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C. Corbetta and C. Dionisi Vici
Guthrie R, Susi A (1963) A simple phenylalanine method for detecting
phenylketonuria in large population of newborn infants. Pediatrics
32:338–343
Millington DS, Kodo N, Norwood DL etal (1990) Tandem mass spec-
trometry: a new method for acylcarnitine proling with potential for
neonatal screening for inborn errors of metabolism. J Inherit Metab
Dis 13:321–324
Pandor A, Eastham J, Beverley C etal (2004) Clinical effectiveness and
cost-effectiveness of neonatal screening for inborn errors of metabolism using tandem mass spectrometry: a systematic review. Health
Technol Assess 8(12):1–121
Price CP, Christenson R (2008) Evaluating new diagnostic technolo-
gies: perspectives in the UK and US.Clin Chem 54:1421–1423
Rinaldo P, Tortorelli S, Matern D (2004) Recent developments and new
applications of tandem mass spectrometry in newborn screening.
Curr Opin Pediat 16:427–433
Saudubray JM, Garcia-Cazorla A (2016) Clinical approach to inborn
errors of metabolism in pediatrics. In: Saudubray JM, Baumgartner
M, Walter J (eds) Inborn metabolic diseases. Springer, Berlin/
Heidelberg
Hyperphenylalaninemias, Tyrosinemias,
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Glycogenosis, Hyperammonemia
MarcelloCiaccio andLuisaAgnello
32
Hyperphenylalaninemias
It refers to all biochemical-clinical conditions characterized
by elevated circulating levels of phenylalanine (>120μmol/L
or >2mg/dL).
Hyperphenylalaninemias represent a heterogeneous
group of autosomal recessive diseases characterized by an
altered phenylalanine metabolism due to enzymatic deciency of phenylalanine hydroxylase (PAH). This hepatic
enzyme catalyzes the hydroxylation of phenylalanine (Phe)
to tyrosine (Tyr), the precursor of several molecules, including dopamine, norepinephrine, and melanin. To perform its
function correctly, PAH requires the presence of a cofactor,
tetrahydrobiopterin (BH4). 2% of hyperphenylalaninemias
are related to a defect in BH4 metabolism due to an alteration
in its synthesis or regeneration.
Phenylalanine is an essential amino acid introduced into
the body through the diet, which can be metabolized in ve
different ways:
1. Protein synthesis
2. Hydroxylation at the level of the benzene ring at the para
position, leadingto tyrosinesynthesis
3. Hydroxylation of the benzene ring at the ortho position,
leading to orthotyrosinesynthesis
M. Ciaccio (*)
Department of Biomedicine, Neurosciences and Advanced
Diagnostics, Institute of Clinical Biochemistry, Clinical Molecular
Medicine and Clinical Laboratory Medicine, and Department of
Laboratory Medicine, University Hospital “P.Giaccone”,
Palermo, Italy
Department of Laboratory Medicine, University Hospital
“P.Giaccone”, Palermo, Italy
e-mail: marcello.ciaccio@unipa.it
L. Agnello
Department of Biomedicine, Neurosciences and Advanced
Diagnostics, Institute of Clinical Biochemistry, Clinical Molecular
Medicine and Clinical Laboratory Medicine, and Department of
Laboratory Medicine, University Hospital “P.Giaccone”,
Palermo, Italy
4. Transamination with α-ketoglutaric acid to form phenylpyruvic acid
5. Decarboxylation, leading to the phenylethylamine synthesis, a biologically active sympathomimetic amine
Under physiological conditions, about 50% of the amino
acid introduced through the diet is transformed into Tyr
byPAH, and the remaining 50% is used for protein synthesis. A small percentage of Phe undergoes transamination,
leading tothe phenylpyruvic acidsynthesis, and decarboxylation, leading to phenylethylaminesynthesis.
Phenylalanine is a toxic amino acid because it can cross
the blood–brain barrier via a neutral amino acid transporter
(LAT1), and its accumulation in the brain may impair psychomotor development. In addition, increased phenylalanine
levels inhibit the intracellular transport of other amino acids
(particularly tyrosine and tryptophan), leading to a maturational and myelination decit in the central nervous system
(CNS). Finally, phenylalanine at high levels causes an
increase in the turnover of the myelin protein fraction that is
not compensated by an increase in its synthesis. The accumulation of catabolic products has a direct toxic action on
the intermediate metabolism of the CNS, as well as inhibits
enzymatic activities related to the metabolism of tryptophan
and tyrosine, causing a decreased synthesis of serotonin,
γ-aminobutyric acid (GABA), dopamine, and sympathomimetic amines that are involved in the regulation of brain
functions. Neuroimaging studies on patienta affected by
hyperphenylalaninemia have shown white matter lesions
associated with altered myelination. Untreated hyperphenylalaninemia manifests progressive retardation of psychomotor development associated with numerous other
manifestations, such as rashes and convulsions.
The PAH gene was cloned in 1983, and since then, mutations in it have been studied. In 1996, McGill University created the “Phenylalanine Hydroxylase Locus Knowledgebase”
(PAHdb) with the intent to collect more information on
mutations in the PAH gene (http://www.pahdb.mcgill.ca/).
To date, this database collects information on more than 500
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. Ciaccio (ed.), Clinical and Laboratory Medicine Textbook, https://doi.org/10.1007/978-3-031-24958-7_32
447
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M. Ciaccio and L. Agnello
mutations in the PAH gene, for 200 of which the residual
enzymatic activity is also known. Of these 564 mutations,
70% are missense, 12% deletions, 11% splicing site mutations, 5% nonsense mutations, and, 2% insertions.
Most patients are compound heterozygotes, i.e., carrying
several mutations in each allele, so the clinical phenotype
depends on the effect and interaction of the mutations.
Hyperphenylalaninemias have an average incidence of
about 1:10,000 infants and can occur in various forms that,
based on phenylalaninemia levels, can be classied into:
– Moderate hyperphenylalaninemia: 120 and 600 μmol/L
(2–10mg/dL)
– Moderate phenylketonuria: 600 and 1200 μmol/L (10–
20mg/dL)
– Classical phenylketonuria: >1200μmol/L (>20mg/dL)
The classic phenylketonuria is the biochemical-clinical
form that, for incidence and severity of the clinical picture,
has greater importance.
Hyperphenylalaninemia is the only metabolic disease for
which early diagnosis, neonatal screening, and effective
therapy have been available throughout Europe for more
than 30years. The early detection and treatment of the disease allow forreaching adulthood, which is infrequentin the
metabolicdiseases eld.
From a biochemical-clinical point of view, the main alterations of phenylketonuria are:
– Signicant increase in circulating levels of Phe;
– Increased urinary excretion of phenylpyruvic acid, phen-
ylacetic acid, and acid orthohydroxyphenylpyruvic;
– Reduction in circulating levels of Tyr,
– Decreased melanin biosynthesis due to competitive inhi-
bition on tyrosinase;
– Decrease in circulating levels of 5-OH-tryptamine, dopa-
mine, and GABA by phenylpyruvic acidainhibition of
specic decarboxylases.
The rst diagnostic investigation involves plasma aminoacidogram to conrm an increase in phenylalanine levels,
normal or low tyrosine values with a consequent Phe/Tyr
ratio >2, and the absolute normality of the levels of other
amino acids.
Once hyperphenylalaninemia is conrmed, it is necessary to verify its etiology, which may or not be due to a
defect in BH4 metabolism. Thus, patients with hyperphenylalaninemia perform a BH4 load test in the neonatal
period and before the start of diet therapy. This standardized test involves administering BH4 at 20 mg/kg.
Phenylalanine is measured before and 8, 16, and 24h after
cofactor administration. Normalization of phenylalanine
levels within 8h of cofactor administration depicts a BH4
defect, whereas lack of or modest reduction in phenylalaninemia identies an alteration in the PAH enzyme. Final
diagnostic conrmation is based on molecular genetic
investigations.
The denition of the clinical phenotype is essential to
dene the most appropriate therapeutic approach, establish
the prognosis, and ensure proper counseling, especially for
future pregnancies.
The treatment is based on the reduction, as early as possible and through a dietary approach, of the high circulatinglevels of Phe, without ever falling below the minimum
requirement to ensure normal protein synthesis in the body.
The intake of Phe must uctuate between a minimum, below
which normal growth is not possible, and a maximum that
represents the limit of individual tolerance understood as the
maximum amount of Phe compatible with maintaining a
phenylalaninemia within the therapeutic target. The personalized diet plan will include:
• The assumption of amino acid mixtures with low or zero
Phe content, enriched with vitamins, trace elements, and
essential fatty acids;
• The restriction in the intake of natural proteins, in particu-
lar meat, eggs, and sh, preferring a vegetarian diet;
• The use of hypo/a-protein products.
Diagnosis andTherapy
Three pivotal dates marked a turning point in the early diagnosis, treatment, and prognosis of hyperphenylalaninemia.In
1934, Dr. Asbjørn Følling identied hyperphenylalaninemia
as a possible cause of neuropsychic decits; in 1953, Dr.
Horst Bickel demonstrated the effectiveness of a low phenylalanine diet in the treatment of phenylketonuria; and, in
1960, Dr. Robert Guthrie developed the rst test that could
be used as mass screening for early detection of hyperphenylalaninemia (Guthrie test).
Infants with hyperphenylalaninemia identied by neonatal screening should undergo in-depth diagnostics to conrm
it.
The target Phe concentrations to be achieved by treatment
are:
– <360μmol/L (<6mg/dL) for patients up to 12years
– <600μmol/L (<10mg/dL) for patients older than 12years.
Maternal Phenylketonuria
Maternal hyperphenylalaninemia exposes the pregnant to the
risk of miscarriage and the fetus to an essential risk of
embryofetopathy, the severity of which is directly related to
maternal phenylalanine values. Phenylalanine can cross the
placenta. In particular, the fetus could develop microcephaly,
32 Hyperphenylalaninemias, Tyrosinemias, Glycogenosis, Hyperammonemia
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449
mental retardation, facial dysmorphia, intrauterine growth
retardation, and structural heart defects.
Phenylketonuric embryofetopathy can be prevented by a
strict phenylalanine-decient diet before conception and
throughout pregnancy. The woman with phenylketonuria
must undergo strict metabolic monitoring during pregnancy,
with phenylalaninemia measured twice weekly.
Phenylalaninemia values should be maintained <360μmol/L
(<6mg/dL).
Tyrosinemias
Tyrosinemia refers to pathological conditions characterized
by increased levels of circulating tyrosine. Tyrosine, a nonessential amino acid, in mammals, is synthesized from phenylalanine by an enzymatic process of hydroxylation. Through
complex metabolic pathways, thyroid hormones, catecholamines, and melanin are synthesized from tyrosine.
Tyrosinemias are distinguished into:
– Tyrosinemia type I
– Tyrosinemia type II
– Neonatal transient tyrosinemia
– Alkaptonuria
Tyrosinemia type I (or hepato-renal tyrosinemia) is due to
the deciency of the enzyme fumarylacetoacetate hydrolase
and has an autosomal recessive inheritance. The deciency
of the enzyme fumarylacetoacetate hydrolase determines the
accumulation of fumarylacetoacetate and maleilacetoacetate
in the liver and kidney and succinylacetone systemically.
These substances are responsible for hepato-renal damage
and neoplastic degeneration of the liver. In addition, succinylacetone inhibits porphobilinogen synthase.
From a clinical point of view, it is characterized by acute
manifestations in newborns and infants, such as severe liver
failure, vomiting, bleeding, sepsis, hypoglycemia, renal
tubulopathy, and chronic manifestations, such as hepatomegaly, growth retardation, rickets, hematomas, tubulopathy and
neurological crisis.
The diagnosis is based on the following ndings:
biochemical point of view, the increase in tyrosine levels,
secondary to TTA deciency, causes the accumulation of
tyrosine crystals, which stimulate an inammatory reaction
and oculo-cutaneous signs; other symptoms include painful corneal lesions, hyperkeratosis (soles of the feet, palms
of the hand), and mental retardation. Diagnosis is based on
the clinical picture, increased plasma and urinary tyrosine
levels, and increased levels of urinary tyrosine metabolites
(such as 4-hydroxyphenylpyruvate, 4-hydroxyphenyllactate,
4-hydroxyphenylacetate, and N-acetyltyrosine), TTA activity assay, and liver biopsy.
Neonatal transient tyrosinemia is due to transient deciency of the enzyme para-hydroxyphenylpyruvic oxidase. It
has an incidence of 0.2–10% in newborns, with a higher frequency in males and preterm infants. From a clinical point of
view, it is often asymptomatic and may be accompanied by
prolonged jaundice and/or reduced motor activity; it resolves
within 2–8weeks after birth. It may be aggravated by highprotein diets.
The diagnosis is based on the following ndings:
– Amino acids in plasma, and in particular, increased tyro-
sine (up to 40–50mg/mL);
– Organic acids in urine: increase in 4- hydroxyphenylpyruvate
and phenylacetate.
Alkaptonuria is due to a deciency of the enzyme homogentisate oxygenase, which causes an increase in circulating
homogentisic acid levels, reaching values above 3 mg/
dL. Homogentisic acid is eliminated by urine and also
excreted by sweat. It is precisely the homogentisic acid on
the skin that, in the presence of oxygen and alkaline pH, is
transformed into a brown pigment, called ochronotic pigment, which causes the typical dark coloration of the skin of
the pubic and axillary region of theaffected patients.
The same ocronotic pigment has a high afnity for cartilage and connective tissues and accumulates in costal cartilages, tendons, and ligaments, laryngeal and tracheal
cartilages, but also in all connective tissues, including endocardium and vascular intima.
The diagnosis is based on nding organic acids in the
urine, and in particular, increased homogentisic acid.
– Organic acids in urine: increase in succinylacetone (diag-
nostic), 4-hydroxyphenylpyruvate, 4-Hydroxyphenylacetate and 4-Hydroxyphenylacetate levels;
– Plasma amino acids: increase in tyrosine and methionine
levels.
Tyrosinemia type II (or oculo-cutaneous tyrosinemia)
is due to a deciency of the enzyme tyrosine transaminase
(TTA) and has an autosomal recessive inheritance. From a
Glycogenosis
It indicates all the clinical pictures referring to diseases due
to hereditary deciency of enzymes involved in glycogen
metabolism. In particular, glycogenosis can be due to:
– Alterations in glycogenolysis;
– Indirect glycogen synthesis increases, resulting in normal
or atypical glycogen accumulation.
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M. Ciaccio and L. Agnello
In vertebrates, glycogen is mainly found in the liver and
skeletal muscle and plays different actions. In particular,
muscle glycogen represents an exclusive source of energy
for the muscle, rapidly usable, which is exhausted in less
than 1h during intense muscular effort. On the other hand,
hepatic glycogen is fundamental for maintaining glucose
homeostasis, acting as a glucose reserve for other tissues
when exogenous glucose is unavailable (in the period
between meals and in fasting); the reserve of hepatic glycogen is exhausted in 12–24h.
The metabolism of glycogen consists of its degradation
and synthesis, each divided into 3 stages catalyzed by a specic enzyme.
The synthesis takes place in the following 3 stages:
1. Glucose activation (UDP-glucose);
2. Addition of UDP-glucose units to the nonreducing ends
on the glycogen by the formation of α-1,4-glycosidic
bonds;
3. Formation of α-1,6-glycosidic bonds to create the
branches.
Degradation takes place in the following 3 stages:
1. Breakage of α-1,4-glycosidic bonds with the release of
glucose 1-phosphate;
2. Breakage of α-1,6-glycosidic bonds;
3. Conversion of glucose 1-phosphate to glucose
6-phosphate.
Depending on the mutated gene, glycogenoses can be
classied into predominantly hepatic or muscular forms
(Table32.1).
All glycogenoses have autosomal recessive inheritance,
except for glycogenosis IX characterized by X-chromosomelinked inheritance.
Glycogenoses have an overall incidence of 1 in every
20–25,000 births. Glycogenoses I and III are the most frequent hepatic glycogenoses, whileglycogenosis IVis rare.
In contrast, glycogenosis II, particularly the severe infantile
Table 32.1 Classication of glycogenosis
Hepatic glycogenosis
I Defect of glucose-6-phosphatase
III Defect of amyl-1,6-glucosidase (deramifying enzyme)
IV Defect of 1,4–1,6-transglucosidase (branching enzyme)
VI Defect of phosphorylase
IX Defect of phosphorylase kinase
Muscle glycogenosis
II Defect of acid alpha-glucosidase (acid maltase)
V Defect of myophosphorylase
VII Defect of phosphofructokinase
form (Pompe disease), is the most common muscle
glycogenoses.
Glycogenosis I, also known as von Gierke’s disease,
named after the Dutch physician who rst described it in
1929, is due to mutations in glucose-6-phosphatase (G6Fase),
an enzyme that is essential for glucose homeostasisregulation. Glucose-6-phosphate resulting from glycogen demolition and gluconeogenesis is transferred by a transporter
(TG6F) from the cytoplasm into the endoplasmic reticulum
(RE), where it is converted to glucose by G6Fase. In the
enzymedeciency, G6F cannot be converted to glucose and,
therefore, cannot escape from the cell and enter circulation.
In glycogenosis Ia, the gene encoding for the G6Fase enzyme
is altered, while in glycogenosis Ib, it is the gene encoding
for the TG6F transporter.
Glycogenosis III is due to a deciency of the enzyme
amylo-1,6-glucosidase or deramifying enzyme, leading to
the hepaticaccumulation of glycogen with an abnormal conformation (glycogen with very short branches) detrimental
to the hepatocyte, progressively leading to periportal brosis
and often to liver cirrhosis.
Glycogenosis II, also known as Pompe disease, is a lysosomal storage disease due to deciency of acid α-glucosidase,
also known as acid maltase.This enzyme hydrolyzes glycogen into glucose units. The deciency results in an intralysosomal accumulation of glycogen. For this reason, this
condition is considered a lysosomal storage disease.
Diagnosis
The diagnostic suspicion of glycogenosis is based on the
biochemical-clinical picture; diagnostic conrmation is
based on molecular genetic investigations and the dosage of
enzymatic activity in muscle or liver tissue. From a
biochemical- clinical point of view, hepatic and muscle forms
present different characteristics. Patients with hepatic glycogenosis have:
– Hypoglycemia;
– Hepatomegaly (due to glycogen accumulation);
– Lactic acid: increased in type I, normal in type III.
Patients with muscle glycogenosis have:
– Intolerance to intense physical exertion with cramps,early
pain, and myoglobinuria;
– No increase in lactic acid and excessive increase in
amoniemia after exercise;
– Increased circulating levels of muscle enzymes, particu-
larly the creatine kinase isoform MM (CK-MM).
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Hyperammonemia
It refers to a clinical condition characterized by elevated
blood levels of ammonium, frequent involvement of the central nervous system, and an uncertain prognosis. The severity
of presentation varies signicantly according tothe patient’s
age, the level of ammonium elevation, and the timeliness of
therapeutic intervention. Hyperammonemia represents a
medical emergency. Ammonium (NH4+) is a low-molecularweight metabolite produced by protein catabolism and eliminated hepatically through the urea cycle. In the urea cycle, a
series of chain reactions in the mitochondrion and cytosol
allows the catabolism of 2 nitrogen (N) atoms and the production of a urea molecule. Altered functioning of the urea
cycle determines a reduced production of urea with an accumulation of the products upstream of the defect; therefore,
hyperammonaemia and deciency of those downstream.
Alteration of the urea cycle can be classied as:
– Primary, due to cycle enzyme deciency;
– Secondary, due to enzymatic inhibition or substrate
deciency.
The severity of hyperammonaemia depends on:
– Enzyme involved;
– Residual activity of the enzyme involved;
– Rate of endogenous protein catabolism;
– Age of onset.
Ammonium is extremely toxic to the CNS due to:
– Glutamine-mediated induction of cerebral edema;
– Alteration of neurotransmitters;
– Energy decit.
Causes of hyperammonaemia can be classied according
to the presence of hypoketotic hypoglycemia (Table32.2).
Among the causes of hyperammonemia with hypochetotic hypoglycemia, defects in beta-oxidation are the most
common. Specically, defects in beta-oxidation are associated with impaired acetyl-CoA synthesis, which in turn
results in:
Table 32.2 Causes of hyperammonemia
Without hypoglycemia With hypochetotic hypoglycemia
-Decit of the urea cycle
- N-acetylglutamate
synthetase inhibitors (e.g.,
therapy with valproic acid
and/or diet low in arginine;
maple syrup urine disease;
etc.)
- HHH syndrome:
Hyperammonemia,
hyperornithinemia,
homocitrullinuria
- Defect in transport of
dibasic aminoacids
- Defects of the
mitochondrial respiratory
chain
-Hepatic insufciency
- Defects in protein
glycosylation
(blood glucose <45mg/dL and
circulating3-OH-butyrate <0.4mM)
- Defects of ketogenesis: 3-OH-3methyl glutaric aciduria
- Defects of mitochondrial betaoxidation of fatty acids
- Multiple defects of acyl-CoA
dehydrogenase or glutaric aciduria
type II, complex deciency ETF-Qo
or ETF-DH inhibits mitochondrial
oxidation of all fatty acids
- Hyperinsulinism with persistent
hyperammonemia
tion of oxaloacetate. Thus, if acetyl-CoA is missing, oxaloacetate will be missing for gluconeogenesis.
Particular care must be observedduring blood collection,
which can be venous or arterial, but it must be avoided prolonged stasis and, therefore, hypoxia (do not compress or
block); the blood must be collected in a tube with EDTA,
stored on ice and analyzed within 1h.
If these rules are not followed, the test must be repeated.
This is a highly URGENT test, which all laboratories must
perform on an urgent/emergency basis. In the presence of
encephalopathy, plasma ammonium levels should be measured, especially before a lumbar puncture.
Hyperammonaemia may occur in acute or subacute,
chronic, and/or relapsing forms. Normal plasma ammonium
values are <50 μmol/L (<90 μg/dL) and in the neonatal
period <110μmol/L (<190μg/dL).
Hyperammoniemias are distinguished into neonatal,
infantile, puberty, and adult forms.
Neonatal Form
– Hyperammoniemia: acetyl-CoA is required for the syn-
thesis of N-acetylglutamate, the cofactor of the enzyme
carbamoyl phosphate synthetase that catalyzes step I of
the urea cycle.
– Hypochetosis: ketogenesis begins from acetyl-CoA.
– Hypoglycemia: acetyl-CoA condenses with oxaloacetate
to start the Krebs cycle, which in turn leads to the forma-
It generally occurs in an acute form in a healthy newborn
who, between 24 and 48h of age, shows weak suction, hypotonia, progressive lethargy, and/or convulsions. Respiratory
alkalosis is present in 50% of patients, allowing a differential
diagnosis with neonatal sepsis, which is characterized by
respiratory acidosis. If adequately treated, patients may survive, with variable risk of mental retardation and epilepsy.
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M. Ciaccio and L. Agnello
Child Form
– Acute: episodes of irritability, refusal of food, drowsiness,
acute encephalopathy, lethargy, altered state of conscious-
ness, ataxia, and convulsions.
– Subacute: poor growth, recurrent vomiting, absence epi-
sodes, psychomotor retardation, and latent neurological
symptoms.
Puberty andAdulthood Forms
Most patients do not have a cognitive delay, although it is
possible to diagnose urea cycle deciency in a patient with
apparently isolated mental retardation or epilepsy.
– Acute: episodes of acute encephalopathy of varying
degrees that may be confused with toxic or infectious
encephalopathy.
– Subacute/chronic: learning disorders, behavioral altera-
tions and/or symptoms of cerebellar lesions, psychic
symptoms, episodes of disorientation, lethargy and/or
acute encephalopathy following protein intake, fever and/
or stress, and postpartum encephalopathy. Headache cri-
ses with repeated vomiting are frequent.
The possibility of an inherited metabolic disorder as a
cause of hyperammonemia must always be considered at any
age. A delay in diagnosis and treatment may result in brain
death and/or irreversible neurological sequelae.
Table 32.3 shows the classication of hyperammonemia
based on ammonium values.
In case of diagnostic suspicion of hyperammonaemia, II
leveltests include plasma aminoacidogram and the measurement of urinary amino acids, and plasma and urinary homocitrullinemia. Diagnostic conrmation can be obtained by
molecular analysis.
After the diagnosis of hyperammonaemia has been made,
treatment should be instituted immediately, even before a
denitive diagnosis of the specic cause of the defect is
established.
The aim of the treatment is to restore and maintain blood
levels of ammonium within normal limits.
The principles of treatment are based on:
– Minimization of endogenous ammonium production;
– Administration of an adequate amount of nonprotein cal-
ories to arrest protein catabolism. Protein and amino acid
intake should be discontinued until ammonium levels
normalize.
Table 32.3
Age Degree
Infants Mild 110–180
>30days Mild 90–150
Infants Moderate 180–350 Lethargy, hypotonia,
>30days Moderate 150–350
At any
age
At any
age
NV normal values
Classication of hyperammonemia based on NH
Levels of
+
NH
4
(μmol/L) Clinical symptoms
Food refusal, hypotonia,
(NV
<110)
(NV <50)
Serious >350 Coma without pain
Very severe, with
certain
neurological
sequelae in case
of survival
>700 Severe coma, retinal
groans, drowsiness, and
progressive lethargy
Drowsiness, irritability,
vomiting, food refusal,
medium-grade ataxia
unresponsive pupils, no
mydriasis. As the
condition progresses,
coma with pain response,
middle pupils, hypotonia
response, hypotonia,
lethargy, mydriasis,
cardio-respiratory arrest
hemorrhage. Breaks from
apnea. Mydriasis. High
risk of herniation after
12months
+
values
4
The hypoproteic diet is also associated with arginine (in
all defects except argininemia) and citrulline, and drugs that
promote the elimination of nitrogen through alternative
routes to the urea cycle, such as sodium benzoate and sodium
phenylbutyrate.
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Circulating Biomarkers inOncology:
https://t.me/medicina_free
Areas ofApplication, Critical Issues,
andPerspectives
MassimoGion, ChiaraTrevisiol, andAlineS.C.Fabricio
33
Biomarkers inOncology: AnEvolving
Concept
The concept of a biomarker as a biochemical signal associated with the presence of a malignancy dates back to 1845,
when Henry Bence-Jones published the article “On a new
substance occurring in the urine of a patient with mollities
ossium.” He accurately described the identication process
of the “substance,” but, more importantly, he hypothesized
the role of the “new substance” as a disease biomarker, suggesting its clinical application and indicating its importance
for understanding the pathophysiology of the associated
disease.
However, the clinical importance of biomarkers in oncology emerged only in the early 1960s of the last century,
thanks to two breakthroughs: the uncovering of immunoassays by Rosalyn S.Yalow and Solomon Berson, which led to
the development of easy-to-use immunometric assays to analyze a large number of samples simultaneously and in a reasonably short working time, and the measurement of estrogen
receptors by Elwood V. Jensen through the development of
radioligand binding assay methods. The determination of
estrogen receptors in the breast cancer tissue was rst used
by clinicians to predict the response to ablative hormonal
therapies, such as ovariectomy, and then to additive therapies
(progestins, antiestrogens, aromatase inhibitors, etc.) in
women with breast cancer. Immunoassays were widely used
to measure circulating levels of an increasing number of biomarkers, such as α-fetoprotein (AFP), carcinoembryonic
antigen (CEA), carbohydrate antigen 125 (CA125), and
many others progressively identied, initially classied as
tumor-associated antigens and conventionally known as
“tumor markers.” The discovery of hybridoma technology by
M. Gion (*)
Regional Center for Biomarkers, Department of Clinical
Pathology, Azienda ULSS 3 Serenissima, Venice, Italy
e-mail: massimo.gion@aulss3.veneto.it
C. Trevisiol · A. S. C. Fabricio
Veneto Institute of Oncology IOV– IRCCS, Padua, Italy
Georges Köhler and César Milstein, and the resulting availability of monoclonal antibodies in the 1980s, fueled the discovery of new biomarkers, the development of more sensitive
and specic immunoassays, and the optimization of assay
techniques. This was followed by a phase of great interest
and enthusiasm, in which it was widely believed that the
early diagnosis of several malignancies could be ultimately
feasible through the determination of “tumor markers.” The
clinical role of tumor markers was progressively scaled down
thereafter as their limitations began to be highlighted. The
rst one is the fact that tumor markers are not specic to
cancer and can be elevated in many non-oncological conditions; the second one is related to the direct relationship
between circulating levels of the marker and the extent of
tumor tissue; therefore, it is unlikely that an early– and thus
a small – malignancy can produce and release signicant
amounts of the marker. The awareness of these limitations
has considerably constrained the potential role of circulating
“tumor markers” for the early diagnosis of cancer. On the
other hand, as concerns predictive biomarkers, the last two
decades have witnessed extraordinary progress in the knowledge of the molecular mechanisms controlling cell growth,
which has led to the development of many new anticancer
drugs directed against specic molecular targets. The
approach to the treatment of malignancies has therefore progressively changed with the introduction of an increasing
number of molecular, targeted anticancer agents. This has
led to an in-depth investigation of the association between
the drugs and their biological targets, nally developing the
concept of a mandatory drug–biomarker association, thus
coining the term “companion diagnostics,” to the extent that
today several molecular targeted anticancer agents can be
prescribed only if the specic target has been determined.
Thus, the concept of a biomarker has undergone a profound change over time, which has extended its biological
meaning and broadened its areas of clinical application. The
American Association for Cancer Research in partnership
with the Food and Drug Administration and the National
Cancer Institute (AACR-FDA-NCI) Cancer Biomarkers
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. Ciaccio (ed.), Clinical and Laboratory Medicine Textbook, https://doi.org/10.1007/978-3-031-24958-7_33
455
456
https://t.me/medicina_free
M. Gion et al.
Collaborative consensus report has operationally dened a
biomarker as “an objectively measurable characteristic and
evaluated as an indicator of a normal biological process, of a
pathological process, or the pharmacological response to a
therapeutic intervention.” From this denition, it follows that
the term “biomarker” also includes, together with molecular
indicators, clinical signs or metabolic information derived
from imaging techniques. This more inclusive denition is
intended to support the development and application of those
new anticancer drugs directed at known biological mechanisms but still lacking a precise molecular target, as in the
case of tumor neoangiogenesis. Thus, hypertension associated with bevacizumab administration has been considered a
predictor of response in patients receiving this drug as the
rst-line therapy for metastatic colorectal cancer. Similarly,
acneiform rash occurring at the rst cycle of cetuximab therapy in patients with non-small cell lung cancer was found to
be a clinical biomarker of response to the drug. Concerning
imaging, changes of tumor metabolic activity evidenced by
positron emission tomography–computed tomography
(PET–CT) seems to be an early predictor of response to
treatment. Humbert etal. reported that a maximum standardized uptake value (SUV) ≤2.0 after the rst cycle of therapy
is the most effective predictor of response to trastuzumabcontaining regimen in women with breast cancer positive for
human epidermal growth factor receptor 2 (HER2).
These biomarkers represent a pragmatic approach to clinical application, but they are only the epiphenomenon of a
complex biological cascade. The need for their use testies
that more specic molecular biomarkers associated with the
different steps of the signaling mechanism of interest suitable for clinical use have not yet been identied.
Critical Issues andRoom forImprovement
Despite decades of research, thousands of published studies,
and a signicant amount of promising, preliminary results,
only a limited number of biomarkers are recommended for
clinical use. Several research groups have examined the
pathway of biomarker translation from discovery to clinical
practice, identifying numerous aspects that may cause delays
or errors in the translation process. Individual research
groups, scientic societies, and institutions have developed a
variety of proposals to improve the quality of studies on biomarkers and accelerate the translational process to clinical
practice.
A great deal of work has been carried out on classifying
biomarkers to make the taxonomy more consistent with
either the biological rationale or the needs for clinical application and to thus dene the boundaries of the different
research areas. Biomarkers are classied according to different criteria: (1) their biochemical nature; (2) the subcellular
site of localization; (3) the biological material where they are
mainly present and measured; and (4) the scope of their clinical application. The most commonly used classication is
the scope of their clinical application, distinguishing between
diagnostic, pharmacodynamic, prognostic, and predictive
biomarkers.
Pepe etal. proposed study protocols organized into different phases, similar to the well-established approach used for
the validation of a new drug. These protocols aim at standardizing the validation process of new biomarkers by reducing the variability of study design and thus improving the
comparability of results from different studies. Other groups
have suggested specic study designs for predictive biomarkers aimed at accelerating the early translation of results
to the clinical practice. Furthermore, the different steps of
the pathway from discovery to clinical application of a biomarker have been carefully examined to identify the points
of weaknesses and confounding factors in the pre-analytical,
analytical, and post-analytical phases.
The evidence provided by these methodological studies
have led scientic societies and boards of experts to develop
and disseminate specic guidelines to facilitate reporting of
studies in a standardized, accurate, and reproducible form,
such as the Standards for Reporting of Diagnostic Accuracy
Studies (STARD) and REporting recommendations for
tumour MARKer (REMARK) initiatives. Numerous scientic journals have adopted these guidelines as recommended
requirements for article submission. An indirect and implicit
objective of these guidelines is also to induce a progressive
improvement of study design.
The briey summarized abovementioned approaches,
aimed at improving the translation of research results on biomarkers to clinical application, have been disseminated and
implemented. They have contributed to the improvement of
the quality of both study design and results reporting.
However, they do not seem to have induced a meaningful
improvement in the pathway of biomarker translation from
discovery to clinical practice. In fact, even though basic
research has discovered thousands of promising candidate
biomarkers for clinical application, Anderson et al. have
shown that the rate of introduction of new molecules into
clinical practice has remained essentially stable with an average of 1.5 new biomarkers approved annually by the Food
and Drug Administration (FDA), which is unsatisfactory for
clinical needs.
Thus, the strategies adopted so far seem to have produced
only a marginal improvement in the biomarker translation
pipeline. The still limited availability of biomarkers for clinical practice suggests that crucial problems, probably not yet
fully identied, persist in the multistage process that, starting
from the discovery of a candidate biomarker, should transfer
it into clinical practice through sequential steps of
validation.
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