Molecular Stratification Research Articles

Clinical Subtype Trajectories in Sepsis Patients Admitted to the ICU: A Secondary Analysis of an Observational Study.

Sepsis is an evolving process and proposed subtypes may change over time. We hypothesized that previously established sepsis subtypes are dynamic, prognostic of outcome, and trajectories are associated with host response alterations. A secondary analysis of two observational critically ill sepsis cohorts: the Molecular diAgnosis and Risk stratification of Sepsis (MARS) and the Medical Information Mart for Intensive Care-IV (MIMIC-IV). ICUs in the Netherlands and United States between 2011-2014 and 2008-2019, respectively. Patient admission fulfilling the Sepsis-3 criteria upon ICU admission adjudicated to one of four previously identified subtypes, comprising 2,416 admissions in MARS and 10,745 in MIMIC-IV. Subtype stability and the changes per subtype on days 2, 4 and 7 of ICU admission were assessed. Next, the associated between change in clinical subtype and outcome and host response alterations. In MARS, upon ICU admission, 6% (n = 150) of the patient admissions were α-type, 3% (n = 70) β-type, 55% (n = 1317) γ-type, and 36% (n = 879) δ-type; in MIMIC-IV, this was α = 22% (n = 2398), β = 22% (n = 2365), γ = 31% (n = 3296), and δ = 25% (2686). Overall, prevalence of subtypes was stable over days 2, 4, and 7. However, 28-56% (MARS/MIMIC-IV) changed from α on ICU admission to any of the other subtypes on day 2, 33-71% from β, 57-32% from γ, and 50-48% from δ. On day 4, overall subtype persistence was 33-36%. γ or δ admissions remaining in, or transitioning to, subtype γ on days 2, 4, and 7 exhibited lower mortality rates compared with those remaining in, or transitioning to, subtype δ. Longitudinal host response biomarkers reflecting inflammation, coagulation, and endothelial dysfunction were most altered in the δ-δ group, followed by the γ-δ group, independent of the day or biomarker domain. In two large cohorts, subtype change to δ was associated with worse clinical outcome and more aberrant biomarkers reflecting inflammation, coagulation, and endothelial dysfunction. These findings underscore the importance of monitoring sepsis subtypes and their linked host responses for improved prognostication and personalized treatment strategies.

Abstract A062: Insightful analysis: Harnessing aqueous humor ctDNA for retinoblastoma stratification

Abstract Retinoblastoma (RB) the most common intraocular malignancy in children poses a significant challenge as traditional tissue biopsies are not feasible. Due to its critical location within the eye and the high risk of tumor seeding, clinicians are limited in performing molecular analysis-based risk stratification prior to enucleation. In this study, we explored the potential of aqueous humor (AH) as a source for minimally invasive liquid biopsies, aiming to establish a molecular profiling methodology for circulating tumor DNA (ctDNA) for risk stratification. We analyzed AH samples and corresponding tumor tissues from 19 enucleated eyes of patients diagnosed with RB. The extracted ctDNA from AH underwent low-coverage whole-genome sequencing (lcWGS) and methylation profiling. These data were then compared with the genomic and methylation landscapes of the matched tumor DNA. Additionally, we assessed the ability of AH- derived ctDNA to predict retinoblastoma methylation cluster. Our findings demonstrate a high concordance between ctDNA from AH and the primary tumor DNA in both methylation patterns and genomic alterations detected by lcWGS allowing the correct prediction of the retinoblastoma molecular group. The shared aberrations underscore the potential of AH as a reliable surrogate for tumor tissue in retinoblastoma. Additionally, the methylation profiles revealed consistent epigenetic modifications, further supporting the accuracy of AH-derived ctDNA in reflecting the tumor’s molecular characteristics. This study highlights the feasibility and effectiveness of using AH for molecular ctDNA genetic analysis in retinoblastoma. The overall high concordance between ctDNA profiles with primary tumor DNA and the ability to accurately predict RB methylation classes, highlights the superiority of the liquid biopsy approach overcoming the limitations of traditional biopsies and transforming the potential for early molecular analysis and risk stratification. This revolutionary technique could redefine pre-enucleation diagnostics, offering unprecedented insights and paving the way for tailored, precision medicine in retinoblastoma care. Citation Format: Sophia H Montigel, Tatsiana Ryl, Stefanie Volz, Elena Afanasyeva, Tatjana Wedig, Nathalie Schwarz, Stefan M Pfister, Kristian W Pajtler, Petra Ketteler, Kendra K Maaß. Insightful analysis: Harnessing aqueous humor ctDNA for retinoblastoma stratification [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A062.

Abstract PR009: Application of Liquid Biopsy-RECIST (LB-RECIST) criteria to track metastatic colorectal cancer (mCRC) molecular response in first-line (1L) setting: Findings from the PLATFORM-B study

Abstract Introduction: Circulating tumor DNA (ctDNA) analysis is a minimally invasive and reproducible method to explore tumor molecular biology. Recently, Gouda et al. proposed the LB-RECIST criteria to monitor molecular response, validating them on a heterogeneous metastatic population. However, data on mCRC are lacking. Here, we report the first attempt to apply LB-RECIST in the multicenter prospective PLATFORM-B study, which enrolled 130 mCRC patients (pts) receiving 1L standard therapy, with ctDNA analysis performed at different time points. Materials and Methods: Baseline (BL) and week 8 (W8) plasma samples were analyzed using a targeted NGS-based approach (Ion S5 system). Changes in ctDNA detectability were assessed to determine molecular response as per qualitative LB-RECIST criteria: Group1 (G1, detectable ctDNA at BL and W8); Group2 (G2, detectable BL and undetectable W8); Group3 (G3, undetectable BL and detectable W8); Group4 (G4, undetectable at BL and W8). Changes in aggregate variant allele frequency (VAF) - the sum of all VAFs of the studied genes in a sample - were used to assess quantitative LB-RECIST responses: ctDNA Complete Response (CCR, clearance after initial detectability), Partial Response (CPR, decrease >10%), Stable Disease (CSD, no change or decrease <10%), Progressive Disease (CPD, increase >10% or new mutations), and Non-measurable Disease (CND, undetectable at BL and W8). Molecular results were correlated with clinical outcomes. Statistically significant differences were assessed across different groups using 2-sided P values with an α ≤ .05 level of significance. Results: Overall, 79 pts had paired BL and W8 samples. Quantitative LB-RECIST identified 32 CCR, 18 CND, 20 CPR, 8 CPD, and 1 CSD cases. Qualitative LB-RECIST found 22 G1, 32 G2, 7 G3, and 18 G4 cases. Radiological RECIST identified 6 CR, 47 PR, 21 SD, and 5 PD. CCR showed the highest survival rates: with a median follow-up of 25 months (mo) (range: 1–49), median progression-free survival (mPFS) was not reached (NR) and median overall survival (mOS) was 41.8 mo. The CND group had similar rates (mPFS NR; mOS 41.1 mo). CPR and CPD had the poorest outcomes: for CPR, mPFS was 12.7 and mOS 16.4 mo; for CPD, mPFS and mOS were 11.9 and 25.5 mo, respectively. In the G1, G2, G3, and G4 groups, mPFS was 11.9, NR, 14.2, and NR, respectively, while mOS was 16.4, 41.8, 33.4, and 41.1 mo. Kaplan-Meier analysis of the quantitative groups showed statistically significant differences (P < .0001) for both PFS and OS curves. The same level of significance was found in the qualitative groups. Conclusion: LB- RECIST can be a rapid and reproducible method to assess molecular response in mCRC. However, concerns remain. The CND group (23% of cases) showed survival rates similar to the CCR group, as observed also between G4 and G2. These data suggest that absence of ctDNA at W8 is the best surrogate for OS. Moreover, CPD had higher OS rates than CPR, but similar PFS. Further investigations in larger populations are needed to refine these criteria and improve molecular response stratification Citation Format: Valentino Martelli, Joana Vidal, Maria Concepción Fernández-Rodríguez, Pilar García-Alfonso, David Páez, Joan Gibert, Jennifer Linares, Annarita Sibilio, Vicente Alonso, Maria Teresa Cano, Cristina Santos, Gema Duran, Elena Élez, José Luis Manzano, Rocio Garcia-Carbonero, Reyes Ferreiro, Ferran Losa, Estela Pineda, Javier Sastre, Auxiliadora Gomez-España, Rodrigo Toledo, Fernando Rivera, Beatriz Bellosillo, Josep Tabernero, Ramon Ramon Salazar, Enrique Aranda, Clara Montagut. Application of Liquid Biopsy-RECIST (LB-RECIST) criteria to track metastatic colorectal cancer (mCRC) molecular response in first-line (1L) setting: Findings from the PLATFORM-B study [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR009.

Abstract 4137646: Transcriptomic Profiling of Human Myocardium at Sudden Death to Define Vulnerable Substrate for Lethal Arrhythmias

Background: While some chronic pathological substrates for sudden cardiac death (SCD) are well-known (e.g., coronary disease and left ventricular [LV] dysfunction), the vulnerable myocardial state predisposing to fatal arrhythmia remains a critical barrier to near-term SCD prevention. Hypothesis: Myocardium of autopsy-defined SCDs exhibit distinct expression profiles vs. non-cardiac sudden deaths and trauma deaths that reflect its acute vulnerable state in the hours to days before SCD, as expression arrests upon death. Goals/Aims: Define the vulnerable myocardial substrate for lethal arrhythmia by targeted RNA profiling. Methods: We used autopsy to adjudicate arrhythmic from non-arrhythmic causes in 1,265 sudden deaths in San Francisco from 2011-2018. We performed a transcriptomic evaluation of LV sampled at the time of SCD from 245 consented cases using a curated panel of 448 genes with known or hypothesized association with SCD. Results: Comparison between Arrhythmic (n=129) and Non-Arrhythmic (n=116) cohorts revealed 31 differentially upregulated and 36 downregulated genes (p-adj<0.05), related to collagen-containing extracellular matrix (upregulation of FAP , FMOD , LTBP2 ), ion transport (upregulation of KCNA5 and KCNN3 , and downregulation of KCNJ8, KCNK1, KCNJ5 ), and contraction (downregulation of MYH6 ). Fibrosis-related genes showed the highest magnitude increased expression in Arrhythmic vs. Non-arrhythmic deaths and vs. published transcriptomes from end-stage heart failure. After molecular stratification by known markers for mature ( COL1A1, COL1A2, COL3A1) and active ( POSTN , MEOX1 ) fibrosis, cases with highest expression of both had the highest proportion of arrhythmic cause of death (27/36 [75%]) vs. cases with low expression of both (87/181 [38%], p=0.006) or vs. mature only (10/14 [71%]) or active only (5/14 [36%]). Activated fibroblast gene expression was enriched in Arrhythmic female vs. Arrhythmic male cases, among other sex-specific differences in ion channel and myosin (upregulation of SCN4B , SCN8A , KCNAB1 in females, KCNJ4 and MYH7B in males) expression. Conclusions: RNA profiling of myocardium at SCD identifies active fibrosis, undetectable by conventional clinical methods, in the presence of fixed scar and selected ion channel dysregulation (more pronounced among female cases) as an acute vulnerable substrate for fatal arrhythmias, and may identify patients at elevated near-term risk for SCD and novel pathways for intervention.

Surrogate immunohistochemistry markers in adult gliomas – Experience in a tertiary care hospital

Objectives: The recent World Health Organization classification has recommended the usage of surrogate immunohistochemical markers for molecular classification of gliomas. However, only a few studies have attempted to study the expression of the entire panel of markers. The current study was undertaken to study the association of recognized surrogate immunohistochemical markers (isocitrate dehydrogenase 1 [IDH-1], alpha-thalassemia mental retardation X-linked [ATRX] and p53) in different histological lineages and grades of adult gliomas. Materials and Methods: This study was conducted on 118 cases of adult gliomas diagnosed on histopathology over a 2-year duration in a tertiary care hospital. The expression of surrogate immunohistochemistry markers (IDH-1, ATRX, and p53) in these cases was studied. Statistical analysis: Descriptive statistical analysis with the Statistical Package for the Social Science system version 17.0. Results: The frequency of IDH-1 positivity was significantly higher in oligodendrogliomas (OG: 76.5%; oligoastrocytoma: 100%) versus astrocytoma (AS) (grades 2 and 3: 48.1%). It was also significantly higher in diffuse gliomas (grades 2 and 3) versus glioblastomas (64% vs. 16.9%). Among IDH-mutant diffuse gliomas, ATRX loss was significantly higher in AS versus OGs (84.6% vs. 7.7%). P53 overexpression correlated significantly with histological subtype (AS 2,3: 55.6% vs. OG: 5.9%). Conclusions: The surrogate immunohistochemical panel of IDH-1, ATRX, and p53 showed significant association with distinct histopathological subtypes and is helpful in molecular stratification. Cut-offs of ≥ 10% nuclear positivity for p53 and 50% loss of nuclear ATRX expression showed a good correlation.

Rapid intraoperative amplicon sequencing of CNS tumor markers

Currently, central nervous system tumors are diagnosed with an integrated diagnostic approach that combines histopathological examination with molecular genetic profiling, which requires days to weeks to achieve a precise and informative classification of CNS tumors. This study demonstrates the feasibility of rapid multiplex amplicon nanopore sequencing for identifying critical mutations relevant to molecular stratification of brain tumors within the timeframe of standard resection surgery. Utilizing live analysis of nanopore sequencing data, we evaluated the brain tumor-associated molecular markers IDH1 R132, IDH2 R172, pTERT C228 and C250, H3F3A K27 and G34, Hist1H3B K27, and BRAF V600. Our method achieved a turnaround time of 105 min at the point-of-care from receipt of a tumor biopsy to result with the potential to impact surgical strategy. Our approach can be integrated with recently developed DNA methylation-based diagnostic classification systems, corroborating diagnoses and even further specifying tumor grades, thus enabling a multimodal diagnostic intraoperative assessment of CNS malignancies.

The impact of integrated genomic analysis on molecular classifications and prognostic risk stratification in endometrial cancer

The impact of integrated genomic analysis on molecular classifications and prognostic risk stratification in endometrial cancer

Advances in Vulvar Cancer Biology and Management.

Vulvar squamous cell carcinoma (VSCC), a rare gynecologic malignancy, has been rising in incidence. Molecular classification on the basis of human papilloma virus (HPV) and tumor protein 53 (p53) status has identified three clinically distinct subtypes, but we still treat all VSCCs the same. Here, we review molecular classification of VSCC, outline treatment landscape, and highlight potential for targeted therapies in advanced VSCC. We conducted a comprehensive review of the literature on treatment of advanced VSCC with particular focus on the implications of molecular stratification on the basis of HPV and p53 status on the treatment landscape of advanced VSCC. Incorporation of HPV and p53 status in locoregional treatment decision making has the potential to advise (de)escalation strategies. The role of immunotherapy, alone and in combination, requires further exploration particularly earlier in the course of the disease. In advanced stages, potential for targeted therapies in VSCCs include inhibitors of vascular endothelial growth factor, endothelial growth factor receptor, cell cycle, and DNA damage response, particularly in HPV-negative (HPV-) VSCCs. Targeting the phosphoinositide 3 kinase/mammalian target of rapamycin pathway is attractive in HPV-positive and HPV-/p53 wildtype VSCCs. Trials incorporating antibody-drug conjugates (eg, trophoblast cell-surface antigen 2, human epidermal growth factor receptor 2) should be considered, and basket trials in perineal squamous cell cancers are warranted. Preclinical models are limited and should be expanded to inform trial design. Like other rare cancers, vulvar cancer lags behind in the identification and optimization of precision medicine strategies. Molecular-based preclinical models and rationally designed clinical trials, incorporating high-quality translational studies, are urgently required. These trials will require international collaboration to ensure feasibility and improvement of outcomes for women diagnosed with this disease.

Prognostic Prediction and Immune Microenvironment Characterization in Uveal Melanoma: A Novel Mitochondrial Metabolism-Related Gene Signature.

Uveal Melanoma (UM), a highly aggressive and metastatic intraocular cancer with a strong propensity for liver metastasis, presents limited therapeutic alternatives and unfavorable survival outcomes. Despite its low incidence, the underlying mechanisms of UM pathogenesis and the precise role of mitochondrial metabolism in UM remain inadequately understood. Utilizing Cox proportional hazards regression analysis was used to assess prognostic relevance, and consensus clustering was employed for molecular subtyping. A risk signature was constructed using Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression. We further conducted comparative analyses on clinicopathological characteristics, somatic mutation profiles, drug sensitivity, gene expression patterns, and tumor microenvironment features across different molecular subtypes. Moreover, a nomogram was developed and evaluated. Among 1234 mitochondria metabolism-related genes (MMRGs), 343 were identified as significantly associated with the prognosis of UM. These prognosis-associated MMRGs facilitated the classification of UM into two distinct molecular subtypes, which displayed notable differences in prognosis and pathological staging. Furthermore, an index termed the MMRGs-derived index (MMI) was derived from eight MMRGs, serving as a quantitative measure for poor prognosis risk in UM. MMI demonstrated significant associations with clinicopathological characteristics, somatic mutations, drug responsiveness, and the tumor microenvironment, where higher MMI levels corresponded to worse prognosis, advanced pathological stages, and increased immune cell infiltration. The nomogram built upon MMI provided a potential tool for clinical prognosis assessment in UM patients. This study demonstrated the potential value of MMRGs in predicting prognosis and molecular stratification within UM; however, additional clinical and basic research is warranted to validate their applicability and elucidate the related mechanisms.

Spatial Profiling of Ovarian Clear Cell Carcinoma Reveals Immune-Hot Features

Ovarian clear cell carcinoma (OCCC) has a high incidence in Asia, with a frequent occurrence at an early stage, but without sufficient data on molecular stratification for high-risk patients. Recently, immune-hot features have been proposed as indicators of poor prognosis in early stage OCCC. Specific patterns of intratumoral heterogeneity associated with immune-hot features must be defined. NanoString Digital Spatial Profiling technology (Cold spring biotech corp.) was used to decipher the spatial distribution of the 18-plex protein panel. Regions of interest (ROIs) were collected based on the reference hematoxylin and eosin–stained morphology. Areas of illumination (AOIs) were defined according to the ROI segmentation using the fluorescence signals of the visualization markers pan-cytokeratin (PanCK), CD45, or DNA. Unsupervised hierarchical clustering of 595 AOIs from 407 ROIs showed that the PanCK segments expressed different combinations of immune markers, suggestive of immune mimicry. The following 3 immune-hot clusters were identified: granzyme B–high, immune signal-high , and immune-like cells; the following 2 immune-cold clusters were identified: fibronectin-high and immune checkpoint–high cells. In tumor samples at the International Federation of Gynecology and Obstetrics stage IC1/2 experiencing recurrence, there was an increased occurrence of PanCK+ AOIs with immune signal-high and immune-like cell groups in the papillary morphology surrounded by macrophage lineage tumor-infiltrating immune cells (TIIs). In contrast, for tumor samples at the International Federation of Gynecology and Obstetrics stage IC3/II with recurrence, PanCK+ AOIs were prevalent in the fibronectin-high group, particularly in those with a tubulocystic morphology surrounded by lymphoid lineage non-TIIs. Our study on the spatial profiling of early stage OCCC tumors revealed that the immune mimicry of tumor cells, presence of TIIs, and morphologic patterns were associated with recurrence, which switched during tumor progression.

Deciphering autism heterogeneity: a molecular stratification approach in four mouse models

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition characterized by impairments in social interaction and communication, as well as restrained or stereotyped behaviors. The inherent heterogeneity within the autism spectrum poses challenges for developing effective pharmacological treatments targeting core features. Successful clinical trials require the identification of robust markers to enable patient stratification. In this study, we identified molecular markers within the oxytocin and immediate early gene families across five interconnected brain structures of the social circuit. We used wild-type and four heterogeneous mouse models, each exhibiting unique autism-like behaviors modeling the autism spectrum. While dysregulations in the oxytocin family were model-specific, immediate early genes displayed widespread alterations, reflecting global changes across the four models. Through integrative analysis, we identified Egr1, Foxp1, Homer1a, Oxt, and Oxtr as five robust and discriminant molecular markers that allowed the successful stratification of the four models. Importantly, our stratification demonstrated predictive values when challenged with a fifth mouse model or identifying subgroups of mice potentially responsive to oxytocin treatment. Beyond providing insights into oxytocin and immediate early gene mRNA dynamics, this proof-of-concept study represents a significant step toward the potential stratification of individuals with ASD. This work has implications for the success of clinical trials and the development of personalized medicine in autism.

Transcriptomic Profiling of Human Myocardium at Sudden Death to DefineVulnerable Substrate for LethalArrhythmias.

While some chronic pathological substrates for sudden cardiac death (SCD) are well known (eg, coronary artery disease and left ventricular [LV] dysfunction), the acute vulnerable myocardial state predisposing to fatal arrhythmia remains a critical barrier to near-term SCD prevention. This study sought to define the distinct myocardial transcriptomic profile of autopsy-defined arrhythmic sudden deaths, compared to nonarrhythmic sudden deaths and trauma deaths, to determine the acute vulnerable state in the hours to days before SCD. We used autopsy to adjudicate arrhythmic from nonarrhythmic causes in 1,265 sudden deaths in San Francisco County from 2011 to 2018. We performed a degradation-tolerant transcriptomic evaluation of LVs sampled at the time of SCD from 245 consented cases using a curated panel of 448 gene probes with known or hypothesized association with SCD. The targeted transcriptome of arrhythmic (n = 129) vs nonarrhythmic (n=90 nonarrhythmic sudden deaths+ 26 trauma deaths) LV samples revealed 31 differentially up-regulated and 36 down-regulated genes (adjusted P< 0.05) related to the collagen-containing extracellular matrix (up-regulation of FAP, FMOD, and LTBP2), regulation of ion transport (up-regulation of KCNA5 and KCNN3 and down-regulation of KCNJ8, KCNK1, and KCNJ5), and contraction (down-regulation of MYH6). Fibrosis-related genes showed the highest magnitude increased expression in arrhythmic vs nonarrhythmic deaths and vs published transcriptomes from end-stage heart failure. After molecular stratification by known markers for mature (COL1A1, COL1A2, COL3A1) and active (POSTN, MEOX1) fibrosis, cases with the highest expression of both had the largest proportion of arrhythmic cause of death (n=27 of 36 [75%]) vs cases with low expression of both markers (n=87 of 181 [38%]) (P = 0.006) or vs mature only (n=10 of 14 [71%]) or active only (n=5 of 14 [36%]). Activated fibroblast gene expression signature was enriched in arrhythmic female vs arrhythmic male cases, among other sex-specific differences in ion-channel and myosin (up-regulation of SCN4B, SCN8A, and KCNAB1 in females and KCNJ4 and MYH7B in males) expression. RNA profiling of the myocardium at SCD identifies active fibrosis, undetectable by conventional clinical methods, in the presence of fixed scar and selected ion-channel dysregulation (more pronounced among female cases) as an acute vulnerable substrate for fatal arrhythmias. These findings may represent novel directions to identify patients at elevated near-term risk for SCD and critical pathways for intervention to reduce acute lethal arrhythmias.

Histopathologic pattern and molecular risk stratification are associated with prognosis in patients with stage IB lung adenocarcinoma.

The benefit of adjuvant therapy remains controversial in completely resected (R0) stage IB non-small cell lung cancer (NCLSC) patients. In this study, we aimed to explore potential prognostic factors in stage IB NSCLC patients. This study included 215 patients with R0 stage IB lung adenocarcinoma (LUAD) (tumor size: 3-4 cm). DNA sequencing was performed with surgical samples of 126 patients using a panel of 9 driver genes. The molecular risk stratification was assessed by a 14-gene quantitative polymerase chain reaction assay. Among the 215 patients, 67.9% had micropapillary/solid (MIP/SOL)-predominant tumors. Epidermal growth factor receptor (EGFR) mutations were detected in 75 of 126 patients (59.5%). MIP/SOL tumors harbored less common EGFR mutations than the other histologic patterns (50.6% vs. 79.5%, P=0.003). Molecular risk stratification was successfully assessed in 99 patients, of whom 37.4%, 26.3%, and 36.4% were high, intermediate, and low risk, respectively. The MIP/SOL pattern was associated with shorter disease-free survival (DFS) [hazard ratio (HR) =2.16, 95% confidence interval: 1.28-3.67; P=0.01]. The molecular high-risk patients had shorter DFS than the low- (HR =2.93, P=0.01) and intermediate-risk patients (HR =2.35, P=0.06). The prognostic value of molecular risk stratification was also significant in the MIP/SOL subset (median DFS high-risk: 45 months, low and intermediate risk: not reached; P=0.03). Our study showed that both the MIP/SOL pattern and molecular high-risk category were adverse prognostic factors in stage IB NSCLC patients. Our results suggest that combining histologic classification and molecular risk stratification may help to identify the subset of patients with poor prognosis.

Abstract B078: A comprehensive spatial atlas of neoadjuvant chemoradiation therapy in resected pancreatic cancer identifies cellular &amp; microenvironmental determinants of persister populations

Abstract Background The molecular pathways involved in the response to radiation therapy (RT) in pancreatic ductal adenocarcinoma (PDAC) remain poorly understood. Despite extensive molecular stratification efforts, improvements in molecularly informed RT-based therapeutic strategies in the neoadjuvant setting have been limited. This study aims to elucidate the adaptive mechanisms and interactions within PDAC to identify new combinatorial therapeutic targets with RT. MethodsWe constructed a high-resolution molecular landscape of the cellular subtypes and spatial communities that constitute PDAC. Single-cell RNA sequencing (scRNA- seq) was performed on 16 PDAC biopsies of matched pre- and post-treatment samples from a Phase I/II dose-escalation clinical trial of pancreatic stereotactic body radiation therapy (SBRT). Additionally, Visium spatial transcriptomics (ST) was conducted on biopsies from primary PDAC patients collected at the time of surgery. These patients received either no neoadjuvant therapy (n = 8) or neoadjuvant chemoradiation (chemoRT) (n = 20). Cell type profiles were determined from scRNA-seq to compare changes in cell type proportions and signatures before and after RT. From these profiles, we employed robust cell type decomposition to analyze cell type signatures in our ST dataset. ResultsChemoRT induced several significant effects compared to controls within the tumor microenvironment, including an increase in the proportion of myofibroblasts, specifically senescent cancer-associated fibroblasts (CAFs), with signatures consistent with immune cell dysfunction. We observed an increase in CD8 and CD4 effector memory cells with RT; however, this occurred alongside a rise in immunosuppressive alternatively activated tumor-associated macrophages. Additionally, RT upregulated pathways related to HIF, cytokine production, B cell proliferation, and negative regulation of lymphocytes. Patients with increased infiltration of NK, memory B, and CD8 effector memory cells demonstrated improved overall survival outcomes. Inference of copy number variations enabled us to identify cancer subclones as either responsive or resistant in matched pre and post RT biopsies. Resistance was characterized by augmented hypoxia, KRAS activation, epithelial- mesenchymal transition, and interferon-gamma response signaling. Conversely, responders exhibited increased oxidative phosphorylation signaling and pathway enrichment associated with cell proliferation and cell cycle progression. Interestingly, RT induced a decrease in cancer cells characterized as harboring a chemoresistant basal-like molecular subtype. ConclusionThrough both longitudinal and therapy stratified tumor biopsies, we were able to observe significant selection pressures on stromal and cancer cells following RT. This includes upregulation of innate and adaptive immune pathways with a compensatory immunosuppressive response driven by myeloid cells and CAFs. This may provide opportunities for development of clinical trials to therapeutically target specific cellular phenotypes and their interactions. Citation Format: Vincent Bernard, Galia Jacobson, Kimal Rajapakshe, Jimin Min, Guangsheng Pei, Daniel Lin, Ayush Suresh, Dadi Jiang, Ching-Wei Tzeng, Manoop S Bhutani, Linghua Wang, Paola A Guerrero, Ethan B Ludmir, Albert C Koong, Anirban Maitra, Cullen M Taniguchi, Eugene J Koay. A comprehensive spatial atlas of neoadjuvant chemoradiation therapy in resected pancreatic cancer identifies cellular &amp; microenvironmental determinants of persister populations [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B078.

Abstract B088: Epigenetic reprograming in ATM-deficient pancreatic cancer: targeting EZH2- mediated oncogenic traits

Abstract Despite extensive research, the complex biology of pancreatic ductal adenocarcinoma (PDAC) continues to impede progress in improving its prognosis. Its resistance to conventional therapies, along with diverse mutations, necessitate improved molecular stratification to develop tailored approaches and to improve survival of PDAC patients. Our previous studies on ATM- deficient models revealed that serine/threonine kinase ATM loss induces homologous recombination deficiency (HRD), promoting invasiveness and desmoplastic reactions, while also sensitizing tumors to platinum-based therapies and PARP1 inhibition. Interestingly, our data also identified the histone-methyltransferase EZH2 as a direct ATM substrate to operate as a crucial regulator of ATM-specific oncogenic traits. In detail, murine and human ATM-deficient tumors accumulated EZH2, suggesting that ATM status contributes to rewiring the epigenetic landscape of pancreatic tumors, subsequently supporting oncogenic features. To investigate how EZH2 impacts ATM-deficient PDAC pathogenesis, we generated conditional double ATM and EZH2-knockout in the Kras G12D/+ ; Ptf1a Cre/+ mice (AKC, Atm fl/fl ; LSL-Kras G12D/+ ; Ptf1a Cre/+ , and EAKC, Ezh2 fl/fl ; Atm fl/fl ; LSL-Kras G12D/+ ; Ptf1a Cre/+ ). EZH2 depletion in AKC mice prolonged overall survival and reinstated a differentiated phenotype. Furthermore, PDAC patients with negative ATM and EZH2 expression from the TCGA-PAAD cohort (PanCancer Atlas) also showed significantly extended progression-free survival compared to ATM neg EZH2 pos patients. In line, CRISPR/Cas9-mediated EZH2 depletion in ATM-deficient pancreatic cancer cells affected their epigenetic landscape and significantly upregulated transcriptomic programs associated with cell differentiation, thereby restoring differentiated tumor features ex vivo. We showed that ATM status significantly impacts EZH2 activity through a post-translational pathway, promoting tumor supportive EZH2 activity in pancreatic cancer. ATM kinase facilitates EZH2 ubiquitination and degradation by recruiting the Cullin1 E3-ubiquitin ligase complex. Additionally, combining an EZH2 inhibitor with PARP inhibition enhances cytotoxicity in ATM- deficient HRD PDAC. Overall, our findings demonstrate that ATM deficiency drives a pro- oncogenic mesenchymal differentiation program via the histone-methyltransferase EZH2. We emphasize the importance of stratifying patients based on ATM status before considering pharmacological interference with EZH2, offering a promising approach for targeting ATM- deficient HRD tumors. Citation Format: Elodie Roger, Anna Härle, Lukas Perkhofer, Elisabeth Hessmann, Alexander Kleger, Johann Gout. Epigenetic reprograming in ATM-deficient pancreatic cancer: targeting EZH2- mediated oncogenic traits [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B088.

Targeted gene sequencing and transcriptome sequencing reveal characteristics of NUP98 rearrangement in pediatric acute myeloid leukemia

BackgroundNUP98 rearrangements (NUP98-r) are rare but overrepresented mutations in pediatric acute myeloid leukemia (AML) patients. NUP98-r is often associated with chemotherapy resistance and a particularly poor prognosis. Therefore, characterizing pediatric AML with NUP98-r to identify aberrations is critically important.MethodsHere, we retrospectively analyzed the clinicopathological features, genomic and transcriptomic landscapes, treatments, and outcomes of pediatric patients with AML.ResultsNine patients with NUP98-r mutations were identified in our cohort of 142 patients. Ten mutated genes were detected in patients with NUP98-r. The frequency of FLT3-ITD mutations differed significantly between the groups harboring NUP98-r and those without NUP98-r (P = 0.035). Unsupervised hierarchical clustering via RNA sequencing data from 21 AML patients revealed that NUP98-r samples clustered together, strongly suggesting a distinct subtype. Compared with that in the non-NUP98-r fusion and no fusion groups, CMAHP expression was significantly upregulated in the NUP98-r samples (P < 0.001 and P = 0.001, respectively). Multivariate Cox regression analyses demonstrated that patients harboring NUP98-r (P < 0.001) and WT1 mutations (P = 0.030) had worse relapse-free survival, and patients harboring NUP98-r (P < 0.008) presented lower overall survival.ConclusionsThese investigations contribute to the understanding of the molecular characteristics, risk stratification, and prognostic evaluation of pediatric AML patients.

Progression from cardiomyopathy to heart failure with reduced ejection fraction: A CORIN deficient course

Cardiomyopathies, encompassing hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM), constitute a diverse spectrum of heart muscle diseases that often culminating in heart failure (HF). The inherent molecular heterogeneity of these conditions has implications for prognosis and therapeutic strategies.Publicly available microarray and RNA sequencing (RNA-seq) data sets of HCM (n = 106 from GSE36961) and DCM (n = 18 from GSE135055 and 166 from GSE141910) patients were employed for our analysis. The Non-negative Matrix Factorization (NMF) algorithm was applied to explore the molecular stratification within HCM and DCM, and enrichment analysis was performed to delineate their biological characteristics. By integrating bulk and single-nucleus RNA-seq (snRNA-seq) data, we identified a potential biomarker for HCM progression and cardiac fibrosis, which was subsequently validated using mendelian randomization and in vitro.Our application of NMF identified two distinct molecular clusters. Particularly, a profibrotic, heart failure with reduced ejection fraction (HFrEF)-resembling Cluster 1 emerged, characterized by diminished expression of CORIN and a high degree of fibroblast activation. This cluster also exhibited lower left ventricular ejection fraction (LVEF) and worse prognostic outcomes, establishing the significance of this molecular subclassification. We further found that overexpression of CORIN could mitigate TGFβ1-induced expression of col1a1 and α-SMA in neonatal rat cardiac fibroblasts.Our results indicated the heterogeneity of HCM population, and further evidenced the participation of corin in the progression of HCM, DCM and HFrEF. Nevertheless, our study is constrained by the lack of corresponding clinical data and experimental validation of the identified subtypes. Therefore, further studies are warranted to elucidate the downstream pathways of corin and to validate these findings in independent patient cohorts.

Monitoring pediatric CNS non-germinomatous germ cell tumors via cerebrospinal fluid circulating tumor DNA.

Accurate molecular and clinical stratification of patients with central nervous system (CNS) non-germinomatous germ cell tumors (NGGCTs) remains challenging, impeding the development of personalized therapeutic approaches. Herein, we investigated the translational significance of cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) in pediatric NGGCTs to identify characteristic features of CNS NGGCTs and to identify a subset of patients for whom the presence of residual disease is a risk factor and an indicator of shorter progression-free survival (PFS) and overall survival (OS). Medical records of patients with CNS NGGCTs between January 1, 2018 and December 31, 2022 were reviewed retrospectively. The cohort consisted of 11 male and six female patients. Tumor markers were elevated in four of the five people who underwent surgery. The remaining 12 patients were diagnosed with malignant NGGCTs according to elevated tumor markers. Among them, ctDNA before chemotherapy as well as ctDNA clearance were consistently associated with PFS and OS (p<.05). By setting a ctDNA positivity threshold of 6%, patients with high ctDNA (above the threshold) levels, which had limitation due to the selection based on optimal statistic from the survival analysis, had significantly inferior 5-year PFS and OS compared to those with low levels (below the threshold). ctDNA or ctDNA clearance combined with the presence of residual disease predicted significantly worse OS and PFS (p<.05). CSF ctDNA might allow the study of genomic evolution and the characterization of tumors in pediatric NGGCTs. CSF ctDNA analysis may facilitate the clinical management of pediatric NGGCT patients, and aid in designing personalized therapeutic strategies.

Clinical features and prognosis of patients with myeloid neoplasms harboring t(7;11)(p15;p15) translocation: a single-center retrospective study

BackgroundFor myeloid neoplasms with t(7;11)(p15;p15) translocation, the prognosis is quite dismal. Because these tumors are rare, most occurrences are reported as single cases. Clinical results and optimal treatment approaches remain elusive. This study endeavors to elucidate the clinical implications and prognosis of this cytogenetic aberration.MethodsThis study retrospectively analyzed 23 cases of myeloid neoplasm with t(7;11)(p15;p15). Clinicopathological characteristics, genetic alterations, and outcomes were evaluated, and the Kaplan-Meier method was employed to construct survival curves.ResultsOf these, nine cases were newly diagnosed acute myeloid leukemia (ND AML), seven presented with relapsed refractory AML (R/R AML), four had myelodysplastic syndrome (MDS), two had secondary AML, and one exhibited a mixed germinoma associated with MDS. Patients with t(7;11)(p15;p15) in AML were primarily younger females who preferred subtype M2. Interestingly, these patients had decreased hemoglobin and red blood cell counts, along with markedly elevated levels of lactic dehydrogenase and interleukin-6, and exhibited the expression of CD117. R/R AML patients exhibited a higher likelihood of additional chromosome abnormalities (ACAs) besides t(7;11). WT1 and FLT3-ITD were the most commonly found mutated genes, and 10 of those instances showed evidence of the NUP98::HOXA9 fusion gene. The composite complete remission rate was 66.7% (12/18), while the cumulative graft survival rate was 100% (4/4). However, the survival outcomes were dismal. Interestingly, the median overall survival for R/R AML patients was 4.0 months (95% CI: 1.7–6.4). Additionally, the type of AML diagnosis or the presence of ACAs or molecular prognostic stratification did not significantly influence clinical outcomes (p = 0.066, p = 0.585, p = 0.570, respectively).ConclusionMyeloid leukemia with t(7;11) exhibits unique clinical features, cytogenetic properties, and molecular genetic characteristics. These survival outcomes were dismal. R/R AML patients have a limited lifespan. For myeloid patients with t(7;11), targeted therapy or transplantation may be an effective course of treatment.

MDB-42. RADIOMIC TEXTURAL AND MORPHOLOGICAL-BASED FEATURES TO RISK-STRATIFY PEDIATRIC MEDULLOBLASTOMA PATIENTS

Abstract BACKGROUND One key determinate in the treatment pathway for pediatric medulloblastoma (MB), the most frequent malignant brain tumor in children, is accurate risk-stratification. MB tumors are classified as standard- or high-risk based on current approaches (Chang’s/molecular stratification). However, there is still a need for additional attributes towards reliable risk-stratification. This work presents a radiomic-based approach that combines textural- and morphological-based attributes to risk-stratify MB patients. Our rationale is that coupling texture and morphology attributes from the intra-tumoral regions that quantify the heterogeneity and disorderly nature of aggressive tumors can transcend the current clinical approaches in accurately stratifying tumors into high- and standard-risk. METHODS T1-weighted MRI scans of 119 MB patients (2–18 years) were collected from Cincinnati Children’s Hospital Medical Center (Site1-n=42), Children’s Hospital Los Angeles (Site2-n=47), and Children’s Hospital of Philadelphia (Site3-n=30), used interchangeably for training and testing. Following preprocessing and performing annotations for the enhancing lesion and edema sub-compartments, 232 textural and morphological features were extracted from each sub-compartment. Namely, 214 textural (gradient, Haralick, intensity, Gabor, Laws, entropy) as well as 34 morphological (4 surface-based (e.g., curvedness and sharpness), 18 global (e.g., roundness, elongation, compactness, flatness)) features were extracted. Features were then fed into Elastic-Net regression models for survival analysis. RESULTS Our analysis revealed that texture and morphology features, combined, yielded Concordance index (CI) and p-value of 0.52, 0.0001, respectively, for the enhancing lesion, using Sites1,3 for training, and CI=0.61, p=0.0065 using Site3 for testing. Additionally, for edema, CI and p-value of 0.514, 0.0001 were obtained when employing Sites2,3 for training and CI=0.61, p=0.00096 using Site1 for testing. Results were not significant when using Chang’s or molecular stratification alone for survival analysis. CONCLUSIONS Our study shows that radiomic-based morphological and textural features show promise towards reliable MB risk-stratification.