Plain Language SummaryEsophageal squamous cell carcinoma (ESCC) is a common type of esophageal cancer in Japan. In rare cases, cancer cells in ESCC develop large, clear vacuoles (empty spaces) in their cytoplasm. These unusual vacuoles do not contain mucus and have been poorly understood because suitable models have been lacking. In this study, we created a new laboratory model, called an organoid, using tumor tissue from a patient with this rare form of ESCC. We named the organoid line ECO_Vac. It successfully mimicked the rare cell features of the original cancer, including the large vacuoles. Using advanced imaging and special dyes, we found that these vacuoles are actually enlarged autolysosomes – cell structures that normally break down waste. These autolysosomes were abnormally large and may be related to how the cancer responds to treatment. We also tested how ECO_Vac responds to chemotherapy drugs and found differences compared with other ESCC models. In addition, ECO_Vac could form tumors in mice while keeping the same vacuole features. This study is the first to report a living model of ESCC with these unique vacuoles. ECO_Vac may help scientists better understand this rare cancer subtype and improve diagnosis and treatment in the future.

Introduction: Oncocytic cell tumours (OCTs), previously referred to as Hürthle cell tumours of the thyroid, are a subset of thyroid and endocrine neoplasms that pose diagnostic and therapeutic challenges owing to their unpredictable clinical behaviour. The transcriptomic and proteomic landscapes of OCTs remain poorly characterized compared with those of mitochondrion-rich neoplasms (MRNs: thyroid tumours with ≥75% oncocytic cells that share similar morphology but harbour nuclear driver mutations consistent with their respective histotypes rather than mitochondrial alterations). Methods: We performed RNA and protein sequencing on 12 OCT samples and 6 MRNs. This study was prompted by the understanding that oncocytic morphology alone does not necessarily predict tumour behaviour. Results: RNA sequencing analysis identified 47 downregulated and 38 upregulated differentially expressed genes (DEGs) in OCTs relative to MRNs, with significant enrichment in pathways related to heme metabolism. Protein sequencing further revealed 20 under-expressed and 64 over-expressed differentially expressed proteins (DEPs) in OCTs. Notably, all oncocytic carcinomas formed a distinct cluster separate from the MRNs, indicating a unique proteomic profile. Conclusion: Most of the DEPs were involved in three key cellular pathways: epigenetic regulation, the tumour microenvironment, and protein biogenesis, suggesting that these processes may underlie the distinctive morphology and behaviour of OCTs. These findings highlight the need for continued research into these molecular mechanisms to improve the diagnostic accuracy and develop targeted therapies for patients with OCTs.

Introduction: Grainyhead-like 2 (GRHL2) regulates epithelial-to-mesenchymal transition (EMT) in cancer. This study analyzed the expression and prognostic role of GRHL2 in high-grade serous carcinoma (HGSC). Methods: GRHL2 protein expression by immunohistochemistry was analyzed in 411 HGSC (198 effusions, 213 surgical specimens). Expression score was generated by combination of staining extent and intensity and was assessed for association with clinicopathologic parameters and survival. Results: GRHL2 expression was significantly higher in effusions compared to surgical specimens in both analysis of all specimens (p < 0.001) and patient-matched tumors (n = 39 patients; p < 0.001). Expression was additionally higher in post-chemotherapy compared to chemo-naive effusions (p < 0.001). Higher GRHL2 score in effusions was associated with a trend for shorter overall survival (OS; p = 0.088). Higher GRHL2 score in surgical specimens was significantly related to nonoptimal (>0 cm) debulking (p = 0.004), non-complete chemoresponse at diagnosis (p = 0.003), primary chemoresistance (p = 0.045), and shorter OS (p = 0.038) and progression-free survival (PFS; p = 0.024). Both OS and PFS findings remained significant in Cox multivariate analysis (OS: p = 0.048; PFS: p = 0.04). Conclusion: GRHL2 is overexpressed in HGSC effusions compared to solid lesions, possibly reflecting altered EMT status. However, high expression in solid lesions is associated with chemoresistance and poor survival, possibly due to mediation of tumor cell migration and invasion.

Myeloid neoplasms (MN) represent genetic disorders with clonal overgrowth of cells bearing mutations of oncogenes and/or tumor suppressor genes. Distinct co-mutation patterns are associated with specific MN. MN progenitor cells retain plasticity to re-shape co-mutational stereotypes and to accumulate genetic damage along the evolution of disease. This evolution is usually branching, which becomes more evident upon recurrences/progressions. Depending on the exact timing, the diagnostic or follow-up biopsies of MN will thus reveal variable co-mutational constellations that may be difficult to interpret. To tackle respective challenges, the EBMWG dedicated a specific workshop on the additive/cumulative effect of molecular genetic alterations in MN at the 22nd Meeting of the EA4HP of Hematopathology. This report summarizes the cases and extracts lessons learned at the workshop, which can be summarized as follows: MN develop by stepwise accumulation of genetic damage; accumulation of this damage is branching and relapsing diseases may originate from a variant branch than the initial tumor; MN often retain significant genetic plasticity and, depending on time, site (of biopsy) and mutational composition, even being progenies of the same clone, later presentations of the initial disease can be multifaced; though mutations of several genes are promiscuously shared between various MN, their exact order of acquisition and their co-mutational pattern may differ between specific disease groups; some co-mutational patterns characteristically "pave the way" to leukemogenesis and even to specific diseases, while other mutations tend to be mutually exclusive, but variation in both, co-mutational patterns and mutual exclusivity, can occur.

Introduction: Cervical cancer, primarily driven by oncogenic HPV16/18, often relapses despite standard treatments. HPV circulating tumor DNA (ctDNA), which reflects tumor-derived genetic material in the bloodstream, has emerged as a promising noninvasive biomarker for monitoring disease progression. Methods: A prospective study was conducted on 20 patients with HPV16/18-associated cervical cancer. Posttreatment blood samples were collected, and HPV ctDNA levels were measured using droplet digital PCR. The correlation between HPV ctDNA levels and disease progression was examined. Results: HPV ctDNA was detected in 21% (18/85) of samples, with 6% (5/85) showing positivity. Patients without disease progression (n = 15) were HPV ctDNA negative, indicating a false positivity rate of zero. HPV ctDNA concentrations appeared higher in samples collected before or during disease progression, suggesting a potential association with disease status. Patients with positive HPV ctDNA tended to have shorter progression-free survival compared to those with negative ctDNA. Conclusions: This study suggests that HPV ctDNA may aid in monitoring disease progression in patients with HPV16/18-associated cervical cancer, highlighting the need for further validation.

Introduction: Metabolites are associated with the biology of cancer; however, no metabolites related to prognosis have been identified in head and neck cancer. This study aimed to identify metabolites associated with prognosis in patients with hypopharyngeal squamous cell carcinoma (HPSCC). Methods: Fifty-two patients who underwent surgery for HPSCC were included and randomly divided into test and validation cohorts of 26 patients each for further metabolome analysis using capillary electrophoresis/mass spectrometry on tumor and non-tumor tissues of the hypopharynx. Twenty-two patients who received adjuvant therapy after surgery were included. The receiver operating characteristic (ROC) and univariate and multivariate analyses were used to explore the relationship between recurrence-free survival (RFS), clinicopathological factors, and differentiated metabolites. Results: ROC analysis revealed six metabolites significantly associated with RFS in both cohorts, and multivariate analysis indicated that 2,3-pyridinedicarboxylate was a significantly independent poorer prognostic factor in the cohorts including patients with HPSCC without any adjuvant therapies (p = 0.017). Conclusion: 2,3-Pyridinedicarboxylate, involved in NAD+ metabolism and genomic stability, suggests the possibility of developing molecular-targeted drugs for the production of metabolites related to prognosis. This study identifies novel prognostic metabolites and their associated metabolic pathways in HPSCC, highlighting potential therapeutic targets for treatment.

Introduction: TP53 mutation is frequently observed in colorectal cancer (CRC) and is often linked to associated with immunohistochemical p53 expression patterns. Recent studies have identified cytoplasmic p53 expression in CRC, but its correlation with TP53 mutation domains and functional properties remains unclear. Methods: We evaluated nuclear and cytoplasmic p53 staining patterns in 429 stage II and III CRC samples. TP53 mutation status was assessed using targeted next-generation sequencing. Correlations among cytoplasmic expression, mutation domains, functional properties, and clinicopathological features were analyzed. Results: Cytoplasmic p53 expression was detected in 21 (4.9%) CRCs. All cytoplasmic expressions were accompanied by nuclear staining. TP53 mutations associated with cytoplasmic p53 predominantly involved nonsense mutations within the tetramerization domain (TD, 61.9%) and nuclear localization signals (NLSs, 14.3%). All functionally characterized mutations associated with cytoplasmic p53 exhibit loss-of-function (LOF) without gain-of-function or dominant-negative effects. NLS and TD mutations were significantly associated with BRAF V600E mutation but not with microsatellite instability status. Conclusion: Aberrant cytoplasmic p53 expression in CRC leads to nonsense mutations in the TD and NLS domains of TP53. These mutations exclusively induced LOF characteristics. Cytoplasmic expression patterns differ functionally and molecularly from classical nuclear staining patterns, highlighting the need for novel interpretation criteria for p53 immunostaining.

Introduction: This study aimed to observe the expression of far upstream element-binding protein 1 (FUBP1) in gastrointestinal stromal tumors (GISTs) and explore its impact on the biological behavior of GIST cells. Methods: Fifty patients with GIST who underwent surgical resection were selected, and cancer tissues and adjacent normal tissues were gathered. The expression level of FUBP1 and its correlation with clinicopathological data and prognosis in patients with GISTs were assessed. GIST-T1 cell lines in the logarithmic growth phase were transfected with lentiviruses overexpressing FUBP1, small hairpin RNA targeting FUBP1, and their respective negative controls. FUBP1 expression levels in each group, cell biological behavior were tested, and the effect of FUBP1 on tumor growth in vivo were tested. Results: Compared to adjacent normal tissues, FUBP1 expression level was elevated in GIST cancer tissues and was concerned with tumor size, NIH risk category, and tumor metastasis in patients. Knockdown of FUBP1 inhibited cell malignant behaviors and promoted cell apoptosis, while overexpression of FUBP1 had the opposite effects. FUBP1 facilitated the growth of GIST cells in vivo. Conclusion: FUBP1 is upregulated in GISTs and facilitates the invasion, migration, and proliferation of GIST cells. This provides a new perspective for understanding the pathogenesis of GIST and lays a foundation for developing potential therapeutic strategies targeting FUBP1.

Introduction: Testicular germ cell tumors (TGCTs) are the most common solid malignancies among young men. Despite good response to cisplatin-based chemotherapy, side effects negatively affect quality of life. Recent development of ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitors opens opportunity for targeted therapy, but ENPP1 expression in TGCTs has not been characterized. We aimed to assess ENPP1 immunoexpression in a cohort of primary and metastatic TGCTs, and in normal testicular parenchyma, looking into differential immunoexpression patterns among subtypes and clinicopathological correlations. Methods: Overall, 90 TGCT individual tumor components from 63 patients diagnosed and treated at a comprehensive cancer center were assessed, using immunohistochemistry to ascertain biomarker expression (combined score of stained tumor cells percentage and intensity). In silico analysis of The Cancer Genome Atlas (TCGA) and Human Protein Atlas (HPA) datasets was performed for additional validation. Results: Significant differences in ENPP1 expression across TGCT subtypes were disclosed. Seminomas and embryonal carcinomas (ECs) showed significantly higher ENPP1 expression compared to other subtypes, the highest levels being found in EC, which was confirmed with in silico analysis of TCGA and HPA. Expression in metastatic samples was overall lower compared to primary TGCTs. ENPP1 was not expressed in germ cells of healthy testicular parenchyma. Conclusion: We demonstrate that ENPP1 is expressed in TGCTs, mostly EC (a frequently aggressive tumor subtype), followed by seminoma (the most common TGCT subtype), suggesting potential responsiveness to novel ENPP1 inhibitors. Absent expression in germ cells of healthy testicular parenchyma suggest cancer cells specificity and low probability of fertility-related toxicity.