Saliva-based microRNA diagnostic signature for the superficial peritoneal endometriosis phenotype.

OBJECTIVE: Patients with superficial peritoneal endometriosis (SPE) present with symptoms suggestive of endometriosis but clinical and imaging exams are inconclusive. Consequently, laparoscopy is usually necessary to confirm diagnosis. The present study aimed to evaluate the accuracy of microRNAs (miRNAs) to diagnose patients with SPE from the ENDOmiARN cohort STUDY DESIGN: This prospective study (NCT04728152) included 200 saliva samples obtained between January and June 2021 from women with pelvic pain suggestive of endometriosis. All patients underwent either laparoscopy and/or MRI to confirm the presence of endometriosis. Among the patients with endometriosis, two groups were defined: an SPE phenotype group of patients with peritoneal lesions only, and a non-SPE control group of patients with other endometriosis phenotypes (endometrioma and/or deep endometriosis). Data analysis consisted of two parts: (i) identification of a set of miRNA biomarkers using next-generation sequencing (NGS), and (ii) development of a saliva-based miRNA signature for the SPE phenotype in patients with endometriosis based on a Random Forest (RF) model. RESULTS: Among the 153 patients with confirmed endometriosis, 10.5 % (n = 16) had an SPE phenotype. Of the 2633 known miRNAs, the feature selection method generated a signature of 89 miRNAs of the SPE phenotype. After validation, the best model, representing the most accurate signature had a 100 % sensitivity, specificity, and AUC. CONCLUSION: This signature could constitute a new diagnostic strategy to detect the SPE phenotype based on a simple biological test and render diagnostic laparoscopy obsolete. PRéCIS: We generated a saliva-based signature to identify patients with superficial peritoneal endometriosis which is the most challenging form of endometriosis to diagnose and which is often either misdiagnosed or requires invasive laparoscopy.

Correction: Dabi et al. Clues for Improving the Pathophysiology Knowledge for Endometriosis Using Plasma Micro-RNA Expression. Diagnostics 2022, 12, 175.

In the original publication [...].

New class of RNA biomarker for endometriosis diagnosis: The potential of salivary piRNA expression.

OBJECTIVES: In contrast to miRNA expression, little attention has been given to piwiRNA (piRNA) expression among endometriosis patients. The aim of the present study was to explore the human piRNAome and to investigate a potential piRNA saliva-based diagnostic signature for endometriosis. METHODS: Data from the prospective "ENDOmiRNA" study (ClinicalTrials.gov Identifier: NCT04728152) were used. Saliva samples from 200 patients were analyzed in order to evaluate human piRNA expression using the piRNA bank. Next Generation Sequencing (NGS), barcoding of unique molecular identifiers and both Artificial Intelligence (AI) and machine learning (ML) were used. For each piRNA, sensitivity, specificity, and ROC AUC values were calculated for the diagnosis of endometriosis. RESULTS: 201 piRNAs were identified, none had an AUC ≥ 0.70, and only three piRNAs (piR-004153, piR001918, piR-020401) had an AUC between ≥ 0.6 and < 0.70. Seven were differentially expressed: piR-004153, piR-001918, piR-020401, piR-012864, piR-017716, piR-020326 and piR-016904. The respective correlation and accuracy to diagnose endometriosis according to the F1-score, sensitivity, specificity, and AUC ranged from 0 to 0.862 %, 0-0.961 %, 0.085-1, and 0.425-0.618. A correlation was observed between the patients' age (≥35 years) and piR-004153 (p = 0.002) and piR-017716 (p = 0.030). Among the 201 piRNAs, four were differentially expressed in patients with and without hormonal treatment: piR-004153 (p = 0.015), piR-020401 (p = 0.001), piR-012864 (p = 0.036) and piR-017716 (p = 0.009). CONCLUSION: Our results support the link between piRNAs and endometriosis physiopathology and establish its utility as a potential diagnostic biomarker using saliva samples. Per se, piRNA expression should be analyzed along with the clinical status of a patient.

Value of non-coding RNAs to assess lymph node status in cervical cancer.

Cervical cancer (CC) is the fourth cancer in women and is the leading cause of cancer death in 42 countries. Lymph node metastasis is a determinant prognostic factor, as underlined in the latest FIGO classification. However, assessment of lymph node status remains difficult, despite the progress of imaging such as PET-CT and MRI. In the specific setting of CC, all data underlined the need for new biomarkers easily available to assess lymph node status. Previous studies have underlined the potential value of ncRNA expression in gynecological cancers. In this review, we aimed to evaluate the contribution of ncRNAs in tissue and biofluid samples to determine lymph node status in CC with potential impact on both surgical and adjuvant therapies. In tissue samples, our analysis found that there are arguments to support the role of ncRNAs in physiopathology, differential diagnosis from normal tissue, preinvasive and invasive tumors. In biofluids, despite small studies especially concerning miRNAs expression, promising data opens up new avenue to establish a non-invasive signature for lymph node status as well as a tool to predict response to neo- and adjuvant therapies, thus improving management algorithm of patients with CC.

Endometriosis-associated infertility diagnosis based on saliva microRNA signatures.

RESEARCH QUESTION: Can a saliva-based miRNA signature for endometriosis-associated infertility be designed and validated by analysing the human miRNome? DESIGN: The prospective ENDOmiARN study (NCT04728152) included 200 saliva samples obtained between January 2021 and June 2021 from women with pelvic pain suggestive of endometriosis. All patients underwent either laparoscopy, magnetic resonance imaging, or both. Patients diagnosed with endometriosis were allocated to one of two groups according to their fertility status. Data analysis consisted of identifying a set of miRNA biomarkers using next-generation sequencing, and development of a saliva-based miRNA signature of infertility among patients with endometriosis based on a random forest model. RESULTS: Among the 153 patients diagnosed with endometriosis, 24% (n = 36) were infertile and 76% (n = 117) were fertile. Small RNA-sequencing of the 153 saliva samples yielded approximately 3712 M raw sequencing reads (from ∼13.7 M to ∼39.3 M reads/sample). Of the 2561 known miRNAs, the feature selection method generated a signature of 34 miRNAs linked to endometriosis-associated infertility. After validation, the most accurate signature model had a sensitivity, specificity and area under the curve of 100%. CONCLUSION: A saliva-based miRNA signature for endometriosis-associated infertility is reported. Although the results still require external validation before using the signature in routine practice, this non-invasive tool is likely to have a major effect on care provided to women with endometriosis.

Saliva microRNA signature to diagnose endometriosis: A cost-effectiveness evaluation of the Endotest®.

OBJECTIVE: To evaluate a saliva diagnostic test (Endotest®) for endometriosis compared with the conventional algorithm. DESIGN: A cost-effectiveness analysis with a decision-tree model based on literature data. SETTING: France. POPULATION: Women with chronic pelvic pain. METHODS: Strategy I is the French algorithm, representing the comparator. For strategy II, all patients have an Endotest®. For strategy III, patients undergo ultrasonography to detect endometrioma and patients with no endometrioma detected have an Endotest®. For strategy IV, patients with no endometrioma detected on ultrasonography undergo pelvic magnetic resonance imaging (MRI) to detect endometrioma and/or deep endometriosis. An Endotest® is then performed for patients with a negative result on MRI. MAIN OUTCOMES MEASURES: Costs and accuracy rates and incremental cost-effectiveness ratios (ICERs). Three analyses were performed with an Endotest® priced at €500, €750, and €1000. Probabilistic sensitivity analysis was conducted with Monte Carlo simulations. RESULTS: With an Endotest® priced at €750, the cost per correctly diagnosed case was €1542, €990, €919 and €1000, respectively, for strategies I, II, III and IV. Strategy I was dominated by all other strategies. Strategies IV, III and II were, respectively, preferred for a willingness-to-pay threshold below €473, between €473 and €4670, and beyond €4670 per correctly diagnosed case. At a price of €500 per Endotest®, strategy I was dominated by all other strategies. At €1000, the ICERs of strategies II and III were €724 and €387 per correctly diagnosed case, respectively, compared with strategy I. CONCLUSION: The present study demonstrates the value of the Endotest® from an economic perspective.

Validation of a Salivary miRNA Signature of Endometriosis - Interim Data.

Salivary miRNA Signature of EndometriosisThis interim analysis of the prospective, multicenter, external validation ENDOmiRNA Saliva Test study, confirms the diagnostic performance and reproducibility of the saliva miRNA signature for endometriosis. At a population prevalence of ∼80%, the miRNA signature had a sensitivity of 96.2%, specificity of 95.1%, and area under the curve of 0.96.

Overview of non-coding RNAs in breast cancers.

Breast cancer in women is the second most common cancer and the fifth leading cause of cancer death worldwide. Although earlier diagnosis and detection of breast cancer has resulted in lower mortality rates, further advances in prevention, detection, and treatment are needed to improve outcomes and survival for women with breast cancer as well as to offer a personalized therapeutic approach. It is now well-established that non-coding RNAs (ncRNAs) represent 98% of the transcriptome but in-depth knowledge about their involvement in the regulation of gene expression is lacking. A growing body of research indicates that ncRNAs are essential for tumorigenesis by regulating the expression of tumour-related genes. In this review, we focus on their implication in breast cancer genesis but also report the latest knowledge of their theragnostic and therapeutic role. We highlight the need for accurate quantification of circulating ncRNAs which is determinant to develop reliable biomarkers. Further studies are mandatory to finally enter the era of personalized medicine for women with breast cancer.

A Bioinformatics Approach to MicroRNA-Sequencing Analysis Based on Human Saliva Samples of Patients with Endometriosis.

Endometriosis, defined by the presence of endometrium-like tissue outside the uterus, affects 2-10% of the female population, i.e., around 190 million women, worldwide. The aim of the prospective ENDO-miRNA study was to develop a bioinformatics approach for microRNA-sequencing analysis of 200 saliva samples for miRNAome expression and to test its diagnostic accuracy for endometriosis. Among the 200 patients, 76.5% (n = 153) had confirmed endometriosis and 23.5% (n = 47) had no endometriosis (controls). Small RNA-seq of 200 saliva samples yielded ~4642 M raw sequencing reads (from ~13.7 M to ~39.3 M reads/sample). The number of expressed miRNAs ranged from 1250 (outlier) to 2561 per sample. Some 2561 miRNAs were found to be differentially expressed in the saliva samples of patients with endometriosis compared with the control patients. Among these, 1.17% (n = 30) were up- or downregulated. Among these, the F1-score, sensitivity, specificity, and AUC ranged from 11-86.8%, 5.8-97.4%, 10.6-100%, and 39.3-69.2%, respectively. Here, we report a bioinformatic approach to saliva miRNA sequencing and analysis. We underline the advantages of using saliva over blood in terms of ease of collection, reproducibility, stability, safety, non-invasiveness. This report describes the whole saliva transcriptome to make miRNA quantification a validated, standardized, and reliable technique for routine use. The methodology could be applied to build a saliva signature of endometriosis.

Endometriosis Associated-miRNome Analysis of Blood Samples: A Prospective Study.

The aim of our study was to describe the bioinformatics approach to analyze miRNome with Next Generation Sequencing (NGS) of 200 plasma samples from patients with and without endometriosis. Patients were prospectively included in the ENDO-miRNA study that selected patients with pelvic pain suggestive of endometriosis. miRNA sequencing was performed using an Novaseq6000 sequencer (Illumina, San Diego, CA, USA). Small RNA-seq of 200 plasma samples yielded ~4228 M raw sequencing reads. A total of 2633 miRNAs were found differentially expressed. Among them, 8.6% (n = 229) were up- or downregulated. For these 229 miRNAs, the F1-score, sensitivity, specificity, and AUC ranged from 0-88.2%, 0-99.4%, 4.3-100%, and 41.5-68%, respectively. Utilizing the combined bioinformatic and NGS approach, a specific and broad panel of miRNAs was detected as being potentially suitable for building a blood signature of endometriosis.

MicroRNome analysis generates a blood-based signature for endometriosis.

Endometriosis, characterized by endometrial-like tissue outside the uterus, is thought to affect 2-10% of women of reproductive age: representing about 190 million women worldwide. Numerous studies have evaluated the diagnostic value of blood biomarkers but with disappointing results. Thus, the gold standard for diagnosing endometriosis remains laparoscopy. We performed a prospective trial, the ENDO-miRNA study, using both Artificial Intelligence (AI) and Machine Learning (ML), to analyze the current human miRNome to differentiate between patients with and without endometriosis, and to develop a blood-based microRNA (miRNA) diagnostic signature for endometriosis. Here, we present the first blood-based diagnostic signature obtained from a combination of two robust and disruptive technologies merging the intrinsic quality of miRNAs to condense the endometriosis phenotype (and its heterogeneity) with the modeling power of AI. The most accurate signature provides a sensitivity, specificity, and Area Under the Curve (AUC) of 96.8%, 100%, and 98.4%, respectively, and is sufficiently robust and reproducible to replace the gold standard of diagnostic surgery. Such a diagnostic approach for this debilitating disorder could impact recommendations from national and international learned societies.

Salivary MicroRNA Signature for Diagnosis of Endometriosis.

BACKGROUND: Endometriosis diagnosis constitutes a considerable economic burden for the healthcare system with diagnostic tools often inconclusive with insufficient accuracy. We sought to analyze the human miRNAome to define a saliva-based diagnostic miRNA signature for endometriosis. METHODS: We performed a prospective ENDO-miRNA study involving 200 saliva samples obtained from 200 women with chronic pelvic pain suggestive of endometriosis collected between January and June 2021. The study consisted of two parts: (i) identification of a biomarker based on genome-wide miRNA expression profiling by small RNA sequencing using next-generation sequencing (NGS) and (ii) development of a saliva-based miRNA diagnostic signature according to expression and accuracy profiling using a Random Forest algorithm. RESULTS: Among the 200 patients, 76.5% (n = 153) were diagnosed with endometriosis and 23.5% (n = 47) without (controls). Small RNA-seq of 200 saliva samples yielded ~4642 M raw sequencing reads (from ~13.7 M to ~39.3 M reads/sample). Quantification of the filtered reads and identification of known miRNAs yielded ~190 M sequences that were mapped to 2561 known miRNAs. Of the 2561 known miRNAs, the feature selection with Random Forest algorithm generated after internally cross validation a saliva signature of endometriosis composed of 109 miRNAs. The respective sensitivity, specificity, and AUC for the diagnostic miRNA signature were 96.7%, 100%, and 98.3%. CONCLUSIONS: The ENDO-miRNA study is the first prospective study to report a saliva-based diagnostic miRNA signature for endometriosis. This could contribute to improving early diagnosis by means of a non-invasive tool easily available in any healthcare system.

Clues for Improving the Pathophysiology Knowledge for Endometriosis Using Serum Micro-RNA Expression.

The pathophysiology of endometriosis remains poorly understood. The aim of the present study was to investigate functions and pathways associated with the various miRNAs differentially expressed in patients with endometriosis. Plasma samples of the 200 patients from the prospective "ENDO-miRNA" study were analyzed and all known human miRNAs were sequenced. For each miRNA, sensitivity, specificity, and ROC AUC values were calculated for the diagnosis of endometriosis. miRNAs with an AUC ≥ 0.6 were selected for further analysis. A comprehensive review of recent articles from the PubMed, Clinical Trials.gov, Cochrane Library, and Web of Science databases was performed to identify functions and pathways associated with the selected miRNAs. In total, 2633 miRNAs were found in the patients with endometriosis. Among the 57 miRNAs with an AUC ≥ 0.6: 20 had never been reported before; one (miR-124-3p) had previously been observed in endometriosis; and the remaining 36 had been reported in benign and malignant disorders. miR-124-3p is involved in ectopic endometrial cell proliferation and invasion and plays a role in the following pathways: mTOR, STAT3, PI3K/Akt, NF-κB, ERK, PLGF-ROS, FGF2-FGFR, MAPK, GSK3B/ß-catenin. Most of the remaining 36 miRNAs are involved in carcinogenesis through cell proliferation, apoptosis, and invasion. The three main pathways involved are Wnt/ß-catenin, PI3K/Akt, and NF-KB. Our results provide evidence of the relation between the miRNA profiles of patients with endometriosis and various signaling pathways implicated in its pathophysiology.

Machine learning algorithms as new screening approach for patients with endometriosis.

Endometriosis-a systemic and chronic condition occurring in women of childbearing age-is a highly enigmatic disease with unresolved questions. While multiple biomarkers, genomic analysis, questionnaires, and imaging techniques have been advocated as screening and triage tests for endometriosis to replace diagnostic laparoscopy, none have been implemented routinely in clinical practice. We investigated the use of machine learning algorithms (MLA) in the diagnosis and screening of endometriosis based on 16 key clinical and patient-based symptom features. The sensitivity, specificity, F1-score and AUCs of the MLA to diagnose endometriosis in the training and validation sets varied from 0.82 to 1, 0-0.8, 0-0.88, 0.5-0.89, and from 0.91 to 0.95, 0.66-0.92, 0.77-0.92, respectively. Our data suggest that MLA could be a promising screening test for general practitioners, gynecologists, and other front-line health care providers. Introducing MLA in this setting represents a paradigm change in clinical practice as it could replace diagnostic laparoscopy. Furthermore, this patient-based screening tool empowers patients with endometriosis to self-identify potential symptoms and initiate dialogue with physicians about diagnosis and treatment, and hence contribute to shared decision making.