Giulia Calafato

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.

Endometrial carcinoma is a biologically and clinically heterogeneous neoplasm, for which molecular classification now plays a central role in determining prognosis and guiding treatment. The aim of this study was to develop and apply an in-house multi-gene NGS panel to expand the molecular characterization of endometrial tumors beyond the markers required for surrogate classification. Seventy primary endometrial carcinomas were selected and analyzed by immunohistochemistry for p53, mismatch repair proteins, ARID1A, and PTEN, and by NGS on DNA extracted from FFPE samples using a panel comprising 28 genes. Molecular classification was assigned according to the WHO algorithm into the subgroups POLE-mutated, MMR-deficient, p53-abnormal, and NSMP (No Specific Molecular Profile). Sixty-eight cases were evaluable by sequencing, and in 93% at least one pathogenic, likely pathogenic, or variant of uncertain significance was identified. The most frequent alterations involved PTEN, PIK3CA, ARID1A, and TP53. POLE-mutated tumors showed the highest mutational burden, while CTNNB1 alterations were predominantly associated with the NSMP subgroup. Overall, the proposed panel proved to be a useful tool for complementing conventional molecular classification and for highlighting additional genomic differences that may be biologically and clinically relevant.

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.

T-cell malignancies represent a complex spectrum of clinically and biologically heterogeneous diseases. Effective translational research and drug development are critically dependent on preclinical models that faithfully recapitulate this diversity. This review analyzes the current preclinical landscape, identifying a profound disparity between the clinical spectrum of T-cell neoplasms and the available in vitro tools. We demonstrate that the existing armamentarium of cell lines is heavily skewed, with an abundance of models for T-cell lymphoblastic leukemia/lymphoma (T-ALL), cutaneous T-cell lymphoma (CTCL), and anaplastic large cell lymphoma (ALCL). This skew is a direct result of a biological selection bias, as these entities are often driven by potent, TME-independent oncogenes (e.g., NOTCH1 mutations, NPM1-ALK fusions) conducive to immortalization. Conversely, the majority of peripheral T-cell lymphoma (PTCL) subtypes, which are frequently TME-dependent and clinically aggressive, remain “preclinical orphans” with few or no authenticated models. This “preclinical void” constitutes a major bottleneck, impeding mechanistic studies and therapeutic progress. We discuss the limitations of 2D cultures and highlight the necessity of adopting advanced platforms, such as patient-derived xenografts (PDX) and 3D organoid systems. These “avatar” models preserve vital tumor heterogeneity and microenvironmental context, offering superior predictive value. The systematic development and integration of these next-generation models are essential to bridge the translational gap and advance precision medicine for all patients with T-cell malignancies.

T-cell malignancies represent a complex spectrum of clinically and biologically heteroge-neous diseases. Effective translational research and drug development are critically de-pendent on preclinical models that faithfully recapitulate this diversity. This review ana-lyzes the current preclinical landscape, identifying a profound disparity between the clin-ical spectrum of T-cell neoplasms and the available in vitro tools. We demonstrate that the existing armamentarium of cell lines is heavily skewed, with an abundance of models for T-cell lymphoblastic leukemia/lymphoma (T-ALL), cutaneous T-cell lymphoma (CTCL), and anaplastic large cell lymphoma (ALCL). This skew is a direct result of a biological se-lection bias, as these entities are often driven by potent, TME-independent oncogenes (e.g., NOTCH1 mutations, NPM1-ALK fusions) conducive to immortalization. Conversely, the majority of peripheral T-cell lymphoma (PTCL) subtypes, which are frequently TME-dependent and clinically aggressive, remain "preclinical orphans" with few or no authenticated models. This "preclinical void" constitutes a major bottleneck, impeding mechanistic studies and therapeutic progress. We discuss the limitations of 2D cultures and highlight the necessity of adopting advanced platforms, such as patient-derived xen-ografts (PDX) and 3D organoid systems. These "avatar" models preserve vital tumor het-erogeneity and microenvironmental context, offering superior predictive value. The sys-tematic development and integration of these next-generation models are essential to bridge the translational gap and advance precision medicine for all patients with T-cell malignancies.

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.