Citations

Background: Exogenous and endogenous polyamines are critical for gastrointestinal (GI) barrier integrity, of which putrescine, spermidine and spermine are most well studied. Our recent work shows n-acetylputrescine, a putrescine derivative, correlates with increased intestinal permeability and loss of microbial diversity. Despite this, bacterial mechanisms of n-acetylputrescine production or direct effects on barrier integrity are unknown. Aim: Determine the role of microbiota in shaping n-acetylputrescine abundance and the effect on barrier integrity. Methods: N-acetylputrescine effects on barrier were assessed both in vitro using Caco-2 and ex vivo in Ussing using colonic strips from conventional mice. Germ-free (GF) mice were monocolonized with wild-type E. coli (SK929) or E.coli SK930 (2-weeks, n=4mice/group), strains given by Dr. Shin Kurihara. E. coli SK930 is deficient in SpeC, SpeF and SpeB, genes generating putrescine, required for n-acetylputrescine production. Untargeted metabolomics (Metabolon) was performed on fecal pellets. Targeted isotope tracing with [13C6, 15N4]-arginine delivered via enema was done to track bacterial production of n-acetylputrescine by Fusobacterium varium, a known producer, in vivo with isotopolog labeling measured by LC/HRMS. Results: In vitro n-acetylputrescine (100mM) addition to Caco-2 monolayers caused significant barrier disruption compared to controls (%Baseline TER, 13.7±6.7 vs 102.8±9.4, 3h postexposure, n=3 p=0.0002), not seen at lower (10mM) concentration. N-acetylputrescine caused increased flux of 300Da fluorescein (12.6±2.0 vs 6.0±0.2 mg/mL, n=3 p=0.004) and 4kDa FITC-Dextran flux on Ussing compared to untreated controls (999±94 vs 562±121 ng/hr/cm2, n=3 p= 0.01). Mutant E. coli SK930 monocolonized mice had similar fecal n-acetylputrescine to GF animals. In contrast, wild type E. coli SK929 monocolonization led to significantly greater n-acetylputrescine (5-fold, FDR< 0.001) compared to SK930, indicating a bulk of n-acetylputrescine production is driven by bacterial mechanisms. Pathway enrichment analysis predicted decreased spermidine and spermine biosynthesis in SK930 monocolonization, indicative of impaired polyamine metabolism, compared to SK929 (FDR< 0.01). Targeted metabolomics on fecal pellets showed a shift in the production of isotopolog [13C1, 15N0]-N-acetylputrescine to increased isotopolog [13C4, 15N2]-N-acetylputrescine concentration after monocolonization with F. varium compared to GF mice (5.9±5.0 vs. 2.1±3.6 pg/mg stool, n=3mice/group p=0.05) suggesting microbiota driven shifts in n-acetylputrescine metabolism. Conclusion: Luminal n-acetylputrescine production and profile is driven by bacteria in the GI tract. Concentration dependent effects of n-acetylputrescine suggest insufficient regulation by intestinal microbiota can lead to accumulation of n-acetylputrescine and the intestinal barrier disruption seen in human studies. Ongoing studies aim to identify bacterial taxa critical for n-acetylputrescine suppression, the mechanisms involved, and impact on GI health. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Abstract Background: Up to 50% of patients with breast cancer experience chemotherapy-inducednausea (CIN) and 95% experience fatigue. A comprehensive understanding of biological mechanisms remains elusive. Herein, we evaluated the association of CIN and fatigue with clinical and multi-omics features, including serum and stool metabolites before (T1) to after chemotherapy (T2). Methods: Patients with breast cancer (n=36) who were to receive chemotherapy, provided symptom assessment using the Memorial Symptom Assessment Scale, stool and blood samples at T1 and T2. Patients provided dietary intake data using a Dietary Screener Questionnaire at T1. Descriptive and univariate analyses evaluated associations of co-occurringsymptoms and diet with CIN and fatigue at T2. Gut microbiome was sequenced using shotgun metagenomics. Targeted metabolomics was performed on stool and serum. Shift in the abundance of taxonomic groups and metabolite levels from T1 to T2 were evaluated in association with CIN and fatigue. Results: Approximately 64% of the patients experienced CIN and 94% experienced fatigue atT2. Three gastrointestinal symptoms co-occurred with CIN and fatigue. Higher daily average intake of calcium (p=0.013) and dairy (p=0.035) were associated with CIN. Levels of hexosylceramides, ceramide, phosphatidylcholine and sphingomyelins were significantly perturbed in association with CIN and fatigue (p&lt;0.05). Conclusion: CIN and fatigue are associated with perturbation in the sphingolipid turnover pathway, which negatively impacts functions in the gut-liver axis that include apoptosis activity and change in bile acid levels. Confirmatory studies are warranted to support the development of interventions to alleviate CIN and fatigue in patients with breast cancer receiving chemotherapy.Keywords: breast cancer; chemotherapy-induced nausea; fatigue; metabolites; sphingolipids Citation Format: Komal Preet Singh, Brenda J. Ernst, Jun Chen, Surendra Dasari, Felipe Batalini, Cindy Tofthagen, Kathryn J. Ruddy, Keenan Pituch, Linda Chlan. Perturbations in gut liver axis functions are associated with chemotherapy induced nausea and fatigue in patients with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6309.

Objective To estimate the prevalence, incidence, and disease burden of autoimmune seizures and epilepsies in Olmsted County. Methods We conducted a retrospective, population‐based study using the Rochester Epidemiology Project (1996–2019). Age‐ and sex‐standardized rates (2019 United States standard population) per 100,000 were calculated with 95% confidence intervals (95% CIs). Temporal trends in incidence were assessed over 3 consecutive 8‐year intervals (1996–2003, 2004–2011, and 2012–2019) using Poisson regression to estimate rate ratios per period and trend. Disability‐adjusted life years (DALYs), years of life lost (YLL), and years lived with disability (YLD) and 95% uncertainty intervals (95% UIs) were estimated for 2019. Results Twenty‐three patients were identified between 1996 and 2019. In 2019, the standardized prevalence of all autoimmune seizures and epilepsies was 9.81 (95% CI, 5.59–15.21) per 100,000, including autoimmune‐associated epilepsy (AAE) 7.27 (3.74–11.95) and acute symptomatic seizures secondary to autoimmune encephalitis (ASSAE) 2.55 (0.69–5.60). From 1996 to 2019, the standardized incidence was 0.50 (0.29–0.77) per 100,000 person‐years overall, with AAE 0.36 (0.19–0.59) and ASSAE 0.15 (0.05–0.30). By period, the standardized incidence of all autoimmune seizures and epilepsies suggested a rising trend from 0.28 (95% CI, 0.06–0.68) in 1996 to 2003 to 0.60 (95% CI, 0.24–1.12) in 2004–2011, then plateaued at 0.53 (95% CI, 0.21–1.00) in 2012 to 2019 (rate ratio per 8‐year interval 1.30, 95% CI, 0.71–2.38), largely driven by the seropositive subgroup. The standardized DALY rate was 26.27 (95% Uncertainly intervals [UI], 5.30–67.09) per 100,000 (YLL 20.07 [0.00–60.21] and YLD 6.20 [5.06–7.39]). Interpretation Although autoimmune seizures and epilepsies remain rare, they result in a disproportionately high disease burden. ANN NEUROL 2026;100:74–83

Abstract Objectives Sarcomatoid urothelial carcinoma (sUC) is associated with poor clinical outcomes. Herein, we evaluated clinicopathologic features (including PD-L1 expression) for institutional cases. Methods Sixty-five patients with sUC treated by radical cystectomy were matched for age, sex, and pathologic stage to 190 patients with pure urothelial carcinoma (UC). Outcomes were evaluated using the Kaplan-Meier method. Immunohistochemistry (IHC) results for tumor-specific PD-L1 expression in sUC were quantified using H-scores (range, 0-300). Cases with constitutive PD-L1 expression (cPD-L1; H-score ≥240) were evaluated for amplification events using fluorescence in situ hybridization (n = 28), mate-pair sequencing (Mpseq; n = 6), RNA sequencing (n = 5), and targeted next-generation sequencing (n = 8). PD-L1 regulatory pathways were evaluated using gene set enrichment analysis in the Cancer Hallmark dataset. Results Most patients with sUC had advanced disease (≥pT2, 62/65, 95%), without significant differences in overall and cancer-specific survival, when matched to pure UC. Median PD-L1 H-scores using different clones were 100 (SP142), 120 (SP263), and 195 (22C3). cPD-L1 was seen in 43% (28/65) of cases of sUC that were enriched for TP53, TERT, and CDKN2A/B alterations. PD-L1 amplification was identified in 7 (of 28, 25%) of these cases. Bionformatics analysis consistently identified IL6-JAK-STAT3 and interferon α/γ signaling in sUC with cPD-L1 expression. Conclusions Our results suggest that cPD-L1 expression in most sUC occurs secondary to IL6-JAK-STAT3 and interferon α/γ signaling. This provides a biologic rationale for evaluating response to immunotherapy in this patient population.

Abstract Background Oxidative phosphorylation (OXPHOS) is a central function and a key indicator of mitochondrial fitness, yet studies in human tissue remain limited. Inclusion body myositis (IBM) is a progressive myopathy that lies at the intersection of aging, inflammation and mitochondrial dysfunction. We aimed to perform a comprehensive profiling of mitochondrial respiration in muscle tissue from patients with IBM. Methods A wide battery of complementary tests from RNA level to high-resolution respirometry on permeabilized muscle fibers was performed. The relationship between respiration, mitochondrial content, mitochondrial DNA (mtDNA) abnormalities and mitophagy was examined, along with the correlation with various clinical parameters to determine the clinical significance of the findings. Results The study included a total of 67 patients with IBM and 45 controls. IBM muscle tissue exhibited reduced maximal respiration per tissue weight in State 3 (high substrates, high ADP) and uncoupled state with decreased coupling efficiency and higher leak control ratios. When adjusting for citrate synthase reflecting mitochondrial content, males had decreased State 3 intrinsic respiration, whereas females had greater intrinsic respiration in leak states. Complex II control ratio strongly correlated with disease duration and severity only in females. IBM was associated with decreased RNA and protein expression of OXPHOS complexes. Complex I activity was decreased mainly in females. IBM samples exhibited lower maximal H 2 O 2 emission, accompanied by a higher total antioxidant capacity that correlated with disease duration in females. In IBM, there was decreased mtDNA content, and impaired mitophagy, both of which strongly correlated with respirometry measures and markers of disease severity, indicating these pathways are likely interconnected and of clinical significance. Conclusion IBM is characterized by multilevel impairments in mitochondrial coupling efficiency, revealing several potential therapeutic targets to improve mitochondrial fitness, while accounting for sex-specific differences.

Abstract Diffuse hemispheric glioma, H3 G34‐mutant (DHG‐H3 G34) has been primarily molecularly characterized by methylation profiling and sequencing studies. We describe an integrated histomolecular evaluation including high‐resolution copy number profiling of a series of 60 DHG‐H3 G34 to further our understanding of the spectrum of genetic changes associated with this tumor type. Cases were clinically tested using an 187‐gene mutation and fusion targeted neuro‐oncology next‐generation sequencing panel ( n = 60) and Oncoscan chromosomal microarray ( n = 26) by a single laboratory (2018–2022). A subset of cases had immunohistochemical results for OLIG2 ( n = 42), p53 ( n = 48), and ATRX ( n = 46), and methylation array data ( n = 8). Median age at testing was 21 years (range, 12–50). No significant difference was noted in clinical, histopathological, and mutational profile between pediatric and adult patients. H3‐3A G34 mutations included G34R ( n = 56; 94%), G34V ( n = 3), and a non‐canonical G34E ( n = 1). Concurrent mutations most often involved TP53 ( n = 55; 92%), ATRX ( n = 50; 83%), and PDGFRA ( n = 34; 57%). A reportedly primary tumor was confirmed to be hypermutant and had a PMS2 mutation. A single case also showed an FGFR3::FAM184B fusion. All cases with available chromosomal microarray data had unbalanced genomes, which were often complex (14/26; 54%). The most frequent recurrent copy number abnormalities were losses involving 3q, 4q, 10q, 13q, and 18q, and 17p copy‐neutral loss of heterozygosity (cnLOH) encompassing TP53 . This copy number profile was reminiscent of that seen in Grade 4 IDH‐mutant astrocytomas. Collectively, a TP53 abnormality at copy number (12/26, all cnLOH), sequence (55/60) and protein expression (46/48) level was detected in all 60 cases. In conclusion, integrated high‐resolution copy number and histomolecular analysis expanded the spectrum of genetic changes associated with DHG‐H3 G34, including the presence of universal TP53 abnormalities with frequent cnLOH—a copy number abnormality that has been largely unrecognized—for this new 2021 World Health Organization central nervous system tumor type.

Abstract Introduction In 2018, our institution implemented a MALDI-TOF mass spectrometry (MS) method (Mass-Fix) to detect myeloma associated M-proteins in blood. Currently, the lab has performed more than 500K tests by Mass-Fix. While Mass-Fix has been found to be more sensitive and specific than IFE, an ESI-Q-TOF (High Resolution Mass-Fix) method has demonstrated the potential to detect M-proteins at significantly lower levels. Blood minimal residual disease (MRD) detection in lieu of bone marrow (BM) is desirable since blood is easier and less expensive to obtain and analyze. This feature can allow for more frequent MRD monitoring for myeloma patients. Methods 66 residual blood samples from myeloma patients with varying M-protein varying isotypes were diluted into healthy blood to estimate the limit of detection of HR-Mass-Fix method. 179 Rochester Mayo Clinic patients who had a clinically ordered BM MRD assessment by next generation flow (NGF) cytometry (2 x 10-6), a blood sample available within 7 days of the BM biopsy, and a pre-treatment measured M-protein mass by Mass-Fix were utilized to assess the concordance of the BM and blood MRD status. Results The average limit of detection (LOD) of the HR-Mass-Fix was found to be significantly lower than Mass-Fix (0.0003 g/dL and 0.01 g/dL, respectively). The treated patient cohort utilized for comparison consisted of 145 myeloma, 21 AL amyloid, 4 LPL lymphoma and 9 other PCDs. Comparing the Mass-Fix and HR-Mass-Fix results, 100% of blood positive MRD results by Mass-Fix were positive by HR-Mass-Fix. HR-Mass-Fix detected residual M-proteins (mass within +/- 1 Da of pre-treatment) in 29 samples (40%) that were negative by Mass-Fix. Thus, HR-Mass-Fix increased the overall blood MRD positivity rate from 57% by Mass-Fix to 78%. The BM NGF MRD positivity was lower at 54%. The overall agreement of blood and BM by Mass-Fix was 71% (kappa 0.4) which decreased to 0.63 (kappa 0.3) with HR-Mass-Fix. Most of the disagreements were HR-Mass-Fix Blood (+)/BM NGF (-). Blood M-protein MRD appeared to be affected by M-protein half-lives. To determine this, the M-protein isotype distribution of Blood/BM MRD status was compared the M-protein isotype distribution of the overall cohort. The half-lives are as follows: IgG 23 days, IgA 6 days, IgM 5 days and FLC 0.25 days. When both blood and BM were positive, the isotype distribution was not significantly different than the overall cohort. For blood(+)/BM(-) patients, IgG (with the longest half-life) were significantly overrepresented compared to the overall cohort (74% vs 51%) . For blood(-)/BM(+) cases, there were no IgG M-proteins, and the majority were FLC patients. These results suggest that patients with shorter half-life M-proteins (non-IgG) are more likely to be Blood MRD(-) than patients with longer half-life M-proteins. M-protein rate changes over time will likely be needed to resolve the M-protein half- life affect. Conclusions HR-Mass-Fix can significantly increase the detection of M-proteins in comparison to our current Mass-Fix method. Ig recycling was found to be a likely reason for the disagreement between BM and blood. Therefore, a negative blood MRD status by HR-Mass-Fix is most likely to be the most significant in predicting patient outcomes.

Abstract Introduction Histiocytic neoplasms (HN) are a rare, heterogeneous group of hematologic disorders derived from myeloid-dendritic cell lineages. The most common forms include Langerhans cell histiocytosis (LCH), Erdheim-Chester disease (ECD), and Rosai-Dorfman disease (RDD). These disorders are driven by recurrent activating mutations in the MAPK pathway, most frequently involving BRAF, MAP2K1, PIK3CA, KRAS, and NRAS. This discovery enabled a major therapeutic advance with the use of BRAF and MEK inhibitors, resulting in durable responses. For patients harboring KRAS mutations, MEK inhibitors (MEKi) remain the primary therapeutic approach. However, the impact of KRAS mutation subtypes on treatment response remains poorly understood. Our study aimed to characterize the spectrum of KRAS mutations in LCH, ECD, and RDD and evaluate the responses to MEKi, providing insights to guide future treatment strategies. Methods We conducted a retrospective review of patients with LCH, ECD, and RDD who were evaluated at Mayo Clinic or University of Alabama at Birmingham. Patients were included if next-generation sequencing identified a KRAS mutation. The response assessment was based on radiographic evaluation under the most recent Histiocyte Society consensus guideline. Results We identified 23 patients with a KRAS mutation: 17 females (73.9%) and 6 males (26.1%), with a median age at diagnosis of 61 years (range, 20–78). RDD accounted for 73.9% (17/23) and ECD for 26.1% (6/23); no KRAS mutations were detected in LCH patients. The most frequent KRAS variants were p.K117N (34.8%), p.G12D (17.4%), p.A146T (13.0%), p.G13C (8.7%), and p.G13D (8.7%), with additional variants including p.T58I, p.A59G, p.A146P, and p.A146V. Mutation distribution differed by disease group: p.G12D was more common in ECD (50%) than RDD (5.9%) (p=0.01), whereas p.K117N occurred in 47.1% of RDD and was absent in ECD. Regarding treatment, 57% (13/23) received MEKi, all initially cobimetinib, with 1 patient switched to trametinib due to progression. Of the 10 patients who did not receive MEKi, 1 was treated with vemurafenib for a concurrent BRAF p.V600E mutation; 3 remained on observation; 2 underwent surgery without recurrence; 3 received other systemic therapies including corticosteroid, methotrexate, rituximab, lenalidomide, or sirolimus; and 1 developed diffuse large B-cell lymphoma, treated with R-CHOP. The overall response rate in the 12 patients on MEKi (excluding 1 patient too early for assessment) was 75%—3 (25%) CR and 6 (50%) PR (including 1 who initially progressed on cobimetinib but responded to trametinib). All 3 non-responders had RDD. Among the 5 patients with p.K117N, 3 had no response to cobimetinib, 1 achieved PR, and 1 achieved CR; notably, 1 non-responder switched to trametinib and achieved PR in 2 months. All 3 patients with codon 146 mutations achieved PR. Of the 2 patients with p.G12D, 1 achieved CR with a co-occurring MAP2K1 mutation, and 1 had no response. No additional genotype–response patterns were observed. Similar heterogeneity has been reported in prior studies. In a phase II cobimetinib trial, an ECD patient with p.R149G achieved PR, whereas another ECD patient with p.G12R and an LCH patient with p.G13C achieved CR in the presence of co-occurring ARAF and NRAS mutations, respectively. A retrospective cohort described an ECD patient with p.K117N achieving PR on dabrafenib and trametinib with a concurrent BRAF mutation and another with p.G12A achieving PR on trametinib. In a multicenter analysis of trametinib, a patient with p.A59T (co-occurring BRAF and NRAS mutations) had no response, whereas another with p.A146P achieved PR. Collectively, these findings, together with our data, underscore that KRAS-mutated histiocytoses exhibit heterogeneous responses to MEKi, likely influenced by variant type and co-mutations. Discussion KRAS mutations represent an important subset of MAPK pathway alterations in HN, although their prevalence varies by disease type and appears extremely rare in LCH, with no cases identified in our cohort. The most common variants include p.K117N and p.G12D, which may be linked to suboptimal response to MEKi in the absence of concurrent MAPK pathway alterations. As MEKi acts downstream, efficacy likely depends on the level of pathway activation conferred by specific KRAS variants. Larger studies are needed to confirm these observations and identify mutation-specific predictors to guide personalized treatment.