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Catecholamine metabolites in neuroblastoma patients

Verly, I.R.N.

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Citation for published version (APA):Verly, I. R. N. (2019). Catecholamine metabolites in neuroblastoma patients.

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Download date: 05 Jul 2020

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CHAPTER 1 General Introduction and outline of the thesis

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Neuroblastoma Epidemiology Neuroblastoma is the most common extra-cranial solid tumour in the paediatric age-group

(<18 years of age) (1, 2) and the most common form of cancer in infants (<1 year of age) (3).

The incidence rate of neuroblastoma decreases with age, starting at 25-50 per million children

(<1 year) and dropping to 1-1.5 per million (>10 years) children (4). Consequently, the average

age at diagnosis is 18 months (5) and 90% of neuroblastoma patients are diagnosed before

10 years of age (6). Although neuroblastoma is much less common than other forms of

paediatric malignancies (e.g. acute lymphoblastic leukaemia), it accounts for 10-15% of the

paediatric oncology related mortality (2, 7, 8).

Pathophysiology and predisposition Neuroblastoma is a malignancy derived from neural crest cells of the developing autonomic

nervous system (9). The location of the primary tumour is also the sites where the sympathetic

ganglia develop, with the adrenal gland medulla as the most common site (~50%) (10).

Although certain paediatric cancers have strong evidence for genetic predisposition (e.g. 45%

of the retinoblastoma cases are caused by a germline mutation in the Rb gene) (11), hereditary

neuroblastoma is very rare (12, 13). It is estimated that 1-2% of the neuroblastoma cases are

hereditary and most of them are linked to germline mutations in the anaplastic lymphoma

kinase (ALK) oncogene (14, 15). Other germline mutations associated with hereditary

neuroblastoma were found in the paired like homeobox 2B (PHOX2B) gene (16).

Sporadic neuroblastoma accounts for 98-99% of the cases and is characterised by

genomic alterations such as gene amplification, chromosomal aberrations and sporadically

mutations in specific genes (17, 18). The most common genetic aberration in neuroblastoma

is amplification of the MYCN oncogene, which is located on small arm of chromosome 2

(2p24.3) and is observed in approximately 20% of all neuroblastoma (8). MYCN, like other

members of the MYC protein family, is regarded as a key-transcription factor of various cellular

processes including proliferation, differentiation and apoptosis (19). For this reason,

expression of the MYC protein family is tightly regulated during embryogenesis and after birth

(19), while aberrant expression of the MYC protein family is associated with malignancies such

as neuroblastoma, medulloblastoma and lymphoma (19, 20). In various solid tumours,

increased MYC-signalling is associated with a more aggressive cancer phenotype (21-23).

The crucial role of MYC-signalling in neuroblastoma was demonstrated by overexpressing

MYCN in neural crest cells of various animal models, which gave rise to a neuroblastoma-like

disease (24, 25). Although in neuroblastoma, increased MYC-signalling is usually due to

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MYCN amplification (MNA), other causes for increased MYC-signalling, such as amplification

of c-MYC or a more stable MYCN protein, have also been described (26, 27).

Activation of the ALK oncogene, usually due to a gain of function mutation, is detected

in approximately 14% of the non-hereditary neuroblastoma cases (28). Although ALK

activation results in increased proliferation, additional aberrations such as MYCN

overexpression is usually required to cause neuroblastoma in animal models (29). The ALK

oncogene is also located on chromosome 2 (2p23.2-p23.1) and due to its close proximity to

MYCN, the two genes are co-amplified in 2-4% of the patients with MNA (28). Because of its

co-amplification with MYCN, the exact role of ALK amplification in neuroblastoma is uncertain

(28, 30).

In neuroblastoma, structural aberrations consisting of losses and gains of complete

chromosomes (numerical aberrations) and/or chromosomal parts (segmental aberrations) are

frequently detected (31). While numerical aberrations and near-triploidy are associated with

favourable neuroblastoma, segmental aberrations such as loss of heterozygosity for 1p

(LOH1p), LOH11q and gain of 17q associated with advanced stage disease and poor clinical

outcome (31-34).

Normal dividing cells have a limited replicative capacity, which is dictated by the

telomeres length (35). Once telomere length becomes too short, cells can no longer divide

and enter cellular senescence in order to prevent apoptosis (35, 36). Cells that must retain

replicative capacity (e.g. stem cells) can escape cellular senescence by increasing the

expression of the enzyme telomerase, which restores the telomere length (37). Also in solid

tumours (85-90%), telomere maintenance is achieved by increased telomerase expression or

by induction of alternative lengthening of the telomeres (ALT) (38). In neuroblastoma, it has

been shown that increased telomerase activity can be the result of MNA, but can also be

caused by rearrangement of the telomerase reverse transcriptase (TERT) gene, which

encodes for the catalytic domain of telomerase (39-41). Furthermore, mutations leading to

loss of function of the ATRX transcriptional regulator gene are associated with ALT (41).

Telomere maintenance, either by increased telomerase activity or by ALT, has been shown to

be a key-feature of aggressive neuroblastoma and associated with poor clinical outcome (40).

Neuroblastoma diagnostic workup Every patient with suspected neuroblastoma is evaluated by means of complete medical

history and physical examination. Clinical symptoms associated with neuroblastoma depend

greatly on the site where the primary tumour and metastases are located. A thoracic/cervical

tumour compressing the trachea or a big abdominal tumour compressing the lungs is likely to

cause dyspnoea. Horner syndrome (ptosis, miosis and anhidrosis) is usually seen in patients

with a cervical tumour that damaged the cervical sympathetic ganglia, while symptoms of

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paralysis are frequently seen in patients with a paraspinal tumour that compresses the anterior

horn of the spinal nerves or the spinal cord itself (17). Metastasis of neuroblastoma cells to

the bone and bone marrow could cause severe bone pain and disruption of normal

haematopoiesis (8). Neuroblastoma can also cause periorbital ecchymosis, causing the

classical racoon eyes, which might be mistaken for child abuse (42). In rare occasion (2-3%),

neuroblastoma can also be accompanied by a paraneoplastic syndrome such as opsoclonus

myoclonus syndrome (OMS), which is characterized by a mixture of neurological symptoms

and is thought to be an autoimmune process induced by neuroblastoma (43).

When the diagnosis neuroblastoma is suspected, urine is collected from the child and

subsequently analysed for the catecholamine metabolites homovanillic acid (HVA) and/or

vanillylmandelic acid (VMA), which are elevated in approximately 85% of the patients (44). In

addition, complete blood count, electrolytes, kidney and liver function, ferritin and lactate

dehydrogenase (LDH) levels are analysed (8, 45). Various imaging modalities including

ultrasound, computed tomography (CT), magnetic resonance imaging (MRI) and iodine-123

metaiodobenzylguanidine (123I-MIBG) scintigraphy are used in order to localise the primary

tumour, assess for image-defined risk factors (IDRFs) and identify distance metastases (46-

48). IDRFs are defined as risk factors that are expected to complicate the complete resection

of the primary tumour (e.g. encasement of major blood vessels) (49).

Bone marrow aspiration from both sides of the iliac crest are analysed by

immunocytology, histopathology and with RNA based quantitative real-time polymerase chain

reaction (qRT-PCR) in order to assess whether neuroblastoma cells have invaded the bone

marrow (50, 51). Biopsy from the primary tumour and/or metastatic sites is obtained for the

confirmation of the diagnosis and for further histological and molecular characterisation of the

tumour (44, 52). Histological characterisation of neuroblastoma is performed according to the

International Neuroblastoma Pathology Classification (INPC), which is based on the balance

between neuroblasts and Schwannian stroma (53, 54). This system distinguishes four main

types: neuroblastoma (NB, Schwannian stroma-poor), ganglioneuroblastoma nodular (GNB

nodular, combination of Schwannian stroma rich/stroma dominant and Schwannian stroma-

poor), ganglioneuroblastoma intermixed (GNB intermixed, Schwannian stroma rich) and

ganglioneuroma (GN, Schwannian stroma dominant) (53, 54). Based on the degree of

Schwannian differentiation, the NB type can be sub-classified as undifferentiated, poorly

differentiated or differentiating, while the GN type can be sub-classified as maturing or mature

(53). In addition, factors such as mitotic index and karyorrexis can also be scored (55).

Molecular characterisation of neuroblastoma usually includes assessment for the amplification

of the MYCN oncogene, DNA index (ploidy), selected structural aberration (LOH1p, LOH11q

and gain 17q) and a selective mutation panel (e.g. ALK mutation) (51). However, broader

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diagnostic platforms including gene expression profiles, complete karyotyping and whole exon

and/or genome are also applied (18, 31, 40, 51, 56).

Neuroblastoma staging and risk assessment Neuroblastoma staging follows the International Neuroblastoma Staging System (INSS),

which was created to classify tumours after surgical resection (44). Stage 1 is defined as

localised disease without lymph node involvement that could be completely resected. On the

other hand, stage 2a, 2b and 3 showed some degree of lymph node involvement and could

not be fully resected due to encasement of vital organs/structures (44). Metastatic

neuroblastoma can be classified as either stage 4 or stage 4 special (stage 4s) depending on

age at diagnosis and the metastatic sites. Stage 4 disease is the main variant of metastatic

neuroblastoma and it includes all patients with metastatic disease, unless they fit the stage 4s

definition. Stage 4s is a rare variant of metastatic neuroblastoma that includes all patients

younger than 12 months with limited metastases to the bone marrow (less than 10%

involvement), skin and/or liver, but no bone metastases (44). Unlike stage 4 patients, the

primary tumour and the metastases in stage 4s patients are expected to undergo spontaneous

regression without medical intervention (57).

In 2009, the International Neuroblastoma Risk Group (INRG) introduced a new staging

system (INRGSS) that would allow patient staging already in the preoperative phase (49). In

this system, patients with localised diseased (INSS stage 1-3) are assessed for Image-Defined

Risk Factors (IDRFs) and subsequently classified as either stage L1 or L2 depending on

whether IDRFs are absent or present, respectively (49). The definition of metastatic disease

(INSS stage 4 and 4s) remained the same, but for INSS 4s, the cut-off age was changed from

12 months to 18 months (49). INSS stage 4 and 4s were renamed M and MS, respectively. It

is worth mentioning that even though the international community has adopted the INRGSS

as its official staging system, the INSS is still widely used (58). After defining the patient’s

stage, other risk factors that are associated with a more aggressive disease and thus poor

outcome are assessed and combined to form a risk assessment model.

Older age at diagnosis is associated with poor clinical outcome in neuroblastoma,

however, the age cut-off that defines “older age” varies between risk assessment models (5,

22). Based on multiple large cohort studies, the INRG concluded that 18 months would be the

optimal age cut-off for stage L1, L2 and MS, while for stage M a more conservative age cut-

off of 12 months was selected (22, 49). Amplification of the MYCN oncogene, defined as >4x

the signal of the chromosome 2 reference probe (51), is considered as a risk factor for poor

clinical outcome because of its association with highly aggressive tumour behaviour (21-23).

For this reason, every neuroblastoma with MNA is regarded as high risk neuroblastoma.

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While there is a consensus about the prognostic value of stage, age at diagnosis and

MNA, other factors such as LOH1p and LOH11q are not included in every risk assessment

model (22). Currently, the risk assessment model applied in the Netherlands (DCOG2009-

GPOH2004) includes INSS stage, age at diagnosis, MNA and LOH1p as risk factors (Table 1). The risk assessment model suggested by the INRG includes INGRSS stage, age at

diagnosis, MNA, histology, LOH11q and DNA ploidy as risk factors (Table 2), however, this

model still requires validation. Most risk assessment models classify neuroblastoma patients

as low, intermediate or high risk, however, the INRG introduced an additional risk group, the

very low risk (22).

Stage Age MYCN 1p Risk group

Local 1 Any Not amplified Any Low 2a and 2b Any Not amplified Normal Low 3 < 24 Not amplified Normal Low 3 ≥ 24 Not amplified Normal Intermediate 2a, 2b and 3 Any Not amplified LOH1p Intermediate 1, 2a, 2b and 3 Any Amplified Any High

Metastatic 4s < 18 Not amplified Any Low 4 < 12 Not amplified Any Intermediate 4 ≥ 12 Not amplified Any High 4 and 4s Any Amplified Any High

Table 1 – Risk stratification according to the DCOG2009-GPOH2004. Primarily, neuroblastoma is diagnosed as either local or metastatic disease. Subsequently, INSS stage, age at diagnosis (months), MYCN status and 1p status are scored. Finally, patients are classified as low risk, intermediate risk or high risk and treated accordingly. LOH1p = loss of heterozygosity for 1p.

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Table 2 – The modified INRG risk assessment model. Primarily, neuroblastoma is diagnosed as either local (L1-L2) or metastatic (M-MS) disease. Subsequently, INRG stage, age at diagnosis (in months), MYCN amplification (MNA) presence, histology & differentiation, 11q status and ploidy are scored. Finally, patients are classified as very low risk, low risk, intermediate risk or high risk and treated accordingly. GN = ganglioneuroma, GNB = ganglioneuroblastoma, NB = neuroblastoma, D = differentiating, PD = poorly differentiating, U = undifferentiated, LOH11q = loss of heterozygosity for 11q, Hyper = hyperdiploidy, Di = diploid.

Treatment and clinical outcome Risk assessment models aim to predict the most likely clinical course of disease and enable

clustering of patients into risk group. Based on these risk groups, neuroblastoma patients are

allocated to the most optimal therapy in order to maximise the effect of the anti-cancer therapy,

while trying to avoid unnecessary harm due to toxicity and therapy-related complications (22,

59).

In low risk patients, neuroblastoma is expected to spontaneously regress or remain

present as uncomplicated stable disease (60, 61). Consequently, a watchful wait-and-see

policy was adopted for these patients with an option to surgically remove the tumour if

complete remission (CR) is not achieved within a year (61-63). Low risk patients with life

threatening symptoms (e.g. dyspnoea, paralysis, etc.) and/or local progression would receive

chemotherapy in order to achieve CR, however, if unsuccessful, the patient would be upstaged

to intermediate risk and treated accordingly. Finally, if a patient progresses to INSS stage 4 or

INRG stage M, the patient is upstaged to the high risk group and treated accordingly. The

expected prognosis of low risk and very low risk patients is excellent with 5-year overall

survival (OS) of ≥90% and 99-100%, respectively (22).

Stage Age MNA Histology Grade 11q Ploidy Risk group L1, L2 Any No GN, GNB intermixed Any Any Any Very Low

L1 Any No GNB nodular, NB Any Any Any Very Low L1 Any Yes GNB nodular, NB Any Any Any High L2 <18 No GNB nodular, NB Any Normal Any Low L2 <18 No GNB nodular, NB Any LOH11q Any Intermediate L2 ≥18 No GNB nodular, NB D Normal Any Low L2 ≥18 No GNB nodular, NB D LOH11q Any Intermediate L2 ≥18 No GNB nodular, NB PD, U Any Any Intermediate L2 ≥18 Yes GNB nodular, NB Any Any Any High M <18 No Any Any Any Hyper Low M <12 No Any Any Any Di Intermediate M 12-18 No Any Any Any Di Intermediate M <18 Yes Any Any Any Any High M ≥18 Any Any Any Any Any High

MS <18 No Any Any Normal Any Very low MS <18 No Any Any LOH11q Any High MS <18 Yes Any Any Any Any High

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Neuroblastoma in intermediate risk patients is likely to progress if treatment is not

given, however, since the tumour is expected to respond well to chemotherapy, aggressive

high risk therapy is not required (64). All intermediate risk patients are treated with alternating

chemotherapy courses, such as the N5 (vindesine, cisplatin and etoposide) and N6

(vincristine, dacarbazine, ifosfamide and doxorubicine) courses of the DCOG2009-

GPOH2004 protocol, followed by surgery and radiotherapy (65). The expected 5-year event-

free survival (EFS) and OS of intermediate risk patients is 50-75% and 70-90%, respectively

(22).

Treatment of high risk patients has tremendously improved over the last 40 years,

resulting in a significant increase of 5-year OS from 29% to 50% (59). The improved life

expectancy of high risk patients is mainly attributed to the introduction of high-dose

chemotherapy in combination with autologous stem cell transplantation and the introduction

of immunotherapy (66-69). Modern high risk therapy can be divided into three phases:

induction, consolidation and maintenance, each with its own goal and requirements in order

to proceed to the next phase (69, 70). The induction phase aims to eliminate the primary

tumour and gross metastatic burden by combining alternating courses of chemotherapy and

surgery. Although, different international protocols generally include the same

chemotherapeutic drugs, there is still variation in drugs combination (course) and frequency

in which every course is given. For example, the DCOG2009-GPOH2004 protocol applies

three alternating courses of N5-N6 (70), while the SIOPEN protocol applies alternation

between A (vincristine, carboplatin and etoposide), B (vincristine and cisplatin) and C

(vincristine, etoposide and cyclophosphamide) courses that are given as A-B-C-B-A-B-C-B

(69). The effectiveness of both induction protocols will be compared in the new high risk trial

of the GPOH-SIOPEN group. During and at the end of induction chemotherapy, the tumour

and its metastases are evaluated for their response to therapy by means of catecholamine

analysis in urine, imaging and bone marrow aspirations (44, 71). If the patient shows signs of

refractory disease or progressive disease, additional chemotherapy courses, different

chemotherapy courses and/or 131I-MIBG therapy are usually given (72, 73). After the tumour

size was reduced by chemotherapy, complete resection of the primary tumour will be

attempted. To ensure local control, the site of the primary tumour is irradiated with a dosage

of 21-36 Gy, depending on the treatment protocol and whether or not complete gross tumour

resection was achieved (74).The consolidation phase includes the high-dose chemotherapy

courses, either BuMel (busulfan and melphalan) or CEM (carboplatin, etoposide and

melphalan), followed by autologous stem cell transplantation (ASCT) (66, 75). Finally, the

maintenance phase combines courses of immunotherapy (anti-GD2 therapy) with or without

cytokines (IL-2, GM-CSF) and retinoic acid (69). Currently, high risk patients are expected to

have a 5-year EFS and OS of <50% and 50%, respectively (22).

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In addition to the standard therapy per risk group, a more targeted therapy based on

biomarkers is also available for neuroblastoma patients. Tyrosine kinase inhibitors such as

crizotinib and lorlatinib can be used in patients with AKL mutation, but their efficacy highly

depends on the type of ALK mutation (28). Similarly, drugs targeting mutated proteins or down-

stream targets in RAS-MAPK pathway, p53 pathway and MYC-signalling pathway are

currently evaluated in clinical trials and/or preclinical settings (76-78).

Relapse and relapse therapy Even with the most modern treatment protocols, approximately 50-60% of the high risk

patients will relapse (17) and their expected 10 year OS is 14.4% (79). As mentioned before,

neuroblastoma rarely contains mutations at diagnosis. However, at relapse, neuroblastoma

shows enrichment for mutations in various pathways, in particularly in RAS-MAPK pathway

(78%) (18). Treatment of patients with relapse includes chemotherapy (similar to patients with

refractory disease), 131I-MIBG therapy and more targeted therapy based on the newly acquired

mutations (28, 72, 80).

Catecholamines and their metabolites Physiological role in multicellular organism Catecholamines are monoamine signal molecules that consist of a benzene ring with two

hydroxyl groups (the catechol nucleus), which is connected to an amine group (81). This class

of signal molecules consists of dopamine, norepinephrine and epinephrine, which can function

as either neurotransmitters (central and peripheral nervous systems) or as hormones

(endocrine system) to enable communication between cells and rapid adaptation to external

and internal signals (82).

Dopamine is mainly produced in the central nervous system (CNS, the midbrain) and

is involved in processes such as motor control, motivation and regulation of the reward system

(83). Outside the CNS, dopamine was shown to play a role in blood pressure regulation,

kidney function and the immune system (84, 85). As dopamine cannot cross the blood-brain

barrier, the exact source of dopamine in plasma remains to be elucidated (86). Norepinephrine

is produced in the pons (CNS), the sympathetic ganglia and the adrenal medulla (sympathetic

nervous system) (87). Norepinephrine prepares the body for a fight-or-flight situation, resulting

in physiological changes such as increase in heart rate, elevation of blood pressure and

increase in alertness (82). Similarly to norepinephrine, epinephrine is also involved in the fight-

or-flight response, however, epinephrine is mainly produced by the adrenal medulla (82). It is

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worth mentioning that catecholamine excretion in humans was mainly studied in healthy adults

under physiological conditions. Based on these studies, it has been shown both in plasma and

urine, that physiological levels of dopamine, norepinephrine and epinephrine not only varies

with physical activity and emotions (88, 89), but also follow a circadian rhythm (90, 91).

Physiological excretion and degradation of catecholamines Catecholamine biosynthesis (Fig. 1) begins with the sequential conversion of the amino acid

tyrosine (TYR) to 3,4-dihydroxy-L-phenylalanine (L-DOPA), dopamine, norepinephrine and

epinephrine by the enzymes tyrosine hydroxylase (TH), dopa decarboxylase (DDC),

dopamine β-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT),

respectively (92). Subsequently, dopamine, norepinephrine and epinephrine are inactivated

by oxidation followed by methylation or vice versa (Fig. 1), which are performed by

monoamine oxidase A (MAOA) and catechol-O-methyltransferase (COMT), respectively (82,

92). Degradation of dopamine, norepinephrine and epinephrine leads to the formation of

various intermediate metabolites (Fig. 1) including their methylated derivatives 3-

methoxytyramine, normetanephrine and metanephrine, respectively, which will eventually be

metabolised to the end products HVA and VMA. Metabolism of norepinephrine and

epinephrine towards VMA (Fig. 1) requires several enzymatic steps present only in the liver

(82, 92).

Under physiological conditions, neurons can be classified as either dopaminergic,

noradrenergic or adrenergic, depending on the neurotransmitter/hormone that they excrete

(Fig. 2A-C). In a dopaminergic neuron (Fig. 2A), cytoplasmic dopamine is transported by the

vesicular monoamine transporter (VMAT) into excretion vesicles, where dopamine is stored

until it is released into the synaptic cleft. Dopamine can be recycled via the dopamine

transporter, which will transport dopamine back into the cytoplasm for storage in excretion

vesicles. In a noradrenergic neuron (Fig. 2B), vesicular dopamine is converted to

norepinephrine and subsequently norepinephrine is stored in vesicles until it is released into

the synaptic cleft. Norepinephrine can be recycled via the norepinephrine transporter (NET),

which will transport norepinephrine back into the cytoplasm for storage in excretion vesicles.

Adrenergic neurons (Fig. 2C), such as in the adrenal medulla, require transport of vesicular

norepinephrine back into the cytoplasm, where norepinephrine is converted to epinephrine

and subsequently released into the circulation.

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Fig. 1 – Simplified catecholamine biosynthesis and degradation pathway. Catecholamines and their precursors are indicated in orange, while their degradation products are indicated in red. Biosynthetic and degradation enzymes are indicated in blue and green, respectively. TYR = tyrosine, L-DOPA = 3,4-dihydroxy-L-phenylalanine, DA = dopamine, NE = norepinephrine, E = epinephrine, 3MT = 3-methoxytyramine, DOPAC = 3,4-dihydroxyphenylacetic acid, NMN = normetanephrine, DHPG = 3,4-dihydroxyphenylglycol, MN = metanephrine, HVA = homovanillic acid, MHPG = 3-methoxy-4hydroxyphenylglycol, MOPEGAL = 4-hydroxy-3-methoxymandelaldehyde, VMA = vanillylmandelic acid, TH = tyrosine hydroxylase, DDC = dopa decarboxylase, DBH = dopamine β-hydroxylase, PNMT = phenylethanolamine-N-methyltransferase, COMT = catechol-O-methyltransferase, MAOA = monoamine oxidase A, ADH = alcohol dehydrogenase, AD = aldehyde dehydrogenase.

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Fig. 2 – Catecholamine biosynthesis, transport and degradation in various neuron subtypes. Panel A: dopamine biosynthesis, transport and degradation in a dopaminergic neuron. TYR = tyrosine,

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TH = tyrosine hydroxylase, L-DOPA = 3,4-dihydroxy-L-phenylalanine, DDC = dopa decarboxylase, DA = dopamine, VMAT = vesicular monoamine transporter, COMT = catechol-O-methyltransferase, 3MT = 3-methoxytyramine, MAOA = monoamine oxidase A, HVA = homovanillic acid. Panel B: norepinephrine biosynthesis, transport and degradation in a noradrenergic neuron. DBH = dopamine β-hydroxylase, NE = norepinephrine, NMN = normetanephrine, NET = norepinephrine transporter, VMA = vanillylmandelic acid. Panel C: epinephrine biosynthesis, transport and degradation in an adrenergic neuron. PNMT = phenylethanolamine-N-methyltransferase, E = epinephrine, MN = metanephrine. Catecholamine excretion as biomarkers for neuroendocrine tumours Due to their neuronal origin, malignancies of the sympathetic nervous system (e.g.

pheochromocytoma, paraganglioma and neuroblastoma) frequently retain the capacity to

produce and excrete catecholamine metabolites (44, 93). Furthermore, catecholamine

excretion in such malignancies is usually increased compared to control subjects and analysis

of catecholamine metabolites in plasma and/or urine provides a relatively simple accurate way

to diagnose those malignancies (44, 93). In the past years, analyses of various catecholamine

metabolites has become more feasible due to the introduction of more precise analytical

methods that are also capable of analysing metabolites in the very low concentration range

(pico- to nanomolar range) (94). Currently, most analyses of catecholamine metabolites in

plasma and urine are performed with high performance liquid chromatography with either

electrochemical detection (HPLC-ECD) or fluorescence detection (HPLC-FD) or with mass

spectrometers in combination with HPLC (HPLC-MS/MS) (95). The current gold standard for

diagnosing pheochromocytoma and paraganglioma is analysis of plasma free metanephrines

(3-methoxytyramine, normetanephrine and metanephrine) (96), while diagnosis of

neuroblastoma mainly relies on analysis of urinary HVA and VMA (44).

Catecholamine excretion in neuroblastoma Neuroblastoma, similarly to pheochromocytoma, has been shown to excrete elevated levels

of catecholamine metabolites in 90% of the patients (97, 98). However, unlike

pheochromocytoma cells, neuroblastoma cells have very low intracellular catecholamine

concentrations (99) and very few catecholamine storage vesicles (100). Therefore,

neuroblastoma cells are believed to continuously release catecholamines and shortly

afterwards inactive them by oxidation and methylation (101). In addition, the DBH activity in

neuroblastoma has been shown to be much lower than in pheochromocytoma and

consequently, plasma norepinephrine and epinephrine concentrations in neuroblastoma

patients are much lower than in pheochromocytoma patients (101). Taken together, it can be

said that catecholamine metabolism and excretion in neuroblastoma differs from

catecholamine metabolism and excretion in pheochromocytoma and normal adrenal medullla

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(101), and consequently, characteristics seen in the latter (e.g. circadian rhythm and pulsatile

excretion) are not necessarily applicable for neuroblastoma.

Urinary catecholamines and neuroblastoma diagnosis Analysis of urinary catecholamine metabolites for the diagnosis of neuroblastoma patients was

already proposed five decades ago (102). However, the first international diagnostic

guidelines only appeared in the 1980 (103). Of all the catecholamine metabolites (Fig. 3),

urinary HVA and VMA were selected by most international groups as biomarkers for

neuroblastoma diagnostics. Because catecholamine metabolites were shown to follow a

circadian rhythm in healthy adults (90, 91), catecholamine metabolites were initially measured

in 24 hours urine collections (44, 104-106). Since the normal levels of catecholamine

metabolites change with the age of the patient, distinct paediatric reference values per age

groups were created (98, 107, 108). Due to practical difficulties with 24 hours urine collections,

analysis of HVA and VMA had to be simplified. This was achieved by introducing the concept

of a relative metabolite concentration by comparing the concentration of a metabolite of

interest to that of creatinine, which enabled analysis of HVA and VMA in a single portion urine

(97, 106, 109). However, many laboratories still claim that 24 hour urine collections are

necessary to avoid false negative and false positive results due to diurnal fluctuation (88-91,

110, 111). Based on various studies, the diagnostic sensitivity of HVA and VMA for

neuroblastoma was estimated to be 67-98% and 57-85%, respectively (97, 98, 106, 108, 110,

112, 113). However, the diagnostic sensitivity of HVA and VMA is significantly lower for

patients with INSS stage 1 disease (44% and 59%, respectively) (97) and patients with 123I-

MIBG non-avid tumours (50% and 16%, respectively) (114). Although catecholamine

metabolites encompass more metabolites than just HVA and VMA (Fig. 3), these other

catecholamine metabolites have been hardly studied. The diagnostic sensitivity of other

catecholamine metabolites have been reported in a few studies, reporting the following

diagnostic sensitivities for dopamine (70-94%), norepinephrine (14-67%), epinephrine (14-

28%), 3-methoxytyramine (87-89%), normetanephrine (72-84%) and metanephrine (17-19%).

However, most of them included less than 50 patients and thus these studies were

underpowered (97, 98, 110, 112, 113).

23

Fig. 3 – Urinary catecholamines used in neuroblastoma diagnostics. The universally applied markers (HVA and VMA) and less frequently applied markers are indicated in red and blue, respectively. The precursors of catecholamine metabolism are indicated in grey. TYR = tyrosine, L-DOPA = 3,4-dihydroxy-L-phenylalanine, DA = dopamine, NE = norepinephrine, E = epinephrine, 3MT = 3-methoxytyramine, NMN = normetanephrine, MN = metanephrine, HVA = homovanillic acid, VMA = vanillylmandelic acid.

Urinary catecholamines, response assessment and disease monitoring Analysis of catecholamine metabolites during therapy and follow up were shown to be

beneficial for assessment of therapy response and monitoring tumour activity, respectively

(61, 115, 116). For this reason, analysis of urinary catecholamine metabolites during therapy

and follow up was also recommended in international guidelines (44, 45). However, due to

potential dietary interference (117, 118), lack of standardisation of the analysis method (95,

104) and the introduction other disease monitoring methods (46, 48, 119), the role of urinary

catecholamines in response assessment and follow up became more questionable, resulting

in their omission from the current international response criteria (71).

Urinary catecholamines and neuroblastoma biology Although usually studied in small cohorts, elevated levels of specific urinary catecholamine

metabolites were also correlated with various clinical characteristics of neuroblastoma patients

(97, 98, 120-123). For example, elevated levels of dopamine, HVA, 3-methoxytyramine and

norepinephrine were all shown to correlate with advanced stage disease (INSS 3-4) and older

age at diagnosis (97, 98, 120-123). Patients with MNA had frequently increased levels of

dopamine and 3-methoxytyramine, while patients with MYCN single copy usually presented

with elevated levels of VMA (120, 121). Similarly, a relative lower excretion of VMA compared

to HVA (VMA/HVA ratio < 0.5) was associated with advanced stage disease, MNA and poor

outcome (44, 97, 98, 120). However, when this VMA/HVA ratio was evaluated in a large cohort

(n = 505), it appeared that a VMA/HVA ratio < 0.5 only predicted poor outcome in patients with

localised disease without MNA (123).

24

The scope of the thesis Catecholamine metabolites play a pivotal role in the diagnosis of neuroblastoma patients,

however, worldwide there is no consensus about how to implement these metabolites in a

clinical setting. The research presented in this thesis focused on improving the current

knowledge about catecholamine metabolites in neuroblastoma patients and exploring

catecholamine metabolites beyond diagnostic biomarkers.

Part I of the of the thesis describes how neuroblastoma diagnostics can be improved.

In chapter 2 we compared the diagnostic sensitivity of the classical catecholamine metabolites

(HVA and VMA) to a panel of six additional catecholamine metabolites (DA, 3MT, NE, NMN,

E and MN) in a large neuroblastoma cohort (n = 301). Special attention was given to clinical

subgroups that are more difficult to diagnose such as HVA and VMA negative patients and

patients with 123I-MIBG non-avid tumours. In chapter 3 we describe how analysis of urinary

catecholamine metabolites can be improved by simplifying urine collection, applying specific

storage conditions, making the extraction and analysis less time consuming. Furthermore, we

investigated whether the panel of eight metabolites catecholamine (described in chapter 2)

can be reduced without comprising the diagnostic sensitivity. Finally, in chapter 4 we explore

the use of plasma as an alternative for urine for the analysis of catecholamine metabolites to

diagnose neuroblastoma patients.

In Part II of the thesis, catecholamine metabolism in neuroblastoma is investigated

both in vitro and in vivo and explores the possibility of using catecholamine metabolites as

prognostic biomarkers. In chapter 5 we introduce the concept of catecholamine excretion

patterns, which are studied both vitro and in vivo and related to neuroblastoma subgroups. In

chapter 6 we tested whether elevation of catecholamine metabolites, in particularly 3-

methoxytyramine, associate with clinical outcome of neuroblastoma patients. Finally, in

chapter 7 we explore the biological rationale underlying the correlation between elevated

urinary 3-methoxytyramine and poor prognosis. Chapter 8 is a general discussion of all the

studies described in this thesis.

25

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