Approximately 15-30 % of patients with colorectal cancer (CRC) present with metastases at diagnosis, while up to 50 % will develop metastases during follow-up. The integration of targeted drugs into standard chemotherapy has significantly improved clinical outcomes, with anti-EGFR monoclonal antibodies playing a crucial role in the treatment of RAS wild-type metastatic CRC. In this context, high-quality RAS testing is essential. A national survey was carried out among 60 Italian laboratories to assess the state of the art for RAS testing. Based on the reported frequency of RAS mutations, laboratories were classified as "in range" (n. 35; RAS mutation frequency >42 % and <56 %) or "outliers" (n. 25; ≤42 % or ≥56 %). Considering the type of institutions, in Community Hospitals the percentage of 'outliers' is significantly higher than in Universities and National Cancer Institutes. Among the laboratories processing fewer than 150 samples per year, 'outliers' are more frequently detected in the group using next generation sequencing (NGS) as prevalent methodology (n = 10) than in the real time PCR (RT-PCR) group (n = 4). In contrast, within laboratories processing more than 150 samples per year, the number of 'within range' laboratories (n = 19) is more than twice that of 'outliers' (n = 8) in the NGS group. The survey underscores the need for continuous training of stakeholders involved in molecular testing and a multidisciplinary approach to patient management. The authors suggest a similar evaluation in other European countries noting that beyond infrastructure and technology, personnel training and improved communication skills are essential for optimizing biomarker testing and personalized treatment approaches.

Objective:Novel therapies for HER2-(ultra)low breast cancer (BC) have changed the traditional binary classification of HER2 status into a multi-tiered system that captures the full spectrum of expression as determined by immunohistochemistry (IHC). Several pre-analytical and analytical variables can influence the accurate identification of HER2 expression within the lowest IHC range. Herein, we present the findings of a national Italian project addressing the diagnostic challenges associated with HER2-(ultra)low BCs and their proper classification. Material and methods:A total of 121 pathologists from various regions and institutions were recruited and asked to: i) complete an online survey addressing key pre-analytical and analytical issues affecting HER2 status reporting in BC; ii) score 8 cases of IHC HER2-(ultra)low whole slide images (WSI) shared online; iii) participate in on-site meetings to discuss the results and evaluate 8 additional challenging WSIs. The assessments were subsequently compared to those of an expert panel. Results:Several pre-analytical and analytical concerns emerged from the questionnaire, such as reporting resection cold ischemia time and decalcification agent of choice and the employment of adequate HER2 controls. Regarding the WSIs, a substantial overall agreement (69%) with the expert panel was observed (74% in the online phase and 64% in the on-site phase), along with substantial to excellent consensus (≥ 61%) in over 60% of the samples. Most discordances emerged in the on- site cohort, which was enriched with challenging cases, particularly within the HER2 0+/ultralow spectrum, which demonstrated an overall agreement of 48%. Conclusions:This nationwide study highlights the complexities of accurately classifying HER2-(ultra)low breast cancers, particularly within the HER2-ultralow subset. While a substantial level of agreement with expert assessments was achieved, the variability observed in more challenging cases underscores the need for standardized interpretation criteria, enhanced training, and continuous quality assurance measures.

The extraction of meaningful information from clinical reports has been an area of growing interest, with a variety of studies leveraging natural language processing (NLP) techniques based on BERT architectures and generative large language models (LLMs). However, identifying the most effective approach remains challenging, especially for text classification, where model architecture, data availability, domain-specific nuances and language play a crucial role in performance. In this study, we present a benchmark analysis of generative LLMs and BERT-based models for the classification of metastasis in Italian clinical reports of breast cancer patients. Our methodology compares the performance of generative LLMs implemented within a structured generation framework, versus BERT-based models fine-tuned on the metastasis classification task, and also applied in a zero-shot learning setting. In our experiments, fine-tuned BERT models achieved the most balanced results (F1 = 0.884, AUC = 0.720). Generative LLMs showed promising performance, with potential for improvement through further adaptation. Finally, our study suggests that both BERT-based models and generative LLMs are potential solutions also in low computational settings, making them accessible for real-world clinical applications, particularly in medical text classification.

Bladder cancer incidence has recently risen, making it the ninth most diagnosed cancer, highlighting an urgent need for novel and effective diagnostic and therapeutic strategies to improve patient outcomes. Here, we report on a secreted glycoprotein, Galectin-3-binding protein (LGALS3BP), as a potential biomarker and therapeutic target for bladder cancer. We found a significantly elevated LGALS3BP expression in bladder cancer tissues, correlating with disease progression. Moreover, urinary and serum levels of LGALS3BP were significantly higher in patients compared to healthy individuals, with a strong correlation observed between elevated urinary protein levels and tumor grade. Of note, LGALS3BP produced by tumor cells treated with a mannosidase I inhibitor, Kifunensine, exhibited increased reactivity to a therapeutic antibody (denoted as "1959"), suggesting that glycosylation of LGALS3BP may influence antibody recognition and protein function. Furthermore, administration of 1959-sss/DM4 antibody-drug conjugate in two xenograft mouse models of human bladder cancer resulted in complete inhibition of tumor growth. In summary, findings presented here highlight LGALS3BP as a promising candidate for further investigation into its potential as a urinary biomarker and a therapeutic target for bladder cancer.

Liquid biopsy (LB) offers a minimally invasive alternative to tissue biopsy by detecting tumor-derived analytes in biological fluids. Nonetheless, its adoption is limited by variability in methodologies. Therefore, this study used a modified RAND/UCLA approach involving 23 experts of the field, providing two questionnaires that assessed agreement on 22 items. Consensus was reached for all the pre- and post-analytical phase items, agreeing on the pivotal role of plasma cfDNA. Conversely, opinions varied regarding other biomarkers and biological samples. Furthermore, turnaround time and disease setting resulted as two of the most important analytical parameters for choosing testing methodology. Particularly, a complementary tissue-liquid approach with sampling interval ≤ 2 weeks was preferred. To conclude, a strong consensus on sample handling, biomarker prioritization, and clinical applications is achieved. However, significant heterogeneity remains regarding novel biomarkers, sampling strategies, and costs. Standardization and validation are needed to enhance the clinical adoption of LB.

BackgroundThe early identification of responsive and resistant patients to androgen receptor-targeting agents (ARTA) in metastatic castration-resistant prostate cancer (mCRPC) is not completely possible with prostate-specific antigen (PSA) assessment and conventional imaging. Considering its ability to determine metabolic activity of lesions, positron emission tomography (PET) assessment might be a promising tool.Patients and methodsWe carried out a monocentric prospective study in patients with mCRPC treated with ARTA to evaluate the role of different PET radiotracers: 49 patients were randomized to receive 11C-Choline, Fluorine 18 fluciclovine (anti-1-amino-3-18F-fluorocyclobutane-1-carboxylic acid - FACBC) (18F-FACBC), or Gallium-68-prostate-specific-membrane-antigen (68Ga-PSMA) PET, one scan before therapy and one 2 months later.The primary aim was to investigate the performance of three novel PET radiotracers for the early evaluation of response to ARTA in metastatic CRPC patients; the outcome evaluated was biochemical response (PSA reduction ≥50%). The secondary aim was to investigate the prognostic role of several semiquantitative PET parameters and their variations with the different radiotracers in terms of biochemical progression-free survival (bPFS) and overall survival (OS).The study was promoted by the Italian Department of Health (code RF-2016-02364809).ResultsRegarding the primary endpoint, at log-rank test a statistically significant correlation was found between metabolic tumor volume (MTV) (P = 0.018) and total lesion activity (TLA) (P = 0.025) percentage variation among the two scans with 68Ga-PSMA PET and biochemical response. As for the secondary endpoints, significant correlations with bPFS were found for 68Ga-PSMA total MTV and TLA at the first scan (P = 0.001 and P = 0.025, respectively), and MTV percentage variation (P = 0.031). For OS, statistically significant correlations were found for different 68Ga-PSMA and 18F-FACBC parameters and for major maximum standardized uptake value at the first 11C-Choline PET scan.ConclusionsOur study highlighted that 11C-Choline, 68Ga-PSMA, and 18F-FACBC semiquantitative PET parameters and their variations present a prognostic value in terms of OS and bPFS, and MTV and TLA variations with 68Ga-PSMA PET a correlation with biochemical response, which could help to assess the response to ARTA.

Objective. The uPath PD-L1 (SP263) is an AI-based platform designed to aid pathologists in identifying and quantifying PD-L1 positive tumor cells in non-small cell lung cancer (NSCLC) samples stained with the SP263 assay. Methods. In this preliminary study, we explored the diagnostic performance of the uPath PD-L1 algorithm in defining PD-L1 tumor proportion score (TPS) and predict clinical outcomes in a series of patients with advanced stage NSCLC treated with single agent PD-1/ PD-L1 checkpoint blockade previously assessed with the SP263 assay in clinical practice. Results. 44 patients treated from August 2015 to January 2019 were included, with baseline PD-L1 TPS of >= 50%, 1-49% and < 1% in 38.6%, 25.0% and 36.4%, respectively. The median uPath PD-L1 score was 6 with a significant correlation with the baseline PD-L1 TPS (r: 0.83, p < 0.01). However, only 27 cases (61.4%) were scored within the same clinically relevant range of expression (>= vs < 50%). In the study population the baseline PD-L1 TPS was not significantly associated with clinical outcomes, while the uPath PD-L1 score showed a good diagnostic ability for the risk of death at the ROC curve analysis [AUC: 0.81 (95%CI: 0.66-0.91), optimal cut-off of >= 3.2], resulting in 19 patients (43.2%) being u-Path low and 25 patients (56.8%) being uPath high. The objective response rate in uPath high and low was 51.6% and 25.0% (p = 0.1), respectively, although the uPath was significantly associated with overall survival (OS, HR 2.45, 95%CI: 1.19-5.05) and progression free survival (PFS, HR 3.04, 95%CI: 1.51-6.14). At the inverse probability of treatment weighting analysis used to balance baseline covariates, the uPath categories confirmed to be independently associated with OS and PFS. Conclusions. This preliminary analysis suggests that AI-based, digital pathology tools such as uPath PD-L1 (SP263) can be used to optimize already available biomarkers for immune-oncology treatment in patients with NSCLC.

Biliary tract cancers (BTCs) represent a spectrum of malignancies associated with a dismal prognosis. Recent genomic profiling studies have provided a deeper understanding of the complex and heterogenous molecular landscape of BTCs, identifying several actionable genetic alterations, and expanding treatment options. Due to the high number and complexity of genetic alterations which require testing, next-generation sequencing (NGS) is currently the preferred approach over conventional methods (i.e., immunohistochemistry, fluorescence in-situ hybridization and PCR) for molecular profiling of BTCs and should be performed upfront in all BTC patients. However, BTC sampling often yields low tumor cellularity tissue, hampering NGS analysis. Future perspectives to overcome this obstacle include liquid biopsy and optimization of biopsy protocols. In this position paper, the authors discuss the current histopathologic, molecular, and therapeutic landscape of BTCs, provide a critical overview of the available testing methods for molecular diagnostics, and propose a practical diagnostic algorithm for molecular testing of BTC samples.

Neoplastic tissue is still considered today, among all biological materials, the gold standard for carrying out molecular tests according to the treatment. Genetic analysis provides information both on driver alterations that favored the development of neoplastic growth and on alterations triggering resistance to treatment. Cancer tissue can be obtained through a small biopsy or a larger surgical resection. It should be emphasized that currently targeted molecular therapies are aimed at metastatic or locally advanced malignancies, and therefore not eligible for surgery. In these cases, it is possible at the time of diagnosis to obtain only small samples for tumor characterization. In this chapter, we aimed to summarize all the modern techniques used in precision oncology.

PDF file - 161K, Ctrl and MLK4 knock down DLD were seeded and allowed to grow for the indicated times in flasks with ultra-low attachment surface to prevent cell adhesion. Cells were harvested and protein lysates were subjected to western blotting with the indicated antibodies.

Molecular biomarker testing is increasingly becoming standard of care for advanced non-small cell lung cancer (NSCLC). Tissue and liquid biopsy-based next-generation sequencing (NGS) is now highly recommended and has become an integral part of the routine management of advanced NSCLC patients. This highly sensitive approach can simultaneously and efficiently detect multiple biomarkers even in scant samples. However full optimization of NGS in clinical practice requires accurate reporting and interpretation of NGS findings. Indeed, as the number of NSCLC biomarkers continues to grow, clinical reporting of NGS data is becoming increasingly complex. In this scenario, achieving standardization, simplification, and improved readability of NGS reports is key to ensuring timely and appropriate treatment decisions. In an effort to address the complexity and lengthy reporting of NGS mutation results, an Italian group of 14 healthcare professionals involved in NSCLC management convened in 2023 to address the content, structure, and ease-of-use of NGS reporting practices and proposed a standard report template for clinical use This article presents the key discussion points addressed by the Italian working group and describes the essential elements of the report template.

PDF file - 99K,List of forward and reverse fusion primers utilized for PCR amplification of EGFR exon 19 and 21

Summary Objective To calculate the full cost of diagnostic pathology tests for Non-Small Cell Lung Cancer (NSCLC) across four Italian Pathology Units. Methods Pathology Units were located in private (2) and public (2) hospitals distributed across the Italian territory (North: 2; Centre: 1; South: 1). Pathologists provided via questionnaire data on tests on NSCLC samples along with the identification and quantification of the necessary healthcare resources (diagnostic technologies, laboratory instruments and personnel). Resources were valued according to hospital-specific unit, yearly and hourly costs (disposables; technologies; professional clusters). Results The full cost per NSCLC tissue sample included histopathological immunophenotypic and required molecular analysis. Overall, it reached € 659.77 and it was mainly composed of direct costs (77.69%). The processing of a NSCLC tissue sample was labour intensive, as a relevant share of the full cost (44.98%) was actually due to personnel costs, with laboratory technicians, biologists and pathologist driving this finding (17.09%,12.43% and 10.81%, respectively). Conclusions The results of this research can facilitate the negotiation of new dedicated tariffs for NSCLC sample processing with the national or local third party-payers.

Endometrial carcinoma (EC) harboring POLE exonuclease domain mutations occurs in 5-15% of ECs and frequently affects young women with low body mass index (BMI). It presents at early stage as high grade endometrioid histotype with intense tumor infiltrating lymphocytes and has good clinical outcomes and favorable prognosis. In this article we report the case of a 32-year-old woman with endometriod EC (EEC) exhibiting a "ultramutated" molecular profile and an excellent prognosis despite tumor size and grading. Herein, to highlight the importance of defining POLE status in ECs for both clinical and therapeutic implications for patients.

PDF file - 422K, A) Ctrl and MLK4 knock down (Sh1 and Sh2) Sw48 cells were stimulated with EGF (2nM-20 nM) at 37 {degree sign}C for 10 minutes. Cell lysates were subjected to western blotting with the indicated antibodies. B) Parental and MLK4 -/- HCT116 cells were stimulated with EGF (2nM-20 nM) at 37 {degree sign}C for 10 minutes. Cell lysates were subjected to western blotting with the indicated antibodies.

Platinum-based chemotherapy is the standard chemotherapy for high grade serous ovarian cancer and primary peritoneal high-grade serous carcinoma. PARP inhibitors have changed the paradigm of the treatment in platinum-sensitive ovarian cancers and primary peritoneal high-grade serous carcinoma with BRCA1/2 mutation or homologous recombination deficiency (HRD). Platinum-resistant ovarian and primary peritoneal high-grade serous carcinoma have a lower chance to treat and have worse outcomes. We described a case of patient with a platinum resistant primary peritoneal high-grade serous carcinoma with a rare somatic BRCA2 amplification. There are no guidelines for the treatment of ovarian cancer or primary peritoneal high-grade serous carcinoma with BRCA2 amplification. BRCA2 amplification could result in extreme homologous recombination repair (HRR) pathway efficiency and in less platinum sensitivity, which could be a molecular signature for platinum resistance. Free platinum chemotherapy regimens could be more effective in cases with BRCA2 amplification. Further studies are necessary to establish better approaches and strategies for oncological management and treatment in BRCA2 amplification high grade ovarian cancer and primary peritoneal high-grade serous carcinoma.