Digital Gene Expression Research Articles

Inter- and intra-tumor heterogeneity of genetic and immune profiles in inherited renal cell carcinoma

Patients with von Hippel-Lindau disease (vHL) are at risk of developing spatially and temporally multiple clear cell renal cell carcinomas (ccRCCs), which offers a valuable opportunity to analyze inter- and intra-tumor heterogeneity of genetic and immune profiles within the same patient. Here, we perform whole-exome and RNA sequencing, digital gene expression, and immunohistochemical analyses for 81 samples from 51 ccRCCs of 10 patients with vHL. Inherited ccRCCs are clonally independent and have less genomic alterations than sporadic ccRCCs. Hierarchical clustering of transcriptome profiles shows two clusters with distinct immune signatures: immune hot and cold clusters. Interestingly, not only samples from the same tumors but also different tumors from the same patients tend to show a similar immune signature, whereas samples from different patients frequently exhibit different signatures. Our findings reveal the genetic and immune landscape of inherited ccRCCs, demonstrating the relevance of host factors in shaping anti-tumor immunity.

Long non-coding RNA mitophagy and ALK-negative anaplastic lymphoma-associated transcript: a novel regulator of mitophagy in T-cell lymphoma.

Long non-coding RNA (lncRNA) are emerging as powerful and versatile regulators of transcriptional programs and distinctive biomarkers of progression of T-cell lymphoma. Their role in the aggressive anaplastic lymphoma kinase-negative (ALK-) subtype of anaplastic large cell lymphoma (ALCL) has been elucidated only in part. Starting from our previously identified ALCL-associated lncRNA signature and performing digital gene expression profiling of a retrospective cohort of ALCL, we defined an 11 lncRNA signature able to discriminate among ALCL subtypes. We selected a not previously characterized lncRNA, MTAAT, with preferential expression in ALK- ALCL, for molecular and functional studies. We demonstrated that lncRNA MTAAT contributes to an aberrant mitochondrial turnover restraining mitophagy and promoting cellular proliferation. Functionally, lncRNA MTAAT acts as a repressor of a set of genes related to mitochondrial quality control via chromatin reorganization. Collectively, our work demonstrates the transcriptional role of lncRNA MTAAT in orchestrating a complex transcriptional program sustaining the progression of ALK- ALCL.

Prognostic impact of HLA‐I neoantigen‐specific CD8+ T cells in limited‐stage follicular lymphoma

Introduction: Follicular lymphoma (FL) is the most common indolent NHL. Recently, we demonstrated in limited and advanced stage FL (LSFL, ASFL), that patients with longer remissions had raised clonally expanded intratumoral CD8+ T cells (Tobin, JCO 2019). Durable remissions occur in ∼50% of LSFL patients whereas ASFL is incurable. However, little is known regarding the immunological features associated with this difference. We hypothesized HLA-I neoantigen (neoAg)-specific CD8+ T cells may play a role. Methods: The discovery cohort comprised 101 patients with diagnostic paraffin embedded tissue from the TROG99.03 LSFL clinical trial (MacManus, JCO 2018), in which stage I/II patients were randomized to involved field radiation (IFRT) only or combined modality therapy between 2000 and 2012 (PET from 2006). Contemporaneous validation cohorts were (a) AusLSFL: 60 PET staged, stage I FL patients from Australia (treatment miscellaneous), and (b) CanLSFL: 60 PET staged, stage I FL patients drawn principally from Canada (IFRT only). Digital gene expression (NanoString), targeted sequencing (330 genes), and germline HLA-typing was performed. Mutations observed by sequencing were used to predict neoAgs in TROG99.03 tissues using 8 algorithms (PVACseq) and filtered according to strong binding affinity to HLA-I over wild-type (TESLA consortium guidelines; Wells, Cell 2020). Results: CD8A gene expression was tested for prognostic significance. In TROG99.03, elevated intratumoral CD8A (by MaxStat) was associated with ∼2-fold improvement in PFS for all patients: HR 0.45 (CI: 0.77–0.26, p = 0.0053) and stage I only patients: HR 2.4 (CI: 1.2–4.6, p = 0.036). In keeping with a relationship between CD8+ T cell infiltration and tumor antigen presentation, raised expression of NLRC5 (a transcriptional HLA-I activator) was also associated with superior PFS: HR 0.48 (CI: 0.99–0.24, p = 0.024). CD8A significance was confirmed in both validation cohorts (whereas NLRC5 was validated in AusLSFL only). In keeping with recent IHC CD8 protein data (Los-de Vries, Bld Adv 2022), CD8A gene expression was raised in stage I LSFL vs. 68 ASFL patients treated with immunochemotherapy (p = 0.02). Mutational profiling was concordant with published LSFL data, with CREBBP and KMT2DA most frequent. NeoAg calling methods including functional assays for neoAg peptide binding were confirmed in a separate cohort of fresh FL tissues. 59% of TROG99.03 tissues had ≥1 neoantigens detected. Importantly, unsupervised hierarchical clustering showed 2-fold enrichment of samples with neoantigens among those with high vs. low HLA-I. Conclusions: Raised CD8A is associated with favorable prognosis in LSFL. Our data suggests disease control involves populations of expanded HLA-I neoAg-specific T cells. These findings have implications for novel immunotherapeutic strategies designed to increase the rate of durable remissions. The research was funded by: National Health and Medical Research Council, Australia; Leukaemia Foundation; Mater Foundation Keywords: Diagnostic and Prognostic Biomarkers, Indolent non-Hodgkin lymphoma, Microenvironment Conflicts of interests pertinent to the abstract. C. Keane Honoraria: Takeda, Roche, AZ, MSD, Beigene C. Cheah Consultant or advisory role: Roche, Janssen, Gilead, AstraZenecca, Lilly, TG therapeutics, Beigene, Novartis, Menarini, Daizai, Abbvie, Genmab. BMS Honoraria: BMS, Roche, Abbvie; MSD, Lilly R. Kridel Research funding: Abbvie Educational grants: Eisai M. K. Gandhi Research funding: Beigene, Janssen

RITUXIMAB‐CONTAINING COMBINED MODALITY THERAPY IN LIMITED STAGE FOLLICULAR LYMPHOMA: MATURE FOLLOW UP AND DERIVATION OF A NOVEL PROGNOSTIC SCORE FROM THE TROG99.03 TRIAL

Introduction: The TROG99.03 represents the only randomised phase III trial of combined modality therapy (CMT) in limited-stage follicular lymphoma ‘LSFL’, reporting a prolonged progression-free survival (PFS) in the CMT arm (MacManus, JCO, 2018). Here, we report extended follow up of this study providing mature data of patients treated with a rituximab-containing CMT regimen and the development of a new gene-expression based prognostic score. Methods: Patients with LSFL, grade 1–3a were randomised (1:1) to either involved-field radiotherapy alone (IFRT) (30–36Gy) or to CMT consisting of identical IFRT followed by 6 cycles of CVP. Reflecting evolving clinical practice, from 2006 onwards (i.e., ‘modern-era’), PET staging was increasingly utilised, and rituximab added to the CMT arm. Digital multiplex gene expression by Nanostring was performed on diagnostic biopsies based on genes previously identified to differentiate LSFL from advanced stage FL ‘ASFL’ (AM Staiger, Blood, 2020). Results: 150 patients were recruited between 2000 and 2012 with 31/75 patients in each arm recruited in the ‘modern-era’. At median follow-up 11.3 years, PFS remained superior for CMT compared to RT (HR 0.6; p = 0.043). Although no significant difference in OS was observed (HR 0.45, p = 0.11), compared with IFRT, patients in the CMT arm experienced fewer composite (deaths and histological transformation ‘HT’) events (HR 0.25; p = 0.045). With additional follow up no new non-malignant late toxicities were observed and incidence of secondary malignancies were similar between both arms (11 IFRT vs. 10 CMT, p = 0.99). Patients treated with a rituximab regimen (i.e., IFRT+R-CVP) had a markedly superior PFS compared to those treated without rituximab (i.e., IFRT, or IFRT+CVP), 8 year PFS rates 81% versus 52%, HR 0.42 p = 0.013 (Figure 1). Amongst PET staged patients the difference between R-CVP/IFRT versus IFRT increased (HR 0.35 p =0.027) suggesting this effect was not due to stage migration. No clinical factors were significantly associated with PFS on multivariate analysis. Nor were prognostic associations found for expression level of any individual genes. However, by penalised Cox regression an 8-gene Lasso-weighted prognosticator was identified, termed the ‘Bio-LSFL-score’. Genes (CACNA2D2, CD69, GZMB, IL7R, MYCT1, SLP1, TNFRSF14, TNFRSF25) reflected both B-cells and the microenvironment. The Bio-LSFL-score was highly significant for PFS (HR 0.25, p < 0.0001) with 100% patients in the high-risk group relapsing before 8 years. Conclusions: Particularly when incorporating rituximab, LSFL patients demonstrated significant increases in PFS and reduction in the rates of death and/or HT when treated with CMT compared with IFRT alone. A novel gene expression prognosticator was identified which in this trial cohort showed ‘ASFL-like’ behaviour by identifying LSFL patients unlikely to experience durable remissions. Keywords: diagnostic and prognostic biomarkers, indolent non-Hodgkin lymphoma, radiation therapy Conflicts of interests pertinent to the abstract C. Cheah Consultant or advisory role: Roche, Janssen, Gilead, Astra Zeneca, Lilly, TG Therapeutics, Beigene, Novartis, Menarini, Daizai, Abbvie, Genmab, BMS Research funding: BMS, Roche, Abbvie, MSD, Lilly J. F. Seymour Consultant or advisory role: Abbvie, Astra Zeneca, Celgene/BMS, Genentech, Genor Bio, Gilead, Janssen, Morphosys, Roche, Sunesis, TG Therapeutics Research funding: Celgene/BMS M. K. Gandhi Research funding: Beigene, Janssen

Growing Hierarchical Self-Organising Representation Map (GHSORM)

The Growing Hierarchical Self-Organising Representation Map (GHSORM) is a model fusing the denoising autoencoder, used to better represent a dataset, and the Growing Hierarchical Self-Organising Map, used for organizing and projecting the input data in clusters of varying detail. It is shown here that the GHSORM is instrumental in sub-grouping clusters that are not fully separable by a single SOM. This combined approach is first tested and illustrated on the problem of clustering handwritten digits where a modification of the Activation Maximisation method for use at the SOM output layer demonstrates the benefit of hierarchical growth in the GHSORM. In particular, the SOM Node Activation Maximisation method is used to visually represent the best approximation of each of the SOM nodes at the output layer. This demonstrates the improvement in representing difficult to separate digits in the hierarchical case. To test and measure the efficacy of the GHSORM hierarchical model in class and sub-class separation the method is applied to complex digital gene expression data. A cancer dataset, comprising of gene expression data that has samples of different classes and sub-classes, is used for this purpose. The GHSORM demonstrates robust capabilities to cluster and sub-cluster the different classes and subclasses of cancer, where the results are superior to both linear methods, currently in use, as well as the methods of its constituent algorithms.

An ecdysteroid-regulated 16-kDa protein homolog participates in the immune response of the crayfish Procambarus clarkii

An ecdysteroid-regulated 16-kDa protein homolog (named Pc-E16), encoding 150 amino acid residues with a conserved MD-2-related lipid-recognition domain, was first identified in Procambarus clarkii. Phylogenetic analyses indicated similarity between Pc-E16 and 16-kDa proteins from Aplysia californica and insects. Recombinant Pc-E16 protein was successfully expressed in BL21 (DE3) Escherichia coli cells, and polyclonal antibodies against purified Pc-E16 proteins were prepared. In comparison with other tissues, Pc-E16 was highly expressed in the intestine; real-time PCR and Western blotting results indicated that Pc-E16 expression was significantly induced by lipopolysaccharides in hepatopancreas and hemocytes. Pc-E16-mediated signaling pathways were investigated by digital gene expression analysis following RNA interference targeting Pc-E16. A total of 6103 differentially expressed genes (DEGs) were identified, of which 3318 were up- and 2785 were downregulated. Many DEGs were involved in binding and catalytic activity. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that DEGs were clustered into 225 pathways, and 15 significantly enriched pathways were identified at the immune system level. In addition, the expression level of Pc-E16 in hemocytes and hepatopancreas was obviously downregulated at 48 h after dsRNA injection, and Pc-E16-RNAi treatment affected the expression levels of immune-related genes. Altogether, our results suggest that Pc-E16 is involved in the innate immune response of P. clarkii.

Intelectin enhances the phagocytosis of macrophages via CDC42-WASF2-ARPC2 signaling axis in Megalobrama amblycephala

Intelectin has been identified in various vertebrates and plays an important role in the host immune system. In our previous studies, recombinant Megalobrama amblycephala intelectin (rMaINTL) protein with excellent bacterial binding and agglutination activities enhances the phagocytic and killing activities of macrophages in M. amblycephala; however, the underlying regulatory mechanisms remain unclear. The present study showed that treatment with Aeromonas hydrophila and LPS induced the expression of rMaINTL in macrophages, and its level and distribution in macrophages or kidney tissue markedly increased after incubation or injection with rMaINTL. The cellular structure of macrophages was significantly affected after incubation with rMaINTL, resulting in an increased surface area and pseudopodia extension, which might contribute to enhancing the phagocytic ability of macrophages. Then, digital gene expression profiling analysis of the kidneys from rMaINTL-treated juvenile M. amblycephala identified some phagocytosis-related signaling factors that were enriched in pathways involved in the regulation of the actin cytoskeleton. In addition, qRT-PCR and western blotting verified that rMaINTL upregulated the expression of CDC42, WASF2, and ARPC2 in vitro and in vivo; however, the expression of these proteins was inhibited by a CDC42 inhibitor in macrophages. Moreover, CDC42 mediated the promotion of rMaINTL on actin polymerization by increasing the F-actin/G-actin ratio, which led to the extension of pseudopodia and remodeling of the macrophage cytoskeleton. Furthermore, the enhancement of macrophage phagocytosis by rMaINTL was blocked by the CDC42 inhibitor. These results suggested that rMaINTL induced the expression of CDC42 as well as the downstream signaling molecules WASF2 and ARPC2, thereby facilitating actin polymerization to promote cytoskeletal remodeling and phagocytosis. Overall, MaINTL enhanced the phagocytosis activity of macrophages in M. amblycephala via activation of the CDC42-WASF2-ARPC2 signaling axis.

Transcriptome analysis of Ganoderma lingzhi (Agaricomycetes) response to Trichoderma hengshanicum infection.

Green mold caused by Trichoderma spp. has become one of the most serious diseases which threatening the production of Ganoderma lingzhi. To understand the possible resistance mechanism of the G. lingzhi response to T. hengshanicum infection, we examined the G. lingzhi transcript accumulation at 0, 12, and 24 h after T. hengshanicum inoculation. The gene expression analysis was conducted on the interaction between G. lingzhi and T. hengshanicum using RNA-seq and digital gene expression (DGE) profiling methods. Transcriptome sequencing indicated that there were 162 differentially expressed genes (DEGs) at three infection time points, containing 15 up-regulated DEGs and 147 down-regulated DEGs. Resistance-related genes thaumatin-like proteins (TLPs) (PR-5s), phenylalanine ammonia-lyase, and Beta-1,3-glucan binding protein were significantly up-regulated. At the three time points of infection, the heat shock proteins (HSPs) genes of G. lingzhi were down-regulated. The down-regulation of HSPs genes led to the inhibition of HSP function, which may compromise the HSP-mediated defense signaling transduction pathway, leading to G. lingzhi susceptibility. Pathway enrichment analyses showed that the main enriched pathways by G. lingzhi after infection were sphingolipid metabolism, ether lipid metabolism, and valine, leucine and isoleucine degradation pathway. Overall, the results described here improve fundamental knowledge of molecular responses to G. lingzhi defense and contribute to the design of strategies against Trichoderma spp.

Trehalose transporter-like gene diversity and dynamics enhances stress response and recovery in Helicoverpa armigera

Trehalose is a primary sugar and its distribution across the insect body, regulated by trehalose transporters (TRETs), is essential for sugar metabolism and energy homeostasis. The large diversity of Tret-like sugar transporters (ST), belonging to SLC2A transporter family, in polyphagous insects probably contributes to their extremely adaptive nature. We aim to study spatio-temporal expression dynamics and functional relevance of ST transcript variants in the lepidopteran model organism, Helicoverpa armigera. Identification of 69 putative Tret-like HaST transcript variants from databases and their digital gene expression analysis indicated tissue and development-specific expression patterns. Phylogenetic and sequence similarity network analysis of HaSTs signify evolutionary divergence, while motif and structure analysis depicted conserved signatures. In vitro gene expression validation for selected genes depicts that HaST09 and 69 are fat body and haemolymph-specific. While, HaST06, 30, 36 and 57 are developmental stage or sex-specific. HaST69 has high expression in the haemolymph of fifth instar larvae. In the presence of trehalose metabolism inhibitors and abiotic stress, HaSTs expression show dysregulation, indicating their possible association with trehalose metabolism and stress recovery. In vivo gene silencing of HaST69 resulted in reduced trehalose accumulation in the insect body, suggesting its plausible role in sugar metabolism. The overall understanding of HaST diversity and expression dynamics highlights their putative roles in sugar transport during adaptation and stress recovery of insects.

Genomic and proteomic characterization of Philadelphia-like B-lineage acute lymphoblastic leukemia: A report of Indian patients.

The gold standard for the identification of Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) patients is gene expression profiling. Because of its diverse nature, its identification is extremely difficult and expensive. On the genomic and proteomic landscape of Ph-like ALL patients, there is a paucity of published literature from developing countries. The authors used digital barcoded nCounter NanoString gene expression profiling for its detection, followed by molecular and proteomic characterization using fluorescence in situ hybridization and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The authors found 32.05% Ph-like ALL patients and their median age at presentation was considerably higher than Ph-negative ALL cases (p=.0306). Furthermore, we identified 20% CRLF2 overexpressed cases having 8.33% CRLF2-IGH translocation with concomitant R683S mutation and 8.33% CRLF2-P2RY8 translocation. In 80% of CRLF2 downregulated cases, we identified 10% as having JAK2 rearrangement. Minimal residual disease-positivity was more common in Ph-like ALL cases (55.55% vs. 25% in Ph-negative ALL cases). Immunoglobulin J chain (Jchain), small nuclear ribonucleoprotein SmD1 (SNRPD1), immunoglobulin κ constant (IGKC), NADH dehydrogenase (ubiquinone) 1 α subcomplex subunit 2 (NDUFA2), histone H2AX (H2AFX), charged multivesicular body protein 4b (CHMP4B), and carbonyl reductase (NADPH) (CBR1) proteins were identified to be substantially expressed in Ph-like ALL patients, using LC-MS/MS. Gene enrichment analysis indicated that involvement of spliceosomal mediated messenger RNA splicing pathway and four microRNAs was statistically significant in Ph-like ALL patients. For the first time, we have described incidence, molecular, and proteomic characterization of Ph-like ALL, in developing nations. In developing countries, detecting Philadelphia (Ph)-like B-lineage acute lymphoblastic leukemia is complicated and challenging due to its diverse genetic landscape. There is no well-defined and cost-effective methodology for its detection. The incidence of this high-risk subtype is very high in adult cases, and there is an urgent need for its accurate detection. We have developed an online PHi-RACE classifier for its rapid detection, followed by delineating the genomic and proteomic landscape of Ph-like acute lymphoblastic leukemias for the first time in Indian patients.

Longitudinal Effects of 1-Year Smoking Cessation on Human Bronchial Epithelial Transcriptome

Longitudinal Effects of 1-Year Smoking Cessation on Human Bronchial Epithelial Transcriptome

Comparative Expression Profiling Reveals the Regulatory Effects of Dietary Mannan Oligosaccharides on the Intestinal Immune Response of Juvenile Megalobrama amblycephala against Aeromonas hydrophila Infection.

Mannan oligosaccharides (MOS) are functional oligosaccharides with beneficial effects on the non-specific immunity of Megalobrama amblycephala, but systematic studies on the immunomodulatory mechanisms of MOS are still lacking. To investigate the protective mechanisms of three different levels of dietary MOS supplementation on the intestinal immunity of juvenile M. amblycephala, comparative digital gene expression (DGE) profiling was performed. In this study, 622 differentially expressed genes (DEGs) were identified, while the similar expression tendency of 34 genes by qRT-PCR validated the accuracy of the DGE analyses. Gene Ontology (GO) enrichment revealed that the DEGs were mainly enriched in two functional categories of biological process and molecular function. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEGs were mainly related to complement and coagulation cascades, coagulation cascades, platelet activation, natural killer cell mediated cytotoxicity, Fc gamma R-mediated phagocytosis and antigen processing and presentation. In addition, the pro-inflammatory, apoptosis and tight junction-related genes were more significantly up-regulated upon infection in the dietary MOS groups to enhance host immune functions and maintain the stability of the intestinal barrier. These results will be helpful to clarify the regulatory mechanism of MOS on the intestinal immunity of M. amblycephala and lay the theoretical foundation for the prevention and protection of fish bacterial diseases.

Comparative transcriptome, digital gene expression and proteome profiling analyses provide insights into the brachyurization from the megalopa to the first juvenile in Eriocheir sinensis

Eriocheir sinensis larva normally experiences 11 stages. The reduced abdomen folded beneath the thorax is the most prominent characteristic of morphological alteration from megalopa to juvenile crab. Up to date, the molecular mechanisms of brachyurization remain a mystery. Here, transcriptome library, digital gene expression (DGE) libraries and proteome libraries at two developmental stages [the megalopa stage of E. sinensis (stage M) and the first stage of juvenile crab (stage J1)] of the Chinese mitten crab larva were constructed for RNA sequencing and iTRAQ approaches followed by bioinformatics analysis, respectively. In total, 1106 genes and 871 proteins were differentially expressed between the stage M and stage J1. Moreover, several important pathways were identified, including biosynthesis of secondary metabolites, metabolic pathways, focal adhesion, and some disease pathways. Besides, muscle contraction, oxidative phosphorylation, calcium signaling, PI3K-Akt, DNA replication pathway, and integrin signaling pathway also had important functions in brachyurization process. Furthermore, the components, actin, actin-related protein, collagens, filamin-A/B, laminin, integrins, paxillin, and fibronectin had up-regulated expression levels in M stage compared to J1 stage.

Assessment of 2-Year Storage Conditions on Protein, RNA, and DNA in Unstained Human Tissue Sections, Including a Novel Multiplex Digital Gene Expression Profiling Method with Implications for Biobanking.

Background: Formalin-fixed, paraffin-embedded (FFPE) tissues are a valuable resource for clinical and basic science research. Paraffin blocks and the resulting unstained sections (USS) are often stored for years before being used. Previous studies have evaluated the effects of time, temperature, humidity, and inert gases on preservation of USS; however, no study has examined all four variables together. Methods: In the current work, we prospectively and blindly assessed time points from 0 to 24 months, room versus refrigerated temperature, and presence of a desiccant and/or nitrogen atmosphere on a variety of benign and malignant tissues from North America and Africa. End points included immunohistochemistry (IHC), in situ hybridization (ISH), extracted RNA and DNA quantity and quality, and messenger RNA performance in a novel, multiplexed digital gene expression profiling assay of both housekeeping and tumor-specific genes. Results: We found that using current methods of antigen retrieval, staining, and extraction, the end points of IHC, ISH, RNA, and DNA were well preserved under the various conditions tested, with implications that pre-embedding factors contribute to variability in subsequent tissue integrity. We also document that spectrophotometric estimations of nucleic acid concentrations were in general estimated to be higher than with fluorimetric methods, which may be pertinent to end assay development. We further describe a new multiplex assay, the PlexSet digital gene expression assay, suitable for evaluating RNA quality in FFPE tissues. Conclusion: Altogether, these results may provide helpful guidance with regard to approaches for long-term storage conditions for USS.

MIR390 Is Involved in Regulating Anthracnose Resistance in Apple

As an important cash crop in China, apple has a good flavor and is rich in nutrients. Fungal attacks have become a major obstacle in apple cultivation. Colletotrichum gloeosporioides is one of the most devastating fungal pathogens in apple. Thus, discovering resistance genes in response to C. gloeosporioides may aid in designing safer control strategies and facilitate the development of apple resistance breeding. A previous study reported that 'Hanfu' autotetraploid apple displayed higher C. gloeosporioides resistance than 'Hanfu' apple, and the expression level of mdm-MIR390b was significantly upregulated in autotetraploid plants compared to that in 'Hanfu' plants, as demonstrated by digital gene expression (DGE) analysis. It is still unclear, however, whether mdm-MIR390b regulates apple anthracnose resistance. Apple MIR390b was transformed into apple 'GL-3' plants to identify the functions of mdm-MIR390b in anthracnose resistance. C. gloeosporioides treatment analysis indicated that the overexpression of mdm-MIR390b reduced fungal damage to apple leaves and fruit. Physiology analysis showed that mdm-MIR390b increased C. gloeosporioides resistance by improving superoxide dismutase (SOD) and peroxidase (POD) activity to alleviate the damage caused by O2- and H2O2. Our results demonstrate that mdm-MIR390b can improve apple plants' anthracnose resistance.

Antigen-Independent, Autonomous B-Cell Receptor Signaling in Diffuse Large B-Cell Lymphoma

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) defines the Cell-of-origin (CoO) classification with two major subtypes, germinal center B-cell (GCB) and the more aggressive activated B-cell (ABC) type. Chronic activation of B-cell receptor (BCR) signaling with constitutive activation of the NF-κB pathway is an essential oncogenic mechanism in ABC DLBCL. BCR pathway activation with increased NF-κB activation can be caused by gain-of-function mutations of the BCR signaling cascade. We investigated whether antigen-independent, i.e. autonomous BCR signaling, akin to its essential pathogenetic role in chronic lymphocytic leukemia, acts as a functional driver mechanism in DLBCL without such gain-of-function mutations. DNA and RNA were extracted from ABC DLBCL cell lines TMD8 and OCI-Ly3 and from fresh-frozen biopsies of 18 histologically confirmed DLBCL cases. Functional BCR transcripts were identified by ARTISAN PCR and transduced into murine triple knock out (TKO) cells. TKO cells lack the recombination activation gene 2 (Rag2) and lambda 5 gene (λ5) and are therefore incapable of expressing an endogenous BCR. In addition, the BCR signal transduction molecule SLP65 is replaced with a tamoxifen-inducible estrogen receptor (ERT2) - SLP65 fusion gene. Upon transduction of both heavy and light BCR chains and staining with Indo-1, background calcium flux can be measured as Indo-1 signal intensity ratio (SIR) by flow cytometry. After restoring functionality of the BCR signaling cascade by tamoxifen, antigen-independent BCR signaling is measured as SIR, followed by maximum BCR stimulation with an isotype-specific crosslinking antibody. Autonomous BCR signaling strength (SSBCR) is quantified by an arithmetic product based on the relative Indo-1 SIR and the relative number of cells with SIR above background in the presence of tamoxifen and calibrated on crosslinking-induced maximum SIR. According to the primary hypothesis, the BCR of CARD11 wild-type, IgM-expressing TMD8 cells demonstrated strong autonomous BCR signaling (SSBCR=0.243), whereas the BCR of CARD11 mutated, IgG-expressing OCI-Ly3 cells completely lacked autonomous signaling (SSBCR=0.022). BCR from 13 primary DLBCL signaled autonomously (SSBCR=0.125-0.619), where 5 samples were negative (SSBCR=0.004-0.023) Autonomous BCR signaling was absolutely dependent on IgM isotype (Fisher's exact test: p=0.0016). CoO classification by digital gene expression (Nanostring) analyses or immunohistochemistry demonstrated autonomous BCR signaling in 8/9 ABC (non-GCB) DLBCL, 2/6 GCB DLBCL, and 2 unclassifiable DLBCL, and a transformed follicular lymphoma DLBCL. Independent of CoO, DLBCL can be assigned to 6-7 clusters based on their genetic aberrations (Chapuy 2018, Wright 2020). Fifteen DLBCL were suited for WES and targeted panel sequencing. Four cases were assigned by a probabilistic calculation to Chapuy cluster C5, 2 cases to C2, and one case to C1, while 8 cases remained unclassifiable. Clustering by the LymphGen tool assigned 3 cases to cluster ST2, one case each into MCD, EZB, and N1, 4 cases into various clusters, and 5 cases unclassified. DLBCL with autonomous BCR signaling fell into clusters C1/unclassified, C2/unclassified, C5/unclassified (n=3), unclassified/EZB, unclassified/ST2, unclassified/N1 and unclassified/unclassified (n=2). Cases lacking autonomous BCR signaling were assigned to unclassified/ST2 (n=2), C5/MCD, and unclassified/unclassified. Eleven of 13 cases with an autonomous signaling BCR lacked activating CARD11 mutations. In conclusion, autonomous BCR signaling is a potential immunological oncogenic driver in DLBCL that cannot be detected by sequencing studies, is independent of external antigen stimulation, and originates from a self-organizing conformation of the surface BCR (Minici 2017). Autonomous BCR signaling is present predominantly in ABC DLBCL and dependent on IgM isotype. Autonomous BCR signaling offers a convincing explanation for oncogenic NF-κB activation in DLBCL. Limited data suggest that the majority of DLBCL with autonomous BCR signaling fall into the ABC-dominated genetic clusters C1/C5, and MCD/N1, respectively. In summary, genetic aberrations, BCR function, and gene expression profile form 3 independent layers of DLBCL pathophysiology that need to be analyzed separately and offer independent targets for therapy.

Tumor Microenvironment and It's Prognostic Value Is Different in Testicular and Systemic Diffuse Large B-Cell Lymphoma

Testicular diffuse large B-cell lymphoma (T-DLBCL) is an aggressive lymphoid cancer with distinct clinical and biological features. Majority of the cases represent non-GCB phenotype, but the genetic landscape with frequent aberrations in MYD88, NFKBIZ, PDCD1LG2, and CD274 differ from systemic non-GCB DLBCL and highlight the significance of immune-escape and tumor microenvironment (TME). We have previously shown that high T-cell gene expression signature and high tumor-infiltrating lymphocyte (TIL) content translate to better survival in testicular but not in systemic DLBCL. We have also described a subpopulation of T-bet+FoxP3+ Tregs that have a significant adverse impact on survival in T-DLBCL but not in in systemic DLBCL. In order to further characterize the differences in the TME between T- and systemic DLBCL, we used NanoString digital gene expression profiling to assess the expression of 770 immune response genes in 60 T-DLBCL and 84 systemic DLBCL samples. In addition, we used multiplex immunohistochemistry (mIHC) to characterize TILs, tumor-associated macrophages (TAMs), NK-cells, checkpoint molecules, cell proportions, and cell interactions in 80 T-DLBCLs and 192 systemic DLBCLs. Unsupervised hierarchical clustering of the gene expression data revealed clusters of genes with differential expression between T- and systemic DLBCLs. In T-DLBCL, 11 HLA genes were downregulated and genes related to B-cell receptor (BCR) signaling upregulated compared to systemic DLBCL. All T-DLBCL patients that clustered among the patients with systemic DLBCL had high T-cell signature expression, and systemic DLBCL patients that clustered among patients with T-DLBCL had significantly higher Ki-67 expression (p=0.00085). Based on the mIHC data, we observed not only a significantly lower proportion of FoxP3+ Tregs altogether but also a higher proportion of T-bet+FoxP3+ double positive Tregs in T-DLBCL compared to systemic DLBCL, confirming our previous findings on the significant role of T-bet+FoxP3+ double positive Tregs in the T-DLBCL TME. We also observed less TAMs in T-DLBCL TME. Unsupervised hierarchical clustering of the cell interaction data verified that distinct interaction clusters are found and separate the patients into different subgroups. Interactions of cytotoxic T-cells were more commonly seen in T-DLBCL whereas interactions of PD-1 expressing cells and interactions between TILs and TAMs were more frequent in systemic DLBCL. Taken together, our results reveal differences in the TME between T- and systemic DLBCL, and further emphasize the impact of immune escape and distinct immune cells in T-DLBCL. Future studies are ongoing in order to further characterize the cell-to-cell interactions, their association with survival, and TME differences between T- and systemic DLBCL, in particular non-GCB DLBCL.

Characterization and Clinical Impact of the Tumor Microenvironment of Post-Transplant Large B-Cell Lymphoma

Post-transplant lymphoproliferative disorders (PTLDs) arise after solid organ or allogeneic hematopoietic stem cell transplantation. They are characterized by abnormal proliferation of lymphoid cells associated with immunosuppression and comprise three histological categories (non-destructive, polymorphic, monomorphic) entailing a heterogeneous clinical behavior. The majority of monomorphic PTLDs fulfill the criteria of aggressive large B-cell lymphomas and are often associated with Epstein-Barr virus (EBV) positivity. The composition and function of the tumor microenvironment (TME) has a major impact on lymphoma development and survival, but to date, only few studies investigating the TME in PTLD have been reported. We used digital gene expression profiling and in silico immunophenotyping to characterize the TME of 75 post-transplant monomorphic aggressive B-cell lymphomas from three Nordic countries and correlated the findings with patient demographics (Table 1) and survival. Unsupervised clustering demonstrated a high degree of heterogeneity and variation in the immune response profile among the PTLDs. EBV+ PTLDs clustered closely together and were enriched for type I interferon signaling and antiviral response genes. In addition, a signature for cytotoxicity associated with EBV-positivity and better overall survival (OS; HR 0.61, 95%CI 0.40-0.92, P=0.019). In silico immunophenotyping utilizing CIBERSORTx suggested that the PTLDs can be divided into two subgroups with distinct immune cell compositions (Figure 1). The patients in the inflamed subgroup with high proportions of T cells and NK cells, as well as high T cell/macrophage ratio, had significantly better OS and PFS compared to the patients in the non-inflamed subgroup (median OS >200.0 vs. 15.2 months, P=0.006; median PFS 89.0 vs. 14.4 months, P=0.049). In multivariable analysis with the EBV-status, International Prognostic Index (IPI) and rituximab-containing treatment, the inflamed TME remained as an independent predictor for favorable OS and PFS. The inflamed subgroup was associated with EBV-positivity (P=0.008) and earlier onset of the disease (P=0.004). We also compared the TME composition between post-transplant and de novo diffuse large B-cell lymphomas (DLBCLs, n=82) and discovered that the PTLD TME was enriched in dendritic cells, NK cells, and activated mast cells, whereas the proportion of T cells, memory B cells and resting mast cells was lower compared to the de novo DLBCL. In conclusion, we provide a comprehensive phenotypic characterization of PTLD, highlighting the importance of TME biology, and in particular T cell infiltration, in the clinical behavior and prognosis of PTLD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Newly Diagnosed, Untreated, Multiple Myeloma (MM) Patient Samples Already Harbor Cereblon (CRBN) Exon 10 Deletions Associated with Drug Resistance

Background: Acquisition of drug resistance, particularly immunomodulatory drugs (IMiDs) in MM is one of the most important challenges in patient treatment today. Early on in the disease, the plasma cells in the MM clone are often sensitive to chemotherapy. However, over time, after exposure to chemotherapy, the cells acquire increasing genomic complexity and mutations leading to resistance to therapy and ultimately patient death. Cereblon (CRBN) is a binding protein, which is required for the action of the widely used IMiDs such as lenalidomide and pomolidomide. CRBN is also essential to the newer CRBN E3 ligase modulator drugs and proteolysis targeting chimeras. We recently described an algorithm based on digital gene expression profiling of purified CD138(+) MM cells from patients at different clinical stages that we term the "MM-IP-7", which correlated with clinical drug resistance, poor survival, and disease progression following combination treatment containing IMIDs and/or PIs (XY Zhu et al, eHaem 2022). Given the recent description of genetic changes in the CRBN gene that are associated with IMiD resistance particularly the exon 10-spliced CRBN transcripts that increase in incidence in parallel with IMiD-refractory states (S. Gooding et al, Blood 2021), we sought to build an integrated, single-platform test to measure both gene expression levels and genetic deletions. Methods: We designed a novel CRBN splicing isoform probe with homology to both exon 9 and exon 11. This probe will thus capture CRBN mRNA sequences in which exon 10 is deleted and portions of exons 9 and 11 and juxtaposed. We also included probes to CRBN overall (Exons 5 and 6). These probes were combined with the MM-IP-7 probes to create a dual gene expression and genetic assay that was used to analyze 30 cases of newly diagnosed MM, 30 cases of end stage MM, and 3 MM cell lines (MM1.S, OPM2, XG1). To define increased exon 10 deleted allele frequency, we calculated the ratio of exon 10 deletion/total exon 10. Cases with apparent exon 10 deletion by expression profiling, were further evaluated with RT-PCR. Results: 40% MM samples in the end stage group versus 23% of samples in the newly diagnosed group were identified with more than 20% exon 10 deleted mRNA as compared to total CRBN mRNA levels. Three cases were shown to have more than 45% exon 10 deleted mRNA, including two newly diagnosed cases and one end stage case. RT-PCR confirmed exon 10 deletion in a newly diagnosed patient sample with sufficient remaining RNA that was tested (cloning and sequencing for confirmation are underway). Cell line results showed very low exon 10 splicing variant, which correlated with our mRNAseq data for these lines. Conclusions: Our findings demonstrate that inclusion of probes bridging CRBN exon 10 deletion with homology to exons 9 and 11, is feasible approach to use on the gene expression platform, which allows for combined evaluation of expression and genetic changes. CRBN exon 10 deletion is a known treatment resistance feature in MM, which may already be present at diagnosis and selected by subsequent therapy with possible therapeutic planning implications. Additional studies to confirm the results and expand the study set are underway.

Effects of DNA methylation and gene expression on rats with protein malnutrition in early life

Although the mechanisms underlying how malnutrition in early life affects the susceptibility to diseases later in life remain unclear, there is considerable interest in the potential role of DNA methylation in the intrauterine programming of diseases. In this study, digital gene expression profiles were used to analyze changes in gene expression of protein-restricted rats early in life, while intergenerational rat models were used to explore differences in whole blood genomic DNA methylation in an environment stimulated by maternal protein intervention. Nine rats were randomly divided into early-life low-protein group (LPE), fetal low-protein group (LPF), and normal control group (CON). The LPE group was fed a low-protein diet on the 1st day of pregnancy until the end of lactation. The LPF group was given low-protein diet during pregnancy. The CON group was given a 20% protein diet from the 1st day of pregnancy. Total mRNA was extracted from the sacrificed rats at the 48th week. The number of differentially expressed genes for LPE versus CON, LPF versus CON, and LPF versus LPE was 178, 223, and 302, respectively. Comparing LPE versus CON and LPF versus CON, the upregulated genes common to the two groups were Gimap-9, Serinc-4, Dnah-2, Sf3b-5, and Sat-2, and the downregulated genes were Ppp1r-3. Comparing LPF versus CON and LPF versus LPE, the upregulated genes were Mgat2 and Cars, and the downregulated genes were Ddx28 and Slc12a9. The differentially expressed genes were mainly related to cell metabolism, immune response, signaling pathway, endocrine metabolism, stress response, ATP binding, and other functions. Early-life protein malnutrition affects gene expression of rat offspring and involves multiple aspects of growth and development, with different stages of early-life malnutrition leading to altered DNA methylation expression of corresponding genes, mainly in mitochondrial genes.