Individual Genes Research Articles

The Immunogenomic Landscape of Peripheral High-Dose IL-2 Pharmacodynamics in Patients with Metastatic Renal Cell Carcinoma: A Benchmark for Next-Generation IL-2-Based Immunotherapies.

High-dose (HD) IL-2 was the first immuno-oncology agent approved for treating advanced renal cell carcinoma and metastatic melanoma, but its use was limited because of substantial toxicities. Multiple next-generation IL-2 agents are being developed to improve tolerability. However, a knowledge gap still exists for the genomic markers that define the target pharmacology for HD IL-2 itself. In this retrospective observational study, we collected PBMC samples from 23 patients with metastatic renal cell carcinoma who were treated with HD IL-2 between 2009 and 2015. We previously reported the results of flow cytometry analyses. In this study, we report the results of our RNA-sequencing immunogenomic survey, which was performed on bulk PBMC samples from immediately before (day 1), during (day 3), and after treatment (day 5) in cycle 1 and/or cycle 2 of the first course of HD IL-2. As part of a detailed analysis of immunogenomic response to HD IL-2 treatment, we analyzed the changes in individual genes and immune gene signatures. By day 3, most lymphoid cell types had transiently decreased, whereas myeloid transcripts increased. Although most genes and/or signatures generally returned to pretreatment expression levels by day 5, certain ones representative of B cell, NK cell, and T cell proliferation and effector functions continued to increase, along with B cell (but not T cell) oligoclonal expansion. Regulatory T cells progressively expanded during and after treatment. They showed strong negative correlation with myeloid effector cells. This detailed RNA-sequencing immunogenomic survey of IL-2 pharmacology complements results of prior flow cytometry analyses. These data provide valuable pharmacological context for assessing PBMC gene expression data from patients dosed with IL-2-related compounds that are currently in development.

Identification of the Molecular Components of Enhancer-Mediated Gene Expression Variation in Multiple Tissues Regulating Blood Pressure.

Inter-individual variation in blood pressure (BP) arises in part from sequence variants within enhancers modulating the expression of causal genes. We propose that these genes, active in tissues relevant to BP physiology, can be identified from tissue-level epigenomic data and genotypes of BP-phenotyped individuals. We used chromatin accessibility data from the heart, adrenal, kidney, and artery to identify cis-regulatory elements (CREs) in these tissues and estimate the impact of common human single-nucleotide variants within these CREs on gene expression, using machine learning methods. To identify causal genes, we performed a gene-wise association test. We conducted analyses in 2 separate large-scale cohorts: 77 822 individuals from the Genetic Epidemiology Research on Adult Health and Aging and 315 270 individuals from the UK Biobank. We identified 309, 259, 331, and 367 genes (false discovery rate <0.05) for diastolic BP and 191, 184, 204, and 204 genes for systolic BP in the artery, kidney, heart, and adrenal, respectively, in Genetic Epidemiology Research on Adult Health and Aging; 50% to 70% of these genes were replicated in the UK Biobank, significantly higher than the 12% to 15% expected by chance (P<0.0001). These results enabled tissue expression prediction of these 988 to 2875 putative BP genes in individuals of both cohorts to construct an expression polygenic score. This score explained ≈27% of the reported single-nucleotide variant heritability, substantially higher than expected from prior studies. Our work demonstrates the power of tissue-restricted comprehensive CRE analysis, followed by CRE-based expression prediction, for understanding BP regulation in relevant tissues and provides dual-modality supporting evidence, CRE and expression, for the causality genes.

BIOINFORMATICS AND MOLECULAR ANALYSES IDENTIFIED THE CONTROL REGION AS THE MOST POWERFUL MITOGENOMIC MARKER FOR DISTINGUISHING THE MAIN MATERNAL HAPLOGROUPS IN GOATS

Identification of genetic markers to distinguish animals within and between species demands extensive genomic and bioinformatics investigation. Previous studies have not carefully taken into consideration the effect of mitogenomic components on the genetic differentiation of the maternal lineages in goats. As a precaution, the complete goat mitogenome was downloaded from the NCBI database and used in the current study to assess the effects of the choice of mitogenomic fragments on phylogenetic studies and to identify any potential polymorphic region by which the main maternal haplogroups of goats can be classified. Phylogenetic results confirmed that all 13 individual mitochondrial protein-coding genes and 2 ribosomal genes are not applicable to differentiate the maternal lineages. Instead, a single novel polymorphic region with a length of 756 bp within the control region was successfully amplified by newly designed primers. Both phylogenetic analysis and principal components analysis of the sequenced mitogenomic region of the mtDNA control region efficiently differentiated the main maternal haplogroups in goats. Higher numbers of polymorphic sites were found in the control region and the mitogenomic marker region. Highly significant correlations were discovered between the polymorphic sites and the length of each individual mitogenomic component. Our results demonstrate useful guidance and cautionary notes for researchers who are interested in the investigation of genetic diversity in animal species using mtDNA sequences. The bioinformatics and molecular methods used herein can be powerful in selecting a minimum amount of data using PCR amplification when the entire sequences of the mitogenome are unavailable.

Conserved role of hnRNPL in alternative splicing of epigenetic modifiers enables B cell activation.

The multifunctional RNA-binding protein hnRNPL is implicated in antibody class switching but its broader function in B cells is unknown. Here, we show that hnRNPL is essential for B cell activation, germinal center formation,and antibody responses. Upon activation, hnRNPL-deficient B cells show proliferation defects and increased apoptosis. Comparative analysis of RNA-seq data from activated B cells and another eight hnRNPL-depleted cell types reveals common effects on MYC and E2F transcriptional programs required for proliferation. Notably, while individual gene expression changes are cell type specific, several alternative splicing events affecting histone modifiers like KDM6A and SIRT1, are conserved across cell types. Moreover, hnRNPL-deficient B cells show global changes in H3K27me3 and H3K9ac. Epigenetic dysregulation after hnRNPL loss could underlie differential gene expression and upregulation of lncRNAs, and explain common and cell type-specific phenotypes, such as dysfunctional mitochondria and ROS overproduction in mouse B cells. Thus, hnRNPL is essential for the resting-to-activated B cell transition by regulating transcriptional programs and metabolism, at least in part through the alternative splicing of several histone modifiers.

Like Father, Like Daughter - Ectodermal Dysplasia-Syndactyly Syndrome: A Case Report.

Ectodermal dysplasia-syndactyly syndrome 1 (EDSS1) is an exceedingly rare condition associated with mutations in the PVL4 gene. It is characterised by sparse, brittle hair, eyebrows and eyelashes, abnormal dentition and nails, along with bilateral cutaneous syndactyly involving the fingers and toes. We report a 2-year-old girl who presented to us with bilateral complete simple syndactyly of the third and fourth web spaces of the hands, along with bilateral syndactyly of both feet involving the second to fourth toes. Upon examination, sparse hair and eyebrows, along with abnormal dentition, were noted. Thorough clinical examination and genetic analysis were conducted on the affected child and her father, who exhibited similar clinical features. Genetic analysis revealed a homozygous nonsense mutation in the PVL4 gene in both individuals. According to the literature, EDSS1 has been reported in only 10 families worldwide, and there are no reported cases from India. Level of Evidence: Level V (Therapeutic).

Identification of an Immunodominant B-Cell Epitope in African Swine Fever Virus p30 Protein and Evidence of p30 Antibody-Mediated Antibody Dependent Cellular Cytotoxicity

Identification of an Immunodominant B-Cell Epitope in African Swine Fever Virus p30 Protein and Evidence of p30 Antibody-Mediated Antibody Dependent Cellular Cytotoxicity

Whole genome sequence analysis of shallot virus X from India reveals it to be a natural recombinant with positive selection pressure

BackgroundShallots are infected by various viruses like Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Shallot latent virus (SLV) and Shallot virus X (ShVX). In India, they have been found to be persistently infected by ShVX. ShVX also infects onion and garlic in combination with other carlaviruses and potyviruses. ShVX is a member of genus Allexivirus of family Alphaflexiviridae. ShVX has a monopartite genome, which is represented by positive sense single-stranded RNA. Globally, only six complete and 3 nearly complete genome sequences of ShV X are reported to date. This number is insufficient to measure a taxon’s true molecular diversity. Moreover, the complete genome sequence of ShVX from Asia has not been reported as yet. Therefore, this study was undertaken to generate a complete genome sequence of ShVX from India.ResultsShallot virus X (ShVX) is one of the significant threats to Allium crop production. In this study, we report the first complete genome sequence of the ShVX from India through Next-generation sequencing (NGS). The complete genome of the ShVX (Accession No. OK104171), from this study comprised 8911 nucleotides. In-silico analysis of the sequence revealed variability between this isolate and isolates from other countries. The dissimilarities are spread all over the genome specifically some non-coding intergenic regions. Statistical analysis of individual genes for site-specific selection indicates a positive selection in NABP region. The presence of a recombination event was detected in coat protein region. The sequence similarity percentage and phylogenetic analysis indicate ShVX Indian isolate is a distinctly different isolate. Recombination and site-specific selection may have a function in the evolution of this isolate. This is the first detailed study of the ShVX complete genome sequence from Southeast Asia.ConclusionThis study presents the first report of the entire genome sequence of an Indian isolate of ShVX along with an in-depth exploration of its evolutionary traits. The findings highlight the Indian variant as a naturally occurring recombinant, emphasizing the substantial role of recombination in the evolution of this viral species. This insight into the molecular diversity of strains within a specific geographical region holds immense significance for comprehending and forecasting potential epidemics. Consequently, the insights garnered from this research hold practical value for shaping ShVX management strategies and providing a foundation for forthcoming studies delving into its evolutionary trajectory.

The association between single-nucleotide polymorphisms within type 1 interferon pathway genes and human immunodeficiency virus type 1 viral load in antiretroviral-naïve participants

BackgroundHuman genetic contribution to HIV progression remains inadequately explained. The type 1 interferon (IFN) pathway is important for host control of HIV and variation in type 1 IFN genes may contribute to disease progression. This study assessed the impact of variations at the gene and pathway level of type 1 IFN on HIV-1 viral load (VL).MethodsTwo cohorts of antiretroviral (ART) naïve participants living with HIV (PLWH) with either early (START) or advanced infection (FIRST) were analysed separately. Type 1 IFN genes (n = 17) and receptor subunits (IFNAR1, IFNAR2) were examined for both cumulated type 1 IFN pathway analysis and individual gene analysis. SKAT-O was applied to detect associations between the genotype and HIV-1 study entry viral load (log10 transformed) as a proxy for set point VL; P-values were corrected using Bonferroni (P < 0.0025).ResultsThe analyses among those with early infection included 2429 individuals from five continents. The median study entry HIV VL was 14,623 (IQR 3460–45100) copies/mL. Across 673 SNPs within 19 type 1 IFN genes, no significant association with study entry VL was detected. Conversely, examining individual genes in START showed a borderline significant association between IFNW1, and study entry VL (P = 0.0025). This significance remained after separate adjustments for age, CD4+ T-cell count, CD4+/CD8+ T-cell ratio and recent infection. When controlling for population structure using linear mixed effects models (LME), in addition to principal components used in the main model, this was no longer significant (p = 0.0244). In subgroup analyses stratified by geographical region, the association between IFNW1 and study entry VL was only observed among African participants, although, the association was not significant when controlling for population structure using LME. Of the 17 SNPs within the IFNW1 region, only rs79876898 (A > G) was associated with study entry VL (p = 0.0020, beta = 0.32; G associated with higher study entry VL than A) in single SNP association analyses. The findings were not reproduced in FIRST participants.ConclusionAcross 19 type 1 IFN genes, only IFNW1 was associated with HIV-1 study entry VL in a cohort of ART-naïve individuals in early stages of their infection, however, this was no longer significant in sensitivity analyses that controlled for population structures using LME.

The complete plastome and phylogenetic analysis of Commelina benghalensis L.1753 (Commelinaceae)

Commelina benghalensis L. 1753, a member of the Commelinaceae family, holds significant medicinal and culinary value. This study represents the first documentation of the sequencing and assembly of the entire plastome of C. benghalensis. The genome spans a total length of 160,663 bp, exhibiting a conventional quadripartite architecture that comprises a large single-copy (LSC) region (87,750 bp), a small single-copy (SSC) region (18,417 bp), and two inverted repeats (IR) regions (both 27,248 bp). In its entirety, the C. benghalensis plastome encompasses 129 genes (with 108 being unique), incorporating 77 individual protein-coding genes, 37 unique tRNA genes, and four unique rRNA genes. Phylogenetic analysis revealed a close resemblance between C. benghalensis and C. communis. The sequencing of this plastome stands to expedite the development of molecular markers and significantly contribute to genetic assays involving this distinctive plant.

Abstract PO2-15-08: Exploring Essential Immune Genes Linked to Pathological Complete Response and Survival in Early-Stage Triple-Negative Breast Cancer (TNBC) after Chemotherapy Treatment

Abstract Background: De-escalation and escalation of systemic therapy in early-stage TNBC will require the implementation of biomarkers to guide therapy. To date, high expression of a variety of immune genes has been associated with higher pathological complete response (pCR) rates following neoadjuvant chemotherapy or with improved survival, or both, in early-stage TNBC. However, it is currently unknown which are the key genes influencing survival (i.e., the Core Immune Genes [CIG]) and if combinations of these genes can improve their association with both clinical endpoints. Methods: The expression of up to 185 genes was evaluated on pre-treatment tumor biopsies from 3 independent publicly available datasets of early-stage TNBC: CALGB40603 (n=388) (NCT00861705), BrighTNess (n=482) (NCT02032277), and SCAN-B (n=498). Patients in CALGB40603 and BrighTNess were treated with neoadjuvant chemotherapy-based therapy and had response data. Patients in CALGB40603 and SCAN-B had survival outcome data (event-free survival [EFS] or relapse-free interval [RFi]). Each individual gene was associated with each clinical endpoint within each dataset using Cox model and logistic regression analyses, adjusting for treatment arm. Genes found in common across datasets were selected as CIG, except for PDCD1, which was not available in BrighTNess. Using the CIGs, we evaluated whether combinations of 2 genes enhance their association with each clinical endpoint using different methods: adding, subtracting, multiplying, and ratio. Likelihood ratio testing was used to evaluate the added contribution of each gene in 2 combined cohorts evaluating pCR (i.e., CALGB40603 and BrighTNess) and survival (i.e., CALGB40603 and SCAN-B). Results: 10 CIGs related to B-cells and T-cells (i.e., IRF4, LAX1, POU2AF1, CD274, CD79A, TNFRSF17, PIM2, PDCD1, CXCR6, and SLAMF1) were associated with higher pCR rates and improved survival outcome across datasets. Of note, 5 (50%) genes are known components of the 14-gene immunoglobulin/B-cell (IGG) signature. The correlation coefficient among the 10 CIG genes was 0.63 (moderate). In general, the sum of 2 CIGs provided more prognostic information than a single gene (p&amp;lt; 0.001). Specifically, the sum of 2 CIGs provides more prognostic information than a single gene in 30 of 45 (66.6%) cases. Two-gene subtraction, multiplication or ratio decreased the amount of prognostic information (p&amp;lt; 0.001). The top 10 2-gene CIGs associated with prognosis (p&amp;lt; 0.001) were CD274_TNFRSF17, IRF4_CD274, PDCD1_TNFRSF17, CD79A_CD274, CD274_POU2AF1, CXCR6_TNFRSF17, LAX1_TNFRSF17, SLAMF1_TNFRSF17, CD274_LAX1 and CD79A_CXCR6. In general, the sum of 2 CIGs provided more information about pCR than a single gene (p&amp;lt; 0.001). Specifically, the sum of 2 CIGs provided numerically more information about pCR than 1 gene in 17 of 36 (47.2%) cases. Two-gene subtraction, multiplication or ratio decreased the amount of information about pCR (p&amp;lt; 0.001). The top 10 2-gene CIGs associated with pCR were CD274_PIM2, CD274_POU2AF1, CXCR6_PIM2, CD274_CXCR6, IRF4_CD274, CXCR6_POU2AF1, CD274_LAX1, IRF4_CXCR6, CXCR6_LAX1, and POU2AF1_SLAMF1. Conclusion: The expression of CIGs associated with B-cell and T-cell adaptive immune response is closely linked to pCR and survival outcomes following (neo)adjuvant chemotherapy. When combining the expression of 2 specific CIGs through addition, we found that this method provides more informative insights into pCR and survival outcomes compared to other combination methods. This approach enhances the predictive power of CIGs, enabling a better understanding of treatment response and patient prognosis, ultimately contributing to improved clinical decision-making and patient outcomes. Citation Format: Fara Brasó-Maristany, Laia Paré, Benedetta Conte, Laura Angelats, Ana Vivancos, Joel S Parker, Charles M. Perou, Patricia Villagrasa, Aleix Prat. Exploring Essential Immune Genes Linked to Pathological Complete Response and Survival in Early-Stage Triple-Negative Breast Cancer (TNBC) after Chemotherapy Treatment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-15-08.

Abstract PO2-13-05: Deciphering pregnancy-associated breast cancer: distinctive molecular profile and clinical implications from GEICAM/2017-07 EMBARCAM study

Abstract Background: Pregnancy-Associated Breast Cancer (PABC) is defined as BC diagnosed during pregnancy, breastfeeding, or within the first year postpartum. This challenging entity has a distinct biology and worst prognosis and presents an increased risk for metastasis and death compared to non-PABC patients. Identification of molecular pathways that define this malignancy is crucial to explain its biological characteristics and potential association with pregnancy as well as serve to identify new biomarkers with preventive and clinical implications. This study assesses the gene expression profile of a PABC cohort from the Registry Study of Pregnancy and Breast Cancer (GEICAM/2017-07 EMBARCAM study, NCT04603820) to identify distinct molecular signatures and altered pathways for this entity. Methods: This was a multicenter, observational and ambispective age-matched cohort of PABC patients (n=46) and non-PABC patients (n=49). We analyzed the expression of 776 genes involved in 23 key breast cancer pathways, using the nCounter® Breast Cancer 360™ Panel (NanoString Technologies) on BC tumor samples, including molecular subtypes by PAM50 gene signature. Associations between individual differential genes and epidemiological and clinical features of these patients were explored. Normalized differential expression values were log2-transformed for statistical analysis in nSolver Software (NanoString Technologies). P-values were adjusted within each gene/signature and on the grouping variable level difference t-test using the Benjamini and Yekutieli False Discovery Rate (FDR) adjustment. Results: PABC patients’ clinical subtypes were distributed as 43,5% HR-positive/HER2-negative, 17,4% HER2-positive and 39,1% triple negative BC. These patients reported higher family history of BC/ovarian cancer compared to non-PABC (52.17% vs 26.53%; p=0.0124). Differential gene expression showed distinct molecular profiles in PABC and non-PABC patients: significantly genes enrichment involved in cell proliferation and p53 signature for PABC patients and higher expression of apoptosis, TGF-β, and PTEN signatures for non-PABC (adj p&amp;lt; 0.05). Furthermore, CDK4 expression signature (adj p=0.124) and DNA damage repair signatures Homologous Recombination Deficiency (HDR) (adj p=0.083) and BRCAness (adj p=0.220) were also upregulated in PABC tumors. The most significantly upregulated genes in PABC patients (CCNA2, DEPDC1, FAM83D, CDC20, CDKN3, TRIP13, TTK, MAD2L1, KIF2C, and UBE2C) (adj p&amp;lt; 0.05) were involved in cell cycle regulation and DNA damage repair. PABC basal-like tumors were found to be significantly more prevalent than non-PABC, as per the PAM50 classification (45.7% vs 20.4%; p=0.020). Interestingly, basal-like PABC showed higher expression of ESR1 than basal-like non-PABC (logFC=1.486; p=0.022). In contrast, luminal A PABC tumors exhibited lower ESR1 expression vs non-PABC (logFC=-1.5; p=0.021). Additionally, HER2-enriched PABC tumors displayed lower B7-H3/CD276 expression compared to HER2-enriched non-PABC (adj p=0.031). Conclusions: Our study shows that PABC is potentially a clinical and molecular different entity with predominance of the basal-like subtype. Moreover, our results suggest the activation of oncogenic pathways related to cell proliferation, DNA damage repair and p53 mutations which may lead to a clinically more aggressive phenotype for PABC patients. Likewise, the enrichment of BRCAness and HRD signatures found in PABC patients in our study may suggest an increased genetic instability due to a breakdown in the DNA damage repair. These findings may be clinically relevant and translate to new treatment options in PABC patients, who may benefit from therapies targeting DNA repair or cell-cycle checkpoints, such as PARP inhibitors. Citation Format: Juan de la Haba-Rodríguez, Regina Peña, Marina Pollan, Yolanda Jerez Gilarranz, Jose Ponce, Antonio Fernández Aramburu, Blanca Cantos, Ana Santaballa Bertrán, Elena Galve, María Eva Pérez, Susana de la Cruz, María Helena López-Ceballos, Yolanda Fernández, Fernando Moreno, Sonia González, Manuel Ruíz - Borrego, Isabel Blancas, José L Alonso-Romero, Álvaro Jiménez-Arranz, Raúl Rincón, Silvia Guil, Alejandra Díaz-Chacón, Rosalía Caballero, Begoña Bermejo, Angel Guerrero. Deciphering pregnancy-associated breast cancer: distinctive molecular profile and clinical implications from GEICAM/2017-07 EMBARCAM study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-13-05.

Phylogenomic discordance is driven by wide-spread introgression and incomplete lineage sorting during rapid species diversification within rattlesnakes (Viperidae: Crotalus and Sistrurus).

Phylogenomics allows us to uncover the historical signal of evolutionary processes through time and estimate phylogenetic networks accounting for these signals. Insight from genome-wide data further allows us to pinpoint the contributions to phylogenetic signal from hybridization, introgression, and ancestral polymorphism across the genome. Here we focus on how these processes have contributed to phylogenetic discordance among rattlesnakes (genera Crotalus and Sistrurus), a group for which there are numerous conflicting phylogenetic hypotheses based on a diverse array of molecular datasets and analytical methods. We address the instability of the rattlesnake phylogeny using genomic data generated from transcriptomes sampled from nearly all known species. These genomic data, analyzed with coalescent and network-based approaches, reveal numerous instances of rapid speciation where individual gene trees conflict with the species tree. Moreover, the evolutionary history of rattlesnakes is dominated by incomplete speciation and frequent hybridization, both of which have likely influenced past interpretations of phylogeny. We present a new framework in which the evolutionary relationships of this group can only be understood in light of genome-wide data and network-based analytical methods. Our data suggest that network radiations, like seen within the rattlesnakes, can only be understood in a phylogenomic context, necessitating similar approaches in our attempts to understand evolutionary history in other rapidly radiating species.

Abstract PO4-14-11: Findings of clinically significant variants (Tier IA) with OmniSeq INSIGHT ® in a breast cancer cohort of 987 patients

Abstract Background: There were an estimated 2.3 million new breast cancer cases worldwide in 2020, with breast cancer now the most diagnosed type of cancer representing 25% of cancer cases and 17% of cancer deaths (1). The GLOBOCAN Cancer Tomorrow prediction tool estimates that incidence will increase by &amp;gt; 45% by 2040 (2). Optimal treatment for breast cancer is increasingly dependent upon knowing a patient’s somatic and germline genomic alteration status. To highlight the importance of these genomic alterations, we provide an overview of the genomic findings in 987 consecutive breast cancer patients tested in the course of routine clinical care. Methods: Comprehensive Genomic and Immune Profiling (CGIP) was performed on 987 qualified breast cancer samples at a CAP/CLIA and NYS CLEP certified reference laboratory with the OmniSeq INSIGHT ® test (3). OmniSeq INSIGHT ® is a next generation sequencing-based laboratory developed test for the detection of genomic variants, signatures, HLA Class I genotypes, and immune gene expression in formalin-fixed paraffin-embedded (FFPE) tumor tissue. DNA is sequenced to detect small variants in the full exonic coding region of 523 genes, copy number alterations in 59 genes (gains and losses), as well as analysis of microsatellite instability (MSI) and tumor mutational burden (TMB). RNA is sequenced to detect fusions and splice variants in 55 genes, in addition to mRNA expression in 64 immune genes. The resultant information, along with PD-L1 protein expression by immunohistochemistry (IHC), is intended for use by qualified health care professionals in accordance with professional guidelines in oncology for management of patients with solid neoplasms. Tier IA variants include variants with strong clinical significance as per AMP–ASCO–CAP recommendations (4). Results: There were 384 Tier IA variants detected in 370 of 987 breast cancer patients (~37.5%) with 251 in PIK3CA (65.3%), 60 in ERBB2 (15.6%), 46 in BRCA2 (11.9%), 19 in BRCA1 (4.9%), 4 in NTRK3 (1.0%), 1 in NTRK1 (0.26%) and 1 in PALB2 (0.26%) including copy number variations (CNV), gene fusion and single nucleotide variants (SNV). At the individual gene level for Tier IA variants, PIK3CA had 251 SNVs detected, ERBB2 had 55 CNVs, 1 gene fusion and 4 SNVs detected, BRCA2 had 39 SNVs and 8 CNVs detected, BRCA1 had 19 SNVs, NTRK3 had 4 gene fusions, NTRK1 had 1 gene fusion and PALB2 had 1 SNV detected. At the variant class level, there were 63 CNVs, 6 gene fusions and 315 SNVs observed. Conclusions: CGIP for breast cancer patients identified one or more clinically significant Tier IA genomic alterations that directs targeted therapy in ~ 37.5% of patients in a cohort of real world patients tested during the standard course of clinical care. This highlights the need for comprehensive genomic testing in breast cancer patients to drive therapeutic decision making.

Abstract PS14-07: Exploring Circulating Leukocyte RNA Expression: Implications for Treatment Outcomes and Immune-Related Adverse Events in Patients with Triple Negative Breast Cancer Enrolled in the GeparNuevo Trial

Abstract Abstract Exploring Circulating Leukocyte RNA Expression: Implications for Treatment Outcomes and Immune-Related Adverse Events in Patients with Triple Negative Breast Cancer Enrolled in the GeparNuevo Trial Background: Significant research has been conducted on the influence of immune checkpoint inhibitor therapy on tumor microenvironment, particularly with regard to tumor-infiltrating immune cells. Nevertheless, our understanding of the circulating immune repertoire and its association with treatment outcomes remains limited. Consequently, our subproject of the GeparNuevo trial aimed to explore the RNA phenotype of circulating leukocytes and its impact on overall survival (OS), and adverse events of patients enrolled in the GeparNuevo trial (Loibl S et al. Annals Oncol 2022). Methods: The GeparNuevo phase II trial focused on the effects of neoadjuvant nab-paclitaxel followed by epirubicin/cyclophosphamide (nabP-EC) chemotherapy combined with the anti-PD-L1 immune checkpoint inhibitor durvalumab versus placebo in patients with non-metastatic triple-negative breast cancer. RNA-stabilizing PAXgene tubes were used to collect blood samples prior to treatment initiation. RNA was extracted from circulating leukocytes of 117 patients and analyzed using a custom NanoString nCounter CodeSet, including 290 immune-related target genes. The associations between 16 immune cell scores, 26 immune signaling scores, 31 individual gene expression patterns, OS, and immune-related adverse events (irAEs) were analyzed. Results: Univariate Cox regression analysis using continuous scores revealed a significant correlation between PIP3 activates AKT signaling, T cells, CDK2, and TIMP1 expression with OS in the placebo arm. Higher expression of PIP3 activates AKT signaling, T cells, and CDK2, as well as lower expression of TIMP1, were associated with prolonged survival. Notably, T cell scores and CDK2 expression exhibited a significant interaction with the treatment arm (p=0.0489 and 0.0210, respectively). Multivariate Cox regression analysis demonstrated a significant association of DPP4, ICOS and MYC expression with OS. Additionally, CDK2, CDKN2A, F5 and HLA-DRA expression were linked to the presence of irAEs. In the durvalumab arm, TNFR2 non canonical NFkB pathway signaling, CDK2 and CDKN2A expression showed an inverse association with the occurrence of irAEs. Conclusions: Our study provides preliminary evidence that RNA derived from circulating leukocytes may serve as a potential biomarker for predicting treatment outcomes and identifying patients prone to develop side effects during standard-of-care chemotherapy or immune checkpoint therapy. These findings highlight the potential utility of peripheral immune cell RNA profiling in improving treatment strategies and patient management. Further research and validation are necessary to fully comprehend the clinical significance and broader implications of these findings. Citation Format: Hanna Hübner, Matthias Ruebner, Andreas Schneeweiss, Carsten Denkert, Hans-Peter Sinn, Michael Braun, Thomas Karn, Bruno V. Sinn, Dirk-Michael Zahm, Jörg Thomalla, Jens Huober, Claus Hanusch, Michael Untch, Christine Solbach, Theresa Link, Natalie Filmann, Julia Rey, Johannes Holtschmidt, Sibylle Loibl, Peter A. Fasching. Exploring Circulating Leukocyte RNA Expression: Implications for Treatment Outcomes and Immune-Related Adverse Events in Patients with Triple Negative Breast Cancer Enrolled in the GeparNuevo Trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS14-07.

Abstract PO5-22-12: Development and Validation of a Genomic Test to Predict Tumor Response of the Axillary Nodes after Neoadjuvant Chemotherapy (NAC) in Patients with HER2-negative Breast Cancer: Results of the AGO-35 trial

Abstract Background: De-escalation of axillary surgery in patients with lymph node-positive breast cancer is currently being tested in ongoing trials for patients with downstaged nodes after neoadjuvant chemotherapy (NAC). A proportion of such patients will achieve an excellent axillary node response to NAC, i.e. conversion to pathologic node-negative status after chemotherapy. Genomic prediction of tumor chemosensitivity before final surgery may identify which patients could safely undergo conservative surgical management of their regional lymph nodes. Methods: The AGO-35 (NCT0203274) is a prospective trial for patients with HER2 negative breast cancer scheduled for neoadjuvant chemotherapy that includes an anthracycline- based regimen followed by 3-4 cycles of taxanes. The LN predictor is a newly developed model for predicting the probability of pathological lymph node-positivity after NAC in triple-negative (TNBC) or hormone-receptor positive (HR+) breast cancer. To calculate the LN predictor, which is developed in microarray, we calibrated the normalized individual gene module and SET ER/PR from RNAseq to the Affymetrix U133A platform using independently combined total of 220 pairs of TNBC samples with both RNAseq and U133A microarray results. The logistic regression model is applied for the prediction of the binary outcome, pathological lymph node-positive or negative after NAC. The cut-points of LN predictor in TNBC and HR+ samples were pre-determined for each subtype of breast cancer and then, independent, blinded evaluation in the AGO-35 trial samples. Results: A total of 251 patients with HER2 negative breast treated with neoadjuvant chemotherapy were included. The LN predictor was validated on 189 qualified samples with wtRNA sequence. Statistical analysis of 181 samples is shown in Table 1. Conclusion: Preliminary results of the AGO-35 trial show that the newly developed molecular LN predictor may accurately predict chemosensitivity response to NAC, in high proportion of patients with TNBC. Supported by: Wissenschaftsfonds (FWF) KLI 406 EUBREAST study group AGO Österreich Breast Cancer Research Foundation (BCRF-0158) Table 1. Genomic prediction of chemosensitivity Citation Format: Florentia Peintinger, Thorsten Kühn, Hans-Christian Kolberg, Roland Reitsamer, Sabine Schmatloch, Elisa Sieghartsleitner, Lili Du, Andrea Berghold, W. Fraser Symmans. Development and Validation of a Genomic Test to Predict Tumor Response of the Axillary Nodes after Neoadjuvant Chemotherapy (NAC) in Patients with HER2-negative Breast Cancer: Results of the AGO-35 trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-22-12.

Metabolomics of Multiple Sclerosis Lesions Demonstrates Lipid Changes Linked to Alterations in Transcriptomics-Based Cellular Profiles.

People with multiple sclerosis (MS) have a dysregulated circulating metabolome, but the metabolome of MS brain lesions has not been studied. The aims of this study were to identify differences in the brain tissue metabolome in MS compared with controls and to assess its association with the cellular profile of corresponding tissue. MS tissues included samples from the edge and core of chronic active or inactive lesions and periplaque white matter (WM). Control specimens were obtained from normal WM. Metabolomic analysis was performed using mass-spectrometry coupled with liquid/gas chromatography and subsequently integrated with single-nucleus RNA-sequencing data by correlating metabolite abundances with relative cell counts, as well as individual genes using Multiomics Factor Analysis (MOFA). Seventeen samples from 5 people with secondary progressive MS and 8 samples from 6 controls underwent metabolomic profiling identifying 783 metabolites. MS lesions had higher levels of sphingosines (false discovery rate-adjusted p-value[q] = 2.88E-05) and sphingomyelins and ceramides (q = 2.15E-07), but lower nucleotide (q = 0.05), energy (q = 0.001), lysophospholipid (q = 1.86E-07), and monoacylglycerol (q = 0.04) metabolite levels compared with control WM. Periplaque WM had elevated sphingomyelins and ceramides (q = 0.05) and decreased energy metabolites (q = 0.01) and lysophospholipids (q = 0.05) compared with control WM. Sphingolipids and membrane lipid metabolites were positively correlated with astrocyte and immune cell abundances and negatively correlated with oligodendrocytes. On the other hand, long-chain fatty acid, endocannabinoid, and monoacylglycerol pathways were negatively correlated with astrocyte and immune cell populations and positively correlated with oligodendrocytes. MOFA demonstrated associations between differentially expressed metabolites and genes involved in myelination and lipid biosynthesis. MS lesions and perilesional WM demonstrated a significantly altered metabolome compared with control WM. Many of the altered metabolites were associated with altered cellular composition and gene expression, indicating an important role of lipid metabolism in chronic neuroinflammation in MS.

Genetic Diversity of Plasmodium vivax Surface Ookinete Protein Pvs25 and Host Genes in Individuals Living along the Thai–Myanmar Border and Their Relationships with Parasite Density

Plasmodium vivax (Pv) accounts for over 50% of malaria cases in Latin America and Asia. Despite a significant reduction in Pv transmission in Thailand, the parasite remains endemic to the border areas. This study aimed to investigate the genetic diversity of the parasites and the host factors, as well as their relation to parasite density in Pvisolates, along the Thai–Myanmar border. Genetic variations in Pv markers, specifically the ookinete surface protein Pvs25, and host genes, including Toll-like receptor 6 (TLR6), TLR9, TIR Domain-containing adaptor protein (TIRAP), Toll-interacting protein (TOLLIP), Duffy antigen receptor for chemokines (DARC), and intercellular adhesion molecule 1 (ICAM-1), were investigated using polymerase chain reaction (PCR) with restriction fragment length polymorphism (RFLP). A total of 548 PCR-positive Pv samples collected from Tak and Kanchanaburi provinces during two periods (2006–2007 and 2014–2016) were included in the study. Pvs25 exhibited four haplotypes, with H1 (EGTKV) being the most prevalent in both provinces. Kanchanaburi isolates exhibited greater genetic diversity than Tak isolates. No significant deviations from neutrality were observed for Pvs25 in either area. ICAM-1 and TOLLIP s3750920 heterozygous carriers had greater median parasite densities than homozygous mutants. The TLR9 rs187084 T genotype had a significantly higher parasite density than the non-T genotype. The findings underscore the significant association between the rs3750920 C/T, rs5498 A/G, and rs187084 T genotypes and high parasite density in patients infected with Pv, highlighting their potentially critical role in malaria susceptibility.

The efficacy of single mitochondrial genes at reconciling the complete mitogenome phylogeny-a case study on dwarf chameleons.

Although genome-scale data generation is becoming more tractable for phylogenetics, there are large quantities of single gene fragment data in public repositories and such data are still being generated. We therefore investigated whether single mitochondrial genes are suitable proxies for phylogenetic reconstruction as compared to the application of full mitogenomes. With near complete taxon sampling for the southern African dwarf chameleons (Bradypodion), we estimated and compared phylogenies for the complete mitogenome with topologies generated from individual mitochondrial genes and various combinations of these genes. Our results show that the topologies produced by single genes (ND2, ND4, ND5, COI, and COIII) were analogous to the complete mitogenome, suggesting that these genes may be reliable markers for generating mitochondrial phylogenies in lieu of generating entire mitogenomes. In contrast, the short fragment of 16S commonly used in herpetological systematics, produced a topology quite dissimilar to the complete mitogenome and its concatenation with ND2 weakened the resolution of ND2. We therefore recommend the avoidance of this 16S fragment in future phylogenetic work.

Cauchy hyper-graph Laplacian nonnegative matrix factorization for single-cell RNA-sequencing data analysis.

Many important biological facts have been found as single-cell RNA sequencing (scRNA-seq) technology has advanced. With the use of this technology, it is now possible to investigate the connections among individual cells, genes, and illnesses. For the analysis of single-cell data, clustering is frequently used. Nevertheless, biological data usually contain a large amount of noise data, and traditional clustering methods are sensitive to noise. However, acquiring higher-order spatial information from the data alone is insufficient. As a result, getting trustworthy clustering findings is challenging. We propose the Cauchy hyper-graph Laplacian non-negative matrix factorization (CHLNMF) as a unique approach to address these issues. In CHLNMF, we replace the measurement based on Euclidean distance in the conventional non-negative matrix factorization (NMF), which can lessen the influence of noise, with the Cauchy loss function (CLF). The model also incorporates the hyper-graph constraint, which takes into account the high-order link among the samples. The CHLNMF model's best solution is then discovered using a half-quadratic optimization approach. Finally, using seven scRNA-seq datasets, we contrast the CHLNMF technique with the other nine top methods. The validity of our technique was established by analysis of the experimental outcomes.

Influence of some abiotic factors on the apple stressassociated protein gene expression profiles

In plants, stress-associated proteins (SAP) play a crucial role in responding to adverse biotic and abiotic factors. In this study, we evaluated the expression profiles of 14 apple SAP genes (MdSAP) under the influence of low and high temperatures, as well as salinity, using the quantitative polymerase chain reaction (qPCR). The results showed that the most significant changes in expression levels were observed in the genes MdSAP11, MdSAP2, and MdSAP3 under high temperature conditions, MdSAP1, MdSAP2, MdSAP4, and MdSAP6 under low temperature conditions, and MdSAP1, MdSAP8, and MdSAP11 under salt stress. Furthermore, there was a tendency for gene expression to increase at 2 and/or 4 hours of exposure followed by a decrease at 24 hours. The analysis of the correlation between the expression levels of MdSAP genes revealed both positive and negative linear relationships.These findings will help elucidate the specific roles of individual SAP genes in shaping the apple stress response to low and high temperatures, as well as salinity.