Update on Paragangliomas and Pheochromocytomas

Genomic studies in the recent decades lead to the identification of new genetic mutations that have been shown to play detrimental roles in the formation of pheochromocytoma or paraganglioma. The majority of these genetic mutations detected affect two major cellular pathways – pseudo hypoxic pathway and kinase signalling pathway. Genetic mutations also resulted in syndromes related to paraganglioma/pheochromocytoma. The classical syndromes comprise neurofibromatosis, multiple neuroendocrine neoplasia (Men) (II and III) syndromes and von Hippel-lindau syndrome. also, mutations in succinate dehydrogenase genes contribute to the understanding of hereditary paragangliomapheochromocytoma syndromes, Carney’s triad and CarneyStratakis syndrome. lesions newly known to be associated with the genetic mutations in pheochromocytoma/ paraganglioma include gastrointestinal stromal tumour and renal cell carcinoma. Pathological features, proliferative index, genetic and biochemical parameters could help to predict the malignant potential of paraganglioma and pheochromocytoma. different predictive systems have been proposed and with the help of immunochemical studies. Pathologist should be aware of the advances in knowledge and contribute to the validation of the pathological features and markers for prediction of malignant potential of this group of tumours.


GeneRAL ConCepTS updATeS
Pheochromocytoma and paraganglioma are neuroendocrine tumours with differentiation of chromaffin cells originated from neural crests.Pheochromocytomas are tumours located in adrenal glands whereas outside the adrenal gland, this group of tumours are labelled paragangliomas.due to the close relationship with neural tissue, composite lesions were reported both in the adrenal and extraadrenal locations.The other component of the composite tumour could be neuroblastoma, ganglioneuroblastoma, ganglioneuroma or malignant peripheral nerve sheath tumour (1-4).
The classical morphological features of pheochromocytoma or paraganglioma are tumour cells having granular cytoplasm with rich vascular networks.S-100 positive sustentacular cells could often be demonstrated.Unusual morphological features like co-existing cortical hyperplasia, vacuolar degeneration of tumour cells, presence of pheochromoblasts (small cells) and calcospherites, melanin pigmentation, cystic and oncocytic changes may be seen (5-9).The clinical and pathological diagnosis of paraganglioma may not be strict forward as they can occur in many different parts of the body.They could mimic other malignancies; negativity for keratins along with positivity for tyrosine hydroxylase and other neuroendocrine marker(s) are useful to confirm the diagnosis.Paragangliomas are sub-grouped based on the locations and properties into 4 groups according to classification by the World Health organization (10)(11)(12)(13).
The first group of paragangliomas are tumours arising from the paraganglia distributed along the parasympathetic nerve in the head, neck and mediastinum.They are tumours in the carotid body, jugulotympanic (middle ear), vagal (base of skull), laryngeal and aortico-pulmonary.Paragangliomas most commonly found in the head/neck region and this group is the most commonly known paraganglioma (14,15).
There are 2 groups of paragangliomas located in ganglia along the sympathetic chain.one group of paragangliomas parallel the distribution of the paravertebral sympathetic chain.Paragangliomas of the retroperitoneum along the aorta including suprarenal, renal hilar and infra-renal sites are the highest in frequency.These paragangliomas may be functionally active with excess secretion of catecholamines (usually norepinephrine) resulting in hypertension.The other group of paragangliomas come from paraganglia that are distributed along the pre-and paravertebral sympathetic chains and the sympathetic nerve fibres innervating the pelvic and retroperitoneal organs.They encompass aorticosympathetic paragangliomas and a subset of "visceralautonomic" paragangliomas.Well-known example of the group is urinary bladder paraganglioma with micturition induced hypertension (14)(15)(16).They were also examples noted in intra-thoracic and cervical paravertebral regions.
The last group of paragangliomas arise outside the usual distribution of sympathetic and parasympathetic paraganglia.They are rare and comprise gangliocytic paraganglioma (small intestine and pancreas), cauda equina paraganglioma, orbital paraganglioma and nasopharyngeal paraganglioma.
Cluster 1 mutations are involved with the pseudo-hypoxic pathway and with reduced oxidative response.also, cluster 1 tumours showed a marked increase in vascularization and in the expression of vascular endothelial growth factor (VeGF) and its receptors.VeGF pathway is important in the angiogenesis of many cancers and drugs targeting VeGF pathway is the standard for treatment for many metastatic cancers (37)(38)(39)(40)(41)(42).Thus, drugs targeting angiogenesis may be theoretically useful to be on trial on this group of lesions.In addition, some members of the group impair dna demethylation and this raise the possibility of innovative epigenetic therapies involving demethylation agents for some of these tumours.examples of the genes in this cluster for the pathogenesis of paraganglioma/ pheochromocytoma comprise VHL, EGLN1, SDHx (SDHC, SDHD, SDHB, SDHAF2, SDHA), IDH, HIF2A, and FH.
Cluster 2 mutations are associated with abnormal activation of kinase signalling pathways and included mutations of RET, NF1, KIF1Bβ, MAX and TMEM127.examples of the kinase pathways involved are PI3Kinase/aKt, raS/ raF/erK and the mtor pathway.The mtor pathway is important in the pathogenesis of many cancers and anticancer agents targeting this pathway is available (43).
Thus, agents targeting mtor pathway may be investigated for the treatment of some paragangliomas/ pheochromocytomas with cluster 2 gene mutations.
pAThoLoGiCAL LeSionS ASSoCiATed wiTh pARAGAnGLiomA oR pheoChRomoCyTomA an important update of knowledge required for pathologist and clinicians is the spectrum of lesions that could be related to paraganglioma or pheochromocytoma.The presence of syndromes related to phaeochromocytoma or paraganglioma often associated with bilateral tumours (Figure 1).

A. Classical Syndromes
Classical lesions associated with paragangliomas or pheochromocytomas occur in the hereditary syndromes -neurofibromatosis, multiple neuroendocrine neoplasia (Men) (II and III) syndromes and von Hippel-lindau syndrome (44).They are autosomal dominantly inherited diseases.In the settings of neurofibromatosis type I (NF1 gene), pheochromocytoma or paraganglioma could be associated with other neurofibromas, malignant peripheral nerve sheath tumours and gliomas as well as café au lait spots, iris hamartoma and dysplasia of the sphenoid bone.less commonly, gastrointestinal tumours, glioma or leukaemia may be noted.In multiple neuroendocrine neoplasia II (RET gene), pheochromocytoma could be found with medullary thyroid carcinoma and parathyroid hyperplasia.In multiple neuroendocrine neoplasia III (RET gene), pheochromocytoma could be seen in the settings of medullary thyroid carcinoma, marfanoid habitus SDHC, and SDHD (50).This syndrome is characterised by association of paragangliomas and gastrointestinal stromal tumour.In Carney's triad, paragangliomas are noted to be associated with gastrointestinal stromal tumour as well as pulmonary chondroma.also, adrenal cortical adenoma, oesophageal leiomyoma and pheochromoctyoma may be noted (51).Hyper-methylation of the promotor region of SDHC was recently reported to be responsible for the syndrome (52).The GISt is of similar pathological characteristics noted in PGl-PHeo syndromes.
It is worth noting that Carney-Stratakis dyad and Carney triad are different entirely from Carney complex in terms of pathogenesis (53).Carney complex is an autosomal dominant caused by mutations in the PRKAR1A (protein kinase a, regulatory subunit, type 1, alpha) gene on chromosome 17q23-q24 or other genes on chromosome 2p16.The complex has myxomas of the heart and skin, hyperpigmentation of skin and pituitary adenoma.The only feature in common in SdH related syndromes is the presence of pituitary adenoma.

updATeS on TeRminoLoGy
Pheochromocytomas have been called the "10% tumour" as many of the characteristics of the tumour could be accounted about ten percent of the time (54).These features may include approximately 10% are bilateral (i.e. in both adrenal glands), 10% are found in children, 10% are genetic (i.e.inherited), 10% are malignant or recurrence, and 10% are found outside the adrenal gland (paraganglioma).In addition, approximately 10% of these tumours are found after the patient has a stroke and 10% noted to be associated with Men syndromes.The "10% features" are noted to useful to arise awareness and remember the characteristics of this group of tumours.However, with the recent advances in knowledge about the tumours, we noted that these 10% features are no longer accurate.
The two 10% features that should definitely be deleted on the list are namely -"10% extra-adrenal" and "10% genetic".Many of the larger series had shown that paraganglioma could occur in different parts of the body and are found in higher prevalence than 10% extra-adrenal.The true prevalence of extra-adrenal lesions (paragangliomas) should be around 30 to 40% (14).also, it is now clear from the genetic studies that up to 40% of this group of tumours are either inherited or having known genetic susceptibility (36).

pRediCATion oF mALiGnAnT poTenTiAL
The prediction of malignant potential for endocrine tumours is always a problem for pathologists and clinicians.as in the case for other endocrine tumours, there is no definite as well as intestinal ganglioneuromas and cutaneous neuromas.Furthermore, in Von Hippel-lindau syndrome (VHL gene), pheochromocytoma or paraganglioma may be noted in the settings of clear cell renal cell carcinoma and central nervous system (cerebellum and spinal cord) haemangioblastomas.

B. Sdh Related Syndromes
In the last decade, mutations in succinate dehydrogenase (SDH) genes contribute to the hereditary paragangliomapheochromocytoma (PGl-PHeo) syndromes, Carney's triad and Carney-Stratakis syndrome.

B1. paraganglioma-pheochromocytoma (pGL-pheo) Syndromes
PGl-PHeo syndromes comprise PGl1, PGl2, PGl3, PGl4 and PGl5 (36,(44)(45)(46)(47)(48).PGl1 consists of pheochromocytoma or head &neck paraganglioma and relates to mutation in SDHD gene.The lesions noted in this condition also include gastrointestinal stromal tumour (GISt) and pituitary adenoma.PGl2 is related to mutation in SDHAF2 gene and consists of head & neck paraganglioma without other known lesions.PGl3 is associated with mutation with SDHC gene and characterized by head & neck paraganglioma and gastrointestinal stromal tumour.PGl4 is due to the mutation in the SDHB gene.The disease could present as pheochromocytoma or head & neck paraganglioma as well as the presence of papillary thyroid carcinoma, breast cancer, gastrointestinal stromal tumour and renal cell carcinoma.The type of renal cell carcinoma is a tumour of specific histological features and is now currently labelled as SDHB negative renal cell carcinoma (48).PGl5 is noted with mutation of SDHA gene.The disease could have paraganglioma and gastrointestinal stromal tumour.
In other words, the most common lesion noted in the PGl-PHeo syndrome is gastrointestinal stromal tumour (49).Pathologists should take notice if a GISt is detected in paediatric or young populations and without c-kit or PdGFr alpha (platelet-derived growth factor receptor alpha) mutation typically noted in majority of GISt.The GISt in this setting is likely to be having epitheloid morphology and have higher incidence of lymph node metastases.The GISt will not be responsive to standard therapy for GISt -Imatinib (tyrosine-kinase inhibitor)

Carney-Stratakis syndrome (Carney dyad) is an autosomal dominant syndrome associated with mutations in SDHB,
Vol. 31, Suppl, 2015; Page 105-112 histological feature that can predict the clinical behaviour of both paraganglioma and pheochromocytoma before metastases occur.In 2002, PaSS (Pheochromocytoma of the adrenal gland Scaled Score) have been developed by Thompson after review of 100 cases of pheochromocytoma (55).PaSS is based on a set of 12 histological features.Benign pheochromocytoma has a score of less than 4 and malignant pheochromocytoma has a PaSS score higher than 6.Patients with a PaSS >4 should be followed closely for recurrence.The drawbacks of using PaSS are the score is based purely on histological features and is only for pheochromocytoma.also, the assessment of 12 histological features is a complex and time consuming task.The prognostic values of PaSS have been tested by different research groups and usefulness of PaSS has been questioned (56,57).
In 2005, Kimura and colleagues from Japan proposed another system for assessment of malignant potential of this group of tumours based on 146 pheochromocytoma and paraganglioma (58).The system was refined in 2014 based on a total of 163 tumours, including 40 metastatic tumours, collected by the Pheochromocytoma Study Group in Japan (PHeo-J) (59).The system is called grading system for adrenal pheochromocytoma and paraganglioma (GaPP).The tumours were classified as well (score= 0-2), moderately (score =3-6), and poorly differentiated (score =7-10) types according to their scores.The differentiation of the pheochromocytoma appears to be correlated with potential for metastases and survival rates.Patients with poorly-differentiated pheochromocytoma are malignant and reported to have 0% survival rate.The system is yet to be validated by other researchers in different groups.
GaPP was based on 4 histological features (histological pattern, cellularity, comedo-type necrosis, capsular/ vascular invasion), proliferative index (Ki-67) and hormones secreted (biochemical phenotype) by the pheochromocytoma/paraganglioma.The GaPP system has the merits on assessing potential reproducible histological parameters.It also takes into account of commonly used parameter, Ki-67 proliferative index.The Ki-67 nuclear antigen has been associated with more aggressive cancers in different cancers.a Ki-67 index >3% is considered a useful parameter predicting malignant potential (60).one potential difficulty in using the GaPP is the range of Ki-67 index adopted is very narrow.The score is: very few cells positive (score=0); 1-3% positive (score= 1) and >3% positive (=2).
GaPP takes into account of biochemical properties of paraganglioma or pheochromocytoma.tumours with norepinephrine (noradrenaline) secretion are given higher score than non-functional tumours or tumours secreting epinephrine (adrenaline).It is worth noting that pheochromocytoma mainly produce adrenaline, while functional paraganglioma secrete noradrenaline.Malignant tumours have been shown to secrete predominantly noradrenaline, but due to less-differentiated catecholamine biosynthetic pathway, they may often produce mainly or exclusively dopamine (60).Therefore, the presence of large predominantly noradrenaline-producing paragangliomas and increased levels of plasma dopamine or its metabolite methoxytyramine may suggest malignancy.It is worth noting that malignancy in this group of tumours is generally associated to very high plasma levels of chromogrannin (61).
Beside histological factors, larger tumour (>5cm) appears to be carry a higher risk of malignancy (60)(61)(62).The survival rate may depend upon sites of metastatic lesions.also, patients with liver or lung metastases tend to have a poorer survival rates than patients with isolated bone lesions (60).different molecular markers have been proposed to affect the survival of patients with pheochromocytoma or paragangliomas (60).For the genes that have been discovered in the pathogenesis of pheochromocytoma/paraganglioma in the last couple years, SdHB and FH mutations have been associated with a high malignant potential in pheochromocytoma/ paraganglioma harbouring the genes.In particular, SdHB mutation has been studied in depth in this group of tumours with immunohistochemistry using commercially available antibody (Figure 2a,B).The Japanese group who proposed the GaPP system has also taken SdHB expression as a prognostic parameter (59).
For tumours that are negative for SdHB protein, it is likely that one of the few SdH genes have been mutated.SdHa protein loss could also be tested by immunohistochemistry. Therefore, SdHB immunohistochemistry could be performed to guide genetic testing in pheochromocytoma/ paraganglioma.The negative staining may be difficult to be assessed probably.nevertheless, the reproductively of the use of SdHB/SdHa in pheochromocytoma/paraganglioma has been validated by multicentre inter-observer variation analysis in europe (63).
Micro-rnas are known to be affected many target genes and are important in pathogenesis and predication of clinical behaviour of different cancers (64)(65)(66)(67)(68)(69)(70).Wholeexome sequencing analysis of pheochromocytoma/ paraganglioma has been presented (71).a group of few micro-rnas related to the genomic results was also noted to be able to predict the behaviour of this group of tumour (71,72).

ConCLuSion RemARkS
It is clear that profound knowledge have been gain in the understanding of pathogenesis and nature of diseases associated with pheochromocytoma/paraganglioma.The findings may help in development and optimization of management strategies for patients with this group of tumours.Pathologist should aware of the updates in knowledge, contribute to validation of the pathological features and markers for prediction of malignant potential of this group of tumours.

ConFLiCT oF inTeReST
The author has declared no conflict of interest.A B

Figure 2 :
Figure 2: The role of SdHB immunohistochemistry. A) Paraganglioma which is positive for SdHB protein expression (Immunohistochemistry; x 14); B) Paraganglioma which is negative for SdHB protein expression (Immunohistochemistry; x14).Genetic testing for SdHx genes should be performed in such case.