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.

Pulmonary neuroendocrine tumors (NETs) include typical and atypical carcinoids, corresponding to low- and intermediate-grade malignancies. Integration of necrosis, mitotic count per 2 mm[2] (MC), and Ki-67 index, a desirable criterion in the current WHO scheme, may enable a classification framework beyond conventional histology. We used a clustering method for mixed-type data, including necrosis, MC and Ki-67 index, on a single-institution cohort of 358 typical and 125 atypical carcinoids with long-term follow-up. An external validation series of 259 additional carcinoids was used. A three-cluster solution was selected, yielding a tiered prognostic stratification model, that included NETG1 (383 cases; mean MC: 0.7; necrosis: 8.1% of cases; mean Ki-67 index: 2.5%), NETG2 (77 cases; mean MC 2.3; necrosis 27.3%; mean Ki-67: 11.4%), and NETG3 (23 cases; mean MC: 5.5; necrosis 60.9%; mean Ki-67: 25.1%). The model was independent of histological classification. Ki-67 index was the strongest discriminator, distinguishing NET G1 tumors with a Ki-67 index < 6% from NET G3 tumors (Ki-67 > 16%), while MC contributed to identifying a subset of NET G2 tumors. The selected cut-offs were derived through a statistical approach evaluating hundreds of decision-tree configurations, following a sequential algorithm from Ki-67 to MC, with 6-16% identified as the most informative Ki-67 range for intermediate stratification. Necrosis added no discriminative value, as it was closely correlated with the Ki-67 index and MC. This classification corresponded to a progressive worsening of overall survival and, more significantly, relapse-free survival, and it was transferable to the external cohort. In conclusion, based on routinely available pathological parameters, this model provides a practical and reproducible classification of resected pulmonary NETs, overcoming the traditional typical-atypical carcinoid dichotomy and is potentially applicable to biopsy material.

Thyroid nodules with indeterminate cytology represent a clinical challenge owing to uncertain malignancy risk, often leading to diagnostic surgery. Molecular testing has emerged as a promising adjunct to improve risk stratification and guide surgical decision-making. However, the clinical utility and impact of different molecular platforms remain debated. The present study evaluates the impact of molecular testing on surgical decision-making in indeterminate thyroid nodules and compares outcomes between first- and second-generation molecular platforms. A PRISMA-guided systematic review and meta-analysis identified 132 studies including 66,448 thyroid nodules, of which 30,292 (45.6%) were indeterminate (Bethesda III/IV). Pooled analyses included studies directly comparing conventional management with molecular testing or different molecular platforms. Primary outcomes were surgical resection rates and risk of malignancy (ROM), stratified by diagnostic approach and test generation. Conventional management demonstrated significantly higher surgical rates than molecular testing group (OR = 2.258, 95% CI: 1.548-3.293, p < 0.001). ROM among resected nodules was lower in the conventional management compared with the molecular testing group (OR = 0.594, 95% CI: 0.388-0.911, p = 0.017). First-generation molecular tests showed higher surgical rates and lower ROM than second-generation assays (OR = 1.709, 95% CI: 1.122-2.601, p = 0.013; OR = 0.665, 95% CI: 0.515-0.858, p = 0.002, respectively). Exclusion of non-invasive follicular thyroid neoplasm with papillary-like nuclear features attenuated differences in malignancy rates. Molecular testing significantly reduces surgical interventions in indeterminate thyroid nodules without evidence of reduced detection of clinically significant malignancy, particularly with second-generation platforms supporting more individualized and evidence-based management strategies.

Well-differentiated neuroendocrine tumors, grade 3 (NETs G3) of the digestive system are high-grade neuroendocrine neoplasms characterized by well-differentiated neuroendocrine morphology and high proliferative activity. Their molecular identity and therapeutic vulnerabilities remain incompletely defined. We performed a transcriptomic analysis and a comprehensive genomic profiling of 40 cases, including 26 NETs G3, 8 NETs G1/G2, and 6 neuroendocrine carcinomas (NECs). Targeted sequencing assessed alterations at both the DNA and RNA level, and gene expression profiling was conducted using a targeted immune-oncology panel. NETs G3 retained recurrent alterations in chromatin-regulatory genes characteristic of well-differentiated tumors and largely lacked the consistent TP53 and RB1 inactivation typical of NECs. Transcriptomic and genomic analysis demonstrated separation of NECs from NETs, with NETs G3 partially overlapping with NETs G1/G2 supporting a model of molecular relatedness. However, when compared with NETs G1/G2, NETs G3 showed upregulation of extracellular matrix remodeling, metabolic reprogramming, and cytokine signaling pathways. NETs G3 were heterogeneous in terms of number of genomic alterations per case and involved genes. A distinctive genomic feature was the predominance of large-segment copy-number losses, frequently involving chromatin-regulatory regions on chromosomes X and 10. Potentially targetable alterations were detected in up to 25% of NETs G3. Analysis of immune microenvironment supported a poorly inflamed tumor status, with individual cases harboring microsatellite instability or enrichment in tumor-infiltrating lymphocytes potentially amenable to immunotherapy.

Pituitary neuroendocrine tumors (PitNETs) are common sellar neoplasms and represent a major component of routine pituitary pathology. In the 2022 World Health Organization (WHO) Classification, transcription factor-defined lineage assignment is central to diagnosis. However, lineage-related morphologic information on routine hematoxylin and eosin (H&E)-stained whole-slide images (WSIs) has not been systematically characterized. We developed an attention-guided graph neural network to predict PitNET lineage directly from H&E-stained WSIs and to identify regions prioritized for classification. Consecutive patients who underwent surgical resection for PitNETs at Beijing Tiantan Hospital between 2021 and 2025 were included. In the internal hold-out set, the five fold-specific models achieved a mean F1-score of 92.78% and a mean balanced accuracy of 94.84%. In a temporally independent validation cohort, the final model achieved an F1-score of 87.64% and a balanced accuracy of 89.48%. Performance varied across lineages and histomorphologic subtypes, and the most common error pattern was misassignment of PIT-1 and T-PIT tumors to SF-1. Attention maps predominantly highlighted tumor-rich regions. Quantitative cell-level morphometry supported lineage-associated patterns, including larger cell and nuclear sizes in PIT-1 tumors, more elongated nuclei in SF-1 tumors, and higher cellular density with reduced intercellular spacing in T-PIT tumors. In six cases with multiple synchronous PitNETs of distinct lineages, patch-level prediction maps corresponded closely to transcription factor immunohistochemistry. In a small exploratory subset with available DNA methylation data, methylation classifier results were concordant with model predictions in 11 of 15 cases and with routine histopathologic diagnoses in 8 of 15 cases. These findings indicate that routine H&E-stained WSIs contain learnable morphologic information related to PitNET lineage. Attention-guided spatial modeling provides an interpretable framework for characterizing lineage-associated patterns in PitNETs.

The clinical behavior and molecular underpinnings of rare cases of poorly differentiated thyroid carcinoma (PDTC) diagnosed in young adults are not established. We evaluated 13 cases of PDTC in adult patients ≤ 45 years of age. The median age at diagnosis was 40 years (range 27 - 45 years). The median tumor size was 3.7 cm. The median mitotic count was 6 per 2 mm[2]. Necrosis was present in 7 (54%) cases. Eleven (85%) cases were either encapsulated with extensive (≥ 4 foci) angioinvasion or widely invasive. Molecular analysis was available for nine (69%) cases and revealed two (22%) with PAX8::PPARG fusions, two (22%) with NRAS alterations, one (11%) with a DICER1 variant, one (11%) with alterations in ATM, CDKN1A and TERT, one (11%) with a TSC2 deletion and TERT copy number gain, and two (22%) with variants of uncertain significance only. Of the 12 (92%) patients with follow-up data available (median 6.2 years, range 1.5-16.4 years), the only patient who died of disease had distant metastases at diagnosis. Of the remaining patients with initial M0 disease, only one had disease recurrence, developing local recurrence and distant metastasis with persistent disease at last follow up. Our findings suggest that PDTC in adults ≤ 45 years of age at diagnosis, may have a better prognosis than is typically associated with PDTC despite high-grade features and invasive growth. Additionally, we found a higher rate of PAX8::PPARG fusions and a lower rate of TERT alterations, suggesting that the molecular profile might explain the improved survival.