Biomarkers in pheochromocytomas and paragangliomas

Pheochromocytomas (PHEOs) derived from adrenal medulla and paragangliomas (PGLs) from sympathetic or parasympathetic paraganglia are rare neuroendocrine tumors. Incidence of PHEOs and PGLs is between 0.4–9.5 cases per one million people per year. In Finland about 10–15 PHEOs are diagnosed per year, but the incidence is rising. PHEOs and sympathetic PGLs can secrete catecholamines, often in bouts, which makes the symptoms associated with these tumors very diverse, with high blood pressure being the leading symptom. During recent years, knowledge of the variable genetic background and pathogenesis of PGLs and PHEOs has increased, and about 30-40% of these tumors are known to be hereditary. However, prognosis and aggressiveness of an individual tumor cannot be unequivocally predicted histologically or with any biomarkers. The aim of this thesis was to find biomarkers in PHEOs and PGLs for diagnostic, prognostic, and predictive purposes. The study cohort consisted of 153 consecutive PHEOs or PGLs operated from 147 patients during the years 1973–2009 at Helsinki University Hospital. Tissue microarray blocks were constructed for immunohistochemistry studies. Matrix-assisted laser desorption/ionization time of flight mass spectrometric profiling of 16 tissue samples was used to analyze N-glycan structures in eight metastasized and eight nonmetastasized tumors. In addition, five thyroid PGLs originating from the population-based European-American-Head-and-Neck-Paraganglioma-Registry (European-American-HNPGL-Registry, Freiburg, Germany) were investigated. Metastasized PHEOs and PGLs expressed significantly more intracytoplasmic human antigen R (HuR) protein immunohistochemically than nonmetastasized tumors. The metastatic potential was also associated with higher proliferation and tumor necrosis. Five somatostatin receptors (SSTR1–5) showed individual and varying SSTR profiles in PHEOs and PGLs. The most abundant SSTRs were SSTR2 and SSTR3. Between metastatic PHEOs and PGLs the SSTR2 expression varied – all PGLs were strongly SSTR2 positive, while most PHEOs were negative. The N-glycan profile differed depending on the metastatic status of the tumor. Metastasized tumors expressed more fucosylation and complex fucosylation in their N-glycans. Based on different N-glycan profiles, metastatic and nonmetastatic tumors could be separated in principal component analysis. Extremely rare thyroid PGLs showed a strong association with succinate dehydrogenase (SDH) mutation. Of five patients with thyroid PGL, two had SDHB mutation and two SDHA mutation. In our Finnish cohort, 10% of PHEO and PGL patients had SDHB mutation and 40% of these a metastatic disease. In conclusion, intracytoplasmic HuR is increased in most metastatic PHEOs and PGLs and can be used in the panel of prognostic markers in these tumors. HuR may have a role in malignant transformation. PHEOs and PGLs have individual variable SSTR1–5 profiles. Investigating the SSTR1–5 profile in PHEOs and PGLs can be beneficial in choosing somatostatin analog based imaging and therapy. Metastasized and nonmetastasized PHEOs and PGLs have differences in N-glycans. Those N-glycans, associated with aggressive disease, may possibly be used in the future as prognostic biomarkers. PHEOs and PGLs have a strong genetic background, and genetic testing is recommended for PHEO and PGL patients.Feokromosytoomat ja paraganglioomat ovat harvinaisia neuroendokriinisiä kasvaimia, joiden esiintyvyydeksi on arvioitu 0.4-9.5/1000000. Lisämunuaisen ytimen kasvaimia kutsutaan feokromosytoomiksi. Paraganglioomiksi kutsutaan histologisesti samankaltaisia lisämunuaisen ytimen ulkopuolisia kasvaimia, jotka saavat alkunsa sympaattisista tai parasympaattisista ganglioista. Suomessa todetaan vuosittain noin 10–15 feokromosytoomaa, mutta esiintyvyyden arvellaan olevan nousussa. Kaikilla feokromosytoomilla ja paraganglioomilla ajatellaan nykyisin olevan enemmän tai vähemmän kykyä lähettää etäpesäkkeitä, mutta histologisin keinoin on mahdotonta arvioida luotettavasti kasvaimen metastaattista potentiaalia, mikä tekee tästä kasvainryhmästä haastavan sekä kliinikolle että patologille. Feokromosytoomista noin 10 % ja paraganglioomista 15–30% lähettää etäpesäkkeitä. Toistaiseksi mikään immunohistokemiallinen värjäys, kuvantamistutkimus tai geneettinen testi ei luotettavasti erottele metastasoivia kasvaimia ei-metastasoivista. Viime vuosina feokromosytoomien ja paraganglioomien geneettisestä taustasta on saatu uutta tietoa ja nykytiedon mukaan 30–40 %: feokromosytoomista ja paraganglioomista on perinnöllisiä, ituradan mutaatiosta johtuvia. Taustalla oleva mutaatio vaikuttaa kasvaimen patogeneesiin ja ennusteeseen. Sukkinaattidehydrogenaasi kompleksin B alayksikön, SDHB-geenin, mutaatioon liittyvistä kasvaimista metastasoi noin 30 %. Väitöskirjatutkimuksen tavoitteena oli löytää primäärituumorin metastasointia ennustavia diagnostisia, prognostisia ja prediktiivisiä biomarkkereita. Materiaalina oli 153 primääriä feokromosytoomaa ja paraganglioomaa, jotka oli leikattu HUS:ssa vuosina 1973–2009. Potilaita materiaalissa oli 147. Kasvaimista tehtiin Tissue Micro Array blokki (TMA) immunohistokemiallisia tutkimuksia varten. SDHB-mutaation esiintymistä selvitettiin immunohistokemiallisella SDHB-värjäyksellä. Kuudentoista tuumorin asparagiiniin liittyvät glykaanirakenteet (N-glykaanit) analysoitiin MALDI-TOF massa spektrometrisellä menetelmällä. Harvinaisista kilpirauhasen paraganglioomista, jotka etsittiin paraganglioomarekisteristä (European-American-Head-and-Neck-Paraganglioma-Registry) analysoitiin pään ja kaulan alueen paraganglioomissa esiintyvät mutaatiot. Totesimme RNA:ta sitovan posttranskriptionaalisen säätelijäproteiini HuR:n lisääntyvän metastasoituneissa feokromosytoomissa ja paraganglioomissa. Totesimme myös merkitsevästi enemmän tuumorinekroosia ja proliferatiivisuutta metastasoituneissa kasvaimissa. Kasvainten somatostatiinireseptoriprofiili oli yksilöllinen. Somatostatiinireseptoriprofiilin immunohistokemiallinen tutkiminen voi auttaa sopivan somatostatiinianalogipohjaisen kuvantamismenetelmän ja hoidon valitsemisessa. Neljä neutraalia N-glykaania ja kolme hapanta N-glykaania, assosioituivat metastasoituneisiin kasvaimiin, joissa oli myös enemmän fukosylaatiota ja kompleksia fukosylaatiota. Harvinaiset kilpirauhasen paraganglioomat assosioituivat vahvasti SDHB- ja SDHA- mutaatioihin. Suomalaisessa potilasmateriaalissamme 10% potilaista oli immunohistokemiallisesti SDHB-negatiivisia, mikä on vahva osoitus SDHB-mutaatiosta

Automated assessment of Ki-67 proliferation index in neuroendocrine tumors by deep learning

The Ki-67 proliferation index (PI) is a prognostic factor in neuroendocrine tumors (NETs) and defines tumor grade. Analysis of Ki-67 PI requires calculation of Ki-67-positive and Ki-67-negative tumor cells, which is highly subjective. To overcome this, we developed a deep learning-based Ki-67 PI algorithm (KAI) that objectively calculates Ki-67 PI. Our study material consisted of NETs divided into training (n = 39), testing (n = 124), and validation (n = 60) series. All slides were digitized and processed in the Aiforia(R) Create (Aiforia Technologies, Helsinki, Finland) platform. The ICC between the pathologists and the KAI was 0.89. In 46% of the tumors, the Ki-67 PIs calculated by the pathologists and the KAI were the same. In 12% of the tumors, the Ki-67 PI calculated by the KAI was 1% lower and in 42% of the tumors on average 3% higher. The DL-based Ki-67 PI algorithm yields results similar to human observers. While the algorithm cannot replace the pathologist, it can assist in the laborious Ki-67 PI assessment of NETs. In the future, this approach could be useful in, for example, multi-center clinical trials where objective estimation of Ki-67 PI is crucial.Peer reviewe

Immunohistochemical Expression of Somatostatin Receptor Subtypes in a Panel of Neuroendocrine Neoplasias

Neuroendocrine neoplasias (NENs) are known to express somatostatin receptors (SSTRs) 1-5, which are G-protein-coupled cell membrane receptors. Somatostatin receptor imaging and therapy utilizes the SSTR expression. Synthetic somatostatin analogs with radioligands are used to detect primary tumors, metastases, and recurrent disease. Receptor analogs are also used for treating NENs. Furthermore, commercially available SSTR antibodies can be used for the immunohistochemical (IHC) detection of SSTRs. We investigated different SSTR antibody clones applying diverse IHC protocol settings to identify reliable clones and feasible protocols for NENs. A tissue microarray including NENs from 12 different primary sites were stained. Only UMB clones were able to localize SSTR on the cell membranes of NENs. SSTR2 (UMB1) emerged as the most common subtype followed by SSTR5 (UMB4) and SSTR1 (UMB7). SSTR3 (UMB5) expression was mainly cytoplasmic. Yet, SSTR4 expression was weak and located primarily in the cytoplasm. Thus, appropriate IHC protocols, including proper positive and negative controls, represent requirements for high-quality NEN diagnostics and for planning personalized therapy.Peer reviewe

Lateralization in C-11-Metomidate PET and outcome of adrenalectomy in primary aldosteronism

Introduction Subtype classification method is essential when considering adrenalectomy as a possible treatment for primary aldosteronism. We aimed to retrospectively evaluate surgical outcomes of primary aldosteronism in patients who had undergone C-11-metomidate positron emission tomography (C-11-MTO-PET) for subtype classification. Methods Postoperative clinical and biochemical cure and histopathological diagnosis from biobank samples were retrospectively evaluated in 44 patients who had all undergone preoperative C-11-MTO-PET with or without adrenal venous sampling (AVS). We compared those operated based on C-11-MTO-PET alone and those with concordant or discordant lateralization in C-11-MTO-PET and AVS studies according to postoperative immunohistochemical findings and biochemical and clinical cure. Results Adrenalectomy side was based on C-11-MTO-PET alone in 14 cases and on AVS in 30 cases of whom 42 achieved complete and two partial biochemical cures. Among those who underwent AVS and were operated according to it, the two lateralization methods were concordant in 22 cases and discordant in 8 cases. Similar immunohistochemical profiles and cure rates were seen after C-11-MTO-PET alone or AVS-based operations. Respectively, those with concordant or discordant C-11-MTO-PET and AVS lateralization did not differ in surgical outcome. Together, we found errors of lateralization diagnostics with C-11-MTO-PET in 18% and with AVS in 3% among those eligible for adrenal surgery. Conclusions Outcomes of adrenalectomy based on clinically significant lateralization in C-11-MTO-PET alone correspond to those based on C-11-MTO-PET with concordant AVS lateralization. However, our results suggest that diagnosis of unilateral PA should be performed with caution with C-11-MTO-PET in case of discordant lateralization studies.Peer reviewe

Max Schottelius : Pioneer in Pheochromocytoma

First descriptions of diseases attract tremendous interest because they reveal scientific insight even in retrospect. Max Schottelius, the pathologist contributing the first histological description of pheochromocytoma, remains anonymous. We reviewed the description by Schottelius and weighed the report in modern context. Schottelius described the classical diagnostic elements of pheochromocytoma, including the brown appearance after exposure to chromate-containing Mueller's fixative. This color change, known as chromaffin reaction, results fromoxidation of catecholamines and is reflected in the name pheochromocytoma, meaning dusky-colored chromate-positive tumor. Thus Schottelius performed the first known histochemical contribution to diagnosis, which is today standard with immunohistochemistry for chromogranin. Copyright (c) 2017 Endocrine Society This article has been published under the terms of the Creative Commons Attribution NonCommercial, No-Derivatives License (CC BY-NC-ND).Peer reviewe

Somatostatin Receptor Expression Is Associated With Metastasis and Patient Outcome in Pulmonary Carcinoid Tumors

Context: Pulmonary carcinoids (PCs) belong to neuroendocrine tumors that often overexpress somatostatin receptors (SSTRs). This overexpression provides a molecular basis for tumor imaging and treatment with somatostatin analogs. Objective: To evaluate SSTR1 to SSTR5 distribution in a large set of PC tumors and to investigate whether the expression is associated with clinicopathological and outcome data. Design, Setting, and Patients: This retrospective study was conducted at Helsinki University Hospital and University of Helsinki. It included 178 PC tumors coupled with patients' clinical data retrieved through Finnish biobanks. After histological reclassification, tissue specimens were processed into next-generation tissue microarray format and stained immunohistochemically with monoclonal SSTR1 to SSTR5 antibodies. Main Outcome Measure: SSTR1 to SSTR5 expression in PC tumors. Results: Expression of SSTR1 to SSTR5 was detected in 52%, 75%, 56%, 16%, and 32% of the tumors, respectively. Membrane-bound staining was observed for all receptors. SSTR2 negativity and SSTR4 positivity was associated with lymph node involvement at the time of surgery (P = 0.014 and P = 0.017, respectively) and with distant metastasis (P = 0.027 and P = 0.015, respectively). SSTR3 and SSTR4 expression was associated with increased risk of shorter survival [P = 0.046, hazard ratio (HR) 4.703, 95% CI 1.027 to 21.533; and P = 0.013, HR 6.64, 95% CI 1.48 to 29.64, respectively], whereas expression of SSTR1 and SSTR2 was associated with improved outcome (P = 0.021, HR 0.167, 95% CI 0.037 to 0.765; and P = 0.022, HR 0.08, 95% CI 0.01 to 0.70, respectively). Conclusion: SSTR1 to SSTR5 expression is observed in PCs. As SSTR expression is associated with the tumor's metastatic potential and patient outcome, these receptors may offer the possibility for individualized prognosis estimation.Peer reviewe

Variable somatostatin receptor subtype expression in 151 primary pheochromocytomas and paragangliomas

Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are neuroendocrine tumors that express somatostatin receptors (SSTRs), a phenomenon that constitutes a basis for tumor imaging and treatment with somatostatin analogues and peptide receptor radionuclide therapy. We studied the immunohistochemical expression of SSTR1-5 in 151 primary tumors, including 14 metastasized and 16 SDHB-deficient tumors. SSTR2 and SSTR3 were most abundantly present in these tumors, whereas the tumors were mostly negative for SSTR1, SSTR4, and SSTR5. All metastasized PGLs (9/9), but only one metastasized PHEO (1/5), were strongly SSTR2 positive. SSTR3 expression was lower in metastatic tumors and tumors with a high proliferation rate (MIB1 >= 5%), but tumors had variable individual SSTR profiles. No correlation was found between SDHB status and SSTR expression. Our results suggest that new SSTR analogues with affinity for several SSTRs could be relevant for a subgroup of patients with these tumors. Better knowledge of tumor SSTR profiles could open the door for personalized imaging and treatment in the future. Because SSTR profiles vary in PHEOs and PGLs, individual analysis is required for each tumor. (C) 2018 The Authors. Published by Elsevier Inc.Peer reviewe

Adrenal androgens versus cortisol for primary aldosteronism subtype determination in adrenal venous sampling

Objective We examined if measurement of adrenal androgens adds to subtype diagnostics of primary aldosteronism (PA) under cosyntropin-stimulated adrenal venous sampling (AVS). Design A prospective pre-specified secondary endpoint analysis of 49 patients with confirmed PA, of whom 29 underwent unilateral adrenalectomy with long-term follow-up. Methods Concentrations of androstenedione, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) were measured during AVS in addition to aldosterone and cortisol. Subjects with lateralisation index (LI) of >= 4 were treated with unilateral adrenalectomy, and the immunohistochemical subtype was determined with CYP11B2 and CYP11B1 stains. The performance of adrenal androgens was evaluated by receiver operating characteristics (ROC) curve analyses in adrenalectomy and medical therapy groups. Results During AVS, the correlations between cortisol and androstenedione, DHEA and DHEAS for LI and selectivity index (SI) were highly significant. The right and left side SIs for androstenedione and DHEA were higher (p < .001) than for cortisol. In ROC analysis, the optimal LI cut-off values for androstenedione, DHEA and DHEAS were 4.2, 4.5 and 4.6, respectively. The performance of these LIs for adrenal androgens did not differ from that of cortisol. Conclusions Under cosyntropin-stimulated AVS, the measurement of androstenedione and DHEA did not improve the cannulation selectivity. The performance of cortisol and adrenal androgens are confirmatory but not superior to cortisol-based results in lateralisation diagnostics of PA.Peer reviewe

Somatostatin receptor expression in parathyroid neoplasms

Introduction: Parathyroid carcinoma represents a rare cause of primary hyperparathyroidism. Distinguishing carcinoma from the benign tumors underlying primary hyperparathyroidism remains challenging. The diagnostic criteria for parathyroid carcinoma are local and/or metastatic spreading. Atypical parathyroid adenomas share other histological features with carcinomas but lack invasive growth. Somatostatin receptors are commonly expressed in different neuro endocrine tumors, but whether this also holds for parathyroid tumors remains unknown. Aim: Our aim is to examine the immunohistochemical expression of somatostatin receptor 1-5 in parathyroid typical adenomas, atypical adenomas and carcinomas. Methods: We used a tissue microarray construct from a nationwide cohort of parathyroid carcinomas (n = 32), age- and gender-matched typical parathyroid adenomas (n = 72) and atypical parathyroid adenomas (n = 27) for immunohistochemistry of somatostatin receptor subtypes 1-5. We separately assessed cytoplasmic, membrane and nuclear expression and also investigated the associations with histological, biochemical and clinical characteristics. Results: All parathyroid tumor subgroups expressed somatostatin receptors, although membrane expression appeared negligible. Except for somatostatin receptor 1, expression patterns differed between the three tumor types. Adenomas exhibited the weakest and carcinomas the strongest expression of somatostatin receptor 2, 3, 4 and 5. We observed the largest difference for cytoplasmic somatostatin receptor 5 expression. Conclusions: Parathyroid adenomas, atypical adenomas and carcinomas all express somatostatin receptor subtypes 1-5. Somatostatin receptor 5 may serve as a potential tumor marker for malignancy. Studies exploring the role of somatostatin receptor imaging and receptor-specific therapies in patients with parathyroid car cinomas are needed.Peer reviewe

Lateralization in 11C-Metomidate PET and outcome of adrenalectomy in primary aldosteronism

IntroductionSubtype classification method is essential when considering adrenalectomy as a possible treatment for primary aldosteronism. We aimed to retrospectively evaluate surgical outcomes of primary aldosteronism in patients who had undergone 11C-metomidate positron emission tomography (11C-MTO-PET) for subtype classification.MethodsPostoperative clinical and biochemical cure and histopathological diagnosis from biobank samples were retrospectively evaluated in 44 patients who had all undergone preoperative 11C-MTO-PET with or without adrenal venous sampling (AVS). We compared those operated based on 11C-MTO-PET alone and those with concordant or discordant lateralization in 11C-MTO-PET and AVS studies according to postoperative immunohistochemical findings and biochemical and clinical cure.ResultsAdrenalectomy side was based on 11C-MTO-PET alone in 14 cases and on AVS in 30 cases of whom 42 achieved complete and two partial biochemical cures. Among those who underwent AVS and were operated according to it, the two lateralization methods were concordant in 22 cases and discordant in 8 cases. Similar immunohistochemical profiles and cure rates were seen after 11C-MTO-PET alone or AVS-based operations. Respectively, those with concordant or discordant 11C-MTO-PET and AVS lateralization did not differ in surgical outcome. Together, we found errors of lateralization diagnostics with 11C-MTO-PET in 18% and with AVS in 3% among those eligible for adrenal surgery.ConclusionsOutcomes of adrenalectomy based on clinically significant lateralization in 11C-MTO-PET alone correspond to those based on 11C-MTO-PET with concordant AVS lateralization. However, our results suggest that diagnosis of unilateral PA should be performed with caution with 11C-MTO-PET in case of discordant lateralization studies.</p