Over the past decade, patient-derived organoids (PDOs) have emerged as powerful in vitro models that closely recapitulate the histological, genetic, and functional features of their parental primary tissues, representing a ground-breaking tool for cancer research and precision medicine. This advancement has led to the development of living PDO biobanks, collections of organoids derived from a wide range of tumor types and patient populations, which serve as essential platforms for drug screening, biomarker discovery, and functional genomics. The classification and global distribution of these biobanks reflect a growing international effort to standardize protocols and broaden accessibility, supporting both basic and translational research. While their relevance to personalized medicine is increasingly recognized, the establishment and maintenance of PDO biobanks remain technically demanding, particularly in terms of optimizing long-term culture conditions, preserving sample viability, and mimicking the tumor microenvironment. In this context, this review provides an overview of the classification and worldwide distribution of tumor and paired healthy tissue-specific PDO biobanks, explores their translational applications, highlights recent advances in culture systems and media formulations, and discusses current challenges and future perspectives for their integration into clinical practice.

TERT gene mutations play critical roles in tumor progression and have important implications in several solid tumors, including thyroid carcinoma. This study aimed to evaluate the association between TERT promoter mutations and histology and clinical features of thyroid carcinoma (TC). We performed an up-to-date systematic review and a comprehensive meta-analysis. Systematic searches were made on MEDLINE (via Pubmed) databases using relevant keywords, and articles published until November 1st, 2024 were selected. A total of 54 studies and 17'021 samples are included in the meta-analysis. Relevant data for the meta-analysis was extracted, and for statistical analysis, chi-square calculation was used. Thyroid carcinomas with the highest frequency of TERT mutations are Anaplastic thyroid carcinoma (55.8 %) and Diffuse sclerosing variant of papillary thyroid carcinoma (60.4 %). No TERT mutations founds in Benign neoplasm, NIFTP and Medullary thyroid carcinoma. Mutations with the highest frequency is c.-124C>T (chr5:1295228 - C228T). TERT mutations are statistically correlated with Stage III&IV, presence of metastasis, overall survival, recurrence and radioiodine-refractory. TERT mutation plays a crucial role as a prognostic biomarker with tumor aggressiveness. Thus, in clinical practice, mutational status assessment of the TERT promoter should be considered for accurate prognostic stratification of TCs.

WNT3A is an intestinal ligand triggering the Wnt/β-catenin (Wnt) pathway, which can be enhanced by R-spondin 1 (RSPO1) through the RSPO1–LGR axis or antagonized by the adenomatous polyposis coli (APC) protein supporting β-catenin-degradation. Wnt interplays with several pathways including PI3K/mTOR (mTOR). In this study, we evaluated the influence of WNT3A-commercial and home-made culture media and RSPO1 protein on the Wnt and mTOR interplay in non-APC and APC-mutated intestinal patient-derived organoids (PDOs). Normal mucosa (NM) of sporadic CRC and FAP PDOs were cultured with: WNT3A-lacking/containing commercial (A/A+B) or home-made (BASAL/WNT3A-conditioned medium (CM)±RSPO1) media. In non-APC-mutated-PDOs (CRC-NM), WNT3A-CM, over commercial A+B, strongly activated Wnt-target-genes CCND1 and c-MYC. Most importantly, the addition of RSPO1 to home-made WNT3A-CM or A+B led to the downregulation of the mTOR-downstream-effector phospho-S6 ribosomal protein (p-S6R), highlighting the activation of the RSPO1–pS6R in both non-APC (CRC-NM) and APC-mutated (FAP-NM) PDOs, independently from LGR5 gene expression modulation. Our work demonstrates that home-made WNT3A-CM strongly impacts the crosstalk between Wnt and mTOR over commercial media, and proposes RSPO1 as a key regulator of the RSPO1–p-S6R axis in both non-APC and APC-mutated PDOs. Together, these findings represent an important methodological guide for scientists working in these fields to select the most appropriate intestinal PDO media.

Sarcomas are very complex and clinically challenging mesenchymal tumors. Although the standard therapeutic approach has improved the 5-year survival rate, many patients experience local relapses and/or distant metastases. To improve patient outcome, new strategies need to be investigated. Immunotoxins (ITs) based on rRNA N-glycosylases (also named ribosome-inactivating proteins, RIPs) are promising tools for cancer therapy because, by combining rRNA-glycosylase’s high cytotoxicity with carrier selectivity, they can specifically eliminate target neoplastic cells. In the last few years, 3D models have been extensively used in cancer research, particularly for target-specific drug screening. This study aimed to evaluate the possibility of utilizing ribosome-inactivating protein (RIP)-containing ITs to selectively target TfR1-, EGFR1- and Her2-expressing sarcoma adherent cells (ACs), spheroids (SSs) and organoids (ORs). To compare Its’ efficacy and ability to induce apoptosis, we performed dose–response viability and caspase 3/7 activation assays on rhabdomyosarcoma and osteosarcoma ACs, SSs and ORs treated with Tf-IT, αEGFR1-IT and αHer2-IT. Our results indicate that, compared to the corresponding unconjugated RIPs, all ITs showed increased cytotoxicity in sarcoma ACs. Despite the increased complexity characterizing 3D models, the higher IC₅₀ differences between ITs and unconjugated RIPs were obtained in ORs, which appeared more resistant to the nonspecific killing of the RIPs than either the ACs or SSs, thus augmenting the therapeutic window between unconjugated and conjugated RIPs. IT induced a more delayed apoptosis in 3D compared to 2D models. Our results provide essential outcomes for the potential use of these RIP-based ITs as a therapeutic strategy to treat sarcoma.

Aims: Pigmented spindle cell naevus of Reed (PSCN) is mainly characterized by MYO5A::NTRK3 fusion. However, the prevalence of NTRK fusions/rearrangements in PSCNs ranges from 15.8% to 57% in the literature, and the agreement among techniques for detecting these alterations has not been analysed. Here, we analysed NTRK fusions/rearrangements in a series of PSCNs using an optimized pan‐TRK protocol, RNA‐NGS fusion panel and FISH ( NTRK1/2/ 3). Methods and results: Seventy‐nine PSCNs were collected, reclassified (2025 WHO criteria and negative for BRAF V600E/RAS Q61R stains), and stained with a modified version of a recently proposed pan‐TRK protocol (additional amplification steps and Red chromogen). In 64 and 57 cases with available material, we performed RNA‐NGS and FISH ( NTRK1/2/3 ) analyses, respectively. Pan‐TRK was positive in 16 (20.3%) cases, all showing diffuse, granular, cytoplasmic staining of moderate intensity. In 59 cases with evaluable RNA‐NGS data, 33 (55.9%) harboured MYO5A::NTRK3 fusion, 3 (5.1%) had NTRK3 imbalance, and 1 (1.7%) case each showed MYO5A::ROS1 and ESR1::CCDC170 fusion. In 51 cases with evaluable FISH ( NTRK3 ) data, 33 (64.7%) were positive for NTRK3 rearrangement. Both the comparison of pan‐TRK versus RNA‐NGS and versus FISH ( NTRK3 ) revealed high values of specificity (SP) and positive predictive value (PPV) (92.3%–96.2%) for the detection of NTRK3 fusions/rearrangements, despite a limited sensitivity (SE) of 36.4%. In the comparison of pan‐TRK versus RNA‐NGS and FISH ( NTRK3 ), both SP and PPV were 100%, with a SE of 33.3%. Conclusions: Our ‘optimized’ pan‐TRK protocol achieved high SP and PPV for the detection of NTRK3 fusions/rearrangements in PSCNs, despite a limited sensitivity compared to molecular assay. These results suggest that this protocol may serve as a diagnostic tool in routine practice, effectively guiding molecular testing and refining the diagnostic workflow for PSCNs.

Background/Objectives: Colorectal cancer (CRC) is one of the most frequently diagnosed malignancies and a leading cause of cancer-related mortality worldwide. Its development and progression are driven by a complex interplay of genetic and epigenetic alterations that contribute to marked biological heterogeneity and variable clinical outcomes. Comprehensive molecular profiling is therefore essential for accurately characterizing CRC and guiding personalized therapeutic strategies. In this context, next-generation sequencing (NGS) has emerged as a powerful tool, enabling high-throughput, simultaneous detection of multiple clinically relevant genomic alterations in a single assay. Methods: We report our experience with a laboratory-designed NGS panel for the routine assessment of advanced-stage CRC. A total of 611 colorectal cancer specimens were analyzed using a laboratory-developed multi-gene NGS panel targeting 229 amplicons across 25 oncogenes and tumor suppressor genes. Results: The panel demonstrated analytical feasibility, with unsuccessful results observed in only 1.3% of the analyzed samples, due to low-quality/quantity DNA. RAS genes (KRAS and NRAS, altered in 51.2% and 2.7% of cases, respectively), together with BRAF (10.6%), PIK3CA (16.1%), SMAD4 (11.8%), and TP53 (64.3%), represented the most frequently altered molecular markers in the studied cohort. The frequency of major actionable genetic alterations in CRC was comparable between primary (n = 482) and metastatic (n = 121) lesions, suggesting similar mutation frequencies across the groups. Conclusions: This study presents a laboratory-developed multi-gene NGS panel specifically designed for molecular profiling of CRC in routine clinical practice.