Non-invasive Molecular Biomarkers Research Articles

Application of serum tumor specific protein 70 for prognostic stratification in acute myeloid leukemia

Objective: To assess the value of serum tumor specific protein 70 (SP70) for prognostic stratification in acute myeloid leukemia (AML). Methods: A cohort study design was adopted. 129 newly diagnosed AML patients from September 2022 to January 2024 at the Hematology Department of the First Affiliated Hospital of Nanjing Medical University were included, as well as a control group consisted of 120 healthy individuals and 7 cases with benign hematologic diseases during the same period (total 127 cases). Clinical data were collected from Electronic Medical Records. According to the 2023 edition of the Chinese Leukemia Diagnosis and Treatment Guidelines, AML patients with good or moderate prognosis were categorized as low-to-intermediate risk, while those with poor prognosis were high-risk group. Univariate and multivariate logistic regression analyses were used to identify variables significantly associated with AML prognostic risk. ROC analysis was used to evaluate diagnostic performance. A nomogram for predicting patient prognostic risk was constructed by R 4.0.2 software, and the internal validation was performed using bootstrapping. Results: Among 129 AML patients, there were 71 males (55.0%) and 58 females (45.0%), with 42 (32.6%) classified as high-risk and 87 (67.4%) as low-intermediate risk. The high-risk group had a significantly higher median age [62 (48, 67) years] compared to the low-intermediate risk group [50 (35, 63) years, Z=-2.381, P=0.017], and a significantly higher proportion of males (30 patients, 71.4%) compared to the low-intermediate risk group (41 patients, 47.1%, χ2=6.760, P=0.009). Multivariate logistic regression analysis indicated that serum SP70 (OR=2.54, 95%CI: 1.68-3.84, P<0.001), hemoglobin (HB) (OR=0.96, 95%CI: 0.93-0.99, P<0.05), and bone marrow blast (BM blast) (OR=1.07, 95%CI: 1.02-1.13, P<0.05) were independent risk factors for high-risk prognosis in AML patients. ROC analysis showed that the area under the curve (AUC) for SP70 predicting high-risk patients was 0.908 (cut-off value of 5.74 ng/ml, 95%CI: 0.845-0.952, sensitivity 90.5%, specificity 82.8%). The combined model of serum SP70, HB, and BM blasts had an AUC of 0.931 (95%CI: 0.890-0.973); C-index=0.925 (95%CI: 0.876-0.963),with no statistically significant difference compared to serum SP70 alone (Z=1.693,P>0.05). Conclusion: Serum SP70 may be a promising non-invasive molecular biomarker for prognostic stratification in AML.

Molecular detection of exosomal miRNAs of blood serum for prognosis of colorectal cancer

Colorectal cancer (CRC) is the third most common cancer affecting people. The discovery of new, non-invasive, specific, and sensitive molecular biomarkers for CRC may assist in the diagnosis and support therapeutic decision making. Exosomal miRNAs have been demonstrated in carcinogenesis and CRC development, which makes these miRNAs strong biomarkers for CRC. Deep sequencing allows a robust high-throughput informatics investigation of the types and abundance of exosomal miRNAs. Thus, exosomal miRNAs can be efficiently examined as diagnostic biomarkers for disease screening. In the present study, a number of 660 mature miRNAs were detected in patients diagnosed with CRC at different stages. Of which, 29 miRNAs were differentially expressed in CRC patients compared with healthy controls. Twenty-nine miRNAs with high abundance levels were further selected for subsequent analysis. These miRNAs were either highly up-regulated (e.g., let-7a-5p, let-7c-5p, let-7f-5p, let-7d-3p, miR-423-5p, miR-3184-5p, and miR-584) or down-regulated (e.g., miR-30a-5p, miR-99-5p, miR-150-5p, miR-26-5p and miR-204-5p). These miRNAs influence critical genes in CRC, leading to either tumor growth or suppression. Most of the reported diagnostic exosomal miRNAs were shown to be circulating in blood serum. The latter is a novel miRNA that was found in exosomal profile of blood serum. Some of the predicted target genes of highly expressed miRNAs participate in several cancer pathways, including CRC pathway. These target genes include tumor suppressor genes, oncogenes and DNA repair genes. Main focus was given to multiple critical signaling cross-talking pathways including transforming growth factor β (TGFβ) signaling pathways that are directly linked to CRC. In conclusion, we recommend further analysis in order to experimentally confirm exact relationships between selected differentially expressed miRNAs and their predicted target genes and downstream functional consequences.

High prevalence of CD44 and its ligand low molecular weight hyaluronan in plasma of HNSCC patients: clinical significance.

This study aims to evaluate the role of cancer stem cell marker, CD44, and its ligand HA as potential molecular biomarker for early detection of HNSCC. The expression profile (mRNA/Protein) of CD44 variants were analysed in primary HNSCC lesions and plasma of the patients. Then, prevalence of HA variants was analysed in plasma of the patients. The mRNA expression of CD44 variants, CD44S and CD44v3, were significantly high in both early (stage I/II) and late (stage III/IV) invasive lesions, with predominant expression of CD44v3 in the late-stage lesions. In plasma of HNSCC patients, increased levels of SolCD44, CD44-ICD and unique 62 KD CD44 variants with respect to standard CD44S were seen, in comparison to their prevalence in plasma of normal individuals. The abundance of CD44-ICD and 62 KD variants were significantly high in plasma of late stage HNSCC patients. Interestingly, significantly high level of low molecular weight HA(LMW HA) with respect to high molecular weight HA(HMW HA) was seen in plasma of HNSCC patients irrespective of clinical stages. On the contrary, high HMW HA level in plasma of normal individuals was seen. The high level of LMW HA in plasma of HNSCC patients might be due to combinatorial effect of increased mRNA expression of HA synthesizing enzyme HAS1/2/3 and HA degrading enzyme HYAL1/2, as seen in the primary HNSCC samples. Thus, our data revealed the importance of specific CD44 and HA variants in plasma of HNSCC patients during its development as potential non-invasive molecular biomarker of the disease.

Non-invasive molecular biomarkers for monitoring solid organ transplantation: A comprehensive overview.

Solid organ transplantation is a life-saving intervention for individuals with end-stage organ failure. Despite the effectiveness of immunosuppressive therapy, the risk of graft rejection persists in all viable transplants between individuals. The risk of rejection may vary depending on the degree of compatibility between the donor and recipient for both human leucocyte antigen (HLA) and non-HLA gene-encoded products. Monitoring the status of the allograft is a critical aspect of post-transplant management, with invasive biopsies being the standard of care for detecting rejection. Non-invasive biomarkers are increasingly being recognized as valuable tools for aiding in the detection of graft rejection, monitoring graft status and evaluating the efficacy of immunosuppressive therapy. Here, we focus on the importance of molecular biomarkers in solid organ transplantation and their potential role in clinical practice. Conventional molecular biomarkers used in transplantation include HLA typing, detection of anti-HLA antibodies, killer cell immunoglobulin-like receptor genotypes, and anti-MHC class 1-related chain A antibodies, which are important for assessing the compatibility of the donor and recipient. Emerging molecular biomarkers include the detection of donor-derived cell-free DNA, microRNAs (regulation of gene expression), exosomes (small vesicles secreted by cells), and kidney solid organ response test, in the recipient's blood for early signs of rejection. This review highlights the strengths and limitations of these molecular biomarkers and their potential role in improving transplant outcomes.

Peak amplitude of the normalized power spectrum of the electromyogram of the uterus in the low frequency band is an effective predictor of premature birth.

The current trends in the development of methods for non-invasive prediction of premature birth based on the electromyogram of the uterus, i.e., electrohysterogram (EHG), suggest an ever-increasing use of large number of features, complex models, and deep learning approaches. These "black-box" approaches rarely provide insights into the underlying physiological mechanisms and are not easily explainable, which may prevent their use in clinical practice. Alternatively, simple methods using meaningful features, preferably using a single feature (biomarker), are highly desirable for assessing the danger of premature birth. To identify suitable biomarker candidates, we performed feature selection using the stabilized sequential-forward feature-selection method employing learning and validation sets, and using multiple standard classifiers and multiple sets of the most widely used features derived from EHG signals. The most promising single feature to classify between premature EHG records and EHG records of all other term delivery modes evaluated on the test sets appears to be Peak Amplitude of the normalized power spectrum (PA) of the EHG signal in the low frequency band (0.125-0.575 Hz) which closely matches the known Fast Wave Low (FWL) frequency band. For classification of EHG records of the publicly available TPEHG DB, TPEHGT DS, and ICEHG DS databases, using the Partition-Synthesis evaluation technique, the proposed single feature, PA, achieved Classification Accuracy (CA) of 76.5% (AUC of 0.81). In combination with the second most promising feature, Median Frequency (MF) of the power spectrum in the frequency band above 1.0 Hz, which relates to the maternal resting heart rate, CA increased to 78.0% (AUC of 0.86). The developed method in this study for the prediction of premature birth outperforms single-feature and many multi-feature methods based on the EHG, and existing non-invasive chemical and molecular biomarkers. The developed method is fully automatic, simple, and the two proposed features are explainable.

Expression of microRNAs ‘let-7d and miR-195’ and Apoptotic Genes ‘BCL2 and Caspase-3’ as Potential Biomarkers of Female Breast Carcinogenesis

Objective: Breast cancer (BC) is the most common cause of cancer-related death among women worldwide. Let-7d and microRNA-195 (miR-195) are members of microRNAs that are known tumor suppressors and are involved in the regulation of apoptosis, invasion, and other cellular functions. However, the roles of these microRNAs in BC progression remain controversial. This study aimed to explore the correlation between the expression of let-7d and miR-195 and apoptosis-related genes (ARGs) “B-cell lymphoma 2 (BCL2) and caspase-3 (CASP3)” as potential biomarkers of breast carcinogenesis. Methods: It was a retrospective case-control study in which expression of let-7d, miR-195, CASP3, and BCL2 was assessed using quantitative real-time PCR (qRT-PCR); and immunohistochemical (IHC) staining was used to determine expression of BCL2 and CASP3 in BC tissue versus normal breast tissue (NT) samples. Results: The expression of let-7d and miR-195 was significantly reduced within BC tissues compared to NT (P: &lt; 0.0001); and there was a statically positive correlation between them (r=0.314, P: 0.005). They have also been correlated to biomarkers’ expression of genes related to apoptosis. There was a statistically significant positive association between CASP3, and both let-7d, and miR-195 relative gene expression (r=0.713, P: &lt;0.0001 and r=0.236, P: 0.03, respectively). In contrast, there was a statistically significant negative association between the relative gene expression of BCL2, with let-7d, and miR-195 (r=-0.221, P: 0.04 and r=-0.311, P: 0.005, respectively). Conclusion: Let-7d and miR-195 have been suggested to be involved in BC through modulation of the ARGs including BCL2 and CASP3. The qRT-PCR and IHC studies demonstrated that decreased expression of let-7d and miR-195 prohibits apoptosis via downregulating CASP3 and increasing BCL2 expressions promoting BC progression. These results also hypothesize that let-7d and miR-195 along with apoptotic biomarkers (BCL2 and CASP3) can be used in the future to introduce novel, non-invasive molecular biomarkers for BC into clinical practice.

Urinary-derived extracellular vesicle microRNAs as non‐invasive diagnostic biomarkers for early-stage renal cell carcinoma

Background and aimsThe potential of urinary-derived extracellular vesicle (uEV) microRNAs (miRNAs) as noninvasive molecular biomarkers for identifying early-stage renal cell carcinoma (RCC) patients is rarely explored. The present study aims to explore the possibility of uEV miRNAs as novel molecular biomarkers for distinguishing early-stage RCC. Materials and methodsuEVs were extracted by ExoQuick-TC™ kit and miRNA concentrations were measured by RT-qPCR. ROC curves and bioinformatics analysis were employed to predict the diagnostic efficacy and regulatory mechanisms of dysregulated miRNAs. ResultsThrough a multiphase case-control study on uEV miRNAs screening, training, and validation in RCC cells (ACHN, Caki-1) and control cells (HK-2) and in uEVs of 125 RCC patients and 128 age- and sex-matched controls, we successfully identified four uEVs miRNAs (miR-135b-5p, miR-196b-5p, miR-200c-3p, and miR-203a-3p) were significantly and stably upregulated in RCC in vitro and in vivo. When adjusted with estimated glomerular filtration rate (eGFR), the AUC of the three-uEV miRNA panel (miR-135b-5p, miR-200c-3p, and miR-203a-3p) was 0.785 (95 % CI = 0.729–0.842, P < 0.0001) for discriminating RCC patients from controls. Notably, this panel exhibited similar performance in distinguishing early-stage (stage Ⅰ) RCC patients, with an AUC of 0.786 (95 %CI = 0.727–0.844, P < 0.0001). Bioinformatics analysis predicted that candidate miRNAs were involved in cancer progressing. ConclusionOur study identified a four uEV miRNAs panel (miR-135b-5p, miR-196b-5p, miR-200c-3p, and miR-203a-3p) may serve as an auxiliary noninvasive indication of early-stage RCC.

Characterization of a miRNA Signature with Enhanced Diagnostic and Prognostic Power for Patients with Bladder Carcinoma.

Bladder carcinoma is globally among the most prevalent cancers and is associated with a high mortality rate at advanced stages. Its detection relies on invasive diagnostic methods that are unpleasant for the patient. Non-invasive molecular biomarkers, such as miRNAs, could serve as alternatives for early detection and prognosis of this malignancy. We designed a computational approach that combines transcriptome profiling, survival analyses, and calculation of diagnostic power in order to isolate miRNA signatures with high diagnostic and prognostic utility. Our analysis of TCGA-BLCA data from 429 patients yielded one miRNA signature, consisting of five upregulated and three downregulated miRNAs with cumulative diagnostic power that outperforms current diagnostic methods. The same miRNAs have a strong prognostic significance since their expression is associated with the overall survival of bladder cancer patients. We evaluated the expression of this signature in 19 solid cancer types, supporting its unique diagnostic utility for bladder carcinoma. We provide computational evidence regarding the functional implications of this miRNA signature in cell cycle regulation, demonstrating its abundance in body fluids, including peripheral blood and urine. Our study characterized a novel miRNA signature with the potential to serve as a non-invasive method for bladder cancer diagnosis and prognosis.

Association of XIST/miRNA155/Gab2/TAK1 cascade with the pathogenesis of anti-phospholipid syndrome and its effect on cell adhesion molecules and inflammatory mediators.

Anti-phospholipid syndrome (APS) is an autoimmune disease characterized by thrombosis and miscarriage events. Still, the molecular mechanisms underlying APS, which predisposes to a wide spectrum of complications, are being explored. Seventy patients with primary and secondary APS were recruited, in addition to 35 healthy subjects. Among APS groups, the gene expression levels of XIST, Gab2, and TAK1 were higher along with declined miRNA155 level compared with controls. Moreover, the sera levels of ICAM-1, VCAM-1, IL-1ꞵ, and TNF-α were highly elevated among APS groups either primary or secondary compared with controls. The lncRNA XIST was directly correlated with Gab2, TAK1, VCAM-1, ICAM-1, IL-1ꞵ, and TNF-α. The miRNA155 was inversely correlated with XIST, Gab2, and TAK1. Moreover, ROC curve analyses subscribed the predictive power of the lncRNA XIST and miRNA155, to differentiate between primary and secondary APS from control subjects. The lncRNA XIST and miRNA155 are the upstream regulators of the Gab2/TAK1 axis among APS patients via influencing the levels of VCAM-1, ICAM-1, IL1ꞵ, and TNF-α which propagates further inflammatory and immunological streams. Interestingly, the study addressed that XIST and miRNA155 may be responsible for the thrombotic and miscarriage events associated with APS and provides new noninvasive molecular biomarkers for diagnosing the disease and tracking its progression.

Urinary MicroRNAs as Biomarkers of Urological Cancers: A Systematic Review.

MicroRNAs (miRNAs) are emerging as biomarkers for the detection and prognosis of cancers due to their inherent stability and resilience. To summarize the evidence regarding the role of urinary miRNAs (umiRNAs) in the detection, prognosis, and therapy of genitourinary cancers, we performed a systematic review of the most important scientific databases using the following keywords: (urinary miRNA) AND (prostate cancer); (urinary miRNA) AND (bladder cancer); (urinary miRNA) AND (renal cancer); (urinary miRNA) AND (testicular cancer); (urinary miRNA) AND (urothelial cancer). Of all, 1364 articles were screened. Only original studies in the English language on human specimens were considered for inclusion in our systematic review. Thus, a convenient sample of 60 original articles was identified. UmiRNAs are up- or downregulated in prostate cancer and may serve as potential non-invasive molecular biomarkers. Several umiRNAs have been identified as diagnostic biomarkers of urothelial carcinoma and bladder cancer (BC), allowing us to discriminate malignant from nonmalignant forms of hematuria. UmiRNAs could serve as therapeutic targets or recurrence markers of non-muscle-invasive BC and could predict the aggressivity and prognosis of muscle-invasive BC. In renal cell carcinoma, miRNAs have been identified as predictors of tumor detection, aggressiveness, and progression to metastasis. UmiRNAs could play an important role in the diagnosis, prognosis, and therapy of urological cancers.

Panel of miR-150 and linc00673, regulators of CCR6/CCL20 may serve as non-invasive diagnostic marker of non-small cell lung cancer

The C–C motif ligand 20 (CCL20) is a chemokine that specifically binds to the chemokine receptor 6 (CCR6) and the CCL20/CCR6 axis has been implicated in the non-small lung cancer (NSCLC) development and progression. Its expression is regulated by mutual interactions of non-coding RNAs (ncRNAs). This goals of presented study was to evaluate the expression level of CCR6/CCL20 mRNA in NSCLC tissue comparative to selected ncRNAs: miR-150, linc00673. The expression level of the studied ncRNAs was also assessed in serum extracellular vesicles (EVs). Thirty patients (n = 30) were enrolled as the study cohort. Total RNA was isolated from tumor tissue, adjacent macroscopically unchanged tissue and serum EVs. The expression level of studied genes and ncRNAs were estimated based on the qPCR method. Higher expression level of CCL20 mRNA but lower expression level of CCR6 mRNA were observed in tumor in comparison to control tissue. Relative to the smoking status, higher CCL20 (p < 0.05) and CCR6 mRNA (p > 0.05) expression levels were observed in current smokers than in never smokers. In serum EVs the expression level of miR-150 has a negative correlation with AJCC tumor staging, whereas the expression level of linc00673 positively correlated (p > 0.05). The lower expression level of miR-150 and higher expression level of linc00673 in serum EVs were observed in NSCLC patients with lymph nodes metastases (p > 0.05). Regarding the histopathological type, significantly lower expression level of miR-150 and higher expression level of linc00673 were observed in the serum EVs of patients with AC compared to patient with SCC. Our findings revealed that smoking significantly changed the expression level of CCL20 mRNA in NSCLC tissue. Changes in expression levels of miR-150 and linc00673 in the serum EVs of NSCLC patients in relation to presence of lymph node metastases and the stage of cancer development may serve as a non-invasive molecular biomarkers of tumor progression. Furthermore, expression levels of miR-150 and linc00673 may serve as non-intrusive diagnostic biomarkers differentiating adenocarcinoma from squamous cell carcinoma.

Seminal extracellular vesicle sncRNA sequencing reveals altered miRNA/isomiR profiles as sperm retrieval biomarkers for azoospermia.

Non-invasive molecular biomarkers for classifying azoospermia by origin into either obstructive or non-obstructive/secretory azoospermia, as well as for inferring the spermatogenic reserve of the testis of non-obstructive/secretory azoospermia patients, are of great interest for testicular sperm retrieval outcome prediction for assisted reproduction. Prior analyses of semen small non-coding RNA expression in azoospermia have focused on microRNAs, but there has been a lack of attention on other regulatory small RNA species. In this regard, studying more in-depth expression changes of small non-coding RNA subtypes in small extracellular vesicles from semen of azoospermic individuals could be useful to select additional non-invasive biomarkers with diagnostic/prognostic purposes. A high-throughput small RNA profiling analysis to determine the expression pattern of seminal small extracellular vesicle microRNAs (analyzed at the isomiR level), PIWI-interacting RNAs, and transfer RNA-derived small RNAs in normozoospermic (n = 4) and azoospermic (obstructive azoospermia because of pathological occurring obstruction in the genital tract, n = 4; secretory azoospermic individuals with positive testicular sperm extraction value, n = 5; secretory azoospermic individuals with negative testicular sperm extraction value, n = 4) individuals was carried out. Reverse transcriptase-quantitative real-time polymerase chain reaction validation analysis of selected microRNAs was additionally performed in a larger number of individuals. Clinically relevant quantitative changes in the small non-coding RNA levels contained in semen small extracellular vesicles can be used as biomarkers for the origin of azoospermia and for predicting the presence of residual spermatogenesis. In this regard, canonical isoform microRNAs (n = 185) but also other isomiR variants (n = 238) stand out in terms of numbers and fold-change differences in expression, underlining the need to consider isomiRs when investigating microRNA-based regulation. Conversely, although transfer RNA-derived small RNAs are shown in our study to represent a high proportion of small non-coding RNA sequences in seminal small extracellular vesicle samples, they are not able to discriminate the origin of azoospermia. PIWI-interacting RNA cluster profiles and individual PIWI-interacting RNAs with significant differential expression were also not able to discriminate. Our study demonstrated that expression values of individual and/or combined canonical isoform microRNAs (miR-10a-5p, miR-146a-5p, miR-31-5p, miR-181b-5p; area under the receiver operating characteristic curve >0.8) in small extracellular vesicles provide considerable clinical value in identifying samples with a high likelihood of sperm retrieval while discriminating azoospermia by origin. Although no individual microRNA showed sufficient discriminating power on its own to identify severe spermatogenic disorders with focal spermatogenesis, multivariate microRNA models in semen small extracellular vesicles have the potential to identify those individuals with residual spermatogenesis. Availability and adoption of such non-invasive molecular biomarkers would represent a great improvement in reproductive treatment decision protocols for azoospermia in clinical practice.

Validation of a microRNA liquid biopsy assay for diagnosis and risk stratification of invasive cutaneous melanoma.

Noninvasive molecular biomarkers are needed to improve the early, accurate and precise diagnosis of invasive cutaneous melanoma. To independently validate a previously identified circulating microRNA signature of melanoma (MEL38), and, secondly, to develop a complementary microRNA signature, optimized for prognostication. MicroRNA expression profiling was performed on plasma samples from a multicentre observational case-control study, involving patients with primary or metastatic melanoma, melanoma in situ, nonmelanoma skin cancer, or benign naevi. MicroRNA profiles from patients with length of survival, treatment and sentinel lymph node biopsy (SLNB) data were used to develop the prognostic signature. The primary outcome of interest for MEL38 was its association with melanoma status, including area under the curve, binary diagnostic sensitivity and specificity, and incidence-adjusted positive and negative predictive values. The prognostic signature was assessed using rates of survival per risk group and relationship to conventional predictors of outcome. Circulating microRNA profiles of 372 patients with invasive melanoma and 210 control individuals were generated. The average age of all participants was 59 years; 49% were male. A MEL38 score > 5.5 indicated the presence of invasive melanoma. Overall, 551/582 (95%) of patients were correctly diagnosed, with 93% sensitivity and 98% specificity. MEL38 score ranged from 0 to 10 with an area under the curve of 0.98 (95% confidence interval 0.97-0.99, P < 0.001). A novel prognostic 12-microRNA signature (MEL12) developed from 232 patients identified low-, standard- or high-risk groups, with 94%, 78% and 58% rates of 10-year melanoma-specificsurvival, respectively (log-rank P < 0.001). MEL12 prognostic risk groups were significantly associated with clinical staging (χ2, P < 0.001) and SLNB status (P = 0.027). Patients who were classified as high risk by MEL12 were approximately three times more likely to have melanoma detected in their sentinel lymph nodes compared to low-risk patients. The circulating MEL38 signature may assist in diagnosing patients with invasive melanoma vs. other conditions associated with a lower - or negligible - risk of mortality. A complementary and prognostic MEL12 signature is predictive of SLNB status, clinical stage and probability of survival. Plasma microRNA profiling may help to optimize existing diagnostic pathways as well as enable personalized, risk-informed melanoma treatment decisions.

Clinical Biomarkers and Prediction Models for Poststroke Epilepsy: Have We Settled the Scores Yet?

In an era of time-dependent reperfusion and recanalization therapy for stroke leading to improved survival, there is a growing population at risk of poststroke epilepsy (PSE). Accumulating evidence suggests a multidirectional interaction among stroke, PSE, and dementia in stroke survivors. There is no evidence to justify prophylactic antiseizure medication (ASM) to reduce these morbidities. Although several predictive molecular biomarkers and scoring models have been proposed, they remain inadequately validated for stratifying risk and indicating who will benefit from prophylactic ASM. Studies leveraging advances in genetics, metabolomics, electrophysiology, imaging, and artificial intelligence (AI) may help to discover noninvasive molecular biomarkers and easy-to-score models. These discoveries should improve our understanding of epileptogenesis in PSE and identify new pharmacologic targets. Besides, accurately identifying high-risk patients and timely initiating prophylactic ASM therapy has the potential to disrupt the feed-forward multidirectional interaction among stroke, PSE, and dementia.

Molecular and Immunohistochemical Alterations in Breast Cancer Patients in Upper Egypt.

Breast cancer (BC) plays a major public health in Egyptian woman. In Upper Egypt, there is an increase in incidence of BC compared to other Egyptian areas. Triple-negative BC, estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-neu-negative, is a high-risk BC that lacks the benefit of specific therapy that targets these proteins. Accurate determination of Caveolin-1(Cav-1), Caveolin-2 (Cav-2) and HER-2/neu status have become of major clinical significance in BC by focusing about its role as a tumor marker for response to different therapies. The present study was performed on 73 female BC patients in the South Egypt Cancer Institute. Blood samples were used for Cav-1, Cav-2, and HER-2/neu genes amplification and expression. In addition, immunohistological analysis of mammaglobin, GATA3, ER, PR, and HER-2/neu was done. There was a statistically significant association between Cav-1, 2 and HER-2/neu genes expression and the age of patients (P< 0.001). There are increase in the level of Cav-1, 2 and increase in HER-2/neu mRNA expression in groups treated with chemotherapy and group treated with both chemotherapy and radiotherapy compared to each group baseline level of genes mRNA expression before treatment. On the contrary, the group treated with chemotherapy, radiotherapy and hormonal therapy revealed increase on the level of Cav-1, 2 and HER-2/neu mRNA expression when compared with their baseline for the same patients before treatment. Noninvasive molecular biomarkers such as Cav-1 and Cav-2 have been proposed for use in the diagnosis and prognosis for women with BC.

Abstract 5174: Utilization of Cell free messenger RNA Sequencing for non-invasive molecular profiling of multiple myeloma and Acute Myeloid Leukemia

Abstract Background and Aims: Hematologic malignancies account for approximately 10% of all cancers in the US. In order to improve the treatment and outcome of patients with hematologic malignancies, integration of accurate noninvasive molecular biomarkers to diagnose, monitor therapeutic response and identify disease subtype are critical. Recent advances in next generation sequencing (NGS) have accelerated the utilization of circulating nucleic acids for the development of non-invasive disease biomarkers. We have developed an RNA-Seq platform that allows robust quantification of the circulating cell-free messenger RNA (cf-mRNA) whole transcriptome. Here, we conducted cf-mRNA profiling on plasma samples collected from multiple myeloma and acute myeloid leukemia patients to investigate the feasibility of cell-free transcriptomic profiling as a monitoring tool for hematologic malignancies following bone marrow transplantation and therapeutic treatment. Methods: We performed cf-mRNA RNA-Sequencing on 96 longitudinally collected plasma samples from 3 multiple myeloma and 3 acute myeloid leukemia patients. Cf-mRNA profiles of patients were evaluated using GTEx (tissue type) and Blueprint (cell type), and dysregulation of gene-expression signatures were assessed following bone marrow transplantation and therapeutic treatment. In addition, we sequenced corresponding whole blood cells and buffy coat for comparison. Results: Plasma cf-mRNA profiling showed substantially higher proportion of transcripts from megakaryocyte and non-hematopoietic cells relative to whole blood cellular transcripts, suggesting that the molecular profile of cf-mRNA is more suitable for the assessment of gene-expression dysregulation of bone marrow than whole blood. We further report that non-invasive cf-mRNA profiling captures gene-expression alterations of cancer patients following chemotherapy and bone marrow transplantation. And we observed dysregulation of hematopoietic lineage-specific transcripts in circulation during bone marrow ablation and reconstitution. Lastly, we used cf-mRNA profiling to identify a malignant Ig clone for multiple myeloma patients and were able to monitor bone marrow reconstitution. Conclusions: We demonstrated that cf-mRNA profiling is an effective means to non-invasively assess molecular characteristics that are associated with multiple clinical parameters of hematological malignancies. Our study highlights the potential of cf-mRNA Seq as a platform to develop patient monitoring biomarkers for hematologic malignancies. Citation Format: Shusuke Toden, David A. Ross, Stephen R. Quake, John J. Sninsky. Utilization of Cell free messenger RNA Sequencing for non-invasive molecular profiling of multiple myeloma and Acute Myeloid Leukemia [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 5174.

Clinicopathological utility of miR-203a-3p in diagnosing colorectal cancer.

Biomarkers, such as microRNAs, are helpful in diagnosing colorectal cancer, regulating disease progression, predicting disease recurrence, and determining therapy success. This research aimed to look at the clinicopathological characteristics of serum miRNA-203a-3p expression in colorectal cancer patients. This case-control study was conducted on 43 patients with colorectal cancer and 43 healthy individuals. After RNA extraction, cDNA was synthesized. The expression of miR-203a-3p was measured using RT-qPCR. Demographic and histochemical data were extracted from patient documents. SPSS and GraphPad Prism software were used to analyze the data. The expression of miR-203a-3p in CRC patients was 2.39 times lower than in the control group (p < 0.0001). The miR-203a-3p expression was significantly lower in the CRC tumor stages, tumor grades, and lymph node metastasis compared to the control group (p < 0.0001 each). The ROC curves showed that the AUC was 0.73, and the best cut-point based on the Youden index was 0.3954, 0.7105, 0.5087, and 0.4868 for detecting colorectal cancer (p = 0.0002), tumor grade (p = 0.006), tumor stage (p = 0.001), and lymph node metastasis (p = 0.0011) compared to the control group, respectively. The binary logistic regression analysis was performed on the correlation between BMI, smoking, and cancer inheritance with miR-203a-3p in cancer and control groups. This study's findings revealed that serum miR-203a-3p is a fair non-invasive molecular biomarker for diagnosing and progressing tumor grade, tumor stage, and lymph node metastasis in colorectal cancer. However, further research with higher statistical numbers is needed to strengthen the correlation and be used for diagnostic applications.

Colorectal Cancer Diagnosis: The Obstacles We Face in Determining a Non-Invasive Test and Current Advances in Biomarker Detection.

Simple SummaryColorectal cancer (CRC) is one of the most common cancers in the western world. CRC originates from precursor adenomatous polyps, which may over time develop into cancer. Endoscopic evaluation remains the gold-standard investigation for the disease. In the absence of molecular tools for early detection, the removal of neoplastic adenomas via polypectomy remains an important measure to prevent dysplastic adenomas from evolving into invasive carcinoma. Colonoscopy is an intrusive procedure that provides an uncomfortable experience for patients. Kits for testing for the presence of blood hemoglobin in the stool are now widely used, and DNA methylation-based detection kits have been approved in the USA for testing the stool and plasma, but few other molecular biomarkers have found their way into medical practice. This review summarizes current trends in the detection and screening of CRC and provides a definitive review of emerging molecular biomarkers for CRC.Globally, colorectal cancer (CRC) is the third most common cancer, with 1.4 million new cases and over 700,000 deaths per annum. Despite being one of the most common cancers, few molecular approaches to detect CRC exist. Carcinoembryonic antigen (CEA) is a known serum biomarker that is used in CRC for monitoring disease recurrence or response to treatment. However, it can also be raised in multiple benign conditions, thus having no value in early detection or screening for CRC. Molecular biomarkers play an ever-increasing role in the diagnosis, prognosis, and outcome prediction of disease, however, only a limited number of biomarkers are available and none are suitable for early detection and screening of CRC. A PCR-based Epi proColon® blood plasma test for the detection of methylated SEPT9 has been approved by the USFDA for CRC screening in the USA, alongside a stool test for methylated DNA from CRC cells. However, these are reserved for patients who decline traditional screening methods. There remains an urgent need for the development of non-invasive molecular biomarkers that are highly specific and sensitive to CRC and that can be used routinely for early detection and screening. A molecular approach to the discovery of CRC biomarkers focuses on the analysis of the transcriptome of cancer cells to identify differentially expressed genes and proteins. A systematic search of the literature yielded over 100 differentially expressed CRC molecular markers, of which the vast majority are overexpressed in CRC. In terms of function, they largely belong to biological pathways involved in cell division, regulation of gene expression, or cell proliferation, to name a few. This review evaluates the current methods used for CRC screening, current availability of biomarkers, and new advances within the field of biomarker detection for screening and early diagnosis of CRC.

Comparing miR-16 and miR-1228 as an optimal endogenous control for quantification of circulating microRNAs in colorectal cancer patients

ABSTRACTObjectives:Circulating microRNAs (miRNAs) have been discovered to play a novel role in intercellular communication and cancer biology. They are emerging candidates for noninvasive molecular biomarkers of cancer and other diseases. However, current translational researches have been limited by the lack of consensus on the optimal endogenous control of circulating miRNAs quantitation. In this study, we compared two promising miRNAs, miR-1228 and miR-16, as an endogenous control. The effects of normalizers on the relative quantification of circulating miR-31 in plasma samples of colorectal cancer (CRC) were also assessed.Materials and Methods:The cel-miR-39 was a spiked-in RNA used as an external control and added to plasma samples before RNA extraction. Quantitative real-time polymerase chain reaction technology was used to analyze the expression levels of circulating miRNAs in plasma samples of 4 healthy controls and 14 CRC patients. The expression stability of the candidate controls was compared by Ct analysis and NormFinder algorithms.Results:There was no significant difference in expression level of miR-16 and miR-1228 between healthy control group and before or after therapy of CRC patient groups. The expression of miR-1228 has smaller the range Ct values (28.25-25.64)compared with those of miR-16 (24.91-20.34). The stability value of miR-1228 (0.102) is lower than that of miR-16 (0.350). The expression of miR-1228 endogenous reference candidate has lower stability value and smaller the range Ct values compared with those in miR-16. According to the range Ct values and stability value, miR-1228 is better than miR-16 as endogenous control in CRC patients. There are significant differences in circulating miR-31 expression between healthy control and CRC patients when miR-1228 was used to standardize miR-31 expression.Conclusions:miR-1228 is recommended as a better endogenous control in quantification of circulating miRNAs in CRC patients.

Oxidative stress-related circulating miRNA-27a is a potential biomarker for diagnosis and prognosis in patients with sepsis

BackgroundOxidative stress plays a critical role on the processes of sepsis, and several microRNAs have been identified that may regulate the occurrence of oxidative stress. However, the relation between oxidative stress-related microRNA 27a (miR-27a) and sepsis is unknown. The present study aimed to determine the value of circulating miR-27a for the diagnosis and prognosis of sepsis.MethodsThis retrospective study included 23 patients with sepsis and 25 without sepsis treated at the emergency intensive care unit (EICU) or our institution between January 2019 and January 2020. Levels of circulating miR-27a and levels of oxidative stress-related indicators were measured and compared between sepsis and non-sepsis patients. Receiver operating characteristic (ROC) curve analysis was used to determine diagnostic efficiency of miR-27a.ResultsCirculating miR-27a levels in sepsis patients were higher than those in non-sepsis patients (p < 0.05), and levels were significantly higher in patients that died than those that lived (p < 0.05). In patients with sepsis, circulating miR-27a level was positively correlated with serum malondialdehyde (MDA) level (rs = 0.529, p = 0.007), and negatively correlated with serum glutathione peroxidase (GSH-Px) level (rs = − 0.477, p = 0.016). No significant correlation was observed between circulating miR-27a and serum superoxide dismutase (SOD) in sepsis patients (rs = − 0.340, p = 0.096). The area under the ROC curve (AUC) of miR-27a level for prediction of sepsis was 0.717 (p = 0.009) and for 28-day mortality was 0.739 (p = 0.003).ConclusionsThis study showed that circulating miR-27a level is correlated with oxidative stress and mortality in patients with sepsis, and may serve as a potential non-invasive molecular biomarker.