Background:Next generation sequencing (NGS) impacted on clinical algorithm of solid tumor patients. A heterogeneous series of NGS platforms have been implemented in clinical practice but challenging handling procedures, high technical costs, and scant affordability on sequencing diagnostic routine specimens can leave behind some patients who could benefit from target drugs. Here, we sought to evaluate technical feasibility of Oxford Nanopore Technologies (ONT) sequencing accurate identification of tumor-associated molecular alterations, in a pilot series of real-world samples. Methods:We developed a technical workflow adapting the SiRe® NGS panel, originally designed for Ion semiconductor sequencing, on MinION platform (Oxford nanopore technologies), a portable, cost effective long read sequencer. The SiRe® panel enables detection of ٥٦٨ clinically actionable somatic mutations across six key genes (EGFR, KRAS, NRAS, BRAF, cKIT, PDGFRα) relevant to targeted therapies in several solid tumors. We implemented a multiplexed assay using pooled and barcoded samples, processed on a single MinION flow cell. Performance was benchmarked from a pilot series of nine FFPE samples against Ion Torrent sequencing data. A single liquid biopsy sample was also analyzed testing accuracy of MinION technology. Results:The adapted ONT workflow demonstrated high concordance ratei in detecting clinically relevant molecular alterations on short-read fragments, achieving comparable accuracy with standardized second generation NGS platforms on tissue and liquid biopsy samples. Conclusions:This proof of concept aimed to integrate ONT sequencing into molecular oncology workflows, providing practical, low-cost, and scalable alternative to conventional NGS platforms. The results support the potential of ONT technology to democratize access to precision oncology, particularly in laboratories with limited resources.

Objective:Traditional pathological examination of lymph nodes is labor-intensive and has shown variability in diagnostic accuracy. Recent advancements in artificial intelligence (AI) provide promising opportunities to enhance and standardize pathological workflows. AI-based image analysis models, particularly those utilizing deep learning algorithms, have demonstrated potential in automating and improving diagnostic accuracy in histopathology. This study aimed to evaluate the performance of a novel AI model known as ChatGPT-4 in detecting metastatic involvement in sentinel lymph nodes (SLNs) from breast cancer cases. Methods:We utilized digital slides from frozen sections, which are commonly employed intraoperatively, to assess the model's diagnostic accuracy. A total of 90 SLNs were retrospectively collected and analyzed using ChatGPT-4. The generated diagnoses were evaluated by two senior pathologists. Results:The AI model achieved an overall accuracy of 92.2%, with a sensitivity of 100% and specificity of 80.6%. The study highlights the practical applicability of AI in diagnosing SLN metastasis, emphasizing the importance of frozen sections in real-world scenarios. Conclusions:These findings suggest that integrating AI models like ChatGPT-4 into pathological workflows could enhance diagnostic accuracy and efficiency in breast cancer treatment.

Despite significant improvements in the outcome of Estrogen Receptor (ER) α-positive breast cancer (BC) following the use of endocrine therapies, resistance remains a major challenge. Clinical studies proved that obesity, in addition to promote BC progression, is associated with a reduced efficacy to these treatments, but mechanisms remain unclear. We used co-culture systems followed by validation through an ‘ex vivo’ model of human mammary obese (Ob) adipocytes and obese endocrine-resistant metastatic Patient-Derived Organoids (PDOs). Transcriptomics with MixOmics-MINT and MetaCore Functional Tools along with lentiviral and pharmacological approaches provide insights into mechanisms. Clinical relevance was investigated using public datasets, transcriptome-based (n = 375), and immunohistochemistry-based (n = 65) evaluations. In a model of co-culture, we demonstrated that conditioned media (CM) released by 3T3-L1A adipocytes reduced the sensitivity of parental MCF-7 BC cells to the inhibitory effects of Tamoxifen (Tam) on growth, motility and invasion and significantly increased the proliferative, motile and invasive phenotype of Tam-resistant (TR) BC cells. Transcriptomics identified TXNIP (Thioredoxin-interacting protein), a known tumor suppressor gene, as a network central hub, that was significantly down-regulated in CM-treated MCF-7 and TR cells. Accordingly, TXNIP expression was negatively correlated with Body Mass Index (BMI) in BC patients. Lentiviral TXNIP overexpression and pharmacological induction of TXNIP (i.e. SAHA) or the blockade of insulin-like growth factor-I (IGF-1) signaling, an obesity hallmark able to affect TXNIP expression, reversed CM-mediated effects. TXNIP down-regulation, proliferation and motility in TR cells were exacerbated by CM derived from Ob 3T3-L1A, and combination of an IGF-1 inhibitor and SAHA abrogated Ob-CM activities. Results were also validated in aromatase inhibitor-resistant BC cells. The effectiveness of IGF-1/TXNIP axis inhibition was confirmed using an ‘ex vivo’ model of human mammary obese adipocytes and PDO models. Finally, retrospective analyses demonstrated that an IGF-1high/TXNIPlow signature was correlated with poorer survival in endocrine-treated BC patients. In conclusion, our study sheds new light on adipocyte/BC cell crosstalk, underscoring the potential of targeting IGF-1/TXNIP axis to block this harmful connection, especially in the context of obesity.

In cancer patients, only a small fraction of circulating cell-free DNA (cfDNA) consists of circulating tumor DNA (ctDNA), which contains tumor-specific features. Detecting ctDNA in peripheral blood through liquid biopsy offers a safe, noninvasive alternative to traditional tissue biopsy, with the added benefit of enabling repeated testing over time. This study investigates the potential of liquid biopsy as an innovative and noninvasive prognostic tool for patients with stage II–III colon cancer. Specifically, we analyzed the presence of cfDNA harboring tumor-specific mutations, previously identified in tumor tissue via next-generation sequencing (NGS), both before and after therapeutic surgery. Our aim was to assess its predictive value for relapse, ultimately guiding therapeutic decisions and improving patient outcomes. Our results demonstrate that the presence of ctDNA before surgery was significantly associated with disease relapse, indicating its potential as a predictive biomarker. In this cohort, ctDNA detection after surgery, during adjuvant chemotherapy, did not maintain the same predictive value. This suggests that preoperative ctDNA analysis may provide critical prognostic information, while post-surgical ctDNA monitoring, in this specific setting, may be influenced by treatment dynamics. In conclusion, we found that combining NGS profiling of the primary tumor tissue with droplet digital PCR (ddPCR)-based analysis of cfDNA provides a comprehensive approach to therapy monitoring in stage II-III colon cancer patients. Liquid biopsy offers valuable insights into treatment response and disease progression while serving as a noninvasive and repeatable method for routine clinical care.

BACKGROUND AND OBJECTIVES:Colorectal cancer (CRC) is the third most diagnosed tumor type and the second leading cause of cancer-related mortality. Despite recent improvements in the clinical management of CRC patients both before and after surgery, disease recurrence remains common, with an incidence of about 20-30% within 5 years. Current tissue biopsy techniques are invasive and inadequate for assessing tumor heterogeneity or capturing real-time disease dynamics. In contrast, liquid biopsy offers a promising, minimally invasive alternative. This study aimed to evaluate extracellular vesicle (EV)-associated protein markers, detected through super-resolution microscopy, as potential indicators of recurrence in CRC patients. METHODS:We employed a novel liquid biopsy approach based on the super-resolution imaging (dSTORM) of specific protein markers carried by EVs isolated from the plasma of CRC patients. We analyzed combinations of both surface and intravesicular proteins, including EpCAM, PD-L1, CD81, IL-6, and Cyclin D1. RESULTS:Specific combinations of EV-associated markers were able to distinguish patients with recurrence from those without residual disease. Additionally, we observed correlations between some marker profiles and tumor stage or lymph node involvement. No association was found with mismatch repair system status. CONCLUSIONS:To our knowledge, this is the first study to propose the use of EV-bound proteins for recurrence detection in CRC using super-resolution microscopy within a liquid biopsy framework. These findings support the potential of this approach as a non-invasive tool for CRC monitoring.

AT-rich interaction domain 1 (ARID1A) is a pivotal gene with a significant role in gastrointestinal tumors which encodes a protein referred to as BAF250a or SMARCF1, an integral component of the SWI/SNF (SWItch/sucrose non-fermentable) chromatin remodeling complex. This complex is instrumental in regulating gene expression by modifying the structure of chromatin to affect the accessibility of DNA. Mutations in ARID1A have been identified in various gastrointestinal cancers, including colorectal, gastric, and pancreatic cancers. These mutations have the potential to disrupt normal SWI/SNF complex function, resulting in aberrant gene expression and potentially contributing to the initiation and progression of these malignancies. ARID1A mutations are relatively common in gastric cancer, particularly in specific adenocarcinoma subtypes. Moreover, such mutations are more frequently observed in specific molecular subtypes, such as microsatellite stable (MSS) cancers and those with a diffuse histological subtype. Understanding the presence and implications of ARID1A mutations in GC is of paramount importance for tailoring personalized treatment strategies and assessing prognosis, particularly given their potential in predicting patient response to novel treatment strategies including immunotherapy, poly(ADP) ribose polymerase (PARP) inhibitors, mammalian target of rapamycin (mTOR) inhibitors, and enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) inhibitors.

Classic Hodgkin’s lymphoma (cHL) is a curable cancer with a disease-free survival rate of over 10 years. Over 80% of diagnosed patients respond favorably to first-line chemotherapy, but few biomarkers exist that can predict the 15–20% of patients who experience refractory or early relapsed disease. To date, the identification of patients who will not respond to first-line therapy based on disease staging and traditional clinical risk factor analysis is still not possible. Three-dimensional (3D) telomere analysis using the TeloView® software platform has been shown to be a reliable tool to quantify genomic instability and to inform on disease progression and patients’ response to therapy in several cancers. It also demonstrated telomere dysfunction in cHL elucidating biological mechanisms related to disease progression. Here, we report 3D telomere analysis on a multicenter cohort of 156 cHL patients. We used the cohort data as a training data set and identified significant 3D telomere parameters suitable to predict individual patient outcomes at the point of diagnosis. Multivariate analysis using logistic regression procedures allowed for developing a predictive scoring model using four 3D telomere parameters as predictors, including the proportion of t-stumps (very short telomeres), which has been a prominent predictor for cHL patient outcome in a previously published study using TeloView® analysis. The percentage of t-stumps was by far the most prominent predictor to identify refractory/relapsing (RR) cHL prior to initiation of adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) therapy. The model characteristics include an AUC of 0.83 in ROC analysis and a sensitivity and specificity of 0.82 and 0.78 respectively.

Background First identified in Drosophila melanogaster , the Hippo pathway is considered a major regulatory cascade controlling tissue homeostasis and organ development. Hippo signaling components include kinases whose activity regulates YAP and TAZ final effectors. In response to upstream stimuli, YAP and TAZ control transcriptional programs involved in cell proliferation, cytoskeletal reorganization and stemness. Main text While fine tuning of Hippo cascade components is essential for maintaining the balance between proliferative and non-proliferative signals, pathway signaling is frequently dysregulated in gastrointestinal cancers. Also, YAP/TAZ aberrant activation has been described in conditions characterized by chronic inflammation that precede cancer development, suggesting a role of Hippo effectors in triggering carcinogenesis. In this review, we summarize the architecture of the Hippo pathway and discuss the involvement of signaling cascade unbalances in premalignant lesions of the gastrointestinal tract, providing a focus on the underlying molecular mechanisms. Conclusions The biology of premalignant Hippo signaling dysregulation needs further investigation in order to elucidate the evolutionary trajectories triggering cancer inititation and develop effective early therapeutic strategies targeting the Hippo/YAP pathway.

Colitis cystica profunda (CCP) is a rare, uncommon and nonneoplastic condition that can occur anywhere in gastrointestinal tract, but its main occurrence is in the rectum and sigmoid colon. It is characterized by the presence of mucin filled cysts, lined by benign epithelium, beneath the muscularis mucosae, usually confined to the submucosa, and it can clinically and radiologically mimic a neoplasm. Here we report a rare case of CCP in a patient with a 2-months history of abdominal pain and severe anemia, associated with diverticulosis. The knowledge of this entity and its differential diagnosis, in particular with the intestinal mucinous adenocarcinoma, is necessary, as it can be a clinically and histological mimic of a malignant neoplasm.

Supplementary Material from PIK3CA Mutations Correlate with Hormone Receptors, Node Metastasis, and ERBB2, and Are Mutually Exclusive with PTEN Loss in Human Breast Carcinoma

Supplementary Information, Methods, Table 1, Figures 1-7 from 3-Phosphoinositide–Dependent Kinase 1 Potentiates Upstream Lesions on the Phosphatidylinositol 3-Kinase Pathway in Breast Carcinoma

Thyroid carcinoma (TC) is the most common malignancy of endocrine organs. The cell subpopulation in the lineage hierarchy that serves as cell of origin for the different TC histotypes is unknown. Human embryonic stem cells (hESCs) with appropriate in vitro stimulation undergo sequential differentiation into thyroid progenitor cells (TPCs-day 22), which maturate into thyrocytes (day 30). Here, we create follicular cell-derived TCs of all the different histotypes based on specific genomic alterations delivered by CRISPR-Cas9 in hESC-derived TPCs. Specifically, TPCs harboring BRAFV600E or NRASQ61R mutations generate papillary or follicular TC, respectively, whereas addition of TP53R248Q generate undifferentiated TCs. Of note, TCs arise by engineering TPCs, whereas mature thyrocytes have a very limited tumorigenic capacity. The same mutations result in teratocarcinomas when delivered in early differentiating hESCs. Tissue Inhibitor of Metalloproteinase 1 (TIMP1)/Matrix metallopeptidase 9 (MMP9)/Cluster of differentiation 44 (CD44) ternary complex, in cooperation with Kisspeptin receptor (KISS1R), is involved in TC initiation and progression. Increasing radioiodine uptake, KISS1R and TIMP1 targeting may represent a therapeutic adjuvant option for undifferentiated TCs.

We review research regarding the epidemiology, risk factors, genetic susceptibility, molecular pathology, and early detection of SCLC, a deadly tumor that accounts for 14% of lung cancers. We first summarize the changing incidences of SCLC globally and in the United States among males and females. We then review the established risk factor (i.e., tobacco smoking) and suspected nonsmoking-related risk factors for SCLC, and emphasize the importance of continued effort in tobacco control worldwide. Review of genetic susceptibility and molecular pathology suggests different molecular pathways in SCLC development compared with other types of lung cancer. Last, we comment on the limited utility of low-dose computed tomography screening in SCLC and on several promising blood-based molecular biomarkers as potential tools in SCLC early detection.

PDF file - 570K, Correlation between tumors histological type and grade and disease stage and patients' survival. Analysis of disease-specific survival of patients was assessed using Kaplan-Meier curves and statistical significance of the difference between the groups was determined using the Log-Rank test.

Supplementary Figure 2 from PIK3CA Mutations Correlate with Hormone Receptors, Node Metastasis, and ERBB2, and Are Mutually Exclusive with PTEN Loss in Human Breast Carcinoma

RAS gene mutational status represents an imperative predictive biomarker to be tested in the clinical management of metastatic colorectal adenocarcinoma. Even if it is one of the most studied biomarkers in the era of precision medicine, several pre-analytical and analytical factors may still impasse an adequate reporting of RAS status in clinical practice, with significant therapeutic consequences. Thus, pathologists should be aware on the main topics related to this molecular evaluation: (i) adopt diagnostic limit of detections adequate to avoid the interference of sub-clonal cancer cell populations; (ii) choose the most adequate diagnostic strategy according to the available sample and its qualification for molecular testing; (iii) provide all the information regarding the mutation detected, since many RAS mutation-specific targeted therapeutic approaches are in development and will enter into routine clinical practice. In this review, we give a comprehensive description of the current scenario about RAS gene mutational testing in the clinic focusing on the pathologist’s role in patient selection for targeted therapies.

PDF file - 78K, Type of KRAS and EGFR mutations in NSCLC tumors of different histological subtypes.

PDF file - 35K, MAGE-A expression score in NSCLC and correlation with tumor grade and disease stage.