Abstract Ovarian clear cell carcinoma (OCCC) is an aggressive subtype of epithelial ovarian cancer, characterized by high recurrence rates and poorer prognosis compared to other ovarian cancers. It has an elevated mortality worldwide, particularly in Taiwan. Strikingly, OCCC cases with an immune-hot microenvironment exhibited worse survival outcomes, challenging the conventional understanding that 'hot' tumors confer a better prognosis. This paradox prompts our investigation into how the immune-hot microenvironment influences OCCC progression and leads to these counterintuitive survival outcomes In this study, we utilized insertion-deletion (indel) variants within hypermutable polyguanine (polyG) sequences (mutation rate: ∼10-6-10-4) to reconstruct the phylogeny of a cohort of 20 early-stage OCCC patients. These patients were evenly stratified into immune-hot and immune-cold phenotypes based on their predefined immune status. From a subset of 11 cases (7 immune-hot and 4 immune-cold), we collected a total of 201 samples. Using the published mutation rate for polyG loci (9.43*10-5), we converted the polyG phylogenies into cell-division trees to objectively quantify the lineages. We further examined phylogenetic patterns and clonality through whole-exome sequencing (WES). By comparing evolutionary processes across phylogenies, we identified a distinct subgroup through total cell divisions and MRCA division within the immune-hot tumors that may explain unexpected survival outcomes. We compared three groups: immune-hot Subgroup 1, immune-hot Subgroup 2, and immune-cold tumors. Phylogenetic analysis revealed that Subgroup 1 underwent significantly more cell divisions from the zygote to the MRCA compared to Subgroup 2 and immune-cold tumors (1744 vs. 742 vs. 936, p = 0.012), despite maintaining similar clonal complexity (CV: 26.04% vs. 29.34% vs. 34.18%, p = 0.52). However, Subgroup 1 exhibited a significantly lower pairwise genetic distance compared to the other groups (0.08 vs. 0.10 vs. 0.10, p = 2.73 × 10-9), implying a recent clonal sweep. This finding was further supported by the sample-wise coalescence ratio (CoaR), a metric that quantifies the shared evolutionary history between samples. Subgroup 1 exhibited a significantly higher ratio than Subgroup 2 and immune-cold tumors (0.96 vs. 0.80 vs. 0.80, p = 1.52 × 10- 46), indicating high phylogenetic similarity. Conversely, when quantifying clonal diversity from WES data (using CONIPHER and the Shannon diversity index), Subgroup 1 displayed higher diversity than Subgroup 2 and immune-cold tumors (1.32 vs. 0.70 vs. 0.87, p = 0.012). This discrepancy, high genomic diversity yet low phylogenetic heterogeneity, suggests intense selection pressure in Subgroup 1 that eliminates neo-clones. Finally, we observed that while Subgroup 2 shared a similar clonal structure to immune-cold tumors, it was associated with the worst survival outcomes (p = 0.0019). This suggests that Subgroup 2 may represent a distinct evolutionary trajectory or intermediate state, possessing adaptive features that facilitate aggressive progression. These findings highlight the heterogeneity within immune-hot tumors and offer a potential explanation for why high immune infiltration does not always correlate with favorable survival in OCCC." Citation Format: YuYi Huang, Yen-Chun Chen, Pei-Chi Cheng, Ko-Chen Chen, Alice Hsiang-Kuo Yang, Hsien-Neng Huang, Ruby Yun-Ju Huang, Lin-Hung Wei, Wei-Ting Hung. Elucidating the impact of the immune microenvironment on the evolutionary dynamics of early-stage ovarian clear cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB235.

Background:Rho GTPases are essential regulators for cellular movement and intracellular membrane trafficking. Their enzymatic activities fluctuate between active GTP-bound and inactive GDP-bound states regulated by GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Arhgap39/Vilse/Porf-2 is a newly identified GAP. The role of Arhgap39 in migration and invasion has not been addressed thoroughly. Methods:The Arhgap39 gene was knocked out by Crispr-Cas9 gene editing in mouse Hepa1-6 and Hepa-1c1c7 cells to analyze the impact of Arhgap39 depletion on migration and invasion. Results:Loss of Arhgap39 noticeably increased the migration and invasive potential. Purified Arhgap39 recombinant protein facilitated the hydrolysis of GTP in RhoA and Rac1 in vitro. RNA-seq analysis revealed that matrix metalloproteinase 13 (MMP13) and Laminin subunit beta 1 (LAMB1) were increased in Arhgap39-/- cells. We further crossed Arhgap39fl/fl with KrasLSL-G12D and p53fl/fl mice under the control of albumin-Cre recombinase to induce the spontaneous development of hepatocellular carcinomas. Intriguingly, the expression levels of MMP13 and the overall survival in Alb-Cre_KrasLSL-G12D_p53fl/fl_Arhgap39fl/fl (KPA) mice were comparable to control Alb-Cre_KrasLSL-G12D_p53fl/fl (KP) mice. The cell migration and invasion of KPA mice were also similar to those of control KP mice. Conclusion:Arhgap39 loss could modulate the migration and invasion in some hepatocellular cancer cells, but not in those isolated from KPA mice.

Abstract Background To estimate the burden of invasive pneumococcal disease, non-bacteremic pneumococcal pneumonia, and acute otitis media before and after inclusion of the 13-valent pneumococcal vaccine (PCV13) into Taiwan’s Childhood Immunization Program in 2015. Methods Episodes of eligible children aged < 18 years hospitalized with invasive pneumococcal disease, non-bacteremic pneumococcal pneumonia, or acute otitis media between 1 January 2011 and 31 December 2019 were identified from the National Health Insurance Research Database. Annual hospitalized incidence rate, case fatality rate, and healthcare resource utilization and costs were estimated. Incidence time trends were assessed with interrupted time series analyses. Results 1,284 invasive pneumococcal disease episodes, 25,074 non-bacteremic pneumococcal pneumonia episodes, and 23,139 acute otitis media episodes were identified. The overall annual incidence rates of invasive pneumococcal disease, non-bacteremic pneumococcal pneumonia, and acute otitis media were 3.31, 64.61, and 59.62 episodes per 100,000 person-years, respectively. Interrupted time series analyses results showed a significantly lower baseline incidence rate (incidence rate ratio [IRR]:0.58, p-value = 0.001) for invasive pneumococcal disease, and significantly higher baseline incidence rate (IRR:1.17, p-value < 0.001) for non-bacteremic pneumococcal pneumonia in the post-PCV13 period. Baseline incidence rates between the two periods were comparable for acute otitis media. A significant increase in trend of incidence rate was observed for all three diseases. Case fatality rate was 1.79%, 0.09%, and 0.00% for invasive pneumococcal disease, non-bacteremic pneumococcal pneumonia, and acute otitis media, respectively. Median length of hospitalization per inpatient visit was comparable between the two periods for invasive pneumococcal disease and non-bacteremic pneumococcal pneumonia, but significantly shorter in the post-PCV13 period for acute otitis media. In the post-PCV13 period, average total costs per episode was lower for invasive pneumococcal disease and non-bacteremic pneumococcal pneumonia, but higher for acute otitis media. Conclusions Residual clinical and economic burden of pneumococcal diseases remained substantial after PCV13 inclusion into Taiwan’s Childhood Immunization Program. To further reduce the disease burden among children, additional research to investigate the cause of increasing trends of hospitalized invasive pneumococcal disease, non-bacteremic pneumococcal pneumonia and acute otitis media in the post-PCV13 era will be required.

Intratumoral heterogeneity is intrinsically comprised of molecular alterations of tumor cells and extrinsically from interconnections with microenvironments. This study explores the spatial heterogeneity of ovarian clear cell carcinoma (OCCC), a rare cancer with significance to East Asian women. We profiled 21 tumors from matched ovarian and metastatic sites with spatial transcriptomic (ST) platforms including GeoMx and Visium CytAssist. Three subclusters of OCCC tumor cells enriched in oxidative phosphorylation, epithelial-mesenchymal transition, and inflammation pathways were identified with preferential geospatial localizations in tumor centers and invasive margins/tumor-stromal interfaces. These ST subclusters correlated with epithelial-mesenchymal gradients and prognostic features in larger cohorts. Single-cell ST in CosMx identified 9 cancer cell populations with organ-specific tropism. Trajectory analysis indicated a NEAT1-high subclone having founder characteristics, progressively dominating the metastatic niche, displaying mesenchymal traits. This founder feature correlated with clinical outcomes in PanCancer datasets. Our findings highlight the adaptive and context-specific interactions between cancer cells and microenvironments within spatial complexities. ### Competing Interest Statement The authors have declared no competing interest.

Purpose Exposure of hydrochlorothiazide (HCTZ) has been linked to the increase of skin cancer in the Caucasian population, especially for the squamous cell carcinoma (SCC) but not for basal cell carcinoma (BCC). This study aimed to evaluate the risks of skin cancer between patients receiving HCTZ and those receiving other antihypertensives. Methods This retrospective cohort study was derived from the National Health Insurance Database in Taiwan. We enrolled patients aged 20 years and older who newly receiving antihypertensive medications between 2004 - 2015. We calculated the medication possession ratio (MPR) of the first two years of treatment for patient enrollment and treatment classification, in which patients with MPR above 80% were enrolled and patients were subsequently categorized into receiving HCTZ, other thiazide diuretics, non-thiazide diuretics, and non-diuretic antihypertensives. The Cox proportional hazards model was used to evaluate the risk of skin cancer, and further divided into SCC, and BCC. Results Our study enrolled 41,086, 27,402, 19,613, and 856,782 patients receiving HCTZ, other thiazide diuretics, non-thiazide diuretics, and non-diuretic antihypertensives, respectively. We found BCC risks were similar when comparing HCTZ with other thiazides (adjusted hazard ratio, 0.84; 95% CI: 0.54-1.33), non-thiazide diuretics (0.93; 0.51-1.67), and non-diuretic antihypertensives (0.91; 0.66-1.26). Higher SCC risk was observed in the HCTZ group, compared to other thiazide (1.24; 0.74-2.08), non-thiazide diuretics (1.32; 0.70- 2.51), and non-diuretic antihypertensives (1.23; 0.87-1.73), although the differences were not statistically significant. Conclusions We conclude that skin cancer need not be of major concern to physicians when prescribing antihypertensives for an Asian population.

ObjectiveSeveral guidelines do not recommend beta-blocker as the first-line treatment for hypertension because of its inferior efficacy in stroke prevention. Combination therapy with beta-blocker is commonly used for blood pressure control. We compared the clinical outcomes in patients treated with amlodipine plus bisoprolol (A + B), a ss 1-selective beta-blocker and amlodipine plus valsartan (A + V).MethodsA population-based cohort study was performed using data from the Taiwan National Health Insurance Research Database. From 2012 to 2019, newly diagnosed adult hypertensive patients who received initial amlodipine monotherapy and then switched to A + V or A + B were included. The efficacy outcomes included all-cause death, atherosclerotic cardiovascular disease (ASCVD) event (cardiovascular death, myocardial infarction, ischemic stroke, and coronary revascularization), hemorrhagic stroke, and heart failure. Multivariable Cox proportional hazards model was used to evaluate the relationship between outcomes and different treatments.ResultsOverall, 4311 patients in A + B group and 10980 patients in A + V group were included. After a mean follow-up of 4.34 +/- 1.79 years, the efficacy outcomes were similar between the A + V and A + B groups regarding all-cause death (adjusted hazard ratio [aHR] 0.99, 95% confidence interval [CI] 0.83-1.18), ASCVD event (aHR 0.97, 95% CI 0.84-1.12), and heart failure (aHR 1.06, 95% CI 0.87-1.30). The risk of hemorrhagic stroke was lower in A + B group (aHR 0.70, 95% CI 0.52-0.94). The result was similar when taking death into consideration in competing risk analysis. The safety outcomes were similar between the 2 groups.ConclusionsThere was no difference of all-cause death, ASCVD event, and heart failure in A + B vs. A + V users. But A + B users had a lower risk of hemorrhagic stroke.

Background Fas-associated factor 1 (FAF1) is a multidomain protein that interacts with diverse partners to affect numerous cellular processes. Previously, we discovered two Small Ubiquitin-like Modifier (SUMO)-interacting motifs (SIMs) within FAF1 that are crucial for transcriptional modulation of mineralocorticoid receptor. Recently, we identified Sin3A-associated protein 130 (SAP130), a putative sumoylated protein, as a candidate FAF1 interaction partner by yeast two-hybrid screening. However, it remained unclear whether SAP130 sumoylation might occur and functionally interact with FAF1. Results In this study, we first show that SAP130 can be modified by SUMO1 at Lys residues 794, 878 and 932 both in vitro and in vivo. Mutation of these three SUMO-accepting Lys residues to Ala had no impact on SAP130 association with Sin3A or its nuclear localization, but the mutations abrogated the association of SAP130 with the FAF1. The mutations also potentiated SAP130 trans-repression activity and attenuated SAP130-mediated promotion of cell growth. Additionally, SUMO1-modified SAP130 was less stable than unmodified SAP130. Transient transfection experiments further revealed that FAF1 mitigated the trans-repression and cell proliferation-promoting functions of SAP130, and promoted SAP130 degradation by enhancing its polyubiquitination in a sumoylation-dependent manner. Conclusions Together, these results demonstrate that sumoylation of SAP130 regulates its biological functions and that FAF1 plays a crucial role in controlling the SUMO-dependent regulation of transcriptional activity and protein stability of SAP130.

Background This study evaluated the utility of using Accreditation Council for Graduate Medical Education (ACGME) Milestones as a formative assessment tool for the fifth- and sixth-grade medical students’ performance in their internal medicine (IM) clerkship and the same students’ performance in their post-graduate year (PGY) IM training. Methods Retrospective data were collected from 65 medical students completing the two-year IM clerkship in the academic years 2019 and 2020 and 26 of the above students completing their PGY-1 training at the same university hospital in the academic year 2021. Data included the assessment results of 7 of the ACGME IM Milestones, information on admitted patients assigned to the students, and surveys of the students’ satisfaction. Results The analysis included 390 assessment results during the IM clerkship and 78 assessment results during the PGY-1 training. Clinical teachers commonly rated level 3 to medical students in the IM clerkship, with PC-2 subcompetency receiving the lowest rating among seven subcompetencies. The levels of most subcompetencies showed stationary in the two-year IM clerkship. Significant improvement was observed in all subcompetencies during the PGY-1 training. The medical students in the second-year IM clerkship expressed higher satisfaction with implementing Milestones than in their first-year IM clerkship and perceived Milestones assessments’ usefulness as learning feedback. Conclusions Using ACGME Milestones as a formative assessment tool in the IM clerkship yielded promising outcomes. Longitudinal follow-up of subcompetencies facilitated tracking students’ development and providing constructive feedback.

Endometrial and ovarian cancers are both fearsome gynecological diseases with high mortality rates worldwide. Due to their histological similarity, it is still a clinical challenge to distinguish between double primary cancer (DPC) and metastasis cancer (MC) in synchronous endometrial and ovarian cancers in which the latter represents a poor prognostic outcome. Clinically, the standard procedure for examining DPC and MC relies on their morphology and histopathology; however, showing poor accuracy even under explicating criteria. Here, we attempt to utilize indel variants within polyguanine (polyG) sequences to generate genetic distances of synchronous endometrial and ovarian tumors to normal tissue in a patient and then calculate the correlation coefficient for them. Polyguanine sequences are hypermutable guanine repeats that acquire insertion or deletion at a high incident rate (mutation rate: ~10-6 - 10-4; ~1000 folds higher than unique sequence) and, therefore, could serve as a very rich resource for comparing the genetic similarity between samples. The correlation between selected clinical variables, e.g., disease-free survival and the described correlation coefficient is then calculated to test whether this protocol can serve as an optimal tool for distinguishing between DPC and MC. We have assembled a cohort of 39 patients with synchronous endometrial and ovarian cancers in which pathologists cannot determine the relationship between endometrial and ovarian cancer in 23 of them. Preliminarily, we have collected DNA from formalin-fixed paraffin-embedded (FFPE) specimens and completed polyG genotyping for four of them. Indel variants, both insertions and deletions, are rich in all cases indicating the approach is technically rigid for this purpose even with FFPE specimens. Subsequently, we evaluate the similarity between endometrial and ovarian cancer by calculating Spearman's correlation with the genetic distances obtained from polyG genotyping. As the result of preliminary cases, we observe the correlation coefficient spreading between -0.14 to 0.95, implying this methodology can determine the genetic similarity between endometrial and ovarian tumor in a case and, therefore, could be a potential tool to supplement the current observation-based approach in the clinic. Our next step is to correlate the correlation coefficients with meaningful clinical variables once we complete genotyping the entire cohort. We anticipate this approach could provide additional information to distinguish DPC and MC and therefore benefit the patients. Citation Format: Yu-Yi Huang, Pei-Chi Cheng, Ching Hsu, Yung-Ming Jeng, Lin-Hung Wei, Wei-Ting Hung. Determine the genetic similarity of synchronous endometrial and ovarian cancers with variants in polyguanine sequences [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 129.

Supplementary Figure 1 from IL-6 Trans-Signaling in Formation and Progression of Malignant Ascites in Ovarian Cancer

PHRF1 is an E3 ligase that promotes TGF-β signaling by ubiquitinating a homeodomain repressor TG-interacting factor (TGIF). The suppression of PHRF1 activity by PML-RARα facilitates the progression of acute promyelocytic leukemia (APL). PHRF1 also contributes to non-homologous end-joining in response to DNA damage by linking H3K36me3 and NBS1 with DNA repair machinery. However, its role in class switch recombination (CSR) is not well understood. In this study, we report the importance of PHRF1 in IgA switching in CH12F3-2A cells and CD19-Cre mice. Our studies revealed that Crispr-Cas9 mediated PHRF1 knockout and shRNA-silenced CH12F3-2A cells reduced IgA production, as well as decreased the amounts of PARP1, NELF-A, and NELF-D. The introduction of PARP1 could partially restore IgA production in PHRF1 knockout cells. Intriguingly, IgA, as well as IgG1, IgG2a, and IgG3, switchings were not significantly decreased in PHRF1 deficient splenic B lymphocytes isolated from CD19-Cre mice. The levels of PARP1 and NELF-D were not decreased in PHRF1-depleted primary splenic B cells. Overall, our findings suggest that PHRF1 may modulate IgA switching in CH12F3-2A cells.

Supplementary Figure 3 from IL-6 Trans-Signaling in Formation and Progression of Malignant Ascites in Ovarian Cancer

Human PUF-A/PUM3 is a RNA and DNA binding protein participating in the nucleolar processing of 7S to 5.8S rRNA. The nucleolar localization of PUF-A redistributes to the nucleoplasm upon the exposure to genotoxic agents in cells. However, little is known regarding the roles of PUF-A in tumor progression. Phosphoprotein database analysis revealed that Y259 phosphorylation of PUF-A is the most prevalent residue modified. Here, we reported the importance of PUF-A’s phosphorylation on Y259 in tumorigenesis. PUF-A gene was knocked out by the Crispr/Cas9 method in human cervix epithelial HeLa cells. Loss of PUF-A in HeLa cells resulted in reduced clonogenic and lower transwell invasion capacity. Introduction of PUF-AY259F to PUF-A deficient HeLa cells was unable to restore colony formation. In addition, the unphosphorylated mutant of PUF-A, PUF-AY259F, attenuated PUF-A protein stability. Our results suggest the important role of Y259 phosphorylation of PUF-A in cell proliferation.

Purpose: We aim to elicit views from individuals at different levels of a medical hierarchy on the effects of the dury hour reform (DHR) on medical education and patient care. We also address the changes in the summative assessment of learning outcomes after the DHR. Methods: We conducted surveys of attending physicians from the Department of Internal Medicine, postgraduate year (PGY) residents, and clerks. Rates of prolonged hospitalization ＞ 30 days, 2-week readmissions, and revisits to the emergency room within 3 days of discharge before and after the DHR were checked. The subcompetence levels of reporting milestones of PGY residents before and after the DHR were compared. Results: More than half of the attending physicians were concerned about declines in the residents' development of skill (66.7%), knowledge (55.6%), and overall achievement (63%). However, none of the PGY residents considered the DHR to have a negative impact on learning. There were no significant declines in the perceptions of medical students on residents' teaching performance after the DHR or on the rates of readmission, emergency room revisits, and prolonged hospitalization. Significantly lower levels of milestones assessment scores in 17 out of 22 subcompetencies were noted after the DHR. Conclusions: A DHR has no significant impact on inpatient care and clerk education. Different from the perceptions of residents, both the assessments of milestones and the perceptions of attending physicians indicated a decline in learning outcomes after the DHR.

Statins possess neuroprotective effects. However, real-world evidence supporting their utility in post-stroke epilepsy (PSE) prevention is limited.