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Preface
ix
edema, angina, or syncope, which is associated with more advanced disease,
complicating diagnosis, definition of incidence, and treatment and
management. A family history of amyloidosis should therefore also be
considered during evaluation and genetic counseling should be recommended
to all patients with a suspicion for or new diagnosis of a condition with a
potential hereditary cause. Genetic counseling provides invaluable education
and support for families with a genetic diagnosis even if testing is not the goal
of the session. The lack of specificity in susceptibility to amyloidosis makes it
a challenge in terms of treatment and management, with treatment options
varying depending on several factors, including the type of amyloidosis,
possible side effects, the patient’s preferences, and their overall health.
Chapter 4 - Apart from a general clinical examination, a tissue biopsy with
the morphological proof of amyloid deposits plays an unsubstitutable role in
making a diagnosis. In cases of systemic diseases, a biopsy could be
untargeted at first (subcutaneous fat, small labial glands, rectal mucosa, and
tongue) and followed by a targeted biopsy of the involved organ (heart or
kidney) when the first untargeted specimen was not diagnostic.
In methods of special histological staining, amyloid reacts positively with
histochemical dyes Congo red or Sirius red, a metachromatic staining (methyl
violet) can also be used. Characteristic properties of amyloid using Congo red
stained slides seen under polarized light are required for the diagnosis.
An ultrastructure of amyloid deposits is demonstrated by using an electron
microscope, where non-branching fibrils composed of protofibrils can be seen.
Nowadays a determination of a specific amyloid type is dominantly based
on immunological methods with an antigen-antibody reaction
(immunohistochemistry, immunofluorescence, or less used immunoelectron
microscopy), that is the indirect evidence of the protein. Several difficulties
have been described in immunohistochemistochemical examination of
formalin-fixed paraffin embedded tissue samples. For example, the
heterogeneity of variable domains of light chains, antigen masking due to
protein folding, or contamination of amyloid deposits by serum proteins could
reduce reliability of the testing using an immunohistochemical method.
To avoid problems with nonspecific protein detection, the new
methodology based on mass spectrometry seems to be highly specific and
sensitive with the direct protein recognition in a small piece of tissue sample.
Liquid chromatography-mass spectrometry is an analytic chemistry technique
and a well established powerful proteomic tool which allows an identification
of the amyloidogenic protein and other amyloid-associated molecules as well
(serum amyloid P component, apolipoproteins). These form usual constituent
Raquel Watts
x
parts of the amyloid and may serve as an ancillary diagnostic approach. Laser
microdissection of amyloid deposit from histological sections significantly
increases the accuracy and reliability of the LC-MS analysis and is preferred.
Chapter 5 - Introduction. AL amyloidosis is a rare disease belonging to
the group of monoclonal gammopathies. The basis of the disease is the
deposition of fibrils formed by molecules of monoclonal immunoglobulin light
chains into the form of amyloid - amorphous proteinaceous material. Detection
and quantification of intact monoclonal immunoglobulin (MIg) or
immunoglobulin light chains is a key aspect in the laboratory diagnosis and
monitoring of patients with AL amyloidosis.
Aim. The content of this contribution is the authors’ own experience with
the determination of monoclonal immunoglobulin in patients with AL
amyloidosis together with other biochemical parameters applied to diagnostics
and monitoring of the disease. In addition to the usual parameters such as urine
analysis or the monoclonal immunoglobulin level assays, the benefits of free
light immunoglobulin chains (FLC) serum level assay and its application to
treatment response evaluation are pointed out, as well as the benefits of heart
function indicators such as troponins and natriuretic peptides and other
biochemical parameters for the stratification of AL amyloidosis patients.
Patients and Methods. The analyzed group included 27 patients with
systemic AL amyloidosis. In all patients the authors performed detection and
quantification of monoclonal immunoglobulins in serum and urine using
electrophoresis and immunofixation techniques, serum immunoglobulin free
light chains levels were determined by immunoturbidimetry FreeLite
TM
system. In group of 13 patients determination of heavy/light chain pairs of
immunoglobulins (HLC), using immunoturbidimetry HevyLite
TM
system was
performed. In part of patients cardiac enzymes and natriuretic peptides on
Roche Cobas system were determined.
Results. The immunofixation and electrophoresis of samples revealed the
presence of monoclonal components in 17 of 27 (63%) patients and the
median concentration was 3.8 g/l (0.3 to 14.2 g/l). 5 findings of complete
molecules of the IgG isotype (1x IgG-κ, 4x IgG-λ), 4 findings IgA-λ and in
one finding of IgD-λ were found by immunofixation. In 7 patients only λ
immunoglobulin light chains in serum were detected. When analyzing the
urine excretion, Bence - Jones protein was detected in 15 (56%) patients. In all
cases the quantity was over 200 mg/24 hours, while in 3 patients the authors
found the presence of complete molecules of monoclonal immunoglobulin in
urine (1x IgA λ, 2x IgG-λ). Analysis of serum immunoglobulin free light
chains showed abnormal values in all patients with median of 310 mg/l (53.6-
Preface
xi
1562 mg/l), pathology of κ/λ ratio was found in 25 patients (92.5%). HLC
analysis was performed in 13 patients, where those with a complete
immunoglobulin molecule (IgGλ, IgAλ) showed pathological levels of HLC
isotype and change in HLC index. In 2 patients with electrophoretic
quantitation of λ light chains only and in 3 patients with electrophoretic
negativity suppression of HLC levels in IgG class was observed. The patient
set monitored for cardiac markers consisted of 22 patients with histologically
confirmed AL amyloidosis, of whom 18 met the criteria for cardiac
involvement. Levels of cardiac biomarkers troponin T (TnT) and N‑terminal
pro‑brain natriuretic peptide (NT‑ProBNP) were determined in all patients. Of
the 4 patients without cardiac involvement, 1 has shown positive levels of TnT
and 2 positive levels of NT‑ProBNP. All cardiac involvement patients
exhibited abnormal levels of NT‑ProBNP (median 4,752 ng/ l; 415.7– 35,000)
as well as positive levels of TnT (median 0.0815 μg/ l; 0.02– 0.986). The
application of the Mayo stratification system to the set had determined 2
patients at stage I, 5 patients at stage II and 15 patients at stage III.
Conclusion. Determination of selected biochemical indicators plays an
irreplaceable role in diagnosis and monitoring of disease progress and requires
a close cooperation of hematologists and biochemical stuff. Advanced
laboratory facilities with modern methods are a necessity for quality care for
patients suffering from AL amyloidosis and other monoclonal gammopathies.
In: Amyloidosis ISBN: 978-1-63485-884-7
Editor: Raquel Watts © 2017 Nova Science Publishers, Inc.
Chapter 1
AMYLOID CARDIOMYOPATHY
Tomas Pika
1,*
, MD, PhD and Jiri Vymetal
2
, MD, PhD
1
Department of Hemato-oncology,
University Hospital Olomouc, Czech Republic
2
3rd Department of Internal Medicine,
University Hospital Olomouc, Czech Republic
ABSTRACT
Light-chain amyloidosis (AL), hereditary transthyretin amyloidosis
and senile systemic amyloidosis are the most common amyloidoses with
cardiac involvement. Cardiac involvement is present in more than 60% of
patients with light-chain amyloidosis, and symptomatic heart failure is a
dominant symptom in 35% of patients. In hereditary transthyretin
amyloidosis, clinical symptoms depend on the type of mutations in the
transthyretin gene. Patients with dominant cardiac involvement usually
carry the Val122Ile mutation.
Advanced stage clinical finding includes symptoms of right-sided
heart failure caused by restrictive and hypertrophic cardiomyopathy with
initial diastolic dysfunction, followed by a relatively rapid progression to
global form of heart failure. Chronic hypotension with ortostatic syncope
is often present with autonomous nervous system involvement. A sudden
death due to arrhythmogenic causes and a chronic refractory heart failure
are the most common causes of death.
*
Corresponding author: tomas.pika@seznam.cz.
Tomas Pika and Jiri Vymetal
2
Cardiac biomarkers assay is an essential part of diagnostic algorithm
for patients with AL amyloidosis. Usually, serum levels of troponins and
N-terminal pro-brain natriuretic peptide are evaluated. The values
determined at the time of diagnosis of the disease serve as a prognostic
parameter, and also enable an effective stratification of patients within the
Mayo Clinic staging system. Typical ECG findings include reduced
voltage in the extremity leads under 5 mm and the presence of QS in the
chest leads (pseudoinfarct pattern). At present, echocardiography is
crucial for the diagnosis and monitoring of cardiac involvement in
amyloidosis. The criterion of impairment is concentric hypertrophy of left
ventricle with end-diastolic thickness of the interventricular septum in
excess of 12 mm. Echocardiography, along with the cardiac biomarkers
assay, are crucial for the evaluation of organ treatment response or
disease progression. Magnetic resonance examination of the heart allows
morphological and functional imaging. The use of gadolinium contrast
examination enables evaluation of the extent of myocardial imbibition by
amyloid masses, using the phenomenon of “delayed enhancement”.
Endomyocardial biopsy is indicated in case of suspected heart condition
when non-targeted biopsies are negative. Cardiac amyloidosis therapy
focuses on influencing the principal process leading to amyloid
production, and on the treatment of cardiac failure.
DEFINITION, PATHOPHYSIOLOGY AND CLASSIFICATION
Amyloidosis is a heterogeneous group of diseases characterized by the
deposition of amyloid. Amyloid is the name of an insoluble fibrillar protein
with β-sheet conformation, which is rather resistant to proteolysis. Amyloid
masses are deposited extracellularly in tissues, resulting in their
disorganization and reduction of normal cellular elements with consequent
disruption of organ function. This is a dynamic, progressive process. More
than 30 different amyloidogenic proteins have been identified so far.
Amyloidosis is principally differentiated to systemic and localized types
depending on the scope of involvement, and to acquired and hereditary types
according to the character of transmission [1, 2]. Majority of systemic
amyloidoses is associated with multi-organ involvement. Cardiac involvement
is very frequent in AL (light chain), transthyretin and senile systemic
amyloidosis, but less frequent in cases of AA (secondary) and lipoprotein
amyloidosis. Isolated involvement of cardiac atria is found in AANF
amyloidosis, where the atrial natriuretic peptide represents the precursor
protein.
Amyloid Cardiomyopathy
3
AL amyloidosis (light chain) is either systemic or organ-limited disease
which belongs to the group of monoclonal gammopathies or plasma cell
dyscrasias. It tends to be associated with multiple myeloma or Waldenström’s
macroglobulinemia in approximately 10–20% of cases. This is a not-so-
frequent disease with incidence of 5–12 patients per million people per year,
but represents more than 70% of cases among all amyloidosis types. The
disease is characterized by deposition of fibrils formed from fragments or
complete molecules of monoclonal light immunoglobulin chains produced by
the clonal plasma cell population. Imbibition of tissues with amyloid leads to a
progressive involvement of organs – most often the kidneys, heart, liver and
the peripheral nervous system. In addition to the infiltration of tissues by
amyloid per se, direct toxic effect of light chains on cells is also anticipated,
apparently via oxidative stress mechanisms. Overwhelming majority of cases
presents obvious multi-organ involvement. The kidneys are the most
frequently affected organ, with clinical image of renal insufficiency and
excessive proteinuria, quite often accompanied by an advanced nephrotic
syndrome [3 – 7]. Cardiac involvement is detected in 50–60% of patients, and
20–35% of patients present clinical signs of cardiac involvement at the time of
diagnosis. On the other hand, isolated cardiac involvement is present in less
than 5% of patients. Cardiac involvement represents the most significant
prognostic factor relative to patient survival, while the severity of cardiac
involvement influences to a certain degree the choice of therapeutic strategy [8
– 10].
Hereditary transthyretin amyloidosis (ATTR, mATTR) counts among less
frequent amyloidoses (7–9%), in which the amyloid fibrils are formed by
transthyretin (prealbumin) molecules. Prealbumin is generated mostly by the
liver, and to a lesser degree also by the choroid plexus cells and retinal cells.
The disease is characterized by autosomally dominant type of heredity with
variable penetration. ATTR presents abnormal transthyretin variants due to
mutations of the transthyretin gene. To date, more than 100 TTR gene
mutations have been identified, and about 44 are associated with cardiac
involvement [8, 11]. The disease has endemic or familial presence; origin due
to a random mutation is less frequent. The most frequent disease type is
familial amyloid polyneuropathy (FAP) type I, characterized in overwhelming
majority of cases by a Val30Met mutation. Primary symptom is a progressive
peripheral sensomotoric polyneuropathy, while involvement of the alimentary
tract or eyes represent secondary symptoms. Cardiac involvement tends to be a
symptom of an advanced disease. Dominant cardiac involvement with minimal
neurological deficit is characteristic for patients carrying mutation Val122Ile
Tomas Pika and Jiri Vymetal
4
(familial amyloid cardiomyopathy, FAC), which is detected in 4% of African
Americans. The disease manifests by a progressive cardiomyopathy with onset
in patients after the age of 60. Other TTR gene mutations associated with
dominant cardiac involvement are rather rare (Ile68Leu, Thr60Ala,
Leu111Met and others) [12, 13]. The presence of TTR gene mutations can be
confirmed or excluded by a molecular genetic examination.
Senile systemic amyloidosis (SSA) is an acquired type of the disease, in
which the fibril aggregates are composed of native transthyretin (wild-type,
wtATTR) molecules. Senile amyloidosis represents predominantly a sectional
finding; clinical manifestation tends to be less frequent and the dominant
clinical symptom is usually cardiac involvement – most often in the form of a
restrictive or hypertrophic cardiomyopathy and supraventricular arrhythmias.
Dominant involvement is typical in males; sectional findings indicate that the
disease is present in as many as 25% of males older than 80 years of age,
however, clinical manifestation is less frequent. As a rule, carpal tunnel
syndrome is present. Confirmation of the diagnosis requires a biopsy with
determination of the type of amyloid masses and exclusion of hereditary forms
by genetic analysis of the TTR gene [12, 14, 15].
Serum amyloid A (SAA), an acute phase protein synthesized in the liver,
is the etiological agent of amyloid deposits in secondary amyloidosis (AA). It
represents 4–7% of all amyloidoses. AA amyloidosis accompanies chronic
inflammatory diseases, usually difficult to control with therapy (rheumatoid
arthritis, Bechterew disease), periodic febrile states (Familial Mediterranean
fever), and may be present also with malignancies (Castelman disease).
Dominant involvement is most often that of kidneys, in the form of the
nephrotic syndrome and/or renal failure, but cardiac involvement is rather rare
(2–3%). Diagnostics and treatment consists of elucidation of the underlying
disease or exclusion of hereditary febrile states (Familial Mediterranean fever,
TRAPS syndrome) [3, 16, 17].
Apolipoprotein AI amyloidosis (AAPO AI) is a rare disease with
autosomal dominant form of heredity. Deposition of amyloidogenic fibrils in
tissues and organs leads most often to renal, hepatic or cardiac damage.
Altogether, more than 50 mutations of the APOAI gene were described, out of
which 19 cause AAPO AI. The mutation type influences clinical symptoms,
organ involvement, rate of progress, prognosis and the age at which disease
begins to manifest. The most often involved organ are the kidneys, in the form
of progressive renal insufficiency, which, unlike with AA or AL amyloidosis,
is usually not associated with excessive proteinuria. Some of the patients
present progressive cardiomyopathy with cardiac failure [18 – 20].
Amyloid Cardiomyopathy
5
Table 1. Systemic amyloidoses affecting the heart
Type
Amyloidogenic protein
Organ involvement
Common:
AL
Kappa or lambda light
chains
Heart, kidney, liver, peripheral
and autonomic nervous system,
gastrointestinal tract
ATTR (mATTR)
Mutant transthyretin
Nervous system, heart
ATTR (wtATTR, SSA)
Wild-type transthyretin
Heart, carpal tunnel
Rarely:
AA
Serum amyloid A
Kidney, liver, spleen,
gastrointestinal tract, heart
AApoAI
Mutant apolipoprotein AI
Kidney, liver, skin, heart
AFib
Mutant fibrinogen
Kidney, spleen, liver, heart,
autonomic nervous system
AANF represents a localized amyloidosis type affecting cardiac atria.
Atrial natriuretic peptide is the precursor protein. Unlike the SSA, AANF
affects dominantly older women. Finding of this amyloidosis type is
predominantly sectional. It is anticipated that AANF may be the cause of atrial
conduction disorders or supraventricular arrhythmias [8, 21].
CLINICAL MANIFESTATION AND DIAGNOSTICS
The initial clinical symptoms of amyloid cardiomyopathy are non-
specific, because amyloid deposition in the myocardium leads to a gradual
thickening of the walls, which primary affects cardiac filling, while left
ventricular ejection fraction (LVEF) is preserved for a relatively long time. An
advanced clinical image includes progressive congestive cardiac failure with
dominant right-sided symptomatology. The patients complain of fatigue,
weakness, frequently also of abdominal discomfort, weight loss (namely with
SSA), or, on the contrary, rapid weight gain. Dyspnea tends to be exertional at
the beginning, but in late stages, in case of global cardiac failure development,
manifests also at rest. Physical examination usually reveals edemas of lower
extremities, ascites, hepatomegaly with blood stasis; pleural exudates are
frequent as well. Peripheral edemas are often enhanced also by
hypoalbuminemia with advanced nephrotic syndrome. Chronic hypotension is
often detected, frequently of orthostatic character, with reduced cardiac output
and vegetative nervous system involvement. Autonomous dysfunction is also
Tomas Pika and Jiri Vymetal
6
associated with the reduction or loss of cardiac frequency variability.
Palpitations or syncope may be the manifestation of supraventricular
arrhythmias, which are often due to dilatation of the left atrium caused by
chronically increased left ventricular filling pressure. This circumstance also
brings up the risk of intraatrial thrombus generation, which can be often
present also in patients with sinus rhythm. Prothrombogenic risk is moreover
often enhanced by the presence of nephrotic syndrome with hemostasis
cofactor deficiency (antithrombin III). Conduction disorders are much more
frequent with TTR amyloidosis and may be an indication for pacemaker
implantation. Valvular defects (mitral and tricuspid insufficiency) are rather
frequent with infiltration of the valvular and suspensory system. Angina-like
pain due to amyloid infiltration of the coronary vessels is less frequent.
Chronic refractory cardiac failure and sudden death due to electromechanical
dissociation or arrhythmogenic states count among the most frequent causes of
death [8 – 10, 12, 15, 22].
A thorough physical examination may indicate to the physician the
possible presence of amyloidosis, because most of the patients has multiple
organ involvement manifesting with an array of clinical symptoms (nephrotic
syndrome, cardiac failure, hypotension, polyneuropathy, macroglossia,
periorbital purpura); likewise, monoclonal gammopathy in patient history may
somewhat simplify diagnostics. Determination of AL amyloidosis diagnosis
itself necessarily requires a complex examination including evidence of
monoclonal immunoglobulin or structural subunits – free light
immunoglobulin chains – in the serum and/or urine, evidence of a productive
B-cell clone (clonal plasmocytes or possible ruling out of association with
multiple myeloma, Waldenström’s macroglobulinemia or other B-
lymphoproliferation) and organ screening (echocardiography or MRI of the
heart, US of the kidneys and liver, proteinuria, cardiac biomarkers and alkaline
phosphatase activity assays) [7, 17, 23 – 25]. Nevertheless, determination of
the diagnosis per se in all types absolutely demands tissue sample collection
with histological proof and consequent determination of amyloid mass type.
Non-targeted biopsies are used primarily (rectum, tongue, subcutaneous fat),
and only in case of negative result and lasting suspicion, a targeted biopsy of
the involved organ system is performed (biopsy of the kidney, liver or
endomyocardium). The principal morphologic method for amyloid proof is a
special Congo red stain showing specific birefringence with dichroism in
polarized light; direct immunofluorescence method can be used as well. Proof