40TH ANNIVERSARY ISSUE

A JH
Thalassemia 2016: Modern medicine battles an ancient
disease
Deborah Rund*

Thalassemia was first clinically described nearly a century ago and treatment of this widespread genetic
disease has greatly advanced during this period. DNA-based diagnosis elucidated the molecular basis of the
disease and clarified the variable clinical picture. It also paved the way for modern methods of carrier
identification and prevention via DNA-based prenatal diagnosis. Every aspect of supportive care, including
safer blood supply, more regular transfusions, specific monitoring of iron overload, parenteral and oral
chelation, and other therapies, has prolonged life and improved the quality of life of these patients.
Significant advances have also been made in allogenic bone marrow transplantation, the only curative
therapy. Recently, there has been a rejuvenated interest in studying thalassemia at the basic science level,
leading to the discovery of previously unknown mechanisms leading to anemia and enabling the
development of novel therapies. These will potentially improve the treatment of, and possibly cure the
disease. Pathways involving activin receptors, heat shock proteins, JAK2 inhibitors and macrophage
targeted therapy, among others, are being studied or are currently in clinical trials for treating thalassemia.
Novel types of genetic therapies are in use or under investigation. In addition to the challenges of treating
each individual patient, the longer survival of thalassemia patients has raised considerations regarding
worldwide control of thalassemia, since prevention is not universally implemented. This review will trace a
number of the original medical milestones of thalassemia diagnosis and treatment, as well as some of the
most recent developments which may lead to innovative therapeutic modalities.
Am. J. Hematol. 91:15–21, 2016. V
C 2015 Wiley Periodicals, Inc.

䊏 Introduction
It is now over 90 years since the first description by Dr. Thomas Cooley of thalassemia in children of Mediterranean origin [1]. Yet, archeologi-
cal evidence [2] and even DNA from remains dating back many generations [3] have suggested that the disease is quite ancient. During the nine
decades following the first clinical description of thalassemia, there have been revolutionary changes in the fields of science and medicine, which
have impacted on the understanding and treatment of the thalassemias. In this review, some of these changes will be discussed, putting into per-
spective the substantial progress which was made both in understanding the pathophysiology of thalassemia and its diagnosis and therapy. This
review will not be all-inclusive, but will touch on the more active and controversial areas, and those with therapeutic implications.

䊏 Diagnosis: From Protein to Genes to Prenatal Diagnosis

Analysis of electrophoresis of hemoglobin provided the first insights into the pathophysiology of the hemoglobinopathies. However, the ability
to analyze genetic information at the level of DNA and RNA led to rapid progress in understanding the disease. By the early 1970s, scientists had
succeeded in isolating mRNA from actual reticulocytes of normal individuals and patients [4], followed by discovery of the quantitative chain
imbalance between the amounts of a- and b-globin RNA [5] in a- and b-thalassemia. Subsequently, restriction enzyme analysis of high molecular
weight DNA with Southern blotting yielded vital information. However, this technique was cumbersome and was unsuitable for analyzing b-
thalassemia, which is generally caused by point mutations. Therefore the development of PCR was truly revolutionary [6]. All of the globin genes
are small, just over 2 kilobases, and as such, are all highly amenable to analysis using PCR-based methodology which has been used to great
advantage for nearly three decades. Various permutations of PCR have revealed hundreds of point mutations causing a- and b-thalassemia, and
gap-PCR can analyze deletions. The large quantity of information amassed on thalassemia mutations has been stored in a freely accessible online
database known as HbVar (http://globin.bx.psu.edu/hbvar). Originally begun in 2001 [7], it was recently updated [8]. This database is one of the
oldest locus-specific databases and is invaluable to clinicians and researchers alike.
The availability of precise genetic diagnostic information enabled progress in diagnosis and furthered understanding the etiology of the varying
severities of thalassemia syndromes. What could crudely be defined as b1- or b0-thalassemia (by the presence of HbA on hemoglobin electropho-
resis) could now be defined by DNA analysis of the specific mutations present in each patient. Subsequently, it became clear that some genotypes
were associated with a variable phenotype, depending on other factors such as fetal hemoglobin levels [9,10]. Furthermore, diseases which are

Hematology Department, Hebrew University-Hadassah Medical Organization, Ein Kerem, Jerusalem, Israel
Conflict of interest: The author has no conflicts of interest to disclose
*Correspondence to: Deborah Rund, Hebrew University-Hadassah Medical Organization, P.O.B. 12000, Ein Kerem, Jerusalem, Israel 91120. E-mail: deborah.
rund@mail.huji.ac.il
Received for publication: 28 September 2015; Revised: 1 November 2015; Accepted: 2 November 2015
Am. J. Hematol. 91:15–21, 2016.
Published online: 4 November 2015 in Wiley Online Library (wileyonlinelibrary.com).
DOI: 10.1002/ajh.24231

C 2015 Wiley Periodicals, Inc.

V

doi:10.1002/ajh.24231 American Journal of Hematology, Vol. 91, No. 1, January 2016 15

Rund
genotypically different could have similar clinical manifestations [11].
This led to reclassification of thalassemia syndromes into transfusion
dependent versus nontransfusion dependent thalassemias. The main
types of non transfusion dependent thalassemia are b-thalassemia
intermedia, HbH disease and HbE/ b-thalassemia [11]. While these
patients do not require lifelong transfusion, they have significant dis-
ease manifestations requiring therapy.
DNA-based analysis also facilitated carrier identification, enabling
determination of couples at risk who could then be referred for early
and rapid (first trimester) prenatal diagnosis by direct detection of
mutations in fetal DNA. This led to a dramatic fall in birthrates of
patients with severe thalassemia syndromes in many countries where
this technology was available [12]. One of the earlier goals of prenatal
diagnosis was to perform it noninvasively using maternal blood sam-
ples which contain minute amounts of fetal material. This would
avoid the small but possible risks of fetal injury or even fetal loss dur-
ing invasive methods including amniocentesis and chorionic villus
sampling. An early report of the use of maternal peripheral blood
sampling for prenatal diagnosis of thalassemia was published in 2002
[13]. However, this report only screened for one particular mutation
which was relatively easy to detect. The methodology was also applied
to deletional a-thalassemia but not sufficiently accurate for most b-
globin point mutations. Recently, more modern techniques of DNA
analysis using next generation sequencing (NGS) were applied toward
the goal of noninvasive fetal diagnosis of thalassemia [14]. Using this
technique, maternal peripheral blood is drawn at 9 to 10 weeks,
before chorionic villus sampling can be performed, and analysis is
performed for a panel of single nucleotide polymorphisms (SNPs)
which are predetermined, using family studies, to be linked to the
paternal mutant allele. Using the technique of NGS, the use of four
SNP pairs has been found to be sufficiently accurate to determine if
the fetus is affected. Thus far the technique is only in experimental
use since it is not diagnostic in all cases and it has required confirma-
tion by CVS testing [14]. Yet, it is probable that in the near future,
this innovative technique will be perfected such that it will be in rou-
tine use.
Accurate DNA diagnosis of fetal DNA has engendered controversy
regarding the use of preimplantation genetic diagnosis (PGD) of tha-
lassemia [15]. This method involves in vitro fertilization of embryos
and diagnosis using an embryo biopsy or polar body DNA [15]. The
fetuses identified as healthy are selected for implantation [15]. This
method seems ideal for couples with religious or ethical proscriptions
against termination of pregnancy of an affected fetus. However, in
principle, the same ethical dilemma exists since only a fraction of the
fertilized embryos are implanted, according to the genetic results.
Therefore this technique is not permitted in some regions of the
world. Although efforts at perfecting the technique are underway,
preimplantation genetic diagnosis is not perfect. Using this technique,
the rate of successful pregnancy is at best around 30% [16], which is
considerably lower than the rate of spontaneous conception in a fer-
tile couple who would not normally need reproductive intervention
other than for embryo selection. Furthermore, it is challenging to
employ this technique for b-thalassemia which is usually due to point
mutations [17]. Since there are hundreds of such mutations, the
methodology would need to be perfected for each particular mutation
with its different primers and PCR conditions. Since there are only
isolated cells as a template, asymmetric amplification of the different
alleles can occur, which can lead to errors in diagnosis. Thus, there
are intense technical demands on multiple disciplines whose coordi-
nated efforts are required to successfully perform this procedure [18].
Furthermore, this technique is considerably less accurate than con-
ventional prenatal diagnosis due to the minute amount of cellular
material available. To add fuel to the controversy, PGD methods have
also been used to select fetuses which are not only unaffected by
16 American Journal of Hematology, Vol. 91, No. 1, January 2016
40TH ANNIVERSARY ISSUE
thalassemia, but are HLA identical to a previously born affected sib-
ling [15]. This ensures that the newborn can be a potential transplant
donor for the sibling patient. Ethical issues have been raised regard-
ing the planned selection of such “savior siblings” [19].
In summary, advanced sophisticated molecular techniques and the
large database of thalassemia mutations have enabled substantial
advances in the field of diagnosis and prenatal diagnosis in terms of
speed, accuracy and noninvasive methodologies. These advances have
evoked some controversies but have furthered efforts at preventing
births of affected children.
䊏 Genetic Modifiers
Paradoxically the availability of more accurate knowledge at the
DNA level regarding thalassemia mutations led to a clinical conun-
drum: patients with the same mutations (sometimes even siblings in
the same family) could have very disparate degrees of clinical severity.
This led to the concept of genetic modifiers of thalassemia. Initially,
the search for such modifiers was based on linkage analysis of large
family pedigrees [20] or analysis of multiple genes in patients with
varying thalassemia phenotypes [21]. This subject has been expertly
reviewed [22]. Besides clarifying and predicting disease severity, the
search for genetic modifiers has another goal: to identify genetic
determinants which can be used for novel therapies, which will be
discussed below.
Modifiers which directly affect the degree of globin chain imbal-
ance are now referred to as “primary modifiers” [22]. These have the
most notable effects on the level of hemoglobin and on the overall
severity of disease. Ironically, despite advances in genomics, the out-
put of the globin genes themselves are still the most potent modifiers.
For instance, coinheritance of excess a-globin genes (five or six rather
than four) can worsen the phenotype of individuals who are geneti-
cally thalassemia trait. These individuals carry only a single b-globin
gene mutation but have a phenotype of nontransfusion dependent
thalassemia. In contrast, a number of cis or transacting elements
influence output of the g-globin genes, raising HbF and ameliorating
the phenotype of transfusion dependent b-thalassemia [22]. Interest-
ingly, HbF elevation has been mapped to loci located both inside (the
XmnI polymorphism 5’ to the Gg-globin gene) and outside the b-
globin gene cluster by family and linkage studies [22,23]. More
recently, the technique of genome wide association studies (GWAS)
has been used to confirm the importance of 2 previously identified
loci. One of these is the BCL11A intron 2 polymorphism at chromo-
some 2p16, and the other is the HBS1L-MYB intergenic polymor-
phism at chromosome 6q23 [24]. The advantage of genome wide
association studies is that it enables pinpointing the specific gene
involved more precisely than linkage studies and furthermore, this
methodology can potentially reveal associations which were not previ-
ously revealed using linkage.
The subject of genetic modifiers has grown quite complex as vari-
ous disease manifestations have been studied for modifier effects.
These include the severity of the anemia or of other accompanying
manifestations such as iron overload, the presence of gallstones, and
so on. These are referred to as “secondary modifiers” and are related
to any number of genes whose functions relate to various organ sys-
tems [22]. Future studies will determine whether there are treatment
implications to these genetic causes for symptom variability.
䊏 Conventional Therapy
Prolonging survival: Transfusions and chelation
Blood transfusions were given in the 1940s for “Mediterranean
anemia” to prolong the lives of severely anemic children. It was only
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40TH ANNIVERSARY ISSUE
recognized in 1955 [25] that blood transfusion also suppresses endog-
enous hematopoiesis in thalassemia. Since ineffective erythropoiesis is
one of the major pathogenic mechanisms of disease manifestations
(bone deformities, hepatosplenomegaly due to extramedullary hema-
topoiesis) it became clear about 50 years ago that transfusion was the
mainstay of therapy. Since patients are transfusion dependent, they
have benefited from advances in blood banking starting with better
crossmatching. Transfusion reactions (some fatal) due to alloantibod-
ies were well documented in the older literature. Unfortunately due
to differences in ethnicity in donors and recipients of transfusions,
alloimmunization will undoubtedly continue to persist even in mod-
ern times [26]. A recent survey found that, even in the industrialized
world, the practice of transfusion medicine is still variable and could
be improved and standardized to reduce alloimmunization [27].
It is clear that thalassemia patients born in the industrialized world
in the last two to three decades have benefited from modern stand-
ards of safe blood banking with much reduced pathogen transmis-
sion. Transfusion transmitted diseases have dealt a lifelong blow to
patients who began receiving transfusions prior to testing for hepatitis
and/or HIV. In the industrialized world, these are either the oldest
patient cohort or immigrants who began their transfusion history in
countries where testing for pathogens is not yet standard practice
[26]. Besides their immediate impact on the patient’s health, transfu-
sion transmitted infections can lead to late complications such as cir-
rhosis and/or hepatocelluar carcinoma (HCC). Approximately 85% of
thalassemia patients with HCC in a recent Italian study showed evi-
dence of infection with hepatitis B virus or hepatitis C virus [28].
Hemosiderosis was long recognized as a complication of prolonged
transfusion therapy and a half a century ago the first reports of the
use of desferrioxamine in thalassemia were published [29,30]. Three
decades ago, desferrioxamine was shown to prevent many of the
complications of iron overload [31]. However, desferrioxamine is not
suitable for all patients and even early on, investigators were working
on new chelators [32]. At the present time there are three chelators,
two oral and one parenteral, in routine use, all of which are effective
to varying degrees and which have differing adverse effects [33,34].
The availability of orally active chelators has greatly improved com-
pliance which is still an issue for patients requiring this treatment on
a lifelong basis. New combination therapy approaches and intensifica-
tion of dosing are promising in terms of increased efficacy [33].
While long hypothesized but not absolutely proven [35], recent in
vitro evidence demonstrates synergistic chelation efficacy using a
combination of different types of chelators [36]. In summary, half a
century of arduous clinical, pharmacological, and laboratory studies
have not yet led to a simple solution to the need for chelation, and
many problems exist in “real life” practice [37]. Even patients with
access to all available drugs, chelation is not always optimal [37]. Yet
despite these problems, chelation has significantly prolonged the lives
of thalassemia patients who can obtain this treatment and who com-
ply with its use [38], mainly by reducing early death from cardiac
events due to iron overload.
Improvements in chelation led to the need for more accurate mon-
itoring of the efficacy of iron chelation therapy. Serum parameters
(serum iron and transferrin saturation) gave way to the widely avail-
able serum ferritin, which is inexpensive and useful for routine clini-
cal assessment. However ferritin is subject to many variations, and
even when measured frequently, may not accurately reflect tissue iron
burden [39]. Recently, scientific studies have led to better understand-
ing of iron trafficking, including how iron accumulates in tissues
which were not intended to store iron [40]. These studies have eluci-
dated the need to monitor not only serum parameters but also tissue
iron: hepatic iron and even iron in other tissues. Liver biopsy (which
is invasive) was previously considered the “gold standard” but both
only measured hepatic iron stores. Currently, there are several types
doi:10.1002/ajh.24231
Thalassemia 2016
of magnetic resonance imaging (MRI) which can noninvasively mea-
sure liver, cardiac, pancreatic, and even pituitary iron [40]. MRI
measures iron indirectly by the influence of iron on nearby water
molecules. Tissue iron disrupts the magnetic field for a period of time
which can be measured in units of time (designated T2 or T2* by
spin echo imaging or gradient echo imaging) or by rate (R2 and R2*
by spin and gradient echo imaging, respectively) [40]. Quantitation of
liver iron by R2* has been validated by liver biopsy results [40]. How-
ever, T2 and T2* have not yet been validated for liver iron concentra-
tion. A recent meta-analysis [41] found that MRI T2 and T2* are
better at excluding liver iron overload than by definitely diagnosing it
in individual patients. Furthermore, thresholds have not been speci-
fied and uniformly used, therefore further studies are needed to
standardize T2/T2* protocols for hepatic iron determination [41].
Since cardiac mortality is the major cause of mortality in nonchelated
patients, cardiac T2*, which has been validated clinically [40] has
become the standard of care in industrialized countries. As such, the
use of cardiac MRI has contributed to prolonging life expectancy in
countries where this tool is available, such as the UK and other parts of
Europe [42]. An algorithm has been developed for routine use of car-
diac MRI to guide long term optimum chelation [40]. Ferritin remains
useful in young children, for whom the use of MRI requiring sedation is
problematic and needs to be weighed individually [33].
Hypercoagulability
One of the factors long known to contribute to mortality and mor-
bidity in thalassemia is the chronic hypercoagulability of this disease,
particularly in splenectomized patients [43]. The contribution of
hypercoagulability to the mortality and morbidity of thalassemia
patients has long been understood [44]. Laboratory studies have eluci-
dated the many factors contributing to hypercoagulability of this dis-
ease [45]. Of concern, hypercoagulability can contribute to cerebral
ischemia which may initially be clinically silent [46]. Nonetheless
despite the importance of these findings, there are still no clearcut
guidelines on prevention of these complications [45], despite the
availability of many new antithrombotic agents. Clearly this is an area
in which further clinical research is urgently needed.
Improving quality of life
Prolonging life in thalassemia has been accomplished. However, a
longer life is not necessarily a better life, and unfortunately, the quality
of life for these patients can be poor. This may be due to discomfort
from the disease and its therapies, psychological issues, or to low func-
tional capacity due to chronic anemia. Recently more attention has
been paid to these types of issues. Among the areas receiving attention
is the chronic bone pain and osteoporosis which can be quite debilitat-
ing in adult patients [47]. The pathogenesis is multifactorial, from bone
expansion due to hyperactivity of the bone marrow, to hypogonadism
[48], and even iron overload is contributory [49]. Recently, an expert
panel has published recommendations for surveillance and therapy
which are quite welcome, as this area has previously been one of contro-
versy. In particular, it is encouraging that the use of biophosphonates
shows beneficial results to date [50,51], since in the past, these drugs
have been questioned regarding their safety in thalassemia.
Recently, concern has been expressed regarding the presence of
neurological complications of thalassemia. As life expectancy
increases, chronic hypoxia, thromboembolic phenomena, neurotoxic
drugs, and other factors, can cause damage which may manifest as
clinically apparent (such as cognitive impairment or peripheral neu-
ropathy) or subclinical findings (abnormal evoked potentials) [52].
This is an important area for future preventative research, in order to
optimize the level of functioning and quality of life of thalassemia
patients.
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Rund
Bone marrow transplantation
Allogeneic bone marrow transplantation (alloBMT) is curative for
thalassemia. Indeed, it has been performed since 1982, with continu-
ous advances in the field in the past decades [53]. There have been
many issues to resolve in order to achieve successful alloBMT specifi-
cally in thalassemia. All patients are heavily transfused leading to
alloimmunization and a high risk of graft rejection. End organ dam-
age from iron overload increases risk of toxicity from conditioning
regimens. The stratification of risk by Lucarelli predicted poor out-
come and enabled selection of those suitable for alloBMT. More
recently, even high risk patients have been successfully transplanted
[54]. For patients at high risk, conditioning needs to be titrated to
reduce toxicity and at the same time, prevent graft rejection. Condi-
tioning regimens for thalassemia have recently been reviewed [55].
Reduced intensity conditioning had generally had disappointing
results due to autologous recovery [56]. However, more favorable
results were recently reported using reduced intensity conditioning
[57].
In the industrialized world, the limiting factor for alloBMT for tha-
lassemia has been donor availability. Traditionally, fully matched
HLA identical sibling donors were used but improved outcome was
reported with matched unrelated [53] and with single locus mismatch
related (not sibling) donors [58]. Recently, success has even been
reported using unrelated cord blood, including with reduced intensity
conditioning [59]. The ability to use unrelated cord blood consider-
ably enlarges the donor pool available for each patient.
AlloBMT is costly and has not been available in most developing
countries for this and other technical reasons. However, recent efforts
have been made to foster international cooperation between industri-
alized and developing countries to increase the utilization of this ther-
apy in the developing world. It has been suggested that alloBMT is
preferable to routine supportive care, since safe blood products and
chelation are not readily available in many parts of the world. In
addition, it has been suggested that the cost could be reduced to
make it economically feasible [60]. Considering the high expenditures
associated with standard lifelong supportive care, alloBMT could be
financially tenable. International cooperation is required to increase
access to alloBMT in areas with the greatest need [61].
䊏 Novel Therapies Already Here or on
the Horizon
While thalassemia was once on the forefront of genetic and scien-
tific research, in the last several decades it fell behind while major
strides were made in the comprehension and therapy of malignancies.
In recent years, thalassemia has returned to the forefront of science.
The study of anemia in general [62] and thalassemia [63] and the b-
hemoglobinopathies in particular [64], have led to increased under-
standing of molecular mechanisms underlying these diseases. Thalas-
semia has thus become a paradigm for discoveries regarding the
pathophysiology of anemia and potential novel therapies. Three excel-
lent reviews comprehensively discuss potential (or actual) novel tar-
gets for therapies for anemia, including thalassemia [62–64]. This
review will only mention seven of these newer therapeutic avenues
which are aimed at alleviating the anemia and/or iron overload. Some
are already in clinical use, others are in clinical trials and some are
potential mechanisms which could be targeted for therapeutic pur-
poses in the future.
Fetal hemoglobin induction
HbF induction has been known for nearly four decades. A number
of agents have been used with varying success to pharmacologically
raise HbF with tolerable adverse effects (reviewed in Ref. 65). Results
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40TH ANNIVERSARY ISSUE
to date have generally been disappointing. Recently, greater compre-
hension of the regulatory pathways of HbF expression, including the
crucial influences of BCL11a and MYB, has suggested new therapeutic
targets for raising HbF [62]. These include interfering with BCL11a
or MYB, which both directly repress g-globin gene expression. These
potential targets were revealed by studies, mentioned above, of genetic
modifier loci on chromosome 6 [66]. However, there are presently no
specific drugs which can target these pathways. There are several rea-
sons for this, including the broad effects on hematopoiesis exerted by
both BCL11a and MYB. Therefore, inhibiting either one could prove
to be problematic. Currently, research in animal models is aimed at
finding erythroid specific methods of targeting these regulators for
the treatment of thalassemia [62].
Activin receptor ligand traps
Despite all that is known about erythropoiesis, new and important
regulatory pathways are still being discovered. Such is the case with
the GDF11 pathway. This gene had been studied predominantly for
its role in organs such as pancreas, intestine, kidney, skeletal muscle,
and developing nervous system. Knockout gdf112/2 mice develop
skeletal and gut abnormalities [67]. A role for this gene in human
erythropoiesis was found indirectly and serendipitously. The first clue
came during a trial of a novel drug designed to treat osteoporosis, in
which it was noted that the subjects developed an average of 12% rise
in Hb [68]. This was unexpected and it quickly led to the hypothesis
that this drug, sotatercept, could be used to treat anemia. Sotatercept
was developed to treat osteoporosis by targeting activin signaling,
which inhibits osteoclast dependent bone resorption. Activin belongs
to the TGFb superfamily. It binds to various receptors which initiate
a cascade culminating in phosphorylation of SMADs which activate
gene expression. Sotatercept acts as an activin IIA receptor ligand
trap. Ligand traps are molecules that inhibit signaling by binding
ligands and preventing them from interacting with their receptors. A
murine version of sotatercept was tested in a mouse model of thalas-
semia [69] and the results were remarkable. Not only was there
increased terminal differentiation of erythroblasts, with correction of
maturation arrest, but RBC morphology improved, with fewer a-
globin precipitates on RBC membranes. Furthermore, splenomegaly
decreased. Remarkably, hepcidin levels also increased and reduced
iron and transferrin saturation were found, which was independent of
GDF15 expression. This is significant since GDF15 has been sus-
pected of being the culprit in downregulating hepcidin in thalassemia,
leading to iron overload [70]. The role of GDF11 in these beneficial
effects was elucidated by a series of elegant molecular studies which
were initially based on results of gene expression studies in the fetal
livers of mice with thalassemia major. These results, found in an
online database dating 2012 (from the laboratory of Dr. Mitchell
Weiss), showed that the murine gdf11 gene was the only gene overex-
pressed in the fetal liver of thalassemic mice at embryonic day 14.5.
From there, additional experiments and deduction led to the role of
the activin type IIA receptor [69].
Luspatercept is a compound which is very similar to Sotatercept
and differs in being an activin IIB (not IIA) receptor ligand trap. Lus-
patercept has been assessed in a Phase I study in normal human vol-
unteers [71] and in a Phase 2 study in thalassemia patients [72]. In
both normal individuals and patients, hemoglobin rose and side
effects were tolerable. Further results of ongoing studies of this excit-
ing novel agent are eagerly awaited.
JAK2 inhibitors
Elevation of erythropoietin levels, as seen in thalassemia and other
anemias, increase the number of erythroid precursors carrying phos-
phorylated Jak2 [73]. This is seen in murine models and in
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40TH ANNIVERSARY ISSUE
thalassemia patients, and it is thought to contribute to ineffective
erythropoiesis, with massive erythroid hyperplasia and extramedullary
hematopoiesis [74]. Therefore it is plausible to administer JAK2
inhibitors to reduce splenomegaly and ineffective erythropoiesis in
nontransfusion dependent thalassemia [63,73]. While this may seem
counterintuitive, as these drugs can cause anemia and other cytope-
nias, this approach is currently being tested in clinical trials for
selected patients.
Macrophage targeted therapy
The concept of the bone marrow microenvironment, known to be
important in many hematological diseases, is increasingly understood
to be a crucial factor in erythropoiesis [75]. The erythroid island has
a central macrophage which interacts with surrounding erythroblasts,
and provides regulatory feedback as erythropoiesis progresses, as well
as supplying nutrients and phagocytosing extruded erythroblast
nuclei. The microenvironment has a particularly important role in
the stress erythropoiesis which characterizes thalassemia, with extra-
medullary hematopoiesis and expansion of the erythroid progenitor
pool [76]. Unexpectedly, macrophages (previously benevolently
known as “nurse cells”) have been found to exert a negative effect on
hematopoiesis in thalassemia. This has been demonstrated in a recent
study in which pharmacological macrophage depletion (by injecting
clodronate liposomes) resulted in marked improvement of clinical
features in a murine model of thalassemia intermedia [77]. There was
improvement in splenomegaly, reduction in ineffective erythropoiesis,
increased Hb with improved RBC morphology (including reduced
accumulation of a-globin chains in the RBC membrane), and
improved iron kinetics [77]. (Astonishingly, the identical macrophage
depletion treatment ameliorated the phenotype of P. vera mice) [77].
Thus bone marrow macrophages regulate not only normal but patho-
logical erythropoiesis [78] and could be a target for therapeutic inter-
vention in thalassemia.
Heat shock protein 70 (HSP70)
Over 20 years ago, it was noted that thalassemic erythroid precur-
sors underwent accelerated apoptosis while normal precursors did not
[79]. Studies of normal erythropoiesis have led to increased under-
standing of these processes. GATA-1, an essential erythroid transcrip-
tion factor, is cleaved by transiently active caspases during late
erythroid differentiation. In erythroid precursors undergoing normal
terminal differentiation, GATA-1 is protected from premature caspase
action by the chaperon protein, heat shock protein (HSP) 70. This
protein translocates to the nucleus and protects GATA-1 from
caspase-mediated proteolysis. However, in b-thalassemia, HSP70
interacts with free a-globin chains in the cytoplasm, which sequester
it there and prevent it from translocating to the nucleus to protect
GATA-1. This results in maturation arrest and apoptosis [80].
Genetic manipulations such as transfecting an uncleavable GATA-1
or an HSP70 targeted to the nucleus, restored normal maturation of
thalassemia erythroblasts [80]. These findings may be applicable to
the development of novel therapies directed at free a-globin chains
or HSP70.
Genetic therapies
Since the b-globin gene was cloned, scientists have been striving
toward the goal of gene therapy to replace nonfunctional b-globin
genes. Though conceptually simple, this has proven to be an onerous
task due to many initially unforeseen factors. These include factors
related to the various vectors, to the complex regulation of expression
of globin genes, as well as the very high level expression which was
needed to correct the genetic defects. At the present time, a handful
of patients in several different centers have been treated on approved
doi:10.1002/ajh.24231
Thalassemia 2016
(and still open) clinical trials for gene therapy of thalassemia [81].
Unforeseen genetic effects were noted in the first successful patient
treated: the integrated lentiviral vector caused transcriptional activa-
tion of HMGA2 in erythroid cells [82], thus this vector had to be
altered. The modified vector has proven successful in the treatment
of two adolescents with transfusion dependent thalassemia, who have
been transfusion free for 11 to 14 months following autologous trans-
plantation using stem cells corrected with the lentiviral vector [83]
The subject of vectors has been expertly reviewed in great detail [84],
including vectors designed for simple gene replacement, newer con-
structs such as vectors aimed to overcome silencing of the g-globin
genes, and vectors for genome editing. This newest genetic modifica-
tion therapy aims to directly repair mutations at the DNA level in
hematopoietic stem cells. While this would be ideal, this approach
has a low level of efficiency of transfecting hematopoietic stem cells
in addition to low efficiency of the homologous recombination neces-
sary for the correction of the mutation(s). Though gene editing will
not be clinically applicable in the near future, a recent report suggests
that these shortcomings can be overcome [85].
Therapies to prevent iron overload: Modulation of
hepcidin
While chelation is an effective and proven therapy, the ideal solu-
tion for iron overload would be its prevention. In addition to transfu-
sional iron overload, thalassemia patients have hyperabsorption of
iron due to insufficient production of the peptide hepcidin. Hepcidin
expression is decreased in response to ineffective erythropoiesis, as is
found in thalassemia. Therefore inappropriately low levels of hepcidin
cause deleterious iron hyperabsorption in thalassemia patients. Several
tactics have been attempted to compensate for hepcidin insufficiency,
including the use of hecpidin mimetic agents (“minhepcidins”), trans-
genic overexpression of hepcidin, and repression of TMPRSS6 (phar-
macologically or using genetic methods such as lipid nanoparticle
encapsulated siRNA). TMPRSS6 is one of the key upstream regula-
tory proteins involved in upregulating hepcidin expression These and
other methods have been recently summarized [86]. Interestingly, in
animal models of thalassemia intermedia, methods leading to
increased hepcidin also had a beneficial effect on ineffective erythro-
poiesis, with improvement of anemia and reduction of splenomegaly
in these animals [86].
䊏 The Future of Thalassemia: Will
Modern Medicine Win the Battle?
In summary, there are many exciting new developments in the
field of thalassemia at the diagnostic but more importantly, at the
therapeutic level. The pace at which new diagnostic and therapeutic
developments have been developed and clinically applied is rapidly
increasing (Table I). Over the last four decades, the life expectancy
for thalassemia patients in the industrialized world has increased dra-
matically. In the United Kingdom, the average life expectancy in 1970
was 17 years, in 1980 it was 27 years and in 1990, it was 37 years,
more than doubling of the life expectancy in two decades [42]. Since
2000, over 80% of thalassemia patients in the United Kingdom could
be expected to live longer than 40 years and the actual upper lifespan
limit cannot be defined due to continued improvements over time
[42]. Lifespan and hopefully, quality of life, should continue to
improve in the industrialized world.
Unfortunately, most of the techniques for diagnosis, proper treat-
ment, and prevention of thalassemia, utilize sophisticated technology
and/or are very costly. It is well known that the world’s distribution
of thalassemia patients is predominantly in developing countries
which do not have the resources (financial and technological) to
American Journal of Hematology, Vol. 91, No. 1, January 2016 19
Rund 40TH ANNIVERSARY ISSUE

TABLE I. Thalassemia: From Early Milestones to the Present and Future

Year Milestone Current status

1925 Cooley’s clinical description DNA-based mutational diagnosis

1946 First transfusion clinic for thalassemia Routine transfusions widely available
1973 Parenteral chelation: desferrioxamine Parenteral and oral chelation widely available;
hepcidin modulation for prevention of iron overload
1975 Liver biopsy as indicator of transfusional hemosiderosis No longer needed
1976 Second trimester DNA-based prenatal diagnosis First trimester DNA-based diagnosis widely
for certain genotypes available for virtually all genotypes;
investigational noninvasive diagnosis at
9 to 10 weeks using maternal blood sample
1982 Allogeneic BMT Routinely available for suitable patients
1984 MRI for liver iron overload MRI simultaneous measurement of heart,
liver, and pancreatic iron content
1985 Pharmacologic HbF induction Butyrates in clinical trials, novel agents
under development
1989 Preimplantation genetic diagnosis Available for selected cases
2005 Gene therapy Clinical trials with improved vectors
2014 Novel therapies: activin ligand traps, JAK-2 inhibitors In early clinical trials
2016 Additional novel therapies Under development

Dates are approximate in some cases and refer to the earliest approved human (not animal) use.

adopt the use of most of these modalities. Tragically, there are many
countries in which the most basic routine care of transfusion and
chelation are either nonexistent or woefully underutilized due to
financial constraints. To those in the industrialized world, it is shock-
ing to read that only about 12% of children born with thalassemia
worldwide are transfused and of these, only about 40% are adequately
chelated [86]. Therefore, the survival of thalassemia patients in low
income countries at present is similar to that in the Europe and the
United States 50 years ago [87]. Annually about 1.33 million preg-
nancies worldwide are at risk for thalassemia [86] but only a small
fraction have access to genetic counseling and prenatal diagnosis. Fur-
thermore, the problem of prevention and treatment of thalassemia is
presently a worldwide issue, due to migration of individuals from
higher prevalence regions to areas of lower prevalence [88]. Since
infection has always been a cause of death of thalassemia patients
[42,88], antibiotic therapy has prevented many deaths of children
with thalassemia syndromes. More patients are surviving into late
childhood and adulthood [88], thus increasing the number of thalas-
semia patients requiring long term therapy. All of these factors
increase the likelihood that thalassemia will constitute a global health
problem in the coming decades [88].
Modern medicine can partially alleviate the suffering of thalassemia
patients and increase the implementation of prevention in regions
where the standard of care is already high. However, it cannot van-
quish thalassemia on a global basis. Thalassemia is, now more than
ever, a disease of imbalance. At the cellular level, there is chain syn-
thesis imbalance, and at the societal level, there is a critical imbalance
between resource availability and resource need. Ultimately, social
policy makers and public health practitioners must enact measures to
maximize prevention of the disease, and provide optimal care for tha-
lassemia patients. These are both sorely lacking in much of the world,
even in the year 2016.

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21