltunnel syndrome as a possible predictor for amyloidheart disease, and the opportunity for incorporatingtenosynovial tissue biopsy for early detection anddiagnosis of amyloidosis in the perioperativeworkflow.

Amyloid cardiomyopathy is an under-recognizedetiology of heart failure with preserved ejectionfraction. According to an international survey,amyloidosis was initially misdiagnosed in 57% of pa-tients with hereditary ATTR, 39% of patients withwild-type ATTR, and 43.8% of patients with AL. Timebetween onset of symptoms and diagnosis was>6 months in 72% of patients with AL. Notably, 44%of patients with ATTR and 81% of patients with ALvisited 3 or more different physicians before receivinga correct diagnosis (21,22). Indeed, our patient #4 hadknown monoclonal gammopathy of undeterminedsignificance and clinically appeared to have signs andsymptoms of heart failure, which were previouslyunevaluated and undiagnosed. It is estimated that upto one-fourth of patients over 85 years of age havewild-type ATTR deposits (23), and 13% of thoseadmitted with heart failure with preserved ejectionfraction and LV wall thickness >1.2 cm on echocar-diography have ATTR (24). In addition, ATTR is oftenfound in patients with aortic stenosis (25), with aprevalence of 6% to 8% in all-comers (26,27) and 16%in those evaluated for transcatheter aortic valvereplacement (28). As the population ages and detec-tion mechanisms improve, cardiac amyloidosis can nolonger be considered a rare entity.

In addition to deposition in the myocardium, a sig-nificant proportion of patients with amyloidosisdevelop fibril deposition in the flexor tenosynovium

and transverse carpal ligament in the hand as well as inother areas, such as the rotator cuff, biceps tendon,and ligamentum flavum in the spine. Prior studiesshowed that soft tissue removed at the time of carpaltunnel surgery stained positively with Congo red in12% to 35% of patients with idiopathic carpal tunnelsyndrome (6,8,9). In these studies, only the ATTR typewas noted, and genetic mutations in the TTR genewere exceedingly rare (7-9,29). These studies did notuse mass spectrometry for confirmation and typing,raising the concern for false positive interpretation ofCongo red staining in some cases. Two older studiesdescribed non-ATTR type amyloidosis in the carpaltunnel, with up to 16% of amyloid deposits being dueto AL (5,30). The remainder were predominantlythought to have localized amyloidosis; however, thesestudies enrolled patients prior to the development ofcurrent diagnostic techniques for ATTR. In our cohort,2 of the 10 patients with biopsy-diagnosed amyloidosiswere found to have AL. This is not surprising, as carpal

TABLE 2 Demographics and Diagnostic Criteria of Patients Diagnosed With Amyloidosis

PatientAge,yrs Sex

AfricanAmerican

BilateralSymptoms

Prior CTSRelease

AmyloidType

GeneticMutation

Kappa,mg/l

Lambda,mg/l

FLCRatio

MonoclonalProtein

#1 73 F No Yes Yes ATTR Ala81Thr 46.0 25.7 1.79 Yes

#2 82 F No Yes Yes ATTR None 21.1 13.9 1.52 No

#3 85 M No Yes Yes AL N/A 33.1 15.6 2.12 No

#4 78 F No Yes Yes AL N/A 20.4 299.6 0.07 Yes

#5 67 M No Yes Yes ATTR None 12.3 10.0 1.23 No

#6 56 M No Yes Yes ATTR Leu58His 15.5 9.9 1.57 No

#7 62 M No Yes No ATTR None 13.9 11.3 1.23 Yes

#8 72 F No Yes Yes Presumed ATTR* None 20.9 11.2 1.87 No

#9 65 M Yes Yes No ATTR None 29.7 26.3 1.13 Yes

#10 80 M No Yes No ATTR None 14.7 18.6 0.79 Yes

*Mass spectrometry and immunohistochemistry without sufficient tissue to accurately subtype sample.

CTS ¼ carpal tunnel syndrome; FLC ¼ free light chain ratio (kappa/lambda) with reference range 0.26 to 1.65.

TABLE 3 Cardiac Eva

PatientAmyloidType

#1 ATTR

#2 ATTR

#3 AL

#4 AL

#5 ATTR

#6 ATTR

#7 ATTR

#8 PresumedATTR‡

#9 ATTR

#10 ATTR

Low voltage defined as alldegree of atrioventricular btroponin T values were no

EF ¼ ejection fraction; GLnatriuretic peptide; RBBB ¼

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tunnel syndrome may be present in 30% of patientswith AL as well (4), although not as common as inATTR. As AL is a much more rapidly progressive dis-ease with a poor prognosis after cardiac involvement isadvanced, the early recognition of this entity is evenmore important to initiate appropriate disease-modifying therapy (31).

ATTR amyloid deposition in the ligamentum fla-vum is associated with lumbar spinal stenosis and ispresent in up to 45% of surgical biopsies (32). In ourstudy, 6 of the 10 amyloid-positive patients had ahistory of lumbar spinal stenosis (4 ATTR, 2 AL).Three of those required surgical intervention,including the 2 patients with AL, both of whom werealso afflicted by cervical spinal stenosis. Two (20%) of

luation of Patients Diagnosed With Amyloidosis

ExaminationCardiac

Biomarkers ECG

JVP, cmH2O Edema

NT-proBNP,pg/ml

TnT,ng/ml

HeartBlock

LimbVoltage

SolokowVoltage,

mm

PseuInfaPatt

5 0 108 <0.01 None Normal 12 No

6 0 N/A† N/A† None Normal 21 No

7 Trace 35 <0.01 None Normal 20 No

17 3þ 1,615 <0.01 None Normal 19 No

5 0 63 <0.01 None Normal 21 No

7 0 61 <0.01 RBBB Normal 15 No

5 0 92 <0.01 RBBB Normal 20 No

4 0 58 <0.01 None Normal 17 Ye

16 1þ 155 0.017 None Normal 13 No

4 0 222 <0.01 None Low 15 No

QRS complexes in limb leads #5 mm. Sokolow voltage defined as Q-wave in V1 þ R-wave ilock or bundle branch block. *Only patient #5 had visual uptake on SPECT imaging. †Baselinet elevated. ‡Mass spectrometry and immunohistochemistry without sufficient tissue to accu

S ¼ global longitudinal strain; H/CL ¼ heart-to-contralateral lung; IVS ¼ interventricular sepright bundle branch block; TnT ¼ Troponin T.

the amyloid-positive patients had a history of bicepstendon rupture (1 wild-type ATTR, 1 hereditaryATTR). While uncommon, biceps tendon rupture isfound much more often in ATTR than in the generalpopulation, perhaps indicative that it is a morespecific extracardiac finding (33).

The prevalence of cardiac involvement at the timeof carpal tunnel release surgery has not previouslybeen established. As several emerging pharmaco-logical treatments are in development that mayslow, halt, or reverse ATTR, earlier diagnosis is ad-vantageous. Based upon several clinical trialsmeeting their primary endpoint, 3 new therapieshave received breakthrough designation from theU.S. Food and Drug Administration, with more in the

Echocardiography TcPYP

dorctern

EF,%

IVS,cm

DiastolicStage

AverageE/eʹ

GLS,%

VisualGrade*

H/CLRatio1 h

H/CLRatio3 h

62 0.9 0 9.6 �19.6 0 1.26 1.39

60 1.0 0 14.8 �16.4 0 1.20 1.25

67 1.1 1 17.1 �16.4 0 1.15 1.16

64 1.5 2 12.9 �19.7 0 1.29 1.32

60 1.4 0 9.6 �15.8 3 1.75 1.67

65 0.9 0 8.9 �23.4 0 1.10 1.34

53 1.4 1 5.7 �15.2 0 1.25 1.37

s 68 1.2 1 13.3 �21.7 0 1.23 1.11

65 1.3 2 7.3 �22.4 0 1.22 1.21

46 0.9 1 7.9 �15.3 0 1.37 1.34

n V6; a value <15 mm is sensitive for cardiac amyloidosis. Heart block defined as anyvalues not available due to insufficient sample; however, subsequent NT-proBNP andrately type sample.

tal thickness; JVP ¼ jugular venous pressure; NT-proBNP ¼ N-terminal pro brain-type

FIGURE 2 Patient Example

This is an example of a patient diagnosed with ATTR amyloidosis at the time of tenosynovial biopsy during carpal tunnel release surgery. (A) An image of the tenosynovial

tissue prior to biopsy. (B)H&E and (C)Congo red stains confirmed amyloid deposition. Echocardiography showed amild increase in LVwall thickness (D) aswell as amildly

abnormal global longitudinal strain of �15.8% (E-F). Technetium pyrophosphate nuclear scintigraphy confirmed a diagnosis of cardiac involvement of ATTR (G).

J A C C V O L . 7 2 , N O . 1 7 , 2 0 1 8 Sperry et al.O C T O B E R 2 3 , 2 0 1 8 : 2 0 4 0 – 5 0 Amyloidosis Screening in the Carpal Tunnel

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FIGURE 3 Plasma TTR Concentration and TTR Tetramer Kinetic Stability

50

p = 0.7569

40

[Nat

ive

TTR]

(mg/

dL)

30

20

10

0CTS with ATTR

(wt or mut)

ATTRwt

ATTRwtATTRL58H

ATTR-A81T

ATTRwtATTRwt

ATTRwt

CTS withoutAmyloid

60A

–4

Ln K

ex (h

–1)

–5

–6

More stable

Less stableB

(A) There was no difference in plasma TTR levels between patients with or without ATTR amyloid found in carpal tunnel biopsy. (B) TTR

kinetic stability measured as the rate of subunit exchange in all patients included in this study. The blue dots represent the kinetic stability of

the 5 patients who were identified as having wild-type TTR amyloid fibrils in tenosynovial tissue biopsy, gray dots represent the 2 patients

with hereditary TTR fibrils (A81T and L58H), and orange dots represent patients without amyloid. The median rate of subunit exchange in

affected patients was found to be numerically less stable than patients without ATTR amyloid, although the difference was not statistically

significant.

Sperry et al. J A C C V O L . 7 2 , N O . 1 7 , 2 0 1 8

Amyloidosis Screening in the Carpal Tunnel O C T O B E R 2 3 , 2 0 1 8 : 2 0 4 0 – 5 0

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pipeline. Identification and implementation of ther-apy for prevention or early disease treatment mayalter the natural history of this progressive systemicdisease.

The native TTR concentration in our cohort wasnot different between patients with or without softtissue ATTR deposition. A recent study in patientswith wild-type ATTR evaluated the association be-tween serum TTR concentration and outcomes (34),and described a median TTR concentration of 23 mg/dl(IQR: 20 to 26 mg/dl). They noted that lower TTRconcentrations were associated with lower survival ina multivariable stepwise model, particularly if levelswere below the lower limit of normal (18 mg/dl). As themedian values for TTR concentration are similar inpatients with normal biopsies, with soft tissue amyloiddeposition, and with wild-type cardiac amyloidosis asnoted in the previous text, it is unlikely that thismarker will aid in the diagnosis of patients. Our studyalso quantified TTR tetramer kinetic stability with asubunit exchange assay, a biochemical assay that hasbeen used to measure endogenous plasma and CSFTTR kinetic stability of patients treated with the

kinetic stabilizer tafamidis (3,17). There was atrend toward less stable TTR tetramers in patientswith ATTR; however, there was significant overlapbetween groups. Retinol binding protein 4 (RBP4)and potentially other proteins might play animportant role in plasma TTR kinetic stability.Moreover, the absolute value of the TTR kineticstability may be less important than the change inkinetic stability over time or with disease modi-fying therapies. Kinetic stability, native TTR con-centration, and RBP4 levels should be testedlongitudinally in a larger group of patients todetermine their usefulness to noninvasively di-agnose and follow ATTR.

This study along with other emerging data sur-rounding soft tissue amyloidosis has led to achange in practice at our institution. A screeningalgorithm has been implemented (Online Figure 1)to guide the hand surgeons regarding the appro-priateness of tenosynovial biopsy at the time ofcarpal tunnel release surgery. If Congo red stainingis positive, prompt typing with mass spectrometryand referral to an amyloidosis specialist is

PERSPECTIVES

COMPETENCY IN MEDICAL KNOWLEDGE: One in 10 older

patients undergoing carpal tunnel release surgery for idiopathic

carpal tunnel syndrome have either TTR or AL amyloidosis in

tenosynovial tissue, and this may be an early marker or precursor

of amyloid heart disease.

TRANSLATIONAL OUTLOOK: Long-term follow-up of pa-

tients with tenosynovial amyloidosis is needed to understand the

natural history, frequency of cardiac involvement, and implica-

tions for management.

J A C C V O L . 7 2 , N O . 1 7 , 2 0 1 8 Sperry et al.O C T O B E R 2 3 , 2 0 1 8 : 2 0 4 0 – 5 0 Amyloidosis Screening in the Carpal Tunnel

2049

recommended. We believe that the low cost ofscreening patients for amyloidosis at the time ofcarpal tunnel release surgery may avert theexpense of progressive heart failure care in pa-tients diagnosed early.

STUDY LIMITATIONS. This study was performedwith an open carpal tunnel release, although somesurgeons used a minimally invasive technique witha smaller incision. Obtaining a sample of tenosy-novial tissue with a nonopen (endoscopic) release ismore challenging and may limit application of thisstrategy for those surgeons who use this surgicalapproach. Inclusion criteria were limited to olderadults given the high prevalence of both types ofcardiac amyloidosis; this acknowledges a decreasein sensitivity of identifying younger patients whomay be more likely to have AL. There was a limitednumber of African Americans in the study cohort.This was due to the demographics of patients un-dergoing carpal tunnel release at our institution anda higher percentage of African-American patientswho declined to participate (10% African Americansin the screened cohort vs. 5% enrolled). It would beimportant to know the prevalence of biopsy-diagnosed carpal tunnel amyloidosis in this cohort,as it is estimated that 3% to 4% of African Ameri-cans in the United States carry the V122I TTR genemutation, which is a known cause of hereditarycardiac amyloidosis (35). Additionally, low RBP4levels have been shown to potentially help identifypatients with V122I ATTR (36), and it has been hy-pothesized that TTR kinetic stability is related tothe relative concentrations of RBP4 to tetramericTTR (3); these hypotheses were not studied in thisanalysis. Last, the progression from soft tissueamyloidosis to the involvement of other organs overtime is unknown. Long-term follow-up of thiscohort may be able to shed light on the naturalhistory of this disease and is already planned as thenext phase of this study.

CONCLUSIONS

We demonstrate that 10.2% of a cohort of menage $50 years and women $60 years undergoingcarpal tunnel release surgery for idiopathic carpaltunnel syndrome had amyloidosis diagnosed fromtenosynovial tissue biopsy. Subsequent cardiacworkup identified patients with both clinical andsubclinical cardiac amyloid involvement of both theAL and ATTR subtypes. TTR concentration andtetramer kinetic stability were not able to classifypatients with biopsy-diagnosed disease. We believethat hand surgeons should be aware of the associationbetween carpal tunnel syndrome and amyloidosisand consider biopsy of tenosynovial tissue with sub-sequent Congo red staining, particularly in patientswith bilateral symptoms. Tenosynovial biopsy is alow-risk procedure that may lead to early diagnosis ofamyloidosis, thereby allowing for timely interventionto combat this life-threatening disease.

ADDRESS FOR CORRESPONDENCE: Dr. Mazen Hanna,Cleveland Clinic, 9500 Euclid Avenue, J3-4, Cleve-land, Ohio 44195. E-mail: hannam@ccf.org. Twitter:@maz_hanna, @BrettSperryMD, @ClevelandClinic.

RE F E RENCE S

1. Vranian MN, Sperry BW, Valent J, Hanna M.Emerging advances in the management ofcardiac amyloidosis. Curr Cardiol Rep 2015;17:100.

2. Sperry BW, Tang WHW. Amyloid heart disease:genetics translated into disease-modifying ther-apy. Heart Br Card Soc 2017;103:812–7.

3. Rappley I, Monteiro C, Novais M, et al. Quanti-fication of transthyretin kinetic stability in humanplasma using subunit exchange. Biochemistry(Mosc) 2014;53:1993–2006.

4. Prokaeva T, Spencer B, Kaut M, et al. Soft tissue,joint, and bone manifestations of AL amyloidosis:clinical presentation, molecular features, and sur-vival. Arthritis Rheum 2007;56:3858–68.

5. Kyle RA, Eilers SG, Linscheid RL, Gaffey TA.Amyloid localized to tenosynovium at carpal tun-nel release. Natural history of 124 cases. Am J ClinPathol 1989;91:393–7.

6. Breda S, Richter HP, Schachenmayr W. [Inci-dence of biopsy-detectable amyloid deposits inthe retinaculum flexorum and in the tenosynovial

tissue in carpal tunnel syndrome.]. ZentralblNeurochir 1993;54:72–6.

7. Gioeva Z, Urban P, Meliss RR, et al. ATTR amyloidin the carpal tunnel ligament is frequently of wild-type transthyretin origin. Amyloid 2013;20:1–6.

8. Sueyoshi T, Ueda M, Jono H, et al. Wild-typetransthyretin-derived amyloidosis in various lig-aments and tendons. Hum Pathol 2011;42:1259–64.

9. Sekijima Y, Uchiyama S, Tojo K, et al. Highprevalence of wild-type transthyretin deposition

Sperry et al. J A C C V O L . 7 2 , N O . 1 7 , 2 0 1 8

Amyloidosis Screening in the Carpal Tunnel O C T O B E R 2 3 , 2 0 1 8 : 2 0 4 0 – 5 0

2050

in patients with idiopathic carpal tunnel syndrome:a common cause of carpal tunnel syndrome in theelderly. Hum Pathol 2011;42:1785–91.

10. Sperry BW, Vranian MN, Hachamovitch R, et al.Are classic predictors of voltage valid in cardiacamyloidosis? A contemporary analysis of electro-cardiographic findings. Int J Cardiol 2016;214:477–81.

11. Senapati A, Sperry BW, Grodin JL, et al.Prognostic implication of relative regional strainratio in cardiac amyloidosis. Heart 2016;102:748–54.

12. Lang RM, Badano LP, Mor-Avi V, et al.Recommendations for cardiac chamber quanti-fication by echocardiography in adults: an up-date from the American Society ofEchocardiography and the European Associationof Cardiovascular Imaging. J Am Soc Echo-cardiogr 2015;28:1–39.e14.

13. Vranian MN, Sperry BW, Hanna M, et al.Technetium pyrophosphate uptake in trans-thyretin cardiac amyloidosis: associations withechocardiographic disease severity and outcomes.J Nucl Cardiol 2018;25:1247–56.

14. Sperry BW, Gonzalez MH, Brunken R,Cerqueira MD, Hanna M, Jaber WA. Non-cardiacuptake of technetium-99m pyrophosphate intransthyretin cardiac amyloidosis. J Nucl Cardiol2018 Jan 17 [E-pub ahead of print].

15. Wiseman RL, Green NS, Kelly JW. Kinetic sta-bilization of an oligomeric protein under physio-logical conditions demonstrated by a lack ofsubunit exchange: implications for transthyretinamyloidosis. Biochemistry 2005;44:9265–74.

16. Choi S, Ong DST, Kelly JW. A stilbene thatbinds selectively to transthyretin in cells and re-mains dark until it undergoes a chemoselectivereaction to create a bright blue fluorescent con-jugate. J Am Chem Soc 2010;132:16043–51.

17. Cho Y, Baranczak A, Helmke S, et al. Person-alized medicine approach for optimizing the doseof tafamidis to potentially ameliorate wild-typetransthyretin amyloidosis (cardiomyopathy). Am-yloid 2015;22:175–80.

18. Phelan D, Collier P, Thavendiranathan P,et al. Relative apical sparing of longitudinalstrain using two-dimensional speckle-tracking

echocardiography is both sensitive and specificfor the diagnosis of cardiac amyloidosis. Heart2012;98:1442–8.

19. Berk JL, Suhr OB, Obici L, et al. Repurposingdiflunisal for familial amyloid polyneuropathy: arandomized clinical trial. JAMA 2013;310:2658–67.

20. Mendell JR, Jiang XS, Warmolts JR,Nichols WC, Benson MD. Diagnosis of Maryland/German familial amyloidotic polyneuropathy usingallele-specific, enzymatically amplified, genomicDNA. Ann Neurol 1990;27:553–7.

21. McCausland KL, White MK, Guthrie SD, et al.Light chain (AL) amyloidosis: the journey to diag-nosis. Patient 2018;11:207–16.

22. Lousada I, Maurer MS, Warner M, Guthrie S,Hsu K, Grogan M. Amyloidosis Research Con-sortium cardiac amyloidosis survey: results frompatients with ATTR amyloidosis and their care-givers. Poster presented at: European ATTRAmyloidosis Meeting for Patients and Doctors.Paris, France. November, 2017.

23. Tanskanen M, Peuralinna T, Polvikoski T, et al.Senile systemic amyloidosis affects 25% of thevery aged and associates with genetic variation inalpha2-macroglobulin and tau: a population-based autopsy study. Ann Med 2008;40:232–9.

24. González-López E, Gallego-Delgado M,Guzzo-Merello G, et al. Wild-type transthyretinamyloidosis as a cause of heart failure with pre-served ejection fraction. Eur Heart J 2015;36:2585–94.

25. Sperry BW, Jones BM, Vranian MN, Hanna M,Jaber WA. Recognizing transthyretin cardiacamyloidosis in patients with aortic stenosis: impacton prognosis. J Am Coll Cardiol Img 2016;9:904–6.

26. Treibel TA, Fontana M, Gilbertson JA, et al.Occult transthyretin cardiac amyloid in severecalcific aortic stenosis: prevalence and prognosisin patients undergoing surgical aortic valvereplacement. Circ Cardiovasc Imaging 2016;9:e005066.

27. Cavalcante JL, Rijal S, Abdelkarim I, et al.Cardiac amyloidosis is prevalent in older patientswith aortic stenosis and carries worse prognosis.J Cardiovasc Magn Reson 2017;19:98.

28. Castaño A, Narotsky DL, Hamid N, et al.Unveiling transthyretin cardiac amyloidosis andits predictors among elderly patients with severeaortic stenosis undergoing transcatheter aorticvalve replacement. Eur Heart J 2017;38:2879–87.

29. Valentino P, Pirritano D, Bono F, et al. Geneticscreening for familial amyloid polyneuropathy inpatients with idiopathic carpal tunnel syndrome.J Peripher Nerv Syst 2008;13:151–2.

30. Yamaguchi DM, Lipscomb PR, Soule EH. Carpaltunnel syndrome. Minn Med 1965;48:22–33.

31. Sperry BW, Ikram A, Hachamovitch R, et al.Efficacy of chemotherapy for light-chainamyloidosis in patients presenting with symp-tomatic heart failure. J Am Coll Cardiol 2016;67:2941–8.

32. Yanagisawa A, Ueda M, Sueyoshi T, et al.Amyloid deposits derived from transthyretin in theligamentum flavum as related to lumbar spinalcanal stenosis. Mod Pathol 2015;28:201–7.

33. Geller HI, Singh A, Alexander KM, Mirto TM,Falk RH. Association between ruptured distal bi-ceps tendon and wild-type transthyretin cardiacamyloidosis. JAMA 2017;318:962–3.

34. Hanson JLS, Arvanitis M, Koch CM, et al. Useof serum transthyretin as a prognostic indicatorand predictor of outcome in cardiac amyloid dis-ease associated with wild-type transthyretin. CircHeart Fail 2018;11:e004000.

35. Quarta CC, Buxbaum JN, Shah AM, et al. Theamyloidogenic V122I transthyretin variant inelderly Black Americans. N Engl J Med 2015;372:21–9.

36. Arvanitis M, Koch CM, Chan GG, et al.Identification of transthyretin cardiac amyloidosisusing serum retinol-binding protein 4 and aclinical prediction model. JAMA Cardiol 2017;2:305–13.

KEY WORDS amyloid, amyloidosis, carpaltunnel syndrome, light chain, transthyretin

APPENDIX For a supplemental figure, pleasesee the online version of this paper.