Neuroendocrine neoplasms (NEN) comprise well-differentiated neuroendocrine tumours (NET) and neuroendocrine carcinomas (NEC), whose distinction is clinically critical. Although c-MYC alterations have been implicated in NEC pathogenesis, c-MYC expression across NEC subtypes and anatomical sites, as well as in NET, remains incompletely defined. We analysed c-MYC immunohistochemically in 1380 resected NEN using the Immunoreactive Score (IRS: negative 0-1, weak 2-3, moderate 4-8, strong 9-12). Overall, c-MYC positivity (IRS ≥ 2) was observed in 13.3% of NEN. Expression was detected in 43% of NEC (164/381), including strong staining in 19.4%, whereas it was rare in NET and pulmonary carcinoids (20/999; 2%; p ≤ 0.001). Within NEC, c-MYC expression was enriched in LCNEC and MiNEN compared with SCNEC and Merkel cell carcinoma (p ≤ 0.001) and occurred more often in gastroenteropancreatic than in pulmonary NEC (57.6% vs. 37.3%; p ≤ 0.001). Among NET, G3 tumours showed the highest positivity rate (6/35; 17.1%), although this was significantly lower than in NEC (p ≤ 0.001), with strong expression observed in only one NET G3 (2.9%). No association between c-MYC expression and survival was identified in either NEC or NET. Our study confirms c-MYC expression as a common event in NEC and highlights differences across histological subtypes and anatomical sites, while demonstrating its absence in most low-proliferative NET. A subset of NET G3 tumours exhibits weak to moderate c-MYC expression at levels far below those seen in NEC, suggesting that strong c-MYC positivity may support a NEC classification in borderline cases but does not represent a definitive discriminatory marker.

Ampullary somatostatin-producing D-cell neuroendocrine tumors are rare neoplasms that may be associated with type 1 neurofibromatosis. The molecular features of sporadic ampullary somatostatin-producing D-cell neuroendocrine tumors (SAMSOM-NETs) remain poorly characterized. We performed an integrated morphological, immunohistochemical, and genomic analysis of a multicenter series of SAMSOM-NETs. Eleven cases were included (73% male; median age: 63 years). All six patients who underwent lymphadenectomy were staged as pN1, and liver metastases were found in three cases; however, no tumor-related deaths occurred (median follow-up: 104 months). Common histologic features that can pose diagnostic challenges in the differential diagnosis with adenocarcinoma included a tubulo-glandular architecture (100%), periodic Acid-Schiff (PAS)-positive intraluminal mucin (73%), MUC1 expression (100%), and carcinoembryonic antigen (45%) expression. All tumors exhibited dot-like cytoplasmic reactivity for cytokeratins (CK) CAM5.2 or CK AE1/AE3, and 82% were CK7-positive. ISL1 and PDX1 were diffusely expressed in all cases, while CDX2 was positive in 54% and ARX showed only focal expression in four tumors. Genomic profiling revealed microsatellite stability and low tumor mutational burden. Alterations in the RAS pathway, including HRAS mutations (4 cases, 36%), a KRAS mutation (1 case), and NF1 alterations (one case), were identified in 54% of cases and in all tumors with liver metastases. Additional molecular findings included a CDK12 splice-site alteration and an NTRK3::PRDM4 fusion. Potentially actionable alterations affecting kinase-related pathways were detected in 64% of tumors. Our findings support SAMSOM-NET as a peculiar neuroendocrine tumor subtype showing distinctive histologic and molecular characteristics with potential diagnostic and therapeutic implications.

Non-functioning pancreatic neuroendocrine tumors (NF-pNETs) show a variable prognosis. Despite 40-60% of patients remaining recurrence-free after surgery, guidelines recommend ≥ 10 years of follow-up. Recent studies identify prognostic subgroups based on ATRX, DAXX, and MEN1 mutations and chromosomal aneuploidy, highlighting a subgroup with favorable prognosis. We aimed to classify resected NF-pNETs into molecular subgroups using ATRX, DAXX, and Menin immunohistochemistry as surrogate markers for genomic status. To do so, we retrospectively collected resected primary sporadic grade 1 and 2 NF-pNETs without synchronous metastases from five international local pathology archives. ATRX, DAXX, and Menin immunohistochemistry was performed, and tumors were categorized into three groups: ATRX-DAXX-loss-group (ATRX or DAXX loss), isolated-Menin-loss-group (Menin loss without ATRX and DAXX loss), and unspecified-group (retained Menin, ATRX and DAXX). Kaplan-Meier analysis evaluated prognostic differences, and multivariable Cox regression assessed the added value of molecular subgroups beyond established prognostic factors. In total, 139 patients with grade 1 (64%) and 2 (36%) NF-pNETs were included. The median follow-up was 28 months (range 0-195) and recurrences occurred in 20% (28/139). No recurrences occurred in the isolated-Menin-loss-group (0/33), versus 12.5% (8/64) in the unspecified-group and 48% (20/42) in the ATRX-DAXX-loss-group. Kaplan-Meier analysis showed better recurrence-free interval in the isolated-Menin-loss-group than other subgroups (P < 0.001). In univariate analysis, the isolated-Menin-loss-group had a lower recurrence hazard (HR 0.03; 95%CI: 0.00-0.55; P = 0.018), than other subgroups, with little change in the hazard ratio after adjustment, but loss of significance. In conclusion, molecular subclassification by immunohistochemistry identifies NF-pNET patients with a low risk of recurrence and lays the groundwork for future prospective studies assessing genetics-based risk stratification as a tool for guiding clinical management.

Neuroendocrine neoplasms (NENs) of the urinary bladder are rare but highly aggressive tumors that account for under 1% of bladder malignancies. The 2022 WHO classification recognizes small cell neuroendocrine carcinoma (SCNEC), large cell neuroendocrine carcinoma (LCNEC), mixed neuroendocrine neoplasms (MiNEN), well-differentiated neuroendocrine tumor (NET), and paraganglioma; however, this framework is largely extrapolated from other organ systems and does not fully capture the biological complexity of bladder NENs. Most cases represent poorly differentiated neuroendocrine carcinomas, frequently admixed with urothelial carcinoma and characterized by recurrent TP53 and RB1 inactivation, TERT promoter mutations, and epigenetic dysregulation. Emerging transcriptomic data further identify lineage-defined subgroups within SCNEC and LCNEC based on ASCL1, NEUROD1, and POU2F3 expression, with potential prognostic and therapeutic relevance. This review integrates histopathologic, immunophenotypic, and molecular data to highlight diagnostic challenges, biologic heterogeneity, and limitations of current classification schemes. We propose a refined, bladder-specific framework that incorporates proliferative indices, molecular alterations, and recognition of mixed neuroendocrine-non-neuroendocrine neoplasms, with the goal of improving diagnostic reproducibility and informing future biomarker-driven therapeutic strategies.

Adrenal and extra-adrenal paragangliomas (PPGLs) are highly heritable non-epithelial neuroendocrine neoplasms. Through a retrospective chart review of 110 individuals diagnosed with PPGLs at the University Health Network in Toronto, Canada (2011-2023), we characterized germline findings, tumor features, self-reported ethnicity, and variant reclassification across a multi-ethnic cohort using targeted germline panel sequencing, whole genome sequencing (WGS), and optical genome mapping (OGM). Panel sequencing identified pathogenic or likely pathogenic (P/LP) germline variants in 28.18% (31/110) of individuals, with five variants of uncertain significance (VUS) in PPGL predisposition genes reclassified to P/LP during the study period (32.7%, 36/110). VUS rates were comparable across ethnic groups (16.28%, 7/43 in the group of non-European ethnicities; 17.24%, 10/58 in the group of European ethnicities), but the non-European group had a lower rate of VUS-to-P/LP reclassification (28.57%, 2/7 vs. 40.0%, 4/10). Uninformative targeted panel testing was supplemented by WGS in seven individuals with succinate dehydrogenase (SDH)-deficient tumors, leading to detection of P/LP in 6/7 cases (85.7%). Broader sequencing also uncovered incidental P/LP variants in genes without an established PPGL association, including TSC1 and PALB2. OGM resolved a complex structural variant (SV) involving SDHA as an inverted tandem duplication that WGS alone could not fully characterize. Our findings support a tiered diagnostic approach: comprehensive germline panels and tumor SDHB immunohistochemistry to maximize solved cases, with WGS and OGM reserved for select unresolved cases, and highlight the importance of equitable variant interpretation across diverse PPGL populations.

Paragangliomas (PGLs) are rare neural crest-derived tumors with highly variable morphology and clinical behavior. These tumors can present with cystic and necrotic alterations that may influence the clinical presentation of affected patients. However, prevalence and clinical significance of these pathological alterations remain poorly understood. In this retrospective study (2011-2021), 103 patients with confirmed PGLs were examined for cystic and necrotic alterations based on imaging and pathology reports as well as pathological re-evaluation. Cystic alterations, including microscopic, macroscopic, and pseudocystic alterations, were observed in 61.8% of PGLs. Regardless of their pathomorphological presentation, cystic alterations did not affect the clinical presentation of these patients. In contrast, necrotic alterations were identified in 3.9% of PGLs and were associated with a higher rate of metastasis (75% vs. 10%, p = 0.0124), larger tumor volume (53.6 cm[3] vs. 6.6 cm[3]p = 0.0339), and more pronounced signs and symptoms, including hypertension, palpitations, and weakness compared to patients without cystic/necrotic lesions. The prevalence of cystic and necrotic lesions was higher in adrenal than extra-adrenal PGLs. An additional meta-analysis of 235 patients with PGLs confirmed the increased rate of metastasis in necrotic PGLs (16% vs. 4.5%, p = 0.0369), which were also significantly more frequently associated with cardiovascular complications (p < 0.0001) than cystic PGLs. Considering pathological alterations, particularly necrotic lesions, may substantially affect the clinical management of patients with PGLs. Incorporating these features into preoperative management and follow-up strategies might be crucial, given the higher metastatic potential and increased cardiovascular risk associated with necrotic PGLs.

A substantial fraction (60-85%) of hereditary primary hyperparathyroidism (hPHPT) lacks an identifiable genetic etiology. We describe fumarate hydratase (FH) mutations as a potential cause of hPHPT, expanding the phenotypic spectrum of FH deficiency tumor predisposition syndromes. In an index patient who presented with asymptomatic hypercalcemia and a chief-to-transitional cell-dominant parathyroid adenoma, whole-exome sequencing revealed two unique heterozygous FH variants (germline p.Gln376fs*2; somatic p.Pro503_Lys504dup). Functional inactivation of FH was supported by diffuse nuclear and cytoplasmic 2-succinocysteine immunoreactivity and elevated fumarate/malate ratio in tumor tissue. This individual did not show classic HLRCC manifestations. Preserved FH protein expression suggested residual enzymatic activity, which may account for an attenuated phenotype. To assess broader relevance, no additional patients with bona fide FH-deficient parathyroid adenoma were identified among 130 individuals with suspected hPHPT of unknown etiology evaluated at our institute. In a complementary cohort of 11 patients with pheochromocytoma/paraganglioma syndrome harboring pathogenic germline heterozygous FH variants, one female (FH p.Thr234Ala) presented with multi-gland disease requiring parathyroidectomy at age 40 years, features suspicious for hPHPT. These findings support fumarate hydratase deficiency as a plausible etiology for a subset of parathyroid adenomatous disease. Thus, consideration of parathyroid function surveillance in patients with fumarate hydratase deficiency tumor predisposition syndromes may be warranted. CLINICAL TRIAL NUMBER: NCT04969926.

Adrenocortical carcinoma (ACC) is a rare malignancy with high recurrence rates. Circulating tumor DNA (ctDNA) is a minimally invasive, blood-based biomarker with multiple oncologic applications. The role of ctDNA in ACC care remains understudied. This retrospective, single-institution, study evaluated real-world use of ctDNA testing in patients with ACC and its correlation with imaging-based tumor burden. Patients with ACC (≥ 18 years) who underwent ctDNA testing via a clinically validated, personalized, tumor-informed assay (Signatera™, Natera) during their disease course were reviewed, and clinical data were obtained from the medical records. ctDNA results were tested for association with disease status and tumor burden determined by concurrent imaging and clinical evaluation. Twenty patients (55% male) were included; the median age at first ctDNA assessment was 58.3 years (range, 23.3-81.1). A total of 87 ctDNA tests were analyzed (median, 4 tests/patient; interquartile range [IQR], 1-6). The median ctDNA level was 1.27 mean tumor molecules/mL (IQR, 0.05-22.86). Detected ctDNA was strongly associated with evidence of disease (odds ratio, 74; 95% confidence interval, 9-3438). The ctDNA assay demonstrated high sensitivity (88%), specificity (91.7%), and overall accuracy (88.5%), with a positive post-test probability of 98% and a negative post-test probability of 45%. In addition, ctDNA levels were positively correlated with tumor burden (Spearman R = 0.844; p < 0.001). These findings support the potential value of ctDNA testing for disease monitoring in ACC and it may support a more personalized approach to this rare disease.

Renal well-differentiated neuroendocrine tumors (RenNETs) are rare primary renal neoplasms with enigmatic histogenesis. To define the molecular features of RenNETs and clarify their relationship to neuroendocrine tumors (NETs) from other sites, we performed whole-exome DNA sequencing and transcriptomic profiling on six RenNETs. In one case, adjacent non-neuroendocrine cystic epithelium and a regional lymph node metastasis were independently analyzed. RenNETs demonstrated a low-to-intermediate tumor mutational burden and recurrent copy number alterations, including losses involving chromosomes 3, 11, and 18 and gains involving chromosomes 1, 2, 8, and 14, partially overlapping those reported in gastroenteropancreatic NETs, in the absence of alterations in canonical NET-associated driver genes. RNA sequencing revealed marked upregulation of genes involved in neuroendocrine differentiation, peptide processing, and pancreatic beta cell function, accompanied by loss of renal lineage markers, indicating a neuroendocrine transcriptional program distinct from other primary renal neoplasms. RenNETs showed heterogeneous activation of hormone related transcriptional programs, rather than conforming to a discrete EC-cell or L-cell signature. Immunohistochemistry confirmed strong membranous expression of somatostatin receptor 2A (SSTR2A) in all cases, supporting established imaging and therapeutic paradigms used for NETs. Notably, two RenNETs were associated with adjacent CDX2-positive epithelial-lined cysts with intestinal differentiation. In one case, a shared somatic mutation was identified across the epithelial cyst, the adjacent RenNET, and a nodal metastasis, providing genetic evidence of a common clonal origin and implicating gastrointestinal-type epithelium as a potential precursor lesion in a subset of cases. Features supporting teratomatous origin, such as chromosome 12p gains or other germ cell tumor elements, were not identified. Together, these findings demonstrate that RenNETs exhibit convergent transcriptomic and phenotypic neuroendocrine programs, along with partial copy number overlap, with well-differentiated gastroenteropancreatic NETs, and support a model in which RenNETs may arise from developmentally displaced or aberrantly differentiated epithelial precursors with gastrointestinal-type differentiation, although this hypothesis requires validation in larger cohorts.

Multifocal fibrosing thyroiditis (MFT) is a poorly understood condition characterized by multiple fibrotic scar-like lesions with paucicellular core and peripheral follicular cell atypia. Described by Dr. Rosai in 1990 in consultation cases, MFT has received limited attention due to its perceived rarity. To define its prevalence and clinicopathologic features in the routine practice, consecutive thyroid resections were analyzed from the archives of two medical institutions in Italy and Portugal. Among 1,736 resections 27 MFT were identified (1.56%), corresponding to a detection rate of 1.34% MTF cases per year in the study cohort. No significant difference between Italian and Portuguese cases were observed. Twenty-three of 27 (85.2%) patients were female. Age range was 34-69 years (median: 52). Autoimmune thyroiditis affected 7/27 patients (25.9%), with clinical hypothyroidism in four (14.8%). Clinical hyperthyroidism was present in 4/27 patients (14.8%), including 2 cases of hyperfunctioning adenoma, 1 case of toxic multinodular goiter, and 1 case of Graves' disease. In 2/27 cases (7.4%) thyroid resection was performed due to fibrotic MFT foci mimicking PTC. Malignant thyroid nodules - papillary thyroid carcinoma (PTC) in all cases - or other benign nodules caused thyroid resection in 10/27 (37.0%) and in 12/27 (44.4%) cases, respectively; Hashimoto thyroiditis (2 cases) and Graves disease (1 case) caused thyroid resection in the remaining cases. Meticulous histologic examination disclosed additional alterations in 25/27 cases (92.6%), often overlapping with each other and with PTC: incidental millimetric PTC in 7/27 cases (25.9%), follicular nodular disease in 17/27 cases (62.9%), chronic lymphocytic thyroiditis/Hashimoto thyroiditis in 14/27 (51.8%). No genetic alteration was identified in any of the 15/27 cases tested. In the routine practice MFT is an uncommon disorder affecting diseased thyroids that simulates PTC. Its cause remains elusive.

Alterations in alternative splicing are emerging as a novel cancer hallmark, offering new insights into tumor biology. However, integrative analyses of splicing are still scarce, particularly in rare cancers like pancreatic neuroendocrine tumors (PanNETs), whose striking heterogeneity complicates patient diagnosis and treatment. Here, we provide the first comprehensive characterization of the RNA splicing landscape in PanNETs through integrative analysis of RNA-seq data from 174 tumor samples. We identified three robust spliceosomic groups (SPN1, SPN2, SPN3) each associated with unique clinical and molecular characteristics. SPN1 displayed intermediate clinical behavior alongside enhanced mTOR signaling; SPN2 was characterized by a less secretory phenotype, enrichment in alpha-cell markers and somatostatin receptors, increased metastasis, and frequent mutations in MEN1 and DAXX/ATRX genes; in contrast, SPN3 was composed mainly by low grade tumors with beta-cell marker expression and the lowest mutational rate, yet it also contained all the highly proliferative neoplasms. Moreover, each group had a specific alternative splicing events signature, revealing an unprecedented discovery: the association between the expression profile of the splicing machinery and its product, the splicing variants. We provide a detailed characterization of the molecular and functional consequences of the splice variants defining each of the spliceosomic groups. These findings underscore the previously unrecognized yet significant impact of RNA splicing on PanNET heterogeneity and suggest that detailed splicing profiles could serve as valuable tools for identifying novel biomarkers and therapeutic targets. Thus, beyond providing crucial insights into PanNET molecular biology, our study offers a foundation for future studies exploring personalized therapeutic strategies based on splicing features.

Oncocytic thyroid carcinomas are characterized by a unique chromosomal landscape and frequent resistance to radioactive iodine therapy, underscoring the need for improved molecular characterization. Although ALK rearrangements, particularly STRN::ALK fusions, have been described in thyroid carcinomas, their biological and clinical significance in oncocytic thyroid neoplasms remains unclear. This study investigated STRN exon 3-ALK exon 20-derived transcripts in 56 oncocytic thyroid neoplasms using RT-PCR, followed by cloning and Sanger sequencing of positive cases. Transcript-positive cases were further evaluated by fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and in silico structural modeling. STRN::ALK-derived transcripts were detected in 9 of 56 tumors; however, their frequency reflected the combined detection of distinct transcript architectures. Only 2 of 56 tumors (3.6%) harbored canonical in-frame STRN::ALK fusions, whereas 7 of 56 tumors (12.5%) contained noncanonical out-of-frame variants. Although ALK rearrangement by FISH was confirmed in all transcript-positive tumors irrespective of transcript architecture, detectable ALK protein expression by IHC was observed only in tumors harboring canonical in-frame fusions, whereas all noncanonical variants lacked detectable ALK protein expression. Structural modeling was concordant with these observations, suggesting preservation of kinase-domain features in canonical fusions and predicted disruption in noncanonical variants. Overall, STRN::ALK transcripts in oncocytic thyroid neoplasms exhibit marked architectural heterogeneity with distinct ALK protein expression patterns. ALK FISH-positive/IHC-negative cases should be interpreted cautiously, as they may not represent biologically equivalent ALK-driven neoplasia, with uncertain therapeutic relevance. Integrated molecular and protein-level assessment may support interpretation of ALK alterations in oncocytic thyroid neoplasms.

Current data on neuroendocrine tumors (NETs) of the gallbladder and cystic duct (GB-NETs) are highly limited, and the available evidence, largely derived from cancer registry data, suggests that these tumors exhibit a substantially more aggressive clinical behavior than NETs arising at other anatomical sites. We analyzed 26 GB-NETs. Female-to-male ratio: 1.9:1; median age: 50 years. They were typically incidental small (median: 0.8 cm, range: 0.08-2.3 cm) tumors, with 81% classified as grade 1 (G1), either pT1 or pT2, and 38% showing a paraganglioma-like pattern at least focally. Body/fundus tumors were mostly polypoid while neck/cystic duct examples showed mural growth. Symplastic pleomorphism was found in 11%, pseudoglandular structures in 50% and psammomatous intraluminal calcifications in 15%. Pancreatic polypeptide, somatostatin, and gastrin were detected in 5/7 (71%), 5/8 (62.5%), and 4/7 (57%) tested cases, respectively. ISLET1 and ARX were diffusely expressed in all 5 tested cases. Cholesterolosis was common, and six appeared to arise within cholesterol polyps. None had any evidence of metastasis and all 12 patients with available follow-up were alive without disease after a median follow-up of 30 months. Critical review of the literature (79 bona-fide GB-NETs) showed that tumors > 2 cm and grade 3 (G3) were enriched among metastatic GB-NETs. In conclusion, GB-NETs are generally indolent neoplasms, in contrast to gallbladder neuroendocrine carcinomas. Tumor size and grade may help predict metastatic potential, while the relatively frequent paraganglioma-like morphology, often associated with diffuse somatostatin expression, can represent a diagnostic pitfall. Their frequent association with cholesterol polyps also suggests a possible etiopathogenetic link.

The mechanisms underlying how adrenocortical carcinoma (ACC) progresses into a metastatic and lethal disease remain poorly understood. To address this, we performed comprehensive genomic analyses to delineate the evolutionary trajectory of advanced ACC. Fresh frozen tumour samples (n = 29) were obtained from nine patients, all of whom had matched primary and relapse specimens, including recurrent (n = 4) and metastatic (n = 11) lesions. In four patients, multiple primary and metastatic samples were available, enabling detailed evolutionary comparisons. All tumours underwent whole-genome sequencing, RNA sequencing, and DNA methylation profiling. Our analyses revealed that seven of nine patients exhibited global loss of heterozygosity (LOH) often followed by whole-genome doubling, resulting in copy-neutral LOH. SNP-based analyses indicated that these alterations occurred as a single catastrophic event that was conserved across all matched samples within each patient, suggesting that this event constitutes a truncal feature of the evolutionary tree. These results support a model in which chromosomal aneuploidy is important in ACC tumourigenesis, potentially distinguishing carcinomas from benign adrenal adenomas. This may explain the rarity of adenoma-to-carcinoma transformation and suggest a diagnostic and therapeutic relevance of chromosomal instability in ACC.