Compared Gene Expression Profiles Research Articles

Abstract PO1-03-09: Transcriptomic profiling of ER-negative and ER-low, HER2-negative breast cancer and its implications on immune regulation

Abstract Background: The recommended cut-off of < 1% positive cells to define estrogen receptor (ER) negative status in breast cancer (BC) is highly debated. Few data exist on transcriptomic features of ER-low BC, suggesting no major differences vs ER-neg. Among HER2-negative BCs, we compared gene-expression profiling (including PAM50 intrinsic subtyping) according to ER status (ER-neg: 0% vs. ER-low: 1-9%) and assessed its impact on Tumor-infiltrating lymphocytes (TILs) levels. Methods: Across two cancer centers, stage I-III HER2-negative BCs FFPE samples were reviewed for ER status. All ER-low (ER 1-9%) samples were identified and a cohort of ER-neg BC samples, matched for age and stage, was also retrieved. A small cohort of pts with intermediate ER expression (ER-int: 10-50%) was included as a control cohort. Expression of 776 BC–related genes was evaluated by nCounter® (Breast Cancer 360TM Panel) and intrinsic molecular subtyping was determined using the PAM50 subtype predictor. A False Discovery Rate (FDR) corrected unpaired two-class SAM was used to identify genes differentially expressed in different subgroups. TILs were evaluated on archival H&E slides following guidelines. Results: Of 116 stage I-III HER2-neg BC pts included, 39 had ER-neg BC, 65 had ER-low BC, and 12 had ER-int disease. PAM50 intrinsic subtype distribution was similar in ER-neg and ER-low BCs, (with an enrichment in basal-like tumors), while both subgroups differed significantly from ER-int samples (Table). As compared to ER-neg tumors, ER-low BCs showed significantly higher expression of GATA3 gene and lower expression of EDN1 and PROM1 genes by SAM analysis (FDR< 5%); however, when the analysis was limited to PAM50 basal-like tumors (N=77) we only identified two genes that were downregulated in ER-low BCs (EDN1, PROM1) as compared to ER-neg BCs. On the contrary, significant differences in transcriptomic regulation were observed between ER-low and ER-int BCs, with ER-low BCs showing significantly higher expression of 53 genes and lower expression of 398 genes, as compared to ER-int (SAM analysis FDR< 5%). We previously reported similar TIL levels in ER-low and ER-neg tumors. In both ER-low and ER-int BCs, PAM50 basal-like subtype was associated with significantly higher TILs: median TIL levels 20 (range 0-80) and 6 (range 1-40) for PAM50 Basal-like BCs versus other PAM50 subtypes respectively in ER-low samples (p< 0.001); median TIL levels 53 (range 25-80) and 5 (range 0-10) for PAM50 Basal-like BCs versus other PAM50 subtypes respectively in ER-int samples (p=0.036). No significant difference in TIL levels according to PAM50 subtyping were observed in ER-neg tumors. No significant difference in mRNA levels for PD-1 (PDCD1) and PD-L1 (CD274) genes was observed between ER-low and ER-neg tumors. Conclusions: Among patients with HER2-neg BC, gene-expression profiling is similar in ER-neg (i.e., < 1%) vs. ER-low tumors with a high prevalence of PAM50 Basal-like tumors and few differentially expressed genes. In both ER-low and ER-int HER2-neg BCs, PAM50 Basal-like subtype is also associated with higher TIL levels. Our results strongly indicate that HER2-neg/ER-low BCs and HER2-neg/ER-neg BCs are similar biological entities, thus further supporting the use of similar treatments in patients with early-stage ER-low and triple-negative BC, including immune checkpoint inhibitors. PAM50 intrinsic subtype distribution according to ER levels Citation Format: Gaia Griguolo, Claudio Vernieri, Davide Massa, Lorenzo Nicolè, Claudia Pinato, Federica Miglietta, Andrea Vingiani, Silvia Brich, Riccardo Lobefaro, Francesca Schiavi, Matteo Fassan, Giancarlo Pruneri, Valentina Guarneri, Maria Vittoria Dieci. Transcriptomic profiling of ER-negative and ER-low, HER2-negative breast cancer and its implications on immune regulation [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 PO1-03-09.

A Principal Components Analysis and Functional Annotation of Differentially Expressed Genes in Brain Regions of Gray Rats Selected for Tame or Aggressive Behavior.

The process of domestication, despite its short duration as it compared with the time scale of the natural evolutionary process, has caused rapid and substantial changes in the phenotype of domestic animal species. Nonetheless, the genetic mechanisms underlying these changes remain poorly understood. The present study deals with an analysis of the transcriptomes from four brain regions of gray rats (Rattus norvegicus), serving as an experimental model object of domestication. We compared gene expression profiles in the hypothalamus, hippocampus, periaqueductal gray matter, and the midbrain tegmental region between tame domesticated and aggressive gray rats and revealed subdivisions of differentially expressed genes by principal components analysis that explain the main part of differentially gene expression variance. Functional analysis (in the DAVID (Database for Annotation, Visualization and Integrated Discovery) Bioinformatics Resources database) of the differentially expressed genes allowed us to identify and describe the key biological processes that can participate in the formation of the different behavioral patterns seen in the two groups of gray rats. Using the STRING- DB (search tool for recurring instances of neighboring genes) web service, we built a gene association network. The genes engaged in broad network interactions have been identified. Our study offers data on the genes whose expression levels change in response to artificial selection for behavior during animal domestication.

Abstract 1798: Glyoxalase 1 (GLO1) as an oncometabolic enabler of prostate cancer progression: CRISPR/Cas9-based GLO1-deletion from human DU145-Luc2 cells blocks EMT and bone metastasis in SCID mice

Abstract Glyoxalase 1 (encoded by GLO1) is a glutathione-dependent enzyme detoxifying the glycolytic byproduct methylglyoxal (MG), an oncometabolite involved in metabolic reprogramming. Recently, we have demonstrated that GLO1 is overexpressed in human prostate cancer cells and patient tumors. In order to inform the ongoing debate on the role of GLO1 as an oncometabolic enabler of tumor glucose metabolism and malignant progression, we performed CRISPR/Cas9-based GLO1-deletion in human DU145-Luc2 malignant prostate cancer cells. NanoString nCounterTM ('PanCancer-Progression-Panel') comparative gene expression profiling (GLO1_KO versus GLO1_wt) revealed a significant downregulation of EMT-related pathways in GLO1_KO cells; concordantly, phenotypical screening indicated a pronounced attenuation of matrigel invasiveness observable in GLO1_KO cells. Likewise, inclusion of MG or a small molecule GLO1 inhibitor (TLSC-702) blocked invasiveness of DU145 GLO1_wt cells. Downregulation of EMT-related genes (including MMP3, SPP1, CXCL8) was accompanied by increased expression of TXNIP (thioredoxin-interacting protein), a master regulator of cellular energy metabolism and redox homeostasis. In a bioluminescent SCID mouse bone metastasis model (intracardial injection of DU145-Luc2 GLO1_wt and GLO1_KO cells), GLO1 expression was necessary to cause mandibular bone metastases (as evidenced by the complete absence of bone metastases after GLO1 deletion). Given the availability of drug-like small molecule inhibitors of GLO1 enzymatic activity these data suggest that GLO1 represents a novel molecular target for the pharmacological suppression of prostate cancer bone metastasis. Citation Format: Jana Jandova, Anne E. Cress, Georg T. Wondrak. Glyoxalase 1 (GLO1) as an oncometabolic enabler of prostate cancer progression: CRISPR/Cas9-based GLO1-deletion from human DU145-Luc2 cells blocks EMT and bone metastasis in SCID mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1798.

Abstract 3919: Targeting tissue-resident macrophage progenitors by anti-IL34 restores anti-tumor immunity and suppresses tumor growth

Abstract Macrophages play important roles in tumor microenvironment, where they can promote immune suppression, tumor growth and resistance to checkpoint inhibitor therapy and are the focus of efforts to develop novel cancer therapeutics. Strategies to inhibit macrophage accumulation to suppress tumor growth by blocking their recruitment from circulation have been only partially effective, leading to the identification of roles in tumor progression for tissue resident macrophages that are seeded during embryonic development. We examined the origins of lung tumor associated macrophages using lineage tracing, flow cytometry, and single cell sequencing. By comparing gene expression profiles of resident F4/80hi Lyve1+ Ly6C- macrophages and Ly6C+ F4/80lo CCR2+ recruited macrophages in subcutaneous, lung, central nervous and renal normal and tumor tissues, we identified generic tissue resident macrophage and recruited macrophage transcriptomic signatures, revealing that resident macrophages are characterized by signatures of Myc driven proliferation, fatty acid and cholesterol metabolism and immune suppression, while recruited macrophages are characterized by signature of inflammation and immune stimulation. Resident macrophages were associated with worse outcomes in cancer patients than recruited macrophages. Single cell sequencing and flow cytometry of genetically engineered mouse models of CC10-Cre KrasG12D;p53-/- and SPC-Cre KrasG12D lung cancer revealed that lung tumor progression was characterized by extensive expansion of immune suppressive, Ki67+ resident alveolar macrophages and loss of T cells in the lung. Single cell transcriptomics identified IL-34 expressed exclusively by tumor cells as a possible driver of alveolar macrophage proliferation and IGF1 expressed by tumor associated alveolar macrophages as a possible regulator of tumor cell proliferation. Accordingly, blockade of IL-34 profoundly reduced tissue resident macrophage proliferation, stimulated recruitment of activated T cells, and suppressed growth of KrasG12D driven tumors. Analysis of TCGA data sets revealed that expression of IGF1 or IL34 was associated with reduced survival in lung, kidney, liver, head and neck and ovarian cancer. These studies identify key roles for IL-34 and IGF1 in solid tumor progression and suggest new therapeutic targets for the development of therapeutic strategies to stimulate anti-cancer immunity. Citation Format: Hui Chen, Jaroslav Zak, Mark Paradise, Guangfang Wang, John Teijaro, Mark Onaitis, Judith Varner. Targeting tissue-resident macrophage progenitors by anti-IL34 restores anti-tumor immunity and suppresses tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3919.

Abstract 541: Establishment of Inotuzumab ozogamicin resistant leukemia cell lines; Focusing on p-gp and DNA damage repair for overcoming drug resistance

Abstract Introduction: Inotuzumab Ozogamicin (IO) is a humanized anti-CD22 monoclonal antibody conjugated with chalicheamicin, which causes single- and double-strand DNA breaks. Although IO shows a high CR/CRi rate in relapsed/refractory CD22-positive B-cell acute lymphoblastic leukemia (ALL), the duration of remission is short due to early acquisition of resistance. Therefore, the present study attempted to enhance the cytotoxicity of IO by focusing on polyadenosine diphosphate-ribose polymerase (PARP) inhibition. Furthermore, IO-resistant cell lines were newly established to investigate the mechanism of IO resistance. Methods: Reh cell line derived from Ph-negative B-ALL cells was used. To establish IO-resistant cell lines, Reh cells were long-term exposed to IO, followed by limiting dilution. FACS analysis evaluated the CD22 positivity and the induction of apoptosis. WTS assay determined the cell growth inhibition effects. The Comet assay quantitatively assessed DNA damage. Gene expression profiles were determined by using DNA microarray. Results: The 50%-inhibitory concentration (IC50) of IO was 2.1 ng/mL in Reh cells. Reh cells were synergistically sensitized to IO by the addition of non-toxic concentrations of PARP inhibitors, olaparib or talazoparib, with the Combination Index of 0.19 and 0.42, respectively. Moreover, these combinations augmented the induction of apoptosis (23.5% in IO alone, 51.7% in IO + olaparib, 66.1% in IO + talazoparib). The Comet assay demonstrated that DNA strand breaks observed 1h after IO administration were repaired 6h later. In the combination of IO with olaparib or talazoparib, DNA damage remained even at 6h, suggesting the inhibition of DNA repair by PARP inhibitors. Next, six IO-resistant cell lines were established with IC50 more than 60-fold that of parental cells. All these cell lines kept CD22 expression. These cell lines were also resistant to apoptosis induced by IO. In addition of olaparib or talazoparib to IO, the IC50 of IO decreased by 40% to 70% in 6 IO-resistant cells. These combinations also augmented the induction of apoptosis (24.2% in IO alone, 45.3% in IO+olaparib, and 56.8% in IO+talazoparib). DNA microarray was performed to compare gene expression profiles between parental and IO-resistant cell lines. ABCB1 (p-gp), a drug efflux pump that causes multi-drug resistance, was overexpressed in resistant cell lines. Moreover, the addition of p-gp inhibitor, PSC-833, to IO enhanced the cell growth inhibition and induction of apoptosis due to more DNA strand break. Conclusion: The present study demonstrated that the combination of IO with PARP inhibitors exerted synergistic cytotoxicity via the inhibition of DNA repair. In addition, it was suggested that overexpression of p-gp caused IO resistance, which was partially reversed with the combination of PARP inhibitors or a p-gp inhibitor. Citation Format: Naoko Ida, Miyuki Okura, Naoko Hosono, Takahiro Yamauchi. Establishment of Inotuzumab ozogamicin resistant leukemia cell lines; Focusing on p-gp and DNA damage repair for overcoming drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 541.

Single-cell transcriptomic sequencing identifies subcutaneous patient-derived xenograft recapitulated medulloblastoma.

Medulloblastoma (MB) is one of the most common malignant brain tumors that mainly affect children. Various approaches have been used to model MB to facilitate investigating tumorigenesis. This study aims to compare the recapitulation of MB between subcutaneous patient-derived xenograft (sPDX), intracranial patient-derived xenograft (iPDX), and genetically engineered mouse models (GEMM) at the single-cell level. We obtained primary human sonic hedgehog (SHH) and group 3 (G3) MB samples from six patients. For each patient specimen, we developed two sPDX and iPDX models, respectively. Three Patch+/- GEMM models were also included for sequencing. Single-cell RNA sequencing was performed to compare gene expression profiles, cellular composition, and functional pathway enrichment. Bulk RNA-seq deconvolution was performed to compare cellular composition across models and human samples. Our results showed that the sPDX tumor model demonstrated the highest correlation to the overall transcriptomic profiles of primary human tumors at the single-cell level within the SHH and G3 subgroups, followed by the GEMM model and iPDX. The GEMM tumor model was able to recapitulate all subpopulations of tumor microenvironment (TME) cells that can be clustered in human SHH tumors, including a higher proportion of tumor-associated astrocytes and immune cells, and an additional cluster of vascular endothelia when compared to human SHH tumors. This study was the first to compare experimental models for MB at the single-cell level, providing value insights into model selection for different research purposes. sPDX and iPDX are suitable for drug testing and personalized therapy screenings, whereas GEMM models are valuable for investigating the interaction between tumor and TME cells.

Transcriptomic analyses to summarize gene expression patterns that occur during leaf initiation of Chinese cabbage.

In Chinese cabbage, rosette leaves expose their adaxial side to the light converting light energy into chemical energy, acting as a source for the growth of the leafy head. In the leafy head, the outer heading leaves expose their abaxial side to the light while the inner leaves are shielded from the light and have become a sink organ of the growing Chinese cabbage plant. Interestingly, variation in several ad/abaxial polarity genes is associated with the typical leafy head morphotype. The initiation of leaf primordia and the establishment of leaf ad/abaxial polarity are essential steps in the initiation of marginal meristem activity leading to leaf formation. Understanding the molecular genetic mechanisms of leaf primordia formation, polar differentiation, and leaf expansion is thus relevant to understand leafy head formation. As Brassica's are mesa-hexaploids, many genes have multiple paralogues, complicating analysis of the genetic regulation of leaf development. In this study, we used laser dissection of Chinese cabbage leaf primordia and the shoot apical meristem (SAM) to compare gene expression profiles between both adaxial and abaxial sides and the SAM aiming to capture transcriptome changes underlying leaf primordia development. We highlight genes with roles in hormone pathways and transcription factors. We also assessed gene expression gradients along expanded leaf blades from the same plants to analyze regulatory links between SAM, leaf primordia and the expanding rosette leaf. The catalogue of differentially expressed genes provides insights in gene expression patterns involved in leaf development and form a starting point to unravel leafy head formation.

Gene expression profiles associated with early relapse during first remission induction in canine multicentric high-grade B-cell lymphoma.

Although chemotherapy using CHOP-based protocol induces remission in most cases of canine multicentric high-grade B-cell lymphoma (mhBCL), some cases develop early relapse during the first induction protocol. In this study, we examined the gene expression profiles of canine mhBCL before chemotherapy and investigated their associations with early relapse during the first whole CHOP-based protocol. Twenty-five cases of mhBCL treated with CHOP-based protocol as first induction chemotherapy were included in this study. Sixteen cases completed the first whole CHOP-based protocol without relapse (S-group), and nine developed relapse during the chemotherapy (R-group). RNA-seq was performed on samples from neoplastic lymph nodes. Differentially expressed genes (DEGs) were extracted by the comparison of gene expression profiles between S- and R-groups, and the differences in the expression levels of these genes were validated by RT-qPCR. Extracted 179 DEGs included the genes related to chemokine CC motif ligand, T-cell receptor signaling pathway, and PD-L1 expression and PD-1 checkpoint pathway. We focused on chemokine CC motif ligand, and CCL4 was confirmed to be significantly downregulated in the R-group (P=0.039). We also focused on the genes related to T-cell signaling pathway, and CD3E (P=0.039), ITK (P=0.023), and LAT (P=0.023) genes were confirmed to be significantly upregulated in the R-group. The current results suggest that both changes in tumor cells and the interactions between tumor cells and immune cells are associated with the efficacy of the chemotherapy for first remission induction.

Single-Cell Sequencing Technology and Its Application in the Study of Central Nervous System Diseases.

The mammalian central nervous system consists of a large number of cells, which contain not only different types of neurons, but also a large number of glial cells, such as astrocytes, oligodendrocytes, and microglia. These cells are capable of performing highly refined electrophysiological activities and providing the brain with functions such as nutritional support, information transmission and pathogen defense. The diversity of cell types and individual differences between cells have brought inspiration to the study of the mechanism of central nervous system diseases. In order to explore the role of different cells, a new technology, single-cell sequencing technology has emerged to perform specific analysis of high-throughput cell populations, and has been continuously developed. Single-cell sequencing technology can accurately analyze single-cell expression in mixed-cell populations and collect cells from different spatial locations, time stages and types. By using single-cell sequencing technology to compare gene expression profiles of normal and diseased cells, it is possible to discover cell subsets associated with specific diseases and their associated genes. Therefore, scientists can understand the development process, related functions and disease state of the nervous system from an unprecedented depth. In conclusion, single-cell sequencing technology provides a powerful technology for the discovery of novel therapeutic targets for central nervous system diseases.

Comparative responses of flocculating and nonflocculating yeasts to cell density and chemical stress in lactic acid fermentation.

While flocculation has demonstrated its efficacy in enhancing yeast robustness and ethanol production, its potential application for lactic acid fermentation remains largely unexplored. Our study examined the differences between flocculating and nonflocculating Saccharomyces cerevisiae strains in terms of their metabolic dynamics when incorporating an exogenous lactic acid pathway, across varying cell densities and in the presence of lignocellulose-derived byproducts. Comparative gene expression profiles revealed that cultivating a nonflocculant strain at higher cell density yielded a substantial upregulation of genes associated with glycolysis, energy metabolism, and other key pathways, resulting in elevated levels of fermentation products. Meanwhile, the flocculating strain displayed an inherent ability to sustain high glycolytic activity regardless of the cell density. Moreover, our investigation revealed a significant reduction in glycolytic activity under chemical stress, potentially attributable to diminished ATP supply during the energy investment phase. Conversely, the formation of flocs in the flocculating strain conferred protection against toxic chemicals present in the medium, fostering more stable lactic acid production levels. Additionally, the distinct flocculation traits observed between the two examined strains may be attributed to variations in the nucleotide sequences of the flocculin genes and their regulators. This study uncovers the potential of flocculation for enhanced lactic acid production in yeast, offering insights into metabolic mechanisms and potential gene targets for strain improvement.

Detection of Occurrent Somatic Hypermutation and Associated Gene Expression Profile in Single B Cells

Introduction Somatic Hypermutation (SHM) is an endogenous mutator mechanism in B lymphocytes that preferentially targets the immunoglobulin V(D)J coding region and facilitates antibody affinity maturation. The exact frequency and impact of SHM in individual B cells remains elusive, as previous quantifications only considered cumulative outcomes at population level. We therefore employed single-cell RNA sequencing on primary follicular lymphoma (FL) cells, a B-cell malignancy retaining the germinal center (GC) B-cell phenotype, to identify and quantify individual SHM events. This approach allowed us to detect neovariants, defined as single nucleotide differences that originate from recent SHM events, and to explore the gene expression profiles of the B lymphoma cells with neovariants, particularly focusing on genes and pathways effectuating SHM. Methods We collected and processed samples from 12 follicular lymphoma (FL) and five chronic lymphocytic leukemia (CLL) patients as non-germinal center controls. Single cell whole cDNA libraries were prepared by 10X Genomics. Immunoglobulin gene single cell libraries were prepared by enrichment with semi-nested amplification using 3′ constant domain primers followed by 10X Genomics prep. Both single cell libraries were sequenced in paired-end mode on Illumina HiSeq 4000. We developed a customized method to preprocess scBCR data designed specifically for highly mutated FL BCR sequences. To ensure data quality and validity of putative neovariants, we applied a comprehensive filtering system, including stringent thresholds in terms of numbers of individual immunoglobulin (IG) mRNA molecules sequenced as defined by their UMI, and reads per position obtained from the median of the estimator's distribution, base quality scores, exclusion of cell doublets, position within the V(D)J region, and proportion of cell-level variants. Based on a reported IG mRNA half-life of 3 h and assuming a first-order decay, the filtering criteria implied that detected neovariants were created between 1 and 7 hours prior to the time of sampling. Results A median of 1112 cells (range: 215-16432) per case were analyzed. 1239 IG neovariants were detected in a total of 334 FL cells in 7 of the 12 FL cases. Neovariants were undetectable in control CLL cells. A remarkably high proportion (95,4%) of neovariants were found within known AID motifs (WRCY, WA, and RCG), suggesting active AID involvement in the hypermutation process. The observed fractions of FL cells carrying recent SHM events indicated initiation of FL pathophysiology at least 11,2 months prior to sampling. To further evaluate the role of AID, we compared gene expression profiles between FL cells with and without neovariants. The results demonstrated not only higher expression of AID but as well of pathways involved in SHM. Mismatch repair (hsa03430, 9 enriched genes with p.adjust < 0.001) and base excision repair (hsa03410, 17 enriched genes with p.adjust = 0.01), together with DNA replication (hsa03030, 14 enriched genes with p.adjust < 0.001), were the three most prominent pathways with upregulated genes. In cells with detectable neovariants, the MLH1/MSH2 genes, associated with the recognition of the deamination process, as well as POLH gene, which is involved in error-prone mutations in MMR, were highly expressed. Overall, we observed a more prominent expression of genes related to MMR than BER in cells with neovariants supported by a higher normalized enrichment score (NES) in MMR (MMR:1.93 and BER:1.6). This suggests not only an active SHM process but also potential interplay between AID and MMR in driving SHM in FL. Conclusions Here we report for the first time quantitative detection of ongoing somatic hypermutation at single cell level in FL. Based on the lifespan of IG mRNA, simultaneous detection of two IG mRNA transcripts differing by one (or a few) nucleotides only within a single cell permits to estimate the timing when this SHM event occurred. The detection of this phenomenon in 58% of FL samples and its association with AID expression suggests that AID-induced mutagenesis might be acting at a much higher rate than expected. Quantification of SHM at the single-cell level should facilitate the understanding of the cellular heterogeneity and dynamics in physiological GC reactions but also may offer novel insights into the AID-dependent shaping of the genomic landscape of germinal center B-cell neoplasms.

Longitudinal Noninvasive Surveillance & Fragmentomic Characterization of Follicular Lymphoma

Introduction While cell-free DNA (cfDNA) plays an increasingly defined role in aggressive lymphomas, its characteristics in indolent lymphomas are less established. Many follicular lymphoma (FL) patients experience durable remissions and late relapses ( Fig A). We therefore explored circulating tumor DNA (ctDNA) kinetics during long-term blood-based surveillance in patients with FL. In addition to noninvasive detection using somatic mutations, we also evaluated gene expression inference from cfDNA utilizing a novel fragmentomic method to detect histological transformation (tFL). Methods We studied 121 serial samples in a cohort of 17 FL patients (median 7 per patient) with long term clinical and blood-based MRD monitoring (median 5.6 years, max 10.8). We considered serial ctDNA status at 4 milestones: baseline/pre-treatment, post-treatment, in radiographic remission (MRD), and at relapse. We also profiled pre-treatment cfDNA for 9 tFL cases. Cases were genotyped to identify somatic mutations from diagnostic tumor or baseline plasma, with matched PBMCs used to censor germline variants and clonal hematopoiesis. ctDNA status in subsequent samples was evaluated via CAPP-Seq (SNVs) and PhasED-Seq (PVs). The cfDNA fragmentation profiles of pre-treatment FL and control samples were evaluated for inferred gene expression via EPIC-seq (Esfahani Nat Biotech 2022) using a novel 1676-gene panel focused on lymphoid neoplasms, including classic and transformed FL. Results Treatment & Relapse Kinetics Most patients had advanced disease (88% stage III-IV) and Low to Intermediate risk FLIPI (71% 0-2). At first cfDNA evaluation, 9/17 (53%) were previously untreated. Median pre-treatment ctDNA levels were 73.4 hGE/mL in FL, as compared to 236 hGE/mL in DLBCL (Kurtz JCO 2018). Post-treatment, median ctDNA levels dropped to 1.2 hGE/mL. Depth of response was similar between rituximab monotherapy and cytotoxic regimens, with median -2.2 log10 fold change from baseline. MRD Detection PVs were observed in all tumors, consistent with expected aberrant somatic hypermutation in FL. To evaluate MRD detection via PhasED-Seq, we considered plasma timepoints with undetectable ctDNA by CAPP-Seq (n=26) from patients (n=9) with subsequent clinical relapse. MRD was detected in 11 additional specimens (42%) by PhasED-Seq. Among FL patients tested for MRD while in radiographic remission, low-level ctDNA was detectable in 62% of plasma specimens (8/13) obtained within 1 month of negative PET/CT, suggesting potential additional utility for surveillance. Long-term Genomic Stability Sequential biopsies of FL tumors have shown genomic heterogeneity between diagnosis and relapse. We therefore evaluated ctDNA concordance across variant classes & regions. Relative AF was calculated pre-treatment and at relapse (median 2 years between samples, range 1-8 years), normalized by total ctDNA levels. The most stable lesions included missense mutations in CREBBP and TP53, nonsense KMT2D mutations, and BCL2 5'-UTR variants. BCL6 intronic mutations and chr2/chr14 SHM varied between diagnosis/relapse. Plasma versus Cellular Concentrations In contrast to DLBCL, circulating FL cells are frequently detectable at low levels in blood. To compare relative FL concentrations in the cellular versus plasma compartments, we profiled 10 paired specimens from patients with radiographic disease. While tumor-confirmed variants were detected in 7 of 10 cellular specimens, mean allelic frequency (AF) was 5.3x higher in plasma. Fragmentomic Assessment of Transformation We compared gene expression profiles inferred from cfDNA from pre-treatment lymphoma (FL=15, tFL=9) versus control (n=20) samples ( Fig B). Considering genes associated with FL (Huet Lancet Onc 2018), FL patients had significantly higher inferred expression than healthy adults (p=0.036). Likewise, patients with tFL showed higher inferred expression of genes associated with histologic transformation (Gentles Blood 2009, p=0.025). Conclusions Sensitive methods are required for effective MRD assessment in FL given lower ctDNA shedding as compared with DLBCL. Frequent detectable MRD in radiographic remission suggests that integrating molecular surveillance may augment serial imaging in the long-term management of FL. Simultaneous inferred gene expression from FL cfDNA appears to hold promise, including for noninvasive detection of transformation.

Leveraging diverse cell-death patterns to predict the prognosis, immunotherapy and drug sensitivity of clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) poses clinical challenges due to its varied prognosis, tumor microenvironment attributes, and responses to immunotherapy. We established a novel Programmed Cell Death-related Signature (PRS) for ccRCC assessment, derived through the Least Absolute Shrinkage and Selection Operator (LASSO) regression method. We validated PRS using the E-MTAB-1980 dataset and created PCD-related clusters via non-negative matrix factorization (NMF). Our investigation included an in-depth analysis of immune infiltration scores using various algorithms. Additionally, we integrated data from the Cancer Immunome Atlas (TCIA) for ccRCC immunotherapy insights and leveraged the Genomics of Drug Sensitivity in Cancer (GDSC) database to assess drug sensitivity models. We complemented our findings with single-cell sequencing data and employed the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and qRT-PCR to compare gene expression profiles between cancerous and paracancerous tissues. PRS serves as a valuable tool for prognostication, immune characterization, tumor mutation burden estimation, immunotherapy response prediction, and drug sensitivity assessment in ccRCC. We identify five genes with significant roles in cancer promotion and three genes with cancer-suppressive properties, further validated by qRT-PCR and CPTAC analyses, showcasing gene expression differences in ccRCC tissues. Our study introduces an innovative PCD model that amalgamates diverse cell death patterns to provide accurate predictions for clinical outcomes, mutational profiles, and immune characteristics in ccRCC. Our findings hold promise for advancing personalized treatment strategies in ccRCC patients.

3-D culture of human lung fibroblasts decreases proliferative and increases extracellular matrix remodeling genes.

Fibroblasts are the main producers of extracellular matrix (ECM) responsible for ECM maintenance and repair, a process often disrupted in chronic lung diseases. The accompanying mechanical changes adversely affect resident cells and overall lung function. Numerous models have been used to elucidate fibroblast behavior that are now evolving toward complex three-dimensional (3-D) models incorporating ECM, aiming to replicate the cells' native environment. Little is known about the cellular changes that occur when moving from two-dimensional (2-D) to 3-D cell culture. This study compared the gene expression profiles of primary human lung fibroblasts from seven subjects with normal lung function, that were cultured for 24 h on 2-D collagen I-coated tissue culture plastic and in 3-D collagen I hydrogels, which are commonly used to mimic ECM in various models, from contraction assays to intricate organ-on-a-chip models. Comparing 3-D with 2-D cell culture, 6,771 differentially expressed genes (2,896 up, 3,875 down) were found; enriched gene sets within the downregulated genes, identified through Gene Set Enrichment Analysis and Ingenuity Pathway Analysis, were involved in the initiation of DNA replication which implied downregulation of fibroblast proliferation in 3-D. Observation of cells for 72 h in 2-D and 3-D environments confirmed the reduced progression through the cell cycle in 3-D. A focused analysis, examining the Hippo pathway and ECM-associated genes, showed differential patterns of gene expression in the 3-D versus 2-D culture. Altogether, the transcriptional response of fibroblasts cultured in 3-D indicated inhibition of proliferation, and alterations in Hippo and ECM pathways indicating a complete switch from proliferation to ECM remodeling.NEW & NOTEWORTHY With the introduction of complex three-dimensional (3-D) lung models, comes a need for understanding cellular behavior in these models. We compared gene expression profiles of human lung fibroblasts grown on two-dimensional (2-D) collagen I-coated surfaces with those in 3-D collagen I hydrogels. RNA sequencing and subsequent pathway analyses showed decreased proliferation, increased extracellular matrix (ECM) remodeling, and altered Hippo signaling and ECM deposition-related gene signatures. These findings highlight unique responses of fibroblasts in 3-D models.

1190P Comparative gene expression and mutational profiling of neuroendocrine neoplasm in relation to clinical outcome

1190P Comparative gene expression and mutational profiling of neuroendocrine neoplasm in relation to clinical outcome

Gene expression signatures of human senescent corneal and conjunctival epithelial cells.

This study aimed to investigate the senescent phenotypes of human corneal and conjunctival epithelial cells. We examined cell morphology, senescence-associated β-galactosidase (SA-β-gal) activity, cell proliferation, and expression of senescence markers (p16 and p21). RNA sequencing analysis was conducted to compare gene expression profiles between senescent and non-senescent cells. Finally, the potential involvement of senescent cells in the pathogenesis of ocular surface diseases was investigated. X-irradiated corneal and conjunctival epithelial cells exhibited typical senescence phenotypes, i.e., flattened morphologies, increased SA-β-gal activity, decreased cell proliferation, and increased expression of senescence markers, p16 and p21. RNA-seq analysis revealed substantial differences in gene expression profiles between senescent corneal (SCo) and conjunctival epithelial cells (SCj). Moreover, SCj were detected in pathological conjunctival tissues associated with limbal stem cell deficiency (LSCD) due to Stevens-Johnson syndrome or chemical burns, potentially being involved in abnormal differentiation. This study highlights the cellular and molecular characteristics of senescent ocular surface cells, particularly in SCj that show abnormal keratin expression, and their potential roles in severe ocular surface diseases and pathology.

Genetic insights into fiber quality, coat color and adaptation in Changthangi and Muzzafarnagri sheep: A comparative skin transcriptome analysis

Changthangi sheep, which inhabit the high-altitude regions of Ladakh, are known for their fine fiber production and are characterized by grey skin and either black or white coats. In contrast, Muzzafarnagri sheep from the plains of Uttar Pradesh produce coarse wool and have white skin and coats. We conducted comparative global gene expression profiling on four biological replicates of skin from each breed. Notably, our analysis identified 149 up-regulated genes and 2,139 down-regulated genes in Changthangi sheep compared to Muzzafarnagri sheep, with a p-adjusted value (padj) of ≤0.05 and a Log2 fold change of ≥1.5. Gene Ontology analysis of the up-regulated genes revealed an enrichment of terms related to melanin biosynthesis and developmental pigmentation. Additionally, enriched KEGG pathways included tyrosine metabolism and metabolic pathways. Among the melanogenesis-related genes that exhibited higher expression in Changthangi sheep were TYR, TYRP1, DCT, SLC45A2, PMEL, MLANA, and OCA2. These findings confirm melanin's role in both the animals' black coat color and UV protection at high-altitude. Furthermore, we observed more pronounced expression of genes related to fiber quality, namely KRTAP6, KRTAP7, KRTAP13, and KRTAP2, in the fine wool-producing sheep from Ladakh. The results of the RNA sequencing were validated using real-time PCR on 10 genes governing fiber quality and coat color, with ACTB and PPIB serving as reference genes. In conclusion, our comparative skin transcriptome analysis of Changthangi and Muzzafarnagri sheep sheds light on the genetic differences associated with distinct phenotypic traits and environmental adaptability, offering valuable insights into the underlying mechanisms.

Identification of a gene set that maintains tumorigenicity of the hepatocellular carcinoma cell line Li-7

The identification and development of therapeutic targets in cancer stem cells that lead to tumor development, recurrence, metastasis, and drug resistance is an important goal in cancer research. The hepatocellular carcinoma cell line Li-7 contains functionally different types of cells. Cells with tumor-forming activity are enriched in cancer stem cell-like CD13+CD166− cells and this cell population gradually decreases during culture in conventional culture medium (RPMI1640 containing 10% fetal bovine serum). When Li-7 cells are cultured in mTeSR1, a medium developed for human pluripotent stem cells, CD13+CD166− cells, and their tumorigenicity is maintained. Here, we sought to identify the mechanisms of tumorigenicity in this sub-population. We compared gene expression profiles of CD13+CD166− cells with other cell sub-populations and identified nine overexpressed genes (ENPP2, SCGN, FGFR4, MCOLN3, KCNJ16, SMIM22, SMIM24, SERPINH1, and TMPRSS2) in CD13+CD166− cells. After transfer from mTeSR1 to RPMI1640 containing 10% fetal bovine serum, the expression of these nine genes decreased in Li-7 cells and they lost tumorigenicity. In contrast, when these genes of Li-7 cells were forcibly expressed in cultures using RPMI1640 containing 10% fetal bovine serum, Li-7 cells maintained tumorigenicity. A metabolome analysis using capillary electrophoresis–mass spectrometry showed that two metabolic pathways, “Alanine, aspartate and glutamate metabolism” and “Arginine biosynthesis” were activated in cancer stem-cell-like cells. Our analyses here showed potential therapeutic target genes and metabolites for treatment of cancer stem cells in hepatocellular carcinoma.

FGF1 supports glycolytic metabolism through the estrogen receptor in endocrine-resistant and obesity-associated breast cancer

BackgroundObesity increases breast cancer risk and breast cancer-specific mortality, particularly for people with estrogen receptor (ER)-positive tumors. Body mass index (BMI) is used to define obesity, but it may not be the best predictor of breast cancer risk or prognosis on an individual level. Adult weight gain is an independent indicator of breast cancer risk. Our previous work described a murine model of obesity, ER-positive breast cancer, and weight gain and identified fibroblast growth factor receptor (FGFR) as a potential driver of tumor progression. During adipose tissue expansion, the FGF1 ligand is produced by hypertrophic adipocytes as a stimulus to stromal preadipocytes that proliferate and differentiate to provide additional lipid storage capacity. In breast adipose tissue, FGF1 production may stimulate cancer cell proliferation and tumor progression.MethodsWe explored the effects of FGF1 on ER-positive endocrine-sensitive and resistant breast cancer and compared that to the effects of the canonical ER ligand, estradiol. We used untargeted proteomics, specific immunoblot assays, gene expression profiling, and functional metabolic assessments of breast cancer cells. The results were validated in tumors from obese mice and breast cancer datasets from women with obesity.ResultsFGF1 stimulated ER phosphorylation independently of estradiol in cells that grow in obese female mice after estrogen deprivation treatment. Phospho- and total proteomic, genomic, and functional analyses of endocrine-sensitive and resistant breast cancer cells show that FGF1 promoted a cellular phenotype characterized by glycolytic metabolism. In endocrine-sensitive but not endocrine-resistant breast cancer cells, mitochondrial metabolism was also regulated by FGF1. Comparison of gene expression profiles indicated that tumors from women with obesity shared hallmarks with endocrine-resistant breast cancer cells.ConclusionsCollectively, our data suggest that one mechanism by which obesity and weight gain promote breast cancer progression is through estrogen-independent ER activation and cancer cell metabolic reprogramming, partly driven by FGF/FGFR. The first-line treatment for many patients with ER-positive breast cancer is inhibition of estrogen synthesis using aromatase inhibitors. In women with obesity who are experiencing weight gain, locally produced FGF1 may activate ER to promote cancer cell metabolic reprogramming and tumor progression independently of estrogen.

A novel reporter mouse line for studying alveolar macrophages.

Alveolar macrophages (AMs) are self-maintained immune cells that play vital roles in lung homeostasis and immunity. Although reporter mice and culture systems have been established for studying macrophages, an accurate and specific reporter line for alveolar macrophage study is still not available. Here we reported a novel Rspo1-tdTomato gene reporter mouse line that could specifically label mouse AMs in a cell-intrinsic manner. Using this reporter system, we visualized the dynamics of alveolar macrophages intravitally under steady state and characterized the alveolar macrophage differentiation under in vitro condition. By performing ATAC-seq, we found that insertion of the tdTomato cassette in the Rspo1 locus increased the accessibility of a PPARE motif within the Rspo1 locus and revealed a potential regulation by key transcription factor PPAR-γ for alveolar macrophage differentiation in vitro and in vivo. Consistently, perturbation of PPAR-γ by its agonist rosiglitazone or inhibitor GW9662 resulted in corresponding alteration of tdTomato expression in alveolar macrophages together with the transcription of PPAR-γ downstream target genes. Furthermore, global transcriptomic analyses of AMs from the wild type mice and the Rspo1-tdTomato mice showed comparable gene expression profiles, especially those AM-specific genes, confirming that the insertion of the tdTomato cassette in the Rspo1 locus does not impact the cell identity and biological function of AMs under normal condition. Taken together, our study provides an alternative tool for in vivo and in vitro labeling of alveolar macrophages with high specificity which could also be utilized as an indicator of PPAR-γ activity for future development of PPAR-γ specific targeting drugs.