Role of epigenetic regulation on catecholamine synthesis in pheochromocytoma and paraganglioma.

INTRODUCTION: Pheochromocytomas and paragangliomas (PPGLs) typically secrete catecholamines and their metabolites (metanephrines [MN] and normetanephrine [NMN]). Catecholamines are synthesized by several enzymes: phenylalanine hydroxylase (encoded by PAH), tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (DDC), dopamine ß-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT). MN/NMN secretion varies between anatomical and molecular subgroups. The aim of this study was to assess the correlation between DNA methylation of catecholamine synthesis genes and MN/NMN secretion. METHODS: Gene promoter methylation of PAH, TH, AADC, DBH, and PNMT were extracted and calculated based on publicly available data. Comparisons and correlation analysis were performed between MN ± NMN (MN/NMN), NMN only, and neither/unknown secretion patterns. Methylation levels and MN/NMN patterns were compared by three genetic alteration subgroups: pseudohypoxia (PH), kinase signaling (KS), and others. RESULTS: A total of 178 cases were included. Methylation of PAH CpGs negatively correlated with probability for MN/NMN secretion (p < .05 for all CpGs) and positively with NMN-only secretion. NMN-only secreting tumors had significantly higher promoter methylation of PAH, DBH, and PNMT compared with MN/NMN-secreting tumors. MN/NMN-secreting PPGLs had mainly KS alterations (52.1%), whereas NMN-only PPGLs had PH alterations (41.9%). PPGLs in the PH versus KS group had gene promoter hypermethylation of PAH (p = .002), DBH (p = .02), and PNMT (p = .003). CONCLUSIONS: Promoter methylation of genes encoding catecholamine synthesis enzymes is strongly and inversely correlated with MN/NMN patterns in PPGLs. KS and PH-related tumors have distinct methylation patterns. These results imply that methylation is a key regulatory mechanism of catecholamine synthesis in PPGLs.

Dihydroxyphenylacetaldehyde Lowering Treatment Improves Locomotor and Neurochemical Abnormalities in the Rat Rotenone Model: Relevance to the Catecholaldehyde Hypothesis for the Pathogenesis of Parkinson's Disease.

The catecholaldehyde hypothesis for the pathogenesis of Parkinson's disease centers on accumulation of 3,4-dihydroxyphenylacetaldehyde (DOPAL) in dopaminergic neurons. To test the hypothesis, it is necessary to reduce DOPAL and assess if this improves locomotor abnormalities. Systemic administration of rotenone to rats reproduces the motor and central neurochemical abnormalities characterizing Parkinson's disease. In this study, we used the monoamine oxidase inhibitor (MAOI) deprenyl to decrease DOPAL production, with or without the antioxidant N-acetylcysteine (NAC). Adult rats received subcutaneous vehicle, rotenone (2 mg/kg/day via a minipump), or rotenone with deprenyl (5 mg/kg/day i.p.) with or without oral NAC (1 mg/kg/day) for 28 days. Motor function tests included measures of open field activity and rearing. Striatal tissue was assayed for contents of dopamine, DOPAL, and other catechols. Compared to vehicle, rotenone reduced locomotor activity (distance, velocity and rearing); increased tissue DOPAL; and decreased dopamine concentrations and inhibited vesicular sequestration of cytoplasmic dopamine and enzymatic breakdown of cytoplasmic DOPAL by aldehyde dehydrogenase (ALDH), as indicated by DA/DOPAL and DOPAC/DOPAL ratios. The addition of deprenyl to rotenone improved all the locomotor indices, increased dopamine and decreased DOPAL contents, and corrected the rotenone-induced vesicular uptake and ALDH abnormalities. The beneficial effects were augmented when NAC was added to deprenyl. Rotenone evokes locomotor and striatal neurochemical abnormalities found in Parkinson's disease, including DOPAL buildup. Administration of an MAOI attenuates these abnormalities, and NAC augments the beneficial effects. The results indicate a pathogenic role of DOPAL in the rotenone model and suggest that treatment with MAOI+NAC might be beneficial for Parkinson's disease treatment.

Plasma Hemoglobin and Red Blood Cell Mass Levels as Dynamic Prognostic Markers for Progression and Survival in Pancreatic Neuroendocrine Tumors.

There are scarce data on readily available markers enabling immediate risk stratification and personalized management in patients with advanced pancreatic neuroendocrine tumors. This study explores the association of red blood cells-related parameters as prognostic markers in patients harboring pancreatic neuroendocrine tumors. Retrospective analysis of a tertiary medical center database, acquiring data of patients with pancreatic neuroendocrine tumors including demographics, tumor-related parameters and consecutive imaging results, vital status at last follow-up, and red blood cells parameters at baseline, last follow-up, and dynamics (last/baseline ratio). Univariate and multivariable analyses were performed. Sixty-seven patients were identified (mean age at diagnosis of 63±11 years, 56.7% males). Patients with disease progression had lower hemoglobin, red blood cells mass values and hematocrit at the last evaluation (p<0.001 for all comparisons), with red blood cells mass level<3.9 m/µl and a 6% and 9% relative reduction in hemoglobin and hematocrit levels, respectively, associated with an increased risk for disease progression. Similarly, patients deceased during the study period had lower hemoglobin, red blood cells mass values and hematocrit (p<0.03 for all) than those alive, at last follow-up. Eleven percent reduction in hemoglobin level was noted indicating a higher mortality risk (p=0.04). Negative hemoglobin and hematocrit dynamics were independently associated with increased risk for disease progression (p=0.03 and 0.049, respectively). In conclusion, decrease in red blood cells mass, hemoglobin and/or hematocrit levels are all associated with poor prognosis in patients with pancreatic neuroendocrine tumors. We suggest utilizing these parameters as complementary follow-up prognostic markers to radiologic imaging in this patients population.

Distinct Prognostic Factors in Sporadic and Multiple Endocrine Neoplasia Type 1-Related Pancreatic Neuroendocrine Tumors.

Pancreatic neuroendocrine tumors (PNET) may develop sporadically or in the context of hereditary syndromes. In patients with multiple endocrine neoplasia type 1 (MEN1), PNET is the leading cause of death. Our aim was to compare the mortality risk in sporadic and MEN1-related PNETs and identify high-risk populations. A retrospective Surveillance, Epidemiology, and End Results database analysis of patients with PNET was used. Patients with MEN1 were defined by syn/metachronous pituitary adenoma. Clinical data were retrieved, and all-cause mortality (ACM) risk was compared in univariate and multivariable analyses. The cohort included 569 patients (46.6% males) with sporadic (n=542) and MEN1-related (n=27) PNETs. Age at diagnosis of MEN1-related PNET was significantly younger than with sporadic PNETs (mean age 49.2±16.7 vs. 61.6±12.7 years, respectively; p < 0.001). Survival analysis showed a trend for a better outcome in patients with MEN1-related vs. sporadic PNET (Log-rank, p=0.09) and in subgroup analysis for patients with advanced disease (p=0.08). Furthermore, among patients followed expectantly, those with MEN1-related PNET had lower ACM risk than their sporadic counterparts (p=0.08). Multivariable analysis demonstrated lower ACM risk in patients diagnosed with MEN1 (hazard ratio 0.35, 95% confidence interval 0.11-1.2, p=0.09), further supporting the trend detected in the univariate analysis. In conclusion, our study demonstrates the distinct clinical profile of patients with MEN1-related PNET compared to sporadic disease and emphasizes the expertise required to accurately manage patients with PNET in this rare context. The cautious decision-making required before embarking on surgical intervention is further emphasized in this robust analysis of a large cancer database.

Impact of Pancreatic Neuroendocrine Tumor on Mortality in Patients With von Hippel-Lindau Disease.

OBJECTIVE: The main causes for morbidity and mortality in von Hippel-Lindau (VHL) disease are central nervous system hemangioblastoma and clear cell renal cell carcinoma, but the effect of VHL-related pancreatic neuroendocrine tumors (PNET) on patient outcome is unclear. We assessed the impact of PNET diagnosis in patients with VHL on all-cause mortality (ACM) risk. METHODS: We used the Surveillance, Epidemiology, and End Results database. Of 16 344 patients, 170 had VHL based on clinical diagnostic criteria, and 510 patients had PNET (91 VHL-related and 419 sporadic). RESULTS: Survival analysis demonstrated a lower ACM among patients with VHL-related PNET compared to patients with sporadic PNET (log-rank test, P = .011). Among patients with VHL, ACM risk was higher with vs without PNET (P = .029). The subgroup analysis revealed a higher ACM risk with metastatic PNET (sporadic P = .0031 and VHL-related P = .08) and a similar trend for PNET diameter ≥3 cm (P = .06 and P = 0.1 in sporadic and VHL-related PNET, respectively). In a multivariable analysis of patients with VHL, diagnosis with PNET by itself was associated with a trend of lower risk for ACM, while presence of metastatic PNET was independently associated with increased ACM risk. CONCLUSION: Diagnosis with PNET is not associated with a higher ACM risk in VHL by itself. The independent association of advanced PNET stage with higher mortality risk emphasizes the importance of active surveillance for detecting high-risk PNET at an early stage to allow timely intervention.

Mid-Treatment Response to 177-Lutetium Dotatate Predicts Overall Outcome in Patients With Advanced Neuroendocrine Tumors.

PURPOSE: Patients with advanced, well-differentiated neuroendocrine tumors (WD-NETs) often require both peptide receptor radionuclide therapy (PRRT) and subsequent chemotherapy. However, no mid-PRRT predictors are available to identify patients who will not benefit from subsequent PRRT to limit their radiation exposure. Our aim is to characterize patients for whom subsequent PRRT is less efficacious on the basis of mid-PRRT evaluation. MATERIALS AND METHODS: A retrospective study of patients with WD-NET who underwent ≥four PRRT cycles. Data gathered included demographics, tumor grade, stage, and response (partial response [PR], stable disease [SD], and progressive disease [PD]) on the basis of RECIST 1.1 criteria and 68Ga-dotatate positron emission tomography-computerized tomography pretreatment, after second and fourth treatment cycle, 6 months after fourth cycle, and at last follow-up. RESULTS: Fifty-one patients (51.6% women; age at diagnosis 66.0 ± 1.65 years), with pancreatic NET (PNET; n = 24), small intestine NET (n = 13), or other NET (n = 14), received PRRT, resulting in PR (n = 21), SD (n = 23), and PD (n = 3). Of the patients reaching PR after PRRT, most reached PR after two treatments (70.4%), with only 11.8% PR occurring between subsequent cycles (P = .001). Furthermore, patients with PR at mid-treatment had higher PR rates after PRRT completion than those with SD (P = .007). Patients harboring PNET who achieved PR had a more pronounced reduction of tumor burden in additional cycles than patients who did not (25.6% v 1.5%; P = .03, respectively). On the multivariable model, adjusted for grade and primary site, PR at mid-treatment evaluation was associated with a 20.9 adjusted odds ratio for additional PR at PRRT completion (P = .002). CONCLUSION: Mid-PRRT assessment predicts subsequent PRRT response in patients with WD-NET, especially those with PNET, informing personalized management and consideration of reduced bone marrow radiation exposure in high-risk patients.

Somatic USP8 alteration affects the immune landscape of corticotroph pituitary adenomas- a pilot study.

INTRODUCTION: Somatic mutations in ubiquitin-specific protease-8 (USP8), encoding a deubiquinating protein, are found in approximately 30% of corticotroph-derived pituitary adenomas (CPAs). Stratifin, a protein encoded by SFN, inhibits USP8 catalytic activity. USP8 has immunomodulating properties that have been demonstrated in non-tumoral diseases. METHODS: We assessed the influence of USP8 on the immune landscape of CPA and validated this effect and its dependency on stratifin in large cohorts of non-pituitary tumors. We analyzed data of CPA samples (n = 20) and additional non-pituitary tumors from the TCGA database, using transcriptome signature-recognition algorithms. Immune tumor microenvironment (iTME) was compared both by USP8 and SFN expression levels (n = 843) and by USP8 mutation status and SFN expression (n = 12,389). RESULTS: CPA with activating USP8 mutations was associated with "cold" iTME compared with wild-type USP8 CPA, as reflected by lower fractions of immune cells, including B cells, CD4, regulatory and gamma/delta T cells, natural killer cells, M0 and M1 macrophages, dendritic cells, and eosinophils (p < 0.05 for all comparisons). Pathways altered by the presence of USP8 mutation, based on the most differentially expressed genes (3061 genes), included microglia pathogen phagocytosis and multiple toll-like receptor signaling pathways (p < 0.0001). In a validation analysis based on large cohorts of non-pituitary tumors, high expression of USP8 was associated with a suppressed iTME effect that was augmented by a low SFN expression. CONCLUSIONS: Our data demonstrate for the first time, to our knowledge, a distinct immune landscape of tumors based on USP8 status and expression and the dependency of this immunological effect on SFN expression.

Serum phosphate levels at diagnosis predict long-term risk for hypopituitarism in patients with acromegaly.

INTRODUCTION: Excess growth hormone (GH) secretion in acromegaly has a major impact on mineral balance and serum phosphate levels. However, the clinical utilization of serum phosphate levels as a marker for long-term disease outcomes in acromegaly has not been evaluated. METHODS: This is a retrospective study of patients with acromegaly who were followed in a tertiary center. Data were retrieved on patient characteristics, endocrine and biochemical evaluation, and tumor parameters. Comparisons were performed by measuring baseline phosphate levels and conducting correlation analysis and multivariable logistic regression. RESULTS: Sixty-one patients were followed for 4.5 years (range 1-21). Patients with hyperphosphatemia (> 4.5 mg/dl) at baseline had larger adenomas (15.0 mm [8.0, 47.0] vs. 10.0 mm [3.0, 24.0], p = 0.001), a rate chance of invasive adenoma (16 [80.0%] vs. 14 [46.7%], p = 0.02), and lower serum cortisol levels (226.0 nmol/l [27.6, 516.0] vs. 294.0 nmol/l [32.0, 610.0], p = 0.02). Baseline serum phosphate levels positively correlated with IGF-1 levels (r = 0.43, p = 0.003) and negatively correlated with morning plasma cortisol levels (r = -0.46, p = 0.002). Regarding long-term impact, baseline phosphate levels correlated with the number of pituitary axes involved 6 months after diagnosis (r-0.34, p = 0.01). In multivariable analysis, baseline plasma phosphate levels were independently associated with risk for disease progression/recurrence (odds ratio [OR] 9.66, 95% confidence interval [CI] 1.5, 105.9, p = 0.03) and for invasive adenoma (OR 6.21, 95% CI 1.6, 28.7, p = 0.01). CONCLUSION: Elevated pretreatment serum phosphate levels are associated with a greater risk of disease persistence and recurrence and with altered pituitary function in patients with acromegaly.

Non-Interventional Management of Advanced Pancreatic Neuroendocrine Neoplasms in Patients with von Hippel-Lindau Disease.

Von Hippel-Lindau (VHL) is a rare autosomal dominant hereditary cancer predisposition syndrome. Patients with VHL have a high risk for developing retinal and central nervous system hemangioblastoma, pheochromocytoma, renal cell carcinoma, and pancreatic neuroendocrine neoplasms (PNEN). About a fifth of patients with VHL will develop PNEN, and only a tenth of them will develop metastatic or unresectable (advanced) PNEN requiring medical intervention. In this review, we performed a literature search for studies, written in English, on the medical interventions for VHL-related localized and advanced PNENs and their clinical outcomes. We detail the various medical interventions for this rare group of patients, including their mode of action and potential efficacy and toxicity. Finally, based on the current literature, we delineate a possible management algorithm for patients with VHL and advanced PNEN. We can conclude that data on the efficacy of various vascular endothelial growth factor (VEGF) receptor inhibitors, and on the efficacy of belzutifan, a novel hypoxia-inducible factor 2 inhibitor, for the management of advanced PNEN in VHL, are scarce. Hence, deduction from the management of sporadic PNEN is required, and is implemented in the proposed management algorithm provided within this review.

A Case of Metastatic Thymoma Responsive to Treatment With 177 Lu-DOTATATE.

ABSTRACT: We describe a case of a 74-year-old woman with germline BRCA2 mutation, with an incidental diagnosis of metastatic thymoma presenting as a mediastinal mass with cardiac muscle and lymph node involvement. Despite surgical and radiotherapy treatment, there was marked advancement with new lung and liver metastases. All lesions demonstrated 68 Ga-DOTATATE PET/CT uptake, and the patient received 4 peptide receptor radionuclide therapy cycles with 177 Lu-DOTATATE, with pronounced reduction in the size of the liver, cardiac, and pleural lesions. This is the first case to demonstrate partial response to peptide receptor radionuclide therapy in metastatic thymoma, thus suggesting possible treatment option to refractory and advancing metastatic thymoma.

Unique Characteristics of Patients with Von Hippel-Lindau Disease Defined by Various Diagnostic Criteria.

Von Hippel-Lindau (VHL) disease diagnosis is based on two criteria sets: International criteria (IC, two hemangioblastomas, one hemangioblastoma plus one visceral lesion, or VHL family history/pathogenic variant plus hemangioblastoma/visceral lesion); or Danish criteria (DC, two clinical manifestations, or VHL family history/pathogenic variant plus hemangioblastoma/visceral lesion). We aimed to compare the characteristics of patients with VHL-related pancreatic neuroendocrine tumor (vPNET) meeting either the clinical Danish criteria only (DOC) or IC to those with sporadic PNET (sPNET). The cohort included 33 patients with VHL (20 vPNETs) and 65 with sPNET. In terms of genetic testing and family history of VHL, 90.0% of the patients with vPNET in the IC group had a germline VHL pathogenic variant, and 70.0% had a family history of VHL vs. 20% and 10% in the DOC group, respectively (p < 0.05 for both). Patients with vPNET were younger at diagnosis compared with sPNET (51.6 ± 4.1 vs. 62.8 ± 1.5 years, p < 0.05). Patients in the IC group were younger at diagnosis with VHL, vPNET, pheochromocytoma, or paraganglioma (PPGL) and renal-cell carcinoma (RCC) than those in the DOC group (p < 0.05 for all comparisons). The most prevalent presenting manifestations were hemangioblastoma (42.8%) and PPGL (33.3%) vs. RCC (58.3%) and PNET (41.7%) in the IC vs. DOC groups. In conclusion, patients with vPNET meeting DOC criteria show greater similarity to sPNET. We suggest performing genetic testing, rather than solely using clinical criteria, for establishing the diagnosis of VHL.

Germline CDKN1B variant type and site are associated with phenotype in MEN4.

Multiple endocrine neoplasia 4 (MEN4) is a rare multiglandular endocrine neoplasia syndrome clinically hallmarked by primary hyperparathyroidism (PHPT), pituitary adenoma (PitAd), and neuroendocrine tumors (NET), clinically overlapping MEN1. The underlying mutated gene - CDKN1B, encodes for the cell-cycle regulator p27. Possible genotype-phenotype correlations in MEN4 have not been thoroughly assessed. Prompted by the findings in three Israeli MEN4 kindreds, we performed a literature review on published and unpublished data from previously reported MEN4/CDKN1B cases. Univariate analysis analyzed time-dependent risks for developing PHPT, PitAd, or NET by variant type and position along the gene. Overall, 74 MEN4 cases were analyzed. PHPT risk was 53.4% by age 60 years (mean age at diagnosis age 50.6 ± 13.9 years), risk for PitAd was 23.2% and risk for NET was 16.2% (34.4 ± 21.4 and 52.9 ± 13.9 years, respectively). The frameshift variant p.Q107fs was the most common variant identified (4/41 (9.7%) kindreds). Patients with indels had higher risk for PHPT vs point mutations (log-rank, P = 0.029). Variants in codons 94-96 were associated with higher risk for PHPT (P < 0.001) and PitAd (P = 0.031). To conclude, MEN4 is clinically distinct from MEN1, with lower risk and older age for PHPT diagnosis. We report recurrent CDKN1B frameshift variants and possible genotype-phenotype correlations.

The rat rotenone model reproduces the abnormal pattern of central catecholamine metabolism found in Parkinson's disease.

Recent reports indicate that Parkinson's disease (PD) involves specific functional abnormalities in residual neurons - decreased vesicular sequestration of cytoplasmic catecholamines via the vesicular monoamine transporter (VMAT) and decreased aldehyde dehydrogenase (ALDH) activity. This double hit builds up the autotoxic metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), the focus of the catecholaldehyde hypothesis for the pathogenesis of PD. An animal model is needed that reproduces this abnormal catecholamine neurochemical pattern. Adult rats received subcutaneous vehicle or the mitochondrial complex 1 inhibitor rotenone (2 mg/kg/day via a minipump) for 10 days. Locomotor activity was recorded, and striatal tissue sampled for catechol contents and catechol ratios that indicate the above abnormalities. Compared to vehicle, rotenone reduced locomotor activity (P=0.002), decreased tissue dopamine concentrations (P=0.00001), reduced indices of vesicular sequestration (3,4-dihydroxyphenylacetic acid (DOPAC)/dopamine) and ALDH activity (DOPAC/DOPAL) (P=0.0025, P=0.036), and increased DOPAL levels (P=0.04). The rat rotenone model involves functional abnormalities in catecholaminergic neurons that replicate the pattern found in PD putamen. These include a vesicular storage defect, decreased ALDH activity and DOPAL build-up. The rat rotenone model provides a suitable in vivo platform for studying the catecholaldehyde hypothesis.