NanoString nCounter Technology Research Articles

Sciatic nerve stimulation alleviates neuropathic pain and associated neuroinflammation in the dorsal root ganglia in a rodent model

BackgroundSatellite glial cells (SGCs) in the dorsal root ganglia (DRG) play a pivotal role in the formation of neuropathic pain (NP). Sciatic nerve stimulation (SNS) neuromodulation was reported to alleviate NP and reduce neuroinflammation. However, the mechanisms underlying SNS in the DRG remain unclear. This study aimed to elucidate the mechanism of electric stimulation in reducing NP, focusing on the DRG.MethodsL5 nerve root ligation (NRL) NP rat model was studied. Ipsilateral SNS performed 1 day after NRL. Behavioral tests were performed to assess pain phenotypes. NanoString Ncounter technology was used to explore the differentially expressed genes and cellular pathways. Activated SGCs were characterized in vivo and in vitro. The histochemical alterations of SGCs, macrophages, and neurons in DRG were examined in vivo on post-injury day 8.ResultsNRL induced NP behaviors including decreased pain threshold and latency on von Frey and Hargreaves tests. We found that following nerve injury, SGCs were hyperactivated, neurotoxic and had increased expression of NP-related ion channels including TRPA1, Cx43, and SGC-neuron gap junctions. Mechanistically, nerve injury induced reciprocal activation of SGCs and M1 macrophages via cytokines including IL-6, CCL3, and TNF-α mediated by the HIF-1α-NF-κB pathways. SNS suppressed SGC hyperactivation, reduced the expression of NP-related ion channels, and induced M2 macrophage polarization, thereby alleviating NP and associated neuroinflammation in the DRG.ConclusionsNRL induced hyperactivation of SGCs, which had increased expression of NP-related ion channels. Reciprocal activation of SGCs and M1 macrophages surrounding the primary sensory neurons was mediated by the HIF-1α and NF-κB pathways. SNS suppressed SGC hyperactivation and skewed M1 macrophage towards M2. Our findings establish SGC activation as a crucial pathomechanism in the gliopathic alterations in NP, which can be modulated by SNS neuromodulation.

G2/M Checkpoint Modulation: Insights from miRNA Profiles in FAM and Breast Cancer.

The aim of this research is to understand the role of microRNA in cell cycle regulation especially on G2M Checkpoint from Luminal A samples Indonesian population. The profile results are used as biomarkers and therapeutic targets for breast cancer. For this reason, analysis was carried out on the comparison of miRNA expression between Luminal A and Fibroadenoma mamae (FAM) using Nanostring nCounter. In this study, 5 (Formalin-Fixed Paraffin-Embedded) FFPE Luminal A tissues and 4 FFPE FAM samples were used. RNA was isolated from cancer tissue samples. Differential expression analysis of miRNA was conducted using Nanostring nCounter technology, subsequently followed by the expression analysis between FAM and Luminal A using nSolver softwere. Elevated expression levels of miRNAs were subjected to pathway and gene regulation analysis using KEGG and GSEA MsigDB databases. Data visualization was performed utilizing Cytoscape, NetworkAnalyst, and SRplot tools. Based on 792 miRNAs detected on Nanostring nCounter, it was found that 60 miRNAs were upregulated and 6 miRNAs were downregulated. The 15 upregulated miRNAs analyzed show their role in the G2M Checkpoint through several pathways. The five miRNAs that significantly regulate the G2M Checkpoint are hsa-miR-196b-5p, hsa-miR-218-5p, hsa-miR-7-5p, hsa-miR-19a-5p, and hsa-miR-18a-5p Where each of these miRNAs regulates the CDKN1B gene. Significant differences in the expression of multiple miRNAs between Luminal A and FAM samples were observed. Furthermore, several of these miRNAs were found to modulate the G2M Checkpoint in Luminal A cancer by suppressing tumor suppressor genes.

A protective effect of lower MHC-II expression in MOGAD

Myelin oligodendrocyte glycoprotein-antibody-associated disease (MOGAD) is a demyelinating central nervous system disorder. We aimed to uncover immune pathways altered in MOGAD to predict disease progression. Using nanostring nCounter technology, we analyzed immune gene expression in PBMCs from MOGAD patients and compare it with healthy controls (HCs). We found 35 genes that distinguished MOGAD patients and HCs. We then validated those results in a larger cohort including MS and NMOSD patients. Expressions of HLA-DRA was significantly lower in MOGAD patients. This reduction in HLA-DRA, correlated with a monophasic disease course and greater brain volume, enhancing our ability to predict MOGAD progression.

Abstract 3258: A potential oncogenic role and immunoregulatory mechanisms of MZB1 in melanoma

Abstract While immune checkpoint blockade (ICB) therapies have drastically improved melanoma treatment, a majority of patients fail to achieve a durable response. Thus, there is a critical need to advance our understanding of melanoma immune evasion mechanisms and identify novel targets capable of enhancing ICB responses. MZB1 (Marginal Zone B and B1 Cell Specific Protein), an immune regulator gene, is differentially expressed in metastatic melanoma compared to primary melanoma or normal skin. In a limited number of cancers, MZB1 has been shown to function as either an oncogene or a tumor suppressor. However, its role and significance in melanoma is not known. In this study, we determined the functional significance and immunoregulatory mechanisms of MZB1 in human melanoma cell lines using MZB1 overexpression (OE) (in SK-MEL-2, and SK-MEL-28) and CRISPR knockout (KO) (in A375 and Hs294T) lines. MZB1 OE significantly increased melanoma cell proliferation and expression of the proliferation markers Ki67 and PCNA, while MZB1 KO yielded the opposite effect. To identify immunoregulatory mechanisms associated with MZB1, we performed differential gene expression analysis of 770 immune-related genes using NanoString nCounter technology. nSolver analysis identified 153 differentially expressed genes (DEGs) as a result of MZB1 knockout in Hs294T cells (adjusted p <0.05, 1.5-fold change cutoff). MZB1 KO strongly reduced the expression of the HLA class-II family genes and altered the expression of various cytokines, suggesting an immunoregulatory role of MZB1. Further, MZB1 KO significantly downregulated genes related to cancer progression and metastasis promotion. Additionally, MZB1 KO reduced expression of common “cancer stem cell” genes, such as RXRG, ALDH1A1, AQP1, NFATC2, LPAR1, indicating MZB1 expression in melanoma may promote a dedifferentiated or neural crest-like state. In addition, an Ingenuity Pathway Analysis (IPA) of the DEGs revealed many cancer-related functions altered upon MZB1 KO (p adjusted < 0.05) and displayed a negative enrichment for cancer-promoting pathways including tumor growth, metastasis, and proliferation. IPA regulatory analysis predicted MZB1 KO to decrease the activation of oncogenic signaling components including PI3K, MEK, and ERK1/2. Further, IPA predicted that MZB1 inhibition could positively sensitize melanoma cells towards higher immune responses by activating mechanisms associated with T cell proliferation and activation, leukocyte migration, and macrophage activation. Overall, our study suggests MZB1 promotes oncogenic signaling in melanoma to drive proliferation and dedifferentiation, as well as regulates a wide range of immune-related genes that may be responsible for immune evasion in melanoma cells. Thus, MZB1 merits further investigation to determine if the immune consequences yielded by targeting MZB1 can complement ICB approaches in the management of melanoma. Citation Format: Gagan Chhabra, Carl A. Shirley, Julia M. Krien, Mary A. Ndiaye, Nihal Ahmad. A potential oncogenic role and immunoregulatory mechanisms of MZB1 in melanoma [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 3258.

Unraveling the MicroRNA tapestry: exploring the molecular dynamics of locoregional recurrent rectal cancer.

Colorectal cancer (CRC) ranks as the third most prevalent malignancy globally, with a concerning rise in incidence among young adults. Despite progress in understanding genetic predispositions and lifestyle risk factors, the intricate molecular mechanisms of CRC demand exploration. MicroRNAs (miRNAs) emerge as key regulators of gene expression and their deregulation in tumor cells play pivotal roles in cancer progression. NanoString's nCounter technology was utilized to measure the expression of 827 cancer-related miRNAs in tumor tissue and adjacent non-involved normal colon tissue from five patients with locoregional CRC progression. These expression profiles were then compared to those from the primary colon adenocarcinoma (COAD) cohort in The Cancer Genome Atlas (TCGA). Intriguingly, 156 miRNAs showed a contrasting dysregulation pattern in reccurent tumor compared to their expression in the TCGA COAD cohort. This observation implies dynamic alterations in miRNA expression patterns throughout disease progression. Our exploratory study contributes to understanding the regulatory landscape of recurrent CRC, emphasizing the role of miRNAs in disease relapse. Notable findings include the prominence of let-7 miRNA family, dysregulation of key target genes, and dynamic changes in miRNA expression patterns during progression. Univariate Cox proportional hazard models highlighted miRNAs associated with adverse outcomes and potential protective factors. The study underscores the need for more extensive investigations into miRNA dynamics during tumor progression and the value of stage specific biomarkers for prognosis.

TGF-β and BMP signaling are associated with the transformation of glioblastoma to gliosarcoma and then osteosarcoma.

Gliosarcoma, an isocitrate dehydrogenase wildtype (IDH-WT) variant of glioblastoma, is defined by clonal biphasic differentiation into gliomatous and sarcomatous components. While the transformation from a glioblastoma to gliosarcoma is uncommon, the subsequent transformation to osteosarcoma is rare but may provide additional insights into the biology of these typically distinct cancers. We observed a patient initially diagnosed with glioblastoma, that differentiated into gliosarcoma at recurrence, and further evolved to osteosarcoma at the second relapse. Our objective was to characterize the molecular mechanisms of tumor progression associated with this phenotypic transformation. Tumor samples were collected at all 3 stages of disease and RNA sequencing was performed to capture their transcriptomic profiles. Sequential clonal evolution was confirmed by the maintenance of an identical PTEN mutation throughout the tumor differentiation using the TSO500 gene panel. Publicly available datasets and the Nanostring nCounter technology were used to validate the results. The glioblastoma tumor from this patient possessed mixed features of all 3 TCGA-defined transcriptomic subtypes of an IDH-WT glioblastoma and a proportion of osteosarcoma signatures were upregulated in the original tumor. Analysis showed that enhanced transforming growth factor-β (TGF-β) and bone morphogenic protein signaling was associated with tumor transformation. Regulatory network analysis revealed that TGF-β family signaling committed the lineage tumor to osteogenesis by stimulating the expression of runt-related transcription factor 2 (RUNX2), a master regulator of bone formation. This unusual clinical case provided an opportunity to explore the modulators of longitudinal sarcomatous transformation, potentially uncovering markers indicating predisposition to this change and identification of novel therapeutic targets.

Structural and functional damage to neuronal nuclei caused by extracellular tau oligomers.

Neuronal nuclei are normally smoothly surfaced. In Alzheimer's disease (AD) and other tauopathies, though, they often develop invaginations. We investigated mechanisms and functional consequences of neuronal nuclear invagination in tauopathies. Nuclear invagination was assayed by immunofluorescence in the brain, and in cultured neurons before and after extracellular tau oligomer (xcTauO) exposure. Nucleocytoplasmic transport was assayed in cultured neurons. Gene expression was investigated using nanoString nCounter technology and quantitative reverse transcription polymerase chain reaction. Invaginated nuclei were twice as abundant in human AD as in cognitively normal adults, and were increased in mouse neurodegeneration models. In cultured neurons, nuclear invagination was induced by xcTauOs by an intracellular tau-dependent mechanism. xcTauOs impaired nucleocytoplasmic transport, increased histone H3 trimethylation at lysine 9, and altered gene expression, especially by increasing tau mRNA. xcTauOs may be a primary cause of nuclear invagination in vivo, and by extension, impair nucleocytoplasmic transport and induce pathogenic gene expression changes. Extracellular tau oligomers (xcTauOs) cause neuronal nuclei to invaginate. xcTauOs alter nucleocytoplasmic transport, chromatin structure, and gene expression. The most upregulated gene is MAPT, which encodes tau. xcTauOs may thus drive a positive feedback loop for production of toxic tau.

Association between Microenvironment-Related Genes and Prognosis of Primary Central Nervous System Lymphoma

Background: Comprehensive gene mutation profiling of primary central nervous system lymphoma (PCNSL) revealed genomic abnormalities associated with the NFκB signaling pathway and immune escape (Chapuy B, et al. Blood 2016). Although this has led to advances in targeted therapy, there are only a few candidate biomarkers for diagnosis and prediction of survival in PCNSL. Recurrence still occurs at high rate of over 60% and therapeutic resistance is a significant challenge in the management of recurrent PCNSL. Until now, the systemic profiling of tumor micro-environment (TME) was performed through gene expression analysis by whole transcriptome analysis (WTA) and single-cell RNA sequencing analysis (Heming M, et al. Genome medicine 2022), and stratification in PCNSL was attempted by using spatial transcriptome analysis (Xia Y, et al. Leukemia 2023). However, TME of PCNSL and their impact on prognosis remain uncertain. Objective: We performed this study to investigate the prognostic impact on survival and establish novel prognostic biomarkers in PCNSL. Methods: We analyzed the expression levels of 770 neuroinflammation-related (NFR) genes by the NanoString nCounter technology in tumor samples from 30 PNCSL patients. The clinical significance of genes and their association with prognosis were assessed. Genes related to “worse prognosis (WP)” or “better prognosis (BP)” were identified. We performed the univariable analysis using a cox proportional hazards model to evaluate the predictive value of expression of genes related to WP and clinical risk factors, such as age, sex, serum lactate dehydrogenase level, Karnofsky Performance Status (KPS), consciousness levels at diagnosis and lymphoma involvement of the deep brain structure. Gene expression data related to WP were subjected to multivariate analysis with clinical variables with p values less than 0.15 in the univariate analysis. The genes associated with WP were further validated using WTA of an independent PCNSL cohort (n=30, previously published by Fukumura K, et al. Acta neuropathologica 2016). Results: The median age at diagnosis was 69 years old (range, 32-83). The median follow-up period was 25 months (range, 1-110 months), with overall survival (OS) at 3 years of 39.7% and progression free survival at 3 years of 25.4%. Notably, 48 of 770 NFR genes were highly expressed in the WP group (3-year OS, 22.2%), compared with the BP group (3-year OS, 66.7%) (Figure 1). We found that oligodendrocyte and astrocyte-related signatures were enriched in the WP-associated gene set. Among clinical prognostic factors, KPS > 70 (p=0.0293), and consciousness levels at diagnosis (other than JCS 0-1) (p=0.104) were relatively associated with poor OS (p < 0.15) in univariate analysis. Multivariate analysis revealed that high expressions of TUBB4A (p=0.028, HR:3.88), S100B (p=0.046, HR:3.093) and SLC6A1 (p=0.034, HR:3.765) were significantly related to death independent from KPS and consciousness levels at diagnosis. Expression levels of these 3 genes were also significantly associated with poor OS in a validation cohort. Conclusion: Our observations suggest high expression of TUBB4A, S100B and SLC6A1 are the clinical indicators to predict poor prognosis in PCNSL patients. Furthermore, these data suggest that genes related to TME may play a crucial role in the pathogenesis of PCNSL, complementing the well-known involvement of the NF-kB signaling pathway. Therefore it is necessary to continue research focused on TME-targeted therapeutics to encounter drug resistance and refractoriness in PCNSL.

Identification and subsequent validation of transcriptomic signature associated with metabolic status in endometrial cancer

Aberrant metabolism has been identified as a main driver of cancer. Profiling of metabolism-related pathways in cancer furthers the understanding of tumor plasticity and identification of potential metabolic vulnerabilities. In this prospective controlled study, we established transcriptomic profiles of metabolism-related pathways in endometrial cancer (EC) using a novel method, NanoString nCounter Technology. Fifty-seven ECs and 30 normal endometrial specimens were studied using the NanoString Metabolic Panel, further validated by qRT-PCR with a very high similarity. Statistical analyses were by GraphPad PRISM and Weka software. The analysis identified 11 deregulated genes (FDR ≤ 0.05; |FC|≥ 1.5) in EC: SLC7A11; SLC7A5; RUNX1; LAMA4; COL6A3; PDK1; CCNA1; ENO1; PKM; NR2F1; and NAALAD2. Gene ontology showed direct association of these genes with ‘central carbon metabolism (CCM) in cancer’. Thus, ‘CCM in cancer’ appears to create one of the main metabolic axes in EC. Further, transcriptomic data were functionally validated with drug repurposing on three EC cell lines, with several drug candidates suggested. These results lay the foundation for personalized therapeutic strategies in this cancer. Metabolic plasticity represents a promising diagnostic and therapeutic option in EC.

C-reactive protein is a prognostic biomarker in pancreatic ductal adenocarcinoma patients.

The 5-year survival rate of pancreatic ductal adenocarcinoma (PDAC) is approximately 11% and has only improved marginally over the last three decades. For operable PDAC, resection and adjuvant FOLFIRINOX chemotherapy is standard of care. There is growing interest in perioperative regimens to improve outcomes. The non-randomized Phase II study "Gemcitabine and Abraxane for resectable Pancreatic cancer" (GAP) demonstrated the feasibility of perioperative gemcitabine/abraxane. Long-term survival in PDAC requires an effective immune response; hence, we undertook this translational study of the GAP trial cohort to identify immune-oncology biomarkers for clinical use. We combined Nanostring nCounter technology with immunohistochemistry to investigate the correlation between gene expression and overall patient survival. Findings were investigated in samples from the International Cancer Genome Consortium (ICGC, n=88) and the Australian Pancreatic Genome Initiative (APGI, n=227). We confirmed that human equilibrative nucleoside transporter 1 (hENT1) expression was not a prognostic marker in PDAC but patients with high levels of hENT1 were more likely to live longer than 24 months post-surgery. Additionally, CD274 (PD-L1) and two novel biomarkers of survival, cathepsin W (CTSW) and C-reactive protein (CRP), were identified in the GAP cohort (n=19). CRP expression was confirmed in data from the ICGC. Although PD-L1 and CTSW proteins were not significant across all three cohorts, results show that low CRP mRNA and protein expression are associated with longer overall survival in all three patient groups. PDAC patients with long survival have higher hENT1 expression levels. Furthermore, CRP expression is a biomarker of poor prognosis following perioperative chemotherapy and resection in PDAC patients and thus may be useful for identifying patients who could benefit from more aggressive adjuvant strategies.

MTHFD1 is critical for the negative regulation of retinoic acid receptor signalling in anencephaly.

Neural tube defects are the most severe congenital malformations that result from failure of neural tube closure during early embryonic development, and the underlying molecular mechanisms remain elusive. Retinoic acid, an active derivative of vitamin A, is critical for neural system development, and retinoic acid receptor (RAR) signalling malfunctions have been observed in human neural tube defects. However, retinoic acid-retinoic acid receptor signalling regulation and mechanisms in neural tube defects are not fully understood. The mRNA expression of RARs and retinoid X receptors in the different human neural tube defect phenotypes, including 11 pairs of anencephaly foetuses, 10 pairs of hydrocephalus foetuses and nine pairs of encephalocele foetuses, was investigated by NanoString nCounter technology. Immunoprecipitation-mass spectrometry was performed to screen the potential interacting targets of retinoic acid receptor γ. The interactions between proteins were confirmed by co-immunoprecipitation and immunofluorescence laser confocal microscopy. Luciferase and chromatin immunoprecipitation with quantitative real-time polymerase chain reaction assays were used to clarify the underlying mechanism. Moreover, a neural tube defect animal model, constructed using excess retinoic acid, was used for further analysis with established molecular biology technologies. We report that level of retinoic acid receptor γ (RARγ) mRNA was significantly upregulated in the brain tissues of human foetuses with anencephaly. To further understand the actions of retinoic acid receptor γ in neural tube defects, methylenetetrahydrofolate dehydrogenase 1 was identified as a specific retinoic acid receptor γ target from IP-MS screening. Additionally, methylenetetrahydrofolate dehydrogenase 1 negatively regulated retinoic acid receptor γ transcription factor activity. Furthermore, low expression of methylenetetrahydrofolate dehydrogenase 1 and activation of retinoic acid receptor signalling were further determined in human anencephaly and a retinoic acid-induced neural tube defect mouse model. This study reveals that methylenetetrahydrofolate dehydrogenase 1, the rate-determining enzyme in the one-carbon cycle, might be a specific regulator of retinoic acid receptors; these findings provide new insights into the functional linkage between nuclear folate metabolism and retinoic acid receptor signalling in neural tube defect pathology.

The trehalose glycolipid C18Brar promotes antibody and T-cell immune responses to Mannheimia haemolytica and Mycoplasma ovipneumoniae whole cell antigens in sheep.

Bronchopneumonia is a common respiratory disease in livestock. Mannheimia haemolytica is considered the main causative pathogen leading to lung damage in sheep, with Mycoplasma ovipneumoniae and ParaInfluenza virus type 3, combined with adverse physical and physiological stress, being predisposing factors. A balance of humoral and cellular immunity is thought to be important for protection against developing respiratory disease. In the current study, we compared the ability of the trehalose glycolipid adjuvant C18Brar (C18-alkylated brartemicin analogue) and three commercially available adjuvant systems i.e., Quil-A, Emulsigen-D, and a combination of Quil-A and aluminium hydroxide gel, to stimulate antibody and cellular immune responses to antigens from inactivated whole cells of M. haemolytica and M. ovipneumoniae in sheep. C18Brar and Emulsigen-D induced the strongest antigen-specific antibody responses to both M. haemolytica and M. ovipneumoniae, while C18Brar and Quil-A promoted the strongest antigen-specific IL-17A responses. The expression of genes with known immune functions was determined in antigen-stimulated blood cultures using Nanostring nCounter technology. The expression levels of CD40, IL22, TGFB1, and IL2RA were upregulated in antigen-stimulated blood cultures from animals vaccinated with C18Brar, which is consistent with T-cell activation. Collectively, the results demonstrate that C18Brar can promote both antibody and cellular responses, notably Th17 immune responses in a ruminant species.

MicroRNA profiling of low concentration extracellular vesicle RNA utilizing NanoString nCounter technology

AbstractExtracellular vesicles (EV) and the microRNAs that they contain are increasingly recognised as a rich source of informative biomarkers, reflecting pathological processes and fundamental biological pathways and responses. Their presence in biofluids makes them particularly attractive for biomarker identification. However, a frequent caveat in relation to clinical studies is low abundance of EV RNA content. In this study, we used NanoString nCounter technology to assess the microRNA profiles of n = 64 EV low concentration RNA samples (180–49125 pg), isolated from serum and cell culture media using precipitation reagent or sequential ultracentrifugation. Data was subjected to robust quality control parameters based on three levels of limit of detection stringency, and differential microRNA expression analysis was performed between biological subgroups. We report that RNA concentrations > 100 times lower than the current NanoString recommendations can be successfully profiled using nCounter microRNA assays, demonstrating acceptable output ranges for imaging parameters, binding density, positive/negative controls, ligation controls and normalisation quality control. Furthermore, despite low levels of input RNA, high‐level differential expression analysis between biological subgroups identified microRNAs of biological relevance. Our results demonstrate that NanoString nCounter technology offers a sensitive approach for the detection and profiling of low abundance EV‐derived microRNA, and may provide a solution for research studies that focus on limited sample material.

Analysis of Copy Number Variation of DNA Repair/Damage Response Genes in Tumor Tissues.

Cells experience increased genome instability through the course of disease development including cancer initiation and progression. Point mutations, insertion/deletions, translocations, and amplifications of both coding and noncoding regions all contribute to cancer phenotypes. Copy number variation (CNV), i.e., changes of the number of copies of nuclear DNA, occurs in the genome of even normal somatic cells. Studies to understand the effects of CNV on tumor development, especially aspects concerning tumor aggressiveness and the influence on outcomes of therapeutic modalities, have been reignited by the breakthrough technologies of the molecular genomics. This section discusses the significance of analyzing CNVs that cause simultaneous increase/decrease of clusters of genes, using the expression profile of XRCC1 with its neighbor genes LIG1, PNKP, and POLD1 as an example. Methods for CNV assay at the individual gene level on formalin-fixed, paraffin-embedded (FFPE) tissues using the NanoString nCounter technology will then be described.

RNA profiling of circulating tumor cells systemically captured from diagnostic leukapheresis products in prostate cancer patients.

The application of circulating tumor cells (CTCs) in both clinical practice and research has been continuously limited by the rare number of targets that can be found in a tube of peripheral blood. Diagnostic leukapheresis (DLA) was used to increase the sampling volume. AdnaTest was used to process the whole leukopak, and the RNAs of captured CTCs was then profiled by NanoString nCounter platform. Spike-in experiments and leukopaks from patients with metastatic prostate cancer were used to validate this new strategy. The whole leukopak was further concentrated five times to reduce the total volume from 150​mL to 30​mL, which enabled it to be processed by 3 separate AdnaTest kits. The spike-in experiment demonstrated a reliable capture when there were more than 100 cancer cells/10​mL of concentrated leukopak. In 1 out of 5 real patient samples, CTCs were only detected in the leukopak, but not in peripheral blood. The RNA profiling of DLA CTCs indicated a more aggressive phenotype of CTCs occurred when the patient was experiencing a disease relapse, even when the serum prostate specific antigen (PSA) level was still relatively low and CTCs in peripheral blood were not detectable. We established a new protocol, integrating DLA, AdnaTest and NanoString nCounter technology, to profile RNAs from CTCs captured from a large blood screening volume. The new protocol can process the whole leukopak with sensitive CTC capture. The RNA profiling of CTCs can provide valuable information for disease monitoring.

Exploring New Prognostic Biomarkers in Mantle Cell Lymphoma: The Best RNA Based Risk Score

INTRODUCTION Mantle Cell Lymphoma (MCL) is characterized by heterogenous disease courses ranging from indolent cases, that do not require therapy for years, to highly aggressive disease with a poor prognosis despite intensive treatment regimens. This emphasizes the need for accurate prognostic biomarkers to guide risk-adapted treatment strategies. The established risk factors used in the clinic today including high Mantle Cell Lymphoma International Prognostic Index (MIPI), high Ki67 proliferation index, high MIPI-c, TP53 mutations, and blastoid morphology, only partly identify patients in need of alternative treatment. Therefore, the search for clinically fit and biologically relevant biomarkers has been intensified. This has led to the proposal of two new NanoString-based, proliferation-associated prognostic biomarkers: the mRNA-based MCL35 score (Scott et al. 2017) and the circular RNA based circSCORE (Dahl et al. 2021). Both biomarkers have shown promising prognostic potential, but a comparison has never been done before. In this study we aimed at comparing MCL35 and circSCORE in 149 patients from the uniformly treated MCL2 and MCL3 cohorts. MATERIALS AND METHODS We included 149 patients from the Nordic patient cohorts MCL2 (70 patients) and MCL3 (79 patients) with sample data on both circSCORE and MCL35 along with long-term follow up data including progression free survival (PFS) and overall survival (OS). Gene expression analysis was obtained with the NanoString nCounter technology after RNA-isolation, using samples originating from both diagnostic lymph-nodes and non-nodal tissue. We also performed individual analyses on the MCL3 cohort only to eliminate the potential risk of overfitting, since the circSCORE is trained upon the MCL2 cohort. RESULTS We report that both circSCORE (PFS log-RANK p-value 0.0048 and OS log-RANK p-value 0.0426) and MCL35 (PFS log-RANK p-value 0.0422 and OS log-RANK p-value 0.0444) in the MCL3 only survival analyses displayed equal significant, prognostic potential in stratifying high and low risk groups. These analyses also revealed inability to significantly stratify the MCL35 standard risk group from the high- and low MCL35 risk groups (figure 1). Neither continuous circSCORE nor continuous MCL35 held prognostic significance when adjusting for known high-risk factors in multivariable cox regression, where only MIPI and TP53 mutation maintained significant results in all settings, except for PFS in MCL3 only (data not shown). The categorial data were analyzed with log-likelihood (LL) using the MIPI and TP53wt (wild type) cox models as baseline models (table 1). The results showed that both circSCORE and Ki67 added significant improvement to the MIPI baseline model in the pooled, MCL2+MCL3 setting, but most interestingly, only circSCORE added significant improvement in MCL3 only for PFS (LL -97.655 vs -100.197, p-value 0.011). Neither circSCORE, MCL35 nor Ki67 added significant improvement to the TP53wt baseline model (table 1). CONCLUSION Both circSCORE and MCL35 demonstrated equal prognostic potential in separating high-and low risk groups in this study. Neither circSCORE, MCL35 nor Ki67 added significant improved value to the TP53wt baseline cox model, but circSCORE added significant improved value to the MIPI baseline cox model, even outperforming Ki67. This implies a new combined prognostic biomarker "MIPI-circSCORE", which could reflect the complexity of MCL through both clinical and biological, proliferation-associated features. However, the overall performance of the MCL35 risk score might be affected due to the inability of separating the standard risk group from the other MCL35 risk groups, the limited number of patients, and the tissue specific criteria of the MCL35 score not being met in this study. Therefore, the findings of this study should be further explored in larger MCL-cohorts to ensure stronger statistical power. Also, testing the biomarkers in MCL-relapse would be of great value to examine the prognostic potential in other settings of the heterogenic course of MCL. This is warranted to clarify the clinical future relevance of these biomarkers in supporting risk-adapted treatment regimens in patients with MCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IMMU-03. ANALYSIS OF EXPRESSION SIGNATURES OF MATCHED PRIMARY GASTROINTESTINAL CANCER AND BRAIN METASTASES USING NANOSTRING NCOUNTER TECHNOLOGY

Abstract BACKGROUND Brain metastases from the gastrointestinal tract are a rare event (less than 10% of all brain lesions), but their impact on prognosis is remarkable. Their incidence is increasing due to earlier diagnosis and prolonged survival because of more effective treatment modalities. Because of the lack of evidence-based recommendations, no optimal treatment strategy has been defined. Hope might come from the discovery of new prognostic and predictive factors and their biological inhibitors. The aim of this study was to create a repository of matched primary cancer and brain metastases samples for clinicopathologic and molecular correlations of gastrointestinal tumors and to analyze changes in expression signatures between both samples. The main goal was to study the metastatic process and characterize the immune response by characterizing features such as invasiveness, vascular co-option, proliferation, stemness and cell-type heterogeneity between the matched tissues. METHODS We searched for patients at Stanford hospital with matched brain metastases from gastrointestinal tumors from 2008 to 2020 using the STARR database (The STAnford Research Repository, or STARR, is Stanford Medicine's approved resource for working with clinical data for research purposes.). Matched specimens mean that both brain metastases and gastrointestinal tumors were studied at Stanford University. FFPE blocks from these matched patients were sectioned and sections analyzed for gene expression analysis using nanoString nCounter technology and the immune-oncology panel. RESULTS Expression data from 32 matched patients with primary tumor and brain metastases will be discussed at the meeting. CONCLUSION We believe that analysis of changes in expression signatures between the matched primary gastrointestinal cancer and brain metastases will provide novel insights into the role for tumor factors, such as invasiveness, proliferation and stemness, and the tumor microenvironment in progression.

Distinct Immune Reconstitution Profiles Captured by Immune Functional Assays at 6 Months Post Allogeneic Hematopoietic Stem Cell Transplantation

Immune reconstitution after allogeneic-hematopoietic-stem-cell transplantation (allo-HSCT) is a complex and individual process. In this cross-sectional study, whole-blood (WB) immune functional assay (IFA) was used to characterize immune function by assessing immune-related gene/pathway alterations. The usefulness of this tool in the context of infection, 6 months after transplantation, was evaluated. Sixty allo-HSCT recipients at 6 months after transplantation and 10 healthy volunteers (HV) were included. WB was stimulated in standardized TruCulture tubes using lipopolysaccharides and Staphylococcal enterotoxin B. Gene expression was quantified using a custom 144-gene panel using NanoString nCounter technology and analyzed using Ingenuity Pathway Analysis. The relationships between immune function and clinical characteristics, immune cell counts, and post-transplantation infections were assessed. Allo-HSCT recipients were able to activate similar networks of the innate and adaptive immune response compared to HV, with, nevertheless, a lower intensity. A reduced number and a lower expression of genes associated with immunoregulatory and inflammatory processes were observed in allo-HSCT recipients. The use of immunosuppressive treatments was associated with a protracted immune reconstitution revealed by transcriptomic immunoprofiling. No difference in immune cell counts was observed among patients receiving or not receiving immunosuppressive treatments using a large immunophenotyping panel. Moreover, the expression of a set of genes, including CCL3/CCL4, was significantly lower in patients with Herpesviridae reactivation (32%, 19/60), which once again was not identified using classical immune cell counts. Transcriptional IFA revealed the heterogeneity among allo-HSCT recipients with a reduced immune function, a result that could not be captured by circulating immune cell counts. This highlights the potential added value of this tool for the personalized care of immunocompromised patients.

Feasibility and Potential of Transcriptomic Analysis Using the NanoString nCounter Technology to Aid the Classification of Rejection in Kidney Transplant Biopsies.

Transcriptome analysis could be an additional diagnostic parameter in diagnosing kidney transplant (KTx) rejection. Here, we assessed feasibility and potential of NanoString nCounter analysis of KTx biopsies to aid the classification of rejection in clinical practice using both the Banff-Human Organ Transplant (B-HOT) panel and a customized antibody-mediated rejection (AMR)-specific NanoString nCounter Elements (Elements) panel. Additionally, we explored the potential for the classification of KTx rejection building and testing a classifier within our dataset. Ninety-six formalin-fixed paraffin-embedded KTx biopsies were retrieved from the archives of the ErasmusMC Rotterdam and the University Hospital Cologne. Biopsies with AMR, borderline or T cell-mediated rejections (BLorTCMR), and no rejection were compared using the B-HOT and Elements panels. High correlation between gene expression levels was found when comparing the 2 chemistries pairwise (r = 0.76-0.88). Differential gene expression (false discovery rate; P < 0.05) was identified in biopsies diagnosed with AMR (B-HOT: 294; Elements: 76) and BLorTCMR (B-HOT: 353; Elements: 57) compared with no rejection. Using the most predictive genes from the B-HOT analysis and the Element analysis, 2 least absolute shrinkage and selection operators-based regression models to classify biopsies as AMR versus no AMR (BLorTCMR or no rejection) were developed achieving an receiver-operating-characteristic curve of 0.994 and 0.894, sensitivity of 0.821 and 0.480, and specificity of 1.00 and 0.979, respectively, during cross-validation. Transcriptomic analysis is feasible on KTx biopsies previously used for diagnostic purposes. The B-HOT panel has the potential to differentiate AMR from BLorTCMR or no rejection and could prove valuable in aiding kidney transplant rejection classification.

Global circRNA expression changes predate clinical and histological improvements of psoriasis patients upon secukinumab treatment.

Psoriasis is a common chronic inflammatory skin disease accompanied by heterogenous clinical and histological features, including a characteristic keratinocyte hyperproliferation and dermal immunogenic profile. In addition, psoriasis is associated with widespread transcriptomic alterations including changes in microRNA (miRNA) and circular RNA (circRNA) abundance, which constitute non-coding RNA (ncRNA) classes with specific regulatory capacities in diverse physiological and pathological processes. However, the knowledge about the expression dynamics of ncRNA during psoriasis treatment is sparse. To elucidate the dynamics of miRNA and circRNA abundance during secukinumab (anti-IL-17A) treatment, we studied their expression patterns in skin biopsies from 14 patients with severe plaque-type psoriasis before and during an 84-day secukinumab therapy at day 0, 4, 14, 42, and 84 using NanoString nCounter technology. We found a comprehensive downregulation of the majority of investigated circRNAs and specific alterations in the miRNA profile, including an upregulation of miR-203a-3p, miR-93-5p, and miR-378i in lesional compared to non-lesional skin before treatment. During treatment, the circRNAs progressively returned to the expression levels observed in non-lesional skin and already four days after treatment initiation most circRNAs were significantly upregulated. In comparison, for miRNAs, the normalization to baseline during treatment was delayed and limited to a subset of miRNAs. Moreover, we observed a strong correlation between multiple circRNAs, including ciRS-7 and circPTPRA, and the psoriasis area and severity index (PASI). Similar pronounced correlations could, however, not be found for miRNAs. Finally, we did not observe any significant changes in circRNA expression in peripheral blood mononuclear cells during treatment. In conclusion, we uncovered a rapid shift in global circRNA abundance upon anti-IL-17A treatment, which predated clinical and histological improvements, and a strong correlation with PASI, indicating a biomarker potential of individual circRNAs.