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Cardiac Amyloid Load

A Prognostic and Predictive Biomarker in Patients WithLight-Chain Amyloidosis

Arnt V. Kristen, MD,a Eva Brokbals, MS,a Fabian aus dem Siepen, MD,a Ralf Bauer, MD,a Selina Hein, MD,a

Matthias Aurich, MD,a Johannes Riffel, MD,a Hans-Michael Behrens, MSC,b Sandra Krüger, BSC,b

Peter Schirmacher, MD,c Hugo A. Katus, MD,a,d Christoph Röcken, PHDb

ABSTRACT

FrobIn

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Dr

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BACKGROUND Cardiac amyloid load has not been analyzed for its effect on mortality in patients with amyloid

light-chain (AL) cardiac amyloidosis.

OBJECTIVES This study retrospectively compared histological amyloid loadwith common clinical predictors ofmortality.

METHODS This study assessed 216 patients with histologically confirmed cardiac amyloidosis at a single center with

electrocardiography, echocardiography, and laboratory testing.

RESULTS AL amyloid deposits were usually distributed in a reticular/pericellular pattern, whereas transthyretin amyloid

(ATTR) more commonly showed patchy deposits. Median amyloid load was 30.5%; no amyloid load was above 70%.

During follow-up (median 19.1 months), 112 patients died. Chemotherapy had a significant effect on overall survival in AL

amyloidosis (16.2 months vs. 1.4 months; p ¼ 0.003). Patients with <20% AL amyloid load who responded to

chemotherapy showed significantly better survival than nonresponders. According to univariate analysis, predictors of

survival in AL amyloidosis included sex, Karnofsky index, New York Heart Association (NYHA) functional class, diastolic

blood pressure, estimated glomerular filtration rate, N-terminal pro–B-type natriuretic peptide, mineralocorticoid re-

ceptor antagonists, low voltage, ineligibility for chemotherapy, response to chemotherapy, and amyloid load. Indepen-

dent predictors of mortality by multivariate analysis included NYHA functional class (III vs. II), estimated glomerular

filtration rate, responders to chemotherapy, and amyloid load. In ATTR amyloidosis, survival correlated with NYHA

functional class, diastolic blood pressure, and use of diuretic agents. Following Cox regression analysis, NYHA functional

class (III vs. II; p < 0.05) remained the only independent predictor of patient survival in ATTR amyloidosis.

CONCLUSIONS Early identification of subjects with AL amyloid is essential given that in late-stage disease with

extensive amyloid load, our data suggested that outcomes are not affected by administration of chemotherapy.

(J Am Coll Cardiol 2016;68:13–24) © 2016 by the American College of Cardiology Foundation.

A myloidosis constitutes a group of diseaseswith different etiologies, characterized byextracellular deposition of proteins oriented

in a b-sheet structure. A total of 31 autologous physi-ological proteins that can form amyloid, each linkedto a unique etiology, have been identified (1).Depending on the anatomical distribution and extentof amyloid formation, it can disrupt normal tissue

m the aDepartment of Cardiology, Angiology, and Respiratory Medicin

stitute of Pathology, Christian-Albrechts-University, Kiel, Germany; cInst

rg, Germany; and the dDZHK (German Center for Cardiovascular Research

. Röcken was supported by grants from the German Research Foundation

ucation and Research (GERAMY). All other authors have reported that th

s paper to disclose.

nuscript received November 23, 2015; revised manuscript received April

architecture and function, leading to progressiveorgan failure and eventually death. The most clini-cally relevant forms are immunoglobulin-derivedlight-chain (AL) and transthyretin-derived (ATTR)amyloidosis (2). Clinical presentation of AL amyloid-osis is multifactorial, involving almost every organwith predominance of kidney and heart involvement(3). ATTR amyloidosis can occur in a hereditary form

e, University of Heidelberg, Heidelberg, Germany;

itute of Pathology, University of Heidelberg, Heidel-

), Site Heidelberg/Mannheim, Heidelberg, Germany.

(Grant-No. Ro 1173/11) and the Federal Ministry of

ey have no relationships relevant to the contents of

3, 2016, accepted April 12, 2016.

ABBR EV I A T I ON S

AND ACRONYMS

AL = light-chain amyloid

ATTR = transthyretin amyloid

eGFR = estimated glomerular

filtration rate

EMB = endomyocardial biopsy

mt-ATTR = mutant-type

transthyretin amyloid

NT-proBNP = N-terminal

pro-B-type natriuretic peptide

NYHA = New York Heart

Association

wt-ATTR = wild-type

transthyretin amyloid

Kristen et al. J A C C V O L . 6 8 , N O . 1 , 2 0 1 6

Amyloid Load and Prognosis in AL Amyloidosis J U L Y 5 , 2 0 1 6 : 1 3 – 2 4

14

due to a point mutation in the TTR gene oras a wild-type variant; the latter almostexclusively affects elderly men. In general,ATTR amyloidosis is characterized by senso-motoric polyneuropathy and/or cardiomy-opathy (4–6). Median survival is about 4months in patients with AL amyloidosisand manifest systolic left ventricular (LV)heart failure and 3 to 6 years in ATTRamyloidosis (7).

In view of the clinical and prognostic sig-nificance of cardiac amyloidosis, severalnoninvasive diagnostic tools have been re-ported for predicting AL amyloidosis mor-tality, including electrocardiography (ECG)

and echocardiography (8–10). In recent years, cardiacmagnetic resonance (CMR) imaging has increasinglybeen used for tissue characterization using late gad-olinium enhancement (LGE) or T1 mapping (11).Skeletal scintigraphy also has demonstrated highsensitivity and specificity for diagnosing ATTRamyloidosis (12,13). Cardiac biomarkers such astroponin T and N-terminal pro–B-type natriureticpeptide (NT-proBNP) provide potent prognostic in-formation in AL amyloidosis (3,14,15).

SEE PAGE 25

However, prior to clinical and serological tests, asound diagnosis and classification of cardiacamyloidosis is mandatory. Clinical management issubtype-dependent and may require, for instance,chemotherapy and autologous bone marrow trans-plantation in AL amyloidosis or liver transplantationin ATTR amyloidosis. Endomyocardial biopsy (EMB)is often crucial for the differential diagnosis of hy-pertrophic cardiomyopathies. Despite the importanceof EMB and thorough clinical assessment of patientsexperiencing cardiac amyloidosis, few studies havecompared cardiac histology with clinical data (16). Inthis study, we carried out a comprehensive retro-spective study testing the following hypotheses: 1) ALand ATTR amyloid show unique distribution patternsin the heart; 2) AL and ATTR amyloid show distinctclinical presentations; 3) amyloid load in tissuespecimens correlates with clinico-pathological pa-tient characteristics and (cardiac) disease severity;and 4) amyloid load is a prognostic and/or predictivebiomarker.

METHODS

This project was approved by the local ethics com-mittee of the University Hospital in Heidelberg con-forming to the Declaration of Helsinki.

PATIENT COHORT AND CLINICAL ASSESSMENT.

From the Heidelberg Amyloidosis Center, we identi-fied all patients with cardiac AL and ATTR amyloid-osis who had undergone EMB and a detailed clinicalassessment and were studied histologically by theAmyloid Registry Kiel. From 2004 to 2015, we iden-tified a total of 216 patients.

Screening for monoclonal gammopathy by serum/urine immunofixation electrophoresis and the serumfree light-chain test was carried out routinely (Bind-ing Site, Schwetzingen, Germany). Patient recordswere analyzed for time of diagnosis and disease-specific parameters, including organ involvement,standard blood tests, 12-lead ECG, and echocardiog-raphy. Cardiac troponin T was measured by either afourth-generation assay or a high-sensitivity assay(Roche Diagnostics, Mannheim, Germany). Due tolack of comparability of the different troponin assays,abnormal values were defined as >0.03 mg/l (fourth-generation assay) or >14 pg/ml (high-sensitivityassay). NT-proBNP was measured using ElecsysproBNP (Roche Diagnostics). In patients with ALamyloidosis, response to treatment was defined ac-cording to international consensus criteria (17,18).

ECGs were analyzed for a low-voltage pattern,defined as QRS complex deflection <0.5 mV in anylimb leads or the sum of the S-wave deflection in V1–2

and R-wave deflection in V5–6 <1.5 mV. Transthoracicechocardiograms were analyzed for surrogatemarkers of cardiac amyloidosis, including left atrialdiameter, diastolic interventricular septum thickness,diastolic posterior wall thickness, LV end-diastolicdiameter, or LV end-systolic diameter. LV end-diastolic volume, LV end-systolic volume, strokevolume, ejection fraction (EF), and LV mass werecalculated as reported previously (19). LVEF <45%was considered markedly impaired. Mitral annularsystolic velocity was measured by pulsed-waveDoppler tissue imaging with sample volume on thelateral mitral annulus in the apical 4-chamber view.LVmyocardial volume was defined as LVmass dividedby the mean density of myocardium (1.05 g/ml).Myocardial contraction fraction was calculated asstroke volume divided by myocardial volume (20).

HISTOLOGY. Formalin-fixed and paraffin-embeddedtissue samples were used throughout this study.Amyloid was detected by Congo red staining, viewedunder polarized light, showing green-yellow-orangebirefringence. Immunohistochemical classificationof amyloid was carried out as described elsewhere(21–23). Additional information on description ofdeoxyribonucleic acid extraction is in the OnlineAppendix.

J A C C V O L . 6 8 , N O . 1 , 2 0 1 6 Kristen et al.J U L Y 5 , 2 0 1 6 : 1 3 – 2 4 Amyloid Load and Prognosis in AL Amyloidosis

15

QUANTIFICATION OF AMYLOID LOAD. Immunohis-tochemically stained slides were scanned using a LeicaSCN400 whole slide scanner (Leica Biosystems,Nussloch, Germany) with 40� magnification. Theslide images were exported as overview andwith 9� magnification (pixel width of about 1.2 mm).The percentage of the amyloid area was evaluatedusing ImageJ version 1.47v (U.S. National Institutesof Health, Bethesda, Maryland) by countingimmunohistochemically stained and nonstainedpixels. Artifacts surrounding the specimen within theoriginal image were removed manually using photoediting software. Processing in Image J was done usingthe “color threshold” function to filter pixels on thebasis of ranges of hue, saturation, and brightnessvalues in the HSB color model. Background wasdetected by filtering pixels of high brightness and lowsaturation. The specimen’s pixel count was calculatedby subtracting the background pixel count from thetotal pixel count. Stained areas (red pixels) weredetected by filtering the corresponding range of huevalues in combination with a lower threshold ofsaturation. Threshold values were adjusted indivi-dually under supervision of a pathologist (C.R.) tocompensate for variations of the staining process.

STATISTICAL ANALYSIS. Statistical analyses werecarried out with SPSS version 20 (IBM Corporation,Armonk, New York) and R Version 3.2.0 (R Founda-tion for Statistical Computing, Vienna, Austria).Continuous data were expressed as median (inter-quartile range [IQR]). Categorical variables wereexpressed as absolute numbers (percentages). Amy-loid load was divided into 3 groups (<20%, 20% to40%, and >40%). To test for significant differencesof continuous variables between amyloid typesand between amyloid load groups, we applied theKruskal-Wallis test and Mann-Whitney U test. Forcategorical clinical variables, we applied Fisher exacttest, and for pairwise comparisons, the 2-sample testfor equality of proportions with continuity correctionusing R. Overall survival, defined as the time betweenEMB and death, was analyzed using the Kaplan-Meiermethod. Differences of median overall survival wereassessed using the log-rank test. We carried out uni-variate Cox regression separately for all continuousand categorical clinical variables. All variables havingp # 0.1 in univariate Cox regression were reportedand included in a multivariate Cox regression toidentify independent prognostic variables. Multivar-iate results are shown after backward-LR methodusing p ¼ 0.05 as the exclusion limit. All p valueswere taken from 2-tailed tests. We assumed a signif-icance level of p < 0.05.

To account for false discoveries due to multipletesting, we applied the Simes (Benjamini-Hochberg)procedure per test group (i.e., separately for com-parisons with amyloid type, amyloid load, and sur-vival tests) (24). All p values were given unadjusted,but were marked where they lost significance afterSimes’ multiple testing procedure.

RESULTS

The characteristics of the EMB cohort are summarizedin Table 1. In total, 81 biopsies were obtained from theleft and 73 from the right ventricular myocardium(unknown in 62 patients). AL amyloidosis was diag-nosed in 107 patients (ALl n ¼ 91 [85%] and ALkn ¼ 16 [15%]) and ATTR in 109 patients. Molecular-genetic testing showed TTR-wild type (wt-ATTR) in76 patients and mutant TTR (mt-ATTR) in 33 patients,including p.Val50Met (n ¼ 9); p.Val40Ile (n ¼ 6);p.Cys30Arg (n ¼ 2); p.Glu74Lys (n ¼ 2); p.Val142Ile(n ¼ 2); p.Thr79Lys (n ¼ 2); and p.Asp38Glu,p.Ala39Asp, p.His51Asn, p.Gly67Glu, p.Ser70Arg,p.Thr80Ala, p.Val114Ala, p.Thr126Asn, p.Ile127Val,and unknown (each n ¼ 1), respectively. No correla-tion was found between biopsy site and amyloid type.No sex-based differences were observed, except forhigher prevalence of males with wt-ATTR.

CLINICAL PRESENTATION OF CARDIAC AMYLOIDOSIS.

The clinical, ECG, and echocardiographic findingsshowed several significant differences between pa-tients experiencing AL, wt-ATTR, and mt-ATTRamyloidosis (Table 1). These differences can be cate-gorized into amyloid-type specific differences (AL vs.ATTR), putative age-dependent differences (mt-ATTRvs. wt-ATTR), and differences probably related toboth amyloid type and patient age (AL vs. wt-ATTR).

Amyloid-type specific differences (AL vs. ATTR)were found with regard to NT-proBNP serum levels(significantly higher in AL) and LV mass (significantlylower in AL) (Table 1). These 2 characteristics showedno differences between wt- and mt-ATTR amyloid.

Differences probably related to patient age(mt-ATTR vs. wt-ATTR) included estimated glomer-ular filtration rate (eGFR) and medication use, withpatients with wt-ATTR showing a significantlylower eGFR and more frequently taking beta-blockeror angiotensin-converting enzyme inhibitors/angiotensin-1 receptor antagonist therapy. Addition-ally, disease-specific characteristics of patients withwt-ATTR included a higher age at diagnosis, morecommonly being male, and an insignificantlyhigher prevalence of atrial fibrillation in wt-ATTRwhen compared with mt-ATTR and AL amyloidosis(Table 1).

TABLE 1 Clinico-Pathological Characteristics

Valid, n TotalAL Amyloid(n ¼ 107)

mt-ATTRAmyloid(n ¼ 33)

wt-ATTRAmyloid(n ¼ 76)

p Values

TotalAL vs.

mt-ATTRAL vs.

wt-ATTRmt-ATTR

vs. wt-ATTR

Age at baseline, yrs (median [range]) 215 67.2 (30.5–86.5) 61.5 (37.0–85.9) 63.2 (30.5–75.9) 72.4 (55.0–86.5) <0.001* 0.887† <0.001† <0.001†

Male 216 163 (75.5) 70 (65.4) 22 (66.7) 71 (93.4) <0.001‡ 1.000‡ <0.001‡ 0.001‡

Female 53 (24.5) 37 (34.6) 11 (33.3) 5 (6.6)

Karnofsky index 192 80 (70–85) 80 (70–80) 80 (72.5–87.5) 80 (80–85) 0.012* 0.326† 0.003† 0.293†

NYHA functional class 207 3.0 (2–3) 3 (2–3) 2.5 (2–3) 3 (2–3) 0.185* 0.085† 0.989† 0.094†

Diastolic blood pressure, mm Hg 202 70 (70–80) 70 (61.3–80.0) 70 (70–80) 75 (70–80) 0.010* 0.112† 0.003† 0.536†

eGFR, ml/min/1.73 m2 211 69.2 (55.0–88.9) 74.0 (56.0–91.4) 81.9 (61.6–107.7) 64.0 (51.4–78.4) 0.012* 0.209† 0.029† 0.005†

NT-proBNP, ng/ml 203 5,510 (2,430–10,341) 7,684 (3,726–14,288) 3,330 (1,109–6,160) 3,710 (1,813–7,249) <0.001* <0.001† <0.001† 0.320†

Abnormal TnT 203 186 (91.6) 89 (89.0) 27 (90.0) 70 (95.9) 0.273‡ 1.000‡ 0.157‡ 0.354‡

Beta-blocker 212 140 (66.0) 57 (54.3) 20 (62.5) 63 (84.0) <0.001‡ 0.542‡ <0.001‡ 0.022‡

ACE inhibitor/AT1 212 116 (54.7) 45 (42.9) 14 (43.8) 57 (76.0) <0.001‡ 1.000‡ <0.001‡ 0.002‡

MR antagonist 212 84 (39.6) 34 (32.4) 14 (43.8) 36 (48.0) 0.090‡ 0.291‡ 0.044‡ 0.833‡

Electrocardiography findings

Heart rate, beats/min 206 79 (68.8–91.3) 83 (75.0–96.5) 77 (68–87) 74 (60.0–81.3) <0.001* 0.091† <0.001† 0.118†

PQ interval, ms 146 180 (156–207) 178 (149–197) 180 (154–198) 195 (173–224) 0.037* 0.532† 0.012† 0.100†

QRS duration, ms 203 106 (94–128) 103 (93–118) 108 (92–128) 111 (98–149) 0.022* 0.313† 0.006† 0.313†

QTc, ms 203 441 (418–468) 436 (416–464) 449 (424–476) 450 (420–471) 0.392* 0.252† 0.293† 0.699†

Low voltage 201 62 (30.8) 40 (39.6) 9 (29.0) 13 (18.8) 0.015‡ 0.396‡ 0.004‡ 0.300‡

Atrial fibrillation 216 46 (21.3) 17 (15.9) 6 (18.2) 23 (30.3) 0.065‡ 0.790‡ 0.029‡ 0.241‡

Echocardiography findings

LA diameter, mm 208 44 (40–48) 43 (40–48) 43 (40.0–47.5) 45 (41–48) 0.505* 0.840† 0.318† 0.320†

Thickness of septal wall, mm 209 18 (16–21) 17 (15–20) 19 (15–22) 20 (17–22) <0.001* 0.115† <0.001† 0.215†

LV end-diastolic volume, ml 207 79.4 (65.0–95.2) 72.0 (61.6–91.1) 79.4 (68.4–104.0) 83.2 (72.0–99.4) 0.005* 0.068† 0.002† 0.670†

Myocardial contraction fraction, % 207 17.3 (12.4–25.4) 17.9 (12.9–25.9) 17.7 (9.9–25.7) 16.2 (12.2–24.3) 0.484* 0.672† 0.228† 0.675†

LV mass, g/m2 207 203 (172–262) 185 (163–218) 237 (189–294) 232 (184–279) <0.001* <0.001† <0.001† 0.817†

LV ejection fraction, % 208 43.8 (34.7–55.6) 43.8 (35.0–54.4) 45.0 (35.1–55.6) 42.7 (34.3–60.6) 0.975* 0.793† 0.986† 0.897†

Mitral annular systolic velocity, m/s 169 5.0 (4–7) 6.0 (4–8) 5.0 (4–7) 5.0 (4–7) 0.387* 0.329† 0.224† 0.819†

RV hypertrophy 160 95 (59.4) 51 (58.0) 15 (68.2) 29 (58.0) 0.709‡ 0.469‡ 1.000‡ 0.446‡

Impaired RV function 161 110 (68.3) 62 (70.5) 13 (56.5) 35 (70.0) 0.443‡ 0.219‡ 1.000‡ 0.296‡

Pericardial effusion 206 68 (33.0) 46 (44.7) 10 (31.3) 12 (16.9) 0.001‡ 0.220‡ <0.001‡ 0.122‡

Voltage to mass ratio 193 0.96 (0.68–1.22) 1.06 (0.78–1.32) 1.01 (0.71–1.17) 0.77 (0.12–1.08) <0.001* 0.352† <0.001† 0.021†

Histological findings and survival

Amyloid load, % 215 30.5 (18.3–42.1) 28.3 (18.5–38.0) 35.0 (25.0–47.7) 34.0 (15.0–44.1) 0.067* 0.022† 0.156† 0.417†

Median overall survival sincebaseline, monthsk(median [95% CI])

213 total/112 events 30.7 (20.5–40.8) 15.7 (11.5–19.9) 38.9 (20.6–57.2) 64.0 (20.5–107.0) <0.001§ 0.028§ <0.001§ 0.638§

Values are n (%) or median (interquartile range) unless otherwise indicated. Bold indicates significant after multiple testing procedure; italics indicates not significant after multiple testing procedure. *Kruskal-Wallis test. †Mann-Whitney U test. ‡Fisher exact test. §Log-rank test. kMedian (95% confidence interval).

ACE ¼ angiotensin-converting enzyme; AL¼ light-chain amyloid; AT1¼ angiotensin 1; CI ¼ confidence interval; eGFR¼ estimated glomerular filtration rate; IQR ¼ interquartile range; LA¼ left atrial; LV ¼ left ventricular; MR ¼mineralocorticoid; mt-ATTR ¼mutant-type transthyretin (amyloid); NT-proBNP ¼ N-terminal pro–B-type natriuretic peptide; NYHA ¼ New York Heart Association; QTc ¼ corrected QT interval; RV ¼ right ventricular; TnT ¼ troponin T; wt-ATTR ¼ wild-type transthyretin (amyloid).

Kristen

etal.

JACC

VOL.68,NO.1,2016

Amyloid

Loadand

Prognosisin

ALAmyloidosis

JULY

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J A C C V O L . 6 8 , N O . 1 , 2 0 1 6 Kristen et al.J U L Y 5 , 2 0 1 6 : 1 3 – 2 4 Amyloid Load and Prognosis in AL Amyloidosis

17

Finally, many differences could not be related toeither age (no significant difference between mt- andwt-ATTR) or amyloid type (no significant differencebetween AL vs. mt-ATTR) and may be related to amixture of both and included Karnofsky index, dia-stolic blood pressure, heart rate, PQ interval, QRSduration, low voltage pattern, thickness of the septalwall, LV end-diastolic volume, and pericardial effu-sion (Table 1).

In total, 94 (88%) patients with AL amyloidosisreceived chemotherapy. The clinico-pathological pa-tient characteristics did not significantly differ be-tween responders (complete response, n ¼ 8; verygood partial response, n ¼ 14; partial response, n ¼ 28)and nonresponders (n ¼ 14). Clinical variables did notdiffer between responders and nonresponders whendivided according to amyloid load (<20% vs. 20% to40% vs. >40%). In 30 patients, early death preventedassessment of hematological response status.

Amyloid was found in the interstitium and vesselwalls with distinct distribution patterns in AL

FIGURE 1 Amyloid Deposition Patterns

The deposition pattern of transthyretin (amyloid) (ATTR) was commonly

(A and B) (antitransthyretin antibody). Light-chain (AL) amyloid was mo

individual cardiomyocytes (C) (antilambda light chain antibody). Origina

and ATTR amyloidosis (25). AL amyloid depositswere usually more evenly distributed in a reticular/pericellular pattern around myocytes, and the widthof the deposits increased with progressive amounts ofAL amyloid. Cardiac ATTR amyloid was patchy, withthe number and size of individual deposits varyingdepending on the overall amount of ATTR amyloid(Figure 1). Progressive amyloid deposition wasaccompanied by an increasing loss of cardiomyocytes.

The median amyloid load was 30.5% (IQR: 18.3% to42.1%) without any significant difference between bi-opsy sites. Most patients had an amyloid load of 30% to40%, with none>60% (AL amyloid) or>70% (wt-ATTRamyloid) (Online Figures 1 and 2). The amyloid loadwas higher in wt- and mt-ATTR amyloidosis comparedwith AL amyloidosis (Table 1). However, this differencewas not statistically significant. No difference wasfound in themedian amyloid load betweenmt- andwt-ATTR (Online Figure 3).

Correlating amyloid load with clinico-pathologicalparameters showed an increase of NT-proBNP in

patchy with a variable number and size of the individual deposits

re evenly distributed in a reticular/pericellular pattern around the

l magnification: �2.5 (A) or �5 (B and C).

Kristen et al. J A C C V O L . 6 8 , N O . 1 , 2 0 1 6

Amyloid Load and Prognosis in AL Amyloidosis J U L Y 5 , 2 0 1 6 : 1 3 – 2 4

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ATTR amyloidosis. Patients with <20% amyloid loadharbored a median serum NT-proBNP of 2,579 ng/ml(IQR: 1,274 to 7,249 ng/ml); a 20% to 40% load, 2,742ng/ml (IQR: 1,201 to 5,280 ng/ml); and a >40% load,4,832 ng/ml (IQR: 2,701 to 10,387 ng/ml) (<20% vs.20% to 40%: p ¼ 0.494; <20% vs. >40%: p ¼ 0.043;20 to 40% vs. >40%: p ¼ 0.002). However, this dif-ference lost statistical significance after multipletesting adjustments. Detailed correlations of amyloidload with clinico-pathological parameters accordingto amyloid type are shown in Online Table 1.

SURVIVAL ANALYSIS. Survival data were availablefor 213 patients. During follow-up (median 19.1months; range 2.5 to 145.7 months), 112 (52.6%) pa-tients died (AL n ¼ 73 [68.2%]; mt-ATTR n ¼ 15[48.4%]; wt-ATTR n ¼ 24 [32.0%]). Mortality wassignificantly lower in wt-ATTR amyloidosis comparedwith AL amyloidosis. In total, 94 patients receivedchemotherapy (autologous stem cell transplantationn ¼ 16). Additional first-line treatment regimenswere melphalan/dexamethasone (n ¼ 27), bortezo-mib/dexamethasone (n ¼ 42), and melphalan/prednisolone (n ¼ 9). Patients deemed ineligible forany type of chemotherapy were excluded from sur-vival analyses to avoid bias. Six patients withmt-ATTR received a liver transplant. Chemotherapyhad a significant effect on overall survival in ALamyloidosis: 16.2 months (95% confidence interval[CI]: 11.3 to 21.1 months) versus 1.4 months (95%confidence interval: 0 to 31 months) (p ¼ 0.003). Nodifference was found in mt-ATTR amyloidosis afterliver transplantation (Online Figure 4).

Patients with AL amyloidosis who had <20% (me-dian 12.5 months [95% CI: 3.5 to 21.4 months]) and>40% amyloid load (median 6.7 months [95% CI: 0 to15.1 months]) had shorter survival than patientsharboring a 20% to 40% load (median 24.5 months[95% CI: 0.6 to 48.4 months]) (p ¼ 0.024) (Figure 2).Survival depended significantly on chemotherapy(data not shown). Responders (complete response,very good partial response, and partial responsepost-chemotherapy) and nonresponders (no res-ponse, progressive disease) with <20% amyloidshowed a significant difference in overall survival(Central Illustration). No difference was found betweenresponders and nonresponders if amyloid load was20% to 40% or >40% (Central Illustration).

UNIVARIATE AND MULTIVARIATE ANALYSIS.

Finally, we correlated patient survival with all clinico-pathological patient characteristics. In univariate an-alyses, survival of patients with AL amyloidosiscorrelated with sex, Karnofsky index, New York HeartAssociation (NYHA) functional class, diastolic blood

pressure, eGFR, NT-proBNP, medication with miner-alocorticoid receptor antagonists, low voltage pattern,ineligibility for chemotherapy, and amyloid load. Allvariables having p < 0.1 in univariate analyses(Table 2) were included in a multivariate Cox regres-sion, which showed NYHA functional class, eGFR,amyloid load, and response to chemotherapy to beindependent predictors of patient survival (Table 2). Aseparate analysis excluding 13 patients ineligible forany chemotherapy revealed similar results (Table 2).

In ATTR amyloidosis (wt-ATTR, mt-ATTR), patientsurvival correlated with NYHA functional class, dia-stolic blood pressure, and medication with diureticagents. Karnofsky index, troponin T, QRS duration,LVEF, and impaired right ventricular functionshowed p values <0.1 and were included in the sub-sequent multivariate Cox regression analysis, whichrendered NYHA functional class as the only inde-pendent predictor of patient survival (Table 2).

DISCUSSION

The present data demonstrate that EMBs revealedimportant information beyond histological diagnosisin AL amyloidosis. Quantification of amyloid load atdiagnosis indicated survival benefit of patients withan amyloid load <20%. In contrast, no survivalbenefit for future chemotherapy was observed ifamyloid load was $20% even with hematologicalresponse. In general, cardiac manifestation is com-mon in AL and ATTR amyloidosis (26). More than one-half of the patients with AL amyloidosis present withcardiac symptoms at diagnosis. Our study confirmedsome distinct differences between AL and ATTRamyloidosis, including lower LV mass but higher NT-proBNP values in AL amyloidosis (7), most likelyrelated to toxic effects of amyloidogenic light chainscausing apoptosis of cardiomyocytes (27). Differ-ences observed between patients with mt- andwt-ATTR, including eGFR and medication use, wereprobably related to patient age. Additionally, disease-specific characteristics of wt-ATTR included higherage at diagnosis, more commonly male sex, andhigher prevalence of atrial fibrillation.

HISTOLOGICAL DIAGNOSIS AND DIFFERENTIATION

OF CARDIAC AMYLOIDOSIS. Despite diagnostic im-provements, including CMR imaging, echocardiogra-phy with speckle tracking, and skeletal scintigraphy(10,28,29), EMB remains a crucial step in the diag-nostic work-up of unexplained cardiac hypertrophyand the gold standard for diagnosing cardiacamyloidosis (30). Moreover, distinct patterns of am-yloid aid in the differentiation of AL (reticular, peri-cellular) and ATTR (patchy) amyloid, to some extent

FIGURE 2 Survival

1.0

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0.8

0.6

0.4

0.2

0.0

0 12 24 36 48 60 72 84 96 108 120 132 144Time Since Biopsy [Months]

Over

all S

urvi

val

1.0

0.8

0.6

0.4

0.2

0.0

0 12 24 36 48 60 72 84 96 108 120 132 144Time Since Biopsy [Months]

Over

all S

urvi

val

# at Risk:284719

14469

5317

5125

553

353

332

222

111

110

110

100

000

# at Risk:612

110

05

05

05

03

03

02

01

01

01

01

00

# at Risk:5

285

20311

28

13

13

12

11

01

01

01

00

00

# at Risk:3

1016

05

04

03

03

02

02

01

00

00

00

00

AL Patients with Therapy Amyloid Load <20%

Amyloid Load 20-40% Amyloid Load >40%

Amyloid Load Response

Response Response

<20%20-40%>40%<20%-censored20-40%-censored>40%-censored

Non-Responder (NR / PD)Responder (CR / VGPR / PR)Non-Responder (NR / PD)-censoredResponder (CR / VGPR / PR)-censored

Non-Responder (NR / PD)Responder (CR / VGPR / PR)Non-Responder (NR / PD)-censoredResponder (CR / VGPR / PR)-censored

Non-Responder (NR / PD)Responder (CR / VGPR / PR)Non-Responder (NR / PD)-censoredResponder (CR / VGPR / PR)-censored

A B

C D

Amyloid load had a significant effect on survival: in the full patient population with light-chain amyloid (AL) independent of any treatment and response to

treatment (n ¼ 107) (A), patients with a 20% to 40% amyloid load had a significantly longer survival than those patients with smaller or larger loads.

Consideration of therapeutic response in 94 patients receiving chemotherapy showed that AL patients with <20% amyloid had a significant difference in

overall survival between responders and nonresponders (B). To the contrary, no difference in overall survival was found between responders and non-

responders in patients with 20% to 40% (C) and >40% amyloid load (D). CR ¼ complete response; NR ¼ nonresponders; PD ¼ progressive disease;

PR ¼ partial response; VGPR ¼ very good partial response.

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even without immunohistochemical staining. How-ever, at least in the setting of ATTR subjects who havea concomitant plasma cell dyscrasia, a misdiagnosisrate of 10% was reported despite application ofimmunohistochemistry (31). In our center, combineduse of clinical examination and specialized pathologyyielded a high accuracy for correct amyloid typing(22).

Due to potential lack of therapeutic implications aswell as the latent risk of pericardial effusion, physi-cians often avoid performing cardiac biopsies despitestrong suspicion of amyloidosis. However, due toimprovement of biopsy forceps, LV EMBs can be per-formed easily and safely (32). In our center, no

fatal complication occurred after restriction to LVbiopsies.

All primary antibodies used in the present studywere highly specific to detect a single amyloid pre-cursor protein (21,33). Digital conversion of immu-nohistochemically stained areas appears to representthe extent of amyloid deposited in this particular vi-sual field. However, whether the amyloid burdenobtained from a visual field of an EMB representswhole cardiac amyloid load precisely remains debat-able. A comparable software-based method forquantification of amyloid deposits has been reportedpreviously in whole hearts obtained from hearttransplantation or autopsies (34). Unfortunately, we

CENTRAL ILLUSTRATION Amyloid Load: Predictive Biomarker in AL Amyloidosis

Kristen, A.V. et al. J Am Coll Cardiol. 2016;68(1):13–24.

In a retrospective analysis of histological amyloid load for effect onmortality, amyloid loaddid not exceed60%,potentially indicating themaximal tolerable cardiac amyloid load.

Patients with light-chain (AL) amyloid with <20% amyloid had a significant difference in overall survival between responders and nonresponders (NR), whereas no overall

survival difference was found at higher levels of amyloid load. CR¼ complete response; PD¼ progressive disease; PR¼ partial response; VGPR¼ very good partial response.

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were unable to compare biopsy and autopsy resultsdue to lack of autopsy cases. Generally, a diffuse,predominantly subendocardial LGE was indicative ofcardiac amyloid (28,35,36). LGE was associated withamyloid deposition in explanted hearts (37), anddiverse patterns of amyloid were reported inexplanted and autopsy hearts, including diffuse,segmental, and subendocardial infiltration (34). Inthis series, the extent of amyloid infiltration of theright ventricle was higher in AL amyloidosis than inATTR amyloidosis. No differences between AL and

ATTR amyloidosis were reported regarding LV amy-loid deposition (34).AMYLOID LOAD AND SEVERITY OF CARDIAC

MANIFESTATION. In the present study, amyloid loadwas equally distributed in the different amyloidsubtypes, but in only 1 specimen (a patient withwt-ATTR amyloidosis) was amyloid load higher than60%, potentially indicating the maximal tolerablecardiac amyloid load. Amyloid load is claimed to behigher in ATTR rather than AL amyloidosis despiteminor symptoms. In the present study, higher

TABLE 2 Prognostic Factors for Cardiac Amyloidosis

AL Amyloid(n ¼ 107)

AL Amyloid With Therapy(n ¼ 94)

ATTR Amyloid (mt þ wt)(n ¼ 109)

UnivariateCox Regression

MultivariateCox Regression

UnivariateCox Regression

MultivariateCox Regression

UnivariateCox Regression

MultivariateCox Regression

HR (95% CI) p Value HR (95% CI) p Value HR (95% CI) p Value HR (95% CI) p Value HR (95% CI) p Value HR (95% CI) p Value

Male 1.981 (1.176–3.338) 0.010 2.246 (1.267–3.984) 0.006

Karnofsky index 0.938 (0.911–0.966) <0.001 0.948 (0.919–0.979) 0.001 0.961 (0.923–1.000) 0.051

NYHA functionalclass

0.001 0.063 0.010 0.059 0.002 0.002

II vs. I 1.965 (0.557–6.933) 0.294 0.679 (0.076–6.026) 0.728 2.083 (0.589–7.366) 0.255 0.675 (0.076–6.002) 0.724 2.919 (0.631–13.51) 0.171

III vs. II 2.559 (1.455–4.501) 0.001 3.814 (1.223–11.900) 0.021 2.231 (1.236–4.027) 0.008 3.876 (1.240–12.11) 0.020 1.534 (0.725–3.246) 0.264 2.391 (0.927–6.166) 0.071

IV vs. III 1.721 (0.235–12.610) 0.593 1.995 (0.270–14.740) 0.499 10.25 (2.246–46.730) 0.003 14.34 (2.723–75.640) 0.002

Diastolic bloodpressure

0.974 (0.956–0.991) 0.004 0.976 (0.957–0.995) 0.014 0.958 (0.927–0.991) 0.014

eGFR 0.985 (0.977–0.993) <0.001 0.962 (0.944–0.981) <0.001 0.986 (0.977–0.995) 0.002 0.961 (0.942–0.980) <0.001

log10 ofNT-proBNP

2.094 (1.181–3.713) 0.011 1.811 (1.002–3.274) 0.049

Abnormal TnT 3.897 (0.915–16.600) 0.066

MR antagonist 2.106 (1.294–3.427) 0.003 1.815 (1.067–3.088) 0.028

Diuretic agents 2.430 (1.016–5.815) 0.046

QRS duration 1.009 (0.998–1.021) 0.099 1.008 (0.999–1.016) 0.075

Low voltage 1.845 (1.143–2.981) 0.012

LA diameter 1.033 (0.995–1.072) 0.092

LV ejectionfraction

0.981 (0.960–1.003) 0.086

Impaired RVfunction

2.467 (0.990–6.147) 0.053

Amyloid load 0.028 0.002 0.013 0.001

20%–40%vs. <20%

0.503 (0.293–0.863) 0.013 0.566 (0.229–1.399) 0.218 0.447 (0.250–0.799) 0.007 0.537 (0.221–1.306) 0.170

>40% vs. <20% 0.923 (0.516–1.652) 0.788 5.615 (1.589–19.840) 0.007 0.946 (0.507–1.766) 0.863 5.287 (1.546–18.080) 0.008

>40% vs.20%–40%

1.837 (1.022–3.304) 0.042 9.913 (2.841–34.590) <0.001 2.118 (1.121–4.003) 0.021 9.849 (2.864–33.860) <0.001

Chemotherapy 0.374 (0.190–0.735) 0.004 0.353 (0.134-0.934) 0.036

Responder 0.516 (0.249–1.072) 0.076 0.516 (0.249–1.072) 0.076 0.350 (0.133–0.923) 0.034

Hazard ratios (HRs) of continuous variables are given per unit of measurement. Variables having p values #0.100 in univariate Cox regressions are listed in the Univariate Cox regression columns and were included into multivariate Cox regressions, respectively.

HR ¼ hazard ratio; other abbreviations as in Table 1.

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amyloid load was observed in ATTR amyloidosis,although the difference was not statistically signifi-cant. This pathological observation aligned withprevious imaging data (12).

In our series, amyloid load correlated with someindicators of severity of cardiac amyloidosis (NT-proBNP and LV wall thickness) in patients with ATTRamyloidosis but not inAL amyloidosis (15). However, inAL amyloidosis, amyloid load correlated with patientsurvival. This might be explained by selection bias, asour patients had exclusively histologically provencardiac involvement in contrast to other studies thatanalyzed patients independent of organ manifesta-tion. Correlation of troponin T would be of remarkableinterest, as it has been reported to be a strong predictorof cardiac amyloidosis severity (14). Unfortunately,results were not available due to a change of troponinassays during the sample collection.

In general, EMBs carry the risk of a sampling error,which might explain the missing correlation withnoninvasive indicators of severity of cardiacamyloidosis. Skeletal scintigraphy and CMR imagingwith T1 mapping were alternative tools for noninva-sive quantification of cardiac amyloid (11). Bonetracers are capable of differentiating and quantifyingcardiac ATTR amyloid. T1 mapping allows detectionof even minor changes of the myocardial texture.Unfortunately, data were not available for the presentcohort due to the long observation period.

AMYLOID LOAD AND SURVIVAL. In the presentstudy, patients with AL amyloidosis had a signifi-cantly worse prognosis over ATTR amyloidosis (me-dian overall survival: 15.7 months vs. $38.9 months)(Table 1). In general, AL exhibits poorer survival thanATTR amyloidosis (38). However, according to morerecent reports on survival of patients with ATTRamyloidosis, distinct differences between individualmutations were reported. Shorter survival wasobserved in patients with mainly cardiac manifesta-tion due to p.Val142Ile transthyretin gene variant,and survival of patients with wt-ATTR amyloidosisappears to be worse than expected (39,40). However,in our cohort, NYHA functional class was the onlyindependent prognosticator of patient survival.Presence of cardiac amyloid itself might be associatedwith poorer survival in ATTR; however, this wasbeyond the scope of the current analysis.

Besides diagnostic purposes (30), our studydemonstrated that quantification of AL amyloid loadin EMBs may have prognostic and predictive value forfuture treatment. Following multivariate analysis,amyloid load remained an independent predictor ofpatient survival: patients with cardiac amyloid >40%

had a worse prognosis. With regard to the predictivevalue, Kaplan-Meier plots illustrated a significantdifference between responders and nonresponderswhen the amyloid load was <20%. No difference wasfound in patients with $20% amyloid load. Thus,early identification of subjects with AL amyloid isessential, as our preliminary data suggested that inlate stages of disease with extensive amyloid load,chemotherapy administration does not affect out-comes. This finding may illustrate different mecha-nisms of cardiac damage. It has long been recognizedthat cardiac impairment in AL amyloidosis is relatedto the toxic effects light chains exert on car-diomyocytes. This effect is probably most effective atthe early stage of the disease (i.e., when amyloid loadis <20%). However, in more advanced cardiac ALamyloidosis, the deposits increasingly exert biome-chanical effects on cardiac function, resulting inimpaired diastolic blood pressure.

On the basis of our data, we hypothesized that 20%to 40% cardiac amyloid load marks a threshold atwhich the primarily toxic and primarily biomechaniceffects of cardiac AL amyloid are temporarilybalanced out. This conjecture was supported by thelower median NT-proBNP serum levels observed inpatients with AL who had 20% to 40% (6,989 ng/ml)amyloid load compared with patients with <20%(8,613 nm/ml) or >40% (8,746 ng/ml) (Online Table 1).Moreover, quantitative amyloid load in EMBs appearsto identify patients who are at high risk for fataloutcome and, hence, might be an additional param-eter for selecting patients for heart transplantation ifsolely cardiac manifestation is present (41).

According to the present study, in ATTR amyloid-osis, EMBs did not reveal any benefit. Noninvasivemethods using skeletal scintigraphy show highsensitivity and specificity for identifying cardiacATTR amyloid, and no additional benefit of amyloidquantification was reported. Thus, EMBs appear notto be needed in these patients, when the diagnosiswas already reached by other measures.

STUDY LIMITATIONS. This was a retrospective anal-ysis of a large cohort of patients with cardiacamyloidosis confirmed by EMB. It was limited by thelack of skeletal scintigraphy, CMR imaging with T1

mapping, echocardiography with speckle tracking,and high-sensitivity troponin T as potential diagnostictools for early manifestation of cardiac amyloidosis.Moreover, evaluation of response status was notachievable for 30 subjects due to limited survival ofpatients with cardiac amyloidosis. Thus, the power ofsurvival analyses needs to be confirmed prospectivelyin an independent, larger patient cohort.

PERSPECTIVES

COMPETENCY IN PATIENT CARE AND PROCEDURAL

SKILLS: In patients with AL amyloidosis, evaluation of the

cardiac amyloid load by EMB has both diagnostic and prognostic

value, as those with high loads gain less benefit from available

chemotherapy.

TRANSLATIONAL OUTLOOK: Further studies are needed

to determine whether selection of patients with AL

amyloidosis for chemotherapy or transplantation can be

guided effectively by quantification of the endomyocardial

amyloid burden.

J A C C V O L . 6 8 , N O . 1 , 2 0 1 6 Kristen et al.J U L Y 5 , 2 0 1 6 : 1 3 – 2 4 Amyloid Load and Prognosis in AL Amyloidosis

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CONCLUSIONS

Early identification of subjects with AL amyloid isessential, as in late stages of disease with extensiveamyloid load, preliminary data suggest that chemo-therapy does not affect outcomes. These results needto be confirmed in an independent cohort of ALamyloidosis and a larger group of patients withdifferent types of ATTR amyloidosis.

REPRINT REQUESTS AND CORRESPONDENCE: Dr.Arnt V. Kristen, Department of Cardiology, Angiology, andRespiratory Medicine, University Hospital Heidelberg, ImNeuenheimer Feld 410, D-69120 Heidelberg, Germany.E-mail: Arnt.Kristen@med.uni-heidelberg.de.

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KEY WORDS amyloidosis, endomyocardialbiopsy, immunohistochemistry, light-chainamyloid, survival, transthyretin

APPENDIX For an expanded Methods sectionas well as supplemental figures and a table,please see the online version of this article.