Normalisation of total body iron load with very intensivecombined chelation reverses cardiac and endocrinecomplications of thalassaemia major

At one time, thalassaemia major, a hereditary anaemia, had

such a poor prognosis that patients died in childhood (Engle

et al, 1964). Although transfusion therapy improved the

severity of the disease, it resulted in a positive iron balance

and secondary haemosiderosis, often leading to vital organ

damage and dysfunction in the second decade of life (Fink,

1964). The systematic use of iron chelation therapy since the

1970s has increased patients’ life expectancy (Modell et al,

2000) and delayed iron-overload complications (Gabutti &

Piga, 1996). However, even with the introduction of desfer-

rioxamine (DFO) as an iron chelator, cardiac failure due to

iron overload still accounted for 67% of deaths in thalassaemia

major, and many patients experienced endocrinopathies

secondary to iron overload (Borgna-Pignatti et al, 2004;

Cunningham et al, 2004).

Desferrioxamine was the first iron chelation treatment used

in thalassaemia. The effectiveness of this parenterally admin-

istered chelator is limited partly by poor patient compliance

(Caro et al, 2002). In addition, because cell membranes are

relatively impermeable to DFO and DFO-iron complexes,

serious cardiac, hepatic and endocrine complications continue

to arise, even in compliant patients (Borgna-Pignatti et al,

2004; Cunningham et al, 2004).

Deferiprone (DFP), an orally administered iron chelator

licenced in Europe in 1999, has a lower molecular weight than

DFO and a neutral charge when complexed with iron;

Kallistheni Farmaki,1 Ioanna Tzoumari,1

Christina Pappa,1 Giorgos Chouliaras2

and Vasilios Berdoukas2

1Transfusion Department and Thalassaemia Unit,

General Hospital of Corinth, Corinth, and2Thalassaemia Unit, 1st Dept of Paediatrics,

University of Athens, ‘‘Aghia Sophia’’ Children’s’

Hospital, Athens, Greece

Received 2 July 2009; accepted for publication

11 September 2009

Correspondence: Dr Kallistheni Farmaki,

Transfusion Department and Thalassaemia

Unit, General Hospital of Corinth, Leoforos

Athinon 53, Corinth 20100, Greece.

E-mail: stheni@otenet.gr

Summary

Cardiac and endocrine disorders are common sequelae of iron overload in

transfused thalassaemia patients. Combined chelation with desferrioxamine

(DFO) and deferiprone (DFP) is well tolerated and produces an additive/

synergistic effect superior to either drug alone. 52 thalassaemia major patients

were transitioned from DFO to combined chelation with DFO and DFP.

Serum ferritin, cardiac and hepatic iron levels were monitored regularly for

up to 7 years, as were cardiac and endocrine function. Patients’ iron load

normalized, as judged by ferritin and cardiac and hepatic magnetic resonance

imaging findings. In all 12 patients receiving treatment for cardiac

dysfunction, symptoms reversed following combined chelation, enabling

nine patients to discontinue heart medications. In the 39 patients with

abnormal glucose metabolism, 44% normalized. In 18 requiring thyroxine

supplementation for hypothyroidism, 10 were able to discontinue, and four

reduced their thyroxine dose. In 14 hypogonadal males on testosterone

therapy, seven stopped treatment. Of the 19 females, who were hypogonadal

on DFO monotherapy, six were able to conceive. Moreover, no patients

developed de novo cardiac or endocrine complications. These results suggest

that intensive combined chelation normalized patients’ iron load and thereby

prevented and reversed cardiac and multiple endocrine complications

associated with transfusion iron overload.

Keywords: thalassaemia, combined chelation, iron overload, cardiomyo-

pathy, endocrinopathy.

research paper

First published online 12 November 2009doi:10.1111/j.1365-2141.2009.07970.x ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 148, 466–475

it therefore mediates the efficient removal of excess cellular and

organelle iron (Sohn et al, 2008). DFP also neutralizes labile

iron in the plasma and within cells, thus preventing formation

of cytotoxic reactive oxygen species (Zanninelli et al, 1997).

A number of clinical studies demonstrated the ability of DFP

to increase iron excretion in patients with iron overload

(Hoffbrand et al, 2003; Hoffbrand, 2005). Compliance was

higher than with DFO and DFP treatment was associated with

an improved cardioprotective effect compared to DFO (Piga

et al, 2003; Borgna-Pignatti et al, 2006).

Combined chelation with DFO and DFP has been used since

1998 (Wonke et al, 1998). Studies in vitro and on urinary iron

excretion demonstrated an additive or synergistic effect of the

two chelators (Giardina & Grady, 2001; Link et al, 2001).

Various combination schemes have been developed in order to

maximize treatment efficacy, tolerability and compliance and

to prevent the development of iron-load complications.

In the present longitudinal, observational study of intensive

combined chelation in thalassaemia major, the primary

endpoint was reversal of total body iron overload. We also

sought to analyse the impact of combined chelation on pre-

existing morbidities and on the development of new transfu-

sion-associated complications.

Patients and methods

Our centre sought and received Ethics Committee approval to

administer intensive chelation therapy to our patients as it had

recently been reported as being the most effective way of

reducing body iron overload (Wonke et al, 1998). All patients

provided informed consent to the treatment. In addition we

sought and received permission to review the charts and analyse

the data on the patients with respect to efficacy and safety.

In our unit, thalassaemic patients are transfused at intervals

of 10–20 d, with white cell depleted, genotyped packed red

blood cells, in order to maintain haemoglobin (Hb) levels

‡100 g/l. Until the start of 2000 all patients received mono-

therapy with DFO (approximately 40 mg/kg per d) by sub-

cutaneous infusions over 8–12 h via portable pumps that were

worn at least 5 d/week. After 2000, based on the evidence of

continued iron overload, several patients were transitioned to

intensive chelation therapy with combination therapy of DFO

(20–60 mg/kg per d) by subcutaneous infusions and DFP

(75–100 mg/kg per d in three divided doses). Subsequently, this

form of therapy was prescribed as a first option for chelation

therapy and, by the end of 2002, all patients in this study were

receiving that therapy. Contrary to other investigators, we

maintained combined chelation until the achievement of

normal ferritin levels and until clearance of heart and liver

iron, as measured by magnetic resonance imaging (MRI). Once

total body iron had normalized, the frequency and dose of DFO

was reduced in order to avoid potential toxicity. In general the

DFP dose was maintained or reduced to 75 mg/kg per d.

Patient life style was taken into account to assist with the

compliance to DFO. Where appropriate, the patients were

offered balloon pumps (Accufusers, Diafuser Pumps). Also

self-management was fortified, and patients were involved in

decision-making. Compliance, defined as the ratio of actual

chelation treatments to recommended treatments, was calcu-

lated on a monthly basis.

Patients were monitored regularly for the emergence of

adverse events, as follows: complete blood count (CBC) with

absolute neutrophil count (ANC), every 7–15 d; liver enzymes

and renal function, bimonthly; audiogram and ophthalmology

testing, yearly. In the event of joint symptoms, gastrointestinal

upset, or increased liver enzymes, DFP dose could be

transiently reduced and symptomatic treatment implemented.

DFO treatment was transiently interrupted in case of tinnitus

or ocular problems. With topical reactions, DFO dose could be

transiently reduced. Combined chelation was withheld in

patients experiencing fevers.

Chelator doses and frequency of DFO infusion were

adjusted at least every 6–12 months, on the basis of patients’

body weight and adverse events, as well as clinical findings on

whole-body or tissue-specific iron load. In patients <20 years

old, the dose of DFO was limited to £40 mg/kg.

Iron load studies

Ferritin was calculated as the mean of monthly determinations

by chemiluminescent microparticule immunoassay (CMIA).

The normal range of ferritin is between 50 and 120 lg/l.

Hepatic and cardiac iron were quantified annually, initially

using Signa-MRI 1.5 Tesla, multiple-echo T2. Subsequently,

T2* sequences were performed (CVi; General Electric Signa,

Milwaukee, WI, USA). In parallel, cardiac function was

examined by cardiac magnetic resonance (CMR). Normal

values for T2 heart (T2H) and T2* heart (T2*H) were >35 and

>20 ms respectively; for T2 liver (T2L) and T2* liver (T2*L),

were >33 and >19 ms, respectively.

Liver Iron concentration (LIC) in mg/g dry weight (dw) was

determined by MRI (St Pierre et al, 2005; Wood et al, 2005).

Normal LIC was <1Æ5 mg/g dw.

Cardiac and endocrine function

Cardiac and endocrine function were longitudinally assessed

from baseline (the time of transition to intensified chelation

regime) until treatment discontinuation or the end of 2007.

Cardiac function was assessed at least annually, with tissue

Echo-Doppler TD (Philips ie33 system, Philips Ultrasound,

Bothell, WA, USA). Patients were classified according to the

New York Heart Association (NYHA) criteria (Dolgin 1994).

At annual intervals, patients’ endocrine function was

assessed, including evaluation of glucose metabolism and the

development of diabetes or related subclinical states (increased

fasting glucose or impaired glucose tolerance).

Except for those with insulin-treated diabetes mellitus

(ITDM), all patients underwent an annual oral glucose

tolerance test (OGTT). Patients were challenged with 1Æ75 g

Complication Reversal in Thalassaemia

ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 148, 466–475 467

glucose/kg body weight, up to a maximum of 75 g, in the

morning after a 12-h fast. Samples were taken at 0, 30, 60, 90

and 120 min for glucose and insulin evaluation. The area

under the curve (AUC) was assessed for glucose and insulin.

Homeostasis model assessment (HOMA) was used to quantify

pancreatic b cell function (insulin secretion; SCHOMA) and

insulin resistance (ISIHOMA) (Angelopoulos et al, 2006).

Patients who were not insulin treated were classified according

to their OGTT status. Thus, Normal Glucose Metabolism was

defined as fasting glucose < 6Æ1 mmol/l and 2-h glucose

level < 7Æ75 mmol/l; Impaired Fasting Glucose (IFG) as

6Æ1 mmol/l ‡ fasting glucose £ 7Æ0 mmol/l; Impaired Glucose

Tolerance (IGT) as 6Æ1 mmol/l ‡ fasting glucose £ 7Æ0 mmol/l

and 7Æ75 mmol/l ‡ 2-h glucose level £ 11Æ1 mmol/l; and

Non-Insulin Dependent Diabetes (NIDDM) as fasting

glucose > 7Æ0 mmol/l and 2 h glucose level > 11Æ1 mmol/l.

Thyroid function was assessed by thyroid-stimulating

hormone (TSH), triiodothyronine (T3) and thyroxine (T4)

screening and by the thyroid releasing hormone (TRH)

stimulation test, conducted at baseline and annually thereafter

for 5–7 years. Following intravenous infusion of 200 lg TRH,

blood samples were taken at 0, 30, 60, 90 and 120 min for

TSH. Patients discontinued thyroxin at least 30 d before the

test. Criteria for the diagnosis of subclinical or compensated

hypothyroidism was an increase of the TSH levels during the

test of more than 20 li/u per ml from the basal value or an

elevated basal TSH concentration (>5 li/u per ml) and for

overt hypothyroidism a further decrease in FT4 and FT3 levels.

Gonadal function was assessed by peripheral hormone levels:

testosterone and free testosterone or oestradiol and progester-

one. In addition, serum basal levels of follicle-stimulating

hormone (FSH) and luteinizing hormone (LH) were assayed.

Gonadotrophin response was also assayed after intravenous

infusion of 200 lg gonadotrophin-releasing hormone

(GnRH). Samples were taken at 0, 30, 60, 90 and 120 for the

measurement of FSH and LH.

All analyses were performed by CMIA technology using the

automatic immuno-analyzer ARCITECT, i2000SR, (Abbott

Laboratories, Abbot Park, IL, USA).

Statistical analysis

Continuous variables are presented as mean ± standard devi-

ation (SD), whereas discrete variables are described using

absolute and relative frequencies. Means were compared by

t-test or Mann–Whitney test in cases of small samples.

Proportions were compared by Fisher’s exact test. Trend

analyses of continuous variables in cases of repeated measure-

ments over time were performed using PROC MIXED with the

sas Statistical Software, Version 8 or the non parametric

Friedman and Wilcoxon test. A P-value of <0Æ05 was considered

significant. All statistical analyses were carried out using the

Statistical Package for the Social Sciences (spss) software (SPSS

release 13.0, Chicago, IL, USA) or sas Statistical Software,

Version 8 (SAS Institute Inc. Cary, North Carolina, USA).

Results

Fifty-two patients, aged 10–49 years at baseline, were followed

over a period of 5–7 years. The mean age at baseline was

25Æ2 ± 8Æ9 years compared to 32Æ9 ± 9Æ8 at the end of the

study. There were 25 males and 27 females at baseline. Two

patients (one male, one female) discontinued prior to the end

of the study (31/12/2007) because of concerns about repeated

episodes of neutropenia. Their observations were censored

from the 14th and 18th month.

Body iron

There was a statistically significant reduction of the total body

iron load, as indicated by the ferritin levels, cardiac and liver

iron (Table I).

Analysis of repeated blood measurements revealed a

cumulative decrease in serum ferritin, with a rate of decline

of 85 lg/l per month (P < 0Æ001). After 1 year on combination

therapy, 53% of our patients had ferritin levels <1000 lg/l, and

after 2 years this increased to 85%. At 5 years, 90% of patients

had ferritin values within normal limits.

At baseline, 98% of patients had hepatic iron overload

(LIC ‡ 1Æ5 mg/g dw), and 64% had severe iron overload

(LIC > 12 mg/g dw). After 3 years of intensive combined

chelation, these proportions declined to 60% and 10%,

respectively. By 5 years, none of the 50 patients remaining

on the study had iron overload. This shift to milder hepatic

iron load over time was significant (Fisher’s exact test

P < 0Æ001). Thus the longer the patients were on intensive

combined chelation, the greater the decrease in body iron.

At baseline, 82% of patients presented with cardiac iron

overload (T2* < 20 ms), and of those, 36% had severe

overload (T2* < 8 ms). By the end of the study, only two

patients had mild to moderate and only one severe cardiac iron

load. The overall change from cardiac iron loaded to non-iron

loaded was significant (Fisher’s exact test P < 0Æ001).

Cardiac function

As judged by left ventricular ejection fraction (LVEF), 18

(35%) of patients had cardiac dysfunction at baseline (six with

Table I. Results (mean ± SD) of iron load assessments in all 52

patients.

Parameter Baseline studies End of study P-value

Ferritin (lg/l) 3421Æ6 ± 882Æ0 87 ± 25 <0Æ001

MRI T2H (ms) 28Æ2 ± 5Æ6 38Æ1 ± 4Æ2 <0Æ001

MRI T2*H (ms) 13Æ8 ± 9Æ8 35Æ5 ± 8Æ1 <0Æ001

MRI T2L (ms) 22Æ7 ± 5Æ2 37Æ2 ± 6Æ6 <0Æ001

MRI T2*L (ms) 1Æ5 ± 8Æ2 34Æ4 ± 5Æ4 <0Æ001

LIC (mg/g dw) 15Æ7 ± 11Æ1 0Æ9 ± 0Æ2 <0Æ001

MRI, magnetic resonance imaging; LIC, liver iron concentration.

K. Farmaki et al

468 ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 148, 466–475

NYHA Class I heart failure (not on medication), seven with

Class II, three with Class III and two with Class IV). In these 18

patients, mean LVEF increased from 54Æ20 ± 0Æ06% to

67Æ60 ± 0Æ07% (P < 0Æ001). The percentage of patients with

normal cardiac function increased from 64% to 94% by the

end of the study (Fisher’s exact test, P = 0Æ001).

In all, 15 of the 18 patients with cardiac dysfunction at

baseline achieved normal function, and the remaining three

(two of Class IV and one of Class III) improved to Class II.

Moreover, none of the 34 patients with normal cardiac

function at baseline deteriorated, and mean LVEF in this

patient subset increased from 63Æ80 ± 0Æ05% to 72Æ10 ± 0Æ07%

(P < 0Æ001).

Twelve patients were on cardiac medications (angiotensin-

converting enzyme [ACE] inhibitors, anti arrhythmics, digoxin

or diuretics), nine of whom were able to discontinue

medications when their symptoms abated and their cardiac

parameters and function returned to normal. The three

patients with residual cardiac dysfunction also failed to achieve

normalisation of their total body iron load, apparently a

consequence of their relatively poor compliance to DFO and

DFP treatment and their failure to follow recommended

chelator dosage. In two of these patients, ferritin levels

decreased to <500 lg/l, but they still had a moderate hepatic

iron load and a severe to moderate iron load in the heart.

Endocrine function

Glucose metabolism. While on DFO monotherapy, 78% of

patients had glucose metabolism abnormalities, either with

insulin treated diabetes or as determined by the OGTT, but

this proportion declined to 34% after 5–7 years of combined

chelation. Overall, patients experienced a significant shift

(Fisher’s exact test, P < 0Æ001) to either less severe forms of

glucose abnormality or to complete normality, according to

World Health Organisation and the American Diabetes

Association criteria (Table II) (Melchionda et al, 2002).

Following intensive combined chelation, we observed a

statistically significant decrease in post-challenge glycaemia

(Fig 1; P < 0Æ001), and a significant increase in insulin

secretion (P < 0Æ005) and SCHOMA.(Fig 2; P < 0Æ005) Insulin

sensitivity, as judged by ISIHOMA, improved in parallel

(P < 0Æ001). This improvement persisted over time for both

glycaemia (P = 0Æ002) and insulin secretion SCHOMA

(P = 0Æ004), despite the fact that patients experienced a

significant increase in body mass index (BMI) from baseline

to the end of the study (21Æ3 ± 2Æ8 kg/m2 vs. 23Æ4 ± 1Æ9 kg/m2,

P < 0Æ001).

Thyroid function. Of the 50 patients completing the study, 18

(36%) were treated with thyroxinee replacement therapy at

baseline while on DFO monotherapy. After combined

chelation and an important decrease in total body iron

overload 14/18 who had subclinical or compensated

Table II. Distribution of patients according to their glucose metabo-

lism status according to the World Health Organisation and the

American Diabetes Association criteria, at the start and end of the

study period.

Glucose metabolism status DFO monotherapy After combined

Insulin-treated diabetes 6 (12%) 6

Non-insulin dependent diabetes 14 (28%) 5 (10%)

Impaired glucose tolerance 16 (32%) 6 (12%)

Impaired fasting glucose 3 (6%) 0 (0%)

Normal 11 (22%) 33 (66%)

DFO, desferrioxamine.

30 000

25 000

20 000

15 000

10 000

5000

0NIDDM, n = 14 IGT & IFG, n = 19 Normal, n = 11 All pts, n = 44

Before combination therapyAfter combination therapy

AU

C G

luco

se

Fig 1. Post-challenge glycaemia before and after intensive combined chelation in patients with thalassaemia major. Based on their oral

glucose tolerance test (OGTT) data at baseline, patients were characterized as having non Insulin-dependent diabetes mellitus (NIDDM), impaired

glucose tolerance (IGT) and impaired fasting glucose (IFG) or normal glucose tolerance. Mean ± SD glycaemia is calculated as the area under

the glucose curve (AUC Glucose) following challenge with 75 g glucose in the fasting state. (Insulin Treated Diabetic patients were not tested by

OGTT).

Complication Reversal in Thalassaemia

ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 148, 466–475 469

hypothyroidism presented a significant increase in mean FT4

(0Æ70 ± 0Æ06 vs. 1Æ07 ± 0Æ12 ng/ml, P < 0Æ001) and mean FT3

(1Æ30 ± 0Æ3 vs. 2Æ50 ± 0Æ6 pg/ml, P < 0Æ001) and an additional

significant decrease in the mean TSH (6Æ27 ± 1Æ08 vs.

4Æ12 ± 0Æ63 li/u per ml P < 0Æ001) and TSH quantitative

secretion, calculated as the AUC (2231 ± 241 vs. 1332 ± 131

P < 0Æ001) in response to TRH stimulation. Among them,

10/18 (56%) discontinued thyroxine therapy (Fisher’s exact

test P < 0Æ001) and 4/18 (22%) reduced their thyroxine

dose. The remaining four (8% of the total study group)

who had biochemically overt hypothyroidism, while they all

improved their TRH stimulation test, only two converted to

compensated hypothyroidism with TSH levels 5–10 mi/u per

ml and normal FT4 and FT3 levels.

In addition, in the other 32/50 euthyroid patients, no new

cases of hypothyroidism were noted after combined chelation

and a significant increase was observed in the mean FT4

(0Æ80 ± 0Æ09 vs. 1Æ10 ± 0Æ09 ng/ml P < 0Æ001) and FT3 levels

(1Æ6 ± 0Æ2 vs. 2Æ9 ± 0Æ5 pg/ml, P < 0Æ001).

Gonadal function. Overall, 70% of adult thalassaemic patients,

including 14/24 males and 19/26 females, were hypogonadal on

DFO monotherapy. Of the 14 males on testosterone replacement

therapy for hypogonadism, only seven remained on this therapy

at the end of the study (Fisher’s exact test P = 0Æ08). The

remaining seven (50%), after combined chelation and a

significant decrease of total body iron overload, achieved

normal mean testosterone levels, normalized their LH–FSH

response as shown by their GnRH response and were able to

discontinue testosterone injections (Table III). Of note, two of

these individuals became fathers (one of twins) without

hormonal stimulation. No new cases of hypogonadism were

observed, and in previously eugonadal males the mean

testosterone level increased significantly (5Æ71 ± 0Æ55 vs.

7Æ67 ± 0Æ63 ng/ml; P < 0Æ001) and LH responses to GnRH

improved (P = 0Æ05), although FSH levels remained unchanged.

At baseline, there were 19 women on hormone replacement

therapy for hypogonadism, nine with primary amenorrhea and

10 with secondary amenorrhea. After intensive combined

240220200180160140120100806040200

Before combination therapyAfter combination therapy

NIDDM, n = 14 IGT & IFG, n = 19 Normal, n = 11 All pts, n = 44

SC

HO

MA

Fig 2. Post-challenge insulin secretion before and after intensive combined chelation in patients with thalassaemia major as shown by SCHOMA, an

index of Homeostasis Model Assessment (HOMA), indicative of pancreatic b cell function and insulin secretion.

Table III. Differences in age, ferritin, liver iron

concentration and hormonal parameters

between male patients whose gonadal function

improved to the point of no longer needing

hormone replacement therapy and those whose

did not.

Males still requiring replacement

therapy (n = 7)

Males no longer requiring

replacement therapy (n = 7)

Baseline After combined Baseline After combined

Age (years) 35Æ3 ± 8Æ5 42Æ3 ± 8Æ5 25Æ1 ± 7Æ1 32Æ1 ± 7Æ1Ferritin (lg/l) 3880 ± 2947 704 ± 1353 2749 ± 2090 85 ± 51

MRI T2L (ms) 4Æ3 ± 3Æ8 25Æ9 ± 12Æ9 7Æ7 ± 4Æ5 36Æ9 ± 4Æ2LIC (mg/g dw) 26Æ7 ± 26Æ2 4Æ4 ± 8Æ8 11Æ2 ± 14Æ8 0Æ8 ± 0Æ1Testosterone (ng/ml) 0Æ6 ± 0Æ3 3Æ8 ± 2Æ0 1Æ5 ± 0Æ5* 4Æ6 ± 0Æ7*

LH (i/u per l) 0Æ5 ± 0Æ5 3Æ6 ± 2Æ0 1Æ4 ± 0Æ8 9Æ0 ± 4Æ8FSH (i/u per l) 0Æ7 ± 0Æ7 2Æ1 ± 1Æ6 2Æ3 ± 0Æ9 4Æ9 ± 1Æ8

MRI T2L, magnetic resonance T2 liver; LIC, liver iron concentration; LH, luteinizing hormone;

FSH, follicle-stimulating hormone.

*P-value for testosterone levels in these patients <0Æ001.

K. Farmaki et al

470 ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 148, 466–475

chelation, six of these patients (32%) became pregnant, two

with primary amenorrhea after in vitro fertilisation (IVF) and

four with secondary amenorrhea, two of them spontaneously

and two after IVF. All delivered healthy infants. Moreover,

hypogonadal women experienced a significant increase in

GnRH test (Table IV). Hormone replacement therapy was

continued in those hypogonadal women who additionally had

osteoporosis. In previously eugonadal females the mean

oestradiol level increased significantly (46 ± 8 vs. 221 ±

63 ng/l) and LH responses to GnRH improved (AUC LH

1047 ± 337 vs. 1929 ± 513), as well as FSH (AUC FSH

571 ± 163 vs. 868 ± 156).

Safety

Two non splenectomised patients withdrew from the study

because of repeated episodes of neutropenia. The episodes

appeared at 14 and 18 months after the start of combined

chelation. One patient had an ANC of approximately

0Æ5 · 109/l and the other 1Æ0 · 109/l; the former patient

presented with tonsillitis, which was managed only with

antibiotics and continued CBC monitoring. DFP therapy was

interrupted for 1 year after which re-challenge was attempted,

leading to a mild neutropenia (0Æ8–1Æ2 · 109/l). Both patients

refused to continue the study protocol.

Combined chelation was otherwise well tolerated. Patients

were advised to reduce their DFP dose temporarily in the event

of joint symptoms (reported in 5% of patients), gastrointes-

tinal upset (8%) or increase in liver enzymes (11%). DFO was

transiently interrupted for 1–2 months in the case of tinnitus

(one patient – 2%) and ocular problems (one patient – 2%)

which reversed, in both cases.

There were no fatalities during the study period.

Discussion

This study demonstrated the efficacy of combined chelation

therapy with DFO and DFP in reducing total body iron load,

endocrine complications and in preventing or reversing iron-

induced cardiac complications. This is the first report that

documents reduction of ferritin to normal levels. It provides

clear evidence that iron-induced tissue damage is reversible,

suggesting that such reductions may improve and maintain

cardiac function and prevent or reverse endocrinopathies.

Our results are consistent with findings by other investiga-

tors, establishing that combined chelation could effect an

improvement in cardiac dysfunction, as estimated by LVEF

(Wu et al, 2004; Tsironi et al, 2005; Tavecchia et al, 2006). We

further found that endocrine dysfunction, which had not been

systematically studied, could also be reversed. In addition, it

appears that new-onset cardiac and endocrine abnormalities

can be prevented with effective chelation.

Previous retrospective and prospective studies suggested that

mortality, due mainly to cardiac damage, was reduced or

completely absent in patients treated with DFP, alone or in

combination with DFO (Piga et al, 2003; Borgna-Pignatti et al,

2006; Telfer et al, 2006; Modell et al, 2008). A prospective

randomized controlled trial comparing DFO monotherapy with

DFP, either as monotherapy or combined with DFO, showed

definite cardiac protection in DFP-treated patients, compared to

those on DFO monotherapy. The authors speculated that DFP

had a protective effect on the heart even before cardiac iron

declined significantly, most likely because of the clearance of

cellular toxic labile iron (Maggio et al, 2009). Increased risk of

death with DFO monotherapy may be partly ascribed to the

adverse cardiovascular effects of diabetes, as well as to direct

cardiac cytotoxicity seen in inadequately chelated patients. This

effect is not solely a matter of compliance, because excess cardiac

iron, as assessed by a T2*H of <20 ms, was also observed in

patients with good compliance to DFO monotherapy (Aessopos

et al, 2007). These studies concur with our results using intensive

combined chelation.

Prior to the availability of DFO, iron-induced myocardial

disease started early in a patient’s life (Borgna-Pignatti et al,

2004). Continuous 24-h parenteral infusion of DFO can reverse

arrhythmias and congestive heart failure in many but not all

patients (Miskin et al, 2003; Anderson et al, 2004). As stated

above, a considerable body of data now exists suggesting that

DFP is superior to DFO in reducing iron in the heart (Pennell

et al, 2006) and reversing cardiac complications. Many studies

have demonstrated that combined chelation (DFP–DFO) can

have a more marked effect in purging cardiac iron (Tanner et al,

2007, 2008), as was observed in this study. Reversal of cardiac

complications and discontinuation of cardiac medication, as

documented here, is a significant achievement, consistent with

other cases of reversal of late-stage heart failure.( Wu et al, 2004;

Tsironi et al, 2005; Tavecchia et al, 2006).

Glucose metabolic disorders (GMDs) are the second most

common class of endocrine complication, occurring in 21–42%

Table IV. Differences in age, ferritin, liver iron concentration and

hormonal parameters between female patients with primary and

secondary amenorrhea.

Females with primary

amenorrhea (n = 9)

Females with secondary

amenorrhea (n = 10)

Baseline

After

combined Baseline

After

combined

Age (years) 33Æ4 ± 8 40Æ4 ± 8 24Æ9 ± 9Æ6 31Æ9 ± 9Æ6Ferritin (lg/l) 2696 ± 660 115 ± 35 1501 ± 273 99 ± 37

MRI T2L (ms) 10Æ4 ± 9Æ9 34Æ2 ± 4Æ8 8Æ7 ± 7 34Æ9 ± 2Æ6LIC (mg/g dw) 9Æ5 ± 10Æ6 1Æ0 ± 0Æ1 9Æ4 ± 9Æ3 0Æ9 ± 0Æ05

Estradiol (ng/l) 9 ± 2 24 ± 5Æ2 20 ± 7Æ6 104 ± 61Æ7LH (i/u per l) 0Æ4 ± 0Æ3 1Æ0 ± 0Æ4 1Æ9 ± 0Æ3 3Æ8 ± 0Æ5AUC LH (GnRH) 77 ± 50 177 ± 74 406 ± 111 712 ± 211

FSH (i/u per l) 1Æ0 ± 0Æ6 2Æ5 ± 0Æ8 3Æ6 ± 1Æ2 6Æ8 ± 1Æ6AUC FSH (GnRH) 109 ± 64 256 ± 75 373 ± 37 733 ± 58

MRI T2L, magnetic resonance T2 liver; LIC, liver iron concentration;

LH, luteinizing hormone; FSH, follicle-stimulating hormone; AUC,

area under the curve; GnRH, gonadotrophin-releasing hormone.

Complication Reversal in Thalassaemia

ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 148, 466–475 471

of thalassaemic patients (Borgna-Pignatti et al, 2004; Cunning-

ham et al, 2004). Not only do they affect quality of life, but there

is also a definite relationship between diabetes and cardiovas-

cular disease (Aessopos et al, 2008). The main mechanisms

underlying GMD development in thalassaemia are insulin

deficiency, due to the direct toxic damage of iron on pancreatic

b cells, as well as longstanding insulin resistance (Cario et al,

2003a; Angelopoulos et al, 2006), secondary to changes in

glucose metabolism occurring in the liver (Cario et al, 2003b),

muscles and adipose tissue (Gamberini et al, 2004).

In most studies, GMD frequency seems to increase with the

prolongation of survival (Borgna-Pignatti et al, 2004; Cunn-

ingham et al, 2004). One study (Kattamis et al, 2004) reported

an increase of GMD during adolescence. In our study, GMD at

baseline was significantly more prevalent in patients >30 years

old than in younger patients, and those who continued to have

diabetes despite combined chelation were also older. With

DFO monotherapy, patients typically experience a gradual

decline in glycaemic control (Merkel et al, 1988), even with

optimal treatment compliance (De Sanctis et al, 2004). As

pancreatic iron seems to correlate well with cardiac iron (Au

et al, 2008), it is likely that the continuing incidence of GMD

reflects the intrinsic limitations of the efficacy of DFO in the

pancreas, as is seen in the heart.

To our knowledge, our group was the first to identify a

beneficial effect of intensive combined chelation on GMD in

patients without insulin-treated diabetes. Not only were GMDs

reversed in a significant proportion of our patients, but the

cumulative glucose response was also significantly improved

with this regimen. There were no new cases of GMDs, and no

patients experienced a deterioration of glucose metabolism.

These findings are consistent with studies by our group and

others (Christoforidis et al, 2006; Farmaki et al, 2006), com-

paring combined chelation with DFO or DFP monotherapy.

However, in the study of Christoforidis et al (2006), differ-

ences failed to reach statistical significance, probably because

chelation was incomplete, with mean ferritin levels remaining

above 1500 lg/l. It was also indicated that mean BMI was

higher in patients with GMDs. Conversely, increased BMI

(P < 0Æ001) did not negatively affect our patients’ glycaemic

control, consistent with the idea that effective iron chelation

per se was sufficient to improve glucose metabolism.

Thyroid hormones are important determinants of physical

and intellectual development in children and play a role in

adult activity, affecting the metabolism of almost all organ

systems. Moreover, hypothyroidism is associated with

increased risk of cardiovascular disease (De Sanctis et al,

2008). As hypothyroidism symptoms are not typical and some,

such as fatigue, cold intolerance, weight gain and constipation,

may be present in normal subjects, the diagnosis of hypothy-

roidism is based upon biochemical results (TSH, FT4, FT3).

TRH stimulation test is a sensitive assay, which may reveal a

dynamic thyroid dysfunction, especially when TSH and FT4

levels are borderline, thus influencing the decision to treat with

thyroid hormone. The TRH stimulation test can also specify if

hypothyroidism is primary (arising from iron deposition in the

thyroid gland), or central (pituitary or hypothalamic dysfunc-

tion) in multi-transfused thalassaemic patients.

Although in the normal population, experts recommend

treatment with thyroid hormone if TSH levels >10 mU/l, in

thalassaemic patients with thyroid replacement therapy more

criteria are taken into consideration (Cooper, 2001; Gharib et al,

2005; De Sanctis et al, 2008) and, in particular, is recommended

because of the potential for reducing the risk of cardiac problems

(Biondi & Cooper, 2008). Therefore in our group of thalassae-

mic patients the decision to treat was individualized and based

on patients’ clinical history and aggravating factors (hyperlip-

idaemia, cardiac dysfunction, diabetes, pregnancy, depression).

For this reason, the overall prevalence in our group (18/50 36%)

is higher than that reported by others (Cunningham et al, 2004;

Borgna-Pignatti et al, 2005). Among them 14/18 (28%) had

subclinical or compensated hypothyroidism and 4/18 (8%),

mostly older patients, had overt hypothyroidism.

Our study suggested that intensive combined chelation and

the important decrease in total body iron overload

(P < 0Æ0001) may reverse most of the cases of subclinical

and compensated hypothyroidism and may prevent progres-

sion to overt hypothyroidism. Some patients with overt

hypothyroidism may also improve, suggesting that even

iron-induced damage of the thyroid pituitary axis might be

ameliorated. The excellent improvement in the subclinical and

compensated patients would be unlikely and the risk of

progression to overt hypothyroidism would remain if the

patients were not on intensive chelation therapy.

Hypogonadism, the most common endocrinopathy in thal-

assaemic patients (40–91%) (Borgna-Pignatti et al, 2004; Cunn-

ingham et al, 2004), dramatically affects quality of life because of

fertility problems and related effects on patients’ psychological

well-being. Hypogonadism may result from iron deposition in

the hypothalamic-pituitary cells (hypogonadotrophic or sec-

ondary hypogonadism) or in the gonads (hypergonadotrophic

or primary hypogonadism). Our results in adult thalassaemic

patients show clearly that iron-induced primary or secondary

hypogonadism may be reversible, and perhaps also preventable,

following the transition to intensive combined chelation.

Particularly in men, the improved gonadal function was

manifested by amelioration of clinical symptoms such as

increase in activity and libido, increased hair and bone mineral

density and the improvement of overall hormonal profile

leading to discontinuation of replacement therapy.

With respect to the women, we acknowledge that hypogo-

nadal thalassaemics are able to achieve pregnancy following

hormonal treatment (Thomas & Skalicka, 1980; Tuck et al,

1998) but it is of note that spontaneous pregnancy in patients

who had secondary amenorrhea and the improvement of

overall hormonal profile was demonstrated in these patients.

From our experience, combined chelation with DFO and

DFP seems to be the treatment of choice in order to achieve a

significant decrease in total body iron load. Combined

chelation can be applied routinely to all patients, provided

K. Farmaki et al

472 ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 148, 466–475

that there is no contraindication to its use, until all clinical and

subclinical complications are reversed. Monotherapy with

whichever chelator is prescribed, usually maintains iron

balance but does not decrease iron that has accumulated over

an extensive period.

The time needed to reverse all complications varies accord-

ing to the patients’ age and iron load status at the time of

starting the combination therapy. A minimum of 4 years is

necessary. After the reversal of all endocrine and other

complications, the DFO dose can be reduced to a range of

20–30 mg/kg, with 2–3 infusion days per week; DFP dose is

maintained at between 75 and 100 mg/kg per d. Nevertheless,

monotherapy with any of the three chelators (including the

recently licenced deferasirox [DFX]) may be considered in

some patients, depending on their individual responses.

In conclusion, intensive chelation therapy using combined

DFO and DFP, because of the additive or synergistic effect,

achieves a negative iron balance in all thalassaemic patients,

allowing the reduction of total body iron load to normal levels.

Our data on patients receiving intensified chelation suggest

that reducing ferritin and total body iron to normal levels may

prevent and/or reverse cardiac and endocrine complications.

Heart failure, GMD, hypothyroidism and hypogonadism all

improved, with a positive impact on patients’ quality of life.

Intensive combined chelation was well tolerated. It remains to

be established, by future clinical trials, whether other inten-

sified regimens, such as combined treatment with the two oral

chelators DFP and DFX, will offer comparable benefits.

Author contributions

KF, IT, CP designed and performed research, entered data on

data base, and were involved in the writing of the paper. VB

analysed data and was involved in writing the paper. GC

analysed data and contributed to the writing of the paper.

Conflicts of interest

IT, CP and GC have no conflicts to declare. KF was a speaker at

ApoPharma key opinion leader meetings and was involved as

an investigator in Novartis Inc. studies for ICL670 (Exjade).

VB is a consultant for ApoPharma Inc., and has a confiden-

tiality agreement with Novartis Inc. with respect to the

development of ICL670 (Exjade).

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