Fraction Of Cells Research Articles

Characterization of TP53 mutations in actionable driver-negative non-small cell lung cancer (NSCLC).

8621 Background: TP53 mutations are common in NSCLC and have been associated with poor prognosis in patients with actionable genomic drivers. Despite the increased prevalence in actionable driver-negative (DN) NSCLC, the role of TP53 mutations in this subtype is not fully explored. We characterize the association of TP53 mutations with the molecular and immune landscapes, as well as outcomes, in DN NSCLC histologic subtypes. Methods: 17,689 NSCLC specimens were analyzed using next-generation sequencing of DNA (DNA-592 panel or whole exome) or RNA (whole transcriptome) at Caris Life Sciences (Phoenix, AZ). Tumors with KRAS, EGFR, BRAF, MET, HER2, ALK, ROS1, NRG1, and NTRK 1-3 alterations were excluded. Specimens were grouped as TP53WT (wild type) or TP53MUT (pathogenic/likely pathogenic mutations). PD-L1 expression was analyzed by IHC (Dako 22c3; PD-L1 positive: TPS ≥1%). TMB was measured by counting all somatic mutations found per tumor (TMB-H: ≥10 mutations/Mb). Composition of the tumor microenvironment (TME) was estimated from bulk RNA sequencing using QuanTIseq method. Overall survival was extracted from insurance claims data and calculated from the time of tissue collection (OS) or initiation of Pembrolizumab treatment (Pembro-OS) to the last contact, using Kaplan-Meier estimates. Significance was determined using chi-square, Fisher’s Exact, Mann Whitney U and adjusted for multiple comparisons (p-value < 0.05). Results: Among DN tumors, the prevalence of TP53MUT was 86%. TP53MUT was more common in men (62% vs 38%), those with a smoking history (99% vs 1%), and those with squamous cell histology (46% vs33.6%, all p<0.05). Interestingly, histological stratification suggested that TP53MUT was associated with poor OS in adenocarcinoma (AC) (HR:1.3, p<0.05), and shorter Pembro-OS in squamous cell carcinoma (SCC) (HR:1.425, p<0.05). In AC, mutations in FAT1, ARID1A, SMARCA4, ARID2 and RBM10 were enriched, while in SCC, mutations in PIK3CA were less common with TP53MUT. Gene sets involved in cell cycle regulation such as G2M, MYC, and E2F targets were enriched in TP53MUT and to a greater extent in AC (1.22-1.29) vs SCC (1.06-1.08, both p<0.05). Looking at immunotherapy-related biomarkers, both AC and SCC TP53MUT tumors had a higher prevalence of TMB-H compared to TP53WT, while PD-L1 positivity was enriched in only AC TP53MUT tumors. Conversely, KEAP1 and STK11 mutations were enriched in AC TP53WT. M2 Macrophage (1.25-fold) and NK cell (1.3-fold) fractions were enriched and B cell fractions (1.25-fold) were less common in TP53MUT SCC TMEs (all p<0.05). Conclusions: TP53MUT were associated with poor OS and Pembro-OS exclusively in AC and SCC, respectively. The distinct molecular and immune landscapes associated with TP53MUT in AC and SCC potentially explain the differences in outcomes mentioned above. Further research is needed to better understand therapeutic implications of TP53 mutations in DN-NSCLC.

The genomic, transcriptomic, and immunological profile of patients with recurrent/refractory NSCLC.

8039 Background: Lung cancer is the leading cause of cancer-related deaths worldwide, and often presents at advanced stages, with > 50% of patients presenting as stage III or IV. Patients with recurrent stage III NSCLC (R/R) previously treated with definitive chemoradiation and immunotherapy consolidation may have a difference in overall survival compared to de novo stage IV (DN). In this study, we aim to compare the molecular and immune landscapes of these two patient populations to identify potential genomic patterns and biomarkers of resistance. Methods: 3728 NSCLC specimens underwent sequencing of DNA (592-gene panel or whole exome) or RNA (whole transcriptome) or immunohistochemistry at Caris Life Sciences (Phoenix, AZ). Patients were classified as R/R (n = 26) if they received Durvalumab and chemoradiation within 12 months (m) before tissue collection and treated with Pembrolizumab within 6 m after tissue collection. Patients treated with Pembrolizumab within 6 months after tissue collection and had no prior (or post) treatment with Durvalumab and chemoradiation at any point in time were classified as DN (n = 3702). Tumor microenvironment (TME) cell fractions were estimated from bulk RNA sequencing using QuanTIseq. Significance was determined using chi-square, Mann Whitney U and adjusted for multiple comparisons where applicable (q < 0.05). Results: TP53 (91 vs 68%) and KEAP1 (30 vs. 14%, both p < 0.05, q > 0.05) mutation prevalence was higher in R/R, whereas a decreased prevalence of KRAS mutations was noted in this group (13 vs. 33%) as compared to the DN group. Compared to DN patients, R/R patients were associated with an increased prevalence of mutations in BCL2 (4.3 vs. 0%, q < 0.05), BCL9 (4.3 vs. 0.2%, q < 0.05), HNF1A (4.3 vs. 0.2%), TNFAIP3 (4.3 vs. 0.2%), AKT1 (4.3 vs. 0.4%), MAP3K1 (4.3 vs. 0.5%), and SPEN (4.3 vs. 0.6%; all p < 0.05, q > 0.05). Further, copy number amplification of NUTM1 (4.5 vs. 0%), SMO (4.3 vs. 0.5%), CHIC2 (4.5 vs. 0.2%; all q < 0.05), PIK3CA (9 vs. 1.2%) and FLCN (4.5 vs. 0.3%; both p < 0.05, q > 0.05) was more common in R/R compared to DN. R/R TME was associated with decreased B cell (2.9 vs 4.4%, q < 0.05) and regulatory T cell fractions (1.3 vs. 2.6%, p < 0.05, q > 0.05). Conclusions: R/R patient tumors had distinct molecular alterations and immune landscapes, including an increased prevalence of biomarkers associated with immunotherapy resistance ( TP53 and KEAP1 mutations) and lower tumor B- and T reg- cell fractions, which may suggest decreased likelihood of response to immunotherapy compared to patients with DN NSCLC. The RR population had a lower-than-expected percentage of KRASmutations. These distinct molecular differences highlight the need for NGS testing in the recurrent setting, despite initial NGS testing at diagnosis. Molecularly targeted interventions may be options in the future for patients with R/R Stage III NSCLC.

The Contribution of Mosaic Chromosomal Alterations to Schizophrenia

BackgroundMosaic chromosomal alterations are implicated in neuropsychiatric disorders, but the contribution to schizophrenia (SCZ) risk for somatic copy number variations (sCNVs) emerging in early developmental stages has not been fully established. MethodsWe analyzed blood-derived genotype arrays from 9715 patients with SCZ and 28,822 control participants of Chinese descent using a computational tool (MoChA) based on long-range chromosomal information to detect mosaic chromosomal alterations. We focused on probable early developmental sCNVs through stringent filtering. We assessed the burden of sCNVs across varying cell fraction cutoffs, as well as the frequency with which genes were involved in sCNVs. We integrated this data with the PGC (Psychiatric Genomics Consortium) dataset, which comprises 12,834 SCZ cases and 11,648 controls of European descent, and complemented it with genotyping data from postmortem brain tissue of 936 participants (449 cases and 487 controls). ResultsPatients with SCZ had a significantly higher somatic losses detection rate than control participants (1.00% vs. 0.52%; odds ratio = 1.91; 95% CI, 1.47–2.49; two-sided Fisher’s exact test, p = 1.49 × 10−6). Further analysis indicated that the odds ratios escalated proportionately (from 1.91 to 2.78) with the increment in cell fraction cutoffs. Recurrent sCNVs associated with SCZ (odds ratio > 8; Fisher’s exact test, p < .05) were identified, including notable regions at 10q21.1 (ZWINT), 3q26.1 (SLITRK3), 1q31.1 (BRINP3) and 12q21.31-21.32 (MGAT4C and NTS) in the Chinese cohort, and some regions were validated with PGC data. Cross-tissue validation pinpointed somatic losses at loci like 1p35.3-35.2 and 19p13.3-13.2. ConclusionsThe study highlights the significant impact of mosaic chromosomal alterations on SCZ, suggesting their pivotal role in the disorder’s genetic etiology.

Immunotherapy-based neoadjuvant therapy followed by concurrent chemoradiotherapy in locally advanced and advanced cervical cancer: A real-world study.

e17509 Background: Locally advanced and advanced cervical cancer (CC), characterized by extensive local invasion and potential distant metastasis, present substantial clinical challenges. Although concurrent chemoradiotherapy (CCRT) is the standard treatment for locally advanced CC, a considerable proportion (23%-34%) of treated pts experienced recurrence or subsequent metastasis. Recently, neoadjuvant therapy (NAT) has been proposed to increase the radiosensitivity and decreases the fraction of hypoxic cells. Particularly, immunotherapy (IO)-based NAT has shown high activity in treating CC. Moreover, IO plus CRT has shown promise in enhancing both local control and systemic response. Hence, we analyzed the efficacy and safety of IO-based NAT followed by CCRT for locally advanced and advanced CC in a real-world setting. Methods: This study retrospectively analyzed data from pts with locally advanced and advanced CC who underwent IO-based NAT followed by CCRT at Shengjing Hospital of China Medical University from January 2022 to January 2024. Outcome measures included complete response (CR), partial response (PR), objective response rate (ORR), progression free survival (PFS), and safety. Tumor responses after neoadjuvant or CCRT therapy were evaluated using RECIST v1.1 and PFS was analyzed with the Kaplan-Meir method. Safety was assessed by adverse events (AEs) referring to CTCAEs v 5.0. Results: A total of 20 pts (median age 57 years, range, 39-72) were included in this analysis, with 15% at stage IIIB, 60% at IIIC, 10% at IVA, and 15% at IVB. Eight (40%) pts received 1 cycle of neoadjuvant IO, 8 (40%) received 1-4 cycles of neoadjuvant IO + chemotherapy, and 4 (20%) received 2 cycles of neoadjuvant IO + bevacizumab (Bev) + chemotherapy. After NAT, all 20 pts received CCRT. In the overall population, ORR after NAT was 55% (11/20), with 5% CR and 50% PR. Regarding different IO-based NAT regimens, objective responses were achieved in 1 of 8 (12.5%) pts receiving neoadjuvant IO, 6 of 8 (75%) pts receiving neoadjuvant IO + chemotherapy, and 4 of 4 (100%) pts receiving neoadjuvant IO + Bev + chemotherapy. After CCRT, 6 (30%) pts had a CR and 7 (35%) pts had a PR, resulting in an ORR of 65.0%. At a median follow-up of 14.9 months, the median PFS was not reached, with a 12-month and 24-month PFS rates of 68% and 56%, respectively. The most common treatment-related AEs during NAT were white blood cell count decreased (50%) and neutrophil count decreased. Conclusions: IO-based NAT followed by CCRT were effective and well-tolerated for pts with locally advanced and advanced CC. This treatment modalities may provide a potential therapeutic strategy for locally advanced and advanced CC.

Subclonal mutational signatures in breast cancer brain metastases.

e13108 Background: Breast cancer is the leading cause of cancer-related deaths among women in Brazil and the cancer type with highest incidence. When diagnosed at early stages, it has an overall five-year survival rate of up to 90%. However, in advanced stages, survival rate is reduced to about 24%, and 90% of women in stage IV die of metastatic complications. Brain is one of the most common metastatic sites in breast cancer patients, and even though it is a site with poor prognosis, the mutational profile of brain tumors is poorly described in the literature. Therefore, we understand that the identification of this profile may contribute to elucidate mechanisms associated with tumor progression. The aim of this study was to reconstruct the subclonal evolution of breast cancer metastases in the brain. Methods: To carry out this study, the whole exome was accessed through Next Generation Sequencing (NGS) using the Illumina platform in DNA samples extracted from buffy coat cryopreserved and paraffin material of breast tumors and paired brain metastases (n=10). The median read coverage was 68x. DNA was aligned to a Human Reference Genome using the Burrows–Wheeler Aligner, Genome Analysis Toolkit was used for germline short variants calling, and Mutect2 and MuSE were used for variant calling with somatic character (SNVs and INDELs). FACETS was used for assessing the somatic DNA copy number (Copy Number Aberrations). PyClone-VI tool was used for the subclonal reconstruction and to infer the phylogeny it was considered the mutation loci, Cancer Cell Fraction (CCF) and clustering assignments, using R Statistical Environment and Stats, Argparse, and Devtools packages. Results: We identified the mutational signature in these patients with subclonal and clonal mutations that segregated in approximately two clusters with a median between ten and forty variants related with tumor progression process. Conclusions: These findings show a set of variants with driver potential for breast cancer brain metastasis, bringing new perspectives in identifying mechanisms associated with disease progression.

PD-L1 and alternative immune checkpoints (AICs) in non-small cell lung cancer (NSCLC): Insights into the tumor immune microenvironment (TIME) and immunotherapy (IO) outcomes.

8531 Background: PD-L1 and recently identified AICs (B7x, B7-H3, and HHLA2) may function as key immune evasion mechanisms in NSCLC and influence IO efficacy. This study explores the expression patterns of PD-L1 and AICs within the TIME and their impact on IO outcomes in NSCLC, including key molecular subtypes. Methods: NSCLC samples (n=27,629) were analyzed (Caris Life Sciences) with NextGen Sequencing on DNA (592 genes or whole exome)/RNA (whole transcriptome). Tumor cell PD-L1 expression (22c3) was assessed by IHC. Gene expression profiles were analyzed for IFN-γ score and QuantiSEQ was used to assess immune cell infiltration in the TIME. Real-world overall survival (rwOS: from time of biopsy to last contact), time on treatment [TOT: from the first pembrolizumab (pembro) treatment to the last], and OS on pembro (OSpem: from pembro start to last contact) were calculated. Mann-Whitney U, X2/Fisher-Exact, and log-rank tests were applied where appropriate. Results: PD-L1 IHC+ NSCLC and cases with high PD-L1 RNA expression had higher IFN-γ score and immune cell fractions (p&lt;0.0001 for both) vs PD-L1 IHC- and low expression, respectively. Additionally, PD-L1 IHC+ NSCLC was associated with (a/w) higher median transcript levels (TPM) of PD-L1 and B7-H3, while PD-L1 IHC- NSCLC was a/w higher TPM of HHLA2 and B7x (p&lt;0.0001 for all). AICs expression varied by molecular subtype, e.g., higher HHLA2/B7x RNA in EGFR mutant vs wild-type (WT) NSCLC. Moreover, PD-L1 IHC+ NSCLC and cases with high PD-L1 RNA and low B7H3 RNA were a/w longer rwOS, OSpem, and TOT; high HHLA2 RNA was a/w longer rwOS and OSpem with similar TOT; and low B7x RNA expression was a/w longer OSpem and TOT with similar rwOS (Table). Conclusions: Both PD-L1 IHC+ NSCLC and cases with high PD-L1 RNA expression are a/w a more inflammatory TIME, and superior survival and IO outcomes. Moreover, consistent with our prior HHLA2 IHC work (PMIDs: 27553831, 29374053), HHLA2 RNA expression was significantly higher in PD-L1 IHC- and EGFR mutant cases. These findings support the potential utility of transcriptomics along with protein expression for assessing novel predictive biomarkers for IO. Furthermore, the distinct expression profiles of AICs identified in our study may represent key subtypes mediating IO response in WT and EGFR mutant NSCLC. [Table: see text]

ITHindex: An integrated tool for quantifying intra-tumor heterogeneity using omics data in cancers.

e14689 Background: Intra-tumor heterogeneity (ITH) is a hallmark of cancer and plays a significant role in tumor evolution, treatment response, and the development of drug resistance. However, there is currently no standardized metric for assessing ITH in clinical practice. Several recent studies have proposed different algorithms of ITH based on multi-omics data. In this study, we have developed a web-based tool for analyzing tumor heterogeneity using diverse omics data and investigated the correlation between different ITH algorithms using TCGA data. Methods: We collected previously published ITH algorithms based on single-/multi-region genomic, transcriptomic, and proteomic data. We integrated these algorithms into a web server called ITHindex, which utilizes Shiny framework. TCGA data was employed for a comprehensive comparison of five ITH algorithms including MATH, CNH, DEPTH2, Shannon index and DITHER. Results: ITHindex (https://xwen.shinyapps.io/ithindex/) is an open-access online tool that encompasses a total of eight algorithms. Based on the type of input data, these algorithms can be categorized into SNV, cancer cell fraction (CCF), copy number variations segment, bulk or single-cell RNAseq, and multi-region mass spectrometry of protein. According to the calculation principles, these algorithms include those based on Shannon entropy, MAD, tumor clonality, ratio, and distance. To demonstrate the practicality of ITHindex, we employed TCGA data to calculate ITH values using five algorithms. The analysis unveiled that the distribution of the same algorithm differs across various tumor types. When considering the same type of input data, different algorithms exhibited a strong correlation. Furthermore, with the exception of MATH, the other ITH values displayed a negative correlation with overall survival. Conclusions: ITHindex is a user-friendly web-based tool that quantifies intra-tumor heterogeneity, empowering researchers to investigate the role of ITH in oncologic biology.

Whole exome sequencing analysis of intratumoral heterogeneity, tumor neoantigen burden and human leukocyte antigen loss of heterozygosity in HCC.

e16003 Background: Hepatocellular Carcinoma (HCC) patients has a high postoperative recurrence within 2 years and a low 5-year survival rate. However, molecular mechanisms for early recurrence are poorly understood. Immune checkpoint blockades (ICBs) show clinical benefits in HCC, with the immune microenvironment influencing ICBs therapeutic effects. Exploring the relationship between intratumoral heterogeneity (ITH), tumor neoantigen burden (TNB), and human leukocyte antigen loss of heterozygosity (HLA LOH) can deepen our understanding of the immune evasion mechanisms. Methods: In this study, whole exome sequencing (WES) was employed to analyze tumor specimens and matched peripheral blood controls derived from a cohort of 235 HCC patients. PD-L1 expression levels were assessed in the samples using immunohistochemistry. NetMHCpan was applied to predict neoantigen and quantified the peptide affinity of MHC-I genes measured by IC50. Neoantigen-MHC affinity was categorized as strong (IC50≤50nM), weak (50nM &lt; IC50≤500nM), and negative (IC50 &gt; 500nM). TNB was defined by the number of high affinity neoantigens per megabase in WES. Analysis of LOH was performed on HLA-I class genes and categorized as strong positive, weak positive, and negative groups. We used PyClone to conduct ITH analysis to assess the distribution of multiple subclones within a tumor. The average Cancer Cell Fraction (CCF) for each somatic mutation cluster was calculated, and these clusters were classified into major clones and subclones. Results: In 235 HCC samples, median TMB and TNB were 2.48 Muts/Mb and 0.30 Neos/Mb. Only 6.81% exhibited TMB-H (TMB≥10 Muts/Mb). TMB and TNB are significantly positively correlated (p &lt; 0.0001). HLA LOH occurred in 19.57% HCC patients, with high LOH samples having more high-affinity neoantigens (mean TNB 1.65 Neos/Mb). ITH analysis showed that 83.83% samples harbored subclonal clusters. HCC samples were divided into high (ITH≥0.908) and low (ITH &lt; 0.908) groups according to the median ITH of 0.908. High ITH group had higher TNB (median 0.43 vs 0.20 Neos/Mb) , more HLA LOH (9.24% vs 5.17% strong positive LOH). The high ITH groups exhibited a lower percentage of samples with elevated PD-L1 expression (14.29% vs 25.86%). The Combined Positive Score (CPS) revealed a significant difference between the groups (Fisher's test p = 0.03). Conclusions: In HCC, the high ITH group exhibited a higher proportion of HLA LOH compared to the low ITH group. Moreover, a decreased proportion of PD-L1 positive samples was observed in the high ITH group, suggesting that high ITH may be a key factor influencing treatment failure in immunotherapy. ITH analysis demonstrated a potential multi-subclonal origin of recurrent tumor cells, which is associated with immune escape, tumor malignancy,as well as the development of resistance.

The role of the ubiquitin ligase UBE4B in neural tumors in adults and children.

e14034 Background: In cancers of the nervous system such as neuroblastoma (NB), the ubiquitin ligase UBE4B directly ubiquitinates growth factor receptors (GFRs), influencing their trafficking and lysosomal degradation. While the diverse functions of UBE4B are acknowledged, we hypothesize that UBE4B expression has distinct associations with outcomes in pediatric versus adult nervous system cancers. We examined the impact of UBE4B expression on glioblastoma (GBM) development, growth, and metastasis by correlating its expression with patient outcomes across age groups. Methods: Glioblastoma samples that were IDH1/2 wild type (n = 2072) underwent DNA (592-gene/whole-exome) and RNA (whole transcriptome) sequencing at Caris Life Sciences. Samples were stratified by UBE4B expression (transcripts per million – TPM) quartiles for all GBM tumors (Q4: H and Q1: L) and by patient age at the time of tissue collection (younger: &lt; 30 years; older: &gt; 60 years). Cell infiltration in the tumor microenvironment was inferred by quanTIseq. Gene expression profiles were analyzed for transcriptomic signature predictive of response to immunotherapy. Overall survival (OS) was derived from insurance claims data, and Kaplan-Meier estimates were calculated. Mann-Whitney U and Chi-square/Fisher-Exact tests were applied were appropriate, with p-values adjusted for multiple comparison. Results: UBE4B expression was similar between younger (n = 57) and older (n = 1115) glioblastoma patients (median: 24.7 vs. 21.6 TPM, p = 0.17). In younger patients, those with high UBE4B expression showed improved OS compared to low expressors (24.9 vs 19.2 months [mos]; HR = 0.41, 95% CI 0.15 – 1.11, p = 0.07). However, UBE4B expression was not associated with OS among older GBM patients (13.5 vs 12.9 mos; HR 1.10 95% CI 0.9 – 1.3, p = 0.33). Within the younger cohorts, higher expression of growth factor receptor genes was observed in high compared to low UBE4B expression ( EGFR: 96.57 vs. 7.07 TPM, p = 0.0092; RET: 0.99 vs. 0.30 TPM, p = 0.018; NTRK2: 236.9 vs. 29.5 TPM, p = 0.084). Concurrent mutation of PDGFRA was also exclusively observed in UBE4B-high tumors (42.9% vs. 0% in UBE4B-L, p = 0.024). Transcriptomic analysis, however, showed that UBE4B-high tumors had lower IFN-g signature scores compared to UBE4B-L (-0.62 vs. -0.41 arbitrary units, p = 0.02). M1 macrophage cell fractions were lower in UBE4B-high compared to UBE4B-low (median: 0.33% vs. 3.38%, p = 0.005), while the fraction of B cells was significantly higher (median: 9.57% vs. 8.14%, p = 0.009). Conclusions: Higher UBE4B expression in younger GBM cases correlated with improved OS suggesting it may have utility as a prognostic biomarker. Increased expression of GFRs in younger patients with high UBE4B expression may provide rationale for new treatment strategies in these patients. Future work may prioritize designing drug targets for UBE4B, offering therapeutic opportunities, especially for younger GBM patients.

The effect of resting dendritic cells on overall survival in the hepatocellular carcinoma tumor microenvironment.

e14559 Background: Hepatocellular carcinoma (HCC) continues to have a poor prognosis even in the advent of new immunotherapy regimens. An area of growing interest in HCC is its tumor immune microenvironment. Classifying the immune microenvironment based on available genomics can lead to insights on prognosis and treatment, especially regarding the role of immunotherapy. Methods: RNA sequencing data was extracted from The Cancer Genome Atlas (TCGA), specifically from the hepatocellular carcinoma section (n = 366). CIBERSORT was utilized to organize the immune cell fractions present for each patient sample. Radiant by Shiny was used to perform a linear regression elucidating the relationship between overall survival and several other variables including: age, gender, tumor stage, race, ethnicity, B-cells naïve, B-cells memory, plasma cells, CD8 T-cells, CD4 T-cells, T-cells follicular, T-cells regulatory, natural killer activated and resting cells, monocytes, macrophages (M0, M1, M2), dendritic cells activated and resting, mast cells activated and resting, eosinophils, and neutrophils. Subgroup analyses were also performed via radiant regarding gender, race, and resting dendritic counts. Results: Resting dendritic cells were independently prognostic of overall survival on multivariable analysis (p = 0.041). The coefficient of correlation was +164.53 showing a positive association between the number of resting dendritic cells and the overall survival. Subgroup analysis showed no difference in resting dendritic counts between males and females (p = 0.526). Also, there was no difference seen in resting dendritic counts between populations of Asians and African Americans (p = 0.873), Asians and Whites (p = 0.223), and between Whites and African Americans (p = 0.811). Conclusions: Patients with a higher number of resting dendritic cells in their tumor immune microenvironment had a better overall survival. There was no difference between males and females in the resting dendritic counts. Likewise, there was no difference in the resting dendritic counts between Asian, African American, and white populations. Resting dendritic cells have been shown to be enriched in hepatocellular carcinomas with a low tumor mutational burden. This is a paradigm that should be explored further in the context of immunotherapy. Resting dendritic cells are also in a state of plasticity as they go from resting to active. Future genomic analysis of the tumor microenvironment regarding this plasticity could also lead to enhancing treatment options.

Immune infiltrate analysis to reveal distinct immune classes for leiomyosarcomas.

11546 Background: Leiomyosarcomas (LMS) are aggressive malignancies of smooth-muscle origin. Few targeted molecular options exist and immunotherapy (IO) agents against T cells have been disappointing. In this study, we examine the immune infiltrate of LMS tumors by immunohistochemistry (IHC) and gene expression. Our objective was to identify actionable targets for immunotherapy in patients with LMS. Methods: Tumor samples from a series of LMS patients were prospectively collected and banked for DNA and RNA sequencing as part of the MD Anderson Patient Mosaic. Associated clinical data for the patients were collected and maintained. IHC was performed on available separate tissue microarrays. Differential gene expression (DGE) analysis was done using the DESeq2 package for R and the Benjamini-Hochberg procedure used to control for false discovery rate. Immune cell fractions were estimated using the MCPcounter package. All gene expression analyses were repeated on The Cancer Genome Atlas (TCGA) dataset as an independent dataset. Results: A total of 104 TCGA samples and 72 samples from Mosaic from 53 patients with uterine or soft tissue LMS were included in the analysis. Tumors in the TCGA group were treatment naive. Of the samples in the Mosaic group, 61 (85%) had been previously treated with chemotherapy, 8 (11%) had been radiated, and 5 (7.5%) had been previously treated with immunotherapy. Thirty-one (43%) tumors were metastatic. Fifty-one (70%) were soft tissue LMS and 21 (30%) were uterine LMS. Four distinct immune classes were seen in both the TGCA and Mosaic: a predominantly B-cell group, a monocyte/macrophage group, a mixed infiltrate group and a group with a neutrophil signature. DGE performed between the subgroups revealed a unique gene expression pattern for tumors in the B cell and monocyte/macrophage subgroups. Interrogation of specific immune-associated genes revealed low levels of expression in the neutrophil group, suggesting this may be the most immunologically “cold” subset. Some of these results were reproduced by IHC using a TMA: the presence of a cluster of samples with B-cells and higher expression of immune checkpoints, a macrophage driven cluster, and an immunologically cold cluster. Conclusions: Analyses of LMS samples across two large datasets demonstrate the existence of 4 distinct immune classes of tumors that persist despite variation in clinical and treatment conditions: B cell, monocytic/macrophage, mixed-infiltrate and neutrophil rich. The B cell and monocytic groups have increased expression of immune markers which may be amenable to targeted therapies.

Characterization of enhancer of zeste homolog 2 (EZH2) expression, activity, and association with the tumor immune microenvironment in olfactory neuroblastoma (ONB).

e14655 Background: ONB is a rare sinonasal cancer with limited therapeutic options. Increased expression and up-regulation of EZH2, a core protein of the epigenetic modifier methyltransferase Polycomb repressive complex 2, have been associated with worse prognosis in several cancer types. EZH2 inhibitors are currently approved in Epithelioid Sarcoma (ES) and Follicular Lymphoma. We examined the expression and activity of EZH2 in ONB, as compared to ES (where EZH2 inhibition is standard-of-care), and its impact on the immune microenvironment. Methods: ONB (N = 35) and ES (N = 78) were tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing of DNA (592 genes or whole exome) and RNA (whole transcriptome). PD-L1 expression (22C3; Positive (+): TPS ≥1%) was assessed by IHC. Transcriptomic signatures predictive of immunotherapy response (T-cell inflamed [TCI] score) and MAPK pathway activation (MPAS) were applied. Immune cell fractions were estimated using quanTIseq. A transcriptional ‘ EZH2 Repression Score' (ERS, arbitrary units: AU) was examined, with high ERS suggestive of lower EZH2 activity (Morel et al, {2021}). ONB were stratified by their median ERS (high -H, N=17 v low -L, N=18). Three additional score variants were applied (equally weighted, immune regulatory genes excluded, or both) and all produced similar results. ONB were stratified by the median EZH2 transcripts per million (TPM, -H v -L). Mann-Whitney U, Fisher’s Exact/χ2 tests were applied as appropriate with p-values adjusted for multiple comparisons ( p &lt;.05). Results: No pathogenic EZH2 mutations were observed in ONB; EZH2 expression was higher compared to ES (44.4 v 14.1 TPM, p &lt; 0.001), yet ERS was not significantly different (5111 v 5143 AU, p = 0.76). There was no association between EZH2 expression and ERS (Pearson’s correlation = .1). In ONB, there was no difference in the prevalence of PD-L1+ between ERS-H v -L (20% v 20%). ERS-H were associated with increased expression of β2M (2110 v 1019 TPM, p = .01) and immune checkpoint genes: CTLA4 (1.4 v 0.5, p = .03), PD-1 (1.1 v 0.3, p = .04), PD-L1 (4.7 v 1.6, p &lt; .001) and VISTA (22.1 v 6.5, p &lt; .001). ERS-H was associated with increased M2 macrophage infiltrate (5.0 v 2.5%, p &lt; .001). ERS-H had increased MPAS (1.99 v -.42 AU, p &lt; .001) and increased prevalence TCI tumors (29 v 0%, p &lt; .001), while EZH2-H tumors were not associated with differences in immune infiltrate, TCI, MPAS, nor with immune checkpoint gene expression compared to EZH2-L. Conclusions: EZH2 mutations were not detected in ONB or ES, though similar EZH2 activity (by ERS) was observed in both cohorts, thus making ONB an intriguing candidate for EZH2 modulating therapies. High ERS in ONB was associated with expression of immune checkpoint genes suggesting that inhibiting EZH2 activity could enhance the susceptibility of ONB to immune checkpoint blockade.

Relationship between thrombus composition and prognosis in patients with acute ischemic stroke undergoing mechanical thrombectomy

BackgroundMechanical thrombectomy has emerged as the primary endovascular treatment for acute ischemic stroke. Numerous studies have investigated the relationship between thrombus composition and factors such as pharmacological thrombolysis, stroke etiology, mechanical thrombectomy, and radiological imaging. However, limited research has explored the association between thrombus composition and clinical outcomes. MethodsThis retrospective analysis examined the histopathological examination of thrombi retrieved from 50 patients with acute ischemic stroke between May 2020 and May 2023. The composition of the retrieved thrombi was assessed using HE staining to quantify the proportions of red blood cells, white blood cells, platelets, and fibrin. Based on the predominant composition of the thrombus, the patients were divided into two groups: erythrocyte-rich and fibrin-rich. Demographics, clinical characteristics, and clinical outcomes assessed by the National Institutes of Health Stroke Scale (NIHSS) score and modified Rankin Scale (mRS) scores were collected retrospectively. ResultOf the 50 patients, 23 were classified in the erythrocyte-rich group, and 27 were classified in the fibrin-rich group. There were no significant differences between the two groups in terms of age, sex, stroke subtype, history of hypertension and diabetes, thrombus location, NIHSS scores, mRS scores on admission, the time interval from symptom onset to hospitalization and reperfusion, or the rate of successful reperfusion. However, erythrocyte-rich thrombi were associated with a shorter time interval from puncture to reperfusion. No significant differences were found in the red blood cell fraction and fibrin/platelet fraction between large artery atherosclerosis and cardioembolism. At the 90-day follow-up, patients with erythrocyte-rich thrombi exhibited lower NIHSS scores and more favorable functional outcomes (mRS scores of 0–2) compared to those with fibrin-rich thrombi. ConclusionErythrocyte-rich thrombi were linked to shorter time intervals from puncture to reperfusion and favorable clinical outcomes in patients with acute ischemic stroke. The composition of the thrombus may influence the thrombectomy strategy for endovascular therapy.

Integrative analyses of multi-omics data constructing tumor microenvironment and immune-related molecular prognosis model in human colorectal cancer

The increasing prevalence and incidence of colorectal cancer (CRC), particularly in young adults, underscore the imperative to comprehend its fundamental mechanisms, discover novel diagnostic and prognostic markers, and enhance therapeutic strategies. Here, we integrated multi-omics data, including gene expression, somatic mutation data and DNA methylation data, to unravel the intricacies of tumor microenvironment (TME) in CRC and search for novel prognostic markers. By calculating the immune score for each patient from the expression profile, we delineated the differential immune cell fraction, constructed an immune-related multi-omics atlas, and identified molecular characteristics. The entire colorectal dataset (n = 343) was randomly divided into training (n = 249) and testing datasets (n = 94). We screened 144 immune-related genes, 6 mutant genes, and 38 methylation probes associated with overall survival (OS). These makers were then incorporated into a 10-gene prognostic model using Lasso and Cox regression in the training dataset, and the model's performance was evaluated in an independent validation dataset. The model exhibited satisfactory results (average concordance index [C-index] = 0.77), with the average 1-year, 3-year, and 5-year AUCs being 0.79, 0.76, and 0.76 in the training dataset and 0.74, 0.80, and 0.90 in the testing dataset. Furthermore, the prognostic model demonstrated applicability in guiding chemotherapy for CRC patients and exhibited a degree of pan-cancer utility in risk stratification. In conclusion, our integrated analysis of multi-omics data revealed immune-related genetic and epigenetic characteristics of the TME. We propose an integrative prognostic model that can stratify risk and guide chemotherapy for CRC patients. The generalizability of the model in risk stratification across different cancer types was validated in Pan-Cancer cohort.

A dynamic network model predicts the phenotypes of multicellular clusters from cellular properties

Cell division without cell separation produces multicellular clusters in budding yeast. Two fundamental characteristics of these clusters are their size (the number of cells per cluster) and cellular composition: the fractions of cells with different phenotypes. Using cells as nodes and links between mother and daughter cells as edges, we model cluster growth and breakage by varying three parameters: the cell division rate, the rate at which intercellular connections break, and the kissing number (the maximum number of connections to one cell). We find that the kissing number sets the maximum possible cluster size. Below this limit, the ratio of the cell division rate to the connection breaking rate determines the cluster size. If links have a constant probability of breaking per unit time, the probability that a link survives decreases exponentially with its age. Modeling this behavior recapitulates experimental data. We then use this framework to examine synthetic, differentiating clusters with two cell types, faster-growing germ cells and their somatic derivatives. The fraction of clusters that contain both cell types increases as either of two parameters increase: the kissing number and difference between the growth rate of germ and somatic cells. In a population of clusters, the variation in cellular composition is inversely correlated (r2= 0.87) with the average fraction of somatic cells in clusters. Our results show how a small number of cellular features can control the phenotypes of multicellular clusters that were potentially the ancestors of more complex forms of multicellular development, organization, and reproduction.

New insight on porcine carboxylesterases expression and activity in lung tissues

Over the course of the last twenty years, there has been a growing recognition of the pig's potential as a valuable model for studying human drug metabolism. This study aimed to investigate the expression, enzymatic activity, inhibitory susceptibility, and cellular localization of carboxylesterases (CES) in porcine lung tissue not yet explored. Our results showed that CESs hydrolysis activity followed Michaelis-Menten kinetics in both cytosolic and microsomal fractions of porcine lung tissues (N = 8), with comparable hydrolysis rates for tested substrates, namely 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD). We also determined the CESs hydrolysis activity in a representative sample of the porcine liver that, as expected, displayed higher activity than the lung ones. The study demonstrated variable levels of enzyme activities and interindividual variability in both porcine lung fractions. Inhibition studies used to assess the CESs' involvement in the hydrolysis of pNPA, 4-MUA, and FD suggested that CESs may be the enzymes primarily involved in the metabolism of ester compounds in the pig lung tissue. Overall, this study provides insight into the distribution and diversity of CES isoforms involved in substrate hydrolysis across different cellular fractions (cytosol and microsomes) in porcine lungs.

The Role of Insulin-like Growth Factor Binding Protein (IGFBP)-2 in DNA Repair and Chemoresistance in Breast Cancer Cells.

The role if insulin-like growth factor binding protein-2 (IGFBP-2) in mediating chemoresistance in breast cancer cells has been demonstrated, but the mechanism of action is unclear. This study aimed to further investigate the role of IGFBP-2 in the DNA damage response induced by etoposide in MCF-7, T47D (ER+ve), and MDA-MB-231 (ER-ve) breast cancer cell lines. In the presence or absence of etoposide, IGFBP-2 was silenced using siRNA in the ER-positive cell lines, or exogenous IGFBP-2 was added to the ER-negative MDA-MB-231 cells. Cell number and death were assessed using trypan blue dye exclusion assay, changes in abundance of proteins were monitored using Western blotting of whole cell lysates, and localization and abundance were determined using immunofluorescence and cell fractionation. Results from ER-positive cell lines demonstrated that upon exposure to etoposide, loss of IGFBP-2 enhanced cell death, and this was associated with a reduction in P-DNA-PKcs and an increase in γH2AX. Conversely, with ER-negative cells, the addition of IGFBP-2 in the presence of etoposide resulted in cell survival, an increase in P-DNA-PKcs, and a reduction in γH2AX. In summary, IGFBP-2 is a survival factor for breast cancer cells that is associated with enhancement of the DNA repair mechanism.

Somatic USP8 alteration affects the immune landscape of corticotroph pituitary adenomas- a pilot study.

Somatic mutations in ubiquitin-specific protease-8 (USP8), encoding a deubiquinating protein, are found in approximately 30% of corticotroph-derived pituitary adenomas (CPAs). Stratifin, a protein encoded by SFN, inhibits USP8 catalytic activity. USP8 has immunomodulating properties that have been demonstrated in non-tumoral diseases. We assessed the influence of USP8 on the immune landscape of CPA and validated this effect and its dependency on stratifin in large cohorts of non-pituitary tumors. We analyzed data of CPA samples (n = 20) and additional non-pituitary tumors from the TCGA database, using transcriptome signature-recognition algorithms. Immune tumor microenvironment (iTME) was compared both by USP8 and SFN expression levels (n = 843) and by USP8 mutation status and SFN expression (n = 12,389). CPA with activating USP8 mutations was associated with "cold" iTME compared with wild-type USP8 CPA, as reflected by lower fractions of immune cells, including B cells, CD4, regulatory and gamma/delta T cells, natural killer cells, M0 and M1 macrophages, dendritic cells, and eosinophils (p < 0.05 for all comparisons). Pathways altered by the presence of USP8 mutation, based on the most differentially expressed genes (3061 genes), included microglia pathogen phagocytosis and multiple toll-like receptor signaling pathways (p < 0.0001). In a validation analysis based on large cohorts of non-pituitary tumors, high expression of USP8 was associated with a suppressed iTME effect that was augmented by a low SFN expression. Our data demonstrate for the first time, to our knowledge, a distinct immune landscape of tumors based on USP8 status and expression and the dependency of this immunological effect on SFN expression.

Sequential drug treatment targeting cell cycle and cell fate regulatory programs blocks non-genetic cancer evolution in acute lymphoblastic leukemia

BackgroundTargeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side-effects of chemotherapy. However, efficacy of precision therapies is limited in part because of tumor cell heterogeneity. A better mechanistic understanding of how drug effect is linked to cancer cell state diversity is crucial for identifying effective combination therapies that can prevent disease recurrence.ResultsHere, we characterize the effect of G2/M checkpoint inhibition in acute lymphoblastic leukemia (ALL) and demonstrate that WEE1 targeted therapy impinges on cell fate decision regulatory circuits. We find the highest inhibition of recovery of proliferation in ALL cells with KMT2A-rearrangements. Single-cell RNA-seq and ATAC-seq of RS4;11 cells harboring KMT2A::AFF1, treated with the WEE1 inhibitor AZD1775, reveal diversification of cell states, with a fraction of cells exhibiting strong activation of p53-driven processes linked to apoptosis and senescence, and disruption of a core KMT2A-RUNX1-MYC regulatory network. In this cell state diversification induced by WEE1 inhibition, a subpopulation transitions to a drug tolerant cell state characterized by activation of transcription factors regulating pre-B cell fate, lipid metabolism, and pre-BCR signaling in a reversible manner. Sequential treatment with BCR-signaling inhibitors dasatinib, ibrutinib, or perturbing metabolism by fatostatin or AZD2014 effectively counteracts drug tolerance by inducing cell death and repressing stemness markers.ConclusionsCollectively, our findings provide new insights into the tight connectivity of gene regulatory programs associated with cell cycle and cell fate regulation, and a rationale for sequential administration of WEE1 inhibitors with low toxicity inhibitors of pre-BCR signaling or metabolism.

Exploring Syndecan-4 and MLP and Their Interaction in Primary Cardiomyocytes and H9c2 Cells.

The transmembrane proteoglycan syndecan-4 is known to be involved in the hypertrophic response to pressure overload. Although multiple downstream signaling pathways have been found to be involved in this response in a syndecan-4-dependent manner, there are likely more signaling components involved. As part of a larger syndecan-4 interactome screening, we have previously identified MLP as a binding partner to the cytoplasmic tail of syndecan-4. Interestingly, many human MLP mutations have been found in patients with hypertrophic (HCM) and dilated cardiomyopathy (DCM). To gain deeper insight into the role of the syndecan-4-MLP interaction and its potential involvement in MLP-associated cardiomyopathy, we have here investigated the syndecan-4-MLP interaction in primary adult rat cardiomyocytes and the H9c2 cell line. The binding of syndecan-4 and MLP was analyzed in total lysates and subcellular fractions of primary adult rat cardiomyocytes, and baseline and differentiated H9c2 cells by immunoprecipitation. MLP and syndecan-4 localization were determined by confocal microscopy, and MLP oligomerization was determined by immunoblotting under native conditions. Syndecan-4-MLP binding, as well as MLP self-association, were also analyzed by ELISA and peptide arrays. Our results showed that MLP-WT and syndecan-4 co-localized in many subcellular compartments; however, their binding was only detected in nuclear-enriched fractions of isolated adult cardiomyocytes. In vitro, syndecan-4 bound to MLP at three sites, and this binding was reduced in some HCM-associated MLP mutations. While MLP and syndecan-4 also co-localized in many subcellular fractions of H9c2 cells, these proteins did not bind at baseline or after differentiation into cardiomyocyte-resembling cells. Independently of syndecan-4, mutated MLP proteins had an altered subcellular localization in H9c2 cells, compared to MLP-WT. The DCM- and HCM-associated MLP mutations, W4R, L44P, C58G, R64C, Y66C, K69R, G72R, and Q91L, affected the oligomerization of MLP with an increase in monomeric at the expense of trimeric and tetrameric recombinant MLP protein. Lastly, two crucial sites for MLP self-association were identified, which were reduced in most MLP mutations. Our data indicate that the syndecan-4-MLP interaction was present in nuclear-enriched fractions of isolated adult cardiomyocytes and that this interaction was disrupted by some HCM-associated MLP mutations. MLP mutations were also linked to changes in MLP oligomerization and self-association, which may be essential for its interaction with syndecan-4 and a critical molecular mechanism of MLP-associated cardiomyopathy.