Plasma Of Colorectal Cancer Patients Research Articles

Evaluation of the Plasma Expression Levels of miR-21 and miR-145 as Potential Non-Invasive Biomarkers for Early Detection of Colorectal Cancer.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in the world. Early detection would be greatly enhanced if accurate and cost-effective diagnostic biomarkers for CRC were accessible. The development of blood tests would evidently lower the screening cost of CRC detection. The aim of the present study was to examine the prospective of plasma miRNAs as non-invasive biomarkers for CRC screening. The expressions of miR-21 and miR-145 in the plasma of colorectal adenocarcinomas and normal healthy controls were quantified by using TaqMan miRNA assays. MiRNA expression levels were also correlated with commonly used clinicopathological features of CRC. Out of 30 CRC patients, 19 were male and 11 were female. The Mean age of patients was 51.3 ±14.6 years. A statistically significant increase in expression of miR-21 was observed in CRC patients' as compared to healthy controls (p<0.001). A significant association between miR-21 expression and age group (p=0.002) was noticed. Also, a statistically significant difference (p=0.015) between miR-21 expression and tumor location in the proximal and distal sites of the colon was observed in CRC patients. Further, a statistically significant downregulation of miR-145 expression was observed in the plasma of CRC patients as compared to healthy controls (p<0.05). This is the first study to report a significant association between miR-21 expression, age group, and tumor location in CRC patients. The present study thus emphasises that the appraisal of miR-21 and miR-145 plasma levels may serve as a promising non-invasive screening tool for the early detection of colorectal cancer.

Tumor-Derived Exosomal Circular RNA Pinin Induces FGF13 Expression to Promote Colorectal Cancer Progression through miR-1225-5p.

: Colorectal cancer (CRC) is a common malignant tumor, and circular RNAs (circRNAs) are abnormally expressed in CRC. However, the function and underlying mechanism of circRNA pinin (circ-PNN; hsa_circ_0101802) in CRC remain unclear. : Exosomes were isolated from the plasma of CRC patients and identified by transmission electron microscopy and Western blotting. The RNA expression levels of circ-PNN, miR-1225-5p, and fibroblast growth factor 13 (FGF13) were measured by quantitative real-time polymerase chain reaction. Cell proliferation was detected by Cell Counting K-8, colony formation, and 5-ethynyl-2'-deoxyuridine assays. Cell apoptosis was assessed by flow cytometry. The expression of apoptosis and metastasis-related proteins was evaluated by Western blotting. The associations among circ-PNN, miR-1225-5p, and FGF13 were confirmed by dual-luciferase report assay and RNA immunoprecipitation assay. A xenograft model was used to verify the function of circ-PNN in tumor formation in vivo. : circ-PNN expression was upregulated in plasmic exosomes derived from CRC patients. The expression of circ-PNN and FGF13 was upregulated, while miR-1225-5p expression was downregulated in CRC cells incubated with plasmic exosomes derived from CRC patients. Tumor-derived exosomes promoted the proliferation, migration, and invasion but inhibited apoptosis of CRC cells. Moreover, the addition of tumor-derived exosomes partly reversed the inhibitory effect of circ-PNN knockdown on CRC tumor progression in vitro and in vivo. Thus, circ-PNN acts as a sponge for miR-1225-5p to regulate FGF13 expression. : Tumor-derived exosomal circ-PNN promoted CRC progression through the regulation of the miR-1225-5p/FGF13 pathway, providing a potential therapeutic target for CRC.

H1Innovative approaches to combat anti-cancer drug resistance: Targeting lncRNA and autophagy.

To date, standardizing clinical predictive biomarkers for assessing the response to immunotherapy remains challenging due to variations in personal genetic signatures, tumour microenvironment complexities and epigenetic onco-mechanisms. Early monitoring of key non-coding RNA (ncRNA) biomarkers may help in predicting the clinical efficacy of cancer immunotherapy and come up with standard predictive ncRNA biomarkers. For instance, reduced miR-125b-5p level in the plasma of non-small cell lung cancer patients treated with anti-PD-1 predicts a positive outcome. The level of miR-153 in the plasma of colorectal cancer patients treated with chimeric antigen receptor T lymphocyte (CAR-T) cell therapy may indicate the activation of T-cell killing activity. miR-148a-3p and miR-375 levels may forecast favourable responses to CAR-T-cell therapy in B-cell acute lymphoblastic leukaemia. In cancer patients treated with the GPC3 peptide vaccine, serum levels of miR-1228-5p, miR-193a-5p and miR-375-3p were reported as predictive biomarkers of good response and improved overall survival. Therefore, there is a critical need for further studies to elaborate on the key ncRNA biomarkers that have the potential to predict early clinical responses to immunotherapy. This review summarises important predictive ncRNA biomarkers that were reported in cancer patients treated with different immunotherapeutic modalities including monoclonal antibodies, small molecule inhibitors, cancer vaccines and CAR-T cells. In addition, a concise discussion on forthcoming perspectives is provided, outlining technical approaches for the optimal utilisation of immune-modulatory ncRNA biomarkers as predictive tools and therapeutic targets.

Non-coding RNAs in cancer immunotherapy: Predictive biomarkers and targets.

To date, standardising clinical predictive biomarkers for assessing the response to immunotherapy remains challenging due to variations in personal genetic signatures, tumour microenvironment complexities and epigenetic onco-mechanisms. Early monitoring of key non-coding RNA (ncRNA) biomarkers may help in predicting the clinical efficacy of cancer immunotherapy and come up with standard predictive ncRNA biomarkers. For instance, reduced miR-125b-5p level in the plasma of non-small cell lung cancer patients treated with anti-PD-1 predicts a positive outcome. The level of miR-153 in the plasma of colorectal cancer patients treated with chimeric antigen receptor T lymphocyte (CAR-T) cell therapy may indicate the activation of T-cell killing activity. miR-148a-3p and miR-375 levels may forecast favourable responses to CAR-T-cell therapy in B-cell acute lymphoblastic leukaemia. In cancer patients treated with the GPC3 peptide vaccine, serum levels of miR-1228-5p, miR-193a-5p and miR-375-3p were reported as predictive biomarkers of good response and improved overall survival. Therefore, there is a critical need for further studies to elaborate on the key ncRNA biomarkers that have the potential to predict early clinical responses to immunotherapy. This review summarises important predictive ncRNA biomarkers that were reported in cancer patients treated with different immunotherapeutic modalities, including monoclonal antibodies, small molecule inhibitors, cancer vaccines and CAR-T cells. In addition, a concise discussion on forthcoming perspectives is provided, outlining technical approaches for the optimal utilisation of immunomodulatory ncRNA biomarkers as predictive tools and therapeutic targets.

Evaluation of C–reactive protein and fibrinogen in comparison to CEA and CA72–4 as diagnostic biomarkers for colorectal cancer

Carcinoembryonic antigen (CEA) and carbohydrate antigen 72–4 (CA72–4) are commonly used markers for colorectal cancer (CRC) in clinical applications. However, low positivity rate and sensitivity limits their clinical effectiveness. In this study, we explored the potential of C–reactive protein (CRP) and fibrinogen to improve the diagnostic efficiency of traditional biomarkers of CRC. The concentrations of CRP and fibrinogen in plasma were significantly higher in CRC patients compared with benign or healthy controls. The area under the ROC curves (AUCs) showed that the diagnostic efficacy of CRP and fibrinogen was 0.745 (95% CI: 0.712–0.779) and 0.699 (95% CI: 0.663–0.734), respectively. AUC increased to 0.750 (95% CI: 0.716–0.784) when CRP and fibrinogen were combined. It also further improved to 0.889 (95% CI: 0.866–0.913) when CRP and fibrinogen were integrated with CEA and CA72–4. Moreover, this combination increased the maximum area under AUC to 0.857 (95% CI: 0.830–0.883), which effective differentiated CRC from benign disease. Overall, this study found that CRP and fibrinogen were highly expressed in the plasma of CRC patients, suggesting their potential to improve the diagnostic efficiency of traditional biomarkers of CRC.

The Composition of Small Extracellular Vesicles (sEVs) in the Blood Plasma of Colorectal Cancer Patients Reflects the Presence of Metabolic Syndrome and Correlates with Angiogenesis and the Effectiveness of Thermoradiation Therapy.

The majority of colorectal cancer patients (CRCPs) develop tumors on the background of "metabolically healthy obesity" or metabolic syndrome. The aim of the work was to study the levels of matrix metalloproteinases (MMPs) and heat shock proteins (HSPs) on the surface of blood plasma CD9-positive and FABP4-positive small extracellular vesicles (sEVs) from CRCPs depending on metabolic status and tumor angiogenesis, as well as to evaluate the sEVs markers as predictors of the effectiveness of thermoradiotherapy. In CRCPs, compared with patients with colorectal polyps (CPPs), the proportion of triple positive EVs and EVs with the MMP9+MMP2-TIMP1+ phenotype increased significantly among FABP4-positive EVs (adipocyte-derived EVs), which in general may indicate the overexpression of MMP9 and TIMP1 by adipocytes or adipose tissue macrophages in CRCPs. The results obtained have prospects for use as markers to clarify cancer risk in CPPs. One can assume that for CRCPs with metabolic syndrome or metabolically healthy obesity, it is the FABP4+MMP9+MMP2-TIMP1- population of circulating sEVs that is the most optimal biomarker reflecting tumor angiogenesis. Determining this population in the blood will be useful in monitoring patients after treatment for the early detection of tumor progression. CD9+MMP9+MMP2-TIMP1- and MMP9+MMP2-TIMP1+ subpopulations of circulating sEVs are the most promising predictors of the efficacy of thermoradiation therapy because their levels at baseline differ significantly in CRCPs with different tumor responses.

Development of Multiplex Drop-Off Digital PCR Assays for Hotspot Mutation Detection of KRAS, NRAS, BRAF, and PIK3CA in the Plasma of Colorectal Cancer Patients

The detection of mutations in KRAS, NRAS, BRAF, and PIK3CA has become essential in managing the treatment of metastatic colorectal cancer (CRC) with the approval of new targeted therapies. We developed novel multiplex drop-off digital PCR (MDO-dPCR) assays by combining amplitude-/ratio-based multiplexing with drop-off/double drop-off strategies that allow for the detection of at least the 69 most frequent hotspot mutations in all four genes with only three reactions. We assessed the analytical performance of the assays using synthetic oligonucleotides, which were further validated on plasma cfDNA samples from a large cohort of CRC patients and compared with next-generation sequencing data. The MDO-dPCR assays showed a high sensitivity with a limit of detection ranging from 0.084% to 0.182% in mutant allelic frequency. The screening of plasma cfDNAs from 106 CRC patients identified mutations in 42.45% of them, with a sensitivity of 95.24%, a specificity of 98.53%, and an accuracy of 96.98% for mutation detection, and a strong correlation of measured mutant allelic frequencies compared with next-generation sequencing results. The high sensitivity and comprehensive mutation coverage of the MDO-dPCR assays make them suitable for rapid and cost-effective detection of KRAS, NRAS, BRAF, and PIK3CA mutations in the plasma of CRC patients, and could be useful in early response assessment and longitudinal disease monitoring.

LncGMDS-AS1 promotes the tumorigenesis of colorectal cancer through HuR-STAT3/Wnt axis

Chronic inflammation promotes the tumorigenesis and cell stemness maintenance of colorectal cancer (CRC). However, the bridge role of long noncoding RNA (lncRNA) in linking chronic inflammation to CRC development and progression needs better understanding. Here, we elucidated a novel function of lncRNA GMDS-AS1 in persistently activated signal transducer and transcription activator 3 (STAT3) and Wnt signaling and CRC tumorigenesis. Interleukin-6 (IL-6) and Wnt3a induced lncRNA GMDS-AS1 expression, which was highly expressed in the CRC tissues and plasma of CRC patients. GMDS-AS1 knockdown impaired the survival, proliferation and stem cell-like phenotype acquisition of CRC cells in vitro and in vivo. We performed RNA sequencing (RNA-seq) and mass spectrometry (MS) to probe target proteins and identify their contributions to the downstream signaling pathways of GMDS-AS1. In CRC cells, GMDS-AS1 physically interacted with the RNA-stabilizing protein HuR, thereby protecting the HuR protein from polyubiquitination- and proteasome-dependent degradation. HuR stabilized STAT3 mRNA and upregulated the levels of basal and phosphorylated STAT3 protein, persistently activating STAT3 signaling. Our research revealed that the lncRNA GMDS-AS1 and its direct target HuR constitutively activate STAT3/Wnt signaling and promote CRC tumorigenesis, the GMDS-AS1-HuR-STAT3/Wnt axis is a therapeutic, diagnostic and prognostic target in CRC.

MiRNA probe integrated biosensor platform using bimetallic nanostars for amplification-free multiplexed detection of circulating colorectal cancer biomarkers in clinical samples

There is a critical need for sensitive and rapid detection technologies utilizing molecular biotargets such as microRNAs (miRNAs), which regulate gene expression and are a promising class of diagnostic biomarkers for disease detection. Here, we present the development and fabrication of a highly reproducible and robust plasmonic bimetallic nanostar biosensing platform to detect miRNA targets using surfaced-enhanced Raman scattering (SERS)-based gene probes called the inverse Molecular Sentinel (iMS). We investigated and optimized the integration of iMS gene probes onto this SERS substrate, achieving ultra-sensitive detection with limits of detection of 6.8 and 16.7 zmol within the sensing region for two miRNA sequences of interest. Finally, we demonstrated the biomedical usefulness of this nanobiosensor platform with the multiplexed detection of upregulated miRNA targets, miR21 and miR221, from colorectal cancer patient plasma. The resulting SERS data are in excellent agreement with PCR data obtained from patient samples and can distinguish between healthy and cancerous patient samples. These results underline the potential of the iMS-integrated substrate nanobiosensing platform for rapid and sensitive diagnostics of cancer biomarkers for point-of-care applications.

Identification of a 5-Hydroxymethylation Signature in Circulating Cell-Free DNA for the Noninvasive Detection of Colorectal Cancer.

Background As a crucial epigenetic modification, DNA 5-hydroxymethylcytosine (5-hmC) plays a key role during colorectal cancer (CRC) carcinogenesis. Nevertheless, the levels of 5-hmC-related genes in the circulating DNA of CRC remain largely unknown. Methods and Results The GSE81314 dataset from the Gene Expression Omnibus (GEO), which was generated by chemical marking-based low-input shotgun sequencing to detect 5-hmC in circulating cell-free DNA (cfDNA) was used in the present study. The GSE81314 dataset includes data for 8 plasma samples from healthy individuals and 4 plasma samples from CRC patients. The difference in the 5-hmC levels in cfDNA between the CRC group and healthy individuals was analyzed by the differentially expressed genes (DEG) package. Weighted gene coexpression network analysis (WGCNA) was conducted to analyze gene coexpression modules associated with sample characteristics. DEG analysis identified 19 upregulated and 9 downregulated 5-hmC-related genes. WGCNA showed that the pink, purple, and brown modules, which contain 531 genes in total, were significantly correlated with CRC (0.66, 0.61, and -0.59, respectively). We used gene set enrichment analysis (GSEA) software to compare 5-hmC-related genes and pathways between CRC patients and healthy controls. We further performed a protein–protein interaction (PPI) analysis and identified 4 nodes (LCN2, LRG1, S100P, and TACSTD2) that played key roles in the network, and we analyzed the expression of these nodes S100P in the GEPIA database. Consistent with the 5-hmC levels in CRC patient plasma, our external validation results from the GEPIA and UALCAN databases showed that LCN2, LRG1, S100P, and TACSTD2 were highly expressed in CRC tissue compared with controls. The DNA promoter methylation levels of LCN2, LRG1, and S100P were lower in CRC tissue than in normal control tissue. Conclusion The present findings suggest that abnormality in cell-free DNA hydroxylation in plasma may be associated with CRC. In addition, the 5-hmC levels of LCN2, LRG1, S100P, and TACSTD2 in circulating cfDNA may be used as potential noninvasive markers for CRC.

Abstract 1484: Emerging microRNAs as biomarkers for colorectal cancer diagnosis: A meta-analysis

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer deaths worldwide. The current method of clinical screening is mainly based on colonoscopy. However, many CRC patients are unwilling to undergo colonoscopy because of the high procedure cost and the discomfort during examination. Therefore, it appears necessary to find novel and non-invasive biomarkers that would improve CRC early detection. MicroRNAs (miRNAs) are a class of non-coding RNAs with critical regulatory roles in fundamental cellular processes, such as proliferation, migration, and apoptosis. They can act as potential oncogenes or tumor suppressors in cancer development. Numerous studies have shown significant differences in miRNAs expression in serum or plasma of CRC patients and healthy subjects. Thus, the potential use of circulating miRNAs as diagnostic biomarkers in CRC seems to be a fast and relevant alternative to traditional and invasive methods. Here, we reviewed selected studies which identified dysregulated circulating miRNAs in CRC and evaluated their diagnostic value. Methods: This meta-analysis study was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The articles were searched in online databases such as PubMed, Google Scholar to identify all eligible studies that evaluated the diagnostic value of miRNAs for CRC. Then we explored the diagnostic accuracy of each miRNA using the random-effects model to estimate the pooled sensitivity (Se), specificity (Sp), Positive likelihood ratio (PLR) and Negative likelihood ratio (NLR) and the area under the curve (AUC), along with their corresponding 95% confidence intervals (CIs). The statistical analyses were performed using the Review Manager (Revman) 5.4 software and the Meta Disc software. Results: Thirteen studies involving five microRNAs were included in our analysis. Our results suggested that serum miR-29a levels showed great potential as a high diagnostic biomarker for CRC. The overall pooled sensitivity, specificity, area under the curve (AUC), PLR and NLR of circulating miR-29a were 69.75%, 88.25%, 0.8351, 5.93 and 0.342 respectively. Conclusion: miRNAs have become increasingly recognized molecular tools in CRC diagnosis. According to our results, circulating miR-29a level has potential value for early detection and might be used as a novel diagnostic biomarker for CRC in combination with other biomarkers. Citation Format: Sofia Fathi, Fadila Guessous, Mehdi Karkouri. Emerging microRNAs as biomarkers for colorectal cancer diagnosis: A meta-analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1484.

Clinical relevance and prognostic role of preoperative cell-free single-stranded DNA concentrations in colorectal cancer patients.

Circulating cell-free single-stranded DNA (ccf-ssDNA) is extracellular DNA and it is a useful biomarker for the diagnosis of tumors and predicting the prognosis of tumors. However, the clinical usefulness of ccf-ssDNA in colorectal cancer (CRC) is not well known. Thus, the purpose of this study was to investigate the clinical usefulness of ccf-ssDNA in CRC. The study was conducted on 44 patients who had undergone surgery for CRC, and ccf-ssDNA level was measured before surgery and statistical analysis was performed on clinical factors. The association between ccf-ssDNA level and clinicopathological factors was analyzed and compared, and these factors included age, sex, body mass index, diabetes mellitus, hypertension, tumor markers (carcinoembryonic antigen and carbohydrate antigen 19-9), tumor location, size, stage (TNM), recurrence, and death. The group with a ccf-ssDNA level of ≥7.5 ng/μL had a lower age (P=0.010), and was associated with diabetes mellitus (P=0.037) and lymph node metastasis (P=0.049). Multivariate analysis of disease-free survival showed that lymph node metastasis and ccf-ssDNA level (hazard ratio, 10.011; 95% confidence interval, 2.269-44.175; P=0.002) were independent prognostic factors for recurrence. In terms of overall survival, there were no statistically significant results except for vascular invasion. This study showed that ccf-ssDNA level in plasma in CRC patients was an independent prognostic factor that could predict recurrence non-invasively. In this regard, further evaluation with a prospective, large sample size study will be needed to obtain additional results.

New Circulating Circular RNAs with Diagnostic and Prognostic Potential in Advanced Colorectal Cancer.

Circular RNAs (circRNAs) are a group of special endogenous long non-coding RNAs which are highly stable in the circulation, and, thus, more suitable as new biomarkers of colorectal cancer (CRC). The aim of our study was to explore the plasma expression levels of four circRNAs: has_circ_0001445, hsa_circ_0003028, hsa_circ_0007915 and hsa_circ_0008717 in patients with CRC and to evaluate their associations with clinicopathological characteristics and the clinical outcome of the patients. CircRNAs were extracted from patients’ plasma obtained prior to chemotherapy. Their expression levels were measured by qPCR and calculated applying the 2−ΔΔCt method. The levels of all four circRNAs were significantly increased in the plasma of CRC patients. At the optimal cut-off values hsa_circ_0001445 and hsa_circ_0007915 in plasma could significantly distinguish between patients with or without metastatic CRC with 92.56% sensitivity and 42.86% specificity, and with 86.07% sensitivity and 57.14% specificity, respectively. The mean overall survival (OS) of patients with high/intermediate expression of hsa_circ_0001445 was 30 months, significantly higher in comparison with the mean OS of the patients with low expression—20 months (log-rank test, p = 0.034). In multivariate Cox regression analysis, the low levels of hsa_circ_0001445 were also associated with shorter survival (HR = 1.59, 95% CI: 1.02–2.47, p = 0.040). A prognostic significance of hsa_circ_0001445 for patients with metastatic CRC was established.

LncRNA ITGB8-AS1 functions as a ceRNA to promote colorectal cancer growth and migration through integrin-mediated focal adhesion signaling

Long non-coding RNAs (lncRNAs) play critical roles in tumorigenesis and progression of colorectal cancer (CRC). However, functions of most lncRNAs in CRC and their molecular mechanisms remain uncharacterized. Here we found that lncRNA ITGB8-AS1 was highly expressed in CRC. Knockdown of ITGB8-AS1 suppressed cell proliferation, colony formation, and tumor growth in CRC, suggesting oncogenic roles of ITGB8-AS1. Transcriptomic analysis followed by KEGG analysis revealed that focal adhesion signaling was the most significantly enriched pathway for genes positively regulated by ITGB8-AS1. Consistently, knockdown of ITGB8-AS1 attenuated the phosphorylation of SRC, ERK, and p38 MAPK. Mechanistically, ITGB8-AS1 could sponge miR-33b-5p and let-7c-5p/let-7d-5p to regulate the expression of integrin family genes ITGA3 and ITGB3, respectively, in the cytosol of cells. Targeting ITGB8-AS1 using antisense oligonucleotide (ASO) markedly reduced cell proliferation and tumor growth in CRC, indicating the therapeutic potential of ITGB8-AS1 in CRC. Furthermore, ITGB8-AS1 was easily detected in plasma of CRC patients, which was positively correlated with differentiation and TNM stage, as well as plasma levels of ITGA3 and ITGB3. In conclusion, ITGB8-AS1 functions as a competing endogenous RNA (ceRNA) to regulate cell proliferation and tumor growth of CRC via regulating focal adhesion signaling. Targeting ITGB8-AS1 is effective in suppressing CRC cell growth and tumor growth. Elevated plasma levels of ITGB8-AS1 were detected in advanced-stage CRC. Thus, ITGB8-AS1 could serve as a potential therapeutic target and circulating biomarker in CRC.

Circulating lncRNA UCA1 and lncRNA PGM5-AS1 act as potential diagnostic biomarkers for early-stage colorectal cancer.

Background: Colorectal cancer (CRC) is one of the most common and significant malignant diseases worldwide. In the present study, we evaluated two long non-coding RNAs (lncRNAs) in CRC patients as diagnostic markers for early-stage CRC.Methods: Using Gene Expression Omnibus (GEO) datasets GSE102340, GSE126092, GSE109454 and GSE115856, 14 differentially expressed lncRNAs were identified between cancer and adjacent tissues, among which, the two most differentially expressed were confirmed using quantitative real-time polymerase chain reaction (qRT-PCR) in 200 healthy controls and 188 CRC patients. A receiver operating characteristic (ROC) analysis was employed to evaluate the diagnostic accuracy for CRC.Results: From four GEO datasets, three up-regulated and eleven down-regulated lncRNAs were identified in CRC tissues, among which, lncRNA urothelial carcinoma-associated 1 (UCA1) and lncRNA phosphoglucomutase 5-antisense RNA 1 (PGM5-AS1) were the most significantly up- and down-regulated lncRNAs in CRC patient plasma, respectively. The area under the ROC curve was calculated to be 0.766, 0.754 and 0.798 for UCA1, PGM5-AS1 and the combination of these two lncRNAs, respectively. Moreover, the diagnostic potential of these two lncRNAs was even higher for the early stages of CRC. The combination of UCA1 and PGM5-AS1 enhanced the AUC to 0.832, and when the lncRNAs were used with carcinoembryonic antigen (CEA), the AUC was further improved to 0.874.Conclusion: In the present study, we identified two lncRNAs, UCA1 and PGM5-AS1, in CRC patients’ plasma, which have the potential to be used as diagnostic biomarkers of CRC.

P-245 Evaluation of colon-specific plasma nanovesicles as new markers of colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide, which is associated with low detection efficiency in the early stages of its development. The traditional methods of CRC screening are not effective enough. The liquid biopsy, based on the identification of extracellular nanovesicles (ENVs) in plasma, appears to be a promising diagnostic technology. Current approaches ENV-based cancer diagnostics suppose either quantification of tumor-derived ENVs or detection of cancer-associated components (e.g. miRNA) in total population of plasma ENVs. The first approach lacks specificity due to an absence of well-established vesicular membrane-associated markers of CRC. The second approach lacks sensitivity due to the extremely heterogeneous composition of plasma ENVs derived mostly from endothelium and blood cells. We hypothesized that development of a well-differentiated adenocarcinoma from colon epithelium should result in increased release of ENVs bearing markers of the intestinal differentiation. If this hypothesis is true, these vesicles might serve as markers of CRC. Plasma samples from CRC patients (n=48) and healthy donors (n.=50) were used. Design of study was confirmed by the local ethics committee, each participant signed a form of informed consent. The total population of ENVs was isolated from plasma using a two-phase polymer system and analyzed in terms of size, concentration, morphology and surface markers using NTA, Cryo-EM and on-bead flow cytometry. To isolate tissue-specific exosomes, we used streptavidin-coated super-paramagnetic particles (SPMP) and biotinylated antibodies to CLRN3, GPA33, GCNT3, PIGY, REG4, and other ten potential markers. The efficiency of the immune-sorption of such exosomes to SPMP was assessed by flow cytometry after labeling with CD63 FITC and/or CD9 PerCP-Cy5.5 antibodies. Potential markers were selected on the basis of their colon-specific expression pattern and cell surface membrane-associated localization. We selected 16 potential markers and evaluated the amount of ENVs positive for these markers in plasma of CRC patients and healthy donors. The amount of ENVs bearing CLRN3, GPA33, GCNT3, PIGY and REG4 differed between groups of patients and donors, difference was statistically significant. The value of AUC varied between 0,6 and 0,81. Multiplexed analysis allowed us to observe an increased difference between compared groups (CRC vs HD) and to achieve AUC = 0.72. The presented study is the first attempt to explore intestine-specific ENVs as a marker of cancer arising from this tissue. Our results confirmed the initial hypothesis and showed the effectiveness of the proposed approach. Further investigations are still required to identify the best markers of colon epithelium cell-derived ENVs, to better understand the link between CRC characteristics and the profile of tumor-released ENVs and to optimize techniques of quantification of the intestine-specific ENVs in plasma.

Abstract LB059: Dual methylation and mutation analysis from a single input of plasma cfDNA without sample splitting

Abstract Genomic and epigenetic changes are known to be the underlying mechanism of cancer development. Assessing the genomic (mutation) and epigenetic (methylation) changes has become increasingly important for cancer detection and treatment selection, especially in the field of cfDNA-based liquid biopsy. Targeted Next-Generation Sequencing (NGS) enables in-depth analysis of specific genomic regions that is essential for the clinical application of liquid biopsy. However, current targeted NGS assays are complicated, lengthy, and inefficient. Two major hurdles of existing targeted NGS methods make them impractical for the clinical setting. First, there is no comprehensive, end to end, kit solution available for targeted methylation sequencing (TMS) from sample to sequencing ready library. Second, the low yield of cfDNA from clinical blood samples presents a major challenge for multi-modal analyses on one sample. Thus, an assay that is capable of both methylation and mutation analysis would be optimal for clinical research and future diagnostic assays.Here, we report the performance of Point-n-Seq, a kit solution that can provide in-depth DNA analysis with highly flexible and customizable focused panels to enable both methylation and mutation analysis without sample splitting. Custom panels of 10 to 1000s of markers can be designed with &amp;gt;99% first-pass success rate. We conducted both performance validation and multi-center, multi-operator, reproducibility studies. With spike-in titration of cancer cell-line gDNA with known mutation and methylation profile, we achieved a reliable detection level down to 0.003% of tumor DNA with a linear relationship between the measured and expected fractions.In a pilot study, we designed a colorectal cancer (CRC) TMS panel covering 560 methylation markers and a mutation panel with &amp;gt;350 hotspot mutations in 22 genes. From only 1ml of plasma from late stage CRC patients, we detected cancer-specific methylation signals in all samples tested, and hotspot mutations with clinical implications. Next, a tumor-informed mutation panel covering approximately 100 personalized mutations for each patient was compared to the tumor-independent CRC methylation panel to analyze 23 stage I and II CRC patient plasma samples. The initial results showed that tumor-independent TMS assay achieved a comparable or superior limit of detection compared to the personalized tumor-informed approach. Moreover, to further improve assay sensitivity, metrics for cfDNA size can be integrated into the analysis of the same workflow without sample splitting. The Point-n-Seq kit, with a highly streamlined workflow and dual methylation and mutation analysis, provides a highly sensitive and robust non-invasive method for cancer detection, monitoring and treatment guidance. Citation Format: Grace Q. Zhao, Yun Bao, Heng Wang, Vanita S. Natu, Jayashree Joshi, Yingmin Wang, Elena Arzumanyan, Wanping Wang, Jianmin Wang, John Coller, Shengrong Lin. Dual methylation and mutation analysis from a single input of plasma cfDNA without sample splitting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB059.

The circular RNA circSPARC enhances the migration and proliferation of colorectal cancer by regulating the JAK/STAT pathway

BackgroundNoncoding RNAs such as circular RNAs (circRNAs) are abundant in the human body and influence the occurrence and development of various diseases. However, the biological functions of circRNAs in colorectal cancer (CRC) are largely unknown.MethodsRT-qPCR was used to detect the expression of circRNAs and mRNA in CRC cells and tissues. Fluorescence in situ hybridization (FISH) was used to analyze the location of circSPARC. Function-based experiments were performed using circSPARC knockdown and overexpression cell lines in vitro and in vivo, including CCK8, colony formation, transwell and metastasis models. Mechanistically, luciferase reporter assay, western blots, RNA immunoprecipitation (RIP), Chromatin isolation by RNA purification (ChIRP) and immunohistochemical stainings were performed.ResultsCircSPARC was upregulated in both the tissues and plasma of CRC patients. High expression of circSPARC was associated with advanced TNM stage, lymph node metastases, and poor survival. Silencing circSPARC inhibited CRC cell migration and proliferation in vitro and vivo. Mechanistically, circSPARC sponged miR-485-3p to upregulate JAK2 expression and ultimately contribute to the accumulation of phosphorylated (p)-STAT3. Besides, circSPARC recruited FUS, which facilitated the nuclear translocation of p-STAT3.ConclusionsThese findings suggest that circSPARC might serve as a potential diagnostic and prognostic biomarker and a therapeutic target for CRC treatment by regulating JAK2/STAT3 pathway.

Selected Articles from This Issue

Angiosarcomas (AS) are rare, aggressive tumors arising from vascular tissues, and harbor extensive genomic events such as driver gene mutations and chromosomal translocations. However, recurrent fusion products and specific genomic events that promote disease progression have thus far proven elusive. In this study, Kim and colleagues employ RNA sequencing data from human angiosarcomas and canine hemangiosarcomas—which closely mirror human AS—and identify 25 novel fusion products among the human and canine samples. Though the fusions were themselves non-recurrent, they co-occured alongside recurrent mutations in TP53, PIK3CA, PIK3R1, and NRAS, suggesting that genomic instability induced by these mutations may play a role in the genomic complexity of the disease. Further, the novel fusions were uniformly associated with increased angiogenic signaling, indicating that diverse genomic events in AS likely converge on the activation of a specific transcriptional program. Taken together, the data nominate a host of new molecular targets for investigation and clarify a unifying role for diverse genomic events in the pathogenesis of AS.Extracellular vesicles (EV) have emerged as a key mediator of intercellular signaling and have been linked to the pathogenesis of cancer. EV are loaded with different “cargo” depending on their source, and the characterization of these cargoes is important to understanding and contextualizing their role. Here, Ikeda and colleagues use proteomics to quantitatively probe the differences between EV from cancerous versus normal colon. Employing an ex vivo method for harvesting tissue-exudative EV (Te-EV) from patient samples, the authors find that the amino acid transporter CAT1 is enriched in Te-EV from colorectal cancer (CRC). CAT1-positive EV were found at higher abundance in the plasma of CRC patients and were shown to mediate CRC pathogenesis: by modulating the metabolism of arginine and nitric oxide in endothelial cells, CAT1-positive EV from CRC tissues promoted vascular cell proliferation and tubule formation. These observations identify a key mechanism underlying angiogenesis in CRC and nominate CAT1-positive EV as a potential plasma biomarker for CRC development and progression.Oncogene signaling is a potent driver of tumorigenesis and progression, but cells inherently resist oncogenic transformation via feedback loops leading to cellular senescence. Understanding how oncogene-induced senescence (OIS) is subverted by tumors is critical to the development of new methods to clinically manage cancers driven by BRAF- and RAS-activating mutations. Here, Garnett and colleagues employ a genetic screen to identify mediators of OIS in the context of BRAF activation. The authors found that Amylin, a 37-amino acid peptide that promotes glycolysis, is a key molecular determinant of the response to oncogene signaling. Amylin-ablated cells underwent a series of metabolomic and epigenetic changes that circumvented the onset of senescence in response to BRAF signaling. However, exogenous supplementation of Amylin to the cell growth medium restored the ability of cells to enter a senescent state. These data identify Amylin as a key control node governing the response to oncogene signaling in cancer cells, and suggest that Amylin treatment may hold clinical benefit for patients with oncogene-driven cancers.Acute myeloid leukemia (AML) is a genetically diverse cancer with over ten different molecular subtypes characterized by specific genomic events. EGFR pathway signaling has also been described in AML cells, but most AML cells paradoxically do not express any EGFR family members on the cell surface. In this study, Kam and colleagues address this discrepancy and identify intracellular expression of truncated ERBB2 isoforms in AML cells and myelodysplastic syndrome, an AML precursor lesion. These ERBB2 isoforms—which are also known to be pathogenic in breast cancer—mediated active signaling via classical EGFR-related pathways and were sensitive to inhibition with ERBB2 tyrosine kinase inhibitors. While ERBB2 inhibition was effective as a single agent, combination of the ERBB2 inhibitor lapatinib with the BCL-2 inhibitor venetoclax resulted in marked synergism at all tested doses. These observations nominate ERBB2 inhibitors for further investigation to complement the growing arsenal of therapeutic options for AML patients.

A novel proteomics approach to epigenetic profiling of circulating nucleosomes

Alteration of epigenetic modifications plays an important role in human cancer. Notably, the dysregulation of histone post-translational modifications (PTMs) has been associated with several cancers including colorectal cancer (CRC). However, the signature of histone PTMs on circulating nucleosomes is still not well described. We have developed a fast and robust enrichment method to isolate circulating nucleosomes from plasma for further downstream proteomic analysis. This method enabled us to quantify the global alterations of histone PTMs from 9 CRC patients and 9 healthy donors. Among 54 histone proteoforms identified and quantified in plasma samples, 13 histone PTMs were distinctive in CRC. Notably, methylation of histone H3K9 and H3K27, acetylation of histone H3 and citrullination of histone H2A1R3 were upregulated in plasma of CRC patients. A comparative analysis of paired samples identified 3 common histone PTMs in plasma and tumor tissue including the methylation and acetylation state of lysine 27 of histone H3. Moreover, we highlight for the first time that histone H2A1R3 citrulline is a modification upregulated in CRC patients. This new method presented herein allows the detection and quantification of histone variants and histone PTMs from circulating nucleosomes in plasma samples and could be used for biomarker discovery of cancer.