Key words
familial - MAX - pancreatic neuroendocrine neoplasm - pituitary - pheochromocytoma
Introduction
Pancreatic neuroendocrine neoplasms (pNEN) have an incidence of 0.48 cases per
100 000, and the frequency is rising [1]. While they are usually sporadic, pNENs can occur in the setting of
multiple endocrine neoplasia type 1 (MEN1) and hence they are the subject of active
surveillance in that setting [2]. Other
genetic syndromes that are rarely associated with pNENs include von
Hippel–Lindau disease, neurofibromatosis type 1 (NF1), MEN4, Lynch and
Cowden syndrome [3]
[4]
[5]
[6]
[7]
[8]
[9].
In 2011, Comino Mendez et al. identified MAX as a risk gene for the
development of hereditary pheochromocytoma [10]. Germline mutations in MAX lead to the development of sporadic
and familial pheochromocytoma-paragangliomas and MAX acts as a tumor
suppressor gene in the MYC/MAX/MXD1 pathway [11]. While germline MAX genetic changes
account for a small proportion of all known genetic forms of
pheochromocytoma-paragangliomas, they appear to have an aggressive phenotype.
Burnichon et al. reported that pheochromocytoma-paragangliomas patients with
MAX mutations had an earlier age at onset as compared with non-mutated
cases and MAX associated tumors are much more frequently bilateral or have
multiple tumors occurring within the same gland [11]. Until recently the tumoral phenotypes associated with germline
MAX mutations and rearrangements were limited to pheochromocytoma,
paraganglioma and kidney neoplasms [12]
[13]. In primary tumors and cell cultures
derived from small cell lung cancer, a neuroendocrine tumor, somatic MAX
mutations and deletions with concurrent loss of heterozygosity (LOH) were found to
occur in 6% of cases [14].
Furthermore, two patients with gastrointestinal intestinal stromal tumors (GIST)
that were negative for
KIT/PDGFRA/BRAF/SDHx
abnormalities (quadruple wild-type) were reported as having somatic truncating
mutations in MAX
[15]
.
An association between MAX and the development of pituitary adenomas
(acromegaly or prolactinoma) has been described recently [16]
[17]. We described three cases of intragenic
germline deletions in MAX that were not identified on Sanger sequencing but
were established with multiplex ligation-dependent probe amplification (MLPA). Those
cases had aggressive features with early onset, recurrence, bilateral
pheochromocytomas or metastatic disease, in keeping with established MAX
related characteristics [11]
[17]. In one kindred, the deletion was
inherited by the patient’s son from his father [17]. Subsequent screening of this 31-year old
male, who had no medical history, was undertaken to identify tumors in known sites
related to MAX mutations. Unexpectedly, abdominal imaging studies revealed a
pancreatic mass, which was further investigated and characterized.
Statement of Ethics
The patient provided informed consent and the study was approved by the Ethics
Committee of the CHU de Liège.
Methods and Results
As we reported previously, the patient’s father had a history of recurrent
pheochromocytoma and a prolactinoma in the setting of a germline intragenic exon 3
deletion in MAX
[17]
. The pheochromocytoma tissue had been shown to have LOH at the MAX
locus that differed between the initial tumor and the recurrence (18 years later),
indicating separate somatic “second hit” events affecting the
wild-type MAX allele [17]. Family
genetic studies including MLPA had identified the son as a carrier of the identical
germline exon 3 MAX deletion as his father ([Fig. 1a]). Screening studies were performed and included biochemical and
hormonal analyses of adrenal and pituitary function, hematological, renal and liver
function tests. All were normal. Abdomino-thoracic and pituitary magnetic resonance
imaging (MRI) were performed and no evidence of
pheochromochromocytoma/paraganglioma, pituitary adenoma, or kidney tumors
was identified. On the abdominal MRI a 1 cm lesion in body of the pancreas was
identified, which was hyperintense on T2 weighted signal ([Fig. 1b]). An
18F-fluorodeoxyglucose-positron emission tomography-CT
(18FDG-PET-CT) scan showed no enhanced uptake. There was hyperfixation of
the tumor on 68Ga-DOTANOC PET-CT images, indicating strong
SST2 expression ([Fig. 1c]).
Neither biochemical evidence nor signs/symptoms of pancreatic hormone excess
were identified. The patient provided informed consent and the study was approved by
the Ethics Committee of the CHU de Liège.
Fig. 1 Panel a shows the genealogical tree of the family. The
father (I1) had a pheochromocytoma at 32 years of age that recurred
at the age of 50 and a prolactinoma that was diagnosed at the age of 49
years. His son (II1) had a pancreatic neuroendocrine tumor discovered
during screening at the age of 32. Both I1 and II1 were
diagnosed with an intragenic deletion of exon 3 in MAX. Other family
members were tested and had a wild-type MAX sequence and MLPA. Panel
b shows the location of the pNEN (arrow) as a hyperintense lesion
in the body of the pancreas on a T2-weighted MRI. Panel c shows
intense uptake in the tumor (arrow) on 68Ga-DOTANOC PET-CT.
To further investigate the lesion, a percutaneous ultrasound-guided fine-needle
aspiration (FNA) biopsy was performed. Hematoxylin and eosin staining showed
aggregations of cells with eccentric nuclei, salt and pepper chromatin pattern and a
granular, eosinophilic cytoplasm ([Fig. 2a]).
The tissue was positive for anti-CD56, Chromogranin A and Synaptophysin and no
mitoses were seen. A pathological diagnosis of a low grade pancreatic neuroendocrine
tumor was made (G1 grade; Ki-67: 1–2%, mitotic index: 0).
Immunohistochemistry of the FNA material for MAX was performed as previously
described [12]; this showed neuroendocrine
cells that exhibited loss of MAX nuclear staining in the setting of other
normally-stained cells ([Fig. 2b]). Genetic
analyses were also performed on the pNEN FNA tissue DNA; MLPA showed LOH and an
apparent homozygous deletion of the exon 3 of MAX gene ([Fig. 2c]). The MLPA results and the paternal
inheritance pattern strongly point copy neutral LOH involving the MAX locus due to
paternal uniparental disomy (UPD) at chromosome 14q as has been demonstrated in
familial cases of MAX-related pheochromocytoma and renal oncocytoma [11]
[12].
Fig. 2 Panel a shows an image of the hematoxylin and eosin
stain of the tissue obtained following fine needle biopsy of the pancreatic
lesion. The biopsy material shows groups of abnormal cells with generally
eccentric nuclei and an eosinophilic cytoplasm. Inset is a high
magnification image of a section of the tumor cells. Arrowed on Panel
b are groups of tumoral cells that were negative for MAX staining
(blue nuclei and negative cytoplasm) interspersed with groups of normal
cells. Inset is an image of a positive control from a normal pancreas
section demonstrating strong positive (brown) nuclear and moderately
positive cytoplasmic staining. Panel c illustrates the MLPA finding
on pNEN tumor DNA showing an apparent homozygous deletion in exon 3 in the
MAX gene. The upper histograms show each probe coverage in the
kit controls (blue) and in the patient’s sample (green).
The patient remains under close clinical follow-up and is currently asymptomatic. On
abdominal MRI at six months post-diagnosis the tumor remains stable and in light of
the low grade, size < 2 cm, low Ki-67 score, non-functional
status and patient wishes, the patient is being managed with active surveillance
[18].
Discussion
This is, to the best of our knowledge, the first case of a gastroenteropancreatic NEN
associated with an inherited germline MAX mutation or deletion. Originally
MAX mutations were described in association with pheochromocytoma, and
subsequent research has further defined the clinical phenotype which can be
bilateral and aggressive [10]
[11]
[19]. Since then MAX has been
implicated in a growing number of sporadic and familial cancers, many which have a
neuroendocrine origin. Emerging evidence suggests that inactivating MAX
genetic abnormalities appears to lead to tumor risk at multiple endocrine and
non-endocrine tissues, including pheochromocytoma, paraganglioma, renal tumors,
pituitary adenomas, and GIST and SCLC [10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[19]
[20]. Clustering of tumors within the same
patient and/or kindred with MAX mutations includes
pheochromocytoma-paraganglioma, pituitary adenoma, and renal tumors [10]
[11]
[12]
[16]
[17].
The past decade has seen a large volume of fundamental research on the genetics and
genomics of NEN in general and pNEN in particular. The study of inherited or
familial disorders provided early and important insights into pNEN pathogenesis,
including sporadic disease [21]. For example,
comprehensive analyses have identified mutations in genes such MEN1,
VHL, TSC1, TSC2, and PTEN, which cause individual
syndromic diseases, as also playing an integral role in the development of sporadic
pNET [21]
[22]
[23]. In addition, mutations in the
ATRX and DAXX genes that are involved in telomere length
regulation via histone 3.3 deposition are frequently found in pNEN [2]. Subsequent work has expanded the list of
recurrent genetic alterations, chromosomal loss/gain patterns and epigenetic
profiles and certain pathway groupings are now evident, including,
MEN1-related alterations, telomeric changes
(ATRX/DAXX), abnormal cell-cycle regulation (e. g.,
CDKN1B), PI3K-mTORpathway disorders, and disordered chromatin remodeling
or DNA and base repair dysregulation [21].
While these large-scale studies have not identified MAX
mutations/deletions as a major contributor to sporadic pNEN pathogenesis, it
remains to be seen if MAX intragenic copy number variations represent a
contributory factor in a subgroup of cases. Taking the findings of the current study
into account, it seems reasonable to suggest that surveillance of previously
identified MAX carriers could be expanded to include a wider range of
potential target tumors. As sporadic pheochromocytoma-paraganglioma cases without
known family history can have unsuspected germline mutations in MAX, similar
tumor risk related to MAX might be present in sporadic cases of NEN,
pituitary adenoma, among others [24]. Genetic
analyses of large NEN and other tumor banks should assess for intragenic deletions
and complex rearrangements of MAX, which can be missed by some sequencing
driven approaches [17].
