A multicenter prospective study of biochemical profiles of monoamine-producing tumors: utility
for diagnosis and determinants of clinical presentation. (The PMT study)

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors
that derive from adrenomedullary tissue (pheochromocytomas) in about 85% of
cases and from extra-adrenal chromaffin tissue (paragangliomas) in 15% of cases.
PPGL*s represent a surgically correctable cause of hypertension, a result of
their capacity to secrete catecholamines. As a consequence of the
unpredictable, often explosive nature of this secretion, the tumors are
potentially lethal if not quickly diagnosed and treated. Fortunately, once
suspected, appropriate biochemical testing now makes it unlikely that the
presence of a catecholamine-producing tumor will be missed. In particular,
recognition that the O-methylated metabolites of catecholamines - the
metanephrines - are produced continuously within chromaffin tumor cells and
independently of variations in secretory activity has led to promulgation of
these analytes as superior for diagnosis of PPGLs compared to other analytes.
PPGL*s occur either sporadically or as a part of several hereditary syndromes:
MEN2), VHL, NF 1, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127.
The clinical presentation and manifestations of PPGL*s are highly variable and
only now becoming understood to be influenced by the underlying mutation. There
is currently no method to predict malignant potential or even diagnose the
presence of malignant disease from immunopathological examination of a resected
tumor. This diagnosis continues to depend on identification of metastases, at
which stage the response to available therapies is limited.
Based on this extensive knowledge on the pathophysiology, diagnosis and
treatment of PPGLs tumors, implementation of innovative approaches aimed at
increasing the positive and negative predictive value of early stage
diagnostics as well as post-therapeutic monitoring is appealing. One of those
approaches is the so-called *miRNA-profiling*. MiRNA-profiling relies on the
detection and monitoring in body fluids of non-coding RNAs, the so-called
micro-RNAs (miRNA). Since the discovery of RNA interference, miRNAs have been
recognized as regulators in numerous developmental and physiological processes.
MiRNAs have been linked to cancer development and progression as well as other
diseases such as diabetes and amyotrophic lateral sclerosis (44). A mammalian
miRNA expression atlas based on small RNA library sequencing has been published
by the NIH. Interestingly, it has been shown that HIF may be involved in
up-regulation of miR-210 and miR-373, possibly implicated in DNA repair
pathways. Recently, miRNAs in serum and blood are emerging as a new and
promising class of biomarkers for the diagnosis of cancer and other diseases.
In this context, a further aim of this project would be to characterize
additional miRNA-biomarkers for PGGL through comparison in a prospective
setting of the miRNA-profiles detected in serum and urine of the patients with
the clinical patterns of disease and the biochemical profiles of the tumors.
Moreovoer, miRNAs could also represent future therapeutic targets.

Primary objective:
to identify new and improved disease biomarkers and establish the biochemical
and molecular basis for variations in the clinical presentation of the
different groups of tumors.

Secondary objectives:
• compare the diagnostic utility of urinary and plasma free metanephrines and
establish an effective strategy for distinguishing true- from false-positive
test results in patients with suspected PPGL.
• to establish relationships between biochemical and metabolic profiles,
underlying genotypes and presentation of disease.
• to compare miRNA-profiles in patients with suspected PPGLs with healthy
individual controls

This is mainly a prospective observational study with no specific extra
interventions for all patients suspected to have a PPGL. Alle diagnostic
methods are already used in regular patient care. The extra measurements
include a blood sample for miRNA profiles in all suspected patients and an
overnight urine sample for measurement of urinary free metanephrines in those
patients in whom a PPGL is possible (equivocal test results) or in whom it is
confirmed.

All patients with suspected PPGLs will follow several diagnostic and follow-up
phases as outlined in the protocol. The first 3 phases are scheduled within
the first 3 years of the study while the last phase will be carried out over
the entire 5-year period. Since these patients will be seen in regular care,
there is no fixed date schedule.
Phase 1: patients enter the study for diagnostic testing.
Phase 2: involves follow-up testing for confirmation of the biochemical
diagnosis of pheochromocytoma in a subset of patients with equivocal test
results.
Phase 3: involves disease characterisation (only patients with biochemically
confirmed PPGL).
Phase 4: involves disease verification and patient follow-up.

In all hypertensive and normotensive subjects, only blood and urine sampling
will be carried out for establishing reference intervals for biochemical tests.

The study will last for five years but all patients will be enrolled in the
first three years of the study. Follow-up will take place over minimum of two
and a maximal five years.
• start date: 1-10-2011
• end date: 1-10-2016

There are no extra risks involved since there are no extra interventional or
diagnostic procedures. This also applies for venous blood sampling or urine
sampling since this should done anyway.

In the normotensives there is a small risk of bruising when venous blood
sampling is done.