Extracellular vesicles associated microRNAs: Their biology and clinical significance as biomarkers in gastrointestinal cancers.

Gastrointestinal (GI) cancers, including colorectal, gastric, esophageal, pancreatic, and liver, are associated with high mortality and morbidity rates worldwide. One of the underlying reasons for the poor survival outcomes in patients with these malignancies is late disease detection, typically when the tumor has already advanced and potentially spread to distant organs. Increasing evidence indicates that earlier detection of these cancers is associated with improved survival outcomes and, in some cases, allows curative treatments. Consequently, there is a growing interest in the development of molecular biomarkers that offer promise for screening, diagnosis, treatment selection, response assessment, and predicting the prognosis of these cancers. Extracellular vesicles (EVs) are membranous vesicles released from cells containing a repertoire of biological molecules, including nucleic acids, proteins, lipids, and carbohydrates. MicroRNAs (miRNAs) are the most extensively studied non-coding RNAs, and the deregulation of miRNA levels is a feature of cancer cells. EVs miRNAs can serve as messengers for facilitating interactions between tumor cells and the cellular milieu, including immune cells, endothelial cells, and other tumor cells. Furthermore, recent years have witnessed considerable technological advances that have permitted in-depth sequence profiling of these small non-coding RNAs within EVs for their development as promising cancer biomarkers -particularly non-invasive, liquid biopsy markers in various cancers, including GI cancers. Herein, we summarize and discuss the roles of EV-associated miRNAs as they play a seminal role in GI cancer progression, as well as their promising translational and clinical potential as cancer biomarkers as we usher into the area of precision oncology.

A Circulating Panel of circRNA Biomarkers for the Noninvasive and Early Detection of Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal malignancies. Delayed manifestation of symptoms and lack of specific diagnostic markers lead patients being diagnosed with PDAC at advanced stages. This study aimed to develop a circular RNA (circRNA)-based biomarker panel to facilitate noninvasive and early detection of PDAC. METHODS: A systematic genome-wide discovery of circRNAs overexpressed in patients with PDAC was conducted. Subsequently, validation of the candidate markers in the primary tumors from patients with PDAC was performed, followed by their translation into a plasma-based liquid biopsy assay by analyzing 2 independent clinical cohorts of patients with PDAC and nondisease controls. The performance of the circRNA panel was assessed in conjunction with the plasma levels of cancer antigen 19-9 for the early detection of PDAC. RESULTS: Initially, a panel of 10 circRNA candidates was identified during the discovery phase. Subsequently, the panel was reduced to 5 circRNAs in the liquid biopsy-based assay, which robustly identified patients with PDAC and distinguished between early-stage (stage I/II) and late-stage (stage III/IV) disease. The areas under the curve of this diagnostic panel for the detection of early-stage PDAC were 0.83 and 0.81 in the training and validation cohorts, respectively. Moreover, when this panel was combined with cancer antigen 19-9 levels, the diagnostic performance for identifying patients with PDAC improved remarkably (area under the curve, 0.94) for patients in the validation cohort. Furthermore, the circRNA panel could also efficiently identify patients with PDAC (area under the curve, 0.85) who were otherwise deemed clinically cancer antigen 19-9-negative (<37 U/mL). CONCLUSIONS: A circRNA-based biomarker panel with a robust noninvasive diagnostic potential for identifying patients with early-stage PDAC was developed.

Synthetical lethality of Werner helicase and mismatch repair deficiency is mediated by p53 and PUMA in colon cancer.

Synthetic lethality is a powerful approach for targeting oncogenic drivers in cancer. Recent studies revealed that cancer cells with microsatellite instability (MSI) require Werner (WRN) helicase for survival; however, the underlying mechanism remains unclear. In this study, we found that WRN depletion strongly induced p53 and its downstream apoptotic target PUMA in MSI colorectal cancer (CRC) cells. p53 or PUMA deletion abolished apoptosis induced by WRN depletion in MSI CRC cells. Importantly, correction of MSI abrogated the activation of p53/PUMA and cell killing, while induction of MSI led to sensitivity in isogenic CRC cells. Rare p53-mutant MSI CRC cells are resistant to WRN depletion due to lack of PUMA induction, which could be restored by wildtype (WT) p53 knock in or reconstitution. WRN depletion or treatment with the RecQ helicase inhibitor ML216 suppressed in vitro and in vivo growth of MSI CRCs in a p53/PUMA-dependent manner. ML216 treatment was efficacious in MSI CRC patient-derived xenografts. Interestingly, p53 gene remains WT in the majority of MSI CRCs. These results indicate a critical role of p53/PUMA-mediated apoptosis in the vulnerability of MSI CRCs to WRN loss, and support WRN as a promising therapeutic target in p53-WT MSI CRCs.

Circulating cell-free DNA methylation patterns as non-invasive biomarkers to monitor colorectal cancer treatment efficacy without referencing primary site mutation profiles.

This study investigates methylation patterns in circulating cell-free DNA (ccfDNA) for their potential role in colorectal cancer (CRC) detection and the monitoring of treatment response. Through methylation microarrays and quantitative PCR assays, we analyzed 440 samples from The Cancer Genome Atlas (TCGA) and an additional 949 CRC samples. We detected partial or extensive methylation in over 85% of cases within three biomarkers: EFEMP1, SFRP2, and UNC5C. A methylation score for at least one of the six candidate regions within these genes' promoters was present in over 95% of CRC cases, suggesting a viable detection method. In evaluating ccfDNA from 97 CRC patients and 62 control subjects, a difference in methylation and recovery signatures was observed. The combined score, integrating both methylation and recovery metrics, showed high diagnostic accuracy, evidenced by an area under the ROC curve of 0.90 (95% CI = 0.86 to 0.94). While correlating with tumor burden, this score gave early insight into disease progression in a small patient cohort. Our results suggest that DNA methylation in ccfDNA could serve as a sensitive biomarker for CRC, offering a less invasive and potentially more cost-effective approach to augment existing cancer detection and monitoring modalities, possibly supporting comprehensive genetic mutation profiling.

The Spigelman Staging System and the Risk of Duodenal and Papillary Cancer in Familial Adenomatous Polyposis: A Systematic Review and Meta-Analysis.

INTRODUCTION: Individuals with familial adenomatous polyposis (FAP) have an almost 20% lifetime risk of duodenal adenocarcinoma, currently the leading cause of death in FAP. The Spigelman staging system provides guidance on the surveillance intervals and timing of prophylactic surgery. Still, its accuracy in predicting duodenal and papillary cancer development has not been systematically evaluated. We investigated the sensitivity and cancer risk of the Spigelman stages. METHODS: We performed a systematic review on PubMed, MEDLINE, EMBASE, and Cochrane and used a random-effects model to pool effect sizes. RESULTS: After removing duplicate entries, we screened 1,170 records and included 27 studies for quantitative analysis. Once duodenal polyposis reaches Spigelman stage IV, the risk of duodenal and papillary cancers increased to 25% (95% confidence interval [CI] 12%-45%). However, the sensitivity of Spigelman stage IV for these cancers was low (51%, 95% CI 42%-60%), especially for papillary adenocarcinoma (39%, 95% CI 16%-68%). We investigated the reasons behind these low values and observed that duodenal cancer risk factors included polyps >10 mm, polyp count >20, and polyps with high-grade dysplasia. Risk factors associated with papillary cancer included a papilla with high-grade dysplasia or >10 mm. The evidence on other risk factors was inconclusive. DISCUSSION: The current Spigelman staging system had a low sensitivity for duodenal and papillary adenocarcinomas. Two Spigelman variables (duodenal villous histology and polyp count) and the lack of papilla-specific variables likely contributed to the low sensitivity values for duodenal and papillary cancers, respectively. While clinicians may be familiar with its current form, there is an urgent need to update it.

Role of serine/threonine protein phosphatase PrpN in the life cycle of Bacillus anthracis.

Reversible protein phosphorylation at serine/threonine residues is one of the most common protein modifications, widely observed in all kingdoms of life. The catalysts controlling this modification are specific serine/threonine kinases and phosphatases that modulate various cellular pathways ranging from growth to cellular death. Genome sequencing and various omics studies have led to the identification of numerous serine/threonine kinases and cognate phosphatases, yet the physiological relevance of many of these proteins remain enigmatic. In Bacillus anthracis, only one ser/thr phosphatase, PrpC, has been functionally characterized; it was reported to be non-essential for bacterial growth and survival. In the present study, we characterized another ser/thr phosphatase (PrpN) of B. anthracis by various structural and functional approaches. To examine its physiological relevance in B. anthracis, a null mutant strain of prpN was generated and shown to have defects in sporulation and reduced synthesis of toxins (PA and LF) and the toxin activator protein AtxA. We also identified CodY, a global transcriptional regulator, as a target of PrpN and ser/thr kinase PrkC. CodY phosphorylation strongly controlled its binding to the promoter region of atxA, as shown using phosphomimetic and phosphoablative mutants. In nutshell, the present study reports phosphorylation-mediated regulation of CodY activity in the context of anthrax toxin synthesis in B. anthracis by a previously uncharacterized ser/thr protein phosphatase-PrpN.

Curcumin and colorectal cancer: An update and current perspective on this natural medicine.

Colorectal cancer (CRC) is one of most common malignancies worldwide and its incidence is still growing. In spite of recent advances in targeted therapies, their clinical efficacy has been limited, non-curative and unaffordable. A growing body of literature indicates that CRC is a multi-modal disease, where a variety of factors within the tumor microenvironment play a significant role in its pathogenesis. For instance, imbalance in gut microbial profiles and impaired intestinal barrier function contribute to the overall intestinal inflammation and initiation of CRC. Moreover, persistent chronic inflammation favors a tumor microenvironment for the growth of cancer. In addition, autophagy or 'self-eating' is a surveillance mechanism involved in the degradation of cellular constituents that are generated under stressful conditions. Cancer stem cells (CSCs), on the other hand, engage in the onset of CRC and are able to endow cancer cells with chemo-resistance. Furthermore, the aberrant epigenetic alterations promote CRC. These evidences highlight the need for multi-targeted approaches that are not only safe and inexpensive but offer a more effective alternative to current generation of targeted drugs. Curcumin, derived from the plant Curcuma longa, represents one such option that has a long history of its use for a variety of chronic disease including cancer, in Indian ayurvedic and traditional Chinese medicine. Scientific evidence over the past few decades have overwhelmingly shown that curcumin exhibits a multitude of anti-cancer activities orchestrated through key signaling pathways associated with cancer. In this article, we will present a current update and perspective on this natural medicine - incorporating the basic cellular mechanisms it effects and the current state of clinical evidence, challenges and promise for its use as a cancer preventative and potential adjunct together with modern therapies for CRC patients.

Ginseng mediates its anticancer activity by inhibiting the expression of DNMTs and reactivating methylation-silenced genes in colorectal cancer.

Developing safe and effective therapeutic modalities remains a critical challenge for improving the prognosis of patients with colorectal cancer (CRC). In this regard, targeting epigenetic regulation in cancers has recently emerged as a promising therapeutic approach. Since several natural compounds have recently been shown to be important epigenetic modulators, we hypothesized that Ginseng might exert its anticancer activity by regulating DNA methylation alterations in CRC. In this study, a series of cell culture studies were conducted, followed by their interrogation in patient-derived 3D organoid models to evaluate Ginseng's anticancer activity in CRC. Genome-wide methylation alterations were interrogated by undertaking MethylationEpic BeadChip microarrays. First, 50% inhibitory concentrations (IC50) were determined by cell viability assays, and subsequent Ginseng treatment demonstrated a significant anticancer effect on clonogenicity and cellular migration in CRC cells. Treatment with Ginseng potentiated cellular apoptosis through regulation of apoptosis-related genes in CRC cells. Furthermore, Ginseng treatment downregulated the expression of DNA methyltransferases (DNMTs) and decreased the global DNA methylation levels in CRC cells. The genome-wide methylation profiling identified Ginseng-induced hypomethylation of transcriptionally silenced tumor suppressor genes. Finally, cell culture-based findings were successfully validated in patient-derived 3D organoids. In conclusion, we demonstrate that Ginseng exerts its antitumorigenic potential by regulating cellular apoptosis via the downregulation of DNMTs and reversing the methylation status of transcriptionally silenced genes in CRC.

An exosome-based liquid biopsy signature for pre-operative identification of lymph node metastasis in patients with pathological high-risk T1 colorectal cancer.

BACKGROUND: According to current guidelines, more than 70% of patients with invasive submucosal colorectal cancer (T1 CRC) undergo a radical operation with lymph node dissection, even though only ~ 10% have lymph node metastasis (LNM). Hence, there is imperative to develop biomarkers that can help robustly identify LNM-positive patients to prevent such overtreatments. Given the emerging interest in exosomal cargo as a source for biomarker development in cancer, we examined the potential of exosomal miRNAs as LNM prediction biomarkers in T1 CRC. METHODS: We analyzed 200 patients with high-risk T1 CRC from two independent cohorts, including a training (n = 58) and a validation cohort (n = 142). Cell-free and exosomal RNAs from pre-operative serum were extracted, followed by quantitative reverse-transcription polymerase chain reactions for a panel of miRNAs. RESULTS: A panel of four miRNAs (miR-181b, miR-193b, miR-195, and miR-411) exhibited robust ability for detecting LNM in the exosomal vs. cell-free component. We subsequently established a cell-free and exosomal combination signature, successfully validated in two independent clinical cohorts (AUC, 0.84; 95% CI 0.70-0.98). Finally, we developed a risk-stratification model by including key pathological features, which reduced the false positive rates for LNM by 76% without missing any true LNM-positive patients. CONCLUSIONS: Our novel exosomal miRNA-based liquid biopsy signature robustly identifies T1 CRC patients at risk of LNM in a preoperative setting. This could be clinically transformative in reducing the significant overtreatment burden of this malignancy.

A liquid biopsy signature for predicting early recurrence in patients with gastric cancer.

BACKGROUND: Gastric cancer (GC) patients who experience recurrence within the first year following surgery (early recurrence [ER]) exhibit worse prognosis. Herein, we established a microRNA-based liquid biopsy assay to predict ER in GC patients. METHODS: A comprehensive biomarker discovery was performed by analysing miRNA expression profiling in 271 primary GC tumours. Thereafter, the expression of these biomarkers was validated in 290 GC cases, which included 218 tissues and 72 pre-treatment sera, from two independent institutions. RESULTS: A panel of 8 miRNAs was identified during the initial biomarker discovery, and this panel could robustly predict ER in a tissue-based clinical cohort (area under the curve [AUC]: 0.81). Furthermore, a model combining the miRNA panel, microsatellite instability (MSI) status and tumour size exhibited superior predictive performance (AUC: 0.86), and was defined as a Prediction of Early Recurrence in GC (PERGC) signature, which was successfully validated in another independent cohort (AUC: 0.82). Finally, the PERGC signature was translated into a liquid biopsy assay (AUC: 0.81), and a multivariate regression analysis revealed this signature to be an independent predictor for ER (odds ratio: 11.20). CONCLUSION: We successfully established a miRNA-based liquid biopsy signature that robustly predicts the risk of ER in GC patients.

Genome-wide methylation profiling identifies a novel gene signature for patients with synchronous colorectal cancer.

BACKGROUND: There are no robust tools for the diagnosis of synchronous colorectal cancer (SyCRC). Herein, we developed the first methylation signature to identify and characterise patients with SyCRC. METHODS: For biomarker discovery, we analysed the genome-wide methylation profiles of 16 SyCRC and 18 solitary colorectal cancer (SoCRC) specimens. We thereafter established a methylation signature risk-scoring model to identify SyCRC in an independent cohort of 38 SyCRC and 42 SoCRC patients. In addition, we evaluated the prognostic value of the identified methylation profile. RESULTS: We identified six differentially methylated CpG probes/sites that distinguished SyCRC from SoCRC. In the validation cohort, we developed a methylation panel that identified patients with SyCRC from not only larger tumour (AUC = 0.91) but also the paired remaining tumour (AUC = 0.93). Moreover, high risk scores of our panel were associated with the development of metachronous CRC among patients with SyCRC (AUC = 0.87) and emerged as an independent predictor for relapse-free survival (hazard ratio = 2.72; 95% CI = 1.12-6.61). Furthermore, the risk stratification model which combined with clinical risk factors was a diagnostic predictor of recurrence (AUC = 0.90). CONCLUSIONS: Our novel six-gene methylation panel robustly identifies patients with SyCRC, which has the clinical potential to improve the diagnosis and management of patients with CRC.

Clinical significance and functional role of adhesion G-protein-coupled receptors in human pancreatic ductal adenocarcinoma.

BACKGROUND: The adhesion G-protein-coupled receptors (GPCRs) play crucial roles in tumour pathogenesis, however, their clinical significance in pancreatic ductal adenocarcinoma (PDAC) remains unclear. METHODS: We analysed 796 PDAC patients, including 331 from public data sets (TCGA, ICGC and GSE57495) and 465 from independent cohorts (training: n = 321, validation: n = 144). Using in-vitro studies, we confirmed the biological function of the candidate GPCRs. RESULTS: Analysis of all 33 adhesion GPCRs, led to identify GPR115, as the only significant prognostic factor in all public data sets. The patients with high GPR115 expression exhibited significantly poorer prognosis for OS and RFS, in training (P < 0.01, P < 0.01) and validation cohort (P < 0.01, P = 0.04). Multivariate analysis indicated that GPR115 high expression was an independent prognostic factor in both cohorts (HR = 1.43; P = 0.01, HR = 2.55; P < 0.01). A risk-prediction model using Cox regression by incorporating GPR115 and clinicopathological factors accurately predicted 5-year survival following surgery. In addition, GPR115 silencing inhibited cell proliferation and migration in PDAC cells. CONCLUSION: We demonstrated that GPR115 has important prognostic significance and functional role in tumour progression; providing a rationale that this may be a potential therapeutic target in patients with PDAC.

A DNA Methylation-based Epigenetic Signature for the Identification of Lymph Node Metastasis in T1 Colorectal Cancer.

OBJECTIVE: This study aimed to unravel the lymph node metastasis (LNM)-related methylated DNA (mDNA) landscape and develop a mDNA signature to identify LNM in patients with T1 colorectal cancers (T1 CRC). BACKGROUND: Considering the invasiveness of T1 CRC, current guidelines recommend endoscopic resection in patients with LNM-negative, and radical surgical resection only for high-risk LNM-positive patients. Unfortunately, the clinicopathological criteria for LNM risk stratification are imperfect, resulting in frequent misdiagnosis leading to unnecessary radical surgeries and postsurgical complications. METHODS: We conducted genome-wide methylation profiling of 39 T1 CRC specimens to identify differentially methylated CpGs between LNM-positive and LNM-negative, and performed quantitative pyrosequencing analysis in 235 specimens from 3 independent patient cohorts, including 195 resected tissues (training cohort: n=128, validation cohort: n=67) and 40 pretreatment biopsies. RESULTS: Using logistic regression analysis, we developed a 9-CpG signature to distinguish LNM-positive versus LNM-negative surgical specimens in the training cohort [area under the curve (AUC)=0.831, 95% confidence interval (CI)=0.755-0.892; P <0.0001], which was subsequently validated in additional surgical specimens (AUC=0.825; 95% CI=0.696-0.955; P =0.003) and pretreatment biopsies (AUC=0.836; 95% CI=0.640-1.000, P =0.0036). This diagnostic power was further improved by combining the signature with conventional clinicopathological features. CONCLUSIONS: We established a novel epigenetic signature that can robustly identify LNM in surgical specimens and even pretreatment biopsies from patients with T1 CRC. Our signature has strong translational potential to improve the selection of high-risk patients who require radical surgery while sparing others from its complications and expense.

A Liquid Biopsy Signature for the Detection of Patients With Early-Onset Colorectal Cancer.

BACKGROUND & AIMS: Early-onset colorectal cancer (EOCRC) is a distinct clinical and molecular entity with poor survival outcomes compared with late-onset CRC. Although the incidence of EOCRC is rising, current CRC screening strategies have several limitations in diagnostic performance for EOCRC. In view of this clinical challenge, novel and robust biomarkers for detection of EOCRC are necessary. The aim of this study was to develop a circulating micro RNA (miRNA) signature for the diagnosis of patients with EOCRC. METHODS: A systematic discovery approach by analyzing a large, publicly available, noncoding RNA expression profiling dataset (GSE115513) was used. A panel of miRNAs was identified, which was subsequently validated in blood samples from patients with EOCRC in 2 independent cohorts (n = 149) compared with controls (n = 110) and pre/postoperative plasma specimens (n = 22) using quantitative reverse-transcription polymerase chain reaction assays. RESULTS: In the discovery phase, 4 miRNAs were found to be expressed in blood samples. A combination signature of these 4 miRNAs (miR-193a-5p, miR-210, miR-513a-5p, and miR-628-3p) yielded an area under the curve of 0.92 (95% confidence interval, 0.85-0.96) for identification of EOCRC in the training cohort. The miRNA panel performance was then confirmed in an independent validation cohort (area under the curve, 0.88; 95% confidence interval, 0.82-0.93). Moreover, the miRNA panel robustly identified patients with early-stage EOCRC (P < .001). The decreased expression of miRNAs in postsurgery plasma specimens indicated their tumor specificity. CONCLUSIONS: Our novel miRNA signature for the diagnosis of EOCRC has the potential to identify patients with EOCRC with high accuracy for clinical application in the noninvasive diagnosis of EOCRC.

An Exosome-based Transcriptomic Signature for Noninvasive, Early Detection of Patients With Pancreatic Ductal Adenocarcinoma: A Multicenter Cohort Study.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) incidence is rising worldwide, and most patients present with an unresectable disease at initial diagnosis. Measurement of carbohydrate antigen 19-9 (CA19-9) levels lacks adequate sensitivity and specificity for early detection; hence, there is an unmet need to develop alternate molecular diagnostic biomarkers for PDAC. Emerging evidence suggests that tumor-derived exosomal cargo, particularly micro RNAs (miRNAs), offer an attractive platform for the development of cancer-specific biomarkers. Herein, genomewide profiling in blood specimens was performed to develop an exosome-based transcriptomic signature for noninvasive and early detection of PDAC. METHODS: Small RNA sequencing was undertaken in a cohort of 44 patients with an early-stage PDAC and 57 nondisease controls. Using machine-learning algorithms, a panel of cell-free (cf) and exosomal (exo) miRNAs were prioritized that discriminated patients with PDAC from control subjects. Subsequently, the performance of the biomarkers was trained and validated in independent cohorts (n = 191) using quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays. RESULTS: The sequencing analysis initially identified a panel of 30 overexpressed miRNAs in PDAC. Subsequently using qRT-PCR assays, the panel was reduced to 13 markers (5 cf- and 8 exo-miRNAs), which successfully identified patients with all stages of PDAC (area under the curve [AUC] = 0.98 training cohort; AUC = 0.93 validation cohort); but more importantly, was equally robust for the identification of early-stage PDAC (stages I and II; AUC = 0.93). Furthermore, this transcriptomic signature successfully identified CA19-9 negative cases (<37 U/mL; AUC = 0.96), when analyzed in combination with CA19-9 levels, significantly improved the overall diagnostic accuracy (AUC = 0.99 vs AUC = 0.86 for CA19-9 alone). CONCLUSIONS: In this study, an exosome-based liquid biopsy signature for the noninvasive and robust detection of patients with PDAC was developed.

Delphi Initiative for Early-Onset Colorectal Cancer (DIRECt) International Management Guidelines.

BACKGROUND & AIMS: Patients with early-onset colorectal cancer (eoCRC) are managed according to guidelines that are not age-specific. A multidisciplinary international group (DIRECt), composed of 69 experts, was convened to develop the first evidence-based consensus recommendations for eoCRC. METHODS: After reviewing the published literature, a Delphi methodology was used to draft and respond to clinically relevant questions. Each statement underwent 3 rounds of voting and reached a consensus level of agreement of ≥80%. RESULTS: The DIRECt group produced 31 statements in 7 areas of interest: diagnosis, risk factors, genetics, pathology-oncology, endoscopy, therapy, and supportive care. There was strong consensus that all individuals younger than 50 should undergo CRC risk stratification and prompt symptom assessment. All newly diagnosed eoCRC patients should receive germline genetic testing, ideally before surgery. On the basis of current evidence, endoscopic, surgical, and oncologic treatment of eoCRC should not differ from later-onset CRC, except for individuals with pathogenic or likely pathogenic germline variants. The evidence on chemotherapy is not sufficient to recommend changes to established therapeutic protocols. Fertility preservation and sexual health are important to address in eoCRC survivors. The DIRECt group highlighted areas with knowledge gaps that should be prioritized in future research efforts, including age at first screening for the general population, use of fecal immunochemical tests, chemotherapy, endoscopic therapy, and post-treatment surveillance for eoCRC patients. CONCLUSIONS: The DIRECt group produced the first consensus recommendations on eoCRC. All statements should be considered together with the accompanying comments and literature reviews. We highlighted areas where research should be prioritized. These guidelines represent a useful tool for clinicians caring for patients with eoCRC.

Role of GSDM family members in airway epithelial cells of lung diseases: a systematic and comprehensive transcriptomic analysis.

Gasdermin (GSDM) family, the key executioners of pyroptosis, play crucial roles in anti-pathogen and anti-tumor immunities, although little is known about the expression of GSDM in lung diseases at single-cell resolution, especially in lung epithelial cells. We comprehensively investigated the transcriptomic profiles of GSDM members in various lung tissues from healthy subjects or patients with different lung diseases at single cell level, e.g., chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), lung adenocarcinoma (LUAD), or systemic sclerosis (SSC). The expression of GSDM members varied among pulmonary cell types (immune cells, structural cells, and especially epithelial cells) and even across lung diseases. Regarding disease-associated specificities, we found that GSDMC or GSDMD altered significantly in ciliated epithelia of COPD or LUAD, GSDMD in mucous, club, and basal cells of LUAD and GSDMC in mucous epithelia of para-tumor tissue, as compared with the corresponding epithelia of other diseases. The phenomic specificity of GSDM in lung cancer subtypes was noticed by comparing with 15 non-pulmonary cancers and para-cancer samples. GSDM family gene expression changes were also observed in different lung epithelial cell lines (e.g., HBE, A549, H1299, SPC-1, or H460) in responses to external challenges, including lipopolysaccharide (LPS), lysophosphatidylcholine (lysoPC), cigarette smoking extract (CSE), cholesterol, and AR2 inhibitor at various doses or durations. GSDMA is rarely expressed in those cell lines, while GSDMB and GSDMC are significantly upregulated in human lung epithelia. Our data indicated that the heterogeneity of GSDM member expression exists at different cells, pathologic conditions, challenges, probably dependent upon cell biological phenomes, functions, and behaviors, upon cellular responses to external changes, and the nature and severity of lung disease. Thus, the deep exploration of GSDM phenomes may provide new insights into understanding the single-cell roles in the tissue, regulatory roles of the GSDM family in the pathogenesis, and potential values of biomarker identification and development.

Non-coding RNA biomarkers in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, which is usually diagnosed at an advanced stage. The late disease diagnosis, the limited availability of effective therapeutic interventions and lack of robust diagnostic biomarkers, are some of the primary reasons for the dismal 5-year survival rates (∼8%) in patients with PDAC. The pancreatic cancer develops through accumulation of a series of genomic and epigenomic alterations which lead to the transformation of normal pancreatic epithelium into an invasive carcinoma - a process that can take up to 15-20 years to develop, from the occurrence of first initiating mutational event. These facts highlight a unique window of opportunity for the earlier detection of PDAC, which could allow timely disease interception and improvement in the overall survival outcomes in patients suffering from this fatal malignancy. Non-coding RNAs (ncRNAs) have been recognized to play a central role in PDAC pathogenesis and are emerging as attractive candidates for biomarker development in various cancers, including PDAC. More specifically, the ncRNAs play a pivotal role in PDAC biology as they affect tumor growth, migration, and invasion by regulating cellular processes including cell cycle, apoptosis, and epithelial-mesenchymal transition. In this review, we focus on three types of well-established ncRNAs - microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) - and discuss their potential as diagnostic, prognostic and predictive biomarkers in PDAC.

An integrated workflow for biomarker development using microRNAs in extracellular vesicles for cancer precision medicine.

EV-miRNAs are microRNA (miRNA) molecules encapsulated in extracellular vesicles (EVs), which play crucial roles in tumor pathogenesis, progression, and metastasis. Recent studies about EV-miRNAs have gained novel insights into cancer biology and have demonstrated a great potential to develop novel liquid biopsy assays for various applications. Notably, compared to conventional liquid biomarkers, EV-miRNAs are more advantageous in representing host-cell molecular architecture and exhibiting higher stability and specificity. Despite various available techniques for EV-miRNA separation, concentration, profiling, and data analysis, a standardized approach for EV-miRNA biomarker development is yet lacking. In this review, we performed a substantial literature review and distilled an integrated workflow encompassing important steps for EV-miRNA biomarker development, including sample collection and EV isolation, EV-miRNA extraction and quantification, high-throughput data preprocessing, biomarker prioritization and model construction, functional analysis, as well as validation. With the rapid growth of "big data", we highlight the importance of efficient mining of high-throughput data for the discovery of EV-miRNA biomarkers and integrating multiple independent datasets for in silico and experimental validations to increase the robustness and reproducibility. Furthermore, as an efficient strategy in systems biology, network inference provides insights into the regulatory mechanisms and can be used to select functionally important EV-miRNAs to refine the biomarker candidates. Despite the encouraging development in the field, a number of challenges still hinder the clinical translation. We finally summarize several common challenges in various biomarker studies and discuss potential opportunities emerging in the related fields.

A combined treatment with melatonin and andrographis promotes autophagy and anticancer activity in colorectal cancer.

Colorectal cancer (CRC) is one of the most frequent malignancies worldwide and remains one of the leading causes of cancer-related deaths in the USA. The high degree of morbidity and mortality associated with this disease is largely due to the inadequate efficacy of current treatments as well the development of chemoresistance. In recent years, several pharmaceutical agents screened from natural products have shown the promise to offer a safe, inexpensive and synergistically multi-targeted treatment option in various cancers. Given the growing evidence of anti-carcinogenic properties of two natural compounds, melatonin (MLT) and andrographis (Andro), we aimed to evaluate their synergistic anticancer effects in CRC. We demonstrate that indeed these two compounds possessed a synergistic anticancer effect in terms of their ability to inhibit cell viability, suppression of colony-formation and induction of apoptosis (P < 0.05). In line with our in vitro findings, we were able to validate this combinatorial anticancer activity in xenograft animal models (P < 0.001) as well as tumor-derived 3D organoids (P < 0.01). RNA-sequencing analysis revealed candidate pathways and genes that mediated antitumor efficacy of MLT and Andro in CRC, among which autophagy pathway and related genes, including NR4A1, CTSL and Atg12, were found to be primarily responsible for the increased anticancer effect by the two natural products. In conclusion, our data reveal a potent and synergistic therapeutic effect of MLT and Andro in the treatment of CRC and provides a rationale for suppressing autophagy in cancer cells as a potential therapeutic strategy for CRC.